Reviewer #1 (Public review):

Summary:

The authors constructed a novel HSV-based therapeutic vaccine to cure SIV in a primate model. The novel HSV vector is deleted for ICP34.5. Evidence is given that this protein blocks HIV reactivation by interference with the NFkappaB pathway. The deleted construct supposedly would reactivate SIV from latency. The SIV genes carried by the vector ought to elicit a strong immune response. Together the HSV vector would elicit a shock and kill effect. This is tested in a primate model.

Strengths and weaknesses:

(1) Deleting ICP34.5 from the HSV construct has a very strong effect on HIV reactivation. The mechanism underlying increased activation by deleting ICP34.5 is only partially explored. Overexpression of ICP34.5 has a much smaller effect (reduction in reactivation) than deletion of ICP34.5 (strong activation); this is acknowledged by the authors that no full mechanistic explanation can be given at this moment.

(2) No toxicity data are given for deleting ICP34.5. How specific is the effect for HIV reactivation? A RNA seq analysis is required to show the effect on cellular genes.

A RNA seq analysis was done in the revised manuscript comparing the effect of HSV-1 and deleted vector in J-LAT cells (Fig S5). More than 2000 genes are upregulated after transduction with the modified vector in comparison with the WT vector. Hence, the specificity of upregulation of SIV genes is questioned. Authors do NOT comment on these findings. In my view it questions the utility of this approach.

(3) The primate groups are too small and the results to variable to make averages. In Fig 5, the group with ART and saline has two slow rebounders. It is not correct to average those with the single quick rebounder. Here the interpretation is NOT supported by the data.

Although authors provided some promising SIV DNA data, no additional animals were added. Groups of 3 animals are too small to make any conclusion, especially since the huge variability in response. The average numbers out of 3 are still presented in the paper, which is not proper science.

No data are given of the effect of the deletion in primates. Now the deleted construct is compared with an empty vector containing no SIV genes. Authors provide new data in Fig S2 on the comparison of WT and modified vector in cells from PLWH, but data are not that convincing. A significant difference in reactivation is seen for LTR in only 2/4 donors and in Gag in 3/4 donors. (Additional question what is meaning of LTR mRNA, do authors relate to genomic RNA??)

Discussion

HSV vectors are mainly used in cancer treatment partially due to induced inflammation. Whether these are suitable to cure PLWH without major symptoms is a bit questionable to me and should at least be argued for.

The RNA seq data add on to this worry and should at least be discussed.

Reviewer #2 (Public review):

Summary:

In this article Wen et. al., describe the development of a 'proof-of-concept' bi-functional vector based out of HSV-deltaICP-34.5's ability to purge latent HIV-1 and SIV genomes from cells. They show that co-infection of latent J-lat T-cell lines with a HSV-deltaICP-34.5 vector can reactivate HIV-1 from a latent state. Over- or stable expression of ICP 34.5 ORF in these cells can arrest latent HIV-1 genomes from transcription, even in the presence of latency reversal agents. ICP34.5 can co-IP with- and de-phosphorylate IKKa/b to block its interaction with NF-k/B transcription factor. Additionally, ICP34.5 can interact with HSF1 which was identified by mass-spec. Thus, the authors propose that the latency reversal effect of HSV-deltaICP-34.5 in co-infected JLat cells is due to modulatory effects on the IKKa/b-NF-kB and PP1-HSF-1 pathway.

Next the authors cleverly construct a bifunctional HSV based vector with deleted ICP34.5 and 47 ORFs to purge latency and avoid immunological refluxes, and additionally expand the application of this construct as a vaccine by introducing SIV genes. They use this 'vaccine' in mouse models and show the expected SIV-immune responses. Experiments in rhesus macaques (RM), further elicit potential for their approach to reactivate SIV genomes and at the same time block their replication by antibodies. What was interesting in the SIV experiments is that the dual-functional vector vaccine containing sPD1- and SIV Gag/Env ORFs effectively delayed SIV rebound in RMs and in some cases almost neutralized viral DNA copy detection in serum. Very promising indeed, however there are some questions I wish the authors explored to answer, detailed below.

Overall, this is an elegant and timely work demonstrating the feasibility of reducing virus rebound in animals, and potentially expand to clinical studies. The work was well written, and sections were clearly discussed.

Strengths:

The work is well designed, rationale explained and written very clearly for lay readers.<br /> Claims are adequately supported by evidence and well designed experiments including controls.

Weaknesses:

(1) It looks like ICP0 is also involved in latency reversal effects. More follow-up work will be required to test if this is in fact true.

(2) It is difficult to estimate the depletion of the latent viral reservoir. The authors have tried to address this issue. A more convincing argument to this reviewer will be data to demonstrate that after the bi-functional vaccine, the animals show overall reduction in the number of circulating latent cells. The feasibility to obtain such a result is not clearly demonstrated.

(3) The authors state that the reduced virus rebound detected following bi-functional vaccine delivery is due to latent genomes becoming activated and steady-state neutralization of these viruses by antibody response. This needs to be demonstrated. Perhaps cell-culture experiments from specimen taken from animals might help address this issue. In lab cultures one could create environments without antibody responses, under these conditions one would expect higher level of viral loads being released in response to the vaccine in question.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary:

The authors constructed a novel HSV-based therapeutic vaccine to cure SIV in a primate model. The novel HSV vector is deleted for ICP34.5. Evidence is given that this protein blocks HIV reactivation by interference with the NF-kB pathway. The deleted construct supposedly would reactivate SIV from latency. The SIV genes carried by the vector ought to elicit a strong immune response. Together the HSV vector would elicit a shock and kill effect. This is tested in a primate model.

Thank you for your kind comments and suggestions, which are very helpful in improving our manuscript. We have carefully revised our manuscript and performed additional experiments accordingly, and we now think this version has been substantially improved for your reconsideration.

Strengths and weaknesses:

(1) Deleting ICP34.5 from the HSV construct has a very strong effect on HIV reactivation. Why is no eGFP readout given in Figure 1C as for WT HSV? The mechanism underlying increased activation by deleting ICP34.5 is only partially explored. Overexpression of ICP34.5 has a much smaller effect (reduction in reactivation) than deletion of ICP34.5 (strong activation); so the story seems incomplete.

Thank you for your careful review and kind reminder.

(1) We are sorry for the misunderstanding of Figure 1C. In the experiment of Figue 1C, we used an HSV-1 17 strain containing GFP (HSV-GFP) and HSV-DICP34.5 (recombinant HSV-1 17 strain with ICP34.5 deletion based on HSV-GFP) to reactivate the HIV latency cell line (J-Lat 10.6 cell). Since detecting GFP cannot distinguish between HSV infection and HIV reactivation, we assessed the reactivation by measuring the mRNA levels of HIV LTR upon stimulation with either HSV-GFP or HSV-ΔICP34.5. Actually, in Figure 1B, we had verified the reactivation efficacy by infecting J-Lat 10.6 cells with the HSV-1 17 strain containing GFP (HSV-GFP) and found significant upregulation of mRNA levels of HIV-1 LTR, Tat, Gag, Vif, and Vpr. We have adjusted the corresponding descriptions accordingly in the revised manuscript.

(2) We agree with your insightful mention that the mechanism underlying increased activation by HSV-ΔICP34.5 is worthy to be further explored in the future study. In this study, we found that ICP34.5 play an antagonistic role with the reactivation of HIV latency by HSV-1 mainly through the modulation of host NF-κB and HSF1 pathways, while HSV-1 (especially HSV-ΔICP34.5) might reactivate HIV latency through NF-κB, HSF1, and other yet-to-be-determined mechanisms. Thus, ICP34.5 overexpression can only a partial effect on the reduction of the HIV latency reactivation by HSV-1. We have mentioned this issue in the revised “Discussion section”. “Intriguingly, these findings collectively indicated that ICP34.5 might play an antagonistic role in the reactivation of HIV by HSV-1, and thus our modified HSV-DICP34.5 constructs can effectively reactivate HIV/SIV latency through the release of imprisonment from ICP34.5. However, ICP34.5 overexpression had only a partial effect on the reduction of the HIV latency reactivation, indicating that HSV-DICP34.5-based constructs can also reactivate HIV latency through other yet-to-be-determined mechanisms.” (Lines 334 to 340).

(2) No toxicity data are given for deleting ICP34.5. How specific is the effect for HIV reactivation? An RNA seq analysis is required to show the effect on cellular genes.

Thank you for your questions and suggestions.

(1) It’s well known that ICP34.5 is a neurotoxicity factor that can antagonize host immune responses, and previous studies (in gene therapy and oncolytic virotherapy) have shown that the safety of recombinant HSV-based vector can be improved by deleting ICP34.5. In this study, we also found that HSV-DICP34.5 exhibited lower virulence and replication ability than its parental strain (HSV-GFP) (Figure 1D, Figure S1). In addition, HSV-DICP34.5 induced a lower level of inflammatory cytokines (including IL-6, IL-1β, and TNF-α) in primary CD4+ T cells from PLWH compared to HSV-GFP stimulation, likely due to its lower virulence and replication ability (Figure 1I-K). In addition, the CD4+ /CD8+ T cell ratio (Figure 5I) and body weight (Figure S9) after treatment were effectively ameliorated in the SIV-infected macaques of the ART+HSV-DICP34.5-sPD1-SIVgag/SIVenv group. Our data also demonstrated that there was no significant effect on the cell composition of peripheral blood in the SIV-infected macaques of ART+HSV-sPD1-SIVgag/SIVenv group (Figure S10). Thus, these data suggest the safety of HSV-DICP34.5 in PLWH might be tolerable. We have added the corresponding description in the revised manuscript.

(2) In our study, we found both adenovirus and vaccinia virus cannot reactivate HIV latency (Figure S3). In addition, the deletion of ICP0 gene from HSV-1 diminished the reactivation effect of HIV latency by HSV-1 (Figure S4). Thus, these data suggested the reactivation of HIV latency by HSV-1 might be virus-specific. Of course, this might be further investigated in future studies. We have added the corresponding description in the revised manuscript.

(3) To explore the mechanism of reactivating viral latency by HSV-DICP34.5-based constructs, we performed RNA-seq analysis (Figure S5). We have added the corresponding description accordingly in the revised manuscript.

(3) The primate groups are too small and the results to variable to make averages. In Figure 5, the group with ART and saline has two slow rebounders. It is not correct to average those with a single quick rebounder. Here the interpretation is NOT supported by the data.

We agree with you that this is a pilot study with limited numbers of rhesus macaques. Although the number of macaques was relatively limited, these nine macaques were distributed evenly based on the background level of age, sex, weight, CD4 count, and viral load (VL) (Table S2). All SIV-infected macaques used in this study had a long history of SIV infection and had several courses of ART therapy, which mimics treatment of chronic HIV-1 infection in humans. These macaques were infected with SIVmac239 for more than 5 years, and highly pathogenic SIV-infected macaques have been well-validated as a stringent model to recapitulate HIV-1 pathogenesis and persistence during ART therapy in humans. Indeed, in our Chinese rhesus model, ART treatment effectively suppressed SIV infection to undetectable levels in plasma, and upon ART discontinuation, virus rapidly rebounded, which is very similar with that in ART-treated HIV patients. We think the results of this pilot study were very promising for further studies which will be expanded the scale of animals and then to preclinical and clinical study in our next projects. Thank you for your understanding.

As for your question regarding “the two animals with low VL and slow rebound”, our explanation is following: As mentioned above, these macaques were distributed evenly based on the background level of CD4 count and VL (Table S2), and then there were different change of viral load and viral rebound in different groups. Thus, we think these data can support our interpretation. Moreover, our conclusion can also be supported from at least three evidences.

(1) The VL in the ART+saline group promptly rebounded after ART discontinuation, with an average 8.63-fold increase in the rebounded peak VL compared with the pre-ART VL (Figure 5A, D and E). However, plasma VL in the ART+HSV-sPD1-SIVgag/SIVenv group exhibited a delayed rebound interval (Figure 5B-D).

(2) There was a lower rebounded peak VL than pre-ART VL in the ART+HSV-sPD1-SIVgag/SIVenv group (average 12.20-fold decrease), while a higher rebounded peak VL than pre-ART VL in the ART+HSV-empty group (average 2.74-fold increase) (Figure 5E).

(3） We found significant suppression of total SIV DNA and integrated SIV DNA provirus in the ART+HSV-sPD1-SIVgag/SIVenv group. However, the copies of the SIV DNA provirus were significantly improved in the ART+HSV-empty group and ART+saline group (Figure 5F-G).

Thank you for your understanding.

Discussion

HSV vectors are mainly used in cancer treatment partially due to induced inflammation. Whether these are suitable to cure PLWH without major symptoms is a bit questionable to me and should at least be argued for.

Thank you for your kind question comment and question. We confirmed the enhanced reactivation of HIV latency by HSV-∆ICP34.5 in primary CD4+ T cells from people living with HIV (PLWH) (Figure S2). As mentioned above, previous studies have shown that the safety of recombinant HSV-based vector can be improved by deleting ICP34.5. In this study, we also found that HSV-DICP34.5 exhibited lower virulence and replication ability than its parental strain (HSV-GFP) (Figure 1D, Figure S1). In addition, HSV-DICP34.5 induced a lower level of inflammatory cytokines (including IL-6, IL-1β, and TNF-α) in primary CD4+ T cells from PLWH compared to HSV-GFP stimulation, likely due to its lower virulence and replication ability (Figure 1I-K). In addition, the CD4+ /CD8+ T cell ratio (Figure 5I) and body weight (Figure S9) after treatment were effectively ameliorated in the SIV-infected macaques of the ART+HSV-DICP34.5-sPD1-SIVgag/SIVenv group. Our data also demonstrated that there was no significant effect on the cell composition of peripheral blood in the SIV-infected macaques of ART+HSV-sPD1-SIVgag/SIVenv group (Figure S10). Thus, these data suggest the safety of HSV-DICP34.5 in PLWH might be tolerable. We have added the corresponding description in the revised manuscript.

Reviewer #2 (Public Review):

Summary:

In this article, Wen et. al. describe the development of a 'proof-of-concept' bi-functional vector based on HSV-deltaICP-34.5's ability to purge latent HIV-1 and SIV genomes from cells. They show that co-infection of latent J-lat T-cell lines with an HSV-deltaICP-34.5 vector can reactivate HIV-1 from a latent state. Over- or stable expression of ICP 34.5 ORF in these cells can arrest latent HIV-1 genomes from transcription, even in the presence of latency reversal agents. ICP34.5 can co-IP with- and de-phosphorylate IKKa/b to block its interaction with NF-k/B transcription factor. Additionally, ICP34.5 can interact with HSF1 which was identified by mass-spec. Thus, the authors propose that the latency reversal effect of HSV-deltaICP-34.5 in co-infected JLat cells is due to modulatory effects on the IKKa/b-NF-kB and PP1-HSF-1 pathway.

Next, the authors cleverly construct a bifunctional HSV-based vector with deleted ICP34.5 and 47 ORFs to purge latency and avoid immunological refluxes, and additionally, expand the application of this construct as a vaccine by introducing SIV genes. They use this 'vaccine' in mouse models and show the expected SIV-immune responses. Experiments in rhesus macaques (RM), further elicit the potential for their approach to reactivate SIV genomes and at the same time block their replication by antibodies. What was interesting in the SIV experiments is that the dual-functional vector vaccine containing sPD1- and SIV Gag/Env ORFs effectively delayed SIV rebound in RMs and in some cases almost neutralized viral DNA copy detection in serum. Very promising indeed, however, there are some questions I wish the authors had explored to get answers to, detailed below.

Overall, this is an elegant and timely work demonstrating the feasibility of reducing virus rebound in animals, with the potential to expand to clinical studies. The work was well-written, and sections were clearly discussed.

Strengths:

The work is well designed, rationale explained, and written very clearly for lay readers.<br /> Claims are adequately supported by evidence and well-designed experiments including controls.

Thank you for your nice comments regarding our work.

Weaknesses:

(1) While the mechanism of ICP34.5 interaction and modulation of the NF-kB and HSF1 pathways are shown, this only proves ICP34.5 interactions but does not give away the mechanism of how the HSV-deltaICP-34.5 vector purges HIV-1 latency. What other components of the vector are required for latency reversal? Perhaps serial deletion experiments of the other ORFs in the HSV-deltaICP-34.5 vector might be revealing.

Thank you for your valuable suggestion. In fact, we are currently further exploring some potential viral genes of HSV-1 that might play a role in the reactivation of HIV latency. We have found that the deletion of ICP0 gene from HSV-1 diminished the reactivation effect of HIV latency by HSV-1 (Figure S4), showing that ICP0 might play a vital role for the reactivation. Of course, this might be further investigated in future studies. We have added the corresponding description in the revised manuscript.

(2) The efficacy of the HSV vaccine vectors was evaluated in Rhesus Macaque model animals. Animals were chronically infected with SIV (a parent of HIV), treated with ART, challenged with bi-functional HSV vaccine or controls, and discontinued treatment, and the resulting virus burden and immune responses were monitored. The animals showed SIV Gag and Env-specific immune responses, and delayed virus rebound (however rebound is still there), and below-detection viral DNA copies. What would make a more convincing argument to this reviewer will be data to demonstrate that after the bi-functional vaccine, the animals show overall reduction in the number of circulating latent cells. The feasibility of obtaining such a result is not clearly demonstrated.

Thank you for your valuable mention. We have now provided more data about this issue. We found significant suppression of total SIV DNA and integrated SIV DNA provirus in the ART+HSV-sPD1-SIVgag/SIVenv group. However, the copies of the SIV DNA provirus were significantly improved in the ART+HSV-empty group and ART+saline group (Figure 5F-G). We have added the corresponding description in the revised manuscript.

(3) The authors state that the reduced virus rebound detected following bi-functional vaccine delivery is due to latent genomes becoming activated and steady-state neutralization of these viruses by antibody response. This needs to be demonstrated. Perhaps cell-culture experiments from specimens taken from animals might help address this issue. In lab cultures one could create environments without antibody responses, under these conditions one would expect a higher level of viral loads to be released in response to the vaccine in question.

Thanks for your kind mention and suggestion. We performed the following cell experiment to address this issue. Primary CD4+ T cells from people living with HIV (PLWH) were isolated, and then infected with HSV or HSV-∆ICP34.5 constructs. As expected, we confirmed the enhanced reactivation of HIV latency by HSV-∆ICP34.5 (Figure S2). Thank you.

(4) How do the authors imagine neutralizing HIV-1 envelope epitopes by a similar strategy? A discussion of this point may also help.

Thank you for your kind comment. We have added the corresponding discussion in the revised manuscript. “The current consensus on HIV/AIDS vaccines emphasizes the importance of simultaneously inducing broadly neutralizing antibodies and cellular immune responses. Therefore, we believe that incorporating the induction of broadly neutralizing antibodies into our future optimizing approaches may lead to better therapeutic outcomes.” (Lines 384 to 388)

(5) I thought the empty HSV-vector control also elicited somewhat delayed kinetics in virus rebound and neutralization, can the authors comment on why this is the case?

