Induction of CTL and nonpolarized Th cell responses by CD8alpha(+) and CD8alpha(-) dendritic cells

Two distinct dendritic cell (DC) subpopulations have been evidenced in mice on the basis of their differential CD8alpha expression and their localization in lymphoid organs. Several reports suggest that CD8alpha(+) and CD8alpha(-) DC subsets could be functionally different. In this study, using a panel of MHC class I- and/or class II-restricted peptides, we analyzed CD4(+) and CD8(+) T cell responses obtained after i.v. injection of freshly purified peptide-pulsed DC subsets. First, we showed that both DC subsets efficiently induce specific CTL responses and Th1 cytokine production in the absence of CD4(+) T cell priming. Second, we showed that in vivo activation of CD4(+) T cells by CD8alpha(+) or CD8alpha(-) DC, injected i.v., leads to a nonpolarized Th response with production of both Th1 and Th2 cytokines. The CD8alpha(-) subset induced a higher production of Th2 cytokines such as IL-4 and IL-10 than the CD8alpha(+) subset. However, IL-5 was produced by CD4(+) T cells activated by both DC subsets. When both CD4(+) and CD8(+) T cells were primed by DC injected i.v., a similar pattern of cytokines was observed, but, under these conditions, Th1 cytokines were mainly produced by CD8(+) T cells, while Th2 cytokines were produced by CD4(+) T cells. Thus, this study clearly shows that CD4(+) T cell responses do not influence the development of specific CD8(+) T cell cytotoxic responses induced either by CD8alpha(+) or CD8alpha(-) DC subsets.     

IL-12 induction by a TH1-inducing adjuvant in vivo: dendritic cell subsets and regulation by IL-10

IL-12 induction is critical for immune responses against many viruses and intracellular bacterial pathogens. Recent studies suggest that IL-12-secreting dendritic cells (DC) are potent Th1-inducing APC. However, controversy exists concerning the function of DC subsets. Murine studies have suggested that CD8(+) DC preferentially induce Th1 responses, whereas CD8(-) DC induce Th2 development; in this model, different DC subsets prime different responses. Alternatively, the propensity of DC subsets to prime a Th1 response could depend upon the type of initial stimulus. We used a prototypic Th1-inducing adjuvant, heat-killed Brucella abortus (HKBA) to assess stimulation of DC subsets, relationship between Ag burden and IL-12 production, and down-regulation of DC subset IL-12 production by IL-10. In this study, we show that DC were sole producers of IL-12, although most HKBA uptake was by splenic macrophages and granulocytes. More CD8(-) than CD8(+) DC produced IL-12 after HKBA challenge, whereas only CD8(+) DC produced IL-12 after injection of another Th1-promoting microbial substance, soluble Toxoplasma gondii Ags. Studies in IL-10-deficient mice revealed that IL-10 down-regulates frequency and duration of IL-12 production by both DC subsets. In the absence of IL-10, IL-12 expression is enabled in CD11c(low) cells, but not in macrophages or granulocytes. These findings support the concept of DC as the major IL-12 producers in spleens, but challenge the notion that CD8(+) and CD8(-) DC are destined to selectively induce Th1 or Th2 responses, respectively. Thus, the nature of the stimulating substance is important in determining which DC subsets are activated to produce IL-12.     

TNF-alpha drives human CD14+ monocytes to differentiate into CD70+ dendritic cells evoking Th1 and Th17 responses

