Ig Fusion Protein Research Articles

A chimeric HLA-A2:β2M:Ig fusion protein for the study of virus-specific CD8+ T-cells

IntroductionThe response mediated by CD8+ T-cells in the context of infection and vaccination has been thoroughly investigated and represents one of the most important branches that allow for the development of immunity against intracellular pathogens and, thus, the establishment of robust antiviral responses. However, there is a lack of methods to assess antigen-specific CD8+ T-cells. ObjectiveSearch for the ideal assays to assess the function of antigen-specific CD8+ T-cells. MethodsIn the present study a chimeric HLA-A2:β2M:Ig fusion protein was produced, purified, and evaluated in functional CD8+ T-cell response studies using samples from Influenza A patients and humanized mice upon adenoviral vaccination. ResultsThe HLA-A2:β2M:Ig molecule, bound to immunodominant viral peptides by passive transfer, was able to induce robust antiviral CD8+ T-cell responses mediated by IFN-γ. The in vitro IFN-γ release assay using the chimeric HLA-A2:β2M:Ig fusion protein detected bona fide human CD8+ T-cells, demonstrating superior production of IFN-γ by human CD8+ T-cells induced by Influenza A immunodominant GILGFVFTL peptide. Removal of antigen-presenting cells and CD8+ T-cell enrichment improved significantly the IFN-γ production. The chimeric HLA-A2:β2M:Ig fusion protein also triggered HLA-A2-restricted CD8+ T-cell response in a humanized mouse model upon vaccination with adenovirus encoding HLA-A2-restricted HIV p24 antigen. The results strongly suggest the use of tailor-made assays for detecting HLA-A2-restricted CD8+ T-cell Responses in the Humanized Mouse Model. ConclusionThe chimeric HLA-A2:β2M:Ig fusion protein-based assays provided a sensitive tool that may be paramount to measure virus-specific CD8+ T-cell response in a range of viral infections of clinical relevance.

Circulating TFH cells as a marker for early therapeutic intervention in T1D.

Costimulatory blockade via the CTLA-4–Ig fusion protein abatacept is beneficial in patients with early-onset type 1 diabetes, but some individuals benefit more than others. A new study reports that the pretreatment abundance of T follicular helper (TFH) cells could predict clinical responses to abatacept.

Jak-Stat Signaling Pathway Related Gene Expressions and Blood Biochemical indicators in Acute Promyelocytic Leukemia

Jak-Stat Signaling Pathway Related Gene Expressions and Blood Biochemical indicators in Acute Promyelocytic Leukemia

Direct Binding Analysis Between C-Type Lectins and Glycans Using Immunoglobulin Receptor Fusion Proteins.

C-type lectins bind to carbohydrate structures in a Ca2+-dependent manner. Some transmembrane forms of lectins act as innate immune receptors and induce signal transduction pathways in macrophages and dendritic cells (DCs). Expressing these receptors in cells bearing a reporter gene is a useful tool to investigate ligand binding and recognition. However, it cannot be used to quantify the precise affinity of the interaction, and the involvement of other proteins remains a possibility. Direct binding between a receptor and its ligand can be investigated using an immunoglobulin receptor (Ig)-fused soluble protein. This binding can be assessed using enzyme-linked immunosorbent assays and flow cytometry, and the fusion protein may also be used in a glycan array. In this chapter, we explain the generation of Ig fusion proteins and subsequent binding assays using these proteins.

Ancient BCMA-like Genes Herald B Cell Regulation in Lampreys.

