MyD88-dependent activation of B220-CD11b+LY-6C+ dendritic cells during Brucella melitensis infection

IFN-gamma is a key cytokine controlling Brucella infection. One of its major function is the stimulation of Brucella-killing effector mechanisms, such as inducible NO synthase (iNOS)/NOS2 activity, in phagocytic cells. In this study, an attempt to identify the main cellular components of the immune response induced by Brucella melitensis in vivo is made. IFN-gamma and iNOS protein were analyzed intracellularly using flow cytometry in chronically infected mice. Although TCRbeta(+)CD4(+) cells were the predominant source of IFN-gamma in the spleen, we also identified CD11b(+)LY-6C(+)LY-6G(-)MHC-II(+) cells as the main iNOS-producing cells in the spleen and the peritoneal cavity. These cells appear similar to inflammatory dendritic cells recently described in the mouse model of Listeria monocytogenes infection and human psoriasis: the TNF/iNOS-producing dendritic cells. Using genetically deficient mice, we demonstrated that the induction of iNOS and IFN-gamma-producing cells due to Brucella infection required TLR4 and TLR9 stimulation coupled to Myd88-dependent signaling pathways. The unique role of MyD88 was confirmed by the lack of impact of Toll-IL-1R domain-containing adaptor inducing IFN-beta deficiency. The reduction of IFN-gamma(+) and iNOS(+) cell frequency observed in MyD88-, TLR4-, and TLR9-deficient mice correlated with a proportional lack of Brucella growth control. Taken together, our results provide new insight into how immune responses fight Brucella infection.     

Endotoxin-induced maturation of MyD88-deficient dendritic cells

LPS, a major component of the cell wall of Gram-negative bacteria, can induce a variety of biological responses including cytokine production from macrophages, B cell proliferation, and endotoxin shock. All of them were completely abolished in MyD88-deficient mice, indicating the essential role of MyD88 in LPS signaling. However, MyD88-deficient cells still show activation of NF-kappaB and mitogen-activated protein kinase cascades, although the biological significance of this activation is not clear. In this study, we have examined the effects of LPS on dendritic cells (DCs) from wild-type and several mutant mice. LPS-induced cytokine production from DCs was dependent on MyD88. However, LPS could induce functional maturation of MyD88-deficient DCs, including up-regulation of costimulatory molecules and enhancement of APC activity. MyD88-deficient DCs could not mature in response to bacterial DNA, the ligand for Toll-like receptor (TLR)9, indicating that MyD88 is differentially required for TLR family signaling. MyD88-dependent and -independent pathways originate at the intracytoplasmic region of TLR4, because both cytokine induction and functional maturation were abolished in DCs from C3H/HeJ mice carrying the point mutation in the region. Finally, in vivo analysis revealed that MyD88-, but not TLR4-, deficient splenic CD11c(+) DCs could up-regulate their costimulatory molecule expression in response to LPS. Collectively, the present study provides the first evidence that the MyD88-independent pathway downstream of TLR4 can lead to functional DC maturation, which is critical for a link between innate and adaptive immunity.     

TNF-alpha-dependent and -independent maturation of dendritic cells and recruited CD11c(int)CD11b+ Cells during oral Salmonella infection

