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The Journal of Immunology, 2006, 177: 2097-2106.
Copyright © 2006 by The American Association of Immunologists

Endogenous Myelin Basic Protein Is Presented in the Periphery by Both Dendritic Cells and Resting B Cells with Different Functional Consequences1

Audrey Seamons2, Antoine Perchellet and Joan Goverman3

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Multiple sclerosis is an inflammatory disease believed to be triggered by erroneous activation of self-reactive T cells specific for myelin proteins such as myelin basic protein (MBP). Inflammation is limited to the CNS, suggesting that the myelin-specific T cells encounter their Ags only after they cross the blood-brain barrier. However, our previous studies in mice showed that MBP epitopes are constitutively presented in lymphoid tissues. Here we identified which APCs in lymph nodes present endogenous MBP epitopes and determined the functional consequences of this presentation for both naive and activated MBP-specific T cells. Both CD8{alpha}+ and CD8{alpha}– dendritic cells were potent stimulators of proliferation for both naive and previously activated/memory MBP-specific T cells. Surprisingly, resting B cells also presented endogenous MBP that was acquired using a BCR-independent mechanism. 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 cytokines and became more refractory to subsequent stimulation. Interestingly, cytokine production by activated/memory T cells was triggered by resting B cells if the number of MBP epitopes presented was increased by adding exogenous MBP peptide. These results suggest that activated MBP-specific T cells may become less pathogenic in vivo following encounter with resting B cells presenting steady-state levels of endogenous MBP but can expand and remain pathogenic if the amount of MBP presented by B cells is increased, which could occur during chronic demyelinating disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Multiple tolerance mechanisms exist to prevent destructive autoimmune responses such as those that occur in multiple sclerosis (MS).4 Many self-reactive T cells are eliminated by exposure to self-Ag in the thymus. Tolerance also occurs in the periphery when self-reactive T cells encounter their Ag under non-inflammatory conditions and are either triggered to die or are prevented from acquiring effector function. Immature dendritic cells (DCs) have been implicated as the primary APC in the periphery responsible for inducing this type of peripheral tolerance (1, 2, 3, 4). Immature DCs residing in peripheral tissues are highly phagocytic but do not express high levels of the co-stimulatory molecules needed to prime T cell responses until they are activated (5). Some of these immature DCs migrate to lymph nodes (LNs) and spleen where they can present tissue-derived self-Ags to naive T cells directly via the MHC class II pathway or through cross-presentation in the MHC class I pathway. Because immature DCs do not provide the necessary co-stimulatory signals, the T cells that interact with these DCs typically proliferate briefly and die or become anergic (2, 3). Among the different DC subsets, CD8{alpha}+ DCs have been implicated as the major cell type responsible for cross-presenting Ags in the MHC class I pathway leading to peripheral tolerance in self-reactive CD8+ T cells (6).

Although most attention has focused on immature DCs as the primary APCs responsible for inducing T cell tolerance, B cells also present Ag. Unlike DCs, B cells are not highly phagocytic and therefore should not present the same wide range of tissue-derived self-Ags that immature DCs can present. However, B cells can efficiently capture specific Ags through their BCRs and present the processed Ag on MHC class II molecules. As is the case for immature DCs, Ag capture alone is not sufficient for B cells to function as immunogenic APCs without receiving additional signals to express co-stimulatory molecules. For resting B cells, these signals are provided via the CD40/CD154 pathway that is engaged when activated T cells recognize Ag on the surface of the resting B cell (7). Naive T cells recognizing Ag presented by resting B cells do not provide the signals required to trigger resting B cells to express co-stimulatory molecules and consequently the T cells become tolerant (8, 9, 10). Tolerance induction of naive T cells by resting B cells in vivo has been shown to involve abortive proliferation followed by disappearance of the T cells (11). The only exceptions to the tolerogenic effect of Ag presentation by resting B cells have been observed under non-physiological conditions when large numbers of naive BCR transgenic (Tg) B cells have been used to present Ag to naive TCR Tg T cells with the same Ag specificity (12, 13). Because the number of B cells expressing a BCR specific for any particular self-Ag should normally be very low, DCs are presumed to play a more predominant role in mediating peripheral tolerance as they do not depend on Ag-specific receptors to acquire Ag.

Our previous studies investigated the tolerance mechanisms that regulate CD4+ T cells specific for myelin basic protein (MBP), one of the predominant protein components of myelin. Understanding how immune responses to MBP are regulated is important because MBP is believed to be one of the self-Ags targeted in MS. There are several isoforms of MBP that are incorporated into myelin. This family of isoforms is referred to as "classic" MBP (14). A separate family of proteins called golli-MBPs are transcribed from a distinct promoter but share many of the exons that encode classic MBPs (15, 16). The function of golli-MBPs is less well understood but they are not incorporated into myelin. T cell tolerance to MBP sequences shared by classic and golli-MBP proteins should be readily achieved because golli-MBPs are expressed in both the thymus and peripheral lymphoid tissues. However, some portions of classic MBP are not found in golli-MBP proteins and these uniquely classic MBP sequences should be generated only in myelin-forming cells. We investigated how tolerance is induced to portions of classic MBP not found in golli-MBP using TCR Tg mice specific for MBP121–140, an epitope contained only in classic MBP and presented by the MHC class II molecule I-Au. Surprisingly, most of the MBP-specific Tg T cells were eliminated during maturation in the thymus. Endogenous MBP was presented in the MHC class II pathway only by bone marrow-derived cells in the thymus and these cells also presented MBP constitutively in the periphery (17). To determine which types of peripheral bone marrow-derived cells presented endogenous MBP, fractionated splenocytes were used to stimulate the MBP-specific TCR Tg T cells directly ex vivo. The majority of MBP-specific T cell proliferation was triggered by DCs, with a very small amount of proliferation inconsistently detected using splenic CD11c–F4/80+ macrophages as APCs (18).

