Oral Presentation CD1-MR1 Workshop 2025

TCRαβ+ double-negative T cells; new members of the innate T cell family? (#24)

Martin Brennan 1 2 , Harry Kane 1 3 , Brenneth Stevens 1 2 , Calvin Xu 4 , Katie Healy 3 5 , Hannah Prendeville 1 , Claire McIntyre 3 , Brittany Bamonte 5 , Jaclyn Kline 3 , Aaron Douglas 1 , Ayantu Temesgen 3 , Karen Slattery 1 , Aine Ni Scanail 3 , Hui-Fern Koay 4 , Lydia Lynch 2 3 5
  1. Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland
  2. Molecular Biology, Princeton University, Princeton, NJ, USA
  3. Brigham and Women's Hospital , Harvard Medical School, Boston, MA, USA
  4. Doherty Institute, University of Melbourne, Melbourne, Australia
  5. Ludwig Cancer Research Institute, Princeton University, Princeton, NJ, USA

Unconventional T cells bridge innate and adaptive immunity, combining features of both arms of the immune system. While iNKT, MAIT and γδ T cells have been extensively studied as innate T cells in both homeostasis and disease, considerably less is known about non-CD1d/MR1-restricted TCRαβ⁺ CD4⁻CD8⁻ double-negative T cells (DNTs). Here, we characterize non-MAIT/iNKT/γδ DNTs across murine tissues and investigate their potential innateness in comparison to other unconventional T cells. Combined single-cell TCR and RNA sequencing revealed a largely polyclonal and heterogeneous TCRαβ⁺ DNT population in the spleen and adipose tissue. Flow cytometric analysis shows DNTs account for 1–5% of T cells in secondary lymphoid organs, but expand to 10–20% in non-lymphoid tissues such as adipose. We identify two dominant subtypes: type-1 DNTs (NK1.1⁺, T-bet⁺) and type-17 DNTs (CXCR6⁺, RORγt⁺), with broad tissue distribution. Phenotypically, DNTs share conserved innate T cell features, including expression of PLZF and Helios, a pre-activated effector-memory–like state, and rapid cytokine-driven effector function independent of TCR engagement. Subtype-specific enrichment of DNTs across various organs, signifies specialised roles for DNTs in certain tissue environments, further authenticated by the ability of DNTs to expand and fill the T cell “niche” or compartment in knockout models which lack other T cell populations. Additionally, we present evidence of distinct developmental requirements for both type-1 and type-17 DNTs in the thymus. Finally, we demonstrate that type-1 DNTs, the most abundant subtype throughout murine tissues, infiltrate multiple syngeneic tumor models. Through ex-vivo expansion and subsequent adoptive therapy, we highlight their role in mediating an anti-tumor response in the MC38 colorectal tumor model. Together, our findings define DNTs as a thymically derived, innate-like T cell population with broad tissue distribution, distinct subtypes, and functional relevance in tumor immunity.

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