Type I IFN Drives Experimental Systemic Lupus ......studies, however, have sought to characterize the mechanism of action of IFN speciﬁcally in SLE. Studies of SLE peripheral blood-derived

CellsErythematosus by Distinct Mechanisms in CD4 T Cells and B Type I IFN Drives Experimental Systemic Lupus

Hoebe and Edith M. JanssenJared Klarquist, Rachel Cantrell, Maria A. Lehn, Kristin Lampe, Cassandra M. Hennies, Kasper

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2020, 4 (3) 140-152ImmunoHorizons

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Type I IFN Drives Experimental Systemic Lupus Erythematosusby Distinct Mechanisms in CD4 T Cells and B Cells

Jared Klarquist,* Rachel Cantrell,†,‡ Maria A. Lehn,†,‡ Kristin Lampe,†,‡ Cassandra M. Hennies,†,‡ Kasper Hoebe,§ andEdith M. Janssen§

*Department of Immunology and Microbiology, University of Colorado, Anschutz Medical Campus, Aurora, CO 80045; †Department of Pediatrics,

University of Cincinnati College of Medicine, Cincinnati, OH 45267; ‡Division of Immunobiology, Cincinnati Children’s Hospital Medical Center,

Cincinnati, OH 45229; and §Janssen Research and Development, Johnson & Johnson, Spring House, PA 19477

ABSTRACT

Myriad studies have linked type I IFN to the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE).

Although increased levels of type I IFN are found in patients with SLE, and IFN blockade ameliorates disease in many mouse models of

lupus, its precise roles in driving SLE pathogenesis remain largely unknown. In this study, we dissected the effect of type I IFN sensing

by CD4 T cells and B cells on the development of T follicular helper cells (TFH), germinal center (GC) B cells, plasmablasts, and

antinuclear dsDNA IgG levels using the bm12 chronic graft-versus-host disease model of SLE-like disease. Type I IFN sensing by

B cells decreased their threshold for BCR signaling and increased their expression of MHC class II, CD40, and Bcl-6, requirements for

optimal GC B cell functions. In line with these data, ablation of type I IFN sensing in B cells significantly reduced the accumulation of

GC B cells, plasmablasts, and autoantibodies. Ablation of type I IFN sensing in T cells significantly inhibited TFH expansion and

subsequent B cell responses. In contrast to the effect in B cells, type I IFN did not promote proliferation in the T cells but protected

them from NK cell–mediated killing. Consequently, ablation of either perforin or NK cells completely restored TFH expansion of

IFNAR2/2 TFH and, subsequently, restored the B cell responses. Together, our data provide evidence for novel roles of type I IFN and

immunoregulatory NK cells in the context of sterile inflammation and SLE-like disease. ImmunoHorizons, 2020, 4: 140–152.

INTRODUCTION

Theoverproductionof type I IFN is aprominent featureassociatedwith the development of systemic lupus erythematosus (SLE) andhas been implicated in the pathology of other autoimmune dis-eases including Sjogren syndrome, systemic sclerosis, adult-onsetrheumatoid arthritis, and type I diabetes mellitus (1, 2). A role fortype I IFN in driving disease pathology has been demonstrated in

several genetic and inducible murine models of SLE (3–7).Moreover, the therapeutic use of IFN to treat patients withhepatitis C virus and cancer has been associatedwith the inductionof SLE (8–10), and the administration of IFN to lupus-prone miceinduced earlier onset, lethal disease (11). Furthermore, a type I IFNgene signature correlates with disease severity in SLE patients (12,13) and approaches blocking type I IFN, or its receptor have shownsome efficacy in clinical trials (14–17). Although these studies

Received for publication January 16, 2020. Accepted for publication February 20, 2020.

Address correspondence and reprint requests to: Dr. Jared Klarquist, Department of Immunology and Microbiology, University of Colorado, Anschutz MedicalCampus, 12800 E 19th Avenue, MS8333, Aurora, CO 80045. E-mail address: [email protected]

ORCIDs: 0000-0002-4072-3962 (J.K.); 0000-0001-8424-2530 (R.C.); 0000-0001-8400-7733 (M.A.L.).

This work was supported by the Lupus Research Institute, National Cancer Institute Grant CA138617, National Institute of Diabetes and Digestive and Kidney DiseasesGrant DK090978, the Charlotte Schmidlapp Award (to E.M.J.), the Albert J. Ryan Fellowship, and National Institute of Allergy and Infectious Diseases Training Grant T32-AI074491-09 (to J.K.).

Study design: J.K., K.H., and E.M.J. Experimental execution and data acquisition: J.K., R.C., M.A.L., C.M.H., and K.L. Data analysis and interpretation: J.K., K.H., and E.M.J.Manuscript drafting: J.K., K.H., and E.M.J. All authors have critically reviewed the manuscript and approved the final manuscript.

Abbreviations used in this article: BM, bone marrow; cGVHD, chronic graft-versus-host; CTV, Cell Trace Violet; DC, dendritic cell; GC, germinal center; LCMV,lymphocytic choriomeningitis virus; MHCII, MHC class II; SLE, systemic lupus erythematosus; TFH, T follicular helper cell; Treg, regulatory T cell; WT, wild type.

The online version of this article contains supplemental material.

This article is distributed under the terms of the CC BY 4.0 Unported license.

Copyright © 2020 The Authors

140 https://doi.org/10.4049/immunohorizons.2000005

RESEARCH ARTICLE

Adaptive Immunity

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strongly implicate type I IFN in promoting SLE pathogenesis,relatively little is known about the precise functions of IFN in SLE.

Type I IFN comprises 13 IFN-a proteins, IFNb, IFNk, andIFNv, all of which signal through a common receptor known asthe IFN a and b receptor, or IFNAR. IFNAR is expressed byessentially all nucleated cells andplayswell-described, key roles inantimicrobial defenses (18). In SLE, one prevailing hypothesis isthat by some combination of genetic and environmental factors,an accumulation of dying cells leads to increased exposure to self-RNA and self-DNA, which induces high levels of IFN productionby plasmacytoid dendritic cells (DCs) (19–21). Relatively fewstudies, however, have sought to characterize the mechanism ofaction of IFN specifically in SLE.