Maturation of dendritic cells (DC) is crucial for their ability to induce adaptive immunity. Although several mediators of DC maturation have been found, their contributions to DC maturation during infection are poorly understood. In this study we show that murine conventional (CD11c(high)) DC up-regulate costimulatory molecules in a subset-specific manner after oral Salmonella infection. Although both CD8alpha+ and CD8alpha- subsets increase CD86 expression, CD40 was preferentially up-regulated on CD8alpha+ DC, and CD80 was preferentially increased on CD8alpha- DC. In addition, high levels of CD80 and CD86 were found on CD11c(int)CD11b+ cells that accumulated in infected organs. Costimulatory molecules were simultaneously induced on CD11c(high) and CD11c(int)CD11b+ cells in Peyer's patches, mesenteric lymph nodes and spleen 5 days after infection despite different kinetics of peak bacterial burden in these organs. Up-regulation of costimulatory molecules occurred on all DC within the respective subset. Moreover, <1% of CD11c-expressing cells associated with Salmonella expressing enhanced GFP in vivo. Thus, DC maturation did not depend on bacterial uptake. Rather, infection-induced up-regulation of CD80, CD86, and CD40 on CD11c-expressing cells of mesenteric lymph nodes was dependent on TNFR type I (TNFRI) signaling. Although indirect up-regulation of costimulatory molecules on DC and CD11c(int)CD11b+ cells was TNFRI dependent, cells directly associated with Salmonella were able to mature independently of TNFRI signaling. Thus, Salmonella-induced TNF-alpha is an important mediator of indirect DC maturation during infection, whereas a TNF-alpha-independent maturation pathway contributes to direct maturation of bacteria-associated DC.     

Newly activated T cells promote maturation of bystander dendritic cells but not IL-12 production

The activation of dendritic cells (DC) leads to increased costimulatory activity (termed DC maturation) and, in some instances, production of immunomodulatory cytokines such as IL-12. Both innate and T cell-derived signals can promote DC activation but it is unclear to what extent the two classes of stimuli are interchangeable or regulate distinct aspects of DC function. In this study, we show that signals from newly activated CD4(+) T cells cannot initiate IL-12 synthesis although they can amplify secretion of bioactive IL-12 p70 by DC exposed to an appropriate innate stimulus. This occurs exclusively in cis and does not influence IL-12 synthesis by bystander DC that do not present Ag. In marked contrast, signals from newly activated CD4(+) T cells can induce an increase in DC costimulatory activity in the absence of any innate priming. This occurs both in cis and in trans, affecting all DC in the microenvironment, including those that do not bear specific Ag. Consistent with the latter, we show that newly activated CD4(+) T cells in vivo can deliver "help" in trans, effectively lowering the number of MHC/peptide complexes required for proliferation of third-party naive CD4(+) T cells recognizing Ag on bystander DC. These results demonstrate that DC maturation and cytokine production are regulated distinctly by innate stimuli vs signals from CD4(+) T cells and reveal a process of trans activation of DC without secretion of polarizing cytokines that takes place during T cell priming and may be involved in amplifying immune responses.     

Central role of MyD88-dependent dendritic cell maturation and proinflammatory cytokine production to control Brucella abortus infection

Brucella abortus is a facultative intracellular bacterium that infects humans and domestic animals. The enhanced susceptibility to virulent B. abortus observed in MyD88 knockout (KO) mice led us to investigate the mechanisms involved in MyD88-dependent immune responses. First, we defined the role of MyD88 in dendritic cell (DC) maturation. In vitro as well as in vivo, B. abortus-exposed MyD88 KO DCs displayed a significant impairment on maturation as observed by expression of CD40, CD86, and MHC class II on CD11c+ cells. In addition, IL-12 and TNF-alpha production was totally abrogated in MyD88 KO DCs and macrophages. Furthermore, B. abortus-induced IL-12 production was found to be dependent on TLR2 in DC, but independent on TLR2 and TLR4 in macrophages. Additionally, we investigated the role of exogenous IL-12 and TNF-alpha administration on MyD88 KO control of B. abortus infection. Importantly, IL-12, but not TNF-alpha, was able to partially rescue host susceptibility in MyD88 KO-infected animals. Furthermore, we demonstrated the role played by TLR9 during virulent B. abortus infection. TLR9 KO-infected mice showed 1 log Brucella CFU higher than wild-type mice. Macrophages and DC from TLR9 KO mice showed reduced IL-12 and unaltered TNF-alpha production when these cells were stimulated with Brucella. Together, these results suggest that susceptibility of MyD88 KO mice to B. abortus is due to impaired DC maturation and lack of IL-12 synthesis. Additionally, DC activation during Brucella infection plays an important regulatory role by stimulating and programming T cells to produce IFN-gamma.     

