MHC class II molecules play a pivotal role for the induction and maintenance of immune responses against pathogens, but are also implicated in pathological conditions like autoimmune diseases or rejection of transplanted organs. Human antigen-presenting cells express three human leukocyte antigen (HLA) class II isotypes (DR, DP, and DQ), which are, with the exception of DRα, composed of highly polymorphic α and β subunits. The combination of α- and β-chains results in a multitude of MHC-II αβ-heterodimers of the same isotype, but also isotype-mixed MHC class II molecules have been identified. Invariant chain chaperones the assembly of MHC-II molecules within the endoplasmatic reticulum and also facilitates the intracellular transport to MHC class II loading compartments (MIICs). MHC-II molecules are loaded with antigenic peptides and shuttled to the cell surface for inspection by CD4 T-cells. Alternatively, class-II molecules enriched on intraluminal vesicles can be released via exosomes into the extracellular space. Since some of the αβ-combinations may yield mismatched nonfunctional heterodimers, it is not entirely clear which type of HLA class II peptide receptors are transported to MIICs and found on the cell surface of antigen-presenting cells. We present techniques to inspect assembly, intracellular transport, cell surface expression, and exosomal release of MHC class II heterodimers.

Herpes simplex virus (HSV)-encoded glycoprotein B (gB) is the most abundant protein in the viral envelope and promotes fusion of the virus with the cellular membrane. In the present study, we found that gB impacts on the major histocompatibility complex (MHC)-II pathway of antigen presentation by fostering homotypic fusion of early endosomes and trapping MHC-II molecules in these altered endosomes. By using an overexpression approach, we demonstrated that transient expression of gB induces giant vesicles of early endosomal origin, which contained Rab5, early endosomal antigen 1 (EEA1), and large amounts of MHC-II molecules [human leukocyte antigen (HLA)-DR, and HLA-DM], but no CD63. In HSV-1-infected and stably transfected cell lines that expressed lower amounts of gB, giant endosomes were not observed, but strongly increased amounts of HLA-DR and HLA-DM were found in EEA1+ early endosomes. We used these giant vesicles as a model system and revealed that gB interacts with Rab5 and EEA1, and that gB-induced homotypic fusion of early endosomes to giant endosomes requires phosphatidylinositol 3-phosphate, the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptors, and the cytosolic gB sequence 889YTQVPN894 We conclude that gB expression alters trafficking of molecules of the HLA-II processing pathway, which leads to increased retention of MHC-II molecules in early endosomal compartments, thereby intercepting antigen presentation.-Niazy, N., Temme, S., Bocuk, D., Giesen, C., König, A., Temme, N., Ziegfeld, A., Gregers, T. F., Bakke, O., Lang, T., Eis-Hübinger, A. M., Koch, N. Misdirection of endosomal trafficking mediated by herpes simplex virus-encoded glycoprotein B.

HLA class II alpha and beta chains form receptors for antigen presentation to CD4(+) T cells. Numerous pairings of class II alpha and beta subunits from the wide range of haplotypes and isotypes may form, but most of these combinations, in particular those produced by isotype mixing, yielded mismatched dimers. It is unclear how selection of functional receptors is achieved. At the atomic level, it is not known which interactions of class II residues regulate selection of matched alpha beta heterodimers and the evolutionary origin of matched isotype mixed dimer formation. In this study we investigated assembly of isotype-mixed HLA class II alpha and beta heterodimers. Assembly and carbohydrate maturation of various HLA-class II isotype-mixed alpha and beta subunits was dependent on the groove binding section of the invariant chain (Ii). By mutation of polymorphic DP beta sequences, we identified two motifs, Lys-69 and GGPM-(8487), that are engaged in Ii-dependent assembly of DP beta with DR alpha. We identified five members of a family of DP beta chains containing Lys-69 and GGPM 84-87, which assemble with DR alpha. The Lys/GGPM motif is present in the DP beta sequence of the Neanderthal genome, and this ancient sequence is related to the human allele DPB1*0401. By site-directed mutagenesis, we inspected Neanderthal amino acid residues that differ from the DPB1*0401 allele and aimed to determine whether matched heterodimers are formed by assembly of DP beta mutants with DR alpha. Because the *0401 allele is rare in the sub-Saharan population but frequent in the European population, it may have arisen in modern humans by admixture with Neanderthals in Europe.

