Ton N. Schumacher

Immune checkpoint inhibitors, which unleash a patient’s own T cells to kill tumors, are revolutionizing cancer treatment. To unravel the genomic determinants of response to this therapy, we used whole-exome sequencing of non–small cell lung cancers treated with pembrolizumab, an antibody targeting programmed cell death-1 (PD-1). In two independent cohorts, higher nonsynonymous mutation burden in tumors was associated with improved objective response, durable clinical benefit, and progression-free survival. Efficacy also correlated with the molecular smoking signature, higher neoantigen burden, and DNA repair pathway mutations; each factor was also associated with mutation burden. In one responder, neoantigen-specific CD8+ T cell responses paralleled tumor regression, suggesting that anti–PD-1 therapy enhances neoantigen-specific T cell reactivity. Our results suggest that the genomic landscape of lung cancers shapes response to anti–PD-1 therapy.

The clinical relevance of T cells in the control of a diverse set of human cancers is now beyond doubt. However, the nature of the antigens that allow the immune system to distinguish cancer cells from noncancer cells has long remained obscure. Recent technological innovations have made it possible to dissect the immune response to patient-specific neoantigens that arise as a consequence of tumor-specific mutations, and emerging data suggest that recognition of such neoantigens is a major factor in the activity of clinical immunotherapies. These observations indicate that neoantigen load may form a biomarker in cancer immunotherapy and provide an incentive for the development of novel therapeutic approaches that selectively enhance T cell reactivity against this class of antigens.

Approximately 75% of objective responses to anti-programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown.We analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti-PD-1 therapy (pembrolizumab) followed by disease progression months to years later.Whole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated loss-of-function mutations in the genes encoding interferon-receptor-associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I.In this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.).

The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell immunogenicity, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion. Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits-including durable responses--to patients with different malignancies. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers1-10 and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity11-15, although these have been less well-studied in ICB treatment16. A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling17 that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter18) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.

Expression of programmed death-ligand 1 (PD-L1) is frequently observed in human cancers. Binding of PD-L1 to its receptor PD-1 on activated T cells inhibits anti-tumor immunity by counteracting T cell-activating signals. Antibody-based PD-1-PD-L1 inhibitors can induce durable tumor remissions in patients with diverse advanced cancers, and thus expression of PD-L1 on tumor cells and other cells in the tumor microenviroment is of major clinical relevance. Here we review the roles of the PD-1-PD-L1 axis in cancer, focusing on recent findings on the mechanisms that regulate PD-L1 expression at the transcriptional, posttranscriptional, and protein level. We place this knowledge in the context of observations in the clinic and discuss how it may inform the design of more precise and effective cancer immune checkpoint therapies.

Therapeutic efficacy of a tumor cell-based vaccine against experimental B16 melanoma requires the disruption of either of two immunoregulatory mechanisms that control autoreactive T cell responses: the cytotoxic T lymphocyte-associated antigen (CTLA)-4 pathway or the CD25(+) regulatory T (Treg) cells. Combination of CTLA-4 blockade and depletion of CD25(+) Treg cells results in maximal tumor rejection. Efficacy of the antitumor therapy correlates with the extent of autoimmune skin depigmentation as well as with the frequency of tyrosinase-related protein 2(180-188)-specific CTLs detected in the periphery. Furthermore, tumor rejection is dependent on the CD8(+) T cell subset. Our data demonstrate that the CTL response against melanoma antigens is an important component of the therapeutic antitumor response and that the reactivity of these CTLs can be augmented through interference with immunoregulatory mechanisms. The synergism in the effects of CTLA-4 blockade and depletion of CD25(+) Treg cells indicates that CD25(+) Treg cells and CTLA-4 signaling represent two alternative pathways for suppression of autoreactive T cell immunity. Simultaneous intervention with both regulatory mechanisms is therefore a promising concept for the induction of therapeutic antitumor immunity.

‘T cell exhaustion’ is a broad term that has been used to describe the response of T cells to chronic antigen stimulation, first in the setting of chronic viral infection but more recently in response to tumours. Understanding the features of and pathways to exhaustion has crucial implications for the success of checkpoint blockade and adoptive T cell transfer therapies. In this Viewpoint article, 18 experts in the field tell us what exhaustion means to them, ranging from complete lack of effector function to altered functionality to prevent immunopathology, with potential differences between cancer and chronic infection. Their responses highlight the dichotomy between terminally differentiated exhausted T cells that are TCF1– and the self-renewing TCF1+ population from which they derive. These TCF1+ cells are considered by some to have stem cell-like properties akin to memory T cell populations, but the developmental relationships are unclear at present. Recent studies have also highlighted an important role for the transcriptional regulator TOX in driving the epigenetic enforcement of exhaustion, but key questions remain about the potential to reverse the epigenetic programme of exhaustion and how this might affect the persistence of T cell populations. In this Viewpoint article, Nature Reviews Immunology invites 18 experts to discuss the nature of T cell exhaustion. How should T cell exhaustion be defined and what are the developmental relationships between exhausted T cell subsets? The contributors share their thoughts on key recent developments in the field. Christian U. Blank is a medical oncologist and principal investigator at the Netherlands Cancer Institute. He is Professor of Haematology/Oncology at the University of Regensburg, Germany, and received an MBA degree from the University of Warwick, UK. His research interests include neoadjuvant immunotherapies, targeted and biological response modifiers, and prognostic markers for cancer immunotherapies. W. Nicholas Haining is a physician–scientist and Vice-President for Discovery Oncology and Immunology at Merck Research Laboratories. His former academic laboratory at the Dana-Farber Cancer Institute and the Broad Institute focused on understanding the transcriptional control of T cell exhaustion and on identifying regulators of the immune response to cancer in tumour and immune cells. Werner Held’s laboratory has a long-standing interest in understanding the development, differentiation and function of natural killer cells and CD8+ T cells. Current work focuses on CD8+ T cell differentiation in response to acute and chronic infections as well as cancer. Patrick G. Hogan’s research centres on mechanisms and regulation of cellular calcium signalling, the biology of the nuclear factor of activated T cells (NFAT) family of transcription factors and the transcriptional control of immune cell development and function. Axel Kallies is a professor at the University of Melbourne, Australia. His laboratory studies the molecular control of CD8+ cytotoxic T cell and regulatory T cell differentiation with a focus on populations residing in non-lymphoid tissue, including healthy tissues and tumours. The Kallies laboratory has developed and applied genetic and molecular approaches to this field, including novel gene reporters, metabolic techniques, transcriptional profiling, chromatin immunoprecipitation and accessible chromatin sequencing. Enrico Lugli’s laboratory is focused on understanding the biological mechanisms at the basis of memory T cell responses and homeostasis in humans and how this information can be exploited to favour antitumour immune responses in patients with cancer. The group is specialized in single-cell technologies, in particular high-dimensional flow cytometry. Rachel C. Lynn is an associate director of research at Lyell Immunopharma. She received her PhD degree from the the University of Pennsylvania, where she developed multiple preclinical chimeric antigen receptor (CAR) T cell therapy platforms. During her postdoctoral work with Crystal Mackall at Stanford University, she developed models to interrogate and strategies to mitigate CAR T cell exhaustion. At Lyell Immunopharma, her research group will continue to investigate optimal strategies for adoptive T cell therapy in cancer. Mary Philip is an assistant professor in the Department of Medicine, Division of Hematology and Oncology and Department of Pathology, Microbiology, and Immunology at Vanderbilt University Medical Center. She did her haematology and oncology fellowship training at the Fred Hutchinson Cancer Research Center and the University of Washington and then joined Andrea Schietinger’s group at Memorial Sloan Kettering Cancer Center investigating the epigenetic and transcriptional regulation of T cell dysfunction and reprogrammability in cancer. She is a physician–scientist with clinical expertise in haematological cancers, and her research group at Vanderbilt University Medical Center is developing innovative models to dissect the regulation of T cell dysfunction in cancer and T cell lymphomas. Anjana Rao’s laboratory studies transcriptional regulation in several cell types, including CD4+ and CD8+ T cells. One current focus is elucidation of the transcriptional and epigenetic regulatory mechanisms operating in T cells and other immune cells within the tumour microenvironment. Nicholas P. Restifo was trained at Memorial Sloan Kettering Cancer Center and was a principal investigator at the National Cancer Institute. His work has focused on the use of adoptively transferred T cells in immunotherapy for cancer for the past 31 years. He recently joined Lyell Immunopharma. Andrea Schietinger is an assistant member in the Immunology Program at Memorial Sloan Kettering Cancer Center. During her PhD studies with Hans Schreiber at the University of Chicago, she studied how aberrant glycosylation of wild-type proteins in cancer cells creates tumour-specific neoantigens. As a postdoctoral fellow in Philip Greenberg’s laboratory at the University of Washington in Seattle, she defined the transcriptional programmes associated with T cell self-tolerance and tumour-specific T cell dysfunction. Her laboratory at Memorial Sloan Kettering Cancer Center studies the regulatory molecular and epigenetic mechanisms underlying T cell differentiation and dysfunction in the context of self-tolerance, autoimmunity and tumours. Ton N. Schumacher is a principal investigator at the Netherlands Cancer Institute and Professor of Immunotechnology at Leiden University. His research focuses on the dissection of tumour-specific T cell responses in human cancer. In addition to his academic role, he is the founder of four biotech companies and a venture partner at Third Rock Ventures. Pamela L. Schwartzberg is a senior investigator at the National Institute of Allergy and Infectious Diseases. She completed her MD and PhD degrees at Columbia University and her postdoctoral studies at the National Cancer Institute. Her laboratory studies signalling pathways in T cells using genetic, cellular, biochemical and genomic approaches to understand how these affect responses to infection and immunization with a focus on molecules and pathways affected by primary immunodeficiencies. Arlene H. Sharpe is Chair of the Department of Immunology at Harvard Medical School. Her laboratory focuses on understanding the diverse roles of costimulatory and coinhibitory signals in infection, autoimmunity and cancer. Daniel E. Speiser’s team has investigated and treated cancer patients with immunotherapy for the past 30 years. The demonstration and mechanistic elucidation of graft-versus-leukaemia effects and autologous T cell reactivity have helped to pave the way for remarkable therapy innovations in oncology. E. John Wherry is the Barbara and Richard Schiffrin President’s Distinguished Professor, Chair of the Department of Systems Pharmacology and Translational Therapeutics in the Perelman School of Medicine and Director of the Penn Institute for Immunology. His research has focused on T cell exhaustion in chronic viral infections and cancer. His work has defined the developmental biology and molecular regulation of T cell exhaustion and used this information to inform immunotherapy for human cancer. Benjamin A. Youngblood is an associate member in the Department of Immunology at St Jude Children’s Research Hospital. His postdoctoral training in Rafi Ahmed’s laboratory focused on the epigenetic regulation of memory CD8+ T cell differentiation. In 2014, he joined the faculty at St Jude Children’s Research Hospital and has developed a research programme studying epigenetic mechanisms that regulate the development of functional and non-functional CD8+ T cells during viral infection, cancer and autoimmunity. Dietmar Zehn is a full professor and Chair of the Division of Animal Physiology and Immunology of the Technical University of Munich. His research focuses on understanding the molecular and cellular mechanisms that control the differentiation of T cells in acute and chronic infection, with specific interests in T cell exhaustion and T cell receptor signal strength.

The T cell infiltrates that are formed in human cancers are a modifier of natural disease progression and also determine the probability of clinical response to cancer immunotherapies. Recent technological advances that allow the single-cell analysis of phenotypic and transcriptional states have revealed a vast heterogeneity of intratumoural T cell states, both within and between patients, and the observation of this heterogeneity makes it critical to understand the relationship between individual T cell states and therapy response. This Review covers our current knowledge of the T cell states that are present in human tumours and the role that different T cell populations have been hypothesized to play within the tumour microenvironment, with a particular focus on CD8+ T cells. The three key models that are discussed herein are as follows: (1) the dysfunction of T cells in human cancer is associated with a change in T cell functionality rather than inactivity; (2) antigen recognition in the tumour microenvironment is an important driver of T cell dysfunctionality and the presence of dysfunctional T cells can hence be used as a proxy for the presence of a tumour-reactive T cell compartment; (3) a less dysfunctional population of tumour-reactive T cells may be required to drive a durable response to T cell immune checkpoint blockade. Recent single-cell RNA-sequencing studies have revealed a range of intratumoural T cell states, both within and between patients. This Review outlines the CD8+ T cell states that have been identified in human tumours and the potential roles they play in tumour control as well as how they are influenced by immune checkpoint blockade.

