Development of a RACE-based RNA-Seq approach to characterize the T-cell receptor repertoire of porcine γδ T cells

Recent data suggest that porcine gamma delta T cells exhibit a similar degree of functional plasticity as human and murine gamma delta T cells. Due to the high frequency of TCR-gamma delta(+) cells in blood and secondary lymphatic organs, the pig is an attractive model to study these cells, especially their combined features of the innate and the adaptive immune system. Using a 5' RACE-like approach, we translated a human/murine NGS library preparation strategy to capture full-length V-(D)-J TRG and TRD clonotypes in swine. After oligo(dT) primed conversion of input RNA, the cDNA population was enriched for full-length V(D)J TCR transcripts with porcine-specific primers including Illumina adaptor sequences as overhangs for Illumina MiSeq analysis. After quality control and processing by FastQC and ea-utils, porcine TRG and TRD sequences were mapped against the human IMGT reference directory. Porcine blood-derived CD2(+) and CD2(-) TCR-gamma delta(+) cells exhibited two distinct clonotypes V gamma llJ gamma P1 (74.6%) and V gamma 10J gamma P1 (57.7%), respectively. Despite the high TCR-8 diversity among CD2(+) cells (39 clonotypes), both subsets shared the same abundant V delta 1D delta xJ delta 4 clonotype at approximately identically frequencies (CD2(+): 31.2%; CD2(-) : 37.0%). The flexible nature of this approach will facilitate the assessment of organ-specific phenotypes of gamma delta T cell subsets alongside with their respective TCR diversity at single cell resolution.