The ins and outs of MHC class II proteins in dendritic cells

One more mechanism for regulating surface expression of MHC class II proteins in dendritic cells (an important process that regulates both tolerance and immunity) has been uncovered, polyubiquitination of Lys225 in the intracytoplasmic region and subsequent endocytosis and proteolysis (Omura-Hoshino et al., 2006; van Neil et al., 2006; Shin et al., 2006).     

Dexamethasone inhibits the antigen presentation of dendritic cells in MHC class II pathway

Glucocorticoids (GC) are physiological inhibitors of inflammatory responses and are widely used as anti-inflammatory and immunosuppressive agents in treatment of many autoimmune and allergic diseases. In the present study, we demonstrated that one of the mechanisms by which GC can suppress the immune responses is to inhibit the differentiation and antigen presentation of dendritic cells (DC). DC were differentiated from murine bone marrow hematopoietic progenitor cells by culture with GM-CSF and IL-4 with or without dexamethasone (Dex). Our data showed that Dex, in a dose dependent manner, down-regulated surface expression of CD86, CD40, CD54 and MHC class II molecules by DC, but the expression of MHC class I, CD80, CD95 and CD95L were not affected. In addition, Dex-treated DC showed an impaired function to activate alloreactive T cells and to secrete IL-Ibeta and IL-12p70. Moreover, Dex inhibited DC to present antigen by MHC class II pathway. However, the endocytotic activity of DC was not affected. The inhibitory effect of Dex on the expression of costimulatory molecules and the antigen-presenting capacity of DC could be blocked by the addition of RU486, a potent steroid hormone antagonist, suggesting the requirement of binding to cytosolic receptors in the above-described action of Dex. Since DC have the unique property to present antigen to responding naive T cells and are required in the induction of a primary response, the functional suppression of DC by Dex may be one of the mechanisms by which GC regulate immune responses in vivo.     

MHC class II signaling function is regulated during maturation of plasmacytoid dendritic cells

Dendritic cells (DC) play a central role in the immune response, linking innate and adaptative responses to pathogens. Myeloid DC (MDC) produce interleukin-12 in response to bacterial stimuli, whereas plasmacytoid DC (PDC) produce high levels of type I interferon upon viral infection. Human leukocyte antigen (HLA)-DR engagement has been shown to induce apoptosis in various antigen-presenting cells (APC). We now report the consequences of HLA-DR molecule engagement in human PDC, which had thus far not been studied as a result of the difficulty in isolating such cells. HLA-DR engagement on PDC, obtained using a two-step, immunomagnetic separation, led to recruitment of HLA-DR molecules at the site of engagement in mature but not immature PDC. In contrast, relocalization of protein kinase C (PKC) isoenzymes, indicating PKC activation, was observed at the site of HLA-DR engagement and was accompanied by relocalization of a lipid raft marker, the ganglioside M1 staining, in immature and mature PDC. Similar to MDC, HLA-DR-mediated apoptosis was regulated throughout PDC maturation. Freshly isolated PDC were resistant, whereas CD40 ligand-matured PDC were sensitive to HLA-DR-mediated apoptosis. Neither caspase activation nor PKC activation was required for HLA-DR-mediated apoptosis. However, the intrinsic pathway of apoptosis was implicated as mature PDC underwent mitochondrial depolarization in response to HLA-DR engagement. These data provide further arguments for considering HLA-DR-mediated apoptosis as a conserved mechanism of regulating survival of diverse APC and support the ongoing development of humanized ligands for HLA class II molecules as therapeutic tools for use in lymphoproliferative disease.     

MHC class II expression is differentially regulated in plasmacytoid and conventional dendritic cells

Major histocompatibility complex (MHC) class II-restricted antigen presentation is essential for the function of dendritic cells (DCs). We show here that plasmacytoid DCs (pDCs) differ from all other DC subsets with respect to expression of CIITA, the 'master regulator' of MHC class II genes. The gene encoding CIITA is controlled by three cell type-specific promoters: pI, pIII and pIV. With gene targeting in mice, we demonstrate that pDCs rely strictly on the B cell promoter pIII, whereas macrophages and all other DCs depend on pI. The molecular mechanisms driving MHC class II expression in pDCs are thus akin to those operating in lymphoid rather than myeloid cells.     

