Knowledge 925206-65-1 proven are PF-915275 representative of three impartial experiments. p<0.001. iNOS: inducible nitric oxide synthase COX: cyclooxygenase.from the 2 week T cell proliferation system did not exactly reproduce the short-term T cell proliferation results measured by CFSE dilution. As shown in Fig 7, the proliferation of OT-II T cells was low in the absence of the inhibitors regardless of OVA and TLR ligand stimulation. During the 2-week incubation, Indo dominantly improved the expansion of OT-II T cells in all stimulations. Dual inhibition of NO and PGE2 synergistically enhanced CD4+ T cell expansion only when stimulated with OVA plus CL097, but not with OVA alone or with OVA plus CpG. This suggests that CL097 may exert adjuvant activity stronger than CpG to enhance CD4+ T cell expansion when both prostaglandins (PGs) and NO are inhibited.Our in vitro observations indicate that higher induction of PGE2 and NO by CL097 than CpG may account for the low adjuvanticity of CL097 compared to CpG (Fig 1). To investigate the in vivo relevance of our in vitro data, we treated mice with AG and Indo to inhibit NO and PGE2 production, respectively. AG was given in drinking water  and Indo was injected i.p. three times at four day intervals . Unlike the in vitro observation that inhibiting both NO and PGE2 synthesis enhanced CD4+ T cell expansion, neither Indo nor AG treatment enhanced CD4+ T cell expansion after immunization with OVA plus CpG (Fig 8A) or CL097 (Fig 8B).Fig 8. Expansion of OT-II CD4+ T cells are not enhanced by systemic treatment with Indo or AG. Mice were adoptively transferred with 1 x 106 splenocytes obtained from OT-II mice and, 24 h later, immunized with 25 g OVA protein with or without 20 g CpG (A) or the same dose of CL097 (B) formulated in IFA. To inhibit NO, mice were given drinking water containing 2.5% AG and 1% glucose. To inhibit PGE2, mice were injected i.p. with Indo at a dose of 2.5 mg/Kg on the day of immunization and two more injections after 4 and 8 days. Mice were sacrificed 11 days post immunization, and the number of expanded OT-II CD4+ T cells were quantified by cell surface staining of CD4 and CD45.1 followed by flow cytometric analysis. The total numbers of OT-II CD4+ T cells in spleens were calculated based on the percentages of OT-II CD4+ T cells and known numbers of splenocytes. Statistical differences were analyzed by one-way ANOVA between treatment groups. p<0.05. AG: aminoguanidine hemisulfate Indo: indomethacin.This negative result with systemic inhibition may reflect the complexity of NO and PGE2 signaling pathways operating in a more complex in vivo environment.Natural and synthetic adjuvants are being actively discovered and developed to support a growing trend preferring better defined subunit vaccines over live/whole vaccines . However, there is currently no strong evidence to inform the rational selection of specific PRR agonists as adjuvants for a particular vaccine. We found that CpG, as an adjuvant, promoted stronger CD4+ T cell expansion than CL097 in mice. This difference in adjuvant potential of CpG and CL097 was delineated in the in vitro studies demonstrating that CpG was a weaker inducer of NO and PGE2 than CL097. This study provides evidence that adjuvant activity of PRR agonists is influenced by concurrent induction of regulatory factors. When comparing different PRR agonists, differential expression of regulatory products, such as NO and PGE2, should be considered and might be utilized as biomarkers of adjuvanticity. One strategy to enhance vaccine efficacy is to increase the size of memory T cell populations. Because the size of memory T cell pools is proportional to the level of initial clonal expansion after antigenic stimulation , increasing the magnitude of the T cell expansion has been suggested to enhance memory generation .