Immunodeficiency disorders have provided much information on the development and interaction of the various B and T lymphoid components in the immune system of man. As the lymphoid system becomes increasingly divided into functional subsets of cells it will be important to find immunodeficiencies affecting newly discovered cell types. Natural killer (NK) cells are a recently described but ill-defined subpopulation of lymphocytes which is thought to play an important part in surveillance against tumour development. Mice homozygous for the beige gene were found to have a selective deficiency in NK function and were more susceptible to transplantation of syngeneic tumours as predicted. We report here that patients carrying the analogous, autosomal recessive Chediak-Higashi (CH) gene have a profound defect in their ability to spontaneously lyse various tumour cells in vitro by either antibody-dependent or independent mechanisms. Since other cell-mediated cytolytic functions were relatively normal, these results suggest that the beige or Chediak-Higashi gene in both man and mouse controls NK function.

We have previously observed that a point mutation on chromosome 13 in the mouse, called beige, leads to a marked, selective impairment in natural-killer (NK) acell function (1). Because beige mice have long been used as an animal model of the Ch~diak-Steinbrinek-Higashi syndrome (C-HS) in man (2) it seemed relevant to assess NK function in humans who bear this rare, autosomal recessive gene. Because of the marked propensity of C-HS patients for developing lymphoproliferative disorders that may be malignant (3) it was predicted that if NK cells in the human were important in immune surveillance, then C-H patients might have low levels of NK activity.In this present paper we show that NK activity in two C-HS donors was profoundly impaired and did not appear to be a result of an alteration of an anti-target selectivity pattern or of the kinetics of lysis, of suppressor cells, of a lack of ability to respond to interferon, or of a lack of target-cell recognition. In the accompanying paper (4) we show that antibody-dependent cell-mediated cytotoxicity against tumor-cell targets is also depressed in C-HS patients with cytolytic activity by monocytes, by polymorphonuclear leukocytes, and by T cells that are relatively normal. Materials and MethodsSubjects. Two male patients, Le. R. (age 26 yr), and La. R. (age: 28 yr), siblings from a consanguinous marriage, and eight different age-and sex-matched normal controls were studied in parallel experiments on three separate occasions over a l-mo period. The detailed clinical history and many previous experiments on these particular patients has been described (5-7). At the time of study the patients were free of overt infections and were not on therapy.

We have isolated a repetitive 1.8 kb KpnI 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 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.

In the preceding (1) paper we showed that human Ch6diak-Steinbrinck-Higashi syndrome (C-HS) 1 donors had a marked impairment in natural-killer (NK)-cell function. It was then relevant to ask whether this defect was selective for NK cells as previously observed in the mouse C-HS model (2) or whether other effector-cell types were also defective. Because the C-HS is thought to involve a single recessive gene (3), it was hoped that these experiments would help clarify the relationship between NK cells and a variety of other cell types capable of lysing tumor-cell targets, including K cells involved in antibody-dependent cell-mediated cytotoxicity (ADCC), T cells, monocytes, and neutrophils. In this paper, we show that lymphocyte-mediated ADCC against tumor-cell targets is also defective, whereas ADCC mediated by both mononuclear cells and polymorphonuclear leukocytes (PMN) against erythrocyte targets is normal. Cytolysis of tumor cells by other effectors (T cells, monocytes, and PMN) was also relatively normal. These results suggest that C-HS patients may have a quite selective impairment in immunologic function. The implication of these findings for immune surveillance is discussed.

To investigate the role of ras gene activity in cellular transformation by polyomavirus, murine C3H1OT1/2 cells were rendered ras deficient by transfection with an antisense ras gene construct. Ras deficiency resulted in a partial suppression of the polyomavirus-induced transformed phenotype. The production of viral middle T antigen and its association with pp60csrc, increased membrane-associated protein kinase C activity, and morphological transformation were unaffected by the downregulation of c-ras gene expression. On the other hand, stimulated proliferation, focus formation on confluent monolayers of normal cells, and colony formation in soft agar were all greatly reduced in cells containing reduced p2lras levels. It is concluded that ras gene activity is needed for full cell transformation by polyomavirus. ICN Pharmaceuticals Inc., Irvine, Calif.Cell lines, culture techniques, and plasmid transfections. All cells were grown in 10% fetal calf serum-90% Dulbecco's modification of Eagle's medium in a humidified CO2 incubator.Plasmid transfections were performed as described previously (42). For expression of the ras antimessage, the pMt-I antisense ras construct containing the human c-H-ras gene 5203 on July 4, 2020 by guest http://jvi.asm.org/ Downloaded from 5204 RAPTIS ET AL.

In classical Hodgkin's lymphoma (cHL), specific changes in the 3D telomere organization cause progression from mononuclear Hodgkin cells (H) to multinucleated Reed-Sternberg cells (RS). In a post-germinal center B-cell in vitro model, permanent latent membrane protein 1 (LMP1) expression, as observed in Epstein-Barr virus (EBV)-associated cHL, results in multinuclearity and complex chromosomal aberrations through downregulation of key element of the shelterin complex, the telomere repeat binding factor 2 (TRF2). Thus, we hypothesized that the three-dimensional (3D) telomere-TRF2 interaction was progressively disturbed during transition from H to RS cells. To this end, we developed and applied for the first time a combined quantitative 3D TRF2-telomere immune fluorescent in situ hybridization (3D TRF2/Telo-Q-FISH) technique to monolayers of primary H and RS cells, and adjacent benign internal control lymphocytes of lymph node biopsy suspensions from diagnostic lymph node biopsies of 14 patients with cHL. We show that H and RS cells are characterized by two distinct patterns of disruption of 3D telomere-TRF2 interaction. Disruption pattern A is defined by massive attrition of telomere signals and a considerable increase of TRF2 signals not associated with telomeres. This pattern is restricted to EBV-negative cHL. Disruption pattern B is defined by telomere de-protection due to an impressive loss of TRF2 signals, physically linked to telomeres. This pattern is typical of, but is not restricted to, LMP1+EBV-associated cHL. In the disruption pattern B group, so-called 'ghost' end-stage RS cells, void of both TRF2 and telomere signals, were identified, whether or not associated with EBV. Our findings demonstrate that two molecularly disparate mechanisms converge on the level of 3D telomere-TRF2 interaction in the formation of RS cells.

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The effect of mutated c-Ha-ras expression on Ca2+ and phospholipid-dependent protein kinase C (PKC) activity during the process of transformation was analysed using an inducible metallothionein-ras hybrid oncogene system. A close correlation was found between the timing of ras expression and the loss of PKC enzymatic activity measured in a cell-free system. Examination of the subcellular distribution of the enzyme in inducible and constitutive ras-transformants revealed that expression of ras was associated with an apparent translocation of PKC to the plasma membrane concomitant with down-regulation of PKC enzymatic activity in particulate as well as cytosolic fractions. Quantitation of PKC protein utilizing a PKC-specific antiserum showed that ras expression was associated with a decrease in the total amount of PKC protein present in the cell. We conclude that transformation by c-Ha-ras is accompanied by down-regulation of PKC activity and that the basis of this effect may, to a large extent, lie in the down-regulation of the amount of PKC protein.