Survival Function CD23 Expression Independent of Its B Cell Cutting

Cutting Edge: BAFF Regulates CD21/35 and CD23 Expression Independent of Its B Cell Survival Function

This information is current as Leonid Gorelik, Anne H. Cutler, Greg Thill, Steven D. of October 4, 2021. Miklasz, Dianna E. Shea, Christine Ambrose, Sarah A. Bixler, Lihe Su, Martin L. Scott and Susan L. Kalled J Immunol 2004; 172:762-766; ; doi: 10.4049/jimmunol.172.2.762 http://www.jimmunol.org/content/172/2/762 Downloaded from

References This article cites 20 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/172/2/762.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on October 4, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts Errata An erratum has been published regarding this article. Please see next page or: /content/172/7/4646.2.full.pdf

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 CUTTING EDGE

Cutting Edge: BAFF Regulates CD21/35 and CD23 Expression Independent of Its B Cell Survival Function Leonid Gorelik,1* Anne H. Cutler,1,2* Greg Thill,† Steven D. Miklasz,* Dianna E. Shea,* Christine Ambrose,‡ Sarah A. Bixler,‡ Lihe Su,§ Martin L. Scott,* and Susan L. Kalled3*

Herein we demonstrate that B cell-activating factor of the The earliest emigrants from BM to the spleen are transitional TNF family (BAFF), a B cell survival factor, also regulates type 1 (T1) B cells. These cells develop into transitional type 2 CD21/35 and CD23 expression. BAFF blockade in wild- (T2), mature follicular, and marginal zone (MZ) B cells. From type mice down-modulates CD21/35 and CD23 on B cells analysis of BAFF KO mice, it was concluded that B cell devel- while survival remains intact, and BAFF exposure causes opment was blocked at the T1 stage (2, 3). However, it was

elevated CD21/35 and CD23 expression. Similar down- shown that while the humoral responses to T-dependent Ags Downloaded from modulation is observed in bcl-2-transgenic mice treated were severely impaired in BAFF KO mice, Ag-specific, class- with a BAFF inhibitor. This is the first evidence that switched Ab was still produced (2, 3). Recently, we showed that BAFF has a function independent of B cell survival. Re- BAFF KO mice form germinal centers (GC) with intact so- ports using CD21/35 and CD23 expression to assess matic hypermutation after Ag challenge (7). These findings splenic B cell subsets in BAFF-null mice concluded a lack suggest that BAFF KO mice possess more differentiated, ma- of B cells beyond the immature stage. Since CD21/35 and ture B cells than originally believed. Therefore, we re-examined http://www.jimmunol.org/ CD23 are inadequate for delineating B cell subpopula- the nature of splenic B cells in BAFF KO mice. During this tions in BAFF-null mice, we used expression of BAFF-R investigation, we found CD21/35 and CD23 expression to be and several B cell markers to identify more mature splenic regulated by BAFF. B cells in these mice. These data broaden our understand- A common method to delineate B cell subsets in the spleen em- ploys surface markers CD21/35 and CD23 since B cells can be ing of BAFF function and correct the view that BAFF-null Ϫ Ϫ ϩ defined as T1, CD21/35 CD23 ; T2, CD21/35highCD23 ; mice lack mature B cells. The Journal of Immunology, low ϩ high Ϫ 2004, 172: 762–766. mature, CD21/35 CD23 ; and MZ, CD21/35 CD23 (8). We and others previously concentrated on CD21/35 and CD23 by guest on October 4, 2021 though the developmental stages of B cell maturation expression to conclude that B cell development in BAFF KO mice 4 was blocked at the T1 stage (2, 3). However, B cell subsets can also from bone marrow (BM) precursors to mature pe- high low/Ϫ ripheral B cells are known, the survival and differen- be defined by IgM and IgD expression: T1, IgM IgD ; T2, A high high low high high low tiation factors required along this pathway have remained elu- IgM IgD ; mature, IgM IgD ; and MZ, IgM IgD sive. B cell-activating factor of the TNF family (BAFF), a TNF (9). Herein we show that BAFF-R, the only BAFF receptor to be family ligand, (also known as BLyS, THANK, TALL-1, and expressed on all peripheral B cells (10), exhibits a differentially reg- zTNF4) (1) was identified as a B cell survival factor and shown ulated pattern of expression on maturing B cells and can be used to to be required for B cell survival since BAFF knockout (KO) identify B cell subsets. Therefore, using expression of IgM, IgD, mice exhibit Ͼ90% loss of peripheral B cells (2, 3). Conversely, BAFF-R, and other B cell markers, we show that BAFF KO mice BAFF-transgenic (Tg) mice, which overexpress BAFF, have have all splenic B cell subsets, but that CD21/35 and CD23 are markedly elevated numbers of peripheral B cells and develop markedly reduced on mature, T2, and MZ B cells. autoimmunity (4–6). Thus, the function of BAFF has signifi- These data led us to hypothesize that BAFF regulates cance in B cell biology as well as disease. BAFF has three known CD21/35 and CD23 expression independently of its survival receptors, BAFF-R (also called BR3), BCMA, and TACI, and function. To test this, we examined B cell CD21/35 and CD23 while BAFF-R binds only to BAFF, TACI and BCMA also bind expression under conditions of BAFF deficiency and excess in APRIL, a related protein (1). vivo and in vitro. We present data that support our hypothesis

