Regulate CD4 T Cell Responses Dependent Red Pulp Macrophages − CSF-1

Home , Red pulp

CSF-1−Dependent Red Pulp Macrophages Regulate CD4 T Cell Responses Daisuke Kurotaki, Shigeyuki Kon, Kyeonghwa Bae, Koyu Ito, Yutaka Matsui, Yosuke Nakayama, Masashi Kanayama, This information is current as Chiemi Kimura, Yoshinori Narita, Takashi Nishimura, of September 29, 2021. Kazuya Iwabuchi, Matthias Mack, Nico van Rooijen, Shimon Sakaguchi, Toshimitsu Uede and Junko Morimoto J Immunol published online 14 January 2011

http://www.jimmunol.org/content/early/2011/01/14/jimmun Downloaded from ol.1001345

Supplementary http://www.jimmunol.org/content/suppl/2011/01/14/jimmunol.100134 Material 5.DC1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

by guest on September 29, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 14, 2011, doi:10.4049/jimmunol.1001345 The Journal of Immunology

CSF-1–Dependent Red Pulp Macrophages Regulate CD4 T Cell Responses

Daisuke Kurotaki,*,† Shigeyuki Kon,† Kyeonghwa Bae,† Koyu Ito,† Yutaka Matsui,* Yosuke Nakayama,† Masashi Kanayama,† Chiemi Kimura,† Yoshinori Narita,‡ Takashi Nishimura,‡ Kazuya Iwabuchi,x Matthias Mack,{ Nico van Rooijen,‖ Shimon Sakaguchi,# Toshimitsu Uede,*,† and Junko Morimoto†

The balance between immune activation and suppression must be regulated to maintain immune homeostasis. Tissue macrophages (MFs) constitute the major cellular subsets of APCs within the body; however, how and what types of resident MFs are involved in the regulation of immune homeostasis in the peripheral lymphoid tissues are poorly understood. Splenic red pulp MF

(RPMs) remove self-Ags, such as blood-borne particulates and aged erythrocytes, from the blood. Although many scattered T cells Downloaded from exist in the red pulp of the spleen, little attention has been given to how RPMs prevent harmful T cell immune responses against self-Ags. In this study, we found that murine splenic F4/80hiMac-1low MFs residing in the red pulp showed different expression patterns of surface markers compared with F4/80+Mac-1hi monocytes/MFs. Studies with puriﬁed cell populations demonstrated that F4/80hiMac-1low MFs regulated CD4+ T cell responses by producing soluble suppressive factors, including TGF-b and IL-10. Moreover, F4/80hiMac-1low MFs induced the differentiation of naive CD4+ T cells into functional Foxp3+ regulatory T cells. hi low F Additionally, we found that the differentiation of F4/80 Mac-1 M s was critically regulated by CSF-1, and in vitro-generated http://www.jimmunol.org/ bone marrow-derived MFs induced by CSF-1 suppressed CD4+ T cell responses and induced the generation of Foxp3+ regulatory T cells in vivo. These results suggested that splenic CSF-1–dependent F4/80hiMac-1low MFs are a subpopulation of RPMs and regulate peripheral immune homeostasis. The Journal of Immunology, 2011, 186: 000–000.

acrophages (MFs) play a critical role in innate and Once the circulating monocytes (Mo) enter various tissue com- acquired immunity and can contribute proinflamma- partments, they exhibit a high degree of heterogeneity and acquire M tory or anti-inflammatory responses (1). Recently, some specific functions (4, 5). Spleen is known to contain various resident MFs populations possessing regulatory function were MF subsets, such as white pulp tingible-body MFs, marginal discovered in the airway interstitium and intestinal lamina propria zone-residing MFs, and red pulp MFs (RPMs) (4–6). RPMs by guest on September 29, 2021 (2, 3), indicating that these suppressive MFs play important roles remove self-Ags, such as blood-borne particulate matter and aged in the maintenance of immune homeostasis. However, in the erythrocytes, from the blood (6). Although T cell responses usu- steady state, the role of resident splenic MFs in T cell immune re- ally occur in the T cell zone of the white pulp, it is well known sponses is poorly understood. that numerous small patches within red pulp parenchyma (making up a total volume comparable to that in the white pulp in humans) contain mainly T cells, B cells, and RPMs (6–8), indicating the possibility of interaction between RPMs and T cells in the red *Division of Matrix Medicine, Institute for Genetic Medicine, Hokkaido University, pulp. Therefore, T cell responses occurring in the red pulp should † Sapporo 060-0815, Japan; Division of Molecular Immunology, Institute for Genetic be strictly regulated, because the majority of Ags are derived from Medicine, Hokkaido University, Sapporo 060-0815, Japan; ‡Division of Immunoreg- ulation, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, self. x Japan; Division of Immunobiology, Institute for Genetic Medicine, Hokkaido Uni- CSF-1, also known as M-CSF, is constitutively produced by versity, Sapporo 060-0815, Japan; {Department of Internal Medicine II, University Hospital Regensburg, 93042 Regensburg, Germany; ‖Department of Molecular Cell several types of cells, including fibroblasts, endothelial cells, stro- Biology, Vrije Universiteit Medical Center, 1081 BT Amsterdam, The Netherlands; mal cells, and MFs (9, 10). The major function of CSF-1 is to elicit # and Department of Experimental Pathology, Institute for Frontier Medical Sciences, the differentiation and development of various tissue-resident Kyoto University, Kyoto 606-8507, Japan MFs (9, 10). CSF-1–deficient mice, which are also known as Received for publication April 26, 2010. Accepted for publication December 14, 2010. op/op mutant mice, have a residual MF population (CSF-1– independent MFs). Previous studies showed that op/op mice were This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T.U.). capable of inducing normal humoral and cellular immunity, Address correspondence and reprints requests to Dr. Toshimitsu Uede and Dr. Junko but they contained lower levels of TNF-a and G-CSF following Morimoto, Division of Molecular Immunology, Institute for Genetic Medicine, Hok- immunization with sheep RBCs, suggesting that the CSF-1– kaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan. E-mail ad- independent MF population is primarily responsible for the dresses: [email protected] and [email protected] classical APC population, whereas the CSF-1–dependent MF The online version of this article contains supplemental material. population has the potential to regulate immune response (11, 12). Abbreviations used in this article: clod-lip, clodronate liposomes; FCM, flow cytometry; GMDC, bone marrow-derived dendritic cells induced by GM-CSF; IHC, im- Consistent with this notion, a recent report showed that in a model munohistochemistry; MF, macrophage; M-MF, bone marrow-derived macrophages of graft-versus-host disease, mice in which CSF-1–dependent induced by CSF-1; Mo, monocyte; MOG, myelin oligodendrocyte glycoprotein; MFs were depleted using an Ab against CSF-1R had a decreased RPM, red pulp macrophage; SSC, side scatter; Treg, regulatory T cell. number of Foxp3+ regulatory T cells (Tregs), which resulted in Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 accelerated pathology and exaggerated donor T cell activation

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001345 2 REGULATORY RED PULP MFs

