These APC were pulsed with OVA peptide and used to stimulate na?ve CD4+ OVA-specific DO11
These APC were pulsed with OVA peptide and used to stimulate na?ve CD4+ OVA-specific DO11.10 transgenic T cells. pathogenic anti-collagen II antibodies. Inhibition of IL-10 reversed the beneficial effects of AC. Passive transfer of B cells from AC-treated mice provided significant protection from arthritis. These data demonstrate that AC exert a profound influence on an adaptive immune response through the generation of CD19+ regulatory B cells, which in turn are able to influence the cytokine profile of antigen-specific effector ATR-101 T cells. induced a populace of antigen-specific IL-10-secreting CD4+ T cells. Their induction depended on AC interacting Rabbit Polyclonal to IP3R1 (phospho-Ser1764) directly with activated B cells, and the secretion of IL-10 by B cells was required to induce these regulatory T cells both and with na?ve BALB/c splenocytes as APC. Supernatants collected after 3 days of culture revealed that CD4+ T cells from AC-treated mice produced significantly more IL-10 and IFN- than controls (Fig. 1 and in the presence of adjuvant induces distinct populations of antigen-specific CD4+ T cells that make either IFN- or IL-10 upon secondary antigen stimulation. Open in a separate windows Fig. 1. T cells generated in the presence of AC. DO11.10 T cells were transferred into BALB/c mice, immunized with OVA/CFA, and treated with AC or vehicle alone on days 0, 1, and 2. CD4+ T cells harvested on day 7 from the spleens and draining lymph nodes were restimulated with na?ve BALB/c splenocytes in the presence of increasing doses of OVA peptide. IL-10 ( 0.005; ***, 0.0007. B Cells Exposed to AC Induce an IL-10-Secreting T Cell Population. We then asked whether AC have a differential effect on APC populations when given with adjuvant. BALB/c mice were immunized with OVA/CFA and given AC i.v. On day 7 the spleens and draining lymph nodes were harvested and CD11c+ and CD19+ cells were isolated. These APC were pulsed with OVA peptide and used to stimulate na?ve CD4+ OVA-specific DO11.10 transgenic T cells. Proliferation of T cells was equally effective with all ATR-101 groups of APC (Fig. 2 and and and 0.03; **, 0.009. AC Interact Directly with B Cells to Induce IL-10-Secreting Effector T Cells. We next asked whether B cells interacted directly with AC to alter B and T cell function. B cells purified from the spleens of BALB/c mice were sorted into marginal zone (MZ) or follicular (FO) B cell subsets. They were then pulsed with OVA peptide and used to stimulate na?ve DO.11.10 T cells. AC at 106 per well were added for the duration of the culture period (72 h), after which supernatants were collected and analyzed for cytokine secretion (Fig. 3 in the presence of CFA, IFN- secretion was equivalent and only a small increase in IL-4 was seen in the AC-treated groups. Using OVA-pulsed CD19+ B cells cocultured with AC and DO11.10 T cells, we confirmed by intracellular cytokine staining that this increase in IL-10 secretion was a product of both B and T cell production (Fig. 3 and results with OTII mice on a C57BL/6 background were entirely similar to those with DO11.10 (on a BALB/c background). Open in a separate windows Fig. 3. B cells interact directly with AC, inducing regulation through IL-10. Splenocytes from BALB/c mice were separated into MZ and FO B cell subsets and cultured with na?ve DO11.10 T cells in the presence of increasing doses of OVA peptide with or without added AC for 3 days after which supernatants were collected ATR-101 and analyzed for secreted IL-10 ( 0.05; **, 0.02; ***, 0.0001. AC Protect Mice from Autoimmune Disease. Given evidence that i.v. AC might influence B cell presentation to CD4+ T cells of a model antigen, we went on to ask whether AC could affect a CD4+ T cell-dependent autoimmune disease, collagen-induced arthritis (CIA). In control, type II collagen (CII)/CFA-immunized DBA-1 mice (treated with PBS), clinical disease was apparent from day 21 and became steadily worse until the end of the experiment (Fig. 4 0.05; **, 0.02. (and to elicit profound suppression of proinflammatory cytokine production by macrophages stimulated through their Toll-like receptors (2). Macrophages are important for the effector phase of joint destruction in CIA (14). However, AC given after the onset of clinical arthritis in the CIA model had no protective effect on limiting the severity of disease (data not shown). This indicated that this mechanism of protection seen in CIA required AC to be present during the phase of induction of autoimmunity rather than the effector phase of clinically apparent.