Evolution of MHC-based technologies used for detection of antigen-responsive T cells.

T cell-mediated recognition of peptide-major histocompatibility complex (pMHC) class I and II molecules is crucial for the control of intracellular pathogens and cancer, as well as for stimulation and maintenance of efficient cytotoxic responses. Such interactions may also play a role in the development of autoimmune diseases. Novel insights into this mechanism are crucial to understanding disease development and establishing new treatment strategies. MHC multimers have been used for detection of antigen-responsive T cells since the first report by Altman et al. showed that tetramerization of pMHC class I molecules provided sufficient stability to T cell receptor (TCR)-pMHC interactions, allowing detection of MHC multimer-binding T cells using flow cytometry. Since this breakthrough the scientific community has aimed for expanding the capacity of MHC multimer-based detection technologies to facilitate large-scale epitope discovery and immune monitoring in limited biological material. Screening of T cell specificity using large libraries of pMHC molecules is suitable for analyses of T cell recognition potentially at genome-wide levels rather than analyses restricted to a selection of model antigens. Such strategies provide novel insights into the immune specificities involved in disease development and response to immunotherapy, and extend fundamental knowledge related to T cell recognition patterns and cross-recognition by TCRs. MHC multimer-based technologies have now evolved from detection of 1-2 different T cell specificities per cell sample, to include more than 1000 evaluable pMHC molecules using novel technologies. Here, we provide an overview of MHC multimer-based detection technologies developed over two decades, focusing primarily on MHC class I interactions.

Simultaneous analysis of pMHC binding and reactivity unveils virus-specific CD8 T cell immunity to a concise epitope set.

CD8 T cells provide immunity to virus infection through recognition of epitopes presented by peptide major histocompatibility complexes (pMHCs). To establish a concise panel of widely recognized T cell epitopes from common viruses, we combined analysis of TCR down-regulation upon stimulation with epitope-specific enumeration based on barcode-labeled pMHC multimers. We assess CD8 T cell binding and reactivity for 929 previously reported epitopes in the context of 1 of 25 HLA alleles representing 29 viruses. The prevalence and magnitude of CD8 T cell responses were evaluated in 48 donors and reported along with 137 frequently recognized virus epitopes, many of which were underrepresented in the public domain. Eighty-four percent of epitope-specific CD8 T cell populations demonstrated reactivity to peptide stimulation, which was associated with effector and long-term memory phenotypes. Conversely, nonreactive T cell populations were associated primarily with naive phenotypes. Our analysis provides a reference map of epitopes for characterizing CD8 T cell responses toward common human virus infections.

T antigen-specific CD8+ T cells associate with PD-1 blockade response in virus-positive Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a highly immunogenic skin cancer primarily induced by Merkel cell polyomavirus, which is driven by the expression of the oncogenic T antigens (T-Ags). Blockade of the programmed cell death protein-1 (PD-1) pathway has shown remarkable response rates, but evidence for therapy-associated T-Ag-specific immune response and therapeutic strategies for the nonresponding fraction are both limited. We tracked T-Ag-reactive CD8+ T cells in peripheral blood of 26 MCC patients under anti-PD1 therapy, using DNA-barcoded pMHC multimers, displaying all peptides from the predicted HLA ligandome of the oncoproteins, covering 33 class I haplotypes. We observed a broad T cell recognition of T-Ags, including identification of 20 T-Ag-derived epitopes we believe to be novel. Broadening of the T-Ag recognition profile and increased T cell frequencies during therapy were strongly associated with clinical response and prolonged progression-free survival. T-Ag-specific T cells could be further boosted and expanded directly from peripheral blood using artificial antigen-presenting scaffolds, even in patients with no detectable T-Ag-specific T cells. These T cells provided strong tumor-rejection capacity while retaining a favorable phenotype for adoptive cell transfer. These findings demonstrate that T-Ag-specific T cells are associated with the clinical outcome to PD-1 blockade and that Ag-presenting scaffolds can be used to boost such responses.

Improved T cell receptor antigen pairing through data-driven filtering of sequencing information from single cells.