The TNF superfamily ligands BAFF and APRIL interact with three receptors, BAFFR, BCMA, and TACI, to play discrete and crucial roles in regulating B cell selection and homeostasis in mammals. The interactions between these ligands and receptors are both specific and redundant: BAFFR binds BAFF, whereas BCMA and TACI bind to either BAFF or APRIL. In a previous phylogenetic inquiry, we identified and characterized a BAFF-like gene in lampreys, which, with hagfish, are the only extant jawless vertebrates, both of which have B-like and T-like lymphocytes. To gain insight into lymphocyte regulation in jawless vertebrates, in this study we identified two BCMA-like genes in lampreys, BCMAL1 and BCMAL2, which were found to be preferentially expressed by B-like lymphocytes. In vitro analyses indicated that the lamprey BAFF-like protein can bind to a BCMA-like receptor Ig fusion protein and to both BCMAL1- and BCMAL2-transfected cells. Discriminating regulatory roles for the two BCMA-like molecules are suggested by their differential expression before and after activation of the B-like lymphocytes in lampreys. Our composite results imply that BAFF-based mechanisms for B cell regulation evolved before the divergence of jawed and jawless vertebrates.

Nonclinical safety assessment of a human interleukin-22FC IGfusion protein demonstrates invitro to invivo 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.

Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

Highly sensitive glycosylamine labelling of O-glycans using non-reductive β-elimination.

When developing biopharmaceuticals, glycans are the most important posttranslational protein modifications that must be addressed because they affect the between-protein interactions that maintain homeostasis. The glycan profile may be defined as a critical quality attribute of a biopharmaceutical. Comprehensive analysis of protein glycosylation must overcome challenges such as the release, labelling, separation and detection of O-glycans. In contrast, N-glycans can be readily released non-reductively from peptide backbones using an enzyme such as peptide N-glycosidase F. We developed a highly sensitive protocol using RapiFluor-MS to label glycosylamines for O-glycan analysis combined with a non-enzyme treatment for efficient release of the reduced O-glycans from the glycoproteins. Here we used the cytotoxic T lymphocyte associated protein 4-immunoglobulin G (Ig) fusion protein and fetuin as models for O-glycan analysis and compared the analytical methods glycopeptide mapping, 2-AB labelling and RapiFluor-MS labelling. The structures of major O-glycans and low-abundance O-glycans were successfully identified using the third technique, which detected the O-glycans with high sensitivity.

Induced Treg Cells Augment the Th17-Mediated Intestinal Inflammatory Response in a CTLA4-Dependent Manner.

Th17 cells and Foxp3+ regulatory T cells (Tregs) are thought to promote and suppress inflammatory responses, respectively. However, whether they counteract each other or synergize in regulating immune reactions remains controversial. To determine their interactions, we describe the results of experiments employing mouse models of intestinal inflammation by transferring antigen-specific Th cells (Th1, Th2, and Th17) differentiated in vitro followed by the administration of the cognate antigen via enema. We show that cotransfer of induced Tregs (iTregs) suppressed Th1- and Th2-mediated colon inflammation. In contrast, colon inflammation induced by transfer of Th17 cells, was augmented by the cotransfer of iTregs. Furthermore, oral delivery of antigen potentiated Th17-mediated colon inflammation. Administration of a blocking antibody against cytotoxic T lymphocyte-associated antigen 4 (CTLA4) abrogated the effects of cotransfer of iTregs, while the injection of a soluble recombinant immunoglobulin (Ig) fusion protein, CTLA4-Ig substituted for the cotransfer of iTregs. These results suggest that antigen-specific activation of iTregs in a local environment stimulates the Th17-mediated inflammatory response in a CTLA4-dependent manner.

Success and failure in translation of findings from experimental autoimmune encephalomyelitis to multiple sclerosis

Despite the differences between multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), most of the knowledge on immune-mediated tissue damage in MS is based on data from EAE. Immune response– modifying therapies such as TNFα inhibitors, glatiramer acetate (GA) or natalizumab have been studied in rodents and have been translated into treatment for patients with varying success. In humans treatment with an anti TNF monoclonal antibody or a recombinant TNF receptor Ig fusion protein resulted in enhanced inflammation. In contrast, findings from GA or natalizumab have been successful in human translation. Findings of EAE studies should be interpreted carefully and should be confirmed using substances (proof of principle). Only if successful, these substances should be studied in humans.

