Marie-Christine Dokhelar

NATURAL KILLER CELL ACTIVITY IN HUMAN BONE MARROW RECIPIENTS EARLY REAPPEARANCE OF PERIPHERAL NATURAL KILLER ACTIVITY IN GRAFT-VERSUS-HOST DISEASE

Natural killer (NK) cell activity toward K562 target cells and antibody-dependent cell-mediated cytotoxicity (ADCC) toward L1210 cells sensitized with anti-L1210 antisera were sequentially tested in peripheral blood lymphocytes (PBLs) from 24 human bone marrow (BM) recipients. Although consistently decreased before the transplant, NK cell activity was restored in all of the patients tested that argues for a bone marrow origin of NK progenitors in humans. In patients without graft-versus-host disease (GVHD), peripheral NK cell activity remained low during the 1st month after the transplant, then rapidly increased and reached normal values usually between days 30 and 50. By contrast, peripheral ADCC appeared earlier restored (since day 13), suggesting that NK and ADCC are two distinct effector mechanisms. When restored, peripheral NK cell activity remained within normal range, except in seven cases with a drastic fall in NK cell values contemporary with a severe viral infection, mainly with cytomegalovirus (CMV). NK cells are thus suggested to play an important role in the control of viral infections in these deeply immunodepressed patients. In patients with acute GVHD, strikingly high NK values were observed early after the transplant, and during the 1st month a strong correlation did exist between high NK values and acute GVHD occurrence. These results suggest that cells involved in GVHD mechanism are able to exert NK cell activity at some stages of their maturation. The assessment of NK cell activity could be an attractive routine procedure for monitoring the prophylaxis of GVHD in human BM recipients.

Low natural killer cell activity in patients with malignant lymphoma

Natural killer (NK) cell activity was found decreased (mean value: 27.5%) in peripheral blood lymphocytes from 59 untreated patients with malignant lymphoma, when compared to 112 healthy subjects (46.3%) and to 75 cancer patients with nonlymphoid tumors (43.7%) (P > 0.001). NK cell values were similar in patients with Hodgkins disease (25.5%) and with non‐Hodgkins lymphoma (28.2%). Decreased NK cell activity was not correlated with age, lymphoma extension or leukocyte counts. A physiological role for NK cells in the immune surveillance against lymphoid neoplasias is discussed.

K562 cells induced to differentiate by phorbol ester tumor promotors resist NK lysis

The effect of various phorbols and phorbol diesters on the NK sensitivity of the human leukemic K562 cells was studied. A marked decrease in K562 cell susceptibility was achieved by culture in the presence of either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or beta-phorbol-dibutyrate. The maximum protection against NK lysis was achieved when K562 cells were cultured in the presence of 160 nM TPA for 48 hr (mean percentage inhibition: 61% of specific lysis). As for untreated targets, the residual killing of K562 cells after TPA treatment was mediated through large granular lymphocytes (LGL). The experimental procedures required to achieve maximal NK protection with TPA resulted simultaneously in marked phenotypical changes in K562 cells: erythroid and early myeloid markers decreased, whereas the expression of megakaryocytic markers was increased as shown by staining with antiplatelet monoclonal antibodies and assessment of platelet peroxidase activity. Chemical phorbol analogs which were unable to induce K562 cell differentiation did not affect K562 cell sensitivity to NK lysis. De novo protein synthesis is involved in the TPA-induced NK resistance, since this effect was abolished by pretreatment of K562 cells with actinomycin D or cycloheximide. TPA has been previously demonstrated to reduce NK effector activity. In our data however, the observed TPA effects were not due to release of TPA acting on effector cells during the NK assay since TPA-treated K562 cell supernatants were unable to inhibit NK activity in control assays. Thus, TPA appears to decrease NK killing of malignant cells, both by depressing NK effector cells functions and by reducing the susceptibility to NK lysis of the target cells. In single-cell agarose assays, TPA-treated K562 cells demonstrated reduced NK-binding capacity and reduced sensitivity to lysis after binding. These defects could not be reversed by activation of the NK effector cells with interferon. The results here reported extend the previously suggested relations between the expression of NK-target structures and the differentiation stage of malignant cells.

Protective effects of differentiation inducers on natural killer sensitivity of K 562 cells: Analysis at a single-cell level.

K 562 cells induced to differentiate by sodium butyrate (SB) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were studied for their capacities to be bound and killed by large granular lymphocytes (LGL) in a single-cell cytotoxicity assay in agarose. After SB treatment, K 562 cells were less efficient in binding to LGL, whereas the frequency of killer cells among bound LGL was unaffected. When TPA was used to induce K 562 differentiation, the binding of LGL to their target and the lytic efficiency of the bound LGL were both diminished when compared to control K 562 cells. It has been demonstrated that the expression of structures involved in the binding of natural killer (NK) effectors to their targets could be correlated with the target-differentiation stage. It is shown that phorbol-ester treatment can also affect NK target structures involved in the killing step.

