The effect of heating and drugs on HL-A antigens and their reactions with cytotoxins]

In some instances the heating of lymphocytes for 2 to 3 minutes at 56 degrees C enhances HL-A antigens or makes it possible to detect these antigens by a twostage microlymphocytotoxic test on preheated lymphocytes only. Similar phenomenons are observed in some tannin (1: 40 000) or 0.1% phenol-treated lymphocytes and after the addition of these solutions during the first stage of the lymphocytotoxic test. Prolonged heating at 56 degrees C leads to nonspecific polyreactivity of lymphocytes, giving false results with all sera. For similar reasons also higher concentrations of tannin and phenol solutions were found dissatisfactory for the pretreatment of lymphocytes. The pretreatment of lymphocytes with 36-0.5% formaline induces full inhibition of specific cytotoxic reactivity of HL-A antigens and their absorption ability with regards to respective HL-A sera. The addition of formaline at 0.06-0.3% concentration during the first and second stages of the test and at the end of the second stage (or simultaneously with eosine) gives also negative results of the cytotoxic test owing to the inhibitory effect of formaline on rabbit complement and HL-A antigens.     

HL-A genotype of patients with acute lymphoblastic leukaemia

Summary 10 patients with acute lymphoblastic leukaemia were tissue-typed for 21 HL-A specificities. Of these, genotypes of 9 pateints were determined by family analyses. Haplotype HL-A1,8 occurred in 5 out of 18 instances. On phenotype basis, a slight increase was observed in the incidence of antigens HL-A1 and HL-A8. No loss of HL-A specificities could be detected on lymphocytes through family analyses.     

Cross-reactivity between human and murine lymphocyte antigens: III. Reactivity of H-2 allo- and xenoantisera with human lymphoid cells

H-2 alloantisera and antimouse lymphocyte xenoantisera react with 14%–100% of human lymphocytes from a panel of at least 80 unrelated people. Population and family studies did not reveal HL-A specificity of such lymphocytotoxic antibodies but indicated that the antibodies are directed against polymorphic antigenic determinants inherited in association with HL-A antigens. H-2 allo- and xenoantisera absorbed with human lymphoid cells and a panel of platelets bearing all the known HL-A specificities were still cytolytic when tested against murine lymphocytes, suggesting that only a small proportion of the heterogeneous population of H-2 antibodies react with human lymphocytes. On the other hand, HL-A alloantisera could be absorbed by lymphocytes from certain murine strains. These results suggest that the crossreactivity between human and murine lymphocytes is caused by antigens common to several HL-A (or H-2) types or by antigens linked to HL-A but not identical with them.     

The identification of HL-A antigens on fresh and cultured human endothelial cells.

Human endothelial cells were obtained from the umbilical cord veins of 16 newborns by methods previously described and tested for HL-A antigens by a microcytotoxicity method. HL-A antigens were present on all endothelial cell lines tested. When the HL-A phenotypes of fresh endothelial cells and autologous fetal lymphocytes were compared, a concordance of 70% was observed. When the HL-A phenotypes of maternal lymphocytes and fresh endothelial cells were compared, a maternal contribution to the endothelial cell phenotype was evident in 72% of the possible commmon antigens. Some HL-A antigens were deleted from 11 of 16 endothelial cell lines that were re-typed after 2 weeks in tissue culture. The majority (90%) of deleted antigens were from the second HL-A locus. When three lines of endothelials cells were again re-typed after 6 weeks in culture, no further changes in antigenicity were noted. These findings: a) demonstrate that HL-A antigens are present on human endothelium and suggest that endothelial cells are actively involved in establishing the immunogenicity of a graft, and b) demonstrate that the HL-A antigens on human endothelial cells may be modulated by in vitro culture.     

Modifications of lymphocyte HL-A antigens as a consequence of therapy in patients with manic-depressive psychosis]

In 54 (= 46.96%) of 115 patients with maniacal-depressive psychosis a HLA modification could be identified. This modification turned out to be temporary and from a serological point of view it revealed a different character. In 29 cases a loss of HLA antigens could be observed, in 3 cases there was a decrease, in 14 cases a combination of both changes, twice a polyreactivity was observed and 6 times a change of the antigen HLA-A 2 in A 28 could be determined. These serological modifications appeared after therapy with lithium as well as with various antidepressive and neuroleptic medicaments. The connection between therapy and development of HLA modification could be ensured statistically. The modifications of HLA antigens A 10 and B 7 developed after administering neuroleptic medicaments, those of HLA antigens A 9, A 11, B 12, and B 13 after therapy with antidepressive medicaments. HLA antigens B 27 and B 40 showed a relative resistance towards therapy. The significance of these findings for the possibility of mistakes in HLA typing and from the standpoint of therapy efficiency in connection with the patient's HLA phaenotype is discussed.     

