Complement-dependent lysis of tumor cells by a baboon IgM antibody to a tumor-associated antigen

Summary We developed a high-titer polyclonal antiserum to a glycoprotein tumor-associated antigen (TAA) by immunization of a baboon with the purified glycoprotein antigen. The baboon serum was fractionated into IgG and IgM components by DEAE Affi-Gel blue chromatography. The ability of the baboon IgM anti-TAA antibody to effect tumor cell lysis in the presence of complement was tested using a chromium-release assay. The baboon antibody was able to lyse melanoma target cells (20.8%–71.4% cytolysis), breast carcinoma cells (36.5%–38.9% cytolysis), and a neuroblastoma cell line (35.5% cytolysis) in the presence of complement but did not effect significant lysis of autologous lymphoblastoid cell lines (4.9% cytolysis) or peripheral blood lymphocytes from healthy volunteers (12.6% cytolysis). Cytolysis of melanoma target cells was completely inhibited by preabsorption of the IgM anti-TAA antibody with UCLA-SO-M14 (M14) cells and partially inhibited by preabsorption with several other melanoma cell lines. There was no significant inhibition of tumor cell lysis after preabsorption of the antibody with lymphoblastoid cell lines. Complement-dependent lysis of M14 targets could be blocked by addition of the purified antigen to the antibody prior to incubation with the tumor cells. Our results suggest that the glycoprotein TAA resides on the tumor cell surface and that the baboon IgM anti-TAA antibody recognizes the antigen on the cell surface and is able to fix complement and effect the lysis of the tumor cells.     

Specific lysis of human tumor cells by T cells coated with anti-T3 cross-linked to anti-tumor antibody

Heteroaggregates containing anti-T3 cross-linked to anti-target cell antibodies have been shown to cause human T cells to lyse target cells that express antigens recognized by the anti-target cell antibody. In this study, we test targeted human T cells for the ability to lyse human tumor cells as a first step toward the application of this phenomenon to tumor immunotherapy. Several monoclonal anti-human tumor antibodies were assayed for binding to a number of human tumor lines and for the ability to promote specific tumor cell lysis when cross-linked with anti-T3. We found that anti-T3 cross-linked to anti-tumor monoclonal antibodies caused cloned human T cells and fresh peripheral blood T cells to lyse the tumor cells with the same specificity as predicted by the binding studies. Peripheral blood T cells were then tested in the presence of various heteroaggregates for the ability to lyse single cell suspensions prepared from fresh tumor or fresh normal tissue. These studies showed that heteroaggregates containing anti-T3 cross-linked to anti-tumor antibody cause fresh human T cells to specifically lyse fresh tumor cells, but not (with one exception) fresh normal cells.     

Human leukemia-associated antigens: detection on cells of established lymphoblastoid lines.

Primate and rabbit antisera to different morphologic classes of human leukemia cells, after appropriate absorptions, detected leukemia-associated antigens present on cultured lymphoblastoid cell lines derived from leukemia patients. The primate antisera distinguished antigens on cells derived from myeloid leukemia patients from those on cells derived from lymphocytic leukemia patients. Of particular interest was the fact that antigens of myeloid leukemia, but not of lymphatic leukemia, were detected on lymphoid cell lines established from blood of patients with myeloid leukemia. One of four lymphoblastoid cell lines derived from normal donors expressed antigens of lymphatic leukemia. Leukemia-associated antigens were not found on the HRIK lymphoblastoid line derived from a Burkitt's lymphoma patient on skin fibroblasts or HeLa cells. Expression of these antigens on cultured cells derived from leukemia patients could not be related to the presence of the EB virus or the EB virus genome. Rabbit antisera detected antigens common to cells from patients with myeloid and lymphocytic leukemia. Absorption experiments demonstrated that the antigens detected on cell lines derived from leukemia patients are similar to those detected by the primate and rabbit antisera on fresh peripheral blood leukemic cells. The serologic detection of leukemia-associated antigens on lymphoblastoid cell lines indicates that some of these cultures contain cells with antigenic properties similar to those of human peripheral blood leukemic cells.     

