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. Author manuscript; available in PMC: 2011 Mar 15.
Published in final edited form as: J Immunol. 2009 Jul 27;183(4):2232–2241. doi: 10.4049/jimmunol.0900191

CD44 co-stimulation promotes FoxP3+ regulatory T-cell persistence and function via production of IL-2, IL-10 and TGF-beta

Paul L Bollyky *,†, Ben A Falk *, Alice Long *, Anton Preisinger *, Kathy R Braun *, Rebecca P Wu *, Stephen P Evanko *, Jane H Buckner *, Thomas N Wight *, Gerald T Nepom *
PMCID: PMC3057032  NIHMSID: NIHMS264915  PMID: 19635906

Abstract

Work by our group and others has demonstrated a role for the extracellular matrix receptor CD44 and it's ligand hyaluronan in CD4+CD25+ regulatory T-cell (Treg) function. Herein we explore the mechanistic basis for this observation. Using mouse FoxP3/GFP+ Treg we find that CD44 co-stimulation promotes expression of FoxP3, in part through production of IL-2. This promotion of IL-2 production was also resistant to Cyclosporine A treatment, suggesting that CD44 costimulation may promote IL-2 production through bypassing FoxP3-mediated suppression of NFAT. CD44 co-stimulation increased production of IL-10 in a partially Il-2 dependant manner and also promoted cell-surface TGF-β expression. Consistent with these findings, Treg from CD44 knock-out mice demonstrated impaired regulatory function ex vivo and depressed production of IL-10 and cell-surface TGF-β. These data reveal a novel role for CD44 cross-linking in the production of regulatory cytokines. Similar salutary effects on FoxP3 expression were observed upon co-stimulation with hyaluronan, the primary natural ligand for CD44. This effect is dependent upon CD44 cross-linking; while both high molecular weight hyaluronan (HMW-HA) and plate-bound anti-CD44 Ab promoted FoxP3 expression, neither low-molecular weight HA (LMW-HA) nor soluble anti-CD44 Ab did so. The implication is that intact HMW-HA can cross-link CD44 only in those settings where it predominates over fragmentary LMW-HA, namely in un-inflamed tissue. We propose that intact but not fragmented ECM is capable of cross-linking CD44 and thereby maintains immunologic tolerance in uninjured or healing tissue.

Introduction

CD4+CD25+ regulatory T-cells (Treg) are a specialized subpopulation of T cells which suppress a range of effector cell types and thereby contribute to the maintenance of immune homeostasis (1,2). Studies have demonstrated an increased frequency or severity of autoimmunity in the absence of Treg and that transfer of Treg is sufficient to protect from or reverse autoimmunity (3–5). Treg –mediated suppression is cell contact dependant but the immunosuppressive cytokines TGF-ß and IL-10 also play a role (6–8). Treg make negligible amounts of IL-2 but nonetheless require this cytokine to maintain their viability and suppressive function (1).

The development and function of Treg requires the transcription factor FoxP3. Spontaneous mutation of FoxP3 leads to widespread lymphocytosis and autoimmunity in the scurfy mouse and in humans with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX)(9,10). It has been proposed that Foxp3 may function as a transcriptional repressor, potentially through the formation of both DNA-protein and protein-protein interactions with NF-AT and also NF-κB(11,12).

The relative expression of CD44 and particular CD44v isoforms was reported by ourselves and others to be associated with FoxP3 expression and Treg function(13,14). CD44 is a cell-surface molecule with important roles in activation, migration and apoptosis(15,16). The diverse roles of CD44 are thought to reflect different CD44v isoforms(17) as well as the range of other cell-surface receptors with which CD44 complexes, including Fas(18) and the CD3 complex(19). As with other cell-surface molecules reported to characterize Treg, CD44 expression is heightened on a range of activated cell types other than Treg.

A key ligand of CD44 is hyalronan (HA), a repeating disaccharide of N-acetylglucosamine and D-glucuronic acid and a prominent component of inflamed tissues (20). The relative amount of HA as well as the size of HA molecules are highly contextual and of physiologic importance (21,22). Low-molecular weight forms of HA (LMW-HA) (<15 saccharides; <3 kDa) predominate during injury and inflammation. LMW-HA breakdown products are generated from intact high-molecular weight HA (HMW-HA) (> 2,000 saccharides; > 400 kDa) by endogenous catabolism, by bacterial hyaluronidases, and by mechanical forces and oxidative stress(23). LMW-HA promotes angiogenesis (24), inflammation (25,26), maturation of antigen presenting cells (27,28) and cell migration (29). Consistent with this role LMW-HA has been shown to be a ligand of TLR4 (28). HMW-HA conversely predominates in steady-state conditions and in healing tissues (20). HMW-HA serves a variety of structural functions in joints (30,31) and tissue repair (30,31)and typically been reported to be either inert or anti-inflammatory (32–35). HA binding-competent CD44v isoforms are expressed on T-cells only after activation via the TCR or with pro-inflammatory cytokines including TNF-α and INF-γ (36,37). Therefore the ability of these cells to interact with the HA is intrinsically related to their activation state.

In our previous work we have asked whether CD44 and ligands such as HA exert direct immuno-modulatory effects on Treg. We observed that intact HMW-HA promotes Treg mediated suppression while LMW-HA, does not. We demonstrated that CD44 expression is correlated with the expression of FoxP3 as well as with HA-binding efficiency (14).

