Effect of interleukin-12 on antitumor activity of human umbilical cord blood and bone marrow cytotoxic cells.

Interleukin (IL)-12, a natural killer (NK) cell stimulatory factor, is a heterodimeric cytokine that is known to be a potent activator of non-major histocompatibility complex-restricted cytotoxicity by peripheral blood-derived NK cells. NK cells (CD3-CD16+/CD56+) represent approximately 15% of human umbilical cord blood mononuclear cells (HUCB MNCs) and are known to be highly sensitive to activation by IL-2. In the present study, we monitored the effect of IL-12 on the cytotoxic activity, proliferation, and phenotypic expression of HUCB-derived resting and IL-2-activated cytotoxic cells and compared these parameters with those of bone marrow (BM)-derived cells. Lymphocytes were separated from HUCB by 3% gelatin sedimentation and incubated with IL-12 and/or IL-2 for 18 hours. At effector:target ratios of 40:1 and 20:1, IL-12 (50 U/mL) significantly increased both resting and IL-2-activated NK cell-mediated cytotoxicity in a standard 51Cr-release assay against both NK-sensitive (K562) and NK-resistant (Colo-205) cell lines. In addition, resting and IL-2-activated cytotoxic cells derived from HUCB exhibited superior cytolytic ability compared with BM-derived cells. This increase was observed in resting cells as well as in those that were preincubated with IL-12. Moreover, HUCB-derived cells were found to be more sensitive to IL-12 activation than cytotoxic cells from BM. To evaluate the involvement of accessory cells, NK cells were purified from HUCB using immunomagnetic beads, and these cells were found to have a lower response to treatment with IL-12 than unpurified populations. HUCB MNCs exhibited a nonsignificant increase in proliferation after IL-12 treatment and were better able to respond to IL-12 activation than BM MNCs. Following an 18-hour incubation, IL-12 was able to cause upregulation of CD25 and CD69 activation antigens, whereas no significant change in expression of CD16 and CD56 NK cell surface antigens, CD3 on T cells, or IL-12 receptor was observed. Similarly, IL-12 did not affect NK cell:target cell conjugation as assessed by fluorescence-activated cell sorting. Our results indicate that HUCB-derived NK-mediated cytotoxic capabilities can be increased by IL-12, a finding that may have clinical relevance.

Ex vivo expansion of CD56+ cytotoxic cells from human umbilical cord blood.

The immune-mediated effect of natural killer (NK) and cytotoxic T cells against residual tumor cells previously was shown to prevent relapse and reinduce remission after bone marrow transplantation. Human umbilical cord blood is a rich source of cytotoxic CD56+ cells including fetal NK cells (CD16(-)CD56+1) with high lytic capabilities upon activation with interleukin-2 (IL-2). Cord blood transplantations are reported to be associated with lower risk of graft-vs-host disease, which may jeopardize the graft-vs-leukemia effect. Therefore, our goal was to expand and amplify, ex vivo, cord blood-derived CD56+ cell-mediated cytotoxic activity. Cord blood-derived CD56+ cells were separated using anti-CD56 monoclonal antibody and immunomagnetic beads. The cells were expanded in the presence of irradiated feeder cells and various concentrations of IL-2. Maximal fold expansion (152 +/- 29) was achieved on day 22 by culturing the cells in the presence of irradiated autologous lymphocytes. Irradiated murine stromal cells yielded 42 +/- fourfold expansion (p < 0.05). FACS analysis at the peak of expansion revealed that the cells were 96% +/- 1% CD56+. Interferon-gamma levels significantly decreased throughout the culture period (from 1,034 +/- 34 pg/mL to 21 +/- 8 pg/mL) as did IL-6 levels (from 11,535 +/- 1,452 pg/mL to 323 +/- 161 pg/mL) whereas tumor necrosis factor-alpha levels did not change. The expanded cells manifested potent lytic capabilities against K562 and Colo-205 cell lines (70.9% +/- 2.0% and 48.2% +/- 4.0%, respectively) (n = 5) (effector-to-target ratio 25:1). Coculturing the expanded NK cells with fresh ALL blasts resulted in 85% +/- 1% inhibition of colony growth in methylcellulose (n = 2). In addition, the CD56+ expanded cells induced 44% +/- 7.5% apoptosis of K562 target cells (n = 3). It is possible to effectively expand cord blood-derived CD56+ cells, ex vivo, while maintaining their antileukemic capablilities.

