Neoantigen-specific CD8 T cells with high structural avidity preferentially reside in and eliminate tumors.

The success of cancer immunotherapy depends in part on the strength of antigen recognition by T cells. Here, we characterize the T cell receptor (TCR) functional (antigen sensitivity) and structural (monomeric pMHC-TCR off-rates) avidities of 371 CD8 T cell clones specific for neoantigens, tumor-associated antigens (TAAs) or viral antigens isolated from tumors or blood of patients and healthy donors. T cells from tumors exhibit stronger functional and structural avidity than their blood counterparts. Relative to TAA, neoantigen-specific T cells are of higher structural avidity and, consistently, are preferentially detected in tumors. Effective tumor infiltration in mice models is associated with high structural avidity and CXCR3 expression. Based on TCR biophysicochemical properties, we derive and apply an in silico model predicting TCR structural avidity and validate the enrichment in high avidity T cells in patients' tumors. These observations indicate a direct relationship between neoantigen recognition, T cell functionality and tumor infiltration. These results delineate a rational approach to identify potent T cells for personalized cancer immunotherapy.

TCR sequencing and cloning methods for repertoire analysis and isolation of tumor-reactive TCRs.

T cell receptor (TCR) technologies, including repertoire analyses and T cell engineering, are increasingly important in the clinical management of cellular immunity in cancer, transplantation, and other immune diseases. However, sensitive and reliable methods for repertoire analyses and TCR cloning are still lacking. Here, we report on SEQTR, a high-throughput approach to analyze human and mouse repertoires that is more sensitive, reproducible, and accurate as compared with commonly used assays, and thus more reliably captures the complexity of blood and tumor TCR repertoires. We also present a TCR cloning strategy to specifically amplify TCRs from T cell populations. Positioned downstream of single-cell or bulk TCR sequencing, it allows time- and cost-effective discovery, cloning, screening, and engineering of tumor-specific TCRs. Together, these methods will accelerate TCR repertoire analyses in discovery, translational, and clinical settings and permit fast TCR engineering for cellular therapies.

Improved predictions of antigen presentation and TCR recognition with MixMHCpred2.2 and PRIME2.0 reveal potent SARS-CoV-2 CD8+ T-cell epitopes.

The recognition of pathogen or cancer-specific epitopes by CD8+ T cells is crucial for the clearance of infections and the response to cancer immunotherapy. This process requires epitopes to be presented on class I human leukocyte antigen (HLA-I) molecules and recognized by the T-cell receptor (TCR). Machine learning models capturing these two aspects of immune recognition are key to improve epitope predictions. Here, we assembled a high-quality dataset of naturally presented HLA-I ligands and experimentally verified neo-epitopes. We then integrated these data in a refined computational framework to predict antigen presentation (MixMHCpred2.2) and TCR recognition (PRIME2.0). The depth of our training data and the algorithmic developments resulted in improved predictions of HLA-I ligands and neo-epitopes. Prospectively applying our tools to SARS-CoV-2 proteins revealed several epitopes. TCR sequencing identified a monoclonal response in effector/memory CD8+ T cells against one of these epitopes and cross-reactivity with the homologous peptides from other coronaviruses.

CD4 T cells target colorectal cancer antigens upregulated by oxaliplatin.

Immune checkpoint blockade has proven its efficacy in hypermutated subtypes of metastatic colorectal cancers (mCRC). Immunogenic potential can also be observed with conventional chemotherapies, but this property has never been explored thoroughly in CRC patients. The CRC therapeutic arsenal includes oxaliplatin, a well-characterized platinum drug already described as immunogenic. Here, we investigated the impact of the oxaliplatin-based treatment on mCRC immunopeptidome. We demonstrated that oxaliplatin-resistant CRC cell lines overexpressed telomerase reverse transcriptase (TERT), colorectal-associated-tumor antigen-1 (COA-1) and mesothelin tumor-associated antigens. We identified new HLA class-II-restricted and promiscuous peptides derived from COA-1 and mesothelin. The two naturally processed peptides COA-1331-345 and Meso366-380 appear to be the most immunogenic in mCRC patients. A prospective cohort of 162 mCRC patients enabled us to explore the impact of oxaliplatin exposure on the antitumor-specific immune response. Interestingly, chemotherapy-naive mCRC patients present high immune CD4 T-cell responses directed against TERT, COA-1 and mesothelin-derived peptides. These antitumor T-cell responses were maintained after 3 months of oxaliplatin-based treatment. Altogether, these findings highlight the interest of immunostimulatory agents to improve the management of chemoresistant mCRC patients. Finally, the high frequency of immune responses targeting the new immunogenic peptides derived from COA-1 and mesothelin support their use in immunomonitoring strategies.

Heparan Sulfate Proteoglycans Promote Telomerase Internalization and MHC Class II Presentation on Dendritic Cells.