Herein we examine the mechanistic basis of these observations. We postulated that the ECM displays tissue integrity signals through CD44 which are seen by Treg as the antithesis of danger signals. We theorized that these signals might be most relevant in tissues with minimal pro-inflammatory cues. Therefore using GFP-FoxP3 knock-in mice we asked whether this co-stimulatory signal is relevant to Treg maintenance and requirement for IL-2. We asked whether this co-stimulatory signal is relevant to Treg function and to production of known regulatory cytokines, IL-10 and TGF-β. Finally using human Treg we explored the hypothesis that HMW-HA but not LMW-HA provides a co-stimulatory signal via cross-linking of CD44.

Materials and Methods

Human blood samples

Human peripheral blood samples were obtained from healthy volunteers with informed consent, participating in a research protocol approved by the institutional review board of the Benaroya Research Institute at Virginia Mason (BRI).

Mice

FoxP3-GFP C57BL/6 mice were the kind gift of Dr. Alexander Rudensky at the University of Washington (Seattle,WA). CD44 deficient C57BL/6 (CD44−/−), IL-10 deficient C57BL/6 (IL-10−/−), and wild type C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, Me). FoxP3-GFP and CD44−/− mouse lines were intercrossed to generate FoxP3-GFP CD44−/− at our institution. All mice were maintained in a specific pathogen-free AAALAC-accredited animal facility at the Benaroya Research Institute and handled in accordance with institutional guidelines.

Reagents

Hyaluronan with a molecular weight of 1.5 × 106 kDa (HMW-HA) was provided by Genzyme (Cambridge, MA). LMW-HA was prepared by digestion of HMW-HA with Streptomyces derived hyaluronidase, followed by filtration through a Centricon microconcentrator (Amicon, Inc., Beverly, MA) to produce fragments less than 3 kDa, as described previously (38). Cyclosporine A was obtained from Sigma-Aldrich (St. Louis, MO).

Human flow cytometry experiments used the following fluorochrome-labeled antibodies: CD4 (RPA-T4), CD25 (M-A251), CD44 (G44-26), CD127 (hIL-7R-M21) from BD-Biosciences (San Jose, CA) and biotinylated polyclonal TGF-β1 antibody from R&D Systems (Minneapolis, MN) with streptavidin-PerCp secondary from eBioscience (San Diego,CA). FoxP3 staining kit & isotype control antibodies from BioLegend (San Diego, CA) were used as per the manufacturer's instructions.

Human T-cell activation studies used antibodies: CD3 (OKT3) and CD28 (CD28.2) from eBioscience, CD44 (G44-26) was from BD Biosciences, and CD44 blocking mAb (Bu75) from Ancell Corp. (Bayport, MN). Recombinant IL-2 was from Chiron (Emeryville, CA) and the anti-IL-2 antibody (MQ1-17H12) was from BD-Biosciences. HMW-HA was conjugated to BSA for using previously described methods (39) for plate-bound activation studies.

Mouse flow cytometry experiments used the following fluorochrome-labeled antibodies: CD4 (RM4-5), CD25 (PC61.5), CD44 (IM7) from BD-Biosciences. FoxP3 (FJK.16a) antibody and staining reagents from eBioscience were used as per the manufacturer's instructions. The same anti-human TGF-β1 polyclonal antibody was used for mouse staining as for the human samples.

Mouse T-cell activation studies used antibodies: CD3e (145-2C11), CD28 (37.51), CD44 (IM7), anti-IL-10 (JES5-2A5), anti-TGF-β1 mAb (1D11), CD25 (PC61.5), CD278 (ICOS) (C398.4A) from BD Biosciences and eBioscience. Recombinant human TGF-β1, mouse IL-10, and mouse IL-2R alpha were from R&D Systems, and recombinant mouse IL-2 was from eBioscience. A BSA-conjugated form of HMW-HA was used for plate-bound activation studies, as noted above.

Isolation of leukocyte populations

Human PBMC were prepared by centrifugation of peripheral blood over Ficoll-Hypaque gradients. CD4+ T cells were isolated using the Dynal CD4 Positive Isolation Kit (Invitrogen) as per the manufacturer's instructions then sorted for CD25 (and with CD127 where noted) on a FACS-Vantage Flow Cytometer Cell Sorter. The top 2.5–5.0% of CD25+ cells were used for Treg studies. Purity of the resulting cell fractions was reliably >98% CD4+CD25+. Cells were cultured in RPMI (Invitrogen) supplemented with 10% pooled human serum, 100μg/ml Penicillin, 100U/ml Streptomycin, and 1mM Na pyruvate (Invitrogen).

