pkh-26 and HIV-Infections

We demonstrate that the FATAL cytolysis assay can be adapted into a rapid and fluorometric antibody-dependent cellular cytotoxicity assay (RFADCC). The RFADCC relies on double-staining target cells with a membrane dye (PKH-26) and a viability dye (CFSE) prior to the addition of antibody and effector cells. We used the RFADCC to assess dose-dependent and envelope-specific anti-human immunodeficiency virus (HIV) ADCC responses mediated by monoclonal antibody-2G12 and human sera. Using the assay, we also detected early anti-simian immunodeficiency virus (SIV) ADCC responses in rhesus macaques infected with pathogenic SIV(mac251). Importantly, the RFADCC was further useful in monitoring anti-HIV and anti-SIV ADCC responses elicited by immunizing chimpanzees and rhesus macaques with replicating adenovirus-based AIDS vaccine candidates. In comparison to the standard chromium release assay, the RFADCC provides a higher cell killing readout and is advantageous in allowing use of viably frozen as well as fresh effector cells, thus facilitating assay standardization. The RFADCC is therefore a simple, reliable, and highly sensitive method that can be applied to assess the ADCC activity of monoclonal antibodies as well as ADCC responses elicited by HIV or SIV infection or by AIDS vaccine candidates.

Topics: AIDS Vaccines; Animals; Antibodies, Monoclonal; Antibodies, Viral; Antibody-Dependent Cell Cytotoxicity; Chromium; Cytotoxicity Tests, Immunologic; Fluoresceins; Fluorescent Dyes; Fluorometry; HIV Infections; Humans; In Vitro Techniques; Macaca mulatta; Organic Chemicals; Pan troglodytes; Simian Acquired Immunodeficiency Syndrome; Succinimides

The 51Cr release assay has been the method of choice in analyzing natural killer cell (NK) function. Previous FCM cytotoxicity assays of NK activity have had numerous disadvantages that discouraged clinicians from attempting to evaluate NK function by flow cytometry. We demonstrate the effectiveness of using PKH-26, a stable membrane dye, to label the K562 target cells and propidium iodide intercalation into killed target cell DNA to determine the percentage of target cells killed by effector NK cells from the peripheral blood or bone marrow. This method compares favorably with the 51Cr release assay and is quicker and easier to perform. The percentage of cytotoxicity of NK cells (CD3- CD56+ and/or CD16+) from 10 normal subjects and 10 HIV-infected children are reported to demonstrate the feasibility of studying NK function in clinical populations by FCM. The potentiation of cytolysis by alpha-interferon and interleukin 2 in vitro was also compared between these two study groups. In addition, a patient whose leukemic blasts expressed CD56+ was also studied for NK activity using this flow cytometric assay. The benefits of using this flow cytometric approach to clinically assess NK function are discussed.

Topics: Adolescent; Adult; Child; Child, Preschool; Chromium Radioisotopes; Cytotoxicity Tests, Immunologic; Flow Cytometry; Fluorescent Dyes; HIV Infections; Humans; Immunophenotyping; Killer Cells, Natural; Leukemia, Myeloid, Acute; Organic Chemicals; Tumor Cells, Cultured