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Supplementary Figure S3. PROS1 is located in GSCs.

Glioblastoma (GBM) is a lethal disease with no effective therapies available. We previously observed upregulation of the TAM (Tyro-3, Axl, and Mer) receptor tyrosine kinase family member AXL in mesenchymal GBM and showed that knockdown of AXL induced apoptosis of mesenchymal, but not proneural, glioma sphere cultures (GSC). In this study, we report that BGB324, a novel small molecule inhibitor of AXL, prolongs the survival of immunocompromised mice bearing GSC-derived mesenchymal GBM-like tumors. We show that protein S (PROS1), a known ligand of other TAM receptors, was secreted by tumor-associated macrophages/microglia and subsequently physically associated with and activated AXL in mesenchymal GSC. PROS1-driven phosphorylation of AXL (pAXL) induced NFκB activation in mesenchymal GSC, which was inhibited by BGB324 treatment. We also found that treatment of GSC-derived mouse GBM tumors with nivolumab, a blocking antibody against the immune checkpoint protein PD-1, increased intratumoral macrophages/microglia and activation of AXL. Combinatorial therapy with nivolumab plus BGB324 effectively prolonged the survival of mice bearing GBM tumors. Clinically, expression of AXL or PROS1 was associated with poor prognosis for patients with GBM. Our results suggest that the PROS1–AXL pathway regulates intrinsic mesenchymal signaling and the extrinsic immune microenvironment, contributing to the growth of aggressive GBM tumors.Significance: These findings suggest that development of combination treatments of AXL and immune checkpoint inhibitors may provide benefit to patients with GBM. Cancer Res; 78(11); 3002–13. ©2018 AACR.

Supplementary figure legends and materials and methods

Tumor-specific T cells likely underpin effective immune checkpoint-blockade therapies. Yet, most studies focus on Treg cells and CD8

Introduction Programmed cell death protein-1 (PD-1) and programmed death-ligand 1 (PD-L1) blockade is currently widely used in the treatment of metastatic NSCLC. Despite available biomarker stratification, clinical responses vary. Thus, the search for novel biomarkers with improved response prediction is ongoing. Previously, using mass cytometry or cytometry by time-of-flight (CyTOF), our group demonstrated that CD39+CD8+ immune cells represent tumor antigen-specific, cytotoxic T cells in treatment-naive NSCLC. We hypothesized that accurate quantitation of this T cell subset would predict immunotherapy outcome. Methods To translate this to a clinical setting, the present study compared CyTOF data with a range of clinically relevant methods, including conventional immunohistochemistry (IHC), multiplex IHC or immunofluorescence (mIHC), and gene expression assay by NanoString. Results Quantification using mIHC but not conventional IHC or NanoString correlated with the CyTOF results. The specificity and sensitivity of mIHC were then evaluated in a separate retrospective NSCLC cohort. CD39+CD8+ T cell proportion, as determined by mIHC, successfully stratified responders and nonresponders to PD-1 or PD-L1 inhibitors (objective response rate of 63.6%, compared with 0% for the negative group). This predictive capability was independent from other confounding factors, such as total CD8+ T cell proportion, CD39+ lymphocyte proportion, PD-L1 positivity, EGFR mutation status, and other clinicopathologic parameters. Conclusions Our results suggest that the mIHC platform is a clinically relevant method to evaluate CD39+CD8+ T cell proportion and that this marker can serve as a potential biomarker that predicts response to PD-1 or PD-L1 blockade in patients with NSCLC. Further validation in additional NSCLC cohorts is warranted.

BackgroundBecause of the shortage of ideal cell surface antigens, the development of T-cell receptor (TCR)-engineered T cells (TCR-T) that target intracellular antigens such as NY-ESO-1 is a promising approach for treating patients with solid tumors. However, endogenous TCRs in vector-transduced T cells have been suggested to impair cell-surface expression of transduced TCR while generating mispaired TCRs that can become self-reactive.MethodsWe conducted a first-in-human phase I clinical trial with the TCR-transduced T-cell product (TBI-1301) in patients with NY-ESO-1-expressing solid tumors. In manufacturing TCR-T cells, we used a novel affinity-enhanced NY-ESO-1-specific TCR that was transduced by a retroviral vector that enables siRNA (small interfering RNA)-mediated silencing of endogenous TCR. The patients were divided into two cohorts. Cohort 1 was given a dose of 5×108 cells (whole cells including TCR-T cells) preconditioned with 1500 mg/m2 cyclophosphamide. Cohort 2 was given 5× 109 cells preconditioned with 1500 mg/m2 cyclophosphamide.ResultsIn vitro study showed that both the CD8+ and CD4+ T fractions of TCR-T cells exhibited cytotoxic effects against NY-ESO-1-expressing tumor cells. Three patients and six patients were allocated to cohort 1 and cohort 2, respectively. Three of the six patients who received 5×109 cells showed tumor response, while three patients developed early-onset cytokine release syndrome (CRS). One of the patients developed a grade 3 lung injury associated with the infiltration of the TCR-T cells. No siRNA-related adverse events other than CRS were observed. Cytokines including interleukin 6 I and monocyte chemotactic protein-1/chemokine (C-C motif) ligand (CCL2)increased in the sera of patients with CRS. In vitro analysis showed these cytokines were not secreted from the T cells infused. A significant fraction of the manufactured T cells in patients with CRS was found to express either CD244, CD39, or both at high levels.ConclusionsThe trial showed that endogenous TCR-silenced and affinity-enhanced NY-ESO-1 TCR-T cells were safely administered except for grade 3 lung injury. The TCR-T cell infusion exhibited significant tumor response and early-onset CRS in patients with tumors that express NY-ESO-1 at high levels. The differentiation properties of the manufactured T cells may be prognostic for TCR-T-related CRS.Trial registration numberNCT02366546.

