of December 22, 2018.This information is current as

Functional ConsequencesCells and Resting B Cells with DifferentPresented in the Periphery by Both Dendritic Endogenous Myelin Basic Protein Is

Audrey Seamons, Antoine Perchellet and Joan Goverman

http://www.jimmunol.org/content/177/4/2097doi: 10.4049/jimmunol.177.4.2097

2006; 177:2097-2106; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/177/4/2097.full#ref-list-1

, 19 of which you can access for free at: cites 36 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2006 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on Decem

ber 22, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

by guest on D

ecember 22, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Endogenous Myelin Basic Protein Is Presented in thePeriphery by Both Dendritic Cells and Resting B Cells withDifferent Functional Consequences1

Audrey Seamons,2 Antoine Perchellet, and Joan Goverman3

Multiple sclerosis is an inflammatory disease believed to be triggered by erroneous activation of self-reactive T cells specific formyelin proteins such as myelin basic protein (MBP). Inflammation is limited to the CNS, suggesting that the myelin-specific T cellsencounter their Ags only after they cross the blood-brain barrier. However, our previous studies in mice showed that MBPepitopes are constitutively presented in lymphoid tissues. Here we identified which APCs in lymph nodes present endogenous MBPepitopes and determined the functional consequences of this presentation for both naive and activated MBP-specific T cells. BothCD8�� and CD8�� dendritic cells were potent stimulators of proliferation for both naive and previously activated/memoryMBP-specific T cells. Surprisingly, resting B cells also presented endogenous MBP that was acquired using a BCR-independentmechanism. Interaction with resting B cells triggered proliferation of both naive and activated MBP-specific T cells. Activated/memory MBP-specific T cells proliferating in response to resting B cells presenting endogenous MBP did not produce cytokinesand became more refractory to subsequent stimulation. Interestingly, cytokine production by activated/memory T cells wastriggered by resting B cells if the number of MBP epitopes presented was increased by adding exogenous MBP peptide. Theseresults suggest that activated MBP-specific T cells may become less pathogenic in vivo following encounter with resting B cellspresenting steady-state levels of endogenous MBP but can expand and remain pathogenic if the amount of MBP presented by Bcells is increased, which could occur during chronic demyelinating disease. The Journal of Immunology, 2006, 177: 2097–2106.

M ultiple tolerance mechanisms exist to prevent destruc-tive autoimmune responses such as those that occur inmultiple sclerosis (MS).4 Many self-reactive T cells

are eliminated by exposure to self-Ag in the thymus. Tolerancealso occurs in the periphery when self-reactive T cells encountertheir Ag under non-inflammatory conditions and are either trig-gered to die or are prevented from acquiring effector function.Immature dendritic cells (DCs) have been implicated as the pri-mary APC in the periphery responsible for inducing this type ofperipheral tolerance (1–4). Immature DCs residing in peripheraltissues are highly phagocytic but do not express high levels of theco-stimulatory molecules needed to prime T cell responses untilthey are activated (5). Some of these immature DCs migrate tolymph nodes (LNs) and spleen where they can present tissue-de-rived self-Ags to naive T cells directly via the MHC class II path-way or through cross-presentation in the MHC class I pathway.Because immature DCs do not provide the necessary co-stimula-tory signals, the T cells that interact with these DCs typically pro-liferate briefly and die or become anergic (2, 3). Among the dif-ferent DC subsets, CD8�� DCs have been implicated as the majorcell type responsible for cross-presenting Ags in the MHC class I

pathway leading to peripheral tolerance in self-reactive CD8� Tcells (6).

Although most attention has focused on immature DCs as theprimary APCs responsible for inducing T cell tolerance, B cellsalso present Ag. Unlike DCs, B cells are not highly phagocytic andtherefore should not present the same wide range of tissue-derivedself-Ags that immature DCs can present. However, B cells canefficiently capture specific Ags through their BCRs and present theprocessed Ag on MHC class II molecules. As is the case for im-mature DCs, Ag capture alone is not sufficient for B cells to func-tion as immunogenic APCs without receiving additional signals toexpress co-stimulatory molecules. For resting B cells, these signalsare provided via the CD40/CD154 pathway that is engaged whenactivated T cells recognize Ag on the surface of the resting B cell(7). Naive T cells recognizing Ag presented by resting B cells donot provide the signals required to trigger resting B cells to expressco-stimulatory molecules and consequently the T cells become tol-erant (8–10). Tolerance induction of naive T cells by resting Bcells in vivo has been shown to involve abortive proliferation fol-lowed by disappearance of the T cells (11). The only exceptions tothe tolerogenic effect of Ag presentation by resting B cells havebeen observed under non-physiological conditions when largenumbers of naive BCR transgenic (Tg) B cells have been used topresent Ag to naive TCR Tg T cells with the same Ag specificity(12, 13). Because the number of B cells expressing a BCR specificfor any particular self-Ag should normally be very low, DCs arepresumed to play a more predominant role in mediating peripheraltolerance as they do not depend on Ag-specific receptors toacquire Ag.

Our previous studies investigated the tolerance mechanisms thatregulate CD4� T cells specific for myelin basic protein (MBP),one of the predominant protein components of myelin. Under-standing how immune responses to MBP are regulated is importantbecause MBP is believed to be one of the self-Ags targeted in MS.

Department of Immunology, University of Washington, Seattle, WA 98195

Received for publication May 3, 2006. Accepted for publication May 22, 2006.

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 research was funded by the National Institutes of Health Grant NS035126.2 Current address: Department of Comparative Medicine, University of Washington,Seattle, WA, 98195.3 Address correspondence and reprint requests to Dr. Joan Goverman, Department ofImmunology, University of Washington, Box 357650, 1959 NE Pacific St., Seattle,WA 98195. E-mail address: goverman@u.washington.edu4 Abbreviations used in this paper: MS, multiple sclerosis; DC, dendritic cell; LN,lymph node; HEL, hen egg lysozyme; MBP, myelin basic protein; Tg, transgenic.

The Journal of Immunology

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00

by guest on Decem

ber 22, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

There are several isoforms of MBP that are incorporated into my-elin. This family of isoforms is referred to as “classic” MBP (14).A separate family of proteins called golli-MBPs are transcribedfrom a distinct promoter but share many of the exons that encodeclassic MBPs (15, 16). The function of golli-MBPs is less wellunderstood but they are not incorporated into myelin. T cell tol-erance to MBP sequences shared by classic and golli-MBP pro-teins should be readily achieved because golli-MBPs are expressedin both the thymus and peripheral lymphoid tissues. However,some portions of classic MBP are not found in golli-MBP proteinsand these uniquely classic MBP sequences should be generatedonly in myelin-forming cells. We investigated how tolerance isinduced to portions of classic MBP not found in golli-MBP usingTCR Tg mice specific for MBP121–140, an epitope contained onlyin classic MBP and presented by the MHC class II molecule I-Au.Surprisingly, most of the MBP-specific Tg T cells were eliminatedduring maturation in the thymus. Endogenous MBP was presentedin the MHC class II pathway only by bone marrow-derived cells inthe thymus and these cells also presented MBP constitutively inthe periphery (17). To determine which types of peripheral bonemarrow-derived cells presented endogenous MBP, fractionatedsplenocytes were used to stimulate the MBP-specific TCR Tg Tcells directly ex vivo. The majority of MBP-specific T cell prolif-eration was triggered by DCs, with a very small amount of pro-liferation inconsistently detected using splenic CD11c�F4/80�

macrophages as APCs (18).In the present study, we observed that LN cells triggered sig-

nificantly more MBP121–140-specific T cell proliferation ex vivothan did splenocytes. This observation suggested that detection ofMBP epitopes on different types of APCs might be easier usingcells isolated from LNs. Similar to our results with splenocytes, wefound that most of the proliferation of MBP-specific T cells trig-gered by LN cells ex vivo was due to presentation of endogenousMBP by both CD8�� and CD8�� DCs. Surprisingly, however,we found that resting B cells purified from LNs also stimulatedproliferation of both naive and previously activated/memory MBP-specific T cells ex vivo. Interestingly, acquisition of endogenousMBP by B cells did not appear to be BCR mediated nor wassynthesis of MBP by B cells required. Despite stimulation of aproliferative response, neither activated/memory nor naive MBP-specific T cells produced significant amounts of cytokines in re-sponse to resting B cells. Furthermore, activated/memory T cellsincubated with B cells presenting endogenous MBP became lessresponsive to subsequent stimulation with bulk LN cells. However,increasing the amount of MBP presented by resting B cells byadding exogenous MBP peptide stimulated activated/memory Tcells to produce effector cytokines. These results suggest that con-stitutive presentation of endogenous MBP by resting B cells mayhave a tolerogenic effect on previously activated/memory MBP-specific T cells. However, if the amount of endogenous MBP ac-quired by B cells increases, as could occur during an ongoingdemyelinating disease, presentation of MBP by resting B cells mayamplify the responses of activated/memory MBP-specific T cells.

Materials and MethodsMice

B10.PL-H2u H2-T18a/(73 NS)Sn (B10.PL) mice, B6.PL-Thy1a/Cy.J(Thy1.1), C57BL/6-Tg(IghelMD4)4Ccg/J (HEL-BCR-Tg) mice (19), andC57BL/6J mice were purchased from The Jackson Laboratory. The Thy1.1allele and Rag1�/� mutation were backcrossed 8 to 10 generations onto theB10.PL background. MBP�/� B10.PL mice, and MBP121–140-specificTCR Tg mice have been described previously (17, 20). All mice were bredand maintained in a specific pathogen-free facility at the University ofWashington (Seattle) and all procedures involving animals were approved

by the Institutional Animal Care and Use Committee at the University ofWashington.

Antibodies

All Abs used in these experiments were purchased from BD Biosciencesexcept anti-Ig-PE which was from Sigma-Aldrich (P8547).

T cell proliferation assays

Naive MBP-specific T cells were purified from spleen and LNs of MBP�/�

MBP121–140 TCR Tg mice by magnetic bead selection after labeling witheither biotinylated anti-V�2.3 and streptavidin-coated microbeads (Fig. 1)or Abs contained in the MACS CD4� T cell isolation kit (Miltenyi Biotec).CD44� TCR Tg T cells were obtained by sorting V�2�CD4�CD44� cellsfrom spleen and LN on a FACSVantage.

Activated/memory MBP-specific T cells were prepared by culturingspleen and LN cells from MBP�/� Tg mice with 5 nM MBP121–140 inClick’s medium supplemented with 10% FBS, penicillin-streptomycin, 4mM L-glutamine, 1 mM sodium pyruvate, and 0.5 mM 2-ME in T-25 tissueculture flasks at 3–5 � 106 cells/ml. After 4 days, cells were centrifugedover Lympholyte-M (Cedarlane Laboratories) to remove dead cells andtransferred to T-75 flasks containing 15 ml of fresh medium supplementedwith 2.5 U/ml IL-2. Twenty-four hours before harvest (day 7–9 poststimu-lation), 10 ml/flask IL-2-supplemented medium was added.

MBP-specific T cells (5 � 104 cells/well unless indicated otherwise infigure legends) were cultured in complete RPMI in triplicate with the in-dicated APCs in 96-well round-bottom plates at 37°C for 48 h, pulsed with[3H]thymidine, and harvested 18 h later. Bulk cells (spleen or LN) andpurified B cells were plated at 1 � 106 and 5 � 105 cells per well, re-spectively, unless indicated otherwise. For certain experiments, 8 �MBrdU (BD Biosciences) was added to each well after 48 h of culture.Eighteen to 24 hours after addition of BrdU, cells were harvested, pooled,and stained with anti-V�2, anti-CD4 and anti-BrdU Abs. Samples wereanalyzed on a FACScan to determine the percent of BrdU� cells in theV�2�CD4� population. For proliferation assays examining the functionaleffects of B cell presentation of MBP, activated/memory MBP-specific Tcells (5 � 105/ml) were incubated for five days with irradiated B cells(2.5 � 106/ml) purified by magnetic bead selection from MBP�/� orMBP�/� mice. Cells were then centrifuged over a Lympholyte-M gradientand were stained to determine numbers of Tg T cells recovered from thecultures. CD4�V�2�V�8� T cells (5 � 104/well) were then plated asdescribed above with LN cells from MBP�/� or MBP�/� mice and pro-liferation determined by [3H]thymidine incorporation. Stimulation index

FIGURE 1. Ex vivo LN cells trigger more proliferation of naive MBP-specific T cells than splenocytes. Single cell suspensions were preparedfrom either pooled LNs (inguinal, axial, and brachial) or spleens and platedat 1 � 106 cells/well. Naive MBP-specific T cells were added and prolif-eration was measured by incorporation of [3H]thymidine. The stimulationindex (SI) was plotted on a log scale with each symbol representing the SIobtained using cells from an individual mouse. Black bars indicate themean SI for each tissue. The fraction of individual samples with SI �2 was12/21 for cultures using splenocytes as APCs and 46/54 for cultures usingLN cells. The mean SI obtained using LN cells significantly differed fromthe mean SI using splenocytes (p � 0.01 by Student’s t test). Data shownare compiled from nine different experiments.

2098 PERIPHERAL APCs PRESENTING MYELIN BASIC PROTEIN

by guest on Decem

ber 22, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

was calculated by dividing the mean cpm of cultures containing MBP�/�

APCs by the mean cpm of cultures containing MBP�/� APCs.

Collagenase digests

LNs or spleens were harvested and subjected to mild collagenase digestion(0.6 Wunsch U/ml Liberase RI; Roche) in the presence of 50 �g/ml DNaseI (grade II; Roche) until tissues appeared fully digested (stirring �45 minat 37°C.). Digestion was stopped with the addition of EDTA (10 mM finalconcentration), RBC were lysed, and the sample was filtered through a 70-�m nylon mesh before the various cell purifications described below. AllDCs were harvested from collagenase-digested tissues unless otherwisenoted.

DC isolation and depletion

For DC purification by magnetic bead selection, spleen or LN cells werelabeled with anti-CD11c-conjugated microbeads (Miltenyi Biotec), andDCs were positively selected on an auto-MACS (Miltenyi Biotec). DCsubsets were purified from spleens (without collagenase digestion) by firstisolating CD11c� cells using anti-CD11c microbeads and magnetic beadselection. Cells were then stained with anti-CD11c-FITC and anti-CD8�-PE-Cy5 Abs and sorted into CD8�� and CD8��CD11c� subsets on aFACSVantage cell sorter (BD Biosciences). Subset purity was �95%. DCsubsets were purified from LN cells by first depleting T and B cells bymagnetic bead selection using anti-TCR and anti-CD19 biotinylated Absand streptavidin-conjugated microbeads (Miltenyi Biotec). Enriched DCswere then stained with anti-CD11c and anti-CD8 and sorted intoCD11c�CD8�� and CD11c�CD8�� subsets on an InFlux Flow Cytom-eter (Cytopeia) (Fig. 2B). LN cells were depleted of DCs or DC subsetsusing Abs specific for CD11c and CD8� and sorting on a FACSVantagecell sorter (BD Biosciences).

