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B ComplexesκRelA-Containing NF-Regulation of Developing B Cell Survival by

Maria Prendes, Ye Zheng and Amer A. Beg

http://www.jimmunol.org/content/171/8/3963doi: 10.4049/jimmunol.171.8.3963

2003; 171:3963-3969; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/171/8/3963.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2003 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Regulation of Developing B Cell Survival by RelA-ContainingNF-�B Complexes1

Maria Prendes, Ye Zheng, and Amer A. Beg2

Mice deficient in the RelA (p65) subunit of NF-�B die during embryonic development. Fetal liver (FL) hemopoietic precursorsfrom these mice were used to generate RelA-deficient lymphocytes by adoptive transfer into lethally irradiated mature lympho-cyte-deficient recombination-activating gene-1�/� mice. Strikingly, RelA�/� lymphocyte generation was greatly diminished com-pared with that of RelA�/� lymphocytes. The most dramatic reduction was noticed in the numbers of developing B cells, whichwere considerably increased when RelA�/� FL cells that were also TNFR1 deficient were used. The role of RelA was furtherinvestigated in FL-derived developing B cells in vitro. Our results show that RelA is a major component of constitutive andTNF-�-induced �B site-binding activity in developing B cells, and provide evidence for a direct role of TNF-� in killing RelA�/�

B cells. The absence of RelA significantly reduced mRNA expression of the antiapoptotic genes cellular FLICE-inhibitory proteinand Bcl-2. Retroviral transduction of RelA�/� B cells with either cFLIP or Bcl-2 significantly reduced TNF-� killing. Together,these results indicate that RelA plays a crucial role in regulating developing B cell survival by inhibiting TNF-� cytotoxicity. TheJournal of Immunology, 2003, 171: 3963–3969.

P rogenitors of B and T lymphocytes originate in the bonemarrow from hemopoietic stem cells. Both B and T cellsexpress Ag-specific receptors that are generated by V-D-J

rearrangement events during development. Commitment to the Bcell lineage is initiated in progenitor (pro) B cells, which have notundergone rearrangement of Ig genes. Successful rearrangement ofIg H chain (HC)3 results in generation of precursor (pre) B cells.Pre-B cells express the pre-B cell receptor (pre-BCR), which com-prises the rearranged HC and the V-preB and �5 molecules. Sig-nificantly, signals transmitted by the pre-BCR are required for Bcell survival and further development (also see below). Thus, micedeficient in the recombination-activating genes (Rag-1 or Rag-2),which cannot undergo rearrangement of Ig genes, do not progressbeyond the pro-B cell stage (1). L chain rearrangement in pre-Bcells transforms them into immature and finally into mature Bcells. Both immature and mature B cells express cell surface Igmolecules (IgM) in a complex with key signaling molecules thattogether make the BCR.

A key feature of lymphocyte development is the high rate ofapoptosis (programmed cell death), which can take place duringseveral distinct developmental stages. Thus, pro-B cells that do notundergo productive rearrangement of Ig HC are removed by apo-ptosis (2). It is thought that antiapoptotic members of the Bcl-2family of proteins, including Bcl-2 and Bcl-xL, play an importantrole in regulating B cell survival (3). Notably, induction of Bcl-xL

expression correlates with expression of the pre-BCR (4). Bcl-xL

may therefore be responsible for survival of B cells that have un-dergone successful rearrangement of Ig HCs (4, 5). In addition,apoptosis plays an important role in removal of lymphocytes thatrecognize self Ags, a process known as negative selection. Nega-tive selection of both B and T cells is thought to help eliminateautoreactive lymphocytes, and thus prevent development of auto-immunity. Elimination of autoreactive lymphocytes occurs in thebone marrow and thymus, and in the periphery. Significantly, re-moval of autoreactive lymphocytes is thought to be conducted bymembers of the TNFR superfamily, in particular Fas and TNFR1(6, 7). Unlike Fas-deficient mice, TNF-�- or TNFR1-deficientmice do not display autoimmune pathology. However, absence ofboth TNFR1 and Fas significantly enhances autoimmune pathol-ogy in mice (8), suggesting a possibly redundant role for these twomolecules in preventing autoimmunity. Whether TNF-�-inducedcytotoxic pathways also affect survival of developing lymphocytesis presently not clear.

The NF-�B transcription factors play an important role in reg-ulating inflammatory, immune, and survival pathways (9, 10).Members of this family include several distinct subunits, includingcRel, RelB, p52, and perhaps the most ubiquitous RelA (p65) andp50 proteins (9, 10). Although these NF-�B subunits may formvirtually any homo- or heterodimer, the common complexespresent in lymphocytes constitute heterodimers of p50/RelA andp50/cRel subunits. Dimeric NF-�B proteins typically reside in thecytoplasm in a complex with inhibitory I-�B proteins. Treatmentof cells with proinflammatory cytokines (TNF-�), bacterial prod-ucts (LPS), or engagement of Ag receptors present on B and Tcells (BCR and TCR) results in activation of NF-�B, through phos-phorylation-triggered degradation of I-�B proteins. This process isinitiated by I-�B kinases (IKK� and IKK�) (10, 11), and allowsnuclear translocation of NF-�B and activation of expression oftarget genes (10).

