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The Journal of Immunology, 2008, 180, 327 -334
Copyright © 2008 by The American Association of Immunologists, Inc.
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Antigen-Driven Interactions with Dendritic Cells and Expansion of Foxp3+ Regulatory T Cells Occur in the Absence of Inflammatory Signals1

Pascal Chappert*, Marylène Leboeuf*, Philippe Rameau*, Daniel Stockholm*, Roland Liblau{dagger}, Olivier Danos*,{ddagger}, Jean M. Davoust2,3,*,§ and David-Alexandre Gross2,3,*

* Genethon, Centre National de la Recherche Scientifique, Unité Mixte de Recherche, 8115, Évry, France; {dagger} Institut National de la Santé et de la Recherche Médicale U563, Université Paul Sabatier, Hôpital Purpan, Toulouse, France; {ddagger} Université Paris Descartes, Institut National de la Santé et de la Recherche Médicale U781 and § Institut National de la Santé et de la Recherche Médicale U580, Hôpital Necker-Enfants Malades, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Foxp3+ regulatory T cells (Tregs) play a pivotal role in the maintenance of peripheral T cell tolerance and are thought to interact with dendritic cells (DC) in secondary lymphoid organs. We analyzed here the in vivo requirements for selective expansion of Ag-specific Treg vs CD4+CD25– effector T cells and engagement of Ag-specific Treg-DC interactions in secondary lymphoid organs. Using i.v. Ag delivery in the absence of inflammation, we found that CD4+CD25+Foxp3+ Tregs undergo vigorous expansion and accumulate whereas naive CD4+CD25–Foxp3– T cells undergo abortive activation. Quantifying directly the interactions between Tregs and CD11c+ DC, we found that Tregs establish cognate contacts with endogenous CD11c+ DC in spleen and lymph nodes at an early time point preceding their expansion. Importantly, we observed that as few as 103 Tregs selectively expanded by i.v. Ag injection are able to suppress B and T cell immune responses in mouse recipients challenged with the Ag. Our results demonstrate that Tregs are selectively mobilized by Ag recognition in the absence of inflammatory signals, and can induce thereafter potent tolerance to defined Ag targets.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Peripheral immune regulation by naturally arising CD4+CD25+ regulatory T cells (Tregs)4 governed by the forkhead family member transcription factor Foxp3 requires cell contacts and Ag recognition both in vitro and in vivo (1, 2, 3). Our group and others have reported that CD4+CD25+Foxp3+ T cells that are Ag specific are much more powerful in regulating transplantation tolerance (7) anti-transgene (6) or autoimmune responses (4, 5) than Tregs of broad specificity. The potency of Ag-specific Tregs is due in all likelihood to their capacity to spread tolerance to several Ag linked to cognate recognition of the Ag initially encountered, and to generate a set of secondary Tregs (8). In our previously described bone marrow transplantation model we showed that low amounts of Ag-specific Tregs are highly effective in conferring tolerance to multiple linked antigenic determinants, a result correlated with the fact that Ag recognition promotes transient expansion of Tregs in secondary lymphoid organs (7). Elucidating the mechanism that controls Ag-specific Tregs expansion and accumulation is now of great importance to unravel how immunological tolerance is achieved and maintained in the periphery, as well as to develop new strategies to treat autoimmunity and other immune-related diseases.

Although inflammatory signals that induce dendritic cells (DC) migration and maturation play a crucial role for effector T cells activation, it is not clear whether Tregs have identical requirements to be efficiently activated in vivo. As shown previously, Ag-driven expansion of functional Tregs populations can be achieved following peptide injection with IFA or after infusion of peptide-pulsed mature DCs, both of which also expand naive T cells (4, 9, 10, 11). Importantly, reports also indicate however that Tregs activation can also result from peptide-MHC presentation under steady-state conditions: 1) Tregs undergo continuous expansion after adoptive transfer into syngenic animals (12, 13), 2) human CD4+CD25highFoxp3+ Tregs are highly proliferative in vivo (14). These observations are consistent with the fact that tolerance induction might result from peptide-MHC presentation by immature DCs or more generally by Ag presenting cells that fail to express costimulatory molecules required for efficient effector T cells activation (15).

In vivo administration of soluble Ag in the absence of inflammation generally leads to immune tolerance (16, 17). Intravenous Ag administration can lead to CD4+ thymocyte apoptosis, lymphocyte anergy or deletion in the periphery (18, 19, 20). Pioneering observations also showed acquisition of immunoregulatory properties by T cells (21). More recently, CD4+CD25– naive TCR transgenic T cells were converted into CD25+ Tregs without extensive proliferation using continuous s.c. peptide delivery with osmotic pumps (22). Refining the role of resident DCs to promote Tregs mediated tolerance (23, 24), Kretschmer and colleagues (25) have shown that DC targeting with DEC-205 conjugates induces extrathymic conversion of CD4+Foxp3– into CD4+Foxp3+ T cells, but again with a limited expansion. Thus, it is likely that Ag presentation by different DC subtypes at the steady state promotes tolerance in several ways, by deletion, anergy induction or conversion of CD4+Foxp3– T cells.

