Characterisation of T cell receptor repertoires in coeliac disease.

AIMS: Characterise T-cell receptor gene (TR) repertoires of small intestinal T cells of patients with newly diagnosed (active) coeliac disease (ACD), refractory CD type I (RCD I) and patients with CD on a gluten-free diet (GFD). METHODS: Next-generation sequencing of complementarity-determining region 3 (CDR3) of rearranged T cell receptor ß (TRB) and γ (TRG) genes was performed using DNA extracted from intraepithelial cell (IEC) and lamina propria cell (LPC) fractions and a small subset of peripheral blood mononuclear cell (PBMC) samples obtained from CD and non-CD (control) patients. Several parameters were assessed, including relative abundance and enrichment. RESULTS: TRB and TRG repertoires of CD IEC and LPC samples demonstrated lower clonality but higher frequency of rearranged TRs compared with controls. No CD-related differences were detected in the limited number of PBMC samples. Previously published LP gliadin-specific TRB sequences were more frequently detected in LPC samples from patients with CD compared with non-CD controls. TRG repertoires of IECs from both ACD and GFD patients demonstrated increased abundance of certain CDR3 amino acid (AA) motifs compared with controls, which were encoded by multiple nucleotide variants, including one motif that was enriched in duodenal IECs versus the PBMCs of CD patients. CONCLUSIONS: Small intestinal TRB and TRG repertoires of patients with CD are more diverse than individuals without CD, likely due to mucosal recruitment and accumulation of T cells because of protracted inflammation. Enrichment of the unique TRG CDR3 AA sequence in the mucosa of patients with CD may suggest disease-associated changes in the TCRγδ IE lymphocyte (IEL) landscape.

A dataset comprised of binding interactions for 104,972 antibodies against a SARS-CoV-2 peptide.

The dataset presented here contains quantitative binding scores of scFv-format antibodies against a SARS-CoV-2 target peptide collected via an AlphaSeq assay that can be used in the development and benchmarking of machine learning models. Starting from three seed sequences identified from a phage display campaign using a human naïve library, four sets of 29,900 antibodies were designed in silico by creating all k = 1 mutations and random k = 2 and k = 3 mutations throughout the complementary-determining regions (CDRs). Of the 119,600 designs, 104,972 were successfully built in to the AlphaSeq library and target binding was subsequently measured with 71,384 designs resulting in a predicted affinity value for at least one of the triplicate measurements. Data include antibodies with predicted affinity measurements ranging from 37 pM to 22 mM. To our knowledge, this dataset is the largest, publicly available dataset that contains antibody sequences, antigen sequence and quantitative measurements of binding scores and provides an opportunity to serve as a benchmark to evaluate antibody-specific representation models for machine learning.

Massively multiplexed affinity characterization of therapeutic antibodies against SARS-CoV-2 variants.

Antibody therapies represent a valuable tool to reduce COVID-19 deaths and hospitalizations. Multiple antibody candidates have been granted emergency use authorization by the Food and Drug Administration and many more are in clinical trials. Most antibody therapies for COVID-19 are engineered to bind to the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein and disrupt its interaction with angiotensin-converting enzyme 2 (ACE2). Notably, several SARS-CoV-2 strains have accrued mutations throughout the RBD that improve ACE2 binding affinity, enhance viral transmission and escape some existing antibody therapies. Here, we measure the binding affinity of 33 therapeutic antibodies against a large panel of SARS-CoV-2 variants and related strains of clinical significance using AlphaSeq, a high-throughput yeast mating-based assay to determine epitopic residues, determine which mutations result in loss of binding and predict how future RBD variants may impact antibody efficacy.

Lymphocytic infiltration in stage II microsatellite stable colorectal tumors: A retrospective prognosis biomarker analysis.

