Non-Redundant Roles of T Cell Costimulation Pathways in Inflammatory Arthritis Revealed by Dual Blockade of ICOS and CD28 with Acazicolcept (ALPN-101).

OBJECTIVE: CD28 and inducible T cell costimulator (ICOS) appear to have nonredundant roles in T cell activation and adaptive immunity. We undertook this study to characterize in vitro and in vivo the therapeutic potential of acazicolcept (ALPN-101), an Fc fusion protein of a human variant ICOS ligand (ICOSL) domain designed to inhibit both CD28 and ICOS costimulation, in inflammatory arthritis. METHODS: Acazicolcept was compared in vitro with inhibitors of either the CD28 or ICOS pathways (abatacept and belatacept [CTLA-4Ig], prezalumab [anti-ICOSL monoclonal antibody]) in receptor binding and signaling assays, and in a collagen-induced arthritis (CIA) model. Acazicolcept was also compared in cytokine and gene expression assays of peripheral blood mononuclear cells (PBMCs) from healthy donors or rheumatoid arthritis (RA) or psoriatic arthritis (PsA) patients stimulated with artificial antigen-presenting cells (APCs) expressing CD28 and ICOS ligands*. RESULTS: Acazicolcept bound CD28 and ICOS, prevented ligand binding, and inhibited human T cell functional interactions, matching or exceeding the activity of CD28 or ICOS costimulatory single-pathway inhibitors tested individually or in combination. Acazicolcept administration significantly reduced disease in the CIA model and more potently than abatacept. Acazicolcept also inhibited proinflammatory cytokine production from stimulated PBMCs in cocultures with artificial APCs and demonstrated unique effects on gene expression distinct from those induced by abatacept, prezalumab, or a combination of both. CONCLUSION: Both CD28 and ICOS signaling play critical roles in inflammatory arthritis. Therapeutic agents such as acazicolcept that coinhibit both ICOS and CD28 signaling may mitigate inflammation and/or disease progression in RA and PsA more effectively than inhibitors of either pathway alone.

Systemic Administration of Acazicolcept, a Dual CD28 and Inducible T cell Costimulator Inhibitor, Ameliorates Experimental Autoimmune Uveitis.

Purpose: Combined inhibition of CD28 and inducible T cell costimulator (ICOS) pathways with acazicolcept (ALPN-101) represents a potential new treatment for uveitis. Here, we evaluate preclinical efficacy using experimental autoimmune uveitis (EAU) in Lewis rats. Methods: Efficacy was tested in 57 Lewis rats treated with either systemic (subcutaneous) or local (intravitreal) administration of acazicolcept and compared to treatment with a matched Fc-only control or corticosteroid. Impact of treatment on uveitis was assessed using clinical scoring, optical coherence tomography (OCT), and histology. Ocular effector T cell populations were determined using flow cytometry, and multiplex ELISA used to measure aqueous cytokine concentrations. Results: When compared to Fc control treatment, systemic acazicolcept led to statistically significant decreases in clinical score (P < 0.01), histologic score (P < 0.05), and number of ocular CD45+ cells (P < 0.01). Number of ocular CD4+ and CD8+ T cells expressing IL-17A+ and IFNγ+ were also decreased with statistical significance (P < 0.01). Similar results were achieved with corticosteroids. Intravitreal acazicolcept decreased inflammation scores when compared to untreated fellow eyes and to Fc control treated eyes, although not statistically significant. Systemic toxicity, measured by weight loss, occurred in the corticosteroid-treated, but not in the acazicolcept-treated animals. Conclusions: Systemic treatment with acazicolcept statistically significantly suppressed EAU. Acazicolcept was well-tolerated without the weight loss associated with corticosteroids. Acazicolcept may be an effective alternative to corticosteroids for use in treating autoimmune uveitis. Additional studies are needed to clarify the optimal dose and route for use in humans. Translational Relevance: We show that T cell costimulatory blockade could be an effective mechanism for treating uveitis.

Povetacicept, an Enhanced Dual APRIL/BAFF Antagonist That Modulates B Lymphocytes and Pathogenic Autoantibodies for the Treatment of Lupus and Other B Cell-Related Autoimmune Diseases.