We searched here how to achieve selective expansion and accumulation of functional CD4+CD25+ Tregs by Ag exposure in vivo. We found that these conditions are uniquely realized after i.v. injection of the Ag performed in the absence of inflammatory signals, and that Tregs interact with lymphoid organ-resident DCs before Ag-driven expansion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

BALB/c mice were obtained from Charles River, congenic Thy1.1 BALB/c mice and TCR-hemagglutinin protein (HA) transgenic mice (26) were purchased from CDTA. Animal experiments were performed according to institutional guidelines for animal care and use.

Synthetic peptides and challenge

HA107–119 (SVSSFERFEIFPK), HA126–138 (HNTNGVTAACSHE) control peptide and HA512–520 (IYSTAVSSL) were synthesized by Epytop. Mice were challenged subcutaneously at the base of the tail with 50 µg of peptide emulsified in IFA (BD-Difco), or injected i.v. with 66 µg of peptide diluted in 0.2 ml of PBS 1x.

Recombinant virus vector challenge and immunohistochemistry

Recombinant adeno-associated viral vector (AAV)-HA vectors were produced by triple transfection into 293 cells as previously described (6) and injected i.m. in a volume of 25 µl into the tibialis anterior of anesthetized mice. Mice were sacrificed at day 22 and their injected tibialis anterior was harvested and snap frozen. For histology, frozen muscle sections were fixed in acetone and incubated with biotinylated H37/38 mAb, directed against a conformational epitope of HA in PBS/2% goat serum, washed, and incubated in avidin-biotin-chromagen solution (Vectastain ABC kit; Vector Laboratories). The slides were revealed in diaminobenzidine (DAB fast; Sigma-Aldrich), counterstained with Mayer’s hematoxylin and analyzed as described previously (6).

Regulatory T cell purification

As previously described (6, 7), splenocytes and lymph node cells were first incubated with biotinylated anti-CD25 (7D4) and streptavidin microbeads (Miltenyi Biotec), followed by magnetic cell separation using LS columns (Miltenyi Biotec), and then sorted on a MoFlow (DakoCytomation). To monitor the fate of equivalent numbers of CD4+ CD25+ Tregs and CD4+ CD25– helper T cells, 3 x 105 CD25+ cells or 106 CD25– cells (the latter containing 30% CD4+ cells) were transferred into BALB/c mice. For in vivo proliferation assays, CD25+ and CD25– cells were labeled for 5 min at 37°C with 5 µM CFSE (Molecular Probes).

Flow cytometry

All reagents and mAbs used for flow cytometry were purchased from BD Biosciences, except for the clonotypic 6.5 mAb produced and biotinylated in our laboratory. Cell suspensions from spleen or lymph nodes were blocked with anti-Fc{gamma}RIII/II (2.4G2) mAb, and then stained with various mAb combinations. Dead cells were excluded using 7-actinomycin D (Sigma-Aldrich) staining. Flow cytometric analysis was performed on a FACSCalibur using CELLQuest software (BD Biosciences).

ELISPOT and ELISA analysis

IFN-{gamma} ELISPOT and ELISA were performed as previously described (6). In brief, for IFN-{gamma} ELISPOT assays, freshly isolated splenocytes (2 x 106/well and serial dilutions) were cultured in complete RPMI 1640 medium with or without 10 µM HA512–520. For each assay, ConA was added (5 µg/ml) as a positive control. After 20 h, spots were revealed and counted using a Bioreader 2000 (BIO-SYS). Spot-forming units are represented after subtraction of background values obtained with unpulsed splenocytes. ELISA was done using influenza virus PR/8/34 as Ag. Serum samples and purified anti-HA Ab standard of IgG isotype (H37/38, 1.4 mg/ml) were serially diluted, detected with peroxidase-conjugated goat anti-mouse IgG (Vector Laboratories) and revealed with TBM solution (Kirkegaard-Perry).

Confocal image analysis

Mice were sacrificed at indicated time points, spleen and lymph nodes were harvested, snap frozen and sectioned (6–8 µm) for fluorescence staining with blocking 2.4G2 mAb for 45 min and with combinations of FITC-anti-CD45R, PE-anti-CD4, PE-anti-CD11c, and allophycocyanin-anti-CD90.2 (anti Thy1.2) for 60 min. Confocal analysis was performed on a LEICA TCS SP2 microscope (Leica Microsystems).

