MHC class II+ exosomes in plasma suppress inflammation in an antigen-specific and Fas ligand/Fas-dependent manner

Exosomes are 50- to 100-nm vesicles that are formed within the late endocytic compartment and released from a variety of cell types. Previously, we demonstrated that exosomes derived from dendritic cells transduced with adenoviral vectors expressing IL-10, IL-4, or Fas ligand (FasL) produce anti-inflammatory exosomes able to reduce inflammation in a murine paw delayed-type hypersensitivity model, suppress the onset on murine collagen-induced arthritis, and reduce the severity of established collagen-induce arthritis. In this study, we examined the ability of endogenous, blood-borne exosomes to regulate the immune response. Exosomes isolated from plasma of mice immunized to keyhole limpet hemocyanin, but not from naive or OVA-immunized mice, were able to suppress the keyhole limpet hemocyanin-specific delayed-type hypersensitivity inflammatory response. The anti-inflammatory effect was mediated by MHC class II(+) plasma exosomes that were also FasL(+) and CD11b(+), but CD11c(-). Moreover, the anti-inflammatory effect of the MHC class II(+) plasma-derived exosomes was, in part, dependent upon the presence of FasL in the exosomes and Fas in the recipient mouse. These results suggest that exosomes in the plasma, produced by MHC class II(+) and CD11b(+) cells, have the ability to suppress the immune response in an Ag-specific manner in part through a Fas/FasL-dependent manner.     

Exosomes derived from IL-10-treated dendritic cells can suppress inflammation and collagen-induced arthritis

We have demonstrated previously that local, adenoviral-mediated gene transfer of viral IL-10 to a single joint of rabbits and mice with experimental arthritis can suppress disease in both the treated and untreated contralateral joints. This contralateral effect is mediated in part by APCs able to traffic from the treated joint to lymph nodes as well as to untreated joints. Moreover, injection of dendritic cells (DC) genetically modified to express IL-4 or Fas ligand was able to reverse established murine arthritis. To examine the ability of exosomes derived from immunosuppressive DCs to reduce inflammation and autoimmunity, murine models of delayed-type hypersensitivity and collagen-induced arthritis were used. In this study, we demonstrate that periarticular administration of exosomes purified from either bone marrow-derived DCs transduced ex vivo with an adenovirus expressing viral IL-10 or bone marrow-derived DCs treated with recombinant murine IL-10 were able to suppress delayed-type hypersensitivity responses within injected and untreated contralateral joints. In addition, the systemic injection of IL-10-treated DC-derived exosomes was able suppress the onset of murine collagen-induced arthritis as well as reduce severity of established arthritis. Taken together, these data suggest that immature DCs are able to secrete exosomes that are involved in the suppression of inflammatory and autoimmune responses. Thus DC-derived exosomes may represent a novel, cell-free therapy for the treatment of autoimmune diseases.     

Liver dendritic cells present bacterial antigens and produce cytokines upon Salmonella encounter

The capacity of murine liver dendritic cells (DC) to present bacterial Ags and produce cytokines after encounter with Salmonella was studied. Freshly isolated, nonparenchymal liver CD11c(+) cells had heterogeneous expression of MHC class II and CD11b and a low level of CD40 and CD86 expression. Characterization of liver DC subsets revealed that CD8alpha(-)CD4(-) double negative cells constituted the majority of liver CD11c(+) ( approximately 85%) with few cells expressing CD8alpha or CD4. Flow cytometry analysis of freshly isolated CD11c(+) cells enriched from the liver and cocultured with Salmonella expressing green fluorescent protein (GFP) showed that CD11c(+) MHC class II(high) cells had a greater capacity to internalize Salmonella relative to CD11c(+) MHC class II(low) cells. Moreover, both CD8alpha(-) and CD8alpha(+) liver DC internalized bacteria with similar efficiency after both in vitro and in vivo infection. CD11c(+) cells enriched from the liver could also process Salmonella for peptide presentation on MHC class I and class II to primary, Ag-specific T cells after internalization requiring actin cytoskeletal rearrangements. Flow cytometry analysis of liver CD11c(+) cells infected with Salmonella expressing GFP showed that both CD8alpha(-) and CD8alpha(+) DC produced IL-12p40 and TNF-alpha. The majority of cytokine-positive cells did not contain bacteria (GFP(-)) whereas only a minor fraction of cytokine-positive cells were GFP(+). Furthermore, only approximately 30-50% of liver DC containing bacteria (GFP(+)) produced cytokines. Thus, liver DC can internalize and process Salmonella for peptide presentation to CD4(+) and CD8(+) T cells and elicit proinflammatory cytokine production upon Salmonella encounter, suggesting that DC in the liver may contribute to immunity against hepatotropic bacteria.     