OBJECTIVE: Dysregulated APRIL/BAFF signaling is implicated in the pathogenesis of multiple autoimmune diseases, including systemic lupus erythematosus and lupus nephritis. We undertook this study to develop and evaluate a high-affinity APRIL/BAFF antagonist to overcome the clinical limitations of existing B cell inhibitors. METHODS: A variant of TACI-Fc generated by directed evolution showed enhanced binding for both APRIL and BAFF and was designated povetacicept (ALPN-303). Povetacicept was compared to wild-type (WT) TACI-Fc and related molecules in vitro and in vivo. RESULTS: Povetacicept inhibited APRIL and BAFF more effectively than all evaluated forms of WT TACI-Fc and selective APRIL and BAFF inhibitors in cell-based reporter assays and primary human B cell assays, mediating potent suppression of B cell proliferation, differentiation, and immunoglobulin (Ig) secretion. In mouse immunization models, povetacicept significantly reduced serum immunoglobulin titers and antibody-secreting cells more effectively than anti-CD20 monoclonal antibodies, WT TACI-Fc, or APRIL and BAFF inhibitors. In the NZB × NZW mouse lupus nephritis model, povetacicept significantly enhanced survival and suppressed proteinuria, anti-double-stranded DNA antibody titers, blood urea nitrogen, glomerulonephritis, and renal immunoglobulin deposition. In the bm12 mouse lupus model, povetacicept significantly reduced splenic plasmablasts, follicular helper T cells, and germinal center B cells. In non-human primates, povetacicept was well tolerated, exhibited high serum exposure, and significantly decreased serum IgM, IgA, and IgG levels after a single dose. CONCLUSION: Enhanced APRIL and BAFF inhibition by povetacicept led to greater inhibition of B cell populations critical for autoantibody production compared to WT TACI-Fc and CD20-, APRIL-, or BAFF-selective inhibitors. Potent, dual inhibition by povetacicept has the potential to significantly improve clinical outcomes in autoantibody-related autoimmune diseases.

The engineered CD80 variant fusion therapeutic davoceticept combines checkpoint antagonism with conditional CD28 costimulation for anti-tumor immunity.

Despite the recent clinical success of T cell checkpoint inhibition targeting the CTLA-4 and PD-1 pathways, many patients either fail to achieve objective responses or they develop resistance to therapy. In some cases, poor responses to checkpoint blockade have been linked to suboptimal CD28 costimulation and the inability to generate and maintain a productive adaptive anti-tumor immune response. To address this, here we utilize directed evolution to engineer a CD80 IgV domain with increased PD-L1 affinity and fuse this to an immunoglobulin Fc domain, creating a therapeutic (ALPN-202, davoceticept) capable of providing CD28 costimulation in a PD-L1-dependent fashion while also antagonizing PD-1 - PD-L1 and CTLA-4-CD80/CD86 interactions. We demonstrate that by combining CD28 costimulation and dual checkpoint inhibition, ALPN-202 enhances T cell activation and anti-tumor efficacy in cell-based assays and mouse tumor models more potently than checkpoint blockade alone and thus has the potential to generate potent, clinically meaningful anti-tumor immunity in humans.

Acazicolcept (ALPN-101), a dual ICOS/CD28 antagonist, demonstrates efficacy in systemic sclerosis preclinical mouse models.

BACKGROUND: Uncontrolled immune response with T cell activation has a key role in the pathogenesis of systemic sclerosis (SSc), a disorder that is characterized by generalized fibrosis affecting particularly the lungs and skin. Costimulatory molecules are key players during immune activation, and recent evidence supports a role of CD28 and ICOS in the development of fibrosis. We herein investigated the efficacy of acazicolcept (ALPN-101), a dual ICOS/CD28 antagonist, in two complementary SSc-related mouse models recapitulating skin fibrosis, interstitial lung disease, and pulmonary hypertension. METHODS: Expression of circulating soluble ICOS and skin-expressed ICOS was investigated in SSc patients. Thereafter, acazicolcept was evaluated in the hypochlorous acid (HOCL)-induced dermal fibrosis mouse model and in the Fra-2 transgenic (Tg) mouse model. In each model, mice received 400 µg of acazicolcept or a molar-matched dose of an Fc control protein twice a week for 6 weeks. After 6 weeks, skin and lung were evaluated. RESULTS: ICOS was significantly increased in the sera from SSc patients and in SSc skin biopsies as compared to samples from healthy controls. Similar body weight changes were observed between Fc control and acazicolcept groups in both HOCL and Fra-2 Tg mice suggesting a good tolerance of acazicolcept treatment. In mice challenged with HOCL, acazicolcept induced a significant decrease in dermal thickness, collagen content, myofibroblast number, and inflammatory infiltrates characterized by B cells, T cells, neutrophils, and macrophages. In the Fra-2 Tg mouse model, acazicolcept treatment reduced lung collagen content, fibrillar collagen, histological fibrosis score, and right ventricular systolic pressure (RVSP). A reduction in frequency of CD4+ and T effector memory cells and an increase in the percentage of CD4+ T naïve cells in spleen and lung of acazicolcept-treated Fra-2 Tg mice was observed as compared to Fc control-treated Fra-2 Tg mice. Moreover, acazicolcept reduced CD69 and PD-1 expression on CD4+ T cells from the spleen and the lung. Target engagement by acazicolcept was demonstrated by blockade of CD28 and ICOS detection by flow cytometry in treated mice. CONCLUSIONS: Our results confirm the importance of costimulatory molecules in inflammatory-driven fibrosis. Our data highlight a key role of ICOS and CD28 in SSc. Using complementary models, we demonstrated that dual ICOS/CD28 blockade by acazicolcept decreased dermal and pulmonary fibrosis and alleviated pulmonary hypertension. These results pave the way for subsequent research on ICOS/CD28-targeted therapies.

IL-31 transgenic mice show reduced allergen-induced lung inflammation.

Interleukin-31 (IL-31) is a Th2 cell-derived cytokine that has been closely linked to pruritic skin inflammation. More recently, enhanced IL-31 serum levels have also been observed in patients with allergic rhinitis and allergic asthma. Therefore, the main aim of this study was to unravel the contribution of IL-31 to allergen-induced lung inflammation. We analyzed lung inflammation in response to the timothy grass (Phleum pratense) pollen allergen Phl p 5 in C57BL/6 wild-type (wt) mice, IL-31 transgenic (IL-31tg) mice, and IL-31 receptor alpha-deficient animals (IL-31RA-/- ). IL-31 and IL-31RA levels were monitored by qRT-PCR. Cellular infiltrate in bronchoalveolar lavage fluid (BALF) and lung tissue inflammation, mucus production as well as epithelial thickness were measured by flow cytometry and histomorphology. While allergen challenge induced IL-31RA expression in lung tissue of wt and IL-31tg mice, high IL-31 expression was exclusively observed in lung tissue of IL-31tg mice. Upon Phl p 5 challenge, IL-31tg mice showed reduced numbers of leukocytes and eosinophils in BALF and lung tissue as well as diminished mucin expression and less pronounced epithelial thickening compared to IL-31RA-/- or wt animals. These findings suggest that the IL-31/IL-31RA axis may regulate local, allergen-induced inflammation in the lungs.

ICOSL+ plasmacytoid dendritic cells as inducer of graft-versus-host disease, responsive to a dual ICOS/CD28 antagonist.

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic cell transplantation (HCT). CD146 and CCR5 are proteins that mark activated T helper 17 (Th17) cells. The Th17 cell phenotype is promoted by the interaction of the receptor ICOS on T cells with ICOS ligand (ICOSL) on dendritic cells (DCs). We performed multiparametric flow cytometry in a cohort of 156 HCT recipients and conducted experiments with aGVHD murine models to understand the role of ICOSL+ DCs. We observed an increased frequency of ICOSL+ plasmacytoid DCs, correlating with CD146+CCR5+ T cell frequencies, in the 64 HCT recipients with gastrointestinal aGVHD. In murine models, donor bone marrow cells from ICOSL-deficient mice compared to those from wild-type mice reduced aGVHD-related mortality. Reduced aGVHD resulted from lower intestinal infiltration of pDCs and pathogenic Th17 cells. We transplanted activated human ICOSL+ pDCs along with human peripheral blood mononuclear cells into immunocompromised mice and observed infiltration of intestinal CD146+CCR5+ T cells. We found that prophylactic administration of a dual human ICOS/CD28 antagonist (ALPN-101) prevented aGVHD in this model better than did the clinically approved belatacept (CTLA-4-Fc), which binds CD80 (B7-1) and CD86 (B7-2) and interferes with the CD28 T cell costimulatory pathway. When started at onset of aGVHD signs, ALPN-101 treatment alleviated symptoms of ongoing aGVHD and improved survival while preserving antitumoral cytotoxicity. Our data identified ICOSL+-pDCs as an aGVHD biomarker and suggest that coinhibition of the ICOSL/ICOS and B7/CD28 axes with one biologic drug may represent a therapeutic opportunity to prevent or treat aGVHD.