Significance Strongly implicated in effective antitumor immune responses, tumor mutation–derived antigens, or neoantigens, are one of the main targets for cancer vaccines despite uncertainty of the efficacy of this approach. Using a high-throughput screening method, we identify an endogenously immunogenic neoantigen in a commonly used mouse lung tumor model. We also found that the endogenous CD8 T cells specific for this neoantigen expand greatly upon treatment with immune checkpoint inhibitors or vaccination despite the lack of associated tumor regression in this model. In addition to informing neoantigen vaccination strategies and providing an accessible system for testing alternative therapeutics, our results provide insights into the mechanisms for the lack of response observed for a majority of patients treated with checkpoint blockade immunotherapies., Neoantigen-specific T cells are strongly implicated as being critical for effective immune checkpoint blockade treatment (ICB) (e.g., anti–PD-1 and anti–CTLA-4) and are being targeted for vaccination-based therapies. However, ICB treatments show uneven responses between patients, and neoantigen vaccination efficiency has yet to be established. Here, we characterize neoantigen-specific CD8+ T cells in a tumor that is resistant to ICB and neoantigen vaccination. Leveraging the use of mass cytometry combined with multiplex major histocompatibility complex (MHC) class I tetramer staining, we screened and identified tumor neoantigen–specific CD8+ T cells in the Lewis Lung carcinoma (LLC) tumor model (mRiok1). We observed an expansion of mRiok1-specific CD8+ tumor-infiltrating lymphocytes (TILs) after ICB targeting PD-1 or CTLA-4 with no sign of tumor regression. The expanded neoantigen-specific CD8+ TILs remained phenotypically and functionally exhausted but displayed cytotoxic characteristics. When combining both ICB treatments, mRiok1-specific CD8+ TILs showed a stem-like phenotype and a higher capacity to produce cytokines, but tumors did not show signs of regression. Furthermore, combining both ICB treatments with neoantigen vaccination did not induce tumor regression either despite neoantigen-specific CD8+ TIL expansion. Overall, this work provides a model for studying neoantigens in an immunotherapy nonresponder model. We showed that a robust neoantigen-specific T-cell response in the LLC tumor model could fail in tumor response to ICB, which will have important implications in designing future immunotherapeutic strategies.

Monitoring the frequency of circulatory CXCR5+ (cCXCR5+) CD4+ T cells in periphery blood provides a potential biomarker to draw inferences about T follicular helper (TFH) activity within germinal center. However, cCXCR5+ T cells are highly heterogeneous in their expression of ICOS, PD1 and CD38 and the relationship between different cCXCR5 subsets as delineated by these markers remains unclear. We applied class II tetramer reagents and mass cytometry to investigate the ontogeny of different subsets of cCXCR5+ T cell following yellow fever immunization. Through unsupervised analyses of mass cytometry data, we show yellow fever virus-specific cCXCR5 T cells elicited by vaccination were initially CD38+ICOS+PD1+, but then transitioned to become CD38+ICOS−PD1+ and CD38−ICOS−PD1+ before coming to rest as a CD38−ICOS−PD1− subset. These results imply that most antigen-specific cCXCR5+ T cells, including the CD38−ICOS−PD1− CXCR5+ T cells are derived from the CXCR5+CD38+ICOS+PD1+ subset, the subset that most resembles preTFH/TFH in the germinal center.

Tumor-specific T cells likely underpin effective immune checkpoint-blockade therapies. Yet, most studies focus on Treg cells and CD8+tumor-infiltrating lymphocytes (TILs). Here we study CD4+TILs in human lung and colorectal cancers and observe that non-Treg CD4+TILs average more than 70% of total CD4+TILs in both cancer types. Leveraging high dimensional analyses including mass cytometry and single-cell sequencing, we reveal that CD4+TILs are heterogeneous at both gene and protein levels, within each tumor and across patients. Consistently, we find different subsets of CD4+TILs showing characteristics of effectors, tissue resident memory (Trm) or exhausted cells (expressing PD-1, CTLA-4 and CD39). In both cancer types, the frequencies of CD39−non-Treg CD4+TILs strongly correlate with frequencies of CD39−CD8+TILs, which we and others have previously shown to be enriched for cells specific for cancer-unrelated antigens (bystanders).Ex-vivo, we demonstrate that CD39−CD4+TILs can be specific for cancer unrelated antigens, such as HCMV epitopes. Overall, our findings highlight that CD4+TILs cells are not necessarily tumor-specific and suggest measuring CD39 expression as a straightforward way to quantify or isolate bystander CD4+T cells.Graphical abstract

