Ankylosing spondylitis,HLA-B27 and Klebsiella. I. Cross-reactivity studies with rabbit antisera.

Sera from rabbits immunized with HLA-B27 lymphocytes showed increased activity against klebsiellal enterobacter antigens using immunodiffusion, bacterial agglutination (P less than 0.025), haemagglutination (P less than 0.001) and radiobinding assays (P less than 0.001). Immunoprecipitin lines were also produced by these antilymphocyte sera against extracts from Yersinia enterocolitica and Shigella sonnci microorganisms. Rabbit anti-klebsiella sera had lymphocytotoxic activity against HLA-B27 lymphocytes obtained from patients with ankylosing spondylitis (P less than 0.001). These results suggest partial cross-reactivity between some antigens found in several Gram-negative microorganisms and HLA-B27 lymphocytes.     

A double determinant immunoassay for HLA class I typing using serum as an antigen source

We have applied a double determinant immunoassay (DDIA) to HLA-A2,A28, and B13 typing, using serum as an antigen source. The results obtained show a correlation of 96% (B13) and 89.1% (A2,A28) with the results obtained by conventional HLA typing. Furthermore, the results obtained were highly reproducible, since testing of 18 sera on two occasions gave concordant results with all samples tested. The variation in the content of HLA-A2 antigens in sera taken at different times from a given donor was less than 5%. A sevenfold variation was found in the serum level of HLA-A2,A28 antigens: the highest level was found in the sera from HLA-A2,A28 donors and in decreasing order in HLA-A2 homozygous, HLA-A28 homozygous, HLA-A2 heterozygous, and HLA-A28 heterozygous donors. The results of this study indicate that the DDIA is a sensitive, simple, and reproducible procedure for HLA class I typing. The DDIA offers the following advantages in comparison with the conventional lymphocytotoxic assay: it provides information not only about the expression of a given alloantigen, but also about its level; it does not require viable cells, thus facilitating retrospective studies and typing of leucopenic patients; it eliminates variability of results caused by abnormal susceptibility of target cells to complement-dependent lysis.     

A reanalysis of the HLA-B7 cross-reactive group

Cross-reactivity between antigens of the HLA-B7 cross-reactive group (B7 CREG) was investigated by the serological analysis of 60 "broad" cytotoxic HLA antisera produced by pregnancy alone, the HLA typing of the antiserum donors and the identification of their immunizing antigens. Thirty-five sera, made in response to B7, covered (as a group) HLA-B7, B27, Bw42, Bw48, Bw54, Bw55, Bw56, Bw60 and Bw61 — the B7 CREG antigens. Bidirectional cross-reactivity occurred between the B7 antigen and B27, Bw55, Bw56 and Bw60 antigens but not between the major B7 CREG antigens B27, Bw22 and B40. HLA-B27 stimulated antisera included B7, B13 and Bw47 within their reaction range and Bw55/Bw56 cross-reacted with Bw42, Bw54, Bw57, Bw58, Bw62 and Bw63. Bidirectional cross-reactivity was observed between Bw55 and Bw57. Fourteen responders possessed an antigen cross-reactive with their immunizing antigen. These findings are discussed in relation to the sharing of determinants by pairs and "families" of HLA antigens.     

HLA and antinuclear antibody incidence in asbestos workers.

The incidence of antinuclear antibodies (ANA) in asbestos textile workers was compared with the results of HLA typing. HLA B8 antigen was more frequent in the ANA-positive group as compared to the ANA-negative group. The relative risk of this associati-n is 2.77 (p less than 0.02) without correction for the number antigens tested. A decrease in frequency of HLA A10 in workers with ANA was also found. The results suggest that the development of ANA under asbestos hazard may depend on inherited predisposition of which HLA B8 is a marker.     

Anti-lymphocytic antibodies in autoimmune chronic active hepatitis starting in childhood.

Anti-lymphocytic antibodies (ALA) have been described in a variety of autoimmune disorders. We have investigated the presence of ALA in autoimmune chronic active hepatitis (aCAH) starting in childhood. Using a modified Terasaki technique ALA were found in 17 of 18 patients with aCAH but in only one of 15 patients with alpha-1-anti-trypsin deficiency or Wilson's disease and three of 27 age-matched healthy controls (P less than 0.0005 for both). Sera from 12 patients with uncontrolled aCAH had significantly higher cytotoxicity values than sera from six children with inactive diseases (P less than 0.01). ALA were directed to T but not B lymphocytes and were not reactive with specific HLA antigens. No preferential killing was observed against CD4 or CD8 positive T lymphocytes. Characterization of ALA revealed them to be cold-reactive IgM. The possible role of ALA in aCAH is discussed.     