IL-13 regulates the immune response to inhaled antigens

The large inhibitory effect of IL-13 blockers on the asthma phenotype prompted us to ask whether IL-13 would play a role in regulating the allergic immune response in addition to its documented effects on structural pulmonary cells. Because IL-13 does not interact with murine T or B cells, but with monocytes, macrophages, and dendritic cells (DCs), we examined the role of IL-13 in the activation of pulmonary macrophages and DCs and in the priming of an immune response to a harmless, inhaled Ag. We found that a majority of cells called "alveolar or interstitial macrophages" express CD11c at high levels (CD11c(high)) and are a mixture of at least two cell types as follows: 1) cells of a mixed phenotype expressing DC and macrophage markers (CD11c, CD205, and F4/80) but little MHC class II (MHC II); and 2) DC-like cells expressing CD11c, CD205, MHC II, and costimulatory molecules. Endogenous IL-13 was necessary to induce and sustain the increase in MHC II and CD40 expression by pulmonary CD11c(high) cells, demonstrated by giving an IL-13 inhibitor as a measure of prevention or reversal to allergen-primed and -challenged mice. Conversely, IL-13 given by inhalation to naive mice increased the expression of MHC II and costimulatory molecules by CD11c(high) cells in an IL-4Ralpha-dependent manner. We found that exogenous IL-13 exaggerated the immune and inflammatory responses to an inhaled, harmless Ag, whereas endogenous IL-13 was necessary for the priming of naive mice with an inhaled, harmless Ag. These data indicate that blockade of IL-13 may have therapeutic potential for controlling the immune response to inhaled Ags.     

Dynamics of dendritic cell phenotype and interactions with CD4+ T cells in airway inflammation and tolerance

An emerging concept is that different types of dendritic cells (DCs) initiate different immune outcomes, such as tolerance vs inflammation. In this study, we have characterized the DCs from the lung draining lymph nodes of mice immunized for allergic airway inflammation or tolerance and examined their interactions with CD4(+) T cells. The DC population derived from tolerized mice was predominantly CD11c(+), B220(+), Gr-1(+), CD11b(-), and MHC class II(low), which resembled plasmacytoid-type DCs whereas DCs from the inflammatory condition were largely CD11c(+), B220(-), Gr-1(-), CD11b(+), and MHC class II(high) resembling myeloid-type DCs. The DCs from the tolerogenic condition were poor inducers of T cell proliferation. DCs from both conditions induced T cell IL-4 production but the T cells cultured with tolerogenic DCs were unresponsive to IL-4 as indicated by inhibition of STAT6 activation and expression of growth factor-independent 1, which has been recently shown to be important for STAT6-activated Th2 cell expansion. Our data suggest that airway tolerance vs inflammation is determined by the DC phenotype in lung draining lymph nodes.     

