Thymic B cells promote thymus-derived regulatory T cell development and proliferation

Journal of Autoimmunity xxx (2015) 1e11

Contents lists available at ScienceDirect

Journal of Autoimmunity journal homepage: www.elsevier.com/locate/jautimm

Thymic B cells promote thymus-derived regulatory T cell development and proliferation Fang-Ting Lu a, Wei Yang a, Yin-Hu Wang a, Hong-Di Ma a, Wei Tang a, Jing-Bo Yang a, Liang Li a, Aftab A. Ansari b, Zhe-Xiong Lian a, c, * a

Liver Immunology Laboratory, Institute of Immunology and The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui 230027, China Department of Pathology, Emory University School of Medicine, Atlanta, GA 30322, USA c Innovation Center for Cell Signaling Network, Hefei National Laboratory for Physical Sciences at Microscale, Hefei, Anhui 230026, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 May 2015 Received in revised form 19 May 2015 Accepted 21 May 2015 Available online xxx

Thymic CD4þ FoxP3þ regulatory T (Treg) cells are critical for the development of immunological tolerance and immune homeostasis and requires contributions of both thymic dendritic and epithelial cells. Although B cells have been reported to be present within the thymus, there has not hitherto been a definition of their role in immune cell development and, in particular, whether or how they contribute to the Treg cellular thymic compartment. Herein, using both phenotypic and functional approaches, we demonstrate that thymic B cells contribute to the maintenance of thymic Treg cells and, using an in vitro culture system, demonstrate that thymic B cells contribute to the size of the thymic Treg compartment via cellecell MHC II contact and the involvement of two independent co-stimulatory pathways that include interactions between the CD40/CD80/CD86 co-stimulatory molecules. Our data also suggest that thymic B cells promote the generation of thymic Treg cell precursors (pre-Treg cells), but not the conversion of FoxP3þ Treg cells from pre-Treg cells. In addition, thymic B cells directly promote the proliferation of thymic Treg cells that is MHC II contact dependent with a minimal if any role for costimulatory molecules including CD40/CD80/CD86. Both pathways are independent of TGFb. In conclusion, we rigorously define the critical role of thymic B cells in the development of thymic Treg cells from non-Treg to precursor stage and in the proliferation of mature thymic Treg cells. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Thymic B cells Thymic Treg cells Development Proliferation Cellecell contact

1. Introduction After positive selection, CD4 and CD8 single-positive (SP) thymocytes are negatively selected against auto-antigens through

Abbreviations: Treg cells, regulatory T cells; mMT, immunoglobulin heavy chain of the class mu knockout mice; IL-7, interleukin-7; IL-2, interleukin-2; MOG, myelin oligodendrocyte glycoprotein; MHCII, major histocompatibility complex class II; CD, cluster of differentiation; GFP, green fluorescent protein; FoxP3, forkhead box P3. * Corresponding author. Liver Immunology Laboratory, Institute of Immunology and The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui 230027, China. Tel./fax: þ86 551 63600317. E-mail addresses: [email protected] (F.-T. Lu), [email protected]. cn (W. Yang), [email protected] (Y.-H. Wang), [email protected] (H.-D. Ma), [email protected] (W. Tang), [email protected] (J.-B. Yang), [email protected] (L. Li), [email protected] (A.A. Ansari), [email protected] (Z.-X. Lian).

deletion of self-reactive thymocytes in the thymic medulla [1,2]. However, some self-reactive thymocytes escape deletion and further differentiate into thymus-derived regulatory T (Treg) cells [3,4]. The Treg lineage specific transcriptional factor FoxP3 is required for the maintenance of immune homeostasis and tolerance [5,6]. Indeed, ablation of Treg cells via FoxP3 deficiency leads to multi-organ lethal autoimmune diseases in both humans and mice [5,7]. The development of thymic Treg cells is a two-step process. First, an initial step, dependent on TCR-MHC II and CD28-CD80/ CD86 interaction, leads to the generation of thymic CD4þ CD25þ Foxp3 Treg cell precursors (pre-Treg cells). Subsequently, an IL-2dependent process drives thymic pre-Treg cells to mature CD25þ FoxP3þ Treg cells [8e11]. Several populations of antigen presenting cells (APCs) in the thymus have shown to contribute to thymic Treg development and including sirpaþ dendritic cells (DCs), CD8aþDCs, plasmacytoid DCs and thymic medullary epithelium [12e18].

http://dx.doi.org/10.1016/j.jaut.2015.05.008 0896-8411/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: F.-T. Lu, et al., Thymic B cells promote thymus-derived regulatory T cell development and proliferation, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.05.008

2

F.-T. Lu et al. / Journal of Autoimmunity xxx (2015) 1e11

The thymus contains a low frequency (0.1e0.3%) population of B cells that have previously been implicated in thymic negative selection [19,20]. The fact that B cells in the periphery have been implicated in the formation of the Treg compartment [21e24], prompted us to hypothesize that this process may indeed be initiated within the thymus. Our view is consistent with the thought that the immunological process of negative selection needs to be controlled and hence our thesis is that thymic B cells have a potentially important role in the development of thymic Treg cells. In the studies reported herein, we demonstrate that thymic B and Treg cells generally co-localize in the thymic medulla. Further, a deficiency of thymic B cells results in a significant decrease in both the frequency and number of thymic Treg cells. In addition, using an in vitro culture system, we demonstrate that thymic B cells contribute to thymic Treg cell number via cellecell contact involving two independent pathways. In the first pathway, thymic B cells promote the generation of thymic pre-Treg cells from CD4 single positive thymocytes. Second, thymic B cells directly promote the proliferation of thymic Treg cells both in vivo and in vitro that is MHC II contact dependent with a minimal-role for the costimulatory molecules CD40/CD80/86. Thus, our data reveal that thymic B cells have a non-redundant role in regulating the size of the Treg compartment in the thymus. 2. Materials and methods 2.1. Mice C57BL/6 (B6) mice were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Science (Shanghai, China). FoxP3-GFP mice (Foxp3tm2Ayr) [25] were kindly provided by Dr. A.Y. Rudensky, and mMT mice (B6.129S2-Igh-6tm1Cgn/J) [26] were from Jackson Laboratory (Bar Harbor, Maine, USA). mMT FoxP3-GFP mice were generated by breeding mMT mice with FoxP3-GFP mice. dnTGFbRII mice (B6.Cg-Tg(Cd4-TGFBR2)16Flv/J) [27] were initially purchased from Jackson Laboratory, and dnTGFbRII FoxP3-GFP mice were obtained by backcrossing dnTGFbRII with FoxP3-GFP mice. All mice were housed in a specific pathogen-free and controlled environment (22  C, 55% humidity, and 12-h day/night rhythm) and care provided according to the regulations of animal care at University of Science and Technology of China (Hefei, Anhui, China). All animal experiments were performed using 5e10 week old mice. 2.2. Phenotyping A single cell suspension were first blocked with Fc-blocking Ab CD16/32 (BioLegend, San Diego, CA, USA) and aliquots stained with a combination of fluorescence-labeled monoclonal antibodies (mAbs) specific for B220 (RA3-6B2), CD19 (6D5), MHC II (M5/ 114.15.2), CD40 (3/23), CD44 (IM7), CD69 (H1.2F3), CD80 (16-10A1), CD86 (GL-1), CD40L (MR1), CD4 (GK1.5), CD8a (53e6.7), CD8b (YTS156.7.7), CD25 (PC61) and TCRb (H57-597) in the dark at 4  C for 20 min. All antibodies were purchased from Biolegend except for anti-B220 (BD Biosciences, San Jose, CA, USA). Anti-rat IgG2a and k Abs (BioLegend) were used as isotype controls. For Ki67 intracellular staining, after staining with APC/Cy7conjugated anti-CD19 for B cells, or PerCP/Cy5.5-conjugated antiCD4 and PE/Cy7-conjugated anti-CD8a for thymic Treg cells, cells were fixed and permeabilized with a Foxp3 staining buffer (eBioscience, San Diego, CA, USA), and then stained with PE-conjugated anti-Ki67 (16A8, BioLegend) for 60 min. Flow cytometry was then performed on a FACSVerse (BD). Acquired data were analyzed with FlowJo Software (Tree Star, Ashland, USA).

