Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis.

T cells are critical effectors of cancer immunotherapies, but little is known about their gene expression programs in diffuse gliomas. Here, we leverage single-cell RNA sequencing (RNA-seq) to chart the gene expression and clonal landscape of tumor-infiltrating T cells across 31 patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma and IDH mutant glioma. We identify potential effectors of anti-tumor immunity in subsets of T cells that co-express cytotoxic programs and several natural killer (NK) cell genes. Analysis of clonally expanded tumor-infiltrating T cells further identifies the NK gene KLRB1 (encoding CD161) as a candidate inhibitory receptor. Accordingly, genetic inactivation of KLRB1 or antibody-mediated CD161 blockade enhances T cell-mediated killing of glioma cells in vitro and their anti-tumor function in vivo. KLRB1 and its associated transcriptional program are also expressed by substantial T cell populations in other human cancers. Our work provides an atlas of T cells in gliomas and highlights CD161 and other NK cell receptors as immunotherapy targets.

Gut microbiome-derived metabolites modulate intestinal epithelial cell damage and mitigate graft-versus-host disease.

The effect of alterations in intestinal microbiota on microbial metabolites and on disease processes such as graft-versus-host disease (GVHD) is not known. Here we carried out an unbiased analysis to identify previously unidentified alterations in gastrointestinal microbiota-derived short-chain fatty acids (SCFAs) after allogeneic bone marrow transplant (allo-BMT). Alterations in the amount of only one SCFA, butyrate, were observed only in the intestinal tissue. The reduced butyrate in CD326(+) intestinal epithelial cells (IECs) after allo-BMT resulted in decreased histone acetylation, which was restored after local administration of exogenous butyrate. Butyrate restoration improved IEC junctional integrity, decreased apoptosis and mitigated GVHD. Furthermore, alteration of the indigenous microbiota with 17 rationally selected strains of high butyrate-producing Clostridia also decreased GVHD. These data demonstrate a heretofore unrecognized role of microbial metabolites and suggest that local and specific alteration of microbial metabolites has direct salutary effects on GVHD target tissues and can mitigate disease severity.

Targeting of the CD161 inhibitory receptor enhances T-cell-mediated immunity against hematological malignancies.

ABSTRACT: The CD161 inhibitory receptor is highly upregulated by tumor-infiltrating T cells in multiple human solid tumor types, and its ligand, CLEC2D, is expressed by both tumor cells and infiltrating myeloid cells. Here, we assessed the role of the CD161 receptor in hematological malignancies. Systematic analysis of CLEC2D expression using the Cancer Cell Line Encyclopedia revealed that CLEC2D messenger RNA was most abundant in hematological malignancies, including B-cell and T-cell lymphomas as well as lymphocytic and myelogenous leukemias. CLEC2D protein was detected by flow cytometry on a panel of cell lines representing a diverse set of hematological malignancies. We, therefore, used yeast display to generate a panel of high-affinity, fully human CD161 monoclonal antibodies (mAbs) that blocked CLEC2D binding. These mAbs were specific for CD161 and had a similar affinity for human and nonhuman primate CD161, a property relevant for clinical translation. A high-affinity CD161 mAb enhanced key aspects of T-cell function, including cytotoxicity, cytokine production, and proliferation, against B-cell lines originating from patients with acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and Burkitt lymphoma. In humanized mouse models, this CD161 mAb enhanced T-cell-mediated immunity, resulting in a significant survival benefit. Single cell RNA-seq data demonstrated that CD161 mAb treatment enhanced expression of cytotoxicity genes by CD4 T cells as well as a tissue-residency program by CD4 and CD8 T cells that is associated with favorable survival outcomes in multiple human cancer types. These fully human mAbs, thus, represent potential immunotherapy agents for hematological malignancies.

Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial.

Neoantigens, which are derived from tumour-specific protein-coding mutations, are exempt from central tolerance, can generate robust immune responses1,2 and can function as bona fide antigens that facilitate tumour rejection3. Here we demonstrate that a strategy that uses multi-epitope, personalized neoantigen vaccination, which has previously been tested in patients with high-risk melanoma4-6, is feasible for tumours such as glioblastoma, which typically have a relatively low mutation load1,7 and an immunologically 'cold' tumour microenvironment8. We used personalized neoantigen-targeting vaccines to immunize patients newly diagnosed with glioblastoma following surgical resection and conventional radiotherapy in a phase I/Ib study. Patients who did not receive dexamethasone-a highly potent corticosteroid that is frequently prescribed to treat cerebral oedema in patients with glioblastoma-generated circulating polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses that were enriched in a memory phenotype and showed an increase in the number of tumour-infiltrating T cells. Using single-cell T cell receptor analysis, we provide evidence that neoantigen-specific T cells from the peripheral blood can migrate into an intracranial glioblastoma tumour. Neoantigen-targeting vaccines thus have the potential to favourably alter the immune milieu of glioblastoma.

