BAFF)-R Is the Principal BAFF Receptor Facilitating BAFF Costimulation of Circulating T and B Cells This Information Is Current As of September 23, 2021

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B Cell-Activating Factor Belonging to the TNF Family (BAFF)-R Is the Principal BAFF Receptor Facilitating BAFF Costimulation of Circulating T and B Cells This information is current as of September 23, 2021. Lai Guan Ng, Andrew P. R. Sutherland, Rebecca Newton, Fang Qian, Teresa G. Cachero, Martin L. Scott, Jeffrey S. Thompson, Julie Wheway, Tatyana Chtanova, Joanna Groom, Ian J. Sutton, Cynthia Xin, Stuart G. Tangye, Susan L. Kalled, Fabienne Mackay and Charles R. Mackay Downloaded from J Immunol 2004; 173:807-817; ; doi: 10.4049/jimmunol.173.2.807 http://www.jimmunol.org/content/173/2/807 http://www.jimmunol.org/

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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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

B Cell-Activating Factor Belonging to the TNF Family (BAFF)-R Is the Principal BAFF Receptor Facilitating BAFF Costimulation of Circulating T and B Cells1

Lai Guan Ng,2* Andrew P. R. Sutherland,2*† Rebecca Newton,* Fang Qian,§ Teresa G. Cachero,§ Martin L. Scott,§ Jeffrey S. Thompson,§ Julie Wheway,* Tatyana Chtanova,*† Joanna Groom,* Ian J. Sutton,* Cynthia Xin,*† Stuart G. Tangye,‡ Susan L. Kalled,§ Fabienne Mackay,*† and Charles R. Mackay3*†

BAFF (B cell-activating factor belonging to the TNF family) is a cell survival and maturation factor for B cells, and overproduction of BAFF is associated with systemic autoimmune disease. BAFF binds to three receptors, BAFF-R, transmembrane activator and Downloaded from calcium modulator and cyclophilin ligand interactor (TACI), and B cell maturation Ag (BCMA). Using speciﬁc mAbs, BAFF-R was found to be the predominant BAFF receptor expressed on peripheral B cells, in both humans and mice, and antagonist mAbs to BAFF-R blocked BAFF-mediated costimulation of anti-␮ responses. The other BAFF receptors showed a much more restricted expression pattern, suggestive of specialized roles. BCMA was expressed by germinal center B cells, while TACI was expressed predominantly by splenic transitional type 2 and marginal zone B cells, as well as activated B cells, but was notably absent from germinal center B cells. BAFF was also an effective costimulator for T cells, and this costimulation occurs entirely through http://www.jimmunol.org/ BAFF-R. BAFF-R, but not TACI or BCMA, was expressed on activated/memory subsets of T cells, and T cells from BAFF-R mutant A/WySnJ mice failed to respond to BAFF costimulation. Thus, BAFF-R is important not only for splenic B cell maturation, but is the major mediator of BAFF-dependent costimulatory responses in peripheral B and T cells. The Journal of Immunology, 2004, 173: 807–817.

he B cell-activating factor from the TNF family (BAFF)4 and salivary glands of Sjo¬gren’s syndrome patients (7). These el- (also known as BLyS, TALL-1, zTNF-4, THANK, evated levels of BAFF in blood and tissues in human autoimmune TNFSF 13b), is emerging as an important regulator of B patients likely affect peripheral B cell survival as well as B cell

T by guest on September 23, 2021 cell and T cell responses. BAFF was originally identified as a maturation and activation in the spleen, similar to effects of BAFF factor responsible for B cell survival and maturation (reviewed in in BAFF transgenic mice. Although high levels of BAFF have Refs. 1Ð3). BAFF was subsequently associated with autoimmune been associated mostly with autoimmune diseases, BAFF is likely disease, because transgenic mice overproducing BAFF produce important for normal immune responses to microbial challenge, by autoantibodies and develop diseases akin to human systemic lupus priming or enhancing T and B cell activity to facilitate clearance erythematosus and Sjo¬gren’s syndrome (4Ð8). In humans, high (11). The baff gene, located in humans on chromosome 13, has levels of BAFF are detectable in the blood of a proportion of been identified as a likely determinant for susceptibility to Ascaris patients with autoimmune rheumatic diseases, particularly sys- infection (12). temic lupus erythematosus and Sjo¬gren’s syndrome (7, 9, 10), and BAFF binds to several receptors. These include transmembrane BAFF is present at high levels in rheumatoid synovial fluid (9), activator and calcium modulator and cyclophilin ligand interactor (TACI), BAFF-R (BR3), and B cell maturation Ag (BCMA) (13Ð 17). Another TNF family member termed a proliferation-inducing *Arthritis and Asthma Research Program, The Garvan Institute of Medical Research, Sydney, †Cooperative Research Center for Asthma, Sydney, ‡Centenary Institute of ligand (APRIL) also binds to TACI and BCMA (14, 18). BAFF-R Cancer Medicine and Cell Biology, Newtown, New South Wales, Australia; and appears to be particularly important for the regulation of B cell ¤Department of Research, Biogen Idec Inc., Cambridge, MA 02142 survival and maturation in the spleen, because A/WySnJ mice ex- Received for publication March 15, 2004. Accepted for publication May 14, 2004. pressing a defective BAFF-R have disrupted B cell maturation, The costs of publication of this article were defrayed in part by the payment of page similar to that seen in BAFF-deficient mice (17, 19). The BCMA charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. receptor appears not to play a role in B cell maturation but is 1 This work was supported by the Wellcome Trust, the Cooperative Research Center involved in plasma cell survival (20). In TACI-deficient mice, for Asthma, and the National Heath and Medical Research Council of Australia. there are increased B cell numbers, marked splenomegaly, and 2 L.G.N. and A.S. contributed equally to this work. mice develop autoimmune disorders (21). Antigenic challenge in 3 Address correspondence and reprint requests to Dr. Charles R. Mackay, Arthritis these mice results in enhanced Ab production (21). Because an and Inflammation Research Program, The Garvan Institute of Medical Research, 384 agonistic mAb to TACI inhibited B cell proliferation, signaling Victoria Street, Sydney, New South Wales 2010, Australia. E-mail address: [email protected] through TACI may in fact serve to down-regulate B cell activity (21). 4 Abbreviations used in this paper: BAFF, B cell-activating factor belonging to the TNF family; BCMA, B cell maturation Ag; h., human; hIgG, human IgG; GC, ger- BAFF and possibly APRIL also act on T cells. In vitro studies minal center; m., mouse; MZ, marginal zone; RBL, rat basophilic leukemia; TACI, of human T cells showed that BAFF provided a complete costimu- transmembrane activator and calcium modulator and cyclophilin ligand interactor; T1, transitional type 1; T2, transitional type 2; WT, wild type; APRIL, a proliferation- latory signal together with anti-TCR stimulation (22, 23). In mice, inducing ligand. a TACI-Fc fusion protein blocked activation of T cells in vitro, and

