|Budget Amount *help
¥7,200,000 (Direct Cost : ¥7,200,000)
Fiscal Year 1999 : ¥2,900,000 (Direct Cost : ¥2,900,000)
Fiscal Year 1998 : ¥4,300,000 (Direct Cost : ¥4,300,000)
The Green Fluorescent Protein (GFP) transgenic mice with B6 background, bm1, or bm12 mice were cleaned up by egg-transfer technique and specific pathogen-free mice were generated. First, whether the GFP acts as transplantation antigen was tested by skin-grafting and we found it only worked as very weak transplantation antigens (i.e.: mean survival>60 days). Thus, GFP transgene are used as marker to track allogeneic cells in vivo in the following studies. Second, the following findings were made by the skin-grafting using non transgenic mice.
A : The survival of class I MHC-disparate bm1 skin grafts were dose-dependently prolonged by intravenous presensitization of B6 host with one to ten million of(B6×bm1)F1 spleen cells.
B : Intravenous presensitization of B6 host with even a hundred million of (B6×bm12) F1 spleen cells did not have any significant effect on the survival of class II MHC-disparate bm12 skin grafts.
Third, the fate of class I or class II MHC-disparate allogeneic cells inje
cted into B6 host was tracked in vivo using GFP-positive B6, (B6×bm1) F1, or (B6×bm12) F1 spleen cells. Ten million spleen cells from each mice were intravenously injected and the host spleen cells were analyzed with FACS 1, 2, 4, or 8 weeks after the injection. The following results were achieved by the investigation.
C : The injected B6 cells consisted of 1-2% of host spleen cells at 1 weeks and the fraction of the injected cells detected in hosts were proportional to those before the injection. The number of injected cells detected gradually decreased to 0.2-0.3% by 4 weeks and became undetectable 8 weeks after the injection. The speed of decay seemed not to be affected by the type of cells injected.
D : The class I MHC-disparate (B6×bm1) F1 cells injected were detected at 1 and 2 weeks after the injection and became undetectable at 4 weeks and thereafter. The numbers of cells detected were constantly smaller than those of injected B6 cells but the speed of decay seemed unaffected by the type of cells detected.
E : The total number of class II MHC-disparate (B6×bm12) F1 cells injected was roughly similar to those of (B6×bm1) F1 cells at 1, 2, 4, or 8 weeks after the injection. However, class II MHC-disparate B cells were disappeared more quickly than the other types of cells included in spleen.
In summary, although we could not perform all the studies planned because of the delay in cleaning-up process of the mice, we could get some interesting results from the novel method which has the potential to clarify the undiscovered mechanisms of transplantation tolerance. In addition, the other molecular biological methods established in this study such as quantitative RT-PCR proved to be useful in the other fields of investigation and the three papers using those methods were published as listed on the other side of the form.