TLR Stimulation Produces IFN-β as the Primary Driver of IFN Signaling in Nonlymphoid Primary Human Cells

Ryan Nistler, Ankur Sharma, Katrina Meeth, Christine Huard, Christine Loreth, Alireza Kalbasi,

Edyta Tyminski, Ryan Bellmore, Anthony J. Coyle, Stefano V. Gullà, Stephen P. Berasi, Steven A. Downloaded from Greenberg and Janet E. Buhlmann

ImmunoHorizons 2020, 4 (6) 332-338 doi: https://doi.org/10.4049/immunohorizons.1800054

http://www.immunohorizons.org/content/4/6/332 http://www.immunohorizons.org/ This information is current as of October 2, 2021.

Supplementary http://www.immunohorizons.org/content/suppl/2020/06/18/4.6.332.DCSupp Material lemental References This article cites 31 articles, 4 of which you can access for free at: http://www.immunohorizons.org/content/4/6/332.full#ref-list-1 by guest on October 2, 2021 Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://www.immunohorizons.org/alerts

ImmunoHorizons is an open access journal published by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. ISSN 2573-7732. RESEARCH ARTICLE

Clinical and Translational Immunology

TLR Stimulation Produces IFN-b as the Primary Driver of IFN Signaling in Nonlymphoid Primary Human Cells

Ryan Nistler,* Ankur Sharma,* Katrina Meeth,* Christine Huard,* Christine Loreth,† Alireza Kalbasi,* Edyta Tyminski,* † ‡ Ryan Bellmore, Anthony J. Coyle,* Stefano V. Gulla`,* Stephen P. Berasi,* Steven A. Greenberg, ,§,2 and Janet E. Buhlmann*,2 †

*Centers for Therapeutic Innovation, Pfizer Inc., Cambridge, MA 02139; Research Business Technology, Pfizer Inc., Cambridge, MA 02139; Downloaded from ‡Department of Neurology, Brigham and Women’s Hospital, Boston, MA 02115; and §Boston Children’s Hospital Computational Health Informatics Program, Harvard Medical School, Boston, MA 02115

ABSTRACT http://www.immunohorizons.org/ Several human autoimmune diseases are characterized by increased expression of type 1 IFN-stimulated in both the peripheral blood and tissue. The contributions of different type I IFNs to this signature are uncertain as the type I IFN family consists of 13 alphas and one each of b, e, k, and v subtypes. We sought to investigate the contribution of various IFNs to IFN signaling in primary human cell types. We stimulated primary skin, muscle, kidney, and PBMCs from normal healthy human donors with various TLR ligands and measured the expression of type I IFN subtypes and activation of downstream signaling by quantitative PCR. We show that IFNB1 is the dominant type I IFN expressed upon TLR3 and TLR4 stimulation, and its expression profile is associated with subsequent MX1 transcription. Furthermore, using an IFN-b–specific neutralizing Ab, we show that MX1 expression is inhibited in a dose-dependent manner, suggesting that IFN-b is the primary driver of IFN-stimulated genes following TLR3 and TLR4 engagement. Stimulation with TLR7/8 and TLR9 ligands induced IFNB1 and IFNA subtypes and MX1 expression only in PBMCs and not in tissue by guest on October 2, 2021 resident cell types. Concordantly, IFN-b neutralization had no effect on MX1 expression in PBMCs potentially because of the combination of IFNB1 and IFNA expression. Combined, these data highlight the potential role for IFN-b in driving local inflammatory responses in clinically relevant human tissue types and opportunities to treat local inflammation by targeting IFN-b. ImmunoHorizons, 2020, 4: 332–338.

INTRODUCTION pathogenesis of autoimmune disorders, including dermatomyositis (DM), lupus nephritis (LN), cutaneous lupus erythematosus Type I IFNs are secreted pleiotropic that play a pivotal (CLE), and systemic lupus erythematosus (SLE) (3–6) as well as role in innate and adaptive immune responses against pathogen monogenic autoimmune disorders such as STING-associated invasion. Among the various type I IFNs known, the most well vasculopathy of infancy and Aicardi-Goutieres` syndrome (7, 8). studied are IFN-a (13 IFN-a subtypes exist, each encoded by Specifically, correlation of type I IFN gene signature with disease adifferent gene) and IFN-b (1). Although these cytokines play severity has been reported in DM, LN, and SLE (6, 9–11). One a crucial role in pathogen defense, their sustained production study found that ectopic stimulation of cultured muscle cells canresultininflammation (2). Type I IFNs are linked to the with purified IFN-a or IFN-b phenocopied the molecular

