Assessment of Skin Toxicity in an in Vitro Reconstituted Human Epidermis Model Using Deep Learning.

Skin toxicity is a common safety concern associated with drugs that inhibit epidermal growth factor receptors as well as other targets involved in epidermal growth and differentiation. Recently, the use of a three-dimensional reconstructed human epidermis model enabled large-scale drug screening and showed potential for predicting skin toxicity. Although a decrease in epidermal thickness was often observed when the three-dimensional reconstructed tissues were exposed to drugs causing skin toxicity, the thickness evaluation of epidermal layers from a pathologist was subjective and not easily reproducible or scalable. In addition, the subtle differences in thickness among tissues, as well as the large number of samples tested, made cross-study comparison difficult when a manual evaluation strategy was used. The current study used deep learning and image-processing algorithms to measure the viable epidermal thickness from multiple studies and found that the measured thickness was not only significantly correlated with a pathologist's semi-quantitative evaluation but was also in close agreement with the quantitative measurement performed by pathologists. Moreover, a sensitivity of 0.8 and a specificity of 0.75 were achieved when predicting the toxicity of 18 compounds with clinical observations with these epidermal thickness algorithms. This approach is fully automated, reproducible, and highly scalable. It not only shows reasonable accuracy in predicting skin toxicity but also enables cross-study comparison and high-throughput compound screening.

Randomized Phase I Healthy Volunteer Study of UTTR1147A (IL-22Fc): A Potential Therapy for Epithelial Injury.

Most treatments for epithelial injury target hematopoietic mechanisms, possibly causing immunosuppression. Interleukin (IL)-22 promotes tissue regeneration, acting directly on epithelial cells. UTTR1147A, a human IL-22Fc (immunoglobulin G (IgG)4) fusion protein, activates IL-22 signaling. This phase I placebo-controlled trial of single, ascending, i.v. (1-120 µg/kg) and s.c (3-120 µg/kg) doses of UTTR1147A analyzed its effects on safety, tolerability, pharmacokinetics, and pharmacodynamic biomarkers in healthy volunteers. Most adverse events (AEs) were mild or moderate. The maximum tolerated i.v. dose in healthy volunteers was 90 µg/kg. Predominant AEs were dose-dependent reversible skin effects consistent with IL-22 pharmacology. UTTR1147A exposure increased approximately dose-proportionally, with a half-life of ~1 week. IL-22 biomarkers (regenerating islet protein 3A (REG3A), serum amyloid A (SAA), and C-reactive protein (CRP)) increased dose-dependently. Neither inflammatory symptoms and signs nor cytokines increased with CRP elevations. UTTR1147A demonstrated acceptable safety, pharmacokinetics, and IL-22R engagement, supporting further clinical development.

Nonclinical safety assessment of a human interleukin-22FC IG fusion protein demonstrates in vitro to in vivo and cross-species translatability.

Although Interleukin-22 (IL-22) is produced by various leukocytes, it preferentially targets cells with epithelial origins. IL-22 exerts essential roles in modulating various tissue epithelial functions, such as innate host defense against extracellular pathogens, barrier integrity, regeneration, and wound healing. Therefore, IL-22 is thought to have therapeutic potential in treating diseases associated with infection, tissue injury or chronic tissue damage. A number of in vitro and in vivo nonclinical studies were conducted to characterize the pharmacological activity and safety parameters of UTTR1147A, an IL-22 recombinant fusion protein that links the human cytokine IL-22 with the Fc portion of a human immunoglobulin. To assess the pharmacological activity of UTTR1147A, STAT3 activation was evaluated in primary hepatocytes isolated from human, cynomolgus monkey, minipig, rat, and mouse after incubation with UTTR1147A. UTTR1147A activated STAT3 in all species evaluated, demonstrating that all were appropriate nonclinical species for toxicology studies. The nonclinical safety profile of UTTR1147A was evaluated in rats, minipigs, and cynomolgus monkeys to establish a safe clinical starting dose for humans in Phase I trials and to support clinical intravenous, subcutaneous and/or topical administration treatment regimen. Results demonstrate the cross-species translatability of the biological response in activating the IL-22 pathway as well as the translatability of findings from in vitro to in vivo systems. UTTR1147A was well tolerated in all species tested and induced the expected pharmacologic effects of epidermal hyperplasia and a transient increase in on-target acute phase proteins. These effects were all considered to be clinically predictable, manageable, monitorable, and reversible.

