γδ T cells are a major subpopulation of circulating T cells in the pig, but their function is still poorly understood. The transcription factors GATA-3, T-bet and Eomesodermin (Eomes) play important roles in T-cell development and functional differentiation. However, their expression has not been studied in porcine γδ T cells yet. By using multi-color flow cytometry we investigated protein expression of these transcription factors in γδ thymocytes and mature γδ T cells as well as sorted and in vitro stimulated γδ T-cell subpopulations.
GATA-3 expression was found in the vast majority of γδ thymocytes. Minor populations of CD2+ γδ thymocytes expressed Eomes and T-bet and the latter was associated with a CD8αhigh phenotype. Similarly, extra-thymic CD2- γδ T cells expressed high levels of GATA-3. In contrast, T-bet expression was restricted to a subpopulation of CD2+ γδ T cells with a uniform CD8αhighCD27dim/-perforin+ phenotype across various analysed organs, including blood, spleen and lung tissue. Eomes+ γδ T cells were also only present among CD2+ cells but had mixed phenotypes for CD8α, CD27 and perforin. Moreover, Eomes+ γδ T cells were strongly enriched in the spleen compared to other analyzed organs. In vitro stimulation experiments with sorted CD2- γδ T cells revealed that following stimulation with ConA, IL-2, IL-12 and IL-18 these cells partially start to express CD2 and T-bet but keep their initial GATA-3 expression. Moreover, first experiments on cytokine production in the supernatants of these cultures indicate IFN-γ production but absence of IL-4, despite high levels of GATA-3 expression.
In conclusion, our data suggest a role for GATA-3 in γδ T cell development in the thymus and a type-1 differentiation of a subpopulation of CD2+ γδ T cells in extra-thymic locations. However, the relevance of the high expression of GATA-3 in extra-thymic CD2- γδ T cells requires further investigation.