Highly diverse TCR δ chain repertoire in bovine tissues due to the use of up to four D segments per δ chain

Tissue-specific distribution of γδ TCRs with limited TCR diversity is a common phenomenon in species with a low percentage of γδ T cells like humans and mice. We set out to investigate whether this is also the case in cattle (Bos taurus), a species with high percentages of γδ T cells. Using a method that was independent of variable (V) segment-specific primers, we generated 65 unique TCR δ chain sequences. We found no evidence for preferential use of certain Vδ segments in lymph node, skin, spleen, small intestine, large intestine, and blood. The δ chain CDR3 length distribution was very wide in each tissue, which was confirmed by spectratyping. The highly variable CDR3 length was due to the use of up to four diversity (D) segments by one bovine δ chain. Human and murine δ chains contain only one or two D segments. The five functional Dδ segments that we describe here were identified at cDNA and genomic level, and are the first ruminant D segments described. Fourteen TCR δ chain sequences used novel Vδ1 segments, and one expressed a novel member of the Vδ3 family. The number of known functional Vδ segments in cattle including these new ones is 42 now, but the total number may be much higher. A high number of Vδ segments in combination with the use of up to four out of five D segments, and the possibility of using non-template encoded (N) nucleotides on either side of these, makes the potential bovine δ chain repertoire much bigger than any known TCR chain. This situation is quite different from the situation in humans and mice, and suggests that the differences between γδ high and γδ low species in distribution, diversity, and function of γδ T cells may be substantial.