Thank you for your careful review and mention. We agree with you that the HSV-1 empty vector does exhibit somewhat a delayed rebound. We think the possible reason is: Although the empty HSV-vector cannot elicit SIV-specific CTL responses, it effectively activates the latent SIV reserviors, and then these activated virions can be partially killed by ART drugs. Therefore, even without carrying HIV/SIV antigens, somewhat delayed kinetics in virus rebound may be observed. Thank you.

Reviewer #1 (Recommendations For The Authors):

(1) The authors should provide toxicity data for HSV transduction after deleting ICP34.5 and provide an explanation of why overexpression of ICP34.5 has such a small effect.

Thank you for your questions and suggestions. As mentioned above, we now provided data for the safety of HSV-DICP34.5-based constructs.

(1) It’s well known that ICP34.5 is a neurotoxicity factor that can antagonize host immune responses, and previous studies (in gene therapy and oncolytic virotherapy) have shown that the safety of recombinant HSV-based vector can be improved by deleting ICP34.5. In this study, we also found that HSV-DICP34.5 exhibited lower virulence and replication ability than its parental strain (HSV-GFP) (Figure 1D, Figure S1). In addition, HSV-DICP34.5 induced a lower level of inflammatory cytokines (including IL-6, IL-1β, and TNF-α) in primary CD4+ T cells from PLWH compared to HSV-GFP stimulation, likely due to its lower virulence and replication ability (Figure 1I-K). In addition, the CD4+ /CD8+ T cell ratio (Figure 5I) and body weight (Figure S9) after treatment were effectively ameliorated in the SIV-infected macaques of the ART+HSV-DICP34.5-sPD1-SIVgag/SIVenv group. Our data also demonstrated that there was no significant effect on the cell composition of peripheral blood in the SIV-infected macaques of ART+HSV-sPD1-SIVgag/SIVenv group (Figure S10). Thus, these data suggest the safety of HSV-DICP34.5 in PLWH might be tolerable. We have added the corresponding description in the revised manuscript.

(2) We agree with your insightful mention that the mechanism underlying increased activation by HSV-ΔICP34.5 is worthy to be further explored in the future study. In this study, we found that ICP34.5 play an antagonistic role with the reactivation of HIV latency by HSV-1 mainly through the modulation of host NF-κB and HSF1 pathways, while HSV-1 (especially HSV-ΔICP34.5) might reactivate HIV latency through NF-κB, HSF1, and other yet-to-be-determined mechanisms. Thus, ICP34.5 overexpression can only a partial effect on the reduction of the HIV latency reactivation by HSV-1. We have mentioned this issue in the revised “Discussion section”. “Intriguingly, these findings collectively indicated that ICP34.5 might play an antagonistic role in the reactivation of HIV by HSV-1, and thus our modified HSV-DICP34.5 constructs can effectively reactivate HIV/SIV latency through the release of imprisonment from ICP34.5. However, ICP34.5 overexpression had only a partial effect on the reduction of the HIV latency reactivation, indicating that HSV-DICP34.5-based constructs can also reactivate HIV latency through other yet-to-be-determined mechanisms.” (Lines 334 to 340).

(2) How specific is the effect for HIV reactivation? An RNA seq analysis is required to show the effect on cellular genes.

Thank you for your questions and suggestions.

(1) In our study, we found both adenovirus and vaccinia virus cannot reactivate HIV latency (Figure S3). In addition, the deletion of ICP0 gene from HSV-1 diminished the reactivation effect of HIV latency by HSV-1 (Figure S4). Thus, these data suggested the reactivation of HIV latency by HSV-1 might be virus-specific. Of course, this might be further investigated in future studies. We have added the corresponding description in the revised manuscript.

(2) To explore the mechanism of reactivating viral latency by HSV-DICP34.5-based constructs, we performed RNA-seq analysis (Figure S5). Results showed that there were numerous differentially expressed genes (DEGs) in response to HSV-ΔICP34.5 infection. Among them, 2288 genes were upregulated, and 611 genes were downregulated. GO analysis showed the enrichment of these DEGs in cellular cycle, cellular development, and cellular proliferation, and KEGG enrichment analysis indicated the enrichment in pathways such as cellular cycle and cytokine-cytokine receptor interaction. We have added the corresponding description accordingly in the revised manuscript.

(3) A comparison in primates has to be given for constructs with or without ICP34.5 to validate cell culture data (what is an empty vector?)

Thank you for your reminder. In the revised manuscript, we performed the following cell experiment to address this issue. Primary CD4+ T cells from people living with HIV (PLWH) were isolated, and then infected with HSV or HSV-∆ICP34.5 constructs. As expected, we confirmed the enhanced reactivation of HIV latency by HSV-∆ICP34.5 (Figure S2). Thank you.

(4) Legends should be improved in writing and content.

Thank you for your kind mention. In the revised version, we have improved both the manuscript content and the legends of all Figures have been carefully revised in writing and content. Thank you.

(5) The primate groups should be enlarged before any reliable conclusions can be made. Inflammatory/tox data should be provided.

Thank you for your question.

(1) As mentioned above, we agree with you that this is a pilot study with limited numbers of rhesus macaques. Although the number of macaques was relatively limited, these nine macaques were distributed evenly based on the background level of age, sex, weight, CD4 count, and viral load (VL) (Table S2). All SIV-infected macaques used in this study had a long history of SIV infection and had several courses of ART therapy, which mimics treatment of chronic HIV-1 infection in humans. These macaques were infected with SIVmac239 for more than 5 years, and highly pathogenic SIV-infected macaques have been well-validated as a stringent model to recapitulate HIV-1 pathogenesis and persistence during ART therapy in humans. Indeed, in our Chinese rhesus model, ART treatment effectively suppressed SIV infection to undetectable levels in plasma, and upon ART discontinuation, virus rapidly rebounded, which is very similar with that in ART-treated HIV patients. We think the results of this pilot study were very promising for further studies which will be expanded the scale of animals and then to preclinical and clinical study in our next projects. Thank you for your understanding.

(2) As well known, ICP34.5 is a neurotoxicity factor that can antagonize host immune responses, and previous studies have shown that the safety of recombinant HSV-based vector can be improved by deleting ICP34.5. In this study, we also found that HSV-DICP34.5 exhibited lower virulence and replication ability than its parental strain (HSV-GFP) (Figure 1D, Figure S1). In addition, HSV-DICP34.5 induced a lower level of inflammatory cytokines (including IL-6, IL-1β, and TNF-α) in primary CD4+ T cells from PLWH compared to HSV-GFP stimulation, likely due to its lower virulence and replication ability (Figure 1I-K). In addition, the CD4+ /CD8+ T cell ratio (Figure 5I) and body weight (Figure S9) after treatment were effectively ameliorated in the SIV-infected macaques of the ART+HSV-DICP34.5-sPD1-SIVgag/SIVenv group. Our data also demonstrated that there was no significant effect on the cell composition of peripheral blood in the SIV-infected macaques of ART+HSV-sPD1-SIVgag/SIVenv group (Figure S10). Thus, these data suggest the safety of HSV-DICP34.5 in PLWH might be tolerable. We have added the corresponding description in the revised manuscript.

(6) Discuss the potential of inflammatory HSV vaccines to be used in PLWH without clinical symptoms.

Thank you for your mention. As discussed above, we found that HSV-DICP34.5 exhibited lower virulence and replication ability than its parental strain (Figure 1D, Figure S1), and we also found that HSV-DICP34.5 induced a lower level of inflammatory cytokines (including IL-6, IL-1β, and TNF-α) in primary CD4+ T cells from PLWH compared to HSV-GFP stimulation, likely due to its lower virulence and replication ability (Figure 1I-K). In addition, the CD4+ /CD8+ T cell ratio (Figure 5I) and body weight (Figure S9) after treatment were effectively ameliorated in the SIV-infected macaques of the ART+HSV-DICP34.5-sPD1-SIVgag/SIVenv group. Our data also demonstrated that there was no significant effect on the cell composition of peripheral blood in the SIV-infected macaques of ART+HSV-sPD1-SIVgag/SIVenv group (Figure S10). Thus, these data suggest the safety of HSV-DICP34.5 in PLWH might be tolerable. We have added the corresponding description in the revised manuscript.

Reviewer #2 (Recommendations For The Authors):

I think the authors have done due diligence to the experimental system, and collected evidence to show the feasibility of delaying virus rebound in macaques. However, I would encourage the authors to perform experiments that can back up the claim that delayed virus rebound is due to neutralization effects, or perhaps due to a reduction in viral reservoir. I believe insights into this process will add rigor, and push the relevance of the study to the next level.

Thank you for your nice comment and valuable suggestion. We have now provided more data about this issue. We found significant suppression of total SIV DNA and integrated SIV DNA provirus in the ART+HSV-sPD1-SIVgag/SIVenv group. However, the copies of the SIV DNA provirus were significantly improved in the ART+HSV-empty group and ART+saline group (Figure 5F-G). We also discussed that incorporating the induction of broadly neutralizing antibodies into our future optimizing approaches may lead to better therapeutic outcomes in the revised Discussion section. We have added the corresponding description in the revised manuscript. Thank you.

Altogether, all of the above comments and suggestions are very helpful in improving our manuscript. We have taken these comments into account seriously and try our best to address these questions point-by-point. After making extensive revisions, we now submit this revised manuscript for your re-consideration. Thank you again for all of your comments and suggestions.

eLife Assessment

This useful study reports on the impact of antibiotic pressure on the genomic stability of the mc2155 strain of Mycobacterium smegmatis, a model for Mycobacterium tuberculosis. The findings of the study indicate that exposure to antibiotics did not lead to the development of new adaptive mutations in controlled laboratory environments, challenging the notion that antibiotic resistance arises from drug-induced microevolution. The genomic analysis provides detailed insights into the stability of M. smegmatis following exposure to standard TB treatment antibiotics, and the evidence suggesting that antibiotic pressure does not contribute to the emergence of new adaptive mutations is solid.

Reviewer #1 (Public review):

Molnar, Suranyi and colleagues have generated a useful dataset characterizing the rate of mutations in Mycobacterium smegmatis - a non-pathogenic model mycobacterial strain, to several antibiotics at sub-lethal dose. The whole genome sequencing approach used is a strength of this study. Overall, the results are consistent with a low rate of mutations, consistent with other reports in Mycobacterium smegmatis and in vitro and clinical studies with Mycobacterium tuberculosis. The data supports phenotypic tolerance rather than genetic mutations as a driver.

The revised manuscript is improved and addresses several concerns raised by the reviewers from the previous rounds. These relate primarily to the presentation of data in the figures, but there is also new data in Figure 2 to show an increased MIC for M. smegmatis under antibiotic pressure. An additional dataset of sequences from ciprofloxacin-treated bacteria has also been generated and made publicly accessible, which will be of interest to the community.

Reviewer #2 (Public review):

Summary

In this study, the authors evaluate the impact of selective pressure from chemotherapeutic drugs on the development of drug resistance in Mycobacteria, specifically through the acquisition of genetic mutations or phenotypic tolerance. Their findings indicate that treatment with sublethal concentrations of first-line antibiotics does not lead to enhanced mutation rates.

Strengths

The use of the mutation accumulation assay demonstrating low spontaneous mutation rates combined with the display of an increased MIC supports drug resistance as a consequence of phenotypic tolerance. Additionally, the use of the ciprofloxacin tolerance assay in combination with whole genome sequencing demonstrating a lack of mutations provides further support of this. The results now support the authors claims.

Weaknesses

Besides an increase in DNA stress response other underlying tolerance mechanisms were not established - increased efflux pump, thickening of the cell wall, decrease in metabolic processes, rerouting of metabolic processes etc.

Reviewer #3 (Public review):

Summary:

This manuscript describes how antibiotics influence genetic stability and survival in Mycobacterium smegmatis. Prolonged treatment with first-line antibiotics did not significantly impact mutation rates. Instead, adaptation to these drugs appears to be mediated by upregulation of DNA repair enzymes. While this study offers robust data, findings remain correlative and fall short of providing mechanistic insights.

Strengths:

The strength of this study is the use of genome-wide approaches to address the specific question of whether or not mycobacteria induce mutagenic potential upon antibiotic exposure.

Comments on revised version:

The authors responded adequately to my comments, and I have no further suggestions for the revised manuscript.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

In this manuscript, Molnar, Suranyi and colleagues have probed the genomic stability of Mycobacterium smegmatis in response to several anti-tuberculosis drugs as monotherapy and in combination. Unlike the study by Nyinoh and McFaddden http://dx.doi.org/10.1002/ddr.21497 (which should be cited), the authors use a sub-lethal dose of antibiotic. While this is motivated by sound technical considerations, the biological and therapeutic rationale could be further elaborated.

In the mutation accumulation experiments, we needed to ensure continuous and reproducible growth of a small number of colonies across multiple passages. This technical requirement necessitated the use of sublethal drug concentrations. However, sublethal doses also have biological relevance. Noncompliance with prescribed antibiotic regimens and the presence of antibiotic residues in food due to the extensive use of antibiotics in agricultural mass production are two obvious sources of prolonged exposure to sublethal antibiotics.

The results the authors obtain are in line with papers examining the genomic mutation rate in vitro and from patient samples in Mycobacterium tuberculosis, in vitro in Mycobacterium smegmatis and in vitro in Mycobacterium tuberculosis (although the study by HL David (PMID: 4991927) is not cited). The results are confirmatory of previous studies.

The two cited studies, along with several others, did not distinguish between genetic mutations and phenotypic responses to drug exposure (the fluctuation test alone is not suitable for this). Therefore, their objectives are not comparable to ours, which specifically investigated whether resistant colonies carry adaptive mutations. Nevertheless, we acknowledge the relevance of these studies and have now cited them in the appropriate sections in the text.

It is therefore puzzling why the authors propose the opposite hypothesis in the paper (i.e antibiotic exposure should increase mutation rates) merely to tear it down later. This straw-man style is entirely unnecessary.  

The phenomenon of stress-inducible mutagenesis in bacterial evolution remains a topic of heated debate. The emergence of genetically encoded resistance may stem from either microevolution or the dissemination of pre-existing variants from polyclonal infections under drug pressure. We believe that the Introduction presents both of these hypotheses in a balanced manner to elucidate the rationale behind our mutation accumulation investigations.  

The results on the nucleotide pools are interesting, but the statistically significant data is difficult to identify as presented, and therefore the new biological insights are unclear.

We now indicate statistical significance in the figure, in addition to the detailed statistical analysis of all dNTP measurements provided in Table S5.

Finally, the authors show that a fluctuation assay generates mutations with higher frequencies that the genetic stability assays, confirming the well-known effect of phenotypic antibiotic resistance.

What we show is that the fluctuation assay generated bacteria that tolerated the applied antibiotic without developing mutations. Conclusions about mutation rates are often drawn from fluctuation assays without confirming genetic-level changes, a discrepancy that persists despite these assays accounting for both phenotypic and genotypic alterations. By combining genome sequencing with fluctuation assays, our approach emphasizes the importance of distinguishing between these changes. While fluctuation assays remain valuable, inexpensive, and simple tools for evaluating the response of bacterial populations to various selective environments, they should not be considered definitive indicators of genetic changes.

Recommendations For The Authors:

The quality of the figures can be significantly improved. In Figure 1, cell lengths can be shown on separate histograms or better still as violin plots to enable better comparisons.

Thank you for the suggestion. We have revised the data presentation accordingly.

Details for statistical tests should be provided in the figure legend.  

Statistical details are now added in the figure legend.

In Figure 2, the number of data points is not mentioned.

Statistical information is now added to the new Figure 2, which has been revised extensively based on suggestions from all Referees.

The data in Figure 3 would be much easier to comprehend as a heatmap.  

The figure we provided is a color gradient table representing different gene expression levels, along with numerical data and statistical significance indicated within the color boxes, expanding the information content of a traditional heatmap. In response to the Referee's suggestion, we also prepared a hierarchical clustering heatmap, demonstrating that the grouping of rows and columns based on functional information in the original figure is consistent with the clustering pattern observed in the heatmap (Figure S5). As the original figure is more informative and better structured, we have included the new figure in the supplementary materials.

No statistical tests are provided for Figure 4.

We now indicate statistical significance in the figure and describe the statistical analysis in the figure legend, as suggested. Additionally, Table S5 is dedicated to the statistical analysis of the dNTP data.  

Reviewer #2 (Public Review):

In this study, the authors assess whether selective pressure from drug chemotherapy influences the emergence of drug resistance through the acquisition of genetic mutations or phenotypic tolerance. I commend the authors on their approach of utilizing the mutation accumulation (MA) assay as a means to answer this and whole genome sequencing of clones from the assay convincingly demonstrates low mutation rates in Mycobacteria when exposed to sub-inhibitory concentrations of antibiotics. Also, quantitative PCR highlighted the upregulation of DNA repair genes in Mycobacteria following drug treatment, implying the preservation of genomic integrity via specific repair pathways.

Even though the findings stem from M. smegmatis exposure to antibiotics under in vitro conditions, this is still relevant in the context of the development of drug resistance so I can see where the authors' train of thought was heading in exploring this. However, I think important experiments to perform to more fully support the conclusion that resistance is largely associated with phenotypic rather than genetic factors would have been to either sequence clones from the ciprofloxacin tolerance assay (to show absence/ minimal genetic mutations) or to have tested the MIC of clones from the MA assay (to show an increase in MIC).

Thank you for acknowledging the values of the manuscript and for the insightful suggestions for improvement. We agree on the necessity to directly connect the mutation accumulation experiments with the tolerance assay, and we have performed both suggested additional experiments.  

(1) We repeated the ciprofloxacin tolerance assay (Figure S6) using a large number of plates to gather enough cells for genomic DNA extraction and whole genome sequencing. The sequencing confirmed the absence of mutations in bacteria grown in both 0.3 and 0.5 ug/ml ciprofloxacin. We integrated this result in the revised manuscript text, while the sequencing data are available at the European Nucleotide Archive (ENA) with PRJEB71590 project number.

(2) We resuscitated three different clones from the MA assays stored at -80°C and tested the MIC of the respective drugs. The results are presented in Figure 2C. Except for EMB, we observed an increase in MIC values across the treatments.

There seems to be a disconnect between making these conclusions from experiments conducted under different conditions, or perhaps the authors can clarify why this was done.  

Molecular biology analysis methods are not easily compatible with long-term mutation accumulation experiments, or at least we could not establish the necessary conditions. When DNA or RNA extraction was required, we had to adjust the experimental scale for further analysis, which could be done in liquid culture. We believe that the suggested critical back-and-forth control experiments have significantly improved the comparability of the results.

With regards to the sub-inhibitory drug concentration applied, there is significant variation in the viability as calculated by CFUs following the different treatments and there is evidence that cell death greatly affects the calculation of mutation rate (PMCID: PMC5966242). For instance, the COMBO treatment led to 6% viability whilst the INH treatment led to 80% cell viability. Are there any adjustments made to take this into account?

We agree with and have been aware of the notion that cell death affects the calculation of the mutation rate. We included treatment optimization data on agar plates (Table 1 and Figure S2), which now demonstrate that the applied subinhibitory drug concentrations resulted in ≤10% viability across all treatments in the MA assay. This minimizes the potential discrepancy in the mutation rate calculation caused by variable cell death.  