Effect of bone marrow T lymphocytes treated with CAMPATH 1G on megakaryocyte colony formation.

Specific populations of lymphocytes play a significant role in the regulation of megakaryocyte (MK) development. CAMPATH 1G (C1G) and CAMPATH 1M (C1M) are T lymphocyte (TL)-specific monoclonal antibodies (MABs) that are routinely employed to reduce graft-vs.-host disease (GVHD) in allogeneic bone marrow transplantation (BMT). Following BMT, engraftment of the erythroid and myeloid lineages is prompt, but prolonged thrombocytopenia often prevails. We therefore studied the effect on MK colony formation of treating donor bone marrow (BM) with the CAMPATH MABs. MK colonies were grown in plasma clots using postirradiation aplastic canine serum (PICS-J) as MK colony-stimulating factor (MK-CSF). C1M, which causes TL destruction by complement-dependent lysis, had no effect on MK cloning efficiency. C1G is not lytic but causes the elimination of TL in the BMT recipients via antibody-dependent cell cytotoxicity (ADCC). Treatment of donor BM with C1G significantly enhanced the number of early burst-forming units (BFU-MK) and late colony-forming units (CFU-MK) and had no effect on granulocyte-macrophage (CFU-GM) or erythroid (BFU-E) colonies. Enhancement of MK cloning efficiency was concentration-dependent between 0.03 and 3 micrograms MAB/10(6) BM mononuclear cells (MNC). Similar results were observed when C1G-treated TL or purified CD4+ TL were co-cultured with untreated autologous BMMNC or peripheral blood (PB) MNC. Conditioned medium (CM) from C1G-treated TL and CD4+ TL contained soluble factors that, when combined with suboptimal doses of PICS-J, potentiated MK growth. C1G in combination with PICS-J also stimulated TL proliferation in a dose-dependent manner. The T cell CM did not contain elevated levels of interleukin-3 (IL-3), IL-6, IL-1 beta, or GM-CSF. Our data provide additional evidence for the involvement of activated TL, and perhaps novel soluble T cell products, in the immunomodulation of megakaryocytopoiesis.

The putative role of HLA-C recognition in graft versus host disease (GVHD) and graft rejection after unrelated bone marrow transplantation (BMT).

We assessed cytotoxic activity of large granular lymphocytes (LGLs) derived from 10 patients transplanted from molecular HLA-C mismatched (5) and matched (5) unrelated donors and compared it to the cytotoxic activity of 10 patients transplanted from HLA-identical siblings. In addition, we correlated clinical outcome with the level of molecular HLA-C disparity in a cohort of 22 patients who underwent unrelated BMT. Cells obtained from patients transplanted (related or unrelated) from fully matched donors did not generate allospecific lysis of patient (pre-BMT) or donor PHA blasts. Five of nine patients who received BMT from HLA-C mismatched unrelated donors developed > grade II graft-vs.-host disease (GVHD), and four developed graft rejection. Cells derived from three of three patients with GVHD lysed patients' pre-BMT PHA blasts. In the patients with GVHD grade III-IV, cytotoxicity was higher (60-70%) than in the patient with grade II GVHD (20%) (p < 0.05). Cytotoxic cells derived from one patient who rejected his graft lysed donor PHA blasts. In one remaining patient who had graft rejection followed by autologous rescue, no in vitro allospecificity was observed. In summary, cytotoxic cells from patients transplanted with marrow mismatched at locus C demonstrated in vitro cytolysis of PHA blasts, and this phenomenon showed positive correlation with the clinical outcome of the BMT. These findings may indicate specific allorecognition. A mismatch at locus C leading to alloreactivity should be considered a risk factor in determining an appropriate match for allogeneic BMT, especially when the donor is unrelated.

