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Estefania Claudio, Keith Brown, Sun Park, Hongshan Wang and Ulrich SiebenlistPublished online: 23 September 2002, doi:10.1038/ni842

NF-κB is usually activated by signal-induced, ubiquitin-mediated degradation of its inhibitor, IκB.Thisprocess is initiated by phosphorylation of IκB by the IκB kinase (IKK) complex, predominantly by theIKKβ catalytic subunit, and requires the regulatory subunit IKKγ (NEMO). Another activationpathway, with no known physiological inducers, involves ubiquitin-mediated processing of the NF-κB2inhibitory protein p100 and is dependent on phosphorylation of p100 by IKKα .We show here that Bcell–activating factor (BAFF) activates this second pathway and that this requires the BAFF receptor(BAFF-R), the NF-κB–inducing kinase (NIK) and protein synthesis, but not NEMO. This NEMO-independent cascade is physiologically relevant for the survival and, hence, progression of maturingsplenic B cells.

Laboratory of Immunoregulation, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1876, USA.Correspondence should be addressed to U. S. (us3n@nih.gov).

BAFF-induced NEMO-independent processing of NF-κB2 in maturing B cells

NF-κB transcription factors are critical for the regulation of immuneand inflammatory processes and for stress responses in general1,2. Thesefactors exist as dimers composed of members of the Rel–NF-κB fami-ly of polypeptides; in mammals these are p65 (also known as RelA), c-Rel (also known as Rel), RelB, the NF-κB1 protein p50 and the NF-κB2 protein p52. Most dimer combinations are possible, except thatRelB dimerizes with p50 or p52 only. p50 and p52 are generated fromthe NH2-terminal portions of the NF-κB1 protein p105 and the NF-κB2protein p100, respectively. In the absence of proper stimulatory signals,NF-κB complexes are normally retained in the cytoplasm by associa-tion with the small inhibitory IκB proteins IκBα, IκBβ and IκBε. p105and p100 can also function as inhibitors, through IκB-like ankyrindomains in their COOH-terminal portions1,2. Inflammatory stimuli ini-tiate the canonical NF-κB activation pathway that leads to rapid phos-phorylation of the small IκB proteins by the IKK complex, followed byubiquitin-dependent degradation. IKK is composed of two catalyticsubunits, IKKα and IKKβ, and the regulatory subunit IKKγ (NEMO).The IKKβ subunit is sufficient for mediating the inflammatory signals,whereas IKKα may serve a regulatory role and participate in otherphysiological roles. The IKK-initiated destruction of IκB inhibitorsleads to translocation of NF-κB into the nucleus and transcription oftarget genes.

Mouse models in which one or more of the NF-κB gene family aredisrupted have been used to determine the distinct and critical functionsof the different encoded polypeptides. Such studies have shown thatNF-κB is not only critical during immune responses but is also impor-tant during the generation and maintenance of secondary lymphoidstructures and during various developmental transitions of hematopoi-etic cells1,2 (see below). For example, NF-κB2–deficient mice aredefective in their T-dependent antigen responses, partly due to abnor-malities in splenic architecture, lymph nodes and Peyer’s patches thatinterfere with germinal center formation. These mutant mice also havepartially reduced numbers of mature naïve B cells3,4. NF-κB1–deficientmice show no discernable defects during the development of mature

recirculating B cells, but subsequent B cell differentiation and functionare partially impaired5. NF-κB1 and NF-κB2 (NF-κB1/2) double-defi-cient mice show more severe phenotypic defects than those with a sin-gle deficiency because of the partly redundant functions of these close-ly related proteins. Double-deficient mice are osteopetrotic due to anearly block in osteoclast development6,7, and they show a completeblock in generation of mature B cells6. Both blocks result from defectsintrinsic to these hematopoietic cell types.

The tumor necrosis factor (TNF) family member B cell activatingfactor (BAFF, also known as BLyS, TALL-1, zTNF4 or THANK) isrequired for generation and maintenance of the mature B cell pool8–11.BAFF can bind three receptors called BAFF receptor (BAFF-R, alsoknown as BR3), B cell maturation antigen (BCMA) and transmem-brane activator and CAML interactor (TACI). BCMA and TACI, butnot BAFF-R, are also recognized by a proliferation-inducing ligand(APRIL), another member of the TNF family8–12. BAFF-R is requiredfor the B cell developmental function of BAFF. Transgene-mediatedoverexpression of BAFF causes a breakdown of B cell tolerance andleads to a systemic lupus erythematosus–like condition in mice.Humans with various autoimmune diseases have increased concentra-tions of BAFF in sera8–11,13–15. B cell development in BAFF-deficientmice is completely blocked at the transitional stage in the spleen; theblock occurs at a critical point for tolerance induction, just before fullB cell maturation to naïve follicular and marginal zone B cells16. In con-trast to its biological activities, relatively little is known about howBAFF mediates its functions within B cells.

Given the phenotypic similarity between BAFF-deficient and NF-κB1/2 double-deficient mice, we examined whether NF-κB is a criticalmediator of BAFF signaling in maturing B cells. We show here thatBAFF induced a second pathway to activate NF-κB by causing pro-cessing of the NF-κB2 protein p100 to p52. Processing was dependenton protein synthesis and was mediated by the kinases NF-κB–inducingkinase (NIK) and IKKα. It did not require NEMO and, hence, wasindependent of the canonical IKK pathway of NF-κB activation. The

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BAFF-induced p100 processing enhanced B cell survival and, in turn,progression of immature transitional to mature B cells. Thus, ourresults show that BAFF is a physiological signal for the recently dis-covered p100 processing pathway that leads to activation of NF-κB andthey demonstrate the importance of this pathway in B cell maturation.