2.3. Cell sorting For thymic CD4 single positive (SP) T cells, single thymocytes from 5- to 8-week-old FoxP3-GFP mice or dnTGFbRII FoxP3-GFP mice were labeled with PE/Cy7-conjugated anti-CD8a, PEconjugated anti-CD25 and PerCP/Cy5.5-conjugated anti-CD4. Thymic CD4 SP T cells were sorted as cells that express CD4þ CD8a CD25 GFP using a FACSAria I (BD). the purity was higher than >95% (Supplementary Fig. 1B). For thymic B cells and DCs, single thymocytes from 5- to 10week-old B6 mice were re-suspended in 60% Percoll gradient. After centrifugation, thymic B cells were initially enriched from the top layer. Dynabeads conjugated with sheep anti-rat IgG (Life Technologies, Carlsbad, CA, USA) were then used to remove T cells as follows. Briefly, cells were re-suspended in media containing 1 mg of purified rat anti-mouse CD4 mAb (GK1.5, BioLegend) per 106 target cells, incubated for 30 min and then washed with buffer. Next, cells were incubated with pre-washed sheep anti-rat IgG conjugated dynabeads at 4  C for 30 min with gentle tilting and rotation. The bead-coated cells were then removed using a magnet (BD), and the remaining cells were stained with PE-conjugated anti-CD11c (N418, Biolegend) and PE/Cy7-conjugated anti-CD19. CD19þ CD11c thymic B cells and CD19 CD11cþ thymic DCs were sorted by using a FACSAria I, the purity were both higher than >90% (Supplementary Fig. 1A). For thymic Treg cells and pre-Treg cells, 106 single thymocytes from 5- to 10-week-old FoxP3-GFP mice were re-suspended in media containing 1 mg of purified rat anti-mouse CD8a mAb (53e6.7, BioLegend), incubated for 30 min and then washed with buffer. Next, cells were incubated with pre-washed dynabeads conjugated with sheep anti-rat IgG at 4  C for 30 min with gentle tilting and rotation. The bead-coated cells were then removed using a magnet and the remaining cells were stained with PerCP/Cy5.5conjugated anti-CD4, PE/Cy7-conjugated anti-CD8b and PEconjugated anti-CD25. Thymic Treg cells were sorted from this population as cells that expressed CD4þ CD8b GFPþ and pre-Treg cells were sorted as CD4þ CD8b CD25þ GFP using a FACSAria I, the purity were both higher than >90% (Supplementary Fig. 2A). 2.4. In vitro Treg induction experiments In vitro Treg cell induction assays were performed in 96-well Ubottom plates with 1.5  104 sorted thymic B cells or thymic DCs from B6 mice and 3  104 sorted CD4þ CD8 CD25 GFP thymic CD4 SP T cells from FoxP3-GFP mice for 5 days. Cells were stimulated with a-CD3 (0.5 mg/ml, BioLegend), a-CD28 (0.5 mg/ml, BioLegend) and recombinant-IL-2 (rIL-2, 2 ng/ml) (PeproTech, Rocky Hill, USA) in complete RPMI 1640 (Life Technologies) medium consisting of RPMI supplemented with 10% heat-inactivated FCS (HyClone, Logan, UT, USA), 100 U/ml penicillin/streptomycin, 10 mM HEPES, 1 mM sodium pyruvate and 50 mM b-mercaptoethanol. After 5 days culture, cells were analyzed by flow cytometry gated on the DAPI CD4þ T cells, and GFP was used as an indicator for the induction of Treg cells. For blocking assays of Treg induction, an optimal dilution (10 mg/ml) of anti-CD80 (16-10A1) [28], antiCD86 (GL-1) [28], anti-MHC class II (M5/114.15.2), anti-CD40 blocking (HM40-3) or their relevant isotype control antibodies were added to the co-cultures. Armenian Hamster IgG (HTK888), Rat IgG2a k (RTK2758), Rat IgG2b k (RTK4530) and Armenian Hamster IgM (HTK204) antibodies were used as their relevant isotype controls. a-CD28 was omitted in the CD80 and CD86 blocking assays. All blocking antibodies were purchased from Biolegend. For the transwell assays, 6  104 sorted CD4þ CD8 CD25 GFP thymic CD4 SP T cells from FoxP3-GFP mice were cultured in the

Please cite this article in press as: F.-T. Lu, et al., Thymic B cells promote thymus-derived regulatory T cell development and proliferation, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.05.008

F.-T. Lu et al. / Journal of Autoimmunity xxx (2015) 1e11

bottom compartment of Transwell-96 System (3 mm pore size, corning, NY), while 3  104 thymic B cells were plated in the top chamber for 5 days in the presence of a-CD3 (0.5 mg/ml), a-CD28 (0.5 mg/ml) and rIL-2 (2 ng/ml). GFPþ Treg cells were analyzed by flow cytometry gated on the DAPI CD4þ T cells. For in vitro Treg induction using supernatants, supernatant fluids were collected from co-cultures of thymic B cells and thymic CD4 SP T cells in the above in vitro Treg cell induction assays. Thence, 3  104 CD4þ CD8 CD25 GFP thymic CD4 SP T cells from FoxP3-GFP mice were cultured with or without supernatant fluids in the presence of a-CD3 (0.5 mg/ml), a-CD28 (0.5 mg/ml) and rIL-2 (2 ng/ml) for 5 days. Treg cells were analyzed for the expression of GFP gated on the DAPI CD4þ T cells. 2.5. In vitro pre-Treg induction experiments In vitro pre-Treg induction assays were also performed in 96well U-bottom plates. 3  104 sorted CD4þ CD8 CD25 GFP thymic CD4 SP T cells from FoxP3-GFP mice were cultured with or without 1.5  104 sorted thymic B cells for 18 h using the same stimulation protocol as utilized in the in vitro Treg cell induction assays. Pre-Treg cells were analyzed by flow cytometry and gated on PI CD4þ CD25þ GFP cells.

3

2.9. BrdU incorporation assay Mice were injected i.p. with 10 mg of the nucleotide analog bromodeoxyuridine (BrdU, BD) for 3 h. Single cell suspensions of thymocytes and splenocytes were then prepared. BrdU incorporation was detected by flow cytometry using a BrdU Flow Kit (BD) [31]. In brief, cells were stained for cell surface phenotypic markers, fixed and then permeabilized. After treating with DNase, cells were stained with APC-conjugated anti-BrdU mAb (BD). Finally, BrdU incorporation was measured by analysis of the cells using FACSVerse. 2.10. Immunohistochemistry Thymic tissues from FoxP3-GFP mice were fixed in 10% neutral formalin overnight, embedded in paraffin, cut into 5 mm slices, then deparaffinized and blocked with 10% goat serum in PBS for 40 min. For B cells, thymic sections were stained with rat anti-mouse CD19 mAb (6D5, Abcam, Cambridge, MA, USA) overnight at 4  C and then incubated with biotin labeled anti-rat antibody (Bioster, WuHan, China) at room temperature for 30 min. Sections were subsequently incubated with AP linked anti-biotin antibody (Bioster) for 30 min, stained with Fast-red chromogen (Abcam). For Treg cells, thymic sections were stained with rabbit anti-GFP mAb (Abcam) overnight at 4  C and visualized with HRP labeled anti-rabbit antibody (ChemMate™ EnVision™ Detection Kit, Dako, Denmark).