Vaccination without autoantigen protects against collagen II-induced arthritis via immune deviation and regulatory T cells

Anti-inflammation immunotherapy has been successfully applied for the treatment of autoimmune diseases. Mucosal vaccines against autoimmune disorders are beneficial by influencing the regulatory compartment of gut and systemic adaptive immune systems. A Salmonella vector expressing colonization factor Ag I (CFA/I), shown to behave as an anti-inflammatory vaccine, stimulates the production of CD4(+)CD25(+) T cells and regulatory cytokines. In this work, we queried whether Salmonella-CFA/I can protect DBA/1 mice from collagen-induced arthritis. The incidence of arthritis and cartilage loss in vaccinated DBA/1 mice was remarkably lower when compared with unprotected mice. Clinical findings were accompanied by the suppression of inflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IL-27. Vaccination evoked a multi-tier response consisting of IL-4 producing Th2 cells, an increased production of TGF-beta by CD4(+) T cells, and suppression of collagen II-specific CD4(+) T cell proliferation. To assess the contribution of Salmonella-CFA/I-primed CD4(+) T cells, adoptive transfer studies with total CD4(+), CD4(+)CD25(-), or CD4(+)CD25(+) T cells were performed 15 days postchallenge. Mice receiving either subset showed reduced disease incidence and low clinical scores; however, mice receiving total CD4(+) T cells showed delayed disease onset by 10 days with reduced clinical scores, reduced IL-17 and IL-27, but enhanced IL-4, IL-10, IL-13, and TGF-beta. Inhibition of TGF-beta or IL-4 compromised protective immunity. These data show that Salmonella-CFA/I vaccination of DBA/1 mice protects against collagen-induced arthritis by stimulating TGF-beta- and IL-4-producing regulatory CD4(+) T cells.     

4-1BB-mediated immunotherapy of rheumatoid arthritis

Collagen type II-induced arthritis is a CD4(+) T-cell-dependent chronic inflammation in susceptible DBA/1 mice and represents an animal model of human rheumatoid arthritis. We found that development of this condition, and even established disease, are inhibited by an agonistic anti-4-1BB monoclonal antibody. Anti-4-1BB suppressed serum antibodies to collagen type II and CD4(+) T-cell recall responses to collagen type II. Crosslinking of 4-1BB evoked an antigen-specific, active suppression mechanism that differed from the results of blocking the interaction between 4-1BB and its ligand, 4-1BBL. Anti-4-1BB monoclonal antibodies induced massive, antigen-dependent clonal expansion of CD11c(+)CD8(+) T cells and accumulation of indoleamine 2,3-dioxygenase in CD11b(+) monocytes and CD11c(+) dendritic cells. Both anti-interferon-gamma and 1-methyltryptophan, a pharmacological inhibitor of indoleamine 2,3-dioxygenase, reversed the anti-4-1BB effect. We conclude that the suppression of collagen-induced arthritis was caused by an expansion of new CD11c(+)CD8(+) T cells, and that interferon-gamma produced by these cells suppresses antigen-specific CD4(+) T cells through an indoleamine 2,3-dioxygenase-dependent mechanism.     

CD154 plays a central role in regulating dendritic cell activation during infections that induce Th1 or Th2 responses

We compared splenic DC activation during infection with either the Th2 response-inducing parasite Schistosoma mansoni or with the Th1 response-inducing parasite Toxoplasma gondii. CD8alpha(+) DC from schistosome-infected mice exhibited a 2- to 3-fold increase in the expression of MHC class II, CD80, and CD40 (but not CD86) compared with DC from uninfected control animals, while CD8alpha(-) DC exhibited a 2- to 3-fold increase in the expression of MHC class II and CD80 and no alteration, compared with DC from uninfected mice, in the expression of CD86 or CD40. Intracellular staining revealed that DC did not produce IL-12 during infection with S. mansoni. In contrast, infection with T. gondii resulted in a more pronounced increase in the expression of activation-associated molecules (MHC class II, CD80, CD86, and CD40) on both CD8alpha(-) and CD8alpha(+) splenic DC and promoted elevated IL-12 production by DC. Analysis of MHC class I and of additional costimulatory molecules (ICOSL, ICAM-1, OX40L, 4-1BBL, and B7-DC) revealed a generally similar pattern, with greater indication of activation in T. gondii-infected mice compared with S. mansoni-infected animals. Strikingly, the activation of DC observed during infection with either parasite was not apparent in DC from infected CD154(-/-) mice, indicating that CD40/CD154 interactions are essential for maintaining DC activation during infection regardless of whether the outcome is a Th1 or a Th2 response. However, the ability of this activation pathway to induce IL-12 production by DC is restrained in S. mansoni-infected, but not T. gondii-infected, mice by Ag-responsive CD11c(-) cells.     