B cell isolation

Resting B cells were purified via negative selection from collagenase-di-gested LNs or spleens using biotinylated anti-CD11c, anti-CD11b, anti-CD43 and streptavidin-labeled microbeads (Miltenyi Biotec). Cells werenegatively selected on an auto-MACS. Cell purity was determined by stain-ing with anti-CD19, anti-CD43, anti-CD11c, and anti-CD11b. For purifi-cation of B cells from BCR-Tg F1 mice, anti-CD11b Ab was omitted fromthe negative selection protocol because BCR-Tg B cells are CD11b� in thismodel. In Fig. 4, resting B cells were positively purified by FACS by stainingLN cells with anti-mouse Ig-PE (F(ab�)2 fragment), anti-CD43-FITC and anti-CD11c-biotin/streptavidin-TriColor (Caltag). Resting B cells were negativelypurified by FACS by staining LN cells with PE-labeled anti-CD11c and anti-CD11b, and anti-CD43-biotin/streptavidin-TriColor. Ig�CD43�CD11c� cells(positive purification) and CD11c�CD11b�CD43� cells (negative purifi-cation) were sorted on a FACSAria (BD Biosciences). Purities for bothwere �98%.

Bone marrow chimeras

Bone marrow chimeras were generated by transferring 1 � 107 bone mar-row cells from MBP�/� donors into lethally irradiated MBP�/� Rag�/�

mice (1000 rad on day �1). Recipient mice were provided neomycin sul-fate (Sigma-Aldrich) in the drinking water (2 mg/ml) from day �2 to day14. Resting B cells were purified 8 weeks later by magnetic bead selectionfrom the LNs of bone marrow chimeric, wild-type MBP�/�, and MBP�/�

mice and used in ex vivo proliferation assays with activated/memory MBP-specific Tg T cells as described above.

ELISPOT

ELISPOT assays were performed in duplicate using anti-cytokine Ab pairs(BD Biosciences) for IFN-� and IL-2 according to the manufacturer’s di-rections. Activated/memory and naive T cells were serially diluted andincubated with the following APCs from MBP�/� mice: bulk LN cells,auto-MACS-purified B cells, peptide-pulsed bulk LN cells, or peptide-pulsed purified B cells. LN cells and purified B cells were also isolatedfrom MBP�/� mice and incubated with T cells at the same serial dilutionsto determine background numbers of spots. Either 5 � 105 purified B cellsor 1 � 106 bulk LN cells were plated per well. Plates containing activatedT cells were incubated for 18 h. Plates containing naive T cells were in-cubated 18 h in experiment 1 and 40 h in experiments 2 and 3. Frequenciesof spots/number of MBP-specific T cells were determined for each welland duplicate well frequencies were averaged (only wells that had �300spots per well were counted). SDs for duplicate wells were calculatedbased on the binomial distribution. The average number of spots in wellscontaining MBP�/� APCs were subtracted from the average number ofspots in wells containing MBP�/� APCs at the specified T cell dilutions.

FIGURE 2. DCs stimulate most of the proliferation observed for bothnaive and activated/memory MBP-specific T cells responding to LN cellsex vivo. A, Purified naive MBP-specific T cells were incubated with eitherbulk LN cells from MBP�/� mice or LN cells depleted of various DCpopulations (1 � 106/well). The DC-depleted populations were obtained byFACS using the indicated Abs. Non-depleted bulk LN cells were alsosorted through a live gate to assure that the samples were treated compa-rably. B, Purified naive MBP-specific T cells were incubated with the in-dicated DC populations isolated from either MBP�/� (black bars) orMBP�/� (white bars) LNs by FACS based on CD8� and CD11c expres-sion; 2 � 104 purified DCs or 1 � 106 bulk LN cells were plated per well.Purity of sorted DCs was �79% in all samples, contaminants were smallCD11c� cells. C, Activated/memory MBP-specific T cells (6 � 104 cells/well) were incubated with either bulk LN or spleen cells or with auto-MACS-purified CD11c� DCs (8 � 104 DCs/well, purity �96% CD11c�).Proliferation was measured by incorporation of [3H]thymidine.

2099The Journal of Immunology

by guest on Decem

ber 22, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

A combined SD was calculated for these values. Data are plotted as theaverage number of specific spots observed versus the number of MBP-specific T cells per well.

ResultsEndogenous MBP is presented by both CD8�� and CD8��

DCs

Our previous studies showed that constitutive presentation of MBPepitopes could be detected in vivo in peripheral lymphoid tissuesof healthy, wild-type mice using TCR Tg T cells specific for theepitope MBP121–140 (17). The MBP-specific T cells also prolif-erated to bulk splenocytes isolated from wild-type mice ex vivobut not to splenocytes isolated from MBP-deficient (MBP�/�)mice. Fractionation of the splenocytes demonstrated that DCs werethe primary APCs in the spleen that stimulated proliferation of theMBP-specific T cells ex vivo (18). Here we show that LN cellsisolated from wild-type mice are more potent than splenocytes instimulating naive MBP121–140-specific Tg T cells ex vivo (Fig.1). The stimulation index for MBP-specific T cells proliferating inresponse to either splenocytes or LN cells varied between individ-ual mice; however, the average stimulation index observed usingLN cells as APCs was significantly greater than the average stim-ulation index obtained using splenocytes as APCs ( p � 0.01). Thisobservation motivated us to investigate which cells in the LN stim-ulate MBP-specific T cells ex vivo.

Depletion of DCs from bulk LN cells reduced ex vivo prolifer-ation of naive MBP-specific T cells by 94% (Fig. 2A), indicatingthat DCs are the predominant APC presenting endogenous MBPepitopes in the LN as was observed for splenocytes. To determinewhether presentation of endogenous MBP is limited to a particularDC subset, bulk LN cells depleted of either CD8�� or CD8��

DCs were used to stimulate naive Tg MBP-specific T cells ex vivo.Depletion of either DC subset substantially reduced the prolifera-tion of naive MBP-specific T cells relative to that seen with bulkLN cells but not to the same degree as the reduction observedwhen all CD11c� cells were depleted (Fig. 2A), suggesting thatboth DC subsets may present MBP. To confirm this,CD11c�CD8�� and CD11c�CD8�� cells were purified from LNcells by flow cytometry and used to stimulate naive MBP-specificT cells ex vivo. As shown in Fig. 2B, both CD8�� and CD8�� LNDCs stimulated the naive MBP-specific T cells to proliferate. Asimilar experiment using CD8�� and CD8�� DCs sorted fromsplenocytes also showed that both of these populations stimulatednaive MBP-specific T cells ex vivo (data not shown). MBP-spe-

cific T cells that had been previously activated in vitro also pro-liferated to endogenous MBP presented ex vivo by DCs (Fig. 2C).As expected, the response of the activated/memory MBP-specificT cells was even stronger than the response of naive MBP-specificT cells to endogenous MBP presented by DCs isolated from eitherLN cells or spleen.