Classical and alternative NF-κB signaling cooperate in regulating adipocyte differentiation and function.

Inflammation of adipose tissue (AT) is a central mediator of insulin resistance. However, the molecular mechanisms triggered by inflammatory cells are not fully understood. The aim of this study was to analyze the metabolic functions of lymphotoxin-β-receptor (LTβR)-mediated alternative NF-κB signaling in adipocytes and to reveal its effects on body weight and insulin sensitivity in vivo. RelB(FatKO) mice and littermate controls were treated with LTβR agonistic antibody (α-LTβR) or a LTβR antagonist (LTβR:Ig fusion protein) after feeding a high-fat diet or standard diet. Mice were analyzed by insulin tolerance and glucose tolerance tests prior to analysis by necropsy and qRT-PCR of abdominal white adipose tissue. 3T3-L1 preadipocytes and mouse embryonic fibroblasts were used for differentiation and expression analysis after treatment with α-LTβR and differentiation to adipocytes. The molecular mechanism was elucidated by chromatin immunoprecipitation and combinatorial treatment with α-LTβR and tumor necrosis factor (TNF). RelB(FatKO) mice showed improved insulin sensitivity despite increased adiposity and adipocyte hypertrophy. LTβR-induced activation of p52-RelB in 3T3-L1 cells attenuated adipogenesis and modulated adipocyte functions via transcriptional downregulation of peroxisome proliferator-activated receptor γ (PPARγ). This LTβR-mediated pathway was synergistically regulated via a TNF-induced increase in p100 and RelB expression and nuclear translocation. Our data describe an anti-adipogenic action of LTβR signaling and a novel synergism of alternative and classical NF-κB signaling in the regulation of adipocytes. In conclusion, this strong synergism between the two NF-κB pathways shows a method to inhibit adipocyte differentiation and to improve insulin sensitivity and can be a potential target to treat metabolic disorders more efficiently than with other known drugs.

Enhanced PD-1 Signalling Downregulated B-Cell Activation and Antibody Responses to Porcine Xenoantigen.

Background: Human T-cells express several receptors like programmed death-1 receptor (PD-1), which is known to negatively regulate T-cell activation. Thus, transgenic overexpression of corresponding ligands on porcine cells and tissues could be a strategy to prevent human immune responses to xenografts. We have previously shown that in vitro human T-cell responses to porcine transfectants overexpressing human PD-Ligand 1 (L23-PD-L1) are reduced. In this study we assessed the role of enhanced PD-1 signalling on B-cell activation and antibody responses to xenoantigen. Methods: Polyclonal activation of isolated human B-cells was induced by CpG-ODN and the effect of a dimeric PD-L1 immunoglobulin (Ig) fusion protein was determined by [3H]-thymidine cell proliferation assay. A xenoreactive response was induced by coculturing human peripheral blood mononuclear cells (hPBMC) with L23-PD-L1 cells or mock-transfected controls in vitro. To characterize the xenospecific antibody responsein vivo, PD-L1.Ig was applied to rats immunized with L23-control cells. Antibody titers in the culture supernatant or the sera of rats and the activation status of B-cells in draining lymph nodes were determined by flow cytometry or ELISA. Results: Stimulation of isolated B-cells with CpG-ODN resulted in a high proliferation rate and upregulation of PD-1. Addition of PD-L1.Ig significantly reduced B-cell proliferation in dose-dependent manner. Moreover, cocultivation of hPBMCs with porcine cells resulted in high levels of anti-pig antibodies, whereas only low titers were observed in the presence of membrane bound PD-L1 (L23-PD-L1 cells) or soluble PD-L1.Ig. Data of rat immunization experiments confirmed that triggering of the PD-1 pathway reduced serum anti-pig antibody levels. Further analysis of B-cell populations in draining lymph nodes revealed a lower frequency of activated B-cells (Ig+CD44hi) after application of PD-L1-Ig. Conclusion: These data suggest a direct inhibitory role of the PD-1 pathway on B-cell activation, as reported for T-cells. Thus, antibody production to porcine xenoantigen can be markedly lower in the presence of high concentrations of the inhibitory ligand PD-L1. Altogether, treatment with PD-L1 might be a strategy to diminish not only T-cell activation, but also the elicited antibody response to xenoantigen, which is a major hurdle to successful xenotransplantation.

