Characterization of an antigen from the myelogenous leukemia cell line K-562.

A protein was solubilized from the myelogenous leukemia cell line K-562 WITh 3 M KCl that specifically inhibited the antibody-dependent, complement-mediated cytolysis of 51Cr-labeled K-562 cells by a monkey antiserum to K-562. When the crude 3 M KCl extract uas fractionated with ammonium sulfate, an eightfold increase in specific activity (U inhibition/mg protein) resulted. This purified fraction migrated as a single protein band after polyacrylamide gel electrophoresis (PGE) with no detectable carbohydrate or lipid. The molecular weight of the denatured protein determined by sodium dodecyl sulfate-PGE was 77,000, similar to that of the native protein (80,000) determined by Sephadex exclusion chromatography. The protein was stable at pH 6-8, with an apparent isoelectric point between pH 5 and 6. In addition to being irreversibly denatured at pH 5 or less, it was unstable at osmolarities below 0.25 M (NaCl). It was denatured at temperatures of 56 degrees C or above. Normal human peripheral blood leukocytes were extracted similarly with 3 M KCl and fractionated with ammonium sulfate. Neither the crude preparation nor any fraction purified as described for the specific antigen inhibited the cytolytic assay, which indicated at least a quantitative lack of the protein on the surfaces of normal leukocytes.

Cytotoxicity of antisera to a myelogenous leukemia cell line with the Philadelphia chromosome.

Rabbit antisera to myelogenous leukemia (ML) cells were raised; ML cells from line K-562 that has the Philadelphia (Ph) chromosome were used as antigen. Antibodydependent, complement-mediated cytotoxicity was demonstrated by the trypan blue test and Cr release assay for cultured ML cells, whereas no cytotoxicity was demonstrated for cells from B (SB) and T (MOLT 4) lymphoblastoid cell lines. The antisera showed no cross-reactivity for normal human peripheral leukocytes or purified granulocytes. A low level (less than 8%) of cytotoxicity was directed against cell membrane associated fetal bovine serum proteins. Absorption of the immune serum with normal human bone marrow cells of first trimester human whole embryo cells reduced the cytotoxic titer to a similar extent; this suggested the possibility of crossreactivity between ML cells and fetal antigen(s). However, the ML antigen(s) was unrelated to carcinoembryonic antigen (CEA), since absorption with CEA had no effect on the serum cytotoxic titer. The anti-ML sera were cytotoxic for cells taken from 10 patients with chronic myelogenous leukemia and from 3 with acute myelogenous leukemia. In contrast, the leukocytes of 1 of 4 patients with acute lymphocytic leukemia, and 3 of 7 with chronic lymphocytic leukemia shared similar antigenic determinants as demonstrated by cytotoxicity tests. The significance of the cross-reactivity of some lymphatic and ML cells may be the result of the use of rabbit sera that did not distinguish antigens common to both granulocytic and lymphocytic cells, or it may reflect an "immature" or "blastic" antigen present on many leukemia cells.

Antibody lysis of human hematopoietic cells in the absence of complement and effector cells.

The incubation of K-562 leukemia cells with specific goat immunoglobulin resulted in gradual cytolysis in the absence of complement and effector cells. Optimal lysis (100%), reached in 3-4 days, occurred when the concentration of the antibody in the culture medium was about 20 micrograms/ml containing an initial inoculum of 15,000 cells. A lower concentration (10 micrograms/15,000 cells/ml) led to partial cytolysis; the surviving cells, cultured in fresh medium, were still vulnerable to antibody lysis (30 micrograms/ml), thus indicating the lack of an apparent resistant cell population. The amount of free antibody in the culture medium diminished rapidly and very little or none was detectable after 2-3 hours of incubation with K-562 cells. The immune gamma-globulin also inhibited colony formation of K-562 cells in soft agar. The immune gamma-globulin, absorbed extensively with normal blood cells, to pluripotent K-562 cells, cross-reacted with several human malignant hematopoietic cell lines tested. These cells included T-lymphoblasts (JM and MOLT-4), promyelocytes (HL-60), Burkitt's lymphoma cells (RAJI), myeloblasts (KG-1 and KG-1a), and multiple myeloma cells (K-737). The absorption of the immune gamma-globulin with an equal number of cells from each of the cell lines assayed diminished but did not completely remove the antibody responsible for cytolysis of K-562 target cells. Thus, the K-562 leukemia blasts appear to possess common and specific antigens that may be only partially expressed on other committed leukemia cell lines studied. For this reason, the cytolytic activity of the immune gamma-globulin could only be removed by absorption with K-562 cells possessing a complete array of antigens solely present on pluripotential hematopoietic cells.

