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Epithelial cells and macrophages represent two major cell populations in the lung. They reside in physical proximity and are influenced by inhaled substances, microbial- and host-derived factors, as well as by crosstalk between each other. Here, we report the first systematic study to compare the effects of apical and basolateral secretomes from primary human small airway epithelial cells (SAEC) on human macrophages. We exposed monocyte-derived macrophages (MDMs) to the secretome supernatants (SN) from the apical and basolateral chamber of SAEC culture in an air-liquid interface (ALI) setting and analyzed expression of macrophage surface markers. We found that the apical SN increased the expression of CD11c and CD16, whereas basolateral SN increased the expression of CD163 and CD300e, consistent with apical and basolateral epithelial secretions inducing an M1-biased and M2-biased macrophage polarization, respectively. Conversely, in the presence of Nontypeable Haemophilus influenzae (NTHi), apical SN from NTHi-exposed SAEC induced CD36, CD163 and CD300e and supressed CD11c expression suggesting a switch towards an M2-biased macrophage polarization. Analysis of SN from polarized epithelium revealed a number of factors with differential expression in the apical and basolateral secretome. Functional neutralization of IL6, IL8 or IL1α in the apical secretome led to a decrease in expression of 'M2-like' surface markers, supporting the concept of epithelial-derived secreted factors influencing macrophage phenotype. In conclusion, we show, for the first time to our knowledge, that SN from polarized epithelium, depending on the side of secretion, apical or basolateral, can elicit a differential influence on the macrophages polarization phenotype.

Chronic hepatitis B virus (HBV) infection is characterized by the presence of defective viral envelope proteins (hepatitis B surface antigen [HBsAg]) and the duration of infection-most patients acquire the infection at birth or during the first years of life. We investigated the effects of these factors on patients' lymphocyte and HBV-specific T-cell populations.We collected blood samples and clinical data from 243 patients with HBV infection (3-75 years old) in the United Kingdom and China. We measured levels of HBV DNA, HBsAg, hepatitis B e antigen, and alanine aminotransferase; analyzed HBV genotypes; and isolated peripheral blood mononuclear cells (PBMCs). In PBMCs from 48 patients with varying levels of serum HBsAg, we measured 40 markers on nature killer and T cells by mass cytometry. PBMCs from 189 patients with chronic infection and 38 patients with resolved infections were incubated with HBV peptide libraries, and HBV-specific T cells were identified by interferon gamma enzyme-linked immune absorbent spot (ELISpot) assays or flow cytometry. We used multivariate linear regression and performed variable selection using the Akaike information criterion to identify covariates associated with HBV-specific responses of T cells.Although T- and natural killer cell phenotypes and functions did not change with level of serum HBsAg, numbers of HBs-specific T cells correlated with serum levels of HBsAg (r = 0.3367; P.00001). After we performed the variable selection, the multivariate linear regression model identified patient age as the only factor significantly associated with numbers of HBs-specific T cells (P = .000115). In patients younger than 30 years, HBs-specific T cells constituted 28.26% of the total HBV-specific T cells; this value decreased to 7.14% in patients older than 30 years.In an analysis of immune cells from patients with chronic HBV infection, we found that the duration of HBsAg exposure, rather than the quantity of HBsAg, was associated with the level of anti-HBV immune response. Although the presence of HBs-specific T cells might not be required for the clearance of HBV infection in all patients, strategies to restore anti-HBV immune responses should be considered in patients younger than 30 years.

In many solid cancers, tumor-associated macrophages (TAM) represent the predominant myeloid cell population. Antigen (Ag) cross-presentation leading to tumor Ag-directed cytotoxic CD8+ T cell responses is crucial for anti-tumor immunity. However, the role of recruited monocyte-derived macrophages, including TAM, as potential cross-presenting cells is not well understood. Here, we show that primary human as well as mouse CD206+ macrophages are effective in functional cross-presentation of soluble self and non-self Ag, including tumor-associated Ag (TAA) as well as viral Ag. To confirm the presence of cross-presenting TAM in vivo, we performed phenotypic and functional analysis of TAM from B16-F10 and CT26 syngeneic tumor models and have identified CD11b+F4/80hiCD206+ TAM to effectively cross-present TAA. We show that CD11b+CD206+ TAM represent the dominant tumor-infiltrating myeloid cell population, expressing a unique cell surface repertoire, promoting Ag cross-presentation and Ag-specific CD8+ T cell activation comparable to cross-presenting CLEC9A+ dendritic cells (cDC1). The presence of cross-presenting CD206+ TAM is associated with reduced tumor burden in mouse syngeneic tumor models and with improved overall survival in cutaneous melanoma patients. Therefore, the demonstration of effective Ag cross-presentation capabilities of CD206+ TAM, including their clinical relevance, expands our understanding of TAM phenotypic diversity and functional versatility.

More than 70% of colorectal, prostate, ovarian, pancreatic and breast cancer specimens show expression of CD276 (B7–H3), a potential immune checkpoint family member. Several studies have shown that high CD276 expression in cancer cells correlates with a poor clinical prognosis. This has been associated with the presence of lower tumor infiltrating leukocytes. Among those, tumor-associated macrophages can comprise up to 50% of the tumor mass and are thought to support tumor growth through various mechanisms. However, a lack of information on CD276 function and interaction partner(s) impedes rigorous evaluation of CD276 as a therapeutic target in oncology. Therefore, we aimed to understand the relevance of CD276 in tumor-macrophage interaction by employing a 3D spheroid coculture system with human cells. Our data show a role for tumor-expressed CD276 on the macrophage recruitment into the tumor spheroid, and also in regulation of the extracellular matrix modulator PAI-1. Furthermore, our experiments focusing on macrophage-expressed CD276 suggest that the antibody-dependent CD276 engagement triggers predominantly inhibitory signaling networks in human macrophages.

During injury, monocytes are recruited from the circulation to inflamed tissues and differentiate locally into mature macrophages, with prior reports showing that cavity macrophages of the peritoneum and pericardium invade deeply into the respective organs to promote repair. Here we report a dual recombinase-mediated genetic system designed to trace cavity macrophages in vivo by intersectional detection of two characteristic markers. Lineage tracing with this method shows accumulation of cavity macrophages during lung and liver injury on the surface of visceral organs without penetration into the parenchyma. Additional data suggest that these peritoneal or pleural cavity macrophages do not contribute to tissue repair and regeneration. Our in vivo genetic targeting approach thus provides a reliable method to identify and characterize cavity macrophages during their development and in tissue repair and regeneration, and distinguishes these cells from other lineages., Body cavity macrophages reside on the serous surfaces of organs and believed to participate in organ repair following injury. Here the authors show with a fate-mapping reporter system that these cells, although accumulate at the surfaces of injured liver or lung, don’t penetrate deeply into the tissue.

