Enhanced GITR/GITRL interactions augment IL-27 expression and induce IL-10-producing Tr-1 like cells.

The glucocorticoid-induced TNFR-related (GITR) protein is a coactivating receptor that is constitutively expressed on Treg cells and induced on activated T cells. To better under-stand the role of long-term GITR signaling, we generated a mouse that constitutively expresses GITR ligand (GITRL) on APCs that mimics the physiological distribution of GITRL in vivo. Despite a five-fold expansion of the Treg-cell pool, there is increased activation and depletion of naive T cells in the transgenic (Tg) mice, suggesting that the increased number of Treg cells cannot fully suppress T-cell activation. Interestingly, GITRL Tg mice have multiorgan lymphocytic infiltrates yet display no overt autoimmunity, indicating the existence of a compensatory immunoregulatory mechanism(s). In the spleens and tissue infiltrates ofGITRL Tg mice, we found increased numbers of Foxp3(-) IL-10-producing type 1 regulatory T (Tr-1)-like cells that suppress naïve T-cell proliferation in an IL-10-dependent fashion. Increased IL-27 production from Tg APCs and activation of c-Maf in the Tr1-like cells suggest a possible mechanism for their induction. Our results demonstrate that enhanced GITR/GITRL interactions have a pleiotropic role on the regulation of T-cell responses, which includes promoting the differentiation of Tr-1-like cells, which contribute to the maintenance of peripheral T-cell tolerance.

Interleukin 21 is a T helper (Th) cell 2 cytokine that specifically inhibits the differentiation of naive Th cells into interferon gamma-producing Th1 cells.

The cytokine potential of developing T helper (Th) cells is directly shaped both positively and negatively by the cytokines expressed by the effector Th cell subsets. Here we find that the recently identified cytokine, interleukin (IL)-21, is preferentially expressed by Th2 cells when compared with Th1 cells generated in vitro and in vivo. Exposure of naive Th precursors to IL-21 inhibits interferon (IFN)-gamma production from developing Th1 cells. The repression of IFN-gamma production is specific in that the expression of other Th1 and Th2 cytokines is unaffected. IL-21 decreases the IL-12 responsiveness of developing Th cells by specifically reducing both signal transducer and activator of transcription 4 protein and mRNA expression. These results suggest that Th2 cell-derived IL-21 regulates the development of IFN-gamma-producing Th1 cells which could serve to amplify a Th2 response.

Regulation and function of the interleukin 13 receptor alpha 2 during a T helper cell type 2-dominant immune response.

Highly polarized type 2 cytokine responses can be harmful and even lethal to the host if they are too vigorous or persist too long. Therefore, it is important to elucidate the mechanisms that down-regulate these reactions. Interleukin (IL)-13 has emerged as a central mediator of T helper cell (Th)2-dominant immune responses, exhibiting a diverse array of functional activities including regulation of airway hyperreactivity, resistance to nematode parasites, and tissue remodeling and fibrosis. Here, we show that IL-13 receptor (R)alpha2 is a critical down-regulatory factor of IL-13-mediated tissue fibrosis induced by the parasitic helminth Schistosoma mansoni. IL-13Ralpha2 expression was induced after the onset of the fibrotic response, IL-10, IL-13, and Stat6 dependent, and inhibited by the Th1-inducing adjuvant IL-12. Strikingly, schistosome-infected C57BL/6 and BALB/c IL-13Ralpha2-deficient mice showed a marked exacerbation in hepatic fibrosis, despite displaying no change in granuloma size, tissue eosinophilia, or mastocytosis. Fibrosis increased despite the fact that IL-13 levels decreased significantly in the liver and serum. Importantly, pathology was prevented when IL-13Ralpha2-deficient mice were treated with a soluble IL-13Ralpha2-Fc construct, formally demonstrating that their exacerbated fibrotic response was due to heightened IL-13 activity. Together, these studies illustrate the central role played by the IL-13Ralpha2 in the down-regulation of a chronic and pathogenic Th2-mediated immune response.

