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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.

Although immune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein-1 (PD-1), can deliver durable antitumor effects, most patients with cancer fail to respond. Recent studies suggest that ICI efficacy correlates with a higher load of tumor-specific neoantigens and development of vitiligo in patients with melanoma. Here, we report that patients with low melanoma neoantigen burdens who responded to ICI had tumors with higher expression of pigmentation-related genes. Moreover, expansion of peripheral blood CD8+ T cell populations specific for melanocyte antigens was observed only in patients who responded to anti-PD-1 therapy, suggesting that ICI can promote breakdown of tolerance toward tumor-lineage self-antigens. In a mouse model of poorly immunogenic melanomas, spreading of epitope recognition toward wild-type melanocyte antigens was associated with markedly improved anti-PD-1 efficacy in two independent approaches: introduction of neoantigens by ultraviolet (UV) B radiation mutagenesis or the therapeutic combination of ablative fractional photothermolysis plus imiquimod. Complete responses against UV mutation-bearing tumors after anti-PD-1 resulted in protection from subsequent engraftment of melanomas lacking any shared neoantigens, as well as pancreatic adenocarcinomas forcibly overexpressing melanocyte-lineage antigens. Our data demonstrate that somatic mutations are sufficient to provoke strong antitumor responses after checkpoint blockade, but long-term responses are not restricted to these putative neoantigens. Epitope spreading toward T cell recognition of wild-type tumor-lineage self-antigens represents a common pathway for successful response to ICI, which can be evoked in neoantigen-deficient tumors by combination therapy with ablative fractional photothermolysis and imiquimod.

As part of the 2018 Immunotherapy Bridge congress (November 28-29, Naples, Italy), the Great Debate session featured counterpoint views from leading experts on four topical clinical issues in immunotherapy today. These were: the relative importance of adaptive versus innate immunity in the anti-cancer immune response; the merits of combination versus sequential immunotherapy regimens in the treatment of cancer; the advantages and disadvantages of murine models of cancer versus humans in order to evaluate immunotherapy; and whether or not mechanisms of resistance to immunotherapy differ between different cancers. Discussion of these important topics are summarised in this report.

Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to many fields of medicine as biomarkers and mediators of or treatments for disease. However, exosomes and small viruses fall below the detection limits of conventional flow cytometers due to the overlap of particle-associated scattered light signals with the detection of background instrument noise from diffusely scattered light. To identify, sort, and study distinct subsets of EVs and other nanoparticles, as individual particles, we developed nanoscale Fluorescence Analysis and Cytometric Sorting (nanoFACS) methods to maximise information and material that can be obtained with high speed, high resolution flow cytometers. This nanoFACS method requires analysis of the instrument background noise (herein defined as the "reference noise"). With these methods, we demonstrate detection of tumour cell-derived EVs with specific tumour antigens using both fluorescence and scattered light parameters. We further validated the performance of nanoFACS by sorting two distinct HIV strains to >95% purity and confirmed the viability (infectivity) and molecular specificity (specific cell tropism) of biological nanomaterials sorted with nanoFACS. This nanoFACS method provides a unique way to analyse and sort functional EV- and viral-subsets with preservation of vesicular structure, surface protein specificity and RNA cargo activity.

