High lytic activity against human leukemia cells after activation of allogeneic NK cells by IL-12 and IL-2.

IL-12 is a novel cytokine with interesting features regarding its potential usefulness in peripheral blood stem cell transplantation and leukemia immunotherapy. We used cryopreserved leukemia cells of 18 patients with acute myelogenous (n= 14) or lymphocytic (n= 4) leukemia to investigate the effect of IL-12, alone or in combination with IL-2, on the cytolytic activity of NK cells against human leukemia targets. Effector cells were peripheral blood mononuclear cells from healthy donors which were depleted from CD3+ T cells by immunomagnetic separation. CD3-negative effector cells (mainly CD56+ NK cells) were treated for 24 h with various concentrations of IL-2 (100 U/ml to 1000 U/ml) and IL-12 (1 U/ml to 100 U/ml). Cytotoxicity was measured in a 4 h 51Cr-release assay. Whereas a two-fold enhancement of cytotoxic activity was observed after incubation with optimal doses of IL-2 or IL-12, the combination of both cytokines (500 U/ml IL-2, 100 U/ml IL-12) increased the lytic activity more than six-fold. This effect was accompanied by increased expression of cellular adhesion molecules (CD2, CD18) and CD25 on CD56+ effector cells. Of 18 leukemias investigated, five were completely resistant to lysis by effector cells activated with IL-2 or IL-12 alone. In three of these five cases, however, high cytolytic activity was observed after coincubation with IL-2 and IL-12. In comparison to allogeneic NK cells, autologous cells of three patients in remission demonstrated significantly lower cytotoxic activity. No killing of nonmalignant cells (PHA blasts) by allogeneic NK cells was observed. Our data demonstrate that IL-12 can enhance or even induce MHC-unrestricted cytotoxicity of IL-2-activated allogeneic natural killer cells. Since IL-12 has also been shown to have stem-cell mobilizing capacities, it could be used for the recruitment of both stem cells and antileukemic effector cells in the context of peripheral blood stem cell transplantation.

Eradication of residual disease by administration of leukemia-specific T cells after experimental allogeneic bone marrow transplantation.

It is now well established that allogeneic lymphocytes can mediate a potent graft-vs.-leukemia (GVL) reaction when administered to bone marrow transplant (BMT) recipients. The benefit of allogeneic lymphocyte transfusion is limited because many patients develop graft-vs.-host disease (GVHD) with prolonged pancytopenia, which sometimes proves fatal. The object of the present study was to determine the antileukemic potential and GVHD risk of in vivo-generated tumor-specific allogeneic T cells given shortly after BMT. BALB/C (H-2d) mice were inoculated with different cell doses (10(5) and 5 x 10(5)) of the A20 leukemia (BALB/C origin) 2 days prior to lethal total-body irradiation (TBI) and transplantation of allogeneic, major histocompatibility complex (MHC)-matched DBA marrow grafts (H-2d, minor difference to BALB/C). Donors of BM grafts and T cells were allogeneic, MHC-matched mice (DBA, H-2d, minor difference to BALB/C). Donor-type T cells were generated from mice immunized with irradiated A20 leukemia cells or nonmalignant BALB/C splenocytes and restimulated in vitro. Whereas no significant immunotherapeutic effect was seen in mice with high tumor burden (5 x 10(5)), allogeneic BMT in mice inoculated with 1 x 10(5) A20 cells resulted in a modest antileukemic effect. This survival rate remained unchanged when 10(6) T cells obtained from donors immunized with nonmalignant BALB/C derived cells were given posttransplantation. In contrast, a single injection of 10(6) T cells from leukemia-immunized donors led to potent GVL effects without mediating clinically overt GVHD. Our data provide evidence for the hypothesis that minimal residual disease can be eradicated without inducing GVHD by administering small amounts of specific allogeneic cytotoxic T cells after BMT.

Abrogation of graft-vs.-leukemia activity after depletion of CD3+ T cells in a murine model of MHC-matched peripheral blood progenitor cell transplantation (PBPCT).

