| The department of Clinical Viro-Immunology is involved in the "Bolder project". This is a database to document studies done by the four institutes that collaborate as the Amsterdam Cohort Studies on HIV/AIDS. The information in this database can be accessed through the Internet: www.amsterdamcohortstudies.org/ 1. Mechanisms of CTL decline during the natural course of HIV-infection. In HIV-infected individuals, cytotoxic T lymphocytes (CTL) eventually decline and HIV-viral load increases ultimately leading to AIDS. We hypothesized that loss of CTL is due to physical depletion, dysfunction, or viral escape mutants. We evaluated CTL number by tetrameric HLA-peptide complexes, and assessed CTL function by antigenic stimulation. Viral epitopes were sequenced to detect escape variants. Epitope mutations were not prominently observed, absolute number of CTL usually were stable, but CTL function decreased in most patients progressing to AIDS. 2. Role of EBV and anti-EBV cellular immunity in the development of AIDS-related non-Hodgkin's lymphoma. Epstein-Barr virus (EBV) is a widespread persistent B cell tropic human herpesvirus, which is controlled by specific CD8+ T cells. In HIV-infected individuals reactivation of EBV-infection may lead to uncontrolled lymphoproliferation. These AIDS-related EBV-positive non-Hodgkin's lymphomas (AIDS-NHL) are thought to arise because of loss of EBV-specific T cell immunity. Our studies aim to unravel the mechanism by which EBV-specific T cells are lost in HIV-1 infection and find parameters that predict the onset of AIDS-NHL. Therefore, we study CD8+ T cell immunity to EBV in the course of HIV-1 infection using tetrameric HLA-EBV-peptide complexes, to directly stain EBV-specific CD8+ T cells, and INF-ELIspot assays, to assess functional capacity of these cells. As a possible predictor, EBV load is measured using a real time quantitative PCR. Since the role of CD4+ T cells in anti-tumor immunity seems to be increasingly important, future studies also focus on EBV-specific CD4+ T helper cells. The decrease in EBV-specific immunity might also result in superinfection with additional EBV-strains, which might contribute to the development of lymphomas. Therefore, we developed a type-specific PCR for direct EBV-typing in PBMC to determine the prevalence of both EBV-types 1 and 2 in groups of HIV-1 infected individuals and uninfected individuals. 3. T cell dynamics in HIV-1 infection. In human immunodeficiency virus (HIV)-1 infection, highly increased T cell turnover was proposed to cause exhaustion of lymphocyte production and subsequently development of AIDS. We have investigated this hypothesis by measuring T cell proliferation rates in HIV-1 infected individuals longitudinally, before and during treatment with highly active anti-retroviral therapy (HAART). T cell division rates were measured by intracellular detection of Ki67 antigen expression, which is a protein that is expressed exclusively by cells in cell cycle. Furthermore, studies are underway to examine T lymphocyte dynamics by labeling dividing cells in vivo with a stable isotope. Alternatively, HIV-1 could interfere with de novo production of T cells, by infection of bone marrow, thymus, or T cell progenitor cells. Recent thymic emigrants can be identified by T cell Receptor Excision Circles (TRECs) formed during T cell receptor rearrangement. To study the influence of HIV-1 on thymic function, we are currently measuring TRECs in purified naive CD4+ and naive CD8+ T cells in HIV-1 infected patients before and during treatment with HAART. 4. Detection of virus-specific CD8+ and CD4+ T cells. Tetrameric complexes of HLA class I molecules containing viral epitopes can be used to visualise antigen-specific CD8+ T cells. To allow the detection of virus-specific CD4+ T cells we are developing HLA class II tetramers. This involves finding the proper conditions for protein expression and purification, as well as defining relevant Th epitopes. We use class I tetramer technology in combination with intracellular cytokine staining to study the HIV-specific immune response that is restricted by HLA-B57, a protective HLA allele associated with long-term asymptomatic infection. In a new PhD project starting this year, cellular immunity against CMV in HIV infected individuals will be evaluated. |