Studies of SLE peripheral blood-derived DCs by Blanco et al.(22) indicated a likely role of IFN sensing by DCs in SLEpathogenesis. It is likely that type I IFN sensing by B cells andCD4T cells also contributes to disease development, given that type IIFN is known to promote B cell activation, class switching, andsupport the generation of Ab-secreting plasma cells (23–25), andtype I IFN sensing by CD4 T cells can dramatically enhance theirpriming in the context of certain viral infections and vaccines(25–27). Further dissection of the relative contribution of type IIFN sensing in different cell types in disease developmentand progression would provide valuable insights that could beexploited for therapeutic gain.

In this study, we investigated the potential role for type I IFNsensing by B cells and CD4 T cells in the bm12 chronic graft-versus-host (cGVHD) disease model of SLE, a well-establishedmurinemodelofSLEwithclinical signs that correspond to thoseofSLE patients, including the development of antinuclear Abs, lupusnephritis, and a recognized role for type I IFN (28, 29). Our datashow that direct sensing of type I IFN by B cells is required forthe maximal development of germinal center (GC) B cells,plasmablasts, and anti-dsDNA IgG through the upregulation ofmembrane-associated molecules that promote T:B interactions,induction of the GC-promoting transcription factor Bcl-6, andincreasing the proliferative capacity to BCR stimuli. Directsensing of type I IFN by CD4 T cells was critically important forT follicular helper cell (TFH) accumulation and subsequentdisease development. However, in this scenario type I IFNsensing was required to protect the TFH from NK cell–mediatedkilling and elimination of NK cells or genetic ablation of perforinallowed for normal accumulation of IFNAR2/2TFH and disease-associated sequelae.

Our data provide evidence for novel roles of type I IFN andimmunoregulatory NK cells in SLE, providing rationale andpotential new targets for the development of future combinationimmunotherapies.

MATERIALS AND METHODS

Mice and cell linesAll experiments involving mice were conducted following pro-tocols approved by the Cincinnati Children’s Hospital MedicalCenter’s InstitutionalAnimalCare andUseCommittee.Micewere

maintained under specific pathogen-free conditions in accordancewith guidelines by the Association for Assessment and Accred-itation of Laboratory Animal Care International. C57BL/6J,B6.SJL-Ptpcra (CD45.1), B6.PL-Thy1a/CyJ (CD90.1), B6.129S2-Ighmtm1Cgn/J (mMT),C57BL/6-Prf1tm1Sdz/J (Prf12/2), B6(C)-H2-Ab1bm12/KhEgJ (bm12), and B6.129(Cg)-Foxp3tm(DTR/GFP)Ayr/J(Foxp3-DTR) mice were originally purchased from The JacksonLaboratory (Bar Harbor, ME) and bred in-house. IFNAR12/2

mice were originally a gift fromDr. J. Sprent and have since beenbred and backcrossed to the C57BL/6 background in our facility(30). Both male and female mice were used, but each experimentconsisted of only one aged-matched gender. ISRE-L929 IFNreporter cells have been described previously (31). Mixed bonemarrow (BM)–chimeric mice were generated using lethallyirradiated mice of indicated strains on a C57BL/6 background.Mice received an initial dose of 700 rad followed by an additional475 rad dose 3 h later using a 137Cs irradiator (JL Shepherd &Associates, San Fernando, CA). BM from indicated strains (ondifferent congenic backgrounds) were mixed at indicated ratiosprior to reconstitution, and 6 million total BM cells weretransferred via tail vein injection. All BM-chimeric mice weregiven 12 wk for hematopoietic reconstitution.

CD4 T cell isolationFor most in vivo and in vitro assays, CD4 T cells were sorted to.97% purity by negative selection using magnetic bead sortingtechnologies fromMiltenyi Biotec (Bergisch Gladbach, Germany)and BioLegend (San Diego, CA). Post sort purity was determinedby flow staining for live cells, CD45, CD3, and CD4. In severalexperiments, cells were labeled with Cell Trace Violet (CTV) orCFSE as proliferation-tracking dyes. For isolation of CD4bm12

T cells for the TFH in vivo–killing assay, CD4bm12 T cells wererepurified from spleens 2 wk after their initial transfer bynegative selection for CD4, followed by positive selection forthe congenic marker using biotinylated Ab to CD45.1 (cloneA20; BioLegend) and antibiotin labeled Miltenyi beads. Flowcytometric analysis showed .95% viability and .95% purity(CD45.1+CD4+), with .95% of the cells expressing TFH markersPD1 and CXCR5.

Bm12 model of cGVHDThe bm12 model experiments were performed as previouslydescribed (7, 28). Briefly, 73 106 naive donor CD4bm12T cells fromindicated strains were injected i.p. into recipient mice of thespecified strains. Fourteen days later, unless indicated otherwise,spleens were harvested, weighed, and processed into single-cellsuspensions for counting and flow staining. Serum was harvestedby retro-orbital bleeding prior to sacrificingmice, processed usingserum separator tubes (BD Biosciences, San Jose, CA) and storedat280°C for analysis of cytokine and anti-dsDNA IgG levels.

In vivo depletion studiesNK cells were depleted via an i.p. injection of 10ml of anti–Asialo-GM1 rabbit antiserum (FUJIFILM Wako Chemicals, Richmond,VA) at 21, 1, 5, and 10 d after CD4bm12T cell transfer. Parallel


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studies used NK1.1 (clone PK136, 25 mg/mouse) on day 21 and 1.CD8 T cells were depleted by i.p. injection of 25 mg of anti-CD8b(clone YTS156.7.7, 20 mg per mouse) 1 d prior to CD4bm12T celltransfer. Regulatory T cell (Treg) depletion in Foxp3-DTR micewas achieved by administration of DT (i.p. 20 mg/kg) 21, 3, and10 d after CD4bm12 T cell transfer. Depletion efficacy was assessedat the end of the experiments by flow cytometry.