BackgroundAllergic asthma causes morbidity in many subjects, and novel precision-directed treatments would be valuable.ObjectiveWe sought to examine the role of a novel innate molecule, repulsive guidance molecule b (RGMb), in murine models of allergic asthma.MethodsIn models of allergic asthma using ovalbumin or cockroach allergen, mice were treated with anti-RGMb or control mAb and examined for airway inflammation and airway hyperreactivity (AHR), a cardinal feature of asthma. The mechanisms by which RGMb causes airways disease were also examined.ResultsWe found that blockade of RGMb by treatment with anti-RGMb mAb effectively blocked the development of airway inflammation and AHR. Importantly, blockade of RGMb completely blocked the development of airway inflammation and AHR, even if treatment occurred only during the challenge (effector) phase. IL-25 played an important role in these models of asthma because IL-25 receptor-deficient mice did not develop disease after sensitization and challenge with allergen. RGMb was expressed primarily by innate cells in the lungs, including bronchial epithelial cells (known producers of IL-25), activated eosinophils, and interstitial macrophages, which in the inflamed lung expressed the IL-25 receptor and produced IL-5 and IL-13. We also found that neogenin, the canonical receptor for RGMb, was expressed by interstitial macrophages and bronchial epithelial cells in the inflamed lung, suggesting that an innate RGMb-neogenin axis might modulate allergic asthma.ConclusionsThese results demonstrate an important role for a novel innate pathway in regulating type 2 inflammation in patients with allergic asthma involving RGMb and RGMb-expressing cells, such as interstitial macrophages and bronchial epithelial cells. Moreover, targeting this previously unappreciated innate pathway might provide an important treatment option for allergic asthma.

Anti-PD-1 immune checkpoint blockers can induce sustained clinical responses in cancer but how they function in vivo remains incompletely understood. Here, we combined intravital real-time imaging with single-cell RNA sequencing analysis and mouse models to uncover anti-PD-1 pharmacodynamics directly within tumors. We showed that effective antitumor responses required a subset of tumor-infiltrating dendritic cells (DCs), which produced interleukin 12 (IL-12). These DCs did not bind anti-PD-1 but produced IL-12 upon sensing interferon γ (IFN-γ) that was released from neighboring T cells. In turn, DC-derived IL-12 stimulated antitumor T cell immunity. These findings suggest that full-fledged activation of antitumor T cells by anti-PD-1 is not direct, but rather involves T cell:DC crosstalk and is licensed by IFN-γ and IL-12. Furthermore, we found that activating the non-canonical NF-κB transcription factor pathway amplified IL-12-producing DCs and sensitized tumors to anti-PD-1 treatment, suggesting a therapeutic strategy to improve responses to checkpoint blockade.

Therapeutic strategies that augment antiviral immunity and reduce the viral reservoir are critical to achieving durable remission of HIV. The coinhibitory receptor programmed death-1 (PD-1) regulates CD8+ T cell dysfunction during chronic HIV and SIV infections. We previously demonstrated that in vivo blockade of PD-1 during chronic SIV infection improves the function of antiviral CD8+ T cells and B cells. Here, we tested the immunological and virological effects of PD-1 blockade combined with antiretroviral therapy (ART) in rhesus macaques. Administration of anti-PD-1 antibody 10 days prior to ART initiation rapidly enhanced antiviral CD8+ T cell function and diminished IFN-stimulated genes. This resulted in faster viral suppression in plasma and better Th17 cell reconstitution in the rectal mucosa following ART initiation. PD-1 blockade during ART resulted in lower levels of cell-associated replication-competent virus. Following ART interruption, PD-1 antibody-treated animals showed markedly higher expansion of proliferating CXCR5+perforin+granzyme B+ effector CD8+ T cells and lower regulatory T cells that resulted in better control of viremia. Our results show that PD-1 blockade can be administered safely with ART to augment antiviral CD8+ T cell function and reduce the viral reservoir, leading to improved control of viral rebound after ART interruption.

