ADCC of Cultured Human Melanoma Cells: Analysis with Monoclonal Antibodies to Human Melanoma-associated Antigens

Four monoclonal antibodies to human melanoma-associated antigens (MAA) were found to be able to mediate specific cell-dependent lysis (ADCC) of cultured human melanoma cells. The extent of specific lysis of melanoma cells was influenced by the effector to target cell ratio, the amount of antibody added to the reaction mixture, and the incubation time. Cultured melanoma cells treated for 16 h with puromycin or cycloheximide (final concentration, 1.0 μg/ml) displayed increased Susceptibility to ADCC even though the cell surface expression of MAA was not changed. On the other hand, treatment of melanoma cells with tunicamycin (final concentration, 1.0 μg/ml) reduced the expression of MAA but did not affect susceptibility to ADCC. These findings suggest that other properties of the target cell membrane besides antigen density play a role in the outcome of ADCC reaction.     

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.     

Protein synthesis is required for expression of anthrax lethal toxin cytotoxicity.

Anthrax lethal toxin, which is composed of two proteins, i.e., protective antigen and lethal factor, is cytolytic to mouse peritoneal macrophages and the macrophage-like cell line J774A.1. After exposure of cells to lethal toxin, inhibition of protein synthesis occurred only slightly before the onset of cytolysis. Thus, cell death did not appear to be due to inhibition of protein synthesis. However, prior treatment of J774A.1 cells with cycloheximide or puromycin, which inhibited protein synthesis, protected them completely against lethal toxin-induced cytolysis, which suggested that continuous protein synthesis is required for the expression of lethal toxin activity. Inhibition of protein synthesis had no appreciable effect on the binding of protective antigen to the cell surface receptor or on proteolytic cleavage of surface-bound protective antigen. Furthermore, inhibition of protein synthesis did not alter the uptake of toxin, which suggested that protein synthesis is required at a later stage of the intoxication process. The protection provided by inhibition of protein synthesis was effective, even up to 1 h after exposure to anthrax lethal toxin. The increased uptake of calcium observed in cells exposed to lethal toxin did not occur when they were protected by blocking protein synthesis. Identifying the protein(s) synthesized during the intoxication process may help to understand the mechanism of cell death produced by anthrax lethal toxin.     

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.     

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.     

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.     

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.     

SPECULATIONS ON THE FUNCTION OF IMMUNE RESPONSE GENES

Immune response (Ir) genes linked to genes encoding histocompatibility antigens affect antibody-formation to synthetic polypeptides, weak native antigens and strong native protein antigens given in limiting doses. It has been proposed that the products of such Ir genes function as receptors for antigen on thymus-derived lymphocytes (T-cells) and these products are not immunoglobulin in nature. Ho wever, recent evidence indicates that (a) Ir genes are probably expressed in both T-cells and in bone-marrow-derived lymphocytes (B-cells); (b) IgM immunoglobulin is present on T-cells and functions in the binding of antigen, and (c) T-cells of nonresponder animals possess the capacity to recognize antigen. These results prompted us to consider alternative hypotheses for the function of Ir genes; namely (1)‘tolerance’hypotheses in which response is dominant over non-response, and (2) the possibility that the Ir gene product can act as an amplifier of T-cell function. These hypotheses are discussed within the context of murine studies and in relation to the association of human diseases, notably immunopathic phenomena, with HL-A.     

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.     

Protein synthesis in Japanese encephalitis virus-infected cells

We studied the effects of actinomycin D, cycloheximide, and puromycin on virus-specified protein synthesis in Japanese encephalitis (JE) virus-infected chick embryo and LLC-MK₂ (rhesus monkey kidney) cells. To prove that the radioactively labeled proteins we had previously identified in infected chick cells were virus-specified, we showed that they electrophoretically comigrated with radioactively labeled proteins from infected, but not from uninfected, LLC-MK₂ cells. We found that the maximum value for the ratio of protein synthesis in infected chick cells to uninfected cells occurred when the cells were treated with actinomycin D and were pulse-inhibited with cycloheximide. Alone, actinomycin D treatment decreased the background radioactivity of high molecular weight in electropherograms of infected chick cells and allowed virus-specified proteins to be prominent. Cycloheximide pulse-inhibition of infected, actinomycin D-treated cells decreased total cellular protein synthesis and slightly decreased the background radioactivity in electropherograms without changing the distribution of radioactivity among virus-specified proteins. Neither drug treatment decreased the yield of infectious virus. These results differ in some respects from the related results of Trent and Qureshi (1971). In contrast to our results with cycloheximide, pulse-inhibition of infected chick embryo cells with puromycin inhibited the synthesis of polypeptides NV-5, NV-4, and NV-1 (virus-specified nonstructural, nonglycosylated proteins) to a greater extent than that of V-3, NV-3, NV-2, and V-2 (virus-specified glycosylated and/or structural proteins). It also generally inhibited the synthesis of large proteins relative to small ones.We then studied the effects of puromycin and cycloheximide in LLC-MK₂ cells. In contrast to our results in chick embryo cells, pulse-inhibition of infected LLC-MK₂ cells with either drug (in the continuous presence of actinomycin D) did not alter the pattern of virus-specified proteins in electropherograms from that obtained without pulse-inhibition. Treatment with continuous levels of either drug (in the presence of actinomycin D) did, however, alter the protein pattern by differentially inhibiting the synthesis of nonstructural, nonglycosylated proteins. By labeling infected cells with one of eight different amino acids, we were unable to find an unusually enriched (or lowered) amino acid content that was common to all nonstructural, nonglycosylated proteins. A possible explanation for the differential inhibition is that the virus directs the formation of functionally different polyribosomes, messengers, or initiation factors which vary in their susceptibility to low levels of inhibitors of protein synthesis.     