Pre-clinical and translational pharmacology of a human interleukin-22 IgG fusion protein for potential treatment of infectious or inflammatory diseases.

Interleukin (IL)-22 plays protective roles in infections and in inflammatory diseases that have been linked to its meditation of innate immunity via multiple mechanisms. IL-22 binds specifically to its heterodimeric receptor, which is expressed on a variety of epithelial tissues. UTTR1147A is a recombinant fusion protein that links the human cytokine IL-22 with the Fc portion of human immunoglobulin (Ig) G4. Here, we report extensive in vitro and in vivo nonclinical studies that were conducted to characterize the pharmacological activity of UTTR1147A. The in vitro activity and potency of UTTR1147A were analyzed using primary human hepatocytes and human colonic epithelial cell lines. Assessment of in vivo efficacy was performed in a mouse colitis model and by measuring relevant pharmacodynamic biomarkers, including antimicrobial peptides REG3A/ß, serum amyloid protein A (SAA) and lipopolysaccharide binding protein (LBP). The pharmacokinetic and pharmacodynamic characteristics of UTTR1147A were assessed in healthy mice, rats and cynomolgus monkeys. UTTR1147A induced STAT3 activation through binding to IL-22 receptor expressed in primary human hepatocytes and human colon cell lines. In both, activation occurred in a concentration-dependent manner with similar potencies. In the mouse colitis model, murine IL-22Fc- (muIL-22Fc) treated groups at doses of 1.25 µg and above had statistically lower average histologic colitis scores compared to the control treated group. Administration of muIL-22Fc or UTTR1147A was associated with a dose-dependent induction of PD markers REG3ß and SAA in rodents as well as REG3A, SAA and LBP in cynomolgus monkeys. The combined data confirm pharmacological activity of IL-22Fc and support potential regenerative and protective mechanisms in epithelial tissues.

IL-22R Ligands IL-20, IL-22, and IL-24 Promote Wound Healing in Diabetic db/db Mice.

Diabetic foot ulcers (DFU) are one of the major complications in type II diabetes patients and can result in amputation and morbidity. Although multiple approaches are used clinically to help wound closure, many patients still lack adequate treatment. Here we show that IL-20 subfamily cytokines are upregulated during normal wound healing. While there is a redundant role for each individual cytokine in this subfamily in wound healing, mice deficient in IL-22R, the common receptor chain for IL-20, IL-22, and IL-24, display a significant delay in wound healing. Furthermore, IL-20, IL-22 and IL-24 are all able to promote wound healing in type II diabetic db/db mice. Mechanistically, when compared to other growth factors such as VEGF and PDGF that accelerate wound healing in this model, IL-22 uniquely induced genes involved in reepithelialization, tissue remodeling and innate host defense mechanisms from wounded skin. Interestingly, IL-22 treatment showed superior efficacy compared to PDGF or VEGF in an infectious diabetic wound model. Taken together, our data suggest that IL-20 subfamily cytokines, particularly IL-20, IL-22, and IL-24, might provide therapeutic benefit for patients with DFU.

Anti-CD22 and anti-CD79b antibody-drug conjugates preferentially target proliferating B cells.

BACKGROUND AND PURPOSE: CD22 and CD79b are cell-surface receptors expressed on B-cell-derived malignancies such as non-Hodgkin's lymphoma (NHL). An anti-mitotic agent, monomethyl auristatin E, was conjugated to anti-CD22 and anti-CD79b antibodies to develop target-specific therapies for NHL. The mechanism of action (MOA) and pharmacological and pharmacokinetic (PK) profiles of these antibody-drug conjugates (ADCs) were investigated in cynomolgus monkeys. EXPERIMENTAL APPROACH: Animals were administered anti-CD22 or anti-CD79b ADCs, respective unconjugated antibodies or vehicle. Pharmacodynamic effects on total and proliferating B cells and serum PK were then assessed. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of the ADCs were evaluated in vitro. KEY RESULTS: Depletion of B cells was observed after administration of either ADC or the respective unconjugated antibodies. An extended duration of depletion was observed in animals administered ADCs. Similarly, preferential depletion of proliferating B cells in blood and germinal centre B cells in spleen were only observed in animals administered ADCs. Serum PK profiles of ADCs and respective unconjugated antibodies were comparable. In vitro, anti-human CD22 and anti-human CD79b antibodies showed no or only moderate ADCC activity, respectively; neither antibody had CDC activity. CONCLUSIONS AND IMPLICATIONS: The findings support the proposed MOA: initial depletion of total B cells by antibody-mediated opsonization, followed by preferential, sustained depletion of proliferating B cells by the auristatin conjugate due to its anti-mitotic action. Delivering potent anti-mitotic agents to B cells via the specificity of monoclonal antibodies provides a means to eliminate pathogenic B cells in NHL with improved risk-benefit profiles over traditional chemotherapeutics.

