Cutting edge: JAM-C controls homeostatic chemokine secretion in lymph node fibroblastic reticular cells expressing thrombomodulin

The development and maintenance of secondary lymphoid organs, such as lymph nodes, occur in a highly coordinated manner involving lymphoid chemokine production by stromal cells. Although developmental pathways inducing lymphoid chemokine production during organogenesis are known, signals maintaining cytokine production in adults are still elusive. In this study, we show that thrombomodulin and platelet-derived growth factor receptor α identify a population of fibroblastic reticular cells in which chemokine secretion is controlled by JAM-C. We demonstrate that Jam-C-deficient mice and mice treated with Ab against JAM-C present significant decreases in stromal cell-derived factor 1α (CXCL12), CCL21, and CCL19 intranodal content. This effect is correlated with reduced naive T cell egress from lymph nodes of anti-JAM-C-treated mice.     

IFN-gamma secreted by CD103+ dendritic cells leads to IgG generation in the mesenteric lymph node in the absence of vitamin A

Although the induction mechanism of secretory IgA has been well studied, that of IgG in the mucosal compartments is not well understood. In this study, vitamin A deficiency was convincingly shown to be associated with increased IgG in serum and intestinal fluid. We found increased numbers of IgG-secreting B cells in the lamina propria of the small intestine and mesenteric lymph node (MLN) of vitamin A-deficient (VAD) mice. Of note, IFN-γ secreted by MLN dendritic cells (DCs) was significantly augmented in VAD mice, unlike control mice, and CD103(+) DCs were the main subsets to secrete IFN-γ. The aberrant increase of IgG in VAD mice can be ascribable to IFN-γ, because IFN-γ(-/-) VAD mice have normal IgG levels and the addition of rIFN-γ increased IgG production by B cells cocultured with MLN DCs from IFN-γ(-/-) VAD mice. Oral feeding of antibiotics resulted in significant reduction of IgG in VAD mice, indicating a critical role for altered commensal bacteria for IgG class-switching recombination in the absence of vitamin A. Collectively, vitamin A deficiency provokes the generation of IFN-γ-secreting CD103(+) DCs, which may be a critical regulator for IgG generation in the MLN.     

Cutting edge: human CD4+CD25+ T cells restrain the maturation and antigen-presenting function of dendritic cells

The characteristics and functions of CD4(+)CD25(+) regulatory cells have been well defined in murine and human systems. However, the interaction between CD4(+)CD25(+) T cells and dendritic cells (DC) remains unclear. In this study, we examined the effect of human CD4(+)CD25(+) T cells on maturation and function of monocyte-derived DC. We show that regulatory T cells render the DC inefficient as APCs despite prestimulation with CD40 ligand. This effect was marginally reverted by neutralizing Abs to TGF-beta. There was an increased IL-10 secretion and reduced expression of costimulatory molecules in DC. Thus, in addition to direct suppressor effect on CD4(+) T cells, regulatory T cells may modulate the immune response through DC.     

Cutting edge: Clec9A+ dendritic cells mediate the development of experimental cerebral malaria

Plasmodium infections trigger strong innate and acquired immune responses, which can lead to severe complications, including the most feared and often fatal cerebral malaria (CM). To begin to dissect the roles of different dendritic cell (DC) subsets in Plasmodium-induced pathology, we have generated a transgenic strain, Clec9A-diphtheria toxin receptor that allows us to ablate in vivo Clec9A(+) DCs. Specifically, we have analyzed the in vivo contribution of this DC subset in an experimental CM model using Plasmodium berghei, and we provide strong evidence that the absence of this DC subset resulted in complete resistance to experimental CM. This was accompanied with dramatic reduction of brain CD8(+) T cells, and those few cerebral CD8(+) T cells present had a less activated phenotype, unlike their wildtype counterparts that expressed IFN-γ and especially granzyme B. This almost complete absence of local cellular responses was also associated with reduced parasite load in the brain.     

Cutting edge: Cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells.

alpha-Galactosylceramide (alpha-GalCer) is a glycolipid with potent antitumor properties that binds to CD1d molecules and activates mouse Valpha14 and human Valpha24 NKT cells. Surprisingly, we found that, as early as 90 min after alpha-GalCer injection in vivo, NK cells also displayed considerable signs of activation, including IFN-gamma production and CD69 induction. NK activation was not observed in RAG- or CD1-deficient mice, and it was decreased by pretreatment with anti-IFN-gamma Abs, suggesting that, despite its rapid induction, it was a secondary event that depended on IFN-gamma release by NKT cells. At later time points, B cells and CD8 T cells also began to express CD69. These findings identify a high-speed communication network between the innate and adaptive immune systems in vivo that is initiated upon NKT cell activation. They also suggest that the antitumor effects of alpha-GalCer result from the sequential recruitment of distinct innate and adaptive effector lymphocytes.     

