Induction of CD4T-cell anergy and apoptosis by activated human B cells

doi:10.1182/blood-2008-02-140087Prepublished online September 18, 2008;2008 112: 4555-4564

and Hanns-Martin LorenzTheresa Tretter, Ram K. C. Venigalla, Volker Eckstein, Rainer Saffrich, Serkan Sertel, Anthony D. Ho cellsInduction of CD4+ T-cell anergy and apoptosis by activated human B

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IMMUNOBIOLOGY

Induction of CD4� T-cell anergy and apoptosis by activated human B cellsTheresa Tretter,1 Ram K. C. Venigalla,1 Volker Eckstein,2 Rainer Saffrich,2 Serkan Sertel,3 Anthony D. Ho,2 andHanns-Martin Lorenz1

Divisions of 1Rheumatology and 2Hematology and Oncology, Department of Medicine V, and 3Department of Otolaryngology, Clinics of Head and Neck Surgery,University of Heidelberg, Heidelberg, Germany

B cells are well-known mediators of hu-moral immunity and serve as costimula-tors in the generation of T cell–mediatedresponses. In several mouse models,however, it was observed that B cells canalso down-regulate immune reactions,suggesting a dual role for B cells. Due tothis discrepancy and so far limited data,we directly tested the effects of primaryhuman B cells on activated CD4� T helpercells in vitro. We found that under optimalcostimulation large, activated CD25�

B cells but not small CD25� B cells in-duced temporary T-cell anergy, deter-mined by cell division arrest and down-regulation of cytokine production. Inaddition, large CD25� B cells directlyinduced CD95-independent apoptosis ina subpopulation of activated T cells. Sup-pression required direct B-T-cell contactand was not transferable from T to T cell,excluding potential involvement of regula-tory T cells. Moreover, inhibitory effectsinvolved an IL-2–dependent mechanism,

since decreasing concentrations of IL-2led to a shift from inhibitory toward co-stimulatory effects triggered by B cells.We conclude that activated CD25� B cellsare able to costimulate or down-regulateT-cell responses, depending on activa-tion status and environmental conditionsthat might also influence their pathophysi-ological impact. (Blood. 2008;112:4555-4564)

Introduction

The adaptive immune response is a tightly regulated process involvingproper initiation and termination of an immune reaction. Both tasks areperformed via a network of cytokines, antigen-presenting cells (APCs),effector cells, and regulatory cell populations. Among the latter,regulatory T cells (Tregs) have been identified, which are able todown-regulate responses of effector T cells toward both foreign andself-antigen, and are mandatory for maintenance of tolerance.1

Little is known so far about other lymphocyte populations, suchas B cells, in this process: B cells perform a wide array of functionsfacilitating the immune response, among them humoral immunity,presentation of antigen and costimulatory molecules, and produc-tion of cytokines.2,3 On the other hand, B cells can participate in theinduction of immune tolerance4,5 and promotion of naive T-celldifferentiation toward a Th2 phenotype.6 In autoimmune diseases,B cells have been widely ascribed a pathogenic role via productionof autoreactive antibodies (Abs), enhanced priming of self-reactiveT cells or blocking of regulatory T cells.7-9 However more and morehints are pointing to the existence of B-cell subsets that candown-regulate autoimmune reactions or promote recovery fromchronic inflammation, suggesting a dual role of B cells in immuneresponses.10,11 This was first demonstrated in respective mousemodels, where absence of B cells led to an exacerbated course ofdiseases such as autoimmune encephalomyelitis, chronic colitis,schistosoma infection, and allergy.12-15 Further research revealeddifferent potential mechanisms, among them signaling via Fc�R,13,14

down-regulation of Th1 response,16,17 production of immunosup-pressive cytokines,18-21 and induction of Tregs.22,23

Unfortunately in many of these in vivo mouse models, “third-party” effects, for example, via dendritic cells could not always be

excluded. Besides, genetically altered mouse strains do not easilyallow translation of the obtained results into a normal backgroundor, even further, into the human system.

Due to the discrepancy of available reports and the so farlimited experimental means, we decided to set up an in vitro modelwith primary human B cells from peripheral blood (PB) to test theireffects on TCR-activated CD4� T-helper cells.

We found that upon appropriate stimulation, activated B cellswere able to down-regulate T-cell responses and induce temporaryanergy as well as apoptosis. We conclude that human B cells areable to develop regulatory and effector functions, which assignsthem an essentially new role as modulators of immune responsesand might influence their pathophysiological impact as well.

Methods

Antibodies, cytokines, and reagents

Antibodies used for flow cytometry are as follows: PE-anti-CD19, -CD25,-CD27, -CD38, -CD69; APC-anti-CD4, -CD19, -CD38; FITC-anti-IgD (allfrom BD Biosciences, San Jose, CA); FITC-anti-CD4, -CD19, -CD20,CD25, -CD38, -CD54, -CD71, -CD80, -CD86; PE-anti-CD4; PeCy5-anti-CD4, -CD19, -CD25 (all from Beckman Coulter, Hialeah, FL); PE-anti-B7-H1 and -PD-1 (eBiosciences, San Diego, CA); and FITC-anti-GITR(R&D systems, Minneapolis, MN).

Antibodies used for blocking and neutralization experiments are asfollows: anti-hIL-10 (clone 23738), anti-hTGF�1,2,3 (clone 1D11), anti-CD25 (IL-2Ra, clone 22722.2), anti-CD122 (IL-2R�, clone 27302),anti-CD95L (clone NOK1 and clone NOK2), anti-CD54 (clone BBIG-I1)(all from R&D systems), anti-hTNFa (clone 1825 [R&D systems], clone

Submitted February 15, 2008; accepted August 26, 2008. Prepublished onlineas Blood First Edition paper, September 18, 2008; DOI 10.1182/blood-2008-02-140087.

