Composition of MHC class II-enriched lipid microdomains is modified during maturation of primary dendritic cells

Dendritic cells (DCs) are the most potent antigen presenting cells. Major histocompatibility complex (MHC) class II molecule expression changes with maturation; immature DCs concentrate MHC class II molecules intracellularly, whereas maturation increases surface expression of MHC class II and costimulatory molecules to optimize antigen presentation. Signal transduction via MHC class II molecules localized in lipid microdomains has been described in B lymphocytes and in the THP-1 monocyte cell line. We have characterized MHC class II molecules throughout human DC maturation with particular attention to their localization in lipid-rich microdomains. Only immature DCs expressed empty MHC class II molecules, and maturation increased the level of peptide-bound heterodimers. Ligand binding to surface human leukocyte antigen (HLA)-DR induced rapid internalization in immature DCs. The proportion of cell-surface detergent-insoluble glycosphingolipid-enriched microdomain-clustered HLA-DR was higher in immature DCs despite the higher surface expression of HLA-DR in mature DCs. Constituents of HLA-DR containing microdomains included the src kinase Lyn and the cytoskeletal protein tubulin in immature DCs. Maturation modified the composition of the HLA-DR-containing microdomains to include protein kinase C (PKC)-delta, Lyn, and the cytoskeletal protein actin, accompanied by the loss of tubulin. Signaling via HLA-DR redistributed HLA-DR and -DM and PKC-delta as well as enriching the actin content of mature DC microdomains. The increased expression of HLA-DR as a result of DC maturation was therefore accompanied by modification of the spatial organization of HLA-DR. Such regulation could contribute to the distinct responses induced by ligand binding to MHC class II molecules in immature versus mature DCs.     

Class II MHC cytoplasmic domain-mediated signaling in B cells: A tail of two signals

In addition to their role in antigen presentation, class II MHC molecules also transmit signals to B lymphocytes. Class II MHC-mediated signals initiate a range of events in B cells, including induction of cell surface proteins, initiation of cell-cycle progression/proliferation, activation of or protection from apoptosis, and antigen-dependent plasma cell differentiation. Although various transmembrane signaling proteins associate with class II MHC molecules, the class II MHC cytoplasmic domains are essential for signals leading to increased intracellular cAMP and activation of protein kinase C (PKC). Although truncation and mutagenesis studies have provided considerable information about the cytoplasmic domain sequences required, how class II MHC molecules elicit cAMP and PKC activation is not known. Further, appropriate T-dependent B cell responses require intact cAMP and PKC signaling, but the extent to which class II MHC signals are involved is also unknown. This review details our current knowledge of class II MHC cytoplasmic domain signaling in B cells with an emphasis on the likely importance of class II MHC signals for T-dependent antibody responses.     

Sustained cross‐presentation capacity of murine splenic dendritic cell subsets in vivo

An exclusive feature of dendritic cells (DCs) is their ability to cross‐present exogenous antigens in MHC class I molecules. We analyzed the fate of protein antigen in antigen presenting cell (APC) subsets after uptake of naturally formed antigen‐antibody complexes in vivo. We observed that murine splenic DC subsets were able to present antigen in vivo for at least a week. After ex vivo isolation of four APC subsets, the presence of antigen in the storage compartments was visualized by confocal microscopy. Although all APC subsets stored antigen for many days, their ability and kinetics in antigen presentation was remarkably different. CD8α⁺ DCs showed sustained MHC class I‐peptide specific CD8⁺ T‐cell activation for more than 4 days. CD8α^? DCs also presented antigenic peptides in MHC class I but presentation decreased after 48 h. In contrast, only the CD8α^? DCs were able to present antigen in MHC class II to specific CD4⁺ T cells. Plasmacytoid DCs and macrophages were unable to activate any of the two T‐cell types despite detectable antigen uptake. These results indicate that naturally occurring DC subsets have functional antigen storage capacity for prolonged T‐cell activation and have distinct roles in antigen presentation to specific T cells in vivo.     

