Gal-9 is likely upregulated early on during cross-presentation of tumor antigens when intratumoral DCs sense danger signals from dying tumor cells, but is only secreted in large quantities after T cells are activated to produce IFNγ.
Is this like the immune system trying to turn off the immune response after the tumor cell is killed?
In fact, PD-1+ Treg cells amplified by PD-1 blockade have been shown to promote hyperprogression of cancer63.
So there is a double edge swordness to the PD-1 blockade, it stops the CD8 T cell exhaustion but it then promotes the PD-1 Treg cells.
Galectin mRNAs lack signal peptide coding sequence, and their proteins are synthesized by cytosolic ribosomes and do not enter the classical ER–Golgi secretory pathway21,50. This raises an important question of how galectins leave the cell to interact with its potential glycan ligands, which are located mostly on the cell surface or in the extracellular matrix. It was recently reported that Gal-9 and TIM-3 are secreted by exocytosis as a complex in acute myeloid leukemia cells that co-express these two molecules51 although how and where the Gal-9/TIM-3 complexes are formed is not clear. Interestingly, we found that IFNβ facilitated Gal-9 secretion from tumor and myeloid cells, and its secretion was further augmented by the presence of IFNγ (Fig. 7h–k).
So the interferon pathway is connected to promoting secretion of Gal-9 so maybe an involvement of the lipid raft proteins are in play here? Like the interferons cause upregulation of Mal proteins which then leads to increased secretion of galectin9??? Not really sure but can look more into this.
h A375 melanoma cells. n = 3 independent experiments. Control vs IFNβ, P = 0.0074; IFNβ vs IFNβ + IFNγ, P = 0.0388; control vs IFNβ + IFNγ, P = 0.0070.
Interesting that the combo of IFNbeta and IFNgamma seems to be synergistic in promoting the expression of galectin9
. Interestingly, while IFNβ strongly induced Gal-9 in human lung cancer cell lines with wildtype EGFR (A549, H441, H1229), it failed to do so in those with mutant EGFR (H1650, H1975, HCC827) (Fig. 7f; Supplementary Fig. 8e), suggesting that IFNβ induction of Gal-9 expression may be affected by EGFR signaling in lung cancer cells. Unlike cancer cells, primary human macrophages constitutively express Gal-9 (Fig. 7g).
So maybe IFNbeta pathway can be targetable as well for therapeutics?
We found that IFNβ strongly upregulated the levels of both Gal-9 protein and mRNA in A375 human melanoma cells, whereas IFNγ only moderately induced Gal-9 and TNFα did not have a detectable effect (Fig. 7b).
Okay nvm, so now they are more specifically looking the cancer cell (in this case a melanoma cell line) expression of LGALS9 in the TME.
LGALS9 gene (encoding Gal-9) was predominately expressed in immune cells, particularly myeloid cells, including dendritic cells and monocytes (Supplementary Fig. 8a. Data from BioGPS). Flow cytometric analysis of human PBMCs showed high Gal-9 expression in monocytes and low expression in T cells (Supplementary Fig. 8b–d). In most cancer cell lines, the expression of Gal-9 was low or undetectable with the exception of some leukemic cell lines, such as Jurkat T cells and THP-1 monocytic cells (Fig. 7a).
So sounds like LGALS9 underexpressed in most cancer cell lines and mostly expressed in APCs, but they really only utilize like 9 cancer cell lines. Is this enough to state that "most cancer cell lines do not express LGALS9????" And in what condition as well? Were these cells just grown on a plate or extracted from tumors in vivo????
To identify changes in immune cells that may contribute to the treatment effects, we utilized mass cytometry (CyTOF) to profile the tumor immune infiltrate from each treatment group, using a panel of 30 antibodies against various lineage and functional markers of immune cells (Supplementary Table 1). Unsupervised clustering analysis of CD45+ immune cells from tumors using viSNE41 and FlowSOM42 identified 8 major immune cell populations or clusters (Supplementary Fig. 5).
Immune cell population coming from the MC-38 mouse colon cancer model
Taken together, such mixed phenotype of CD8 T_1 is similar to what was observed in CD8 T cells that respond to other immune checkpoint therapies and are critical for tumor control6,10,48, and is indicative of transitory T cells in the process of differentiation from precursor exhausted T cells to terminally exhausted T cells8,33.