Nonselective inhibition of the epigenetic transcriptional regulator BET induces marked lymphoid and hematopoietic toxicity in mice.

Bromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4 and BRDT) are epigenetic transcriptional regulators required for efficient expression of growth promoting, cell cycle progression and antiapoptotic genes. Through their bromodomain, these proteins bind to acetylated lysine residues of histones and are recruited to transcriptionally active chromatin. Inhibition of the BET-histone interaction provides a tractable therapeutic strategy to treat diseases that may have epigenetic dysregulation. JQ1 is a small molecule that blocks BET interaction with histones. It has been shown to decrease proliferation of patient-derived multiple myeloma in vitro and to decrease tumor burden in vivo in xenograft mouse models. While targeting BET appears to be a viable and efficacious approach, the nonclinical safety profile of BET inhibition remains to be well-defined. We report that mice dosed with JQ1 at efficacious exposures demonstrate dose-dependent decreases in their lymphoid and immune cell compartments. At higher doses, JQ1 was not tolerated and due to induction of significant body weight loss led to early euthanasia. Flow cytometry analysis of lymphoid tissues showed a decrease in both B- and T-lymphocytes with a concomitant decrease in peripheral white blood cells that was confirmed by hematology. Further investigation with the inactive enantiomer of JQ1 showed that these in vivo effects were on-target mediated and not elicited through secondary pharmacology due to chemical structure.

Preclinical Safety Profile of a Depleting Antibody against CRTh2 for Asthma: Well Tolerated Despite Unexpected CRTh2 Expression on Vascular Pericytes in the Central Nervous System and Gastric Mucosa.

CRTh2 is expressed on immune cells that drive asthma pathophysiology. Current treatment options for severe asthma are inadequate and therapeutic antibody-mediated depletion of CRTh2-expressing cells represents a promising new therapeutic strategy. Here we report for the first time that CRTh2 is not only expressed on immune cells, but also on microvasculature in the central nervous system (CNS) and gastric mucosa in humans. Microvascular expression of CRTh2 raises a safety concern because a therapeutic antiCRTh2 antibody with enhanced depletion capacity could lead to vascular damage. To evaluate this safety risk, we characterized microvascular expression in human and in transgenic mice expressing human CRTh2 protein (hCRTh2.BAC.Tg) and found that CRTh2 is not localized to microvascular endothelium that is directly exposed to circulating therapeutic antibody, but rather, to pericytes that in the CNS are shielded from direct circulatory exposure by the blood-brain barrier. Immunohistochemical visualization of an intravenously administered antiCRTh2 antibody in transgenic mice revealed localization to microvascular pericytes in the gastric mucosa but not in the CNS, suggesting the blood-brain barrier effectively limits pericyte exposure to circulating therapeutic antibody in the CNS. Repeated dosing with a depleting antiCRTh2 antibody in hCRTh2.BAC.Tg mice revealed linear pharmacokinetics and no drug-related adverse findings in any tissues, including the CNS and gastric mucosa, despite complete depletion of CRTh2 expressing circulating eosinophils and basophils. Collectively, these studies demonstrate that the likelihood of drug-related CNS or gastrointestinal toxicity in humans treated with a therapeutic depleting antiCRTh2 antibody is low despite pericyte expression of CRTh2 in these tissues.

An Overview of the Pathogenesis of Immune-mediated Skin Injury.

