Anti-CD4 Induced Transplantation Tolerance in the Rat: Molecular and Cellular Mechanisms

Jaques, Bryon C (1997) Anti-CD4 Induced Transplantation Tolerance in the Rat: Molecular and Cellular Mechanisms. PhD thesis, University of Glasgow.

Abstract

Anti-CD4 induced transplant tolerance in the rat: molecular and cellular mechanisms Monoclonal antibodies (mAb) directed against the Cluster of Differentiation (CD)4 molecule, expressed on the surface of T helper cells, are a potentially important tool for manipulating the in vivo immune response to a tissue or organ allograft. In rodent models of organ transplantation, administration of anti-CD4 mAb prolongs allograft survival and may even induce permanent transplant tolerance. However, clinical application of anti-CD4 mAb requires a better understanding of the molecular and cellular mechanisms by which anti-CD4 mAb promotes graft survival. Early mechanisms may include CD4 T cell depletion and impairment of residual CD4 T cell function by blocking interaction of CD4 with Class II Major Histocompatibility (MHC) antigen or disrupting T cell signalling pathways. The experiments described in this thesis were undertaken to provide new insight into how one particular anti-CD4 mAb, (Medical Research Council (MRC) Oxford University Cellular Immunology Unit (0X)38), prolongs graft survival in a rat model of cardiac transplantation. Initial studies showed that 0X38, a mouse Immunoglobulin G (IgG)2a mAb, which binds to the membrane distal domain of rat CD4, when given as a brief treatment preoperatively, was able to prolong survival of fully allogeneic Lewis (RTla) heterotopic cardiac grafts in DA (RTl1) recipients (Median Graft Survival Time (MST) >100 days). Moreover, recipients bearing long-standing heart grafts developed specific tolerance to donor alloantigen since they accepted a second donor graft but rejected third party grafts. Tolerance induction following 0X38 mAb was associated with partial depletion (approximately 50%) of peripheral CD4 T cells. Initially, residual CD4 T cells were shown on the basis of phenotype, to consist of predominantly Recent Thymic Emigrant (RTE) (CD4+ve CD45RClow Thy-1high), but over several weeks, peripheral CD4 T cell numbers recovered to near normal levels. Non-rejecting heart grafts in OX38 mAb treated recipients were heavily infiltrated with mononuclear cells, including numerous CD4 cells. Analysis of intragraft cytokine messenger Ribonucleic Acid (mRNA) transcripts by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) revealed the presence of both T helper 1 (Th1) and T helper 2 (Th2) cytokine mRNA in non-rejecting grafts with a pattern similar to that seen during unmodified rejection. Moreover, measurement of alloantibody isotypes in OX38 mAb treated animals did not support the view that anti-CD4 induced tolerance was attributable to a dominant Th2 cytokine response. Residual CD4 T cells, following anti-CD4 mAb treatment, remained only transiently (2 days) coated with OX38 mAb. Encounter of CD4 T cells with alloantigen whilst they were still coated with mAb was not essential for tolerance induction. A small 'window of opportunity' was detected (up to 4 days following mAb treatment) during which cardiac transplantation failed to trigger allograft rejection. CD4+ve T cells obtained from OX38 mAb treated animals during this 'window of opportunity', showed altered tyrosine phosphorylation of a 36-38 KILO DALTON (kDa) protein on subsequent activation in vitro with immobilised anti-T CELL RECEPTOR (TCR) mAb when compared with anti-TCR activated, untreated CD4 T cells. The altered phosphorylation pattern of in vivo OX38 mAb treated cells was similar to that seen in naive CD4 T cells activated with anti-TCR after in vitro OX38 treatment. In addition, the pattern of phosphorylation observed in anti-CD4 treated cells was similar to that previously described in anergic CD4 T cells, suggesting that OX38 mAb treated cells may be functionally anergic. An unexpected role for the thymus in OX38 mAb induced transplantation tolerance was identified. Anti-CD4 mAb treated recipients which had undergone thymectomy several weeks prior to transplantation, rapidly rejected their heart grafts, despite OX38 therapy. This observation suggested that CD4 T cell depletion alone was not, in itself, sufficient to prevent allograft rejection and that a product from the thymus was necessary. A role for RTE in anti-CD4 mAb induced transplantation tolerance was indirectly explored in experiments using immunopurified single positive (CD4+ve alphabetaTCR+ve) thymocytes and Lymph NODE Cells (LNC). In reconstitution experiments using thymectomised anti-CD4 treated animals, adoptive transfer of purified single positive thymocytes on days 2 and 4 after heart grafting, restored the ability of OX38 mAb to prolong allograft survival in thymectomised animals. Furthermore, in anti- TCR proliferation assays, single positive thymocytes were less affected by in vitro OX38 mAb treatment than single positive LNC and were found to produce both Th1 and Th2 cytokine mRNA. A possible interpretation of these findings, is that in this experimental model, RTE may be analogous to naive T helper 0 (Th0) T cells and may have an essential role in tolerance induction. By adding to current understanding of the molecular and cellular mechanisms of anti-CD4 induced transplantation tolerance, the above observations may provide a more rational basis from which to further develop strategies for using anti-CD4 mAb in clinical transplantation.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Andrew J Bradley
• Keywords: Molecular biology
• Date of Award: 1997
• Depositing User: Enlighten Team
• Unique ID: glathesis:1997-74796
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Sep 2019 16:18
• Last Modified: 27 Sep 2019 16:18
• URI: https://theses.gla.ac.uk/id/eprint/74796