Document Type

Dissertation

Date of Degree

Spring 2011

Degree Name

PhD (Doctor of Philosophy)

Degree In

Immunology

First Advisor

Thomas S. Griffith

Abstract

Billions of cells die everyday as a result of normal tissue turnover, infection, trauma or injury. These dead cells are taken up, processed and presented to T cells by antigen presenting cells resulting in tolerance or immunity. Apoptotic cells induce tolerance; however, the precise mechanisms are still unknown. Previous studies have shown that direct infusion of apoptotic cells induce tolerance mediated by TRAIL-expressing CD8+ T cells. We hypothesized that immunologic tolerance induced by apoptotic cells is dependent on the activation status of apoptotic cells and mediated by direct killing of target cells by TRAIL-expressing CD8+ T cells. Three different experimental systems were used to elucidate the mechanisms by which apoptotic cells regulate immune responses.

Using a classical system of tolerance induction, we examined the immunological consequence of intravenous (i.v.) delivery of ex vivo-generated naïve or activated apoptotic cells. Naïve apoptotic cells induced tolerance when injected i.v.; however, previously activated apoptotic cells induced immunity. Further analysis revealed a key role for CD154 in the tolerogenic or immunogenic nature of the naïve or activated apoptotic cells, respectively, as tolerance resulted after i.v. injection of either naïve or activated apoptotic CD154-/- cells, while co-injection of an agonistic anti-CD40 mAb with naïve apoptotic T cells induced robust immunity.

The infusion of large numbers of apoptotic cells has limited physiological relevance, so the investigation of the influence of apoptotic cells on the immune system turned to another experimental tolerance model where soluble peptide antigen is injected systemically to induce the peripheral deletion of a population of antigen-specific T cells. Using this system, we investigated how apoptotic cells generated in vivo leads to T cell tolerance. Following adoptive transfer of OT-II cells, wild-type mice injected with soluble OVA323-339 became unresponsive to subsequent CFA/OVA immunization. Interestingly, Trail-/- or Dr5-/- mice developed robust immunity; even though all strains displayed peripheral deletion of OVA-specific T cells. Subsequent investigation found the mechanism of action of the CD8+ T cells was TRAIL-mediated deletion of the OVA-responsive T cells in a TCR-specific manner.

The experimental systems used above have some clinical relevance but are still not physiologic. To study the impact of apoptotic cells in a physiologic setting, we took advantage of the medical condition sepsis, which is accompanied by massive apoptosis of multiple immune cell populations. Thus, the final set of experiments in this thesis examined the tolerance induced during sepsis using a clinically-relevant cecal-ligation and puncture (CLP) model that included a secondary bacterial infection. CLP-treated WT mice had a reduced ability to control the secondary bacterial infection, which was paralleled by suppressed T cell responses, compared to sham-treated WT mice. In contrast, CLP- and sham-treated Trail-/- and Dr5-/- mice were able to similarly control the bacterial infection and generated bacterial antigen-specific T cell responses. The ability of CLP-treated wild-type mice to control the secondary infection and generate T cell immunity could be restored by the administration of a blocking anti-TRAIL mAb. These results suggest the importance of TRAIL in the induction of sepsis-induced immune suppression, such that TRAIL neutralization may be a potential therapeutic target to restore cellular immunity in septic patients.

Keywords

Apoptosis, CD8+ T regulatory cells, Sepsis, Tolerance

Pages

ix, 185 pages

Bibliography

Includes bibliographical references (pages 165-185).

Copyright

Copyright 2011 Prajwal Gurung

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