In the present study, we observed that LN cells triggered significantly more MBP121–140-specific T cell proliferation ex vivo than did splenocytes. This observation suggested that detection of MBP epitopes on different types of APCs might be easier using cells isolated from LNs. Similar to our results with splenocytes, we found that most of the proliferation of MBP-specific T cells triggered by LN cells ex vivo was due to presentation of endogenous MBP by both CD8{alpha}+ and CD8{alpha}– DCs. Surprisingly, however, we found that resting B cells purified from LNs also stimulated proliferation of both naive and previously activated/memory MBP-specific T cells ex vivo. Interestingly, acquisition of endogenous MBP by B cells did not appear to be BCR mediated nor was synthesis of MBP by B cells required. Despite stimulation of a proliferative response, neither activated/memory nor naive MBP-specific T cells produced significant amounts of cytokines in response to resting B cells. Furthermore, activated/memory T cells incubated with B cells presenting endogenous MBP became less responsive to subsequent stimulation with bulk LN cells. However, increasing the amount of MBP presented by resting B cells by adding exogenous MBP peptide stimulated activated/memory T cells to produce effector cytokines. These results suggest that constitutive presentation of endogenous MBP by resting B cells may have a tolerogenic effect on previously activated/memory MBP-specific T cells. However, if the amount of endogenous MBP acquired by B cells increases, as could occur during an ongoing demyelinating disease, presentation of MBP by resting B cells may amplify the responses of activated/memory MBP-specific T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

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), and C57BL/6J mice were purchased from The Jackson Laboratory. The Thy1.1 allele and Rag1–/– mutation were backcrossed 8 to 10 generations onto the B10.PL background. MBP–/– B10.PL mice, and MBP121–140-specific TCR Tg mice have been described previously (17, 20). All mice were bred and maintained in a specific pathogen-free facility at the University of Washington (Seattle) and all procedures involving animals were approved by the Institutional Animal Care and Use Committee at the University of Washington.

Antibodies

All Abs used in these experiments were purchased from BD Biosciences except 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 with either biotinylated anti-V{alpha}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{alpha}2+CD4+CD44– cells from spleen and LN on a FACSVantage.


Figure 1
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  		FIGURE 1. Ex vivo LN cells trigger more proliferation of naive MBP-specific T cells than splenocytes. Single cell suspensions were prepared from either pooled LNs (inguinal, axial, and brachial) or spleens and plated at 1 x 106 cells/well. Naive MBP-specific T cells were added and proliferation was measured by incorporation of [3H]thymidine. The stimulation index (SI) was plotted on a log scale with each symbol representing the SI obtained using cells from an individual mouse. Black bars indicate the mean SI for each tissue. The fraction of individual samples with SI >2 was 12/21 for cultures using splenocytes as APCs and 46/54 for cultures using LN cells. The mean SI obtained using LN cells significantly differed from the mean SI using splenocytes (p = 0.01 by Student’s t test). Data shown are compiled from nine different experiments.

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

MBP-specific T cells (5 x 104 cells/well unless indicated otherwise in figure legends) were cultured in complete RPMI in triplicate with the indicated 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) and purified B cells were plated at 1 x 106 and 5 x 105 cells per well, respectively, unless indicated otherwise. For certain experiments, 8 µM BrdU (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{alpha}2, anti-CD4 and anti-BrdU Abs. Samples were analyzed on a FACScan to determine the percent of BrdU+ cells in the V{alpha}2+CD4+ population. For proliferation assays examining the functional effects of B cell presentation of MBP, activated/memory MBP-specific T cells (5 x 105/ml) were incubated for five days with irradiated B cells (2.5 x 106/ml) purified by magnetic bead selection from MBP+/+ or MBP–/– mice. Cells were then centrifuged over a Lympholyte-M gradient and were stained to determine numbers of Tg T cells recovered from the cultures. CD4+V{alpha}2+Vbeta8+ T cells (5 x 104/well) were then plated as described above with LN cells from MBP+/+ or MBP–/– mice and proliferation determined by [3H]thymidine incorporation. Stimulation index 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 Wünsch U/ml Liberase RI; Roche) in the presence of 50 µg/ml DNase I (grade II; Roche) until tissues appeared fully digested (stirring ~45 min at 37°C.). Digestion was stopped with the addition of EDTA (10 mM final concentration), RBC were lysed, and the sample was filtered through a 70- µm nylon mesh before the various cell purifications described below. All DCs were harvested from collagenase-digested tissues unless otherwise noted.

