CD11c-Expressing B Cells Are Located at the / Border in and Are Potent APCs

This information is current as Anatoly V. Rubtsov, Kira Rubtsova, John W. Kappler, of September 25, 2021. Jordan Jacobelli, Rachel S. Friedman and Philippa Marrack J Immunol published online 1 June 2015 http://www.jimmunol.org/content/early/2015/05/30/jimmun ol.1500055 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 1, 2015, doi:10.4049/jimmunol.1500055 The Journal of Immunology

CD11c-Expressing B Cells Are Located at the T Cell/B Cell Border in Spleen and Are Potent APCs

Anatoly V. Rubtsov,*,†,1 Kira Rubtsova,†,1 John W. Kappler,*,†,‡,x Jordan Jacobelli,† Rachel S. Friedman,† and Philippa Marrack*,†,x,{

In addition to the secretion of Ag-specific Abs, B cells may play an important role in the generation of immune responses by ef- ficiently presenting Ag to T cells. We and other investigators recently described a subpopulation of CD11c+ B cells (Age/ autoimmune-associated B cells [ABCs]) that appear with age, during virus infections, and at the onset of some autoimmune diseases and participate in autoimmune responses by secreting autoantibodies. In this study, we assessed the ability of these cells to present Ag and activate Ag-specific T cells. We demonstrated that ABCs present Ag to T cells, in vitro and in vivo, better than do follicular B cells (FO cells). Our data indicate that ABCs express higher levels of the chemokine receptor CCR7, have higher

responsiveness to CCL21 and CCL19 than do FO cells, and are localized at the T/B cell border in spleen. Using multiphoton Downloaded from microscopy, we show that, in vivo, CD11c+ B cells form significantly more stable interactions with T cells than do FO cells. Together, these data identify a previously undescribed role for ABCs as potent APCs and suggest another potential mechanism by which these cells can influence immune responses and/or the development of autoimmunity. The Journal of Immunology, 2015, 195: 000–000. http://www.jimmunol.org/ t is well known that B cells contribute to immune responses the border between these two zones (6, 7). Such directed move- via their descendants, Ab-secreting plasma cells. In addition, ment is tightly regulated by interactions of multiple chemokines B cells are also professional APCs and, therefore, can par- produced by stromal cells and chemokine receptors expressed on I + ticipate in immune responses by activating Ag-specific CD4 the surface of . In particular, it was demonstrated T cells. Interactions between B and T cells occur mainly in sec- that B cells upregulate the expression of CCR7 upon encounter ondary lymphoid organs (spleen and lymph nodes) whose archi- with Ag. CCR7 expression drives the cells to move toward the tecture is favorable for such events. B cells migrate to the B cell chemokines CCL19 and CCL21 that are produced by stromal zones in secondary lymphoid organs where they may encounter cells in the T cell zone (8). In addition, the formation of stable Ags and become activated (1). Migration to B cell follicles is and motile Ag-specific B cell/CD4 T cell conjugates was detected by guest on September 25, 2021 driven by a chemokine gradient of CXCL13, which is produced by using multiphoton microscopy, indicating that B cells may indeed follicular stromal cells and is recognized by the CXCR5 receptor act as APCs during the initiation of an immune response (8). on B cells (2). In the follicles, B cells can encounter Ags from the In support of this statement, it was demonstrated that B cells surface of neighboring cells, such as follicular dendritic cells (3) are able to prime naive CD4 T cells both in vitro (9) and in vivo or macrophages (4). Alternatively, small soluble Ags can be ac- (10, 11). quired by B cells directly from the lymph (5). Although B and Several well-characterized subsets of B cells are found in the T cells are organized into separate zones in resting lymphoid spleen and/or lymph nodes. These are distinguished by the ex- organs, upon activation they move and interact with each other at pression of distinct surface markers. In addition to phenotypic differences, each B cell subset has a unique function. The ability to present Ag also differs among the different subsets of B cells. For *Howard Hughes Medical Institute, Department of Biomedical Research, National example, it was demonstrated that marginal zone (MZ) B cells are Jewish Health, Denver, CO 80206; †Department of Immunology and Microbiology, more potent activators of naive CD4 T cells than are follicular University of Colorado Denver, Anschutz Medical Campus, Denver, CO 80206; B cells (FO cells) (12). Enhanced Ag-presenting capabilities also ‡Department of Pharmacology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045; xDepartment of Medicine, University of Colorado Den- were demonstrated for B cells (13). { ver, Anschutz Medical Campus, Aurora, CO 80045; and Department of Biochem- We (14, 15) and other investigators (16) recently described istry and Molecular Genetics, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045 a novel subset of B cells (age/autoimmune-associated B cells 1 [ABCs]) in the of elderly female mice that is character- A.V.R. and K.R. contributed equally to this work. ized by the expression of CD11c and the transcription factor T-bet. Received for publication January 13, 2015. Accepted for publication May 4, 2015. B cells with a similar phenotype appear in autoimmune-prone This work was supported in part by U.S. Public Health Service Grants AI-18785, AI-22295, AI-046374, and T32 AI-074491. mice, at about the time that the symptoms of their disease ap- pear, as well as in animals suffering from acute virus infections Address correspondence and reprint requests to Dr. Anatoly V. Rubtsov and Dr. Philippa Marrack, Department of Biomedical Research, National Jewish Health, 1400 Jackson (14, 15, 17). Gene-expression analysis, as well as surface staining Street, K519, Denver, CO 80206. E-mail addresses: [email protected] (A.V.R.) and of these cells, indicated that the cells express high levels of the [email protected] (P.M.) costimulatory molecules CD80 and CD86, as well as MHC class The online version of this article contains supplemental material. II (MHCII) (14). These characteristics led us to hypothesize that Abbreviations used in this article: ABC, autoimmune-associated B cell; B6, C57BL/6; ABCs can serve as efficient APCs to prime CD4 T cells. FO cell, follicular B cell; MHCII, MHC class II; MZ, marginal zone. In this article, we demonstrate that CD11c+T-bet+ B cells ac- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 quired from aged or autoimmune female mice present Ag more