Therapeutic reinvigoration of tumor-specific T cells has greatly improved clinical outcome in cancer. Nevertheless, many patients still do not achieve durable benefit. Recent evidence from studies in murine and human cancer suggest that intratumoral T cells display a broad spectrum of (dys-)functional states, shaped by the multifaceted suppressive signals that occur within the tumor microenvironment. Here we discuss the current understanding of T cell dysfunction in cancer, the value of novel technologies to dissect such dysfunction at the single cell level, and how our emerging understanding of T cell dysfunction may be utilized to develop personalized strategies to restore antitumor immunity. Therapeutic reinvigoration of tumor-specific T cells has greatly improved clinical outcome in cancer. Nevertheless, many patients still do not achieve durable benefit. Recent evidence from studies in murine and human cancer suggest that intratumoral T cells display a broad spectrum of (dys-)functional states, shaped by the multifaceted suppressive signals that occur within the tumor microenvironment. Here we discuss the current understanding of T cell dysfunction in cancer, the value of novel technologies to dissect such dysfunction at the single cell level, and how our emerging understanding of T cell dysfunction may be utilized to develop personalized strategies to restore antitumor immunity.

Tumor immune cell compositions play a major role in response to immunotherapy, but the heterogeneity and dynamics of immune infiltrates in human cancer lesions remain poorly characterized. Here, we identify conserved intratumoral CD4 and CD8 T cell behaviors in scRNA-seq data from 25 melanoma patients. We discover a large population of CD8 T cells showing continuous progression from an early effector “transitional” into a dysfunctional T cell state. CD8 T cells that express a complete cytotoxic gene set are rare, and TCR sharing data suggest their independence from the transitional and dysfunctional cell states. Notably, we demonstrate that dysfunctional T cells are the major intratumoral proliferating immune cell compartment and that the intensity of the dysfunctional signature is associated with tumor reactivity. Our data demonstrate that CD8 T cells previously defined as exhausted are in fact a highly proliferating, clonal, and dynamically differentiating cell population within the human tumor microenvironment.

Evidence from mouse chronic viral infection models suggests that CD8+ T cell subsets characterized by distinct expression levels of the receptor PD-1 diverge in their state of exhaustion and potential for reinvigoration by PD-1 blockade. However, it remains unknown whether T cells in human cancer adopt a similar spectrum of exhausted states based on PD-1 expression levels. We compared transcriptional, metabolic and functional signatures of intratumoral CD8+ T lymphocyte populations with high (PD-1T), intermediate (PD-1N) and no PD-1 expression (PD-1-) from non-small-cell lung cancer patients. PD-1T T cells showed a markedly different transcriptional and metabolic profile from PD-1N and PD-1- lymphocytes, as well as an intrinsically high capacity for tumor recognition. Furthermore, while PD-1T lymphocytes were impaired in classical effector cytokine production, they produced CXCL13, which mediates immune cell recruitment to tertiary lymphoid structures. Strikingly, the presence of PD-1T cells was strongly predictive for both response and survival in a small cohort of non-small-cell lung cancer patients treated with PD-1 blockade. The characterization of a distinct state of tumor-reactive, PD-1-bright lymphocytes in human cancer, which only partially resembles that seen in chronic infection, provides potential avenues for therapeutic intervention.

PD-1 plus CTLA-4 blockade is highly effective in advanced-stage, mismatch repair (MMR)-deficient (dMMR) colorectal cancers, yet not in MMR-proficient (pMMR) tumors. We postulated a higher efficacy of neoadjuvant immunotherapy in early-stage colon cancers. In the exploratory NICHE study (ClinicalTrials.gov: NCT03026140), patients with dMMR or pMMR tumors received a single dose of ipilimumab and two doses of nivolumab before surgery, the pMMR group with or without celecoxib. The primary objective was safety and feasibility; 40 patients with 21 dMMR and 20 pMMR tumors were treated, and 3 patients received nivolumab monotherapy in the safety run-in. Treatment was well tolerated and all patients underwent radical resections without delays, meeting the primary endpoint. Of the patients who received ipilimumab + nivolumab (20 dMMR and 15 pMMR tumors), 35 were evaluable for efficacy and translational endpoints. Pathological response was observed in 20/20 (100%; 95% exact confidence interval (CI): 86-100%) dMMR tumors, with 19 major pathological responses (MPRs, ≤10% residual viable tumor) and 12 pathological complete responses. In pMMR tumors, 4/15 (27%; 95% exact CI: 8-55%) showed pathological responses, with 3 MPRs and 1 partial response. CD8+PD-1+ T cell infiltration was predictive of response in pMMR tumors. These data indicate that neoadjuvant immunotherapy may have the potential to become the standard of care for a defined group of colon cancer patients when validated in larger studies with at least 3 years of disease-free survival data.

The evidence for T-cell–mediated regression of human cancers such as non–small-cell lung carcinoma, renal cell carcinoma, and—in particular—melanoma after immunotherapy is strong. Anti-CTLA4 (ipilimumab) treatment has been approved for treatment of meta-static melanoma,1 and antibody-mediated blockade of PD-1, a second inhibitory receptor on T cells, has shown highly encouraging results in early clinical trials.2,3 Although the clinical activity of these treatments is apparent, it is still unknown which T-cell reactivities are involved in immunotherapy-induced cancer regression.4 T-cell reactivity against nonmutated tumor-associated self-antigens has been analyzed in patients treated with ipilimumab or with autologous tumor-infiltrating T cells, but the magnitude of the T-cell responses observed has been relatively modest.5,6 In part on the basis of such data, recognition of patient-specific mutant epitopes (hereafter referred to as neoantigens) has been suggested to be a potentially important component.7 A potential involvement of mutated epitopes in T-cell control would also fit well with the observation that the mutation load in sun-exposed melanomas is particularly high.8-10 Intriguingly, on the basis of animal model data, it has recently been suggested that (therapy-induced) analysis of T-cell reactivity against patient-specific neoantigens may be feasible through exploitation of cancer genome data.11,12 However, human data have thus far been lacking. Here we report a case of a patient with stage IV melanoma who exhibited a clinical response to ipilimumab treatment. Cancer exome–guided analysis of T-cell reactivity in this patient revealed reactivity against two neoantigens, including a dominant T-cell response against a mutant epitope of the ATR (ataxia telangiectasia and Rad3 related) gene product that increased strongly after ipilimumab treatment. These data provide the first demonstration (to our knowledge) of cancer exome–guided analysis to dissect the effects of melanoma immunotherapy.

Cancer immunotherapies have shown substantial clinical activity for a subset of patients with epithelial cancers. Still, technological platforms to study cancer T-cell interactions for individual patients and understand determinants of responsiveness are presently lacking. Here, we establish and validate a platform to induce and analyze tumor-specific T cell responses to epithelial cancers in a personalized manner. We demonstrate that co-cultures of autologous tumor organoids and peripheral blood lymphocytes can be used to enrich tumor-reactive T cells from peripheral blood of patients with mismatch repair-deficient colorectal cancer and non-small-cell lung cancer. Furthermore, we demonstrate that these T cells can be used to assess the efficiency of killing of matched tumor organoids. This platform provides an unbiased strategy for the isolation of tumor-reactive T cells and provides a means by which to assess the sensitivity of tumor cells to T cell-mediated attack at the level of the individual patient.

Visualizing the state of cancer–immune system interactions may spur personalized therapy

The clinical benefit for patients with diverse types of metastatic cancers that has been observed upon blockade of the interaction between PD-1 and PD-L1 has highlighted the importance of this inhibitory axis in the suppression of tumour-specific T-cell responses. Notwithstanding the key role of PD-L1 expression by cells within the tumour micro-environment, our understanding of the regulation of the PD-L1 protein is limited. Here we identify, using a haploid genetic screen, CMTM6, a type-3 transmembrane protein of previously unknown function, as a regulator of the PD-L1 protein. Interference with CMTM6 expression results in impaired PD-L1 protein expression in all human tumour cell types tested and in primary human dendritic cells. Furthermore, through both a haploid genetic modifier screen in CMTM6-deficient cells and genetic complementation experiments, we demonstrate that this function is shared by its closest family member, CMTM4, but not by any of the other CMTM members tested. Notably, CMTM6 increases the PD-L1 protein pool without affecting PD-L1 (also known as CD274) transcription levels. Rather, we demonstrate that CMTM6 is present at the cell surface, associates with the PD-L1 protein, reduces its ubiquitination and increases PD-L1 protein half-life. Consistent with its role in PD-L1 protein regulation, CMTM6 enhances the ability of PD-L1-expressing tumour cells to inhibit T cells. Collectively, our data reveal that PD-L1 relies on CMTM6/4 to efficiently carry out its inhibitory function, and suggest potential new avenues to block this pathway.

After an infection, pathogen-specific tissue-resident memory T cells (T(RM) cells) persist in nonlymphoid tissues to provide rapid control upon reinfection, and vaccination strategies that create T(RM) cell pools at sites of pathogen entry are therefore attractive. However, it is not well understood how T(RM) cells provide such pathogen protection. Here, we demonstrate that activated T(RM) cells in mouse skin profoundly alter the local tissue environment by inducing a number of broadly active antiviral and antibacterial genes. This "pathogen alert" allows skin T(RM) cells to protect against an antigenically unrelated virus. These data describe a mechanism by which tissue-resident memory CD8(+) T cells protect previously infected sites that is rapid, amplifies the activation of a small number of cells into an organ-wide response, and has the capacity to control escape variants.

MHC class I molecules devoid of peptide are expressed on the cell surface of the mouse mutant lymphoma cell line RMA-S upon culture at reduced temperature. Empty class I molecules are thermolabile at the cell surface and in detergent lysates, but can be stabilized by the addition of presentable peptide; peptide binding appears to be a rapid process. Furthermore, class I molecules on the surface of RMA-S (H-2b haplotype) cells cultured at 26 degrees C can efficiently and specifically bind iodinated peptide presented by H-2Kb. Binding of iodinated peptide is also observed at a lower level for nonmutant cells (RMA) cultured at 26 degrees C. These experiments underscore the role for peptide in maintenance of the structure of class I molecules and, more importantly, provide two assay systems to study the interactions of peptides with MHC class I molecules independent of the availability of T cells that recognize a particular peptide-MHC class I complex.

Outsourcing cancer immunotherapy Successful cancer immunotherapy depends on a patient's T cells recognizing tumor-specific mutations and then waging a lethal attack. Despite tumors harboring many mutations, most individuals have very few T cells that respond to these so-called “neo-antigens.” Strønen et al. isolated T cells from healthy donors that responded to predicted neo-antigens expressed by melanomas taken from three patients, sometimes including neo-antigens that the patient's own T cells ignored (see the Perspective by Yadav and Delamarre). Testing whether such an outsourcing strategy could improve clinical outcomes will be an important next step. Science , this issue p. 1337 ; see also p. 1275

Malignant transformation of cells depends on accumulation of DNA damage. Over the past years we have learned that the T cell–based immune system frequently responds to the neoantigens that arise as a consequence of this DNA damage. Furthermore, recognition of neoantigens appears an important driver of the clinical activity of both T cell checkpoint blockade and adoptive T cell therapy as cancer immunotherapies. Here we review the evidence for the relevance of cancer neoantigens in tumor control and the biological properties of these antigens. We discuss recent technological advances utilized to identify neoantigens, and the T cells that recognize them, in individual patients. Finally, we discuss strategies that can be employed to exploit cancer neoantigens in clinical interventions.

T cells detect infection of cells by recognizing peptide fragments of foreign proteins bound to class I molecules of the major histocompatibility complex (MHC) on the surface of the infected cell. MHC class I molecules bind peptide in the endoplasmic reticulum, and analysis of mutant cells has demonstrated that an adequate supply of peptides requires the presence of two genes in the MHC class II locus that encode proteins called transporters associated with antigen processing (TAP) 1 and 2. TAP1 and TAP2 are members of the ATP-binding cassette family of membrane translocators. In this study, we demonstrate in a cell-free system that TAP1 is part of an ATP-dependent, sequence-specific, peptide translocator.

