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Progenitor effects and unique transcriptomic signatures linked to differentiation phenotype in clonally expanded antigen-specific CD8 T memory stem cells (TSCM)

ABSTRACT: Memory stem CD8 T cells (TSCM) are a long-lived T cell subset displaying stem cell-like properties, which play a significant role in anti-viral defense and exhibit a gene expression profile between naïve and central memory T cells. Yet, comprehensive transcriptional profiling of antigen-specific CD8 TSCM cells at the single cell level has not previously been undertaken. In this analysis, we utilized an in vitro single cell colony expansion protocol to study human CD8 T cell clones specific for Cytomegalovirus (CMV) epitopes - phenotypically, functionally, and in transcriptomes. We characterized clonal lineages from 3 donors by single cell sorting dextramer positive CD8 T cells of varied effector and memory surface phenotypes, all recognizing one of two HLA restricted CMV epitopes. Phenotypic and functional characterization of clonal lineages derived from antigen-specific TSCM revealed differentiated memory and effector subsets (TCM, TEM, TEFF) as well as TSCM, with the latter subset featuring multi-potentiality and multi-cytokine production. Studying TSCM-derived progeny of these varied differentiation phenotypes in single cell transcriptomic analysis revealed strong progenitor-to-progeny effects, whereby daughter cells from a shared progenitor clustered closely regardless of progeny differentiation phenotype, suggesting a dominant impact associated with heritable genes. The TSCM-derived TSCM-progeny revealed distinct transcriptomic profiles featuring expression of both recognized early differentiation memory T cell genes, such as IL7R, CCR7, SELL and CD27; as well as several novel genes which were shared across subjects and epitopes. The transcriptional features of clonal lineages are strongly dependent on the progenitor cell, but TSCM have an additional transcriptomic signature likely underpinning the unique differentiation and functional characteristics. These findings have important implications for identification of long-lived and multi-potent CD8 T cells for immunotherapy and immunization against viral infections.

ORGANISM(S): Homo sapiens

PROVIDER: GSE280113 | GEO | 2025/10/20

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:Reconstitution of cytomegalovirus (CMV)-specific immunity following transplant remains a primary clinical objective to prevent CMV disease, and adoptive immunotherapy of CMV-specific T cells can be an effective therapeutic approach. Due to the persistence of CMV, most CMV-specific CD8+ T cells become terminally differentiated effector cells (TEFF). However, a minor subset retains a memory phenotype (TM). Interestingly, recent studies suggest that CMV-specific CD8+ T cells with different phenotypes may have different abilities to reconstitute sustained immunity following transfer. The immunology of human CMV (HCMV) infections is reflected in the mouse model of MCMV infection. We found that HCMV- and MCMV-specific T cells displayed shared genetic programs, validating the MCMV model for studies of CMV-specific T cells in vivo. After transfer, the proliferative capacity of MCMV-specific TM cells was vastly superior to TEFF cells. Strikingly, TM cells expanded and established sustained and diverse T cell populations even after multiple challenges. Although both TEFF and TM cells could protect Rag-/- mice, only TM cells could consistently survive after transfer into immune replete, latently infected recipients and respond if recipient immunity was lost. These data show that CMV-specific TM cells retain memory function during persistent infection and can re-establish CMV immunity when necessary. C57BL/6 mice were infected intraperitoneally (i.p.) with MCMV strain MW97.01 between 6-16 weeks of age. Splenocytes were isolated from chronically-infected mice and co-stained with three PE-conjugated tetramers loaded with the antigenic peptides from M38, m139 and IE3, all of which promote memory inflation. Cells were then stained with fluorescently conjugated antibodies and sorted on a MoFlo cell sorter. Naïve CD8+ cells were identified as CD44lo. MCMV-specific T cells were identified as CD8+, CD44hi and tetramer binding and then further segregated into memory and effector cells subsets by their expression of KLRG1 and CD127.

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Progenitor effects and unique transcriptomic signatures linked to differentiation phenotype in clonally expanded antigen-specific CD8 T memory stem cells (TSCM)

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