(13). Although RPMs in op/op mice were reduced to ∼50% of those negative selection system, and the cells were irradiated by x-ray (3000 in the normal littermates, these mice still had F4/80+ RPMs, sug- rad). gesting that RPMs are composed of CSF-1–dependent and -inde- Cell culture pendent populations (14–16). However, the phenotypical and func- 3 6 tional differences between CSF-1–dependent and -independent Isolated MFs were adjusted to 1.5 or 2.0 10 cells/ml in TIL media (IBL), supplemented with 10% FCS and cultured in 96-well flat-bottom RPMs remain largely unknown. dishes at 37˚C at 5% CO for 48 or 72 h. The cultured supernatants were hi low 2 Previous studies showed that splenic F4/80 Mac-1 MFs exist used for ELISA (48 h) and T cell-stimulation experiments (72 h). For TLR in the red pulp of the spleen (3, 5, 17–20). In this study, we first stimulation of MFs, purified F4/80hiMac-1low MFs and F4/80+Mac-1hi hi low 3 6 isolated splenic F4/80 Mac-1 MFs and analyzed how T cell Mo/MFs were adjusted to 1.0 10 cells/ml in TIL media, supplemented m responses are regulated by F4/80hiMac-1low MFs in vivo and with 10% FCS and LPS (1 g/ml; IBL) or CpG-B dinucleotides hi low (ODN1668, 3 mg/ml; system science) and cultured for 48 h. The culture in vitro. F4/80 Mac-1 MFs showed a different expression of supernatant was harvested and used for ELISA. To generate M-MFsor + hi surface molecules compared with splenic F4/80 Mac-1 Mo/MFs, bone marrow-derived dendritic cells (GMDCs), bone marrow cells were which are capable of inducing strong T cell immune responses. isolated from the femurs and tibias of mice and cultured in RPMI 1640 Purified F4/80hiMac-1low MFs produced suppressive cytokines, media, supplemented with 10% FCS, 100 U/ml penicillin, and 100 mg/ml + streptomycin in the presence of 20 ng/ml CSF-1 or GM-CSF (WAKO). such as TGF-b and IL-10, and suppressed CD4 T cell prolifera- After 3 d, the adherent cells were collected and cultured for 2 more days in hi low tion in vitro. Moreover, F4/80 Mac-1 MFs induced the differ- the presence of fresh CSF-1 or GM-CSF (20 ng/ml) and then the cells were entiation of naive CD4+ T cells into Foxp3+ Tregs via a TGF-b– collected by cell scrapers and used in subsequent experiments. For the hi low dependent mechanism. Additionally, the differentiation of F4/80hi stimulation of lymphocytes, APCs, such as purified F4/80 Mac-1 MFs, F4/80+Mac-1hi M /MFs, CD4 T cell-deleted splenocytes as positive low hi o Downloaded from Mac-1 MFs was highly regulated by CSF-1, because F4/80 3 low control, M-MFs, or GMDCs, were cultured with 2D2-CD4 T cells (1 Mac-1 MFs were not present in op/op mice. In vitro-generated 5 10 ) and MOG35255 peptide (MEVGWYRSPFSRVVHLYRNGK: 20 mg/ bone marrow-derived MFs induced by CSF-1 (M-MFs) showed an ml) in 96-well round-bottom plates. For measuring the proliferation, [3H] expression pattern of surface molecules similar to F4/80hiMac-1low methyl thymidine was added to cells during the last 18 h of culture. In MFs. Furthermore, M-MFs suppressed CD4+ T cell proliferation some studies, anti–TGF-b mAb (1D11 from R&D Systems), isotype control Ab, or human rTGF-b (PeproTech) was included in the cultures at and induced the generation of Foxp3+ Tregs in vitro and in vivo. the indicated concentration. In other experiments, splenic Mo/MFs were hi low + hi These data suggested that CSF-1–dependent F4/80 Mac-1 MFs cultured with 2D2 naive CD4 CD62L T cells and MOG35–55 peptide in http://www.jimmunol.org/ are a subpopulation of RPMs and that they regulate T cell immune the absence or presence of F4/80hiMac-1low MFs or culture supernatant + + responses by several distinct mechanisms and maintain peripheral (100 ml). For suppression assays, sorted CD4 EGFP Tregs were cultured 3 4 + 2 3 4 immune homeostasis. with 5 10 freshly isolated CD4 CD25 naive T cells, 5 10 irradiated APCs, and 0.5 mg/ml anti-CD3 mAb (Biolegend). Cells were cultured for 96 h, and the T cell proliferation was determined as described above. Materials and Methods In vivo CD4+ T cell priming Animals For in vivo CD4+ T cell priming, 5 3 106 MACS-sorted naive 2D2-CD4+ C57BL/6 and BALB/c male mice were purchased from Japan SLC (Shi- T cells were injected i.v. into normal C57BL/6 recipients. In some by guest on September 29, 2021 3 6 + zuoka, Japan). C57BL/6-Tg (Tcra2D2,Tcrb2D2)1Kuch/J mice (transgenic experiments (Foxp3-expression assay), 2 10 FACS-sorted CD4 CD25- for the TCR Va3.2 and Vb11 chains reactive to myelin oligodendrocyte (2D2) T cells were injected into the C57BL/6 recipients. At 24, 48, and 72 h after T cell transfer, the recipient mice received 0.5–1 3 106 M-MFs glycoprotein [MOG]35–55), commonly known as 2D2 mice (21), and B6; C3Fe a/a-Csf1op/J mice, heterozygous for the osteopetrotic mutation (op/ or GMDCs pulsed with MOG35–55 peptide. These cells were prepared as wt) (14), were purchased from Jackson Laboratory. Foxp3-EGFP knockin described above and pulsed with MOG35–55 peptide (50 mg/ml) for 2.5 h at 37˚C before transfer. At 7 d following T cell transfer, the spleens were mice (BALB/c background), produced by a standard method based on + a previous report (22), were obtained from Kyoto University. All mice used removed, and Ag-specific CD4 T cells were detected by flow cytometry (FCM) using TCR Va3.2 and Vb11 mAbs. In another experiment, 2 3 106 were 7–9 wk old and were maintained under specific pathogen-free con- + ditions in our animal facility and were studied using a protocol approved MACS-sorted naive 2D2-CD4 T cells were injected i.v. into normal C57BL/6 recipients. On days 1–3 after T cell transfer, recipient mice re- by the Hokkaido University Committee for Animal Use and Care. 6 ceived 0.5–1 3 10 M-MFs pulsed with MOG35–55 peptide, followed by 6 Cell isolation GMDC injection (1 3 10 ) on days 7–9. Fourteen days after T cell transfer, the spleens were removed, and the numbers of Ag-specific CD4+ hi low + hi To obtain F4/80 Mac-1 MFs and F4/80 Mac-1 Mo/MFs, splenocytes T cells were analyzed by FCM. were prepared by collagenase D treatment (Roche) and incubated with biotinylated anti-mouse a9 integrin mAb for 20 min. After washing, IMag Flow cytometry Streptavidin Particles (BD Bioscience) were added to the cell suspension. a The tube containing this labeled cell suspension was placed within the mAbs to 9 integrin (18R18D) (23) and CCR2 (clone MC21) (24) were magnetic field of a BD IMagnet. The a9 integrin+ fraction contained F4/ used. Anti-mouse F4/80 (Cl:A3-1) and Dectin-2 (D2.11E4) mAbs were hi low + 2 a 80 Mac-1 a9 MFs with ∼80% purity. Because the a9integrin purchased from Serotec. Anti-mouse CD8 (53-6.7), CD11b (M1/70), fraction contained many non-M /MF cells, Mac-1+ cells were enriched CD49d (R1-2), CD62L (MEL-14), and ICAM-1 (3E2) mAbs were pur- o chased from BD Bioscience. Anti-mouse MHC class I (28-14-8), MHC using mouse CD11b (Mac-1) MicroBeads (Miltenyi Biotec), according to the manufacturer’s protocol (these cells are Mac-1+a92 cells). Next, these class II (M5/114.15.2), and TLR9 (M9.D6) mAbs were purchased from two populations were stained with anti-F4/80 and Mac-1 mAbs, and the eBioscience. Anti-mouse CD44 (IM7), CD80 (16-10A1), CD86 (GL-1), F4/80hiMac-1lowa9+ (F4/80hiMac-1low MFs) and F4/80+Mac-1hia92side CD11a (M17/4), VCAM-1 (429), TLR-4 (MTS510), and Foxp3 (150D) low + hi mAbs were purchased from Biolegend (San Diego, CA). For intracellular scatter (SSC) (F4/80 Mac-1 Mo/MFs) cells were purified using a FACS Vantage SE (BD Bioscience). F4/80+Mac-1hi cells are composed of staining, cells were incubated for 20 min at 4˚C in Cytofix/Cytoperm so- low hi lution (BD Bioscience) and were stained with anti-Foxp3, TLR9, and SSC Mo/MFs and SSC eosinophils (18). Because the two populations cannot be distinguished only by the expression of F4/80 and Mac-1, we isotype-control mAbs. Splenic cells were evaluated with FACSCalibur and hi + hi FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo excluded SSC eosinophils from F4/80 Mac-1 Mo/MFs using an SSC parameter in all experiments. Naive CD4+ T cells were isolated from 2D2 software (Tree Star). mice with anti-CD4 microbeads (Miltenyi Biotec), according to the Immunohistochemistry analysis manufacturer’s protocol, and then were stained with anti-CD4, CD62L, or CD25 mAbs. The CD4+CD62Lhi (2D2-CD4) cells were sorted using a mAbs used for immunohistochemistry (IHC) included anti mouse-F4/80 FACSVantage SE. In some experiments, we sorted the CD4+CD252 cells (Cl:A3-1) from Biolegend (San Diego, CA), anti-CD169 (MOMA-1) from the spleen of 2D2 mice. Purity of all sorted populations was .95%. from Serotec, and anti-mouse CD3ε (145-2C11), CD11c (HL3), and The splenic APCs that we used were obtained using a CD4-microbeads B220 (RA3-6B2) from BD Bioscience. Goat anti-Syrian hamster IgG- The Journal of Immunology 3 biotin and goat anti-rat IgG-biotin for secondary Abs were purchased from Jackson ImmunoResearch Laboratories. Spleens were embedded in OCT compound (SAKURA), and 10-mm sections were prepared. The sections were fixed in acetone for 7 min, washed three times with PBS, and blocked with 10% normal goat serum for 30 min, followed by avidin/biotin blocking (Nichirei) for 10 min. After washing three times, the sections were stained with primary Abs for 1 h. Endogenous peroxidase was depleted by incubating sections for 5 min in 0.6% H2O2 in PBS. Bound Abs were detected with biotin-conjugated goat secondary Abs. Staining was amplified using a Vectastain-ABC kit (Vector Laboratories), according to the manufacturer’s recommendations, followed by diaminobenzidine (DAKO) visualization of peroxidase activity.

