Antibody-induced redistribution of HL-A antigens at the cell surface

The in vitro binding of anti-HL-A antibodies to the membrane of human lymphocytes induces important changes in the distribution of HL-A antigens on the cell surface. Following either direct or indirect immunofluorescence staining at 0 °C, cell-bound anti-HL-A antibodies are dispersed all over the cell surface. When the washed, stained lymphocytes are warmed and incubated at 37 °C, fluorescent antibodies cluster progressively at the cell surface. They form large spots of fluorescence, and sometimes single caps at one pole of the cell, outside which HL-A antigens are no longer detectable, but other antigens can still be found. Similar findings were made in electron microscopy, following indirect labeling of HL-A antigens on human lymphocytes, with ferritin or plant virus as the markers. With the indirect immunofluorescence technique, anti-human thymocyte antibodies and anti-mouse H-2 antibodies were found to induce a similar redistribution of the corresponding antigens. The mechanism and the interpretation of this displacement of surface antigens by antibodies are still unclear and are discussed in terms of membrane structure and immunological significance.     

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.     

Spontaneous Release of Soluble HL-A Antigens from Platelets During Conservation

Experiments with the aim of studying the solubilisation of HL-A antigens from blood platelets by methods which do not involve any biologically active processes (moderate, discontinuous agitation of a low concentration of platelets suspended in a saline medium, in the presence of an antiseptic; supernatants collected at frequent intervals) have shown that platelets release membrane proteins, including HL-A antigens, spontaneously. Optimal conditions for the treatment of membrane proteins have been perfected. The great stability of HL-A antigens under these conditions permits prolonged treatment. The products extracted are soluble and extremely complex. The molecular weight of the HL-A antigens is between 40,000 and 70,000.     

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.     

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.     

Spontaneous release of soluble HL-A antigens from platelets during conservation.

Experiments with the aim of studying the solubilisation of HL-A antigens from blood platelets by methods which do not involve any biologically active processes (moderate, discontinuous agitation of a low concentration of platelets suspended in a saline medium, in the presence of an antiseptic; supernatants collected at frequent intervals) have shown that platelets release membrane proteins, including HL-A antigens, spontaneously. Optimal conditions for the treatment of membrane proteins have been perfected. The great stability of HL-A antigens under these conditions permits prolonged treatment. The products extracted are soluble and extremely complex. The molecular weight of the HL-A antigens is between 40,000 and 70,000.     

Spontaneous Release of Soluble HL-A Antigens from Platelets During Conservation

Experiments with the aim of studying the solubilisation of HL-A antigens from blood platelets by methods which do not involve any biologically active processes (moderate, discontinuous agitation of a low concentration of platelets suspended in a saline medium, in the presence of an antiseptic; supernatants collected at frequent intervals) have shown that platelets release membrane proteins, including HL-A antigens, spontaneously. Optimal conditions for the treatment of membrane proteins have been perfected. The great stability of HL-A antigens under these conditions permits prolonged treatment. The products extracted are soluble and extremely complex. The molecular weight of the HL-A antigens is between 40,000 and 70,000.     

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.     

Escape from Sensitization to HL-A Antibodies

Human lymphocytes sensitized with HL-A antibodies could not be killed by the addition of complement if the lymphocytes were incubated for periods of 1 to 5 hours at 37° C. This phenomenon was not produced by simple dissociation of the antibodies or by loss of antigens (modulation), but rather is postulated to result from an active release or pinocytosis of HL-A antigens together with the attached antibody. "Escape from sensitization" is followed closely by reformation of antigens, for the cells can readily be resensitized and killed by addition of complement. Loss of sensitization is an active process, since one-day-aged lymphocytes or lymphocytes treated with actinomycin D or puromycin were unable to express this activity. A differential escape rate for different specificities was encountered indicating that HL-A9 was produced more rapidly than several other specificities present on the same lymphocytes. A correlation was noted between the rate of HL-A antigen synthesis by lymphocytes (indicated by escape rate) and the quantity of HL-A antigen in the serum.
Inhibition of lymphocyte cytotoxicity was used to detect the presence of HL-A antigens in serum. Certain specificities, such as HL-A9, were generally present in serum whereas others, such as HL-A8, could not be detected. Cross-inhibition tests with over 100 sera clearly showed an association of inhibition with specificity, although nonspecific inhibition was often observed. Sephadex fractions of sera tested at different concentrations revealed the greatest inhibitory activity in the 19S and 7S fractions.     

Independent expression of the two HL-A antigen polypeptide chains.

