L. Uharek

Graft-versus-leukaemia activity can be predicted by natural cytotoxicity against leukaemia cells

We have investigated graft‐versus‐leukaemia (GVL) effects after allogeneic bone marrow transplantation (BMT), using three murine leukaemia models, A20 (B lymphocytic), WEHI‐3 (myelomonocytic) and PU5‐1R (myeloid). Injection of leukaemia cells in a high number (106 cells) into syngeneic Balb/c mice (H‐2d) invariably led to death with a median survival time of 22 d (A20), 18 d (WEHI‐3) and 45 d (PU5‐1R). A lower tumour load of A20 (5 × 105 cells) was used in some experiments resulting in a leukaemic death rate of 94%. Lethal total‐body irradiation followed by syngeneic BMT prolonged survival (P < 0.05) for animals bearing the leukaemia A20 and WEHI‐3 but was unsuccessful for animals injected with cells from the monocytic leukaemia PU5‐1R. Graft‐versus‐host (GVH)‐nonreactive marrow of (C57 × Balb/c)F1 mice (H2bxd) exerted a significant GVL‐effect with reduced relapse rate and improved survival in mice receiving the leukaemia cell line A20. In animals with low tumour load a significant reduction of the relapse rate from 82% following syngeneic BMT to 47% following allogeneic, GVH‐nonreactive BMT could be achieved. Depletion of natural killer (NK) cells from the GVL‐reactive semi‐allogenic bone marrow graft enhances the relapse rate of the leukaemia A20 to 65%. In mice bearing the leukaemias WEHI‐3 or PU5‐1R allogeneic GVH‐nonreactive BMT did not improve survival compared to syngeneic BMT. Transplantation of GVH‐reactive bone marrow from DBA mice (MHC identical to Balb/c, minor difference) caused only a limited and insignificant reduction of relapse rate for animals with the leukaemia A20. These in vivo data are in close correlation with in vitro natural killer cell (NK) activity of the donor strains against the respective leukaemia targets. Depletion of NK cells from the GVL‐reactive (C57 × Balb/c)F1 bone marrow resulted in a significant loss of GVL activity. We conclude that NK cells are involved in graft‐versus‐leukaemia effects independent of graft‐versus‐host disease (GVHD).

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.

Gaft-versus-leukemia activity after bone marrow transplantation does not require graft-versus-host disease

SummaryClinical data have suggested that graft-versus-host disease (GVHD) plays a crucial role in the antileukemic effects of bone marrow grafts. We investigated (a) whether bone marrow cells unable to induce GVHD can effect graft-versus-leukemia (GVL) activity and (b) whether such antileukemic capacity depends on the presence of T lymphocytes in the graft. Balb/c mice were inoculated with A 20 cells, a B-cell lymphoma/leukemia of Balb/c origin. Four weeks after tumor inoculation the animals were lethally irradiated and received a bone marrow graft. Cells from (Balb/c × C57) F1 or (C3H × Balb/c) F1 hybrids were transplanted into parental-strain Balb/c mice. Since lymphocytes from F1 hybrids are unable to cause graft-versus-host reactivity against a parental-strain animal, we used this experimental setting to explore GVL effects in a GVHD-free system. In vitro incubation with monoclonal anti-Thy-1.2 antibody plus complement was used to eliminate Thy-1+ cells. After syngeneic transplantation, the death rate due to leukemia remained unchanged (91%) compared with that among untreated animals (86%). Following transplantation of F1 marrow cells of either (C57 × Balb/c) F1 or (C3 H × Balb/c) F1 origin, death rates of 40% and 50% were observed; these were significantly lower. Depletion of Thy 1+ cells from bone marrow graft caused only a slight increase in the leukemic death rate after transplantation of bone marrow of (C57 × Balb/c) F1 hybrid origin (50%), but a high leukemic death rate was seen after transplantation of (C3H × Balb/c) F1 bone marrow (100%). Additional experiments with fully allogeneic, T-cell-depleted C57 bone marrow transplantation suggest an antileukemic effect that is comparable to that seen after transplantation of unmanipulated F1 bone marrow. Taken together, our results indicate that GVL activity can be dissociated from graft-versus-host reaction.

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.

Abstract 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.