MyD88-dependent activation of dendritic cells and CD4(+) T lymphocytes mediates symptoms, but is not required for the immunological control of parasites during rodent malaria

We investigated the role of different TLRs and MyD88 in host resistance to infection and malaria pathogenesis. TLR2(-/-), TLR4(-/-), TLR6(-/-), TLR9(-/-) or CD14(-/-) mice showed no change in phenotypes (parasitemia, body weight and temperature) when infected with Plasmodium chabaudi chabaudi (AS). MyD88(-/-) mice displayed comparable ability to wild type animals in controlling and clearing parasitemia. Importantly, MyD88(-/-) mice exhibited impaired production of TNF-alpha and IFN-gamma as well as attenuated symptoms, as indicated by changes in body weight and temperature during parasitemia. Consistently, CD11b(+) monocytes and CD11c(+) dendritic cells from infected MyD88(-/-) mice were shown impaired for production of pro-inflammatory cytokines, and in initiating CD4(+) T cell responses. Importantly, the inhibition of T cell activation with anti-CD134L, mostly inhibited IFN-gamma, partially inhibited TNF-alpha production, and protected the animals from malaria symptoms. Our findings suggest that MyD88 and possibly its associated TLRs expressed by dendritic cells play an important role in pro-inflammatory responses, T cell activation, and pathogenesis of malaria, but are not critical for the immunological control of the erythrocytic stage of P. chabaudi.     

Survival, maturation, and function of CD11c- and CD11c+ peripheral blood dendritic cells are differentially regulated by cytokines.

Two types of dendritic cells (DC) are circulating in human blood and can be identified by their differential expression of the myeloid Ag CD11c. In this study, we show that CD11c- peripheral blood (PB)-DC correspond to plasmacytoid DC of lymphoid tissue not only by their surface Ag expression profile but, more impressively, by their peculiar ultramorphology. We also demonstrate that CD11c- and CD11c+ DC differ in the quality of their response to and in their requirement for certain cytokines. Freshly isolated CD11c- cells depend on IL-3 for survival and use autocrine or exogenous TNF-alpha as maturation signal, leading to the appearance of a highly dendritic phenotype, the up-regulation and redistribution of MHC class II from lysosomal compartments to the plasma membrane, the increased expression of costimulatory molecules, and the switch from a high Ag-processing to a low Ag-processing/potent accessory cell mode. Surprisingly, IL-4 efficiently killed freshly isolated CD11c- PB-DC, but did not impair the viability of CD11c+ PB-DC and, together with GM-CSF, induced maturation of these cells. A direct functional comparison revealed that neo-Ag-modified and subsequently matured CD11c- but to a lesser extent CD11c+ DC were able to prime naive Ag-specific CD4+ T cells. Our findings show that two diverse DC types respond to certain T cell-derived cytokines in a differential manner and, thus, suggest that suppression or activation of functionally diverse DC types may be a novel mechanism for the regulation of the quantity and quality of immune responses.     

CD36 is differentially expressed by CD8+ splenic dendritic cells but is not required for cross-presentation in vivo

Cross-presentation allows the processing of Ags from donor cells into the MHC class I presentation pathway of dendritic cells (DCs). This is important for the generation of cytotoxic T cell immunity and for induction of self tolerance. Apoptotic cells are reported to be efficient targets for cross-presentation, and in vitro studies using human DCs have implicated CD36 in their capture. In support of a role for CD36 in cross-presentation, we show that this molecule is differentially expressed by CD8(+) splenic DCs, which previously have been identified as responsible for cross-presentation in the mouse. Three different cross-presentation models were examined for their dependence on CD36. These included cross-priming to OVA-coated spleen cells and cross-tolerance to OVA transgenically expressed in the pancreatic islet beta cells under constitutive conditions or during beta cell destruction. In these models, CD36 knockout DCs were equivalent to wild-type DCs in their capacity to cross-present either foreign or self Ags, indicating that CD36 is not essential for cross-presentation of cellular Ags in vivo.     

