Rat plasmacytoid dendritic cells are an abundant subset of MHC class II+ CD4+CD11b-OX62- and type I IFN-producing cells that exhibit selective expression of Toll-like receptors 7 and 9 and strong responsiveness to CpG

We have identified in the rat a new subset of MHC class II(+) CD4(+)CD3(-)CD11b(-) leukocytes that produce high amounts of type I IFN upon viral stimulation and that appeared homologous to plasmacytoid DC (pDC) previously described in humans and mice. These cells exhibited the following phenotype: CD5(+),CD90(+),CD45R(+),CD45RC(+),CD11c(-),CD161a(+),CD200(+),CD172a(+),CD32(+),CD86(+). Rat pDC did not express the DC-specific marker OX62 and were more abundant in the spleen than the classical CD4(+) and CD4(-) subsets of OX62(+)CD11b(+) DC we previously described that produced very little, if any, type I IFN. Spleen pDC exhibited an undifferentiated morphology and rapidly died in vitro, but showed extensive dendrite formation, survival, maturation, and moderate type I IFN production upon stimulation by oligonucleotides containing type B CpG motifs (CpG ODN). Type A CpG ODN and CD40 ligand induced pDC to produce large amounts of type I IFN, but did not promote maturation. CpG ODN and CD40 ligand, but not influenza virus, induced IL-12p40 and IL-6 secretion. Spleen pDC did not produce IL-12p70, TNF-alpha, IL-1beta, or IL-10 using these stimulation conditions. Correlating with their strong responsiveness to virus and CpG ODN, rat pDC specifically expressed Toll-like receptor 7 and 9 mRNA. Fresh spleen pDC were poor stimulators of allogenic CD4(+) and CD8(+) T cells, but became potent inducers of allogenic T cell proliferation as well as Th1 differentiation after stimulation by type B CpG. Therefore, rat pDC appear very similar to human pDC, indicating that the specific phenotype and functions of pDC have been highly conserved between species.     

Expression of the adaptor protein hematopoietic Src homology 2 is up-regulated in response to stimuli that promote survival and differentiation of B cells

Analysis of hematopoietic Src homology 2 (HSH2) protein expression in mouse immune cells demonstrated that it is expressed at low levels in resting B cells but not T cells or macrophages. However, HSH2 expression is up-regulated within 6-12 h in response to multiple stimuli that promote activation, differentiation, and survival of splenic B cells. HSH2 expression is increased in response to anti-CD40 mAb, the TLR ligands LPS and CpG DNA, and B lymphocyte stimulator (BLyS), a key regulator of peripheral B cell survival and homeostasis. Stimulation of B cells with anti-CD40 mAb, LPS, CpG DNA, or BLyS has previously been shown to induce activation of NF-kappaB. In agreement with this finding, up-regulation of HSH2 expression in response to these stimuli is blocked by inhibitors of NF-kappaB activation and is potentiated by stimulation with PMA, suggesting that HSH2 expression is dependent on NF-kappaB activation. In contrast to CD40, BAFF receptor, TLR4, and TLR9 mediated signaling, stimulation of splenic B cells via the BCR was not observed to induce expression of HSH2 unless the cells had been stimulated previously through CD40. Finally, HSH2 expression is down-regulated in splenic B cells in response to stimulation with IL-21, which has been shown to induce apoptosis, even in the presence of anti-CD40 mAb, LPS, or CpG DNA. IL-21 stimulation also results in down-regulation of antiapoptotic proteins such as Bcl-x(L) and up-regulation of proapoptotic proteins like Bim. Therefore, HSH2 expression is coordinately up-regulated with known antiapoptotic molecules and directly correlates with B cell survival.     

Bruton's tyrosine kinase separately regulates NFkappaB p65RelA activation and cytokine interleukin (IL)-10/IL-12 production in TLR9-stimulated B Cells

