Relationship Between HLA Antigens and Infectious Agents in Contributing Towards the Development of Graves' Disease

Graves' disease (GD) is an autoimmune thyroid disorder which is associated with the human leucocyte antigens HLA-DR3 and DQA1^* O501 in Caucasians. We have explored the possibility that some patients with certain HLA specificities develop anti-HLA antibodies which are correlated with environmental factors that may contribute to the development of GD. We studied 40 GD patients and 157 healthy individuals (controls). Serology was used to type HLA-A, -B, -Cw, and -DR antigens. The frequencies of these antigens in relation to lymphocytotoxic anti-HLA-A-B-Cw-DR antibodies and two environmental factors (Yersinia enterocolitica and Coxsackie B virus) were determined. The frequencies of HLA-B15, -B21 and DR3 antigens were increased, whereas HLA-DR5 antigen was decreased in GD patients. A significant association between HLA-DR3 antigen and lymphocytotoxic antibodies was observed, i. e., IgGs from GD patients were cytotoxic to HLA-DR3+ normal B cells. Following absorption with Yersinia enterocolitica or Coxsackie-B-virus, only Coxsackie-B virus completely inhibited the lymphocytotoxic reactions against HLA-DR3⁺ B cells. Besides confirming the association of HLA-DR3 with GD, this study also suggests the role of Coxsackie-reative HLA-DR3 antibodies as contributing factors to the pathogenesis of the disease.     

Non-polarized cell surface expression of HLA-A,B,C and HLA-DR antigens in Graves' thyroid follicle cells.

The intrathyroidal distribution and cell surface location of HLA-A,B,C and HLA-DR antigens was studied in polarized thyroid follicle cells from Graves' (n = 11) and normal (n = 3) thyroid tissue, using light and electron microscopy. Cryosections and isolated, open follicle segments were incubated with monoclonal antibodies against HLA-A,B,C and HLA-DR antigens and with patient sera containing autoantibodies against the microsomal antigen/thyroperoxidase. Immunoreactivity for HLA-A,B,C and HLA-DR on isolated thyroid follicle cells was frequently detected in Graves' disease, but absent in normal glands. There was a large variation in the immunolabelling between follicles as well as between different glands. Both HLA-A,B,C and HLA-DR immunoreactivity were detected on the apical and the basal surface of the follicle cells. Microsomal antigen/thyroperoxidase immunoreactivity was restricted to the apical cell surface. In contrast to normal tissue, HLA-DR positive cells with a dendritic or macrophage-like morphology were frequent in Graves' tissue. These cells adhered directly to the basal surface of isolated follicle segments. We conclude that HLA antigens are, unlike thyroid-specific plasma membrane constituents, expressed in a non-polarized manner at the surface of the follicular epithelium. These observations might have implications on the immune recognition of thyroidal autoantigens in Graves' disease.     

HLA-D and -DR antigens on human amniotic fluid cells. II. Heterogeneous expression of HLA-DR and other cell surface markers

To evaluate further the feasibility of HLA typing for prenatal diagnosis, we tested human amniotic fluid cells (AFC), known to express HLA-A, -B, and -C antigens, for the presence of HLA-DR antigens using type-specific antisera in the microcytotoxicity assay and a monoclonal antibody directed against the common HLA-DR structure (cDR) in indirect immunofluorescence. Prenatal typing of HLA-DR on AFC in the microcytotoxicity test was possible in only one out of eight families studied. The detected DR2 antigen was confirmed by postnatal typings of cord blood lymphocytes. Thereafter, 23 different AFC cultures were tested with monoclonal antibodies in indirect immunofluorescence. Only six cultures were partially positive (23-35% fluorescent cells) with the monoclonal cDR antibody while all AFC cultures demonstrated strong positive fluorescence (68-100%) with a monoclonal antibody against the common HLA-A, -B, and -C structure (cHLA). These data suggest that only a small subpopulation of AFC expresses class II (HLA-DR) antigens in contrast to the nearly ubiquitous expression of class I (HLA-A, -B, and -C) antigens. Furthermore, the heterogeneous expression of cell surface antigens within the various AFC cultures was substantiated with monoclonal antibodies directed toward cell surface antigens of the OKT, OKM, and Lyt series that have been found to be characteristic for subpopulations of lymphoid and hemapoetic cells. Thus, at present, HLA-DR typing is not reliable for prenatal diagnosis.     

