The Establishment Of Mouse Model Of Acute Graft-versus-host Disease And Study On Relationship Between Memory CD8～+T Cell And GVHD

Graft-versus-host disease (GVHD) remains a major complication after allogeneic bone marrow transplantation (allo-BMT), producing multiple-organ damage, immune deficiency and infection. Even when siblings are matched at the human leukocyte antigen (HLA) locus, GVHD is induced by donor T cells recognizing minor histocompatibilty Ags (miHAs). Mouse model of allo-BMT is a good tool for study of pathogenesis and prophylaxis. The MHC-identical, multiple miHA mismatched murine model of allo-BMT is analogous to most human allo-BMTs. Acute GVHD evolve over several weeks to months. Although it is well known that the persistence of alloreactive T cells is necessary for host tissue damage and subsequent GVHD, previous studies have shown that massive apoptotic death of alloreactive effector T cells occurs in GVHD recipients of T cell-replete allo-BMT. Uptill now, GVHD persistence is still not well understood. Thus, it is very important to establish a mouse model of miHAs-initiated acute GVHD, and further study on the relationship between memory CD8~+ T and the mechanism of GVHD will be much more benefited from this model.Part I The establishment and identification of mouse model of acute graft-versus-host diseaseAfter transplantation with bone marrow and splenic cells, the clinical signs of recipient mice is analogous to the clinic patients. Experimental mouse model is various, which is determined by BMT conditioning regimen, mouse strain, gender, age and MHC-matching. In mouse models of human allo-BMT, both CD4+ and CD8+ T cells contribute to mediate GVHD. However, while CD4+ T cells can cause lethal GVHD in only a few H-2-matched, miHA-mismatched mouse strain combinations, donor CD8+ T cells can induce lethal GVHD in most strain combinations. This suggests that CD8+ T cells are the most important T cell subset contributing to miHA-initiated GVHD. B6/SJL (H-2b, CD45.1+) recipient mice and C3H.SW (H-2b, CD45.2+) donor mice are miHA-mismatched.B6 recipients received 9.5 Gy TBI administered in three treatments from a 137Cs source. C3H.SW CD8+ T cells, with or without C3H.SW T cell-depleted BM (TBM) cells, were transplanted into the recipients by tail vein injection immediately after irradiation. B6 mice without transplantation died within 6-10 days after irradiation. B6 mice which were injected with C3H.SW TBM mixed with C3H.SW CD8+T cells died after 28daysfollowing allo-BMT. B6 mice which were injected with C3H.SW TBM were still alive within 70 days after transplantation. Flow cytometric analysis showed that mononuclear cells from the bone marrow, spleens and livers of B6 mice derived from donor, and part of mononuclear cells from the spleens and livers was CD45.2+CD8+. Pathologic examination of the liver showed that the accumulation of mononuclear cells was in the portal areas of the liver. The structure of skin was destroyed. The above results suggested that the miHA-mismatched mouse model was CD8+T cell-mediated acute GVHD model.Part II Study on the relationship between memory CD8+T cell and GVHDAlthough it is well known that the persistence of alloreactive T cells is necessary for host tissue damage and subsequent GVHD, previous studies have shown that massive apoptotic death of alloreactive effector T cells occurs in GVHD recipients of T cell-replete allo-BMT. Which alloreactive T cell subsets are responsible for GVHD persistence is still not well understood.Using miHA-mismatched mouse GVHD models of human allo-BMT, we analyzed the quantitative changes of donor-derived CD8+T cells in the spleens and livers. At different time point, mononuclear cells were recovered from the spleens and livers of B6 mice (CD45.1) receiving C3H.SW TBMand CD8+T cells (CD45.2), numerated, and stained with anti-CD45.2 and anti-CD8. The number of donor CD8+T cells was calculated after flow cytometric analysis. Donor lymphocytes that were separately recovered from the spleens and livers of B6 mice receiving C3H.SW T"BM and CD8+T cells were cultured in the presence of PMA and ionomycin for 6 h to measure the production of IFN-y by intracellular cytokine staining. The number of alloreactive CD8+ T cells secreting high levels of IFN-y was calculated by flow cytometric analysis. The analysis showed that in parallel to GVHD development, donor (CD45.2+) CD8+T cells and CD8+T cells secreting high levels of IFN-y peaked in the livers and spleens of these B6 mice (CD45.1+) by day 14, considerably declined by day 28, increased again by day 42 after transplantation. In contrast, B6 mice receiving C3H.SW TBM alone did not develop any signs of GVHD and had 3-10 fold less donor CD8+T cells that produced IFN-y by day 42 after transplantation. Lymphocytes were recovered from the spleens and livers of B6 mice receiving C3H.SW TBM and CD8+ T cells on days 14 and 21, respectively, after transplantation, pooled, and stained with anti-CD45.2, anti-CD8 Abs, and FITC-annexin V for flow cytometric analysis. The decline in donor CD8+ T cells in these B6 mice was accompanied with increased annexin V-positive donor CD8+T cells in vivo.These results suggest that a typical memory T cell response, as manifested by T cell expansion, contraction, and reexpansion, develops inrecipients during the GVHD process, and a small population of alloreactive CD8+ T cells survives after the death phase of effector cells.