Thymic Stromal Lymphopoietin-Activated Plasmacytoid Dendritic Cells Induce the Generation of FOXP3+...

Thymic Stromal Lymphopoietin-Activated PlasmacytoidDendritic Cells Induce the Generation of FOXP3+ Regulatory TCells in Human Thymus

Shino Hanabuchi*, Tomoki Ito†, Woon-Ryon Park*, Norihiko Watanabe‡, Joanne L. Shaw*,Eulogia Roman§, Kazuhiko Arima*, Yui-Hsi Wang¶, Kui Shin Voo*, Wei Cao*, and Yong-JunLiu*

*Department of Immunology and Center for Cancer Immunology Research, University of TexasM. D. Anderson Cancer Center§Department of Pathology and Laboratory Medicine, University of Texas Health Sciences Center,Houston, TX 77030†First Department of Internal Medicine, Kansai Medical University, Osaka‡Department of Gastroenterology and Hepatology, Graduate School of Medicine, KyotoUniversity, Kyoto, Japan¶Division of Allergy and Immunology, University of Cincinnati, Cincinnati Children’s HospitalMedical Center, Cincinnati, OH 45229

AbstractHuman thymus contains major dendritic cell (DC) subsets, myeloid DCs (mDCs), andplasmacytoid DCs (pDCs). We previously showed that mDCs, educated by thymic stromallymphopoietin (TSLP) produced by the epithelial cells of the Hassall’s corpuscles, induceddifferentiation of CD4+CD25− thymocytes into Forkhead Box P3+ (FOXP3+) regulatory T cells(TR) within the medulla of human thymus. In this study, we show that pDCs expressed the TSLPreceptor and IL-7 receptor a complexes upon activation and became responsive to TSLP. TSLP-activated human pDCs secrete macrophage-derived chemokine CCL-22 and thymus- andactivation-regulated chemokine CCL-17 but not Th1- or Th2-polarizing cytokines. TSLP-activatedpDCs induced the generation of FOXP3+ TR from CD4+CD8−CD25− thymocytes, which could bestrongly inhibited by Th1-polarizing cytokine IL-12 or Th2-polarizing cytokine IL-4.Interestingly, the FOXP3+ TR induced by the TSLP-pDCs expressed more IL-10 but less TGF-bthan that induced by the TSLP-mDCs. These data suggest that TSLP expressed by thymicepithelial cells can activate mDCs and pDCs to positively select the FOXP3+ TR with differentcytokine production potential in human thymus. The inability of TSLP to induce DC maturationwithout producing Th1- or Th2-polarizing cytokines may provide a thymic niche for TRdevelopment.

Nondeletional tolerance leading to the generation of CD4+ CD25+FOXP3+ regulatory Tcells (TR) within the thymus represents a key mechanism to establish immunologic self-tolerance (1–6). How some of the medium- to high-affinity self-reactive TR precursors

Copyright © 2010 by The American Association of Immunologists, Inc.Address correspondence and reprint requests to Dr. Yong-Jun Liu, Department of Immunology and Center for Cancer ImmunologyResearch, University of Texas M. D. Anderson Cancer Center, 7455 Fannin Street, Unit 901, Houston, TX [email protected] The authors have no financial conflicts of interest.

NIH Public AccessAuthor ManuscriptJ Immunol. Author manuscript; available in PMC 2012 April 13.

Published in final edited form as:J Immunol. 2010 March 15; 184(6): 2999–3007. doi:10.4049/jimmunol.0804106.

NIH

-PA Author Manuscript

NIH


NIH


escape negative selection and what type of APCs positively selects them to become TRwithin the thymus are unsolved questions.

Pioneering studies using three different models suggested that thymic epithelial cells arecritical for the induction of nondeletional tolerance or the generation of TR. The first modelwas based on xeno- or allotransplantation of embryotic thymic rudiments of chick, quail, ormice into a developing embryo before the colonization of hematopoietic progenitors (7–9).These embryonic thymic rudiments at this stage were called precolonized embryotic thymicrudiments and were believed to give rise to thymic epithelial cells but not hematopoieticcells. Therefore, it has been generally accepted that the resulting chimeric thymus containsepithelial cells of donor origin and hematopoeitic cells of host origin. The results of theseexperiments suggested that thymic epithelial cells, but not hematopoietic cells, areresponsible for the induction of central tolerance, either by deletion or by a nondeletionalmechanism involving the induction of TR (1, 10, 11). A major question is whether theprecolonized thymic rudiments contain hematopoietic progenitors that will give rise tothymic dendritic cells (DCs) in the medulla. Because the window during which precolonizedthymic rudiments can be isolated without colonization of the hematopoietic stem cells isvery narrow and variable in all three species, it is likely that the hematopoietic progenitorswithin these thymic rudiments were contaminated in these early studies. In particular, donorDCs might have been mistakenly classified as reticular cells or stromal cells in these earlierstudies.

The second model involved the transfer of thymic epithelial cells or thymic stroma cellsderived from thymic grafts treated with irradiation or with deoxyguanosine that kills thecycling hematopoietic cells within the thymus (12). However, it is unclear whether suchtreatment would have efficiently eliminated all the noncycling thymic DCs in the medulla.

The third model involved targeted expression of MHC class II (MHC-II) molecules onthymic cortical epithelial cells in class II-deficient mice and showed that the CD4+CD25+

TR are selected by thymic cortical epithelial cells (5, 13, 14). The questions posed were: howdid the CD4+CD25+ TR that were selected by cortical epithelial cells escape the negativeselection mediated by medullary DCs, and if cortical epithelial cells selected theCD4+CD25+ TR at the CD4+CD8+ double-positive stage, why were so few T cellsexpressing Forkhead Box P3+ (FOXP3+)TR found in the cortex (15, 16)?

We have recently shown that a subset of myeloid DCs (mDCs) educated by thymic stromallymphopoietin (TSLP) produced by epithelial cells of the Hassall’s corpuscles induceddifferentiation of CD4+CD25− thymocytes into FOXP3+ TR within the medulla of humanthymus (16). The ability of TSLP-activated mDCs to induce TR appeared to be linked withtheir unique features, including: 1) expression of high MHC-II and costimulatory molecules(CD80, CD86); 2) sparse production of proinflammatory cytokines IL-1, IL-6, and IL-12,which inhibit TR development; and 3) prolonged formation of stable conjugate with T cellsthat provide sustained TCR signaling and costimulation (17). We believe that DCs haveseveral advantages over epithelial cells as the APCs for selecting TR in thymus: 1) DCs cantransport peripheral Ags into the thymus; 2) DCs have the ability to cross-present self-Ags,including epithelial cell-derived Ags; and 3) TSLP-matured DCs can provide better qualityof costimulation to prevent the developing T cells’ pro-pensity for apoptosis when they aresignaled through medium- to high-affinity self-reactive TCR.

