Primary leukemia cells resistant to alpha-interferon in vitro are defective in the activation of the DNA-binding factor interferon- stimulated gene factor 3

Cells from one-third of chronic lymphocytic leukemia (CLL) patients are resistant to alpha-interferon (alpha-IFN) as measured by induction of blast transformation. We have previously shown that all CLL clones express alpha/beta-IFN receptors, but that the resistant cells are defective in the induction of the enzyme 2',5'-oligoadenylate synthetase (2',5-A synthetase). Thus, the deficiency in IFN sensitivity is localized somewhere between the interaction of the IFN molecule with its receptor and induction of 2',5'-A synthetase. We have now further characterized the resistance of CLL clones to IFN by investigating whether it is associated with a defect in the activation of IFN- stimulated gene factor 3 (ISGF3), which is involved in the activation of alpha-IFN-stimulated genes (ISGs). A defect induction of ISGF3 after alpha-IFN treatment was found in 4 of 12 CLL patients. There was a close correlation between defective induction of ISGF3 and a lack of enhancement of 2',5'-A synthetase as well as induction of blast transformation. Pretreatment with gamma-IFN and mixing experiments with extracts from IFN-sensitive cells indicate that a lack of the gamma- component of ISGF3 was the reason for defect in activation in 2 of the patients. We conclude that a defect in activation of ISGF3 is a possible cause for resistance in CLL cells to IFN-induced blast transformation in vitro.     

Suppression of normal human erythropoiesis by human recombinant DNA-produced alpha-2-interferon in vitro

Interferons (IFN) have been shown to suppress the proliferation of human erythroid progenitors (BFU-E, CFU-E) in vitro. We have previously demonstrated that the inhibition of erythroid colony formation by gamma-IFN in vitro is mediated, in part, through the activation of monocytes and T-lymphocytes. In order to examine the mechanism(s) underlying the inhibitory action of one type of recombinant alpha-IFN (alpha-2-IFN) on erythropoiesis, the effect of different doses (80-10,000 U) of alpha-2-IFN on erythroid colony formation by normal human bone marrow cells in the presence or absence of monocytes and/or T cells was studied. The addition of alpha-2-IFN to whole marrow caused the suppression of BFU-E (10%-68%) and CFU-E (5%-75%) in a dose-dependent fashion. This inhibition occurred with the direct addition of alpha-2-IFN to culture plates but not with brief preincubation of marrow cells with alpha-2-IFN followed by washing of the cells. By contrast, brief exposure of marrow cells to gamma-IFN resulted in significant suppression of erythroid colony formation. The inhibitory action of alpha-2-IFN was not influenced by erythropoietin. Removal of monocytes and/or T cells prior to the addition of alpha-2-IFN failed to significantly reduce the suppressive effects of this molecule (BFU-E: 21%-66%; CFU-E: 20%-83%). Coculture of purified monocytes or T-lymphocytes preexposed to alpha-2-IFN with autologous bone marrow cells did not cause suppression of erythropoiesis; monocytes or T cells similarly treated with gamma-IFN, however, inhibited autologous BFU-E and CFU-E in vitro. These results demonstrate that, unlike gamma-IFN, the inhibitory effect of alpha-2-IFN on erythroid colony formation in vitro is not mediated to any significant degree through monocytes and T-lymphocytes. The suppressive effect of alpha-2-IFN occurs either directly at the erythroid progenitor(s) level and/or through accessory cell(s) other than monocytes and T cells.     

Interferon production by human marrow stromal cells

We have demonstrated that normal human bone marrow stromal cells can be induced to produce high levels of beta-interferon (IFN). One of three randomly selected human stromal cell lines produced beta-IFN in similar amounts to one of the best beta-IFN producer cell lines produced beta-IFN in similar amounts to one of the best beta-IFN producer cell lines (MG-63 osteosarcoma cells). The marrow stromal cell lines did not produce gamma-IFN, though a low level of alpha-IFN was apparently produced. The stromal cell lines differ from usual producer cell lines in that they can be subcultured for a much longer growth period and also maintain the ability to producer IFN. The cells are karyotypically normal and are not virus transformed. Such cell lines may be useful in the production of human IFN as well as allowing studies on the role of IFN in stromal cell-haemopoietic cell interactions.     