BackgroundElevated counts of alveolar macrophages and attenuated phagocytic capacity are associated with chronic obstructive pulmonary disease (COPD). Factors governing macrophage phagocytosis are poorly understood. In this study we aimed to compare the influence of airway epithelial cell secretions from individuals with COPD and without COPD (non-COPD) on macrophage phagocytic activity, and the role of antimicrobial peptides (AMPs).MethodsSupernatants from non-COPD and COPD small airway epithelial cell (SAEC) cultures exposed to non-typeable Haemophilus influenzae (NTHi) were applied to human monocyte-derived macrophages (MDMs) to assess their influence on phagocytosis. SAECs were analysed for changes in AMP expression by quantitative reverse transcription PCR, and the influence of select AMPs on macrophage phenotype and function was assessed by flow cytometry and metabolic activity assay.ResultsSecretions from the apical and basolateral surface of NTHi-exposed SAECs from non-COPD donors elicited superior phagocytic capacity in MDMs. Moreover, NTHi exposure led to a rapid increase in the expression of a range of AMPs by non-COPD SAECs, but this response was delayed in COPD SAECs. We demonstrate that treatment with AMPs β-defensin 2 and S100 calcium binding protein A8/S100 calcium binding protein A9 (S100A8/A9) improved the phagocytic capacity of MDMs. In-depth analysis of the influence of S100A8/A9 on MDMs revealed a role for this AMP in macrophage phenotype and function. Furthermore, we show that the expression of S100A8 and S100A9 is directly regulated by WNT/β-catenin signalling, a known deregulated pathway in COPD.ConclusionIn conclusion, for the first time, we demonstrate that airway epithelium from patients with COPD has a reduced capacity to support the phagocytic function of macrophages in response to acute NTHi exposure, and we identify the WNT/β-catenin signalling-modulated and epithelium-derived S100A8/A9 as a potent regulator of macrophage phenotype and function.

HIV infection is controlled immunologically in a small subset of infected individuals without antiretroviral therapy (ART), though the mechanism of control is unclear. CD8(+) T cells are a critical component of HIV control in many immunological controllers. NK cells are also believed to have a role in controlling HIV infection, though their role is less well characterized. We used mass cytometry to simultaneously measure the levels of expression of 24 surface markers on peripheral NK cells from HIV-infected subjects with various degrees of HIV natural control; we then used machine learning to identify NK cell subpopulations that differentiate HIV controllers from noncontrollers. Using CITRUS (cluster identification, characterization, and regression), we identified 3 NK cell subpopulations that differentiated subjects with chronic HIV viremia (viremic noncontrollers [VNC]) from individuals with undetectable HIV viremia without ART (elite controllers [EC]). In a parallel approach, we identified 11 NK cell subpopulations that differentiated HIV-infected subject groups using k-means clustering after dimensionality reduction by t-neighbor stochastic neighbor embedding (tSNE) or linear discriminant analysis (LDA). Among these additional 11 subpopulations, the frequencies of 5 correlated with HIV DNA levels; importantly, significance was retained in 2 subpopulations in analyses that included only cohorts without detectable viremia. By comparing the surface marker expression patterns of all identified subpopulations, we revealed that the CD11b(+) CD57(−) CD161(+) Siglec-7(+) subpopulation of CD56(dim) CD16(+) NK cells are more abundant in EC and HIV-negative controls than in VNC and that the frequency of these cells correlated with HIV DNA levels. We hypothesize that this population may have a role in immunological control of HIV infection. IMPORTANCE HIV infection results in the establishment of a stable reservoir of latently infected cells; ART is usually required to keep viral replication under control and disease progression at bay, though a small subset of HIV-infected subjects can control HIV infection without ART through immunological mechanisms. In this study, we sought to identify subpopulations of NK cells that may be involved in the natural immunological control of HIV infection. We used mass cytometry to measure surface marker expression on peripheral NK cells. Using two distinct semisupervised machine learning approaches, we identified a CD11b(+) CD57(−) CD161(+) Siglec-7(+) subpopulation of CD56(dim) CD16(+) NK cells that differentiates HIV controllers from noncontrollers. These cells can be sorted out for future functional studies to assess their potential role in the immunological control of HIV infection.

Chronic obstructive pulmonary disease (COPD) is characterized by emphysema, chronic inflammation and increased bacterial colonization of airways associated with exacerbations. While the number of macrophages is elevated in COPD, their phagocytic capacity is attenuated. Antimicrobial peptides (AMP) are naturally occurring proteins with anti-pathogen and proposed immunomodulatory functions. We hypothesize that the airway epithelium-derived AMPs regulate the function of macrophages in COPD. We stimulated non-COPD and COPD small airway epithelial cells (SAEC) with heat-inactivated non-typeable Haemophilus influenzae (Nthi) in air-liquid-interface cultures. We subjected the secretome from apical and basolateral compartment to mass spectrometry and identified a number of secreted AMPs, differentiating between the site of secretion, but not with the Nthi challenge or disease status. We found, however, that Nthi regulated the mRNA expression of AMPs. We observed upregulation of S100A8 and S100A9 after 3h stimulation in non-COPD- to a higher extent than in COPD-derived SAEC but the difference was leveled out after 24h stimulation. This suggests the delayed response in COPD epithelium. Additionally, we used the supernatants collected from SAECs to stimulate primary human monocyte-derived macrophages (MDM). Supernatants from non-COPD cultures had higher impact on phagocytosis in comparison to COPD after 3h Nthi stimulation, but again after 24h this effect was diminished. We next stimulated MDM with S100A8/A9 and observed significant improvement in phagocytosis. Thus, lung epithelium-derived S100A8/A9 could improve phagocytosis in macrophages and this pathway could be explored for therapeutic utility in COPD.

