Cross‐presenting dendritic cells are required for control of Leishmania major infection

Leishmania major infection induces self‐healing cutaneous lesions in C57BL/6 mice. Both IL‐12 and IFN‐γ are essential for the control of infection. We infected Jun dimerization protein p21SNFT (Batf3 ^?/? ) mice (C57BL/6 background) that lack the major IL‐12 producing and cross‐presenting CD8α⁺ and CD103⁺ DC subsets. Batf3^?/? mice displayed enhanced susceptibility with larger lesions and higher parasite burden. Additionally, cells from draining lymph nodes of infected Batf3^?/? mice secreted less IFN‐γ, but more Th2‐ and Th17‐type cytokines, mirrored by increased serum IgE and Leishmania‐specific immunoglobulin 1 (Th2 indicating). Importantly, CD8α⁺ DCs isolated from lymph nodes of L. major‐infected mice induced significantly more IFN‐γ secretion by L. major‐stimulated immune T cells than CD103⁺ DCs. We next developed CD11c‐diptheria toxin receptor: Batf3^?/? mixed bone marrow chimeras to determine when the DCs are important for the control of infection. Mice depleted of Batf‐3‐dependent DCs from day 17 or wild‐type mice depleted of cross‐presenting DCs from 17–19 days after infection maintained significantly larger lesions similar to mice whose Batf‐3‐dependent DCs were depleted from the onset of infection. Thus, we have identified a crucial role for Batf‐3‐dependent DCs in protection against L. major.     

IL‐18Rα‐deficient CD4+ T cells induce intestinal inflammation in the CD45RBhi transfer model of colitis despite impaired innate responsiveness

IL‐18 has been implicated in inflammatory bowel disease (IBD), however its role in the regulation of intestinal CD4⁺ T‐cell function remains unclear. Here we show that murine intestinal CD4⁺ T cells express high levels of IL‐18Rα and provide evidence that IL‐18Rα expression is induced on these cells subsequent to their entry into the intestinal mucosa. Using the CD45RB^hi T‐cell transfer colitis model, we show that IL‐18Rα is expressed on IFN‐γ⁺, IL‐17⁺, and IL‐17⁺IFN‐γ⁺ effector CD4⁺ T cells in the inflamed colonic lamina propria (cLP) and mesenteric lymph node (MLN) and is required for the optimal generation and/or maintenance of IFN‐γ‐producing cells in the cLP. In the steady state and during colitis, TCR‐independent cytokine‐induced IFN‐γ and IL‐17 production by intestinal CD4⁺ T cells was largely IL‐18Rα?dependent. Despite these findings however, IL‐18Rα?deficient CD4⁺ T cells induced comparable intestinal pathology to WT CD4⁺ T cells. These findings suggest that IL‐18‐dependent cytokine induced activation of CD4⁺ T cells is not critical for the development of T‐cell‐mediated colitis.     

Selective depletion of CD11c+CD11b+ dendritic cells partially abrogates tolerogenic effects of intravenous MOG in murine EAE

Intravenous (i.v.) injection of a soluble myelin antigen can induce tolerance, which effectively ameliorates experimental autoimmune encephalomyelitis (EAE). We have previously shown that i.v. myelin oligodendrocyte glycoprotein (MOG) induces tolerance in EAE and expands a subpopulation of tolerogenic CD11c⁺CD11b⁺ dendritic cells (DCs) with an immature phenotype having low expression of IA and co‐stimulatory molecules CD40, CD86, and CD80. Here, we further investigate the role of tolerogenic DCs in i.v. tolerance by injecting clodronate‐loaded liposomes, which selectively deplete CD11c⁺CD11b⁺ and immature DCs, but not CD11c⁺CD8⁺ DCs and mature DCs. I.v. MOG‐induced suppression of EAE was partially, yet significantly, blocked by CD11c⁺CD11b⁺ DC depletion. While i.v. MOG inhibited IA, CD40, CD80, CD86 expression and induced TGF‐β, IL‐27, IL‐10 production in CD11c⁺CD11b⁺ DCs, these effects were abrogated after injection of clodronate‐loaded liposomes. Depletion of CD11c⁺CD11b⁺ DCs also precluded i.v. autoantigen‐induced T‐cell tolerance, such as decreased production of IL‐2, IFN‐γ, IL‐17 and numbers of IL‐2⁺, IFN‐γ⁺, and IL‐17⁺ CD4⁺ T cells, as well as an increased proportion of CD4⁺CD25⁺Foxp3⁺ regulatory T cells and CD4⁺IL‐10⁺Foxp3^? Tr1 cells. CD11c⁺CD11b⁺ DCs, through low expression of IA and costimulatory molecules as well as high expression of TGF‐β, IL‐27, and IL‐10, play an important role in i.v. tolerance‐induced EAE suppression.     