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 808 BAFF-R IS THE PRINCIPAL BAFF RECEPTOR FOR B AND T CELLS inhibited T cell priming in vivo (24). Also, treatment with hIgG (Novartis Pharmaceuticals, East Hanover, NJ), h.BCMA-Fc, or anti- TACI-Fc substantially inhibited inflammation, as well as bone and mouse BAFF-R (m.BAFF-R) mAbs (29). For Ag-specific T cell assays, cartilage destruction, in a mouse model of rheumatoid arthritis splenic T cells from DO11.10 TCR transgenic mice were purified by magnetic separation, and 5 ϫ 104 cells/well were cultured with 1 ϫ 105 cells/ (24). T cells from transgenic mice that overexpressed human well of mitomycin C-inactivated APC, together with OVA peptide in 96- APRIL showed greatly enhanced survival in vitro and in vivo (25). well U-bottom plates. BAFF costimulation of T cells was performed by In autoimmune patients, inappropriate costimulation of T and B adding 4 ␮g/ml soluble BAFF or denatured BAFF to wells. For inhibition cells by BAFF may be an important component of disease patho- assays, cells were cultured with anti-m.BAFF-R mAbs (2 ␮g/ml), h.B- CMA-Fc, or hIgG (2 ␮g/ml). genesis. The receptor for BAFF on T cells is largely unknown, For anti-CD3 T cell proliferation assay, human T cells were activated although one study reported TACI expression on a subset of acti- using plate-bound anti-human CD3 mAb (mAb TR66), and mouse T cells vated T cells using a polyclonal Ab (26). Expression of BCMA and were activated with anti-mouse-CD3 mAb 145-2C11 (BD Pharmingen). BAFF-R is thought to be restricted to B cells (16, 17, 27, 28). Anti-CD3 mAb and BAFF were coated onto plates overnight in PBS at 4¡C either separately or in combination, followed by two PBS washes. A total The critical functions of BAFF for B and T cell biology are ϩ of 2 ϫ 106 PBMC (human) or CD3 (human or mouse) cells/ml were facilitated by the regulated expression of BAFF receptors. Accord- added to tissue culture plates containing immobilized anti-CD3 Ϯ plate- ingly, we have investigated the capacity of BAFF to stimulate B bound or soluble BAFF, and harvested after 72 h. For all mouse and human and T cells, and have used specific antagonistic mAbs as well as proliferation measurements, cultures were pulsed with [3H]thymidine (1 strains of mice with mutant BAFF receptors to ascertain precise ␮Ci/well) 18 h before harvesting and quantified using a beta-scintillation counter. expression patterns and functional roles for the three receptors.