Control of MHC class II antigen presentation in dendritic cells: a balance between creative and destructive forces

Summary: The antigen capturing and presenting abilities of dendritic cells (DCs) are developmentally regulated in a process known as maturation. During maturation, DCs increase several fold their surface expression of major histocompatibility complex class II (MHC II) molecules. This increase is accompanied with a dramatic change in localization of MHC II molecules, which are abundant in endosomal structures in immature DCs but located mostly on the plasma membrane in mature DCs. How these changes relate to antigen processing, generation of MHC II–peptide complexes, and trafficking of MHC II molecules, in the immature and mature states of DC development, has been a matter of debate. Here, we discuss the work carried out to characterize the biochemical and cell biological mechanisms that control MHC II antigen presentation in mouse and human DCs, and how these mechanisms relate to the function of the DC network in vivo. We conclude that the control checkpoints operate downstream of MHC II–peptide complex formation and expression on the plasma membrane, acting in accord with control of MHC II synthesis. Therefore, immature and mature DCs present antigens to T cells under steady state and inflammatory conditions. We advocate that the mechanisms regulating MHC II–peptide complex turnover should be emphasized as an important theme for future DC research.     

Murine neutrophils present Class II restricted antigen

Neutrophils were originally described as short lived, terminally differentiated phagocytes that contribute only to the innate immune response. Recent evidence of neutrophil cytokine production and expression of numerous cell surface proteins has suggested that neutrophils are likely to influence adaptive responses and may satisfy the criteria of antigen presenting cells. Under certain inflammatory conditions human neutrophils express major histocompatibilty complex (MHC) Class II and the costimulatory molecules CD80 and CD86. We have employed a murine T cell hybridoma with a transgenic T cell receptor specific for ovalbumin peptide 323-339 (OVA(323-339)), and a green fluorescent reporter of T cell receptor ligation, to directly investigate neutrophil-T cell interactions. These cells provide an ideal model system, allowing precise identification of antigen specificity and a clear readout of T cell activation. Additionally, whilst murine neutrophils have previously been shown to stimulate MHC Class I-dependent CD8(+) T cell activation, CD4(+) T cells stimulation via MHC Class II-expressing neutrophils has not been investigated. We addressed this by isolating murine neutrophils, loading with OVA(323-339) and co-culturing with T cells specific for the OVA(323-339)/MHC Class II complex, and this resulted in T cell activation, as determined by expression of the green-fluorescent protein reporter. Antigen-pulsed neutrophils were also able to prime na?ve OVA-specific CD4(+) T cells in a contact-dependent manner, as shown by proliferation and cytokine production. Activation of lymphocytes was not due to contaminating macrophages. These studies demonstrate that murine neutrophils present MHC Class II-restricted peptides and induce T cell proliferation, confirming findings in human neutrophils, and demonstrate a novel pro inflammatory effect of murine neutrophils.     

Interactions of dendritic cells with cancer cells and modulation of surface molecules affect functional properties of CD8+ T cells

To understand the interaction of dendritic cells (DCs) with cancer cells, we investigated molecular changes in DCs following co-culture with cancer cells. DCs co-cultured with Jurkat cancer cells showed remarkable down-regulation of MHC class I molecules, while DCs co-cultured with MCF-7 cancer cells showed minimal changes. Interestingly, down-regulation of MHC class I on DCs was not observed upon treatment with Jurkat cell lysate or culture supernatant, suggesting the importance of direct cell-cell interactions. The expressions of CD40, CD80, CD83, MHC class II, and IL-12p40 on DCs co-cultured with Jurkat cells were only slightly affected. In contrast, DCs co-cultured with MCF-7 cells showed increased expressions of CD80, CD83, CD86, and IL-12p40. Furthermore, DCs co-cultured with Jurkat cells showed a down-regulation of low molecular weight polypeptides (LMP) 7, and of transporter associated with antigen processing (TAP) 1 and 2 at the mRNA expression level. LMP7, TAP2 and β2-microglobulin (β2M) were also down-regulated at the protein level. We further demonstrated how altered expression of MHC class I on DCs caused by co-culture with cancer cells affected autologous CD8(+) T cells, using the model MHC class I-presented HSV antigen. We found that DCs that had been HSV-treated and co-cultured with Jurkat cells showed a reduced potency to activate CD8(+) T cells. In contrast, HSV-treated DCs that had been co-cultured with MCF-7 cells induced activation of CD8(+) T cells, including high expression of CD25, CD69, granzyme B and cytokines, TNF-α and IFN-γ.     