BACKGROUND: Identifying stage II patients with colorectal cancer (CRC) at higher risk of progression is a clinical priority in order to optimize the advantages of adjuvant chemotherapy while avoiding unnecessary toxicity. Recently, the intensity and the quality of the host immune response in the tumor microenvironment have been reported to have an important role in tumorigenesis and an inverse association with tumor progression. This association is well established in microsatellite instable CRC. In this work, we aim to assess the usefulness of measures of T-cell infiltration as prognostic biomarkers in 640 stage II, CRC tumors, 582 of them confirmed microsatellite stable. METHODS AND FINDINGS: We measured both the quantity and clonality index of T cells by means of T-cell receptor (TCR) immunosequencing in a discovery dataset (95 patients with colon cancer diagnosed at stage II and microsatellite stable, median age 67, 30% women) and replicated the results in 3 additional series of stage II patients from 2 countries. Series 1 and 2 were recruited in Barcelona, Spain and included 112 fresh frozen (FF, median age 69, 44% women) and 163 formalin-fixed paraffin-embedded (FFPE, median age 67, 39% women) samples, respectively. Series 3 included 270 FFPE samples from patients recruited in Haifa, Northern Israel, as part of a large case-control study of CRC (median age 73, 46% women). Median follow-up time was 81.1 months. Cox regression models were fitted to evaluate the prognostic value of T-cell abundance and Simpson clonality of TCR variants adjusting by sex, age, tumor location, and stage (IIA and IIB). In the discovery dataset, higher TCR abundance was associated with better prognosis (hazard ratio [HR] for ≥Q1 = 0.25, 95% CI 0.10-0.63, P = 0.003). A functional analysis of gene expression on these tumors revealed enrichment in pathways related to immune response. Higher values of clonality index (lower diversity) were not associated with worse disease-free survival, though the HR for ≥Q3 was 2.32 (95% CI 0.90-5.97, P = 0.08). These results were replicated in an independent FF dataset (TCR abundance: HR = 0.30, 95% CI 0.12-0.72, P = 0.007; clonality: HR = 3.32, 95% CI 1.38-7.94, P = 0.007). Also, the association with prognosis was tested in 2 independent FFPE datasets. The same association was observed with TCR abundance (HR = 0.41, 95% CI 0.18-0.93, P = 0.03 and HR = 0.56, 95% CI 0.31-1, P = 0.042, respectively, for each FFPE dataset). However, the clonality index was associated with prognosis only in the FFPE dataset from Israel (HR = 2.45, 95% CI 1.39-4.32, P = 0.002). Finally, a combined analysis combining all microsatellite stable (MSS) samples demonstrated a clear prognosis value both for TCR abundance (HR = 0.39, 95% CI 0.26-0.57, P = 1.3e-06) and the clonality index (HR = 2.13, 95% CI 1.44-3.15, P = 0.0002). These associations were also observed when variables were considered continuous in the models (HR per log2 of TCR abundance = 0.85, 95% CI 0.78-0.93, P = 0.0002; HR per log2 or clonality index = 1.16, 95% CI 1.03-1.31, P = 0.016). LIMITATIONS: This is a retrospective study, and samples had been preserved with different methods. Validation series lack complete information about microsatellite instability (MSI) status and pathology assessment. The Molecular Epidemiology of Colorectal Cancer (MECC) study had information about overall survival instead of progression-free survival. CONCLUSION: Results from this study demonstrate that tumor lymphocytes, assessed by TCR repertoire quantification based on a sequencing method, are an independent prognostic factor in microsatellite stable stage II CRC.

Clonality and antigen-specific responses shape the prognostic effects of tumor-infiltrating T cells in ovarian cancer.

