Intracranial tumors present unique challenges for immunotherapy, which can include both local and systemic immune suppression whose mechanistic underpinnings are incompletely understood. Here, we reveal that tumors harbored intracranially elicit systemic increases in catecholamines and chronic sympathetic hyperactivity that drives T cell dysfunction and limits immunotherapeutic success. Conversely, treatment with β-adrenergic receptor blockade increases NF-κB activity in immune cells, restores T cell polyfunctionality, modifies the tumor microenvironment, and licenses immune-based therapies in murine models of GBM to extend survival. Extended survival was also observed in GBM patients receiving β-blockers for any indication, as well as in patients with melanoma and lung cancer brain metastases who received concomitant immune checkpoint inhibition and β-adrenergic blockade compared to immune checkpoint inhibition alone. These data suggest roles for increased adrenergic activity in facilitating systemic immune dysfunction in the setting of intracranial tumors, specifically, and advance a role for β-adrenergic blockade in licensing immunotherapeutic responses within the intracranial compartment.### Competing Interest StatementThe authors have declared no competing interest.

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 (TAMs)—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 αPD1 immunotherapy. Thus, targeting TAM-T cell interactions may further license checkpoint blockade responses.

Abstract Checkpoint inhibitors have been successful in various tumors. However, these treatments have failed in glioblastoma. Approaches harnessing the immune response are hindered by multiple factors, including T cell exhaustion. TOX is particularly important for the transcriptional and epigenetic reprogramming of exhausted T cells. While it is known that NFAT is upstream of TOX, our understanding of the upregulation of TOX remains incomplete. We hypothesized that tumor necrosis factor (TNF) could be involved in the upregulation of TOX as TNF can lead to the translocation of NFAT into the nucleus. We observed significant upregulation of the anti-inflammatory TNF receptor type II (TNFR2) in tumor-infiltrating T cells. This local upregulation mimics expression patterns of canonical exhaustion markers. TNFR2 expression is correlated with markers of exhaustion, including PD1, TIM3, and TOX. Furthermore, TNFR2 knock out (KO) CD8 T cells have significantly lower TOX expression, without the concomitant decrease of TIM3. Whereas previous studies have linked TIM3 and TOX expression, these data suggest that TIM3 and TOX are regulated independently. We utilized bulk RNA-sequencing to assess transcriptional regulation of WT and KO T cells and detected a significant reduction of T cell exhaustion and immune checkpoint pathways in TNFR2 KO T cells. Various exhaustion-related transcription factors and coinhibitory markers were significantly reduced. In contrast, a significant increase in AP1 transcription factors, commonly associated with T cell effector functions, was detected. Given this reduced exhaustion profile, we subsequently investigated the influence of TNFR2 on tumor burden. TNFR2 KO mice had significantly lower tumor burdens following subcutaneous tumor challenges. We subsequently treated mice with a TNFR2 antagonist. While the antagonist alone was not sufficient to improve tumor control, combination with anti-PD1 significantly reduced tumor volumes. These data provide evidence for a novel marker of exhaustion that could result in a unique therapeutic strategy.

Abstract Glioblastoma (GBM) is the most common primary brain cancer in adults with a median survival less than 15 months. Approaches harnessing the immune response are hindered by multiple factors, including T cell exhaustion. Exhausted T cells are less capable of limiting tumor progression. As cells progress along the exhaustion pathway, SLAM Family Member 6 (SLAMF6) gets downregulated and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) and TOX are upregulated. TOX is both necessary and sufficient to initiate the terminal exhaustion program. However, our understanding of the pathways that lead to the upregulation of TOX remains incomplete. We hypothesized that tumor necrosis factor (TNF) could be involved in the upregulation of TOX, as TNF has been shown to induce exhaustion. We observed upregulation of the TNF receptor type II (TNFR2) in tumor-infiltrating T cells, but not in lymphoid organs. Furthermore, TNFR2 expression is correlated with canonical exhaustion markers. Interestingly, TNFR2 knock out (KO) CD8 T cells express TIM3 but exhibit significantly less TOX. These data suggest that TIM3 and TOX are regulated independently. Finally, TNFR2 KO mice have significantly better CD8-mediated tumor control against subcutaneous GBM tumors. Further studies will need to be performed to determine whether TNFR2 expression on CD8 T cells is sufficient to mediate this anti-tumor effect and whether TNFR2 blockade could improve the efficacy of current treatments.