It would also be useful to the reader to include a supplementary table of the SNPs detected from the lineages of each treatment - to determine if at any point rifampicin treatment led to mutations in rpoB, isoniazid to katG mutations, etc.  

Overall, while this study is tantalizingly suggestive of phenotypic tolerance playing a leading role in drug resistance (and perhaps genetic mutations a sub-ordinate role) a more substantial link is needed to clarify this.

The SNPs identified from the lineages of each treatment are compiled in the 'unique_muts.xls' file within the Figshare document bundle that was originally enclosed with the manuscript. In response to your suggestion, we have now added a simplified version of this data set in Table S2, listing the detected SNPs. Notably, no confirmed adaptive mutation developed in our experiments; rifampicin treatment did not result in mutations in rpoB, nor did isoniazid lead to mutations in katG.

Recommendations For The Authors:

I would suggest moving Figure 1 to the supplementary - it shows that cell wall targeting drugs cause cell shortening and DNA replication targeting drugs cause cell elongation as would be expected and this is simply a secondary observation, not one that is central to the paper.  

We agree that this is not a novel or unexpected observation. However, we used it as an indicator of drug effectiveness, particularly for bacteriostatic cell wall-targeting drugs in liquid culture that induced moderate cell death. Following Reviewer 1's suggestions, we extensively revised the figure to better convey our intended message. We believe the updated version now more clearly demonstrates the drugs' impact, and for this reason, we have opted to keep it in the main text.

Figure 2 and Table 2 show the same data so this can be combined as a paneled figure or one moved to the supplementary. It would be useful to include a diagram of how the MA assay was conducted, similar to the CIP tolerance assay figure.

Thank you for the suggestions. We have added a diagram to Figure 2 explaining the MA assay (Figure 2A), as well as the MIC experiment conducted on the MA cells (Figure 2C). To avoid redundancy, Table 2 has been removed.

Reviewer #3 (Public Review):

Summary:

This manuscript describes how antibiotics influence genetic stability and survival in Mycobacterium smegmatis. Prolonged treatment with first-line antibiotics did not significantly impact mutation rates. Instead, adaptation to these drugs appears to be mediated by upregulation of DNA repair enzymes. While this study offers robust data, findings remain correlative and fall short of providing mechanistic insights.

Strengths:

The strength of this study is the use of genome-wide approaches to address the specific question of whether or not mycobacteria induce mutagenic potential upon antibiotic exposure.

Weaknesses:

The authors suggest that the upregulation of DNA repair enzymes ensures a low mutation rate under drug pressure. However, this suggestion is based on correlative data, and there is no mechanistic validation of their speculations in this study.

Furthermore, as detailed below, some of the statements made by the authors are not substantiated by the data presented in the manuscript.

Finally, some clarifications are needed for the methodologies employed in this study. Most importantly, reduced colony growth should be demonstrated on agar plates to indicate that the drug concentrations calculated from liquid culture growth can be applied to agar surface growth. Without such validations, the lack of induced mutation could simply be due to the fact that the drug concentrations used in this study were insufficient.

Thank you for appreciating the manuscript's merits and for the instructive suggestions. We agree that demonstrating reduced colony growth on agar plates is important to validate the relevance of the drug concentrations used in the study. In response, we have added the treatment optimization data on agar plates in Figure S2 and reorganized Table 1 to show the decrease in CFU achieved with the applied subinhibitory drug concentrations.

We acknowledge that the observed upregulation of DNA repair enzymes and the low mutation rates under drug pressure represent correlative data. We removed the reference to mechanism from the abstract and avoided presenting the qPCR results as a mechanistic explanation in the text. We have only raised the possibility that correlation could be a causal relationship: "The observed upregulation of the relevant DNA repair enzymes might account for the low mutation rate even under drug pressure." We recognize the necessity for a new series of targeted experiments to provide mechanistic explanations. We added the following text to the Discussion:

“The observed activation of DNA repair processes likely mitigates mutation pressure, ensuring genome stability. However, to confirm this hypothesis, these investigations should be conducted using genetically modified DNA repair mutant strains.”

In the current manuscript, we aim to convincingly demonstrate that long-term antibiotic pressure did not induce the occurrence of new adaptive mutations.

Recommendations For The Authors:

Additional specific comments are:

Page 2. Do not italicize "Mycobacteria", which is not considered a scientific name.

Corrected.

Page 4. "Bacto pepcone" is a typo.

Corrected.

Page 6. "Quiagen" is a typo.

Corrected.

Page 9. In Table 1, RIF being described as a protein synthesis inhibitor is misleading.

Corrected.

Page 9. The statement "Specifically, following RIF, CIP, and MMC treatments, we observed cells elongating by more than twofold, whereas INH and EMB treatments led to a reduction in cell length." cannot be justified by Figure 1, as the cell length information is not conveyed in this figure.

Thank you for pointing this out, the revised Figure 1 conveys the cell length information.

Page 10. If the experiment shown in Figure S1 was done in an acidic growth condition, the figure legend should clearly indicate the fact. Additionally, the assay condition should be described in detail in the Methods section.

Thank you, the required information is now included in both the figure legend and the Methods section.

Page 10. If PZA does not work against M. smegmatis, it seems pointless to add it to the COMBO treatment. Please clarify why it was included in the drug combination experiment.

We added the following text to clarify the use of PZA: “Regardless of its inefficacy as a monotherapy, we included PZA in the combination treatment, as we could not rule out the possibility that PZA interacts with the other three drugs or that PZA elimination mechanisms are equally active in M. smegmatis under this regimen.”

Page 10. Generation times calculated from liquid culture cannot be applied to colony growth on an agar plate. The growth behaviors on a solid surface will be totally different from planktonic suspension growth. The numbers of generations indicated here will be inaccurate.

You are absolutely right. We conducted an experiment to calculate the number of generations on plates under the same conditions as used in the MA assay. We found, indeed, a different (doubled) generation time from what was determined in liquid culture. We have adjusted the mutation rates accordingly.

Page 12. Was the experiment shown in Figure 3 done in a liquid culture? If so, the transcriptional profile could be different from the experiment shown in Figure 2, which was done on an agar plate.

Yes, the experiment shown in Figure 3 was conducted in liquid culture. We acknowledge that the transcriptional profile could differ from the experiment shown in Figure 2, which was performed on an agar plate. However, technical limitations required us to use liquid cultures for these experiments.

Page 14. Regarding the statement "INH and EMB coincided with a decreased concentration of these [dCTP and dTTP] nucleotides", by examining Table S5, I do not see any statistical reductions in dCTP and dTTP levels.

Thank you for bringing this to our attention. We have made the necessary corrections to ensure that the text and data are now aligned.

Page 14. Similarly to the comment above, the statement "RIF, CIP and MMC treatments promoted an increase in the dCTP and dTTP pools" is misleading as each drug seems to increase either dCTP or dTTP, not both.

Same as above.

Page 14. The authors state, "a larger overall dNTP pool size coincides with a larger cell size and vice versa (Figure 4H)". Please indicate the unit of the pool size for the graph shown in Figure 4H. According to the legend, I assume that it refers to the concentration. The term "pool size" may be misleading as it implies quantity rather than concentration.

Page 15. Figure 4H is impossible to understand. The left y-axis label looks as if it is a ratio of cell length to volume. There is no point in having these three data on a single graph. Please separate them into individual graphs. Also, what is the spacing between the tick marks? The data also seem inconsistent with the values given in Table S1. For example, the mean volume of COMBO is larger than the control (according to Table S1), and yet the graph in Figure 4H indicates that COMBO's relative length is less than 1.

Thank you for your feedback. We have corrected these and created what we hope is a clearer figure.

Figure S1. Clarify what the gray shade in the graph represents.

The gray shade was unnecessary, so we removed it when recoloring the figure to ensure a more coherent color scheme across the different treatments.

Figure S1. Relative viability cannot be determined by OD600. CFU needs to be determined to assess cell viability.

Thank you. We changed the incorrect term viability to growth inhibition.

This is an example of speculation because it is inferencing what is expected to happen.

This is another example of an attribution.

This fact is important to include because it shows the impact this outbreak is actually having on specific regions. Since one person has died because of it, this makes the case a bigger deal.

Being that they have multiple suppliers for their sliver onions, it is hard to determine who exactly is causing this E. coli outbreak. This statement also appears to be a fact since they have yet to draw a conclusion.

This is an example of an attribution. The writer is clearly stating where they got this information from, so it does not imply opinion.

I want to highlight on the word, "may," this implies they have yet to reach a distinct conclusion.

I once heard a lecture from a cybersecurity expert, and they had us understand that cybersecurity is a matter of balancing needs against profit. The example he gave was that if you had $100, you wouldn't buy a $100 locked box to keep it safe, maybe a 5 or 15 dollar lock. So it's never a matter of what is the "most" secure, but what can be the most practically secured.

This is so fascinating to me. As they were mentioning risks they used real-world situations and linked the articles to learn more about the event. Each one of these events seems crazy and I can't believe I haven't heard of most of them. This shows that even when you think you are being private some things are just out of your control.

This really emphasizes that cybersecurity goes beyond just technology; it also involves human behavior. People often reuse passwords for convenience, unaware of how easily that habit can be exploited. It’s both fascinating and a bit frightening how trust can be manipulated—take the example of the NSA impersonating Google. Social engineering serves as a perfect reminder that hackers don’t always need sophisticated tools; sometimes, they just need to deceive people into trusting the wrong thing. Phishing emails and fake QR codes are particularly clever because they depend on people acting quickly without thinking. The reference to Frank Abagnale from Catch Me If You Can re

Restoration to 18th Century Notes Restoration and the Glorious Revolution Death of Cromwell: Leads to political instability. Charles II's Return: Parliament invites him to rule, marking the start of the Restoration. Initial Anarchy: Political turmoil follows; Charles II serves as a "figurehead." James II's Absolute Rule: Attempts to restore Catholicism create insecurity in Parliament. William of Orange's Intervention: Invited to intervene, leading to the Glorious Revolution (bloodless). The Bill of Rights: Establishes Parliament as the de facto ruler, with the king as titular head, ending conflict between king and Parliament. Jonathan Swift (1667-1745) Background: Irish author, clergyman, and master of satire. Faced hardships: father died, raised in poverty by an uncle. Moved to England during the Glorious Revolution. Career Highlights: Became an Anglican priest. Wrote political tracts, poems, and notable satirical works. Notable Works: A Modest Proposal: Satirical essay addressing poverty in Ireland. Gulliver’s Travels: Most popular work, blending adventure with social critique. A Tale of a Tub: Another significant satire. Themes: Ranges from love/beauty to death/revenge; darker tones reflect personal struggles. A Modest Proposal (1729) Overview: Addresses poverty and overpopulation in Ireland through an absurd proposal. Key Elements: Humor: Uses absurdity to draw attention. Criticism: Critiques societal neglect of the poor. Moral Voice: Presents a moral argument ironically. Irony and Sarcasm: Highlights the ridiculousness of the proposal. Taboo Topics: Engages with sensitive issues to provoke discussion. Five Elements of Satire Ridicule: Makes subjects seem absurd, inviting scorn and amusement. Seriousness: Addresses significant societal issues humorously. Problem Identification: Aims to highlight and correct flaws in society. Mock-Heroic Tone: The speaker may be oblivious to their own absurdity. Modern Satire Examples The Onion: Satirical news outlet that parodies journalism. Comments on real and fictional events humorously. The Colbert Report: Satirical TV show featuring Stephen Colbert as a conservative pundit. Uses satire to critique political and media landscapes.

Another example of "private" data you want companies to have access to may be your user meta-data. In the case that you lose access to your account, you would want to be able to recover it via a password change.

One excellent example is the European Union's GDPR(General Data Protection Regulation), which protects privacy as a fundamental right. The "right to be forgotten," which enables people to ask for the removal of personal information, is one noteworthy clause that reflects the idea that people should have control over their information in the digital age. However, the U.S. lacks the comprehensive, overarching structure found in the EU, and privacy rights are frequently sector-specific (e.g., health, financial).

I believe that being overly private is better than being excessively public. You do not know what information someone is looking for or needs from you, so giving the bare minimum allows you to stay more private. Another thing I think is that while you are signing up for most social media, you are willingly giving up some forms of privacy. It may be small or unuseful, but social media weren't created to be private, so it's exactly what you're signing up for.

I am happy that some governments make it a priority to protect individuals privacy rights. The European unions general data protection regulation is a great piece of legislation and is a step in the right direction.

Restoration Reading & Supplementary Materials The Restoration in England (1660) The Restoration period marks the reinstatement of the monarchy in England with Charles II, following the Commonwealth under Oliver Cromwell. This era saw the revival of the Church of England and the arts, which had been stifled during Cromwell’s Puritan rule. Charles II's reign (1660-1685) allowed for flourishing sports, science, and cultural pursuits.

In-depth Overview: YouTube Video on the Restoration Puritanism Humor: Horrible Histories Clip Culinary Insights: Supersizer’s Go...Restoration The Georgian Period in England (1714-1830) This period, named for the four Hanoverian kings named George, features influential writers and thinkers:

Samuel Pepys (d. 1703)

Known for his Diary, which offers a personal account of life from 1660-1669. Read an excerpt: Pepys Diary Great Plague Context: Diary of Samuel Pepys & Covid-19 Alexander Pope (d. 1744)

Renowned poet and satirist, famous for works like "An Essay on Man." Read Epistles I and VIII: Essay on Man Samuel Johnson (d. 1784)

Noted lexicographer known for A Dictionary of the English Language (1755). Explore Johnson's Dictionary: Johnson's Dictionary Online Jonathan Swift (d. 1745)

Famous for Gulliver's Travels and A Modest Proposal, a satirical commentary on poverty in Ireland. Read A Modest Proposal: A Modest Proposal Read Chapters 1 and 2 of Gulliver's Travels: Gulliver's Travels Key Themes and Concepts Satire: Understanding Swift’s use of satire is crucial. His extreme proposals highlight societal indifference to poverty, using irony to provoke thought and action. Cultural Revival: The Restoration and Georgian periods marked a significant cultural revival, influencing literature, arts, and social thought.

Another fact/statistic in the article

The writer is backing up his opinion as not a simple analysis

A start'em sit'em article will most likely be all opinion based because it is the writer giving you his opinion on who to start in fantasy football and who to sit

eLife Assessment

Yamamoto and Matano provide convincing evidence that a G63E CD8+ T-cell escape mutation in the accessory viral protein Nef facilitates the induction of neutralizing antibody (nAb) responses in rhesus macaques infected with SIVmac239. Functional analyses support that this mutation specifically impairs Nef`s ability to stimulate PI3K/Akt/mTORC2 signalling. This important study suggests that the accessory viral Nef protein impairs B cell function and effective humoral immune responses and is of interest for researchers and physicians interested in HIV/AIDS and vaccine development.

Reviewer #1 (Public review):

Human and simian immunodeficiency viruses (HIV and SIV, respectively) evolved numerous mechanisms to compromise effective immune responses but the underlying mechanisms remain incompletely understood. Here, Yamamoto and Matano examined the humoral immune response in a large number of rhesus macaques infected with the difficult-to-neutralize SIVmac239 strain and identified a subgroup of animals showing significant neutralizing Ab responses. Sequence analyses revealed that in most of these animals (7/9) but only a minority in the control group (2/19) SIVmac variants containing a CD8+ T-cell escape mutation of G63E/R in the viral Nef gene emerged. Functional analyses revealed that this change attenuates the ability of Nef to stimulate PI3K/Akt/mTORC2 signalling. The authors propose that this improved induction of SIVmac239 nAb is reciprocal to antibody dysregulation caused by a previously identified human PI3K gain-of-function mutation associated with impaired anti-viral B-cell responses. Altogether, the results suggest that PI3K signalling plays a role in B-cell maturation and generation of effective nAb responses. Preliminary data indicate that Nef might be transferred from infected T cells to B cells by direct contact. However, the exact mechanism and the relevance for vaccine development requires further studies

Strengths of the study are that the authors analyzed a large number of SIVmac-infected macaques to unravel the biological significance of the known effect of the interaction of Nef with PI3K/Akt/mTORC2 signaling. This is interesting and may provide a novel means to improve humoral immune responses to HIV. In the revised version the authors made an effort to address previous concerns. Especially, they provide data supporting that Nef might be transferred to B cells by direct cell-cell contact. In addition, the provide some evidence that G63R that also emerged in most animals does not share the disruptive effect of G63G although experimental examination and discussion why G63R might emerge remains poor. Another weakness that remains is that some effects of the G63E mutation are modest and effects were not compared to SIVmac constructs lacking Nef entirely. The evidence for a role of Nef G63E mutation on PI3K and the association with improved nAb responses was largely convincing and it is appreciated that the authors provide additional evidence for a potential impact of "soluble" Nef on neighboring B cells. However, the experimental set-up and the results are difficult to comprehend. It seems that direct cell-cell contact is required and membranes are exchanged. Since Nef is associated with cellular membranes this might lead to some transfer of Nef to B cells. However, the immunological and functional consequences of this remain largely elusive. Alternatively, Nef-mediated manipulation of helper CD4 T cells might also impact B cell function and effective humoral immune responses. As previously noted, the presentation of the results and conclusions was in part very convoluted and difficult to comprehend. While the authors made attempts to improve the writing parts of the manuscript are still challenging to follow. This applies even more to the rebuttal (complex words combined with poor grammar), which made it difficult to assess which concerns have been satisfactory addressed.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

This work describes the induction of SIV-specific NAb responses in rhesus macaques infected with SIVmac239, a neutralization-resistant virus. Typically, host NAb responses are not detected in animals infected with SIVmac239. In this work, seventy SIVmac239-infected macaques were retrospectively screened for NAb responses and a subset of nine animals were identified as NAb-inducers. The viral genomes from 7/9 animals that induced NAb responses were found to encode nonsynonymous mutation in the Nef gene (amino acid G63E). In contrast, Nef G63E mutation was found only in 2/19 NAb non-inducers - implicating that the Nef G63E mutation is selected in NAb inducers. Measurement of Nef G63E frequencies in plasma viruses suggested that Nef G63E selection preceded NAb induction. Nef G63E mutation was found to mediate escape from Nef-specific CD8+ T-cell responses. To examine the functional phenotype of Nef G63E mutant, its effect on downmodulation of Nef-interacting host proteins was examined. Infection of rhesus and cynomolgus macaque CD4+ T cell lines with WT or Nef G63E mutant SIV suggested that Nef mutant reduces S473 phosphorylation of AKT. Using flow cytometry-based proximity ligation assay, it was shown that Nef G63E mutation reduced binding of Nef to PI3K p85/p110 and mTORC2 GβL/mLST8 and MTOR components - kinase complex responsible AKT-S473 phosphorylation. In vitro B-cell Nef invasion and in vivo imaging/flow cytometry-based assays were employed to suggest that Nef from infected cells can target Env-specific B cells. Lastly, it was determined that NAb inducers have significantly higher Env-specific B-cells responses after Nef G63E selection when compared to NAb non-inducers. Finally, a corollary was drawn between the Nef G63E-associated B-cell/NAb induction phenotype and activated PI3K delta syndrome (APDS), which is caused by activating GOF mutations in PI3K, to suggest that Nef G63E-meidated induction of NAb response is reciprocal to APDS.

