The impact of H2-DM on humoral immune responses.

H2-DM (DM, previously H2-M) facilitates the exchange of peptides bound to MHC class II molecules. In this study, we have used H2-DM-deficient (DM(-/-)) mice to analyze the influence of DM in the priming of B cell responses in vivo and for Ag presentation by B cells in vitro. After immunization, IgG Abs could be raised to a T-dependent Ag, 4-hydroxy-5-nitrophenylacetyl-OVA, in DM(-/-) mice, but closer analysis revealed the IgG response to be slower, diminished in titer, and composed of low-affinity Abs. The Ab response correlated with a vast reduction in the number of germinal centers in the spleen. The presentation of multiple epitopes by H2-A(b) from distinct Ags was found to be almost exclusively DM-dependent whether B cells internalized Ags via fluid phase uptake or using membrane Ig receptors. The poor B cell response in vivo could be largely, but not completely restored by expression of a H2-Ea(d) transgene, despite the fact that Ag presentation by H2-E(d/b) molecules was found to be highly DM dependent. Hence, while substantial Ab responses can be raised in the absence of DM, this molecule is a crucial factor both for Ag processing and for the normal maturation of T-dependent humoral immune responses in vivo.     

Expression patterns of H2-O in mouse B cells and dendritic cells correlate with cell function

In the endosomes of APCs, the MHC class II-like molecule H2-M catalyzes the exchange of class II-associated invariant chain peptides (CLIP) for antigenic peptides. H2-O is another class II-like molecule that modulates the peptide exchange activity of H2-M. Although the expression pattern of H2-O in mice has not been fully evaluated, H2-O is expressed by thymic epithelial cells, B cells, and dendritic cells (DCs). In this study, we investigated H2-O, H2-M, and I-A(b)-CLIP expression patterns in B cell subsets during B cell development and activation. H2-O was first detected in the transitional 1 B cell subset and high levels were maintained in marginal zone and follicular B cells. H2-O levels were down-regulated specifically in germinal center B cells. Unexpectedly, we found that mouse B cells may have a pool of H2-O that is not associated with H2-M. Additionally, we further evaluate H2-O and H2-M interactions in mouse DCs, as well as H2-O expression in bone marrow-derived DCs. We also evaluated H2-O, H2-M, I-A(b), and I-A(b)-CLIP expression in splenic DC subsets, in which H2-O expression levels varied among the splenic DC subsets. Although it has previously been shown that H2-O modifies the peptide repertoire, H2-O expression did not alter DC presentation of a number of endogenous and exogenous Ags. Our further characterization of H2-O expression in DCs, as well as the identification of a potential free pool of H2-O in mouse splenic B cells, suggest that H2-O may have a yet to be elucidated role in immune responses.     

HLA-DO is a lysosomal resident which requires association with HLA-DM for efficient intracellular transport.

The murine MHC class II molecule H2-O is expressed in B-cells and in thymic epithelium but the human equivalent, HLA-DO (DO), has not been detected, though the corresponding genes, HLA-DNA and HLA-DOB, are well known. Here we show DO to be a lysosomal resident in B-cells. Surprisingly, DO forms stable complexes with HLA-DM (DM), another lysosomal class II-like molecule which is important for class II-restricted antigen presentation. Association with DM is necessary for efficient exit of DO from the endoplasmic reticulum (ER) and thus for accumulation in lysosomes. The association is evolutionarily conserved and in mice lacking H2-M, the mouse equivalent of DM, the amount of intracellular H2-O is decreased and only minor amounts of H2-O appear to leave the ER. The DO-DM complexes survive in the lysosomal system suggesting that DO and DM functions may be intertwined.     

H2-O inhibits presentation of bacterial superantigens, but not endogenous self antigens

H2-O/HLA-DO are MHC class II accessory molecules that modulate exogenous Ag presentation. Most class II accessory molecules are expressed in all professional APC; however, H2-O is only expressed in B cells and medullary thymic epithelial cells. Because B cells present exogenous Ags and superantigens (SAgs), and medullary thymic epithelial cells are specialized APC for self Ags during negative selection in the thymus, we have hypothesized that H2-O might play a role in MHC class II-restricted SAg and self Ag presentation. In this study, we demonstrate that H2-O expression inhibits presentation of the bacterial SAgs staphylococcal enterotoxins A and B to four SAg-reactive T hybridoma cells. In contrast, H2-O has no effect on presentation of endogenous self Ags, as measured by tumorigenicity in vivo and Ag presentation to three self Ag-specific T hybridoma cells. Additional experiments suggest that H2-O inhibits presentation of exogenous Ags by both newly synthesized and recycling MHC class II molecules. These data suggest H2-O may have a physiological role in tolerance induction and SAg-mediated toxic shock.     