EBV transformation and cell culturing destabilizes DNA methylation in human lymphoblastoid cell lines

Recent research suggests that epigenetic alterations involving DNA methylation can be causative for neurodevelopmental, growth and metabolic disorders. Although lymphoblastoid cell lines have been an invaluable resource for the study of both genetic and epigenetic disorders, the impact of EBV transformation, cell culturing and freezing on epigenetic patterns is unknown. We compared genome-wide DNA methylation patterns of four white blood cell samples, four low-passage lymphoblastoid cell lines pre and post freezing and four high-passage lymphobastoid cell lines, using two microarray platforms: Illumina HumanMethylation27 platform containing 27,578 CpG sites and Agilent Human CpG island Array containing 27,800 CpG islands. Comparison of genome-wide methylation profiles between white blood cells and lymphoblastoid cell lines demonstrated methylation alterations in lymphoblastoid cell lines occurring at random genomic locations. These changes were more profound in high-passage cells. Freezing at low-passages did not have a significant effect on DNA methylation. Methylation changes were observed in several imprinted differentially methylated regions, including DIRAS3, NNAT, H19, MEG3, NDN and MKRN3, but not in known imprinting centers. Our results suggest that lymphoblastoid cell lines should be used with caution for the identification of disease-associated DNA methylation changes or for discovery of new imprinted genes, as the methylation patterns seen in these cell lines may not always be representative of DNA methylation present in the original B-lymphocytes of the patient.     

Multiple epitopes on human and murine cells expressing HLA-B7 as defined by specific murine cytotoxic T cell clones

Eleven cytotoxic T lymphocyte (CTL) clones were derived from C57BL/6 spleen cells immunized with HLA-B7 expressing human lymphoblastoid cell lines. Reactivity against HLA-B7 was initially established because the clones lysed 2 target cells that shared only HLA-B7 with the immunizing cell line and they did not lyse five other cell lines that were HLA-B7 negative but expressed other class I or class II antigens found on the immunizing cell. Six of the clones were subsequently shown to lyse all tested HLA-B7-positive B and T lymphoid cell lines, peripheral blood lymphocytes, and a murine L cell that expressed HLA-B7 as a consequence of DNA-mediated gene transfer. On the basis of the inability of the clones to lyse a panel of HLA-B7-negative cell lines, up to 18 other class I antigens could be eliminated as being cross-reactively recognized. However, two of the clones recognized a single HLA-B7-negative cell line. It is suggested that in these cases the clones were cross-reactively recognizing the HLA-B27 or HLA-B40 antigens that were present on these target cells. The remaining five CTL clones failed to lyse one out of seven tested HLA-B7-positive lymphoid lines (either RPMI-1788 or DR1B) and failed to lyse peripheral blood lymphocytes from one out of three tested HLA-B7-positive individuals. These five clones also did not recognize the HLA-B7-positive murine L cell. However, based on analysis with a large target cell panel, the reactivity pattern of these five clones could only be correlated with recognition of HLA-B7. This conclusion is further supported by antibody-blocking studies to be reported elsewhere. As before, lysis of single HLA-B7-negative target cells by two of the clones could be ascribed to recognition of HLA-B27 or HLA-B40. The results show that murine clones raised against HLA-B7 exhibit a high degree of specificity for determinants that are unique or largely confined to the HLA-B7 alloantigen. In addition, these clones define different antigenic determinants on the molecule. Thus, such clones appear to be excellent candidates for use as human tissue typing reagent. The results further show that there is a strong correlation between recognition of particular HLA-B7-positive human cell lines and recognition of the HLA-B7 expressing murine L cell. Possible reasons for such a correlation and their relationship to the general phenomenon of CTL recognition are discussed.     

