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The Journal of Immunology, 2006, 176: 5779-5787.
Copyright © 2006 by The American Association of Immunologists

Regulatory T Cells Inhibit Protein Kinase C{theta} Recruitment to the Immune Synapse of Naive T Cells with the Same Antigen Specificity1

Adriana Sumoza-Toledo*, Alfred D. Eaton{dagger} and Adelaida Sarukhan2,*

* Department of Immunology, Institute of Biomedical Investigation, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico; and {dagger} Institut National de la Santé et de la Recherche Médicale, Unité 591, Centre Hospitalier Universitaire Necker, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The precise mechanisms by which regulatory T cells operate, particularly their effect on signaling pathways leading to T cell activation, are poorly understood. In this study we have used regulatory T (Treg) cells of known Ag specificity, generated in vivo, to address their effects on early activation events occurring in naive T cells of the same Ag specificity. We found that the Treg cells need to be present at the moment of priming to suppress activation and proliferation of the naive T cell. Furthermore, the Treg cells significantly inhibit the recruitment of protein kinase C{theta} (PKC{theta}) to the immune synapse of the naive T cell as long as both T cells are of the same Ag specificity and are contacting the same APC. Finally, naturally occurring CD4+25+ T cells seem to have the same effect on PKC{theta} recruitment in CD25– T cells of the same Ag specificity. These results suggest that although additional mechanisms of regulation are likely to exist, inhibition of PKC{theta} recruitment in the effector T cell may be a common regulatory pathway leading to the absence of NF-{kappa}B activation and contributing to the block of IL-2 secretion characteristic of immune suppression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ever since CD4+25+ T cells with suppressor function were identified and characterized in mice (1) and humans (2, 3), it has become clear that they present a powerful tool for the prevention of and/or interference with unwanted immune responses (i.e., autoimmunity and allergy). For this, however, it is necessary to understand in detail how they are generated and how they operate.

This has not proven easy, because it has become evident that regulatory {alpha}beta T cells are heterogeneous with respect to their phenotype, their ontogeny, and most probably their mechanisms of action. It has been shown that CD4+25+ T regulatory (Treg)3 cells are generated in the thymus (4), seem to be selected by agonist self ligands (5, 6), and express high levels of the Forkhead/winged helix transcription factor 3 (FoxP3) (7, 8, 9). CD25+ FoxP3-expressing Treg cells have also been generated in periphery from mainstream peripheral {alpha}beta T cells using self (6) or foreign Ags (10). These CD25+ FoxP3-expressing cells act in a contact-dependent manner, seem to depend, at least in some studies, on TGF-beta production (11, 12), and do not require IL-10 for their generation or function in vitro (6), although suppression of colitis by these cells does seem to require IL-10 (13, 14). It has been proposed that immune suppression by CD4+CD25+ T cells involves granzyme B secretion by the Treg cell (15) and CTLA-4-CD80/86 interactions between the Treg and the naive T cell (16). In addition, CD4+ and CD8+ Treg cells that express FoxP3, but not CD25, have recently been described using an elegant knockin murine model (17), and their mechanism of action is not known, although some could be acting via TGF-beta (18). Finally, Treg cells that express neither CD25+ nor FoxP3 have been generated by chronic Ag stimulation in vivo (19) or in vitro (20) and in some cases seem to suppress via IL-10.

Although Treg cells may act on the effector arm of the immune response (21), it is nevertheless clear that they have a major effect on the priming of the naive T cell, resulting in an impaired proliferation and IL-2 secretion.

It is known that to achieve full activation, the formation of supramolecular activation clusters (SMAC) at the interface between a T cell and an APC must occur after initial engagement of relatively few TCR molecules. The cell contact site containing these SMAC has been termed the immunological synapse (IS) and coordinates cytoskeletal dynamics with the TCR, the engagement of accessory receptors, and membrane-proximal signaling molecules (22). Importantly, in the absence of SMAC formation, productive T cell activation is not observed (reviewed in Ref. 23).

Surprisingly, few studies have addressed the mechanisms of suppression at very early points of T cell priming. More specifically, possible quantitative and/or qualitative changes in the immune synapse of the naive T cell in the presence of regulatory T cells have not been addressed, partly because naturally occurring CD4+25+ T cells are polyclonal and of unknown Ag specificity.

To bypass this obstacle, we have exploited a murine transgenic model in which a relatively homogenous population of Treg cells can be generated in vivo and with known Ag specificity. These Treg cells are specific for a neo-Ag (the influenza virus hemagglutinin (HA)) expressed in both thymus and periphery, share common features with naturally occurring CD4+25+ T cells, such as high levels of CTLA-4, programmed death-1, and IL-10 (24); inhibit immune responses by naive T cells specific for HA both in vitro and in vivo independently of IL-10 (6, 25); and suppress in an Ag-specific manner (26) through as yet unidentified mechanisms.

In this study we have addressed events involved in the early interaction between naive T cells and Ag-loaded APC in the presence of Treg cells of the same Ag specificity. We have found qualitative changes in IS formation that may explain the drastic effect of immune suppression in terms of proliferation and IL-2 secretion.


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

TCR-HA transgenic mice expressing a TCR{alpha}beta specific for peptide 111–119 from influenza HA presented by I-Ed have been previously described (15) and are on the BALB/c background. For some experiments, TCR-HA mice expressing the Thy1.1 allele were used; in other experiments, Thy1.2 TCR-HA mice deficient for the RAG gene were used. Similar results were obtained with RAG-deficient and wild-type TCR-HA transgenic mice. IG-HA mice, expressing the HA transgene under control of the Ig{kappa} promoter and enhancer elements (18), were crossed with TCR-HA mice to generate TCR-HA x IG-HA double-transgenic mice. 5CC7 mice express a TCR{alpha}beta specific for peptide 81–104 pigeon cytochrome c (PCC) presented by I-Ek and are on a RAG-deficient, B10.A background. All mice were bred in our animal facilities in accordance with institutional guidelines.