Using a murine transplantation model, we simulated a clinical situation in which major histocompatibility complex (MHC)-identical allogeneic peripheral blood progenitor cells (PBPCs) are transplanted for the treatment of a malignant disease that is resistant to resting natural killer (NK) cells but sensitive to cytokine-activated NK cells and T cell-mediated antitumor activity. We determined the influence of selective T cell depletion of allogeneic PBPC grafts on graft-vs.-leukemia (GVL) activity and investigated the effectiveness of ex vivo treatment with NK cell-activating cytokines to compensate for the putative loss of T cell-derived factors stimulating natural cytotoxicity. After pretreatment of Balb/c (H-2d) recipients with 7.5 Gy of total body irradiation, 2x10(7) rhG-CSF-mobilized PBPCs of splenectomized syngeneic or MHC-identical DBA (H-2d) mice were transferred. Selective T cell depletion (TCD) was performed by immunomagnetic purging with a mononoclonal antibody directed against CD3. In some experimental groups, T cell-depleted PBPCs were incubated with 200 U/mL interleukin (IL)-2 and 100 U/mL IL-12 for 24 hours. To investigate antileukemic activity in vivo, recipient mice were inoculated with 1x10(5) A20 cells (a B-lymphoblastic leukemia of Balb/c origin) 2 days before PBPC transplantation (PBPCT). After transplantation of unmanipulated allogeneic cells, 25% of the animals died with signs of graft-vs.-host disease (GVHD) but 71% were free from relapse 100 days after PBPCT. After TCD of allogeneic grafts with anti-CD3, the incidence of GVH-related mortality was below 5% but the leukemia-free survival rate was significantly (p < 0.05) decreased to 25% and thus was similar to that observed after syngeneic PBPCT (17%). When CD3-depleted grafts were incubated with IL-2 and IL-12, 45% of the animals remained free from leukemia; however, the difference was not statistically significant. Our results suggest that ex vivo activation of residual NK cells with IL-2 and IL-12 does not fully compensate for the abrogation of GVL activity after depletion of CD3+ T cells from MHC-matched PBPCT.

Phase III trial of bortezomib, cyclophosphamide and dexamethasone (VCD) versus bortezomib, doxorubicin and dexamethasone (PAd) in newly diagnosed myeloma.

We aimed at demonstrating non-inferiority of bortezomib/cyclophosphamide/dexamethasone (VCD) compared to bortezomib/doxorubicin/dexamethasone (PAd) induction therapy with respect to very good partial response rates or better (⩾VGPR) in 504 newly diagnosed, transplant-eligible multiple myeloma patients. VCD was found to be non-inferior to PAd with respect to ⩾VGPR rates (37.0 versus 34.3%, P=0.001). The rates of progressive disease (PD) were 0.4% (VCD) versus 4.8% (PAd; P=0.003). In the PAd arm, 11 of 12 patients with PD had either renal impairment (creatinine ⩾2 mg/dl) at diagnosis or the cytogenetic abnormality gain 1q21, whereas no PD was observed in these subgroups in the VCD arm. Leukocytopenia/neutropenia (⩾3°) occurred more frequently in the VCD arm (35.2% versus 11.3%, P<0.001). Neuropathy rates (⩾2°) were higher in the PAd group (14.9 versus 8.4%, P=0.03). Serious adverse events, both overall and those related to thromboembolic events, were higher in the PAd group (32.7 versus 24.0%, P=0.04 and 2.8 versus 0.4%, P=0.04). Stem cell collection was not impeded by VCD. VCD is as effective as PAd in terms of achieving ⩾VGPR rates with fewer PD and has a favorable toxicity profile. Therefore, VCD is preferable to PAd as induction therapy.

Idiotype protein-pulsed dendritic cells produce strong anti-myeloma effects after syngeneic stem cell transplantation in mice.