MyD88-mediated instructive signals in dendritic cells regulate pulmonary immune responses during respiratory virus infection

Respiratory syncytial virus (RSV) is the leading cause of respiratory disease in infants worldwide. The induction of innate immunity and the establishment of adaptive immune responses are influenced by the recognition of pathogen-associated molecular patterns by TLRs. One of the primary pathways for TLR activation is by MyD88 adapter protein signaling. The present studies indicate that MyD88 deficiency profoundly impacts the pulmonary environment in RSV-infected mice characterized by the accumulation of eosinophils and augmented mucus production. Although there was little difference in CD4 T cell accumulation, there was also a significant decrease in conventional dendritic cells recruitment to the lungs of MyD88(-/-) mice. The exacerbation of RSV pathophysiology in MyD88(-/-) mice was associated with an enhanced Th2 cytokine profile that contributed to an inappropriate immune response. Furthermore, bone marrow-derived dendritic cells (BMDC) isolated from MyD88(-/-) mice were incapable of producing two important Th1 instructive signals, IL-12 and delta-like4, upon RSV infection. Although MyD88(-/-) BMDCs infected with RSV did up-regulate costimulatory molecules, they did not up-regulate class II as efficiently and stimulated less IFN-gamma from CD4(+) T cells in vitro compared with wild-type BMDCs. Finally, adoptive transfer of C57BL/6 BMDCs into MyD88(-/-) mice reconstituted Th1 immune responses in vivo, whereas transfer of MyD88(-/-) BMDCs into wild-type mice skewed the RSV responses toward a Th2 phenotype. Taken together, our data indicate that MyD88-mediated pathways are essential for the least pathogenic responses to this viral pathogen through the regulation of important Th1-associated instructive signals.     

Bacterial CpG-DNA triggers activation and maturation of human CD11c-, CD123+ dendritic cells

Human plasmacytoid precursor dendritic cells (ppDC) are a major source of type I IFN upon exposure to virus and bacteria, yet the stimulus causing their maturation into DCs is unknown. After PBMC activation with immunostimulatory bacterial DNA sequences (CpG-DNA) we found that ppDC are the primary source of IFN-alpha. In fact, either CpG-DNA or dsRNA (poly(I:C)) induced IFN-alpha from purified ppDC. Surprisingly, only CpG-DNA triggered purified ppDC survival, maturation, and production of TNF, GM-CSF, IL-6, and IL-8, but not IL-10 or IL-12. Known DC activators such as CD40 ligation triggered ppDC maturation, but only IL-8 production, while bacterial LPS was negative for all activation criteria. An additional finding was that only CpG-DNA could counteract IL-4-induced apoptosis in ppDC. Therefore, CpG-DNA represents a pathogen-associated molecular pattern for ppDC. In contrast to these finding, CpG-DNA, like LPS, caused TNF, IL-6, and IL-12 release from PBMC and purified monocytes; however, differentiation of monocytes into DCs with GM-CSF and IL-4 unexpectedly resulted in refractoriness to CpG-DNA, but not LPS. Taken together, these results suggest that within a DC subset a multiplicity of responses can be generated by distinct environmental stimuli and that responses to a given stimulus may be dissimilar between DC subsets.     

Autoimmunity stimulated by adoptively transferred dendritic cells is initiated by both alphabeta and gammadelta T cells but does not require MyD88 signaling

