Peritoneal Cavity Regulatory B Cells (B10 Cells) Modulate IFN-γ+CD4+ T Cell Numbers during Colitis Development in Mice

“T cells, B cells, monocytes, macrophages, mast cells, and eosinophils can all secrete IL-10 that suppresses inflammatory cytokine production, Th1/Th2 polarization, and Ag presentation (5, 13, 14).”

“Given that T cells also play critical regulatory roles during IBD development in mice, as well as in humans (1, 3), it is likely that B10 and regulatory Foxp3+ CD4+ T cells share complementary regulatory functions for IBD, as previously shown for experimental autoimmune encephalomyelitis (29).”

“VH-D-JH and VK-JK transcript alignments and phylogenetic trees based on average percent identity were constructed using ClustalW2 (45).”

“Rag2 and 10BiT reporter mice were as described (39, 40).”

“T cells, B cells, monocytes, macrophages, mast cells, and eosinophils can all secrete IL-10 that suppresses inflammatory cytokine production, Th1/Th2 polarization, and Ag presentation (5, 13, 14).”

“That peritoneal cavity B10 cells can alter the development and severity of colitis was further verified using the inducible T cell– transfer model of colitis (10).”

“B10 cells represent a small and functionally distinct B cell subset that shares some overlapping phenotypic markers with multiple B cell subpopulations, including spleen B1 cells, marginal zone progenitor cells, marginal zone cells, memory cells and plasmablasts (23, 24, 46, 51-53).”

“Productive Ig rearrangements were compared against germline Ig sequences, according to the Ig Basic Local Alignment Search Tool database (National Center for Biotechnology Information, Bethesda, MD), and analyzed using the Immunogenetics V-query and Standardization tool (44) to determine V(D)J gene family usage.”

“Consistent with their in vivo Ag stimulation, spleen B10 cells are hyper-responsive to mitogens (23, 28) and can give rise to Ag-specific, self-reactive or natural antibodies (46).”

“Given that T cells also play critical regulatory roles during IBD development in mice, as well as in humans (1, 3), it is likely that B10 and regulatory Foxp3+ CD4+ T cells share complementary regulatory functions for IBD, as previously shown for experimental autoimmune encephalomyelitis (29).”

“T cells, B cells, monocytes, macrophages, mast cells, and eosinophils can all secrete IL-10 that suppresses inflammatory cytokine production, Th1/Th2 polarization, and Ag presentation (5, 13, 14).”

“Human and mouse B10 cell IL-10 production is central to their ability to negatively regulate innate and Ag-specific adaptive immune responses as well as inflammation and autoimmune disease (23-25, 28-33).”

“Mice deficient in IL10, a potent immunoregulatory cytokine with anti-inflammatory properties (5), are highly susceptible to chronic enterocolitis that is spontaneously triggered by intestinal microbiota (6, 7).”

“Although the induction of colitis requires both spontaneous and Ag-specific T cell proliferation, driven in part by microbiota-derived innate signals (1, 70, 71), the intestinal microbiota also trigger innate immune responses that can modify the T cell compartment and prevent IBD (20, 70, 72).”

“LPS was added to the 5-h PMA, ionomycin, and monensin (PIM)-stimulated cultures because it marginally enhances B10 cell enumeration (28).”

“2A-B) that are functionally distinct (48).”

“Human IBD is also genetically linked with il10 locus polymorphisms or altered serum IL-10 concentrations (11, 12).”

“LPS was added to the 5-h PMA, ionomycin, and monensin (PIM)-stimulated cultures because it marginally enhances B10 cell enumeration (28).”

“Receptors or pathways that positively or negatively regulate BCR signaling such as CD19, CD22 and CD40 can also significantly modulate B10 cell numbers (28, 54-57).”

“1) or within the spleen or peripheral lymph nodes (26).”