Involvement of cytoplasmic membranes in the non-lytic infection of K-562 cells by Semliki Forest virus.

An analysis of the human leukemia cell line, K-562, infected with Semliki Forest virus, has been made with transmission electron microscopy. In contrast to the usual surface budding of the enveloped virus on the plasma membrane of vertebrate cells leading to cytolysis within 20 h, K-562 cells do not show surface budding, and the cells remain intact for periods of several months. Several unusual features of the infection include: 1) the rough endoplasmic reticulum arranges early into continuous perinuclear chains; 2) during the time of virus replication and release, the nucleocapsids aggregate on the cytoplasmic side of internal vesicles in the region of the cell where the Golgi complex is normally located; and 3) during this same time period, the vesicles are seen to contain enveloped virions and rod-like formations, a result suggesting that budding has occurred into these vesicles. Viruses are presumably released from the cell as these vesicles fuse with the plasma membrane. By 12 days post-infection and thereafter, the intact cells show electron-dense aggregates of chromatin, large vacuoles and lipid inclusions throughout the cytoplasm, and only a few virion-containing vesicles.

Monoclonal antibodies that cross-react with the E1 glycoprotein of different alphavirus serogroups: characterization including passive protection in vivo.

A panel of four monoclonal antibodies (mAb) were produced that cross-react with representatives of two different togavirus serogroups, namely sindbis (SIN) and Semliki Forest (SF) viruses, by ELISA and ADCMC assays. Three of these mAb, IgG2a and IgG2b isotypes, passively protected C3H/Hej mice against 10 and 100 LD50 of SF challenge and one, IgM, did not protect against either challenge dose, or even at 1 LD50. All these mAb were cross-reactive with the E1 glycoprotein of the viruses by immunoblotting in which three different patterns of reactivity were evident, suggesting that three epitopes were involved. The patterns depended upon whether the mAb recognized E1 extracted from purified virions or infected cells and whether SDS-PAGE and immunoblotting were done in the presence or absence of beta-mercaptoethanol. One mAb (IgM) reacted with nonreduced or reduced E1 from either virions or cells suggesting recognition of a linear epitope. The other three mAb reacted with nonreduced but not reduced E1 from virions suggesting that recognition depends upon conformational epitopes. These three mAb reacted also with nonreduced E1 extracted from SF-infected cells whereas only one reacted with nonreduced E1 extracted from SIN-infected cells.

Cell death in the human leukemia cell line, K-562, induced by antiserum and monoclonal antibodies.

Rabbit polyclonal antiserum, or derived gamma globulin, to K-562 cells induces decreased TdR uptake within hours and cell death without cytolysis in 2-4 days. A panel of nine mAb, reactive with K-562 cells, was grouped on the basis of no effect on growth or TdR uptake, increased uptake, or decreased uptake. Treatment of cells with antiserum, gamma globulin, or mAb of the last group caused single-strand, but not double-strand, DNA fragmentation at a time when the cells were still viable. Cycloheximide did not inhibit the antibody effect suggesting that protein synthesis was not required. Aurintricarboxylic acid at certain concentrations markedly enhanced TdR uptake and protected the cells when antiserum was used but did not protect from mAb treatment.

Diversity of cell surface hematopoietic antigens on K-562 sublines identified with monoclonal antibodies.

The surface antigen profile of 8 sublines of K-562 cells, the original line, and the clone RA6 was determined with a panel of 12 monoclonal antibodies reactive with hematopoietic cell differentiation antigens. Cells from all sublines expressed the precursor hematopoietic antigen reactive with RFB-1, the T-cell antigen reactive with OKT17, the B cell/granulocyte antigen reactive with BA-1, the My-1 antigen, and glycophorin A which reacted with R10. A low percentage of cells in some of the sublines expressed platelet/monocyte glycoprotein I binding AN51, monocyte antigen binding 63D3, and an erythroblast/monocyte/platelet antigen binding 5F1. The use of a panel of K-562 sublines demonstrates that K-562 cells do share several "common" antigens but express a marked diversity and variability of other hematopoietic antigens.