The publication costs of this article were defrayed in part by page charge

payment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2008 by The American Society of Hematology

4555BLOOD, 1 DECEMBER 2008 � VOLUME 112, NUMBER 12




D2E7 [adalimumab], and clone cA2 [remicade]), anti-B7-H1 (clone MIH1;eBiosciences), and CTLA4-Ig (Impact Biotechnologies, Hamburg, Ger-many), each added to cultures at 10 �g/mL if not indicated otherwise.

Antibodies used for stimulation are as follows: human CD3-specificantibody OKT3 (ATCC, Manassas, VA) was used at a final concentration of1.25 �g/mL; CD28 mAb, at 0.1 �g/mL (clone CD28.2; eBiosciences); andanti-IgM/IgG goat-�-human F(ab�)2 (Jackson ImmunoResearch Laborato-ries, West Grove, PA), at a final concentration of 20 �g/mL. FormalinizedCowan I strain of Staphylococcus aureus (SAC, Pansorbin; Calbiochem,San Diego, CA) was used at a final concentration of 1/10 000 (vol/vol) andpurified phosphorothioate-modified CpG-oligo ODN2006 (5�-tcgtcgtttt-gtcgttttgtcgtt-3�; TIB Molbiol, Berlin, Germany), at a concentration of2 �M.

Human rIL-2 and rIL-4 were obtained from Peprotech (Rocky Hill, NJ)and used at 100 U/mL, if not indicated otherwise.

Separation procedures

Peripheral blood mononuclear cells (PBMCs) from heparinized bloodsamples were isolated via centrifugation over Ficoll-Hypaque (LSM;density, 1.077 g/mL; PAA, Pasching, Austria). Viability was determined bytrypan blue exclusion. B cells were positively selected by CD19-coated magneticbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany), according to the manu-facturer’s instructions and were more than 98% pure, as assessed by immunoflu-orescence. When indicated, subfractions were sorted on a FACS-Vantage-SE cellsorter (BD Biosciences) after incubation with fluorochrome-labeled Abs. Highlypurified CD4� cells were isolated with a negative CD4� T-cell isolation kit(Miltenyi Biotec) and were more than 97% CD4�.

Tonsils were provided by routine tonsillectomy from the clinics forHead and Neck Surgery University of Heidelberg. Briefly, fresh tonsils wereminced, diluted in PBS, and filtered through a 70-�m pore size nylon filter (BDBiosciences). The cell suspension was centrifuged over Ficoll-Hypaque andfurther cell separations were performed as described in the preceding paragraphfor PBMCs.

Cell cultures

RPMI 1640 medium (PAA) was supplemented with 10% heat-inactivatedfetal calf serum (FCS), 100 U/mL penicillin, 100 �g/mL streptomycin (allfrom Gibco-BRL, Carlsbad, CA), 1 mM sodium-pyruvate, 2 mM L glu-tamine, 10 mM Hepes, 0.55 mM MEM nonessential amino acids (all fromPAA), and 0.05 mM 2-mercaptoethanol (Sigma-Aldrich, St Louis, MO), at37°C and 5% CO2 in a fully humidified atmosphere.

B cells were cultured in 24-well plates at 5 � 105 B cells/mL inpresence of respective stimuli. All stimuli were tested for possible effects onT-cell activation and found to be negative. For selective blocking experi-

ments, B cells were incubated with Fc-Block (intravenous immuneglobulin, 1:10 [vol/vol]) diluted in phosphate-buffered saline (PBS) (PAA)for 10 minutes at 4°C, followed by 20 �g/mL of respective Abs for25 minutes in complete media and followed by several washes in PBS. ForT-cell activation, culture plates were coated with OKT3 in PBS at 4°Covernight and washed before use.

Proliferation assays

B cells were seeded at a concentration of 2.5 � 104/well with 2.5 � 104

CD4� T cells in a final volume of 200 �L/well in OKT3-coated (cCD3)96-well round-bottom plates with or without IL-2 in triplicates for 96 hours,unless indicated otherwise. Cells were pulsed with 0.5 �Ci (.0185 MBq)/well [3H]thymidine (TdR; Hartmann Analytik, Braunschweig, Germany)for the last 16 hours of culture and harvested on an semiautomatic cellharvester (Tomtec, Hamden, CT), and [3H]TdR incorporation was quanti-fied in a TopCount Scintillation Counter (Perkin-Elmer, Waltham, MA).B cells were irradiated at first (30 Gy; Figures 1;2), but this was suspendedfor further cultures exceeding 4 days, since controls showed that effectswere comparable (proliferation rate of nonirradiated B cells did not exceed3000 cpm anymore within the cocultures), while B-cell viability wasretained.

Alternatively, T cells were stained with the membrane dye PKH-26(Sigma-Aldrich), and fluorescence intensity at day 0 on a flow cytometerwas set to 100%. Percentage of dividing T cells was calculated bysubtraction of percentage of PKH-26� T cells at day x from PKH-26�

T cells at day 0 (� 100%) after variable incubation times. Counterstainingwith fluorochrome-labeled Abs for CD19 and CD19/CD4, respectively, wasperformed during each measurement.

