IDENTIFICATION OF FUNCTIONAL SUBSETS BY FLOW-CYTOMETRY - INTRACELLULAR DETECTION OF CYTOKINE EXPRESSION

Methods for analysis of T cell function have traditionally relied upon measurements of proliferation or cytokine expression in hulk cultures of PBMC in long term incubations with polyclonal mitogens or putative antigen. These techniques suffer from the drawback that they do not e nable analysis of single cell responses in the context of unselected c ellular backgrounds. In addition these methods are not sensitive enoug h to rapidly assess rare event responses characteristic of cognate mem ory T cell responses. This review discusses recently developed flow cy tometric methods designed to rapidly assess leukocyte subset cytokine responses to polyclonal activators and specific antigen in PBMC and wh ole blood samples. These procedures determine the percentages of activ ated cells and the identification of leucocyte subsets capable of expr essing various cytokines and cell surface antigens. The ability to ass ess key intracellular functional markers by multiparameter flow cytome try offers some unique advantages in a number of clinical applications . The technical simplicity and rapidity of the flow cytometric intrace llular cytokine detection techniques described in this report, as well as the widespread availability of appropriate flow cytometers and cel l surface directed antibodies in clinical laboratories, suggests the p ossibility that this technique could be broadly applicable to the clin ical evaluation of immune status. Since any cell type can be identifie d with this approach, responses to a variety of clinically relevant st imuli in virtually any leukocyte subset can be evaluated including mon ocyte responses to LPS, and T cell responses to mitogens and a variety of bacterial and viral antigens, The significance of measuring low fr equency antigen-specific responses with respect to clinical significan ce in assessing immune status in a variety of clinical conditions and determining efficacy or immunotoxicity of drugs and vaccine antigens i s discussed. (C) 1998 Wiley-Liss, Inc.