DC isolation and depletion

For DC purification by magnetic bead selection, spleen or LN cells were labeled with anti-CD11c-conjugated microbeads (Miltenyi Biotec), and DCs were positively selected on an auto-MACS (Miltenyi Biotec). DC subsets were purified from spleens (without collagenase digestion) by first isolating CD11c+ cells using anti-CD11c microbeads and magnetic bead selection. Cells were then stained with anti-CD11c-FITC and anti-CD8{alpha}-PE-Cy5 Abs and sorted into CD8{alpha}+ and CD8{alpha}–CD11c+ subsets on a FACSVantage cell sorter (BD Biosciences). Subset purity was >95%. DC subsets were purified from LN cells by first depleting T and B cells by magnetic bead selection using anti-TCR and anti-CD19 biotinylated Abs and streptavidin-conjugated microbeads (Miltenyi Biotec). Enriched DCs were then stained with anti-CD11c and anti-CD8 and sorted into CD11c+CD8{alpha}+ and CD11c+CD8{alpha}– subsets on an InFlux Flow Cytometer (Cytopeia) (Fig. 2B). LN cells were depleted of DCs or DC subsets using Abs specific for CD11c and CD8{alpha} and sorting on a FACSVantage cell sorter (BD Biosciences).


Figure 2
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  		FIGURE 2. DCs stimulate most of the proliferation observed for both naive and activated/memory MBP-specific T cells responding to LN cells ex vivo. A, Purified naive MBP-specific T cells were incubated with either bulk LN cells from MBP+/+ mice or LN cells depleted of various DC populations (1 x 106/well). The DC-depleted populations were obtained by FACS using the indicated Abs. Non-depleted bulk LN cells were also sorted through a live gate to assure that the samples were treated comparably. B, Purified naive MBP-specific T cells were incubated with the indicated DC populations isolated from either MBP+/+ (black bars) or MBP–/– (white bars) LNs by FACS based on CD8{alpha} and CD11c expression; 2 x 104 purified DCs or 1 x 106 bulk LN cells were plated per well. Purity of sorted DCs was >79% in all samples, contaminants were small CD11c– cells. C, Activated/memory MBP-specific T cells (6 x 104 cells/well) were incubated with either bulk LN or spleen cells or with auto-MACS-purified CD11c+ DCs (8 x 104 DCs/well, purity ≥96% CD11c+). Proliferation was measured by incorporation of [3H]thymidine.

 
B cell isolation

Resting B cells were purified via negative selection from collagenase-digested LNs or spleens using biotinylated anti-CD11c, anti-CD11b, anti-CD43 and streptavidin-labeled microbeads (Miltenyi Biotec). Cells were negatively selected on an auto-MACS. Cell purity was determined by staining with anti-CD19, anti-CD43, anti-CD11c, and anti-CD11b. For purification of B cells from BCR-Tg F1 mice, anti-CD11b Ab was omitted from the negative selection protocol because BCR-Tg B cells are CD11b+ in this model. In Fig. 4, resting B cells were positively purified by FACS by staining LN cells with anti-mouse Ig-PE (F(ab')2 fragment), anti-CD43-FITC and anti-CD11c-biotin/streptavidin-TriColor (Caltag). Resting B cells were negatively purified 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 purification) were sorted on a FACSAria (BD Biosciences). Purities for both were ≥98%.


Figure 4
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  		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 were purified by positive selection using flow cytometry to sort LN cells stained with anti-mouse Ig (non-activating Fab), anti-CD43, and anti-CD11c Abs. Cells were 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-sorted cells 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 cells with anti-CD43, CD11b, and CD11c Abs and collecting cells through a CD43–CD11c–CD11b– gate (top panel). Sorted cells were 99% pure (bottom panel). C, Proliferation of activated/memory or purified naive MBP-specific T cells incubated with MBP+/+ (black bars) or MBP–/– (white bars) sorted B cells or bulk LN cells was determined via BrdU incorporation. Data are expressed as the percentage of V{alpha}2+CD4+ T cells that were BrdU+ at the end of the incubation. The percentage of V{alpha}2+CD4+ T cells that incorporated BrdU after incubation with bulk LN cells is under-represented due to the presence of some non-Tg CD4+V{alpha}2+ T cells present in the LN cells used as APCs.

 
Bone marrow chimeras

Bone marrow chimeras were generated by transferring 1 x 107 bone marrow cells from MBP–/– donors into lethally irradiated MBP+/+ Rag–/– mice (1000 rad on day –1). Recipient mice were provided neomycin sulfate (Sigma-Aldrich) in the drinking water (2 mg/ml) from day –2 to day 14. Resting B cells were purified 8 weeks later by magnetic bead selection from 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-{gamma} and IL-2 according to the manufacturer’s directions. Activated/memory and naive T cells were serially diluted and incubated 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 isolated from MBP–/– mice and incubated with T cells at the same serial dilutions to determine background numbers of spots. Either 5 x 105 purified B cells or 1 x 106 bulk LN cells were plated per well. Plates containing activated T cells were incubated for 18 h. Plates containing naive T cells were incubated 18 h in experiment 1 and 40 h in experiments 2 and 3. Frequencies of spots/number of MBP-specific T cells were determined for each well and duplicate well frequencies were averaged (only wells that had <300 spots per well were counted). SDs for duplicate wells were calculated based on the binomial distribution. The average number of spots in wells containing MBP–/– APCs were subtracted from the average number of spots in wells containing MBP+/+ APCs at the specified T cell dilutions. A combined SD was calculated for these values. Data are plotted as the average number of specific spots observed versus the number of MBP-specific T cells per well.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Endogenous MBP is presented by both CD8{alpha}+ and CD8{alpha}– DCs

