BAFF Suppresses IL-15 Expression in B Cells

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BAFF Suppresses IL-15 Expression in B Cells Ning Ma, Chen Xing, He Xiao, Youdi He, Gencheng Han, Guojiang Chen, Chunmei Hou, Bernadette Marrero, Yujuan Wang, Shengquan Zhang, Beifen Shen, Yan Li and Renxi This information is current as Wang of September 28, 2021. J Immunol 2014; 192:4192-4201; Prepublished online 26 March 2014; doi: 10.4049/jimmunol.1302132 http://www.jimmunol.org/content/192/9/4192 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2014/03/26/jimmunol.130213 Material 2.DCSupplemental http://www.jimmunol.org/ References This article cites 39 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/192/9/4192.full#ref-list-1

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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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

BAFF Suppresses IL-15 Expression in B Cells

Ning Ma,*,†,1 Chen Xing,*,1 He Xiao,*,1 Youdi He,‡ Gencheng Han,* Guojiang Chen,* Chunmei Hou,* Bernadette Marrero,x Yujuan Wang,{ Shengquan Zhang,‖ Beifen Shen,* Yan Li,* and Renxi Wang*

Clinical trials have shown that BAFF inhibitors do not reduce memory B cell levels but can reduce the number of mature B cells. It remains uncertain whether BAFF affects memory-maintaining cytokines such as IL-15. We found that BAFF suppressed IL-15 expression in B cells from lupus-like or experimental allergic encephalomyelitis mice. When BAFF was blocked with atacicept- IgG, IL-15 expression was upregulated in lupus-like or experimental allergic encephalomyelitis mice. Finally, we showed that BAFF suppressed IL-15 expression in transitional 2 B cells by reducing Foxo1 expression and inducing Foxo1 phosphorylation. This study suggests that BAFF suppresses IL-15 expression in autoimmune diseases, and this opens up the possible opportunity for the clinical application of BAFF- and IL-15–speciﬁc therapeutic agents. The Journal of Immunology, 2014, 192: 4192–4201. Downloaded from

cell–activating factor is a crucial factor that regulates cells (9). Atacicept (TACI)-IgG shows similar clinical results to B cell maturation, survival, and function. An excess of belimumab by binding a portion of the receptor TACI to block the B BAFF has been shown to lead to the development of effects of survival factors BAFF and a proliferating-inducing li- autoimmune disorders in animal models, and BAFF concentra- gand (APRIL). It reduces levels of circulating mature B cells and tions are known to be higher in patients with various autoim- plasma cells in the spleen and bone marrow as well as inhibiting mune conditions compared with normal subjects (1). The pathogenic T cell activation, but not memory B cells (10, 11). As long as the http://www.jimmunol.org/ role of BAFF in systemic lupus erythematosus (SLE) was revealed reactive memory is maintained, it is probably acceptable that in in a study that showed BAFF-transgenic mice develop a lupus-like principle humoral immunological response can be restored from illness with the production of anti-DNA Abs and development immunological memory (12). of glomerulonephritis (2–4). Increased levels of BAFF reported IL-15 provided survival signals that maintain memory T and in some studies correlate with disease activity (5). In multiple B cells in the absence of Ag (13–15). Therefore, we propose that sclerosis (MS) patients, BAFF is expressed by astrocytes that are BAFF suppresses memory B cells by downregulating IL-15 ex- closely associated with BAFFR–expressing cells (6) within ec- pression. In the current study, we found that BAFF suppressed IL- topic lymphoid follicles of the meninges (7). BAFF has been 15 expression in B cells from lupus-like or experimental allergic by guest on September 28, 2021 regarded as a new therapeutic target in many autoimmune dis- encephalomyelitis (EAE) mice. When BAFF was blocked with eases (8). TACI-IgG, IL-15 expression was upregulated. The study provides On March 9, 2011, the Food and Drug Administration approved hints for the clinical application of BAFF- and IL-15–speciﬁc belimumab, a fully human anti-BAFF mAb, as a new B cell– therapeutic agents. speciﬁc treatment for SLE. Belimumab selectively reduces the number of CD20+ naive B cells, activated B cells, and plasma- Materials and Methods blasts and results in a transient increase in the number of memory Ethics committee approval The blood samples were taken with the approval of the local ethical committee (Clinical Trial Center, Beijing Institute of Basic Medical Sci- *Laboratory of Immunology, Institute of Basic Medical Sciences, Beijing 100850, ence), and all participants gave written informed consent to participate in the China; †Department of Rheumatology, First Hospital of Jilin University, Changchun 130021, China; ‡Department of Neurology, Beijing Chaoyang Hospital, Capital Med- current study. ical University, Beijing 100020, China; xMolecular Immunology Section, Laboratory Care, use, and treatment of mice in this study were in strict agreement of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD with international guidelines for the care and use of laboratory animals. This 20892; {Immunopathology Section, Laboratory of Immunology, National Eye Insti- study was approved by Animal Ethics Committee of Beijing Institute of ‖ tute, National Institutes of Health, Bethesda, MD 20892; and Department of Bio- Basic Medical Sciences. chemistry and Molecular Biology, Anhui Medical University, Hefei 230032, China 1N.M., C.X., and H.X. contributed equally to this work. Peripheral blood from normal human subjects, MS patients, Received for publication August 12, 2013. Accepted for publication February 26, and SLE patients 2014. Blood samples were obtained after the approval from the Beijing Institute of This work was supported by National Basic Research Program 973 Grant 2013CB530506 Basic Medical Sciences, consent from 15 normal human subjects and 11 SLE and Beijing Natural Science Foundation Grant 7132139. patients from Clinical Trial Center (Beijing 307 Hospital), and consent from Address correspondence and reprint requests to Dr. Renxi Wang and Dr. Yan Li, 15 MS patients from Department of Neurology, Beijing Chaoyang Hospital. Laboratory of Immunology, Institute of Basic Medical Sciences, P.O. Box 130 (3), Taiping Road #27, Beijing 100850, China. E-mail addresses: wang_renxi@yahoo. Culture of human B cells com (R.W.) and [email protected] (Y.L.) The online version of this article contains supplemental material. PBMCs were isolated from the heparin-treated whole blood by density gradient centrifugation, and CD19+ B cells were isolated using human Abbreviations used in this article: APRIL, a proliferating-inducing ligand; EAE, CD19 MicroBeads (AutoMACS; Miltenyi Biotec). PBMC or CD19+ experimental allergic encephalomyelitis; FB, follicular B; LN, lymph node; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; MZB, marginal zone B cells were stimulated with 1 mg/ml LPS or 10 mg/ml goat F(ab9)2 anti- B; qPCR, quantitative PCR; shRNA, short hairpin RNA; SLE, systemic lupus ery- human IgM (Southern Biotechnology Associates, Birmingham, AL) to thematosus; TACI, atacicept; T1B, transitional 1 B; T2B, transitional 2 B; WT, wild- activate the cells in the presence of 50 ng/ml human rBAFF (PeproTech, type. Rocky Hill, NJ). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302132 The Journal of Immunology 4193