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500055 2 CD11c+ B CELLS ARE POTENT APCs efficiently than do FO cells, both in vitro and in vivo. Moreover, Cells were stained under saturating conditions with Abs to mouse CD4 these cells localize in spleens at the T cell/B cell border where Ag (clone GK1.5), CD8 (clone 53-6.7), B220 (clone RA3-6B2), CD11b (clone presentation takes place. In addition, upon Ag encounter, ABCs M1/70), CD11c (clone N418), and CD19 (clone 1D3), which were pur- chased from eBioscience or BD Pharmingen or generated in-house. form more stable conjugates with T cells than do FO cells. Taken Cells were analyzed by flow cytometry on a CyAn (Beckman Coulter) together, these data indicate a previously undescribed function for instrument, and data were analyzed using FlowJo software (TreeStar). ABCs as potent APCs and suggest an additional way in which they may contribute to the development of autoimmunity. Chemotaxis assay Recombinant murine CXCL13, CCL19, or CCL21 (PeproTech) was added to the bottom well of a 24-well plate in a total of 500 ml migration medium Materials and Methods (Iscove’s medium supplemented with GlutaMAX, 25 mM HEPES buffer, Isolation of distinct B cell populations and 1% fatty acid–free BSA). Total splenic cells from aged (18–20 mo old) or young B6 mice were depleted of RBCs and added to the upper insert of Splenic B cells were purified by negative enrichment using biotinylated 6 TER-119, NK1.1, and anti-TCRab Abs, followed by anti-biotin MicroBeads a Transwell (5 mm pore; Costar) at a concentration of 10 cells/100 ml. (Miltenyi Biotec, Bergisch Gladbach, Germany). All donor mice for ABCs After 3 h of incubation at 37˚C, cells were removed from the upper and were 18 to 20 mo old. ABCs were purified with a MoFlo sorter (Dako lower wells, pooled (two to six wells were used for each condition in each Cytomation) as B220+CD19+CD11c+ to .95% purity. FO cells were experiment), stained on ice, and analyzed by flow cytometry. For relative identified as B220+CD19+CD11c2CD21intCD1dint, and MZ B cells were cell counts, a CyAn was used to acquire cells for 60 s, at which time isolated as B220+CD19+CD11c2CD21highCD1dhigh. After sorting, cells $250,000–500,000 cells were collected from upper wells and 20,000– were stained for CD21 and CD1d surface expression to confirm the 90,000 cells were collected from lower wells. The percentage of cells distinction among three B cell populations: ABCs, FO cells, and MZ migrating into the bottom chamber was calculated: (number of cells in

