Disruption of Pathogenic Cellular Networks by IL-21 Blockade Leads to Disease Amelioration in Murine Lupus

This information is current as Jin-Young Choi, Abhinav Seth, Michael Kashgarian, Sonia of October 1, 2021. Terrillon, Emma Fung, Lili Huang, Li Chun Wang and Joe Craft J Immunol published online 20 February 2017 http://www.jimmunol.org/content/early/2017/02/18/jimmun ol.1601687 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 20, 2017, doi:10.4049/jimmunol.1601687 The Journal of Immunology

Disruption of Pathogenic Cellular Networks by IL-21 Blockade Leads to Disease Amelioration in Murine Lupus

Jin-Young Choi,* Abhinav Seth,* Michael Kashgarian,† Sonia Terrillon,‡ Emma Fung,‡ Lili Huang,‡ Li Chun Wang,‡ and Joe Craft*,x

Systemic lupus erythematosus (lupus) is characterized by autoantibody-mediated organ injury. Follicular Th (Tfh) cells orchestrate physiological germinal center (GC) B cell responses, whereas in lupus they promote aberrant GC responses with autoreactive memory B cell development and plasma cell–derived autoantibody production. IL-21, a Tfh cell–derived cytokine, provides instruc- tional cues for GC B cell maturation, with disruption of IL-21 signaling representing a potential therapeutic strategy for autoantibody-driven diseases such as systemic lupus erythematosus. We used blockade of IL-21 to dissect the mechanisms by which this cytokine promotes autoimmunity in murine lupus. Treatment of lupus-prone B6.Sle1.Yaa mice with an anti–IL-21 blocking Ab reduced titers of autoantibodies, delayed progression of glomerulonephritis and diminished renal-inﬁltrating Tfh and Downloaded from Th1 cells, and improved overall survival. Therapy inhibited excessive accumulation of Tfh cells coexpressing IL-21 and IFN-g, and suppressed their production of the latter cytokine, albeit while not affecting their frequency. Anti–IL-21 treatment also led to a reduction in GC B cells, CD138hi plasmablasts, IFN-g–dependent IgG2c production, and autoantibodies, indicating that Tfh cell–derived IL-21 is critical for pathological B cell cues in lupus. Normalization of GC responses was, in part, caused by uncoupling of Tfh–B cell interactions, as evidenced by reduced expression of CD40L on Tfh cells and reduced B cell proliferation in treated mice. Our work provides mechanistic insight into the contribution of IL-21 to the pathogenesis of murine lupus, while http://www.jimmunol.org/ revealing the importance of T–B cellular cross-talk in mediating autoimmunity, demonstrating that its interruption impacts both cell types leading to disease amelioration. The Journal of Immunology, 2017, 198: 000–000.

ystemic lupus erythematosus (SLE; lupus) is an inflam- (2, 4, 5). The number of circulating Tfh-like cells likewise increases matory disorder characterized by the generation of auto- in human SLE compared with control subjects, with correlation to S antibodies that promote tissue injury. Both adaptive and innate autoantibody production and disease severity (6, 7). Defining the immune cells contribute to the aberrant immune response in SLE, with mechanisms by which Tfh cells promote autoimmunity either follicular Th (Tfh) cells playing a central role given their direct effects through autoreactive B cell responses and/or influencing the func- in promoting the proliferation and maturation of B cells in germinal tion of other immune cells is critical for understanding the molec- by guest on October 1, 2021 centers (GCs) of secondary lymphoid organs (1, 2). Although much is ular and cellular origins of autoimmunity and, ultimately, for known about how Tfh cells function and interact with other immune developing novel treatment strategies. IL-21, the signature effector cytokine secreted by Tfh cells in components during normal immune responses, such as in infection or secondary lymphoid organs (1), promotes B cell proliferation, Ig immunization, less is understood about their function in SLE (3). class switching, and plasma cell differentiation (8–11). IL-21R– In murine models of lupus, GCs expand with concomitant in- deficient mice have weakened humoral responses to T-dependent crease of Tfh cells in both follicular and extrafollicular compartments Ags, with reduced GC B cell formation caused by diminished induction of the transcription factor B cell lymphoma 6 protein *Section of Rheumatology, Department of Internal Medicine, Yale School of Med- (Bcl6) necessary for GC B cell proliferation and Ig gene mutation, icine, New Haven, CT 06520; †Department of Pathology, Yale School of Medicine, New Haven, CT 06520; ‡AbbVie Bioresearch Center, Worcester, MA 01605; and defective plasma cell formation and impaired development of and xDepartment of Immunobiology, Yale School of Medicine, New Haven, CT memory B cells (8, 10, 11). Accordingly, IL-21 transgenic mice 06520 exhibit expansion of plasma cells, hypergammaglobulinemia, and ORCIDs: 0000-0002-7867-2427 (J.-Y.C.); 0000-0003-0362-7688 (M.K.). an increased frequency of class-switched Igs (12). IL-21 is Received for publication September 29, 2016. Accepted for publication January 26, markedly elevated in autoimmune-prone mice, including the 2017. lupus-prone BXSB-Yaa strain (12–14). Moreover, lupus severity is This work was supported by grants from AbbVie (to J.C.) and the Alliance for Lupus Research (to J.C.), as well as by National Institutes of Health Grants R37AR040072, diminished in the absence of IL-21 or IL-21R signaling in the lpr lpr R01AR068994, and P30AR053495 (to J.C.). A.S. was supported by a Scientist De- lupus-prone strains BXSB-Yaa and MRL/MpJ-Fas (MRL/Fas ) velopment Award from the American College of Rheumatology Research Founda- (13–15). Treatment of MRL/Faslpr mice with an IL-21R–Fc fusion tion, as well as by National Institutes of Health Grant T32 AR07107 and a grant from the Robert E. Leet and Clara Guthrie Patterson Trust. protein as an IL-21 sequestering agent lowered levels of circu- Address correspondence and reprint requests to Dr. Joe Craft, Section of Rheuma- lating autoantibodies and diminished deposition of glomerular tology, Department of Internal Medicine and Department of Immunobiology, Yale immune complexes (16). Although a subsequent study of BXSB- School of Medicine, P.O. Box 208031, S-525 TAC, 300 Cedar Street, New Haven, CT 06520. E-mail address: [email protected] Yaa mice treated with an IL-21 blocking agent demonstrated minimal beneficial effect (17), a lack of IL-21R expression on Abbreviations used in this article: B6, C57BL/6J; Bcl6, B cell lymphoma 6 protein; DPBS, Dulbecco’s PBS; GC, germinal center; gMFI, geometric mean fluorescence B cells, but not T cells, nonetheless protected BSXB-Yaa mice intensity; MSD, Meso Scale Discovery; SLE, systemic lupus erythematosus; Tfh, from disease, thereby demonstrating a B cell–intrinsic require- follicular Th. ment for IL-21 signaling to support GC formation and plasma cell Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 differentiation in autoimmunity (14). The protective effect of

