A single-cell profile of the porcine immune repertoire: concurrent profiling of T cell receptor and B cell receptor V(D)J sequences in health

Abstract Adaptive immune diversity is shaped by the complex organization of T cell receptor (TCR) and B cell receptor (BCR) repertoires across tissues. However, a systematic cross-tissue immune repertoire profiling in healthy pigs is still lacking. In this study, we used 3 14-day-old Duroc × Landrace × Yorkshire (DLY) crossbred piglets, all of which were male. After sample purification, a total of 71,731 cells were retained. We established single-cell profiling integrating paired BCR and TCR repertoires across 11 immune-associated tissues using single-cell RNA sequencing combined with V(D)J sequencing based on DLY crossbred piglets. Diverse populations of immune cells were identified, with significant tissue-specific heterogeneity being revealed in lymphatic, mucosal, and circulatory organs. Immune repertoire analysis demonstrated biased V(D)J gene usage, conserved complementarity-determining region 3 (CDR3) length distributions, preferential receptor pairing patterns, and extensive clonotype diversity. Cross-tissue analyses further revealed coordinated clonotype sharing and distinct clonal expansion patterns among immune cell subsets. In addition, using the Pig-specific V(D)J primers we designed, the sequencing results have achieved a modest improvement in the detection of immunoglobulin alleles compared to previous studies. It is expected to provide a more reliable technical solution for related research. Collectively, this work provides a comprehensive reference profiling of the healthy DLY crossbred piglet adaptive immune system and offers valuable resources for future studies of porcine immunology, infectious diseases, and vaccine responses.