Our previous studies showed that constitutive presentation of MBP epitopes could be detected in vivo in peripheral lymphoid tissues of healthy, wild-type mice using TCR Tg T cells specific for the epitope MBP121–140 (17). The MBP-specific T cells also proliferated to bulk splenocytes isolated from wild-type mice ex vivo but not to splenocytes isolated from MBP-deficient (MBP–/–) mice. Fractionation of the splenocytes demonstrated that DCs were the primary APCs in the spleen that stimulated proliferation of the MBP-specific T cells ex vivo (18). Here we show that LN cells isolated from wild-type mice are more potent than splenocytes in stimulating naive MBP121–140-specific Tg T cells ex vivo (Fig. 1). The stimulation index for MBP-specific T cells proliferating in response to either splenocytes or LN cells varied between individual mice; however, the average stimulation index observed using LN cells as APCs was significantly greater than the average stimulation index obtained using splenocytes as APCs (p = 0.01). This observation motivated us to investigate which cells in the LN stimulate MBP-specific T cells ex vivo.

Depletion of DCs from bulk LN cells reduced ex vivo proliferation of naive MBP-specific T cells by 94% (Fig. 2A), indicating that DCs are the predominant APC presenting endogenous MBP epitopes in the LN as was observed for splenocytes. To determine whether presentation of endogenous MBP is limited to a particular DC subset, bulk LN cells depleted of either CD8{alpha}+ or CD8{alpha}– DCs were used to stimulate naive Tg MBP-specific T cells ex vivo. Depletion of either DC subset substantially reduced the proliferation of naive MBP-specific T cells relative to that seen with bulk LN cells but not to the same degree as the reduction observed when all CD11c+ cells were depleted (Fig. 2A), suggesting that both DC subsets may present MBP. To confirm this, CD11c+CD8{alpha}+ and CD11c+CD8{alpha}– cells were purified from LN cells by flow cytometry and used to stimulate naive MBP-specific T cells ex vivo. As shown in Fig. 2B, both CD8{alpha}+ and CD8{alpha}– LN DCs stimulated the naive MBP-specific T cells to proliferate. A similar experiment using CD8{alpha}+ and CD8{alpha}– DCs sorted from splenocytes also showed that both of these populations stimulated naive MBP-specific T cells ex vivo (data not shown). MBP-specific T cells that had been previously activated in vitro also proliferated to endogenous MBP presented ex vivo by DCs (Fig. 2C). As expected, the response of the activated/memory MBP-specific T cells was even stronger than the response of naive MBP-specific T cells to endogenous MBP presented by DCs isolated from either LN cells or spleen.

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

The data described above indicate that DCs are the major APC in both spleen and LN that present endogenous MBP epitopes. Indeed, DCs were the only APCs isolated from the spleen that consistently stimulated significant proliferation of MBP-specific T cells ex vivo (18). Surprisingly, this was not the case for cells isolated from LNs. Resting B cells isolated from LNs triggered Ag-specific proliferation of both naive and previously activated MBP-specific T cells ex vivo (Fig. 3). The amount of proliferation stimulated by resting B cells was less than that triggered by DCs for both activated and naive T cells, reflecting either more acquisition of MBP by DCs in vivo and/or their greater efficiency as APCs for T cell activation. As expected, the proliferative response to resting B cells ex vivo is lower for naive T cells compared with activated/memory T cells but is still significantly above background levels (stimulation index = 21). In these proliferation assays, the naive T cells were obtained by isolating V{alpha}2+ T cells from MBP–/– Rag+/+ TCR Tg mice. Because some of the V{alpha}2+ population could contain T cells that express a second endogenous V{alpha}-chain, it was possible that this population contained T cells that had been activated in vivo by environmental Ags. To confirm that naive MBP-specific T cells could respond to purified, resting B cells, V{alpha}2+CD44low T cells were sorted from the TCR Tg MBP–/– mice and incubated with resting B cells. The sorted naive T cells proliferated to resting B cells as well as V{alpha}2+ T cells that were not sorted for low CD44 expression (data not shown), confirming that resting B cells present sufficient MBP ex vivo to trigger proliferation of naive T cells.


Figure 3
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  		FIGURE 3. Resting B cells trigger Ag-specific proliferation of MBP-specific T cells ex vivo. Proliferative responses of MBP-specific T cells to bulk LN cells (left panel) or purified B cells (right panel) are shown. Purified B cells were 98% CD19+ and <1% CD11c+. Black and white bars represent the responses of activated T cells to APCs from MBP+/+ and MBP–/– mice, respectively, and gray and striped bars indicate the responses of naive T cells. Proliferation was measured by incorporation of [3H]thymidine. Data are representative of two independent experiments.