Departments of *Immunology and Inflammation, †Gene Expression, ‡Gene Discovery, 3 Address correspondence and reprint requests to Dr. Susan L. Kalled, Biogen Idec, and §Protein Chemistry, Biogen Idec Inc., Cambridge Center, Cambridge, MA 02142 Inc., 12 Cambridge Center, Cambridge, MA 02142. E-mail address: [email protected] Received for publication September 11, 2003. Accepted for publication November 11, 2003. 4 Abbreviations used in this paper: BM, bone marrow; BAFF, B cell-activating factor of the TNF family; MZ, marginal zone; T1, transitional type 1; T2, transitional type 2; GC, The costs of publication of this article were defrayed in part by the payment of page germinal center; KO, knockout; Tg, transgenic; hIgG, human IgG; sBAFF, soluble BAFF; charges. This article must therefore be hereby marked advertisement in accordance WT, wild type; MFI, mean fluorescence intensity; FDC, follicular DC; MAdCAM-1, mu- with 18 U.S.C. Section 1734 solely to indicate this fact. cosal addressin cell adhesion molecule 1. 1 L.G. and A.H.C. contributed equally to this work. 2 Current address: Avanir Pharmaceuticals, 11388 Sorrento Valley Road, San Diego, CA 92121.

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 763 and discuss the implications of diminished CD21/35 and CD23 expression on B cell biology and immunity. Materials and Methods Mice Female BALB/c mice (The Jackson Laboratory, Bar Harbor, ME), BAFF KO, and wild-type (WT) littermates (2) were housed in the Biogen Animal Facility under barrier conditions. Protocols were approved by the Biogen Idec Institu- tional Animal Care and Use Committee. BAFF inhibitor reagents and treatment Human BCMA-Fc (11) and BAFF-R:Fc (12) were described previously. Mice received i.p. 250 ␮g of BAFF-R:Fc, BCMA-Fc, or human IgG (hIgG; Novartis, Basel, Switzerland) as indicated.

