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Single-cell RNA-seq of human CD4+ and CD8+ T cells transduced with a TAA-specific low-avidity TCR and the CD8αβ co-receptor

ABSTRACT: Most naturally occurring major histocompatibility class I restricted T cell receptors (TCRs) that target over-expressed tumor-associated self-antigens (TAAs) are of low avidity due to selection and tolerance in the host and are CD8 co-receptor dependent. Adoptive T cell transfer with TCR-engineered T cells thus rely on the function of CD8+ T cells and do not exploit beneficial CD4+ T cell functions. Hence, we developed a novel strategy that combines expression of a TAA-specific low-avidity TCR with the CD8ab co-receptor and explored the properties of purified transgenic CD8+ and CD4+ T cells separately, in vitro and in vivo. We found that CD8ab co-transfer enhanced TCR+ CD8+ T cell function by increasing their serial tumor killing capacity, indicating that limited availability of endogenous CD8 co-receptors impedes full deployment of their functional potential. Engineered CD4+ T cells were efficiently reprogrammed into hybrid multifunctional cytotoxic and helper T cells at the single-cell level: they recognized and killed cells expressing the cognate class I restricted tumor antigen, became serial killers, produced mostly TH1 and preserved some TH2 cytokines, and showed superior anti-tumor function in vivo in a leukemia xenograft model. CD8ab co-transfer restored the TCR-pMHC interaction in CD4+ T cells and enhanced early TCR signaling events. Single-cell RNA-seq profiling suggests that co-transferred CD4+ T cells acquired a cytotoxic gene expression program and at the same time retained CD4 lineage specific features. In conclusion, we present a novel approach that allows us to (1) enhance the function of TCR-transgenic CD8+ T cells and (2) to manufacture class I pMHC targeted hybrid cytotoxic and helper T cells with both CD8+ and CD4+ T cell functions readily available at the single-cell level.

ORGANISM(S): Homo sapiens

PROVIDER: GSE132164 | GEO | 2020/07/13

REPOSITORIES: GEO

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Project description:The avidity of the T-cell receptor (TCR) for antigenic peptides presented by the MHC (pMHC) on cells is an essential parameter for efficient T cell-mediated immunity. Yet, whether the TCR-ligand avidity can drive the clonal evolution of virus antigen-specific CD8 T cells, and how this process is determined in latent Cytomegalovirus (CMV)- against Epstein-Barr virus (EBV)-mediated infection remains largely unknown. Here, we quantified monomeric TCR-pMHC dissociation rates on CMV- and EBV-specific individual TCR-alpha-beta clonotypes and polyclonal CD8 T cell populations in healthy donors over a follow-up time of 15-18 years. Within CMV/pp65-specific T cell repertoires, a progressive contraction of clonotypes with high TCR-pMHC avidity and low CD8 binding dependency was observed, leading to an overall avidity decline during long-term antigen exposure. We identified a unique transcriptional signature preferentially expressed by high-avidity CMV/pp65-specific T cell clonotypes, including the inhibitory receptor LILRB1. Interestingly, T cell clonotypes of high-avidity showed higher LILRB1 expression than the low-avidity ones and LILRB1 blockade moderately increased T cell proliferation. Similar findings were made for CD8 T cell repertoires specific for the CMV/IE-1 epitope. There was a gradual in vivo loss of high-avidity T cells with time for both CMV specificities, corresponding to virus-specific CD8 T cells expressing enhanced LILRB1 levels. In sharp contrast, the EBV/BMFL1-specific T cell clonal composition and distribution, once established, displayed an exceptional stability, unrelated to TCR-pMHC binding avidity or LILRB1 expression. Together, these findings reveal an overall long-term avidity decline of CMV- but not EBV-specific T cell clonal repertoires, highlighting the differing role played by TCR-ligand avidity over the course of these two latent herpesvirus infections. Our data suggest that the inhibitor receptor LILRB1 potentially restricts the clonal expansion of high-avidity CMV-specific T cell clonotypes during latent infection. We propose that the mechanisms regulating the long-term outcome of CMV- and EBV-specific memory CD8 T cell clonotypes in humans are distinct.

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-TCRÎ±/Î²-GFP co-cultured on OP9-DL1 for 48 h, ALL-SIL-TCRÎ±/Î²-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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Single-cell RNA-seq of human CD4+ and CD8+ T cells transduced with a TAA-specific low-avidity TCR and the CD8αβ co-receptor

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