Dendritic cell (DC) vaccination represents an interesting immunotherapeutic option in the treatment of several malignancies. In multiple myeloma (MM) patients, vaccination with autologous idiotype (Id) protein-pulsed DC is feasible, but their antitumoral effectiveness was rather limited. To improve the therapeutic potential of DC therapy, we studied the immunological effects of syngeneic peripheral blood stem cell transplantation (PBSCT) given in conjunction with Id-loaded DC. Balb/c mice were inoculated i.p. with 5 x 10(5) of HOPC myeloma cells (Balb/c origin). Animals were immunized with three injections of 5 x 10(5) DC pulsed with the IgG2a(HOPC) or with a control immunoglobulin (Ig(control)). Some experimental groups of myeloma-bearing animals received total body irradiation (7.5 Gy) and a subsequent transplant of 2 x 10(7)syngeneic peripheral blood progenitor cells (PBPC) followed by DC therapy beginning at day 10 post transplant. Animals receiving DC therapy or syngeneic PBPCT alone neither induce long-term survival nor tumor-specific CTL reactivity in vitro. In marked contrast, combination of syngeneic PBPC transplantation and subsequent DC therapy resulted in 78% survival after a follow-up of 180 days. In addition, this treatment modality conferred a generation of Id peptide-specific CD8-mediated T cell reactivity. These data provide a rationale for DC-based vaccination in multiple myeloma patients administered post syngeneic transplantation.

Immunotherapeutic aspects of allogeneic peripheral progenitor cells.

In a newly developed murine model of allogeneic peripheral progenitor transplantation (PBPCT) we investigated the immunotherapeutic potential of allogeneic peripheral stem cells. The following topics were addressed by our experiments: (1) comparison of the graft-versus-leukemia effect exerted by allogeneic PBPCT compared to allogeneic BMT; (2) the influence of T-lymphocytes on GVL activity; (3) the possibility to enhance the GVL activity of allogeneic PBPCT grafts by ex vivo cytokine incubation. Balb/c mice received cells of the syngeneic B-lymphatic leukemia A20 2 days prior to TBI (7.5 Gy) and the respective graft. The recipients received allogeneic bone marrow grafts or allogeneic peripheral progenitor cells obtained after mobilization of the donors (DBA/2) with either G-CSF in a dose of 250 microg/kg/day for 5 days. In some experiments T lymphocytes were removed by immunomagnetic depletion with CD3-coated beads. An additional group received T cell-depleted and IL-2/IL12-activated PBPCT grafts. The antileukemic activity of an allogeneic PBPCT graft was significantly greater than the antileukemic activity of an allogeneic BMT graft of the same size. Relapse rates were 80% in syngeneic PBPCT, 60% after allogeneic BMT and 34% after allogeneic PBPCT. This rise in antileukemic activity is not accompanied by a rise in GVHD mortality. Depletion of T lymphocytes by CD3-coated beads resulted in a nearly complete loss of the GVL activity with a relapse rate of 75%. Incubation of the T-depleted graft with IL-2 and IL-12 to enhance NK-based GVL activity has only limited success after MHC-matched transplantation with a relapse rate of 55%. Allogeneic PBPC exert a pronounced antileukemic effect. After MHC-matched PBPCT, this GVL effect resides mostly on the T cells of the graft. Ex vivo activation of T cell-depleted grafts by IL-2 and IL-12 is accompanied by an only limited reduction of relapse rate. PBPC are a valuable modality for primary transplantation in situations with high risk of relapse and for the treatment of relapse after BMT.

Superior antileukemic activity of murine peripheral blood progenitor cell (PBPC) grafts mobilized by G-CSF and stem cell factor (SCF) as compared to G-CSF alone.

We have established a murine model to compare the antileukemic effect of PBPC grafts obtained after treatment with SCF + G-CSF and G-CSF alone. C57/BL6, DBA and Balb/c mice were splenectomized and injected with optimal doses of rhG-CSF (250 microg/kg/day s.c.) or rrSCF (100 microg/kg/day s.c.) or with a combination thereof. On day 5, we determined the hematopoietic potential (number of CD34+ cells, CFUs, total CFC, CFU-gm), the proportion of lymphoid (T, NK and B cells) and myeloid components and graft-versus-leukemia activity after allogeneic and syngeneic PBPCT and BMT in Balb/c mice bearing a B-lymphoblastic leukemia cell line (A20). The absolute number of progenitor cells increased two-fold after administering a combination of G-CSF and SCF as compared to G-CSF alone (1500 vs 940 CD34+ cells/microl; 190 vs 70 total CFC/microl; 150 vs 50 CFU-gm/microl and 6600 vs 3000 CFUs/ml). Although no differences could be detected in the cellular composition, especially in the number of T cells, PBPC grafts mobilized by the combination of G-CSF + SCF demonstrated significantly higher antileukemic activity compared to G-CSF alone (94% vs 71% freedom from leukemia, P < 0.05). Because the incidence of lethal GVHD was similar in both groups, improved GVL activity resulted in superior overall survival. Our data suggest that the higher number of progenitor cells can be harvested after G-CSF + SCF and that grafts mobilized by G-CSF + SCF exert significantly enhanced antileukemic activity compared to those harvested after treatment with G-CSF alone.

