Immunology

Flow cytometry has been "instrumental" for many of the advances in Immunology over the last 20 years.

The coupling of monoclonal antibody technology with flow cytometry resulted in characterization of cells based on cell surface expression (see website: http://gryphon.jr2.ox.ac.uk -- for latest Leukocyte Differentiation Antigen Workshop information). Because many of the antibodies identify cells with important functions or cells of distinct lineages, immune cells can be analyzed for distribution, important in the case of immunophenotyping in HIV (1-5). In addition, lymphocytes can be sorted into subsets based on cell surface characteristics and then tested for immune function (6,7).

More recent flow cytometric assays, which have revolutionized our understanding of antigen specific immunity and memory development, include assays for intracellular cytokine production after stimulation in vitro and direct measurement of antigen specific cells using tetramers (8-10). Cell tracking dyes, such as CFSE, can be used to follow dividing cells (11). This coupled with functional analysis and cell surface characterization has the opportunity to significantly advance the study of immunity in the future.

Functional characteristics of activated lymphocytes. Lymphocytes in the histograms to the right were labeled with 1.5 mm of CFSE for 10 mins at 37°C, washed and then cultured for 3 days with or without a stimulating signal, the mitogen PHA.
The unstimulated cells remain uniformly labeled (left panel), whereas the stimulated lymphocytes (right panel) are distributed into 4 peaks of fluorescence representing the number of times the cells have divided. The cells in the first peak on the right have not divided (0), the cells labeled "1" have divided once, the cells labeled "2" have divided twice and the cells labeled "3" have divided 3 times since stimulation.
Four days after stimulation with PHA, lymphocytes were examined for both CFSE and the ability to make IFN-g after restimulation in vitro.
Cells in the left panel were not initially stimulated and hence do not divide or produce IFN-g. However, cells in the right panel have undergone cell divisions and produce IFN-g upon restimulation. Note that the cells that have divided the most (lost the most CFSE) make the most IFN-g. This technique can, therefore, be used to study functional attributes of dividing lymphocytes, which is important in the study of immune response disorders and autoimmunity.