T cell exhaustion is a state of T cell dysfunction associated with expression of programmed death 1 (PD-1). Exhausted [CD8.sup.+] T cells are maintained by self-renewing stem-like T cells that provide differentiated [TIM3.sup.+] cells, a part of which possesses effector-like properties. PD-1-targeted therapies enhance T cell response by promoting differentiation of stem- like T cells toward [TIM3.sup.+] cells, but the role of mTOR during T cell exhaustion remains elusive. Here, we showed that mTOR inhibition has distinct outcomes during the beginning of and after the establishment of chronic viral infection. Blocking mTOR during the T cell expansion phase enhanced the T cell response by causing accumulation of stem-like T cells, leading to improved efficacy of PD-1 immunotherapy; whereas, after exhaustion progressed, mTOR inhibition caused immunosuppression, characterized by decreased [TIM3.sup.+] cells and increased viral load with minimal changes in stem-like T cells. Mechanistically, a cell- intrinsic mTOR signal was vital for differentiation of stem-like T cells into the [TIM3.sup.+] state in the early and late phases of chronic infection as well as during PD-1 immunotherapy. Thus, PD-1 blockade worked after cessation of mTOR inhibition, but simultaneous treatment failed to induce functional [TIM3.sup.+] cells, reducing efficacy of PD-1 immunotherapy. Our data demonstrate that mTOR regulates T cell exhaustion and have important implications for combination cancer therapies with PD-1 blockade., Introduction T cell exhaustion is a state of T cell dysfunction; it is characterized by poor effector function, impaired proliferative capacity, and expression of multiple inhibitory receptors, most notably programmed [...]

Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1-/- donors. PD-L1-deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1-/- donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell-mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD.

STK11/LKB1 is among the most commonly inactivated tumor suppressors in non-small cell lung cancer (NSCLC), especially in tumors harboring KRAS mutations. Many oncogenes promote immune escape, undermining the effectiveness of immunotherapies, but it is unclear whether the inactivation of tumor suppressor genes, such as STK11/LKB1, exerts similar effects. In this study, we investigated the consequences of STK11/LKB1 loss on the immune microenvironment in a mouse model of KRAS-driven NSCLC. Genetic ablation of STK11/LKB1 resulted in accumulation of neutrophils with T-cell-suppressive effects, along with a corresponding increase in the expression of T-cell exhaustion markers and tumor-promoting cytokines. The number of tumor-infiltrating lymphocytes was also reduced in LKB1-deficient mouse and human tumors. Furthermore, STK11/LKB1-inactivating mutations were associated with reduced expression of PD-1 ligand PD-L1 in mouse and patient tumors as well as in tumor-derived cell lines. Consistent with these results, PD-1-targeting antibodies were ineffective against Lkb1-deficient tumors. In contrast, treating Lkb1-deficient mice with an IL6-neutralizing antibody or a neutrophil-depleting antibody yielded therapeutic benefits associated with reduced neutrophil accumulation and proinflammatory cytokine expression. Our findings illustrate how tumor suppressor mutations can modulate the immune milieu of the tumor microenvironment, and they offer specific implications for addressing STK11/LKB1-mutated tumors with PD-1-targeting antibody therapies.

Esophageal adenocarcinoma is an increasingly common disease with a dismal 5-year survival rate of 10% to 15%. In the first systematic evaluation of the PD-1 pathway in esophageal adenocarcinoma, we identify expression of PD-L2 in cancer cells in 51.7% of esophageal adenocarcinomas. Epithelial PD-L1 was expressed on only 2% of cases, although PD-L1(+) immune cells were observed in 18% of esophageal adenocarcinomas. We also evaluated expression in the precursor lesion of esophageal adenocarcinoma, Barrett's esophagus, which emerges following gastric reflux-induced esophageal inflammation, and found PD-L2 expression in Barrett's esophagus but not in non-Barrett's esophagus esophagitis. Because the progression from squamous esophagitis to Barrett's esophagus is accompanied by a transition from a TH1 to TH2 immune response, we hypothesized that the TH2 cytokines IL4/IL13 could contribute to PD-L2 induction. We confirmed that these cytokines can augment PD-L2 expression in esophageal adenocarcinoma cell lines. These results suggest that the inflammatory environment in Barrett's esophagus and esophageal adenocarcinoma may contribute to the expression of PD-L2. Furthermore, the potential for PD-1 receptor blockade to be effective in esophageal adenocarcinomas with epithelial PD-L2 or immune cell PD-L1 expression should be evaluated in clinical trials.