Biochemical responses of PCs to mpCCL2. (A) Antagonist CCL2 acts as a dominant-negative molecule for AKT phosphorylation (pAKT). Lane 1 indicates unstimulated; lane 2, rCCL2; lane 3, rhMMP1; lane 4, mpCCL2; lane 5, rCCL2 plus mpCCL2 (1:1). rCCL2 stimulation of CD138⁺ PCs leads to pAKT, whereas mpCCL2 alone or in combination with full-length CCL2 leads to a reduced AKT activation demonstrating antagonistic activities occurring at the level of CCR2. (B) Antagonist CCL2 blocks STAT3 activation. Whole-cell lysate of CD138⁺ sorted cells was tested for pSTAT3 as follows: lane 1 indicates unstimulated control; lane 2, rCCL2; lane 3, cCCL2; lane 4, MSC CM plus isotypes; lane 5, MSC CM in the presence of CCL2-neutralizing antibody. pSTAT3 decreased after mpCCL2 stimulation but was completely absent on the addition of MSC CM. The pSTAT3 was induced after the addition of CCL2-neutralizing antibody. In addition, PCs derived from TC-PTP^−/− splenocytes were refractory to STAT3 dephosphorylation by mpCCL2 as no changes were noticed after treatment. (C) mpCCL2 induces PAX5 expression in PC. Lane 1 indicates unstimulated; lane 2, MSC CM + iso; lane 3, MSC CM + α-CCL2; lane 4, rhMMP1; lane 5, rCCL2 + rhMMP1 + iso; lane 6, rCCL2 + rhMMP1 + α-CCL2; lane 7, A2B5 (positive control for PAX5). PAX5 was mainly detected in the lysate of sorted PCs after cCCL2 and MSC CM treatments, whereas CCL2 neutralization significantly decreases PAX5 levels. IgG blockade in PCs is not the result of apoptosis. To confirm that IgG inhibition in PCs is not the result of apoptosis induced by antagonist CCL2, PCs treated with media or mpCCL2 led to the same amount of cell death (25% vs 26%; data not shown).