Graft-vs-leukemia activity and graft-vs-host disease induced by allogeneic Th1- and Th2-type CD4+ T cells in mice.

INTRODUCTION: The transfer of allogeneic lymphocytes contained in a hematopoietic stem cell graft confers an immune-mediated antileukemic effect, termed the graft-vs-leukemia (GVL) effect. Graft-vs-host disease (GVHD), the most detrimental complication of allogeneic BMT, largely resides within the same lymphocyte population. Therefore, separation of GVL- and GVH-reactions is a long-standing goal of experimental studies dealing with allogeneic transplantation of hematopoietic stem cells. MATERIALS AND METHODS: The objective of the current study was to assess the potential of Th1- and Th2-type CD4+ T cells in mediating GVHD and GVL effects in a fully allogeneic murine transplant model. BALB/c (H-2d) mice were given a dose of A20 (H-2d, B-cell leukemia) cells two days prior to lethal total body irradiation (TBI) and transplantation of fully mismatched (C57BL/6, H-2b) T-cell depleted (anti-Thy1.2, CD90) bone marrow (TCD-BM) cells. Graded numbers of either unmanipulated, Th1- or Th2-polarized highly enriched CD4+ donor type T cells (10(6) or 10(7)) were administered 2 h posttransplant. Infusion of 10(6) of unmanipulated, Th1-, or Th2-primed CD4+ T cells resulted in moderate GVHD-related mortality (40%, 50%, 10%) and significantly improved long-term survival (50%, 45%, 46% surviving the observation period of 120 days) as compared to animals receiving TCD-BM alone (18%). RESULTS: The administration of 10(7) unmanipulated or Th1-type CD4+ T cells given shortly after transplantation led to death of all mice within 50 days due to fatal acute GVHD. In contrast, the adoptive transfer of 10(7) Th2-primed CD4+ T cells resulted in significant improvement of long-term survival (80%) compared to the TCD-BM group. This powerful GVL effect was associated with a substantially lower incidence of lethal acute GVHD (10%) if compared to the results of transplantation of Th1-type CD4+ T cells. CONCLUSION: These results demonstrate that allogeneic Th2-type CD4+ T cells given post BMT can induce GVL effects in a cell-dose-dependent manner without increasing the risk of severe acute GVHD.

Transfer of idiotypic protein primed allogeneic marrow grafts elicits potent graft-versus-myeloma effects in mice.

The active immunization of bone marrow (BM) donors with myeloma immunoglobulin (Ig) results in an idiotypic T cell response that can be transferred to the recipient. Using a murine model we evaluated the effectiveness, side-effects and underlying mechanisms of this approach. Balb/c (H-2d) mice were given a dose of HOPC-1F myeloma cells secreting the monoclonal IgG2a followed by lethal total body irradiation (7.5 Gy) 2 days later and a subsequent transplantation of 2 x 10(7) allogeneic MHC-matched DBA/2-derived marrow cells. Donors were pre-immunized with three i.p. injections of HOPC(IgG2a) or control Ig given with incomplete Freund's adjuvants (IFA) spaced 1 week apart. In some experiments, donor-spleen cells were additionally transferred 2 h post transplant. Injection of HOPC-myeloma led to death of all animals after a median survival time (MST) of 42 days. A lethal dose of TBI followed by transfer of unmanipulated marrow grafts plus splenocytes resulted in moderate antimyeloma effects with 8% of mice achieving long-term survival. Nearly the same results were obtained after transplantation of BM immunized with the control Ig. In contrast, transplantation of marrow grafts from HOPC(IgG2a) immunized donors exerted a significant GVM effect with 63% long-term survival for more than 180 days. The additional transfer of 2 x 10(7) immune splenocytes derived from the same donor resulted in even stronger anti-myeloma effects (FFR 87%). No increase in the incidence of severe acute GVHD was observed. In vitro data suggest that allogeneic CD8+ idiotype-specific T cells may be the major effector cells. Our results demonstrate that active immunization of the donor with the myeloma-specific Ig can induce powerful graft-versus-myeloma effects after allogeneic BMT.