Human thymus contains at least two major subsets of DCs, including conventional mDCsand plasmacytoid DCs (pDCs) (18, 19). We have previously shown that TSLP selectivelyactivates ex vivo-derived peripheral blood mDCs but not other blood cell types (20).Recently, we found that although resting pDCs do not express the TSLP receptor (TSLPR),

Hanabuchi et al. Page 2

J Immunol. Author manuscript; available in PMC 2012 April 13.

NIH


NIH


NIH


they do rapidly express mRNA encoding for TSLPR and IL-7 receptor α (IL-7Rα) followingactivation, suggesting that pDCs may acquire the ability to respond to TSLP followingactivation.

In our current study, we showed that pDCs rapidly expressed surface TSLPR and IL-7Rαcomplexes and became responsive to TSLP activation. TSLP-activated human pDCsexpressed higher levels of CD80 and CD86 and secreted thymus and activation-regulatedchemokine CCL-17 and macrophage-derived chemokine CCL-22 but not Th1- or Th2-polarizing cytokines. TSLP-activated pDCs could efficiently induce the generation ofFOXP3+ TR from the CD4+CD8−CD25− thymocytes, which could be strongly inhibited byTh1-polarizing cytokine IL-12 or Th2-polarizing cytokine IL-4. Interestingly, the FOXP3+

TR induced by the TSLP-activated pDCs appeared to produce more IL-10 and to expressless TGF-β than that induced by the TSLP-activated mDCs. Our study suggests that thereare multiple types of APCs such as TSLP-activated mDCs and TSLP-activated pDCs inthymus that are responsible for the selection of functional distinct subsets of FOXP3+ TR.The inability of TSLP to induce DC maturation without producing Th1- or Th2-polarizingcytokines may provide a suitable thymic niche for TR development.

Materials and MethodsThymic and peripheral DC purification and culture

This study was approved by the Institutional Review Board for Human Research at TheUniversity of Texas M. D. Anderson Cancer Center, Houston, TX. Thymuses from humannewborns and children 0 d to 2 y old were obtained from the Texas Children’s Hospital,Houston, Texas. Adult blood buffy coats from healthy donors were obtained from the GulfCoast Regional Blood Center, Houston, TX. For isolation of thymic pDCs, thymuses werecut into small pieces and digested as previously described (18). After separation of themononuclear cells by Ficoll centrifugation, blood DC Ag 4+ (BDCA-4+)cells were obtainedfrom thymus or PBMCs using a Microbeads-BDCA-4 isolation kit (Miltenyi Biotec,Bergisch Gladbach, Germany). The enriched cells were stained with FITC-conjugated anti-CD3, CD14, CD16, CD56, CD19, CD20, PE-conjugated anti-CD11c, allophycocyanin-conjugated anti-CD123, and allophycocyanin-Cy7–conjugated anti-CD4 Abs (BDBiosciences, San Jose, CA), and then linage−CD11c−CD4+CD123+ cells were purifiedusing FACS on an FACS Aria (BD Biosciences) to reach >99% purity, as described (21).Isolated pDCs were cultured for 48 h with various stimuli with or without TSLP (50 ng/ml;recombinant protein was prepared in house using an adenovirus vector system) as described(17). After 48 h of culture, we determined the cell-surface marker characteristics of activatedpDCs also as described (17).

Isolation of thymic T cellsFor isolation of T cell linage thymocytes, thymuses were digested, and mononuclear cellswere separated by Ficoll centrifugation as described (17). Briefly, T cell linage thymocyteswere obtained by negative deletion using a mixture of mouse mAbs against the linagemarkers CD11c, CD14, CD15, CD20, CD56, and CD235a (produced in-house). This wasfollowed by incubation of these thymocytes with goat anti-mouse IgG-coated magneticbeads (M-450; Dynal, Oslo, Norway) and microbeads goat anti-mouse IgG (MiltenyiBiotec). Enriched cells were stained with FITC-conjugated anti-CD11c, CD14, CD16,CD19, CD20, CD56, and BDCA-2, PE-conjugated anti-CD8, PE-Cy7–conjugated anti-CD25, and allophycocyanin-Cy7 conjugated anti-CD4 Abs (BD Biosciences), and thenLinage−CD4+CD8− CD25− cells were purified using FACS on an FACSAria (BDBiosciences) to reach >99% purity.



NIH


NIH


NIH


DCs and T cell cocultureAfter 2 d of culture, pDCs were collected and washed three times to remove any cytokines.Viable DCs were counted by trypan blue exclusion of dead cells. The remaining cells werecocultured with 2 × 104 freshly isolated allogeneic thymocytes or peripheral blood-naiveCD4+ T cells in round-bottomed 96-well culture plates in IMDM containing 2% human ABserum and 10 ng/ml of IL-7. Cultures were done in triplicate at a 1:2 or 1:1 ratio of DCs:Tcells. Neutralizing anti–HLA-DR (BD Biosciences), anti-CD80, anti-CD86, and anti–IL-2(R&D Systems, Minneapolis, MN) mAbs were used in culture at a concentration of 30 μg/ml. The labeling of T cells with CFSE (Molecular Probes, Carlsbad, CA) or PKH26 (Sigma-Aldrich, St. Louis, MO) was performed as described (17). In some experiments, isolated Tcells were cultured with plate-bound anti-CD3 Ab (OKT-3; 2 μg/ml), soluble anti-CD28(28.2; 1 μg/ml), 20 U/ml IL-2, and 10 ng/ml IL-7 with or without 10 μg/ml TGF-β (R&DSystems).

T cell proliferation and expansion assayAfter 7 d of culture, cells were collected and resuspended in an EDTA-containing mediumto dissociate the clusters. Viable cells were counted by trypan blue exclusion of dead cells.The remaining cells were stained with PE-conjugate anti-CD25, and Alexa Fluor 647-conjugated anti-FOXP3 Abs (259D or 236A/E7) (Biolegend, San Diego, CA, andeBioscience, San Diego, CA) and analyzed with a LSR-II (BD Biosciences). Dead cellswere excluded on the basis of side- and forward-scatter characteristics, and viableCD4+CD25+ and FOXP3+ T cell numbers were calculated as: the percentage of cells in thecell type 3 the number of viable cells.

Suppression assayAfter 7 d of culture with TSLP-DCs, CD4+ thymocytes were stained with PE-conjugatedanti-CD25 (Miltenyi Biotec), and then CD25high cells and CD25dim cells, which divided >5times, were isolated by cell sorting. The mixture of these sorted cells and peripheralCD4+CD25− T cells at a 1:1 ratio or multiple ratios were stimulated with immobilized anti-CD3 (OKT-3, 2 μg/ml) and anti-CD28 Abs (CD28.2, 1 μg/ml) for 4 d. Cellular proliferationwas assessed by [3H]thymidine incorporation, as described in Ref. 17. PeripheralCD4+CD25− T cells were also labeled with CFSE and cultured with sorted CD25high cellsand CD25dim cells at a 1:1 ratio in the presence of immobilized anti-CD3 (OKT-3, 2 μg/ml)and soluble anti-CD28 Abs (CD28.2, 0.5 μg/ml). After 4 d of cultures, cell division ofCFSE-labeled CD4+CD25− T cells was evaluated by flow cytometry.