RelA subunit knockout mice die during embryogenesis becauseof apoptosis of hepatocytes (12, 13). Importantly, hepatocyte ap-optosis and embryonic lethality in RelA�/� mice can be preventedin RelA�/�TNF-��/� (14) or RelA�/�TNFR1�/� mice (15).Studies with p50�/� and cRel�/� mice have shown that NF-�B

Department of Biological Sciences, Columbia University, New York, NY 10027

Received for publication February 7, 2003. Accepted for publication August 6, 2003.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health R01 CA074892 to A.A.B.2 Address correspondence and reprint requests to Dr. Amer A. Beg, 1110 FairchildCenter, Department of Biological Sciences, 1212 Amsterdam Avenue, Columbia Uni-versity, New York, NY 10027. E-mail address: aab41@columbia.edu3 Abbreviations used in this paper: HC, H chain; BCR, B cell receptor; BM, bonemarrow; cFLIP, cellular FLICE-inhibitory protein; cFLIP(L), cFLIP (long); FL, fetalliver; GFP, green fluorescence protein; IKK, I-�B kinase; MIG, mouse stem cell virus2.2-internal ribosomal entry site-GFP 3M; PI, propidium iodide; Rag, recombination-activating gene; RPA, RNase protection assay.

The Journal of Immunology

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00

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activation following BCR engagement in mature B cells is impor-tant for mediating proliferative and survival responses (16–20) andfor development of marginal zone B cells (21). Interestingly, cRelwas found to be specifically important for preventing Ag receptor-induced, but not Fas-induced cell death (19). Our recent studies ofCD4 T cells deficient in p50 and cRel NF-�B proteins have iden-tified an important role for these proteins in regulating mature Tcell survival (22). In addition, studies with mice lacking p50 �RelA or IKK� have shown impaired lymphocyte generation (23,24). Significantly, the generation of T cells in IKK��/� mice canbe rescued in a TNFR1-deficient background (24). However, TNF-�-dependent mechanisms responsible for impaired lymphocytegeneration and the precise role of NF-�B in this process are notknown.

In this study, we have studied the role of the NF-�B RelA sub-unit in lymphocyte generation. Our in vivo and in vitro resultsindicate that RelA plays a key role in regulating developing B cellsurvival, and provide evidence for a direct role of TNF-� in killingRelA�/� developing B cells. The absence of RelA significantlyreduces �B site-binding activity and mRNA expression of cellularFLICE-inhibitory protein (cFLIP) and Bcl-2. Retroviral transduc-tion of RelA�/� B cells with either cFLIP or Bcl-2 significantlyreduces TNF-� killing. These results indicate that RelA plays akey role in regulating developing B cell survival by inhibitingTNF-�-induced cell death.

Materials and MethodsAdoptive transfer experiments

TNFR1�/� and Rag-1�/� mice were purchased from The Jackson Labo-ratory (Bar Harbor, ME). Fetal liver (FL) adoptive transfer experimentswere performed, as previously described (25). Briefly, 1 � 106 viable FLcells were injected i.v. into lethally irradiated Rag-1�/� mice (2 doses of800 and 400 rad, 3 h apart). Mice were sacrificed 6–8 wk after transplan-tation. Spleen and bone marrow cells were analyzed by FACS. The studiesshown in this work represent results from three independent experiments.All experiments with mice were carried out in accordance with institutionalguidelines.

Generation of B cells from FL precursors

FL from mouse embryos were obtained on day 13 of development. Single-cell suspensions were cultured in the presence of 5 ng/ml IL-7 in Opti-MEM medium containing 10% FBS and 50 �M 2-ME. On day 8 of culture,nonadherent cells were removed and analyzed by FACS. For TNF-� treat-ments, mouse rTNF-� was added to RelA�/� and RelA�/� cultures at afinal concentration of 10 ng/ml. Following this incubation, the B cells werestained with propidium iodide (PI), and FACS analysis was performed todetermine the percentage of cell death.

FACS

Single-cell suspensions of B cells were first incubated with Fc-Block (anti-mouse CD16/CD32), followed by staining with biotin-conjugated mAb(pre-BCR), followed by staining with CyChrome-conjugated streptavidin,PE-conjugated mAb (B220, CD19), and FITC-conjugated mAb. To deter-mine the surface expression of TNFR1, the cells were preincubated withFc-Block, followed by incubation with a purified TNFR1 Ab. The cellswere washed and incubated with a biotin-conjugated cocktail of anti-IgGsecondary Abs. After washing, the cells were incubated with PE-conju-gated streptavidin. Stained cells were fixed in 4% paraformaldehyde, andanalyzed using a BD Biosciences (San Jose, CA) flow cytometer. For in-tracellular staining, the cells were first fixed in 2% paraformaldehyde inPBS for 1 h at 4°C, permeabilized in 0.2% Tween 20 in PBS, washed, andstained, as described above. All Abs and streptavidin conjugates used werefrom BD PharMingen (San Diego, CA).

EMSA, Northern blotting, and RNase protection assay (RPA)

Nuclear extracts from in vitro developed B cells were made, as describedpreviously (26). The �B site oligonucleotide probe was from the mouseMHC class I promoter. As control, we show binding to the constitutiveOct-1 transcription factor site (5-TGTCGAATGCAAATCACTAGAA-3).A total of 5 �g of nuclear extracts was incubated with the 32P-labeled

probes for 15 min and loaded on Tris-base/glycine/EDTA gels. The gelswere quantified by using a PhosphorImager Storm 860 from MolecularDynamics (Sunnyvale, CA) and the computer software ImageQuaNT.