The outcome of patients with macroscopic stage III melanoma is poor. Neoadjuvant treatment with ipilimumab plus nivolumab at the standard dosing schedule induced pathological responses in a high proportion of patients in two small independent early-phase trials, and no patients with a pathological response have relapsed after a median follow up of 32 months. However, toxicity of the standard ipilimumab plus nivolumab dosing schedule was high, preventing its broader clinical use. The aim of the OpACIN-neo trial was to identify a dosing schedule of ipilimumab plus nivolumab that is less toxic but equally effective.OpACIN-neo is a multicentre, open-label, phase 2, randomised, controlled trial. Eligible patients were aged at least 18 years, had a WHO performance status of 0-1, had resectable stage III melanoma involving lymph nodes only, and measurable disease according to the Response Evaluation Criteria in Solid Tumors version 1.1. Patients were enrolled from three medical centres in Australia, Sweden, and the Netherlands, and were randomly assigned (1:1:1), stratified by site, to one of three neoadjuvant dosing schedules: group A, two cycles of ipilimumab 3 mg/kg plus nivolumab 1 mg/kg once every 3 weeks intravenously; group B, two cycles of ipilimumab 1 mg/kg plus nivolumab 3 mg/kg once every 3 weeks intravenously; or group C, two cycles of ipilimumab 3 mg/kg once every 3 weeks directly followed by two cycles of nivolumab 3 mg/kg once every 2 weeks intravenously. The investigators, site staff, and patients were aware of the treatment assignment during the study participation. Pathologists were masked to treatment allocation and all other data. The primary endpoints were the proportion of patients with grade 3-4 immune-related toxicity within the first 12 weeks and the proportion of patients achieving a radiological objective response and pathological response at 6 weeks. Analyses were done in all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, number NCT02977052, and is ongoing with an additional extension cohort and to complete survival analysis.Between Nov 24, 2016 and June 28, 2018, 105 patients were screened for eligibility, of whom 89 (85%) eligible patients were enrolled and randomly assigned to one of the three groups. Three patients were excluded after randomisation because they were found to be ineligible, and 86 received at least one dose of study drug; 30 patients in group A, 30 in group B, and 26 in group C (accrual to this group was closed early upon advice of the Data Safety Monitoring Board on June 4, 2018 because of severe adverse events). Within the first 12 weeks, grade 3-4 immune-related adverse events were observed in 12 (40%) of 30 patients in group A, six (20%) of 30 in group B, and 13 (50%) of 26 in group C. The difference in grade 3-4 toxicity between group B and A was -20% (95% CI -46 to 6; p=0·158) and between group C and group A was 10% (-20 to 40; p=0·591). The most common grade 3-4 adverse events were elevated liver enzymes in group A (six [20%)]) and colitis in group C (five [19%]); in group B, none of the grade 3-4 adverse events were seen in more than one patient. One patient (in group A) died 9·5 months after the start of treatment due to the consequences of late-onset immune-related encephalitis, which was possibly treatment-related. 19 (63% [95% CI 44-80]) of 30 patients in group A, 17 (57% [37-75]) of 30 in group B, and nine (35% [17-56]) of 26 in group C achieved a radiological objective response, while pathological responses occurred in 24 (80% [61-92]) patients in group A, 23 (77% [58-90]) in group B, and 17 (65% [44-83]) in group C.OpACIN-neo identified a tolerable neoadjuvant dosing schedule (group B: two cycles of ipilimumab 1 mg/kg plus nivolumab 3 mg/kg) that induces a pathological response in a high proportion of patients and might be suitable for broader clinical use. When more mature data confirm these early observations, this schedule should be tested in randomised phase 3 studies versus adjuvant therapies, which are the current standard-of-care systemic therapy for patients with stage III melanoma.Bristol-Myers Squibb.

In vivo ‘cellular barcoding’ shows that early haematopoietic progenitors are heterogeneous in the cell types that they produce, and this is partly due to an ‘imprinting’ of fate in progenitors, including for a separate dendritic cell lineage. A fundamental question in biology is that of how different tissue types develop from single stem cells. This study tackles the question by following the fate of presumed 'multipotent' lymphoid-primed progenitors (LMPPs) at the single-cell level in mice. Shalin Naik and colleagues used a highly sensitive barcoding method to track the lineage output of hundreds of single LMPPs and haematopoietic stem cells in vivo. They find that not all presumed 'multipotent' progenitors are especially 'multi-outcome': rather, many LMPPs produce a single lineage of cells. The authors also found an unexpectedly large number of LMPPs with a unique dendritic cell potential, which suggests that the dendritic cell lineage is distinct from the myeloid and B-cell branches. Haematopoietic stem cells (HSCs) and their subsequent progenitors produce blood cells, but the precise nature and kinetics of this production is a contentious issue. In one model, lymphoid and myeloid production branch after the lymphoid-primed multipotent progenitor (LMPP)1, with both branches subsequently producing dendritic cells2. However, this model is based mainly on in vitro clonal assays and population-based tracking in vivo, which could miss in vivo single-cell complexity3,4,5,6,7. Here we avoid these issues by using a new quantitative version of ‘cellular barcoding’8,9,10 to trace the in vivo fate of hundreds of LMPPs and HSCs at the single-cell level. These data demonstrate that LMPPs are highly heterogeneous in the cell types that they produce, separating into combinations of lymphoid-, myeloid- and dendritic-cell-biased producers. Conversely, although we observe a known lineage bias of some HSCs11,12,13,14, most cellular output is derived from a small number of HSCs that each generates all cell types. Crucially, in vivo analysis of the output of sibling cells derived from single LMPPs shows that they often share a similar fate, suggesting that the fate of these progenitors was imprinted. Furthermore, as this imprinting is also observed for dendritic-cell-biased LMPPs, dendritic cells may be considered a distinct lineage on the basis of separate ancestry. These data suggest a ‘graded commitment’ model of haematopoiesis, in which heritable and diverse lineage imprinting occurs earlier than previously thought.

Ectopic lymphoid aggregates, termed tertiary lymphoid structures (TLSs), are formed in numerous cancer types, and, with few exceptions, their presence is associated with superior prognosis and response to immunotherapy. In spite of their presumed importance, the triggers that lead to TLS formation in cancer tissue and the contribution of these structures to intratumoral immune responses remain incompletely understood. Here, we discuss the present knowledge on TLSs in cancer, focusing on (i) the drivers of TLS formation, (ii) the function and contribution of TLSs to the antitumor immune response, and (iii) the potential of TLSs as therapeutic targets in human cancers.

Anti-CTLA-4 treatment improves the survival of patients with advanced-stage melanoma. However, although the anti-CTLA-4 antibody ipilimumab is now an approved treatment for patients with metastatic disease, it remains unknown by which mechanism it boosts tumor-specific T cell activity. In particular, it is unclear whether treatment amplifies previously induced T cell responses or whether it induces new tumor-specific T cell reactivities. Using a combination ultraviolet (UV)-induced peptide exchange and peptide-major histocompatibility complex (pMHC) combinatorial coding, we monitored immune reactivity against a panel of 145 melanoma-associated epitopes in a cohort of patients receiving anti-CTLA-4 treatment. Comparison of pre- and posttreatment T cell reactivities in peripheral blood mononuclear cell samples of 40 melanoma patients demonstrated that anti-CTLA-4 treatment induces a significant increase in the number of detectable melanoma-specific CD8 T cell responses (P = 0.0009). In striking contrast, the magnitude of both virus-specific and melanoma-specific T cell responses that were already detected before start of therapy remained unaltered by treatment (P = 0.74). The observation that anti-CTLA-4 treatment induces a significant number of newly detected T cell responses-but only infrequently boosts preexisting immune responses-provides strong evidence for anti-CTLA-4 therapy-enhanced T cell priming as a component of the clinical mode of action.

Adoptive T cell transfer for cancer, chronic infection, and autoimmunity is an emerging field that shows promise in recent trials. Using the principles of synthetic biology, advances in cell culture and genetic engineering have made it possible to generate human T cells that display desired specificities and enhanced functionalities compared with the natural immune system. The prospects for widespread availability of engineered T cells have changed dramatically, given the recent entry of the pharmaceutical industry to this arena. Here, we discuss some of the challenges--such as regulatory, cost, and manufacturing--and opportunities, including personalized gene-modified T cells, that face the field of adoptive cellular therapy.

Upon infection, antigen-specific CD8(+) T lymphocyte responses display a highly reproducible pattern of expansion and contraction that is thought to reflect a uniform behavior of individual cells. We tracked the progeny of individual mouse CD8(+) T cells by in vivo lineage tracing and demonstrated that, even for T cells bearing identical T cell receptors, both clonal expansion and differentiation patterns are heterogeneous. As a consequence, individual naïve T lymphocytes contributed differentially to short- and long-term protection, as revealed by participation of their progeny during primary versus recall infections. The discordance in fate of individual naïve T cells argues against asymmetric division as a singular driver of CD8(+) T cell heterogeneity and demonstrates that reproducibility of CD8(+) T cell responses is achieved through population averaging.

Cancer-associated systemic inflammation is strongly linked to poor disease outcome in patients with cancer1,2. For most human epithelial tumour types, high systemic neutrophil-to-lymphocyte ratios are associated with poor overall survival3, and experimental studies have demonstrated a causal relationship between neutrophils and metastasis4,5. However, the cancer-cell-intrinsic mechanisms that dictate the substantial heterogeneity in systemic neutrophilic inflammation between tumour-bearing hosts are largely unresolved. Here, using a panel of 16 distinct genetically engineered mouse models for breast cancer, we uncover a role for cancer-cell-intrinsic p53 as a key regulator of pro-metastatic neutrophils. Mechanistically, loss of p53 in cancer cells induced the secretion of WNT ligands that stimulate tumour-associated macrophages to produce IL-1β, thus driving systemic inflammation. Pharmacological and genetic blockade of WNT secretion in p53-null cancer cells reverses macrophage production of IL-1β and subsequent neutrophilic inflammation, resulting in reduced metastasis formation. Collectively, we demonstrate a mechanistic link between the loss of p53 in cancer cells, secretion of WNT ligands and systemic neutrophilia that potentiates metastatic progression. These insights illustrate the importance of the genetic makeup of breast tumours in dictating pro-metastatic systemic inflammation, and set the stage for personalized immune intervention strategies for patients with cancer.

The cytotoxic activity of myeloid cells is regulated by a balance of signals that are transmitted through inhibitory and activating receptors. The Cluster of Differentiation 47 (CD47) protein, expressed on both healthy and cancer cells, plays a pivotal role in this balance by delivering a “don’t eat me signal” upon binding to the Signal-regulatory protein alpha (SIRPα) receptor on myeloid cells. Here, we review the current understanding of the role of the CD47-SIRPα axis in physiological tissue homeostasis and as a promising therapeutic target in, among others, oncology, fibrotic diseases, atherosclerosis, and stem cell therapies. We discuss gaps in understanding and highlight where additional insight will be beneficial to allow optimal exploitation of this myeloid cell checkpoint as a target in human disease.

Distinct types of CD4(+) T cells protect the host against different classes of pathogens. However, it is unclear whether a given pathogen induces a single type of polarized T cell. By combining antigenic stimulation and T cell receptor deep sequencing, we found that human pathogen- and vaccine-specific T helper 1 (T(H)1), T(H)2, and T(H)17 memory cells have different frequencies but comparable diversity and comprise not only clones polarized toward a single fate, but also clones whose progeny have acquired multiple fates. Single naïve T cells primed by a pathogen in vitro could also give rise to multiple fates. Our results unravel an unexpected degree of interclonal and intraclonal functional heterogeneity of the human T cell response and suggest that polarized responses result from preferential expansion rather than priming.

B cells are well documented as APC; however, their role in supporting and programming the T cell response in vivo is still unclear. Studies using B cell-deficient mice have given rise to contradictory results. We have used mixed BM chimeric mice to define the contribution that B cells make as APC. When the B cell compartment is deficient in MHC class II, while other APC are largely normal, T cell clonal expansion is significantly reduced and the differentiation of T cells into cytokine-secreting effector cells is impaired (in particular, Th2 cells). The development of the memory T cell populations is also decreased. Although MHC class II-mediated presentation by B cells was crucial for an optimal T cell response, neither a B cell-specific lack of CD40 (influencing costimulation) nor lymphotoxin alpha (influencing lymphoid tissue architecture) had any effect on the T cell response. We conclude that in vivo B cells provide extra and essential Ag presentation capacity over and above that provided by dendritic cells, optimizing expansion and allowing the generation of memory and effector T cells.

Somatic recombination and accumulation of mutations in V-D-J segments result in vast heterogeneity of T-cell receptor (TCR) and immunoglobulin repertoires1,2. High-throughput profiling of immune receptors has become an important tool for studies of adaptive immunity and for the development of diagnostics, vaccines, and immunotherapies3,4,5,6,7. There are efficient molecular and software tools for the targeted sequencing of TCR and immunoglobulin repertoires6,8, including MiXCR, developed by our team9. However, sufficient amount and quality of tissue or extracted RNA or DNA are not always available for analysis.

Recent work has demonstrated that following the clearance of infection a stable population of memory T cells remains present in peripheral organs and contributes to the control of secondary infections. However, little is known about how tissue-resident memory T cells behave in situ and how they encounter newly infected target cells. Here we demonstrate that antigen-specific CD8(+) T cells that remain in skin following herpes simplex virus infection show a steady-state crawling behavior in between keratinocytes. Spatially explicit simulations of the migration of these tissue-resident memory T cells indicate that the migratory dendritic behavior of these cells allows the detection of antigen-expressing target cells in physiologically relevant time frames of minutes to hours. Furthermore, we provide direct evidence for the identification of rare antigen-expressing epithelial cells by skin-patrolling memory T cells in vivo. These data demonstrate the existence of skin patrol by memory T cells and reveal the value of this patrol in the rapid detection of renewed infections at a previously infected site.

The immune system can mount T cell responses against tumors; however, the antigen specificities of tumor-infiltrating lymphocytes (TILs) are not well understood. We used yeast-display libraries of peptide-human leukocyte antigen (pHLA) to screen for antigens of "orphan" T cell receptors (TCRs) expressed on TILs from human colorectal adenocarcinoma. Four TIL-derived TCRs exhibited strong selection for peptides presented in a highly diverse pHLA-A∗02:01 library. Three of the TIL TCRs were specific for non-mutated self-antigens, two of which were present in separate patient tumors, and shared specificity for a non-mutated self-antigen derived from U2AF2. These results show that the exposed recognition surface of MHC-bound peptides accessible to the TCR contains sufficient structural information to enable the reconstruction of sequences of peptide targets for pathogenic TCRs of unknown specificity. This finding underscores the surprising specificity of TCRs for their cognate antigens and enables the facile indentification of tumor antigens through unbiased screening.