DC and T cell cytokine productionDC culture supernatants were collected at 24 h, and chemokine and cytokine production wasassessed as described Ref. 17. CD4+CD25− T cells from adult peripheral blood, fetal, orneonatal thymuses were cultured for 7 d with TSLP-DCs and IL-7 or with immobilized anti-CD3, soluble anti-CD28 Abs, IL-2, and IL-7. T cells were then washed and restimulatedwith 50 ng/ml PMA and 2 μg/ml ionomycin for 6 h (Sigma-Aldrich). A total of 10 μg/ml ofbrefeldin A (Sigma-Aldrich) was added during the last 2 h. The T cells were stained withPE-conjugated anti–IL-10 (BD Biosciences) and Alexa Fluor 647-conjugated anti-FOXP3(259D or 236A/E7) Abs using CALTAG FIX and PERM kit (Invitrogen, Carlsbad, CA) andthe FOXP3 staining kit (eBioscience). The T cells were also stimulated with plate-bound 2μg/ml of anti-CD3 and 1 μg/ml of soluble anti-CD28 Abs at a concentration of 106 cells/mlfor 24 h. The level of IL-10 in the supernatants was measured by ELISA (R&D Systems).For detection of membrane-bound latency associated peptide (LAP) (TGF-β1) on thesurface, cells were stained with LAP (TGF-β1) Ab (27232, R&D Systems), followed bystaining with PE-conjugated anti-mouse IgG1 Abs (BD Biosciences).



NIH


NIH


NIH


Intracellular FOXP3 stainingAfter 7 d of culture with DCs, T cells were stained with PE-conjugated anti-CD25 and thenfixed with Fixation/Permeabilization working solution (eBioscience) for 1 h. After washing,the cells were treated with Per-meabilization Buffer (eBioscience) in the presence of AlexaFluor 647-conjugated anti-FOXP3 Ab (259D or 236A/E7) for 30 min.

ImmunohistochemistryFor immunofluorescence staining, the slides were incubated with biotinylated mouse anti-CD123 mAb (BD Biosciences) at room temperature for 1 h. After washing, anti-CD123mAb-dependent tissue deposition of biotin was increased using a tyramide signal biotinamplification system and then was visualized by Alexa Fluor 549-conjugated streptavidin(Molecular Probes). The slides were then stained with FITC-conjugated anti-FOXP3 mAb(eBioscience) for 1 h. Images were acquired by using an inverted microscope, BX41(Olympus, Tokyo, Japan). Final image processing was performed by using Photoshop(Adobe, San Jose, CA).

ResultsTSLP costimulates pDCs

We have previously shown that TSLP could strongly activate mDCs to expresscostimulatory molecules CD80 and CD86 and to produce Th2 chemokines CCL-17 andCCL-22 without producing Th1-polarizing cytokine IL-12 (20). Through gene expressionanalyses, we recently found that pDCs rapidly express mRNA encoding for TSLPR andIL-7Rα following activation through TLR-7 or TLR-9 (data not shown). This stimulated usto further investigate whether pDCs have the ability to express surface TSLPR at the proteinlevel and to become functionally responsive to TSLP. We found that whereas pDCsconstitutively expressed surface IL-7Ra, pDCs expressed surface TSLPR followingactivation by TLR7-ligand R848 and TLR9-ligand CpG-B (Fig. 1A). IL-3 plus CD40L,CpG-C, HSV, and influenza A moderately upregulated the expression of TSLPR by pDCs.We found that TSLP strongly upregulated surface expression of CD80 and CD86 on pDCsactivated by CpG-B or R848 (Fig. 1B) and promoted pDCs to produce CCL-17 and CCL-22without inducing or effecting IFN-α or IL-12 production in culture (Fig. 1C). These datasuggest that, unlike CD40L or ligands for different TLRs, TSLP represents a very uniquestimulus that induces activation of both mDCs and pDCs to upregulate costimulatorymolecules without producing either Th1-polarizing cytokines or proinflammatory cytokines.

TSLP-conditioned CpG-B preactivated pDCs induce generation of FOXP3+ TRIn the human system, we have recently shown that TSLP is expressed by epithelial cells ofthe Hassall’s corpuscles in the thymic medulla, and TSLP-conditioned mDCs could inducethe differentiation of CD4+ CD25− thymocytes into CD4+CD25+FOXP3+ TR cells (16).Because human thymus contains both mDC and pDC, we next investigated whether TSLP-activated human pDCs could induce the differentiation of CD4+CD25− thymocytes intoCD4+CD25+FOXP3+ TR cells. CD4+ CD25−FOXP3− thymocytes isolated from humanthymus were labeled with CFSE and cultured for 7 d with allogeneic pDCs precultured for 2d with CpG-B in the presence or absence of TSLP. The sorted CD4+CD25− thymocytescontained <0.2% of FOXP3+ cells as assessed by staining with anti-human FOXP3 mAbs259D (Fig. 2A) and 236A/E7 (data not shown). We found that TSLP-conditioned CpG-Bpreactivated pDCs (TSLP-pDCs) induced a vigorous expansion of CD4+ thymocytes (Fig.2B, 2D, 2F) and the generation of CD4+CD25+ FOXP3+ cells (Fig. 2B, 2E, 2G), whereaspDCs activated by CpG-B alone induced less expansion and few generation of FOXP3+

cells. The number of the FOXP3+ cells induced by TSLP-pDCs followed the number of



NIH


NIH


NIH


TSLP-pDCs added in the cultures (Fig. 2G). In contrast, whereas CD4+CD25− thymocytesdifferentiated into FOXP3+ cells by stimulating with anti-CD3, anti-CD28, IL-2, and TGF-β–like peripheral naive CD4+ T cells (22), stimulation of CD4+CD25− thymocytes withanti-CD3 and IL-2 or anti-CD3, anti-CD28, and IL-2 induced vigorous expansion but not thedifferentiation of CD4+CD25− cells into FOXP3+ cells (Fig. 2C). These data suggest thatthe stimulation by anti-CD3, anti-CD28, and IL-2 is not sufficient to induce FOXP3+ cellsfrom human CD4+CD25− thymocytes.

We then examined whether TSLP-pDCs induced the differentiation of CD4+CD25−FOXP3−cells into CD4+CD25+FOXP3+ cells or the expansion of a small number ofCD4+CD25+FOXP3+ TR contaminated in sorted CD4+CD25− thymocytes. To determinethis, we performed cell-mixed culture experiments with CFSE-labeled CD4+CD25+ TR cellsand PKH26-labeled CD4+CD25− cells (16). Sorted CD4+CD25+ thymocytes were labeledwith CFSE and mixed with PKH26-labeled CD4+CD25− thymocytes at a 1:9 ratio. Thisratio was chosen because the original CD4+ thymocyte population contains CD25+ andCD25− cells at 1:9 ratios (Fig. 2A) (16). These mixed cells were then cultured together withTSLP-pDCs, and the proliferative response of the two populations was compared (Fig. 3A,3B). We found that thymic CD4+CD25+ TR in mixed culture expanded ~4-fold, which is asimilar extent as the expansion of CD4+CD25− cells. Thus, it seems unlikely that thecontaminated CD4+CD25+FOXP3+ TR, which is <0.2% in CD4+CD25− cells, can reach to4.8%. Based on the cell number, contaminated CD4+ CD25+FOXP3+ cells have to expand>30–200-fold to reach 4.8% of cultured cells. Therefore, these data indicate that TSLP-pDCs preferentially induce CD4+CD25− thymocytes to differentiate intoCD4+CD25+FOXP3+ T cells.