Received for publication August 3, 2018. Accepted for publication May 20, 2020. Address correspondence and reprint requests to: Dr. Steven A. Greenberg or Dr. Janet E. Buhlmann, Brigham and Women’s Hospital, Department of Neurology, and Children’s Hospital Informatics Program, Harvard Medical School, 75 Francis Street, Boston, MA 02115 (S.A.G.) or Centers for Therapeutic Innovation, Pfizer Inc., 610 Main Street, Cambridge, MA 02139 (J.E.B.). E-mail addresses: [email protected] (S.A.G.) or [email protected] (J.E.B.) ORCIDs: 0000-0002-6712-5241 (R.N.); 0000-0002-6657-4950 (A.K.); 0000-0002-9319-7652 (R.B.); 0000-0002-6305-5354 (J.E.B.). 2S.A.G. and J.E.B. contributed equally to the work. Abbreviations used in this article: CLE, cutaneous lupus erythematosus; Ct, cycle threshold; DM, dermatomyositis; GMC, glomerular mesangial cell; LN, lupus nephritis; NHDF, normal human dermal fibroblast; poly I:C, polyinosinic:polycytidylic acid; SLE, systemic lupus erythematosus; SMC, skeletal muscle cell. The online version of this article contains supplemental material. This article is distributed under the terms of the CC BY-NC-ND 4.0 Unported license. Copyright © 2020 The Authors

332 https://doi.org/10.4049/immunohorizons.1800054

ImmunoHorizons is published by The American Association of Immunologists, Inc. ImmunoHorizons TLR-STIMULATED PRIMARY CELLS PREDOMINATELY PRODUCE IFN-b 333 signature found in DM muscle biopsies with stronger correlation IFN-b. Isotype control Ab was generated in-house and showed to IFN-b stimulation than IFN-a (12). Elevated blood IFN-b no binding to any IFN. All procedures performed on animals levels, but not IFN-a, also correlate with high type I IFN gene were in accordance with regulations and established guidelines signature and DM disease activity (5, 9, 13). Elevated skin IFNB1 and were reviewed and approved by an institutional animal care transcript, but not IFNA, IFNK,orIFNO transcripts, also and use committee. correlate with skin type I IFN gene signature in DM (14). The correlation of IFN-stimulated genes with disease severity Specificity of anti–IFN-b Ab has led to recent clinical studies to treat SLE by neutralizing IFN-a IFN-b (300-02BC; Peprotech) and IFN-a2 (11100-1; PBL Assay subtypes (, ) or by neutralizing IFNAR1 Science) were diluted with anti–IFN-b in RPMI. The final Ab () (15–17). Phase IIb clinical studies with the various concentrations ranged from 100 to 1 3 107 nM. The IFNs were mAbs showed mixed results, with rontalizumab failing to meet diluted to a final concentration of 20 pM (IFN-b)or50pM(IFN- its primary and secondary endpoints (18). Sifalimumab met its a2) with medium alone or a dose curve of the anti–IFN-b Ab in a primary, but not secondary endpoints, with modest therapeutic final volume of 50 ml and incubated for 2 h at 37°C. U-937 cells effect (19). Anifrolumab met both primary and secondary endpoints, were cultured in RPMI supplemented with 10% FBS, 13 Glutamax fi and treated patients showed improvement over patients receiving (35050061; Thermo Fisher Scienti c) and harvested just before Downloaded from placebo, although the result was not dose dependent and only stimulation. Cells were counted and resuspended at 2 3 106 cells/ml observed in patients with a high IFN gene signature at baseline in RPMI. Fifty microliters of cells was added to 50 ml of diluted (20, 21). Combined, these results suggest that targeting IFN-a a-IFNAR Ab and incubated for 2 h. After the incubations, the alone may not be sufficient to reduce modest to severe SLE. IFNs and diluted Abs were mixed with cells (100,000 per well) We sought to identify which type I IFNs are induced in and incubated at 37°C for 15 min. clinically relevant primary human cells when challenged with TLR At the conclusion of the incubation, an equal volume of Cytofix http://www.immunohorizons.org/ ligands. Heightened expression of TLRs in local tissue and (554655; BD Biosciences) was added per well, and the plate was infiltrating cells has been observed in DM and polymyositis (PM), returned to 37°C for 15 min. After fixation, plates were centrifuged highlighting a potential feedback loop for inflammation (22). In at 1500 rpm for 5 min to pellet cells. Cells were washed with 200 ml this study, we used primary human cells from healthy donors that of PBS and resuspended in 100 mlof13 Phosflow Perm Buffer IV represent tissues involved in DM, CLE, LN, and SLE pathologies: (612597; BD Biosciences) for 15 min at room temperature. After dermal fibroblasts, skeletal muscle cells (SMC), glomerular incubation, plates were centrifuged and washed as described mesangial cells (GMC), and PBMCs. We measured the expression above. Cells were resuspended in 100 mlofPBSwith5%FBS profile of IFNB1 and IFNA subtypes in response to TLR3, TLR4, (FACS buffer) containing TruStain FcX (422302; BioLegend) and TLR7/8, and TLR9 activation. To evaluate downstream IFNAR incubated for 10 min at 4°C. After incubation, 10 mlofAF647 signaling, we measured expression of MX1, a clinical marker for mouse anti-STAT1 (pY701) Ab (612597; BD Biosciences) was by guest on October 2, 2021 type I IFN activity that is overexpressed in DM, CLE, LN, and SLE addedperwellandincubatedfor20min.Next,100mlofFACS patients (23–26). In addition, we used a high-affinity anti–IFN-b buffer was added per well and centrifuged as described above. The Ab to assess the specificroleofIFN-b in driving MX1 expression wash was repeated, and samples were resuspended in a final in the various cell types. This study identifies the expression volume of 120 mlofFACSbuffer for data acquisition on the pattern of type I IFN species in human disease-relevant tissues Fortessa flow cytometer (BD Biosciences). and suggests a novel role for IFN-b in driving local inflammation Data were analyzed using FlowJo software (TreeStar); forward and tissue injury that may contribute to the autoimmune disease scatter () channel versus side scatter (light) channel was used phenotype. to identify cells, and the geometric mean fluorescence intensity of