Summary Growing evidence indicates a role for the gut microbiota in modulating anti-tumor treatment efficacy in human cancer. Here we study mucosa-associated invariant T (MAIT) cells to look for evidence of bacterial antigen recognition in human colon, lung, and kidney carcinomas. Using mass cytometry and single-cell mRNA sequencing, we identify a tumor-infiltrating MAIT cell subset expressing CD4 and Foxp3 and observe high expression of CD39 on MAIT cells from colorectal cancer (CRC) only, which we show in vitro to be expressed specifically after TCR stimulation. We further reveal that these cells are phenotypically and functionally exhausted. Sequencing data show high bacterial infiltration in CRC tumors and highlight an enriched species, Fusobacteria nucleatum, with capability to activate MAIT cells in a TCR-dependent way. Our results provide evidence of a MAIT cell response to microbial antigens in CRC and could pave the way for manipulating MAIT cells or the microbiome for cancer therapy., Graphical Abstract, Highlights Comprehensive profiling of tumor-infiltrating MAIT cells in colorectal cancer (CRC) Identification of a tumor-infiltrating MAIT cell subset expressing CD4 and Foxp3 TCR-driven CD39 expression is enriched in tumor-infiltrating MAIT cells CRC-related Fusobacterium nucleatum can activate MAIT cells in a TCR-dependent way, By investigating human colon, lung, and kidney carcinomas, Li et al. propose MAIT cells as part of the missing link between the gut microbiota and tumor biology. This work will open new avenues in cancer therapy by manipulating MAIT cells or MAIT-related bacterial ligands in the context of microbiota-influenced cancers.

SUMMARY High-throughput single-cell RNA sequencing (scRNA-seq) has become a frequently used tool to assess immune cell heterogeneity. Recently, the combined measurement of RNA and protein expression was developed, commonly known as cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq). Acquisition of protein expression data along with transcriptome data resolves some of the limitations inherent to only assessing transcripts but also nearly doubles the sequencing read depth required per single cell. Furthermore, there is still a paucity of analysis tools tovisualize combined transcript-protein datasets. Here, we describe a targeted transcriptomics approach that combines an analysis of over 400 genes with simultaneous measurement of over 40 proteins on 2 × 104 cells in a single experiment. This targeted approach requires only about one-tenth of the read depth compared to a whole-transcriptome approach while retaining high sensitivity for low abundance transcripts. To analyze these multi-omic datasets, we adapted one-dimensional soli expression by nonlinear stochastic embedding (One-SENSE) for intuitive visualization of protein-transcript relationships on a single-cell level., Graphical Abstract, In Brief Mair et al. describe a targeted transcriptomics approach combined with surface protein measurement to capture immune cell heterogeneity at a low sequencing depth. One-SENSE is used as a visualization tool to intuitively explore the relationship of protein and transcript expression on the single-cell level.

Various forms of immunotherapy, such as checkpoint blockade immunotherapy, are proving to be effective at restoring T cell-mediated immune responses that can lead to marked and sustained clinical responses, but only in some patients and cancer types1–4. Patients and tumours may respond unpredictably to immunotherapy partly owing to heterogeneity of the immune composition and phenotypic profiles of tumour-infiltrating lymphocytes (TILs) within individual tumours and between patients5,6. Although there is evidence that tumour-mutation-derived neoantigen-specific T cells play a role in tumour control2,4,7–10, in most cases the antigen specificities of phenotypically diverse tumour-infiltrating T cells are largely unknown. Here we show that human lung and colorectal cancer CD8+ TILs can not only be specific for tumour antigens (for example, neoantigens), but also recognize a wide range of epitopes unrelated to cancer (such as those from Epstein–Barr virus, human cytomegalovirus or influenza virus). We found that these bystander CD8+ TILs have diverse phenotypes that overlap with tumour-specific cells, but lack CD39 expression. In colorectal and lung tumours, the absence of CD39 in CD8+ TILs defines populations that lack hallmarks of chronic antigen stimulation at the tumour site, supporting their classification as bystanders. Expression of CD39 varied markedly between patients, with some patients having predominantly CD39− CD8+ TILs. Furthermore, frequencies of CD39 expression among CD8+ TILs correlated with several important clinical parameters, such as the mutation status of lung tumour epidermal growth factor receptors. Our results demonstrate that not all tumour-infiltrating T cells are specific for tumour antigens, and suggest that measuring CD39 expression could be a straightforward way to quantify or isolate bystander T cells. Human lung and colorectal tumours contain a population of tumour-infiltrating lymphocytes that are specific for tumour-unrelated antigens and, unlike tumour-antigen-specific tumour-infiltrating lymphocytes, do not express CD39.

Advancement in methodologies for single cell analysis has historically been a major driver of progress in immunology. Currently, high dimensional flow cytometry, mass cytometry and various forms of single cell sequencing-based analysis methods are being widely adopted to expose the staggering heterogeneity of immune cells in many contexts. Here, we focus on mass cytometry, a form of flow cytometry that allows for simultaneous interrogation of more than 40 different marker molecules, including cytokines and transcription factors, without the need for spectral compensation. We argue that mass cytometry occupies an important niche within the landscape of single-cell analysis platforms that enables the efficient and in-depth study of diverse immune cell subsets with an ability to zoom-in on myeloid and lymphoid compartments in various tissues in health and disease. We further discuss the unique features of mass cytometry that are favorable for combining multiplex peptide-MHC multimer technology and phenotypic characterization of antigen specific T cells. By referring to recent studies revealing the complexities of tumor immune infiltrates, we highlight the particular importance of this technology for studying cancer in the context of cancer immunotherapy. Finally, we provide thoughts on current technical limitations and how we imagine these being overcome.

