John C. Roder

The biology of the human natural killer cell

Natural killer (NK) cells in the human are a population of large granular lymphocytes (LGL) with at least one unique surface antigen not expressed on cells of other lineages. NK-target-cell interaction appears to involve carbohydrate recognition and, following binding, the NK cells are induced to generate O2−, transmethylate membrane phospholipids, and activate phospholipase A2. Some or all of these activities trigger a cascade of events which ultimately leads to the secretion of a substance toxic to the target cell. A variety of genes controls various steps in this cytolytic pathway. There is a good deal of evidence in the mouse, and some in the human, that NK cells play a role in host surveillance against tumor development, resistance to viral infections, and, possibly, hematopoietic regulation.

Human monoclonal antibodies.

SummaryThe technology for the production of murine monoclonal antibodies has been refined enormously since its introduction in 1975. However, the technology for generating human monoclonal antibodies has only recently come into its own. In this review, three currently available approaches to the production of human monoclonal antibodies are described. These include the hybridoma technique, based on the fusion of antibody-producing human B lymphocytes with either mouse or human myeloma or lymphoblastoid cells; the EBV immortalization technique, based on the use of Epstein-Barr virus (EBV) to ‘immortalize’ antigen-specific human B lymphocytes; and the EBV-hybridoma technique, based on a combination of the first two methods.The EBV-hybridoma system retains the advantageous features of the other two systems while overcoming their pitfalls and may be the current method of choice for producing human monoclonal antibodies with a defined specificity.

Organization of a repetitive human 1.8 kb KpnI sequence localized in the heterochromatin of chromosome 15

We have isolated a repetitive 1.8 kb Kpnl DNA sequence which is amplified in the homogeneously staining regions of a human melanoma cell line. Under low stringency conditions this sequence (D15Z1) hybridized in situ to the centromeric heterochromatin of chromosomes 1, 9, 15p, 16, and distal Yq as well as to the the short arms of the other acrocentric chromosomes. Under conditions of high stringency, labelling was predominantly on the short arm of chromosome 15. D15Z1 was shown to be present at approximately 3,000 copies per haploid genome and organized in long tandem arrays showing restriction site heterogeneity. Sequences homologous to D15Z1 were highly enriched in the less dense shoulder region of a Ag+—Cs2SO4 gradient. Analysis of D15Z1 indicated that this sequence is composed of tandemly arranged imperfect repeats of the consensus 5′ AATGG 3′ similar to previously identified satellite III sequences. Digestion of D15Z1 with HinfI resulted in a series of restriction fragments making up a subset of the HinfI ladder components of satellites III and IV. These data suggest that D15Z1 represents a chromosome 15 specific domain of human satellites III or IV and that it makes up the major fraction of the heterochromatin of this chromosome. Possible relationships between this sequence and the cytochemical staining properties of human chromosomes with distamycin A/DAPI, D280/170, and antiserum to 5-methylcytosine are discussed.

Specific immunoglobulin production and enhanced tumorigenicity following ascites growth of human hybridomas

Human X human hybridomas constructed with the B6 lymphoblastoid clone, which produces antitetanus toxoid (TT) antibody, and the lymphoblastoid cell line KR-4 or human hybrid myeloma KR-12, were adapted to growth as ascites in pristane-treated BALB/c nude mice by a single prior passage as a solid subcutaneous (s.c.) tumor in irradiated nude mice followed by in vitro culture. Both B6 X KR-4 and B6 X KR-12 hybrids produced anti-TT antibody and phenotypically resembled the lymphoblastoid KR-4, or the hybrid myeloma KR-12 parent, respectively. Growth as ascites increased the tumorigenicity of both hybrids in nude mice as measured by tumor incidence and rate of tumor growth. The observed increase in tumorigenicity of these hybrid cells after ascites growth was associated with a substantial loss of chromosomes. Passage of the B6 X KR-4 lymphoblastoid hybrid resulted in several reversible morphological changes characteristic of myeloma cells. These changes correlated with increased human Ig production. These observations provide a system for greatly amplifying human monoclonal antibody production.

Adhesion properties of a neuronal epitope recognized by the monoclonal antibody HNK-1

A carbohydrate epitope on adhesion proteins of the developing nervous system, and on myelin-associated glycoprotein, is recognized by the monoclonal antibody HNK-1. The HNK-1 epitope bearing proteins and the monoclonal antibody alter, in a dose-dependent manner, the interaction between neurons and neurite-promoting substrate-attached materials released from cultured neural cells.

Do NK cells play a role in anti-tumor surveillance?

A class of lymphocytes found in several mammalian species including man will kill cells of many tumor lines invitro(1-4). There is growing evidence, derived mainly from the work of Kiessling and co-workers, that these natural killer (NK) cells play a role in surveillance against tumor developmentin vivo. In this article John Roder and Tina Haliotis discuss a hypothesis for anti-tumor surveillance which integrates all of the potentially important immunological systems in the host and gives to NK cells the role of foremost barrier against developing tumors.

The effect of unphosphorylated and phosphorylated sugar moieties on human and mouse natural killer cell activity: IS there selective inhibition at the level of target recognition and lytic acceptor site?

