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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.

Inhibitors of the PD-1–PD-L1 axis have been approved as therapy for many human cancers. In spite of the evidence for their widespread clinical activity, little is known about the immunological alterations that occur in human cancer tissue after PD-1 blockade. We developed and employed a patient-derived tumor fragment platform to dissect the early immunological response of human tumor tissue to ex vivo PD-1 blockade. We observed that the capacity of immune cells to be reactivated ex vivo was predictive of clinical response, and perturbation analyses identified tumor-resident T cells as a key component of this immunological response. In addition, through combined analysis of baseline properties and immune response capacity, we identified a new subgroup of infiltrated tumors that lacks the capacity to respond to PD-1 blockade. Finally, the baseline presence of tertiary lymphoid structures and their components correlated with the capacity of cancers to undergo intratumoral immune cell reactivation. An ex vivo platform of patient-derived tumor fragments enables the assessment of intratumoral immune reactivation after PD-1 blockade that is predictive of clinical outcomes in patients with cancer.

Adoptive transfer of genetically modified or donor-derived T cells can efficiently eradicate human tumors but is also frequently associated with major toxicity. There are several switches that can be used to kill the infused cell pool in the case of major toxicity, but the irreversible nature of these suicide switches means that the therapeutic effect is lost when they are used. To address this issue, we engineered a small-molecule responsive genetic safety switch that in the absence of drug robustly blocked cytotoxicity and cytokine expression of primary human T cells. Upon administration of drug, T-cell functions were restored in a reversible and titratable manner. We showed that this T-cell switch was universal, as it could be combined with endogenous or transduced T-cell receptors (TCR), as well as chimeric antigen receptors. The modular nature of the Chemically Regulated - SH2-delivered Inhibitory Tail (CRASH-IT) switch concept, in which inhibitory domains are brought to activating immune receptors in a controlled manner, makes it a versatile platform to regulate the activity of cell products that signal through immunoreceptor tyrosine-based activation motif (ITAM)–containing receptors.

Human skin harbors various immune cells that are crucial for the control of injury and infection. However, the current understanding of immune cell function within viable human skin tissue is limited. We developed an ex vivo imaging approach in which fresh skin biopsies are mounted and then labeled with nanobodies or antibodies against cell surface markers on tissue-resident memory CD8+ T cells, other immune cells of interest, or extracellular tissue components. Subsequent longitudinal imaging allows one to describe the dynamic behavior of human skin-resident cells in situ. In addition, this strategy can be used to study immune cell function in murine skin. The ability to follow the spatiotemporal behavior of CD8+ T cells and other immune cells in skin, including their response to immune stimuli, provides a platform to investigate physiological immune cell behavior and immune cell behavior in skin diseases. The mounting, staining and imaging of skin samples requires ~1.5 d, and subsequent tracking analysis requires a minimum of 1 d. The optional production of fluorescently labeled nanobodies takes ~5 d.

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) have revolutionized the treatment of melanoma patients. Based on early studies addressing the mechanism of action, it was assumed that PD-1 blockade mostly influences T cell responses at the tumor site. However, recent work has demonstrated that PD-1 blockade can influence the T cell compartment in peripheral blood. If the activation of circulating, tumor-reactive T cells would form an important mechanism of action of PD-1 blockade, it may be predicted that such blockade would alter either the frequency and/or the breadth of the tumor-reactive CD8 T cell response. To address this question, we analyzed CD8 T cell responses toward 71 melanoma-associated epitopes in peripheral blood of 24 melanoma patients. We show that both the frequency and the breadth of the circulating melanoma-reactive CD8 T cell response was unaltered upon PD-1 blockade. In contrast, a broadening of the circulating melanoma-reactive CD8 T cell response was observed upon CTLA-4 blockade, in concordance with our prior data. Based on these results, we conclude that PD-1 and CTLA-4 blockade have distinct mechanisms of action. In addition, the data provide an argument in favor of the hypothesis that anti-PD-1 therapy may primarily act at the tumor site.

Adoptive transfer of allogeneic and genetically modified T cells, such as CAR-T and TCR-T cells, can induce profound immune reactivity against cancer tissue. At the same time, these therapies are associated with severe off-target and on-target toxicities. For this reason, the development of genetic safety switches that can be used to control the activity of T cells in vivo has become an active field of research. With the spectrum of technologies developed, reversible control of cell products either by supply or removal of small molecules, by supply of protein-based regulators, or by physical stimuli such as light, ultrasound or heat, has become feasible. In this review, we describe the mechanistic classes of genetic safety switches, such as transcription-based or protein-based control of antigen receptors, split receptors, small molecule responsive antibodies, as well as universal remote controls, and discuss their advantages and limitations.

Many viral antigens have been identified in patients with COVID-19 patients, but which of these result in meaningful immune responses is unclear. Here the authors identify a range of SARS-CoV-2 CD8(+) T cell responses across patients including a response targeting an epitope of ORF1ab with immunodominant properties.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.

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.

Infiltration of human cancers by T cells is generally interpreted as a sign of immune recognition, and there is a growing effort to reactivate dysfunctional T cells at such tumor sites1. However, these efforts only have value if the intratumoral T cell receptor (TCR) repertoire of such cells is intrinsically tumor reactive, and this has not been established in an unbiased manner for most human cancers. To address this issue, we analyzed the intrinsic tumor reactivity of the intratumoral TCR repertoire of CD8+ T cells in ovarian and colorectal cancer—two tumor types for which T cell infiltrates form a positive prognostic marker2,3. Data obtained demonstrate that a capacity to recognize autologous tumor is limited to approximately 10% of intratumoral CD8+ T cells. Furthermore, in two of four patient samples tested, no tumor-reactive TCRs were identified, despite infiltration of their tumors by T cells. These data indicate that the intrinsic capacity of intratumoral T cells to recognize adjacent tumor tissue can be rare and variable, and suggest that clinical efforts to reactivate intratumoral T cells will benefit from approaches that simultaneously increase the quality of the intratumoral TCR repertoire. Intratumoral T cells that are capable of recognizing adjacent tumor tissue antigens can be exceedingly rare.