The generation of CD4+CD25+FOXP3+ TR induced by TSLP-pDCs was completelyblocked by the addition of Abs to MHC-II (HLA-DR), CD80/CD86, or IL-2 (Fig. 4A, 4B),suggesting the critical role of the interaction of TCR and Ag/MHC-II complex, CD28signaling through CD80/CD86, and IL-2 in the generation and expansion ofCD4+CD25+FOXP3+ TR in thymus, as previously observed in both humans and mice (23–26).

We next examined the phenotype, cytokine profile, and suppressive activity of FOXP3+

cells generated from CD4+CD25−FOXP3− thymocytes by TSLP-pDCs.CD4+CD25−FOXP3− thymocytes were labeled with CFSE and cultured with TSLP-pDCs.After 7 d of culture, we evaluated the surface phenotype of FOXP3+ or FOXP3− cells in thesame culture, which went through similar rounds of cell division after culture with pDCs. Asshown in Fig. 5A, FOXP3+ cells expressed much higher levels of CD25, CTLA-4, and ICOSthan FOXP3− cells and a similar level of glucocorticoid-induced TNF receptor to thatexpressed by FOXP3− cells. Furthermore, FOXP3+ cells induced by TSLP-pDCs did notproduce IL-2 and IFN-γ, whereas FOXP3− cells in the same culture produced significantlevels of IL-2 and IFN-γ (Fig. 5B).

We next examined whether CD4+CD25+FOXP3+ T cells generated by TSLP-pDCsexhibited suppressive function. After 7 d of culture, we sorted both CD25high and CD25dim

cells, which went through >5 times of division. More than 60% of the sorted CD25high cellsand <5% of CD25dim cells expressed FOXP3 (Fig. 6A). These cells were cultured withCD4+CD25− T cells isolated from peripheral blood in the presence of immobilized anti-CD3and soluble anti-CD28. We found that CD4+CD25highFOXP3+ cells induced by TSLP-pDCswere anergic and displayed the ability to strongly inhibit the proliferation of CD4+CD25− Tcells (Fig. 6B, 6D). In contrast, CD4+CD25dimFOXP3− cells were neither anergic norsuppressive (Fig. 6C, 6D). They even promoted the expansion of responder CD4+CD25− Tcells rather than inhibited their proliferation. We confirmed these results by CFSE-labeling



NIH


NIH


NIH


experiments. The proliferation of CFSE-labeled responder CD4+CD25− T cells wassignificantly inhibited by CD25+CD25highFOXP3+ cells but not CD4+CD25dimFOXP3−cells (Fig. 6E).

These findings show that newly generated CD4+CD25+FOXP3+ T cells by TSLP-pDCspossess some of the key features of naturally occurring TR developed in mice and humanthymus.

FOXP3+ TR induced by TSLP-pDCs or by TSLP-mDCs express different levels of IL-10 andLAP (TGF-β1)

We have recently found that human thymus contains two distinct FOXP3+ TR subsetsdefined by surface expression of the costimulatory molecule ICOS (27). Although theICOS+FOXP3+ TR produced high levels of IL-10 that suppressed DC function andexpressed low levels of LAP (TGF-β1) that directly suppressed T cell proliferation, theICOS−FOXP3+ TR expressed high levels of LAP (TGF-β1) but produced low levels ofIL-10, suggesting that there may be different populations of APCs selecting the FOXP3+ TR.Thus, we next questioned whether there are phenotypic differences between TSLP-pDC–induced and TSLP-mDC– induced TR. Interestingly, the FOXP3+ TR generated by TSLP-pDCs expressed lower LAP (TGF-β) (Fig. 7A) and produced higher IL-10 levels (Fig. 7B,7C) than did the FOXP3+ TR induced by TSLP-mDCs, suggesting that TSLP-pDCs andTSLP-mDCs may play different roles in TR development.

Th1/Th2 cytokines inhibit the ability of TSLP-pDCs to induce the generation of FOXP3+ TRfrom CD4+CD25− thymocytes

Unlike CD40L or ligands for different TLRs that all induce mDCs to produce Th1-polarizing cytokine IL-12, TSLP does not induce mDCs or pDCs to produce IL-12. Wehypothesized that the absence of Th1-polarizing cytokines provided by TSLP-activated DCsmay provide a permissive environment that allows the development of TR. To test thishypothesis, CFSE-labeled CD4+CD25−FOXP3− thymocytes were cultured for 7 d withTSLP-pDCs or TSLP-mDCs in the presence or absence of IL-4, IFN-γ, IL-12, TNF-α, orTGF-β. IL-12 and IL-4 strongly inhibited the differentiation of CD4+CD25− thymocytesinto CD4+CD25+FOXP3+ cells (Fig. 8A, 8B). However, IFN-γ and TNF-α did not inhibit thegeneration of FOXP3+ cells. Interestingly, TGF-β, which is known to induce FOXP3 inperipheral CD4+ T cells activated by DCs (Fig. 8C, 8D) (28) or by anti-CD3 and anti-CD28(22, 29, 30) and seems to be important to convert peripheral T cells to FOXP3+ T cells andto maintain the expression of FOXP3 in peripheral TR, significantly inhibited the generationof FOXP3+ thymocytes induced by TSLP-pDCs. These data suggest that both Th1-polarizing cytokine IL-12 and Th2-polarizing cytokine IL-4 can inhibit the ability of TSLP-pDCs to induce the generation of FOXP3+ TR from thymocytes in culture and that theexpression of FOXP3 in thymus and in periphery may be regulated by different molecularmechanisms.

TSLPR+ pDCs are present in human thymus and colocalize with FOXP3+ TRWe next investigated whether human thymic pDCs expressed TSLPR and searched forevidence of in vivo interaction between pDCs and FOXP3+ TR in situ. pDCs were enrichedfrom total thymocytes by anti-BDCA4 magnetic bead sorting. The pDCs were furtherdefined by a phenotype of linage−CD4+CD123+ using flow cytometry. These cells showedthe typical phenotype of peripheral pDCs, including expression of BDCA-2, BDCA-4,CD45RA, and IL-7Rα (Fig. 9A). Freshly isolated thymic pDCs also had the ability toproduce IFN-α but not IL-12 and increased the expression of HLA-DR, CD80, and CD86 inresponse to HSV or CpGs (data not shown). As shown in Fig. 9B, significant numbers ofthymic pDCs (5–20%) expressed TSLPR without any stimulation. Importantly, these



NIH


NIH


NIH


TSLPR+ pDCs expressed higher levels of CD80, CD86, CD40, and HLA-DR than TSLP−pDCs. These results clearly indicate that TSLPR is expressed on pDCs residing in humanthymus.