Flow cytometric analysis FIGURE 1. Splenic B cell subsets in BAFF KO and WT mice. A, Splenocytes were stained with mAbs (BD PharMingen, San Diego, CA) di- Splenocytes from 12-wk-old BAFF KO and WT mice were stained with rected against various markers: IgM-allophycocyanin (11/41), CD21/35-FITC mAbs specific for IgM and IgD for FACS analysis. B cell subsets, T1, T2 (7G6), CD23-biotin (B3B4), B220-FITC (RA3-6B2), CD43-PE (S7), CD22- MZ/T1, and mature (M) are evident in both BAFF KO and WT mice. B, B biotin (Cy34.1), CD24-FITC (M1/69), CD40-FITC (HM40-3), CD19- cell subsets, as defined in A, were examined for CD21/35 and CD23 ex- FITC (1D3), IgD-PE (11-26; Southern Biotechnology Associates, Birming- pression. C, Frozen spleen sections from BAFF KO mice were stained with Downloaded from ham, AL). Anti-BAFF-R-biotin (P1B8 and B9C11) was generated at Biogen. anti-MAdCAM-1 (blue) and anti-IgM (brown) to visualize B cells within Streptavidin-PerCP (BD PharMingen) was used to visualize biotin-labeled the MZ. Arrows point to B cells in the MZ area (magnification, ϫ400). mAbs. Data depicted in A–C are representative of four mice analyzed per group in In vitro assays two separate experiments. Splenocytes from 5- to 6-wk-old BAFF KO and WT mice were prepared under sterile conditions and plated in 24-well plates (Corning, Corning, NY) at 3 ϫ high http://www.jimmunol.org/ 6 ␮ 35 MZ B cells were sparse in BAFF KO mice (Fig. 1B, MZ/T1 10 cells/ml in RPMI 1640/10% FBS at 37°C. Some wells received 2 g/ml Ϯ ϩ soluble human BAFF (sBAFF). Cells were harvested at 24, 48, and 72 h for gate) and made up only 1.9 0.6% of total IgM splenic B cells analysis. compared with 9 Ϯ 0.8% in WT mice and had a low level of surface CD21/35. To identify MZ B cells by localization, BAFF Immunohistochemistry KO spleen sections were stained for MAdCAM-1 to illuminate the Frozen spleen sections were prepared as described previously (13). Anti-muco- marginal sinus and demarcate the B cell follicle and MZ area, and sal addressin cell adhesion molecule 1 (MAdCAM-1)-biotin (Southern Bio- anti-IgM to visualize B cells. In Fig. 1C, a small number of B cells technology Associates) followed by streptavidin-alkaline phosphatase and are visible in the MZ, confirming that some MZ B cells in BAFF 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Vector Labora- KO mice can be defined geographically. This is consistent with the tories, Burlingame, CA), and anti-IgM-HRP (Jackson ImmunoResearch Lab- by guest on October 4, 2021 oratories, West Grove, PA) followed by 3,3Ј-diaminobenzidine (Vector Labo- fact that CD21/35-null mice also possess MZ B cells (14). ratories) were used. Examination of CD19, CD22, CD24, and CD40 on mature and Statistical analysis T1 B cells in WT and BAFF KO mice further indicate that the IgMlowIgDhigh B cells found in BAFF KO mice resemble WT Statistical analysis was performed using Student’s t test. mature, not T1, B cells. One difference observed was the higher level of CD24 on BAFF KO mature B cells, although this is much Results lower than that found on WT T1 B cells (Table I). This is due to IgM and IgD expression identifies mature B cells in BAFF KO mice a greater proportion of IgMlowIgDhigh B cells in KO mice that are Using four-color flow cytometric analysis with mAbs specific for CD24high (43.5 Ϯ 3.1%) when compared with WT mice (19.1 Ϯ IgM, IgD, CD21/35, and CD23, we found that by the IgD vs IgM 1.8%). Combined, the data show that the majority of splenic IgMϩ profile, like age-matched WT controls, 12-wk-old BAFF KO mice B cells in BAFF KO mice are beyond the T1 stage and suggest that appear to possess all splenic B cell subsets (Fig. 1A). Furthermore, BAFF may play a role in regulating CD21/35 and CD23 expression. when comparing the frequencies in the context of total splenic IgMϩ B cells, mature B cells represent the largest population in BAFF-R expression as a tool to identify splenic B cell subsets BAFF KO and WT mice (33.8 Ϯ 6.7% and 53.5 Ϯ 2.1%, respec- BAFF-R is expressed on all murine splenic B220ϩ B cells (10). To tively) followed by IgMhighIgDlow cells (KO ϭ 29.8 Ϯ 6%, WT ϭ determine whether expression varies during development and 21.9 Ϯ 3.2%) which include MZ and IgDlow T1 B cells (referred to as MZ/T1). The frequencies of T2 B cells in BAFF KO (6.9 Ϯ 0.3%) and WT mice (9.7 Ϯ 0.2%) do not differ greatly, while the Table I. B cell surface phenotype frequency of T1 B cells is elevated in BAFF KO mice compared with WT controls (KO ϭ 12.7 Ϯ 1.8%, WT ϭ 4.5 Ϯ 0.1%). WT KO Previous CD21/35 and CD23 expression analyses of splenic B Mature CD22 368.3 Ϯ 4.7a 306.7 Ϯ 8.3 cells from BAFF KO mice concluded that mature B cells were CD19 33.3 Ϯ 17 40.4 Ϯ 1.1 absent. Results using IgM/IgD to define B cells in BAFF KO mice CD24 96.3 Ϯ 6.3 140.5 Ϯ 9.2 Ϯ Ϯ are in conflict with this conclusion; therefore, IgD vs IgM gated CD40 19.1 1.4 17.8 1.1 T1 CD22 118 Ϯ 3.7 121.2 Ϯ 3.5 cells were further examined for CD21/35 and CD23 expression. As CD19 ND ND expected, CD21/35 and CD23 expression was minimal or nonex- CD24 298.8 Ϯ 23 293.4 Ϯ 16.8 istent on T2 and mature B cells (Fig. 1B). Although by IgM/IgD CD40 13.2 Ϯ 0.5 9.7 Ϯ 0.7 gating MZ B cells appear to exist at similar frequencies (MZ/T1 a MFI Ϯ SD using three mice per group. Representative of two separate gate), as described above, by examination of CD21/35, CD21/ experiments. 764 CUTTING EDGE: BAFF REGULATES CD21/35 AND CD23