Signal regulatory protein alpha (SIRPα) regulates the homeostasis of CD103+CD11b+ DCs in the intestinal lamina propria

Signal regulatory protein alpha (SIRPα/CD172a) is a conserved transmembrane protein thought to play an inhibitory role in immune function by binding the ubiquitous ligand CD47. SIRPα expression has been used to identify dendritic cell subsets across species and here we examined its expression and function on intestinal DCs in mice. Normal mucosa contains four subsets of DCs based on their expression of CD103 and CD11b and three of these express SIRPα. However, loss of SIRPα signaling in mice leads to a selective reduction in the CD103⁺CD11b⁺ subset of DCs in the small intestine, colon, and among migratory DCs in the mesenteric lymph node. In parallel, these mice have reduced numbers of T_H17 cells in steady‐state intestinal mucosa, and a defective T_H17 response to Citrobacter infection. Identical results were obtained in CD47KO mice. DC precursors from SIRPα mutant mice had an enhanced ability to generate CD103⁺CD11b⁺ DCs in vivo, but CD103⁺CD11b⁺ DCs from mutant mice were more prone to die by apoptosis. These data show a previously unappreciated and crucial role for SIRPα in the homeostasis of CD103⁺CD11b⁺ DCs in the intestine, as well as providing further evidence that this subset of DCs is critical for the development of mucosal T_H17 responses.     

Critical role of CCL22/CCR4 axis in the maintenance of immune homeostasis during apoptotic cell clearance by splenic CD8α+ CD103+ dendritic cells

Macrophages and dendritic cells (DCs) in murine spleen are essential for the maintenance of immune homeostasis by elimination of blood‐borne foreign particles and organisms. It has been reported that splenic DCs, especially CD8α⁺ CD103⁺ DCs, are responsible for tolerance to apoptosis‐associated antigens. However, the molecular mechanism by which these DCs maintain immune homeostasis by blood‐borne apoptotic cell clearance remains elusive. Here, we found that the CCL22/CCR4 axis played a critical role in the maintenance of immune homeostasis during apoptotic cell clearance by splenic CD8α⁺ CD103⁺ DCs. The present results revealed that systemic administration of apoptotic cells rapidly induced a large number of CCL22 and CCR4⁺ regulatory T (Treg) cells in the spleen of C57BL/6J mice. Further study demonstrated that CD8α⁺ CD103⁺ DCs dominantly produce much higher CCL22 than CD8α⁺ CD103^? DCs. Moreover, the transient deletion of CD8α⁺ CD103⁺ DCs caused a decrease in CCL22 levels together with CCR4⁺ Treg cell percentage. Subsequently, the levels of some pro‐inflammatory cytokines, such as interleukin‐17 and interferon‐γ in the spleen with the absence of CD8α⁺ CD103⁺ DCs increased in response to the administration of apoptotic cells. Hence, intravenous injection of apoptotic cells induced a subsequent increase in CCL22 expression and CCR4⁺ Treg cells, which contribute to the maintenance of immune homeostasis at least partially by splenic CD8α⁺ CD103⁺ DCs.     