Materials and Methods Production and specificity of mAbs to human and mouse BAFF Downloaded from Reagents and flow cytometry receptors Soluble forms of human BAFF and BAFF receptors were supplied by An expression DNA construct containing the human TACI cDNA se- Apotech (Epalinges, Switzerland) and P. Schneider (Institute of Biochem- quence was kindly provided by P. Schneider, and a human BAFF-R ex- istry, BIL Biomedical Research Center, University of Lausanne, Epalinges, pression construct was developed using the p-Tracer vector, as per man- Switzerland). Denatured BAFF controls were prepared by incubation at ufacturer’s protocol (Invitrogen, Mt. Waverley, Australia). Cell surface 95¡C for 2 h. Unconjugated and FITC-, PE-, CyChrome-, PerCP-, PE- receptor-expressing transfectants were made using the rat basophilic leu- Cy7-, allophycocyanin-Cy7- and allophycocyanin-conjugated mAbs to kemia (RBL) mast cell line (human and mouse TACI, mouse BAFF-R), or http://www.jimmunol.org/ various cell surface markers were from BD Biosciences (San Diego, CA), mouse B cell lymphoma L1.2 cells (human BAFF-R). Anti-h.TACI mAb ϫ 7 with the exception of CCR7-FITC (R&D Systems, Minneapolis, MN). Im- 1A1 (rat IgG1) was generated by immunizing Wistar rats with 2 10 munofluorescent staining was performed using standard procedures with irradiated TACI-transfected RBL cells, 6 times at 2-wk intervals. Cell fu- appropriate secondary staining reagents (Jackson ImmunoResearch Labo- sion was performed as described (31). Similarly, anti-h.BAFF-R mAb ratories, West Grove, PA), and cells were analyzed using BD FACSCalibur 11C1 (mouse IgG1) was generated using the same procedures except that or LSRII flow cytometers (BD Biosciences). Six-color flow cytometric BAFF-R-L1.2 transfectants were used to immunize C57BL/6 mice. In ad- analysis to assess BAFF-R expression on naive and memory subsets of dition, anti-h.BAFF-R (clone 9-1) and hamster anti-human BCMA (clone CD4ϩ and CD8ϩ cells used anti-human BAFF-R (h.BAFF-R) biotin (mAb C4E2.2) mAbs were used. These were generated by immunizing with re- 9-1) and streptavidin-PE, anti-CCR7-FITC, anti-CD3-PerCP, anti-CD45RO- ceptor-Ig fusion proteins, as detailed previously (28). The rat anti-mouse TACI mAbs, 8F10 (IgG2a) and 5L7 (IgG1), were generated by immuniz- allophycocyanin, anti-CD4-PE-Cy7, and anti-CD8-allophycocyanin-Cy7. A by guest on September 23, 2021 biotinylated mIgG1 (BD Pharmingen, San Diego, CA) was used as an isotype ing Wistar rats with soluble TACI extracellular domain protein, 6 times at control for the BAFF-R Ab. 2-wk intervals. Anti-m.BAFF-R mAbs B2G1 and P1B8 were produced by immunizing hamsters with murine BAFF-R-Fc protein. Rat anti- Animals, lymphocyte preparations, and T and B cell stimulations m.BAFF-R mAbs 7H22-E16 and 3I4 (both IgG1) were produced by immunizing Wistar rats with soluble m.BAFF-R-Fc protein, 5 times at 2-wk All human and mouse experiments were performed with approval of St. intervals. Vincent’s campus human or animal ethics committees. Human PBMCs For immunohistochemical visualization of BAFF-R expression, Ag re- were isolated from human blood by Ficoll gradient centrifugation. Human trieval was performed by immersing 4-␮M-thick paraffin sections of pal- splenocytes or tonsil cells were obtained from resected human spleen or ϩ atine tonsil in an EDTA-based retrieval solution (pH 9.0) and heating for tonsil, and were prepared by gentle teasing with forceps. Human CD3 T 20 min at 95Ð99¡C in a water bath. After cooling, sections were immuno- cells were isolated from PBMC preparations by magnetic separation stained using a DakoCytomation Autostainer (DakoCytomation, Carpinte- (MACS; Miltenyi Biotec, Sydney, Australia). CD3ϩ cells were then iso- Ͼ ria, CA); following 5 min incubation with 3% hydrogen peroxide, sections lated in a magnetic field, to 98% purity. For human B cell stimulation, were incubated sequentially for 30 min with anti-BAFF-R mAb (11C1) and 5 ␮ PBLs were incubated in 96-well plates (10 cells/well in 100 l RPMI Mouse EnVisionϩ HRP (DakoCytomation). Anti-BAFF-R binding was 1640 supplemented with 10% FBS) for 48 h with 75 ng/ml soluble BAFF ϩ ␮ Ј ␮ visualized using Liquid diaminobenzidine (DakoCytomation). in the presence of 5 g/ml goat F(ab )2 anti-human -chain Ab (Jackson ImmunoResearch Laboratories), and with different concentrations of anti- h.BAFF-R mAb, h.BAFF-R-Fc, or human IgG (hIgG) control (29). GeneChip microarray analysis (Affymetrix, Millenium Science, Homozygous TACIϪ/Ϫ mice were generated as previously described for Victoria, Australia), and real-time LightCycler PCR (Roche targeting the BAFF and BCMA loci (30). Briefly, a tailless human CD2 Molecular Biochemicals, Sydney, Australia) reporter cDNA was inserted in frame at the ATG that normally initiates TACI translation and just upstream of a phosphoglycerate kinase 1-pro- Total RNA was isolated from harvested cells using Qiagen RNeasy Total moted neomycin resistance cassette. The resulting deletion of the TACI RNA Isolation kit (Valencia, CA). Total RNA (2 ␮g) was then used for genomic locus spanned 6.12 kb of DNA immediately downstream of the cDNA synthesis, with 4.5 U AMV reverse transcriptase and MgCl2-con- initiating ATG. This mutation introduced premature stop codons in the taining buffer (Promega, Madison, WI), 20 nmol dNTPs (Promega), and three possible reading frames downstream of the CD2 reporter and elim- 0.02 nmol oligo-p(dT)15 primer (Roche Molecular Biochemicals) and in- inated all the nucleotides encoding aa 1Ð89 of normal mouse TACI (Gen- cubated at 42¡C for 90 min. cDNA was used for LightCycler PCR (Roche Bank accession number NM_021349). The A/WySnJ mouse strain (17) Molecular Biochemicals) with the LightCycler FastStart Master SYBR was obtained from The Jackson Laboratory (Bar Harbor, ME). Mouse Green I kit (Roche Molecular Biochemicals), using 3 mM MgCl2 and 0.5 CD3ϩ cells were isolated from spleen via magnetic separation using Pan T ␮M individual primers with the following specific protocol: 10 min 95¡C cell isolation kit (MACS; Miltenyi Biotec). For mouse B cell proliferation activation of FastStart TaqDNA polymerase (Roche Molecular Biochemi- assays, B cells were isolated from spleens of 2-mo old C57BL/6 mice using cals), and 40 cycles of 95¡C for 15 s, 63¡C for 5 s, and 72¡C for 21 s. The B cell recovery columns (Accurate Chemical & Scientific, Westbury, NY). primers used were a combination of original sequences designed using Prim- Mouse B cells were incubated in 96-well plates (105 cells/well in 50 ␮l er3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). Light- RPMI 1640 supplemented with 10% FBS) for 72 h with 75 ng/ml BAFF Cycler analyses used the crossing point data for each gene during the loga- (Biogen Idec, Cambridge, MA), in the presence of 2 ␮g/ml goat anti-mouse rithmic amplification program. The crossing point for each gene in each ␮-chain Ab (Jackson ImmunoResearch Laboratories), and with the indi- sample was normalized to the crossing point of GAPDH. Respective genes cated concentrations of previously described reagents, including polyclonal were then compared between two samples and expressed as a fold change. The Journal of Immunology 809