Modulation of responsiveness of chronic myelogenous leukemia granulocyte-macrophage colony-forming cells to growth regulation following in vivo treatment with recombinant gamma-interferon

A patient with Philadelphia chromosome (Ph) chronic myelogenous leukemia (CML), in chronic phase, was treated with recombinant gamma-interferon (r gamma-IFN) in a phase I clinical trial. Prior to treatment, analysis of in vitro agar culture parameters indicated hyporesponsiveness of granulocyte-macrophage colony-forming cells (CFU-GM) to inhibition by prostaglandin E and acidic isoferritins and diminished expression of class II major histocompatibility complex (MHC) antigens (HLA-DR). Treatment was associated with no change in bone marrow cellularity or in the percentage of Ph cells. However, in vitro cultures of bone marrow cells showed a return to normal levels of both expression of CFU-GM class II antigen and of sensitivity to inhibition by prostaglandin E and acidic isoferritins which predicted and/or confirmed clinical response. Throughout the course of interferon therapy, white blood cell counts (WBC) and the percentage of bone marrow blast cells were maintained at normal levels. Onset of aggressive-phase disease was associated with increased WBC, an increase in bone marrow blast cells, a secondary chromosomal abnormality, loss of CFU-GM sensitivity to inhibition by putative negative growth regulators, and markedly diminished MHC class II antigen expression. Following a bone marrow transplant from a matched sibling, all hematologic parameters studied were found to be normal. These findings indicate that treatment with r gamma-IFN can modulate some of the abnormal growth characteristics of CFU-GM observed in CML.     

Serum levels of alpha-interferon and gamma-interferon in patients with acute and chronic viral hepatitis

We measured activities of alpha- and gamma-interferon simultaneously in 198 sera of 70 patients with acute and chronic viral hepatitis using specific and sensitive enzyme immunoassay and immunoradiometric assay. The results were compared with those in patients with influenza and in healthy controls. Twelve out of 28 patients with acute viral hepatitis showed positive alpha-IFN and/or gamma-IFN activities. alpha-IFN was detectable throughout the clinical course while gamma-IFN levels rose in the convalescent phase regardless of etiology. Conversely, in patients with influenza, both alpha-IFN and gamma-IFN levels of initial samples tended to be higher than those of late samples. Six out of 12 patients with chronic active type B hepatitis showed increased alpha-IFN and/or gamma-IFN values during acute deterioration with marked elevation of serum alanine aminotransferase. However, the two interferons did not always appear simultaneously, although either was detectable in both acute and chronic hepatitis. Enhanced alpha-IFN or gamma-IFN activity was not found in asymptomatic chronic hepatitis B carriers or in patients with chronic persistent hepatitis and liver cirrhosis with chronic hepatitis B virus infection, with the exception of 2 cases. Our results indicated that circulating multiple IFN species were present during the clinical course in some patients with acute and chronic viral hepatitis.     

Differential expression of HLA-DR antigens in subsets of human CFU-GM

Expression of HLA-DR surface antigens by granulocyte/monocyte colony- forming cells (CFU-GM) may be important in the regulation of proliferation of these cells. Using immunological techniques to enrich for progenitor cells, we investigated the expression of HLA-DR in subsets of CFU-GM. "Early" (day 14) CFU-GM express higher levels of HLA- DR than do "late" (day 7) CFU-GM. Among late CFU-GM, cells destined to form monocyte (alpha-naphthyl acetate esterase-positive) colonies express higher levels of HLA-DR than do CFU-GM destined to form granulocyte (chloroacetate esterase-positive) colonies. Because high- level expression of DR antigen was a marker for monocyte differentiation, we examined several lymphokines for their effects on both DR expression and in vitro commitment to monocyte differentiation by myeloid precursor cells. DR antigen density could be increased by more than twofold over 48 hours upon exposure to gamma-interferon (gamma-IFN), whereas colony-stimulating factors had no effect. This was associated with a dose-dependent inhibition of total CFU-GM number, and a relative, but not absolute, increase in the ratio of monocyte colonies to granulocyte colonies. Similarly, in day 7 suspension cultures of purified myeloid precursor cells, gamma-IFN inhibited cell proliferation and increased the ratio of monocytes to granulocytes. Thus, despite the induction of high levels of HLA-DR antigen on precursor cells (a marker of monocyte commitment), the dominant in vitro effect of gamma-IFN was inhibition of granulocyte differentiation.     

