Interpreting T-Cell Cross-reactivity through Structure: Implications for TCR-Based Cancer Immunotherapy

Immunotherapy has become one of the most promising avenues for cancer treatment, making use of the patient\u27s own immune system to eliminate cancer cells. Clinical trials with T-cell-based immunotherapies have shown dramatic tumor regressions, being effective in multiple cancer types and for many different patients. Unfortunately, this progress was tempered by reports of serious (even fatal) side effects. Such therapies rely on the use of cytotoxic T-cell lymphocytes, an essential part of the adaptive immune system. Cytotoxic T-cells are regularly involved in surveillance and are capable of both eliminating diseased cells and generating protective immunological memory. The specificity of a given T-cell is determined through the structural interaction between the T-cell receptor (TCR) and a peptide-loaded major histocompatibility complex (MHC); i.e., an intracellular peptide-ligand displayed at the cell surface by an MHC molecule. However, a given TCR can recognize different peptide-MHC (pMHC) complexes, which can sometimes trigger an unwanted response that is referred to as T-cell cross-reactivity. This has become a major safety issue in TCR-based immunotherapies, following reports of melanoma-specific T-cells causing cytotoxic damage to healthy tissues (e.g., heart and nervous system). T-cell cross-reactivity has been extensively studied in the context of viral immunology and tissue transplantation. Growing evidence suggests that it is largely driven by structural similarities of seemingly unrelated pMHC complexes. Here, we review recent reports about the existence of pMHC hot-spots for cross-reactivity and propose the existence of a TCR interaction profile (i.e., a refinement of a more general TCR footprint in which some amino acid residues are more important than others in triggering T-cell cross-reactivity). We also make use of available structural data and pMHC models to interpret previously reported cross-reactivity patterns among virus-derived peptides. Our study provides further evidence that structural analyses of pMHC complexes can be used to assess the intrinsic likelihood of cross-reactivity among peptide-targets. Furthermore, we hypothesize that some apparent inconsistencies in reported cross-reactivities, such as a preferential directionality, might also be driven by particular structural features of the targeted pMHC complex. Finally, we explain why TCR-based immunotherapy provides a special context in which meaningful T-cell cross-reactivity predictions can be made

NLRC5/MHC class I transactivator is a target for immune evasion in cancer

Cancer cells develop under immune surveillance, thus necessitating immune escape for successful growth. Loss of MHC class I expression provides a key immune evasion strategy in many cancers, although the molecular mechanisms remain elusive. MHC class I transactivator (CITA), known as “NLRC5” [NOD-like receptor (NLR) family, caspase recruitment (CARD) domain containing 5], has recently been identified as a critical transcriptional coactivator of MHC class I gene expression. Here we show that the MHC class I transactivation pathway mediated by CITA/NLRC5 constitutes a target for cancer immune evasion. In all the 21 tumor types we examined, NLRC5 expression was highly correlated with the expression of MHC class I, with cytotoxic T-cell markers, and with genes in the MHC class I antigen-presentation pathway, including LMP2/LMP7, TAP1, and β2-microglobulin. Epigenetic and genetic alterations in cancers, including promoter methylation, copy number loss, and somatic mutations, were most prevalent in NLRC5 among all MHC class I-related genes and were associated with the impaired expression of components of the MHC class I pathway. Strikingly, NLRC5 expression was significantly associated with the activation of CD8(+) cytotoxic T cells and patient survival in multiple cancer types. Thus, NLRC5 constitutes a novel prognostic biomarker and potential therapeutic target of cancers

MHC Class I Endosomal and Lysosomal Trafficking Coincides with Exogenous Antigen Loading in Dendritic Cells