So it seems that this galectin9 TIM3 CD8 killing is very specific to a subset of CD8s that have lots of the T cell coinhibitory molecules which make sense. But at the same time these subset of CD8s have a lot of T cell activation and memory markers as well so meaning that these are the CD8s that are probably more able to promote cancer cell killing over other subsets of CD8s. So we really need to then categorize CD8s into CD8 PD-1 TIM3 pos vs CD8 PD-1 TIM3 negs??? with the former being the most important in killing cancer and most important targets for immunotherapy.
Tumor-infiltrating Treg cells may be especially susceptible to Gal-9 killing as they express high levels of TIM-3 compared with their counterparts in the periphery44,45.
So Galectin-9 can promote this tumor killing by killing off the Treg cells but why does that happen? Galectin-9 seems to have opposing functions since 1) it promotes CD8 killing but it 2) plays a role in T cell activation through the TNFR pathway and a role in killing intratumor Treg cells. But the interesting thing is that anti-galectin9 preserves these Treg cells but the addition of pro-GITR causes this phenotype of anti-galectin9 to disappear and cause there to be a lowering of these Tregs, so very strange.
We postulated that this may be due to impaired T cell co-stimulation after Gal-9 inhibition, as Gal-9 has been shown to be required for the signaling of 4-1BB36, a T cell co-stimulatory receptor of the tumor necrosis factor receptor superfamily (TNFRSF).
So Gal-9 may also play a role in T cell activation then through the 4-1BB signaling pathway.
Taking together, the data so far are consistent with the notion that Gal-9 promotes TIM-3-mediated T cell apoptosis by crosslinking TIM-3 and facilitating TIM-3 aggregation; co-expressed PD-1 attenuates Gal-9/TIM-3-induced apoptosis by promoting the formation of TIM-3/Gal-9/PD-1 lattices.
Interesting why this happens since we would think that PD-1 would promote cell death in the context of Gal-9 TIM3 mediated T cell apoptosis so PD-1 has a T cell survival aspect? Is it that PD-1 causes T cell survival but still causes T cell inactivity or activity suppression? Not sure.
To determine the glycosylation sites on PD-1 contributing to Gal-9 binding, we mutated the asparagine (N) residues in the four putative glycosylation sites (N49, N58, N74, and N116) to glutamine (Q) individually and assessed the effects on Gal-9 binding. We found that binding of PD-1 to Gal-9 was largely abolished by the N116Q mutation, although binding was also moderately reduced by the other three mutations (Fig. 2d). We concluded that Gal-9/PD-1 interaction is primarily mediated by the C-CRD of Gal-9 and the N116-linked glycan of PD-1
Here they determine the specific site that the C-terminal binds to on PD-1 which is the N116-linked glycan. This is important most likely since if an antibody therapy was to be created, it would be important to identify the specific binding sites of these two proteins.
In accordance with the observation that Gal-9 mainly uses its C-CRD to interact with PD-1 (Fig. 2b, c) and thus could not efficiently crosslink the PD-1 molecules, PD-1 did not form insoluble lattices with Gal-9 in the absence of TIM-3 (Fig. 3j).
Key to understanding is that C-CRDs have lower effectiveness in making strong cross-links between proteins since they have lower affinity for protein-protein interactions compared to carbohydrate binding while N-CRDs have higher effectiveness in making strong cross-links between proteins
f Jurkat cells expressing PD-1 (myc tagged) and TIM-3 (3xFlag tagged) individually or together were incubated with or without 2 μg/ml exogenous Gal-9 followed by IP/western blotting with indicated antibodies.
For Figure 3 experiments, not only is there a TIM3-GAL9-PD-1 extracellular triplet interaction going on but they show in figure 3F that there may be something intracellular between TIM3 and PD-1 despite an absence of GAL9 since in figure 3F, the FLAG tag was only on TIM3 so if TIM3 didn't interact with PD-1 without GAL9's help, there would be no PD-1 bands in 4th lane in the top most blot since the IP is only targeting TIM3. Since PD-1 is interacting with TIM3 somehow, PD-1 is present regardless.