Enhanced interleukin (IL)-13 responses in mice lacking IL-13 receptor alpha 2.

Interleukin (IL)-13 has recently been shown to play important and unique roles in asthma, parasite immunity, and tumor recurrence. At least two distinct receptor components, IL-4 receptor (R)alpha and IL-13Ralpha1, mediate the diverse actions of IL-13. We have recently described an additional high affinity receptor for IL-13, IL-13Ralpha2, whose function in IL-13 signaling is unknown. To better appreciate the functional importance of IL-13Ralpha2, mice deficient in IL-13Ralpha2 were generated by gene targeting. Serum immunoglobulin E levels were increased in IL-13Ralpha2-/- mice despite the fact that serum IL-13 was absent and immune interferon gamma production increased compared with wild-type mice. IL-13Ralpha2-deficient mice display increased bone marrow macrophage progenitor frequency and decreased tissue macrophage nitric oxide and IL-12 production in response to lipopolysaccharide. These results are consistent with a phenotype of enhanced IL-13 responsiveness and demonstrate a role for endogenous IL-13 and IL-13Ralpha2 in regulating immune responses in wild-type mice.

The human IL-17F/IL-17A heterodimeric cytokine signals through the IL-17RA/IL-17RC receptor complex.

IL-17A and IL-17F, produced by the Th17 CD4(+) T cell lineage, have been linked to a variety of inflammatory and autoimmune conditions. We recently reported that activated human CD4(+) T cells produce not only IL-17A and IL-17F homodimers but also an IL-17F/IL-17A heterodimeric cytokine. All three cytokines can induce chemokine secretion from bronchial epithelial cells, albeit with different potencies. In this study, we used small interfering RNA and Abs to IL-17RA and IL-17RC to demonstrate that heterodimeric IL-17F/IL-17A cytokine activity is dependent on the IL-17RA/IL-17RC receptor complex. Interestingly, surface plasmon resonance studies indicate that the three cytokines bind to IL-17RC with comparable affinities, whereas they bind to IL-17RA with different affinities. Thus, we evaluated the effect of the soluble receptors on cytokine activity and we find that soluble receptors exhibit preferential cytokine blockade. IL-17A activity is inhibited by IL-17RA, IL-17F is inhibited by IL-17RC, and a combination of soluble IL-17RA/IL-17RC receptors is required for inhibition of the IL-17F/IL-17A activity. Altogether, these results indicate that human IL-17F/IL-17A cytokine can bind and signal through the same receptor complex as human IL-17F and IL-17A. However, the distinct affinities of the receptor components for IL-17A, IL-17F, and IL-17F/IL-17A heterodimer can be exploited to differentially affect the activity of these cytokines.

A simple high-throughput purification method for hit identification in protein screening.

Phage and ribosome display technologies have emerged as important tools in the high-throughput screening of protein pharmaceuticals. However, a challenge created by the implementation of such tools is the need to purify large numbers of proteins for screening. While some assays may be compatible with crude bacterial lysates or periplasmic extracts, many functional assays, particularly cell-based assays, require protein of high purity and concentration. Here we evaluate several methods for small-scale, high-throughput protein purification. From our initial assessment we identified the HIS-Select 96-well filter plate system as the method of choice for further evaluation. This method was optimized and used to produce scFvs that were tested in cell-based functional assays. The behavior of HIS-Select purified scFvs in these assays was found to be similar to scFvs purified using a traditional large-scale 2-step purification method. The HIS-Select method allows high-throughput purification of hundreds of scFvs with yields in the 50-100 microg range, and of sufficient purity to allow evaluation in a cell-based proliferation assay. In addition, the use of a similar 96-well-based method facilitates the purification and subsequent screening of large numbers of IgGs and Fc fusion proteins generated through reformatting of scFv fragments.