Background & Aims GS-9620 is an oral agonist of toll-like receptor 7, a pattern-recognition receptor whose activation results in innate and adaptive immune stimulation. We evaluated the safety, pharmacokinetics, and pharmacodynamics of GS-9620 in patients with chronic hepatitis B. Methods In two double-blind, phase 1b trials of identical design, 49 treatment-naive and 51 virologically suppressed patients were randomized 5:1 to receive GS-9620 (at doses of 0.3mg, 1mg, 2mg, 4mg) or placebo as a single dose or as two doses seven days apart. Pharmacodynamic assessment included evaluation of peripheral mRNA expression of interferon-stimulated gene 15 ( ISG15 ), serum interferon gamma-induced protein 10 and serum interferon (IFN)-alpha. Results Overall, 74% of patients were male and 75% were HBeAg negative at baseline. No subject discontinued treatment due to adverse events. Fifty-eight percent experienced ⩾1 adverse event, all of which were mild to moderate in severity. The most common adverse event was headache. No clinically significant changes in HBsAg or HBV DNA levels were observed. Overall, a transient dose-dependent induction of peripheral ISG15 gene expression was observed peaking within 48hours of dosing followed by return to baseline levels within seven days. Higher GS-9620 dose, HBeAg positive status, and low HBsAg level at baseline were independently associated with greater probability of ISG15 response. Most patients (88%) did not show detectable levels of serum IFN-alpha at any time point. Conclusions Oral GS-9620 was safe, well tolerated, and associated with induction of peripheral ISG15 production in the absence of significant systemic IFN-alpha levels or related symptoms.

Individualized assessment of hepatocellular carcinoma (HCC) risk in chronic liver disease remains challenging. Serum biomarkers including cytokines may offer helpful adjuncts to standard parameters for risk prediction. Our aim was to identify markers associated with increased HCC incidence. This was a prospective cohort study of 282 patients with both viral or non-viral chronic liver disease. Baseline serum cytokines and other markers were measured in multiplex with a commercially-available Luminex-based system. Patients were followed until death or HCC diagnosis. We performed Lasso-based survival analysis to determine parameters associated with HCC development. Cytokine mean florescence intensity (MFI) was the primary predictor and HCC development the primary outcome. 25 patients developed HCC with total follow-up of 1,363 person-years. Parameters associated with increased HCC incidence were cirrhosis, hepatic decompensation, and soluble serum intercellular adhesion molecule 1 (sICAM-1) MFI. No other molecules increased predictive power for HCC incidence. On univariate analysis, the parameters associated with HCC incidence in patients with cirrhosis were age, antiviral treatment, and high sICAM-1 MFI; on multivariate analysis, sICAM-1 remained associated with HCC development (adjusted HR = 2.75). On unbiased screening of serum cytokines and other markers in a diverse cohort, baseline sICAM-1 MFI is associated with HCC incidence.

Background GS-9620 is a potent oral agonist of toll-like receptor 7, a key modulator of the innate immune response. In healthy volunteers, low doses of GS-9620 (2, 4 and 6 mg) induced significant expression of peripheral interferon-stimulated-gene (ISG) mRNA in the absence of detectable serum interferon-α and systemic adverse events (AEs). We evaluated the safety, pharmacokinetics and pharmacodynamics of GS-9620 in treatment-naive patients chronically infected with HCV genotype 1. Methods In this double-blind, placebo-controlled study, 51 patients were randomized 5:1 (active:placebo) to receive either a single dose or two once-weekly doses of GS-9620 at four dose levels (0.3, 1, 2 and 4 mg) or placebo. Pharmacodynamic assessments included peripheral ISG15 mRNA expression, serum interferon-α and interferon-γ-inducible protein (IP)-10 levels and HCV RNA quantification. Results GS-9620 was well-tolerated at all doses. Most AEs were mild or moderate in severity. GS-9620 exhibited dose-linear pharmacokinetics with a median half-life in plasma of 18 h. Transient, dose-dependent ISG15 induction was observed at 1, 2 and 4 mg, with peak mean fold change within 48 h followed by a decline to baseline levels within 7 days of dosing. Serum interferon-α induction post-baseline was detected in 16.7% (8/48) of patients. No clinically significant reductions in HCV RNA were observed. Conclusions GS-9620 was safe, well-tolerated and biologically active in patients with HCV infection. Induction of ISG15 occurred in the absence of detectable serum interferon-α or systemic AEs in most patients, supporting a pre-systemic mechanism of action. ClinicalTrials.gov identifier: NCT01591668.

Summary The immune microenvironment of hepatocellular carcinoma (HCC) is poorly characterized. Combining two single-cell RNA sequencing technologies, we produced transcriptomes of CD45+ immune cells for HCC patients from five immune-relevant sites: tumor, adjacent liver, hepatic lymph node (LN), blood, and ascites. A cluster of LAMP3+ dendritic cells (DCs) appeared to be the mature form of conventional DCs and possessed the potential to migrate from tumors to LNs. LAMP3+ DCs also expressed diverse immune-relevant ligands and exhibited potential to regulate multiple subtypes of lymphocytes. Of the macrophages in tumors that exhibited distinct transcriptional states, tumor-associated macrophages (TAMs) were associated with poor prognosis, and we established the inflammatory role of SLC40A1 and GPNMB in these cells. Further, myeloid and lymphoid cells in ascites were predominantly linked to tumor and blood origins, respectively. The dynamic properties of diverse CD45+ cell types revealed by this study add new dimensions to the immune landscape of HCC.

GS-9620 [8-(3-(pyrrolidin-1-ylmethyl)benzyl)-4-amino-2-butoxy-7,8-dihydropteridin-6(5H)-one] is a potent, orally bioavailable small-molecule agonist of Toll-like receptor 7 (TLR7) developed for finite treatment of chronic hepatitis B viral (HBV) infection, with the goal of inducing a liver-targeted antiviral effect without inducing the adverse effects associated with current systemic interferon-α (IFN-α) therapies. We characterized the pharmacodynamic response of GS-9620 in CD-1 mice and cynomolgus monkeys following intravenous or oral administration and showed that GS-9620 induces the production of select chemokines and cytokines, including IFN-α and interferon-stimulated genes (ISGs). It is noteworthy that we also demonstrated that, in animals and healthy human volunteers, oral administration of GS-9620 can induce a type I interferon-dependent antiviral innate immune response, as measured by whole-blood mRNA of the ISGs 2'5'-oligoadenylate synthetase 1 (OAS1) and myxovirus resistance 1 (MX1), without the induction of detectable systemic IFN-α, i.e., a presystemic response. Additionally, presystemic induction of hepatic OAS1 and MX1 mRNA was observed in CD-1 mice in the absence of detectable systemic IFN-α. We propose that the mechanism of this presystemic response is likely its high intestinal absorption, which facilitates localized activation of TLR7, probably in plasmacytoid dendritic cells at the level of gut-associated lymphoid tissue and/or the liver. This localized response is further supported by data that indicate only minimal contributions of systemic immune stimulation to the overall pharmacodynamic response to orally administered GS-9620. These data demonstrate that GS-9620 can induce an antiviral innate immune response without inducing a systemic IFN-α response and thus suggest the therapeutic potential of this approach in the treatment of chronic HBV infection.