To provide further anatomical evidence that pDCs are involved in FOXP3+ TR developmentin thymus, we investigated the localization of thymic pDCs and FOXP3+ TR in humanthymus by microscopic analysis using specific Abs to pDCs (anti-CD123 mAb) and toFOXP3+ TR (anti-FOXP3 mAb). Both CD123+ pDCs and FOXP3+ TR were found in thymicmedulla, and some pDCs were in close contact with FOXP3+ cells (Fig. 9C). These findingssuggest that thymic pDCs as well as mDCs instructed by TSLP produced by Hassall’scorpuscles may have a critical role in inducing the generation and expansion ofCD4+CD25+FOXP3+ TR through direct interaction in thymus.

DiscussionHuman thymus contains two major DC subsets, mDCs and pDCs (18, 19). We havepreviously shown that mDCs from human peripheral blood and thymus expressed theTSLPR/IL-7Ra complex and could be activated by TSLP (20). We presented experimentalevidence suggesting that thymic mDCs educated by TSLP produced by the epithelial cells ofthe Hassall’s corpuscles may induce the differentiation of CD4+CD25− thymocytes intoFOXP3+ TR within the medulla in human thymus (16).

In this study, we found that the subpopulation of pDCs isolated from human thymusexpressed TSLPR at steady state. Furthermore, this pDC subpopulation is located close toand associated with FOXP3+ cells in the thymic medulla. We also found that pDCs inperipheral blood express both TSLPR and IL-7Rα after activation through TLRs. TSLPactivated pDCs to express higher levels of costimulatory molecule and to producechemokines CCL-17 and CCL-22, which are important in guiding the traffic of developingimmature thymic T cells into the medulla (31–33). We showed that TSLP-activated pDCsinduced the expansion and differentiation of CD4+CD8−CD25− FOXP3− thymocytes intoCD4+CD25+FOXP3+ TR. The TR generated by TSLP-pDCs possess a typical feature ofnaturally occurring TR as previously reported in mice and humans (3, 34).

Interestingly, the FOXP3+ TR induced by TSLP-pDCs showed distinct cytokine expressionpotential compared with the FOXP3+ TR induced by TSLP-mDCs. The TSLP-pDC–inducedTR were IL-10high /TGF-βlow and the TSLP-mDC–induced TR were IL-10low/TGF-βhigh

after activation. These data are consistent with our recent observation that human thymus,peripheral blood, and secondary lymphoid tissues contain two subsets of CD25highFOXP3+

TR according to ICOS expression (27). Whereas the ICOS+ TR subset had the potential toexpress high IL-10 and less TGF-β,theICOS− TR subset expressed high TGF-β but lowerIL-10. These results suggest that pDCs and mDCs in thymus not only play a critical role inthe selection of TR but may also imprint the two cell subsets of TR to produce differentsuppressive cytokines, IL-10 and TGF-β, in response to self-Ag in the periphery.

Previous studies have shown that circulating immature DCs may migrateinto the thymusandinduce nondeletional tolerance (35–37). A more recent study by Proietto et al. (38) hasdemonstrated that the circulating peripheral Sirpa+CD8low DC subset migrates into thethymus and selects the FOXP3+ TR. Another interesting study provided direct evidence thatpDCs are able to acquire and process allogeneic cell-derived Ags and to cross-present thealloantigen to induce FOXP3+ TR cells in periphery (39). In our study, only ~4–20% ofpDCs in the thymus expressed TSLPR. This together with the observation that the peripheralblood pDCs expressed TSLPR only following activation through TLR-7/TLR-9 suggeststhat peripheral pDCs that carry the peripheral Ags may migrate into the thymus upon



NIH


NIH


NIH


activation and participate in the selection of TR together with TSLP-mDCs (16) andmedullar epithelial cells (40).

Differentiation of T cells into distinct cell linages is controlled by several key transcriptionfactors that have pivotal roles in cell fate choice during the early stage of lymphoid celldevelopment. These linage specification factors not only promote a particular T cell fate butalso are responsible for repressing alternative differentiation pathways. In our experiment,IL-12 and IL-4 actively repressed the adoption of CD4+ thymocytes’ fate to be TR. It seemsthat transcription factors induced by these cytokines, which are critical to promote Th1 orTh2 differentiation, override FOXP3-dependent development of TR. Our current studysuggests that primary CD4+ thymocytes have less ability to produce cytokines, includingIL-4 and IFN-γ, by stimulation with DCs or anti-CD3 plus anti-CD28 than do peripheralnaive CD4+ T cells (S. Hanabuchi and Y.J. Liu, unpublished observation). Furthermore,both TSLP-pDCs and TSLP-mDCs produce little Th1-polarizing (IL-12) or Th2-polarizing(IL-4) cytokines that inhibit TR development. These features of DCs and thymocytes arelikely to be essential for the differentiation of FOXP3+ TR in the thymic microenvironment.Although the existence of a unique niche in thymus has been proposed, its exact nature hasremained unclear (40, 41). Decoding the molecular signature of DCs, including surfacemolecules and soluble factors such as cytokines that may positively or negatively regulatethymic selection, should be very important to accomplish the differentiation of TR inthymus. Thus, we speculate that TR differentiation in thymus is regulated in a very subtleway by DCs, providing a suitable niche that allows TR to develop.

In conclusion, this study provides experimental evidence showing thatactivatedpDCs rapidlyexpress TSLPRand IL-7Ra and thatTSLP could strongly activate human pDCs to enhancecostimulatory molecules and to initiate production of CCL-17 and CCL-22. Furthermore,TSLP-activated pDCs could efficiently induce the generation and expansion of FOXP3+ TR.Interestingly, the FOXP3+ TR induced by TSLP-pDCs produced more IL-10 and less TGF-βthan that of the FOXP3+ TR induced by TSLP-mDCs. These data suggest that two subsets ofTR with different cytokine production potential can be selected by TSLP-activated mDC orpDCs, respectively, in thymus. The inability of TSLP to induce DC maturation withoutproducing Th1- or Th2-polarizing cytokines may provide a thymic niche for TRdevelopment.

AcknowledgmentsWe thank Karen Ramirez, Zhiwei He, and Eric Wieder for cell sorting and support and M.J. Fingold and K.Sternberg for tissue materials.

This work was supported by National Institutes of Health Grant AI062888-01 (to Y.-J.L.) and the Keck Foundation(to Y.-J. L.).

