Targeting of multiple tumor-associated antigens by individual T cell receptors during successful cancer immunotherapy.

The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual T cells to attack cancer in several ways simultaneously. Such "multipronged" T cells exhibited superior recognition of cancer cells compared with conventional T cell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies.

Author Correction: Genome-wide CRISPR-Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genome-wide CRISPR-Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1.

Human leukocyte antigen (HLA)-independent, T cell-mediated targeting of cancer cells would allow immune destruction of malignancies in all individuals. Here, we use genome-wide CRISPR-Cas9 screening to establish that a T cell receptor (TCR) recognized and killed most human cancer types via the monomorphic MHC class I-related protein, MR1, while remaining inert to noncancerous cells. Unlike mucosal-associated invariant T cells, recognition of target cells by the TCR was independent of bacterial loading. Furthermore, concentration-dependent addition of vitamin B-related metabolite ligands of MR1 reduced TCR recognition of cancer cells, suggesting that recognition occurred via sensing of the cancer metabolome. An MR1-restricted T cell clone mediated in vivo regression of leukemia and conferred enhanced survival of NSG mice. TCR transfer to T cells of patients enabled killing of autologous and nonautologous melanoma. These findings offer opportunities for HLA-independent, pan-cancer, pan-population immunotherapies.

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination.

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR-CD3 complexes. We found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

Structural Mechanism Underpinning Cross-reactivity of a CD8+ T-cell Clone That Recognizes a Peptide Derived from Human Telomerase Reverse Transcriptase.

T-cell cross-reactivity is essential for effective immune surveillance but has also been implicated as a pathway to autoimmunity. Previous studies have demonstrated that T-cell receptors (TCRs) that focus on a minimal motif within the peptide are able to facilitate a high level of T-cell cross-reactivity. However, the structural database shows that most TCRs exhibit less focused antigen binding involving contact with more peptide residues. To further explore the structural features that allow the clonally expressed TCR to functionally engage with multiple peptide-major histocompatibility complexes (pMHCs), we examined the ILA1 CD8+ T-cell clone that responds to a peptide sequence derived from human telomerase reverse transcriptase. The ILA1 TCR contacted its pMHC with a broad peptide binding footprint encompassing spatially distant peptide residues. Despite the lack of focused TCR-peptide binding, the ILA1 T-cell clone was still cross-reactive. Overall, the TCR-peptide contacts apparent in the structure correlated well with the level of degeneracy at different peptide positions. Thus, the ILA1 TCR was less tolerant of changes at peptide residues that were at, or adjacent to, key contact sites. This study provides new insights into the molecular mechanisms that control T-cell cross-reactivity with important implications for pathogen surveillance, autoimmunity, and transplant rejection.

Engineering of Isogenic Cells Deficient for MR1 with a CRISPR/Cas9 Lentiviral System: Tools To Study Microbial Antigen Processing and Presentation to Human MR1-Restricted T Cells.

The nonclassical HLA molecule MHC-related protein 1 (MR1) presents metabolites of the vitamin B synthesis pathways to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells. This new class of Ags represents a variation on the classical paradigm of self/non-self discrimination because these T cells are activated through their TCR by small organic compounds generated during microbial vitamin B2 synthesis. Beyond the fundamental significance, the invariant nature of MR1 across the human population is a tantalizing feature for the potential development of universal immune therapeutic and diagnostic tools. However, many aspects of MR1 Ag presentation and MR1-restricted T cell biology remain unknown, and the ubiquitous expression of MR1 across tissues and cell lines can be a confounding factor for experimental purposes. In this study, we report the development of a novel CRISPR/Cas9 genome editing lentiviral system and its use to efficiently disrupt MR1 expression in A459, THP-1, and K562 cell lines. We generated isogenic MR1(-/-) clonal derivatives of the A549 lung carcinoma and THP-1 monocytic cell lines and used these to study T cell responses to intracellular pathogens. We confirmed that MAIT cell clones were unable to respond to MR1(-/-) clones infected with bacteria whereas Ag presentation by classical and other nonclassical HLAs was unaffected. This system represents a robust and efficient method to disrupt the expression of MR1 and should facilitate investigations into the processing and presentation of MR1 Ags as well as into the biology of MAIT cells.

