In control CD47−/− and WT mice fed

PBS, a similar frequency of adoptively transferred cells was found in MLN (Fig. 2a). Three days after feeding OVA, the fraction of DO11.10 T cells that had entered division was reduced by 50% in the MLN of CD47−/− mice, when compared with WT mice (Fig. 2b,c). However, intravenous OVA administration did not affect proliferation of DO11.10 T cells in the spleen of CD47−/− mice (Fig. 2d). Addition of CT did not alter the reduced proliferation signaling pathway of DO11.10 T cells in MLN (data not shown) or PP of CD47−/− mice (Fig. 2e,f). These experiments show that CD47−/− mice have a reduced ability to induce proliferation of CD47-expressing CD4+ T cells in GALT after feeding OVA in the presence or absence of an adjuvant. However, the expansion of CD4+ T cells in CD47−/− mice is not compromised after parenteral immunization. We next assessed the capability of CD47−/− mice to induce oral tolerance. CD47−/− and WT mice were fed 50 mg OVA or PBS. Two weeks later, mice were challenged subcutaneously with OVA + IFA, and 1 week later draining LN were harvested. The antigen-specific proliferative response of LN cells was then determined in vitro after re-stimulation with OVA. The OVA-fed CD47−/− and WT mice buy Ixazomib exhibited a similar capacity to inhibit the

OVA-specific proliferative response in vitro (approximately 75% suppression; Fig. 3a). As feeding a high dose of OVA may conceal differences in the efficacy of tolerance induction between mouse strains, the experiment was repeated using a 10-fold lower dose of OVA. This reduced antigen dose resulted in efficient tolerance induction in CD47−/− mice that was not significantly Etofibrate different from what was seen in WT mice (Fig. 3b). These results show that although

CD47−/− mice have a reduced frequency of CD11b+ DC in LP and MLN, and a reduced capacity to induce T cell proliferation in the MLN following OVA feeding, they maintain the capacity to induce oral tolerance. CD4+ T cell help is required for the generation of antigen-specific antibodies following oral immunization with CT.1,2 As feeding OVA + CT resulted in reduced proliferation of OVA-specific CD4+ T cells in PP of CD47−/− mice, we next assessed OVA-specific antibody titres in intestinal tissues and serum after three oral immunizations with OVA + CT. CD47−/− mice generated significantly lower intestinal anti-OVA IgA titres than WT mice (Fig. 4a), whereas total intestinal IgA and OVA-specific serum IgA and IgG titres did not differ between CD47−/− and WT mice (Fig. 4b–d). In support of this, the frequency of OVA-specific IgA-producing cells in the intestine is reduced in CD47−/− mice following immunization with OVA and CT (531 ± 102/1 × 106 cells in WT and 219 ± 49/1 × 106 cells in CD47−/− mice, n = 10 and P < 0·05).