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The 4-trifluoromethyl analog 4c shown moderate activity against Pim-1, but was surprisingly effective when tested against Pim-3 (residual activities 51% and 24%, respectively) The overall yield for the preparation of the C8 methyl derivative 17 from the common aldehyde starting material was 18%

After 6 d, the T cells were restimulated with anti-CD3 (10 g/mL plate-bound) and anti-CD28 (1 g/mL soluble). CT and irradiated anthrax spores induced spore-specific CD4 T cells that produced IL-17 but not IFN-, IL-5, or IL-10 (Fig. 1and mice were incubated with CT or OVA over night before incubation with OT-II CD4+ T cells. After restimulation, supernatants were analyzed by ELISA. Data demonstrated are representative of three self-employed experiments. All graphs Ro 3306 display the mean of triplicate samples and error bars reflect standard deviation. *, < 0.05 compared to CT-untreated group; n.d., not recognized. Because CT was present when T cells were added in the experiments above, we evaluated whether CT induces IL-17 by acting via DCs or directly on T cells. Treatment of T cells with CT during activation by formalin-fixed, OVA-pulsed DCs did not induce IL-17 (Fig. 2bar), much like CT-untreated cells (Fig. 2bar). In contrast, OVA-pulsed DCs treated with CT before fixation, and then incubated in their initial CT-conditioned medium with T cells, induced IL-17 (Fig. 2bar). Therefore, CT does not take action directly on T cells but instead Ro 3306 via DCs to induce IL-17 production by T cells. Nod2 and Toll-like receptors (TLR) influence IL-17 production (9, 16). DCs from and mice Ro 3306 retained the ability to elicit IL-17-secreting T cells after exposure to CT (Fig. 2DCs further confirmed TLR-independence (Fig. 2and < 0.05 compared with media-treated group. Th17 Differentiation by CT Depends on a Combination of Secreted Factors. To investigate the mechanisms by which CT-treated DCs induce T-cell IL-17 production, we first decided whether induction of T-cell IL-17 by DCs requires cognate conversation via induced cell-surface molecules or depends on secreted factors. Compared with unfixed DCs (Fig. 4bars), DCs that were formalin-fixed after incubation with CT and OVA failed to induce IL-17 secretion in T cells (Fig. 4bars), indicating that cognate conversation was insufficient. Adding back supernatant from CT-treated DCs restored the ability of fixed DCs to induce IL-17 (Fig. 4bars), implicating one or more secreted factors. Addition of medium from CT-treated DCs (CT-conditioned medium, CT-CM) directly to sorted naive CD4 T cells (>99% purity) (Fig. S4and Fig. S4< 0.05 compared with media-treated group; n.d., not detected. To investigate the mechanisms by which CT-CM induces Th17 differentiation, we first tested the role of factors known to Ro 3306 influence Th17 differentiation. Blockade of IL-6 using a combination of neutralizing antibodies against IL-6 and IL-6 receptor inhibited the ability of CT-CM to induce IL-17 from naive T cells (Fig. 5and Fig. S5and Fig. S5and Fig. S5and Fig. S5and Fig. S5and and < 0.05 compared with CT-CM (and Fig. S5DCs were capable of inducing differentiation of naive T cells into Th17 cells (Fig. 5and Fig. S5and Fig. S5heat-labile toxin also induced IL-17 (36C39), but the mechanisms involved and the role of IL-17 in mediating adjuvant effects (i.e., antibody responses and protection against contamination) Mouse monoclonal to EGR1 were not explored. In addition, our data show that a natural biological molecule such as CT induces DCs to secrete a complex mixture of products that are required to act in combination to determine T-cell fate (Fig. S8). The Th17 phenotype induced by this mixture of products differs from that induced by IL-6 and TGF-, suggesting that the specific combination of factors influences the stability and characteristics of Th17 cells that are induced. The CT-CM system described, where IL-6 levels generated by CT are low (Fig. S7) and the use of 10% CT-CM further lowers cytokine concentrations to limiting levels, likely enhanced our ability to detect the contribution of molecules such as IL-1 and -CGRP to Th17 differentiation by CT. Our data suggest that CT activates cAMP-dependent pathways in DCs to drive Th17 differentiation. Further studies will be needed to address whether cAMP activation in vivo is sufficient to mimic the adjuvant effects of CT. Interestingly, ATP can stimulate purinergic receptors to induce both cAMP (40) and IL-1 (41), suggesting involvement of these molecules in the ability of bacterially-derived ATP to drive Ro 3306 Th17 responses in the gut (42)..