Enhanced antileukemic activity of allogeneic peripheral blood progenitor cell transplants following donor treatment with the combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) in a murine transplantation model.

Allogeneic peripheral blood progenitor cells (PBPCs) have mostly been mobilized by granulocyte colony-stimulating factor (G-CSF). There is neither clinical nor experimental data available addressing the question if other hematopoietic growth factors or combinations thereof might influence engraftment, graft-versus-host disease (GvHD), and graft-versus-leukemia (GvL) effects after allogeneic peripheral blood progenitor cell transplantation (PBPCT). We used a murine model to investigate these parameters after transplantation of PBPCs mobilized with G-CSF and SCF either alone or in combination. Treatment of splenectomized DBA and Balb/c mice with 250 microg/kg/day G-CSF for 5 days resulted in an increase of CFU-gm from 0 to 53/microl. The highest progenitor cell numbers (147/microl) were observed after treatment with 100 microg/kg/day SCF administered in conjunction with G-SCF. No differences were detected with regard to the number of T cells (CD3+), T cell subsets (CD4+, CD8+), B cells (CD19+) and NK cells (NK1.1+) in PBPC grafts mobilized by G-CSF plus SCF compared to those mobilized with G-CSF alone. The antileukemic activity of syngeneic and MHC-identical allogeneic PBPC grafts was investigated in lethally irradiated Balb/c mice bearing the B-lymphatic leukemia cell line A20. In this model, PBPCs mobilized by G-CSF plus SCF exerted a significantly higher antileukemic activity compared to grafts mobilized by G-CSF alone (94 vs 71% freedom from leukemia at day 100, P<0.05). The antileukemic effect was lowest after BMT (38% freedom from leukemia). Since significant differences in the incidence of lethal GvHD were not observed, improved GVL-activity resulted in superior overall survival. Our data demonstrate that the utilization of specific hematopoietic growth factors not only improve the yield of hematopoietic progenitor cells but can also significantly enhance the immunotherapeutic potential of allografts.

Induction of graft-versus-leukemia (GVL) activity in murine leukemia models after IL-2 pretreatment of syngeneic and allogeneic bone marrow grafts.

To investigate GVL effects, Balb/c mice (H-2d) received 5 x 10(5) A20 (B cell leukemia) or 1 x 10(6) WEHI-3 (myelomonocytic leukemia) cells. These cell lines lead to death after a median of 19 (WEHI-3) or 30 days (A20). A lethal dose of total body irradiation followed by syngeneic BMT resulted in significantly prolonged survival of leukemia-bearing animals. Transplantation of (C57 x Balb/c)F1 (H-2bxd) allogeneic, but GVH-non-reactive marrow grafts differentially influenced the relapse rates in the two leukemia models. Whereas allogeneic BMT reduced the relapse rates in A20-bearing mice, the leukemia-free survival was not improved in mice bearing the leukemia WEHI-3 compared with syngeneic BMT. Pre-treatment of allogeneic (C57 x Balb/c)F1 or syngeneic Balb/c marrow cells with 200 U/ml IL-2 for 24 h did not reduce relapse rates in animals inoculated with A20 leukemia cells compared with unmanipulated bone marrow. In contrast, IL-2 treatment of syngeneic or allogeneic GVH non-reactive donor marrow significantly decreased the relapse rate in mice inoculated with WEHI-3 leukemia cells. The NK cell-mediated lysis of cultured leukemia cells was determined in vitro using a conventional 56Cr-release assay. Our data revealed a strong correlation between the level of natural killer activity determined in vitro and GVL activity in vivo.