B lymphocytes express both B cell receptor and Toll-like receptors (TLR). We show here that Bruton's tyrosine kinase (Btk), a critical component in B cell receptor signaling, is also involved in TLR9 signaling in B cells. Stimulation of B cells with TLR9 ligand CpG oligodeoxynucleotide (ODN) leads to transient phosphorylation of Btk, and in the absence of Btk, TLR9-induced proliferation of B cells is impaired. Interestingly, Btk(-/-) B cells secrete significantly more interleukin (IL)-12 but much less IL-10 compared with wild type B cells upon TLR9 stimulation. Immunization of Btk(-/-) mice with CpG ODN also leads to elevated levels of IL-12 in vivo and consequently, a greater -fold increment in the production of Th1 type IgG2b and IgG3 antibodies in these mice compared with wild type controls. The addition of exogenous recombinant IL-10 could suppress IL-12 production by TLR9-activated Btk(-/-) B cells, suggesting that in B cells, Btk negatively regulates IL-12 through the induction of autocrine IL-10 production. TLR9 signaling also leads to the activation of NFkappaB, including the p65RelA subunit in wild type B cells. The lack of Btk signaling affects the activation of NFkappaB and impairs the translocation of the p65RelA subunit to the nucleus of B cells upon TLR9 stimulation. However, p65RelA(-/-) B cells could respond similarly to wild type B cells in terms of IL-10 and IL-12 secretion when stimulated with CpG ODN, suggesting that the defect in NFkappaB p65RelA activation is additional to the impairment in cytokine production in TLR9-activated Btk(-/-) B cells. Thus, Btk plays an important role in TLR9 signaling and acts separately to regulate NFkappaB RelA activation as well as IL-10 and IL-12 production in B cells.     

Presentation of soluble antigens to CD8+ T cells by CpG oligodeoxynucleotide-primed human naive B cells

Naive B lymphocytes are generally thought to be poor APCs, and there is limited knowledge of their role in activation of CD8(+) T cells. In this article, we demonstrate that class I MHC Ag presentation by human naive B cells is enhanced by TLR9 agonists. Purified naive B cells were cultured with or without a TLR9 agonist (CpG oligodeoxynucleotide [ODN] 2006) for 2 d and then assessed for phenotype, endocytic activity, and their ability to induce CD8(+) T cell responses to soluble Ags. CpG ODN enhanced expression of class I MHC and the costimulatory molecule CD86 and increased endocytic activity as determined by uptake of dextran beads. Pretreatment of naive B cells with CpG ODN also enabled presentation of tetanus toxoid to CD8(+) T cells, resulting in CD8(+) T cell cytokine production and granzyme B secretion and proliferation. Likewise, CpG-activated naive B cells showed enhanced ability to cross-present CMV Ag to autologous CD8(+) T cells, resulting in proliferation of CMV-specific CD8(+) T cells. Although resting naive B cells are poor APCs, they can be activated by TLR9 agonists to serve as potent APCs for class I MHC-restricted T cell responses. This novel activity of naive B cells could be exploited for vaccine design.     

Impaired naive and memory B-cell responsiveness to TLR9 stimulation in human immunodeficiency virus infection

Toll-like receptor 9 (TLR9) agonists such as unmethylated bacterial CpG DNAs activate B lymphocytes directly, potentially influencing their function and homeostasis. To assess B-cell responsiveness to TLR9 agonists in human immunodeficiency virus (HIV) disease, we examined the ability of naive and memory B cells to proliferation and to increase surface expression of CD80 in response to CpG oligonucleotides (ODN). CpG ODN induced expression of CD80 similarly in B cells from HIV-infected persons and from healthy controls. In contrast, proliferation responses to CpG ODN were markedly impaired in both naive and memory B-cell subsets from HIV-infected persons. Naive B-cell proliferation defects were related to plasma HIV RNA and, among memory B cells, to the frequencies of CD21-negative cells. Importantly, TLR9 mRNA levels were significantly diminished in freshly prepared naive B cells and especially so in memory B cells from HIV-positive viremic donors, suggesting a possible underlying mechanism for the observed functional impairments. Dose-response studies indicated that optimal induction of CD80 expression was achieved with much lower concentrations of CpG ODN than optimal induction of proliferation. We propose that the relatively low threshold of activation that is required for CD80 induction by CpG ODN might explain the preservation of this response in B cells from HIV-infected persons despite diminished TLR9 expression. Impaired responsiveness to TLR9 agonists may contribute to defects in humoral immunity in HIV infection.     

