Nonimmune human cells can express MHC class II antigens in the absence of invariant chain--an immunohistological study on normal and chronically inflamed small intestine

The expression of HLA-DR, HLA-DP and HLA-DQ antigens and of the associated invariant chain (Ii) was studied immunohistologically in sessile cells of normal ileum and ileum affected by Crohn's disease which was taken as a model for chronic inflammation. Corresponding to the local inflammatory cell density, a considerable neo-expression of MHC class II antigens and Ii was observed in epithelial cells, vascular endothelial cells, and Schwann cells of the enteric nerve plexus. While HLA-DP and HLA-DQ antigens were undetectable in sessile cells of normal ileum, class II antigens expression in ileitis followed the order HLA-DR greater than or equal to HLA-DP greater than or equal to HLA-DQ. In various cell types a differential expression of Ii and class II antigens was noted. In normal crypt enterocytes and in arterial endothelial cells of inflamed ileum, Ii was found in the absence of class II antigens. On the other hand, most Schwann cells and internodal nerve strands of the submucous and myenteric plexus exhibited HLA-DR (-DP, -DQ) antigens in the absence of detectable Ii. Moreover, HLA-DR+ endothelial cells in normal tissue specimens were Ii-, and in inflamed intestine HLA-DR expression of venous/venular and capillary endothelium greatly exceeded Ii expression. The observation of both Ii+/HLA-DR- and Ii-/HLA-DR+ (HLA-DP+, HLA-DQ+) cells questions the previously assumed close association of Ii and class II antigen expression.     

Immunohistological evaluation of MHC class I and II antigen expression on nevi and melanoma: relation to biology of melanoma

MHC antigen expression on 20 nevi, and 35 primary and 95 metastatic melanomas was studied by immunoperoxidase techniques using monoclonal antibodies to identify the antigens on frozen tissue sections. DR antigens were not detected on nevi but were detected on 71% of primary melanomas and 56% of metastases, suggesting that this antigen may be a useful marker of malignant transformation of nevi. Expression of class II antigen could not be related to other prognostic histological features of primary melanoma such as tumour thickness, but comparison of the common phenotypes of primary and metastatic melanoma suggested that expression of DR antigens alone in the absence of DP, DQ and ABC antigens may be an indicator of metastatic potential. Class I (HLA-A,B,C) antigens were also expressed infrequently on nevi but were detected on 43% of primary melanomas and 34% of metastases. HLA-A,B,C expression was inversely related to thickness of the primary melanoma. This as well as the lower expression of class I antigens on metastases, may indicate that growth and spread of melanoma may be inhibited by MHC (class I) dependent cytotoxic T cell responses. Expression of class I MHC antigens was unrelated to class II antigens. Expression of DR was more common than DP or DQ, but the latter with one exception, were not expressed in the absence of DR antigens. Significant differences were not found in MHC antigen expression on metastases in lymph nodes compared to those in subcutaneous sites, but further studies are needed to determine whether such differences may exist between metastases in other visceral sites.     

Differential expression of HLA-DR,HLA-DP,HLA-DQ and associated invariant chain (Ii) in normal colorectal mucosa,adenoma and carcinoma

Summary The expression of MHC class II antigens (HLA-DR, HLA-DP and HLA-DQ) and the associated invariant chain (Ii) was studied in epithelial cells of normal colorectal mucosae, colorectal adenomas and carcinomas, using a sensitive immunoperoxidase technique with monoclonal antibodies on frozen sections. In contrast to class II antigens, Ii was detected in some normal mucosae distant from the tumour. In residual non-neoplastic mucosa adjacent to carcinomas, Ii and class II antigens were induced in the order Ii HLA-DRHLA-DPHLA-DQ, the reactions being most pronounced in cases with inflammatory alteration of the crypts. In 22/37 adenomas and 77/123 carcinomas, Ii expression clearly exceeded class II antigen expression. Class II antigens were found in 20/37 adenomas and 62/123 carcinomas, mostly in a non-coordinate manner, following the above order. A detailed analysis of the expression patterns in normal and neoplastic colon epithelial cells revealed a closer association of HLA-DP with HLA-DQ than of HLA-DR with HLA-DP, or HLA-DQ.     

