Natural killing and antibody-dependent cellular cytotoxicity: characterization of effector cells by E-rosetting and monoclonal antibodies

Recent investigations have indicated that the OKM1 hybridoma monoclonal antibody reactive with cells of the myelomonocytic series identifies a subpopulation of human peripheral blood mononuclear cells (PBMNC) which mediate natural and antibody-dependent cellular cytotoxicity (ADCC). However, it was not clear whether this OKM1+ group was heterogeneous with regard to cytotoxic function or the presence of receptors for sheep erythrocytes. Thus, the purpose of the present study was to further define the phenotype of the ADCC effector cell and natural killer (NK) cell using a combination of reactivity with hybridoma antibodies and separation of subsets by sheep erythrocyte rosette (E+) formation. Furthermore, the phenotypes of the NK population were assessed directly by performing two-color immunofluorescent staining on tumor cell conjugates. These studies led to the following conclusions: (1) that NK activity is mediated by both E+ OKM1+ and E- OKM1+ cells; the E+ OKT3+ cell possessed essentially no ADCC or NK activity; (2) that E+ OKM1+ cells mediated more NK activity on a per cell basis than E- OKM1+ cells; this was verified by separating OKM1+ cells on a cell sorter into E+ and E- with the OKT11 monoclonal antibody (anti-E-receptor antibody); (3) that E+ OKM1+ cells mediated both ADCC and NK activity; (4) that the phenotypes of PBMNC forming tumor cell conjugates were (a) OKM1+ (both E-receptor positive and negative) and (b) OKM1- E-receptor positive.     

Natural cytotoxicity in rats: Strain distribution and genetics

The term natural cytotoxicity (NC) describes a phenomenon in rats whereby significant numbers of intravenously injected ⁵¹Cr-labeled lymph node cells are rapidly destroyed by unsensitized allogeneic hosts. Cell death is reflected in a decreased accumulation of labeled cells in the host lymph nodes, with a corresponding increase in the label excreted by the kidney. Natural cytotoxicity has been studied in 95 allogeneic donor-host combinations among inbred rats and in a segregating population of F1 backcross animals. On the basis of lymphocyte distribution patterns, the individual donor-host combinations have been categorized as exhibiting high NC (13 strain combinations), intermediate NC (63 strain combinations), or low NC (19 strain combinations). Analysis of the segregating F1 backcross population showed NC to be controlled by at least two independently segregating genes, one of which was linked to the MHC, and the other of which was possibly, but by no means certainly, X linked. No linkage was demonstrated with respect to coat color loci (C, A, H) or to kappa chain allotype (RI-1). Natural cytotoxicity appears to belong to a group of several phenomena characterized by the rapid destruction of allogeneic cells by apparently unsensitized hosts.     

Natural killing and antibody-dependent cellular cytotoxicity are mediated by different mechanisms and by different cells.

Natural killing (NK) in humans, as well as in other species, has been shown to be specific for antigenic determinants present on the surfaces of a variety of tumor cells. Physical separation of NK cells from K cells, which mediate antibody-dependent cellular cytotoxicity (ADCC), has not been successful; however, there is indirect evidence suggesting that these activities are distinct. To further study the relationship between NK and K cells, competitive inhibition techniques were employed. NK cells can be blocked via two mechanisms: 1) by direct inhibition with NK-sensitive tumor cells binding to NK receptor sites present on the effector cells and 2) by steric inhibition resulting from the binding of antibody-coated cells to the FcR on the effector cells. K cells, however, lack the NK receptor site(s) but are FcR+, and can therefore be blocked only by antibody-coated cells. We therefore postulate that NK and K cells are two separate lymphoid populations. NK cells bear receptor site(s) for NK determinants and FcR, whereas K cells bear only FcR.     