Selective CD4+ T-cell depletion does not prevent graft-versus-host disease.

Donor-derived CD4+ T cells may play a role in the development of graft-versus-host disease (GVHD) and graft-versus-leukemia reaction after allogeneic bone marrow transplantation (BMT). Therefore, we evaluated the effect of CD4+ T-cell depletion on GVHD and graft-versus-leukemia reaction after HLA-matched BMT. CD4 depletion was performed using anti-CD4 monoclonal antibodies and immunomagnetic beads, initially in small-scale experiments on bone marrow and granulocyte colony-stimulating factor-mobilized peripheral blood apheresis products. The result was elimination of the CD4+ T cells from both sources (0% and 2+/-1.4% CD4+ cells, respectively). Subsequently, we used this technique for large-scale negative selection of CD4+ T cells from bone marrow grafts of four consenting leukemic patients in relapse (ALL-3, ANLL-1) (M-3, F-1). The large-scale CD4+ T-cell depletion resulted in >98% (n=4) elimination of CD4+ cells. The resulting population included 17.7+/-4.6% CD3+ T cells, 8.9+/-2.5% CD8+ T cells, 0.1+/-0.1% CD16+ natural killer cells, and 2.3+/-3.2% CD34+ hematopoietic progenitor cells. Patients were transplanted with 2.84+/-1.31 x 10(8) viable cells/kg. They received cyclosporine starting on day -1 as GVHD prophylaxis. Engraftment was fast with a white blood cell count of >1 x 10(9)/L on day 13.2+/-0.5, an absolute neutrophil count of >0.5 x 10(9)/L on day 13.8+/-0.5, and a platelet count of >25 x 10(9)/L on day 26.5+/-6.8. Immunological reconstitution was normal, and peripheral blood phenotyping 3 weeks after BMT disclosed 49.0+/-5.0% CD3, 14.3+/-12.4% CD4, and 59.5+/-7.8% CD8 T cells in addition to 17.0+/-3.0% CD16+ and 9.0+/-3.0% CD56 natural killer cells. Three out of four patients developed very early grade IV GVHD beginning on day 12 (10-13) and died 2-4 months after BMT. One patient is alive and well with a follow-up of 36 months. We conclude that selective CD4 T-cell depletion does not prevent GVHD.

ASTA-Z 7557 impairs human natural killer (NK) cell activity.

Mafosfamide (ASTA-Z 7557) is a chemotherapeutic agent currently used for purging human bone marrow cells prior to autologous bone marrow transplantation. Adoptive cell-mediated immunotherapy has been shown to have a positive effect on the control of minimal residual disease and reinduction of remission post-bone marrow transplantation. Large granular lymphocytes and natural killer (NK) cells are believed to play a role in this effect. In the present work, we assessed the effect of ASTA-Z on the cytotoxic and proliferative capabilities of large granular lymphocytes (LGLs). The ASTA-Z significantly inhibited peripheral blood and bone marrow-derived LGL proliferation and cytotoxic capabilities, while this effect was less pronounced post-IL-2 treatment. We conclude that ASTA-Z purging may significantly impair the NK cell-mediated graft versus leukemia effect and therefore should be preceded by IL-2 therapy.

The novel immunomodulator, Linomide, stimulates interleukin-2-induced human natural killer (NK) cell and PHA-stimulated T cell proliferation from normal donors.

