Characterization of avian T-cell receptor gamma genes

Abstract

In birds and mammals T cells develop along two discrete pathways characterized by expression of either the alpha beta or the gamma delta T-cell antigen receptors (TCRs). To gain further insight into the evolutionary significance of the gamma delta T-cell lineage, the present studies sought to define the chicken TCR gamma locus. A splenic cDNA library was screened with two polymerase chain reaction products obtained from genomic DNA using primers for highly conserved regions of TCR and immunoglobulin genes. This strategy yielded cDNA clones with characteristics of mammalian TCR gamma chains, including canonical residues considered important for proper folding and stability. Northern blot analysis with the TCR gamma cDNA probe revealed 1.9-kb transcripts in the thymus, spleen, and a gamma delta T-cell line, but not in B or alpha beta T-cell lines. Three multimember V gamma subfamilies, three J gamma gene segments, and a single constant region C gamma gene were identified in the avian TCR gamma locus. Members of each of the three V gamma subfamilies were found to undergo rearrangement in parallel during the first wave of thymocyte development. TCR gamma repertoire diversification was initiated on embryonic day 10 by an apparently random pattern of V-J gamma recombination, nuclease activity, and P-and N-nucleotide additions to generate a diverse repertoire of avian TCR gamma genes early in ontogeny.

Figure 1

Nucleotide and predicted amino acid sequence of 1.9-kb TCRγ G186cDNA (GenBank accession no. U22666U22666). Boundaries between the leader peptide (L), variable (V), junctional (J), extracellular (Cex), transmembrane (TM), and cytoplasmic (Cyt) regions are indicated by arrow heads. Boundary and canonical residues are numbered according to mammalian Vγ gene segment convention (6, 7). The 51-residue leader peptide was predicted by the psort program (http://psort.nibb.ac.jp) based on the criteria of von Heijne (19). Putative CDR1 and CDR2 regions are indicated. Asp₁₁₇ is the first position assigned to the Cγ region, although in TCR γ-chains using Jγ3 the aspartic acid is replaced by an asparagine (Fig. 3B). Residues conserved in mammalian Cγ genes are underlined. One potential N-glycosylation site (★) is found at position 237. A consensus polyadenylylation sequence is boldfaced.

Figure 2

Northern blot analysis of different tissues and cell lines with the candidate TCR Cγ cDNA probe. RNA (10 μg) from liver (L), thymus (T), spleen (S), blood lymphocytes (Bl), intestinal cecal tonsils (I), bursa (Bu), DT40 B cell line (B), 857–7 γδ cell line (γδ), and CU15 αβ cell line (αβ) was electrophoresed in agarose, blotted on nylon membrane, and sequentially hybridized with ³²P-labeled Cγ (G186cDNA origin) and glyceraldehyde-3-phosphate dehydrogenase probes.

Figure 3

TCR Vγ and Jγ nucleotide and amino acid sequences. (A) Comparison of amino acid sequences of Vγ1, Vγ2, and Vγ3 subfamily members characterized according to sequence data of genomic gene segments, cDNA clones from the cDNA library, and from anchored-PCR or specific PCR. Residues identical to the prototypic sequence are indicated by dashes. Vγ1.1b, Vγ1.3b, and Vγ2.5 are partial amino sequences. (B) Nucleotide sequences of Vγ–Jγ junctions. Vγ usage is indicated on the left and Jγ usage on the right. Vγ, Jγ1, and Jγ2 gene segment assignment is based on germ-line sequences. Dashes indicate identity to the germ-line Vγ sequence. Assignment of nucleotides to the Jγ3 gene segment is based on a consensus sequence of TCR γ-chains using the Jγ3 segment. Nucleotides that cannot be assigned to either Vγ or Jγ gene segments are indicated as N- or P-additions. Putative P nucleotides are underlined. (C) Recombination signal sequences of Vγ1, Vγ2, Vγ3, Jγ1, and Jγ2 gene segments. Heptamer/nonamer sequences, indicated in boldface type, were identified by comparison to the consensus sequences of mammalian TCR/Ig genes (27). Positions matching the consensus sequences are underlined.

Figure 4

Southern blot analysis of chicken genomic DNA with TCRγ constant and variable region probes. DNA (15 μg) from bursa (Bu), spleen (S), and thymus (T) was digested with the PstI restriction enzyme, electrophoresed in agarose, and blotted onto nylon membranes. Hybridization was performed with the Cγ probe and, after stripping, with Vγ1, Vγ2, and Vγ3 probes. Sizes of λ HindIII DNA marker fragments are indicated on the left. Digestion with EcoRI, XbaI, and HindIII restriction enzymes gave similar results (data not shown).

Figure 5

Ontogenetic pattern of Vγ and Vβ expression in the thymus. Thymi were collected from individual embryos at 10 to 18 days of incubation. RNA was extracted from homogenized tissues and first-strand cDNA was synthesized. PCR assays were conducted with relevant primers to detect TCR Cγ, Vγ1-Cγ, Vγ2-Cγ, Vγ3-Cγ, Vβ1-Cβ, and Vβ2-Cβ transcripts. PCR products were detected after agarose gel electrophoresis and staining with ethidium bromide.