the pSTAT1 signal was determined. EC50,EC90,andIC50 analyses were done using GraphPad Prism v 6.0. Data were transformed MATERIALS AND METHODS (concentration X = log [x]), then a nonlinear regression analysis using a 4-parameter slope of log (inhibitor/stimulator) versus Reagents response (geometric mean fluorescence intensity) was performed Polyinosinic:polycytidylic acid (poly I:C; P9582) and LPS (L2755) (Supplemental Fig. 1). were purchased from Sigma-Aldrich. R848 (Tlrl-r848) and ODN2216 (Tlrl-2216) were purchased from Invivogen. Anti– Cells and cell culture IFN-b Ab was generated in-house using licensed commercially Normal human dermal fibroblasts (NHDF) (CC-2511; Lonza) were available IFN-b (Peprotech) as Ag. Anti-human IFN-b Abs were cultured in FGM-2 media (CC-3132; Lonza), and GMC (CC-2559; generated by immunizing BALB/c mice with IFN-b and fusing Lonza) were cultured in MsGM media (CC-3146; Lonza). SMC mouse splenocytes with NS-1 myeloma cells. Hybridomas were (SK-1111; Cook Myosite) were cultured in Myotonic Growth screened for IFN-b binders, and leading candidates were Medium (MK-4444; Cook Myosite). At least five donors of each humanized and underwent affinity maturation to improve affinity. cell type were purchased for experiments. PBMCs were isolated The final IgG1 candidate was shown to have a neutralizing affinity from healthy donor blood using Ficoll-Paque Plus separation ,20 pM to human IFN-b and no cross-reactivity to mouse or rat medium (17-1440-03; GE Healthcare) in Leucosep tubes (227290;

https://doi.org/10.4049/immunohorizons.1800054 334 TLR-STIMULATED PRIMARY CELLS PREDOMINATELY PRODUCE IFN-b ImmunoHorizons

Greiner Bio-One) and cultured in RPMI 1640 (11875-093; Life IFN ELISA Technologies) supplemented with 10% FBS (16140-071; Life Cell supernatants were collected and run neat (IFN-b) or diluted Technologies). Informed consent was obtained from all human 1:10 (IFN-a). IFN-b ELISA (41415-1; PBL Assay Science) and IFN-a subjects in accordance with Pfizer Institutional Review Board multisubtype (41105-1; PBL Assay Science) were run according to guidelines. the manufacturer’sspecifications.