We used correlation maps as previously described (27, 28). This method identifies local similarities between two fluorescence profiles from the computation of a correlation coefficient on a local scale for each pixel in the images. The local correlation coefficient is either positive for colocalized profiles, null for unrelated profiles, or negative for inverse profiles. These correlation maps were initially designed to visualize the sites of fluorescence colocalization where fluorescence profiles are highly similar (30). In this study, we computed the correlation coefficient of raw images comparing CD11c (red channel) and Thy1.2 (blue channel) using a Gaussian window of 5 pixels equivalent to a 3.6 µm diameter. We displayed the negative component in the green channel for each given pixel on the raw image. Large fields of over 1 mm2 surface areas per tissue section (2048 x 2048 micrographs) were inspected using a high numerical aperture 10x objective to visualize rare HA-Tregs, from 5 to 90 per section depending on the expansion conditions. We calculated the local correlation maps for all images acquired under standardized conditions and counted the pairs of CD11c/Thy1.2 interacting cells exhibiting a local peak of correlation at their interface on these maps. A total of 1123 cells on 46 images for CD25+ and 1986 cells on 45 images for CD25– were counted. Statistical analysis of obtained proportions of interacting cells was done using the prop.test function in R software (http://www.r-project.org/).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Transient HA-Tregs expansion and accumulation during in vivo suppression

We showed previously that Ag-specific CD4+CD25+ Tregs derived from TCR-HA mice (26) (HA-Tregs) can maintain peripheral tolerance to HA expressed in muscle following injection of an HA-encoding AAV (AAV-HA) (6). Although the CD8 response normally peaks between day 14 and day 21, suppression is already efficient at day 14 and is still operative at day 35. Using this system, we find here that as few as 103 HA-Tregs totally suppress the CD8 response directed against the immunodominant HA512–520 epitope at day 21 (Fig. 1, A and B) and promotes successful HA engraftment in muscle (data not shown). We then monitored the fate of HA-Tregs after AAV-HA injection by transferring Thy1.2 HA-Tregs into congenic Thy1.1 mice and we found that they rapidly expanded, peaking at day 6 before returning to a basal level (Fig. 1, C and D). Interestingly, the transient nature of this accumulation did not impair the long-term tolerance to HA and could not be attributed to HA-Tregs migration to the inflamed site, because none of them could be retrieved from the injected muscle (data not shown). Our results indicate that Tregs-mediated suppression is associated with their transient expansion. Under steady-state conditions, Tregs were shown to undergo continuous expansion (12, 13) and to prevent autoimmunity through the lifespan of mice (29). Together with our results, this led us to explore whether Tregs can be activated in the presence of the Ag delivered under noninflammatory conditions and still exert potent suppressive functions once expanded.


Figure 1
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  		FIGURE 1. Tregs expand transiently during in vivo suppression. A and B, BALB/c mice transferred i.v. by different doses of HA-Tregs were injected 1 day later in the tibialis anterior with 1011pp of AAV-HA viral vector. Mice were sacrificed at day 21 and their splenocytes tested in a standard IFN-{gamma} ELISPOT assay against the HA512–520 epitope. Data represent the mean of 6–12 mice per dose ± SEM, pooled from four independent experiments. C and D, Congenic Thy1.1 mice received 3 · 105 HA-Tregs (Thy1.2) 1 day before i.m. injection of either AAV-HA or AAV-LacZ. Blood samples were regularly collected and frequency of transferred HA-Tregs (CD4+Thy1.2+) quantified by FACS. C, Day 6 dot plots; D, data represent the mean of 3 mice per group ± SEM. One of three independent experiments is shown.

