Semester

Spring

Date of Graduation

2004

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

James M. Sheil.

Abstract

In general, CD8+ T lymphocytes respond to a single class I MHC/peptide complex, a phenomenon known as "MHC self-restriction". We previously reported that CD8+ CTLs from C57BL/6 mice were restricted to both H-2Kb and H-2Db when responding to the horse cyt c-derived peptide, p41--49. In Part I of this dissertation, we examine structural features of H-2Kb/cyt c and H-2Db/cyt c complexes responsible for dual recognition. B6.H-4.1c, a representative cloned CTL, was induced by similar concentrations of native peptide added to either H-2Kb- or H-2Db-expressing targets. Furthermore, the dissociation rates for both H-2Kb/cyt c and H-2D b/cyt c were comparable, but the optimal concentration of native peptide significantly increased for both H-2Kb/cyt c and H-2Db/cyt c in peptide competition assays. Based on computer-generated models, we proposed that the Pro 44-Gly45 sequence was critical for B6.H-4.1c recognition and therefore constructed single Ala substitution analogues of cyt c, designated p41--49/44A and p41--49/45A. Although p41--49/44A binds as well to H-2Kb and H-2Db as the native peptide, B6.H-4.1c recognition was ablated. The H-2Kb/p41--49/45A and H-2Db/p41--49/45A complexes were lysed, but required significantly higher peptide concentrations than the native peptide. Molecular models for cyt c, p41--49/44A and p41--49/45A demonstrated that disrupting the Pro44-Gly45 sequence altered peptide configuration and inhibited B6.H-4.1c recognition. Nonspecific immunosuppressive agents are critical in clinical transplantation success, but cause generalized T cell inhibition and leave patients susceptible to elevated rates of infection and malignancy. In part II of this dissertation, we propose that removing allospecific CTLs that selectively expand in vitro will ablate the alloreactive response. We show that five CD8+ Vbeta families in bm19 anti-C57BL/6 cultures proliferate, but cytolytic analysis implicates only Vbeta9+ and Vbeta12 + CTLs as responders against H-2Kb-expressing targets. Removal of both Vbeta9/12+ alloreactive CTLs did not affect the response to Kb-restricted antigens, nor to unrelated H-2d alloantigens. Depletion of both Vbeta9/12 + families significantly prolonged B6 allograft survival in bm19 mice. In addition, bm19 mice mounted a cell-mediated response against L. monocytogenes despite removing Vbeta9/12+ CTLs. These studies demonstrate the synergistic effect of two Vbeta families on alloreactivity and confirm that depletion of allograft-specific Vbeta+ CTLs prolong graft survival without disrupting host immune responsiveness.

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