Chemokine programming dendritic cell antigen response: part II – programming antigen presentation to T lymphocytes by partially maintaining immature dendritic cell phenotype

In a companion article to this study,¹ the successful programming of a JAWSII dendritic cell (DC) line's antigen uptake and processing was demonstrated based on pre‐treatment of DCs with a specific ‘cocktail’ of select chemokines. Chemokine pre‐treatment modulated cytokine production before and after DC maturation [by lipopolysaccharide (LPS)]. After DC maturation, it induced an antigen uptake and processing capacity at levels 36% and 82% higher than in immature DCs, respectively. Such programming proffers a potential new approach to enhance vaccine efficiency. Unfortunately, simply enhancing antigen uptake does not guarantee the desired activation and proliferation of lymphocytes, e.g. CD4⁺ T cells. In this study, phenotype changes and antigen presentation capacity of chemokine pre‐treated murine bone marrow‐derived DCs were examined in long‐term co‐culture with antigen‐specific CD4⁺ T cells to quantify how chemokine pre‐treatment may impact the adaptive immune response. When a model antigen, ovalbumin (OVA), was added after intentional LPS maturation of chemokine‐treated DCs, OVA‐biased CD4⁺T‐cell proliferation was initiated from ~ 100% more undivided naive T cells as compared to DCs treated only with LPS. Secretion of the cytokines interferon‐γ, interleukin‐1β, interleukin‐2 and interleukin‐10 in the CD4⁺ T cell : DC co‐culture (with or without chemokine pre‐treatment) were essentially the same. Chemokine programming of DCs with a 7 : 3 ratio of CCL3 : CCL19 followed by LPS treatment maintained partial immature phenotypes of DCs, as indicated by surface marker (CD80 and CD86) expression over time. Results here and in our companion paper suggest that chemokine programming of DCs may provide a novel immunotherapy strategy to obviate the natural endocytosis limit of DC antigen uptake, thus potentially increasing DC‐based vaccine efficiency.     

Enrichment of IL‐17A+IFN‐γ+ and IL‐22+IFN‐γ+ T cell subsets is associated with reduction of NKp44+ILC3s in the terminal ileum of Crohn's disease patients

Crohn's disease (CD) is a chronic inflammatory condition of the human gastrointestinal tract whose aetiology remains largely unknown. Dysregulated adaptive immune responses and defective innate immunity both contribute to this process. In this study, we demonstrated that the interleukin (IL)‐17A⁺interferon (IFN)‐γ⁺ and IL‐22⁺IFN‐γ⁺ T cell subsets accumulated specifically in the inflamed terminal ileum of CD patients. These cells had higher expression of Ki‐67 and were active cytokine producers. In addition, their proportions within both the IL‐17A‐producer and IL‐22‐producer populations were increased significantly. These data suggest that IL‐17A⁺IFN‐γ⁺ and IL‐22⁺IFN‐γ⁺ T cell subsets might represent the pathogenic T helper type 17 (Th17) population in the context of intestinal inflammation for CD patients. In the innate immunity compartment we detected a dramatic alteration of both phenotype and function of the intestinal innate lymphoid cells (ILCs), that play an important role in the maintenance of mucosal homeostasis. In the inflamed gut the frequency of the NKp44^–CD117^–ILC1s subset was increased significantly, while the frequency of NKp44⁺ILC3s was reduced. Furthermore, the frequency of human leucocyte antigen D‐related (HLA‐DR)‐expressing‐NKp44⁺ILC3s was also reduced significantly. Interestingly, the decrease in the NKp44⁺ILC3s population was associated with an increase of pathogenic IL‐17A⁺IFN‐γ⁺ and IL‐22⁺IFN‐γ⁺ T cell subsets in the adaptive compartment. This might suggest a potential link between NKp44⁺ILC3s and the IL‐17A⁺IFN‐γ⁺ and IL‐22⁺IFN‐γ⁺ T cell subsets in the terminal ileum of CD patients.     

