Serologic Specificity and Crossreactivity of Soluble HL-A Alloantigens

The serologic activity of soluble human histocompatibility (HL-A) antigens extracted from four human cultured lymphoid cell lines by the 3M KCI method has been evaluated by their ability to inhibit specifically the cytotoxicity of 38 operationally monospecific alloantisera recognizing 18 HL-A specificities. Several HL-A alloantisera directed against the same HL-A specificity, although used at functionally comparable levels of activity, varied in their susceptibility to inhibition by the same soluble HL-A alloantigen preparation. Similarly, lymphocytes from a number of subjects, when used as target cells for the same alloantiserum, detected quantitatively different activities of a given soluble HL-A alloantigen preparations in the inhibition test. Soluble HL-A alloantigens can block the cytotoxicity of antisera directed against HL-A specificities either crossreacting with or present on the cultured lymphoid cells used as source for extraction of soluble HL-A alloantigens. Crossreactivity was exhibited only by some of the soluble alloantigens and alloantisera with a given HL-A specificity tested. Quantitative studies indicate that the amount of soluble HL-A antigens necessary to inhibit the cytotoxicity of operationally monospecific alloantisera (used at the highest dilution producing 95% lysis) directed against crossreacting specificities is significantly greater than that necessary to inhibit alloantisera directed against HL-A determinants present on the soluble HL-A antigens. Since the alloantisera are used at relatively high dilutions, these data suggest that the crossreactivity in the HL-A system is not caused by contamination of operationally monospecific alloantisera with antibodies of low avidity. On the contrary, these data substantiate the hypothesis that crossreactivity is caused by structural similarities among different HL-A antigenic determinants.     

Common antigen structures of HL-A antigens: I. Antigenic determinants recognizable by rabbits on papain- solubilized HL-A molecular fragments

Antigenic determinants recognizable by rabbits were found to be present on the molecular fragments (48,000 Daltons) which were obtained by papain-solubilization of the membrane fractions of cultured human lymphoid cells and which carried the HL-A determinants. Results were obtained which suggest that these antigenic determinants are present in common on these molecular fragments carrying HL-A determinants regardless of their HL-A specificity and are restricted to the molecular fragments which carry HL-A determinants.

The study was made by use of radioimmune methods involving the binding of radioiodine-labelled soluble HL-A antigen preparations by anti-HL-A alloantisera and by rabbit antisera raised against the membrane fractions of cultured human lymphoid cells.

     

Xenoantisera against Lymphoid Cells: Specificity and Use in Monitoring Purification of Mouse and Human Histocompatibility Antigens

Rabbit xenoantisera against membranes of mouse spleen cells and cultured human lymphocytes were used to monitor the purification of solubilized mouse H–2 and human HL–A antigens in parallel with routine serological methods employing alloantisera. Antigenic material was subjected to gel filtration, ion exchange chromatography and electrophoresis in polyacrylamide gel, and in all cases the only material which inhibited the lymphocytotoxic action of the xenoantisera was that which inhibited defined alloantisera. Conversely, all alloantigenic material inhibited xenoantisera. Highly purified H–2 and HL–A preparations completely inhibited the lymphocytotoxic action of xenoantisera to crude membrane antigen. The specificity of the anti-mouse xenoantisera was examined. Antibodies specific for the immunizing mouse strains were revealed only after so-called species-specific antibodies were removed by judicious absorption of the serum with cells of a mouse of another H–2 type. The residual strain-discriminating antibodies reacted with target cells from a large panel of different mouse strains with an intensity directly proportional to the number of reacting H–2 specificities. It was not possible, however, to detect monospecific H–2 antibodies in any absorbed sera It is concluded that the lymphocytotoxic antibodies in xenoantisera predominantly are directed against determinants intimately associated with H–2 and HL–A alloantigens.     

Evaluation of two sources of soluble HL-A antigens: platelets and serum

Platelets and human serum have been evaluated as sources for extraction of soluble HL-A antigens. 3 M KCl was found to efficiently solubilize HL-A antigens from platelets with a recovery ranging between 50 and 100 %. However, because of the low density of HL-A determinants on platelets, the yield of soluble antigens is low, as only 1 mg of protein can be recovered from 1 × 10⁹ platelets. Thus, while it is difficult to solubilize sufficient antigens from platelets for chemical characterization, it is possible to use these materials for biological applications such as pretreatment of kidney recipients. Soluble HL-A antigen is present in human serum, since following extensive ultracentrifugation, it can still effectively inhibit the cytotoxic activity of HL-A alloantisera. The same HL-A specificities were found to be present in varying amounts among different donors. Partial purification of such antigens can be achieved by ion-exchange chromatography of serum on QAE-Sephadex.     