Regulation of MHC class II antigen presentation by sorting of recycling HLA-DM/DO and class II within the multivesicular body.

MHC class II molecules bind antigenic peptides in the late endosomal/lysosomal MHC class II compartments (MIIC) before cell surface presentation. The class II modulatory molecules HLA-DM and HLA-DO mainly localize to the MIICs. Here we show that DM/DO complexes continuously recycle between the plasma membrane and the lysosomal MIICs. Like DMbeta and the class II-associated invariant chain, the DObeta cytoplasmic tail contains potential lysosomal targeting signals. The DObeta signals, however, are not essential for internalization of the DM/DO complex from the plasma membrane or targeting to the MIICs. Instead, the DObeta tail determines the distribution of both DM/DO and class II within the multivesicular MIIC by preferentially localizing them to the limiting membrane and, in lesser amounts, to the internal membranes. This distribution augments the efficiency of class II antigenic peptide loading by affecting the efficacy of lateral interaction between DM/DO and class II molecules. Sorting of DM/DO and class II molecules to specific localizations within the MIIC represents a novel way of regulating MHC class II Ag presentation.     

Ectopic expression of HLA-DO in mouse dendritic cells diminishes MHC class II antigen presentation

The MHC class II-like molecule HLA-DM (DM) (H-2M in mice) catalyzes the exchange of CLIP for antigenic peptides in the endosomes of APCs. HLA-DO (DO) (H-2O in mice) is another class II-like molecule that is expressed in B cells, but not in other APCs. Studies have shown that DO impairs or modifies the peptide exchange activity of DM. To further evaluate the role of DO in Ag processing and presentation, we generated transgenic mice that expressed the human HLA-DOA and HLA-DOB genes under the control of a dendritic cell (DC)-specific promoter. Our analyses of DCs from these mice showed that as DO levels increased, cell surface levels of A(b)-CLIP also increased while class II-peptide levels decreased. The presentation of some, but not all, exogenous Ags to T cells or T hybridomas was significantly inhibited by DO. Surprisingly, H-2M accumulated in DO-expressing DCs and B cells, suggesting that H-2O/DO prolongs the half-life of H-2M. Overall, our studies showed that DO expression impaired H-2M function, resulting in Ag-specific down-modulation of class II Ag processing and presentation.     

Memory B cell survival and function in the absence of secreted antibody and immune complexes on follicular dendritic cells

Ag, in the form of immune complexes retained on follicular dendritic cells, has been implicated in the development and maintenance of B cell memory. We addressed this question using a H chain transgenic (Tg) mouse model that lacks secreted Ig (mIg), and thus does not deposit Ag-containing immune complexes. We compared the ability of the mIg strain and a control Tg strain, which secretes IgM, to develop and maintain long-lived memory cells. After immunization, there was an increase of Ag-specific B cells in both strains that was maintained for at least 20 wk. We labeled the long-lived Ag-specific cells with BrdU and found that this population was similarly maintained. In addition, both Tgs were able to maintain a functional memory response as measured by secondary germinal center reactions. Our studies indicate that localization of Ag on follicular dendritic cells is not necessary for development and maintenance of B cell memory.     

What to do with HLA-DO?

Antigenic peptide binding to MHC class II molecules in the endocytic pathway occurs via a multifactorial process that requires the support of a specialized lysosomal chaperone called HLA-DM. DM shows both in primary amino acid sequence and quaternary structure a high homology to both MHC class I and class II molecules. Like the peptide presenting class II molecules, DM is expressed in all professional antigen presenting cells. DM catalyzes the dissociation of peptides that do not bind stably to the class II peptide-binding groove, thereby leading to the preferential presentation of stably binding antigenic peptides. The recently discovered HLA-DO molecule is mainly expressed in B cells and associates with DM, thereby markedly affecting DM function. Like DM, the genes encoding the HLA-DO heterodimer lie within the MHC class II region and exhibit strong homology to classical class II molecules. This review evaluates the unique effects of DO on DM-mediated antigen presentation by MHC class II molecules and discusses the possible physiological relevance for the B cell-specific expression of DO and its function.     