Mice to the following grading scale: 0 = no signs; 1 = distal tail weakness; 1.5 = tail weakness and some hind limb weakness; 2 = complete tail paralysis; 2.5 = Nine-week-old C57BL/6, BALB/c mice, and (New Zealand Black/New complete tail paralysis and partial hind limb weakness; 3 = complete hind Zealand White)F1 mice (Chinese Academy of Medical Sciences, Bei- limb weakness; 3.5 = inability to right when placed on back or significant jing, China) were bred in our animal facilities under specific pathogen- forelimb weakness; 4 = euthanized or spontaneous death (17, 18). Mice free conditions. were euthanized if they lost 20% of their starting weight, displayed a clinical score of 3 for 72 h, or reached a clinical score of 3.5. Mice were EAE induction examined for up to 21 d postimmunization. Number of mice per treatment group was 12. EAE induction was performed, as previously described (16). Briefly, C57BL/6 mice at 9 wk of age received a s.c. injection of 125 mg myelin Treatment of EAE or lupus-like mice with TACI-IgG and/or oligodendrocyte glycoprotein (MOG)35–55 peptide (Mimotopes) emulsified anti–IL-15 Ab 1:1 (v/v) in CFA containing 4 mg/ml Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI), to the base of the tail and both flanks. Pertussis toxin EAE and lupus-like mice were divided into the following six groups: 1, (300 ng pertussis toxin in PBS; List Biological) was injected i.p. at the time control mice; 2, untreated; 3, human IgG treated; 4, TACI-IgG treated; 5, of induction, and a second dose was administered 3 d later. The same volume TACI-IgG plus rabbit IgG treated; 6, TACI-IgG plus anti–IL-15. Twelve of CFA and pertussis toxin was injected into each mouse in the control EAE mice per group were injected i.v. with 2 mg/kg TACI-IgG and/or 0.5 group. Animals were weighed, monitored, and clinically assessed according mg/kg anti-mouse IL-15 neutralizing Ab (R&D Systems) on days 4, 8, 12, Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. TACI-IgG treatment upregulated IL-15 expression in lupus-like mice. (A) TACI-IgG treatment upregulated memory B cell number in lupus-like mice. Twelve lupus-like mice per group were injected i.p. with 5 mg/kg TACI-IgG and/or 1 mg/kg anti–IL-15 neutralizing Ab at 1, 2, 3, and4wk(twiceperweek)afterthemicehadreached6moofage.Ondays4–6 after therapy, the mice were euthanized and lymphocytes were collected from the spleen. Memory B cells were isolated from the spleen by a memory B cell isolation kit, according to the manufacturer’s protocol. The average total cell number in each spleen of the six mice per group is shown. The data represent at least three independent experiments (**p , 0.01, ***p , 0.001). (B) IL-15 levels were increased in the serum from TACI-IgG–treated lupus-like mice. The serum was collected from WT (None lupus) mice, IgG-, and TACI-IgG–treated lupus-like mice (12 mice per group) on days 4–6 after therapy. IL-15 levels were determined by ELISA. The data represent at least three independent experiments (****p , 0.0001). (C) IL-15 mRNA was increased in the spleen and LN from TACI-IgG–treated lupus-like mice. Lymphocytes were collected from the spleen and LN in IgG- and TACI-IgG–treated lupus-like mice (12 mice per group) on days 4–6 after therapy and subjected to qPCR. The data represent at least five independent experiments (*p , 0.05, ***p , 0.001, ****p , 0.0001). (D) TACI-IgG treatment increased IL-15 expression in B cells. Lymphocytes were collected from the spleen and LN in IgG- or TACI-IgG–treated lupus-like mice (12 mice per group) on days 4–6 after therapy. The cells were stained with anti-B220 and anti–IL-15 and then analyzed by FACS. The percentage of IL-15–expressing B cells, statistical analysis of the percentage of IL-15+ B cells, and the absolute number of IL-15+ B cells in each spleen were shown in (D), (E), and (F), respectively. The data represent at least four independent experiments (*p , 0.05, **p , 0.01, ***p , 0.001). 4194 BAFF SUPPRESSES IL-15 and 16 (one time per day) after EAE was induced with MOG33–55. allophycocyanin, followed by FACS analysis. Flow cytometric analysis Twelve (New Zealand Black/New Zealand White)F1 mice (lupus-like demonstrated .90% memory B cells (data not shown). Finally, we counted mice) per group were injected i.p. with 5 mg/kg TACI-IgG and/or 1 mg/kg the absolute number of memory B cells per mouse. rabbit anti-mouse IL-15 neutralizing Ab (R&D Systems) at 1, 2, 3, and 4 wk (twice per week) after the mice reached 6 mo of age. In vitro B cell cultures Isolation of memory B cells CD19+ B cells were isolated using mouse CD19 MicroBeads (AutoMACS; Miltenyi Biotec) and cultured in RPMI 1640 medium containing 10% There is no marker for memory B cells in mice (12). Thus, we used a two- FBS, 2 mM glutamine, penicillin (100 IU/ml), streptomycin (100 mg/ml), step procedure to isolate the memory B cells from six mice per group by and 50 mM 2-ME. Cells were stimulated with 1 mg/ml LPS, 10 mg/ml goat memory B cell isolation kit (MACS Miltenyi Biotec; Order 130-095-838), F(ab9)2 anti-mouse IgM (Southern Biotechnology Associates), MOG35–55 according to the manufacturer’s protocol (Miltenyi Biotec). Briefly, we peptide (20 mg/ml precoated in 96-well plates), or dsDNA (20 mg/ml first depleted nonmemory B cells with nonmemory B cell biotin–Ab precoated in 96-well plates) in the presence of 50 ng/ml rBAFF mixture and anti-biotin microbeads. Additional labeling was done with (PeproTech). anti-IgG1 allophycocyanin and anti-IgG2ab allophycocyanin. The mag- netically labeled cells were passed through a column placed in the magnetic Cytometric analysis and intracellular cytokine staining field of a MACS separator. Secondly, anti-allophycocyanin microbeads were used for indirect magnetic labeling of memory B cells. Positive selection All cell experiments were strictly prepared on ice, unless otherwise stated 6 of memory B cells was sorted by the magnetic field of a MACS separator. in other specific procedures. Cells (1 3 10 cells/sample) were washed with Memory B cells were fluorescently stained with B220-VioBlue (130-094- FACS staining buffer (PBS, 2% FBS or 1% BSA, 0.1% sodium azide). All 287), CD38 PE (130-097-087), anti-IgG1 allophycocyanin, and anti-Ig2ab samples were incubated with 2.4G2 anti-FcRs (BD Pharmingen), prior to Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. TACI-IgG treatment upregulated IL-15 expression in EAE mice. (A) TACI-IgG treatment upregulated memory B cell number in EAE mice. EAE were induced in female C57BL/6 mice at 9 wk of age by MOG35–55 peptide in CFA. Twelve EAE mice per group were injected i.v. with 2 mg/kg TACI-IgG and/or 0.5 mg/kg anti–IL-15 neutralizing Ab on days 4, 8, 12, and 16 (once per day) after EAE was induced. On day 21, mice were euthanized and the lymphocytes were collected from the spleen. Memory B cells were isolated from the spleen by a memory B cell isolation kit, according to the manufacturer’s protocol. The average total cell number in each spleen of the six mice per group is shown. The data represent at least three independent experiments (**p , 0.01, ***p , 0.001). (B) IL-15 levels were increased in the serum from TACI-IgG–treated EAE mice. The serum was collected from CFA-, IgG-, and TACI-IgG– treated mice (16 mice per group) on days 4–6 after therapy. IL-15 levels were determined by ELISA. The data represent at least four independent experiments (***p , 0.001). (C) IL-15 mRNA was increased in B cells of the LN and spleen from TACI-IgG–treated EAE mice. B cells were sorted from the LN and spleen in IgG- and TACI-IgG–treated EAE mice (16 mice per group) on days 4–6 after therapy and subjected to qPCR. The data represent at least six independent experiments (*p , 0.05, ***p , 0.001). (D–F) TACI-IgG treatment increased IL-15 expression in B cells. B cells were sorted by B220 beads, stained with anti- B220 and anti–IL-15, and then analyzed by FACS. The percentage of IL-15–expressing B cells, statistical analysis of the percentage of IL-15+ B cells, and the absolute number of IL-15+ B cells in each spleen are shown in (D), (E), and (F), respectively. The data represent at least four independent experiments (*p , 0.05). The Journal of Immunology 4195 incubation with other Abs diluted in FACS buffer supplemented with 2% with 1 mg/ml LPS and analyzed by FACS. Cells were also sorted by flow anti-FcR Ab. For intracellular cytokine staining, 50 ng/ml PMA and 1 mg/ cytometry. cDNA synthesis and qPCR analyses were done to detect IL-15 ml ionomycin (all from Sigma-Aldrich) were added, and then 10 mg/ml expression. brefeldin A and 2 mM monensin were added 3 h later. Another 3 h later, cells were collected and fixed for 20 min with 1 mL fixation buffer (Fix IL-15 promoter reporting gene analysis and Perm cell permeabilization kit; eBioscience). After washing, the fixed To clone series of the human IL-15 gene promoter and to construct six cells were stained. The samples were filtered immediately before analy- luciferase reporter gene vectors pGL3 containing IL-15 promoter regions, sis or cell sorting to remove remaining clumps. Dead cell exclusion/ six DNA fragments of the 59-flanking region of human IL-15 gene were discrimination dyes were used to eliminate dead cells from analysis and isolated from genomic DNA of HaCaT cells by PCR with IL-15 promoter sorted. For nonfixed cells, we used DAPI, SYTOX Blue, Green, or Red; for primer sets (Table I). Empty vector or pEGFP-N1/Foxo1 and luciferase fixed cells, we use LIVE/DEAD Fixable Blue, Green, or Red. Choice of reporter vector pGL3/IL-15 promoter were cotransduced into 293T cells. dead cell exclusion dye depended on the color combination of fluo- Dual luciferase reporter gene expression was analyzed, and the results rochromes within the sample. The following Abs were purchased from were shown as the ratio of firefly to Renilla luciferase activity. eBioscience: anti-mouse CD27, CD3, B220, CD19, CD93, CD21, CD23, and CD4 Abs. Anti–IL-15 Ab was purchased from R&D Systems. Data Western blot analyses collection and analyses were performed on a FACSCalibur flow cytometer + using CellQuest software. CD19 B cells were stimulated with 10 mg/ml goat F(ab9)2 anti-mouse IgM (Southern Biotechnology Associates) in the presence of 50 ng/ml Cytokine analysis by ELISA human rBAFF (PeproTech). Cells were stimulated for 72 h. For Foxo1 phosphorylation analysis, CD19+ B cells were stimulated for 0, 30, and 60 The concentration of cytokines (IL-15, BAFF, and dsDNA) was measured min with 10 mg/ml goat F(ab9)2 anti-mouse IgM (Southern Biotechnology by ELISA kits. Anti-mouse IL-15, BAFF, and dsDNA ELISA kits were Associates) in the presence of 50 ng/ml human rBAFF (PeproTech). The purchased from R&D Systems. Briefly, diluted supernatants were added in whole-cell lysates were prepared. Blots were probed with anti–b-actin, Downloaded from triplicate to the plate for 1 h at 37˚C. Then after washing, biotin rat anti- Foxo1, and p-Foxo1 (Santa Cruz Biotechnology). Preimmune serum was mouse IL-15, BAFF, and dsDNA (4 mg/ml) Abs were added to the plate used in parallel as controls and signals were detected with HRP-conjugated and were incubated for another hour at 37˚C. Thereafter, unbinding Abs secondary F(ab9)2 (Zymed Laboratories, San Francisco, CA) using ECL were washed off, followed by addition of avidin-HRP (1/1000 diluted) system (Amersham, Arlington Heights, IL). (all Abs were obtained from eBioscience). Plates were incubated for 1 h at 37˚C. Finally, the color was developed by incubation with o-phenylenedi- Statistics amine. The OD was read at 492 nm with an ELISA reader (Bio-Rad).