On day 9, B cells were left untreated or treated with TNF-� (10 ng/ml)for 2 or 6 h. Total RNA was extracted using TRIzol reagent (Life Tech-nologies-BRL, Gaithersburg, MD), according to the manufacturer’s in-structions, and resuspended in 20 �l deionized water. Total RNA (10 �g)was used per sample for Northern blot analysis with mouse I-�B� or �-ac-tin 32P-labeled probes.

The same RNA was also used for RPA. Antisense RNA probes forBcl-xL, Bcl-2, and cFLIP were prepared using the T7 promoter in pBlue-script. The probes were labeled with [32P]UTP using Ambion (Austin, TX)RPA kit. RPA analysis was conducted according to manufacturer’s rec-ommendations (Ambion).

Retroviral infection of developing B cells

The mouse stem cell virus 2.2-internal ribosomal entry site-GFP 3M (MIG)retroviral vector (27) was used for experiments shown in this work (kindlyprovided by L. Van Parijs, Massachusetts Institute of Technology (Cam-bridge, MA)). This vector allows simultaneous expression of a gene ofinterest (Bcl-2 or cFLIP) and green fluorescence protein (GFP). Retrovirusstocks were generated using the ecotropic packaging cell line BOSC23(28). MIG vectors were cotransfected with PCLEco (encoding retroviralproteins), and supernatants were collected after 48 h. B cells from RelA�/�

embryos cultured with IL-7 were infected on days 6 and 7. A total of 500�l of viral supernatant was used per well in a total volume of 1 ml of B cellculture medium and polybrene at a final concentration of 10 �g/ml. Ret-roviral infection was performed using centrifugal enhancement (centri-fuged for 1 h at 1000 � g at 30°C). FACS analysis and TNF-� treatmentswere performed on day 9 of culture, as described in the text.

To measure TNF-�-specific apoptosis, cells were stained with PI, andthe percentage of infected living cells (GFP� PI�) in untreated samples(Pu) and TNF-�-treated samples (Pt) was determined. The TNF-�-specificapoptosis rate was calculated according to the following formula: (1 �(Pt/Pu)) � 100.

Results and DiscussionImpaired RelA�/� B lymphocyte generation in Rag-1�/� mice

Although absence of RelA results in embryonic lethality, RelA�/�

lymphocytes can be obtained after transfer of CD45.2 RelA�/� FLcells into irradiated congenic C57BL/6 CD45.1 recipient mice (23,25) (CD45.2 and CD45.1 are different isoforms that can be distin-guished from each other by Abs). However, one of the problemsencountered following this procedure was the presence of a small,but significant number of radioresistant recipient CD45.1 T cells(�15% of total T cells; data not shown) (25). We wanted to use asystem in which RelA�/� lymphocytes were generated in the ab-sence of contaminating recipient T cells. To this end, we usedRag-1-deficient mice, which completely lack mature T and B lym-phocytes (1).

Rag-1�/� mice were subjected to a lethal dose of 1200 rad ofgamma-radiation to completely destroy hemopoiesis (usingCD45.1 mice, we have found that this dose eliminates all recipientmouse B cells (B220� cells); and all B220� cells detected origi-nate from donor hemopoietic precursors). The studies described inthis work are based on three independent experiments in whichmice were examined 6–8 wk after adoptive transfer. As expected,injection of RelA�/� FL cells into irradiated Rag-1�/� mice re-sulted in reconstitution of splenic B cells (Fig. 1) and T cells (datanot shown). In contrast, the numbers of RelA�/� lymphocytesgenerated in Rag-1�/� mice were greatly reduced. Thus, Rag-1�/�

mice injected with RelA�/� FL cells had 10 times less splenic Blymphocytes than RelA�/� FL-injected Rag-1�/� mice (Fig. 1).FACS analysis of bone marrow (BM) cells from Rag-1�/� miceinjected with RelA�/� FL cells revealed an even more significantreduction in B220�IgM� developing B cells (�40-fold less thanin RelA�/� FL-injected mice) (Fig. 1). In addition, RelA�/� FL-injected Rag-1�/� mice typically had atrophied thymi, whileRelA�/� FL-injected Rag-1�/� mice had normal thymi. However,

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the RelA�/� splenic T cell population was not as significantlyreduced (2-fold less than RelA�/�) as the splenic B cell population(data not shown). We have therefore primarily focused on RelAfunction in B cells in this study. In addition, myelopoiesis wasunaffected, and in fact RelA�/� FL-injected Rag-1�/� miceshowed elevated numbers of granulocytes both in blood and tis-sues (see below). These results contrast with previous studies usingCD45.1 recipient mice, which showed that RelA�/� lymphocytegeneration is relatively normal (23, 25, 29). Thus, the geneticbackground of the recipient mouse also plays a crucial role inhemopoietic reconstitution.

