TRAF6 is a critical factor for dendritic cell maturation and development

IL-1 receptor (IL-1R)/Toll-like receptor (TLR) family and TNF receptor (TNFR) superfamily members are critical for regulating multiple aspects of dendritic cell (DC) biology. Several signaling pathways associated with each family utilize the adapter molecule, TRAF6, but its role in DCs is unclear. By examining TRAF6-deficient mice and bone marrow (BM) chimeras reconstituted with TRAF6-deficient fetal liver cells, we show that proper DC maturation requires TRAF6. In response to either microbial components or CD40L, TRAF6-deficient DCs fail to upregulate surface expression of MHCII and B7.2, or produce inflammatory cytokines. Moreover, LPS-treated TRAF6-deficient DCs do not exhibit an enhanced capacity to stimulate naive T cells. Interestingly, a major population of splenic DCs, the CD4(+)CD8alpha(-) subset, is nearly absent in both TRAF6-deficient mice and BM chimeras. Together these results indicate that TRAF6 regulates the critical processes required for maturation, activation, and development of DCs, the primary cellular bridge between innate and adaptive immunity.     

Intrinsic lymphotoxin-beta receptor requirement for homeostasis of lymphoid tissue dendritic cells

The factors regulating dendritic cell (DC) development and homeostasis are incompletely understood. Here, we demonstrate that DCs express the lymphotoxin (LT)-beta receptor (LT beta R) and that in mice lacking the LT beta R in hematopoietic cells, spleen, and lymph node, CD8- DC numbers are reduced. B cells are a key source of LT alpha 1 beta 2 for splenic DC homeostasis, and transgenic overexpression of LT alpha 1 beta 2 on B cells leads to expansion of the CD8- DC compartment. Furthermore, we find that about 5% of splenic DCs are undergoing cell division, and the number of dividing CD8- DCs is disproportionately reduced in the absence of the LT beta R. In parabiosis experiments, splenic DCs were only partially replaced by circulating precursors over a 6 week period. We conclude that LT alpha 1 bet a2 acts on DCs or DC precursors to promote DC homeostasis, and we suggest that DC proliferation is an important pathway for locally maintaining these cells in the steady state.     

Qualitative and quantitative abnormalities in splenic dendritic cell populations in NOD mice

The phenotype and function of splenic DC populations from diabetes-prone NOD mice were characterized and compared to DC from diabetes-resistant strains in the presence or absence of Flt3 ligand (FL) treatment. NOD mice were found to have significantly fewer CD8alpha+ DC than both B10.BR and C57BL/6 mice, and this defect was reversed by FL treatment. Freshly isolated CD8alpha+ and CD8alpha- DC from all three strains were found to express similar levels of costimulatory molecules and this was similar in both FL-treated and untreated animals. IL-12 p40 production was significantly lower in purified CD11c+ DC from NOD mice compared to DC from C57BL/6 or B10.BR mice. CD8alpha+ DC isolated from NOD mice produced lower levels of IL-12p40 than CD8alpha+ DC from C57CBL/6 and this was dependent on the nature of the stimulus given. In contrast both CD8alpha+ and CD8alpha- DC from FL-treated mice produced high levels of IL-12p40 following activation, but only the CD8alpha- DC produced IL-12p70. Functionally, freshly isolated CD8alpha- DC were more stimulatory than CD8alpha+ DC in a primary allogeneic mixed lymphocyte reaction. However, DC maturation resulted in increased T cell stimulatory capacity for both DC subsets, and this pattern was seen in all strains. These results demonstrate significant differences in phenotype and function of splenic NOD CD8alpha+ DC, and further suggest that FL treatment may reverse some of these abnormalities.     

