Human natural killer cells (NK) require accessory cell-derived contact and soluble factors for maximal expansion. However, it is unclear whether increased recruitment of clonogenic NK, increased proliferation on a per cell basis, or a combination of both is responsible for the increased expansion. We show that expansion of both CD56+dim and CD56+bright NK from normal donors is increased in the presence of M2- 10B4 accessory cell-soluble factors. In contrast, the addition of M2- 10B4 stromal ligands further augments only the expansion of CD56+bright NK. Using single-cell sorting of CD56+bright NK, M2–10B4-soluble and contact factors independently increase both recruitment of clonogenic NK and proliferation on a per cell basis. This well-defined M2–10B4 accessory cell system was used to investigate potential defects in NK from patients with CML. Although we have previously shown diminished interleukin-2 (IL-2)-activated NK outgrowth and function from patients with chronic myelogenous leukemia (CML) as their disease progresses, it has been unclear if this is due to a defect in an accessory cell function or an inherent abnormality of CML NK themselves. CD56+/CD3- NK purified by fluorescence-activated cell sorting from 21 patients (7 early chronic phase [ECP] patients, 10 late chronic and accelerated phase [LCP/AP], and 4 blast crisis [BC] patients) were studied. The proliferative capacity, clonogenic frequency, and cytotoxic capacity of CML NK were compared with NK from normal donors. The absolute number of circulating NK per milliliter of peripheral blood is significantly decreased in patients with CML compared with normal donors (normal, 63,700 +/- 6,400; ECP, 40,700 +/- 6,700; LCP/AP, 31,900 +/- 6,000; BC, 10,700 +/- 5,200). Additionally, the unique CD56+bright NK subset, analyzed as a percentage of the total circulating NK pool, is significantly reduced in all patients with CML (normal, 5.7% +/- 0.8% v CML [all stages combined], 2.5% +/- 0.5%, P = .001]. After purification of NK to correct for differences in circulating NK number, resting NK cytotoxicity against K562 tumor targets is significantly reduced in patients with CML on or recently on hydroxyurea therapy. However, this reduced cytotoxicity can be corrected by 18 hours of incubation with 1,000 U/mL recombinant IL-2. When plated in limiting dilution on viable M2–10B4, which maximally stimulates NK from normal donors, we show that both NK clonogenic frequency and proliferative capacity are significantly reduced as CML progresses, demonstrating an inherent defect in their ability to respond to normal NK stimuli. Although NK cloning efficiency between normal donors and ECP CML patients was the same, significant differences were observed in (1) the absolute number of circulating CD56+/CD3- NK, (2) the absolute number of circulating CD56+bright NK, and (3) proliferation on a per cell basis. Unlike resting NK function, prior cytotoxic therapy alone did not account for these observed abnormalities. These data suggest that, although NK are not derived from the malignant clone, they are inherently affected by their malignant microenvironment.

A graft-versus-leukemia effect has been well documented to prevent relapse in chronic myelogenous leukemia (CML) after allogeneic marrow transplantation. One type of lymphocytes that may contribute to this effect are natural killer cells (NK), which after activation with interleukin (IL)-2, exhibit a broad range of cytolytic activity against allogeneic and autologous cells. We have previously demonstrated that IL-2-activated NK (ANK) can be generated from blood of patients with CML and are benign in origin. Their proliferation and function, however, diminish with disease progression in CML, suggesting a role in tumor surveillance. We studied the effect of IL-2-activated NK (ANK) on normal and malignant primitive and committed progenitors in a novel long-term bone marrow culture (LTBMC) assay. Because ANK destroy marrow stromal layers, the use of classic stroma-dependent long-term cultures is not possible. Therefore, we used the stroma noncontact LTBMC system developed in our laboratory to analyze the effect of autologous ANK cells on primitive hematopoietic progenitors. Autologous ANK (CD56+/CD3- ) were generated from the peripheral blood of 10 patients with chronic phase CML and from six normal individuals by culturing CD5/CD8-depleted mononuclear cells for 14 days in 1,000 U/mL IL-2. At the same time ANK cultures were initiated, sorted normal (CD34+/DR+) marrow populations were plated in Transwell inserts of the stroma noncontact culture. On day 15, hydrocortisone, which rapidly inhibits ANK function, was removed, and autologous ANK were added to the Transwell inserts with fresh LTBMC medium without hydrocortisone but supplemented with 1,000 U/mL IL-2. After 48 hours, the number of colony-forming cells (CFC) was enumerated in methylcellulose culture. To determine the effect of ANK on more primitive long-term culture-initiating cells (LTCIC), the IL-2- supplemented LTBMC medium was replaced with fresh hydrocortisone containing LTBMC medium, and cultures were maintained for an additional 5 weeks. We demonstrate that autologous ANK did not suppress normal CFC or LTCIC. In contrast, ANK from eight patients with CML with potent cytotoxicity against NK-sensitive (K562) NK-resistant (Raji) tumor targets exhibited an ANK dose-dependent suppression of both CFC and LTCIC. Interestingly, ANK from two patients with CML who exhibited diminished cytotoxicity also did not suppress autologous CFC and LTCIC. These studies indicate that ANK with potent major histocompatibility complex unrestricted cytotoxic activity suppress malignant hematopoiesis. This effect was not mediated by soluble factors and was absolutely dependent on direct cell-to-cell contact. We further demonstrate that the beta2 integrin receptor is involved in ANK recognition of CML targets. These observations support the use of autologous ANK therapy to prevent relapse of CML after autologous marrow transplantation or use of ANK to purge CML marrow for autologous transplantation.