In experiments using cell culture inserts, 1.25 � 105 B cells werecultured with equal numbers of PKH-26–stained T cells in OKT3-coated24-well plates at a total volume of 1 mL. B cells were physically separatedfrom T cells by placing them into cell culture inserts with a 0.4-�msemipermeable PCF-membrane (Millicell; Millipore, Billerica, MA).

Flow cytometry

Cells were stained with combinations of fluorochrome-labeled Abs accord-ing to standard procedures, including a blocking step with Fc-Block for10 minutes at 4°C before incubation with fluorochrome-labeled Abs.Expression of respective surface markers was quantified on a double laser(488 nm and 637 nm) 5-color FACScan flow cytometer (BD Biosciences,upgraded by Cytek, Fremont, CA). Propidium iodide (PI)–positive deadcells were gated out. Cell clusters were separated by reverse pipetting, andremaining CD4�CD19� cell doublets (3%-7%) were excluded from

B

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Figure 1. Activated B cells mediate inhibition of Th-cell proliferation. (A) Effect of differently activated B cells on CD4� Th-cell proliferation: Equal amounts of B cells wereadded unstimulated, or after prestimulation for 3 days with SAC or �Ig to Th cells and cCD3� IL-2. [3H]TdR incorporation was measured after 4 days of culture. Mean plus orminus SEM of 3 representative experiments. (B) Separation of SAC-activated B cells into 2 major populations, according to cell size distribution and activation status. Shown is1 representative sort gate for separation of small CD25 (R2 and R4 and not R6) and large CD25� (R3 and R5 and not R6) B cells after prestimulation for 3 days

4556 TRETTER et al BLOOD, 1 DECEMBER 2008 � VOLUME 112, NUMBER 12




analysis. Sorting of different cell populations was performed on a FACS-Vantage-SE cell sorter (BD Biosciences) and viability was checked duringreanalysis.

Measurement of apoptosis by flow cytometry

Fluorochrome-labeled cells were washed in annexin V labeling buffer(10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2), followed by addition of1 �L annexin V-FITC, 0.5 �g PI, and incubation for 30 minutes at 4°C.During subsequent analysis, viable lymphocytes were gated using forward-scatter versus side-scatter characteristics, and T-cell apoptosis (in %) wasdetermined by gating for the annexin V-FITC–positive cells within theCD4�CD19PI population.

Immunofluorescence microscopy

Cocultures were stained with respective surface Abs and annexin V,according to respective standard protocols, immobilized by addition ofglycerol and visualized by a Olympus IX70 inverted fluorescence micro-scope (Olympus Optical, Hamburg, Germany) equipped with a digitalimage acquisition and processing system (analySIS 3.2; Soft ImagingSystem, Munster, Germany). Appropriate fluorescence filter sets for FITC,CY3, and APC were used for the detection of antibodies labeled with thecorresponding fluorescent dye. Cells were observed with a 40� immersionobjective (40� HI; Olympus Optical), connected to a video camera(Colorview XS; Soft Imaging Systems; NA of 40� objective: 1.0).

Enzyme-linked immunosorbent assay

IL-10 and IFN� enzyme-linked immunosorbent assay (ELISA, duo set;R&D Systems) were performed from cell culture supernatants after 48-hourincubation in doublets, and cytokine concentrations were quantified accord-ing to the standard curves.

Statistical analysis

Data from individual experiments are expressed as mean plus or minusSEM. Statistical significance was determined using the paired Student t testand P value less than .05 was considered statistically relevant.

Results

Polyclonally activated B cells inhibit CD4� T-cell proliferation

In preliminary experiments, purified human B cells from PB wereactivated via their BCR by anti-IgG/IgM-cross-linking (�Ig) orS aureus Cowan I antigen (SAC). After 3 days, B cells werewashed, irradiated, and cocultured in equal numbers with freshlyisolated autologous CD4� T cells, serving as responder cells inpresence of CD3-mAb. Since the activated B-cell populations wereexpressing variable high numbers of IL-2R, all cocultures wereprovided with exogenous IL-2 to assure equal and unlimitedavailability of IL-2 for the T cells and optimal stimulatoryconditions. After 4 days, T-cell proliferation was determined by3H-thymidine ([3H]-TdR) incorporation (Figure 1A): Whereasunstimulated B cells had no significant effect on T-cell proliferation,�Ig-stimulated B cells inhibited T-cell proliferation to about 30% andSAC-stimulated B cells to about 50%. Since SAC stimulation led to astronger activated phenotype than �Ig, according to size and CD25(IL-2R�) expression (SAC: 48% 8%, �Ig: 31% 4%, unstimulated:8% 5%,), we asked whether highly activated B cells could be the truesource of inhibitory activity.

The large CD25� but not the small CD25� B-cell population hassuppressive activity

In the following experiments, B cells were activated for 3 days withSAC and IL-2 and separated by flow cytometric cell sorting into2 major fractions according to cell size and CD25 expression(Figure 1B): large FSChi CD25� B cells (lgB25�) and small FSClow

CD25 B cells (smB25). These different B-cell subpopulationswere cocultured with responder Th cells as described. The results(Figure 2A) show that only the fraction of activated large B cellswas able to suppress T-cell proliferation effectively(40 103 9289 cpm vs 134 942 15 159 cpm), compared withthe small B cells (115 112 9915 cpm), which had no biologicallysignificant effect. Therefore enrichment of these cells showed thatthe suppressive effect had increased up to 72%. In contrast,stimulation with �CD3 alone led to an overall low proliferation rateand a slightly costimulatory effect by the B cells, independent from

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Figure 2. The large CD25� B cells mediate suppression of activated Th cells.(A) Proliferation assay of cCD3-stimulated Th cells alone or in presence of lgB25� orsmB25 cells after 4 days in presence (f) or absence ( ) of IL-2. Bars representmean of 9 independent experiments plus or minus SEM. (B) Proliferation ofprestimulated ( ) or freshly isolated (f) Th cells in presence of lgB25� or smB25

B cells after 4-day culture with IL-2. Mean plus or minus SEM of 3 representativeexperiments. (C) Proliferation of Th cells in presence of different concentrations oflgB25� cells, diluted in cell culture media (f) or in B25 cells ( ). Data expressed asmean plus or minus SEM of triplicates from one representative experiment.