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 TOX and expression of TIM3. Our understanding of factors regulating TOX expression and the transition from progenitor to terminal exhaustion, however, remains incomplete. We reveal here that T cell upregulation of tumor necrosis factor receptor type II (TNFR2) coincides with the gain of phenotypic markers and functions reflective of terminal exhaustion. Meanwhile, knocking out TNFR2 affords a novel population of T cells that express TIM3 but possess diminished TOX levels and 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 viral (cLCMV) infection, while pharmacologic antagonism of TNFR2 licenses responses to checkpoint blockade in multiple tumor models. ### Competing Interest Statement The authors have declared no competing interest.

SUMMARYWhile terminally exhausted T cells (Tex_term) retain important anti-tumor cytotoxic function, it is the relative preservation of renewable, stem-like progenitor exhaustion (Tex_prog) that better indicates immunotherapeutic responsivity. Although restraining the progression from Tex_prog to Tex_term thus takes on clinical significance, the cellular interactions in a tumor microenvironment (TME) governing such progression remain less established. Employing glioblastoma (GBM) and other solid tumors as models of severe exhaustion, we provide a detailed characterization of the progression from Tex_prog to Tex_term within the TME, where we observe a striking and disproportionate loss of Tex_prog over time, leading to a low progenitor exhaustion to terminal exhaustion ratio (PETER). We find exhaustion concentrated within tumor-specific T cell subsets, with cognate antigenic exposure requisite for acquisition of the Tex_term phenotype. However, we implicate tumor-associated macrophages (TAM), and not tumor cells, as the source of antigenic exposure governing the Tex_prog to Tex_term transition. Using cell – cell interaction analysis, we additionally highlight candidate receptor–ligand communications that may be specifically mediating the progression to Tex_term and resultant decline in PETER within the TME.GRAPHICAL ABSTRACT

Abstract The long-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, in contrast to the decades old model of T cell immunity, we instead 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 NKG2D and tumor NKG2D ligands (NKG2DL), the latter of which are highly expressed on MHC-loss variants. 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 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.

Abstract Peripheral nervous system innervation of various organs has been associated with the development and maintenance of the immune system. Interestingly, various neurological insults including brain tumors, intracranial metastases, and neurotropic infections have been shown to have profound impacts on peripheral immunity, including thymic size and cellularity. Therefore, we sought to determine whether the thymus was directly innervated. Using flow cytometry and confocal microscopy, we identified a population of cells within the thymus that contained both neuronal markers and morphology. Subsets expressed tyrosine hydroxylase, BIII tubulin and/or NeuN, distinguishing them from thymic epithelial cells. These cells do not express AIRE, further highlighting their neuronal identity. Interestingly, a subset of these neurons also express MCH-II, implying a potential role in T cell selection. Neuronal cell bodies and/or axonal processes were identified within the thymus. Using retrograde rabies virus-derived neurotracers, we confirmed axonal connections and the identity of thymic neurons. Finally, both neuronal and epithelial cells were absent in RAG-deficient mice, suggesting a shared differentiation or survival pathway. Both populations could be restored by introducing healthy bone marrow. To summarize, we describe a novel population of neuronal cells within the thymus that interact with developing thymocytes to establish optimal thymic architecture.