CD8+ tumor-infiltrating lymphocytes (TILs) are not all specific for tumor antigens, but can include bystander TILs that are specific for cancer-irrelevant epitopes, and it is unknown whether the T-cell repertoire affects prognosis. To delineate the complexity of anti-tumor T-cell responses, we utilized a computational method for de novo assembly of sequences from CDR3 regions of 369 high-grade serous ovarian cancers from TCGA, and then applied deep TCR-sequencing for analyses of paired tumor and peripheral blood specimens from an independent cohort of 99 ovarian cancer patients. Strongly monoclonal T-cell repertoires were associated with favorable prognosis (PFS, HR = 0.65, 0.50-0.84, p = 0.003; OS, HR = 0.61, 0.44-0.83, p = 0.006) in TCGA cohort. In the validation cohort, we discovered that patients with low T-cell infiltration but low diversity or focused repertoires had clinical outcomes almost indistinguishable from highly-infiltrated tumors (median 21.0 months versus 15.9 months, log-rank p = 0.945). We also found that the degree of divergence of the peripheral repertoire from the TIL repertoire, and the presence of detectable spontaneous anti-tumor immune responses are important determinants of clinical outcome. We conclude that the prognostic significance of TILs in ovarian cancer is dictated by T-cell clonality, degree of overlap with peripheral repertoire, and the presence of detectable spontaneous anti-tumor immune response in the patients. These immunological phenotypes defined by the TCR repertoire may provide useful insights for identifying "TIL-low" ovarian cancer patients that may respond to immunotherapy.

Analytical evaluation of the clonoSEQ Assay for establishing measurable (minimal) residual disease in acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma.

BACKGROUND: The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines. METHODS: Sensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay's ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels. RESULTS: LoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low. CONCLUSIONS: These studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.

Comprehensive T cell repertoire characterization of non-small cell lung cancer.

Immunotherapy targeting T cells is increasingly utilized to treat solid tumors including non-small cell lung cancer (NSCLC). This requires a better understanding of the T cells in the lungs of patients with NSCLC. Here, we report T cell repertoire analysis in a cohort of 236 early-stage NSCLC patients. T cell repertoire attributes are associated with clinicopathologic features, mutational and immune landscape. A considerable proportion of the most prevalent T cells in tumors are also prevalent in the uninvolved tumor-adjacent lungs and appear specific to shared background mutations or viral infections. Patients with higher T cell repertoire homology between the tumor and uninvolved tumor-adjacent lung, suggesting a less tumor-focused T cell response, exhibit inferior survival. These findings indicate that a concise understanding of antigens and T cells in NSCLC is needed to improve therapeutic efficacy and reduce toxicity with immunotherapy, particularly adoptive T cell therapy.

T Cell Repertoire Dynamics during Pregnancy in Multiple Sclerosis.

Identifying T cell clones associated with human autoimmunity has remained challenging. Intriguingly, many autoimmune diseases, including multiple sclerosis (MS), show strongly diminished activity during pregnancy, providing a unique research paradigm to explore dynamics of immune repertoire changes during active and inactive disease. Here, we characterize immunomodulation at the single-clone level by sequencing the T cell repertoire in healthy women and female MS patients over the course of pregnancy. Clonality is significantly reduced from the first to third trimester in MS patients, indicating that the T cell repertoire becomes less dominated by expanded clones. However, only a few T cell clones are substantially modulated during pregnancy in each patient. Moreover, relapse-associated T cell clones identified in an individual patient contract during pregnancy and expand during a postpartum relapse. Our data provide evidence that profiling the T cell repertoire during pregnancy could serve as a tool to discover and track "private" T cell clones associated with disease activity in autoimmunity.

Longitudinal immunosequencing in healthy people reveals persistent T cell receptors rich in highly public receptors.

BACKGROUND: The adaptive immune system maintains a diversity of T cells capable of recognizing a broad array of antigens. Each T cell's specificity for antigens is determined by its T cell receptors (TCRs), which together across all T cells form a repertoire of millions of unique receptors in each individual. Although many studies have examined how TCR repertoires change in response to disease or drugs, few have explored the temporal dynamics of the TCR repertoire in healthy individuals. RESULTS: Here we report immunosequencing of TCR ß chains (TCRß) from the blood of three healthy individuals at eight time points over one year. TCRß repertoires of all peripheral-blood T cells and sorted memory T cells clustered clearly by individual, systematically demonstrating that TCRß repertoires are specific to individuals across time. This individuality was absent from TCRßs from naive T cells, suggesting that the differences resulted from an individual's antigen exposure history, not genetic background. Many characteristics of the TCRß repertoire (e.g., diversity, clonality) were stable across time, although we found evidence of T cell expansion dynamics even within healthy individuals. We further identified a subset of "persistent" TCRßs present across all time points. These receptors were rich in clonal and highly public receptors and may play a key role in immune system maintenance. CONCLUSIONS: Our results highlight the importance of longitudinal sampling of the immune system, providing a much-needed baseline for TCRß dynamics in healthy individuals. Such a baseline will improve interpretation of changes in the TCRß repertoire during disease or treatment.

