Prenatal development of the porcine TCR δ repertoire: dominant expression of an invariant T cell receptor Vδ3‐Jδ3 chain

Abstract

The prenatal development of the porcine + /ˇTCR repertoire was studied by complementarity-determining region 3 (CDR3) spectratyping and sequencing of TRDV1-DV5 transcripts. Specimens from the small and large intestine, spleen, thymus, liver, bone marrow and PBMC from fetal piglets between 38 and 114 days of gestation (DG) were examined. The TCRˇrepertoire was highly restricted early in gestation (DG38-DG57) and an invariant TRDV3 transcript, lacking the N/D region, was found in different fetuses throughout gestation and dominated the TRDV3 repertoires of all organs at mid gestation (˚DG55). Near the end of gestation, this invariant TRDV3 transcript was absent from the thymus but was still present, in a less dominant manner, in the intestine and spleen. The average CDR3 length of all Vˇsubgroups increased with ontogeny, suggesting an increase in activity of TdT. Thus, the persistence of fetal + /ˇT cells expressing an invariant TRDV3 chain throughout development is especially surprising since TdT is active early in gestation in swine. We speculate that these + /ˇT cells might have been selectively expanded by (self)-ligands and may have an important function throughout fetal development. IntroductionThe swine is one of the most important large-animal models belonging to " + /ˇhigh" species. They have an abundance of + /ˇT lymphocytes in their peripheral lymphoid pool [1][2][3][4], which makes them an attractive and useful model for the study of + /ˇT cells. However, only limited molecular data are available about porcine + /ˇT cells. Previously we have characterized the development and compartmentalization of the porcine TCRˇreper-toire at mucosal and extraintestinal sites after birth [5]. We have also demonstrated that the TCRˇrepertoire is highly polyclonal in young pigs and becomes increasingly oligoclonal and compartmentalized with age. These findings resemble those in humans [6] and non-human primates [7, 8]. In this study we characterized the prenatal development of the porcine TCRˇrepertoire and compared it with published data from human [6, 9, 10] and murine fetuses [11][12][13][14][15].Flow cytometric studies showed that + /ˇT cells are the earliest detectable T cell population in swine, developing first in the thymus around day of gestation (DG)40 and subsequently populating the periphery at about DG45 [2,[16][17][18]. This is also the case for mice [19], chicken [20,21] and humans [10,22,23] but not for sheep [24] and rats [25]. The proportion of + /ˇT cells decreases later in ontogeny (DG50-55) as § / g T cells expand and become the predominant CD3 + lymphocyte subset. This results in a predominance of § / g T cells in the second half of gestation in both the thymus and periphery [2,16,18]. Until DG90, peripheral + /ˇT cells account for only 3-5% of all lymphocytes [2,16]. However, near the end of gestation (DG90-114), there is a rapid expansion of + /ˇT cells in the periphery [2,16], so that + /ˇT cells in newborn and young piglets account for about 20% of all peripheral lymphocytes [2]. The prop...

Highly restricted diversity of TCR delta chains of the amphibian Mexican axolotl (Ambystomamexicanum) in peripheral tissues

“…A canonical TRD transcript that completely lacks N nucleotides and D regions and contains the TRDV3 gene directly rearranged to the TRDJ3 gene has recently been described in swine. This canonical TRD chain is recovered from the first cd T cells that emerge from the fetal thymus and dominates the TRD repertoire of all organs at midgestation [43]. At late gestation, this canonical TRDV3 transcript is absent from the thymus but is still present, in a less dominant manner, in the spleen and the intestine.…”

Porcine γδ T Lymphocytes Can Be Categorized into Two Functionally and Developmentally Distinct Subsets according to Expression of CD2 and Level of TCR

“…Thus, separation of CDR3 regions for TCRd on polyacrylamide sequencing gels provides a clonotypic analysis of porcine gd T cells showing their level of diversification (19). This procedure for measuring CDR3 polymorphism is called spectratyping and was performed essentially as described previously (5,12,13,19). Briefly, 50,000 or 100,000 sorted cells were immediately after sort dissolved in 0.5 ml TRI Reagent (Sigma-Aldrich).…”

“…7). Only the Vd1 family was studied because it is the most varied family (containing .30 members) whereas the rest of the Vd families (Vd2-Vd5) contains one to two members (19). Analyses of sorted TCRgd med and TCRgd hi (Fig.…”

“…Our previous studies revealed the basic distribution and phenotypic profile of porcine gd T cells (3,12,18), their ontogeny (3,11), their development in the thymus (5,11,13), and the repertoire diversification of their TCR (19). In this study, we used high-speed flow cytometry sorting and cultivation techniques to characterize different subpopulation of gd T cells defined by TCRgd, CD2, and CD8 expression.…”

Development of the neonatal B and T cell repertoire in swine: implications for comparative and veterinary immunology

“…Using the same molecular tools described above [48], we analyzed the CDR3 regions of TRDV1 to TRDV5 transcripts from multiple fetal organs from DG38 to DG114 since the first γδ T cells can be detected on DG38 [49] (see above). These studies revealed an invariant TRDV3 transcript in all fetuses from early gestation until birth.…”