Multicolor flow cytometric analysis of CD4+ T-cells for CD25 and Foxp3 expression in human Prostate Cancer (50.21)

Abstract

Abstract CD4+CD25+ T regulatory cells (Treg) are postulated to regulate immune responses against self-antigen, infectious agents, tumor antigens and transplantation antigens. The objective of the study is to examine whether PSA146–154 peptide vaccination impacts the frequency and/or phenotype of Treg in peripheral blood mononuclear cells (PBMC) of patients with prostate cancer (CaP). Frozen PBMC were rapidly thawed, enumerated, checked for viability and directly stained with fluorescent labeled antibodies to CD3, CD4, CD8, CD25 and Foxp3 and analyzed on a flow cytometer. Based on morphology, gates were set to include, (a) whole PBMC population but exclude debris and clumps or, (b) predominantly large lymphocyte population and then back gated on CD3+CD4+ T-cells. Quadrant analysis demonstrated the presence of 1–5% of CD4+CD25+Foxp3−, less than 1% of CD4+CD25−Foxp3+ and CD4+CD25+Foxp3+ cells in the whole PBMC population. While, 10–44% of CD4+CD25−Foxp3+, 1–2% of CD4+CD25+Foxp3−, 1–3% of CD4+CD25+Foxp3+ cells were observed in the large lymphocyte population. However, there was no significant difference between before and post vaccine samples. CD4+CD25−Foxp3+ cells may represent a distinct T-cell subset and warrants further investigation. Funding: The Illinois Department of Public Health (IDPH# 4328301)