Suppressor of cytokine signaling-1 is essential for suppressing dendritic cell activation and systemic autoimmunity

Suppressor of cytokine signaling-1 (SOCS1/JAB) negatively regulates not only the cytokine-signaling pathway but also lipopolysaccharide (LPS)-induced macrophage activation. We found that SOCS1-deficient dendritic cells (DCs) were also hyperresponsive to interferon-gamma and interleukin-4. To define the role of SOCS1-deficient DCs in vivo, we generated mice in which the SOCS1 expression was restored in T and B cells on a SOCS1(-/-) background. In these mice, DCs were accumulated in the thymus and spleen and produced high levels of BAFF/BLyS and APRIL, resulting in the aberrant expansion of B cells and autoreactive antibody production. SOCS1-deficient DCs efficiently stimulated B cell proliferation in vitro and autoantibody production in vivo. These results indicate that SOCS1 plays an essential role in the normal DC functions and suppression of systemic autoimmunity.     

Intrasplenic steady-state dendritic cell precursors that are distinct from monocytes

Immediate precursors of the many subtypes of dendritic cells (DCs) remain obscure. Here we purified a splenic precursor population that produced all splenic CD8+ and CD8- conventional DCs (cDCs) but not plasmacytoid DCs or other lineages. This 'pre-cDC' population included cells 'precommitted' to form either CD8+ or CD8- cDCs. The pre-cDCs, which comprised 0.05% of splenocytes, expressed a CD11c(int) CD45RA(lo) CD43(int) SIRP-alpha(int) CD4- CD8- major histocompatibility complex class II-negative surface phenotype. The pre-cDCs were not monocytes. Monocytes generated few cDCs in steady-state recipient mice. However, when transferred into mice with an inflammatory milieu dependent on granulocyte-macrophage colony-stimulating factor, monocytes produced a distinct type of splenic DC. Thus, the inflammatory status of the host influences the developmental origin and type of DC present in lymphoid tissues.     

Dendritic cell expansion occurs in mesenteric lymph nodes of B10.BR mice infected with the murine nematode parasite Trichuris muris

Dendritic cells (DCs) are a crucial element in the immune system and bridge innate and adaptive immunity. CD11c+ B220- DCs residing in Peyer's patches (PPs) have the ability to produce interleukin 10 (IL-10) and induce T helper (Th2) development. Evidence suggests that CD11c+ B220- DCs maintain the gut environment by suppressing Th1 responses with IL-10, resulting in a Th2-dominat gut environment. Th2 effectors are required for protection against the murine nematode parasite Trichuris muris, and thus CD11c+ B220- DCs may be involved in the induction of Th2 cells in T. muris infection. In the present study, the kinetics of CD11c+ B220- DCs were analyzed in mesenteric lymph nodes of B10.BR mice infected with the E-J isolate of T. muris, and the cellular expansion of CD11c+ B220- DCs was also observed. As well, the DC expansion was consistent with the occurrence of worm expulsion augmented by IL-4 and IL-13. The evidence here suggests the involvement of CD11c+ B220- DCs in protective Th2 responses to T. muris infection.     

Rv0315, a novel immunostimulatory antigen of Mycobacterium tuberculosis, activates dendritic cells and drives Th1 immune responses

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is one of the most deadly infectious diseases, with approximately two million people dying of TB annually. An effective therapeutic method for activating dendritic cells (DCs) and driving Th1 immune responses would improve host defenses and further the development of a TB vaccine. Given the importance of DC maturation in eliciting protective immunity against TB, we investigated whether Rv0315, a newly identified Mtb antigen, can prompt DC maturation. We found that Rv0315 functionally activated DCs by augmenting the expression of the co-stimulatory molecules CD80 and CD86 as well as MHC class I/II molecules. Moreover, it increased DC secretion of the pro-inflammatory cytokines IL-6, IL-1β, and TNF-α. Unlike LPS, however, Rv0315 induced the secretion of IL-12p70, but not IL-10. In addition, Rv0315-treated DCs accelerated the proliferation of CD4(+) and CD8(+) splenic T cells from Mtb-infected mice, with increased levels of IFN-γ, in syngeneic and allogeneic mixed lymphocyte reactions, indicating that Rv0315 contributes to Th1 polarization of the immune response. Importantly, both mitogen-activated protein kinases and nuclear factor κB signaling mediated the expression of DC surface markers and cytokines. Taken together, our results indicate that Rv0315 is a novel DC maturation-inducing antigen that drives T cell immune responses toward Th1 polarization, suggesting that Rv0315 plays a key role in determining the nature of the immune response to TB.     