“Adoptively transferred peritoneal cavity B10 cells can also regulate skin inflammation during contact hypersensitivity responses (37), and B10 cell numbers expand significantly within the CNS during experimental autoimmune encephalomyelitis (29).”

“Tiger mice have an IRES-GFP element inserted following the il10 locus (38).”

“B10 cells represent a small and functionally distinct B cell subset that shares some overlapping phenotypic markers with multiple B cell subpopulations, including spleen B1 cells, marginal zone progenitor cells, marginal zone cells, memory cells and plasmablasts (23, 24, 46, 51-53).”

“Spleen B10 cells are predominantly found within the minor CD1dhiCD5+ B cell subpopulation along with B10 progenitor (B10pro) cells that are induced to acquire IL-10 competence during in vitro culture with agonistic CD40 mAb or LPS (28, 30, 32).”

“However, peritoneal B cells can also migrate into the periphery under certain conditions or following stimulation, particularly through TLRs (65).”

“Th1 and Th17 cells are major disease contributors in both the IL-10-deficient (IL-10−/−) mouse model of spontaneous disease and the CD4+ T cell-induced model of colitis, with IFN-γ– and IL-17-competent T cells detectable at all stages of disease in mice and humans (1-4).”

“B10 cells represent a small and functionally distinct B cell subset that shares some overlapping phenotypic markers with multiple B cell subpopulations, including spleen B1 cells, marginal zone progenitor cells, marginal zone cells, memory cells and plasmablasts (23, 24, 46, 51-53).”

“Receptors or pathways that positively or negatively regulate BCR signaling such as CD19, CD22 and CD40 can also significantly modulate B10 cell numbers (28, 54-57).”

“Mature B cell depletion in humans with ulcerative colitis using CD20 mAb was ineffective in a placebo-controlled study (15), and has even been suggested to exacerbate colonic inflammation in some patients (16, 17).”

“For immunofluorescence analysis, single-cell leukocyte suspensions (1 3 10(6) cells) were stained on ice for 20–30 min using predetermined optimal concentrations of mAbs, as described (41).”

“fined in humans and mice by their ability to express IL-10 following 5 h of ex vivo phorbol ester and ionomycin stimulation (23–25), which distinguishes them from regulatory B cells that modulate immune responses through other mechanisms (26, 27).”

“Given that T cells also play critical regulatory roles during IBD development in mice, as well as in humans (1, 3), it is likely that B10 and regulatory Foxp3+ CD4+ T cells share complementary regulatory functions for IBD, as previously shown for experimental autoimmune encephalomyelitis (29).”

“C hronic inflammatory disorders of the intestine are collectively referred to as inflammatory bowel disease (IBD), with ulcerative colitis and Crohn’s disease being the most prevalent in humans (1).”

“Thereby, peritoneal cavity B10 cell VH use was similar to that observed for spleen B10 cells (46) and conventional B cells (49), but it also included sequences commonly associated with peritoneal cavity B1 cells (58, 59).”

“Human and mouse B10 cell IL-10 production is central to their ability to negatively regulate innate and Ag-specific adaptive immune responses as well as inflammation and autoimmune disease (23-25, 28-33).”

“VH3 and VH12 were the most frequently observed VH families, consistent with the predominance of these families within the B1 cell repertoire (49, 50).”

“In studies by other investigators, mesenteric lymph node B cells, in combination with CD8 T cells, protected mice from colitis induced by Gai2 CD4 T cells through the formation of regulatory T cells (68).”

“Lymphocytes were isolated on day 3 or 7 and assayed for reporter protein expression, as described (46).”

“The adoptive transfer of spleen B10 cells dramatically reduces DSS-induced colon inflammation in CD19 mice, which are B10 cell deficient (22).”

“Mature B cell depletion in humans with ulcerative colitis using CD20 mAb was ineffective in a placebo-controlled study (15), and has even been suggested to exacerbate colonic inflammation in some patients (16, 17).”