Monocytes enhance gamma-interferon-induced inhibition of myeloid progenitor cell growth through secretion of tumor necrosis factor

In this study, we have examined the effects of autologous monocytes and T-lymphocytes on gamma-interferon (gamma-IFN)-induced inhibition of granulocyte-monocyte progenitor cells (CFU-GM) in vitro. Depletion of adherent cells from the mononuclear fraction of normal bone marrow (NBM) resulted in a significant reduction in the inhibitory effects of gamma-IFN on CFU-GM growth, whereas T-lymphocyte depletion had no effect. Adding back autologous monocytes to the underlayer fraction of agar culture resulted in a concentration-dependent enhancement of gamma-IFN-induced CFU-GM inhibition that did not require cell-cell contact. Adding back autologous T-lymphocytes had no effect and did not synergize with monocytes in enhancing gamma-IFN-induced inhibition. Based on the use of indomethacin and the pattern of CFU-GM subset growth, it was determined that prostaglandin E was unlikely to be the humoral inhibitory factor involved in this process. However, the effects of monocytes were completely reversed in the presence of a neutralizing monoclonal antibody to tumor necrosis factor (TNF), suggesting that monocyte-derived TNF was responsible for the enhancement of gamma-IFN-induced CFU-GM inhibition. This observation was further supported by the ability of gamma-IFN to induce an eightfold increase of baseline monocyte TNF secretion in agar culture. These data suggest that gamma-IFN may inhibit progenitor cell growth in vitro through indirect humoral mechanisms involving monocyte-derived TNF, as well as through direct inhibitory effects on CFU-GM proliferation. Because monocytes are a component of the bone marrow microenvironment, the ability of gamma-IFN to induce biologically relevant levels of monocyte-derived TNF may play an important role in the negative regulation of hematopoiesis.     

Alpha-interferon broadens the difference between surviving fractions of normal and leukemic progenitor cells in vitro by heat: its application to marrow purging.

Alpha-interferon (IFN) may inhibit the proliferation of human leukemic progenitor cells (L-CFU) in vitro and enhance the anti-tumor effects by heat. In this study, the combined effects of IFN and hyperthermia on the growth of L-CFU and human granulocyte-macrophage progenitors (CFU-GM) were examined to determine if this combination resulted in a greater selective killing of L-CFU than that obtained by heat treatment alone. The survival of normal CFU-GM without IFN decreased at elevated temperatures (42-44 degrees C). However, IFN added during heating (42 and 43 degrees C) appeared significantly to protect against the hyperthermic killing of CFU-GM in vitro leaving over 50% of CFU-GM surviving. The optimal dose to protect CFU-GM in vitro dropped to a rather low dose (100 U/ml). On the other hand, the addition of IFN to leukemic cell suspensions enhanced the hyperthermic killing of myeloid leukemic cell lines (HEL and KG-1) as well as a T lymphoblastic cell line (CEM) in a dose-related manner. In addition, similar results were observed in the study of L-CFU from patients with acute myelogenous leukemia. These results suggest that IFN can be used to broaden the difference between surviving fractions of CFU-GM and L-CFU by heat. Thus, this combination could be applied effectively and safely for the elimination of residual clonogenic leukemic cells in autologous remission marrow graft before autologous bone marrow transplantation.     