 
Selective expansion of Ag-specific Tregs in the absence of inflammation

CFSE-labeled HA-Tregs were adoptively transferred into Thy1.1 mice that were further challenged i.v. by the cognate HA107–119 peptide. HA-Tregs were systematically compared with the CD4+CD25– subset (HA-Th), which contained <2% of Foxp3+ cells (data not shown). We first control the tolerogenic effect of i.v. peptide on naive T cells. After i.v. injection of HA107–119 peptide, HA-Th in the spleen remained either undivided or underwent a limited number of divisions at day 3 and were unable to accumulate thereafter (Fig. 2A), consistent with the previously reported activation-induced cell death of Th cells in such conditions (18). The addition of LPS to the i.v. injected HA107–119 peptide restored a vigorous proliferative response of HA-Th (Fig. 2B), indicating that the lack of HA-Th accumulation resulted from the lack of proper costimulation (30). Exploring the fate of HA-Tregs after i.v. infusion of HA107–119 peptide, we found that they divided extensively and accumulated up to day 6 in marked contrast with HA-Th (Fig. 2C). At this time, over 90% of donor-derived HA-Tregs from both spleen and lymph nodes had divided, and up to 75% of them accumulated at least seven divisions (Fig. 2C), whereas injection of control peptide had no effect (Fig. 2D). In agreement with this result, the proportion of HA-Tregs increased up to 9-fold at day 6 in the spleen and lymph nodes (Fig. 3A) and up to 50-fold in the blood (data not shown). After day 6, the number of HA-Tregs started to decrease, but they were still enriched in the spleen and lymph nodes at day 12 (3-fold increase; Fig. 3A), and were detectable up to 4 wk after i.v. peptide delivery (data not shown). Similar results were obtained following s.c. peptide injection in IFA (Fig. 3A) and i.v. peptide injection with LPS (data not shown), revealing that the level of expansion and its transient nature are independent of the inflammatory (IFA, LPS) or noninflammatory (i.v.) stimuli. In all conditions, HA-Tregs required the presence of HA107–119 peptide, because Tregs failed to accumulate in the presence of a control peptide at an equivalent dose (Fig. 2D). In contrast to Tregs, which expand and accumulate in the presence of a cognate peptide but in the absence of inflammatory signals, CD4+CD25– Th need both the cognate peptide and inflammation to accumulate (Fig. 3A). Moreover this selective expansion of Tregs over Th occurred for a large range of i.v. peptide doses. No significant expansion of HA-Th was observed from day 3 to day 6 at all peptide doses tested from 0.6 to 600 µg, while HA-Tregs underwent efficient expansion at day 6 with a peptide dose of 6 µg, and was maximal above a dose of 60 µg (Fig. 3B).


Figure 2
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  		FIGURE 2. Selective expansion of HA-Tregs in the absence of inflammation. Thy1.1 mice received CFSE-labeled HA-Tregs or HA-Th and were injected i.v. with PBS (no injection) or HA107–119 peptide (66 µg). At indicated time points, spleen and peripheral lymph nodes were harvested and stained for FACS analysis. Histograms gated on CD4+Thy1.2 plus 7-aminoactinomycin D cells show the dilution of CFSE in CD25– (A and B) or CD25+ donor-derived CD4+ T cells (C and D) in one representative experiment of four. A, Lack of expansion of CD4+CD25– cells. B, Expansion of CD4+CD25– cells with 30 µg of LPS added to the specific HA107–119 peptide compared with the control HA126–138 peptide. C, Expansion of CD4+CD25+ cells. D, Lack of expansion of CD4+CD25+ cells with the control HA126–138 peptide.

 

Figure 3
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  		FIGURE 3. Accumulation kinetics of HA-Tregs in the absence of inflammation. Thy1.1 mice received Thy1.2 HA-Tregs (full symbol) or HA-Th (open symbol) and were injected i.v. with HA107–119 peptide (66 µg) or PBS (no injection) or s.c. with HA107–119 peptide plus IFA. At indicated time points, spleen and inguinal lymph nodes were harvested and stained for FACS analysis as in Fig. 2. A, Fold expansion of CD25+ or CD25– donor-derived population relative to day 3 PBS injection condition. Data represent the mean of 3 mice per time point (2 mice at day 12) ± SEM, pooled from two of four independent experiments. B, Fold expansion of CD25– and CD25+ donor-derived population relative to day 0 in the PBL as a function of the dose of i.v. injected HA107–119 peptide.

 
Finally, the Ag specificity of expanded HA-Tregs was ascertained by the preferential expansion of HA-Tregs bearing the HA107–119/I-Ed-specific TCR using the clonotypic 6.5 mAb (Fig. 4). Although only 30% of the initially injected HA-Tregs (and HA-Th, data not shown) expressed high levels of the transgenic TCR, this value increased to over 80% after either i.v. or s.c peptide injection (Fig. 4, A and B). We observed a basal proliferation of HA-Tregs in the absence of HA peptide (Fig. 2) and effectively found here that a fraction of the injected HA-Tregs underwent homeostatic expansion independently of the expression of the HA-specific TCR (Fig. 4C). This probably results from endogenous {alpha}-chain rearrangement and subsequent second TCR expression allowing HA-Tregs interactions with self-Ags. Altogether, our results prove that recognition of the cognate peptide under noninflammatory conditions is necessary and sufficient to drive the expansion and accumulation of Ag-specific Tregs.


