Defining T cell receptor repertoires using nanovial-based binding and functional screening
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE252830 | GEO | 2024/01/12
REPOSITORIES: GEO
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Project description:Programming T lymphocytes to distinguish self from non-self is a vital, multi-step process arising in the thymus. Signalling through the pre-T cell receptor (preTCR), a CD3-associated heterodimer comprising an invariant pTα chain and a clone-specific β chain, constitutes a critical early checkpoint in thymocyte development within the αβ T-cell lineage. Recent work demonstrates that preTCRs arrayed on double negative (DN) thymocytes, like αβ TCRs appearing on double positive (DP) thymocytes, ligate peptides bound to MHC molecules (pMHC) on thymic stroma but via a different molecular docking strategy. Here we show the consequences of those distinctive interactions for thymocyte progression, using synchronized fetal thymic progenitor cultures differing in the presence or absence of pMHC on support stroma, determining single cell transcriptomes at key thymocyte developmental transitions. Although MHC negative stroma fosters αβ T lymphocyte differentiation, the absence of pMHC-preTCR interplay leads to deviant thymocyte transcriptional programming associated with de-differentiation. Highly proliferative DN and DP subsets with antecedent characteristics of T cell lymphoblastic and myeloid malignancies emerge. Thus, in addition to fostering β chain repertoire broadening for subsequent αβ TCR utilization, preTCR-pMHC interaction limits cellular plasticity to facilitate normal thymocyte differentiation and proliferation that, if absent, introduces significant developmental vulnerabilities.
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Project description:The T cell receptor (TCR) determines the specificity and affinity for both foreign and self-peptides presented by MHC. It is established that self-pMHC reactivity impacts T cell function, but it has been challenging to identify TCR sequence features that predict T cell fate. To discern patterns distinguishing TCRs from naïve CD4+ T cells with low versus high self-pMHC reactivity, we used data from 42 mice to train a machine learning (ML) algorithm that predicts self-reactivity directly from TCRβ sequences. This approach revealed that n-nucleotide additions and acidic amino acids weaken selfreactivity. We tested our ML predictions of TCRβ sequence self-reactivity using retrogenic mice. Extrapolating our analyses to independent datasets, we found high predicted self-reactivity for regulatory CD4+ T cells and low predicted self-reactivity for T cells responding to chronic infection. Our analyses suggest a potential trade-off between repertoire diversity and self-reactivity intrinsic to the architecture of a TCR repertoire.
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Project description:Developmental checkpoints in stem/progenitor cells are critical to the determination, commitment and differentiation into distinct lineages. Cancer cells often retain expression of lineage-specific checkpoint proteins, but their potential impact in cancer remains elusive. T lymphocytes mature in the thymus following a highly orchestrated developmental process that entails the successive rearrangements and expression of T-cell receptor (TCR) genes. Low affinity recognition of self-peptide/MHC complexes (self-pMHC) presented by thymic epithelial cells by the TCR of CD4+CD8+ (DP) cortical thymocytes transduces positive selection signals that ultimately shape the developing T cell repertoire. DP thymocytes not receiving these signals die by lack of stimulation whereas those that recognize self-pMHC with high affinity undergo TCR-mediated apoptosis and negative selection. In T-cell acute lymphoblastic leukaemia (T-ALL), leukaemic transformation of maturating thymocytes results from the acquisition of multiple genetic and epigenetic alterations in oncogenes and tumour suppressor genes, that disrupt the normal regulatory circuits and drive clonal expansion of differentiation-arrested lymphoblasts. We show here that TCR triggering by negatively-selecting self-pMHC prevented T-ALL development and leukaemia maintenance in mice. Induction of TCR signalling by high affinity self-pMHC or treatment with monoclonal antibodies to the CD3 signalling chain (anti-CD3) caused massive leukaemic cell death and a gene expression program resembling that of thymocyte negative selection. Importantly, anti-CD3 treatment hampered leukaemogenesis in mice transplanted with either mouse or patient-derived T-ALLs. These data provide a rationale for targeted therapy based on anti-CD3 treatment of T-ALL patients and demonstrate that endogenous developmental checkpoint proteins are amenable to therapeutic intervention in cancer cells. Gene expression data from four culture conditions was performed for the following cells: ALL-SIL-TCRα/β-GFP co-cultured on OP9-DL1 for 48 h, ALL-SIL-TCRα/β-GFP without co-culture, ALL-SIL cells transduced with TLX shRNA (sh-TLX), and ALL-SIL transduced with sh-control vectors. All conditions are performed in two replicates.
