We report on the role of specific CD8(+) T cells in the pathogenesis of a highly lethal human viral disease, hantavirus pulmonary syndrome (HPS). HPS is a zoonotic disease caused by transmission of Sin Nombre virus (SNV) from chronically infected deer mice. In humans, this fulminant infection is characterized by lung capillary leakage, respiratory failure, and cardiogenic shock. Individuals with HLA-B*3501 have an increased risk of developing severe HPS, suggesting that CD8(+) T cell responses to SNV contribute to pathogenesis. We identified three CD8(+) T cell epitopes in SNV presented by HLA-B*3501 and quantitated circulating SNV-specific CD8(+) T cells in 11 acute HPS patients using HLA/peptide tetramers. We found significantly higher frequencies of SNV-specific T cells in patients with severe HPS requiring mechanical ventilation (up to 44.2% of CD8(+) T cells) than in moderately ill HPS patients hospitalized but not requiring mechanical ventilation (up to 9.8% of CD8(+) T cells). These results imply that virus-specific CD8(+) T cells contribute to HPS disease outcome. Intense CD8(+) T cell responses to SNV may be induced by the encounter of the unnatural human host to this zoonotic virus without coevolution. This may also be the immunopathologic basis of other life-threatening human virus infections.

The focus of this dissertation is the role of specific CD8+ T cells in the pathogenesis of a highly lethal human viral disease, hantavirus pulmonary syndrome (HPS). HPS is a zoonotic disease caused by transmission of Sin Nombre virus (SNV) from chronically infected deer mice. In humans, this fulminant infection is characterized by lung capillary leakage, respiratory failure and cardiogenic shock. Individuals with HLA-B*3501 have an increased risk of developing severe HPS, and the majority of defined CD8+ T cell epitopes in SNV are presented by this HLA allele, suggesting that CD8+ T cell responses to SNV contribute to pathogenesis. We speculate that CD8+ T cell mediated immune responses to SNV antigens in pulmonary endothelial cells contribute to the pathology of HPS. Specifically, we hypothesize that there are quantitative and/or qualitative differences in SNV-specific CD8+ T cell responses in HPS patients with moderate vs. severe disease. In this dissertation I measured the frequencies of SNV-specific CD8+ T cells during acute HPS. Using HLA/peptide tetramers, I quantitated circulating SNV-specific CD8+ T cells of all the available HLA-B35+ patients with HPS caused by SNV. This is the first time hantavirus-specific T cells have been quantitated during acute infection. I report that between 2.9% and 44.2% of the CD8+ T cells were specific for the three SNV epitopes in combination during acute disease in the patients analyzed in this study. These levels are very high in comparison to the frequencies reported in the literature for other acute human viral infections. Furthermore, I report significantly higher frequencies of SNV-specific T cells in patients with severe HPS requiring mechanical ventilation (up to 44.2% of CD8+ T cells) than in moderately ill HPS patients hospitalized but not requiring mechanical ventilation (up to 9.8% of CD8+ T cells). These results imply that virus-specific CD8+ T cells contribute to HPS disease outcome. In this dissertation I also provide preliminary data on qualitative aspects of SNV-specific T cells. Analysis of the TCR repertoire of SNV-specific T cell lines isolated from the PBMC of acute HPS patients raises the possibility that SNV-specific T cells express a limited number of TCR Vβ alleles; however, this is quite speculative because it is based on the analysis of only seven CTL lines. Analysis of cytokine expression by the CTL lines in response to in vitro antigen-specific stimulation indicate that SNV-specific T cells are capable of secreting IFN-γ, TNF-α, and IL-13 upon stimulation. The data presented in this dissertation extend previous studies, which suggested a role for virus-specific T cells in HPS pathogenesis and support our hypothesis that virus-specific CD8+ T cells contribute to HPS disease outcome. The results of this study will be useful in the design of future therapeutic strategies for this emerging human pathogen. The conclusions of this study may also benefit the study of other human viral hemorrhagic fevers. Improved understanding of the mechanism of pathogenesis of severe viral zoonoses will result in better treatment and prevention strategies.

Immunization with vaccinia virus resulted in long-lasting protection against smallpox and was the approach used to eliminate natural smallpox infections worldwide. Due to the concern about the potential use of smallpox virus as a bioweapon, smallpox vaccination is currently being reintroduced. Severe complications from vaccination were associated with congenital or acquired T cell deficiencies, but not with congenital agammaglobulinemia, suggesting the importance of T cell immunity in recovery from infection. In this report, we identified two CD8+ T cell epitopes restricted by the most common human major histocompatibility complex (MHC) class I allele, HLA-A*0201. Both epitopes are highly conserved in vaccinia and variola viruses. The frequency of vaccinia-specific CD8+ T cell responses to these epitopes measured by interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) assay and HLA/peptide tetramer staining peaked 2 wk after primary immunization and then declined, but were still detectable 1 to 3 yr after primary immunization. 2 wk after immunization, IFN-gamma-producing cells specific to these two epitopes were 14% of total vaccinia virus-specific IFN-gamma-producing cells in one donor, 35% in the second donor, and 6% in the third donor. This information will be useful for studies of human T cell memory and for the design and analyses of the immunogenicity of experimental vaccinia vaccines.

Fas and the tumor necrosis factor receptor (TNFR)1 regulate the programmed cell death of lymphocytes. The death domain kinase, receptor interacting protein (rip), is recruited to the TNFR1 upon receptor activation. In vitro, rip-/- fibroblasts are sensitive to TNF-induced cell death due to an impaired nuclear factor kappaB response. Because rip-/- mice die at birth, we were unable to examine the effects of a targeted rip mutation on lymphocyte survival. To address the contribution of RIP to immune homeostasis, we examined lethally irradiated mice reconstituted with rip-/- hematopoietic precursors. We observed a decrease in rip-/- thymocytes and T cells in both wild-type C57BL/6 and recombination activating gene 1-/- irradiated hosts. In contrast, the B cell and myeloid lineages are unaffected by the absence of rip. Thus, the death domain kinase rip is required for T cell development. Unlike Fas-associated death domain, rip does not regulate T cell proliferation, as rip-/- T cells respond to polyclonal activators. However, rip-deficient mice contain few viable CD4+ and CD8+ thymocytes, and rip-/- thymocytes are sensitive to TNF-induced cell death. Surprisingly, the rip-associated thymocyte apoptosis was not rescued by the absence of TNFR1, but appears to be rescued by an absence of TNFR2. Taken together, this study implicates RIP and TNFR2 in thymocyte survival.