2.6. In vitro the conversion of pre-Treg into Treg experiments 2.11. Immunofluorescence For the conversion of pre-Treg into Treg assays, 2  104 sorted thymic CD4þ CD8 CD25þ GFP pre-Treg cells from FoxP3-GFP mice were cultured with or without 2  104 sorted thymic B cells for 24 h in the presence of rIL-2 (10 ng/ml) [9]. Treg cells were analyzed by the expression of GFP gated on DAPI CD4þ T cells. 2.7. In vitro Treg proliferation experiments Cell Sorter enriched thymic CD4þ CD8 GFPþ Treg cells from FoxP3-GFP mice were labeled with CellTrace Violet proliferation dye (Life Technologies). Thence, 2  104 labeled Treg cells were cocultured with or without 2  104 sorted thymic B cells for 3 days in U-bottom 96-well plates that were pre-coated with a-CD3 (1 mg/ ml) in the presence of soluble a-CD28 (1 mg/ml) antibodies and rIL2 (100 ng/ml) [29]. After 3 days culture, cells were stained with Propidium Iodide solution for 15 min to exclude dead cells, and then PI GFPþ Treg cells proliferation was assessed by CellTrace Violet dilution. For blocking assays of Treg proliferation, predetermined optimal dilutions (10 mg/ml) of anti-CD40, anti-CD80, anti-CD86 and anti-MHC II blocking or their relevant isotype control antibodies were added to the co-cultures. For CD80 and CD86 blocking, a-CD28 was omitted in the co-culture of thymic Treg cells and thymic B cells. 2.8. In vitro T cell suppression experiments Splenic CD4þ GFP responder cells were sorted from 5- to 8week-old FoxP3-GFP mice. Thymic B cell-induced Treg cells or thymic DC-induced Treg cells were sorted from the co-cultures of Treg induction assays, and then co-cultured with CellTrace Violet labeled CD4þ GFP responder cells (5  104) at indicated ratios in the presence of soluble a-CD3 (1 mg/ml), a-CD28 (1 mg/ml) and Mitomycin C (Sigma, St. Louis, MO, USA)-treated splenic CD4 negative supporter cells (1  105) [30]. On day 3, cells were stained with Propidium Iodide solution for 15 min to exclude dead cells, and then PI GFP CD4þ T responder cells proliferation was assessed by analysis of CellTrace Violet dilution using FACSVerse.

Thymic tissues from FoxP3-GFP mice were embedded in TissueTek OCT and cut into 7 mm slices, fixed in acetone for 10 min at 4  C, and then preserved at 70  C. Thymic sections were brought to room temperature, fixed in acetone for 10 min at 4  C, and then placed in PBS for 15 min to remove OCT. All sections were blocked with 10% goat serum for 40 min. Next, sections were incubated with rabbit anti-GFP antibody (Abcam) and rat anti-mouse CD19 antibody (Abcam) overnight in a humidified chamber at 4  C, and then sections were washed five times with PBS for 5 min, incubated with appropriate secondary Abs for 1 h. For B cells, sections were stained with R-phycoerythrin goat anti-rat IgG (Life Technologies). For Treg cells, sections were stained with Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies). Finally, sections were observed and visualized under confocal microscopy (LSM 710, Cral Zeiss). 2.12. Statistics All data shown are described as mean ± standard deviation (SD). Statistical significance was assessed using 2-tailed Student's t-test. P-values <0.05 were considered statistically significant. 3. Results 3.1. Thymic B cells co-locate with regulatory T cells in the thymus medulla Consistent with previously published data [19,32,33], thymic B cells were readily detected in the medulla and cortico-medullary area of the thymus (Fig. 1A). More specifically, we noted that thymic Treg cells were not only localized to the same thymic medullary area (Fig. 1A), but, in addition, there was co-localization of thymic B cells and Treg cells (Fig. 1B). 3.2. Thymic B cells display a highly activated phenotype Thymic DCs and thymic epithelial cells function as efficient APCs and have been shown to contribute to thymic Treg cell

Please cite this article in press as: F.-T. Lu, et al., Thymic B cells promote thymus-derived regulatory T cell development and proliferation, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.05.008

4

F.-T. Lu et al. / Journal of Autoimmunity xxx (2015) 1e11

Fig. 1. Microscopic analysis of thymic B cells and Treg cells. (A) Representative immunohistochemical staining patterns of thymic tissues from FoxP3-GFP mice using mAbs against CD19 (B cells, left column) and GFP (Tregs, right column) shown on the top panel (100; scale bars, 100 mm) and lower panel (400; scale bars, 25 mm). (B) Representative immunofluorescence staining patterns of frozen sections of thymic tissues from FoxP3-GFP mice on the top panel (100; scale bars, 100 mm) and bottom panel (200; scale bars, 50 mm), stained with fluorescence-labeled secondary antibody to anti-GFP (left column, Tregs, green) and anti-CD19 (middle column, B cells, red) and the merged images (right column) with white arrowheads indicating the anatomic co-localization of Treg cells and B cells within the thymus.

development. TCR-MHC II, CD40-CD40L and CD28-CD80/CD86 interactions are known to be involved in this process [12e18,34]. To determine if thymic B cells express these markers, we assessed their expression and that of other co-stimulatory molecules. Splenic B cells were examined in parallel for purposes of comparison. Strikingly, thymic B cells expressed higher levels of MHC II, CD80 and CD86 compared with splenic B cells (Fig. 2A, B), suggesting that thymic B cells express the requisite molecules to efficiently present antigen perhaps even more efficiently than splenic B cells. Thymic B cells expressed CD40 at levels comparable to levels expressed by splenic B cells, but neither of them expressed CD40L (data not shown). In addition, thymic B cells expressed increased relative levels of the activation markers CD44 and CD69 (Fig. 2A, B) compared to splenic B cells. Finally, thymic B cells expressed 2-fold

higher levels of Ki67 and incorporated four times more Brdu than splenic B cells in wild type mice (Fig. 2C, D). These data, in concert, suggest that thymic B cells express the requisite molecules for functional antigen presentation and reflect a constitutively highly activated phenotype. 3.3. Thymic B cells are required for normal numbers of regulatory T cells in the thymus In efforts to define whether the co-localization of Treg cells requires the presence of thymic B cells, we examined tissues from mMT FoxP3-GFP mice. This strain lacks B lymphocytes and GFPtagged Treg cells are readily identified based on GFP expression. As expected, there were no detectable CD19þ B220þ cells in thymic

Please cite this article in press as: F.-T. Lu, et al., Thymic B cells promote thymus-derived regulatory T cell development and proliferation, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.05.008

F.-T. Lu et al. / Journal of Autoimmunity xxx (2015) 1e11

5

(A)

Cell count

Sp B Thy B

CD86

MHCII

CD44

(B)

4

80 40

2

Sp B

250

Thy B

40.0

Empty

16.9

500

50

***

90 60

20

Sp B Thy B

12 8

30

4

0

0

(D) 15

***

12

40 30

***

16

120

750

0

0

0

(C))

120

20

***

Brdu+ (%)

6

160

150

***

CD44 RFI

CD86 RFI

CD80 RFI

8

1000

***

Ki67+ (%)