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601687 2 IL-21 BLOCKADE IN MURINE LUPUS

B cell–restricted IL-21R deficiency was not simply limited to parameter logistic model was used for curve fitting. The regression modulation of autoantibody titers, because these animals also had equation for the calibration curve was then used to back-calculate the improvement in non–B cell features, such as reduced numbers of measured concentrations. The lower limit of quantitation was 0.021 mg/ml. splenic myeloid cells, which have been associated with lupus Anti–IL-21 treatment in vivo severity (18, 19). Together these studies demonstrate that effective Eight-week-old male B6.Sle1.Yaa mice were separated randomly into two targeting of a specific population of immune cells may have ripple groups and injected i.p. with anti–IL-21 mAb (4H11-B4; AbbVie Phar- effects throughout the immune system that can lead to greater maceutical) at 30 mg/kg, two times a week for the first 4 wk followed by therapeutic impact. Based on these data, we hypothesized that IL- three times a week for 3 mo. Control mice were injected with PBS in the 21 signaling from Tfh cells to B cells represents a critical node in same manner. Serum samples were collected by retro-orbital bleeding 2 mediating the pathologic cellular networks that lead to disease using heparinized capillary tubes, and sera were stored at 80˚C. pathology and severity in murine lupus. Preparation of single-cell suspensions To better understand the temporal molecular and cellular re- Spleens were homogenized by crushing between two frosted glass slides sponses to IL-21 in autoimmunity, we have used a novel anti–IL-21 followed by straining through a 40-mm cell strainer. RBCs were lysed by mAb to interrupt IL-21 signaling in lupus-prone C57BL/6J (B6) hypotonic shock and washed with Dulbecco’s PBS (DPBS) before Sle1.Yaa mice (E. Wakeland, University of Texas Southwestern counting cells. To isolate renal lymphocytes, we perfused mice with DPBS, Medical Center). These mice harbor an introgressed Sle1 locus and kidneys were removed, minced, and strained through a 70-mm cell from the NZM2410 strain and carry a duplication of Tlr7 (TLR7) strainer. Cell suspensions were loaded onto Ficoll-Paque, followed by density-gradient centrifugation. Cells were harvested from the opaque via the Y-linked autoimmune accelerating (Yaa) locus (20–23). layer in the interface and washed twice with DPBS before staining for flow The combination of these genetic components causes spontaneous cytometry. immunological aberrations, including abnormal GC responses; Downloaded from Flow cytometry expansion of splenic T, B, and myeloid cells; and development of autoantibody-mediated, fatal lupus glomerulonephritis (13, 22, Abs and dilutions used for staining included CD4 (RM4-5, 1:200), CD44 24). Anti–IL-21 treatment improved disease parameters in these (IM7, 1:200), ICOS (C398.4A, 1:100), TCRb (H57-597, 1:100), F4/80 animals, including blockade of pathological B cell expansion and (BM8, 1:400), and T-bet (c4B10, 1:50), all from eBioscience; PSGL-1 (2PH1, 1:1000), B220 (RA3-6B2, 1:400), GL-7 (GL7, 1:400), CD95 organ damage. Although we anticipated that the effects of anti–IL- (Jo2, 1:400), CD138 (281-2, 1:400), CD11b (M1/70, 1:400), Gr-1 (RB6- 21 therapy would primarily derive from downmodulation of B cell 8C5, 1:400), CD8 (53-6.7, 1:200), Bcl6 (K112-91, 1:50), and CD40L http://www.jimmunol.org/ responses, we found that, despite maintenance of overall splenic (MR1, 1:50), all from BD Biosciences; PD-1 (RMP1-30, 1:200), IgD (11– Tfh cell frequency, IL-21 blockade significantly inhibited the 26, 1:400), CXCR5 (L138D7, 1:1000), Ki-67 (16A8, 1:100), and IFN-g (XMG1.2, 1:100), all from BioLegend; granzyme B (GB11, 1:200; Invi- disease-associated expansion of Tfh cells that produced both IL- trogen) and IL-21R–Fc (1:50; R&D Systems). For intracellular cytokine 21 and IFN-g, in particular reducing IFN-g synthesis and con- staining, cells were stimulated with PMA (50 ng/ml) and ionomycin (1 mg/ml) comitant pathogenic IgG2c autoantibody production. These results for 2 h with GolgiPlug (BD Biosciences) for an additional 2 h. Cells demonstrate that IL-21 signaling plays an essential role in the were fixed (BD Cytofix/Cytoperm) and permeabilized (BD Perm/Wash) pathological cellular cross-talk that drives autoimmunity in mu- following the manufacturer’s protocol. IL-21 was detected by PE- conjugated F(ab9) (1:400; Jackson ImmunoResearch). For intracellular rine lupus, including the aberrant expansion of pathogenic IFN-g– 2 transcription factor staining, surface molecule–stained cells were fixed and by guest on October 1, 2021 producing Tfh cells. Our findings will help develop a better permeabilized using the buffer (eBioscience) without stimulation and framework for understanding current lupus therapies and for followed by staining for transcription factors. Stained cells were analyzed evaluating and designing new therapeutics for autoimmune dis- using an LSRII multilaser flow cytometry (BD) and followed by FlowJo software (Tree Star). eases. ELISA and ELISPOT analysis Materials and Methods To determine anti-dsDNA titers, we coated ELISA plates with methylated Mice BSA, followed by calf thymus DNA. Diluted sera were added to the plates and allowed to bind overnight. Anti-dsDNA Abs were detected with goat B6.Sle1.Yaa mice (22) were provided by E. Wakeland (University of Texas anti-mouse IgG conjugated with HRP (Southern Biotechnology). MRL/ Southwestern Medical Center), bred and maintained in specific pathogen- Faslpr serum samples were used as an internal standard. Plates were de- free conditions, and handled according to protocols approved by Yale veloped with TMB Microwell Peroxidase Substrate (SureBlue; KPL), and Institutional Animal Care and Use Committee. the OD was read at 450 nm. ELISPOT analysis was carried out similarly to determine autoantibody-forming cells. MultiScreen filter plates (EMD Generation of anti-mouse IL-21 mAb Millipore) were coated with either chromatin or DNA. Single-cell sus- The rat anti-mouse IL-21 hybridoma mAb was generated by immunization pensions of splenocytes and kidney cells were prepared in 10% FBS 3 6 of rats with cDNA expressing mouse IL-21. Clone BFJ-4H11-B4 was containing RPMI 1640 media at 2 10 /ml concentration. Cells were selected as the hybridoma lead based on its strong neutralization activity on plated directly onto coated plates in triplicate and incubated for 24 h at 37˚C. mouse IL-21 on a STAT3 phosphorylation bioassay. A rat/mouse chimeric Spots were developed with Vector Blue Substrate Kit (Vector Laboratories) mAb 4H11-B4 mIgG2a/k was constructed and generated in HEK293 cells and read by AID ELISPOT Reader. via transient transfection to enable evaluation in chronic mouse lupus Histology models. 4H11-B4 mIgG2a/k exhibits strong neutralization activity on mouse IL-21, with an IC50 of ∼30 pM. Kidneys were removed, bisected, formalin-fixed, paraffin-embedded, and H&E-stained. Kidneys were scored for glomerulonephritis by M.K. in a Quantification of the anti–IL-21 Ab in serum samples blinded manner, on a scale of 0 to 6 performed as previously reported 2 Murine anti–IL-21 serum samples were analyzed with a Meso Scale (25). For immunofluorescence staining, frozen kidneys stored in 80˚C m Discovery (MSD) assay employing biotinylated murine IL-21 (PR- were cut into 7- m sections, fixed in acetone, and stained with FITC- 1464132, lot 2130004; 0.05 mg/ml) for capture and goat anti-mouse conjugated anti-mouse IgG2a (R19-15, 1:200; BD Biosciences) and SULFO-TAG Ab for detection (MSD catalog no. R32AC-1; 0.5 mg/ml). FITC-conjugated anti-mouse C3 Abs (1:200; MP Biomedicals). Ab de- Samples were diluted 1:100 in assay buffer (1% MSD blocker A in TBS posits within glomeruli were analyzed using a fluorescence microscope with 0.02% Tween 20), then further diluted in assay buffer containing (Olympus BX40). serum for a 1% final matrix concentration. MSD standard curve fitting and Statistical analysis data evaluation were performed using XLfit4 software (version 4.2.1 Build 16). A calibration curve was prepared in serum and was plotted from MSD All data were presented as the mean 6 SEM. The significance of the luminescence units versus theoretical standard concentrations. A four- difference between two groups was evaluated by the two-tailed Student The Journal of Immunology 3 t test. Each dot in a scatterplot corresponds to an individual mouse. Control B6.Sle1.Yaa mice had enlarged spleens with increased Pearson correlation coefficient with two-tailed p value was determined in cellularity, compared with animals treated with anti–IL-21 , the analysis of correlations. The p values 0.05 were considered signif- (Fig. 1A, 1B). Anti–IL-21–treated mice had significant reduction icant. Data were analyzed with Prism software (version 5.0d for Macintosh; GraphPad Software). in nephritis at ages 4 and 6 mo (Fig. 1C, right panel, 1D), in contrast with kidneys from controls that exhibited hypercellu- lar glomeruli with crescent formation, suggestive of necrosis Results (Fig. 1C, left panel). As expected, spleen weights correlated IL-21 blockade inhibits disease progression in B6.Sle1.Yaa with glomerulonephritis scores (Fig. 1E). Serum creatinine mice concentrations, used as a measure of renal function, were Twenty-one 8-wk-old male B6.Sle1.Yaa mice were injected i.p. similar between both groups before treatment, but were sig- with anti–IL-21 mAb (4H11-B4) at 30 mg/kg, two times a week nificantly elevated in control mice at 4 mo of age, suggesting for 4 wk followed by three times a week for an additional 12 wk. compromised kidney function in the untreated animals, whereas Mice were selected for intervention at 8 wk of age, because ani- they remained low in anti–IL-21–treated mice (Fig. 1F). Serum mals at this age have developed evidence of aberrant T and B creatinine levels and kidney histopathology scores were correlated activation, albeit without apparent deep organ injury (22). As a (Fig. 1G). Control B6.Sle1.Yaa mice had significant cumulative control, 18 age-matched male B6.Sle1.Yaa mice were injected mortality beginning at approximately 4 mo of age and .50% with PBS. Nine mice from each group were sacrificed at 4 mo of mortality rate at 6 mo (22). By contrast, nearly all mice treated with age (after 8 wk of treatment), and all surviving animals were anti–IL-21 survived to age 6 mo (Fig. 1H). Moreover, clinical sacrificed at age 24 wk (16 wk of treatment). phenotypes in treated animals had an inverse correlation with Downloaded from