Donor-derived cell-mediated immunotherapy has been shown to be an effective tool for reinduction of remission in chronic myeloid leukaemia (CML) patients who have relapsed post-bone marrow transplantation (BMT). Linomide, quinoline-3-carboxamine (LS 2616), is a new immunomodulator shown to increase the number of NK precursors in mice in addition to upregulating the quantity of CD56(+), CD3(-) and CD16(+) NK cells in the peripheral blood of patients following autologous BMT (ABMT). We investigated the in vitro effects of Linomide on NK activity of normal human donors. Large granular lymphocytes (LGLs) and NK cells were incubated overnight with Linomide (0.02-4.8 mg/ml), recombinant human interleukin-2 (IL-2, 75 IU/ml), or a combination of both. Linomide, at 0.02-0.3 mg/ml, augmented IL-2-induced proliferation of LGLs and NK cells in an inversely proportional manner. In contrast, Linomide at 0.6-4.8 mg/ml inhibited IL-2-induced proliferation of LGLs and NK cells in a dose-dependent manner. Linomide was able to potentiate phytohemaglutinin-induced CD3(+) cell proliferation. In addition, supernatants derived from Linomide treated CD3(+) T cells were able to mimic the direct stimulatory effect of Linomide on activated NK cell proliferation. These supernatants were found to have low levels of tissue necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) and therefore Linomide stimulation of NK and T cell proliferation may be due to its inhibitory effect on the secretion of these cytokines by activated CD3(+) T cells. Linomide had no effect on cytotoxicity nor on the phenotypic expression of resting and IL-2-activated LGLs or NK cells. In view of our results, Linomide could possibly play a potential role in adoptive cell-mediated immunotherapy post-BMT.

Campath-1G impairs human natural killer (NK) cell-mediated cytotoxicity.

Campath-1 is a rat anti-human (CDw52) monoclonal antibody (MoAb) which is currently used for T cell depletion of allogeneic bone marrow and more recently peripheral blood stem cells prior to transplantation to prevent graft-versus-host disease (GVHD). In addition, in vivo Campath-1 is presently administered for the purpose of achieving increased immunosuppression during pre-transplant conditioning to aid in the prevention of graft rejection following T cell-depleted bone marrow transplantation (BMT). Graft-versus-leukemia (GVL) effect is an immunological effect that is of importance in controlling minimal residual disease (MRD) and reinducing remission post-BMT. It is thought that large granular lymphocytes (LGLs) and natural killer (NK) cells play a role in GVL. However, no data are available on the GVL effect post-Campath-mediated T cell-depleted allogeneic peripheral blood stem cell transplantation (alloPBSCT). In the present work, we assessed the effect of Campath-1G on the cytotoxic and proliferative capabilites of peripheral blood derived LGLs and NK cells. Campath-1G significantly inhibited binding of LGLs to tumor cells as well as resting and interleukin-2 (IL-2)-activated LGL and NK cell cytotoxic capabilites, which may be of clinical importance.

Enhancement of megakaryocytopoiesis by Campath-1G-treated natural killer cells.

Campath-1G is a CD52 (rat IgG2b) moAb used in bone marrow transplantation (BMT) to prevent graft-versus-host disease (GVHD) by the elimination of T cells via antibody-dependent cell cytotoxicity (ADCC) in vivo. We have previously reported that Campath-1G induces T cell proliferation, activation, and production of cytokines which in turn causes an enhancement of megakaryocytopoiesis in vitro. In view of the fact that recent studies have indicated that natural killer (NK) cells may also be involved in the regulation of megakaryocytopoiesis, we undertook the study of the in vitro effect of Campath-1G-treated NK cells on the regulation of megakaryocytopoiesis. Early burst-forming BFU-MK and late colony-forming CFU-MK were grown from 2 x 10(5) peripheral blood non-adherent mononuclear cells (NAMC) in plasma clots in the presence of aplastic canine plasma (PICS-J) which was used as megakaryocyte colony-stimulating factor (MK-CSF). The first step in elucidating this series of events was to investigate the direct influence of NK cells on megakaryocytopoiesis. Co-culturing NK cells (>85% CD16+) with autologous NAMC at a ratio of 1:1 resulted in a significant increase in the proliferation of CFU-MK and BFU-MK over NAMC cultured alone. This effect was further enhanced upon exposure of NK to Campath-1G (0.1-3 microg/ml). To investigate the possible influence of soluble factors released from NK cells treated with Campath-1G on MK maturation, conditioned medium (CM) derived from Campath-1G-treated-enriched populations of NK cells was found to enhance MK progenitor growth. Our data demonstrate that resting and Campath-1G-treated NK may be involved in the immunomodulation of megakaryocytopoiesis.

