Figure 3

Lack of B cell-derived IL-10 results in decreased Tr1 responses, however FoxP3⁺ Tregs are unaffected. A. A total of 35 days after CIA induction, draining lymph node cells were excised and cultured with PMA plus ionomycin in the presence of Brefeldin A for five hours. The CD4+ T cell population was identified using anti-CD4 FITC mAb and intracellular levels of IL-10 were measured. B. Numbers indicate percentages of cells in the quadrants. Data shown mean ± SEM (n = 4), are representative of four experiments. Data were compared by statistical analysis using the unpaired t test. *** P < 0.001. In addition, supernatant was collected from lymph node cells stimulated in vitro for 48 hours with anti-CD3, and then secreted IL-10 was measured by cytokine FlowCytomix kit. Data show mean ± SEM (n = 4), and are representative of four experiments. Data were compared by statistical analysis using the unpaired t test. *** P < 0.001. C. Tregs were assessed using anti-CD25, anti-CD4 and anti-FoxP3 mAb. Numbers indicate percentages of CD4 gated FoxP3⁺ cells or the absolute number of cells in a draining lymph node. Data show mean ± SEM (n = 4), and are representative of four experiments. D. Spleens were taken from IL-10^-/- B cell and WT B cell animals and used to isolate CD25+ Treg cells using Milteyi Biotech magnetic beads. T-effector cells were isolated from immunized WT B6 mice using the same process. The cells were cultured for 72 hours with anti-CD3 antibody (1 μg/ml). For 12 hours before harvesting cells were pulsed with (³H) thymidine. Data shown are mean ± SEM of triplicate wells and are representative of two independent experiments. E. Additionally, a BD in vivo cytokine capture assay was used to assess in vivo levels of IL-2 over a 12-hour period. The mean levels of IL-2-conjugated to antibody in serum are shown ± SEM (n = 4), are representative of two separate experiments. Data were compared by statistical analysis using the unpaired t test.