Stimulation with autologous lymphoblastoid cell lines: lysis of Epstein-Barr virus-positive and -negative cell lines by two phenotypically distinguishable effector cell populations

Human lymphocytes, stimulated in vitro for 6 days with x-irradiated or glutaraldehyde-treated autologous Epstein-Barr (EB) virus-transformed lymphoblastoid cell lines (LCL), are cytotoxic for autologous and allogeneic EB+ LCLs as well as for several EB- cell lines that are also susceptible to lysis by interferon-activated natural killer (NK) cells. To determine whether the apparent nonspecific lysis mediated by LCL-stimulated cells is due to a mixture of effector cells directed against different target cells, advantage was taken of our recent finding that monoclonal antibody OKT8 reacts with human cytotoxic T lymphocytes but not with NK cells or NK-like cells generated in mixed leukocyte cultures. The depletion of OKT8+ cells from LCL-stimulated cultures by treatment with OKT8 and complement abolished or markedly depleted cytotoxicity against all EB+ target cells tested, whereas cytotoxicity against EB-, NK-sensitive cell lines including K562, MOLT-4 and HSB-2 was not or only minimally reduced. These results indicate that stimulation with autologous LCL results in the generation of OKT8+ cytotoxic T lymphocytes that lyse EB virus-transformed LCL and OKT8- NK-like cells that lyse EB-, NK-sensitive cells.     

Lysosomal acid hydrolases in established lymphoblastoid cell lines,transformed by Epstein-Barr virus,from patients with genetic lysosomal storage diseases

Summary Lysosomal acid hydrolases were determined in established lymphoblastoid cell lines, transformed in vitro by Epstein-Barr virus (EBV) from lymphocyte-rich cell populations isolated from the peripheral blood of patients with genetic lysosomal storage diseases—Hurler syndrome, Scheie syndrome, G_M1-gangliosidosis type 1 and type 2, Tay-Sachs disease, and I-cell disease—and from obligate heterozygotes for these diseases.The respective enzyme activity was undectectable in lymphoblastoid cells from the patients, but not from controls. Obligate heterozygotes could not always be distinguished from controls in lymphoblastoid cells as well as in leukocytes. These results suggest that established lymphoblastoid cell lines are useful material for the enzymatic study of genetic lysosomal storage diseases.     

Generation of natural killer cell lines from murine long-term bone marrow cultures

Functionally active natural killer (NK) cells with the ability to lyse 51Cr-labeled YAC-1 lymphoma target cells are no longer detectable by 1 wk of culture in cultured marrow cells harvested from Dexter-type long-term marrow cultures (LMC). Interferon, which enhances NK cell-mediated target cell lysis, fails to induce NK activity from LMC cells even at high effector to target cell ratios. However, such LMC cells, when placed in secondary cultures in the presence of Con A-splenic leukocyte-conditioned medium (spleen-CM) generated a population of cells with NK activity within 1 wk. Kinetic studies showed that the generation of NK activity was not due simply to proliferation of a few surviving NK cells, but suggested derivation from NK precursors through clonal expansion and functional maturation. This NK activity was further shown to be associated with a subpopulation of cells bearing surface Thy-1, Ly-5, and NK-1 as well as asialo-GM1 antigens but lacking Ly-1 antigen. The expression of Ly-2 antigen, however, was variable. Electron microscopy studies of isolated asialo-GM1-positive cells showed a uniform lymphoblastoid morphology with large cytoplasmic to nuclear ratios and prominent electron dense cytoplasmic granules characteristic of large granular lymphocytes. In support of the NK nature of such cultured cells was the ability of anti-asialo-GM1 and complement to abrogate, and of interferon to augment, target cell lysis. Isolated cell lines also showed target selectivity similar to NK cells. The implications of the studies on further analysis of the nature of NK precursors is discussed.     

Group C adenovirus DNA sequences in human lymphoid cells.