For GeneChip assessment of transcript expression, cRNA was prepare- Results dusing the methods as described (32). Hybridization to the Affymetrix Production of mAbs to human and mouse BAFF receptors U133A and B GeneChips and subsequent scanning and analysis was con- ducted exactly according to Affymetrix protocols. GeneChip analyses in- mAbs were raised to human TACI, BAFF-R, and BCMA, and to cluded human Th1 and Th2, purified eosinophils, cultured mast cells, IgE- activated mast cells, purified neutrophils, and splenic memory B cells. A full mouse TACI and BAFF-R, to determine the functional signifi- description of the preparation of these cells, and access to the full GeneChip cance of these three receptors for B and T cells, and to determine results, is available at http://www.garvan.unsw.edu.au/public/microarrays. their precise expression pattern. Various immunization strategies Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 1. Production and specificity of mAbs to human and mouse BAFF receptors. A, Reactivity of various mAbs against transfectants of human TACI and BAFF-R. The different shaded profiles represent different BAFF-R transfectants; lighter profiles are human BAFF-R transfectants (in mouse L1.2 cells, a B lymphoma line), and darker profiles are human TACI transfectants (in rat RBL cells). Negative control staining used mAbs of irrelevant specificity, and anti-h.TACI or anti-h.BAFF-R staining on irrelevant transfectants resembled this staining (data not shown). B, Mouse TACI and BAFF-R transfectants (both in rat RBL cells); lighter profiles are m.BAFF-R transfectants and darker profiles are m.TACI transfectants. In all instances, Ab staining of nontransfected cells resembled negative control staining. C, ELISA of anti-human BAFF-R, TACI, and BCMA mAbs against human TACI-Fc, BAFF-R-Fc, or BCMA-Fc fusion proteins. The mAbs used were anti-h.TACI mAb 1A1, anti-h.BCMA mAb C4E2.2, and anti-h.BAFF-R mAb 9-1. Values are shown as relative absorbance after subtraction of background values. 810 BAFF-R IS THE PRINCIPAL BAFF RECEPTOR FOR B AND T CELLS were used, including receptor fusion proteins and transfectants ex- BAFF-R blocked proliferation to the same level as did the positive pressing high levels of these receptors. All of the ensuing mAb control BCMA-Fc (Fig. 2B), indicating that in mice, BAFF-R is raised against either human or mouse BAFF-R or TACI stained the principal costimulatory BAFF receptor for resting mature B their respective receptor transfectants (Fig. 1, A and B) or reacted cells. specifically against receptor-Ig fusion proteins by ELISA (Fig. TACI expression was detected on T cells using a polyclonal Ab 1C), without cross-reacting with the other BAFF receptors. The (26), however, a kinetic analysis of TACI expression on T cells specificity of a mAb raised against human BCMA-Fc, C4E2.2, has failed to detect TACI expression on 48-h-activated T cells (Fig. been reported previously (28). Anti-human BAFF-R mAb 9-1 2C), or on resting T cells or T cells at other activation time points blocked BAFF binding to BAFF-R expressing cells (data not (data not shown). However, TACI was expressed on a subset of shown). Similarly, anti-murine BAFF-R mAb B2G1 was blocking, ϩ peripheral B220 B cells, and, as expected, mAb to TACI was while mAb P1B8 was nonblocking (see below). Ϫ Ϫ unreactive with all B cell subsets from TACI / mice. The most BAFF-R is the predominant BAFF receptor expressed on mouse notable expression of TACI was by maturing subsets of splenic B T and B cells, whereas TACI marks maturing splenic B cell cells. Transitional type 1 (T1), transitional type 2 (T2), and mar- subsets ginal zone (MZ) B cells were distinguished using multicolor flow mAb to mouse BAFF-R stained the vast majority of mouse splenic cytometry using IgM, CD21, and CD23 (see Ref. 33). In wild-type B cells (CD4-negative cells, Fig. 2A) and lymph node B cells (data (WT) mice, T2 and MZ B cells expressed very high levels of TACI not shown). A few resting T cells were clearly BAFF-Rϩ (Fig. 2A) (Ͼ95% positive, Fig. 2D), whereas on other splenic B cell subsets and PCR analysis of mouse T cells and T cell lines revealed ex- such as follicular B cells and T1 B cells, TACI expression was low Downloaded from pression of BAFF-R and absence of TACI and BCMA (data not or absent, respectively. However, TACI expression by T2 or MZ shown). In experiments using BAFF costimulation of anti- B cells was not essential for B cell maturation in the spleen, be- ␮-mediated B cell proliferation, an antagonistic mAb to mouse cause all B cell subsets developed in TACIϪ/Ϫ mice, although http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. BAFF-R is the principal BAFF receptor for mouse B cells, and is expressed on a subset of T cells while TACI is expressed on T2 and MZ B cells. A, Expression of BAFF-R (mAb P1B8) by the vast majority of splenocytes (comprising mostly B cells), and also by a small subset of CD4ϩ T cells. B, Inhibition of BAFF costimulatory effects on anti-␮-mediated (2 ␮g/ml) B cell proliferation by anti-m.BAFF-R mAb B2G1. hIgG as well as the nonblocking mAb, P1B8, were used as negative controls, and BCMA-Fc was used as a positive BAFF neutralizing agent. Each point was done in triplicate and the experiment was performed three times with identical results. C, TACI is expressed on a small subset of B cells in the spleen, but is absent from T cells. The left panels shows TACI expression on a subset of B220ϩ splenocytes from WT but not TACIϪ/Ϫ mice. The right panels shows activated splenic CD4ϩ T cells, either from WT or TACIϪ/Ϫ mice, stained with anti-m.TACI mAb 8F10 (y-axis) and with anti-CD69 mAb (x-axis). Activated CD4ϩ T cells were produced following T cell stimulation in vitro with anti-CD3 (4 ␮g/ml, optimal concentration) and anti-CD28 (2 ␮g/ml) for 48 h. D, On murine splenic B cells, TACI is expressed highly by T2 and MZ B cells. Mature B cells, T1, T2, and MZ phenotypes were identified using multicolor flow cytometry by staining for IgM, CD21, and CD23, as described (33). Control mAb staining resembled TACIϪ/Ϫ mouse splenocyte staining (dotted line). The Journal of Immunology 811 these mice displayed splenomegaly and peripheral B cell hyper- least in part, through the up-regulation of the survival factor Bcl-2 plasia, as described for other strains of TACIϪ/Ϫ mice (21, 34, 35). (33, 37). Bound BAFF, but not denatured BAFF, also resulted in When splenic B cells were stimulated with anti-␮ for 24 h, TACI an up-regulation of Bcl-2 in purified mouse T cells suboptimally was strongly up-regulated (36) and the expression profile resem- stimulated for 72 h with anti-CD3 (Fig. 3C). bled that shown for MZ or T2 B cells. To confirm that BAFF-R was facilitating BAFF-mediated effects on T cells, we studied BAFF costimulation of anti- BAFF costimulation of T cells in mice is mediated by BAFF-R CD3-mediated T cell proliferation using two mutant strains of We next assessed BAFF responses by mouse T cells, using an mice, TACI-deficient mice, and A/WySnJ mice with a defective OVA peptide-specific proliferation by DO11.10 TCR transgenic BAFF-R (17, 19). Fig. 3D shows that the costimulatory effect of mouse T cells. Fig. 3A shows that an antagonistic mAb recogniz- BAFF on purified T cells was abolished in A/WySnJ mice, in ing murine BAFF-R (mAb B2G1) inhibited T cell proliferation by contrast to T cells from WT mice of the same genetic background ϳ20%, indicating that endogenous BAFF participates in T cell expressing a functional BAFF-R, which responded to BAFF co- costimulation, for an optimal response (Fig. 3A), as previously stimulation. T cells from A/WySnJ mice were costimulated by shown by others (23). As a control, BCMA-Fc inhibited prolifer- anti-CD28, similar to T cells from control A/J mice (Fig. 3D) ation to the same extent. Furthermore, addition of exogenous sol- indicating that T cells from A/WySnJ mice were competent and uble BAFF also increased T cell proliferation in the presence of responded normally. Therefore, the defect in the ability to respond APC (Fig. 3B). The effects of BAFF on B cells in mice occur, at to BAFF costimulation was due to a lack of functional BAFF-R Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. BAFF costimulates mouse T cells through BAFF-R. A, Inhibition of BAFF costimulation by h.BCMA-Fc and anti-m.BAFF-R blocking mAb B2G1. Purified DO11.10 T cells were stimulated with 0.05 ␮g/ml OVA peptide, together with 2 ␮g/ml anti-m.BAFF-R mAbs (P1B8, nonblocking; B2G1, blocking), or 2 ␮g/ml h.BCMA-Fc. B, Soluble BAFF (BAFF), but not denatured BAFF (DN BAFF), costimulates Ag-specific T cell proliferation at suboptimal concentrations of OVA peptide (0.01 ␮g/ml). BAFF was used at 4 ␮g/ml. C, Up-regulation of Bcl-2 in 72 h anti-CD3-stimulated, purified splenic T cells upon incubation with bound BAFF (B BAFF). Shaded and dotted profiles are similarly treated T cells, stimulated with denatured BAFF or no BAFF. D, T cell costimulation by BAFF is compromised in BAFF-R mutant mice (strain A/WySnJ). T cell costimulation by BAFF of purified T cells from the relevant A/J control background (f), and lack of costimulation of A/WySnJ T cells (Ⅺ). T cells obtained from A/WySnJ mice responded well to anti-CD28 (2 ␮g/ml) costimulation. These T cells were purified from spleens by magnetic sorting, and cultures were activated with 1 ␮g/ml plate-bound anti-CD3, Ϯ an optimal amount of plate-bound BAFF (4 ␮g/ml). E, T cells form TACIϪ/Ϫ mice, stimulated with anti-CD3 Ϯ BAFF (Ⅺ), compared with T cells from control littermates (f) (mixed B6/129 background). 812 BAFF-R IS THE PRINCIPAL BAFF RECEPTOR FOR B AND T CELLS expression, rather than an intrinsic defect in T cell function. In express low levels of TACI (Fig. 4A); however, TACI is known to addition, others have reported that APC function and T cell pro- repress B cell activation, and TACI is dispensable for BAFF stim- liferation in A/WySnJ mice is normal (38). It is noteworthy that ulation of B cell responses to anti-␮ (21). proliferation of T cells from A/J mice, without addition of exogenous BAFF, was consistently higher than for A/WySnJ mice, sug- B cells with a germinal center (GC) phenotype show altered gesting that activated T cells may be a source of BAFF (see mi- expression of BAFF receptors croarray data below). In contrast, T cells from TACI-deficient The staining of human tonsil cells with mAbs to BAFF-R, BCMA, mice were unaffected in their response to BAFF costimulation and TACI revealed that BAFF-R was also the predominant recep- (Fig. 3E), indicating that BAFF does not costimulate T cells tor expressed on tonsil B cells. Nevertheless, BAFF-R did show a through this receptor. Similar to the previous study (22), we also variation in staining intensity between different B cell subsets (Fig. found that anti-CD3-stimulated T cells responded to BAFF only 5A). B cells with a GC phenotype (CD38ϩ, CD27ϩ, CD39Ϫ, when it was immobilized to plastic, although soluble BAFF was CD24Ϫ, and IgMϪ) expressed lower levels of BAFF-R, and this able to costimulate T cells in the Ag-specific assay in the presence was clearly evident through immunohistochemical staining of B of APCs (Fig. 3B). cell follicles in tonsil (Fig. 5B). In contrast to the blood, where no B cells expressed BCMA, a proportion of tonsil B cells did express BAFF-R is the predominant BAFF receptor expressed on human low levels, and multicolor flow cytometry revealed that these blood B cells BCMAϩ B cells displayed a phenotype consistent with GC B cells The studies described above established BAFF-R as the predom- (Fig. 5A). Strikingly, TACI and BCMA were expressed on differ- ϩ ϩ inant BAFF receptor for mouse B and T cells. The expression ent subsets of the CD19 B cell population, with the TACI sub- Downloaded from pattern of BAFF-R, BCMA, and TACI was also assessed on subset being CD38Ϫ, CD27Ϫ, CD39ϩ, CD24ϩ, and IgMϩ (i.e., a sets of blood and tonsil B cells in humans. On blood B cells, non-GC phenotype). The distinct difference between the TACIϩ BAFF-R was expressed at a high level on all CD19ϩ B cells, and BCMAϩ subsets was further illustrated by a direct two-color whereas BCMA was absent (Fig. 4A). TACI was expressed, but analysis (Fig. 5C), which showed that the vast majority of only on a proportion of blood B cells, and at a much lower level BCMAϩ B cells were TACIϪ; this was particularly evident when than BAFF-R. We confirmed that BAFF-R was also the principal B cell blasts were gated and analyzed (Fig. 5C). http://www.jimmunol.org/ stimulatory receptor for human blood B cells, by inhibition of BAFF costimulation of anti-␮-treated blood B cells using a block- Expression of BAFF-R by human T cells ing mAb to BAFF-R. Fig. 4B shows that anti-BAFF-R mAb 9-1, BAFF has been reported to costimulate T cell responses in humans which interferes with binding of BAFF to BAFF-R, inhibited (22), as well as in mice (see above). However, mAb staining sug- BAFF costimulation of B cells. A proportion of these B cells did gested that TACI and BCMA were absent from human blood T by guest on September 23, 2021