Quantitative and nonquantitative RT-PCR Total RNA was isolated using TRIzol reagent (Invitrogen). Reverse transcription was performed on 5 mg total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics), according to the manufacturer’s directions. From these cDNA pools, specific targets amplified by PCR were quantified with a Lightcycler FastStart DNA Master SYBR FIGURE 1. The phenotype of F4/80hiMac-1low MFs. A, The expressions Green I kit (Roche Diagnostics). The results were normalized to G3PDH. of F4/80 and Mac-1 on splenocytes were determined by FCM. Numbers The following primers were used: G3PDH, 59-ACC ACA GTC CAT GCC hi low + hi indicate the percentages of F4/80 Mac-1 MFs or F4/80 Mac-1 Mo/ Downloaded from ATC AC-39 (sense) and 59-TCC ACC ACC CTG TTG CTG TA-39 (anti- MFs within the splenocytes. SSChi eosinophils were excluded in all sense); Foxp3, 59-TTC ATG CAT CAG CTC TCC AC-39 (sense) and 59- experiments. B, The expressions of various surface molecules and in- CTG GAC ACC CAT TCC AGA CT-39 (antisense); and TGF-b2, 59-CCA + hi hi low CCT CCC CTC CGA AAA-39 (sense) and 59-AGACATCAAAGCG- tracellular receptors by F4/80 Mac-1 Mo/MFs and F4/80 Mac-1 MFs GACGATTCT-39 (antisense). were analyzed by FCM. Shaded graphs represent isotype-matched mAb- stained control. Data are representative of three independent experiments. Phagocytosis + For phagocytosis assays, fluorescein-conjugated zymosan A (Saccharo- Listeria monocytogenes induced strong Ag-specific CD4 and http://www.jimmunol.org/ + myces cerevisiae) (Molecular Probes) was opsonized by fresh mouse se- CD8 T cell responses in vitro, indicating that these cells possess rum at 37˚C for 1 h. Isolated MFs(13 105 cells) were plated onto 96-well a strong Ag-priming activity in vivo (27–29). First, we examined plates and adhered for 1 h at 37˚C. Then, opsonized zymosans were added the expression of surface and intracellular molecules on F4/80hi to wells at a concentration of 20 mg/ml and incubated for 3 h at 37˚C, and low nonphagocytosed particles were washed out with PBS containing EDTA Mac-1 MFs. The expression levels of classes I and II MHC and trypsin. The cells were then detached and analyzed by FCM (25). molecules, costimulatory molecules (CD80 and CD86), TLRs Surface-attached zymosans were quenched by trypan blue solution. FITC- (TLR4 and TLR9), a4 integrin, and ICAM-1 were very similar labeled zymosan (400 mg) was injected i.v. in normal mice for in vivo hi low + hi between F4/80 Mac-1 MFs and F4/80 Mac-1 Mo/MFs. F4/ phagocytosis experiments. The animals were killed at 27 h following the + hi hi low 80 Mac-1 Mo/MFs, but not F4/80 Mac-1 MFs, expressed i.v. injection of FITC-labeled zymosan, and the splenocytes were re- by guest on September 29, 2021 covered. Then, the cells were stained with anti-F4/80 and Mac-1 mAbs and LFA-1 and inflammatory chemokine receptor CCR2 at high levels, hi analyzed by FCM. The uptake was measured by cellular fluorescence. as previously reported (3). In contrast, a majority of F4/80 Mac- 1low MFs expressed VCAM-1 (20), dectin-2 (18), and a9 integrin Clodronate liposome injection + hi with autofluorescence, whereas F4/80 Mac-1 Mo/MFs did not Clodronate was a gift of Roche Diagnostics (Mannheim, Germany). C57BL/ (Fig. 1B, Supplemental Fig. 1). 6 mice received i.v. administration of 0.2 ml clodronate liposomes (clod-lip) Because we detected a9 integrin expression on F4/80hiMac-1low and were killed at 1 or 10 d thereafter. Spleens were removed and processed. MFs by FCM analysis, we performed IHC using a mAb for a9 integrin to confirm whether a9 integrin can be useful as a marker ELISA for F4/80hiMac-1low MFs. As expected, the staining of a9 integrin Cytokine levels in supernatants of in vitro-cultured cells were measured by was restricted to red pulp cells, and the staining pattern of a9 ELISA (IL-1a, IL-2, IL-4, IL-6, IL-10, IL-12p40, IL-12p70, IFN-g, and integrin was very similar to that of F4/80, with a typical RPM TGF-b from BD Biosciences). ELISA assays were performed according to the manufacturer’s protocol. pattern (Supplemental Fig. 2), indicating that a9 integrin can be useful as a marker for F4/80hiMac-1low MFs in the red pulp. Statistical analysis F4/80hiMac-1low MFs have a strong phagocytic activity A statistical comparison between the groups was performed with a two- + tailed paired Student t test. Data were considered statistically significant A previous report demonstrated that F4/80 RPMs are required for at p , 0.05. clearance of RBCs (6). To examine the activity of phagocytosis by F4/80hiMac-1low MFs in vitro, we purified F4/80hiMac-1low MFs + hi Results and F4/80 Mac-1 Mo/MFs from the spleen using anti-a9 integrin, F4/80, and Mac-1 mAbs and incubated them with opsonized hi low F a F4/80 Mac-1 M s express APC-related molecules and 9 FITC-labeled zymosan. Although F4/80hiMac-1low MF and F4/80+ integrin hi Mac-1 Mo/MF populations phagocytosed FITC-labeled zymo- It was reported that splenic F4/80hiMac-1low cells are MFs that are san, the phagocytic activity of F4/80hiMac-1low MFs was stronger + hi + located in the red pulp (3, 5, 17–20). We performed FCM analysis than that of F4/80 Mac-1 Mo/MFs, as evidenced by FITC cells to examine the role of F4/80hiMac-1low MFs on T cell responses. (Supplemental Fig. 3A). Furthermore, to examine the phagocytic Consistent with the previous reports, we detected F4/80hiMac-1low activity of F4/80hiMac-1low MFs in vivo, we injected FITC-labeled MFs and F4/80+Mac-1hi cells in the spleen (Fig. 1A). It is well zymosan into mice. Twenty-seven hours later, FITC-fluorescence + hi hi low + hi known that F4/80 Mac-1 cells are a splenic Mo (17–19) and MF was detectable in F4/80 Mac-1 MFs and F4/80 Mac-1 Mo/ (3, 26) population that can produce inflammatory cytokines and MFs (Supplemental Fig. 3B). These results suggested that F4/80hi have a strong Ag-presenting ability (3, 26). Freshly isolated Mac-1low MFs have a strong phagocytic activity in vitro and splenic F4/80+Mac-1hi MFs from mice that were infected with in vivo. 4 REGULATORY RED PULP MFs