Modulation of immune responses to porcine xenoantigen by targeting stimulatory and inhibitory signalling pathways

Genetic modification of pigs (e.g. transgenic expression of human complement regulatory molecules or inactivation of α1,3 galactosyltransferase) [1] enabled the development of promising strategies to overcome hyperacute rejection after pig‐to‐primate xenotransplantation. However, cellular rejection still remains a hurdle for successful xenograft survival. Cellular rejection of porcine cells in xenotransplantation models is mediated by monocytes/macrophages, natural killer (NK) cells, and T cells.Research in our laboratory focuses on receptor‐ligand interactions regulating the responses of human cells to porcine tissues and thus could be targets for immunomodulation. NK cell activation is tightly regulated by different inhibitory and activating receptors and their ligands. Transgenic pigs overexpressing HLA‐E have been generated [2]. In vitro experiments revealed that cells from these pigs are only partially protected from NK cell‐mediated lysis. This might be due to the fact that the inhibitory receptor for HLA–E (CD94/NKG2A) is not expressed on all human NK cells. We are currently exploring the concept of downregulating NK cell activity by enhancing CD161/KLRB1‐mediated signalling. CD161 is a C‐type lectin‐like receptor which is expressed on the great majority of human NK cells and can transmit inhibitory signals after binding of its ligand LLT1. The data obtained so far suggest, that porcine cells genetically engineered to overexpress human LLT1 posses reduced potential to activate human NK cells.Activation of T cells by antigen presenting cells (APC) requires interactions between T cell receptor (TCR) and MHC/peptide (“signal one” of T cell activation) as well as interactions between costimulatory receptors and their ligands (“signal two” of T cell activation). T cells receiving “signal one” alone without “signal two” are not activated but achieve a state of unresponsiveness (anergy). In vitro and in vivo data suggest that effective inhibition of human T cell activation can be obtained by blocking the costimulatory CD28‐CD80/CD86 interaction using monoclonal antibodies (mAb) or an antagonistic CTLA‐4.Ig fusion protein [3]. Furthermore, blocking of TCR‐MHC and CD154(CD40L)‐CD40 interactions by mAb to porcine MHC class‐II or CD40 significantly reduced human T cell activation. New approaches for the modulation of anti‐pig immune responses may result from the finding that enhancement of inhibitory signals diminishes T cell activation. We have recently shown that overexpression of the human inhibitory ligand PD‐L1 on porcine APC markedly decreased their capacity to activate human T cells and promotes regulatory T cells [4, 5]. Current studies suggest that enhanced inhibitory signalling does not only downregulate T cell reactivity but also dampens induced antibody responses to porcine xenoantigen. Thus, disruption of stimulatory receptor‐ligand interactions (e.g. by blocking antibodies or “knock‐out/down” technologies) combined with transgenic overexpression of inhibitory ligands in porcine cells and tissues could be an effective approach to downregulate human anti‐pig immunity.

Clec12a: Quieting the Dead

Immune activation as a result of the recognition of damage-associated molecular patterns needs to be controlled. In this issue of Immunity, Neumann etal. (2014) demonstrates that Clec12a is a receptor for dead cells through the recognition of uric acid crystals and contributes to the dampening of the responses.