Production of Anti-W24 Xenoantisera in Rabbits1

One out of four rabbits injected with partially purified soluble HL-A antigen from serum produced cytotoxic antibodies to human lymphocytes. These antibodies appear to be directed to the specificity W24 as determined by correlation studies with HL-A alloantisera and by absorption-inhibition experiments with human platelets and with serum soluble HL-A antigens. In the sera from the other three immunized rabbits, no antibodies to human lymphoid cells could be detected by the cytotoxic test, blocking of T cell rosette formation, stimulation of DNA synthesis and inhibition of mixed lymphocyte reaction.     

The preparation and purification of HL-A monospecific antigens using platelets and various lymphoid sources

HL-A-active protein fragments have been isolated from lymphoid sources (tonsillar tissue, cells from lymphoblastoid cell lines (LCL cells)) and from platelets. The yields and specific activities of products obtained by enzymic chaotropic treatment indicate the superiority of 3 M KCl extraction of platelets. Individual HL-A antigenic specificities were further distinguished in fractions separated by polyacrylamide gel electrophoresis. Antigens were separated between the LA and FOUR loci, and within the LA locus. Purified monospecific HL-A antigenic preparations were conjugated with aminoethyl-Sepharose-4B, and used to remove corresponding specificities from alloantisera.     

HLA antigens: rabbit antisera reacting with all A series or all B series specificities

Papain-solubilized HLA antigens giving only two bands of 34 000 mol.wt. and 11 000 mol.wt. by sodium dodecyl sulfate (SDS) gel electrophoresis and isolated from the cultured human lymphoblastoid cell line RPMI 4265, have been used to prepare antisera in rabbits. Antisera were raised against soluble products of the A (or 1st) series of HLA, bearing the determinant HLA-A2 (A2 substance), or against a mixture of products of the B (or 2nd) series of HLA, bearing the determinants HLA-B7 and HLA-B 12 (B7-12 substances). Rabbit antisera to A2 substance reacted primarily with A2 substance on Ouchterlony analysis, showing an apparent spur of cross-reactivity with B7-12 substances. Rabbit antisera to B7-12 substances reacted primarily with B7-12 substances, giving a spur of cross-reactivity with the A2 material. Neither antiserum precipitated β-₂microglobulin. Both types of sera reacted with membrane molecules of 43 000 mol.wt. and 11 000 mol.wt. by immune precipitation and gel electrophoresis in SDS of detergent-solubilized radiolabeled membranes from cultured cell lines, as predicted for sera directed towards HLA antigens. F(ab′)₂ fragments of the antibodies blocked the complement-mediated cytotoxicity of all HLA alloantisera tested for human peripheral blood lymphocytes. After absorption with B7-12 substances, F(ab′)₂ fragments of antisera to A2 substance only blocked the cytotoxicity of HLA alloantisera to A series specificities. After absorption with A2 substance, F(ab′)₂ fragments of rabbit antisera to B7-12 substances only blocked the cytotoxicity of HLA alloantisera to B series specificities. The results prove the existence of shared antigenic determinants between all members of the same series. These findings support the genetic evidence that A series HLA antigens are allelic products of a single locus, while B series HLA antigens are allelic products of a separate locus, by establishing some invariance of structure, presumably amino acid sequence, between members of the same series. The apparent cross-reactivity between A2 substance and B7-12 substances, and the ability of the unabsorbed F(ab′)₂, preparations to block the cytotoxicity of all HLA alloantisera, suggests that some determinants are common to both HLA loci. This may be considered to support the hypothesis that the two loci arose by gene duplication.     

Serological Cross-reactivity in the HL-A System

New cross-reactions between allelic antigens of the HL-A system have been identified by examining new and well characterized HL-A alloantisera. Sixteen "monospecific" and 25 "duospecific" antisera were absorbed with various known platelets and examined for inhibition of cytotoxicity to lymphocytes from different individuals. Up-to-date diagrammatic representations of the most frequently observed cross-reactivities are given.     

Stimulation of lymphocytes by allogeneic lymphocytes and lymphoblasts in the presence of anti-HLA antisera.