Many mechanisms involving TNF-alpha, Th1 responses, and Th17 responses are implicated in chronic inflammatory autoimmune disease. Recently, the clinical impact of anti-TNF therapy on disease progression has resulted in re-evaluation of the central role of this cytokine and engendered novel concept of TNF-dependent immunity. However, the overall relationship of TNF-alpha to pathogenesis is unclear. Here, we demonstrate a TNF-dependent differentiation pathway of dendritic cells (DC) evoking Th1 and Th17 responses. CD14(+) monocytes cultured in the presence of TNF-alpha and GM-CSF converted to CD14(+) CD1a(low) adherent cells with little capacity to stimulate T cells. On stimulation by LPS, however, they produced high levels of TNF-alpha, matrix metalloproteinase (MMP)-9, and IL-23 and differentiated either into mature DC or activated macrophages (M phi). The mature DC (CD83(+) CD70(+) HLA-DR (high) CD14(low)) expressed high levels of mRNA for IL-6, IL-15, and IL-23, induced naive CD4 T cells to produce IFN-gamma and TNF-alpha, and stimulated resting CD4 T cells to secret IL-17. Intriguingly, TNF-alpha added to the monocyte culture medium determined the magnitude of LPS-induced maturation and the functions of the derived DC. In contrast, the M phi (CD14(high)CD70(+)CD83(-)HLA-DR(-)) produced large amounts of MMP-9 and TNF-alpha without exogenous TNF stimulation. These results suggest that the TNF priming of monocytes controls Th1 and Th17 responses induced by mature DC, but not inflammation induced by activated M phi. Therefore, additional stimulation of monocytes with TNF-alpha may facilitate TNF-dependent adaptive immunity together with GM-CSF-stimulated M phi-mediated innate immunity.     

Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo

Dendritic cells (DCs) have a unique ability to stimulate naive T cells. Recent evidence suggests that distinct DC subsets direct different classes of immune responses in vitro and in vivo. In humans, the monocyte-derived CD11c+ DCs induce T cells to produce Th1 cytokines in vitro, whereas the CD11c- plasmacytoid T cell-derived DCs elicit the production of Th2 cytokines. In this paper we report that administration of either Flt3-ligand (FL) or G-CSF to healthy human volunteers dramatically increases distinct DC subsets, or DC precursors, in the blood. FL increases both the CD11c+ DC subset (48-fold) and the CD11c- IL-3R+ DC precursors (13-fold). In contrast, G-CSF only increases the CD11c- precursors (>7-fold). Freshly sorted CD11c+ but not CD11c- cells stimulate CD4+ T cells in an allogeneic MLR, whereas only the CD11c- cells can be induced to secrete high levels of IFN-alpha, in response to influenza virus. CD11c+ and CD11c- cells can mature in vitro with GM-CSF + TNF-alpha or with IL-3 + CD40 ligand, respectively. These two subsets up-regulate MHC class II costimulatory molecules as well as the DC maturation marker DC-lysosome-associated membrane protein, and they stimulate naive, allogeneic CD4+ T cells efficiently. These two DC subsets elicit distinct cytokine profiles in CD4+ T cells, with the CD11c- subset inducing higher levels of the Th2 cytokine IL-10. The differential mobilization of distinct DC subsets or DC precursors by in vivo administration of FL and G-CSF offers a novel strategy to manipulate immune responses in humans.     

Cytokines regulate the capacity of CD8alpha(+) and CD8alpha(-) dendritic cells to prime Th1/Th2 cells in vivo

Prior studies have shown that subclasses of dendritic cells (DC) direct the development of distinct Th populations in rodents and in humans. In the mouse, we have recently shown that administration of Ag-pulsed CD8alpha(-) DC induces a Th2-type response, whereas injection of CD8alpha(+) DC leads to Th1 differentiation. To define the DC-derived factors involved in the polarization of Th responses, we injected either subset purified from mice genetically deficient for IFN-gamma, IL-4, IL-12, or IL-10 into wild-type animals. In this work, we report that DC-derived IL-12 and IFN-gamma are required for Th1 priming by CD8alpha(+) DC, whereas IL-10 is required for optimal development of Th2 cells by CD8alpha(-) DC. The level of IL-12 produced by the DC appears to determine the Th1/Th2 balance in vivo. We further show that the function of DC subsets displays some flexibility. Treatment of DC with IL-10 in vitro induces a selective decrease in the viability of CD8alpha(+) DC. Conversely, incubation with IFN-gamma down-regulates the Th2-promoting capacities of CD8alpha(-) DC and increases the Th1-skewing properties of both subsets.     