Novel Immunomodulatory Proteins Generated via Directed Evolution of Variant IgSF Domains.

Immunoglobulin superfamily member (IgSF) proteins play a significant role in regulating immune responses with surface expression on all immune cell subsets, making the IgSF an attractive family of proteins for therapeutic targeting in human diseases. We have developed a directed evolution platform capable of engineering IgSF domains to increase affinities for cognate ligands and/or introduce binding to non-cognate ligands. Using this scientific platform, ICOSL domains have been derived with enhanced binding to ICOS and with additional high-affinity binding to the non-cognate receptor, CD28. Fc-fusion proteins containing these engineered ICOSL domains significantly attenuate T cell activation in vitro and in vivo and can inhibit development of inflammatory diseases in mouse models. We also present evidence that engineered ICOSL domains can be formatted to selectively provide costimulatory signals to augment T cell responses. Our scientific platform thus provides a system for developing therapeutic protein candidates with selective biological impact for treatments of a wide array of human disorders including cancer and autoimmune/inflammatory diseases.

Conditioned media from the renal cell carcinoma cell line 786.O drives human blood monocytes to a monocytic myeloid-derived suppressor cell phenotype.

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells critical in mediating immune suppression in cancer patients. To develop an in vitro assay system that functionally mimics the tumor microenvironment, we cultured human monocytes with conditioned media from several cancer cell lines. Conditioned media from five tumor cell lines induced survival and differentiation of monocytes into cells characteristically similar to macrophages and MDSCs. Notably, media from the 786.O renal cell carcinoma line induced monocytes to acquire a monocytic MDSC phenotype characterized by decreased HLA-DR expression, increased nitric oxide production, enhanced proliferation, and ability to suppress autologous CD3+ T cell proliferation. We further demonstrated that these in vitro MDSCs are phenotypically and functionally similar to patient-derived MDSCs. Inhibitors of STAT3, CK2, and GM-CSF resulted in partial reversal of the MDSC phenotype. MDSCs generated in vitro from 786.O tumor conditioned media represent a platform to identify potential therapeutics that inhibit MDSC activities.

Correction: IL-31-Driven Skin Remodeling Involves Epidermal Cell Proliferation and Thickening That Lead to Impaired Skin-Barrier Function.

[This corrects the article DOI: 10.1371/journal.pone.0161877.].

Interleukin-21 combined with PD-1 or CTLA-4 blockade enhances antitumor immunity in mouse tumor models.

Recent advances in cancer treatment with checkpoint blockade of receptors such as CTLA-4 and PD-1 have demonstrated that combinations of agents with complementary immunomodulatory effects have the potential to enhance antitumor activity as compared to single agents. We investigated the efficacy of immune-modulatory interleukin-21 (IL-21) combined with checkpoint blockade in several syngeneic mouse tumor models. After tumor establishment, mice were administered recombinant mouse IL-21 (mIL-21) alone or in combination with blocking monoclonal antibodies against mouse PD-1 or CTLA-4. In contrast to monotherapy, IL-21 enhanced antitumor activity of mCTLA-4 mAb in four models and anti-PD-1 mAb in two models, with evidence of synergy for one or both of the combination treatments in the EMT-6 and MC38 models. The enhanced efficacy was associated with increased intratumoral CD8+ T cell infiltrates, CD8+ T cell proliferation, and increased effector memory T cells, along with decreased frequency of central memory CD8+ T cells. In vivo depletion of CD8+ T cells abolished the antitumor activities observed for both combination and monotherapy treatments, further supporting a beneficial role for CD8+ T cells. In all studies, the combination therapies were well tolerated. These results support the hypothesis that the combination of recombinant human IL-21 with CTLA-4 or PD-1 monoclonal antibodies could lead to improved outcomes in cancer patients.

IL-31-Driven Skin Remodeling Involves Epidermal Cell Proliferation and Thickening That Lead to Impaired Skin-Barrier Function.

