Student Research: Michael Rosato

MS, Toxicology (Tox), 1999
Faculty Advisor: Elaine M. Faustman

Multiparameter Analysis of Surface Receptor Expression and Cell Cycle Progression in Peripheral Blood Lymphocytes



Specific immune responses are characterized by activation of the appropriate immunocompetent cells, and their proliferation and differentiation into effector cells that function in eliminating foreign antigens. In vitro lymphocyte proliferation is often used in examining cell-mediated responses to various stimuli as well as an endpoint in assessing functional activity of cells in immunotoxicity assays [1-5]. Furthermore, combining analysis of proliferation with simultaneous identification of cellular subsets is a powerful approach that allows for a more thorough examination of the cellular activity within a cell population in vitro in response to treatment [1, 6-9].

Lymphocyte proliferation has traditionally been examined by measuring incorporation of tritiated 3[H] thymidine into DNA, but this method has several limitations. Most notably, this approach does not allow for examination of proliferation at the single cell level and is generally unsuitable for multiparameter analysis [2]. Alternatively, flow cytometry allows examination of highly heterogeneous cell populations at the single cell level and is a useful instrument in studying the cell-mediated immune [7, 10].

Flow cytometric methods have been developed that allow simultaneous detection of cell-surface antigens and proliferation in stimulated lymphocytes. However, the usefulness of these methods is compromised by their limited analysis of cell proliferation. Flow cytometric examination of cell proliferation has commonly been approached by DNA staining with a dye such as a ethidium bromide (EB), propidium iodide (PH), Hoechst 33258 or 33342, or 7-Aminoactinomycin D (7-AAD) [2, 6, 7, 9]. These dyes stoichometrically bind DNA and their fluorescence is proportional to DNA content, allowing identification of the G0/1, S and G2 phases of the cell cycle. Alternatively, stimulated cells are pulse labeled with the thymidine analogue bromodeoxyuridine (BrdU) and detection of analog incorporation with an Anti-BrdU antibody allows identification of cells actively undergoing DNA synthesis [1, 2, 7, 11]. However, in using these methods it is difficult to follow the proliferative history of cells through multiple cell cycles.

An improved method for proliferation analysis involves continuous labeling with BrdU and detection of analog uptake by an indirect method involving the BrdU-mediated stoichiometric quenching of the DNA intercalating dye Hoechst 33258 [10, 12-14]. In this approach, proliferating cells that have incorporated BrdU can be identified by their decreased Hoechst fluorescence when compared to non-proliferating cells, and the fluorescence intensity provides information at the single cell level regarding the number of mitotic cycles completed during the culture period. The position of the cells within the cell cycle can be identified by counter staining with a second DNA dye (e.g. 7-ADD, PI, EB) whose fluorescence is unaffected by BrdU. Thus, this “BrdU-Hoechst” assay provides detailed information about the proliferative history of cells and their position within the cell cycle. This assay is commonly used in examining proliferative alone but has been combined with additional parameters such ass expression of nuclear proteins for multiparameter analysis [15]. By modifiying the existing BrdU-Hoechst assay, we combined this assay together with cell surface immunolabeling with monoclonal antibodies to cell surface receptors for simultaneous analysis of proliferation and cell phenotype.

Lymphocyte subsets examined included CD19+B cells, and CD3+T cells and their CD4+ and CD8+ subsets. Furthermore, T helper (CD4+) cells are divided into functionally distinct Th1 and Th2 subpopulations that are most often distinguished based on their cytokine profiles. In general, Th1 cells produce IL-2, TNF-alpha, and INF-gamma and are thought to be involved in phagocyte-dependent host response; whereas Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13 and are involved in reactions primarily mediated by immunoglobulin E, eosinophils, and basophils [16]. Recent evidence suggests that expression of the chemokine receptor CCR5 among CD4+ cells is associated with the Th1 phenotype [17-21]. Therefore, examination of CCR5 expression was included in our analysis.

We describe here a method combing the BrdU-Hoechst assay with examination of cell surface receptor expression for multiparameter analysis of proliferation and cell phenotype in peripheral blood lymphocytes. Furthermore, the utility of this assay is demonstrated by examining immunophenotype specific proliferation among cells stimulated with tetanus toxoid or IL-2. We demonstrate that this method allows both examination of proliferation among different subsets of cells and comparison of immunophenotype composition among proliferating and non-proliferating cells.