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.

Influence of donor lymphocytes on the incidence of primary graft failure after allogeneic bone marrow transplantation in a murine model.

We used a murine model to determine the impact of donor lymphocyte subsets on the incidence of primary marrow graft failure after transplantation of lymphocyte-depleted bone marrow. After lethal irradiation with 7.5 Gy, Balb/c mice received 1 x 10(5) to 4 x 10(7) GvH-nonreactive (C57 x Balb)F1 or GvH-reactive C57Bl/6 marrow cells. Pretreatment with anti-Thy-1.2, anti-CD4/CD8, anti-asialo-GM1 or L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) was employed to eliminate T lymphocytes and/or natural killer cells. Primary graft failure was defined as death with neutrophils < 0.5 x 10(9)/l. To assess long-term chimaerism, the percentage of H-2b-positive spleen cells was determined. Pretreatment with anti-Thy-1.2, anti-CD4/CD8 or Leu-Leu-OMe successfully eliminated GvHR-induced mortality. Graft failure rates gradually declined from 88% after transplantation of 1 x 10(5) cells to 0% after transplantation of 4 x 10(7) C57Bl/6 cells. The incidence of graft failure, however, was not altered by T-cell depletion, provided that the unspecific loss of marrow cells was compensated for. After transplantation of GvH-nonreactive (C57 x Balb)F1 bone marrow, neither ex-vivo treatment with anti-Thy-1.2 and anti-asialo GM1 nor addition of 1 x 10(7) donor thymocytes to the allograft significantly influenced engraftment. The data obtained in our animal model suggest that the total number of marrow cells is of critical importance for successful marrow engraftment and not the presence or absence of T cells, NK cells or GvHR.

The influence of graft-versus-host reactivity, lymphocyte depletion, and cell dose on allogeneic bone marrow engraftment.

Graft rejection has hampered the use of T cell depletion (TCD) in allogeneic bone marrow transplantation. A model of host-versus-graft (HVGR) and graft-versus-host reaction (GVHR) as two inversely related processes has been proposed. We investigated graft rejection rates in graft-versus-host-reactive and graft-versus-host-nonreactive situations in a rat and a mouse model. Model 1: LEW rats were pretreated with a fixed myeloablative dose of busulfan and increasing doses of the immunosuppressive cyclophosphamide. The animals received different doses of semiallogeneic GvH-nonreactive BM cells. Graft rejection rates were dependent on the bone marrow cell number transplanted and on the pretransplant immunosuppression. Graft rejection rates following transplantation of GvH-reactive CAP marrow and genetically GvH-nonreactive (CAP x LEW)F1 marrow were the same. In conclusion, there was no advantage with respect to engraftment for the GvH-reactive marrow. Model 2: In irradiated Balb/c mice, graft rejection rates following T cell-depleted and unmanipulated transplantation of GvH-reactive or GvH-nonreactive bone marrow grafts were identical. All experiments were done with graded numbers of BM cells and revealed a strong impact of the BM cell dose on engraftment. In our experiments the cell loss during the ex-vivo manipulation was approximately 50% and, in contrast to the clinical situation, we readjusted to the intended number after TCD. Our experiments demonstrate that neither GvHR nor T cells but the BM cell dose has a strong impact on engraftment of allogeneic bone marrow.

Natural killer cells as effector cells of graft-versus-leukemia activity in a murine transplantation model.

The transfer of cytotoxic effector cells reduces the risk of relapse after allogeneic BMT. Two murine leukemia cell lines, A20 (B lymphocytic) and WEHI-3 (myelomonocytic), were used to investigate antileukemic effector mechanisms operating independently from graft-versus-host disease (GVHD). Different results were obtained with the two leukemia models. After injection of A20 cells, the majority of Balb/c recipients treated with syngeneic BMT died due to leukemia relapse (89%). The transplantation of MHC-matched DBA marrow resulted in chronic GvHD but did not reduce the risk of relapse (86%). Grafting of MHC-mismatched (but GvH-nonreactive) marrow cells from (C57xBalb)F1 hybrids, however, led to a significantly lower relapse rate (47%, p < 0.05). In vitro testing revealed that F1 cells but not Balb/c or DBA cells exert NK cell activity against A20. The elimination of NK 1.1-positive cells from the graft reduced the antileukemic activity of (C57xBalb)F1 marrow against A20 in vivo. After injection of WEHI-3 leukemia cells, syngeneic BMT cured most of the recipients (62%) and transplantation of (C57xBalb)F1 marrow provided no additional benefit. In contrast to unmanipulated Balb/c and (C57xBalb)F1 cells, which showed no NK activity against WEHI-3 in vitro, IL-2 treated effector cells were highly cytotoxic. Transfer of IL-2 preincubated grafts significantly decreased the relapse rate of WEHI-3 (19 vs. 38%) after syngeneic and allogeneic BMT. Our data indicate that GvL activity can be separated from GvHD. In our murine model, GvL activity appears to depend more on the donors NK/LAK cell activity than on the presence or absence of GvHD.