Sensitive detection of Foxp3 expression in bovine lymphocytes by flow cytometry

Abstract

The transcription factor forkhead-box p3 (Foxp3) has been designated as a master regulator for the function of regulatory T cells (Treg). Therefore, the identification of Foxp3 expression in T cells is indispensable for the study of Treg. However, studies on Foxp3 expression in bovine lymphocytes are still sparse, probably due to a lack of Foxp3-specific antibodies with reliable performance in flow cytometry. Our group recently demonstrated that a monoclonal antibody (FJK-16s) developed against murine Foxp3 also binds to porcine Foxp3 and performs well in flow cytometry. A protein sequence alignment of the binding region of the FJK-16s antibody revealed, that within this region the sequences of porcine and bovine Foxp3 are identical. Therefore, we tested this antibody for its suitability in flow cytometry with bovine peripheral blood mononuclear cells (PBMC). By using nonspecific isotype-matched antibodies and competition labeling with non-fluorescent FJK-16s antibodies as negative controls, we readily observed a specific staining of a small subpopulation of CD25 high lymphocytes within PBMC. Co-staining with monoclonal antibodies against CD3, CD4, CD8β and TCR-γδ revealed that all Foxp3 + cells co-expressed CD3, and were in their vast majority CD4 +. However, minor populations of Foxp3 +CD8β + and Foxp3 +TCR-γδ + lymphocytes could also be identified. In summary, our data demonstrate that the FJK-16s antibody is a valuable tool to promote the study of Foxp3 + T cells and their biological relevance in cattle.