F4/80hiMac-1low MFs produce suppressive cytokines MFs produced IL-12p40 in response to CpG-B stimulation and spontaneously or in response to TLR stimuli IL-6 in response to LPS and CpG-B stimulation, as expected (3, hi low Recent evidence points to a central role of APCs in fine-tuning the 26). Importantly, F4/80 Mac-1 MFs constitutively produced quality of the immune response by their cytokine secretion (30). TGF-b1, and the production of TGF-b1 was not altered, even after LPS and CpG-B stimulation (Fig. 2B). These data indicated that Therefore, we evaluated the ability of cytokine expression in F4/ hi low hi low + hi F4/80 Mac-1 MFs produced suppressive cytokines, such as 80 Mac-1 MFs. Freshly isolated splenic F4/80 Mac-1 Mo/ MFs spontaneously produced IL-6 and IL-12p40 in vitro, con- TGF-b and IL-10. hi low sistent with previous reports (3, 26). In contrast, F4/80 Mac-1 F4/80hiMac-1low MFs inhibit CD4+ T cell proliferation and MFs spontaneously produced IL-1a, IL-12p70, and, importantly, induce the differentiation of Foxp3+ Tregs via TGF-b TGF-b1 (Fig. 2A). Additionally, constitutive TGF-b2 mRNA ex- hi low pression was detected in F4/80hiMac-1low MFs at high levels Because of the data showing that F4/80 Mac-1 MFs had phagocytic activity and produced anti-inflammatory cytokines, (Supplemental Fig. 4). hi It was shown that F4/80+ RPMs and marginal zone MFs se- such as TGF-b and IL-10, we further investigated whether F4/80 Mac-1low MFs can regulate T cell responses. To address this creted TGF-b following injection of zymosan, which is a stimulus + for TLR2/6 (27). Therefore, we next evaluated the cytokine pro- question, we used naive CD4 T cells from 2D2-TCR transgenic hi low mice, expressing transgenic TCR specific for MOG35–55 epitope files of purified F4/80 Mac-1 MFs after stimulation with LPS + hi low hi low (21). Naive 2D2-CD4 T cells were cultured with F4/80 Mac-1 (TLR4 ligand) and CpG-B (TLR9 ligand). F4/80 Mac-1 MFs + hi showed marked production of IL-1a and IL-10 in response to LPS MFs or F4/80 Mac-1 Mo/MFs in the presence of MOG35–55 + hi peptide; T cell proliferation and IL-2 production were assessed. Downloaded from and CpG-B stimulation (Fig. 2B). In contrast, F4/80 Mac-1 Mo/ + hi As previously reported (3), F4/80 Mac-1 Mo/MFs induced strong T cell proliferation, which was comparable to that induced by splenic APCs containing dendritic cells, and showed strong IL- 2 production (Fig. 3A). 2D2-CD4+ T cells activated by F4/80+ hi Mac-1 Mo/MFs also produced large amounts of IFN-g and IL-4 hi low (Fig. 3B). In sharp contrast, F4/80 Mac-1 MFs failed to induce http://www.jimmunol.org/ the proliferation of 2D2-CD4+ T cells, consistent with small amounts of IL-2 production and undetectable levels of IFN-g and IL-4 production (Fig. 3A,3B). It should be pointed out that F4/80hi Mac-1low MFs are not simply incapable of presenting Ag, because a majority of 2D2-CD4+ T cells cultured with F4/80hiMac-1low MFs showed a CD44hi phenotype (Fig. 3C). Next, we tried to understand the reason why F4/80hiMac-1low MFs were unable to induce Ag-specific CD4+ T cell proliferation. Because F4/80hi Mac-1low MFs spontaneously produced a large amount of TGF-b by guest on September 29, 2021 (Fig. 2, Supplemental Fig. 4), we hypothesized that TGF-b may be involved in poor T cell proliferation. Indeed, T cell proliferation induced by F4/80hiMac-1low MFs was partially, but significantly, rescued when TGF-b was neutralized. Meanwhile, T cell pro- + hi liferation induced by F4/80 Mac-1 Mo/MFs was not affected by the anti–TGF-b Ab (Fig. 3D). Because F4/80hiMac-1low MFs also secreted suppressive cytokine IL-10 (Fig. 2), we tested the suppressive role of IL-10. We found that the T cell proliferation was partially, but significantly, rescued by anti–IL-10R mAb treatment (Supplemental Fig. 5), indicating the additional involvement of IL-10 in suppression of the T cell response. We next examined whether F4/80hiMac-1low MFs have a suppressive effect on T cell + hi hi proliferation induced by F4/80 Mac-1 Mo/MFs. F4/80 Mac- 1low MFs were capable of inhibiting the proliferation of 2D2- + + hi CD4 T cells induced by F4/80 Mac-1 Mo/MFs in a cell concentration-dependent manner (Fig. 3E). We then cultured na- + + hi ive 2D2-CD4 T cells with Ag-pulsed F4/80 Mac-1 Mo/MFs plus culture supernatant from nonpulsed F4/80hiMac-1low MFs. We observed that the culture supernatant from nonpulsed F4/80hi Mac-1low MFs markedly inhibited the 2D2-CD4+ T cell prolif- + hi eration induced by Ag-pulsed F4/80 Mac-1 Mo/MFs (Supple- mental Fig. 6). These data suggested that F4/80hiMac-1low MFs are capable of suppressing CD4+ T cell proliferation by producing soluble suppressive factors, including TGF-b and IL-10, with- FIGURE 2. The cytokine expression profile of F4/80hiMac-1low MFs. out Ag presentation. Purified F4/80hiMac-1low MFs and F4/80+Mac-1hi M /MFs were cultured o It was demonstrated that, upon Ag stimulation in the presence of for 48 h with medium alone (A), LPS (1 mg/ml) (B) or CpG-B (3 mg/ml). + The amounts of IL-1a, IL-6, IL-12p40, IL-12p70, IL-10, and TGF-b1in TGF-b, peripheral naive CD4 T cells are forced to differentiate hi the culture supernatants were determined by ELISA (n $ 4 per group). into Tregs expressing Foxp3 (31). To test whether F4/80 Mac- low Data are presented as the mean 6 SD. *p , 0.05, **p , 0.01, ***p , 1 MFs are capable of inducing the differentiation of naive + hi hi low + + 0.001, F4/80 Mac-1 Mo/MFs versus F4/80 Mac-1 MFs. CD4 T cells into Tregs, we cultured naive 2D2-CD4 T cells with The Journal of Immunology 5

FIGURE 3. F4/80hiMac-1low MFssuppressT cell proliferation. F4/80hiMac-1low MFs, F4/80+ hi Mac-1 Mo/MFs, and APCs (as a positive control) were purified from the spleen. MOG35–55-specific naive 2D2-CD4+ T cells were cultured with APCs, hi low + hi F4/80 Mac-1 MFs, or F4/80 Mac-1 Mo/ MFs intheabsence(white bars) orpresence(black bars) of MOG peptide (20 mg/ml) for 66 h (for proliferationassay)or72h(forELISA).A, T cell proliferation was determined by [3H]thymidine uptake during the final 18 h of culture. Cytokine levels of IL-2 (A), IFN-g, and IL-4 (B) in culture supernatants were determined by ELISA. Data are representative of three independent experiments. C, After 4 d of coculture in the presence of MOG peptide, the expression of CD44 by 2D2-CD4+ T cells was analyzed by FCM. Numbers indicate the percentage of CD44hi 2D2-CD4+ T cells within 2D2-CD4+ T cells. Data are representative of two independent experiments. D, Naive 2D2-CD4+ T cells were cultured with F4/80hiMac-1low MFs Downloaded from hi low and MOG peptide or F4/80 Mac-1 Mo/MFs and MOG peptide in the presence of anti–TGF-b or isotype-matched control Ab for 66 h, and T cell proliferation was determined by [3H]thymidine uptake during the final 18 h of culture. Data are representative of three independent experi- http://www.jimmunol.org/ ments. E, Naive 2D2-CD4+ T cells were cultured with indicated numbers of F4/80+Mac-1hi

Mo/MFs and MOG peptide in the presence of the indicated numbers of F4/80hiMac-1low MFs for 66 h, and T cell proliferation was determined by [3H]thymidine uptake during the ﬁnal 18 h of culture. Data are representative of three independent experiments. Data are presented as the mean 6 SD. *p , 0.05, **p , 0.01, ***p , + hi hi 0.001, F4/80 Mac-1 Mo/MFs versus F4/80 by guest on September 29, 2021 Mac-1low MFs. ND, not detectable.