Abbreviations used in this paper

BDCA blood dendritic cell Ag

DC dendritic cell

FOXP3+ Forkhead Box P3+

IL-7Rα IL-7 receptor α

LAP latency associated peptide

mDC myeloid dendritic cell

MFI mean fluorescence intensity



NIH


NIH


NIH


MHC-II MHC class II

pDC plasmacytoid dendritic cell

TR regulatory T cell

TSLP thymic stromal lymphopoietin

TSLPR thymic stromal lymphopoietin receptor

References1. Modigliani Y, Thomas-Vaslin V, Bandeira A, Coltey M, Le Douarin NM, Coutinho A, Salaün J.

Lymphocytes selected in allogeneic thymic epithelium mediate dominant tolerance toward tissuegrafts of the thymic epithelium haplotype. Proc. Natl. Acad. Sci. USA. 1995; 92:7555–7559.[PubMed: 7638230]

2. Modigliani Y, Coutinho A, Pereira P, Le Douarin N, Thomas-Vaslin V, Burlen-Defranoux O,Salaün J, Bandeira A. Establishment of tissue-specific tolerance is driven by regulatory T cellsselected by thymic epithelium. Eur. J. Immunol. 1996; 26:1807–1815. [PubMed: 8765025]

3. Sakaguchi S. Naturally arising CD4+ regulatory t cells for immunologic self-tolerance and negativecontrol of immune responses. Annu. Rev. Immunol. 2004; 22:531–562. [PubMed: 15032588]

4. Shevach EM, McHugh RS, Piccirillo CA, Thornton AM. Control of T-cell activation by CD4+CD25+ suppressor T cells. Immunol. Rev. 2001; 182:58–67. [PubMed: 11722623]

5. Bensinger SJ, Bandeira A, Jordan MS, Caton AJ, Laufer TM. Major histocompatibility complexclass II-positive cortical epithelium mediates the selection of CD4(+)25(+) immunoregulatory Tcells. J. Exp. Med. 2001; 194:427–438. [PubMed: 11514600]

6. Fontenot JD, Rudensky AY. A well adapted regulatory contrivance: regulatory T cell developmentand the Forkhead family transcription factor FOXP3. Nat. Immunol. 2005; 6:331–337. [PubMed:15785758]

7. Le Douarin NM, Jotereau FV. Tracing of cells of the avian thymus through embryonic life ininterspecific chimeras. J. Exp. Med. 1975; 142:17–40. [PubMed: 239088]

8. Guillemot FP, Oliver PD, Peault BM, Le Douarin NM. Cells expressing Ia antigens in the avianthymus. J. Exp. Med. 1984; 160:1803–1819. [PubMed: 6595343]

9. Fontaine-Perus JC, Calman FM, Kaplan C, Le Douarin NM. Seeding of the 10-day mouse embryothymic rudiment by lymphocyte precursors in vitro. J. Immunol. 1981; 126:2310–2316. [PubMed:6971895]

10. Houssaint E, Torano A, Ivanyi J. Split tolerance induced by chick embryo thymic epitheliumallografted to embryonic recipients. J. Immunol. 1986; 136:3155–3159. [PubMed: 3514753]

11. Salaün J, Bandeira A, Khazaal I, Calman F, Coltey M, Coutinho A, Le Douarin NM. Thymicepithelium tolerizes for histocompatibility antigens. Science. 1990; 247:1471–1474. [PubMed:2321009]

12. Ramsdell F, Lantz T, Fowlkes BJ. A nondeletional mechanism of thymic self tolerance. Science.1989; 246:1038–1041. [PubMed: 2511629]

13. Laufer TM, DeKoning J, Markowitz JS, Lo D, Glimcher LH. Unopposed positive selection andautoreactivity in mice expressing class II MHC only on thymic cortex. Nature. 1996; 383:81–85.[PubMed: 8779719]

14. Laufer TM, Fan L, Glimcher LH. Self-reactive T cells selected on thymic cortical epithelium arepolyclonal and are pathogenic in vivo. J. Immunol. 1999; 162:5078–5084. [PubMed: 10227976]

15. Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG, Rudensky AY. Regulatory T celllineage specification by the Forkhead transcription factor fOXP3. Immunity. 2005; 22:329–341.[PubMed: 15780990]

16. Watanabe N, Wang YH, Lee HK, Ito T, Wang YH, Cao W, Liu YJ. Hassall’s corpuscles instructdendritic cells to induce CD4+CD25+ regulatory T cells in human thymus. Nature. 2005;436:1181–1185. [PubMed: 16121185]



NIH


NIH


NIH


17. Watanabe N, Hanabuchi S, Soumelis V, Yuan W, Ho S, de Waal Malefyt R, Liu YJ. Humanthymic stromal lymphopoietin promotes dendritic cell-mediated CD4+ T cell homeostaticexpansion. Nat. Immunol. 2004; 5:426–434. [PubMed: 14991051]

18. Bendriss-Vermare N, Barthélémy C, Durand I, Bruand C, Dezutter-Dambuyant C, Moulian N,Berrih-Aknin S, Caux C, Trinchieri G, Brière F. Human thymus contains IFN-alpha-producingCD11c(−), myeloid CD11c (+), and mature interdigitating dendritic cells. J. Clin. Invest. 2001;107:835–844. [PubMed: 11285302]

19. Wu L, Shortman K. Heterogeneity of thymic dendritic cells. Semin. Immunol. 2005; 17:304–312.[PubMed: 15946853]

20. Soumelis V, Reche PA, Kanzler H, Yuan W, Edward G, Homey B, Gilliet M, Ho S, Antonenko S,Lauerma A, et al. Human epithelial cells trigger dendritic cell mediated allergic inflammation byproducing TSLP. Nat. Immunol. 2002; 3:673–680. [PubMed: 12055625]

21. Ito T, Kanzler H, Duramad O, Cao W, Liu YJ. Specialization, kinetics, and repertoire of type 1interferon responses by human plasmacytoid predendritic cells. Blood. 2006; 107:2423–2431.[PubMed: 16293610]

22. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM. Conversion ofperipheral CD4+CD25-naive T cells to CD4+ CD25+ regulatory T cells by TGF-beta induction oftranscription factor FOXP3. J. Exp. Med. 2003; 198:1875–1886. [PubMed: 14676299]

23. Salomon B, Lenschow DJ, Rhee L, Ashourian N, Singh B, Sharpe A, Bluestone JA. B7/CD28costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells thatcontrol autoimmune diabetes. Immunity. 2000; 12:431–440. [PubMed: 10795741]

24. Jordan MS, Boesteanu A, Reed AJ, Petrone AL, Holenbeck AE, Lerman MA, Naji A, Caton AJ.Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat.Immunol. 2001; 2:301–306. [PubMed: 11276200]

25. Malek TR, Yu A, Vincek V, Scibelli P, Kong L. CD4 regulatory T cells prevent lethalautoimmunity in IL-2Rbeta-deficient mice. Implications for the nonredundant function of IL-2.Immunity. 2002; 17:167–178. [PubMed: 12196288]

26. Tai X, Cowan M, Feigenbaum L, Singer A. CD28 costimulation of developing thymocytes inducesFOXP3 expression and regulatory T cell differentiation independently of interleukin 2. Nat.Immunol. 2005; 6:152–162. [PubMed: 15640801]