Dendritic cells from the human female reproductive tract rapidly capture and respond to HIV

Dendritic cells (DCs) throughout the female reproductive tract (FRT) were examined for phenotype, HIV capture ability and innate anti-HIV responses. Two main CD11c⁺ DC subsets were identified: CD11b⁺ and CD11b^low DCs. CD11b⁺CD14⁺ DCs were the most abundant throughout the tract. A majority of CD11c⁺CD14⁺ cells corresponded to CD1c⁺ myeloid DCs, whereas the rest lacked CD1c and CD163 expression (macrophage marker) and may represent monocyte-derived cells. In addition, we identified CD103⁺ DCs, located exclusively in the endometrium, whereas DC-SIGN⁺ DCs were broadly distributed throughout the FRT. Following exposure to GFP-labeled HIV particles, CD14⁺ DC-SIGN⁺ as well as CD14⁺ DC-SIGN⁻ cells captured virus, with ∼30% of these cells representing CD1c⁺ myeloid DCs. CD103⁺ DCs lacked HIV capture ability. Exposure of FRT DCs to HIV induced secretion of CCL2, CCR5 ligands, interleukin (IL)-8, elafin, and secretory leukocyte peptidase inhibitor (SLPI) within 3 h of exposure, whereas classical pro-inflammatory molecules did not change and interferon-α2 and IL-10 were undetectable. Furthermore, elafin and SLPI upregulation, but not CCL5, were suppressed by estradiol pre-treatment. Our results suggest that specific DC subsets in the FRT have the potential for capture and dissemination of HIV, exert antiviral responses and likely contribute to the recruitment of HIV-target cells through the secretion of innate immune molecules.     

IFN‐γ regulates survival and function of tumor‐induced CD11b+Gr‐1high myeloid derived suppressor cells by modulating the anti‐apoptotic molecule Bcl2a1

Myeloid derived suppressor cells (MDSCs) play a critical role in suppression of immune responses in cancer and inflammation. Here, we describe how regulation of Bcl2a1 by cytokines controls the suppressor function of CD11b⁺Gr‐1^high granulocytic MDSCs. Coculture of CD11b⁺Gr‐1^high granulocytic MDSCs with antigen‐stimulated T cells and simultaneous blockade of IFN‐γ by the use of anti‐IFN‐γ blocking antibody, IFN‐γ^?/? effector T cells, IFN‐γR^?/? MDSCs or STAT1^?/? MDSCs led to upregulation of Bcl2a1 in CD11b⁺Gr‐1^high cells, improved survival, and enhanced their suppressor function. Molecular studies revealed that GM‐CSF released by antigen‐stimulated CD8⁺ T cells induced Bcl2a1 upregulation, which was repressed in the presence of IFN‐γ by a direct interaction of phosphorylated STAT‐1 with the Bcl2a1 promotor. Bcl2a1 overexpressing granulocytic MDSCs demonstrated prolonged survival and enhanced suppressor function in vitro. Our data suggest that IFN‐γ/ STAT1‐dependent regulation of Bcl2a1 regulates survival and thereby suppressor function of granulocytic MDSCs.     

Immunotherapy using lipopolysaccharide‐stimulated bone marrow‐derived dendritic cells to treat experimental autoimmune encephalomyelitis

Lipopolysaccharide (LPS) produced by Gram‐negative bacteria induces tolerance and suppresses inflammatory responses in vivo; however, the mechanisms are poorly understood. In this study we show that LPS induces apoptosis of bone marrow‐derived dendritic cells (DCs) and modulates phenotypes of DCs. LPS treatment up‐regulates expression of tolerance‐associated molecules such as CD205 and galectin‐1, but down‐regulates expression of Gr‐1 and B220 on CD11c⁺ DCs. Moreover, LPS treatment regulates the numbers of CD11c⁺CD8⁺, CD11c⁺CD11b^low and CD11c⁺CD11b^hi DCs, which perform different immune functions in vivo. Our data also demonstrated that intravenous transfer of LPS‐treated DCs blocks experimental autoimmune encephalomyelitis (EAE) development and down‐regulates expression of retinoic acid‐related orphan receptor gamma t (ROR‐γt), interleukin (IL)‐17A, IL‐17F, IL‐21, IL‐22 and interferon (IFN)‐γ in myelin oligodendrocyte glycoprotein (MOG)‐primed CD4⁺ T cells in the peripheral environment. These results suggest that LPS‐induced apoptotic DCs may lead to generation of tolerogenic DCs and suppress the activity of MOG‐stimulated effector CD4⁺ T cells, thus inhibiting the development of EAE in vivo. Our results imply a potential mechanism of LPS‐induced tolerance mediated by DCs and the possible use of LPS‐induced apoptotic DCs to treat autoimmune diseases such as multiple sclerosis.     