Radiation-induced changes in the cell membrane of cultured human endothelial cells

We investigated the effect of irradiation on the kinetic characteristics of amino acid and glucose transport, and the effect on the activity of the cell membrane-bound enzyme 5'-nucleotidase and on the receptor-mediated stimulation of cyclic adenosine monophosphate synthesis by prostaglandin E1. Irradiation inhibited the sodium-dependent amino acid transport by a reduced binding of the amino acid to the transport unit. The transport of glucose, which appeared to be a sodium-independent process, was temporarily stimulated by increased maximal velocity of the transport. No effect was found on the binding to the transport unit. Irradiation increased the 5'-nucleotidase activity and decreased the prostaglandin E1-stimulated cyclic adenosine monophosphate synthesis 48 h after exposure to 20 Gy. It is concluded that irradiation decreases sodium-dependent transport by impairment of the transport unit, does not impair a sodium-independent process, and has opposite effects on membrane-bound enzyme activity and a receptor-mediated process.     

Complement-mediated serum cytotoxicity for Leishmania major amastigotes: killing by serum deficient in early components of the membrane attack complex

Leishmania major, the agent of Oriental sore, is an obligate intracellular parasite of macrophages in mammalian hosts. Man's immune defense against this organism requires participation of specifically sensitized lymphocytes and activated macrophages. Recent studies, however, have demonstrated that as little as 1/120 concentration of normal human serum is highly cytotoxic for the amastigote form of L. major. Initiation of the lethal process occurs rapidly, requiring only 30 sec of parasite exposure to serum, and is mediated by antibody-independent activation of the alternate complement pathway. The molecular mechanism of cytotoxicity is not known, but may require participation of the membrane attack complex, C5b-9. We investigated this possibility by treating amastigotes with human sera genetically deficient in complement components C5, 6, 7, 8, or 9. We then measured viability of treated parasites by amastigote-promastigote conversion. Our results were quite unexpected: not only did C9-deficient serum kill organisms, but sera singly deficient in each of the preceding components C6 to C8 were also cytotoxic. The degree of cytotoxicity was related both to serum concentration and to the point in the complement cascade at which deficiency occurred. Sera lacking C6 or C7 were less cytotoxic than those deficient in C8, which were less toxic than those deficient in C9. Cytoxicity of deficient sera was abolished by heating serum to 56 degrees C for 30 min. These findings indicate that an incomplete membrane attack complex may mediate cytotoxicity for L. major amastigotes. Moreover, our results raise important questions regarding the mechanism by which the complex is assembled on the surface of a living, unicellular eukaryotic organism.     

Induction of preferential cytotoxicity against allogeneic mouse lymphoma cells: in vitro and in vivo studies

The aim of this study was to activate, in mixed leukocyte/tumor cell cultures (MLTC), cytotoxic lymphocytes exhibiting preferential activity in vitro and in vivo towards allogeneic mouse lymphoma cells. Whereas the lymphoma target cells were readily lysed by the MLTC-derived lymphocytes, the cytotoxicity against the corresponding allogeneic concanavalin-A(ConA)-induced lymphoblasts was more than tenfold lower. Both activities were mediated by CD3⁺, TCR⁺, CD8⁺, CD4⁻ cytotoxic T cells (CTL). ConA-induced lymphoblasts were readily lysed by anti-Thy1.2 antibodies and complement, by CTL derived from mixed leukocyte cultures (MLC) and by the MLTC-derived CTL in the presence of ConA, indicating that the lymphoblasts are not merely less lysable than the lymphoma cells but that the latter are specifically recognized by the CTL. Lymphoblasts poorly competed with ⁵¹Cr-labeled lymphoma cells in a “cold”-target competition assay, suggesting that the MLTC-derived CTL largely recognize epitopes expressed only by the lymphoma cells. Furthermore, analysis of the cytotoxic activity of more than 500 MLTC-derived CTL oligoclones and over 30 clones revealed that one-third of them were cytotoxic only against the allogeneic lymphoma cells, one-third were reactive against both the lymphoma and the allogeneic lymphoblast target cells and the remainder were not cytotoxic at all. Upon injection into sublethally irradiated, lymphoma-bearing allogeneic mice, the MLTC-derived CTL cured 56% of the recipients and caused graft versus host disease (GVHD) is only 22%, whereas CTL activated in MLC against allogeneic splenocytes were therapeutically ineffective and caused lethal GVHD in 89% of the recipients. Although the therapeutic efficacy of the in vitro-generated antitumor CTL was demonstrated against experimental lymphoma lines, this strategy might prove effective in tumor immunotherapy in conjunction with other modalities. Received: 24 December 1998 / Accepted: 5 April 1999     