Antigen-specific T cells play a crucial role for the host protective immunity against viruses and other diseases. The use of mass cytometry together with a combinatorial multiplex tetramer staining has successfully been applied for probing and characterization of multiple antigen-specific CD8+ T cells in human blood samples. The present study shows that this approach can also be used to rapidly assess the magnitude of influenza-specific CD8+ T cell epitope dominance across lymph nodes and lungs in a murine model of a highly pathological influenza infection. Moreover, we show feasibility of extending this approach to include concurrent identification of virus-specific CD4+ T cells. By using a double coding approach, we probed for five influenza-specific MHCI-peptide complexes as well as one influenza-specific MHCII-peptide complex in the presence of irrelevant control peptides and show that this approach is capable of tracking antigen-specific T cells across individual lymph nodes and lungs. The simultaneous staining with 26 surface maker molecules further facilitated an in-depth characterization of T cells reacting with influenza epitopes and revealed tissue specific phenotypic differences between CD4+ T cells targeting the same pathogenic epitope. In conclusion, this approach provides the possibility for a rapid and comprehensive analysis of antigen-specific CD8+ and CD4+ T cells in different disease settings that might be advantageous for subsequent vaccine formulation strategies.

Innate lymphoid cells (ILCs) have been divided into three distinct groups based on functional capacities, cytokine profiles and transcription factor expression. Studies performed mainly in mice have demonstrated the importance of ILCs in chronic inflammation, infection, allergy and cancer. In this review, we discuss the heterogeneity of human ILC and focus primarily on the taxonomy of human ILC cell subsets and their phenotypical and functional diversity. We summarize recent findings concerning the diversity of ILCs between and within the major subsets [natural killer (NK), ILC1, intra-epithelial ILC1 (ieILC1), ILC2, ILC3, lymphoid tissues inducer (LTi) and ILC progenitor (ILCP)], as well as the abundance of each in human tissues. We also discuss the similarities observed between groups of cells in term of receptors expressed and cytokines produced, and how these relate to the pleiotropic properties of each subset.

The analysis of neoantigen-specific CD8+ T cells in tumour-bearing individuals is challenging due to the small pool of tumour antigen-specific T cells. Here we show that mass cytometry with multiplex combinatorial tetramer staining can identify and characterize neoantigen-specific CD8+ T cells in mice bearing T3 methylcholanthrene-induced sarcomas that are susceptible to checkpoint blockade immunotherapy. Among 81 candidate antigens tested, we identify T cells restricted to two known neoantigens simultaneously in tumours, spleens and lymph nodes in tumour-bearing mice. High-dimensional phenotypic profiling reveals that antigen-specific, tumour-infiltrating T cells are highly heterogeneous. We further show that neoantigen-specific T cells display a different phenotypic profile in mice treated with anti-CTLA-4 or anti-PD-1 immunotherapy, whereas their peripheral counterparts are not affected by the treatments. Our results provide insights into the nature of neoantigen-specific T cells and the effects of checkpoint blockade immunotherapy.Immune checkpoint blockade (ICB) therapies can unleash anti-tumour T-cell responses. Here the authors show, by integrating MHC tetramer multiplexing, mass cytometry and high-dimensional analyses, that neoantigen-specific, tumour-infiltrating T cells are highly heterogeneous and are subjected to ICB modulations.

Objectives Lymphoepithelioma‐like carcinoma (LELC) is an uncommon lung cancer, typically observed in young, non‐smoking Asian populations. LELC is associated with Epstein–Barr virus (EBV) infection of lung tumor cells of epithelial origin, suggesting a carcinogenic role of EBV as observed in nasopharyngeal carcinoma (NPC). Here, we studied the antigen specificity and phenotype of EBV‐specific CD8+ T cells in blood and tumor of one LELC patient positive for EBV infection in lung tumor cells. Methods Using multiplex MHC class I tetramers, mass cytometry and mRNA sequencing, we studied EBV‐specific CD8+ T cells at the transcriptomic and phenotypic levels in blood and tumor tissues of the LELC patient. Results Lymphoepithelioma‐like carcinoma lung tumor cells were positive for EBV infection. In both blood and tumor tissues, we detected two populations of EBV‐specific CD8+ T cells targeting the EBV lytic cycle proteins: BRLF1 and BMLF1. Transcriptomic analyses of these two populations in the tumor, which can be considered as tumor‐specific, revealed their distinct exhausted profile and polyclonal TCR repertoire. High‐dimensional phenotypical analysis revealed the distinct phenotype of these cells between blood and tumor tissues. In tumor tissue, EBV‐specific CD8+ TILs were phenotypically heterogeneous, but consistently expressed CD39. Unexpectedly, although the LELC tumor cells expressed abundant PD‐L1, these tumor‐specific CD8+ tumor‐infiltrating lymphocytes (TILs) mostly did not express PD‐1. Conclusion Epstein–Barr virus‐specific CD8+ TILs in EBV‐driven tumor are heterogeneous and partially lack PD‐1 expression, suggesting that anti‐PD1/PD‐L1 immunotherapy may not be an appropriate strategy for disinhibiting EBV‐specific cells in the treatment of LELC patients., We studied the antigen specificity and phenotype of CD8+ tumor‐infiltrating lymphocytes (TILs) in one LELC patient positive for EBV infection in lung tumor cells. Transcriptomic analyses of EBV‐specific CD8+ TILs reveal their distinct exhausted profiles and polyclonal TCR repertoire. Although the LELC tumor cells expressed abundant PD‐L1, EBV‐specific CD8+ TILs mostly did not express PD‐1, but express CD39.

