Expression and Segregation of HL-A Antigens in D981AH-2 by Lymphocyte and Fibroblast Hybrids

Somatic cell hybrids between D98/AH-2 and fibroblasts or peripheral blood lymphocytes were analysed for the expression of HL-A antigens. The hybrids express the antigens of the donor lymphocytes or fibroblasts as well as other HL-A specificities from D98/AH-2, a HeLa derivative on which no HL-A could be detected by direct cytotoxicity. Segregant hybrid clones could be used to determine HL-A haplotypes and define the linkage relationships between these haplotypes and the PGM3 alleles expressed in the hybrids. An analysis of the HL-A antigens on D98/AH-2 and other HeLa derivates was done using absorption and a modified cytotoxicity assay.     

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.     

Human intraspecific somatic cell hybrids: A genetic and karyotypic analysis of crosses between lymphocytes and D98/AH-2

A number of human intraspecific hybrids were produced by fusing the 8-azaguanine-resistant cell line D98/AH -2 with PHA-stimulated lymphocytes from a normal human male, followed by selection in HAT medium. The parent cells differed in zymogram patterns for 4 enzyme systems. Hypoxanthine-guanine phophoribosyltransferase was missing in D98/AH -2 and was determined in the hybrids by the normal gene derived from the lymphocyte donor's X chromosome. The HL-A antigens of the lymphocyte donor as well as the W28 specificity from HeLa were easily recognized by a cytotoxicity assay on the hybrid cells, while D98/AH -2 itself was not killed in the normal way by any HL-A typing sera. The initial hybrid karyotype in all lines was relatively stable, but slow loss of chromosomes occurred following extended growth in culture. The importance of the culture conditions for the rate of chromosome loss was demonstrated. The behavior of several chromosomes was followed in the hybrids and their derivatives. There was relatively nonspecific loss of small numbers of chromosomes, showing that loss of chromosomes from both the D98/AH -2 and the normal lymphocyte parent can occur. Cell lines resistant to 6-thioguanine were selected from the sensitive hybrids. Most had lost the lymphocyte donor's X chromosome, thereby losing the only active allele for HGPRT present in the initial hybrids. However, one line, DMR41, apparently retained the X chromosome and may have a mutated allele for HGPRT. Two lines that are the products of spontaneous segregation are also described: DM4CS and DM17A.     

Ultrastructural localization of human HL-A membrane antigens by use of hybrid antibodies

The localization of HL-A histocompatibility antigens at the surface of human lymphocytes in electron microscopy has been studied using hybrid antibodies to bind electron-dense particles (ferritin and plant viruses) to anti-HL-A antibody. A discontinuous distribution of the markers is observed at the cell surface, which is identical with that described for H-2 antigens on mouse lymphocytes with the same technique. Double labelling experiments suggest that the areas of the cell surface where HL-A antigens are detected contain also the heterologous lymphocyte antigens detected by an anti-thymocyte serum and that HL-A antigens are not renewed at a detectable level during the period of the labelling procedure in the areas of the cell surface which are not labelled primarily with ferritin-anti-IgG-anti-HL-A complexes. The interpretation of the discontinuous labelling of HL-A antigens with direct immunoferritin techniques is discussed.     

The Serologic Detection of HL-A Antigens in Human Milk

The whey prepared from human milk has been shown to contain the HL-A antigens of the milk donor. The inhibition of specific and nonspecific anti-HL-A alloantisera by the antigens in whey was assessed by a microcytotoxicity assay employing ⁵¹Cr release. After centrifugation at 100,000 g for 1 h, significant serologic activity could be demonstrated by the antigens contained in both the insoluble fraction and the soluble fraction. The serologic activity of the HL-A antigens in whey was specific and dose-dependent. The HL-A antigens were not equally represented within the soluble fraction when compared to their distribution in the insoluble fraction, and this may indicate some antigen destruction upon solubilization.     

Solubilization of HL-A Antigens from Peripheral Blood Leukocytes

Peripheral blood leukocytes, isolated by continuous flow leukapheresis and purification, are an excellent source of HL-A antigen. 3M KCI extraction releases the HL-A antigenic mosaic with good yield and specificity. This technique will allow a selective choice of HL-A representation in source material for studies of biochemical characteristics of the HL-A antigens. The technique would allow preparation of antigen from a proposed allograft donor and the pretreatment of the recipient in attempts to induce specific immunoIogic unresponsiveness.     

Solubilization of HL-A Antigens from Peripheral Blood Leukocytes

Peripheral blood leukocytes, isolated by continuous flow leukapheresis and purification, are an excellent source of HL-A antigen. 3M KCI extraction releases the HL-A antigenic mosaic with good yield and specificity. This technique will allow a selective choice of HL-A representation in source material for studies of biochemical characteristics of the HL-A antigens. The technique would allow preparation of antigen from a proposed allograft donor and the pretreatment of the recipient in attempts to induce specific immunoIogic unresponsiveness.     

