PMC

Purity of isolated AMs. AMs were isolated from BAL fluids of immunosuppressed, uninfected mice (AMs/L3T4) and Pneumocystis-infected mice (AMs/PcP) using biotin-anti-mouse CD11c antibody and anti-biotin magnetic microbeads. An aliquot of the purified cells was cytospun on a slide; the cells were stained with Giemsa stain and examined by microscopy.

Reduced phagocytic activity in AMs incubated with MDSCs. AMs cocultured with control Gr1BM cells (A) or MDSCs (B) overnight were incubated with fluorescein-conjugated zymosan beads for 1 h. The nuclei of AMs were counterstained with DAPI. (C) The number of zymosan beads phagocytosed by AMs incubated with MDSCs or Gr1BM cells were counted under a confocal microscope. Data are presented as means ± SD from three independent experiments.

Increased PD-L1 expression in MDSCs from PcP mice. MDSCs (MDSCs/PcP) were isolated from PcP mice at 5 weeks post-Pneumocystis infection. Control Gr1BM cells were isolated from uninfected mice immunosuppressed by weekly injection of anti-CD4 (L3T4) antibody. (A) Total RNA was isolated from the cells, and PD-L1 gene expression was determined by real-time RT-PCR. The average PD-L1 expression level in Gr1BM cells was set as 1, and that in MDSCs was compared to it. Data are presented as means ± SD from three independent experiments. (B) The cells were examined by flow cytometry using anti-Gr-1 and anti-PD-L1 antibodies. The result shown is representative of three independent experiments.

Increased DNA methylation of PU.1 promoter in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM, AM genomic DNA was isolated and assessed for CpG methylation by digestion with methylation-dependent and methylation-sensitive restriction enzymes using the EpiTect methyl II enzyme kit (Qiagen). Real-time PCR then was performed to amplify a 100-bp region of the PU.1 promoter. The resulting C_T values were entered into the data analysis spreadsheet of the kit to calculate the relative amount of methylated DNA in each sample. Data are presented as means ± SD from three independent experiments.

Decreased PU.1 expression in AMs incubated with MDSCs. AMs from uninfected mice were cocultured with MDSCs or Gr1BM cells at a ratio of 1:5 for 16 h in a 37°C incubator with 5% CO₂. After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, total RNA of AMs was isolated, and PU.1 mRNA levels were determined by real-time PCR. The level of PU.1 expression in AMs that were not incubated with MDSCs or Gr1BM cells was set as 1, and that in AMs incubated with either type of cell was compared to it. Data are presented as means ± SD from three independent experiments.

Increased PD-1 expression in AMs incubated with MDSDs. AMs isolated from uninfected mice were cocultured with MDSCs or Gr1BM cells at a ratio of 1:5 for 16 h in a 37°C incubator with 5% CO₂. (A) After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, total RNA was isolated from AMs of each group. PD-1 gene expression was determined by real-time RT-PCR. The level of AMs/Gr1BM was set as 1, and that in AMs/MDSCs was compared to it. Data are presented as means ± SD from three independent experiments. (B) AMs incubated with MDSCs or Gr1BM cells were analyzed for surface PD-1 expression by flow cytometry.

Increased histone deacetylation of PU.1 gene in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, the AMs were treated with 0.1% formaldehyde to cross-link histone proteins to DNA, lysed, and sonicated to generated chromatin fragments. Chromatin immunoprecipitation was performed using anti-H3K4me3, anti-H3ac, and anti-H3K27me3 antibodies in separate reactions. DNA in the precipitated chromatin was isolated and used as the template for real-time PCR to amplify the 3′URE, 5′URE, and promoter regions of the PU.1 gene. The C_T values obtained were used to determine the ratios of percent input of H3K4me3 to H3K27me3 and H3Ac to H3K27me3. Data are presented as means ± SD from three independent experiments.

Loss of suppressive effect of MDSCs pretreated with anti-PD-L1 antibody on AMs. A total of 1 × 10⁵ AMs were cocultured with 5 × 10⁵ MDSCs that were treated with anti-PD-L1 antibody or control IgG. (A) After 16 h of incubation, AMs were isolated, and the PU.1 mRNA levels were determined by real-time RT-PCR. Data are presented as means ± SD from three independent experiments. The level of PU.1 expression in AMs that were not incubated with MDSCs or Gr1BM cells was set as 1, and that in AMs incubated with MDSCs that were pretreated with or without anti-PD-L1 antibody was compared to it. (B) Phagocytosis was assayed, and the number of zymosan beads phagocytosed by AMs incubated with MDSCs pretreated with anti-PD-L1 antibody or control IgG was counted under a confocal microscope.

Increased PD-1 expression in AMs from PcP mice. AMs (AMs/PcP) were isolated from PcP mice at 5 weeks post-Pneumocystis infection. Control AMs (AMs/L3T4) were from uninfected mice immunosuppressed by weekly injection of anti-CD4 (L3T4) antibody. (A) Total RNA was isolated from AMs, and PD-1 gene expression was determined by real-time RT-PCR. The average PD-1 expression level in AMs/L3T4 was set as 1, and that in AMs/PcP was compared to it. Data are presented as means ± SD from three independent experiments. (B) The AMs were examined by flow cytometry using anti-CD11c (Alexa Flour 647 conjugated), rat IgG isotype control (phycoerythrin [PE] conjugated), and anti-PD-1 (phycoerythrin conjugated) antibodies. The result shown is representative of three independent experiments. (C) PD-1 expression in AMs from PcP mice was further confirmed by Western blotting using anti-PD-1 antibody. The expression of GAPDH was examined similarly as a loading control.