A modified flow cytometry method for objective estimation of human CD4+ regulatory T cells (CD4+ Tregs) in peripheral blood, via CD4/CD25/CD45RO/FoxP3 labeling.

Abstract

Several methods exist for flow-cytometric estimation of human peripheral blood CD4+ T regulatory cells (CD4+ Tregs). We report our experience with the estimation of human CD4+ Tregs via three different characterizations using flow cytometry (CD25high FoxP3+ , CD25high CD127low/- FoxP3+ , and CD4+ CD25high/int CD45ROFoxP3+ ) in normal subjects. We have used these methods on the control populations from two studies (32 and 36 subjects, respectively), the latter two methods retrospectively on the subjects of the first study. The six CD4+ T cell fractions obtained by the third method were differentially colored to ascertain the distribution of these cell fractions in the CD25/FoxP3, CD45RO/FoxP3, and CD25/CD127 dot plots from CD4/CD25/CD45RO/FoxP3 and CD4/CD25/CD45RO/CD127 panels. Each approach gives significantly different estimates of Tregs (expressed as percentage of CD4+ T cells), with the second almost invariably yielding higher percentages than the other two. Only the third approach can distinguish among effector and naïve Tregs and FoxP3+ non-Tregs. Analysis of CD25/CD127 dot plots reveals that Treg delineation via the widely used definition of CD4+ CD25high CD127low/- cells unavoidably yields a mixture of nearly all effector and most of naïve Tregs, as well as FoxP3+ non-Tregs plus other cells. Delineation of effector/naïve Tregs and FoxP3+ non-Tregs is possible via CD45RO/CD25 dot plots but not by CD45RO/FoxP3 counterparts (as done previously) because of overlapping FoxP3 intensities among Tregs and non-Tregs. Our comparison shows that CD4/CD25/CD45RO/FoxP3 panels are an objective means of estimating effector and naïve Tregs via colored dot plots, aiding thus in Treg delineation in health and detecting aberrations in disease.