3 Downloaded from B cells (Supplemental Fig. 1). For analysis, events were collected on a lower well)/(number of cells in lower + upper wells) 100. CyAn ADP (Beckman Coulter), and data were analyzed using FlowJo Mice version 8.8 (Tree Star). B6 mice were obtained from The Jackson Laboratory. In Ag-presentation In vitro Ag-presentation assay assays, T cells were isolated from transgenic mice expressing the F508abTCR (T cells are specific for 3K peptide presented by I-Ab) (21) or 5 B cell populations were isolated, as described above, and 10 cells were OT-II (T cells are specific for OVA peptide presented by I-Ab) (22). B6/ 5 mixed with either 10 OVA-specific H-2b–restricted T cell hybridomas UBI-GFP (23) mice were generated and maintained at the National Jewish http://www.jimmunol.org/ + (BO 80.10) or CD4 T cells from OT-II–transgenic mice. Cells were in- Health animal facility. B6.Nba2 (24) mice were purchased from The cubated in the presence of whole OVA protein or OVA peptide (aa 323– Jackson Laboratory (B6.NZB-(D1Mit47-D1Mit209)/BkotJ). Female mice 339) for 24 h at 37˚C. The presence of IL-2 in the supernatants was de- were used for all experiments. All animals were handled in strict accor- termined by MTT assay using IL-2–dependent HT-2 cells, as described dance with good animal practice, as defined by the relevant national and/or previously (18). IL-2 titers are expressed in U/ml. local animal welfare bodies, and all animal work was approved by the National Jewish Health Animal Care and Use Committee. In vitro proliferation assay OVA-specific CD4+ T cells were isolated from OT-II–transgenic mice, Multiphoton microscopy labeled with CFSE, and incubated with either FO cells or ABCs (isolated 3K-specific CD4+ T cells were isolated from 508 TCR-transgenic mice from 18–20 mo old mice) in the presence of whole OVA protein (10 mg/ml) 2 that were crossed to Rag B6 mice (bred at National Jewish Health) (19). by guest on September 25, 2021 m or OVA peptide (aa 323–339; 10 g/ml) for 4 h at 37˚C. CFSE dilution of Cells were labeled with 20 mMCMTMR,2mM CFSE, or 2 mMVPD; CD4+ T cells was determined by flow cytometry. washed three times; and injected i.v. into wild-type B6 recipients. Twenty- In vivo proliferation assay four hours later, ABCs and FO cells were isolated from 18–20-mo-old mice by flow cytometric sorting, as described above; incubated in the presence of 3K-specific CD4+ T cells were isolated using a CD4 T Cell Isolation Kit 3K peptide for 4 h at 37˚C; labeled with 20 mM CMTMR, 2 mMCFSE,or (Miltenyi Biotec) from 508 TCR-transgenic mice crossed to Rag-negative 2 mM VPD; and injected into recipient mice that received labeled CD4+ background (bred at National Jewish Health) (19). Cells were labeled with T cells. Twelve hours following injection, mice were sacrificed, and their CFSE, according to the manufacturer’s protocol, and injected i.v. into spleens were surgically removed for imaging and immobilized on cov- C57BL/6 (B6) mice (2 3 106 cells/mouse). Twenty-four hours later, ABCs erslips. During imaging, spleens were maintained at 35–37˚C in or FO cells were isolated from 18–20-mo-old mice by flow cytometric a flow chamber perfused with RPMI 1640 medium without phenol red sorting, as described above, incubated in the presence of 3K peptide or 3K- (Life Technologies) saturated with 95% O2/5% CO2. Multiphoton OVA protein for 4 h at 37˚C, and injected into mice that received labeled imaging was done using an Olympus FV1000MPE microscope with an CD4+ T cells. Dilution of CFSE by T cells was determined 4 d later. XLPLN25XWMP Super 253/1.05 NA water-immersion objective and a Spectra Physics 10 W Mai-Tai HP DeepSee-OL laser. The 450–490, 500– Immunofluorescence histology 550, 575–640, and 645–685-nm filters were used for blue, green, red, and far ABCs or FO cells from 18–20-mo-old GFP-expressing B6 mice were red emission channel acquisition, respectively. To avoid potential effects of isolated as described above and injected i.v. into B6 mice. Three days after the fluorescent dyes on viability and motility of ABCs and FO cells, we injection, spleens were harvested, incubated in 4% paraformaldehyde and swapped the dyes used to label the two transferred B cell populations be- tween experimental repeats. 10% sucrose for 2 h at room temperature, and incubated in 30% sucrose 3 overnight at 4˚C. Spleens were frozen at 280˚C in OCT compound (EM For time-lapse image acquisition, each x–y plane spanned 509 509 m m Sciences). Tissues were cut into 5–7-mm sections and dried at room m at a resolution of 0.994 m/pixel. Images of up to 22 x–y planes with m temperature overnight. Sections were rehydrated with PBS for 20 min and 3- m Z-spacing were acquired every 30 s for 30 min. Data were visualized blocked for 30 min with PBS, 2% BSA, 0.05% Tween 20. Ab mixtures and analyzed using Imaris (Bitplane) and MATLAB (MathWorks). To were added and incubated for 45 min, followed by three 5-min washes isolate each fluorophore to a single channel, linear unmixing was per- with PBS. Sections were allowed to dry and were mounted, and analyzed formed. The fluorescence intensity of a given fluorophore in its optimal with a Zeiss Axiovert 200M microscope (3i Marianas System) using channel was determined. The bleed-through fluorescence of the same SlideBook 4.0 software (Intelligent Imaging Innovations). fluorophore in each of the other channels was then assessed. The per- centage “bleed” into each channel was calculated by dividing the fluo- Flow cytometry rescence in the nonoptimal channel by the fluorescence in the optimal channel. The fluorescence in all nonoptimal channels was then subtracted Lymph nodes or spleens were prepared at the indicated times, and RBCs out on a pixel-by-pixel basis with MATLAB and the ImarisXT “Image were lysed. Single-cell suspensions were stained with MHC tetramers at Arithmetic” function using the percentage bleed determined. Surfaces 37˚C for 2 h. Allophycocyanin-Db/NP366–74 and PE-Db/PA224–38 were made in Imaris (using the “Surface” function) to identify the T cells, were produced as described (20), and PE-IAb/NP311–25 tetramer was FO cells, and ABCs. Based on these surface objects, individual B cells provided by the National Institutes of Health Tetramer Core Facility. Abs were identified and tracked by Imaris, and cellular speed and displace- to surface proteins were added, and the cells were incubated for an ad- ment were calculated from the tracks. Only cells that were tracked for ditional 20 min at 4˚C. $5 min were included in analyses of motility and displacement, and only The Journal of Immunology 3 cells that were tracked for $10 min were included in analyses of inter- demonstrate that, unlike conventional B cells, ABCs are primarily actions. Using a custom automated Matlab script, T cell/B cell inter- localized in the T cell zone. actions were quantified; T cells and B cells were scored as interacting if their cellular surfaces were within 0.994 mm (1 pixel) of each other. ABCs express T cell–specific chemokine receptors and migrate toward both T cell– and B cell–attracting chemokines Statistical analysis Next, we tried to determine which factors are responsible for ABCs’ All statistical analyses were performed with Prism software using the localization at the T cell/B cell border. It is commonly accepted that t Student test. chemokines and chemokine receptors determine the localization and migration of lymphocytes within secondary lymphoid organs (26). Results FO cells express the chemokine receptor CXCR5, and thus, they ABCs are localized to the T cell/B cell border in spleen respond to the chemokine CXCL13, which is produced in the B cell We demonstrated previously that ABCs obtained from aged mice follicle by follicular stromal cells. In contrast, T cells generally ex- express high levels of MHCII, CD80, and CD86 on their surface press the CCR7 receptor and migrate toward CCL19 and CCL21 compared with FO cells (Fig. 1A) (14), suggesting that these cells chemokines (27). To understand how ABCs are localized to the might be efficient at stimulating T cells. Because activated B cells T cell zone, we first examined the expression of CXCR5 and CCR7 are known to upregulate costimulation molecules and migrate by these cells and compared the results with those from FO cells. toward the T cell/B cell border to present Ag to T cells (25), we Flow cytometric analysis revealed that ABCs express levels of decided to determine the localization of ABCs in the spleen. CXCR5 that are similar to those observed on FO cells (Fig. 2A).

