The C-type lectin MGL expressed by dendritic cells detects glycan changes on MUC1 in colon carcinoma

The epithelial mucin MUC1 is a high molecular weight membrane glycoprotein frequently overexpressed and aberrantly glycosylated in adenocarcinoma. Mucins normally contain high amounts of O-linked carbohydrate structures that may influence immune reactions to this antigen. During malignant transformation, certain glyco-epitopes of MUC1, such as Tn-antigen, TF-antigen and their sialylated forms become exposed. The role of these glycan structures in tumor biology is unknown, but their presence is known to correlate with poor prognosis in several adenocarcinomas. We analyzed the potency of MUC1 containing Tn-antigens (MUC1-Tn) to target C-type lectins that function as carbohydrate recognition and uptake molecules on dendritic cells (DC). We identified the macrophage galactose type C-type lectin (MGL), expressed by both DC and macrophages, as the receptor for recognition and binding of MUC1-Tn. To validate the occurrence of MGL–MUC1 interactions in situ, we studied the binding of MGL to MUC1 in primary colon carcinoma tissue. Isolation of MUC1 out of colon carcinoma tissue showed strong binding activity to MGL. Interestingly, MGL binding to MUC1 was highly correlated to binding by the lectin Helix pomatia agglutinin (HPA), which is associated with poor prognosis in colorectal cancer. The detection of MGL positive cells in situ at the tumor site together with the modified glycosylation status of MUC1 to target MGL on DC suggests that MGL positive antigen presenting cells may play a role in tumor progression.     

Monitoring of in vivo function of superparamagnetic iron oxide labelled murine dendritic cells during anti-tumour vaccination

Dendritic cells (DCs) generated in vitro to present tumour antigens have been injected in cancer patients to boost in vivo anti-tumour immune responses. This approach to cancer immunotherapy has had limited success. For anti-tumour therapy, delivery and subsequent migration of DCs to lymph nodes leading to effective stimulation of effector T cells is thought to be essential. The ability to non-invasively monitor the fate of adoptively transferred DCs in vivo using magnetic resonance imaging (MRI) is an important clinical tool to correlate their in vivo behavior with response to treatment. Previous reports of superparamagnetic iron oxides (SPIOs) labelling of different cell types, including DCs, have indicated varying detrimental effects on cell viability, migration, differentiation and immune function. Here we describe an optimised labelling procedure using a short incubation time and low concentration of clinically used SPIO Endorem to successfully track murine DC migration in vivo using MRI in a mouse tumour model. First, intracellular labelling of bone marrow derived DCs was monitored in vitro using electron microscopy and MRI relaxometry. Second, the in vitro characterisation of SPIO labelled DCs demonstrated that viability, phenotype and functions were comparable to unlabelled DCs. Third, ex vivo SPIO labelled DCs, when injected subcutaneously, allowed for the longitudinal monitoring by MR imaging of their migration in vivo. Fourth, the SPIO DCs induced the proliferation of adoptively transferred CD4(+) T cells but, most importantly, they primed cytotoxic CD8(+) T cell responses to protect against a B16-Ova tumour challenge. Finally, using anatomical information from the MR images, the immigration of DCs was confirmed by the increase in lymph node size post-DC injection. These results demonstrate that the SPIO labelling protocol developed in this study is not detrimental for DC function in vitro and in vivo has potential clinical application in monitoring therapeutic DCs in patients with cancer.     

Lung neutrophils facilitate activation of naive antigen-specific CD4+ T cells during Mycobacterium tuberculosis infection