Identification of HLA-linked antigens by pregnancy-associated non-cytotoxic alloantisera

Non-cytotoxic sera obtained from post-partum primiparous and multiparous women were examined by a rosette inhibition technique for the presence of antibodies mediating blockade of human B lymphocyte Fc receptors. Selective activity was demonstrated against a panel of normal human B lymphocytes and lymphocytes from patients with chronic lymphocytic leukaemia (CLL). A pattern of specific activity was found in sera and in their IgG fractions, which was not accounted for by antibodies directed to known HLA-A, -B or -DR antigens. Several sera were identified with selective activity in this assay. As the results of testing sera in a direct binding assay correlated with those of the EA inhibition assay, and since EA inhibitory activity occurred in F(ab')2 fractions of sera, it is possible that these non-cytotoxic antibodies bind directly to B cell surface antigens. Sera may therefore have been identified which possess antibodies to hitherto undefined HLA antigens.     

HLA Determinants in Idiopathic Hemochromatosis

HLA-A and B antigens were defined in 154 unrelated idiopathic hemochromatosis patients. The study confirmed the highly significant positive association with HLA antigens A3 (corrected P less than 10(-10)) and B14 (corrected P less than 10(-9)). HLA-DR typing showed increased frequency of the specificity DRw6, which was frequently associated with the phenotype A3, B14 and antigen B14, suggesting linkage disequilibrium. This was borne out by PLT data.     

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-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.     

HLA and IgA deficiency in blood donors

We have identified 67 IgA deficient healthy blood donors in our region by systematic screening of 24,782 blood samples. HLA typing results on 36 of these donors indicated a significant association with both HLA-A1 and HLA-B14 antigens. The frequency of A29 and B8 antigens was also increased. However, B8 association may be secondarily involved due to linkage disequilibrium (e.g., A1-B8-DR3). The frequency of HLA-A1 and HLA-B8 antigens was increased in the group of IgA deficient donors who developed anti-IgA antibodies (53.9%) compared to those who did not (26.1%). Although the sample size appears to be too small to show a statistical significance (chi 2 = 2.76, 0.05 less than p less than 0.1), this is the first report to imply a possible HLA association with anti-IgA antibody formation by IgA deficient healthy individuals.     

中国人HLA-B27、B7、B13和B40的检测及其相互杂合与交叉的分析

目的　检测 1194例初诊怀疑患强直性脊柱炎 (AS)和其它关节性疾病患者的HLA B2 7、B7、B13和B40位点抗原的分布 ,并分析这些位点的相互杂合和其抗原的交叉反应性。方法　微量细胞毒反应法 ,在同一块反应盘上同时检测。结果　 1.在 1194例患者中 ,HLA B2 7抗原阳性率达38 6 % ;B7的抗原阳性率为 7 4% ;B13为 14 6 % ;B40为 13 4%。2 .在HLA B2 7阳性人群中 ,B7抗原的阳性率为 14 3% ,明显升高 ,而在HLA B2 7阴性人群中 ,B7的阳性率仅为 3 2 % ,明显降低 ;3.在44 7例HLA B2 7阳性人群中 ,未检测到HLA B2 7、B7、B13三种抗原皆为阳性者 ,说明B7和B13之间没有交叉 ;4.据此 ,在HLA B2 7阴性人群中 ,查到的HLA B7/B13阳性率 0 5 4%应代表这两个位点的杂合 ;5 .无论在HLA B2 7阳性还是阴性人群中 ,HLA B13/B40的抗原阳性率远高于HLA B7/B40的阳性率 ,说明B13和B40位点的杂合或交叉 ,远大于B7与B40之间的杂合或交叉。结论　检测HLA B2 7及其相关B位点抗原的频度有助于分析这些位点的杂合及其抗原的交叉反应性的特点     

Association of RFLP of HLA class I genes with Chinese ankylosing spondylitis patients

By serum typing, it is indicated that HLA B27 may be associated with the susceptibility to ankylosing spondylitis (AS). We analyzed DNA restriction fragment length polymorphism (RFLP) in 28 Chinese AS patients and 99 healthy controls, using a 1.4 Kb HLA class I cDNA probe. The results showed that the frequencies of the 8.1 Kb EcoRI, 5.2 Kb EcoRI and 21.9 Kb XbaI fragments were found to be significantly increased in affected patients (P less than 0.0001, P = 0.0015, respectively), but that of 19.2 Kb XbaI fragment was decreased (P = 0.0021). The data suggest that AS may be a polygenic disease; furthermore, B27 and 8.1 Kb EcoRI band may be two different factors responsible for the susceptibility or just in linkage disequilibrium with the susceptible gene(s).     