Vaccination of nonautoimmune prone mice with syngeneic dendritic cells (DC) readily induces anti-DNA autoantibodies but does not trigger systemic disease. We observed that anti-DNA autoantibody generation absolutely required alphabeta T cells and that gammadelta T cells also contributed to the response, but that regulatory T cells restrained autoantibody production. Although both NZB/W F(1) mice and DC vaccinated C57/BL6 mice produced autoantibodies against dsDNA, vaccinated mice had higher levels of Abs against H1 histone and lower levels of antinucleosome Abs than NZB/W F(1) mice. Despite a 100-fold increase in IL-12 and Th1 skewing to a foreign Ag, OVA, synergistic TLR activation of DC in vitro failed to augment anti-DNA Abs or promote class switching beyond that induced by LPS alone. TLR stimulation was not absolutely required for the initial loss of B cell tolerance because anti-DNA levels were similar when wild-type (WT) or MyD88-deficient DC were used for vaccination or WT and MyD88-deficient recipients were vaccinated with WT DC. In contrast, systemic administration of LPS, augmented anti-DNA Ab levels and promoted class switching, and this response was dependent on donor DC signaling via MyD88. LPS also augmented responses in the MyD88-deficient recipients, suggesting that LPS likely exerts its effects on both transferred DC and host B cells in vivo. These results indicate that both the alphabeta and gammadelta subsets are necessary for promoting autoantibody production by DC vaccination, and that although TLR/MyD88 signaling is not absolutely required for initiation, this pathway does promote augmentation, and Th1-mediated skewing, of anti-DNA autoantibodies.     

Langerhans cells require MyD88-dependent signals for Candida albicans response but not for contact hypersensitivity or migration

Langerhans cells (LC) are a subset of skin-resident dendritic cells (DC) that reside in the epidermis as immature DC, where they acquire Ag. A key step in the life cycle of LC is their activation into mature DC in response to various stimuli, including epicutaneous sensitization with hapten and skin infection with Candida albicans. Mature LC migrate to the skin-draining LN, where they present Ag to CD4 T cells and modulate the adaptive immune response. LC migration is thought to require the direct action of IL-1β and IL-18 on LC. In addition, TLR ligands are present in C. albicans, and hapten sensitization produces endogenous TLR ligands. Both could contribute to LC activation. We generated Langerin-Cre MyD88(fl) mice in which LC are insensitive to IL-1 family members and most TLR ligands. LC migration in the steady state, after hapten sensitization and postinfection with C. albicans, was unaffected. Contact hypersensitivity in Langerin-Cre MyD88(fl) mice was similarly unaffected. Interestingly, in response to C. albicans infection, these mice displayed reduced proliferation of Ag-specific CD4 T cells and defective Th17 subset differentiation. Surface expression of costimulatory molecules was intact on LC, but expression of IL-1β, IL-6, and IL-23 was reduced. Thus, sensitivity to MyD88-dependent signals is not required for LC migration, but is required for the full activation and function of LC in the setting of fungal infection.     

TLR4 and Toll-IL-1 receptor domain-containing adapter-inducing IFN-beta, but not MyD88, regulate Escherichia coli-induced dendritic cell maturation and apoptosis in vivo

Dendritic cells (DC) are short-lived, professional APCs that play a central role in the generation of adaptive immune responses. Induction of efficient immune responses is dependent on how long DCs survive in the host. Therefore, the regulation of DC apoptosis in vivo during infection remains an important question that requires further investigation. The impact of Escherichia coli bacteremia on DCs has never been analyzed. We show here that i.v. or i.p. administration of live or heat-killed E. coli in mice induces splenic DC migration, maturation, and apoptosis. We further characterize which TLR and Toll-IL-1R (TIR)-containing adaptor molecules regulate these processes in vivo. In this model, DC maturation is impaired in TLR2(-/-), TLR4(-/-) and TIR domain-containing adapter-inducing IFN-beta (TRIF)(-/-) mice. In contrast, DC apoptosis is reduced only in TLR4(-/-) and TRIF(-/-) mice. As expected, DC apoptosis induced by the TLR4 ligand LPS is also abolished in these mice. Injection of the TLR9 ligand CpG-oligodeoxynucleotide (synthetic bacterial DNA) induces DC migration and maturation, but only modest DC apoptosis when compared with LPS and E. coli. Together, these results suggest that E. coli bacteremia directly impacts on DC maturation and survival in vivo through a TLR4-TRIF-dependent signaling pathway.     