Engagement of B cell receptor regulates the invariant chain-dependent MHC class II presentation pathway

The intracellular sites in which Ags delivered by the B cell receptor (BCR) are degraded and loaded onto class II molecules remain poorly defined. To address this issue, we generated wild-type and invariant chain (Ii)-deficient H-2k mice bearing BCR specific for hen egg lysozyme. Our results show that, 1) unlike Ags taken up from the fluid phase, Ii is required for presentation of hen egg lysozyme internalized through the BCR in a manner independent of the peptide analyzed; 2) BCR ligation induces intracellular accumulation of MHC class II molecules only in Ii-positive B cells; and 3) these class II molecules reach intracellular compartments where BCR targets exogenous Ag. No differences in expression of adhesion and costimulatory molecules or in the presentation of soluble peptides were detectable between Ii-positive and -negative B cells. Therefore, the BCR delivers its ligand to compartments containing MHC class II-Ii complexes and bypasses the Ii-independent presentation pathway. The linked roles of Ag internalization and B cell activation of the BCR leads to potent Ii-dependent presentation in splenic B cells.     

Differential MHC class II-mediated presentation of rheumatoid arthritis autoantigens by human dendritic cells and macrophages

Rheumatoid arthritis is characterized by synovial joint infiltration of activated CD4(+) T cells and MHC class II(+) APC, and is linked to specific HLA-DR alleles. Candidate autoantigens in synovial fluid and cartilage include type II collagen (CII) and cartilage gp39 (HCgp39). Using preparations of native Ag and T cells derived from Ag-immunized DR4-transgenic mice, we determined that human ex vivo differentiated DR4(+) dendritic cells (DC) and macrophages (Mphi) can mediate MHC class II presentation of CII or HCgp39 epitopes. The form of the Ag (soluble, partially degraded, or particulate) delivered to the APC influenced its presentation by DC and Mphi. DC efficiently presented partially degraded, but not native CII alpha-chains, while Mphi presentation was most efficient after phagocytosis of bead-conjugated CII. Both DC and Mphi presented soluble HCgp39, and activated Mphi from some donors presented epitopes derived from endogenously synthesized HCgp39. When synovial fluid from rheumatoid arthritis patients was used as a source of Ag, DC presentation of HCgp39 and CII epitopes was efficient, indicating that synovial fluid contains soluble forms of CII and HCgp39 amenable to internalization, processing, and presentation. These data support the hypothesis that CII and HCgp39 are autoantigens and that their class II-mediated presentation by DC and Mphi to T cells in vivo has a critical role in the pathogenesis of human rheumatoid arthritis.     

Human T cell clones present antigen

Two human T cells clones are described which react with influenza virus hemagglutinin type H3 and synthetic peptides of H3 when presented by PBMC APC. Both T cell clones also responded to peptide Ag in the absence of additional APC suggesting that T cells can simultaneously present and respond to Ag. T cell clones could only present peptide Ag and not an appropriate strain of inactivated whole influenza virus thus indicating an inability to process Ag conventionally. Peptide presentation by T cells was dose dependent, restricted by MHC class II Ag and was dependent on the number of Ag presenting T cells per culture. Experiments with nested peptides showed that the same epitope was recognized in the presence and absence of PBMC APC. No Ag or IL-2 from the propagation procedure was carried over into assays and two-color fluorescence-activated cell sorter analysis of each clone detected no contaminating cells with the phenotype of monocytes, macrophages or B cells; in each T cell clone, all cells expressing MHC class II Ag co-expressed CD3. These date therefore provide strong evidence that human T cell clones can simultaneously present and respond to appropriate forms of Ag.     

HLA-DO as the Optimizer of Epitope Selection for MHC Class II Antigen Presentation

Processing of antigens for presentation to helper T cells by MHC class II involves HLA-DM (DM) and HLA-DO (DO) accessory molecules. A mechanistic understanding of DO in this process has been missing. The leading model on its function proposes that DO inhibits the effects of DM. To directly study DO functions, we designed a recombinant soluble DO and expressed it in insect cells. The kinetics of binding and dissociation of several peptides to HLA-DR1 (DR1) molecules in the presence of DM and DO were measured. We found that DO reduced binding of DR1 to some peptides, and enhanced the binding of some other peptides to DR1. Interestingly, these enhancing and reducing effects were observed in the presence, or absence, of DM. We found that peptides that were negatively affected by DO were DM-sensitive, whereas peptides that were enhanced by DO were DM-resistant. The positive and negative effects of DO could only be measured on binding kinetics as peptide dissociation kinetics were not affected by DO. Using Surface Plasmon Resonance, we demonstrate direct binding of DO to a peptide-receptive, but not a closed conformation of DR1. We propose that DO imposes another layer of control on epitope selection during antigen processing.     