Absence of TNFR1 rescues RelA�/� B lymphocyte generation inRag-1�/� mice

The development of RelA�/� lymphocytes (e.g., in CD45.1 mice)suggests that there is no cell-autonomous function for RelA inlymphocyte generation. Thus, one possibility is that RelA�/� Blymphocytes generated in Rag-1�/� mice fail to survive. BecauseRelA�/� hepatocyte loss and embryonic lethality are TNF-� de-pendent (14, 15), we determined whether loss of RelA�/� B lym-phocytes was also dependent on a TNF-�-induced mechanism. Totest this possibility, we injected RelA�/�TNFR1�/� FL cells intoirradiated Rag-1�/� mice (previous studies have indicated thatTNFR1 mediates most TNF-�-induced responses, including em-bryonic lethality in RelA�/� mice) (15). Interestingly, greatly en-hanced numbers of B cells were generated after injection ofRelA�/�TNFR1�/� FL cells into Rag-1�/� mice, with no differ-ence in the splenic B220� population compared withRelA�/�TNFR1�/� FL-transplanted mice (Fig. 2). In the BM, theB220� population was also considerably increased, although stillless than in RelA�/�TNFR1�/� FL-transplanted mice (Fig. 2).Thus, loss of RelA�/� B lymphocytes occurs in large part by aTNFR1-dependent mechanism. Between 6 and 8 wk, Rag-1�/�

mice transplanted with RelA�/�, but not RelA�/� FL cells alsoshowed severe infiltration of neutrophils into tissues in the appar-ent absence of infection (data not shown). Significantly, Rag-1�/�

mice in which RelA�/�TNFR1�/� FL cells had been injected alsoshowed this phenotype, suggesting that B lymphocyte loss and

neutrophil infiltration occur by different mechanisms, with only theformer being TNFR1 dependent.

In vitro development of RelA�/� B cells is normal despitesignificantly reduced �B site-binding activity

Our results suggest that the disappearance of RelA�/� B cells maybe due to TNF-�-induced loss of developing B cells (Figs. 1 and2). To test this possibility, we generated RelA�/� B lymphocytesfrom FL hemopoietic precursors in vitro. FL cells from RelA�/�

and RelA�/� embryos were obtained at day 13 of developmentand cultured in the presence of IL-7 for 8 days, after which non-adherent cells were analyzed by FACS. On day 8 of culture, �90%of the cells stained positive for B220 (Fig. 3A). The same resultswere obtained using CD19 as a marker for the B cells (data notshown). Significantly, double staining with anti-IgM HC showedless than 5% cells to be B220�IgM� in both RelA�/� andRelA�/� cell populations (Fig. 3A). Both RelA�/� and RelA�/�

showed very little cell surface expression of pre-BCR, but highintracellular expression. These results therefore indicate that thevast majority of cells are at the early pre-B cell stage. Significantly,these results also show normal in vitro development of RelA�/� Bcells.

We then analyzed �B site-binding activity in RelA�/� andRelA�/� cells, and in particular, the effect of TNF-� treatment on�B site-binding activity. FL-derived B cells incubated overnightwithout IL-7 were left untreated or treated with TNF-� for 2 h.Significantly, nuclear extracts from untreated RelA�/� B cellsshowed considerably decreased constitutive �B site-binding activ-ity compared with RelA�/� B cells (6.4-fold) (Fig. 3B). Aftertreatment with TNF-� for 2 h, a moderate increase in DNA-bind-ing activity was observed in RelA�/� (1.4-fold), but not inRelA�/� B cells (Fig. 3B). Our results also demonstrate equivalentOctamer 1 site binding, indicating that nuclear extracts fromRelA�/� B lymphocytes are not degraded. These results indicatethat RelA is a major component of �B site-binding activity presentin FL-derived pre-B cells. Notably, these results also suggest that

FIGURE 1. Impaired RelA�/� B lymphocyte generation in Rag-1�/�

mice. RelA�/� and RelA�/� FL cells were transplanted into Rag-1�/�

mice. Seven weeks after transplantation, recipient mice were sacrificed.Spleen and bone marrow cells were stained with anti-B220 and anti-IgMAbs before FACS analysis. B220�IgM� and B220�IgM� populations areshown in boxes along with the respective percentages.

FIGURE 2. Absence of TNFR1 rescues RelA�/� B lymphocyte gener-ation in Rag-1�/� mice. RelA�/�TNFR1�/� and RelA�/�TNFR1�/� FLcells were transplanted into Rag-1�/� mice. Seven weeks after transplan-tation, recipient mice were sacrificed. Spleen and bone marrow cells werestained with anti-B220 and anti-IgM Abs before FACS analysis.B220�IgM� and B220�IgM� populations are shown in boxes along withthe respective percentages.

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decreased �B site-binding activity in RelA�/� B cells has no ap-parent effect on pre-B cell development.

Sensitivity of developing RelA�/� B cells to TNF-� killing invitro

The results shown above indicate that loss of RelA�/� B cellsoccurs in a TNFR1-dependent manner. We next determinedwhether this was due to direct killing of RelA�/� B cells byTNF-�. To this end, B cells were removed from plates on day 8and cultured without IL-7 for 16 h. On day 9, mouse TNF-� wasadded to both RelA�/� and RelA�/� B cells for 48 h. Cells werethen stained with propidium iodide (PI), and FACS analysis wasperformed to determine the percentage of cell death. Our resultsrevealed significant differences between RelA�/� and RelA�/� B

cells in susceptibility to TNF-� killing (Fig. 4A). RelA�/� B cellsshowed 58% cell death after TNF-� treatment compared with 17%cell death in untreated cells. RelA�/� cells treated with TNF-�, incontrast, showed only 19% killing, while untreated cells showed12% cell death (Fig. 4A). Thus, RelA�/� B cells show 3-fold morecell death after TNF-� treatment compared with RelA�/� B cells.We also analyzed the surface expression of TNFR1 on RelA�/�

and RelA�/� B cells (Fig. 4B). The surface expression of TNFR1in developing B cells was low, but detectable. Nevertheless,RelA�/� and RelA�/� B cells were found to have similar levels ofTNFR1 cell surface expression. The mean fluorescence intensityvalues for TNFR1 were 20.11 in RelA�/� and 21.30 in RelA�/�

(the mean values in unstained samples were 12.63 in RelA�/� and12.71 in RelA�/�). We have found that the kinetics of cell deathof TNF-�-treated RelA�/� cells are rather slow, compared withmouse embryonic fibroblasts (30). Very little cell death was de-tected until 24 h, which typically progressed to 50–75% by 48 h indifferent experiments (Fig. 4 and data not shown). Wild-type Bcells also reproducibly show some cytotoxicity, but which wassignificantly less than in RelA�/� B cells. In contrast, untreatedRelA�/� and RelA�/� cells did not reproducibly show differences

FIGURE 3. In vitro development of RelA�/� B cells is normal despitesignificantly reduced �B site-binding activity. A, FL-derived RelA�/� andRelA�/� B cells were stained with anti-B220, anti-IgM, and anti-pre-BCRAbs. The percentage of different populations of cells is indicated. For pre-BCR, both cell surface and intracellular staining is shown. B, EMSA anal-ysis of nuclear extracts from FL-derived RelA�/� and RelA�/� pre-B cellswas performed after incubation of cells for 2 h with or without TNF-� (10ng/ml). The mouse H-2 binding site was used. As control, the constitutiveOct-1 binding site was used.

FIGURE 4. A, Sensitivity of developing RelA�/� B cells to TNF-� kill-ing in vitro. In vitro developed B cell precursors from RelA�/� andRelA�/� embryos were kept untreated or treated with TNF-� (10 ng/ml)for 48 h. The cells were then stained with PI, and FACS analysis wasperformed to determine the percentage of cell death. B, Surface expressionof TNFR1 in in vitro developed B cells. FL-derived RelA�/� and RelA�/�

B cells were stained with purified TNFR1 Abs. Filled histograms showunstained cells, while open histograms show TNFR1 Ab-stained cells.

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in amount of spontaneous cell death (12 and 17%, in the resultsshown in this study). Therefore, these results demonstrate that inthe absence of RelA, TNF-� can induce cell death of developingB cells. The disappearance of developing RelA�/� B cells in Rag-1�/� mice is thus most likely the result of direct killing by TNF-�.

Reduced levels of cFLIP and Bcl-2 in RelA�/� B cells

The TNF-� sensitivity and greatly reduced �B site-binding activityin RelA�/� B cells suggest that NF-�B-regulated gene expression,including expression of antiapoptotic genes, may be compromisedin these cells. To test this possibility, we first determined expres-sion levels of I-�B�, a well-characterized NF-�B target gene (31,32), in RelA�/� B cells. Northern blot analysis showed that theconstitutive levels of I-�B� mRNA were significantly reduced inRelA�/� B cells (Fig. 5A). Induction of I-�B� expression byTNF-� was also significantly reduced in RelA�/� cells (Fig. 5A).These results indicate that overall reduction in �B site-bindingactivity in RelA�/� B cells significantly impairs expression of theNF-�B target gene, I-�B�.

We next determined mRNA expression of three putative NF-�Btarget genes (33–35), which are also thought to be important inregulating lymphocyte survival: Bcl-2, Bcl-xL, and cFLIP (long)(cFLIP(L)). Notably, previous studies have shown that cFLIP(L)can prevent both Fas and TNFR1-induced cell death by blockingactivation of caspase 8 (36, 37). Strikingly, constitutive expressionof Bcl-2 and cFLIP(L) was significantly reduced in RelA�/� cells,while, in contrast, Bcl-xL levels were not significantly affected(Fig. 5B). Shorter exposure showed that Bcl-xL expression wasalso slightly reduced in untreated RelA�/� B cells, which wasincreased to levels similar to RelA�/� B cells after TNF-� treat-ment (data not shown). In contrast, no significant increase inmRNA levels of Bcl-2 and cFLIP was observed after TNF-� treat-ment in either cell type. These results thus indicate that the absenceof RelA considerably reduces constitutive Bcl-2 and cFLIP ex-

pression levels, consistent with reduced constitutive �B site-bind-ing activity in RelA�/� B cells. Thus, susceptibility of RelA�/� Bcells to TNF-� may be due to reduced expression of these anti-apoptotic genes.

Retrovirus-mediated expression of Bcl-2 or cFLIP inhibitsTNF-� killing of RelA�/� B cells

To further study the potential role of Bcl-2 and cFLIP in regulatingTNF-�-induced cell death, we infected RelA�/� B cells with ret-roviruses expressing these genes. For these studies, we used theMIG retroviral vector (27). B cells from two different RelA�/�

embryos were obtained, as described above. On days 6 and 7 ofculture, the developing B cells were infected with MIG, MIG-Bcl-2, or MIG-cFLIP retroviral supernatants. The efficiency of in-fection of B cells (i.e., GFP� cells) is shown in Fig. 6A. In com-parison with MIG-infected RelA�/� B cells, Bcl-2 and cFLIPexpression significantly reduced TNF-�-specific killing ofRelA�/� B cells (Fig. 6B). These results therefore suggest thatsusceptibility of RelA�/� B cells to TNF-�-induced cell deathmay be due to reduced expression of Bcl-2 and cFLIP.

