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

https://doi.org/10.1016/j.dci.2019.103575Get rights and content

Highlights

  • Establishment of a NGS-based strategy for γ/δ TCR profiling of porcine γδ T cells.

  • Porcine blood-derived CD2+ TCR-γδ+ cells showed a high TCR-δ diversity (39 clonotypes).

  • Both subsets shared the abundant Vδ1DδxJδ4 clonotype (CD2+: 31.2%; CD2-: 37.0%).

  • The most abundant TRG clonotype in CD2+ TCR-γδ+ cells was Vγ11JγP1 (74.6%).

  • In CD2 TCR-γδ+ cells, the clonotype Vγ10JγP1 occurred at highest frequency (57.7%).

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‾ 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‾: 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.

Introduction

In humans and rodents, γδ T cells show a high variability in their functional capacities depending on their tissue distribution and respective T-cell receptor (TCR) recombination. Their potential functions are ranging from protective immunity against extracellular and intracellular pathogens to tumor surveillance, modulation of innate and adaptive immune responses, tissue healing and epithelial cell maintenance as well as regulation of physiological organ function (reviewed in Mair et al., 2014). The frequency and distribution of γδ T cells in diverse species has been found to be different, distinguishing them generally into two classes, namely so called γδ ‘high’ and γδ ‘low’ species. Unlike in human and rodents, which show a low frequency of γδ T cell in blood, species like chicken, sheep, cattle, and swine possess a high frequency of γδ T cells circulating in peripheral blood or residing in the skin and spleen (Sedlak et al., 2014a; Holderness et al., 2013). In pigs, the earliest T-cell population emerging in the thymus are the γδ T cells around day 40 of gestation and five days later, these cells are found in the periphery (Sinkora et al., 2005). After birth and in young piglets, they are making up to 20% of the T cells. Later on, during the age period of 4–12 months their number can reach up to 50% in the peripheral blood, before decreasing with adolescence (Saalmüller and Gerner, 2016; Sedlak et al., 2014b; Talker et al., 2015; Gerner et al., 2009).

Also different from murine and human γδ T cells, porcine γδ T cells divide into a CD2+ and CD2 subset (Stepanova and Sinkora, 2012; Yang and Parkhouse, 1996; Saalmüller et al., 1990). The CD2‾ subset dominates in blood and liver, while CD2+ γδ T cells were preferentially found in spleen and thymus (Sedlak et al., 2014a). CD2 γδ T cells are mainly CD8α (Yang and Parkhouse, 1996) and differ from CD2+CD8α‾ and CD2+CD8α+ γδ T cells in regard to their expression level of the TCR-γδ: CD2+CD8α‾ and CD2+CD8α+ cells have a lower expression of the TCR in comparison to CD2‾CD8α‾ cells (Stepanova and Sinkora, 2013).

For the genomic organisation of the T cell receptor γ and δ chains based on the Scrofa 11.1 genome build (GCA_000003025.6, 2017/02/07), initial annotations report the existence of 8 TRGV, 7 TRGJ and 4 TRGC gene segments for the TRG locus (Schwartz et al., in preparation; Thome et al., 1993, Thome et al., 1994). The porcine TRD locus shows a complex structure with 28 TRDV, 6 TRDD, 4 TRDJ, and 1 TRDC gene segments (Uenishi et al., 2003, 2009).

It has to be noted that for porcine αβ T cells the expressed TCR repertoire has been studied by cDNA libraries, which were enriched for TCR transcripts prior to Next Generation Sequencing (NGS) or CDR3 spectratyping analysis (Yang et al., 2019; Massari et al., 2018; Wang et al., 2017). To our knowledge, such comprehensive studies are still missing for a detailed characterization of the TCR-γδ repertoire in swine. The aim of this was study was to translated a human TCR profiling approach (Ravens et al., 2017, 2018) to characterize the TCR repertoire in sorted TCR-γδ CD2+ and TCR-γδ CD2‾cells. A 5’ RACE-like approach was used to generate and enrich full-lengths cDNA transcripts of the porcine TCR γ/δ chains being present in peripheral blood derived TCR-γδ+ cells. With respect to TCR-δ chain diversity, TCR-γδ CD2‾ cells exhibited a lower number of clonotypes compared with the CD2+ subset. In comparison, both subpopulations shared approximately identically TCR-γ chain diversities. In the course of this initial proof-of-principle approach, we successfully established a NGS-based strategy for future γ/δ TCR profiling of compartment-specific γδ T-cell subsets alongside with a detailed phenotypic characterization and assessment of their functional capacity.

Section snippets

Ethics statement

Experimental procedures were approved by the institutional ethics and animal welfare committee and the national authority according to §26 of Animal Experiments Act 2012 (Tierversuchsgesetz 2012 – TVG 2012), reference number GZ 68.205/0005V/3b/2018.

Isolation of porcine PBMC

For this pilot study, PBMC were isolated from heparinized blood of two four-month-old commercial pig (pig A and B), being raised at the VetFarm of the University of Veterinary Medicine Vienna (Vetmeduni Vienna, Austria) and subsequently housed at the

Results and discussion

Lymphocytes of a four-month-old conventional pig (pig A: frequency of γδ T cells 19.6% within PBL) were FACS sorted and the purity of the subsets was assessed by reanalysis in the flow cytometer. Purities of the CD2+ and CD2‾ subsets were 98.1% and 99.3%, respectively (Supplementary Fig. 1A and 1B). The outcome of total RNA extractions were as follows: From sorted TCR-γδ CD2+ (pig A) we obtained 23.2 ng/μl, (RINe = 9.3, 28S/18S area = 1.4) and TCR-γδ CD2‾ (pig A) 42.4 ng/μl (RINe = 9.2, 28S/18S

Author contributions

SEH, KHM, WG and AS were responsible for the design of the study and conceived experiments. SEH supervised research, designed and performed experiments, interpreted the results and wrote the manuscript with input from all authors. ML, L-MP, KHM and SR performed experiments and analysed data. JCS and JAH annotated porcine TRG and TRD gene loci. All authors contributed to the editing of the manuscript. All authors read and approved the final manuscript before submission.

Funding

JCS and JAH were supported by funding from the UKRI-BBSRC awards BBS/E/I/00007031, BBS/E/I/00007038 and BBS/E/I/00007039.

Data availability statement

FASTQ files of TRG and TRD sequences are deposited under BioProject accession code: PRJNA563689 (TCR profiling of porcine γδ T cells). Further information about data and reagents used is available by request to the corresponding author.

Declaration of competing interest

The authors declare no competing interests.

Acknowledgments

The authors are indebted to the University Clinic for Swine of the University of Veterinary Medicine Vienna (Austria) for providing sample material. We acknowledge the support from the Institute of Immunology, Hannover Medical School, Hannover, Germany for conducting the library construction, and sequencing of collected samples.

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