New GvHD Mouse Model for Assessing Both Acute and Chronic Phase of the Disease.

Graft versus host disease (GvHD) is still a very serious problem in hematopoietic stem cells transplantation which makes searching for new possibilities of its prevention and treatment necessary. Animal models are very useful tools for such research. We present a mouse model of GvHD allowing observation of both acute and chronic phase of the disease.

C57Bl/6 mice (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. Sex mismatched transplants were performed and chimerism of transplanted animals was confirmed by detection of sry gene with PCR. As a control group for blood examination after transplantation C3H syngeneic transplantations were performed. After transplantation, mice were weighted and physically examined by looking for changes in skin, posture, physical activity and peripheral blood parameters. Histopathological examination was performed on day +8, +16, +31 in some of the animals. Autopsy was also performed on mice which died during the experiment or which body weight decreased below 65% of the initial weight.

We observed a characteristic pattern of physical symptoms of GvHD including weight loss, skin desquamation, hunching and loss of activity, diarrhea. Weight loss to 88.2% of the initial body weight on day +7 was followed by a return to the initial weight (101.1%) on day +14 and then another decrease either strong and leading to the death of the animal or moderate and leading to a long time plateau (the average body weight on days +31, +59 and +101 was 94.4%, 91.7% and 93.6%). On day +7 desquamation of the skin of paws was very well visible and since day +10 gradually intensifying desquamative changes of the skin of whole body were observed - the mean level of changes in the population was highest on days +21 to + 24. Interestingly areas of the skin which were nude before TBI and transplantation were affected earlier (changes were visible on day +7) and more severly than other regions of the skin suggesting that local more intensive damage caused by irradiation can locally aggravate the course of GvHD. The physical symptoms were accompanied with histopathological changes of liver, skin, spleen and gut. Neither physical nor histopathological symptoms of the disease were observed in mice transplanted with syngeneic cells. In fluorocytometric analysis of peripherial blood performed on days +17, +31 and +45 severe lymphopenia was observed. The average number of CD3⁺CD4⁺, CD3⁺CD8⁺ and B220⁺ cells was strongly decreased in animals transplanted with allogeneic cells as compared to syngeneic graft recipients. At the same time expression of CD69 activation antigen on CD4⁺ and CD8⁺ cells was strongly increased in allograft recipients as compared to syngeneic controls.

The course of the disease, including weight loss and skin changes, was generally less severe in the population of mice transplanted with BM + 4×10⁶ splenocytes as compared to BM + 10×10⁶ of splenocytes recipients. The 25^th, 50^th and 75^th percentiles of survival function for both populations were162, 301, 405 and 45, 88, 277 days, respectively.