200

***

MHCII RFI

10

CD69

CD69 RFI

CD80

9 6

10

3

0

0

Ki67 Fig. 2. Comparative phenotypic analysis of thymic and splenic B cells. (A) Representative flow cytometric profiles of CD80 (n ¼ 8), CD86 (n ¼ 4), MHC II (n ¼ 4), CD44 (n ¼ 8) and CD69 (n ¼ 8) expression on the gated population of B220þ CD19þ (B cells) in thymocytes (Thy) and splenic cells (Sp) from B6 mice. (B) The expression levels of surface molecules presented in (A) are illustrated as the mean ± SD of the relative fluorescence intensity (RFI) for each group. RFI was calculated by dividing the mean fluorescence intensity (MFI) of surface molecules by the MFI of isotype control. The values for thymic B cells are shown in black and the values for splenic B cells are shown in white. (C) The flow cytometric profiles of Ki67 expression by the CD19þ gated population of thymic and splenic B cells (n ¼ 3) (left two panels) along with the mean ± SD of the frequencies (%) of Ki67þ thymic and splenic B cells are shown in right panel. (D) The mean ± SD (n ¼ 4) of the percentage of thymic and splenic B cells that incorporated BrdU are shown. ***p < 0.001.

tissues from mMT FoxP3-GFP mice (data not shown). However, the absence of CD19þ B220þ cells did not lead to any significant change in the total number of thymocytes in mMT FoxP3-GFP compared with Foxp3-GFP mice (Fig. 3A). Furthermore, the absence of thymic B cells did not lead to any changes in the number of either CD4 or CD8 single positive thymocytes (Fig. 3B). However, the frequency and number of thymic Treg cells were significantly decreased in mMT FoxP3-GFP mice compared with FoxP3-GFP mice (Fig. 3C, D). Taken together, these results demonstrate that the absence of thymic B cells correlates with a reduction of Treg cells in the thymus.

Treg cells induced by co-culture with thymic B cells suppressed the proliferation of CD4þ GFP T cells in vitro (Fig. 4C). Since thymic DCs also contribute to the development of thymusderived Treg cells [12e16], we compared the number of Treg cells induced by thymic B cells with thymic DCs under the same culture conditions. We demonstrated that the potential capacity of promoting Treg cells was similar in these two cell populations (Supplementary Fig. 1C, D). Moreover, CD4þ GFPþ Treg cells induced by both of these cell lineages in vitro had an equivalent suppressive capacity against CD4þ GFP T cell proliferation (Supplementary Fig. 1E).

3.4. Thymic B cells promote the development of regulatory T cells in vitro

3.5. The ability of thymic B cells to contribute to the development of regulatory T cells is dependent on cellecell contact

An in vitro functional assay was performed in attempts to define the mechanisms for the reduced number of Treg cells in the thymus of B cell deficient mice. Thus, CD4þ CD8 CD25 GFP thymic CD4 SP T cells (Supplementary Fig. 1B) from FoxP3-GFP mice were cocultured with or without thymic B cells (Supplementary Fig. 1A) in the presence of a-CD3, a-CD28 and rIL-2; GFPþ Treg cells were analyzed by flow cytometry 5 days later. Consistent with the in vivo data, very few Treg cells developed in co-cultures lacking thymic B cells. However, there was a highly significant emergence of FoxP3GFP positive cells when thymic B cells were included in the cocultures (Fig. 4A, B). Upon further analysis, the resulting Treg cells that emerged were shown to be functional. Thus, the resulting Treg cells were sorted as CD4þ GFPþ cells and co-cultured with dye labeled splenic CD4þ GFP responder T cells for 3 days. CD4þ GFPþ

TGF-b and retinoic acid (RA) have been shown to be critical for the generation of Treg cells [35e37]. Thus, we determined whether thymic B cells affect Treg cell number by secreting these cytokines. First, thymic B cells were co-cultured in the presence of a-CD3/ CD28 þ rIL-2 with thymic CD4 SP T cells from dnTGFbRII mice that have specific impairment of TGFb signaling in T cells [38]. Results of this study demonstrate that thymic B cells were able to promote the development of Treg cells (Fig. 5A), suggesting that there was no requirement for TGFb signaling for the in vitro development of Treg cells. Second, we collected supernatant fluids from thymic B cells co-cultured with thymic CD4 SP T cells in the presence of a-CD3/ CD28 þ rIL-2. We incubated such supernatant fluids with cultures of freshly isolated thymic CD4 SP T cells. However, the addition of such supernatant fluids did not promote the generation of Treg cells

Please cite this article in press as: F.-T. Lu, et al., Thymic B cells promote thymus-derived regulatory T cell development and proliferation, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.05.008

F.-T. Lu et al. / Journal of Autoimmunity xxx (2015) 1e11

(A)

(B) CD4 SP cells number ( 106)

Thymocytes number ( 107)

12 9 6 3

FoxP3-GFP

FoxP3-GFP

12 9 6 3

3 2 1 0

0

0

(C)

4

15

15

CD8 SP cells number ( 106)

6

MT FoxP3-GFP

3.67

2.61

CD4 FoxP3-GFP 5 4 3 2 1 0

5

*** Tregs number in thymus ( 105)

Tregs (%) in CD4 SP cells

(D)

*

μMT FoxP3-GFP

4 3 2 1 0

Fig. 3. Frequencies of Treg cells in thymocytes from FoxP3-GFP mice and B cell-deficient FoxP3-GFP mice. Thymocytes were isolated from 8- to 10-week-old FoxP3-GFP mice (n ¼ 8, white bars) and mMT FoxP3-GFP (B cell-deficient FoxP3-GFP) mice (n ¼ 6, black bars), stained with antibodies to CD8, CD4 and TCRb, and analyzed by flow cytometry. The absolute number of (A) thymocytes (B) CD4 single positive (CD4þ CD8) and CD8 single positive (CD4 CD8þ) thymocytes. (C) Representative flow cytometric profiles of CD4þGFPþ Treg cells in thymic CD4þ CD8 TCRbþ subset. (D) The frequencies (%) and absolute number of thymic Treg cells. Data are illustrated as the mean ± SD. *p < 0.05, ***p < 0.001.

(Fig. 5B). In addition, when thymic B cells were cultured in the top chamber with thymic CD4 SP T cells in the bottom chamber along with a-CD3/CD28 þ rIL-2 using a transwell system, the absolute number of Treg cells in the bottom chamber was decreased significantly compared with co-cultures allowing direct cellecell contact (Fig. 5C). These data indicate that thymic B cell cytokines alone are not optimal and/or sufficient to promote Treg development or maintenance in vitro. Thus, we speculate that the optimal induction of Treg cells by thymic B cells requires cellecell contact. To identify cell surface molecules that are involved in the interactions between thymic B cells and thymic CD4 SP T cells, we utilized in vitro Treg cell induction assays in combination with various blocking Abs, including defined optimal concentrations of antibodies targeting MHC II or CD40 molecules in thymic B cell/ thymic CD4 SP T cell co-cultures. Data obtained clearly suggest that the addition of anti-MHC II or CD40 antibodies significantly reduced Treg cell induction (Fig. 5D, E). We next examined whether CD80/CD86 co-stimulation provided by thymic B cells also participates in Treg cell induction. Once again, the addition of previously defined optimal concentrations of anti-CD80, anti-CD86, or both in co-cultures of thymic B cells and thymic CD4 SP T cells, had a significant impact on the induction of Treg cells (Fig. 5F). Taken together, the promotion of Treg cell development in vitro appears to require an interaction between thymic B cells and thymic CD4 SP T

cells that is regulated by MHC II contact and CD40, CD80/CD86 costimulation. 3.6. Thymic B cells promote the development of regulatory T cells from CD4 single positive thymocytes to pre-Treg cells only The development of thymic Treg cells occurs in two stages. CD4 single positive thymocytes mature into pre-Treg cells and pre-Treg cells then develop into functional Foxp3þ Treg cells [9]. We examined the effects of thymic B cells on each of these transitions. We first sorted thymic pre-Treg cells (Fig. 6A, Supplementary Fig. 2A), co-cultured them with thymic B cells in the presence of rIL-2 in vitro, and analyzed the generation of thymic Treg cells. The frequencies of Foxp3-GFPþ Treg cells were similar in the presence or absence of thymic B cells (Fig. 6B). These data suggest that the co-culture of pre-Treg cells with thymic B cells does not lead to further development of thymic Treg cells. In contrast, co-cultures of thymic B cells and thymic CD4 SP T cells in the presence of a-CD3/ CD28 þ rIL-2 for 18 h in vitro led to a significantly higher percentage of CD4þ CD25þ Foxp3 pre-Treg cells as compared with thymic CD4 SP T cells cultured alone (Fig. 6C, D). Taken together, these results indicate that thymic B cells promote the development of thymic CD4 SP T cells to pre-Treg cells but not the maturation of pre-Treg cells into Foxp3þ Treg cells.