F4/80hiMac-1low MFs pulsed with MOG peptide and analyzed F4/80hiMac-1low MFs have the ability to induce the generation of Foxp3 expression in 2D2-CD4+ T cells by RT-PCR and FCM. functional Foxp3+ Tregs from naive CD4+T cells. Indeed, F4/80hiMac-1low MFs, but not F4/80+Mac-1hi M /MFs, o hi low F induced Foxp3 expression in 2D2-CD4+ T cells (Fig. 4A,4B). As F4/80 Mac-1 M s are a CSF-1–dependent RPM shown in Fig. 4C, Foxp3 expression in 2D2-CD4+ T cells induced subpopulation by F4/80hiMac-1low MFs was inhibited when anti–TGF-b Ab was It was reported that op/op mice exhibited ∼50% reduction in F4/ added to the culture. Furthermore, when rTGF-b1 was exoge- 80+ RPMs, as evaluated by IHC (14–16). We used op mutant mice nously added to the cultures, F4/80hiMac-1low MFs enhanced the to examine the necessity of CSF-1 for F4/80hiMac-1low MF de- generation of Foxp3+ T cells from naive CD4+ T cells. In contrast, velopment. F4/80hiMac-1low MF proportion and numbers were the generation of the Foxp3+ T cell population by F4/80+Mac-1hi substantially reduced in op mutant mice, depending on their hi low + hi Mo/MFs was signiﬁcantly less than that by F4/80 Mac-1 MFs, genotypes (Fig. 5A). In contrast, F4/80 Mac-1 Mo/MF pro- even after rTGF-b1 was added (Fig. 4D). However, it remains to portion was unaffected, although their absolute numbers were be clariﬁed whether Foxp3+ Tregs generated by F4/80hiMac-1low slightly reduced in op mutant mice, as previously reported (Fig. MFs have suppressive functions. Because live Foxp3+ cells can- 5A) (9). These results clearly indicated that the generation and not be isolated based on Foxp3 expression in the absence of a development of splenic F4/80hiMac-1low MFs are dependent on reporter protein, we could not examine the suppressive function of CSF-1. To understand an additional aspect of F4/80hiMac-1low the Foxp3+CD4+ T cells generated using the experimental setup MF development, we injected clod-lip i.v. into normal mice. As described above. Therefore, to test the ability of Foxp3+ T cells expected, this completely eliminated F4/80+ RPMs by day 1, as induced by F4/80hiMac-1low MFs to suppress the proliferation of demonstrated by IHC (Fig. 5B) (16). In addition, by using FCM, + 2 + hi low + hi naive CD4 T cells, EGFP CD4 T cells were isolated from we found that F4/80 Mac-1 MF and F4/80 Mac-1 Mo/MF Foxp3-EGFP mice and were activated in the presence of F4/80hi populations were almost completely depleted compared with those Mac-1low MFs and anti-CD3 mAb. F4/80hiMac-1low MFs, but not in mice treated with PBS-encapsulated liposomes. At day 10, we + hi + + + F4/80 Mac-1 Mo/MFs, led to the generation of Foxp3 CD4 detected the substantial, but not complete, repopulation of F4/80 T cells (Supplemental Fig. 7A). Then, we sorted the Foxp3+CD4+ RPMs by using IHC, despite the fact that the F4/80hiMac-1low MF population and used it to examine the suppressive activity of Tregs population still was not detectable by FCM (Fig. 5B). In contrast, hi low + hi induced by F4/80 Mac-1 MFs (Supplemental Fig. 7B). We the F4/80 Mac-1 Mo/MF population reappeared at day 10 using found that induced Foxp3+CD4+ T cells potently inhibited naive FCM, again indicating that the appearance of two splenic MF and + CD4 T cell proliferation (Supplemental Fig. 7C). Taken together, Mo/MF populations is differentially regulated. Thus, our present 6 REGULATORY RED PULP MFs

MFs failed to elicit a CD4+ T cell-proliferative response (Fig. 6B), whereas GMDCs induced a strong proliferative response, as previously reported (10). In addition, M-MFs inhibited the proliferation of CD4+ T cells induced by GMDCs (Fig. 6C). Similar to the ﬁndings in splenic F4/80hiMac-1low MFs (Fig. 3C), M-MFs were able to present Ag to 2D2-CD4+ T cells, because CD44 expression was upregulated in 2D2-CD4+ T cells (Fig. 6D). Fur- thermore, M-MFs, but not GMDCs, induced the expression of Foxp3 in CD4+ T cells via a mechanism partially dependent on TGF-b (Fig. 6E, Supplemental Fig. 8), consistent with previous reports on human Mo-derived MFs induced by CSF-1 (33). Thus, in vitro-generated M-MFs were phenotypically and functionally similar to splenic F4/80hiMac-1low MFs. We then decided to use M-MFs to analyze whether the in vitro suppressive effect of splenic F4/80hiMac-1low MFs on the CD4+ T cell response could also operate in vivo. To address this question, normal C57BL/6 recipients were transferred with naive 2D2- CD4+ T cells, followed by M-MFs or GMDCs pulsed with MOG

peptide. On day 7 after T cell transfer, the absolute number of Downloaded from 2D2-CD4+ T cells in the spleen was assessed (Fig. 6F). We found that Ag-speciﬁc T cell proliferation was not induced in recipients treated with M-MFs, because the number of 2D2-CD4+ T cells determined by TCR Va3.2 and Vb11 expression was comparable to that in recipient mice treated with PBS (Fig. 6G). The inability

of M-MFs to induce in vivo T cell proliferation was not due to the http://www.jimmunol.org/ absence of function for Ag presentation, because a signiﬁcant proportion of 2D2-CD4+ T cells in recipient mice treated with M- MFs showed the CD44hi phenotype (Fig. 6H). In contrast, 2D2- CD4+ T cells transferred into recipient mice treated with GMDCs hi low FIGURE 4. F4/80 Mac-1 MFs induce generation of Foxp3-ex- showed robust expansion and the CD44hi phenotype (Fig. 6G, pressing Tregs from naive CD4+ T cells. Naive 2D2-CD4+ T cells were 6H). Furthermore, 2D2-CD4+ T cells primed by M-MFsinvivo cultured with F4/80hiMac-1low MFs and MOG peptide or F4/80+Mac-1hi expressed Foxp3, but T cells primed by GMDCs did not (Fig. 6I). Mo/MFs and MOG peptide. A, At 3 d after coculture, Foxp3 mRNA levels in T cells were assessed by RT-PCR (normalized to G3PDH). Data are Finally, we examined whether the difference in the proportion of