Phenotypic heterogeneity of HLA products on human melanoma cells

The expression of class I and class II HLA antigens has been studied on primary and metastatic human melanomas, and on cell clones derived from the latter. A panel of monoclonal antibodies and flow cytofluorometric analysis were used to evaluate the presence of HLA-A, B, C and -DR, DQ antigens on freshly isolated tumour cells. HLA class I antigens were present on 91% and 93% of primary and metastatic tumours, respectively. Sixty per cent of primary and 50% of metastatic melanomas expressed HLA-DR antigens, whereas 38% and 21% of cases were positive for HLA-DQ. A marked heterogeneity was evident among primary and metastatic lesions for expression of class I and II antigens. Similar findings were obtained by analysing the phenotype of melanoma clones which indicates that a marked antigenic heterogeneity for class I and II HLA antigens occurs even among clones isolated from short-term cultures of metastatic melanomas.     

Enhanced expression of HLA antigens and beta 2-microglobulin on interferon-treated human lymphoid cells.

Human leukocyte interferon increased the expression of HLA-A, B antigens and beta 2-microglobulin on two lines of human lymphoblastoid cells and on peripheral blood lymphocytes. No effect was observed on the expression of HLA-DR antigens.     

Expression of HLA-like structures on a permanent human tumor line PC-93

A permanent human tumor-line PC-93 was HLA allotyped with the use of a complement-dependent cytotoxicity test supplemented with absorption experiments. In comparison with the peripheral blood lymphocytes of the patient, whose adenocarcinoma of the prostate was the origin of PC-93, remarkable differences were observed. The tumor line lacked the expression of the genetically appropriate HLA-A1, A2 and B15 antigens. Instead of these antigens, cross-reactive HLA antigens seemed to be expressed on PC-93, i.e. All, A28 and B17. Absorption experiments showed that the antibodies reactive with the neo-HLA antigens were not the same as those reactive with the specific HLA antigens. This alien expression of HLA-like structures was specific for the patients' prostatic tumor line, as EB-virus transformed B-lymphocytes of the patient expressed the genetically appropriate HLA antigens.     

Techniques used to define human MHC antigens: serology

The classical, routine test employed for definition of HLA antigens expressed in humans (tissue typing) is the complement-mediated cytotoxicity assay developed by Terasaki and McClelland in the early 1960s. In both healthy persons and patients, the assay target cells are usually lymphocytes obtained from peripheral blood, but when typing cadaver donors, splenic or lymph node lymphocytes can be used. HLA-A, B, Cw (class I) antigens are expressed on all nucleated cells while HLA-DR, DQ (class II) are restricted to B lymphocytes and immune activated cells. Tissue typing has been achieved using culture cells from amniocentesis and typing of cell lines is possible with small modifications to the standardised cytotoxicity assay. Usually, target cells are incubated under oil with typing antisera at 22 degrees C in a 60- or 72-well Terasaki tray. After 30 min rabbit serum is added as a source of complement. After a further 60 min incubation the test is stained. A positive reaction results in target cell death. There are local variations to this test. Automation of the assay is now commonplace, from reagent dispensing to automated reading of finished assay. The use of antibody-coated magnetisable microspheres has enabled separation of pure B lymphocyte samples for class II typing and has reduced incubation times through antigen modulation. It is possible to define antibodies to HLA antigens in the same assay using target cells with known HLA phenotypes.     

HLA antigens in hemophiliacs A with or without factor VIII antibodies in a Venezuelan Mestizo population

Twenty-eight unrelated hemophilia A patients, seven of them with anti-factor VIII antibodies were typed for HLA-A, B, C antigens and 25 of them for HLA-DR. The results show a significant difference in HLA-DR4 frequency between hemophiliacs with antibodies who lack this antigen and hemophiliacs without antibodies, in whom HLA-DR4 is increased as compared to a healthy control series. This data suggests that DR4 may be associated with a factor preventing anti-factor VIII immunization.     

Distribution of blood group antigens in adult pancreas

Numerous blood group antigens are present in different human tissues where they appear as immunological markers of certain structures. The Pr antigen is the only antigen present in the islet of Langerhans. The A, B, H and Lewis antigens are present in the centro acinar cells and the Pr and Lewis antigens are found on the membrane of the pancreatic ducts. A, B, H and Pk antigens are expressed in the capillaries. These kinds of studies could be important for the transplantation of the endocrine pancreas.     