Please cite this article in press as: F.-T. Lu, et al., Thymic B cells promote thymus-derived regulatory T cell development and proliferation, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.05.008

F.-T. Lu et al. / Journal of Autoimmunity xxx (2015) 1e11

-Thy B

+Thy B

(B)

Thy CD4SP T

1.0

24.7

Tregs (%)

FoxP3-GFP

30

Thy CD4SP T + Thy B 6000

*** Tregs number

(A)

7

24 18 12 6 0

4800

***

3600 2400 100 0

CD4 (C) Tregs: Tresp 0:1

1/4:1

1/2:1

37.1

27.0

22.1

Events

78.9

1/8:1

CellTrace Violet Fig. 4. Thymic B cells induce thymic Treg cell generation in vitro. 3  104 cell sorter enriched population of CD4þ CD8 CD25 GFP thymic CD4 SP T cells (Thy CD4SP T) from FoxP3GFP mice were co-cultured with (þThy B, black bars) or without (- Thy B, white bars) 1.5  104 thymic B cells for 5 days to induce Treg cells. (A) Flow cytometric profiles of GFP expression by thymic CD4 SP T cells co-cultured with or without thymic B cells. (B) The frequencies (%) and absolute number of Treg cells induced in vitro in the assays are displayed (A). The data shown are mean ± SD of four separate experiments. (C) Representative profiles of the suppression assay of thymic B cells induced-Treg cells (Thy B-Tregs). Thymic B cell-induced Treg cells were co-cultured with 5  104 CellTrace Violet labeled splenic CD4þ GFP responder cells (Tresp) from FoxP3-GFP mice at indicated ratios in the presence of 1  105 Mitomycin C-treated splenic CD4 negative supporter cells for 3 days, then responder cells proliferation was assessed by CellTrace Violet dilution. Data shown are representative of three independent experiments. ***p < 0.001.

3.7. Different roles of MHC II and CD40/CD80/86 of thymic B cells on thymic regulatory T cell proliferation As noted in Fig. 7A, the levels of Ki67 expressed by thymic Treg cells from mMT FoxP3-GFP mice were significantly lower than thymic Treg cells from FoxP3-GFP mice (Fig. 7A, B) consistent with a requirement for the presence of thymic B cells. The use of CellTrace Violet-labeling technique in such in vitro co-cultures incubated for 3 days confirmed our observation (Fig. 7C, D). Similar to the studies outlined above, we used mAbs to block MHC II and CD40/CD80/ CD86 co-stimulatory molecules in such co-culture assays. However, while the addition of anti-MHC II reduced the frequencies of proliferating Treg cells (Fig. 7E), the addition of anti-CD40 or antiCD80 or anti-CD86 molecules to such co-cultures had no detectable effect on Treg cell proliferation (Fig. 7F, G). Thus, we conclude that the proliferation of thymic Treg cells that is promoted by thymic B cells is dependent on MHC II contact, but not costimulation by CD40/CD80/CD86 molecules. 4. Discussion The development of thymic Treg cells occurs in two sequential steps. In the first step, CD4 SP thymocytes undergo TCR-MHC II and CD28-CD80/CD86 dependent selection that give rise to the generation of CD4þ CD25þ GITRhi FoxP3 thymic pre-Treg cells. A subsequent IL-2-dependent step results in the conversion of thymic pre-Treg cells into mature FoxP3 positive Treg cells. Several types of APCs including thymic DCs and thymic epithelial cells contribute to thymic Treg cell development [12e18]. Hitherto, the role for other thymic APCs, and, in particular, thymic B cells, in the development of thymic Treg cells has not been clearly defined. These issues are critical to autoimmunity [39e41]. In the periphery, B cell deficiency or depletion leads to a marked decrease in both the frequency and number of peripheral Treg cells as exemplified in both the mMT or B cell depleting anti-CD20 treated mice [21,22,42]. Two hypotheses have been forwarded to

explain the mechanisms that are required for B cell dependent, Treg cells maintenance in the periphery. First, B cells are antigen presenting cells and therefore promote the generation of peripheral Treg cells [22,23]. Second, B cells regulate peripheral Treg cells through production of several factors, including IL-10, TGF-b and the GITR ligand [21,24,43e46]. Similar to DCs and medullary epithelial cells in the thymus, thymic B cells constitute only 0.1e0.3% of total thymocytes [19,33] and are primarily located in the thymus medulla and corticomedullary junction [19,32,33]. Several groups have reported that both fetal liver cells and bone marrow cells have the capacity to reconstitute thymic B cells. However, unlike splenic B cells, most thymic B cells develop in the thymus from B cell progenitors [19,32,33,47]. Thymic B cells present peptides derived from autoantigens captured by their B-cell receptors (BCRs) and potentially eliminate auto-reactive thymocytes [19]. This view is exemplified by the finding that MOG35-45 peptide presenting thymic B cells have been shown to induce the deletion of MOG-specific T cells in the thymus of BMOG/2D2 mice [20]. These data demonstrate that thymic B cells contribute to the negative selection of self-reactive thymocytes. We demonstrate herein that thymic B cells promote thymic Treg cell development. In the absence of thymic B cells, thymic Treg cells are significantly decreased in frequency and absolute number. The in vitro culture system used herein demonstrate that thymic B cells require cellecell MHC II contact and CD40/CD80/CD86 costimulation for thymic Treg cell development. Furthermore, thymic B cells contribute to the development of thymic Treg cells from CD4 single positive thymocytes to pre-Treg cells, but have no action on the further development of pre-Treg cells to Foxp3þ Treg cells. Further, thymic B cells promote the proliferation of thymic Treg cells in vivo and in vitro. However, different from thymic Treg cell development, this process is independent on CD40/CD80/CD86 molecules. Thus, signals required for thymic Treg cell development may be distinct from those required for thymic Treg cell proliferation. It is also important to note that unlike thymic Treg cell

Please cite this article in press as: F.-T. Lu, et al., Thymic B cells promote thymus-derived regulatory T cell development and proliferation, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.05.008

8

F.-T. Lu et al. / Journal of Autoimmunity xxx (2015) 1e11

(B)

(A) dnTGF RII mice: CD4SP T

Thy Thy CD4SP T + Thy B

***

Tregs number

Tregs number

5000

4000

3000 50 25 0

30 20 10

(D) Untreated Transwell

***

8000 6000 4000 2000

4000

Tregs number

10000

Tregs number

-Sup +Sup

40

0

(C)

IgG

***

-MHCII

3200 2400 1600 800 0

0

(F)