Anti-HLA alloantisera inhibit mixed lymphocyte responses in which normal lymphocytes are used as stimulator cells. These same antisera are unable to inhibit lymphocyte proliferative responses stimulated by lymphoblastoid cells from cultured lymphoid cell lines. They also fail to inhibit either the generation of cytotoxic effector cells by lymphoblastoid cells or lymphocyte-mediated cytotoxicity against the lymphoblasts. Although the number of HLA antigens on the surface of lymphoblasts is reported to be greater than on normal lymphocytes, the failure of alloantisera to inhibit lymphoblast-induced responses in vitro does not appear to be due to insufficient amounts of antiserum to react with the antigenic sites. Rather, the data are interpreted to suggest that antigens which are not HLA and are not closely associated with HLA on the lymphocyte membrane are responsible for the stimulation of allogeneic lymphocytes by lymphoblastoid cells. Although lymphoid cell lines are known to contain the genome of the Epstein-Barr virus, antisera against products of the viral genome fail to inhibit proliferative responses to lymphoblastoid cells, suggesting that these antigens do not directly participate in lymphocyte activation.     

Soluble HL-A antigens in serum. I. Isolation and Purification

A new method was developed which utilizes fractional salting out, gel filtration, ion exchange chromatography and polyacrylamide gel electrophoresis for the isolation and purification of soluble HL-A antigens from serum. The method produced purified antigens possessing HL-A 5,9 specificities with yield up to 60 % of that detected in the original serum. Up to 150-fold increase in specific HL-A antigenic activity above that in the starting material could be achieved as measured by the ability of the purified antigens to specifically inhibit the cytotoxic activity of HL-A alloantisera against selected target cells. Electrophoretically purified HL-A antigens with different specificities appear to have a similar molecular size, i.e. 33 000, but differ in their electric charge properties. β₂-microglobulin (β₂m) seems to be noncovalently associated with HL-A antigens present in serum, but during purification β₂m is progressively lost until at the final purification by polyacrylamide gel electrophoresis, the material is completely devoid of β₂m.     

Antibody Induced Cell-Mediated Cytotoxicity. Studies of Human Alloantibodies in a Micro-Plate-Test

Human alloantisera were investigated for their ability to induce antibody-dependent cell-mediated cytotoxicity (AICC) in a micro-plate-system. Ten to 20 × 10³ fresh (non-stimulated) lymphocytes were used as targets, and an effector: target cell ratio of 33:1 was usually satisfactory. Harvesting was performed after 8–16 h of incubation. This system proved as reliable as tests in tubes with larger volumes and cell numbers. The HL-A specificity of the sera determined by complement-dependent cytotoxicity (CDC) was found to be completely included in the specificity determined by the AICC test. The latter specificity, however, was broader and the AICC test also revealed much higher titers of alloantibody activity.
When antibodies reacted with HL-A antigens of the effector cells, as revealed by CDC tests, the AICC test was inhibited.
By running the test system in medium not containing serum with alloantibody activity, the presence of cytotoxic (T) lymphocytes in the effector cell population could also be detected. Thus, the micro-test system is a sensitive test for both pre-existing cellular and humoral immunity against human alloantigens.     

Studies of human alloantigens on man-mouse hybrids: possible syntheny between hl-a and p systems

The expression of HL-A, P and H (ABO) systems has been studied on man-mouse hybrid clones and their subclones. Using the microcomplement fixation technique, we detected antigens of the HL-A, P and H systems in some clones corresponding to the phenotype of the human cell donor. Absorption tests were used to provide the specificity of the alloantisera. We also found a positive correlation between the presence of HL-A and P1/P antigens in these clones, but no correlation was found between HL-A and P^k specificities and between HL-A and H specificities. It was thought that the positive correlation might be due to a syntheny between HL-A and P loci or to the presence of HL-A and P specificities on a unique molecule. Therefore, preliminary experiments were made using antibody-induced redistribution phenomena (capping) to study the relationships between P and HL-A antigens at the cell surface. They indicated that the two molecules are independent of one another.     

