Human CD4
+Foxp3
+ Treg cells are composed of three phenotypically and functionally distinct subpopulations: CD25
highCD45RA
− active Tregs, CD25
lowCD45RA
+ resting Tregs, and CD25
lowCD45RA
− nonsuppressive T cells.
13 The combination of CD25 and CD45RA staining of CD4
+ RPE-induced Tregs revealed five subpopulations, two of which, Gate 1 (G1) and Gate 2 (G2), are shown in
Figure 4A. Gate 1 includes CD25
highCD45RA
− active Tregs. RPE-induced Tregs included significant numbers of these active Tregs, compared with control T cells without RPE supernatants (middle histogram) and naïve T cells (left histogram). On the other hand, both RPE-induced Tregs and control T cells included CD25
lowCD45RA
− nonsuppressive T cells (Gate 2 in
Fig. 4A). Next, we assessed whether CD25
highCD45RA
− active RPE-induced Tregs express Foxp3 and CD152 (CTLA-4) and produce inflammatory cytokines (IFN-γ and IL-17). By using the sorting system, CD25
highCD45RA
− active Tregs (Gate 1) and CD25
lowCD45RA
− nonsuppressive T cells (control: Gate 2) were separated from human RPE–induced Tregs. RPE-induced active Tregs (CD25
highCD45RA
−) greatly expressed Foxp3 and CD152 as compared with RPE-induced CD25
lowCD45RA
− nonsuppressive T cells (
Fig. 4B). As expected, RPE-induced CD25
lowCD45RA
− nonsuppressive T cells also expressed these molecules, but in lower amounts. Importantly, RPE-induced active Tregs produced less IFN-γ and IL-17, whereas CD25
lowCD45RA
− nonsuppressive T cells produced significantly higher levels of these cytokines (
Fig. 4B). Based on the mean fluorescence intensity determined by flow cytometric analysis, active Tregs (CD25
highCD45RA
−) expressed high levels of Foxp3 and CD152 as compared with CD25
lowCD45RA
− nonsuppressive T cells (
Fig. 4C). These results suggest that separated active Tregs do not include inflammatory cytokine–secreting nonsuppressive T cells and significantly express Treg cell markers.