Initiation of the adaptive immune response to Mycobacterium tuberculosis occurs in the lung-draining mediastinal lymph node and requires transport of M. tuberculosis by migratory dendritic cells (DCs) to the local lymph node. The previously published observations that 1) neutrophils are a transiently prominent population of M. tuberculosis-infected cells in the lungs early in infection and 2) that the peak of infected neutrophils immediately precedes the peak of infected DCs in the lungs prompted us to characterize the role of neutrophils in the initiation of adaptive immune responses to M. tuberculosis. We found that, although depletion of neutrophils in vivo increased the frequency of M. tuberculosis-infected DCs in the lungs, it decreased trafficking of DCs to the mediastinal lymph node. This resulted in delayed activation (CD69 expression) and proliferation of naive M. tuberculosis Ag85B-specific CD4 T cells in the mediastinal lymph node. To further characterize the role of neutrophils in DC migration, we used a Transwell chemotaxis system and found that DCs that were directly infected by M. tuberculosis migrated poorly in response to CCL19, an agonist for the chemokine receptor CCR7. In contrast, DCs that had acquired M. tuberculosis through uptake of infected neutrophils exhibited unimpaired migration. These results revealed a mechanism wherein neutrophils promote adaptive immune responses to M. tuberculosis by delivering M. tuberculosis to DCs in a form that makes DCs more effective initiators of naive CD4 T cell activation. These observations provide insight into a mechanism for neutrophils to facilitate initiation of adaptive immune responses in tuberculosis.     

Dendritic cells and C-type lectin receptors: coupling innate to adaptive immune responses

Dendritic cells (DCs) have an important function in the initiation and differentiation of immune responses, linking innate information to tailored adaptive responses. Depending on the pathogen invading the body, specific immune responses are built up that are crucial for eliminating the pathogen from the host. Host recognition of invading microorganisms relies on evolutionarily ancient, germline-encoded pattern recognition receptors (PRRs) that are highly expressed on the cell surface of DCs, of which the Toll-like receptors (TLRs) are well characterized and recognize bacterial or viral components. Moreover, they bind a variety of self-proteins released from damaged tissues including several heat-shock proteins. The membrane-associated C-type lectin receptors (CLRs) recognize glycan structures expressed by host cells of the immune system or on specific tissues, which upon recognition allow cellular interactions between DCs and other immune or tissue cells. In addition, CLRs can function as PRRs. In contrast to TLRs, CLRs recognize carbohydrate structures present on the pathogens. Modification of glycan structures on pathogens to mimic host glycans can thereby alter CLR interactions that subsequently modifies DC-induced polarization. In this review, we will discuss in detail how specific glycosylation of antigens can dictate both the innate and adaptive interactions that are mediated by CLRs on DCs and how this balances immune activation and inhibition of DC function.     

Prion protein expression by mouse dendritic cells is restricted to the nonplasmacytoid subsets and correlates with the maturation state

Expression of the physiological cellular prion protein (PrP(C)) is remarkably regulated during differentiation and activation of cells of the immune system. Among these, dendritic cells (DCs) display particularly high levels of membrane PrP(C), which increase upon maturation, in parallel with that of molecules involved in Ag presentation to T cells. Freshly isolated mouse Langerhans cells, dermal DCs, and DCs from thymus, spleen, and mesenteric lymph nodes expressed low to intermediate levels of PrP(C). Highest levels of both PrP(C) and MHC class II molecules were displayed by lymph node CD8alpha(int) DCs, which represent fully mature cells having migrated from peripheral tissues. Maturation induced by overnight culture resulted in increased levels of surface PrP(C), as did in vivo DC activation by bacterial LPS. Studies on Fms-like tyrosine kinase 3 ligand bone marrow-differentiated B220(-) DCs confirmed that PrP(C) expression followed that of MHC class II and costimulatory molecules, and correlated with IL-12 production in response to TLR-9 engagement by CpG. However, at variance with conventional DCs, B220(+) plasmacytoid DCs isolated from the spleen, or in vitro differentiated, did not significantly express PrP(C), both before and after activation by TLR-9 engagement. PrP knockout mice displayed higher numbers of spleen CD8alpha(+) DCs, but no significant differences in their maturation response to stimulation through TLR-4 and TLR-9 were noticed. Results are discussed in relation to the functional relevance of PrP(C) expression by DCs in the induction of T cell responses, and to the pathophysiology of prion diseases.     