“Continuous rIL-10 treatment attenuates pathology in the T cell–transfer model of colitis following the adoptive transfer of CD25(2)CD45RBCD4 T cells into lymphocyte-deficient Rag2 mice (4, 9, 10).”

“Mature B cell depletion in humans with ulcerative colitis using CD20 mAb was ineffective in a placebo-controlled study (15), and has even been suggested to exacerbate colonic inflammation in some patients (16, 17).”

“The cellular origin of B10 cells is unknown, while the origin of CD5+ B1 cells has been a matter of debate for over 30 years (62, 63).”

“LPS was added to the 5-h PMA, ionomycin, and monensin (PIM)-stimulated cultures because it marginally enhances B10 cell enumeration (28).”

“A modified scoring system was used to evaluate histological changes based on three parameters of disease, as described (47).”

“autoimmune encephalomyelitis and during Listeria infections (32, 33).”

“Because functionally matured B10pro cells and ex vivo B10 cells both express IL-10 after these cultures, B10+B10pro cell frequencies are measured (42).”

“Mizoguchi and colleagues (67) recently demonstrated a regulatory role for peritoneal cavity B1 cells from TCRa mice during chronic colitis, possibly through generating natural Abs in response to microbial flora.”

“Although the fetal liver was once thought to be the exclusive source of B1 cells, adult bone marrow precursor cells can become fully functional CD5 cells in adult mice (64).”

“Receptors or pathways that positively or negatively regulate BCR signaling such as CD19, CD22 and CD40 can also significantly modulate B10 cell numbers (28, 54-57).”

“Spontaneous ex vivo secretion of IL-10 by peritoneal cavity B cells has been well described (36, 60), with B1a, B1b, and B2 cells variably contributing to IL-10 secretion in vitro (Fig.”

“Thus, spontaneous ex vivo IL-10 production by peritoneal cavity B10 cells may reflect their prior in vivo interactions with T cells, although it remains possible that innate- or pathogeninduced signals may also induce B10 cell IL-10 production in vivo (46, 61).”

“3A) (69), B10 cells may be functionally important both centrally within the spleen and within the peritoneal cavity and GALTs during colitis.”

“Receptors or pathways that positively or negatively regulate BCR signaling such as CD19, CD22 and CD40 can also significantly modulate B10 cell numbers (28, 54-57).”

“Thereby, peritoneal cavity B10 cell VH use was similar to that observed for spleen B10 cells (46) and conventional B cells (49), but it also included sequences commonly associated with peritoneal cavity B1 cells (58, 59).”

“The mAbs used in this study are as follows: CD21/35 (7G6), CD23 (B3B4), CD24 (M1/69), CD25 (PC61), CD38 (90/CD38), CD43 (S7), CD44 (IM7), CD138 (281-2), and CD11b (M1/70) (all from BD Pharmingen, San Diego, CA); IgM (11/41), CD4 (GK1.”

“cDNA was synthesized, and Ig H and L chain transcripts were amplified using nested PCR primers, as described (43).”

“Human IBD is also genetically linked with il10 locus polymorphisms or altered serum IL-10 concentrations (11, 12).”

“Given that T cells also play critical regulatory roles during IBD development in mice, as well as in humans (1, 3), it is likely that B10 and regulatory Foxp3 CD4 T cells share complementary regulatory functions for IBD, as previously shown for experimental autoimmune encephalomyelitis (29).”

“Spleen B cells exposed to enterobacterial components can also acquire IL-10-dependent suppressive functions and inhibit experimental colitis (21).”

“These data are reminiscent of animal studies where Breg cells have been shown to lead to the resolution of colitis in mice [88].”

“Besides, murine IL-10 Bregs can prevent the recruitment of neutrophils to the sites of inflammation in a model of colitis (42).”

“Whether these regulatory activities and mechanisms of action are attributable to a single B cell subset or functionally diverse B cell populations during IBD is unresolved.”