Unfortunately, direct immunofluorescent staining of aged spleen However, they express significantly higher levels of the T cell–spe- Downloaded from sections was not able to distinguish definitively between ABCs cific chemokine receptor CCR7 (Fig. 2A). Next, we tested whether and other B cell and dendritic cell populations. Therefore, we this difference in expression levels reflects a functional difference isolated ABCs and FO cells from old B6 female mice in which all in the ability of these cells to migrate toward specific chemokines. cells express GFP (UBI-GFP mice) and injected them i.v. into Using migration Transwells, we assessed the responsiveness of ABCs naive B6 mice. After 72 h, we determined the localization of GFP+ and FO cells to three chemokines: the B cell–specific chemokine

B cells by immunofluorescent histology. Spleen sections were CXCL13 and two nominally T cell–specific chemokines (CCL19 and http://www.jimmunol.org/ examined because ABCs are found preferentially in this organ CCL21). Although migration toward CXCL13 was comparable be- rather than in lymph nodes. Although FO cells were primarily tween ABCs and FO cells (Fig. 2B), ABCs migrated significantly distributed homogenously in B cell follicles, ABCs were localized better toward CCL19 and CCL21 than did FO cells (Fig. 2C, 2D). either in T cell zones or at the T cell/B cell border (Fig. 1B–D). These Transwell migration data are consistent with the expression These data were quantified based on the absolute number of ABCs patterns of chemokine receptors by ABCs and explain their locali- or FO cells localized to the T cell zone or B cell follicle (Fig. 1C) zation to the T cell/B cell border in the spleen. During their con- or normalized to the area of each zone (Fig. 1D). The data clearly version from FO cells (16), ABCs retain responsiveness to CXCL13 by guest on September 25, 2021

FIGURE 1. ABCs localize at the T cell/B cell border. (A) ABCs and FO cells were analyzed for the expression of MHCII, CD80, and CD86. (B) Localization of GFP+ ABCs or FO cells (green cells) in spleen 3 d after i.v. transfer in B6 mice. Data represent one of three independent experi- ments. Quantitation of histology either by absolute number of cells in T cell or B cell zones (C)orby normalizing to the area of each zone (D). Data are mean 6 SEM. *p , 0.01, t test. 4 CD11c+ B CELLS ARE POTENT APCs