Human peripheral blood lymphocytes from healthy adults, cord blood lymphocytes, and lymphoblastoid cell lines were screened by hybridization for the presence of group C adenovirus DNA sequences. In 13 of 17 peripheral blood lymphocyte samples from adults, 1 of 10 cord blood samples, and seven of seven lymphoblastoid cell lines tested, results were positive for Group C adenovirus DNA (adenovirus 1 [Ad1], Ad2, Ad5, or Ad6). About 1 to 2% of the lymphocytes carried 50 to 100 viral genome copies per positive cell, as estimated by in situ hybridization. Infectious virus representing all members of group C were recovered, but cultivation in the presence of adenovirus antibody did not cure the cells of free viral genomes. Viral DNA was found in B, T, and N cells but only in 1 of 10 cord blood samples. The results suggest that group C adenovirus infections in childhood result in the persistence of the viral genome in circulating lymphocytes.     

A comparison of fragile X expression in lymphocyte and lymphoblastoid cultures

This study compares fragile X expression in peripheral blood lymphocyte cultures with expression in lymphoblastoid cell lines established from 23 individuals from families in which the fragile X is segregating. Most patients expressed the fragile X in lymphoblastoid cell lines treated with FUdR under optimal conditions at approximately the same frequency as in peripheral blood cultures from the same individual. No fragile X cells were seen in the lymphoblastoid cell lines from three phenotypically normal males who had transmitted the fragile X gene to offspring or in the lines from three phenotypically normal obligate-carrier females, all of whom were also negative in peripheral blood cultures. Two individuals, however, who expressed at high levels in peripheral blood lymphocytes expressed in lymphoblastoid cells only at low levels or not at all. We describe the considerations needed for the consistent demonstration of the fragile X in lymphoblastoid cell lines.     

Major histocompatibility complex-unrestricted cytolytic activity of human T cells. Analysis of precursor frequency and effector phenotype

The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. These conditions were shown to expand a mean of 96% of cells cultured. All of the 198 clones generated by this method were T cells (CD2+, CD3+, CD4+ or CD2+, CD3+, CD8+) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. In addition, the activity was not inhibited by monoclonal antibodies directed against class I or class II nonpolymorphic MHC determinants. Killing, however, was inhibited by soluble monoclonal antibodies against the CD3 complex. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur or K562 was not mediated by a soluble factor secreted by the clones. Some of the clones retained their cytotoxic activity when grown in rIL-2 alone for 4 to 6 wk, whereas others exhibited markedly diminished cytotoxicity after maintenance in this manner. Clones that exhibited diminished or no cytotoxic activity after prolonged maintenance in rIL-2 could be induced to kill by stimulation with immobilized but not soluble monoclonal antibodies to CD3 in the absence of lectin. All of the clones examined expressed NKH1 and CD11b but none were CD16 positive. The degree of cytotoxicity of resting or activated clones could not be correlated with expression of these markers. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype.     

Serological analysis of xenogeneic anti-lymphoblastoid cell-line sera with specificity against HLA-B12

In view of the importance of potent anti-HLA sera with narrow reaction patterns against defined HLA antigens, two xenogeneic antisera were raised in rabbits following immunization with human lymphoblastoid cell lines from HLA-nonidentical donors homozygous for HLA-B12. After absorption with lymphoblastoid cell lines of an appropriate HLA phenotype, the antisera were purified over DEAE-cellulose ion exchange chromatography and reconcentrated. Both antisera recognized HLA-B12-positive peripheral blood cells of unrelated donors tested in the microcytotoxicity assay. The two rabbit antisera revealed a high degree of similarity in their anti-HLA-B12 antibody specificity. One antiserum showed some cross reactivity with HLA-B13 as has been reported in allo-anti-HLA-B12 sera. The other antiserum revealed some activity against HLA-DRw7-positive donors. Antibody activity could be removed completely from two further rabbit anti-HLA antisera by absorption with lymphoblastoid cell lines from related and unrelated HLA-identical donors. The advantages of using lymphoblastoid cell lines as immunogens and absorption material for the production of heterologous anti-HLA typing sera are discussed.     

