Unlike mice and humans, porcine γδ T cells represent a prominent subset of T cells in blood and secondary lymphatic organs. GATA-3, T-bet and Eomesodermin (Eomes) are transcription factors with crucial functions in T-cell development and functional differentiation, but their expression has not been investigated in porcine γδ T cells so far. We analyzed the expression of these transcription factors in γδ thymocytes, mature γδ T cells from blood, spleen, lymph nodes, and lung tissue as well as in vitro stimulated γδ T cells on the protein level by flow cytometry. GATA-3 was present in more than 80% of all γδ-thymocytes. Extra-thymic CD2^- γδ T cells expressed high levels of GATA-3 in all investigated organs and had a CD8α^-/dimCD27⁺perforin^- phenotype. T-bet expression was mainly found in a subset of CD2⁺ γδ T cells with an opposing CD8α^highCD27^dim/-perforin⁺ phenotype. Eomes⁺ γδ T cells were also found within CD2⁺ γδ T cells but were heterogeneous in regard to expression of CD8α, CD27, and perforin. Eomes⁺ γδ T cells frequently co-expressed T-bet and dominated in the spleen. During aging, CD2^-GATA-3⁺ γδ T cells strongly prevailed in young pigs up to an age of about 2 years but declined in older animals where CD2⁺T-bet⁺ γδ T cells became more prominent. Despite high GATA-3 expression levels, IL-4 production could not be found in γδ T cells by intracellular cytokine staining. Experiments with sorted and ConA + IL-2 + IL-12 + IL-18-stimulated CD2^- γδ T cells showed that proliferating cells start expressing CD2 and T-bet, produce IFN-γ, but retain GATA-3 expression. In summary, our data suggest a role for GATA-3 in the development of γδ-thymocytes and in the function of peripheral CD2^-CD8α^-/dimCD27⁺perforin^- γδ T cells. In contrast, T-bet expression appears to be restricted to terminal differentiation stages of CD2⁺ γδ T cells, frequently coinciding with perforin expression. The functional relevance of high GATA-3 expression levels in extra-thymic CD2^- γδ T cells awaits further clarification. However, their unique phenotype suggests that they represent a thymus-derived separate lineage of γδ T cells in the pig for which currently no direct counterpart in rodents or humans has been described.

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