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.     

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.     

Expression of HLA-A, -B, -C, -DR, -DP, -DQ, and of HLA-D-associated invariant chain (Ii) in non-neoplastic mammary epithelium, fibroadenoma, adenoma, and carcinoma of the breast

Non-neoplastic mammary gland, 20 benign tumors and 206 carcinomas of the breast were immunohistochemically examined for expression of HLA-A, -B, -C, HLA-DR, -DP, and -DQ molecules and the HLA-D associated invariant chain (Ii). In contrast to cells from benign lesions, tumor cells of 51.2% of carcinomas had an abnormally low content of HLA-A, -B, and -C determinants ranging from reduction of antigenic density per cell (28.8%) over an incomplete (15.6%) to complete loss of antigens (6.8%). Associated with lymphohistiocytic stromal infiltrates, HLA-D/Ii determinants were found to be induced in benign duct and acinar epithelium after the order Ii greater than or equal to HLA-DR greater than or equal to HLA-DP greater than or equal to HLA-DQ. These antigens were also expressed, mostly noncoordinately, in 55.5% of carcinomas, and in 98 cases according to the above order. In 28.6%, Ii expression clearly exceeded HLA-D antigen expression; conversely, 6.2% contained HLA-DR+/Ii- tumor cell subsets. In breast carcinoma, the association of reduced HLA-A, -B, and -C expression and a noninduction of HLA-DR was highly significant (P less than 0.0009), suggesting an abnormal signal acting down-regulating on the expression of both classes of antigens. Because the modality of HLA-A, -B, and -C and HLA-D/Ii expression correlated with neither tumor type nor grade, it might be an independent parameter.     

HLA Antibodies Cytotoxic only for B Lymphocytes

Of 28 unabsorbed sera from polytransfused individuals or multiparous women, which were not cytotoxic for total peripheral blood lymphocytes, 13 were found to react positively against B cell-enriched suspensions. These 13 sera were further characterized after platelet absorption-elution, as well as by pretreatment with turkey anti-human-beta2-microglobulin serum. Different patterns were found: five sera behaved as pure anti-B cell reagents; four seemed to contain different antibody populations, directed against both B cell determinants and HLA-A, -B or -C antigens; four only contained antibodies directed against HLA-A, -B or -C specificities. Absorption experiments with purified T and B lymphocytes, showed that these last sera, although noncytotoxic for T cells, can be absorbed by them. It was concluded that not all sera reacting only with B lymphocytes recognize specifities absent from T cells, and that all sera should be exhaustively absorbed with platelets before being tested as anti-B cell-specific reagents.     

HLA-D typing in Austria. A panel study using 26 newly defined homozygous typing cells

A panel of 120 HLA-A, -B, -C and -DR typed Austrians has been typed for HLA-D by the use of 26 Homozygous Typing Cells (HTC). The new Austrian HTC, partly defined by the 9th International Histocompatibility Workshop (9WS), partly by a checkerboard experiment with internationally well defined reference HTC, type for HLA-Dw1 to -Dw7 and an obviously new, so far unknown HLA-DR2 related HLA-D determinant. Associations of HLA-DR and HLA-D antigens in Austria and their frequencies are determined. Antigen frequencies in Austria are compared to frequencies in other Caucasoid populations.     

HLA-A, -B, -C and -DR antigen profile in pulmonary tuberculosis in North India

Investigations for the HLA-A, -B, -C and -DR antigens were conducted on 124 random North Indian patients with confirmed diagnosis of pulmonary tuberculosis by the demonstration of acid fast bacilli in the sputum. 109 appropriately matched controls from the same ethnic background were also tissue typed. No significant deviation was observed in the HLA-A, -B, and -C locus antigens. With the HLA-DR typing, there was a marginal increase in DR2 and a concurrent significant decrease in DRw6 in the patient group. These deviations were, however, insignificant when correction for the P value was made. ABO blood group typing results indicate that blood group 'O' may afford protection against TB. The involvement of both DR2 and DRw6 is interesting as it is also implicated in leprosy, another mycobacterial disease. The results suggest the possibility of a common gene in the MHC for both tuberculosis and leprosy.     