Antibodies to surface IgM and IgD increase the expression of various class II antigens on human B cells

Antibodies to surface IgM and IgD were found to induce increased expression of class II antigens on normal and neoplastic human B cells within 24 h of stimulation. Antigens associated with different class II sub-locus genes (DC, DR and SB) were all found to be increased as determined by monoclonal antibodies (Leu-10 and B 3/4 for DC, D 1/12 for DR and MHM4 for SB-associated antigens). The increased expression of class II antigens was selective as anti-immunoglobulins failed to increase expression of other surface antigens such as B1 and beta 2-microglobulin. The effect of anti-mu and anti-delta could be blocked specifically by corresponding myeloma proteins suggesting that antibodies to surface IgM and IgD, respectively, were responsible for the effect observed. Moreover, antibodies to another surface antigen (B1) failed to induce such changes. Increased class II antigen expression appeared to be dependent on protein synthesis, and early changes in ion fluxes, but could not be elicited by membrane depolarization as reported in murine systems.     

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.     

Alteration of the HL-A Antigenic site in Situ

The chemical nature of the HL-A antigenic sites on peripheral blood lymphocytes was studied by treatment of these cells with glycolytic enzymes and with sodium metaperiodate, and monitoring the residual antigen expression either by the lymphocyte cytotoxicity test or by quantitative microabsorption of monospecific anti-HL-A sera. Neither the gentle enzymatic nor the chemical treatment of lymphocytes altered expression of the HL-A antigens indicating that carbohydrates are not involved, in a major way, in the HL-A antigenic sites.     

Alteration of the HL-A antigenic site in situ.

The chemical nature of the HL-A antigenic sites on peripheral blood lymphocytes was studied by treatment of these cells with glycolytic enzymes and with sodium metaperiodate, and monitoring the residual antigen expression either by the lymphocyte cytotoxicity test or by quantitative microabsorption of monospecific anti-HL-A sera. Neither the gentle enzymatic nor the chemical treatment of lymphocytes altered expression of the HL-A antigens indicating that carbohydrates are not involved, in a major way, in the HL-A antigenic sites.     

THE INHERITANCE OF HL-A ANTIGENS IN MYASTHENIA GRAVIS

Thirty-one patients with myasthenia gravis were studied for their HL-A antigen expression. Eighteen patients had family members available for HL-A typing, and full genotyping for HL-A antigens was possible. The inheritance of HL-A8 was studied in detail, and it was noted that this did not correlate exactly with the development of myasthenia gravis. The multifactorial nature of the genetic component of this disease was confirmed by these studies.     

Alteration of the HL-A Antigenic site in Situ

The chemical nature of the HL-A antigenic sites on peripheral blood lymphocytes was studied by treatment of these cells with glycolytic enzymes and with sodium metaperiodate, and monitoring the residual antigen expression either by the lymphocyte cytotoxicity test or by quantitative microabsorption of monospecific anti-HL-A sera. Neither the gentle enzymatic nor the chemical treatment of lymphocytes altered expression of the HL-A antigens indicating that carbohydrates are not involved, in a major way, in the HL-A antigenic sites.     

Phosphorylation of a ribosomal protein and of virus-specific proteins in cells infected with herpes simplex virus.

In cells infected with herpes simplex virus a protein associated with the small subunit of ribosomes became phosphorylated. It was not detectably labelled with 14C-amino acids added after infection and is therefore probably a cellular protein. The phosphorylated ribosomal proteins from HSV-1- and HSV-2-infected cells were indistinguishable electrophoretically and had an apparent mol. wt. of about 48 000. Phosphorylation of the 48K protein was detected 2 to 3 h after infection and reached a maximum rate at 4 to 5 h. It was prevented by adding cycloheximide at 2 h, or actinomycin at 1.5 h p.i., or azetidine at the beginning of infection. The phosphorylation did not occur on reversal of a cycloheximide block in the presence of actinomycin, confirming that it is not caused by a virus alpha-polypeptide. Virus that had been irradiated with u.v. light, although still able to suppress synthesis of cellular protein and DNA, did not induce phosphorylation of the 48K ribosomal protein. Therefore the phosphorylation is not responsible for the suppression of host synthesis. The alpha polypeptides ICP 4, 0, 22 and 27 are also phosphorylated but, in contrast to that of the ribosomal protein, their phosphorylation does not depend on the synthesis of beta and gamma polypeptides. It is probably mediated by a host enzyme.     

Common antigenic structures of HL-A antigens: II. Small fragments derived from papain-solubilized HL-A antigen molecules

A distinctive fragment of the HL-A antigen molecule which retains characteristic structural features of the intact molecule has been obtained from soluble HL-A antigens by mild degradation procedures. When molecular fragments of 48,000 Daltons which are derived from cultured human lymphoid cells and carry HL-A alloantigenic activity are exposed to a pH of 2.4 in glycine buffer for 15 minutes at 0–4°, they split and a fragment of 11,000 Daltons can be isolated by gel filtration. This fragment does not carry HL-A alloantigenic activity but does carry HL-A common activity. This common activity had previously been found on papain-solubilized HL-A antigens as well as on intact lymphoid cells. It is immunogenic in rabbits and reacts with antiserum raised in rabbits against lymphoid cell membrane fractions. These small fragments appear to be characteristic, common structural components of HL-A antigen molecules of 48,000 Daltons and hence are designated `HL-A common-portion fragments'. Similar molecular fragments are found in solutions of the HL-A molecular fragments of 48,000 Daltons that have been stored in buffer at pH 7.8 at 4° for some time.     

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.