Review21 December 2012free access The cancer antigenome Bianca Heemskerk Bianca Heemskerk Department of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Pia Kvistborg Pia Kvistborg Department of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Ton N M Schumacher Corresponding Author Ton N M Schumacher Department of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Bianca Heemskerk Bianca Heemskerk Department of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Pia Kvistborg Pia Kvistborg Department of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Ton N M Schumacher Corresponding Author Ton N M Schumacher Department of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Author Information Bianca Heemskerk1, Pia Kvistborg1 and Ton N M Schumacher 1 1Department of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands *Corresponding author. Department of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands. Tel.:+31 20 5122072; Fax:+31 20 5122057; E-mail: [email protected] The EMBO Journal (2013)32:194-203https://doi.org/10.1038/emboj.2012.333 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cancer cells deviate from normal body cells in two immunologically important ways. First, tumour cells carry tens to hundreds of protein-changing mutations that are either responsible for cellular transformation or that have accumulated as mere passengers. Second, as a consequence of genetic and epigenetic alterations, tumour cells express a series of proteins that are normally not present or present at lower levels. These changes lead to the presentation of an altered repertoire of MHC class I-associated peptides. Importantly, while there is now strong clinical evidence that cytotoxic T-cell activity against such tumour-associated antigens can lead to cancer regression, at present we fail to understand which tumour-associated antigens form the prime targets in effective immunotherapies. Here, we describe how recent developments in cancer genomics will make it feasible to establish the repertoire of tumour-associated epitopes on a patient-specific basis. The elucidation of this ‘cancer antigenome’ will be valuable to reveal how clinically successful immunotherapies mediate their effect. Furthermore, the description of the cancer antigenome should form the basis of novel forms of personalized cancer immunotherapy. Introduction It has long been known that T cells have the capacity to eliminate human cancer cells, as shown by the anti-leukaemic effects of allogeneic haematopoietic stem cell transplantation and donor lymphocyte infusions (Barnes et al, 1956; Sprangers et al, 2007). In this setting, the presence of polymorphic antigens on the surface of tumour cells that are foreign to the infused T cells forms the basis of tumour recognition. However, in recent years, it has become apparent that also autologous T cells can—at least in some cases—show profound anti-tumour reactivity in cancer patients. In particular in melanoma, the clinical activity of tumour-reactive T cells has now been documented in a large number of clinical trials. Specifically, treatment of patients with the antibody ipilimumab, which interferes with the function of the T-cell inhibitory receptor CTLA4, has been shown to result in a significant effect on patient survival (Hodi et al, 2010). More recently, treatment of patients with antibodies that block signalling through PD-1, a second inhibitory receptor on T cells, has also shown considerable clinical promise (Brahmer et al, 2012; Topalian et al, 2012). As an alternative to these antibody-based strategies, regression of even large tumour masses has been observed upon the adoptive transfer of autologous, ex vivo expanded, tumour-infiltrating lymphocytes (TILs) obtained from metastatic melanoma lesions. Objective response rates for anti-CTLA4 therapy and TIL treatment are ∼10–15% in randomized phase III studies and ∼50% in multiple non-randomized phase I–II studies, respectively, and both treatments can induce complete tumour regressions in a subset of treated patients (Rosenberg et al, 2008; Hodi et al, 2010; Robert et al, 2011). These clinical data provide clear evidence that human tumour cells can express antigenic determinants—epitopes—that can be the target of autologous T cells, and that enhancement of such reactivity can lead to cancer regression. Importantly, in the majority of patients responding to these immunotherapeutic strategies, we do not know which antigens are the target(s) in the observed tumour regression. Such knowledge would be of obvious value, as it could allow one to steer reactivity towards antigens of interest. Within this review, we will distinguish two main classes of tumour-specific antigens that together make up the cancer antigenome, the class of ‘neo-antigens’ and the class of non-mutated ‘self-antigens’ (Figure 1). The presentation of neo-antigens by tumour cells is a direct consequence of the large number of somatic mutations that are found in human tumours. Such neo-antigens may be newly displayed at the surface of tumour cells because a mutation increases the efficiency with which a peptide is presented by MHC molecules, for instance by increasing its binding affinity. Alternatively, generation of neo-antigens that can be recognized by T cells may occur when a mutation alters the T-cell receptor (TCR)-exposed area of a peptide that is also presented by MHC molecules in its non-mutated form. Figure 1.Distribution of self- versus neo-antigens and their tolerance and toxicity profile. Schematic overview of the different types of antigens that can be potentially targeted by T cells: self-antigens and neo-antigens. Blue denotes the shared antigens between different patients and red represents unique, patient-specific antigens. At present, there is little evidence that shared antigens can be truly patient specific, although some of the shared antigens are only expressed in a fraction of a given human tumour type. Download figure Download PowerPoint Presentation of the second class of tumour-associated epitopes, the non-mutated ‘self-antigens’, involves the display of epitopes from gene products that are normally only expressed in a restricted set of cell types. Thus, rather than being a direct consequence of mutations, presentation of self-antigens is a consequence of the tissue-specific or transformation-induced gene expression profile of tumour cells. An important distinction between these two classes of antigens is that T-cell reactivity against self-antigens can only occur when T-cell tolerance towards a given antigen is incomplete, and there is strong data to suggest that for at least part of the tumour-associated self-antigens, the T-cell repertoire available for tumour recognition is of a lower avidity. In contrast, as neo-antigens are fully tumour specific, central T-cell tolerance does not form a concern. By the same token, T-cell responses against neo-antigens are not expected to result in autoimmune toxicity against healthy tissues, making immunotherapeutic manipulation highly attractive from a theoretical point of view (Figure 1). In spite of the significant appeal of neo-antigen-specific T-cell reactivity, our current understanding of tumour-specific T-cell immunity is in large part restricted to the class of non-mutated self-antigens. This strong bias is a consequence of the fact that the majority of the mutations in human tumours that could lead to neo-antigens are unique to that tumour (see below). Thus, contrary to the non-mutated self-antigens that are to a substantial extent shared between patients, most neo-antigens are patient specific (Figure 1). Importantly, with recent advances in cancer genomics and immunomonitoring, the analysis of the full repertoire of tumour-associated antigens on a patient-specific basis has now become a realistic goal. Current knowledge of the repertoire of human tumour antigens Our first understanding of the molecular determinants on tumour cells that can be recognized by human T cells came in 1991 when Boon and co-workers isolated the MAGE-1 antigen via cDNA expression cloning, using recognition by tumour-reactive cytotoxic T lymphocytes (CTLs) from a melanoma patient as a readout system (van der Bruggen et al, 1991). In the following decades, a large number of self-antigens that are aberrantly expressed in human tumours has been discovered, either using tumour-reactive CTL or using patient sera (serological expression cloning, SEREX) as a readout system. In addition, a large set of tumour-associated epitopes has been identified by analysing which peptides from candidate tumour antigens (i.e., proteins that are highly/aberrantly expressed in tumours) could lead to the induction of a tumour-reactive T-cell response, an approach termed as ‘reverse immunology’ (Kawakami et al, 2004). The self-antigens that have been discovered—predominantly in melanoma—by these different approaches can be divided into several subclasses: A first class represents epitopes for which expression is normally largely restricted to male germline cells. These cancer-germline (C/G) antigens are frequently overexpressed in tumours due to demethylation events, as has been shown for the prototypic MAGE antigens (A, B and C) (Chomez et al, 2001). A second class of self-antigens consists of the tissue differentiation antigens, antigens that are shared by tumour cells and the tissue it originated from. A well-known example of this class is the Melan-A/MART-1 antigen that is expressed in melanoma but also in healthy melanocytes (Coulie et al, 1994; Kawakami et al, 1994). A third class of epitopes is derived from proteins that are overexpressed in tumours, but that are also expressed in healthy tissues, such as the Her-2/Neu and PRAME antigens (Fisk et al, 1995; Kessler et al, 2001). While we now know the identity of a large number of non-mutated self-antigens that are expressed in human cancer (e.g., for HLA-A2, some 150 epitopes from non-mutated self-antigens have been identified; (Andersen et al, 2012b), it is not immediately clear whether recognition of these antigens is associated with cancer regression, and arguments can be made both in favour and against their importance. Specifically, a large series of clinical trials has evaluated whether induction of T-cell responses against non-mutated self-antigens was associated with clinical efficacy. While occasional clinical responses have been observed in these trials, clinical response rates have on general been disappointingly low (around 3–5%, reviewed in Rosenberg et al, 2004 and Boon et al, 2006), in some cases even when high numbers of circulating peptide-reactive T cells could be obtained (Rosenberg et al, 2005). In more recent years, the potential of targeting non-mutated self-antigens has also been evaluated in clinical studies in which T cells were infused that had been genetically engineered to express a tumour-reactive TCR. In trials that utilized TCRs specific for the melanocyte differentiation antigens MART-1 and gp100, a modest clinical efficacy was observed (response rates of ∼15–20%; Morgan et al, 2006; Johnson et al, 2009) and T-cell infusion was accompanied by significant toxicity due to recognition of healthy tissues expressing the same antigen. More encouraging, in a trial in which a TCR specific for the NY-ESO-1 C/G antigen was used in patients with metastatic melanoma and synovial sarcoma, clinical response rates were higher (∼50%), although these responses were not always durable (Robbins et al, 2011). Based on these data, but also based on recent analyses of T-cell reactivity in patients that received ipilimumab or TIL therapy (see below), it seems possible that a significant part of the clinically relevant T-cell activity in human cancers does not involve recognition of self-antigens, but could involve recognition of patient-specific neo-antigens. A small number of studies have aimed to address the potential importance of neo-antigen recognition by (highly involved) expression cloning approaches, and these studies led to the identification of a number of tumour-specific neo-antigens that are recognized by autologous T cells. Furthermore, in a seminal study by Lennerz et al (2005) an unbiased analysis was performed for antigens recognized by different T-cell cultures from a single melanoma patient. This analysis led to the identification of a series of neo-antigens, and T-cell reactivity against these neo-antigens dominated the tumour-specific T-cell response in this patient (Lennerz et al, 2005). Based on these early studies, it is clear that recognition of human neo-antigens can occur in patients, even in the absence of immunotherapy. However, data have been lacking to reveal whether such responses are enhanced by therapies. Likewise, whether such responses are a crucial component of therapeutic efficacy or can be selectively enhanced has not been established. The cancer genome The development of second-generation sequencing technology has made it feasible to describe the full mutation load (i.e., the ‘genetic landscape’) of human tumours (Meyerson et al, 2010; Zhao and Grant, 2011). Specifically, comparison of the genome sequence of cancer tissue to that of non-transformed tissue from the same patient has been used to reveal the full range of genomic alterations within a tumour—including nucleotide substitutions, structural rearrangements and copy number alterations (Meyerson et al, 2010). In early studies that described cancer genomes, analysis was still restricted to subsets of the genome (e.g., all kinase genes; the kinome), because of high costs and limited sequencing capacity. However, with technology advancing and costs dropping, analysis of the entire protein-encoding part of the genome (the exome) or the entire cancer genome has become feasible and will soon become routine. Exome sequencing and whole genome sequencing each have their specific advantages and disadvantages in the detection of mutations that could potentially be relevant for the immune system. Exome sequencing (which covers only the ∼1% coding region of the genome) has the clear advantage that it can provide a higher sequence coverage and consequently a higher ability to detect mutations, including mutations that are only expressed by part of the tumour cells. As a downside, this method is still somewhat limited by our knowledge of the protein-coding parts of the genome and by uneven capture efficiency across exons and, as a consequence, some mutations may be missed. In one study, a hepatocellular cancer was analysed by both whole genome and exome sequencing. The results obtained showed that a significant fraction (25 of the 63) of the mutations that were identified by whole genome sequencing could not be detected by exome sequencing, with the missed mutations primarily being present in areas with low coverage (Totoki et al, 2011). On the other hand, data from the Sanger Institute have demonstrated that of a set of mutations that had previously been identified in cell lines by conventional sequence analysis, the vast majority (313 out of 326 mutations, 97%) was also identified by whole exome sequencing (S Behjati and M Stratton, personal communication). As a third alternative to whole genome and exome sequencing, tumour-specific mutations within the set of expressed genes (the transcriptome) may be identified by RNA sequencing. This approach has the advantage over exome sequencing that it is not limited to known genes, and thereby has the potential to also detect novel transcripts, for instance formed by intragenic fusions or, in case of pathogen-induced cancers, non-human genes. As a downside, it is difficult to identify a matched control, as mRNA expression profiles of tumour and normal tissue will not be identical, and this makes it challenging to distinguish tumour-specific mutations from polymorphisms. As a second concern, the ability to reliably call mutations within RNA species that are only present at a low level, either because of low level gene expression or because of low mRNA stability (for instance due to non-sense-mediated RNA decay) will be limited. Mutation rates and patterns in human cancers Within a period of only a few years, second-generation sequencing has allowed a description of the genomic changes within thousands of human cancer exomes and genomes (Stratton, 2011; (http://www.sanger.ac.uk/genetics/CGP/cosmic). The genome changes encountered include single-base substitutions, small insertions and deletions (together referred to as indels), copy number changes, DNA rearrangements, but also structural variants at the level of the chromosome (e.g., amplifications, deletions and fusion/translocation events). Single-base substitutions (single-nucleotide variants, SNVs) comprise the vast majority of mutations that are encountered in human cancers and range in number from ∼1000 to 100 000 in the entire genome (Stratton, 2011). However, the number of mutations that is relevant to immune recognition—mutations that lead to the production of a protein sequence that is absent in the germline—is much smaller. This type of mutations includes indels that alter the open reading frame of proteins, intragenic fusions that lead to the production of a fusion sequence, splice site mutations and, in particular, non-synonymous SNVs within exons. Interestingly, sequencing studies have revealed a substantial variability between the number of non-synonymous changes between different tumour types (Greenman et al, 2007; Stratton, 2011). For instance, the description of the first cancer genome sequence in 2008 revealed a total of 10 non-synonymous mutations in the coding regions of an acute myeloid leukaemia (Ley et al, 2008). Somewhat higher numbers of non-synonymous mutations within exonic regions have been reported for a basal-like breast cancer (28, n=1) (Ding et al, 2010), glioblastoma multiforme (average of 36, n=21) (Parsons et al, 2008), pancreatic tumours (average of 48, n=24) (Jones et al, 2008b), hepatocellular cancer (63, n=1) (Totoki et al, 2011), and colon and breast tumours (average of 76 and 84, respectively, n=11 each) (Sjoblom et al, 2006; Wood et al, 2007). Furthermore, very high mutation rates have been observed in cancers with substantial exogenous mutagenic exposures, such as ultraviolet light in the case of melanoma (187 non-synonymous mutations in a case report and an average of 201 mutations in 14 tumours) (Pleasance et al, 2010a; Wei et al, 2011), or exposure to tobacco smoke carcinogens in lung cancers (94 non-synonymous mutations in a lung cancer cell line and >300 mutations in one primary tumour) (Lee et al, 2010; Pleasance et al, 2010b). Finally, as expected, tumours with mismatch repair deficiencies also carry large numbers of mutations (Greenman et al, 2007). As an example, whereas microsatellite stable (MSS) colon cancers carried on average ∼100 mutations, the number of mutations in two microsatellite instable (MSI) colon cancers was 532 and 915 (Timmermann et al, 2010). The above data provide ballpark figures on the number of mutations that are present in a high proportion of the cells within different types of human tumours at a given point in time. However, tumour cells continue to accumulate mutations throughout the stages of tumour progression. Furthermore, metastasis can involve the distal outgrowth of tumour cells that were derived from a subclone that was small within the primary tumour. Because of this potential for genetic heterogeneity, it is important to also understand the degree of kinship between different areas within the same tumour, and between different metastatic lesions within a patient. Heterogeneity within primary tumours and between tumour metastases Comparative sequence analysis of multiple intrapatient lesions has now been performed for a number of human tumour types. In a study by Jones et al (2008 a,b), it was evaluated to what extent mutations found in an index metastatic lesion were also present in the matched primary tumours and at other metastatic sites within the same patient. Sanger sequencing revealed that only a minor fraction (around 3%) of the mutations that were identified within the index lesions were not present in the primary tumour or other metastases (Jones et al, 2008a). More recently, whole genome sequencing has been employed to compare the mutation profile of a primary basal-like breast cancer and a brain metastasis (Ding et al, 2010). In these tumours, a total of 50 mutations (including SNV and indels) were found, of which 48 were shared between the two. Furthermore, similar results were obtained in a hepatocellular cancer, in which 205 out of 214 mutations (96%) were present in both the primary tumour and two metastases (Tao et al, 2011). In contrast to these data that show a high degree of kinship between different tumour sites, one report documented that 19 out of 32 mutations within a metastasized lobular breast cancer were not present in the primary tumour that was resected 9 years earlier, prior to radiotherapy. Furthermore, of the 11 mutations that were shared, only 5 were abundant with the primary tumour, whereas the remainder had a very low allele frequency, in between 1 and 13% (Shah et al, 2009). Likewise, a substantial fraction (around 35%) of mutations observed in different metastatic lesions of patients with pancreatic cancer was not shared between individual metastases (Yachida et al, 2010). Finally, a recent study that analysed heterogeneity between different metastases and also between different regions of the primary tumour in patients with renal cell carcinoma demonstrated that, in these cases, the fraction of mutations that were detected in only some of the lesions was very high (∼65%). Furthermore, substantial heterogeneity was even observed within the primary tumour mass. Thus, for a randomly identified mutation, the likelihood that this mutation would be present at all tumour sites within these patients was only one in three (Gerlinger et al, 2012). Collectively, these data show a striking degree of variability between patients with respect to the genetic kinship between primary and metastasized tumours, with in some cases different lesions being nearly identical, whereas in other cases the majority of mutations being private. It seems plausible that kinship between different tumour sites will in part differ in a systematic way between tumour types, for instance depending on whether metastasis is likely to occur early or late in the disease process. In addition, disparity between different lesions is likely to be influenced by therapy, both by the direct DNA damaging effect of for instance radiation therapy or alkylating agents, but also by the genetic bottleneck that an efficient therapy will form. Finally, the kinship between different lesions will in part be governed by chance; whether the cell that forms a given metastasis happens to derive from a dominant or minor clone within the primary tumour. More data will be required to understand which of the above factors is most dominant. However, even with the limited data currently available, it is already apparent that genetic heterogeneity with human tumours will be an important factor to take into account when targeting tumour-specific neo-antigens. Driver and passenger mutations Classically, the mutations that are found in cancer cells are divided into two categories, ‘Drivers’ and ‘Passengers’, according to their role in cancer development. Driver mutations are those mutations that confer a selective advantage to the cells that carry them, and these include inactivating mutations in tumour suppressor genes and activating mutations in oncogenes. All other mutations, which are neutral with respect to cell division or death, are considered as ‘passengers’. Such passengers were either already present in the ancestor cancer cell at the moment it acquired one of its driver mutations, or were acquired (by a subclone of tumour cells) during subsequent tumour growth (Stratton et al, 2009). The number of human genes for which a role in tumour development has been shown or is suspected is substantial. To date, ∼400 (2%) of the ∼22 000 protein-coding genes have been reported to have recurrent mutations in human cancer and are therefore likely to confer a selective advantage (Lee et al, 2010; Stratton, 2011). Furthermore, as many of these driver mutations only occur in a low fraction of tumours (Wood et al, 2007), our knowledge of recurring—and hence presumed to be driver—mutations in human cancer is likely to still be incomplete. Nevertheless, even though the number of driver mutations that can occur in human tumours is substantial, it is important to realize that most of the mutations that are found in a given tumour are likely to be passengers that are neutral to tumour growth (estimated at 85% in a study by Wood et al, 2007). This implies that most of the potential T-cell reactivity towards neo-antigens will be directed against mutated gene products that are dispensable for tumour growth. The consequence of T-cell targeting of drivers and passengers is described below. In addition, we will introduce a third class of mutations, called ‘essential passengers’. From cancer genomes to cancer antigenomes The mutational landscapes described above indicate that there is a clear opportunity for the immune system to distinguish tumour cells from healthy tissue. How can genomic information on human cancers be utilized to understand which mutations may result in T-cell recognition? As discussed above, only those mutations that result in the expression of a non-germline protein sequence can lead to the formation of neo-antigens. As for most tumours the bulk of such mutations are formed by non-synonymous SNV we will here focus on this category of mutations, but the same analysis pipeline applies to for instance indels or gene fusions that alter germline protein sequences. The majority of MHC class I binding peptides is nine amino acids long. Thus, when we for now ignore the contribution of longer peptides, nine neo-peptides can be formed for each non-synonymous SNV, in which the mutated residue is present at either one of the positions within the peptide. However, of those neo-peptides, only a small fraction (roughly a few %) will bind with high affinity to a given HLA allele. Importantly, through seminal work of Rammensee and colleagues in the early nineties (Falk et al, 1991) and work by many groups since then, we have obtained a very detailed understanding of the ligand preference of human MHC class I alleles. Therefore, with the repertoire of protein-changing mutations in a cancer genome identified, it is straightforward to predict with reasonable accuracy which encoded peptides are likely to bind to the MHC class I alleles expressed by that patient, using algorithms such as NetMHC (Buus et al, 2003). As an example of such an in silico epitope prediction, Segal et al (2008) have analysed the potential for neo-antigen formation using a set of 1152 mutations that were found in 11 colon and 11 breast cancers. The results obtained showed that, on average, 7–10 mutated peptides were predicted as ligands for the human MHC class I alleles (HLA-A0201) for these 2 tumour types. Extrapolation of these data to the 6 different HLA class I alleles (-A, -B and -C, 2 each) that can be expressed results in a total number of ∼40 to ∼60 potential targets for T-cell recognition per tumour. However, a number of factors that will determine which of these predicted MHC ligands can actually be seen by T cells should be taken into account. (i) Mutations in genes that are not expressed within a given tumour is a non-event from an immunological point of view. Thus, the number of mutations that can be expected to be of immunological interest can be obtained by correcting for the fraction of genes expressed in the average tumour, and perhaps even somewhat more, as DNA repair is more efficient for transcribed genes. (ii) While binding of mutated peptides to MHC molecules is probably the most significant bottleneck in epitope presentation, it is not the only one. Specifically, the protein that contains the mutated residue needs to be processed—primarily by the proteasome—such that the peptide that has the potential to bind to MHC class I molecules is actually produced. Furthermore, this peptide then has to be transported into the ER lumen by the TAP1/TAP2 transporter to allow assembly with MHC class I, providing another—albeit minor—bottleneck. (iii) Even if a given mutated peptide is presented by MHC class I at the cell surface, this does not guarantee T-cell recognition. Specifically, T-cell recognition can only occur when TCRs that have the ability to recognize the mutant epitope but not the parental peptide exist within the T-cell repertoire. While prior data indicate that the immune system has a high ability to distinguish even minor variations in MHC-bound peptides, certain types of mutation, such as alterations at the N-terminal peptide residue or conservative substitutions at other positions can be missed (Kessels et al, 2004). Because of the above factors, the number of mutated epitopes that will actually be presented at the cell surface of tumour cells and can also be re