Differential MHC class II expression on human peripheral blood monocytes and dendritic cells

Both monocytes (MO) and dendritic cells (DC) in human peripheral blood are of a plastic-adherent nature. The expression of the MHC class II sublocus products HLA-DP, -DQ and -DR on human peripheral blood transiently adherent cells (TA) was examined by an immunocytochemical staining technique. While most TA showed strong expression of molecules of the HLA-DR subtype, only a small proportion of cells (2-6%) showed strong HLA-DP or -DQ positivity. This strong expression of the HLA-DP and HLA-DQ sublocus products by a subset of TA was seen only after short-term culture; freshly isolated cells expressed comparatively low levels of these molecules. Enrichment for Fc receptor-negative or low-density cells from TA produced populations with strong HLA-DQ and -DP expression. Such co-enrichment of the strongly HLA-DQ+ and strongly HLA-DP+ cells suggests that the same cells express high levels of both types of MHC class II molecule. Immunocytochemical analysis of TA indicated that the strongly HLA-DQ+ cells, at least, were only weakly or non-reactive with the MO-specific monoclonal antibodies OKM1, UCHM1, MO2 and EB11. In addition, strongly HLA-DQ- or -DP-positive cells were poorly phagocytic in comparison with the majority of adherent cells. The apparent FcR-negative, low-density and weakly phagocytic nature of the strongly HLA-DQ/DP+ cells, combined with their lack of reactivity with several MO-specific antibodies, suggests that they may represent the DC component of TA. Such strong HLA-DQ/DP expression by DC may aid their positive identification in human peripheral blood and may be of relevance to DC function in antigen presentation.     

ATPase and MHC class II molecules co-expression in Rana pipiens dendritic cells.

Mammalian Langerhans cells are antigen-presenting cells located in different epithelia. These cells have a characteristic ultrastructural pattern, present a plasmatic membrane ATPase activity and constitutively express class II molecules of the major histocompatibility complex. ATPase-positive dendritic cells that are morphologically similar to Langerhans cells have also been found in amphibian epidermis. In order to demonstrate that ATPase-positive dendritic cells of amphibian epidermis express class II molecules and are present in other stratified epithelia, histochemical and immunohistochemical as well as ultrastructural analysis were performed. ATPase-positive dendritic cells and class II-positive dendritic cells were observed in epidermis, nictitant membrane and cornea. In epidermis the number of ATPase-positive dendritic cells was 656+/-186/mm2 while class II-positive dendritic cells was 119+/-45/mm2. Some ATPase-positive dendritic cells showed co-expression of class II molecules. These results suggest the existence of dendritic cell subsets in amphibians as is clearly demonstrated in mammals.     

Contrasting cytoskeletal regulation of MHC class II peptide presentation by human B cells or dendritic cells

MHC class II-mediated antigen presentation by B lymphocytes or dendritic cells (DC) initiates CD4+ T lymphocyte activation. In B lymphocytes, MHC class II peptide presentation has been characterised by recruitment of MHC class II, F-actin and lipid rafts to the B cell-T cell immunological synapse. We now show that MHC class II engagement in B lymphocytes induced lipid raft-independent Rho and Rac activation and that inhibition of either Rho-GTPase activation or actin polymerisation in the B cell abrogated T cell activation without altering B cell-T cell conjugate formation. Short-hairpin RNA studies excluded a role for the Cdc42 effector Wiskott-Aldrich syndrome protein. In contrast, antigen presentation by DC was Rho-GTPase-independent although actin was recruited to the DC-T cell interaction site. Moreover, actin depolymerisation in the DC significantly increased T cell activation without altering the number of DC-T cell conjugates. Finally we show that stable recruitment of HLA-DR to the site of the immunological synapse is not a uniform observation in DC and demonstrate reduced HLA-DR expression at the site of microtubule organising centre polarization. Therefore although actin accumulates in DC and B lymphocytes at the immunological synapse with antigen-specific T lymphocytes, this does not reflect comparable functional roles of their actin cytoskeletons in antigen presentation.     