Strengths:

This study aims to understand the viral-host interaction that governs NAb induction in SIVmac239-infected macaques - this could enable identification of determinants important for induction of NAb responses against hard-to-neutralize tier-2/3 HIV variants. The finding that SIV-specific B-cell responses are induced following Nef G63E CD8+ T-cell escape mutant selection argue for an evolutionary trade-off between CTL escape and NAb induction. Exploitation of such a cellular-humoral immune axis could be important for HIV/AIDS vaccine efforts.

Although more validation and mechanistic basis are needed, the corollary between PI3K hyperactive signaling during autoimmune disorders and Nef-mediated abrogated PI3K signaling could help identify novel targets and modalities for targeting immune disorders and viral infections.

We are grateful for the supportive and insightful comments. The work did seem to unintendedly highlight a conceptual link between extrinsic and intrinsic immune perturbations. We will keep working on both wings, aiming to evoke synergisms.

Weaknesses:

Although the paper does have strengths in principle, the weaknesses of the paper are that the mechanistic basis of Nef-mediated induction of NAb responses are not directly examined. For example, it remains unclear whether SIVmac239 with engineered G63E mutation in Nef would induce faster and potent NAb responses. A macaque challenge study is needed to address this point.

We appreciate the point. We do have certain difficulties in availability of macaques for de novo experiments. As partially discussed in ver1, the identified Nef phenotype selected post-acute infection confers an enhanced CD4+ T cell-killing effect (revised Fig 4F), and it is likely that de novo infection with the mutant would redirect the trajectory of infection to rapid disease/AIDS progression accompanying generalized immune failure by boosting acute-phase CD4 destruction. In other words, mutant de novo infection may not necessarily be directly discussable as an attempt for reconstitution. It appears equally critical to understand the mutant in vitro on an immunosignaling basis, and in the current work we have focused on depicting this as the first step. We will work on reconstitution experiments with emphasis on pharmacology in our future study.

As presented, the central premise of the paper involves infected cell-generated Nef (WT or G63E mutant) being targeted to adjacent Env-specific B cells. However, it remains unclear how this is transfer takes place. A direct evidence demonstrating CD4+ T cell-associated and/or cell-free Nef being transferred to B-cell is needed to address this concern.

We appreciate the point, also pointed out by Reviewers 2 and 3. We have performed three sets of in vitro reconstitution experiments graphically/functionally addressing how Nef transfer from CD4+ T cells to B cells can be modulated (new Fig 6) and edited text accordingly.

The interaction between Nef and PI3K signaling components (p85, p110, GβL/mLST8, and MTOR) has been explored using PLA assay, however, this requires validation using additional biochemical and/or immunoprecipitation-based approaches. For example, is Nef (WT or mutant form) sufficient to affect PI3K-induced phosphorylation of Akt in an in vitro kinase assay? Moreover, the details regarding the binding events of WT vs mutant Nef with PI3K signaling components is lacking in this study. Lastly, it is unclear whether the interaction of Nef with PI3K signaling components is a conserved function of all primate lentiviruses or is this SIV-specific phenotype.

We appreciate the point. Co-immunoprecipitation analysis via pulldown with the mTORC2-intrinsic cofactor Sin1 (revised Fig 4E), showing decreased G63E-Nef binding, should confer robustness to the statement combined with initial manipulation results (Fig 4C). As Sin1 is mTORC2- and not mTORC1-intrinsic, results should be strengthened. Phosflow may be a standard readout nowadays for pAkt itself. Related with sequence variation, conservation will be addressed in studies ahead. We concisely mentioned on this in the revision (Lines 390-391).

It has been previously reported that the region of Nef encoding glycine at position 63 is not conserved in HIV-1 (Schindler et al, Journal of Virology 2004). Thus, does HIV-1 Nef also function in induction of NAb responses in humans? or the observed phenotype specific to SIV?

We appreciate the point, and do not have an answer at the moment. We will explore in our HIV-1-infected patient cohort (Hau et al, AIDS 2022) and other occasions whether corresponding phenotypes may exist. We have mentioned on this point in the revised manuscript (Line 392-393).

Reviewer #2 (Public Review):

It is well known that human and simian immunodeficiency viruses (HIV and SIV, respectively) evolved numerous mechanisms to compromise effective immune responses but the underlying mechanisms remain incompletely understood. Here, Yamamoto and Matano examined the humoral immune response in a large number of rhesus macaques infected with the difficult-to-neutralize SIVmac239 strain. They identified a subgroup of animals that showed significant neutralizing Ab responses. Sequence analyses revealed that in most of these animals (7/9) but only a minority in the control group (2/19) SIVmac variants containing a CD8+ T-cell escape mutation of G63E/R in the viral Nef gene emerged. They further show that this change attenuates the ability of Nef to stimulate PI3K/Akt/mTORC2 signaling. The authors propose that this induction of SIVmac239 nAb induction is reciprocal to antibody dysregulation caused by a previously identified human PI3K gain-of-function (Ref). Altogether, the results suggest that PI3K signaling plays a key role in B-cell maturation and generation of effective nAb responses.

Strengths of the study are that the authors analyzed a large number of SIVmac-infected macaques to unravel the biological significance of the known effect of the interaction of Nef with PI3K/Akt/mTORC2 signaling. This is interesting and may provide a novel means to improve humoral immune responses to HIV. Weaknesses are that only G63E and not G63R that also emerged in most animals was examined in most functional assays. Some effects of the G63E mutation seem modest and comparison to a grossly nef-defective SIVmac construct would be desirable to better assess to impact of the mutation of Nef-mediated stimulation of PI3K. While the impact of this Nef mutations on PI3K and the association with improved nAb responses is largely convincing, the results on the potential impact of soluble Nef on neighboring B cells is much less clear. SIVmac239 infects and manipulates helper CD4 T cells and these are essential for the activation and differentiation of B cells into antibody-producing plasma cells and effective humoral immune responses. Without additional functional evidence that Nef indeed specifically targets and manipulated B cells these results and conclusions should be made with much greater caution. Finally, the presentation of the results and conclusions is partly very convoluted and difficult to comprehend. Editing to improve clarity is highly recommended.

We are very grateful for the supportive and visionary review and suggestions. Experiments have been performed to improve the points raised. This work inevitably involved interdisciplinary factors to even hit on the schematic (NAbs, B cells, CD4+T, CD8+T, viral escape, immunosignaling, IEI as extrapolation & microscopy implementations) and convoluted sections should have existed. We attempted streamlining of certain portions and edited writing throughout, and hope that it became more straightforward.

Reviewer #2 (Recommendations For The Authors):

As outlined in the public review, I found the results potentially very interesting but parts of the manuscript much more complex and confusing than necessary. In addition, the methods on the potential impact of soluble Nef on neighboring B cells in vivo was difficult to assess but altogether this part was not convincing. Have the following specific suggestions:

We are very grateful for the scholarly review, and encouraging and suggestive comments on this orphan work. In the revision we designed experiments to address the properties of Nef transfer to append understanding on the in vivo B-cell data. Recommendations have been addressed as follows.

(1) Title: "AIDS virus-neutralizing antibody induction reciprocal to a PI3K gain-of-function disease". Think this title hardly reflects the data; SIVmac cause simian AIDS and is not the "AIDS virus" the 2nd part is more appropriate for discussion than for the title (and the abstract).

We appreciate the point. The original intent of the title was to conceptually bridge two differing fields of virus-host interaction and inborn errors of immunity/immunosignaling on an original article basis. Certain papers (Mudd et al, Nature 2012 etc) do utilize the term AIDS virus, and we similarly chose the term for simplification to non-virologists at initial submission.

That being said, we understand the scholarly point raised, and feel that the initial aim can be well attained by retaining the key host effector PI3K in the title, as in the revised submission titled “SIV-specific neutralizing antibody induction following selection of a PI3K drive-attenuated nef variant”.

(2) Abstract and throughout: As the authors show, SIVmac is not generally "neutralization resistant"; difficult to neutralize is more appropriate and should be used throughout. Also, the abstract and other parts are more complicated than necessary.

We appreciate the point. HIV/SIV Env immunology work utilizes “neutralization-resistant” for SIVmac239 (e.g., Mason et al, PLoS Pathog 2016), and autologous titer positivity of ~10% at this size of examination does appear low amongst lentiviruses. Nevertheless, as recommended, “difficult-to-neutralize” better describes the nature, and we have switched the term accordingly.

Linked with title modification, we reflected the comment on abstract structure and switched the main introductory sentence (Here we…) to a more data-based one instead of depicting extrapolation, and have modified phrasings in the latter half.

(3) The intro seems a bit biased. Immune evasion due to mutations and proviral integration that play key roles in viral persistence are not mentioned. nAbs are not known to efficiently control HIV or SIV replication in vivo (not even in the present study). Thus, a more "balanced" presentation of the role of nAbs in vivo is desirable.

We agree with the comment. Introduction in ver1 submission was compressed to just display humoral immune perturbation examples across persistence-prone viral infections, and indeed it should be much better to layout the multiscale strategies of lentiviruses in manifesting viral persistence. We have appended two sets of texts, one on the fundamental integrating retroviral life cycle and another on the wide spectrum of accessory protein-driven perturbation. As pointed out, the current endogenous induction is of course not early enough to exert suppressive impact on replication as like in exogenous Ab passive infusions. We have accordingly modulated text to improve the balance.

(4) Lines 73-76: rephrase for clarity.

We acknowledge the comment and have rephrased accordingly.

(5) Line 92: "linked with sustained Env-specific B-cell responses after the mutant Nef selection". After or during in one case; the time frame varies enormously and this should be discussed.

We appreciate the comment. The six Nef-G63E mutant-selecting NAb inducers subjected to B-cell analysis were the ones that showed precedence in Fig 2D (mutant before induction). That being said, we modified text as suggested (Line 104 in revised uploaded text). Text related to temporal deviation has been appended (Lines 378-383 in revised uploaded text).

(6) The authors should discuss G63R and include it in the functional analyses.

We appreciate the comment. Discussion on Nef-G63R in ver1 submission was kept minimal because statistical significance for selection was marginal. We generated a Nef-G63R mutant and results are appended in Fig 4-Figure Supplement 2.

(7) Lines 124/5: conservation only applies to SIVsmm/mac Nefs and this region is also frequently deleted/length-variable in primary HIV-1 Nefs.

We appreciate the comment. We modified description of the region accordingly (Lines 139-141 in revised text).

(8) Lines 153-155: Statement doesn't seem to make sense. The triple mutant Nef SIVmac construct was not attenuated for replication but specifically disrupted in CD3 down-modulation.

We acknowledge the comment. It had meant that the consequent plasma viral load showed a trend of decrease (as in the Graphical Abstract of the work) which should (in a simplistic view) influence antigenicity for humoral immune responses. Yet it is very true that virological replicative capacity was comparable with wild-type as in Fig.1. We have taken down the related text and rephrased it (Ref remains cited in introduction).

(9) Lines 178/9: levels in PI3K gain-of-function mice "with full disease phenotype (Avery et al., 2018)". This needs more information, e.g. what disease exactly are they talking about?

We are grateful for the correction, and have appended text and introduced the mentioned congenital disease in the Introduction section in advance. In-detail description is also appended in the Discussion section.

(10) Lines 186/7: "Env-stimulating high-MOI infection also accelerated phenotype appearance, with enhanced 50% reduction (Figure 4C, right)". Modify text and corresponding figure for clarity.

We acknowledge the comment. We revised as: “A high-MOI SIV infection, comprising higher initial concentration of extracellular Env stimuli, also accelerated phenotype appearance from day 3 to day 1 post-infection with stronger pAkt reduction”.

(11) The validity of the results described in the section "Targeting of lymph node Env-specific B cells by Nef in vivo" was difficult to assess. Altogether, however, I didn't find them convincing, especially since a negative control (e.g. macaques infected with nef-deleted SIVmac) are missing.

We acknowledge the comment. As a pure experimental control, whole-Nef deletion may assist for subtracted baselines. Within this work, the staining per se at least should be highly specific (mAb multiply verified in other applications and cytometry panel also designed for minimal spillover into AF488 channel). On in vivo basis, direct comparison may be somewhat frustrated by the fact that reduction in other pleiotropic effects of Nef seem to more dominate upon Nef deletion, as a set of reduced viremia, robust CD8 responses, killer CD4 responses and increased binding Ab titers (Johnson et al, J Virol 1997, Gauduin et al, J Exp Med 2006, Fukazawa et al, Nat Med 2012, Adnan et al, PLoS Pathog 2016 etc) leading to altered trajectory. We promise that we will work on refinement of the methodology in studies ahead.

(12) Lines 309-319: This paragraph made little sense to me (as did lines 328-331).

We acknowledge the comment and have edited both sections.

Reviewer #3 (Additional Reviewer):

In this manuscript, Hiroyuki Yamamoto et al examined virus-specific antibody responses and identified a subgroup of nine individuals, out of seventy SIVmac239 rhesus macaques of Burmese origin infected with SIVmac239, that develop neutralizing antibodies (NAb). The authors propose the emergence of a nef mutant (Nef-G63E) that impacts on B cell maturation resulting in PI3K gain-of-function.

My major concerns are:

The authors by different aspect addressed the role of the emergence of Nef-G63E mutant in individuals developing NAb. The manuscript is confused and the rational not always clearly stated. This reflects the two aspects of the manuscript (i) NAb identification in a subgroup of macaque and (2) the identification this nef mutation.

We are grateful for the comprehensive and scholarly comments. As pointed out, the work did need to confront potential bifurcation of the influence of the obtained viral immunosignaling phenotype for CD4-intrinsic (which might be your specialty) and B-cell-intrinsic impact. Based on your suggestions we have acquired additional data and revised the manuscript as attached.

The authors used both males (n=57) and females (n=13). However, there is no indication related to the sex regarding NAb inducers versus non-NAb Inducers. The notion of "highly pathogenic" is certainly not correct (see the introduction). Pathogenicity is also depending on monkey origin. Thus, cynomolgus are less sensitive to SIVmac239 or SIVmac251 compared to rhesus macaques (Ling B Aids 2002; Reimann KA, J Virol 2005; Cumont MC, J Virol 2008), or to pigtails used in US. Indeed, the authors used Burmese macaques, and therefore the dynamics of pathogenicity is different to rhesus macaque (Indian origin) housed in US. How many animals have been sacrificed out of the 61 animals? Herein, the animals are surviving longer (more than one year), and therefore the notion of "highly pathogenic" merits to be modulated.

We appreciate the comment. We have accordingly appended sex information (M/F: 8/1 versus 49/12 in NAb inducers vs non-inducers, p > 0.99 by Fisher’s exact test) in the methods section. As pointed out there are differences in the frequency and rate of AIDS progression among macaques of differing origin, whereas we have also previously reported reproducible AIDS progression dependent on MHC-I genotypes in the Burmese rhesus macaques utilized (Nomura, Yamamoto et al., J Virol 2012). Adhering to advice, we have attenuated the term to “pathogenic” in the revised manuscript and appended one reference showing pathogenesis gradation from a cell-death perspective (Cumont 2008).

Furthermore, no indication is provided regarding CD4 T cell dynamics, or CD8 T cells. In particular, the extent of T cell immunodeficiency may compromise humoral response. Therefore, this data needs to be shown. Indeed, previous reports have indicated that early CD4 T cell depletion is associated with defective humoral response. Furthermore, Tfh cell depletion was reported in several immune tissues, which are essential for B cell immune response like the spleen. Thus, this should be discussed as an alternative mechanism to the absence of NAb. Indeed, the authors found higher and persistent env-specific plasmablast cells in NAb inducers than that observed in non-NAb inducers figure 6. Why to have selected twelve individuals out of 61 individuals for assessing anti-env response (Supplemental S3 for figure 1, panel 1), and only eleven for western blots. The explanation in the text is absent. This requires to be clearly stated. See lines 108-110.

We appreciate the comment. As in other sections, this study utilized available cryopreserved samples from a retrospective cohort, also having heterogeneity in data acquisition along the way. We acknowledge that some supplemental data are particularly limited in information, which is also a reason they are presented in SI. We felt that one important core was to secure samples for Nef-G63E-selecting NAb inducers versus viremic non-inducers, for which we acquired six versus twelve in the B-cell analysis.

We (Nakane et al, PLoS ONE 2013) and others (Hirsch et al, J Virol 2004) have already reported on western blotting-basis that SIV-infected rapid progressors tend to manifest serological failure (impaired binding Ab-WB bands). Therefore, to compare quantitative traits at this basal stage (Fig 1), we judged that NAb inducer comparison with more non-rapid-progressing (>60 wk survival) non-inducers would be a criterion. We have mentioned on this in the revised manuscript (results/methods). Additionally, we have replaced the immunoblotting result with one more non-inducer (n = 12) to enhance results. Please note that there are lot deviations in strip-coated antigen (e.g., gp160) but the result is comparable (now covers 12/13 of animals with >60-wk survival).

The authors indicated the frequencies of Nef-G63E mutant in figure 2 panel C. However. no information is indicated in the legend about the number of NAb non-inducers used to calculate this frequency. The authors indicated line 127, "only in two of the nineteen NAb non-inducers, including one rapid progressor". Thus, different numbers of individuals are used through the manuscript. For the readers, this is clearly a statement that needs to be clarify and to refer to what. This is not homogeneous along the text and the analyses performed.

We appreciate the comment, and have appended the number in the revised Fig 2C. As aforementioned, heterogeneity of sample number in different sections is indeed a limitation of the work, and have mentioned this in the Discussion.

The rational related to the sentence lines 140-142. Please clarify.. "NAb induction is not associated with these MHC-I genotypes (P = 0.25 by Fisher's exact test, data not shown) but with the Nef-G63E mutation itself".

We appreciate the comment. We have rephrased it as:

“Ten of nineteen NAb non-inducers also had either of these alleles (Figure 1-figure supplement 1). This did not significantly differ with the NAb inducer group (P = 0.25 by Fisher’s exact test, data not shown), indicating that NAb induction was not simply linked with possession of these MHC-I genotypes but instead required furthermore specific selection of the Nef-G63E mutation.” (Lines 159-162).

In supplemental figure 3, only 7 individuals have been tested, while the authors indicated "Ten of nineteen NAb non-inducers also had either of these alleles". Why only seven? In NAb Burmese monkeys, the authors indicate specific T cells capable to recognize WT nef peptide, but not G63E peptide mutant. Thus, nef is immunogenic in vivo generating T cells despite to be mutated.

In contrary, non-NAb-inducers demonstrate the absence of nef specific T cells (supplemental figure 3, excepted R01-011 panel A). Although, the authors propose an escape mutant for CD8 T cells, this is not associated with the absence of immunogenicity and not with a difference in viral load in comparison to NAb inducers (panel C). Therefore, the conclusions merit to be revised. Thus, this part of the manuscript is confusing. Please clarify the rational to link NAb and Nef specific CD8 T cells.

We appreciate the comment. 7 out of 8 non-inducers positive for the allele and not selecting for the Nef-G63E mutant was available for analysis. The relative contribution of this single Nef62-70 epitope-specific CTL response is speculated not to be largely impacting viral control, among the many induced. This is basally discussed in a previous paper (Nomura, Yamamoto et al., J Virol 2012), more suggestive of an MHC-I haplotype-level correlation with plasma viral load. We assume that the CTL pressure-driven selection of Nef-G63E mutant was a rather pure immunosignaling trigger under persistent viremia. We appended this in the revised text (Line 172).