SAG/Rbx2-Dependent Neddylation Regulates T-Cell Responses.

Neddylation is a crucial post-translational modification that depends on the E3 cullin ring ligase (CRL). The E2-adapter component of the CRL, sensitive to apoptosis gene (SAG), is critical for the function of CRL-mediated ubiquitination; thus, the deletion of SAG regulates neddylation. We examined the role of SAG-dependent neddylation in T-cell-mediated immunity using multiple approaches: a novel T-cell-specific, SAG genetic knockout (KO) and chemical inhibition with small-molecule MLN4924. The KO animals were viable and showed phenotypically normal mature T-cell development. However, in vitro stimulation of KO T cells revealed significantly decreased activation, proliferation, and T-effector cytokine release, compared with WT. Using in vivo clinically relevant models of allogeneic bone marrow transplantation also demonstrated reduced proliferation and effector cytokine secretion associated with markedly reduced graft-versus-host disease. Similar in vitro and in vivo results were observed with the small-molecule inhibitor of neddylation, MLN4924. Mechanistic studies demonstrated that SAG-mediated effects in T cells were concomitant with an increase in suppressor of cytokine signaling, but not NF-κB translocation. Our studies suggest that SAG is a novel molecular target that regulates T-cell responses and that inhibiting neddylation with the clinically available small-molecule MLN4924 may represent a novel strategy to mitigate T-cell-mediated immunopathologies, such as graft-versus-host disease.

Sugar polymers exacerbate lung GVHD.

In this issue of Blood, Uryu et al demonstrate that recognition of a fungal cell wall component, α-mannan (Mn), an Mn (sugar) polymer, by the C-type lectin receptor Dectin-2 on host macrophages leads to a lung chemokine environment conducive to donor T helper (Th)17 accumulation resulting in severe pulmonary graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT). These findings shed new light on a long-standing gap in our understanding of the mechanistic link between infections, specifically fungal, and GVHD severity following allo-HSCT.

BET bromodomain inhibition suppresses graft-versus-host disease after allogeneic bone marrow transplantation in mice.

Acute graft-versus-host disease (GVHD) is the major obstacle of allogeneic bone marrow transplantation (BMT). Bromodomain and extra-terminal (BET) protein inhibitors selectively block acetyl-binding pockets of the bromodomains and modulate histone acetylation. Here, we report that inhibition of BET bromodomain (BRD) proteins with I-BET151 alters cytokine expression in dendritic cells (DCs) and T cells, including surface costimulatory molecules, in vitro and in vivo cytokine secretion, and expansion. Mechanistic studies with I-BET151 and JQ1, another inhibitor, demonstrate that these effects could be from disruption of association between BRD4 and acetyl-310 RelA of nuclear factor kappa B. Short-term administration early during BMT reduced GVHD severity and improved mortality in two different allogeneic BMT models but retained sufficient graft-versus-tumor effect. Thus inhibiting BRD proteins may serve as a novel approach for preventing GVHD.

Siglec-G-CD24 axis controls the severity of graft-versus-host disease in mice.

Activation of sialic-acid-binding immunoglobulin-like lectin-G (Siglec-G) by noninfectious damage-associated molecular patterns controls innate immune responses. However, whether it also regulates T-cell-mediated adaptive immune responses is not known. Graft-versus-host reaction is a robust adaptive immune response caused by allogeneic hematopoietic cell transplantation that have been activated by antigen-presenting cells (APCs) in the context of damaged host tissues following allogeneic hematopoietic cell transplantation. The role of infectious and noninfectious pattern recognition receptor-mediated activation in the induction and aggravation of graft-versus-host disease (GVHD) is being increasingly appreciated. But the role of pathways that control innate immune responses to noninfectious stimuli in modulating GVHD has heretofore not been recognized. We report that Siglec-G expression on host APCs, specifically on hematopoietic cells, negatively regulates GVHD in multiple clinically relevant murine models. Mechanistic studies with various relevant Siglec-G and CD24 knockout mice and chimeric animals, along with rescue experiments with novel CD24 fusion protein demonstrate that enhancing the interaction between Siglec-G on host APCs with CD24 on donor T cells attenuates GVHD. Taken together, our data demonstrate that Siglec-G-CD24 axis, controls the severity of GVHD and suggest that enhancing this interaction may represent a novel strategy for mitigating GVHD.

