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α2,6-Sialoglycan-regulated anti-tumor immunity of tumor-infiltrating CD8+ T cells in the tumor microenvironment [CD8+T]

ABSTRACT: Cell-surface sialoglycans overexpressed on cancer cells suppress the activation of tumor-infiltrating immune cells (TIICs), and have emerged as a promising target for cancer immunotherapy. However, the functional roles of sialylation in TIICs remain incompletely understood. Here, we develop the single-cell correlative analysis of α2,3- and α2,6-linked sialoglycans and transcriptomes (scCAST_3&6) strategy to correlate cell-surface sialylation in a linkage-specific manner with gene expression of the same cells by single-cell RNA sequencing. Applying scCAST_3&6, we discover that α2,3- and α2,6-sialoglycans are distinctively regulated in specific TIIC subpopulations. Importantly, downregulated α2,6-sialoglycan is demonstrated as a cell-surface marker for labeling and isolating CD8+ T cells with high reactivity to tumor-specific antigens (TSAs) from the tumor. Mechanistically, α2,6-sialyltransferase ST6Gal-I is transcriptionally suppressed upon the activation of intratumoral CD8+ T cells with high-avidity T cell receptors towards tumor antigens, which downregulates α2,6-sialylation and exposes galectin-1 ligands. Functionally, increased galectin-1 binding to TSA-reactive CD8+ T cells promotes T cell apoptosis and cancer immune evasion. This work underscores the functional significance and therapeutic promise of specific sialoglycans on TIICs.

ORGANISM(S): Mus musculus

PROVIDER: GSE316541 | GEO | 2026/08/01

REPOSITORIES: GEO

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Project description:Tumor-associated macrophages (TAMs) form functionally diverse populations of innate immune cells in the tumor microenvironment. While the pro- and anti-inflammatory TAMs are recognized to shape inflammation and impact disease progression in cancer, the molecular and cellular perturbations of the glycocalyx accompanying TAM polarization remain unknown. Taking a systems glycobiology approach, we here firstly used cell surface-focused glycomics and lectin flow cytometry of ex vivo polarized monocyte-derived macrophages to demonstrate profound sialyl linkage switching of the surface N-glycome in pro-inflammatory (α2,3-NeuAc-favored) and anti-inflammatory (α2,6-NeuAc-dominant) macrophages. No polarization-induced alterations in sialylation were observed in the surface O-glycome. Expression of ST6GAL1 that adds α2,6-sialylation to N-glycans was elevated in anti-inflammatory compared to levels in pro-inflammatory macrophages providing a mechanistic basis for the sialyl linkage switching which was supported by ST6GAL1 silencing. Interestingly, SNA-focused lectin cytochemistry of anti-inflammatory macrophages revealed dense networks of α2,6-sialylated nanotubules forming proteinaceous inter-connecting cellular structures that were absent in pro-inflammatory macrophages. Temporal ST6GAL1 silencing caused nanotubule fragmentation in the anti-inflammatory macrophages. Moreover, live cell recordings of anti-inflammatory macrophages cultured alone and with colorectal cancer (CRC) cells showed reduced macrophage motility and attenuated inter-macrophage and macrophage-CRC cell interactions upon ST6GAL1 disruption indicating functional roles of the α2,6-sialylated nanotubules. Finally, sialyl linkage switching was observed in pro- and anti-inflammatory TAMs using lectin histochemistry on tumor tissues from CRC patients with stage 4 disease. We report on the mechanistic basis for and functional consequences of the profound glycocalyx remodeling accompanying TAM polarization.

Project description:The goal of this study was to examine differences in gene expression of tumor specific CD8 T cells in an in vivo tumor mouse model after inhibition of galectin-3 protein expression by genetic knockout. Galectin-3 is thought to modulate CD8 T cell response by cross-linking cell surface glycoproteins Galectin-3 is a 31 kD carbohydrate-binding lectin that is over-expressed by many human malignancies. It also modulates T cell responses through a diverse array of mechanisms including induction of apoptosis, TCR cross linking in CD8+ T cells, and T cell receptor (TCR) down regulation in CD4+ T cells. We found that patients responding to a granulocyte-macrophage colony-stimulating factor (GM-CSF) secreting allogeneic pancreatic tumor vaccine developed post immunization antibody responses to galectin-3 on a proteomic screen. We used the HER-2/neu (neu-N) transgenic mouse model to study galectin-3 binding on adoptively transferred high avidity neu-specific CD8+ T cells derived from TCR transgenic mice. Here, we show that galectin-3 binds preferentially to activated antigen-committed CD8+ T cells only in the tumor microenvironment (TME). Galectin-3 deficient mice exhibit improved CD8+ T cell effector function and increased expression of several inflammatory genes when compared with wild type (WT) mice. We also show that galectin-3 binds to LAG-3, and LAG-3 expression is necessary for galectin-3 mediated suppression of CD8+ T cells in vitro. Lastly, galectin-3 deficient mice have significantly elevated levels of circulating plasmacytoid dendritic cells (pDCs), which are superior to conventional dendritic cells (cDCs) in activating CD8+ T cells. Binding of galectin-3 to cell-surface glycoproteins on immune cells suppresses a pro-inflammatory immune response. Thus, inhibiting galectin-3 in conjunction with CD8+ T cell directed immunotherapies should enhance the tumor specific immune response. 3 different experimental groups were studied. Galectin-3 WT CD8 T cells adoptively transferred into Galectin-3 WT mice, galectin-3 WT CD8 T cells transferred into galectin-3 KO mice, and finally galectin-3 KO CD8 T cells transferred into galectin-3 KO mice. Galectin-3 WT CD8 T cells transferred into Galectin-3 WT mice were used as the reference group. Four biological replicates were submitted for each group, and adoptively transfered CD8 T cells were isolated 5 days post-adoptive transfer into tumor-bearing mice treated with a whole cell GM-CSF secreting vaccine. Cells were purified by cell sorting on the Thy1.2 surface marker.

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α2,6-Sialoglycan-regulated anti-tumor immunity of tumor-infiltrating CD8+ T cells in the tumor microenvironment [CD8+T]

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