The findings presented in this work have revealed a key role forthe RelA subunit of NF-�B in regulating B cell survival. To studythe function of RelA in lymphocyte development, we adoptivelytransferred RelA�/� FL cells into lethally irradiated mature lym-phocyte-deficient Rag-1�/� mice. Our results indicate greatly im-paired generation of RelA�/� B lymphocytes in Rag-1�/� mice.Significantly, in the absence of TNFR1 signaling, the disappear-ance of B cells was largely rescued. Our in vitro results show thatin RelA�/� developing B cells, both constitutive and TNF-�-in-duced NF-�B activity is dramatically reduced. Significantly,RelA�/�, but not RelA�/� B cells, underwent cell death in thepresence of TNF-�, demonstrating that TNF-� is directly cytotoxicto RelA�/� B cells. Our results further suggest that susceptibilityto TNF-� is due to reduced constitutive expression of Bcl-2 andcFLIP in RelA�/� cells. Interestingly, while TNF-� treatmentmoderately increased NF-�B activity in RelA�/� cells, expressionof Bcl-2 or cFLIP was not affected by TNF-�. The reason for thisis presently unclear, but may suggest a requirement for additionaltranscriptional factors for enhancement of expression of thesegenes, which may not be induced by TNF-�. Together, these re-sults suggest that a key function of RelA-containing NF-�B com-plexes in developing B cells is to prevent TNF-� killing by en-hancing expression of antiapoptotic genes.

Interestingly, previous studies have shown that, unlike cRel,RelA is not required for mature B cell proliferation (29). Further-more, cRel-containing complexes are predominant in mature Bcells, most likely due to inefficient removal of cRel from nuclei(38, 39). Our results indicate that RelA-containing complexes arepredominant in pre-B cells (Fig. 3). In addition, we have alsofound that pre-B cells lacking cRel (and p50) are not susceptible toTNF-� killing (data not shown). Together, these results suggestdistinct functions for the RelA and cRel in B cells at differentdevelopmental stages: RelA inhibits TNF-� cytotoxicity in devel-oping B cells, but is not essential for mature B cell proliferation,while for cRel, the converse is true.

In contrast to Bcl-2 and cFLIP, we have found that significantexpression of Bcl-xL still occurs in RelA�/� B cells. Successfulrearrangement of the Ig HC leading to pre-BCR expression is es-sential for in vivo pre-B cell survival. Significantly, induction ofBcl-xL expression by the pre-BCR may be one of the key mech-anisms for pre-B cell survival (4). Our results indicate that RelA isnot essential for Bcl-xL expression in pre-B cells. In addition, thereis no cell-intrinsic requirement for RelA or p50 � RelA (23, 29)

FIGURE 5. Reduced levels of cFLIP and Bcl-2 in RelA�/� B cells. A,Northern blotting using I-�B� and �-actin-specific probes. Total RNA (10�g) was used per sample. B, RPA was performed using 40 �g of total RNAfrom RelA�/� and RelA�/� in in vitro developed B cells. The cells wereuntreated or treated with TNF-� (10 ng/ml) for 2 or 6 h. Mobilities cor-responding to Bcl-xL, Bcl-2, cFLIP, and �-actin are indicated.

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in B cell development. Together, these findings suggest that thekey function of RelA-containing complexes during B cell devel-opment may be to prevent TNF-� killing, but not to regulate sur-vival pathways that play an integral role in B cell development,such as pre-BCR-induced Bcl-xL expression.

The source of cytotoxic TNF-� that kills developing B cells ispresently unclear. However, it is likely that stromal cells in thebone marrow, especially macrophages, may be an importantsource. These cells are present in close proximity to developing Bcells within the BM microenvironment and are known to be im-portant producers of TNF-�. Our results also indicate an importantrole for the recipient mouse background in determining the out-come of hemopoietic reconstitution. In contrast to Rag-1�/�-in-jected mice, significant numbers of RelA�/� B lymphocytes weregenerated after transfer of RelA�/� FL cells into CD45.1 congenicmice (�2-fold less than in RelA�/� FL-injected mice; data notshown) (23, 25, 29). These results suggest that RelA�/� lympho-

cytes are generated and survive after transfer of RelA�/� FL cellsinto lymphocyte-containing mice (CD45.1 mice), but not in micedevoid of lymphocytes (Rag-1�/� mice). Significantly, we havefound that even after receiving a lethal dose of radiation, CD45.1mice retain a small, but significant number of radioresistant recip-ient CD45.1 T cells (�15% of total T cells; data not shown) (25).Thus, it is possible that radioresistant WT T cells may enhanceRelA�/� lymphocyte survival by inhibiting TNF-� generationand/or cytotoxic mechanisms.

In conclusion, our findings have revealed a key role for the RelAsubunit of NF-�B in regulating B cell survival. Although RelA isapparently not required for B cell development, it plays a crucialrole in preventing TNF-� killing of B cells. Our results furthersuggest that RelA may inhibit TNF-� killing by regulating expres-sion of the key antiapoptotic genes, Bcl-2 and cFLIP. Together,these findings help define a key mechanism that plays a crucial rolein developing B cell survival.

