Regulatory B cells (Breg cells) are a set of activated B cells that appear during inflammatory conditions and exhibit immune suppressive functions. Their regulatory function may be directly accomplished by the production of regulatory cytokines such as IL-10 and TGF-β and/or by the ability of B cells to interact with pathogenic T cells to dampen harmful immune responses [Mizoguchi and Bhan, 2006].
B cells are known to have a pathogenic role in arthritis, as B-cell deficient mice do not develop CIA [Mizoguchi and Bhan, 2006]. Recent studies indicate the co-existence of Breg cell subsets along with pathogenic subsets, which suppress the progression of and/or enhance the recovery from acquired immune-mediated inflammations. Transfer of these IL-10-producing B cells could ameliorate established arthritis and therefore this approach may represent a potential strategy to treat autoimmune diseases, as the IL-10 can be delivered by the B cells in an antigen-specific fashion to the crucial site of priming or inflammation [Mauri and Ehrenstein, 2008].
Breg cells exhibit immune suppressive action in an antigen-specific manner (B cells need BCR specificity in addition to TLR signals for their regulatory function) and can induce regulatory T cells [Mauri and Ehrenstein, 2008]. This confers an advantage of using Breg cells over Treg cell-based approaches in treating autoimmune diseases.
c) A survey of work done in the research area and the need for more research
CIA model of rheumatoid arthritis helps to understand the way by which B cells modulate the severity of the disease. The incidence of CIA is altered by stimulation of splenocytes with anti-CD40 antibody and it is shown to be B cell-dependent. Thus, when B cells were depleted before CD40 stimulation, all of the mice developed arthritis [Mauri et al., 2003]. CD40 stimulation resulted in a three-fold increase in the number of IL-10-producing B cells, while the number of B cells that produce IFN-γ decreased. Recent reports state that B cells depend on stimulation via BCR and CD40 to ameliorate CIA [Evans et al., 2007]. Activated B cells are shown to have regulatory effect in various autoimmune diseases other than CIA; however, the action is always reported to be mediated through IL-10. LPS-activated cells, which produce cytokines other than IL-10, were shown to exhibit suppressive activity in autoimmune mouse diabetic model [Tian et al., 2001].
All B cells can make IL-10, given the correct activatory context and time [Gray and Gray, 2010]. Although a definitive ''Breg'' subset may not exist, what is emerging is the importance of the stimulus. Among the strongest stimuli for IL-10 production are TLR ligands [Lampropoulou et al., 2008]. Few studies stated that the immune suppressive role of B cells require signals from both BCR and TLR [Yanaba et al., 2008], in addition to a co stimulatory one from CD40 [Fillatreau et al., 2008]. A comparative study of suppressive effects of differentially activated B cells on development of CIA, and understanding the mechanisms might help to improve the strategies of cell-based therapies.
Regulatory B cells have neither a unique set of cell surface markers that define functionality nor a defined transcription factor that controls their development and/or function. In mice, Tedder and colleagues [Yanaba et al., 2008] have described a ''B10'' subset contained within the splenic CD5+CD1d+ B-cell population that uniquely secretes IL-10 in response to LPS activation. The precise cellular origin of B10 cells remains unknown, although some studies have shown that they originate from an as yet uncharacterized subset of B cells [DiLillo et al., 2010] and this is a critical issue to be addressed. Further insight is required to understand their physiological role under normal and autoimmune conditions.
As of now, only one factor B-cell activating factor (BAFF) is known to induce Bregs, both in vitro and in vivo [Min Yang et al., 2010]. Search for other such factors is important as the numbers of naturally existing different B cell subsets are limited.
d) Aims and Objectives
1) To evaluate the role of differentially activated B cells in immune suppression in vitro.
2) To further evaluate the role of activated B cells (regulatory B cells) in amelioration of CIA.
3) To delineate mechanisms of action of regulatory B cells.