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have revolutionized the treatment of melanoma patients. Based on early studies addressing the mechanism of action, it was assumed that PD-1 blockade mostly influences T cell responses at the tumor site. However, recent work has demonstrated that PD-1 blockade can influence the T cell compartment in peripheral. blood. If activation of circulating tumor-reactive T cells would form an important mechanism of action of PD-1 blockade, it may be predicted that such blockade would alter either the frequency and/or the breadth of the tumor-reactive CD8 T cell response. To address this question, we analyzed CD8 T cell responses towards 71 melanoma associated epitopes in peripheral blood of 24 melanoma patients. We show that both the frequency and the breadth of the melanoma-reactive CD8 T cell response in peripheral blood was unaltered upon PD-1 blockade. In contrast, a broadening of the melanoma-reactive CD8 T cell response was observed upon CTLA-4 blockade, in concordance with our prior data. On the basis of these results, we conclude that PD-1 and CTLA-4 blockade impact the tumor-reactive CD8 T cell response in a distinct manner. In addition, the data provide an argument in favor of the hypothesis that anti-PD-1 therapy may primarily act at the tumor site.One sentence summaryWe demonstrate that, contrary to CTLA-4 blockade, PD-1 blockade in melanoma patients does not lead to an increase in the breadth or magnitude of the melanoma-reactive CD8 T cell response in peripheral blood, thereby arguing for a dominant effect of PD-1 blockade at the tumor site.

Many approaches to identify therapeutically relevant neoantigens couple tumor sequencing with bioinformatic algorithms and inferred rules of tumor epitope immunogenicity. However, there are no reference data to compare these approaches, and the parameters governing tumor epitope immunogenicity remain unclear. Here, we assembled a global consortium wherein each participant predicted immunogenic epitopes from shared tumor sequencing data. 608 epitopes were subsequently assessed for T cell binding in patient-matched samples. By integrating peptide features associated with presentation and recognition, we developed a model of tumor epitope immunogenicity that filtered out 98% of non-immunogenic peptides with a precision above 0.70. Pipelines prioritizing model features had superior performance, and pipeline alterations leveraging them improved prediction performance. These findings were validated in an independent cohort of 310 epitopes prioritized from tumor sequencing data and assessed for T cell binding. This data resource enables identification of parameters underlying effective anti-tumor immunity and is available to the research community.

Background: A large global effort is ongoing to develop vaccines against SARS-CoV-2, the causative agent of COVID-19. While there is accumulating information on the antibody response against SARS-CoV-2, less is known about the SARS-CoV-2 antigens that are targeted by CD8 T cells. Such knowledge will be of high value to gain fundamental insights into the antigenic landscape of SARS-CoV-2 recognized by CD8 T cells, to develop tool allowing focused analysis of the SARS-CoV-2 specific T cell responses directly ex vivo, and to understand whether current vaccine designs are covering the CD8 T cell recognized antigens. Methods: To address this issue, we have analyzed samples from 18 COVID-19 patients for CD8 T cell recognition of 500 predicted SARS-CoV-2-derived epitopes restricted to 10 common HLA-A and HLA-B alleles. For each HLA allele, the top 50 epitopes were selected based on predicted binding affinity and likelihood of successful proteasomal processing. To probe for CD8 T cell recognition of the selected epitope-HLA complexes, we made use of our in-house technology based on multiplexing of peptide HLA (pHLA) multimers conjugated to fluorescent dyes. Results: In addition to previous studies showing CD8 T cell reactivity towards epitopes derived from the spike protein of SARS-CoV-2, we have identified several CD8 T cell recognized epitopes derived from the ORF1ab, including one epitope displaying clear immunodominant properties across patients positive for HLA-A*01:01. Investigation of the functional status of part of the identified responses (including 4 responses specific for the immunodominant epitope) revealed that the T cell responses were highly dysfunctional. In addition the SARS-CoV-2 specific CD8 T cell responses displayed an increased expression of NKG2A in comparison with bulk CD8 T cells, which may explain their dysfunctional state. Conclusions: Our data suggest that part of the ORF1ab encodes multiple CD8 T cell antigens including one immunodominant epitope. Noteworthy these epitopes were derived from a part of the viral genome that is not included in the majority of vaccine candidates in development, and this may potentially influence their clinical activity and safety profile. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

Following local inflammation, CD8+ T cells develop into tissue-resident memory T cells (TRM). Through combined lineage tracing and single-cell transcriptome analysis, the authors show that the circulating T cell pool harbors TRM precursors that commit to the TRM fate before tissue entry., 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., Graphical Abstract

Reporter proteins have become an indispensable tool in biomedical research. However, exogenous introduction of these reporters into mice poses a risk of rejection by the immune system. Here, we describe the generation, validation and application of a multiple reporter protein tolerant ‘Tol' mouse model that constitutively expresses an assembly of shuffled reporter proteins from a single open reading frame. We demonstrate that expression of the Tol transgene results in the deletion of CD8+ T cells specific for a model epitope, and substantially improves engraftment of reporter-gene transduced T cells. The Tol strain provides a valuable mouse model for cell transfer and viral-mediated gene transfer studies, and serves as a methodological example for the generation of poly-tolerant mouse strains., Bresser and Dijkgraaf et al. develop the ‘Tol’ strain, a genetically modified mouse model that expresses a range of shuffled reporter and modifier proteins from a single open reading frame. This strain is immunologically tolerant to these reporter and modifier proteins, providing a valuable model system for cell transfer studies and virus-mediated gene transfer studies.