 
The resting B cells used in these assays were purified using negative selection by magnetic beads. Contaminating DCs were <1% of the purified B cell population. An experiment titrating the number of DCs needed to detect T cell proliferation indicated that the proliferative response stimulated by purified B cells could not be due to the small number of potentially contaminating DCs in the assays (data not shown). To further eliminate the possibility that contaminating DCs accounted for the T cell response to purified B cells, B cells were sorted by flow cytometry using either positive or negative selection. B cells were positively purified using anti-CD43 and anti-mouse Ig F(ab')2 Abs (Fig. 4A) or negatively purified using Abs specific for CD43, CD11b, and CD11c (Fig. 4B). B cells purified by either method stimulated proliferation of both activated and naive MBP-specific Tg T cells (Fig. 4C). To confirm that incubation with the anti-mouse Ig F(ab')2 fragment did not activate the B cells, expression of CD40, CD86, and MHC class II was analyzed following overnight incubation with this Ab. No increases in expression of these molecules were observed in B cells incubated overnight in tissue culture plates with or without anti-mouse Ig F(ab')2 compared with B cells analyzed directly ex vivo (data not shown).

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

The observation that resting B cells purified from wild-type mice constitutively present sufficient endogenous MBP to stimulate proliferation of both naive and activated MBP-specific T cells ex vivo was very surprising because effective Ag presentation by B cells is believed to depend on the efficient capture of Ag through a specific BCR. Consistent with this, activation of naive T cells by resting B cells in vivo has been shown to require a very high frequency of B cells specific for the same Ag as the T cells (12, 13). Thus, one explanation for our results could be that wild-type B10.PL mice contain an unusually high frequency of MBP-specific B cells despite the fact that these self-reactive B cells should undergo tolerance in vivo. To test this hypothesis, B cells isolated from hen egg lysozyme (HEL)-specific BCR Tg mice were used as APCs to stimulate MBP-specific T cells ex vivo. Although these BCR-Tg mice were not on a Rag–/– background, expression of the BCR transgenes should significantly limit the repertoire of endogenous BCRs specific for other Ags. Because the BCR Tg mice were on the C57BL/6 (H-2b) background, they were bred to B10.PL (H-2u) mice to obtain F1 B cells expressing both the HEL-specific Tg BCR and the appropriate MHC molecule recognized by the MBP-specific T cells (I-Au). As expected, both activated/memory and naive MBP-specific T cells proliferated in response to both F1 BCR Tg and non-Tg LN cells ex vivo, as this population contains DCs that are efficient at presenting endogenous MBP and are not affected by expression of the BCR transgenes (Fig. 5A). The MBP-specific T cells did not proliferate in response to LN cells isolated from wild-type C57BL/6 mice, demonstrating that their proliferation to the F1 LN cells required the I-Au MHC molecule and was not due to alloreactivity directed toward B6 Ags (Fig. 5A). Importantly, purified B cells isolated from either BCR-Tg or non-Tg F1 mice stimulated comparable and significant levels of proliferation of both activated and naive MBP-specific T cells (Fig. 5B). These results indicate that the ability of resting B cells to trigger proliferation of MBP-specific T cells directly ex vivo does not depend on a high precursor frequency of B cells expressing MBP-specific BCRs.


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

 
The ability of B cells isolated from HEL-specific BCR Tg mice to activate MBP-specific T cells ex vivo suggested that the acquisition of MBP by resting B cells occurred in a BCR-independent manner. Because previous reports suggested that some immune cells synthesize classic MBP (21), we investigated whether synthesis of MBP by B cells is necessary to trigger Ag-specific proliferation of MBP-specific T cells. Bone marrow chimeric mice were generated by transplanting bone marrow from MBP–/– mice into irradiated Rag–/– MBP+/+ B10.PL mice. B cells that mature in these recipients can only be derived from the MBP–/– donor bone marrow cells. B cells isolated from these bone marrow chimeras, as well as from wild-type MBP+/+ mice and MBP–/– mice, were used ex vivo to stimulate activated/memory MBP-specific T cells. As shown in Fig. 6, resting B cells purified from either the bone marrow chimeras or from wild-type mice stimulated proliferation of MBP-specific T cells ex vivo to the same extent. This result indicates that resting B cells obtain enough MBP from the host to trigger proliferation of MBP-specific T cells ex vivo without a requirement for de novo synthesis of the protein.


Figure 6
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  		FIGURE 6. Resting MBP–/– B cells acquire sufficient MBP from the periphery to stimulate activated MBP-specific T cells ex vivo. Activated/memory MBP-specific T cells were cultured with resting B cells purified from MBP+/+, MBP–/–, or MBP–/– -> MBP+/+Rag–/– bone marrow chimeric (BMC) mice. Proliferation was measured by incorporation of [3H]thymidine. Data are representative of two independent experiments.