MHC class II expression is regulated in dendritic cells independently of invariant chain degradation.

We have investigated the mechanisms that control MHC class II (MHC II) expression in immature and activated dendritic cells (DC) grown from spleen and bone marrow precursors. Degradation of the MHC II chaperone invariant chain (Ii), acquisition of peptide cargo by MHC II, and delivery of MHC II-peptide complexes to the cell surface proceeded similarly in both immature and activated DC. However, immature DC reendocytosed and then degraded the MHC II-peptide complexes much faster than the activated DC. MHC II expression in DC is therefore not controlled by the activity of the protease(s) that degrade Ii, but by the rate of endocytosis of peptide-loaded MHC II. Late after activation, DC downregulated MHC II synthesis both in vitro and in vivo.     

MHC class II signaling in antigen-presenting cells

The MHC class II molecules have been recognized as signaling receptors for more than a decade, and recent work has revealed the importance of their signaling for the immune response. Today, we know that the function of MHC class II molecules on antigen-presenting cells (APCs) is not limited to their role as antigen-presenting structures; they are flexible receptors that, by triggering a variety of signaling pathways, can regulate APC activities from proliferation and maturation to apoptosis. Recent advances have provided insights into how these molecules might accommodate such regulation.     

MHC class II molecules transferred between allogeneic dendritic cells stimulate primary mixed leukocyte reactions.

Presentation of antigen to T cells is generally restricted by MHC type but the mixed leukocyte reaction (MLR) was thought to involve direct stimulation by dendritic cells (DC) of allogeneic T cells. However, here we showed that DC bearing allogeneic MHC class II acted synergistically with responder-type DC. Removal of residual DC from 'purified' responder T cell populations was achieved using treatment with DC-specific antibody and complement. These DC-depleted cells showed a significantly reduced response to allogeneic DC which was restored by addition of DC syngeneic with responder T cells. The studies support the concept that a major component of the MLR is the secondary presentation of alloantigens acquired from stimulator DC by DC of responder type. To investigate the reasons why DC and not other cells stimulate an MLR, synergy between DC and other cell types was investigated. Synergy was found exclusively between DC; macrophages, B cells or L cells transfected with MHC class II molecules did not contribute. When allogeneic DC were mixed in culture, transfer of MHC molecules between DC was observed as assessed by flow cytometry. Freshly obtained cell-free supernatants from cultured DC contained MHC class II and stimulated primary allogeneic MLR. DC of responder type acquired allogeneic MHC molecules from the supernatants and stimulated proliferation in syngeneic T cells. The capacity of DC both to shed and to acquire MHC molecules may contribute to their potency in stimulating primary responses, and could explain why passenger DC within allografts provide a potent stimulus for graft rejection.     

Differential regulation of maturation and apoptosis of human monocyte-derived dendritic cells mediated by MHC class II

Antigen-driven interaction of dendritic cells (DC) with CD4(+) T(h) cells results in the exchange of bidirectional activating signals. Cross-linking of TCR by MHC class II-bound antigen activates T(h) cells, resulting in their up-regulation of CD40 ligand. Here we show that MHC class II molecules, in addition to their passive role in DC-T(h) cell interaction, can also actively induce DC maturation. Cross-linking of MHC class II molecules on human monocyte-derived DC results in the up-regulation of the surface expression of CD83, CD80, CD86, CD54, CD1a and CD40 molecules, the typical DC maturation-associated markers. It also promotes a rapid homotypic aggregation of DC paralleled by the up-regulation of such adhesion molecules as VLA-4, tissue transglutaminase, CD54 and CD11c. The impact of MHC class II cross-linking upon DC was context dependent. The outcome of MHC class II signaling depends on the maturation status of DC. While the cross-linking of MHC class II on immature DC promoted their maturation, the dominant effect upon the DC that were previously matured was the induction of DC apoptosis. Our current observations indicate that, in addition to the previously reported negative impact of MHC class II-mediated signaling on DC function, it also promotes DC maturation, participating in the enhancement of DC stimulatory function. Importantly, MHC class II-induced DC maturation and apoptosis are mediated by different signaling pathways, sensitive to different sets of inhibitors. This opens the possibility of differential regulation of each of these events in immunotherapy.     

