David W. Houck

CD4+CD8dim T lymphocytes exhibit enhanced cytokine expression, proliferation and cytotoxic activity in response to HCMV and HIV‐1 antigens

CD4+CD8dim T cells represent a minor subset of the total CD3+ T cell population in peripheral blood. Although transient and persistent expansions of these cells havebeen reported in both healthy and diseased individuals, the functional properties of the CD4+CD8dim population are largely unknown. In this study, we examined antigen‐specific cytokine and proliferative responses of the CD4+CD8dim subset. In whole blood cultures stimulated with the viral antigens HCMV and HIV‐1, a significant fraction of the CD4+CD8dim subset exhibited cytokine expression and proliferation in response to antigen activation. Typically, the CD4+CD8dim population contained two‐ to eightfold higher frequencies of antigen‐specific cytokine producing cells than the CD4+CD8‐ population. Phenotypic analysis of the cytokine expressing CD4+CD8dim population indicated that these cells are memory T cells, with a high frequency of this population expressing the cytotoxic markers CD56 and perforin. Furthermore, the CD4+CD8dim cytokine responses to CMV were shown to be MHC class II dependent. Significantly, purified CD4+CD8dim T cells were found to possess higher CMV‐specific cytotoxic activity than purified CD4+CD8– T cells in a standard 51Cr‐release CTL assay. Thus, CD4+CD8dim T cells appear to be MHC class II dependent, are capable of cytolytic effector activity, and are highly enriched within the CD4+ cell populations specific for HCMV and HIV‐1.

SLIT-SCAN FLOW CYTOMETRY FOR CONSISTENT HIGH RESOLUTION DNA ANALYSIS OF X-AND Y-CHROMOSOME BEARING SPERM

This paper describes the application of slit-scan flow cytometry for accurate DNA analysis of X- and Y-chromosome bearing sperm. The introduction of the slit-scanning technique was initiated to improve the consistency in resolution of the X and Y population from donor to donor. An optimal resolution is essential for high purity sorting of X and Y sperm, as the difference in DNA content is small (3-4%) in most mammals. This difference is the discriminatory parameter for the flow cytometric sorting of the two populations. Our approach was to focus on the role of the sperm tail in the detection process. Slit-scan flow cytometric analysis allows the whole sperm to be spatially analyzed along the direction of flow. Sperm were stained with Dansyl Lysine, a UV excitable fluorescent membrane dye, which stained the head, midpiece, and principal piece. Analysis of these stained sperm showed that there was no difference between the relative number of sperm that travel headfirst or tailfirst through the detection zone of the flow cytometer. The influence of sperm with coiled tails on DNA analysis was also investigated. The proportion of sperm with coiled tails influences semen quality. The standard X-Y separation procedure uses Hoechst 33342, which stains all intact sperm, both living and dead. Propidium iodide was added to discriminate the dead sperm population. Slit-scan analysis showed that measurement of a sample containing a high proportion of living sperm with coiled tails results in an inferior DNA histogram and reduced X-Y resolution. Sperm with coiled tails can result in a lower detected fluorescence intensity, but the reason for this is unclear. Slit-scan flow cytometry allows exclusion of sperm with coiled tails from the analysis, resulting in a restoration of high resolution of X- and Y-chromosome bearing sperm populations.

Light scattered at two wavelengths can discriminate viable lymphoid cell populations on a fluorescence-activated cell sorter.

Erythrocytes could be distinguished from nucleated cells by both uv and 488 nm light scattering. By correlating the two colors of light scattering a third population of bone marrow cells, predominantly large lymphocytes, was identified. Among splenic lymphocytes, T cells could be distinguished from B cells based on the differences in scattering of these two wavelengths. These data indicate that light scattering patterns change drastically when different colors of light are used to analyze the population. In addition, the correlation of light scattered at two different � wavelengths can aid in distinguishing populations of cells � that could not be identified by a single wavelength of