Use of Toll-Like Receptor Chimeras to Dissect Mechanisms of Receptor Localization and Signaling

Retroviruses ..................................................................................... 119 7.3.4 Determination of Infection Efficiency by Flow Cytometry ............ 120

7.4 Characterization of TLRs by Using TLR Chimeras ..................................... 121 7.4.1 Examination of the Subcellular Localization of TLRs by Flow

Cytometry ........................................................................................ 121 7.4.2 Examination of the Subcellular Localization of TLRs by

Microscopy ......................................................................................124 7.4.3 Determination of the Intracellular Targeting Elements of TLRs

by Random Mutagenesis Approach .................................................124 7.4.4 Examination of the Signaling Properties of TLRs ......................... 127

References .............................................................................................................. 129

Immune protection in mammals results from the combination of innate immune recognition molecules that provide genome-encoded sensing of conserved components of infectious organisms1 with the somatic recombination-generated antigen recognition molecules of lymphocytes that recognize individualized features of particular infecting organisms. Among the molecules that provide hardwired recognition of infection by bacteria, fungi, parasites, and viruses, the Toll-like receptors (TLRs) are perhaps the most important for immune function. They are present in the three types of immune cells resident in tissues-the macrophage, the immature dendritic cell, and the mast cell-all of which act as sentinels that respond to recognition of infection by TLRs by inducing inflammation and/or taking antigen to the draining lymph node to initiate an adaptive immune response. TLRs are also present in a variety of other cell types, including B lymphocytes, endothelial cells, and epithelial cells, the latter two of which may also contribute to initial recognition of infection in some circumstances.