In the next part of the manuscript, the authors assessed the function of this Nef-G63E mutant. The rational to introduce Ferritin in this part of the document is not clear for the reader. Furthermore, a subgroup for each (NAb+ versus NAb-) is shown: 4 for NAbneg versus 6 for NAbpos.

We appreciate the point. As introduced, Swingler et al Cell Host Microbe 2008 reported HIV-infected macrophage-derived ferritin as a potentially B cell-disrupting factor. In that paper, viral load, ferritin and binding antibody titers positively correlated. Current data shows that SIVmac239-specific NAb induction is distinct from such kinetics already versus viral load (Fig 3-Supplement 1C), and ferritin levels were measured for some available samples more simply for confirmation. We appended three more available samples in the NAb- group. (The six NAb+/G63E animals correspond to the ones with B-cell data in Figure 7.) Statistical results appear unaffected and robust, as shown in this version. The revised manuscript incorporates appended explanation for the former.

Similarly, whereas the authors observed a role of nef mutant on pAkt Ser473 (less induced) in comparison to WT, the authors suggest that this may have an impact on T cell survival.

We appreciate the point. In the first submission we obtained peripheral memory Tfh decrease, whereas it is true that this is indirect. In the current revision we have addressed apoptotic cell death, shown to increase with Nef-G63E mutation (Figure 4F).

The rational to analyze CXCR3-CXCR5+PD-1+ memory follicular Th (Tfh) is not clear. Moreover, the references used are not the adequately cited. Indeed, these papers show an expansion. See the literature for a depletion (Xu H, J Immunol. 2015; Moukambi F, PLoS Pathog. 2015; Yamamoto T, Sci Transl Med. 2015; Xu H, J Immunol. 2018 Moukambi F, Mucosal Immunol. 2019).

We appreciate these points on in vivo CD4+ T cells.

Peripheral memory Tfh was reported to correlate with Ab cross-reactivity in one human cohort (Locci et al, Immunity 2013) and we concisely examined the subset in the current NAb induction. We mentioned this in the revised manuscript.

Moukambi F et al, PLoS Pathog 2015 & Mucosal Immunol 2019 are demonstrative work on acute-phase destruction. We have cited non-neonatal/vaccine-related ones suggested, including these two, in the revised manuscript. The biphasic dysregulation of Th (acute-phase destruction and chronic-phase adverse hyper-expansion) may indeed have a unique role with the current phenotype, which is beyond aim of the current analysis. We have concisely mentioned on this in the Discussion.

Then, the authors assess the potential B-cell-intrinsic influence of the G63E-Nef phenotype. The rational here is clearly indicated, making sense with figure 1. Furthermore, this part is clearer. The dot-plots merit to be revised and the markers used better stated. The authors indicate that Nef invasion upregulates pAkt Ser473 assuming aberrant PI3K/mTORC2 signaling. What is the impact of Nef-G63E mutant on pAkt Ser473 using in vitro model of transfer. This is not addressed for comparison.

We appreciate the remarks/suggestions, also pointed out by Reviewers 1 and 2. We have performed three sets of in vitro reconstitution experiments visually and functionally addressing how Nef transfer to B cells can be modulated (new Fig 6), and edited text accordingly.

Minor points are:

- the presence of references in the legend.

-some Ab clones are in the table, however they are not used such CD38 and CD138, which are well known to be non-valid B cell markers for monkeys."

We appreciate the suggestions.

Mentioning on reference have been removed from the legend (Fig.1, Fig. 3) and moved to the corresponding Methods section (Fig. 1).

We also understood this well in advance (CD38/CD138), and incorporated them in the memory B-cell panel just to check whether they ever behave in a specific pattern. As expected, no notable behavior was observed in these NAb inducers.

Platforms are vulnerable to cyber-attacks, users' information may leak. Also, while platforms collect users' preferences, users' info may be kind of transparent on the internet.

There is potential profit comes from violating users' privacy. For example, these information can be sell to ad companies to make targeting ads. Or sell these data to third parties. And it is hard for users to find out social media sell their information, the risks involve is low.

Because it shows how businesses might produce new information on users without their knowledge or agreement, the idea of inferred data—such as shadow profiles made by social media companies—is worrisome. This type of privacy violation frequently takes place in the background and is challenging for users to keep an eye on or manage. Furthermore, Facebook's collection of information on users without accounts demonstrates how widespread data tracking has become. The broad ramifications of contemporary data surveillance are demonstrated by the fact that even non-users' information can be collected without their agreement.Because it shows how businesses might produce new information on users without their knowledge or agreement, the idea of inferred data—such as shadow profiles made by social media companies—is worrisome.

I think that all major social media companies should make there privacy laws clear when signing up for them. Unclear privacy rules can result in a misunderstanding between the user and the corporation and it can lead to the users privacy being violated.

Question 7) The season for Dungeness crab starts December 1st according to the 2012 article. Only male crab the size of 6.25(in) or 159(mm) minimum may be harvested during this season and the season ends August 14th. The fall months are avoided because this is when male molting season is. According to the ODFW website, the updated minimum size for harvest is 5.75(in) or 146.05(mm). The updated open season dates start December 1st and end October 15th. In order to go crabbing you must be 12 years or older and have a shellfish license. When catching the crab you must release any female crab immediately and any male crab must be measured. When measuring you want to use a crab gauge and you measure across their back horizontally, not including their spike on each side. When determining sex, on the underside of the male crab you will see a long skinny triangle and on female it will be more wide and round. over the past years, not much has changed regulation-wise, only a slight change in size and dates. For commercial crabbing the regulations have stayed the same since 2012, minimum size being 6.25(in) or 159(mm) and the season starting December 1st and ending August 14th. Although if you are commercial crabbing, you must have a commercial crabbing license and no more than 15 crab on each vessel.

Question: If crabbing for females was aloud, only for recreational crabbing, and only at females maximum size, would this drop the population of Dungeness crab too much? or would it not affect the population significantly?

This is a telling sign that many in Paine's audience are Christian. It illustrates Paine's understanding of who his audience is, and takes advantage of that knowledge to drive home the points he's making to the part of his audience.

This is a significant in that a system that had long been accepted by many is being challenged here. It shows a difference in belief many colonists have in contrast to their predecessors.

Why is the cause of America great for the cause of mankind? What benefits does rebellion does it bring for everybody else in the world?

Why does Paine avoid getting personal? Is there a downside to expressing something personal? What being personal weaken the argument Paine is trying to lay out?

This shows an understanding from Paine that many colonists are not fully for rebellion, even with it looming on the horizon. Thus Paine adds this part in to show understanding in the fact that he likely won't gain any favor for writing, at least not yet.

This highlights the time Paine lived, where most continents were ruled by monarchies, or if not that something similar. This shows how much of a step the colonies are taking, which in effect was fighting a system of government that was in charge of almost every nation on Earth.

Oppression is normally a situation in which people governed unfairly and cruelly, with rights and freedoms being suppressed. Paine is trying to get across that the colonists are being oppressed because of the amount of power Britain have over them.

It's important to highlight that the answers are in the people who live in the neighborhoods, they reflect the problems and the ways we can improve. These communities benefit economically and the redline system decides their future in an unfair way .

I wonder if the women from other nations (outside of America) had the same problem post WWII of not being properly compensated for their service

I never had historical context for the origin of this meme so this was cool to learn

The tragedy of these acts of sexual violence and their abuse of comfort women, including drug-induced compliance, continues to affect Chinese people (women in particular)

Did they not see how useless trench warfare was?

It's interesting how big this war was. There was so any different fronts, it feels bigger than WW1 definitely but just by far so large in comparison to the wars before this time.

I was under the impression that America had a really strong military. Was it weak (in comparison) because of WW1? Or just because they weren't looking to get involved in the war? (Unlike Japan who was looking to prove strength)

It's really crazy to me that they were being so drastically violent that it wasn't even believable.

Did he do this because he wanted to be the sole leading power? Or because they were communist? (Or both?)

This new strategy completely changed how war was fought. The fact Germany completely destroyed every country they fought against with this strategy was shocking to me. Germany was a superpower but they did do things that would hurt them in the long run and they then were fighting some of the best superpowers which were Great Britain, U.S. and the USSR.

Seeing this now really is shocking that we now have one of the highest military spending in the entire world.

Japan was the ready to go ally Germany needed and they had no threats to worry about in Asia or the Pacific. Both countries did as they please and they were simply gaining strength and not another country tried to limit them or stop them.

This is a very important part because Japan started to threaten U.S islands in the Pacific and they ended up attacking Hawaii but this conquering turned Japan into a feared force in the Pacific Area and in Asia too.

It is evident that the Indigenous did not wish to kill John, as they commended him for defending himself against the attack. Earlier in the text, we can also see that they aided him in minimizing the damage to his frostbitten leg, and provided him with snowshoes to use in the heavy snowfall. These all demonstrate that although he was a prisoner, John was still being taken care of to a reasonable extent.

This excerpt shows the non-sedentary lifestyle of the Indigenous. Rather than set up a settlement, they were setting up camp wherever they made a kill, as we can see here. In both articles, travel is a major theme, showing that Indigenous groups kept captives with them while traveling, as they did not possess dungeons or prisons in which to keep prisoners.

It is interesting that the captors only let John eat once he has done certain tasks (supply them with wood and water), whereas Mary was given broth without conducting any tasks. This could be due to gender differences, as keeping John weak to prevent him from revolting would have been a possible strategy.

This passage shows us the close knit nature of Indigenous societies, as no man or woman was left behind. When they were unable to carry the bier through the thick woods, they took turns carrying the man through the woods, rather than just leaving him.

Religious syncretism

Even Kim Kardashian has come to defend the brothers, influencing her followers to advocate for them too. This case has also been in lots of young people's media, especially through TikTok.

This signifies that the American mindset has developed greatly over the past couple of decades and is more understanding.

I think this has become very relevant today. Even recently with the Abercrombie CEO sex trafficking scandal, people often tend to believe only women are victims of sexual abuse which is just completely false.

All this detail was explicitly shown in the Netflix series.

These brothers being young and conventionally attractive displayed a different side of America; a side that supports murderers because of their good looks and interesting background.

Both of these cases are engraved in the nations mind. Many people can look back at when these cases first began and describe in detail what happened. People love the drama, with case being wealthy, white men, and from Hollywood all eyes all over America were on a television screen. Furthermore, these cases being televised and becoming so popular signifies that the nation is obsessed with murder, drama, and sexual appeal.

Both of these shows that aired on Netflix got huge media attention, and caused an uproar on social media, often with people defending the two brothers. Many people even took to social media to call for a retrial to show their support and love to the brothers.

Times are very different now compared to how they were in the 1980s. Sex crimes and offenses were often overlooked and doubted highly in the courts. Now, with the rise of these cases the criminal justice system handles these cases very seriously due to how common these crimes have unfortunately become.

It is important to note that this case has had an unprecedented amount of attention since it first came about. People feel very strongly about this case, and the brothers have thousands upon thousands of supporters.

Crash course on typewriter maintenance and repair

A list of resources and references for the budding typewriter repair person. There is a lot here that I've compiled and consumed over the last six months, so don't be overwhelmed. Half the battle is figuring out where to find all these things, so if nothing else, this should shave off a month of reading and researching.

Basic Introductory Material

Get a notebook and be ready to take some notes so you'll remember where you found the random information you're bound to pick up over time and are able to occasionally review it.

Work your way through Sarah Everett's excellent Typewriter 101 videos (at least the first five): https://www.youtube.com/playlist?list=PLJtHauPh529XYHI5QNj5w9PUdi89pOXsS

Read Richard Polt's book which is a great overview to the general space:<br /> Polt, Richard. The Typewriter Revolution: A Typist’s Companion for the 21st Century. 1st ed. Woodstock, VT: Countryman Press, 2015.

Next watch the documentary California Typewriter. Documentary. Gravitas Pictures, 2016. https://www.imdb.com/title/tt5966990/. It has some interesting subtle material hiding within it, but it will give you a good idea of where you're headed off to.

Get a machine (or four) you can practice on. Get a flat head screwdriver and maybe a small adjustable wrench. Buy some mineral spirits and a small headed toothbrush and clean out your first machine. Buy some light sewing machine oil and try oiling it. Search YouTube for videos about how to repair anything that may be wrong with it.

Basic restoration advice: https://site.xavier.edu/polt/typewriters/tw-restoration.html

On colloquial advice for degreasing, cleaning, and oiling manual typewriters https://boffosocko.com/2024/08/09/on-colloquial-advice-for-degreasing-cleaning-and-oiling-manual-typewriters/

Repair Manuals

Create an account on typewriterdatabase.com which will give you some additional access to catalogs, manuals, and dealer catalogs.

They also have some openly accessible material like:<br /> * https://typewriterdatabase.com/manuals.php

Printed manuals: https://www.lulu.com/search?adult_audience_rating=00&contributor=Ted+Munk&page=1&pageSize=50 PDF manuals: https://sellfy.com/twdb

Ted Munk's website also has a plethora of ephemera that is often useful * https://munk.org/typecast/

Richard Polt's list of service manuals, which also includes some correspondence course typewriter repair classes: https://site.xavier.edu/polt/typewriters/tw-manuals.html#servicemanuals

Tools

In rough order of increasing complexity:

• Typewriter Tool Kit from the DOLLAR TREE by Just My Typewriter https://www.youtube.com/watch?v=R-7E4fM6gBw
• Some simple basics and where to get them: https://boffosocko.com/2024/08/11/adding-to-my-typewriter-toolset/
• Typewriter tools of the trade: https://www.youtube.com/watch?v=x5bKk93rtxs
• Lucas Dul's Toolset presentation: https://virtualhermans.com/lucas-dul
• Tour of an advanced hobbyist/semi-pro shop: https://www.youtube.com/watch?v=lZjf6InMlgU

Tools can be expensive, so start out small with just a few things and expand as you need them. You'll be amazed at what you can accomplish with a single thin bladed flathead screwdriver, an adjustable wrench, a rag, a bottle of Simple Green cleaning solution, and a bottle of isopropyl alcohol.

Videos

Subscribe to and become acquainted with YouTube channels like the following:

• Duane Jensen's Phoenix Typewriter https://www.youtube.com/@phoenixtypewriter2136
• Gerren Balch's HotRod Typewriter Company https://www.youtube.com/@HotRodTypewriter
• Joe Van Cleave https://www.youtube.com/@Joe_VanCleave
• Sarah Everett's Just My Typewriter https://www.youtube.com/@JustMyTypewriter

While watching a variety of videos is great, as you're doing specific repairs search YouTube and you're likely to find full demos of the repairs you're doing yourself.

I've compiled a playlist of videos for repair of an Olympia SM3 which, while specific to the SM3, is a an excellent outline/overview of how to disassemble a portable typewriter, where many of the adjustment points are as well as an outline of the order to do them in.

If you're not a good typist or don't have experience in the area, try out some of the following short films which will also provide some useful historical perspective:

• Basic Typing: Methods. Vol. MN-1512a. United States Navy Training Film, 1943. https://www.youtube.com/watch?v=ztyzGit1dTI.
• Basic Typing: Machine Operation. Vol. MN-1512b. United States Navy Training Film, 1943. https://www.youtube.com/watch?v=b-REJEArnjE.
• Advanced Typing: Shortcuts. Vol. MN-1512c. United States Navy Training Film, 1943. https://www.youtube.com/watch?v=JUJfCfqgsX0.
• Advanced Typing: Duplicating and Manuscript. Vol. MN-1512d. United States Navy Training Film, 1943. https://www.youtube.com/watch?v=7ve5JnTUzvo.
• Maintenance Of Office Machines. Vol. MN-1513. United States Navy Training Film, 1943. https://www.youtube.com/watch?v=ocdxgkxKAKo.

Internships & Apprenticeships

If you have the time and flexibility try arranging an internship or apprenticeship with a local typewriter repair shop. Meet your local repair people even if you can't spend the time on an internship. You'll learn a lot and create relationships with businesses who will more easily swap/supply you with machines they're parting out or access to tools which may otherwise be difficult to source.

• https://site.xavier.edu/polt/typewriters/tw-repair.html

Podcasts

• Austin Typewriter Ink https://www.austintypewriterink.com/ati-the-podcast.html
• Charm Type Repair https://charmtypepodcast.podbean.com/

Some useful Bibliography

• Athey, Ralph S. Typewriter Repair Training Course. Tarentum, PA: Typewriter Repair Training, 1957. https://site.xavier.edu/polt/typewriters/AtheyTypewriterRepair.pdf.
• Atkinson, Annelise. Typewriter SOS: The DIY Guide to Fixing Common Problems with Typewriters, 2014. https://www.amazon.com/Typewriter-SOS-Fixing-Problems-Typewriters/dp/1520902700/.
• Hausrath, Alfred H., and Eugene L. Dahl. Typewriter Care. Edited by Walter K.M. Slavik. Federal Work Improvement Program United States Civil Service Commission and Government Division, U.S. Treasury Department, 1945. http://archive.org/details/twcare-1945.
• Jones, Clarence LeRoy. The Manual Typewriter Repair Bible. Edited by Theodore Munk. The Typewriter Repair Bible Series, 2017. https://www.lulu.com/shop/ted-munk/the-manual-typewriter-repair-bible/paperback/product-1vgk72jp.html.
• Kasten, R. M. “First Aid for Typewriters.” Popular Science Monthly, May 1941.
• Kravitz, Bryan. Hints for a Happy Typewriter. Bryan Kravitz, 1983. https://site.xavier.edu/polt/typewriters/Hints-Happy-TW.pdf.
• Hutchison, Howard. The Typewriter Repair Manual. 1st ed. Blue Ridge Summit, Pa: Tab Books, 1981. https://www.amazon.com/typewriter-repair-manual-Howard-Hutchison/dp/0830600345.
• Munk, Theodore. “The Typewriter Database,” 2012. https://typewriterdatabase.com/.
• Pearce, H. G. Complete Instructions: How to Repair, Rebuild, and Adjust Underwood Typewriters With Handy Reference for Locating Trouble Quickly. Bridgeport, CT: Typewriter Mechanics Publishing Co., 1920. https://johnesimmons.com/Typewriter/Articles/Manualpdf/Underwood_Repair_Manual.pdf.
• Polt, Richard. “The Classic Typewriter Page : All About Typewriters,” 2009. https://site.xavier.edu/polt/typewriters/index.html.
• Scadden, David T. Approved Home Study Course in Typewriter Repair and Service. Little Falls, NJ: Typewriter Repair School, 1959. https://site.xavier.edu/polt/typewriters/homestudycourse.pdf.

Good luck on your journey!

reply to u/fontinalispluma at https://old.reddit.com/r/typewriters/comments/1gaza5x/learning_typewriter_maintenance_and_repair/

If ligand I weak field ligand then it will be S.P>

More and more people are getting college degrees, which that high school degrees are less valuable or the college degree will worth more. Thinking about jobs such as "secretary" that we read about in Tuesday's reading. The job description and responsibilities can also change so that it requires a higher degree. Now, so many people are getting bachelor's degrees that further education or a lot of experience is needed to be competitive.

