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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2022)

The adaptive immune system provides protection against disease and maintains memory of past exposures. Randomly rearranged antigen receptors can recognize virtually any pathogen. T cells recognize processed peptides that originate from protein fragments and are presented on major histocompatibility complex molecules. Human T cell receptor (TCR) function is mostly studied in the context of model antigens. TCRs against the majority of presented epitopes are not defined. Single cell sequencing allowed for paired TCRα/β sequencing at a scale not previously imaginable, which increased interest in identification of antigen specific TCRs.

Antigen specific TCR discovery efforts are challenging because of the repertoire diversity. Up to 10^19 possible TCRs can be randomly generated in the thymus. Antigen specific T cells can be selected by a range of techniques based on either physical staining of the TCR or selection of cells activated with specific antigen. Antigen specific T cell rarity and abundance of weakly reactive cells leads to selection of large numbers of false positive clones. The TCRα/β needs to be sequenced and genetically reconstructed in allogeneic T cells to confirm antigen recognition. The qualities of an antigen specific TCR include the ability to direct the killing of target cell lines, stimulating the production of multiple cytokines, and driving cell division. Such data can then be used to rank TCRs and identify highly functional clones. More sensitive and specific selection prior to sequencing can reduce the amount of labor involved when profiling TCRs.

This thesis work describes a new technology for sequencing antigen specific TCRs, which is used to study T cell responses against SARS-CoV-2. We developed a protocol that allows for TCR sequencing in cells that have been identified as functional based on cytokine production in response to stimulation with specific antigens. TCR engagement drives cytokine production which is one of the fundamental properties of a T cell. For this reason, intracellular cytokine staining is one of the most commonly used techniques to quantify antigen specific T cells. We then identified SARS-CoV-2 polymerase specific TCRs in unexposed human donors. Polymerase specific TCRs are able to recognize multiple human coronaviruses, indicating the potential to provide immunity regardless of the SARS-CoV-2 variant. Broadly reactive T cells provide heterosubtypic immunity against Influenza, another respiratory virus. Based on this work our laboratory, in collaboration with other laboratories at UCLA, is developing a novel COVID-19 vaccine strategy.

Our SARS-CoV-2 work illustrates the utility of studying single TCRs to discover immune responses, but another approach is to use TCRs directly as therapeutics. TCR engineered T cells are effective in the clinic and cure late-stage tumors. Projects described in this thesis are helping drive other efforts in our laboratory that are directed at identifying TCRs against novel prostate cancer antigens.

    Cover page: Characterizing the T cell immune response through the receptor-ligand interaction
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2021)
    T cell receptors (TCRs) are generated by somatic recombination of V/D/J segments to produce up to 1015 unique sequences. Highly sensitive and specific techniques are required to isolate and identify the rare TCR sequences that respond to antigens of interest. Here, we describe the use of mRNA sequencing via cross-linker regulated intracellular phenotype (CLInt-Seq) for efficient recovery of antigen-specific TCRs in cells stained for combinations of intracellular proteins such as cytokines or transcription factors. This method enables high-throughput identification and isolation of low-frequency TCRs specific for any antigen. As a proof of principle, intracellular staining for TNFα and IFNγ identified cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-reactive TCRs with efficiencies similar to state-of-the-art peptide-MHC multimer methodology. In a separate experiment, regulatory T cells were profiled based on intracellular FOXP3 staining, demonstrating the ability to examine phenotypes based on transcription factors. We further optimized the intracellular staining conditions to use a chemically cleavable primary amine cross-linker compatible with current single-cell sequencing technology. CLInt-Seq for TNFα and IFNγ performed similarly to isolation with multimer staining for EBV-reactive TCRs. We anticipate CLInt-Seq will enable droplet-based single-cell mRNA analysis from any tissue where minor populations need to be isolated by intracellular markers.
      Cover page: Droplet-based mRNA sequencing of fixed and permeabilized cells by CLInt-seq allows for antigen-specific TCR cloning
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2021)
      Cross-reactivity and direct killing of target cells remain underexplored for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific CD8+ T cells. Isolation of T cell receptors (TCRs) and overexpression in allogeneic cells allows for extensive T cell reactivity profiling. We identify SARS-CoV-2 RNA-dependent RNA polymerase (RdRp/NSP12) as highly conserved, likely due to its critical role in the virus life cycle. We perform single-cell TCRαβ sequencing in human leukocyte antigen (HLA)-A∗02:01-restricted, RdRp-specific T cells from SARS-CoV-2-unexposed individuals. Human T cells expressing these TCRαβ constructs kill target cell lines engineered to express full-length RdRp. Three TCR constructs recognize homologous epitopes from common cold coronaviruses, indicating CD8+ T cells can recognize evolutionarily diverse coronaviruses. Analysis of individual TCR clones may help define vaccine epitopes that can induce long-term immunity against SARS-CoV-2 and other coronaviruses.
        Cover page: HLA-A∗02:01 restricted T cell receptors against the highly conserved SARS-CoV-2 polymerase cross-react with human coronaviruses
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2022)
        Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.
          Cover page: Physical and in silico immunopeptidomic profiling of a cancer antigen prostatic acid phosphatase reveals targets enabling TCR isolation
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2023)
          Alternative splicing (AS) is prevalent in cancer, generating an extensive but largely unexplored repertoire of novel immunotherapy targets. We describe Isoform peptides from RNA splicing for Immunotherapy target Screening (IRIS), a computational platform capable of discovering AS-derived tumor antigens (TAs) for T cell receptor (TCR) and chimeric antigen receptor T cell (CAR-T) therapies. IRIS leverages large-scale tumor and normal transcriptome data and incorporates multiple screening approaches to discover AS-derived TAs with tumor-associated or tumor-specific expression. In a proof-of-concept analysis integrating transcriptomics and immunopeptidomics data, we showed that hundreds of IRIS-predicted TCR targets are presented by human leukocyte antigen (HLA) molecules. We applied IRIS to RNA-seq data of neuroendocrine prostate cancer (NEPC). From 2,939 NEPC-associated AS events, IRIS predicted 1,651 epitopes from 808 events as potential TCR targets for two common HLA types (A*02:01 and A*03:01). A more stringent screening test prioritized 48 epitopes from 20 events with "neoantigen-like" NEPC-specific expression. Predicted epitopes are often encoded by microexons of ≤30 nucleotides. To validate the immunogenicity and T cell recognition of IRIS-predicted TCR epitopes, we performed in vitro T cell priming in combination with single-cell TCR sequencing. Seven TCRs transduced into human peripheral blood mononuclear cells (PBMCs) showed high activity against individual IRIS-predicted epitopes, providing strong evidence of isolated TCRs reactive to AS-derived peptides. One selected TCR showed efficient cytotoxicity against target cells expressing the target peptide. Our study illustrates the contribution of AS to the TA repertoire of cancer cells and demonstrates the utility of IRIS for discovering AS-derived TAs and expanding cancer immunotherapies.
            Cover page: IRIS: Discovery of cancer immunotherapy targets arising from pre-mRNA alternative splicing
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