 
Interaction with resting B cells presenting endogenous MBP causes activated/memory T cells to become less responsive to subsequent stimulation

Ag presentation by resting B cells has been reported to have a tolerogenic effect on naive T cells. However, activated/memory MBP-specific T cells that recognize MBP on resting B cells may have a different fate. To investigate the effect of endogenous MBP presentation by resting B cells on activated/memory T cells, activated/memory T cells were incubated with B cells isolated from either wild-type or MBP–/– mice for 5 days. During this incubation, 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 proliferative potential of the MBP-specific T cells was assessed by re-stimulating them with bulk LN cells. Although T cells from both groups proliferated in response to the LN cells, the T cells that had been exposed to MBP–/– B cells proliferated much more than T cells that had been exposed to wild-type B cells (Fig. 7). Thus, encounter of activated T cells with resting B cells presenting endogenous MBP appears to decrease the proliferative potential of the activated/memory T cells upon subsequent restimulation.


Figure 7
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  		FIGURE 7. Activated/memory T cells are less responsive to stimulation with LN cells following exposure to B cells presenting endogenous MBP. Activated/memory MBP-specific T cells were incubated with purified B cells from either MBP+/+ or MBP–/– mice. Cells were incubated for 5 days at a density of 3 x 106 total cells/ml with a T cell to B cell ratio of 5:1. Following incubation, live cells were harvested, and 5 x 104 CD4+V{alpha}2+Vbeta8+ cells (determined by flow cytometry) from each culture were set up in a stimulation assay with irradiated MBP+/+ (black bars) or MBP–/– (white bars) whole LN cells. Proliferation was measured by incorporation of [3H]thymidine. The stimulation index obtained with LN cells as APCs for activated/memory T cells incubated previously with MBP+/+ or MBP–/– B cells is 59 and 101, respectively. Data are representative of two experiments.

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

To determine whether resting B cells presenting endogenous MBP can stimulate T cell effector function as well as proliferation, we measured cytokine production by MBP-specific T cells incubated with wild-type B cells. Both activated/memory and naive Tg T cells were incubated with either purified resting B cells or bulk LN cells, and the number of T cells producing cytokines was measured using ELISPOT analyses for IL-2 and IFN-{gamma}. For each ELISPOT assay, the T cells were plated at serial dilutions to confirm a dose/response relationship between the number of T cells plated and the number of spots observed. Background levels of cytokine production (≤5 spots) were measured by plating the same numbers of T cells with B cells or LN cells isolated from MBP–/– mice instead of wild-type mice. Because naive T cells typically produce very low levels of cytokines during their initial stimulation, larger numbers of naive T cells were plated compared with activated T cells and the incubations were conducted for both 18 and 40 h. An example of ELISPOT data generated when activated/memory MBP-specific T cells were incubated with B cells is shown in Fig. 8. For both cytokines, increasing the number of T cells plated per well resulted in increased numbers of observed spots. Addition of exogenous MBP peptide to the resting B cells resulted in increased cytokine production compared with B cells presenting only endogenous MBP. ELISPOT data such as those shown in Fig. 8 were used to calculate the percentages of MBP-specific T cells that produced cytokines in response to either B cells or bulk LN cells, with or without exogenous peptide (Table I). Because of the large difference in numbers of activated/memory versus naive T cells plated in each assay, calculating the percentages of responding T cells provided the most straightforward way to compare results.


Figure 8
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  		FIGURE 8. B cells trigger little cytokine production by activated/memory MBP-specific T cells ex vivo. The number of spots/well detected by ELISPOT for the indicated cytokines is shown for increasing numbers of activated/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 of data used to generate Table I.

 

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

 
The data in Table I show that naive T cells responding to resting B cells did not produce any detectable cytokines. Even when exogenous MBP peptide was added to the B cells, only a very small percentage of naive T cells produced IL-2 in experiments 2 and 3 (percentages over 0.1% indicate values that were calculated from data that followed an approximately linear titration with increasing numbers of T cells and are likely to reflect accurate detection of cytokine-producing T cells). Similarly, only low percentages of naive T cells produced cytokines in response to bulk LN cells even though the predominant APCs presenting MBP in this population are DCs. Addition of exogenous MBP peptide to the bulk LN cells resulted in some increase in the percentage of IL-2-producing T cells but little change in production of IFN-{gamma}.

