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.     

Purification and characterization of HL-A antigens from human platelets, solubilized by the non-ionic detergent NP-40

Soluble HL-A antigens can be extracted with high yields from whole human platelets or platelet membrane preparations by the non-ionic detergent NP-40. The active molecules have high molecular weights ( 200 000) and the HL-A specificities present in the preparation were not separated by electrofocusing. An increase of the detergent/protein ratio gives rise to active molecules with lower molecular weight (15 000–30 000). In this case the two specificities studied (HL-A2 and HL-A7) could be separated by electrofocusing.     

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.     

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.     

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.     

Purification of papain-solubilized histocompatibility antigens from a cultured human lymphoblastoid line, RPMI 4265.

HL-A antigens having specificities HL-A2, HL-A7, HL-A12 have been solubilized by papain treatment of membrane preparations from the cultured human lymphoblastoid cell line RPMI 4265 and purified about 80-fold by chromatography on carboxymethylcellulose, Sephadex G-150, and diethylaminoethylcellulose columns. Separation of HL-A2 from a mixture of HL-A7 and HL-A12 was achieved on the final DEAE-cellulose column. The yield was about 1 mg of protein of each antigen preparation per 100 g of packed, frozen cells. On sodium dodecyl sulfate gel electrophoresis both preparations showed two polypeptide bands. The smaller subunit of 12,000 daltons is common to all HL-A preparations and has been shown to be identical with beta2-microglobulin. The larger subunit is a glycopeptide and in the HL-A7, 12 preparation was resolved into a duplex of 34,000 and 37,000 daltons. The HL-A2 preparation showed a single band at 34,000 daltons. On isoelectric focusing under nondenaturing conditions, the preparation showed multiple bands, all of which contained both subunits and retained antigenic activity. On isoelectric focusing in the presence of 6 M urea a single band for beta2-microglobulin and multiple bands for the larger subunit were seen. This charge heterogeneity of the larger subunit has been shown to be due to variable amounts of sialic acid. When HL-A antigen preparations were subjected to Sephadex G-100 chromatography in the presence of 3 M KCl, no separation of the two subunits was observed.     

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.     

Fate of HL-A antigens in aging cultured human diploid cell strains : II. Quantitative absorption studies

The expression of histocompatibility antigens on cultured human fibroblasts was studied utilizing a quantitative microabsorption assay. Trypsin treatment of cultured human embryonic and adult fibroblasts did not change their capacity to absorb selected HL-A alloantisera as compared with cells harvested by scraping. The density of HL-A antigens was found to remain unchanged throughout the finite in vitro lifetime of two human embryonic diploid cell strains (WI-38 and WI-26) and ten adult skin fibroblast cultures. Cultured fibroblasts derived from skin, lung, heart, and liver of one donor showed similar quantitative expression of HL-A1, 9, W5 and W16. These experiments support the contention that the HL-A marker system is at present the only system by which human fibroblasts derived from different normal human donors can be distinguished in vitro.     

HL-A Inhibiting Activity in Human Seminal Plasma

THE HL-A antigens of man have been described on a number of somatic (diploid) cells¹ and demonstrated on spermatozoa, suggesting an expression of the haploid genome on spermatozoan membrane². Soluble HL-A antigens, HL-A2, HL-A7 and 7b, have been reported in serum^3,4. We have now found in human seminal plasma substances that inhibit specific anti-HL-A antibodies. This has important biological and clinical implications, especially for the characterization of HL-A antigens and for the induction of specific tolerance.     

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.     

Alpha 1 antitrypsin deficiency: a study of the relationship between the Pi system and genetic markers.

Twenty-four members (4 generations) of a family with alpha 1 antitrypsin deficiency were studied in an attempt to determine the chromosomal location of the Pi system locus. Three alpha 1 antitrypsin alleles (PiM, PiI, and PiZ) and five phenotypes (MM, MZ, MI, IZ, and ZZ) were detected in family members. The quinacrine fluorescent banding technique was successfully utilized to reveal eight polymorphic chromosomal markers in family members. Eight red cell antigens and HL-A antigens were identified for each family member. No linkage between the Pi system and chromosomal markers, four polymorphic red cell antigens, and HL-A antigens was detected. On the basis of this family study, the Pi locus as defined by alpha 1 antitrypsin deficiency does not appear to be on chromosomes 2, 3, 13, 14, 21, or 22 within measurable distance of the markers used.     

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.     

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.     

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.     

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.     

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.     

Studies on changes in lymphocyte HL-A antigens during the course of malignant diseases]

The presence of HL-A antigens 1, 2, 5, 7, 8 and 12 on the lymphocytes of 26 patients with blood diseases and malignant tumours was examined by means of the two-step microcytotoxicity test. The studies carried out several times in the course of the disease and with 4 to 5 sera of the same specificity. Two types of the serological modifications were found: 1. Transient loss of HL-A antigens in 6 patients, 2. Evidence of the polyreactivity of lymphocytes in 3 patients. The polyreactivity was later changed to the loss of HL-A antigens. In one patient, the destruction of lymphocytes in the course of the testing was proved. The importance of the results is discussed. The additional serological, morphological and clinical investigations seems to be necessary.     

The immune response to a synthetic amino acid terpolymer in man: relationship to HL-A type.

The immune response to the synthetic amino acid terpolymer (L-glutamic acid-55 L-lysine-33 L-tyrosine15)n (GLT) was studied in normal human volunteers. Delayed skin test reactivity to this antigen was seen in 34 of 61 subjects immunized with 150 mug of GLT. No antibody to GLT was detected in these responding individuals. There was a close correlation between the in vivo skin reactivity of volunteers to GLT and the ability of their lymphocytes to produce migration inhibitory factor (MIF) in response to GLT in vitro. However, a similar correlation was not seen when the in vitro proliferative response of lymphocytes to GLT, as measured by [methyl-3H] thymidine ([3H] T dR) incorporation, was assayed. HL-A typing of volunteers was studied to determine if responsiveness to GLT was correlated to HL-A type. No statistical association was seen after correction was made for the number of individual HL-A antigens.     

HL-A frequencies in Down's syndrome.

HL-A antigen frequencies were examined in 76 Down's syndrome individuals and 733 normal Caucasians. 10 antigens of the first locus and 15 antigens of the second locus were defined, using a microlymphocytotoxicity technique. No significant differences were observed between the normal and Down's syndrome samples, in contrast to a previous report (Boxer and Yokoyama, 1972) of decreased HL-A antigen frequencies in Down's syndrome individuals. Our results therefore suggest that there is no relationship between trisomy 21-associated immune aberrations and altered HL-A antigen frequencies.     

Immunological selection of HL-A variants of cultured progeric fibroblasts and their identification by quinacrine fluorescence

HL-A2 deficient clones of cultured fibroblasts from a boy with progeria were selected immunologically from a thousand-fold excess of HL-A2 positive normal female cells in mixed cultures. Identification of surviving male cells was both confirmed and facilitated by quinacrine fluorescence of the Y chromosome in metaphase plates within several clones. These results support previous findings that progeric cells have altered HL-A antigens and also provide a more generally applicable method of selection for genetic studies on cultured somatic cells.