Novel single-cell-based technologies hold the promise of matching T cell receptor (TCR) sequences with their cognate peptide-MHC recognition motif in a high-throughput manner. Parallel capture of TCR transcripts and peptide-MHC is enabled through the use of reagents labeled with DNA barcodes. However, analysis and annotation of such single-cell sequencing (SCseq) data are challenged by dropout, random noise, and other technical artifacts that must be carefully handled in the downstream processing steps. We here propose a rational, data-driven method termed ITRAP (improved T cell Receptor Antigen Paring) to deal with these challenges, filtering away likely artifacts, and enable the generation of large sets of TCR-pMHC sequence data with a high degree of specificity and sensitivity, thus outputting the most likely pMHC target per T cell. We have validated this approach across 10 different virus-specific T cell responses in 16 healthy donors. Across these samples, we have identified up to 1494 high-confident TCR-pMHC pairs derived from 4135 single cells.

TCR-engaging scaffolds selectively expand antigen-specific T-cells with a favorable phenotype for adoptive cell therapy.

BACKGROUND: Adoptive cell therapy (ACT) has shown promising results for the treatment of cancer and viral infections. Successful ACT relies on ex vivo expansion of large numbers of desired T-cells with strong cytotoxic capacity and in vivo persistence, which constitutes the greatest challenge to current ACT strategies. Here, in this study, we present a novel technology for ex vivo expansion of antigen-specific T-cells; artificial antigen-presenting scaffolds (Ag-scaffolds) consisting of a dextran-polysaccharide backbone, decorated with combinations of peptide-Major Histocompatibility Complex (pMHC), cytokines and co-stimulatory molecules, enabling coordinated stimulation of antigen-specific T-cells. METHODS: The capacity of Ag-scaffolds to expand antigen-specific T-cells was explored in ex vivo cultures with peripheral blood mononuclear cells from healthy donors and patients with metastatic melanoma. The resulting T-cell products were assessed for phenotypic and functional characteristics. RESULTS: We identified an optimal Ag-scaffold for expansion of T-cells for ACT, carrying pMHC and interleukin-2 (IL-2) and IL-21, with which we efficiently expanded both virus-specific and tumor-specific CD8+ T cells from peripheral blood of healthy donors and patients, respectively. The resulting T-cell products were characterized by a high frequency of antigen-specific cells with high self-renewal capacity, low exhaustion, a multifunctional cytokine profile upon antigen-challenge and superior tumor killing capacity. This demonstrates that the coordinated stimuli provided by an optimized stoichiometry of TCR engaging (pMHC) and stimulatory (cytokine) moieties is essential to obtain desired T-cell characteristics. To generate an 'off-the-shelf' multitargeting Ag-scaffold product of relevance to patients with metastatic melanoma, we identified the 30 most frequently recognized shared HLA-A0201-restricted melanoma epitopes in a cohort of 87 patients. By combining these in an Ag-scaffold product, we were able to expand tumor-specific T-cells from 60-70% of patients with melanoma, yielding a multitargeted T-cell product with up to 25% specific and phenotypically and functionally improved T cells. CONCLUSIONS: Taken together, the Ag-scaffold represents a promising new technology for selective expansion of antigen-specific CD8+ T cells directly from blood, yielding a highly specific and functionally enhanced T-cell product for ACT.

A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo.

Acute myeloid leukemia (AML), the most frequent leukemia in adults, is driven by recurrent somatically acquired genetic lesions in a restricted number of genes. Treatment with tyrosine kinase inhibitors has demonstrated that targeting of prevalent FMS-related receptor tyrosine kinase 3 (FLT3) gain-of-function mutations can provide significant survival benefits for patients, although the efficacy of FLT3 inhibitors in eliminating FLT3-mutated clones is variable. We identified a T cell receptor (TCR) reactive to the recurrent D835Y driver mutation in the FLT3 tyrosine kinase domain (TCRFLT3D/Y). TCRFLT3D/Y-redirected T cells selectively eliminated primary human AML cells harboring the FLT3D835Y mutation in vitro and in vivo. TCRFLT3D/Y cells rejected both CD34+ and CD34- AML in mice engrafted with primary leukemia from patients, reaching minimal residual disease-negative levels, and eliminated primary CD34+ AML leukemia-propagating cells in vivo. Thus, T cells targeting a single shared mutation can provide efficient immunotherapy toward selective elimination of clonally involved primary AML cells in vivo.