Anti-ICOSL New Antigen Receptor Domains Inhibit T Cell Proliferation and Reduce the Development of Inflammation in the Collagen-Induced Mouse Model of Rheumatoid Arthritis.

Lymphocyte costimulation plays a central role in immunology, inflammation, and immunotherapy. The inducible T cell costimulator (ICOS) is expressed on T cells following peptide: MHC engagement with CD28 costimulation. The interaction of ICOS with its sole ligand, the inducible T cell costimulatory ligand (ICOSL; also known as B7-related protein-1), triggers a number of key activities of T cells including differentiation and cytokine production. Suppression of T cell activation can be achieved by blocking this interaction and has been shown to be an effective means of ameliorating disease in models of autoimmunity. In this study, we isolated specific anti-ICOSL new antigen receptor domains from a synthetic phage display library and demonstrated their ability to block the ICOS/ICOSL interaction and inhibit T cell proliferation. Anti-mouse ICOSL domains, considered here as surrogates for the use of anti-human ICOSL domains in patient therapy, were tested for efficacy in a collagen-induced mouse model of rheumatoid arthritis where they significantly decreased the inflammation of joints and delayed and reduced overall disease progression and severity.

IL-21 and IL-21 receptor: a new cytokine pathway modulates innate and adaptive immunity.

Interleukin (IL)-21 is a new member of the type I cytokine superfamily. Although it is most homologous to IL-15, it has a unique receptor chain, IL-21R, that pairs with the gamma-common cytokine receptor chain. The first experiments examining the biology of the IL-21 pathway reveal that it is a cytokine with effects on natural killer (NK) cells, T cells, and B cells. Mice deficient in the IL-21R have also been made, and are being examined for the effects of the IL-21/IL-21R pathway in vivo. Here we summarize our current knowledge of this new cytokine pathway, and its role in innate and adaptive immunity.

CDR-restricted engineering of native human scFvs creates highly stable and soluble bifunctional antibodies for subcutaneous delivery.

While myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulation. We focused on an scFv-Fc-scFv format, with a flexible (A4T)3 linker coupling an additional scFv to the C-terminus of an scFv-Fc. While one of the lead scFvs isolated directly from a naïve library was well-behaved and sufficiently potent, the parental anti-CXCL13 scFv 3B4 required optimization for affinity, stability, and cynomolgus ortholog cross-reactivity. To achieve this, we eschewed framework-based stabilizing mutations in favor of complementarity-determining region (CDR) mutagenesis and re-selection for simultaneous improvements in both affinity and thermal stability. Phage-displayed 3B4 CDR-mutant libraries were used in an aggressive "hammer-hug" selection strategy that incorporated thermal challenge, functional, and biophysical screening. This approach identified leads with improved stability and>18-fold, and 4,100-fold higher affinity for both human and cynomolgus CXCL13, respectively. Improvements were exclusively mediated through only 4 mutations in VL-CDR3. Lead scFvs were reformatted into scFv-Fc-scFvs and their biophysical properties ranked. Our final candidate could be formulated in a standard biopharmaceutical platform buffer at 100 mg/ml with<2% high molecular weight species present after 7 weeks at 4 °C and viscosity<15 cP. This workflow has facilitated the identification of a truly manufacturable scFv-based bispecific therapeutic suitable for subcutaneous administration.

Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin-17 immunity.

Chronic mucocutaneous candidiasis disease (CMCD) is characterized by recurrent or persistent infections of the skin, nails, and oral and genital mucosae caused by Candida albicans and, to a lesser extent, Staphylococcus aureus, in patients with no other infectious or autoimmune manifestations. We report two genetic etiologies of CMCD: autosomal recessive deficiency in the cytokine receptor, interleukin-17 receptor A (IL-17RA), and autosomal dominant deficiency of the cytokine interleukin-17F (IL-17F). IL-17RA deficiency is complete, abolishing cellular responses to IL-17A and IL-17F homo- and heterodimers. By contrast, IL-17F deficiency is partial, with mutant IL-17F-containing homo- and heterodimers displaying impaired, but not abolished, activity. These experiments of nature indicate that human IL-17A and IL-17F are essential for mucocutaneous immunity against C. albicans, but otherwise largely redundant.