Progression of autoimmune damage in primary biliary cirrhosis: an immunohistochemical study

Aberrant MHC Class II antigen expression and the nature of the infiltrating lymphoid cells were studied by immunohistochemical techniques in liver biopsies from 37 patients with Primary biliary cirrhosis (PBC) (11 histological stage I, 13 stage II-III, 13 stage IV) and 15 patients with chronic non autoimmune liver disease. Bile duct epithelial cells expressed HLA-DR, DP and DQ antigens in biopsies from patients with early (Stage I) PBC and less frequently in the late cirrhotic phases of the disease (Stage IV); these observations support the hypothesis that induction of Class II antigens on epithelial cells may be involved in initiating autoimmune responses towards bile duct components. The presence of cytotoxic/suppressor T cells around the bile ducts in Stage I suggests a role for cell mediated destruction of the ducts at this early stage. The nature of the chronic inflammatory cell infiltrate in the portal tracts, periportal areas and lobular parenchyma does not establish the mechanism(s) involved in disease progression. However, the lack of Class II antigen expression on hepatocytes is compatible with the hypothesis that hepatocellular damage is non-specific and may be secondary to the initial bile duct injury.     

MHC antigen expression in sequential biopsies from cardiac transplant patients--correlation with rejection

Class I induction on the myocardium of transplanted heart was investigated with regard to its temporal relationship to rejection episodes, how it is affected by anti-rejection therapy and whether it is dependent upon the presence of a T cell infiltrate in the biopsy. Sequential cardiac biopsies (total 114) from 11 patients from the time of transplant to 1 year after transplant were studied using immunocytochemical techniques. The effect of different immunosuppressive regimens on MHC antigen expression was also studied. All the biopsies diagnosed as showing rejection for the first time showed induction of Class 1 on the myocardium with 79% during subsequent rejection episodes. Class I induction was associated with a leucocyte infiltrate, not always containing T cells, and disappeared in 47% of biopsies taken 3-4 weeks after treatment with steroids and/or ATG. Increased expression of Class II, in particular DQ antigens on interstitial structures, paralleled Class 1 induction. MHC antigen expression returned to normal in 8/9 patients, at 1 year after transplant. Different immunosuppressive regimens affected the number of biopsies showing Class 1 induction on the myocardium. Our results suggest that in clinical heart transplantation class I induction is related to the rejection process.     

Distribution of Human Leukocyte Antigen-ABC and -D/DR Antigens in the Unfixed Human Testis

ABSTRACT: The distribution of the major histocompatibility complex (MHC) antigens in the unfixed human testicle was studied by indirect immunofluorescence. Three murine monoclonal antibodies to the common determinants of class I MHC antigens (human leukocyte antigen [HLA]-ABC) and three against class II MHC antigens (HLA-D/DR antigens), respectively, were utilized. No class I MHC antigens were identified on developing testicular germ cells including spermatozoa, but interstitial cells between the seminiferous tubules (including Leydig cells) and blood vessel endothelium expressed the antigen. Class II MHC antigens were not found on any cells within the seminiferous tubules. However, the class II antigen was identified on dendritic-like cells between the seminiferous tubules and on vessel endothelium, although its expression was expectedly limited. These findings indicate that human testicular germ cells express minimal or no MHC antigens.     

Invariant chains with the class II binding site replaced by a sequence from influenza virus matrix protein constrain low-affinity sequences to MHC II presentation