Allogenic transplantation of mobilized peripheral blood progenitor cells: towards tailored cell therapy.

Mobilized peripheral blood prognitor cells (PBPC) are increasingly being used instead of bone marrow for allogeneic transplantation. The present article will briefly review important aspects of allogeneic PBPCT including, donor safety, timing of leukapheresis, factors influencing the yield, cellular composition of PBPC allografts, hematopoietic capacity of allogeneic PBPC, and graft-versus-host and graft-versus-leukemia activities. It will particularly focus on the perspectives opened by PBPC for graft engineering and cell therapy.

Needle-localized breast biopsy for mammographic abnormalities: a community hospital experience.

Increased awareness of benefits of early detection of breast cancer has resulted in increased numbers of screening mammographies and breast biopsies for nonpalpable lesions. Tertiary hospital studies have demonstrated positive biopsy rates from abnormal mammographic findings at 18 to 32 per cent. We examined the effectiveness of needle biopsy for nonpalpable radiographic abnormalities in our community hospital. We reviewed 167 records of patients biopsied over a 2-year period. Mammographic assessment, biopsy, and pathological assessment were performed using accepted methods. Malignancy was detected in 34 of 167 biopsies (20%). The biopsy yield rate was highest for mammographic findings of spiculated or stellate masses (75%, P < 0.01). Most biopsies (83%) were performed because of mammographic findings of microcalcifications or circumscribed enlarging masses/nodular developing densities for a positive biopsy yield rate of 16 per cent. Rates were higher in patients with personal (44%) or family history (30%) of breast cancer and in postmenopausal women (30%). These results demonstrate that 1) factors such as age, personal or family history of breast cancer, and certain mammographic features of breast lesions are associated with high biopsy yield rates, and 2) the biopsy yield rate in our community setting is comparable to tertiary hospital experience.

Activation of natural killer cells with interleukin 2 (IL-2) and IL-12 increases perforin binding and subsequent lysis of tumour cells.

Natural killer (NK) cells can lyse a variety of different tumour cells by exocytosis of perforin, subsequent binding of perforin to the target cell membrane and formation of lytic pores. Some tumour cells, however, are resistant to cellular cytotoxicity. Using the NK-resistant tumour cell lines ML-2, MONOMAC-1, RPMI and L540Cy, we demonstrated that activation of NK cells with interleukin 2 (IL-2) and IL-12 resulted in significant lysis of these tumour targets. To investigate the underlying mechanisms, we isolated the cytotoxic granules from non-activated and IL-2-/IL-12-activated NK cells and compared the killing of K562 leukaemia cells (sensitive to NK cell-mediated lysis) and ML-2 leukaemia cells (resistant to NK cell-mediated lysis). In contrast to K562 cells, which were easily killed by NK-cell granules, ML-2 cells were resistant to granules from non-activated NK cells. However, granules from NK cells activated with IL-2 and IL-12 were able to induce significant tumour cell lysis. Cell death of both K562 and ML-2 cells by granules from activated NK cells was completely blocked by anti-perforin antibodies, indicating that perforin mainly accounts for the lysis induced by NK granules. Comparing granules from non-activated and IL-2-/IL-12-activated NK cells, the increased cell death of ML-2 cells was caused by an improved binding of perforin to the target cell membrane. Functional assays, however, indicated that the differences in perforin binding were not as a result of an augmented production of perforin by activated NK cells. We conclude that activation of NK cells results in an increased binding of perforin and subsequent lysis of tumour cells.

Impaired binding of perforin on the surface of tumor cells is a cause of target cell resistance against cytotoxic effector cells.

