Cellular localization of class I (HLA-A, B, C) and class II (HLA-DR and DQ) MHC antigens on the epithelial cells of normal human jejunum

HLA class I and class II (HLA-DR (human I-E equivalent) and DQ (human I-A equivalent] antigens were localized by immunofluorescence technique on thin frozen sections of normal human jejunum using a panel of monomorphic monoclonal antibodies. HLA class I (A, B and C) and HLA-DR molecules were found in the basolateral membrane of enterocytes; HLA-DR were also detected in a patchy distribution in the apical part of enterocytes; HLA-DQ molecules (the human equivalent of the murine I-A molecular subset) were not detected on normal enterocytes. All three molecules were detected on the membrane of lymphocytes and monocytes present in the lamina propria.     

Modulation of the expression of histocompatibility antigens class I (HLA-A,B,C) in the human pancreas

In this study the expression of HLA class I molecules was analysed on caudal portions of ten pancreata from cadaver donors by means of indirect immunoperoxidase and immunophosphatase techniques. In 7 out of ten pancreata the results showed that islets tissue was almost negative for the expression of HLA Class I antigens as opposed to exocrine tissue that appeared positive. Within exocrine tissue and large sized islets strongly positive interstitial cells were also detected. Double stainings showed that the strongly positive interstitial cells expressed also Leu M1 antigens. Preliminary studies on the remaining three pancreata demonstrate an increase of hematic interstitial cells together with a parallel increase of HLA class I antigens by endocrine parenchyma. The above data suggest that an increase of interstitial cells within pancreas may influence islets antigenicity.     

Aberrant expression of HLA-DR antigens of the MHC class II in bronchial epithelial cells in asthmatic patients

The study of cellular events in the bronchi of asthmatic patients shows significant epithelial destruction. The ciliated cells are more often destroyed than others in the respiratory epithelium. Using highly specific monoclonal antibodies, we detected that the epithelial cells were positive for HLA-DR antigen expression, whereas those from healthy subjects were negative. The aberrant expression could be explained by the presence of T lymphocytes in the asthmatic bronchial mucosa. The activated lymphocytes (CD4+) are known to release gamma interferon, which is a lymphokine that induces or enhances the expression of HLA-DR antigens. These cells, which are known to mediate cytotoxicity in an Ag-specific and Ia-restricted way, could take part in the shedding process of epithelial cells in asthma.     

Expression of MHC class I, MHC class II, and cancer germline antigens in neuroblastoma

Background: Neuroblastoma is the most common solid extracranial tumor in childhood, still with poor survival rates for metastatic disease. Neuroblastoma cells are of neuroectodermal origin and express a number of cancer germline (CG) antigens. These CG antigens may represent a potential target for immunotherapy such as peptide-based vaccination strategies. Objective: The purpose of this study was to analyze the presence of MAGE-A1, MAGE-A3/A6, and NY-ESO-1 on an mRNA and protein level and to determine the expression of MHC class I and MHC class II antigens within the same tumor specimens. Methods: A total of 68 tumors were available for RT-PCR, and 19/68 tumors were available for immunohistochemical (IHC) analysis of MAGE-A1, MAGE-A3/A6, and NY-ESO-1. In parallel, the same tumors were stained with a panel of antibodies for MHC class I and MHC class II molecules. Results: Screening of 68 tumor specimens by RT-PCR revealed expression of MAGE-A1 in 44%, MAGE-A3/A6 in 21%, and NY-ESO-1 in 28% of cases. Immunohistochemistry for CG antigens of selected tumors showed good agreement between protein and gene expression. However, staining revealed a heterogeneous expression of CG antigens. None of the selected tumors showed MHC class I or MHC class II expression. Conclusions: mRNA expression of MAGE-A1, MAGE-A3/A6, and NY-ESO-1 is congruent with the protein expression as determined by immunohistochemistry. The heterogeneous CG-antigen expression and the lack of MHC class I and II molecules may have implications for T-cell–mediated immunotherapy in neuroblastoma.     