Mouse leukocyte populations were isolated from inguinal, axial and brachial lymph nodes and spleen cells from 6 to 8 wk old mice. CD4+CD25+ and CD4+CD25− T-cell populations were isolated using a CD4+ T Regulatory Cell Isolation Kit (Miltenyi Biotec, Auburn, CA) as per the manufacturer's instructions. CD4+FoxP3/GFP+ and CD4+FOXP3/GFP− T-cells were isolated by pre-selection with a Dynal CD4+ T-cell Negative Isolation Kit (Invitrogen, Carlsbad, CA) and then sorted into both FoxP3/GFP+ and FoxP3/GFP− fractions using a FACS-Vantage Flow Cytometer Cell Sorter. Purity of the resulting cell fractions was reliably >99.9% FoxP3/GFP+. Cells were cultured in DMEM-10 (Invitrogen) supplemented with 10% FBS (Hyclone, Logan Utah), 100μg/ml Penicillin, 100U/ml Streptomycin, 50μM βme, 2mM glutamine and 1mM Na pyruvate (Invitrogen).

T-cell Activation and phenotyping assays

Human T-cell experiments were performed as follows: 96 well flat bottom tissue culture plates were pre-coated with CD3 antibody (0.5 μg/ml) and CD44 antibody (1ug/ml) where relevant. Anti-CD28 Ab was not used in the human Treg experiments unless otherwise noted. Coating with HMW-HA was treated as a second step. Plates were washed with PBS and then either 100 μl of 100 μg/ml HMW-HA or 100 μl of 10% BSA in PBS was added and the plates were incubated at 37°C for 2 hours. Plates were again washed with PBS prior to the addition of 150,000 T-cells. No exogenous IL-2 was added unless otherwise noted. T-cells cultures were analyzed by flow cytometry after 4 days. Other cell culture reagents were added at the initiation of the cultures as follows: human IL-2 antibody (10ug/ml), soluble CD44 antibody (5ug/ml) clone BU75 (blocks HA binding), soluble LMW-HA (20 μg/ml), recombinant human IL-10 (5 ng/ml), and IL-10 antibody (5ug/ml). Where noted Cyclosporine A was added at 50 ng/ml. Where noted Rapamycin was added at 100uM/ml.

Low-dose IL-2 experiments used freshly isolated human CD4+CD25+CD127- T-cells activated with anti-CD3/28 beads (1 bead/10 cells) (Invitrogen). Exogenous IL-2 was added at the concentrations indicated only at the initiation of the culture. After 36 hours the beads were removed with a magnet and the cells were transferred to wells with HMW-HA (20 μg/ml), or LMW-HA (20 μg/ml). After an additional 24 hours cells were analyzed by flow cytometry.

Mouse T-cell activation experiments were performed as follows: 96 well flat bottom tissue culture plates were pre-coated with CD3 (0.5ug/ml) and CD44 (1ug/ml) antibodies. CD28 (0.2ug/ml) antibody was added with 100,000 T-cells per well in DMEM-10 complete medium and cultured for 3 days. The CD278 (ICOS) control co-stimulation was performed by pre-coating the well with CD278 antibody (1ug/ml) in combination with anti-CD3. Other cell culture reagents were added at the initiation of the cultures as follows: mouse IL-10 (5ng/ml), mouse IL-2 (100U/ml), human TGF-B (3ng/ml), IL-10 antibody (5ug/ml), TGF-B antibody (5ug/ml), and soluble mouse IL-2R alpha (5ug/ml).

FACS samples were stained in media on ice for 45 min, washed once, resuspended in FACS stain buffer (PBS containing 1% FBS, 0.1% Na-azide), and run on a FACSCaliber flow cytometer (Becton Dickinson). Analysis was performed using CELLQuest (BD) and FlowJo (Treestar Inc., Ashland OR) software.

Cytokine analysis

Both mouse and human cell culture supernatants were analyzed for cytokines using Meso Scale Discovery (Gaithersburg, MD) MSD 96 well Multi-Array Cytokine TH1/TH2 assays for human and mouse and read on a SECTOR Imager 2400 as per the manufacturer's instructions.

Suppression Assays

Human suppression assays were performed as follows: CD4+CD25+ Treg were titrated into a combination of CFSE (0.8uM) (Molecular Probes) labeled CD4+CD25− responder T-cells (200,000 cells/ well) and irradiated CD4- cells (600,000 cells/ well) in 5ml FACS tubes (Falcon-BD), then stimulated with soluble CD3 (5ug/ml) and CD28 (2.5ug/ml) antibodies. After 4 days T-cells were analyzed by flow cytometry.

Mouse Treg suppression assays were performed as follows: purified CD4+CD25+ or CD4+FOXP3/GFP+ T-cells were titrated into a combination of CD4+CD25− or CD4+FOXP3/GFP− T-cells (4×104 cells/ well) and irradiated (2,000 rad) splenocytes (2×105/well) depleted of CD4+ T-cells, then stimulated for 72hrs with 1ug/ml Con A in 96 well round bottom plates. During the final 16 hours cells were pulsed with 1uCi/ well of [3H] thymidine, then harvested onto filter mats and counted on a 1450 Wallac microbeta scintillation plate counter (Perkin Elmer, Waltham,MA).