FIGURE 1. Treatment of lupus- http://www.jimmunol.org/ prone B6.Sle1.Yaa mice with anti– IL-21 inhibits disease progression and promotes survival. (A and B) Spleen weight (A) and total number of splenocytes (B) from control (closed circles) or anti–IL-21–treated (open circles) B6.Sle1.Yaa mice at indicated ages. (C and D) Scores of glomerulonephritis on a scale of 0 to 6(D) and representative images of by guest on October 1, 2021 H&E-stained kidney sections from 4- mo-old control (C, left) and anti–IL- 21–treated animals (C, right), original magnification 3200. (E) Correlation between glomerulonephritis and spleen weights from all animals sacrificed at ages 4 and 6 mo. Each circle represents an individual animal; controls are represented by closed circles and anti–IL-21–treated animals by open circles. (F) Measure- ment of creatinine using QuantiChrom Creatinine Assay Kit (BioAssay Sys- tems) in sera from all animals at indicated age (months). (G) Correlation between glomerulonephritis and serum creatinine from all animals sacrificed at age 4 mo. (H)Survival of B6.Sle1.Yaa mice treated with anti–IL-21 (n = 12), compared with PBS-treated controls (n = 9), using Mantel-Cox test. Correlations were determined using Pearson’s correlation analyses. (I) Correlation between the amount of anti–IL-21 in sera and spleen weights at 6 mo of age. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 4 IL-21 BLOCKADE IN MURINE LUPUS the sera concentrations of the anti–IL-21 Ab, as evidenced by animals, .60% of activated B220+IgDlo splenic B cells expressed decreases in spleen weights in treated mice (Fig. 1I). These data intracellular Ki-67 as a marker of proliferation at age 6 mo, demonstrate that treatment of B6.Sle1.Yaa mice with anti–IL-21 whereas only 20% of B cells from mice treated with anti–IL-21 decreases the severity of lupus glomerulonephritis and improves were Ki-67+ (Fig. 2A), suggesting IL-21 blockade suppressed survival. their proliferation. Moreover, numbers of TCRb2B220+CD95hi GL7hi GC B cells were significantly reduced by IL-21 blockade IL-21 blockade inhibits GC maturation and autoantibody (Fig. 2B). IL-21 blockade also caused significant reduction in production formation of TCRb2 B220loCD138hi plasmablasts (Fig. 2C). In Because of the necessary role that IL-21 plays in B cell differ- concert with anti–IL-21–mediated reduction in GC B cells and entiation and GC responses (8–11), we asked whether IL-21 plasmablasts in B6.Sle1.Yaa mice, treated animals had a trend blockade impaired B cell proliferation, maturation in GCs, and toward reduced formation of IgG1 and IgG2c anti-chromatin Abs development into plasmablasts in B6.Sle1.Yaa mice. In control as measured by ELISPOT assays, compared with controls (Fig. 2D), Downloaded from