GM-CSF is an essential regulator of T cell activation competence in uterine dendritic cells during early pregnancy in mice

Uterine dendritic cells (DCs) are critical for activating the T cell response mediating maternal immune tolerance of the semiallogeneic fetus. GM-CSF (CSF2), a known regulator of DCs, is synthesized by uterine epithelial cells during induction of tolerance in early pregnancy. To investigate the role of GM-CSF in regulating uterine DCs and macrophages, Csf2-null mutant and wild-type mice were evaluated at estrus, and in the periconceptual and peri-implantation periods. Immunohistochemistry showed no effect of GM-CSF deficiency on numbers of uterine CD11c(+) cells and F4/80(+) macrophages at estrus or on days 0.5 and 3.5 postcoitum, but MHC class II(+) and class A scavenger receptor(+) cells were fewer. Flow cytometry revealed reduced CD80 and CD86 expression by uterine CD11c(+) cells and reduced MHC class II in both CD11c(+) and F4/80(+) cells from GM-CSF-deficient mice. CD80 and CD86 were induced in Csf2(-/-) uterine CD11c(+) cells by culture with GM-CSF. Substantially reduced ability to activate both CD4(+) and CD8(+) T cells in vivo was evident after delivery of OVA Ag by mating with Act-mOVA males or transcervical administration of OVA peptides. This study shows that GM-CSF regulates the efficiency with which uterine DCs and macrophages activate T cells, and it is essential for optimal MHC class II- and class I-mediated indirect presentation of reproductive Ags. Insufficient GM-CSF may impair generation of T cell-mediated immune tolerance at the outset of pregnancy and may contribute to the altered DC profile and dysregulated T cell tolerance evident in infertility, miscarriage, and preeclampsia.     

Characterization of distinct conventional and plasmacytoid dendritic cell-committed precursors in murine bone marrow

The developmental pathways and differentiation relationship of dendritic cell (DC) subsets remain unclear. We report that murine CD11c(+)MHC II(-) bone marrow cells, which are immediate DC precursors of CD8 alpha(+), CD8 alpha(-), and B220(+) DC in vivo, can be separated into B220(+) and B220(-) DC precursor subpopulations. Purified B220(-) DC precursors expand, and generate exclusively mature CD11c(+)CD11b(+)B220(-) DC in vitro and after adoptive transfer. B220(+) DC precursors, which resemble plasmacytoid pre-DC, have a lower proliferative potential than B220(-) DC precursors and generate both CD11b(-) B220(+) and CD11b(+)B220(-) DC populations. Both DC precursor populations can give rise to CD8 alpha(+) and CD8 alpha(-) DC subtypes. Our findings indicate that CD11c(+)MHC II(-)B220(+) and CD11c(+)MHC II(-)B220(-) bone marrow cells are distinct DC lineage-restricted precursors.     

Differential regulation of human blood dendritic cell subsets by IFNs

Based on the relative expression of CD11c and CD1a, we previously identified subsets of dendritic cells (DCs) or DC precursors in human peripheral blood. A CD1a(+)/CD11c(+) population (CD11c(+) DCs), also called myeloid DCs, is an immediate precursor of Langerhans cells, whereas a CD1a(-)/CD11c(-) population (CD11c(-) DCs), sometimes called lymphoid DCs but better known as plasmacytoid DCs, is composed of type I IFN (IFN-alpha beta)-producing cells. Here, we investigate the effects of IFN-alpha beta and IFN-gamma as well as other cytokines on CD11c(+) and CD11c(-) DC subsets, directly isolated from the peripheral blood, instead of in vitro-generated DCs. IFN-gamma and IFN-alpha, rather than GM-CSF, were the most potent cytokines for enhancing the maturation of CD11c(+) DCs. Incubation of CD11c(+) DCs with IFN-gamma also resulted in increased IL-12 production, and this IL-12 allowed DCs to increase Th1 responses by alloreactive T cells. In contrast, IFN-alpha did not induce IL-12 but, rather, augmented IL-10 production. IFN-alpha-primed matured CD11c(+) DCs induced IL-10-producing regulatory T cells; however, this process was independent of the DC-derived IL-10. On the other hand, IFN-alpha by itself neither matured CD11c(-) DCs nor altered the polarization of responding T cells, although this cytokine was a potent survival factor for CD11c(-) DCs. Unlike IFN-alpha, IL-3 was a potent survival factor and induced the maturation of CD11c(-) DCs. The IL-3-primed CD11c(-) DCs activated T cells to produce IL-10, IFN-gamma, and IL-4. Thus, CD11c(+) and CD11c(-) DC subsets play distinct roles in the cytokine network, especially their responses to IFNs.     