Neoadjuvant ipilimumab plus nivolumab showed high pathologic response rates (pRRs) in patients with macroscopic stage III melanoma in the phase 1b OpACIN ( NCT02437279 ) and phase 2 OpACIN-neo ( NCT02977052 ) studies1,2. While the results are promising, data on the durability of these pathologic responses and baseline biomarkers for response and survival were lacking. After a median follow-up of 4 years, none of the patients with a pathologic response (n = 7/9 patients) in the OpACIN study had relapsed. In OpACIN-neo (n = 86), the 2-year estimated relapse-free survival was 84% for all patients, 97% for patients achieving a pathologic response and 36% for nonresponders (P < 0.001). High tumor mutational burden (TMB) and high interferon-gamma-related gene expression signature score (IFN-γ score) were associated with pathologic response and low risk of relapse; pRR was 100% in patients with high IFN-γ score/high TMB; patients with high IFN-γ score/low TMB or low IFN-γ score/high TMB had pRRs of 91% and 88%; while patients with low IFN-γ score/low TMB had a pRR of only 39%. These data demonstrate long-term benefit in patients with a pathologic response and show the predictive potential of TMB and IFN-γ score. Our findings provide a strong rationale for a randomized phase 3 study comparing neoadjuvant ipilimumab plus nivolumab versus standard adjuvant therapy with antibodies against the programmed cell death protein-1 (anti-PD-1) in macroscopic stage III melanoma.

T cells are key players in cancer immunotherapy, but strategies to expand tumor-reactive cells and study their interactions with tumor cells at the level of an individual patient are limited. Here we describe the generation and functional assessment of tumor-reactive T cells based on cocultures of tumor organoids and autologous peripheral blood lymphocytes. The procedure consists of an initial coculture of 2 weeks, in which tumor-reactive T cells are first expanded in the presence of (IFNγ-stimulated) autologous tumor cells. Subsequently, T cells are evaluated for their capacity to carry out effector functions (IFNγ secretion and degranulation) after recognition of tumor cells, and their capacity to kill tumor organoids. This strategy is unique in its use of peripheral blood as a source of tumor-reactive T cells in an antigen-agnostic manner. In 2 weeks, tumor-reactive CD8+ T-cell populations can be obtained from ~33–50% of samples from patients with non-small-cell lung cancer (NSCLC) and microsatellite-instable colorectal cancer (CRC). This enables the establishment of ex vivo test systems for T-cell-based immunotherapy at the level of the individual patient. Tumor-reactive T cells are generated by coculturing tumor organoids and autologous peripheral blood lymphocytes and are evaluated for their capacity to carry out effector functions after recognition of tumor cells and whether they kill tumor organoids.