Identification of an interleukin 17F/17A heterodimer in activated human CD4+ T cells.

IL-17F and IL-17A are members of the IL-17 pro-inflammatory cytokine family. IL-17A has been implicated in the pathogenesis of autoimmune diseases. IL-17F is a disulfide-linked dimer that contains a cysteine-knot motif. We hypothesized that IL-17F and IL-17A could form a heterodimer due to their sequence homology and overlapping pattern of expression. We evaluated the structure of recombinant IL-17F and IL-17A proteins, as well as that of natural IL-17F and IL-17A derived from activated human CD4+ T cells, by enzyme-linked immunosorbent assay, immunoprecipitation followed by Western blotting, and mass spectrometry. We find that both IL-17F and IL-17A can form both homodimeric and heterodimeric proteins when expressed in a recombinant system, and that all forms of the recombinant proteins have in vitro functional activity. Furthermore, we find that in addition to the homodimers of IL-17F and IL-17A, activated human CD4+ T cells also produce the IL-17F/IL-17A heterodimer. These data suggest that the IL-17F/IL-17A heterodimer may contribute to the T cell-mediated immune responses.

Blockade of the interleukin-21/interleukin-21 receptor pathway ameliorates disease in animal models of rheumatoid arthritis.

OBJECTIVE: Interleukin-21 (IL-21) is a T cell-derived cytokine that modulates T cell, B cell, and natural killer cell responses. In this study, the effects of blocking IL-21 were examined in 2 rodent models of rheumatoid arthritis (RA) to determine whether IL-21 contributes to their pathologic processes. METHODS: DBA/1 mice were immunized with bovine type II collagen and then treated with murine IL-21 receptor Fc fusion protein (IL-21R.Fc), which was initiated after the onset of arthritis symptoms in 10% of the cohort. The mice were assessed 3 times per week for signs of disease, including histologic features as well as serum cytokine, Ig, and cytokine messenger RNA (mRNA) levels in the paws. In a separate experiment, Lewis rats were immunized with Freund's complete adjuvant followed by administration of IL-21R.Fc at the peak of inflammation in the joints. Rats were assessed daily for histologic features and for scoring of arthritis severity. In addition, the effects of IL-21R.Fc on the production of interferon-gamma (IFNgamma) by T cells were examined. RESULTS: Treatment of DBA/1 mice with IL-21R.Fc reduced the clinical and histologic signs of collagen-induced arthritis. Nonspecific IgG1 levels were decreased in response to treatment. The levels of IL-6 mRNA in the paws and the serum IL-6 levels were decreased after treatment with IL-21R.Fc. IFNgamma mRNA levels were increased in the paws, and the addition of IL-21R.Fc to collagen-activated lymph node cultures enhanced the levels of IFNgamma. Collagen-specific spleen cell responses in IL-21R.Fc-treated mice were observed as reduced levels of IFNgamma and increased levels of IL-6. Treatment of Lewis rats with IL-21R.Fc after induction of adjuvant-induced arthritis resulted in reversal of disease signs and improvements in histologic parameters. CONCLUSION: These findings demonstrate a pathogenic role for IL-21 in animal models of RA, and support consideration of IL-21 as a therapeutic target in human RA.

An IL-17F/A heterodimer protein is produced by mouse Th17 cells and induces airway neutrophil recruitment.