Standard curves were established to quantitate the amounts of the re- Statistics were generated using t test in GraphPad Prism (version 5.0; http://www.jimmunol.org/ spective cytokines. GraphPad Software), and values are represented as mean 6 SEM. Results were considered statistically signiﬁcant at p , 0.05. Cell proliferation assays Lymphocytes were collected from lymph nodes (LN) in different groups of EAE. The cells (5 3 105 cells/well) were stimulated for 72 h with 0, 1, 5, 15, and 30 mg/ml MOG35–55. Forty-eight hours later, the cultures were pulsed with 3H-thymidine (0.5 mCi), and data were expressed as mean cpm 6 SE of responses of ﬁve replicate cultures. The results were expressed as the stimulation index (cpm with Ag divided by cpm with medium alone). The data shown were from only one of the Ag concentrations; however, results by guest on September 28, 2021 were obtained with various concentrations of the Ags.

Quantitative PCR analysis All RNA samples were DNA free. cDNA synthesis and quantitative PCR (qPCR) analyses were performed, as previously described (19). Each gene- specific primer pair used for qPCR analysis spanned at least an intron. Primers and probes used for qPCR were purchased from Applied Bio- systems, and mRNA expression was normalized to the levels of b-actin gene. B cell subpopulation sorting For analysis of transitional B cells, multicolor flow cytometry (three, four, or five colors) was performed by gating on CD19+CD93+ B cells that were either CD212/CD23+ (transitional 1 B [T1B] cells) or CD21+CD23+ (transitional 2 B [T2B] cells). For analysis of mature B cells, multicolor flow cytometry (three, four, or five colors) was performed by gating on CD19+CD932 B cells that were either CD21+/CD23+ (follicular B [FB] cells) or CD21+ CD232 (marginal zone B [MZB] cells). All flow cytometry data were acquired with FACSCanto, FACSCantoII, or FACSAria (BD Biosciences), gated on live lymphocyte-sized cells on the basis of forward and side scatter, and analyzed using FlowJo software (Tree Star, Ashland, OR). The following Abs were purchased from eBioscience: PerCP-conjugated anti- mouse CD19, FITC-conjugated anti-mouse CD93, PE-conjugated anti- mouse CD21, and allophycocyanin-conjugated anti-mouse CD23. FIGURE 3. IL-15 was mainly expressed in the spleen. (A) IL-15 was Reduced expression or overexpression of Foxo1 increased mainly in the spleen from lupus-like mice. Lymphocytes were collected from the spleen and LN in 6-mo-old controls and lupus-like mice Foxo1-specific short hairpin RNA (shRNA) with GFP (Santa Cruz Bio- (6 mice per group) and then subjected to qPCR. The data represent at least + technology, Santa Cruz, CA) were transfected into CD19 B cells. The three independent experiments (**p , 0.01). (B–D) IL-15 was increased B cells were stimulated for 72 h with 1 mg/ml LPS. The GFP+ (Foxo1 was 2 mainly in the spleen from EAE mice. Lymphocytes were collected from reduced) and GFP T2B cells were sorted from cultured cells by flow cytometry. All RNA samples were DNA free. cDNA synthesis and qPCR the spleen and LN in CFA and EAE mice (12 mice per group) on day 21 analyses were done to detect Foxo1 and IL-15 expressions. Mouse after EAE induction (8 mice per group), and the cells were subjected to recombinant Foxo1 construct pEGFP-N1/Foxo1 was generated by rPCR. qPCR (B) and FACS analysis (C). The statistical analysis of the percentage Plasmid pEGFP-N1/Foxo1 was transduced into CD19+ B cells by Lipo- of IL-15+ cells was shown in (D). The data represent at least three inde- fectamine 2000 (Invitrogen, Carlsbad, CA). Cells were stimulated for 72 h pendent experiments (*p , 0.05, ***p , 0.001). 4196 BAFF SUPPRESSES IL-15 Downloaded from http://www.jimmunol.org/