Neoantigen-reactive CD8+ T cells affect clinical outcome of adoptive cell therapy with tumor-infiltrating lymphocytes in melanoma.

BACKGROUNDNeoantigen-driven recognition and T cell-mediated killing contribute to tumor clearance following adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs). Yet how diversity, frequency, and persistence of expanded neoepitope-specific CD8+ T cells derived from TIL infusion products affect patient outcome is not fully determined.METHODSUsing barcoded pMHC multimers, we provide a comprehensive mapping of CD8+ T cells recognizing neoepitopes in TIL infusion products and blood samples from 26 metastatic melanoma patients who received ACT.RESULTSWe identified 106 neoepitopes within TIL infusion products corresponding to 1.8% of all predicted neoepitopes. We observed neoepitope-specific recognition to be virtually devoid in TIL infusion products given to patients with progressive disease outcome. Moreover, we found that the frequency of neoepitope-specific CD8+ T cells in TIL infusion products correlated with increased survival and that neoepitope-specific CD8+ T cells shared with the infusion product in posttreatment blood samples were unique to responders of TIL-ACT. Finally, we found that a transcriptional signature for lymphocyte activity within the tumor microenvironment was associated with a higher frequency of neoepitope-specific CD8+ T cells in the infusion product.CONCLUSIONSThese data support previous case studies of neoepitope-specific CD8+ T cells in melanoma and indicate that successful TIL-ACT is associated with an expansion of neoepitope-specific CD8+ T cells.FUNDINGNEYE Foundation; European Research Council; Lundbeck Foundation Fellowship; Carlsberg Foundation.

Neoantigen-specific CD8 T cell responses in the peripheral blood following PD-L1 blockade might predict therapy outcome in metastatic urothelial carcinoma.

CD8+ T cell reactivity towards tumor mutation-derived neoantigens is widely believed to facilitate the antitumor immunity induced by immune checkpoint blockade (ICB). Here we show that broadening in the number of neoantigen-reactive CD8+ T cell (NART) populations between pre-treatment to 3-weeks post-treatment distinguishes patients with controlled disease compared to patients with progressive disease in metastatic urothelial carcinoma (mUC) treated with PD-L1-blockade. The longitudinal analysis of peripheral CD8+ T cell recognition of patient-specific neopeptide libraries consisting of DNA barcode-labelled pMHC multimers in a cohort of 24 patients from the clinical trial NCT02108652 also shows that peripheral NARTs derived from patients with disease control are characterised by a PD1+ Ki67+ effector phenotype and increased CD39 levels compared to bystander bulk- and virus-antigen reactive CD8+ T cells. The study provides insights into NART characteristics following ICB and suggests that early-stage NART expansion and activation are associated with response to ICB in patients with mUC.

NetTCR-2.0 enables accurate prediction of TCR-peptide binding by using paired TCRα and ß sequence data.

Prediction of T-cell receptor (TCR) interactions with MHC-peptide complexes remains highly challenging. This challenge is primarily due to three dominant factors: data accuracy, data scarceness, and problem complexity. Here, we showcase that "shallow" convolutional neural network (CNN) architectures are adequate to deal with the problem complexity imposed by the length variations of TCRs. We demonstrate that current public bulk CDR3ß-pMHC binding data overall is of low quality and that the development of accurate prediction models is contingent on paired α/ß TCR sequence data corresponding to at least 150 distinct pairs for each investigated pMHC. In comparison, models trained on CDR3α or CDR3ß data alone demonstrated a variable and pMHC specific relative performance drop. Together these findings support that T-cell specificity is predictable given the availability of accurate and sufficient paired TCR sequence data. NetTCR-2.0 is publicly available at https://services.healthtech.dtu.dk/service.php?NetTCR-2.0 .

Tumor-Infiltrating T Cells From Clear Cell Renal Cell Carcinoma Patients Recognize Neoepitopes Derived From Point and Frameshift Mutations.