Figure 4
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  		FIGURE 4. Preferential expansion of Tregs bearing the clonotypic anti-HA TCR. Thy1.1 mice received CFSE-labeled Thy1.2 HA-Tregs and were injected i.v. with 66 µg of HA107–119 peptide or PBS, or s.c. with 50 µg of HA107–119 peptide in IFA. At indicated time points, spleen were harvested and stained for CD4, Thy1.2 and 6.5. A, Histogram shows the expression of HA-specific TCR on donor-derived CD4+Thy1.2+ T cells at day 6 (full line) vs freshly purified HA-Tregs (gray). B, Percentage of 6.5high cells in this population as a function of time. The mean of 2–3 mice per time point, pooled from two independent experiments, is shown. C, Dot plots show the expression of 6.5 vs CFSE dilution at day 6 in the donor-derived T cells (CD4+Thy1.2+).

 
In vivo function of expanded HA-Tregs

Anergy and deletion of helper T cells are thought to be the two main mechanisms of peripheral tolerance induction by i.v. Ag delivery. As Tregs undergo vigorous proliferation and accumulate under these conditions, it was of high importance to determine whether newly expanded Tregs could exert full immunosuppressive properties.

At day 5 after i.v. peptide delivery, proliferating (CFSEint) HA-Tregs exhibited a characteristic activated CD62Lhigh CD44high CD45RBlow CD25high phenotype, which was lost following division because CFSElow cells tended to down-regulate CD62L and up-regulate CD45RB (data not shown). This result was confirmed at day 12, with a phenotype of expanded HA-Tregs closer to that of freshly purified HA-Tregs, with however a marked down-regulation of the CD25 marker, reminiscent of the phenotype previously described for CD25–Foxp3+ Tregs (31). Importantly, Foxp3 expression was maintained throughout divisions, as revealed by intracellular staining (Fig. 5A) or quantitative RT-PCR on sorted cells (data not shown).


Figure 5
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  		FIGURE 5. In vivo function of expanded HA-Tregs. Thy1.1 mice received i.v. 6 · 105 CFSE-labeled HA-Tregs and 66 µg of HA107–119 peptide. A, Foxp3 expression analyzed by intracellular staining at day 6. B, Spleen and lymph node from 2 mice were pooled and CFSElow Thy1.2+ sorted (expanded HA-Tregs) cells transferred into secondary recipients to test their regulatory properties. C–F, Secondary recipients were either untreated (no cells) or injected i.v. with 103 or 104 expanded HA-Tregs, 1 day before AAV-HA i.m. injection. At day 22, mice were sacrificed. C, Their splenocytes were tested by IFN-{gamma} ELISPOT against the HA512–520 epitope. D, Anti-HA IgG were assayed in the sera by ELISA. E, Muscles were frozen and HA expression was measured by immunohistochemistry using biotinylated H37/38 mAb, directed against a conformational epitope of HA, HRP/DAB staining (brown) and counterstained with Mayer’s hematoxylin (blue) which stains the nuclei of muscle fibers and infiltrating lymphoid cells. Images are representative of 3 mice per group. F, Positive cells were manually counted on three sections per mice. Results represent the mean of 3 mice per group and are expressed as mean ± SEM. *, p ≤ 0.05.

 
Thy1.2+CFSElow cells (expanded HA-Tregs) were then purified by cell sorting and either tested in a standard in vitro suppression assay, revealing their ability to inhibit HA-Th proliferation (data not shown), or re-infused into secondary recipient mice injected with AAV-HA (Fig. 5B). We found that as few as 103 expanded HA-Tregs were sufficient to inhibit the CD8 T cell response against HA512–520 epitope as determined by IFN-{gamma} ELISPOT assay (Fig. 5C), and the humoral anti-HA response as revealed by ELISA (Fig. 5D). We further analyzed HA expression in muscle and found that as few as 103 expanded HA-Tregs completely inhibit the rejection of transduced muscle fibers (Fig. 5, E and F), as in control experiments using 103 freshly purified HA-Tregs (Fig. 1). In this cell dose range, it was not feasible to determine whether expanded Tregs are more efficient than fresh Tregs.