27. Ito T, Hanabuchi S, Wang YH, Park WR, Arima K, Bover L, Qin FX, Gilliet M, Liu YJ. Twofunctional subsets of FOXP3+ regulatory T cells in human thymus and periphery. Immunity. 2008;28:870–880. [PubMed: 18513999]

28. Luo X, Tarbell KV, Yang H, Pothoven K, Bailey SL, Ding R, Steinman RM, Suthanthiran M.Dendritic cells with TGF-beta1 differentiate naive CD4+CD25-T cells into islet-protectiveFOXP3+ regulatory T cells. Proc. Natl. Acad. Sci. USA. 2007; 104:2821–2826. [PubMed:17307871]

29. Rao PE, Petrone AL, Ponath PD. Differentiation and expansion of T cells with regulatory functionfrom human peripheral lymphocytes by stimulation in the presence of TGF-beta. J. Immunol.2005; 174:1446–1455. [PubMed: 15661903]

30. Rubtsov YP, Rudensky AY. TGFbeta signalling in control of T-cell-mediated self-reactivity. Nat.Rev. Immunol. 2007; 7:443–453. [PubMed: 17525753]

31. Campbell JJ, Pan J, Butcher EC. Cutting edge: developmental switches in chemokine responsesduring T cell maturation. J. Immunol. 1999; 163:2353–2357. [PubMed: 10452965]

32. Chantry D, Romagnani P, Raport CJ, Wood CL, Epp A, Romagnani S, Gray PW. Macrophage-derived chemokine is localized to thymic medullary epithelial cells and is a chemoattractant forCD3(+), CD4(+), CD8(low) thymocytes. Blood. 1999; 94:1890–1898. [PubMed: 10477717]

33. Annunziato F, Romagnani P, Cosmi L, Beltrame C, Steiner BH, Lazzeri E, Raport CJ, Galli G,Manetti R, Mavilia C, et al. Macrophage-derived chemokine and EBI1-ligand chemokine attracthuman thymocytes in different stage of development and are produced by distinct subsets ofmedullary epithelial cells: possible implications for negative selection. J. Immunol. 2000;165:238–246. [PubMed: 10861057]



NIH


NIH


NIH


34. Sakaguchi S. Naturally arising FOXP3-expressing CD25+CD4+ regulatory T cells inimmunological tolerance to self and non-self. Nat. Immunol. 2005; 6:345–352. [PubMed:15785760]

35. Donskoy E, Goldschneider I. Two developmentally distinct populations of dendritic cells inhabitthe adult mouse thymus: demonstration by differential importation of hematogenous precursorsunder steady state conditions. J. Immunol. 2003; 170:3514–3521. [PubMed: 12646612]

36. Beschorner WE, Yao X, Divic J. Recruitment of semiallogeneic dendritic cells to the thymusduring post-cyclosporine thymic regeneration. Transplantation. 1995; 60:1326–1330. [PubMed:8525529]

37. Goldschneider I, Cone RE. A central role for peripheral dendritic cells in the induction of acquiredthymic tolerance. Trends Immunol. 2003; 24:77–81. [PubMed: 12547504]

38. Proietto AI, van Dommelen S, Zhou P, Rizzitelli A, D’Amico A, Steptoe RJ, Naik SH, LahoudMH, Liu Y, Zheng P, et al. Dendritic cells in the thymus contribute to T-regulatory cell induction.Proc. Natl. Acad. Sci. USA. 2008; 105:19869–19874. [PubMed: 19073916]

39. Ochando JC, Homma C, Yang Y, Hidalgo A, Garin A, Tacke F, Angeli V, Li Y, Boros P, Ding Y,et al. Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts.Nat. Immunol. 2006; 7:652–662. [PubMed: 16633346]

40. Aschenbrenner K, D’Cruz LM, Vollmann EH, Hinterberger M, Emmerich J, Swee LK, Rolink A,Klein L. Selection of FOXP3+ regulatory T cells specific for self antigen expressed and presentedby Aire+ medullary thymic epithelial cells. Nat. Immunol. 2007; 8:351–358. [PubMed: 17322887]

41. Fontenot JD, Dooley JL, Farr AG, Rudensky AY. Developmental regulation of FOXP3 expressionduring ontogeny. J. Exp. Med. 2005; 202:901–906. [PubMed: 16203863]



NIH


NIH


NIH


FIGURE 1.TSLP expressed on pDCs enhances the expression of costimulatory molecules CD80 andCD86 and initiates the production of chemokines CCL-17 and CCL-22. A, Expression ofTSLPR and IL-7Rα by pDCs stimulated with each stimuli for 36 h was determined by flowcytometry. Open histograms represent isotype control, and filled histograms representstaining of TSLPR and IL-7Rα on pDCs. B, mDCs were stimulated with TSLP orpolyinosinic-polycytidylic acid, or cultured in medium only. pDCs were stimulated withCpG-B or R848 in the presence or absence of TSLP. After 48 h of culture, CD80 and CD86expression of mDCs and pDCs were determined by flow cytometry. Open histogramsrepresent isotype control, and filled histograms represent CD80 or CD86. The number in thehistograms indicates the mean fluorescence intensity (ΔMFI). All data are representative ofthree experiments. C, The production of CCL-17, CCL-22, IFN-α, and IL-12p70 by mDCsand pDCs cultured with each stimuli for 48 h was determined by ELISA.



NIH


NIH


NIH


FIGURE 2.TSLP-pDCs induce CD4+CD25−FOXP3− thymocytes to differentiate intoCD4+CD25+FOXP3+ T cells. A, Flow cytometric analysis of unsorted T cell linagethymocytes and FOXP3 expression of sorted CD4+CD25− thymocytes before culturing withDCs. B, FOXP3 expression of CFSE-labeled CD4+CD25−FOXP3− thymocytes wasanalyzed after culturing with allogeneic medium-mDCs, TSLP-mDCs, CpG-B–pDCs, orCpG-B plus TSLP-pDCs for 7 d at 1:2 ratio of DCs:thymocytes. The number in dot plotsindicates the percentage of CD4+FOXP3+ cells. C, FOXP3 expression of CFSE-labeledCD4+CD25−FOXP3− thymocytes after culturing with anti-CD3 and IL-2, anti-CD3, anti-CD28, and IL-2, or anti-CD3, anti-CD28, IL-2, and TGF-β for 7 d. The number in dot plotsindicates the percentage of CD4+FOXP3+ cells. D and E, CD4+CD25−FOXP3− thymocyteswere cultured for 7 d with CpG-B–pDCs or CpG-B plus TSLP-pDCs at a 1:1 ratio ofDCs:thymocytes. Filled circles indicate the total number of CD4+ cells per well (D) and thenumber of CD4+CD25+FOXP3+ cells per well (E) in five individual experiments.Horizontal bars indicate the mean. F and G, CD4+CD25−FOXP3− thymocytes were culturedfor 7 d at various DC:thymocyte ratios with CpG-B–pDCs (open circles) or CpG-B plusTSLP-pDCs (closed circles), and the total number of CD4+ cells per well (F) and thenumber of CD4+CD25+FOXP3+ cells per well (G) were assessed in five individualexperiments.