FIGURE 4. BAFF suppressed IL-15 expression in autoantigen-stimulated B cells. (A) BAFF but not April suppressed IL-15 in B cells. Splenic B cells were collected from lupus-like mice (8 mice per group) at 6 mo of age and stimulated for 48 h with precoated dsDNA with or without 20 ng/ml BAFF or April. The cells were then stained with PerCP-conjugated anti–IL-15, and the percentage of IL-15–expressing cells is shown. The data represent at least three independent experiments. BAFF suppressed IL-15 mRNA in dsDNA (B)- and MOG35–55 (C)-stimulated lymphocytes. Splenic lymphocytes were collected from controls and lupus-like mice (8 mice per group) at 6 mo of age (B) or CFA and EAE mice (8 mice per group) on day 21 after EAE induction (C). Lymphocytes were stimulated for 48 h with precoated dsDNA (B) or MOG35–55 (C) with or without APRIL or BAFF. Cells were subjected to qPCR. by guest on September 28, 2021 The data represent at least six independent experiments (*p , 0.05, **p , 0.01, ***p , 0.001). (D) BAFF suppressed IL-15 expression in B cells but not T cells. Lymphocytes were collected from CFA and EAE mice (10 mice per group) on day 21 after EAE induction and stimulated for 72 h with precoated MOG35–55 with or without BAFF. The cells stained with anti-CD4, anti-CD19, and anti–IL-15. The percentage of IL-15–expressing CD4+ T(upper panel) and CD19+ B(lower panel) cells and their statistical analyses are shown in the left and right panels, respectively. The data represent at least ﬁve independent experiments (*p , 0.05, **p , 0.01).

Results that TACI-IgG does not reduce memory B cells (10, 11). How- TACI-IgG upregulated IL-15 in lupus-like and EAE mice ever, there is no marker for memory B cells in mice (12). Thus, BAFF levels have been shown to be increased in many auto- we used a two-step procedure to isolate the memory B cells from immune diseases, such as SLE, MS, and their animal models six mice per group with a memory B cell isolation kit. The ab- (Supplemental Fig. 1). When BAFF was blocked with TACI- solute memory B cell number per mouse was counted, and the IgG, numbers of mature B cells (Supplemental Fig. 2A–C) and results suggested that TACI-IgG treatment signiﬁcantly upreg- the titer of anti-dsDNA autoantibodies (Supplemental Fig. 2D) ulated memory B cell number in lupus-like mice (Fig. 1A). were decreased in lupus-like mice. Previous studies have shown These results are in accordance with the clinical data suggesting

FIGURE 5. BAFF suppressed IL-15 expression in nonspecific reagent-stimulated B cells. B cells were sorted using CD19 beads from the spleen in 9-wk-old naive C57BL/6 mice. B cells were stimulated for 48 h with LPS (A) or anti-mouse IgM (B) with or without BAFF. (C) B cells from the peripheral blood cells of MS patients were sorted by CD19 beads and stimulated for 48 h with anti-human IgM with or without BAFF. Cells were subjected to qPCR. The data represent at least five independent experiments (**p , 0.01, ***p , 0.001). The Journal of Immunology 4197 that the total number of circulating memory B cells is preserved IL-15 in lupus-like mice. As expected, TACI-IgG treatment up- (or sometimes even increased) in belimumab- or TACI-IgG–treated regulated IL-15 expression in the serum (Fig. 1B), spleen, and LN patients. (Fig. 1C) from lupus-like mice. Because BAFF, APRIL, and their IL-15 is an important cytokine for the survival and maintenance receptors play important roles in the B cell arm of the immune of memory T and B cells (13–15). Thus, we propose that blocking system (20), we assessed whether TACI-IgG upregulated B cells BAFF with TACI-IgG upregulates memory B cells by inducing to express IL-15 by qPCR and flow cytometry (FACS) analysis. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. BAFF suppressed T2B cells to secrete IL-15. (A and B) BAFF suppressed IL-15 expression in T2B cells. Splenic B cells were sorted from CFA and EAE mice (6 mice per group) on day 21 after EAE induction and then stimulated for 72 h with precoated MOG35–55 with or without BAFF. The cells were stained with anti-CD19 or anti-CD93, anti-CD21, anti-CD23, and anti–IL-15 Abs. The percentage of IL-15–expressing T1B (CD19+CD93+ CD212CD23+), T2B (CD19+CD93+CD21+CD23+), FB (CD19+CD932CD21+CD23+), and MZB (CD19+CD932CD21+CD232) cells and statistical analysis for IL-15–expressing T2B cells are shown in (A) and (B), respectively. The data represent at least six independent experiments (*p , 0.05, **p , 0.01, ***p , 0.001). (C) BAFF suppressed IL-15 mRNA expression in T2B cells. T1B, T2B, FB, and MZB were sorted (see Supplemental Fig. 4) from the medium and BAFF-treated B cells described as in (A) and subjected to qPCR. The data represent at least three independent experiments (****p , 0.0001). (D and E) BAFF induced B cell maturation. (D) Splenic B cells were sorted from CFA and EAE mice on day 21 after EAE induction and then stimulated for 72 h with precoated MOG35–55 with or without BAFF. Cells were stained with anti-CD19 or anti-CD93, anti-CD21, and anti-CD23; T1B (CD19+CD93+ CD212CD23+), T2B (CD19+CD93+CD21+CD23+), FB (CD19+CD932CD21+CD23+), and MZB (CD19+CD932CD21+CD232) cells (see Supplemental Fig. 4) were analyzed. The transforming ratio from T1B to T2B to FB cells was shown in (E). The data represent at least three independent experiments. 4198 BAFF SUPPRESSES IL-15 Downloaded from