+ by guest on September 29, 2021 representative of two independent experiments, B, Foxp3 protein expres- Foxp3 T cells between M-MF– and PBS-treated mice correlated sions in T cells were analyzed by FCM. Plots were gated on CD4+ cells. with the in vivo immune-inhibitory activity when rechallenged Numbers represent the percentage of Foxp3+CD4+ T cells within CD4+ with GMDCs (Supplemental Fig. 9A). We found that PBS-primed T cells. Data are representative of four independent experiments. C, Naive mice, which contained smaller proportions of Foxp3+ T cells, + + hi + 2D2-CD4 T cells were cultured with F4/80 Mac-1 Mo/MFs and MOG exhibited significant expansion of Ag-specific CD4 T cells. In hi low peptide or F4/80 Mac-1 MFs and MOG peptide in the presence of contrast, Ag-pulsed M-MF–primed mice, which contained a anti–TGF-b mAb or isotype-matched control Ab for 4 d, and Foxp3 greater proportion of Foxp3+ cells, exhibited little, if any, expan- expressions in CD4+ T cells were analyzed by FCM. Plots are gated on + + + sion of Ag-specific T cells (Supplemental Fig. 9B). Collective- CD4 T cells. Numbers represent the percentages of Foxp3 CD4 T cells hi low + F within CD4 T cells. Data are representative of four independent experi- ly, these data strongly supported that splenic F4/80 Mac-1 M s + + hi are able to regulate immune responses, and their generation is ments. D, Naive 2D2-CD4 T cells were cultured with F4/80 Mac-1 Mo/ MFs and MOG peptide or F4/80hiMac-1low MFs and MOG peptide in the regulated by CSF-1. presence of the indicated concentration of recombinant human TGF-b1 (rhTGF-b) for 4 d, and Foxp3 expressions in CD4+ T cells were analyzed by FCM. Plots are gated on CD4+ T cells. Numbers represent the per- Discussion centages of Foxp3+CD4+ T cells within CD4+ T cells. Data are repre- In the spleen, the blood vascular system is designed to allow most of sentative of four independent experiments and represent the mean 6 SD. the blood to flow directly into the red pulp, and RPMs actively , + hi hi low *p 0.05, F4/80 Mac-1 Mo/MFs versus F4/80 Mac-1 MFs. phagocytose and remove copious amount of aging and injured RBCs and blood-borne particulates from the blood (6, 34). However, many data clearly demonstrated that F4/80hiMac-1low MFs, previously scattered T cells exist in the red pulp (Supplemental Fig. 10) (7, 8). recognized as RPMs (3, 5, 17–20), are a subpopulation of RPMs, Therefore, T cell responses against such self-Ags must be regulated and their development is dependent on CSF-1. to avoid harmful autoimmune responses. In this regard, it was reported that the injection of yeast zymosan with specific Ag into F + In vitro-generated M-M s suppress CD4 T cell response mice resulted in Ag-specific T cell tolerance (30). Although this To confirm the above notion that generation of F4/80hiMac-1low tolerance induction is partially explained by TGF-b produced by MFs is CSF-1 dependent, we examined the characteristics of M- zymosan-stimulated RPMs, detailed analyses of RPMs had not MFs and generated GMDCs as a positive control for Ag pre- been performed. In this study, we analyzed how splenic F4/80hi sentation (10). The majority of M-MFs was F4/80hiMac-1+, a9 Mac-1low MFs residing in red pulp regulate T cell responses, and integrin+, but CCR2low, which is very similar to the phenotype found the potential importance of splenic F4/80hiMac-1low MFsin found in splenic F4/80hiMac-1low MFs. In contrast, GMDCs were the regulation of peripheral immune homeostasis. F4/80+Mac-1hi and expressed CCR2 (10, 32) but not a9 integrin Although it was reported that splenic F4/80hiMac-1low cells are (Fig. 6A). We next analyzed the function of M-MFs in vitro. M- MFs that are found in red pulp (5, 17, 18, 20), there was no The Journal of Immunology 7

FIGURE 5. F4/80hiMac-1low MFs are a subpopulation of RPMs, whose generation is CSF-1 dependent. A, The expressions of F4/80 and Mac-1 on splenocytes obtained from wild-type (wt), op heterozygous (op/wt), and op homozygous mice (op/op) were analyzed by FCM, and absolute numbers of F4/80hi low + hi Mac-1 MFs and F4/80 Mac-1 Mo/MFs were de- Downloaded from termined (n $ 4 per group). Numbers represent the percentages of F4/80hiMac-1low MFs and F4/80+Mac- hi 1 Mo/MFs within total splenocytes. Data are presented as the mean 6 SD. *p , 0.05, **p , 0.01, ***p , 0.001. B, Mice were injected i.v. with PBS-encapsulated liposomes (PBS-lip) or clod-lip. Spleens were removed at the indicated time points, and splenocytes were http://www.jimmunol.org/ stained with anti-F4/80 and Mac-1 mAbs, followed by FCM analysis. Numbers indicate the percentages of F4/ hi low hi low 80 Mac-1 MFs or F4/80 Mac-1 Mo/MFs within the splenocytes. Spleen sections were stained with anti- F4/80 mAb (brown). The sections were further coun- terstained with hematoxylin (blue). The borders between red pulp and white pulp are marked by black dashed lines. WP, white pulp; RP, red pulp. Data are representative of three independent experiments. Scale by guest on September 29, 2021 bars, 50 mm

detailed report on the immunological phenotype and function of that the generation and differentiation of F4/80hiMac-1low MFs these cells. In this study, we demonstrated the surface profile for were critically regulated by CSF-1, and F4/80hiMac-1low MFs are F4/80hiMac-1low MFs; they expressed MHC molecules, costim- a subpopulation of RPMs. Other resident MF populations, in- ulatory molecules, and TLRs. F4/80hiMac-1low MFs also expres- cluding muscle, kidney, and synovium MFs, are reduced in op/op sed a relatively minor integrin (a9 subunit). Splenic F4/80+Mac- mice (10, 14), indicating that factors other than CSF-1 contribute hi hi low 1 Mo/MFs expressed high levels of chemokine receptor CCR2 to the generation of splenic F4/80 Mac-1 MFs in red pulp, as (35), which is a dominant receptor for MCP-1 and a key che- well as other tissue-resident MFs. Interestingly, a recent study mokine receptor that regulates migration and infiltration of Mo and demonstrated that mice deficient in Spi-C, belonging to the Ets MFs (24). In contrast, F4/80hiMac-1low MFs did not express transcription factor family, specifically lacked splenic F4/80+ CCR2, suggesting that following the capture of Ags, they remain RPMs (20), which indicated that Spi-C might be critical for the inside the red pulp rather than migrating to inflammatory sites and development of CSF-1–dependent and -independent RPMs. In participating in inflammation. addition, M-MFs and splenic F4/80hiMac-1low MFs possess Earlier IHC studies showed that the number of F4/80+ RPMs similar phenotypes and functions. They commonly expressed a9 was reduced by half in op/op mice compared with normal litter- integrin but not CCR2. Because freshly isolated bone marrow mates, suggesting that RPMs are composed of CSF-1–dependent myeloid cells did not express a9 integrin (Supplemental Fig. 11), and -independent populations (14, 15). In this study, we found the expression of a9 integrin by splenic F4/80hiMac-1low MFs 8 REGULATORY RED PULP MFs Downloaded from http://www.jimmunol.org/