MHC-Associated and MHC-lndependent Urinary Chemosignals in Mice Are Expressed via the Hematopoietic System

The change in genetically determined urine odors which appears after experimental bone marrow transplantations in mice was examined in order to test whether the HMC is the only group of genes that influences the chemosensory identity via the hematopoietic system. 5 female rats were trained in a computer-controlled olfactometer to discriminate urine odors of two MHC congenic or background congenic inbred strains of mice. Transfer-of-training tests with urine samples of irradiated mice which were restored with bone marrow either from an MHC congenic or a background congenic inbred strain reveal a change of urinary chemosignals after both types of experimental bone marrow transplantation. Thus, both MHC-associated as well as MHC-independent urinary chemosignals are expressed via the hematopoietic system.

High‐dose cytostatic agents in allogeneic bone marrow transplantation: comparison of the engraftment promoting potential

Summary. We investigated the potential of various cytostatic agents for preventing graft rejection following allogeneic bone marrow transplantation. LEW rats received a lethal dose (35 mg/kg) of busulfan followed by injection of 1 x 108 F1(CAP x LEW) marrow cells, which are unable to induce a graft‐versus‐host reaction in LEW recipients. Rejection of the marrow graft was assessed by monitoring haematocrit and granulocyte counts. Due to its weak immunosuppressive activity, busulfan by itself is unable to allow engraftment of allogeneic marrow. Therefore, agents administered in addition to busulfan can be tested for their capacity to prevent marrow graft rejection. 120 mg/kg of cyclophosphamide, 20 mg/kg of ACNU and 240 mg/kg of ifosfamide completely prevented rejection of the allogeneic marrow. Maximum doses of BCNU applicable in conjunction with busulfan reduced the rejection rate to 12% (30 mg/kg) and 17% (40 mg/kg), whereas the antitumour agents thiotepa, melphalan, and carboplatin exhibited a very limited engraftment‐promoting potential in this experimental setting. Thus, BCNU (carmustine), ACNU (nimustine), and ifosfamide might be suitable candidates for conditioning of allogeneic bone marrow graft recipients.

Adoptive Transfer of NK Cells Can Eradicate Residual Leukemia After BMT

The transfer of allogeneic lymphocytes given after allogeneic bone marrow transplantation (BMT) provides a beneficial antileukemic effect, the so-called graft-versus-leukemia (GVL) effect. However, T cell mediated severe graft-vs-host-disease (GVHD) remains a major problem. The aim of the present study was to determine the antileukemic potential of IL-2 activated NK cells given shortly after BMT. BALBc/mice were given a lethal dose of A20 (H-2d, B cell leukemia) cells 2 days prior to lethal total body irradiation (TBI) and transplantation of either syngeneic or allogeneic bone marrow cells. After depletion of Thy-1.2-positive T cells, either syngeneic or allogeneic IL-2 activated spleen cells were given 24 h after BMT. Injection of A20 leukemia led to death after a median of 30 days. A lethal dose of TBI followed by either syngeneic or allogeneic Thy 1.2 depleted BMT resulted in a slight antileukemic effect. The adoptive transfer of syngeneic enriched and IL-2 preincubated NK cells at the time of BMT exerted a significant GVL effect. Although the animals demonstrated no signs of GvHD, the strongest GVL effect was observed after infusion of allogeneic MHC-mismatched NK cells. The results clearly demonstrate that allogeneic NK cells offer superior antileukemic activity without mediating GVHD.

Natural Killer Cell Alloreactivity Against Acute Leukemia Blasts: The Level of Activity Depends on the Individual Target-Effector Pair

Natural killer (NK) cells appear to be involved in graft-versus-leukemia activity after allogeneic bone marrow transplantation. Since recent findings suggest that NK cells can exert specific activity against allogeneic leukocytes, we tested 10 subjects for differences in their NK and lymphokine-activated killer (LAK) cell activity against 4 allogeneic leukemia cell targets and the cell line K562. Our results support the hypothesis that NK cells can react against allogeneic leukemia cells specifically. Only three of the donors demonstrated either generally high or low NK cell activity and none of the leukemias turned out to be principally resistant or sensitive towards NK cell-mediated lysis. Thus, most of the variance must be explained by specific NK cell/target cell interactions resulting in a complex pattern of high or low NK cell-mediated cytotoxicity. There was no clear correlation between lytic activity against a certain leukemia target and lysis of K562 or between lytic activity and the percentage of CD16- or CD56-positive cells. Future studies with larger data samples might allow the definition of distinct groups according to the pattern of alloreactivity observed. Our findings are of relevance for the application of allogeneic NK cells in the context of cellular immunotherapy for hematological malignancies.

