Study on the molecular mechanisms of induction and suppressive activity of Treg-of-B cells
Date Issued
2015
Date
2015
Author(s)
Lau, Ka-Yi
Abstract
B cells are capable to induce immunologic tolerance despite their primary role in humoral immunity. Accumulating evidence with a particular focus on defining conventional B-2 cells can serve as antigen presenting cells to convert naïve CD4+CD25- T cells into regulatory T cells (so-called Treg-of-B cells) in vitro. The B cells primed regulatory T were found to exert a suppressive function on T-cell proliferation. Previous study has already revealed that the mechanism involved in the generation of Treg-of-B cells was in a contact-dependent manner. There was a reversal of suppressive function of Tregs when B cells cultured with T cells were separated by a trans-well membrane. According to the two-signal model of T-cell activation, activation of naïve antigen-specific CD4+ T cells is thought to require at least both stimulation of the T cell receptor (TCR) (Signal 1), and stimulation of costimulatory molecules (Signal 2). The cytokine signal (Signal 3) is additional signal which plays critical role for generating a robust and specialized T-cell response. Many cytokine signaling is transmitted via Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathway. In this study, we aim to (1) investigate whether costimulatory blockade by single blocking antibody could boost or dampen Treg-of-B-cell activation and (2) identify the phosphorylated STAT proteins which are involved in induction and suppressive function of Treg-of-B cells. To induce regulatory T cells stimulated by B-2 cells, naïve B cells were isolated from splenoctyes of BALB/c mice and then pulsed with OVA-peptide for a day. After that, OVA peptide-loaded B cells were co-cultured with naïve CD4+ T cells isolated from splenocytes of DO11.10 mice for 3 days without addition of exogenous cytokines. After a 3 day co-culture, CD4+ T cells were purified after depletion of B220+ cells. The suppressive function of isolated CD4+ T cells was further examined. The effect of costimulatory blockade on B cells generated regulatory T cell was examined by using single neutralizing blocking antibody. On the other hand, to study the involvement of phosphorylated STAT proteins during induction and suppressive function of Treg-of-B cells, naïve B cells were co-cultured with naïve CD4+ T cells which both isolated from splenocytes of BALB/c mice under the stimulation of anti-CD3/CD28 for 3 days. The cell lysates of CD4+ T cells and re-stimulated CD4+ T cells were harvested after depletion of B220+ cells during induction and suppressive function at various time points respectively. We found that OVA-peptide loaded B-2 cells can serve as antigen presenting cells to induce CD4+ T cells into regulatory T cells (Treg-of-B cells). We further demonstrated that B-2 cells expressed GITRL, ICAM-1, ICOSL, CD70 and OX40L as antigen presenting cells to CD4+ T cells. However, there was no reversal or enhancement of suppressive function of Treg-of-B cells on T-cell proliferation under the treatment of particular single blocking antibody during induction. Only under anti-GITR, the expression of GITR on Treg-of-B cells was significantly down-regulated. On the other hand, we identified that the phosphorylated Stat1, Stat3, Stat5 and Stat6 were involved in the induction and suppressive function of Treg-of-B cells. Our results confirmed that conventional B-2 cells can modulate immune responses through priming T cells into regulatory T cells. Also, we demonstrated that B cells generated regulatory T cells was not through single costimulatory pathways. We further demonstrated that the expression of GITR on Treg-of-B cells was not a critical molecule to exert suppressive function on T-cell proliferation. On the other hand, we have shown that phosphorylated Stat1, Stat3, Stat5 and Stat6 were involved in the induction and suppressive activity of Treg-of-B cells which is different from nTregs and other inducible Tregs. These findings might help us on identifying Treg-of-B cells into distinct subsets of Treg cells.
Subjects
B-2 cells
regulatory T cells
co-stimulatory molecules
pStats
immune regulation
Type
thesis