New opportunities are needed to increase immune checkpoint blockade (ICB) benefit. Whereas the interferon (IFN)γ pathway harbors both ICB resistance factors and therapeutic opportunities, this has not been systematically investigated for IFNγ-independent signaling routes. A genome-wide CRISPR/Cas9 screen to sensitize IFNγ receptor-deficient tumor cells to CD8 T cell elimination uncovered several hits mapping to the tumor necrosis factor (TNF) pathway. Clinically, we show that TNF antitumor activity is only limited in tumors at baseline and in ICB non-responders, correlating with its low abundance. Taking advantage of the genetic screen, we demonstrate that ablation of the top hit, TRAF2, lowers the TNF cytotoxicity threshold in tumors by redirecting TNF signaling to favor RIPK1-dependent apoptosis. TRAF2 loss greatly enhanced the therapeutic potential of pharmacologic inhibition of its interaction partner cIAP, another screen hit, thereby cooperating with ICB. Our results suggest that selective reduction of the TNF cytotoxicity threshold increases the susceptibility of tumors to immunotherapy.

Cancer cells can evade immune surveillance through the expression of inhibitory ligands that bind their cognate receptors on immune effector cells. Expression of programmed death ligand 1 in tumor microenvironments is a major immune checkpoint for tumor-specific T cell responses as it binds to programmed cell death protein-1 on activated and dysfunctional T cells1. The activity of myeloid cells such as macrophages and neutrophils is likewise regulated by a balance between stimulatory and inhibitory signals. In particular, cell surface expression of the CD47 protein creates a 'don't eat me' signal on tumor cells by binding to SIRPα expressed on myeloid cells2-5. Using a haploid genetic screen, we here identify glutaminyl-peptide cyclotransferase-like protein (QPCTL) as a major component of the CD47-SIRPα checkpoint. Biochemical analysis demonstrates that QPCTL is critical for pyroglutamate formation on CD47 at the SIRPα binding site shortly after biosynthesis. Genetic and pharmacological interference with QPCTL activity enhances antibody-dependent cellular phagocytosis and cellular cytotoxicity of tumor cells. Furthermore, interference with QPCTL expression leads to a major increase in neutrophil-mediated killing of tumor cells in vivo. These data identify QPCTL as a novel target to interfere with the CD47 pathway and thereby augment antibody therapy of cancer.

Abstract The COVID-19 pandemic caused by SARS-CoV-2 is a continuous challenge worldwide, and there is an urgent need to map the landscape of immunogenic and immunodominant epitopes recognized by CD8 + T cells. Here, we analyze samples from 31 patients with COVID-19 for CD8 + T cell recognition of 500 peptide-HLA class I complexes, restricted by 10 common HLA alleles. We identify 18 CD8 + T cell recognized SARS-CoV-2 epitopes, including an epitope with immunodominant features derived from ORF1ab and restricted by HLA-A*01:01. In-depth characterization of SARS-CoV-2-specific CD8 + T cell responses of patients with acute critical and severe disease reveals high expression of NKG2A, lack of cytokine production and a gene expression profile inhibiting T cell re-activation and migration while sustaining survival. SARS-CoV-2-specific CD8 + T cell responses are detectable up to 5 months after recovery from critical and severe disease, and these responses convert from dysfunctional effector to functional memory CD8 + T cells during convalescence.

Abstract Identifying strategies to improve the efficacy of immune checkpoint blockade (ICB) remains a major clinical need. Here, we show that therapeutically targeting the COX2/PGE2/EP2-4 pathway with widely used nonsteroidal and steroidal anti-inflammatory drugs synergized with ICB in mouse cancer models. We exploited a bilateral surgery model to distinguish responders from nonresponders shortly after treatment and identified acute IFNγ-driven transcriptional remodeling in responder mice, which was also associated with patient benefit to ICB. Monotherapy with COX2 inhibitors or EP2-4 PGE2 receptor antagonists rapidly induced this response program and, in combination with ICB, increased the intratumoral accumulation of effector T cells. Treatment of patient-derived tumor fragments from multiple cancer types revealed a similar shift in the tumor inflammatory environment to favor T-cell activation. Our findings establish the COX2/PGE2/EP2-4 axis as an independent immune checkpoint and a readily translatable strategy to rapidly switch the tumor inflammatory profile from cold to hot. Significance: Through performing in-depth profiling of mice and human tumors, this study identifies mechanisms by which anti-inflammatory drugs rapidly alter the tumor immune landscape to enhance tumor immunogenicity and responses to immune checkpoint inhibitors. See related commentary by Melero et al., p. 2372. This article is highlighted in the In This Issue feature, p. 2355

CD8+ tissue resident memory T cells (TRM cells) are essential for immune defence against pathogens and malignancies, and the molecular processes that lead to TRM cell formation are therefore of substantial biomedical interest. Prior work has demonstrated that signals present in the inflamed tissue micro-environment can promote the differentiation of memory precursor cells into mature TRM cells, and it was therefore long assumed that TRM cell formation adheres to a ‘local divergence’ model, in which TRM cell lineage decisions are exclusively made within the tissue. However, a growing body of work provides evidence for a ‘systemic divergence’ model, in which circulating T cells already become preconditioned to preferentially give rise to the TRM cell lineage, resulting in the generation of a pool of TRM cell-poised T cells within the lymphoid compartment. Here, we review the emerging evidence that supports the existence of such a population of circulating TRM cell progenitors, discuss current insights into their formation and highlight open questions in the field. CD8+ tissue resident memory T cells (TRM cells) are essential for defence against pathogens and malignancies. Prior work had indicated that these cells form within inflamed tissue, but there is emerging evidence that a pool of TRM cell precursors exists within the circulation. This Review examines the processes and signals within the lymphoid compartment that determine lineage decisions towards the formation of TRM cells.

DNA mismatch repair-deficient (MMR-d) cancers present an abundance of neoantigens that is thought to explain their exceptional responsiveness to immune checkpoint blockade (ICB)1,2. Here, in contrast to other cancer types3-5, we observed that 20 out of 21 (95%) MMR-d cancers with genomic inactivation of β2-microglobulin (encoded by B2M) retained responsiveness to ICB, suggesting the involvement of immune effector cells other than CD8+ T cells in this context. We next identified a strong association between B2M inactivation and increased infiltration by γδ T cells in MMR-d cancers. These γδ T cells mainly comprised the Vδ1 and Vδ3 subsets, and expressed high levels of PD-1, other activation markers, including cytotoxic molecules, and a broad repertoire of killer-cell immunoglobulin-like receptors. In vitro, PD-1+ γδ T cells that were isolated from MMR-d colon cancers exhibited enhanced reactivity to human leukocyte antigen (HLA)-class-I-negative MMR-d colon cancer cell lines and B2M-knockout patient-derived tumour organoids compared with antigen-presentation-proficient cells. By comparing paired tumour samples from patients with MMR-d colon cancer that were obtained before and after dual PD-1 and CTLA-4 blockade, we found that immune checkpoint blockade substantially increased the frequency of γδ T cells in B2M-deficient cancers. Taken together, these data indicate that γδ T cells contribute to the response to immune checkpoint blockade in patients with HLA-class-I-negative MMR-d colon cancers, and underline the potential of γδ T cells in cancer immunotherapy.

Genetically encoded libraries of peptides and oligonucleotides are well suited for the identification of ligands for many macromolecules. A major drawback of these techniques is that the resultant ligands are subject to degradation by naturally occurring enzymes. Here, a method is described that uses a biologically encoded library for the identification of D-peptide ligands, which should be resistant to proteolytic degradation. In this approach, a protein is synthesized in the D-amino acid configuration and used to select peptides from a phage display library expressing random L-amino acid peptides. For reasons of symmetry, the mirror images of these phage-displayed peptides interact with the target protein of the natural handedness. The value of this approach was demonstrated by the identification of a cyclic D-peptide that interacts with the Src homology 3 domain of c- SRC. Nuclear magnetic resonance studies indicate that the binding site for this D-peptide partially overlaps the site for the physiological ligands of this domain.

Mice deficient in the gene encoding the peptide transporter associated with antigen processing (TAP1) have drastically reduced levels of surface expression of MHC class I molecules and few CD8+ T cells. Addition of class I binding peptides to cultured fetal thymi from TAP1 mutant mice invariably allowed the rescue of class I expression, while only some of these peptides promoted the positive selection of CD8+ T cells, which were polyclonal and specific for the peptide-MHC complex. A nonselecting peptide was converted to a mixture of selecting peptides when the residues involved in the interaction with TCRs were altered. A mixture of self-peptides derived from C57BL/6 thymi induced positive selection of CD8+ T cells at concentrations that gave relatively low class I surface expression. The implication of these observations is that self-peptides determine, in part, the repertoire of specificities exhibited by CD8+ T cells.

Abstract During immunosuppression, cytomegalovirus (CMV) can reactivate and cause serious clinical problems. Normally, abundant virus replication is suppressed by immune effector mechanisms. To study the interaction between CD8+ T cells and persisting viruses, frequencies and phenotypes of CMV-specific CD8+ T cells were determined in healthy individuals and compared to those in renal transplant recipients. In healthy donors, function of circulating virus-specific CD8+ T cells, as measured by peptide-induced interferon γ (IFN-γ) production, but not the number of virus-specific T cells enumerated by binding of specific tetrameric peptide/HLA complexes, correlated with the number of CMV-specific IFN-γ–secreting CD4+ helper T cells. Circulating CMV- specific CD8+ T cells did not express CCR7 and may therefore not be able to recirculate through peripheral lymph nodes. Based on coexpression of CD27 and CD45R0 most CMV-specific T cells in healthy donors appeared to be memory-type cells. Remarkably, frequencies of CMV-specific CD8+ T cells were significantly higher in immunosuppressed individuals than in healthy donors. In these patients CMV-specific cells predominantly had an effector phenotype, that is, CD45R0+CD27−CCR7− or CD45RA+CD27−CCR7− and contained both granzyme B and perforin. Our data show that in response to immunosuppressive medication quantitative and qualitative changes occur in the CD8+ T-cell compartment. These adaptations may be instrumental to maintain CMV latency.

Dendritic cells (DCs) play a central role in the immune system as they drive activation of T lymphocytes by cognate interactions. However, as DCs express high levels of major histocompatibility complex class I, this intimate contact may also result in elimination of DCs by activated cytotoxic T lymphocytes (CTLs) and thereby limit induction of immunity. We show here that immature DCs are indeed susceptible to CTL-induced killing, but become resistant upon maturation with anti-CD40 or lipopolysaccharide. Protection is achieved by expression of serine protease inhibitor (SPI)-6, a member of the serpin family that specifically inactivates granzyme B and thereby blocks CTL-induced apoptosis. Anti-CD40 and LPS-induced SPI-6 expression is sustained for long periods of time, suggesting a role for SPI-6 in the longevity of DCs. Importantly, T helper 1 cells, which mature DCs and boost CTL immunity, induce SPI-6 expression and subsequent DC resistance. In contrast, T helper 2 cells neither induce SPI-6 nor convey protection, despite the fact that they trigger DC maturation with comparable efficiency. Our data identify SPI-6 as a novel marker for DC function, which protects DCs against CTL-induced apoptosis.

In vitro selection experiments have produced nucleic acid ligands (aptamers) that bind tightly and specifically to a great variety of target biomolecules. The utility of aptamers is often limited by their vulnerability to nucleases present in biological materials. One way to circumvent this problem is to select an aptamer that binds the enantiomer of the target, then synthesize the enantiomer of the aptamer as a nuclease-insensitive ligand of the normal target. We have so identified a mirror-image single-stranded DNA that binds the peptide hormone vasopressin and have demonstrated its stability to nucleases and its bioactivity as a vasopressin antagonist in cell culture.

Type 1 diabetes results from selective T-cell-mediated destruction of the insulin-producing beta-cells in the pancreas. In this process, islet epitope-specific CD8(+) T-cells play a pivotal role. Thus, monitoring of multiple islet-specific CD8(+) T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive.Here, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B(10-18), prepro-insulin (PPI)(15-24), islet antigen (IA)-2(797-805), GAD65(114-123), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(265-273), and prepro islet amyloid polypeptide (ppIAPP)(5-13)-specific CD8(+) T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients.Using this kit, islet autoreactive CD8(+) T-cells recognizing insulin B(10-18), IA-2(797-805), and IGRP(265-273) were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI(15-24) proved to be the most sensitive epitope. Applying the "Diab-Q-kit" to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell-derived epitopes that were associated with disease activity and correlated with clinical outcome.A kit was developed that allows simultaneous detection of CD8(+) T-cells reactive to multiple HLA-A2-restricted beta-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples.

