Irreversible cancer cell-induced functional anergy and apoptosis in resting and activated NK cells.

The interaction of natural killer (NK) cells with target cells, such as K562, results in NK functional inactivation and apoptosis. The role of NK-activating cytokines, IL-2, IL-12, and IL-15, in the regulation of NK inactivation and programmed cell death by target cells was examined. Purified natural killer cells were obtained from human peripheral blood and either co-incubated with K562 target cells and cytokines or the NK cells were pretreated with cytokines for 18 h prior to co-culture with K562 cells. Sorted NK cells were examined for cytotoxic activity and NK co-cultured with K562 were examined for cytokine secretion, phenotyping and DNA fragmentation. The cytotoxic activity was inhibited and was not alleviated by cytokine treatment. Whereas the cytokine treatment maintained NK cell viability for several days, NK cell viability was decreased significantly in the presence of K562 target cells. Downregulation of CD16 and upregulation of CD69 on NK cells were induced by K562 target cells and no modulation of these antigens was observed with cytokine treatment. A subpopulation of target-treated NK cells succumbed to cell death by apoptosis and cell death was not rescued by the activating cytokines. These findings demonstrate that target-induced functional inactivation and apoptosis of NK cells were not rescued by the activating cytokines IL-2, IL-12, and IL-15 regardless of whether the NK cells were pretreated with cytokines prior to exposure to K562 or the cytokines were added to the NK-K562 mixtures. These results also suggest that signals triggered by the target cells and resulting in NK cell anergy and apoptosis override cytokine-mediated signals for activation, cell proliferation, and survival.

Generation of activated natural killer (A-NK) cells in patients with chronic myelogenous leukaemia and their role in the in vitro disappearance of BCR/abl-positive targets.

Activated natural killer (A-NK) cells, a subset of CD56(dim)CD3- lymphocytes, are obtained from PBMC of normal donors by adherence to plastic and culture in the presence of IL2. In this study we tested the feasibility of generating A-NK cells in patients with Ph+ chronic myeloid leukaemia (CML). Cultures obtained from patients with early chronic phase (ECP; n=7) contained a mean (+/-SD) of83 +/- 7% of CD3- cells, and those from patients with advanced chronic phase (ACP; n=7) contained 27+/-33% CD56+CD3- cells. In three patients with leukaemia in a blastic phase (BP) it was only possible to obtain one culture enriched in CD56+CD3- cells (81%). Cellular aggregates of myeloid cells and large granular lymphocytes were observed in early A-NK cell cultures. Paired freshly-adherent and cultured A-NK cells were tested for the presence of BCR/abl mRNA by RT-PCR. The BCR/abl+ cells were detected in all 12 preparations of the freshly adherent A-NK cells tested. In 6/12 the BCR/abl+ cells were no longer detectable by RT-PCR on day 14 of culture. Both proliferation and antileukaemic cytotoxicity were significantly higher (P=0.002 and P=0.029, respectively) in the BCR/abl- cultures than those in the six BCR/abl+ cultures. 5/6 BCR/abl- cultures were highly enriched in A-NK cells on day 14, and 1/6 contained predominantly CD56+CD3+ cells. Only 2/6 BCR/abl + cultures were enriched in A-NK cells on day 14, but they had poor cytotoxicity and a low proliferative index. Myeloid cells (CD33+) were more frequently detected in the BCR/abl+ than BCR/abl- A-NK cell cultures (P=0.028). These observations suggest that: (1) populations of benign A-NK cells can be generated from the peripheral blood of CML patients; (2) the ability to generate A-NK cells is impaired in patients with advanced CML; and (3) the ability to generate A-NK cells with antileukaemic activity correlates with the disappearance of BCR/abl+ cells from these cultures.

Unimpaired ability to generate adherent lymphokine-activated killer (A-LAK) cells in patients with primary or metastatic liver tumors.

Adherent lymphokine-activated killer cells (A-LAK cells) obtained from human peripheral blood mononuclear cells represent a population of potent antitumor effectors enriched in interleukin-2(IL-2)-activated natural killer cells. This study shows that A-LAK cells can be successfully generated from the blood of patients with liver cancer not treated with adjuvant chemotherapy or irradiation. Mononuclear cells were isolated from the blood of 33 patients with liver tumors (6 benign, 10 primary malignant, 17 metastatic) at the time of liver resection. A-LAK cells were separated by adherence to plastic following activation of peripheral blood mononuclear cells in 1000 U/ml recombinant IL-2. A-LAK cells (enriched up to 92% in CD3-CD56+ cells) showed better subsequent expansion and two to six times higher antitumor cytotoxicity per cell than unseparated LAK cells cultured under the same conditions. The ability to generate A-LAK cells with superior in vitro cytotoxicity from the blood of most patients with liver cancer indicates that adoptive cellular immunotherapy may be a feasible and new way of treatment for primary and secondary hepatic neoplasms in man.