Development of a RACE-based RNA-Seq approach to characterize the T-cell receptor repertoire of porcine γδ T cells.
Dev Comp Immunol
; 105: 103575, 2020 04.
Article
em En
| MEDLINE
| ID: mdl-31846687
ABSTRACT
Recent data suggest that porcine γδ T cells exhibit a similar degree of functional plasticity as human and murine γδ T cells. Due to the high frequency of TCR-γδ+ 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⾠TCR-γδ+ cells exhibited two distinct clonotypes Vγ11JγP1 (74.6%) and Vγ10JγP1 (57.7%), respectively. Despite the high TCR-δ diversity among CD2+ cells (39 clonotypes), both subsets shared the same abundant Vδ1DδxJδ4 clonotype at approximately identically frequencies (CD2+ 31.2%; CD2⾠37.0%). The flexible nature of this approach will facilitate the assessment of organ-specific phenotypes of γδ T cell subsets alongside with their respective TCR diversity at single cell resolution.
Palavras-chave
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Suínos
/
Linfócitos T
/
Receptores de Antígenos de Linfócitos T gama-delta
/
Análise de Sequência de RNA
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Dev Comp Immunol
Ano de publicação:
2020
Tipo de documento:
Article