Background The human lymphocyte antigen (HLA) encoded BAT3/BAG6 recently attracted interest as a regulator of protein targeting and degradation, a function that could be exerted in the cytosol and in the nucleus. The BAT3 gene was described to consist of 25 exons. Diversity of transcripts can be generated by alternative RNA splicing, which may control subcellular distribution of BAT3. Methodology/Principal Findings By cDNA sequencing we identified a novel alternatively spliced sequence of the BAT3 gene located between exons 11 and 12, which was designated as exon 11B. Using PCR and colony hybridization we identified six cDNA variants, which were produced by RNA splicing of BAT3 exons 5, 11B and 24. In four examined cell types the content of BAT3 splice variants was examined. Most of the cDNA clones from monocyte-derived dendritic cells contain exon 11B, whereas this sequence was almost absent in the B lymphoma Raji. Exon 5 was detected in most and exon 24 in approximately half of the cDNA clones. The subcellular distribution of endogenous BAT3 largely correlates with a cell type specific splicing pattern. In cells transfected with BAT3 variants, full-length and Δ24 BAT3 displayed nearly exclusive nuclear staining, whereas variants deleted of exon 11B showed substantial cytosolic expression. We show here that BAT3 is mainly expressed in the cytosol of Raji cells, while other cell types displayed both cytosolic and nuclear staining. Export of BAT3 from the nucleus to the cytosol is inhibited by treatment with leptomycin B, indicating that the Crm1 pathway is involved. Nuclear expression of BAT3 containing exon 11B suggests that this sequence plays a role for nuclear retention of the protein. Conclusions/Significance Cell type-specific subcellular expression of BAT3 suggests distinct functions in the cytosol and in the nucleus. Differential expression of BAT3 variants may reconcile the multiple roles described for BAT3.

Human antigen presenting cells express three human leukocyte antigen (HLA) class II isotypes (DR, DP, and DQ), which are composed of polymorphic α and β subunits. The combination of polymorphic α- and β-chains results in cis (encoded on the same chromosome) or trans (encoded on different chromosomes) combinations. Since some of the α-β combinations may yield mismatched non-functional α-β heterodimers, it is not entirely clear which type of HLA class II peptide receptors are found on the cell surface of antigen presenting cells. We have developed a combination of biochemical techniques for inspection of the assembly and intracellular transport of isotype matched and mismatched class II heterodimers.

Antigen presentation by human lymphocyte antigen (HLA) class II peptide receptors alerts the immune system to infections. In antigen-presenting cells (APCs), HLA class II, HLA-DM, and associated invariant chain-encoding genes are exclusively regulated by the interferon gamma (IFN gamma)-inducible class II transactivator (CIITA). Control of CIITA expression could therefore govern expression of class II peptide receptors in the diverse group of APCs. We discovered that elevation of the HLA class III region encoded B-associated transcript 3 (BAT3) increases and depletion of BAT3 decreases expression of HLA class II, HLA-DM, and invariant chain. IFN gamma strongly elevates BAT3 transcription in various tumor cell lines and in primary macrophages. BAT3 chaperones the simultaneously IFN gamma-induced CIITA. Following IFN gamma-treatment, both CIITA and BAT3 translocate from the cytosol to the nucleus. The nuclear import of CIITA mediated by IFN gamma controls activation of HLA class II genes. BAT3 is a novel key regulator of components of the HLA class II processing pathway. We present a mechanism explaining how parallel IFN gamma-mediated regulation of CIITA and of its chaperone BAT3 controls the level of components of the HLA class II processing pathway.-Kamper, N., Franken, S., Temme, S., Koch, S., Bieber, T., Koch, K. gamma-Interferon-regulated chaperone governs human lymphocyte antigen class II expression. FASEB J. 26, 104-116 (2012). www.fasebj.org

BACKGROUND:The HLA gene complex encodes three class II isotypes, DR, DQ, and DP. HLA class II molecules are peptide receptors that present antigens for recognition by T lymphocytes. In antigen presenting cells, the assembly of matched α and β subunits to heterodimers is chaperoned by invariant chain (Ii). Ii forms a homotrimer with three binding sites for class II heterodimers. The current model of class II and Ii structure states that three αβ heterodimers bind to an Ii trimer.METHODOLOGY/PRINCIPAL FINDINGS:[corrected] We have now analyzed the composition and size of the complexes of class II and Ii using epitope tagged class II subunits and density gradient experiments. We show here that class II-Ii oligomers consist of one class II heterodimer associated with one Ii trimer, such that the DR, DQ and DP isotypes are contained within separate complexes with Ii.CONCLUSION/SIGNIFICANCE:We propose a structural model of the class II-Ii oligomer and speculate that the pentameric class II-Ii complex is bent towards the cell membrane, inhibiting the binding of additional class II heterodimers to Ii.