Human CD4+CD25high regulatory T cells modulate myeloid but not plasmacytoid dendritic cells activation

Human CD4(+)CD25(+) regulatory T cells (Treg) play an essential role in the prevention of autoimmune diseases. However, the mechanisms of immune suppression and the spectrum of cells they target in vivo remain incompletely defined. In particular, although Treg directly suppress conventional T cells in vitro, they have been shown to inhibit the Ag-presenting functions of macrophage- and monocyte-derived dendritic cells (DC). We have now studied the maturation of human blood-derived myeloid DC and plasmacytoid DC activated with TLR ligands in the presence of Treg. Preactivated Treg suppressed strongly TLR-triggered myeloid DC maturation, as judged by the blocking of costimulatory molecule up-regulation and the inhibition of proinflammatory cytokines secretion that resulted in poor Ag presentation capacity. Although IL-10 played a prominent role in inhibiting cytokines secretion, suppression of phenotypic maturation required cell-cell contact and was independent of TGF-beta and CTLA-4. In contrast, the acquisition of maturation markers and production of cytokines by plasmacytoid DC triggered with TLR ligands were insensitive to regulatory T cells. Therefore, human Treg may enlist myeloid, but not plasmacytoid DC for the initiation and the amplification of tolerance in vivo by restraining their maturation after TLR stimulation.     

The lipopolysaccharide adjuvant effect on T cells relies on nonoverlapping contributions from the MyD88 pathway and CD11c+ cells

Bacterial LPS is a natural adjuvant that induces profound effects on T cell clonal expansion, effector differentiation, and long-term T cell survival. In this study, we delineate the in vivo mechanism of LPS action by pinpointing a role for MyD88 and CD11c(+) cells. LPS induced long-term survival of superantigen-stimulated CD4 and CD8 T cells in a MyD88-dependent manner. By tracing peptide-stimulated CD4 T cells after adoptive transfer, we showed that for LPS to mediate T cell survival, the recipient mice were required to express MyD88. Even when peptide-specific CD4 T cell clonal expansion was dramatically boosted by enforced OX40 costimulation, OX40 only synergized with LPS to induce survival when the recipient mice expressed MyD88. Nevertheless, these activated, but moribund, T cells in the MyD88(-/-) mice acquired effector properties, such as the ability to synthesize IFN-gamma, demonstrating that effector differentiation is not automatically coupled to a survival program. We confirmed this notion in reverse fashion by showing that effector differentiation was not required for the induction of T cell survival. Hence, depletion of CD11c(+) cells did not affect LPS-driven specific T cell survival, but CD11c(+) cells were paramount for optimal effector T cell differentiation as measured by IFN-gamma potential. Thus, LPS adjuvanticity is based on MyD88 promoting T cell survival, while CD11c(+) cells support effector T cell differentiation.     

CD11c+CD11b+ dendritic cells play an important role in intravenous tolerance and the suppression of experimental autoimmune encephalomyelitis

The central role of T cells in the induction of immunological tolerance against i.v. Ags has been well documented. However, the role of dendritic cells (DCs), the most potent APCs, in this process is not clear. In the present study, we addressed this issue by examining the involvement of two different DC subsets, CD11c(+)CD11b(+) and CD11c(+)CD8(+) DCs, in the induction of i.v. tolerance. We found that mice injected i.v. with an autoantigen peptide of myelin oligodendrocyte glycoprotein (MOG) developed less severe experimental autoimmune encephalomyelitis (EAE) following immunization with MOG peptide but presented with more CD11c(+)CD11b(+) DCs in the CNS and spleen. Upon coculturing with T cells or LPS, these DCs exhibited immunoregulatory characteristics, including increased production of IL-10 and TGF-beta but reduced IL-12 and NO; they were also capable of inhibiting the proliferation of MOG-specific T cells and enhancing the generation of Th2 cells and CD4(+)CD25(+)Foxp3(+) regulatory T cells. Furthermore, these DCs significantly suppressed ongoing EAE upon adoptive transfer. These results indicate that CD11c(+)CD11b(+) DCs, which are abundant in the CNS of tolerized animals, play a crucial role in i.v. tolerance and EAE and may be a candidate cell population for immunotherapy of autoimmune diseases.     