Resting B cells present endogenous MBP to both naive andactivated MBP-specific T cells

The data described above indicate that DCs are the major APC inboth spleen and LN that present endogenous MBP epitopes. In-deed, DCs were the only APCs isolated from the spleen that con-sistently stimulated significant proliferation of MBP-specific Tcells ex vivo (18). Surprisingly, this was not the case for cellsisolated from LNs. Resting B cells isolated from LNs triggeredAg-specific proliferation of both naive and previously activatedMBP-specific T cells ex vivo (Fig. 3). The amount of proliferationstimulated by resting B cells was less than that triggered by DCsfor both activated and naive T cells, reflecting either more acqui-sition of MBP by DCs in vivo and/or their greater efficiency asAPCs for T cell activation. As expected, the proliferative responseto resting B cells ex vivo is lower for naive T cells compared withactivated/memory T cells but is still significantly above back-ground levels (stimulation index � 21). In these proliferation as-says, the naive T cells were obtained by isolating V�2� T cellsfrom MBP�/� Rag�/� TCR Tg mice. Because some of the V�2�

population could contain T cells that express a second endogenousV�-chain, it was possible that this population contained T cellsthat had been activated in vivo by environmental Ags. To confirmthat naive MBP-specific T cells could respond to purified, restingB cells, V�2�CD44low T cells were sorted from the TCR TgMBP�/� mice and incubated with resting B cells. The sorted naiveT cells proliferated to resting B cells as well as V�2� T cells thatwere not sorted for low CD44 expression (data not shown), con-firming that resting B cells present sufficient MBP ex vivo to trig-ger proliferation of naive T cells.

The resting B cells used in these assays were purified usingnegative selection by magnetic beads. Contaminating DCs were�1% of the purified B cell population. An experiment titrating thenumber of DCs needed to detect T cell proliferation indicated thatthe proliferative response stimulated by purified B cells could notbe due to the small number of potentially contaminating DCs in theassays (data not shown). To further eliminate the possibility thatcontaminating DCs accounted for the T cell response to purified B

FIGURE 3. Resting B cells trigger Ag-specific pro-liferation of MBP-specific T cells ex vivo. Proliferativeresponses of MBP-specific T cells to bulk LN cells (leftpanel) or purified B cells (right panel) are shown. Pu-rified B cells were 98% CD19� and �1% CD11c�.Black and white bars represent the responses of acti-vated T cells to APCs from MBP�/� and MBP�/� mice,respectively, and gray and striped bars indicate the re-sponses of naive T cells. Proliferation was measured byincorporation of [3H]thymidine. Data are representativeof two independent experiments.

2100 PERIPHERAL APCs PRESENTING MYELIN BASIC PROTEIN

by guest on Decem

ber 22, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

cells, B cells were sorted by flow cytometry using either positiveor negative selection. B cells were positively purified using anti-CD43 and anti-mouse Ig F(ab�)2 Abs (Fig. 4A) or negatively pu-rified using Abs specific for CD43, CD11b, and CD11c (Fig. 4B).B cells purified by either method stimulated proliferation of bothactivated and naive MBP-specific Tg T cells (Fig. 4C). To confirmthat incubation with the anti-mouse Ig F(ab�)2 fragment did notactivate the B cells, expression of CD40, CD86, and MHC class IIwas analyzed following overnight incubation with this Ab. Noincreases in expression of these molecules were observed in Bcells incubated overnight in tissue culture plates with or withoutanti-mouse Ig F(ab�)2 compared with B cells analyzed directly exvivo (data not shown).

B cells acquire sufficient exogenous MBP in a BCR-independentmanner to stimulate T cells ex vivo

The observation that resting B cells purified from wild-type miceconstitutively present sufficient endogenous MBP to stimulate pro-liferation of both naive and activated MBP-specific T cells ex vivowas very surprising because effective Ag presentation by B cells isbelieved to depend on the efficient capture of Ag through a specificBCR. Consistent with this, activation of naive T cells by resting Bcells in vivo has been shown to require a very high frequency of Bcells specific for the same Ag as the T cells (12, 13). Thus, oneexplanation for our results could be that wild-type B10.PL micecontain an unusually high frequency of MBP-specific B cells de-spite the fact that these self-reactive B cells should undergo toler-ance in vivo. To test this hypothesis, B cells isolated from hen egglysozyme (HEL)-specific BCR Tg mice were used as APCs tostimulate MBP-specific T cells ex vivo. Although these BCR-Tgmice were not on a Rag�/� background, expression of the BCRtransgenes should significantly limit the repertoire of endogenousBCRs specific for other Ags. Because the BCR Tg mice were onthe C57BL/6 (H-2b) background, they were bred to B10.PL (H-2u)mice to obtain F1 B cells expressing both the HEL-specific TgBCR and the appropriate MHC molecule recognized by the MBP-specific T cells (I-Au). As expected, both activated/memory andnaive MBP-specific T cells proliferated in response to both F1BCR Tg and non-Tg LN cells ex vivo, as this population contains

DCs that are efficient at presenting endogenous MBP and are notaffected by expression of the BCR transgenes (Fig. 5A). The MBP-specific T cells did not proliferate in response to LN cells isolatedfrom wild-type C57BL/6 mice, demonstrating that their prolifera-tion to the F1 LN cells required the I-Au MHC molecule and wasnot due to alloreactivity directed toward B6 Ags (Fig. 5A). Impor-tantly, purified B cells isolated from either BCR-Tg or non-Tg F1mice stimulated comparable and significant levels of proliferation ofboth activated and naive MBP-specific T cells (Fig. 5B). These resultsindicate that the ability of resting B cells to trigger proliferation ofMBP-specific T cells directly ex vivo does not depend on a highprecursor frequency of B cells expressing MBP-specific BCRs.

The ability of B cells isolated from HEL-specific BCR Tg miceto activate MBP-specific T cells ex vivo suggested that the acqui-sition of MBP by resting B cells occurred in a BCR-independentmanner. Because previous reports suggested that some immunecells synthesize classic MBP (21), we investigated whether syn-thesis of MBP by B cells is necessary to trigger Ag-specific pro-liferation of MBP-specific T cells. Bone marrow chimeric micewere generated by transplanting bone marrow from MBP�/� miceinto irradiated Rag�/� MBP�/� B10.PL mice. B cells that maturein these recipients can only be derived from the MBP�/� donorbone marrow cells. B cells isolated from these bone marrow chi-meras, as well as from wild-type MBP�/� mice and MBP�/�

mice, were used ex vivo to stimulate activated/memory MBP-spe-cific T cells. As shown in Fig. 6, resting B cells purified from eitherthe bone marrow chimeras or from wild-type mice stimulated pro-liferation of MBP-specific T cells ex vivo to the same extent. Thisresult indicates that resting B cells obtain enough MBP from thehost to trigger proliferation of MBP-specific T cells ex vivo with-out a requirement for de novo synthesis of the protein.