Natural selection and early changes of phenotype of tumor cells in vivo: acquisition of new defense mechanisms

This review summarizes results obtained in the author's and collaborating laboratories within the last decade and is designed to attract the attention of researchers to discrete biochemical mechanisms of protection acquired in vivo by cells of malignant tumors against effectors of innate antitumor immunity. Tumor progression in vivo is associated with the appearance and selection of tumor cells with new specific characteristics: a high level of H(2)O(2)-catabolizing (antioxidant) activity (H(2)O(2)CA) and the ability for immediate release of E2-type prostaglandin (PGES) on contact with natural killers, macrophages, and neutrophils; the expression of the [H(2)O(2)CA + PGES] phenotype provides the tumor cells with two mechanisms of local protection against effectors of innate and acquired antitumor immunity. This results in a 10-100-fold less effective rejection of tumor cells in immune and normal animals and corresponding increase of tumorigenicity. The in vitro transformation of normal fibroblasts, spontaneous or induced by oncogenes LTSV40, E1a,b, Ha-ras, myc, and also by p53(175) and bcl-2 does not result in the [H(2)O(2)CA + PGES] phenotype expression, but during subsequent in vivo growth of the above-mentioned transformants the selection of tumor cells of the [H(2)O(2)CA + PGES] phenotype is correlated with a 30-200-fold increase in their tumorigenicity (accompanied or not accompanied by spontaneous metastatic activity). Unlike the transformation induced by the above-mentioned oncogenes, the transformation of normal cells by the v-src gene results in the [H(2)O(2)CA + PGES] phenotype expression. The data presented confirm the determining role of the v-src gene in the expression of the [H(2)O(2)CA + PGES] phenotype. In various primary viral carcinogenesis (SV40, SA7(C8)) the natural selection of cells expressing the [H(2)O(2)CA + PGES] phenotype begins even within the latent period and can be completed by the appearance of primary tumors.     

Natural killing in immunodeficient patients

Natural killing (NK) capacity was evaluated in peripheral blood mononuclear cells from 14 patients with well defined primary immunodeficiency disorders and compared with the activity of those cells in antibody-dependent cell-mediated cytotoxicity (ADCC) assays against antibody-coated erythrocyte (killed primarily by monocytes) and lymphoid or tumor targets (killed exclusively by lymphoid cells). A selective inability to lyse antibody-coated lymphocyte targets was observed with cells from patients with x-linked agammaglobulinemia, suggesting the involvement of either a different lymphocyte subpopulation or membrane receptor for NK and ADCC, or that a different functional susceptibility exists for the two types of killing. The only immunodeficiency state in which lymphocyte NK activity was found to be lacking was severe combined immunodediciency disease.     