SFRP5 Inhibits Early Stages Of B-Cell Differentiation and Modulates Estrogen-Related Changes In The Immune System

A large body of research has demonstrated that the maternal immune system is elaborately regulated during pregnancy to establish immunological tolerance to the fetus. Although our previous works have revealed that female sex hormones, particularly estrogen, play pivotal roles in suppressing maternal B-lymphopoiesis, the precise molecular mechanisms that mediate their functions are largely unknown. Because T and B lymphocytes function coordinately in the adaptive immune system, the inhibition of B-lymphopoiesis during pregnancy should be involved, at least in part, in “maternal-fetal immune tolerance.” Understanding the molecular mechanisms of tolerance would contribute to the development of new methods to inhibit immune responses after organ transplantation, such as rejection by the host or graft-versus-host diseases. The goal of our present study is to identify the molecular pathways through which estrogen exerts its suppressive effect on B-lymphopoiesis.We performed global analyses of estrogen-inducible genes in bone marrow (BM) stromal cells and identified the secreted frizzled-related protein (sFRP) family. A sFRP1-immunoglobulin G (Ig) fusion protein inhibited early differentiation of B-cells originating from BM-derived hematopoietic stem/progenitor cells (HSPC) in culture (Yokota T. et al. Journal of Immunol, 2008). Conversely, sFRP1 deficiency in vivo caused dysregulation of HSPC homeostasis in BM and aberrant increase of peripheral B lymphocytes (Renström J. et al. Cell Stem Cell, 2009). Therefore, in the present study we generated sFRP1 transgenic chimera (TC) mice that produced high levels of circulating sFRP1 after birth to examine the influence of sFRP1 on adult lymphopoiesis in vivo. Further, we generated sFRP5 TC mice using the same procedure to determine whether there were functional differences or redundancies between sFRP1 and sFRP5. The two are most closely related isoforms among the sFRP family and are known to play redundant roles during embryonic development; however, their physiological function in the immune system is largely unknown.Unexpectedly, while only subtle change was detected in the lymphoid lineage of sFRP1 TC mice, we found that the number of B cells was significantly reduced in the sFRP5 TC mice. The frequency of B cells, which normally account for approximately 50% of peripheral leukocytes of wild-type (WT) mice, was reduced to less than 20% in the sFRP5 TC mice. The suppression was likely specific to the B lineage, because overexpression of sFRP5 did not affect myeloid, T, or NK cells. Compared with WT littermates, the body size of sFRP5 TC mice was slightly, but significantly smaller. Thymocyte counts were not affected. In contrast, the number of splenocytes, particularly those of the B lineage, significantly decreased. In BM of sFRP5 TC mice, early B-cell differentiation was inhibited, resulting in the accumulation of cells whose phenotype corresponds to those of common lymphoid progenitors (CLPs). Gene array analyses of the accumulated CLPs indicated that sFRP5 affects the expression of adaptive immune system-related genes. Further, the sFRP5 overexpression was found to induce the expression of Wnt and Notch-related molecules that regulate the integrity of HSPCs.To determine the physiological involvement of sFRP5 in the inhibition of early B-cell differentiation, we exploited mice lacking sFRP5. It is noteworthy that, although the level of sFRP5 expression was minimal in steady-state BM, it was markedly induced after estrogen treatment. We injected water-soluble β-estradiol into WT or sFRP5-null mice for 4 days and evaluated their lympho-hematopoiesis 12 h after the last injection. While the highly HSPC-enriched Lineage- Sca-1+ c-kitHi Flt3- fraction of WT mice was resistant to the treatment, the same fraction of sFRP5-null mice showed a declining trend. Further, although the CLP fraction was significantly reduced in both strains, CLPs of sFRP5-null mice were more sensitive to estrogen than those of WT. We also performed gene expression analyses of WT and sFRP5-null mice after the estrogen treatment. We found that estrogen induced the expression of Hes1 in HSPCs of WT but not sFRP5-null mice. Thus, we conclude that estrogen-inducible sFRP5 blocks the differentiation of HSPCs in BM to B-lymphocytes in the presence of high levels of estrogen, at least in part by activation of the Notch pathway. Disclosures:No relevant conflicts of interest to declare.