SummaryLiver fibrosis is a consequence of chronic liver diseases and thus a major cause of mortality and morbidity. Clinical evidence and animal studies suggest that local tissue homeostasis is disturbed due to immunological responses to chronic hepatocellular stress. Poorly defined stress-associated inflammatory networks are thought to mediate gradual accumulation of extracellular-matrix components, ultimately leading to fibrosis and liver failure. Here we have reported that hepatic expression of interleukin-33 (IL-33) was both required and sufficient for severe hepatic fibrosis in vivo. We have demonstrated that IL-33’s profibrotic effects related to activation and expansion of liver resident innate lymphoid cells (ILC2). We identified ILC2-derived IL-13, acting through type-II IL-4 receptor-dependent signaling via the transcription factor STAT6 and hepatic stellate-cell activation, as a critical downstream cytokine of IL-33-dependent pathologic tissue remodeling and fibrosis. Our data reveal key immunological networks implicated in hepatic fibrosis and support the concept of modulation of IL-33 bioactivity for therapeutic purposes.

Although the causes of inflammatory arthritis elude us, aberrant cytokine expression has been linked to joint pathology. Consequently, several approaches in the clinic and/or in clinical trials are targeting cytokines, e.g. tumor necrosis factor (TNF), interleukin 23 (IL-23) and interleukin 17 (IL-17), with the goal of antagonizing their respective biologic activity through therapeutic neutralizing antibodies. Such, cytokine signaling-dependent molecular networks orchestrate synovial inflammation on multiple levels including differentiation of myeloid cells to osteoclasts, the central cellular players in arthritis-associated pathologic bone resorption. Hence, understanding of the cellular and molecular mechanisms elicited by synovial cytokine networks that dictate recruitment, differentiation and activation of osteoclast precursors and osteoclasts, respectively, is central to shaping novel therapeutic options for inflammatory arthritis patients. In this article we are discussing the complex signaling interactions involved in the regulation of inflammatory arthritis and it’s associated bone loss with a focus on interleukin 27 (IL-27). The present review will discuss the primary bone-degrading cell, the osteoclast, and on how IL-27, directly or indirectly, modulates osteoclast activity in autoimmune-driven inflammatory joint diseases.

Cytokines play an important role in the pathogenesis of cirrhosis and hepatocellular carcinoma (HCC), most cases of which are related to either hepatitis B virus (HBV) or hepatitis C virus (HCV). Prior studies have examined differences in individual cytokine levels in patients with chronic liver disease, but comprehensive cytokine profiling data across different clinical characteristics are lacking. We examined serum cytokine profiles of 411 patients with HCC (n = 102: 32% HBV, 54% HCV, 14% non-viral) and without HCC (n = 309: 39% HBV, 39% HCV, 22% non-viral). Multiplex analysis (Luminex 200 IS) was used to measure serum levels of 51 common cytokines. Random forest machine learning was used to obtain receiver operator characteristic curves and to determine individual cytokine importance using Z scores of mean fluorescence intensity for individual cytokines. Among HCC and non-HCC patients, cytokine profiles differed between HBV and HCV patients (area under curve (AUC) 0.82 for HCC, 0.90 for non-HCC). Cytokine profiles did not distinguish cirrhotic HBV patients with and without HCC (AUC 0.503) or HCV patients with and without HCC (AUC 0.63). In conclusion, patients with HBV or HCV infection, with or without HCC, have distinctly different cytokine profiles, suggesting potential differences in disease pathogenesis and/or disease characteristics.

Maintaining detectable levels of antibodies to hepatitis B surface antigen (HBsAg) in serum after HBsAg sero-conversion is the key clinical endpoint indicative of recovery from infection with hepatitis B virus (HBV). As HBV-infected hepatocytes secrete HBsAg subviral particles in vast excess over HBV virions, detectable hepatitis B surface antibody (anti-HBs) titres imply complete elimination of HBV virions as well as HBsAg particles. Although intrahepatic phagocytes, for example Kupffer cells, are thought to mediate clearance of HBsAg via antibody (Ab)-dependent and Ab-independent mechanisms, the relative contributions of circulating phagocytic cell types to HBsAg elimination are poorly characterized. Understanding the role of various immune cell subsets in the clearance of HBsAg is important because Ab-dependent or Ab-independent phagocytic HBsAg uptake may modulate presentation of HBsAg-derived epitopes to antigen-specific T cells and hence plays a critical role in adaptive immunity against HBV. This study aims to characterize phagocytic leucocyte subsets capable of internalizing HBsAg immune complexes (HBsAg:IC) or un-complexed HBsAg particles in whole blood directly ex vivo. The data show that uptake of HBsAg:IC occurs most prominently in monocytes, B cells, dendritic cells and in neutrophils. In contrast, B cells, and to a lesser degree also monocytes, seem to be effective phagocytes for un-complexed HBsAg. Importantly, a similar pattern of phagocytic HBsAg uptake was observed in blood from chronic hepatitis B (CHB) patients compared to healthy controls, suggesting that phagocytosis-related cellular functions are not altered in the context of CHB.

GS-9620 is an orally administered agonist of Toll-like receptor (TLR)7 currently being evaluated in clinical studies for the treatment of chronic HBV and HIV patients. GS-9620 has shown antiviral efficacy in preclinical models of chronic hepadnavirus infection in woodchuck as well as chimpanzee. However, the molecular determinants of GS-9620-dependent activation of TLR7 are not well defined. The studies presented here elucidate GS-9620 subcellular distribution and characterize its molecular interactions with human TLR7 using structure-guided mutational analysis. Based on our results we present a molecular model of TLR7 bound to GS-9620. We also determine that several coding SNPs had no effect on GS-9620-dependent TLR7 activation. In addition, our studies provide evidence that TLR7 exists in a ligand-independent oligomeric state and that, TLR7 activation by GS-9620 is likely associated with compound-induced conformational changes. Finally, we demonstrate that activation of NF-κB and Akt pathways in primary plasmacytoid dendritic cells occur as immediate downstream cellular responses to GS-9620 stimulation. The data presented here further our understanding of the molecular parameters governing TLR7 activation by GS-9620, and more generally by nucleos/tide-related ligands.