IL-10 secreting CD21+ B cells are present in jejunal Peyer’s patches of sheep during fetal development

We recently reported a novel interleukin-10 (IL-10)-secreting CD21⁺ B cell population in jejunal Peyer’s patches (JPP) of sheep with a regulatory function (B_regs) suppressing Toll-like receptor 9 (TLR9)-induced cytokine responses. However, little is known about the development of these cells. Therefore, we investigate their existence in JPP cells from fetal and newborn lambs. CD21⁺ B cells were purified from JPP cells by magnetic cell sorting and subsequently stimulated with the TLR9 agonist, CpG ODN (CpG oligodeoxynucleotide). Lymphocyte proliferative responses, cytokine production (IL-10, IL-12 and interferon-γ [INF-γ]) and antibody secretion were assayed. We found that fetal and neonatal CD21⁺ B cells spontaneously secreted high levels of IL-10 regardless of CpG stimulation but that these cells did not produce any IL-12 or INF-γ upon stimulation with CpG. The observed responses are consistent with those previously reported for B_regs characterized in JPP of older lambs. Surprisingly, unlike in older lambs, fetal and neonatal JPP CD21⁺ B cells proliferated in response to CpG stimulation. Our investigations of fetal and neonatal lambs provide evidence for the development of IL-10-secreting CD21⁺ B cells in PPs prior to antigen exposure.     

Heterozygous carriage of a dysfunctional Toll-like receptor 9 allele affects CpG oligonucleotide responses in B cells

Toll-like receptors (TLR) are employed by the innate immune system to detect microbial pathogens based on conserved microbial pathogen molecules. For example, TLR9 is a receptor for CpG-containing microbial DNA, and its activation results in the production of cytokines and type I interferons from human B cells and plasmacytoid dendritic cells, respectively. Both are required for mounting an efficient antibacterial or antiviral immune response. These effects are mimicked by synthetic CpG oligodeoxynucleotides (ODN). Although several hyporesponsive TLR9 variants have been reported, their functional relevance in human primary cells has not been addressed. Here we report a novel TLR9 allele, R892W, which is hyporesponsive to CpG ODN and acts as a dominant-negative in a cellular model system. The R892W variant is characterized by increased MyD88 binding and defective co-localization with CpG ODN. Whereas primary plasmacytoid dendritic cells isolated from a heterozygous R892W carrier responded normally to CpG by interferon-α production, carrier B cells showed impaired IL-6 and IL-10 production. This suggests that heterozygous carriage of a hyporesponsive TLR9 allele is not associated with complete loss of TLR9 function but that TLR9 signals elicited in different cell types are regulated differently in human primary cells.     

CpG oligodeoxynucleotides enhance neonatal resistance to Listeria infection

Infection by Listeria monocytogenes causes serious morbidity and mortality during the neonatal period. Previous studies established that immunostimulatory CpG oligodeoxynucleotides (ODN) can increased the resistance of adult mice to many infectious pathogens, including Listeria. This work examines the capacity of CpG ODN to stimulate a protective immune response in newborns. Results indicate that dendritic cells, macrophages, and B cells from 3-day-old mice respond to CpG stimulation by secreting IFN-gamma, IL-12, and/or TNF-alpha. Spleen cells from CpG-treated neonates produce large amounts of cytokine and NO when exposed to bacteria in vitro. Newborns treated with CpG ODN are protected from lethal Listeria challenge and generate Ag-specific CD4 and CD8 T cells that afford long-term protection against subsequent infection. These results demonstrate that cellular elements of the neonatal immune system respond to stimulation by CpG ODN, thereby reducing host susceptibility to infectious pathogens.     

Immune complex negatively regulates Toll‐like receptor 9‐mediated immune responses in B cells through the inhibitory Fc‐gamma receptor IIb

Because inappropriate activation of Toll‐like receptor 9 (TLR9) may induce pathological damage, negative regulation of the TLR9‐triggered immune response has attracted considerable attention. Nonpathogenic immune complex (IC) has been demonstrated to have beneficial therapeutic effects in some kinds of autoimmune diseases. However, the role of IC in the regulation of TLR9‐triggered immune responses and the underlying mechanisms remain unclear. In this study, it was demonstrated that IC stimulation of B cells not only suppresses CpG‐oligodeoxynucleotide (CpG‐ODN)‐induced pro‐inflammatory IL‐6 and IgM κ production, but also attenuates CD40 and CD80 expression. Furthermore, our results suggest that the receptor for the Fc portion of IgG (FcγR) IIb is involved in the suppressive effect of IC on TLR9‐mediated CD40, CD80 and IL‐6 expression. Finally, it was found that IC down‐regulates TLR9 expression in CpG‐ODN activated B cells. Our results provide an outline of a new pathway for the negative regulation of TLR9‐triggered immune responses in B cells via FcγRIIb. A new mechanistic explanation of the therapeutic effect of nonpathogenic IC on inflammatory and autoimmune diseases is also provided.     

Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides

The Toll-like receptor (TLR)9 is critical for the recognition of immunostimulatory CpG motifs but may cooperate with other TLRs. We analyzed TLR1-10 mRNA expression by using quantitative real-time PCR in highly purified subsets of human PBMC and determined the sensitivity of these subsets to CpG oligodeoxynucleotides (ODN). TLR1 and TLR6 were expressed in all cell types examined. TLR10 was highly expressed in B cells and weakly expressed in plasmacytoid dendritic cells (PDC). High expression of TLR2 was characteristic for monocytes. PDC and B cells expressed marked levels of TLR7 and TLR9 and were directly sensitive to CpG ODN. In CpG ODN-stimulated PDC and B cells, TLR9 expression rapidly decreased, as opposed to TLR7, which was up-regulated in PDC and decreased in B cells. In monocytes, NK cells, and T cells, TLR7 was absent. Despite low expression of TLR9, monocytes, NK cells, and T cells did not respond to CpG ODN in the absence of PDC but were activated in the presence of PDC. In conclusion, our studies provide evidence that PDC and B cells, but not monocytes, NK cells, or T cells, are primary targets of CpG ODN in peripheral blood. The characteristic expression pattern of TLR1-10 in cellular subsets of human PBMC is consistent with the concept that TLR9 is essential in the recognition of CpG ODN in PDC and B cells. In addition, selective regulation of TLR7 expression in PDC and B cells by CpG ODN revealed TLR7 as a candidate TLR potentially involved in modulating the recognition of CpG motifs.     

Analysis of maturation states of rat bone marrow-derived dendritic cells using an improved culture technique

In this study, we examined in more detail the development of rat bone marrow-derived dendritic cells (BMDC). A two-stage culture system was used to propagate BMDC from rat bone marrow precursors. BMDC developed within clusters of proliferating cells after repetitive addition of rat granulocyte/macrophage colony-stimulating factor and rat interleukin (IL)-4 at a concentration of 5 ng/ml to the cultures. Fluorescence-activated cell sorter analysis performed at an early stage of development (day 6) revealed an immature phenotype with intermediate levels of major histocompatibility complex (MHC) class II expression and low levels of the costimulator molecules CD80 and CD86. Upon further culture, a strong upregulation of MHC class II, costimulatory and adhesion molecules could be observed, whereas macrophage marker antigens were downregulated. Late-stage BMDC (day 10) showed a high expression of MHC class I and II, ICAM-1, Ox62 and CD11c, and revealed a split pattern of B7-1 and B7-2. The cell yield was about 40% of the initially plated bone marrow cells with 80% MHC class II-high and less than 20% MHC class II-low positive cells. Full maturation of rat BMDC (day 12) with an almost uniform expression of B7 was achieved by subsequent subculture and further stimulation with rat tumour necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS) or soluble CD40 ligand (CD40L). Analysis of the cell supernatant revealed a strong IL-12 production after LPS or CD40L, and to a lesser extent after TNF-alpha stimulation. Additionally, LPS-treated, but not CD40L-treated BMDC secreted TNF-alpha into the supernatant. Early-stage BMDC sufficiently triggered a T cell receptor (TCR) downregulation, but did not stimulate naive T cells in an allogeneic mixed leukocyte reaction (MLR) and revealed a low stimulatory capacity in an antigen-specific T cell assay. In contrast, late-stage BMDC and especially fully mature BMDC strongly induced TCR internalisation, elicited high T cell responses in the allogeneic MLR similar to those obtained by mature rat spleen dendritic cells and efficiently activated antigen-specific T cells. In conclusion, this protocol allows easy access to large numbers of rat BMDC at defined maturation stages and selective studies for the manipulation of immune responses in rat models.     