It is now well established that beta2-microglobulin constitutes one of the two HL-A antigen subunits. In this study support was obtained for the previous notion that the human lymphoma Daudi does not produce beta2-microglobulin (beta2m). Papain-solubilized as well as Nonidet P-40-solubilized Daudi HL-A antigens do not contain any beta2m or any detectable structural analogue of this protein. The chemical and physico-chemical characteristics of highly purified HL-A antigens derived from Daudi cells are indistinguishable from those of the HL-A antigen-carrying polypeptide chain isolated from the P3HRIK cell line. Like P3HRIK-derived HL-A antigens, the HL-A antigens derived from Daudi cells are composed of two identical heavy, alloantigenic polypeptide chains with a molecular weight of about 50 000 each, which are held together by disulfide bridge(s). The HL-A antigens of P3HRIK cells contain, in contrast to Daudi HL-A antigens, two molecules of beta2m. Although no evidence was obtained suggesting any beta2m synthesis in Daudi cells it was apparent that these cells express the HL-A alloantigenic polypeptide chain in amounts similar to those of other cell lines which produce beta2m. The present data suggest [1] that beta2m and the alloantigenic HL-A polypeptide chain are under separate genetic regulation [2], that the cell surface integration of the HL-A antigen-carrying polypeptide chain is independent of the presence of beta2m and [3] that beta2m does not constitute a membrane component absolutely necessary to the integrity of the cell membrane.     

Lymphocyte transformation in vitro. III. Mechanism of stimulation in the mixed lymphocyte culture

In view of the correlation between the mixed lymphocyte culture and the HL-A locus, experiments were performed to determine whether the reactivity in the mixed lymphocyte culture is elicited by the HL-A antigens as such.

Fresh lymphocytes were mixed with either allogeneic fibroblasts or allogeneic lymphocytes treated with various metabolic inhibitors or by heating which abolishes their capacity to tranfsorm in vitro.

No stimulation of the normal untreated lymphocytes was observed in any of these mixed cultures.

However, the HL-A antigens 4b and 7b were not affected quantitatively by this treatment as determined serologically.

The conclusion is drawn that mixed lymphocyte culture reactivity is not merely a reaction to HL-A antigens, and the consequences of this are discussed.

     

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.     

Independence of HL-A antigens and immunoglobulin determinants on the surface of human lymphoid cells

Membrane-bound HL-A antigens and μ-chain determinants were studied on normal and leukemic lymphocytes and on a tissue culture cell line derived from a Burkitt's tumor, using membrane immunofluorescence on living cells. A redistribution of both antigens at the cell surface occurred when cells labeled in the cold by conjugated antisera were incubated at 37 °C. Double labeling experiments showed that HL-A antigens, either of the first or the second sublocus, were unaffected by the redistribution of IgM determinants and vice versa. This result strongly suggests molecular independence of these two systems. When redistribution was induced with a mixture of conjugated sera to HL-A and Ig, the caps showing each of the two specificities were located at the same pole of the cells.     

Independent expression of the two HL-A antigen polypeptide chains

It is now well established that β₂-microglobulin constitutes one of the two HL-A antigen subunits. In this study support was obtained for the previous notion that the human lymphoma Daudi does not produce β₂-microglobulin (β₂m). Papain-solubilized as well as nonidet P-40-solubilized Daudi HL-A antigens do not contain any β₂m or any detectable structural analogue of this protein. The chemical and physico-chemical characteristics of highly purified HL-A antigens derived from Daudi cells are indistinguishable from those of the HL-A antigen-carrying polypeptide chain isolated from the P₃HRIK cell line. Like P₃HRIK-derived HL-A antigens, the HL-A antigens derived from Daudi cells are composed of two identical, heavy, alloantigenic polypeptide chains with a molecular weight of about 50000 each, which are held together by disulfide bridge(s). The HL-A antigens of P₃HRIK cells contain, in contrast to Daudi HL-A antigens, two molecules of β₂m. Although no evidence was obtained suggesting any β₂m synthesis in Daudi cells it was apparent that these cells express the HL-A alloantigenic polypeptide chain in amounts similar to those of other cell lines which produce β₂m The present data suggest [1] that β₂m and the alloantigenic HL-A polypeptide chain are under separate genetic regulation [2], that the cell surface integration of the HL-A antigen-carrying polypeptide chain is independent of the presence of β₂m and [3] that β₂m does not constitute a membrane component absolutely necessary to the integrity of the cell membrane.     

HL-A antigens on man-mouse hybrid cells

Cultures of hybrid cells produced by fusion of human peripheral leukocytes with murine cell cultures were studied by means of mixed agglutination tests. All three hybrids studied contain human species-specific antigens. HL-A antigens corresponding to the phenotype of the leukocyte donor were demonstrated in two hybrids. With one of these hybrids, cloning experiments were performed. Five of six clones contained HL-A antigen controlled by one haplotype. In the remaining clone, which did not have human species-specific antigens, no HL-A antigens were detected. The third hybrid contained human alloantigens, but none of the HL-A antigens detected on the donor leukocytes.     