FIGURE 6. Retrovirus-mediated expression of Bcl-2 or cFLIP inhibits TNF-� killing of RelA�/� B cells. A, FL-derived B cells from two differentRelA�/� embryos were obtained as above. On days 6 and 7 of culture, they were infected with MIG, Bcl-2-MIG, and cFLIP-MIG retroviral supernatants.The percentage of infection of B cell from embryos 1 and 2 was determined by FACS from GFP fluorescence on day 9. The cells were also stained withB220 as marker for B cells. B, After infection, TNF-� was added at a final concentration of 10 ng/ml for 48 h. Following this incubation, the B cells wereremoved from the wells and stained with PI, and FACS analysis was performed to determine TNF-�-specific apoptosis of the infected cells (see Materialsand Methods). The percentage of TNF-�-specific cell death is shown in MIG-, Bcl-2-MIG-, and cFLIP-MIG-infected cells.

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AcknowledgmentsWe thank Dr. Christopher Roman (Down State University, New York, NY)for very helpful discussions. We also thank Dr. Luk Van Parijs (MIT) forproviding the MIG vectors, Fang Li for technical assistance, and membersof the lab for discussions.

References1. Mombaerts, P., J. Iacomini, R. S. Johnson, K. Herrup, S. Tonegawa, and

V. E. Papaioannou. 1992. RAG-1-deficient mice have no mature B and T lym-phocytes. Cell 68:869.

2. Loffert, D., S. Schaal, A. Ehlich, R. R. Hardy, Y. R. Zou, W. Muller, andK. Rajewsky. 1994. Early B-cell development in the mouse: insights from mu-tations introduced by gene targeting. Immunol. Rev. 137:135.

3. Chao, D. T., and S. J. Korsmeyer. 1998. BCL-2 family: regulators of cell death.Annu. Rev. Immunol. 16:395.

4. Grillot, D. A., R. Merino, J. C. Pena, W. C. Fanslow, F. D. Finkelman,C. B. Thompson, and G. Nunez. 1996. bcl-x exhibits regulated expression duringB cell development and activation and modulates lymphocyte survival in trans-genic mice. J. Exp. Med. 183:381.

5. Fang, W., D. L. Mueller, C. A. Pennell, J. J. Rivard, Y. S. Li, R. R. Hardy,M. S. Schlissel, and T. W. Behrens. 1996. Frequent aberrant immunoglobulingene rearrangements in pro-B cells revealed by a Bcl-xL transgene. Immunity4:291.

6. Van Parijs, L., and A. K. Abbas. 1998. Homeostasis and self-tolerance in theimmune system: turning lymphocytes off. Science 280:243.

7. Lenardo, M., K. M. Chan, F. Hornung, H. McFarland, R. Siegel, J. Wang, andL. Zheng. 1999. Mature T lymphocyte apoptosis: immune regulation in a dy-namic and unpredictable antigenic environment. Annu. Rev. Immunol. 17:221.

8. Zhou, T., C. K. Edwards, P. Yang, Z. Wang, H. Bluethmann, and J. D. Mountz.1996. Greatly accelerated lymphadenopathy and autoimmune disease in lpr micelacking tumor necrosis factor receptor I. J. Immunol. 156:2661.

9. Baldwin, A. S., Jr. 1996. The NF-�B and I�B proteins: new discoveries andinsights. Annu. Rev. Immunol. 14:649.

10. Ghosh, S., M. J. May, and E. B. Kopp. 1998. NF-�B and Rel proteins: evolu-tionarily conserved mediators of immune responses. Annu. Rev. Immunol.16:225.

11. Karin, M., and M. Delhase. 2000. The I�B kinase (IKK) and NF-�B: key ele-ments of proinflammatory signalling. Semin. Immunol. 12:85.

12. Beg, A. A., W. C. Sha, R. T. Bronson, S. Ghosh, and D. Baltimore. 1995. Em-bryonic lethality and liver degeneration in mice lacking the RelA component ofNF-�B. Nature 376:167.

13. Doi, T. S., T. Takahashi, O. Taguchi, T. Azuma, and Y. Obata. 1997. NF-�BRelA-deficient lymphocytes: normal development of T cells and B cells, impairedproduction of IgA and IgG1 and reduced proliferative responses. J. Exp. Med.185:953.

14. Doi, T. S., M. W. Marino, T. Takahashi, T. Yoshida, T. Sakakura, L. J. Old, andY. Obata. 1999. Absence of tumor necrosis factor rescues RelA-deficient micefrom embryonic lethality. Proc. Natl. Acad. Sci. USA 96:2994.

15. Alcamo, E., J. P. Mizgerd, B. H. Horwitz, R. Bronson, A. A. Beg, M. Scott,C. M. Doerschuk, R. O. Hynes, and D. Baltimore. 2001. Targeted mutation ofTNF receptor I rescues the RelA-deficient mouse and reveals a critical role forNF-�B in leukocyte recruitment. J. Immunol. 167:1592.

16. Kontgen, F., R. J. Grumont, A. Strasser, D. Metcalf, R. Li, D. Tarlinton, andS. Gerondakis. 1995. Mice lacking the c-rel proto-oncogene exhibit defects inlymphocyte proliferation, humoral immunity, and interleukin-2 expression.Genes Dev. 9:1965.

17. Sha, W. C., H. C. Liou, E. I. Tuomanen, and D. Baltimore. 1995. Targeteddisruption of the p50 subunit of NF-�B leads to multifocal defects in immuneresponses. Cell 80:321.

18. Grumont, R. J., I. J. Rourke, L. A. O’Reilly, A. Strasser, K. Miyake, W. Sha, andS. Gerondakis. 1998. B lymphocytes differentially use the Rel and nuclear factor�B1 (NF-�B1) transcription factors to regulate cell cycle progression and apo-ptosis in quiescent and mitogen-activated cells. J. Exp. Med. 187:663.