Protective adaptive immune responses rely on TCR-mediated recognition of Ag-derived peptides presented by self-MHC molecules. However, self-Ag (tumor)-specific TCRs are often of too low affinity to achieve best functionality. To precisely assess the relationship between TCR-peptide-MHC binding parameters and T cell function, we tested a panel of sequence-optimized HLA-A(*)0201/NY-ESO-1(157-165)-specific TCR variants with affinities lying within physiological boundaries to preserve antigenic specificity and avoid cross-reactivity, as well as two outliers (i.e., a very high- and a low-affinity TCR). Primary human CD8 T cells transduced with these TCRs demonstrated robust correlations between binding measurements of TCR affinity and avidity and the biological response of the T cells, such as TCR cell-surface clustering, intracellular signaling, proliferation, and target cell lysis. Strikingly, above a defined TCR-peptide-MHC affinity threshold (K(D) < approximately 5 muM), T cell function could not be further enhanced, revealing a plateau of maximal T cell function, compatible with the notion that multiple TCRs with slightly different affinities participate equally (codominantly) in immune responses. We propose that rational design of improved self-specific TCRs may not need to be optimized beyond a given affinity threshold to achieve both optimal T cell function and avoidance of the unpredictable risk of cross-reactivity.

Major histocompatibility complex (MHC) class I molecules associate with peptides that are delivered from the cytosol to the lumen of the endoplasmic reticulum by the transporter associated with antigen processing (TAP). Liver microsomes of SHR and Lewis rats, which express different alleles of TAP (cimb and cima, respectively), accumulate different sets of peptides. Use of MHC congenic rats assigned this difference to the MHC, independent of the class I products expressed. Both the cima and cimb TAP complexes translocate peptides with a hydrophobic carboxyl terminus, but translocation of peptides with a carboxyl-terminal His, Lys, or Arg residue is unique to cima. Thus, the specificity of the TAP peptide translocator restricts the peptides available for antigen presentation.

Regulatory T cells (Tregs) can suppress a wide range of immune cells, making them an ideal candidate for the treatment of autoimmunity. The potential clinical translation of targeted therapy with antigen-specific Tregs is hampered by the difficulties of isolating rare specificities from the natural polyclonal T cell repertoire. Moreover, the initiating antigen is often unknown in autoimmune disease. Here we tested the ability of antigen-specific Tregs generated by retroviral gene transfer to ameliorate arthritis through linked suppression and therefore without cognate recognition of the disease-initiating antigen. We explored two distinct strategies: T cell receptor (TCR) gene transfer into purified CD4+CD25+ T cells was used to redirect the specificity of naturally occurring Tregs; and co-transfer of FoxP3 and TCR genes served to convert conventional CD4 + T cells into antigen-specific regulators. Following adoptive transfer into recipient mice, the gene-modified T cells engrafted efficiently and retained TCR and FoxP3 expression. Using an established arthritis model, we demonstrate antigen-driven accumulation of the gene modified T cells at the site of joint inflammation, which resulted in a local reduction in the number of inflammatory Th17 cells and a significant decrease in arthritic bone destruction. Together, we describe a robust strategy to rapidly generate antigen-specific regulatory T cells capable of highly targeted inhibition of tissue damage in the absence of systemic immune suppression. This opens the possibility to target Tregs to tissue-specific antigens for the treatment of autoimmune tissue damage without the knowledge of the disease-causing autoantigens recognized by pathogenic T cells.

The mechanism by which the immune system produces effector and memory T cells is largely unclear. To allow a large-scale assessment of the development of single naive T cells into different subsets, we have developed a technology that introduces unique genetic tags (barcodes) into naive T cells. By comparing the barcodes present in antigen-specific effector and memory T cell populations in systemic and local infection models, at different anatomical sites, and for TCR-pMHC interactions of different avidities, we demonstrate that under all conditions tested, individual naive T cells yield both effector and memory CD8+ T cell progeny. This indicates that effector and memory fate decisions are not determined by the nature of the priming antigen-presenting cell or the time of T cell priming. Instead, for both low and high avidity T cells, individual naive T cells have multiple fates and can differentiate into effector and memory T cell subsets.

Cell dynamics in subcutaneous and breast tumors can be studied through conventional imaging windows with intravital microscopy. By contrast, visualization of the formation of metastasis has been hampered by the lack of long-term imaging windows for metastasis-prone organs, such as the liver. We developed an abdominal imaging window (AIW) to visualize distinct biological processes in the spleen, kidney, small intestine, pancreas, and liver. The AIW can be used to visualize processes for up to 1 month, as we demonstrate with islet cell transplantation. Furthermore, we have used the AIW to image the single steps of metastasis formation in the liver over the course of 14 days. We observed that single extravasated tumor cells proliferated to form "pre-micrometastases," in which cells lacked contact with neighboring tumor cells and were active and motile within the confined region of the growing clone. The clones then condensed into micrometastases where cell migration was strongly diminished but proliferation continued. Moreover, the metastatic load was reduced by suppressing tumor cell migration in the pre-micrometastases. We suggest that tumor cell migration within pre-micrometastases is a contributing step that can be targeted therapeutically during liver metastasis formation.

Activated CD8+ T cells detect virally infected cells and tumor cells by recognition of major histocompatibility complex class I-bound peptides derived from degraded, endogenously produced proteins. In contrast, CD8+ T cell activation often occurs through interaction with specialized antigen-presenting cells displaying peptides acquired from an exogenous cellular source, a process termed cross-priming. Here, we observed a marked inefficiency in exogenous presentation of epitopes derived from signal sequences in mouse models. These data indicate that certain virus- and tumor-associated antigens may not be detected by CD8+ T cells because of impaired cross-priming. Such differences in the ability to cross-present antigens should form important considerations in vaccine design.

Induction of immunity after DNA vaccination is generally considered a slow process. Here we show that DNA delivery to the skin results in a highly transient pulse of antigen expression. Based on this information, we developed a new rapid and potent intradermal DNA vaccination method. By short-interval intradermal DNA delivery, robust T-cell responses, of a magnitude sufficient to reject established subcutaneous tumors, are generated within 12 d. Moreover, this vaccination strategy confers protecting humoral immunity against influenza A infection within 2 weeks after the start of vaccination. The strength and speed of this newly developed strategy will be beneficial in situations in which immunity is required in the shortest possible time.

A number of immunotherapies, in particular immune checkpoint targeting antibodies and adoptive T-cell therapies, are starting to transform the treatment of advanced cancers. The likelihood to respond to these immunotherapies differs strongly across tumor types, with response rates for checkpoint targeting being the highest in advanced melanoma, renal cell cancer and non-small cell lung cancer. However, also non-responsiveness is observed, indicating the presence of intrinsic resistance or naturally acquired resistance. In addition, a subgroup of patients that do initially respond to immunotherapy will later recur, thereby also pointing towards a role of therapy-induced acquired resistance. Here, we review our current understanding of both intrinsic and acquired resistance mechanisms in cancer immunotherapy, and discuss potential strategies to overcome them.

There is strong evidence that both adoptive T cell transfer and T cell checkpoint blockade can lead to regression of human melanoma. However, little data are available on the effect of these cancer therapies on the tumor-reactive T cell compartment. To address this issue we have profiled therapy-induced T cell reactivity against a panel of 145 melanoma-associated CD8(+) T cell epitopes. Using this approach, we demonstrate that individual tumor-infiltrating lymphocyte cell products from melanoma patients contain unique patterns of reactivity against shared melanoma-associated antigens, and that the combined magnitude of these responses is surprisingly low. Importantly, TIL therapy increases the breadth of the tumor-reactive T cell compartment in vivo, and T cell reactivity observed post-therapy can almost in full be explained by the reactivity observed within the matched cell product. These results establish the value of high-throughput monitoring for the analysis of immuno-active therapeutics and suggest that the clinical efficacy of TIL therapy can be enhanced by the preparation of more defined tumor-reactive T cell products.

<h2>Summary</h2> Development of mature blood cell progenies from hematopoietic stem cells involves the transition through lineage-restricted progenitors. The first branching point along this developmental process is thought to separate the erythro-myeloid and lymphoid lineage fate by yielding two intermediate progenitors, the common myeloid and the common lymphoid progenitors (CMPs and CLPs). Here, we use single-cell lineage tracing to demonstrate that so-called CMPs are highly heterogeneous with respect to cellular output, with most individual CMPs yielding either only erythrocytes or only myeloid cells after transplantation. Furthermore, based on the labeling of earlier progenitors, we show that the divergence between the myeloid and erythroid lineage develops within multipotent progenitors (MPP). These data provide evidence for a model of hematopoietic branching in which multiple distinct lineage commitments occur in parallel within the MPP pool.

Preparation for Cell Wars When T cells encounter an infection, they proliferate to create a larger army to fight the invader. The overall magnitude of the T cell response depends on the severity of infection and is determined by the number of T cells of a particular antigen specificity that are initially recruited, as well as the magnitude of the proliferative response. The extent to which these two components contribute to the response is unknown. By using DNA barcoding to track the responses of individual T cells, van Heijst et al. (p. 1265 ) showed that the recruitment of T cells of a particular antigen specificity is similar and nearly complete, but that the extent of the proliferative response differed, and this determined the overall magnitude of the T cell response.

Abstract Tumor-infiltrating lymphocytes (TIL) isolated from melanoma patients and expanded in vitro by interleukin (IL)-2 treatment can elicit therapeutic response after adoptive transfer, but the antigen specificities of the T cells transferred have not been determined. By compiling all known melanoma-associated antigens and applying a novel technology for high-throughput analysis of T-cell responses, we dissected the composition of melanoma-restricted T-cell responses in 63 TIL cultures. T-cell reactivity screens against 175 melanoma-associated epitopes detected 90 responses against 18 different epitopes predominantly from differentiation and cancer-testis antigens. Notably, the majority of these responses were of low frequency and tumor-specific T-cell frequencies decreased during rapid expansion. A further notable observation was a large variation in the T-cell specificities detected in cultures established from different fragments of resected melanoma lesions. In summary, our findings provide an initial definition of T-cell populations contributing to tumor recognition in TILs although the specificity of many tumor-reactive TILs remains undefined. Cancer Res; 72(7); 1642–50. ©2012 AACR.

Major histocompatibility complex (MHC) class I multimer technology has become an indispensable immunological assay system to dissect antigen-specific cytotoxic CD8(+) T cell responses by flow cytometry. However, the development of high-throughput assay systems, in which T cell responses against a multitude of epitopes are analyzed, has been precluded by the fact that for each T cell epitope, a separate in vitro MHC refolding reaction is required. We have recently demonstrated that conditional ligands that disintegrate upon exposure to long-wavelength UV light can be designed for the human MHC molecule HLA-A2. To determine whether this peptide-exchange technology can be developed into a generally applicable approach for high throughput MHC based applications we set out to design conditional ligands for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Here, we describe the development and characterization of conditional ligands for this set of human MHC molecules and apply the peptide-exchange technology to identify melanoma-associated peptides that bind to HLA-A3 with high affinity. The conditional ligand technology developed here will allow high-throughput MHC-based analysis of cytotoxic T cell immunity in the vast majority of Western European individuals.

Antibodies against T cell checkpoint molecules have started to revolutionize cancer treatment. Nevertheless, less than half of all patients respond to these immunotherapies. Recent work supports the potential value of biomarkers that predict therapy outcome and inspires the development of assay systems that interrogate other aspects of the cancer-immunity cycle.

T cell responses display two key characteristics. First, a small population of epitope-specific naive T cells expands by several orders of magnitude. Second, the T cells within this proliferating population take on diverse functional and phenotypic properties that determine their ability to exert effector functions and contribute to T cell memory. Recent technological advances in lineage tracing allow us for the first time to study these processes in vivo at single-cell resolution. Here, we summarize resulting data demonstrating that although epitope-specific T cell responses are reproducibly similar at the population level, expansion potential and diversification patterns of the offspring derived from individual T cells are highly variable during both primary and recall immune responses. In spite of this stochastic response variation, individual memory T cells can serve as adult stem cells that provide robust regeneration of an epitope-specific tissue through population averaging. We discuss the relevance of these findings for T cell memory formation and clinical immunotherapy.

The development of targeted inhibitors, like vemurafenib, has greatly improved the clinical outcome of BRAF(V600E) metastatic melanoma. However, resistance to such compounds represents a formidable problem. Using whole-exome sequencing and functional analyses, we have investigated the nature and pleiotropy of vemurafenib resistance in a melanoma patient carrying multiple drug-resistant metastases. Resistance was caused by a plethora of mechanisms, all of which reactivated the MAPK pathway. In addition to three independent amplifications and an aberrant form of BRAF(V600E), we identified a new activating insertion in MEK1. This MEK1(T55delins) (RT) mutation could be traced back to a fraction of the pre-treatment lesion and not only provided protection against vemurafenib but also promoted local invasion of transplanted melanomas. Analysis of patient-derived xenografts (PDX) from therapy-refractory metastases revealed that multiple resistance mechanisms were present within one metastasis. This heterogeneity, both inter- and intra-tumorally, caused an incomplete capture in the PDX of the resistance mechanisms observed in the patient. In conclusion, vemurafenib resistance in a single patient can be established through distinct events, which may be preexisting. Furthermore, our results indicate that PDX may not harbor the full genetic heterogeneity seen in the patient's melanoma.

