Graft-versus-leukemia in rat MHC-mismatched bone marrow transplantation is merely an allogeneic effect.

One of the major problems in the treatment of leukemia with bone marrow transplantation (BMT) remains leukemia relapse. It has been clearly shown that graft-versus-host disease (GVHD) is accompanied by a graft-versus-leukemia reaction (GVLR) which reduces the incidence of leukemia relapse. To date, discussion is still going on as to whether GVHD and GVLR are either two different reactions or are exerting their effects through the same mechanism(s). In two rat leukemia models, namely the Brown Norway acute myelocytic leukemia (BNML) and the WAG/Rij acute lymphocytic leukemia L4415, total body irradiation (TBI) was given to induce a state of so-called minimal residual disease (MRD). Subsequently, it was attempted to evoke a GVLR distinct from GVHD by using semi-allogeneic hybrid-to-parent or parent-to-hybrid BMT, with or without the addition of graded numbers of lymphocytes. In both leukemia models applying hybrid-to-parent BMT, the addition of high numbers of semi-allogeneic lymphocytes to the semi-allogeneic graft had no antileukemic effect. In parent-to-hybrid BMT, the grafts sharing their alloantigens with the leukemia cells did not induce an anti-leukemic effect, irrespective of the number of lymphocytes present in the graft or the induction of evident GVHD. When the parental graft was histoincompatible with the leukemia cells, transplantation of bone marrow alone induced a significant increase in life span correlating with 2.8 log leukemia cell kill (LCK).(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-2 therapy after allogeneic bone marrow transplantation for acute myelocytic leukemia: studies in a relevant rat model for AML.

One of the major problems in the treatment of leukemia with BMT remains leukemia relapse. It has generally been established that allogeneic BMT, compared with autologous BMT, gives rise to a graft-versus-leukemia reaction (GVLR), usually associated with GVHD. To explore a possible role for post-BMT immunotherapy, recombinant human IL-2 therapy has been studied in the Brown Norway acute myelocytic leukemia (BNML), a rat leukemia model relevant for human AML. The antileukemic efficacy of rhIL-2 therapy is studied applying different doses of rhIL-2 after syngeneic or allogeneic BMT. rhIL-2 treatment post-syngeneic BMT showed a small, borderline significant GVLR. Repeated rhIL-2 treatment after allogeneic BMT resulted either in no significant antileukemic effect or in lethal GVHD when 'low' or 'high' doses were administered, respectively. An intermediate dose, however, induced a significant GVLR without the induction of (lethal) GVHD. Transplantation of allogeneic rat BM, which contains only a few lymphocytes, does not result in a significant GVLR or GVHD and thus resembles human HLA-matched allogeneic T cell-depleted (TCD) BMT. In conclusion, from the rat studies presented it appears that the GVLR lost by TCD of the allogeneic graft, may be more than fully compensated by IL-2 treatment post-allogeneic TCD BMT.

Quantitative studies on graft-versus-leukemia after allogeneic bone marrow transplantation in rat models for acute myelocytic and lymphocytic leukemia.

The major shortcoming of present day treatment of leukemia by bone marrow transplantation (BMT) remains leukemia relapse. It has become clear that a graft-versus-host reaction (GVHR) is accompanied by a graft-versus-leukemia reaction (GVLR) which may prevent leukemia relapse. In two non-immunogenic rat leukemia models, the Brown Norway acute myelocytic leukemia (BNML) and the WAG/Rij acute lymphocytic leukemia L4415, total body irradiation (TBI) was given to induce 'minimal residual disease' (MRD). Subsequently, it was attempted to evoke a GVLR by using syngeneic or allogeneic BMT, with or without addition of graded numbers of lymphocytes. In both leukemia models the addition of high numbers of syngeneic lymphocytes to the syngeneic graft had no antileukemic effect. Allogeneic marrow grafts, which contain at the most 8% lymphocytes, only resulted in a GVLR when splenocytes were added. The therapeutic window was found to be narrow, i.e. in fully mismatched BMT the number of allogeneic splenocytes resulting in a significant GVLR (2-3 log leukemic cell kill) without inducing (lethal) acute GVHD was critical. Increasing the number of allogeneic spleen cells added to the allogeneic BM graft induced lethal acute GVHD. To date, our data indicate that the GVLR is an allogeneic effect, inseparable from GVHD.

In vitro resistance of the brown Norway rat acute myelocytic leukemia (BNML) to lymphokine-activated killer activity.