FIGURE 2. BAFF-R expression on B cell subsets. A, FACS analysis of BM from WT mice. Gates were set to identifying pre-B, pro-B, immature, and mature B cell populations before analysis of BAFF-R expression (solid line). Dashed line indicates isotype control. B, FACS analysis of splenocytes from BAFF KO and WT mice. Gates were set on T1, T2, MZ/T1, and mature (M) B cells based on IgM/IgD. The data in A and B are representative of three to four mice assessed in each group. Downloaded from thereby useful for defining B cell subsets, we examined BAFF-R mice were cultured with or without sBAFF for 24, 48, and 72 h. At levels on BM B cell precursors and splenic B cell populations in each time point, cells were collected and subjected to four-color WT mice. As seen in Fig. 2A, pro-B (B220lowCD43ϩIgMϪ) and flow cytometric analysis to examine the level of CD21/35 and pre-B (B220lowCD43ϪIgMϪ) cells lack surface BAFF-R. A low CD23 expression on T2 and mature B cells defined by IgM and level of BAFF-R was observed on immature B cells IgD. The MZ/T1 gate was not examined since we could not rule http://www.jimmunol.org/ (B220lowCD43ϪIgMϩ) in the BM, thus BAFF-R appears to coin- out increasing CD21/35 and CD23 expression due to maturation of cide with the appearance of surface IgM. Circulating, mature B T1 B cells. Compared with freshly prepared (time ϭ 0 h) BAFF cells in the BM exhibited the highest level of BAFF-R (Fig. 2A). KO splenocytes, the expression level of CD21/35 increased with Using IgM/IgD gates of splenic B cells, we observed that T1 time, with ϳ3.5-fold increase on mature and T2 BAFF KO B cells cells exhibit a level of BAFF-R similar to immature B cells in BM by 72 h when cultured with sBAFF (Fig. 4, left panel). WT mature (Fig. 2B). T2 and mature B cells exhibit the highest level, although and T2 B cells also increased CD21/35 expression when cultured the mean fluorescence intensity (MFI) is greater for T2 than ma- with sBAFF, with a 2.3- and 2-fold increase, respectively, ob- ture B cells. The B cells within the MZ/T1 gate have an interme- served at 72 h. BAFF KO and WT B cells in medium-only cultures diate level of BAFF-R, and T1 B cells express little, or no, exhibited no or Ͻ2-fold increase in CD21/35 expression (Fig. 4, by guest on October 4, 2021 BAFF-R. Given that BAFF-R expression increases with matura- left panel). sBAFF also mediated increased CD23 expression with tion beyond the T1 stage, we used BAFF-R expression as an ad- the maximum expression observed at 72 h. At this time point, ditional marker, along with IgM and IgD, to identify splenic B cell cultures of BAFF KO splenocytes plus sBAFF had the greatest subsets that lack CD21/35 and CD23 in BAFF KO mice. As seen increase in CD23 expression compared with freshly prepared cells in Fig. 2B, the T1, T2, mature, and MZ/T1 B cell subsets in BAFF (3.8-fold vs 1.6-fold medium alone; Fig. 4, right panel). Mature B KO mice have identical patterns of BAFF-R expression when cells from BAFF KO mice exhibited a 1.8-fold increase in CD23 compared with corresponding subsets in WT mice. T2 cells have the highest level followed by mature, MZ/T1, then T1 B cells. Although the pattern of BAFF-R expression on B cell subsets in BAFF KO is similar to that of WT animals, BAFF KO mice have a slightly reduced MFI for BAFF-R on all B cells.

CD21/35 and CD23 expression on B cells from BAFF Tg mice Since B cells in a BAFF-deﬁcient environment exhibit reduced levels of CD21/35 and CD23, we speculated that the converse would be true in BAFF Tg mice. Therefore, we examined B cells from BAFF Tg mice for CD21/35 and CD23 expression. Consis- tent with the notion of CD21/35 regulation by BAFF, mature, MZ, and T2 B cells from BAFF Tg mice have a markedly increased MFI for CD21/35 when compared with WT controls (Fig. 3, upper panel). No increase in CD23 expression was observed in BAFF Tg mice (Fig. 3, lower panel).