IL-33 (IL-1F11) is a recently described member of the IL-1 family of cytokines that stimulates the generation of cells, cytokines, and Igs characteristic of a type 2 immune response. IL-33 mediates signal transduction through ST2, a receptor expressed on Th2 and mast cells. In this study, we demonstrate that IL-33 and ST2 form a complex with IL-1R accessory protein (IL-1RAcP), a signaling receptor subunit that is also a member of the IL-1R complex. Additionally, IL-1RAcP is required for IL-33-induced in vivo effects, and IL-33-mediated signal transduction can be inhibited by dominant-negative IL-1RAcP. The implications of this shared usage of IL-1RAcP by IL-1(α and β) and IL-33 are discussed.

Activation of immune cells has to be tightly controlled to prevent detrimental hyperactivation. In this regulatory process molecules of the C-type lectin-like family play a central role. Here we describe a new member of this family, CLEC12B. The extracellular domain of CLEC12B shows considerable homology to the activating natural killer cell receptor NKG2D, but unlike NKG2D, CLEC12B contains an immunoreceptor tyrosine-based inhibition motif in its intracellular domain. Despite the homology, CLEC12B does not appear to bind NKG2D ligands and therefore does not represent the inhibitory counterpart of NKG2D. However, CLEC12B has the ability to counteract NKG2D-mediated signaling, and we show that this function is dependent on the immunoreceptor tyrosine-based inhibition motif and the recruitment of the phosphatases SHP-1 and SHP-2. Using monoclonal anti-CLEC12B antibodies we found de novo expression of this receptor on in vitro generated human macrophages and on the human myelo-monocytic cell line U937 upon phorbol 12-myristate 13-acetate treatment, suggesting that this receptor plays a role in myeloid cell function.

Inhibition of the interleukin-2 (IL-2) pathway has potent immunosuppressive activity in humans as is evident from the broad therapeutic utility of cyclosporine, rapamycin, tacrolimus, and monoclonal antibodies blocking the high-affinity subunit of the IL-2 receptor (CD25). Here we describe a humanized antibody, MT204, interfering with IL-2 signaling by a novel mechanism. Although MT204 did not prevent IL-2 from binding to CD25, it potently antagonized downstream signaling events of IL-2 at sub-nanomolar concentrations, such as STAT3 tyrosine phosphorylation, expression of CD124, production of gamma-interferon and cell proliferation. While MT204 and the anti-CD25 mAb daclizumab were equally effective in inhibiting autocrine growth of human CD4(+) T cells, MT204 was far superior in preventing proliferation of NKL lymphoma cells, production of gamma-interferon by natural killer (NK) cells and proliferation of primary NK cells. MT204 has potential as a novel immunosuppressive and anti-proliferative therapy with an apparently broader spectrum of activities than anti-CD25 antibodies.

Interleukin (IL)-27 is a novel heterodimeric cytokine of the IL-12 family that is composed of two subunits, Epstein-Barr virus (EBV)-induced gene 3 (EBI3) and p28. EBI3 is expressed at high levels in EBV-transformed B-cell lines and is induced in vitro by the EBV oncogene LMP1 in a nuclear factor (NF)-κB-dependent manner. We show here that EBI3 expression is up-regulated in human T-cell leukemia virus type 1 (HTLV-1)-infected cell lines and IL-2-dependent leukemic cells from adult T-cell leukemia/lymphoma (ATL) patients, compared to normal activated T cells. EBI3 expression was decreased in HTLV-1-transformed cells after treatment with the NF-κB inhibitor BAY11-7082 and was induced in Jurkat cells by expression of HTLV-1 wild-type Tax oncoprotein, but not by the Tax mutant M22, which is defective for NF-κB activation. In situ analysis of EBI3 and p28 expression in Hodgkin's lymphomas (HLs), in various EBV-associated lymphoproliferative disorders (LPDs) (including post-transplant LPDs and nasal-type NK/T-cell lymphomas), and in ATL showed that EBI3 was expressed by neoplastic cells in all cases of HL and of LMP1-positive EBV-associated LPD, at variable levels in ATL cases, but rarely in control T-cell lymphomas. In contrast, in all lymphomas tested, no or few tumoral cells expressed p28. Consistent with these data, no significant p28 or IL-27 expression was detected in HL-derived cell lines, or in EBV- or HTLV-1-transformed cell lines. This selective overexpression of EBI3 by transformed cells suggests that EBI3 may play a role, independently from its association to p28, in regulating anti-viral or anti-tumoral immune responses.

The recently discovered cytokine IL-27 belongs to the IL-6/IL-12 family of cytokines and induced proliferation of naive CD4+ T cells and the generation of a Th1-type adaptive immune response. Although binding of IL-27 to the cytokine receptor WSX-1 was demonstrated, this interaction proved insufficient to mediate cellular effects. Hence, IL-27 was believed to form a heteromeric signaling receptor complex with WSX-1 and another, yet to be identified, cytokine receptor subunit. In this study, we describe that WSX-1 together with gp130 constitutes a functional signal-transducing receptor for IL-27. We show that neither of the two subunits itself is sufficient to mediate IL-27-induced signal transduction, but that the combination of both is required for this event. Expression analysis of WSX-1 and gp130 by quantitative PCR suggests that IL-27 might have a variety of cellular targets besides naive CD4+ T cells: we demonstrate gene induction of a subset of inflammatory cytokines in primary human mast cells and monocytes in response to IL-27 stimulation. Thus, IL-27 not only contributes to the development of an adaptive immune response through its action on CD4+ T cells, it also directly acts on cells of the innate immune system.

Originally the only known heterodimeric cytokine, IL-12 is now part of a family of five cytokines and shares important functions in the regulation of both innate and adaptive immunity with two of them, IL-23 and IL-27. Although initially these three cytokines were considered to have largely overlapping immunological functions, more recent studies, including two articles in this issue of Immunity (Hamano et al., 2003; Villarino et al., 2003), indicate that they mediate complex and well-differentiated functions.

