Figure 4. LTA and ATP act synergistically to promote IL-23 and IL-1β dependent Th17-activation.

Overnight cultures of monocytes were treated with ATP (100 uM) for 4–6 hrs followed by application of 10 ug/ml LTA. Other groups received either no treatment (iDC) or a standard DC1 polarizing regimen (see Materials and Methods). Cells were harvested 5–6 h after TLR ligand application to serve as stimulators. (A) Monocytes were co-cultured with purified CD4^pos (naïve+memory) or CD4^pos/CD45RO^neg (naïve) T cells at 1∶10 stimulator:responder ratios. After 6 days co-culture, T cells were harvested and re-cultured on tissue culture plates pre-coated with anti-CD3 and anti-CD28 antibodies, with supernatants harvested 24 h later and analyzed by ELISA for IFN-γ and IL-17. Results shown from 6 allogeneic APC:T cell pairings +/− SEM. (B) Culture supernatants removed from initial co-cultures of total CD4^pos populations on day 5 (prior to cell harvest and antibody re-stimulation) were analyzed by ELISA for IL-17 and IL-22. Results for (B) expressed as mean percent maximums from 6 allogeneic APC:T cell pairings +/− SEM. (C) Co-cultures of APCs and total CD4^pos T cells were initiated in the presence of IL-23/12 (p40), IL-12 (p70) or IL1β –neutralizing MoAb, or isotype controls, with culture supernatants harvested on day 5 and analyzed for IL-17 production by ELISA Results from 3 allogeneic APC:T cell pairings +/− SEM.