ResultsB cell maturation dependent on NF-κB1/2To identify the stage at which the development of B cells from NF-κB1/2 double-deficient mice was blocked, we investigated the expres-sion of surface markers on their splenic B cells. These were comprisedentirely of early immunoglobulin M–positive (IgM+)CD21– transitional1 (T1) B cells, the most recent splenic immigrants from bone marrow(BM), whereas the more advanced IgM+CD21+ T2, marginal zone andmature B cell populations present in wild-type mice were absent in themutant mice (Fig. 1a, for details of the marker expression see17). Basedon four separate experiments, essentially no T2 or mature B cells couldbe detected in any of the double-deficient mice; the total numbers ofwild-type and double-deficient T1 B cells were 4.3 × 106 ± 2.7 × 106 and1.22 × 106 ± 0.7 × 106, respectively. NF-κB1/2 double-deficient splenicB cells also lacked expression of CD23 or CD62L, confirming theirearly transitional stage17 (data not shown). The inability of these mutantB cells to progress further was intrinsic to B cells and cell-autonomous(unpublished results). In contrast to NF-κB1/2 double-deficient B cells,Bruton’s tyrosine kinase (Btk)-deficient B cells were partially blockedat the more advanced T2 stage18 (and unpublished data). Btk is requiredto relay B cell receptor (BCR) signals that activate NF-κB in mature Bcells, but it does not appear to be required for progression of T1 cells.Because BAFF-deficient mice also lack T2 and mature B cells, but docontain T1 B cells16, their B cells may be blocked at or about the samestage of development as those of NF-κB1/2 double-deficient mice.

NF-κB1/2 extends B cell lifetimesTo assess whether NF-κB1/2 could mediate the survival and maturationfunctions of BAFF8–12,16, we determined whether these NF-κB subunitswere required for the survival of maturing B cells. Cultured NF-κB1/2double-deficient early transitional B cells were intrinsically more apop-totic than wild-type cells (Fig. 1b), as judged by their more rapid rateof increase in numbers of Trypan blue–staining dead cells and highernumbers of merocyanine-staining early apoptotic cells among remain-ing live cells. Consistent with this, two anti-apoptotic genes—Bcl2a1(encoding A1) and Bcl2 (encoding Bcl2)—that have been implicated in

B cell survival19 were both expressed in lower amounts in NF-κB1/2double-deficient early T1 B cells (Fig. 1c). These data further demon-strate the similarity of B cells deficient in BAFF signaling to those lack-ing NF-κB1/2.

NF-κB1/2–dependent BAFF functionNext we asked whether BAFF function in transitional B cells requiresNF-κB1/2. Freshly generated transitional B cells were obtained from invitro BM cultures, which were established without stromal cells20. Afterseveral days in interleukin 7 (IL-7), essentially pure B cell populationswere obtained, which consisted primarily of pro-B and late pro-B stagecells, which lacked T2 or mature B cells20 (and data not shown).Withdrawal of IL-7 stopped expansion of precursor cells and increasedthe proportion of immature IgM+ and transitional IgM+ and IgD+ Bcells. Based on their surface marker expression (CD23–CD21–CD62L–

IgMhiIgDlo), the most advance-staged cells in BM cultures resemblethose leaving the BM and entering spleens in vivo17,20 and thus representa good model system with which to investigate the possible effects ofBAFF on early transitional B cells.

Exposure of wild-type BM cultures to BAFF for 4 days (after IL-7withdrawal) increased the fraction of IgD+ cells relative to that inuntreated cultures (Fig. 2a). Relative to no treatment, BAFF increasedthe percentage of IgD+ cells by 4.6 ± 1.8-fold (n = 9). It also increasedthe total number of IgD+ cells in the population by 15.33 ± 2.9-fold (n= 3) compared to the numbers present at the start (after IL-7 withdraw-al). In contrast, BAFF failed to increase the numbers of IgD+ cells pre-sent in NF-κB1/2 double-deficient BM cultures (Fig. 2a). Even in theabsence of BAFF, numbers of IgD+ cells were already consistentlylower in mutant compared to WT cultures. In addition to increasing thenumbers of transitional B cells, BAFF also induced expression of high-er amounts of IgD and B220 in WT cultures (Fig. 2b). The relativelyhigher percentages of immature and transitional B cells we observedwere due to the long incubation time of over 6 days after IL-7 with-drawal, a time at which the culture had lost most of its earlier-stagedcells. An increase in expression of B220 and IgD is also seen during thematuration of transitional splenic B cells in vivo21. Neither TNF norRANK ligand (RANKL) had any of these effects on transitional B cellsin the BM cultures (Fig. 2b). These results therefore showed that BAFFallowed the transitional B cells in culture to advance toward a more dif-ferentiated state, possibly due to prolonged survival times. However,these cells did not appear to progress fully to the T2 stage under ourculture conditions, as shown by their lack of detectable CD23 surface

Figure 1. NF-κB1/2 double-deficient B cells fail to progress past the T1 stage and are intrinsically more apoptotic. (a) FACS analysis of splenic B cells from3-week-old WT and age-matched NF-κB1/2 double-deficient (dKO) mice, stained with antibodies to CD21and IgM. Because NF-κB1/2 double-deficient mice begin to dieafter weaning, these mice had to be analyzed by 3 weeks of age. M, mature B cells; MZ, marginal zone B cells. (b) (Left panel) Negatively selected WT and dKO T1 B cellswere placed in culture and cell viability was measured by Trypan blue exclusion at 0 and 24 h. (b) (Right panel) Merocyanine staining marks apoptotic cells in WT and dKOnegatively selected T1 B cells placed in culture for 16 and 48 h.Analysis was gated on B220+ live cells (see Methods). Data are from representative experiments, each donein triplicate. Results were confirmed with two additional independent experiments. (c) Real-time PCR analysis of Bcl-2 and A1 expression in negatively selected T1 WT anddKO B cells. Data represent the means of three independent experiments.