HL-A antigens in juvenile rheumatoid arthritis.

HL-A antigens were examined in sixty-two patients, thirty-one boys and thirty-one girls, diagnosed as having JRA. HL-A 27 was the only antigen with a significantly increased frequency. This increase concerned predominantly male patients in whom the disease developed at the beginning of puberty. In this group of sixteen boys, the polyarticular form was more frequent and tended to be associated with sacroliitis, while the rheumatoid factor was negative in all but one. The frequency of HL-A 27 in this group was 65.5%. The patients were also examined by the lymphotoxic test with anti H-2 alloimmune sera known to exhibit a high degree of association with some HL-A antigens. On JRA lymphocytes these associations were confirmed for HL-A 2 and anti-H-2f and for HL-A 27 and anti-H-2p. The strongest reactions were observed with lymphocytes from male patients around the age of puberty. These data indicate that steroid hormones influence the expressivity of some HL-A associated cell plasma membrane structures.     

Observations on the cytotoxicity of lymphocytes to a target cell system

It is known that after mixed culture lymphocytes exhibit an increase in cytotoxicity to a target cell. The present study was undertaken to see if the degree of cytotoxicity was also related to HL-A differences.

A cytotoxic effect was found where there was antigenic disparity. The degree was unrelated to the number of different antigens at the HL-A locus but cytotoxic effect was absent in cultures where there was complete HL-A identity using lymphocytes from twins, siblings and unrelated persons.

     

Serological studies of HL-A on continuous lymphoblastoid cell lines (CLC) and the definition of an antigen on CLC determined by a heat-labile antibody

Continuous lymphoblastoid cell lines (CLC) are more reactive with HL-A antisera in a complement-dependent cytotoxic test than are peripheral blood lymphocytes (PBL). This additional reactivity leads to assignment to a given CLC of more than four HL-A antigens, the maximum allowable under the two locus concept of the genetic control of HL-A. However, absorption of antisera by CLC shows that no more than four HL-A antigens exist on any of the CLC used in this laboratory. The additional reactivity of these cells lines can be explained in three ways. Firstly, it may be due to the presence of sublytic amounts of HL-A antibody in operationally monospecific antisera. Secondly, it may be due to cross-reactivity between HL-A antigens. Both these findings can be explained on the basis of the increased quantity of HL-A antigens on CLC compared to PBL. Thirdly, it may be due to the presence of a heat-labile (56° for 30 min) complement-dependent cytotoxic antibody which is present in 90% of normal human sera, and detects an antigen group tentatively labelled `D'.     

Expression of HL-A Antigens on the Surface of Cultured Human Lymphoid Cells: Effect of Inhibitors of Protein and Nucleic Acid Synthesis

Cultured human lymphoid cells (WI-L2), when incubated in mid-log phase with different concentrations of puromycin, showed a 70% decrease in the expression of HL-A antigens on their surface as measured by quantitative microabsorption. This decrease in surface antigen expression was accompanied by a marked inhibition of the rate of incorporation of ¹⁴C leucine into cellular polypeptides suggesting a correlation between protein synthesis and HL-A antigen expression on cultured human lymphoid cells. The inhibitory effects of puromycin on HL-A expression were reversible. Actinomycin D did not affect HL-A antigen expression, suggesting that the messenger RNA coding for these antigens is relatively long-lived. Cytosine arabinoside, 5-bromodeoxyridine, ethidium bromide and rifampicin did not affect HL-A antigen expression. By contrast, cycloheximide at a concentration which inhibited the rate of polypeptide synthesis by at least 95% did not change HL-A antigen expression. The differential effects of puromycin and cycloheximide on the synthesis of HL-A polypeptides are similar to the effect of these drugs on mitochondrial protein synthesis.     

Permanent Lymphoid Lines from Genetically Marked Lymphocytes: Success with Lymphocytes Recovered from Frozen Storage

Permanent human lymphoid cell lines were established successfully from peripheral blood lymphocytes which had been separated for HL-A typing and stored in liquid nitrogen for two years. Frozen lymphocytes were chosen from two siblings who were homozygous at the LA and FOUR HL-A loci. Thawed lymphocytes were transformed with EB virus produced by the marmoset lymphoid line B95-8. No chromosome abnormalities were seen on karyotypes prepared on cells from the established human lymphoid lines using G and Q banding techniques. HL-A typing showed the expected HL-A antigens plus a considerable number of additional reactions. Separation of lymphocytes and freezing them for possible future use requires a relatively small investment. This method of preserving cells can be applied to patients with interesting genetic disorders or other biochemical markers to provide cells which can be transformed and propagated years later.