Dendritic cells and the regulation of a granulomatous immune response in the lung

To investigate the contribution of dendritic cells (DC) in a pulmonary granulomatous immune response, C57BL/l6 mice, nonimmunized or immunized with purified protein derivative (PPD) of Mycobacterium bovis, were intravenously injected with PPD-coated Sepharose-4B beads. One and three days later lungs were harvested, granuloma size was measured, and immunolabeled cells in granulomas were counted. On Day 1, granulomas in immunized mice were 3-fold larger and contained more major histocompatibility complex class II+, CD11c+ DCs than nonimmunized mice. By Day 3, these differences had diminished. In all granulomas MHC class II+, CD11c+ DCs were in contact with the beads. By in situ hybridization these DCs expressed interleukin (IL)-12 p40 mRNA. MOMA2+ macrophages were present throughout the granulomas, whereas CD4+ and CD8alpha+ T cells were localized at the granuloma periphery. DCs isolated from granulomatous lungs at Day 1, and from thoracic lymph nodes (LNs) at Days 1 and 3, stimulated PPD-specific T cell proliferation without exogenously added antigen, indicating that they had acquired bead-bound antigen. By Day 3, however, granuloma DCs presented little antigen, suggesting that newly immigrated DC lacked access to antigen or that antigen uptake/processing was inhibited. RNase protection assays of whole-lung mRNA showed increased interferon-gamma, IL-1beta, IL-1 receptor antagonist, IL-6, and macrophage inhibitory factor, but no IL-10 mRNA on Days 1 and 3. These observations support the premise that DCs are key in initiating granulomatous cell-mediated immunity. However, factors generated within the granuloma downregulate the antigen presenting function of DC by Day 3 in this experimental model.     

CD44 is dispensable for B lymphopoiesis

Several studies have implicated a role for the cell surface glycoprotein CD44 in lymphocyte differentiation, adhesion to the matrix, homing, activation and apoptosis. However, the requirement of CD44 in B cell maturation remains elusive. To address this point, we analyzed the generation and activation of B lymphocytes in CD44-deficient mice. Unexpectedly, both maturation and autoreconstitution of early bone marrow B cell progenitors after sublethal irradiation as well as frequencies of splenic B cell subsets are indistinguishable in wild-type and CD44-deficient mice. Furthermore, splenic CD44-deficient B lymphocytes show a normal activation pattern after stimulation with LPS, anti-IgM/IL-4 or anti-CD40/IL-4. These results show for the first time that CD44 is clearly dispensable for development, reconstitution and activation of B lymphocytes.     

The CC chemokine ligand 3 regulates CD11c+CD11b+CD8alpha- dendritic cell maturation and activation following viral infection of the central nervous system: implications for a role in T cell activation

The role of CC chemokine ligand 3 (CCL3) in activation of dendritic cells (DCs) following mouse hepatitis virus (MHV) infection of the central nervous system (CNS) was examined. The results indicate that CCL3 participates in an effective host response to MHV infection by contributing to CD11c+CD11b+CD8alpha- DC maturation, activation, and migration to cervical lymph nodes (CLN). Diminished CD8alpha- DC activation correlated with reduced IFN-gamma expression by virus-specific T cells accompanied by increased IL-10 production suggesting that CCL3 contributes to an effective host response to viral infection by enhancing the T cell activation potential of DC.     