HLA linked immune response to S and pre-S2 gene products in hepatitis B vaccination

In order to detect a possible HLA linked genetic control of human immune responses to hepatitis B virus, forty healthy adult persons of the same age typed for HLA-A, -B and -DR antigens, were vaccinated against virus hepatitis B and sequentially tested for anti-HBs and anti-pre-S2 antibodies. They received three injections of Hevac-B Pasteur vaccine, the second 1 month and the third 3 months after the first. Following the third immunization, 38 individuals (95%) had a protective level of anti-HBs antibodies and 17 (42.3%) had a positive level of anti-pre-S2 antibodies. HLA-A11 antigen was significantly more frequent (pc = 0.007) among anti-HBs high responders than low responders. In addition, anti-HBs high responders were more frequently HLA-DR1, and less frequently HLA-DR4 and DR7 positive; corrected values, however, were not significant. Anti-pre-S2 high responders showed an apparent increase of HLA-B7, B14 or DR3 antigens, when compared to low responders (pc not significant).     

Helix pomatia agglutinin (HpA) affinity chromatography: the isolation of pure B and T cell populations and their use for the routine HLA-DR (Ia) serology

The anti-A1 agglutinin (HpA) of the albumin gland of the snail Helix pomatia binds specifically to neuraminidase-treated T lymphocytes. After its immobilization on Sepharose-6MB an affinity matrix is obtained which is able to separate T and B lymphocytes. In 40 independent experiments enriched T and B cell populations were isolated either by this HpA affinity technique or by E-rosettes incubated with sheep red blood cells for 2 h or 15 h. The results showed that with the HpA affinity matrix within 3 h a highly enriched B cell population is isolated with a purity of 80% and a yield of 65%. Since the separation is done within a short period of time and under gentle conditions the percentage of dead cells is not more than 5%, which is a requirement when the cells are to be used for immunofluorescence studies of surface markers and serological determinations involving microscopical scoring. Typing for HLA-A, B, C antigens and for HLA-DR alloantigens can be done on the same day by cytotoxicity assay. The method also provides a clear-cut distinction between the HLA-A, B, C antibodies and the HLA-DR antibodies.     

HLA-DR and HLA-A, B, C typing of human fetal tissue

In anticipation of clinical trials of fetal pancreas transplantation we have investigated the feasibility of performing HLA-DR and HLA-A, B, C typing on fetal lymphoid cells other than PBL. Using the standard NIH microcytotoxicity test modified for HLA-DR typing it was possible to demonstrate HLA-DR antigens on subpopulations of bone marrow cells and splenocytes but not on thymocytes or hepatocytes. In contrast, HLA-A, B, C antigens could be detected on all four tissues. Excellent HLA-DR typing, confirmed by maternal typing, was obtained for 19 fetuses (14 to 23 weeks old) using bone marrow cells isolated by two-fold purification on discontinuous Percoll buoyant density gradients. Similar purification of splenocytes resulted in weak reactions with anti-DR sera; however, adherent splenocytes recovered from nylon wool columns proved to be primarily DR-bearing and also provided excellent DR typing. As a corollary to these results, non-adhering splenocytes depleted of DR-bearing cells were ideal for HLA-A, B, C typing since spurious reactions due to DR antigens were greatly diminished, whereas strong specific reactions were obtained with anti-HLA-A, B, C sera. Despite weaker reactions with HLA-A, B, C antisera obtained for thymocytes, reliable HLA-A, B, C typing could be obtained when results from thymocytes were evaluated together with typing from bone marrow cells or splenocytes. The possible benefits of fetal HLA typing for fetal pancreas transplantation are discussed.     