Neutralizing Abs play an important role for immunity against HSV-1 infection. This branch of the immune response is initiated by MHC class II Ag presentation and activation of T cell help. In this study, we show that the HSV-1 encoded glycoprotein B (gB) manipulates the class II processing pathway by perturbing endosomal sorting and trafficking of HLA-DR (DR) molecules. Expression of gB in the human melanoma cell line Mel JuSo results in formation of enlarged DR(+) intracellular vesicles. Costaining of the vesicles revealed the presence of DR, gB, and the late endosomal marker CD63. The lumen of these late endosomal membranes shows a variable content, containing either gB or CD63, or both CD63 and gB. gB targets DR molecules on their biosynthetic route, after the MHC class II invariant chain is released from the DR heterodimer. gB-DR complexes were detected in a post-Golgi compartment and in exosomes, but not on the cell surface. Interestingly, increasing expression of gB strongly elevated the amount of DR and CD63 released into the exosome pathway. In conclusion, this is a previously undescribed mode of viral immune evasion involving hijacking of DR from its normal transport route to the cell surface, followed by viral-mediated release of DR into the exosome pathway.

SummaryRat major histocompatibility complex (MHC) class II molecules RT1.Bl (DQ‐like) and RT1.Dl (DR‐like) were cloned from the LEW strain using reverse transcription–polymerase chain reaction and expressed in mouse L929 cells. The transduced lines bound MHC class II‐specific monoclonal antibodies in an MHC‐isotype‐specific manner and presented peptide antigens and superantigens to T‐cell hybridomas. The T‐cell‐hybridomas responded well to all superantigens presented by human MHC class II, whereas the response varied considerably with rat MHC class II‐transduced lines as presenters. The T‐cell hybridomas responded to the pyrogenic superantigens Staphylococcus enterotoxin B (SEB), SEC1, SEC2 and SEC3 only at high concentrations with RT1.Bl‐transduced and RT1.Dl‐transduced cells as presenters. The same was true for streptococcal pyrogenic exotoxin A (SPEA), but this was presented only by RT1.Bl and not by RT1.Dl. SPEC was recognized only if presented by human MHC class II. Presentation of Yersinia pseudotuberculosis superantigen (YPM) showed no MHC isotype preference, while Mycoplasma arthritidis superantigen (MAS or MAM) was presented by RT1.Dl but not by RT1.Bl. Interestingly, and in contrast to RT1.Bl, the RT1.Dl completely failed to present SEA and toxic shock syndrome toxin 1 even after transduction of invariant chain (CD74) or expression in other cell types such as the surface MHC class II‐negative mouse B‐cell lymphoma (M12.4.1.C3). We discuss the idea that a lack of SEA presentation may not be a general feature of RT1.D molecules but could be a consequence of RT1.Dlβ‐chain allele‐specific substitutions (arginine 80 to lysine, asparagine 82 to aspartic acid) in the extremely conserved region flanking the Zn2+‐binding histidine 81, which is crucial for high‐affinity SEA‐binding.

We report the generation of a monoclonal antibody (MAb) that reacts to the N-terminus of the denatured HLA-DRalpha chain. The 1C4.6 MAb was raised against a peptide corresponding to amino acid residues 10 to 32 of a highly conserved region within the alpha1 domain of HLA-DR. This region partially overlaps with the epitope recognized by the conformationally dependent L243 MAb. In Western blot analysis, MAb 1C4.6 reacted with denatured HLA-DRalpha chains, but failed to bind the HLA-DRbeta chain expressed individually by transfectant cells, confirming that it recognizes an epitope on the alpha-chain of HLA-DR. In addition, this antibody was found to be isotype specific to HLA-DRalpha, as it did not cross-react to HLA class II proteins HLA-DP and-HLA-DQ. The 1C4.6 MAb is a valuable addition to existing reagents used to probe the structure and function of MHC class II molecules. This anti-HLA-DRalpha1 domain MAb may prove valuable for studies of HLA class II heterodimer assembly, structure, and function, as well as for studies into the release of soluble MHC class II.