FIGURE 6. In vitro-generated M-MFs are functionally and phenotypically similar to F4/80hiMac-1low MFs. A, Bone marrow cells were cultured with CSF-1 (M-MFs) or GM-CSF (GMDCs) for 5 d, and the expressions of F4/80, Mac-1, a9 integrin, and CCR2 were analyzed by FCM. Histograms are gated on each population. Shaded histograms represent isotype-matched mAb-stained control. Numbers indicate the percentages of M-MFs or GMDCs within the total cells. Data are representative of four independent experiments. B, Naive 2D2-CD4+ T cells were cultured with M-MFs or GMDCs in the absence (white bars) or presence (black bars) of MOG peptide for 66 h, and T cell proliferation was determined by [3H]thymidine uptake during the last 18 h of culture. C, Naive 2D2-CD4+ T cells were cultured with the indicated numbers of GMDCs and MOG peptide in the presence of the indicated numbers of M- 3 + MFs for 66 h, and T cell proliferation was determined by [ H]thymidine uptake during the last 18 h of culture. D, CD44 expressions by 2D2-CD4 T cells by guest on September 29, 2021 were determined by FCM at 4 d after coculture. Numbers indicate the percentage of CD44hiCD4+ T cells within CD4+ T cells. Data are representative of two independent experiments. E, Naive 2D2-CD4+ T cells were cultured with M-MFs or MOG peptide in the presence of anti–TGF-b or isotype matched- control mAb for 4 d, and Foxp3 expressions in CD4+ T cells were analyzed by FCM. Plots are gated on CD4+ T cells. Numbers represent the percentages of Foxp3+CD4+ T cells within the CD4+ T cells. Data are representative of three independent experiments. F, B6 recipients were injected i.v. with naive 2D2- CD4+ T cells; 24, 48, and 72 h later, recipients received MOG peptide-loaded M-MFs or GMDCs i.v. Seven days after T cell transfer, spleens were collected and stained with CD4, CD44, Foxp3, TCRVa3.2, and TCRVb11 mAbs. G, The proportion of MOG-specific CD4+ T cells (Va3.2+Vb11+CD4+ cells) in the spleen was analyzed by FCM, and absolute numbers of MOG-specific CD4+ T cells were determined. H, The expression of CD44 by MOG- specific CD4+ T cells was analyzed by FCM. Numbers represent the percentage of CD44hi Va3.2+ Vb11+CD4+ T cells within the Va3.2+Vb11+CD4+ T cells. I, Foxp3 expressions in MOG-specific CD4+ T cells were analyzed by FCM and absolute numbers of MOG-specific Foxp3+CD4+ T cells were determined (n = 4 per group). Data are representative of three independent experiments and are expressed as the mean 6 SD. *p , 0.05, ***p , 0.001. may be regulated by CSF-1 during their development. It was might be cases in which F4/80hiMac-1low MFs interact with other reported that Spi-C expression in RPMs induced the expression of APCs in the red pulp to regulate excessive immune responses. VCAM-1 (20), which is a ligand for a9 integrin (36), suggesting F4/80hiMac-1low MFs are also capable of suppressing CD4+ the possibility of an interaction between a9 integrin and VCAM-1 T cell responses through induction of Foxp3+ Tregs. It is known on F4/80hiMac-1low MFs (Fig. 1B). However, we do not know that the differentiation of Tregs from naive CD4+ T cells is re- whether a9 integrin-mediated signaling plays a role in the gen- quired for TGF-b and stimulation from TCRs (31). The fact that eration and function of splenic F4/80hiMac-1low MFs. splenic F4/80hiMac-1low MFs and in vitro-generated M-MFs In this study, we found that F4/80hiMac-1low MFs are capable could induce the differentiation of naive CD4+ T cells into func- of suppressing CD4+ T cell responses by the mechanism dependent tional Foxp3+ Tregs (Figs. 4, 6E,6I) seems reasonable, because on suppressive soluble factors, because culture supernatant from these MFs have the ability to present Ag and produce a large F4/80hiMac-1low MFs efficiently suppressed T cell proliferation amount of TGF-b. We also found that Foxp3+ Tregs induced by in vitro. F4/80hiMac-1low MFs produced large amounts of anti- M-MFs strongly suppressed Ag-specific CD4+ T cell proliferation inflammatory cytokines, such as TGF-b and IL-10 (IL-10 pro- induced by GMDCs in vivo (Supplemental Fig. 9B). It should be duction by F4/80hiMac-1low MFs was further augmented following pointed out that Tregs, as well as soluble factors produced by M- TLR ligation); however, these cytokines had only a partial sup- MFs, contributed to suppression of T cell proliferation in this pressive effect on the T cell response, suggesting that other sup- experiment. pressive factor(s) might be secreted by F4/80hiMac-1low MFs. The F4/80hiMac-1low MFs induced the generation of Foxp3+ Tregs fact that F4/80hiMac-1low MFs suppressed CD4+ T cell pro- via a TGF-b–dependent mechanism (Fig. 4C). The contribution of + hi + liferation induced by F4/80 Mac-1 Mo/MFs suggested that there Foxp3 Tregs to suppression of the T cell response seemed minor The Journal of Immunology 9 in our in vitro coculture experiment (Fig. 3D), because the effect ginal zone macrophages in the spleen of osteopetrosis (op) mutant mice lacking functional M-CSF activity. J. Leukoc. Biol. 55: 581–588. of anti–TGF-b Ab treatment was partial. However, it should be 15. Cecchini, M. G., M. G. Dominguez, S. Mocci, A. Wetterwald, R. Felix, + noted that it takes .72 h to generate Foxp3 Tregs from naive H. Fleisch, O. Chisholm, W. Hofstetter, J. W. Pollard, and E. R. Stanley. 1994. CD4+ T cells following TCR and TGF-b stimulation (37, 38), and Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development of the mouse. Development 120: the regulatory event induced by Tregs occurs even before the first 1357–1372. division of target T cell (39). In our in vitro coculture experiment, 16. Yamamoto, T., C. Kaizu, T. Kawasaki, G. Hasegawa, H. Umezu, R. Ohashi, we cultured naive T cells and F4/80hiMac-1low MFs for 3 d and J. Sakurada, S. Jiang, L. Shultz, and M. Naito. 2008. Macrophage colony- + stimulating factor is indispensable for repopulation and differentiation of analyzed T cell proliferation. Therefore, it is likely that Foxp3 Kupffer cells but not for splenic red pulp macrophages in osteopetrotic (op/op) Tregs induced by F4/80hiMac-1low MFs are not capable of con- mice after macrophage depletion. Cell Tissue Res. 332: 245–256. 17. Taylor, P. R., G. D. Brown, A. B. Geldhof, L. Martinez-Pomares, and S. Gordon. tributing to the suppression of T cell proliferation because of the 2003. Pattern recognition receptors and differentiation antigens define murine delay in Treg generation. myeloid cell heterogeneity ex vivo. Eur. J. Immunol. 33: 2090–2097. In summary, our results demonstrated that the F4/80hiMac-1low 18. Taylor, P. R., D. M. Reid, S. E. Heinsbroek, G. D. Brown, S. Gordon, and + S. Y. Wong. 2005. Dectin-2 is predominantly myeloid restricted and exhibits MFs suppress autoimmune responses through regulating CD4 unique activation-dependent expression on maturing inflammatory monocytes T cell responses and inducing the generation of Tregs. It seems elicited in vivo. Eur. J. Immunol. 35: 2163–2174. likely that F4/80hiMac-1low MF also regulate excessive immune 19. Idoyaga, J., N. Suda, K. Suda, C. G. Park, and R. M. Steinman. 2009. Antibody to Langerin/CD207 localizes large numbers of CD8alpha+ dendritic cells to the responses by suppressing T cell responses induced by other red marginal zone of mouse spleen. Proc. Natl. Acad. Sci. USA 106: 1524–1529. pulp professional APCs. The generation of F4/80hiMac-1low MFs 20. Kohyama,M.,W.Ise,B.T.Edelson,P.R.Wilker, K. Hildner, C. Mejia, W. A. Frazier, T. L. Murphy, and K. M. Murphy. 2009. Role for Spi-C in the development of red pulp is strictly regulated by CSF-1, and in vitro-generated M-MFs macrophages and splenic iron homeostasis. Nature 457: 318–321. showed suppressive functions in vivo. Thus, our findings provide 21. Bettelli, E., M. Pagany, H. L. Weiner, C. Linington, R. A. Sobel, and Downloaded from a new aspect for F4/80hiMac-1low MFs and raise the possibility V. K. Kuchroo. 2003. Myelin oligodendrocyte glycoprotein-specific T cell receptor transgenic mice develop spontaneous autoimmune optic neuritis. J. Exp. that CSF-1–induced regulatory MFs may be useful for the treat- Med. 197: 1073–1081. ment of autoimmune disorders. 22. Fontenot, J. D., J. P. Rasmussen, L. M. Williams, J. L. Dooley, A. G. Farr, and A. Y. Rudensky. 2005. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22: 329–341. Acknowledgments 23. Kanayama, M., D. Kurotaki, J. Morimoto, T. Asano, Y. Matsui, Y. Nakayama,