Immune function in ankylosing spondylitics and their relatives: influence of disease and HLA B27.

So as to distinguish the separate influences of ankylosing spondylitis (AS) and possible HLA B27 associated immune response genes on immune response patterns, a battery of immunological tests were performed on fourteen patients with AS and their first-degree relatives. Previously unrecognized AS was detected by clinical and radiological means. Individuals with ankylosing spondylitis had significantly higher serum IgG and IgA concentrations than both their B27 positive and B27 negative relatives. B27 positive relatives had significantly lower phytohaemagglutinin (PHA) lymphocyte transformations than B27 negative relatives (P less than 0.01), while there was no difference between the ankylosing spondylitic and B27 positive groups. Antibody titres to Streptokinase/Streptodornase were significantly higher in the B27 positive individuals, with or without AS, than their B27 negative relatives (P less than 0.005 and P less than 0.02 respectively). These results show that serum immunoglobulin differences were associated with disease, while differences in PHA stimulation and varidase antibody titres were associated with the B27 antigen. These findings may indicate the presence of HLA associated immune response genes including those involved with reactions to a particular antigenic component of Streptokinase/Streptodornase.     

B15 Heterogeneity in East African Blacks

One-hundred-forty-one Blacks (135 unrelated) from Kenya and Tanzania have been tissue-typed (HLA–A, B and C loci) as part of a study of host factors involved in Burkitt's lymphoma and naso-pharyngeal carcinoma. Evidence is presented for the existence in this population of several B15-related antigens which together occur with a relatively high frequency of 30% in unrelated individuals. It is likely that these variants may include the antigens SV and perhaps Bu recently defined with population frequencies of under 1% in Caucasians. In the absence of monospecific typing sera, identification of these variants may be helped by their apparently strong association with C-locus antigens in Blacks. Recognition of these B15 variants has been largely responsible for reducing the proportion of unidentified or "blank" B-locus antigens in this population to only 6%. These findings substantiate and amplify previous reports suspecting the presence of such antigens in Blacks, and should facilitate studies of possible associations of disease with HLA in these populations.     

Normal Human Sera Cytotoxic to Cells of Human Acute Leukemia

Eight human sera from healthy individuals with no history of immunization with human transplantation antigens have demonstrated complement dependent cytotoxicity to cells of some patients with active acute leukemia. The antigen(s) detected by these sera are absent from normal and remission lymphocytes and appear most often in ALL patients with a high peripheral lymphoblast count. Some B and T lymphoblastoid cell lines carry the antigen(s) as evidenced by their ability to react with and absorb the antileukemia activity. The data support the existence of at least two overlapping specificities detected by these antileukemia sera. The leukemia antigen(s) show no strong correlation with any known HLA antigen. These observations provide evidence for a human leukemia blast associated antigen or set of antigens which may be immunogenic in man. Their relationship to normal HLA antigens of loci other than A, B or C is unknown.     

Histocompatibility class I antigens loci A and B in familial sarcoidosis in Poland

Thirty three affected members from 17 Polish sarcoidosis families and their healthy relatives (61 subjects) have been typed for HLA class I (A and B). Controls consisted of 101 healthy Polish subjects. The HLA typing was performed by the serological method using the standard microcytotoxicity test. No significant differences were observed between group 1 (33 affected family members), group 2 (61 healthy relatives) and group 3 (healthy controls) considering the HLA class I locus A antigens. The frequencies of HLA B8, HLA B16 and HLA B40 were significantly higher in affected sarcoidosis families members compared to control subjects (p < 0.05). When comparing all families members (affected and non-affected as a one group) to the control group we found a significant overrepresentation of HLA B12 and B35 in the control group. We concluded that HLA antigens locus B: B12 and B35 may have a protective function against the disease; the role of HLA class I antigens in the pathogenesis of sarcoidosis needs further evaluation.     

