Long-term survival of cardiac transplant recipients continues to be severely limited by the development of a pathological, chronic rejection process, termed allograft vasculopathy (AV). This remains to be the case despite dramatic improvements in the areas of surgical techniques, pre- and post-operative care, and immunosuppression. To model the clinical setting we used calcineurin inhibitor (CNI) immunosuppression, the cornerstone of post-transplant immunosuppression, in a murine aortic interposition transplant model for our analysis of AV development. This model mimics the presentation of AV in human cardiac transplants through the development of a progressively occlusive neointimal lesion. Our previous work in this model has demonstrated that CD8+, but not CD4+, T cells play a role in neointimal lesion formation. Further investigation also highlighted a specific requirement for either CD8+ T cell-derived IFN-γ or direct cytotoxicity in the development of lesion formation. In the current study we confirmed that CD8+ T cell-derived IFN-γ also leads to the loss of medial smooth muscle cells, an event which inversely correlates with lesion formation. The Fas/FasL direct cytotoxic pathway was also significantly involved in neointimal lesion formation and medial remodeling. This work clarified the pathways utilized by CD8+ T cells in their role as mediators of AV development. Recognizing the threat that CD8+ T cells pose to cardiac transplant recipients in the presence of CNI immunosuppression, and a growing concern with the presence of anti-donor memory T cells in transplant recipients, we next explored the development of memory CD8+ T cells in the presence of CNI immunosuppression. We first established that memory CD8+ T cells could not develop when CNI immunosuppression was initiated immediately post-challenge. Next, we hypothesized that the clinical practice of CNI delay post-transplant would permit the development of de novo memory CD8+ T cells. Immediate and early initiation was sufficient to prevent the development of de novo memory CD8+ T cells. However, later delay to within a clinically practiced timeframe did permit the development of de novo memory CD8+ T cells. Our analysis revealed that this population demonstrated equivalent functionality to de novo memory CD8+ T cells generated in the absence of CNI immunosuppression.

The mechanism of T cell recognition is the central but unsolved puzzle of adaptive immunology. The difficulties come from the multichain structure of TCR/CD3, the binate binding structure of the pMHC molecule, the diversity of the peptides presented on the APC, the critical role of coreceptor CD4/8, the communication between TCR and coreceptor CD4/8, the complex environment of interactions taking place and the binding and signaling coupled process of recognition. Most studies were using the 3D kinetic measurements or biological functional assays to address the mechanism of the T cell recognition. However, those assays are usually either lacking of physiology relevance or missing of the initial recognition signals. Here a 2D micropipette adhesion assay with high temporal resolution (-second) was used to address the in situ kinetics of molecular interaction at the membrane of live T cells. The aim of this project is to advance our understanding to the T cell recognition mechanism. The micropipette adhesion assay was firstly used to address a simple case, the resting state pMHC-CD8 interaction. In the absence of TCR-pMHC interaction, the pMHC-CD8 interaction has a very low affinity that depends on the MHC alleles and the lipid rafts of the T cell membrane where CD8 resides, but not on the peptide complexed to the MHC and whether the CD8 is an a a homodimer or an αβ heterodimer. For cognate pMHC, following the initial observation in the F5 T cell system, the binding also displays a two-step curve in the OTI T cell system. The first-step binding occurs before one second and has a very fast on-rate and off-rate (>2s ⁻¹), and the secondstep binding follows immediately but reaches a much higher level of binding. It was identified that the first-step binding is mediated by the TCR-pMHC interaction, and the second-step binding is triggered by the TCR-pMHC interaction but mediated by CD8- pMHC binding. The two-step binding is the unique property of cognate pMHC, and it can be abolished by disrupting the lipid rafts, inhibiting the Src family protein tyrosine kinases (PTK) or protein tyrosine phosphatase (PTP). The finding of two-step binding identifies a CD8-dependent signaling amplification pathway. The data also indicated the active communication between TCR and CD8 in the antigen recognition. The crosstalk between TCR and CD8 was further dissected using two anti-CD8 antibodies 53.6.7 and CT-CD8a. 53-6.7 can significantly enhance the binding of pMHC to the T cell. Although the enhancement is directly mediated by MHC-CD8 interaction, the enhancing role of this antibody is TCR dependent. Blocking the TCR-pMHC interaction on OTI T cell or expressing CD8 alone on a hybridoma abolished the enhancement. The enhancement is also dependent on the integrity of lipid rafts and the normal function of PTP. In contrast, the antibody CT-CD8 can inhibit the binding of pMHC to the T cells and interfere with the TCR-pMHC interaction. The enhancing or inhibitory role of these two anti-CD8 antibodies is reversely correlated with the affinities of TCR-pMHC interactions. Only 53-6.7, but not CT-CD8 antibody, can phosphorylate and activate Lck. The data demonstrated a dual way crosstalk between TCR and CD8, and indicated the importance of cooperation of TCR and CD8 in antigen recognition. In the physiology condition, the TCR must accurately and efficiently recognize the cognate peptide from thousands of surrounding endogenous peptides. There is an argument regarding whether the endogenous peptides plays a role in helping the TCR recognition. Our results demonstrated that the nonstimulatory peptides can significantly enhance the T cell recognition sensitivity. In the presence of nonstimulatory peptide, the TCR can efficiently detect a single antigenic pMHC. The enhancement of recognition is due to the CD8 binding to the nonstimulatory pMHC. Blocking the CD8 binding can paralyze the enhancement. In contrast, it was found that the presence of antagonist can inhibit the binding of agonist pMHC to the T cells, and the inhibition occurs in the initial recognition step. Based on the data, an "amplification and competition" model was proposed to explain the molecular mechanism of the enhancement and inhibition function of the nonstimulatory and antagonist peptides in the T cell recognition, respectively.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 135-160).

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 115-126).

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 129-144).

Thesis (Ph. D.)--Cornell University, 1989. / Vita. Includes bibliographical references.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p.156-190

Thesis (M.S.)--University of Missouri--Columbia, 2004. / "May 2004." Typescript. Includes bibliographical references (leaves 97-111). Also issued on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / "May 1999." Typescript. Vita. Includes bibliographical references (leaves 199-250). Also available on the Internet.