FIGURE 7. Foxo1 controlled IL-15 expression in T2B cells. (A) Foxo1 expression was positively associated with IL-15 level. T1B (CD19+CD93+CD212 CD23+), T2B (CD19+CD93+CD21+CD23+), FB (CD19+CD932CD21+CD23+), and MZB (CD19+CD932CD21+CD232) cells were sorted by flow http://www.jimmunol.org/ cytometry (see Supplemental Fig. 4) and subjected to qPCR. Foxo1 and IL-15 mRNA expressions were shown in the upper and lower panels, respectively. The data represent at least three independent experiments (****p , 0.0001). (B) IL-15 expression was reduced in Foxo1-specific shRNA-transfected T2B cells. CD19+ B cells were transfected with Foxo1-specific shRNA with GFP and then stimulated for 72 h with LPS. GFP+ (Foxo1 was reduced) and GFP2 (Foxo1 was not reduced) T2B cells were sorted by flow cytometry and subjected to qPCR. Foxo1 and IL-15 mRNA expressions are shown in the upper and lower panels, respectively. The data represent at least four independent experiments (*p , 0.05). (C) Foxo1 overexpression induced IL-15 expression. CD19+ B cells were transduced with empty vector or pEGFP-N1/Foxo1 and then stimulated for 72 h with LPS. GFP+ B cells were analyzed and sorted by flow cytometry. Cells were gated on enhanced GFP with numbers in the quadrants indicating the percentage of IL-15–expressing B cells (upper panel), and the sorted cells were subjected to qPCR (lower panel). The data represent at least four independent experiments (****p , 0.0001). (D) Foxo1 directly

regulated IL-15 promoter. Six DNA fragments (described in Materials and Methods) of the 59-flanking region of human IL-15 gene were isolated from by guest on September 28, 2021 genomic DNA of HaCaT cells by PCR. Six luciferase reporter gene vectors pGL3 containing IL-15 promoter regions were constructed. Empty vector or pEGFP-N1/Foxo1 and luciferase reporter vector pGL3/IL-15 promoter were cotransduced into 293T cells. Dual luciferase reporter gene expression was analyzed, and the results were shown as the ratio of firefly to Renilla luciferase activity. The results for IL-15 F0 and IL-15 F3 are shown. The results of IL-15 F0 and F1 as well as IL-15 F2–F6 were similar. The data represent at least four independent experiments (***p , 0.001).

We showed that IL-15 expression was upregulated mainly in B cells The evidence for the role of IL-15 in autoimmune condition is from TACI-IgG–treated lupus-like mice (Fig. 1D–F). compelling. ELISA has demonstrated that IL-15 levels are increased To further prove the effect of TACI-IgG on IL-15 expression in in many autoimmune diseases, such as SLE, MS, and their animal B cells, TACI-IgG was also used to treat EAE mice, resulting in the models (Supplemental Fig. 1). qPCR and FACS analysis have fur- reduction of B cell numbers (Supplemental Fig. 3A–C), clinical ther shown that IL-15 was upregulated mainly in the spleen in scores (Supplemental Fig. 3D), and autoantigen-induced immune autoimmune diseases (Fig. 3). IL-15/IL-15R has been used as response (Supplemental Fig. 3E). However, TACI-IgG treatment a therapeutic target in inﬂammatory autoimmune diseases (21). In also upregulated both the memory B cell number (Fig. 2A) and addition, our results presented above suggest that TACI-IgG treat- IL-15 expression in the serum (Fig. 2B) of EAE mice. Further ment upregulates memory B cells by inducing IL-15 in B cells. experiments showed that TACI-IgG induced B cells to upregulate Thus, the combination of TACI-IgG and anti–IL-15 Ab was used the expression of IL-15 (Fig. 2C–F). to treat lupus-like and EAE mice. Once IL-15 was blocked with

Table I. PCR primers’ sequences

Common Downstream Primer 59-CTCATTACTCAAAGCCACGGTAA-39 Primers’ Sequence Production (bp) F0 59-ATGCAGTTATGGAGGGAGTGAG-39 1021 F1 59-GATTCAGGCAGCAAGCAGAG-39 1264 F2 59-CTCCCGACATCAGGTGATCC-39 1519 F3 59-GTGAAAATCCATTGTGACAT-39 1769 F4 59-TTGAAGCACTGAGATAATAT-39 2021 F5 59-CAGAAACATCTGAAAAGTATA-39 2279 F6 59-CTCAAATGATTAACAATGGTTTC-39 2559 F, front (upstream) primer. The Journal of Immunology 4199 neutralizing anti–IL-15 Ab, the memory B cell number was sig- T2B cells than Foxo1-unreduced T2B cells (Fig. 7B, lower panel). nificantly reduced in TACI-IgG–treated lupus-like (Fig. 1A) and When overexpressed in B cells, Foxo1 upregulated IL-15 mRNA EAE (Fig. 2A) mice. This study provides hints for the clinical ap- (Fig. 7C, lower panel) and protein (Fig. 7C, upper panel) expres- plication of BAFF- and IL-15–specific therapeutic agents. sion. These results suggest that Foxo1 regulates IL-15 expression in T2B cells. To prove that Foxo1 directly regulates IL-15 expres- BAFF suppressed IL-15 expression in B cells sion, we constructed six luciferase reporter gene vectors pGL3 The above experiments demonstrated that IL-15 was upregulated containing IL-15 promoter regions. By dual luciferase reporter mainly in the spleen but not in LN (Figs. 1C–F, 2C–F, 3). Thus, we gene expression analysis, we found that Foxo1 bound the region focused on splenic B cells. To determine the effect of TACI ligand (BAFF or APRIL) on IL-15 expression in B cells, we used autoantigen dsDNA (precoated in 96-well plate) plus BAFF or APRIL to stimulate B cells from lupus-like mice. FACS assay showed that BAFF but not APRIL significantly suppressed IL-15 expression in dsDNA-stimulated B cells from lupus-like mice (Fig. 4A). Fur- thermore, qPCR assay also proved that BAFF but not APRIL significantly suppressed IL-15mRNAexpressioninBcells (Fig. 4B). To further prove the effect of BAFF on IL-15 expression in

B cells, we used MOG35–55 (precoated in 96-well plates) plus Downloaded from BAFF to stimulate B cells from EAE mice. BAFF signiﬁcantly suppressed IL-15 expression in MOG35–55-stimulated B cells from EAE mice (Fig. 4C, 4D). In comparison, BAFF also slightly suppressed IL-15 expression in B cells from wild-type (WT) mice (Fig. 4A, 4B). To further prove these results, we also