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. In this Perspective, we describe the current state of knowledge on the role of the CD47-SIRPα axis in physiological tissue homeostasis and as a promising therapeutic target in, amongst others, oncology, fibrotic diseases, atherosclerosis and stem cell therapies. In addition, we highlight where additional insight will be beneficial to allow optimal exploitation of this myeloid cell checkpoint as a target in human disease.

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.

Multiomic profiling of several cohorts of patients treated with immune checkpoint blockade highlights the presence and potential role of B cells and tertiary lymphoid structures in promoting therapy response.Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers(1-10) and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity(11-15), although these have been less well-studied in ICB treatment(16). A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling(17) 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-counter(18)) 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.

BackgroundThe profound disparity in response to immune checkpoint blockade (ICB) by cutaneous melanoma (CM) and uveal melanoma (UM) patients is not well understood. Therefore, we characterized metastases of CM and UM from the same metastatic site (liver), in order to dissect the potential underlying mechanism in differential response on ICB.MethodsTumor liver samples from CM (n=38) and UM (n=28) patients were analyzed at the genomic (whole exome sequencing), transcriptional (RNA sequencing) and protein (immunohistochemistry and GeoMx Digital Spatial Profiling) level.ResultsComparison of CM and UM metastases from the same metastatic site revealed that, although originating from the same melanocyte lineage, CM and UM differed in somatic mutation profile, copy number profile, tumor mutational burden (TMB) and consequently predicted neoantigens. A higher melanin content and higher expression of the melanoma differentiation antigen MelanA was observed in liver metastases of UM patients. No difference in B2M and human leukocyte antigen-DR (HLA-DR) expression was observed. A higher expression of programmed cell death ligand 1 (PD-L1) was found in CM compared with UM liver metastases, although the majority of CM and UM liver metastases lacked PD-L1 expression. There was no difference in the extent of immune infiltration observed between CM and UM metastases, with the exception of a higher expression of CD163 (pConclusionsWhile TMB was different between CM and UM metastases, tumor immune infiltration was similar. The greater dependency on PD-L1 as an immune checkpoint in CM and the identification of higher exhaustion ratios in UM may both serve as explanations for the difference in response to ICB. Consequently, in order to improve current treatment for metastatic UM, reversal of T cell exhaustion beyond programmed cell death 1 blockade should be considered.

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.PurposeExecute 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.ExperimentalTen 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.ResultsFive 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.ConclusionThe 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 cells are a key player in cancer immunotherapy, but strategies to expand tumor-reactive cells and study their interaction with tumor cells at the level of an individual patient are limited. This protocol describes the generation and functional assessment of tumor-reactive T cells based on co-cultures of tumor organoids and autologous peripheral blood lymphocytes. The procedure consists of an initial co-culture of two weeks in which tumor-reactive T cells are first expanded in the presence of (IFNγ-stimulated) autologous tumor cells. Subsequently, T cells may be evaluated for their capacity to carry out effector functions (IFNγ secretion, degranulation) upon 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 two weeks, tumor-reactive CD8(+) T cell populations can be obtained for ~33-50% of non-small cell lung cancer (NSCLC) and microsatellite instable (MSI) colorectal cancer (CRC) patient samples. It thereby allows the establishment of ex vivo test systems for T cell-based immunotherapy at the level of the individual patient.

Tick-borne encephalitis virus (TBEV) is a leading cause of viral meningoencephalitis in many parts of Europe and eastwards in Asia, with high morbidity and often long-term neurologic sequelae. With no treatment available, studies of the immune response to TBEV are essential for the understanding of the immunopathogenesis of tick-borne encephalitis and for the development of therapeutics. We have previously demonstrated that CD8+ T cell responses in peripheral blood in patients with acute TBEV peak at around 7 d after hospitalization in the neuroinvasive phase of the disease. In this study, we identified six novel TBEV HLA-A2– and HLA-B7–restricted epitopes, all derived from the nonstructural proteins of TBEV. This identification allowed for a comprehensive phenotypic and temporal analysis of the HLA-A2– and HLA-B7–restricted Ag-specific CD8+ T cell response during the acute stages of human TBEV infection. HLA-A2– and HLA-B7–restricted TBEV epitope–specific effector cells predominantly displayed a CD45RA−CCR7−CD27+CD57− phenotype at day 7, which transitioned into separate distinct phenotypes for HLA-A2– and HLA-B7–restricted TBEV-specific CD8+ T cells, respectively. At day 21, the most prevalent phenotype in the HLA-A2–restricted CD8+ T cell populations was CD45RA−CCR7−CD27+CD57+, whereas the HLA-B7–restricted CD8+ T cell population was predominantly CD45RA+CCR7−CD27+CD57+. Almost all TBEV epitope–specific CD8+ T cells expressed α4 and β1 integrins at days 7 and 21, whereas the bulk CD8+ T cells expressed lower integrin levels. Taken together, human TBEV infection elicits broad responses to multiple epitopes, predominantly derived from the nonstructural part of the virus, establishing distinct maturation patterns for HLA-A2– and HLA-B7–restricted TBEV epitope–specific CD8+ T cells.

