Figure 3. Combined therapy with T_reg and rapamycin inhibits CD4⁺ and CD8⁺ T-cell proliferation and potentiates apoptosis of CD4+ lymphocytes in vitro.

CFSE-labeled PBMC (10⁵ per well) have been incubated with anti-CD3/anti-CD28 beads (cells:bead ratio 5:1) in the presence or absence of 10 nM rapamycin and/or 10⁴ ex vivo expanded CD127^lo T_reg cells per well. (A) Representative plots depicting CFSE dilution in CD4⁺ and CD8⁺ lymphocytes after 5 day of culture. The numbers represent percentage of undivided CFSE^hi cells in the gated populations. (B) Percentage of undivided CFSE^hi lymphocytes of the total lymphocyte population, as shown in A. (C) Representative plots demonstrating the binding of the apoptosis marker Annexin V to CD4⁺ and CD8⁺ T cells. The numbers represent percentage of AnnexinV⁺ cells of the total lymphocyte population. (D) Absolute numbers and percentage of AnnexinV⁺ apoptotic cells within the CD4⁺ and CD8⁺ gates as demonstrated in panel C.