used different conditions such as LPS or anti-IgM to stimulate http://www.jimmunol.org/ B cells from WT mice. qPCR analysis showed that BAFF suppressed IL-15 expression in LPS (Fig. 5A)- or anti-IgM (Fig. 5B)– stimulated B cells from WT mice. Similarly, BAFF also suppressed IL-15 expression in anti-human IgM-stimulated B cells from healthy donors (Fig. 5C). These results suggest that BAFF suppresses IL-15 expression in B cells induced by different stimuli. Furthermore, BAFF suppresses the secretion of IL-15 by CD19+ B cells but not CD4+ T cells (Fig 4C). by guest on September 28, 2021 BAFF suppressed T2B cells to secrete IL-15 Next, we determined which population of splenic B cells was crucial in the BAFF suppression of IL-15. We used autoantigen MOG35–55 (precoated in 96-well plates) plus BAFF to stimulate B cells from control and EAE mice. Different populations of B cells were analyzed by FACS (Fig. 6A). The results showed that T2B cells expressed a high level of IL-15, whereas T1B cells, MZB cells, and FB cells expressed low levels of IL-15. Among these cell types, BAFF suppressed the expression of IL-15 from FIGURE 8. BAFF suppressed IL-15 expression by downregulating T2B cells (Fig. 6A, 6B). Furthermore, we sorted T1B, T2B, FB, Foxo1. BAFF suppressed Foxo1 mRNA in dsDNA (A)- and MOG35–55 and MZB cells from BAFF-treated B cells, as described in (B)-stimulated B cells. Splenic B cells were sorted by CD19 beads from Fig. 6A, by FACS (Supplemental Fig. 4) and then used qPCR to control and lupus-like mice (10 mice per group) at 6 mo of age (A)orCFA detect IL-15 expression. The results suggest that IL-15 is and EAE mice (6 mice per group) on day 21 after EAE induction (B). B A expressed mainly in T2B cells but not in T1B, MZB, or FB cells cells were stimulated for 48 h with precoated dsDNA ( ) and MOG35–55 (B) with or without BAFF. Cells were subjected to qPCR. The data rep- (Fig. 6C). In addition, BAFF suppressed IL-15 expression in resent at least four independent experiments (**p , 0.01). (C) BAFF re- T2B cells (Fig. 6C). Meanwhile, we found that BAFF promoted duced Foxo1 protein expression in B cells. Splenic B cells were stimulated the transformation of T1B cells into T2B cells and T2B cells into for 72 h with anti-IgM with or without BAFF. The whole-cell lysates were FB cells (Fig. 6D, 6E). These results suggest that BAFF sup- subjected to Western blot. The data represent at least six independent presses T2B cells from secreting IL-15 and promotes the trans- experiments. (D) BAFF induced Foxo1 phosphorylation in B cells. Splenic formation of T2B cells into mature B cells. B cells were stimulated for 0, 30, and 60 min with anti-IgM in the presence of BAFF, and the whole-cell lysates were subjected to Western blot. The BAFF suppression of Foxo1 downregulates IL-15 expression in data represent at least four independent experiments. TACI-IgG treatment B cells induced Foxo1 expression in lupus-like (E) and EAE (F) mice. (E) Eight Next, we examined which transcription factor is responsible for lupus-like mice per group were i.p. injected with 5 mg/kg IgG or TACI- IgG at 1, 2, 3, and 4 wk (twice per week) after mice reached 6 mo of age. IL-15 expression in T2B cells. Foxo1 was mainly expressed in (F) Ten EAE mice per group were i.v. injected with 2 mg/kg IgG or TACI- T2B cells but not in T1B, MZB, or FB cells (Fig. 7A, upper panel), IgG on days 4, 8, 12, and 16 (once per day) after EAE mice were induced which is in accordance with IL-15 expression (Fig. 7A, lower in 9-wk-old C57BL/6 mice by MOG35–55 peptide. On days 4–6 after panel). We used Foxo1-specific shRNA to reduce Foxo1 expres- therapy, the mice were sacrificed. B cells were collected and subjected to sion in B cells (Fig. 7B, upper panel) and then sorted the T2B cells. qPCR. The data represent at least three independent experiments (**p , qPCR analysis showed a lower IL-15 expression in Foxo1-reduced 0.01, ***p , 0.001). 4200 BAFF SUPPRESSES IL-15 from 1500 to 1800 bp upstream of the IL-15 promoter (Fig. 7D, of mature B cells. However, memory B cells were upregulated Table I). in TACI-IgG–treated lupus-like (Fig. 1A) and EAE (Fig. 2A) mice. Subsequently, we detected whether BAFF suppressed Foxo1 These data showed a strong correlation with previous studies expression. We used autoantigen dsDNA or MOG35–55 (precoated suggesting that belimumab and TACI-IgG effectively reduce in 96-well plates) plus BAFF to stimulate B cells from lupus-like the number of circulating mature B cells but not memory B cells or EAE mice, respectively. BAFF significantly suppressed Foxo1 (10, 11). expression in dsDNA-stimulated B cells from lupus-like mice Previous studies have shown that the survival signals main- (Fig. 8A) and MOG35–55-stimulated B cells from EAE mice taining memory T and B cells in the absence of Ag are provided (Fig. 8B). To further prove the results, we used different con- by IL-15 (13–15). Our studies demonstrate that blockade of ditions such as LPS or anti-IgM to stimulate B cells from WT BAFF with TACI-IgG upregulates IL-15 levels in B cells but not mice. qPCR analysis showed that BAFF suppressed Foxo1 ex- in other cells, such as T cells (Figs. 1–3). These results suggest pression in LPS (Fig. 9A)- or anti-IgM (Fig. 9B)–stimulated that BAFF inhibitors upregulate IL-15 expression in B cells so B cells from WT mice. Similarly, BAFF also suppressed Foxo1 that they could not control memory B cells. Once the combi- expression in anti-human IgM-stimulated B cells from healthy nation of TACI-IgG and anti–IL-15 Ab was used to treat lupus- donors (Fig. 9C). These results suggest that BAFF suppresses like and EAE mice, anti–IL-15 Ab could effectively reduce memory Foxo1 expression induced by different stimuli. Further Western B cells in lupus-like (Fig. 1A) and EAE (Fig. 2A) mice. IL-15 was blot analysis also showed that BAFF suppressed Foxo1 expression upregulated mainly in the spleen (Figs. 1C–F, 2C–F) but not in LN, in CD19+ B cells (Fig. 8C). Additionally, we found that BAFF which is in accordance with previous publications suggesting that induced Foxo1 phosphorylation (Fig. 8D), which suggests that memory cells reside in the spleen (24, 25). Downloaded from BAFF could also reduce Foxo1 level in the nuclei by inducing IL-15 is an inflammatory cytokine, and disordered IL-15 expres- Foxo1 phosphorylation. Furthermore, blocking BAFF with TACI- sion has been reported in patients with an array of inflammatory IgG induced Foxo1 expression in B cells from lupus-like (Fig. 8E) autoimmune diseases (26–28). Our study showed that IL-15 was and EAE (Fig. 8F) mice. These results altogether suggest that increased in autoimmune diseases and EAE or lupus-like mice BAFF suppresses IL-15 in B cells by downregulating Foxo1 ex- (Supplemental Fig. 1). A series of therapeutic agents that inhibit

pression. IL-15 action have been introduced, including anti–IL-15 neutral- http://www.jimmunol.org/ izing Abs (29). Further studies show that IL-15 was expressed Discussion mainly in T2B cells and BAFF suppressed IL-15 expression in BAFF demonstrates sp. act. toward B cells and supports B cell T2B cells (Fig. 5). These results suggest that BAFF suppresses late proliferation, differentiation, and survival (22). BAFF transgenic immune response (memory B cells) by regulating IL-15 expres- mice harbor increased numbers of B220+ B cells and plasma cells sion when BAFF positively activates earlier stage B cells. The in the spleen and LN; they also develop anti-dsDNA Abs, pro- basic mechanism in the immune response is a self-limited process, teinuria, and glomerulonephritis consistent with a systemic lupus- and, based on this, the possible harmful effects are self-limited like autoimmunity as they age (2–4). By contrast, BAFF knockout (30). CD4+ T cells can also mediate pathologic immune responses, mice have a markedly reduced mature B cell population and de- such that Th1 cells self-limit their inflammatory activity by IFN-g by guest on September 28, 2021 creased serum Ig levels (22, 23). This study demonstrates that (31). When it expands B cell responses, BAFF limits the late BAFF is increased in autoimmune diseases, such as SLE, MS, and response (memory B cells) by suppressing IL-15 expression in lupus-like and EAE animal models (Supplemental Fig. 1). BAFF B cells. is regarded as a potential new therapeutic target in autoimmune TACI-IgG neutralizes both BAFF and APRIL. To determine the diseases (8). These studies certainly suggest that BAFF is a posi- effect of BAFF and APRIL on IL-15 expression in B cells, we tive immune regulator in autoimmune diseases; however, our used autoantigen dsDNA (precoated in 96-well plates) plus BAFF current study shows that BAFF could suppress IL-15 expression in or APRIL to stimulate B cells from lupus-like mice. The results B cells stimulated with autoantigens (Fig. 4) as well as nonspecific suggest that BAFF but not APRIL significantly suppresses IL-15 stimulation reagents (such as LPS and anti-IgM) (Fig. 5), which expression in dsDNA-stimulated B cells from lupus-like mice implies that BAFF has a different role in autoimmune response. (Fig. 4A). Moreover, we found that IL-15 was expressed mainly in Many trials have been done to study the effects of BAFF-specific T2B cells and BAFF suppressed IL-15 expression in these cells inhibition, such as belimumab and TACI-IgG on SLE and other (Fig. 5). Thus, our data suggest that BAFF but not APRIL could autoimmune diseases. Our data demonstrate that TACI-IgG treat- suppress IL-15 expression in earlier B cells, such as T2B cells. ment effectively controls the disease in lupus-like (Supplemental This is in accordance with previous studies suggesting that the Fig. 2) and EAE (Supplemental Fig. 3) mice by reducing the number interaction of BAFF and BAFFR is essential for viability of B cells,