Real-time quantitative PCR, data analyses, and heatmap generation RESULTS RNA was isolated using RNeasy Micro Kit (74004; Qiagen), and cDNA was synthesized using a high-capacity cDNA reverse IFNB1 is the dominant type I IFN produced in somatic transcription (4368813; Applied Biosystems). TaqMan assays cell types were purchased from Applied Biosystems. For heatmap genera- Primary human cells were chosen based on their relevance to a tion, four independent donors of each cell type were assayed and variety of autoimmune disorders. We obtained primary human the results were averaged. For neutralization studies, at least five cells from normal healthy donors and exposed them to a range of

independent donors were used except where noted. Samples from stimulatory ligands to mimic various pathogen- or danger- Downloaded from each donor were run in duplicate and averaged. Data are displayed associated molecular patterns that may trigger the production of as the average with each individual donor plotted. Relative an immune response. In NHDF, GMC, and SMC samples, both the quantification was obtained using the DD cycle threshold (Ct) TLR3 ligand poly I:C and the TLR4 ligand LPS induced a robust method. Individual primer efficiency was tested and ranged IFNB1 response, whereas IFNA expression was mostly unaffected from 91.99 to 109.37%. (Fig. 1). The highest expression of IFNA was in the NHDF, where

one a subtype, IFNA5, showed a 6-fold expression increase over http://www.immunohorizons.org/ Neutralization studies medium alone, whereas the corresponding IFNB1 increase was Primary human cells were stimulated with LPS, poly I:C, R848, .2200-fold (Fig. 1A). Following poly I:C and LPS stimulation, the or ODN2216 at the minimal dose that generated robust MX1 IFNB1 transcript was upregulated and trailed at later time points expression (determined empirically for each cell line and by a concomitant increase in MX1 expression. With the TLR 7/8 stimulant). Neutralizing IFN-b Ab (100 nM) or a dose curve of and 9 ligands, R848 and ODN2216, respectively, little IFNB1 or 0.1–100 nM was added to cells concurrently with TLR ligands IFNA expression was seen, and therefore, no MX1 expression was and incubated with cells for varying times (see figures for seen either. details). RNA was isolated and analyzed by TaqMan quantitative To assess the contribution of IFN-b to drive expression PCR as described above. Expression of MX1 was measured as a of MX1, we stimulated all the cell types in the presence of a relative amount per 10 ng of RNA per sample. neutralizing anti–IFN-b Ab (0.1–100 nM). The time frame and by guest on October 2, 2021

FIGURE 1. IFNB1 is the primary IFN generated in nonhematopoietic cells. Heat map depiction of IFNB1, MX1, and IFNA subtype transcript expression on a log scale in (A) donor-derived NHDF, (B) GMC, and (C) SMC. Cells were stimulated with the following: 25 mg/ml (NHDF, GM) or 125 mg/ml (SMC) of poly I:C, 5 ng/ml (NDHF, GMC) or 25 ng/ml (SMC) of LPS, 5 mg/ml of R848, and 5 mM of ODN 2216. The number following the ligand name is the length of stimulation. Each cell in the heat map represents the average result of at least four individual donors. Results are displayed as log2 of DDCt of the gene of interest compared with the geometric mean of GUSB and B2M as endogenous controls.