Differential requirement for the CD40-CD154 costimulatory pathway during Th cell priming by CD8 alpha+ and CD8 alpha- murine dendritic cell subsets

Dendritic cells (DCs) regulate the development of distinct Th populations and thereby provoke appropriate immune responses to various kinds of Ags. In the present work, we investigated the role CD40-CD154 interactions play during the process of Th cell priming by CD8 alpha(+) and CD8 alpha(-) murine DC subsets, which have been reported to differently regulate the Th response. Adoptive transfer of Ag-pulsed CD8 alpha(+) DCs induced a Th1 response and the production of IgG2a Abs, whereas transfer of CD8 alpha(-) DCs induced Th2 cells and IgE Abs in vivo. Induction of distinct Th populations by each DC subset was also confirmed in vitro. Although interruption of CD80/CD86-CD28 interactions inhibited Th cell priming by both DC subsets, disruption of CD40-CD154 interactions only inhibited the induction of the Th1 response by CD8 alpha(+) DCs in vivo. CD40-CD154 interactions were not required for the proliferation of Ag-specific naive Th cells stimulated by either DC subset, but were indispensable in the production of IL-12 from CD8 alpha(+) DCs and their induction of Th1 cells in vitro. Taken together, in our immunization model of Ag-pulsed DC transfer, CD40-CD154 interactions play an important role in the development of CD8 alpha(+) DC-driven Th1 responses but not CD8 alpha(-) DC-driven Th2 responses to protein Ags.     

Distinct and overlapping effector functions of expanded human CD4+, CD8α+ and CD4-CD8α- invariant natural killer T cells

CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+), CD8α(+) and CD4(-)CD8α(-) double-negative (DN) subsets. CD4(+) iNKT cells expanded more readily than CD8α(+) and DN iNKT cells upon mitogen stimulation. CD8α(+) and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+) cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4(+) and CD8α(+) fractions, respectively. Only CD4(+) iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α(+), DN or CD4(+) iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.     

Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo.

The adaptive immune system has evolved distinct responses against different pathogens, but the mechanism(s) by which a particular response is initiated is poorly understood. In this study, we investigated the type of Ag-specific CD4(+) Th and CD8(+) T cell responses elicited in vivo, in response to soluble OVA, coinjected with LPS from two different pathogens. We used Escherichia coli LPS, which signals through Toll-like receptor 4 (TLR4) and LPS from the oral pathogen Porphyromonas gingivalis, which does not appear to require TLR4 for signaling. Coinjections of E. coli LPS + OVA or P. gingivalis LPS + OVA induced similar clonal expansions of OVA-specific CD4(+) and CD8(+) T cells, but strikingly different cytokine profiles. E. coli LPS induced a Th1-like response with abundant IFN-gamma, but little or no IL-4, IL-13, and IL-5. In contrast, P. gingivalis LPS induced Th and T cell responses characterized by significant levels of IL-13, IL-5, and IL-10, but lower levels of IFN-gamma. Consistent with these results, E. coli LPS induced IL-12(p70) in the CD8alpha(+) dendritic cell (DC) subset, while P. gingivalis LPS did not. Both LPS, however, activated the two DC subsets to up-regulate costimulatory molecules and produce IL-6 and TNF-alpha. Interestingly, these LPS appeared to have differences in their ability to signal through TLR4; proliferation of splenocytes and cytokine secretion by splenocytes or DCs from TLR4-deficient C3H/HeJ mice were greatly impaired in response to E. coli LPS, but not P. gingivalis LPS. Therefore, LPS from different bacteria activate DC subsets to produce different cytokines, and induce distinct types of adaptive immunity in vivo.     