FIGURE 2. Anti–IL-21 treatment of B6. http://www.jimmunol.org/ Sle1.Yaa mice inhibits GC B cell maturation, plasmablast differentiation, and autoantibody production. (A–C) Representative ﬂow cytometry plots of control (left, closed circles) and anti–IL-21–treated (middle, open circles) animals and scatterplot (right) of the percentage of Ki-67+ among IgDlo B220hi activated B cells in mice at 6 mo (A) and GL7hiCD95hi GC B cells among B220+

B cells at 4 mo (B), and the percentage of by guest on October 1, 2021 TCRb2B220loCD138hi plasmablasts among total splenocytes at 4 mo (C). Histogram in gray in (A) is IgDloB220hi naive B cells. (D) Representative ELISPOT analysis (left, original magnification 320) and number of splenocytes secreting anti-chromatin Abs in 4-mo-old animals (right). (E) IgG anti- dsDNA IgG autoantibodies from 4-mo-old control and anti–IL-21–treated B6.Sle1.Yaa mice. Each dot indicates an individual animal. Each scatterplot is a compilation of two to three independent experiments. (F) Immunofluorescent staining for IgG2c and C3 deposits in the glomeruli of control and anti–IL-21–treated mice at 4 mo of age (original magnification 3400). The significance of the difference between two groups was evaluated by two-tailed Student t test. *p , 0.05, **p , 0.01, ****p , 0.0001. The Journal of Immunology 5 as well as a reduction in IgG anti-dsDNA Ab amounts as deter- among experiments, we also normalized Bcl6 expression to the mined by ELISA (Fig. 2E). Accordingly, glomerular immune degree of expression in naive CD4+ T cells (Fig. 4A, right panel). complex deposition was reduced in anti–IL-21–treated animals, Regardless of whether we normalized its expression, there was compared with controls, as determined by immunofluorescence significantly less Bcl6 in Tfh cells of control animals compared analysis for IgG2c and C3 deposition (Fig. 2F). These data indicate the treated cohort. Bcl6 expression also was inversely correlated that IL-21 blockade prevents proliferation and expansion of B cells, with spleen weights and kidney disease (Fig. 4B, left and right, GC maturation, and plasmablast differentiation, as well as autoan- respectively). T-bet expression in splenic Tfh cells from control tibody generation in B6.Sle1.Yaa mice. animals was also significantly lower than that observed in cells

+ + from anti–IL-21–treated animals (Fig. 4C), albeit not different IL-21 blockade blocks accumulation of IL-21 IFN-g Tfh between Th1 cells in either group (Fig. 4D). These results cells in B6.Sle1.Yaa mice suggest that Tfh cells in B6.Sle1.Yaa mice with advanced dis- Activated CD4+ T cells expand as B6.Sle1.Yaa mice age, with ease, as manifested by splenomegaly and glomerulonephritis, do robust expression of transcripts for Il21, Ifng, and Il4, with ex- not conform to the canonical Tfh cell phenotype, as evidenced pansion of a population bearing Tfh cell surface markers (22). We by lower Bcl6 and T-bet expression, and by expression of the likewise found that .90% of splenic CD4+ T cells from 4-mo-old inflammatory cytokine IFN-g. The lack of correlation between control lupus mice were activated, as evidenced by CD44hi T-bet expression and IFN-g production is surprising and sug- staining, with ,10% CD44lo naive, finding similar percentages at gests there are T-bet–independent mechanisms of IFN-g pro- age 6 mo (Fig. 3A, top). By contrast, mice treated with anti–IL-21 duction in lupus Tfh cells. For example, in the chronic had significant reduction in percentages of activated CD44hi cells inflammatory milieu of active lupus characterized by robust cy- (∼60%) at age 4 mo, with a concomitant increase in their naive tokine production, chromatin modifications necessary for Ifng Downloaded from counterparts (∼40%), with similar results at age 6 mo (Fig. 3A, expression can be acquired in the absence of T-bet (J.S. Weinstein top). In a similar manner, anti–IL-21–treated animals had signif- and J. Craft, unpublished observations). Although treatment icant reduction in total CD4+ T cell numbers compared with with anti–IL-21 blockade did not affect the generation of Tfh controls at age 4 mo, with fewer such cells also found at age 6 mo cells, it significantly altered their phenotypic and functional (Fig. 3A, bottom). By contrast with the ameliorative effects of IL-21 properties, reverting them toward a more classical Tfh cell blockade on numbers and percentages of activated CD4+ T cells, phenotype with enhanced Bcl6 expression and reduced num- http://www.jimmunol.org/ the frequency and number of CD4+CD44hiPSGL-1loCXCR5hiPD-1hi bers of IL-21 IFN-g double-positive cells compared with cells Tfh cells were relatively unaltered by anti–IL-21 treatment (Fig. 3B, in control mice.