Continuous or modulation expression of hematopoietic cell antigens in sublines of the leukemia cell line, K-562.

K-562 is described as a pluripotent leukemic cell line which expresses a diversity of hematopoietic cell differentiation antigens. With the use of monoclonal antibodies (MoAb), we show that the expression of these antigens is either "modulated", i.e. they are not expressed in early logarithmic growth but are expressed in late logarithmic growth, or "continuous", i.e. they are not affected by proliferation and phase of culture growth. Whether an antigen is modulated or continuously expressed appears to be an inherent property of the subline, since the expression of myeloid antigen binding the MoAb, PM81, was modulated on cells from a clone, whereas, the expression of this antigen was continuous by the parent subline and an independent subline F-1. Continuously expressed antigens appear to be an integral component of the K-562 cell surface matrix, while modulated antigen expression appears to be influenced by culture growth conditions.

Levels of homocytotropic antibody in hereditarily asplenic, splenectomized and normal mice.

No significant differences were detected in circulating levels of IgE or IgG1 in hereditarily asplenic (Dh/+) or splenectomized mice compared to normal controls (+/+). These findings suggest that the spleen plays a minor role in the regulation of IgG1 and IgE production in this mouse line.

Comparison of three hemolytic plaque assays using sheep and mouse erythrocytes as antigens in rats.

Several hemolytic plaque assays using spleen cells from rats immunized with sheep or mouse erythrocytes were compared. Modifications of the assays were made to define optimal conditions. Mrbc in assays with guinea pig complement yield a much reduced number of plaques than is observed when human complement is used. In contrast, no difference in numbers is noted with Srbc regardless of complement source. The use of Srbc in an assay technique that does not employ agarose is more sensitive than techniques that require agarose; the result is a 50-100% increase in the number of plaques. This increase in plaque number was observed with either guinea pig or human complement. In contrast, there was considerable clumping of Mrbc in the absence of agarose which tended to obscure distinct plaque formation. Increased sensitivity of plaque development for Srbc in the absence of agarose was due to increased numbers of direct (IgM) plaque detection and not to concomitant detection of cells producing IgG (indirect plaques). Maximal detection of plaques with Mrbc was observed when human complement was used in assays containing agarose. These assays gave a threefold increase in plaque numbers when compared with assays without agarose. It is evident from the above that the most sensitive and reproducible hemolytic plaque assay to be used by an investigator will depend primarily upon the antigen, and on reagents selected for the test.

Preliminary characterization of "immunogenic" ribonucleic acid derived from rat peritoneal exudate cells.

An "immunogenic" ribonucleic acid (Im-RNA) has been extracted from peritoneal exudate (PE) cells of rats that were immunized with sheep erythrocytes (SRBC). Following multiple phenol extractions and deoxyribonuclease treatment, the material obtained from PE cells was eluted from diethylaminoethyl-cellulose at 0.55 M NaCl concentration and partially purified in this procedure by a factor of 7- to 10-fold. After column chromatography, Im-RNA was found to be free of antigen based on results using (51)Cr-labeled SRBC or (14)C-dinitrophenol coupled to methylated bovine serum albumin as antigens. The Im-RNA showed a biphasic hyperchromicity curve when heated. The first phase, from 30 C to 90 C was gradual, accounting for 15.2% hyperchromicity suggestive of transfer RNA melting. No loss in immunogenic activity was observed when the Im-RNA was heated to 90 C. The second phase, from 90 C to 102 C, accounting for 15.2% further hyperchromicity, had a calculated T(m) of 96 C. Heating above 90 C resulted in an irreversible loss of immunogenic activity. These results strongly suggested that the RNA fraction contained a highly ordered secondary structure such as might be found with double-stranded nucleic acid. The nature and function of the Im-RNA is discussed.

Peritoneal barrier to the spread of Semliki forest virus in mice.