Quantitiative PCR

Total RNA was harvested from freshly isolated and cultured mouse CD4+FOXP3/GFP+ and CD4+FOXP3/GFP− T-cells using the RNeasy mini kit from Qiagen (Valencia, CA). cDNA was prepared from 350ng total RNA reverse transcribed in a 40ul reaction mix with random primers using the High-Capacity cDNA Archive Kit according to manufacturer's instructions. Relative quantitation of TGFbeta-1 gene expression was performed using Taqman Gene Expression Assay Mm03024053_m1 and eukaryotic 18S rRNA Endogenous Control part no.4333760. Briefly, 1.2ul cDNA was amplified in 1XTaqman Fast Universal PCR Mix with 250nM Taqman probe in a 20ul reaction using the Fast program for 50 cycles on an ABI7900HT thermocycler. (All qPCR reagents were from Applied Biosystems, Foster City, CA) All samples were done in duplicate and data was analyzed using the Comparative Ct Method with software from Applied Biosystems. Estimated copy numbers were generated from a standard curve created by using a selected reference cDNA template and Taqman probe (40).

Statistical Analysis

Statistical comparison of samples stimulated with either anti-CD3/28 alone versus in conjunction with anti-CD44 co-stimulation were made using the Wilcoxon Signed Rank test. Values of p < 0.05 were considered significant.

Results

CD44 co-stimulation promotes expression of FoxP3 and CD25 by GFP/FoxP3+ mouse Treg

To evaluate the effects of CD44 co-stimulation on Treg we first isolated FoxP3/GFP+ Treg from CD4+ T-cells (Figure 1A). These were then activated with plate-bound anti-CD3 Ab, soluble aCD28 Ab +/− plate-bound anti-CD44 Ab without exogenous IL-2 for three days prior to staining. We found that anti-CD44 co-stimulation promoted expression of FoxP3/GFP and CD25 (Figure 1B). Both anti-CD3/28 and anti-CD3/28/44 treated GFP/FoxP3+ Treg maintained GFP/Foxp3 expression to a greater degree than GFP/FoxP3− controls (Supplemental Figure 1). We found that anti-CD44 Ab alone did not promote GFP/FoxP3 expression; earlier or concomitant stimulation with anti-CD3 Ab was also required (Figure 1C). Notably only plate-bound anti-CD44 had this effect while soluble anti-CD44 did not (data not shown). While exogenous IL-2, added at 100 U/ml, had a similar effect, anti-ICOS-1 Ab treatment conversely did not (Figure 1D&E). ICOS-1 was chosen because, similar to CD44, it is a co-stimulatory molecule with important roles in T-cell activation and IL-2 production (41). It was possible to negate this effect with soluble recombinant IL-2Rα protein. This reagent was used because it has the advantage of making IL-2 unavailable for binding to cell-surface IL-2Rα without a propensity to form bioactive IL-2 dimers or signal through CD25. These data implicate a role for IL-2 in CD44 cross-linking effects on FoxP3 expression. Co-stimulation of mouse FoxP3/GFP+ Treg with anti-CD44 Ab led to enhanced Treg suppressive function (Figure 1F). HMW-HA, a natural ligand of CD44, also promoted expression of GFP/FoxP3 (Figure 1G). Interestingly the capacity of HMW-HA to promote GFP/FoxP3 required supplementation with 5% mouse serum. Without this there was no effect (data not shown).

Figure 1. CD44 cross-linking of mouse GFP/FoxP3+ Treg enhances FoxP3 and CD25 expression as well as suppressor function.

Figure 1

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(A & B) Treg expression of GFP-FoxP3 immediately upon isolation on Day 0 (A) and 3 days after (B) activation with either anti-CD3/28 or anti-CD3/28/44. Data are representative of five experiments. (C) FoxP3 MFI of Treg activated with anti-CD44 alone or in conjunction with anti-CD3/28. Expression of (D) FoxP3/GFP and (E) CD25 on mouse Treg following activation with anti-CD3/28 versus anti-CD3/28/44 or controls. Data are included from seven experiments. Mouse IL-2 was added at 100 IU/ml where noted. (F) Suppression assay comparing freshly isolated Treg (no pre-activation) against Treg pre-activated with or without plate-bound anti-CD44 co-stimulation. No data are shown for Treg activated with anti-CD3/CD28 stimulation because too few were viable after 72 hours of culture (data not shown). (G) GFP/FoxP3 MFI following activation with anti-CD3/28 alone or in conjunction with anti-CD44 or HMW-HA, a natural ligand of CD44. All conditions for the experiments in this sub-figure alone also included 5% mouse serum. Data from three experiments are included.

CD44 crosslinking promotes production of IL-2 by Treg in a manner which bypasses cyclosporine A treatment

Given the well-established role of IL-2 in Treg function and persistence, these data raised the obvious question of whether CD44 co-stimulation was promoting the availability of IL_2 or reducing the requirement of GFP/FoxP3+ Treg for this cytokine. Although small in magnitude, FoxP3/GFP+ Treg co-stimulated with anti-CD44 Ab demonstrated a modest but reproducible increase in IL-2 production over such cells stimulated with anti-CD3/28 alone (Figure 2A). Co-stimulation with anti-ICOS-A Ab did not promote IL-2 production (Figure 2B). Consistent with these data mRNA harvested from FoxP3/GFP Treg pooled from 5 mice contained increased IL-2 message in CD3/28/44 activated cells relative to CD3/28 activated controls (Figure 2C). IL-2 mRNA was not detectable pre-activation (data not shown). GFP/FoxP3+ Treg were used in order to exclude any contribution by FoxP3− cells to the effects described here. These data support the conclusion that CD44 cross-linking promoted modest IL-2 production by cells isolated on the basis of FoxP3/GFP expression.