Natural killer (NK) cells prevent virus production in cell culture.

Natural killer (NK) cells (CD3-/CD16+/CD56+ lymphocytes) play an important role in early immune defense against viral infection, a fact which is of prime significance for heavily immunosuppressed patients after bone marrow transplantation. In this study we demonstrate that NK cells preferentially lyse human colon adenocarcinoma (Colo-205) tumor cells infected with herpes simplex virus type 1 (HSV-1) and vaccinia virus (VV) and autologous T cells infected with VV. This phenomenon was assessed by the viral infectious center (IC) method and compared with the results obtained by means of the standard 51Cr-release assay. Using the IC assay, we found that NK cells lyse virus infected cells at an early stage of infection, thereby preventing viral dissemination to neighboring cells. 51Cr-release assay verified by propidium iodide (PI) penetration showed that the early effects of NK mediated anti-viral activity are not the result of membrane damage. The effect of NK cells on HSV-1 infected Colo-205 cells appears to be independent of the level of expression of major histocompatibility complex (MHC) class I molecules while the killing of autologous VV-infected T cells correlates with a reduction in MHC class I expression. Our results suggest that additional factors besides MHC play a role in the regulation of NK cell-mediated lysis of virus infected cells. This may be of clinical importance in patients who are heavily immunosuppressed after bone marrow transplantation.

Hyperoxia damages phagocytic defenses of neonatal rabbit lung.

The effect of hyperoxia on phagocytic defenses of neonatal rabbit lung was ascertained by exposure to a fractional inspired O2 concentration of 0.95 + or 0.21 for 48, 96, or 168 h. Intrapulmonary clearance of inhaled staphylococci was reduced by 67 and 74% after 96 and 168 h in hyperoxia (P less than 0.05). Impaired phagocytic killing was not due to diminished bacterial ingestion. Alveolar macrophages (AM) lavaged from pups reared in normoxia had a progressive ability to release superoxide (O-2) and showed increasing cyanide-sensitive O2 consumption during the 1st wk of life. Conversely, AM recovered from litters housed in hyperoxia for 48 h produced 190% more O-2 than normoxic controls (P less than 0.005), but this capacity to generate O-2 fell by 43% after 96 h of exposure (P less than 0.05). After 96 h of hyperoxia, AM had a significant shift toward cyanide-insensitive metabolism compared with normoxic cells (P less than 0.05). Polymorphonuclear leukocytes (PMN) entered the alveoli after 96 h of hyperoxia, and mortality rose abruptly in animals exposed for 168 h (16%) vs. 96 h (3%). Our findings indicate neonatal hyperoxia induces metabolic and bactericidal dysfunction in the primary pulmonary phagocyte, the AM, and this injury is followed by additional lung insult during PMN migration into the airways.

Detection of minimal residual disease (MRD) after bone marrow transplantation (BMT) by multi-parameter flow cytometry (MPFC).