**

5000

IgM

4000

**

3000

-CD40

3000 2000 1000

Tregs number

(E) Tregs number

50

**

2400

***

1800

IgG -CD80 -CD86 -CD80 + -CD86

1200 600 0

0

Fig. 5. Thymic B cells promote thymic Treg cell generation in a cellecell contact manner. (A) The absolute number of Treg cells induced by thymic B cells. 3  104 CD4þ CD8 CD25 GFP thymic CD4 SP T cells from dnTGFbRII FoxP3-GFP mice were co-cultured with (black bar) or without (white bar) 1.5  104 thymic B cells as described in Fig. 4A. (B) The absolute number of Treg cells induced by supernatant fluids, which were collected from co-cultures of thymic B cells and thymic CD4 SP T cells. “ Sup” means no supernatant in cultures, “þ Sup” means addition of supernatant in cultures. (C) 6  104 thymic CD4 SP T cells were co-cultured with 3  104 thymic B cells for 5 days either in direct contact (Untreated, white bar) or separated by a transwell membrane (Transwell, black bar), and the number of Treg cells induced by thymic B cells were determined by flow cytometry. (DeF) 3  104 thymic CD4 SP T cells from FoxP3-GFP mice were co-cultured with 1.5  104 thymic B cells for 5 days to induce Treg cells, and an optimal dilution of blocking antibodies or their relevant isotype control antibodies were added to the co-cultures. The number of Treg cells were determined by flow cytometry. (DeE) The number of Treg cells induced by thymic B cells in the presence of anti-rat IgG2b,k isotype control (IgG, white bar) or anti-MHC II blocking antibodies (a-MHC II, black bar) (D) and Armenian Hamster IgM isotype control (IgM, white bar) or anti-CD40 blocking antibodies (a-CD40, black bar) (E). (F) The number of Treg cells induced by thymic B cells in combination with anti-CD80 (aCD80, light grey bar), anti-CD86 (a-CD86, dark grey bar) or both blocking antibodies (black bar). IgG isotype controls (white bar) of both a-CD80 and a-CD86 were added. All data shown are mean ± SD of cultures performed in triplicate. **p < 0.01, ***p < 0.001.

(B)

CD4

93.7

Pre-Treg 2.82

CD25 -Thy B

+Thy B

0.301

98.8

CD25

0.701

1.31

88.2

43.2

+Thy B

44.3

CD4 (D) 15 Pre-Tregs (%)

(C)

FoxP3-GFP

3.14

-Thy B

FoxP3-GFP

CD8

8.03

FoxP3-GFP

(A)

10.3

12

Thy CD4SP T

***

Thy CD4SP T + Thy B

9 6 3 0

Fig. 6. Thymic B cells promote the generation of pre-Treg cells. (A) Representative dot plots shown represent a population of CD4þ CD8 gated thymocytes from FoxP3-GFP mice (left), then Treg cell precursors (pre-Treg cells) were gated on CD25þ GFP (right). (B) Representative flow cytometric profiles of GFP expression by pre-Treg cells from FoxP3-GFP mice co-cultured with (þThy B) or without ( Thy B) thymic B cells for 24 h. (C) Representative flow cytometric profiles of GFP and CD25 expression by thymic CD4 SP T cells cocultured with or without thymic B cells for 18 h as described in Fig. 4A. (D) The mean ± SD of the frequencies of CD25þ GFP pre-Treg cells induced in vitro in the assays are displayed (C). All data shown are representative of three independent experiments. ***p < 0.001.

Please cite this article in press as: F.-T. Lu, et al., Thymic B cells promote thymus-derived regulatory T cell development and proliferation, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.05.008

F.-T. Lu et al. / Journal of Autoimmunity xxx (2015) 1e11

FoxP3-GFP

MT FoxP3-GFP 58.7

CD4

72.3

(B)

100

Ki67+ (%) in thymus Tregs

(A)

FoxP3-GFP MT FoxP3-GFP

*

80 60 40 20 0

Ki67 +Thy B

17.0

65.1

(D)

100

Tregs proliferation (%)

-Thy B

Events

(C)

9

Thy Treg Thy Treg + Thy B

***

80 60 40 20 0

CellTrace Violet

80 60 40 20 0

***

IgG -MHCII

(F) 100 80 60 40 20 0

IgM -CD40

(G) 100

Tregs proliferation (%)

Tregs proliferation (%)

100

Tregs proliferation (%)

(E)

IgG -CD80 -CD86

80 60 40 20 0

Fig. 7. Thymic B cells promote the proliferation of thymic Treg cells both in vivo and in vitro. (A) The flow cytometric profiles of Ki67 expression by the CD4þ CD8 TCRbþ GFPþ gated population of thymic Treg cells from 8- to 10-week-old FoxP3-GFP (n ¼ 4) and mMT FoxP3-GFP mice (n ¼ 5). (B) Summary graph of data shown in (A) are illustrated as the mean ± SD of the frequencies of Ki67þ Treg cells. (C) Representative profiles of thymic Treg cell proliferation in vitro. CellTrace Violet-labeled thymic Treg cells from FoxP3-GFP mice were co-cultured with (þThy B) or without ( Thy B) thymic B cells for 3 days. Treg cells proliferation was assessed by CellTrace Violet dilution. (D) Summary graph of data shown in (C). The data shown are mean ± SD of four separate experiments. (E) The frequencies (%) of thymic Treg cells that underwent proliferation promoted by thymic B cells in the presence of rat IgG2b, k isotype control (white bar) or a-MHC II blocking antibodies (black bar). (F) The frequencies (%) of thymic Treg cells that underwent proliferation promoted by thymic B cells in the presence of Armenian Hamster IgM isotype control (white bar) or a-CD40 blocking antibodies (black bar). (G) The frequencies (%) of thymic Treg cells that underwent proliferation promoted by thymic B cells in combination with a-CD80 (grey bar) or a-CD86 (black bar) blocking antibodies. IgG indicates the isotype control (white bar) for both a-CD80 and a-CD86. Data (E and G) shown are mean ± SD of cultures performed in triplicate. *p < 0.05, ***p < 0.001.

development, the maintenance of thymic B cells requires CD40/ CD40L but not MHC class II interaction [48]. Hence, signals required for thymic Treg cell development are distinct from those required for thymic B cell maintenance. Previous data has indicated that the percentage and absolute number of Treg cells in the periphery are significantly reduced in mMT mice, which, for example, subsequently impacts the ability to resolve EAE [21,42,49,50]. Our data also indicate that B cell deficiency leads to substantial decreases in the number of both splenic Helios Treg cells (periphery-derived Treg cells, p-Treg cells) and Heliosþ Treg cells (thymus-derived Treg cells, t-Treg cells) (data not shown). Thus, the decline of both p-Treg and t-Treg cells could account for the reduction of total Treg cells in the periphery of mMT mice. These data also reinforce the multi-complex roles of regulation required for immune tolerance [51e54]. We have relied on an in vitro culture system to explore the role of thymic B cells. Interestingly, and contrary to our data, it has previously been reported that the capacity of thymic B cells to induce the development of Treg cells is negligible in vitro [13]. We speculate this may due to the different culture systems being utilized. In the previous report, thymic B cells were co-cultured with

CD4þ CD8 CD25 thymocytes in the presence of IL-7. However, IL7 is not essential for the functional maturation of thymic Treg cells [9,55]. We reasoned that culturing T cells in the presence of a-CD3 and a-CD28 þ rIL-2 may be a more relevant and physiologically optimal culture system to study the development of thymic Treg cells in vitro. Compared with splenic B cells, thymic B cells are constitutively activated associated with the expression of relatively higher levels of MHC II and co-stimulatory molecules [19,56,57]. The activated state of thymic B cells correlates with their contribution to negative selection. We report herein that this activated state of thymic B cells contribute to the size of the thymic Treg cell compartment. These findings are consistent with a recent study report that documents the role of thymic B cells in the generation of thymic Treg cells via MHC II contact [58]. Similar to thymic DCs and medullary epithelial cells, interactions between TCR-MHC II, CD40-CD40L and CD28CD80/CD86 function in the crosstalk between thymic B cells and CD4 single positive thymocytes and are essential for the development of Treg cells. Clearly, we cannot exclude the role of other molecules that maybe potentially involved in Treg cell development. Nonetheless, our data demonstrate that thymic B cells, in

