Th eJournal of Brief Reviews Immunology B10 Cells: A Functionally Defined Regulatory B Cell Subset Thomas F. Tedder B cells are commonly thought to enhance inflamma- particularly those that influence immune responses through tory immune responses. However, specific regulatory mechanistically distinct pathways. In many cases, regulatory B cell subsets recently were identified that downregu- B cells inhibit inflammation and disease through the pro- late adaptive and innate immunity, inflammation, and duction of either IL-10 alone or in combination with other autoimmunity through diverse molecular mechanisms. immunomodulatory cytokines or cell surface receptors. However, In both mice and humans, a rare, but specific, subset of IL-10 expression by B cells is not always necessary to suppress regulatory B cells is functionally characterized by its inflammation, and multiple regulatory B cell subsets and capacity to produce IL-10, a potent inhibitory cyto- mechanisms for reducing inflammation are likely to exist. The kine. For clarity, this regulatory B cell subset has been complexity of this issue was well demonstrated in studies of IBD. Mizoguchi et al. (24) first showed that regulatory B cells labeled as B10 cells, because their ability to downregu- 2/2 late immune responses and inflammatory disease is from the mesenteric lymph nodes of TCRa mice and their Ab products suppress colitis by enhancing the clearance of fully attributable to IL-10, and their absence or loss apoptotic cells. Ab blockade of the CD40 or B7-2 costimu- exacerbates disease symptoms in mouse models. This latory molecules on adoptively transferred B cells also elimi- review preferentially focuses on what is known about nates their suppressive regulatory effects on pathogenic T cells mouse B10 cell development, phenotype, and effector (1). This group subsequently showed that IL-10 production function, as well as on mechanistic studies that demon- by CD1dhigh B cells within the mesenteric lymph nodes of 2 2 strated their functional importance during inflamma- TCRa / mice inhibits chronic intestinal inflammation (2). tion, autoimmune disease, and immune responses. The However, they also demonstrated a regulatory role for perito- 2 2 Journal of Immunology, 2015, 194: 1395–1401. neal cavity B1 cells from TCRa / mice during chronic colitis, possibly through natural Ab generation in response to micro- cells are generally thought to augment immune bial flora (25). In studies by other investigators, mesenteric responses. However, B cells also can suppress immune lymph node B cells in combination with CD8+ T cells protect 2 2 B responses in a variety of mouse models of autoim- mice from colitis induced by Gai2 / CD4+ T cells by in- munity and inflammation. Mizoguchi et al. (1, 2) first coined stigating the formation of immunosuppressive T cells (26). the term regulatory B cell to describe B cells that suppress Whether these regulatory activities and mechanisms of action disease in a mouse inflammatory bowel disease (IBD) model. are attributable to a single B cell subset or functionally diverse Similarly, the absence or loss of B cells with regulatory ac- B cell populations during IBD is unresolved. tivities exacerbates disease symptoms in models of experi- Wolf et al. (3) first demonstrated a requirement for B cells mental autoimmune encephalomyelitis (EAE) (3–7), type 1 during recovery from EAE, with mice rarely returning to diabetes (8), collagen-induced arthritis (9–11), contact hy- normal in the absence of B cells. Dittel and colleagues (27) persensitivity (12, 13), lupus (14–16), and allergy (17). The subsequently demonstrated that B cell deficiency and the identification and evolving characterization of regulatory B cells absence of B7 expression delayed the emergence of Foxp3+ and their potential mechanisms of action were comprehensively regulatory T cells (Tregs) and IL-10 in the CNS during EAE. reviewed elsewhere (18–22). Therefore, this review specifically However, most recently, they demonstrated a novel IL-10–, focuses on mouse regulatory B cells that produce IL-10, a po- B7-, and MHC class II–independent regulatory role for tent inhibitory cytokine with pleiotropic activities in vitro and B cells in suppressing autoimmunity by the maintenance of in vivo, whereas it is appreciated that monocytes, T cell subsets, Tregs via GITR ligand (28). In contrast, B cell IL-10 pro- and other cells also can produce IL-10 (23). duction dramatically modulated EAE resolution in a separate study (4). IL-10–competent B cells control EAE initiation (6), Regulatory B cells whereas Tregs and IL-10–competent B cells independently Because the existence of regulatory B cells is well documented, influence EAE resolution (7, 29, 30). Thus, functionally the current challenge is