FIGURE 4.

TLT2 expressing monocytes negatively regulated Th1 response against Mtb infection. (A) CD14⁺ monocytes isolated from PBMCs of the healthy controls were treated with recombinant chimeric human TLT2-Fc (4 μg/mL) and human IgG1-Fc (4 μg/ml) for 24 h followed by H37Rv infection at an MOI of 5 for 12 h. The TLT2⁺ CD3^– CD14⁺ cells were analyzed for the expression of IL-6 by flow cytometry. (B–D) Naïve CD4⁺ T cell purified from human PBMCs of healthy controls using magnetic separation. CD4⁺ T cell were activated with plate-bound anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 3 days co-culture with CD14⁺ monocytes in the presence of recombinant chimeric human TLT2-Fc (4 μg/ml) and human IgG1-Fc (4 μg/ml). CD14⁺ monocytes were pre-incubated with heat-killed H37Rv followed by co-cultured with CD4⁺ T cell. IFN-γ (B), IL-4 (C), and IL-17A (D) expressions in CD4⁺ T cells were measured by flow cytometry, representative flow cytometric plots and the percentages of cells were shown. (E,F) Naïve CD4⁺ T cell co-cultured with CD14⁺ monocytes as described in (B), but in the presence of neutralizing anti-IL-6 mAbs (10 μg/ml; anti-IL-6). IFN-γ and IL-4 expressions in CD4⁺ T cells were measured by flow cytometry, representative flow cytometric plots and the percentages of cells were shown. (G) Monocytes were infected with H37Rv at an MOI of 10 for 2 h. After washing, H37Rv -infected monocytes were cultured with T cells in the presence of anti-human IgG1-Fc, or anti-human TLT2-Fc. CFU were calculated on Day 0, 3, 5. (H) H37Rv-infected monocytes were cultured with T cells as described in (G), but in the presence of anti-IL-6 mAbs. CFU were calculated on Day 0, 3, 5. Data are a representative of at least three independent experiments. ns, no significance; **p < 0.01; and ***p < 0.001.