Non HLA antigenic determinants expressed on activated T and B human lymphocytes

Human antigenic determinants present on activated T and B lymphocytes and not on resting B and T cells defined by serological studies are reported. The reactivity of 13 sera against human PBM activated by various agents was tested using microlymphocytotoxicity technique. Results showed that generally similar reactions are present on B and T blasts whatever the technique of activation (PHA, ConA, PWM, TCGF, EBV and allo activation). Absorption elution studies with 5 sera on 10 alloactivated cells clearly demonstrated their independence from DR determinants. The analysis of results obtained by these 15 sera on blasts from 50 donors showed no correlation with HLA-A, -B, -C, DR and -MB antigens, neither can it be HT (Qa-like) antigens. Furthermore, 5 clusters of sera are reported. The weak correlation coefficients cannot affirm the existence of a system with an allelic distribution although each cell except one out of a panel of 50 individuals carries at most 2 specificities. The segregation of 3 of these specificities within 5 informative families showed no linkage with HLA genes.     

HLA-A, -B, and -DR specificities on human monocytes

Monocyte-enriched cell suspensions obtained by a plate adherence method from 57 blood donors were typed by microcytotoxicity for HLA-A, -B and -DR determinants. Parallel assays were performed with autologous unfractionated lymphocytes for HLA-A and -B determinants and with autologous B-lymphocytes for HLA-DR determinants. Correlation coefficients (r values) were calculated for nine HLA-A specificities (r > 0.79), 16 HLA-B specificities (r > 0.56) and seven HLA-DR specificities (r > 0.81). For certain HLA-A and -B specificities detection was less readily achieved on monocytes than on autologous lymphocytes. Extra reactions were observed in some instances. With sera defining HLA—DR specificities, monocytes and B-cells showed reactivity of almost identical strength and reliability.     

The HLA-DR phenotype of the responder is predictive of humoral response against HLA class I antigens

Recent studies suggest that the immunogenicity of an human leukocyte antigen (HLA) incompatibility should be considered in the context of the HLA phenotype of the recipient. The HLA-DR phenotype of the responder is thought to be predictive for the strength of the alloimmune response. In order to analyze the humoral response against HLA class I antigens in the context of the HLA-DR phenotype of the responder, we selected all HLA-DR homozygous Dutch patients that were present on the Eurotransplant waiting list between 1967 and 2000 (n=1,317 patients). By logistic regression it was determined whether antibody production against a specific HLA class I antigen is associated with a particular HLA-DR antigen in the patient. Furthermore, it was analyzed whether a patient, expressing a particular HLA-DR antigen, preferentially produces antibodies against particular HLA class I antigens. The results demonstrate that patients, homozygous for a certain HLA-DR antigen, cannot be considered high or low responders when analyzing the antibody response in terms of panel reactive antibody (PRA) value. However, a correlation can be found between the HLA-DR phenotype of the patient and the specific antibody response against HLA class I antigens. For example, antibodies against HLA-A10, -A11, -A19, and -B35 are produced more frequently by HLA-DR6 positive individuals, whereas antibodies against HLA-A3, -B5, -B7, -B8, and -B12 are produced more frequently by HLA-DR4 positive individuals. These data confirm that the HLA-DR phenotype of the responder plays a determinative role in the immunogenicity of mismatched HLA antigens. The results indicate that selection of HLA class I mismatches of the donor in the context of the HLA-DR phenotype of the responder might reduce the incidence of humoral graft rejection and minimize the sensitization grade of retransplant candidates.     

Cytotoxic Human HLA Class II Restricted Purified Protein Derivative-Reactive T-Lymphocyte Clones

Human T-lymphocyte clones specific for antigenic components of purified protein derivative (PPD) of tuberculin were generated by limiting dilution using in vitro PPD-activated peripheral blood mononuclear cells from a single donor. The HLA restriction specificity of eight clones that were cytotoxic against autologous PPD-pulsed monocyte targets, was examined against a panel of allogeneic PPD pulsed targets. In agreement with our findings with bulk-expanded PPD-reactive cytotoxic T lymphocytes, all clones were restricted by HLA class II antigens: seven by HLA-DR 2 and one by HLA-DRw10--the other HLA-DR antigen of the donor. All clones were CD3+, CD4+, CD8-. One clone exhibited, in addition to HLA-DR2 restriction, unrestricted cytotoxic alloreactivity against HLA-DR1. In monoclonal antibody-blocking experiments the latter clone was the only one that was blocked. Its lytic ability was abolished by two monoclonal antibodies against monomorphic HLA-DR determinants. The antigen specificity of the clones was studied by using autologous monocyte targets pulsed with antigens prepared from a range of different mycobacterial species. All seven HLA-DR2-restricted clones reacted with the majority of antigens tested. In contrast, the HLA-DRw10-restricted clone reacted exclusively with an antigen unique to PPD.     

