In addition to demonstrating strain independence, experiments were performed to show that Treg-cell control of GC responses was also antigen independent. Figure 3 summarizes the effect of anti-GITR mAb treatment on splenic GC responses induced by i.p challenge of BALB/c mice with IAV. Whereas SRBC induce a Th2-biased response,5 IAV invokes a Th1-polarized reaction.56Figure 3(a) shows that mice immunized i.p. with IAV generate MAPK Inhibitor Library high throughput a robust splenic GC response which peaks at day 12 (Fig. 3b). Similar to Th2 antigens,5,6 the GC reaction induced by

IAV was characterized by a steady ratio of IgM+ to switched GC B cells (Fig. 3c). Importantly, anti-GITR mAb administration resulted in a higher frequency and total number of splenic GC B cells at several time-points (Fig. 3b), and significantly increased the proportion of switched GC B cells throughout the entire reaction (Fig. 3c). As opposed to GCs induced with SRBC immunization, we observed no significant difference CP-673451 mouse in the distribution of IgG isotypes within the switched GC B-cell pool at any time-points after IAV challenge (data not shown). The results generated above demonstrated the role of Treg cells in controlling both the size of SRBC-induced and IAV-induced GC responses, and the ratio of IgM+ to switched B cells within the

GC population. In these experiments, however, total splenic GC B cells were enumerated because the B220+ PNAhi B-cell population induced after SRBC or IAV injection was presumed to be specific for the challenge antigen. (Please note that specific pathogen-free mice do not exhibit splenic GCs in the absence of immunization, Fig. 1.) We therefore sought to confirm the role of Treg cells in governing GC reactions by tracking antigen-binding GC B cells, instead of the entire B220+ PNAhi splenic B-cell pool. To perform these studies, PE was used as the challenge antigen,57–59 and PE-binding GC B cells were analysed in anti-GITR mAb or control rIgG-treated mice. As shown in Fig. 4(a), i.p. immunization with PE precipitated in alum induced splenic B220+ PNAhi GC B cells, a Etomidate sub-set of which retained the ability to bind native

PE. In control animals, the PE-binding GC B-cell response peaked at day 12 (Fig. 4b) and like other normal splenic GC responses, displayed a relatively steady ratio of IgM+ to switched B cells (Fig. 4c). As expected, disruption of Treg cells with anti-GITR mAb administration resulted in an increased total PE-binding GC response, and a progressive increase in the proportion and total number of switched PE-binding GC B cells. In Figs 1–4, splenic GC responses were dysregulated when anti-GITR mAb was given before and soon after immunization. To assess whether already established GCs can be altered by late-stage Treg-cell disruption, mice were challenged with SRBC at day 0 and treated with either anti-GITR mAb or control rIgG on days 8 and 12, or days 12 and 16 post-immunization. Splenic GCs from both groups were examined on days 18 and 24.