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expression (data not shown). This suggested that in vivo, BAFF mayfunction in concert with another unknown signal to completely drive Bcells into the T2 and mature B cell stages. The effect of BAFF in BMcultures was confirmed with freshly isolated immatureCD43–CD62L–BM cells. A 4-day incubation with BAFF increased thepercentage and total number of IgD+ B cells approximately twofoldcompared to untreated cultures (Fig. 2c). These results demonstratedthat the BAFF-induced increase in T1 B cell numbers in BM cultureswas dependent on NF-κB factors.

BAFF function requires BAFF-R, NF-κB2 and NIKThe loss of either BCMA or TACI receptors has no apparent effect onB cell maturation, but a natural mutation in BAFF-R severely impairsB cell maturation in A/WySnJ mice12. To determine whether this recep-tor mediated the survival effects of BAFF, we established BM culturesfrom A/WySnJ mice and treated these with BAFF. BAFF did notmarkedly increase IgD+ cell numbers in BM cultures from this mutant,which indicated that a wild-type BAFF-R is required for normal BAFFaction in these cells (Fig. 3a) We verified that BAFF-R mRNA isexpressed from the immature B cell stage onwards in all mice strainswe treated with BAFF (data not shown).

To determine whether NF-κB1, NF-κB2 or both mediated the effectsof BAFF, we tested single-knockout mice. The addition of BAFF to BMcultures from NF-κB1–deficient mice increased relative and total IgD+

cell numbers, consistent with the apparently normal B cell developmentin these mice (apart from a reported reduction in marginal zone Bcells)22. However, BM cultures from NF-κB2–deficient mice failed toshow this response, which suggested that NF-κB2, but not NF-κB1,was critical for the increase in T1 B cells in response to BAFF (Fig. 3a).Additional experiments confirmed the results obtained with the threemutant mice in Fig. 3a (data not shown).

In apparent contrast to the BM cultures, NF-κB2 appears not beabsolutely required in vivo because NF-κB2–deficient mice do gener-ate mature B cells, albeit in reduced amounts3. Mature B cells were alsogenerated after long-term adoptive BM transfers from these mutantmice into recombination-activating gene 1–deficient (RAG-1–/–) mice,although again the mature pool was only partially reconstituted (datanot shown). Therefore we directly tested the extent of the B cell defectassociated with lack of NF-κB2. Isolated splenic early transitional

B cells were transferred into RAG-1–deficient mice and their short-term fate was analyzed. Whereas wild-type Ig-bearing B transitionalcells could be readily detected in recipient spleens 40 h after transfer,with a considerable fraction of these cells having progressed to themature IgDhiIgMlo stage, only a few NF-κB2–deficient Ig-bearing Bcells could be detected in recipient spleens (Fig. 3b). Based on fourindependent and parallel 40-h transfers, the following percentages wererecorded: WT mature cells 0.73 ± 0.47%, transitional 1.22 ± 0.72%;NF-κB2–deficient mature cells 0.09 ± 0.03%, transitional 0.13 ±0.04%. Analysis of total cell numbers revealed ratios that were similarto the ratios derived from the percentages, with more than sevenfoldmore wild-type than NF-κB2–deficient B cells detected. The resultswere confirmed with additional experiments analyzed 48 h and 18 hafter transfer (data not shown). Slightly more of the transferred NF-κB2–deficient B cells were still detectable by 18 h compared to 40 hafter transfer, but their numbers were already reduced relative to thewild-type transfers.

These results revealed an important contribution(s) of NF-κB2 toearly transitional B cell survival, although this function appears not tobe absolutely essential in vivo, where compensatory mechanismsappear to permit an eventual partial repopulation of the mature B cellpool. The key contribution made by NF-κB2 to the survival of trans-ferred T1 B cells in vivo is consistent with the need for NF-κB2 in theBAFF-induced accumulation of IgD+ B cells in BM cultures.

The survival effects of BAFF must therefore be mediated, at least inpart, by NF-κB2. In most cells, the p100 form of NF-κB2 predomi-nates, whereas little of the p52 form is present1. We therefore postulat-ed that BAFF might act by inducing the processing of p100 to p52. Thiswould not only facilitate the generation of p52-containing NF-κBdimers, but also relieve p100-mediated cytoplasmic sequestration andinhibition of NF-κB complexes23. Ubiquitin-dependent processing ofp100 is reported to be dependent on NIK24 and on IKKα-mediatedphosphorylation of p100 at the COOH-terminal domain25, in a manneranalogous to the IKK complex-dependent phosphorylation and degra-dation of IκBα. Thus, we investigated whether NIK is involved inBAFF function. A naturally occurring mutation in NIK, found in alymice, is unable to induce p100 processing in transfection experiments24.aly/aly B cells have partially impaired development because adoptivetransfer of aly/aly BM resulted in poor reconstitution of B cell follicles