Cutting edge: differential chemokine production by myeloid and plasmacytoid dendritic cells

To examine the different roles of myeloid dendritic cells (M-DCs) and plasmacytoid dendritic cells (P-DCs) in the induction and regulation of immune response, we have studied chemokine secretion by freshly isolated DC subsets in response to bacterial, viral, and T cell-derived stimuli. M-DCs selectively produced very high levels of the homeostatic chemokines CC chemokine ligand (CCL)17 and CCL22, while P-DCs produced very little if any. In contrast, the proinflammatory chemokine CCL3 was secreted mostly by P-DCs, whereas CCL4 and CXC chemokine ligand 8 were produced by both subsets. The selective production of CCL17 and CCL22 by M-DCs but not P-DCs was confirmed in vivo by immunohistology on human reactive lymph node sections. The high production of CCR4 ligands by M-DCs suggests their capacity to selectively recruit at sites of inflammation T cells with regulatory properties or with a Th2 phenotype, whereas P-DCs, by preferentially secreting CCR1/CCR5 ligands, would mostly recruit effector T cells and, in particular, Th1-type cells.     

Cutting edge: central nervous system plasmacytoid dendritic cells regulate the severity of relapsing experimental autoimmune encephalomyelitis

Plasmacytoid dendritic cells (pDCs) have both stimulatory and regulatory effects on T cells. pDCs are a major CNS-infiltrating dendritic cell population during experimental autoimmune encephalomyelitis but, unlike myeloid dendritic cells, have a minor role in T cell activation and epitope spreading. We show that depletion of pDCs during either the acute or relapse phases of experimental autoimmune encephalomyelitis resulted in exacerbation of disease severity. pDC depletion significantly enhanced CNS but not peripheral CD4(+) T cell activation, as well as IL-17 and IFN-gamma production. Moreover, CNS pDCs suppressed CNS myeloid dendritic cell-driven production of IL-17, IFN-gamma, and IL-10 in an IDO-independent manner. The data demonstrate that pDCs play a critical regulatory role in negatively regulating pathogenic CNS CD4(+) T cell responses, highlighting a new role for pDCs in inflammatory autoimmune disease.     

Ca2+ signals in CD4+ T cells during early contacts with antigen-bearing dendritic cells in lymph node

T cell activation by APC requires cytosolic Ca(2+) ([Ca(2+)](i)) elevation. Using two-photon microscopy, we visualized Ca(2+) signaling and motility of murine CD4(+) T cells within lymph node (LN) explants under control, inflammatory, and immunizing conditions. Without Ag under basal noninflammatory conditions, T cells showed infrequent Ca(2+) spikes associated with sustained slowing. Inflammation reduced velocities and Ca(2+) spiking in the absence of specific Ag. During early Ag encounter, most T cells engaged Ag-presenting dendritic cells in clusters, and showed increased Ca(2+) spike frequency and elevated basal [Ca(2+)](i). These Ca(2+) signals persisted for hours, irrespective of whether T cells were in contact with visualized dendritic cells. We propose that sustained increases in basal [Ca(2+)](i) and spiking frequency constitute a Ca(2+) signaling modality that, integrated over hours, distinguishes immunogenic from basal state in the native lymphoid environment.     

Cutting edge: analysis of human V alpha 24+CD8+ NK T cells activated by alpha-galactosylceramide-pulsed monocyte-derived dendritic cells

Human Valpha24(+) NKT cells constitute a counterpart of mouse Valpha14(+) NKT cells, both of which use an invariant TCR-alpha chain. The human Valpha24(+) NKT cells as well as mouse Valpha14(+) NKT cells are activated by glycolipids in a CD1d-restricted manner and produce many immunomodulatory cytokines, possibly affecting the immune balance. In mice, it has been considered from extensive investigations that Valpha14(+)CD8(+) NKT cells that express invariant TCR do not exist. Here we introduce human Valpha24(+)CD8(+) NKT cells. These cells share important features of Valpha24(+) NKT cells in common, but in contrast to CD4(-)CD8(-) (double-negative) or CD4(+) Valpha24(+) NKT cells, they do not produce IL-4. Our discovery may extend and deepen the research field of Valpha24(+) NKT cells as well as help to understand the mechanism of the immune balance-related diseases.     