BACKGROUND: Cross-presentation by dendritic cells (DCs) is a crucial prerequisite for effective priming of cytotoxic T-cell responses against bacterial, viral and tumor antigens; however, this antigen presentation pathway remains poorly defined. METHODOLOGY/PRINCIPAL FINDINGS: In order to develop a comprehensive understanding of this process, we tested the hypothesis that the internalization of MHC class I molecules (MHC-I) from the cell surface is directly involved in cross-presentation pathway and the loading of antigenic peptides. Here we provide the first examination of the internalization of MHC-I in DCs and we demonstrate that the cytoplasmic domain of MHC-I appears to act as an addressin domain to route MHC-I to both endosomal and lysosomal compartments of DCs, where it is demonstrated that loading of peptides derived from exogenously-derived proteins occurs. Furthermore, by chasing MHC-I from the cell surface of normal and transgenic DCs expressing mutant forms of MHC-I, we observe that a tyrosine-based endocytic trafficking motif is required for the constitutive internalization of MHC-I molecules from the cell surface into early endosomes and subsequently deep into lysosomal peptide-loading compartments. Finally, our data support the concept that multiple pathways of peptide loading of cross-presented antigens may exist depending on the chemical nature and size of the antigen requiring processing. CONCLUSIONS/SIGNIFICANCE: We conclude that DCs have 'hijacked' and adapted a common vacuolar/endocytic intracellular trafficking pathway to facilitate MHC I access to the endosomal and lysosomal compartments where antigen processing and loading and antigen cross-presentation takes place

The Role of Antigen Cross-presentation From Leukemia Blasts on Immunity to the Leukemia-associated Antigen PR1

Cross-presentation is an important mechanism by which exogenous tumor antigens are presented to elicit immunity. Since neutrophil elastase (NE) and proteinase-3 (P3) expression is increased in myeloid leukemia, we investigated whether NE and P3 are cross-presented by dendritic cells (DC) and B-cells, and whether the NE and P3 source determines immune outcomes. We show that NE and P3 are elevated in leukemia patient serum and that levels correlate with remission status. We demonstrate cellular uptake of NE and P3 into lysosomes, ubiquitination and proteasome processing for cross-presentation. Using anti-PR1/HLA-A2 monoclonal antibody, we provide direct evidence that B-cells cross-present soluble and leukemia-associated NE and P3, while DCs cross-present only leukemia-associated NE and P3. Cross-presentation occurred at early time points but was not associated with DC or B-cell activation, suggesting that NE and P3 cross-presentation may favor tolerance. Furthermore, we show aberrant subcellular localization of NE and P3 in leukemia blasts to compartments that share common elements of the classical MHC class I antigen-presenting pathway, which may facilitate cross-presentation. Our data demonstrate distinct mechanisms for cross-presentation of soluble and cell-associated NE and P3, which may be valuable in understanding immunity to PR1 in leukemia

A Novel HLA-A*0201 Restricted Peptide Derived from Cathepsin G Is an Effective Immunotherapeutic Target in Acute Myeloid Leukemia

Immunotherapy targeting aberrantly expressed leukemia associated antigens (LAA) has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy

Peptide/MHC Tetramer-Based Sorting of CD8+ T Cells to a Leukemia Antigen Yields Clonotypes Drawn Nonspecifically from an Underlying Restricted Repertoire

Testing of T cell-based cancer therapeutics often involves measuring cancer antigen-specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous

MHC class 1 cytoplasmic domain : defining a role for conserved amino acid residues in class 1 expression, trafficking, and antigen presentation

