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Chapter 100 : Investigation of Signal Transduction Defects
This chapter discusses antigen-receptor signal transduction pathways for both T cells and B cells and describes methods to evaluate these pathways in children with inherited immune deficiencies. ZAP-70 deficiency was the first described T-cell receptor (TCR)-associated protein tyrosine kinases (PTKs) defect in humans. Moreover, intracellular cytokine expression using fluorescence-activated cell sorter (FACS) analysis may be useful in characterizing the scope of T-cell defects. Fluorimetric methods require more cells but do not require concomitant cell staining with potentially stimulatory MAbs. This assay is best done using fresh peripheral blood mononuclear cells (PBMC), but bone marrow-derived lymphocytes, B-cell lines (BCL), or T-cell lines (TCL) rested overnight in medium without IL-2 or mitogen may also be used. Distal TCR signal transduction events, as well as the interleukin 2 (IL-2) pathway, may be analyzed by measurement of IL-2 production in response to TCR-mediated stimuli. In vitro T-cell proliferative studies are useful in the screening of immunodeficient patients for proximal signal transduction defects. TCL and BCL may be the only source of patient cells; therefore, analysis of signal transduction defects using transformed lines may be complicated by the effects of HTLV-1 and EBV, and possibly HVS, on lymphocyte signaling pathways. The described methods in this chapter are valuable both to screen and to diagnose signaling defects in patients with inherited T- or B-cell immune deficiencies. Signaling pathways resulting in abnormal T- or B-cell activation and development of immune deficiency are the focus of much investigation at present.