Figure 2. CD44 cross-linking of GFP-Foxp3+ Treg promotes IL-2 production.

Figure 2

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(A) IL-2 concentrations from FoxP3/GFP+ Treg culture supernatants co-stimulated with anti-CD3/28/44 versus anti-CD3/28 alone. Supernatants were taken 72 hours after activation. Data are shown for 5 experiments. Each experiment utilized Treg pooled from 4–5 mice. (B) Fold change in IL-2 production following activation with anti-CD3/28 alone or in conjunction with anti-CD44 Ab, or anti-ICOS Ab. Data shown are for 5 experiments. (C) Fold change in IL-2 mRNA expression by FoxP3/GFP+ cells upon activation with anti-CD3/28 Ab versus anti-CD3/28/44 Ab. mRNA was isolated from CD4+FoxP3/GFP+ cells pooled from 5 animals. mRNA copy number was normalized relative to 105 18S mRNA.

Given that FoxP3 is thought to inhibit IL-2 production via inhibition of NFAT it seemed reasonable to asked whether CD44 treatment promoted Il-2 production in an NFAT independent manner. To ascertain this we added cyclosporine A to the cultures on the grounds that this molecule inhibits IL-2 production via effects on calcineurin and hence NFAT activation. We found that the addition of cyclosporine A depressed GFP/FoxP3 MFI on Treg activated with aCD3/28 but had minimal effects on GFP/FoxP3 MFI on Treg activated with aCD3/28/44 (Figure 3A). Similar effects were observed vis-à-vis Il-2 production (Figure 3B). These data indicate that anti-CD44 treatment bypasses cyclosporine A mediated inhibition of IL-2.

Figure 3. CD44 mediated effects on GFP/FoxP3 MFI and on Treg production of IL-2 are resistant to cyclosporine A treatment.

Figure 3

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(A). Fold change in FoxP3 MFI for Treg activated with anti-CD3/28 alone or in conjunction with anti-CD44 and both with or without cyclosporine A treatment. Data are for 4 experiments. B. (B) Fold change in IL-2 production following activation with anti-CD3/28 alone or in conjunction with anti-CD44 Ab and both with or without cyclosporine A treatment. Data shown are for 3 experiments.

CD44 cross-linking and exogenous IL-10 promote cell-surface TGF-β

The modest amounts of IL-2 produced and the fact that soluble IL-2R in excess did not completely abrogate the effects of CD44 co-stimulation (Figure 1D) prompted us to consider other mechanisms known to have salutary effects on Treg. One possibility was concomitant effects on TGF-β, a cytokine known to be important for Treg number and function (42–44). Co-stimulation through CD44 promoted a substantial increase in the amount of surface-bound TGF-β on the FoxP3/GFP Treg (Figure 4A). The addition of IL-2 did not markedly increase cell-surface TGF-β and soluble IL-2Rα treatment did not substantially diminish it (Figure 4B). Interestingly the addition of exogenous IL-10 also strongly promoted cell-surface TGF-β. This was the case for normal (Figure 4B) as well as CD44−/− Treg (Figure 4C). The effect of CD44 cross-linking was not decreased by anti-IL-10 Ab, suggesting that both CD44 co-stimulation and IL-10 may independently promote cell-surface TGF-β. CD44 cross-linking neither promoted TGFB1 mRNA nor did it significantly alter the amount of soluble mature-form TGF-β present (data not shown). Together these data suggest that the increase in TGF-β may be particular to the cell surface and may involve the conversion of latent TGF-β to an active form.

Figure 4. CD44 cross-linking and exogenous IL-10 promote cell-surface TGF-β.

Figure 4

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(A) Expression of cell-surface TGF-β and by FoxP3/GFP+ Treg following 72 hours activation. (B) Fold change in the percentage of FoxP3/GFP+ Treg positive for cell-surface TGF-β following activation with anti-CD44 co-stimulation and controls. Included are data from 11 experiments. Each experiment utilized Treg pooled from 4–5 mice. (C) Fold change in the percentage of CD44−/− FoxP3/GFP+ Treg positive for cell-surface TGF-β following activation with anti-CD44 co-stimulation and controls. Data are representative of three experiments.

It should be noted that even without CD44 co-stimulation a small fraction of Treg do express activated TGF-β on their cell-surface. The results here are generally consistent with previously published reports in terms of the percentage of Treg which are cell-surface TGF-β+ (45,46).

CD44 cross-linking and exogenous IL-2 promote production of IL-10

CD44 cross-linking strongly promoted IL-10 production (Figure 5A)(p = <0.05). This effect appears to be fairly specific and furthermore do not support a departure from the phenotype of differentiated natural Treg as negligible amounts of other TH1or TH2 cytokines were detected including IFNg, IL-4, and IL-5 and minimal TNF-α or IL-1β was seen (data not shown). Treg from CD44−/− mice produced diminished levels of IL-10 upon activation with anti-CD3/28 (Figure 5A)(p = < 0.05). Consistent with these data IL-10 mRNA from FoxP3/GFP+ Treg harvested 24 hours after activation likewise demonstrated enhanced IL-10 production upon CD44 cross-linking (Figure 5B). Our data this suggests that IL-10 production may occur in a partially IL-2 dependent manner as addition of exogenous IL-2 at the inception of the culture alone and in an additive manner with CD44 costimulation increased IL-10 production (Figure 5C). As with our earlier data on IL-2 production, the addition of cyclosporine A did not diminish IL-10 production in the setting of CD44 costimulation (Figure 5D).