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-1 T ), intermediate (PD-1 N ) and no PD-1 expression (PD-1 - ) from non-small-cell lung cancer patients. PD-1 T T cells showed a markedly different transcriptional and metabolic profile from PD-1 N and PD-1 - lymphocytes, as well as an intrinsically high capacity for tumor recognition. Furthermore, while PD-1 T 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-1 T 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.

BackgroundIL-2 and currently available engineered variants are of interest for solid tumor treatment, but their efficacy and toxicity profiles remain suboptimal. These results reflect the pleiotropic signaling via IL-2 receptors on different cell types that may simultaneously drive desired and undesired responses. We hypothesized that restricting IL-2’s activity to CD8+ T cells would improve efficacy while also lowering its toxicity profile.MethodsWe developed a cis-targeted IL-2 that selectively acts on CD8+ T cells (CD8-IL2) and assessed its activity using the T3 progressor MCA sarcoma model, which was selected because (a) it is sensitive to anti-PD-1 therapy when tumors are small but develops insensitivity as tumor size increase, (b) rejection requires both CD4+ and CD8+ T cells and (c) rejection is dependent on tumor expression of two neoantigens: mItgb1 (MHC-II) and mLama4 (MHC-I).ResultsWhereas mice bearing 8-day T3 tumors had become insensitive to anti-PD-1 mediated tumor rejection, 90% of mice treated with single dose CD8-IL2 monotherapy rejected their tumors, while high dose IL-2 produced minimal efficacy. Efficacy occurred without body weight loss. These results suggest that CD8-IL2 can induce therapeutic effects at a time when tumors became insensitive to anti-PD-1. To assess this possibility in a more controlled manner, we used a tumor neoantigen vaccine model that depends on CD4+ T cell help for development of functional CD8+ T cells at both the priming stage in the lymph node as well as the effector stage at the tumor site. Mice bearing T3 tumors were vaccinated with a synthetic long peptide (SLP) containing the mLama4 neoepitope and either a high or low dose of an SLP containing the mItgb1 neoepitope. Whereas 85% of tumor bearing mice that received the vaccine containing mLama4 plus low dose mItgb1 SLP rejected their tumors, surprisingly none of the mice receiving high dose mItgb1 underwent tumor rejection. This high dose inhibition was reversed when CD8-IL2 was administered after high dose vaccination and at concentrations that had only modest activity in tumor bearing, non-vaccinated mice. With CD8-IL2 treatment, antigen specific T cells were expanded and displayed increased expression of activation-associated markers and reduced expression of exhaustion-associated markers.ConclusionsCD8-IL2 outperformed other forms of engineered IL-2 in anti-tumor efficacy, showed a significantly improved toxicity profile, and rescued deficient CD8 T cell responses resulting from poor CD4 help. In sum, we demonstrate high level antitumor efficacy and tolerability with a new form of targeted IL-2.Ethics ApprovalMice used in this study were between 8 and 12 weeks of age and were maintained in accordance with procedures approved by the Association for Assessment and Accreditation of Laboratory Animal Care and Accredited Animal Studies Committee of Washington University in St. Louis

'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.

Cancer cells are able to evade immune surveillance through the expression of inhibitory ligands that bind their cognate receptors on immune effector cells. Expression of Programmed Death-Ligand 1 (PD-L1) in tumor micro-environments is a major immune checkpoint for tumor-specific T cell responses, by binding to Programmed Cell Death protein-1 (PD-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 has been shown to form 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 (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. Both 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 tumor cell killing in vivo. These data identify QPCTL as a novel target to interfere with the CD47 pathway, and thereby augment antibody therapy of cancer.

To increase cancer immunotherapy success, PD-1 blockade must be combined with rationally selected treatments. Here, we examined in a poorly immunogenic mouse breast cancer model the potential of antibody-based immunomodulation and conventional anti-cancer treatments to collaborate with anti-PD-1 treatment. One important requirement to improve anti-PD-1-mediated tumor control was to promote tumor-specific cytotoxic T cell (CTL) priming, which was achieved by stimulating the CD137 costimulatory receptor. A second requirement was to overrule PD-1-unrelated mechanisms of CTL suppression in the tumor micro-environment (TME). This was achieved by radiotherapy and cisplatin treatment. In the context of CD137/PD-1-targeting immunotherapy, radiotherapy allowed for tumor elimination by altering the TME, rather than intrinsic CTL functionality. Combining this radioimmunotherapy regimen with low-dose cisplatin improved CTL-dependent regression of a contralateral tumor outside the radiation field. Thus, systemic tumor control may be achieved by combining immunotherapy protocols that promote T cell priming with (chemo)radiation protocols that permit CTL activity in both the irradiated tumor and (occult) metastases.Summary statementThis study reveals that radiotherapy and cisplatin can be ‘re-purposed’ to improve antibody-based immunotherapy success in poorly immunogenic breast cancer by overruling PD-1 unrelated mechanisms of T cell suppression in the tumor micro-environment.

Experimental and clinical observations have highlighted the role of cytotoxic T cells in human tumor control. However, the parameters that control tumor cell sensitivity to T cell attack remain incompletely understood. To identify modulators of tumor cell sensitivity to T cell effector mechanisms, we performed a whole genome haploid screen in HAP1 cells. Selection of tumor cells by exposure to tumor-specific T cells identified components of the interferon-γ (IFN-γ) receptor (IFNGR) signaling pathway, and tumor cell killing by cytotoxic T cells was shown to be in large part mediated by the pro-apoptotic effects of IFN-γ. Notably, we identified schlafen 11 (SLFN11), a known modulator of DNA damage toxicity, as a regulator of tumor cell sensitivity to T cell-secreted IFN-γ. SLFN11 does not influence IFNGR signaling, but couples IFNGR signaling to the induction of the DNA damage response (DDR) in a context dependent fashion. In line with this role of SLFN11, loss of SLFN11 can reduce IFN-γ mediated toxicity. Collectively, our data indicate that SLFN11 can couple IFN-γ exposure of tumor cells to DDR and cellular apoptosis. Future work should reveal the mechanistic basis for the link between IFNGR signaling and DNA damage response, and identify tumor cell types in which SLFN11 contributes to the anti-tumor activity of T cells.