https://doi.org/10.4049/immunohorizons.1800054 ImmunoHorizons TLR-STIMULATED PRIMARY CELLS PREDOMINATELY PRODUCE IFN-b 335 dose of stimulant were chosen empirically for each cell type, based little to no expression of the IFNA subtypes. To explore the on minimum dose of agonist required to observe maximal MX1 contribution of IFN-b or IFN-a subtypes to MX1 expression induction (Supplemental Fig. 2A, 2B). Addition of the neutralizing following stimulation with all the ligands, we inhibited IFN-b with Ab resulted in a dose-dependent decrease in MX1 expression, a dose curve of anti–IFN-b neutralizing Ab. With both TLR3 and 4 whereas IFNB1 expression levels were unchanged (data not stimulation, we observed a significant reduction of MX1 expres- shown). A nonneutralizing control IgG had no effect on MX1 levels sion, further demonstrating the significance of IFN-b expression (Fig. 2, Supplemental Fig. 3A–C, 3E–G). following TLR3 and 4 engagement. (Fig. 3B, 3C, Supplemental Fig. 3D,3H).However,wewereunabletoalterMX1 expression in PBMCs generate IFNB1 and IFNA in a PBMCs stimulated with R848 or ODN2216, likely because of the ligand-dependent manner abundant expression of IFN-a subtypes (Fig. 3D, 3E, Supplemental As PBMCs have been widely studied and validated as a major Fig. 3I, 3J). To confirm expression of IFN-b and IFN-a subtypes, source of IFN-a in blood (27), we investigated IFNA, IFNB1, we measured protein concentration by ELISA (Fig. 3F). Superna- and MX1 transcript levels following TLR activation. We tant from stimulated cells of two donors was collected after 8 h and stimulated PBMCs with all four TLR ligands in a dose- and analyzed by an IFN-b–specificELISAandapan–IFN-a ELISA. b time-dependent manner to measure IFNA, IFNB1,andMX1 IFN- was only detected in cultures from one donor stimulated Downloaded from expression. Of all cell types tested, only PBMCs responded to with ODN2216 and was present at 1/50th of the pan–IFN-a levels R848 and ODN stimulation and upregulated IFNB1 and IFNA observed with the same donor. IFN-a was observed from both transcripts in response to the ligands. These data show that donors with both stimuli. The contribution of individual IFN-a among the cell types tested, only PBMCs respond to TLR7/8/9 subtypes is difficult to determine because of the lack of selective ligands and express both IFNB1 and IFNA subtypes (Fig. 3A, reagents. This result, combined with the inability of the anti–IFN-

Supplemental Fig. 2C, 2D). b Ab to impact MX1 expression, suggests that IFN-a is the primary http://www.immunohorizons.org/ When PBMCs were stimulated with poly I:C and LPS, type I IFN driving MX1 expression following TLR7/8/9 stimula- however, it was predominately IFNB1 that was upregulated with tion in PBMCs. by guest on October 2, 2021

FIGURE 2. Anti–IFN-b Ab blocks IFN signaling in nonhematopoietic cells. NHDF (A and B), GMC (C and D), and SMC (E and F) were stimulated with LPS for 5 h (A, C, and E) or poly I:C for 6 h (B, D, and F) in the presence of medium, control IgG, or anti–IFN-b Ab. The TLR ligands and Ab were given at the same time. MX1 expression was measured, and the relative quantification (RQ) was calculated by transforming the log2 value of DDCt of MX1 compared with the geometric mean of GUSB and B2M as endogenous controls. As there was donor-to-donor variation, the response of each donor to ligand only was set to 1, and the results are displayed as a percentage of the ligand-only response. Each data point represents the average of two to three independent biological replicates of five independent healthy donors per condition (except LPS-stimulated GMC, which only had three donors).

https://doi.org/10.4049/immunohorizons.1800054 336 TLR-STIMULATED PRIMARY CELLS PREDOMINATELY PRODUCE IFN-b ImmunoHorizons Downloaded from http://www.immunohorizons.org/ by guest on October 2, 2021

FIGURE 3. PBMCs generate IFN-b and IFN-a in a ligand-dependent manner. Heat map depiction of IFNB1, MX1,andIFNA subtype transcript expression on a log scale in PBMCs (A). Cells were stimulated with the following: 5 mg/ml of poly I:C, 5 ng/ml of LPS, 5 mg/ml of R848, and 0.25 mM of ODN 2216. The number following the ligand name is the length of stimulation. Each cell in the heat map represents the average result of at least five individual donors. Results are displayed as log2 of DDCt of the gene of interest compared with the geometric mean of GUSB and B2M as endogenous controls. PBMCs were stimulated with LPS for 5 h (B), poly I:C for 6 h (C), R848 for 24 h (D), or ODN for 24 h (E)inthe presence of medium, control IgG, or anti–IFN-b Ab. The TLR ligands and Ab were given at the same time. MX1 expression was measured, and the relative quantification (RQ) was calculated by transforming the log2 value of DDCt of MX1 compared with the geometric mean of GUSB and B2M as endogenous controls. As there was donor-to-donor variation, the response of each donor to ligand only was set to 1,andtheresultsaredisplayedasapercentageofthe ligand-only response. Each data point represents the average of two to three independent biological replicates of six independent healthy donors per condition. (F) Supernatants were taken from two donors following R848 and ODN stimulation to measure the concentrations of IFN-b and IFN-a.

