FOXP3 and CD25 double staining antibody cocktails identify regulatory T cells in different types of tumor tissues using tissue microarrays.

Regulatory T cells (Tregs) are CD4+ T cells that express CD25 and transcription factor Forkhead box P3 (FOXP3), and Tregs play a central role in regulation of tumor immunity. FOXP3 immunohistochemistry has been widely used to study Tregs in paraffin embedded tissue, and flow cytometry using fresh tissue has been used to identify FOXP3+/CD25+ double positive Tregs. In our study, we validated the FOXP3/CD25 double staining antibody cocktails for detecting Tregs in paraffin-embedded tissue. Tissue microarrays (TMA) included 115 malignant tumors, 3 ovarian mucinous borderline tumors and 15 benign tissues. Digital image analysis was performed using ImageJ software. Our results showed that FOXP3/CD25 double positive lymphocytes, a subset of FOXP3+ lymphocytes, accounted for variable percentage of the total FOXP3+ lymphocytes and they were positively correlated with FOXP3+ cell counts. Tumors from different sites had variable FOXP3+/CD25+ and FOXP3+ lymphocyte counts. Tumors from lung, head & neck and colon had more and renal cell carcinoma had minimal FOXP3+/CD25+ and FOXP3+ lymphocytes. In conclusion, FOXP3/CD25 double staining antibody cocktails can be easily applied to paraffin-embedded tissue, and FOXP3+/CD25+ Tregs count was positively correlated with FOPX3+ Tregs count but they were not interchangeable. We recommend using CD25/FOXP3 double staining for studying Tregs in tumor tissue.