HLA antigens in IGA deficient paediatric patients

HLA antigens (A, B, C and DR loci) were studied in 62 IgA-deficient (IgAd) paediatric patients: 17 with coeliac disease (CD), 13 with juvenile arthritis (JA), 27 with frequent respiratory tract infections (RTI) and five with other diseases. The frequencies of HLA antigens in IgAd patients were compared with those in healthy blood donors, and in CD and JA patients with normal serum IgA levels. The IgA deficiency in the patients showed significant associations with HLA A1, B8, B13, Cw6, DR3 and DR7 (P less than 0.0005, P corr less than 0.02 vs controls) and decreased frequencies of DR2 (P less than 0.0005, P corr less than 0.02 vs controls). The HLA associations typical of coeliac disease, increased frequencies of HLA-B8 and DR3, were evident among the IgAd coeliacs; in contrast to the coeliacs with normal IgA levels, the IgAd coeliacs showed a significant increase of the HLA-Cw6 allele (P less than 0.0005, P corr less than 0.02 vs control coeliacs). Increased frequencies of HLA-A1, B8, B13, Cw6, DR3 and DR7 were noted in the patients with RTI, which can be explained by the frequent occurrence of the haplotypes A1, B8, DR3 and B13, DR7, the latter haplotype often also having the Cw6 allele. Among the IgAd JA patients, the antigen frequencies were similar to those in the JA patients with normal serum immunoglobulins.     

HLA-B27基因检定技术

用聚合酶链反应和血清学方法检测ＨＬＡ－Ｂ２７，并进行比较。方法：采用ＰＣＲ技术７８例可疑为强直性脊椎炎患者样本的ＨＬＡ－Ｂ２７基因，并与血清学比较。结果：３６例具有Ｂ２７基因者中２４例同时作血清学试验，５例血清学难以判定结果，并与ＰＣＲ结果不一致。     

Production in vitro of antibodies directed against alloantigen-specific recognition sites on T cells and on lymphocytotoxic HLA antibodies.

We have examined the mechanism of immunological unresponsiveness in a recipient (P.S.) with a long-term functioning renal allograft. P.S., whose HLA type is A1, A30; B14, B18; DR1, w8; DRw52; DQw1 and in whose serum we had earlier demonstrated the presence of antiidiotypic antibodies, received a kidney from a cadaver donor of HLA type A1, A10, B8 in March, 1970. Peripheral blood B lymphocytes from the patient were transformed with Epstein-Barr virus (EBV), and by the cluster-picking technique a B cell line was propagated with continuous production of antibodies. Antiidiotypic antibodies with two distinct biological functions were demonstrable; one specifically inhibiting the lymphocytotoxic activity of anti-HLA-B8, B5, and DR3 reference typing sera, and the other specifically inhibiting proliferative responses in MLC of the recipient's lymphocytes and of third party cells sharing B14, DR1, DQw1 with the patient against stimulator cells carrying B8, DR3 antigens. Immunodepletion experiments demonstrated that the inhibitory activity was associated with the IgM fraction. Absorption experiments suggested that different antibodies may be responsible for the inhibition of lymphocytotoxic activity of anti-HLA sera and of the proliferative responses in MLC. Antiidiotypic antibodies have been postulated to be important in maintaining allograft tolerance in vivo, thereby enhancing renal allograft survival. The availability of such antibodies in large quantities, produced in vitro, could provide antisera for the immunochemical characterization of specific idiotypic receptors on immunoglobulins and T lymphocytes.     

Tissue Typing of Cells in Culture. IV. Cell Surface Antigens of Tumor Cell Lines, not Obviously Belonging to the HLA-System

Studies on the HLA pattern of several human tumor cell lines by the mixed hemadsorption test and also studies on the discriminative patterns formed by a collection of sera on a number of cell lines and diploid cells have been reported elsewhere. It was noted during these studies that some sera reacted in a fashion indicating that they did not represent any of the HLA-A or -B series and probably not the HLA-C series either. The corresponding antigenic determinants were tentatively designated Ek-1 to Ek-11. The pattern of these antigens among the cell lines is given in a table and it seems apparent that the Ek-series considerably increases the cell identification potential of the typing results. So far, five of the sera determining the Ek-antigens have failed to be blocked by anti-beta-2-microglobulin, indicating that the antigens do not belong to the HLA-A, B or C series. Preparatory work for HLA-D typing is under way.