In vitro cytotoxicity by human lymphocytes from individuals immunized against histocompatibility antigens. II. Relation to HL-A incompatibility between effector and target cells

Lymphocytes from humans immunized by allogeneic skin grafts destroyed fibroblast monolayer cultures derived from the skin donor. Cytotoxicity also developed on several allogeneic fibroblast monolayers from unrelated persons. HL-A typing showed that all of these allogeneic fibroblasts shared one or more HL-A antigens with the skin donor. The intensity of the cytotoxic reaction increased with the number of these antigens present on the fibroblast targets, whereas no reaction occurred on allogeneic targets lacking these antigens or on the autochthonous fibroblasts. It is suggested, therefore, that the cytotoxic reaction reflects immunization against antigens within the HL-A system. An analogous correlation between the response of immunized lymphocytes and the number of immunizing HL-A antigens present was demonstrated in mixed lymphocyte cultures.

Lymphocytes from kidney grafted patients were not cytotoxic to any fibroblasts tested, including those from the kidney donor, not even during periods of clinical rejection. Humoral antibodies directed against the donor cells were demonstrated in one patient, but still no cytotoxicity occurred on the donor fibroblasts. Similarly negative results were obtained with lymphocytes from bone grafted patients.

     

Assessment of the cellular immune response to HL-A antigens in human renal allograft recipients

The cellular response to HL-A antigens has been studied in thirty-one patients who had received a renal allograft from either a cadaveric or living donor, utilizing the leucocyte migration technique. The results indicate that inhibition of migration develops prior to or during the onset of a clinical rejection episode. This inhibition of migration reverts to non-inhibition in autologous serum when the rejection crisis is reversed. Inhibition of migration is still noted in allogeneic serum following this clinical reversal, but after varying time intervals the inhibition reaction also decreases in this serum. The abrogation of inhibition in autologous serum is specific to the HL-A antigens of the donor. These observations suggest that survival of human renal allografts depends on a blocking substance in the serum initially; subsequently, the loss of inhibition of migration with HL-A antigens in both autologous and allogeneic serum suggests an inactivation of specific antigen sensitive cells to the histocompatibility antigens of the donor.     

In vitro assay of cell-mediated immunity: the inhibition of migraton of sensitized human lymphocytes by HL-A antigens

Human blood lymphocytes from donors sensitized by skin grafting were inhibited in their migration from capillary tubes by antigens from the skin graft donor. Lymphocytes from non-sensitized human subjects showed no or a very slight inhibition with similar antigens. When confronted with cell antigens from individuals which did not possess any of the detectable HL-A specificities of the donor, the sensitized lymphocytes exhibited a marginal degree of inhibition (15·3±2·0%). The degree of migration inhibition increased to an average of 24·9±2·0% when the sensitized lymphocytes were confronted with one HL-A antigen similar to that of the immunizing donor. When there were two and three HL-A antigens involved, inhibition increased to 29·4±4·1% and 37·8±4·6%, respectively.     

Repeated Platelet Transfusions from HL-A Compatible Unrelated and Sibling Donors

The results of prolonged platelet transfusions in two patients with aplastic anaemia and thrombocytopenia are presented. One of them has been supported for more than 1 1/2 years with weekly platelet transfusions from an unrelated donor. Lymphocyte typing of donor and recipient revealed incompatibility only for HL-A12, but this antigen was not detected on donor platelets. The other patient had strong multispecific HL-A antibodies in his serum, and only his HL-A identical brother was found compatible. After 10 months of support by platelet transfusions from his brother, the mixed lymphocyte culture test demonstrated strong cellular immunity probably against non HL-A transplantation antigens of the donor. No circulating anti non-HL-A antibodies could be demonstrated with certainty.     

The Assessment of Histocompatibility by Mixed Lymphocyte Reaction as Measured by the Macrophage Electrophoretic Mobility (MEM) Test

The mixed lymphocyte reaction (MLR) between donor and recipient lymphocytes has been measured by the macrophage electrophoretic mobility (MEM) test and the modified (MOD-MEM) test. Its value as a measure of compatibility has been assessed by comparison with conventional HL-A serotyping and with the outcome of renal transplantation. Thirty-six living donor/recipient pairs and 59 cadaver donor/recipient pairs for transplantation have been studied. Whilst uniovular twins gave lymphocyte interactions, measured as macrophage slowings of about 1%, the slowing produced by paired allogeneic lymphocytes ranged from 2% to 26% depending on the number of HL-A matches. The test measurement of lymphocytic interaction was significantly correlated with histocompatibility measured by HL-A serotyping, in both living and cadaver donors. One way MEM-MLR showed the dominant role of the second HL-A sublocus in mixed lymphocyte reactivity. The long term success of the renal graft correlated with the pre-transplant initial reaction between donor and recipient lymphocytes. The test has advantages in the field of human histocompatibility assessment since no particular reference to individual antigens is made and it may be performed in a matter of hours.     