B cells from BAFF KO mice up-regulate CD21/35 and CD23 when exposed to BAFF FIGURE 3. Analysis of CD21/35 and CD23 expression on B cells in BAFF Tg mice. Splenocytes from BAFF Tg (n ϭ 3) and WT mice (n ϭ 3) To determine a direct relationship between BAFF and CD21/35 were prepared and subjected to FACS analysis. Gates were set on mature, and CD23 expression, we asked whether BAFF could stimulate MZ/T1, and T2 B cells based on IgM/IgD. Each B cell subset was assessed p Ͻ ,ء .CD21/35 and CD23 expression to WT levels on T2 and mature B for CD21/35 (upper panel) and CD23 (lower panel) expression cells from BAFF KO mice. Splenocytes from WT and BAFF KO 0.05. The data are representative of three separate experiments. The Journal of Immunology 765

Table II. BAFF/APRIL blockade in bcl-2 Tg mice

B cellsa CD21/35b Treatment (ϫ106) (MFI) CD23b (MFI)

BCMA-Fc 4.0 13.7 145 BCMA-Fc 4.3 13.0 136 BCMA-Fc 6.1 12.1 174 Mean Ϯ SD 5.2 Ϯ 1.0 12.9 Ϯ 0.7 151.7 Ϯ 16 hIgG 5.7 16.9 256 hIgG 5.7 19.2 279 Mean Ϯ SD 6.3 Ϯ 0.8 18.1 Ϯ 1.2 268 Ϯ 12

a Assessed on day 8 after two doses of BCMA-Fc. b Assessed 2 days after a single dose of BCMA-Fc.

