Simultaneous cytometric analysis for the expression of cytoplasmic and surface antigens in activated T cells

Abstract

A method of two-colour immunofluorescence staining has been developed to allow the simultaneous analysis of both surface and cytoplasmic antigens. This involves the use of direct fluorochrome antibody conjugates for cell-surface antigen staining, followed by cell permeabilization and the staining of cytoplasmic antigens with biotinylated antibodies and streptavidin-fluorochrome conjugates.Fluorochrome-antibody conjugates bound to cell-surface epitopes were found not to be affected by the subsequent permeabilisation and cytoplasmic staining. This method was used to examine the surface phenotype of T cells expressing a cytoplasmic antigen, STA.STA is a unique determinant detected in activated human T cells by the monoclonal antibody K-1-21, which also recognizes a crossreactive conformation-dependent epitope on human free kappa light chains. Cytometric analysis showed that STA is found in both Leu 2a+ cytotoxidsuppressor T cells and Leu 3a+ helpedinducer T cells but is not induced in the Leu 15" population which contains suppressor T cells. STA was also shown to be an activation antigen in murine T cells. Key terms: Intracytoplasmic and surface immunofluorescence, flow cytometry analysisIn recent years several studies have shown that intracellular antigens can be readily investigated by flow cytometry. Highly specific staining has been achieved for cytoskeletal structures such as intermediate filaments (171, for intranuclear proteins such as simian virus 40 T antigen (7) and cytoplasmic immunoglobulin (5, 17,221. Situations may arise when it would be advantageous to be able t o simultaneously examine both surface and cytoplasmic cell antigens. One example would be to establish the relationship between a cytoplasmic antigen and the subset derivation of a given lymphocyte population which is largely defined by the surface expression of characteristic epitopes (8). Previous approaches to determine the existence of such relationships have involved the separation of cell subpopulations by either panning on antibody-coated petri dishes (21) or flow cytometric sorting before analysis for cytoplasmic antigen expression. However, the length of time required and the physical nature of such cellular separation techniques may result in a decrease in the number of viable cells and cannot guarantee discrete subset separations. For these reasons a two-colour immunofluorescence staining method was developed to allow the simultaneous analysis of both surface and cytoplasmic cellular antigens.Previously, we reported the detection of a unique cytoplasmic T cell activation protein, Sezary T cell antigen (STA), using flow cytometry (20). STA is a high molecular weight protein (> 200 kilodaltons unreduced) which is detected with a monoclonal antibody, K-1-21. Interestingly, K-1-21 also reacts with a structurally distinct conformational epitope expressed in the constant domain of free, but not heavy chain-associated, human kappa light chains (15) by negative selection panning (21) before cytometric analysis for surface antigens or for...

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