Abstract
B cells are generated every day in the bone marrow, but only a small fraction integrates the peripheral B-cell pool. In the murine spleen, we can find several B-cell subsets representing various maturation stages and/or cell functions. The spleen is a complex lymphoid organ organized in two main structures with different functions: the red and white pulp. The red pulp is flowed with blood while the white pulp is organized in primary follicles, with a B-cell area composed of follicular B cells and a T-cell area surrounding a periarterial lymphatic sheath. The frontier between the red and white pulp is defined as the marginal zone and contains the marginal zone B cells. Because B cells, localized in different areas, are characterized by distinct expression levels of B-cell receptor (BCR) and other surface markers, splenic B-cell subsets can be easily identified and purified by flow cytometry analyses and cell sorting (FACS).
Here, we will focus on marginal zone B cells and their precursors giving some experimental hints to identify, generate, and isolate these cells. We will combine the use of FACS analysis and confocal microscopy to visualize marginal zone B cells in cell suspension and tissue sections, respectively.
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References
Godin I, Cumano A (2002) The hare and the tortoise: an embryonic haematopoietic race. Nat Rev Immunol 2:593–604
Ghosn EE, Sadate-Ngatchou P, Yang Y, Herzenberg LA, Herzenberg LA (2011) Distinct progenitors for B-1 and B-2 cells are present in adult mouse spleen. Proc Natl Acad Sci U S A 108:2879–2884
Rosado MM, Aranburu A, Capolunghi F, Giorda E, Cascioli S, Cenci F, Petrini S, Miller E, Leanderson T, Bottazzo GF, Natali PG, Carsetti R (2009) From the fetal liver to spleen and gut: the highway to natural antibody. Mucosal Immunol 2:351–361
Carsetti R, Kohler G, Lamers MC (1995) Transitional B cells are the target of negative selection in the B cell compartment. J Exp Med 181:2129–2140
Rolink AG, Andersson J, Melchers F (2004) Molecular mechanisms guiding late stages of B-cell development. Immunol Rev 197:41–50
Carsetti R, Rosado MM, Wardmann H (2004) Peripheral development of B cells in mouse and man. Immunol Rev 197:179–191
Freitas AA, Rosado MM, Viale AC, Grandien A (1995) The role of cellular competition in B cell survival and selection of B cell repertoires. Eur J Immunol 25:1729–1738
Casola S, Otipoby KL, Alimzhanov M, Humme S, Uyttersprot N, Kutok JL, Carroll MC, Rajewsky K (2004) B cell receptor signal strength determines B cell fate. Nat Immunol 5:317–327
Chen X, Martin F, Forbush KA, Perlmutter RM, Kearney JF (1997) Evidence for selection of a population of multi-reactive B cells into the splenic marginal zone. Int Immunol 9:27–41
Mebius RE, Kraal G (2005) Structure and function of the spleen. Nat Rev Immunol 5: 606–616
Oliver AM, Martin F, Gartland GL, Carter RH, Kearney JF (1997) Marginal zone B cells exhibit unique activation, proliferative and immunoglobulin secretory responses. Eur J Immunol 27:2366–2374
Oliver AM, Martin F, Kearney JF (1999) IgMhighCD21high lymphocytes enriched in the splenic marginal zone generate effector cells more rapidly than the bulk of follicular B cells. J Immunol 162:7198–7207
De Togni P, Goellner J, Ruddle NH, Streeter PR, Fick A, Mariathasan S, Smith SC, Carlson R, Shornick LP, Strauss-Schoenberger J et al (1994) Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin. Science 264:703–707
Alimzhanov MB, Kuprash DV, Kosco-Vilbois MH, Luz A, Turetskaya RL, Tarakhovsky A, Rajewsky K, Nedospasov SA, Pfeffer K (1997) Abnormal development of secondary lymphoid tissues in lymphotoxin beta-deficient mice. Proc Natl Acad Sci U S A 94:9302–9307
