Transcriptional repression by HDAC3 mediates T cell exclusion from Kras mutant lung tumors.

Histone Deacetylase 3 (HDAC3) function in vivo is nuanced and directed in a tissue-specific fashion. The importance of HDAC3 in Kras mutant lung tumors has recently been identified, but HDAC3 function in this context remains to be fully elucidated. Here, we identified HDAC3 as a lung tumor cell-intrinsic transcriptional regulator of the tumor immune microenvironment. In Kras mutant lung cancer cells, we found that HDAC3 is a direct transcriptional repressor of a cassette of secreted chemokines, including Cxcl10. Genetic and pharmacological inhibition of HDAC3 robustly up-regulated this gene set in human and mouse Kras, LKB1 (KL) and Kras, p53 (KP) mutant lung cancer cells through an NF-κB/p65-dependent mechanism. Using genetically engineered mouse models, we found that HDAC3 inactivation in vivo induced expression of this gene set selectively in lung tumors and resulted in enhanced T cell recruitment at least in part via Cxcl10. Furthermore, we found that inhibition of HDAC3 in the presence of Kras pathway inhibitors dissociated Cxcl10 expression from that of immunosuppressive chemokines and that combination treatment of entinostat with trametinib enhanced T cell recruitment into lung tumors in vivo. Finally, we showed that T cells contribute to in vivo tumor growth control in the presence of entinostat and trametinib combination treatment. Together, our findings reveal that HDAC3 is a druggable endogenous repressor of T cell recruitment into Kras mutant lung tumors.

Linoleoyl-lysophosphatidylcholine suppresses immune-related adverse events due to immune checkpoint blockade.

Immune related adverse events (irAEs) after immune checkpoint blockade (ICB) therapy occur in a significant proportion of cancer patients. To date, the circulating mediators of ICB-irAEs remain poorly understood. Using non-targeted mass spectrometry, here we identify the circulating bio-active lipid linoleoyl-lysophosphatidylcholine (LPC 18:2) as a modulator of ICB-irAEs. In three independent human studies of ICB treatment for solid tumor, loss of circulating LPC 18:2 preceded the development of severe irAEs across multiple organ systems. In both healthy humans and severe ICB-irAE patients, low LPC 18:2 was found to correlate with high blood neutrophilia. Reduced LPC 18:2 biosynthesis was confirmed in preclinical ICB-irAE models, and LPC 18:2 supplementation in vivo suppressed neutrophilia and tissue inflammation without impacting ICB anti-tumor response. Results indicate that circulating LPC 18:2 suppresses human ICB-irAEs, and LPC 18:2 supplementation may improve ICB outcomes by preventing severe inflammation while maintaining anti-tumor immunity.

ACLY and ACSS2 link nutrient-dependent chromatin accessibility to CD8 T cell effector responses.

Coordination of cellular metabolism is essential for optimal T cell responses. Here, we identify cytosolic acetyl-CoA production as an essential metabolic node for CD8 T cell function in vivo. We show that CD8 T cell responses to infection depend on acetyl-CoA derived from citrate via the enzyme ATP citrate lyase (ACLY). However, ablation of ACLY triggers an alternative, acetate-dependent pathway for acetyl-CoA production mediated by acyl-CoA synthetase short-chain family member 2 (ACSS2). Mechanistically, acetate fuels both the TCA cycle and cytosolic acetyl-CoA production, impacting T cell effector responses, acetate-dependent histone acetylation, and chromatin accessibility at effector gene loci. When ACLY is functional, ACSS2 is not required, suggesting acetate is not an obligate metabolic substrate for CD8 T cell function. However, loss of ACLY renders CD8 T cells dependent on acetate (via ACSS2) to maintain acetyl-CoA production and effector function. Together, ACLY and ACSS2 coordinate cytosolic acetyl-CoA production in CD8 T cells to maintain chromatin accessibility and T cell effector function.

ARID1A suppresses R-loop-mediated STING-type I interferon pathway activation of anti-tumor immunity.