In vivo NK cell–killing assayTo assess the susceptibility of theTFH toNK cell–mediated killing,naive wild type (WT), and IFNAR2/2 CD45.1 CD4bm12 T cellswere injected into Prf2/2 mice. After 14 d, TFH were sorted andmixed in a 1:1 ratio with naive CD4 T cells from CD90.1 mice. Atotal of 43 106 cells were injected i.v. into intact andNK-depleted(anti–Asialo-GM1–treated) WT recipients. After 24 h, spleenswere collected and the ratio of CD45.1 CD4bm12T cells to CD90.1control CD4 T cells was determined by flow cytometry.

Serum cytokine and anti-dsDNA lgG analysesCirculating cytokines were analyzed in the serum of mice byLuminex Multiplex Technology (Austin, TX). Serum type I IFNwas measured using the highly sensitive ISRE-L929 bioassay.Serum levels of anti-dsDNA were determined using ELISAessentially as described previously (7, 28). Anti-dsDNA IgG levelsare plotted as relative units toward a reference standard thatwas used in all experiments.

In vitro T and B cell assaysSpleens and lymph nodes were harvested from indicated strains,andCD4T cells or B cells were isolated by positive selection usingmagnetic bead-sorting technology (CD4+ T cell and CD19+ B cellIsolation Kits; Miltenyi). Cells were labeled with a proliferationtracking dye (CTV or CFSE), per the manufacturer’s protocol,and cultured in IMDM supplemented with 10% FBS, penicillin,streptomycin, and L-glutamine. CD4 T cells were stimulated withCD3 (1 mg/ml plate-bound, clone 17A2; BioLegend) and solubleCD28 (1mg/ml, clone 37.51; BioLegend) in the absence or presenceof 50–200U/ml rIFN-a or IFN-b (PBLAssay Science, Piscataway,NJ). B cells were stimulated with goat anti-mouse IgM F (ab’)2fragment (10 mg/ml, unless otherwise noted; Jackson Immuno-Research Laboratories, West Grove, PA) and anti-mouse CD40(10mg/ml, unless otherwise noted, clone FGK4.5; BioXCell). Anal-ysis was performed by flow cytometry at indicated timepoints.

Flow cytometryUnless otherwise specified, cells were collected and immediatelystained directly ex vivo. For the bm12 model, cells were stainedessentially as described previously (7, 28) using combinations of aFixable Viability dye and Abs to CD3, CD4, PD1, CXCR5, ICOS,CD45.1/CD45.2, CD90.1/CD90.2, andCD25. Intracellular stainingfor Bcl-6, Foxp3, Bcl-2, Bcl-X, Bim, Bcl2A1, Ki67, and pH2AXwasperformed using the Foxp3 Staining Buffer Set (eBioscience, SanDiego, CA). NK cells were defined as NK1.1+NKp46+ and wereevaluated further using combinations of Abs toward CD27, CD11b,NKG2A, NKG2D, Ly49A, C, D, F, G2, H, I, CD244, and CD122.

Staining for NK cell ligands included CD48, ICAM1, ICAM2,H2-Kb/Db,Rae1,Mult1, andNCR1-Fc, followedbya secondaryAb.B cellswere stained for viability, B220, CD19,GL7, Fas, andCD138.In vitro assessment of B cells included MHC class II (MHCII),CD40, and Bcl-6. For in vivo depletion studies, efficacy wasassessed by staining for NK cells by NK1.1 and NKp46 and CD8T cells by CD8a, CD3, and CD4. All Abs and dyes were purchasedfrom BioLegend or eBioscience, (except for NCR1-Fc, whichpurchased from R&D Systems). Samples were collected on aFortessa Flow Cytometer with FACSDiva software (BD Biosci-ences), and data were analyzed with FlowJo software (Tree Star,Ashland, OR).

Statistical analysisData were analyzed using Prism software (GraphPad Software).Unless statedotherwise, thedata are represented asmeans6SEMand group sizes for each experiment are provided in the legends.Data were evaluated using a Student t test unless otherwisespecified. A p value#0.05 was considered statistically significant.

RESULTS

Type I IFN sensing by the hematopoietic compartment isrequired for disease development in the bm12 cGVHDmodel of SLEAnalysis of serum cytokines showed that type I IFN levelsincreased over time, concomitant with an increase in the serumlevels of the type I IFN–inducible chemokines CXCL9 andCXCL10 (Fig. 1A). Additional inflammatory cytokines includingTNF-a, IL-12, and IL-6 were also induced, although these werepresent in relatively low levels and at generally delayed kineticscomparedwith the type I IFNs and the IFN-inducible chemokines(Fig. 1A).

To assess the relative contribution of type IFN sensing in thehematopoietic and nonhematopoietic compartments to diseasedevelopment, we generatedBM-chimericmice forwhichWT andIFNAR2/2micewere reconstitutedwith eitherWTor IFNAR2/2

BM. After 12 wk, CD45.1 CD4bm12 T cells were transferred andsplenic mass, development of TFH (PD1+CXCR5+ within liveCD4+CD45.1+ cells), GC B cells (live, CD19+Fas+GL7+), plasma-blasts (live, B220med/low CD138+), and anti-dsDNA IgG levelswereassessed 2 wk later (Fig. 1B, 1C) (28).

WT→WT and WT → IFNAR2/2 recipients showed compa-rable increases in splenicmass, frequencies, and absolute numbersof TFH, GC B cells, plasmablasts, and anti-dsDNA IgG levels,indicating that type I IFN sensing by the nonhematopoietic com-partment was not required for disease development (Fig. 1D).In contrast, IFNAR2/2 → WT mice showed significantly di-minished disease compared with mice reconstituted with WTBM. There was no added effect of loss of IFNAR on thenonhematopoietic compartment (IFNAR2/2 → IFNAR2/2 com-pared with IFNAR2/2 → WT) (Fig. 1D). Together, these dataidentify type I IFN sensing by the hematopoietic compartment asthe critical component in disease development.


