Comparative transcriptome analysis of T lymphocyte subpopulations from porcine thymus and lymph nodes

The development and migration of T cells in the thymus and peripheral tissues are crucial for maintaining adaptive immunity in mammals. However, the regulatory mechanisms underlying T cell development and thymocyte identity formation in pigs remain largely underexplored. Here, by integrating bulk and single-cell RNA-sequencing data, we investigated regulatory signatures of porcine thymus and lymph node T cells. The comparison of T cell subpopulations derived from porcine thymus and lymph nodes revealed that their transcriptomic differences were influenced more by tissue origin than by T cell phenotypes, and that lymph node cells exhibited greater transcriptional diversity than thymocytes. Through weighted gene co-expression network analysis (WGCNA), we identified the key modules and candidate hub genes regulating the heterogeneity of T cell subpopulations. Further, we integrated the porcine thymocyte dataset with peripheral blood mononuclear cell (PBMC) dataset to systematically compare transcriptomic differences between T cell types from different tissues. Based on single-cell datasets, we further identified the key transcription factors (TFs) responsible for maintaining porcine thymocyte identity and unveiled that these TFs coordinately regulated the entire T cell development process. Finally, we performed GWAS of cell type-specific differentially expressed genes (DEGs) and 30 complex traits, and found that the DEGs in thymus-related and peripheral blood-related cell types, especially CD4_SP cluster and CD8-related cluster, were significantly associated with pig productive and reproductive traits. Our findings provide an insight into T cell development and lay a foundation for further exploring the porcine immune system and genetic mechanisms underlying complex traits in pigs. Overall design: Fresh thymic and mesenteric lymph node tissues were obtained 3 healthy Large White pigs from the experimental farm of Huazhong Agricultural University (Wuhan, China) Porcine thymocytes were initially were divided into CD3-positive and CD3-negative fractions using FACS. The CD3-positive fraction was subjected to FACS gating based on forward scatter (FSC) and side scatter (SSC) parameters, further divided into 3 distinct populations according to CD4 and/or CD8 marker expression, namely, CD4-CD8+ (Q1), CD4+CD8+ (Q2), and CD4+CD8- (Q4) T cells. The CD4-CD8- (Q3) cell population was enriched through negative sorting.Using the above-mentioned sorting strategy, we divided porcine mesenteric lymph node cells into 3 cell populations including CD4-CD8- (Q3), CD4+CD8- (Q4), and CD4-CD8+ (Q1) T cells To investigate the transcriptomic differences of T lymphocyte across different tissues, we conducted RNA-seq of a total of 7 distinct T cell subpopulations isolated from porcine thymus and lymph nodes.