Interaction with resting B cells presenting endogenous MBPcauses activated/memory T cells to become less responsive tosubsequent stimulation

Ag presentation by resting B cells has been reported to have atolerogenic effect on naive T cells. However, activated/memoryMBP-specific T cells that recognize MBP on resting B cells mayhave a different fate. To investigate the effect of endogenous MBP

FIGURE 4. Stimulation of activated and naive MBP-specific T cells by purified B cells is not due to contaminating non-B cell APCs. A, B cells werepurified by positive selection using flow cytometry to sort LN cells stained with anti-mouse Ig (non-activating Fab), anti-CD43, and anti-CD11c Abs. Cellswere collected through a live gate, two gates to remove doublets and an Ig�CD43� gate (top left panel) and a CD11c� gate (top right panel). Post-sortedcells are shown in the bottom panels. Cells were 98% pure through the live gate. B, B cells were also purified via negative selection by staining LN cellswith anti-CD43, CD11b, and CD11c Abs and collecting cells through a CD43�CD11c�CD11b� gate (top panel). Sorted cells were 99% pure (bottompanel). C, Proliferation of activated/memory or purified naive MBP-specific T cells incubated with MBP�/� (black bars) or MBP�/� (white bars) sortedB cells or bulk LN cells was determined via BrdU incorporation. Data are expressed as the percentage of V�2�CD4� T cells that were BrdU� at the endof the incubation. The percentage of V�2�CD4� T cells that incorporated BrdU after incubation with bulk LN cells is under-represented due to the presenceof some non-Tg CD4�V�2� T cells present in the LN cells used as APCs.

2101The Journal of Immunology

by guest on Decem

ber 22, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

presentation by resting B cells on activated/memory T cells, acti-vated/memory T cells were incubated with B cells isolated fromeither wild-type or MBP�/� mice for 5 days. During this incuba-tion, the T cells exposed to wild-type B cells expanded 1.5-fold,whereas the T cells exposed to MBP�/� B cells did not expand.After incubation with either MBP�/� or MBP�/� B cells, the pro-liferative potential of the MBP-specific T cells was assessed byre-stimulating them with bulk LN cells. Although T cells fromboth groups proliferated in response to the LN cells, the T cells thathad been exposed to MBP�/� B cells proliferated much more thanT cells that had been exposed to wild-type B cells (Fig. 7). Thus,encounter of activated T cells with resting B cells presenting en-dogenous MBP appears to decrease the proliferative potential ofthe activated/memory T cells upon subsequent restimulation.

Resting B cells stimulate proliferation but not cytokineproduction by MBP-specific T cells

To determine whether resting B cells presenting endogenous MBPcan stimulate T cell effector function as well as proliferation, wemeasured cytokine production by MBP-specific T cells incubated

with wild-type B cells. Both activated/memory and naive Tg Tcells were incubated with either purified resting B cells or bulk LNcells, and the number of T cells producing cytokines was measuredusing ELISPOT analyses for IL-2 and IFN-�. For each ELISPOTassay, the T cells were plated at serial dilutions to confirm a dose/response relationship between the number of T cells plated and thenumber of spots observed. Background levels of cytokine produc-tion (�5 spots) were measured by plating the same numbers of Tcells with B cells or LN cells isolated from MBP�/� mice insteadof wild-type mice. Because naive T cells typically produce verylow levels of cytokines during their initial stimulation, larger num-bers of naive T cells were plated compared with activated T cellsand the incubations were conducted for both 18 and 40 h. Anexample of ELISPOT data generated when activated/memoryMBP-specific T cells were incubated with B cells is shown in Fig.8. For both cytokines, increasing the number of T cells plated perwell resulted in increased numbers of observed spots. Addition ofexogenous MBP peptide to the resting B cells resulted in increasedcytokine production compared with B cells presenting only endog-enous MBP. ELISPOT data such as those shown in Fig. 8 wereused to calculate the percentages of MBP-specific T cells that pro-duced cytokines in response to either B cells or bulk LN cells, withor without exogenous peptide (Table I). Because of the large dif-ference in numbers of activated/memory versus naive T cellsplated in each assay, calculating the percentages of responding Tcells provided the most straightforward way to compare results.

The data in Table I show that naive T cells responding to restingB cells did not produce any detectable cytokines. Even when ex-ogenous MBP peptide was added to the B cells, only a very smallpercentage of naive T cells produced IL-2 in experiments 2 and 3(percentages over 0.1% indicate values that were calculated fromdata that followed an approximately linear titration with increasingnumbers of T cells and are likely to reflect accurate detection ofcytokine-producing T cells). Similarly, only low percentages ofnaive T cells produced cytokines in response to bulk LN cells eventhough the predominant APCs presenting MBP in this populationare DCs. Addition of exogenous MBP peptide to the bulk LN cellsresulted in some increase in the percentage of IL-2-producing Tcells but little change in production of IFN-�.

Unlike naive MBP-specific T cells, activated/memory T cellsproduced cytokines when incubated with bulk LN cells. Although

FIGURE 5. Presentation of endogenous MBP by B cells does not de-pend on an MBP-specific BCR. A, Activated/memory (black bars) or naive(white bars) MBP-specific T cells were incubated with bulk LN cells pre-pared from either HEL-BCR-Tg (B6 � B10.PL) F1 MBP�/� mice, non-BCR-Tg F1 litter mates, wild-type B6 or MBP�/� B10.PL mice. B, Acti-vated and naive MBP-specific T cells (as indicated) were incubated withresting B cells purified as described in Materials and Methods. B cells were�95% CD19� in all samples and the contamination of DCs in each samplewas �1%. Proliferation was measured by incorporation of [3H]thymidine.Error bars represent one SD of counts from triplicate wells. The experimentwas performed twice with similar results.

FIGURE 6. Resting MBP�/� B cells acquire sufficient MBP from theperiphery to stimulate activated MBP-specific T cells ex vivo. Activated/memory MBP-specific T cells were cultured with resting B cells purifiedfrom MBP�/�, MBP�/�, or MBP�/� 3 MBP�/�Rag�/� bone marrowchimeric (BMC) mice. Proliferation was measured by incorporation of[3H]thymidine. Data are representative of two independent experiments.

2102 PERIPHERAL APCs PRESENTING MYELIN BASIC PROTEIN

by guest on Decem

ber 22, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

the largest percentage of these cells produced IL-2 in experiment2, the size of the spots in the IFN-� assay was much larger than thesize of the spots in the IL-2 assay. Addition of exogenous MBPpeptide to the bulk LN cells did not significantly increase the per-centage of T cells producing some cytokines in experiment 2 andonly doubled cytokine production seen in experiment 3. In contrastto the response to LN cells, however, �1% of activated T cellsproduced cytokines in response to resting B cells ex vivo. Thisresult indicates that the proliferation triggered by exposure to rest-ing B cells was not accompanied by stimulation of effector func-tion in the activated/memory T cells. Interestingly, addition ofMBP peptide to the B cells increased the percentage of activated/memory MBP-specific T cells producing cytokines, such that thepercentage of IFN-�-producing T cells was similar to that seen inresponse to LN cells. Thus, the amount of MBP that is constitu-tively presented by resting B cells is sufficient to trigger activated/memory MBP-specific T cells to proliferate but is not sufficient toinduce production of effector cytokines. However, when the num-ber of MBP/MHC complexes presented by resting B cells is in-creased by addition of exogenous MBP peptide, activated/memoryMBP-specific T cells both proliferate and produce cytokines, sug-gesting that the amount of MBP available to resting B cells affectsthe outcome of their interaction with activated MBP-specific Tcells.