Dynamics of the membrane–cytoskeleton interface in MHC class II-restricted antigen presentation

Antigen presentation refers to the ability of cells to show MHC-associated determinants to T lymphocytes, leading to their activation. MHC class II molecules mainly present peptide-derived antigens that are internalized by endocytosis in antigen-presenting cells (APCs). Here, we describe how the interface between cellular membranes and the cytoskeleton regulates the various steps that lead to the presentation of exogenous antigens on MHC class II molecules in the two main types of APCs: dendritic cells (DCs) and B lymphocytes. This includes antigen uptake, processing, APC migration, and APC–T cell interactions. We further discuss how the interaction between APC-specific molecules and cytoskeleton elements allows the coordination of antigen presentation and cell migration in time and space.     

Evidence that protein kinase C differentially regulates the human T lymphocyte CD2 and CD3 surface antigens

The purpose of the present study was to explore the effects of protein kinase C (PKC) stimulation on two cell surface receptors that regulate T cell growth: the T cell antigen receptor/CD3 complex and the CD2 antigen. The data show that PKC differentially regulates the expression and functions of CD2 and CD3 molecules. Thus, activation of PKC induced a decrease in cell surface levels of CD3 molecules but an increase in the expression of CD2 antigens. Additionally, prolonged stimulation of PKC inhibited subsequent T cell activation via CD3 but promoted activation via CD2 molecules. These results suggest that the CD2 cellular activation pathway would be preferred in T cells which have been exposed to stimulators of PKC. The molecular basis for the regulatory effects of PKC on CD3 and CD2 molecules and its physiological significance are discussed.     

The functional role of class II-associated invariant chain peptide (CLIP) in its ability to variably modulate immune responses

During the process of class II MHC assembly and cell surface expression, the class II-associated invariant chain peptide (CLIP) is removed from the peptide-binding groove of MHC, a task mediated by H-2M. This allows binding and presentation of peptide epitopes. We have previously shown that exogenously added CLIP interferes with this process and down-regulates the cell surface expression of class II molecules. In this study, we explored the effect of exogenously added CLIP on antigen-specific immune responses. In vivo studies with CLIP and various peptide and protein antigens with different affinities for I-A(d) molecules demonstrated that CLIP variably affects the T cell-mediated immune responses. Immunization with CLIP along with the antigen induced a shift from a T(h)1- to T(h)2-like response as determined by the cytokine profile and antibody isotype. These results suggest that the presence of exogenous CLIP can significantly influence the presentation of antigen by class II MHC molecules to CD4 T cells and thereby modulate immune responses. Exogenously added CLIP rapidly localized into the subcellular compartment of antigen-presenting cells where MHC class II molecules are present. We suggest that exogenous CLIP reduces the loading of peptides on the class II molecules, thus down-regulating MHC-peptide complexes on the cell surface. Alternatively, CLIP may bind to cell surface class II molecules and this complex is rapidly internalized resulting in reduced cell surface MHC class II expression. The reduced level of MHC-peptide complexes favors the activation of T(h)2 cells over T(h)1 cells. These results have implications in the regulation of immune responses, particularly the prevention of certain autoimmune diseases where T(h)1-type responses are pathogenic and T(h)2-type responses are protective.     

