T cells in cryptopatch aggregates share TCR γ variable region junctional sequences with γδ cells in the small intestinal epithelium of mice

The role of cryptopatch aggregates in the development of intestinal intraepithelial lymphocytes (IEL) is a matter of controversy. Therefore, an important question is whether T cells in cryptopatch aggregates are lineally related to IEL. We hypothesized that if γδ⁺ IEL derive from T cells in cryptopatch aggregates, then a clonal relationship would exist between the two populations. To test this hypothesis, we compared the sequence of rearranged TCR gamma variable region 5 genes in γδ⁺ IEL and cryptopatch cells. We purified IEL by FACS and cryptopatch cells were isolated from frozen sections of the intestine by laser-assisted microdissection. PCR showed that TCR gamma variable region 5 was rearranged in γδ⁺ IEL and in CD3⁺ cryptopatch cells, but not in CD3^- cryptopatch cells. DNA sequence analysis showed that the frequency of in-frame junctions in cryptopatch aggregates was at a level consistent with positive selection in both wild-type and athymic nude mice. In addition, the predicted amino acid sequences of V-J junctions present in γδ⁺ IEL and cryptopatch cells were encoded by identical nucleotide sequences. By contrast, the frequency of in-frame joints was significantly reduced in cryptopatch cells isolated from TCR δ-deficient mice, indicating that the enrichment of in-frame joints in cryptopatch cells must normally depend on expression of surface γδ TCR. Our results are consistent with the hypothesis that a subset of γδ⁺ IEL are related to T cells in cryptopatch aggregates. The precise role of cryptopatch aggregates in intestinal γδ⁺ T cell homeostasis still needs to be determined.