Activation of Toll‐like receptor 9 inhibits lipopolysaccharide‐induced receptor activator of nuclear factor kappa‐ B ligand expression in rat B lymphocytes

B lymphocytes express multiple TLRs that regulate their cytokine production. We investigated the effect of TLR4 and TLR9 activation on receptor activator of NF‐κB ligand (RANKL) expression by rat spleen B cells. Splenocytes or purified spleen B cells from Rowett rats were cultured with TLR4 ligand Escherichia coli LPS and/or TLR9 ligand CpG‐oligodeoxynucleotide (CpG‐ODN) for 2 days. RANKL mRNA expression and the percentage of RANKL‐positive B cells were increased in rat splenocytes challenged by E. coli LPS alone. The increases were less pronounced when cells were treated with both CpG‐ODN and E. coli LPS. Microarray analysis showed that expressions of multiple cyclin‐dependent kinase (CDK) pathway‐related genes were up‐regulated only in cells treated with both E. coli LPS and CpG‐ODN. This study suggests that CpG‐ODN inhibits LPS‐induced RANKL expression in rat B cells via regulation of the CDK pathway.     

Autoreactive preplasma cells break tolerance in the absence of regulation by dendritic cells and macrophages

The ability to induce Ab responses to pathogens while maintaining the quiescence of autoreactive cells is an important aspect of immune tolerance. During activation of TLR4, dendritic cells (DCs) and macrophages (MFs) repress autoantibody production through their secretion of IL-6 and soluble CD40L (sCD40L). These soluble mediators selectively repress B cells chronically exposed to Ag, but not naive cells, suggesting a means to maintain tolerance during TLR4 stimulation, yet allow immunity. In this study, we identify TNF-α as a third repressive factor, which together with IL-6 and CD40L account for nearly all the repression conferred by DCs and MFs. Similar to IL-6 and sCD40L, TNF-α did not alter B cell proliferation or survival. Instead, it reduced the number of Ab-secreting cells. To address whether the soluble mediators secreted by DCs and MFs functioned in vivo, we generated mice lacking IL-6, CD40L, and TNF-α. Compared to wild-type mice, these mice showed prolonged anti-nuclear Ab responses following TLR4 stimulation. Furthermore, adoptive transfer of autoreactive B cells into chimeric IL-6(-/-) × CD40L(-/-) × TNF-α(-/-) mice showed that preplasma cells secreted autoantibodies independent of germinal center formation or extrafollicular foci. These data indicate that in the absence of genetic predisposition to autoimmunity, loss of endogenous IL-6, CD40L, and TNF-α promotes autoantibody secretion during TLR4 stimulation.     

Extracellular ATP induces a distorted maturation of dendritic cells and inhibits their capacity to initiate Th1 responses

Dendritic cells (DCs) express functional purinergic receptors, but the effects of purine nucleotides on DC functions have been marginally investigated. In this study, we report on the ability of micromolar concentrations of ATP to affect the maturation and Ag-presenting function of monocyte-derived DCs in vitro. Chronic stimulation (24 h) of DCs with low, noncytotoxic ATP doses increased membrane expression of CD54, CD80, CD86, and CD83, slightly reduced the endocytic activity of DCs, and augmented their capacity to promote proliferation of allogeneic naive T lymphocytes. Moreover, ATP enhanced LPS- and soluble CD40 ligand-induced CD54, CD86, and CD83 expression. On the other hand, ATP markedly and dose-dependently inhibited LPS- and soluble CD40 ligand-dependent production of IL-1alpha, IL-1beta, TNF-alpha, IL-6, and IL-12, whereas IL-1 receptor antagonist and IL-10 production was not affected. As a result, T cell lines generated from allogeneic naive CD45RA(+) T cells primed with DCs matured in the presence of ATP produced lower amounts of IFN-gamma and higher levels of IL-4, IL-5, and IL-10 compared with T cell lines obtained with LPS-stimulated DCs. ATP inhibition of TNF-alpha and IL-12 production by mature DCs was not mediated by PGs or elevation of intracellular cAMP and did not require ATP degradation. The inability of UTP and the similar potency of ADP to reproduce ATP effects indicated that ATP could function through the P2X receptor family. These results suggest that extracellular ATP may serve as an important regulatory signal to dampen IL-12 production by DCs and thus prevent exaggerated and harmful immune responses.     