Effect of a synthetic thymic factor (facteur thymique serique) on natural killer cell activity in humans

The effect of the serum thymic factor, FTS, on human NK cells was studied. NK cell activity was measured in a 51Chromium-release assay in which effector cells were peripheral blood lymphocytes (PBL) or bone marrow (BM) lymphocytes from healthy individuals and patients, and targets were K562 cells. When added in vitro in this assay, FTS modulated NK cell activity of normal PBL. Low concentrations of FTS (10(-2) ng/ml) increased NK activity (P less than 0.001) whereas higher concentrations decreased it (P less than 0.01) for 10 and 10(2) ng/ml. FTS exhibited no effect on NK cell activity of BM lymphocytes. When administered in vivo to 4 cancer patients (10 micrograms/kg i.v. every 3 days), FTS progressively increased peripheral NK activity in two patients with low pre-treatment NK values, whereas it decreased NK activity in two patients with previously normal or high NK values. The mechanism by which FTS modulates NK cell activity is still unknown but such modulation suggests that NK cells belong in part to the T-lineage.

NK CELL ACTIVITY IN PATIENTS WITH HIGH RISK FOR TUMORS AND IN PATIENTS WITH CANCER

Publisher Summary This chapter explores the NK cell activity in patients with high risk for tumors and in patients with cancer. In a study described in the chapter, effector cells were peripheral blood lymphocytes isolated by centrifugation on Eicoll–Paque and tested for spontaneous cytotoxicity against 51Cr-labeled K562 cells. Effector to target cell ratio was 50:1. Incubation was at 37°C for 4 h. Supernatants were harvested and radioactivity was counted. Percent specific lysis (% SL) was calculated. Results were compared by statistical methods with mean specific lysis calculated in control populations tested in the same conditions. Peripheral blood lymphocytes of a series of patients with various ID were tested for natural killer (NK) cell activity. Individual patients with NK cell activity in the normal range were affected with X-linked agammaglobulinemia, hypogammaglobulinemia, or some common variable ID mainly involving humoral immunity. ID patients with low NK cell activity are listed in the chapter. These included patients with severe combined ID, ataxia-telangectasia, common variable ID and Wiskott–Aldrich syndrome. All these patients shared severe defects in various T cell-mediated functions, in agreement with experimental data suggesting that NK cells could be of T lineage.

RECOVERY OF NK CELL ACTIVITY AFTER BONE-MARROW TRANSPLANTATION

Publisher Summary This chapter presents a study analyzing the recovery of natural killer (NK) cell activity after bone-marrow transplantation. Twenty-four patients undergoing bone-marrow transplantation were studied. They were all grafted at the Hopital St Louis, Paris, bone-marrow transplant unit. Thirteen patients had idiopathic severe aplastic anemia, 4 post-hepatitic aplastic anemia, 1 Fanconi anemia, and 6 various leukemias. In 17 cases, the donor was an HLA-identical sibling and an identical twin in 4 cases. The conditioning regimen in most cases included total body irradiation (700 to 1,000 rads) and cyclophosphamide (120 mg/kg). After the transplant, 14 patients received methotrexate and 6 patients cyclosporin A for GVHD prophylaxis. Patients who had identical twins received no treatment after grafting. During the first 30 days, nine patients had evidence of GVHD. No rejection was observed, and complete chimerism was demonstrated in 17 patients. NK cell activity was depressed in most cases studied before the conditioning regimen, especially in patients with aplastic anemia. Such low NK values had already been observed and could reflect some stem cell defect involving NK precursors.

EFFECT OF SODIUM BUTYRATE AND HEMIN ON NK SENSIVITY OF K562 CELLS

Publisher Summary This chapter describes the effect of sodium butyrate and hemin on natural killer (NK) sensitivity of K562 cells. In human NK cell assays, the leukemic K562 cells are commonly used as targets for their high and reproducible sensitivity to NK lysis. The K562 cell line was originally considered as a highly undifferenciated cell line of the granulocytic lineage. Several erythroid markers such as glycophorin A are also expressed in K562 cells, and hemoglobin synthesis has been demonstrated either spontaneously in various culture conditions and after sodium butyrate or hemin induction. Exposure to hemin increases Hb synthesis four- to fivefold but does not permit K562 cells to reach the final maturation steps of the erythroid lineage. A complete abrogation of K562 sensitivity to NK lysis after SB treatment could never be achieved. It was found that with regard to various hematopoietic markers, the K562 line appeared quite heterogeneous, some cells exhibiting erythroid features either spontaneously or after induction and some other cells carrying megakarycytic markers. Cloning of K562 cells resulted in different subclones appearing more homogenous in their cellular commitment.