Carbohydrate binding mechanism of the macrophage galactose-type C-type lectin 1 revealed by saturation transfer experiments

Macrophage galactose-type C-type lectins 1 and 2 (MGL1/2) are expressed on the surfaces of macrophages and immature dendritic cells. Despite the high similarity between the primary sequences of MGL1 and MGL2, they display different ligand specificities. MGL1 shows high affinity for the LewisX trisaccharide, whereas MGL2 shows affinity for N-acetylgalactosamine. To elucidate the structural basis for the ligand specificities of the MGLs, we performed NMR analyses of the MGL1-LewisX complex. To identify the LewisX binding site on MGL1, a saturation transfer experiment for the MGL1-LewisX complex where sugar-CH/CH2-selective saturation was applied was carried out. To obtain sugar moiety-specific information on the interface between MGL1 and the LewisX trisaccharide, saturation transfer experiments where each of galactose-H5-, fucose-CH3-, and N-acetylglucosamine-CH3-selective saturations was applied to the MGL1-LewisX complex were performed. Based on these results, we present a LewisX binding mode on MGL1 where the galactose moiety is bound to the primary sugar binding site, including Asp-94, Trp-96, and Asp-118, and the fucose moiety interacts with the secondary sugar binding site, including Ala-89 and Thr-111. Ala-89 and Thr-111 in MGL1 are replaced with arginine and serine in MGL2, respectively. The hydrophobic environment formed by a small side chain of Ala-89 and a methyl group of Thr-111 is a requisite for the accommodation of the fucose moiety of the LewisX trisaccharide within the sugar binding site of MGL1.     

MGL2+ Dermal Dendritic Cells Are Sufficient to Initiate Contact Hypersensitivity In Vivo

Background

Dendritic cells (DCs) are the most potent antigen-presenting cells in the mammalian immune system. In the skin, epidermal Langerhans cells (LCs) and dermal dendritic cells (DDCs) survey for invasive pathogens and present antigens to T cells after migration to the cutaneous lymph nodes (LNs). So far, functional and phenotypic differences between these two DC subsets remain unclear due to lack of markers to identify DDCs.

Methodology/Principal Findings

In the present report, we demonstrated that macrophage galactose-type C-type lectin (MGL) 2 was exclusively expressed in the DDC subset in the skin-to-LN immune system. In the skin, MGL2 was expressed on the majority (about 88%) of MHCII⁺CD11c⁺ cells in the dermis. In the cutaneous LN, MGL2 expression was restricted to B220⁻CD8α^loCD11b⁺CD11c⁺MHCII^hi tissue-derived DC. MGL2⁺DDC migrated from the dermis into the draining LNs within 24 h after skin sensitization with FITC. Distinct from LCs, MGL2⁺DDCs localized near the high endothelial venules in the outer T cell cortex. In FITC-induced contact hypersensitivity (CHS), adoptive transfer of FITC⁺MGL2⁺DDCs, but not FITC⁺MGL2⁻DCs into naive mice resulted in the induction of FITC-specific ear swelling, indicating that DDCs played a key role in initiation of immune responses in the skin.

Conclusions/Significance

These results demonstrated the availability of MGL2 as a novel marker for DDCs and suggested the contribution of MGL2⁺ DDCs for initiating CHS.     

Identification of sialoadhesin as a dominant lymph node counter-receptor for mouse macrophage galactose-type C-type lectin 1

In the sensitization phase of contact hypersensitivity in mice, dermal macrophages (MOs) expressing MO galactose-type C-type lectin1 (MGL1) are known to migrate from the dermis to lymph nodes (LNs) where they accumulate in the subcapsular sinus, interfollicular regions, and areas surrounding high endothelial venules. We hypothesize that the interactions between MGL1 and its ligands determine the localizations of MGL1-positive cells within the LNs. In the present study, our major aim was to isolate MGL1 counter-receptor(s) from lysates of LNs using affinity chromatography with immobilized recombinant MGL1. Fractions bound and eluted with EDTA were analyzed by SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. One of the predominant components was sialoadhesin (Sn, Siglec-1). Sn from lysates of LNs was immobilized on microtiter plates precoated with anti-Sn monoclonal antibody, and binding of recombinant MGL1 and adhesion of cells expressing MGL1 were tested. The binding of recombinant MGL1 to Sn was shown to be dependent on Ca2+ and N-glycans on Sn. MGL1-transfected Chinese hamster ovary cells adhered to the Sn-coated plates, whereas mock transfectants did not. Immunohistochemical localization of anti-Sn monoclonal antibody in LN coincided with the subcapsular sinus area to which recombinant MGL1 was bound. Furthermore, the distribution of MGL1+ cells after sensitization with FITC was demonstrated to overlap with that of Sn within the subcapsular sinus of draining LNs. These results suggest that Sn acts as an endogenous counter-receptor for MGL1.     