3C). These results show that although IL-21 blockade inhibits the + overall activation and expansion of CD4+ T cells, the Tfh cell Effect of IL-21 blockade on myeloid cells and CD8 T cells in population appears largely unchanged numerically. B6.Sle1.Yaa mice We next asked whether IL-21 blockade affected cytokine Cellular abnormalities exhibited by B6.Sle1.Yaa mice are not production by splenic Tfh cells, determining expression of IL- limited to CD4+ T and B lymphocytes, but are also apparent in the 21 and IFN-g in CD44hiPSGL-1loPD-1hiCXCR5hi Tfh and myeloid cell compartment (24), the expansion of which is caused by guest on October 1, 2021 CD44hi PSGL-1hiPD-1loCXCR5lo Th1 cells. IL-21 was ro- in part by altered expression of IL-6 (26, 27). We assessed ex- bustly produced by Tfh cells in control mice, findings similar pression of myeloid cell markers, including CD11b, F4/80, and to those earlier reported for Il21 transcripts in BXSB-Yaa mice Gr-1, among TCRb2 B2202 splenocytes using flow cytometry. At (13), with IL-21 protein also produced by Th1 cells (Fig. 3D, 4 mo of age, TCRb2 B2202 cells constituted ∼40% of the 3E), which was also consistent with earlier findings of its splenocytes in control animals, increasing to 60% at 6 mo of age transcript in BXSB-Yaa Th1 cells (13). IL-21 producers from (Fig. 5A). The expanded TCRb2 B2202 cell pool included both populations commonly expressed IFN-g, with more CD11bhiF4/80hiGr-1med monocytes (Fig. 5B) and CD11bhiF4/80lo IL-21+ IFN-g+ double-positive cells in the Th1 population Gr-1hi granulocytes (Fig. 5C). By contrast, TCRb2 B2202 cells (∼40%) than in the Tfh group (∼30%) in control animals constituted ,20% of the splenocytes, and the proportion remained (Fig. 3D, 3E), whereas relatively few cells produced only IFN-g. unchanged at 6 mo of age in the anti–IL-21–treated animals. Treatment with anti–IL-21 led to a significant decrease in cyto- Accordingly, monocyte and granulocyte populations were signif- kine double-positive cells among both the Tfh and the Th1 subsets icantly reduced by anti–IL-21 treatment (Fig. 5B, 5C). at age 4 mo, and for Tfh cells, also at age 6 mo with a trend toward IL-21 signaling is necessary for sustained function of CD8+ a decrease in Th1 cells, without significantly impacting the fre- T cells during chronic viral infection (28–30) and for expansion of quency of IL-21 single-positive cells (Fig. 3D, 3E). The frequency central memory and suppressor CD8+ T cells in autoimmune of Tfh cells expressing CD40L was also significantly reduced BXSB-Yaa mice (14). We therefore characterized splenic CD8+ in animals treated with IL-21 blockade (55 versus 45%, control T cells in control and treated B6.Sle1.Yaa mice at age 6 mo. CD8+ versus IL-21 blockade, respectively) (Fig. 3F). Hence, IL-21 T cells from control mice comprised .70% of CD44hi cells, blockade resulted in the reduction of the expression of IFN-g whereas anti–IL-21–treated animals had significant reduction to by Tfh and Th1 cells in B6.Sle1.Yaa mice, as well as that 40% of CD44hi cells (Fig. 5D). Expression of PD-1 on CD44hi of CD40L, which is critical for GC B maturation to plasma CD8+ T cells, which was correlated with CD8+ T cell activation, cells (9). was high in control animals, but decreased to that of naive CD8+ To further investigate the phenotype of splenic Tfh cells in B6. T cells in treated animals (Fig. 5E). CD8+CD44hi T cells from Sle1.Yaa mice, we measured the flow cytometric expression of control animals commonly expressed the cytolytic molecule Bcl6 and T-box transcription factor Tbx21 (T-bet), subset- granzyme B (Fig. 5F, top). By contrast, granzyme B–expressing associated transcription factors for Tfh and Th1 cells, respec- CD8+CD44hi T cells were markedly reduced in anti–IL-21–treated tively. Bcl6 expression by Tfh cells, determined as geometric animals (Fig. 5F, bottom), suggesting that CD8+ T cell expansion mean fluorescence intensity (gMFI), was lower in control animals in murine lupus is dependent upon IL-21, as in viral infection (28– compared with those treated with anti–IL-21 (Fig. 4A, left and 30), with IL-21 blockade reducing the cytolytic potential of this center panels). To compensate for differences in Bcl6 staining population. 6 IL-21 BLOCKADE IN MURINE LUPUS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Altered phenotype of splenic Tfh cells in B6.Sle1.Yaa mice is restored by anti–IL-21 treatment. (A) Quantification of percentage (top) and total numbers (bottom) of CD44loPSGL-1hi naive and CD44hi activated CD4+ T cell subsets from 4- to 6-mo-old B6.Sle1.Yaa mice. (B and C) Strategy for identification of Tfh cells from control (top, closed circles) or anti–IL-21–treated (bottom, open circles) animals by CD44hiPSGL-1lo gating of CD4+ T cells (left) followed by that of CXCR5hiPD-1hi cells (right), with quantification of percentage (left) and total number (right) of Tfh cells from 4- to 6-mo-old B6. Sle1.Yaa mice. (D and E) Representative flow cytometry plot from control (top) and anti–IL-21–treated (bottom) animals showing intracellular IL-21 and IFN-g staining in Tfh (D) or Th1 cells (E) in black dot plots overlaid on naive CD4+ T cells staining in gray contour plot. The percentages of total IL-21+ cells (red box) or IL-21+ IFN-g+ double-positive cells (blue box) from control (closed circles) versus anti–IL-21–treated (open circles) B6.Sle1.Yaa mice are shown at indicated ages. (F) The percentages of CD40Lhi Tfh cells from control compared with anti–IL-21–treated animals at 6 mo of age. The gate was set based on CD40L expression on naive CD4+ cells. Each dot indicates an individual animal. Scatterplots are compilations of two to three independent experiments. The significance of the difference between two groups was evaluated by a two-tailed Student t test. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