IL-33 is a novel IL-1 family member with a putative role in inflammatory skin disorders and a complex biology. Therefore, recent conflicting data regarding its function in experimental models justify a close assessment of its tissue expression and regulation. Indeed, we report here that there are strong species differences in the expression and regulation of epidermal IL-33. In murine epidermis, IL-33 behaved similar to an alarmin, being constitutively expressed in keratinocyte nuclei and rapidly lost during acute inflammation. By contrast, human and porcine IL-33 were weakly expressed or absent in keratinocytes of noninflamed skin but induced during acute inflammation. To this end, we observed that expression of IL-33 in human keratinocytes but not murine keratinocytes was strongly induced by IFN-γ, and this upregulation completely depended on the presence of EGFR ligands. Accordingly, IFN-γ increased the expression of IL-33 in the basal layers of the epidermis in human ex vivo skin cultures only, despite good evidence of IFN-γ activity in cultures from both species. Together these findings demonstrate that a full understanding of IL-33 function in clinical settings must take species-specific differences into account.

Glycoprotein 130 (gp130) is a type I transmembrane protein and serves as the common signal-transducing receptor subunit of the interleukin-6-type cytokines. Whereas the membrane-distal half of the gp130 extracellular part confers ligand binding and has been subject to intense investigation, the structural and functional features of its membrane-proximal half are poorly understood. On the basis of predictions of tertiary structure, the membrane-proximal part consists of three fibronectin-type-III-like domains D4, D5 and D6. Here we describe the bacterial expression of the polypeptides predicted to comprise each of these three domains. The recombinant proteins were refolded from solubilized inclusion bodies in vitro, purified to homogeneity and characterized by means of size-exclusion chromatography and CD spectroscopy. For the first time the prediction of three individual membrane-proximal protein domains for gp130has been verified experimentally. The three domains do not show intermediate-affinity or high-affinity interactions between each other. Mapping of a neutralizing gp130 monoclonal antibody against D4 suggested a particular functional role of this domain for gp130 activation, because above that an intrinsic tendency for low-affinity oligomerization was demonstrated for D4.

A panel of 14 hybridoma cell lines secreting monoclonal antibodies against the human interleukin-11 receptor alpha chain (hIL-11Ralpha) was obtained using two different approaches. Two antibodies were raised against peptides of the N- and C-terminal sequences, respectively, of the extracellular part of the hIL-11Ralpha. Another group of 12 antibodies was generated against a hybrid protein consisting of the extracellular part of the hIL-11Ralpha fused to mature full-length human IL-2. All these antibodies recognized native hIL-11Ralpha and most also recognized the denatured receptor on immunoblots after SDS-PAGE. Four different epitopes were identified on the extracellular part of the hIL-11Ralpha. One epitope, defined by the E27 antibody, is located at the N-terminus and the other three epitopes are clustered in the membrane-proximal, C-terminal region. The antibodies defining epitopes I and II recognized membrane-bound hIL-11Ralpha expressed in gp130/hIL-11Ralpha-co-transfected Ba/F3 cells. The E27 antibody cross-reacted with murine IL-11Ralpha, in agreement with the fact that the N-terminal region is highly conserved between species. The other 13 antibodies all recognized a region between amino acids 319 and 363, which is the membrane-proximal part of the hIL-11Ralpha. This region, which is less conserved between mouse and human, is shown here to be an immunodominant region. Anti-IL-11Ralpha monoclonal antibodies, which have not been described previously enabled us to explore the expression and tissue distribution of IL-11Ralpha on human peripheral blood mononuclear cells and cell lines. The antibodies provide powerful tools for the study of the regulation and function of the receptor.

Gp130 is the common signal transducing receptor subunit of interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor and cardiotrophin-1. IL-6 and IL-11 induce gp130 homodimerization whereas the others lead to the formation of heterodimers with LIFR or OSMR. Binding epitopes for IL-6 and IL-11 are located in the immunoglobulin-like domain and the cytokine binding module (CBM). Here we show that a gp130 mutant lacking domain 1, although unresponsive to IL-6 and IL-11, can still activate signal transducer and activator of transcription (STAT) transcription factors in response to LIF or OSM. Moreover, point mutations in the CBM of gp130 (F191E and V252D) that severely impair signal transduction in response to IL-6 and IL-11 differentially interfere with gp130 activation in response to LIF and OSM. Thus, epitopes involved in gp130 homodimerization are distinct from those leading to the formation of gp130/LIFR or gp130/OSMR heterodimers. These findings may serve as the base for rational design of gp130 antagonists that specifically interfere with bioactivity of distinct IL-6-type cytokines.

IL-33 is an IL-1-related cytokine which has been implicated in T(h)2-associated biology and allergic diseases in humans and mice. IL-33 stimulates T(h)2 cells, mast cells, eosinophils, basophils, iNKT cells and circulating CD34(+) stem cells to proliferate and produce pro-allergic cytokines such as IL-5 and IL-13. IL-33 mediates its cytokine effects through a receptor consisting of ST2 and IL-1RAcP. Whereas IL-1RAcP is ubiquitously expressed, ST2 expression is cell-type restricted and determines responsiveness to IL-33. Studies employing ST2-deficient mice have reported variable results on the role of this receptor, and consequently IL-33, with regards to allergic lung inflammation. In this study, we demonstrate that IL-33 is important for allergic lung inflammation. Intra-nasal administration of IL-33 triggered an immediate allergic response in the airways, and more importantly, we show that endogenous IL-33 contributes to airway inflammation and peripheral antigen-specific responses in ovalbumin-induced acute allergic lung inflammation using IL-33-deficient mice. Our results suggest that IL-33 is sufficient and required for severe allergic inflammation in the lung and support the concept of IL-33 as a therapeutic target in allergic lung inflammation.

C17 was first described about ten years ago as a gene expressed in CD34+ cells. A more recent study has suggested a role for C17 in chondrogenesis and development of cartilage. However, based on sequence analysis, we believe that C17 has homology to IL-2 and hence we present the hypothesis that C17 is a cytokine possessing immune-regulatory properties. We provide evidence that C17 is a secreted protein preferentially expressed in chondrocytes, hence in cartilage-rich tissues. Systemic expression of C17 in vivo reduces disease in a collagen antibody-induced arthritis model in mice (CAIA). Joint protection is evident by delayed disease onset, minimal edema, bone protection and absence of diverse histological features of disease. Expression of genes typically associated with acute joint inflammation and erosion of cartilage or bone is blunted in the presence of C17. Consistent with the observed reduction in bone erosion, we demonstrate reduced levels of RANKL in the paws and sera of mice over-expressing C17. Administration of C17 at the peak of disease, however, had no effect on disease progression, indicating that C17's immune-regulatory activity must be most prominent prior to or at the onset of severe joint inflammation. Based on this data we propose C17 as a cytokine that s contributes to immune homeostasis systemically or in a tissue-specific manner in the joint.