In in vivo allogeneic bone marrow transplantation studies with the Brown Norway (BN) rat as recipient and the WAG/Rij rat as allogeneic donor a significant graft-versus-leukemia (GVL) effect is observed. Studies were performed to investigate whether lymphokine-activated killer (LAK) cells play a role in this GVL effect. Splenocytes from WAG/Rij and BN rats were activated in vitro by recombinant human interleukin-2 (rhIL-2) for 5-6 days. The cytolytic activity of these LAK cells was tested on four rat solid tumor cell lines, i.e. an ureter carcinoma, a rhabdomyosarcoma, and two lung tumors, and on leukemic cells derived from the BN rat acute myelocytic leukemia (BNML) and the WAG/Rij acute lymphocytic leukemia (L4415). The panel of target cells also included the murine cell lines P815 and YAC. Both WAG/Rij and BN LAK cells were not capable of lysing the leukemic cells in contrast to significant cytolytic activity on the rat solid tumor cell lines and P815 and YAC. BNML cells showed to be resistant to lysis by human NK cells. Phenotypical analysis of the rat LAK population revealed a decrease in the CD4/CD8 ratio compared to the unstimulated splenocyte population. Rat LAK cells displayed no antibody-dependent cellular cytotoxicity (ADCC) on the leukemic cells, whereas IL-2-stimulated human peripheral blood cells showed moderate ADCC activity on the leukemic cells. To investigate whether cytokines play a role in lysis of leukemic target cells, graded numbers of LAK cells and leukemic cells were co-cultivated for seven days in an agar-based colony culture system. This resulted in moderate suppression of leukemic colony formation. From the current in vitro studies it appears that the graft-versus-leukemia observed in in vivo allogeneic bone marrow transplantation studies is probably not due to a direct leukemic cell kill by LAK cells.

L4415: further characterization of the rat model for human acute lymphocytic leukemia.

The biological properties of a transplantable lymphocytic leukemia, L4415 in the WAG/Rij rat, are described. The radiation-induced L4415 leukemia is characterized as a relatively slowly growing, non-immunogenic, immature T-cell leukemia which shows a reproducible growth pattern upon intravenous (i.v.) transfer. Survival time following i.v. inoculation is inversely related to the number of leukemic cells in the inoculum, which allows a quantitative estimate in terms of log leukemic cell kill of the effect of treatment. The first signs of leukemic growth are found in the bone marrow, the spleen, and the liver. Leukemic cells can be detected in the peripheral blood 13 days after inoculation. Due to replacement of normal hemopoietic tissue by leukemic cells and their number increasing exponentially thereafter, normal hemopoiesis is inhibited in the later stages of the disease as indicated by severe thrombocytopenia and anemia. Death is caused by a combination of splenic rupture, gastrointestinal and pulmonary hemorrhage, and impaired functions of heavily infiltrated organs. Hepatosplenomegaly and lymphadenopathy are prominent features at autopsy. Cyclophosphamide- and radiosensitivity of the clonogenic leukemic cells have been determined, a 2.9 log cell kill could be induced by single dose cyclophosphamide inoculation and a dosage giving a surviving fraction of 0.37 (D0) of 0.99 Gy with an extrapolation number (N) of 8.5 were calculated. Based on these data, the L4415 rat leukemia may be regarded as a relevant model for human acute lymphocytic leukemia and may thus serve to explore new treatment strategies.

Assessment of the inhibitory effect of immunosuppressive agents on rat T cell interferon-gamma production using an ELISPOT assay.

The immunospot (ELISPOT) assay has proven to be an efficient and sensitive method for the enumeration of single cells secreting antibodies or cytokines. Here we show that the generation of interferon-gamma producing cells (IFN-gamma pc) in rat spleen cell cultures stimulated with concanavalin A (ConA) is dose-dependently inhibited by a wide variety of immunosuppressants such as cyclosporin A, FK506, hydrocortisone, dexamethasone, azathioprine and ART-18, a monoclonal antibody (mAb) with established immunosuppressive activity in organ transplantation and autoimmunity. The minimal inhibitory concentration (m.i.c.) correlated well with the reported m.i.c. in the mixed lymphocyte reaction (MLR) assay and the therapeutically effective plasma levels in vivo. Our data indicate that the IFN-gamma-specific immunospot assay is a powerful tool for determining the potency of immunosuppressive agents in vitro and provides a simple and accurate method for screening large numbers of agents with suspected immunosuppressive properties. The assay may additionally prove to be of value for determining the therapeutically effective doses of immunosuppressants that should be administered in vivo.