Differential gene expression of TLRs in primary human cells was observed only in PBMCs, TLR9 transcripts were detectable in We assessed TLR transcript levels in unstimulated cells to determine all cell types, albeit at levels far reduced compared with PBMCs. if TLR expression correlated with response (Supplemental Fig. 4). Consistent with the capacity of all these cell types to respond to poly I:C and LPS, TLR3 and TLR4 transcript was detected in all DISCUSSION cells. In contrast, basal TLR7 was only detected in PBMCs, and a small amount was detected in GMC. TLR8 was undetectable in all Clinical evidence has implicated circulating levels of type I IFNs in the cell types except PBMCs. Whereas a response to ODN2216 thepathogenesisofautoimmunediseaseslikeSLE,CLE,andLN

https://doi.org/10.4049/immunohorizons.1800054 ImmunoHorizons TLR-STIMULATED PRIMARY CELLS PREDOMINATELY PRODUCE IFN-b 337

(6). However, the potential for IFN-b to contribute to substantial result in a decrease of pathogen-associated molecular pattern local inflammation and tissue destruction remained unexplored. receptors, therefore increasing the threshold of signaling required Measuring IFNs in diseased tissue faces technical and biological to initiate or maintain a response. Further profiling of DM and challenges that have largely prohibited researchers from identi- lupus tissues may elucidate the source and effect of localized IFN fying IFN-b or distinguishing between IFN-a subtypes in serum induction. and tissues from patients with autoimmune disorders. Given the In summary, our results indicate that IFN-b is the dominant heterogenous nature of autoimmune disease, the underlying type I IFN induced in human-derived primary tissue resident cells triggers for the type I IFN response are unknown. Thus, only when challenged with various TLR ligands. These findings suggest measuring circulating IFNs may be a poor clinical biomarker for that IFN-b may have a critical role in local tissue damage and determining the contribution of those IFNs to disease. This study autoimmune disease modulation that has previously been un- identifies IFNB1 as the major type I IFN produced by primary derappreciated. Furthermore, specificneutralizationofIFN-b human disease-relevant cells when challenged with various TLR resulted in an attenuation of MX1 expression, indicating that ligands. Furthermore, we show that neutralizing the production of IFN-b inhibition could be a novel therapy in the treatment of IFN-b in nonlymphoid primary cells can abrogate downstream DM, lupus, and other IFNopathies.

IFN signaling. Downloaded from Of note, the primary cells used in this study were from healthy DISCLOSURES donors. Cells from diseased tissue may amplify the type I IFN response through upregulation of TLR3, TLR7, and TLR9 as R.N., A.S., K.M., C.H., C.L., A.K., E.T., R.B., A.J.C., S.P.B., and J.E.B. reported in PBMCs from SLE patients (28), overexpression of are or were employees of Pfizer and may hold stock in Pfizer. S.A.G. TLR4 and TLR9 as seen in DM muscle tissues (29), and glomerular fi fi

receives nancial support from P zer. C.H., A.J.C., S.V.G., S.A.G., http://www.immunohorizons.org/ expression of TLR9 as observed in LN but not in normal kidneys and J.E.B. are patent authors of the anti–IFN-b Ab. (30). These changes may render tissues susceptible to additional upregulation of IFN-b.Usinganti–IFN-b Ab to block downstream IFNAR signaling (as evidenced by loss of MX1 expression) ACKNOWLEDGMENTS following stimulation in nonhematopoietic cells may interfere with a cascade of inflammatory signaling (Fig. 2). To address this We thank Leigh Zawel, Brian Bates, Robert Martinez, and Henry Wang for question in vivo, we have an ongoing phase II clinical trial in critical discussions and helpful comments. humans with DM comparing anti–IFN-b Ab to placebo control fi with a readout expected in 2021 (ClinicalTrials.gov identi er: REFERENCES NCT03181893).

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