Radioiodinated soluble HL-A antigens: HL-A alloantigenic characterization and use in radioimmunoassay

A method is decribed for the quantitative radioimmunoassay of HL-A antigens which is based on the degree of inhibition of the direct binding of radiolabeled soluble HL-A antigen and its alloantibody by the sample to be assayed. The assay is easily carried out, the results are highly quantitative and reproducible. The method can be applied to the quantitation of HL-A antigens in the insoluble from including those on intact cells as well as in the soluble form. The method has an advantage over the cytotoxic method since it does not require viable cells and complement for the primary test system.The radiolabeled HL-A antigens are prepared by radioiodinating purified, soluble HL-A antigens isolated from human lymphoid cells in long-term culture. The radiolabeled HL-A antigens show high binding activities with the corresponding alloantisera. The degree of binding is determined by precipitating the radioactive HL-A antigen-alloantibody complex with goat anti-human IgG antibody, i.e., the double antibody technique.     

The Immune Reactivity of Heterologous Antisera Against a Solubilized Human Lymphoid Membrane Component

Purified HL-A antigen bearing cell membrane components were prepared from human lymphoid cells by papain digestion and physicochemical fractionation. Rabbits immunized with these components produced antisera which had high cytotoxic titers against human lymphoid cells and other human tissue culture cells. The cytotoxicity was inhibited only by those membrane fractions which contained HL-A activity. Absorption and cytotoxicity inhibition studies showed that the antisera possessed antibodies against antigens shared by humans and monkeys, antigens common to different human tissues, lymphoid antigens and allotypic antigens.
To determine if the heterologous antisera reacted with immunoglobulin-like receptor sites on the lymphoid cell surface, purified human immunoglobulins were tested for their ability to inhibit the cytotoxic antisera. The evidence obtained from complement and noncomplement dependent systems indicated that the surface components recognized by the antisera did not include immunoglobulins. Addition of IgM to the complement dependent cytotoxicity assay, however, did interfere with the reaction by binding complement.     

Immunosuppressive ALS. II. Antibody to Ia antigens in heterologous anti-lymphocyte serum.

The relationship between Ia alloantigens and xenoantigens detected by immunosuppressive heterologous anti-lymphocyte sera has been investigated. Three rabbit anti-rat lymphocyte sera were examined for the presence of antibodies to Ia antigens. Two of these sera, an anti-thymus membrane and anti-lymphocyte sera detected labelled cell-surface Ia antigens (mol. wt 35,000 and 27,000) present on rat spleen B cells. The third antiserum, prepared against fractionated soluble lymphocyte antigens, was essentially non-reactive with these antigens. Of these three heterologous antisera, the anti-membrane serum reacted with the same antigens detected by two alloantisera. It seemed possible that such an antiserum could modify a recipient animal's immune response in vivo in a fashion identical to alloantibody to Ia antigens. In fact, all three heterologous antisera, including one devoid of antibody to Ia proved immunosuppressive in vivo. These results suggest that antibodies to antigens other than Ia can induce prolonged allograft survival. Since heterologous sera bind Ia antigens, it remains to be determined whether monospecific heterologous antisera to Ia antigens can allograft survival. The results raise the prospect that more than one antibody specificity may contribute to the immunosuppression achieved with ALS.     

Cross-Reactivity of Primate Alloantigens: Absorption of Anti-HL-A Reactivity from Human Alloantisera by Chimpanzee Lymphocytes

Similarity between human and chimpanzee alloantigens was investigated by absorbing human alloantisera with chimpanzee lymphocytes and testing the absorbed antisera with appropriate HL–A-phenotyped human lymphocytes. Chimpanzee lymphocytes were consistently negative in cytotoxicity assays with monospecific human HL–A2, 3, 9, and 13 antisera. Chimpanzee lymphocytes failed to remove reactivity for these specificities in absorption studies. Positive cytotoxic reactions were observed with chimpanzee lymphocytes and anti-HL–A1, 11, 12, and W27 human alloantisera. Absorption with chimpanzee lymphocytes removed reactivity removed reactivity for these specificities as shown by tests with human lymphocytes of the appropriate HL–A phenotype. Non-specific absorption or a CYNAP-type phenomenon was seen in chimpanzee lymphocyte absorptions with anti-HL–A1, 5, and 11 antisera.     