FIGURE 4. BAFF-R is the predominant BAFF receptor expressed on human blood B cells, and facili- tates the vast majority of BAFF costimulation of anti-␮ proliferation of B cells. A, Two-color expression analysis of BAFF-R, TACI, and BCMA on CD19ϩ B cells from human blood. Cells were stained with CD19 Ab and respective biotinylated BAFF receptor mAbs followed by streptavidin-PE. B, Inhibition of BAFF costimulatory effects on anti- ␮-mediated (5 ␮g/ml) B cell proliferation by anti-h.BAFF-R mAb 9-1 (2 ␮g/ml) in the presence of 75 ng/ml BAFF. Anti-h.BAFF-R mAb (F) vs hIgG (E). h.BAFF-R Fc was used as a positive BAFF-neutralizing agent (Œ) The Journal of Immunology 813 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 5. B cells regulate BAFF receptors upon differentiation to GC phenotype. A, Three-color FACS analysis of B cells (performed by gating on CD19ϩ B cells from tonsil) showing expression of BAFF-R, BCMA, and TACI in relation to the B cell phenotypic markers CD38, CD39, CD24, CD27, and IgM. BAFF-R expression was down-regulated on B cells with a phenotype of GC B cells (CD38ϩ, CD27ϩ, CD39Ϫ, and IgMϪ), whereas BCMA was expressed on these cells. B, Immunohistochemical staining of human tonsil with anti-h.BAFF-R mAb 11C1, showing intense staining of B cell follicles, and weaker staining of GC (magnification, ϫ200). Anti-h.TACI and anti-h.BCMA mAbs failed to work in immunohistochemistry, presumably because epitopes were not retained by the fixation process. C, Two-color analysis of TACI and BCMA on gated CD19ϩ B cells from tonsil. The left panel shows the entire CD19ϩ B cell gate, and right panel shows CD19ϩ B cell blasts, gated according to high forward and side scatter. The mAbs used were 9-1 (anti-h.BAFF-R), 1A1 (anti-h.TACI), and C4E2.2 (anti-h.BCMA). cells, and that BAFF-R was expressed at very low levels (Fig. 6A). Affymetrix data-mining tool to assess the presence of BCMA and To further define the precise expression pattern of the receptors, TACI transcripts in numerous T cell subsets (BAFF-R was not particularly BAFF-R, a sensitive multicolor flow cytometric anal- represented on the Affymetrix human U133 chips). TACI and ysis was performed using a BD LSRII or FACSCalibur. All human BCMA were absent from all human T cell subsets assessed, in- T cell populations examined, including blood T cells, in vitro anti- cluding effector memory and central memory T cells, Th1 and Th2 CD3-activated T cells (24 and 48 h, data not shown), and tonsillar cells generated in vitro, and specialized subsets such as T follic- T cells were found to be consistently negative for BCMA and ular-homing cells (Fig. 6D). The GeneChip results also showed TACI (Fig. 6A, and data not shown). In contrast, a fraction of T that TACI and BCMA were undetectable in all nonlymphoid leu- cells expressed BAFF-R, especially T cells activated for 72 h with kocyte types, such as mast cells, neutrophils, and eosinophils, and anti-CD3 (Fig. 6A). This led us to examine expression of BAFF-R staining by flow cytometry with the specific mAbs confirmed these on naive, central memory, and effector memory CD4ϩ and CD8ϩ results (data not shown). BCMA and TACI were detectable in cells as defined using the markers CCR7 and CD45RO. Expression certain B cell populations, particularly memory cell subsets (Fig. of BAFF-R was determined for the various populations and is 6D). Interestingly, all of the T cell subsets analyzed did show represented by different shaded profiles, as indicated (Fig. 6B). expression of BAFF. Transcripts of BAFF were also expressed BAFF-R was expressed predominantly by central and effector strongly in mast cells, eosinophils, and particularly neutrophils, memory T cells, and not naive T cells, in keeping with the up- which is consistent with a recent report on production of BAFF by regulation of BAFF-R on activated T cells. neutrophils (39). Transcripts of APRIL were also detected in mast RT-PCR showed that mRNA for BAFF-R, but not TACI or cells, eosinophils, and neutrophils but at much lower levels than BCMA, was demonstrable in several T cell RNA samples, BJAB for BAFF (Fig. 6D); transcripts of APRIL were absent from all T and RAJI cells were used as control (Fig. 6C). We also used the cell subsets. 814 BAFF-R IS THE PRINCIPAL BAFF RECEPTOR FOR B AND T CELLS Downloaded from http://www.jimmunol.org/