Transforming growth factor-beta differentially inhibits MyD88-dependent, but not TRAM- and TRIF-dependent, lipopolysaccharide-induced TLR4 signaling

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional, potent anti-inflammatory cytokine produced by many cell types that regulates cell proliferation, apoptosis, and immune responses. Toll-like receptors (TLRs) recognize various pathogen-associated molecular patterns and are therefore a pivotal component of the innate immune system. In this study we show that TGF-beta1 blocks the NF-kappaB activation and cytokine release that is stimulated by ligands for TLRs 2, 4, and 5. We further show that TGF-beta1 can specifically interfere with TLR2, -4, or -5 ligand-induced responses involving the adaptor molecule MyD88 (myeloid differentiation factor 88) but not the TRAM/TRIF signaling pathway by decreasing MyD88 protein levels in a dose- and time-dependent manner without altering its mRNA expression. The proteasome inhibitor epoxomicin abolished the MyD88 degradation induced by TGF-beta1. Furthermore, TGF-beta1 resulted in ubiquitination of MyD88 protein, suggesting that TGF-beta1 facilitates ubiquitination and proteasomal degradation of MyD88 and thereby attenuates MyD88-dependent signaling by decreasing cellular levels of MyD88 protein. These findings importantly contribute to our understanding of molecular mechanisms mediating anti-inflammatory modulation of immune responses by TGF-beta1.     

Macrophages and myeloid dendritic cells, but not plasmacytoid dendritic cells, produce IL-10 in response to MyD88- and TRIF-dependent TLR signals, and TLR-independent signals

We have previously reported that mouse plasmacytoid dendritic cells (DC) produce high levels of IL-12p70, whereas bone marrow-derived myeloid DC and splenic DC produce substantially lower levels of this cytokine when activated with the TLR-9 ligand CpG. We now show that in response to CpG stimulation, high levels of IL-10 are secreted by macrophages, intermediate levels by myeloid DC, but no detectable IL-10 is secreted by plasmacytoid DC. MyD88-dependent TLR signals (TLR4, 7, 9 ligation), Toll/IL-1 receptor domain-containing adaptor-dependent TLR signals (TLR3, 4 ligation) as well as non-TLR signals (CD40 ligation) induced macrophages and myeloid DC to produce IL-10 in addition to proinflammatory cytokines. IL-12p70 expression in response to CpG was suppressed by endogenous IL-10 in macrophages, in myeloid DC, and to an even greater extent in splenic CD8alpha(-) and CD8alpha(+) DC. Although plasmacytoid DC did not produce IL-10 upon stimulation, addition of this cytokine exogenously suppressed their production of IL-12, TNF, and IFN-alpha, showing trans but not autocrine regulation of these cytokines by IL-10 in plasmacytoid DC.     

CD4+ CD25+ regulatory T cells inhibit the maturation but not the initiation of an autoantibody response

To investigate the mechanism by which T regulatory (Treg) cells may control the early onset of autoimmunity, we have used an adoptive transfer model to track Treg, Th, and anti-chromatin B cell interactions in vivo. We show that anti-chromatin B cells secrete Abs by day 8 in vivo upon provision of undeviated, Th1- or Th2-type CD4+ T cell help, but this secretion is blocked by the coinjection of CD4+ CD25+ Treg cells. Although Treg cells do not interfere with the initial follicular entry or activation of Th or B cells at day 3, ICOS levels on Th cells are decreased. Furthermore, Treg cells must be administered during the initial phases of the Ab response to exert full suppression of autoantibody production. These studies indicate that CD25+ Treg cells act to inhibit the maturation, rather than the initiation, of autoantibody responses.