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Figure 2. BAFF promotes accumulation of IgD+ transitional B cells in in vitro BM cultures from wild-type but not from NF-κB1/2 double-deficient mice. (a) FACS analysis of wild-type (WT) and NF-κB1/2 double-deficient (dKO) BM cultures (see Methods).The percentages of IgM+IgD+ cells are indicated; one representative of three independent matched experiments is shown. BAFF treatmentlasted for 4 days. (b) FACS analysis of WT BM cultures that were left untreated or treated with BAFF as in a (except that treatment wasextended to just over 6 days) or with TNF-α or RANKL. One representative of three independent matched experiments is shown; the per-centages of IgM+ and IgD+ cells are indicated. (c) Freshly isolated B cells from BM enriched for immature (CD43–CD62L–) B cells wereplaced in culture with or without BAFF for 4 days.The percentages of IgD+ cells are indicated. Compared to the starting population, BAFFalso increased the total numbers of IgD+ cells more than twofold; similar data were obtained in a second independent experiment.

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and functions26 (and data not shown). BM cultures from aly/aly micewere tested and found, based on the failure to accumulate IgD+ cells, tobe nearly unresponsive to BAFF treatment; in contrast, aly/+ controlcultures behaved essentially like the wild-type cultures describedabove, showing increases in both the percentages and total numbers ofIgD+IgM+ cells (Fig. 3c). These data implied that the BAFF-inducedincrease in transitional B cells in BM cultures requires NIK.

Together our results indicated that BAFF acts to promote the survivalof transitional B cells through a signaling pathway that requires BAFF-R, NF-κB2 and NIK. In addition, NIK’s activities are likely mediatedby IKKα, as B cell precursors that are mutated or deficient in IKKαalso fail to fully reconstitute the mature B cell pool25,27.

BAFF induces Bcl-2 in transitional B cellsTo confirm the ability of BAFF to promote survival in transitional B cells,its effects on anti-apoptotic gene expression were tested. This analysiswas inspired by reports that BAFF failed to promote proliferation ofmature B cells but did increase their expression of anti-apoptoticgenes8–11,28. We found that prolonged exposure to BAFF led to increasedexpression of the surface marker B220 and the anti-apoptotic factor Bcl-2 in IgD+ cells in BM cultures, as analyzed by intracellular staining (Fig.4). Real-time polymerase chain reaction (PCR) also showed a BAFF-induced increase in Bcl-2 and Bcl-xL mRNA. NF-κB1–deficient B cellsresponded to BAFF in a similar manner to wild-type B cells, whereasNF-κB2–deficient cells did not (data not shown). BAFF also reducedexpression of an early apoptotic marker (merocyanine staining) on IgD+

cells in BM cultures (data not shown). These results supported the ideathat BAFF induces anti-apoptotic factors and acts as a survival factor intransitional B cells as represented in the in vitro BM model.

BAFF induces p100 processingTo test whether BAFF induced processing of NF-κB2 p100 to p52, westimulated freshly isolated immature BM cells (which were depleted inmature and pro-B cells) and total splenic B cells (primarily mature Bcells, which were preincubated in medium for 24–36 h before treat-ment) ex vivo with BAFF. Without preincubation, splenic B cells con-tained relatively high amounts of p52 at the time of isolation, presum-ably due to BAFF exposure in vivo. Preincubation was not necessary inthe case of BM B cells. In both B cell populations, BAFF induced p100processing, which led to a corresponding loss in p100 (Fig. 5a). Therewas no apparent change in the amount of p50, which was constitutive-ly high in the presence or absence of BAFF (data not shown). Neitherlipopolysaccharide (LPS) nor anti-IgM induced detectable processingto p52 in these cells (Fig. 5b). Thus, BAFF induced p100 processing inboth immature and mature B cells.

To identify the requirements for BAFF-induced p100 processing, wetested mutants lacking intact NIK and BAFF-R. In contrast to het-erozygous aly/+ and wild-type B cells, BAFF failed to induce process-ing in splenic or immature BM B cells from aly/aly mice with mutantNIK (Fig. 5c). This was consistent with the severely reduced amountsof p52 previously noted in aly/aly splenocytes26. In addition, BAFF didnot induce processing in splenic B cells derived from A/WySNJ mice

Figure 4. BAFF induces B220 and Bcl-2 in transitional Bcells generated in BM cultures. BM cultures (see Methods)were treated or not with BAFF for 3 or 6 days and analyzed forB220 or intracellular Bcl-2 (in cells gated for expression of B220and IgD) expression by FACS.

Figure 3. BAFF functionrequires BAFF-R, NF-κB2 and NIK. (a) FACSanalysis of BM culturesfrom WT, NF-κB2–defi-cient, NF-κB1–deficient(NF-κB2 KO and NF-κB1KO, respectively) andA/WySnJ mice treated withBAFF or left untreated as inFig.2a. Data are representa-tive of two independentmatched experiments; thepercentages of IgD+IgM+

cells are indicated. BAFFincreased the percentagetotal numbers of IgD+IgM+

cells in WT and NF-κB1–deficient, but not NF-κB2–deficient or A/WySnJ, BM cul-tures. (b) NF-κB2–deficient T1 splenic B cells show severely impaired in vivo sur-vival compared to WT T1 B cells upon adoptive transfer into RAG-1–/– (RAG KO)mice. FACS analysis of the splenocytes of recipient mice 40 h after transfer (upperpanels) or no transfer (lower panel) is shown. (c) BAFF-induced accumulation ofIgD+ B cells in in vitro BM cultures depends on NIK. FACS analysis of BM culturesfrom aly/+ and aly/aly mice that were left untreated or treated with BAFF as in Fig.2a. Data are representative of two independent matched experiments; the per-centages of IgM+IgD+ cells are indicated.