Molecular associations of class II MHC antigens in mitogen-stimulated B cells

Previously, we showed that murine B cell membrane proteins undergo rearrangements in the plasma membrane to form new molecular associations in response to mitogenic stimulation. These complexes were covalently stabilized by photoreactive cross-linking agents and were analyzed by SDS PAGE. We have now identified certain complexes that involve class II MHC products, the Ia antigens. Upon stimulation of B cells with LPS, Ia surface molecules (as identified by radioimmunoprecipitation with polyclonal anti-Ia antiserum) enter into a molecular complex with a 95-kd membrane-associated protein (p95) to form a 200-kd complex that may be stabilized by the cross-linking agent dithiobisphenylazide (DTPA). This molecular association is not observed upon stimulation with mitogenic anti-Ig reagents, nor with the polyclonal B cell activator 8-bromoguanosine. p95 is not a disulfide-linked molecule itself, and by separate immunoprecipitation experiments we have established that it is not a component of surface Ig, transferrin receptor, the B cell Fc receptor, or CR1, the receptor for complement component C3b. Further analysis of the association of Ia antigens with surface proteins, with the use of monoclonal antibodies directed against I-A or I-E, has demonstrated that each subregion gene product forms a unique molecular association. Precipitation of radiolabeled lysates from LPS-activated B cells with anti-I-A reveals the aforementioned association with p95. In contrast, the I-E antigen apparently forms complexes with a multimer of a 15-kd protein to give complexes of 45, 60, 75, and 90 kd. When analyzed by two-dimensional diagonal gels (nonreducing/reducing), only the I-E bands are revealed by autoradiography, indicating that the putative p15 that associates with I-E may not be accessible to surface labeling. The disparate molecular associations for I-A and I-E suggest that the formation of these distinct protein complexes may be functionally related to a different role in the process of cellular activation for each of these Ia subregion gene products.     

Human class I histocompatibility antigens (HLA-A,B,C). A small proportion only is phosphorylated

Human Class I HLA antigens (HLA-A,B,C) were isolated by immune precipitation from cells labelled with 32P, [35S]methionine or 125I (by lactoperoxidase-catalysed cell-surface iodination) and were analysed using both one- and two-dimensional electrophoretic systems. In several B-lymphoblastoid cell lines and in human peripheral blood lymphocytes the electrophoretic mobility of the 32P-labelled HLA-A,B,C heavy chains consistently differed from that of molecules labelled by other means. Thus the 32P-labelled heavy chains appeared to be larger and to possess a more acidic pI than did heavy chains labelled with [35S]methionine or 125I, or identified by Coomassie Blue staining. Phosphatase treatment of immunoprecipitates, under conditions where 32P-labelled antigens were shown to be dephosphorylated, did not affect the mobilities of the [35S]methionine-labelled heavy chains. On glycosidase treatment, the positions of the 32P-labelled heavy chains were affected by neuraminidase but not by endo-beta-N-acetylglucosaminidase H. These results imply that phosphorylated HLA-A,B,C antigens comprise only a small proportion (relative to the total cellular HLA-A,B,C antigens) of the biosynthetically mature molecules. The possible significance of such heterogeneity is discussed.     

Expression of MHC class II antigens in human lung cancer cells.

Surgical specimens of lung cancers were examined immunopathologically for the expression of major histocompatibility complex class II (MHC-II) antigens in the tumor cells and their relationship to the lymphocytic infiltration. A lymphocytic infiltrate was frequently observed in the tumor tissue, though its intensity differed among the various histological types. MHC-II antigens were often demonstrated in tumors with a lymphocytic infiltrate. They were detected predominantly in the cytoplasm of tumor cells and to a lesser extent on the cell membranes. The emergence of the MHC-II-positive tumor cells was closely related to a local infiltration by lymphocytes including interferon-gamma (IFN-gamma)-producing T-cells. On the basis of the histological findings, an in vitro experiment was carried out. Four types of lung cancer cells were incubated with recombinant IFN-gamma in order to induce MHC-II antigens. MHC-II antigens (HLA-DR as well as HLA-DQ and HLA-DP antigens) were elicited in three cancer cell lines depending on the concentration of IFN-gamma. Immunoelectron microscopic study revealed that they were expressed on the surface of the cell membrane, though to a lesser extent than in the cytoplasm. It was considered that MHC-II antigens could be induced in some tumor cells in the immunological environment where IFN-gamma was secreted from T-cells and concentrated locally.     

