Cellular Requirements for Renal Allograft Rejection

Medicine, Surgery

Bolton, Eleanor Mary

University:University of Glasgow

关键词: Medicine, Surgery;   

Others  :  http://theses.gla.ac.uk/77765/1/10999298.pdf

来源: University of Glasgow

【 摘 要 】

Graft rejection remains a major problem in clinical renal transplantation despite recent improvements in immunosuppressive therapy. While it is accepted that T lymphocytes play an essential role in acute rejection, the relative contributions of the different effector pathways have not been established. The aim of this thesis was to examine the cellular effector mechanisms of renal allograft rejection in the rat. In initial experiments, the characteristic features of unmodified rejection were observed in a serial immunohistological study of rejecting DA allografts transplanted into PVG recipients. The progressive mononuclear cell infiltration of the grafts initially comprised predominantly CD4 lymphocytes and subsequently CD8 cells. In addition to T cells, many of the infiltrating cells were of a phenotype consistent with NK cells and macrophages. This was associated with a striking increase in the expression, within the graft, of donor MHC class I and II antigens, together with the early disappearance from the graft of class II positive donor interstitial cells. Mononuclear cells harvested from the grafts and spleens of these animals displayed moderate levels of in vitro allospecific cytotoxicity against donor strain ConA blasts as well as high levels of nonspecific cytotoxicity against the NK susceptible Y3 target. Following on from these basic observations, subsequent experiments investigated the ability of CD4 or CD8 T lymphocyte subpopulations (prepared by negative selection) to mediate allogeneic kidney damage in different experimental models. One approach was to examine the ability of adoptively transferred lymphocyte subpopulations to cause renal allograft rejection in rats rendered lymphocyte deficient by a sublethal dose of whole body gamma irradiation. Acutely irradiated (8.5 Gy) Lewis recipients were unable to reject DA renal allografts unless reconstituted with syngeneic lymph node cells (LNC). Whereas transfer of 5x10e6 naive Lewis T lymphocytes rapidly restored graft rejection, similar numbers of either CD4 or CDS lymphocytes were relatively ineffective. Immunohistological examination of day 7 allografts in reconstituted recipients revealed, in all groups, a moderate leukocytic infiltrate of similar phenotypic composition, irrespective of the phenotype of the reconstituting cells, and with broad similarity to the infiltrate observed in unmodified rejection. When harvested infiltrating cells and splenocytes were tested in functional assays, only effector cells from CD4+CD8 T cell reconstituted animals, and not those from animals receiving either separated subpopulation, demonstrated allospecific cytotoxicity. Interestingly, splenocytes from all animals (including unreconstituted rats) showed nonspecific cytotoxic activity against Y3, but graft infiltrating cells from the same groups showed none. Collectively, these experiments suggested that both T cell subpopulations were necessary for optimal graft rejection, and that in this particular strain and model, graft rejection correlated with specific cytotoxic I cell lysis rather than nonspecific cytotoxic activity. The second approach examined the ability of T cell subpopulations to mediate allogeneic tissue damage in the renal graft versus host reaction (GVHR). Renal subcapsular injection of parental CD4 lymphocytes in F1 recipients was sufficient to produce, by day 7, a grossly observable renal GVHR, while CD8 lymphocytes (even if specifically sensitised) were ineffective. CD4 lymphocytes required the essential participation of radiosensitive F1 host, bone marrow derived cells to cause tissue damage. This occurred in the absence of demonstrable specific T cell lysis and appeared to be a DTH reaction. Experiments with PVG recombinant rats showed that an isolated MHC class II, but not a class I incompatibility was sufficient to provoke a response. The final group of experiments examined the ability of adoptively transferred lymphocyte subpopulations to restore renal allograft rejection in the congenitally athymic PVG-rnu/rnu rat. CD4 lymphocytes alone were able to restore the first-set rejection response, while CD8 cells alone (naive or specifically sensitised) were ineffective, although the addition of CD8 cells to the inoculum had a synergistic effect on the ability of CD4 cells to restore rejection. Immunohistological studies revealed moderate cellular infiltration of non-rejecting grafts and increased cellular infiltration of the rejecting grafts, with a significant increase in the number of MRC 0X8 positive cells. An interesting finding was the presence, in unmodified PVG-rnu/rnu rats, of extrathymically derived cells with wide alloreactivity as detected by in vitro cytotoxicity assays against a range of allogeneic ConA blasts and NK susceptible targets. Treatment of the effector cells with anti-asialo GM1, and cold target inhibition assays together suggested the presence of populations of atypical, widely reactive NK cells, with the additional ability to preferentially recognise a specific target. These cells were also present in the rejecting grafts of CD4 reconstituted recipients. Overall therefore, the transfer of CD8 lymphocytes alone was insufficient to cause tissue damage in allogeneic kidneys in any of the experimental models studied. In contrast, CD4 lymphocytes alone were able to cause extensive parenchymal damage in the renal GVHR, and were sufficient to initiate allograft rejection in athymic recipients, but required the additional presence of CD8 cells to restore rejection in acutely irradiated animals.