mTORC1-Activated Monocytes Increase Tregs and Inhibit the Immune Response to Bacterial Infections

Figure 4

mTORC1 influences the numbers of Tregs and Th17 and Th1 cells in lymph nodes and spleen. (a) The percentage and number of Tregs increased in TSC1 KO lymph nodes and spleens compared to WT mice (four mice per group, three representative experiments). (b) The percentage of Ki67-positive Tregs increased in TSC1 KO lymph nodes and spleens compared to WT mice (four mice per group, three representative experiments). (c) The percentage and number of Th17 cells (CD4⁺IL-17⁺) decreased in lymph nodes and spleens in TSC1 KO mice when compared to WT mice (four mice per group, three representative experiments). (d) The number and percentage of Th1 cells (CD4⁺IFN-gamma⁺) decreased in the LNs and spleen of TSC1 KO mice compared to those of WT mice (four mice per group, two representative experiments). (e) 2 × 10⁵ Tregs (CD4⁺CD25⁺) labeled with CFSE (5 μM; eBioscience) were cocultured with 1 × 10⁵ sorted monocytes (CD11b⁺Ly6C⁺) from WT or TSC1 KO mice in DMEM containing IL-2 (100 ng/mL) in 96-well flat-bottomed plates for 6 days. There was no significant difference in proliferation of Tregs cocultured with monocytes obtained from TSC1 KO or WT mice ( representative experiments). (f) 2 × 10⁵ naïve CD4⁺ T cells (CD4⁺CD4CD62⁺) were cocultured with 1 × 10⁵ sorted monocytes (CD11b⁺Ly6C⁺) from WT or TSC1 KO mice in DMEM with anti-mouse CD3e (10 μg/mL, BD Biosciences, USA) and CD28 antibody (10 μg/mL, BD Biosciences, USA) in 96-well flat-bottomed plates for 6 days. The percentage of Th17 cells of naïve CD4⁺ T cells (CD4⁺CD4CD62L⁺) cocultured with monocytes obtained from TSC1 KO mice was lower than the WT group at day 6. However, the percentage of Tregs was higher in the TSC1 KO group than in the WT group at day 6 ( representative experiments). (g) 2 × 10⁵ sorted naïve CD4⁺ T cells (CD4⁺CD4CD62L⁺) were cocultured with 1 × 10⁵ sorted monocytes (CD11b⁺Ly6C⁺) from WT mice in DMEM with or without IL-10 (20 ng/mL, Peprotech, UK). After 6 days, cells were used to estimate Th17 cells using flow cytometry (BD FACSCanto II, USA). and compared to WT mice or between the indicated groups. values were determined using Student’s -tests.

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