Mutation-derived neoantigens are important targets for T cell-mediated reactivity toward tumors and, due to their unique tumor expression, an attractive target for immunotherapy. Neoepitope-specific T cells have been detected across a number of solid cancers with high mutational burden tumors, but neoepitopes have been mostly selected from single nucleotide variations (SNVs), and little focus has been given to neoepitopes derived from in-frame and frameshift indels, which might be equally important and potentially highly immunogenic. Clear cell renal cell carcinomas (ccRCCs) are medium-range mutational burden tumors with a high pan-cancer proportion of frameshift mutations. In this study, the mutational landscape of tumors from six RCC patients was analyzed by whole-exome sequencing (WES) of DNA from tumor fragments (TFs), autologous tumor cell lines (TCLs), and tumor-infiltrating lymphocytes (TILs, germline reference). Neopeptides were predicted using MuPeXI, and patient-specific peptide-MHC (pMHC) libraries were created for all neopeptides with a rank score < 2 for binding to the patient's HLAs. T cell recognition toward neoepitopes in TILs was evaluated using the high-throughput technology of DNA barcode-labeled pMHC multimers. The patient-specific libraries consisted of, on average, 258 putative neopeptides (range, 103-397, n = 6). In four patients, WES was performed on two different sources (TF and TCL), whereas in two patients, WES was performed only on TF. Most of the peptides were predicted from both sources. However, a fraction was predicted from one source only. Among the total predicted neopeptides, 16% were derived from frameshift indels. T cell recognition of 52 neoepitopes was detected across all patients (range, 4-18, n = 6) and spanning two to five HLA restrictions per patient. On average, 21% of the recognized neoepitopes were derived from frameshift indels (range, 0-43%, n = 6). Thus, frameshift indels are equally represented in the pool of immunogenic neoepitopes as SNV-derived neoepitopes. This suggests the importance of a broad neopeptide prediction strategy covering multiple sources of tumor material, and including different genetic alterations. This study, for the first time, describes the T cell recognition of frameshift-derived neoepitopes in RCC and determines their immunogenic profile.

Human endogenous retroviruses form a reservoir of T cell targets in hematological cancers.

Human endogenous retroviruses (HERV) form a substantial part of the human genome, but mostly remain transcriptionally silent under strict epigenetic regulation, yet can potentially be reactivated by malignant transformation or epigenetic therapies. Here, we evaluate the potential for T cell recognition of HERV elements in myeloid malignancies by mapping transcribed HERV genes and generating a library of 1169 potential antigenic HERV-derived peptides predicted for presentation by 4 HLA class I molecules. Using DNA barcode-labeled MHC-I multimers, we find CD8+ T cell populations recognizing 29 HERV-derived peptides representing 18 different HERV loci, of which HERVH-5, HERVW-1, and HERVE-3 have more profound responses; such HERV-specific T cells are present in 17 of the 34 patients, but less frequently in healthy donors. Transcriptomic analyses reveal enhanced transcription of the HERVs in patients; meanwhile DNA-demethylating therapy causes a small and heterogeneous enhancement in HERV transcription without altering T cell recognition. Our study thus uncovers T cell recognition of HERVs in myeloid malignancies, thereby implicating HERVs as potential targets for immunotherapeutic therapies.

T-cell-receptor cross-recognition and strategies to select safe T-cell receptors for clinical translation.

Adoptive transfer of T-cell-receptor (TCR)-transduced T cells has shown promising results for cancer treatment, but has also produced severe immunotoxicities caused by on-target as well as off-target TCR recognition. Off-target toxicities are related to the ability of a single T cell to cross-recognize and respond to several different peptide-major histocompatibility complex (pMHC) antigens; a property that is essential for providing broad antigenic coverage despite a confined number of unique TCRs in the human body. However, this degeneracy makes it incredibly difficult to account for the range of targets that any TCR might recognize, which represents a major challenge for the clinical development of therapeutic TCRs. The prospect of using affinity-optimized TCRs has been impeded due to observations that affinity enhancement might alter the specificity of a TCR, thereby increasing the risk that it will cross-recognize endogenous tissue. Strategies for selecting safe TCRs for the clinic have included functional assessment after individual incubations with tissue-derived primary cells or with peptides substituted with single amino acids. However, these strategies have not been able to predict cross-recognition sufficiently, leading to fatal cross-reactivity in clinical trials. Novel technologies have emerged that enable extensive characterization of the exact interaction points of a TCR with pMHC, which provides a foundation from which to make predictions of the cross-recognition potential of individual TCRs. This review describes current advances in strategies for dissecting the molecular interaction points of TCRs, focusing on their potential as tools for predicting cross-recognition of TCRs in clinical development.

