MODULATION OF ANTIGEN-PROCESSING AND PRESENTATION BY COVALENTLY-LINKED COMPLEMENT C3B FRAGMENT

Ligands such as complement fragments (C3, C4), IgG or alpha(2)-macrogl obulin, which bind antigen (Ag) before their uptake by antigen-present ing cells (APC), are likely to modulate the different steps of Ag proc essing and presentation. These ligands contribute to internalization a nd endosomal targeting of Ag; they also influence its processing and, consequently, the binding of resulting peptides to major histocompatib ility complex (MHC) class II molecules before presentation to T cells. Complement protein C3 contains, like other members of the alpha(2)-ma croglobulin family, an intrachain thiolester bond. Conformational alte ration or limited proteolysis of C3 into C3b leads to breaking of the thiolester with transient capacity of the revealed carbonyl group to e sterify hydroxyl groups of Ag. Ester-linked complexes including tetanu s toxin (TT) and C3b were prepared to analyse the influence of bound C 3b on TT processing and presentation by APC. Covalent binding of C3b t o Tr resulted in increased and prolonged stimulation of specific T-cel l proliferation. This effect was observed with non-specific B cells, a s well as with a TT-specific B-cell clone, as APC. On the other hand, SDS-PAGE analysis of proteolysates of TT or C3b-TT, obtained with endo some/lysosome-enriched subcellular fractions prepared from human Epste in-Barr virus (EBV)-transformed B cells, indicated a delay of TT prote olysis when TT was associated to C3b. Treatment of APC with protease i nhibitors, before and during exposure of the cells to Ag, resulted in differences in the inhibition of TT and C3b-TT proteolysis. Using puri fied cathepsins B and D, we demonstrated that covalent binding of C3b to TT totally abolished TT proteolysis by cathepsin D, while proteolys is by cathepsin B was preserved. This finding and the absence of cathe psin B in endosomes may explain a delay in TT processing when it is as sociated to C3b. Confirming these data, presentation by formaldehyde-f ixed cells of C3b-TT proteolysates showed higher stimulation of specif ic T-cell clones than formaldehyde-fixed TT proteolysates.