Lymphoepithelioma-like carcinoma (LELC) is an uncommon lung cancer, typically observed in young, non-smoking Asian populations. LELC is associated with Epstein-Barr virus (EBV) infection of lung tumor cells of epithelial origin, suggesting a carcinogenic role of EBV as observed in nasopharyngeal carcinoma (NPC). Here, we studied the antigen specificity and phenotype of CD8+tumor infiltrating lymphocytes (TILs) in one LELC patient positive for EBV infection in lung tumor cells. Using MHC class I tetramers, we detected two populations of EBV-specific CD8+TILs, which can be considered as tumor-specific CD8+T cells, in the tumor of this patient. Transcriptomic analyses of these two populations reveal their distinct exhausted profiles and polyclonal TCR repertoire. High dimensional analyses at single cell level using mass cytometry showed showed that populations of tumor specific CD8+TILs are phenotypically heterogeneous, although they consistently express CD39. Unexpectedly, although the LELC tumor cells expressed abundant PD-L1, these tumor-specific CD8+TILs mostly did not express PD-1, suggesting that anti-PD1/PD-L1 immunotherapy may not be an appropriate strategy for disinhibiting EBV-specific cells for the treatment of LELC patients. These results might also help to explain low rates of checkpoint blockade immunotherapy response for NPC, despite the antigenicity of EBV for both tumor types.

Antigen-specific CD8

Innate lymphoid cells (ILCs) play a central role in the response to infection by secreting cytokines crucial for immune regulation, tissue homeostasis, and repair. Although dysregulation of these systems is central to pathology, the impact of HIV-1 on ILCs remains unknown. We found that human blood ILCs were severely depleted during acute viremic HIV-1 infection and that ILC numbers did not recover after resolution of peak viremia. ILC numbers were preserved by antiretroviral therapy (ART), but only if initiated during acute infection. Transcriptional profiling during the acute phase revealed upregulation of genes associated with cell death, temporally linked with a strong IFN acute-phase response and evidence of gut barrier breakdown. We found no evidence of tissue redistribution in chronic disease and remaining circulating ILCs were activated but not apoptotic. These data provide a potential mechanistic link between acute HIV-1 infection, lymphoid tissue breakdown, and persistent immune dysfunction. Keywords: innate lymphoid cells; HIV-1 infection

e15014 Background: Recently, using human colorectal and lung cancer, we showed that CD8+ T cells infiltrating tumor tissue (TILs) are not only specific for tumor antigens, but are also composed of CD8+ TILs specific for cancer unrelated epitopes – called bystander – such as HCMV, EBV or flu epitopes. We also showed that the surface marker CD39 can be useful for discriminating bystander (CD39−) from tumor-specific (CD39+) CD8+ TILs (Simoni et al, Nature, 2018). Methods: Here, our aim was to test these findings in human Renal cell Carcinoma (RCC) and to better understand the biology of these bystander CD8+ TILs. Results: Surprisingly, our primary CYTOF analyses showed heterogeneity within bystander CD8 TILs that possess various phenotypes including effector (CD45RO+), memory (CD45RO+ CCR7+), Trm (CD69+ CD103+/–), and senescent (CD57+ KLRG1+) cell features. Using targeted mRNA single-cell sequencing combined with BD AbSeq assay, we analyzed sorted CD8+ TILs from RCC patients and identified bystander CD8+ TILs using oligo-tagged tetramers. Gene signature analysis of these different subsets revealed transcriptomic similarities with the tumor-specific CD8+ TILs. Conclusions: Our data provide a comprehensive perspective of cancer unrelated CD8+ TILs in RCC and suggest functional roles for these cells in tumor immune responses, which could lead to new diagnostic and therapeutic strategies.

