Liposomal Alpha-Galactosylceramide Ameliorates Graft-Versus-Host Disease with Remaining Intensified GVL Gained By Dose-Reduction of Posttransplant Cyclophosphamaide after Haploidentical HSCT

Allogeneic hematopoietic stem cell transplantation (HSCT) is the curative treatment for many hematological malignancies. Clinical studies have shown that HSCT from haplo-identical donors with posttransplant cyclophosphamide (PTCy) can widely provide alternative donor sources and is an effective prophylaxis for both acute and chronic graft-versus-host disease (GVHD). However, on the other hands, it is concerned that the immune suppressive effect by PTCy also might diminish graft-versus-leukemia (GVL) effect and increase the relapse rate in patients with high tumor burden at transplant. Indeed, recent clinical studies suggested that a certain extent of dose-reduction of PTCy could sufficiently prevent GVHD, but whether the dose-reduction can lead to enhance GVL effect has not been clarified. In this study, we investigated the effects of dose-reduction of PTCy on GVHD and GVL by using murine haploidentical BMT model. Also, we explored the immune-modulating impacts of liposomal alpha-galactosylceramide (liposomal a-galcer; RGI2001) on reduced PTCy. Liposomal a-galcer is a synthetic glycolipid, a ligand stimulating invariant natural killer T (NKT) cell in CD1d restricted manner, which is shown to prevent GVHD via expansion of regulatory T cell (Treg) in murine experiments. First, we tested the effects of dose-reduction of PTCy on GVL effect, we transplanted from C57BL/6J donor to B6D2F1 recipients bearing with luciferase-expressing P815 leukemia cells. The impact of PTCy dose-reduction on GVL was evaluated by using in vivo bioluminescence system. Lethally irradiated B6D2F1 mice were transplanted with 5x10⁶ T cell depleted (TCD) BM, 10x10⁶ whole splenic cells from C57BL/6J donor mice and 5x10⁵ luciferase-expressing P815 leukemia cells. PTCy or control vehicle was administered at day3 after BMT. As for PTCy, 50mg/kg of cyclophosphamide (Cy) has already shown as standard dose to sufficiently suppress GVHD, thus we tested effect of 25mg/kg of Cy comparing with 50mg/kg of Cy. In vivo imaging system (IVIS) was followed weekly since day10 after BMT to evaluate tumor burden. A trend toward lower levels of radiance was measured by IVIS in reduced-dose group than full-dose group through the experimental period, suggesting reduced-dose of Cy contribute to leave alloagressive T cell activity and result in the enhancement of GVL effect. However, the benefit of GVL enhancement in reduced PTCy group was counterbalanced by the deterioration of GVHD when we used higher dose of total body irradiation (TBI) for the aggressive GVHD-prone experimental setting. To overcome the negative impact of PTCy-reduction on GVHD, we next tested the immune-modulating effect of liposomal a-galcer on this system. Liposomal a-galcer 1mg/kg or control vehicle was administered at day3, 5, 7, after BMT. Our data indicated that reduced PTCy plus liposomal a-galcer significantly improved acute GVHD in overall survival (vs control group, p < 0.01). In comparison with those treated with PTCy plus liposomal a-galcer, the others kept on losing weight and worsening GVHD score to the end beyond the third week without recovery. Importantly, liposomal a-galcer did not influence the effect of GVL when we evaluated tumor-bearing recipents with IVIS, suggesting that add-on of liposomal a-galcer improved GVHD with remaining intensified GVL effect which was gained by dose-reduction of PTCy.　Flowcytometric analysis of splenic cells at day 14 showed that %Treg of CD4+ Tcell was significantly higher in reduced PTCy plus liposomal a-galcer group than control group (5.30 vs 2.75, p < 0.05) and reduced PTCy alone group (5.30 vs 2.57, p < 0.05). Additionally, in PTCy plus liposomal a-galcer group, both Treg/CD4+ conventional Tcell (Tcon) ratio and Treg/CD8+ Tcell ratio were significantly higher than control group (p < 0.05) and reduced PTCy alone group (p < 0.05), respectively. Analysis of MLN cells also showed the selective increase of Treg in PTCy plus liposomal a-galcer group more than in other groups. Analysis of splenic cells and MLN cells at day28 showed similar trends with at day14. Taken together, these data demonstarated that liposomal a-galcer could efficiently compensate the immune-suppressive effect after reduced PTCy by preferentially promoting Treg recovery with remaining GVL. Our data might provide the important information for developing new strategy of haploidentical HSCT with PTCy for patient with high-tumor burden.