Application of the Fluorescent Antibody Method for the Identification of HL-A Antibodies

A method for the immunofluorescent (IF) identification of antibodies to HL-A antigens on human peripheral blood lymphocytes is described. Pending further study, the detection of pre-existing humoral sensitization to donor alloantigens, particularly weakly reactive or non-complement fixing HL-A antibodies by IF is suggested as a means of augmenting present methodology.     

Chromosome mapping of human cell surface molecules: Monoclonal anti-human lymphocyte antibodies 4F2, A3D8, and A1G3 define antigens controlled by different regions of chromosome 11

Monoclonal antibodies 4F2, A3D8, and A1G3, directed against cell surface antigens present on subsets of human cells, were used to identify the human chromosome regions that code for the antigenic determinants. Human fibroblasts expressed all three antigens, and no cross-reactivity with Chinese hamster or mouse cells was found. Fourteen rodent x human somatic cell hybrids, derived from six different human donors and from two different Chinese hamster and one mouse cell line, were studied simultaneously for human chromosome content and for antibody binding as detected by indirect immunofluorescence. Concordancy with binding of all three antibodies was observed only for human chromosome 11. All other chromosomes were excluded by three or more discordant hybrid clones. Data from six hybrids containing three different regions of chromosome 11 indicate that it is the long arm of chromosome 11 which is both necessary and sufficient for expression of the human antigen defined by 4F2 while the antigen(s) defined by A3D8 and A1G3 map to short arm.     

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.     

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.     

Expression of human CD antigens, including CD1 and CD25, by human x mouse interlineage leukaemia hybrids

The expression of human cell-membrane antigens by hybrid cell lines derived by fusing a human B-ALL and mouse BW 5147 T-lymphoma cells has been studied. Using monoclonal antibodies (mAb), the phenotypes of 19 of the 24 hybrids which grew 11-44 days post-fusion have been analysed by indirect membrane immunofluorescence (IF). These uncloned hybrid cells were assayed early after outgrowth, prior to extensive human chromosome and antigen loss. Nonetheless, cytogenetic analysis showed that all hybrids contained variable numbers of human chromosomes. Phenotypic analysis showed that the hybrids could be grouped as follows: a high frequency expressing CD25 (IL-2 receptor), human T200, HLA class I alpha and beta 2microglobulin, and reacting with the mAb H207 and 12E7; an intermediate frequency expressing CD1 and CD2; and a low frequency expressing CD3, CD4, CD5, CD7, CD8 and CD9. This pattern of antigen expression resembled the frequency of these cells in the human B-ALL parent line. Cell sorting was used to immunoselect hybrids expressing CD1 and CD2, but CD1 expression was unstable during subsequent culture.     

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.     

HL-A TYPING IN DRIED BLOOD STAINS 1. SPECIFICITY AND RELIABILITY OF THE INHIBITION TEST

It is described that HL-A inhibiting substances can be detected in dried human blood stains. An inhibition test is used to evaluate stability and specificity of soluble HL-A antigens in one- and two-stage methods. With few exceptions, the occurrence of inhibition and non-inhibition of stains from different subjects in respect to given HL-A antisera is in agreement with specificities present on the donor's cells. Some‘weak inhibitors’were observed. Extra-reactions with given antisera can be explained by cross-inhibition. One- and two-stage methods are evaluated with regard to the inhibition test for human blood stain HL-A typing.     

Lymphopenic immunologic deficiency in identical twins: Lymphocyte allografting and graft-versus-host disease following treatment with albumin-gradient-separated paternal bone marrow cells

The clinical, immunologic and pathologic features of the first recorded examples of lymphopenic immunologic deficiency in twins are presented. Eleven-month-old male identical twin infants were found to be severely lymphopenic and lacked demonstrable cell-mediated immunity and antibody formation. Each first-degree relative differed from the probands by one chromosome at the major human histocompatibility locus, HL-A. Because of their rapidly deteriorating clinical conditions, allogeneic bone marrow transplantation was undertaken. Circumvention of graft-versus-host disease was attempted by separation of donor bone marrow cells on a discontinuous-albumin-gradient and administration of only 5×10⁶ immature nucleated marrow cells per kilogram infant body weight. Additionally, immunologic enhancement was attempted by pretreating the infants with human isoantisera to identifiable infant HL-A antigens not present in the marrow donor. Paternal lymphocyte allografting occurred, as demonstrated by lymphocyte cytotoxicity testing, but both infants succumbed to graft-versus-host disease at the end of 3 weeks.