Differential control of T regulatory cell proliferation and suppressive activity by mature plasmacytoid versus conventional spleen dendritic cells

Anergy and suppression are cardinal features of CD4(+)CD25(+)Foxp3(+) T cells (T regulatory cells (Treg)) which have been shown to be tightly controlled by the maturation state of dendritic cells (DC). However, whether lymphoid organ DC subsets exhibit different capacities to control Treg is unclear. In this study, we have analyzed, in the rat, the role of splenic CD4(+) and CD4(-) conventional DC and plasmacytoid DC (pDC) in allogeneic Treg proliferation and suppression in vitro. As expected, in the absence of exogenous IL-2, Treg did not expand in response to immature DC. Upon TLR-induced maturation, all DC became potent stimulators of CD4(+)CD25(-) T cells, whereas only TLR7- or TLR9-matured pDC induced strong proliferation of CD4(+)CD25(+)Foxp3(+) T cells in the absence of exogenous IL-2. This capacity of pDC to reverse Treg anergy required cell contact and was partially CD86 dependent and IL-2 independent. In suppression assays, Treg strongly suppressed proliferation and IL-2 and IFN-gamma production by CD4(+)CD25(-) T cells induced by mature CD4(+) and CD4(-) DC. In contrast, upon stimulation by mature pDC, proliferating Treg suppressed IL-2 production by CD25(-) cells but not their proliferation or IFN-gamma production. Taken together, these results suggest that anergy and the suppressive function of Treg are differentially controlled by DC subsets.     

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.     

Antigen presentation capacity and cytokine production by murine splenic dendritic cell subsets upon Salmonella encounter

Salmonella typhimurium is an intracellular bacterium that replicates in the spleen and mesenteric lymph nodes (MLN) of orally infected mice. However, little is known about the Ag presentation and cytokine production capacity of dendritic cells (DC), particularly CD8alpha(+), CD8alpha(-)CD4(-), and CD8alpha(-)CD4(+) DC, from these organs in response to SALMONELLA: Infection of purified splenic DC with S. typhimiurium expressing green fluorescent protein (GFP) and OVA revealed that all three splenic DC subsets internalize bacteria, and splenic as well as MLN DC process Salmonella for peptide presentation. Furthermore, presentation of Salmonella Ags on MHC-I and MHC-II was evident in both CD8alpha(+) and CD8alpha(-) splenic DC subsets. Direct ex vivo analysis of splenic DC from mice infected with GFP-expressing Salmonella showed that all three subsets harbored bacteria, and splenic DC purified from mice given Salmonella-expressing OVA presented OVA-derived peptides on MHC-I and MHC-II. Cytokine production analyzed by intracellular staining of splenic DC infected with GFP-expressing Salmonella revealed that TNF-alpha was produced by a large percentage of CD8alpha(-) DC, while only a minor proportion of CD8alpha(+) DC produced this cytokine following bacterial exposure. In contrast, the greatest number of IL-12p40-producing DC were among CD8alpha(+) DC. Experiments inhibiting bacterial uptake by cytochalasin D as well as use of a Transwell system revealed that bacterial contact, but not internalization, was required for cytokine production. Thus, DC in sites of Salmonella replication and T cell activation, spleen and MLN, respond to bacterial encounter by Ag presentation and produce cytokines in a subset-specific fashion.     

Cytokine production by mature and immature CD4-CD8- T cells. Alpha beta-T cell receptor+ CD4-CD8- T cells produce IL-4.