Radiotherapy induces responses of lung cancer to CTLA-4 blockade.

Focal radiation therapy enhances systemic responses to anti-CTLA-4 antibodies in preclinical studies and in some patients with melanoma1-3, but its efficacy in inducing systemic responses (abscopal responses) against tumors unresponsive to CTLA-4 blockade remained uncertain. Radiation therapy promotes the activation of anti-tumor T cells, an effect dependent on type I interferon induction in the irradiated tumor4-6. The latter is essential for achieving abscopal responses in murine cancers6. The mechanisms underlying abscopal responses in patients treated with radiation therapy and CTLA-4 blockade remain unclear. Here we report that radiation therapy and CTLA-4 blockade induced systemic anti-tumor T cells in chemo-refractory metastatic non-small-cell lung cancer (NSCLC), where anti-CTLA-4 antibodies had failed to demonstrate significant efficacy alone or in combination with chemotherapy7,8. Objective responses were observed in 18% of enrolled patients, and 31% had disease control. Increased serum interferon-ß after radiation and early dynamic changes of blood T cell clones were the strongest response predictors, confirming preclinical mechanistic data. Functional analysis in one responding patient showed the rapid in vivo expansion of CD8 T cells recognizing a neoantigen encoded in a gene upregulated by radiation, supporting the hypothesis that one explanation for the abscopal response is radiation-induced exposure of immunogenic mutations to the immune system.

A Diverse Lipid Antigen-Specific TCR Repertoire Is Clonally Expanded during Active Tuberculosis.

Human T cells that recognize lipid Ags presented by highly conserved CD1 proteins often express semi-invariant TCRs, but the true diversity of lipid Ag-specific TCRs remains unknown. We use CD1b tetramers and high-throughput immunosequencing to analyze thousands of TCRs from ex vivo-sorted or in vitro-expanded T cells specific for the mycobacterial lipid Ag, glucose monomycolate. Our results reveal a surprisingly diverse repertoire resulting from editing of germline-encoded gene rearrangements analogous to MHC-restricted TCRs. We used a distance-based metric (TCRDist) to show how this diverse TCR repertoire builds upon previously reported conserved motifs by including subject-specific TCRs. In a South African cohort, we show that TCRDist can identify clonal expansion of diverse glucose monomycolate-specific TCRs and accurately distinguish patients with active tuberculosis from control subjects. These data suggest that similar mechanisms govern the selection and expansion of peptide and lipid Ag-specific T cells despite the nonpolymorphic nature of CD1.

Early Stage HER2-Positive Breast Cancers Not Achieving a pCR From Neoadjuvant Trastuzumab- or Pertuzumab-Based Regimens Have an Immunosuppressive Phenotype.