MHC class II (MHC-II) molecules are capable of transducing signals with the help of associated molecules. Although the search to find associated molecules over the past few years has been fruitful, it remains clear that not all signaling components and their mechanisms of action have been identified. In this study, we investigated calcium and MAPK signaling pathways using the BJAB and Raji human B cell lines. We demonstrate that calcium mobilization is an isotype-independent event that triggers the dephosphorylation of NFAT. We also show that BCR activation followed by MHC-II ligation increases the activation of NFAT. This signaling pathway differs from MHC-II-mediated MAP activation, where MEK1/2 and ERK1/2 phosphorylation are isotype-specific events, which correspond to the induction of c-Fos and formation of AP-1. Future studies should elucidate the intertwined, intricate signaling cascades triggered by BCR and MHC-II leading to humoral immune responses.

Exosomes are lipid-bound nanovesicles formed by inward budding of the endosomal membrane and released following fusion of the endosomal limiting membrane with the plasma membrane. We show here that primary leukocytes do not release exosomes unless subjected to potent activation signals, such as cytokine or mitogen stimulation. In particular, high levels of exosomes were released when murine splenic B cells were stimulated via CD40 and the IL-4 receptor. This property was shared by B cells from different anatomic locations, as newly formed marginal zone and follicular B cells were capable of secreting exosomes upon CD40/IL-4 triggering. B cell exosomes expressed high levels of MHC class I, MHC class II, and CD45RA (B220), as well as components of the BCR complex, namely, surface Ig, CD19, and the tetraspanins CD9 and CD81. Ig on the plasma membrane of primary B cells was targeted to the exosome pathway, demonstrating a link between the BCR and this exocytic pathway. IgD and IgM were the predominant Ig isotypes associated with CD40/IL-4 elicited exosomes, though other isotypes (IgA, IgG1, IgG2a/2b, and IgG3) were also detected. Together, these results suggest that exosome release is not constitutive activity of B cells, but may be induced following cell: cell signaling.

OBJECTIVES:The major histocompatibility complex class II chaperone invariant chain (Ii) is widely used as a carrier for inserted antigenic sequences and their introduction into the class II processing pathway. The tumor-associated antigen core 2beta 1,6 N-acetylglucosaminyltransferase (C2GnT), a glycosyltransferase present in human pancreatic tumor cells, is not expressed by normal pancreatic tissues. METHODS:A set of expression vectors was engineered where the class II binding region of Ii was replaced by C2GnT-derived sequences. We investigated in vitro whether dendritic cells transfected with Ii-C2GnT constructs were capable to stimulate proliferation of CD4 T cells. We also tested whether vaccination with Ii-C2GnT would protect mice from tumor development. RESULTS:Invariant chain-C2GnT fusion proteins bind to human DR1, DR3, DR4 and to mouse I-A molecules. Our results demonstrate that the plasmid DNA encoding the C2GnT epitope embedded in Ii induces tumor-specific T-cell responses. Mice immunized with the Ii constructs showed reduced growth of Panc02 pancreatic tumor cells. CONCLUSIONS:Therefore, Ii clipped with the tumor-associated antigen C2GnT shows promise for the treatment of pancreatic cancer.

Human HLA-DM or mouse H2-DM plays a vital role for presentation of antigenic sequences by MHC class II peptide receptors. These non-classical MHC class II molecules catalyze the release of the invariant chain (Ii) fragment CLIP from the class II cleft and facilitate acquisition of antigenic peptides by MHC class II peptide receptors. H2-DM- or Ii-deficient mice display an impaired ability of their antigen-presenting cell to present peptides to CD4+ T cells and a molecular link between the immunodeficiencies of these mouse strains may exist. We show that in transfected cells the presence of HLA-DM molecules in endocytic vesicles was strongly reduced when HLA-DM was accompanied by HLA-DR. Exclusion of HLA-DM from endocytic vesicles is explained by mixed association of HLA-DM with HLA-DR subunits and retention of the aggregates in the endoplasmic reticulum. Expression of Ii, however, impairs formation of mixed HLA-DR and HLA-DM complexes and directs matched pairing of HLA-DR and HLA-DM heterodimers. In Ii gene-deficient mice, aberrant association of H2A with H2-DM polypeptides was detected. Low expression of Ii in transgenic mice inhibits interaction of H2A with H2-DM subunits and facilitates formation of H2-DM alphabeta heterodimers. A role of Ii for assembly of H2-DM heterodimers partially explains the immunodeficient phenotype of Ii-/- mice.