Kuprash DV, Alimzhanov MB, Tumanov AV, Anderson AO, Pfeffer K, Nedospasov SA (1999) TNF and lymphotoxin beta cooperate in the maintenance of secondary lymphoid tissue microarchitecture but not in the development of lymph nodes. J Immunol 163:6575–6580
Kuprash DV, Alimzhanov MB, Tumanov AV, Grivennikov SI, Shakhov AN, Drutskaya LN, Marino MW, Turetskaya RL, Anderson AO, Rajewsky K, Pfeffer K, Nedospasov SA (2002) Redundancy in tumor necrosis factor (TNF) and lymphotoxin (LT) signaling in vivo: mice with inactivation of the entire TNF/LT locus versus single-knockout mice. Mol Cell Biol 22:8626–8634
Futterer A, Mink K, Luz A, Kosco-Vilbois MH, Pfeffer K (1998) The lymphotoxin beta receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues. Immunity 9:59–70
Koike R, Nishimura T, Yasumizu R, Tanaka H, Hataba Y, Watanabe T, Miyawaki S, Miyasaka M (1996) The splenic marginal zone is absent in alymphoplastic aly mutant mice. Eur J Immunol 26:669–675
Yamada T, Mitani T, Yorita K, Uchida D, Matsushima A, Iwamasa K, Fujita S, Matsumoto M (2000) Abnormal immune function of hemopoietic cells from alymphoplasia (aly) mice, a natural strain with mutant NF-kappa B-inducing kinase. J Immunol 165:804–812
Weih F, Caamano J (2003) Regulation of secondary lymphoid organ development by the nuclear factor-kappaB signal transduction pathway. Immunol Rev 195:91–105
Cariappa A, Liou HC, Horwitz BH, Pillai S (2000) Nuclear factor kappa B is required for the development of marginal zone B lymphocytes. J Exp Med 192:1175–1182
Weih DS, Yilmaz ZB, Weih F (2001) Essential role of RelB in germinal center and marginal zone formation and proper expression of homing chemokines. J Immunol 167:1909–1919
Guinamard R, Okigaki M, Schlessinger J, Ravetch JV (2000) Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nat Immunol 1:31–36
Cariappa A, Tang M, Parng C, Nebelitskiy E, Carroll M, Georgopoulos K, Pillai S (2001) The follicular versus marginal zone B lymphocyte cell fate decision is regulated by Aiolos, Btk, and CD21. Immunity 14:603–615
Pabst O, Forster R, Lipp M, Engel H, Arnold HH (2000) NKX2.3 is required for MAdCAM-1 expression and homing of lymphocytes in spleen and mucosa-associated lymphoid tissue. EMBO J 19:2015–2023
Kitamura D, Roes J, Kuhn R, Rajewsky K (1991) A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature 350:423–426
Hayakawa K, Hardy RR, Honda M, Herzenberg LA, Steinberg AD, Herzenberg LA (1984) Ly-1 B cells: functionally distinct lymphocytes that secrete IgM autoantibodies. Proc Natl Acad Sci U S A 81:2494–2498
Wardemann H, Boehm T, Dear N, Carsetti R (2002) B-1a B cells that link the innate and adaptive immune responses are lacking in the absence of the spleen. J Exp Med 195:771–780
Baumgarth N (2011) The double life of a B-1 cell: self-reactivity selects for protective effector functions. Nat Rev Immunol 11:34–46
Acknowledgements
We thank Dr. Claudio Pioli for his critical review of the manuscript. M.M.R. is supported by a Merieux Starting Grant.
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Rosado, M.M., Scarsella, M., Cascioli, S., Giorda, E., Carsetti, R. (2014). Purification and Immunophenotypic Characterization of Murine MZ and T2-MZP Cells. In: Vitale, G., Mion, F. (eds) Regulatory B Cells. Methods in Molecular Biology, vol 1190. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1161-5_1
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DOI: https://doi.org/10.1007/978-1-4939-1161-5_1
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