T-CELL SUPPRESSION BY ACTIVATED B CELLS 4557BLOOD, 1 DECEMBER 2008 � VOLUME 112, NUMBER 12




their activation status. Inhibitory effects were reproducible alsowith �CD28 or �CD28 � IL-2 (data not shown) and with T cellsprestimulated by �CD3 � IL-2 (Figure 2B) or �CD28, suggestingthat initial activation does not abolish susceptibility to the negativeregulatory effects exerted by the B cell.

To determine the amount of B cells necessary for suppression,we created different ratios of activated B cells to T cells by mixinglgB25� cells with smB25 cells in increasing dilutions and bykeeping a constant number of 1:1 B/T ratio. Alternatively, lgB25�

cells were diluted in cell culture media (Figure 2C). The resultsshowed clearly that even when the total B-cell number remainedconstant, the extent of suppression was dependent on the amount ofthe lgB25� cells within the population. Remarkably, addition oflgB25� cells in a 2:1 ratio to the T cells further increased inhibitionup to 90%. In experiments with sorted CD25� �Ig-stimulatedB cells, a similar dependency was found, though the effects werestill not as striking (maximum 50% inhibition, data not shown),indicating that—apart from CD25�—differences in phenotype andfunction of B-cell populations might influence regulatory capabili-ties. This hypothesis was also supported by the fact that CD25�

B cells, which comprise about 5% to 15% of the total B cells, sorteddirectly out of PB, had no significant impact in our system. In addition,sorting of SAC-activated B cells for CD25high, as successfully per-formed for enrichment of human natural regulatory T cells,24 did notincrease inhibition significantly more (data not shown).

Interestingly, also prestimulation of B cells by use of CpG-oligos (ODN2006) led to T-cell suppression comparable with SAC(Table 1): Here again, only large activated B cells suppressed toabout 70% in a 1:1 ratio (30 358 10 048 cpm vs 99 449 11 593cpm; n � 3) and about 84% in a 2:1 ratio (16 323 7824 cpm),indicating that also bacterial DNA (containing unmethylated CpGmotifs) can lead to B-cell–mediated T-cell suppression as soon as itleads to sufficient B-cell activation.

Inhibitory effects involve long-lasting cell division arrest

Measurement of [3H]-TdR incorporation at different time pointsrevealed that inhibitory activity increased over time: Althoughinhibition started with 38% at day 3, it increased up to 77% at day 6of the cocultures (Figure 3A). Additional staining of T cells withPKH-26 at day 0 of the culture revealed that the majority of T cellsremained under cell division arrest (Figure 3B). Long-term suppres-sion was also confirmed in an MLR, where autologous B andT cells were mixed with allogeneic irradiated PBMCs (Figure 3C)for more than 7 days, confirming that our observations were notsolely based on the CD3 antibody stimulus but could also bereproduced in the context of recognition of foreign MHC II. Theslightly reduced suppression (50%) could be caused by the PBMCs

in the culture that might impede close B-T-cell contact. Interest-ingly, a slight suppression by the initially smB25 population couldbe observed over time. Since smB25 cells started to up-regulateCD25 in the cocultures, it is tempting to speculate that some ofthese might have acquired regulatory functions as well, perhaps viaanother mechanism. Although no �CD3 was added to the MLRcultures, exogenous IL-2 had to be present for suppressive activity.This was consistent with CD3 stimulation and led us to wonderabout the requirements for suppressor activity.

B cell–mediated suppression is critically dependent on IL-2

To investigate the correlation between activation signals andsuppression, we tested different concentrations of IL-2 (Figure4A). Under influence of low IL-2 concentrations (up to 10 U/mL),lgB25� cells had a slightly costimulatory effect on T-cell prolifera-tion. Concentrations from IL-2 higher than 50 U/mL led to astriking increase of DNA replication among T cells cultured aloneor with smB25 cells, whereas presence of lgB25� cells kept T-cellproliferation below 30%, as observed before. PKH-26 labelingmoreover revealed that initial percentages of dividing T cells inpresence of lgB25� cells decreased with increasing IL-2 concentra-tions (Figure 4B). These data suggest that inhibition of T cellscorrelates directly with the amount of IL-2 offered and cannotresult from competition for IL-2 with the B cells, as sometimesclaimed in context with Treg function.25

To test whether IL-2 might rather serve the suppressivecapabilities of the B cell or susceptibility to inhibition by the T cell,a selective preincubation of B cells with blocking Abs againstCD25 and CD122 subunits of IL-2R was performed. With viability

Table 1. Inhibitory properties of CpG-activated B cells

Ratio B cell/T cell

% T-cell inhibition

B large B small

0.062:1 17 4 6 4

0.125:1 22 3 9 5

0.25:1 36 3 15 4

0.5:1 41 2 17 1

1:1 69 9 20 3

2:1 84 7 31 7

Data represent mean inhibition of T-cell proliferation SD from 3 donors after4-day culture with CD3/IL-2 and autologous B cells, prestimulated with CpG and IL-2for 3 days and FACS-sorted according to cell size. Inhibition was calculated asproliferation of T cells in presence of B cells in relation to proliferation of T cells alone.