Gal-9 consists of two CRDs, N-terminal CRD (N-CRD) and C-terminal CRD (C-CRD), with similar but distinct specificities for glycans21,28. To determine which CRD mediates Gal-9 binding to PD-1, we purified the two CRDs individually as GST-fusion proteins (Fig. 2a) and found that GST-9C (C-CRD) exhibited greater binding to PD-1 compared to GST-9N (N-CRD) (Fig. 2b and Supplementary Fig. 1e), whereas the two CRDs exhibited largely equal binding to TIM-3.
To get even more specific, to determine which end of the Galectin 9 protein bound PD-1, they created a N-terminal galectin-9 and glutathione transferase fusion protein and a C-terminal counterpart. The glutathione transferase parts are needed since its needed for purification. *Question is can you just create non fusion N terminal and C terminal halves of the galectin-9 and then do a binding purification using PD-1 and then analyze the purified protein with proteomics and determine whether the purified protein is the C-terminal or N-terminal by looking at the amino acid order? (Sounds more difficult than using gluthathione purification) In Figure 2b, they applied PD-1, TIM-3, and PD-L1 to the immobilized fusion proteins and saw that the C-terminal plate had more signal when applying PD-1 protein.
Immune checkpoint molecules can be regulated at multiple levels. For instance, in addition to transcriptional regulation, post-translational modifications of PD-116 and PD-L117 have been shown to regulate their protein stability and interaction. The turnover of PD-1 is also regulated by its interaction with the E3 ubiquitin ligase FBXO3818. Recently, CD80, the ligand for both CD28 and CTLA-4, was found to interact with PD-L1 in cis on antigen-presenting cells (APCs) to repress the PD-1 and CTLA-4 co-inhibitory pathways while promoting CD28 co-stimulation19,20. Those findings reveal interesting crosstalk between immune checkpoint pathways in regulating T cell activity.
The foundational question of this paper is what are other proteins that interact with PD1 and how can we use that for improving immune checkpoint blockade therapy
To further understand the function of PD-1, we sought to identify additional PD-1-binding proteins by expressing a C-terminal 3×FLAG-tagged PD-1 (PD-1.3F) in Jurkat T cells using a doxycycline-inducible retro-lentiviral system27 followed by immunoprecipitation (IP) with a FLAG antibody.
How they proved that PD-1 is bound by Galectin-9 1. Overexpressed PD-1 in model Jurkat TG cells with a Tet on system -> precipitated protein -> analyzed the protein cluster (PD-1 and any protein attached to PD-1) with mass spec -> found Gal-9 as a major binder. So experiments include 1) Mass Spec, 2) Western blot, 3) Pull down assay to show how galectin and PD-1 bind together. 2. To show that they are specific aka only galectin-9 is the bounder and not other galectins like galectin-1 and galectin-8 + the galectin-9 PD-1 bind site is unique -> protein assays used above for galectin 1 and 8 were negative and nivo and pembro treatment did not interfere with galectin-9 and PD-1 binding so galectin-9 does not bind in the same place as PD-L1. 3. Summary is that if you want to say that protein A binds protein B, should try to show they bind each other in both directions -> should try to show that their interaction is specific by testing other proteins in their family or different binding sites.
Jurkat PD-1 cell lysates were incubated with glutathione-Sepharose (control) or Gal-9-Sepharose beads with or without sucrose or lactose. Bound proteins were eluted and western blotted with anti-PD-1 antibody.
In figure 1Bs experiment, you are showing the interaction in the opposite way. Instead of using PD-1 to fish out galectin-9, you are using galectin-9 to fish out PD-1 which gives even more credence to the idea that they interact with each other.
Lysates of Jurkat cells transduced with control lentivirus or PD-1 tagged at the C-terminus with 3× FLAG tag (PD-1.3F) were immunoprecipitated with anti-FLAG magnetic beads and the associated proteins were subjected to immunoblotting with Gal-9 or PD-1 antibodies. The three Gal-9 bands (L, M, S) represent different isoforms resulted from alternative pre-mRNA splicing.