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Type I IFN sensing by B cells is required for optimal GC andplasmablast formationAs it is likely that multiple components in the hematopoieticcompartment require type I IFN sensing to confer diseasedevelopment, we next assessed the contribution of type I IFN

sensing on the development of GC B cells, plasmablasts, and anti-dsDNA IgG levels.

To generate mice that lacked IFNAR only on the B cellcompartment,WTmicewere reconstitutedwith amixofBMfromB cell–deficientmMTmice togetherwith eitherWTor IFNAR2/2

FIGURE 1. Type I IFN sensing by hematopoietic cells is critical for disease development.

(A). C57BL/6 mice were injected with 7 3 106 purified WT CD45.1 CD4bm12 T cells. At the indicated days after injection, serum type I IFN was

analyzed by bioassay, and additional cytokines and chemokines were analyzed by multiplex technology. The dotted line indicates the limit of

detection for each analyte. Data are shown as mean 6 SEM (n = 2–3 per timepoint). (B) Schema of BM-chimeric mice experiments. All BM donors

and recipients were on a CD45.2 background. BM-chimeric mice were injected with purified CD45.1+ WTbm12 CD4 T cells (n = 6–7 per group), and

mice were sacrificed after 14 d. (C) Representative flow cytometry plots depict the gating strategy for analysis of splenic T cell (in total live

splenocytes), GC B cells (in live CD19+ cells), and plasmablasts (in total live splenocytes). (D) Splenic mass and the absolute splenic number of

TFH (live, CD4+CD45.1+PD1+CXCR5+), GC B cells, plasma B cells, and anti-dsDNA IgG levels 14 d after CD45.1+ WTbm12 CD4 T cell transfer. Naive WT

→ WT represents WT mice reconstituted with WT BM that did not receive CD45.1+ WTbm12 CD4 T cells. Data from one representative

experiment (out of two experiments with n = 6–7 per group) are shown as mean 6 SEM. *p # 0.05, **p , 0.01, ***p , 0.001.




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BMin a 10:1 ratio, effectively generatingmice inwhich theB cellsare eitherWTor IFNAR2/2 (Fig. 2Ai). Transfer ofCD45.1CD4bm12

T cells into the WT/mMT mice resulted the expansion of TFH

and induction of GC B cells, plasmablasts, and anti-dsDNAIgG. However, in the IFNAR2/2/mMT recipients, GC B cells,plasmablasts, and anti-dsDNA IgG levels were significantly re-duced, suggesting a direct effect of type I IFN sensing onB cells (Fig. 2B). However, IFNAR2/2/mMT recipient mice

also had lower levels of TFH thanWT/mMTrecipientmice. TFH

and GC B cells are reciprocally supportive of each other: thefrequency of TFH is associated with the magnitude of the B cellresponse, and GC B cells have been shown to promote TFH

expansion (32–34). Therefore, the observed reduction in B cellresponses in the IFNAR2/2/mMT mice could result fromchanges in reciprocal TFH–B cell interactions, rather than a soleeffect on B cells.

FIGURE 2. B cell IFN sensing augments plasmablast and GC B cell development.

(Ai and Aii) Schemas used for the mixed BM chimeras in (B) and (C), respectively. (B) BM from WT or IFNAR2/2 CD45.1 mice was combined with BM

of B cell–deficient CD45.2 mMT mice at a 1:10 ratio and transplanted into lethally irradiated C57BL/6 mice. After 12 wk, mice received CD45.1/2

WTbm12 CD4 T cells. B cell and donor TFH response 2 wk after T cell transfer presented as absolute number of cells per spleen. (C) BM from WT

(CD45.2) and IFNAR2/2 (CD45.1) mice was combined at a 1:1 ratio and transplanted into lethally irradiated C57BL/6 mice. Twelve weeks later, mice

received CD45.1/2 WTbm12 CD4 T cells and composition of the B cell compartments in each part of the graft was assessed. Depicted are the

percentages of WT versus IFNAR2/2 cells within GC or plasmablast B cell subsets normalized to the percentage of each genotype within the total

B cell graft. For (B) and (C), data from one representative experiment (out of two experiments with n = 4–6 per group) are shown as mean 6 SEM.

(D) CTV-labeled purified B cells were cultured with 1 or 10 mg/ml anti-IgM and anti-CD40 with or without 100 U/ml IFN-a, and proliferation was

assessed 96 h later. (E and F) Purified B cells were cultured under the indicated conditions with or without 50 U/ml IFN-a. Expression of CD40 and

MHCII was assessed over time, and Bcl-6 expression was assessed after 3 d by flow cytometry. Data are shown as mean 6 SEM (n = 3–4 per group

for each timepoint). One of two experiments is shown. *p # 0.05, ***p , 0.001.




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To circumvent this problem,we generated chimericmicewithWT(CD45.2) and IFNAR2/2 (CD45.1)BMmixed ina 1:1 ratio (Fig.2Aii). In this setting, the WT B cells facilitate the development ofTFH, allowing the assessment of the direct effect of type I IFNsensing on B cell responses during disease by the comparison ofWT and IFNAR2/2 B cells in the same animal. Upon transfer ofCD45.1/2 CD4bm12 T cells, all mice developed high frequenciesof TFH and anti-dsDNA IgG levels (data not shown). However,compared with the WT B cell compartment, the developmentof both GC B cells and plasmablasts was significantly reduced inthe IFNAR2/2 B cell compartment (Fig. 2C). Together, these datademonstrate a critical role for type I IFN signaling in the B cellresponse that cannot be overcome by other mediators involved inthe disease development.