固相MHCI类相关抗原A刺激的NK细胞对树突状细胞活性的影响

目的:观察固相MHCI类相关抗原A(iMICA)刺激的NK细胞对树突状细胞(DCs)活性的影响.方法:首先取新鲜分离及受固相MICA刺激的异体NK细胞,或IL-2、及IL-2联合iMICA刺激的自体NK细胞与未成熟DCs(iDCs)按5:1比例孵育24 h后,用流式细胞术(FCM)分析HLA-DR+或CD86+频率的DCs.然后取自体NK细胞以iMICA刺激后,按1:5的比例与iDCs孵育24 h后,FCM检测DCs上HLA-DR、CD86的表达.最后在NK细胞与DCs的共培养体系中加人抗IFN-γ抗体,观察DCs上HLA-DR、CD86表达的变化.结果:当NK细胞与iDCs孵育比例为5:1时,异体新鲜分离的与自体活化的NK细胞均能杀伤iDCs;而MICA无协同作用.当NK细胞与iDCs孵育的比例为1:5时,iMICA刺激的NK细胞可促进DCs表达HLA-DR和CD86;而加人抗IFN-γ抗体可抑制 NK细胞诱导DCs表面HLA-DR和CD86表达的上调.结论:异体新鲜分离的或自体活化的NK细胞杀伤iDCs时,无需iMICA的刺激;但当NK细胞的数目明显低于iDCs时,iMICA可刺激NK细胞分泌IFN-γ促进DCs成熟.     

Presentation of self-antigens on MHC class II molecules during dendritic cell maturation

Little is known about how dendritic cells (DCs) maintain a balance between tolerance and immunity for antigens synthesized by DCs themselves. Using transgenic DCs expressing a model self-antigen, in vitro self-peptide-MHC class II complex formation and presentation increased with DC maturation, as for exogenous antigens. In vivo, however, even 'immature' DCs isolated from steady-state lymph nodes expressed MHC at mature cell levels, although many were also CD86 low. Adoptive transfer of naive specific T cells into unstimulated transgenic mice resulted in tolerance. If the mice were also injected with anti-CD40 or Listeria monocytogenes, there was robust specific T cell expansion and inflammation. Thus, DC-endogenous antigens may induce tolerance, but only in the absence of potent maturation stimuli.     

MHC class II antigens on canine bronchoalveolar cells

To evaluate the expression of MHC (major histocompatibility complex) antigens on canine bronchoalveolar cells (BAC), bronchoalveolar lavages (BAL) were performed in mongrel and German shepherd dogs. MHC class II antigens on canine BAC and peripheral blood mononuclear cells (PBMC) were detected by monoclonal antibodies (mAbs) B1F6, 7.5.10.1 and Q5/13 recognising canine MHC class II antigens, using cytofluorometry. These mAbs reacted with more than 20% of BAC and PBMC in both breeds of dog. The percentage of MHC class II positive cells in BAC were lower than those in PBMC. There was no significant difference in the percentages of MHC class II positive BAC and PBMC in mongrel and German shepherd dogs. To further identify the expression of MHC class II antigens on BAC, the cells were separated into adherent and nonadherent cells by petri dish adherence. The percentages of MHC class II positive cells in adherent and non-adherent cell populations were similar. Nearly half the lymphocytes in normal BAC were T cells detected by mAbs F3-20-7 and 1A1; B cells were scarce and represented less than 10% of nonadherent cells. Immunoprecipitation by anti-MHC class II mAbs, and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed MHC class II-like molecules on canine BAC and PBMC. After stimulation with phytohaemagglutinin (PHA), the percentages of class II positive cells in BAC and PBMC were significantly increased. Thus, these anti-MHC class II mAbs may prove to be of advantage in experiments designed to evaluate the changes in class II antigen expression on canine BAC during the course of immune response in the lung, as in pulmonary allograft rejection.     