Neddylation plays an important role in the regulation of murine and human dendritic cell function.

Posttranslational protein modifications (PTMs) are necessary for cells to function properly. The role of PTMs in regulating immune responses, specifically those mediated by dendritic cells (DCs), which are critical for both innate and adaptive immunity, is not well understood. Utilizing multiple but complementary approaches, we determined the role of an important but less understood type of PTM, namely, neddylation, in regulating DC functions. Inhibition of neddylation suppressed the release of proinflammatory cytokines by DCs in response to Toll-like receptor, nucleotide oligomerization domain-like receptor, and noninfectious CD40L stimulation. These effects were more profound than those mediated by the proteasome inhibitor bortezomib or a commonly used antiinflammatory agent, dexamethasone. Targeting neddylation also suppressed the ability of DCs to stimulate murine allogeneic T cells in vitro and in vivo and human allogeneic T-cell responses in vitro. Mechanistic studies demonstrated that inhibition of neddylation reduced both canonical and noncanonical nuclear factor-κB (NF-κB) activity. Neddylation inhibition prevented the degradation of inhibitor-κB and thus reduced the translocation and activation of NF-κB, but without perturbation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Thus, blocking neddylation could be a novel strategy for mitigating immune-mediated disease processes.

Host-derived CD8+ dendritic cells are required for induction of optimal graft-versus-tumor responses after experimental allogeneic bone marrow transplantation.

The graft-versus-tumor (GVT) effect after allogeneic hematopoietic cell transplantation (allo-HCT) represents an effective form of immunotherapy against many malignancies. Meaningful separation of the potentially curative GVT responses from graft-versus-host disease (GVHD), the most serious toxicity following T-cell replete allo-HCT, has been an elusive goal. GVHD is initiated by alloantigens, although both alloantigens and tumor-specific antigens (TSAs) initiate GVT responses. Emerging data have illuminated a role for antigen-presenting cells (APCs) in inducing alloantigen-specific responses. By using multiple clinically relevant murine models, we show that a specific subset of host-derived APCs-CD8(+) dendritic cells (DCs)-enhances TSA responses and is required for optimal induction of GVT. Stimulation of TLR3, which among host hematopoietic APC subsets is predominantly expressed on CD8(+) DCs, enhanced GVT without exacerbating GVHD. Thus, strategies that modulate host APC subsets without direct manipulation of donor T cells could augment GVT responses and enhance the efficacy of allo-HCT.

PU.1-dependent transcriptional regulation of miR-142 contributes to its hematopoietic cell-specific expression and modulation of IL-6.

MicroRNAs (miRs) have emerged as critical modulators of immune responses, but little is known about their transcriptional regulation and tissue specificity. miR-142 is specifically expressed in hematopoietic tissues and plays an important role in regulating immunity. In this study we identified the key transcriptional elements for regulation of miR-142 and its impact on TLR4-mediated expression of IL-6. The PU.1, C/EBPß, and Runx1 transcription factor binding sites are conserved and constitutively occupied by the respective transcription factors in the miR-142 gene promoter only in the hematopoietic cells. Specific knockdown experiments in hematopoietic cells and rescue experiments in nonhematopoietic cells show that PU.1 is critical for miR-142 gene expression and that it synergizes with Runx1, C/EBPß, and CBFß. Furthermore, TLR4 stimulation enhanced miR-155 whereas experiments with knockdown and mimic expression of miR-155 demonstrated that miR-155 negatively regulates miR-142-3p expression by targeting PU.1. Thus, TLR4 stimulation represses PU.1, resulting in downregulation of miR-142 and increased expression of IL-6. These results collectively reveal the direct cis-acting sequences of miR-142 specific promoter and that transcription factor PU.1 is necessary for its exclusive expression in hematopoietic cells and regulation of IL-6.

IL2 Targeted to CD8+ T Cells Promotes Robust Effector T-cell Responses and Potent Antitumor Immunity.

IL2 signals pleiotropically on diverse cell types, some of which contribute to therapeutic activity against tumors, whereas others drive undesired activity, such as immunosuppression or toxicity. We explored the theory that targeting of IL2 to CD8+ T cells, which are key antitumor effectors, could enhance its therapeutic index. To this aim, we developed AB248, a CD8 cis-targeted IL2 that demonstrates over 500-fold preference for CD8+ T cells over natural killer and regulatory T cells (Tregs), which may contribute to toxicity and immunosuppression, respectively. AB248 recapitulated IL2's effects on CD8+ T cells in vitro and induced selective expansion of CD8+T cells in primates. In mice, an AB248 surrogate demonstrated superior antitumor activity and enhanced tolerability as compared with an untargeted IL2Rßγ agonist. Efficacy was associated with the expansion and phenotypic enhancement of tumor-infiltrating CD8+ T cells, including the emergence of a "better effector" population. These data support the potential utility of AB248 in clinical settings. Significance: The full potential of IL2 therapy remains to be unlocked. We demonstrate that toxicity can be decoupled from antitumor activity in preclinical models by limiting IL2 signaling to CD8+ T cells, supporting the development of CD8+ T cell-selective IL2 for the treatment of cancer. See related article by Kaptein et al. p. 1226.