Type I IFN sensing byBcells has beenshown topromoteB cellsurvival and lower the threshold for BCR signaling, enhancing theproliferation of naive B cells when stimulatedwith anti-IgM [(35),Fig. 2D]. Indeed, B cells exposed to IgM and type I IFN alsoincrease expression of MHCII and CD40, molecules involvedwith cognate T:B interaction and requirements for optimal GCformation (Fig. 2E). Moreover, type I IFN sensing in IgM- andCD40-stimulated B cells increased the expression of Bcl-6, thetranscription factor that is associated with GC B cell formation(Fig. 2F).

Together, these data indicate that type I IFNhas a direct effecton B cells and promotes B cell responses on the transcriptionallevel as well as stimulatory capacity toward T cells.

TFH require type I IFN for their accumulationGenetic ablation of IFNAR can completely prevent disease devel-opment in various experimental models of SLE (4–6). Althoughour data show that a lack of IFN sensing by the hematopoieticcompartment was critically important for disease development,the transfer of IFNAR-sufficient CD45.1 CD4bm12T cells intoIFNAR recipients still resulted in partial disease (Fig. 1D and (7)).As these transferred cells are the drivers of disease, we next testedthe contribution of IFN sensing by the donor CD4bm12T cells ondisease development.

Compared with the transfer of WT CD4bm12 T cells, thetransfer of IFNAR2/2CD45.1CD4bm12T cells (intoWTrecipients)resulted in a significantly reduced disease, illustrated by reducedTFH, GC B cells, plasmablasts, and anti-dsDNA IgG levels (Fig. 3A,3C). Importantly, transfer of IFNAR2/2 CD45.1 CD4bm12 T cellsinto IFNAR2/2 recipients completely abrogated disease develop-ment. Because TFH and GC B cell responses are reciprocallysupportive of one another,we considered the possibility that type IIFN may have acted in trans to promote TFH expansion. Wereasoned that type I IFN sensing by CD4bm12 T cells may increasetheir expression of costimulatory molecules, such as IL-21, thatsupport amore robustGCBcell response.AnaugmentedGCBcellresponse may subsequently drive greater TFH expansion. To testthis hypothesis,wecotransferredcongenicallymarkedWTCD4bm12

T cells (CD45.2) and IFNAR2/2CD4bm12 T cells (CD45.1) intoCD45.1/2 WT recipients (Fig. 3B). In this model the WT CD4bm12

T cells drive comparable GC B cell and plasmablast responses to

those found inmice that receivedWTCD4 bm12T cells only. DirectcomparisonbetweentheWTandIFNAR2/2CD4bm12Tcells in thesame animals still showed a significant reduction in TFH develop-ment in the IFNAR2/2CD4bm12 T cells (Fig. 3C), indicating acritical role of type I IFN sensing by the T cells that could not beovercome by other inflammatory mediators.

Type I IFN is not required for proliferation orTFH developmentTo identify themechanisms that drive the reduced IFNAR2/2TFH

accumulation, we first assessed when TFH accumulation of WTandIFNAR2/2CD45.1CD4bm12T started todiverge.A timecourseindicated that small differences were apparent within 3 d oftransfer and became significant within 5 d after transfer (Fig. 4A).Proliferation dye indicated that only a proportion of transferredWT and IFNAR2/2CD4bm12 T cells proliferated and that theWTand IFNAR2/2CD4bm12 T cells that did proliferate showed asimilar loss of the dye, indicating similar proliferation rates (Fig.4B). The absolute number of nonproliferating cells remainedsimilar betweenWT and IFNAR2/2CD4bm12 T cells, whereas theabsolute number of proliferating cells was significantly reducedfor the IFNAR2/2 CD4bm12 T cells (Fig. 4B). Analysis of Ki67expression was low/absent in undivided cells and high in dividedcells, resulting in a trend toward reduced Ki67 levels in the bulkIFNAR2/2CD4bm12 T cell population (Fig. 4C). A comparablepercentage of WT and IFNAR CD4bm12 T cells also expressedphospho-gH2AX (pgH2AX), awell-characterizedmarker for theDNA damage response, shown to be highly elevated in in vivoactivated and proliferating T cells (36).

Upon transfer, bothWTandIFNAR2/2CD4bm12Tcells rapidlyadopted a TFH phenotype and both genotypes expressedcomparable levels of PD1, CXCR5, ICOS, and Bcl-6 (Fig. 4D).Together, these data suggest that differences in proliferationrate, either directly driven by type I IFN or as a consequence ofdifferent phenotype development, were not responsible for thedifferences in TFH accumulation.

In vivo proliferation data can be skewed by silent clearanceof dying cells or migration away from target organs. Analysis of awide variety of lymphoid and nonlymphoid tissues eliminated thedifferences in migratory capacity as a critical contributor to thephenotype, as all lymphoid tissues and the peritoneal cavityshowed similar reduction in IFNAR2/2 CD4bm12 T cells and thetransferred cells (WT and IFNAR2/2) were not detectable innonlymphoid tissues at this timepoint (data not shown).

To assess the effect of IFN sensing on CD4 T cell proliferationand survival in vitro, purified CD4bm12 T cells fromWT (CD45.2)and IFNAR2/2 (CD45.1) donors were combined at a 1:1 ratio,labeledwith aproliferation-trackingdye, and stimulatedwith anti-CD3/CD28 in the absence or presence of type I IFN. Type I IFNinhibited theproliferationof activatedWTbutnot IFNAR2/2CD4T cells, demonstrating that the antiproliferative effect of IFN-involved direct signaling in the CD4bm12 T cells (Fig. 4E). As thesedata were seemingly at odds with the phenomenon observed invivo, inwhich IFNsensing allowed for the increased accumulationofWT CD4bm12 T cells, we therefore shifted our focus toward the




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potential effect of type I IFN on the survival of these cells.However, analysis of pro- and antiapoptotic molecules did notshow a difference between WT and IFNAR2/2CD4bm12 T cells5 d after transfer, and IFNAR2/2 CD45.1 CD4bm12 T cell hadcomparable levels of pgH2AX+ cells (Fig. 4C, 4F). In linewith thisfinding, bothWT and IFNAR2/2CD45.1 CD4bm12 T cells showedsimilar rates of cell death when cultured ex vivo in medium or inthe presence of type I IFN (data not shown).