Circulating progenitor cells in myelofibrosis: the effect of recombinant α2b interferon in vivo and in vitro

We have studied circulating progenitor cells in patients with myelofibrosis, the effects of recombinant alpha 2b interferon (IFN) treatment on these cells in vivo and in vitro and the patients' clinical response to IFN treatment. A 75-fold increase in circulating granulocyte macrophage colony forming units (CFU-GM) and a 25-fold increase in multilineage colonies (CFU-GEMM) were seen in the patients compared with controls. Patients who had undergone splenectomy had circulating progenitors within the normal range. IFN treatment of two patients resulted in clinical improvement and reduction in spleen size, but was complicated by a fall in platelet count and persistent malaise. Whilst on treatment the circulating progenitor cells increased up to 5-fold. In contrast, the addition of IFN in vitro to the CFU-GM and CFU-GEMM culture plates resulted in a dose-dependent inhibition of colony growth which was unaffected by the removal of T-cells and monocytes. Thus we confirm that circulating progenitors are raised in patients with myelofibrosis, IFN may be of benefit clinically, reducing spleen size but may increase levels of these cells in vivo. This is in contrast to the inhibitory effect of IFN in vitro on CFU-GM and CFU-GEMM growth.     

Modulation of the expression of major histocompatibility antigens on splenic hairy cells--differential effect upon in vitro treatment with alpha-2b-interferon, gamma-interferon, and interleukin-2

The mechanism of action responsible for the beneficial effect of alpha-interferon (alpha-IFN) in patients with hairy cell leukemia (HCL) is still unknown. Direct antineoplastic and immunomodulating effects on both the host immune system and the hairy cells themselves have been implicated. To evaluate whether lymphokines have any regulatory effect on antigens of the major histocompatibility complex (MHC) on hairy cells and whether this corresponds to prognostic clinical parameters, we studied splenic hairy cells from ten previously untreated patients. The samples were incubated with recombinant human alpha-IFN, gamma-IFN (gamma-IFN), and interleukin-2 (IL-2). In an indirect staining procedure, cells were labeled with monoclonal antibodies (MoAb) to HLA ABC and HLA DR surface structures and subjected to cytofluorimetric analysis. Results concerning the expression of MHC class I and HLA DR antigens were mixed for incubation of hairy cells with gamma-IFN and IL-2, whereas alpha-IFN had a distinct effect on HLA DR antigen expression. alpha-IFN strongly enhanced the intensity of staining with HLA DR MoAb in six patients, and it increased the percentage of MoAb-positive cells in five of these samples. In contrast, the staining intensity in samples from four patients was reduced considerably on alpha-IFN treatment. In this group, two samples showed a sharp alpha-IFN-induced decrease in the number of HLA DR MoAb-positive cells from originally high values, and in one sample the very low percentage of positive cells was unaffected by alpha-IFN exposure. These two groups of patients whose hairy cells displayed contrasting HLA DR expression on incubation with alpha-IFN in vitro, were found to differ in their subsequent clinical course.     

Antigenic determinants on myeloid leukaemia colony-forming cells resemble those of normal myeloid progenitor cells and differ from those of circulating blast cells

We studied the antigenic characteristics of leukaemic colony-forming cells (CFU-L) from the blood of patients with chronic granulocytic leukaemia (CGL) in blastic transformation (BT) and acute myeloid leukaemia (AML) by in vitro culture techniques after complement-mediated lysis with one anti-DR and 10 selected myeloid monoclonal antibodies (McAbs), all of which were cytotoxic in the presence of complement. At the same time we studied the antigenic characteristics of the circulating blast cells from the same patients using in addition one non-complement fixing antibody (BI.3C5) with standard immunofluorescence and immunoalkaline phosphatase techniques. We also used myeloid progenitor cell assays in conjunction with cytotoxic McAbs to investigate the antigenic determinants on Day 7 CFU-GM, Day 14 CFU-GM and BFU-E from the blood of patients with CGL in chronic phase (CP) and from normal bone marrow. We found that two of the McAbs, S4-7 and WGHS29.1, recognized a higher proportion of CFU-L from the blood of AML patients than from patients with CGL-BT. However, the patterns of reactivity for CFU-L from CGL-BT and AML patients with the other McAbs quite closely resembled those observed in CFU-GM and BFU-E from normal individuals and patients with CGL in CP. A McAb with DR specificity and one of the myeloid McAbs, 54/39, recognized both CFU-L from CGL-BT and AML and reacted also with circulating blast cells from the same patients. In contrast, six of the other myeloid McAbs that recognized CFU-L failed to label the corresponding blast cells. We conclude that the antigenic properties of CFU-L in CGL-BT and AML are very similar to, but perhaps not identical with, those of normal CFU-GM and BFU-E. There was a major discrepancy in the antigenic profiles of CFU-L and of the blast cells predominating in the blood.     