FIGURE 6. BAFF-R but not TACI or BCMA is expressed on human T cells. A, TACI, BAFF-R, and BCMA expression on resting PBMC and 72 h anti-CD3-activated T cells. The x-axis shows CD69 expression. B, Expression of BAFF-R on naive, central memory (Tcm), and effector memory (Tem) by guest on September 23, 2021 CD4ϩ and CD8ϩ cells. PBMC were gated first on CD3ϩ lymphocytes, and then CD4ϩ cells or CD8ϩ T cells, and assessed for CCR7 vs CD45RO to define naive (CCR7ϩROϪ), central memory (Tcm, CCR7ϩROϩ), and effector memory (Tem, CCR7ϪROϩ) T cells (top panels). Expression of BAFF-R (middle panels) was determined for the various populations and are represented by different shaded profiles, as indicated. Isotype control staining was used to determine background staining (bottom panels). C, PCR for BAFF-R, BCMA, and TACI in pure cell populations/lines. GAPDH was used a positive control. D, Data mining was performed on a series of Affymetrix GeneChip experiments, performed on various purified leukocyte populations. Affymetrix U133A and B chips contain probes for BAFF, APRIL, BCMA, and TACI, (but not BAFF-R), and semiquantitative expression values, expressed as a heat map, are shown. Affymetrix algorithms made calls of presence or absence for each gene, absence is indicated by an “A” in each square.