Human platelets as a source of HL-A antigens : a study of various solubilization techniques.

The efficiency of various methods of solubilizing HL-A platelet antigens was investigated. The yield of soluble material was compared with that obtained from lymphocytes in culture in order to judge the quality of platelets as a source of HL-A antigens. The conclusion was reached that platelets, easily obtainable, can be considered as a good source of HL-A antigens.     

HL-A and Kidney Transplantation

AT present the role of HL-A antigens in the rejection of organ transplants is uncertain; we therefore wish to report the results of renal transplantations, under known HL-A compatibility conditions, performed in France since 1959. The 179 cases already published² have now increased to 416 cases³ including 84 sibling (SS), parent-to-child (PC) and 253 unrelated (UR) allografts. Only three cases were excluded from the analysis, which was similar to that previously used². We calculated the probability of incompatibility for the non-determined HL-A antigens, assuming the presence of four HL-A antigens of similar strength in each individual. Differences between antigens having serological similarities (cross-reactions) were considered in a first analysis as incompatibilities and in a second analysis as identities.     

The transient loss of HLA antigens on lymphocytes in patients following administration of penicillin and tetracyclin.

Ten patients with inflammatory affections of urinary, respiratory and bile tract, as well as otitis patients were administered Penicillin. Six or seven days after the start of the treatment, four of them showed weakening or loss of minimally two earlier determined HLA antigens. The loss of HLA antigen was likewise demonstrated in a patient treated with Tetracycline. The HLA antigens were reliably demonstrated again six to seven days after the end of the treatment. The results suggest a possible false typing of HLA antigens in patients who had been administered Penicillin or Tetracycline. In accordance with Ben-David et al. [1] transient loss of two HLA antigens with concomitant development of lymphocyte polyreactivity was found in one of four patients treated with Chloramphenicol.     

Inhibition of T-lymphocyte rosetting by HL-A alloantisera and complement.

The relationship between HL-A antigens and rosetting of sheep red blood cells (SRBC) with peripheral human lymphocytes has been investigated by incubating them with HL-A antibodies. Although sensitizing the lymphocytes with HL-A alloantisera had no effect on their ability to form rosettes with SRBC, further sensitization with C6 deficient rabbit serum as a source of early complement components inhibited the formation of rosettes with SRBC. The involvement of HL-A alloantibodies in the inhibition of rosette formation was shown first by correlating the HL-A phenotype of the lymphocytes and the HL-A specificity of the alloantisera and, second, by specifically absorbing the HL-A alloantibodies from the alloantisera. Complement was needed to inhibit rosette formation since this effect was lost when rabbit serum was treated to inactivate complement. The participation of complement's classical pathway in rosette inhibition was shown by chelating the Ca2+ ions by EGTA treatment of the C6 deficient rabbit serum. Perhaps, binding of HL-A antibodies and early complement components to the lymphocyte surface disturbs the distribution of the receptors or affects the charge of the cell membrane, thus inhibiting the rosette formation with SRBC.     

Genetic relationships between the HL-A system, the blood group systems (Rh, ABO) and the immune reactivity]

The authors examined in immunized healthy persons the correlations between the ability of immune response, the value of their different immunological parameters, and the HL-A blood-group antigens by computer analysis. Immune reactivity showed mosaic-like correlation against the HL-A system. The most definite negative correlation was noticed between the HL-A 3 and 7 antigens and the cytotoxic activity of lymphocytes. Remarkable and definite correlation was found between the Rh system and immune reactivity. The level of the natural antibodies, the immunoglobulins and the functions of lymphocytes were generally decreased in males in comparison to females.     

Myasthenia Gravis,Autoantibodies, and HL-A Antigens

The serum of 100 patients with myasthenia gravis and 441 of their first-degree relatives was studied for the presence of autoantibodies against several antigens. Antibodies to skeletal muscle were present in 22% of the patients and in 2% of the relatives. Both these frequencies were significantly higher than those in matched control subjects. Also, antinuclear antibodies were present more often both in the patients and in the relatives. Typing for HL-A antigens had shown a positive correlation between HL-A 8 and myasthenia gravis which was significantly higher in women than in men. Antibodies to skeletal muscle and thymomas were found to be much rarer in HL-A 8-positive patients than in HL-A 8-negative patients; HL-A 8-positive patients acquired the disease at an earlier age.HL-A 2-positive patients more often had thymomas and antibodies to skeletal muscle than HL-A 2-negative patients; HL-A 2-positive patients acquired myasthenia gravis at a later age.The fact that the clinical aspects of the HL-A 8-negative and HL-A 2-positive patients were different from those of the HL-A 8-positive and HL-A 2-negative patients justifies the hypothesis that there are two forms of myasthenia gravis.     

