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. 2023 Jan 26;14:1039244. doi: 10.3389/fimmu.2023.1039244

Figure 4.

Figure 4

Suppression of T-cell responses by MDSCs. To evaluate the ability of MDSCs to suppress T cell proliferation, MDSCs were obtained from the lungs of P. brasiliensis-infected mice after two weeks of infection (Lung-MDSCs). Alternatively, bone marrow-derived MDSCs (BM-MDSCs) were generated in vitro and challenged overnight or not by P. brasiliensis at a ratio of 1:50 (yeast: MDSCs), as indicated in the material and methods. Both Lung-MDSCs (A, B) and BM-MDSCs (C, D) were sorted and subsequently cocultured with CFSE-labeled CD3+ T cells, previously activated with anti-CD3/CD28 antibodies. Following coculture for four days (ratios of 1:10 and 1:5 -MDSC: lymphocytes- for L-MDSCs and ratio of 1:10 for BM-MDSCs), the proliferation of the CD4 and CD8 T cells was characterized by flow cytometric analysis, and the cell division index (CDI) was obtained (A, C). To better evaluate the ability of Lung-MDSCs (B) and BM-MDSCs (D) to suppress lymphoproliferation, the percentage of T cell suppression was calculated from CDI results. To evaluate the ability of BM-MDSCs to suppress activated T cells (E) and Th/Tc lymphocytes differentiation (F, G), spleen lymphocytes from naïve mice were cocultured with BM-DCs that were previously challenged with P. brasiliensis yeasts (ratio 1:50; yeast-DCs). After 24 hours of DC-Lymphocyte coculture, the BM-MDSCs were added to the coculture (ratio of 1:10; MDSC: T cells) and T cell responses were compared to cocultures without MDSCs. After four days of coculture, lymphocytes were evaluated by flow cytometry for total and activated T cells (CD4+, CD4+CD25+, CD8+ and CD8+CD69+) (E), and for intracellular expression of Th-signature cytokines by CD4+ T cells (Th1: CD4+IFN-γ+; Th2: CD4+IL-4+, Th17: CD4+IL-17+) (F), and CD8+ T signature cytokines (Tc1: CD8+IFN-γ+, Tc2: CD8+IL-4+, Tc17: CD8+IL-17+ (G). Data represent two independent experiments. For comparisons between two groups, the mean ± SEM was obtained and analyzed by the unpaired Student’s t-test. Differences were considered significant when: *p<0.05; **p<0.01; ***p<0.001 and ****p<0.0001.