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with mutant BAFF-R (Fig. 5c). Together, these results indicated BAFF-specific induction of p100 processing in mature and earlier staged Bcells. Processing was dependent on BAFF-R and NIK and thereforetightly correlated with the biological effects of BAFF in BM cultures.Finally, the intrinsic impairment of B cells in mice lacking componentsrequired for processing underscored the physiological relevance of thispathway, even if it is not absolutely required to generate mature B cells.

On examining its kinetics, we found that p100 processing in wild-type mature B cells was induced slowly—it was detected only after 2–3h of BAFF treatment (data not shown)—and persisted over long periods(Fig. 5). Also it required new protein synthesis because the reaction wassensitive to cycloheximide treatment (Fig. 5c). Thus a protein importantfor processing needs to be continuously or inducibly synthesized.

NEMO-independent regulation of NF-κBWe asked next whether the requirements for BAFF induction of NF-κB matched those for p100 processing. BAFF induced limited butsustained NF-κB activation in splenic B cells, as assessed by electro-mobility shift assay (EMSA) after 8 h of stimulation (Fig. 6a) and inagreement with published data8–11,28. Whereas NF-κB was activated byBAFF in wild-type splenic B cells, it was not activated in NF-κB2–deficient or aly/aly B cells. We did note slightly higher basalNF-κB activity in NF-κB2–deficient cells, which could have been due

to the absence of the p100 inhibitor (which is present in WT andaly/aly B cells). Therefore, like processing, NF-κB induction byBAFF required intact NF-κB2 and NIK.

Nuclear entry of RelB may be regulated by p100 processing29; inagreement with this we found that BAFF induced higher amounts ofnuclear RelB and p52 in wild-type but not NF-κB2–deficient B cells(Fig. 6b). Note that the cytoplasmic fraction was under-representedbecause much more protein was extracted from the cytoplasm thanfrom the nucleus. Therefore, the total amounts of RelB in stimulatedcells may not have changed much. We also noted some increase in theamounts of nuclear p50 and c-Rel in wild-type cells treated with BAFF(data not shown). The nuclear translocation data were confirmed byEMSA supershifts with subunit-specific antibodies (data not shown).

These results argue that the increased NF-κB binding activityobserved after 8 h of treatment with BAFF depended on processing ofthe p100 inhibitor and release of p52, RelB and, to a lesser extent, c-Rel and p50. This highlights the importance of this second NF-κB acti-vation pathway. Although we cannot strictly rule out the possibility thatthe canonical pathway may also have been weakly activated by BAFF,especially at early times, we were unable to detect BAFF-induceddegradation of IκBα or IκBβ (data not shown). Therefore, BAFFappears to activate NF-κB primarily via processing of p100 during sus-tained exposure of the cells analyzed here.

Figure 6. BAFF induces NF-κB binding and nucleartranslocation. (a) BAFF induces NF-κB binding activity in WT,but not in NF-κB2-deficient or aly/aly, splenic B cells. EMSAs weredone with whole-cell extracts prepared after 8 h of stimulation.Asa negative control, Oct-1 binding is shown. The upper diffuseEMSA shift contained several NF-κB dimers, whereas the lowershift contained predominantly p50 homodimers. (b) BAFF inducednuclear translocation of p52 and RelB in WT but not NF-κB2–defi-cient splenic B cells. Cytoplasmic (cyto.) and nuclear (nuc.)extracts were prepared from B cells stimulated for 8 h with BAFF;30 and 22 µg protein, respectively, were loaded per lane. ns cyto-plasm, nonspecific cytoplasmic protein detected with anti-p52; nsnucleus, nonspecific nuclear protein detected with anti-RelB(these proteins were controls for the efficiency of nuclear andcytoplasmic separation and for protein loading).

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Figure 5. BAFF induces processing of NF-κB2 p100 to p52 in mature splenic and immature BM WT B cells. (a,b) B220+ splenic B cells and B cells from BMenriched for immature cells were analyzed by immunoblotting. Immunoblots were done with cells that were left untreated or treated with BAFF, LPS or anti-IgM; 25 µg ofprotein was loaded per lane.The p100 and p52 bands are indicated and were confirmed by analysis of NF-κB2–deficient B cells. n.s., a nonspecific band of variable strengththat was not detected with other p52 antibodies and was present in NF-κB2–deficient cells. (c) B220+ splenic aly/+ or aly/aly B cells,WT and aly/aly BM cells enriched forimmature B cells, B220+ splenic WT and A/WySnJ B cells and B220+ splenic WT B cells were analyzed. Cells were also treated with 10 µg/ml of cycloheximide (CHX), as indi-cated. Protein loading as in a,b.

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To analyze the mechanism underlying p100 processing, we tested the70Z/3 small pre-B, immature-like B cell line and its NEMO-deficientderivative line 1.3E2, which is blocked in activation of IKK complexand NF-κB by numerous signals30. BAFF, but not 4 h of LPS treatment,induced p100 processing in both the parental and NEMO-deficientderivative cell line (Fig. 7). However, we noted increased amounts ofp100 in WT in response to 4-h LPS treatment, which could eventuallylead to higher basal amounts of p52 with prolonged stimulation. BAFFactivated moderate amounts of NF-κB in both wild-type and NEMO-deficient cells, as assessed by EMSA, whereas LPS only activated NF-κB in wild-type cells (data not shown). This confirmed that activation byLPS, but not BAFF, depends on the canonical pathway.