Anti–IL-21 treatment abrogates infiltration of inflammatory action. We therefore characterized the nature of the T cell infil- CD4+ T cells in the kidney tration in kidneys of B6.Sle1.Yaa mice. At 4 mo of age, the total Infiltration of inflammatory cells into the kidney is a key feature of number of kidney-infiltrating leukocytes, as determined by for- murine lupus nephritis (31), with the diseased human kidney ward and side scatter gating followed by a live cell gate, was containing infiltrates of B cells and Tfh cells located in close significantly increased in control B6.Sle1.Yaa mice compared with proximity (32), suggesting local, pathological Tfh–B cell inter- animals treated with anti–IL-21 (Fig. 6A). Infiltrating CD4+ The Journal of Immunology 7

FIGURE 4. Altered phenotype of splenic Tfh cells in B6.Sle1.Yaa mice is correlated with disease progression. (A) Representative flow cytometry plots from control (top, closed circles) and anti–IL-21–treated (bottom, open circles) animals showing Bcl6 expressions by Tfh cells (black line) overlaid on naive cells (filled gray) and scatterplots of gMFI of Bcl6 in Tfh cells (middle) and normalized Bcl6 gMFI in Tfh cells (right). (B) Correlation between the ratio of Bcl6 gMFI and spleen weights (left), and the ratio of Bcl6 gMFI and kidney disease scores (right). Correlations were determined using Pearson’s correlation analyses. (C and D) Representative flow Downloaded from cytometry plots from control (top) and anti–IL-21–treated (bottom) animals showing T-bet expression by Tfh (C) and Th1 cells (D) in black overlaid on naive cells (filled gray) and scatterplots of normalized T-bet gMFI in Tfh (C) and http://www.jimmunol.org/ Th1 cells (D). Each dot indicates an individual animal. Scatterplots are compilations of two to three independent experiments. The significance of the difference between two groups was evaluated by two-tailed Student t test. *p , 0.05, **p , 0.01, ****p , 0.0001. n.s., not significant. by guest on October 1, 2021

T cells mostly bore a CD44hiPSGL-1hi Th1 phenotype, whereas (Fig. 7C), CD138hi plasmablasts were dramatically diminished in CD44hiPSGL-1lo Tfh cells were less commonly observed animals treated with IL-21 blockade (Fig. 7D). Likewise, poten- (Fig. 6B). Kidney-infiltrating Th1 cells produced IL-21, with IL- tially pathogenic IgG2c plasmablasts from anti–IL-21–treated 21 IFN-g double-positive cells also increased compared with those animals were significantly reduced compared with those from observed in the spleen (60 versus 40%; Fig. 6C, compare with control animals (10 versus 30%, respectively), with both groups Fig. 3E). CD44hiPSGL-1lo Tfh cells in the kidney likewise con- having similar numbers of IgG1 plasmablasts (Fig. 7E, 7F). tained IL-21 IFN-g double producers (Fig. 6D). Although the total Numbers of such cells producing anti-dsDNA Abs were also numbers of renal Th1 and Tfh cells were reduced by anti–IL-21 significantly reduced by anti–IL-21 treatment (Fig. 7G), suggest- therapy, their cytokine phenotypes did not change (Fig. 6C, 6D), ing that IL-21 signaling is required for the expansion and main- nor did the proliferative capacity of the Th1 pool (Fig. 6E). These tenance of autoantibody-secreting plasmablasts even in advanced data indicate that infiltrating renal CD4+ T cells exhibit a Th1 disease. Yet, the frequency of B cells with CD38hiGL7lo memory phenotype in B6.Sle1.Yaa mice, with fewer Tfh-like CD44hi phenotype was significantly increased by treatment (Fig. 7I), PSGL-1lo cells, with both populations reduced numerically by suggesting IL-21 signaling is dispensable in memory B cell for- anti–IL-21 therapy. mation in murine lupus as it appears to be in normal physiological contexts (10, 11). At this later stage of disease, 1 mo of anti–IL-21 Effect of IL-21 blockade in B6.Sle1.Yaa mice with advanced blockade had no significant effect on renal disease, because se- disease rum creatinine concentrations had increased from 0.13 6 0.03 6 6 We next assessed the effect of anti–IL-21 blockade in older B6. mg/dl (mean SD)atage2mo(Fig.1F)to0.17 0.03 and 6 6 Sle1.Yaa mice, initiating therapy in twelve 3-mo-old mice. Six 0.18 0.07 mg/dl (mean SD) at ages 3 and 4 mo (Fig. 7H). animals were treated with either 30 mg/kg anti–IL-21 or PBS two times a week for 4 wk, with all animals sacrificed at age 4 mo; one Discussion PBS-treated animal died during treatment. Although anti–IL-21 Lupus-prone B6.Sle1.Yaa mice spontaneously develop expansion therapy did not affect spleen weights and total number of of T and B cells, with an abnormal GC output leading to patho- splenocytes (Fig. 7A and 7B, respectively), or the overall fre- logical autoantibody-mediated glomerulonephritis (22). We show quency and numbers of splenic Tfh (data not shown) or GC B cells that treatment of these animals, before onset of pathological renal 8 IL-21 BLOCKADE IN MURINE LUPUS Downloaded from http://www.jimmunol.org/

