IL10 -Transduced Mesenchymal Stem Cells Improve the Acute Graft-Versus-Host Disease Protection in a Murine Model.

Recent research has suggested that mesenchymal stem cells(MSCs) have some immunosuppressive properties which may be of interest to lessen GVHD after transplant. Although encouraging, very limited systematic study has been performed to assess the ability of MSCs to suppress GVHD in vivo. In the early study, we failed to demonstrate murine MSCs attenuate the acute GVHD in an established murine model of acute GVHD (C57BL/6 mice → CB6F1 mice). Because of its anti-inflammatory properties and its association with dendritic cells and regulatory T cells, IL10 may has substantial benefit on the induction of immune tolerance and graft survival. In this study, we investigated whether the co-transplantation of genetically transduced MSCs expressing IL10 could improve the GVHD protection.

Methods: 8–9 week old male CB6F1 mice (C57BL/6×Balb/c) were given 12Gy (100cGy/min) total body irradiation (6mv,X-ray). 4 hours after the lethal irradiation, the mice received one of four groups of donor mice cells (8–10 week old female C57BL/6 mice) via tail vein injection:

1. 1×10⁷ bone marrow MNC+3×10⁷ splenic MNC (n=10) (aGVHD inoculum, control);

2. 1×10⁷ bone marrow MNC+3×10⁷ splenic MNC+1×10⁵ murine marrow-derived MSCs (n=15);

3. 1×10⁷ bone marrow MNC+3×10⁷ splenic MNC+1×10⁵ IL10-transduced MSCs (transfected with IL10- recombinant adenoviral vector)(n=15);

4. 1×10⁷ bone marrow MNC+3×10⁷ splenic MNC+5×10⁵ IL10-transduced MSCs (n=15).

The weight, survival time, WBC count and chimerism of recipient mice were observed, the severity of aGVHD was assessed with both an aGVHD scoring system for physical signs and a semi-quantitative score for histologic examination. The serum of recipient mice was collected on the day 14, 28, 35,or the brink of death after transplant, then be used to detect the levels of cytokines (IL10,IL4,INF-γ and TNF-α) by ELISA.

Results: The injection of 1×10⁷ bone marrow MNCs plus 3×10⁷ splenic MNCs from C57BL/6 donor into CB6F1 recipient following 12Gy irradiation resulted in a reliable model of aGVHD: the clinical signs and histologic degree was relatively coincident, the incidence of aGVHD were 100% and all mice died within +27d∼+33d. The co-transplant of 1×10⁵ MSCs failed to decrease the incidence and severity of aGVHD significantly(81.8% vs 100%, score 6.0±2.83 vs 9.0±0.38, P>0.05).Compared with the group of 1×10⁵ MSCs or control, 1×10⁵ IL10-transduced MSCs demonstrated significantly reduced the incidence and severity of aGVHD(0%,score 0.5±0.05,P<0.01), while increased cell dose(5×10⁵) failed to enhance the effect significantly. Serum levels of cytokines showed the significant increase of IL-10 (46.7±3.4 pg/ml vs 17.5 ±2.6 pg/ml, P<0.05), the corresponding increase of IL-4 (20.7±2.5 pg/ml vs 17.7±3.8 pg/ml, P=0.13),and more dramatic decrease of INF-γ (90.4±16.1 pg/ml vs 234.0 ±83.2 pg/ml, P<0.01) in the group of IL10-transduced MSCs.

Conclusion: Benefical effects on aGVHD protection could be observed when MSCs were engineered to expressed IL10. The significant increase of Th2 type cytokines and decrease of INF-γ may be one of the mechanisms for the aGVHD protection in this murine model.