Radiation-induced changes in oral carcinoma cells – a multiparametric evaluation

The aim of this study was to see whether serial cytological evaluation of various cellular abnormalities in tumours from patients receiving fractionated radiotherapy can predict radio-response in oral carcinoma. Cytological assessment was carried out in scrape smears collected prior to and during the course of radiotherapy in 68 patients with squamous cell carcinoma of the oral cavity planned for radical radiotherapy with accelerated fraction schedule. Smears were evaluated for a set of 15 radiation-induced cellular abnormalities. The relationship between the cellular alterations and the cumulative radiation dose was analysed by Kruskal-Wallis one-way anova. The results showed that among the various quantifiable changes that occur in irradiated cancer cells, karyolysis, karyorrhexis, pyknosis, cytolysis, multinucleation, micronucleation and nuclear budding show significant increase depending on the dose of radiation. The radio-resistant group of patients exhibited a lesser degree of change compared with the radio-sensitive group. This suggests that radio-resistance may be due to the defective induction of cell damage and that these cytological features may have potential use as predictive markers of radio-sensitivity in oral carcinoma.     

Cytotoxic studies in human newborns: lessened allogeneic cell-induced (augmented) cytotoxicity but strong lymphokine-activated cytotoxicity of cord mononuclear cells

Nonspecific cytotoxic responses such as natural killer activity can be increased in vitro by incubating effector cells with soluble factors or allogeneic cells. We sought to determine if newborn cells, known to be deficient in most cytotoxic responses, including resting NK activity, could develop enhanced cytotoxic responses following incubation with allogeneic cells (augmented cytotoxicity) or with lymphokines (lymphokine-activated cytotoxicity). Cord whole mononuclear cells (WMC) incubated with irradiated Raji cells for 5 days develop lower levels of cytotoxicity toward K562 targets at both a 20:1 effector:target (E:T) ratio (39 +/- 2.7% vs 49 +/- 3.6%) and a 10:1 E:T ratio (29 +/- 2.6% vs 40 +/- 3.6%) than do adult cells. Lessened specific cytotoxicity of cord cells developed toward the sensitizing Raji cells was also observed at both E:T ratios. Attempts to enhance the induced cytotoxicity by incubation with interferon or isoprinosine were unsuccessful. In contrast, lymphokine (i.e., interleukin 2)-activated killer (LAK) cytotoxicity is not deficient in cord WMC. Indeed, the level of LAK cytotoxicity is equivalent to that observed with similarly treated adult cells despite a lower baseline level of cytotoxicity toward the target cells. In the presence of purified IL-2 for 5 days, cord WMC cytotoxicity against K562 cells increased from 12 +/- 2.6 to 71 +/- 4.2% and against Raji cells increased from 9.6 +/- 2.5 to 48 +/- 6.7%. Similarly treated adult cells increased their killing against K562 from 23 +/- 4.2 to 61 +/- 4.5% and against Raji from 12 +/- 3.0 to 36 +/- 5.3%. This substantial lymphokine-activated cytotoxicity of newborn cells suggests the possibility of therapeutic intervention with purified lymphokines in neonatal infections or neoplasms.     

Antibody-dependent cell cytotoxicity and spontaneous cytotoxicity of peripheral blood lymphocytes of organ recipients and cells infiltrating the kidney allogeneic transplant

It is supposed that determination of antibody-dependent cellular-mediated cytotoxicity and spontaneous cellular-mediated cytotoxicity in the peripheral blood is non-informative for early diagnosis of acute rejection crisis. Activity of K- and NK-cells in peripheral blood is observed to increase during a peak of the rejection crisis. While studying cellular infiltrate obtained from the transplanted kidney, activity of K- and NK-cells is shown to considerably increase in the rejected transplants, which can testify that they contribute much in the loss of the allotransplant function.     

Radiation-induced, irreparable, hereditary changes in cells promoting their neoplastic transformation

In experiments with model plant tumors (Kalanchoe-ti plasmid Agrobact. tumefaciens C-58D) it was shown that exposure of the recipient plant to low-level gamma-radiation of 2 Gy induced changes in cells that were not repaired over two months promoting tumoral transformations in them. Those changes were shown to persist in the offspring of the exposed somatic cells.     

