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.

In fact, PD-1+ Treg cells amplified by PD-1 blockade have been shown to promote hyperprogression of cancer63.

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).

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.

. 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).

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).

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).

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).

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.

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.

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).

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.

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

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).

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.

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.

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.

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.

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.

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.

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.

Leukocytes from patients with SLE were treated with 17β-estradiol in vitro, which led to production of anti-dsDNA antibodies, especially IgG type.57

A shift toward Th2 in pregnancy can lead to increased antigen stimulation and production of NMO-IgG (123).

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).

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.

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.

CD45RBlow

Shown are Lck-specific bands at 56 (*) and 59 (**) kDa, the latter indicative of Lck also phosphorylated at S59 by ERK upon TCR induction.

“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)

“Do you ever things other people don't see?”

What age? How much? History of blackouts?

What was school like for you?

Friends? Family? Co-workers?

“What kind of jobs did you have?”

blunted

Loud, soft, monotone, weak, strong