19. Owyang, A. M., J. R. Tumang, B. R. Schram, C. Y. Hsia, T. W. Behrens,T. L. Rothstein, and H. C. Liou. 2001. c-Rel is required for the protection of Bcells from antigen receptor-mediated, but not Fas-mediated, apoptosis. J. Immu-nol. 167:4948.

20. Andjelic, S., C. Hsia, H. Suzuki, T. Kadowaki, S. Koyasu, and H. C. Liou. 2000.Phosphatidylinositol 3-kinase and NF-�B/Rel are at the divergence of CD40-mediated proliferation and survival pathways. J. Immunol. 165:3860.

21. Cariappa, A., H. C. Liou, B. H. Horwitz, and S. Pillai. 2000. Nuclear factor �Bis required for the development of marginal zone B lymphocytes. J. Exp. Med.192:1175.

22. Zheng, Y., M. Vig, J. Lyons, L. Van Parijs, and A. A. Beg. 2003. Combineddeficiency of p50 and cRel in CD4� T cells reveals an essential requirement forNF-�B in regulating mature T cell survival and in vivo function. J. Exp. Med.197:861.

23. Horwitz, B. H., M. L. Scott, S. R. Cherry, R. T. Bronson, and D. Baltimore. 1997.Failure of lymphopoiesis after adoptive transfer of NF-�B-deficient fetal livercells. Immunity 6:765.

24. Senftleben, U., Z. W. Li, V. Baud, and M. Karin. 2001. IKK� is essential forprotecting T cells from TNF�-induced apoptosis. Immunity 14:217.

25. Zheng, Y., F. Ouaaz, P. Bruzzo, V. Singh, S. Gerondakis, and A. A. Beg. 2001.NF-�B RelA (p65) is essential for TNF-�-induced fas expression but dispensablefor both TCR-induced expression and activation-induced cell death. J. Immunol.166:4949.

26. Beg, A. A., T. S. Finco, P. V. Nantermet, and A. S. Baldwin. 1993. Tumornecrosis factor and interleukin-1 lead to phosphorylation and loss of I�B: a mech-anism for NF-�B activation. Mol. Cell. Biol. 13:3301.

27. Van Parijs, L., Y. Refaeli, A. K. Abbas, and D. Baltimore. 1999. Autoimmunityas a consequence of retrovirus-mediated expression of c-FLIP in lymphocytes.Immunity 11:763.

28. Pear, W. S., G. P. Nolan, M. L. Scott, and D. Baltimore. 1993. Production ofhigh-titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci.USA 90:8392.

29. Horwitz, B. H., P. Zelazowski, Y. Shen, K. M. Wolcott, M. L. Scott,D. Baltimore, and C. M. Snapper. 1999. The p65 subunit of NF-�B is redundantwith p50 during B cell proliferative responses, and is required for germline CH

transcription and class switching to IgG3. J. Immunol. 162:1941.30. Beg, A. A., and D. Baltimore. 1996. An essential role for NF-�B in preventing

TNF-�-induced cell death. Science 274:782.31. Scott, M. L., T. Fujita, H.-C. Liou, G. P. Nolan, and D. Baltimore. 1993. The p65

subunit of NF-�B regulates I�B by two distinct mechanisms. Genes Dev. 7:1266.32. Sun, S.-C., P. A. Ganchi, D. W. Ballard, and W. C. Greene. 1993. NF-�B controls

expression of inhibitor I�B�: evidence for an inducible autoregulatory pathway.Science 259:1912.

33. Chen, C., L. C. Edelstein, and C. Gelinas. 2000. The Rel/NF-�B family directlyactivates expression of the apoptosis inhibitor Bcl-xL. Mol. Cell. Biol. 20:2687.

34. Grossmann, M., L. A. O’Reilly, R. Gugasyan, A. Strasser, J. M. Adams, andS. Gerondakis. 2000. The anti-apoptotic activities of Rel and RelA required dur-ing B-cell maturation involve the regulation of bcl-2 expression. EMBO J.19:6351.

35. Micheau, O., S. Lens, O. Gaide, K. Alevizopoulos, and J. Tschopp. 2001. NF-�Bsignals induce the expression of c-FLIP. Mol. Cell. Biol. 21:5299.

36. Yeh, W. C., A. Itie, A. J. Elia, M. Ng, H. B. Shu, A. Wakeham, C. Mirtsos,N. Suzuki, M. Bonnard, D. V. Goeddel, and T. W. Mak. 2000. Requirement forCasper (c-FLIP) in regulation of death receptor-induced apoptosis and embryonicdevelopment. Immunity 12:633.

37. Irmler, M., M. Thome, M. Hahne, P. Schneider, K. Hofmann, V. Steiner,J. L. Bodmer, M. Schroter, K. Burns, C. Mattmann, et al. 1997. Inhibition ofdeath receptor signals by cellular FLIP. Nature 388:190.

38. Tam, W. F., W. Wang, and R. Sen. 2001. Cell-specific association and shuttlingof I�B� provides a mechanism for nuclear NF-�B in B lymphocytes. Mol. Cell.Biol. 21:4837.

39. Liou, H.-C., W. C. Sha, M. L. Scott, and D. Baltimore. 1994. Sequential induc-tion of NF-�B/Rel family proteins during B-cell terminal differentiation. Mol.Cell. Biol. 14:5349.

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