Type I IFN sensing protects TFH from perforin-dependentcell deathBecause type I IFN imparted no obvious cell-intrinsic survivaladvantage, we hypothesized that IFNmay be protecting TFH from

cell-extrinsic pressure, either control by Tregs or killing by NKcells or CD8 T cells.

The frequency and total number of CD4+Foxp3+CD25+ cells inthe spleens did increase slightly between day 3 and day 7 aftertransfer, but no difference between WT and IFNAR2/2 recipientgroups was seen. Similarly, 7 d after transfer, WT and IFNAR2/2

CD45.1CD4bm12 T cells contained comparable proportions ofTregs,and elimination of recipient Tregs before and during the T celltransfer did not restore the IFNAR2/2 CD4bm12 TFH accumulation(Supplemental Fig. 1). These observations argue against a specificrole for Treg-mediated suppression of the IFNAR2/2 TFH.

To assess susceptibility to killing, WT and IFNAR2/2CD4bm12

T cells were transferred into WT or perforin-deficient (Prf12/2)

FIGURE 3. T cells require type I IFN sensing for TFH accumulation.

WT and IFNAR2/2 CD45.1+CD4bm12 T cells were transferred into WT or IFNAR2/2 recipients, and responses were assessed 2 wk later. Data from

naive WT and naive IFNAR mice are included as reference. (A) Splenic mass and the absolute number of splenic donor TFH, GC B cells, plasmablasts,

and anti-dsDNA IgG levels are plotted. Data from one representative experiment (out of two experiments with n = 4–5 per group) are shown as

mean 6 SEM. (B) WT (CD45.2) and IFNAR2/2 (CD45.1) CD4bm12 T cells were mixed in a 1:1 ratio and transferred into WT CD45.1/2 recipients. As a

control, a parallel group of mice received only WT or only IFNAR2/2 CD4bm12 T cells. All mice received a total of 7 3 106 CD4bm12 T cells. Flow data

show cells before injection and 2 wk after injection (gated on live CD4+ cells). (C) Absolute number of splenic GC B cells, plasmablasts, and donor

TFH in mice that received either WT, IFNAR2/2 CD4bm12 T cells, or the mixture. Data are from one representative experiment (out of three

experiments with n = 11–12 per group). *p # 0.05, **p , 0.01, ***p , 0.001.




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FIGURE 4. Normal development but lack of accumulation of IFNAR2/2 TFH.

(A) CTV-labeled WT and IFNAR2/2 CD45.1 CD4bm12 T cells were transferred into CD45.2 WT recipients, and the absolute number of splenic donor

cells was assessed over time. Data are shown as mean 6 SEM (with n = 5–6 per group) and exhibit significant interaction by a two-way ANOVA.

(B–D) CTV-labeled WT, IFNAR2/2 CD45.1+ CD4+ bm12 T cells, or control CTV-labeled CD45.1+ CD4+ H-2Kb T cells were transferred into B6 mice.

Spleens were harvested 5 d later. (B) Representative CTV-dilution plots (gated on live CD4+ CD45.1+) and absolute numbers of divided and

undivided donor CD4bm12 T cells 5 d after transfer. Data are shown as mean 6 SEM (with n = 4–5 per group) and exhibit significant interaction by a

two-way ANOVA. (C) Proportion of Ki67+ and pgH2AX+ cells within the proliferating cells 5 d after transfer. (D) Comparable expression of TFH-

associated molecules in proliferating donor WT and IFNAR2/2 CD45.1+ CD4bm12 T cells 5 d after transfer. Data are gated on live CD45.1+ CD4+

bm12 CTV-diluted cells or CD45.1+ CD4+ undiluted control cells. (E) Inhibition of proliferation by type I IFN. Purified naive WT (CD45.2) and

IFNAR2/2 (CD45.1) CD4bm12 T cells were mixed (1:1 ratio), CTV labeled, and cocultured with CD3/CD28 in the absence or presence of type I IFN.

Proliferation was assessed 24, 48, and 72 h later. Representative CTV dilution plots are shown from one of three independent experiments. Day 3

proliferation data for varying concentrations of IFN-a and IFN-b are also plotted using the Divisional Index calculated by the FlowJo proliferation

modeling tool. (F) Expression of pro- and antiapoptotic markers in proliferating WT (black bar) and IFNAR2/2 (white bar) CD4bm12 T cells 3 d after

transfer (gating on live CD45.1+ CD4+ CTV-diluted cells). Data are shown as mean 6 SEM (with n = 3–4 per group). **p , 0.01, ***p , 0.001.




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recipients. Transfer of IFNAR2/2CD4bm12 T cells into Prf12/2

recipientscompletely restoredaccumulationof IFNAR2/2CD4bm12

TFH concomitant with increases in plasmablasts, GC, and anti-dsDNA IgG levels (Fig. 5).

Type I IFN protects TFH from NK cell–mediated killingBased on the kinetic accumulation data, both CD8 T cells and NKcells could be involved in the elimination of the IFNAR2/2 TFH.Depletion of CD8T cells before IFNAR2/2CD4bm12 T cell transferdid not restore TFH accumulation, nor the number of GC B cells,plasmablasts and anti-dsDNA IgG levels (Fig. 6A).

In contrast, NK cell depletion by either NK1.1 or anti–Asialo-GM1 prior to IFNAR2/2CD4bm12 T cell transfer restored TFH

accumulation and subsequent GC B cell, plasmablast and anti-dsDNAIgGleveldevelopment (Fig. 6B, 6C).Toeliminate theeffectof the changes on the GC B cell response on the accumulation ofthe TFH, WT (CD45.2) and IFNAR2/2 (CD45.1) CD4bm12 T cellswere transferred together into CD45.1/2 NK cell–depleted mice(as in Fig. 3). Again, NK cell depletion restored IFNAR2/2 TFH

accumulation, further confirming that direct type I IFN sensing byTFH protected them from NK cell–mediated killing (Fig. 6D).