Alloantigens expressed on activated human T cells different from HLA-A,B, C,and DR antigens

This study investigates alloantisera containing antibodies directed against antigens which are expressed on alloactivated human T lymphocytes but are absent on resting T and B cells. Among 39 defined anti-HLA-DR sera from multiparous women we found six sera giving positive reactions (more than 25 percent cytotoxicity) on in vitro alloactivated T cells, though negative reactions with resting B or T cells from the donors of either the responding or stimulating cell populations used for alloactivation. Two such sera were submitted to absorption and elution studies. Absorption of these sera with activated T cells did not remove the anti-HLA-DR activity. Furthermore, the antibodies eluted from activated T cells did not react with B cells but were positive only on activated T cells. In addition, we absorbed the sera with B cells and observed that they remained positive on activated T cells. The positive reactions do not seem to be due to either the passive acquisition of antigens from the stimulating population or to low levels of HLA-specific antibodies. As one of the sera we studied intensively gave clear positive and negative reactions on a panel of activated T lymphocytes, we believe it may recognize an antigen of an allogeneic system expressed on alloactivated human T cells.     

Allelic variants of the human MHC class I chain-related B gene (MICB)

 The human major histocompatibility complex (MHC) is located within a 4 megabase segment on chromosome 6p21.3. Recently, a highly divergent MHC class I chain-related gene family, MIC was identified within the class I region. The MICA and MICB genes in this family have unique patterns of tissue expression. The MICA gene is highly polymorphic, with more than 20 alleles identified to date. To elucidate the extent of MICB allelic variations, we sequenced exons 2 (α1), 3 (α2), 4 (α3), and 5 (transmembrane) as well as introns 2 and 4 of this gene in 46 HLA homozygous B-cell lines. We report the identification of eleven alleles based on seven non-synonymous, two synonymous, and four intronic nucleotide variations. Interestingly, one allele has a nonsense mutation resulting in a premature termination codon in the α2 domain. Thus, MICB appears to have fewer alleles than MICA, not unlike the allelic ratio between the HLA-C and -B loci. A preliminary linkage analysis of the MICB alleles with those of the closely located MICA and HLA-B genes revealed no conspicuous linkage disequilibrium between them, implying the presence of a potential recombination hotspot between the MICB and MICA genes. Received: 16 April 1997 / Revised: 19 May 1997     

NetMHCIIpan-3.0, a common pan-specific MHC class II prediction method including all three human MHC class II isotypes, HLA-DR, HLA-DP and HLA-DQ

Major histocompatibility complex class II (MHCII) molecules play an important role in cell-mediated immunity. They present specific peptides derived from endosomal proteins for recognition by T helper cells. The identification of peptides that bind to MHCII molecules is therefore of great importance for understanding the nature of immune responses and identifying T cell epitopes for the design of new vaccines and immunotherapies. Given the large number of MHC variants, and the costly experimental procedures needed to evaluate individual peptide–MHC interactions, computational predictions have become particularly attractive as first-line methods in epitope discovery. However, only a few so-called pan-specific prediction methods capable of predicting binding to any MHC molecule with known protein sequence are currently available, and all of them are limited to HLA-DR. Here, we present the first pan-specific method capable of predicting peptide binding to any HLA class II molecule with a defined protein sequence. The method employs a strategy common for HLA-DR, HLA-DP and HLA-DQ molecules to define the peptide-binding MHC environment in terms of a pseudo sequence. This strategy allows the inclusion of new molecules even from other species. The method was evaluated in several benchmarks and demonstrates a significant improvement over molecule-specific methods as well as the ability to predict peptide binding of previously uncharacterised MHCII molecules. To the best of our knowledge, the NetMHCIIpan-3.0 method is the first pan-specific predictor covering all HLA class II molecules with known sequences including HLA-DR, HLA-DP, and HLA-DQ. The NetMHCpan-3.0 method is available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.0.     

