Figure 6.

Blockade of the PD-1/PD-L1 signaling pathway can augment Mtb-specific IFN-γ secretion, but not CD4 T cell proliferative capacity. Proliferation assays were performed using freshly isolated PBMC labeled with Oregon Green (OG) and incubated for 6 days under the following conditions: media alone (negative control), CFP-10/ESAT-6 peptide pool, and SEB (positive control). Proliferation assays were performed in the presence of either anti-PD-L1 blocking Ab, or an IgG2b isotype-matched control Ab (n = 13 LTBI; n = 13 smear⁺ TB). (A) Flow cytometry data indicating the frequency of proliferating CD4 T cells following stimulation with CFP-10/ESAT-6 peptide pool, in the presence of anti-PD-L1 Ab or IgG2b isotype control Ab. Dot plots are shown gated on viable CD3⁺CD4⁺ lymphocytes from an individual with LTBI (left) and a patient with smear⁺ TB (right). (B,C) Comparison of the frequency of proliferating (OG^lo) CD4 T cells stimulated with CFP-10/ESAT-6 peptide pool, in the presence of anti-PD-L1 Ab or IgG2b isotype control Ab. (D,E) Levels of IFN-γ in cell culture supernatants of PBMC stimulated with CFP-10/ESAT-6 peptide pool for 6 days in the presence of anti-PD-L1 Ab or IgG2b isotype control Ab. Levels of IFN-γ were quantified by ELISA; data are shown after subtraction of background IFN-γ production in the negative control (media alone) conditions. Differences in proliferative capacity and IFN-γ secretion in the presence and absence of anti-PD-L1 Ab were determined using the Wilcoxon matched pairs signed rank test.