Clinical trials have identified ARID1A mutations as enriched among patients who respond favorably to immune checkpoint blockade (ICB) in several solid tumor types independent of microsatellite instability. We show that ARID1A loss in murine models is sufficient to induce anti-tumor immune phenotypes observed in ARID1A mutant human cancers, including increased CD8+ T cell infiltration and cytolytic activity. ARID1A-deficient cancers upregulated an interferon (IFN) gene expression signature, the ARID1A-IFN signature, associated with increased R-loops and cytosolic single-stranded DNA (ssDNA). Overexpression of the R-loop resolving enzyme, RNASEH2B, or cytosolic DNase, TREX1, in ARID1A-deficient cells prevented cytosolic ssDNA accumulation and ARID1A-IFN gene upregulation. Further, the ARID1A-IFN signature and anti-tumor immunity were driven by STING-dependent type I IFN signaling, which was required for improved responsiveness of ARID1A mutant tumors to ICB treatment. These findings define a molecular mechanism underlying anti-tumor immunity in ARID1A mutant cancers.

EGFR-driven lung adenocarcinomas coopt alveolar macrophage metabolism and function to support EGFR signaling and growth.

The limited efficacy of currently approved immunotherapies in EGFR-driven lung adenocarcinoma (LUAD) underscores the need to better understand alternative mechanisms governing local immunosuppression to fuel novel therapies. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophage (TA-AM) proliferation which supports tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF-PPARγ signaling and inhibition of airway GM-CSF or PPARγ in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPARγ in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases proinflammatory immune responses. These results reveal new therapeutic combinations for immunotherapy resistant EGFR-mutant LUADs and demonstrate how cancer cells can metabolically co-opt TA-AMs through GM-CSF-PPARγ signaling to provide nutrients that promote oncogenic signaling and growth.

EGFR-Driven Lung Adenocarcinomas Co-opt Alveolar Macrophage Metabolism and Function to Support EGFR Signaling and Growth.

The limited efficacy of currently approved immunotherapies in EGFR-driven lung adenocarcinoma (LUAD) underscores the need to better understand alternative mechanisms governing local immunosuppression to fuel novel therapies. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophage (TA-AM) proliferation, which supports tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF-PPARγ signaling and inhibition of airway GM-CSF or PPARγ in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPARγ in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases proinflammatory immune responses. These results reveal new therapeutic combinations for immunotherapy-resistant EGFR-mutant LUADs and demonstrate how cancer cells can metabolically co-opt TA-AMs through GM-CSF-PPARγ signaling to provide nutrients that promote oncogenic signaling and growth. SIGNIFICANCE: Alternate strategies harnessing anticancer innate immunity are required for lung cancers with poor response rates to T cell-based immunotherapies. This study identifies a targetable, mutually supportive, metabolic relationship between macrophages and transformed epithelium, which is exploited by tumors to obtain metabolic and immunologic support to sustain proliferation and oncogenic signaling.

A bedside to bench study of anti-PD-1, anti-CD40, and anti-CSF1R indicates that more is not necessarily better.

BACKGROUND: Stimulating inflammatory tumor associated macrophages can overcome resistance to PD-(L)1 blockade. We previously conducted a phase I trial of cabiralizumab (anti-CSF1R), sotigalimab (CD40-agonist) and nivolumab. Our current purpose was to study the activity and cellular effects of this three-drug regimen in anti-PD-1-resistant melanoma. METHODS: We employed a Simon's two-stage design and analyzed circulating immune cells from patients treated with this regimen for treatment-related changes. We assessed various dose levels of anti-CSF1R in murine melanoma models and studied the cellular and molecular effects. RESULTS: Thirteen patients were enrolled in the first stage. We observed one (7.7%) confirmed and one (7.7%) unconfirmed partial response, 5 patients had stable disease (38.5%) and 6 disease progression (42.6%). We elected not to proceed to the second stage. CyTOF analysis revealed a reduction in non-classical monocytes. Patients with prolonged stable disease or partial response who remained on study for longer had increased markers of antigen presentation after treatment compared to patients whose disease progressed rapidly. In a murine model, higher anti-CSF1R doses resulted in increased tumor growth and worse survival. Using single-cell RNA-sequencing, we identified a suppressive monocyte/macrophage population in murine tumors exposed to higher doses. CONCLUSIONS: Higher anti-CSF1R doses are inferior to lower doses in a preclinical model, inducing a suppressive macrophage population, and potentially explaining the disappointing results observed in patients. While it is impossible to directly infer human doses from murine studies, careful intra-species evaluation can provide important insight. Cabiralizumab dose optimization is necessary for this patient population with limited treatment options. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03502330.