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Figure 3. lgB25� cells induce long lasting T-cell division arrest. (A) T cells werestimulated alone (rhombus), with unstimulated B cells (square), smB25 cells (cross),and lgB25� cells (triangle) as usual in presence of cCD3 and IL-2. Data are expressedas mean plus or minus SEM of triplicates from 1 representative experiment of at least3. (B) T-cell division after 3 and 6 days, measured by PKH-26 dilution. M1 relates to100% PKH-26–positive cells at day 0. (C) MLR with autologous B and T cells (each25 000) and allogeneic irradiated PBMCs (50 000) in presence of IL-2. Data areexpressed as mean plus or minus SEM of triplicates from 1 representativeexperiment of at least 3 (symbols as in panel A)





of the B cells retained, T-cell proliferation was partly reconstitutedby about 50% (from 22% back to 46%, Figure 4C). It isconceivable that preblocking with Ab is not sufficient to perma-nently block signaling via IL-2 receptors, up-regulated on theB cells later on. However the limited availability of IL-2 apparentlyhad alleviated suppressive effects, whereas high concentrations (orfully available receptors) rather promoted it, altogether assigning aspecial role to this cytokine in generation of inhibitory B cells.

Interestingly addition of the important B-cell survival factor and� chain cytokine IL-4 instead of IL-2 did not lead to observed cell

division arrest and, applied together with IL-2, it did not interferewith inhibition—neither did addition of neutralizing Abs againstimmunosuppressive IL-10 or TGF�. Besides, IL-10 was greatlydiminished in the supernatants of B-T cocultures in parallel withIFN �, and B cell–specific production was negligible (data notshown).

Suppression is cell contact dependent and requires constantpresence of SAC-activated B cells

Since supernatants from SAC-stimulated B cells alone or from B-Tcocultures had no inhibitory effect on the T cells (data not shown),we tested whether the suppressive effects are dependent on cell

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Figure 5. T-cell inhibition requires cell contact and constant presence ofactivated B cells. (A) PKH assay of Th cells stimulated alone or together withSAC-activated B cells, or separated by cell culture insert (T/Insert/B-SAC) after 3 and6 days. (B) Surface expression of CD71, CD69, and CD25 on gated CD4� T cells after24-hour stimulation with cCD3/IL-2 in presence or absence of lgB25� cells. (C) T cellsprestimulated alone (T-prestim) or with lgB25� cells (T-prestimB) for 48 hours andrestimulated with IL-2 or IL-2� lgB25� cells. Data are expressed as mean plus or minusSEM of triplicates from 1 representative experiment. (D) Coculture of PKH-26� responderT cells (Tresps) with Tprestim or with TprestimB for 24 or 48 hours. Tresps were used fresh( ) or prestimulated for 48 hours (f). PKH-26 was measured after 3 days.

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Figure 4. B cell–mediated Th-cell suppression is critically dependent on IL-2.(A-C) Th cells were incubated alone (f), with lgB25� ( ) or smB25 (z) cells inpresence of cCD3 and different concentrations of IL-2 or without IL-2 (med).(A) [3H]TdR incorporation of T cells. Data are expressed as mean plus or minus SEMof triplicates from 1 representative experiment. (B) PKH-26 assay at day 6(mean SEM of 3 independent experiments). (C) Blockade of IL-2R�� by addition ofAbs directly to culture or by preincubation of lgB25� cells (B*) alone; cultures providedwith cCD3 and IL-2 (100 U/mL). Mean plus or minus SEM and P value of5 independent experiments.





contact: SAC-stimulated B cells were cocultured with PKH-26–stained T cells or separated by semipermeable cell culture inserts,under presence of �CD3/IL-2. Although in T-B coculture themajority of T cells remained suppressed as expected (Figure 5A),separation from B cells did not affect T-cell proliferation, but rathersupported it at first, suggesting that direct cell contact is aprerequisite for regulatory activity and could potentially outbalancesoluble costimulatory factors. In addition, keeping the cocultures inflat-bottom plates instead of round-bottom plates reduced theinhibitory effects, though did not abrogate them, probably due tolesser cell contact at the cell concentrations used (60 000 cells).

Interestingly, the expression of early activation markers on theT cell was not inhibited in presence of lgB25� cells, as the T-cellpopulation expressed homogenously CD25, CD69, and CD71within 24 hours (Figure 5B). To determine whether these T cellscould recover from suppression and continue cell cycling, wepreincubated CD4� T cells with �CD3/IL-2 with or without lgB25�

B cells as usual. After 48 hours (Figure 5C), CD19� B cells anddead cells were removed by fluorescence-activated cell sorting(FACS) sorting, and the T cells were set into culture again inparallel with their counterparts that had been precultured withoutB cells. The proliferation assay after 4 days showed that the“B-primed” T cells achieved a growth rate comparable with their“unprimed” counterparts, suggesting that inhibition was reversible.In addition, these lgB25�-“primed” T cells were not able to transferanergizing effects to PKH-26� T responders (Figure 5D), suggest-ing that constant presence of the B cell is mandatory for suppres-sion. It also confirms that the B cells do not mediate suppressiveeffects indirectly via regulatory T-cell populations, such as naturalor induced Tregs. This was further supported by the observationthat the extent of inhibition was still the same, when all CD25�

T cells containing natural Tregs were depleted by cell sortingbefore coculture (data not shown).

