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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.

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.

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.