Mechanism of Marked Reduction In the Severity of Graft-Versus-Host Disease by Reduced-Intensity Conditioning In Murine MHC-Haploidentical BMT Model

Abstract 4707

Reduced-intensity stem cell transplantation (RIST) has come to be generally accepted as a method of allogeneic stem cell transplantation (SCT) for patients considered ineligible for myeloablative preparative regimens because of advanced age or comorbidities. We have recently reported unmanipulated nonmyeloablative HLA-haploidentical SCT using a conditioning treatment consisting of fludarabine, busulfan and anti-T-lymphocyte globulin and graft-versus-host disease (GVHD) prophylaxis consisting of tacrolimus and methylprednisolone (1 mg/kg) (Biol Blood Marrow Transplant 2006; 12:1073). In that study, the incidence of severe GVHD was only 10%. One of the mechanisms for such a low incidence of GVHD may have been caused by a reduced intensity of conditioning. Less intensive regimens should be associated with lower toxicity, a lower release of inflammatory cytokines, and potentially less GVHD; however, the mechanisms remain to be determined. Thus, using a murine MHC-haploidentical BMT model, BDF1(H-2^b/d)®B6C3F1 (H-2^b/k), that we established, we examined the influence of an intensity of conditioning treatment on GVHD.

Recipient mice received T-cell-depleted bone marrow (5×10⁶) and spleen cells (2×10⁷) after total body irradiation (TBI) 13 Gy (myeloablative group) or 5 Gy (RIST group). Both groups of mice rapidly achieved donor engraftment. Recipients in the RIST group showed significantly fewer GVHD signs than those in the myeloablatve group. Histopathological examination of the myeloablative group on day 14 revealed various pathological changes in intestine (in particular large intestine) compatible to GVHD. In contrast, intestine samples from the RIST group showed few pathological changes with much less infiltration of donor T cells. Consequently, all recipients in the myeloablative group had died of GVHD by day 60, while all recipients survived for more than 3 months. These results clearly showed that the intensity of conditioning treatment influenced on the severity of GVHD and survival of recipients.

Next, we investigated the mechanisms by which reduced intensity of conditioning ameliorated GVHD. Transplantation was performed using spleen cells that were labeled with the fluorescent cytoplasmic dye, carboxyfluorescein diacetate succinimidyl ester (CFSE), and cells in secondary lymphoid organs were analyzed by flow cytometry. The number of donor T cells in mesenteric lymph nodes on day 7 of the RIST group was significantly lower than that of the myeloablative group. In addition, a significantly increased number of host CD4⁺ T cells were recruited to secondary lymphoid organs on day 4 in the RIST group compared with the myeloablative group. An increased number of donor or host regulatory (Foxp3⁺CD4⁺) T cells were also observed in the RIST group. The levels of IFN gamma or IL-4 in lymphoid organs of the RIST group were higher than those of the myeloablative group. These results strongly suggest that host immune cells that survived conditioning treatment or cytokine milieu in secondary lymphoid organs contributed to the suppression of donor T cells during the initiation of GVHD. In addition, the expression of Th1 chemokine receptor, CXCR3, on donor T cells in secondary lymphoid organs and the expression levels of CXCL9, CXCL10, and CXCL11, ligands for CXCR3, in the large intestines were relatively lower in the RIST group, suggesting that the migration ability of donor T cells into GVHD target organs was negatively influenced by the intensity of conditioning.

In conclusion, we showed that reduced intensity of conditioning improved the severity of GVHD, and that recipient immune cells, including regulatory T cells, together with reduced expression of inflammatory cytokines or chemokines, contributed to the improvement of GVHD in RIST.