Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens.

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies

Diversity within the adenovirus fiber knob hypervariable loops influences primary receptor interactions

Adenovirus based vectors are of increasing importance for wide ranging therapeutic applications. As vaccines, vectors derived from human adenovirus species D serotypes 26 and 48 (HAdV-D26/48) are demonstrating promising efficacy as protective platforms against infectious diseases. Significant clinical progress has been made, yet definitive studies underpinning mechanisms of entry, infection, and receptor usage are currently lacking. Here, we perform structural and biological analysis of the receptor binding fiber-knob protein of HAdV-D26/48, reporting crystal structures, and modelling putative interactions with two previously suggested attachment receptors, CD46 and Coxsackie and Adenovirus Receptor (CAR). We provide evidence of a low affinity interaction with CAR, with modelling suggesting affinity is attenuated through extended, semi-flexible loop structures, providing steric hindrance. Conversely, in silico and in vitro experiments are unable to provide evidence of interaction between HAdV-D26/48 fiber-knob with CD46, or with Desmoglein 2. Our findings provide insight into the cell-virus interactions of HAdV-D26/48, with important implications for the design and engineering of optimised Ad-based therapeutics

The nature of the human T cell response to the cancer antigen 5T4 is determined by the balance of regulatory and inflammatory T cells of the same antigen-specificity: implications for vaccine design

The oncofoetal antigen 5T4 is a promising T cell target in the context of colorectal cancer, as demonstrated by a recent clinical study where 5T4-specific T cell responses, induced by vaccination or cyclophosphamide, were associated with a significantly prolonged survival of patients with metastatic disease. Whilst Th1-type (IFN-γ+) responses specific to 5T4, and other oncofoetal antigens, are often readily detectable in early stage CRC patients and healthy donors, their activity is suppressed as the cancer progresses by CD4+CD25hiFoxp3+ regulatory T cells (Treg) which contribute to the immunosuppressive environment conducive to tumour growth. This study mapped the fine specificity of Th1 and Treg cell responses to the 5T4 protein. Surprisingly, both immunogenic peptides and those recognised by Tregs clustered in the same HLA-DR transcending epitope-rich hotspots within the 5T4 protein. Similarly, regions of low Th1-cell immunogenicity also did not contain peptides capable of stimulating Tregs, further supporting the notion that Treg and Th1 cells recognise the same peptides. Understanding the rules which govern the balance of Th1 and Treg cells responding to a given peptide specificity is, therefore, of fundamental importance to designing strategies for manipulating the balance in favour of Th1 cells, and thus the most effective anti-cancer T cell responses

Cancer antigen discovery is enabled by RNA-sequencing of highly purified malignant and non-malignant cells

Purpose: Broadly expressed, highly differentiated tumor-associated antigens (TAA) can elicit anti-tumor immunity. However, vaccines targeting TAAs have demonstrated disappointing clinical results, reflecting poor antigen selection and/or immunosuppressive mechanisms. Experimental design: Here, a panel of widely expressed, novel colorectal TAAs were identified by performing RNA sequencing of highly purified colorectal tumor cells in comparison to patient-matched colonic epithelial cells; tumor cell purification was essential to reveal these genes. Candidate TAA protein expression was confirmed by immunohistochemistry, and pre-existing T cell immunogenicity towards these antigens tested. Results: The most promising candidate for further development is DNAJB7 [DnaJ heat shock protein family (Hsp40) member B7], identified here as a novel cancer-testis antigen. It is expressed in many tumors and is strongly immunogenic in patients with cancers originating from a variety of sites. DNAJB7-specific T cells were capable of killing colorectal tumor lines in vitro, and the IFN-gamma+ response was markedly magnified by control of immunosuppression with cyclophosphamide in cancer patients. Conclusion: This study highlights how prior methods that sequence whole tumor fractions (i.e. inclusive of alive/dead stromal cells) for antigen identification may have limitations. Through tumor cell purification and sequencing, novel candidate TAAs have been identified for future immunotherapeutic targeting

VDJdb in 2019: database extension, new analysis infrastructure and a T-cell receptor motif compendium