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.

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.

T cell responses upon infection display a remarkably reproducible pattern of expansion, contraction, and memory formation. If the robustness of this pattern builds entirely on signals derived from other cell types or if activated T cells themselves contribute to the orchestration of these population dynamics-akin to bacterial quorum regulation-is unclear. Here, we examined this question using time-lapse microscopy, genetic perturbation, bioinformatic predictions, and mathematical modeling. We found that ICAM-1-mediated cell clustering enabled CD8+ T cells to collectively regulate the balance between proliferation and apoptosis. Mechanistically, T cell expressed CD80 and CD86 interacted with the receptors CD28 and CTLA-4 on neighboring T cells; these interactions fed two nested antagonistic feedback circuits that regulated interleukin 2 production in a manner dependent on T cell density as confirmed by in vivo modulation of this network. Thus, CD8+ T cell-population-intrinsic mechanisms regulate cellular behavior, thereby promoting robustness of population dynamics.

Upon infection, antigen-specific T lymphocytes become activated, proliferate, differentiate, and acquire various effector functions. Much of our understanding of the molecular mechanisms underlying these processes derives from studies leveraging gene deletion, RNAi, and overexpression approaches. However, these perturbations do not inform on the regulation of gene activity under physiological conditions. Genetic reporter systems that couple biological events to detectable output signals are capable of providing this information. Here, we review the reporter approaches being currently used to investigate various aspects of T cell behavior, and discuss advantages and disadvantages inherent to different designs. We outline emerging applications based on recent advances in other fields, and highlight the potential of synthetic biology and genome engineering to address open questions in the field.

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.

For T cells, variety is the spice of life CD4 + helper T cells come in a variety of flavors. This allows them to respond in a manner that is tailored to the pathogen they encounter. Becattini et al. wondered whether multiple “flavors” of human CD4 + T cells respond to specific stimuli or if just one flavor dominates. To find out, they stimulated human memory CD4 + T cells with a fungus, a bacteria, or a vaccine antigen. Multiple helper cell subsets participated in each response. T cell receptor sequencing revealed that in some cases, T cells with the same specificity acquired different helper cell fates. Thus, there is more heterogeneity in human T cell responses than previously appreciated. Science , this issue p. 400

Infusion of tumor-infiltrating T-lymphocytes was developed as treatment for metastatic melanoma by Dr. S. Rosenberg (Surgery Branch, NIH, Bethesda, MD, USA) in the late 90's of the past century. This therapy was based on the finding that melanomas oftentimes have infiltrates of CD8 T cells that show recognition of melanoma cells. Many tumor antigens have been characterized for which specific T cells can be found within the tumor or in peripheral blood. As tumors may use mechanisms to induce local T cell unresponsiveness, isolation of TILs and in vitro activation and expansion was shown to overcome this anergic state both in preclinical models and in clinical trials. In order for the TILs to survive, persist, and be cytolytic after infusion, culture time should be as short as possible (to obtain cells with a less terminally differentiated phenotype), patients should receive lymphodepleting treatment (chemotherapy +/- TBI) prior to infusion and interleukin-2 as a survival factor post infusion. Up to date several hundreds of metastatic melanoma patients have been treated with TIL therapy in centers within the US, Europe and Israel. Objective response rates vary between 38 and 72% depending on the phase I/II trial with 10-20% of patients acquiring a CR, which were most often very durable. Many of these oftentimes heavily pretreated patients have benefitted from TIL therapy. As melanomas are heavily mutated cancers, the idea that T cells may react to epitopes derived from mutated proteins could be investigated with new technologies developed at NKI. Using whole exome sequencing, the total mutational burden could be measured for each melanoma. Only those mutations present in expressed genes were used for epitope prediction for every MHC class I molecule of the patient using validated prediction algorithms. Filters were used to select those epitopes with high enough binding affinity for MHC that were subsequently synthesized for production of peptide-MHC multimers that could be used to screen for neo-antigen specific T cells among peripheral blood T- cells or TIL using a multiplex flow cytometric technology. Together with the NCI, using a somewhat different approach, we were able to show that in the vast majority of melanoma TIL, CD8 T cells specific for epitopes resulting from UV light induced DNA damage can be found, sometimes in high frequencies (up to 25-30%), strongly suggesting that neo-antigen-specific T cells play an important role in the success of TIL therapy and possibly other immunotherapies, including checkpoint inhibitors. So far TIL therapy has been mostly used in metastatic melanoma patients pretreated with BRAF inhibitors, anti-CTLA4, high-dose IL-2, and demonstrated activity irrespective of these treatments, although isolation of TIL appeared less successful in patients progressing upon BRAFi. Early results in patients pretreated with PD1 blocking antibodies are promising as well, although the objective response rate is still unknown. Next to metastatic melanoma, TIL can be grown from other cancers as well and promising results have been obtained in patients with cervical cancer and in a patient with cholangiocarcinoma. Trials are planned for patients with epithelial ovarian cancer as well. Citation Format: John B.A.G. Haanen, Joost Van den Berg, Maaike Van Zon, Noor Bakker, Marnix Geukes, Hans Van Thienen, Christian Blank, Pia Kvistborg, Ton Schumacher. TIL therapy in the era of immune checkpoint blockade. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL01-03.