CD8α+ DC are not the sole subset cross‐presenting cell‐associated tumor antigens from a solid tumor

One of the clear paradoxes in tumor immunology is the fact that cross‐presentation of cell‐associated tumor antigens to CD8⁺ T cells is efficient, yet CTL generation is weak, and tumors continue to grow. We examined, for the first time whether this may be due to alterations in the phenotype or function of cross‐presenting DC using a solid tumor model expressing a membrane bound neo‐antigen (hemagglutinin, HA). Tumor antigen was constitutively cross‐presented in the tumor‐draining LN throughout tumor progression by CD11c⁺ DC. Further analysis revealed that both CD8α⁺ and CD8α^? DC subsets, but not plasmacytoid DC, were effective at cross‐presenting HA tumor antigen. The proportions of DC subsets in the tumor‐draining LN were equivalent to those seen in the LN of naïve mice; however, a significant increase in the expression of the potential inhibitory B7 molecule, B7‐DC, was noted and appeared to be restricted to the CD8α^– DC subset. Therefore LN resident CD8α⁺ DC are not the sole DC subset capable of cross‐presenting cell‐associated tumor antigens. Migratory tumor DC subsets with altered co‐stimulatory receptor expression may contribute to induction and regulation of tumor‐specific responses.     

Species‐dependent role of type I IFNs and IL‐12 in the CTL response induced by humanized CpG complexed with β‐glucan

CpG oligodeoxynucleotide (ODN) is one of promising nucleic acid‐based adjuvants. We recently improved its ability to enhance CD8⁺ T‐cell responses to coadministered protein antigen without conjugation or emulsion, by forming a nanoparticulate complex between CpG ODN (K3) and mushroom‐derived β‐glucan schizophyllan (SPG), namely K3‐SPG. Here, we sought to elucidate the cellular immunological mechanisms by which K3‐SPG induce such potent CD8⁺ T‐cell responses to coadministered antigen. By focusing on two DC subsets, plasmacytoid DCs and CD8α⁺ DCs, as well as the secreted cytokines, IFN‐α and IL‐12, we found that K3‐SPG strongly activates mouse plasmacytoid DCs to secrete IFN‐α and CD8α⁺ DCs to secrete IL‐12, respectively. Although a single cytokine deficiency had no impact on adjuvant effects, the lack of both type I IFN and IL‐12 in mice resulted in a significant reduction of Th1 type immune responses and CD8⁺ T‐cell responses elicited by protein vaccine model. By sharp contrast, type I IFN, but not IL‐12, was required for the production of IFN‐γ by human PBMCs as well as antigen‐specific CD8⁺ T‐cell proliferation. Taken together, K3‐SPG may overcome the species barrier for CpG ODN to enhance antigen‐specific CD8⁺ T‐cell responses despite the differential role of IL‐12 between human and mice.     