Lysate (top blot) should have all the galectin in all cells (control and PD-1.3F) but the middle blot is only IP of the PD-1 protein so if galectin9 did not bind to PD-1 protein, then there should theoretically only be PD-1 protein in the sample aka the bottom blot. However, since galectin 9 does bind to PD-1, there is present galectin 9 per the middle blot in the PD-1.3F sample.
High estrogen levels during pregnancy can increase activation-induced cytidine deaminase expression, which is responsible for immunoglobulin production. Additionally, sex hormones may influence antibody glycosylation, with effects on antibody function. Estrogen decreases apoptosis of self-reactive B cells, through upregulation of antiapoptotic molecules. Furthermore, high estrogen levels during pregnancy can boost B-cell activating factor and type 1 interferon (IFN) production, facilitating development of self-reactive peripheral B cells in association with increased disease activity. Elevated levels of estrogen during pregnancy decrease IFN-γ generation, which causes a shift toward T helper (Th) 2 immunity, thereby propagating NMO pathogenesis.
Mechanisms of how estrogen leads to increased IgG
Leukocytes from patients with SLE were treated with 17β-estradiol in vitro, which led to production of anti-dsDNA antibodies, especially IgG type.57
Non human in vivo study that shows estrogen treated leukocytes leads to increase in anti dsDNA antibodies.
A shift toward Th2 in pregnancy can lead to increased antigen stimulation and production of NMO-IgG (123).
Evidence of pregnancy related increase in autoimmune disease related antibodies
In patients undergoing hepatic resection of liver-limited metastases from colorectal cancer (CRC), multiple retrospective series demonstrated 5-year disease-free survival (DFS) of 15–23% and 5-year overall survival (OS) of 28–37%22,23,24. In a systematic review of adrenalectomy for isolated metastases from non-small-cell lung cancer (NSCLC), 5-year OS was 25% and 26% in patients with metachronous and synchronous metastases, respectively, despite 21% of patients having disease recurrence in the adrenal bed or retroperitoneum25. Curative-intent pulmonary metastasectomy is associated with 5-year OS of 34–40% (and 10-year OS of 20%) in patients with soft-tissue sarcoma (STS)26,27,28. Notably, these historical studies were published during an era of systemic therapies with limited efficacy and of poor-quality radiographic cancer staging, including limited utilization of CT, PET and MRI. Nevertheless, such retrospective series have generally suggested that approximately one-fifth of patients remain disease-free and one-third remain alive at 5 years after local therapy for limited-extent metastatic disease, across various cancer types (Table 1).
To summarize, data from patients with colorectal cancer to liver mets, NSCLC to adrenal mets, and soft tissue sarcoma to lung mets show that a significant minority about a 3rd survive beyond 5 years, showing support for the oligometastatic hypothesis, but mind that these data were from retrospective studies
The oligometastasis hypothesis suggests a spectrum of metastatic virulence where some metastases are limited in extent and curable with focal therapies. A subset of patients with metastatic colorectal cancer achieves prolonged survival after resection of liver metastases consistent with oligometastasis. Here we define three robust subtypes of de novo colorectal liver metastasis through integrative molecular analysis. Patients with metastases exhibiting MSI-independent immune activation experience the most favorable survival. Subtypes with adverse outcomes demonstrate VEGFA amplification in concert with (i) stromal, mesenchymal, and angiogenic signatures, or (ii) exclusive NOTCH1 and PIK3C2B mutations with E2F/MYC activation. Molecular subtypes complement clinical risk stratification to distinguish low-risk, intermediate-risk, and high-risk patients with 10-year overall survivals of 94%, 45%, and 19%, respectively. Our findings provide a framework for integrated classification and treatment of metastasis and support the biological basis of curable oligometastatic colorectal cancer. These concepts may be applicable to many patients with metastatic cancer.
There is evidence that among oligometastatic cancer, there is a subset of tumors (identified using molecular methods such as next generation sequencing) that exhibit favourable outcomes in colorectal liver metastatic cancer.