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 identification of tumor antigens through unbiased screening.

Resident memory T cells sound the alarm Immunological memory protects against reinfection. Resident memory T cells (T RM ) are long-lived and remain in the tissues where they first encountered a pathogen (see the Perspective by Carbone and Gebhardt). Schenkel et al. and Ariotti et al. found that CD8 + T RM cells act like first responders in the female reproductive tissue or the skin of mice upon antigen reencounter. By secreting inflammatory proteins, T RM cells rapidly activated local immune cells to respond, so much so that they protected against infection with an unrelated pathogen. Iijima and Iwasaki found that CD4 + T RM cells protected mice against reinfection with intravaginal herpes simplex virus 2. Science , this issue p. 98 , p. 101 , p. 93 ; see also p. 40

Upon primary infection, naïve T cells that recognize their cognate antigen become activated, proliferate, and simultaneously differentiate into various subsets. A long-standing question in the field has been how this cellular diversification is achieved. Conceptually, diverse cellular output may either arise from every single cell or only from populations of naïve cells. Furthermore, such diversity may either be driven by cell-intrinsic heterogeneity or by external, niche-derived signals. In this review, we discuss how recently developed technologies have allowed the analysis of the mechanisms underlying T cell diversification at the single cell level. In addition, we outline the implications of this work on our understanding of the formation of immunological memory, and describe a number of unresolved key questions in this field.

e14010 Background: The key driver for effective immune cell therapies is the overall binding strength of the immune cell and the target cell (e.g. tumor cells). The overall strength is known as ‘avidity’, a parameter reflecting interaction efficiency. The key to success for immune cell therapies is generating effective and long-lasting immune responses. The avidity of an immune cell to its target is predicative of its function, but current techniques to measure avidity are low-throughput and ineffective. Herein, we describe the use of acoustic forces to discriminate immune cells based on their avidity to tumor cells. The force required to separate a cell from its target is called the ‘rupture force’, and in this study, we were able to identify the rupture forces of tumor specific and non-specific T cells and enrich these different populations for downstream characterization. Methods: T cells from a healthy donor were transduced with either a non-relevant, or a melanoma recognizing T cell receptor and selected with puromycin resistance. Melanoma cells were seeded in the flow cell and allowed to adhere overnight to form a monolayer. For confocal experiments CFSE and Cell Trace far red stained T cells were mixed in a 1:1 ratio before co-culturing them in the flow cell. An acoustic force ramp was applied within the flow cell and cell detachment was monitored. Results: T cells engineered with a melanoma antigen-recognizing T-cell receptor needed 6 times more force than non-specific T cells to be separated from the melanoma target cells. Furthermore, 1.4 to 3.6-fold enrichment of high-avidity T cells was obtained from a mixed population of specific and non-specific T cells using acoustic forces. Conclusions: These findings indicate that melanoma-specific T cells bind with a higher avidity than non-specific T cells and that they can be separated with this approach. In conclusion, we demonstrate a novel method to measure cell avidity and sort cells by utilizing acoustic forces.

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 the accumulated DNA damage causing the malignant transformation. 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. From the efforts dissecting the neoantigen-specific T-cell response it has become clear that only a very minor fraction of the accumulated mutations is recognized by the immune system, and the challenge to unravel the neoantigen-specific T-cell response lies in identifying which neoantigens are more likely to be true T-cell epitopes. We have analyzed neoantigen-specific T-cell reactivity in 12 melanoma patients using an in silico epitope prediction pipeline based on RNA expression, predicted HLA binding affinity, proteasomal processing and self-similarity to predict potential neoepitopes. We screened for T-cell recognition of 7000 epitopes from these 12 patients (average ~550 epitopes per patient, range: 96-1902) using our pMHC multimer combinatorial encoding technology and found 19 epitopes to be recognized by T-cells (hits) and 6981 to be “non-hits.” Based on these data we have examined the properties of T-cell recognized neoantigens. An intriguing observation is an enrichment within T-cell recognized epitopes of epitopes with the mutation positioned within the last 4 amino acids (C-terminal end of the peptide) compared to the screened set of epitopes. Fifteen out of 19 hits (approximately 80%) harbored a mutation within the last 4 amino acids of the peptide, whereas within the full set of screen epitopes it is 43%. While it is currently unclear what the reason is for this, this could reflect a biologic importance in T-cell recognition of the C-terminal part of the epitope. Furthermore, RNA expression and predicted binding affinity to HLA are important informative parameters for selecting T-cell recognized epitopes. A striking observation is that predicted binding affinity not only correlates with likelihood of observing a T-cell response but also the magnitude of this T-cell response, suggesting a hierarchy within neoantigens, and that not all neoantigens are of equal immunologic quality. In summary, our findings indicate that T-cell recognized neoantigens may differ from the neoantigen pool not recognized. In particular regarding position of the mutation with the epitope, RNA abundance and predicted HLA binding affinity. Importantly, our data reveal a hierarchy within neoantigens comparable to immunodominance known from viral infections. This hierarchy appears to depend mostly on binding affinity. These observations are likely to be highly relevant when selecting neoantigens for therapeutic manipulation such as vaccines. Citation Format: Pia Kvistborg, Marit M. van Buuren, Daisy Philips, Nienke van Rooij, Arno Velds, Sam Behjati, Marlous van den Braber, Mireille Toebes, Lorenzo Fanchi, Maarten Slagter, Marie Stentoft Svane, Patrick Hwu, Joost van den Berg, Michael Stratton, Christian Blank, John B.A.G. Haanen, Can Kesmir, Ton N.M. Schumacher. Properties of T-cell-recognized neoantigens [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B022.