This is a sad reality for many schools such as Santa Ana. Constant faculty turnover would result in less experienced teachers and rookie teachers taking their place. Teachers can also be discouraged because they want to teach and help the students, but instead have to "baby-sit" or grow to have an apathetic attitude because nothing seems to work.

This is interesting. There is definitely a difference in top and bottom socioeconomic schools, but what makes more of a difference in gaps are nonschool factors. We've been reading about this in previous readings. Where you live matters and overlaps with SES which also afffects where you go to school and the opportunities you are exposed to. Cognitive stimulation can be affected by family structure and how much support and attention a child recieves.

16:30 "But the only fatwa that has, in fact, said that no images of Mohammed are permitted, and that includes Islamic paintings, not just the cartoons, came out in 2013 in Saudi Arabia by a Salafi cleric whose name is Al-Munajid. And there are other fatwas like Asistani, the Shi'i cleric, who says these images are perfectly fine, as long as they're respectful."

20:00 Fatwas can be issued (like the above) in a vacuum without any real conversation within the Islamic community. Few years back even building a snowman fatwa as haram. Animals are decapitated in Saudi textbooks. People in 20th century having 14th century book that depicts a head, decapitating it (al-ras).

15:30 The advent of media allowing for more images to be produced of the prophet. This in turn led to more anxiety under the Islamic community. See The Message (and the other movies) as examples of this increase of anxiety.

05:00 Gruber discusses how the canon should be decolonised (how eurocentrism should be replaced with different centres of interests) and how showing the prophet actually contributes to that purpose (including 14th century Iran that portrays these depictions of Muhammad).

07:30 Wow, interesting point: The people back then struggled with the question of how to depict prophetic and divine figures. How to do that? Certain anxieties underlying this.

08:30 A week before the incident, Lopez showed art in India and of the Buddha and so on. A whole course on how the divine is presented, and struggled with, in different regions and cultures, other than the West (an Eurocentric view).

Put tracks between each pair of songs to highlight the journey with a natural flow.

About four landmarks for an hour long playlist.

Go from the end to the beginning. Tell a story

This shows the effect of finances on a family dynamic because although both Mrs. McAllister and Ms. Williams are good mothers who provide for their kids, the difference shows in their parenting styles. Mrs. McAllister does not possess the time and money that allows Ms. Williams to think Alexander is more than just a child, his opinions need to be cultivated and developed.

It's interesting to note the family dynamic differences between Harold McAllister's family and Alexander Williams'. One important note is the lack of a typical family structure, because Harold's father and mother never married. Therefore, the father figure is not a permanent fixture in Harold's life as his visits, though regular, are not the same as living in the same household.

Children born into wealthy or middle class families are not ever exposed to the harsh reality that children born into poverty are, which is that their families will not be able to afford basic necessities, much less extracurricular activities. This is why children born into wealthy or middle class families often have a sheltered "bubble" in which they even complain about the multiple opportunities offered to them that other children cannot even dream of.

This goes to show that it tends to be a generational issue in terms of negative parent attitudes towards school. If a parent has negative experiences in school, his attitude may reflect on his child as they try to "protect" their child from the same things they had to go through. This can lead to their child not participating in school functions or activities, which can have a negative effect on their social behavior.

Eh, what stops them to come to consensus regarding some topic?

Swiss folks interact peer-to-peer, passing laws.

Why so? They're not in the KEL anymore.

And those who learned them will accept superseeding recovery, so maliciously issued certificates out they go.

I understand this author's approach to her teaching students at a "failing" school with lower-income students. She not only attempts to understand her students living in poverty, she tries to change small things to assist their situations. No matter what, she believes in them as she notes "All students are capable of learning".

I appreciate how the author does recognize that she does fit a lot of the typical stereotypes that a white female would incur, and yet there's more beyond what the eye can see. When she goes into detail about her living experiences, I was shocked by the level of harsh reality she was thrust in. It allows her students to recognize that she is not just their white teacher, she is her own person who has also gone through difficulties in life.

I love this quote because it not only shows the author's willingness to be open to learning from his students, it conveys his gratefulness towards his pupils for giving him a priceless education. He even goes on to say they may not have "walked in with a learning objective that matched the common core standards", but he appreciates the beauty of students teaching the teacher, calling it "organic and spontaneous".

https://www.etsy.com/shop/HotRodTypewriterCo

Gerren Balch is the eminence gris behind HotRod Typewriter Company

This quote recognizes the importance of a healthy family environment for a student to be able to thrive in a school setting. The author's father feels suspicious and untrusting of authority due to an unstable home life and thus this translates into his raising of his son, as he refuses to show up to school functions unless he has to be.

Severe lack of context of the incident, and the historical context of portraying Mohammed, is very evident here. The imam says "that we Muslims don't show images of the prophet" which is partially true, some Muslims don't. An interesting parallel with the general outcry of support for the teacher.

Jaylani Hussein says that most Muslims don't support portraying the prophet, whilst acknowledging that some do. (But portraying the prophet thus is not good). Even says it is Islamophobia at 2:00

"The president of the Muslim student association" Aram Wedetella also in the class speaks up.

JAN. 17,2023 "Based on all that we have learned, we have determined that our usage of the term 'Islamophobic' was therefore flawed." Ellen Watters, Chair, Hamline University Board of Trustees Fayneese Miller, President, Hamline University

04:30 Question of how these images are now marginalised in the Islamic community. Came from the Ottoman Empire? Safi blames the rise of Salafi and Wahhabi movements. These images seem to portray mainly the mi'raj, ie spiritual and mystical dimensions. And the prophet portrayed in companion with other prophets (ie the universality of Islam).

"We believe in academic freedom, but it should not and cannot be used to excuse away behavior that harms others." (STATEMENT FROM HAMLINE UNIVERSITY) The above statements shows the struggle that Hamline faced when deciding to fire Erika Lopez Prater. A tension between academic freedom and insulting others.

1:05 A good point by Safi is that the statements of the director was assuming that the freedom of speech was in direct opposition to the feelings of Muslims. This is not the case. A difference between iconical devotion within the Islamic tradition (both Sunni and Shia) and Charlie Hebdo examples.

03:00 Both to respect students and expand their horizons. Not everyone needs to agree with it.

Usually the keys on most typewriters are friction fit on and come off pretty easily. The tougher part is that the slugs are soldered onto the typebars, so you'll need to remove them and swap them with a soldering arm. Even if you have the soldering tools, the more trying part is aligning them properly when putting them back on. Many old school shops have/had custom jigs made for properly aligning slugs when soldering them on.

If you don't have the tools, patience or facility, this is usually something best left to your favorite shop: https://site.xavier.edu/polt/typewriters/tw-repair.html

reply to u/fontinalispluma at https://old.reddit.com/r/typewriters/comments/1gb3dwc/change_the_keys_and_type_slugs/

God does mean for the commandments to be controlling... JPII thinks it is moral to follow the rules and sees the rules as a baseline and then challenges us to find other things to make life more flourishing

morality is based on the upholding of human dignity

JP II understands that abortion is more tempting given certain situations especially when the women is left alone and the father leaves.

JP II is very set on the idea that rules will lead you to live a holy and fulfilling life

The audience are judges who are caught up in the witchcraft time. Only believing what they hear not what is factual.

Ann felt bad that she falsely accused others who now she believed in their innocence. Wondering if Sarah looked back on her life feeling any guilt in the death of her mother.

Providence is God's guidance for the universe, destiny God's will.

This shows that they knew nothing about disease. The Phelps child could've gotten sick from a number of things.

This could be the child's imagination. There is no scientific proof that the mother was a cat. This testimony condemned her mother to death.

This sounds like the Grimm Reaper the man of death, Christianity was a big part of their culture. This is before the great awakening.

Sarah Carrier is 6 years old, being asked questions by the judge

Part 4 is the hint for HW4 Q3.2.

A reaction that extends the carbon chain of a molecule

A carbon-hydrogen bond in a cyclohexane ring that is parallel to the ring axis, pointing straight up or down.

An organic compound formed by the reaction of an alcohol with carbamic acid

A chemical reaction in which the reaction rate is directly proportional to the concentration of a single reactant.

A solvent that does not react with any reactant or product and functions only as medium for the reaction to occur.

Two positions away from the reference point

specific group of atoms in an organic compound that are responsible for the compound's physical and chemical properties

Hindrance to movement and/or chemical reaction of a molecule due to the position of bulky groups

omega position refers to the terminal position, the farthest position from the reference point.

An atom or a functional group that withdraws electron density from the rest of the molecule.

A C-H bond located on the benzene ring, at a position that is two carbons away from a reference substituent.

A reactant that is first completely consumed in a reaction, thereby, limiting the amount of product formed.

A term used to describe the attachment of a ligand to a metal center.

The component present in larger amount and serves as the medium for the chemical reaction to occur.

A chemical reaction that involves the introduction of a boron containing group into an organic molecule.

welfare states: not saying people do or do not deserve dignity but also at the same time you can't show dignity to one person and not the other

social mortgage: using these gifts but there is a responsibility to carry with it.

Private property: intellectual abilities, technology and skill

trying to say that capitalism should not be way to turn away from socialism. he disagrees with state capitalism but he does agree with the right to voluntarily participate in whichever way they want to in the economic state.

violates the humanistic viewpoint bc seeing someone as a consumers sees them as a means to an end and just trying to sell their products. This defeats human dignity and lessens the inherent human dignity given by God

-trying to capture natives to colonize them

It's saying that there's so much information at our fingertips that we're being bombarded with. We need to be more careful with where we spend the time we have.

FINAL THOUGHTS: This paper discussed the Feminist Theory, talking about the ways in which women suffer due to their gender alone. They discussed the cultural norms associated with each gender, and how humans will decide what gender means. Also, they discussed peace post-war, and questioned if it could be labeled as "peace" when violence against women persists. Overall, I think we need to change our viewpoint, and instead recognize violence against women as a hard issue, as this is a fundamental human rights violation. If the UN does recognize the importance of addressing violence against women in conflict and post-conflict situations, then this issue should be actively being worked on, as its a critical issue. Questions I have after reading this paper are: what can we do as individuals to help encourage people in power to take this issue more seriously? If there was a resolution to help women in the UN that didn't work, shouldn't violence against women be seen as a critical issue that needs immediate attention? Additionally, how can we get more women in these roles when they deal with these stereotypes?

This theory is not only about inequality, but it also studies the powers among genders in relation to global politics.

Military/International organization is always prioritized over an individual. However, it's crazy this is seen as an individualistic problem, when so many of the population is struggling from this violence.

"Peacekeepers" not only allowing, but contributing to the sexual violence.

Not only are they not benefiting from making decisions, they also aren't getting the political, economic, and social gains that come from being a person in power.

Despite the resolution, women still face the same violence.

How can we categorize a state as in peace when half of their population is being denied the rights they deserve.

Despite them attempting to bring more peace postwar, they are heavily ignoring the violence that still persists throughout half of their population.

This reminds me of the example that was given in class, where female politicians would wear masculine suits to appear more serious and less feminine.

Since women don't have access to be in the room while making decisions, their experiences are not heard.

Women are left out of a lot of the decision making in "High Politics".

Doesn't acknowledge the struggles of women.

Liberal and Realist IR theory are the mainstream theories, Others are critical theories.

The current workflow (as outlined) in Jira should be followed. If any resources need to be changed, Himangi can handle it through Strapi. Arif, please confirm with Riza and let us know.

KIV: As discussed - Pls combine all partners into a single section titled “Key Partners”. There will be no separate categories, such as "School and Education Partners" or "Corporate Partners." This section will feature all key partners together in a slider like the testimonials section.

Himangi to add the remaining team members.

The last slide is missing from the timeline. I’ve already notified Astitva about it. I believe we can simply duplicate a slide in strapi and add the content accordingly. Pls confirm

Observation: Pope Gregory is saying that a woman's husband may not approach her until after the child has been born Interpretation: During this time woman were seen as impure while they were menstruating and while they were with child. Context and Change over time: I connect it to connect because people would need to know how men and the church saw women while they were in "that time of the month", they were seen as "dirty", with this context they would clearly see why they would need to stay away. As for change over time we currently we are used to having husbands be there with their wives throughout their pregnancy and for the most part most religions allow them to be together during that time.

Observation: Pope Gregory informs Augustine that they are to punish those who steal from the church Interpretation: They are to punish the person because they should behave like the parents that wish to teach them right from wrong in order to make them better people. Causality: If they steal from the church then they will get punsihed.

new tech advancements

This is an aspect of the story that differs from typical naturalism. In naturalism, there is a big emphasis on Darwins idea of "survival of the fittest." It would be unlikely to see a bunch of men struggling to survive all befriend each other and form such a "subtle brotherhood."

This detailed description of the birds and how the men in the boat envied them as well as felt annoyed by them adds a pessimistic/cynical tone to the story.

I believe this highlighted section follows the characteristic of naturalism that states, "settings in extremely harsh nature (deserts, the Yukon, the open ocean) or cities (the urban jungle); nature is either indifferent or hostile to the plight of humanity"

"the upper edge of the side of a boat or ship"

While The Open Boat is a fictional work, it was based on Stephen Crane's real life experience being shipwrecked.

We utilize our media literacy skills to choose the proper media to learn from.

People can share their ideas and beliefs through technology.

Bringing together information to form an idea.

As part of the younger generation, I can attest that technology is an instinct and we do not even notice how often we utilize it.

A persons interactions form who they are as a person. The media that people interact with will inherently shape their life.

Society is now entangled with technology and it is vital to implement media literacies.

This would give people different perspectives and result in a more diverse and inclusive society.

The main concern of media literacies should be teaching how to detect underlying messages. Underlying messages can be very impactful on their young impressionable audiences.

Since young people are utilizing technology for around 10hrs a day it is important that they are educated on the good usages and the negative effects.

I think this is a great quote and would be perfect to catch the readers attention in the beginning of the article.

In order to keep the consumers attention, media companies may publish untrue and biased information. This is why we need to think about where we are getting our information.

We as a society have become so immersed in society that we forget to critically think on our own and question the sources in which we are getting the information.

Younger generations prefer to get their information from the internet and social media rather than television news sources. This shift requires more media literacies to avoid the spread of false information.

The printing press is known to have sparked a paradigmatic shift. A paradigmatic shift is a major change in society, learning, technology and knowledge. This invention shifted the way humans communicate.

How are humans being transformed by the technology of a museum visit.

This framework will advance media literacies by implementing critical thinking which will allow media users to reclaim their life.

Even with all the tragedy the U.S paved a way for the economy

this type of strategic spending benefited the nation and uplifted the economy

I feel like this is a great idea when trying to understand the information you are being presented with. By limiting distractions you are able understand the material better. This way you can understand what is being said and grasp the information better.

Bastille day - marks the anniversary of the fall of the Bastille in Paris, France, on July 14, 1789.

He then references how it is now 1959 at 12:20 pm. Bastille day is a very celebrated holiday, and a very important one too, so much that a New Yorker is referencing it. Three days after this day a tragic event happens, and Billie Holiday's death shakes the world, stopping everyone's breath and movement.

This poem also signifies how he 'remembers where he was the day ___ happened'

Very little punctuation where there should be in this poem. The lack of punctuation highlights the urgency and the anxiety behind the writing of his actions, leading up to the moment where it all stops, "and I stopped breathing". New York is a busy place and he shows this through his many actions, portraying through his writing that it is a nonstop day of moving. The anxiousness behind the writing also implies that there is already some sort of buildup to this final moment

This is a subtle foreshadow that the day is different than usual - for once in her life the teller does not check his balance as she usually does

thats scary

I speculate that this is dismissive behavior. I do believe that North Korea has troops in Russia and they plan to use them. While this is not fact, it is my opinion. I don't trust either country and neither does the US, South Korea, Ukraine, or most other countries in the world for that matter.

A very scary idea. These two form a diabolical duo.

More fact. Would Russia call in this pledge even if they have the upper hand? this also feels like a one sided deal in which Russia is getting more out of the relationship. Russia is receiving weapons and allegedly, troops. While North Korea isn't really under any threat to my knowledge. Is North Korea planning on needing Russian assistance in the near future?

Unless the Northern troops are just training, there still isn't really any evidence that this is happening. Just suspicion.

This is a big difference in numbers. I don't know how they have come to that estimation. The end of the year is in just 2 months.

So far this is all speculation. No facts have really been stated. If Putin is using North Korean troops, I feel like it could mean many things. Maybe instead of being in trouble, they're tryin to solidify their upper hand position by pulling in more troops.

This and the other new additions listed at https://onlinebooks.library.upenn.edu/new.html aren't listed on Wikisource and need to be added.

BIOblogs

the color gradation should be like figma

the gradation should be like figma

the border too rounder

// The advantage of this approach is that the link between the news wave and a key event is unambiguous since it is (mostly) pre-defined by case selection. This disadvantage is that the number of cases that can be surveyed is limited and that cases are selected on an ad-hoc or strategic logic that may induce biases and blind spots.

// Finding news waves and linking them to key events can be extremely useful for Communication Researchers, particularly those interested in identifying turning points in debates, focusing periods of discourse, and building of collective memory and collective identity. // Before introducing our own procedure for extracting, labelling, and validating news waves, we will investigate which procedures have been used in the past, what they achieve, what their limitations are, and which common problems and challenges need to be tackled by any procedure detecting and labelling news waves.

// We delve into both concepts and their relationship to later be able to validate the results of news wave detection and labelling as to whether they capture actual news waves and these actual news waves are linked to key events. It also helps understand the social and scientific significance of a procedure that automatically detects and labels such news waves.

in the bellow text should have button and another text " Simplify tech integration with award-winning lesson plans, micro-learning courses, and a supportive community."

Different people were advocating for these different theories from the start. The thing that evolved was just the average credence for each theory. This also suggests Hiltzik did not read the book, as infection while doing fieldwork is only one of the possible vectors. Another major one is infection in the lab (which, as the authors explain, happened before at WIV with less-deadly coronaviruses).

Another instance of Hiltzik simply asserting his thesis, which he has come nowhere close to proving in his rhetoric-filled page-and-a-half review.

eLife Assessment

This important study presents a significant methodological advance by leveraging previously discarded, unmapped DNA sequence reads to estimate pest infestation loads across plant accessions, and map variation in these apparent pest loads to defense genes. The bioinformatics approach is compelling, and the results should bear broad implications for phenotype-genotype prediction, especially regarding the use of unmapped reads for GWAS.

eLife Assessment

This important study examines the effects of NFKB2 mutations on pituitary gland development through hypothalamic-pituitary organoids. The evidence supporting the main conclusions is solid, although analysis of additional clones to exclude inter-clone variability would strengthen the conclusions. This is a revised study, but insight into the mechanism of action of NFKB2 during pituitary development is incomplete. This work will be of interest to endocrinologists and biologists working on pituitary gland development and disease.

Reviewer #1 (Public review):

Summary:

NFKB mutations are thought to be one of the causes of pituitary dysfunction, but until now they could not be reproduced in mice and their pathomechanism was unknown. The authors used the differentiation of hypothalamic-pituitary organoids from human pluripotent stem cells to recapitulate the disease in human iPS cells carrying the NFKB mutation.