BACKGROUND: Stromal tumor-infiltrating lymphocytes (TILs) might predict pathologic complete response (pCR) in patients with HER2-positive (HER2+) breast cancer treated with trastuzumab (H). Docetaxel (T), carboplatin (C), H, and pertuzumab (P) have immune-modulating effects. Pre- and post-treatment immune biomarkers in cancers treated with neoadjuvant TCH with or without P are lacking. In this study we quantified baseline and changes in TILs, cluster of differentiation (CD) 4+, CD8+, FoxP3+, and PD-L1+ cells using immunohistochemistry (IHC) and quantified productive T-cell receptor ß (TCRß) rearrangements and TCRß clonality using next-generation sequencing (NGS) in 30 HER2+ breast cancer tissues treated with neoadjuvant H with or without P regimens. MATERIALS AND METHODS: Thirty pre- and post-neoadjuvant TCH (n = 4) or TCHP (n = 26) breast cancer tissues were identified. TILs were quantified manually using hematoxylin and eosin. CD4, CD8, FoxP3, and PD-L1 were stained using IHC. TCRß was evaluated using NGS. Immune infiltrates were compared between pCR and non-pCR groups using the Wilcoxon rank sum test. RESULTS: A pCR occurred in 15 (n = 15; 50%) cancers (TCH n = 2; TCHP, n = 13). Pretreatment TILs, CD4+, CD8+, FoxP3+, and PD-L1+ cells were not associated with response (P = .42, P = .55, P = .19, P = .66, P = .87, respectively. Pretreatment productive TCRß and TCRß clonality did not predict response, P = .84 and P = .40, respectively). However, post-treatment CD4+ and FoxP3+ cells (T-regulatory cells) were elevated in the non-pCR cohort (P = .042 and P = .082, respectively). CONCLUSION: An increase in regulatory T cells in non-pCR tissues suggests the development of an immunosuppressive phenotype. Further investigation in a larger cohort of samples is warranted to validate these findings.

Role of high-throughput sequencing in the diagnosis of cutaneous T-cell lymphoma.

AIMS: Substantial clinicopathological overlap exists between cutaneous T-cell lymphoma (CTCL) and benign conditions, leading to diagnostic difficulties. We sought to delineate the utility of high-throughput sequencing (HTS) across a spectrum of histological findings in CTCL and reactive mimics. METHODS: One hundred skin biopsies obtained for clinical concern for CTCL were identified, comprising 25 cases each from four histological categories: 'definitive CTCL', 'atypical lymphoid infiltrate, concerning for CTCL', 'atypical lymphoid infiltrate, favour reactive' or 'reactive lymphoid infiltrate'. T-cell receptor gamma chain gene (TRG) PCR and T-cell receptor beta chain gene HTS were performed on both skin biopsy and concurrently collected peripheral blood; most peripheral blood samples were also analysed by flow cytometry. RESULTS: Histologically defined CTCL specimens had significantly higher clonality scores and T-cell fractions via HTS than all other groups (all p<0.002 and p<0.03, respectively). HTS was more diagnostically specific than TRG PCR in skin (100% vs 88%), while diagnostic sensitivity (68% vs 72%) and accuracy (84% vs 80%) were similar. TRG PCR and flow cytometry performed on blood were the least diagnostically useful assays. Some identically sized peaks detected by TRG PCR in concurrent skin and peripheral blood specimens were non-identical by HTS analysis. CONCLUSIONS: HTS, by assessing both clonality and T-cell fractions in skin biopsies, is a powerful tool to aid in the diagnosis of CTCL. It is more specific than TRG PCR in distinguishing definitive CTCL from reactive and indeterminate histology. Identically sized peaks by TRG PCR, typically interpreted to be clonally related, are not always clonally identical by sequencing.

CD1b Tetramers Identify T Cells that Recognize Natural and Synthetic Diacylated Sulfoglycolipids from Mycobacterium tuberculosis.

Mycobacterial cell wall lipids bind the conserved CD1 family of antigen-presenting molecules and activate T cells via their T cell receptors (TCRs). Sulfoglycolipids (SGLs) are uniquely synthesized by Mycobacterium tuberculosis, but tools to study SGL-specific T cells in humans are lacking. We designed a novel hybrid synthesis of a naturally occurring SGL, generated CD1b tetramers loaded with natural or synthetic SGL analogs, and studied the molecular requirements for TCR binding and T cell activation. Two T cell lines derived using natural SGLs are activated by synthetic analogs independently of lipid chain length and hydroxylation, but differentially by saturation status. By contrast, two T cell lines derived using an unsaturated SGL synthetic analog were not activated by the natural antigen. Our data provide a bioequivalence hierarchy of synthetic SGL analogs and SGL-loaded CD1b tetramers. These reagents can now be applied to large-scale translational studies investigating the diagnostic potential of SGL-specific T cell responses or SGL-based vaccines.