Identification, isolation, and culture of HLA-A2-specific B lymphocytes using MHC class I tetramers

Characterizing the individual B cells that participate in the production of anti-HLA Abs requires isolation and culture of these cells and a suitable assay for detection of Abs produced in these B cell cultures. We previously showed that B cell precursors, programmed for anti-HLA Ab secretion, are present at measurable frequencies in peripheral blood of women immunized by pregnancy. In this study, we show that tetrameric HLA-A2, although designed for characterization of CTLs, provides a suitable affinity ligand for isolation of allospecific B cells, which subsequently can be induced to produce HLA-A2 Ab in a CD40-driven culture system. The validity of this concept was established by assaying human hybridomas, producing anti-HLA Abs, for specific tetrameric HLA-A2 binding. The availability of anti-HLA Ab-producing B cell cultures that are established without immortalization will be of value when T-B cell interaction is studied at an alloantigen-specific level.     

MHC class II antigen processing in B cells: accelerated intracellular targeting of antigens.

Processing and presentation by Ag-specific B cells is initiated by Ag binding to the B cell Ag receptor (BCR). Cross-linking of the BCR by Ag results in a rapid targeting of the BCR and bound Ag to the MHC class II peptide loading compartment (IIPLC). This accelerated delivery of Ag may be essential in vivo during periods of rapid Ag-driven B cell expansion and T cell-dependent selection. Here, we use both immunoelectron microscopy and a nondisruptive protein chemical polymerization method to define the intracellular pathway of the targeting of Ags by the BCR. We show that following cross-linking, the BCR is rapidly transported through transferrin receptor-containing early endosomes to a LAMP-1+, beta-hexosaminadase+, multivesicular compartment that is an active site of peptide-class II complex assembly, containing both class II-invariant chain complexes in the process of invariant chain proteolytic removal as well as mature peptide-class II complexes. The BCR enters the class II-containing compartment as an intact mIg/Igalpha/Igbeta complex bound to Ag. The pathway by which the BCR targets Ag to the IIPLC appears not to be identical to that by which Ags taken up by fluid phase pinocytosis traffick, suggesting that the accelerated BCR pathway may be specialized and potentially independently regulated.     

Role of HLA class I and class II antigens in activation and differentiation of B cells

The monoclonal antibodies (MoAb) CR10-214, CR11-115, and Q1/28 to distinct monomorphic determinants of HLA class I antigens, the MoAb CL413 and PTF29.12 recognizing monomorphic determinants of HLA-DR antigens, the anti-HLA-DQw1 MoAb KS11, the anti-HLA-DPw1 MoAb B7/21, and the anti-HLA-DR,DP MoAb CR11-462 were tested for their ability to modulate human B-lymphocyte proliferation and maturation to IgM-forming cells. Purified tonsillar B cells were stimulated with Staphylococcus aureus bacteria of the Cowan first strain (SAC) or anti-human mu-chain xenoantibodies, as well as in growth factor- or T-cell-dependent activation cultures. The B-cell proliferative responses induced by SAC or by mitogenic concentrations of anti-mu-chain xenoantibodies were inhibited by some of the anti-HLA class I and anti-HLA class II monoclonal antibodies tested. The same antibodies were effective inhibitors of the proliferation of B cells stimulated with interferon-gamma (IFN-gamma) or interleukin-2 (IL-2) and with submitogenic concentrations of anti-mu-chain xenoantibodies. The proliferation induced by IL-2 of SAC-preactivated B cells was inhibited by some of the anti-HLA class II monoclonal antibodies, but not by the anti-HLA class I monoclonal antibodies tested. This inhibition appeared to reflect at least in part a direct effect on later events of the B-cell activation cascade, since some anti-HLA class II monoclonal antibodies still exerted considerable inhibitory activity when added together with IL-2 to SAC-preactivated B cells after the third day of culture. Anti HLA-DR, DQ, and DP monoclonal antibodies consistently inhibited the IgM production induced in B cells by T cells alone, T cells plus pokeweed mitogen (PWM), SAC plus IL-2, or IL-2 alone. In contrast, two of the three anti-HLA class I monoclonal antibodies tested inhibited the IgM production in cultures stimulated with SAC plus IL-2 and one the IgM production induced by IL-2 alone, but none of them had inhibitory effects on T-cell dependent IgM production. The results reported herein indicate that HLA class II molecules directly participate in different phases of the B-cell activation cascade. In addition, our data also suggest that HLA class I molecules can be involved in the events leading to B-cell proliferation and differentiation into immunoglobulin-secreting cells.     