DiscussionMBP is unique among myelin proteins in that it is a component ofboth central and peripheral myelin. This expression pattern pre-sumably accounts for the ability to detect constitutive presentationof MBP epitopes in peripheral lymphoid tissues in vivo (17, 22,23). By analyzing different splenocyte populations, we previouslyshowed that only DCs presented sufficient endogenous MBP totrigger proliferation of naive MHC class II-restricted MBP-specific

T cells ex vivo (18). Here we showed that bulk LN cells stimulateon average more proliferation of MBP-specific T cells directly exvivo compared with splenocytes. This observation suggests thatdegradation of myelin in innervated tissues is a significant sourceof MBP presented in the periphery because MBP derived from thissource should accumulate at higher levels in lymph compared withblood. An alternative possibility is that LNs contain DC popula-tions not found in the spleen that express high levels of MHC classII and therefore may be more efficient at stimulating naive T cells(24, 25). These possibilities are not mutually exclusive, and bothmay contribute to the increased ability of LN cells to stimulatenaive MBP-specific T cell proliferation compared with spleno-cytes. Among individual mice, there is more variation in theamount of T cell proliferation triggered by LN cells compared withthe proliferation triggered by splenocytes, suggesting that eitherthe amount of endogenous MBP transported to LNs at any giventime varies from mouse to mouse and/or the number of DCs ex-pressing high levels of MHC class II present in peripheral LNs isvariable. Overall, the data indicate that MBP-specific T cells invivo are more likely to encounter APCs presenting endogenousMBP in LNs than in the spleen.

FIGURE 7. Activated/memory T cells are less responsive to stimulationwith LN cells following exposure to B cells presenting endogenous MBP.Activated/memory MBP-specific T cells were incubated with purified Bcells from either MBP�/� or MBP�/� mice. Cells were incubated for 5days at a density of 3 � 106 total cells/ml with a T cell to B cell ratio of5:1. Following incubation, live cells were harvested, and 5 � 104

CD4�V�2�V�8� cells (determined by flow cytometry) from each culturewere set up in a stimulation assay with irradiated MBP�/� (black bars) orMBP�/� (white bars) whole LN cells. Proliferation was measured by in-corporation of [3H]thymidine. The stimulation index obtained with LNcells as APCs for activated/memory T cells incubated previously withMBP�/� or MBP�/� B cells is 59 and 101, respectively. Data are repre-sentative of two experiments.

FIGURE 8. B cells trigger little cytokine production by activated/mem-ory MBP-specific T cells ex vivo. The number of spots/well detected byELISPOT for the indicated cytokines is shown for increasing numbers ofactivated/memory MBP-specific T cells incubated with purified B cells(top panel) or B cells pulsed with MBP121–140 peptide (bottom panel).Data are representative of two experiments and demonstrate the type ofdata used to generate Table I.

2103The Journal of Immunology

by guest on Decem

ber 22, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

We analyzed the ability of LN APCs to stimulate activated/memory as well as naive MBP-specific T cells in order to detectpresentation of endogenous MBP epitopes by APCs that might notprovide adequate co-stimulatory signals to trigger naive T cells todivide. Our results showed that most of the proliferation by bothnaive and activated/memory MBP-specific T cells ex vivo was dueto presentation of MBP by DCs, and that CD8�� and CD8�� LNDCs stimulated MBP-specific T cells equally well. This result is incontrast to studies of DCs presenting MHC class I- and MHC classII-restricted epitopes of islet Ags in which only CD8�� DCs (6) orCD11b� DCs (4) isolated from pancreatic LNs were found to stim-ulate Ag-specific T cells ex vivo. However, presentation of endog-enous MBP by both CD8�� and CD8�� DC subsets is consistentwith another report documenting presentation of self-Ags by bothCD8�� and CD8�� DCs in draining LNs (26). Interestingly, thisstudy suggested that CD8�� and CD8�� DCs may acquire self-Ags in different locations in vivo. Scheinecker et al. found thatepitopes of H�/K� ATPase, a self-Ag synthesized in the stomach,are presented by both CD8�� and CD8�� DCs in the draininggastric LN but are presented only by CD8�� DCs in the gastricmucosa (26). This observation suggested that Ag carried byCD8�� DCs migrating from tissues might be transferred toCD8�� DCs residing in LNs. This process of transfer of Ag frommigratory to resident DCs has been demonstrated in vitro as well,and may be a strategy for increasing the range and number of DCsin LNs presenting Ag derived from peripheral tissues (27). Wecannot determine whether initial capture of MBP in peripheral tis-sues is limited to the CD8�� subset because most peripheral tis-sues are innervated and therefore there is no unique site of MBPsynthesis. MBP may also be present in lymph and blood as a sol-uble Ag and could gain access to resident LN DCs without beingtransported there by tissue DCs. In addition to DCs, it is possiblethat some MBP may be captured by macrophages in peripheraltissues and presented in lymphoid organs as we observed marginalstimulation of naive T cells (stimulation index �3) by macro-phages purified from the spleen in one of three experiments in ourprevious study (18). The small number of macrophages in LNsprevented us from determining if LN macrophages also presentendogenous MBP ex vivo.

Although DCs were responsible for the majority of the prolif-erative response of MBP-specific T cells in both the LN andspleen, we were surprised to discover that resting B cells isolatedfrom LNs also stimulated proliferation of both activated and naive

MBP121–140-specific T cells ex vivo. Our previous studiesshowed that B cells isolated from the spleen did not trigger pro-liferation of naive MBP-specific T cells, which may reflect thelower amount of MBP present in the spleen. The bone marrowchimeric mice demonstrated that peripheral MBP acquired byMBP�/� B cells in an MBP�/� mouse is sufficient to trigger T cellproliferation, suggesting that MBP obtained from myelin is a sig-nificant source of MBP epitopes. These data do not exclude thepossibility that B cells synthesize some classic MBP and present itin the MHC class II pathway. However, we believe that this is notlikely to be the major source of MBP presented by B cells becauseB cell autonomous synthesis of MBP should have been detected onB cells isolated from the spleen. The acquisition of MBP by B cellsappears to occur in a BCR-independent manner because HEL-spe-cific BCR Tg B cells triggered equivalent T cell proliferation asobserved with non-Tg B cells. This result was also surprising be-cause previous studies have shown that BCR-independent acqui-sition of soluble Ag by B cells in vitro is approximately 1,000–10,000 times less efficient compared with BCR-mediatedinternalization of Ag (28). In vivo, Zhong et al. demonstrated thati.v. injection of very large amounts (milligram quantities) of sol-uble protein associated with induction of high-dose tolerance re-sulted in presentation of processed Ag by Ag-nonspecific, restingB cells and DCs. Under these conditions, DCs and B cells pre-sented similar amounts of MHC class II-restricted epitopes fromthe injected Ag (when the number of MHC complexes present onthe two different cell types was taken into consideration) (29).However, it is unlikely that such a large amount of soluble MBPis available in the lymphatic system draining peripheral tissues,suggesting that a cell-mediated mechanism is more likely to ex-plain B cell acquisition of MBP.