The differential diagnosis of hairy cell leukemia with a panel of monoclonal antibodies

A panel of monoclonal antibodies reactive with normal human lymphoid cells and with hairy cells has been applied to the immunocytochemical analysis of hairy cell leukemia. Staining was performed by immunoenzymatic methods on frozen sections of bone marrow trephines and extramedullary tissues and on cell smears. Hairy cells reacted with antibodies against HLA-DR, leukocyte common antigen, B-cell antigens (antibodies To15 and B1) and with three anti-hairy cell monoclonal antibodies (S-HCL3, HC1, and HC2). Neoplastic cells in other B-cell lymphoproliferative disorders also expressed HLA-DR, leukocyte common, and B-cell antigens but were consistently negative for the antigen detected by monoclonal antibody S-HCL3. Furthermore, hairy cells differed from other neoplastic B-cells in that they were unreactive with monoclonal antibodies against C3b receptors, anti-Leu-1, Tü1, Tü33, and lacked a meshwork of dendritic reticulum cells. These findings establish a distinctive antigenic phenotype for hairy cell leukemia and indicate that it may be diagnosed reliably by immunoenzymatic labeling of tissue sections or cell smears.     

Human liver allograft acceptance and the "tolerance assay". II. Donor HLA-A, -B but not DR antigens are able to trigger regulation of DTH

In our initial study of liver transplant recipients using the trans vivo delayed-type hypersensitivity (DTH) assay, we found that in donor derived B-LCL or sonicates of donor leukocytes triggered linked suppression of the response to recall antigens tetanus toxoid (TT) or Epstein-Barr virus (EBV). Since both donor antigen sources contain HLA class I and class II proteins, we wished to determine which donor HLA proteins were responsible for the linked suppression effect. PBMC from four liver transplant recipients with donor-specific DTH regulation were studied. Surprisingly, we found that single donor HLA-A or B antigens (4/4 patients) but not single HLA-DR (0/4) donor antigens triggered linked suppression of DTH. A dose response study of two patients revealed that donor-type HLA-DR antigens (0.5-500ng) were not capable of triggering any linked suppression; however, as little as 500pg of donor-type HLA-class I protein triggered linked suppression of DTH response to a recall antigen. These findings may have implications for the differential impacts of class I vs class II mismatching in organ transplantation. On a practical level, they indicate that soluble HLA-A and B antigens are the proper choice for detection of DTH regulation as part of a "tolerance assay" in human liver transplant recipients.     

Sequential induction of MHC antigens on autochthonous cells of ileum affected by Crohn''s disease.

Changes were examined in the expression of Class I and II major histocompatibility complex (MHC) antigens by autochthonous cells of the terminal ileum affected by Crohn's disease. The study was based on the analysis of transmural specimens from terminal ileum segments obtained in the course of ileocolectomy for colon cancer and Crohn's disease. Serial sections were immunostained using monoclonal antibodies directed against monomorphic determinants of HLA-A,B,C, DR, DP, DQ, and the invariant chain (Ii) associated with Class II molecules. Compared with the normal state, the only change in Class I antigen expression occurring in Crohn's disease was the induction of HLA-A,B,C antigens in lymphatic endothelium. Changes in Class II antigen expression were more substantial. Enhancement of HLA-DR expression was found in enterocytes; DR induction was observed in glial cells of the visceral nervous plexus and in venular and venous endothelium. HLA-DP and DQ antigens were induced in enterocytes, glial cells, and capillary and venular endothelium, although this induction was restricted to areas of moderate or high inflammatory activity. The tissue distribution of Ii closely resembled that of HLA-DR, although this association was not strict: on the one hand, arterial endothelium contained low amounts of Ii in the absence of DR antigens; on the other hand, glial cells expressed Class II molecules in the absence of Ii. The extent of local enhancement/induction of MHC antigens was positively correlated with the local density of the cellular infiltrate. These data suggest that altered MHC antigen expression by autochthonous structures might be mediated by factors released from the lymphohistiocytic infiltrate, which is itself attracted by an unknown signal. In conjunction with an unknown antigen, the enhanced expression of Class II antigens might trigger an autoaggressive immune response.     

Planned immunization of human volunteers with HLA-DR incompatible lymphocytes

Aiming at the production of anti HLA-DR test sera, eight healthy human volunteers were immunized by repeated intradermal injections of lymphocytes which were selected to be incompatible for one HLA-DR antigen, and matched as well as possible for HLA-A,-B,-C antigens. One out of 3 recipients immunized exclusively against HLA-DR produced lyrnphocytotoxic HLA-DR antibodies. The remaining 5 recipients were immunized against 1 or more HLA-A,-B,-C antigens in addition to one HLA-DR antigen. After 3 immunizations, 3 of these reacted with strong HLA-A or -B antibody production; however, only one showed a parallel anti HLA-DR antibody response detectable by complement dependent lymphocytotoxicity.
Testing of the recipient sera in the antibody dependent cell-mediated cytotoxicity (ADCC) assay revealed that 6 of the 8 recipients did react early to the immunizations with HLA specific antibody production. However, in spite of repeated booster injections it was not possible to obtain more than the above-mentioned 2 sera with HLA-DR antibodies strong enough to react in the lymphocytotoxicity microtechique.     