Some biological properties of beta2-microglobulin and its antibody.

beta2-Microglobulin on the surface of lymphocytes exists in two molecular forms, first as part of the HL-A antigen complex, and second as a free molecule unbound to other membrane macromolecules. Quantitative determinations of the numbers of molecules of beta2-microglobulin per lymphocyte when compared to published values for the numbers of HL-A molecules per cell are consistent with an excess of beta2-microglobulin compared to HL-A antigens on the cell surface. Turnover studies of beta2-microglobulin from the lymphocyte surface indicated that both beta2-microglobulin and HL-A components are metabolized at similar rates. beta2-Microglobulin appears to be released in the free form. HL-A antigens, if released from the cell surface, appear to be released unbound to beta2-microglobulin. The effects of anti-beta2-microglobulin antibody on lymphocyte activation, namely on the mixed lymphocyte reaction and on antigen induced proliferative response, were studied. Anti-beta2-microglobulin completely inhibited the mixed lymphocyte reaction and the antigen induced proliferative response.     

The human mixed-lymphocyte culture (MLC) interaction after in vivo allo-immunization. II. HL-A specificity of early proliferative response

Human volunteers were immunized with a single intradermal dose of allogeneic buffycoat cells. When donor and recipient differed for four HL-A antigens belonging to the first and second series, an increased MLC proliferation early in the cultures was observed when responding lymphocytes from the immunized individual were confronted with stimulating cells from his donor. These findings were not observed when the incompatibility between donor and recipient involved only one second series HL-A antigen. The specificity of the altered response was studied by confronting responding lymphocytes from the immunized individual with different third-party stimulating cells. Most of these combinations revealed an early increased proliferation compared to control combinations with responding lymphocytes from nonsensitized individuals. However, the early increased responsiveness was significantly more pronounced in combinations where the stimulating cells shared two or more “private” HL-A determinants with the immunizing donor. It is concluded that a major part of the early increased responsiveness is due to proliferation of lymphocytes with receptors for HL-A (SD) determinants.     

Exclusion gene mapping utilizing patients with chromosome imbalance: the HL-A system as a prototype.

17 chromosomally unbalanced patients, their siblings and parents were tested for HL-A types and for up to 25 other polymorphic systems to determine whether there was gain or loss of an allele concurrent with the gain or loss of chromosome material. 5 patients had trisomy of part or all of a chromosome; 2 had trisomy of a segment and also deletion of chromosome material. All 7 were due to a familial translocation. The remaining patients had small deletions; 5 had ring chromosomes, 4 had rod deletions and 1 had missing chromosome material due to a heritable translocation. All cases were informative at the HL-A loci because of the high degree of polymorphism of the system whereas only some of the other systems were informative. None of the 17 patients showed unusual inheritance of HL-A or any other of the polymorphic systems examined. These results provide evidence excluding the HL-A and other loci from a number of possible locations in the human genome.     

Analysis of an HL-A antiserum by iso-electric focusing.

An anti-HL-A 3 antiserum with cross-reacting activity for HL-A 1 and HL-A 11 was subjected to isoelectric focusing over a pH 5-8 gradient. The cytotoxic activity of the serum focused into three distinct peaks, one at the basic end of the gradient, one between pH 6 and pH 7 and one at the acidic end. The first and second peaks reacted with HL-A 3 positive cells and HL-A 3 negative cells positive for cross-reacting antigens. The third peak reacted only with HL-A 3 positive cells.     

Alteration of the antigenicity of human lymphocytes by plant lectins and short-term tissue culture.

Human lymphocytes studied after being placed in culture for 1–6 wk progressively lost stimulating ability, i.e., lymphocyte defined antigens, when tested in one way mixed lymphocyte culture (MLC) but retained several other identifiable membrane components as well as the capacity to respond to mitogenic stimuli. Lymphocytes placed in culture with motogenic doses of PHA and Con-A after 1 and 2 wk strongly stimulated autologous responding fresh lymphocytes, but the MLC response of allogeneic fresh lymphocytes to stimulating lectin treated cells was even lower than the response to stimulating allogeneic cultured lymphocytes. The HL-A antigens on lectin treated cells or on lymphocytes through 6 wk in culture were clearly identifiable. Assays for T cell rosettes and B cell surface immunoglobulin showed both cell types to be present in numbers equal to fresh lymphocytes for up to 5 wk after culturing. However, the Fc receptor site on B cells was lost from cultured lymphocytes at the same time that MLC stimulation was lost. It is concluded that plant lectins can unmask new mitogenic sites on the cell surface as well as mask or delete existing sites, and that culturing lymphocytes for 1–6 wk will produce somewhat similar modulations. Modulation of surface membrane components by tissue culture or lectins may, therefore, have a profound effect in altering transplantation immunogenicity.     