FIGURE 2. ABCs express CCR7 and migrate toward both B cell– and T cell–attracting chemokines. (A) ABCs and FO cells were analyzed for the B expression of CXCR5 and CCR7. ABCs’ and FO cells’ chemotactic abilities toward B cell–specific chemokine CXCL13 ( ) and T cell–specific che- Downloaded from mokines CCL21 (C) and CCL19 (D). Data represent one of three independent experiments. and upregulate the expression of CCR7, thereby gaining respon- female B6 mice and incubated them with Ag-specific T cells in the siveness to the T cell zone chemokines CCL19 and CCL21 and presence of either whole protein Ag (OVA) or peptide Ag from resulting in their localization to the T cell/B cell border in the spleen. the same protein. ABCs and FO cells were compared for their ability to induce IL-2 production by OVA-specific T cell hybrid- ABCs present Ag more efficiently than FO cells in vitro omas (Fig. 3A), as well as primary OVA-specific CD4+ T cells http://www.jimmunol.org/ To compare the Ag-presenting abilities of ABCs and FO cells isolated from OT-II–transgenic mice (Fig. 3B, 3C). As shown in in vitro, we isolated these cell subsets from the spleens of aged Fig. 3, in the presence of OVA peptide, ABCs were more efficient by guest on September 25, 2021

FIGURE 3. Ag presentation by ABCs and FO cells in vitro. (A) ABCs and FO cells were analyzed for their ability to present peptide and protein Ags to Ag- specific hybridoma T cells. Data are mean 6 SEM (n = 3 mice/group). ABCs and FO cells were tested for their ability to present different concentrations of protein (B) or peptide (C) to primary OT-II T cells. (D) ABCs and FO cells were tested for their ability to stimulate proliferation of CFSE-labeled Ag-spe- cific T cells after 3 d of coculture in the presence of peptide or protein Ag. Data represent one of three independent experiments. *p , 0.01, t test. The Journal of Immunology 5 than FO cells at inducing secretion of IL-2 by both Ag-specific These data demonstrate that ABCs have an increased ability to T cell hybridoma cells and primary OT-II T cells. This difference take up, process, and present Ag to T cells and induce T cell was probably due to the higher levels of MHCII and costimulatory proliferation both in vivo and in vitro. proteins on the ABCs. Similar results were obtained when intact ABCs form long and stable interactions with Ag-specific T cells OVA was used as the source of Ag and detection was by OVA- specific T cell hybridomas. The results were even more dramatic There are several possible explanations for the robust Ag-present- when naive OT-II cells were used as detectors and intact OVAwas ing abilities of ABCs. These include their localization to the T cell/B used as Ag. In this case, ABCs were still effective APCs, whereas cell border, their ability to process Ag, and their high expression FO cells failed completely to stimulate the T cells. This result may of costimulatory and MHCII molecules. All of these properties sug- have been due to differences in the levels of MHCII and costim- gest that T cell contacts with ABCs may differ in quantity or quality ulatory proteins, as well as to the possibility that ABCs take up from their contacts with other B cells (e.g., FO cells). To test this and process whole OVA protein more efficiently as a result of their idea, we used multiphoton microscopy to compare the interactions high levels of expression of genes involved in vesicular transport between Ag-pulsed B cells (FO cells or ABCs) and Ag-specific and cytoskeletal rearrangement (14). We also assessed T cell T cells in spleen explants. proliferation in vitro in response to Ag presentation by monitor- Fluorescent dye–labeled T cells were transferred into B6 mice, ing CFSE dilution by OT-II T cells 3 d after incubation with Ag- and Ag-pulsed B cells (FO cells and ABCs, labeled with different pulsed FO cells or ABCs (Fig. 3D). Interestingly, the highest doses fluorescent dyes) were injected 24 h later. B cell/T cell inter- of Ag (either protein or peptide) led to equal T cell stimulation by actions in the spleen were tracked by time-lapse multiphoton

FO cells and ABCs. However, ABCs were better T cell stimulators microscopy 12 h later. We observed that ABC/T cell interactions Downloaded from at lower concentrations of Ag. This result contrasts with our were significantly more stable than were FO cell/T cell inter- observations of IL-2 production for which the maximum differ- actions (Fig. 5A–C, Supplemental Video 1), resulting in longer ences were observed in the presence of the highest amount of Ag contact times between ABCs and T cells. Previous studies on (Fig. 3B, 3C). The discrepancy is probably due to consumption of interactions between APCs and T cells demonstrated that the IL-2 by the proliferating T cells, causing the IL-2 assays (Fig. 3B, duration of interactions is critical for the fate of T cells (28). Long . 3C) to underestimate the amounts of IL-2 produced by the T cells APC/T cell interactions, often lasting 12 h, led to sustained http://www.jimmunol.org/ in each assay. T cell activation, whereas aborted or transient interactions led to These results demonstrate that in vitro ABCs are more efficient tolerogenic signaling in T cells (29–31). Therefore, our results APCs than are FO cells. suggest that ABC/T cell interactions are more likely to lead to T cell activation than are FO cell/T cell interactions. As a control, ABCs present Ag more efficiently than FO cells in vivo we also injected T cells that were specific for an unrelated Ag. Next we explored whether the efficient Ag-presenting activity of Neither ABCs nor FO cells interacted with these T cells, con- ABCs in vitro is also evident in vivo. To this end, we transferred firming the Ag-specific nature of the presenting cell/T cell inter- CFSE-labeled OT-II T cells into B6 mice 1 d before they received actions that we observed (Supplemental Video 2).

