LIMITING DILUTION ANALYSIS OF ANTIGEN-STIMULATED IL-4 AND IFN-GAMMA PRODUCTION IN HUMAN MONONUCLEAR CELL-POPULATIONS

Limiting dilution analysis (LDA) of fresh human mononuclear cell popul ations has previously been used to estimate the frequency of specific B cells, CTL, proliferative T cells, or cells capable of IL-2 producti on in various clinical situations. Such approaches evaluate the intens ity of the response but provide little information concerning the bala nce between Th1-vs. Th2-like patterns of cytokine gene expression. Her e, we describe development of an LDA method to obtain quantitative est imates of the frequency of antigen-specific IFN-gamma or IL-4 producin g cells in human peripheral blood. The approach utilizes 3-4 day antig en-mediated restimulation of mononuclear cell populations freshly deri ved from grass pollen sensitive allergic rhinitis subjects. IFN-gamma and IL-4 production in culture supernatants are determined by ELISA an d CT.h4S bioassay. Cytokine production elicited in this assay is CD4 d ependent and antigen specific. As such, it provides a useful non-invas ive approach for rapid evaluation of low frequency, antigen-induced cy tokine production in the circulating repertoire. This method can readi ly be extended to analysis of other cytokines in other immunologic dis orders or in infectious disease states, allowing longitudinal analysis of individuals and facilitating efforts to establish clear correlatio ns between in vivo patterns of cytokine gene expression and disease ex acerbation and remission.