Mouse TIGIT inhibits NK‐cell cytotoxicity upon interaction with PVR

The activity of natural killer (NK) cells is controlled by a balance of signals derived from inhibitory and activating receptors. TIGIT is a novel inhibitory receptor, recently shown in humans to interact with two ligands: PVR and Nectin2 and to inhibit human NK-cell cytotoxicity. Whether mouse TIGIT (mTIGIT) inhibits mouse NK-cell cytotoxicity is unknown. Here we show that mTIGIT is expressed by mouse NK cells and interacts with mouse PVR. Using mouse and human Ig fusion proteins we show that while the human TIGIT (hTIGIT) cross-reacts with mouse PVR (mPVR), the binding of mTIGIT is restricted to mPVR. We further demonstrate using surface plasmon resonance (SPR) and staining with Ig fusion proteins that mTIGIT binds to mPVR with higher affinity than the co-stimulatory PVR-binding receptor mouse DNAM1 (mDNAM1). Functionally, we show that triggering of mTIGIT leads to the inhibition of NK-cell cytotoxicity, that IFN-γ secretion is enhanced when mTIGIT is blocked and that the TIGIT-mediated inhibition is dominant over the signals delivered by the PVR-binding co-stimulatory receptors. Additionally, we identify the inhibitory motif responsible for mTIGIT inhibition. In conclusion, we show that TIGIT is a powerful inhibitory receptor for mouse NK cells.

Therapeutic role of the novel checkpoint regulator VISTA in murine autoimmune disease models. (P5174)

Abstract Negative checkpoint regulators are critical in triggering the immune response and in the clinic, therapeutics targeting the CTLA-4 and PD-L1 pathway have been successful in treating human cancers and marginal therapeutic efficacy noted in autoimmune diseases. Our lab recently identified a novel checkpoint regulator termed V-region Immunoglobulin-containing Suppressor of T cell Activation (VISTA). VISTA is structurally distinct from other members of the Ig superfamily. This study aimed to determine the therapeutic efficacy of VISTA in autoimmunity is effective. To address this, we investigated the role of a VISTA Ig fusion protein (VISTA-Ig) in autoimmune disease models of Multiple Sclerosis (Experimental Autoimmune Encephalomyelitis (EAE)) and Systemic Lupus Erythematosus (SLE). In sick EAE mice, therapeutic treatment with VISTA-Ig suppressed disease severity compared with untreated mice in vivo. In the lupus prone mouse strain NZBW-F1 which closely resembles human SLE, the prophylactic and therapeutic effect of VISTA-Ig was studied. Prophylactic treatment with VISTA-Ig prevented the onset of proteinuria and weight loss compared with untreated mice and therapeutic treatment reversed proteinuria in sick mice. Clinical evaluation of kidneys from the VISTA-Ig treated group showed minimal to mild glomerulonephritis compared with untreated mice. In conclusion we demonstrate that VISTA-Ig is a novel effective therapeutic in treating established autoimmune diseases.

PD-1, B7-H1 and IL-10 in protection from kidney ischemia reperfusion injury (P1022)

Abstract Regulatory T cells (Tregs) protect the kidney from inflammation, tissue damage and loss of function induced by ischemia-reperfusion (IR) in an IL-10 and PD-1 dependent manner, but the mechanisms of action for Tregs in IR are not completely understood. We hypothesized that Treg PD-1 is engaged by its ligand B7-H1, leading to increased IL-10 production by Tregs, which suppresses inflammation and protects from kidney IR. Similar to our previous observation that WT Tregs lose their ability to protect mice from kidney IR if treated with a PD-1 blocking antibody (ex vivo, prior to adoptive transfer), PD-1 KO Tregs did not offer any protection from kidney IR. In a model of mild renal IR that does not result in significant renal dysfunction in isotype control antibody-treated mice (plasma creatinine (PCr) level: 0.5±0.1, n=5 vs. sham PCr: 0.3±0.1, n=3) blockade of B7-H1 prior to IR causes a marked reduction in renal function (PCr: 1.3±0.3, n=6) accompanied by increased acute tubular necrosis and accumulation of neutrophils in the kidney. Stimulation of isolated WT Tregs with plate-bound anti-CD3, in the presence of IL-2, resulted in significant production of IL-10. In contrast to our hypothesis, addition of plate-bound B7-H1 Ig fusion protein dose-dependently inhibited IL-10 production by WT Tregs. Taken together these results suggest PD-1 and its ligand B7-H1 interact to protect from ischemic kidney injury, but argue against enhanced IL-10 production by Tregs as the mechanism.