This study follows our previous investigation describing the production of four cytokines (IL-2, IL-4, IFN-gamma, and TNF-alpha) by subsets of thymocytes defined by the expression of CD3, 4, 8, and 25. Here we investigate in greater detail subpopulations of CD4-CD8- double negative (DN) thymocytes. First we divided immature CD25-CD4-CD8-CD3- (CD25- triple negative) (TN) thymocytes into CD44+ and CD44- subsets. The CD44+ population includes very immature precursor T cells and produced high titers of IL-2, TNF-alpha, and IFN-gamma upon activation with calcium ionophore and phorbol ester. In contrast, the CD44- subset of CD25- TN thymocytes did not produce any of the cytokines studied under similar activation conditions. This observation indicates that the latter subset, which differentiates spontaneously in vitro into CD4+CD8+, already resembles CD4+CD8+ thymocytes (which do not produce any of the tested cytokines). We also subdivided the more mature CD3+ DN thymocytes into TCR-alpha beta- and TCR-gamma delta-bearing subsets. These cells produced cytokines upon activation with solid phase anti-CD3 mAb. gamma delta TCR+ DN thymocytes produced IL-2, IFN-gamma and TNF-alpha, whereas alpha beta TCR+ DN thymocytes produced IL-4, IFN-gamma, and TNF-alpha but not IL-2. We then studied alpha beta TCR+ DN T cells isolated from the spleen and found a similar cytokine production profile. Furthermore, splenic alpha beta TCR+ DN cells showed a TCR V beta gene expression profile reminiscent of alpha beta TCR+ DN thymocytes (predominant use of V beta 8.2). These observations suggest that at least some alpha beta TCR+ DN splenocytes are derived from alpha beta TCR+ DN thymocytes and also raises the possibility that these cells may play a role in the development of Th2 responses through their production of IL-4.     

Role of CD25+ dendritic cells in the generation of Th17 autoreactive T cells in autoimmune experimental uveitis

In the current study, we showed that in vivo administration of an anti-CD25 Ab (PC61) decreased the Th17 response in C57BL/6 mice immunized with the uveitogenic peptide interphotoreceptor retinoid-binding protein, while enhancing the autoreactive Th1 response. The depressed Th17 response was closely associated with decreased numbers of a splenic dendritic cell (DC) subset expressing CD11c(+)CD3(-)CD25(+) and decreased expansion of γδ T cells. We demonstrated that ablation of the CD25(+) DC subset accounted for the decreased activation and the expansion of γδ T cells, leading to decreased activation of IL-17(+) interphotoreceptor retinoid-binding protein-specific T cells. Our results show that an enhanced Th17 response in an autoimmune disease is associated with the appearance of a DC subset expressing CD25 and that treatment of mice with anti-CD25 Ab causes functional alterations in a number of immune cell types, namely DCs and γδ T cells, in addition to CD25(+)αβTCR(+) regulatory T cells.     

Differential involvement of dendritic cell subsets during acute Salmonella infection

Within murine CD11c(+) dendritic cells (DC), CD8alpha+, CD8alpha-CD4+, and CD8alpha-CD4- subsets are defined. This study characterized the localization, number, and function of these subsets during acute Salmonella typhimurium infection. Immunohistochemical and flow cytometric analyses of spleens from mice orally infected with virulent S. typhimurium revealed that in situ redistribution and alteration in the absolute number and function of DC occurred in a subset-specific manner during infection. CD8alpha-CD4+ DC present at B cell follicle borders in the spleen of naive mice were absent 5 days post-Salmonella infection, despite no overall change in the absolute number of CD8alpha-CD4+ splenic DC. CD8alpha+ and CD8alpha-CD4- DC were prominently associated with the red pulp, and the frequency of these cells increased strikingly 5 days post-Salmonella infection. Significant quantitative increases in both CD8alpha+ and CD8alpha-CD4- subsets were associated with the in situ redistribution. Examination of Salmonella-infected TAP1(-/-)/beta(2)-microglobulin(-/-) mice, which lack CD8alpha+ T cells, confirmed the differential subset-specific modulations in the DC populations both in situ and quantitatively. Ex vivo intracellular cytokine analysis showed significantly increased frequencies of CD8alpha(+) DC producing TNF-alpha at days 2 and 5 postinfection. In contrast, CD4+ DC producing TNF-alpha were transiently increased followed by a significant reduction. No significant increase in IL-12p40 or IL-10 production by splenic DC was detected during the first 5 days post-S. typhimurium infection. Together these data reveal differential modulation of splenic DC subsets with regard to organization, number, and cytokine production during the course of acute Salmonella infection.     