Interleukin-31 (IL-31) is a type 2 helper T-cell-derived cytokine that has recently been shown to cause severe inflammation and tissue remodeling in multiple chronic diseases of the skin and lungs. IL-31 is upregulated in allergic and inflammatory diseases, including atopic dermatitis, asthma, cutaneous T-cell lymphomas, and allergic rhinitis, as well as autoimmune diseases such as systemic erythematosus. Overexpression of IL-31 in T cells causes severe inflammation, with histological features similar to skin lesions of patients with atopic dermatitis. However, the molecular mechanisms involved in IL31-driven pathological remodeling in skin diseases remain largely unknown. Here, we studied the role of IL-31 in skin damage as a result of intradermal administration of recombinant IL-31 into mice. Notably, IL-31 was sufficient to increase epidermal basal-cell proliferation and thickening of the epidermal skin layer. Our findings demonstrate a progressive increase in transepidermal water loss with chronic administration of IL-31 into the skin. Further, analysis of the skin transcriptome indicates a significant increase in the transcripts involved in epidermal-cell proliferation, epidermal thickening, and mechanical integrity. In summary, our findings demonstrate an important role for IL-31 signaling in epidermal cell proliferation and thickening that together may lead to impaired skin-barrier function in pathological remodeling of the skin.

The pruritus- and TH2-associated cytokine IL-31 promotes growth of sensory nerves.

BACKGROUND: Pruritus is a cardinal symptom of atopic dermatitis, and an increased cutaneous sensory network is thought to contribute to pruritus. Although the immune cell-IL-31-neuron axis has been implicated in severe pruritus during atopic skin inflammation, IL-31's neuropoietic potential remains elusive. OBJECTIVE: We sought to analyze the IL-31-related transcriptome in sensory neurons and to investigate whether IL-31 promotes sensory nerve fiber outgrowth. METHODS: In vitro primary sensory neuron culture systems were subjected to whole-transcriptome sequencing, ingenuity pathway analysis, immunofluorescence, and nerve elongation, as well as branching assays after IL-31 stimulation. In vivo we investigated the cutaneous sensory neuronal network in wild-type, Il31-transgenic, and IL-31 pump-equipped mice. RESULTS: Transgenic Il31 overexpression and subcutaneously delivered IL-31 induced an increase in the cutaneous nerve fiber density in lesional skin in vivo. Transcriptional profiling of IL-31-activated dorsal root ganglia neurons revealed enrichment for genes promoting nervous system development and neuronal outgrowth and negatively regulating cell death. Moreover, the growth cones of primary small-diameter dorsal root ganglia neurons showed abundant IL-31 receptor α expression. Indeed, IL-31 selectively promoted nerve fiber extension only in small-diameter neurons. Signal transducer and activator of transcription 3 phosphorylation mediated IL-31-induced neuronal outgrowth, and pharmacologic inhibition of signal transducer and activator of transcription 3 completely abolished this effect. In contrast, transient receptor potential cation channel vanilloid subtype 1 channels were dispensable for IL-31-induced neuronal sprouting. CONCLUSIONS: The pruritus- and TH2-associated novel cytokine IL-31 induces a distinct transcriptional program in sensory neurons, leading to nerve elongation and branching both in vitro and in vivo. This finding might help us understand the clinical observation that patients with atopic dermatitis experience increased sensitivity to minimal stimuli inducing sustained itch.

Th2 Cytokines Augment IL-31/IL-31RA Interactions via STAT6-dependent IL-31RA Expression.

Interleukin 31 receptor α (IL-31RA) is a novel Type I cytokine receptor that pairs with oncostatin M receptor to mediate IL-31 signaling. Binding of IL-31 to its receptor results in the phosphorylation and activation of STATs, MAPK, and JNK signaling pathways. IL-31 plays a pathogenic role in tissue inflammation, particularly in allergic diseases. Recent studies demonstrate IL-31RA expression and signaling in non-hematopoietic cells, but this receptor is poorly studied in immune cells. Macrophages are key immune-effector cells that play a critical role in Th2-cytokine-mediated allergic diseases. Here, we demonstrate that Th2 cytokines IL-4 and IL-13 are capable of up-regulating IL-31RA expression on both peritoneal and bone marrow-derived macrophages from mice. Our data also demonstrate that IL-4Rα-driven IL-31RA expression is STAT6 dependent in macrophages. Notably, the inflammation-associated genes Fizz1 and serum amyloid A (SAA) are significantly up-regulated in M2 macrophages stimulated with IL-31, but not in IL-4 receptor-deficient macrophages. Furthermore, the absence of Type II IL-4 receptor signaling is sufficient to attenuate the expression of IL-31RA in vivo during allergic asthma induced by soluble egg antigen, which may suggest a role for IL-31 signaling in Th2 cytokine-driven inflammation and allergic responses. Our study reveals an important counter-regulatory role between Th2 cytokine and IL-31 signaling involved in allergic diseases.

RNA sequencing atopic dermatitis transcriptome profiling provides insights into novel disease mechanisms with potential therapeutic implications.

BACKGROUND: Genomic profiling of lesional and nonlesional skin of patients with atopic dermatitis (AD) using microarrays has led to increased understanding of AD and identification of novel therapeutic targets. However, the limitations of microarrays might decrease detection of AD genes. These limitations might be lessened with next-generation RNA sequencing (RNA-seq). OBJECTIVE: We sought to define the lesional AD transcriptome using RNA-seq and compare it using microarrays performed on the same cohort. METHODS: RNA-seq and microarrays were performed to identify differentially expressed genes (criteria: fold change, ≥ 2.0; false discovery rate ≤ 0.05) in lesional versus nonlesional skin from 18 patients with moderate-to-severe AD, with real-time PCR (RT-PCR) and immunohistochemistry used for validation. RESULTS: Both platforms showed robust disease transcriptomes and correlated well with RT-PCR. The common AD transcriptome identified by using both techniques contained 217 genes, including inflammatory (S100A8/A9/A12, CXCL1, and 2'-5'-oligoadenylate synthetase-like [OASL]) and barrier (MKi67, keratin 16 [K16], and claudin 8 [CLDN8]) AD-related genes. Although fold change estimates determined by using RNA-seq showed somewhat better agreement with RT-PCR (intraclass correlation coefficient, 0.57 and 0.70 for microarrays and RNA-seq vs RT-PCR, respectively), bias was not eliminated. Among genes uniquely identified by using RNA-seq were triggering receptor expressed on myeloid cells 1 (TREM-1) signaling (eg, CCL2, CCL3, and single immunoglobulin domain IL1R1 related [SIGIRR]) and IL-36 isoform genes. TREM-1 is a surface receptor implicated in innate and adaptive immunity that amplifies infection-related inflammation. CONCLUSIONS: This is the first report of a lesional AD phenotype using RNA-seq and the first direct comparison between platforms in this disease. Both platforms robustly characterize the AD transcriptome. Through RNA-seq, we unraveled novel disease pathology, including increased expression of the novel TREM-1 pathway and the IL-36 cytokine in patients with AD.

A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: Involvement of TRPV1 and TRPA1.

BACKGROUND: Although the cytokine IL-31 has been implicated in inflammatory and lymphoma-associated itch, the cellular basis for its pruritic action is yet unclear. OBJECTIVE: We sought to determine whether immune cell-derived IL-31 directly stimulates sensory neurons and to identify the molecular basis of IL-31-induced itch. METHODS: We used immunohistochemistry and quantitative real-time PCR to determine IL-31 expression levels in mice and human subjects. Immunohistochemistry, immunofluorescence, quantitative real-time PCR, in vivo pharmacology, Western blotting, single-cell calcium imaging, and electrophysiology were used to examine the distribution, functionality, and cellular basis of the neuronal IL-31 receptor α in mice and human subjects. RESULTS: Among all immune and resident skin cells examined, IL-31 was predominantly produced by TH2 and, to a significantly lesser extent, mature dendritic cells. Cutaneous and intrathecal injections of IL-31 evoked intense itch, and its concentrations increased significantly in murine atopy-like dermatitis skin. Both human and mouse dorsal root ganglia neurons express IL-31RA, largely in neurons that coexpress transient receptor potential cation channel vanilloid subtype 1 (TRPV1). IL-31-induced itch was significantly reduced in TRPV1-deficient and transient receptor channel potential cation channel ankyrin subtype 1 (TRPA1)-deficient mice but not in c-kit or proteinase-activated receptor 2 mice. In cultured primary sensory neurons IL-31 triggered Ca(2+) release and extracellular signal-regulated kinase 1/2 phosphorylation, inhibition of which blocked IL-31 signaling in vitro and reduced IL-31-induced scratching in vivo. CONCLUSION: IL-31RA is a functional receptor expressed by a small subpopulation of IL-31RA(+)/TRPV1(+)/TRPA1(+) neurons and is a critical neuroimmune link between TH2 cells and sensory nerves for the generation of T cell-mediated itch. Thus targeting neuronal IL-31RA might be effective in the management of TH2-mediated itch, including atopic dermatitis and cutaneous T-cell lymphoma.

