Characterization Of The Peking Duck T Cell Receptor Genes

T cells, which play a major role in regulation and effector functions of immune response, have T-cell receptor (TCR) heterodimers expressed on their surface to react with antigens presented by major histocompatibility complex molecules. So far four typical TCR genes (α,β,γandδ) have been found in jawed vertebrates. As an important poultry, ducks are more and more frequently studied as an avian model for their resistance to various pathogens. So, a thorough analysis of the genomic organization and repertoire diversity of the duck TCR genes may provide basic data and theoretical basis for further understanding of the avian adaptive immune system. In this study, the TRA/D, TRB, and TRG gene loci of Peking duck were mapped by screening a BAC library, and the expressed repertoires of each TCR gene were obtained by5’RACE assay. Finally, the mRNA levels of each TCR gene in different tissues were measured by quantitative PCR.The duck TRB locus in the obtained BAC contains10Vp segments (seven functional and three pseudo genes), a single Dp segment and two functional Cβ genes, which have highly conserved Ig domains and each preceded by two and four functional Jp segments, respectively. Three extra Vβ segments were cloned by5’ RACE assay, and the13Vp gene segments can be grouped into four subgroups. The duck TRG locus has a translocon organization, including13Vγ segments (eight functional and five pseudo genes), five functional Jγ segments, and a single Cγ segment in BAC sequence. Two extra Vγ gene segments were cloned from5’ RACE assay and all15duck Vγ gene segments can be divided into six subgroups. The duck TRD locus is located within the TRA locus, resembling the genomic organization of TRA/D locus in other tetrapods. Within this locus, we identified a single Cα and Cδ gene and33V gene segments, including nine Vα at the5’ end and24Vδ located downstream of the Va group. Three Va and one Vδ segments are pseudogenes. Using5’RACE PCR,40extra Va and six extra Vδ segments were cloned from TRA and TRD cDNA clones. All49(9+40) Va and30(24+6) Vδ segments can be grouped into three and five subgroups, respectively. There are at least68functional Jα gene segments located between the Cδ and Cα genes, and furthermore, two D8and two functional Jδ gene segments were identified upstream of the single Cδ gene, In duck genome sequence, we also identified a second TRD locus, which contains a single VH-like Vδ, Dδ, Jδand Cδ segment. Genes flanking this locus are also conserved in chickens.N and P nucleotide additions and trimming of V, D, and J genes were frequently observed during the V(D)J rearrangement for all four TCR genes. Length distributions of CDR3for all four TCR genes are similar to those of human and mice. The CDR3lengths of TRD chains are longer but constrained, whereas those of the TRA, TRB, and TRG chains are short but variable. For TRB and TRG, the most frequently used V gene subgroup is VpIII and VγI, respectively, whereas the subgroup VβI and VγV, was not observed in all cDNA clones. For TRA and TRD, members of the subgroup VαⅢ occupied more than half of the expressed Va repertoire, and more than60%Vδ repertoire utilized20Va segments. One, two, and none Dδ segments were incorporated within a single CDR3, and a new allele of Dδ2segment was identified in most of the CDR3sequences. We also found that the second TRD gene was highly expressed in mucosal tissue, indicating its important role in mucosal immunity.Based on the genome data of both duck and chicken, we discovered a novel gene of immunoglobulin superfamily characterized by a prejoined VJ in germline sequence. This gene is organized as nine tandem clusters in duck but only a single copy in chicken. V region of this gene shared high sequence identity to V segments of both TCRs and IgLs, and shows no mutations when expressed. This TCR-like gene is highly expressed in both thymus and spleen, as well as in chicken B and T cells lines (DT40and MSB1). Furthermore, the protein encoded by this gene was detected on the surface of MSB1cell by immunofluorescent.In summary, we had thoroughly analysed the genomic organization, the repertoire diversity and V-D-J recombinatorial pattern of five duck TCR genes, these basic data may provide new clues to further understanding of the avian adaptive immune system.