Tumor‐induced myeloid‐derived suppressor cell subsets exert either inhibitory or stimulatory effects on distinct CD8+ T‐cell activation events

Tumor growth coincides with an accumulation of myeloid‐derived suppressor cells (MDSCs), which exert immune suppression and which consist of two main subpopulations, known as monocytic (MO) CD11b⁺CD115⁺Ly6G^?Ly6C^high MDSCs and granulocytic CD11b⁺CD115^?Ly6G⁺Ly6C^int polymorphonuclear (PMN)‐MDSCs. However, whether these distinct MDSC subsets hamper all aspects of early CD8⁺ T‐cell activation — including cytokine production, surface marker expression, survival, and cytotoxicity — is currently unclear. Here, employing an in vitro coculture system, we demonstrate that splenic MDSC subsets suppress antigen‐driven CD8⁺ T‐cell proliferation, but differ in their dependency on IFN‐γ, STAT‐1, IRF‐1, and NO to do so. Moreover, MO‐MDSC and PMN‐MDSCs diminish IL‐2 levels, but only MO‐MDSCs affect IL‐2Rα (CD25) expression and STAT‐5 signaling. Unexpectedly, however, both MDSC populations stimulate IFN‐γ production by CD8⁺ T cells on a per cell basis, illustrating that some T‐cell activation characteristics are actually stimulated by MDSCs. Conversely, MO‐MDSCs counteract the activation‐induced change in CD44, CD62L, CD162, and granzyme B expression, while promoting CD69 and Fas upregulation. Together, these effects result in an altered CD8⁺ T‐cell adhesiveness to the extracellular matrix and selectins, sensitivity to FasL‐mediated apoptosis, and cytotoxicity. Hence, MDSCs intricately influence different CD8⁺ T‐cell activation events in vitro, whereby some parameters are suppressed while others are stimulated.     

Interleukin‐2‐mediated inhibition of dendritic cell development correlates with decreased CD135 expression and increased monocyte/macrophage precursors

We have previously shown that interleukin-2 (IL-2) inhibits dendritic cell (DC) development from mouse bone marrow (BM) precursors stimulated with the ligand for FMS-like tyrosine kinase 3 receptor (Flt3L), and have provided evidence that this inhibition occurs at the monocyte DC precursor stage of DC development. Here, we explored the mechanism of IL-2-mediated inhibition of DC development. First, we showed that these in vitro cultures accurately model DCs that develop in vivo by comparing gene and protein expression of the three main Flt3L-induced DC subsets from the BM, CD11b⁺ and CD24⁺ conventional DCs (cDCs) and plasmacytoid DCs (pDCs) with their respective ex vivo spleen DC subsets (CD11b⁺, CD8⁺ and pDCs). Next, gene expression changes were quantified in Flt3L DC subsets that developed in the presence of IL-2. These changes included increased expression of Bcl2l11, which encodes the apoptosis-inducing protein Bim, and decreased expression of Flt3 (CD135), the receptor that initiates DC development. Interleukin-2 also significantly reduced Flt3 protein expression on all three Flt3L DC subsets, and attenuated Flt3L-induced STAT3 phosphorylation in DCs. Based on these data, we hypothesized that decreased Flt3 signalling may divert BM precursors down monocyte and macrophage lineages. Indeed, addition of IL-2 led to increases in Flt3⁻ cells, including cKit⁺ Ly6C⁺ CD11b⁻ populations consistent with the recently identified committed monocyte/macrophage progenitor. Therefore, IL-2 can inhibit DC development via decreased signalling through Flt3 and increased monocyte/macrophage development.     

Gal‐3 regulates the capacity of dendritic cells to promote NKT‐cell‐induced liver injury

