Figure 4.

The CXCR2^Hi MDSC suppressed the proliferation of CD4⁺ T cells depending upon ARG2-arginase activities in vitro. (A) Experiment design to compare immunosuppressive functions of CXCR2^Low and CXCR2^Hi MDSC subsets. (B) Histogram overly exhibited CD4⁺/CD8⁺ T cell proliferation labeled by CFSE, as measured by flow cytometry. The ratios of co-culture numbers of MDSC and T cells were 1:1. (C) Quantitative bar charts showed the percentage of CD4⁺ and CD8⁺ T cell proliferation normalized to stimulated control. (D) The normalized percentage of CD4⁺ T cell proliferation at co-culture numbers of MDSC and T cells were 1:1, 1:2, and 1:4. (E) The abundance of Arg1 and Arg2 transcripts in PB and BM by bulk-RNA sequencing. (F) RT-PCR assay revealed remarkably high Arg2 expression and low Arg1 expression in CXCR2^Hi MDSC. Statistical significance was analyzed by unpaired t-test. (G) Western blotting assay showed extremely low protein content of ARG1 in both CXCR2^Low and CXCR2^Hi MDSC. Cell lines MH-S (mouse alveolar macrophage) and LLC (mouse Lewis lung cancer cell) were positive controls. (H and I) The H arginine concentration and I normalized percentage of CD4⁺ T cell proliferation elevated with BEC concentration increasing. The ratio of co-culture numbers of MDSC and T cells was 1:1. (J) Quantitative bar charts showing the normalized percentage of CD4⁺ T cell proliferation with treatment of BEC (10 μM) and L-Argine (100 μM). Statistical significances were calculated using the Brown-Forsythe and Welch analysis of variance (ANOVA) test unless otherwise indicated. Data were presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.