Endogenously expressed nef uncouples cytokine and chemokine production from membrane phenotypic maturation in dendritic cells

Immature dendritic cells (DCs), unlike mature DCs, require the viral determinant nef to drive immunodeficiency virus (SIV and HIV) replication in coculture with CD4(+) T cells. Since immature DCs may capture and get infected by virus during mucosal transmission, we hypothesized that Nef associated with the virus or produced during early replication might modulate DCs to augment virus dissemination. Adenovirus vectors expressing nef were used to introduce nef into DCs in the absence of other immunodeficiency virus determinants to examine Nef-induced changes that might activate immature DCs to acquire properties of mature DCs and drive virus replication. Nef expression by immature human and macaque DCs triggered IL-6, IL-12, TNF-alpha, CXCL8, CCL3, and CCL4 release, but without up-regulating costimulatory and other molecules characteristic of mature DCs. Coincident with this, nef-expressing immature DCs stimulated stronger autologous CD4(+) T cell responses. Both SIV and HIV nef-expressing DCs complemented defective SIVmac239 delta nef, driving replication in autologous immature DC-T cell cultures. In contrast, if DCs were activated after capturing delta nef, virus growth was not exacerbated. This highlights one way in which nef-defective virus-bearing immature DCs that mature while migrating to draining lymph nodes could induce stronger immune responses in the absence of overwhelming productive infection (unlike nef-containing wild-type virus). Therefore, Nef expressed in immature DCs signals a distinct activation program that promotes virus replication and T cell recruitment but without complete DC maturation, thereby lessening the likelihood that wild-type virus-infected immature DCs would activate virus-specific immunity, but facilitating virus dissemination.     

Human cytomegalovirus-encoded interleukin-10 homolog inhibits maturation of dendritic cells and alters their functionality

Interleukin-10 (IL-10) suppresses the maturation and cytokine production of dendritic cells (DCs), key regulators of adaptive immunity, and prevents the activation and polarization of na?ve T cells towards protective gamma interferon-producing effectors. We hypothesized that human cytomegalovirus (HCMV) utilizes its viral IL-10 homolog (cmvIL-10) to attenuate DC functionality, thereby subverting the efficient induction of antiviral immune responses. RNA and protein analyses demonstrated that the cmvIL-10 gene was expressed with late gene kinetics. Treatment of immature DCs (iDCs) with supernatant from HCMV-infected cultures inhibited both the lipopolysaccharide-induced DC maturation and proinflammatory cytokine production. These inhibitory effects were specifically mediated through the IL-10 receptor and were not observed when DCs were treated with supernatant of cells infected with a cmvIL-10-knockout mutant. Incubation of iDCs with recombinant cmvIL-10 recapitulated the inhibition of maturation. Furthermore, cmvIL-10 had pronounced long-term effects on those DCs that could overcome this inhibition of maturation. It enhanced the migration of mature DCs (mDCs) towards the lymph node homing chemokine but greatly reduced their cytokine production. The inability of mDCs to secrete IL-12 was maintained, even when they were restimulated by the activated T-cell signal CD40 ligand in the absence of cmvIL-10. Importantly, cmvIL-10 potentiates these anti-inflammatory effects, at least partially, by inducing endogenous cellular IL-10 expression in DCs. Collectively, we show that cmvIL-10 causes long-term functional alterations at all stages of DC activation.     