Presentation of antigenic peptides by MHC II molecules is required to initiate CD4 T(h) cell responses. Some peptides, however, because of low affinity for MHC II, are not efficiently presented. A segment of the MHC II chaperon molecule, invariant chain (Ii), is known to bind early in biosynthesis with low affinity to the peptide binding groove. Here we have exploited the properties of Ii to manipulate the MHC II-loading pathway and to present low-affinity sequences. We used a deletion mutant of Ii where the promiscuous binding site to MHC II, which is adjacent to the groove binding segment, was deleted. A recombinant Ii (rIi) chimera, derived from this construct, was made in which the class II binding segment was exchanged for wild-type or single amino acid substitution variants of an HLA-DR1-restricted sequence from influenza matrix protein (MAT), which leads to MHC II allotype-specific binding. This rIi was expressed in antigen-presenting cells (APC) and introduced the MAT sequence into the MHC II-processing pathway. As expected, rIiMAT elicited antigen-specific, DR1-restricted T cell cytokine production and proliferation. Significantly, rIiMAT, that binds the HLA-DR4 allele with low affinity, elicited DR4-restricted IL-2 production but not proliferation. In contrast, exogenously provided MAT peptide failed to elicit any responses from DR4-restricted T cells. Compatible results were obtained with a single amino acid substitution variant (MAT(T)), which binds with high affinity to DR4 but low affinity to DR1. We conclude that loading of MHC II with antigenic peptides from endogenously synthesized rIi chimeras allows presentation of low-affinity sequences that cannot be presented if provided exogenously as peptides. Ii fusion proteins containing low-affinity antigenic sequences might be useful for vaccination with tumor antigens to overcome deficiencies in antigen presentation.     

MHC class II antigen expression in human vascular smooth muscle cells is induced by interferon-gamma and modulated by tumour necrosis factor and lymphotoxin

Arterial smooth muscle cells (SMC) express major histocompatibility complex (MHC) class II antigens in experimental vasculitis and in the human atherosclerotic plaque. We have therefore studied the regulation of expression of MHC antigens in cultured human arterial SMC, using immunofluorescence, radioimmunoprecipitation and a quantitative cell-surface immunoradiometric assay. SMC expressed class I, but not class II, antigens on their cell surfaces under basal conditions. Treatment of SMC with recombinant or natural interferon-gamma (IFN-gamma) induced expression of class II antigens in the following order of intensity, DR greater than DP greater than DQ. HLA-DR protein in SMC showed the same MW as that synthesized by B-lymphoblastoid cells. Antibodies to IFN-gamma blocked all HLA-DR-inducing activity in mixed leucocyte reaction (MLR) supernatants and PHA-stimulated peripheral blood mononuclear cell (PBMC)-conditioned media, indicating that IFN-gamma is the only lymphokine secreted under these conditions that is capable of de novo induction of HLA-DR expression in SMC. Treatment of SMC with recombinant human tumour necrosis factor-alpha (TNF) or lymphotoxin (LT) did not per se induce class II antigen expression. However, both TNF and LT substantially enhanced IFN-gamma-induced expression of HLA-DQ while decreasing that of HLA-DP. TNF, but not LT, increased HLA-DR expression. Also, in dermal fibroblasts, IFN-gamma-induced HLA-DP expression was significantly inhibited in the presence of TNF. These data demonstrate that TNF and LT differentially modulate IFN-gamma-induced MHC antigen expression in mesenchymal cells. The fact that SMC can express MHC class II antigens suggests that this cell type may serve as an accessory cell in the initiation of the immune response.     

Depletion by monolayer binding of specific precursors of antibody-forming cells directed against cellular antigens

Eight cases of the recently reported ''primary mediastinal clear cell lymphoma of B-cell type'' (Möller et al., 1986) were examined immunohistologically for the expression of cytoplasmic and/or surface antigens of MHC class I and II with mAbs directed against framework determinants of HLA-A,B,C (W6/32; B9.12.1), HLA-DP,DR,DQ (2.06), -DQ (Leu 10; Tü22), -DR (Tü34) gene products, and with mAbs specific for beta 2-microglobulin (BBM-1) and the HLA-D associated invariant chain (Vic-Y1). Besides the reported Ig-deficiency, the neoplastic B-cells of 7/8 tumours have variable defects in MHC antigen expression. Three lack both class I and class II antigens, one tumour lacks class I antigens but expresses HLA-DQ and -DR on the majority of neoplastic cells, three others contain varying proportions of MHC-antigen deficient tumour cells. The expression of Ii is closely correlated with HLA-D(R) expression and its antigenic sites are strictly located in the cytoplasm. Against the background of current knowledge, the variable and occasionally severe defects in MHC antigen expression within the herein presented series of B-cell lymphomas suggest that this unusual feature might be another characteristic of a novel lymphoma type.     

Expression of class II MHC gene products by macrophages in human uteroplacental tissue.