CpG oligodeoxynucleotides modulate the osteoclastogenic activity of osteoblasts via Toll-like receptor 9

Regulation of osteoclastogenesis by lipopolysaccharide (LPS) is mediated via its interactions with toll-like receptor 4 (TLR4) on both osteoclast- and osteoblast-lineage cells. We have recently demonstrated that CpG oligodeoxynucleotides (CpG ODNs), known to mimic bacterial DNA, modulate osteoclastogenesis via interactions with osteoclast precursors. In the present study we characterize the interactions of CpG ODNs with osteoblasts, in comparison with LPS. We find that, similar to LPS, CpG ODNs modulate osteoclastogenesis in bone marrow cell/osteoblast co-cultures, although in a somewhat different pattern. Osteoblasts express receptors for both LPS and CpG ODN (TLR4 and TLR9, respectively). The osteoblastic TLR9 transmits signals into the cell as demonstrated by NFkappaB activation as well as by extracellular-regulated kinase (ERK) and p38 phosphorylation. Similar to LPS, CpG ODN increases in osteoblasts the expression of tumor necrosis factor (TNF)-alpha and macrophage-colony stimulating factor (M-CSF). The two TLR ligands do not affect osteoprotegerin expression in osteoblasts. CpG ODN does not significantly affect receptor activator of NFkappaB ligand (RANKL) expression, in contrast to LPS, which induces the expression of this molecule. In the co-cultures CpG ODN induces RANKL expression in osteoblasts as a result of the more efficient TNF-alpha induction. CpG ODN activity (modulation of osteoclastogenesis, gene expression, ERK and p38 phosphorylation, and nuclear translocation of NFkappaB) is specific, because the control oligodeoxynucleotide, not containing CpG, is inactive. Furthermore, these effects (unlike the LPS effects) are inhibited by chloroquine, suggesting a requirement for endosomal maturation/acidification, the classic CpG ODN mode of action. We conclude that CpG ODN, upon TLR9 ligation, induces osteoblasts osteoclastogenic activity.     

High mobility group box protein 1: an endogenous signal for dendritic cell maturation and Th1 polarization

High mobility group box protein 1 (HMGB1), a DNA binding nuclear and cytosolic protein, is a proinflammatory cytokine released by monocytes and macrophages. This study addressed the hypothesis that HMGB1 is an immunostimulatory signal that induces dendritic cell (DC) maturation. We show that HMGB1, via its B box domain, induced phenotypic maturation of DCs, as evidenced by increased CD83, CD54, CD80, CD40, CD58, and MHC class II expression and decreased CD206 expression. The B box caused increased secretion of the proinflammatory cytokines IL-12, IL-6, IL-1alpha, IL-8, TNF-alpha, and RANTES. B box up-regulated CD83 expression as well as IL-6 secretion via a p38 MAPK-dependent pathway. In the MLR, B box-activated DCs acted as potent stimulators of allogeneic T cells, and the magnitude of the response was equivalent to DCs activated by exposure to LPS, nonmethylated CpG oligonucleotides, or CD40L. Furthermore, B box induced secretion of IL-12 from DCs as well as IL-2 and IFN-gamma secretion from allogeneic T cells, suggesting a Th1 bias. HMGB1 released by necrotic cells may be a signal of tissue or cellular injury that, when sensed by DCs, induces and/or enhances an immune reaction.     

Anti-CD180 (RP105) activates B cells to rapidly produce polyclonal Ig via a T cell and MyD88-independent pathway

CD180 is homologous to TLR4 and regulates TLR4 signaling, yet its function is unclear. We report that injection of anti-CD180 mAb into mice induced rapid Ig production of all classes and subclasses, with the exception of IgA and IgG2b, with up to 50-fold increases in serum IgG1 and IgG3. IgG production after anti-CD180 injection was not due to reactivation of memory B cells and was retained in T cell-deficient (TCR knockout [KO]), CD40 KO, IL-4 KO, and MyD88 KO mice. Anti-CD180 rapidly increased both transitional and mature B cells, with especially robust increases in transitional B cell number, marginal zone B cell proliferation, and CD86, but not CD80, expression. In contrast, anti-CD40 induced primarily follicular B cell and myeloid expansion, with increases in expression of CD80 and CD95 but not CD86. The expansion of splenic B cells was due, in part, to proliferation and occurred in wild-type and TCR KO mice, whereas T cell expansion occurred in wild-type, but not in B cell-deficient, mice, indicating a direct role for B cells in CD180 stimulation in vivo. Combination of anti-CD180 with various MyD88-dependent TLR ligands biased B cell fate because coinjection diminished Ig production, but purified B cells exhibited synergistic proliferation. Anti-CD180 had no effect on cytokine production from B cells, but it increased IL-6, IL-10, and TNF-α production in combination with LPS or CpG. Thus, CD180 stimulation induces intrinsic B cell proliferation and differentiation, causing rapid increases in IgG, and integrates MyD88-dependent TLR signals to regulate proliferation, cytokine production, and differentiation.