In an attempt to define the mechanism by which this kind ofreversible anergy was maintained, we examined the expression ofcostimulatory molecules on the B cell since lack of these is knownto render T cells susceptible to anergy induction.26,27 We found thatthe lgB25� cells were fully capable of costimulation, as confirmedby their high expression of markers such as CD80, CD86, andCD54 (Table 2). However markers with known inhibitory activitywere also found, such as B7-H1.28 Blocking of important interac-tion pathways between B and T cells by using blocking Abs againstB-7H1, CD54, or surface TGF� or by using CTLA-4-Ig revealedthat none of them was able to effectively restore T-cell proliferation(data not shown).

Suppression involves both inhibition of cell division andinduction of apoptosis

To better characterize the mechanism of suppression, we askedwhether cell death played a role in our system. PKH-26–stainedresponder T cells were added to the lgB25� cells and apoptosis wasdetermined by annexin V staining versus PI. As observed before,80% and more of the T cells stimulated alone underwent 3 andmore cell divisions within 4 days, whereas in the cocultures with

lgB25� cells T cells were undergoing 1 cell division, at the most(Figure 6A). Instead, a remarkable proportion of suppressed T cellsunderwent apoptosis: more than 20% within the first 24 hours ofcoculture and more than 50% after 72 hours of culture. Cell deathstarted almost immediately, as an increase in annexin V� T cellswas measurable already within 4 hours of incubation with lgB25�

cells (data not shown). In contrast, apoptosis of T cells alone orwith smB25 cells rarely exceeded 10% to 20% within 3 days(Figure 6B). Remarkably, immunofluorescence microscopy showedmany of those apoptotic T cells located within clusters, preferen-tially formed between T cells and lgB25� cells, confirming actionvia direct B-T contact (Figure 6C).

There was also a correlation between IL-2 concentration and theapoptosis rate in all 3 groups, with differences between T cellsalone and T cells plus lgB25� cells most prominent at doses higherthan 50 U/mL. Low apoptosis rates in absence of �CD3 revealedthat stimulation via the TCR was indispensable for T-cell suscepti-bility to B cell–mediated apoptosis (data not shown).

Due to the known correlation between T-cell activation andsusceptibility to apoptosis, we wondered whether activation-induced cell death (AICD) could play a role in our system. Weperformed blocking experiments with selective �FasLAbs, success-fully proven for blocking the CD95-CD95L interaction (clonesNOK-1 and NOK-2) by prelabeling of the B cells or by addition ofexcessive amounts into the coculture. The �FasL Abs, however,given each alone or in combination did not abolish apoptosis, andneither did �TNF� Abs or the pan-caspase inhibitor z-vad,suggesting that other mechanisms were involved (data not shown).

B cells with inhibitory capabilities can be identified in vivo

Finally, we wanted to test whether inhibitory B cells can also befound at the site of antigen response in vivo. We therefore repeatedthe suppressor assays with B-cell fractions isolated from humanpalatine tonsils, gained by routine tonsillectomy. Whereas suppres-sor assays were set up as before with autologous T cells andCD3/IL-2 stimulation, B cells were left unstimulated, assumingthey had previous antigen contact. From the different tonsillarpopulations sorted according to expression of CD38 and IgD29

(Figure 7B), we found that tonsillar IgD� CD38�� B cells—amajority of them representing the large B-cell population (Figure7A)—reduced proliferation of autologous T cells to a level below50% of control (43% 17%, n � 3). To a lesser degree, IgD�

CD38� B cells had also some impact (65% 15%, n � 3; Figure7C). Other fractions, such as CD38�� IgD centroblasts/centrocytes revealed heterogeneous results, probably at least partlydue to different amounts of further B-cell subpopulations withinand different requirements on their environment: Centrocytes, forexample, have been shown to quickly differentiate into memoryB cells upon T-cell contact and centroblasts have been demon-strated to be sensitive to apoptosis.30,31

CD38�� IgD� B cells have been reported to populate preferen-tially the dark zone of the GC, to express the proliferation markerKi67, and to carry unmutated genes32 (according to B matureclassification: Bm2-Bm3�4�). More work has to be done to further

Table 2. Phenotypical analysis of SAC-activated B cells after 3-day culture

% CD25 CD69 CD80 CD86 CD54 B7-H1 CD95 CD38 CD27

B large 91 6 33 9 82 7 85 5 71 8 45 11 61 15 74 7 29 6

B small 15 4 3 2 22 6 22 9 17 5 23 6 12 4 13 4 18 6

Data represent mean plus or minus SD from purified B cells of 3 donors after 3-day culture with SAC and IL-2. PI� cells were excluded and for analysis 2 gates were setaccording to cell size.





characterize the physiological counterpart of our in vitro–generatedinhibitory B cells, but those cells might indeed most resemble theSAC-stimulated, initially naive IgD� B cells from peripheral bloodthat we had used in our experiments.

Discussion

In the current study, we have shown for the first time that activated,primary human B cells from peripheral blood can regulate T helpercell responses via a new mechanism involving IL-2 signaling anddirect cell contact: In presence of IL-2, polyclonally activated largeCD25� B cells down-regulated T-cell proliferation and inducedapoptosis, whereas the smaller CD25 B-cell population had nosignificant effects, resembling freshly isolated B cells from periph-eral blood in phenotype and function.