FIGURE 4. BAFF stimulates CD21/35 and CD23 expression on B cells from BAFF KO mice. Splenocytes from BAFF KO (E, ‚) and WT control later for flow cytometric analysis. Forty-eight hours posttreatment (F, Œ) were prepared and cultured with (E, F) or without (‚, Œ) sBAFF. no difference in the number of total splenic B cells was observed After 24, 48, and 72 h, cells were harvested, B cell subsets were gated (Fig. 5A). Mature, MZ and T2 B cells were gated based on IgM/ based on IgM/IgD, and assessed for CD21/35 and CD23 expression. The IgD. CD23 (Fig. 5B) and CD21/35 (Fig. 5C) were significantly data are representative of two experiments. reduced on all B cell subsets examined when compared with hIgG- treated mice. CD21/35 expression was also found to be signifi- Downloaded from cantly diminished after only 24 h post-BAFF-R:Fc treatment, expression with no increase seen in the medium-alone culture (Fig. while no change was observed for CD23 (data not shown). 4, right panel). A second system utilized bcl-2 Tg mice for in vivo blockade These data show that BAFF can increase expression of CD21/35 with BCMA-Fc. bcl-2 Tg mice contain the human bcl-2 gene un- and CD23 on B cells from WT and BAFF KO mice. Although the der the control of the E␮ promoter, allowing B cell-specific ex- significance of lower CD21/35 expression on B cells from BAFF pression. bcl-2, an antiapoptotic factor, was shown to enhance B http://www.jimmunol.org/ KO mice in culture is unclear, B cells from BAFF KO mice still cell survival when overexpressed (15). bcl-2 Tg mice were treated can reach the level observed on freshly isolated WT cells. with a single dose of 250 ␮g BCMA-Fc or hIgG and splenic B cells were examined for CD21/35 and CD23 expression 2 days BAFF regulates CD21/35 and CD23 expression independent of B cell later. A separate cohort received 250 ␮g BCMA-Fc or hIgG on survival function days 0 and 4, with splenic B cell analysis on day 8. On day 8, To determine whether BAFF regulates CD21/35 and CD23 ex- BCMA-Fc and hIgG-treated mice had similar numbers of splenic pression independent of its survival function, we used two in vivo B220ϩ B cells (Table II). However, diminished levels of CD21/35 settings. The first utilized WT mice and a receptor-Fc decoy to and CD23 were observed on mature B cells in BCMA-Fc-treated block BAFF for a brief period so that B cell survival remained mice, when compared with controls, as early as 2 days after a by guest on October 4, 2021 intact. BALB/c mice were given a single i.p. dose of 250 ␮g single dose (Table I). BAFF-R:Fc or hIgG as a control, and spleens were harvested 48 h Discussion Although the role of BAFF in B cell survival is well established, it is unclear whether BAFF is required for B cell differentiation and/or function. Despite a presumed dominance of T1 B cells, BAFF KO mice mount class-switched, Ag-specific immune responses, albeit at reduced levels (2). Furthermore, WT mice treated with a BAFF inhibitor produce high-affinity Ab, although the quantity is diminished (16, 17). Recently we showed that BAFF KO and BCMA-Fc-treated WT mice form GCs with intact somatic hypermutation (7). Together, these data suggest the existence of mature B cells in BAFF KO mice. Our expression data of IgM, IgD, and other B cell markers to define mature B cells is in agree- ment with this notion. The differentially regulated expression of BAFF-R, first appearing coincident with surface IgM, provided an additional means to define B cell subsets and allowed us to show that BAFF KO mice have mature B cells, but with diminished CD21/35 and CD23 levels. The finding that BM B cell precursors lack BAFF-R expression likely explains the BAFF-independent nature of these cells (2, 3). Most importantly, our data provide the first evidence that BAFF has a function in addition to and independent of B cell survival. Although others demonstrated that T1 B cells in culture with FIGURE 5. Brief BAFF blockade results in intact B cell survival but reduced CD21/35 and CD23 expression. BALB/c mice (n ϭ 3/group) re- BAFF acquire CD21/35 and CD23, it was presumed that expres- ceived BAFF-R:Fc or hIgG 48 h before FACS analysis of splenic B cells. sion of these molecules was related to differentiation of immature A, Number of splenic B cells. Expression levels of CD23 (B) and CD21/35 B cells (18, 19). Although this may be true, our in vitro results -p Ͻ 0.05. The data are show that BAFF can directly mediate CD21/35 and CD23 expres ,ء .(C) on B cell subsets (f, hIgG; Ⅺ, BAFF-R:Fc) representative of two separate experiments. sion on more mature B cells. The elevated level of CD21/35 on B 766 CUTTING EDGE: BAFF REGULATES CD21/35 AND CD23 cells from BAFF Tg mice correlates with these data. That BAFF our data correct a misperception that B cells fail to develop beyond Tg mice do not express elevated CD23 on B cells suggests an the T1 stage in a BAFF-deficient environment and broaden what intrinsic factor in vivo that may prohibit excessive CD23 surface was a narrow view of the role of BAFF in B cell biology to include expression or cause down-regulation when CD23 is overexpressed. regulation of important B cell surface proteins, CD21/35 and BAFF-R:Fc treatment of WT mice and BCMA-Fc treatment of CD23. bcl-2 Tg mice confirmed the disassociation between BAFF-regulated CD21/35 and CD23 expression and B cell survival. Although Acknowledgments BCMA-Fc also blocks APRIL, the results with BAFF-R:Fc treat- We thank Y.-M. Hsu, D. Gong, T. Cachero, F. Qian, K. Miatkowski, ment of WT mice demonstrate that BAFF alone can regulate K. Strauch, and C. Mullen for BAFF-R:Fc, BCMA-Fc, and sBAFF reagents; the CD21/35 and CD23 expression. FACS Laboratory for technical assistance; and S. Rao, M. Dobles, F. Mackay, It may be argued that BAFF-mediated regulation of CD21/35 and T. Novobrantseva for critical reading of this manuscript. and CD23, markers used to distinguish differentiated B cells, con- stitutes BAFF as a differentiation factor. From a strict phenotypic References definition, this may be accurate. However, IgMlowIgDhighCD21/ Ϫ Ϫ 1. Kalled, S. L., C. Ambrose, and Y. M. Hsu. 2003. BAFF: B cell survival factor and 35 CD23 B cells in BAFF KO mice are functional and can be emerging therapeutic target for autoimmune disorders. Expert Opin. Ther. Targets activated to form GCs, somatically mutate, and produce high-af- 7:115. 2. Schiemann, B., J. L. Gommerman, K. Vora, T. G. Cachero, S. Shulga-Morskaya, finity Ab (2, 3, 7), and our recent experiments indicate that Ag- M. Dobles, E. Frew, and M. L. Scott. 2001. An essential role for BAFF in the normal ϫ specific immune responses in (bcl-2 Tg BAFF KO)F1 mice, development of B cells through a BCMA-independent pathway. Science 293:2111. possessing mature CD21/35ϪCD23Ϫ B cells, are normalized.5 It is 3. Gross, J. A., S. R. Dillon, S. Mudri, J. Johnston, A. Littau, R. Roque, M. Rixon,