The most promising cancer immunotherapies to date target immune checkpoint receptors, such as PD-1 and CTLA-4. However, T cell checkpoint blockade has no inherent tumor specificity and shows uneven response, with up to 70–80% of patients resistant to these therapies. Here, we use a spontaneous mouse model resistant to immunotherapy to explore how immune checkpoint blockade (ICB) affects tumor-specific T cells and understand why these cells fail to eradicate tumor cells after treatment. Using combinatorial tetramer staining by CyTOF, we identified a new tumor-specific neoantigen in Lewis Lung Carcinoma (LLC). These tumor-specific CD8+ tumor-infiltrating lymphocytes (TILs) strongly exhibit an activation phenotype and can also be detected in different peripheral compartments at lower frequency. Surprisingly, despite an absence of tumor regression, we observed a remarkable expansion of tumor-specific CD8+ TILs after ICB with anti-PD1, anti-CTLA4, or neoantigen peptide vaccination. This expansion is associated with phenotypic changes, including enrichment of exhaustion markers and CD39 expression indicating chronic TCR activation and confirming recognition of tumor cells. Current single-cell RNA sequencing and TCR profiling analysis will correlate transcriptomics and clonality of these tumor-specific T cells after cancer therapies with anti-tumor response and elucidate their functionalities within the tumor microenvironment. Overall this work provides a new model for studying neoantigens in tumors and revisits the role of neoantigen-specific T cells in the context of ICB, which will have important implications in designing future immunotherapeutic strategies.

Recently, using human colorectal and lung cancer, we showed that CD8+ T cells infiltrating tumor tissue (TILs) are not only specific for tumor antigens, but are also composed of CD8+TILs specific for cancer unrelated epitopes – called bystander – such as HCMV, EBV or flu epitopes. We also showed that the surface marker CD39 can be useful for discriminating bystander (CD39−) from tumor-specific (CD39+) CD8+TILs (Simoni et al, Nature, 2018). Here, our aim was to test these findings in human Renal cell Carcinoma (RCC) and to better understand the biology of these bystander CD8+TILs. Surprisingly, our primary CYTOF analyses showed heterogeneity within bystander CD8+TILs that possess various phenotypes including effector (CD45RO+), memory (CD45RO+ CCR7+), Trm (CD69+ CD103+/–), and senescent (CD57+ KLRG1+) cell features. Using targeted mRNA single-cell sequencing combined with BD AbSeq assay, we analyzed sorted CD8+TILs from RCC patients and identified bystander CD8+TILs using oligo-tagged tetramers. Gene signature analysis of these different subsets revealed transcriptomic similarities with the tumor-specific CD8+TILs. Our data provide a comprehensive perspective of cancer unrelated CD8+TILs in RCC and suggest functional roles for these cells in tumor immune responses, which could lead to new diagnostic and therapeutic strategies.

Tumor-specific T cells act as major actors in underpinning effective immune checkpoint-blockade therapies in cancer, but most of the studies focus on CD8+ tumor-infiltrating lymphocytes (TILs). Although roles of CD4+ T cells and especially Tregs in cancer have been established, the basis for their phenotypic heterogeneity in human tumor infiltrates is not well determined. Here we profile ex-vivounexpanded CD4+ TILs in human lung and colorectal cancer using single-cell and mass-cytometry analysis. Surprisingly, up to 80% of CD4+ TILs are composed of non-Treg cells (FoxP3−). Non-Treg CD4+ TILs are highly heterogeneous and consist of different populations of effector (CD45RO+), cytotoxic (Granzyme B+), senescent (CD57+) and exhausted cells at different levels (PD-1+/++, CTLA-4+/−, TIGIT+, CD39+/−). Recently, we showed that the surface marker CD39 can accurately discriminate between bystander (CD39−) and tumor-specific (CD39+) CD8+ TILs. Across all tumors tested, more than 90% of Tregs cells expressed CD39. However, frequencies of CD39+ among non-Treg CD4+ TILs varied dramatically from 0% to 90% and strongly correlated (r2=0.8) with the frequency of CD39+CD8+ TILs in our cohort of patients. These observations suggest the presence of bystander CD4+ TILs and a high degree of coordination between the tumor-specific CD4 and CD8 T cell responses. Using in-vitroassay, we aim to confirm the presence of these bystander CD4+ TILs specific for cancer-unrelated antigens (HCMV, EBV, Influenza). Overall, our findings could highlight that not all CD4+ TILs are tumor-specific and could suggest measuring CD39 expression as a straightforward way to quantify or isolate tumor-specific T cells, thus opening new diagnostic and therapeutic avenues.

High throughput single-cell RNA sequencing (sc-RNAseq) has become a frequently used tool to assess immune cell function and heterogeneity. Recently, the combined measurement of RNA and protein expression by sequencing was developed, which is commonly known as CITE-Seq. Acquisition of protein expression data along with transcriptome data resolves some of the limitations inherent to only assessing transcript, but also nearly doubles the sequencing read depth required per single cell. Furthermore, there is still a paucity of analysis tools to visualize combined transcript-protein datasets. Here, we describe a novel targeted transcriptomics approach that combines analysis of over 400 genes with simultaneous measurement of over 40 proteins on more than 25,000 cells. This targeted approach requires only about 1/10 of the read depth compared to a whole transcriptome approach while retaining high sensitivity for low abundance transcripts. To analyze these multi-omic transcript-protein datasets, we adapted One-SENSE for intuitive visualization of the relationship of proteins and transcripts on a single-cell level.