Differential production of IL-12, IFN-alpha, and IFN-gamma by mouse dendritic cell subsets

Dendritic cells (DC) not only stimulate T cells effectively but are also producers of cytokines that have important immune regulatory functions. In this study we have extended information on the functional differences between DC subpopulations to include differences in the production of the major immune-directing cytokines IL-12, IFN-alpha, and IFN-gamma. Splenic CD4(-)8(+) DC were identified as the major IL-12 producers in response to microbiological or T cell stimuli when compared with splenic CD4(-)8(-) or CD4(+)8(-) DC; however, all three subsets of DC showed similar IL-12 regulation and responded with increased IL-12 p70 production if IL-4 was present during stimulation. High level CD8 expression also correlated with extent of IL-12 production for DC isolated from thymus and lymph nodes. By using gene knockout mice we ruled out any role for CD8alpha itself, or of priming by T cells, on the superior IL-12-producing capacity of the CD8(+) DC. Additionally, CD8(+) DC were identified as the major producers of IFN-alpha compared with the two CD8(-) DC subsets, a finding that suggests similarity to the human plasmacytoid DC lineage. In contrast, the CD4(-)8(-) DC produced much more IFN-gamma than the CD4(-)8(+) or the CD4(+)8(-) DC under all conditions tested.     

Distinct subsets of dendritic cells regulate the pattern of acute xenograft rejection and susceptibility to cyclosporine therapy

We determined whether distinct subclasses of dendritic cells (DC) could polarize cytokine production and regulate the pattern of xenograft rejection. C57BL/6 recipients, transplanted with Lewis rat hearts, exhibited a predominantly CD11c(+)CD8alpha(+) splenic DC population and an intragraft cytokine profile characteristic of a Th1-dominant response. In contrast, BALB/c recipients of Lewis rat heart xenografts displayed a predominantly CD11c(+)CD8alpha(-) splenic DC population and IL-4 intragraft expression characteristic of a Th2 response. In addition, the CD11c(+)IL-12(+) splenic DC population in C57BL/6 recipients was significantly higher than that in BALB/c recipients. Adoptive transfer of syngeneic CD8alpha(-) bone marrow-derived DC shifted a Th1-dominant, slow cell-mediated rejection to a Th2-dominant, aggressive acute vascular rejection (AVR) in C57BL/6 mice. This was associated with a cytokine shift from Th1 to Th2 in these mice. In contrast, transfer of CD8alpha(+) bone marrow-derived DC shifted AVR to cell-mediated rejection in BALB/c mice and significantly prolonged graft survival time from 6.0 +/- 0.6 days to 14.2 +/- 0.8 days. CD8alpha(+) DC transfer rendered BALB/c mice susceptible to cyclosporine therapy, thereby facilitating long-term graft survival. Furthermore, CD8alpha(+) DC transfer in IL-12-deficient mice reconstituted IL-12 expression, induced Th1 response, and attenuated AVR. Our data suggest that the pattern of acute xenogeneic rejection can be regulated by distinct DC subsets.     

Differential pattern recognition receptor expression but stereotyped responsiveness in rat spleen dendritic cell subsets

Dendritic cells (DC) are a heterogeneous population of APC endowed with specific functions. The nature of the DC subset involved in the course of an immune response to a specific pathogen might be important for inducing the appropriate effectors. In addition, each DC subset might also exhibit intrinsic functional plasticity. In the rat, spleen DC can be separated into three morphological and phenotypical distinct subsets, namely CD4+, CD4-, and plasmacytoid DC (pDC), whose frequencies are strain dependent. We correlated the expression of TLR and nucleotide-binding oligomerization domain 2 (NOD2) in these DC subsets to their in vitro responsiveness to specific ligands. CD4- DC expressed high levels of TLR1, 2, 3, and 10 mRNA, low TLR4, 5, 6, 7, and 9, and very low, if any, TLR8. pDC had a restricted repertoire characterized by high TLR7 and 9. CD4+ DC expressed all TLR and 10-fold higher levels of NOD2 mRNA than CD4- and pDC. Upon stimulation by TLR and NOD2 ligands, each DC subset responded in quite a stereotyped fashion. TLR2/6, 3, 4, 5, 9, and NOD2 triggering induced CD4- DC to mature and produce high IL-12p40, low IL-10, and TNF-alpha. TLR7/8 and 9 triggering induced pDC to mature and produce copious amounts of IL-6, IL-12p40, and TNF-alpha and low IFN-alpha. CD4+ DC were very poor producers of inflammatory cytokines. This study suggests that the nature of spleen DC responses to pathogens is dependent on subset specific-stimulation rather than intrinsic plasticity.     