Cellular Immunotherapy of Acute Leukemias After High Dose Chemotherapy with Cytarabin (ARA-C) and Cyclophosphamide (CY) in a Murine Model

Allogeneic lymphocytes are able to eradicate resistant leukemia cells after bone marrow transplantation. We developed a murine model to investigate their effectiveness for the prevention of leukemia relapse after high dose chemotherapy. Method: 1 × 105 A20 leukemia cells (H-2d, B cell neoplasm) were injected into Balb/c (H-2d) mice. Five days later, animals were treated with ARA-C (2 × 150 mg/kg i.p. daily; day 1–3) which provides high antileukemic activity against A20. To ensure sufficient immunosuppression, increasing doses of CY (60 to 125 mg/kg i.p.) were additionally given at day 4+5. At day 8 and 11, 2 — 107 either allogeneic MHC-mismatched (C57Bl/6, H-2b) or allogeneic, MHC-matched (DBA, H-2d) spleen cells were injected intravenously. Leukocyte counts were determined every three days. Leukemia relapse was denned as death with spleen weight > 0.15g and visible tumor nodules. Results: Without further therapy, relapse free survival was 0% with a median survival time (MST) of 21 days. Treatment with ARA-C and 2 × 60, 2 × 100, or 2 × 125 mg/kg CY cured 29%, 38% and 75% of the animals (MST = 45, 56, >200 days), respectively. After additional injection of MHC-mismatched allogeneic spleen cells, similar leukemia free survival (LFS) rates were observed: 38%, 45%, and 86% (MST = 52, 47, >200 days). Analysis of leukocyte counts revealed that 2 × 125 mg/kg CY were necessary to induce severe lymphocytopenia (<500/nl) in the majority of animals. Thus, at least in the groups treated with lower doses of CY, our negative findings could be explained by insufficient immunosuppression of the recipient (resulting in rapid rejection of allogeneic effector cells). When cells with a lower immunogenetic barrier to the recipient were used (DBA) after immunosuppression with 2 × 60 mg CY, a significant reduction of the relapse rate from 71% to 56% was observed. Conclusions: Our preclinical model allows the investigation of cellular immunotherapy after chemotherapy. Future studies will focus on the development of preparative regimens with better immunosuppressive potential. The separation and stimulation of allogeneic NK cells with antileukemia activity will enable us to transfer large effector cell numbers without GvHR.

Allogeneic NK cells can eradicate leukemia after allogeneic bone marrow transplantation in mice

REGULATORY ROLE OF INTERLEUKIN 2 AND INTERLEUKIN 4 IN TUMOR REGRESSION OF A MOUSE T-LYMPHOMA K. Jurianz and P. von Hoegen Cytokines have important regulatory functions in the immune system. Particularly in the generation of an anti-Leishmania immune response the activation of Thl or Th2 cells correlates with resistence or susceptibility to the parasites. We now studied in a mouse tumor model system the induction of different cytokines and their receptors with regard to T-cell activation comparing situations of tumorresistence and tumorsusceptibility. Live tumor cells were injected into the pinna of the ear (tumorresistence) or s.c. (tumorsusceptibility). At different time points spleen and lymph nodes were removed and the eytokine patterns were investigated by Slot Blot Analysis. We observed an increase of IL-2 mRNA expression in spleen cells with a peak on day 2 in all experimental groups. On day 5 the IL2 mRNA expression was decreased to control levels. Tumor cells were found in the spleen as early as 3 hours after injection, explaining the rapid induction of/L-2. The/L2Ra mRNA was upregulated in the tumorsusceptible group. A similar kinetic was seen in the peripheral draining lymph nodes. Therefore we conclude, that the failure of an effective antitumor immune response after s.c. inoculation of tumor cells is not due to the defective expression or utilization of IL2. In contrast to these in vivo data the failure to induce cytotoxic T-lymphocytes in vitro in MLTC correlates with a decreased level of IL2 in these cultures as determined in a biological assay. The expression of the typical Th2 cytokine tL4 in spleen cells showed an early (dl or d2) and short ( one day ) peak in the groups with subcutanously growing tumors. Thus, one can speculate, that there is a primary activation of T-helper lymphocytes in all groups resulting in secretion of IL2. In the sucseptible group this cytokine preferentially activates Th2-type CD4 cells. The secretion of Th2 cytokines ( 13.,4 ) leads to inhibition of the Thl cells, which play the important role in generation of cell mediated immunity.