Immunophenotyping of pediatric brain tumors: correlating immune infiltrate with histology, mutational load, and survival and assessing clonal T cell response.

There is little known regarding the immune infiltrate present in pediatric brain tumors and how this compares to what is known about histologically similar adult tumors and its correlation with survival. Here, we provide a descriptive analysis of the immune infiltrate of 22 fresh pediatric brain tumor tissue samples of mixed diagnoses and 40 peripheral blood samples. Samples were analyzed using a flow cytometry panel containing markers for immune cell subtypes, costimulatory markers, inhibitory signals, and markers of activation. This was compared to the standard method of immunohistochemistry (IHC) for immune markers for 89 primary pediatric brain tumors. Both flow cytometry and IHC data did not correlate with the grade of tumor or mutational load and IHC data was not significantly associated with survival for either low grade or high grade gliomas. There is a trend towards a more immunosuppressive phenotype in higher grade tumors with more regulatory T cells present in these tumor types. Both PD1 and PDL1 were present in only a small percentage of the tumor infiltrate. T cell receptor sequencing revealed up to 10% clonality of T cells in tumor infiltrates and no significant difference in clonality between low and high grade gliomas. We have shown the immune infiltrate of pediatric brain tumors does not appear to correlate with grade or survival for a small sample of patients. Further research and larger studies are needed to fully understand the interaction of pediatric brain tumors and the immune system.

Robust Antitumor Responses Result from Local Chemotherapy and CTLA-4 Blockade.

Clinical responses to immunotherapy have been associated with augmentation of preexisting immune responses, manifested by heightened inflammation in the tumor microenvironment. However, many tumors have a noninflamed microenvironment, and response rates to immunotherapy in melanoma have been <50%. We approached this problem by utilizing immunotherapy (CTLA-4 blockade) combined with chemotherapy to induce local inflammation. In murine models of melanoma and prostate cancer, the combination of chemotherapy and CTLA-4 blockade induced a shift in the cellular composition of the tumor microenvironment, with infiltrating CD8+ and CD4+ T cells increasing the CD8/Foxp3 T-cell ratio. These changes were associated with improved survival of the mice. To translate these findings into a clinical setting, 26 patients with advanced melanoma were treated locally by isolated limb infusion with the nitrogen mustard alkylating agent melphalan followed by systemic administration of CTLA-4 blocking antibody (ipilimumab) in a phase II trial. This combination of local chemotherapy with systemic checkpoint blockade inhibitor resulted in a response rate of 85% at 3 months (62% complete and 23% partial response rate) and a 58% progression-free survival at 1 year. The clinical response was associated with increased T-cell infiltration, similar to that seen in the murine models. Together, our findings suggest that local chemotherapy combined with checkpoint blockade-based immunotherapy results in a durable response to cancer therapy. Cancer Immunol Res; 6(2); 189-200. ©2018 AACR.

Staged development of long-lived T-cell receptor αß TH17 resident memory T-cell population to Candida albicans after skin infection.

