Mechanisms Responsible for Indefinite Survival of Cardiac Allografts Induced by R73 Monoclonal Antibody in a Rat Model

Casey, John J (2001) Mechanisms Responsible for Indefinite Survival of Cardiac Allografts Induced by R73 Monoclonal Antibody in a Rat Model. PhD thesis, University of Glasgow.

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The principle obstacle to successful organ transplantation is the failure of modern immunosuppressive protocols to result in indefinite allograft survival and the concurrent side effects of such protocols such as infection and malignancy. As a means of overcoming these difficulties monoclonal antibodies have been used experimentally and in clinical practice to target specific molecules involved in the rejection response in the hope that operational tolerance to a graft can be induced by a short peri transplant treatment. The central role of the alpha/beta T cell receptor in allorecognition and graft rejection makes it an ideal target for intervention. In this thesis, experiments were carried out using a monoclonal antibody to the rat alpha/beta T cell receptor (R73) to investigate its action in a model of rat heterotopic cardiac transplantation. In preliminary experiments, the mouse IgG1 monoclonal antibody R73 was administered intraperitoneally at a dose of 200mug on days -2 and -1 relative to receiving a fully allogeneic heart graft (transplantation taking place on day 0). Prolongation of heart graft survival was seen in the BN-Lewis strain combination however indefinite graft survival (>100days) was only obtained in the Lewis-DA strain combination. In order to obtain the optimal treatment regime in this strain combination, antibody doses of 50mug, 200mug and 500mug were administered to separate groups. 200mug and 500mug doses produced permanent graft survival and so the 200mug dose on days -2 and -1 was used in all further experiments. A group of animals underwent thymectomy prior to transplantation under cover of R73. The majority of animals accepted their grafts permanently, suggesting a peripheral mechanism of action for R73. This was further supported by the observation that exogenous IL-12 administered after heart transplantation to R73 treated animals resulted in reduced graft survival. Studies were then carried out to evaluate the effect of R73 on the peripheral T cell pool. Flow cytometric analysis of lymphocytes in the peripheral blood and lymph nodes of R73 treated graft recipients revealed marked depletion of CD4 and CDS T cells at days 4 and 7 post transplant with reconstitution of the T cell pool by day 21. Depletion was not complete, however, and 50% of T cells were present at day 7. Delaying heart grafting until this time still resulted in indefinite graft survival in the majority of recipients. This would suggest that R73 treatment not only partially depletes peripheral T cells but also renders the remaining cells unresponsive or results in the emergence of a regulatory population of cells. Flow cytometry also revealed the presence of T cell receptor modulation after R73 treatment. Immunohistochemical staining of the heart grafts themselves was also carried out at day 7 post transplant in R73 treated and rejecting control animals. This revealed a marked reduction in the graft infiltrate of R73 treated hearts compared to rejecting untreated hearts. In further in vitro experiments, lymph node cells from R73 treated graft recipients were harvested at various time points post transplant and stimulated in allogeneic MLR. Cells from treated animals failed to proliferate at day 4 post transplant but proliferated normally at days 7, 21 and 100 to both allogeneic and third party stimulators. Analysis of the cytokines in the supernatants of the MLR's revealed reduced IL-2 production by R73 treated LNC compared to rejecting animals and this was observed even as long as 100 days post transplant. Cytokine gene transcripts detected by RT-PCR in the grafts removed from R73 treated and rejecting animals revealed reduced IL-2 and IFNgamma gene transcription in treated animals but comparable production of IL-4, IL-10 and IL-13. It would seem therefore that the cells which remain after R73 treatment are unable to produce IL-2 normally and the absence of this critical cytokine may be an important mechanism in the action of R73. Finally, experiments were carried out to investigate whether R73 ligation of the TCR would result in alterations in apoptotic activity in graft recipients. Propidium iodide staining and flow cytometric analysis of LNC revealed increased apoptotic activity in the lymph nodes of R73 treated graft recipients at day 7 post transplant compared to rejecting and normal animals. This increased activity depended on the presence of a heart graft as DA animals given R73 alone displayed baseline apoptotic activity. Interestingly, TUNEL staining of heart grafts from R73 treated and rejecting animals revealed increased apoptotic activity in the heart grafts of rejecting animals suggesting that apoptosis occurs both as a tolerogenic mechanism perhaps by altered signalling via the TCR and as a mechanism of graft destruction by infiltrating T cells.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Eleanor Bolton
Keywords: Medicine, Surgery, Immunology
Date of Award: 2001
Depositing User: Enlighten Team
Unique ID: glathesis:2001-76038
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 17:05
Last Modified: 19 Nov 2019 17:05

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