Abstract
Regulatory B cells that produce IL-10 are now recognized as an important component of the immune system. We have identified a rare antigen-specific regulatory B-cell subset with a unique CD1dhiCD5+CD19hi phenotype in the spleens of wild-type mice. We call these cells B10 cells because they are responsible for most B cell IL-10 production, they appear to only produce IL-10 after 5 h of in vitro stimulation, and to distinguish them from other potential regulatory B cell subsets. B10 progenitor (B10pro) cells have also been identified within the spleen CD1dhiCD5+CD19hi B-cell subset, and within other lymphoid tissues. Herein, four methods for identifying and isolating regulatory IL-10-producing B10 cells in mice are provided. The first two methods are used to identify and enumerate B10 and B10pro cells based on their cell surface phenotypes and cytoplasmic IL-10 staining. The last two methods are used to isolate viable B10 cells for adoptive transfer and functional studies. These methods should facilitate the study of B10 cells in inflammation, autoimmune disease, immune responses, and cancer therapy.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
LeBien, T. W., and Tedder, T. F. (2008) B-lymphocytes: How they develop and function. Blood 112, 1570–1579.
Liu, Y., Wu, Y., Ramarathinam, L., Guo, Y., Huszar, D., Trounstine, M., et al. (1995) Gene-targeted B-deficient mice reveal a critical role for B cells in the CD4 T cell response. Int. Immunol. 7, 1353–1362.
O’Neill, S. K., Cao, Y., Hamel, K. M., Doodes, P. D., Hutas, G., and Finnegan, A. (2007) Expression of CD80/86 on B cells is essential for autoreactive T cell activation and the development of arthritis. J. Immunol. 179, 5109–5116.
Linton, P. J., Bautista, B., Biederman, E., Bradley, E. S., Harbertson, J., Kondrack, R. M., et al. (2003) Costimulation via OX40L expressed by B cells is sufficient to determine the extent of primary CD4 cell expansion and Th2 cytokine secretion in vivo. J. Exp. Med. 197, 875–883.
Linton, P. J., Harbertson, J., and Bradley, L. M. (2000) A critical role for B cells in the development of memory CD4 cells. J. Immunol. 165, 5558–5565.
Crawford, A., Macleod, M., Schumacher, T., Corlett, L., and Gray, D. (2006) Primary T cell expansion and differentiation in vivo requires antigen presentation by B cells. J. Immunol. 176, 3498–3506.
Bouaziz, J. D., Yanaba, K., Venturi, G. M., Wang, Y., Tisch, R. M., Poe, J. C., et al. (2007) Therapeutic B cell depletion impairs adaptive and autoreactive CD4+ T cell activation in mice. Proc. Natl. Acad. Sci. U. S. A. 104, 20882–20887.
Homann, D., Tishon, A., Berger, D. P., Weigle, W. O., von Herrath, M. G., and Oldstone, M. B. (1998) Evidence for an underlying CD4 helper and CD8 T-cell defect in B-cell-deficient mice: failure to clear persistent virus infection after adoptive immunotherapy with virus-specific memory cells from μMT/μMT mice. J. Virol. 72, 9208–9216.
Bergmann, C. C., Ramakrishna, C., Kornacki, M., and Stohlman, S. A. (2001) Impaired T cell immunity in B cell-deficient mice follo wing viral central nervous system infection. J. Immunol. 167, 1575–1583.
Katz, S. I., Parker, D., and Turk, J. L. (1974) B-cell suppression of delayed hypersensitivity reactions. Nature 251, 550–551.
Neta, R., and Salvin, S. B. (1974) Specific suppression of delayed hypersensitivity: the possible presence of a suppressor B cell in the regulation of delayed hypersensitivity. J. Immunol. 113, 1716–1725.
Mizoguchi, A., and Bhan, A. K. (2006) A case for regulatory B cells. J. Immunol. 176, 705–710.
Serra, P., and Santamaria, P. (2006) To ‘B’ regulated: B cells as members of the regulatory workforce. Trends Immunol. 27, 7–10.
Mauri, C., and Ehrenstein, M. R. (2008) The ‘short’ history of regulatory B cells. Trends Immunol. 29, 34–40.
Lund, F. E. (2008) Cytokine-producing B lymphocytes-key regulators of immunity. Curr. Opin. Immunol. 20, 1–7.
Bouaziz, J.-D., Yanaba, K., and Tedder, T. F. (2008) Regulatory B cells as inhibitors of immune responses and inflammation. Immunol. Rev. 224, 201–214.
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.
Matsushita, T., Yanaba, K., Bouaziz, J. D., Fujimoto, M., and Tedder, T. F. (2008) Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression. J. Clin. Invest. 118, 3420–3430.
Harris, D. P., Haynes, L., Sayles, P. C., Duso, D. K., Eaton, S. M., Lepak, N. M., et al. (2000) Reciprocal regulation of polarized cytokine production by effector B and T cells. Nat. Immunol. 1, 475–482.
Yanaba, K., Bouaziz, J.-D., Matsushita, T., Tasubata, T., and Tedder, T. F. (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.
DiLillo, D. J., Matsushita, T., and Tedder, T. F. (2010) B10 and regulatory B cells balance immune responses during inflammation, autoimmunity and cancer. Ann. New York Acad. Sci. 1183, 38–57.
Kuhn, R., Lohler, J., Rennick, D., Rajewsky, K., and Muller, W. (1993) Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75, 263–274.
Acknowledgments
This work was supported by grants from the National Institutes of Health CA105001, CA96547, AI56363, and AI057157. We thank Drs. Guang Yang, Regina Lin, Kathleen Candando, and David DiLillo for their input and review of this manuscript.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press
About this protocol
Cite this protocol
Matsushita, T., Tedder, T.F. (2010). Identifying Regulatory B Cells (B10 Cells) that Produce IL-10 in Mice. In: Cuturi, M., Anegon, I. (eds) Suppression and Regulation of Immune Responses. Methods in Molecular Biology, vol 677. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-869-0_7
Download citation
DOI: https://doi.org/10.1007/978-1-60761-869-0_7
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-868-3
Online ISBN: 978-1-60761-869-0
eBook Packages: Springer Protocols