Ag-preloaded FO cells or ABCs. CFSE dilution by T cells was Overall, our multiphoton experiments demonstrated that ABCs by guest on September 25, 2021 determined 4 d after the injection of B cells; as shown in Fig. 4, not only localize to the area most suitable for interactions with Ag- ABCs were significantly more potent in stimulating T cell pro- specific T cells, they also possess some cell-intrinsic features that liferation. ABCs were equally good T cell stimulators when given allow them to form more productive interactions with Ag-specific either whole protein or peptide (Fig. 4A, 4B). In contrast, FO cells T cells, ultimately leading to more efficient T cell activation. presented OVA peptide quite well (Fig. 4B) but intact OVA protein poorly (Fig. 4A). ABCs obtained from autoimmune mice also possess more efficient Ag-presenting capabilities than FO cells All of the data described above indicate that ABCs obtained from aged B6 female mice are more efficient APCs than are FO cells. This is explained by their expression of several surface markers, including chemokine receptors and costimulatory molecules, their localization in T cell zones in the spleen, and their ability to process and present protein Ags effectively. B cells expressing T-bet, CD11b, and CD11c are also found in the spleens of autoimmune mice, appearing at about the same time as the first symptoms of autoimmunity (14, 17). These cells also have been termed ABCs; however, they may not share all of the properties of the related population in aged female animals. To determine whether ABCs from autoimmune mice have Ag-presenting capa- bilities similar to those of ABCs from elderly animals, ABCs were obtained from the spleens of the autoimmune-prone strain B6.Nba2. The donor animals were 8–12 mo old and already had autoanti- bodies in their sera. Expression of chemokine receptors on the surface of ABCs and FO cells was compared using flow cytometry. As shown in Fig. 6A, ABCs obtained from B6.Nba2 mice expressed FIGURE 4. Ag presentation by ABCs and FO cells in vivo. (A) CFSE dilution by Ag-specific T cells was determined in mice that received either higher levels of CCR7 and similar levels of CXCR5 compared with ABCs or FO cells pulsed with Ag (protein, peptide, or media). (B) Per- FO cells obtained from the same mice. A comparison of the ability centage of T cells divided more than three times after mice received ABCs of the cells to migrate in CXCL13, CCL19, and CCL21 chemokine or FO cells pulsed with Ag (protein, peptide, or media). Bars represent the gradients showed that ABCs and FO cells from autoimmune mice means 6 SEM of n = 5 mice/group. *p , 0.05, t test. migrated equally well toward CXCL13 (a chemokine expressed in 6 CD11c+ B CELLS ARE POTENT APCs

and activated primary Ag-specific T cells significantly better than did FO cells (Fig. 6C). Taken together, these data demonstrate that ABCs from auto- immune mice possess characteristics that are similar to those of ABCs from aged wild-type mice. Thus, ABCs may influence the development and progress of autoimmunity through secretion of autoantibodies (14, 17), as well as through presentation of self- Ags and activation of autoreactive T cells.

Discussion We recently identified ABCs as cells that are able to produce self- reactive Abs and that accumulate both with age and during the onset of autoimmunity (14, 17). We also described a similar subset of B cells in autoimmune patients; however, the function of these cells in both aged and autoimmune mice remained largely unknown. B cells can contribute to the development of autoimmune disease in various ways. The most obvious and direct way is by

the secretion of autoantibodies that target and destroy the ani- Downloaded from mal’s own organs. Our previously published data showed that ABCs, after activation, are capable of secreting self-reactive Abs (14, 17). In addition, we showed that ABCs are responsi- ble for the appearance of autoantibodies and that the depletion of these cells leads to a reduction in autoreactive Abs in the

