Helper T (Th) cells are critical regulators of adaptive immunity. The cytokines produced by Th cells are powerful bioactive agents that control the outcome of an immnune response to most foreign protein antigens. Cytokine production occurs in complex waves that serve to regulate antigen-specific clonal expansion and subsequent differentiation of effector and memory lymphocytes in vivo. It is our hypothesis that development of sensitive miniaturized sensors able to detect and quantify multiple molecular species in real-time would significantly impact our fundamental understanding of immune regulation. The proposed study will focus on antigen-specific Th cell-dependent B cell development. A view of cytokine-regulated antibody production will be developed that is quantitative and provides a kinetic dimension to the production of multiple cytokines (IL2, IL-4, IL-5, IL-6, IL-lO, IFN-y and TNF-a) and subsequent secretion of multiple immunoglobulin (Ig) isotypes (IgM, IgGi, IgG2a, IgG2b, IgG3, IgE and IgA). The main objective is development of sensitive biosensors able to detect minute quantities of multiple cytokines over time. The first specific aim is proof of concept of an innovative application of capacitance-based biosensing in which microfabricated field effect transistors are modified with specific receptors. This proposed device amplifies the change in gate double-layer capacitance that occurs upon ligand binding - resulting in sensitivity at least three orders of magnitude greater than existing assays while generating a signal that is independent of the sensor area (allowing miniaturization and arrays). The second specific aim is evaluation of sensitivity, specificity and dynamic range of the proposed sensor for individual cytokine and Ig components described above using purified protein sources and timed addition in vitro. The production of these cellular products from defined populations of Th cells and B cells in vitro will then be quantified. Finally, multiple sensors will be fabricated in arrays to allow simultaneous detection of different species and admix purified proteins or mixtures of cells to evaluate the fidelity of measurement in more complex environments. The third specific aim is analysis of the more complex process of cytokine-regulated antibody production using a multiple array sensor in vitro.