CD8+ T cells in the cancer-immunity cycle.

CD8+ T cells are end effectors of cancer immunity. Most forms of effective cancer immunotherapy involve CD8+ T cell effector function. Here, we review the current understanding of T cell function in cancer, focusing on key CD8+ T cell subtypes and states. We discuss factors that influence CD8+ T cell differentiation and function in cancer through a framework that incorporates the classic three-signal model and a fourth signal-metabolism-and also consider the impact of the tumor microenvironment from a T cell perspective. We argue for the notion of immunotherapies as "pro-drugs" that act to augment or modulate T cells, which ultimately serve as the drug in vivo, and for the importance of overall immune health in cancer treatment and prevention. The progress in understanding T cell function in cancer has and will continue to improve harnessing of the immune system across broader tumor types to benefit more patients.

Memory T Cells in the Immunoprevention of Cancer: A Switch from Therapeutic to Prophylactic Approaches.

Cancer immunoprevention, the engagement of the immune system to prevent cancer, is largely overshadowed by therapeutic approaches to treating cancer after detection. Vaccines or, alternatively, the utilization of genetically engineered memory T cells could be methods of engaging and creating cancer-specific T cells with superb memory, lenient activation requirements, potent antitumor cytotoxicity, tumor surveillance, and resilience against immunosuppressive factors in the tumor microenvironment. In this review we analyze memory T cell subtypes based on their potential utility in cancer immunoprevention with regard to longevity, localization, activation requirements, and efficacy in fighting cancers. A particular focus is on how both tissue-resident memory T cells and stem memory T cells could be promising subtypes for engaging in immunoprevention.

Manipulating mitochondrial electron flow enhances tumor immunogenicity.

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.

The ß1-adrenergic receptor links sympathetic nerves to T cell exhaustion.

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 exhaustion1. 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 ß1-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 ß1-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, ß-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 patients2,3, 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 ß-adrenergic signalling in CD8+ T cells rejuvenates anti-tumour functions.

CTLA-4 blockade induces a microglia-Th1 cell partnership that stimulates microglia phagocytosis and anti-tumor function in glioblastoma.

The limited efficacy of immunotherapies against glioblastoma underscores the urgency of better understanding immunity in the central nervous system. We found that treatment with αCTLA-4, but not αPD-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. αCTLA-4 treatment increased frequencies of intratumoral IFNγ-producing CD4+ T cells, and IFNγ blockade negated the therapeutic impact of αCTLA-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 IFNγ-dependent microglia activation and phagocytosis via the AXL/MER tyrosine kinase receptors, which were necessary for tumor suppression. Thus, αCTLA-4 blockade in mesenchymal-like glioblastoma promotes a CD4+ T cell-microglia circuit wherein IFNγ triggers microglia activation and phagocytosis and microglia in turn act as antigen-presenting cells fueling the CD4+ T cell response.

Combinatorial Immunotherapy with Agonistic CD40 Activates Dendritic Cells to Express IL12 and Overcomes PD-1 Resistance.