Deficit of CD47 results in a defect of marginal zone dendritic cells, blunted immune response to particulate antigen and impairment of skin dendritic cell migration

CD47 is a ubiquitously expressed cell surface glycoprotein that associates with integrins and regulates chemotaxis, migration, and activation of leukocytes. CD47 is also a ligand for signal regulatory protein alpha, a cell surface receptor expressed on monocytes, macrophages, granulocytes, and dendritic cell (DC) subsets that regulates cell activation, adhesion, and migration. Although the function of CD47 in macrophages and granulocytes has been studied in detail, little is known about the role of CD47 in DC biology in vivo. In this study we demonstrate that CD47(-/-) mice exhibit a selective reduction of splenic CD11c(high)CD11b(high)CD8alpha(-)CD4(+) DCs. These DCs correspond to marginal zone DCs and express signal regulatory protein alpha, possibly explaining their selective deficiency in CD47(-/-) mice. Deficiency of marginal zone DCs resulted in impairment of IgG responses to corpusculate T cell-independent Ags. Although epidermal DCs were present in normal numbers in CD47(-/-) mice, their migration to draining lymph nodes in response to contact sensitization was impaired, while their maturation was intact. In vitro, CD47(-/-) mature DCs showed normal CCR7 expression but impaired migration to CCL-19, whereas immature DC response to CCL-5 was only slightly impaired. These results demonstrate a fundamental role of CD47 in DC migration in vivo and in vitro and in the function of marginal zone DCs.     

Mast cell migration from the skin to the draining lymph nodes upon ultraviolet irradiation represents a key step in the induction of immune suppression

The UV radiation in sunlight is the primary cause of skin cancer. UV is also immunosuppressive and numerous studies have shown that UV-induced immune suppression is a major risk factor for skin cancer induction. Previous studies demonstrated that dermal mast cells play a critical role in the induction of immune suppression. Mast cell-deficient mice are resistant to the immunosuppressive effects of UV radiation, and UV-induced immune suppression can be restored by injecting bone marrow-derived mast cells into the skin of mast cell- deficient mice. The exact process however, by which mast cells contribute to immune suppression, is not known. In this study, we show that one of the first steps in the induction of immune suppression is mast cell migration from the skin to the draining lymph nodes. UV exposure, in a dose-dependent manner, causes a significant increase in lymph node mast cell numbers. When GFP(+) skin was grafted onto mast cell-deficient mice, we found that GFP(+) mast cells preferentially migrated into the lymph nodes draining the skin. The mast cells migrated primarily to the B cell areas of the draining nodes. Mast cells express CXCR4(+) and UV exposure up-regulated the expression of its ligand CXCL12 by lymph node B cells. Treating UV-irradiated mice with a CXCR4 antagonist blocked mast cell migration and abrogated UV-induced immune suppression. Our findings indicate that UV-induced mast cell migration to draining lymph nodes, mediated by CXCR4 interacting with CXCL12, represents a key early step in UV-induced immune suppression.     

Opsonization with C1q and mannose-binding lectin targets apoptotic cells to dendritic cells

Deficiencies of early components of the classical complement pathway, particularly C1q, are strongly associated with susceptibility to systemic lupus erythematosus. Recent data link this predisposal to autoimmunity to an inappropriate clearance of apoptotic cells, which could lead to a loss of self-tolerance. In the present study, we demonstrate that opsonization of apoptotic cells with C1q and mannose-binding lectin allows and facilitates their uptake not only by macrophages but also by human immature dendritic cells (DCs). Both C1q and mannose-binding lectin enhance the uptake of apoptotic cells by DCs in a dose-dependent way. The uptake of C1q-opsonized apoptotic cells, but not nonopsonized apoptotic cells, by DCs stimulated the production of IL-6, IL-10, and TNF-alpha, without an effect on IL-12p70. We conclude that these recognition molecules of the complement system do not sequester apoptotic cells from DCs, but rather promote their uptake by immature DCs. Therefore, we propose that early complement components support safe clearance of cellular debris by facilitating phagocytosis and possibly by immunomodulatory mechanisms, thus preventing autoimmunity.     

