Glioblastoma (GBM) is the most common adult brain cancer. Recent therapeutic advances, including immunotherapies that enhance anti-tumor immune responses, have failed to significantly improve survival. To address these failures, deeper studies on the cellular interactions that orchestrate whether GBM resists or regresses during immunotherapy are needed. Our group recently discovered that anti-CTLA-4 therapy initiated a novel microglia-CD4 T cell partnership. Following anti-CTLA-4, tumor-bearing mice showed a significant increase in survival and enhanced tumor regression. Further investigation showed that CD4 T cells, not cytotoxic CD8 T cells, were necessary for this response and that microglia were responsible for both sustaining tumoricidal CD4 T cell activity via MHC-II antigen presentation and phagocytosing debilitated tumor cells. With the rise of spatial profiling platforms, such as NanoString’s CosMx, we can now capture snapshots of the cellular dynamics that lead to a successfully mounted anti-tumor response in a continually evolving tumor microenvironment. Using both murine models and human GBM specimens, we are exploring the cellular networks that support beneficial CD4 T cell-microglia interactions and tumor regression. Conversely, we are also examining the role of regulatory T cells in the periphery and tumor microenvironment, and how these cells may interact with other immune suppressive niches, including GBM-associated microglia and infiltrating macrophages, to support GBM progression. Through this work, we are mapping the GBM cellular niches that support or suppress anti-tumor responses, with the goal of exposing vulnerabilities that can be exploited with better tailored immunotherapies. Supported by grants from NIH (R01 CA240909, R01 CA230275)

The limited efficacy of immunotherapies against glioblastoma underscores the urgency of better understanding immunity in the central nervous system. We found that treatment with degrees cCTLA-4, but not degrees cPD-1, prolonged survival in a mouse model of mesenchymal-like glioblastoma. This effect was lost upon the depletion of CD4+ T cells but not CD8+ T cells. degrees cCTLA-4 treatment increased frequencies of intratumoral IFNy-producing CD4+ T cells, and IFNy blockade negated the therapeutic impact of degrees cCTLA-4. The anti-tumor activity of CD4+ T cells did not require tumor-intrinsic MHC-II expression but rather required conventional dendritic cells as well as MHC-II expression on microglia. CD4+ T cells interacted directly with microglia, promoting IFNy-dependent microglia activation and phagocytosis via the AXL/MER tyrosine kinase receptors, which were necessary for tumor suppression. Thus, degrees cCTLA-4 blockade in mesenchymal-like glioblastoma promotes a CD4+ T cell-microglia circuit wherein IFNy triggers microglia activation and phagocytosis and microglia in turn act as antigen-presenting cells fueling the CD4+ T cell response.

Although tumor growth requires the mitochondrial electron transport chain (ETC), the relative contribution of complex I (CI) and complex II (CII), the gatekeepers for initiating electron flow, remains unclear. In this work, we report that the loss of CII, but not that of CI, reduces melanoma tumor growth by increasing antigen presentation and T cell–mediated killing. This is driven by succinate-mediated transcriptional and epigenetic activation of major histocompatibility complex–antigen processing and presentation (MHC-APP) genes independent of interferon signaling. Furthermore, knockout of methylation-controlled J protein (MCJ), to promote electron entry preferentially through CI, provides proof of concept of ETC rewiring to achieve antitumor responses without side effects associated with an overall reduction in mitochondrial respiration in noncancer cells. Our results may hold therapeutic potential for tumors that have reduced MHC-APP expression, a common mechanism of cancer immunoevasion.

CD8 +T cells are critical components of the immune reaction against viral infections and cancer and have the potential to eliminate infected or malignant cells. However, when the antigen persists, CD8 +T cells enter an exhausted state. Chronic disease can lead to increased systemic noradrenaline (NA) levels, but it is currently still unclear how NA impacts the differentiation and function of exhausted CD8 +T cells. We here set out to characterize the effects of β adrenergic signaling on CD8 +T cells in chronic viral infection with LCMV-clone 13 and in murine cancer models. We observed that chronically infected mice had elevated systemic NA levels and CD8 +T cells expressed higher levels of the β-1 adrenergic NA receptor, Adrb1. NA impaired T cell receptor signaling of ADRB1-expressing CD8 +T cells, and reduced T cell proliferation and function. Conversely, genetic ablation of ADRB1 prevented terminal CD8 +T cell differentiation in chronic viral infection and ADRB1-blockade enhanced T cell functionality in combination with immune checkpoint blockade (ICB) in an ICB-sensitive melanoma model. Expanding these observations to an ICB-resistant model of pancreatic cancer, we found that pharmacological blockade of adrenergic receptors synergized with ICB to genetically reprogram CD8 +T cells towards a tissue resident memory T cell-like state and improved T cell functionality, resulting in decreased tumor size. In summary, our data suggest that β adrenergic receptors represent a novel immune checkpoint that modulates CD8 +T cell differentiation and function in the context of chronic antigen exposure and that targeting adrenergic receptors may synergize with ICB in cancer patients. AMG was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, GL 991/1-1). SZ was supported by K00CA222741. This work was supported by grants from the NIH 5 R01 CA240909 (SMK) and 5 R01 CA216101 (SMK).