Figure 5. CD44 cross-linking of FoxP3/GFP+ Treg and exogenous IL-2 promote IL-10 production.

Figure 5

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(A) IL-10 concentrations in WT and CD44−/− Treg culture supernatants activated with anti-CD3/28/44 versus anti-CD3/28 alone (p = 0.007) and also for WT Treg stimulated with anti-CD3/28/44 (p = 0.012 for the comparison vs. WT Treg stimulated with anti-CD3/28 alone). Data are shown for 8 separate experiments. Each experiment utilized Treg pooled from 4−5 mice. (B) IL-10 mRNA copy number/105 18S mRNA for FoxP3/GFP+ cells and FoxP3/GFP- cells immediately upon isolation and for FoxP3/GFP+ Treg following 24 hours activation with either anti-CD3/28 Ab or anti-CD3/28/44 Ab. mRNA was isolated from FoxP3/GFP+ and – cells pooled from 5 animals. Data from three separate such experiments are shown. (C) Fold change in the concentrations of IL-10 in Treg culture supernatants upon stimulation with anti-CD44 Ab and controls. Included are data for 8 experiments. (D) Fold change in the concentrations of IL-10 in Treg culture supernatants upon activation with or without anti-CD44 Ab with or without CSA at 50 ug/ml. Included are data for 3 experiments.

Our data also suggest that there is an IL-2 independent component of CD44 mediated promotion of IL-10 production. The addition of soluble TGF-β did not promote IL-10 production (Figure 5C). Another candidate was mTOR signaling, which has been reported to upregulate IL-10 production via effects on the transcription factor STAT3 (47,48). To evaluate the role of mTOR in this system we tested the effects of Rapamycin addition. We found that CD44 upregulation of IL-10 production also bypasses Rapamycin treatment (Supplemental Figure 2).

To evaluate the functional role of IL-10 in aCD44 co-stimulation effects we isolated CD4+CD25+ Treg from both IL-10−/− and WT mice and stimulated these with aCD3/28/44 (Supplemental Figure 3). The difference in Treg suppression observed can be attributed to differences in IL-10 production.

CD44−/− mice have functionally impaired Treg

We theorized that because HMW-HA cross-linking of CD44 on Treg promoted FoxP3 persistence, CD44−/− mice might have diminished numbers of FoxP3+ cells. This was not the case. CD44−/− mice did not have significantly diminished numbers of CD4+FoxP3+ cells in their lymph nodes and spleens in vivo (Supplemental Figure 4). However equivalent numbers of Treg from CD44−/− animals demonstrated impaired regulatory function in vitro compared to their normal counterparts (Figure 6). These data suggested that the absence of CD44 expression adversely impacted the ability of Treg to suppress effector T cell proliferation.

Figure 6. CD44−/− mice have functionally impaired Treg.

Figure 6

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CFSE based suppression assay utilizing pooled freshly isolated (not pre-activated) CD4+CD25+ cells from four pooled CD44−/− mice (dashed line) and four pooled WT mice (solid line). WT responder cells and APC were used for all conditions.

Co-stimulation with either HMW-HA or CD44 Ab promote persistent FOXP3 expression in Treg

We sought to ascertain whether the effects of CD44 co-stimulation observed in mice upon stimulation with antibodies were also relevant to human Treg and natural ligands of CD44. As with our mouse activation protocol, we activated human CD4+CD25+ cells with plate-bound anti-CD3 Ab +/− HMW-HA and controls without the addition of exogenous IL-2. After 4 days cells were stained for CD25 and FOXP3 expression. From the time of isolation (Figure 7A) to 96 hours afterwards (Figure 7B) without the addition of exogenous IL-2 the level of FOXP3 and CD25 expression falls. However FOXP3 expression can be rescued by co-stimulation with HMW-HA, anti-CD44 Ab, or anti-CD28 Ab. This was the case for Treg isolated from multiple individuals (Figure 7C). Interestingly we find that with human Treg the incorporation of anti-CD28 does not seem to markedly impact FoxP3 expression in these assays (data not shown) whereas this antibody was essential in the experiments with mouse Treg. Whether this is due to a real difference in human/mouse biology or alternatively whether this reflects the greater relative purity of mouse GFP/FoxP3+ Treg is unclear.

Figure 7. HMW-HA and anti-CD44 Ab promote persistent FoxP3 expression by human Treg.

Figure 7

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(A&B) Treg expression of FoxP3 and CD25 immediately upon isolation on Day 0 (noted in the boxed region) (A) and 4 days after (B) activation with plate-bound anti-CD3 and co-stimulation as indicated above the FACS plots. Data are representative of three experiments. (C) Fold change in FoxP3 expression for pooled data from four experiments under the same experimental conditions as in (B).