Unlike naive MBP-specific T cells, activated/memory T cells produced cytokines when incubated with bulk LN cells. Although the largest percentage of these cells produced IL-2 in experiment 2, the size of the spots in the IFN-{gamma} assay was much larger than the size of the spots in the IL-2 assay. Addition of exogenous MBP peptide to the bulk LN cells did not significantly increase the percentage of T cells producing some cytokines in experiment 2 and only doubled cytokine production seen in experiment 3. In contrast to the response to LN cells, however, <1% of activated T cells produced cytokines in response to resting B cells ex vivo. This result indicates that the proliferation triggered by exposure to resting B cells was not accompanied by stimulation of effector function in the activated/memory T cells. Interestingly, addition of MBP peptide to the B cells increased the percentage of activated/memory MBP-specific T cells producing cytokines, such that the percentage of IFN-{gamma}-producing T cells was similar to that seen in response to LN cells. Thus, the amount of MBP that is constitutively presented by resting B cells is sufficient to trigger activated/memory MBP-specific T cells to proliferate but is not sufficient to induce production of effector cytokines. However, when the number of MBP/MHC complexes presented by resting B cells is increased by addition of exogenous MBP peptide, activated/memory MBP-specific T cells both proliferate and produce cytokines, suggesting that the amount of MBP available to resting B cells affects the outcome of their interaction with activated MBP-specific T cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
MBP is unique among myelin proteins in that it is a component of both central and peripheral myelin. This expression pattern presumably accounts for the ability to detect constitutive presentation of MBP epitopes in peripheral lymphoid tissues in vivo (17, 22, 23). By analyzing different splenocyte populations, we previously showed that only DCs presented sufficient endogenous MBP to trigger proliferation of naive MHC class II-restricted MBP-specific T cells ex vivo (18). Here we showed that bulk LN cells stimulate on average more proliferation of MBP-specific T cells directly ex vivo compared with splenocytes. This observation suggests that degradation of myelin in innervated tissues is a significant source of MBP presented in the periphery because MBP derived from this source should accumulate at higher levels in lymph compared with blood. An alternative possibility is that LNs contain DC populations not found in the spleen that express high levels of MHC class II and therefore may be more efficient at stimulating naive T cells (24, 25). These possibilities are not mutually exclusive, and both may contribute to the increased ability of LN cells to stimulate naive MBP-specific T cell proliferation compared with splenocytes. Among individual mice, there is more variation in the amount of T cell proliferation triggered by LN cells compared with the proliferation triggered by splenocytes, suggesting that either the amount of endogenous MBP transported to LNs at any given time varies from mouse to mouse and/or the number of DCs expressing high levels of MHC class II present in peripheral LNs is variable. Overall, the data indicate that MBP-specific T cells in vivo are more likely to encounter APCs presenting endogenous MBP in LNs than in the spleen.

We analyzed the ability of LN APCs to stimulate activated/memory as well as naive MBP-specific T cells in order to detect presentation of endogenous MBP epitopes by APCs that might not provide adequate co-stimulatory signals to trigger naive T cells to divide. Our results showed that most of the proliferation by both naive and activated/memory MBP-specific T cells ex vivo was due to presentation of MBP by DCs, and that CD8{alpha}+ and CD8{alpha}– LN DCs stimulated MBP-specific T cells equally well. This result is in contrast to studies of DCs presenting MHC class I- and MHC class II-restricted epitopes of islet Ags in which only CD8{alpha}+ DCs (6) or CD11b+ DCs (4) isolated from pancreatic LNs were found to stimulate Ag-specific T cells ex vivo. However, presentation of endogenous MBP by both CD8{alpha}+ and CD8{alpha}– DC subsets is consistent with another report documenting presentation of self-Ags by both CD8{alpha}+ and CD8{alpha}– DCs in draining LNs (26). Interestingly, this study suggested that CD8{alpha}+ and CD8{alpha}– DCs may acquire self-Ags in different locations in vivo. Scheinecker et al. found that epitopes of H+/K+ ATPase, a self-Ag synthesized in the stomach, are presented by both CD8{alpha}+ and CD8{alpha}– DCs in the draining gastric LN but are presented only by CD8{alpha}– DCs in the gastric mucosa (26). This observation suggested that Ag carried by CD8{alpha}– DCs migrating from tissues might be transferred to CD8{alpha}+ DCs residing in LNs. This process of transfer of Ag from migratory to resident DCs has been demonstrated in vitro as well, and may be a strategy for increasing the range and number of DCs in LNs presenting Ag derived from peripheral tissues (27). We cannot determine whether initial capture of MBP in peripheral tissues is limited to the CD8{alpha}– subset because most peripheral tissues are innervated and therefore there is no unique site of MBP synthesis. MBP may also be present in lymph and blood as a soluble Ag and could gain access to resident LN DCs without being transported there by tissue DCs. In addition to DCs, it is possible that some MBP may be captured by macrophages in peripheral tissues and presented in lymphoid organs as we observed marginal stimulation of naive T cells (stimulation index <3) by macrophages purified from the spleen in one of three experiments in our previous study (18). The small number of macrophages in LNs prevented us from determining if LN macrophages also present endogenous MBP ex vivo.

Although DCs were responsible for the majority of the proliferative response of MBP-specific T cells in both the LN and spleen, we were surprised to discover that resting B cells isolated from LNs also stimulated proliferation of both activated and naive MBP121–140-specific T cells ex vivo. Our previous studies showed that B cells isolated from the spleen did not trigger proliferation of naive MBP-specific T cells, which may reflect the lower amount of MBP present in the spleen. The bone marrow chimeric mice demonstrated that peripheral MBP acquired by MBP–/– B cells in an MBP+/+ mouse is sufficient to trigger T cell proliferation, suggesting that MBP obtained from myelin is a significant source of MBP epitopes. These data do not exclude the possibility that B cells synthesize some classic MBP and present it in the MHC class II pathway. However, we believe that this is not likely to be the major source of MBP presented by B cells because B cell autonomous synthesis of MBP should have been detected on B cells isolated from the spleen. The acquisition of MBP by B cells appears to occur in a BCR-independent manner because HEL-specific BCR Tg B cells triggered equivalent T cell proliferation as observed with non-Tg B cells. This result was also surprising because previous studies have shown that BCR-independent acquisition of soluble Ag by B cells in vitro is approximately 1,000–10,000 times less efficient compared with BCR-mediated internalization of Ag (28). In vivo, Zhong et al. demonstrated that i.v. injection of very large amounts (milligram quantities) of soluble protein associated with induction of high-dose tolerance resulted in presentation of processed Ag by Ag-nonspecific, resting B cells and DCs. Under these conditions, DCs and B cells presented similar amounts of MHC class II-restricted epitopes from the injected Ag (when the number of MHC complexes present on the two different cell types was taken into consideration) (29). However, it is unlikely that such a large amount of soluble MBP is available in the lymphatic system draining peripheral tissues, suggesting that a cell-mediated mechanism is more likely to explain B cell acquisition of MBP.