Mechanisms of killing of measles virus-infected cells by human lymphocytes: interferon associated and unassociated cell-mediated cytotoxicity

The recent interest in natural killer (NK) cells in immunosurveillance and the ability of infection with certain organisms to modulate NK activity led us to examine the influence of Toxoplasma gondii infection on mouse NK cells. Infection of BALB/c mice with 5 × 10³ virulent Toxoplasma intraperitoneally (ip) resulted in significantly enhanced NK activity in peritoneal exudate cells (PC) and in spleen cells (SC). Intravenous (iv) and subcutaneous (sc) challenge of BALB/c mice with Toxoplasma also resulted in enhanced natural killer (NK) activity in PC and SC. In BALB/c mice, as well as in other strains (A/J, C57BL/6, C3H/HeJ, CeH/HeN, [A/J × C3H]F₁), peak augmentation of PC and SC NK activity was observed 3 days following ip Toxoplasma challenge. Administration of silica to mice abolished Toxoplasma-induced NK cytotoxicity. BALB/c mice chronically infected with Toxoplasma had significantly higher endogenous NK activity than did controls in PC but not in SC. Chronically infected BALB/c mice boosted with virulent Toxoplasma ip exhibited significantly enhanced NK activity in PC but not in SC. Thus, acute and chronic infection with Toxoplasma modulates NK activity in addition to macrophage activation and thereby provides a system that should facilitate study of the relative contribution of NK cells and activated macrophages in resistance to tumor growth and spread.     

Dexamethasone-induced killing of neoplastic cells of lymphoid derivation: lack of early calcium involvement

The role of calcium influx in dexamethasone-induced fragmentation of DNA was studied in the glucocorticoid-sensitive human lymphoid line of T cell derivation (CEM-C7). Reduction of calcium content in the medium or the use of EGTA increased DNA fragmentation and appeared to slightly enhance the effect of dexamethasone. Incubation of isolated nuclei in the presence of high concentrations of calcium did not bring about significant DNA fragmentation. Calmidazolium, an antagonist of calmodulin dependent reactions did not reduce the sensitivity of CEM-C7 cells to dexamethasone nor did it modify the response to dexamethasone of the resistant CEM-C1 line. It appears that in contrast to rodent thymocytes, massive calcium influx is not per se responsible for the initiation of directed cell killing (apoptosis).     

Radiation-induced changes in the structure of erythrocyte membrane proteins

Structural changes in proteins of erythrocyte membranes induced by gamma-radiation at doses of 10-10(3) Gy were studied using the method of tryptophan fluorescence quenching by acrylamide. It was found that the exposure to ionizing radiation leads to a decrease in intramolecular dynamics of membrane proteins.     

Natural cytotoxicity in lymphatic metastasis

Summary The survival of cloned variants of the BSp73 tumor differing in susceptibility to natural killer (NK) and macrophage-mediated cytotoxicity in vitro was evaluated in syngeneic animals. The cytocidal effect was assayed by whole-body determination of (¹²⁵I)5-iodo-2-deoxyuridine (¹²⁵IUdR) retention in intact animals and in animals depleted of phagocytes and/or radiation-sensitive lymphocytes. The following results were obtained: (1) In the peritoneal cavity, survival of the susceptible tumor cells (variant AS) was significantly higher in rats pretreated with radiation and silica than in untreated rats. (2) Cold target competition, response modification by C. parvum, and correlation of label excretion with survival of animals supported the notion that excretion rates of radioactivity were in fact determined by natural cytotoxicity in vivo. (3) Tumor cells resistant to natural cytotoxicity in vitro (variant ASML) were nevertheless killed in vivo (IP) by NK cells and macrophages. (4) Additional elements of tumor cell destruction were active upon IV injection of both AS- and ASML-derived cells, since rapid label excretion was observed irrespective of irradiation and silica treatment or addition of excess unlabeled competitor cells.The apparent increase in susceptibility of ASML cells in vivo might be due to a shift in phenotype induced by microenvironmental factors. No evidence was gained that differences in metastatic capacity exhibited by the variants might relate to differential action of natural cytotoxicity on these cells.