Simultaneous Detection of Circulating Autoreactive CD8+ T-Cells Specific for Different Islet Cell–Associated Epitopes Using Combinatorial MHC Multimers

textabstractOBJECTIVE - Type 1 diabetes results from selective T-cell-mediated destruction of the insulin-producing β-cells in the pancreas. In this process, islet epitope-specific CD8+T-cells play a pivotal role. Thus, monitoring of multiple islet-specific CD8+T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive. RESEARCH DESIGN AND METHODS - Here, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B10-18, prepro-insulin (PPI)15-24, islet antigen (IA)-2797-805, GAD65114-123, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)265-273, and pre-pro islet amyloid polypeptide (ppIAPP)5-13-specific CD8+T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients. RESULTS - Using this kit, islet autoreactive CD8+T-cells recognizing insulin B10-18, IA-2797-805, and IGRP265-273were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI15-24proved to be the most sensitive epitope. Applying the "Diab-Q-kit" to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell-derived epitopes that were associated with disease activity and correlated with clinical outcome. CONCLUSIONS - A kit was developed that allows simultaneous detection of CD8+T-cells reactive to multiple HLA-A2-restricted β-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples

The Immune System Strikes Back: Cellular Immune Responses against Indoleamine 2,3-dioxygenase

The enzyme indoleamine 2,3-dioxygenase (IDO) exerts an well established immunosuppressive function in cancer. IDO is expressed within the tumor itself as well as in antigen-presenting cells in tumor-draining lymph nodes, where it promotes the establishment of peripheral immune tolerance to tumor antigens. In the present study, we tested the notion whether IDO itself may be subject to immune responses.The presence of naturally occurring IDO-specific CD8 T cells in cancer patients was determined by MHC/peptide stainings as well as ELISPOT. Antigen specific cytotoxic T lymphocytes (CTL) from the peripheral blood of cancer patients were cloned and expanded. The functional capacity of the established CTL clones was examined by chrome release assays. The study unveiled spontaneous cytotoxic T-cell reactivity against IDO in peripheral blood as well as in the tumor microenvironment of different cancer patients. We demonstrate that these IDO reactive T cells are indeed peptide specific, cytotoxic effector cells. Hence, IDO reactive T cells are able to recognize and kill tumor cells including directly isolated AML blasts as well as IDO-expressing dendritic cells, i.e. one of the major immune suppressive cell populations.IDO may serve as an important and widely applicable target for anti-cancer immunotherapeutic strategies. Furthermore, as emerging evidence suggests that IDO constitutes a significant counter-regulatory mechanism induced by pro-inflammatory signals, IDO-based immunotherapy holds the promise to boost anti-cancer immunotherapy in general

Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade

As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8(+) tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non–small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy–induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens