A Camelid-Derived STAT-Specific Nanobody Inhibits Neuroinflammation and Ameliorates Experimental Autoimmune Encephalomyelitis (EAE).

Proinflammatory T-lymphocytes recruited into the brain and spinal cord mediate multiple sclerosis (MS) and currently there is no cure for MS. IFN-γ-producing Th1 cells induce ascending paralysis in the spinal cord while IL-17-producing Th17 cells mediate cerebellar ataxia. STAT1 and STAT3 are required for Th1 and Th17 development, respectively, and the simultaneous targeting of STAT1 and STAT3 pathways is therefore a potential therapeutic strategy for suppressing disease in the spinal cord and brain. However, the pharmacological targeting of STAT1 and STAT3 presents significant challenges because of their intracellular localization. We have developed a STAT-specific single-domain nanobody (SBT-100) derived from camelids that targets conserved residues in Src homolog 2 (SH2) domains of STAT1 and STAT3. This study investigated whether SBT-100 could suppress experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We show that SBT-100 ameliorates encephalomyelitis through suppressing the expansion of Th17 and Th1 cells in the brain and spinal cord. Adoptive transfer experiments revealed that lymphocytes from SBT-100-treated EAE mice have reduced capacity to induce EAE, indicating that the immunosuppressive effects derived from the direct suppression of encephalitogenic T-cells. The small size of SBT-100 makes this STAT-specific nanobody a promising immunotherapy for CNS autoimmune diseases, including multiple sclerosis.

IL-27-containing exosomes secreted by innate B-1a cells suppress and ameliorate uveitis.

Introduction: IL-27 is a heterodimeric cytokine composed of Ebi3 and IL-27p28 and can exert proinflammatory or immune suppressive effects depending on the physiological context. Ebi3 does not contain membrane-anchoring motifs, suggesting that it is a secreted protein while IL-27p28 is poorly secreted. How IL-27p28 and Ebi3 dimerize in-vivo to form biologically active IL-27 is unknown. Major impediment to clinical use of IL-27 derives from difficulty of determining exact amount of bioavailable heterodimeric IL-27 needed for therapy. Methods: To understand how IL-27 mediates immune suppression, we characterized an innate IL-27-producing B-1a regulatory B cell population (i27-Breg) and mechanisms i27-Bregs utilize to suppress neuroinflammation in mouse model of uveitis. We also investigated biosynthesis of IL-27 and i27-Breg immunobiology by FACS, immunohistochemical and confocal microscopy. Results: Contrary to prevailing view that IL-27 is a soluble cytokine, we show that i27-Bregs express membrane-bound IL-27. Immunohistochemical and confocal analyses co-localized expression of IL-27p28 at the plasma membrane in association with CD81 tetraspanin, a BCR-coreceptor protein and revealed that IL-27p28 is a transmembrane protein in B cells. Most surprising, we found that i27-Bregs secrete IL-27-containing exosomes (i27-exosomes) and adoptive transfer of i27-exosomes suppressed uveitis by antagonizing Th1/Th17 cells, up-regulating inhibitory-receptors associated with T-cell exhaustion while inducing Treg expansion. Discussion: Use of i27-exosomes thus obviates the IL-27 dosing problem, making it possible to determine bioavailable heterodimeric IL-27 needed for therapy. Moreover, as exosomes readily cross the blood-retina-barrier and no adverse effects were observed in mice treated with i27-exosome, results of this study suggest that i27-exosomes might be a promising therapeutic approach for CNS autoimmune diseases.

Photoreceptor Cells Constitutively Express IL-35 and Promote Ocular Immune Privilege.

Interleukin-27 is constitutively secreted by microglia in the retina or brain, and upregulation of IL-27 during neuroinflammation suppresses encephalomyelitis and autoimmune uveitis. However, while IL-35 is structurally and functionally similar to IL-27, the intrinsic roles of IL-35 in CNS tissues are unknown. Thus, we generated IL-35/YFP-knock-in reporter mice (p35-KI) and demonstrated that photoreceptor neurons constitutively secrete IL-35, which might protect the retina from persistent low-grade inflammation that can impair photoreceptor functions. Furthermore, the p35-KI mouse, which is hemizygous at the il12a locus, develops more severe uveitis because of reduced IL-35 expression. Interestingly, onset and exacerbation of uveitis in p35-KI mice caused by extravasation of proinflammatory Th1/Th17 lymphocytes into the retina were preceded by a dramatic decrease of IL-35, attributable to massive death of photoreceptor cells. Thus, while inflammation-induced death of photoreceptors and loss of protective effects of IL-35 exacerbated uveitis, our data also suggest that constitutive production of IL-35 in the retina might have housekeeping functions that promote sterilization immunity in the neuroretina and maintain ocular immune privilege.

miR-204-containing exosomes ameliorate GVHD-associated dry eye disease.

Graft-versus-host disease (GVHD)­associated dry eye disease is characterized by extensive inflammatory destruction in the ocular surface and causes unbearable pain and visual impairment. Current treatments provide limited benefits. Here, we report that exosomes from mesenchymal stromal cells (MSC-exo) administered as eye drops notably alleviate GVHD-associated dry eye disease by suppressing inflammation and improving epithelial recovery in mice and humans. In a prospective clinical trial, 28 eyes with refractory GVHD­dry eye disease exhibited substantial relief after MSC-exo treatment, showing reduced fluorescein scores, longer tear-film breakup time, increased tear secretion, and lower OSDI scores. Mechanistically, MSC-exo reprogramed proinflammatory M1 macrophages toward the immunosuppressive M2 via miR-204­mediated targeting of the IL-6/IL-6R/Stat3 pathway. Blockade of miR-204 abolished the effects of MSC-exo, while overloading L929-exo with miR-204 markedly attenuated dry eye. Thus, this study suggests that MSC-exo are efficacious in treating GVHD-associated dry eye disease and highlights miR-204 as a potential therapeutic agent.

IL-27-producing B-1a cells suppress neuroinflammation and CNS autoimmune diseases.

Regulatory B cells (Breg cells) that secrete IL-10 or IL-35 (i35-Breg) play key roles in regulating immunity in tumor microenvironment or during autoimmune and infectious diseases. Thus, loss of Breg function is implicated in development of autoimmune diseases while aberrant elevation of Breg prevents sterilizing immunity, exacerbates infectious diseases, and promotes cancer metastasis. Breg cells identified thus far are largely antigen-specific and derive mainly from B2-lymphocyte lineage. Here, we describe an innate-like IL-27-producing natural regulatory B-1a cell (i27-Breg) in peritoneal cavity and human umbilical cord blood. i27-Bregs accumulate in CNS and lymphoid tissues during neuroinflammation and confers protection against CNS autoimmune disease. i27-Breg immunotherapy ameliorated encephalomyelitis and uveitis through up-regulation of inhibitory receptors (Lag3, PD-1), suppression of Th17/Th1 responses, and propagating inhibitory signals that convert conventional B cells to regulatory lymphocytes that secrete IL-10 and/or IL-35 in eye, brain, or spinal cord. Furthermore, i27-Breg proliferates in vivo and sustains IL-27 secretion in CNS and lymphoid tissues, a therapeutic advantage over administering biologics (IL-10, IL-35) that are rapidly cleared in vivo. Mutant mice lacking irf4 in B cells exhibit exaggerated increase of i27-Bregs with few i35-Bregs, while mice with loss of irf8 in B cells have abundance of i35-Bregs but defective in generating i27-Bregs, identifying IRF8/BATF and IRF4/BATF axis in skewing B cell differentiation toward i27-Breg and i35-Breg developmental programs, respectively. Consistent with its developmental origin, disease suppression by innate i27-Bregs is neither antigen-specific nor disease-specific, suggesting that i27-Breg would be effective immunotherapy for a wide spectrum of autoimmune diseases.

STAT3-Specific Single Domain Nanobody Inhibits Expansion of Pathogenic Th17 Responses and Suppresses Uveitis in Mice.

STAT3 activates transcription of genes that regulate cell growth, differentiation, and survival of mammalian cells. Genetic deletion of Stat3 in T cells has been shown to abrogate Th17 differentiation, suggesting that STAT3 is a potential therapeutic target for Th17-mediated diseases. However, a major impediment to therapeutic targeting of intracellular proteins such as STAT3 is the lack of efficient methods for delivering STAT3 inhibitors into cells. In this study, we developed a novel antibody (SBT-100) comprised of the variable (V) region of a STAT3-specific heavy chain molecule and demonstrate that this 15 kDa STAT3-specific nanobody enters human and mouse cells, and induced suppression of STAT3 activation and lymphocyte proliferation in a concentration-dependent manner. To investigate whether SBT-100 would be effective in suppressing inflammation in vivo, we induced experimental autoimmune uveitis (EAU) in C57BL/6J mice by active immunization with peptide from the ocular autoantigen, interphotoreceptor retinoid binding protein (IRBP651-670). Analysis of the retina by fundoscopy, histological examination, or optical coherence tomography showed that treatment of the mice with SBT-100 suppressed uveitis by inhibiting expansion of pathogenic Th17 cells that mediate EAU. Electroretinographic (ERG) recordings of dark and light adapted a- and b-waves showed that SBT-100 treatment rescued mice from developing significant visual impairment observed in untreated EAU mice. Adoptive transfer of activated IRBP-specific T cells from untreated EAU mice induced EAU, while EAU was significantly attenuated in mice that received IRBP-specific T cells from SBT-100 treated mice. Taken together, these results demonstrate efficacy of SBT-100 in mice and suggests its therapeutic potential for human autoimmune diseases.

Uveitis: Molecular Pathogenesis and Emerging Therapies.

The profound impact that vision loss has on human activities and quality of life necessitates understanding the etiology of potentially blinding diseases and their clinical management. The unique anatomic features of the eye and its sequestration from peripheral immune system also provides a framework for studying other diseases in immune privileged sites and validating basic immunological principles. Thus, early studies of intraocular inflammatory diseases (uveitis) were at the forefront of research on organ transplantation. These studies laid the groundwork for foundational discoveries on how immune system distinguishes self from non-self and established current concepts of acquired immune tolerance and autoimmunity. Our charge in this review is to examine how advances in molecular cell biology and immunology over the past 3 decades have contributed to the understanding of mechanisms that underlie immunopathogenesis of uveitis. Particular emphasis is on how advances in biotechnology have been leveraged in developing biologics and cell-based immunotherapies for uveitis and other neuroinflammatory diseases.

Deletion of Irf4 in T Cells Suppressed Autoimmune Uveitis and Dysregulated Transcriptional Programs Linked to CD4+ T Cell Differentiation and Metabolism.

Interferon regulatory factor-4 (IRF4) and IRF8 regulate differentiation, growth and functions of lymphoid and myeloid cells. Targeted deletion of irf8 in T cells (CD4-IRF8KO) has been shown to exacerbate colitis and experimental autoimmune uveitis (EAU), a mouse model of human uveitis. We therefore generated mice lacking irf4 in T cells (CD4-IRF4KO) and investigated whether expression of IRF4 by T cells is also required for regulating T cells that suppress autoimmune diseases. Surprisingly, we found that CD4-IRF4KO mice are resistant to EAU. Suppression of EAU derived in part from inhibiting pathogenic responses of Th17 cells while inducing expansion of regulatory lymphocytes that secrete IL-10 and/or IL-35 in the eye and peripheral lymphoid tissues. Furthermore, CD4-IRF4KO T cells exhibit alterations in cell metabolism and are defective in the expression of two Ikaros zinc-finger (IKZF) transcription factors (Ikaros, Aiolos) that are required for lymphocyte differentiation, metabolism and cell-fate decisions. Thus, synergistic effects of IRF4 and IkZFs might induce metabolic reprogramming of differentiating lymphocytes and thereby dynamically regulate relative abundance of T and B lymphocyte subsets that mediate immunopathogenic mechanisms during uveitis. Moreover, the diametrically opposite effects of IRF4 and IRF8 during EAU suggests that intrinsic function of IRF4 in T cells might be activating proinflammatory responses while IRF8 promotes expansion of immune-suppressive mechanisms.

Analysis of Regulatory B Cells in Experimental Autoimmune Uveitis.

Regulatory B cells (Bregs) that produce IL-35 and IL-10 (i35-Bregs) regulate central nervous system (CNS) autoimmune diseases including uveitis. In the mouse model of uveitis, i35-Breg cells suppress intraocular inflammation by inducing expansion of IL-10-producing B cells (B10), IL-10-producing T cells (Tregs), and IL-35-producing T cells (iTR35), suggesting that i35-Bregs orchestrate an immune-suppressive milieu that regulates immunity during autoimmune diseases. In this chapter, we discuss uveitis and therapeutic challenges that necessitate the development of cell-based therapies for the treatment of these potentially blinding diseases that cause 10% visual handicap. We then describe the methods we set up for ex vivo generation of i35-Breg cells employed in i35-Breg immunotherapy in uveitis and in other CNS autoimmune diseases.

Interleukin 35 Regulatory B Cells.

B lymphocytes play a central role in host immunity. They orchestrate humoral immune responses that modulate activities of other immune cells and produce neutralizing antibodies that confer lasting immunity to infectious diseases including smallpox, measles and poliomyelitis. In addition to these traditional functions is the recent recognition that B cells also play critical role in maintaining peripheral tolerance and suppressing the development or severity of autoimmune diseases. Their immune suppressive function is attributed to relatively rare populations of regulatory B cells (Bregs) that produce anti-inflammatory cytokines including interleukin 10 (IL-10), IL-35 and transforming growth factor-ß. The IL-35-producing B cell (i35-Breg) is the newest Breg subset described. i35-Bregs suppress central nervous system autoimmune diseases by inducing infectious tolerance whereby conventional B cells acquire regulatory functions that suppress pathogenic Th17 responses. In this review, we discuss immunobiology of i35-Breg cell, i35-Breg therapies for autoimmune diseases and potential therapeutic strategies for depleting i35-Bregs that suppress immune responses against pathogens and tumor cells.

STAT3 deficiency in B cells exacerbates uveitis by promoting expansion of pathogenic lymphocytes and suppressing regulatory B cells (Bregs) and Tregs.

STAT3 transcription factor induces differentiation of naïve T cells into Th17 cells and loss of STAT3 in T cell prevents development of CNS autoimmune diseases. However, function of STAT3 in the B lymphocyte subset is not well understood. In this study, we have generated mice lacking STAT3 in CD19+ B cells (CD19-STAT3KO) and investigated intrinsic and extrinsic functions of STAT3 in B cells and its potential role in resistance or pathogenesis of organ-specific autoimmune diseases. We show that STAT3 regulates metabolic mechanisms in B cells with implications for bioenergetic and metabolic pathways that control cellular homeostasis in B cells. Thus, loss of STAT3 in CD19-STAT3KO cells perturbed growth and apoptosis by inducing rapid entry of B cells into the S-phase of the cell cycle, decreasing expression of cyclin-dependent kinase inhibitors and upregulating pro-apoptotic proteins. We further show that the CD19-STAT3KO mice develop severe experimental autoimmune uveitis (EAU), an animal model of human uveitis. Exacerbated uveitis in CD19-STAT3KO mice derived in part from enhanced expression of costimulatory molecules on B cells, marked increase of Th17 responses and increased recruitment of granulocytes into the neuroretina. The enhanced autoimmunity upon deletion of STAT3 in B cells is also recapitulated in experimental autoimmune encephalitis, a mouse model of multiple sclerosis and thus support our conclusion that STAT3 deletion in B cells enhanced inflammation and the effects observed are not model specific. Our data further indicate that STAT3 pathway modulates interactions between B and T cells during EAU resulting in alteration of lymphocyte repertoire by increasing levels of autoreactive pathogenic T cells while suppressing development and/or expansion of immune-suppressive lymphocytes (Bregs and Tregs). Taken together, STAT3 exerts diametrically opposite effects in lymphocytes, with loss of STAT3 in B cells exacerbating uveitis whereas Stat3 deletion in T cells confers protection.

Interleukin 35-Producing Exosomes Suppress Neuroinflammation and Autoimmune Uveitis.