Thus, BAFF activates an NF-κB pathway that is independent of theclassical IKK complex but—as discussed above—dependent on NIKand—as inferred from published reports25,27—dependent on IKKα. Thedata imply the existence of a nonclassical IKK-like activity that is inde-pendent of NEMO.

Processing commences in T1 B cellsBased on our ex vivo results, BAFF-induced processing of NF-κB2may contribute to B cell survival in vivo, starting with T1 B cells—oreven earlier—and promoting their developmental progression.However, it is not known whether T1 B cells are already exposed toBAFF in vivo. To obtain further information on the stage(s) of B cellmaturation in vivo at which BAFF acts, we compared the amounts of

p100 processing at three stages. We found higher amounts of p52 infreshly isolated splenic T1 B cells compared to their immature BM Bcell precursors, with the highest amounts in the mixed T2 plus mature(T2/mature) B cell fraction (Fig. 8). This finding supported the idea thatBAFF and p100 processing may play a role throughout the maturationof splenic B cells in vivo.

DiscussionWe have identified an NF-κB activation pathway in which BAFF con-tributes to the survival and progression of maturing B cells.Engagement of the BAFF-R by BAFF triggered processing of the NF-κB2 p100 inhibitor in a NIK and IKKα–dependent25,27 manner. In addi-tion, BAFF-induced processing proceeded independently of NEMO,the obligatory component of the classical IKK complex. Therefore,although IKKα—a component of classical IKKs—is required for p100processing, the classical IKK complex is not involved in this pathway.It remains to be determined whether proteins other than NIK combinewith IKKα to target p100.

We also found that BAFF-induced p100 processing required ongoingprotein synthesis. Although this could involve induced synthesis of anunknown protein, it is possible that continuous or induced synthesis ofNIK and/or p100 is required. The NIK protein appears to be expressedin low amounts (unpublished observations), whereas transfected over-expressed NIK readily induces processing without any further signals24.NIK binds to p10024 and likely activates IKKα25, but the precise rolesand order of these and possibly additional proteins in the p100-pro-cessing pathway remain to be established.

The in vitro BM culture system generated transitional B cells from apopulation that consisted almost entirely of B cell precursors20. Thisallowed us to investigate the effects of BAFF on newly generated tran-sitional B cells without the influence of non-B cells. In vivo, the matur-ing B cells are likely subject to additional factors, which may obscurethe effects of BAFF. Such factors may partially compensate for the lackof BAFF-induced processing in NF-κB2–deficient cells in vivo. Wefound that transitional B cells isolated from NF-κB2–deficient micesurvive poorly after short-term adoptive transfers into wild-type hosts,whereas a compensatory mechanism appeared to enable small numbersof surviving NF-κB2–deficient B cells to partially repopulate themature B cell population in long-term adoptive transfers. The underly-ing mechanism may depend on NF-κB1, which would explain the totalblock observed at the early transitional stage in NF-κB1/2 double-defi-cient mice.

The biological effects of BAFF in BM cultures were dependent onBAFF-R, NIK and NF-κB2, which are all components of the p100 pro-cessing–second activation pathway. The data suggest that p100 process-ing makes a key contribution to prolonging the life of maturing B cells,a view that is supported by the identification of NF-κB2–dependentBAFF induction of the anti-apoptotic factors Bcl-2 and Bcl-xL. TheBAFF-induced increased expression of IgD and B220 may be an indi-rect consequence of the extended life of these cells, although a moredirect effect of BAFF on the differentiation program remains possible.

The likely importance of BAFF-induced processing of p100 duringsplenic maturation of B cells in vivo is supported by several observa-tions. T2/mature B cells contain high amounts of p52, which is prob-ably a consequence of prolonged exposure to BAFF. Splenic T1 Bcells contain less p52 than T2 and mature B cells, but express sub-stantially higher amounts than immature BM cells; this suggests thatat least some B cells encounter BAFF during or soon after entry intothe spleen. Because BAFF could induce p100 processing in immatureBM B cells in vitro, these cells had presumably not been exposed to

Figure 8.p100 processing begins inT1 B cells in vivo. BM B cells enrichedfor immature cells (see Methods) con-tained much less p52 than splenic T1and T2/mature B cells; the highestamounts were expressed by T2/matureB cells. For each lane, 40 µg of whole-cell extract was loaded; the exceptionwas the last lane,which contained twiceas much protein extract to show thateven with higher loading, immature BMB cells have less p52 than splenic T1 Bcells.

Figure 7. BAFF-induced p100 processing is independent of classical IKKcomplexes. BAFF induced processing of p100 to p52 in 70Z/3 B cells and in 1.3E2mutant 70Z/3 B cells that lacked NEMO.After 4 h of stimulation, LPS failed to induceprocessing.Whole-cell extracts (∼ 60 µg) were loaded per lane and then stimulated.The 24-h untreated 70Z/3 lane contained relatively more protein, but the ratio ofp100 to p52 was unchanged.

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this factor in the BM in vivo. The total block in the maturation of NF-κB1/2 double-deficient B cells at the early T1 stage in the spleenpoints to a critical contribution by NF-κB2 at this stage, as the absenceof NF-κB1 has no effect on the progression of transitional B cells22.The contribution by NF-κB2 is presumably dependent on BAFF-induced processing because little p52 exists in immature B cells with-out such a signal.