FIGURE 5. IL-21 blockade prevents abnormal expansion of leukocytes in B6.Sle1.Yaa mice. (A–C) Quantification of the percentage of TCRb2B2202 cells (A) from control (closed circles) or anti–IL-21–treated (open circles) mice, and total numbers of monocytes (B) and granulocytes (C) among total splenocytes from control (closed circles) or anti–IL-21–treated (open circles) B6.Sle1.Yaa mice at indicated ages. (D and E) The percentages of CD44hi cells among splenic CD8+ T cells (D) and representative flow cytometry plots from control (top) and anti–IL-21–treated (bottom) animals showing PD-1 expression by CD44hiCD8+ cells in black overlaid on naive CD44loCD8+ cells (filled gray) and scatterplots of PD-1 gMFI in CD44hiCD8+ cells at age of 6 mo (E). (F) Representative flow cytometry plots showing intracellular staining for IFN-g and granzyme B among splenic CD8+ T cells from 6-mo-old B6.Sle1. Yaa animals treated with PBS (top) or anti–IL-21 (bottom) and the fraction of the total CD8+ T cells expressing IFN-g, granzyme B, or both (right). Each dot indicates an individual animal. The significance of the difference between two groups was evaluated by two-tailed Student t test. *p , 0.05, **p , 0.01, by guest on October 1, 2021 ***p , 0.001, ****p , 0.0001. disease but after that of immunological abnormalities with T and conclusion is at odds with our findings that earlier intervention B cell activation (22), with a novel anti–IL-21 Ab reduced B cell with anti–IL-21 dramatically reduced renal inflammatory changes proliferation, GC responses, CD138+ plasma cell formation, and in conjunction with reduction in abnormal B cell responses and autoantibody secretion. These findings were in parallel with re- glomerular Ab and complement deposition. It is possible that duction in renal immune-complex deposits and cellular infiltrates, longer-term treatment of older animals is needed to achieve a resulting in amelioration of histological kidney disease and im- significant therapeutic effect. provement in survival. Although B cells with a CD38+GL7lo Pathogenic autoantibodies play a fundamental role in patho- memory phenotype were relatively unaffected by IL-21 blockade, genesis of human lupus (34), and the production of such autoan- this finding is consistent with previous experiments demonstrating tibodies requires T cell–dependent GC B cell responses (3). SLE unaltered formation of memory B cells in the absence of IL-21 patients have elevated blood concentrations of IL-21 compared signaling (10, 11). In aggregate, our results support earlier studies with healthy controls (35, 36) in association with increased in other lupus models using anti–IL-21 blockade, or interruption numbers of circulating IL-21+ Tfh-like cells and disease activity in IL-21 signaling using genetic approaches, that this pathway is (36). Evidence from both human and murine lupus studies un- essential to the pathogenesis of lupus, most notably through equivocally indicates that IL-21 plays a critical role in the path- driving aberrant B cell responses (13–17, 33). ogenesis of lupus and, consequently, is an attractive therapeutic IL-21 blockade was also effective when applied to an older target in human SLE. A fully humanized anti–IL-21R Ab (ART- cohort with advanced renal disease as evidenced by elevated blood 107) has been used safely in a small number of healthy subjects, creatinine levels. Treatment of older animals suppressed production albeit with bioavailability concern (37). IL-21 blockade has also of CD138+ plasma cells that secrete IgG2c pathogenic autoanti- been evaluated in a phase I clinical trial for SLE, albeit with early bodies, although this cohort was too small to effectively evaluate termination (ClinicalTrials.gov Identifier: NCT01689025) (38). changes in renal infiltrates or histological renal disease. None- Our work provides preclinical rationale for these approaches. theless, creatinine concentrations were not altered compared with IL-21 blockade also inhibited aberrant development of Th1 and control mice, despite significant reduction in pathological B cell Tfh cells. B6.Sle1.Yaa mice spontaneously develop and accumu- responses, suggesting that renal inflammation and/or damage had late Tfh cells that promote aberrant GC responses with concom- progressed to a point independent of immune-complex triggering. itant production of pathogenic IgG2c autoantibodies (22, 24). We It is also possible these later changes were independent of found that Tfh cells in diseased B6.Sle1.Yaa mice robustly pro- autoantibody-triggered inflammation altogether; however, this duced IL-21 and IFN-g, perhaps surprisingly in parallel with The Journal of Immunology 9 Downloaded from