Galectin‐3 (Gal‐3), an endogenous lectin, exhibits pro‐ and anti‐inflammatory effects in various disease conditions. In order to explore the role of Gal‐3 in NKT‐cell‐dependent pathology, we induced hepatitis in C57BL/6 WT and Gal‐3‐deficient mice by using specific ligand for NKT cells: α‐galactosylceramide, glycolipid Ag presented by CD1d. The injection of α‐galactosylceramide significantly enhanced expression of Gal‐3 in liver NKT and dendritic cells (DCs). Genetic deletion or selective inhibition of Gal‐3 (induced by Gal‐3‐inhibitor TD139) abrogated the susceptibility to NKT‐cell‐dependent hepatitis. Blood levels of pro‐inflammatory cytokines (TNF‐α, IFN‐γ, IL‐12) and their production by liver DCs and NKT cells were also downregulated. Genetic deletion or selective inhibition of Gal‐3 alleviated influx of inflammatory CD11c⁺CD11b⁺ DCs in the liver and favored tolerogenic phenotype and IL‐10 production of liver NKT and DCs. Deletion of Gal‐3 attenuated the capacity of DCs to support liver damage in the passive transfer experiments and to produce pro‐inflammatory cytokines in vitro. Gal‐3‐deficient DCs failed to optimally stimulate production of pro‐inflammatory cytokines in NKT cells, in vitro and in vivo. In conclusion, Gal‐3 regulates the capacity of DCs to support NKT‐cell‐mediated liver injury, playing an important pro‐inflammatory role in acute liver injury.     

BDCA3 expression is associated with high IFN‐λ production by CD34+‐derived dendritic cells generated in the presence of GM‐CSF,IL‐4, and/or TGF‐β

High BDCA3 expression is associated with a specific human IFN‐λ‐producing dendritic cell (DC) subset. However, BDCA3 has also been detected on other DC subsets. Thus far, development and function of BDCA3 expression on DCs remains poorly understood. Human Langerhans cells (LCs) and interstitial DCs (intDCs) can be generated in vitro by differentiation of CD34⁺ hematopoietic progenitors via distinct precursor DCs (preDCs), CD1a⁺ preDCs, and CD14⁺ preDCs, respectively. Here, we identified BDCA3 expression in this well‐known GM‐CSF/TNF‐α‐driven culture system and described the effect of IL‐4 and/or TGF‐β on induction of BDCA3 expression. In control or TGF‐β cultures, BDCA3 was only detected on CD14⁺ preDC‐derived intDCs. IL‐4 induced BDCA3 expression in both CD14⁺‐derived and CD1a⁺‐derived cultures. TGF‐β and IL‐4 together further increased CD14⁺‐derived and CD1a⁺‐derived BDCA3⁺ DC frequencies, which partly expressed CLEC9A, but were not identical to the BDCA3^highCLEC9A⁺ DC subset in vivo. Importantly, BDCA3⁺ cells, but not BDCA3^? cells, in this system produced high IFN‐λ levels upon polyinosinic:polycytidylic acid (polyI:C) stimulation. This culture system, in which BDCA3 expression is preferentially associated with the intDC lineage and IFN‐λ‐producing capacity, will greatly contribute to further research on the function and regulation of BDCA3 expression and IFN‐λ production by DCs.     

Functional specialization of intestinal dendritic cell subsets during Th2 helminth infection in mice

Dendritic cells (DCs) are essential in dictating the nature and effectiveness of immune responses. In the intestine DCs can be separated into discrete subsets, defined by expression of CD11b and CD103, each with different developmental requirements and distinct functional potential. Recent evidence has shown that different intestinal DC subsets are involved in the induction of T helper (Th)17 and regulatory T cell responses, but the cells that initiate Th2 immune responses are still incompletely understood. We show that in the Th2 response to an intestinal helminth in mice, only CD11b⁺ and not CD11b^? DCs accumulate in the local lymph node, upregulate PDL2 and express markers of alternative activation. An enteric Th1 response instead activated both CD11b⁺ and CD11b^? DCs without eliciting alternative activation in either population. Functionally, only CD11b⁺ DCs activated during helminth infection supported Th2 differentiation in naive CD4⁺ T cells. Together our data demonstrate that the ability to prime Th2 cells during intestinal helminth infection, is a selective and inducible characteristic of CD11b⁺ DCs.     

