Hematopoietic Allotransplant Studies in Complement Deficient Mice Reveal Beneficial Role of Innate Immunity in Ameliorating Consequences of GVHD.

Graft versus host disease (GvHD) is the main complication after allogeneic hematopoetic stem cell transplantation. Immunosuppressive regiments used in prevention and treatment of GVHD are only partially effective, which makes research focused on new possibilities of controlling GVHD necessary. The aim of this study was to evaluate the role of complement system in pathogenesis of GvHD. We employed as a model C3 complement factor knock-out animals.

C3^+/+ and C3^−/− mice (C57Bl/6 background, H-2^b) were transplanted, one day after ablative TBI with 5×10⁶ BM cells and 4×10⁶ or 10×10⁶ splenocytes isolated from allogeneic C3H. He (H-2^k) or from syngeneic animals. Mismatched transplants were performed and chimerism of transplanted mice was confirmed 6 weeks after transplantation by detection of sry gene with PCR. As a control group for hematological and immunological examination after transplantation C3H syngeneic transplantations were also performed. During the experiment mice were weighted and physically examined. In parallel, in some of the animals the histopathological examination and flow cytometric blod analysis was performed. Autopsy was also performed on mice which died during the experiment or which body weight decreased below 65% of the initial weight.

The physical (e.g., weight loss, skin desquamation, diarrhea) and the histopathological (e.g., involvement of liver, skin and gut) symptoms of GVHD were observed in almost all mice transplanted with allogeneic cells but not in mice transplanted with syngeneic cells. A characteristic pattern of weight loss was observed: fall on day +7 was followed by return to the initial weight on days +14 to +17 and then another decrease leading either to the death of the animal or to a long time plateau. In the population of C3 deficient mice the weight loss was higher and the return was significantly slower as compared to C3^+/+ animals - the average body weight of C3^−/− and C3^+/+ mice was: 86,6% and 88,2% of initial weight on day +7; 90,9% and 94,6% (p<0,01) on day +10; 101,8% and 99,4% on day +14; 93,4% and 89,4% on day +31; 93,6% and 93,5% on day +100. Skin changes tended to be less severe in C3^+/+ animals, difference in the level of skin changes of paws was strongly significant on day +10 (p<0,002). In the fluorocytometric analysis of peripheral blood performed on days +17, +31 and +45 severe lymphopenia affecting both B and T cells was observed. At the same time expression of CD69 activation antigen on CD4⁺ and CD8⁺ cells was increased in both C3^+/+ and C3^−/− allograft recipients as compared to syngeneic control groups. However no significant difference between C3^+/+ and C3^−/− mice was observed.

Interestingly, significant difference in mortality was observed in Kaplan-Meier analysis including 40 C3^+/+ and 54 C3^−/− mice from 7 long-time experiments lasting more than 1 year the estimates of the 25^th, 50^th and 75^th percentiles of the survival function were 59, 137 and 367 days for C3^+/+ and 47, 118 and 264 days for C3^−/− mice (Mantel-Cox test: p<0,01). The difference in better survival in C3^+/+ mice as compared to C3^−/− mice as well as in dynamics and severity of physical symptoms of the disease suggests that complement system may have a beneficial role in ameliorating consequences of GVHD.