The expression of class II MHC determinants by fetal and maternal macrophages in human uteroplacental tissues was examined with monoclonal antibodies directed against HLA-DR, DP and DQ antigens. Maternal macrophages in early and full-term pregnancy decidua were HLA-DR positive, and a substantial proportion also expressed DP and DQ antigens. Fetal macrophages in chorionic villous stroma in first-trimester pregnancy were rarely HLA-DR positive, and DQ and DP antigens were never expressed. In term placental tissues, groups of villous stromal macrophages were HLA-DR positive, as were fetal macrophages within amniotic mesenchyme. In contrast, DP and DQ antigens were detected on a small minority of fetal macrophages in term placental tissues. DP and DQ antigens were not detected in the absence of DR antigens.     

Class II HLA antigen expression in familial polyposis coli is related to the degree of dysplasia

Class II HLA antigen expression was studied in 30 polyps from 3 patients who were diagnosed with familial polyposis coli. The highest levels of this expression were associated with the most severe grades of dysplasia (p less than 0.00001), the sequence of positivity being HLA-DR greater than DQ greater than DP. No association was observed between the expression of these antigens and the presence of a specific inflammatory leukocytic infiltrate. Our results imply that HLA class II molecule expression is somehow related to malignant transformation in familial polyposis coli in accordance with the adenoma-dysplastic adenoma-adenocarcinoma sequence. Thus these antigens may be useful markers to tumoral progression.     

Phenotypic expression of histocompatibility antigens in human primary tumours and metastases

HLA class I and II expression was studied on 244 (177 primary and 67 metastatic) solid human tumours of different origin. Alkaline immunophosphatase (APAAP) and immunoperoxidase were used on cryostatic sections to stain MHC antigens. Monomorphic MoAbs were used against class I heavy chain, 2-microglobulin, DR, DQ and DP molecules.Class I expression was homogeneous on colon, melanoma and epidermoidal primitive tumours. Loss of HLA class I antigens was more frequent on basal cell carcinomas and sarcomas and was related to tumour differentiation on larynx carcinoma. Class I expression was heterogeneous on breast, larynx and stomach primitive neoplasias. Class I negative tumours were more frequent on metastatic than on primitive melanomas. Divergence of class I between primary tumours and autologous metastases was observed on melanomas, larynx and colorectal carcinomas.Class II expression was heterogeneous on all tumours and in a large number of cases was associated with high intensity of leukocytic infiltrate. HLA-DR expression was higher than HLA-DP and HLA-DQ (DR>DP>DQ) and was related to tumour progression. Four human tumour cell lines were modulated with recombinant interferon- for HLA class I and II antigens. Different HLA profiles were obtained: increased class I and II expression, increased class II or a low response.Finally, class I genes from 22 tumours were compared with autologous normal cells by Southern blot analysis: 12 tumours were class I positive and 10 negative. No clear differences in RFLP were observed that could be associated with class I rearrangement. The results are discussed in relation to the role that histocompatibility antigens may play in tumour progression and invasiveness.     

Regulation of genes for HLA class II antigens in cell lines from patients with severe combined immunodeficiency

HLA Class II-negative severe combined immunodeficiency (SCID) results from a congenital defect characterized by an absence of HLA Class II antigens. Patients with the disorder have no HLA-DR, DQ, or DP antigens or mRNAs in their peripheral-blood lymphocytes. The affected gene is a recessive, transacting regulatory gene that controls the expression of Class II genes. We studied the regulation of HLA Class II gene expression with the use of established Epstein-Barr virus-transformed B-cell lines and skin fibroblast lines from a group of patients with SCID. Lymphoblastoid B-cell lines from the patients contained no mRNA for HLA-DR, DQ, and DP alpha and beta polypeptides, but did express mRNA for the HLA-associated invariant chain, which is normally coregulated with HLA Class II antigens. In the B-cell line from one patient, a very low amount of DR mRNA could be detected, indicating some heterogeneity in SCID. The lymphokine gamma-interferon, a strong inducer of Class II genes in a variety of normal cells, did not restore Class II gene expression in any of the SCID B-cell lines. More important, gamma-interferon was unable to induce any Class II mRNA in fibroblast lines from patients with SCID, in contrast to the efficient induction observed in normal fibroblasts. The invariant-chain gene, however, was induced in the SCID fibroblasts, confirming a unique uncoupling in the regulation of invariant and Class II genes. Thus, the genetic defect in patients with SCID affects not only the B-cell lineage but also the inducible expression of HLA Class II genes that is normally observed in Class II-negative cells, such as fibroblasts. This unresponsiveness to gamma-interferon in vitro indicates that patients with SCID will not respond to treatment with this lymphokine. Our data also increase understanding of the normal mechanisms regulating the genes for the HLA Class II cell-surface glycoproteins.     