A regulatory effect of B cells was already suspected in earlierreports. However, many of those studies used unstimulated B cells

or B cells after contact with soluble antigen. Those B cells were effectivein Ag presentation via MHC II but weak in providing costimulatoryhelp, leading to the well-described induction of T-cell tolerance.4,26 Inour system, B cells first underwent BCR cross-linking, then expressed afull set of costimulatory markers, and formed strong clusters withT cells, followed by T-cell anergy and apoptosis, confirming thatobserved effects were based on a different mechanism.

A few studies have been performed, by use of immunized miceor stimulated B cells that imply the existence of different inhibitoryB-cell subsets depending on mode of activation: One reportdescribes inactivation of effector T cells and prevention of �-celldestruction in autoimmune diabetes after treatment with LPS-stimulated B cells.17 Of note, in a B cell–deficient model for thisdisease, B cells had first been identified to be essential for initiationof the autoimmune reaction.7 LPS-stimulated murine B cells havealso been described in vitro to anergize CD8� T cells, probably viaTGF�.21 In several autoimmune disease models, such as inflamma-tory bowel disease, experimental autoimmune encephalomyelitis,

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Figure 6. B cell–mediated Th-cell suppression in-volves induction of T-cell apoptosis. (A) Annexinbinding of PKH-26–labeled Th cells cultured alone orwith lgB25� cells � cCD3/IL-2 after different incubationtimes between 24 and 96 hours, 1 representativeexperiment of 4 independent experiments shown.(B) Annexin staining of T cells cultured alone or withlgB25� or smB25 cells at different time points. Meanplus or minus SEM of 6 independent experimentsshown. (C) Immunofluorescence microscopy after24-hour incubation time; 40� magnification. Sixteenadjacent areas in a 4 � 4 array were imaged andstitched together. Images were acquired with respec-tive filters for CD4-APC, CD19-FITC, and annexin-PE;overlay colors: green indicates B cells; blue, T cells;purple, Ax-positive T cells; and yellow, Ax-positiveB cells. One representative area with T� lgB25� andT� smB25 is shown.





or collagen-induced arthritis, IL-10–producing B cells appear toplay a protective role,18-20 probably via a CD40-dependentmechanism. B-T-cell contact was also necessary in our system, andcould take place in form of a reciprocal dialogue. This wouldcontribute to survival of the B cell and help to reduce bystandereffects in a physiological context. Contrary to previously citedreports, however, we did not identify IL-10 or TGF�, but dididentify IL-2, to be of crucial importance for regulatory effects.Under low doses of IL-2, costimulatory effects were prominent,whereas under increasing doses inhibitory effects became obvious.This stands in contrast to Tregs, where—although certain amountsof IL-2 are necessary for maintenance—increasing IL-2 concentra-tions cause their inactivation.25 Based on our data, includingselective blocking of IL-2R on the B cell, we suggest that highconcentrations of IL-2 might sensitize the lgB25� cell to itssuppressive function and cause a switch from costimulatory toinhibitory signals. Since the CD25� B cells directly isolatedfrom PB—a majority of them CD27� memory B cells (see alsoAmu et al33 and T.T., R.K.C.V., V.E., R.S., S.S., A.D.H.,

unpublished observations, 2008)—did not suppress, whereas theless mature, predominantly CD27-naive B cells from PB did, wesuggest that maturation status of the B cell represents anothercritical parameter in this process. This hypothesis is alsosupported by an observation during our experiments with B-cellstimulatory CpG-rich oligonucleotides (CpG-ODNs): Althoughafter treatment again only the large B-cell fraction (representingup to 40% of the total B-cell population) exhibited a stronginhibitory effect toward CD4� T cells, comparable with SAC,the great majority of the B cells (� 80%) expressed CD25 on thecell surface (T.T., R.C.K.V., V.E., R.S., S.S., A.D.H., andH.-M.L., unpublished observations, 2008).

Stimulation with CpG-ODNs via TLR9 represents anotherpathway for T cell–independent B-cell activation and can act aloneor synergistically with BCR signaling.34 Similar to BCR cross-linking, CpG-ODNs activate downstream signals though MAPkinases, resulting in NF- B activation and expression of costimula-tory molecules, production of cytokines, and antibody secretion. Ofnote, although CpG-ODNs induce immune activation, reportsdescribing inhibitory effects have been published previously:Systemic and repeated treatment with CpG-ODNs led to immuno-suppression, including destruction of lymphoid follicles,35 andspecifically abrogation of T-cell expansion in the spleen ofimmunized mice.36 Taken together, this indicates an additionalpotential role for CpG-ODNs in downmodulation of immuneresponses in vivo. However, TLR9 is expressed by various celltypes, including dendritic cells and macrophages. Therefore, respec-tive in vivo models could not provide direct evidence for aninvolvement of B cells in this process, yet.

Future experiments will also show whether rather naive B cells,reacting to T-independent bacterial antigen or bacterial DNA(CpG-ODNs), might be most susceptible to convert into regulatoryB cells (Bregs). This hypothesis would be supported by the fact thatour experiments with tonsillar B cells identified a population withinhibitory properties, which interestingly belonged to an earliermaturation stage (IgD� and CD38�� IgD�, Bm2-Bm3�4�) thanthe IgD germinal center centrocytes and centroblasts that havealready had contact with the T cell and undergo somatic mutationand class switching.31 The tonsillar CD38�� IgD� B cells havebeen reported not to have completed class switching, but to secreteIgM, and to generate “atypical” germinal centers as reactions onbacterial carbohydrate Ag, which are likely involved in pathogene-sis of chronic tonsillitis (TI2 antigens).32