The skin has a relatively limited range of responses to injury regardless of the specific mechanism underlying the insult. When the skin's barrier function is disrupted, it mounts an inflammatory and proliferative response in an effort to restore this essential function. The epidermal keratinocyte is central to the initiation of the skin's response, triggering an immunologic cascade that leads to the stereotypic morphologic responses that we encounter as pathologists. Drug-induced immune-mediated cutaneous injuries or "drug eruptions" are relatively common, sometimes with overlapping mechanisms, and it is often possible to classify these based on the classical hypersensitivity-type reactions. A specific type of immune-mediated skin injury is psoriasis. The pathogenesis of psoriasis is multifactorial but involves the interaction of environmental factors with a genetic predisposition. The initial stimulus triggering the development of psoriatic lesions involves activation of epidermal keratinocytes, with subsequent amplification driven by cross talk between the adaptive and innate immune systems. Several cytokines produced by Th17 T helper cells have recently been shown to be important in the pathogenesis of psoriasis, namely, interleukin-23 (IL-23) and IL-17, due to demonstrated clinical efficacy of cytokine blockade; and IL-22, based on its effects in both in vitro and in vivo models.

In Vitro Skin Models and Their Predictability in Defining Normal and Disease Biology, Pharmacology, and Toxicity.

In vitro skin model systems are increasingly being used both in the early evaluation of therapeutic drug candidates and in confirmatory mechanistic studies. The most commonly used of these in vitro model systems are reconstituted human epidermis (RHE) models. These RHE models consist solely of epidermal keratinocytes, which comes with some limitations but also with the advantage of focusing toxicologic and pharmacologic evaluation on keratinocytes alone. RHE models can generally be implemented more quickly, easily, and reproducibly than in vivo models and can thus be used for high throughput compound screening while potentially reducing the need for animal studies. Histologic evaluation of RHE sections can be done quite easily, and the sections are very amenable to quantification via image analysis, including automated analysis. RHE model systems can provide very valuable early indications of therapeutic candidate biology, pharmacology, and toxicity; and early results have demonstrated that RHE models have been quite predictive for in vivo pharmacologic and toxicologic effects on the skin, including clinical skin toxicity.

Therapeutic Antibody-Induced Vascular Toxicity Due to Off-Target Activation of Nitric Oxide in Cynomolgus Monkeys.

PRO304186, a humanized monoclonal antibody targeting soluble interleukin-17 A and F, was developed for autoimmune and inflammatory disease indications. When administered to cynomolgus monkeys PRO304186 induced unexpected adverse effects characterized by clinical signs of hematemesis, hematochezia, and moribundity. Pathology findings included hemorrhage throughout the gastrointestinal tract without any evidence of vascular wall damage or inflammatory cellular infiltration. Mechanistic investigation of these effects revealed mild elevations of serum MCP-1 and IL-12/23 but without a classical proinflammatory profile in PRO304186-treated animals. In vitro studies demonstrated off-target effects on vascular endothelial cells including activation of nitric oxide synthase leading to production of nitric oxide (NO) accompanied by increased mitochondrial membrane depolarization, glutathione depletion, and increased paracellular permeability. Additionally, endothelial cell-PRO304186-conditioned medium reduced myosin light chain phosphorylation in vascular smooth muscle cells. Furthermore, an ex vivo study utilizing segments from cynomolgus aorta and femoral artery confirmed PRO304186-induced endothelium-dependent smooth muscle relaxation and vasodilation mediated via NO. Finally, a single dose of PRO304186 in cynomolgus monkeys induced a rapid and pronounced increase in NO in the portal circulation that preceded a milder elevation of NO in the systemic circulation and corresponded temporally with systemic hypotension; findings consistent with NO-mediated vasodilation leading to hypotension. These changes were associated with non-inflammatory, localized hemorrhage in the gastrointestinal tract consistent with hemodynamic vascular injury associated with intense local vasodilation. Together, these data demonstrate that PRO304186-associated toxicity in monkeys was due to an off-target effect on endothelium that involved regional NO release resulting in severe systemic vasodilation, hypotension, and hemorrhage.

Polar solvents decrease the viscosity of high concentration IgG1 solutions through hydrophobic solvation and interaction: formulation and biocompatibility considerations.