O. Schou, K. P. Foley, H. Haugen, et al. 2001. TACI-Ig neutralizes molecules critical Downloaded from possible these cells represent a unique BAFF-independent B cell for B cell development and autoimmune disease. impaired B cell maturation in mice population, or lineage, and are too sparse to detect in normal mice. lacking BLyS. Immunity 15:289. 4. Gross, J. A., J. Johnston, S. Mudri, R. Enselman, S. R. Dillon, K. Madden, W. Xu, However, some specific functions, as discussed below, may be J. Parrish-Novak, D. Foster, C. Lofton-Day, et al. 2000. TACI and BCMA are recep- impaired similar to CD21/35 KO and CD23 KO mice, and this tors for a TNF homologue implicated in B-cell autoimmune disease. Nature 404:995. remains to be tested. 5. Mackay, F., S. A. Woodcock, P. Lawton, C. Ambrose, M. Baetscher, P. Schneider, J. Tschopp, and J. L. Browning. 1999. Mice transgenic for BAFF develop lymphocytic The Cr2 locus encodes complement receptors CD21/35 which disorders along with autoimmune manifestations. J. Exp. Med. 190:1697. are expressed on B cells and follicular DCs (FDCs). Through the 6. Khare, S. D., I. Sarosi, X. Z. Xia, S. McCabe, K. Miner, I. Solovyev, N. Hawkins, http://www.jimmunol.org/ generation of Cr2 KO mice, the importance of complement and M. Kelley, D. Chang, G. Van, et al. 2000. Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice. Proc. Natl. Acad. Sci. USA 97:3370. complement receptors in adaptive immunity was established. 7. Vora, K. A., L. C. Wang, S. P. Rao, Z.-Y. Liu, G. R. Majeau, A. H. Cutler, CD21/35, CD19, and CD81 comprise a signaling complex on B P. S. Hochman, M. L. Scott, and S. L. Kalled. 2003. Germinal centers formed in the cells that along with the B cell receptor lowers the threshold for absence of B cell-activating factor belonging to the TNF family exhibit impaired maturation and function. J. Immunol. 171:547. activation and, unlike WT B cells, Cr2-null B cells with low af- 8. Hardy, R. R., K. Hayakawa, A. Cariappa, M. Tang, C. Parng, E. Nebelitskiy, finity to Ag are not maintained in the B cell follicle. Furthermore, M. Carroll, K. Georgopoulos, and S. Pillai. 2001. B cell development pathways. Annu. Rev. Immunol. 19:595. it was found that B cells require Cr2 for sufficient signaling to 9. Cariappa, A., M. Tang, C. Parng, E. Nebelitskiy, M. Carroll, K. Georgopoulos, and survive in GCs (14). That is of particular interest since GCs formed

S. Pillai. 2001. The follicular versus marginal zone B lymphocyte cell fate decision is by guest on October 4, 2021 in a BAFF-deficient environment are unstable, and a mature FDC regulated by Aiolos, Btk, and CD21. Immunity 14:603. 10. Thompson, J. S., S. A. Bixler, F. Qian, K. Vora, M. L. Scott, T. G. Cachero, reticulum fails to form in these GCs (7). This may be due to the C. Hession, P. Schneider, I. D. Sizing, C. Mullen, et al.. 2001. BAFF-R, a newly loss of CD21/35 on B cells and an impaired cosignal. Interestingly, identified TNF receptor that specifically interacts with BAFF. Science 293:2108. expression of the CD35 isoform of Cr2 remains intact on FDCs in 11. Thompson, J. S., P. Schneider, S. L. Kalled, L. Wang, E. A. Lefevre, T. G. Cachero, F. MacKay, S. A. Bixler, M. Zafari, Z. Y. Liu, et al. 2000. BAFF binds to the tumor a BAFF-deficient environment (7). necrosis factor receptor-like molecule B cell maturation antigen and is important for CD23, the low-affinity IgE receptor, is expressed on B cells, maintaining the peripheral B cell population. J. Exp. Med. 192:129. FDCs, and a subset of T cells. Data suggest that B cell CD23 is 12. Pelletier, M., J. S. Thompson, F. Qian, S. A. Bixler, D. Gong, T. Cachero, K. Gilbride, E. Day, M. Zafari, C. Benjamin, et al. 2003. Comparison of soluble decoy involved in Ag presentation and that CD23 KO mice exhibit en- IgG fusion proteins of BAFF-R and BCMA as antagonists for BAFF. J. Biol. Chem. hanced IgE responses (20). Interestingly, human CD21 is a ligand 9:5977. 13. Kalled, S. L., A. H. Cutler, and L. C. Burkly. 2001. Apoptosis and altered dendritic for human CD23. Therefore, loss of B cell-expressed CD23 and/or cell homeostasis in lupus nephritis are limited by anti-CD154 treatment. J. Immunol. CD21/35 may contribute to compromised humoral immunity in a 167:1740. BAFF-deficient environment. Given the importance of B cell-FDC 14. Barrington, R., M. Zhang, M. Fischer, and M. C. Carroll. 2001. The role of complement in inflammation and adaptive immunity. Immunol. Rev. 180:5. interaction in humoral immunity, the expression of CD21/35 and 15. Strasser, A., A. W. Harris, D. L. Vaux, E. Webb, M. L. Bath, J. M. Adams, and CD23 on B cells, and our current understanding of the functions of S. Cory. 1990. Abnormalities of the immune system induced by dysregulated bcl-2 these proteins, independent from survival activity, BAFF may be expression in transgenic mice. Curr. Top. Microbiol. Immunol. 166:175. 16. Xia, X. Z., J. Treanor, G. Senaldi, S. D. Khare, T. Boone, M. Kelley, L. E. Theill, important for B cell function through regulation of CD21/35 A. Colombero, I. Solovyev, F. Lee, et al. 2000. TACI is a TRAF-interacting receptor and CD23. for TALL-1, a tumor necrosis factor family member involved in B cell regulation. The exact nature of the BAFF-RϩIgMlowIgDhighCD21/ J. Exp. Med. 192:137. Ϫ Ϫ 17. Yan, M., S. A. Marsters, I. S. Grewal, H. Wang, A. Ashkenazi, and V. M. Dixit. 2000. 35 CD23 mature B cells in BAFF KO mice is unclear, and ad- Identification of a receptor for BLyS demonstrates a crucial role in humoral immunity. ditional studies are required to fully elucidate the survival pathway Nat. Immunol. 1:37. 18. Batten, M., J. Groom, T. G. Cachero, F. Qian, P. Schneider, J. Tschopp, and functional capacity of these cells in BAFF KO mice and to J. L. Browning, and F. Mackay. 2000. BAFF mediates survival of peripheral immature determine the existence of such cells in WT animals. Nevertheless, B lymphocytes. J. Exp. Med. 192:1453. 19. Rolink, A. G., J. Tschopp, P. Schneider, and F. Melchers. 2002. BAFF is a survival and maturation factor for mouse B cells. Eur. J. Immunol. 32:2004. 5 M. Dobles, L. Gorelik, K. Gilbride, S. L. Kalled, and M. L. Scott. BAFF-indepen- 20. Lamers, M. C., and P. Yu. 1995. Regulation of IgE synthesis: lessons from the study dent differentiation and function of mature B cells. Submitted for publication. of IgE transgenic and CD23-deficient mice. Immunol. Rev. 148:71. The Journal of Immunology