Interleukin-23 (IL-23) is a heterodimeric cytokine, a central factor in chronic/autoimmune inflammation. It signals through a heterodimeric receptor consisting of IL-23r, which is heavily glycosylated. The structural characterization of IL-23r has not been reported. In this work, glycosylation profiles of soluble recombinant human IL-23r (rhIL-23r) were established using mass spectrometry (MS), which included defining glycosylation sites, degree of glycosylation occupancy of each site and structure of attached oligosaccharides. Specifically, precursor ion scan of oxonium ion protonated N-acetylglucosamine (GlcNAc+) (m/z 204) was performed using a triple quadrupole MS instrument to locate the retention time of glycopeptides. Both the glycopeptides and their corresponding deglycosylated forms in each collected HPLC fraction were studied by liquid chromatography-tandem mass spectrometry (LC–MS/MS) (LTQ-Orbitrap) for glycosylation site profiling. The attached glycan structures were elucidated by collision-induced dissociation (CID) fragmentation of target glycopeptides in combination with accurate mass measurement. Eight glycosylation sites were identified on IL-23r (Asn24, Asn209, Asn239, Asn157, Asn118, Asn250, Asn58 and Asn6). Most of the glycosylation sites were > 95% occupied except Asn250 and Asn6. Those two sites were 88% and 45% occupied by estimation from trypsin digestion and were 55% and 42% occupied from LysC digestion. Multiple glycoforms were observed in IL-23r. Most of them were bi-, tri- or tetra-antennary complex type structures with fucose and sialic acid. High mannose and hybrid type glycans were only observed on Asn157. The structural characterization on IL-23r glycosylation provides useful information for better understanding of the biological function of IL-23r. Copyright © 2010 John Wiley & Sons, Ltd.

NKG2D is a surface receptor expressed on NK cells but also on CD8(+) T cells, gammadelta T cells, and auto-reactive CD4(+)/CD28(-) T cells of patients with rheumatoid arthritis. Various studies suggested that NKG2D plays a critical role in autoimmune diseases, e.g., in diabetes, celiac disease and rheumatoid arthritis (RA), rendering the activating receptor a potential target for antibody-based therapies. Here, we describe the generation and characteristics of a panel of human, high-affinity anti-NKG2D IgG1 monoclonal antibodies (mAbs) derived by phage display. The lead molecule mAb E4 bound with an affinity (KD) of 2.7 +/- 1.4 x 10(-11) M to soluble and membrane-bound human NKG2D, and cross-reacted with NKG2D from cynomolgus macaque, indicating potential suitability for studies in a relevant primate model. MAb E4 potently antagonized the cytolytic activity of NKL cells against BaF/3-MICA cells expressing NKG2D ligand, and blocked the NKG2D ligand-induced secretion of TNFalpha, IFNgamma and GM-CSF, as well as surface expression of CRTAM by NK cells cultured on immobilized MICA or ULBP-1 ligands. The antibody did not show a detectable loss of binding to NKG2D after seven days in human serum at 37 degrees C, and resisted thermal inactivation up to 70 degrees C. Based on these results, anti-human NKG2D mAb E4 provides an ideal candidate for development of a novel therapeutic agent antagonizing a key receptor of NK and cytotoxic T cells with implications in autoimmune diseases.

muS110 is a BiTE antibody bispecific for murine EpCAM (CD326) and murine CD3. MT110, its human-specific analog, is in a clinical phase 1 trial for treatment of patients with adenocarcinoma of the lung or gastrointestinal tract. Recent studies have shown a therapeutic window for muS110, have explored single-dose toxicity of muS110, and have found that a 1-week low-dose treatment dramatically increased the tolerability of mice to very high doses of muS110 (Cancer Immunol. Immunother. 2009;58:95-109). Here we analyzed the impact of long-term, high-dose treatment of mice with muS110 on antitumor activity and functionality of T cells. After an initial self-limiting cytokine release, the 1-week adaptation period effectively blunted further cytokine production in response to a subsequent high-dose treatment with muS110. The much-increased tolerability of mice adapted to muS110 was not because of anergy of T cells. T cells isolated from chronically muS110-treated mice fully retained their cytotoxic potential, proliferative capacity, and responsiveness to stimulation by either muS110 or anti-CD3/anti-CD28/interleukin-2 when compared with T cells from control mice. Unimpaired T-cell performance was also evident from the effective prevention of orthotopic 4T1 breast tumor outgrowth in mice treated long term with escalating doses of muS110. Finally, we show that muS110 and MT110 recognize orthologous epitopes on mouse and human EpCAM proteins, suggesting that the target-related safety profile of muS110 in mice may be predictive for MT110 in humans.

Bispecific antibodies have been extensively studied in vitro and in vivo for their use in redirected tumor cell lysis. A particular challenge of bispecific antibody constructs that recognize the invariant CD3 signaling complex is a controlled polyclonal activation of T cells that, ideally, is exquisitely dependent on the presence of target cells. Otherwise, overt production of inflammatory cytokines and secondary reactions may occur as side effects, as can be observed with constitutively T-cell activating monoclonal antibodies to CD3 or CD28, and with bispecific antibodies bearing Fc gamma portions. Here we analyzed 2 distinct bispecific single-chain antibody constructs of the BiTE class, called MT110 and MT103 (or MEDI-538), for conditional T-cell activation. In the presence of target-expressing cell lines, low picomolar concentrations of the BiTE molecules were sufficient to stimulate a high percentage of peripheral human T cells to express cytokines and surface activation markers, enter into cell cycle, and induce redirected lysis of target cells. However, in the absence of target cells, the 2 BiTE molecules even at high concentrations did not detectably activate T cells. Our data show that T cell activation by monomeric forms of MT110 and MT103 is highly conditional in that it is strictly dependent on the presence of cells expressing the proper target antigen. BiTE molecules therefore qualify for a highly controlled polyclonal T-cell therapy of cancer.