Strengths:

The authors achieved their primary goal of recapitulating the disease in human cells. In particular, the differentiation of the pituitary gland is closely linked to the adjacent hypothalamus in embryology, and the authors have again shown that this method is useful when the hypothalamus is suspected to be involved in pituitary abnormalities caused by genetic mutations.

Weaknesses:

On the other hand, the pathomechanism is still not fully understood. This study provides some clues to the pathomechanism, but further analysis of NFKB expression and experiments investigating the relevant factors in more detail may help to clarify it further.<br /> As for the revised manuscript, it is still insufficient for understanding the role of NFKB2 in pituitary development although their additional experiments have improved the manuscript. The strength of the hypothalamus-pituitary organoid lies in its ability to recapitulate the differentiation process including not only the pituitary cells but also neighbouring non-pituitary cells, such as hypothalamic cells in vitro. It is necessary to determine "at which stages" and "in which localizations" NFKB2 expression is critical for pituitary development.

Reviewer #2 (Public review):

Summary:

DAVID syndrome is a rare autosomal dominant disorder characterized by variable immune dysfunction and variable ACTH deficiency. Nine different families have been reported, and all have heterozygous mutations in NFKB2. The mechanism of NFKB2 action in the immune systems has been well-studied, but nothing is known about its role in pituitary gland.

The DAVID mutations cluster in the C-terminus of the NFKB2 and interfere with cleavage and nuclear translocation. The mutations are likely dominant negative, by affecting dimer function. ACTH deficiency can be life-threatening in neonates and adults, thus, understanding the mechanism of NFKB2 action in pituitary development and/or function is important.

The authors use CRISPR/Cas gene editing of human iPSC derived pituitary-hypothalamic organoids to assess the function of NFKB2 and TBX19 in pituitary development. Mutations in TBX19 are the most common, known cause of pituitary ACTH deficiency, and the mechanism of action has been studied in mice, which phenocopy the human condition. Thus, the TBX19 organoids can serve as a positive control. The Nfkb2 mouse model has a p.Y868* mutation that impairs cleavage of NFKB2 p100, and the immune phenotype mimics the patients with DAVID mutations, but no pituitary phenotype was evident. Thus, a human organoid model might be the only approach suitable to discover the etiology of the pituitary phenotype.

Overall, the authors have selected an important problem, and the results suggest that the pituitary insufficiency in DAVID syndrome is caused by a developmental defect rather than an autoimmune hypophysitis condition. The use of gene editing in human iPSC derived hypothalamic-pituitary organoids is significant, as there is only one example of this previously, namely studies on OTX2. Only a few laboratories have demonstrated the ability to differentiate iPSC or ES cells to these organoids, and the authors have improved the efficiency of differentiation, which is also significant.

The strength of the evidence is excellent. The authors have thoroughly analyzed the genetically engineered organoids compared to isogenic controls. They have validated their findings with analysis of RNA and proteins. They have studied the time course of differentiation in the organoids and have a robust experimental design involving many replicates. Analysis of additional clones could strengthen the evidence.

Strengths:

The authors make mutations in TBX19 and NFKB2 that exist in affected patients. The TBX19 p.K146R mutation is recessive and causes isolated ACTH deficiency. Mutations in this gene account for 2/3 of isolated ACTH deficiency cases. The NFKB2 p.D865G mutation is heterozygous in a patient with recurrent infections and isolated ACTH deficiency. NFKB2 mutations are a rare cause of ACTH deficiency, and they can be associated with loss of other pituitary hormones in some cases. However, all reported cases are heterozygous.<br /> The developmental studies of organoid differentiation are rigorous in that 200 organoids were generated for each hiPSC line, and 3-10 organoids were analyzed for each time point and genotype. Differentiation analysis relied on both RNA transcript measurements and immunohistochemistry of cleared organoids using light sheet microscopy. Multiple time points were examined, including seven times for gene expression at the RNA level and two times in the later stages of differentiation for IHC.<br /> TBX19 deficient organoids exhibit reduced levels of PITX1, LHX3, and POMC (ACTH precursor) expression at the RNA and IHC level, and there are fewer corticotropes in the organoids, as ascertained by POMC IHC.<br /> The NFKB2 deficient organoids have normal expression of the early pituitary transcription factor HESX1, but reduced expression of PITX2, LHX3 and POMC. Because there is no immune component in the organoid, this shows that NFKB2 mutations can affect corticotrope differentiation to produce POMC. RNA sequencing analysis of the organoids reveals potential downstream targets of NFKB2 action, including a potential effect on epithelial to mesenchymal like transition and selected pituitary and hypothalamic transcription factors and signaling pathways.

It is important to note that all NFKB2 patients are heterozygous for what appear to be dominant negative mutations that affect protein cleavage and nuclear localization of processed protein as homo or heterodimers. The organoids are homozygous for this mutation.

Weakness:

There could be variation between individual iPSC lines that is unrelated to the genetically engineered change. The work would be strengthened by analysis of independently engineered clones or by correcting the engineered clone to wild type and demonstrating that the phenotypic effects are reversed. The authors do check for off target effects of the guide RNA at predicted sites using WGS.

Reviewer #3 (Public review):

Summary:

This manuscript by Mac et al addresses the causes of pituitary dysfunction in patients with DAVID syndrome which is caused by mutations in the NFKB2 gene and leads to ACTH deficiency. The authors seek to determine whether the mutation directly leads to altered pituitary development, as opposed to an autoimmune defect, by using mutating human iPSCs and then establishing organoids that differentiate into pituitary tissue. They first seek to validate the system using a well-characterised mutation of the transcription factor TBX19, which also results in ACTH deficiency in patients. Then they characterise altered pituitary cell differentiation in mutant NFKB2 organoids and show that these lack corticotrophs, which would lead to ACTH deficiency. Importantly, the findings here suggest the effects of mutant NFKB2 on pituitary organoid differentiation are direct and not a result of altered noncanonical NF-κB signalling, which has been shown to be a mechanism leading to immunodeficiency in DAVID patients.

Strengths:

The conclusion of the paper that ACTH deficiency in DAVID syndrome is independent of an autoimmune input is strong.

Weaknesses:

(1) The authors correctly emphasise the importance of establishing the validity of an iPSC-based model in being able to recapitulate in vivo dysfunctional pituitary development through characterisation of a TBX19 knock-in mutation. Whilst this leads to the expected failure of functional corticotroph differentiation, other aspects of the normal pituitary differentiation pathway upstream of cortocotroph commitment seem to have been affected in surprising ways. In particular, the loss of LHX3 and PITX1 in TBX19 mutant organoids compared with wild type requires explanation, especially as the mutant protein would only be expected to be expressed in a small proportion of anterior pituitary lineage cells. This may identify a difference between human and mouse pituitary development and emphasises the importance of further establishing the developmental programme in human pituitary.

(2) It is notable that the manipulation of iPSC cells used to generate mutants through CRISPR/Cas9 editing is not applied to the control iPSC line. It is possible that these manipulations, including electroporation and puromycin selection may lead to changes to the iPSC cells that is independent of the mutations introduced and this may change the phenotype of the cells. The authors have established that there are no off-target mutations through whole genome sequencing but the iPSC manipulation could have led to changes through epigenetic mechanisms or through non-genomic alterations of developmental potential. A better control in all experiments would have been an iPSC line with a benign knock-in (such as GFP into the ROSA26 locus) or use of a selected line where editing failed. The authors also ackowledge that use of a single clone is not ideal in these studies and characterisation of multiple clones would strengthen the conclusions of the study.

Author response:

The following is the authors’ response to the original reviews.

eLife Assessment

This valuable study examines the effects of NFKB2 mutations on pituitary gland development through hypothalamic-pituitary organoids. The evidence supporting the main conclusions is solid, although analysis of additional clones to exclude inter-clone variability would strengthen the conclusions. Insight into the mechanism of action of NFKB2 during pituitary development is incomplete. This work will be of interest to endocrinologists and biologists working on pituitary gland development and disease.

We agree with these considerations and the summary and thank the Editors for their assessment. Although we indeed share the idea that reproduction of the experiments on a second clone would be a useful confirmatory step, we have not been able to reach this goal within a reasonable time frame for the reason mentioned above (unavailability of the main research engineer knowledgeable in the challenging methods involved for organoids differentiation) and due to the long turnaround time of this kind of experiments (3 months for the whole differentiation starting form iPSC). We therefore decided to publish on a single clone while we are still aiming at reproducing our results on at least a second one and will hopefully be able to provide these additional data in a subsequent revised version. We now acknowledge this limitation in the final part of the Discussion.

Revised text: “Conversely, a limitation of this model is the long duration of the differentiation period (approximately 3 months) and the fact that not all hiPSC clones lead to full differentiation of hypothalamo-pituitary organoids despite similar conditions of culture. For these reasons, we could not include confirmation of our results on an independent clone in the present paper.”

Public Reviews:

Reviewer #1 (Public Review):

Summary:

NFKB mutations are thought to be one of the causes of pituitary dysfunction, but until now they could not be reproduced in mice and their pathomechanism was unknown. The authors used the differentiation of hypothalamic-pituitary organoids from human pluripotent stem cells to recapitulate the disease in human iPS cells carrying the NFKB mutation.

Strengths:

The authors achieved their primary goal of recapitulating the disease in human cells. In particular, the differentiation of the pituitary gland is closely linked to the adjacent hypothalamus in embryology, and the authors have again shown that this method is useful when the hypothalamus is suspected to be involved in pituitary abnormalities caused by genetic mutations.

Weaknesses:

On the other hand, the pathomechanism is still not fully understood. This study provides some clues to the pathomechanism, but further analysis of NFKB expression and experiments investigating the relevant factors in more detail may help to clarify it further.

We thank this reviewer for acknowledging that we've reached our primary objective, in particular the fact that the HPO (hypothalamo-pituitary organoid) model allows recapitulation of the disease in human cells, including hypothalamic-pituitary interactions. Regarding the pathophysiological mechanism of the disease, we must admit that it remains incompletely understood. However, we have analysed more samples by RT-qPCR and further analysed RNASeq data from NFKB2 KI organoids, which provided with more insights into the different levels where NFKB2 may play a role. We have now provided several additional figures derived from these analyses, including a synthetic figure to summarize the most relevant observed effects (Fig. 14). 

Reviewer #2 (Public Review):

We also thank this reviewer for the detailed analysis of our manuscript, for the valuable comments, suggestions and questions that are addressed point-by point below. 

Summary:

DAVID syndrome is a rare autosomal dominant disorder characterized by variable immune dysfunction and variable ACTH deficiency. Nine different families have been reported, and all have heterozygous mutations in NFKB2. The mechanism of NFKB2 action in the immune systems has been well-studied, but nothing is known about its role in the pituitary gland.

The DAVID mutations cluster in the C-terminus of the NFKB2 and interfere with cleavage and nuclear translocation. The mutations are likely dominant negative, by affecting dimer function. ACTH deficiency can be life-threatening in neonates and adults, thus, understanding the mechanism of NFKB2 action in pituitary development and/or function is important.

The authors use CRISPR/Cas gene editing of human iPSC-derived pituitary-hypothalamic organoids to assess the function of NFKB2 and TBX19 in pituitary development. Mutations in TBX19 are the most common, known cause of pituitary ACTH deficiency, and the mechanism of action has been studied in mice, which phenocopy the human condition. Thus, the TBX19 organoids can serve as a positive control. The Nfkb2<Lym1/Lym1> mouse model has a p.Y868* mutation that impairs cleavage of NFKB2 p100, and the immune phenotype mimics the patients with DAVID mutations, but no pituitary phenotype was evident. Thus, a human organoid model might be the only approach suitable to discover the etiology of the pituitary phenotype.

Overall, the authors have selected an important problem, and the results suggest that the pituitary insufficiency in DAVID syndrome is caused by a developmental defect rather than an autoimmune hypophysitis condition. The use of gene editing in human iPSC-derived hypothalamic-pituitary organoids is significant, as there is only one example of this previously, namely studies on OTX2. Only a few laboratories have demonstrated the ability to differentiate iPSC or ES cells to these organoids, and the authors have improved the efficiency of differentiation, which is also significant.

The strength of the evidence is excellent. However, the two ACTH-deficient organoid models use a single genetically engineered clone, and the potential for variability amongst clones makes the conclusions less compelling. Since the authors obtained two independent clones for NFKB2 it is not clear why only one clone was studied.

We experienced difficulties obtaining an hiPSC population devoid of spontaneous differentiation while purifying this second clone, and did not want to delay the start of the experiments. This clone will be analysed in a follow-up study.

Finally, the effect of TBX19 on early pituitary fate markers is somewhat surprising given the phenotype of the knockout mice and patients with mutations. Thus, the use of a single clone for that study is also worrisome.

We agree that the effect of the TBX19 mutant on early pituitary progenitor development is rather puzzling. In our model, TBX19 is expressed throughout the whole experiment, although it is at very low levels in undifferentiated hiPSCs compared to peak expression (over 50-fold difference).

During the CRISPR-Cas9 gene edition, we obtained a clone with a homozygous one base insertion at the cutting site, leading to a frameshift and a premature stop codon 48 bases downstream. This would result in an expected protein of 163 amino acids instead of 488, but with potentially still functional DNA-binding ability. This mutation had a similar effect on LHX3 and PITX1 as the TBX19 KI mutation, although it was even more severe. Our most likely explanation is that the two TBX19 mutants we generated have dominant negative effects. Contrary to mouse, little is known about TBX19 expression in early human pituitary development, but scRNA-seq data on human embryonic pituitaries (Zhang et al.) show low expression in undifferentiated pituitary progenitors between 7 and 9 weeks of gestation. Therefore, early expression of these dominant negative proteins could perturb differentiation in the organoids. Future development of hiPSCs lines with total absence of TBX19 should help clarify these questions.

Strengths:

The authors make mutations in TBX19 and NFKB2 that exist in affected patients. The TBX19 p.K146R mutation is recessive and causes isolated ACTH deficiency. Mutations in this gene account for 2/3 of isolated ACTH deficiency cases. The NFKB2 p.D865G mutation is heterozygous in a patient with recurrent infections and isolated ACTH deficiency. NFKB2 mutations are a rare cause of ACTH deficiency, and they can be associated with the loss of other pituitary hormones in some cases. However, all reported cases are heterozygous.

The developmental studies of organoid differentiation seem rigorous in that 200 organoids were generated for each hiPSC line, and 3-10 organoids were analyzed for each time point and genotype. Differentiation analysis relied on both RNA transcript measurements and immunohistochemistry of cleared organoids using light sheet microscopy. Multiple time points were examined, including seven times for gene expression at the RNA level and two times in the later stages of differentiation for IHC.<br /> TBX19 deficient organoids exhibit reduced levels of PITX1, LHX3, and POMC (ACTH precursor) expression at the RNA and IHC level, and there are fewer corticotropes in the organoids, as ascertained by POMC IHC.

The NFKB2 deficient organoids have a normal expression of the early pituitary transcription factor HESX1, but reduced expression of PITX2, LHX3, and POMC. Because there is no immune component in the organoid, this shows that NFKB2 mutations can affect corticotrope differentiation to produce POMC. RNA sequencing analysis of the organoids reveals potential downstream targets of NFKB2 action, including a potential effect on epithelial-to-mesenchymal-like transition and selected pituitary and hypothalamic transcription factors and signaling pathways.

Weaknesses:

There could be variation between individual iPSC lines that is unrelated to the genetically engineered change. While the authors check for off-target effects of the guide RNA at predicted sites using WGS, a better control would be to have independently engineered clones or to correct the engineered clone to wild type and show that the phenotypic effects are reversed.

All NFKB2 patients are heterozygous for what appear to be dominant negative mutations that affect protein cleavage and nuclear localization of processed protein as homo or heterdimers. The organoids are homozygous for this mutation. Supplemental Figure 4 indicates that one heterozygous clone and two homozygous mutant clones were obtained. Analysis of these additional clones would give more strength to the conclusions, showing reproducibility and the effect of mutant gene dosage.

The main goal of this work was to evaluate if and how NFKB2D865G mutation affects hypothalamic-pituitary organoids development, in order to determine if these organoids would constitute a valuable model to study DAVID syndrome.

We thank this reviewer for noting that we identified an important question and have used appropriate novel and not widely used methods to address it, including CRISPR/Cas9 genome editing of iPSCs and disease modelling in iPSC-derived HPOs that had not previously been reported by a team other than the one that initially described it, allowing to confirm our working hypothesis that DAVID syndrome is caused by a developmental defect rather than an autoimmune hypophysitis condition. We also agree that analysing more clones, generated from same or different hiPSC lines, carrying homozygous or heterozygous mutations, and corrected mutations will be necessary in the future.

Reviewer #3 (Public Review):

We also thank this reviewer for the detailed analysis of our manuscript, for the valuable comments, suggestions and questions that are addressed point-by point below. 

Summary:

This manuscript by Mac et al addresses the causes of pituitary dysfunction in patients with DAVID syndrome which is caused by mutations in the NFKB2 gene and leads to ACTH deficiency. The authors seek to determine whether the mutation directly leads to altered pituitary development, as opposed to an autoimmune defect, by using mutating human iPSCs and then establishing organoids that differentiate into pituitary tissue. They first seek to validate the system using a well-characterised mutation of the transcription factor TBX19, which also results in ACTH deficiency in patients. Then they characterise altered pituitary cell differentiation in mutant NFKB2 organoids and show that these lack corticotrophs, which would lead to ACTH deficiency.

Strengths:

The conclusion of the paper that ACTH deficiency in DAVID syndrome is independent of an autoimmune input is strong.

Weaknesses:

(1) The authors correctly emphasise the importance of establishing the validity of an iPSC-based model in being able to recapitulate in vivo dysfunctional pituitary development through characterisation of a TBX19 knock-in mutation. Whilst this leads to the expected failure of functional corticotroph differentiation, other aspects of the normal pituitary differentiation pathway upstream of corticotroph commitment seem to have been affected in surprising ways. In particular, the loss of LHX3 and PITX1 in TBX19 mutant organoids compared with wild type requires explanation, especially as the mutant protein would only be expected to be expressed in a small proportion of anterior pituitary lineage cells.

If the developmental expression profile of key transcription factors in mutant organoids does not recapitulate that which occurs in vivo, any interpretation of the relevance of expression differences in the NFKB2 organoids to the mechanism(s) leading to corticotroph function in vivo has to be questionable.

See response to Reviewer #2

It is notable that the manipulation of iPSC cells used to generate mutants through CRISPR/Cas9 editing is not applied to the control iPSC line. It is possible that these manipulations lead to changes to the iPSC cells that are independent of the mutations introduced and this may change the phenotype of the cells. A better control would have been an iPSC line with a benign knock-in (such as GFP into the ROSA26 locus).

We agree that the issue of off-target mutations should be addressed. However, we performed whole genome sequencing on TBX19 KI and did not observe any pathogenic variants other than the intended edition. We also checked that clones isolated during the screening procedure but that returned negative for editing still had the ability to generate pituitary cells. However, we made the choice to use the isogenic original hiPSC line as it could be compared to both TBX19 KI and NFKB2 KI simultaneously, therefore reducing workload and cost of the experiments. Any other knock-in mutation, such as GFP into the ROSA26 locus would imply the same risk of off-target mutations, but presumably at other sites in the genome.

