Forced expression of terminal deoxynucleotidyl transferase in fetal thymus resulted in a decrease in gammadelta T cells and random dissemination of Vgamma3Vdelta1 T cells in skin of newborn but not adult mice

Abstract

The repertoire of lymphocyte receptor genes encoded in a germline is further diversified by a number of processes, including the template-independent addition of nucleotides (N regions) by means of terminal deoxynucleotidyl transferase (TdT). Normally, mouse gammadelta T cells in the early fetal thymus, whose T-cell receptor (TCR) genes lack N regions and are encoded by Vgamma3-Jgamma1 and Vdelta1-Ddelta2-Jdelta2 with canonical junctions (invariant Vgamma3Vdelta1), are thought to be the precursors of dendritic epidermal T cells (DETC). We generated mutant mice whose endogenous TdT promoter was replaced with the lck promoter through homologous recombination. These mutant mice expressed TdT in fetal thymus, had abundant N regions and infrequent canonical junctions in gamma and delta rearrangements, and showed a decreased number of gammadelta T cells. Various Vgamma3Vdelta1 T cells, most of which had N regions in their TCR genes, were found to disseminate in the skin of newborn mutant mice, whereas normal numbers of DETCs with the invariant Vgamma3Vdelta1 rearrangement were observed in adult mutants. These data demonstrate that the regulation of TdT expression during fetal development is important for the generation of gammadelta T cells, and that Vgamma3Vdelta1 T cells, which have various junctional sequences in their TCR genes, randomly disseminate in skin, but invariant Vgamma3Vdelta1 T cells have a great advantage for proliferation in skin.

Figure 1

Generation of mutant mice in which the terminal deoxynucleotidyl transferase (TdT) promoter was replaced by the lck promoter. (a) Structure of the targeting vector and partial restriction map of the genomic TdT locus and mutated allele after homologous recombination. Exons 1–4 and 6 are depicted as black boxes. The location of exon 5 (white box) has not been determined. RI, EcoRI; H, HindIII; X, XbaI; S, SalI; Xh, XhoI; K, KpnI. (b) Southern blot analysis of representative mouse tail DNA. Genomic DNA was isolated from tails. DNA was digested with EcoRI and hybridized with the SalI–XbaI probe indicated. WT, wild-type littermate. (c) Expression of TdT in lckTdT^+/+ and control C57BL/6 mice. RNA prepared from each tissue was examined by Northern blot analysis using a probe of full-length cDNA of TdT. Fetal thymus RNA was prepared from embryonic day (E)14.5–E18.5 embryos. Lane 1, C57BL/6 thymus (T) at 8 weeks; lanes 2–7, lckTdT^+/+ thymuses (lane 2, E14.5; lane 3, E15.5; lane 4, E16.5; lane 5, E17.5; lane 6, E18.5; lane 7, 8 weeks). RNA from spleen (S), bone marrow (B), lymph node (L), kidney (K), brain (Br), liver (Li) and lung (Lu), was extracted from 8-week-old lckTdT^+/+ mice. 28S and 18S ribosomal bands stained with ethidium bromide are shown in the lower panel.

Figure 2

Flow cytometric analysis of skin. Epidermal cells from wild-type (WT) and lck-promoter terminal deoxynucleotidyl transferase (lckTdT)^+/+ mice at birth and at 4 and 24 weeks of age were stained for αβ/γδ, Vγ3/heat stable antigen (HSA) and Vγ3/γδ, and analysed by fluorescence-activated cell sorter (FACScan). All nucleated cells gated by forward scatter and side scatter were analysed. Numbers indicate the percentages of cells stained for a particular phenotype in the respective boxed regions. Representative data are shown. NB, newborn.

Figure 3

Reverse transcription–polymerase chain reaction (RT–PCR) analysis of T-cell receptor (TCR) γ and δ mRNA. RNA was extracted from thymocytes (at embryonic day [E]16.5 and 10 weeks old) and skin (at birth and 4 weeks) of wild-type (WT) and lck-promoter terminal deoxynucleotidyl transferase (lckTdT)^+/+ mice. cDNA was amplified by PCR using a Cγ primer and one of five Vγ (Vγ1-Vγ5) primers (a) or with a Cδ primer and one of seven Vδ (Vδ1-Vδ7) primers (b), and an aliquot was separated by electrophoresis and transferred onto nylon membranes. The blotted membranes were hybridized with a Cγ probe (a) or a Cδ probe (b). Hypoxanthine-guanine phosphoribosyl transferase (HPRT) was used as an internal control for loading equal amounts of cDNA for analysis. NB; newborn. (a) 1, Vγ1; 2, Vγ2; 3, Vγ3; 4, Vγ4; 5, Vγ5. (b) 1, Vδ1; 2, Vδ2; 3, Vδ3; 4, Vδ4; 5, Vδ5; 6, Vδ6; 7, Vδ7.

Figure 4

Junctional sequences of Vγ3-Jγ1 and Vδ1-Dδ-Jδ2 cDNA from skin of newborn lck-promoter terminal deoxynucleotidyl transferase (TdT)^+/+ mice. Vγ3-Jγ1 junctional sequences (a) and Vδ1-Dδ-Jδ2 junctional sequences (b) were obtained using cDNA from skin of lckTdT^+/+ mice. Two mice (a and b) were analysed, and two separate polymerase chain reaction (PCR) amplification reactions were performed for each mouse. The sequences are aligned with the germline sequences of the T-cell receptor (TCR) Vγ3 segmentand Vδ1 segment.The frequency of each junction is listed to the left of the sequence. Overlapping nucleotides that could be encoded by either germline segment (including P nucleotides) are underlined. In-frame and out-of-frame sequences are indicated as + and –, respectively, on the right of the sequence. Canonical sequence is indicated by an asterisk.