Influence of donor microbiota on the severity of experimental graft-versus-host-disease.

The link between microbial flora and the shaping of immune responses is being increasingly appreciated, and recent data have uncovered a role for recipient microbiota in the severity of graft-versus-host disease (GVHD). The impact of donor microbiota on T cell-mediated alloresponses and GVHD is not known, however. Using multiple clinically relevant murine models, we analyzed the effect of donor microbiota on the severity of GVHD induced by T cells from specific pathogen-free and germ-free donors, and found that donor microbiota does not alter the expansion or differentiation of alloreactive T cells or the severity of GVHD.

Systematic Interrogation of Tumor Cell Resistance to Chimeric Antigen Receptor T-cell Therapy in Pancreatic Cancer.

Chimeric antigen receptor (CAR) T-cell therapy can lead to dramatic clinical responses in B-cell malignancies. However, early clinical trials with CAR T-cell therapy in non-B-cell malignancies have been disappointing to date, suggesting that tumor-intrinsic features contribute to resistance. To investigate tumor-intrinsic modes of resistance, we performed genome scale CRISPR-Cas9 screens in mesothelin (MSLN)-expressing pancreatic cancer cells. Co-culture with MSLN-targeting CAR T cells identified both antigen-dependent and antigen-independent modes of resistance. In particular, loss of the majority of the genes involved in the pathway responsible for GPI-anchor biosynthesis and attachment abrogated the ability of CAR T cells to target pancreatic cancer cells, suggesting that disruption of this pathway may permit MSLN CAR T-cell evasion in the clinic. Antigen-independent mediators of CAR T-cell response included members of the death receptor pathway as well as genes that regulate tumor transcriptional responses, including TFAP4 and INTS12. TFAP4-mediated CAR T resistance depended on the NFκB transcription factor p65, indicating that tumor resistance to CAR T-cell therapy likely involves alterations in tumor-intrinsic states. Overall, this study uncovers multiple antigen-dependent and -independent mechanisms of CAR T-cell evasion by pancreatic cancer, paving the way for overcoming resistance in this disease that is notoriously refractory to immunotherapy. SIGNIFICANCE: The identification and validation of key determinants of CAR T-cell response in pancreatic cancer provide insights into the landscape of tumor cell intrinsic resistance mechanisms and into approaches to improve therapeutic efficacy.

BAF Complex Maintains Glioma Stem Cells in Pediatric H3K27M Glioma.

Diffuse midline gliomas are uniformly fatal pediatric central nervous system cancers that are refractory to standard-of-care therapeutic modalities. The primary genetic drivers are a set of recurrent amino acid substitutions in genes encoding histone H3 (H3K27M), which are currently undruggable. These H3K27M oncohistones perturb normal chromatin architecture, resulting in an aberrant epigenetic landscape. To interrogate for epigenetic dependencies, we performed a CRISPR screen and show that patient-derived H3K27M-glioma neurospheres are dependent on core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex. The BAF complex maintains glioma stem cells in a cycling, oligodendrocyte precursor cell-like state, in which genetic perturbation of the BAF catalytic subunit SMARCA4 (BRG1), as well as pharmacologic suppression, opposes proliferation, promotes progression of differentiation along the astrocytic lineage, and improves overall survival of patient-derived xenograft models. In summary, we demonstrate that therapeutic inhibition of the BAF complex has translational potential for children with H3K27M gliomas. SIGNIFICANCE: Epigenetic dysregulation is at the core of H3K27M-glioma tumorigenesis. Here, we identify the BRG1-BAF complex as a critical regulator of enhancer and transcription factor landscapes, which maintain H3K27M glioma in their progenitor state, precluding glial differentiation, and establish pharmacologic targeting of the BAF complex as a novel treatment strategy for pediatric H3K27M glioma. See related commentary by Beytagh and Weiss, p. 2730. See related article by Mo et al., p. 2906.

Integrin αvß6-TGFß-SOX4 Pathway Drives Immune Evasion in Triple-Negative Breast Cancer.