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.     

Immune interactions with CD4+ T cells promote the development of functional osteoclasts from murine CD11c+ dendritic cells

Dendritic cells (DC) are innate immune effectors and are critically involved in regulating T cell immunity. Osteoclasts (OC) are bone-resorbing cells derived from the monocyte/macrophage lineage in response to receptor activator of NF-kappaB ligand (RANKL). DC and T cells form aggregates in the inflammatory infiltrates at active disease sites in human and in experimental rheumatoid arthritis and periodontitis. We investigated whether DC interactions with T cells in the bone environment can support the development of functional OC. In the present study, we demonstrate that upon proper activation by microbial or protein Ags (namely Actinobacillus actinomycetemcomitans, bovine insulin, and outer membrane protein-1) and during immune interactions with CD4+ T cells in vitro, murine BM-derived and splenic CD11c+ DC (CD11b- F4/80- Ly-6C- CD31-) develop into TRAP+ CT-R+ cathepsin-k+ functional OC in a RANKL/RANK-dependent manner. Rescue and blocking experiments using CD11c+ DC derived from Csf-1(-/-) op/op mice show that M-CSF is required "before" developing such osteoclastogenic potential upstream of RANKL/RANK signaling, suggesting that immature CD11c+ DC can indeed act like OC precursors. In addition, these CD11c+ DC-derived OC are capable of inducing bone loss after adoptive transfer in vivo. These data suggest a direct contribution of DC during immune interactions with CD4+ T cells to inflammation-induced osteoclastogenesis. Therefore, our findings not only provide further evidence for DC plasticity, but also extend the current paradigm of osteoimmunology.     

NOD bone marrow-derived dendritic cells are modulated by analogs of 1,25-dihydroxyvitamin D3

The immune effects of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) are mainly mediated through dendritic cells (DCs). In vitro, 1,25(OH)(2)D(3) treatment renders murine bone marrow (BM)-derived DCs more tolerogenic, indirectly altering behavior and fate of T lymphocytes. In vivo, treatment with 1,25(OH)(2)D(3) or its analogs prevents diabetes in NOD mice. The aim of this study was to investigate the effects of the 1,25(OH)(2)D(3)-analog TX527 on the expression of antigen-presenting and costimulatory/migratory molecules on BM-derived DCs from NOD mice. After culture with 20 ng/ml GM-CSF + 20 ng/ml IL-4 (8 days) followed by 1000 ng/ml LPS + 100 U/ml IFN-gamma (2 days), with or without 10(-8)M TX527, cells were counted and analyzed by FACS for MHC II, CD86, CD40 and CD54 expression within the CD11c(+) DC population. Upon TX527 treatment, cell recovery was significantly reduced whereas the CD11c(+) DC fraction remained constant. On CD11c(+) DCs, MHC II, CD86 and CD54 were significantly down-regulated and CD40 was twofold upregulated. Globally, BM-derived DCs from NOD mice become more tolerogenic upon TX527 treatment, confirming the effects of 1,25(OH)(2)D(3) on murine DCs and possibly explaining the protective effects of 1,25(OH)(2)D(3) and its analogs from diabetes in NOD mice.     

Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells

Current immunization protocols in cancer patients involve CTL-defined tumor peptides. Mature dendritic cells (DC) are the most potent APCs for the priming of naive CD8(+) T cells, eventually leading to tumor eradication. Because DC can secrete MHC class I-bearing exosomes, we addressed whether exosomes pulsed with synthetic peptides could subserve the DC function consisting in MHC class I-restricted, peptide-specific CTL priming in vitro and in vivo. The priming of CTL restricted by HLA-A2 molecules and specific for melanoma peptides was performed: 1) using in vitro stimulations of total blood lymphocytes with autologous DC pulsed with GMP-manufactured autologous exosomes in a series of normal volunteers; 2) in HLA-A2 transgenic mice (HHD2) using exosomes harboring functional HLA-A2/Mart1 peptide complexes. In this study, we show that: 1). DC release abundant MHC class I/peptide complexes transferred within exosomes to other naive DC for efficient CD8(+) T cell priming in vitro; 2). exosomes require nature's adjuvants (mature DC) to efficiently promote the differentiation of melanoma-specific effector T lymphocytes producing IFN-gamma (Tc1) effector lymphocytes in HLA-A2 transgenic mice (HHD2). These data imply that exosomes might be a transfer mechanism of functional MHC class I/peptide complexes to DC for efficient CTL activation in vivo.     

CD4+ NKT cells,but not conventional CD4+ T cells,are required to generate efferent CD8+ T regulatory cells following antigen inoculation in an immune-privileged site

Following inoculation of Ag into the anterior chamber (a.c.), systemic tolerance develops that is mediated in part by Ag-specific efferent CD8(+) T regulatory (Tr) cells. This model of tolerance is called a.c.-associated immune deviation. The generation of the efferent CD8(+) Tr cell in a.c.-associated immune deviation is dependent on IL-10-producing, CD1d-restricted, invariant Valpha14(+) NKT (iNKT) cells. The iNKT cell subpopulations are either CD4(+) or CD4(-)CD8(-) double negative. This report identifies the subpopulation of iNKT cells that is important for induction of the efferent Tr cell. Because MHC class II(-/-) (class II(-/-)) mice generate efferent Tr cells following a.c. inoculation, we conclude that conventional CD4(+) T cells are not needed for the development of efferent CD8(+) T cells. Furthermore, Ab depletion of CD4(+) cells in both wild-type mice (remove both conventional and CD4(+) NKT cells) and class II(-/-) mice (remove CD4(+) NKT cells) abrogated the generation of Tr cells. We conclude that CD4(+) NKT cells, but not the class II molecule or conventional CD4(+) T cells, are required for generation of efferent CD8(+) Tr cells following Ag introduction into the eye. Understanding the mechanisms that lead to the generation of efferent CD8(+) Tr cells may lead to novel immunotherapy for immune inflammatory diseases.     

α-1 antitrypsin promotes semimature, IL-10-producing and readily migrating tolerogenic dendritic cells

Tolerogenic IL-10-positive CCR7-positive dendritic cells (DC) promote T regulatory (Treg) cell differentiation upon CCR7-dependent migration to draining lymph nodes (DLN). Indeed, in human DC deficiencies, Treg levels are low. α-1 antitrypsin (AAT) has been shown to reduce inflammatory markers, promote a semimature LPS-induced DC phenotype, facilitate Treg expansion, and protect pancreatic islets from alloimmune and autoimmune responses in mice. However, the mechanism behind these activities of AAT is poorly understood. In this study, we examine interactions among DC, CD4(+) T cells, and AAT in vitro and in vivo. IL-1β/IFN-γ-mediated DC maturation and effect on Treg development were examined using OT-II cells and human AAT (0.5 mg/ml). CCL19/21-dependent migration of isolated DC and resident islet DC was assessed, and CCR7 surface levels were examined. Migration toward DLN was evaluated by FITC skin painting, transgenic GFP skin tissue grafting, and footpad DC injection. AAT-treated stimulated DC displayed reduced MHC class II, CD40, CD86, and IL-6, but produced more IL-10 and maintained inducible CCR7. Upon exposure of CD4(+) T cells to OVA-loaded AAT-treated DC, 2.7-fold more Foxp3(+) Treg cells were obtained. AAT-treated cells displayed enhanced chemokine-dependent migration and low surface CD40. Under AAT treatment (60 mg/kg), DLN contained twice more fluorescence after FITC skin painting and twice more donor DC after footpad injection, whereas migrating DC expressed less CD40, MHC class II, and CD86. Intracellular DC IL-10 was 2-fold higher in the AAT group. Taken together, these results suggest that inducible functional CCR7 is maintained during AAT-mediated anti-inflammatory conditions. Further studies are required to elucidate the mechanism behind the favorable tolerogenic activities of AAT.     