Multi-parameter flow cytometry (MPFC) was used to detect minimal residual disease (MRD) following bone marrow transplantation (BMT) in 21 patients. Bone marrow (BM)was analyzed pre-transplant and 3-4 months post-BMT while the patients were in clinical and morphological remission. MRD was detected by identifying cells with aberrant antigen expression and/or leukemia-associated phenotype (LAP) using MPFC. Prior to BMT, 8 out of 21 patients exhibited normal antigen expression based on normal BM samples while 13 BM aspirates had abnormal MPFC. Pre-BMT MPFC was abnormal in all 10 patients who were not in complete remission (CR) (>5% blasts in BM) as well as 3 patients acute lymphoblastic leukemia (ALL) who were in CR. In BM from ALL patients, an abnormal uniform B cell population was observed however antigen expression patterns varied greatly between patients. BM from acute myeloblastic leukemia (AML) patients showed an abnormal distribution of CD34+ cells. In addition, a correlation was observed between pre-BMT cytogenetics and MPFC. Only 2 out of 8 (25%) patients with normal MPFC pre-autologous bone marrow transplantation (ABMT) relapsed (AML), while 6 out of 13 (46%) patients with abnormal pre-BMT MPFC relapsed including 2 out of 3 patients who were transplanted in clinical CR. Pre-BMT MPFC may thus be an effective tool for detection of MRD by detection of a pre-transplant MPFC abnormality.

Sensitization of resting T cells to autologous natural-killer-cell-mediated lysis by phytohemagglutinin.

Natural killer (NK) cells are non-T, non-B cell lymphocytes that lyse a variety of tumor and virus-infected cells. In this study, we demonstrated that phytohemagglutinin (PHA) rendered resistant autologous T cells extremely sensitive to natural-killer(NK)-cell-mediated lysis. The sensitization was very rapid and concentration-dependent (0.01-1 microg/ml); 62% and 95% of autologous T cells were lysed by interleukin-2-activated NK cells 5 min and 18 h respectively after treatment with PHA (1 microg/ml). The maximal decrease in the level of MHC class I molecules observed on T cells was 22%. Induction of susceptibility to NK-mediated lysis was correlated with the expression of activation markers on T cells treated for relatively long intervals (more than 18 h) with high concentrations of PHA (more than 0.1 microg/ml). Sensitization of T cells required RNA and protein synthesis, although DNA synthesis was not essential. We propose that this unique system is suitable for studying the mechanisms involved in recognition and killing of target cells by NK cells.

Natural killer (NK) and T cell-associated surface marker expression following allogeneic and autologous bone marrow transplantation (BMT).

Natural killer (NK) and T cell development was studied after allogeneic and autologous BMT. We determined the phenotypic expression and lytic ability of these subpopulations after BMT. Following T cell-depleted (TCD) BMT, the number of CD16+ and CD56+ cells peaked at 39 and 46 days, respectively, and constituted the majority of peripheral blood lymphocytes (PBL). Coexpression of CD3 and CD16 was <10% up to 14.5 weeks after transplant. Following allogeneic non-T cell-depleted (NTCD) BMT, the number of CD16+ and CD56+ cells peaked at 6 weeks. CD3 expression was normal (70%-80%), % CD8+ cells was high (40%), and % CD4+ cells was low (20%). Following autologous BMT (ABMT), % CD3+ T cells was 80%, of which 70% expressed the CD8 marker. In contrast, CD4 expression was low (20%). CD16+ cells appeared 2.5-3 weeks after ABMT but with low frequency (20%), at which point 20%-30% of the CD3+ cells coexpressed CD16. A positive correlation was found between CD16 expression and cytotoxic capability. In conclusion, a marked difference was observed in NK and T cell-associated markers following TCD BMT, NTCD BMT, and ABMT. Following NTCD or ABMT, but not TCD BMT, a high percentage of cells co-express CD16 and CD3, which may indicate the possibility of a common NK and T cell progenitor.

Inhibition of glomerular mesangial cell proliferation and extracellular matrix deposition by halofuginone.