Sequential treatment of Ph-positive chronic myeloid leukemia with interferon gamma and interferon alpha

Several clinical observations have shown that alpha-IFN is presently the most interesting investigational agent for the treatment of Ph+ chronic myeloid leukemia (CML). Gamma-IFN is also effective, and experimental data as well as preliminary clinical observations suggest that the combination of the two IFNs is worth investigating. No comparative data are available on the effects of the two IFNs, given alone, in the same patients. In this study 11 patients with PH+ CML were first treated with gamma-IFN, up to a maximum period of 35 weeks, and after a short rest period were retreated with alpha-IFN. Both IFNs were ineffective in 3 patients in accelerated or instable chronic phase. Both IFNs were equally effective in 8 patients in stable chronic phase, but none of these patients achieved a karyotypic conversion with either IFN. This study did not show any measurable differences in the therapeutic response to gamma-IFN and alpha-IFN given consecutively to the same patients.     

The exceptional responsiveness of certain human myeloid leukemia cells to colony-stimulating activity.

We have studied the marrow cells from a patient with acute myeloid leukemia (AML) for their responsiveness to colony-stimulating activity (CSA) in vitro. The AML cells were stimulated by CSA to rapid and extended growth in liquid culture. In the absence of CSA, the majority of cells died. CSA also stimulated the clonal growth of AML cells, and the minimum requirement for CSA was one-tenth to one-fiftieth that required to stimulate the growth of normal marrow CFU-C. CSA for AML cells was eluted from Sephacryl S-200 columns in fractions that represented an apparent molecular weight of 45,000 daltons. This fraction also produced optimal stimulation of normal human marrow. During remission, the patient's marrow cells did not grow in liquid culture and produced normal numbers of granulocytic and erythroid colonies in response to CSA and erythropoietin. Extended culture of the AML cells resulted in cell differentiation evidenced by decreasing proliferative capacity and by morphological and histochemical changes. These studies indicate that certain AML cells are extraordinarily responsive to CSA, an in vitro mediator of normal granulopoiesis.     

Divergent effects of interleukin-4 (IL-4) on the granulocyte colony-stimulating factor and IL-3-supported myeloid colony formation from normal and leukemic bone marrow cells.

Human recombinant interleukin-4 (IL-4) was studied for its effects on myeloid progenitor cells from normal and leukemic bone marrow cells in the presence and absence of additional growth factors. IL-4 itself did not support myeloid cluster or colony formation (CFU-GM). However, cultures supplied with IL-4 (300 U/mL) and IL-3 demonstrated a significant decline in myeloid colony numbers (CFU-GM) compared with the effects of IL-3 alone: (48 +/- 27 v 88 +/- 27 CFU-GM/10(5) MNC). In contrast, IL-4 augmented the G-CSF-supported CFU-GM: (80 +/- 31 v 148 +/- 52 CFU-GM/10(5) MNC). The effects of IL-4 were not mediated by accessory cells because similar results were obtained with and without T-cell, B-cell, or adherent depleted cell fractions. Morphologic analysis of clusters (day 7) and the colonies (day 14) demonstrated that IL-4 enhanced myeloid colony formation in the presence of G-CSF, whereas the cultures supplied with IL-3 and IL-4 did not show a lineage-restricted decline of CFU-GM. A heterogeneity in growth response was observed in the leukemic counterpart. With the 3H-thymidine proliferation assay, IL-4 augmented the G-CSF-induced proliferation of acute myeloid leukemic (AML) cells in 4 of the 12 cases, while the IL-3-supported proliferation was antagonized in 3 of the 12 cases. In the blast colony assay, IL-4 suppressed the IL-3-supported AML-CFU in the majority of cases, but enhanced the G-CSF stimulated AML-CFU in 3 of 6 cases. These data demonstrate divergent effects of IL-4 on the normal myeloid progenitor cell in the presence of IL-3 or G-CSF, while a variability in responsiveness is observed in the leukemic counterpart.     