Checkpoint inhibitors have revolutionized cancer treatment, but resistance remains a significant clinical challenge. Myeloid cells within the tumor microenvironment can modulate checkpoint resistance by either supporting or suppressing adaptive immune responses. Using an anti-PD-1-resistant mouse melanoma model, we show that targeting the myeloid compartment via CD40 activation and CSF1R blockade in combination with anti-PD-1 results in complete tumor regression in a majority of mice. This triple therapy combination was primarily CD40 agonist-driven in the first 24 hours after therapy and showed a similar systemic cytokine profile in human patients as was seen in mice. Functional single-cell cytokine secretion profiling of dendritic cells (DC) using a novel microwell assay identified a CCL22+CCL5+ IL12-secreting DC subset as important early-stage effectors of triple therapy. CD4+ and CD8+ T cells are both critical effectors of treatment, and systems analysis of single-cell RNA sequencing data supported a role for DC-secreted IL12 in priming T-cell activation and recruitment. Finally, we showed that treatment with a novel IL12 mRNA therapeutic alone was sufficient to overcome PD-1 resistance and cause tumor regression. Overall, we conclude that combining myeloid-based innate immune activation and enhancement of adaptive immunity is a viable strategy to overcome anti-PD-1 resistance.

Canonical BAF complex activity shapes the enhancer landscape that licenses CD8+ T cell effector and memory fates.

CD8+ T cells provide host protection against pathogens by differentiating into distinct effector and memory cell subsets, but how chromatin is site-specifically remodeled during their differentiation is unclear. Due to its critical role in regulating chromatin and enhancer accessibility through its nucleosome remodeling activities, we investigated the role of the canonical BAF (cBAF) chromatin remodeling complex in antiviral CD8+ T cells during infection. ARID1A, a subunit of cBAF, was recruited early after activation and established de novo open chromatin regions (OCRs) at enhancers. Arid1a deficiency impaired the opening of thousands of activation-induced enhancers, leading to loss of TF binding, dysregulated proliferation and gene expression, and failure to undergo terminal effector differentiation. Although Arid1a was dispensable for circulating memory cell formation, tissue-resident memory (Trm) formation was strongly impaired. Thus, cBAF governs the enhancer landscape of activated CD8+ T cells that orchestrates TF recruitment and activity and the acquisition of specific effector and memory differentiation states.

EGFR + lung adenocarcinomas coopt alveolar macrophage metabolism and function to support EGFR signaling and growth.

The limited efficacy of currently approved immunotherapies in EGFR-mutant lung adenocarcinoma (LUAD) underscores the need to better understand mechanisms governing local immunosuppression. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophages (TA-AM) to proliferate and support tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF-PPARγ signaling and inhibition of airway GM-CSF or PPARγ in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPARγ in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases T cell effector functions. These results reveal new therapeutic combinations for immunotherapy resistant EGFR-mutant LUADs and demonstrate how such cancer cells can metabolically co-opt TA-AMs through GM-CSF-PPARγ signaling to provide nutrients that promote oncogenic signaling and growth.

Immunometabolism at the crossroads of obesity and cancer-a Keystone Symposia report.

Immunometabolism considers the relationship between metabolism and immunity. Typically, researchers focus on either the metabolic pathways within immune cells that affect their function or the impact of immune cells on systemic metabolism. A more holistic approach that considers both these viewpoints is needed. On September 5-8, 2022, experts in the field of immunometabolism met for the Keystone symposium "Immunometabolism at the Crossroads of Obesity and Cancer" to present recent research across the field of immunometabolism, with the setting of obesity and cancer as an ideal example of the complex interplay between metabolism, immunity, and cancer. Speakers highlighted new insights on the metabolic links between tumor cells and immune cells, with a focus on leveraging unique metabolic vulnerabilities of different cell types in the tumor microenvironment as therapeutic targets and demonstrated the effects of diet, the microbiome, and obesity on immune system function and cancer pathogenesis and therapy. Finally, speakers presented new technologies to interrogate the immune system and uncover novel metabolic pathways important for immunity.

Immunoediting instructs tumor metabolic reprogramming to support immune evasion.