Epicutaneous immunization with type II collagen inhibits both onset and progression of chronic collagen-induced arthritis

Epicutaneous immunization is a potential non-invasive technique for antigen-specific immune-modulation. Topical application of protein antigens to barrier-disrupted skin induces potent antigen-specific immunity with a strong Th2-bias. In this study, we investigate whether the autoimmune inflammatory response of chronic collagen-induced arthritis (CCIA) in DBA/1-TCR-beta Tg mice can be modified by epicutaneous immunization. We show that epicutaneous immunization with type II collagen (CII) inhibited development and progression of CCIA and, importantly, also ameliorated ongoing disease as indicated by clinical scores of disease severity, paw swelling and joints histology. Treated mice show reduced CII-driven T cell proliferation and IFN-gamma production, as well as significantly lower levels of CII-specific IgG2a serum antibodies. In contrast, CII-driven IL-4 production and IgE antibody levels were increased consistent with skewing of the CII response from Th1 to Th2 in treated mice. IL-4 production in treated mice was inversely correlated with disease severity. Moreover, T cells from treated mice inhibited proliferation and IFN-gamma production by T cells from CCIA mice, suggesting induction of regulatory T cells that actively inhibit effector responses in arthritic mice. The levels of CD4(+)CD25(+) T cells were however not increased following epicutaneous CII treatment. Together, these results suggest that epicutaneous immunization may be used as an immune-modulating procedure to actively re-programme pathogenic Th1 responses, and could have potential as a novel specific and simple treatment for chronic autoimmune inflammatory diseases such as rheumatoid arthritis.     

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.     

Brain dendritic cells and macrophages/microglia in central nervous system inflammation

Microglia subpopulations were studied in mouse experimental autoimmune encephalomyelitis and toxoplasmic encephalitis. CNS inflammation was associated with the proliferation of CD11b(+) brain cells that exhibited the dendritic cell (DC) marker CD11c. These cells constituted up to 30% of the total CD11b(+) brain cell population. In both diseases CD11c(+) brain cells displayed the surface phenotype of myeloid DC and resided at perivascular and intraparenchymatic inflammatory sites. By lacking prominent phagocytic organelles, CD11c(+) cells from inflamed brain proved distinct from other microglia, but strikingly resembled bone marrow-derived DC and thus were identified as DC. This brain DC population comprised cells strongly secreting IL-12p70, whereas coisolated CD11c(-) microglia/brain macrophages predominantly produced TNF-alpha, GM-CSF, and NO. In comparison, the DC were more potent stimulators of naive or allogeneic T cell proliferation. Both DC and CD11c(-) microglia/macrophages from inflamed brain primed naive T cells from DO11.10 TCR transgenic mice for production of Th1 cytokines IFN-gamma and IL-2. Resting microglia that had been purified from normal adult brain generated immature DC upon exposure to GM-CSF, while CD40 ligation triggered terminal maturation. Consistently, a functional maturation of brain DC was observed to occur following the onset of encephalitis. In conclusion, these findings indicate that in addition to inflammatory macrophage-like brain cells, intraparenchymatical DC exist in autoimmune and infectious encephalitis. These DC functionally mature upon disease onset and can differentiate from resident microglia. Their emergence, maturation, and prolonged activity within the brain might contribute to the chronicity of intracerebral Th1 responses.