Although our studies have not precisely defined the mechanismof B cell acquisition of endogenous MBP, previous studies suggestthat interactions between DCs and resting B cells result in transferof unprocessed Ag between the two cell types. Studies in the rathave shown that resting B cells cluster with DCs, allowing Agtransfer from the DC to the B cell independent of BCR specificity(30, 31). The mechanism of this transfer has not been identified.One intriguing hypothesis for the mechanism by which B cellsacquire MBP in a BCR-independent fashion is through uptake ofMBP-containing exosomes secreted by DCs. These secreted ves-icles are formed by generation of multivesicular bodies in the lateendocytic compartments of both immature and mature DCs and are

Table I. Percentages of MBP-specific T cells producing effector cytokines in response to different APCsa

Purified B Cells Bulk LN

Activated Naive Activated Naive

Endog. � Peptide Endog. � Peptide Endog. � Peptide Endog. � Peptide

Expt. 1IFN-� 0.19 ND 0.00 ND 3.84 ND 0.00 NDIL-2 0.26 ND 0.00 ND 12.61 ND 0.36 ND

Expt. 2IFN-� 0.72 13.09 0.00 0.06 9.15 9.99 0.19 0.49IL-2 0.26 3.59 0.01 0.19 10.66 12.42 0.55 3.13

Expt. 3IFN-� 0.04 8.35 0.00 0.00 4.167 9.09 0.00 0.28IL-2 0.03 0.48 0.00 0.10 2.69 4.80 0.5 3.36

a ELISPOT assays were performed in duplicate wells containing a constant number of MBP�/� or MBP�/� APCs and serial dilutions of activated/memory or naive T cells.The percentage of T cells producing cytokines in response to each type of APC (purified B cells or bulk LN cells with or without exogenous MBP121-140 peptide) was calculatedusing the average number of spots from dilutions in which 15–175 spots were observed in response to the MBP�/� APCs. The number of spots obtained using T cells at thesame serial dilution incubated with MBP�/� APCs were averaged between the duplicate wells and subtracted as background from the average number of spots observed withthe MBP�/� APCs. In Expt. 1, both activated/memory and naive T cells were stimulated for 18 h. In Expts. 2 and 3, activated T cells were stimulated for 18 h and naive T cellswere stimulated for 40 h.

2104 PERIPHERAL APCs PRESENTING MYELIN BASIC PROTEIN

by guest on Decem

ber 22, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

released into the extracellular environment by fusion with theplasma membrane (32). The exosomes secreted by DCs appear toreflect the functional status of the DC, such that only exosomessecreted by mature DCs are able to stimulate T cell effector func-tion (33, 34). Thus, transport of MBP from peripheral tissues byDCs, even if this function is restricted to only one subset of DCs,may result in transfer of MBP not only to other DC subsets but toB cells as well. Because the DCs transporting MBP from periph-eral tissues in healthy mice should be immature, the uptake ofexosomes from these DCs should deliver the Ag in the absence ofother molecules associated with immunogenic signals. This pro-cess would serve to increase the number of APCs in LNs thatpresent MBP in a tolerogenic context. If DCs are the major sourceof MBP acquired by B cells, then our inability to detect MBPpresentation by splenic B cells may reflect the lower amount ofMBP present on DCs migrating to the spleen versus the LN.

Our analyses indicated that both bulk LN cells and resting Bcells presenting endogenous MBP triggered naive T cells to pro-liferate but not produce cytokines. In contrast, MBP-specific acti-vated/memory T cells respond to APCs presenting endogenousMBP differently: they proliferate and produce cytokines in re-sponse to endogenous MBP presented by LN cells containing DCs,but they respond to resting B cells by proliferating without pro-ducing cytokines. Activated/memory MBP-specific T cells ex-posed in vitro to resting B cells presenting endogenous MBP arealso more refractory to subsequent stimulation by bulk LN cellscompared with T cells that were not first exposed to the B cells.Thus, presentation of steady-state levels of endogenous MBP byresting B cells appears to be a tolerogenic event for activated/memory T cells. This outcome may depend on the amount of MBPthat is available to B cells to take up rather than on the quality ofthe signals transmitted by resting B cells, as increasing the amountof MBP on the B cell surface by addition of MBP peptide triggeredcytokine production by activated/memory T cells that was com-parable to that triggered by bulk LN cells.

Our findings have interesting implications for the pathogenesisof CNS autoimmune disease. These studies demonstrate that clas-sic MBP is a very ubiquitous Ag, detectable by both naive andactivated/memory MBP-specific T cells on the surface of DCs andB cells in healthy mice with steady-state levels of MBP in periph-eral tissues. In the absence of autoimmune disease, any MBP-specific T cells circulating in the periphery should have a naivephenotype and encounter with either DCs or B cells presentingMBP should be a tolerogenic event as neither type of APC triggersnaive T cells to acquire effector function. However, MBP-specificT cells that have differentiated into effector T cells during thecourse of CNS autoimmune disease will respond differently to anencounter with APCs presenting endogenous MBP in the periph-ery. Activated/memory T cells will proliferate and retain effectorfunction when interacting with DCs presenting endogenous MBP.Interaction with resting B cells presenting steady-state levels ofMBP, on the other hand, would render the activated T cells lesspathogenic as they will not produce cytokines and will becomemore refractory to further stimulation. Given the numerical advan-tage of B cells compared with DCs in LNs, this dampening effectby B cells may be physiologically significant. This phenomenonmay explain why the presence of endogenous B cells has beenimplicated in modulating experimental autoimmune encephalomy-elitis, an animal model of MS, even though disease can be inducedin B cell-deficient mice (35, 36). A different scenario mightemerge, however, if the amount of endogenous MBP available inthe periphery increases due to degradation of myelin in the CNS.In this case, the amount of MBP presented by B cells could in-crease, causing them to function like DCs in stimulating both T

cell proliferation and cytokine production, thus abrogating theirtolerogenic effect.

AcknowledgmentsWe thank Dr. T. Brabb, I. Stromnes, S. Cabbage, H. Sumerfield, and N.Mausolf for critical reading of the manuscript and assistance with labora-tory experiments, and N. Mausolf, R. Rowe, and H. Sumerfield for excel-lent management of the mouse colony and laboratory resources.

DisclosuresThe authors have no financial conflict of interest.

References1. Kurts, C., M. Cannarile, I. Klebba, and T. Brocker. 2001. Dendritic cells are

sufficient to cross-present self-antigens to CD8 T cells in vivo. J. Immunol. 166:1439–1442.

2. Iyoda, T., S. Shimoyama, K. Liu, Y. Omatsu, Y. Akiyama, Y. Maeda,K. Takahara, R. M. Steinman, and K. Inaba. 2002. The CD8� dendritic cellsubset selectively endocytoses dying cells in culture and in vivo. J. Exp. Med.195: 1289–1302.

3. Hawiger, D., K. Inaba, Y. Dorsett, M. Guo, K. Mahnke, M. Rivera, J. V. Ravetch,R. M. Steinman, and M. C. Nussenzweig. 2001. Dendritic cells induce peripheralT cell unresponsiveness under steady state conditions in vivo. J. Exp. Med. 194:769–779.

4. Hugues, S., E. Mougneau, W. Ferlin, D. Jeske, P. Hofman, D. Homann,L. Beaudoin, C. Schrike, M. Von Herrath, A. Lehuen, and N. Glaichenhaus.2002. Tolerance to islet antigens and prevention from diabetes induced by limitedapoptosis of pancreatic beta cells. Immunity 16: 169–181.