To further confirmthat IFNAR2/2CD4bm12TFHare target cellsfor NK cells, Prf2/2 mice were injected with WT or IFNAR2/2

CD4bm12 T cells and TFH were harvested 2 wk later. Cells wereadded ina 1:1 ratiowithCD90.1CD4controlTcellsand transferredinto intact orNK-depletedWTmice. The ratio of CD4bm12 T cells/CD90.1 control cells was assessed 24 h later (Fig. 6E, 6F). Asexpected, the ratio ofWTCD4bm12 T cells/control T cells in intactand NK-depleted recipients did not change significantly. Transferof IFNAR2/2CD4bm12 TFH into intact WT recipients resulted in asignificant distortion of the TFH/control T cell ratio. In support ofour in vivo studies, the IFNAR2/2CD4bm12TFH/controlCD4Tcellratio remained unaltered and comparable to theWT TFH/controlT cell ratio when cells were transferred into NK-depleted WTmice (Fig. 6G, 6H).

Importantly, the protective effect of type I IFN sensing by theTFH was not shared by the B cells. In mixed BM-chimeric micefrom Fig. 2 (WT/IFNAR 1:1), NK-depletion did not restore

GC B cell and plasmablast numbers in the IFNAR2/2 B cellcompartment (Supplemental Fig. 2). These data indicate thatalthough both the T cell and the B cell response required type IIFN sensing for their accumulation, the underlying mechanismsare distinct.

DISCUSSION

A connection between the overproduction of type I IFN and thedevelopment of SLE has been well established, yet the mecha-nisms by which IFN promotes disease pathology have not beenfully elucidated. In this study,we showed that type I IFNenhancesdevelopment of pathogenic T and B cell responses in the bm12cGVHD model of SLE-like disease through two different mecha-nisms: directly promoting B cell proliferation and GC differenti-ation and protecting TFH from NK cell–mediated killing.

Our observations that type I IFN enhancesB cell responsesfitswellwithexistingdataon thedirect and indirect rolesof type I IFNand induction of humoral responses (23–25, 35, 37). Type I IFNshave been shown to promote B cell survival and isotype switchingthrough the induction of BLyS andAPRIL inDCandmacrophages(24, 38). However, our mixed BM chimera models indicate thatthe lack of type I IFN sensing by B cells cannot be overcome byother inflammatory mediators, implicating a more-importantcontribution of direct type I IFN sensing in the B cell response.Intrinsic direct effects of type I IFN sensing encompass reducingBCR signaling threshold, increasing expression of moleculesinvolved in cognate and reciprocal T–B cell interactions, cytokineproduction, migration, survival, as well as plasmablast develop-ment (25, 35, 37, 39–41). Although our BM chimera experimentwould indicate a limited role for increased cytokine production, itis likely that combinations of several above-mentioned mecha-nisms are involved in the type I IFN–associated boosting of theB cell response inSLE. IFNAR2/2Bcellswere still able tomount apartial response, pointing to the involvement of other stimulatorymechanisms in the autoantibody response. Further dissectionof the relative contribution of these mechanisms might point to

FIGURE 5. Type I IFN protects TFH from perforin-mediated destruction.

WT (filled symbols) or IFNAR2/2 (open symbols) CD4bm12 T cells were transferred into C57BL/6 (+) or perforin-deficient (2) animals. Two weeks

after transfer, splenic weight and the total numbers of splenocytes, donor TFH, GC B cells, and plasmablasts were determined. Total anti-dsDNA IgG

levels in the serum were determined by ELISA. A representative experiment (of four) is shown as mean 6 SEM (with n = 4 per group). *p # 0.05,

**p , 0.01, ***p , 0.001.




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FIGURE 6. Type I IFN protects TFH from NK cell-mediated killing.

(A–C) WT (black bar) or IFNAR2/2 (white bar) CD45.1 CD4bm12 T cells were transferred into intact or Ab-depleted CD45.2 mice. Two weeks after transfer,

mice were assessed for the absolute splenic number of donor TFH, GC B cells, and plasmablasts in each graft. Mice were kept intact or (Continued)




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additional pathways that canbe therapeutically targeted indisease,either with or without type I IFN–interfering therapy.

Our studies show that type I FN sensing by TFH protects themfrom NK cell–mediated killing, which is crucial for their ac-cumulation and promotion of the B cell response. The potentialfor immunoregulation of T cell activity by NK cells has beenuncovered in recent years. Mounting evidence has established arole forNK cells in limiting T cell responses toward lymphocyticchoriomeningitis virus (LCMV) by direct killing of virus-specificT cells (42–45). Recently, NK cytotoxicity toward LCMV-specificT cells was shown to be inhibited by type I IFN (44, 46). Thesereports were consistent with data published over four decadesearlier, inwhichHansson,Welsh, and colleagues (47, 48) showedthat either LCMV infection, the induction of type I IFN by poly(I:C), orpretreatmentwith exogenous IFNreduced the sensitivityof normalmouse thymocytes toNK cells in vitro. In this study, wedemonstrate that type I IFN also inhibits in vivo regulation ofactivated CD4 T cells by NK cells in a sterile model of systemicautoimmune disease. Taken together, these data suggest theintriguing possibility that peripheral tolerance may be main-tained inpart by thedefaultNKkilling of highly activeT cells andthat activatedT cells can be licensed by type I IFNduring certainconditions. Aberrant overproduction of type I IFN in a healthyindividual could contribute to the inadvertent licensing of self-reactive T cells and might thus be capable of initiating autoimmunedisease. This may be one mechanism behind the induction ofautoimmunity, including SLE,which occurs in somepatients afterthe administration of type I IFN to treat conditions such aschronic hepatitis C viral infection (8–10).