Immunoediting sculpts immunogenicity and thwarts host anti-tumor responses in tumor cells during tumorigenesis; however, it remains unknown whether metabolic programming of tumor cells can be guided by immunosurveillance. Here, we report that T cell-mediated immunosurveillance in early-stage tumorigenesis instructs c-Myc upregulation and metabolic reprogramming in tumor cells. This previously unexplored tumor-immune interaction is controlled by non-canonical interferon gamma (IFNγ)-STAT3 signaling and supports tumor immune evasion. Our findings uncover that immunoediting instructs deregulated bioenergetic programs in tumor cells to empower them to disarm the T cell-mediated immunosurveillance by imposing metabolic tug-of-war between tumor and infiltrating T cells and forming the suppressive tumor microenvironment.

Multiomics atlas-assisted discovery of transcription factors enables specific cell state programming.

The same types of cells can assume diverse states with varying functionalities. Effective cell therapy can be achieved by specifically driving a desirable cell state, which requires the elucidation of key transcription factors (TFs). Here, we integrated epigenomic and transcriptomic data at the systems level to identify TFs that define different CD8 + T cell states in an unbiased manner. These TF profiles can be used for cell state programming that aims to maximize the therapeutic potential of T cells. For example, T cells can be programmed to avoid a terminal exhaustion state (Tex Term ), a dysfunctional T cell state that is often found in tumors or chronic infections. However, Tex Term exhibits high similarity with the beneficial tissue-resident memory T states (T RM ) in terms of their locations and transcription profiles. Our bioinformatic analysis predicted Zscan20 , a novel TF, to be uniquely active in Tex Term . Consistently, Zscan20 knock-out thwarted the differentiation of Tex Term in vivo , but not that of T RM . Furthermore, perturbation of Zscan20 programs T cells into an effector-like state that confers superior tumor and virus control and synergizes with immune checkpoint therapy. We also identified Jdp2 and Nfil3 as powerful Tex Term drivers. In short, our multiomics-based approach discovered novel TFs that enhance anti-tumor immunity, and enable highly effective cell state programming. One sentence summary: Multiomics atlas enables the systematic identification of cell-state specifying transcription factors for therapeutic cell state programming.

Clonal lineage tracing reveals mechanisms skewing CD8+ T cell fate decisions in chronic infection.

Although recent evidence demonstrates heterogeneity among CD8+ T cells during chronic infection, developmental relationships and mechanisms underlying their fate decisions remain incompletely understood. Using single-cell RNA and TCR sequencing, we traced the clonal expansion and differentiation of CD8+ T cells during chronic LCMV infection. We identified immense clonal and phenotypic diversity, including a subset termed intermediate cells. Trajectory analyses and infection models showed intermediate cells arise from progenitor cells before bifurcating into terminal effector and exhausted subsets. Genetic ablation experiments identified that type I IFN drives exhaustion through an IRF7-dependent mechanism, possibly through an IFN-stimulated subset bridging progenitor and exhausted cells. Conversely, Zeb2 was critical for generating effector cells. Intriguingly, some T cell clones exhibited lineage bias. Mechanistically, we identified that TCR avidity correlates with an exhausted fate, whereas SHP-1 selectively restricts low-avidity effector cell accumulation. Thus, our work elucidates novel mechanisms underlying CD8+ T cell fate determination during persistent infection and suggests two potential pathways leading to exhaustion.

Early Chromatin Remodeling Events in Acutely Stimulated CD8+ T Cells.

T cells undergo extensive chromatin remodeling over several days following stimulation through the T cell receptor. However, the kinetics and gene loci targeted by early remodeling events within the first 24 hours of T cell priming to orchestrate effector differentiation have not been well described. We identified that chromatin accessibility is rapidly and extensively remodeled within 1 hour of stimulation of naïve CD8+ T cells, leading to increased global chromatin accessibility at many effector T cell-associated genes that are enriched for AP-1, early growth response (EGR), and nuclear factor of activated T cells (NFAT) binding sites, but this short duration of stimulation is insufficient for commitment to clonal expansion in vivo. Sustained 24-hour stimulation led to further chromatin remodeling and was sufficient to enable clonal expansion. These data suggest that the duration of antigen receptor signaling is intimately coupled to chromatin remodeling and activation of genes involved in effector cell differentiation and highlight a potential mechanism that helps CD8+ T cells discriminate between foreign- and self-antigens.