Two distinct human cloned T cell lines that exhibit natural killer-like and anti-human effector activities

Cloned T cell lines from mixed lymphocyte cultures stimulated with autologous Epstein Barr virus- (EBV) transformed lymphoblastoid cell line (LCL) cells were established using a limiting dilution technique in the presence of T cell growth factor (TCGF). The T cell lines included two distinct clones of cytotoxic T cells (Tc) in addition to EBV-specific Tc. A cytotoxic profile of one cloned line was similar to that of endogenous NK cells in peripheral blood. The other cloned Tc line showed an anti-human cytotoxicity. The susceptible targets for this latter Tc line were various human cells, including autologous LCL and peripheral blood lymphocytes (PBL), stimulated with pokeweed mitogen, along with NK-sensitive and NK-resistant cell lines. Weak cytotoxic activity was detected against various xenogeneic cell lines. Furthermore, autologous and allogeneic cloned T cell lines were resistant to killing by the anti-human effector clone. These t wo distinct cloned Tc lines expressed the Leu-1 and Leu-2a antigens, which are markers of suppressor/cytotoxic T cells.     

Alkaline phosphatase phenotypes in tumour and non-tumour cell lines: not an invariable marker for neoplastic transformation

The cytochemical localisation and presumed isoenzyme type (based on selective inhibition experiments) of alkaline phosphatase in 5 cell lines derived frrom normal human, rat, mouse and hamster tissues, 6 human lymphoblastoid lines and 6 human and mouse tumour-derived cell lines are described. Enzyme activity varied between the cell lines. An isoenzyme inhibited by L-phenylalanine was present in 3 normal lines, 3 lymphoblastoid lines and 2 tumour lines. The presence of this isoenzyme cannot be used as a marker of neoplastic transformation.     

Activation of the alternative complement pathway by lymphoblastoid cell lines derived from patients with Burkitt's lymphoma and infectious mononucleosis.

Cultured human lymphoblastoid cell lines derived from patients with Burkitt's lymphoma or infectious mononucleosis were shown to activate the alternative pathway of complement fixation. This reaction does not require any conventional antibody directed against the cells. Although the reaction showed an absolute dependence on the presence of factor B it was relatively independent of the presence of factor D or of properdin. To this extent activation of the alternative pathway by lymphoblastoid cells resembles that produced by “C3-nephritic factor.” Rat and mouse complement were activated in a manner similar to human complement, but guinea pig complement was inactive. Chicken complement, unlike any of the mammalian complements tested, was able to bring about lysis of the lymphoblastoid cell lines by the alternative pathway.     

Fibrinolytic activity associated with established lymphoblastoid cell lines and fresh peripheral blood lymphocytes of human and nonhuman primate origin.

Established lymphoblastoid cell lines and normal peripheral blood lymphocytes were examined for their ability to induce fibrinolysis, a property associated with oncogenic transformation, using a 3H-fibrin plate technique. Fibrinolytic activity showed serum preferences with dog serum being most active. Most cell lines (14/18) induced greater than 40% release, while normal lymphocytes were generally less active. Only one cell line tested released plasminogen activator into the medium. No correlation was shown between fibrinolytic activity and growth in soft agar. Normal rhesus lymphocytes showed fibrinolytic activity in B cell-enriched populations with no evidence of interaction between B cells and T cells.     