Several studies in SLE patients and experimental mousemodels of SLE-like disease indicate that NK cells are dysregulatedand decreased in number and cytolytic capacity (49–55). In thebm12 cGVHD model of SLE, NK cells are reduced and display amore immature phenotype (Supplemental Fig. 3A–C). In additionto therelativepaucityof intermediatematurity (CD11b+CD27+)NKcells, the NK cells remaining showed reductions in a number ofactivatingand inhibitoryNKcell receptors (SupplementalFig. 3D).However, the remaining NK cells were still able to kill IFNAR2/2

TFH, indicating that at least part of their cytolytic machinery wasfunctional. As this remaining cytolytic capacity was not sufficientto affect WT TFH accumulation and WT TFH numbers were

maintained in the in vivoNKcell–killing assay (Fig. 6), this raisesthe possibility that WT TFH are more resistant to NK cell–mediated killing through the differential expression of activatingand inhibitory NK cell ligands. Comparing expression of variousknown NK cell–activating and –inhibiting ligands on WT andIFNAR2/2TFH in intactmice showednotable shifts in expressionof activating (Rae1, Mult-1, CD48, ICAM-1/2) and inhibitory(H-2Db/Kb) NK ligands. We consistently observed increases inthe expression of several NK cell receptor–activating ligandsin CD4bm12 TFH compared with CD4 T cells from naive mice;however, these changes were not unique to IFNAR2/2 TFH fromNK-depleted mice, suggesting the primary mechanism of pro-tection from NK killing mediated by type I IFN was not throughdecreasing these particular signals (Supplemental Fig. 4). Class IMHC (H2-Db and H2-Kb) was increased in WT TFH comparedwith IFNAR2/2 TFH and naive CD4 T cells, indicating onepossible mechanism for type I IFN–induced protection from NKcell killing, although these shifts were relatively small and werenot completely restored in the IFNAR2/2 TFH upon NK celldepletion. One of the recent studies on NK immunoregulation inthe context of LCMV showed that type I IFN protected SMARTAand P14 T cells by upregulating an, as of yet, unidentified Ncr1ligand or ligands (46). However, we were unable to detectdifferences inNcr1 ligands onour polyclonal endogenousWTandIFNAR2/2 TFH upon staining with an Ncr1-hIgG fusion protein.These data suggest that the lack ofWTTFH sensitivity toNK cellscould involve a complex combination of signals, a critical effectorligand or ligands that is/are still unknown, a TFH-intrinsicintracellular compensatory mechanism for the cytolytic assaults,or a combination of these.

With the rise of therapeutics that target the type I IFNpathway, it will be important to evaluate the relative effect ofthe treatment on the different immune cells. Although one canenvision that simply eliminating or reducing type I IFN wouldrender the TFH susceptible toNKcell–mediated killing, it does notnecessarily reinvigorate the disease-affected NK cells. Thecapacity of the host to properly kill the TFH is critically importantas our in vitro and in vivo data indicates that type I IFN has anantiproliferative effect. In vitro stimulation of human and mouseT cells in the presence of type I IFN significantly inhibits celldivision [Fig. 4, (56, 57)]. Moreover, in the absence of perforin,

treated with anti-CD8b (A), anti–Asialo-GM1 (B), or anti-NK1.1 (C) Abs. (D) WT (CD45.2, black circles) and IFNAR 2/2 (CD45.1, white circles) CD4bm12 T cells

were mixed in a 1:1 ratio and transferred into intact or anti–Asialo-GM1–treated (DNK) CD45.1/2 recipients. The absolute splenic number of donor TFH in

each graft was determined after 2 wk. A representative experiment for each depletion approach is shown (out of two to five individual experiments/

depletion approach with each experiment n = 4–6 per group) as mean 6 SEM. (E–H) WT and IFNAR2/2 CD45.1+ CD90.2+ CD4bm12 T cells were

transferred into Prf2/2 CD45.2+ CD90.2+ recipients. After 2 wk, CD45.1 CD4 bm12 T cells were sorted, CFSE labeled, and mixed in a 1:1 ratio with CFSE-

labeled control CD90.1+ CD4 T cells. A total of 43 106 T cells was transferred into intact or NK-depleted (anti–Asialo-GM1) CD45.2+ CD90.2+ WT mice.

The ratio of CD45.1+ CD4 bm12 T cells to control cells was assessed 24 h later by flow cytometry. (E) Schema of transfer approach. (F) Example of flow

cytometric data upon gating on live and CD4+ cells. (G) Examples of histograms of CD90.1 control CD4 T cells and WT and IFNAR2/2 CD45.1 CD4 bm12

T cells in different recipients (gated on live CD4+ CFSE+ cells). (H) Ratio of CD45.1 CD4 bm12 T cells/CD90.1 CD4 T cells in each mouse. Black circles,

WT CD4bm12 T cells; white circles, IFNAR2/2 CD4bm12 T cells. Data are expressed as mean 6 SEM (with n = 4–6 per group). *p # 0.05, **p , 0.01,

***p , 0.001.




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IFNAR2/2TFH showed significantlymore accumulation thanWTTFH (Fig. 5), illustrating the in vivo proliferative potential of TFH inthe absence of type I IFN sensing. This raises the interestingpossibility that bolstering NK cell numbers or their functionalitymight be an appropriate therapeutic approach when combinedwith type I IFN–interfering drugs.

Although the current data set justify targeting type I IFN inSLE-like disease, additional studies are needed to more preciselyappreciate the many functions of type I IFN to enable the rationaldesign of more targeted therapies for SLE and other autoimmunediseases with pathological type I IFN production.

DISCLOSURES

The authors have no financial conflicts of interest.

ACKNOWLEDGMENTS

We thank Dr. Stephen Waggoner for providing reagents and discussion,Jeff Bailey and Victoria Sumney in the Comprehensive Mouse and CancerCore for help with BM chimera experiments, Alyssa Sproles for technicalsupport with the Luminex assays, and Monica DeLay and the CincinnatiChildren’s Hospital Medical Center Flow Core for flow cytometry and cell-sorting support.

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Type I IFN Drives Experimental Systemic Lupus ......studies, however, have sought to characterize the mechanism of action of IFN speciﬁcally in SLE. Studies of SLE peripheral blood-derived