Cancer immunotherapy shows limited efficacy against many solid tumors that originate from epithelial tissues, including triple-negative breast cancer (TNBC). We identify the SOX4 transcription factor as an important resistance mechanism to T cell-mediated cytotoxicity for TNBC cells. Mechanistic studies demonstrate that inactivation of SOX4 in tumor cells increases the expression of genes in a number of innate and adaptive immune pathways important for protective tumor immunity. Expression of SOX4 is regulated by the integrin αvß6 receptor on the surface of tumor cells, which activates TGFß from a latent precursor. An integrin αvß6/8-blocking monoclonal antibody (mAb) inhibits SOX4 expression and sensitizes TNBC cells to cytotoxic T cells. This integrin mAb induces a substantial survival benefit in highly metastatic murine TNBC models poorly responsive to PD-1 blockade. Targeting of the integrin αvß6-TGFß-SOX4 pathway therefore provides therapeutic opportunities for TNBC and other highly aggressive human cancers of epithelial origin.

Interactions between cancer cells and immune cells drive transitions to mesenchymal-like states in glioblastoma.

The mesenchymal subtype of glioblastoma is thought to be determined by both cancer cell-intrinsic alterations and extrinsic cellular interactions, but remains poorly understood. Here, we dissect glioblastoma-to-microenvironment interactions by single-cell RNA sequencing analysis of human tumors and model systems, combined with functional experiments. We demonstrate that macrophages induce a transition of glioblastoma cells into mesenchymal-like (MES-like) states. This effect is mediated, both in vitro and in vivo, by macrophage-derived oncostatin M (OSM) that interacts with its receptors (OSMR or LIFR) in complex with GP130 on glioblastoma cells and activates STAT3. We show that MES-like glioblastoma states are also associated with increased expression of a mesenchymal program in macrophages and with increased cytotoxicity of T cells, highlighting extensive alterations of the immune microenvironment with potential therapeutic implications.

CARM1 Inhibition Enables Immunotherapy of Resistant Tumors by Dual Action on Tumor Cells and T Cells.

A number of cancer drugs activate innate immune pathways in tumor cells but unfortunately also compromise antitumor immune function. We discovered that inhibition of CARM1, an epigenetic enzyme and cotranscriptional activator, elicited beneficial antitumor activity in both cytotoxic T cells and tumor cells. In T cells, Carm1 inactivation substantially enhanced their antitumor function and preserved memory-like populations required for sustained antitumor immunity. In tumor cells, Carm1 inactivation induced a potent type 1 interferon response that sensitized resistant tumors to cytotoxic T cells. Substantially increased numbers of dendritic cells, CD8 T cells, and natural killer cells were present in Carm1-deficient tumors, and infiltrating CD8 T cells expressed low levels of exhaustion markers. Targeting of CARM1 with a small molecule elicited potent antitumor immunity and sensitized resistant tumors to checkpoint blockade. Targeting of this cotranscriptional regulator thus offers an opportunity to enhance immune function while simultaneously sensitizing resistant tumor cells to immune attack. SIGNIFICANCE: Resistance to cancer immunotherapy remains a major challenge. Targeting of CARM1 enables immunotherapy of resistant tumors by enhancing T-cell functionality and preserving memory-like T-cell populations within tumors. CARM1 inhibition also sensitizes resistant tumor cells to immune attack by inducing a tumor cell-intrinsic type 1 interferon response.This article is highlighted in the In This Issue feature, p. 1861.

Opposing immune and genetic mechanisms shape oncogenic programs in synovial sarcoma.

Synovial sarcoma (SyS) is an aggressive neoplasm driven by the SS18-SSX fusion, and is characterized by low T cell infiltration. Here, we studied the cancer-immune interplay in SyS using an integrative approach that combines single-cell RNA sequencing (scRNA-seq), spatial profiling and genetic and pharmacological perturbations. scRNA-seq of 16,872 cells from 12 human SyS tumors uncovered a malignant subpopulation that marks immune-deprived niches in situ and is predictive of poor clinical outcomes in two independent cohorts. Functional analyses revealed that this malignant cell state is controlled by the SS18-SSX fusion, is repressed by cytokines secreted by macrophages and T cells, and can be synergistically targeted with a combination of HDAC and CDK4/CDK6 inhibitors. This drug combination enhanced malignant-cell immunogenicity in SyS models, leading to induced T cell reactivity and T cell-mediated killing. Our study provides a blueprint for investigating heterogeneity in fusion-driven malignancies and demonstrates an interplay between immune evasion and oncogenic processes that can be co-targeted in SyS and potentially in other malignancies.