Single-cell transcriptome delineating asymmetric dynamics of CD4+ T and CD8+ T cell lineage commitment in human prenatal thymus.

INTRODUCTION

The thymus provides a specialized microenvironment for T cell development and selection, yet the cellular heterogeneity and molecular dynamics that govern human prenatal thymopoiesis remain incompletely characterized.

METHODS

We constructed an integrative single-cell atlas of human prenatal thymocytes from 7 to 23 post-conception weeks by combining five published datasets. Selection intermediates were classified based on coreceptor expression patterns. Pseudotime analysis, regulon profiling, metabolic analysis, and cell-cell communication modeling were applied to characterize developmental dynamics.

RESULTS

We identified three transitional populations, Sel. int. DP, Sel. int. CD4, and Sel. int. CD8, positioned between DP and single-positive stages. Critically, our findings reveal that CD4/CD8 lineage commitment in the developing human thymus is not a single event, but an asymmetric, multi-stage dynamic process. This asymmetry manifests in three distinct dimensions. First, at the signaling level, Sel. int. CD4 cells exhibit enriched TCR and cytokine signaling activities compared to their Sel. int. CD8 counterparts. Second, at the temporal level, CD4 lineage traits emerge coincident with cellular activation, whereas CD8 lineage characteristics appear only after activation subsides. Third, at the microenvironmental level, Sel. int. CD8 and CD8+ T cells display the most extensive interaction networks with thymic stromal cells. Pseudotime analysis delineated two developmental paths branching at the Sel. int. DP stage toward the CD4+ and CD8+ lineages, revealing four distinct gene expression patterns encompassing activation, viral response, differentiation, and apoptotic programs.

DISCUSSION: Collectively, this atlas provides a comprehensive resource for understanding the asymmetric, multi-stage dynamics of human prenatal T cell development and the cellular crosstalk that orchestrates CD4/CD8 lineage commitment.