Technical Reference #3209

3209.

A requirement for Fc-gamma-R in antibodymediated bacterial toxin neutralization Nareen Abboud; Siu-Kei Chow; Carolyn Saylor; Alena Janda; Jeffery V. Ravetch; Matthew D. Scharff; and Arturo Casadevall, Albert Einstein College of Medicine, Journal of Experimental Medicine, 207(3209), (2010)
Link To Paper

Abstract:
One important function of humoral immunity is toxin neutralization. The current view posits that neutralization results from antibody-mediated interference with the binding of toxins to their targets a phenomenon viewed as dependent only on antibody specificity. To investigate the role of antibody constant region function in toxin neutralization we generated IgG2a and IgG2b variants of the Bacillus anthracis protective antigen–binding IgG1 monoclonal antibody (mAb) 19D9. These antibodies express identical variable regions and display the same specificity. The efficacy of antibody-mediated neutralization was IgG2a > IgG2b > IgG1 and neutralization activity required competent Fc receptor (FcR). The IgG2a mAb prevented lethal toxin cell killing and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase cleavage more efficiently than the IgG1 mAb. Passive immunization with IgG1 and IgG2a mAb protected wild-type mice but not FcR-deficient mice against B. anthracis infection. These results establish that constant region isotype influences toxin neutralization efficacy of certain antibodies through a mechanism that requires engagement of FcR. These findings highlight a new parameter for evaluating vaccine responses and the possibility of harnessing optimal FcR interactions in the design of passive immunization strategies.

Materials & Methods:
PA and IgG internalization assays. BMMs from wild-type and FcR// RIIB/ mice were plated on MatTek plates labeled with serum-free medium containing 25 μg/ml Alex Fluor 488–conjugated IgG2a and Alexa Fluor 568–conjugated PA and incubated at 4°C for 2 h to enable binding. The cells were washed warmed to 37°C for varying amounts of time washed and then fixed with 2% paraformaldehyde in PBS for 10 min at room temperature. The cells were subsequently imaged by epifluorescence microscopy on an inverted microscope (Axioskop 200; Carl Zeiss Inc.) equipped with a cooled charge-coupled device using a 63× 1.4-NA objective with a 1.6× optovar. Images were acquired using the same exposure times and microscopic settings and processed by AxioVision software (version 4.6; Carl Zeiss Inc.).

Microscopic Technique
epifluorescence microscopy

Cell Type(s)
BM-derived macrophage