Corticosteroids are effective therapy for autoimmune diseases but serious adverse effects preclude their prolonged use. However, immune-suppressive biologics that inhibit lymphoid proliferation are now in use as corticosteroid sparing-agents but with variable success; thus, the need to develop alternative immune-suppressive approaches including cell-based therapies. Efficacy of ex-vivo-generated IL-35-producing regulatory B-cells (i35-Bregs) in suppressing/ameliorating encephalomyelitis or uveitis in mouse models of multiple sclerosis or uveitis, respectively, is therefore a promising therapeutic approach for CNS autoimmune diseases. However, i35-Breg therapy in human uveitis would require producing autologous Bregs from each patient to avoid immune-rejection. Because exosomes exhibit minimal toxicity and immunogenicity, we investigated whether i35-Bregs release exosomes that can be exploited therapeutically. Here, we demonstrate that i35-Bregs release exosomes that contain IL-35 (i35-Exosomes). In this proof-of-concept study, we induced experimental autoimmune uveitis (EAU), monitored EAU progression by fundoscopy, histology, optical coherence tomography and electroretinography, and investigated whether i35-Exosomes treatment would suppress uveitis. Mice treated with i35-Exosomes developed mild EAU with low EAU scores and disease protection correlated with expansion of IL-10 and IL-35 secreting Treg cells with concomitant suppression of Th17 responses. In contrast, significant increase of Th17 cells in vitreous and retina of control mouse eyes was accompanied by severe choroiditis, massive retinal-folds, and photoreceptor cell damage. These hallmark features of severe uveitis were absent in exosome-treated mice and visual impairment detected by ERG was modest compared to control mice. Absence of toxicity or alloreactivity associated with exosomes thus makes i35-Exosomes attractive therapeutic option for delivering IL-35 into CNS tissues.

Persistent Activation of STAT3 Pathway in the Retina Induced Vision Impairment and Retinal Degenerative Changes in Ageing Mice.

Neurotrophic factors can promote the survival of degenerating retinal cells through the activation of STAT3 pathway. Thus, augmenting STAT3 activation in the retina has been proposed as potential therapy for retinal dystrophies. On the other hand, aberrant activation of STAT3 pathway is oncogenic and implicated in diverse human diseases. Furthermore, the STAT3/SOCS3 axis has been shown to induce the degradation of rhodopsin during retinal inflammation. In this study, we generated and used mice with constitutive activation of STAT3 pathway in the retina to evaluate the safety and consequences of enhancing STAT3 activities in the retina as a potential treatment for retinal degenerative diseases. We show that long-term activation of the STAT3 pathway can induce retinal degenerative changes and also exacerbate uveitis and other intraocular inflammatory diseases. Mechanisms underlying the development of vision impairment in the STAT3c-Tg mice derived in part from STAT3-mediated inhibition of rhodopsin and overexpression of SOCS3 in the retina. These results suggest that much caution should be exercised in the use of STAT3 augmentation therapy for retinal dystrophies.

Production of IL-35 by Bregs is mediated through binding of BATF-IRF-4-IRF-8 complex to il12a and ebi3 promoter elements.

IL-10 and IL-35 suppress excessive immune responses and therapeutic strategies are being developed to increase their levels in autoimmune diseases. In this study, we sought to identify major cell types that produce both cytokines in-vivo and to characterize mechanisms that regulate their production. Experimental autoimmune uveitis (EAU) is a CNS autoimmune disease that serves as model of human uveitis. We induced EAU in C57BL/6J mice and investigated whether T cells, B lymphocytes, or myeloid cells are the major producers of IL-10 or IL-35 in blood, lymph nodes (LNs), spleen, and at the site of ocular inflammation, the neuroretina. Analysis of these tissues identified B cells as the major producers of IL-10 and IL-35 in-vivo. Compared to regulatory T cells (Tregs), IL-10- or IL-35-producing regulatory B cells (Bregs) are substantially expanded in blood, LNs, spleen, and retina of mice with EAU. We performed EMSA and chromatin immunoprecipitation (ChIP) assays on activated B cells stimulated with IL-35 or TLR agonists. We found that BATF, IFN regulatory factor (IRF)-4, and IRF-8 transcription factors were recruited and bound to AP1-IRF-composite elements (AICEs) of il12a, ebi3, and/or il10 loci, suggesting their involvement in regulating IL-10 and IL-35 transcriptional programs of B cells. Showing that B cells are major source of IL-10 and IL-35 in-vivo and identifying transcription factors that contribute to IL-10 and IL-35 expression in the activated B-cell, suggest that the BATF/IRF-4/IRF-8 axis can be exploited therapeutically to regulate physiological levels of IL-10/IL-35-Bregs and that adoptive transfer of autologous Bregs might be an effective therapy for autoimmune and neurodegenerative diseases.

