Single Cell and Spatially Resolved Transcriptome and Immune Repertoire of Mouse Thymus During Aging Reveal Immunological Heterogeneity and Direction of Thymic Selection Pressure.

Abstract

The thymus is the central organ where T cells differentiate and proliferate. T cells experience the TCR recombination to generate diversity, self-antigen tolerance, and immune response. However, the spatial heterogeneity of transcriptomics and immune repertoire of the thymus during aging haven't been studied. We constructed the thymus's spatial transcriptomics and spatial immune repertoire atlas by TCR-specific amplification. The integrated multi-omics analysis of the scRNA-seq, ST-seq, spatial-TCR-seq, scTCR-seq, and TCR-seq was developed to investigate the thymus during aging. ST-seq demonstrated that the heterogeneity of the cortex and a novel cortex subset associated with progenitor cell differentiation was identified. Proliferating CD69negDN T cells decreased and regulatory CD4SP T cells increased during aging. Using deconvolution analyses, the spatial distribution of T cell subsets was revealed. The spatial distribution of TCR recombination and distinct thymic selection processes were illustrated. Pseudo-time analyses demonstrated differentiation order and spatial migration route of T cells. The novel spatial-TCR-seq demonstrated the overall spatial distribution of TCR, the TCR pairing process, and their dynamic features under thymic selection pressure which revealed that TRB clonotypes with hydrophilic and low-pI CDR3s tended to pass the negative selection opposite to the positive selection. The positive selection pressure of the thymus diminishes during the aging process. A TCR maturation scoring system using TCR-seq was developed to identify TCR clonotypes that tended to mature. All boost the thymus study into the era of spatial immune repertoire and transcriptome and widen our knowledge about T cell development.