Telomerase is a prototype-shared tumor Ag and represents an attractive target for anticancer immunotherapy. We have previously described promiscuous and immunogenic HLA-DR-restricted peptides derived from human telomerase reverse transcriptase (hTERT) and referred as universal cancer peptide (UCP). In nonsmall cell lung cancer, the presence of spontaneous UCP-specific CD4 T cell responses increases the survival of chemotherapy-responding patients. However, the precise mechanisms of hTERT's uptake, processing, and presentation on MHC-II molecules to stimulate CD4 T cells are poorly understood. In this work, by using well-characterized UCP-specific CD4 T cell clones, we showed that hTERT processing and presentation on MHC-II involve both classical endolysosomal and nonclassical cytosolic pathways. Furthermore, to our knowledge, we demonstrated for the first time that hTERT's internalization by dendritic cells requires its interaction with surface heparan sulfate proteoglycans. Altogether, our findings provide a novel mechanism of tumor-specific CD4 T cell activation and will be useful for the development of novel cancer immunotherapies that harness CD4 T cells.

Prognostic value of baseline seric Syndecan-1 in initially unresectable metastatic colorectal cancer patients: a simple biological score.

In first-line metastatic colorectal cancer (mCRC), baseline prognostic factors allowing death risk and treatment strategy stratification are lacking. Syndecan-1 (CD138) soluble form was never described as a prognostic biomarker in mCRC. We investigated its additional prognostic value for overall survival (OS). mCRC patients with unresectable disease at diagnosis were treated with bevacizumab-based chemotherapy in two independent prospective clinical trials (development set: n = 126, validation set: n = 51, study NCT00489697 and study NCT00544011, respectively). Serums were collected at baseline for CD138 measurement. OS determinants were assessed and, based on the final multivariate model, a prognostic score was proposed. Two independent OS prognostic factors were identified: Lactate Dehydrogenase (LDH) high level (p = 0.0066) and log-CD138 high level (p = 0.0190). The determination of CD138 binary information (cutoff: 75 ng/mL) allowed the assessment of a biological prognostic score with CD138 and LDH values, identifying three risk groups for death (median OS= 38.9, 30.1 and 19.8 months for the low, intermediate and high risk groups, respectively; p < 0.0001). This score had a good discrimination ability (C-index = 0.63). These results were externally confirmed in the validation set. Our study provides robust evidence in favor of the additional baseline soluble CD138 prognostic value for OS, in mCRC patients. A simple biological scoring system is proposed including LDH and CD138 binary status values.

IL-21-Induced MHC Class II+ NK Cells Promote the Expansion of Human Uncommitted CD4+ Central Memory T Cells in a Macrophage Migration Inhibitory Factor-Dependent Manner.

NK cells are critical for innate immunity-mediated protection. The main roles of NK cells rely on their cytotoxic functions or depend on the tuning of Th1 adaptive immunity by IFN-γ. However, the precise influence of inflammatory cytokines on NK cell and CD4 T lymphocyte interactions was never investigated. In this study, we provide evidence that IL-21, a cytokine produced during chronic inflammation or infectious diseases, promotes the differentiation of a specific subset of NK cells coexpressing CD86 and HLA-DR and lacking NKp44. More importantly, IL-21-propagated HLA-DR(+) NK cells produce macrophage migration inhibitory factor and provide costimulatory signaling during naive CD4(+) T cell priming inducing the differentiation of uncommitted central memory T cells. Central memory T cells expanded in the presence of HLA-DR(+) NK cells are CXCR3(+)CCR6(-)CCR4(-)CXCR5(-) and produce IL-2, as well as low levels of TNF-α. Costimulation of CD4(+) T cells by HLA-DR(+) NK cells prevents the acquisition of effector memory phenotype induced by IL-2. Moreover, we identified this population of NK HLA-DR(+) macrophage migration inhibitory factor(+) cells in inflammatory human appendix. Collectively, these results demonstrate a novel function for IL-21 in tuning NK and CD4(+) T cell interactions promoting a specific expansion of central memory lymphocytes.

Rapalogs Efficacy Relies on the Modulation of Antitumor T-cell Immunity.

The rapalogs everolimus and temsirolimus that inhibit mTOR signaling are used as antiproliferative drugs in several cancers. Here we investigated the influence of rapalogs-mediated immune modulation on their antitumor efficacy. Studies in metastatic renal cell carcinoma patients showed that everolimus promoted high expansion of FoxP3 (+)Helios(+)Ki67(+) regulatory CD4 T cells (Tregs). In these patients, rapalogs strongly enhanced the suppressive functions of Tregs, mainly in a contact-dependent manner. Paradoxically, a concurrent activation of spontaneous tumor-specific Th1 immunity also occurred. Furthermore, a high rate of Eomes(+)CD8(+) T cells was detected in patients after a long-term mTOR inhibition. We found that early changes in the Tregs/antitumor Th1 balance can differentially shape the treatment efficacy. Patients presenting a shift toward decreased Tregs levels and high expansion of antitumor Th1 cells showed better clinical responses. Studies conducted in tumor-bearing mice confirmed the deleterious effect of rapalogs-induced Tregs via a mechanism involving the inhibition of antitumor T-cell immunity. Consequently, the combination of temsirolimus plus CCR4 antagonist, a receptor highly expressed on rapalogs-exposed Tregs, was more effective than monotherapy. Altogether, our results describe for the first time a dual impact of host adaptive antitumor T-cell immunity on the clinical effectiveness of rapalogs and prompt their association with immunotherapies. Cancer Res; 76(14); 4100-12. ©2016 AACR.

