4c), when RA was added to CD19+ cells enriched from the PBMC of all individuals, the Selleck Epacadostat frequency of CD19+CD24+CD38+ cells was increased significantly after 3 days (Fig. 4c). This increase, however, was not due to proliferation, as the frequency and numbers of viable BrdU+ cells

that are represented in the CD19+CD24+CD38+ population were not statistically different among control and RA-treated cultures of CD19+ cells from two individuals (data not shown). In order to determine whether the iDC-induced proliferation of CD19+CD24+CD38+ B cells was due exclusively to RA, or if additional mechanisms are involved we co-cultured 2 × 105 iDC in the presence of an equal number of syngeneic CD19+ B cells enriched from thawed, viable PBMC in the presence or absence of 1 mM of ER-50891, a pan-RAR selective antagonist [48] for 72 h. In Fig. 4d we demonstrate that the frequency of CD19+CD24+CD38+ Bregs is significantly lower in iDC co-cultures in the presence of the RAR inhibitor. The inhibitor did not alter viability, as the % of live cells were statistically indistinguishable between inhibitor and control-treated cultures (data not shown). We have shown that administration into humans of autologous DC generated ex vivo under conditions promoting and stabilizing a tolerogenic state is safe and without any detectable side effects [31]. In the same study, we also discovered that administration

of autologous DC with tolerogenic ability generated under ‘conventional’ GM-CSF/IL-4 conditions resulted in an increased frequency of B220+CD11c– cells, Caspase inhibitor preferentially in iDC recipients. We also demonstrated the suppressive ability of B cells whose frequency increased in the DC recipients. The surface phenotype of these B cells, however, as assessed by flow cytometry, suggested that they were heterogeneous Thiamine-diphosphate kinase in character and thus could consist, in principle, of more

than one suppressive subpopulation. How tolerogenic DC could mobilize one or more populations of Bregs is the question we have begun to address. Herein, we demonstrate that the tolerogenic DC used in our Phase I trial, when co-cultured with freshly obtained PBMC, induce an increase in frequency of CD19+CD24+CD38+ B cells in vitro, which are suppressive in allogeneic MLC. By virtue of their surface phenotype these cells are most probably identical, if not related to the Bregs reported by Mauri and colleagues [32, 40]. The increase in their frequency appears to be due to proliferation of existing CD19+CD24+CD38+ Bregs as well as conversion of CD19+ B cells in PBMC into CD19+CD24+CD38+ Bregs. We also show that suppression of T cells in allogeneic MLC in vitro is conferred by the CD19+CD24+ constituent of the B220+CD11c– population from freshly obtained PBMC and more specifically by the known CD19+CD24+CD38+ Bregs. In the process of identifying mechanisms of how the tolerogenic DC could promote suppressive B cell activity, we have discovered that CD19+CD24+CD38+ Bregs express retinoic acid (RA) receptors.