Figure 5.

TmTNF-dependent vesicular trafficking of humanized anti-TNFs and detection of cell surface anti-TNF peptides in dendritic cells. (A) Compartmentalization of anti-TNF: Day 5 human CD14+ monocyte-derived DCs were treated with LPS for 2 h and the cells ‘pulsed’ on ice using Alexa 488-conjugated anti-TNF (0 min, ice). The fluorescent label on the anti-TNF was ‘chased’ for up to 1 h at 37°C. Endosomes and lysosomes were stained with Alexa 647-conjuated anti-EEA1 and anti-LAMP1, respectively. Nucleus was stained with DAPI. Images were acquired using confocal microscopy. (B) Identification of cell surface-associated anti-TNF peptides: Day 5 human CD14+ monocyte-derived DCs were treated with LPS for 2 h and incubated with either an anti-TNF mAb or a DVD-Ig containing one anti-TNF domain for 6 h. Cell surface displayed peptides were eluted under mild acidic conditions and the identity of anti-TNF peptides was obtained using LC-MS/MS. (C) Identification of HLA-DR-associated anti-TNF peptides: Day 5 human CD14+ monocyte-derived DCs were treated with LPS for 2 h and incubated with anti-TNF for up to 8 h. HLA-DR was immunoprecipitated and the identity of HLA-DR-associated anti-TNF peptides was obtained using LC-MS/MS (underlined: linker peptide sequence of anti-TNF DVD-Ig).