In contrast to our results, it is suggested that BAFF contributes to thesurvival of T2 and mature, but not purified, T1 B cells31 and/or to thedifferentiation from T1 to T211,16. The simplest interpretation of our datasuggests BAFF primarily affects B cell survival from the T1 stageonwards and, as a consequence, differentiation. However, it is possiblethat the accumulating IgD+ B cells in our BAFF-treated BM cultureswere actually more T2- than T1-like, despite the absence of readilydetectable amounts of CD23 on their surfaces. On the other hand, it isalso possible that BAFF did not appear to affect purified T1s31 becauseof their rapid death in culture, with too few cells rescued by BAFF tomeasure. In contrast, BM cultures continue to generate early transi-tional cells and, with time, detectable numbers may accumulate inresponse to BAFF, even if only a small fraction is rescued at any onetime. A similar scenario may apply in vivo, where small numbers of res-cued T1 cells may rapidly progress to the T2 stage where they may con-tinue to depend on BAFF for optimal survival. In support of a role forBAFF in T2 cells, we confirmed the reported survival effects of BAFFon isolated T2 cells31 (data not shown). Together, our results indicate asurvival function for BAFF in splenic transitional B cells, but it remainsto be determined at what precise stage the transitional B cells becomeexposed to BAFF, where in the spleen this occurs and whether BAFFalso promotes the differentiation program.

After B cell maturation, p100 processing may continue to contributeto the survival and maintenance of the mature B cell pool. Mature Bcells show constitutive NF-κB activation via unknown mechanisms.BAFF-induced p100 processing in these and in activated B cells couldhelp overcome negative feedback inhibition mediated by the NF-κB–induced synthesis of the p100 inhibitor analogous to NF-κB–induced synthesis of the IκBα inhibitor. Thus, BAFF-induced pro-cessing may contribute to the survival of maturing and mature B cellsvia the activation of NF-κB dimers, especially p52-RelB heterodimers,and by prolonging NF-κB activation induced via the canonical pathway.

BAFF is a physiologically important signal that induces processingof the NF-κB2 protein p100, and preliminary evidence indicates thatseveral other members of the TNF family of ligands may induce pro-cessing, including CD40 ligand and lymphotoxin αβ (unpublishedobservations). The pathway by which BAFF induces p100 processingand NF-κB activation is independent of NEMO—and therefore of theclassical IKK complex—but is dependent on NIK and IKKα. It is pro-posed that BAFF-induced p100 processing aids the survival of transi-tional B cells and that this facilitates the developmental progression ofB cells in the spleen. Our results suggest that the extent of p100 pro-cessing could be critically involved in setting the threshold for self-tol-erance during transitional stages in the spleen, when very few B cellsare positively selected to the long-lived pool. This processing pathwaycould therefore represent an important target for therapeutic strategiesto control autoimmune diseases and possibly other B cell–related dis-eases, such as lymphomas.

MethodsMouse strains and cell lines. CBA/CaHN-Btkxid, B6;129S-RAG-1–/– and A/WySnJ micewere from Jackson Laboratories (Bar Harbor, ME). NF-κB1– and NF-κB2–deficient andNF-κB1/2 double-deficient mice were as described3,6. aly/aly mice were from Clea Japan

(Tokyo, Japan). Mice were used in accordance with NIH guidelines and under an animalstudy protocol approved by the Animal Care and Use Committee of the National Institutesof Allergy and Infectious Diseases.

Flow cytometry. Single-cell suspensions from the BM or spleen were depleted of erythro-cytes with ACK lysis buffer, then 106 cells were incubated with various antibodies for three-or four-color fluorescence surface staining. Data were collected in a FACScalibur flowcytometer and analyzed with CellQuest software (Becton Dickinson, San Jose, CA). Thefollowing monoclonal antibodies (mAbs) were from BD Pharmingen (San Diego, CA):anti-IgM (clone II/41), anti-B220 (clone RA3-6B2), anti-CD21 (clone 7G6), anti-CD23(clone B3B4) and anti–L-selectin (clone MEL-14). Anti-IgD (clone 11-26c.2a) was fromSouthern Biotechnology Associates (Birmingham AL). Intracellular staining: after surfacestaining (for B220 and IgD), 106 cells were fixed with fixation buffer (Caltag Laboratories,Burlingame CA) for 15 min on ice. Subsequently, cells were washed with PBS and incu-bated in permeabilization buffer (Caltag Laboratories) for 15 min at room temperature with20 µl of fluorescein isothiocyanate–labeled anti–Bcl-2 or isotype-negative control antibod-ies (BD Pharmingen). Cells were washed in PBS and data were collected and analyzed.