IL-17A and IL-17F are related homodimeric proteins of the IL-17 family produced by Th17 cells. In this study, we show that mouse Th17 cells also produce an IL-17F/A heterodimeric protein. Whereas naive CD4(+) T cells differentiating toward the Th17 cell lineage expressed IL-17F/A in higher amounts than IL-17A/A homodimer and in lower amounts than IL-17F/F homodimer, differentiated Th17 cells expressed IL-17F/A in higher amounts than either homodimer. In vitro, IL-17F/A was more potent than IL-17F/F and less potent than IL-17A/A in regulating CXCL1 expression. Neutralization of IL-17F/A with an IL-17A-specific Ab, and not with an IL-17F-specific Ab, reduced the majority of IL-17F/A-induced CXCL1 expression. To study these cytokines in vivo, we established a Th17 cell adoptive transfer model characterized by increased neutrophilia in the airways. An IL-17A-specific Ab completely prevented Th17 cell-induced neutrophilia and CXCL5 expression, whereas Abs specific for IL-17F or IL-22, a cytokine also produced by Th17 cells, had no effects. Direct administration of mouse IL-17A/A or IL-17F/A, and not IL-17F/F or IL-22, into the airways significantly increased neutrophil and chemokine expression. Taken together, our data elucidate the regulation of IL-17F/A heterodimer expression by Th17 cells and demonstrate an in vivo function for this cytokine in airway neutrophilia.

IL-21 induces the apoptosis of resting and activated primary B cells.

Cytokines play an important role in regulating the development and homeostasis of B cells by controlling their viability. In this study, we show that the recently described T cell-derived cytokine IL-21 induces the apoptosis of resting primary murine B cells. In addition, the activation of primary B cells with IL-4, LPS, or anti-CD40 Ab does not prevent IL-21-mediated apoptosis. The induction of apoptosis by IL-21 correlates with a down-regulation in the expression of Bcl-2 and Bcl-x(L), two antiapoptotic members of the Bcl-2 family. Furthermore, the reconstitution of Bcl-x(L) or Bcl-2 expression protects primary B cells from IL-21-induced apoptosis. In addition, a short-term preactivation of B cells with anti-CD40 Ab confers protection from IL-21-mediated apoptosis through the up-regulation of Bcl-x(L). These studies reveal a novel pathway that mediates B cell apoptosis via the IL-21R and suggest that IL-21 may play a role in regulating B cell homeostasis.

Tumor cells secreting IL-13 but not IL-13Ralpha2 fusion protein have reduced tumorigenicity in vivo.

IL-13 is a Th2 cytokine that plays crucial roles in the pathophysiology of allergy, asthma and helminth infection. The high affinity receptor for IL-13, IL-13Ralpha2, may act as a decoy receptor for IL-13. The anti-tumor effect of IL-13 and its soluble receptor IL-13Ralpha2 have been examined in different tumor systems. Previous studies have shown that IL-13 enhances anti-tumor responses in some model systems, whereas IL-13Ralpha2Fc prevents IL-13 mediated suppression of tumor immuno-surveillance in a different model system. In this study, we have used a cytokine (receptor) gene therapy approach and studied the immune responses mediated by IL-13 and IL-13Ralpha2Fc in poorly immunogenic B16F1 melanoma and immunogenic MethA fibrosarcoma tumor models. We find that IL-13 reduces the tumorigenicity of B16F1 melanoma and MethA fibrosarcoma cells in vivo, most likely through the recruitment of neutrophils and macrophages. IL-13 mediated anti-tumor responses do not lead to the generation of tumor-specific T cells. Neither IL-13Ralpha2Fc gene transduction nor in vivo treatment with soluble IL-13Ralpha2Fc has a statistically significant effect of tumor growth. IL-13Ralpha2 deficient host background does not alter tumor growth, suggesting that endogenous levels of IL-13 do not contribute to an anti-tumor response in these models. We conclude that IL-13, but not soluble IL-13Ralpha2, has anti-tumor activity in the models described here, possibly by enhancing innate anti-tumor immunity.

