Abstract 1342: Expression of Galectin-1 in exhausted CD8+T cells restrains their antitumor effector functions.

Abstract Antitumor immune responses are constrained by a plethora of inhibitory mechanisms that promote immune evasion. Galectin-1 (GAL1), a glycan-binding protein recognizing N-acetyllactosamine residues in complex N- and O-glycans promotes immunosuppression by expanding regulatory T cells, inducing tolerogenic dendritic cells and activating immunosuppressive circuits in myeloid-derived suppressor cells (MDSCs). Through single-cell RNA sequencing analysis, we identified elevated GAL1 expression in exhausted CD8+ tumor-infiltrating T cells (Tex) compared to effector cells. We validated this observation at the protein level in in vitro-generated Tex (iTex) compared to early-activated T cells. Lgals1-/- CD8+ Tex cells infiltrating B16 tumors displayed decreased expression of exhaustion-associated transcription factor TOX (p0.01), suggesting a potential role of GAL1 in promoting CD8+ T cell terminal differentiation. To test this hypothesis in vivo, we used CRISPR-Cas9 editing on OVA-restricted OT-I CD8+ T cells to generate control and GAL1-deficient antigen-specific T cells (sgLgals1), which we adoptively transferred into B16-OVA tumor-bearing Rag2-/- mice. Flow cytometry and RNA-sequencing analysis revealed that sgLgals1 tumor-infiltrating Tex conserved effector functions as evidenced by elevated Granzyme B, IFN-γ and TNF-α expression compared to control T cells (p0.001). Mechanistically, we explored whether soluble GAL1 may contribute to T cell exhaustion by interacting with immune checkpoint receptors. Using lectin blot and solid-phase binding assays, we demonstrated that Gal1 differentially binds to a set of immune checkpoint receptors (including VISTA, PD-1, CTLA-4, LAG3, and TIGIT, but not TIM3) in a glycan-dependent manner, as binding was inhibited by lactose. To further pursue this observation, we performed isothermal titration calorimetry and confirmed GAL1-VISTA interaction with a dissociation constant (Kd) of 2.78 µM and GAL1-LAG3 interaction with a Kd of 12.75 µM. Then, we profiled VISTA expression on B16 tumor-infiltrating immune cells and found it to be highly expressed within the myeloid compartment, with minimal or absent expression in the lymphoid compartment and in tumor cells. Collectively, our results suggest a dual function for GAL1 in the tumor microenvironment. Endogenous GAL1 expression by CD8+ Tex cells may promote a terminal dysfunctional state in an autocrine fashion, while simultaneously driving an immunosuppressive phenotype on myeloid cells through paracrine engagement of checkpoint receptors. Thus, targeting Gal1 may have therapeutic implications in cancer immunotherapeutic regimens including immune checkpoint blockade and adoptive cell transfer. Our future perspectives will be to focus both on signaling pathways triggered by GAL1 engagement of checkpoint receptors and on GAL1 intracellular functions during T cell exhaustion. Citation Format: Marco A. Scheidegger, Joaquin P. Merlo, Mora Massaro, Juan M. Pérez Saez, Sabrina G. Gatto, Rosa M. Morales, Alejandro J. Cagnoni, Tomás Dalotto-Moreno, Gabriel A. Rabinovich. Expression of Galectin-1 in exhausted CD8+T cells restrains their antitumor effector functions abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1342.