Fresh Leukemic Blasts Research Articles

Generation of cytotoxic NK cells in peripheral blood and bone marrow of patients with acute myelogenous leukemia after continuous infusion with recombinant interleukin‐2

We have studied the cytotoxic profile and distribution of lymphocyte subsets of patients with acute myelogenous leukemia in second remission, after continuous infusion with recombinant interleukin-2 (IL-2). The patients received repetitive cycles of 1-1.25 x 10(6) U/m2/day of IL-2, given as 4 days continuous intravenous infusion followed by a 3-day treatment-free interval for the first 4 weeks. Patients receiving greater than 4 cycles were treated with the same dose of IL-2 continuously for 4 days, followed by a 10-day treatment-free interval. These studies showed that IL-2 treatment resulted in the generation of peripheral blood cytotoxic activity against both NK-susceptible, K-562, and NK-resistant Daudi cell lines. In most patients, enhancement of lytic activity increased with the number of IL-2 infusions. The cytotoxicity in some patients increased as much as 700-fold and 830-fold against K-562 and Daudi cells, respectively. It is of importance that oncolytic activity was also induced in bone marrow compartment (up to 182-fold against K-562). Some decline in cytotoxicity was observed within 14 days after initiation of IL-2 infusion in peripheral blood, but high levels of lytic activity persisted at this time in bone marrow. It is of interest to note that the cytotoxicity of in vivo IL-2 primed lymphocytes was further potentiated by IL-2 in vitro. Importantly, the cytotoxic cells induced in vitro displayed lytic activity against fresh leukemic blasts. Phenotypic analysis demonstrated that CD3-, CD56+ NK cells were significantly increased by in vivo IL-2 treatment (34 to 47-fold in absolute numbers), while CD3-, CD56+ T-cell subset remained low. Characterization of cytotoxic cells using the complement-dependent assay and monoclonal antibodies indicated that both the in vivo-induced and ex vivo-potentiated lytic function was mediated by CD3-, CD56+, CD16- -NK cells.

Effects of Interleukin‐6 and Granulocyte Colony‐stimulating Factor on the Proliferation of Leukemic Blast Progenitors from Acute Myeloblastic Leukemia Patients

The effects of recombinant human interleukin‐6 (rh IL‐6), which has homology with rh granulocyte colony‐stimulating factor (rh G‐CSF) at the amino acid sequence level, and rh G‐CSF on normal human bone marrow cells, fresh leukemic blast progenitors from 16 acute myeloblastic leukemia (AML) patients, and G‐CSF‐dependent human AML cell line (OCI/AML 1a) were investigated. rh G‐CSF stimulated the proliferation of leukemic blast progenitors from 13 out of 16 AML patients tested. rh IL‐6 stimulated the proliferation of blasts from eight AML patients and enhanced the G‐CSF‐dependent proliferation of the fresh AML blasts from two out of eight patients tested. On the other hand, rh IL‐6 suppressed the blast colony formation from two AML patients and OCI/AML 1a cells and also reduced the G‐CSF‐dependent proliferation of the blast progenitors from one of the two patients and the cell line. rh IL‐6 had no effect on the colony formation of normal granulocyte‐macrophage colony‐forming units (CFU‐GM) with or without rh G‐CSF. Differentiation‐induction by rh IL‐6 was not observed in the fresh AML blasts but was observed in OCI/AML 1a. The effect of IL‐6 on the blast colony formation and G‐CSF‐dependent blast cell growth was complicated and heterogenous among the AML cases; IL‐6 stimulated blast colony formation in some cases and suppressed it in others. The heterogeneity of the response was supposed to be derived from the heterogeneity of the characteristics of AML cells. Although G‐CSF simply stimulated the blast colony formation, IL‐6 had a bimodulatory effect on the proliferation of leukemic blast progenitors from AML patients. IL‐6 might be involved in the regulation of the proliferation of AML cells in vivo as well as in vitro.