IL-6 production by pulmonary dendritic cells impedes Th1 immune responses

Mucosal tissues, such as the lung, are continually exposed to both foreign and environmental Ags. To counter the potential inflammatory tissue injury of chronic Th1-mediated responses against these Ags, mucosal sites may skew toward Th2 immune responses. However, the mechanism by which this occurs is unknown. Dendritic cells (DC), as orchestrators of the immune response, skew Th1/Th2 differentiation by cytokine secretion and expression of specific cell surface markers. We compared DC from mucosal and systemic locations. In this study, we show that the lung lacks a CD8alpha(+) DC subpopulation and contains DC that appear less mature than splenic DC. Furthermore, we demonstrate that pulmonary DC produce significant levels of IL-6 and fail to produce the Th1-polarizing cytokine IL-12. Importantly, we demonstrate that IL-6 negatively regulates IL-12 production, as pulmonary DC from IL-6(-/-) mice produce significant levels of IL-12 and induce Th1 polarization of naive CD4(+) T cells. Furthermore, we demonstrate that IL-6 is sufficient to explain the differential polarizing abilities of pulmonary and splenic DC, as splenic DC cocultures supplemented with IL-6 polarize naive T cells toward Th2, and pulmonary DC cultures in which IL-6 was removed with neutralizing Ab resulted in more Th1 polarization, pointing to IL-6 as the mechanism of Th2 polarization in the lung. We propose that the Th2 response seen in the lung is due to DC-mediated inhibition of Th1 responses via IL-6 production, rather than enhanced Th2 responses, and that this regulation decreases the likelihood of chronic inflammatory pathology in the lung.     

CD4-8- dendritic cells prime CD4+ T regulatory 1 cells to suppress antitumor immunity

It is clear that dendritic cells (DCs) are essential for priming of T cell responses against tumors. However, the distinct roles DC subsets play in regulation of T cell responses in vivo are largely undefined. In this study, we investigated the capacity of OVA-presenting CD4-8-, CD4+8-, or CD4-8+ DCs (OVA-pulsed DC (DC(OVA))) in stimulation of OVA-specific T cell responses. Our data show that each DC subset stimulated proliferation of allogeneic and autologous OVA-specific CD4+ and CD8+ T cells in vitro, but that the CD4-8- DCs did so only weakly. Both CD4+8- and CD4-8+ DC(OVA) induced strong tumor-specific CD4+ Th1 responses and fully protective CD8+ CTL-mediated antitumor immunity, whereas CD4-8- DC(OVA), which were less mature and secreted substantial TGF-beta upon coculture with TCR-transgenic OT II CD4+ T cells, induced the development of IL-10-secreting CD4+ T regulatory 1 (Tr1) cells. Transfer of these Tr1 cells, but not T cells from cocultures of CD4-8- DC(OVA) and IL-10-/- OT II CD4+ T cells, into CD4-8+ DC(OVA)-immunized animals abrogated otherwise inevitable development of antitumor immunity. Taken together, CD4-8- DCs stimulate development of IL-10-secreting CD4+ Tr1 cells that mediated immune suppression, whereas both CD4+8- and CD4-8+ DCs effectively primed animals for protective CD8+ CTL-mediated antitumor immunity.