Cutting edge: peripheral neuropeptides attract immature and arrest mature blood-derived dendritic cells

Dendritic cells (DC) are highly motile and play a key role in mediating immune responses in various tissues and lymphatic organs. We investigated locomotion of mononuclear cell-derived DC at different maturation stages toward gradients of sensory neuropeptides in vitro. Calcitonin gene-related peptide, vasoactive intestinal polypeptide, secretin, and secretoneurin induced immature DC chemotaxis comparable to the potency of RANTES, whereas substance P and macrophage-inflammatory protein-3beta stimulated immature cell migration only slightly. Checkerboard analyses revealed a true chemotactic response induced by neuropeptides. Upon maturation of DC, neuropeptides inhibited spontaneous, macrophage-inflammatory protein-3beta- and 6Ckine-induced cell migration. Maturation-dependent changes in migratory behavior coincided with distinct neuropeptide-induced signal transduction in DC. Peripheral neuropeptides might guide immature DC to peripheral nerve fibers where high concentrations of these peptides can arrest the meanwhile matured cells. It seems that one function of sensory nerves is to fasten DC at sites of inflammation.     

T regulatory cells control numbers of NK cells and CD8alpha+ immature dendritic cells in the lymph node paracortex

The spleen contains numerous NK cells whose differentiation profile is characterized by a preponderance of mature elements located mainly in the red pulp. In contrast, lymph nodes (LNs) contain few NK cells and they are sited mostly in T cell zones and skewed toward immature developmental stages. We show that, in mice, naturally occurring CD4+ Foxp3+ regulatory T (Treg) cells are both necessary and sufficient to repress accumulation of NK cells in resting LNs. Moreover, we present evidence that Treg cells hamper generation of mature NK cells through short-range interactions with NK precursors. In turn, mature NK cells specifically regulate the amount of CD8alpha+ phenotypically immature dendritic cells present in LN T cell zones. We propose that the dominant influence of Treg cells on NK cell precursors and CD8alpha+ immature dendritic cells explains why "quiescent" LNs in the absence of infection function as privileged sites for induction and maintenance of tolerance to peripheral Ags.     

Cutting edge: innate immune system discriminates between RNA containing bacterial versus eukaryotic structural features that prime for high-level IL-12 secretion by dendritic cells

RNA derived from bacterial but not eukaryotic sources, when transfected into human monocyte-derived dendritic cell precursors, induces high-level IL-12 secretion in conjunction with dendritic cell maturation stimuli. In vitro-transcribed mRNA that mimics the structure of bacterial mRNA in the lack of a long 3'-poly(A) tail likewise induces IL-12 secretion, but this property is lost upon efficient enzymatic 3'-polyadenylation. Among other tested RNAs, only polyuridylic acid induced IL-12 p70. This RNA response phenomenon appears biologically distinct from the classically defined response to dsRNA. RNA-transfected APC also polarize T cells in an IL-12-dependent manner toward the IFN-gamma(high)IL-5 (low) Th1 phenotype, suggesting a link between the detection of appropriately structured RNA and the skewing of immune responses toward those best suited for controlling intracellular microbes. RNA structured to emulate bacterial patterns constitutes a novel vaccine strategy to engender polarized Th1-type immune responses.     

Cutting edge: conventional CD8 alpha+ dendritic cells are generally involved in priming CTL immunity to viruses

Dendritic cells (DCs) play a central role in initiating immune responses. Despite this, there is little understanding how different DC subsets contribute to immunity to different pathogens. CD8alpha(+) DC have been shown to prime immunity to HSV. Whether this very limited capacity of a single DC subset priming CTL immunity is restricted to HSV infection or is a more general property of anti-viral immunity was examined. Here, we show that the CD8alpha(+) DCs are the principal DC subset that initiates CTL immunity to s.c. infection by influenza virus, HSV, and vaccinia virus. This same subset also dominated immunity after i.v. infection with all three viruses, suggesting a similar involvement in other routes of infection. These data highlight the general role played by CD8alpha(+) DCs in CTL priming to viral infection and raises the possibility that this DC subset is specialized for viral immunity.     

Cutting edge: a novel role for Fas ligand in facilitating antigen acquisition by dendritic cells

Fas ligand (FasL)-expressing tumor cells are found to effectively mediate rejection of the coinoculated FasL negative parental cells while having no effect on the growth of histologically distinct tumor cells. These observations indicate that FasL induces a specific immune response against Ag derived from FasL-bearing tumors and suggest a possible role for FasL in tumor Ag presentation. Indeed, tumor cells expressing FasL can efficiently interact with dendritic cells (DCs) and this interaction requires the expression of membrane-bound FasL on tumors and Fas on DCs. Moreover, DCs cocultured with FasL-expressing tumors are able to elicit a tumor-specific immune response in vivo, suggesting that DCs acquire tumor Ag during the Fas/FasL-mediated DC-tumor contact. These results identify a novel role for FasL in augmenting tumor-DC interactions and subsequent tumor Ag acquisition by DCs, and suggest that FasL-expressing tumor cells could be used to generate tumor-specific DC vaccines.     

