Hellmann, M. | bioRxiv Science

bioRxiv2026

Chitinase-3-like protein 1 decodes chitosan acetylation patterns into toll-like receptor 2 signaling through heparan sulfate

Chitinase-3-like protein 1 (CHI3L1), which is associated with a wide range of inflammatory diseases, lacks chitinase activity but retains the ability to bind chitin and chitosan. Chitin is a major component of fungal cell walls, whereas chitosan is used in biomedicine. In addition to chitosan, CHI3L1 has been proposed to interact with heparan sulfate (HS), a highly sulfated glycosaminoglycan on mammalian cell surfaces. Here, we investigated how interactions with chitosan and HS regulate the pro-inflammatory activity of CHI3L1. Mapping of the chitin-binding cleft revealed preferential binding of CHI3L1 to chitosans with a regular acetylation pattern that, together with CHI3L1, promoted toll-like receptor 2 signaling. We further identified a dominant HS-binding site that recognizes a distinct HS sulfation code containing a coherent motif of N- and 6-O-sulfations. Mutation of this HS-binding site or impaired HS biosynthesis prevented CHI3L1 accumulation at the cell surface and abolished CHI3L1-mediated cell activation. Together, our findings establish HS as a critical co-receptor for CHI3L1 and reveal a pro-inflammatory cross-talk between HS, CHI3L1, and chitosan that may contribute to host defense against fungal pathogens and responses to chitosan-based biomaterials. These findings identify HS- and chitosan-dependent CHI3L1 signaling as a potential target for modulating inflammatory responses.

Molecular Biology↗

bioRxiv2023

Intratumoral immune triads are required for adoptive T cell therapy-mediated elimination of solid tumors

Tumor-reactive CD8 T cells found in cancer patients are frequently dysfunctional, unable to halt tumor growth. Adoptive T cell transfer (ACT), the administration of large numbers of in vitro-generated cytolytic tumor-reactive CD8 T cells, is an important cancer immune therapy being pursued. However, a limitation of ACT is that transferred CD8 T cells often rapidly lose effector function, and despite exciting results in certain malignancies, few ACT clinical trials have shown responses in solid tumors. Here, we developed preclinical cancer mouse models to investigate if and how tumor-specific CD4 T cells can be enlisted to overcome CD8 T cell dysfunction in the setting of ACT. In situ confocal microscopy of color-coded cancer cells, tumor-specific CD8 and CD4 T cells, and antigen presenting cells (APC), combined with functional studies, revealed that the spatial positioning and interactions of CD8 and CD4 T cells, but not their numbers, dictates ACT efficacy and anti-tumor responses. We uncover a new role of antigen-specific CD4 T cells in addition to the known requirement for CD4 T cells during priming/activation of naive CD8 T cells. CD4 T cells must co-engage with CD8 T cells and APC cross-presenting CD8-and CD4-tumor antigens during the effector phase, forming a three-cell-cluster (triad), to license CD8 T cell cytotoxicity and mediate cancer cell elimination. Triad formation transcriptionally and epigenetically reprogram CD8 T cells, prevent T cell dysfunction/exhaustion, and ultimately lead to the elimination of large established tumors and confer long-term protection from recurrence. When intratumoral triad formation was disrupted, adoptively transferred CD8 T cells could not be reprogrammed, and tumors progressed despite equal numbers of tumor-infiltrating CD8 and CD4 T cells. Strikingly, the formation of CD4 T cell::CD8 T cell::APC triads in tumors of patients with lung cancers treated with immune checkpoint blockade was associated with clinical responses, but not CD4::APC dyads or overall numbers of CD8 or CD4 T cells, demonstrating the importance of triads in non-ACT settings in humans. Our work uncovers intratumoral triads as a key requirement for anti-tumor immunity and a new role for CD4 T cells in CD8 T cell cytotoxicity and cancer cell eradication.

immunology↗