CD154 Resistant to Cleavage from Intracellular Milieu and Cell Surface Induces More Potent CD40-Mediated Responses

Abstract

In addition to the membrane-bound form, CD154 also exists as a soluble molecule originating from an intracellular and membrane cleavage. We have previously shown that CD154 cleavage from T cell surface is mediated by CD40 and involves the action of ADAM10/ADAM17 enzymes. In the aim of defining the importance of CD154 maintained on cell surface, we generated a CD154 mutated at the cleavage site. Our data show that the double mutation of E112 and M113 residues of CD154 abolishes its spontaneous release and the CD40-mediated cleavage from cell surface but does not affect its binding to CD40. We also demonstrated that both the release of CD154 from the intracellular milieu and its CD40-mediated cleavage from cell surface are highly dependent on ADAM10/ADAM17 enzymes. The CD154-EM mutant was shown capable of inducing a more prominent apoptotic response in susceptible B cell lines than the wild-type (WT) form of the molecule. In addition, human B cells cultured in the presence of the CD154-EM mutant exhibited upregulated proliferative responses compared with the CD154-WT. The CD154-EM mutant was also shown to trigger differentiation of human B cells, reflected by an increased Ig production, more significantly than CD154-WT. Thus, our data strongly suggest that cleavage-resistant CD154 is a more prominent stimulant than the cleavable form of the molecule. Therefore, a maintained expression of CD154 on cell membrane and a disturbed cleavage of the molecule could be a mechanism by which CD154 is involved in some pathological conditions and should be revisited.

Monoclonal Antibody Targeting the CD154 Cleavage Site Inhibits CD40-Dependent and -Independent Cleavage of CD154 from the Cell Surface

“…Such cleavage-resistant membrane-bound CD154 was shown to trigger a significantly higher response in CD40-positive cells as compared with the cleavable form of the molecule (23). Indeed, the CD154 mutant resistant to cleavage upregulated the apoptotic response of susceptible B cell lymphoma cells as well as enhanced the proliferation and activation of immune cells (i.e., human B cells) (23). These observations strongly support a biological significance of our Clone 8 mAb, inhibiting CD154 cleavage from the cell surface and probably leading to a significant enhancement of CD40-mediated responses.…”

“…In support of this latter observation, we have recently demonstrated that CD154 mutated at the cleavage site itself, namely the E112 and the M113 residues (CD154-EM), is resistant to being spontaneously released from cells or cleaved from their surface upon the interaction with CD40. Such cleavage-resistant membrane-bound CD154 was shown to trigger a significantly higher response in CD40-positive cells as compared with the cleavable form of the molecule (23). Indeed, the CD154 mutant resistant to cleavage upregulated the apoptotic response of susceptible B cell lymphoma cells as well as enhanced the proliferation and activation of immune cells (i.e., human B cells) (23).…”

“…The three complementary approaches used in the current study clearly show that Clone 8 mAb does not alter the interaction of CD154 with CD40. Indeed, our anti-CD154 mAb is aimed at enhancing the availability and thus the immune function of CD154 [as suggested by our previous work (23)] rather than at inhibiting its interaction with CD40. Not only that, the interaction of CD154 and CD40 is of particular importance at this point, as CD40 is the only one among CD154 receptors capable of inducing cleavage of the molecule, as previously shown by our group (7).…”

“…Clone 8 mAb is directed against the residues required for CD154 cleavage To determine whether any of the derived mAbs is directed against the CD154 cleavage site, we first evaluated their recognition of our cleavage-resistant CD154 mutant (CD154-EM mutated at glutamic acid at position 112 and methionine at position 113) described previously (23). For this purpose, we transfected HEK 293 cells with CD154-WT and CD154-EM and assessed their binding to our above-generated mAbs using flow cytometry.…”

“…Our group has recently demonstrated that cleavage-resistant CD154 mutant (mutated at the cleavage sites E112 and M113) exhibited an increased biological function as compared with the cleavable CD154 (23). These findings solicited our interest in developing tools to inhibit cleavage of CD154 from the cell surface.…”