Mesangial cell proliferation, increased deposition of collagen, and expansion of the mesangial extracellular matrix (ECM) are key features in the development of mesangioproliferative diseases. Halofuginone, a low molecular weight anti-coccidial quinoazolinone derivative, inhibits collagen type alpha 1(I) gene expression and synthesis. We investigated the effect of halofuginone on both normal and SV40 transformed mesangial cell proliferation, collagen synthesis, and ECM deposition. Proliferation of both cell types was almost completely inhibited in the presence of 50 ng/ml halofuginone. The cells were arrested in the late G1 phase of the cell cycle and resumed their normal growth rate following removal of the compound from the culture medium. The antiproliferative effect of halofuginone was associated with inhibition of tyrosine phosphorylation of cellular proteins. Similar results were obtained whether the mesangial cells were seeded on regular tissue culture plastic or in close contact with a naturally produced ECM resembling their local environment in vivo. Halofuginone also inhibited synthesis and deposition of ECM by mesangial cells as indicated by a substantial reduction in 14C-glycine and Na2(35)SO4 incorporation into the ECM, and by the inhibition of collagen type I synthesis and gene expression. It is proposed that by inhibiting collagen type I synthesis and matrix deposition, halofuginone exerts a potent antiproliferative effect that may be applied to inhibit mesangial cell proliferation and matrix expansion in a variety of chronic progressive glomerular diseases.

Activated long-term peripheral blood cultures as preparation for adoptive alloreactive cell therapy in cancer patients.

Different modes of in vitro activation of peripheral blood mononuclear cells (PBMC) were compared for their effect on long-term propagation. PBMC cultures were activated by short exposures to the mitogen phytohemagglutinin (PHA) and the CD3 complex, with or without secondary signals provided by ligands of CD28 costimulatory molecules. Activation and long-term cultures were carried out in the presence of recombinant interleukin-2 (rIL-2). Addition of supernatant derived from IL-2-activated PBMC improved culture cell yield. Cumulative fold expansions ranged between 10(3) and 10(5) within 21 days. The highest cell yield was found after PHA activation. Fewer cells were obtained after activation with a combination of CD3 and CD28, and even fewer were obtained after CD3 activation alone. An increase in CD8+ and CD56+ cells, without change in CD4+ cells, was found in activated cultures when compared with fresh PBMC. Non-MHC-restricted cytotoxic activity was documented in all activated cultures. Cytotoxic activity per culture was highest in PHA-activated PBMC because of the high cell yield on the day of harvest. Successful in vitro expansion of PBMC might be helpful for gene transfer into T lymphocytes, as well as for the induction of an antitumor response, particularly for prevention and treatment of relapse of hematologic malignancies following allogeneic or autologous bone marrow transplantation.

Effect of various cytokine combinations on induction of non-MHC-restricted cytotoxicity.

Efforts were directed to achieve an increased lymphokine-activated non-MHC-restricted killing (LAK) activity greater than that induced by rIL-2 alone. Human peripheral blood (PB) and bone marrow (BM)-derived mononuclear cells (MC) were exposed in vitro to multiple cytokine combinations, including rIL-6, rIL-7, rIFN-alpha and rIFN-gamma in the presence of either suboptimal or optimal doses of rIL-2. Our results have shown that BMMC are a potential source for induction of increased LAK activity upon exposure to multiple cytokine combinations, whereas PBMC could not be successfully stimulated under the same conditions. Fifty-five to 62% of BM-derived samples stimulated with high dose rIL-2 + rIL-7 or rIL-2 + rIL-7 + rIL-6 + rIFN-gamma exhibited a higher degree of cytotoxicity than BM samples stimulated with rIL-2 alone. Exposure of PB-derived large granular lymphocytes (LGL) to various cytokine combinations led to increased proliferation after stimulation with suboptimal dose of rIL-2 in the presence of rIL-6 and rIL-7. This increase was not observed in induction of cytotoxicity. We suggest that BMMC activated by multiple cytokine combinations could play an active role in improving antitumor response in vivo by contributing to the control of minimal residual tumor cell growth, particularly post-BM transplantation.