The molecules recognized by anti-MT2 alloantisera and anti-MT2-like (DRw52) monoclonal antibodies are different

The human class II alloantigens include the HLA-DR, DQ, and MT determinants. Previous reports in the literature suggest that while the DQ determinants appear to be on a molecule separate from DR, the MT determinants are variably present on DR or DQ molecules. We have previously reported, using the homozygous DR5 cell line Swei, that the MT4 determinant defined by the allosera, MGH88B, was only on the DQw3 molecule, while MT2, defined by the functionally monospecific anti-MT2 alloantiserum MGH87B, was present on both the DR5 and DQw3 molecules. We now report using the monoclonal antibody ILR2 directed against an MT2-like determinant DRw52, that DRw52 is present on the DR molecules only. The MT2 determinant(s) recognized by the functionally monospecific alloantisera MGH87B appear to include the DRw52 determinant(s) recognized by the monoclonal antibody ILR2.     

The interaction of normal lymphocytes and cells from lymphoid cell lines: IV. HL-A typing of the cell line cells

Cells from thirty-three human lymphoid cell lines and sublines have been typed for HL-A antigens by a microcytotoxicity test. Similar patterns of HL-A antigens were found for the cell line cells and for the fresh lymphocytes of the donor of the line (eleven cases). However, certain typing sera gave positive reactions with the cell line cells which were not found with the fresh lymphocytes. No correlation was noted between the pattern of these `extra' reactions and the HL-A typing of the cells. These same typing sera often gave positive reactions with blood lymphocytes cultured for several days in conventional media. These positive reactions were quantitatively more pronounced and sometimes quantitatively different if the cells had been stimulated. A range of normal sera failed to react with cell line cells suggesting that the HL-A typing sera giving `extra' reactions are detecting antigens in some way related to a histocompatibility system. Absorption studies performed with two of the lines confirmed the HL-A typing by the direct cytotoxicity test. Two sera giving `extra' reactions were also tested in the absorption experiments. The results indicated that antibodies other than those of the HL-A specificity designated for these sera were responsible for the `extra' reactions. It is suggested that `extra' reactions indicate a change in the apparent antigenic expression of lymphoid cells reflecting altered membrane characteristics as they adapt to a culture environment.     

Detection and Quantitation of Human Ia-Type Antigens with Iodinated Protein A and Specific Purification of Antibodies Against Ia-Type Alloantigens

Antibodies directed against specific human Ia-type antigens can easily be detected and quantitated by an improved radioimmunoassay using iodinated protein A bound to a specific antibody-Ia-antigen complex on the surface of freshly drawn peripheral human leukocytes, cultured human cell lines, or lymphoid cells fixed with glutardialdehyde or formaldehyde. The same principle can also be used for the detection of Ia alloantigens on human lymphocytes when testing them with specific antisera known to contain antibodies against transplantation antigens. These anti-Ia-alloantigen antibodies had been purified by a two-step procedure involving ion-exchange chromatography on DEAE-cellulose at pH 6.3 and the specific absorption on formaldehyde-fixed Ia-alloantigen-carrying homozygous cell lines, followed by elution of these antibodies with isotonic citrate buffer at pH 3.0. In this way an about 90-fold purification could be achieved. After such a purification the highly enriched antibody fraction still reacted selectively with one specificity of the Ia antigen system.     

Shared SstI RFLPs by HLA-Aw19, A23/24 and A3/11 crossreacting groups

Three novel restriction fragment length polymorphisms (RFLPs) have been identified using a pan-HLA class I probe and the endonuclease SstI. This study, in conjunction with previously reported SstI RFLPs, now allows the identification of the HLA-crossreacting antigens Aw19 (A29/30/31/32/w33), A23/24 and A3/11 by specific hybridization patterns with a single enzyme/probe combination. Three of the corresponding polymorphic SstI restriction sites map within the HLA-A gene and generate two allelic RFLPs (5.06, 5.92 kb) and one single RFLP (5.92 kb) that show an absolute correlation with HLA-A23/24 and A29/32 crossreacting antigens, respectively. However, other SstI RFLPs (7.97, 9.4, 9.6, 9.8 and 13.34 kb), also linked to HLA-A crossreacting antigens, map outside the HLA-A gene and probably correspond to non-HLA-A,B,C class I genes in strong linkage disequilibrium with the HLA-A gene. These data show that HLA-A crossreacting antigens share more SstI RFLPs than neighboring non-HLA-A,B,C class I genes or pseudogenes; also, this has raised the possibility that some crossreacting HLA-A alloantisera might additionally recognize shared antigenic determinants in non-HLA-A,B,C proteins since the HLA-Aw19 crossreactivity cannot be fully explained by analyzing the HLA-A amino acid sequence.