Figure 2. CD8 Tregs regulate CD4 T cell expansion in vitro and in vivo.

(A) CD8 Tregs form an immune synapse when contacting CD4 T cells. CD8 Tregs were labeled with CellTracker Red, mixed with naive CD4 T cells (1:1 ratio), and stimulated with anti-CD3/CD28 beads. Cells were fixed, permeabilized, stained with Alexa Fluor 488–labeled anti-CD3 antibody, and analyzed by microscopy. Two representative samples are shown. Scale bar: 5 μm. (B and C) The suppressive effect of CD8 Tregs is long lasting. Naive CD4 T cells were CFSE labeled and incubated with or without CD8 Tregs (1:1 ratio) for 30 minutes. CD8 Tregs were removed with magnetic beads, CD4 T cells were stimulated with anti-CD3/CD28 beads for 4 days, and proliferation was analyzed by assessing CFSE dilution with flow cytometry. Data are mean ± SD from 3 independent experiments. (D–F) CD8 Tregs were adoptively transferred into NSG mice that were reconstituted with naive CD4 T cells and monocytes. Proliferative expansion of CD4 T cells was quantified after 9 days by enumerating the frequency and number of human CD4 T cells in the murine spleen by flow cytometry. CD4 and CD8 T cells were derived from the same human donor. Results from 5 independent experiments examining 5 different donors are shown as mean ± SD. (G–I) NSG mice were reconstituted with CD4 T cells, monocytes, and CD8 Tregs, as above. CD4 T cells were CFSE labeled prior to the transfer. Proliferation of CD4 T cells was assessed by flow cytometric analysis of CFSE dilution in splenocytes harvested after 9 days. A representative CFSE dilution curve is shown, and results from 5 independent experiments are summarized as mean ± SD of division index and proliferation index. Unpaired 2-tailed Student’s t test was used for comparisons.