The LD50 for encephalitis caused by Semliki forest virus in 6- to 8-week-old mice is 1 plaque-forming unit (PFU) in C3H/Ten strain of mice when injected intracerebrally, iv, or in the footpad; however, the LD50 by the ip route is 4 x 10(3) PFU. In the ICR strain of mice at the same age, the LD50 for the intracerebral route is 1 PFU, 10(3) PFU for the iv and footpad routes, and 4 x 10(3) PFU for the ip route. A number of in vivo and in vitro experiments were done to explain the relative resistance to Semliki forest virus injection by the ip route. The results suggest that the viruses are adsorbed to and enter adherent cells of the peritoneal cavity but do not replicate and release progeny virus. After inoculation with the virus, viral antigens could only be observed in methanol-treated cells as a halo by immunofluorescence at or just below the plasma membrane of only a small fraction (less than 0.5%) of peritoneal adherent cells. Naturally occurring interferon-alpha/beta (less than 1 unit/ml) was found to probably play a marginal role, if any, in the resistance.

Identification of immunologically cross-reactive proteins of Sindbis virus: evidence for unique conformation of E1 glycoprotein from infected cells.

Hyperimmune antisera to purified Sindbis (SIN) or Semliki Forest (SF) virus were used to identify alphavirus-specific and cross-reactive proteins in virions and infected cells. The hyperimmune sera participated in homologous and cross-cytolysis of alphavirus-infected cells, and the use of monospecific antisera to SIN structural proteins suggested that E1 and E2 could serve as target proteins in cytolysis. Proteins from purified virions or infected cells were extracted with Nonidet P-40, denatured by procedures for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose solid supports, and reacted with hyperimmune sera and 125I-labeled protein A (immunoblotting on denatured proteins). Alternatively, native proteins extracted by mild Nonidet P-40 treatment were precipitated with hyperimmune sera before denaturation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After immunoblotting, homologous antiserum reacted with the virus structural proteins E1, E2, capsid extracted from purified virions, and the counterparts of these proteins extracted from infected cells. In addition, PE2 and a 92,000-molecular-weight protein from infected cells reacted with homologous antiserum. These proteins were also immunoprecipitated with homologous antiserum. After immunoblotting, the Sindbis capsid protein was shown to be cross-reactive whether derived from purified virions or from infected cells; no cross-reactivity was observed with PE2 or E2 from either source, and the E1 glycoprotein was shown to be cross-reactive only when obtained from virions. However, the E1 glycoprotein could be cross-immunoprecipitated from infected cells (as well as from disrupted virions), and, in addition, capsid and a 92,000-molecular-weight protein were cross-immunoprecipitated from infected cells. These results suggest that a native conformation of the cell-associated E1 glycoproteins may be required for immunological cross-reactivity (immune precipitation), whereas virion but not cell-associated E1 retains immunological cross-reactivity after denaturation (immunoblot technique). The findings extend our previously published evidence which suggested that alphavirus maturation is accompanied by a change in immunological cross-reactivity with respect to E1.

Cross-reactive target antigen in cell-mediated cytolysis of cells infected with a temperature-sensitive mutant of Sindbis virus.

Cytotoxic T lymphocytes (CTL) against alphavirus-infected L929 cells were generated in mice by two in vivo immunizations of one virus or by in vivo immunization followed by in vitro stimulation of splenocytes with infected peritoneal exudate cells or splenocytes. These CTL caused specific H-2-restricted cytolysis equally well with homologous, heterologous or Sindbis virus ts20 mutant-infected cells. Thus, specific CTL appear to be cross-reactive components in normal immunity to alphaviruses and the E1 glycoprotein is implicated as the target antigen.

In vitro heterologous cytotoxicity by T effector cells from mice immunized with Sindbis virus.

An in vitro correlate of cell-mediated cross-protection among alpha-viruses was demonstrated by cytotoxicity of Sindbis-immune spleen cells from mice to both Sindbis and Semliki Forest virus (SFV)-infected target cells. This cytotoxicity was shown to be mediated by the T cell population of the spleen and was independent of the presence of macrophages or B cells. The time when the level of the lymphocyte-mediated cytotoxicity (LMC) to SFV-infected cells was maximal coincides with the time when immunity to SFV is maximal in vivo, as reported previously, and when adoptive immunity to SFV can be transferred. After one i.p. injection of Sindbis virus, the level of homologous LMC was higher than the level of heterologous LMC. However, following a second injection of Sindbis virus as immunogen, at a time when the mice are cross-protected to SFV, the heterologous LMC was considerably higher than homologous LMC. We propose that there is suppression of the effector T cells specific for Sindbis-infected cells after the second immunizing injection, probably by homologous antibody. In contrast, there appears to be an anamnestic cell-mediated response to SFV.