Large-scale ex vivo expansion of human natural killer (NK) cells for adoptive immunotherapy requires assurance of good manufacturing practices. However, maximal expansion of NK is also desired to facilitate clinical trials with large numbers of IL-2-activated NK (ANK). A closed-system stirred-tank bioreactor is amenable to computer control of culture variables, thereby reducing the risk of contamination. We demonstrate that NK cultured in 250-ml spinner flasks expand 2.5-fold more than NK cultured in stationary tissue culture wells. We further show that during 33 days of culture, it is feasible to control the pH between 7.0 and 7.2 and the dissolved oxygen concentration at 40% of air saturation via direct on-line computer control in a 750-ml stirred-tank bioreactor. On-line measurement of optical density by a laser turbidity sensor, as a measure of cell concentration, correlated well with actual cell count data. NK expansion in the 750-ml bioreactor was 7-fold greater than in stationary tissue culture controls and 3-fold greater than in spinner flask controls. Consumption rates of glucose and oxygen and the production rate of lactate were measured and will be used to develop a nutrient feeding strategy to convert the batch reactor experiment into closed-system fed-batch or continuous flow modes. Computer-controlled stirred-tank bioreactors may facilitate clinical trials with high-purity ANK populations for adoptive immunotherapy.

We studied the effects of basal medium and human serum on the ex vivo expansion of CD56+/CD3- natural killer cells (NK). We demonstrated that sorted NK cultured for 18 days with 10% human AB serum without accessory cells in a 2:1 DMEM/F12 basal medium expanded significantly greater (6.4 +/- 0.9-fold, n = 14) than when cultured in standard RPMI 1640 basal medium (3.7 +/- 0.67-fold, n = 16; p = 0.019). Supplementation of the DMEM/F12 mixture with 2-mercaptoethanol (2ME), ethanolamine, L-ascorbic acid, and sodium selenite significantly augmented NK proliferation (16.3 +/- 2.5-fold, n = 11; p < 0.001) compared with DMEM/F12 without supplements. NK growth kinetics demonstrated that both the growth rate and the duration of exponential growth were increased by medium supplements. Addition of 2ME-containing supplements to cultures of NK with accessory cells augments NK proliferation, recruits NK progenitors, and has a serum-sparing effect. For the large-scale expansion of NK, we demonstrate a greater than 30-fold increase (n = 7) in the number of activated natural killer cells (ANK) derived from CD5/CD8-depleted PBMNC when cultured for 35 days in supplemented DMEM/F12 versus RPMI 1640 basal medium (197.6 +/- 95.0-fold versus 6.3 +/- 2.1-fold, respectively). Serum-free media (AIM-V and X-VIVO 10) were unable to support NK proliferation. These data suggest that utilizing 2:1 DMEM/F12 + 2ME-containing supplements instead of standard RPMI 1640 as a basal medium can increase the proliferation of cytotoxic NK while reducing the culture interval and the amount of serum needed.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic myelogenous leukemia (CML) is a lethal disease of the hematopoietic stem cell. Bone marrow transplantation has highlighted the importance of allogeneic disparity in maintaining remissions in CML. However, it has been unclear whether the immune effect against CML is mediated by T cells, natural killer cells (NK) or a combination of both. We have previously demonstrated that autologous activated NK are capable of selectively lysing malignant CML progenitors while sparing benign progenitors. NK effectors may play an important role in CML since NK lytic function, clonogenic frequency and proliferative capacity decrease as CML progresses from chronic phase to advanced phase and blast crisis. Incubation of CML NK with IL-2 is capable of restoring cytolytic activity to normal levels. We hypothesize that activated NK represent a potential therapy against CML to maintain remissions in a minimal residual disease setting induced by autologous transplantation. Clinical trials are in progress to test whether IL-2 based immunotherapy and activated cell infusions play a therapeutic role in CML.