to comprehend the diversity of B cell distinct B cell subsets may regulate autoimmune responses at subsets with regulatory function that have been described, different times. Department of Immunology, Duke University Medical Center, Durham, NC 27710 Abbreviations used in this article: B10pro, progenitor B10 cell; EAE, experimental autoimmune encephalomyelitis; IBD, inflammatory bowel disease; PIM, PMA, iono- Received for publication May 23, 2014. Accepted for publication November 19, 2014. mycin, and monensin; TIM-1, T cell Ig domain and mucin domain protein 1; Treg, This work was supported by the Lymphoma Research Foundation. Foxp3+ regulatory T cell. Address correspondence and reprint requests to Dr. Thomas F. Tedder, Box 3010, Ó Department of Immunology, Room 353 Jones Building, Research Drive, Duke Univer- Copyright 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 sity Medical Center, Durham, NC 27710. E-mail address: [email protected] www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401329 1396 BRIEF REVIEWS: MOUSE REGULATORY B10 CELLS The reality that diverse molecular mechanisms and cell plasmic IL-10 (31). Reliable B10pro cell numbers are difficult subsets contribute to regulatory B cell suppression of disease to extrapolate from .48-h cultures because some B cells has created confusion, which is exacerbated by the different proliferate, whereas dead cell frequencies also increase signifi- experimental approaches, phenotypes, and assays used to de- cantly. LPS similarly induces B10pro cell functional matura- scribe these cells. In particular, most studies fractionated B cells tion ex vivo, but it also induces B10 cell IL-10 secretion, which into subsets using phenotypic markers and then characterized reduces subsequent cytoplasmic IL-10 staining. B10+B10pro these B cell subsets based on their level of regulatory activity in cells normally represent 3–8% of spleen B cells (31). Al- different disease models (21). An alternative approach was though B10 cell numbers are dynamic during inflammation used that specifically characterizes regulatory B cell subsets and autoimmunity (6, 7, 13, 15, 31), B10pro cell numbers based on their functional capacity. In particular, we focused are relatively stable. on characterizing the B cells that selectively produce IL-10, B10 effector cells actively secrete IL-10 (Fig. 1A). However, regardless of their cell surface phenotype or tissue location. the intrinsic instability of il10 mRNA and transient low-level For clarity, this IL-10–producing regulatory B cell subset was IL-10 protein expression hinder their enumeration. To cir- labeled as B10 cells to highlight that IL-10 predominantly cumvent this, IL-10 reporter mice were engineered (36). Tiger accounts for their regulatory function during inflammation, mice have an IRES-GFP element inserted following the il10 autoimmune disease, and other adaptive and innate immune locus (37), allowing for enhanced IL-10 visualization due to responses (6, 7, 13, 15, 31–33), as well as to distinguish them the longer intracellular half-life of GFP versus IL-10 (38). from other regulatory B cell subsets that function through GFP+ B cells represent ,0.2% of total B cells in naive mice, different molecular pathways. This review highlights our but GFP expression mimics cytoplasmic IL-10 induction current understanding of mouse B10 cell function with following PIM stimulation ex vivo. 10BiT mice have multiple remarks on tolerance regulation and therapeutic potential. bacterial artificial chromosome transgene insertions that drive Thy1.1 expression under the control of il10 regulatory ele- B10 cell identification by function ments (39). Cell surface Thy1.1 expression is not measurable Currently, there are no phenotypic, transcription factor, or on B cells from naive mice and is delayed relative to IL-10 lineage markers that are unique to B10 cells. Therefore, B10 expression following ex vivo PIM stimulation, but it persists cells in mice and humans are functionally defined and enu- on the cell surface following the termination of IL-10 ex- merated by their competence to express measurable IL-10 pression, allowing for the surveillance of B10 effector cells following 5 h of ex vivo stimulation with PMA and ion- post–IL-10 production (38). B10 cell frequencies increase omycin (13, 32, 34). This combination of stimuli induces and 2–4-fold in Tiger and 10BiT mice following in vivo stimula- enhances lymphocyte translation of cytokine genes that are in tion (38). Only 1% of peritoneal cavity B cells from Tiger mice an accessible configuration for transcription, resulting in express GFP ex vivo, whereas up to 3% express GFP following measurable IL-10 protein production and secretion by B10 intestinal injury, demonstrating that gut inflammation induces