CD8 + T cells are essential components of the immune response against viral infections and tumours, and are capable of eliminating infected and cancerous cells. However, when the antigen cannot be cleared, T cells enter a state known as exhaustion 1 . Although it is clear that chronic antigen contributes to CD8 + T cell exhaustion, less is known about how stress responses in tissues regulate T cell function. Here we show a new link between the stress-associated catecholamines and the progression of T cell exhaustion through the β 1 -adrenergic receptor ADRB1. We identify that exhausted CD8 + T cells increase ADRB1 expression and that exposure of ADRB1 + T cells to catecholamines suppresses their cytokine production and proliferation. Exhausted CD8 + T cells cluster around sympathetic nerves in an ADRB1-dependent manner. Ablation of β 1 -adrenergic signalling limits the progression of T cells towards the exhausted state in chronic infection and improves effector functions when combined with immune checkpoint blockade (ICB) in melanoma. In a pancreatic cancer model resistant to ICB, β-blockers and ICB synergize to boost CD8 + T cell responses and induce the development of tissue-resident memory-like T cells. Malignant disease is associated with increased catecholamine levels in patients 2 , 3 , and our results establish a connection between the sympathetic stress response, tissue innervation and T cell exhaustion. Here, we uncover a new mechanism by which blocking β-adrenergic signalling in CD8 + T cells rejuvenates anti-tumour functions.

Summary The limited efficacy of immunotherapies against glioblastoma illustrates the urgent need to better understand the interactions between the central nervous system and the immune system. Here, we showed that a protective response to αCTLA-4 therapy depended on a mutualistic relationship between microglia and CD4 + T cells. Suppression of gliomas by CD4 + T cells did not require tumor-intrinsic MHC-II expression, but rather was dependent on the selective expression of MHC-II and antigen presentation by local microglia that in turn, sustained CD4 + T cell tumoricidal effector functions. CD4 + T cell secretion of IFNγ made the glioma cells vulnerable to enhanced tumor surveillance and phagocytosis by microglia via the AXL/MER tyrosine kinase receptors that were necessary for tumor suppression. This work illustrates a novel partnership between CD4 + T cells and microglia that unleashes the tumoricidal properties of microglia that can be harnessed to improve immunotherapies for glioblastoma.

A common metabolic alteration in the tumor microenvironment (TME) is lipid accumulation, a feature associated with immune dysfunction. Here, we examined how CD8+ tumor infiltrating lymphocytes (TILs) respond to lipids within the TME. We found elevated concentrations of several classes of lipids in the TME and accumulation of these in CD8+ TILs. Lipid accumulation was associated with increased expression of CD36, a scavenger receptor for oxidized lipids, on CD8+ TILs, which also correlated with progressive T cell dysfunction. Cd36-/- T cells retained effector functions in the TME, as compared to WT counterparts. Mechanistically, CD36 promoted uptake of oxidized low-density lipoproteins (OxLDL) into T cells, and this induced lipid peroxidation and downstream activation of p38 kinase. Inhibition of p38 restored effector T cell functions in vitro, and resolution of lipid peroxidation by overexpression of glutathione peroxidase 4 restored functionalities in CD8+ TILs in vivo. Thus, an oxidized lipid-CD36 axis promotes intratumoral CD8+ T cell dysfunction and serves as a therapeutic avenue for immunotherapies.

T cell metabolic fitness plays a pivotal role in anti-tumor immunity and metabolic deregulation causes T cell dysfunction (i.e., ‘exhaustion’) in cancer. We identify that the scavenger receptor CD36 limits anti-tumor CD8+ T cell effector functions through lipid peroxidation. In murine tumors, oxidized phospholipids (OxPLs) were highly abundant and CD8+ TILs increased uptake and accumulation of lipids and lipid peroxidation. Functionally ‘exhausted’ CD8+ TILs substantially increased CD36 expression and CD36-deficient CD8+ TILs had more robust anti-tumor activity and cytokine production than wild-type cells. We further show that CD36 promotes uptake of oxidized low-density lipoproteins (OxLDL) and induces lipid peroxidation in CD8+ TILs, and OxLDL inhibits CD8+ T cell functions in a CD36-dependent manner. Moreover, glutathione peroxidase 4 (GPX4) over-expression lowers lipid peroxidation and restores functionalities in CD8+ TILs. These results define a key role for an oxidized lipid-CD36 axis in promoting intratumoral CD8+ T cell dysfunction. ### Competing Interest Statement G.C. receives research funding from Bayer AG and Boehringer Ingelheim, but the funding is not relevant to the current study. J.L.W and X.S. are named inventors on patent applications or patents related to the use of oxidation-specific antibodies held by UCSD. All other authors declare no conflict of interest.