Serological analysis of xenogeneic anti-lymphoblastoid cell-line sera with specificity against HLA-B12

In view of the importance of potent anti-HLA sera with narrow reaction patterns against defined HLA antigens, two xenogeneic antisera were raised in rabbits following immunization with human lymphoblastoid cell lines fromHLA-nonidentical donors homozygous for HLA-B12. After absorption with lymphoblastoid cell lines of an appropriate HLA phenotype, the antisera were purified over DEAE-cellulose ion exchange chromatography and reconcentrated. Both antisera recognized HLA-B12-positive peripheral blood cells of unrelated donors tested in the microcytotoxicity assay. The two rabbit antisera revealed a high degree of similarity in their anti-HLA-B12 antibody specificity. One antiserum showed some cross reactivity with HLA-B13 as has been reported in allo-anti-HLA-B12 sera. The other antiserum revealed some activity against HLA-DRw7-positive donors. Antibody activity could be removed completely from two further rabbit anti-HLA antisera by absorption with lymphoblastoid cell lines from related and unrelatedHLA-identical donors. The advantages of using lymphoblastoid cell lines as immunogens and absorption material for the production of heterologous anti-HLA typing sera are discussed.     

In vitro sensitization of human lymphocytes against histiocytic lymphoma cell lines. I. Primary sensitization of lymphocyte subpopulations.

Peripheral blood lymphocytes from normal donors expressed spontaneous cytotoxic activity against human diffuse histiocytic lymphoma cell lines. In the unfractionated state, they could not be further sensitized in vitro against these cell lines. By applying cell separation techniques before culture, subpopulations of lymphocytes were obtained which could be sensitized in vitro and manifested cytotoxic activity against human histiocytic lymphoma cells. Three methods of separation were found effective: E rosette enrichment; elimination of Fc receptor positive cells; and removal of nylon wool adherent cells. Under these conditions, cross-reactive cytotoxicity was observed against non-neoplastic lymphoblastoid cell lines, but not against normal lymphocytes.     

B lymphoblastoid cell lines with rearranged T cell receptor beta-chain genes retain conventional B cell surface antigen and Ig expression

Transformation of peripheral blood lymphocytes by co-incubation with EBV produces B lymphoblastoid cell lines, but rearrangement of TCR beta-chain genes was observed in three different cell lines derived from two individuals. Because rearrangement of TCR genes in B lymphocytes is considered a rare event, these B lymphoblastoid cell lines with rearranged TCR beta-genes were examined in detail to determine whether there were any additional characteristics to distinguish them from B lymphoblastoid cell lines with germ-line TCR beta-genes. All B lymphoblastoid cell lines contained rearranged Ig H and kappa L chain genes, secreted Ig, and expressed B and not T cell surface markers. Cell lines with rearranged TCR beta-genes had rearranged both IgH genes and had rearranged and subsequently deleted both kappa C region genes. Furthermore all three B lymphoblastoid cell lines with rearranged TCR beta-genes produced small amounts of Ig with lambda-L chains. Although the cellular mechanisms maintaining lineage-specific rearrangement events remain unknown, extensive Ig gene rearrangement and inefficient Ig production by B cells may be indicators of a cellular status where normally stringent lineage-specific control elements fail to function efficiently.     

Induction of proliferation in vitro of resting human natural killer cells: expression of surface activation antigens

In this report, we show that resting human peripheral blood NK cells, positively enriched with antibody B73.1, can be induced to proliferate in vitro in short-term cultures, and newly express membrane activation antigens. B73.1+ NK cells, cultured for 6 days in the presence of conditioned medium containing IL 2 and irradiated B lymphoblastoid cells, show significant [3H]TdR incorporation beginning on day 4. At that time, a large proportion of the cells express HLA-DR and 4F2 antigens, and transferrin and IL 2 receptors, all detectable at high density by indirect immunofluorescence with the use of monoclonal antibodies. These cells maintain their ability to lyse target cells spontaneously or in the presence of antibodies. By two-color immunofluorescence and cell cycle analysis combined with indirect immunofluorescence, we demonstrate directly that the activation antigens are expressed on all NK (B73.1+) cells in the S/G2/M phases of the cell cycle, and on only a proportion of those in the G0/G1 phases.