serum (17). http://www.jimmunol.org/ However, there are other ways in which B cells can contribute to autoimmunity, including secretion of pro- or anti-inflammatory cytokines and presentation of self-Ags to autoreactive T cells (32). B cells express MHCII and costimulatory proteins and, therefore, may present Ag to CD4 T cells. The consequences of such presentation for the T cells depend on the state of the B cells. In some cases, B cells, particularly naive resting B cells, present Ag and tolerize the responding T cells (28). In other cases, the target T cells are productively activated. Despite the fact that by guest on September 25, 2021 B cells were demonstrated to be potent APCs, several reports suggest that the presence of B cells in mice is not required for the generation of T cell responses to Ags, such as keyhole limpet hemocyanin, alloantigens, influenza virus, and human g globulin (33–35). Thus, it was suggested that DCs, and not B cells, are responsible for Ag presentation and activation of CD4 T cells (36). However, further studies demonstrated that this is not always the case, and B cells, as well as other APCs, can play various roles in Ag presentation depending on the nature of Ag (11, 37). Overall, it was suggested that, especially at low concentrations of an Ag, Ag- specific B cells uptake and present Ag more efficiently than do other cells (38). This might be explained by the fact that Ag- FIGURE 5. ABCs form more stable interactions with Ag-specific T cells specific B cells can capture Ag via their BCR, whereas DCs and than do FO cells in vivo. (A) Total time for which each analyzed B cell was in contact with Ag-specific T cell(s). (B) Bar graph shows mean (6 SD) macrophages use pinocytosis, which is 1,000–10,000 times less number of breaks each interacting T cell analyzed made during the ob- efficient (39). servation period. (C) Bar graph shows average (6 SD) duration of each The fact that B cells can present Ags efficiently makes them good interaction between a B cell (ABCs or FO cells) and an Ag-specific T cell. candidates to function as APCs during autoimmunity, because self- Data are mean 6 SEM of at least three independent time-lapse multi- Ags are usually present at low concentrations. In support of this, it photon experiments. **p , 0.001, t test. was demonstrated that B cells are the major APCs during the induction of various autoimmune diseases (40, 41). Several reports indicated that the Ag-presenting function of B cells is crucial for B cell follicles). However, autoimmune ABCs migrated better to- the development of type 1 diabetes (42–44). Moreover, NOD mice ward CCL19 and CCL21 (T cell zone chemokines) than did FO that contain B cells that can react only with hen egg lysozyme fail cells. Together, these data suggest that, like ABCs from elderly to develop diabetes. Conversely, disease is accelerated in NOD animals, the ABCs in autoimmune animals may also be localized to mice that express an IgH that is prone to react with insulin (45). the T cell/B cell border (Fig. 6B). These results suggest that autoreactivity of the BCRs in NOD Finally, the Ag-presenting capabilities of ABCs and FO cells mice is critical for presentation of Ag in this model (46). The role from B6.Nba2 autoimmune mice were tested in vitro. Similar to the of B cells as APCs in autoimmunity also was suggested by an data obtained with cells from aged female mice (Fig. 3), ABCs MRLlpr model of lupus-like disease by generating MRLlpr mice from autoimmune mice presented Ag (peptide and whole protein) that contain no B cells or that have B cells that are unable to The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. ABCs from autoimmune mice exhibit increased Ag-presenting properties compared with FO cells. (A) ABCs and FO cells from Nba2 autoimmune mice were analyzed for the expression of CXCR5 and CCR7. (B) ABCs and FO cells from Nba2 mice were tested for chemotactic migration toward CXCL13, CCL21, and CCL19. (C) ABCs and FO cells were tested for Ag presentation, at different concentrations of peptide (left panel) or protein (right panel), to primary OT-II T cells. All data represent one of three independent experiments. secrete Abs (47). In the absence of B cells, the mice do not de- presentation. These intrinsic features could include enhanced Ag velop lupus. In contrast, if B cells are present the animals develop uptake and/or processing by ABCs. To test this idea, we cultured disease, even if the B cells cannot give rise to soluble Abs (47). ABCs and FO cells with DQ-OVA and compared their ability to However, the particular subset of B cells that is responsible for generate fluorescent Ag. We did not see any difference in the rate of the presentation of the self-Ag in autoimmune settings has never Ag processing between ABCs and FO cells (data not shown). been established. In this study, we investigated the role of ABCs Therefore, ABCs probably present Ag to T cells in vitro more in Ag presentation in aged wild-type or autoimmune B6.Nba2 efficiently than do FO cells because ABCs express higher levels of mice. We explored the localization of ABCs in the spleen and MHCII and of the costimulatory proteins CD80 and CD86. their ability to present Ag and activate Ag-specific T cells. Our We showed previously that ABCs from autoimmune-prone mice data indicate that, as a result of the expression of both T and can give rise to cells that secrete autoantibodies (14, 17), indicating B cell–specific chemokine receptors, ABCs are localized at the the specificity of their BCRs for self-Ags. As such, ABCs can take T cell/B cell border in the spleen as opposed to FO cells, which up autoantigens through their AgRs and are perfect candidates for localize primarily to B cell follicles. Thus, ABCs’ localization activating autoreactive T cells, leading to the onset of autoim- provides a greater opportunity to form interactions with Ag- munity. specific T cells. Moreover, multiphoton data demonstrate that ABCs form sig- Coincidental with their localization, ABCs also have a greater nificantly more stable interactions with T cells compared with FO ability to present soluble protein Ag to Ag-specific T cells both cells. The stability of APC/T cell contacts was shown to be critical in vitro and in vivo. The fact that this is true in in vivo assays could for the fate of the T cells because more stable interactions usually be due to the fact that ABCs, but not FO cells, localize to the T cell/ lead to T cell activation, whereas less stable ones often lead to B cell border. However, ABCs are also more potent than FO cells in tolerance (28). Thus, interactions between ABCs and T cells have activating Ag-specific T cells in vitro, indicating that ABCs possess a better chance of leading to activation of the T cell than do cell-intrinsic features that allow them to be more efficient at Ag FO/T cell interactions. 8 CD11c+ B CELLS ARE POTENT APCs