Reinvigoration of tumor-specific T-cells by cancer immunotherapies, in particular PD-1/PD-L1 blocking agents, has greatly improved clinical outcome in multiple cancer types. Nevertheless, durable clinical benefit is currently limited to a small number of patients. To achieve a better understanding of the immunologic determinants of response to anti-PD-1 therapy, we assessed transcriptional and functional profiles of tumor-infiltrating lymphocyte (TIL) subsets from non-small cell lung cancer specimens. Thereby, we identified a transcriptionally distinct CD8 TIL pool with enriched capacity for tumor recognition. This TIL pool, termed PD-1T TILs, is characterized by bright PD-1 expression and constitutive CXCL13 secretion, which can mediate immune cell recruitment to tertiary lymphoid structures. Notably, the presence of PD-1T TILs correlates with response and survival in a small cohort of lung cancer patients treated with PD-1 blockade. To assess the role of PD-1T TILs and CXCL13 for response to PD-1 blockade in other cancer types, we developed a platform using human tumor explants to visualize immunologic responses to anti-PD-1 on a patient-specific level. Analysis of the cellular and soluble tumor microenvironment composition as well as of treatment-induced changes in tumor-infiltrating immune cells revealed immunologic responses to anti-PD-1 in 5 different cancer types. Of note, responding tumors in different tumor types were characterized by a clear enrichment in both PD-1T TILs and CXCL13 production. Collectively, our data reveal a distinct state of PD-1 bright lymphocytes that are enriched for tumor-reactivity in human cancer, making them an attractive proxy for the antitumor potential of the intratumoral T-cell pool. Furthermore, we established technology using human tumor explants to measure the immunologic response to T-cell checkpoint inhibition on a personalized basis. Finally, with this approach we identified PD-1T TILs and CXCL13 as determinants for response to anti-PD-1 in multiple cancer types, opening potential new avenues for therapeutic intervention and improved patient selection. Citation Format: Daniela S. Thommen, Viktor H. Koelzer, Petra Herzig, Marjolein de Bruijn, Paula Voabil, Marlous van den Braber, Karlijn Hummelink, Kim Monkhorst, Kirsten D. Mertz, Alfred Zippelius, John B.A.G. Haanen, Ton N.M. Schumacher. Identification of PD-1T TILs and CXCL13 as determinants for response to anti-PD-1 treatment using human tumor explants [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B050.

Checkpoint blockade therapies that aim to reactivate antitumor immune responses have revolutionized cancer treatment, resulting in durable responses in a significant proportion of patients with advanced tumor progression. Nevertheless, many patients fail to reach long-term clinical benefit due to lack of response or acquired resistance. Inconsistency in therapy outcomes may be explained in part by recent findings suggesting that immune cell infiltrates in tumors are highly heterogeneous among patients. Therefore, comprehensive characterization of the diverse functional states exhibited by immune cell infiltrates is critical for the development of more effective immunotherapies. Here, we characterized immune cell infiltrates within tumors derived from 28 metastatic melanoma patients by single-cell RNA-seq of ~100,000 immune cells, and parallel T-cell receptor (TCR) sequencing, thereby generating an unbiased map of the expression signatures of immune cells, as well as clonality of T-cells within and between metastases. We identified within the tumor infiltrates naïve, semi- and fully-activated effector, dysfunctional, and regulatory T-cells, as well as NK cells, and various myeloid subsets. While various immune cell types and cellular states are shared among patients, their frequency in each is highly heterogeneous even among similar tumor progression stages and treatment background. We noticed that clonally expanded T-cells predominantly adapted similar expression profiles. The frequencies of certain sub-populations were found to be correlated; notably, dysfunctional CD8 T-cell states were associated with prevalence of regulatory T-cells and follicular helper cells. The high-resolution map demonstrated gradient transitions between activation and dysfunctional states of CD8 T-cells, as well as variability within regulatory T-cell states. We used computational modeling to find the key transcription factors driving these gradients of expression states. Our analysis also puts forward novel candidate genes, which are highly correlate with known checkpoint targets within specific single-cell clusters, and may prove to be effective targets for checkpoint blockade. In order to validate our results, we performed in vitro reactivity assays (for available samples), which allowed us to determine autologous-tumor reactive and nonreactive TCRs, adding the reactive potential of T-cells as another layer of information to the single-cell expression signatures, facilitating inference of the functionality of clonally expanded T-cell populations in the tumor. Linking autologous tumor reactivity with single-cell expression profiles, we found that T-cells presenting reactive capability in vitro show a highly dysfunctional signature in the original tumor. The lack or presence of such reactive CD8 T-cells was also associated with patient response to immunotherapy (n=10). In conclusion, we present an atlas of immune cell infiltrates of human melanoma, revealing intra- and inter-patient heterogeneity of tumor immune infiltrates, highlighting regulatory circuits underlying different immune populations and their interactions. Our findings demonstrate how single-cell analysis may serve in the future as a tool for predicting response to therapy, provide rapid and effective tumor-immune state characterization, and ultimately lead to optimization of personalized immunotherapy. Citation Format: do Yofe, Hanjie Li, Anne van der Leun, Yaniv Lubling, Dikla Gelbard, Alexander C. J. van Akkooi, Amos Tanay, Ton N.M. Schumacher, Ido Amit. Single-cell analysis illuminates the gradients of immune cell functional states within human melanoma tumors, and facilitates characterization of tumor-reactive T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B054.