NIH


NIH


NIH


FIGURE 3.TSLP-pDCs induce the generation of FOXP3+ TR from CD4+CD25−FOXP3− thymocytesbut not from CD4+CD25+FOXP3+ cells contaminated in sor-ted CD4+CD25− thymocytes.A, Flowcytometric analysis of FOXP3 expression on sorted CD4+CD25+ and CD4+

CD25−thymocytes before culture. The number in dot plots indicates the percentage ofFOXP3+ cells. B, CFSE-labeled CD4+CD25+ thymocytes, PKH26-labeled CD4+CD25−thymocytes, and the mixture of two populations were co-cultured with TSLP-pDCs at 1:9ratio of CD25+:CD25− cells. After 5 d culture, FOXP3 expression on each population wasanalyzed by flow cytometry. The number in dot plots indicates the percentage of PKH26-labeled FOXP3+ cells (gate II) (upper panel). The fold expansion of PKH26-labeledFOXP3+ cells (gate II) and CFSE-labeled FOXP3+ cells (gate III) is shown in the bar graph(lower panel). All data shown are representative of four experiments.



NIH


NIH


NIH


FIGURE 4.Signaling through TCR, CD80, and IL-2 are critical for the development ofCD4+CD25+FOXP3+ thymocytes induced by TSLP-pDCs. CFSE-labeledCD4+CD25−FOXP3− thymocytes were cultured with TSLP-pDCs at a 1:2 ratio ofDCs:thymocytes in the presence of neutralizing anti– HLA-DR, anti-CD80 plus anti-CD86,anti–IL-2 Abs, or isotype control. A, On day 7, CD4+ cells were assessed for FOXP3expression by flow cytometry. B, The number of CD4+FOXP3+ cells is shown in the bargraph.



NIH


NIH


NIH


FIGURE 5.Phenotype of CD4+CD25+FOXP3+ cells induced by TSLP-pDCs. CFSE-labeledCD4+CD25− thymocytes were cultured with TSLP-pDCs for 7 d. A, Expression ofCD25, CTLA-4, ICOS, and glucocorticoid-induced TNF receptor on FOXP3+ and FOXP3−cells in the same culture were analyzed by flow cytometry. The number in the histogramsindicates the ΔMFI. B, Cultured cells were stimulated with PMA and ionomycin, andexpression of intracellular IL-2 and IFN-γ at the single-cell level was analyzed by flowcytometry.



NIH


NIH


NIH


FIGURE 6.Suppressive function of CD4+CD25highFOXP3+ cells induced by TSLP-pDCs. A, CFSE-labeled CD4+CD25− thymocytes were cultured with TSLP-pDCs for 7 d. FOXP3 expressionon CD4+CD25high cells and CD4+CD25dim cells, which went through >5 times of division,were analyzed after sort. The number in dot plots indicates the percentage of FOXP3+ cell. Band C, Suppressive function of TSLP-pDC–induced CD25highFOXP3+ cells (B) andCD25dimFOXP3− cells (C). Sorted CD4+CD25high cells and CD4+CD25dim cells werecultured with or without CD4+CD25−T cells isolated from peripheral blood in the presenceof immobilized anti-CD3 and soluble anti-CD28 Abs for 4 d. Cell proliferation wasmeasured by [3H]thymidine incorporation. All data shown are representative of fourexperiments. D, CD4+ CD25+TR isolated from peripheral blood, TSLP-pDC–inducedCD4+CD25high cells, or CD4+CD25dim cells were cultured with CD4+CD25− T cells atvarious ratios in the presence of immobilized anti-CD3 and soluble anti-CD28 Abs for 4 d.Cell proliferation was measured by [3H]thymidine incorporation. The data shown arerepresentative of six experiments. E, CFSE-labeled peripheral CD4+CD25− T cells werecultured with CD4+CD25high cells and CD4+CD25dim cells in the presence of immobilizedanti-CD3 and soluble anti-CD28 Abs. After 4 d of culture, division of CFSE-labeledresponder CD4+CD25− T cells was analyzed by flow cytometry.



NIH


NIH


NIH


FIGURE 7.FOXP3+ TR induced by TSLP-pDCs and TSLP-mDCs show different cytokine profiles.CFSE-labeled CD4+CD25−FOXP3− thymocytes were cultured for 7 d. A, Surfaceexpression of LAT (TGF-β1) on FOXP3+ cells or FOXP3− cells, which both went throughsimilar rounds of cell division after culture with DCs, was evaluated by flow cytometry.Open histograms represent isotype control, and filled histograms represent LAP (TGF-β1).The number in the histograms indicates the ΔMFI. All data are representative of sixexperiments. B, The cells were stimulated with PMA and ionomycin for 6 h, and then IL-10expression at the single-cell level was assessed by flow cytometry. C, The cells were alsostimulated with immobilized anti-CD3 and soluble anti-CD28 Abs for 24 h, and IL-10 in theculture supernatant was assessed by ELISA.



NIH


NIH


NIH


FIGURE 8.Th1/Th2 cytokines inhibit the generation of FOXP3+ TR by TSLP-pDCs. A and B, CFSE-labeled CD4+CD25−FOXP3− thymocytes were cultured for 7 d with TSLP-pDCs or TSLP-mDCs at a 1:4 ratio of DCs: thymocytes in the presence or absence of IL-4, IFN-γ, IL-12,TNF-α, and TGF-β. FOXP3 expression (A) and the number of CD4+FOXP3+ cells (B) wereassessed. C and D, CFSE-labeled peripheral naive CD4+ T cells were cultured for 7 d withTSLP-pDCs at a 1:4 ratio of DCs:T cells in the presence or absence of TGF-β, and FOXP3expression (C) and the number of CD4+ FOXP3+ cells (D) were assessed.



NIH


NIH


NIH


FIGURE 9.TSLPR+ pDCs are present in human thymus and colocalize with FOXP3+ TR. pDCs wereenriched from total thymocytes by anti-BDCA4 magnetic-bead sorting. pDCs were furtherdefined by a phenotype of linage−CD4+CD123+ by flow cytometry. A, pDCs were assessedfor expression of BDCA-2, BDCA-4, CD45RA, and IL-7-Ra by flow cytometry. B,Enriched pDCs were further stained with anti-TSLPR mAb together with anti-CD80, anti-CD86, anti-CD40, or anti-HLA-DR mAb. C, Double immunofluorescence staining ofFOXP3 (green) and CD123 (red) was performed on human thymus (original magnification×40).



NIH


NIH


NIH


Date post:	01-Feb-2023
Category:	Documents
Upload:	independent
View:	1 times
Download:	0 times

Thymic Stromal Lymphopoietin-Activated Plasmacytoid Dendritic Cells Induce the Generation of FOXP3+...

Documents