4) To investigate the factors involved in expansion of regulatory B cells.
e) Methodologies and techniques to be used
Techniques such as cell culture, B cell differentiation and purification, T cell proliferation assay, cytokine analysis using ELISA and CBA. RT-PCR, real-time PCR, FACS, Western blotting, in vivo cell transplantation experiments etc. would be used in the proposed project.
f) The kinds of conclusions expected and their possible value
The proposed studies would generate valuable information on the contribution of differentially activated B cells in the regulation of CIA. Identification of the specific triggers will help in fine tuning the cell-based therapeutic approaches for autoimmune diseases. Characterization of the function and development of the regulatory B cells would contribute to our understanding of immunologic tolerance as an acquired process and of the cause of various autoimmune diseases including RA.
g) Plan of research
B cells will be isolated either by positive selection using B220 beads or by negative selection using B cell enrichment cocktail. Different subsets of B cells will be isolated by sorting (FACS) using subset-specific markers. Cells will be activated by either TLR or by BCR ligands and checked for the levels of various cytokines, both secreted as well as intracellular by ELISA and flow cytometry, respectively. B-cell-mediated suppression of immune responses would be studied using a set of in vitro co-culture experiments comprising activated B cells and other immune cells. Transwell culture experiments will show if the effect if any, is contact-dependant or not.
Activated B cell subsets that showed immune suppression in vitro would be used for in vivo experiments by administering them into CIA mice and their therapeutic potential would be evaluated.
Mechanisms underlying regulatory B cell-mediated immune suppression would be studied by employing high-density arrays and studying various signaling pathways involved in activation. Search for molecular signatures, if any would be performed using mass spectrometry.
Effect of cytokines on in vitro regulatory B cell expansion would be studied by analyzing changes in the expression of various surface and intracellular signaling molecules.
h) Bibliography
David Gray, and Mohini Gray, 2010. What are regulatory B cells? Eur. J. Immunol.. 40: 2677-2679.
DiLillo, D. J., T. Matsushita, and T. F. Tedder. 2010. B10 cells and regulatory B cells balance immune responses during inflammation, autoimmunity, and cancer. Ann. NY Acad. Sci. 1183: 38-57.
Evans, J. G., Chavez-Rueda, K. A., Eddaoudi, A., Meyer-Bahlburg, A., Rawlings, D. J., Ehrenstein, M. R. and Mauri, C., 2007. Novel suppressive function of transitional 2 B cells in experimental arthritis. J. Immunol.. 178 7868-7878.
Fillatreau, S., Gray, D. and Anderton, S. M., 2008. Not always the bad guys: B cells as regulators of autoimmune pathology. Nat. Rev. Immunol.. 8:391-397.
Lampropoulou, V., Hoehlig, K., Roch, T., Neves, P., Calderon Gomez, E., Sweenie, C. H., Hao, Y. et al., 2008. TLR-activated B cells suppress T cell-mediated autoimmunity. J. Immunol.. 180: 4763-4773.
Mauri, C., Gray, D., Mushtaq, N. and Londei, M., 2003. Prevention of arthritis by interleukin 10-producing B cells. J. Exp. Med. 197: 489-501.
Mauri C, Ehrenstein MR. 2008. The ''short'' history of regulatory B cells. Trends Immunol; 29:34-40.
Min Yang, Lingyun Sun, Shengjun Wang, King-Hung Ko, Huaxi Xu, Bo-Jian Zheng, Xuetao Cao, and Liwei Lu. 2010. Novel Function of B Cell-Activating Factor in the Induction of IL-10-Producing Regulatory B Cells. . J. Immunol. 184: 3321-3325.
Mizoguchi, A., and A. K. Bhan. 2006. A case for regulatory B cells. J.Immunol.176:705-710.
Tian, J., Zekzer, D., Hanssen, L., Lu, Y., Olcott, A. and Kaufman, D. L., 2001. Lipopolysaccharide-activated B cells down-regulate Th1 immunity and prevent autoimmune diabetes in nonobese diabetic mice. J. Immunol.. 167: 1081-1089.
Yanaba, K., Bouaziz, J. D., Haas, K. M., Poe, J. C., Fujimoto, M. and Tedder,T. F., 2008. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses. Immunity. 28:639-650.
Yanaba K, Bouaziz JD, Matsushita T, Tsubata T, Tedder TF. 2009. The development and function of regulatory B cells expressing IL-10 (B10 cells) requires antigen receptor diversity and TLR signals. J Immunol; 182:7459-7472.