In situ tracking of HA-Tregs during in vivo expansion

To assess whether resident DCs could interact selectively with HA-Tregs in lymphoid organs, we first localized HA-Tregs and HA-Th in lymph nodes and spleen sections of Thy1.1 recipient mice at 16, 40, or 136 h post i.v. peptide exposure (Fig. 6A). We analyzed concurrently the frequencies of Thy1.2+CD4+ 7-aminoactinomycin D donor-derived HA-Tregs in the same organs by flow cytometry. At 16 h, corresponding to early events of peptide presentation by APCs, a large proportion of Thy1.2+ HA-Tregs are present in T cells areas (81 ± 3% and 83 ± 11% in spleen and lymph nodes, respectively, data not shown), and we gained similar results for Thy1.2+ HA-Th (90 ± 1%, in spleen T cell areas, data not shown). The total number of HA-Tregs did not increase between 16 and 40 h in response to the cognate HA107–119 peptide, but we found a clear 9-fold expansion of HA-Tregs in lymph nodes and 6-fold in the spleen between 40 and 136 h (Fig. 6, B and C). These results correlate with those found by flow cytometry, demonstrating that in situ inspection by confocal imaging could be used for further quantification of DC/HA-Tregs interactions after Ag-driven expansion in noninflammatory conditions. Thy1.2+ HA-Tregs were mostly localized in the T cell area in spleen and lymph node organs (Fig. 6D and data not shown), but it was also apparent that few HA-Tregs are located at the marginal zone between T cell and B cell areas after 5 days (Fig. 6D).


Figure 6
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  		FIGURE 6. In situ tracking of HA-Tregs during expansion. Thy1.1 mice received i.v. HA-Tregs and 66 µg of HA107–119 peptide. Spleen and lymph nodes were harvested at indicated times. For each individual mice, half of the spleen and lymph nodes was stained for FACS analysis as in Fig. 2 and half was frozen and sectioned for confocal microscopy with Thy1.2 (red), CD4 (blue), B220/CD45R and DAPI staining (data not shown). A, Axillary lymph node at 136 h; arrows point to Thy1.2+ cells. B and C, Histograms of Thy1.2+ cells/mm2 (gray bar) and CD4+ Thy1.2+ cells/106 cells from FACS analysis (X symbols). Histological results represent the mean of four micrographs (mean ± SEM). One of two independent experiments is shown. D, Spleen at 136 h after i.v. HA107–119 peptide injection. Confocal analysis with Thy1.2 (blue), CD11c (red), and CD45R/B220 (green) staining; white arrows indicate Thy1.2+ cells.

 
Interaction of HA-Tregs with DCs in secondary lymphoid organs

CD11c and Thy1.2 markers give rise to a membrane-specific staining of resident DCs and HA-Tregs, respectively. We quantified the number of cell-cell contacts between CD11c+ DCs and Thy1.2+ HA-Tregs by analysis of CD11c and Thy1.2 fluorescence profiles with image correlation methods (27, 28). The sites of cell contacts between Thy1.2+ HA-Tregs and CD11c+ cells were revealed on correlation maps (green) evidencing the inverse fluorescence profiles of CD11c (red) and Thy1.2 (blue) labeling. This allowed us to count directly the number of interacting HA-Tregs (Fig. 7, A–D).


Figure 7
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  		FIGURE 7. Interaction of HA-Tregs with CD11c+ cells. Thy1.1 mice received i.v. HA-Tregs or CD8-CD25– cells, and 66 µg of HA107–119 or control HA126–138 peptides. Spleens were harvested at indicated time points and processed for confocal analysis as in Fig. 6 with Thy1.2 (blue) and CD11c (red). A, Original image with CD45R/B220 staining (green), white arrows indicate Thy1.2+ cells. B, Same field with the correlation map (green), white arrows indicate interacting Thy1.2+ cells. C, HA-Tregs/DCs contact with inversely related Thy1.2 and CD11c fluorescence profiles or lack of contact between distant cells, with superimposed correlation levels (green) with corresponding one-dimensional intensity profiles on a segment (white) (D). E, Number of conjugates between HA-Tregs and CD11c+ DCs per image (mean of 4 to 14 images counted ± SEM). F, Percentage of HA-Tregs interacting with DCs per image (one symbol per image with mean percentage ± SEM). Results from two separate experiments (triangles and diamonds) have been pooled and compared with prop statistical tests: *, p ≤ 0.01. G and H same as E and F with the CD25– HA-Th compartment: **, p ≤ 0.001.

 
Applying these correlation maps on spleen sections, we numbered Thy1.2+ HA-Tregs and HA-Tregs in close contact with resident CD11c+ cells at 40 and 136 h after injection of either the cognate HA107–119 or the control peptide. Two distinct phases were observed. At 40 h after HA107–119 peptide injection, the absolute number of interacting HA-Tregs increased (Fig. 7E), and the percentage of interacting HA-Tregs reached 56 ± 8% compared with 38 ± 7% in the presence of the control peptide (p < 0.01, Fig. 7F). At 136 h, the absolute number of interacting HA-Tregs per image increased even more (Fig. 7E), but their proportion dropped to 43 ± 11% similar to the control peptide condition (Fig. 7F). This indicates that after an early phase of interaction with resident CD11c+ cells, HA-Tregs enter into a phase of a transient proliferative burst. This initial phase of interaction between Tregs and DCs may also reflect the limited persistence of HA peptide on MHC class II molecules of CD11c+ cells.

