Murine models of acute GVHD

The animal experiments are approved by the Institutional Animal Care and Use Committee at the University of Texas M.D. Anderson Cancer Center.

All recipient mice were age-matched females at 2–6 months of age at the time of BMT. We used two different murine models of GVHD for our studies. In the first model, GVHD was induced by the disparity in MHC class I and II antigens between BALB/c (H-2^d) donor C57BL/6 (H-2^b) recipient mice. To generate BMT chimeras, recipient wild-type (WT) B6 or C3^−/− (C57BL/6 background, Jackson Lab) mice received 12 Gy TBI (¹³⁷Cs source split into 2 doses) on day −1, and received 10×10⁶ T cell depleted (TCD) bone marrow (BM) cells plus 15×10⁶ splenocytes from BALB/c (NCI-Frederick) mice on day 0 ⁹. The WT control mice received only T-cell depleted BM cells. Mice were monitored for clinical signs of GVHD (hair loss, hunched back and diarrhea) and weighed twice a week. Serum concentration of blood urea nitrogen (BUN) and creatinine was measured in blood samples collected by retro-orbital route on days 7 and 10 post-transplant. For histopathological studies, tissue samples were collected from the skin, liver, intestine, lung, and kidney, and fixed in 10% formalin. The tissue samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Tissue slides were graded by a pathologist according to the published GVHD scoring system ¹⁰.

In the second model GVHD model (Parent to F1), 50 × 10⁶ splenocytes from female wild-type C5BL/6 (H-2b) donors were infused to B6D2F1 (H-2b/d) recipient mice. In this model, GVHD was manifested in the recipient mice in 2 weeks. The blood and tissue samples from GVHD and control mice were assessed at 2, 4 and 8 weeks post-transplant.