Detection of Foxp3 protein expression in porcine T lymphocytes

Regulatory T cells (Tregs) are potent regulators of various immune reactions. Due to the lack of Treg-specific markers their analysis had often been challenging until the discovery of the transcription factor Forkhead-box p3 (Foxp3) which serves as this highly demanded marker. So far, antibodies staining human and murine Foxp3 have been developed. This study describes the analysis of four commercially available anti-Foxp3 antibodies for reactivity with their specific antigen in cells derived from porcine lymphoid tissues. One out of the four antibodies showed selective reactivity with porcine CD25+ T lymphocytes. The intracellular antigen was expressed on a small subset of CD25dim cells and the majority of the CD25high positive T-cell subpopulation. Intracellular antigen positive cells showed a heterogeneous expression of other leukocyte differentiation antigens. The majority belonged to the CD4+CD8+ T-lymphocyte subpopulation, but were also found in the CD4+CD8− subpopulation. Another small minority was included in the CD4−CD8+ T-lymphocyte subpopulation. Additionally, a small fraction of the putative Foxp3+ cells showed an expression of MHC-II molecules. These staining patterns in three and four colour flow cytometry analyses indicated that the cells detected by a rat anti-mouse/rat-Foxp3 antibody expressed the porcine Foxp3. The expression of the putative Foxp3 protein in distinct leukocyte subsets was confirmed by molecular analysis of Foxp3 mRNA expression. Using Western blot analysis specific protein bands could only be detected in fractions that also exhibited the corresponding Foxp3 mRNA expression. These experiments also revealed that the antibody recognized a single chain protein with a molecular mass of about 45 kDA similar to Foxp3 described for other species. In summary, these data strongly indicate the reactivity of this antibody with porcine Foxp3. Thereby, this rat anti-mouse/rat Foxp3 antibody presents a powerful tool for the identification of porcine regulatory T cells.