Interestingly, HA specific Th cells behaved initially as HA-Tregs, establishing frequent Ag-dependent contacts at 40 h (59 ± 5% of interacting cells) above the level of nonspecific interactions measured with the irrelevant peptide at 40 h (41 ± 8% of interacting cells, Fig. 7H). However, in agreement with previous results (32, 33), and as expected from our FACS results, the expansion phase was aborted and accompanied by a diminution in the number of interacting cells (Fig. 7G). Altogether the increasing percentages of HA-Tregs and HA-Th interacting with CD11c+ cells indicate that resident DCs can present the cognate peptide to both Tregs and naive T cells under noninflammatory conditions. However, this leads to the sole expansion and accumulation of functional Ag-specific Tregs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
There is a broad consensus that the state of DC activation is crucial for the outcome of an antigenic challenge, i.e., the development of productive T cell immunity or tolerance. Resident DCs have the capacity to directly tolerize CD4+ effector T cells (23) or to convert CD4+CD25– Foxp3– T cells into CD4+CD25+ Foxp3+ Tregs (24, 25). Moreover, an important fraction of endogenous Tregs is activated and undergoes homeostatic expansion at the steady state (12, 13). One important question was to assess the fate and the function of Foxp3+ T cell directly after Ag-driven activation performed under steady-state conditions. Previous studies have shown that different in vivo challenges, used to activate DCs and prime naive T cells, induce efficient proliferation of Ag-specific Tregs (4, 9, 10, 11). Encountering here the Ag in the absence of inflammation, CD25– and CD25+ T cells specific for the same Ag responded differently to the same stimuli. Although, naive T cells became unresponsive, due to anergy or induced cell death after an abortive initial expansion (18, 19) (Fig. 2), a tolerizing process could be reinforced by the efficient mobilization of the Treg compartment, either by peripheral conversion of naive T cells into Tregs (22, 24, 25) or, as shown here, by amplification of the Tregs population (Figs. 1–4).

To gain insight into the early phase preceding selective Tregs expansion, we used confocal imaging and tracked HA-Treg/DC and HA-Th/DC interactions in the secondary lymphoid organs during the course of peptide-induced tolerization. A clear increase in the percentages of both HA-Tregs and naive HA-Th in close contact with CD11c+ DCs was observed at early time points, 16 h (data not shown) and 40 h (Fig. 7), following HA peptide injection. It should be noted that the percentage of interacting cells in our experiments reflects both transient and long-lived interactions, explaining in part the basal percentage of interacting cells in absence of cognate Ag. The first hours of DC/T cell interactions leading to tolerance induction have been recently analyzed using dynamic in situ imaging of OVA-specific CD8+ and CD4+ T cells following OVA delivery to DCs using anti-DEC205 conjugates (32, 33). If most of the OVA-specific naive T cells slow down during the first few hours while regaining higher level of motility at later time point in both tolerizing and priming conditions, a decrease in long-lived interactions with steady-state tolerizing DCs could be observed. Despite this, over 80% of T cells still repeatedly contacted DCs in both conditions and this for up to 48 h (33). This is in agreement with the high percentage of HA-Th-DC interactions observed here 16 and 40 h after i.v. peptide injection (Fig. 7 and data not shown). Comparative in vivo imaging of Th-DC and Tregs-DC interactions under noninflammatory conditions will be necessary to determine whether HA-Tregs could selectively establish long-lasting interactions with steady-state DCs. The persistence of the Ag on APCs, which is unknown here for the HA peptide is certainly of importance in this process. Moreover, the nature of the Ag being either a peptide or a protein fragment requiring a processing step might also influence the type of APCs involved and the fine localization of Tregs in lymphoid organs.

The outcome of Ag recognition leading to the selective expansion of Tregs in the absence of inflammatory signals is presumably related to intrinsic properties of this T cell subset. This could be due to constitutive expression of CTLA-4 of higher avidity to CD80 and CD86 than CD28, or to their apparently "Ag-primed" state resulting in a lower threshold for TCR activation (2). As recently pointed out, regulation of diacylglycerol metabolism may also be critical in determining whether activation or anergy ensues after TCR stimulation (34, 35). In fact, CD4+CD25+ Tregs thymocytes are more resistant to clonal deletion than their CD4+CD25– counterparts, which may also explain the relative enrichment of self-reactive Tregs (10, 36, 37, 38). It remains now to be determined whether HA-Tregs preferentially interact with particular subsets of DCs (CD8+, CD8–CD4+, pDC) after i.v. peptide administration. Along this line, a recent set of experiments performed in vitro, underlined the importance of CD8+ DCs to limit background proliferation of polyclonal Tregs and selectively expand Ag-specific Tregs (39).