Taken together, our data strongly suggest that Ag presentation is mulation of a novel CD11c⁺ B-cell population is important for the development of autoimmunity. 118: 1305–1315. one of the major functions of ABCs in both aged and autoimmune 15. Rubtsova, K., A. V. Rubtsov, L. F. van Dyk, J. W. Kappler, and P. Marrack. 2013. mice. This conclusion leads to several questions that have to be T-box transcription factor T-bet, a key player in a unique type of B-cell activation explored in the future. For example, how does the depletion of essential for effective viral clearance. Proc. Natl. Acad. Sci. USA 110: E3216– E3224. ABCs affect T cell activation during autoimmunity? We already 16. Hao, Y., P. O’Neill, M. S. Naradikian, J. L. Scholz, and M. P. Cancro. 2011. A demonstrated that depletion of ABCs leads to a reduction in the titer B-cell subset uniquely responsive to innate stimuli accumulates in aged mice. of autoantibodies, and we proposed that ABCs themselves were the Blood 118: 1294–1304. 17. Rubtsov, A. V., K. Rubtsova, J. W. Kappler, and P. Marrack2013. TLR7 drives main source of autoantibodies (17). However, in light of this study, accumulation of ABCs and autoantibody production in autoimmune-prone mice. + it is possible that, in the absence of ABCs, CD4 T cells do not Immunol. Res. 55: 210–216. become activated and, therefore, do not provide help to other 18. Kappler, J. W., B. Skidmore, J. White, and P. Marrack. 1981. Antigen-inducible, H-2-restricted, interleukin-2-producing T cell hybridomas. Lack of independent autoreactive B cells. antigen and H-2 recognition. J. Exp. Med. 153: 1198–1214. We recently reported the appearance of ABCs at the peak of 19. MacLeod, M. K., A. David, A. S. McKee, F. Crawford, J. W. Kappler, and antiviral humoral responses (15). Our data indicated that, in the P. Marrack. 2011. Memory CD4 T cells that express CXCR5 provide accelerated help to B cells. J. Immunol. 186: 2889–2896. absence of ABCs, there is a reduction in antiviral IgG2a titers and 20. Crawford, F., H. Kozono, J. White, P. Marrack, and J. Kappler. 1998. Detection inefficient viral clearance. It will be interesting to determine of antigen-specific T cells with multivalent soluble class II MHC covalent whether the Ag-presenting capabilities of ABCs acquired from peptide complexes. Immunity 8: 675–682. 21. Huseby, E. S., J. White, F. Crawford, T. Vass, D. Becker, C. Pinilla, P. Marrack, infected animals are similar to those of aged and autoimmune and J. W. Kappler. 2005. How the T cell repertoire becomes peptide and MHC ABCs. If this is the case, ABCs might play several important roles specific. Cell 122: 247–260. during viral infection. 22. Barnden, M. J., J. Allison, W. R. Heath, and F. R. Carbone. 1998. Defective TCR Downloaded from expression in transgenic mice constructed using cDNA-based alpha- and beta- In summary, the data presented in this article identify a previ- chain genes under the control of heterologous regulatory elements. Immunol. ously unappreciated function for ABCs as potent APCs. The ap- Cell Biol. 76: 34–40. pearance of ABCs in autoimmune mice and autoimmune patients 23. Schaefer, B. C., M. L. Schaefer, J. W. Kappler, P. Marrack, and R. M. Kedl. 2001. Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions leads to the suggestion that their superior Ag-presenting capa- in vivo. Cell. Immunol. 214: 110–122. bilities might be critical for the development of autoimmunity. 24. Rozzo, S. J., J. D. Allard, D. Choubey, T. J. Vyse, S. Izui, G. Peltz, and B. L. Kotzin. 2001. Evidence for an interferon-inducible gene, Ifi202, in the susceptibility to systemic lupus. Immunity 15: 435–443. http://www.jimmunol.org/ Acknowledgments 25. Reif, K., E. H. Ekland, L. Ohl, H. Nakano, M. Lipp, R. Fo¨rster, and J. G. Cyster. We thank Dr. M. Phillips and L. Noges for critical review of the manuscript 2002. Balanced responsiveness to chemoattractants from adjacent zones deter- mines B-cell position. Nature 416: 94–99. and Dr. P. Beemiller and B. Leavitt for programming of image analysis 26. Cyster, J. G. 1999. Chemokines and cell migration in secondary lymphoid Matlab scripts. organs. Science 286: 2098–2102. 27. Cyster, J. G. 2005. Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs. Annu. Rev. Immunol. 23: 127–159. Disclosures 28. Jacobelli, J., R. S. Lindsay, and R. S. Friedman. 2013. Peripheral tolerance and The authors have no financial conflicts of interest. autoimmunity: lessons from in vivo imaging. Immunol. Res. 55: 146–154. 29. Hugues, S., L. Fetler, L. Bonifaz, J. Helft, F. Amblard, and S. Amigorena. 2004.

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