Pseudomonas exotoxin-mediated delivery of exogenous antigens to MHC class I and class II processing pathways

Peptides associated with class II MHC molecules are normally derived from exogenous proteins, whereas class I MHC molecules normally associate with peptides from endogenous proteins. We have studied the ability of Pseudomonas exotoxin A (PE) fusion proteins to deliver exogenously added antigen for presentation by both MHC class I and class II molecules. A MHC class II-restricted antigen was fused to PE; this molecule was processed in a manner typical for class II-associated antigens. However, a MHC class I-restricted peptide fused to PE was processed by a mechanism independent of proteasomes. Furthermore, we also found that the PE fusion protein was much more stable in normal human plasma than the corresponding synthetic peptide. We believe that effective delivery of an antigen to both the MHC class I and class II pathways, in addition to the increased resistance to proteolysis in plasma, will be important for immunization.     

Induction and functional characterization of class II MHC (Ia) antigens on murine keratinocytes

To induce Ia molecules on the surface of murine keratinocytes (KC), healthy mice were treated daily with i.p. injections of rIFN-gamma at a dose of 50,000 U/day for 6 days. This resulted in strong Ia expression by KC as determined by immunofluorescence of epidermal sheets or cell suspensions with anti-class II mAb. To obtain a population of Ia-bearing KC devoid of Langerhans cells, a method of depleting Langerhans cells from such suspensions was developed. Although Ia+ KC were unable to stimulate allogeneic T cells in a primary epidermal cell-lymphocyte reaction (less than 5% control), they did induce a proliferative response in an allospecific T cell line. Ia+ KC were unable to present native peptide molecules to class II restricted, Ag-specific T cell hybridomas. However, Ia+ KC were able to present a peptide fragment of pigeon cytochrome c to a hybridoma, suggesting that although these cells cannot process native protein Ag, they can present antigenic peptides. Ia+ (but not Ia-) KC also served as targets for class II restricted cytolytic T cell clones. These data indicate that the Ia expressed by KC is a functional molecule, and that Ia+ KC can participate in some immunologic reactions.     

Expression of CD1 and class I MHC antigens by human thymocytes

The acquisition of surface class I MHC molecules is associated with the maturation of thymocytes. Here, surface expression of class I MHC and CD1, which represents a family of MHC-related molecules, was analyzed on various human immature and mature thymocyte subpopulations. Class I expression was inversely related to the expression of CD1. The majority of CD4+ CD8+ cortical type thymocytes expressed low levels of class I MHC Ag, the previously described CD4+ CD8+ thymocyte subpopulation with low CD8 expression exhibited intermediate levels of class I MHC, whereas most of the single positive CD4 and CD8 thymocytes displayed high levels of class I MHC. Biochemical comparison of CD1 and class I showed that thymic class I molecules were post-translationally modified by phosphorylation, whereas CD1 was not phosphorylated. Furthermore, our studies suggested that in addition to CD1/CD8 complexes, thymocytes bear CD8/class I complexes. Chemical cross-linking and peptide mapping studies clearly identified the CD8-associated protein on thymic clones as the class I MHC molecule.