Our ability to analyze adaptive immunity and engineer its activity has long been constrained by our limited ability to identify native pairs of heavy-light antibody chains and alpha-beta T-cell receptor (TCR) chains--both of which comprise coupled "halves of a key", collectively capable of recognizing specific antigens. Here, we report a cell-based emulsion RT-PCR approach that allows the selective fusion of the native pairs of amplified TCR alpha and beta chain genes for complex samples. A new type of PCR suppression technique was developed that makes it possible to amplify the fused library with minimal noise for subsequent analysis by high-throughput paired-end Illumina sequencing. With this technique, single analysis of a complex blood sample allows identification of multiple native TCR chain pairs. This approach may be extended to identify native antibody chain pairs and, more generally, pairs of mRNA molecules that are coexpressed in the same living cells.

Abstract Primary triple negative breast cancers (TNBC) are prone to dissemination but sub-clonal relationships between tumors and resulting metastases are poorly understood. Here we use cellular barcoding of two treatment-naïve TNBC patient-derived xenografts (PDXs) to track the spatio-temporal fate of thousands of barcoded clones in primary tumors, and their metastases. Tumor resection had a major impact on reducing clonal diversity in secondary sites, indicating that most disseminated tumor cells lacked the capacity to ‘seed’, hence originated from ‘shedders’ that did not persist. The few clones that continued to grow after resection i.e. ‘seeders’, did not correlate in frequency with their parental clones in primary tumors. Cisplatin treatment of one BRCA1 -mutated PDX model to non-palpable levels had a surprisingly minor impact on clonal diversity in the relapsed tumor yet purged 50% of distal clones. Therefore, clonal features of shedding, seeding and drug resistance are important factors to consider for the design of therapeutic strategies.

Treatment of metastatic melanoma with autologous tumor infiltrating lymphocytes (TILs) is currently applied in several centers. Robust and remarkably consistent overall response rates, of around 50% of treated patients, have been observed across hospitals, including a substantial fraction of durable, complete responses. Purpose Execute a phase I/II feasibility study with TIL therapy in metastatic melanoma at the Netherlands Cancer Institute, with the goal to assess feasibility and potential value of a randomized phase III trial. Experimental Ten patients were treated with TIL therapy. Infusion products and peripheral blood samples were phenotypically characterized and neoantigen reactivity was assessed. Here, we present long-term clinical outcome and translational data on neoantigen reactivity of the T cell products. Results Five out of 10 patients, who were all anti-PD-1 naïve at time of treatment, showed an objective clinical response, including two patients with a complete response that are both ongoing for more than 7 years. Immune monitoring demonstrated that neoantigen-specific T cells were detectable in TIL infusion products from three out of three patients analyzed. For six out of the nine neoantigen-specific T cell responses detected in these TIL products, T cell response magnitude increased significantly in the peripheral blood compartment after therapy, and neoantigen-specific T cells were detectable for up to 3 years after TIL infusion. Conclusion The clinical results from this study confirm the robustness of TIL therapy in metastatic melanoma and the potential role of neoantigen-specific T cell reactivity. In addition, the data from this study supported the rationale to initiate an ongoing multicenter phase III TIL trial.

T cell-secreted IFNγ can exert pleiotropic effects on tumor cells that include induction of immune checkpoints and antigen presentation machinery components, and inhibition of cell growth. Despite its role as key effector molecule, little is known about the spatiotemporal spreading of IFNγ secreted by activated CD8+ T cells within the tumor environment. Using multiday intravital imaging, we demonstrate that T cell recognition of a minor fraction of tumor cells leads to sensing of IFNγ by a large part of the tumor mass. Furthermore, imaging of tumors in which antigen-positive and -negative tumor cells are separated in space reveals spreading of the IFNγ response, reaching distances of >800 µm. Notably, long-range sensing of IFNγ can modify tumor behavior, as both shown by induction of PD-L1 expression and inhibition of tumor growth. Collectively, these data reveal how, through IFNγ, CD8+ T cells modulate the behavior of remote tumor cells, including antigen-loss variants.

Here, we describe a fatal serious adverse event observed in a patient infused with autologous T-cell receptor (TCR) transduced T cells. This TCR, originally obtained from a melanoma patient, recognizes the well-described HLA-A*0201 restricted 26–35 epitope of MART-1, and was not affinity enhanced. Patient 1 with metastatic melanoma experienced a cerebral hemorrhage, epileptic seizures, and a witnessed cardiac arrest 6 days after cell infusion. Three days later, the patient died from multiple organ failure and irreversible neurologic damage. After T-cell infusion, levels of IL-6, IFN-γ, C-reactive protein (CRP), and procalcitonin increased to extreme levels, indicative of a cytokine release syndrome or T-cell-mediated inflammatory response. Infused T cells could be recovered from blood, broncho-alveolar lavage, ascites, and after autopsy from tumor sites and heart tissue. High levels of NT-proBNP indicate semi-acute heart failure. No cross reactivity of the modified T cells toward a beating cardiomyocyte culture was observed. Together, these observations suggest that high levels of inflammatory cytokines alone or in combination with semi-acute heart failure and epileptic seizure may have contributed substantially to the occurrence of the acute and lethal event. Protocol modifications to limit the risk of T-cell activation-induced toxicity are discussed. Here, we describe a fatal serious adverse event observed in a patient infused with autologous T-cell receptor (TCR) transduced T cells. This TCR, originally obtained from a melanoma patient, recognizes the well-described HLA-A*0201 restricted 26–35 epitope of MART-1, and was not affinity enhanced. Patient 1 with metastatic melanoma experienced a cerebral hemorrhage, epileptic seizures, and a witnessed cardiac arrest 6 days after cell infusion. Three days later, the patient died from multiple organ failure and irreversible neurologic damage. After T-cell infusion, levels of IL-6, IFN-γ, C-reactive protein (CRP), and procalcitonin increased to extreme levels, indicative of a cytokine release syndrome or T-cell-mediated inflammatory response. Infused T cells could be recovered from blood, broncho-alveolar lavage, ascites, and after autopsy from tumor sites and heart tissue. High levels of NT-proBNP indicate semi-acute heart failure. No cross reactivity of the modified T cells toward a beating cardiomyocyte culture was observed. Together, these observations suggest that high levels of inflammatory cytokines alone or in combination with semi-acute heart failure and epileptic seizure may have contributed substantially to the occurrence of the acute and lethal event. Protocol modifications to limit the risk of T-cell activation-induced toxicity are discussed.

Recent data suggest that T-cell reactivity against tumor-specific neo-antigens may be central to the clinical efficacy of cancer immunotherapy. The development of personalized vaccines designed to boost T-cell reactivity against patient specific neo-antigens has been proposed largely on the basis of these findings. Work from several groups has demonstrated that novel tumor-specific antigens can be discovered through the use of cancer exome sequencing data, thereby providing a potential pipeline for the development of patient-specific vaccines. Importantly though, it has not been established which fraction of cancer neo-antigens that can be recognized by CD8+ T cells is successfully uncovered with the current exome-based epitope prediction strategies. Here, we use a data set comprising human cancer neo-antigens that was previously identified through the use of unbiased, computational-independent strategies to describe the potential of cancer exome-based neo-antigen discovery. This analysis shows a high sensitivity of exome-guided neo-antigen prediction of approximately 70%. We propose that future research should focus on the analysis and optimization of the specificity of neo-antigen prediction, and should undoubtedly entail the clinical evaluation of patient-specific vaccines with the aim of inducing immunoreactivity against tumor-displayed neo-antigens in a physiologically relevant context.

The relative affinity of T cell receptors for different peptide–MHCs is measured at high throughput, revealing T cell cross-reactivity. The promiscuous nature of T-cell receptors (TCRs) allows T cells to recognize a large variety of pathogens, but makes it challenging to understand and control T-cell recognition1. Existing technologies provide limited information about the key requirements for T-cell recognition and the ability of TCRs to cross-recognize structurally related elements2,3. Here we present a 'one-pot' strategy for determining the interactions that govern TCR recognition of peptide–major histocompatibility complex (pMHC). We measured the relative affinities of TCRs to libraries of barcoded peptide–MHC variants and applied this knowledge to understand the recognition motif, here termed the TCR fingerprint. The TCR fingerprints of 16 different TCRs were identified and used to predict and validate cross-recognized peptides from the human proteome. The identified fingerprints differed among TCRs recognizing the same epitope, demonstrating the value of this strategy for understanding T-cell interactions and assessing potential cross-recognition before selection of TCRs for clinical development.

The identification of immunogenic neoantigens and their cognate T cells represents the most crucial and rate-limiting steps in the development of personalized cancer immunotherapies that are based on vaccination or on infusion of T cell receptor (TCR)-engineered T cells. Recent advances in deep-sequencing technologies and in silico prediction algorithms have allowed rapid identification of candidate neoepitopes. However, large-scale validation of putative neoepitopes and the isolation of reactive T cells are challenging because of the limited availablity of patient material and the low frequencies of neoepitope-specific T cells. Here we describe a standardized protocol for the induction of neoepitope-reactive T cells from healthy donor T cell repertoires, unaffected by the potentially immunosuppressive environment of the tumor-bearing host. Monocyte-derived dendritic cells (DCs) transfected with mRNA encoding candidate neoepitopes are used to prime autologous naive CD8+ T cells. Antigen-specific T cells that recognize endogenously processed and presented epitopes are detected using peptide–MHC (pMHC) multimers. Single multimer-positive T cells are sorted for the identification of TCR sequences, after an optional step that includes clonal expansion and functional characterization. The time required to identify neoepitope-specific T cells is 15 d, with an additional 2–4 weeks required for clonal expansion and downstream functional characterization. Identified neoepitopes and corresponding TCRs provide candidates for use in vaccination and TCR-based cancer immunotherapies, and datasets generated by this technology should be useful for improving algorithms to predict immunogenic neoantigens. The percentage of cancer neoantigens that are spontaneously recognized by T cells is generally very low. This protocol describes how CD8+ T cells from healthy donors can be used for enhanced targeting of these neoantigens.

Cytotoxic CD8 + T cells can effectively kill target cells by producing cytokines, chemokines, and granzymes. Expression of these effector molecules is however highly divergent, and tools that identify and preselect CD8 + T cells with a cytotoxic expression profile are lacking. Human CD8 + T cells can be divided into IFN-γ– and IL-2–producing cells. Unbiased transcriptomics and proteomics analysis on cytokine-producing fixed CD8 + T cells revealed that IL-2 + cells produce helper cytokines, and that IFN-γ + cells produce cytotoxic molecules. IFN-γ + T cells expressed the surface marker CD29 already prior to stimulation. CD29 also marked T cells with cytotoxic gene expression from different tissues in single-cell RNA-sequencing data. Notably, CD29 + T cells maintained the cytotoxic phenotype during cell culture, suggesting a stable phenotype. Preselecting CD29-expressing MART1 TCR-engineered T cells potentiated the killing of target cells. We therefore propose that CD29 expression can help evaluate and select for potent therapeutic T cell products.

An increasing body of evidence emphasizes the role of tissue-resident memory T cells (TRM) in the defense against recurring pathogens and malignant neoplasms. However, little is known with regard to the origin of these cells and their kinship to other CD8+ T cell compartments. To address this issue, we followed the antigen-specific progeny of individual naive CD8+ T cells to the T effector (TEFF), T circulating memory (TCIRCM), and TRM pools by lineage-tracing and single-cell transcriptome analysis. We demonstrate that a subset of T cell clones possesses a heightened capacity to form TRM, and that enriched expression of TRM–fate-associated genes is already apparent in the circulating TEFF offspring of such clones. In addition, we demonstrate that the capacity to generate TRM is permanently imprinted at the clonal level, before skin entry. Collectively, these data provide compelling evidence for early stage TRM fate decisions and the existence of committed TRM precursor cells in the circulatory TEFF compartment.

Acute myeloid leukemia (AML) remains a clinical challenge due to frequent chemotherapy resistance and deadly relapses. We are exploring the immunotherapeutic potential of peripheral blood Vδ1+ T cells, which associate with improved long-term survival of stem-cell transplant recipients but have not yet been applied as adoptive cell therapy. Using our clinical-grade protocol for expansion and differentiation of "Delta One T" (DOT) cells, we found DOT cells to be highly cytotoxic against AML primary samples and cell lines, including cells selected for resistance to standard chemotherapy. Unlike chemotherapy, DOT-cell targeting did not select for outgrowth of specific AML lineages, suggesting a broad recognition domain, an outcome that was consistent with the polyclonality of the DOT-cell T-cell receptor (TCR) repertoire. However, AML reactivity was only slightly impaired upon Vδ1+ TCR antibody blockade, whereas it was strongly dependent on expression of the NKp30 ligand, B7-H6. In contrast, DOT cells did not show reactivity against normal leukocytes, including CD33+ or CD123+ myeloid cells. Adoptive transfer of DOT cells in vivo reduced AML load in the blood and target organs of multiple human AML xenograft models and significantly prolonged host survival without detectable toxicity, thus providing proof-of-concept for DOT-cell application in AML treatment.