BMSC and MC activatory outcome depend on CD40/CD40L interaction. The addition of a blocking anti-CD40 antibody (αCD40) to the BMSC-MC coculture reduces the upregulation of CD40 on BMSC (A) and significantly decreases the release of TNF and IL-6 in the coculture supernatants (B). Consistently, soluble CD40L (sCD40L) induces significant CD40 upregulation (C) and increased IL-6 release by BMSC (D), although it has no effect on TNF release. (E) BMSC stimulation with rTNF causes a slight increase in CD40 expression on BMSC. In contrast, CD40 expression is not significantly modified by BMSC stimulation with rIL-6. (F) The addition of blocking anti-TNF antibody (αTNF) to the BMSC-MC coculture reduces the expression of CD40 induction on BMSC and (G) significantly decreases IL-6 levels. (H) Immunohistochemistry for pSTAT3, showing a higher number of cells with pSTAT3 nuclear localization within the BM infiltrates of cases with high CD40 stromal expression. Immunohistochemistry, STREPTavidin-biotin-peroxidase complex method, DAB chromogen; original magnification ×400. Microphotographs are relative to 2 representative cases with high (score 3) and low (score 1) CD40 stromal expression out of the 30 evaluated. (I) In cases with elevated CD40 stromal expression, double immunofluorescent analysis revealed the presence of tryptase⁺ MCs (red signal) expressing IL-6 (green signal). Immunofluorescence with Alexa-488 and Alexa-568 fluorochromes; original magnification ×400. Microphotograph is relative to 1 representative case with high (score 3) CD40 stromal expression. *P < .05; **P < .01; ***P < .001.