Influenza A virus elevates active cathepsin B in primary murine DC

Dendritic cells (DCs) act as a first-line recognition system for invading pathogens, such as influenza A. The interaction of DC with influenza A virus results in DC activation via endosomal Toll-like receptors and also leads to presentation of viral peptides on MHC class II molecules. Prior work demonstrated that influenza A virus (A/HKx31; H3N2) infection of BALB/c mice activates lung DCs for antigen presentation, and that the enhanced function of these cells persists long after viral clearance and resolution of the virus-induced inflammatory response. Whether influenza A virus has acute or longer-lasting effects on the endo/lysosomal antigen-processing machinery of DCs has not been studied. Here, we show that antigen presentation from intact protein antigen, but not peptide presentation, results in increased T cell stimulation by influenza-exposed lung DCs, suggesting increased antigen processing/loading in these DCs. We find that cathepsin (Cat) B levels and activity are substantially up-regulated in murine lung DCs, harvested 30 days after A/HKx31 infection. CatB levels and activity are also increased in murine splenic and bone marrow-derived DCs, following short-term in vitro exposure to UV-inactivated influenza A virus. Modest effects on CatX are also seen during in vivo and in vitro exposure to influenza A virus. Using a cell permeable Cat inhibitor, we show Cats in influenza-exposed DCs to be functional and required for generation of a T cell epitope from intact ovalbumin. Our findings indicate that influenza A virus affects the MHC class II antigen-processing pathway, an essential pathway for CD4(+) T cell activation.     

Activation of murine dendritic cells and macrophages induced by Salmonella enterica serovar Typhimurium

Macrophages and dendritic cells (DCs) are antigen-presenting cells (APCs), and the direct involvement of both cell types in the immune response to Salmonella has been identified. In this study we analysed the phenotypic and functional changes that take place in murine macrophages and DCs in response to live and heat-killed Salmonella enterica serovar Typhimurium. Both types of cell secreted proinflammatory cytokines and nitric oxide (NO) in response to live and heat-killed salmonellae. Bacterial stimulation also resulted in up-regulation of costimulatory molecules on macrophages and DCs. The expression of major histocompatibility complex (MHC) class II molecules by macrophages and DCs was differentially regulated by interferon (IFN)-gamma and salmonellae. Live and heat-killed salmonellae as well as lipopolysaccharide (LPS) inhibited the up-regulation of MHC class II expression induced by IFN-gamma on macrophages but not on DCs. Macrophages as well as DCs presented Salmonella-derived antigen to CD4 T cells, although DCs were much more efficient than macrophages at stimulating CD4 T-cell cytokine release. Macrophages are effective in the uptake and killing of bacteria whilst DCs specialize in antigen presentation. This study showed that the viability of salmonellae was not essential for activation of APCs but, unlike live bacteria, prolonged contact with heat-killed bacteria was necessary to obtain maximal expression of the activation markers studied.     

Dendritomas formed by fusion of mature dendritic cells with allogenic human hepatocellular carcinoma cells activate autologous cytotoxic T lymphocytes

Mature dendritic cells (DCs) have highly expressed CD1a, MHC class I, MHC class II, B7-1, B7-2 and ICAM-I molecules, all of which are essential for activation of na?ve T cells. In this study, dendritomas were formed by fusion of hepatocellular carcinoma (HCC) SMMC-7721 cells with autologous DCs in vitro. DCs were obtained from adherent monocytes cultured in the presence of GM-CSF and IL-4 and were matured in monocyte-conditioned media. Expression of MHC class II and HCC-specific antigen by these dendritomas were determined using a specific murine anti-HCC monoclonal antibody (mAb) specific for HCC cell line SMMC-7721, and a murine anti-human HLA-DR mAb, and was also confirmed using bi-dimensional flow cytometry and immuno-histostaining. Dendritomas were co-cultured with autologous T cells, resulting in activation of T cell proliferation and priming of na?ve T cells to induce MHC class I restricted lysis of HCC SMMC-7721 cells. The results imply that these dendritomas may have potential for use in HCC immunotherapy.     

