PD-L1 blockade increases Treg cell proliferation but not its suppressive capacity per cell.

In panels A to E, PBMC from HIV-infected individuals were stimulated with Gag peptides for 6 days in the presence of a PD-L1 blocking antibody or an isotype control antibody. (A) Flow cytometry histograms from an HIV-infected individual with 2000 viral RNA copies/mL blood showing CFSE dilution on Treg (including eTreg and rTreg), CD4- and CD8- T cells in presence of PD-L1 blocking antibody (black line) or isotype control antibody (grey line). In panels B, C and E, each dot represents the result from one individual. FC (fold change) is calculated as the ratio between PD-L1 blockade conditions and isotype control conditions. The dashed line in panels B and E (FC = 1) indicates no change due to PD-L1 blockade. The mean is shown. (B) Fold changes in the proliferation of Treg (black circles), CD4 (empty squares) and CD8 (empty triangles) cell populations were determined by CFSE assay. (C) Correlation between the fold change in proliferating Treg cells and HIV viral load. (D) Representative flow cytometry dot plots showing gating of CD39, CTLA4 and Helios on Treg cells for one individual (eTreg cells in red and rTreg cells in black). Numbers indicate the frequencies of CD39, CTLA4 or Helios expressing Treg cells (including eTreg and rTreg). (E) Fold change in the frequencies of CD39, CTLA4 or Helios expressing Treg cells. (F) PBMC from HIV-infected individuals were stimulated with Gag peptides in the presence of a PD-L1 blocking antibody or an isotype control antibody, after 6 days Treg cells were isolated and co-cultured with CFSE-labelled-PBMC at different PBMC-to-Treg cell ratios. The percentage of inhibition of CD8 proliferation after anti-CD3/anti-CD28 T cell stimulation is shown as a function of different PBMC-to-Treg cell ratios. Black dots connected with a solid line correspond to suppression assays performed with Treg cells from PD-L1 blockade conditions whereas empty dots connected with a dashed line correspond to suppression assays performed with Treg cells from isotype control conditions. Mean and SEM from 4 independent experiments are indicated.