MHC class I molecules contain within their cytoplasmic domains a tyrosine and two or three serines that demonstrate a remarkable level of conservation between all mammals and extending to species as evolutionarily distant as the shark and zebrafish. In order to explore the role of these amino acids both in vitro and in vivo, cytoplasmic mutants of the murine H-2 Kb gene were constructed and expressed in murine L cell fibroblasts and transgenic mice. In L cell fibroblast transfectants, Kb mutant molecules reached higher cell surface levels than Kb wild-type (KbWT) molecules and demonstrated a higher capacity for binding exogenously-added peptides. While cytoplasmic mutations had no effect on β2- microglobulin binding nor Kb transport rates to the cell surface, they caused a delay in the ability of the molecules to present an immunodominant vesicular stomatitis virus (VSV) epitope following viral infection. In the transgenic mice, FACScan analysis of peripheral blood leukocytes and splenocytes revealed that cytoplasmic mutant Kb molecules reached significantly higher surface levels compared to wild-type Kb and interfered less with endogenous class I maturation and surface expression. Constitutive endocytosis of class I molecules from the surface of activated T cells was also shown to be significantly impaired by cytoplasmic point mutations. Functional studies on antigen presentation in the transgenic mice revealed that mice containing a point mutation to the conserved tyrosine were severely compromised in their ability to generate a Kb-restricted CTL response to VSV infection compared to KbWT-expressing and C57/BL control mice. Mice containing mutations to the conserved serines by contrast, demonstrated a more vigorous VSV CTL response compared to KbWT-expressing transgenic animals. The results demonstrate that the conserved tyrosine and serine residues are important for regulating lymphocyte and fibroblast MHC class I cell surface expression both in vitro and in vivo, and provide evidence of an inducible tyrosine-based endosomal targeting motif in the cytoplasmic tail of class I molecules. The functional studies suggest that the tyrosine motif in particular may be important for the generation of class I-restricted CTL responses in vivo.Science, Faculty ofZoology, Department ofGraduat

Evolution of CD8+ T Cell Receptor (TCR) Engineered Therapies for the Treatment of Cancer

Engineered T cell receptor T (TCR-T) cell therapy has facilitated the generation of increasingly reliable tumor antigen-specific adaptable cellular products for the treatment of human cancer. TCR-T cell therapies were initially focused on targeting shared tumor-associated peptide targets, including melanoma differentiation and cancer-testis antigens. With recent technological developments, it has become feasible to target neoantigens derived from tumor somatic mutations, which represents a highly personalized therapy, since most neoantigens are patient-specific and are rarely shared between patients. TCR-T therapies have been tested for clinical efficacy in treating solid tumors in many preclinical studies and clinical trials all over the world. However, the efficacy of TCR-T therapy for the treatment of solid tumors has been limited by a number of factors, including low TCR avidity, off-target toxicities, and target antigen loss leading to tumor escape. In this review, we discuss the process of deriving tumor antigen-specific TCRs, including the identification of appropriate tumor antigen targets, expansion of antigen-specific T cells, and TCR cloning and validation, including techniques and tools for TCR-T cell vector construction and expression. We highlight the achievements of recent clinical trials of engineered TCR-T cell therapies and discuss the current challenges and potential solutions for improving their safety and efficacy, insights that may help guide future TCR-T studies in cancer

NLRC5/CITA expression correlates with efficient response to checkpoint blockade immunotherapy

Checkpoint blockade-mediated immunotherapy is emerging as an effective treatment modality for multiple cancer types. However, cancer cells frequently evade the immune system, compromising the effectiveness of immunotherapy. It is crucial to develop screening methods to identify the patients who would most benefit from these therapies because of the risk of the side effects and the high cost of treatment. Here we show that expression of the MHC class I transactivator (CITA), NLRC5, is important for efficient responses to anti-CTLA-4 and anti-PD1 checkpoint blockade therapies. Melanoma tumors derived from patients responding to immunotherapy exhibited significantly higher expression of NLRC5 and MHC class I-related genes compared to non-responding patients. In addition, multivariate analysis that included the number of tumor-associated non-synonymous mutations, predicted neo-antigen load and PD-L2 expression was capable of further stratifying responders and non-responders to anti-CTLA4 therapy. Moreover, expression or methylation of NLRC5 together with total somatic mutation number were significantly correlated with increased patient survival. These results suggest that NLRC5 tumor expression, alone or together with tumor mutation load constitutes a valuable predictive biomarker for both prognosis and response to anti-CTLA-4 and potentially anti-PD1 blockade immunotherapy in melanoma patients