We thank K. Danzaki and D. Ohta (Hokkaido University) for preparation of Y. Saito, K. Ito, C. Kimura, N. Iwasaki, et al. 2009. Alpha9 integrin and its http://www.jimmunol.org/ ligands constitute critical joint microenvironments for development of autoim- mice and tissue sections. mune arthritis. J. Immunol. 182: 8015–8025. 24. Mack, M., J. Cihak, C. Simonis, B. Luckow, A. E. Proudfoot, J. Plachy´, H. Bru¨hl, M. Frink, H. J. Anders, V. Vielhauer, et al. 2001. Expression and Disclosures characterization of the chemokine receptors CCR2 and CCR5 in mice. J. The authors have no financial conflicts of interest. Immunol. 166: 4697–4704. 25. Lehmann, A. K., S. Sornes, and A. Halstensen. 2000. Phagocytosis: measure- ment by flow cytometry. J. Immunol. Methods 243: 229–242. 26. Hirotani, T., P. Y. Lee, H. Kuwata, M. Yamamoto, M. Matsumoto, I. Kawase, References S. Akira, and K. Takeda. 2005. The nuclear IkappaB protein IkappaBNS se- 1. Gordon, S. 2007. The macrophage: past, present and future. Eur. J. Immunol. 37 lectively inhibits lipopolysaccharide-induced IL-6 production in macrophages of (Suppl. 1): S9–S17. the colonic lamina propria. J. Immunol. 174: 3650–3657. by guest on September 29, 2021 2. Bedoret, D., H. Wallemacq, T. Marichal, C. Desmet, F. Quesada Calvo, E. Henry, 27. Skoberne, M., R. Holtappels, H. Hof, and G. Geginat. 2001. Dynamic antigen R. Closset, B. Dewals, C. Thielen, P. Gustin, et al. 2009. Lung interstitial presentation patterns of Listeria monocytogenes-derived CD8 T cell epitopes macrophages alter dendritic cell functions to prevent airway allergy in mice. J. in vivo. J. Immunol. 167: 2209–2218. Clin. Invest. 119: 3723–3738. 28. Skoberne, M., and G. Geginat. 2002. Efficient in vivo presentation of Listeria 3. Denning, T. L., Y. C. Wang, S. R. Patel, I. R. Williams, and B. Pulendran. 2007. monocytogenes- derived CD4 and CD8 T cell epitopes in the absence of IFN- Lamina propria macrophages and dendritic cells differentially induce regulatory gamma. J. Immunol. 168: 1854–1860. and interleukin 17-producing T cell responses. Nat. Immunol. 8: 1086–1094. 29. Skoberne, M., S. Schenk, H. Hof, and G. Geginat. 2002. Cross-presentation of 4. Gordon, S., and P. R. Taylor. 2005. Monocyte and macrophage heterogeneity. Listeria monocytogenes-derived CD4 T cell epitopes. J. Immunol. 169: 1410–1418. Nat. Rev. Immunol. 5: 953–964. 30. Dillon, S., S. Agrawal, K. Banerjee, J. Letterio, T. L. Denning, K. Oswald- 5. Taylor, P. R., L. Martinez-Pomares, M. Stacey, H. H. Lin, G. D. Brown, and Richter, D. J. Kasprowicz, K. Kellar, J. Pare, T. van Dyke, et al. 2006. Yeast S. Gordon. 2005. Macrophage receptors and immune recognition. Annu. Rev. zymosan, a stimulus for TLR2 and dectin-1, induces regulatory antigen- Immunol. 23: 901–944. presenting cells and immunological tolerance. J. Clin. Invest. 116: 916–928. 6. Mebius, R. E., and G. Kraal. 2005. Structure and function of the spleen. Nat. Rev. 31. Sakaguchi, S., T. Yamaguchi, T. Nomura, and M. Ono. 2008. Regulatory T cells Immunol. 5: 606–616. and immune tolerance. Cell 133: 775–787. 7. van Krieken, J. H., J. Te Velde, J. Hermans, and K. Welvaart. 1985. The splenic 32. Nguyen, M. T., S. Favelyukis, A. K. Nguyen, D. Reichart, P. A. Scott, A. Jenn, red pulp; a histomorphometrical study in splenectomy specimens embedded in R. Liu-Bryan, C. K. Glass, J. G. Neels, and J. M. Olefsky. 2007. A subpopulation methylmethacrylate. Histopathology 9: 401–416. of macrophages infiltrates hypertrophic adipose tissue and is activated by free 8. van Krieken, J. H., and J. te Velde. 1988. Normal histology of the human spleen. fatty acids via Toll-like receptors 2 and 4 and JNK-dependent pathways. J. Biol. Am. J. Surg. Pathol. 12: 777–785. Chem. 282: 35279–35292. 9. Dai, X. M., X. H. Zong, V. Sylvestre, and E. R. Stanley. 2004. Incomplete 33. Savage, N. D., T. de Boer, K. V. Walburg, S. A. Joosten, K. van Meijgaarden, restoration of colony-stimulating factor 1 (CSF-1) function in CSF-1-deficient A. Geluk, and T. H. Ottenhoff. 2008. Human anti-inflammatory macrophages Csf1op/Csf1op mice by transgenic expression of cell surface CSF-1. Blood 103: induce Foxp3+ GITR+ CD25+ regulatory T cells, which suppress via 1114–1123. membrane-bound TGFbeta-1. J. Immunol. 181: 2220–2226. 10. Hamilton, J. A. 2008. Colony-stimulating factors in inflammation and autoim- 34. Cesta, M. F. 2006. Normal structure, function, and histology of the spleen. munity. Nat. Rev. Immunol. 8: 533–544. Toxicol. Pathol. 34: 455–465. 11. Wiktor-Jedrzejczak, W., A. A. Ansari, M. Szperl, and E. Urbanowska. 1992. 35. Auffray, C., M. H. Sieweke, and F. Geissmann. 2009. Blood monocytes: de- Distinct in vivo functions of two macrophage subpopulations as evidenced by velopment, heterogeneity, and relationship with dendritic cells. Annu. Rev. studies using macrophage-deficient op/op mouse. Eur. J. Immunol. 22: 1951– Immunol. 27: 669–692. 1954. 36. Taooka, Y., J. Chen, T. Yednock, and D. Sheppard. 1999. The integrin alpha9- 12. Wiktor-Jedrzejczak, W., B. Dzwigala, M. Szperl, M. Maruszynski, beta1 mediates adhesion to activated endothelial cells and transendothelial E. Urbanowska, and P. Szwech. 1996. Colony-stimulating factor 1-dependent neutrophil migration through interaction with vascular cell adhesion molecule-1. resident macrophages play a regulatory role in fighting Escherichia coli fecal J. Cell Biol. 145: 413–420. peritonitis. Infect. Immun. 64: 1577–1581. 37. Wan, Y. Y., and R. A. Flavell. 2005. Identifying Foxp3-expressing suppressor 13. Macdonald, K. P., J. S. Palmer, S. Cronau, E. Seppanen, S. Olver, N. C. Raffelt, T cells with a bicistronic reporter. Proc. Natl. Acad. Sci. USA 102: 5126–5131. R. Kuns, A. R. Pettit, A. Clouston, B. Wainwright, et al. 2010. An antibody 38. Chen, W., W. Jin, N. Hardegen, K. J. Lei, L. Li, N. Marinos, G. McGrady, and against the colony-stimulating factor 1 receptor (CSF1R) depletes the resident S. M. Wahl. 2003. Conversion of peripheral CD4+CD252 naive T cells to subset of monocytes and tissue- and tumor-associated macrophages but does not CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor inhibit inflammation. Blood 116: 3955–3963. Foxp3. J. Exp. Med. 198: 1875–1886. 14. Takahashi, K., S. Umeda, L. D. Shultz, S. Hayashi, and S. Nishikawa. 1994. 39. Sojka, D. K., A. Hughson, T. L. Sukiennicki, and D. J. Fowell. 2005. Early Effects of macrophage colony-stimulating factor (M-CSF) on the development, kinetic window of target T cell susceptibility to CD25+ regulatory T cell activity. differentiation, and maturation of marginal metallophilic macrophages and mar- J. Immunol. 175: 7274–7280.