Autologous activated natural killer cells suppress primitive chronic myelogenous leukemia progenitors in long-term culture

Abstract

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.

Hsp12p and PAU genes are involved in ecological interactions between natural yeast strains

“…Using transwell plates, a well‐known approach to show cell to cell communication non‐mediated by contact (Cervantes et al ., ; Cheng et al ., ), we determined whether the Sgu165 interference on Sgu421 growth depends on molecules released into the culture medium or on the contact between the cells. We cultivated for 6 days Sgu421H11 on the basal surface of 24 transwell plate and on the upper side Sgu165E10 in SWM (Fig.…”

Sustained upregulation of effector natural killer cells in chronic myeloid leukemia after discontinuation of imatinib

“…(16) In CML, CD8 + T cells reportedly recognize leukemic cells, (30,31) and autologous activated NK cells have been shown to efficiently suppress the growth of malignant hematopoietic progenitors in vitro. (32) Some patients with CML and Philadelphia-positive acute lymphoblastic leukemia who were treated with the second-generation TKI dasatinib showed elevation of circulating large granular lymphocytes and developed numerical expansion of NK cells, CD8 + T cells, or both, which is associated with superior therapeutic responses. (33,34) In contrast, dasatinib has also been shown to suppress NK cell cytotoxicity.…”

Granulocyte colony‐stimulating factor given in addition to interferon‐α to mobilize peripheral blood stem cells for autologous transplantation in chronic myeloid leukaemia

“…Although only 14 patients have been currently autografted, the confirmation in our series of a recently described correlation (Carlo-Stella et al, 1994a;Talpaz et al, 1995) between the percentage of Ph ¹ cells in the graft and the degree of cytogenetic response upon haemopoietic recovery post graft might also indicate that this method, allowing the harvest of partially Ph ¹ PBSC samples, could help to improve the antileukaemic effect of the PBSC transplantation . As shown by the antileukaemic effect of graft-versus-host disease following allogeneic transplant in CML (Goldman et al, 1988), and by recent studies showing the role of immune competent cells in the control of the leukaemic clone (Bhatia & McGlave, 1995;Cervantes et al, 1996;Coleman et al, 1996;Lewalle et al, 1996;Pawelec et al, 1996;Silla et al, 1996), the immune system appears to be very important in preventing evolution of the disease post graft. In this respect, a potential interest of priming the harvested PBSC with interferon would be to achieve a better antileukaemic immunity post graft.…”