Abstract Glioblastoma (GBM) is an aggressive form of brain cancer with poor survival despite standard of care. Checkpoint inhibitors aimed to revive dysfunctional T cells have significantly improved outcomes in other cancers, but have failed GBM. GBM patients have severe peripheral immunosuppression, including severe lymphopenia, immune organ atrophy, and defective T cell responses. This immunosuppression is a critical barrier to patient survival and the success of immunotherapies. Using parabiosis, we demonstrated that serum-derived factors were sufficient to drive hallmark features of immunosuppression, including inhibition of ex vivo T cell proliferation. This factor was nonsteroidal in nature and had molecular weights greater than 100kDa. Mass-spectrometry and pathway analysis of the suppressive serum implicated proteins involved in DNA response elements, cell death, and DNA-histone complexes. We hypothesized that the factor is cell-free DNA (cfDNA). Confirming this, serum levels of cfDNA are increased in glioma-bearing mice. Moreover, exposure to cfDNA from serum of glioma-bearing mice was sufficient to directly and potently inhibit T cell proliferation ex vivo. Interestingly, exposure to genomic DNA was not sufficient to induce proliferation defects in T cells, highlighting the unique features of cfDNA. Importantly, reducing cfDNA content using DNaseI partially restored proliferation capacity. We determined that the T cell proliferation defects were independent of DNA sensing mechanisms through AIM2, implicating a novel pathway of DNA sensing in T cells. Finally, to determine the origin of the cfDNA, we employed methyl-seq. The chromatin signature most closely represented neutrophils. This highlights a potential role for neutrophils-derived cfDNA as a targetable strategy in GBM. Together, we contend that cfDNA induces immunosuppressive effects in GBM and devising strategies to reduce circulating cfDNA could be a therapeutic approach to improve T cell functions and outcomes in GBM patients.

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.

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.### Competing Interest StatementThe authors have declared no competing interest.

Background The nervous and immune systems interact in a reciprocal manner, both under physiologic and pathologic conditions. Literature spanning various CNS pathologies including brain tumors, stroke, traumatic brain injury and de-myelinating diseases describes a number of associated systemic immunologic changes, particularly in the T-cell compartment. These immunologic changes include severe T-cell lymphopenia, lymphoid organ contraction, and T-cell sequestration within the bone marrow. Methods We performed an in-depth systematic review of the literature and discussed pathologies that involve brain insults and systemic immune derangements. Conclusions In this review, we propose that the same immunologic changes hereafter termed ‘systemic immune derangements’, are present across CNS pathologies and may represent a novel, systemic mechanism of immune privilege for the CNS. We further demonstrate that systemic immune derangements are transient when associated with isolated insults such as stroke and TBI but persist in the setting of chronic CNS insults such as brain tumors. Systemic immune derangements have vast implications for informed treatment modalities and outcomes of various neurologic pathologies.

Abstract Glioblastoma (GBM) and brain metastases remain largely resistant to immunotherapeutic intervention. T cell exhaustion, a differentiation state characterized by loss of function and persistence, contributes to this resistance. Two exhaustion subsets, progenitor (Tex_prog) and terminal (Tex_term), have been identified, where only Tex_prog remain responsive to immunotherapy. To date, the dynamics and characteristics of these exhausted populations in GBM and brain metastases remain unclear. Herein, we identify a striking loss of Tex_prog in a murine model of GBM throughout tumor progression. We elucidate the requirements for Tex_prog to Tex_term transition through characterization of T cell exhaustion progression at the RNA (paired scRNA and TCR sequencing) and protein level (flow-cytometry). Tex_prog are enriched for pathways of migration, cell-cell adhesion, and differentiation, whereas Tex_term primarily upregulate cytotoxic pathways. TCRseq revealed that clonal expansion is concentrated to the Tex_term cluster, suggesting a link between Tex_prog transition and proliferation in response to antigen. Likewise, we defined the necessity of hematopoietic antigen presentation, but not tumor-derived antigen presentation for the Tex_prog to Tex_term transition. Of the antigen presenting cells in the TME, tumor-associated macrophages (TAM) had the greatest expression of tumor-antigen loaded MHC I, illustrating their capacity to cross-present. By depleting TAM, we observed disruption of the Tex_prog transition. Similar results were seen in subcutaneous and intracranial melanoma, highlighting the role of TAM in this process across tumor histology and location. Lastly, we identified additional inflammatory cell-cell interactions between T cells and TAM in mice and humans that may contribute to this phenomenon. Taken together, we define T cell exhaustion dynamics in models of primary and metastatic brain tumors, where tumor-antigen presentation by TAM is a critical mediator of the loss of Tex_prog. We extend these findings to human data to better define these complex interactions, which we aim to leverage to overcome immunotherapy resistance.