Cytokines produced at the fetal-maternal interface play a key role in regulating maternal tolerance to the fetus and successful pregnancy. Previously, we showed that EBV-induced gene 3 (EBI3), an interleukin (IL)-12 p40 homologue, was expressed at very high levels by syncytiotrophoblasts and extravillous trophoblasts throughout human pregnancy. EBI3 was recently shown to associate with a novel ligand, p28, to form a new heterodimeric cytokine with important immunoregulatory functions, IL-27. In this study, we investigated whether EBI3 expression by trophoblast cells is associated with that of p28 to form IL-27. We found that genes encoding IL-27 (EBI3 and p28) and its receptor (IL-27R and gp130) were expressed in the placenta at various stages of pregnancy. Co-immunoprecipitation experiments performed from placental lysates, and ELISA of culture supernatants from placental explants, showed that IL-27 heterodimer was produced and released from placental cells. In situ studies of placentae of first, second and third trimester of pregnancy, and of choriocarcinomas, demonstrated that syncytiotrophoblast cells co-expressed EBI3 and p28. Similarly, extravillous trophoblast cells invading the decidua were found to co-express both subunits of IL-27. These data suggest that IL-27 may be part of the cytokine network regulating local immune responses and angiogenesis during human pregnancy.

Introduction: Idelalisib is a highly selective oral inhibitor of the phosphoinositide 3-kinase delta (PI3Kδ) that is hyperactive in many B-cell malignancies and is critical for the activation, proliferation, survival and trafficking of B lymphocytes. Idelalisib is approved in the US for the treatment of chronic lymphocytic leukemia (CLL) in combination with rituximab and as monotherapy for patients with relapsed follicular B-cell non-Hodgkin lymphoma and small lymphocytic lymphoma who have received at least two prior systemic therapies. Obinutuzumab (GA101) is a glycoengineered type II, CD20 antibody that induces a high level of direct cell death. As a result of glycoengineering, obinutuzumab has increased affinity for FcγRIII on innate immune effector cells resulting in enhanced induction of ADCC and ADCP. Obinutuzumab has been approved for first line treatment of CLL patients in combination with chlorambucil in the US and Europe and is currently in pivotal clinical trials in indolent NHL and DLBCL. Previous work has shown the covalent BTK inhibitor ibrutinib can interfere with the immune effector function and ultimately in vivo efficacy of rituximab in preclinical models (Kohrt et al., Blood, 2014). As PI3K isoforms also play a role in immune effector cells and FcγR signaling we investigated the impact of PI3Kδ inhibition by the PI3Kδ selective inhibitor idelalisib on the immune effector function of obinutuzumab and rituximab. Experimental methods: The impact of idelalisib on NK cell mediated ADCC induction by obinutuzumab and rituximab was investigated in LDH release assays using WIL2-S, SU-DHL4 and Z138 target cells at plasma protein-binding adjusted clinically relevant concentrations mimicking exposure in patients. As a surrogate for NK cell activation CD16 levels and up-regulation of the degranulation marker CD107a were assessed by FACS. The impact on monocyte-derived macrophage mediated ADCP of WIL2-S cells was measured in a flow cytometry-based phagocytosis assay. Finally, depletion of CD19 positive B cells was determined in whole blood from healthy volunteers in flow cytometry-based whole blood assay. Results: In ADCC assays, no impact of idelalisib on ADCC at saturating concentration of obinutuzumab or rituximab (>1ug/ml) can be detected in LDH release assays with tumor cells targets (N=9 donors for WIL2-S, N>3 donors for SU-DHL-4 and Z138). Idelalisib did not alter obinutuzumab or rituximab ability to kill tumor cells by ADCC at low E:T ratio. Little to no increase of obinutuzumab or rituximab EC50 for LDH release, CD16 down regulation, or degranulation of NK cells could be detected depending on donor effector cells. ADCP assays were conducted with M2c polarized macrophages using WIL2-S as targets. Less than 30% inhibition of ADCP was observed in this assay at idelalisib concentration at protein binding-adjusted clinical Cmax. At idelalisib Cmax (4200 nM) the EC50 of obinutuzumab-mediated B cell depletion in healthy human whole blood was increased 3 to 5 times, whereas at Cmin (760 nM) idelalisib did not significantly influence obinutuzumab EC50 or maximal B cell depletion. Conclusions: PI3Kδ inhibition by idelalisib has minimal impact on the immune effector function of obinutuzumab (GA101) and rituximab as measured in NK cell-mediated ADCC, macrophage-mediated ADCP and whole blood B-cell depletion. Disclosures Herter: Roche: Employment. Palazzo:Gilead Sciences: Employment. Bacac:Roche: Employment. Grosmaire:Gilead Sciences: Employment. Frey:Gilead Sciences: Employment. Pflanz:Gilead Sciences: Employment. Liu:Gilead Sciences: Employment. Tannheimer:Gilead Sciences: Employment. Umana:Roche: Employment. Klein:Roche: Employment, Equity Ownership, Patents & Royalties. Queva:Gilead Sciences: Employment.

Interleukin (IL)-27 is a novel heterodimeric cytokine of the IL-12 family that is composed of two subunits, Epstein-Barr virus (EBV)-induced gene 3 (EBI3) and p28. EBI3 is expressed at high levels in EBV-transformed B-cell lines and is induced in vitro by the EBV oncogene LMP1 in a nuclear factor (NF)-kappaB-dependent manner. We show here that EBI3 expression is up-regulated in human T-cell leukemia virus type 1 (HTLV-1)-infected cell lines and IL-2-dependent leukemic cells from adult T-cell leukemia/lymphoma (ATL) patients, compared to normal activated T cells. EBI3 expression was decreased in HTLV-1-transformed cells after treatment with the NF-kappaB inhibitor BAY11-7082 and was induced in Jurkat cells by expression of HTLV-1 wild-type Tax oncoprotein, but not by the Tax mutant M22, which is defective for NF-kappaB activation. In situ analysis of EBI3 and p28 expression in Hodgkin's lymphomas (HLs), in various EBV-associated lymphoproliferative disorders (LPDs) (including post-transplant LPDs and nasal-type NK/T-cell lymphomas), and in ATL showed that EBI3 was expressed by neoplastic cells in all cases of HL and of LMP1-positive EBV-associated LPD, at variable levels in ATL cases, but rarely in control T-cell lymphomas. In contrast, in all lymphomas tested, no or few tumoral cells expressed p28. Consistent with these data, no significant p28 or IL-27 expression was detected in HL-derived cell lines, or in EBV- or HTLV-1-transformed cell lines. This selective overexpression of EBI3 by transformed cells suggests that EBI3 may play a role, independently from its association to p28, in regulating anti-viral or anti-tumoral immune responses.