Differential MHC class II synthesis and ubiquitination confers distinct antigen-presenting properties on conventional and plasmacytoid dendritic cells

The importance of conventional dendritic cells (cDCs) in the processing and presentation of antigen is well established, but the contribution of plasmacytoid dendritic cells (pDCs) to these processes, and hence to T cell immunity, remains unclear. Here we showed that unlike cDCs, pDCs continued to synthesize major histocompatibility complex (MHC) class II molecules and the MHC class II ubiquitin ligase MARCH1 long after activation. Sustained MHC class II-peptide complex formation, ubiquitination and turnover rendered pDCs inefficient in the presentation of exogenous antigens but enabled pDCs to continuously present endogenous viral antigens in their activated state. As the antigen-presenting abilities of cDCs and pDCs are fundamentally distinct, these two cell types may activate largely nonoverlapping repertoires of CD4(+) T cells.     

Synaptotagmin II negatively regulates MHC class II presentation by mast cells

Synaptotagmins (Syts), comprise a gene family of proteins, implicated in the control of protein traffic. Rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mucosal mast cells (MMC), express at least four distinct Syt homologues, including Syt II, Syt III, Syt V and Syt IX. Synaptotagmin II is located at the late/endosomal/lysosomal compartment, where it negatively regulates lysosomal exocytosis. Mast cells may contribute to immune defense mechanisms by presenting MHC class II/antigen complexes and triggering T cell-dependent immune responses. We now demonstrate that RBL-2H3 mast cells, which express reduced levels of Syt II (<5%) by transfection with Syt II antisense cDNA, are able to release MHC class II molecules. We further show that release of both MHC class II molecules and of the lysosomal enzyme cathepsin D is stimulated by lipopolysaccharide (LPS, 1 microg/ml, 48h). We show further that LPS reduces by >40% the level of Syt II expression in both RBL-2H3 and bone marrow-derived mast cells (BMMC). This effect is both dose and time-dependent. These results indicate that Syt II can be down-regulated by external inflammatory signals, resulting in the amplification of mast cell function. Finally, our results implicate Syt II as an important and novel regulator of MHC class II presentation.     

Increase of the stimulatory effect of dendritic cells by pulsing with apoptotic bodies transfected with the MHC class II gene

Dendritic cells (DC) are capable of capturing and processing antigens and can stimulate co-cultured effector cells in a specific manner. Here, we pulsed DC with apoptotic bodies (apb) from colorectal carcinoma. For enhancement of the immunogenic potential of apb, we transduced the tumor cells with the MHC class II gene before irradiation. After transfection, tumor cells, which are normally MHC class II negative, expressed MHC class II in 26.3%. Staining apb with PKH-2 and DC with PKH-26, we determined an apb-uptake of 27.6% by DC. Lytic activity of effector cells cocultured with DC pulsed with MHC class II transduced apb against the donor cell line at an effector to target ratio of 40:1 was 43.8% compared to 35.3% for pulsing with mock transduced cells. Secretion of interleukin-12 (IL-12) by DC was significantly enhanced after pulsing with MHC class II transduced apb compared to DC pulsed with mock transduced apb. Coculture with apb-pulsed DC led to an increase of proliferation rate and can stimulate effector cells in a specific manner. The immunogenic potential could be enhanced by transducing tumor cells with the MHC class II gene.