Please cite this article in press as: F.-T. Lu, et al., Thymic B cells promote thymus-derived regulatory T cell development and proliferation, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.05.008

10

F.-T. Lu et al. / Journal of Autoimmunity xxx (2015) 1e11

addition to thymic epithelial cells and DCs, contribute to thymusderived Treg cell development. Thymic B cells can induce thymic Treg cells as well as promote the proliferation of thymic Treg cells and thus our studies emphasize a new and critical role of thymic B cells on T cell development within the thymus. Financial support Financial support provided by the National Basic Research Program of China (973 Program-2013CB944900, 2010CB945300), the National Natural Science Foundation of China (81130058, 81430034) and Research Fund for the Doctoral Program of Higher Education of China (RFDP 20133402110015). Conflict of interest The authors declare no competing financial interests. Acknowledgment We thank Dr. Yu Zhang (Peking University, Beijing, China) for

mMT mice. Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.jaut.2015.05.008. References [1] S. Gerondakis, T.S. Fulford, N.L. Messina, R.J. Grumont, NF-kappaB control of T cell development, Nat. Immunol. 15 (2014) 15e25. [2] K. Alexandropoulos, N.M. Danzl, Thymic epithelial cells: antigen presenting cells that regulate T cell repertoire and tolerance development, Immunol. Res. 54 (2012) 177e190. [3] Y.J. Liu, A unified theory of central tolerance in the thymus, Trends Immunol. 27 (2006) 215e221. [4] N. Ohkura, Y. Kitagawa, S. Sakaguchi, Development and maintenance of regulatory T cells, Immunity 38 (2013) 414e423. [5] J.D. Fontenot, M.A. Gavin, A.Y. Rudensky, Foxp3 programs the development and function of CD4þCD25þ regulatory T cells, Nat. Immunol. 4 (2003) 330e336. [6] S. Sakaguchi, T. Yamaguchi, T. Nomura, M. Ono, Regulatory T cells and immune tolerance, Cell 133 (2008) 775e787. [7] R.S. Wildin, F. Ramsdell, J. Peake, F. Faravelli, J.L. Casanova, N. Buist, et al., Xlinked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy, Nat. Genet. 27 (2001) 18e20. [8] X. Tai, M. Cowan, L. Feigenbaum, A. Singer, CD28 costimulation of developing thymocytes induces Foxp3 expression and regulatory T cell differentiation independently of interleukin 2, Nat. Immunol. 6 (2005) 152e162. [9] C.W. Lio, C.S. Hsieh, A two-step process for thymic regulatory T cell development, Immunity 28 (2008) 100e111. [10] C.W. Lio, L.F. Dodson, C.M. Deppong, C.S. Hsieh, J.M. Green, CD28 facilitates the generation of Foxp3(-) cytokine responsive regulatory T cell precursors, J. Immunol. 184 (2010) 6007e6013. [11] L. Klein, K. Jovanovic, Regulatory T cell lineage commitment in the thymus, Semin. Immunol. 23 (2011) 401e409. [12] N. Watanabe, Y.H. Wang, H.K. Lee, T. Ito, W. Cao, Y.J. Liu, Hassall's corpuscles instruct dendritic cells to induce CD4þCD25þ regulatory T cells in human thymus, Nature 436 (2005) 1181e1185. [13] A.I. Proietto, S. van Dommelen, P. Zhou, A. Rizzitelli, A. D'Amico, R.J. Steptoe, et al., Dendritic cells in the thymus contribute to T-regulatory cell induction, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 19869e19874. [14] E. Martin-Gayo, E. Sierra-Filardi, A.L. Corbi, M.L. Toribio, Plasmacytoid dendritic cells resident in human thymus drive natural Treg cell development, Blood 115 (2010) 5366e5375. [15] S. Hanabuchi, T. Ito, W.R. Park, N. Watanabe, J.L. Shaw, E. Roman, et al., Thymic stromal lymphopoietin-activated plasmacytoid dendritic cells induce the generation of FOXP3þ regulatory T cells in human thymus, J. Immunol. 184 (2010) 2999e3007. [16] J.M. Coquet, J.C. Ribot, N. Babala, S. Middendorp, G. van der Horst, Y. Xiao, et al., Epithelial and dendritic cells in the thymic medulla promote CD4þFoxp3þ regulatory T cell development via the CD27-CD70 pathway, J. Exp. Med. 210 (2013) 715e728.