Antibody stabilization of peptide-MHC multimers reveals functional T cells bearing extremely low-affinity TCRs.

Fluorochrome-conjugated peptide-MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II-restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs. In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity. Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents. Importantly, when combined with protein kinase inhibitor treatment, Ab stabilization allowed pMHC tetramer staining of T cells even when the cognate TCR-pMHC affinity was extremely low (KD >1 mM) and produced the best results that we have observed to date. We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining. In summary, Ab stabilization of pMHC multimers during T cell staining extends the range of TCR affinities that can be detected, yields considerably enhanced staining intensities, and is compatible with using reduced amounts of these expensive reagents.

Uncoupling CD4+ TIL-Mediated Tumor Killing from JAK-Signaling in Melanoma.

PURPOSE: Impaired MHCI-presentation and insensitivity to immune effector molecules are common features of immune checkpoint blockade (ICB)-resistant tumors and can be, respectively, associated with loss of ß2 microglobulin (B2M) or impaired IFNγ signaling. Patients with ICB-resistant tumors can respond to alternative immunotherapies, such as infusion of autologous tumor-infiltrating lymphocytes (TIL). CD4+ T cells can exert cytotoxic functions against tumor cells; however, it is unclear whether CD4+ T-cell responses can be exploited to improve the clinical outcomes of patients affected by ICB-resistant tumors. EXPERIMENTAL DESIGN: Here, we exploited CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing to reproduce immune-resistant tumor phenotypes via gene knockout (KO). To determine the role of cytotoxic CD4+ TILs in ICB-resistant tumors, we investigated CD4+ TIL-mediated cytotoxicity in matched pairs of TILs and autologous melanoma cell lines, used as a model of patient-specific immune-tumor interaction. Around 40% of melanomas constitutively express MHC Class II molecules; hence, melanomas with or without natural constitutive MHC Class II expression (MHCIIconst+ or MHCIIconst-) were used. RESULTS: CD4+ TIL-mediated cytotoxicity was not affected by B2M loss but was dependent on the expression of CIITA. MHCIIconst+ melanomas were killed by tumor-specific CD4+ TILs even in the absence of IFNγ-mediated MHCII upregulation, whereas IFNγ was necessary for CD4+ TIL-mediated cytotoxicity against MHCIIconst- melanomas. Notably, although tumor-specific CD4+ TILs did not kill JAK1KO MHCIIconst- melanomas even after IFNγ stimulation, sensitivity to CD4+ TIL-mediated cytotoxicity was maintained by JAK1KO MHCIIconst+ melanomas. CONCLUSIONS: In conclusion, our data indicate that exploiting tumor-specific cytotoxic CD4+ TILs could help overcome resistance to ICB mediated by IFNγ-signaling loss in MHCIIconst+ melanomas. See related commentary by Betof Warner and Luke, p. 3829.

Ligand Identification for Orphan MHC-Agnostic T-Cell Receptors by Whole Genome CRISPR-Cas9 Screening.

Killer T-cells play important roles in immunity to infection and cancer by detecting intracellular anomalies at the cell surface and destroying the cells that bear them. Conventional killer T-cells scan the intracellular proteome by sampling peptides presented at the cell surface by major histocompatibility complex (MHC) molecules. It is becoming apparent that some T-cells can also respond to pathogens and neoplasms by sensing intracellular changes through molecules other than MHC. We describe an unbiased methodology for T-cell receptor ligand discovery that requires no a priori knowledge regarding the nature of the antigen.

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.

The burgeoning role of MR1-restricted T-cells in infection, cancer and autoimmune disease.

MR1 is a ubiquitously expressed, monomorphic antigen presenting molecule that has been largely preserved throughout mammalian evolution. The primary role of MR1 is to present conserved microbial metabolites to highly abundant mucosal-associated invariant T (MAIT) cells. The role of MAIT cells and other MR1-restricted T cells (MR1T) has been recently extended to immunomodulation during cancer. MR1Ts have also been implicated in autoimmune disease. The highly conserved nature of MR1 across the human population is in stark contrast to the MHC molecules recognised by conventional αß T-cells, therefore MR1Ts may form fertile ground for the development of pan-population T-cell immunotherapeutics for a wide range of important morbidities.