Low-volume protein dosage forms for subcutaneous injection pose unique challenges to the pharmaceutical scientist. Indeed, high protein concentrations are often required to achieve acceptable bioavailability and efficacy for many indications. Furthermore, high solution viscosities are often observed with formulations containing protein concentrations well above 150 mg/mL. In this work, we explored the use of polar solvents for reducing solution viscosity of high concentration protein formulations intended for subcutaneous injection. An immunoglobulin, IgG1, was used in this study. The thermodynamic preferential interaction parameter (Γ23 ) measured by differential scanning calorimetry, as well as Fourier transform infrared, Raman, and second-derivative UV spectroscopy, were used to characterize the effects of polar solvents on protein structure and to reveal important mechanistic insight regarding the nature of the protein-solvent interaction. Finally, the hemolytic potential and postdose toxicity in rats were determined to further investigate the feasibility of using these cosolvents for subcutaneous pharmaceutical formulations. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1182-1193, 2013.

Minipig as a potential translatable model for monoclonal antibody pharmacokinetics after intravenous and subcutaneous administration.

Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.

The yin and yang of immunomodulatory biologics: assessing the delicate balance between benefit and risk.

A number of therapeutic immunomodulatory biologics, including antibodies, fusion proteins, and recombinant proteins, have been causally linked with serious adverse effects in humans. In nearly all cases, these serious adverse effects have been directly associated with the immunomodulatory biologic's intended pharmacologic activity or exaggerated pharmacology. Examples of immunomodulatory biologics known to cause serious adverse effects in the clinic ranging from immunostimulation and cytokine release syndrome (e.g., TGN1412) to immunosuppression with increased risk of opportunistic infections (e.g., TNF-α antagonists, anti-integrins) are presented. Specific examples of the nonclinical testing strategy used for the clinical risk assessment of these immunomodulatory biologics are discussed, with an emphasis on the clinical relevance and predictivity of the models. Infectious challenge animal models, in particular, were critically evaluated for their utility in evaluating clinical risk assessment versus understanding mechanism of action. The nonclinical safety testing strategy for an immunomodulatory biologic should be custom tailored to interrogate the biology of the immunologic target in order to best assess potential clinical risk. This nonclinical strategy should include mechanistic and efficacy models of pharmacologic activity and immunologic signaling pathways, in vitro immunologic assays such as cytokine release, and immunophenotypic assessment by flow cytometry, immunohistochemistry, and/or immunofluorescence, as appropriate.

A two-in-one antibody against HER3 and EGFR has superior inhibitory activity compared with monospecific antibodies.

Extensive crosstalk among ErbB/HER receptors suggests that blocking signaling from more than one family member may be essential to effectively treat cancer and limit drug resistance. We generated a conventional IgG molecule MEHD7945A with dual HER3/EGFR specificity by phage display engineering and used structural and mutational studies to understand how a single antigen recognition surface binds two epitopes with high affinity. As a human IgG1, MEHD7945A exhibited dual action by inhibiting EGFR- and HER3-mediated signaling in vitro and in vivo and the ability to engage immune effector functions. Compared with monospecific anti-HER antibodies, MEHD7945A was more broadly efficacious in multiple tumor models, showing that combined inhibition of EGFR and HER3 with a single antibody is beneficial.

Actin-bundling protein L-plastin regulates T cell activation.

Engagement of TCRs induces actin rearrangements, which are critical for T cell activation. T cell responses require new actin polymerization, but the significance of higher-order actin structures, such as microfilament bundles, is unknown. To determine the role of the actin-bundling protein leukocyte-plastin (L-plastin; LPL) in this process, T cells from LPL(-/-) mice were studied. LPL(-/-) T cells were markedly defective in TCR-mediated cytokine production and proliferation. LPL(-/-) T cells also spread inefficiently on surfaces with immobilized TCR ligands and formed smaller immunological synapses with APCs, likely due to defective formation of lamellipodia. LPL(-/-) mice showed delayed rejection of skin allografts after release from immunosuppression. Moreover, LPL(-/-) mice developed much less severe neurologic symptoms in experimental autoimmune encephalomyelitis, which correlated with impaired T cell responses to Ag, manifested by reduced proliferation and production of IFN-γ and IL-17. Thus, LPL-dependent actin bundling facilitates the formation of lamellipodia and normal immunological synapses and thereby enables T cell activation.