Dendritic cells during polymicrobial sepsis rapidly mature but fail to initiate a protective Th1-type immune response

Polymicrobial sepsis is associated with immunosuppression caused by the predominance of anti-inflammatory mediators and profound loss of lymphocytes through apoptosis. Dendritic cells (DC) are potent antigen-presenting cells and play a key role in T cell activation. We tested the hypothesis that DC are involved in sepsis-mediated immunosuppression in a mouse cecal ligation and puncture (CLP) model, which resembles human polymicrobial sepsis. At different time-points after CLP, DC from the spleen and peripheral lymph nodes were characterized in terms of expression of costimulatory molecules, cytokine synthesis, and subset composition. Splenic DC strongly up-regulated CD86 and CD40 but not CD80 as soon as 8 h after CLP. In contrast, lymph node DC equally increased the expression of CD86, CD40, and CD80. However, this process of maturation occurred later in the lymph nodes than in the spleen. Splenic DC from septic mice were unable to secrete interleukin (IL)-12, even upon stimulation with CpG or lipopolysaccharide+CD40 ligand, but released high levels of IL-10 in comparison to DC from control mice. Neutralization of endogenous IL-10 could not restore IL-12 secretion by DC of septic mice. In addition, the splenic CD4+CD8- and CD4-CD8+ subpopulations were lost during sepsis, and the remaining DC showed a reduced capacity for allogeneic T cell activation associated with decreased IL-2 synthesis. Thus, during sepsis, splenic DC acquire a state of aberrant responsiveness to bacterial stimuli, and two DC subtypes are selectively lost. These changes in DC behavior might contribute to impaired host response against bacteria during sepsis.     

MIP-3alpha and MIP-1alpha rapidly mobilize dendritic cell precursors into the peripheral blood

Acquisition of dendritic cells (DCs) or DC precursors in vitro is critical for DC-based immunotherapy. We reported previously that administration of MIP-1alpha mobilized a population of F4/80(-)B220(-)CD11c+ DC precursors into peripheral blood by the expression of CCR1 and CCR5. In this study, we identified a new subset of CCR6+CCR1(-)CCR5(-)B220(-)CD11c(+) cells in MIP-1alpha-administered mice. When cultured with GM-CSF, IL-4, and TNF-alpha, these cells differentiated into mature DCs, possessing the typical morphologic characteristics, phenotypes, and antigen-presenting function (termed CCR6+ DC precursors). Although it did not directly drive the CCR6+ DC precursors, MIP-1alpha could recruit a population of F4/80+CD11c(-) monocyte/macrophage-producing MIP-3alpha in the peripheral blood to mobilize a CCR6+ DC precursor subset of B220(-)CD11c+ DC precursors. Importantly, exogenous administration of MIP-3alpha significantly enhanced MIP-1alpha-induced mobilization of DC precursors. Moreover, these MIP-3alpha- and MIP-1alpha-mobilized DC precursors could be prepared for a DC vaccine capable of eliciting CTL responses to tumor cells, leading to tumor rejection in vitro and in vivo. Taken together, this study further demonstrates the mechanism of DC precursor mobilization induced by MIP-1alpha; that is, besides mobilizing DC precursors with CCR1 and CCR5 expressions, MIP-1alpha recruited F4/80+CD11c(-) monocyte/macrophage-producing MIP-3alpha, which finally mobilized the CCR6+ DC precursor subset to amplify the B220(-)CD11c+ DC precursor population. Furthermore, combined administration of MIP-3alpha and MIP-1alpha may be an efficient strategy for collecting a large number of DCs appropriate for immunotherapy.     