HLA and Japanese MS.

Determination of HLA-A, -B and -C types in 43 Japanese patients with multiple sclerosis (MS) and of DR type in 25 MS patients was carried out using antisera from the 7th International Histocompatibility Workshop. The results were compared with 46 controls typed simultaneously. Twenty-three patients were also tested for HLA-Dw2. The conclusions were: 1) There were no significantly higher occurrences of HLA-A3, B7, Dw2 or DRw2 in Japanese MS. 2) Japanese MS might nevertheless be associated with the human major histocompatibility complex, because HLA-B40 was significantly less frequent in MS and two anti HLA-DRw sera, 7w008 and 034, reacted positively more often against lymphocytes from MS patients.     

Activation of cytotoxic T lymphocytes in HLA-A, -B and -C-identical responder-stimulator pairs I

Cytotoxic T lymphocytes were activated in primary one-way mixed lymphocyte cultures of cells matched for serologically defined HLA-A, -B and -C antigens. In 16 out of the 29 combinations mismatched for the HLA-D/DR antigens, cell-mediated lympholysis of the stimulator cells occurred. The specificity of 5 selected cytotoxic T lymphocytes was studied in detail. Three of these cytotoxic T lymphocytes recognize antigenic determinants associated with HLA-Bw35 (Breuning et al. 1984, II). The 2 other cytotoxic T lymphocytes failed to lyse T-target cells enriched by rosetting with sheep red blood cells, whereas target cells from the 'non-T' fraction were strongly lysed, indicating that antigenic determinants associated with Class-II HLA molecules were the targets recognized by these cytotoxic T lymphocytes. This notion was supported by a study of a panel of HLA-typed third-party target cells. One cytotoxic T-lymphocyte population preferentially lysed HLA-DR2-positive target cells. Family studies, including a family with a recombination between HLA-B and -D, showed that the target antigen recognized by the latter cytotoxic T lymphocyte segregated with DR2. The second cytotoxic T-lymphocyte population recognized a determinant associated with DRw8. However, in 13 of the 29 HLA-A-, -B- and -C-identical, D/DR-different combinations, cell-mediated lympholysis of stimulator target cells could not be detected, not even on enriched 'non-T' target cells. Thus, after primary mixed lymphocyte culture of HLA-A-, -B- and C-identical, HLA-D/DR-non-identical cells, cytotoxic T lymphocytes directed against sensitizing Class-II molecules can be detected in some combinations, but not in others.     

HLA antigens in Bali (Indonesia) with a special reference to an isolated community

One hundred eighty-two Balinese were typed for HLA-A and -B locus antigens. From these, 103 were also typed for HLA -C, 51 for HLA-DR, 172 for Bf and 173 for GLO. These results and the significant phenotypic associations are situated with respect to other South-East Asian populations. In addition to this first study, 175 individuals from an isolated Balinese village typed for HLA-A, -B, -DR, Bf and GLO are presented, The effect of isolation on haplotype (HLA-A/-B/Bf/-DR) variability is discussed.     

Molecular analysis of the HLA-DRS haplotype

A panel of eleven HLA-DR5 homozygous lymphoblastoid cell lines was investigated for structural heterogeneity on the product level. HLA class II antigens were isolated by immunoprecipitation with different anti-class II monoclonal antibodies and separated by two-dimensional (2-D) gel electrophoresis. As a result, three distinct DRB1, one commonly expressed DRB3, and two distinct DQ gene products could be identified that combined to four different haplotypes associated with HLA-DR5. A hitherto serologically undetected split of HLA-DRw11 was presented by three cell lines. HLA-DRw11 and HLA-DRw12 were found to be related allospecifities that differ only in their DRB1 locus products, but are closely associated with the supertypic DRB3 allele HLA-DRw52b and with HLA-DQw7. The DRB3 alleles HLA-DRw52a and DRw52c were not detected in our cell line panel, indicating that these supertypic determinants are in negative linkage disequilibrium with HLA-DR5. Our data suggest that intra HLA-DR/DQ crossing-over events contribute to the development of the HLA class II polymorphism. Evidence is presented that the T cell defined HLA-D allospecifities are commonly determined by DRB1 and DQ gene products.     