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.

Abstract BACKGROUND Glioblastoma (GBM) is the most common primary brain cancer in adults and remains universally lethal. Median survival remains a bleak 15-17 months from time of diagnosis, and current immunotherapeutic efficacy continues to be hindered by the robust immunosuppression present in the GBM microenvironment. T cells, critical for tumor clearance, are particularly affected, and many take on a functionally exhausted phenotype within the tumor. Importantly, two exhaustion states, progenitor and terminal, have been identified in models of chronic infection and cancer. This distinction is particularly relevant, as progenitor exhausted T cells can respond favorably to immune checkpoint blockade, while terminally exhausted T cells are resistant. To date, the dynamics and characteristics of these exhausted populations in GBM remain unclear. RESULTS In an orthotopic murine model of GBM, progenitor and terminal exhausted CD8 T cells were identified by flow cytometry as PD1+SLAMF6+ and PD1+TIM3+, respectively. Using a time-course approach, we detected progenitor exhaustion by day 8 in the tumor, but not in draining lymph nodes. Additionally, we show that the frequency of progenitor exhaustion is highest during early tumor progression, while terminal exhaustion is the most abundant in more advanced tumors ( >14 days). Functional differences between subsets were evaluated via intracellular staining of IFNγ, TNFα, granzyme B, and Ki67. Terminally exhausted T cells displayed higher cytotoxic molecule expression than progenitor exhausted T cells, similar to what has been documented in melanoma models. CONCLUSIONS Our findings identify T cell exhaustion subsets within GBM that require further investigation and may be relevant to overcome current barriers to immunotherapeutic efficacy.

Successful cancer immunotherapies rely on a replete and functional immune compartment. Within the immune compartment, T cells are often the effector arm of immune-based strategies due to their potent cytotoxic capabilities. However, many tumors have evolved a variety of mechanisms to evade T cell-mediated killing. Thus, while many T cell-based immunotherapies, such as immune checkpoint inhibition (ICI) and chimeric antigen receptor (CAR) T cells, have achieved considerable success in some solid cancers and hematological malignancies, these therapies often fail in solid tumors due to tumor-imposed T cell dysfunctions. These dysfunctional mechanisms broadly include reduced T cell access into and identification of tumors, as well as an overall immunosuppressive tumor microenvironment that elicits T cell exhaustion. Therefore, novel, rational approaches are necessary to overcome the barriers to T cell function elicited by solid tumors. In this review, we will provide an overview of conventional immunotherapeutic strategies and the various barriers to T cell anti-tumor function encountered in solid tumors that lead to resistance. We will also explore a sampling of emerging strategies specifically aimed to bypass these tumor-imposed boundaries to T cell-based immunotherapies.

In chronic infections and in cancer, persistent antigen stimulation under suboptimal conditions can lead to the induction of T-cell exhaustion. Exhausted T cells are characterized by an increased expression of inhibitory markers and a progressive and hierarchical loss of function. Although cancer-induced exhaustion in CD8 T cells has been well-characterized and identified as a therapeutic target (i.e., via checkpoint inhibition), in-depth analyses of exhaustion in other immune cell types, including CD4 T cells, is wanting. While perhaps attributable to the contextual discovery of exhaustion amidst chronic viral infection, the lack of thorough inquiry into CD4 T-cell exhaustion is particularly surprising given their important role in orchestrating immune responses through T-helper and direct cytotoxic functions. Current work suggests that CD4 T-cell exhaustion may indeed be prevalent, and as CD4 T cells have been implicated in various disease pathologies, such exhaustion is likely to be clinically relevant. Defining phenotypic exhaustion in the various CD4 T-cell subsets and how it influences immune responses and disease severity will be crucial to understanding collective immune dysfunction in a variety of pathologies. In this review, we will discuss mechanistic and clinical evidence for CD4 T-cell exhaustion in cancer. Further insight into the derivation and manifestation of exhaustive processes in CD4 T cells could reveal novel therapeutic targets to abrogate CD4 T-cell exhaustion in cancer and induce a robust antitumor immune response.