FIGURE 9. BAFF suppressed Foxo1 expression in B cells. Splenic CD19+ B cells were sorted using CD19 beads from naive C57BL/6 mice and then stimulated for 48 h with LPS (A) or anti-IgM (B) with or without BAFF. (C) B cells from the peripheral blood cells of MS patients were sorted using CD19 beads and then stimulated for 48 h with anti-IgM with or without BAFF. (A–C) Cells were subjected to qPCR. The data represent at least four independent experiments (*p , 0.05, **p , 0.01). The Journal of Immunology 4201 whereas APRIL-B cell maturation Ag and APRIL-TACI inter- P. Sańchez-Mateos, E. Martıń-Mola, and M. E. Miranda-Caru´s. 2012. IL-15 expression on RA synovial fibroblasts promotes B cell survival. PLoS One 7: e40620. actions are implicated more in later processes that culminate in Ab 14. Purton, J. F., J. T. Tan, M. P. Rubinstein, D. M. Kim, J. Sprent, and C. D. Surh. 2007. production (32–36). Antiviral CD4+ memory T cells are IL-15 dependent. J. Exp. Med. 204: 951–961. A recent study has shown that Foxo3a expression does not 15. Weng, N. P., K. Liu, M. Catalfamo, Y. Li, and P. A. Henkart. 2002. IL-15 is a growth factor and an activator of CD8 memory T cells. Ann. N. Y. Acad. Sci. 975: 46–56. change in B cell differentiation (37). Foxo1 can directly regulate 16. Stromnes, I. M., and J. M. Goverman. 2006. Active induction of experimental the transcription of RAGs during B cell development (38). Our allergic encephalomyelitis. Nat. Protoc. 1: 1810–1819. study showed that Foxo1 directly regulates the IL-15 gene tran- 17. Butzkueven, H., B. Emery, T. Cipriani, M. P. Marriott, and T. J. Kilpatrick. 2006. Endogenous leukemia inhibitory factor production limits autoimmune demye- scription in T2B cells (Fig. 7). BAFF suppresses memory B cells lination and oligodendrocyte loss. Glia 53: 696–703. by regulating the Foxo1–IL-15 signal pathway (Fig. 7) when it 18. Gresle, M. M., G. Shaw, B. Jarrott, E. N. Alexandrou, A. Friedhuber, T. J. Kilpatrick, and H. Butzkueven. 2008. Validation of a novel biomarker for promoted B cell maturation (Fig. 6D, 6E). These results are consistent acute axonal injury in experimental autoimmune encephalomyelitis. J. Neurosci. with a recent study showing that Foxo1 actively represses effec- Res. 86: 3548–3555. tors or terminal differentiation processes to promote the memory 19. Amadi-Obi, A., C. R. Yu, X. Liu, R. M. Mahdi, G. L. Clarke, R. B. Nussenblatt, + I. Gery, Y. S. Lee, and C. E. Egwuagu. 2007. TH17 cells contribute to uveitis and CD8 T cell development (39). scleritis and are expanded by IL-2 and inhibited by IL-27/STAT1. Nat. Med. 13: In conclusion, BAFF suppressed IL-15 expression in T2B cells 711–718. by downregulating the Foxo1 signaling pathway. Once BAFF was 20. Bossen, C., and P. Schneider. 2006. BAFF, APRIL and their receptors: structure, function and signaling. Semin. Immunol. 18: 263–275. blocked with belimumab or TACI-IgG, Foxo1 and IL-15 levels 21. Waldmann, T. A. 2004. Targeting the interleukin-15/interleukin-15 receptor increased, leading to an increase in the number of memory B cells system in inflammatory autoimmune diseases. Arthritis Res. Ther. 6: 174–177. 22. Gross, J. A., S. R. Dillon, S. Mudri, J. Johnston, A. Littau, R. Roque, M. Rixon, in autoimmune diseases. As long as the immune memory B cells O. Schou, K. P. Foley, H. Haugen, et al. 2001. TACI-Ig neutralizes molecules are maintained, humoral response can in principle be restored critical for B cell development and autoimmune disease: impaired B cell mat- Downloaded from from it (12). If essential components of the reactive memory are uration in mice lacking BLyS. Immunity 15: 289–302. 23. Schiemann, B., J. L. Gommerman, K. Vora, T. G. Cachero, S. Shulga-Morskaya, eliminated through combination therapy targeting memory B cells M. Dobles, E. Frew, and M. L. Scott. 2001. An essential role for BAFF in the (40), patients will become immunologically naive. As a result, it is normal development of B cells through a BCMA-independent pathway. Science necessary to use BAFF inhibitors in combination with anti–IL-15 293: 2111–2114. 24. Bradley, L. M., G. G. Atkins, and S. L. Swain. 1992. Long-term CD4+ memory Abs to treat autoimmune diseases. This provides hints for the T cells from the spleen lack MEL-14, the lymph node homing receptor. J. clinical application of combination of BAFF and IL-15 inhibitor Immunol. 148: 324–331. http://www.jimmunol.org/ 25. Mamani-Matsuda, M., A. Cosma, S. Weller, A. Faili, C. Staib, L. Garçon, in human autoimmune diseases. O. Hermine, O. Beyne-Rauzy, C. Fieschi, J. O. Pers, et al. 2008. The human spleen is a major reservoir for long-lived vaccinia virus-specific memory B cells. Disclosures Blood 111: 4653–4659. 26. Azimi, N., M. Nagai, S. Jacobson, and T. A. Waldmann. 2001. IL-15 plays The authors have no financial conflicts of interest. a major role in the persistence of Tax-specific CD8 cells in HAM/TSP patients. Proc. Natl. Acad. Sci. USA 98: 14559–14564. 27. McInnes, I. B., B. P. Leung, R. D. Sturrock, M. Field, and F. Y. Liew. 1997. References Interleukin-15 mediates T cell-dependent regulation of tumor necrosis factor- 1. Mackay, F., and P. Schneider. 2009. Cracking the BAFF code. Nat. Rev. alpha production in rheumatoid arthritis. Nat. Med. 3: 189–195. Immunol. 9: 491–502. 28. Mention, J. J., M. Ben Ahmed, B. Be`gue, U. Barbe, V. Verkarre, V. Asnafi,