Exocytosis of perforin, subsequent binding of perforin to the target cell membrane, and formation of lytic pores form an important pathway involved in the induction of tumor cell death by cytotoxic effector cells. Here we describe a novel escape mechanism employed by tumor cells to protect themselves from granule-mediated cell death: We were able to demonstrate that the resistance of the human leukemia cell line ML-2 to natural killer (NK)-cell-mediated killing is not caused by impaired NK-cell activation but by resistance against effector molecules contained in the granules of cytotoxic cells. No resistance was observed against other pore-forming agents like complement and streptolysin O. By using the NK-susceptible leukemia cell line K562, we could show that the induction of cell death by cytotoxic granules can be blocked completely by anti-perforin antibodies, indicating that perforin is essentially involved in this process. Flow cytometric data revealed that an impaired binding of perforin on the tumor cell membrane is mainly responsible for target cell resistance, because perforin turned out to bind well on K562 cells but is not able to attach to the surface of ML-2 cells. After impaired binding of perforin was identified as a potential mechanism of tumor cell resistance, leukemia cells from 6 patients with acute myeloid leukemia (AML) were examined. As predicted, AML cells that failed to bind perforin on their surface demonstrated complete resistance toward NK-cell-mediated cytotoxicity. Thus, perforin resistance could represent an important tumor escape mechanism that should be considered when cytotoxic effector cells are used for cellular immunotherapy. (Blood. 2000;96:594-600)

Allogeneic MHC-mismatched activated natural killer cells administered after bone marrow transplantation provide a strong graft-versus-leukaemia effect in mice.

Allogeneic lymphocytes administered with an unmanipulated bone marrow transplant provide a strong antileukaemic effect, the so-called graft-versus-leukaemia (GVL) effect. On the other hand, T-cell-mediated graft-versus-host-disease (GVHD) observed after transplantation of unmanipulated BM graft causes substantial morbidity and mortality. The aim of the present study was to determine the antileukaemic potential of enriched IL-2 activated NK cells administered 2 h after BMT. Balb/c (H-2d) mice were given a dose of A20 (H-2d, B-cell leukaemia) cells 2 d prior to lethal total body irradiation (TBI) and transplantation of either syngeneic or allogeneic anti-Thy1.2 (CD90) depleted bone marrow cells. Either syngeneic (Balb/c, H-2d) or allogeneic (C57BL/6, H-2b) enriched and IL-2 (200 U/ml for 24 h) activated NK cells were given 2 h after BMT. Injection of A20 leukaemia into normal Balb/c recipients led to death after a median of 14 d. A lethal dose of TBI followed by either syngeneic or allogeneic Thy1.2-depleted BMT resulted in a modest antileukaemic effect. The adoptive transfer of syngeneic enriched and IL-2 preincubated NK cells given at time of BMT exerted a significantly better GVL effect. However, the infusion of allogeneic enriched NK cells resulted in a stronger GVL effect. These results clearly demonstrate that allogeneic NK cells are superior to syngeneic NK cells in their potential to eradicate residual leukaemia cells after BMT without mediating clinical overt GVHD. This experimental setting may offer a strategy for treatment of haematological malignancies in a phase of minimal residual disease.

Investigating the lysis of small-cell lung cancer cell lines by activated natural killer (NK) cells with a fluorometric assay for NK-cell-mediated cytotoxicity.

Activation of natural killer (NK) cells with interleukin-2 (IL-2) and IL-12 leads to an enhanced lysis of tumour cells. We investigated the ability of NK cells, with or without prior activation, to lyse a variety of small-cell lung cancer (SCLC) target cells. Specific lysis was measured with a fluorometric assay for NK-cell-mediated cytotoxicity: target cells were labelled with 3,3'-dioctadecyloxacarbocyanine, a green membrane dye. After co-incubation with NK cells, dead target cells were stained with propidium iodide, a red DNA dye that only penetrates dead cells. Of all eight SCLC cell lines tested, three were susceptible to lysis by non-activated NK cells, three were only susceptible to lysis by NK cells activated with IL-2 and IL-12 and two were not even susceptible to lysis by activated NK cells. The differences in target cell susceptibility showed no correlation with the expression of MHC-I on the surface of the target cells or with the expression of the adhesion molecules CD50, CD54, CD58 or CD102. Comparing the kinetics of the lysis of one SCLC cell line sensitive to non-activated NK cells and one sensitive only to activated NK cells, we found that maximum lysis of the former was obtained after 1 h, whereas significant lysis of the latter was only obtained after 4 h of incubation. This might be due to different mechanisms engaged in target cell lysis.