Search for genes responsible for the remarkably high acetic acid tolerance of a Zygosaccharomyces bailii-derived interspecies hybrid strain.

BACKGROUND: Zygosaccharomyces bailii is considered the most problematic acidic food spoilage yeast species due to its exceptional capacity to tolerate high concentrations of weak acids used as fungistatic preservatives at low pH. However, the mechanisms underlying its intrinsic remarkable tolerance to weak acids remain poorly understood. The identification of genes and mechanisms involved in Z. bailii acetic acid tolerance was on the focus of this study. For this, a genomic library from the highly acetic acid tolerant hybrid strain ISA1307, derived from Z. bailii and a closely related species and isolated from a sparkling wine production plant, was screened for acetic acid tolerance genes. This screen was based on the transformation of an acetic acid susceptible Saccharomyces cerevisiae mutant deleted for the gene encoding the acetic acid resistance determinant transcription factor Haa1. RESULTS: The expression of 31 different DNA inserts from ISA1307 strain genome was found to significantly increase the host cell tolerance to acetic acid. The in silico analysis of these inserts was facilitated by the recently available genome sequence of this strain. In total, 65 complete or truncated ORFs were identified as putative determinants of acetic acid tolerance and an S. cerevisiae gene homologous to most of them was found. These include genes involved in cellular transport and transport routes, protein fate, protein synthesis, amino acid metabolism and transcription. The role of strong candidates in Z. bailii and S. cerevisiae acetic acid tolerance was confirmed based on homologous and heterologous expression analyses. CONCLUSIONS: ISA1307 genes homologous to S. cerevisiae genes GYP8, WSC4, PMT1, KTR7, RKR1, TIF3, ILV3 and MSN4 are proposed as strong candidate determinants of acetic acid tolerance. The ORF ZBAI_02295 that contains a functional domain associated to the uncharacterised integral membrane proteins of unknown function of the DUP family is also suggested as a relevant tolerance determinant. The genes ZbMSN4 and ZbTIF3, encoding a putative stress response transcription factor and a putative translation initiation factor, were confirmed as determinants of acetic acid tolerance in both Z. bailii and S. cerevisiae. This study provides valuable indications on the cellular components, pathways and processes to be targeted in order to control food spoilage by the highly acetic acid tolerant Z. bailii and Z. bailii-derived strains. Additionally, this information is essential to guide the improvement of yeast cells robustness against acetic acid if the objective is their use as cell factories.

Prognostic value of angiopoietin-2 for death risk stratification in patients with metastatic colorectal carcinoma.

BACKGROUND: Baseline prognostic biomarkers stratifying treatment strategies in first-line metastatic colorectal cancer (mCRC) are lacking. Angiopoietin-2 (Ang-2) is proposed as a potential biomarker in several cancers. We therefore decided to establish the additional prognostic value of Ang-2 for overall survival (OS) in patients with first-line mCRC. METHODS: We enrolled 177 patients treated with a bevacizumab containing chemotherapy in two prospective phase II clinical trials. Patient plasma samples were collected at baseline. ELISAs were used to measure Ang-2. RESULTS: The multivariable Cox model identified increased lactate dehydrogenase [HR, 1.60; 95% confidence interval (CI), 1.04-2.45; P = 0.03] and Ang-2 log-transformation level (HR, 1.59; 95% CI, 1.14-2.21; P = 0.0065) as two significant independent OS prognostic factors. It exhibited good calibration (P = 0.8) and discrimination (C-index: 0.64; 95% CI, 0.58-0.68). Ang-2 parameter inclusion in the GERCOR reference model significantly and strongly improved its discriminative ability because the C-statistic increased significantly from 0.61 to 0.63 (bootstrap mean difference = 0.07; 95% CI, 0.069-0.077). Interestingly, the addition of Ang-2 binary information with a 5 ng/mL cutoff value to the GERCOR model allowed the reclassification of intermediate-risk profile patients (41%) into two subsets of low and high risks. CONCLUSIONS: Our study provides robust evidence in favor of baseline Ang-2 prognostic value for OS adding to the conventional factors. Its assessment appears to be useful for the improvement in risk stratification for patients with intermediate-risk profile. IMPACT: Ang-2 ability to predict OS at diagnosis could be of interest in the selection of patients eligible for intermittent or sequential therapeutic strategies dedicated to the optimization of patients' quality of life and chemotherapy cost-effectiveness. Cancer Epidemiol Biomarkers Prev; 24(3); 603-12. ©2015 AACR.