2. Khare, S. D., I. Sarosi, X. Z. Xia, S. McCabe, K. Miner, I. Solovyev, N. Hawkins, J. F. Colombel, P. H. Cugnenc, F. M. Ruemmele, E. McIntyre, et al. 2003. In- by guest on September 28, 2021 M. Kelley, D. Chang, G. Van, et al. 2000. Severe B cell hyperplasia and autoimmune terleukin 15: a key to disrupted intraepithelial lymphocyte homeostasis and disease in TALL-1 transgenic mice. Proc. Natl. Acad. Sci. USA 97: 3370–3375. lymphomagenesis in celiac disease. Gastroenterology 125: 730–745. 3. Gross, J. A., J. Johnston, S. Mudri, R. Enselman, S. R. Dillon, K. Madden, 29. Baslund, B., N. Tvede, B. Danneskiold-Samsoe, P. Larsson, G. Panayi, W. Xu, J. Parrish-Novak, D. Foster, C. Lofton-Day, et al. 2000. TACI and BCMA J. Petersen, L. J. Petersen, F. J. Beurskens, J. Schuurman, J. G. van de Winkel, are receptors for a TNF homologue implicated in B-cell autoimmune disease. et al. 2005. Targeting interleukin-15 in patients with rheumatoid arthritis: Nature 404: 995–999. a proof-of concept study. Arthritis Rheum. 52: 2686-2692. 4. Mackay, F., S. A. Woodcock, P. Lawton, C. Ambrose, M. Baetscher, 30. Schmolke, M., and A. Garcıá-Sastre. 2010. Evasion of innate and adaptive im- P. Schneider, J. Tschopp, and J. L. Browning. 1999. Mice transgenic for BAFF mune responses by influenza A virus. Cell. Microbiol. 12: 873–880. develop lymphocytic disorders along with autoimmune manifestations. J. Exp. 31. Feuerer, M., K. Eulenburg, C. Loddenkemper, A. Hamann, and J. Huehn. 2006. Self- Med. 190: 1697–1710. limitation of Th1-mediated inflammation by IFN-g. J. Immunol. 176: 2857–2863. 5. Petri, M., W. Stohl, W. Chatham, W. J. McCune, M. Chevrier, J. Ryel, V. Recta, 32. Varfolomeev, E., F. Kischkel, F. Martin, D. Seshasayee, H. Wang, D. Lawrence, J. Zhong, and W. Freimuth. 2008. Association of plasma B lymphocyte stimu- C. Olsson, L. Tom, S. Erickson, D. French, et al. 2004. APRIL-deficient mice lator levels and disease activity in systemic lupus erythematosus. Arthritis have normal immune system development. Mol. Cell. Biol. 24: 997–1006. Rheum. 58: 2453–2459. 33. Avery, D. T., S. L. Kalled, J. I. Ellyard, C. Ambrose, S. A. Bixler, M. Thien, 6. Krumbholz, M., D. Theil, T. Derfuss, A. Rosenwald, F. Schrader, R. Brink, F. Mackay, P. D. Hodgkin, and S. G. Tangye. 2003. BAFF selectively enhances the survival of plasmablasts generated from human memory B cells. J. C. M. Monoranu, S. L. Kalled, D. M. Hess, B. Serafini, F. Aloisi, et al. 2005. Clin. Invest. 112: 286–297. BAFF is produced by astrocytes and up-regulated in multiple sclerosis lesions 34. Cancro, M. P., D. P. D’Cruz, and M. A. Khamashta. 2009. The role of and primary central nervous system lymphoma. J. Exp. Med. 201: 195–200. B lymphocyte stimulator (BLyS) in systemic lupus erythematosus. J. Clin. In- 7. Magliozzi, R., S. Columba-Cabezas, B. Serafini, and F. Aloisi. 2004. Intrace- vest. 119: 1066–1073. rebral expression of CXCL13 and BAFF is accompanied by formation of lym- 35. Scholz, J. L., J. E. Crowley, M. M. Tomayko, N. Steinel, P. J. O’Neill, phoid follicle-like structures in the meninges of mice with relapsing W. J. Quinn, III, R. Goenka, J. P. Miller, Y. H. Cho, V. Long, et al. 2008. BLyS experimental autoimmune encephalomyelitis. J. Neuroimmunol. 148: 11–23. inhibition eliminates primary B cells but leaves natural and acquired humoral 8. Vincent, F. B., E. F. Morand, and F. Mackay. 2012. BAFF and innate immunity: immunity intact. Proc. Natl. Acad. Sci. USA 105: 15517–15522. new therapeutic targets for systemic lupus erythematosus. Immunol. Cell Biol. 36. O’Connor, B. P., V. S. Raman, L. D. Erickson, W. J. Cook, L. K. Weaver, 90: 293–303. C. Ahonen, L. L. Lin, G. T. Mantchev, R. J. Bram, and R. J. Noelle. 2004. 9. Stohl, W., F. Hiepe, K. M. Latinis, M. Thomas, M. A. Scheinberg, A. Clarke, BCMA is essential for the survival of long-lived bone marrow plasma cells. J. C. Aranow, F. R. Wellborne, C. Abud-Mendoza, D. R. Hough, et al; BLISS-52 Exp. Med. 199: 91–98. Study Group; BLISS-76 Study Group. 2012. Belimumab reduces autoantibodies, 37. Rumfelt, L. L., Y. Zhou, B. M. Rowley, S. A. Shinton, and R. R. Hardy. 2006. normalizes low complement levels, and reduces select B cell populations in patients Lineage specification and plasticity in CD192 early B cell precursors. J. Exp. with systemic lupus erythematosus. Arthritis Rheum. 64: 2328–2337. Med. 203: 675–687. 10. Nestorov, I., A. Munafo, O. Papasouliotis, and J. Visich. 2008. Pharmacokinetics 38. Amin, R. H., and M. S. Schlissel. 2008. Foxo1 directly regulates the transcription and biological activity of atacicept in patients with rheumatoid arthritis. J. Clin. of recombination-activating genes during B cell development. Nat. Immunol. 9: Pharmacol. 48: 406–417. 613–622. 11. Carbonatto, M., P. Yu, M. Bertolino, E. Vigna, S. Steidler, L. Fava, C. Daghero, 39. Rao, R. R., Q. Li, M. R. Gubbels Bupp, and P. A. Shrikant. 2012. Transcription B. Roattino, M. Onidi, M. Ardizzone, et al. 2008. Nonclinical safety, pharma- factor Foxo1 represses T-bet-mediated effector functions and promotes memory cokinetics, and pharmacodynamics of atacicept. Toxicol. Sci. 105: 200–210. CD8(+) T cell differentiation. Immunity 36: 374–387. 12. Yoshida, T., H. Mei, T. Do¨rner, F. Hiepe, A. Radbruch, S. Fillatreau, and B. F. Hoyer. 40. DiLillo, D. J., Y. Hamaguchi, Y. Ueda, K. Yang, J. Uchida, K. M. Haas, 2010. Memory B and memory plasma cells. Immunol. Rev. 237: 117–139. G. Kelsoe, and T. F. Tedder. 2008. Maintenance of long-lived plasma cells and 13. Benito-Miguel, M., Y. Garcıá-Carmona, A. Balsa, M. B. Bautista-Caro, serological memory despite mature and memory B cell depletion during CD20 I. Arroyo-Villa, T. Cobo-Ibań˜ez, M. G. Bonilla-Hernań, C. Pe´rez de Ayala, immunotherapy in mice. J. Immunol. 180: 361–371.