Presentation of antigens internalized through the B cell receptor requires newly synthesized MHC class II molecules

Exogenous Ags taken up from the fluid phase can be presented by both newly synthesized and recycling MHC class II molecules. However, the presentation of Ags internalized through the B cell receptor (BCR) has not been characterized with respect to whether the class II molecules with which they become associated are newly synthesized or recycling. We show that the presentation of Ag taken up by the BCR requires protein synthesis in splenic B cells and in B lymphoma cells. Using B cells transfected with full-length I-Ak molecules or molecules truncated in cytoplasmic domains of their alpha- or beta-chains, we further show that when an Ag is internalized by the BCR, the cytoplasmic tails of class II molecules differentially control the presentation of antigenic peptides to specific T cells depending upon the importance of proteolytic processing in the production of that peptide. Integrity of the cytoplasmic tail of the I-Ak beta-chain is required for the presentation of the hen egg lysozyme determinant (46-61) following BCR internalization, but that dependence is not seen for the (34-45) determinant derived from the same protein. The tail of the beta-chain is also of importance for the dissociation of invariant chain fragments from class II molecules. Our results demonstrate that Ags internalized through the BCR are targeted to compartments containing newly synthesized class II molecules and that the tails of class II beta-chains control the loading of determinants produced after extensive Ag processing.     

Tumor cells present MHC class II-restricted nuclear and mitochondrial antigens and are the predominant antigen presenting cells in vivo

MHC class II-restricted tumor Ags presented by class II(+) tumor cells identified to date are derived from proteins expressed in the cytoplasm or plasma membrane of tumor cells. It is unclear whether MHC class II(+) tumor cells present class II-restricted epitopes derived from other intracellular compartments, such as nuclei and/or mitochondria, and whether class II(+) tumor cells directly present Ag in vivo. To address these questions, a model Ag, hen egg lysozyme, was targeted to various subcellular compartments of mouse sarcoma cells, and the resulting cells were tested for presentation of three lysozyme epitopes in vitro and for presentation of nuclear Ag in vivo. In in vitro studies, Ags localized to all tested compartments (nuclei, cytoplasm, mitochondria, and endoplasmic reticulum) are presented in the absence invariant chain and H-2M. Coexpression of invariant chain and H-2M inhibit presentation of some, but not all, of the epitopes. In vivo studies demonstrate that class II(+) tumor cells, and not host-derived cells, are the predominant APC for class II-restricted nuclear Ags. Because class II(+) tumor cells are effective APC in vivo and probably present novel tumor Ag epitopes not presented by host-derived APC, their inclusion in cancer vaccines may enhance activation of tumor-reactive CD4(+) T cells.     

Cutting edge: editing of recycling class II:peptide complexes by HLA-DM.

HLA-DM catalyzes the exchange and selection of ligands for MHC class II molecules within mature endosomal/lysosomal compartments. Here, evidence is provided that DM edits peptides in early endosomes, thus influencing presentation via recycling class II molecules. Maximal class II-restricted presentation of an albumin-derived peptide, dependent on endocytosis and recycling class II molecules, was observed in cells lacking HLA-DM. DM editing of this epitope was observed in early endocytic compartments as shown using inhibitors of early to late endosomal transport. Editing was tempered by coexpression of HLA-DO, suggesting that DM:DO ratio may be important in guiding epitope editing in early endosomal compartments. Thus, HLA-DM appears to interact with, and edit epitopes displayed by, recycling class II molecules.     

Functional HLA-DM on the surface of B cells and immature dendritic cells

HLA-DM (DM) plays a critical role in antigen presentation through major histocompatibility complex (MHC) class II molecules. DM functions as a molecular chaperone by keeping class II molecules competent for antigenic peptide loading and serves as an editor by favoring presentation of high-stability peptides. Until now, DM has been thought to exert these activities only in late endosomal/lysosomal compartments of antigen-presenting cells. Here we show that a subset of DM resides at the cell surface of B cells and immature dendritic cells. Surface DM engages in complexes with putatively empty class II molecules and controls presentation of those antigens that rely on loading on the cell surface or in early endosomal recycling compartments. For example, epitopes derived from myelin basic protein that are implicated in the autoimmune disease multiple sclerosis are down-modulated by DM, but are presented in the absence of DM. Thus, this novel concept of functional DM on the surface may be relevant to both protective immune responses and autoimmunity.     