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Project description:Chimeric antigen receptor-modified (CAR) T cell therapy targeting highly expressed lineage antigens is effective for B cell malignancies. Achieving durable efficacy for hematological malignancies and extending this therapeutic approach to solid tumors will require T cell recognition and elimination of tumor cells that may express lower levels of the CAR target antigen. Realizing this goal is challenging because current approaches to CAR design are largely empiric and detailed information on CAR signaling is only beginning to emerge. Synthetic CARs typically require hundreds of molecules on the target cell to initiate signaling, whereas natural T cell receptors (TCRs) can recognize less than ten peptide-MHC (pMHC) antigen complexes. We reasoned that in depth comparison of TCR and CAR stimulation-induced signaling events in primary T cells might guide rationale adaptations to CAR design that would improve antigen sensitivity. Bi-specific T cells possessing an endogenous TCR and exogenous CAR of defined specificity were formulated from healthy HLA-B8+ Epstein-Barr virus-seropositive donors. Bi-specific T cells were stimulated with magnetic microbeads coated with recombinant TCR or CAR antigen for 10, 45, or 90 minutes. Some bi-specific T cells were also left unstimulated and harvested at each timepoint to serve as controls. Altogether, 9 unique conditions were tested in an experiment and three independent experiments were performed.
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2023-05-18 | GSE232446 | GEO
Project description:The success of cancer immunotherapy depends in part on the strength of antigen recognition by T cells. We characterized the T cell receptor (TCR) functional (antigen sensitivity) and structural (monomeric pMHC-TCR off-rates) avidities of 371 CD8 T cell clones specific for neoantigens, tumor-associated antigens (TAAs) or viral antigens isolated from tumors or blood of patients and healthy subjects. T cells from tumors exhibit stronger functional and structural avidity than their blood counterparts. Relative to TAA, neoantigen-specific T cells are of higher structural avidity and, consistently, are preferentially detected in tumors. Effective tumor infiltration in mice models is associated with high structural avidity and CXCR3 expression. Based on TCRs biophysicochemical properties, we derived and applied an in silico model predicting TCR structural avidity and validated the enrichment in high avidity T cells in patients’ tumors. These observations indicate a direct relationship between neoantigen recognition, T cell functionality and tumor infiltration. These results delineate a rational approach to identify potent T cells for personalized cancer immunotherapy.
2023-05-18 | GSE232444 | GEO
Project description:Background Clinical success of T cell receptor (TCR) gene therapy has previously been demonstrated for NY-ESO-1 TCR gene therapy. To increase numbers of cancer patients that can be treated with TCR gene therapy we aimed to identify a novel set of high-affinity cancer specific TCRs targeting different cancer testis (CT) antigens in prevalent HLA class I alleles. Methods In this study, we selected based on publicly available gene expression databases the most promising CT genes to target. From these selected genes we identified by HLA peptidomics the naturally processed and presented HLA class I peptides. With these peptide-HLA tetramers were generated, and by single cell sorting CT specific CD8+ T cells were selected, and expanded from the allo-HLA repertoire of healthy donors. By several functional assays high avidity CT-specific T cell clones with safe recognition pattern were selected. To evaluate the potential for clinical application in TCR gene therapy, TCRs were sequenced and transferred into peripheral blood derived CD8+ T cells. Results In total we identified, 7 novel CT-specific TCRs that effectively target MAGE-A1, MAGE-A3, MAGE-A6 and MAGE-A9 in the context of human leukocyte antigen(HLA) -A1, -A2, -A3, -B7, -B35 and -C7. TCR gene transfer into CD8⁺ T cells resulted in efficient cytokine production and cytotoxicity of variety of different tumor types without detectable cross-reactivity. In addition, major in vivo antitumor effects of MAGE-A1 specific TCR engineered CD8⁺ T cells was observed in an orthotopic xenograft model for established multiple myeloma, in which bone marrow located tumor cells were completely eradicated after T cell injection. Conclusion The identification of 7 novel CT-specific TCRs, reactive against CT antigens presented in a variety of different HLA class I alleles, allows selection of therapeutic TCRs for an increased number of cancer patients, and will improve development of personalized TCR gene therapy.
2023-01-23 | PXD038862 | Pride