Early activation markers of human peripheral dendritic cells

Two major populations of dendritic cells (DCs), myeloid and plasmacytoid, can be isolated from human peripheral blood, and are distinguished by differential expression of the cell surface markers CD11c and CD123. These two populations of DCs also are different in their expression of Toll-like receptor (TLRs), which are involved in their activation. To investigate the early events during activation of peripheral DCs, the cells were stimulated in vitro with ligands for TLR-4 (as in lipopolysaccharides [LPS]) or TLR-9 (CpG-containing oligonucleotide [CpG]). The earliest change in protein expression detected after stimulating peripheral DCs with lipopolysaccharide (LPS) or CpG was increased production of the chemokine interleukin (IL)-8. Enhanced production of IL-8 occurred already within 2 hours of stimulation in both myeloid dendritic cells (M-DCs) and plasmacytoid dendritic cells (P-DCs), and preceded expression of the well established activation marker CD40. Although both populations of DCs secreted IL-8 upon activation, the levels of IL-8 produced was several times higher within the M-DCs compared with the P-DCs population. Before activation, both subsets of DCs expressed the IL-8 receptor type B (CD128b); but after stimulation the IL-8 receptor was down-regulated in both populations of DCs. Increased expression of MHC class II molecules is generally regarded as an early activation marker of DCs. However, only the P-DCs showed a significant up-regulation of MHC class II after stimulation. The M-DC population up-regulated MHC class II without any prior activation; thus care should be taken using increased expression of MHC class II molecules as an early activation marker of peripheral M-DCs after activation in vitro. In conclusion, we propose that during activation of human DCs the production of IL-8 and loss of CD128b are the earliest signs of activation preceding both MHC class II, CD40, CD80, and CD86 expression.     

Early induction of MHC antigens in human liver grafts. An immunohistologic study.

The present study documents major histocompatibility complex (MHC) Class I and II expression during early acute rejection of human liver grafts. Serial graft biopsies (pretransplant, time zero, and 1 week) were studied. Ten patients received azathioprine (AZA) and prednisone; the other six patients were treated with quadruple therapy (azathioprine, cyclosporine A, prednisone, and cyclophosphamide). To study the specificity of changes in MHC antigen expression, biopsies of six patients with minor or no morphologic abnormalities served as controls. In addition, phenotypes of inflammatory cells present during rejection were analyzed using a panel of monoclonal antibodies. The results show that during acute rejection expression of MHC Class I and II antigens increased significantly in the AZA-treated patients, in a pattern similar to that seen in the patients treated with quadruple therapy, showing enhanced MHC Class I expression on hepatocytes, bile duct epithelium, and sinusoidal endothelium, and Class II antigen on Kupffer cells and sinusoidal endothelium. Bile duct epithelium was consistently positive for Class II antigen; no significant difference with the nonrejection group was observed. T cells are the predominant inflammatory cells during rejection with equal quantities of CD4+ and CD8+ cells. A majority of the infiltrating T cells show expression of Class II antigen but do not react with anti-interleukin-2 receptor antibody. This may be the result of immunosuppressive therapy or a simple reflection of the temporary expression of interleukin-2 receptors during lymphocyte activation. The authors hypothesize that the induction of MHC antigens on bile duct epithelium leads to rejection whereas the expression on hepatocytes represents an epiphenomenon.     

Simultaneous in vivo tracking of dendritic cells and priming of an antigen-specific immune response

We report the fabrication of a one-pot antigen system that delivers antigen to dendritic cells (DCs) and tracks their in vivo migration after injection. Multifunctional polymer nanoparticles containing ovalbumin protein, magnetic resonance imaging contrast agents (iron oxide nanoparticles), and near-infrared fluorophores (indocyanine green, ICG), MPN-OVA, were prepared using a double emulsion method. The MPN-OVA was efficiently taken up by the dendritic cells and subsequently localized in the lysosome. Flow cytometry analysis revealed an increase in the uptake of OVA antigen by MPN-OVA at 37 °C, when compared with soluble OVA protein. We found that MPN-OVA had no effect on DC surface expression of MHC class I, costimulatory (CD80, CD86) or adhesion (CD54) molecules or the ability of DCs to mature in response to LPS. Following the uptake of MPN-OVA, exogenous OVA antigen was delivered to the cytoplasm, and OVA peptides were presented on MHC class I molecules, which enhanced OVA antigen-specific cross-presentation to OT-1 T cells and CD8OVA1.3 T cell hybridoma in vitro. The immunization of mice with MPN-OVA-treated DCs induced OVA-specific CTL activity in draining lymph nodes. The presence of MPN allowed us to monitor the migration of DCs via lymphatic drainage using NIR fluorescence imaging, and the homing of DCs into the lymph nodes was imaged using MRI. This system has potential for use as a delivery system to induce T cell priming and to image DC-based immunotherapies.