T-Cell Gene Therapy in Cancer Immunotherapy: Why It Is No Longer Just CARs on The Road.

T-cells have a natural ability to fight cancer cells in the tumour microenvironment. Due to thymic selection and tissue-driven immunomodulation, these cancer-fighting T-cells are generally low in number and exhausted. One way to overcome these issues is to genetically alter T-cells to improve their effectiveness. This process can involve introducing a receptor that has high affinity for a tumour antigen, with two promising candidates known as chimeric-antigen receptors (CARs), or T-cell receptors (TCRs) with high tumour specificity. This review focuses on the editing of immune cells to introduce such novel receptors to improve immune responses to cancer. These new receptors redirect T-cells innate killing abilities to the appropriate target on cancer cells. CARs are modified receptors that recognise whole proteins on the surface of cancer cells. They have been shown to be very effective in haematological malignancies but have limited documented efficacy in solid cancers. TCRs recognise internal antigens and therefore enable targeting of a much wider range of antigens. TCRs require major histocompatibility complex (MHC) restriction but novel TCRs may have broader antigen recognition. Moreover, there are multiple cell types which can be used as targets to improve the "off-the-shelf" capabilities of these genetic engineering methods.

GPU-Accelerated Discovery of Pathogen-Derived Molecular Mimics of a T-Cell Insulin Epitope.

The strong links between (Human Leukocyte Antigen) HLA, infection and autoimmunity combine to implicate T-cells as primary triggers of autoimmune disease (AD). T-cell crossreactivity between microbially-derived peptides and self-peptides has been shown to break tolerance and trigger AD in experimental animal models. Detailed examination of the potential for T-cell crossreactivity to trigger human AD will require means of predicting which peptides might be recognised by autoimmune T-cell receptors (TCRs). Recent developments in high throughput sequencing and bioinformatics mean that it is now possible to link individual TCRs to specific pathologies for the first time. Deconvolution of TCR function requires knowledge of TCR specificity. Positional Scanning Combinatorial Peptide Libraries (PS-CPLs) can be used to predict HLA-restriction and define antigenic peptides derived from self and pathogen proteins. In silico search of the known terrestrial proteome with a prediction algorithm that ranks potential antigens in order of recognition likelihood requires complex, large-scale computations over several days that are infeasible on a personal computer. We decreased the time required for peptide searching to under 30 min using multiple blocks on graphics processing units (GPUs). This time-efficient, cost-effective hardware accelerator was used to screen bacterial and fungal human pathogens for peptide sequences predicted to activate a T-cell clone, InsB4, that was isolated from a patient with type 1 diabetes and recognised the insulin B-derived epitope HLVEALYLV in the context of disease-risk allele HLA A*0201. InsB4 was shown to kill HLA A*0201+ human insulin producing ß-cells demonstrating that T-cells with this specificity might contribute to disease. The GPU-accelerated algorithm and multispecies pathogen proteomic databases were validated to discover pathogen-derived peptide sequences that acted as super-agonists for the InsB4 T-cell clone. Peptide-MHC tetramer binding and surface plasmon resonance were used to confirm that the InsB4 TCR bound to the highest-ranked peptide agonists derived from infectious bacteria and fungi. Adoption of GPU-accelerated prediction of T-cell agonists has the capacity to revolutionise our understanding of AD by identifying potential targets for autoimmune T-cells. This approach has further potential for dissecting T-cell responses to infectious disease and cancer.

Thermal Stability of Heterotrimeric pMHC Proteins as Determined by Circular Dichroism Spectroscopy.

T cell receptor (TCR) recognition of foreign peptide fragments, presented by peptide major histocompatibility complex (pMHC), governs T-cell mediated protection against pathogens and cancer. Many factors govern T-cell sensitivity, including the affinity of the TCR-pMHC interaction and the stability of pMHC on the surface of antigen presenting cells. These factors are particularly relevant for the peptide vaccination field, in which more stable pMHC interactions could enable more effective protection against disease. Here, we discuss a method for the determination of pMHC stability that we have used to investigate HIV immune escape, T-cell sensitivity to cancer antigens and mechanisms leading to autoimmunity.