The enormous number of allelic MHC class II glycoproteins provides the immune system with a large set of heterodimeric receptors for the binding of pathogen-derived peptides. How do inherited allo- or isotypic subunits of MHC class II combine to produce such a variety of functional peptide receptors? We propose a new mechanism in which pairing of matched MHC class II alpha- and beta-subunits is coordinated by the invariant chain chaperone. The assembly is proposed to occur in a sequential fashion, with a matched beta-chain being selected by the alpha-chain-invariant chain 'scaffold' complex that is formed first. This sequential assembly is a prerequisite for subsequent intracellular transport of the a-chain-invariant chain-beta-oligomer and its maturation into a functional peptide receptor.

The human lymphocyte antigen (HLA) class II region encodes highly polymorphic peptide receptors, which associate in the ER to the chaperone invariant chain (Ii). Ii facilitates assembly of class II subunits to functional peptide receptors. We searched for a conserved structure on HLA-DR polypeptides that mediates contact to a previously identified proline-rich class-II-binding sequence of Ii. Major histocompatibility complex (MHC) class II beta chain sequences exhibit two conserved tryptophan residues separated by 22 amino acids. Inspection of this motif in the X-ray structure of DR3 showed TrpTyr residues in the vicinity of the Ii-derived fragment CLIP. Five DRbeta mutants were produced. Mutation at Tyr123, Trp153 and Asp152 residues abolished interaction to the proline-rich sequence of Ii. All mutants formed heterodimers with DRalpha, were capable of binding an antigenic sequence and were expressed on the cell surface of transfected cells. In the presence of endogenous DRbeta chain however, the TyrAspTrp mutant was not cell-surface exposed and did not co-isolate with Ii or DRalpha. The competition of the mutant with the endogenous DRbeta for binding to DRalpha indicates that a structure on DRbeta chain regulates assembly of DR subunits. Hence, the chaperone function of Ii is mediated through a conserved region on the beta2 domain of class II.

In vitro studies have shown that soluble MHC (sMHC) released by cell lines is bound to nano-vesicles termed exosomes. It is thought that exosomes may represent the major reservoir of sMHC class I and II molecules in biological fluids. However, most studies have been confined to in vitro assays performed with cell lines. We show here that sMHC in the serum or plasma differs from exosome-bound sMHC in five ways: In contrast to exosome-associated sMHC, circulating sMHC is of low density, has a low apparent molecular mass (40-300 kDa) and is not detergent-labile. Moreover, the majority of MHC class II isoforms and MHC class I in blood are not physically linked and circulating HLA-DR is accessible to an antibody specific for the HLA-DR a-chain intracellular epitope, which is masked by its association with cellular or exosomal membranes. Finally, utilizing transcriptional activator of murine MHC class II (C2ta) promoter-mutant mice, we showed that the release of sMHC class II into the circulation is dependent on the C2ta pI promoter, but not pIII or pIV. This suggests that myeloid dendritic cells and/or macrophages, which preferentially use promoter pI of the C2ta gene, produce most of the sMHC class II found in the circulation.

The highly polymorphic major histocompatibility complex class II (MHCII) polypeptides assemble in the ER with the assistance of invariant chain (Ii) chaperone. Ii binds to the peptide‐binding pocket of MHCII heterodimers. We explored the mechanism how MHCII subunits attach to Ii. Expression with single α or β subunits from three human HLA and two mouse H2 class II isotypes revealed that Ii co‐isolates predominantly with the α polypeptide. Co‐isolation with α chain requires the groove binding Ii‐segment and depends on M91 of Ii. Immunoprecipitation of Ii from pulse chase labeled cells showed sequential assembly of α and β chains.

Recombinant vaccines containing Ii sequences were employed to elicit an antibody response. Gene gun immunisation of mice with the recombinant Ii-antigen-encoding vectors induced antigen-specific antibodies. Antibody levels were substantially elevated when the DNA construct was extended by a sequence encoding the protease inhibitory domain of the invariant chain isoform Ii41. Employing this approach, we raised antibodies specific for a novel MHC class III encoded protein. The antibodies identify the 216 kDa BAT2 polypeptide. Immunostaining of embryonic tissue sections showed specific expression, especially in central nervous tissue, and suggests BAT2 as a novel differentiation marker.