FIGURE 6. Infiltration of inflammatory CD4+ T cells into the kidney is abrogated by treatment with anti–IL-21 in B6.Sle1.Yaa animals. (A and B) Quantification of total number of leukocytes (A), from control (closed circles) or anti–IL-21–treated (open circles), and CD4+ Tcellsubsets(B) isolated http://www.jimmunol.org/ from kidneys from 4-mo-old B6.Sle1.Yaa mice treated with PBS (closed circle) or anti–IL-21 (open circle). (C and D) Scatterplot of the percentages of IL-21+ IFN-g+ cells among Th1 cells (C) and PSGL-1lo cells (D) isolated from kidney from control or anti–IL-21–treated B6.Sle1.Yaa mice at 4 mo of age. (E) Representative flow cytometry plots from control (top) and anti–IL-21–treated (bottom) animals showing Ki-67 expression in Th1 cells (black line) overlaid on naive cells (filled gray) and the percentage of Ki67+ cells in Th1 cells (right) at 6 mo of age. Each dot indicates an individual mouse. Each scatterplot is compiled from two independent experiments. The significance of the difference between two groups was evaluated by two-tailed Student t test. *p , 0.05. by guest on October 1, 2021 downregulation of Bcl6 and T-bet expression, with expression of phenotype in treated animals from pathogenic potential to one the former transcription inversely correlated with renal disease more conventional. Tfh cells in treated animals also had signifi- and spleen weight, indicative of an atypical Tfh cell phenotype in cant reduction in CD40L expression. Its expression on GC Tfh diseased B6.Sle1.Yaa mice. IL-21+ Tfh cells that coproduce IFN-g cells is regulated by Ag-dependent ICOS triggering via GC B cell with pathogenic drive have also been reported in other murine ICOSL (53). Thus, IL-21 blockade interrupts bidirectional col- lupus models (22, 39, 40), and in our hands, in circulating Tfh-like laboration between Tfh cells and GC B cells, affecting both cell cells in patients with SLE (J.-Y. Choi and J. Craft, unpublished populations. We speculate that the effects of IL-21 blockade on observations). Tfh cells with a similar phenotype are found after B cells are direct, given previous results (14), whereas the effects viral challenge in mice (41, 42) and humans (43), albeit they are on Tfh cell cytokine production are indirectly mediated via short-lived. By contrast, they may persist in chronic type I im- B cells, as shown in our recent collaborative studies (54). Thus, mune challenge of mice (44) and humans (43, 45). IFN-g pro- our observation of pleiotropic immunological effects of IL-21 duction by Tfh cells promotes protective immunity via generation blockade suggests that IL-21 plays a critical role in maintaining of high-affinity isotype-specific Abs appropriate to the invading the pathologic cellular interactions that drive persistent autoim- pathogen (46). By contrast, its effects in chronic infection munity in lupus. Along these lines, anti–IL-21–associated down- or autoimmunity may be detrimental. Tfh-derived IFN-g pro- regulation of IFN-g expression by Tfh cells may be of particular motes Ig isotype switching by autoreactive B cells to inflam- benefit in controlling the pathogenic inflammation characteristic matory IgG2a (or IgG2c in C57Bl6 background) (47) and IgG3, of lupus. with Fc receptor activation and complement fixation, poten- IFN-g+ Th1 cells were significantly diminished in treated mice, tially contributing to disease severity (48, 49). Moreover, with a 10-fold decrease in Th1 cell infiltrates in the kidney. We Th1-derived IFN-g, and presumably that derived from Tfh also found that blockade of IL-21 signaling reduced potentially cells, contributes locally to macrophage activation and to renal cytolytic CD8+ T cells, which require IL-21 for their survival and inflammation and tissue damage (50, 51), including in human function in chronic inflammatory states (28–30), as well as IL-6– lupus nephritis (52). dependent myeloid cells (24), with the latter result consistent with Anti–IL-21 treatment suppressed CD4 T cell activation, leading the role of CD4+ T cell–secreted IL-21 in promoting myeloid cell to a decrease in the number and frequency of activated CD44hi migration to survival niches and activation in experimental type 1 CD4+ T cells. Although Tfh cell numbers were unchanged by diabetes mellitus (55). treatment, they nonetheless underwent phenotypic change. Tfh In summary, we report that blocking IL-21 signaling with anti– cells in treated animals produced significantly less IFN-g, albeit IL-21 in lupus-prone B6.Sle1.Yaa mice ameliorated disease with with no change in IL-21 production and with increased Bcl6 ex- improved survival, via downmodulation of pathological B cell pression. These changes revealed reversion of the lupus Tfh cell responses, but also by regulation of pathogenic Tfh and Th1 cell 10 IL-21 BLOCKADE IN MURINE LUPUS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 7. Reduced autoantibody response by a brief treatment with IL-21 blockade in B6.Sle1.Yaa mice with advanced disease. (A and B) Spleen weight (A) and total number of splenocytes (B) from control (closed circles) or anti–IL-21–treated (open circles) B6.Sle1.Yaa mice at age of 4 mo. (C) Scatterplots of the percentage of GL7hiCD95hi GC B cells among B220hiIgDlo B cells. (D) Scatterplot of the percentage of TCRb2B220loCD138hi plasmablasts among total splenocytes. (E and F) Representative flow cytometry plots from control (top) and anti–IL-21–treated (bottom) animals showing intracellular IgG1 and IgG2a staining among TCRb2B220loCD138hi plasmablasts (E) and scatterplots of the percentage of intracellular IgG1+ (left) or IgG2a+ (right) cells among TCRb2B220loCD138hi plasmablasts in control versus anti–IL-21–treated B6.Sle1.Yaa mice at age of 4 mo (F). (G) Number of splenocytes secreting anti-dsDNA Abs in 4-mo-old animals. (H) Measurement of creatinine in sera from control (closed circles) or anti–IL-21–treated (open circles) B6.Sle1.Yaa mice at indicated ages (months). (I) Scatterplots of the percentage of CD38hiGL7lo memory B cells among B220hiIgDlo B cells. Each dot indicates an individual mouse. The significance of the difference between two groups was evaluated by two-tailed Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. n.s., not significant. function. These results add to the rationale for anti–IL-21 thera- 2. Linterman, M. A., R. J. Rigby, R. K. Wong, D. Yu, R. Brink, J. L. Cannons, P. L. Schwartzberg, M. C. Cook, G. D. Walters, and C. G. Vinuesa. 2009. Fol- peutic intervention in SLE and offer insight into the mechanism by licular helper T cells are required for systemic autoimmunity. J. Exp. Med. 206: which IL-21 promotes bidirectional collaboration between Tfh 561–576. and GC B cells and disease progression in lupus. 3. Vinuesa, C. G., I. Sanz, and M. C. Cook. 2009. Dysregulation of germinal centres in autoimmune disease. Nat. Rev. Immunol. 9: 845–857. 4. Vinuesa, C. G., M. C. Cook, C. Angelucci, V. Athanasopoulos, L. Rui, Acknowledgments K. M. Hill, D. Yu, H. Domaschenz, B. Whittle, T. Lambe, et al. 2005. A RING- We thank Stephen Clarke, David Duignan, and Christine Grinnell (AbbVie) type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity. Nature 435: 452–458. and members of the Craft laboratory for helpful discussions and review 5. Odegard, J. M., B. R. Marks, L. D. DiPlacido, A. C. Poholek, D. H. Kono, of the manuscript. We also thank Martha Mayo and Donna McCarthy C. Dong, R. A. Flavell, and J. Craft. 2008. ICOS-dependent extrafollicular helper (AbbVie) for help with in vitro and in vivo assays. T cells elicit IgG production via IL-21 in systemic autoimmunity. J. Exp. Med. 205: 2873–2886. 6. Simpson, N., P. A. Gatenby, A. Wilson, S. Malik, D. A. Fulcher, S. G. Tangye, Disclosures H. Manku, T. J. Vyse, G. Roncador, G. A. Huttley, et al. 2010. Expansion of The authors have no financial conflicts of interest. circulating T cells resembling follicular helper T cells is a fixed phenotype that identifies a subset of severe systemic lupus erythematosus. Arthritis Rheum. 62: 234–244. 7. Choi, J. Y., J. H. Ho, S. G. Pasoto, V. Bunin, S. T. Kim, S. Carrasco, E. F. Borba, References C. R. Gonçalves, P. R. Costa, E. G. Kallas, et al. 2015. Circulating follicular 1. Craft, J. E. 2012. Follicular helper T cells in immunity and systemic autoim- helper-like T cells in systemic lupus erythematosus: association with disease munity. Nat. Rev. Rheumatol. 8: 337–347. activity. Arthritis Rheumatol. 67: 988–999. The Journal of Immunology 11

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