Methimazole blocks Graves' IgG but not interferon-gamma HLA-DR expression by thyroid cells

We have expanded our early observation that a potent Graves' IgG preparation induces HLA-DR expression on thyroid cells, by showing that four randomly-selected Graves' IgG's were also capable of inducing DR antigens on thyroid cells. The effect of Graves' IgG was specific to thyroid cells, as it did not induce MHC Class II molecule expression on endothelial cells whereas interferon-gamma and immune complexes did so. The anti-thyroid drug methimazole was capable of rapidly reducing Graves' IgG-induced DR expression but to a much lesser extent than brought about by interferon-gamma. We conclude that Graves' IgG propagates thyroid-specific autoaggression by continued induction of DR antigens and than an important means whereby methimazole brings about remission is by reducing this induction.     

Presentation of a soluble bacterial antigen and cell-surface alloantigens by large granular lymphocytes (LGL) in comparison with monocytes.

The ability of large granular lymphocytes (LGL) to function as antigen-presenting cells (APC) in the proliferative response to the soluble bacterial antigen streptolysin O (SLO) was investigated. Despite the fact that a subset of LGL isolated by sorting peripheral blood lymphocytes with the B73.1 monoclonal antibody on a fluorescence-activated cell sorter (FACS-IV) expressed MHC Class II molecules of the DP, DQ and DR subregion loci, presentation of SLO by LGL was not demonstrated. Thus, T-cell populations containing LGL but carefully depleted of monocytes, isolated either by sorting using the FACS-IV or by SRBC-rosetting, were unresponsive to antigenic stimulation with SLO. Application of exogenous interleukin-1 to FACS-IV-isolated LGL-containing T-cell populations did not elicit presentation of SLO by the LGL. In vitro activation with phytohaemagglutinin and interleukin-2, which induced Class II expression in T-cell populations, resulted in an increased expression of Class II molecules of the DP, DQ and DR specificities on LGL. Although such activated T-cell and LGL populations were incapable of presenting SLO to freshly isolated antigen-non-responsive T cells, both activated populations were able to act as stimulators in an allogeneic mixed lymphocyte reaction. The ability of highly Class II-positive activated LGL to present membrane-bound antigens suggests that their inability to present a soluble antigen may be related to the absence of effective antigen sequestration and/or processing mechanisms.     

Expression of MHC class I and class II antigens in colonic carcinomas

Malignant and non-malignant ('normal') colonic tissues from patients with colonic carcinoma were examined for the expression of MHC class I and class II antigens by immunoenzymatic staining using monoclonal antibodies. The amount of class I antigen as detected by 2 monoclonal antibodies, FMC 16 or W6/32 was clearly diminished in 11 of 14 tumours when compared to the amount present on 'normal' colonic tissue from the same individual. The loss of class I antigen did not correlate with tumour stage or differentiation. The reactivities of FMC 16 and W6/32 with these tissues were not identical, which indicates that the 2 monoclonal antibodies may recognize different epitopes on the HLA class I molecule. Class II antigens were absent from 'normal' colonic epithelium but were present on 20 of 28 tumours, with DR being detected more often than DP, and DQ found only on 4 of 28 tumours. When present, staining for class II antigens was heterogeneous within the tumour, in that all tumour cells did not stain equally. DR and DP antigens were found more often on moderately or poorly differentiated adenocarcinomas and on stage B, C and D tumours in that order of frequency. Thus tumours with a better prognosis were less likely to express DR and DP. The expression of DQ was unrelated to staging or differentiation.     

