IMMU-35. CD3⁺CD4⁻CD8⁻ (Double-Negative) T-cells Provide Inflammatory Signals That Influence Myeloid Cells and Enhance Survival in Glioblastoma

Abstract Despite responses in other malignancies, immune checkpoint inhibitor immunotherapy does not significantly improve outcomes for most glioblastoma (GBM) patients. Our group previously showed that anti-PD-1 therapy extended survival in CD8-knockout (CD8-KO) mice, implicating that another immune population may mediate the therapeutic effect. Now, depleting the CD4 T-cell in the CD8-KO mice significantly extended survival (medOS=66) in syngeneic gliomas compared to CD8-WT mice treated with IgG (med OS=39.5, p-val=0.0256) or anti-CD4 alone (medOS=35.5, p-val=0.0008). Immune-enriched single-cell RNA sequencing from the gliomas during the therapeutic window (P45) revealed a previously underappreciated CD3⁺CD4⁻CD8⁻ double-negative T-cell (DNTs) population expanded in the tumor of the CD4-depleted CD8-KO mice. These DNTs exhibited upregulation of pro-inflammatory cytokines (Tnf, Ccl12), activation markers (Cd40lg, Il7r, Cd69, Cd226), and reduced expression of exhaustion markers (Pdcd1, Ctla4, Tox), distinguishing them transcriptionally from conventional T-cells. The DNT population lacks markers consistent with NK-cells, NK T-cells, or gamma-delta T-cells. In vitro, DNTs secrete TNF-α at baseline and in response to glioma stem cells (GSCs). DNT–GSC co-culture suppresses GSC proliferation by 20-36%. The DNTs co-cultured with M0 macrophages increased macrophage phagocytosis by 44%, as assessed by fluorescent E. coli uptake assays. Glioma-associated myeloid cells from DNT-enriched mice showed upregulated gene ontologies related to inflammation, antigen presentation, and phagocytic activation, contrasting with the immune tolerance signatures in the CD8-WT background mice. Moreover, anti-PD-1 treatment in the CD4-depleted CD8-WT background and the CD8-KO only mice blunted the survival difference, suggesting that global T-cell elimination is a prerequisite for the emergence of antitumor DNT function. In summary, our findings identify a novel immunoregulatory and antitumor role for DNTs in glioma, characterized by TNFα-driven inflammatory signaling and myeloid activation. These data uncover a previously unrecognized immune axis that may be leveraged to overcome immunotherapy resistance in GBM.