Costimulatory effects of BAFF on human T cell proliferation unlike the mouse experiments described above, anti-h.BAFF-R ␮ Similar to the mouse results and previous study in humans (22), mAb (2Ð30 g/ml used) failed to inhibit T cell proliferation, sug- BAFF increased cell proliferation of suboptimally anti-CD3- gesting that the way BAFF-R signals in B and T cells is different treated human T cells, to a level similar to that observed with and possibly activated by a different interaction between BAFF anti-CD28 stimulation (Fig. 7A). However, we observed that the and BAFF-R. ability of BAFF to costimulate human T cell responses was vari- able, and was highly dependent on T cell purity. We next used Ig fusion proteins of TACI, BCMA, and BAFF-R to block BAFF Discussion (and APRIL) activity in a suboptimal anti-CD3 T cell activation The expression of BAFF receptors by subsets of B cells and T cells assay (Fig. 7B). All three fusion proteins inhibited T cell prolifer- regulates survival or costimulation at critical stages of maturation ation in response to anti-CD3, demonstrating the importance of or functional responses. Discerning the precise role of the various endogenous BAFF for normal T cell stimulation. Neutralization of BAFF receptors is important to understanding how BAFF and BAFF rather than APRIL appeared to be responsible for the ob- APRIL influence immune responses, and how and why overpro- served effect, because BAFF-R-Fc (BAFF specific) reduced production of BAFF causes autoimmune disease. This study estab- liferation to the same extent as TACI-Fc or BCMA-Fc. Although lishes BAFF-R as the important BAFF costimulatory receptor on it has been reported BCMA-Fc has a lower affinity for BAFF (29), circulating T and B cells, and shows that BCMA and TACI display we found all three fusion proteins showed a similar ability to block restricted expression patterns suggestive of specialized roles. BAFF activity, presumably because these fusion proteins were The role of BAFF-R in peripheral B cell survival and costimu- used at high concentrations (30 ␮g/ml). We did observe a degree lation has been difficult to gauge using BAFF-R mutant mice, be- of person to person variation, as TACI-Fc failed to inhibit T cell cause splenic B cell maturation is halted at the T1 stage (17). As proliferation in one individual of five (data not shown). In addition, BAFF-R was the only known BAFF receptor detectable on most The Journal of Immunology 815 Downloaded from http://www.jimmunol.org/

FIGURE 7. BAFF costimulates human T cells. A, Human T cells were puriﬁed from blood by magnetic separation and activated with anti-CD3 (1 ␮g/ml) for 72 h in the presence or absence of BAFF. BAFF was used either as plate-bound (B), soluble (S), or denatured (DN) at 5 ␮g/ml. B, Inhibition of BAFF costimulation by BAFF receptor fusion proteins. PBMCs were activated with anti-CD3 for 72 h, together with fusion proteins of h.BAFF-R-Fc, h.BCMA-

Fc, TACI-Fc, or a hIgG control (30 ␮g/ml). C, Anti-h.BAFF-R failed to inhibit T cell proliferation. PBMCs were activated with anti-CD3 in the presence by guest on September 23, 2021 of anti-h.BAFF-R, h.TACI-Fc, and h.IgG (30 ␮g/ml) were used as controls. All cultures were pulsed with [3H]thymidine for 18 h.

