Sialic acid inhibits engager-mediated T cell activation

Background: Although T cell-based therapies have led to a significant success in certain tumor types, a large proportion of patients receiving cancer immunotherapy still progress with the disease. Thus, there is interest in identifying and blocking alternative pathways mediating immune suppression. Beyond canonical protein-based immune checkpoints, tumor-associated glycosylation-particularly hypersialylation-has emerged as a key modulator of immune suppression. Sialic acid-containing glycans on tumor cells are recognized as glyco-immune checkpoints that engage inhibitory Siglec receptors on immune cells. While the immunosuppressive role of tumor sialylation is well established [1] , new evidence indicates that sialylation of the T cells themselves [2] may also contribute to immune dysfunction, suggesting a previously underappreciated layer of glyco-regulation in the tumor microenvironment. Methods: Human T cells were treated with pan-sialidase to enzymatically remove surface sialic acids. Desialylated T cells were then stimulated with anti-CD3/anti-CD28 or co-cultured with bispecific T cell engagers and tumor target cells, followed by flow cytometry. Primary samples from patients with chronic lymphocytic leukemia (CLL) were analyzed through single-cell RNA sequencing combined with lectin-based glycan profiling. Results: We discovered that T cells treated with sialidase exhibited enhanced proliferation and activation. In a co-culture system with bispecific T cell engagers and target cells, we demonstrated that sialidase-treated T cells showed enhanced cytotoxicity, including increased cytokine production, T cell proliferation, and tumor cell killing. When studying T cell subsets, we observed that sialidase treatment lowered the activation threshold particularly in naïve T cells. Our findings were validated using primary samples from patients with chronic lymphocytic leukemia (CLL): sialic acid remodeling led to improved immune function. Conclusion: Sialic acids on T cells significantly modulate their function. Enzymatic removal of these glycans enhances T cell response and may help overcome resistance mechanisms in cancer immunotherapy. Future studies will investigate whether distinct glycan signatures among T cell subtypes correlate with functional heterogeneity. [1] Dobie, C., Skropeta, D. Br J Cancer 2021 ,124, 76-90. [2] Edgar, L. J.; Paulson J. C et al. ACS Cent Sci 2021 , 7, 1508-1515.