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… Click to show full 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.