mature resting B cells, and anti-BAFF-R mAbs completely inhib- cells (43). The MZ has been suggested as a refuge for autoreactive ited BAFF-mediated costimulation of B cells, we conclude that B cells (44, 45), and increased TACI expression facilitating re- positive BAFF responses up to the stage of CD38ϩ plasmablasts pression of B cell proliferation might help contain autoreactive occur exclusively through this receptor. An assessment of BCMA cells within this population. and TACI deficient mice supports this conclusion. Early studies Although BAFF and its receptors have been associated mostly with BCMA-deficient mice found no unusual phenotype (30, 40, with B cell responses, we showed that BAFF also had profound 41), although recent studies showed impaired survival of long- effects on T cell costimulation, occurring through BAFF-R. How- lived bone marrow plasma cells (20). Our expression analysis also ever, immobilized BAFF rather than soluble BAFF provided these showed that BCMA is most likely relevant for later stages of B cell costimulatory signals to T cells (this report and Ref. 22) suggesting maturation or survival, i.e., for CD38ϩ plasmablasts (28) and GC that BAFF signaling through BAFF-R on T cells requires mem- B cells (this report). BCMA is also up-regulated on mouse plasma brane expression by APC. It is also conceivable that APCs immo- cells (20). Likewise, TACI was expressed only weakly on a small bilize and present soluble BAFF. BAFF-R has been identified as a subset of peripheral B cells. TACI-deficient mice showed in- survival receptor for B cells and it may serve a similar role for T creased B cell numbers, and splenomegaly (Refs. 21, 34, 35, and cells, rather than act as a classic costimulatory receptor. For in- this report). TACI has been proposed to play a role as a negative stance, signals through BAFF-R increase Bcl-2 expression in T regulator for B cells, rather than as an essential survival-related cells (Fig. 3) as they do for B cells (37), which may result in receptor (21). However, TACI may play an important role in some enhanced basal T cell survival, particularly by BAFF-Rϩ T cells aspects of B cell maturation or function within the spleen, because such as effector and central memory subsets. Enhanced T cell sur- T2 and MZ B cells expressed high levels of TACI. MZ B cells are vival would then augment the number of T cells capable of acti- essential for T-independent immune responses (42), and TACI vation. The activity of BAFF as an important component of co- knockout mice have impaired T-independent but normal T-depen- stimulation (or survival factor) for T cell proliferation is intriguing dent responses (34). TACI was clearly dispensable for splenic B because of the very high levels of BAFF in inflammatory lesions cell maturation, as shown by the production of mature B cells in and blood of certain patients with autoimmune diseases (7, 9, 10, TACIϪ/Ϫ mice. Nevertheless, BAFF (or APRIL) signaling through 46), although whether BAFF in such patients affects T cell biology TACI at the T2 or MZ stages may constrain B cell proliferation at is uncertain. Interestingly, the cell types that are expanded in critical stages of tolerance induction. The T1-T2 maturation step is BAFF transgenic mice include mature B cells, splenic T2 and MZ likely an important immune tolerance checkpoint for maturing B B cells, and also effector memory T cells (4, 33). Our in vivo data 816 BAFF-R IS THE PRINCIPAL BAFF RECEPTOR FOR B AND T CELLS has also shown that BAFF enhances the delayed-type hypersensi- tional relevance for T and B cell responses, represents an attractive tivity reaction, which is a classical T-dependent immune response target for intervention in autoimmune diseases. (our unpublished data). Taken together, these results clearly indicate that BAFF is not only an important B cell factor; it is also a Acknowledgments critical factor for T cell responses. The reason why only immobi- We gratefully thank Ian Mackay and Shane Grey for suggestions; lized BAFF costimulated T cells is unresolved; it is possible that Sabine Zimmer, Sue Liu, Mary Sisavanh, Michael Rolph, Kim Good, and immobilization of BAFF on the plastic plate reproduces elements Melinda Frost for Affymetrix expression data; Pascal Schneider for sharing of membrane expression by APCs. A final feature of BAFF-R DNA constructs; Eric Schmied and Carrie Fletcher for animal husbandry; worthy of comment is that BAFF-R binds BAFF but not APRIL, and Jenny Thatcher for assistance in Ab purification. and so the reported effects of APRIL on T cells (25) presumably occur through an unidentified receptor, or through indirect References mechanisms. 1. Mackay, F., P. Schneider, P. Rennert, and J. Browning. 2003. BAFF and APRIL: BAFF receptors were regulated during B cell differentiation to a tutorial on B cell survival. Annu. Rev. Immunol. 21:231. 2. Mackay, F., and C. Ambrose. 2003. The TNF family members BAFF and APRIL: GC cells. The most obvious expression of BCMA was by B cells the growing complexity. Cytokine Growth Factor Rev. 14:311. ϩ in tonsil with a phenotype consistent with GC B cells, i.e., CD38 , 3. Kalled, S. L., C. Ambrose, and Y. M. Hsu. 2003. BAFF: B cell survival factor and CD27ϩ, CD39Ϫ, and IgMϪ (47, 48). BAFF is important for the emerging therapeutic target for autoimmune disorders. Expert Opin. Ther. Tar- gets 7:115. GC reaction, because blocking BAFF in vivo attenuates the GC 4. Mackay, F., S. A. Woodcock, P. Lawton, C. Ambrose, M. Baetscher, reaction (49), and those GCs that do form in BAFF-deficient mice P. Schneider, J. Tschopp, and J. L. Browning. 1999. Mice transgenic for BAFF

develop lymphocytic disorders along with autoimmune manifestations. J. Exp. Downloaded from have impaired maturation and function (50). BAFF-R was also Med. 190:1697. expressed on GC B cells, but at lower levels compared with mature 5. Gross, J. A., S. R. Dillon, S. Mudri, J. Johnston, A. Littau, R. Roque, M. Rixon, B cells. Interestingly, TACI was largely absent from GC B cells, O. Schou, K. P. Foley, H. Haugen, et al. 2001. TACI-Ig neutralizes molecules critical for B cell development and autoimmune disease: impaired B cell matu- suggesting that its proposed role as a negative regulator of B cell ration in mice lacking BLyS. Immunity 15:289. activation (21, 35) does not extend to the GC reaction. Its loss from 6. Khare, S. D., I. Sarosi, X.-Z. Xia, S. McCabe, K. Miner, I. Solovyev, N. Hawkins, GC B cells would be consistent with the down-regulation by GC B M. Kelley, D. Chang, D. Chang, et al. 2000. Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice. Proc. 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