Alloreactive Natural Killer Cells Reduce Graft-Versus-Host Disease and Enhance Engraftment in Mice Bone Marrow Transplantation Model.

Bone marrow transplantation (BMT) has become an important approach to cure many hematological malignancies. Unfortunately, graft-versus-host disease (GVHD) and graft rejection are still the major drawbacks of allogeneic BMT. GVHD fundamentally depends on the interaction of donor T cells with antigen-presenting-cells (APC) and it subsequently effect T cells activation and proliferation. Natural killer (NK) cells exert a variety of immunological functions and accommodation capacity to alter the course of GVHD and graft rejection. In this study, we established a mice GVHD model and with this model explored whether NK cells could reduce GVHD and enhance engraftment.

Methods:C57BL/6(H-2^b) mice were as bone marrow cells donors, and BALB/c (H-2^d) mice as recipients. NK cells from donors and recipients were enriched with anti-CD49b immuno-magnetic microbeads and MiniMACS separator respectively, and been reactive by culture in complete DMEM medium supplemented with recombinant human IL-2(rhIL-2). The recipients were divided into four groups: Control group 1 was treated with nothing, Control group 2 with BMT alone, Experiment group 1 with BMT plus autoreactive NK cells, and Experiment group 2 with BMT plus alloreactive NK cells. Each recipient mouse received 1×10⁷ donor bone marrow cells and 1×10⁷ spleen cells combination infusion after 7Gy ⁶⁰Co total-body irradiation, and in Experiment groups, each mouse was infused 5×10⁵ autoreactive NK cells (group 1)or donor activated NK cells (group 2).

Results: The survival time in groups of the BMT alone, the BMT with alloreactive NK cells, and the BMT with syngeneic NK cells were 25.6±1.2 days, 33.7±1.5 days, and 19.4±1.8 days; the mortality of these three groups at Day +30 were 100%, 20%, and 100%, and the chimerism of donor cells were 51.9±3.3%, 72.9±2.47%, and 41.8±2.19% respectively. Histopathological study in liver, small intestine, and skin of the recipients received BMT alone and BMT with syngeneic NK cells showed extensive infiltration of inflammatory lymphocytes associated with cellular necrosis, and a GVHD score was 7.6±0.47 and 8.6±0.47 respectively at 4 weeks after BMT. However, the mice received BMT with alloreactive NK cells showed little infiltration of inflammatory cells with normal structure of liver, small intestine, and skin, and a GVHD scores was 2.4±0.53(P <0.01). When allogeneic NK cells reduced GVHD and enhanced engraftment, the content of host APC or DCs and host mature APC or DCs were decreased and serum TGF-β1 level were increased, and when syngeneic NK cells aggravated GVHD and reduced engraftment, the contents of host APC or DCs were not changed and host mature APC or DCs were increased and the TGF-β1 level were decreased.

Conclusions:Our study suggested that alloreactive NK cells can reduce GVHD and enhance engraftment; and syngeneic reactive NK cells can aggravate GVHD and reduce engraftment. The mechanism of allogeneic NK cells reducing GVHD and enhancing engraftment is related to decreasing of host APC or DCs, and to increasing of TGF-β1 level, and in the other hand, the mechanism of syngeneic NK cells aggravating GVHD and reducing engraftment is related to increasing of host APC or DCs, and to decreasing of TGF-β1 level. Our results provide a theoretic proof of NK cells reducing GVHD and graft rejection in allogeneic BMT, especially in unrelated donor BMT, and may be related to the mismatch of Ly49-MHCIantigen.