IL-12p35 Inhibits Neuroinflammation and Ameliorates Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease in which cytokines produced by immune cells that infiltrate the brain and spinal cord play a central role. We show here that the IL-12p35, the alpha subunit of IL-12 or IL-35 cytokine, might be an effective biologic for suppressing neuroinflammatory responses and ameliorating the pathology of experimental autoimmune encephalomyelitis (EAE), the mouse model of human MS. We further show that IL-12p35 conferred protection from neuropathy by inhibiting the expansion of pathogenic Th17 and Th1 cells and inhibiting trafficking of inflammatory cells into the brain and spinal cord. In addition, in vitro exposure of encephalitogenic cells to IL-12p35 suppressed their capacity to induce EAE by adoptive transfer. Importantly, the IL-12p35-mediated expansion of Treg and Breg cells and its amelioration of EAE correlated with inhibition of cytokine-induced activation of STAT1/STAT3 pathways. Moreover, IL-12p35 inhibited lymphocyte proliferation by suppressing the expressions of cell-cycle regulatory proteins. Taken together, these results suggest that IL-12p35 can be exploited as a novel biologic for treating central nervous system autoimmune diseases and offers the promise of ex vivo production of large amounts of Tregs and Bregs for immunotherapy.

IL-12p35 induces expansion of IL-10 and IL-35-expressing regulatory B cells and ameliorates autoimmune disease.

Interleukin 35 (IL-35) is a heterodimeric cytokine composed of IL-12p35 and Ebi3 subunits. IL-35 suppresses autoimmune diseases while preventing host defense to infection and promoting tumor growth and metastasis by converting resting B and T cells into IL-10-producing and IL-35-producing regulatory B (Breg) and T (Treg) cells. Despite sharing the IL-12p35 subunit, IL-12 (IL-12p35/IL-12p40) promotes inflammatory responses whereas IL-35 (IL-12p35/Ebi3) induces regulatory responses, suggesting that IL-12p35 may have unknown intrinsic immune-regulatory functions regulated by its heterodimeric partner. Here we show that the IL-12p35 subunit has immunoregulatory functions hitherto attributed to IL-35. IL-12p35 suppresses lymphocyte proliferation, induces expansion of IL-10-expressing and IL-35-expressing B cells and ameliorates autoimmune uveitis in mice by antagonizing pathogenic Th17 responses. Recapitulation of essential immunosuppressive activities of IL-35 indicates that IL-12p35 may be utilized for in vivo expansion of Breg cells and autologous Breg cell immunotherapy. Furthermore, our uveitis data suggest that intrinsic immunoregulatory activities of other single chain IL-12 subunits might be exploited to treat other autoimmune diseases.IL-12p35 is common to IL-35 and IL-12, which have opposing effects on inflammation. Here the authors show that the IL-12p35 subunit induces regulatory B cells and can be used therapeutically to limit autoimmune uveitis in mice.

SOCS1 Mimetic Peptide Suppresses Chronic Intraocular Inflammatory Disease (Uveitis).

Uveitis is a potentially sight-threatening disease characterized by repeated cycles of remission and recurrent inflammation. The JAK/STAT pathway regulates the differentiation of pathogenic Th1 and Th17 cells that mediate uveitis. A SOCS1 mimetic peptide (SOCS1-KIR) that inhibits JAK2/STAT1 pathways has recently been shown to suppress experimental autoimmune uveitis (EAU). However, it is not clear whether SOCS1-KIR ameliorated uveitis by targeting JAK/STAT pathways of pathogenic lymphocytes or via inhibition of macrophages and antigen-presenting cells that also enter the retina during EAU. To further investigate mechanisms that mediate SOCS1-KIR effects and evaluate the efficacy of SOCS1-KIR as an investigational drug for chronic uveitis, we induced EAU in rats by adoptive transfer of uveitogenic T-cells and monitored disease progression and severity by slit-lamp microscopy, histology, and optical coherence tomography. Topical administration of SOCS1-KIR ameliorated acute and chronic posterior uveitis by inhibiting Th17 cells and the recruitment of inflammatory cells into retina while promoting expansion of IL-10-producing Tregs. We further show that SOCS1-KIR conferred protection of resident retinal cells that play critical role in vision from cytotoxic effects of inflammatory cytokines by downregulating proapoptotic genes. Thus, SOCS1-KIR suppresses uveitis and confers neuroprotective effects and might be exploited as a noninvasive treatment for chronic uveitis.