Tick saliva inhibits dendritic cell migration, maturation, and function while promoting development of Th2 responses

Similarly to other blood-feeding arthropods, ticks have evolved immunosuppressive mechanisms enabling them to overcome the host immune system. Although the immunomodulatory effect of tick saliva on several cell populations of the immune system has been extensively studied, little is known about its impact on dendritic cells (DCs). We have examined the effect of Ixodes ricinus tick saliva on DC function in vitro and in vivo. Exposure of DCs to tick saliva in vitro resulted in impaired maturation, upon CD40 or TLR9, TLR3 and TLR7 ligation, as well as reduced Ag presentation capacity. Administration of tick saliva in vivo significantly inhibited maturation and early migration of DCs from inflamed skin to draining lymph nodes, and decreased the capacity of lymph node DCs to present soluble Ag to specific T cells. Moreover, saliva-exposed DCs failed to induce efficient Th1 and Th17 polarization and promoted development of Th2 responses. Our data reveal a complex inhibitory effect exerted by tick saliva on DC function. Given the role of DCs as the key instigators of adaptive immune responses, alteration of their function might represent a major mechanism of tick-mediated immune evasion.     

Skin langerin+ dendritic cells transport intradermally injected anti-DEC-205 antibodies but are not essential for subsequent cytotoxic CD8+ T cell responses

Incorporation of Ags by dendritic cells (DCs) increases when Ags are targeted to endocytic receptors by mAbs. We have previously demonstrated in the mouse that mAbs against C-type lectins administered intradermally are taken up by epidermal Langerhans cells (LCs), dermal Langerin(neg) DCs, and dermal Langerin(+) DCs in situ. However, the relative contribution of these skin DC subsets to the induction of immune responses after Ag targeting has not been addressed in vivo. We show in this study that murine epidermal LCs and dermal DCs transport intradermally injected mAbs against the lectin receptor DEC-205/CD205 in vivo. Skin DCs targeted in situ with mAbs migrated through lymphatic vessels in steady state and inflammation. In the skin-draining lymph nodes, targeting mAbs were found in resident CD8α(+) DCs and in migrating skin DCs. More than 70% of targeted DCs expressed Langerin, including dermal Langerin(+) DCs and LCs. Numbers of targeted skin DCs in the nodes increased 2-3-fold when skin was topically inflamed by the TLR7 agonist imiquimod. Complete removal of the site where OVA-coupled anti-DEC-205 had been injected decreased endogenous cytotoxic responses against OVA peptide-loaded target cells by 40-50%. Surprisingly, selective ablation of all Langerin(+) skin DCs in Langerin-DTR knock-in mice did not affect such responses independently of the adjuvant chosen. Thus, in cutaneous immunization strategies where Ag is targeted to DCs, Langerin(+) skin DCs play a major role in transport of anti-DEC-205 mAb, although Langerin(neg) dermal DCs and CD8α(+) DCs are sufficient to subsequent CD8(+) T cell responses.     

Variable requirement of dendritic cells for recruitment of NK and T cells to different TLR agonists

TLRs initiate the host immune response to microbial pathogens by activating cells of the innate immune system. Dendritic cells (DCs) can be categorized into two major groups, conventional DCs (including CD8(+) and CD8(-) DCs) and plasmacytoid DCs. In mice, these subsets of DCs express a variety of TLRs, with conventional DCs responding in vitro to predominantly TLR3, TLR4, TLR5, and TLR9 ligands, and plasmacytoid DCs responding mainly to TLR7 and TLR9 ligands. However, the in vivo requirement of DCs to initiate immune responses to specific TLR agonists is not fully known. Using mice depleted of >90% of CD11c(+) MHC class II(+) DCs, we demonstrate that cellular recruitment, including CD4(+) T cell and CX5(+)DX5(+) NK cell recruitment to draining lymph nodes following the footpad administration of TLR4 and TLR5 agonists, is dramatically decreased upon reduction of DC numbers, but type I IFN production can partially substitute for DCs in response to TLR3 and TLR7 agonists. Interestingly, TLR ligands can activate T cells and NK cells in the draining lymph nodes, even with reduced DC numbers. The findings reveal considerable plasticity in the response to TLR agonists, with TLR4 and TLR5 agonists sharing the requirement of DCs for subsequent lymph node recruitment of NK and T cells.     