Serum from patients with SLE instructs monocytes to promote IgG and IgA plasmablast differentiation.

The development of autoantibodies is a hallmark of systemic lupus erythematosus (SLE). SLE serum can induce monocyte differentiation into dendritic cells (DCs) in a type I IFN-dependent manner. Such SLE-DCs activate T cells, but whether they promote B cell responses is not known. In this study, we demonstrate that SLE-DCs can efficiently stimulate naive and memory B cells to differentiate into IgG- and IgA-plasmablasts (PBs) resembling those found in the blood of SLE patients. SLE-DC-mediated IgG-PB differentiation is dependent on B cell-activating factor (BAFF) and IL-10, whereas IgA-PB differentiation is dependent on a proliferation-inducing ligand (APRIL). Importantly, SLE-DCs express CD138 and trans-present CD138-bound APRIL to B cells, leading to the induction of IgA switching and PB differentiation in an IFN-α-independent manner. We further found that this mechanism of providing B cell help is relevant in vivo, as CD138-bound APRIL is expressed on blood monocytes from active SLE patients. Collectively, our study suggests that a direct myeloid DC-B cell interplay might contribute to the pathogenesis of SLE.

Stromal endothelial cells establish a bidirectional crosstalk with chronic lymphocytic leukemia cells through the TNF-related factors BAFF, APRIL, and CD40L.

Chronic lymphocytic leukemia (CLL) is a clonal B cell disorder of unknown origin. Accessory signals from the microenvironment are critical for the survival, expansion, and progression of malignant B cells. We found that the CLL stroma included microvascular endothelial cells (MVECs) expressing BAFF and APRIL, two TNF family members related to the T cell-associated B cell-stimulating molecule CD40L. Constitutive release of soluble BAFF and APRIL increased upon engagement of CD40 on MVECs by CD40L aberrantly expressed on CLL cells. In addition to enhancing MVEC expression of CD40, leukemic CD40L induced cleavases that elicited intracellular processing of pro-BAFF and pro-APRIL proteins in MVECs. The resulting soluble BAFF and APRIL proteins delivered survival, activation, Ig gene remodeling, and differentiation signals by stimulating CLL cells through TACI, BAFF-R, and BCMA receptors. BAFF and APRIL further amplified CLL cell survival by upregulating the expression of leukemic CD40L. Inhibition of TACI, BCMA, and BAFF-R expression on CLL cells; abrogation of CD40 expression in MVECs; or suppression of BAFF and APRIL cleavases in MVECs reduced the survival and diversification of malignant B cells. These data indicate that BAFF, APRIL, and CD40L form a CLL-enhancing bidirectional signaling network linking neoplastic B cells with the microvascular stroma.

Generation and characterization of human anti-human IL-21 neutralizing monoclonal antibodies.

Interleukin-21 (IL-21) is a type I four-helical bundle cytokine that exerts a variety of significant effects on many hematopoietic cells, including T and B lymphocytes and natural killer cells. IL-21 is produced predominantly by CD4+ T cells and natural killer T cells and, when aberrantly overexpressed, appears to play important roles in a wide variety of autoimmune disorders. To generate potential therapeutic reagents capable of inhibiting IL-21 for clinical use, we immunized human immunoglobulin transgenic mice with IL-21 and then identified and cloned a panel of human anti-human IL-21 binding monoclonal antibodies. IL-21 neutralizing and IL-21-binding, non-neutralizing antibodies were assigned to distinct epitope "bins" based on surface plasmon resonance competition studies. The most potent neutralizing antibodies had extremely high (sub pM) affinity for IL-21 and were able to block IL-21 activity in various biological assays using either an IL-21R-transfected pre-B-cell line or primary human B cells, and their neutralizing activity was, in some cases, superior to that of a soluble form of the high affinity heterodimeric IL-21 receptor. Characterization of this panel of IL-21 antibodies provided the basis for the selection of a therapeutic candidate antibody capable of inhibiting IL-21 activity for the treatment of autoimmune and inflammatory diseases.