Efficient in vivo presentation of Listeria monocytogenes- derived CD4 and CD8 T cell epitopes in the absence of IFN-gamma

IFN-gamma is an essential component of the early Listeria monocytogenes-specific immune response, and is also an important regulator of Ag processing and presentation. Ag presentation is required for the induction and also the effector function of antimicrobial T cells. To evaluate the effect of IFN-gamma on bacterial Ag presentation in vivo, macrophages and dendritic cells were separated from L. monocytogenes-infected tissues and analyzed with peptide-specific CD4 and CD8 T cell lines in a sensitive ELISPOT-based ex vivo Ag presentation assay. The comparison of professional APCs isolated from infected IFN-gamma-deficient and wild-type mice revealed different peptide presentation patterns of L. monocytogenes-derived CD8 T cell epitopes, while the presentation pattern of CD4 T cell epitopes remained unchanged. The further in vitro analysis of the generation of CD8 T cell epitopes revealed a peptide-specific effect of IFN-gamma on MHC class I-restricted Ag presentation. These results show that despite this modulation of the Ag presentation pattern of CD8 T cell epitopes, IFN-gamma is not generally required for the MHC class I- and MHC class II-restricted presentation of L. monocytogenes-derived antigenic peptides by professional APCs in vivo.     

Cutting edge: H-2DM is responsible for the large differences in presentation among peptides selected by I-Ak during antigen processing

We quantitated the amounts of peptides from hen egg-white lysozyme presented by I-A(k) molecules in APC lines. The large chemical gradient of presentation of the four hen egg-white lysozyme epitopes observed in cell lines expressing HLA-DM or H-2DM (referred to in this study as DM) was significantly diminished in the T2.A(k) line lacking DM. Differences in levels of presentation between wild-type and DM-deficient APC were observed for all four epitopes, but differences were most evident for the highest affinity epitope. As a result of these quantitative differences in display, presentation of all four epitopes to T cells was impaired in the line lacking DM. The binding affinity of the pool of naturally processed peptides from DM-expressing lines was higher than that from the DM-deficient line. Thus, using a direct biochemical approach in APC, we demonstrate that DM influences the selection of peptides bound to MHC class II by favoring high affinity peptides.     

Presentation of endogenously synthesized MHC class II-restricted epitopes by MHC class II cancer vaccines is independent of transporter associated with Ag processing and the proteasome

Cell-based vaccines consisting of invariant chain-negative tumor cells transfected with syngeneic MHC class II (MHC II) and costimulatory molecule genes are prophylactic and therapeutic agents for the treatment of murine primary and metastatic cancers. Vaccine efficacy is due to direct presentation of endogenously synthesized, MHC II-restricted tumor peptides to CD4+ T cells. Because the vaccine cells lack invariant chain, we have hypothesized that, unlike professional APC, the peptide-binding groove of newly synthesized MHC II molecules may be accessible to peptides, allowing newly synthesized MHC II molecules to bind peptides that have been generated in the proteasome and transported into the endoplasmic reticulum via the TAP complex. To test this hypothesis, we have compared the Ag presentation activity of multiple clones of TAP-negative and TAP-positive tumor cells transfected with I-Ak genes and the model Ag hen egg white lysozyme targeted to the endoplasmic reticulum or cytoplasm. Absence of TAP does not diminish Ag presentation of three hen egg white lysozyme epitopes. Likewise, cells treated with proteasomal and autophagy inhibitors are as effective APC as untreated cells. In contrast, drugs that block endosome function significantly inhibit Ag presentation. Coculture experiments demonstrate that the vaccine cells do not release endogenously synthesized molecules that are subsequently endocytosed and processed in endosomal compartments. Collectively, these data indicate that vaccine cell presentation of MHC II-restricted endogenously synthesized epitopes occurs via a mechanism independent of the proteasome and TAP complex, and uses a pathway that overlaps with the classical endosomal pathway for presentation of exogenously synthesized molecules.     

Induction of vigorous helper and cytotoxic T cell as well as B cell responses by dendritic cells expressing a modified antigen targeting receptor-mediated internalization pathway

Efficient Ag presentation is essential to induce effective cellular and humoral immune responses. Thus, one central goal of current immunotherapy and vaccine development is to enhance Ag presentation to induce potent and broad immune responses. Here, a novel Ag presentation strategy is developed by transducing dendritic cells (DCs) to produce an Ag for presentation as an exogenous Ag to efficiently induce both humoral and cellular immunity. The principle of this strategy is illustrated by genetically modifying DCs to secrete a model hepatitis B virus Ag fused with a cell-binding domain and to process the fusion Ag as an exogenous Ag after receptor-mediated internalization for MHC class I and II presentation. Vigorous Ag-specific CD4(+) helper and CD8(+) cytotoxic T cell, as well as B cell, responses were induced by the transduced DCs in mouse models. Thus, this novel strategy uses a receptor-mediated internalization process to efficiently induce all arms of the adaptive immunity and may provide a powerful means to develop potent vaccines and immunotherapies.