Regulation of MHC class I transport in human dendritic cells and the dendritic-like cell line KG-1

Dendritic cells (DCs) progress through distinct maturational phases; immature DCs capture Ag while mature DCs are optimized for Ag presentation. Proper control of immunity requires regulated compartmentalization of MHC class II molecules. We report that DCs also regulate MHC class I trafficking throughout maturation. Although mature human DCs express high levels of surface MHC class I, immature DCs exhibit lower surface levels while retaining MHC class I-peptide complexes in the Golgi. A cell line, KG-1, behaves similarly. We confirm the similarity of KG-1 to DCs by demonstrating its capacity to present exogenous Ags in an MHC class I-restricted fashion to CD8(+) T cell hybridomas, a phenomenon called cross-presentation. Biochemical characterization of MHC class I trafficking throughout maturation showed that, in early KG-1 dendritic-like cells, surface arrival of MHC class I-peptide complexes is delayed by their retention in the Golgi. In mature dendritic-like cells, these complexes relocate to the surface and their stability increases, concomitant with up-regulation of costimulatory molecules. Maturation induces qualitative changes in the MHC class I-associated peptide repertoire demonstrated by increased thermostability. The differential processing of MHC class I throughout maturation may prevent premature immune activation while promoting T cell responses in lymph nodes to Ags acquired at sites of inflammation.     

Preferential infection of mature dendritic cells by mouse hepatitis virus strain JHM

Mouse hepatitis virus strain JHM (MHV-JHM) causes acute encephalitis and acute and chronic demyelinating diseases in mice. Dendritic cells (DCs) are key cells in the initiation of innate and adaptive immune responses, and infection of these cells could potentially contribute to a dysregulated immune response; consistent with this, recent results suggest that DCs are readily infected by another strain of mouse hepatitis virus, the A59 strain (MHV-A59). Herein, we show that the JHM strain also productively infected DCs. Moreover, mature DCs were at least 10 times more susceptible than immature DCs to infection with MHV-JHM. DC function was impaired after MHV-JHM infection, resulting in decreased stimulation of CD8 T cells in vitro. Preferential infection of mature DCs was not due to differential expression of the MHV-JHM receptor CEACAM-1a on mature or immature cells or to differences in apoptosis. Although we could not detect infected DCs in vivo, both CD8(+) and CD11b(+) splenic DCs were susceptible to infection with MHV-JHM directly ex vivo. This preferential infection of mature DCs may inhibit the development of an efficient immune response to the virus.     

Macrophage C-type lectin on bone marrow-derived immature dendritic cells is involved in the internalization of glycosylated antigens

Bone marrow-derived dendritic cells (DCs) were examined for the expression of the murine macrophage C-type lectin specific for galactose and N-acetylgalactosamine (mMGL). Flow cytometric analysis after double staining for MHC class II and mMGL with specific monoclonal antibodies indicated that mMGL was expressed on immature DCs with low to moderate levels of MHC class II and down-regulated during maturation. Immature DCs bound and internalized alpha-N-acetylgalactosaminides conjugated to soluble polyacrylamide (alpha-GalNAc polymers), whereas mature DCs and bone marrow cells did not. The two-color flow cytometric profiles indicated that the degree of alpha-GalNAc polymer bindings exactly coincided with the intensity of the binding of a mMGL-specific monoclonal antibody LOM-14. The internalized alpha-GalNAc polymers seemed to be transported to MHC class II compartments. Thus, mMGL is transiently expressed on bone marrow-derived DCs during their development and maturation and suggested to be involved in the uptake of glycosylated antigens for presentation.