IL-27 supports germinal center function by enhancing IL-21 production and the function of T follicular helper cells.

Maturation and selection of high-affinity B cell clones in the germinal center (GC) relies on support from T follicular helper (T(FH)) cells. T(FH) cells are characterized by their localization to the B cell follicle and their high expression of the costimulatory molecules ICOS and PD1 and the cytokine IL-21, which promotes immunoglobulin (Ig) class switching and production by B cells. We show that the heterodimeric cytokine IL-27 is critical for the function of T(FH) cells and for normal and pathogenic GC responses. IL-27 signaling to T cells results in the production of IL-21, a known autocrine factor for the maintenance of T(FH) cells, in a STAT3-dependent manner. IL-27 also enhances the survival of activated CD4(+) T cells and the expression of T(FH) cell phenotypic markers. In vivo, expression of the IL-27Rα chain is required to support IL-21 production and T(FH) cell survival in a T cell-intrinsic manner. The production of high-affinity antibodies is reduced, and pristane-elicited autoantibodies and glomerulonephritis are significantly diminished, in Il27ra(-/-) mice. Together, our data show a nonredundant role for IL-27 in the development of T cell-dependent antibody responses.

B-cell activation influences T-cell polarization and outcome of anti-CD20 B-cell depletion in central nervous system autoimmunity.

OBJECTIVE: Clinical studies indicate that anti-CD20 B-cell depletion may be an effective multiple sclerosis (MS) therapy. We investigated mechanisms of anti-CD20-mediated immune modulation using 2 paradigms of experimental autoimmune encephalomyelitis (EAE). METHODS: Murine EAE was induced by recombinant myelin oligodendrocyte glycoprotein (rMOG), a model in which B cells are considered to contribute pathogenically, or MOG peptide (p)35-55, which does not require B cells. RESULTS: In EAE induced by rMOG, B cells became activated and, when serving as antigen-presenting cells (APCs), promoted differentiation of proinflammatory MOG-specific Th1 and Th17 cells. B-cell depletion prevented or reversed established rMOG-induced EAE, which was associated with less central nervous system (CNS) inflammation, elimination of meningeal B cells, and reduction of MOG-specific Th1 and Th17 cells. In contrast, in MOG p35-55-induced EAE, B cells did not become activated or efficiently polarize proinflammatory MOG-specific T cells, similar to naive B cells. In this setting, anti-CD20 treatment exacerbated EAE, and did not impede development of Th1 or Th17 cells. Irrespective of the EAE model used, B-cell depletion reduced the frequency of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg), and increased the proinflammatory polarizing capacity of remaining myeloid APCs. INTERPRETATION: Our study highlights distinct roles for B cells in CNS autoimmunity. Clinical benefit from anti-CD20 treatment may relate to inhibition of proinflammatory B cell APC function. In certain clinical settings, however, elimination of unactivated B cells, which participate in regulation of T cells and other APC, may be undesirable. Differences in immune responses to MOG protein and peptide may be important considerations when choosing an EAE model for testing novel B cell-targeting agents for MS.

IL-17RC is required for IL-17A- and IL-17F-dependent signaling and the pathogenesis of experimental autoimmune encephalomyelitis.

It has been suggested that IL-17RC forms a complex with IL-17RA to mediate the functions of IL-17A and IL-17F homodimers as well as IL-17A/F heterodimers. It is still unclear whether IL-17RC is absolutely required for the signaling of IL-17 cytokines in vivo. By using Il-17rc-deficient mice, we show that IL-17RC is essential for the signaling of IL-17A, IL-17F, and IL-17A/F both in vitro and in vivo. IL-17RC does not preassociate with IL-17RA on the cell surface; rather IL-17A can induce the formation of an IL-17RC and IL-17RA complex. This process is not dependent on the intracellular similar expression to fibroblast growth factor genes and IL-17Rs (SEFIR) domain of IL-17RC, but the SEFIR is essential in IL-17A signal transduction. Finally, Il-17rc(-/-) mice develop much milder disease in an experimental autoimmune encephalomyelitis model, supporting an essential role for IL-17RC in mediating immune-mediated CNS inflammation.