Analysis of CD8low T cell Defects in HIV Infection
Marc Schweneker, J. David Gladstone Institutes
Biomedical and Clinical Sciences
Background: Infection with the human immunodeficiency virus type 1 (HIV) usually results in progressive disease that cannot be controlled by the immune system. Recent studies have demonstrated that infection is associated with the development of CD3+CD4-CD8+T cells with low CD8 expression and impaired effector function. Preliminary results indicate that such"CD8low T cells"are defective in signaling across theT cell receptor (TCR) complex. Since these cells can comprise a large fraction of the CD8+T cell compartment in both non-human primates infected with the simian immunodeficiency virus (SIV) and in humans infected with HIV, their presence may in large part explain the ineffectiveness of the anti-viral immune response.
Methods: To better understand functional deficits we will assess signaling pathways in human CD8lowT cells. Conventional methods for the analyses of signaling pathways (e. g.,Western blots) are limited in their ability to simultaneously measure multiple cellular parameters (e. g., cell lineage, phenotype, cellular activation, and protein phosphorylation status) within complex populations of cells of the immune system. Therefore, we implement and optimize a method to study multiple cellular parameters by flow cytometry, which will allow rapid and efficient analyses of signal transduction pathways in combination with phenotypical characterization of single cells within heterogeneous cell populations derived from primary sources. Using this method, TCR-mediated signal transduction will be analyzed in human CD8low T cells generated in the HIV-infected SCID-hu Thy/Liv mouse and in CD8low T cells from HIV-infected patients in varying stages of disease progression.
Results or Expected Results: Different conditions for the flow cytometric-based method were tested and optimized because different fixation and permeabilization protocols have been shown to affect measurements of phosphorylation events and cell surface staining. Optimized conditions will now allow us to analyze defects of TCR-mediated signal transduction in human CD8low T cells generated in the HIV-infected SCIDhu Thy/Liv mouse and to determine whether similar signaling defects are present in CD8low T cells from HIV-infected patients in varying stages of disease progression.
Conclusion: Results of the experiments will enable us to identify altered signal transduction events of CD8low T cells and specifically correlate these with cellular dysfunctions in the context of HIV infection. Information obtained may also suggest strategies by which such CD8+ T cell dysfunction might be prevented or reversed during the course of HIV disease progression. Thus, we will not only be able to assign a more precise molecular mechanism to the observed phenomenon ofT cell dysfunction in HIV disease. We will hopefully be able to later use this information to suggest specific new therapies for HIV disease.