Interferon Regulator Factor 8 (IRF8) Limits Ocular Pathology during HSV-1 Infection by Restraining the Activation and Expansion of CD8+ T Cells.

Interferon Regulatory Factor-8 (IRF8) is constitutively expressed in monocytes and B cell lineages and plays important roles in immunity to pathogens and cancer. Although IRF8 expression is induced in activated T cells, the functional relevance of IRF8 in T cell-mediated immunity is not well understood. In this study, we used mice with targeted deletion of Irf8 in T-cells (IRF8KO) to investigate the role of IRF8 in T cell-mediated responses during herpes simplex virus 1 (HSV-1) infection of the eye. In contrast to wild type mice, HSV-1-infected IRF8KO mice mounted a more robust anti-HSV-1 immune response, which included marked expansion of HSV-1-specific CD8+ T cells, increased infiltration of inflammatory cells into the cornea and trigeminal ganglia (TG) and enhanced elimination of virus within the trigeminal ganglion. However, the consequence of the enhanced immunological response was the development of ocular inflammation, limbitis, and neutrophilic infiltration into the cornea of HSV-1-infected IRF8KO mice. Surprisingly, we observed a marked increase in virus-specific memory precursor effector cells (MPEC) in IRF8KO mice, suggesting that IRF8 might play a role in regulating the differentiation of effector CD8+ T cells to the memory phenotype. Together, our data suggest that IRF8 might play a role in restraining excess lymphocyte proliferation. Thus, modulating IRF8 levels in T cells can be exploited therapeutically to prevent immune-mediated ocular pathology during autoimmune and infectious diseases of the eye.

A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice.

Interleukin-12 family cytokines have emerged as critical regulators of immunity with some members (IL-12, IL-23) associated with disease pathogenesis while others (IL-27, IL-35) mitigate autoimmune diseases. Each IL-12 family member is comprised of an α and a ß chain, and chain-sharing is a key feature. Although four bona fide members have thus far been described, promiscuous chain-pairing between alpha (IL-23p19, IL-27p28, IL-12/IL-35p35) and beta (IL-12/IL-23p40, IL-27/IL-35Ebi3) subunits, predicts six possible heterodimeric IL-12 family cytokines. Here, we describe a new IL-12 member composed of IL-23p19 and Ebi3 heterodimer (IL-39) that is secreted by LPS-stimulated B cells and GL7(+) activated B cells of lupus-like mice. We further show that IL-39 mediates inflammatory responses through activation of STAT1/STAT3 in lupus-like mice. Taken together, our results show that IL-39 might contribute to immunopathogenic mechanisms of systemic lupus erythematosus, and could be used as a possible target for its treatment.

Pre-existing CD19-independent GL7(-) Breg cells are expanded during inflammation and in mice with lupus-like disease.

Interleukin 10 (IL-10)-producing regulatory B-cells (Bregs) suppress inflammatory responses that mediate autoimmune diseases. However, it is unknown whether Bregs derive from a pre-existing dedicated B-cell lineage or if any B-cell can differentiate into Bregs in response to BCR or TLR activation. GL7(+) B-cells are antigen-experienced differentiated B-cells while GL7(-/lo) are at an early stage of B-cell differentiation. While both GL7(-/lo) and GL7(+) B cells can produce IL-10, differentiation of GL7(-) B-cells into Bregs does not require CD19- or Bcl6-induced signals, suggesting that BCR-induced proliferation or Ig class-switching is not necessary for generation of Breg cells. Of particular importance, we show that GL7(-) Breg cells are dramatically expanded in lupus-like mice and GL7(-) Bregs suppressed inflammatory responses in lupus-like mice by inducing expansion of Foxp3(+)Treg cells. Taken together, these results suggest that pre-existing GL7(-)IL-10(+) cells are expanded during inflammation, differentiate into GL7(+) Bregs and contribute to immune-regulation in lupus-like mice.