Administration of alpha-galactosylceramide impairs the survival of dendritic cell subpopulations in vivo

In this study, we examine whether recognition of α-GalCer presented on CD1d-expressing DCs and B cells in vivo elicits the cytotoxic activity of iNKT cells and elimination of α-GalCer-presenting cells. We report that i.v. injection of α-GalCer induced a decrease in the percentage and number of splenic CD8(+)Langerin(+) DCs, while CD8(-) DCs were not affected. The decline in CD8(+) DC numbers was clearly detectable by 15 h after α-GalCer injection, was maximal at 24-48 h, returned to normal by day 7, and was accompanied by a reduced cross-presentation of OVA protein given i.v. to specific CD8(+) T cells in vitro. The decrease in the numbers of CD8(+) DCs required iNKT cells but was independent of perforin, Fas, or IFN-γ, as it was observed in mice deficient in each of these molecules. In contrast, treatment with a TNF-α-neutralizing antibody was effective at reducing the decline in CD8(+) DC numbers and DC activation. Treatment with immunostimulatory CpG ODN also resulted in DC activation and a decreased number of CD8(+) DCs; however, the decline in DC number was a result of down-regulation of CD11c and CD8 and did not require iNKT cells or TNF-α. Although CD8(+)Langerin(+) DCs appeared to be selectively affected by α-GalCer treatment, they were not required for early iNKT cell responses, as their prior depletion did not prevent the increase in serum TNF-α and IL-4 observed after α-GalCer treatment. Thus, iNKT cells regulate the survival of CD8(+) DCs through a mechanism that does not appear to involve direct cell killing.     

Dendritic cell-associated lectin 2 (DCAL2) defines a distinct CD8α- dendritic cell subset

CLRs on DCs play important roles in immunity and are expressed selectively on certain DC subsets. Murine DCAL2 (myeloid inhibitory C-type lectin/Clec12a) is a type-II CLR with an ITIM. Using a mouse DCAL2-specific mAb, we found that DCAL2 is expressed at relatively high levels on APCs and that DCAL2 expression can be used to divide CD8α- DCs into DCAL2+DCIR2- and DCAL2-DCIR2+ subpopulations. CD8α-DCAL2+ DC, CD8α-DCIR2+ DC, and CD8α+DCAL2+ DC subsets each express different levels of TLRs and respond to unique classes of TLR ligands by producing distinct sets of cytokines. Whereas CD8α-DCAL2+ DCs robustly produce cytokines, including IL-12, in response to CpG, CD8α-DCIR2+ DCs produce only TNF-α and IL-10 in modest amounts when stimulated with zymosan. However, CD8α-DCIR2+DCs, unlike the other DC subsets, strongly up-regulate OX40L when stimulated with bacterial flagellin. As predicted from their cytokine expression, CD8α-DCAL2+ DCs efficiently induced Th1 responses in the presence of CpG in vitro and in vivo, whereas CD8α-DCIR2+ DCs induced Th2 cells in response to flagellin. Thus, CD8α-DCAL2+ DCs comprise a distinct CD8α- DC subset capable of supporting Th1 responses. DCAL2 is a useful marker to identify a Th1-inducing CD8α- DC population.     

Cholera toxin adjuvant promotes a balanced Th1/Th2/Th17 response independently of IL-12 and IL-17 by acting on Gsα in CD11b+ DCs

Despite an extensive literature on the mechanism of action of cholera toxin (CT), we still lack critical information about how the toxin acts as an adjuvant and, especially, which dendritic cells (DCs) are the target cells. Although a T helper type 2 (Th2)-skewing effect of CT is most commonly reported, effective priming of Th17 cells as well as suppression of Th1 responses are well documented. However, the ability of CT to block interferon regulatory factor 8 (IRF8) function and interleukin (IL)-12 production in DCs, which blocks CD8α DC and Th1 cell development, is inconsistent with priming of Th1 and CD8 T cells in many other reports. This prompted us to investigate the adjuvant effect of CT in wild-type, IL-12p40−/−, Batf3−/−, and IL-17A−/− mice and in mice that selectively lack the Gsα target protein for CT adenosine diphosphate (ADP)-ribosylation in DCs. We found that CT promoted Th1 priming independently of IL-12, and whereas Th2 and also Th17 responses were augmented, the gut IgA responses did not require IL-17A. Adjuvanticity was intact in Batf3−/− mice, lacking CD8α⁺ DCs, but completely lost in mice with Gsα-deficient CD11c cells. Thus, our data demonstrate that the adjuvant effect requires Gsα expression in CD11b⁺ DCs, and that priming of mucosal IgA and CD4 T cells appears unbiased and is independent of IL-12 and IL-17A.     

