Kynurenine 3-monooxygenase mediates inhibition of Th17 differentiation via catabolism of endogenous aryl hydrocarbon receptor ligands.

The aryl hydrocarbon receptor (AhR) is a key transcriptional regulator of Th17-cell differentiation. Although endogenous ligands have yet to be identified, evidence suggests that tryptophan metabolites can act as agonists for the AhR. Tryptophan metabolites are abundant in circulation, so we hypothesized that cell intrinsic factors might exist to regulate the exposure of Th17 cells to AhR-dependent activities. Here, we find that Th17 cells preferentially express kynurenine 3-monooxygenase (KMO), which is an enzyme involved in catabolism of the tryptophan metabolite kynurenine. KMO inhibition, either with a specific inhibitor or via siRNA-mediated silencing, markedly increased IL-17 production in vitro, whereas IFN-γ production by Th1 cells was unaffected. Inhibition of KMO significantly exacerbated disease in a Th17-driven model of autoimmune gastritis, suggesting that expression of KMO by Th17 cells serves to limit their continuous exposure to physiological levels of endogenous AhR ligands in vivo.

Mycophenolic acid differentially impacts B cell function depending on the stage of differentiation.

Production of pathogenic Abs contributes to disease progression in many autoimmune disorders. The immunosuppressant agent mycophenolic acid (MPA) has shown clinical efficacy for patients with autoimmunity. The goal of these studies was to elucidate the mechanisms of action of MPA on B cells isolated from healthy individuals and autoimmune patients. In this study, we show that MPA significantly inhibited both proliferation and differentiation of primary human B cells stimulated under various conditions. Importantly, MPA did not globally suppress B cell responsiveness or simply induce cell death, but rather selectively inhibited early activation events and arrested cells in the G0/G1 phase of the cell cycle. Furthermore, MPA blocked expansion of both naive and memory B cells and prevented plasma cell (PC) differentiation and Ab production from healthy controls and individuals with rheumatoid arthritis. Finally, whereas MPA potently suppressed Ig secretion from activated primary B cells, terminally differentiated PCs were not susceptible to inhibition by MPA. The target of MPA, IMPDH2, was found to be downregulated in PCs, likely explaining the resistance of these cells to MPA. These results suggest that MPA provides benefit in settings of autoimmunity by directly preventing activation and PC differentiation of B cells; however, MPA is unlikely to impact autoantibody production by preexisting, long-lived PCs.

IL-9 is a Th17-derived cytokine that limits pathogenic activity in organ-specific autoimmune disease.

IL-9 is a pleiotropic cytokine with key functions in tolerance and inflammation, and its expression is considered a hallmark of Th2-lineage cells. Here, we report that human and mouse Th17 cells are a significant source of IL-9. The expression of IL-9 by Th17 cells was strictly dependent on the presence of TGF-ß and IL-1ß, and inhibited by IL-4. IL-9-deficient Th17 cells induced more severe autoimmune gastritis following transfer to nu/nu recipient mice. Th17 cells did not appear to be the target of IL-9 bioactivity as Th17 expansion and differentiation was comparable using IL-9-deficient CD4(+) cells or when IL-9 was neutralized with antibodies in vitro. However, reduced mast cell activity was associated with the increased pathogenicity of IL-9-deficient Th17 cells. Together, these results demonstrate a previously unappreciated role for IL-9 in dampening the pathogenic activities of Th17 cells.

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG.

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.

Complement C5a induces PD-L1 expression and acts in synergy with LPS through Erk1/2 and JNK signaling pathways.

Severe bacterial infection results in both uncontrolled inflammation and immune suppression in septic patients. Although there is ample evidence that complement activation provokes overwhelming pro-inflammatory responses, whether or not it plays a role in immune suppression in this case is unclear. Here, we identify that complement C5a directly participates in negative regulation of immune responses to bacteria-induced inflammation in an ex vivo model of human whole blood. Challenge of whole blood with heat-killed Pseudomonas aeruginosa induces PD-L1 expression on monocytes and the production of IL-10 and TGF-ß, which we show to be inhibited by C5a blockade. The induction of PD-L1 expression by C5a is via C5aR1but not C5aR2. Furthermore, C5a synergises with P. aeruginosa LPS in both PD-L1 expression and the production of IL-10 and TGF-ß. Mechanistically, C5a contributes to the synergy in PD-L1 expression by specifically activating Erk1/2 and JNK signaling pathways. Our study reveals a new role for C5a in directly promoting immunosuppressive responses. Therefore, aberrant production of complement C5a during bacterial infection could have broader effect on compromising host defense including the induction of immune suppression.

Identification and characterization of a very low density lipoprotein receptor-binding peptide from tissue factor pathway inhibitor that has antitumor and antiangiogenic activity.

Tissue factor pathway inhibitor (TFPI) is the major physiologic inhibitor of the extrinsic coagulation pathway. We have previously shown that TFPI is also a potent inhibitor of endothelial proliferation in vitro and of primary and metastatic tumor growth in vivo. Surprisingly, the antitumor activity of TFPI was demonstrated to be independent of its anticoagulant activity, suggesting a possible nonhemostatic mechanism of action for TFPI in these models. This antitumor mechanism may involve the very low density lipoprotein (VLDL) receptor because the in vitro antiproliferative activity of TFPI is mediated through interaction with the VLDL receptor. In the current study, we identify a 23-amino acid fragment of TFPI (TFPIc23) localized to the C-terminus, which mediates binding to the VLDL receptor. The TFPIc23 peptide inhibits endothelial cell proliferation through an apoptotic mechanism and blocks vessel outgrowth in the in vitro assays, and this activity is mediated through interaction with the VLDL receptor. In vivo, this peptide potently inhibits angiogenesis in Matrigel and chick chorioallantoic membrane models and also inhibits metastatic tumor growth. Our data demonstrate that this VLDL receptor-binding fragment of the TFPI molecule has apoptotic, antiangiogenic, and antitumor activity and suggests a possible mechanism whereby TFPI can regulate angiogenesis and tumor growth independently of its anticoagulant activity.