SPECIFIC RECOGNITION OF HUMAN LEUKEMIC CELLS BY ALLOGENEIC T CELL LINES

Clinical and experimental data suggest a role for the immune response in preventing leukemic relapses following allogeneic bone marrow transplantation the graft-versus-leukemia (GVL) effect. In the context of an allogeneic BMT, a number of different immune mechanisms mediated by donor cells may be responsible for the GVL effect. We have approached this question by using limiting dilution cultures of alloactivated human lymphocytes to analyze the in vitro allogeneic cytolytic response against fresh allogeneic leukemia. Initial results in the limiting dilution assays with split culture analyses demonstrated frequent alloreactive cytolytic T lymphocyte precursors that destroyed remission peripheral blood lymphocytes and leukemic cells from the allogeneic leukemic patient. These assays also demonstrated frequent lymphokine-activated killer (LAK) cell precursors that lysed both the LAK sensitive Daudi line and the allogeneic leukemia. In these experiments, isolated cultures also showed cytolytic activity directed against the allogeneic leukemic blasts without activity against remission PBL, or the LAK-sensitive Daudi cell line. Two T cell lines (ABL1 and ABL2) isolated from an LDA, demonstrated this form of specificity, mediating destruction specifically against the allogeneic acute lymphoblastic leukemic cells. Both cell lines ABL1 and ABL2 were CD3+, TCR alpha beta +, and CD4+. These 2 cell lines mediated little or no cytotoxicity against a large panel of other targets tested (natural killer sensitive and resistant cell lines, allogeneic PBL, and allogeneic fresh leukemic blasts). Antibody-blocking experiments revealed a role for the CD3-TCR receptor of both cell lines in lysis of leukemic cells; the CD4 and MHC class II molecules were clearly involved in the lysis by the ABL1 cell line. Specificity of recognition for the allogeneic leukemic blasts was further confirmed by unlabeled target competitive inhibition studies. The mechanism of the preferential lysis of leukemia by the alloactivated T cell lines described in this paper remains uncertain. Nevertheless, these leukemic-specific populations provide a means by which the human GVL effect may be further studied in vitro.

Use of the mtt assay for rapid determination of chemosensitivity of human leukemic blast cells

A microcytotoxicity assay employing a tetrazolium salt has been adapted for testing the response of human leukemic blast cells to a variety of chemotherapeutic agents. After exposure to various concentrations of drugs, the viability of fresh leukemic blast cells was measured using a tetrazolium salt, MTT, which is converted to blue formazan crystals by living cells. The amount of formazan produced was quantitated using a microtitre plate spectrophotometer. In the present study, optimal conditions for chemosensitivity testing of human leukemia samples were determined, and the relative chemosensitivity of five patient samples was tested.

Effects of interferon and retinoic acid on the growth and differentiation of clonogenic leukemic cells from acute myelogenous leukemia patients treated with recombinant leukocyte-αA interferon

Studies with human myeloid leukemia cell lines indicate that combined interferon (INF) and retinoic acid (RA) have greater effects in inhibiting cell growth and in inducing terminal differentiation than either agent alone. Consequently, we studied the effects of these agents, singly and in combination, on fresh leukemic blast cells obtained from 13 acute myelogenous leukemia (AML) patients, most of whom were subsequently treated with recombinant leukocyte-αA interferon (rINF-αA). The in-vitro response to rINF-αA and RA was assessed in an established myeloid leukemic blast cell clonogenic assay containing conditioned medium from phytohemagglutinin-stimulated lymphocytes. Strong inhibition of colony cell growth ( ▪50%) was observed in 4 10 cases treated with rINF-αA alone, but only at high concentration ( ▪2500 U/ml) and in 4 10 cases treated with RA alone (5 × 10 −8M or 5 × 10 −7M). Combined rINF-αA and RA augmented the inhibition of primary or secondary colony cell growth in 5 8 evaluable cases. Stimulation of leukemic cell differentiation was observed in 1 8 cases by rINF-αA alone and in 4 7 cases by RA alone. Combined rINF-αA and RA enhanced cell differentiation in 4 7 cases. In addition, increased inhibition of clonal cell growth and/or differentiation by RA alone was observed in 2 5 cases following in-vivo rINF-αA treatment. These results suggest that treatment with combined rINF-αA and RA may be rewarding in some cases of AML.