Specific subsets of murine dendritic cells acquire potent T cell regulatory functions following CTLA4-mediated induction of indoleamine 2,3 dioxygenase

Murine dendritic cells (DCs) expressing indoleamine 2,3 dioxygenase (IDO) catabolize tryptophan and can suppress T cell responses elicited in vivo. Here, we identify specific subsets of splenic (CD11c+) dendritic cells competent to mediate IDO-dependent T cell suppression following CTLA4-mediated ligation of B7 molecules. IDO-competent DC subsets acquired potent and dominant T cell suppressive properties as a consequence of IDO up-regulation, as they blocked the ability of T cells to respond to other stimulatory DCs in the same cultures. Soluble CTLA4 (CTLA4-Ig) and cloned CTLA4+ regulatory T cells (Tr1D1) up-regulated IDO selectively in DC subsets co-expressing B220 or CD8alpha. The ability of Tr1D1 T cells to suppress CD8+ T cell responses was completely dependent on their ability to induce tryptophan catabolism in DCs. Selective IDO up-regulation in DCs did not inhibit T cell activation, but prevented T cell clonal expansion due to rapid death of activated T cells. T cell responses were restored by genetic or pharmacologic inhibition of IDO enzyme activity, or by adding excess tryptophan. DCs from interferon gamma (IFNgamma)-receptor-deficient mice were effective in promoting IDO-dependent T cell suppression following CTLA4-Ig exposure in vivo, indicating that IFNgamma signaling was not necessary for IDO up-regulation in this model. These findings suggest that IDO-competent DCs provide a regulatory bridge, mediated by CTLA4-B7 engagement, between certain regulatory T cell subsets and naive responder T cells.     

Late dendritic cells are still able to evoke a potent alloreactive CTL response

On exposure to maturation stimuli, immature dendritic cells (DCs) undergo changes that turn them into potent amplifiers of innate immunity and into antigen-presenting cells (APCs) able to prime na?ve T cells. However, their progression through the maturation process is very rapid and finally ends in apoptosis. The aim of our study was to investigate the importance of the maturation stage of DCs, defined by morphology, expression of surface markers and IL-12 production, for their immunostimulatory capacity. DCs were matured with LPS, monocyte-conditioned medium (MCM) or TNF-alpha, sampled several times during a 3-day long maturation period and used as stimulators of allogeneic T cells over a wide range of DC/T cell ratios. T-cell response was assessed by cell proliferation, CTL generation and IFN-gamma production. Our results indicate that the in vitro T cell response is determined mainly by the level of expression of co-stimulatory molecules on DCs and the DC/T cell ratio in the culture. Thus, DCs matured for over 20h, with high expression of co-stimulatory molecules, can still induce a potent CTL response at DC/T cell ratios of 1:10 and 1:20, although their IL-12 production, as well as their ability to induce IFN-gamma production by T cells, are both decreased. In contrast, the CTL response at DC/T cell ratios of 1:2 and 1:5 can be profoundly decreased. Notably, the proportion of proliferating CD4+ T cells in these cultures is reduced. This could well be the reason for the absence of CTL response, since we showed that, even in the case of high expression of co-stimulatory molecules on DCs, generation of CTLs still depends on CD4+ T cells. Our study emphasizes the importance of strong expression of co-stimulatory molecules on DCs and of their ability to activate CD8+ and CD4+ T cells concomitantly in order to initiate a potent cell-mediated immune response. We therefore suggest that a combination of early DCs, which are strong producers of cytokines, and late DCs, which have high expression of co-stimulatory molecules, could prove beneficial in the attempt to initiate in vitro and in vivo cell-mediated immune responses for therapeutic purposes.