5. Shortman, K., and Y. J. Liu. 2002. Mouse and human dendritic cell subtypes. Nat.Rev. Immunol. 2: 151–154.

6. Belz, G. T., G. M. Behrens, C. M. Smith, J. F. Miller, C. Jones, K. Lejon,C. G. Fathman, S. N. Mueller, K. Shortman, F. R. Carbone, and W. R. Heath.2002. The CD8�� dendritic cell is responsible for inducing peripheral self-tol-erance to tissue-associated antigens. J. Exp. Med. 196: 1099–1104.

7. Baumgarth, N. 2000. A two-phase model of B-cell activation. Immunol. Rev. 176:171–174.

8. Eynon, E. E., and D. C. Parker. 1992. Small B cells as antigen-presenting cellsin the induction of tolerance to soluble protein antigens. J. Exp. Med. 175:131–138.

9. Ryan, J. J., R. E. Gress, K. S. Hathcock, and R. J. Hodes. 1984. Recognition andresponse to alloantigens in vivo. II. Priming with accessory cell-depleted donorallogeneic splenocytes: induction of specific unresponsiveness to foreign majorhistocompatibility complex determinants. J. Immunol. 133: 2343–2350.

10. Fuchs, E. J., and P. Matzinger. 1992. B cells turn off virgin but not memory Tcells. Science 258: 1156–1159.

11. Townsend, S. E., and C. C. Goodnow. 1998. Abortive proliferation of rare T cellsinduced by direct or indirect antigen presentation by rare B cells in vivo. J. Exp.Med. 187: 1611–1621.

12. Constant, S. L. 1999. B lymphocytes as antigen-presenting cells for CD4� T cellpriming in vivo. J. Immunol. 162: 5695–5703.

13. Rodriguez-Pinto, D., and J. Moreno. 2005. B cells can prime naive CD4� T cellsin vivo in the absence of other professional antigen-presenting cells in a CD154-CD40-dependent manner. Eur. J. Immunol. 35: 1097–1105.

14. Lemke, G. 1988. Unwrapping the genes of myelin. Neuron 1: 535–534.15. Givogri, M. I., E. R. Bongarzone, and A. T. Campagnoni. 2000. New insights on

the biology of myelin basic protein gene: the neural-immune connection. J. Neu-rosci. Res. 59: 153–159.

16. Seamons, A., A. Perchellet, and J. Goverman. 2003. Immune tolerance to myelinproteins. Immunol.Res. 28: 201–204.

17. Huseby, E. S., B. Sather, P. G. Huseby, and J. Goverman. 2001. Age-dependentT cell tolerance and autoimmunity to myelin basic protein. Immunity 14:471–481.

18. Seamons, A., J. Sutton, D. Bai, E. Baird, N. Bonn, B. F. Kafsack, J. Shabanowitz,D. F. Hunt, C. Beeson, and J. Goverman. 2003. Competition between two MHCbinding registers in a single peptide processed from myelin basic protein influ-ences tolerance and susceptibility to autoimmunity. J. Exp. Med. 197:1391–1397.

19. Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill,R. A. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael,et al. 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334: 676–682.

20. Harrington, C. J., A. Paez, T. Hunkapiller, V. Mannikko, T. Brabb, M. Ahearn,C. Beeson, and J. Goverman. 1998. Differential tolerance is induced in T cellsrecognizing distinct epitopes of myelin basic protein. Immunity 8: 571–580.

21. Liu, H., A. J. MacKenzie-Graham, K. Palaszynski, S. Liva, and R. R. Voskuhl.2001. “Classic” myelin basic proteins are expressed in lymphoid tissue macro-phages. J. Neuroimmunol.116: 83–93.

22. Huseby, E. S., and J. Goverman. 2000. Tolerating the nervous system: a delicatebalance. J. Exp. Med. 191: 757–754.

23. Perchellet, A., I. Stromnes, J. M. Pang, and J. Goverman. 2004. CD8� T cellsmaintain tolerance to myelin basic protein by ‘epitope theft’. Nat. Immunol. 5:606–614.

2105The Journal of Immunology

by guest on Decem

ber 22, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

24. Henri, S., D. Vremec, A. Kamath, J. Waithman, S. Williams, C. Benoist,K. Burnham, S. Saeland, E. Handman, and K. Shortman. 2001. The dendritic cellpopulations of mouse lymph nodes. J. Immunol. 167: 741–744.

25. Anjuere, F., P. Martin, I. Ferrero, M. L. Fraga, G. M. del Hoyo, N. Wright, andC. Ardavin. 1999. Definition of dendritic cell subpopulations present in thespleen. Peyer’s patches, lymph nodes, and skin of the mouse. Blood 93: 590–598.

26. Scheinecker, C., R. McHugh, E. M. Shevach, and R. N. Germain. 2002. Consti-tutive presentation of a natural tissue autoantigen exclusively by dendritic cells inthe draining lymph node. J. Exp. Med. 196: 1079–1090.

27. Carbone, F. R., G. T. Belz, and W. R. Heath. 2004. Transfer of antigen betweenmigrating and lymph node-resident DCs in peripheral T-cell tolerance and im-munity. Trends Immunol. 25: 655–658.

28. Lanzavecchia, A. 1990. Receptor-mediated antigen uptake and its effect on an-tigen presentation to class II-restricted T lymphocytes. Annu. Rev. Immunol. 8:773–793.

29. Zhong, G., C. Reis e Sousa, and R. N. Germain. 1997. Antigen-unspecific B cellsand lymphoid dendritic cells both show extensive surface expression of processedantigen-major histocompatibility complex class II complexes after soluble proteinexposure in vivo or in vitro. J. Exp. Med. 186: 673–682.

30. Wykes, M., A. Pombo, C. Jenkins, and G. G. MacPherson. 1998. Dendritic cellsinteract directly with naive B lymphocytes to transfer antigen and initiate classswitching in a primary T-dependent response. J. Immunol. 161: 1313–1319.

31. Kushnir, N., L. Liu, and G. G. MacPherson. 1998. Dendritic cells and resting Bcells form clusters in vitro and in vivo: T cell independence, partial LFA-1 de-pendence, and regulation by cross-linking surface molecules. J. Immunol. 160:1774–1781.

32. Thery, C., L. Zitvogel, and S. Amigorena. 2002. Exosomes: composition, bio-genesis and function. Nat. Rev. Immunol. 2: 569–564.

33. Segura, E., S. Amigorena, and C. Thery. 2005. Mature dendritic cells secreteexosomes with strong ability to induce antigen-specific effector immune re-sponses. Blood Cells Mol. Dis. 35: 89–93.

34. Quah, B. J., and H. C. O’Neill. 2005. Maturation of function in dendritic cells fortolerance and immunity. J. Cell Mol. Med. 9: 643–654.

35. Wolf, S. D., B. N. Dittel, F. Hardardottir, and C. A. Janeway, Jr. 1996. Experi-mental autoimmune encephalomyelitis induction in genetically B cell-deficientmice. J. Exp. Med. 184: 2271–2278.

36. Hjelmstrom, P., A. E. Juedes, J. Fjell, and N. H. Ruddle. 1998. B-cell-deficientmice develop experimental allergic encephalomyelitis with demyelination aftermyelin oligodendrocyte glycoprotein sensitization. J. Immunol. 161: 4480–4483.

2106 PERIPHERAL APCs PRESENTING MYELIN BASIC PROTEIN

by guest on Decem

ber 22, 2018http://w

ww

.jimm

unol.org/D

ownloaded from