The length of HA is of critical importance. HMW-HA, but not LMW-HA, promoted the FOXP3 expression (Figure 8A). LMW-HA was derived from the same HMW-HA used in these experiments and was therefore identical to HMW-HA except in terms of length. The maintenance of FOXP3 expression by HMW-HA co-stimulation can be inhibited by an anti-CD44 Ab known to inhibit HA binding, suggesting that HMW-HA is acting through this receptor. Consistent with a putative requirement for CD44 cross-linking, plate-bound but not soluble anti-CD44 Ab promoted FOXP3 expression (Figure 8B). We similarly observed in the mouse activation assays that plate-bound but not soluble anti-CD44 Ab promoted FoxP3 expression. Commensurate with this increase in FOXP3 expression and consistent with the analogous mouse Treg data, human Treg which received plate-bound anti-CD3/44 co-stimulation demonstrated increased suppression of effector T cell proliferation relative to that seen with Treg stimulated with anti-CD3 alone (Figure 8C).

Figure 8. HMW-HA promotes persistent FoxP3 expression and suppressor function by human Treg via cross-linking of CD44.

Figure 8

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(A). FoxP3 expression after 4 days of co-stimulation with media versus plate-bound HMW-HA and controls. (B) FoxP3 expression in response to co-stimulation with plate-bound versus soluble anti-CD44 mAb. Data are representative of 3 experiments. (C) CFSE-based suppression assay comparing Treg activated for 4 days with plate-bound anti-CD3 Ab and media alone or in conjunction with plate-bound anti-CD44 co-stimulation. FoxP3 staining for the Treg used in this suppression assay are shown in (A & B).

HMW-HA treatment and CD44 cross-linking promote FOXP3 and CD25 expression in an IL-2 dependent manner

Both HMW-HA and anti-CD44 Ab had analogous effects on human Treg as those observed with mouse Treg vis-à-vis expression of both IL-10 (Figure 9A) and IL-2. (Figure 9B). Notably minimal cell-surface TGF-β production was observed on human Treg under these same conditions (data not shown). To assess the functional relevance of IL-2 production in this setting we activated Treg with 0, 2 or 20 IU/ml IL-2 using anti-CD3/28 coated beads for 48 hours. Cells were subsequently treated with HMW-HA or an equivalent volume of PBS for an additional 24 hours before assessment of FOXP3 expression. Interestingly, differences in FOXP3 expression upon HMW treatment were seen in cells which received 0 or 2 but not 20 IU/ml IL-2 (Figure 9C). At the higher dose, IL-2 itself was sufficient for supporting Treg activity. HMW-HA effects were therefore most pronounced at low concentrations of IL-2. To evaluate the effect of HMW-HA on Treg viability we stained Human Treg for Annexin V/PI following treatment with HMW-HA treatment versus LMW-HA or PBS as controls. We did indeed observe enhanced viability of human Treg (Supplemental Figure 5).

Figure 9. CD44 cross-linking promotes persistent expression of FOXP3.

Figure 9

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(A) HMW-HA or anti-CD44 costimulation promotes IL-10 production by human CD4+CD25+ cells. (B). HMW-HA or anti-CD44 costimulation promotes IL-2 production by human CD4+CD25+ cells. (C). FoxP3 expression (MFI) 72 hours after activation in the setting of 0, 2, or 20 IU/ml of exogenous IL-2 with or without co-stimulation with HMW-HA (administered only for the final 24 hours).

Discussion

The inflammatory milieu plays a major role in the generation and regulation of adaptive immune responses through a variety of mechanisms yet the contribution of the ECM towards regulation of adaptive immunity is poorly understood. In earlier work we demonstrated that the size and amount of HA modulate Treg function. Here we elucidate the salient mechanisms, focusing on the primary HA receptor CD44. We draw four conclusions from these data.

First we conclude that CD44 signaling delivers a stimulatory signal to Treg in the context of TCR activation. In this setting CD44 co-stimulation potently upregulated CD25 expression and maintains expression of FoxP3. The requirement for a TCR signal suggests that Treg may interact with the ECM in inflamed and healing tissues in a manner which is relevant to particular antigens. CD44 co-stimulation has previously been suggested to promote T cell activation in the presence of a sub-threshold stimulus (49); one way this might occur via the apposition of lck into the proximity of the TCR/CD3 complex (50).

Our data suggest that this observation is relevant to both mouse as well as human Treg. Furthermore HMW-HA, a natural ligand of CD44, stimulates human and mouse Treg in an analogous, albeit less potent, manner to anti-CD44 Ab. Interestingly the capacity of HMW-HA to promote GFP/FoxP3 in mouse Treg required the presence of mouse serum as without this there was no effect. The identity of the permissive factor in mouse serum is as yet unclear. There are several candidate soluble factors which promote HA binding. These include inter-alpha trypsin inhibitor and TSG6 (51,52). Whatever the salient molecule is, it is evidently not present in commercially available fetal calf serum. This may be either because it is species specific or due to some aspect of processing.

Second these data suggest that receptor cross-linking is necessary for these effects. While both plate-bound anti-CD44 Ab and HMW-HA promoted FoxP3 persistence, neither soluble anti-CD44 Ab nor LMW-HA did so. This is consistent with previous reports of cross-linking being integral to several functions attributed to CD44 (53–55). We propose that the size of HA is directly related to the capacity of this molecule to cross-link spatially separated CD44 on the cell surface. Indeed these data suggest that LMW-HA inhibits CD44 crosslinking. This may occur via competitive exclusion, in which case the relative molar ratios of HMW-HA versus LMW-HA may determine the extent of CD44 cross-linking in a given environment. Given the relationship between HA size and the stage of inflammation, the extent of CD44 cross-linking by HMW-HA and other ligands may provide contextual cues to Treg and T-cells regarding the inflammatory milieu.