T cell recognition of novel shared breast cancer antigens is frequently observed in peripheral blood of breast cancer patients.

Advances within cancer immunotherapy have fueled a paradigm shift in cancer treatment, resulting in increasing numbers of cancer types benefitting from novel treatment options. Despite originally being considered an immunologically silent malignancy, recent studies encourage the research of breast cancer immunogenicity to evaluate immunotherapy as a treatment strategy. However, the epitope landscape in breast cancer is minimally described, limiting the options for antigen-specific, targeted strategies. Aromatase, never in mitosis A-related kinase 3 (NEK3), protein inhibitor of activated STAT3 (PIAS3), and prolactin are known as upregulated proteins in breast cancer. In the present study, these four proteins are identified as novel T cell targets in breast cancer. From the four proteins, 147 peptides were determined to bind HLA-A*0201 and -B*0702 using a combined in silico/in vitro affinity screening. T cell recognition of all 147 peptide-HLA-A*0201/-B*0702 combinations was assessed through the use of a novel high-throughput method utilizing DNA barcode labeled multimers. T cell recognition of sequences within all four proteins was demonstrated in peripheral blood of patients, and significantly more T cell responses were detected in patients compared to healthy donors for both HLA-A*0201 and -B*0702. Notably, several of the identified responses were directed toward peptides, with a predicted low or intermediate binding affinity. This demonstrates the importance of including low-affinity binders in the search for epitopes within shared tumor associated antigens (TAAs), as these might be less subject to immune tolerance mechanisms. The study presents four novel TAAs containing multiple possible targets for immunotherapy of breast cancer.

CD8+ T cells from patients with narcolepsy and healthy controls recognize hypocretin neuron-specific antigens.

Narcolepsy Type 1 (NT1) is a neurological sleep disorder, characterized by the loss of hypocretin/orexin signaling in the brain. Genetic, epidemiological and experimental data support the hypothesis that NT1 is a T-cell-mediated autoimmune disease targeting the hypocretin producing neurons. While autoreactive CD4+ T cells have been detected in patients, CD8+ T cells have only been examined to a minor extent. Here we detect CD8+ T cells specific toward narcolepsy-relevant peptides presented primarily by NT1-associated HLA types in the blood of 20 patients with NT1 as well as in 52 healthy controls, using peptide-MHC-I multimers labeled with DNA barcodes. In healthy controls carrying the disease-predisposing HLA-DQB1*06:02 allele, the frequency of autoreactive CD8+ T cells was lower as compared with both NT1 patients and HLA-DQB1*06:02-negative healthy individuals. These findings suggest that a certain level of CD8+ T-cell reactivity combined with HLA-DQB1*06:02 expression is important for NT1 development.

Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes.

Identification of the peptides recognized by individual T cells is important for understanding and treating immune-related diseases. Current cytometry-based approaches are limited to the simultaneous screening of 10-100 distinct T-cell specificities in one sample. Here we use peptide-major histocompatibility complex (MHC) multimers labeled with individual DNA barcodes to screen >1,000 peptide specificities in a single sample, and detect low-frequency CD8 T cells specific for virus- or cancer-restricted antigens. When analyzing T-cell recognition of shared melanoma antigens before and after adoptive cell therapy in melanoma patients, we observe a greater number of melanoma-specific T-cell populations compared with cytometry-based approaches. Furthermore, we detect neoepitope-specific T cells in tumor-infiltrating lymphocytes and peripheral blood from patients with non-small cell lung cancer. Barcode-labeled pMHC multimers enable the combination of functional T-cell analysis with large-scale epitope recognition profiling for the characterization of T-cell recognition in various diseases, including in small clinical samples.