[17] K. Aschenbrenner, L.M. D'Cruz, E.H. Vollmann, M. Hinterberger, J. Emmerich, L.K. Swee, et al., Selection of Foxp3þ regulatory T cells specific for self antigen expressed and presented by Aireþ medullary thymic epithelial cells, Nat. Immunol. 8 (2007) 351e358. [18] J.E. Cowan, S.M. Parnell, K. Nakamura, J.H. Caamano, P.J. Lane, E.J. Jenkinson, et al., The thymic medulla is required for Foxp3þ regulatory but not conventional CD4þ thymocyte development, J. Exp. Med. 210 (2013) 675e681. [19] J. Perera, L. Meng, F. Meng, H. Huang, Autoreactive thymic B cells are efficient antigen-presenting cells of cognate self-antigens for T cell negative selection, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 17011e17016. [20] F. Frommer, A. Waisman, B cells participate in thymic negative selection of murine auto-reactive CD4þ T cells, PLoS One 5 (2010) e15372. [21] A. Ray, S. Basu, C.B. Williams, N.H. Salzman, B.N. Dittel, A novel IL-10independent regulatory role for B cells in suppressing autoimmunity by maintenance of regulatory T cells via GITR ligand, J. Immunol. 188 (2012) 3188e3198. [22] S. Morlacchi, C. Soldani, A. Viola, A. Sarukhan, Self-antigen presentation by mouse B cells results in regulatory T-cell induction rather than anergy or clonal deletion, Blood 118 (2011) 984e991. [23] J. Zheng, Y. Liu, Y.L. Lau, W. Tu, CD40-activated B cells are more potent than immature dendritic cells to induce and expand CD4(þ) regulatory T cells, Cell. Mol. Immunol. 7 (2010) 44e50. [24] B. Wei, P. Velazquez, O. Turovskaya, K. Spricher, R. Aranda, M. Kronenberg, et al., Mesenteric B cells centrally inhibit CD4þ T cell colitis through interaction with regulatory T cell subsets, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 2010e2015. [25] J.D. Fontenot, J.P. Rasmussen, L.M. Williams, J.L. Dooley, A.G. Farr, A.Y. Rudensky, Regulatory T cell lineage specification by the forkhead transcription factor foxp3, Immunity 22 (2005) 329e341. [26] D. Kitamura, J. Roes, R. Kuhn, K.A. Rajewsky, B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene, Nature 350 (1991) 423e426. [27] L. Gorelik, R.A. Flavell, Abrogation of TGFbeta signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease, Immunity 12 (2000) 171e181. [28] T.S. Lim, J.K. Goh, A. Mortellaro, C.T. Lim, G.J. Hammerling, P. Ricciardi-Castagnoli, CD80 and CD86 differentially regulate mechanical interactions of Tcells with antigen-presenting dendritic cells and B-cells, PLoS One 7 (2012) e45185. [29] Y. Zheng, S. Josefowicz, A. Chaudhry, X.P. Peng, K. Forbush, A.Y. Rudensky, Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate, Nature 463 (2010) 808e812. [30] A.M. Kruisbeek, E. Shevach, A.M. Thornton, Proliferative assays for T cell function, Curr. Protoc. Immunol. (2004) (Chapter 3):Unit 3.12.1-3.12.20. [31] Z.X. Lian, K. Kikuchi, G.X. Yang, A.A. Ansari, S. Ikehara, M.E. Gershwin, Expansion of bone marrow IFN-alpha-producing dendritic cells in New Zealand Black (NZB) mice: high level expression of TLR9 and secretion of IFNalpha in NZB bone marrow, J. Immunol. 173 (2004) 5283e5289. [32] C. Joao, B.M. Ogle, C. Gay-Rabinstein, J.L. Platt, M. Cascalho, B cell-dependent TCR diversification, J. Immunol. 172 (2004) 4709e4716. [33] K. Akashi, L.I. Richie, T. Miyamoto, W.H. Carr, I.L. Weissman, B lymphopoiesis in the thymus, J. Immunol. 164 (2000) 5221e5226. [34] P.J. Spence, E.A. Green, Foxp3þ regulatory T cells promiscuously accept thymic signals critical for their development, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 973e978. [35] W. Chen, W. Jin, N. Hardegen, K.J. Lei, L. Li, N. Marinos, et al., Conversion of peripheral CD4þCD25- naive T cells to CD4þCD25þ regulatory T cells by TGFbeta induction of transcription factor Foxp3, J. Exp. Med. 198 (2003) 1875e1886. [36] D. Mucida, Y. Park, G. Kim, O. Turovskaya, I. Scott, M. Kronenberg, et al., Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid, Science 317 (2007) 256e260. [37] J.A. Hill, J.A. Hall, C.M. Sun, Q. Cai, N. Ghyselinck, P. Chambon, et al., Retinoic acid enhances Foxp3 induction indirectly by relieving inhibition from CD4þCD44hi Cells, Immunity 29 (2008) 758e770. [38] S. Oertelt, Z.X. Lian, C.M. Cheng, Y.H. Chuang, K.A. Padgett, X.S. He, et al., Antimitochondrial antibodies and primary biliary cirrhosis in TGF-beta receptor II dominant-negative mice, J. Immunol. 177 (2006) 1655e1660. [39] K. Vrolix, J. Fraussen, M. Losen, J. Stevens, K. Lazaridis, P.C. Molenaar, et al., Clonal heterogeneity of thymic B cells from early-onset myasthenia gravis patients with antibodies against the acetylcholine receptor, J. Autoimmun. 52 (2014) 101e112. [40] P. Cufi, N. Dragin, N. Ruhlmann, J.M. Weiss, E. Fadel, A. Serraf, et al., Central role of interferon-beta in thymic events leading to myasthenia gravis, J. Autoimmun. 52 (2014) 44e52. [41] G. Cambridge, H.C. Perry, L. Nogueira, G. Serre, H.M. Parsons, I. De La Torre, et al., The effect of B-cell depletion therapy on serological evidence of B-cell and plasmablast activation in patients with rheumatoid arthritis over multiple cycles of rituximab treatment, J. Autoimmun. 50 (2014) 67e76. [42] J.B. Sun, C.F. Flach, C. Czerkinsky, J. Holmgren, B lymphocytes promote expansion of regulatory T cells in oral tolerance: powerful induction by antigen coupled to cholera toxin B subunit, J. Immunol. 181 (2008) 8278e8287. [43] S. Shah, L. Qiao, Resting B cells expand a CD4þCD25þFoxp3þ Treg population via TGF-beta3, Eur. J. Immunol. 38 (2008) 2488e2498.

Please cite this article in press as: F.-T. Lu, et al., Thymic B cells promote thymus-derived regulatory T cell development and proliferation, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.05.008

F.-T. Lu et al. / Journal of Autoimmunity xxx (2015) 1e11 [44] N.A. Carter, R. Vasconcellos, E.C. Rosser, C. Tulone, A. Munoz-Suano, M. Kamanaka, et al., Mice lacking endogenous IL-10-producing regulatory B cells develop exacerbated disease and present with an increased frequency of Th1/Th17 but a decrease in regulatory T cells, J. Immunol. 186 (2011) 5569e5579. [45] J.B. Sun, C. Czerkinsky, J. Holmgren, B lymphocytes treated in vitro with antigen coupled to cholera toxin B subunit induce antigen-specific Foxp3(þ) regulatory T cells and protect against experimental autoimmune encephalomyelitis, J. Immunol. 188 (2012) 1686e1697. [46] M. Yang, K. Rui, S. Wang, L. Lu, Regulatory B cells in autoimmune diseases, Cell. Mol. Immunol. 10 (2013) 122e132. [47] Y. Hashimoto, E. Montecino-Rodriguez, H. Leathers, R.P. Stephan, K. Dorshkind, B-cell development in the thymus is limited by inhibitory signals from the thymic microenvironment, Blood 100 (2002) 3504e3511. [48] C. Fujihara, J.A. Williams, M. Watanabe, H. Jeon, S.O. Sharrow, R.J. Hodes, T cell-b cell thymic cross-talk: maintenance and function of thymic B cells requires cognate CD40-CD40 ligand interaction, J. Immunol. 193 (2014) 5534e5544. [49] A. Suto, H. Nakajima, K. Ikeda, S. Kubo, T. Nakayama, M. Taniguchi, et al., CD4(þ)CD25(þ) T-cell development is regulated by at least 2 distinct mechanisms, Blood 99 (2002) 555e560. [50] S. Fillatreau, C.H. Sweenie, M.J. McGeachy, D. Gray, S.M. Anderton, B cells regulate autoimmunity by provision of IL-10, Nat. Immunol. 3 (2002) 944e950.

11

[51] S. Sattler, G.S. Ling, D. Xu, L. Hussaarts, A. Romaine, H. Zhao, et al., IL-10producing regulatory B cells induced by IL-33 (Breg(IL-33)) effectively attenuate mucosal inflammatory responses in the gut, J. Autoimmun. 50 (2014) 107e122. [52] C. Chang, Autoimmunity: from black water fever to regulatory function, J. Autoimmun. 48e49 (2014) 1e9. [53] W. Huang, K. Kachapati, D. Adams, Y. Wu, P.S. Leung, G.X. Yang, et al., Murine autoimmune cholangitis requires two hits: cytotoxic KLRG1(þ) CD8 effector cells and defective T regulatory cells, J. Autoimmun. 50 (2014) 123e134. [54] L. Muratori, M.S. Longhi, The interplay between regulatory and effector T cells in autoimmune hepatitis: implications for innovative treatment strategies, J. Autoimmun. 46 (2013) 74e80. [55] S. Tani-ichi, A. Shimba, K. Wagatsuma, H. Miyachi, S. Kitano, K. Imai, et al., Interleukin-7 receptor controls development and maturation of late stages of thymocyte subpopulations, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 612e617. [56] L. Guerri, I. Peguillet, Y. Geraldo, S. Nabti, V. Premel, O. Lantz, Analysis of APC types involved in CD4 tolerance and regulatory T cell generation using reaggregated thymic organ cultures, J. Immunol. 190 (2013) 2102e2110. [57] I. Ferrero, F. Anjuere, P. Martin, G. Martinez del Hoyo, M.L. Fraga, N. Wright, et al., Functional and phenotypic analysis of thymic B cells: role in the induction of T cell negative selection, Eur. J. Immunol. 29 (1999) 1598e1609. [58] S.N. Walters, K.E. Webster, S. Daley, S.T. Grey, A role for intrathymic B cells in the generation of natural regulatory T cells, J. Immunol. 193 (2014) 170e176.

Please cite this article in press as: F.-T. Lu, et al., Thymic B cells promote thymus-derived regulatory T cell development and proliferation, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.05.008