Third our data suggest CD44 cross-linking promotes modest IL-2 production. This was the case for cells previously isolated on the basis of FoxP3/GFP expression as well as for human Treg. These results are consistent with reports of enhanced IL-2 production by other T-cell subsets and NKT cells upon CD44 co-stimulation (56,57) and with reports of requirements for HA and CD44 in certain IL-2 dependent processes (58–61). As Treg are not known to produce substantial quantities of IL-2, it seems likely that at least some FoxP3/GFP+ cells acquire the capacity to produce IL-2 upon activation in the setting of CD44 costimulation.

These data raise the possibility that CD44 costimulation bypasses the negative regulatory influence of FoxP3 on IL-2 production. How might this occur? FoxP3 is known to down regulate IL-2 production through inhibitory effects on NFAT (12,62). We demonstrate that CD44 mediated IL-2 production by Treg occurs irrespective of cyclosporine A treatment, a potent inhibitor of NFAT. This suggests that CD44 costimulation has the capacity to bypass the inhibition of NFAT; whether CD44 signaling indeed bypasses FoxP3 mediated inhibition of NFAT and by what alternative signaling pathway are the subject of current investigation. If so this may help explain how this highly IL-2 dependent cell type (63,64) persists in healing or uninjured tissues where IL-2 concentrations are presumably low. This question is important because many models of autoimmunity invoke the inappropriate persistence of immune responses in former sites of inflammation.

It should be noted that while our sorting methodology yields purities >99.9% GFP/FoxP+ cells, we cannot absolutely exclude the possibility that IL-2 is produced by a small number of FoxP3/GFP- cells conceivably contaminating our sorting protocol. However we do not observe any expansion of GFP- negative cells over the course of the assay, such as would occur were there a quantifiable contaminating population of these cells (data not shown).

Fourth and finally our data supports a previously unreported role for CD44 cross-linking in enhancing expression of the anti-inflammatory cytokines IL-10 and TGF-β. CD44 cross-linking promotes IL-10 production in a partially IL-2 dependant manner. Furthermore we find that CD44−/−Treg produce diminished amounts of this cytokine. (47,48). IL-10 production is reported to be one mechanism by which Treg maintain immune tolerance (65) and expression of this cytokine by CD4+CD25+ cells has been shown to be partially dependant on Il-2 (66). Enhanced IL-10 production may also reduce the dependency of Treg on exogenous IL-2, by independently promoting Treg viability (67). Consistent with a role for CD44 in Treg function, we find that Treg taken from CD44−/− mice demonstrated impaired regulatory function ex vivo.

CD44 cross-linking also promotes production of TGF-β. This cytokine has well-characterized roles in promoting Foxp3 expression (68,69) as well as Treg function (42–44). Increased TGF-beta+ in a Treg population has been shown to enhance suppression of activated T cell proliferation through induction of FoxP3 expression in responder cells (68). Our data suggest that both CD44 cross-linking and exogenous IL-10 promote cell-surface TGF-β. A role for IL-10 in TGF-β production has been previously reported (70–72). HA has previously been demonstrated to promote CD44 interactions with TGF-β receptor I, thereby enhancing TGF-β1 signaling (73). Others have reported that CD44−/− mice have diminished levels of TGF-β (44).

In sum our data support the conclusion that cross-linking of CD44 by HMW-HA and potentially other ECM components promotes Treg persistence and function. Our findings are most consistent with a two-step model, in which the initial activation of Treg through the TCR is sufficient for FOXP3 induction, followed by a requirement for a costimulatory signal to support FOXP3 persistence. This latter step is heavily dependent on environmental cues, such that in the absence of high concentrations of IL-2, the CD44-IL-2-IL-10 pathway we describe can contribute to the support of peripheral immune tolerance. This mechanism may be one way in which viable Treg populations persist in healing or uninjured tissues to bring inflammatory processes to a close and maintain peripheral tissue tolerance. We propose that the HMW-HA molecular composition of the ECM conveys a tissue integrity signal which functions as the opposite of a danger signal, in that adaptive Treg receive an important costimulatory signal in situ, promoting immune homeostasis.

Supplementary Material

Figures 1-5

Acknowledgments

We would like to thank Nathan Standifer and John Gebe for their helpful comments and suggestions.

This work was supported by grants from the NIH (DK46635, HL18645, and DK53004) and the JDRF (The Center for Translational Research at BRI). PLB is supported by NIH K-08 grant DK080178-01 and an NIH LRP grant.

Non-standard abbreviations used

ECM

extra-cellular matrix

HA

hyaluronan

HMW-HA

high-molecular-weight hyaluronan

LMW-HA

low molecular weight hyaluronan

APC

antigen presenting cell

Treg

CD4+CD25+ regulatory T-cell

Footnotes

The authors have no conflicting financial interests.

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Supplementary Materials

Figures 1-5
NIHMS264915-supplement-Figures_1-5.pdf (358.4KB, pdf)

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