CORRECTIONS

Bernhard Banas, Markus Wo¨rnle, Thorsten Berger, Peter J. Nelson, Clemens D. Cohen, Matthias Kretzler, Jochen Pﬁrstinger, Matthias Mack, Martin Lipp, Hermann-Josef Gro¨ne, and Detlef Schlo¨ndorff. Roles of SLC/CCL21 and CCR7 in Human Kidney for Mesangial Proliferation, Migration, Apoptosis, and Tissue Homeostasis. The Journal of Immunol- ogy 2002;168:4301–4307.

In Materials and Methods, under the heading RT-PCR analysis, the primer sequences for CCR7 are incorrect. The other data on the probes (accession number and positions of the primers) are correct as published. The correct primer sequences are shown below.

sense: 5Ј-GCTCCAGGCACGCAACTTT-3Ј antisense: 5Ј-ACCACGACCACAGCGATGA-3Ј probe (FAM): 5Ј-AGCGCAACAAGGCCATCAAGGTG-3Ј

Leonid Gorelik, Anne H. Cutler, Greg Thill, Steven D. Miklasz, Dianna E. Shea, Christine Ambrose, Sarah A. Bixler, Lihe Su, Martin L. Scott, and Susan L. Kalled. Cutting Edge: BAFF Regulates CD21/35 and CD23 Expression Inde- pendent of Its B Cell Survival Function. The Journal of Immunology 2004;172:762–766.

In Results, there were errors in Table II. The legend and conclusion are correct as published. The revised table is shown below.

Table II. BAFF/APRIL blockade in bcl-2 Tg mice

B Cellsa CD21/35b CD23b Treatment (ϫ107) (MFI) (MFI)

BCMA-Fc 15.8 13.7 145 BCMA-Fc 16.0 13.0 136 BCMA-Fc 24.3 12.1 174 Mean Ϯ SD 18.7 Ϯ 4.9 12.9 Ϯ 0.8 151.7 Ϯ 19.9 hIgG 21.8 16.9 256 hIgG 20.7 19.2 279 Mean 21.3 18.1 268

a Assessed on day 8 after 2 doses of BCMA-Fc. b Assessed 2 days after a single dose of BCMA-Fc.

Sergei Kusmartsev, Yulia Nefedova, Daniel Yoder, and Dmitry I. Gabrilovich. Antigen-Speciﬁc Inhibition of CD8ϩ T Cell Response by Immature Myeloid Cells in Cancer Is Mediated by Reactive Oxygen Species. The Journal of Immu- nology 2004;172:989–999.

In Results, the ﬂow cytometry histogram labels MFI and M1 are incorrect in three of the four panels in Figure 6A. The revised ﬁgure is shown below.