Expression of T-Bet, Eomesodermin, and GATA-3 Correlates With Distinct Phenotypes and Functional Properties in Porcine γδ T Cells

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.

Intraepithelial T cells diverge by intestinal location as pigs age

“…CD3e was used to identify T cells, and further characterization of total T cells by expression of cell surface markers γδTCR, CD2, CD4, CD8α, and CD27 was assessed. CD2 and CD8α expression are commonly used to identify functional porcine γδ T cell populations (45, 46); CD4 and CD8α expression identify functional porcine αβ T cell populations (47,48); and CD27 expression is indicative of T cell activation and memory states (49)(50)(51)(52). In total, this gating strategy yielded 16 discrete, nonoverlapping IET subpopulations based on CD27 +/expression within each of the 8 defined IET populations ( Fig.…”

“…However, differing compositions and abundances of microbial species present in the large intestine compared to the small intestine may play a role in IET activation. Moreover, large intestinal IETs may be exposed to a different repertoire of soluble factors than IETs of the small intestine, such as microbially-derived components including lipopolysaccharide (LPS) or microbially-constructed metabolite products including short-chain fatty acids (SCFAs).In peripheral blood and non-intestinal tissues, the presence or absence of CD8α expression within porcine CD2 + γδ T cells is associated with different functional states.CD2 + CD8α + γδ T cells exhibit greater expression of the effector T cell transcription factor T-bet(52), greater cytokine production (45), and expression patterns of cell surface molecules indicative of T cell activation(29,49) when compared to CD2 + CD8αγδ T cells. Resultingly, CD2 + CD8αγδ T cells are a proposed naïve or memory cell population, while CD2 + CD8α + γδ T cells are proposed to be a terminally differentiated population (45, 52).…”

NK and T Cell Differentiation at the Maternal-Fetal Interface in Sows During Late Gestation

“…Pigs are one of the species, including cattle, sheep, and chicken, where γδ T cells represent a substantial proportion of the total T cell population in the blood circulation and secondary lymphoid organs (44,55). When considering the expression of CD2, a positive and negative γδ T cell subset can be identified and it was suggested that these are two different lineages that are already established in the thymus (44,56).…”

“…Pigs are one of the species, including cattle, sheep, and chicken, where γδ T cells represent a substantial proportion of the total T cell population in the blood circulation and secondary lymphoid organs (44,55). When considering the expression of CD2, a positive and negative γδ T cell subset can be identified and it was suggested that these are two different lineages that are already established in the thymus (44,56). Based on the role of transcription factors involved in T cell polarization, a recent study addressing the expression of GATA-3 and T-bet in porcine γδ T cells revealed striking differences between the two lineages (44,49).…”

“…3 the mean frequency was 83.2% and for the other two sows this was lower than 60%. This difference might be attributed to a combination of animal-to-animal variation and the age (44). In addition, it should be noted that in the fetal compartments, including FP and fSpln, most CD2 − γδ T cells did not express CD8α (Figure 5C).…”