Skin conventional dendritic cells (cDCs) exist as two distinct subsets, cDC1s and cDC2s, which maintain the balance of immunity to pathogens and tolerance to self and microbiota. Here, we examined the roles of dermal cDC1s and cDC2s during bacterial infection, notably Propionibacterium acnes (P. acnes). cDC1s, but not cDC2s, regulated the magnitude of the immune response to P. acnes in the murine dermis by controlling neutrophil recruitment to the inflamed site and survival and function therein. Single-cell mRNA sequencing revealed that this regulation relied on secretion of the cytokine vascular endothelial growth factor alpha (VEGF-alpha) by a minor subset of activated EpCAM(+)CD59(+) Ly-6D(+) cDC1s. Neutrophil recruitment by dermal cDC1s was also observed during S. aureus, bacillus Calmette-Guerin (BCG), or E. coli infection, as well as in a model of bacterial insult in human skin. Thus, skin cDC1s are essential regulators of the innate response in cutaneous immunity and have roles beyond classical antigen presentation.

Antigen-specific CD8+ T cells play a crucial role in the host protective immune response against viruses, tumors, and other diseases. Major histocompatibility complex (MHC) class I tetramers allow for a direct detection of such antigen-specific CD8+ T cells. Using mass cytometry together with multiplex MHC class I tetramer staining, we are able to screen more than 1000 different antigen candidates simultaneously across tissues in health and disease, while retaining the possibility to deliver an in-depth characterization of antigen-specific CD8+ T cells and associated phenotypes. Here we describe the method for a MHC class I tetramer multiplexing approach together with intracellular antibody staining for a parallel phenotypic cell characterization using mass cytometry in human specimens.

Glioblastoma (GBM) remains the deadliest of all primary brain tumors with very few effective treatment options. Recently, we reported that high AXL expression is correlated with poor prognosis in GBM patients and demonstrated the therapeutic benefits of targeting AXL, a member of TAM receptor tyrosine kinase family using a novel small molecule inhibitor, BGB324 in immunocompetent mouse GBM models and xenografts of patient-derived glioma stem cells(GSCs). The promise of BGB324 in tumor burden management prompted us to develop a clinical trial with BGB324 as a single agent therapeutic with the goal to extend it as a combinatorial therapy in the future. Our surgical PK/PD clinical trial with BGB324 in recurrent GBM has been approved by the Brain Malignancy Steering Committee at the National Cancer Institute. Study treatment will consist of 2 cohorts of adult GBM patients, one (Group A) receiving the treatment pre-operatively and the other (Group B) receiving no treatment at all prior to surgery. First 5 patients recruited to Group A will be checked for the desired intra-tumoral drug concentration achieved to continue the trial. Group A will be supplemented by an additional 5 patients bringing the number to n=10 in each arm of the trial. Following surgical resection, patients in both cohorts will receive BGB324 daily in 21-day cycles. Treatment will be continued unless patients exhibit significant toxicity or substantial tumor progression. Our preclinical findings show the upregulation of AXL and its role in apoptosis induction in mesenchymal GBM as well as its association with MLK4, a serine threonine kinase we previously characterized as a mesenchymal GSC molecular target. Inhibition of phosphorylation of AXL and concomitant NF-kB activation in mesenchymal GSCs was found to be the nodal target of the drug action. An up-to-date information of the trial will be presented in detail.

Glioblastoma (GBM) is a lethal disease with no effective therapies available. We previously observed upregulation of the TAM (Tyro-3, Axl, and Mer) receptor tyrosine kinase family member AXL in mesenchymal GBM and showed that knockdown of AXL induced apoptosis of mesenchymal, but not proneural, glioma sphere cultures (GSC). In this study, we report that BGB324, a novel small molecule inhibitor of AXL, prolongs the survival of immunocompromised mice bearing GSC-derived mesenchymal GBM-like tumors. We show that protein S (PROS1), a known ligand of other TAM receptors, was secreted by tumor-associated macrophages/microglia and subsequently physically associated with and activated AXL in mesenchymal GSC. PROS1-driven phosphorylation of AXL (pAXL) induced NFκB activation in mesenchymal GSC, which was inhibited by BGB324 treatment. We also found that treatment of GSC-derived mouse GBM tumors with nivolumab, a blocking antibody against the immune checkpoint protein PD-1, increased intratumoral macrophages/microglia and activation of AXL. Combinatorial therapy with nivolumab plus BGB324 effectively prolonged the survival of mice bearing GBM tumors. Clinically, expression of AXL or PROS1 was associated with poor prognosis for patients with GBM. Our results suggest that the PROS1–AXL pathway regulates intrinsic mesenchymal signaling and the extrinsic immune microenvironment, contributing to the growth of aggressive GBM tumors. Significance: These findings suggest that development of combination treatments of AXL and immune checkpoint inhibitors may provide benefit to patients with GBM. Cancer Res; 78(11); 3002–13. ©2018 AACR.

Motivation Recent flow and mass cytometers generate datasets of dimensions 20 to 40 and a million single cells. From these, many tools facilitate the discovery of new cell populations associated with diseases or physiology. These new cell populations require the identification of new gating strategies, but gating strategies become exponentially more difficult to optimize when dimensionality increases. To facilitate this step, we developed Hypergate, an algorithm which given a cell population of interest identifies a gating strategy optimized for high yield and purity. Results Hypergate achieves higher yield and purity than human experts, Support Vector Machines and Random-Forests on public datasets. We use it to revisit some established gating strategies for the identification of innate lymphoid cells, which identifies concise and efficient strategies that allow gating these cells with fewer parameters but higher yield and purity than the current standards. For phenotypic description, Hypergate’s outputs are consistent with fields’ knowledge and sparser than those from a competing method. Availability and implementation Hypergate is implemented in R and available on CRAN. The source code is published at http://github.com/ebecht/hypergate under an Open Source Initiative-compliant licence. Supplementary information Supplementary data are available at Bioinformatics online.

