Phenotypic analysis and gamma delta-T cell receptor repertoire of murine T cells associated with the vaginal epithelium.

T cells expressing alpha beta- and gamma delta-TCR are associated with the murine vaginal epithelium. A combination of phenotypic analysis of cell surface Ag and molecular analysis of the gamma- and delta-genes was used to demonstrate that vaginal gamma delta-T cells have several features that distinguish them from gamma delta-T cells present in other tissues. Three color flow cytofluorometric analysis demonstrated that freshly isolated vaginal gamma delta-T cells are CD4-CD8- and their expression of Thy-1 is strain dependent. Furthermore, specific differences in CD5, CD28, and p55IL-2 receptor expression were found between alpha beta- and gamma delta-T cells isolated from vaginal tissue. The vaginal gamma delta-T cells are predominantly CD45+, pgp-1+, HSA-, Ly-6C-, and MEL-14-. Both alpha beta- and gamma delta-T cells were absent in vaginal tissue from nude mice, although Thy-1-positive cells are present. It was also demonstrated that V gamma 4 and V delta 1 are the only gamma- and delta-genes that are expressed by vaginal cells. Sequence analysis of their junctions revealed that they express the V gamma 4 and V delta 1 sequences also found in fetal thymocytes. Furthermore, the V delta 1 sequence is identical in the vaginal cells and the gamma delta-T cells from the epidermal epithelium. We conclude that the vagina, like the skin, is a site where gamma delta-T cells with an invariant TCR exist.     

T cell receptor gamma and delta gene rearrangements in scid thymocytes. Similarity to those in normal thymocytes.

The nature of TCR gamma and delta gene rearrangements in 4- to 6-week-old scid thymocytes was examined by using the polymerase chain reaction technique, cloning, and DNA sequencing. Analysis of 78 sequences indicates that TCR gamma and delta gene rearrangements in scid mice generally resemble those in thymocytes from normal young adult mice. V gamma 1, V gamma 2, and V gamma 5 rearrangements are heterogeneous, with extensive N region addition and nucleotide excision from the recombining coding segments. In addition, homogeneous and fetal-like V gamma 3, V gamma 4, and V delta 1 rearrangements are observed. These rearrangements are currently difficult to interpret but may be significant with respect to whether certain homogeneous joints in normal mice are due to cellular selection or to the rearrangement process. scid TCR gamma and delta gene nucleotide sequences also reveal direct V-J delta joining, inter-(V-J-C gamma) cluster joining, and the possibility of inversional rearrangement at the gamma locus. Short sequence homologies may contribute to V(D)J recombination and to the rescue of blocked coding joints.     

Preferential activation of an IL-2 regulatory sequence transgene in TCR gamma delta and NKT cells: subset-specific differences in IL-2 regulation

A transgene with 8.4-kb of regulatory sequence from the murine IL-2 gene drives consistent expression of a green fluorescent protein (GFP) reporter gene in all cell types that normally express IL-2. However, quantitative analysis of this expression shows that different T cell subsets within the same mouse show divergent abilities to express the transgene as compared with endogenous IL-2 genes. TCR gamma delta cells, as well as alpha beta TCR-NKT cells, exhibit higher in vivo transgene expression levels than TCR alpha beta cells. This deviates from patterns of normal IL-2 expression and from expression of an IL-2-GFP knock-in. Peripheral TCR gamma delta cells accumulate GFP RNA faster than endogenous IL-2 RNA upon stimulation, whereas TCR alpha beta cells express more IL-2 than GFP RNA. In TCR gamma delta cells, IL-2-producing cells are a subset of the GFP-expressing cells, whereas in TCR alpha beta cells, endogenous IL-2 is more likely to be expressed without GFP. These results are seen in multiple independent transgenic lines and thus reflect functional properties of the transgene sequences, rather than copy number or integration site effects. The high ratio of GFP: endogenous IL-2 gene expression in transgenic TCR gamma delta cells may be explained by subset-specific IL-2 gene regulatory elements mapping outside of the 8.4-kb transgene regulatory sequence, as well as accelerated kinetics of endogenous IL-2 RNA degradation in TCR gamma delta cells. The high levels and percentages of transgene expression in thymic and splenic TCR gamma delta and NKT cells, as well as skin TCR gamma delta-dendritic epidermal T cells, indicate that the IL-2-GFP-transgenic mice may provide valuable tracers for detecting developmental and activation events in these lineages.