Human recombinant multilineage colony stimulating factor (interleukin- 3): stimulator of acute myelocytic leukemia progenitor cells in vitro

Acute myeloid leukemia colony forming cells (AML-CFU) require the addition of colony stimulating factors (CSFs) for in vitro proliferation. Recently, we isolated a human recombinant multilineage CSF (hMulti-CSF). We investigated the ability of hMulti-CSF to stimulate AML clonogenic cells in seven patients in direct comparison with the effects of human granulocyte CSF (hG-CSF), human granulocyte- macrophage CSF (hGM-CSF), and feeder leukocytes. We show that hMulti- CSF is an efficient stimulator of AML colony formation in four of seven cases. In these patients, hGM-CSF was also capable of stimulating AML colonies in vitro. In two of seven cases hMulti-CSF appeared to be a weak stimulus of AML-CFU proliferation. In these latter two cases, however, hG-CSF and in one case hGM-CSF effectively stimulated AML-CFU growth. In one patient none of the hCSFs, either alone or in combination, induced AML colony formation, whereas AML colonies consistently appeared in the phytohemagglutinin (PHA) leukocyte feeder assay. This finding suggests that PHA stimulated leukocytes produce components other than the tested hCSFs that may have a role in the proliferation of AML cells in vitro. Multi-CSF, like hGM-CSF, revealed a limited capacity to induce progressive maturation during AML colony growth, ie, not beyond the promyelocytic stage. On the other hand, in one case, hG-CSF stimulated the growth of AML colonies containing (meta)myelocytes and granulocytes. We conclude that hMulti-CSF is a regulator of AML-CFU proliferation in a significant number of cases. The patterns of responsiveness of AML precursors to the three hCSFs in different patients show a striking variability, which may indicate that AML-CFU are the neoplastic representatives of normal bone marrow progenitors at different stages of maturation and with distinct CSF requirements.     

Acidic isoferritins (leukemia-associated inhibitory activity) fail to inhibit blast proliferation in acute myelogenous leukemia

Cell-free extracts of bone marrow and blood cells from patients with leukemia contain an inhibitor of normal granulocyte/macrophage progenitor (CFU-GM) proliferation (leukemia-associated inhibitory activity, LIA) identified as acidic isoferritins. A comparison was made of the action of crude LIA prepared from frozen-thawed leukemic blood cells and purified spleen ferritin from a patient with chronic myelogenous leukemia, on the proliferation of blast progenitors from patients with acute myelogenous leukemia (AML), and on the promyelocytic leukemia cell line, HL-60. Crude LIA showed no inhibition of blast progenitor or HL-60 proliferation at low concentrations, but inhibited the proliferation of CFU-GM. At higher concentrations, crude LIA inhibited both blast cells and CFU-GM. Purified spleen ferritin failed to inhibit blast progenitors or HL-60 cells at any concentration tested, but inhibited both 70-day and 14-day CFU-GM. Using the thymidine "suicide" technique, the action of LIA was confirmed as being on CFU-GM in S-phase, but it failed to affect the proliferation of blast cell in S-phase. It is concluded that acidic isoferritins inhibit normal CFU-GM but not blast cells from patients with AML. Acidic isoferritins could confer a proliferative advantage of the leukemic clone over its normal counterparts.     

Lymphokine-activated killer cells selectively kill tumor cells in bone marrow without compromising bone marrow stem cell function in vitro

