Abstract
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 CD8αhighCD27dim/−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.
Highlights
Research in murine models has shown that various transcription factors (TFs) are relevant for the development of distinct immune cell lineages and functional subsets by activating or repressing their target genes, frequently alongside the action of particular cytokines
Since γδ T cells in swine have been divided into three subsets on the basis of their CD2/CD8α expression, including CD2−CD8α−, CD2+CD8α+, and CD2+CD8α− [31, 32], we analyzed expression of these two molecules in total γδ thymocytes, T-bet+, Eomes+ or GATA3+ γδ thymocytes (Figure 1A, third row)
Similar to what we previously found for TCR-αβ thymocytes [28], these data suggest that GATA-3 is of relevance in the maturation process of porcine γδ thymocytes
Summary
Research in murine models has shown that various transcription factors (TFs) are relevant for the development of distinct immune cell lineages and functional subsets by activating or repressing their target genes, frequently alongside the action of particular cytokines. The TF GATA-3 is well-known for its function as a master regulator of Th2 responses [17] This TF is involved in T-cell lineage commitment [18], having a pivotal role already during the transition of thymus-seeding progenitor cells into early T-lineage progenitors [19]. Not much is known about the relevance of GATA-3 in mature γδ T cells; an early study with murine γδ T cells from the spleen could identify GATA-3 expression only after in vitro stimulation with IL-4 [16] Despite these findings, to our knowledge the expression of GATA-3, T-bet and Eomes has not been investigated in porcine γδ T cells. This suggests that CD2− γδ T cells differ substantially from other γδ T-cell subsets, their functional properties still await a thorough investigation
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