One biochemical definition of lipid rafts is their insolubility in non-ionic detergents at 4°C - a condition that yields detergent-resistant membranes (DRMs). Because of their high lipid-to-protein ratio, DRMs have a low density and can thus be isolated by flotation on sucrose-density gradients. It is a commonly held view in the field, as recently discussed extensively at the EURESCO Conference on Microdomains, Lipid Rafts and Caveolae [15], that progress has been hindered by the use of numerous detergents under different solubilization conditions. A recent report [16] stresses that the use of most detergents can result in the transferrin receptor, the archetypal non-lipid-raft marker, appearing in the DRM fraction; the only two detergents among those tested that demonstrated specificity for the lipid-raft markers cholesterol and sphingomyelin were CHAPS and Triton X-100. Extraction with any detergent results in a low-density fraction, however, so this criterion alone cannot be used as a definition of a lipid raft. Frequently, DRMs are isolated at the 5%-30% sucrose interface, to which any lipid-rich membrane material would float, even if the cells from which they are derived have never been subjected to any detergents. This also means that non-detergent methods for preparing 'lipid rafts' are likely to generate a floating fraction containing membranes in general, from a number of sub-cellular sites, but not necessarily one enriched specifically in lipid rafts.It is unlikely that the addition of detergents to cells will faithfully preserve particular membrane structures. For instance, concentrations of Triton X-100 that are too low to lyse cells cause cell membranes to vesiculate and fuse [10]. DRMs prepared after Triton X-100 extraction of Jurkat cells (TX-DRMs) are a mixture of vesicles ranging in diameter between 50 nm and several micrometers (Figure (Figure1).1). Considering that individual lipid rafts on the cell surface are estimated to be under a few hundred nanometers in diameter [10], it is clear that TX-DRMs are substantially aggregated structures and are, therefore, likely to contain molecules that would not be in close proximity in an intact cell. Consequently, it is not valid to say that proteins are associated in the same domain in the cell solely because of their appearance in DRMs. Also, Triton X-100 can itself promote the formation of distinct lipid domains [17,18]. On the other hand, a failure to recover a protein in DRMs does not necessarily mean that that protein is not found in lipid rafts in the intact cell. This seems to be the case for the T-cell receptor [19] and the epidermal growth factor receptor [20], which have been shown to be in lipid rafts by means of fluorescence microscopy codistribution or biochemical isolation, respectively, although they are not always present in DRMs.
Proposed biochemical definition of lipid rafts: membranes that are insoluble in non ionic detergents. These insoluble membranes are considered detergent resistant membranes which can be isolated in the detergent resistant membrane fraction using a sucrose gradient. However this definition is not good enough since there are other things that can meet this criteria by also being insoluble in non ionic detergents and being isolated in the DRM fraction as well, essentially there are contaminants that "should not" be in DRMs. This is a call to caution to not believe that the DRMs are specific for lipid rafts.
CD45RBlow
CD45RB marks naive T cells which are downregulated as naive T cells turn into memory T cells as they are activated. CD45RO marks activated and memory T cells and enhances T cell receptor activation
Shown are Lck-specific bands at 56 (*) and 59 (**) kDa, the latter indicative of Lck also phosphorylated at S59 by ERK upon TCR induction.
I am very confused on why there is equal amounts of double phos lck in the Y394 western blot while there is a difference in the pan lck western blot? Because if the first western blot is saying that there is equal amounts single phos, the blot is also showing that there is equal amounts of double phos since the bands appear to be equal in intensity. But on the same blot, after stripping, there is less of the double phos for some reason
“Are the voices outside or inside your head?” (auditory hallucinations are more likely to be heard “outside,” and often patients will look for the voice)
Is there one or more voices? What are the voices saying? How loud are the voices? Are the voices telling negative things about you?
“Do you ever things other people don't see?”
Visual and tactile hallucinations - associated with substance or organic disease
What age? How much? History of blackouts?
When is the last time you drank?
What was school like for you?
Any challenges at school?
Friends? Family? Co-workers?
Are you in a relationship? Are you married? Do you have any children?
“What kind of jobs did you have?”
If not employed, "How do you make a living?", and if disability causing unemployment, "What is the nature of your disability?"
blunted
Blunted different from flat affect, blunted patients shows some sort of affect unlike flat affects, patients show a decreased form of that affect such as a odd smile that isnt really restricted but fairly decreased, pathognomonic for psych disorder such as schizo
Loud, soft, monotone, weak, strong
Sarcastic, childlike, irritable