Although our studies have not precisely defined the mechanism of B cell acquisition of endogenous MBP, previous studies suggest that interactions between DCs and resting B cells result in transfer of unprocessed Ag between the two cell types. Studies in the rat have shown that resting B cells cluster with DCs, allowing Ag transfer 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 cells acquire MBP in a BCR-independent fashion is through uptake of MBP-containing exosomes secreted by DCs. These secreted vesicles are formed by generation of multivesicular bodies in the late endocytic compartments of both immature and mature DCs and are released into the extracellular environment by fusion with the plasma membrane (32). The exosomes secreted by DCs appear to reflect the functional status of the DC, such that only exosomes secreted by mature DCs are able to stimulate T cell effector function (33, 34). Thus, transport of MBP from peripheral tissues by DCs, 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 to B cells as well. Because the DCs transporting MBP from peripheral tissues in healthy mice should be immature, the uptake of exosomes from these DCs should deliver the Ag in the absence of other molecules associated with immunogenic signals. This process would serve to increase the number of APCs in LNs that present MBP in a tolerogenic context. If DCs are the major source of MBP acquired by B cells, then our inability to detect MBP presentation by splenic B cells may reflect the lower amount of MBP present on DCs migrating to the spleen versus the LN.

Our analyses indicated that both bulk LN cells and resting B cells presenting endogenous MBP triggered naive T cells to proliferate but not produce cytokines. In contrast, MBP-specific activated/memory T cells respond to APCs presenting endogenous MBP differently: they proliferate and produce cytokines in response to endogenous MBP presented by LN cells containing DCs, but they respond to resting B cells by proliferating without producing cytokines. Activated/memory MBP-specific T cells exposed in vitro to resting B cells presenting endogenous MBP are also more refractory to subsequent stimulation by bulk LN cells compared with T cells that were not first exposed to the B cells. Thus, presentation of steady-state levels of endogenous MBP by resting B cells appears to be a tolerogenic event for activated/memory T cells. This outcome may depend on the amount of MBP that is available to B cells to take up rather than on the quality of the signals transmitted by resting B cells, as increasing the amount of MBP on the B cell surface by addition of MBP peptide triggered cytokine production by activated/memory T cells that was comparable to that triggered by bulk LN cells.

Our findings have interesting implications for the pathogenesis of CNS autoimmune disease. These studies demonstrate that classic MBP is a very ubiquitous Ag, detectable by both naive and activated/memory MBP-specific T cells on the surface of DCs and B cells in healthy mice with steady-state levels of MBP in peripheral tissues. In the absence of autoimmune disease, any MBP-specific T cells circulating in the periphery should have a naive phenotype and encounter with either DCs or B cells presenting MBP should be a tolerogenic event as neither type of APC triggers naive T cells to acquire effector function. However, MBP-specific T cells that have differentiated into effector T cells during the course of CNS autoimmune disease will respond differently to an encounter with APCs presenting endogenous MBP in the periphery. Activated/memory T cells will proliferate and retain effector function when interacting with DCs presenting endogenous MBP. Interaction with resting B cells presenting steady-state levels of MBP, on the other hand, would render the activated T cells less pathogenic as they will not produce cytokines and will become more refractory to further stimulation. Given the numerical advantage of B cells compared with DCs in LNs, this dampening effect by B cells may be physiologically significant. This phenomenon may explain why the presence of endogenous B cells has been implicated in modulating experimental autoimmune encephalomyelitis, an animal model of MS, even though disease can be induced in B cell-deficient mice (35, 36). A different scenario might emerge, however, if the amount of endogenous MBP available in the periphery increases due to degradation of myelin in the CNS. In this case, the amount of MBP presented by B cells could increase, causing them to function like DCs in stimulating both T cell proliferation and cytokine production, thus abrogating their tolerogenic effect.


   Acknowledgments
 
We thank Dr. T. Brabb, I. Stromnes, S. Cabbage, H. Sumerfield, and N. Mausolf for critical reading of the manuscript and assistance with laboratory experiments, and N. Mausolf, R. Rowe, and H. Sumerfield for excellent management of the mouse colony and laboratory resources.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was funded by the National Institutes of Health Grant NS035126. Back

2 Current address: Department of Comparative Medicine, University of Washington, Seattle, WA, 98195. Back

3 Address correspondence and reprint requests to Dr. Joan Goverman, Department of Immunology, University of Washington, Box 357650, 1959 NE Pacific St., Seattle, WA 98195. E-mail address: goverman{at}u.washington.edu Back

4 Abbreviations used in this paper: MS, multiple sclerosis; DC, dendritic cell; LN, lymph node; HEL, hen egg lysozyme; MBP, myelin basic protein; Tg, transgenic. Back

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


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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