Analysis of novel phospho-ITAM specific antibodies in a S2 reconstitution system for TCR–CD3 signalling

Abstract

The T cell antigen receptor (TCR–CD3) complex contains 12 different cytoplasmic tyrosines, each of which is part of an immunoreceptor tyrosine-based activation motif and thus occurs in similar sequence context. Since phosphorylation of individual tyrosines can be correlated with the quality of the T cell response, monitoring their phosphorylation is important. We thus generated novel antibodies against phospho-tyrosines of the TCR–CD3 complex and tested the specificity in a synthetic biology approach. We utilized the Drosophila S2 reconstitution system testing several kinases and stimulation conditions that lead to optimal phosphorylation of the TCR–CD3 subunit ζ. Expressing TCR–CD3 subunits and tyrosine mutants thereof we tested the specificity of the novel antibodies in Western blot and immunopurification experiments. In particular, we generated and characterized the monoclonal antibody EM-26 that specifically recognizes phosphorylation of the membrane proximal tyrosine of ζ (phospho-ζY1) and antisera raised against the first and the second phospho-tyrosine of CD3ɛ (phospho-ɛY1 and phospho-ɛY2).