Frequency of alloantibodies reacting with PHA-activated T lymphocytes, unexplainable by known HLA activities

Two hundred and three sera obtained from multiparous women were screened to determine the frequency of the appearance of antibodies reactive with PHA-activated T lymphocytes, but not with resting B or T cells from the same donor. The panel of cells on which the sera were tested consisted of lymphocytes from 38 unrelated donors known to be homozygous at the HLA-D locus. Forty-two sera were shown to react uniquely with PHA-activated T cells. The reactivities of these sera could not be explained in terms of the defined HLA-A,-B,-C, or DR antigen specificities on the panel cells, nor on the basis of known cross-reactivities among these antigens. Most of the antisera showed strong cytotoxicity with the activated T cells. Such sera may define antigens presented to the maternal immune system during differentiation and development, which are not expressed on adult T cells unless they are activated.     

Detection of HLA antigens on blood platelets and lymphocytes by means of monoclonal antibodies in an ELISA technique

The expression of HLA-A and -B antigens on peripheral blood lymphocytes and blood platelets was measured using monoclonal antibodies in a semi-quantitative ELISA technique. Reactively of monoclonal anti-HLA-A2 and anti-HLA-B7, with lymphocytes as well as platelets, was in agreement with the presence of these antigens as detected by conventional HLA typing of lymphocytes. When the actual amount of HLA antigens was measured, a gene-dose effect was seen: cells from HLA-B7-homozygous individuals bound more monoclonal anti-HLA-B7 antibodies compared to their HLA-B7-heterozygous siblings. At the same time, cells of different donors showed only very small differences in binding of monoclonal antibody against an HLA-"backbone" determinant. Relative to total HLA-A, -B and -C expression, the amounts of HLA-A2 on lymphocytes and platelets were similar. On the other hand, the expression of HLA-B7 on platelets was diminished compared to that on peripheral blood lymphocytes.     

HLA polymorphisms in Saudi Arabs

The HLA-A, -B, -C, -DR, Bf and GLO phenotypes of 109 unrelated Saudi Arab males have been determined. HLA-A and -B antigen frequencies were compared with data reported for European Caucasoids and various Arab populations. Most similarities in antigen frequencies were seen between Saudi Arab and Iraqi populations. A high frequency of Bw50 was observed in Saudi Arabs. The frequencies of HLA-DR antigens in Saudi Arabs were compared to European Caucasoids. HLA-DR7 was at high frequency in Saudi Arabs. Linkage disequilibria between alleles of HLA loci was examined. Many instances of previously reported antigen associations were seen in Saudi Arabs, together with a number of associations which have not been described elsewhere. HLA-Cw6-Bw50-DR7-BfS0.7 is suggested as being a common haplotype in Saudi Arabs.     

Activation of cytotoxic T lymphocytes in HLA-A, -B and -C-identical responder-stimulator pairs II

We compared five cytotoxic T lymphocytes raised by primary mixed lymphocyte cultures of HLA-A, -B and -C serologically identical Bw35-positive responder-stimulator combinations. When tested on a panel of third-party target cells, the reactivity pattern of these cytotoxic T lymphocytes allowed the distinction of three subtypes of HLA-Bw35. Cold-target inhibition experiments and analysis of CTL activity at the clonal level showed the existence of subsets of CTLs directed against distinct antigenic determinants associated with HLA-Bw35.     

Fetus as an allograft: noncytotoxic maternal antibodies to HLA-linked paternal antigens

A cellular enzyme-linked immunospecific assay (CELISA) was used to monitor maternal humoral responses in human pregnancy. Non-cytotoxic IgG antibodies to paternal lymphocytes were detected in sera from 6 of 20 normal first trimester primigravidae and 6 of 13 multiparae. No antibody activity against lymphocytes from their partners was detected in sera from any of the 15 nulliparous women. The differences in antibody response between primigravidae and nulliparae (P = 0.024) and between multiparae and nulliparae (P = 0.005) were statistically significant. Lymphocytotoic antibodies to T- and B-lymphocytes were present in sera from three multiparae, but from none of the women in the other two groups. Family studies indicated that the non-cytotoxic pregnancy-associated maternal antibodies were directed to HLA-linked antigens (P less than 0.001). Evidence obtained using cell panels and platelet absorption suggested, however, that these antibodies were not directed to the currently recognized HLA specificities (HLA-A, -B, -C, or -DR).