The accepted paradigm for both cellular and anti-tumor immunity relies upon tumor cell killing by CD8⁺T cells recognizing cognate antigens presented in the context of target cell major histocompatibility complex (MHC) class I (MHC-I) molecules. Likewise, a classically described mechanism of tumor immune escape is tumor MHC-I downregulation. Here, we report that CD8⁺T cells maintain the capacity to kill tumor cells that are entirely devoid of MHC-I expression. This capacity proves to be dependent instead on interactions between T cell natural killer group 2D (NKG2D) and tumor NKG2D ligands (NKG2DLs), the latter of which are highly expressed on MHC-loss variants. Necessarily, tumor cell killing in these instances is antigen independent, although prior T cell antigen-specific activation is required and can be furnished by myeloid cells or even neighboring MHC-replete tumor cells. In this manner, adaptive priming can beget innate killing. These mechanisms are active in vivo in mice as well as in vitro in human tumor systems and are obviated by NKG2D knockout or blockade. These studies challenge the long-advanced notion that downregulation of MHC-I is a viable means of tumor immune escape and instead identify the NKG2D–NKG2DL axis as a therapeutic target for enhancing T cell-dependent anti-tumor immunity against MHC-loss variants.

Glioblastoma (GBM) is notorious for its immunosuppressive tumor microenvironment (TME) and is refractory to immune checkpoint blockade (ICB). Here, we identify calmodulin-dependent kinase kinase 2 (CaMKK2) as a driver of ICB resistance. CaMKK2 is highly expressed in pro-tumor cells and is associated with worsened survival in patients with GBM. Host CaMKK2, specifically, reduces survival and promotes ICB resistance. Multimodal profiling of the TME reveals that CaMKK2 is associated with several ICB resistance-associated immune phenotypes. CaMKK2 promotes exhaustion in CD8⁺ T cells and reduces the expansion of effector CD4⁺ T cells, additionally limiting their tumor penetrance. CaMKK2 also maintains myeloid cells in a disease-associated microglia-like phenotype. Lastly, neuronal CaMKK2 is required for maintaining the ICB resistance-associated myeloid phenotype, is deleterious to survival, and promotes ICB resistance. Our findings reveal CaMKK2 as a contributor to ICB resistance and identify neurons as a driver of immunotherapeutic resistance in GBM.

Purpose: Exhaustion represents a collection of programmed T-cell differentiation states and an important mode of T-cell dysfunction. T-cell progression from progenitor to terminal exhaustion is associated with upregulation of the transcription factor thymocyte selection–associated HMG box (TOX). Our understanding of factors regulating TOX expression and the transition from progenitor to terminal exhaustion, however, remains incomplete. Experimental Design: Single-cell RNA sequencing was used to evaluate the expression of TNF receptors on human and murine tumor-infiltrating CD8+ T cells. Flow cytometry was utilized to assess exhaustion markers and TNF receptors on CD8+ T cells. Bulk RNA sequencing was used to demonstrate the role of TNFR2 on the overall exhaustion profile. Finally, the effect of TNFR2 on the overall antitumor response was established using TNFR2-knockout (KO) mice and an antagonist. Results: We reveal that upregulation of TNFR2 coincides with the gain of phenotypic markers and functions reflective of terminal exhaustion. Loss of TNFR2 affords a novel population of T cells that expresses TIM3 but possesses diminished TOX levels and contains functional characteristics of both progenitor and terminally exhausted cells. TIM3+ TNFR2-KO T cells exhibit reduced exhaustion transcriptional programs and enhanced AP1 pathway signatures. Finally, TNFR2-KO mice demonstrate improved T cell–dependent control of tumor and chronic lymphocytic choriomeningitis infection, whereas pharmacologic antagonism of TNFR2 licenses responses to checkpoint blockade in multiple subcutaneous and intracranial tumor models. Conclusions: Our data place TNFR2 signaling as a potential upstream regulator of TOX expression in T cells and propose TNFR2 antagonism as a novel immunotherapeutic strategy.

The accepted paradigm for both cellular and antitumor immunity relies upon tumor cell kill by CD8 ⁺ T cells recognizing cognate antigens presented in the context of target cell major histocompatibility complex class I (MHC I) molecules. Likewise, a classically described mechanism of tumor immune escape is tumor MHC-I downregulation. Here, we report that CD8 ⁺ T cells maintain the capacity to kill tumor cells that are entirely devoid of MHC-I expression. This capacity proves to be dependent on interactions between T cell NKG2D and tumor NKG2D ligands (NKG2DL). Necessarily, tumor cell kill in these instances is antigen-independent, although prior T cell antigen-specific activation is required and can be furnished by myeloid cells or even neighboring MHC-replete tumors cells. These mechanisms are active in vivo in mice, as well as in vitro in human tumor systems, and are obviated by NKG2D knockout or blockade. Tumor cell killing following T cell NKG2D engagement is Fas-independent and appears to involve granzyme. These studies potentially obviate the long-advanced notion that downregulation of MHC-I is a viable means of tumor immune escape, and instead identify the NKG2D/NKG2DL axis as a novel therapeutic target for enhancing T cell-dependent anti-tumor immunity against MHC loss variants.

SUMMARY: Whereas terminally exhausted T (Tex_term) cells retain anti-tumor cytotoxic functions, the frequencies of stem-like progenitor exhausted T (Tex_prog) cells better reflect immunotherapeutic responsivity. Here, we examined the intratumoral cellular interactions that govern the transition to terminal T cell exhaustion. We defined a metric reflecting the intratumoral progenitor exhaustion-to-terminal exhaustion ratio (PETER), which decreased with tumor progression in solid cancers. Single cell analyses of Tex_prog cells and Tex_term cells in glioblastoma (GBM), a setting of severe T cell exhaustion, revealed disproportionate loss of Tex_prog cells over time. Exhaustion concentrated within tumor-specific T cell subsets, with cognate antigen exposure requisite for acquisition of the Tex_term phenotype. Tumor-associated macrophages (TAM) - not tumor cells - were the primary source of antigenic exposure governing the Tex_prog to Tex_term transition. TAM depletion increased frequencies of Tex_prog cells in multiple tumor models, increased PETER, and promoted responsiveness to αPD-1 immunotherapy. Thus, targeting TAM – T cell interactions may further license checkpoint blockade responses.

Bone marrow is a primary immune organ responsible for hematopoiesis, yet its role in shaping adaptive immune responses remains incompletely understood. In