MHC class II antigen and immunoglobulin expression in spontaneous phenotypic variants of the Burkitt's lymphoma cell line Namalwa

Phenotypic variant sublines of the Burkitt's lymphoma cell line Namalwa were examined with cDNA probes for the different MHC class II beta chain genes and with monoclonal antibodies specific for the corresponding cell surface antigens (DP, DQ and DR antigens). Expression of MHC class II antigens in the Namalwa sublines (known as CSN/70, IPN/45, PNT and KN2) was compared with that of the B-lymphoblastoid cell line DEW1, which is identical to Namalwa in DR allotype (DR 2,4). There were markedly different levels of expression of MHC class II antigens among the cell lines: in DEW1 and the Namalwa KN2 subline DP, DQ and DR antigens were expressed on almost all the cells. On the PNT and IPN/45 sublines, DR antigens were expressed on all the cells, and DP and DQ antigens were expressed at detectable levels on only a proportion of cells. On CSN/70, there was weak expression of DR antigens on a minority of cells and no detectable expression of DP and DQ antigens. When examined with MHC class II-specific cDNAs, restriction fragment patterns of DNA were identical for all the cell lines, suggesting that they had structurally identical MHC class II genes. In the Namalwa cell lines the synthesis of Ig and the expression of MHC class II antigens were coordinately regulated.     

Presentation of soluble antigen to human T cells by products of multiple HLA-linked loci: analysis of antigen presentation by a panel of cloned, autologous, HLA-mutant Epstein-Barr virus-transformed lymphoblastoid cell lines

Epstein-Barr virus-transformed human B lymphoblastoid cell lines (EBV-LCL) can present soluble antigens to antigen-primed T lymphocytes. In this study, we used HLA antigen-loss mutants of an EBV-LCL line (LCL 721) to demonstrate that the presentation of a soluble antigen from Candida albicans (CAN) by EBV-LCL to primed T cells can be restricted by multiple HLA determinants. Haplotype-deletion mutants that contained only the maternal or only the paternal HLA-haplotype were used to demonstrate the preferential role of autologous HLA antigens in presenting soluble antigens to Candida-primed T cells from the donor of LCL-721, and to T cells from her mother and father. Immunoselected mutants of LCL-721 showing a variety of distinct phenotypes that are deficient in HLA-DR, DQ, or DP antigen expression were tested as antigen-presenting cells. The antigen-presenting ability of these class II deficient EBV-LCL variants weakened with progressive loss of class II HLA determinants expressed on the cell surface. Our study, therefore, provides evidence for multiple HLA restriction determinants, including HLA-DR, DQ, and DP. Furthermore, LCL lacking all HLA-DR, DQ, and DP expression because of homozygous deletion of these MHC class II genes still presented CAN and Tetanus toxid (TET), although to a much lesser degree than presented by LCL-721. This suggests that determinants other than DR, DQ, and DP which are expressed on these EBV-LCL may also function as restriction elements for the proliferative T-cell response to soluble antigens.     

Expression and function of HLA-DR3 and DQ8 in transgenic mice lacking functional H2-M

H2-M or HLA-DM are non-classical class II molecules encoded by the MHC and play an important role during antigen presentation. They catalyze exchange of CLIP (Class II-associated invariant chain peptide) or other low-affinity peptides bound to class II molecules for peptides capable of more efficient binding. The phenotype of mice lacking H2-M is determined by the allotype of the MHC class II molecules expressed. In general, H2-M deficiency does not affect the surface expression of mature class II molecules. The class II molecules in such cases predominantly contain CLIP in their peptide-binding groove. In some mice strains, H2-M deficiency results in defective CD4+ T-cell development accompanied by defective responses to conventional antigens and superantigens. Even though the HLA class II molecules show similar dependency for HLA-DM for presenting antigens in vitro, their interaction in vivo is not known. By using transgenic approach we show here that DQ8 and DR3 are expressed at normal levels in H2-M-deficient mice and the CD4+ T-cell development is unaltered. However, the ability of DQ8 molecules to present peptide antigens is compromised in a H2-M-deficient state. Presentation of exogenous bacterial superantigens by both DQ8 and DR3 is unaffected in H2-M-deficient mice. Unexpectedly, Staphylococcal Enterotoxin B-induced systemic IFN-gamma production was significantly higher in H2-M-deficient DQ8/DR3 transgenic mice and these mice were susceptible to SEB-induced toxic shock at doses that are non-lethal to H2-M-sufficient counterparts.