A number of different sugars were investigated for their effect on human and mouse natural killer cell (NK)-mediated cytolysis. From the pool of nonphosphorylated sugars, D-mannose, N-acetyl-D-glucosamine (NAcGlc), D-glucose, and, to a lesser extent, beta-gentiobiose were found to inhibit human NK cytolysis. Mouse NK activity against YAC-1 target cells was reduced consistently in the presence of D-mannose and NAcGlc only. The sugars, NAcGlc, D-glucose, and beta-gentiobiose, were specifically inhibitory against NK-mediated cytolysis with no inhibitory effects being observed against ADCC, monocyte-mediated cytolysis, or CTL activity. Pretreatment and washing at either the target or effector cell level as well as direct target binding assays using Percoll-purified NK cells indicated that at least NAcGlc and beta-gentiobiose function at the recognition stage of NK cytolysis. D-Mannose, which was the most effective nonphosphorylated sugar inhibitor, was capable of inhibiting all cell-mediated cytotoxic mechanisms tested (NK, ADCC, monocyte, and CTL) and its action did not appear to be solely due to an impairment in the recognition event. All the phosphorylated sugars caused significant inhibition of human and mouse NK-mediated cytolysis, although repeated analyses of sugar titration curves consistently showed mannose-6-phosphate (Man-6-P) to be the most effective inhibitor. Inhibition with the phosphorylated sugars was apparent against all cytotoxic mechanisms investigated. It is possible that these sugars may function as general metabolic inhibitors or may activate a common signal which negatively regulates cell-mediated cytotoxic mechanisms. Nevertheless, the relative degree of inhibition with the majority of these sugars (particularly Man-6-P) was greater against NK and ADCC activity than against monocyte and CTL activity. Furthermore, studies with selected well-characterized human and mouse NK-resistant target cells strongly indicated that these sugars, particularly Man-6-P, compete at an acceptor site responsible for the uptake of the NK lytic factor, which is independent of the recognition structure(s).

Establishment of a human large cell lung tumor line (QU‐DB) with metastatic properties in athymic mice

A continuous human cell line was established from a patient with large cell anaplastic lung carcinoma. This cell line, designated QU‐DB, has been in culture for over 36 months and grows as an adherent monolayer with a doubling time of 10–12 hours. Its morphology, ultrastructure, karyotype, ability to grow in soft agar and heterotransplantability, indicate it is a large‐cell lung tumor cell line of human origin. Three cell lines were established from metastatic tumors in nude mice receiving subcutaneous injections of QU‐DB cells. The morphology and growth characteristics exhibited by these cell lines were similar to the primary cell line. Karyotypic analysis of cell lines derived from the primary tumor and a metastasis to the diaphragm were similar, but cells from a liver metastasis culture showed additional karyotypic changes. This large cell lung tumor cell line may prove useful as a model system for studies of human tumor progression and metastasis.

Tumor cell differentiation modulates susceptibility to natural killer cells

Abstract Induced differentiation in three human cell lines altered their sensitivity specifically to human natural killer (NK) cells by affecting their expression of NK target antigens. Differentiation of HL-60, a promyelocytic leukemia cell line, and the erythroleukemic cell line K562 was accompanied by a concomitant decrease in susceptibility to NK-mediated lysis whereas induction of MeWo melanoma cells resulted in an enhanced sensitivity to lysis. Our findings suggest that target cell susceptibility to NK-mediated lysis may in part be dependent on the stage of differentiation of the tumor cell target.

Rearrangements of chromosomal regions containing ribosomal RNA genes and centromeric heterochromatin in the human melanoma cell line MeWo

A chromosomal examination of cells from the earliest available passage of the human melanoma cell line MeWo revealed the presence of seven hypodiploid cell types that shared common complex marker chromosomes. Two of the cell types had long homogeneously staining regions (HSR) by Q-banding on three different chromosomes. Distamycin A/DAPI staining and silver staining for active nucleolar organizing regions (NOR) confirmed that the HSR were derived from chromosome #15. All HSR-containing cells had 4-9 pairs of large NOR distributed along the length of each HSR, with all acrocentric chromosomes being negative. The HSR-lacking cells differed primarily with respect to the morphology of the short arm of one #13 chromosome and NOR activity. One cell type had four chromosomes with active NOR, whereas all other cell types had a single active NOR on one #13. One of these cell types had a satellited #8 with NOR. Cells from three other MeWo cultures at higher passages were examined. Two of these contained both hypodiploid and hypotetraploid cells, some of which had satellited X chromosomes or satellited #3 chromosomes with active NOR. The majority of the new chromosomal rearrangements in cells from the later cultures involved the NOR-containing regions, many of which were associated with the distamycin A/DAPI-positive centromeric heterochromatin from chromosome #15. These results indicate that the chromosomal instability in the MeWo cultures is mainly limited to sequences containing active NOR and centromeric heterochromatin from chromosomes #13 and #15. This may be due to a selective pressure to increase the number of active NOR in the MeWo cells. If this is so, it would appear that amplification of active NOR occurs more readily than the activation of the many silent NOR present in these cells.