Type I interferon potentiates T‐cell receptor mediated induction of IL‐10‐producing CD4+ T cells

Type I interferons (IFNs) have the dual ability to promote the development of the immune response and exert an anti‐inflammatory activity. We analyzed the integrated effect of IFN‐α, TCR signal strength, and CD28 costimulation on human CD4⁺ T‐cell differentiation into cell subsets producing the anti‐ and proinflammatory cytokines IL‐10 and IFN‐γ. We show that IFN‐α boosted TCR‐induced IL‐10 expression in activated peripheral CD45RA⁺CD4⁺ T cells and in whole blood cultures. The functional cooperation between TCR and IFN‐α efficiently occurred at low engagement of receptors. Moreover, IFN‐α rapidly cooperated with anti‐CD3 stimulation alone. IFN‐α, but not IL‐10, drove the early development of type I regulatory T cells that were mostly IL‐10⁺ Foxp3^? IFN‐γ^? and favored IL‐10 expression in a fraction of Foxp3⁺ T cells. Our data support a model in which IFN‐α costimulates TCR toward the production of IL‐10 whose level can be amplified via an autocrine feedback loop.     

Interferon-α mRNA in Splenic CD11b+ Marginal Zone Macrophages of C4-Deficient Mice

The absence of early complement components (C1, C4 and C2 but not C3) is a predisposing factor for systemic lupus erythematosus (SLE). Recently, we demonstrated that, in C4‐deficient (C4 def.) mice, IgM‐containing immune complexes (IgM‐IC) are filtered by the splenic barrier of marginal zone macrophages (MZM), resulting in an increased immune response against antigens within these IgM‐IC, but this could not be observed in wildtype or C3 def. mice. We hypothesized that splenic CD11b⁺ MZM play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mRNA was isolated, and real‐time PCR was performed with specific primers for murine IFN‐γ (IFN‐γ), interleukin‐12 (IL‐12) and IFN‐α (IFN‐α). We observe a moderate increase of IL‐12 and IFN‐γ mRNA in CD11b⁺ cells of C4 def. mice compared to wildtype cells. Surprisingly, the concentration of IFN‐α mRNA is six times higher in C4 def. mice. Preliminary results suggest that mRNA in CD11b⁺ cells of C3 def. mice is even lower than that in wt. Six hours following i.v. application of 20 µg of a murine monoclonal IgM anti‐dsDNA antibody, production of IL‐12, IFN‐γ and IFN‐α mRNA is increased in CD11b⁺ cells of both C4 def. and wt mice. Several references described increased levels of INF‐α in patients with SLE. Dendritic cells are discussed as a major source of IFN‐α. Our observation that C4‐deficient, SLE‐susceptible mice demonstrate an increased spontaneous IFN‐α production by splenic CD11b⁺ marginal zone macrophages could be an early sign and a trigger for the development of SLE. This is supported by the fact that the absence of C3 is not a predisposing factor for SLE and our observation that C3 def. animals display low levels of IFN‐α mRNA.     

Differential recruitment and activation of natural killer cell sub‐populations by Mycobacterium bovis‐infected dendritic cells

Through complex interplay with APCs, subsets of NK cells play an important role in shaping adaptive immune responses. Bovine tuberculosis, caused by Mycobacterium bovis, is increasing in incidence and detailed knowledge of host–pathogen interactions in the natural host is essential to facilitate disease control. We investigated the interactions of NK‐cell sub‐populations and M. bovis‐infected DCs to determine early innate mechanisms in the response to infection. A sub‐population of NK cells (NKp46⁺CD2^?) selectively expressing lymphoid homing and inflammatory chemokine receptors were induced to migrate towards M. bovis‐infected DCs. This migration was associated with increased expression of chemokines CCL3, 4, 5, 20 and CXCL8 by M. bovis‐infected DCs. Activation of NKp46⁺CD2^? NK cells and secretion of IFN‐γ was observed, a response reliant on localised IL‐12 release and direct cellular interaction. In a reciprocal manner, NKp46⁺CD2^? cells induced an increase in the intensity of cell surface MHC class II expression on DCs. In contrast, NKp46⁺CD2⁺ NK cells were unable to secrete IFN‐γ and did not reciprocally affect DCs. This study provides novel evidence to demonstrate distinct effector responses between bovine NK‐cell subsets during mycobacterial infection.