Cutting edge: Protective effect of CX3CR1+ dendritic cells in a vaccinia virus pulmonary infection model

The protective host immune response to viral infections requires both effective innate and adaptive immune responses. Cross-talk between the two responses is coordinated by the chemokine network and professional APCs such as dendritic cells (DCs). In mice, subpopulations of myeloid DCs in peripheral tissues such as lungs and in blood express CX3CR1 depending on the inflammation state. We thus examined the host response of mice deficient in the chemokine receptor CX3CR1 to an intranasal vaccinia virus infection. CX3CR1-deficient mice displayed significantly more severe morbidity and mortality compared with control wild-type mice within 10 d following vaccinia virus infection. CX3CR1(-/-) mice had increased viral loads and a reduced T cell response compared with wild-type mice. Finally, an adoptive transfer of CX3CR1(+/+) DCs completely protected CX3CR1(-/-) mice to a previously lethal infection. This study therefore opens up the possibility of novel antiviral therapeutics targeting lung DC recruitment.     

Secretory leukoprotease inhibitor in mucosal lymph node dendritic cells regulates the threshold for mucosal tolerance

The notion that the mucosal immune system maintains a tolerogenic response to harmless Ags while continually being challenged with microbial products seems an enigma. The aim of this study was to unravel mechanisms that are involved in regulating the development of tolerance under constant microbial pressure. The tolerogenic response to Ags administered via the nasal mucosa is dependent on the organized lymphoid tissue of the cervical lymph nodes (LN). We show that cervical LN differentially express secretory leukoprotease inhibitor (SLPI) compared with peripheral LN. SLPI was expressed by dendritic cells (DCs) and because SLPI is known to suppress LPS responsiveness, it was hypothesized that its expression in mucosal DCs may be required to regulate cellular activation to microbial products. Indeed, compared with wild-type controls, bone marrow-derived DCs from SLPI(-/-) mice released more inflammatory cytokines and enhanced T cell proliferation after stimulation with low dose LPS. This increased sensitivity to LPS was accompanied by increased NF-kappaB p65 activation in SLPI(-/-) DCs. In vivo, nasal application of OVA with LPS to SLPI(-/-) mice resulted in enhanced DC activation in the cervical LN reflected by increased costimulatory molecule expression and release of inflammatory cytokines. This led to failure to maintain tolerance to nasal OVA application in the presence of low doses of LPS. We propose that expression of SLPI functions as a rheostat by controlling the level of bacterial stimuli that induce mucosal DC activation. As such, it regulates the quality of the ensuing Ag-specific immune response in the mucosa draining LN.     

Cutting edge: impairment of dendritic cells and adaptive immunity by Ebola and Lassa viruses

Acute infection of humans with Ebola and Lassa viruses, two principal etiologic agents of hemorrhagic fevers, often results in a paradoxical pattern of immune responses: early infection, characterized by an outpouring of inflammatory mediators such as TNF-alpha, IL-1 beta, and IL-6, vs late stage infections, which are associated with poor immune responses. The mechanisms underlying these diverse outcomes are poorly understood. In particular, the role played by cells of the innate immune system, such as dendritic cells (DC), is not known. In this study, we show that Ebola and Lassa viruses infect human monocyte-derived DC and impair their function. Monocyte-derived DC exposed to either virus fail to secrete proinflammatory cytokines, do not up-regulate costimulatory molecules, and are poor stimulators of T cells. These data represent the first evidence for a mechanism by which Ebola and Lassa viruses target DC to impair adaptive immunity.     

Cutting edge: resistance to apoptosis and continuous proliferation of dendritic cells deficient for TNF receptor-1

The individual roles of the two TNFRs on dendritic cells (DC) are poorly understood. Investigating bone marrow-derived DC from TNFR-deficient mice, we found that cultures from TNFR1(-/-) mice continue to form proliferating clusters for 6-9 mo. In contrast, DC derived from wild-type, TNFR2(-/-), or TNFR1/2(-/-) mice survived for only 3-4 wk. DC obtained from these TNFR1(-/-) long term cultures (LTC) mice show an unusual mixed immature/mature phenotype. The continuous proliferation of the LTC is GM-CSF dependent and correlates with decreased protein levels of the cyclin-dependent kinase inhibitors p27(KIP1) and p21(CIP1). Prolonged survival of TNFR1(-/-) DC appears to be independent from NF-kappaB and Bcl-2 pathways and is rather enabled by the down-regulation of CD95, resulting in the resistance to CD95 ligand-induced apoptosis. These data point to proapoptotic signals mediated via TNFR1 and antiapoptotic signals mediated via TNFR2 in DC.