B cell isolations and immunoblottting. B cells were isolated from spleen or BM suspensionsvia negative and/or positive selection. For the experiments in Fig. 1b,c, T1 B cells were isolat-ed by negative selection from the spleens of 2–3-week-old mice (young mice have more T1cells, relatively) as follows: 107 cells were incubated for 20 min on ice with 15 µl of anti-CD43+ 15 µl of anti-CD62L MACS magnetic bead solution (Miltenyi Biotec, Auburn, CA) in 100µl final volume of MACS buffer (0.5% bovine serum albumin + 2 mM EDTA in PBS). Cellswere then sorted by passage through a MS magnetic column (Miltenyi Biotec). For theimmunoblots in Figs. 5 and 6, splenic B cells were isolated by positive selection, whereasimmature BM B cells were enriched by both negative and positive selection. For positive selec-tions, 107 cells (from adult spleen suspensions or negatively selected BM) were incubated withanti-B220 MACS magnetic beads and sorted by passage through a MS magnetic column(Miltenyi Biotec). Bone marrow suspensions were first negatively selected with anti-CD43 andanti-CD62L to enrich for immature B cells. These negatively selected immature B cells wereused directly in Fig. 2c. For the immunoblots in Fig. 8, splenocytes from 6-week-old animalswere first negatively selected with anti-CD43, followed by positive and negative selection withanti-CD62L to obtain T2/mature and T1 B cells, respectively. Cell-free extracts were preparedfor immunoblotting as described32. For the immunoblots in Fig. 6, nuclear and cytoplasmicextracts were prepared as described33, except that 0.5% Triton X100 was used to lyse cells.Blots were probed with rabbit polyclonal anti-p52 (directed against amino acids 1–399) or anti-RelB (SC226, Santa Cruz Biotechnology, Santa Cruz, CA). BAFF (2 µg/ml, PeproTech, RockyHill, NJ) and LPS (10 µg/ml, Sigma Chemical Co., St. Louis, MO) were used to stimulate cells.

Cell death assays. To test T1 B cell survival in tissue culture (Fig. 1b, left panel), 5 × 105

or 1 × 106 negatively selected B cells were plated in a 96-well U-bottomed plate inDulbecco’s modified Eagle’s medium (4.5 g/l of glucose, 13 µM folic acid and 250 µM L-asparagine) supplemented with 50 µM β-mercaptoethanol and 10% fetal bovine serum(FBS). Cell viability was determined after 24 h of incubation by staining with Trypan blue.

Negatively selected T1 splenic B cells (106) were also plated in a 96-well U-bottomedplate in RPMI medium and incubated for 16 or 48 h (Fig.1b, right panel). Cells were thenwashed with PBS and incubated with 1 µg/ml of merocyanine (Sigma Chemical Co) for 15min at room temperature. To exclude dead cells, 10 µg/ml of 7-amino-actinomycin D (7-AAD Probe, BD PharMingen) was added. Cells were also stained with anti-B220 and ana-lyzed by flow cytometry.

Real-time PCR analysis. Total RNA was isolated from negatively selected B cells withTRIzol (Gibco-BRL, Gaithersburg MD). Reverse transcriptions were done with 1 µg oftotal RNA, 0.5 µg of oligo-dT and 1 U of Superscript (Gibco-BRL) in a 20-µl final volumefor 1 h at 42 °C. For quantification of relative gene expressions, real-time PCR assays weredone with a Lightcycler rapid thermal system (Roche Diagnostic, Lewes, UK) according tomanufacturer’s instructions. The reverse transcription reaction (0.2 µl) was amplified in a20-µl final volume containing 0.5 µM of primers and MgCl2 at a concentration optimizedfor each primer pair (β-actin and Bcl-2 were amplified with 3 mM and A1 with 4 mMMgCl2). Primer pairs were as follows. A1: sense 5′-ATTTGCCTTTGGGGGTGTTC-3′,antisense 5′-GATAACCATTCTCGTGGGAGCC-3′; Bcl-2: sense 5′-AAACAGAGGTCG-CATGCTG-3′, antisense 5′-TCGCTACCGTCGTGACTTC-3′; β-actin: sense 5′-GTGGGC-CGCTCTAGGCACCAA-3′ , antisense 5′-CTCTTTGATGTCACGCACGATTTC-3′ .Nucleotides, TaqDNA polymerase and buffer were part of the lightcycler-DNA master mixSYBR Green I mix. The fluorescent products were analyzed after a prescribed 72 °C exten-sion period. To confirm the amplification specificity, PCR products from each primer pairwere subjected to a melting curve analysis and subsequent agarose gel electrophoresis.Quantification was done by comparison with an external standard curve.

BM culture. Total BM single-cell suspensions were depleted of erythrocytes and 3 × 106–4× 106 cells were incubated for 4–7 days with 20 ng/ml of IL-7 (R&D Systems, MinneapolisMN) in Optimem medium (Gibco-BRL), supplemented with 10% FBS, L-glutamine (2mM), penicillin (100 U/ml) and streptomycin (100 µg/ml); this resulted in >90% B220+

cells. After IL-7 incubation, cells were collected, washed once in medium and replatedwith or without 2 µg/ml of BAFF (PeproTech), 10 ng/ml of mouse TNF-α (RocheMolecular Biochemicals, Mannheim, Germany) or 1 µg/ml of human RANKL(PeproTech) for 4–6 days.

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Adoptive transfer experiments. T1 B cells from pools of six wild-type and six NF-κB2–deficient spleens were negatively selected with anti-CD43 and anti-CD62L on mag-netic beads and passage through a LS column (Miltenyi Biotec); the enrichment was con-firmed by flow cytometric analyses. Cells (2 × 106 per mouse) were injected into the tailveins of RAG-1–/– mice that had been irradiated (500 rad).

EMSAs. Whole-cell extracts were prepared as described34 from spleen B cells isolated bypositive selection. EMSAs were done with 2 µg of extract protein per assay and thePromega NF-κB and Oct-1 probes and gel-shift assay system (Promega, Madison, WI).

Acknowledgments

We thank C. Sibley and C. Scheidereit for the 70Z/3 and 1.3E2 cells, K. Kelly for criticalreading of the manuscript and A. Fauci for continued support.

Competing interests statementThe authors declare that they have no competing financial interests.

Accepted 8 August 2002.

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