Presence of the CC-chemokine co-receptors CCR5 on the cell surface of CD4+ T-lymphocytes is necessary for successful HIV-1 infection. However, the interaction of CCR5 by its ligand CCL3L1 provides strong counteraction against HIV-1 infection. Increasing evidence suggests that there is a genetic variability of the CCL3L1 gene copy number among different individuals and racial populations. High genomic copy number of CCL3L1 and its genetic interaction with CCR5 are associated with a reduced susceptibility to HIV-1 and slow progression to AIDS. This finding raises a possible strategy with easy, sensitive, and accurate quantification of CCR5 and CCL3L1 molecules on the surface of CD4+ T-lymphocytes that may provide a diagnostic marker for assessing individual risk or susceptibility to HIV-1 infection. In addition, it may also provide a prognostic tool to predict disease progression of HIV-infected patients and a possible surrogate marker for testing efficacy of future CCR5-based anti-HIV therapies. Objective of this proposal is to develop a new strategy about the single cell imaging using fluorescent metal nanoparticles as probes to investigate the number, distribution, and interaction of CCR5 and CCL3L1 molecules on the surfaces of CD4+ T-lymphocytes. Specific monoclonal antibodies (mAb) to CCR5 or CCL3L1 will be conjugated on the fluorescent metal nanoparticles or first fluorescently labeled and then bound on the metal nanoparticles without fluorophores. The mAb- bound metal particles, which are known to have stronger emission, different lifetime from the cellular autofluorescence, better photostability, and less photoblinking, will be used as fluorescence imaging reagents to immuno-conjugate with the target CCR5 and/or CCL3L1 on the surfaces of CD4+ T- lymphocytes. The cell fluorescence images will be recorded by confocal microscopy, and the number and distribution of CCR5 or CCL3L1 on the cell surfaces will be estimated from the emission intensity or lifetime over the cell images at single cell level. A reasonable statistic approach will be developed to analyze the empirical data. The results will be calibrated with the genomic copy number method, flow cytometry, and immunometal electron microscopy images, as well as the fluorescent images of synthetic beads bound with known amounts of CCR5 and CCL3L1 on surfaces. The interaction of CCR5 and CCL3L1 will be investigated. The established fluorescence images approach will be used to study the CCR5 and CCL3L1 molecules on the surfaces of CD+ T-lymphocytes isolated from the fast and slow disease progressing HIV-infected patients, and furthermore allow us to evaluate individual susceptibility to HIV-1 infection and correlate disease progression of HIV-infected patients.
Presence of the CC-chemokine co-receptors CCR5 on the cell surface of CD4+ T-lymphocytes is necessary for successful HIV-1 infection. The protective interaction of CCR5 by its ligand CCL3L1 counteracts viral invasion. Increasing evidence suggests that copy numbers of CCR5 and CCL3L1 are associated with individual susceptibility to HIV-1 infection and disease progression of HIV- infected patients. Objective of this proposal is to develop a novel approach to quantify and map the target molecules of CCR5 and CCL3L1 on the cell surface by the fluorescence image of single cell using metal plasmon-coupled probes (PCPs). Specific monoclonal antibodies (mAb) to CCR5 and/or CCL3L1 will be conjugated on the labeled metal particles or labeled with the fluorophores before binding to the metal particles. The mAb-metal particles will be bound with the CCR5 or/and CCL3L1 on the surfaces of CD4+ T-lymphocytes. Confocal microscopy will be utilized to record fluorescence intensity and lifetime images of the labeled cells. The amount of the CCR5 or CCL3L1 molecules on the cell surfaces will be detected from the cell images at single cell level. Quantification of CCR5 and CCL3L1 will be calibrated by using an analytic simulation model to ensure accuracy and further validated using established CCR5-positive cell lines with known copy numbers of CCR5. The validated method will be compared with other commonly used methods such as flow cytometric analysis and applied to measure the molecular level of CCR5 and its interaction with CCL3L1 on HIV-infected CD4+ T-lymphocytes isolated from the established fast and slow disease progressing patients.
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