γδ T cells and αβ T cells are present together in all but the most primitive vertebrates. In most adult animals, αβ T cells are the predominant T-cell population and also perform many of the well-defined functions attributed to T cells. Nevertheless, in experimental systems where αβ T cell and/or γδ T cell deficient mice are infected with pathogens, the absence of both T-lymphocyte populations generally results in a more severe infection. In particular, γδ T cell-deficient mice usually fare worse in neutrophil-dominated inflammatory responses, heat-, ozone- or chlorine-induced injuries and in bacterial infections (Nocardia asteroids, Klebsiella pneumonia
) (King et al., 1999
; Koohsari et al., 2007
; Moore et al., 2000
; Toth et al., 2004
). In these cases, fewer infiltrating neutrophils, increased bacterial load, early dissemination and higher mortality rates are noted. Furthermore, patients with bacterial, parasitic and viral infections often have increased numbers of γδ T cells in the peripheral blood (from <5% in healthy individuals to >45% in patients) (De Paoli et al., 1990
; Ho et al., 1990
; Jason et al., 2000
). It was also reported that γδ T cells are over-represented among infiltrating T cells in the early but not in the late lesions of MS patients (up to 20-30% of the total number of T cells) (Wucherpfennig et al., 1992
). These observations suggest that γδ T cells play a unique role in the initial host response to tissue damage and infection. However, it is unclear why and how γδ T cells are preferentially suited for this task.
γδ T cells, like αβ T cells, develop in the thymus before entering the periphery. In the case of αβ T cells, thymic development entails ligand driven positive and negative selection, which determine what αβ T cell can recognize (Huseby et al., 2005
; Van Laethem et al., 2007
); and whether these T cells will develop into CD4+
helper or CD8+
cytolytic T cells. Thus, understanding γδ T cell selection in the thymus could provide valuable clues as to their likely targets and function.
Previous analysis of the role of thymic selection in the establishment of a functional γδ T cell repertoire has focused mostly on the studies of KN6 and G8 γδ TCR transgenic mice. KN6 and G8 are two independently derived γδ T cell clones that recognize the same closely related, β2
m-associated non-classical MHC class I molecules, T10 and T22 (Ito et al., 1990
; Schild et al., 1994
; Weintraub et al., 1994
). In both systems, transgenic mice were crossed to the C57BL/6 (B6) background, which express both the inducible T10 and constitutively expressed T22; BALB/c mice which only express T10, or to the β2
m-/- background which do not have cell surface T10/T22 expression. It was reported that in C57BL/6 mice, there were significantly lower numbers of or no transgenic T cells in the spleens of BALB/c mice. There were also fewer G8 γδ thymocytes (Dent et al., 1990
). While KN6 thymocytes were present, their ability to secrete IL-2/IL-4 and to proliferate was much reduced (Bonneville et al., 1990
). When G8 and KN6 transgenic T cells were expressed in β2
m-/- mice (B6 background), there were fewer transgenic cells in the periphery and transgenic thymocytes showed a reduced ability to secrete cytokine and proliferate when stimulated in vitro
(Pereira et al., 1992
; Wells et al., 1991
). Based on these observations, it was concluded γδ T cells, similar to αβ T cells, undergo ligand driven positive and negative selection in the thymus. However, analyzing the same G8 transgenic mice, Schweighoffer and Fowlkes found that G8 T cells were able to mature in β2
m-/- mice, contradicting the conclusion that positive selection is required (Schweighoffer and Fowlkes, 1996
In addition to the KN6 and G8 transgenic systems, the role of ligand recognition in the development of murine skin-dendritic epidermal T cells (DETCs) has also been analyzed. These γδ T cells express the same TCR and are the first to appear during fetal thymic development (Havran and Allison, 1988
). While the ligand of these cells has yet to be identified, DETCs are reactive to keratinocytes in a TCR dependent manner (Havran et al., 1991
). Here, all experimental results suggest that encountering thymic ligand is necessary for DETCs to migrate to the skin and to acquire their ability to react to keratinocytes (Lewis et al., 2006
; Mallick-Wood et al., 1998
; Xiong et al., 2004
Previously, we found that a sizable population (0.1-1%) of γδ T cells in normal un-immunized mice recognize T10/T22 (Crowley et al., 2000
). Surprisingly, a comparable frequency of T10/T22-specific γδ T cells was also found in β2
m-/- mice (Crowley, 1998
). Moreover, in analyzing the antigen recognition determinant of T10/T22-specific γδ T cells, we found that the T10/T22 specificity is largely encoded by amino acid residues on Vδ and Dδ gene elements which are brought together by rearrangement; and that 0.85% of the non-selected TCRδ sequences (from CD3ε-deficient murine thymocytes and from out-of-frame VDJ recombination events) contain the T10/T22 recognition motif (Shin et al., 2005
). This is within the observed range of T10/T22-specific γδ T cells in normal mice. Therefore, this repertoire seems to be determined largely by gene rearrangement instead of ligand dependent selection-observations, which present a significant departure from the analysis of T10/T22-specific γδ TCR transgenic mice.
If γδ T cells require no ligand driven positive or negative selection to develop, then the repertoire of γδ T cell antigens will be significantly enlarged to include pathogens, which do no cross-react to host thymic molecules, as well as infection- or stress-induced antigens which express in the thymus, such as T10/T22. Furthermore, although γδ T cells and αβ T cells secrete similar cytokines and mount cytolytic responses, there is very little information on how γδ T cell effector functions develop. Since we have developed a T22 tetrameric staining reagent (Crowley et al., 2000
), which allows us to follow and analyze this substantial population of T10/T22-specific γδ T cells in normal non-transgenic mice, we decided to re-evaluate these issues.
Here, we find that (1) encountering antigen in the thymus is neither required nor inhibitory for the development of T10/T22-specific γδ T cells, (2) self-dimerization of γδ TCRs may be sufficient to drive γδ thymocytes development, (3) a sizable number of the γδ T cells in normal mice are phenotypically and functionally similar to β2m-/- T10/T22-specific cells, suggesting that most γδ T cells in the periphery have yet to encounter antigen, and (4) when activated through the TCR, cells with prior antigen exposure produce IFNγ, while cells that develop in the absence of ligand make IL-17, a major initiator of inflammation and that is elicited without prior antigen. Indeed, we find that γδ T cells are major IL-17 producers in the draining lymph nodes after peptide/Complete Freund’s Adjuvant (CFA) immunization. These results suggest that a functional γδ T repertoire can be divided into two subsets, influenced by ligand recognition, and uniquely equipped to initiate and regulate the inflammatory response.