Expression of HLA-DR antigens by colonic epithelium in inflammatory bowel disease

The expression of HLA-DR and HLA-A,B,C antigens by human colonic epithelium has been examined in tissue sections of patients with inflammatory bowel disease using an immunohistological technique. Colonic epithelial cells from all 21 control subjects with histologically normal colonic mucosa were HLA-DR-. In contrast, in nine of 13 patients with active ulcerative colitis and 11 of 12 with active Crohn's disease the epithelium of involved colonic mucosa was HLA-DR+. HLA-DR antigens were found on the epithelium of only one of six patients with ulcerative colitis in remission and one of three with inactive Crohn's disease. Moreover, these antigens were not present on the epithelium of non-inflamed colonic mucosa in two patients with Crohn's disease in whom adjacent involved mucosa showed strong epithelial reactivity. This difference between patients with active and those with inactive disease is highly significant (P less than 0.005). These findings provide further evidence of the importance of cell-mediated immune mechanisms in the pathogenesis of inflammatory bowel disease.     

Inhibition of HLA-DR antigen expression and of the allogeneic mixed leukocyte reaction by photochemical treatment

Treatment of human peripheral blood mononuclear cells with UVB light, the photosensitizing system 8-methoxypsoralen plus UVA light (PUVA), or hematopor-phyrin derivative plus visible light leads to an inhibition of their stimulatory capacity in an allogeneic mixed leukocyte reaction despite unaltered expression of HLA-DR antigens when tested immediately after irradiation. However, HLA-DR positive cells disappear among mononuclear cells in the interval between 4 and 8 h after treatment with either UVB, PUVA, hematoporphyrin derivative and light, or heating to 45°C. The expression of HLA-DQ, but not HLA-A,B,C, antigens, was similarly affected by these treatments. The significance of these results is discussed.     

Analysis of DR-like molecules on a marmoset Epstein-Barr virus-induced cell line using a monomorphic anti-human HLA-DR monoclonal antibody

Mouse anti-HLA D region-related (DR) monoclonal antibodies have been found to cross-react with peripheral blood leukocytes from one primate species, the common marmoset (Callithrix jacchus). Immunoprecipitates of radioactively labeled cells extracted from a marmoset Epstein-Barr virus-induced cell line were analyzed by one- and two-dimensional gel electrophoresis and compared with the DR antigens of a human lymphoblastoid B cell line. Two chains of estimated molecular weights of 34 000 (alpha) and 28 000 (beta), similar to the human alpha and beta chains, have been observed in marmoset immunoprecipitates. Additionally, a set of spots located in the same area as the set of invariant spots found in human HLA-DR antigens is shown by two-dimensional gel electrophoresis. Thus, cross-reacting anti-human HLA-DR monoclonal antibodies could be used to analyze the expression and the structure of marmoset DR-like antigens.     

The pattern of activation antigen expression on T-lymphocyte subpopulation in infectious mononucleosis.

The peripheral blood mononuclear cells of patients with infectious mononucleosis (IM) have been characterized by the determination of activation antigens using a panel of 11 monoclonal antibodies (MoAbs) belonging to 9 clusters. The activation of CD8+ peripheral blood mononuclear cells of IM patients was found to be determined by HLA-DR and CD45RO MoAbs. The antibodies of the CD30, CD40 and CD70 termed antigens also showed an increased expression as compared to the controls. In contrast, the CD25, CD69, CD71, and CD45 antigens were expressed at a low rate on the surface of the CD8+ cells. We found, on the other hand, a very low percentage of B lymphocytes in the peripheral blood, which reflects on the virus caused antigen modulation and on the effectivity of activated CD8 cells with the characteristic expression of the above outlined markers in IM. In six asymptomatic patients, the percentage of CD8+HLA-DR+, as well as of CD8+CD45RO+ cells proved to be lower than that in the active phase of the disease. The number of B cells showed normal value in the clinically asymptomatic cases.