Although it has been demonstrated that lymphokine-activated killer (LAK) cells kill tumor cells in a selective way without being toxic to a variety of normal cells, contradictory results exist about the possible toxicity of natural killer (NK) and LAK cells for hematopoietic progenitor cells. Therefore, the cytolytic activity and growth inhibitory effects of LAK cells on normal bone marrow progenitor cells and the ability of LAK cells to eliminate neoplastic hematopoietic cells from populations of bone marrow cells in vitro was studied. The results of these experiments show the following: (1) LAK cells have little cytolytic activity against normal bone marrow cells; (2) normal bone marrow cells fail to cold target compete for the killing of the hematopoietic tumor cell lines K562 and HL60 or freshly frozen acute myelocytic leukemia (AML) blast cells by LAK cells; (3) LAK cells inhibit the growth of K562 and HL60 to more than 90% in clonogenic assays; (4) LAK cells have no inhibitory effect on hematopoietic progenitor growth in CFU-GM (colony-forming unit- granulocytes, macrophages), CFU-E (colony-forming unit-erythrocytes), BFU-E (burst-forming units-erythrocytes), or CFU-GEMM (colony-forming unit-granulocytes, erythrocytes, macrophages, megakaryocytes) assays. These results indicate that LAK cells have low toxicity for normal bone marrow and that LAK activity against tumor cells is not adversely affected by the presence of normal bone marrow cells. The differences in cytolysis and growth inhibition of neoplastic hematopoietic cells and hematopoietic progenitor cells by LAK cells in vitro could create a therapeutic index that might allow the use of LAK cells for cleansing of the autologous bone marrow graft and for adjuvant therapy in combination with autologous bone marrow transplantation without compromising the reconstitution of the bone marrow in the host.     

Comparison of in vivo and in vitro effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with acute myeloid leukemia.

We studied the in vitro effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) in 13 patients with acute myeloid leukemia (AML) and one patient with refractory anemia with excess of blasts in transformation using the AML blast (AML colony-forming units, AML-CFU) and mixed (granulocyte erythrocyte macrophage megakaryocyte colony-forming units, CFU-GEMM) colony culture assays. In parallel, these patients received GM-CSF s.c. at 125 micrograms/m2/day, or in escalated doses starting with 10 micrograms/m2/day for a week or until circulating blast counts reached 50 x 10(9)/liter, in an effort to sensitize leukemic blasts to cell-cycle-specific agents. Results of in vivo GM-CSF treatment were correlated with those of in vitro assays. In 9 of 12 patients (75%), GM-CSF treatment increased peripheral blood blast counts (in vivo effect). GM-CSF also stimulated in vitro AML blast colony proliferation in these nine patients and increased the S+G2M phases of the cell cycle in five out of five of these patients' samples. Two of three patients in whom an in vivo response could not be demonstrated also failed to have a detectable in vitro response. These observations suggest that the AML blast colony culture assay may be useful in predicting the response of AML to cytokine therapy. Finally, GM-CSF stimulated granulocyte-macrophage (granulocyte-macrophage colony-forming units, CFU-GM) and erythroid (erythroid burst-forming units, BFU-E) colony proliferation in 14 and 11 patients, respectively, including the 3 individuals who demonstrated no clinical effect on blast counts. It is, therefore, possible that GM-CSF may be used to stimulate proliferation of progenitors that differentiate into mature granulocyte, monocyte-macrophage, and erythroid cells.     

Interferon system in primary acute lymphocytic leukemia cells with or without deletions of the alpha-/beta-interferon genes.

Various aspects of the interferon (IFN) system were studied in malignant cells from 37 unselected patients with acute lymphocytic leukemia (ALL). It was found that leukemic cells from two of 37 patients had a complete loss of alpha- and beta-IFN genes, whereas cells from four of 37 had lost one of the alpha-/beta-IFN alleles. In 25 cases, viable cells were also available for functional studies. Cell clones with loss of one of the alpha-/beta-IFN alleles produced low amounts of IFN after virus induction in vitro. Some clones with an apparently normal set of IFN genes were unable to produce detectable amounts of IFN. All clones studied were found to carry high-affinity alpha-IFN receptors. In clones carrying deletions of IFN genes, the cells were sensitive to IFN in vitro as measured by alpha-IFN-induced enhancement of 2',5'-oligoadenylate synthetase (2',5'-A synthetase). Cells from four patients with an apparently normal set of IFN genes were insensitive to this effect of IFN. We conclude that of the 17 patients in which IFN genes, IFN production, alpha-IFN receptors, and IFN-induced enhancement of 2',5'-A synthetase were studied, nine (53%) showed some abnormality in their IFN system. This finding may add some support to the hypothesis that defects in the IFN system could be a step on the path to malignant transformation in ALL. Moreover, patients whose malignant cells carry IFN gene deletions or other defects in their IFN-producing capacity, but are still sensitive to exogenous IFN, could represent a subgroup of ALL with a greater likelihood of responding to IFN therapy.