Another intrinsic difference between Tregs and Th, which appeared throughout our study, is the poor maintenance of expanded HA-Tregs in secondary lymphoid organs, as compared with memory Th cells. Strikingly, this was true under both noninflammatory (i.v. peptide) and inflammatory (IFA, LPS) conditions (Fig. 3A and data not shown). Rapid clearance of cognate Ag following peptide injection could be an explanation, yet the use of osmotic pumps to deliver the peptide continuously for up to 14 days did not modify the observed kinetics and the final contraction of this population (data not shown). Such disappearance of expanded Tregs from day 6 was also observed during the course of anti-AAV-HA immune response suppression (Fig. 1), despite at least 35 days of HA Ag expression (6). It is also possible that once expanded homeostatically in peripheral lymphoid organs, Tregs acquire the capacity to redistribute in nonlymphoid tissues (40, 41, 42) but here no HA-Tregs could ever be detected in the inflamed muscle at day 14 after AAV-HA injection (data not shown). This correlates with recent studies in a type 1 diabetes model showing complete loss of transferred Ag-specific Tregs at day 40 despite efficient protection (43), or showing a high turn over of skin infiltrating Ag-specific Tregs in the leishmania model, with higher amount of apoptotic Tregs compared with Th counterparts (44). As hypothesized by Belkaid et al. (12), this mechanism could control the in vivo function of Tregs by limiting temporally and spatially their suppressive activity. It could also explain the absence of global expansion of the Treg compartment despite continuous activation and division in the periphery (12).

In our model, it is important to note that as low as 103 expanded HA-Tregs transferred adoptively into new recipients were potent suppressor of B and T cell responses (Fig. 5), implying that once expanded, HA-Tregs can be further mobilized by APCs in lymph nodes draining the site of AAV-HA injection. Thus, our results establish for the first time that in vivo expanded Tregs maintain their suppressive properties. They exhibit stable Foxp3 expression and an activated phenotype reminiscent of the one found by Fontenot et al. (31) describing the CD25–Foxp3+ effector enriched Tregs compartment. The role and function of such populations remain to be analyzed, because most of current literature on Tregs has focused on the CD25+ subpopulation. We noticed also that newly expanded Tregs tend to down-regulate the CD25 marker after 12 days, but were insufficiently represented for purification and adoptive transfer. To analyze nevertheless the function of Tregs under these conditions, we challenged the mice with AAV-HA at day 21 and found that potent suppression was still operative 3 wk after the initiation of Ag-mediated Tregs expansion (data not shown). Our findings are in agreement with two recent reports analyzing the consequences of cognate interactions taking place between Tregs and DCs in vivo (45, 46). These authors showed that Tregs appear to restrict the ability of APCs to form stable interactions with self-reactive naive effectors, thus preventing naive T cell priming, and differentiation into effectors and/or memory T cells. As Ag-specific Tregs expand at the steady state (Figs. 2–4) and remain functional (Fig. 5), they can therefore inhibit an increasing number of APCs. This is consistent with the hypothesis that sustained disease suppression by polyclonal Tregs depends on endogenous Ag stimulation, occurring in a location where potent Ag-specific Tregs accumulate and continuously inhibit pathogenic T cell response (29, 47).

Ag-specific Tregs were shown to be highly potent in different settings preventing type 1 autoimmune diabetes (4, 5), transgene rejection (6) or bone-marrow rejection (7). Because a simple modification of the balance between Tregs and Th could lead to autoimmunity, as evidenced in an experimental autoimmune encephalomyelitis model (48), our results support the hypothesis that i.v. administration of specific peptide might be beneficial as an effective treatment for autoimmune disorders and more generally should open the path for new therapeutic interventions to induce Ag-specific tolerance.


   Acknowledgments
 
We thank S. Jimenez, B. Gjata, T. Marais, and L. van Wittenberghe for immunohistology and mice preparation, and L. Leserman and C. Boitard for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Association Française contre les Myopathies and by an Action Thématique Incitative du Génopole d’Evry grant from the Genopole Public Interest Group. P.R.C. is supported by the French Ministry of Research. Back

2 J.M.D. and D.-A.G. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. David-Alexandre Gross and Dr. Jean M. Davoust, Genethon Centre National de la Recherche Scientifique FRE3018, 1 Bis Rue de L’internationale, Evry 91002, France. E-mail addresses: gross{at}genethon.fr and jean.davoust{at}necker.fr Back

4 Abbreviations used in this paper: Tregs, regulatory T cells; AAV, adeno-associated virus; HA, hemagglutinin. Back

Received for publication March 20, 2007. Accepted for publication October 22, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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