Senescence of cultured human fibroblasts: mitotic versus metabolic time

To explore the relative importance of mitotic versus non-mitotic (metabolic) factors in determining the finite lifespan of human fibroblasts, mass cultures and 5 clones from 2 normal adults were followed as cohort pairs monitoring calendar time, mean population doublings (MPD), plating efficiency and expression of HL-A antigens. One member of each pair was split serially while the other was held in relative stationary phase with weekly refeeding using standard medium and culture conditions throughout. First cohorts became senescent after 52–62 MPD and 105–170 days while plating efficiencies declined and some HL-A antigens lost reactivity with specific antisera. At this time second cohorts were released from stationary phase and split serially. After initial loss of viability they generally showed higher plating efficiencies and normal reactivity of HL-A antigens till cell death within a calendar time of 195–240 days. The total MPD were reduced only marginally if allowance was made for low grade mitosis occurring during prolonged stationary phase. The results indicate that continuously replicating cells lose viability significantly before mitotically inhibited but actively metabolizing cohorts and suggest that factors which increase cellular turnover accelerate senescence and its pathological sequelae.     

Histocompatibility Antigens in Active Chronic Hepatitis and Primary Biliary Cirrhosis

The frequency of antigens HL-A 1 (48%) and HL-A 8 (52%) in 54 patients with active chronic hepatitis from south-east England was significantly higher than in 89 control subjects from the same region (22% and 17% respectively). No correlation could be detected with the age and sex of the patients or with the presence of a particular immunological abnormality but the frequency of HL-A 1 and HL-A 8 was much lower in the nine patients who were positive for HBAg than in the 45 HBAg-negative cases. These results provide further evidence of the importance of genetic factors in active chronic hepatitis. In contrast the frequency of HL-A 1 and HL-A 8 in primary biliary cirrhosis, both in 45 patients from south-east England and in 28 patients from western Scotland, was not significantly different from that found in control groups from the same regions.     

Characterization of a serum inhibitor of MLC reactions. III. Specificity: HL-A-related antibodies.

A mixed leukocyte culture (MLC)-inhibitory serum from a healthy multipara, JH, has been characterized with regard to the specificity of its inhibitory antibody. When added directly to MLC, JH serum will inhibit most combinations. However, when lymphocytes intended as responder or stimulator cells in MLC were preincubated with this serum, the specificity narrowed considerably. Four groups of lymphocyte donors were recognized, depending upon whether their lymphocytes were inhibited as responders, as stimulators, as both, or as neither. Absorptions of inhibitory activity, followed by assay of the absorbed sera in pretested MLC combinations, yielded reliable data for determining which donors' cells shared pertinent antigens. An association of MLC inhibition by JH serum with the HL-A types of the involved lymphocytes was observed and these relationships are summarized in Table 4. The three HL-A specificities identified, W19, W29, and 12, correspond with the HL-A typing of the husband of the serum donor. Various cell types absorbed relevant inhibitory activity (against responder and stimulator functions) in the following order of efficiency: LCL cells, B lymphocytes, T lymphocytes, fibroblasts, and erythrocytes. When the above three HL-A specificities were removed by absorption, the serum was no longer inhibitory in any combinations. Whether the inhibitory activity of JH serum is directly related to anti-HL-A antibodies or to antibodies against closely related MLR determinants will depend to a large extent upon the degree of linkage disequilibria found between W19, W29, and 12 antigens and the MLR locus.     

Eliminating antibody polyreactivity through addition of N-linked glycosylation

Antibody polyreactivity can be an obstacle to translating a candidate antibody into a clinical product. Standard tests such as antibody binding to cardiolipin, HEp-2 cells, or nuclear antigens provide measures of polyreactivity, but its causes and the means to resolve are often unclear. Here we present a method for eliminating antibody polyreactivity through the computational design and genetic addition of N-linked glycosylation near known sites of polyreactivity. We used the HIV-1-neutralizing antibody, VRC07, as a test case, since efforts to increase VRC07 potency at three spatially distinct sites resulted in enhanced polyreactivity. The addition of N-linked glycans proximal to the polyreactivity-enhancing mutations at each of the spatially distinct sites resulted in reduced antibody polyreactivity as measured by (i) anti-cardiolipin ELISA, (ii) Luminex AtheNA Multi-Lyte ANA binding, and (iii) HEp-2 cell staining. The reduced polyreactivity trended with increased antibody concentration over time in mice, but not with improved overall protein stability as measured by differential scanning calorimetry. Moreover, glycan proximity to the site of polyreactivity appeared to be a critical factor. The results provide evidence that antibody polyreactivity can result from local, rather than global, features of an antibody and that addition of N-linked glycosylation can be an effective approach to reducing antibody polyreactivity.