IL-21 activates both innate and adaptive immunity to generate potent antitumor responses that require perforin but are independent of IFN-gamma.

IL-21 is a key factor in the transition between innate and adaptive immune responses. We have used the cytokine gene therapy approach to study the antitumor responses mediated by IL-21 in the B16F1 melanoma and MethA fibrosarcoma tumor models in mice. Retrovirally transduced tumor cells secreting biologically functional IL-21 have growth patterns in vitro similar to that of control green fluorescent protein-transduced cells, but are completely rejected in vivo. We show that IL-21 activates NK and CD8(+) T cells in vivo, thus mediating complete rejection of poorly immunogenic tumors. Rejection of IL-21-secreting tumors requires the presence of cognate IL-21R and does not depend on CD4(+) T cell help. Interestingly, perforin, but not IFN-gamma or other major Th1 and Th2 cytokines (IL-12, IL-4, or IL-10), is required for the IL-21-mediated antitumor response. Moreover, IL-21 results in 50% protection and 70% cure of nonimmunogenic tumors when given before and after tumor challenge, respectively, in C57BL/6 mice. We conclude that IL-21 immunotherapy warrants clinical evaluation as a potential treatment for cancer.

Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells.

Nonactivated CD4+CD25+ regulatory T cells constitutively express glucocorticoid-induced TNFR family-related receptor (GITR), a TNFR family member whose engagement was presumed to abrogate regulatory T cell-mediated suppression. Using GITR-/- mice, we report that GITR engagement on CD25-, not CD25+ T cells abrogates T cell-mediated suppression. Mouse APCs constitutively express GITR ligand (GITR-L), which is down-regulated following TLR signaling in vivo. Although GITR-/-CD25- T cells were capable of mounting proliferative responses, they were incapable of proliferation in the presence of physiological numbers of CD25+ T cells. Thus, GITR-L provides an important signal for CD25- T cells, rendering them resistant to CD25+ -mediated regulation at the initiation of the immune response. The down-regulation of GITR-L by inflammatory stimuli may enhance the susceptibility of effector T cells to suppressor activity during the course of an infectious insult.

CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor.

CD4(+)CD25(+) immunoregulatory T cells represent a unique lineage of thymic-derived cells that potently suppress both in vitro and in vivo effector T cell function. We analyzed CD4(+)CD25(+) and CD4(+)CD25(-) T cells by DNA microarray, identifying 29 genes differentially expressed in the resting subpopulations, and 77 that were differentially expressed following activation. Most of these genes were elevated in the CD4(+)CD25(+) population, suggesting a previously activated phenotype. Among these were a number of genes that antagonize signaling, including members of the SOCS family, which may contribute to their anergic phenotype. Multiple cell surface receptors also had increased expression in CD4(+)CD25(+) cells, including GITR, a member of the TNF receptor superfamily. Importantly, antibodies to GITR abrogated suppression, demonstrating a functional role for this receptor in regulating the CD4(+)CD25(+) T cell subset.

IL-21 limits NK cell responses and promotes antigen-specific T cell activation: a mediator of the transition from innate to adaptive immunity.

IFNalpha/beta, IL-12, and IL-15 regulate NK cell activation and expansion, but signals triggering resolution of the NK response upon induction of adaptive immunity remain to be defined. We now report that IL-21, a product of activated T cells, may serve this function. Mice lacking IL-21R (IL-21R(-/-)) had normal NK cell development but no detectable responses to IL-21. IL-21 enhanced cytotoxic activity and IFNgamma production by activated murine NK cells but did not support their viability, thus limiting their duration of activation. Furthermore, IL-21 blocked IL-15-induced expansion of resting NK cells, thus preventing the initiation of further innate responses. In contrast, IL-21 enhanced the proliferation, IFNgamma production, and cytotoxic function of CD8(+) effector T cells in an allogeneic MLR. These observations suggest that IL-21 promotes the transition between innate and adaptive immunity.