Studies in human aging mainly focus on αβ T cells while γδ T cells have been neglected despite their role in immunosurveillance. In this study, we investigated γδ T cells and its subsets (i.e. Vδ1 , Vδ2 and DN γδ (γδ Vδ1-Vδ2-)) by deep immunophenotyping. Our data shows that peripheral Vδ2 are differentially susceptible to life-long stresses against all other αβ and γδ T cells. Using aging and cytomegalovirus history (the main confounding factor in immune-aging studies) as immunological models and via functional assays, telomere length determination, epigenetic methylome profiling and DNA damage response capacity, we show that Vδ2 T cells are resistant from cellular senescence. Our results highlight the differential impact of stress on γδ T cells subsets, reveal surface markers that are functionally relevant for γδ T cells subsets and highlight possible mechanisms that enable Vδ2 to be resistant to cellular aging. This new finding reinforces the concept that Vδ2 have an “innate-like” behavior and are more resilient to the environment as compared to “adaptive-like” Vδ1 T cells. Funding Statement: Weili Xu is funded by A*STAR Graduate Academy (AGA). The work was funded by the Singapore Immunology Network, the Agency for Science Technology and Research (JCO grant #1434m00115) and the Skin Research Institute of Singapore (SRG grant #14018). Declaration of Interests: The authors have no conflict of interests. Ethics Approval Statement: Participants of the study were recruited under National University of Singapore Institutional Review Board (IRB code NUS-IRB 01-256). Cone blood was obtained from Health Science Authority (HSA) Singapore, approved under NUS-IRB 10-250.

Repetitive stimulation by persistent pathogens such as human cytomegalovirus (HCMV) or human immunodeficiency virus (HIV) induces the differentiation of natural killer (NK) cells. This maturation pathway is characterized by the acquisition of phenotypic markers, CD2, CD57, and NKG2C, and effector functions—a process regulated by Tim-3 and orchestrated by a complex network of transcriptional factors, involving T-bet, Eomes, Zeb2, promyelocytic leukemia zinc finger protein, and Foxo3. Here, we show that persistent immune activation during chronic viral co-infections (HCMV, hepatitis C virus, and HIV) interferes with the functional phenotype of NK cells by modulating the Tim-3 pathway; a decrease in Tim-3 expression combined with the acquisition of inhibitory receptors skewed NK cells toward an exhausted and cytotoxic phenotype in an inflammatory environment during chronic HIV infection. A better understanding of the mechanisms underlying NK cell differentiation could aid the identification of new immunological targets for checkpoint blockade therapies in a manner that is relevant to chronic infection and cancer.

Various forms of immunotherapy, such as checkpoint blockade immunotherapy, are proving to be effective at restoring T cell-mediated immune responses that can lead to marked and sustained clinical responses, but only in some patients and cancer types

Natural killer T (NKT) cells are non-conventional T lymphocytes expressing a TCRαβ and several NK cell markers. Once activated, they can rapidly secrete large amounts of cytokines such as IFN-γ and IL-4. As a result they can favor both Th1 and Th2 immune responses and play a critical role in anti-pathogenic immune responses as well as in regulation of autoimmune diseases. It has now been clearly established that iNKT cells can be subdivided into three subpopulations: iNKT1, iNKT2 and iNKT17 cells. Each of these populations is characterized by the expression of a particular transcription factor, surface markers and cytokines making them functionally distinct. Interestingly, NOD mice developing autoimmune diabetes exhibit a high frequency of iNKT17 cells, which can participate in the disease.

Invariant NKT (iNKT)-cell stimulation with exogenous specific ligands prevents the development of type 1 diabetes (T1D) in NOD mice. Studies based on anti-islet T-cell transfer showed that iNKT cells prevent the differentiation of these T cells into effector T cells in the pancreatic lymph nodes (PLNs). We hypothesize that this defective priming could be explained by the ability of iNKT cells to induce tolerogenic dendritic cells (DCs) in the PLNs. We evaluated the effect of iNKT-cell stimulation on T1D development by transferring naive diabetogenic BDC2.5 T cells into proinsulin 2(-/-) NOD mice treated with a long-lasting α-galactosylceramide regimen. In this context, iNKT cells induce the conversion of BDC2.5 T cells into Foxp3(+) Treg cells in the PLNs accumulating in the pancreatic islets. Furthermore, tolerogenic plasmacytoid DCs (pDCs) characterized by low MHC class II molecule expression and TGF-β production are critical in the PLNs for the recruitment of Treg cells into the pancreatic islets by inducing CXCR3 expression. Accordingly, pDC depletion in α-galactosylceramide-treated proinsulin 2(-/-) NOD mice abrogates the protection against T1D. These findings reveal that upon repetitive iNKT-cell stimulation, pDCs are critical for the recruitment of Treg cells in the pancreatic islets and the prevention of T1D development.