The Stalk Domain and the Glycosylation Status of the Activating Natural Killer Cell Receptor NKp30 Are Important for Ligand Binding

The natural cytotoxicity receptors are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The human natural cytotoxicity receptor family comprises the three type I membrane proteins NKp30, NKp44, and NKp46. Especially NKp30 is critical for the cytotoxicity of NK cells against different targets including tumor, virus-infected, and immature dendritic cells. Although the crystal structure of NKp30 was recently solved (Li, Y., Wang, Q., and Mariuzza, R. A. (2011) J. Exp. Med. 208, 703-714; Joyce, M. G., Tran, P., Zhuravleva, M. A., Jaw, J., Colonna, M., and Sun, P. D. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 6223-6228), a key question, how NKp30 recognizes several non-related ligands, remains unclear. Therefore, we investigated the parameters that impact ligand recognition of NKp30. Based on various NKp30-hIgG1-Fc fusion proteins, which were optimized for minimal background binding to cellular Fcγ receptors, we identified the flexible stalk region of NKp30 as an important but so far neglected module for ligand recognition and related signaling of the corresponding full-length receptor proteins. Moreover, we found that the ectodomain of NKp30 is N-linked glycosylated at three different sites. Mutational analyses revealed differential binding affinities and signaling capacities of mono-, di-, or triglycosylated NKp30, suggesting that the degree of glycosylation could provide a switch to modulate the ligand binding properties of NKp30 and NK cell cytotoxicity.

Mouse β-Defensin 14 (Defb14) Promotes Tumor Growth by Inducing Angiogenesis in a CCR6-Dependent Manner

β-Defensins are known for their antimicrobial activity and belong to the molecular barrier of the innate immune system against invading pathogens. In addition, it has been shown that some members of the β-defensin superfamily have the capacity to promote local innate inflammatory and systemic adaptive immune responses, mediated in part by the interaction with CCR6. We found that mouse β-defensin 14 (mBD14, Defb14), a newly identified member of the mouse β-defensin superfamily, is expressed in mouse fibrosarcoma tumor tissue. Tumor cells overexpressing mBD14 demonstrated enhanced solid tumor growth in syngeneic C57BL/6 mice concomitant with increased vascularization of these tumors. Furthermore, mBD14-overexpressing tumors demonstrated increased expression of proangiogenic MIP-2 (CXCL2) ex vivo. In contrast, vascular endothelial growth factor expression was not affected. Cellular analysis of tumor-infiltrating leukocytes revealed a significant increase of CCR6(+) B220(+) lymphocytes in solid tumors derived from mBD14-overexpressing tumor cells. Enhanced tumor growth of mBD14-overexpressing fibrosarcomas was abolished in CCR6-deficient mice, which was paralleled by decreased infiltration of CCR6(+) B220(+) lymphocytes, indicating the requirement of CCR6 expression on host cells. Previously, the interaction of activated, LTαβ(+), lymphocytes with lymphotoxin β-receptor-expressing fibrosarcoma tumor cells has been identified as a new CXCL2-dependent proangiogenic pathway. Coexpression of a soluble lymphotoxin β-receptor:Ig fusion protein, an inhibitor of CXCL2-dependent angiogenesis, in mBD14-overexpressing fibrosarcoma tumor cells abolished enhanced solid tumor growth. Thus, we conclude that mBD14 expression by tumor-infiltrating host cells results in the chemoattraction of CCR6(+) B220(+) lymphocytes, which in turn initiates a proangiogenic pathway leading to enhanced angiogenesis and organized tumor tissue development.