The infiltration of human tumors by T cells is a common phenomenon, and over the past decades, it has become increasingly clear that the nature of such intratumoral T-cell populations can predict disease course. Furthermore, intratumoral T cells have been utilized therapeutically in clinical studies of adoptive T-cell therapy. In this review, we describe how novel methods that are either based on T-cell receptor (TCR) sequencing or on cancer exome analysis allow the analysis of the tumor reactivity and antigen-specificity of the intratumoral TCR repertoire with unprecedented detail. Furthermore, we discuss studies that have started to utilize these techniques to probe the link between cancer exomes and the intratumoral TCR pool. Based on the observation that both the cancer epitope repertoire and intratumoral TCR repertoire appear highly individual, we outline strategies, such as 'autologous TCR gene therapy', that exploit the tumor-resident TCR repertoire for the development of personalized immunotherapy.

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), with both branches subsequently producing dendritic cells. However, this model is based mainly on in vitro clonal assays and population-based tracking in vivo, which could miss in vivo single-cell complexity. Here we avoid these issues by using a new quantitative version of 'cellular barcoding' 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 HSCs, 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.

Peptide–MHC (pMHC) multimers have become one of the most widely used tools to measure Ag-specific T cell responses in humans. With the aim of understanding the requirements for pMHC-based personalized immunomonitoring, in which individuals expressing subtypes of the commonly studied HLA alleles are encountered, we assessed how the ability to detect Ag-specific T cells for a given peptide is affected by micropolymorphic differences between HLA subtypes. First, analysis of a set of 10 HLA-A*02:01–restricted T cell clones demonstrated that staining with pMHC multimers of seven distinct subtypes of the HLA-A*02 allele group was highly variable and not predicted by sequence homology. Second, to analyze the effect of minor sequence variation in a clinical setting, we screened tumor-infiltrating lymphocytes of an HLA-A*02:06 melanoma patient with either subtype-matched or HLA-A*02:01 multimers loaded with 145 different melanoma-associated Ags. This revealed that of the four HLA-A*02:06–restricted melanoma-associated T cell responses observed in this patient, two responses were underestimated and one was overlooked when using subtype-mismatched pMHC multimer collections. To our knowledge, these data provide the first demonstration of the strong effect of minor sequence variation on pMHC-based personalized immunomonitoring, and they provide tools to prevent this issue for common variants within the HLA-A*02 allele group.

The development of peptide loaded major histocompatibility complexes (MHC) conjugated to fluorochromes by Davis and colleagues 20 years ago provided a highly useful tool to identify and characterize antigen-specific T cells. In this chapter we describe a multiplexing strategy that allows detection of high numbers of T cell responses in parallel.

Recognition of neoantigens that are formed as a consequence of DNA damage is likely to form a major driving force behind the clinical activity of cancer immunotherapies such as T-cell checkpoint blockade and adoptive T-cell therapy(1-7). Therefore, strategies to selectively enhance T-cell reactivity against genetically defined neoantigens(1,8-11) are currently under development. In mouse models, T-cell pressure can sculpt the antigenicity of tumours, resulting in the emergence of tumours that lack defined mutant antigens(12,13). However, whether the T-cell-recognized neoantigen repertoire in human cancers is constant over time is unclear. Here we analyse the stability of neoantigen-specific T-cell responses and the antigens they recognize in two patients with stage IV melanoma treated by adoptive T-cell transfer. The T-cell-recognized neoantigens can be selectively lost from the tumour cell population, either by overall reduced expression of the genes or loss of the mutant alleles. Notably, loss of expression of T-cell-recognized neoantigens was accompanied by development of neoantigen-specific T-cell reactivity in tumour-infiltrating lymphocytes. These data demonstrate the dynamic interactions between cancer cells and T cells, which suggest that T cells mediate neoantigen immunoediting, and indicate that the therapeutic induction of broad neoantigen-specific T-cell responses should be used to avoid tumour resistance

The new intradermal DNA delivery technique, termed DNA tattooing might overcome the discrepancy between the encouraging immunogenicity results obtained with DNA vaccines in murine studies and the poor results obtained in non-human primates and humans, the so called "simian barrier". Here, we demonstrate a 10- to 100-fold increase in the magnitude of vaccine specific T-cell responses in peripheral blood from DNA tattooed rhesus macaques, as compared to T-cell responses in animals immunized via intramuscular (IM) route. A marked increase in the magnitude of the antigen specific T-cell responses as well as an increase in the number of animals responding to the immunogens was observed. These findings in non-human primates suggest that similar results may be observed in humans. Clinical trials are planned to validate tattooing as an optimal method of DNA vaccine delivery in humans. © 2008 Elsevier Ltd. All rights reserved.

Somatic mutations in the genome represent one of the major drivers of malignancy. However, non-synonymous mutations are also a source of mutated peptides that are presented by HLA molecules to induce protective CD4 and CD8 T cell responses. Consistent with this notion, the mutation burden of a tumor is correlated with local immunity as well as outcome of therapy and patient survival. Furthermore, neoantigen-specific T cells appear sufficient to control tumors prophylactically and therapeutically. While the role of neoantigens as a determinant of the foreignness of human cancers is now well established, major questions, including the relative importance of clonal vs subclonal neoantigens, and CD4 vs CD8 T cells, remain unanswered. We expect continued animal studies to address some of the open issues and ongoing clinical trials to establish the utility of therapeutic strategies to enhance neoantigen-specific T cell responses in human cancer.