In this context, it appears interesting that some autoimmunediseases seem to be accompanied by defects in B-cell homeostasis,for example by overrepresentation of a more mature memoryphenotype or by a specific lack of naive B cells, for example, asdescribed in rheumatoid arthritis (RA) and systemic lupus erythem-atosus (SLE).37,38

It is plausible to suggest that presence of IL-2 might alsocontribute to sensitivity of the T cell to inhibitory effects, as isalready known from AICD, by shifting them to a highly activatedstate.39 In our system, expression of the IL-2R� on T cells was notinterrupted and even increased in presence of lgB25� cells and sowere other early activation markers. This phenomenon has beenobserved with certain forms of anergy before40,41 and can beaccompanied by a few cell cycles. In our system, the majority ofT cells did not start any cell division at all, speaking for animmediate action in context with TCR signaling, which could helpto explain why receptors such as CTLA-4 or PD-1, which take timeto be up-regulated, were not involved in this process. The observedanergy was reversible, not transferable, and not accompanied by

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Figure 7. Activated B cells with inhibitory properties can be found in humantonsils. (A) Representative example for phenotype of purified tonsillar B cellsaccording to CD38 and IgD expression. Upper quadrant separates CD38� fromCD38�� B cells. Large cells gated on R5; small cells gated on R4; PI� B cells (R2)gated out. (B) Sort gates for separation of IgD� CD38�� B cells (R6 and R1 and notR2) and IgD� CD38� B cells (R7 and R1 and not R2); R1 and R2 refer to the gates ofpanel A. (C) Suppressor assay with freshly sorted autologous cell populations andCD3 alone or CD3� IL-2. f indicate T cells alone; , T� B-IgD� CD38��; and , T�B-IgD� CD38�. Data represent mean plus or minus SEM of tonsils from 3 differentdonors.





induction of Tregs. Noteworthy in the “Breg” mouse modelsmentioned, where activated B cells were used, an induction ofTregs was not described either and T-cell transfer experiments werenot successful, whereas adoptive Breg transfer was,17,42 as evi-denced by the amount of T-cell apoptosis afterward. ActivatedB cells have been reported to up-regulate CD95L43 and itsexpression has been correlated, though not fully evidenced, toB cell–mediated T-cell apoptosis in at least 2 models.17,42,44 Apopto-sis, and specifically AICD, is widely accepted to be required formaintenance of self-tolerance45,46 and plays a critical role duringa variety of physiological conditions and diseases from sepsis-induced lymphopenia47 to systemic autoimmune disorders.48

However, although the lgB25� cells in our system obviouslydeveloped cytotoxic abilities toward the T cells, we did not findan involvement of the CD95 pathway. Recently apoptosis wasalso demonstrated in context with immune regulation byTregs.49,50 Here, cytokine deprivation within the responderT-cell population due to consumption of �-chain cytokines byTregs was revealed to not only suppress their proliferation but alsoto induce apoptosis. However since apoptosis was detectable in oursystem already within few hours of coculture with inhibitoryB cells under presence of sufficient amounts of exogenous IL-2,this possibility can be excluded. Another difference to the citedTreg report was also that the apoptosis pathway induced in oursystem was not sensitive to the pan-caspase inhibitor z-vad.However, alternative apoptosis pathways have been describedbefore51-53 and should be considered for future research. This mightalso help in clarifying how the decision is made between anergyand apoptosis in the T cell.

According to our hypothesis, in a physiological environment,such as the secondary lymphoid organs, the activated B cell wouldbe able to down-regulate T-cell responses as soon as a certain levelof effector activity is reached, determined by accumulation of acritical parameter such as IL-2. This would define B cells as anadditional tool for IL-2 in maintaining peripheral T-cell toler-ance,1,54 next to the expansion of Tregs. The suppressor B cellswould be helpful especially after confrontation with bacterialantigen, which in extreme cases can lead to sepsis or—afterdefective removal of effector T cells—to abnormal outgrowth ofclones with accidentally self-reactive potential, as suggested for

pathogenesis of several systemic autoimmune diseases. Inductionof T-cell apoptosis would prevent survival of such T cells or T cellswith potentially dangerous mutations, which could even lead tocell-autonomous growth. Temporary anergy on the other handwould guarantee to preserve the memory T-cell pool and would befully reversible as soon as the B cells leave the lymph node or as theIL-2 concentration is decreasing. Which characteristics deter-mine T-cell fate toward apoptosis or anergy we were not able todefine, yet. However, we could define activation and composi-tion of the microenvironment as crucial factors for the conver-sion of the B cell into a Breg, and this might help to explain thediscrepant results about B-cell function gained in variousexperimental settings before. Overall, our findings provideevidence for a new function of human B cells during an immuneresponse. Depending on the individual disease, effects ofinhibitory B cells might appear rather desirable or detrimental tothe physician. Further research in this area could put newperspectives on current treatments and offer alternatives fordevelopment of future immunotherapies.

Acknowledgments

The authors thank Kerstin Woerner for excellent flow cytometriccell sorting and Ziya Kaya for fruitful discussions and criticalreading of this paper.

Authorship

Contribution: T.T., R.K.C.V., V.E., and R.S. performed the labora-tory research, and analyzed and interpreted the data; V.E., R.S.,S.S., and A.D.H. contributed analytical tools; and T.T. and H.L.designed the research and drafted the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Theresa Tretter, Division of Rheumatology, Depart-ment of Medicine V, University of Heidelberg, 69120 Heidelberg,Germany; e-mail: [email protected].

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Induction of CD4T-cell anergy and apoptosis by activated human B cells

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