BACKGROUND: Candida albicans is a dimorphic fungus to which human subjects are exposed early in life, and by adulthood, it is part of the mycobiome of skin and other tissues. Neonatal skin lacks resident memory T (TRM) cells, but in adults the C albicans skin test is a surrogate for immunocompetence. Young adult mice raised under specific pathogen-free conditions are naive to C albicans and have been shown recently to have an immune system resembling that of neonatal human subjects. OBJECTIVE: We studied the evolution of the adaptive cutaneous immune response to Candida species. METHODS: We examined both human skin T cells and the de novo and memory immune responses in a mouse model of C albicans skin infection. RESULTS: In mice the initial IL-17-producing cells after C albicans infection were dermal γδ T cells, but by day 7, αß TH17 effector T cells were predominant. By day 30, the majority of C albicans-reactive IL-17-producing T cells were CD4 TRM cells. Intravital microscopy showed that CD4 effector T cells were recruited to the site of primary infection and were highly motile 10 days after infection. Between 30 and 90 days after infection, these CD4 T cells became increasingly sessile, acquired expression of CD69 and CD103, and localized to the papillary dermis. These established TRM cells produced IL-17 on challenge, whereas motile migratory memory T cells did not. TRM cells rapidly clear an infectious challenge with C albicans more effectively than recirculating T cells, although both populations participate. We found that in normal human skin IL-17-producing CD4+ TRM cells that responded to C albicans in an MHC class II-restricted fashion could be identified readily. CONCLUSIONS: These studies demonstrate that C albicans infection of skin preferentially generates CD4+ IL-17-producing TRM cells, which mediate durable protective immunity.

Radiotherapy and CTLA-4 Blockade Shape the TCR Repertoire of Tumor-Infiltrating T Cells.

Immune checkpoint inhibitors activate T cells to reject tumors. Unique tumor mutations are key T-cell targets, but a comprehensive understanding of the nature of a successful antitumor T-cell response is lacking. To investigate the T-cell receptor (TCR) repertoire associated with treatment success versus failure, we used a well-characterized mouse carcinoma that is rejected by CD8 T cells in mice treated with radiotherapy (RT) and anti-CTLA-4 in combination, but not as monotherapy, and comprehensively analyzed tumor-infiltrating lymphocytes (TILs) by high-throughput sequencing of the TCRΒ CDR3 region. The combined treatment increased TIL density and CD8/CD4 ratio. Assessment of the frequency of T-cell clones indicated that anti-CTLA-4 resulted in fewer clones and a more oligoclonal repertoire compared with untreated tumors. In contrast, RT increased the CD8/CD4 ratio and broadened the TCR repertoire, and when used in combination with anti-CTLA-4, these selected T-cell clones proliferated. Hierarchical clustering of CDR3 sequences showed a treatment-specific clustering of TCRs that were shared by different mice. Abundant clonotypes were commonly shared between animals and yet treatment-specific. Analysis of amino-acid sequence similarities revealed a significant increase in the number and richness of dominant CDR3 motifs in tumors treated with RT + anti-CTLA-4 compared with control. The repertoire of TCRs reactive with a single tumor antigen recognized by CD8+ T cells was heterogeneous but highly clonal, irrespective of treatment. Overall, data support a model whereby a diverse TCR repertoire is required to achieve tumor rejection and may underlie the synergy between RT and CTLA-4 blockade. Cancer Immunol Res; 6(2); 139-50. ©2017 AACR.

TCR Repertoire Intratumor Heterogeneity in Localized Lung Adenocarcinomas: An Association with Predicted Neoantigen Heterogeneity and Postsurgical Recurrence.

Genomic intratumor heterogeneity (ITH) may be associated with postsurgical relapse of localized lung adenocarcinomas. Recently, mutations, through generation of neoantigens, were shown to alter tumor immunogenicity through T-cell responses. Here, we performed sequencing of the T-cell receptor (TCR) in 45 tumor regions from 11 localized lung adenocarcinomas and observed substantial intratumor differences in T-cell density and clonality with the majority of T-cell clones restricted to individual tumor regions. TCR ITH positively correlated with predicted neoantigen ITH, suggesting that spatial differences in the T-cell repertoire may be driven by distinct neoantigens in different tumor regions. Finally, a higher degree of TCR ITH was associated with an increased risk of postsurgical relapse and shorter disease-free survival, suggesting a potential clinical significance of T-cell repertoire heterogeneity.Significance: The present study provides insights into the ITH of the T-cell repertoire in localized lung adenocarcinomas and its potential biological and clinical impact. The results suggest that T-cell repertoire ITH may be tightly associated to genomic ITH and disease relapse. Cancer Discov; 7(10); 1088-97. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1047.