Here, we report an update of the VDJdb database with a substantial increase in the number of T-cell receptor (TCR) sequences and their cognate antigens. The update further provides a new database infrastructure featuring two additional analysis modes that facilitate database querying and real-world data analysis. The increased yield of TCR specificity identification methods and the overall increase in the number of studies in the field has allowed us to expand the database more than 5-fold. Furthermore, several new analysis methods are included. For example, batch annotation of TCR repertoire sequencing samples allows for annotating large datasets on-line. Using recently developed bioinformatic methods for TCR motif mining, we have built a reduced set of high-quality TCR motifs that can be used for both training TCR specificity predictors and matching against TCRs of interest. These additions enhance the versatility of the VDJdb in the task of exploring T-cell antigen specificities. The database is available at https://vdjdb.cdr3.net

CD4+ T Cells Recognize Conserved Influenza A Epitopes through Shared Patterns of V-Gene Usage and Complementary Biochemical Features

T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8+ T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4+ T cells. Here, we investigate CD4+ T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4+ T cells in five HLA-DR1+ subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies. © 2020 The Author(s)CD4+ T cells orchestrate protection from severe influenza. However, knowledge of epitopes and the molecular patterns associated with recognition across the population is lacking. Greenshields-Watson et al. identify several influenza epitopes from internal proteins and use them to explore the biochemical features that underpin CD4+ T cell responses to influenza. © 2020 The Author(s

Structural definition of HLA class II-presented SARS-CoV-2 epitopes reveals a mechanism to escape pre-existing CD4+ T cell immunity

Summary: CD4+ T cells recognize a broad range of peptide epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which contribute to immune memory and limit COVID-19 disease. We demonstrate that the immunogenicity of SARS-CoV-2 peptides, in the context of the model allotype HLA-DR1, does not correlate with their binding affinity to the HLA heterodimer. Analyzing six epitopes, some with very low binding affinity, we solve X-ray crystallographic structures of each bound to HLA-DR1. Further structural definitions reveal the precise molecular impact of viral variant mutations on epitope presentation. Omicron escaped ancestral SARS-CoV-2 immunity to two epitopes through two distinct mechanisms: (1) mutations to TCR-facing epitope positions and (2) a mechanism whereby a single amino acid substitution caused a register shift within the HLA binding groove, completely altering the peptide-HLA structure. This HLA-II-specific paradigm of immune escape highlights how CD4+ T cell memory is finely poised at the level of peptide-HLA-II presentation

VDJdb in 2019 : database extension, new analysis infrastructure and a T-cell receptor motif compendium

Here, we report an update of the VDJdb database with a substantial increase in the number of T-cell receptor (TCR) sequences and their cognate antigens. The update further provides a new database infrastructure featuring two additional analysis modes that facilitate database querying and real-world data analysis. The increased yield of TCR specificity identification methods and the overall increase in the number of studies in the field has allowed us to expand the database more than 5-fold. Furthermore, several new analysis methods are included. For example, batch annotation of TCR repertoire sequencing samples allows for annotating large datasets on-line. Using recently developed bioinformatic methods for TCR motif mining, we have built a reduced set of high-quality TCR motifs that can be used for both training TCR specificity predictors and matching against TCRs of interest. These additions enhance the versatility of the VDJdb in the task of exploring T-cell antigen specificities. The database is available at https://vdjdb.cdr3.net

Cancer Antigen Discovery Is Enabled by RNA Sequencing of Highly Purified Malignant and Nonmalignant Cells

Abstract Purpose: Broadly expressed, highly differentiated tumor-associated antigens (TAA) can elicit antitumor immunity. However, vaccines targeting TAAs have demonstrated disappointing clinical results, reflecting poor antigen selection and/or immunosuppressive mechanisms. Experimental Design: Here, a panel of widely expressed, novel colorectal TAAs were identified by performing RNA sequencing of highly purified colorectal tumor cells in comparison with patient-matched colonic epithelial cells; tumor cell purification was essential to reveal these genes. Candidate TAA protein expression was confirmed by IHC, and preexisting T-cell immunogenicity toward these antigens tested. Results: The most promising candidate for further development is DNAJB7 [DnaJ heat shock protein family (Hsp40) member B7], identified here as a novel cancer-testis antigen. It is expressed in many tumors and is strongly immunogenic in patients with cancers originating from a variety of sites. DNAJB7-specific T cells were capable of killing colorectal tumor lines in vitro, and the IFNγ+ response was markedly magnified by control of immunosuppression with cyclophosphamide in patients with cancer. Conclusions: This study highlights how prior methods that sequence whole tumor fractions (i.e., inclusive of alive/dead stromal cells) for antigen identification may have limitations. Through tumor cell purification and sequencing, novel candidate TAAs have been identified for future immunotherapeutic targeting