(2) In the results section of the manuscript the authors acknowledge that hypothalamic tissue in the NFKB2 mutant organoid may be having an effect on the development of pituitary tissue. However, in the discussion the emphasis is entirely on pituitary autonomous mechanisms such as pituitary HESX1 expression or POMC gene regulation; in the conclusion of the abstract, a direct role for NFKB2 in pituitary differentiation is described. Whilst the data here may suggest a non-immune mediated alteration in pituitary function in DAVID syndrome, if this is due to alteration of the developing hypothalamus then this is not direct. A fuller discussion of the potential hypothalamic contribution and/or further characterisation of this aspect is warranted.

We agree with this reviewer that contributions of both hypothalamic and pituitary developing tissues should be taken into account. We performed more experiments and analysed the effect of both mutations on hypothalamic growth factors expression. These results are displayed in new figure 10. The role of the hypothalamus is now clearly mentioned and highlighted in the Discussion.

(3) qRT-PCR data presented in Figure 6A shows negligible alteration of HESX1 expression at all time points in NFKB2 mutant organoids. This is not consistent with the 2-fold increase in HESX1 expression described in day 48 organoids found by bulk RNA sequencing.

How do the authors reconcile these results and why is one result focused on in the discussion where a potential mechanism for a blockade of normal pituitary cell differentiation is suggested? Further confirmation of HESX1 expression is required.

In the previous version on the manuscript, the HESX1 fold-change ratio between NFKB2 KI and WT at d48 was of 2.06 (p=0.22). However, the type of representation for expression kinetics (values relative to the expression peak in WT) and the scale used made it difficult to see. In the new version of the manuscript, we analysed more samples from the same experiments, and new figure (now 6B) shows significant increase of HESX1 expression (Fc = 2.46, p=0.019) in NFKB2 KI.

Also, qPCR results come from at least two different experiments whereas RNAseq come from a single one. For RT-qPCR, 6 HPOs per genotype were picked and further analysed. As we found that only 60-70% of organoids show signs of pituitary cell differentiation, we chose to perform a preselection of organoids, based on RT-qPCR expression of selected markers (SOX2, HESX1, PITX1, LHX3, TBX19, POU1F1 and POMC) in order to avoid having “empty” HPOs sent for bulk RNAseq. We compared HESX1 expression ratios obtained by the two different techniques on the same samples (the ones used for RNA-seq) and found values of 2.19 (p=0.03) and 1.83 (p=0.061) for RNA-seq and RT-qPCR respectively. This is illustrated in Supplementary Figure 7. Our new results thus clearly demonstrate the increase in HESX1 expression in NFKB2 KI from d27 to d75.

(4) Throughout the authors focus on POMC gene expression and ACTH antibody immunopositive as being indicative of corticotroph cell identity. In the human fetal pituitary melanotrophs are present and most ACTH antibodies are unable to distinguish these cells from corticotrophs. Is the antibody used specifically for ACTH rather than other products of the POMC gene? It is unlikely that all the ACTH-positive cells are melanotrophs, nevertheless, it is important to know what the proportions of the 2 POMC-positive cell types are. This could be distinguished by looking for the expression of NeuroD1, which would also define whether corticotrophs are committed but not fully differentiated in the NFKB2 mutant organoids. In support of an effect on corticotrophs, it is notable that CRHR1 expression (which would be expected to be restricted to this cell type) is reduced by 84% in bulk RNAseq data (Table 1) and this may be an indicator of the loss of corticotrophs in the model.

The antibody we used is directed against ACTH. In HPOs, PAX7 expression was barely detected during the whole experiment. Moreover, although PCSK2 transcripts were observed, their expression started very early (d27) and remained constant, suggesting that an expression of this gene in hypothalamic cells rather than pituitary cells. All these observations suggest that melanotrophs are very unlikely to be present in HPOs.

(5) Notwithstanding the caveats about whether the organoid model recapitulates in vivo pituitary differentiation (see 1 above) and whether the bulk RNAseq accurately reflects expression levels (see 3 above), there are potentially some extremely interesting changes in gene expression shown in Table 1 which warrant further discussion. For example, there is a 25-fold reduction in POU1F1 expression which may be expected to reflect a loss of somatotrophs in the organoid (and possibly lactotrophs) and highlights the importance of characterising the effect of NFKB2 on other anterior pituitary cell types within the organoid. If somatotrophs are affected, this may be relevant to the organoids as a model of DAVID syndrome as GH deficiency has been described in some individuals with NFKB2 mutations. The huge increase in CGA expression may reflect a switch in cell fate to gonadotrophs, as has been described with a loss of TPIT in the mouse. These are examples of the changes that warrant further characterisation and discussion.

We performed a more in-depth analysis of other pituitary lineages (mainly somatotrophs). We confirmed the strong reduction in PROP1 and POU1F1 expression in NFKB2 KI organoids. Although the strong increase in CGA expression in the mutant may raise the possibility of a redirection towards gonadotroph lineage, the lack of change in NR5A1 expression may suggest otherwise.

These results are now illustrated in figure 12 and discussed in a full paragraph.

(6) How do the authors explain the lack of effect of NFKB2 mutation on global NFKB signalling?

The most likely explanation is that p100/p52 is not involved in controlling the expression of other members of NFKB signalling. Therefore, the absence of global alteration of NFKB signaling pathway shows that mutant p100/p52 protein is directly responsible for the observed phenotype.

Recommendations for the authors:

Reviewing editor summary of recommendation to authors:

The use of hypothalamic-pituitary organoids can provide a fundamental understanding of pituitary gland development and differentiation. Their use to study human pituitary insufficiency is important, gaining insight into the aetiology of disease and if it implicates the hypothalamus or anterior pituitary. To this end, there is only one other example of their use in the literature, where Matsumoto et al, (2019), used OTX2-mutant hypothalamic-pituitary organoids to understand the aetiology of pituitary hypoplasia driven by OTX2 mutations. This being the second example of using gene editing in human iPSC-derived hypothalamic-pituitary organoids, these studies have improved the efficiency of differentiation previously published by Suga et al. (2011) for ES cells, and Matsumoto et al. (2019) for iPS cells. In addition, it has solidified that this method is useful, especially when studying hypothalamic involvement in human pituitary anomalies, due to the concerted development of these two structures.

The reviewers recognise the valuable insight provided into the mechanism of NFKB2 action during pituitary development and how this human organoid model might be one of the few or only approaches suitable to discover the aetiology of the pituitary phenotype.

The reviewers agree that both the evidence provided from the organoid model, as well as the characterisation of the phenotype are incomplete. In particular, the strength of evidence would be improved by analysing additional independent clones for both NFKB2 as well as TBX19 gene-edited iPSCs. Additionally, analysis of NFKB2 expression both in vivo and in the organoids, as well as analysis for the NFKB2 targets put forward, would be a lot more informative to help understand this phenotype.

The main recommendations discussed are summarised here and the reviewers have elaborated on these points in their individual reviews:

The two ACTH-deficient organoid models use a single genetically engineered clone, and the potential for variability amongst clones, unrelated to the mutation, makes the conclusions less compelling. Two independent homozygous clones were obtained for NFKB2 but only one was used, so analysis of the second clone would strengthen the findings. A heterozygous clone was also obtained and given all NFKB2 patients are heterozygous for what appears to be dominant negative mutations, the heterozygous clone ought to be analysed. Analyses of these additional clones would give more strength to the conclusions, showing reproducibility and the effect of mutant gene dosage. The reviewers provide excellent suggestions for alternative controls for the engineered iPSC lines in their specific comments.

The effect of TBX19 mutation on early pituitary fate markers LHX3 and PITX1 is surprising given the phenotype of the knockout mice and patients with mutations. If the developmental profile of essential transcription factors does not recapitulate the in vivo expression in this well-characterised mutant, this brings the organoid model into question. Thus, analysis of a further clone for the study of mutant TBX19 would be crucial. The validity of this control affects the interpretations relying on expression differences in the NFKB2-mutant organoids.

The study has implicated NFKB2 in pituitary development, but more insight is needed to fully understand disease pathogenesis. The authors presented potential downstream targets of NFKB2 action, including transcription factors and key signalling pathway components; further analyses of NFKB2 expression and experiments investigating the relevant factors in more detail will help elucidate this point.

Discerning between the hypothalamus and pituitary tissue is fundamental to interpreting phenotypes: (i) To pinpoint the primary tissue affected by NFKB2 deficiency, staining for NFKB2 during development in vivo will determine if this is expressed both in the developing hypothalamus and anterior pituitary gland or only one of these tissues. (ii) Using markers of hypothalamus and pituitary to discern between these two tissues in organoids, will provide a lot of valuable information where expression changes are presented. This would help discern the contribution of the developing hypothalamus as this is still unclear and has not been discussed. Knowing which tissue compartments NFKB2 is expressed in the organoids would also be of great value.

The organoids provide an opportunity to characterise the effects of NFKB2 on other pituitary cell types, since the bulk RNAseq presents intriguing changes indicating that not only corticotrophs may be affected. This may be of relevance to patients, which can have additional pituitary hormone deficiencies. If NFKB2 is expressed in the pituitary, demonstrating expression in the different cell types in vivo as well as in the organoids would help interpret the phenotype. Is this expressed only in corticotrophs/corticotroph precursors, or in additional endocrine cells?

We agree with these considerations and the summary and thank the Editors for their assessment. Although we indeed share the idea that reproduction of the experiments on a second clone would be a useful confirmatory step, we have not been able to reach this goal within a reasonable time frame for the reason mentioned above (unavailability of the main research engineer knowledgeable in the challenging methods involved for organoids differentiation) and due to the long turnaround time of this kind of experiments (3 months for the whole differentiation starting form hiPSC). We therefore decided to publish on a single clone while we are still aiming at reproducing our results on at least a second one and will hopefully be able to provide these additional data in a subsequent revised version. We now acknowledge this limitation in the final part of the Discussion.

We have analysed more samples by RT-qPCR and further analysed RNASeq data from NFKB2 KI organoids, which provided with more insights into the different levels where NFKB2 may play a role. Specifically, we now show the effect of NFKB2 mutation on hypothalamic growth factors and pituitary progenitor differentiation (figure 10), different stages of corticotroph maturation (figure 11) and effects on PROP1/POU1F1-dependent lineages (figure 12). We confronted our results to publicly available ChIPseq data concerning p52 transcriptional targets (figure 13). We have now provided several additional figures derived from these analyses, including a synthetic figure to summarize the most relevant observed effects (Fig. 14). 

Reviewer #1 (Recommendations For The Authors):

In organoids, it is essential to stain for NFKB: is it the hypothalamus or the pituitary that expresses NFKB, and if the pituitary, is it the corticotroph itself or the surrounding cells? If immunostaining is not available, FISH or RNAscope can be used to look at expression.

Figure 7 shows stronger expression of p100/p52 in pituitary progenitors, and some expression in the hypothalamic part of the organoid. Due to current lack of biological material and length of experimental procedure, we could not yet determine which differentiated cell types express p100/p52, but this is clearly something we will look at in further experiments.

Regarding Figure 7, NFKB2 (D865G/D865G) shows no LHX3 expression already at day 48. It would be better to look at expression including PITX1 at an earlier time point to see at what point differentiation is impaired.

RT-qPCR results show no statistically significant changes in PITX1 (Fc=0.58, p=0.25) or LHX3 (Fc = 0.15; p=0.22) expression at d27, although there was a tendency towards downregulation.

Is it really just a species difference that NFKB2-deficient mice do not have abnormal pituitary function? This needs to be discussed in the manuscript.

Nfkb2_Lym1/Lym1 mice and _NFKB2 KI model have different but functionally very similar mutations, as they both lead to an abnormal processing of p100 and a strong reduction of p52 content. In mice, these mutations are more severe than the complete absence of Nfkb2 gene product, and they have been called “super repressors”. It is therefore surprising that no pituitary phenotype as been observed in mice. In our opinion, this constitutes a strong argument in favour of an inter-species difference, at least for the pathogenicity of this type of mutations.

This point is now addressed in the Discussion

Just looking at changes in gene expression by qPCR and bulk RNA-seq does not give enough information about localisation. We wish RNA-seq had at least been separated by FACS first. For example, FACS can separate the anterior pituitary and hypothalamus by EpCAM positivity/negativity (PMID: 35903276), so we would like to see gene expression in such separated samples.

This is a pertinent suggestion. We are aware of these techniques and we hope we will be able to include them in future studies

For Figures 2 and 6, just looking at changes in gene expression by qPCR does not provide localisation information, so either (1) immunostaining for LHX3 and NKX2.1 should be shown in each aggregate as in FigS3, or (2) qPCR should be performed on the FACSed cells. (2) qPCR on FACSed cells.

PITX1, LHX3 (as confirmed by our immunofluorescence data) and HESX1 are only expressed in non-neural tissue. TBX19 could be expressed in the hypothalamic part of the organoid, but we observed very little immunostaining outside the outermost layers of organoids (i.e. pituitary tissue). The antibody we used to detect corticotrophs only recognizes ACTH, and therefore only marks pituitary cells.

In addition, pathway and gene ontology analyses should be performed.

Pathways and gene ontology have been performed. However, as organoids consist of two different tissues, the analysis of over 4800 differentially expressed genes did not give us very informative results, apart from an impairment of retinoic acid signalling that we are currently investigating

Reviewer #2 (Recommendations For The Authors):

The differentiation of iPSC to organoids could be variable. The authors indicate that 200 organoids were analyzed for each line, and 3-10 organoids were analyzed per time point, genotype, and assay. Is it clear that 100% of the organoids differentiate to produce corticotropes? Please clarify.

In our experiments, almost 90% of organoids give rise to non-neural ectoderm, as demonstrated by PITX1 expression. However, depending on experiments, only 60-70% of organoids give rise to pituitary progenitors (LHX3+) and subsequently to corticotropes. This has been clarified in the text.

For TBX19, it seems surprising that there is an effect on PITX1 and LHX3 expression, since TBX19 expression is normally activated after these genes are expressed. An effect of TBX19 on EMT would also be surprising as the knockout mice do not have dysmorphology of the stem cell niche. The only evidence for an effect is the reduced IHC for E-cadherin. If this is an important point, the authors should examine other EMT markers such as Zeb2. The TBX19 knockout mice appear to form corticotropes based on the expression of NeuroD1, even though they lack TBX19 and POMC expression. It would be reassuring to see that NeuroD1 is normally expressed in the TBX19 mutant organoids.

We agree that the effect of the TBX19 mutant on early pituitary progenitor development is rather puzzling. In our model, TBX19 is expressed throughout the whole experiment, although it is at very low levels in undifferentiated hiPSCs compared to peak expression (over 50-fold difference).

During the CRISPR-Cas9 gene edition, we obtained a clone with a homozygous one base insertion at the cutting site, leading to a frameshift and a premature stop codon 48 bases downstream. This would result in an expected protein of 163 amino acids instead of 488, but with potentially still functional DNA-binding ability. This mutation had a similar effect on LHX3 and PITX1 as the TBX19 KI mutation, although it was even more severe. Our most likely explanation is that the two TBX19 mutants we generated have dominant negative effects. Contrary to mouse, little is known about TBX19 expression in early human pituitary development, but scRNA-seq data on human embryonic pituitaries (Zhang et al.) show low expression in undifferentiated pituitary progenitors between 7 and 9 weeks of gestation. Therefore, early expression of these dominant negative proteins could perturb differentiation in the organoids. Future development of hiPSCs lines with total absence of TBX19 should help clarify these questions.

Apart from the lack of change in ZEB2 expression in TBX19 KI (Fc = 1.15; p = 0.35), we did not look further for changes in EMT markers in TBX19 KI. However, we added a more detailed analysis for EMT markers expression in NFKB2 KI based on RNAseq results (see table 2).

Due to lack of material, we could not confirm NEUROD1 expression by immunostaining. However, RT-qPCR showed there was no change in NEUROD1 expression in TBX19 KI (Fc = 0.81; p = 0.64)

NFKB2 IHC was markedly reduced in NFKB2 D865G/D865G organoids. Based on previous experiments, the mutant protein should be expressed but not activated by proteolytic cleavage. It is possible that the antibody has a different affinity for the mutant protein and/or the uncleaved protein may be unstable. Can this be clarified? The mRNA for mutant NFKB2 appears unchanged in Table 1.

This is puzzling indeed. We did not notice any change in NFKB2 from d27 to d105, and no significant change either between WT and NFKB2 KI. Although the antibody we used recognizes both p100 and p52, we cannot rule out the possibility that p100/p52 is degraded by pathways other than proteasome. Another possibility is that p100 interactions with other proteins may decrease the accessibility of the antibody to the epitope

The RNA sequencing data from the NFKB2 organoids is intriguing. It suggests that the NFKB2 mutation may have a modest effect on Tbx19 transcription but not Neurod1. It also suggests there are hypothalamic effects, i.e. altered expression of hypothalamic markers in mutant organoids. Is NFKB2 expressed in the developing hypothalamus? Can normal NEUROD1 IHC be confirmed? It is also intriguing that there may be an effect on EMT. However, there seem to be some discrepancies in the direction of effect on these markers. Please clarify.

This is related to the point just above. P100/p52 is described as a ubiquitously expressed protein. We think that it is expressed in the hypothalamic part of the organoids, but at a lower level compared to pituitary progenitors.

As mentioned before, we could not yet confirm NEUROD1 expression by immunostaining, but RT-qPCR clearly showed there was no change in NEUROD1 expression in TBX19 KI (Fc = 0.81; p = 0.64) or NFKB2 KI (Fc = 0.88; p = 0.5). However, we investigated other markers of different stages of corticotroph differentiation (see figure 11) and found that the later stages are most affected.

Concerning the EMT, we also found changes in the expression of other markers that are shown in Table 2 and discussed further in the text.

Cytokines have been proposed to play important roles in pituitary differentiation, i.e. IL6. Is there any evidence for an altered cytokine or chemokine expression in the NFKB2 organoids?

We didn’t see any change in IL6 expression NFKB2 KI (Fc = 2.34; p = 0.55), but RNAseq shows a strong increase in IL6R (Fc = 8.89; p = 2.13e-09). But at this point, the relevance of these observations remains elusive.

Minor:

Some patients with DAVID syndrome have pituitary hypoplasia. The authors measure organoid size and find no differences based on genotype. However, each organoid probably has a variable amount of tissue differentiated to pituitary and hypothalamic fates, therefore, the volume of the whole organoid may not be a good proxy for the amount of pituitary tissue.

We are aware of this issue. However, for most pituitary genes measured by RT-qPCR (PITX1, LHX3, TBX19), the deltaCt values did not drastically vary for a given time point/genotype, suggesting a stable pituitary/hypothalamic ratio.

Figure 9 shows whole transcriptome data for the NFKB2 organoids, and Table 1 lists the data for selected genes. There appears to be disagreement between the significance cut-offs used in the figure and the table. Please adjust.

We removed the fold-change cut-offs to improve clarity

elife120868_0_supp_2945725_rxl2z4. "haft" appears several times, but it should be "half".

подогревает мотивацию