Fig 2: TmTNF-dependent uptake of humanized anti-TNF mAb and generation of a memory T cell recall response by dendritic cells. (A) Scheme of generating anti-TNF-TT fusion IgGs: Sequence of tetanus toxin (TT) peptides (S1 or S2) were fused to the C-terminus of the heavy chains of an anti-TNF to obtain anti-TNF-S1 or anti-TNF-S2 antibodies. (B) Testing the potency of anti-TNF-TT fusion IgGs: The potency of the parental anti-TNF and anti-TT-fusion IgGs (anti-TNF-S1 and anti-TNF-S2) to neutralize soluble TNF were tested using the L929 assay. (C) Measuring T cell recall response in DCs: Day 5 human CD14+ monocyte-derived DCs were treated with LPS for 2 h and incubated either with tetanus toxin (TT antigen), anti-TNF-S1 or anti-TNF-S2. DCs were co-cultured with CFSE-labeled autologous T cells, and T cell proliferation assessed using flow cytometry.

Fig 3: Kinetics of TNF expression in human monocytes and macrophages post LPS treatment. Human CD14+ monocytes (A) or day 7 human CD14+ monocyte-derived macrophages (B) were either untreated or treated with LPS in the presence or absence of TACE inhibitor, TAPI-2, for the indicated periods of time. Cell surface proteins were labeled with cell-impermeable Sulfo-NHS-SS-biotin. Biotinylated surface proteins were precipitated with streptavidin-conjugated agarose beads. Cell surface biotinylated and total proteins were subjected to immunoblotting using anti-TNF IgG. Na, K-ATPase and GAPDH protein expressions were used as total protein loading and cell surface biotinylation controls. Cell-free culture supernatants from monocytes (C) and macrophages (D) as treated in (A) and (B), respectively, were assayed for the presence of soluble TNF (sTNF) by ELISA.

Fig 4: Kinetics of TNF expression in monocyte-derived dendritic cells post LPS treatment. Day 5 human CD14+ monocyte-derived DCs were either untreated or treated with LPS in the absence (A) or presence (B) of 20 µM TACE inhibitor, TAPI-2, for the indicated periods of time. Cell surface proteins were labeled with cell-impermeable Sulfo-NHS-SS-biotin. Biotinylated surface proteins were precipitated with streptavidin-conjugated agarose beads. Cell surface biotinylated and total proteins were subjected to immunoblotting using anti-TNF IgG. Cadherins and GAPDH protein expressions were used as total protein and cell surface biotinylation loading controls. Cell-free culture supernatants of DCs as treated in (A) and (B) above were assayed for the presence of soluble TNF (sTNF) (C) by ELISA.

Fig 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).