Antibodies

The 6.5 Ab, which recognizes the transgenic TCR-HA, was produced in our laboratory and was used coupled to biotin, PE, or FITC. All other Abs for flow cytometry were purchased from BD Pharmingen. Cells were analyzed on a flow cytometer (FACSCalibur; BD Biosciences), using CellQuest and FlowJo software.

Cell isolation

Naive CD4+ T cells were obtained from the lymph nodes of TCR-HA mice; depleted of CD8+, CD19+, and CD11b+ cells using a mixture of mAbs and anti-rat Dynabeads (Dynal Biotech); and then positively selected for expression of the transgenic TCR by magnetic sorting. For this, they were incubated with the biotinylated 6.5 mAb, washed, incubated with streptavidin-MACS microbeads (Miltenyi Biotec), and positively selected according to the manufacturer’s instructions with purity always >95%. Regulatory HA-specific T cells were obtained from the pooled spleens and lymph nodes of 3- to 6-mo-old TCR-HA x IG-HA mice. After magnetic sorting with the 6.5 mAb, cells were stained with CD4-FITC Ab and sorted on a FACSAria (BD Biosciences) to eliminate the contaminating CD4–8– cells expressing the transgenic TCR. CD25+ and CD25– T cells were obtained from pooled spleens and lymph nodes of TCR-HA mice, MACS sorted for 6.5 expression, stained with CD25-FITC Ab, and sorted on a FACSAria (BD Biosciences) for negative or high expression of CD25.

Dendritic cells (DC) were obtained from the bone marrow of BALB/c mice and were grown in the presence of GM-CSF. Between days 8 and 9, cells were incubated overnight with LPS and different doses of HA peptide and were washed before using. For some experiments, bone marrow from (B10D.2 x B10.A)F1 mice was used, and DC were incubated with equimolar doses of PCC and HA peptides.

In vitro regulation assays

All assays were performed in complete IMDM, supplemented with 10% FCS. Naive T cells (2 x 104) were stained with CFSE and incubated with DC (5 x 104). In some wells, FACS-sorted CD4+6.5+ regulatory cells were added at a 1:1 or 1:2 ratio as indicated.

CD69 expression was measured 20 h after incubation by flow cytometry gating on CFSE-positive T cells. IL-2 mRNA expression by MACS-separated Thy1.1 naive T cells was determined by RT-PCR 20 h after incubation.

Cell conjugates

Sorted 6.5+ naive T cells (3 x 104), mixed or not with sorted 6.5+ Treg cells at a 1:2 ratio, were added to DC (also 3 x 104) that had been previously pulsed with no peptide or with 0.5–1.0 µg/ml HA peptide overnight. The cells were centrifuged briefly, incubated at 37°C for 15 min, pipetted several times to eliminate unstable conjugates, transferred to coverslips previously treated with poly-L-lysine (Sigma-Aldrich), and incubated for an additional 15 min at 37°C. Conjugates were fixed with 3% paraformaldehyde for 15 min at room temperature.

To differentiate Treg cells from naive T cells or CD25+ Treg cells from CD25– T cells, cells were stained with CellTracker Green (Molecular Probes) following the manufacturer’s instructions before they were mixed with naive T cells and DC. When both naive HA- and PCC-specific cells were incubated in the presence of Treg cells, the PCC-specific cells were stained previously with CellTracker Orange (Molecular Probes).

Confocal microscopy

For actin and microtubules, conjugates were permeabilized with 0.05% Triton X-100 (Sigma-Aldrich) for 10 min at room temperature and extensively washed with 1x PBS. Samples were then stained with rhodamine-phalloidin (Sigma-Aldrich) for 20 min at room temperature, followed by mouse anti-beta tubulin (Zymed Laboratories) for 1 h at 37°C. For protein kinase C{theta} (PKC{theta}), conjugates were permeabilized with 0.05% saponin (Sigma-Aldrich) plus 2% BFS for 20 min at room temperature, then stained with mouse-anti PKC{theta} (BD Transduction Laboratories) for 1 h at 37°C and counterstained with rhodamine-phalloidin. Alexa Fluor-633-conjugated anti-mouse IgG (Molecular Probes) was used as the secondary Ab for beta-tubulin and PKC{theta}. Finally, conjugates were mounted with Vectashield (Vector Laboratories) and analyzed by confocal microscopy. The sections were observed in a LSM5 Pascal confocal microscope (Zeiss).

Statistical analysis was performed using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Immune suppression requires the presence of Treg cells at the moment of T cell priming

We first tried to determine whether suppression, at least in vitro, required the presence of Treg cells at the moment of priming or whether they were still capable of suppressing T cell proliferation after successful priming of naive T cells had occurred.

For this, we incubated naive T cells from a TCR-HA transgenic mice with peptide-loaded DC in the absence or the presence of HA-specific Treg cells, obtained from a TCR-HA x IG-HA double-transgenic mouse, as described in Materials and Methods. These Treg cells were added at different time points. When naive and Treg cells were added at the same time, a significant decrease in expression of the early activation marker CD69 was observed after 20 h of incubation (Fig. 1A). In parallel, IL-2 gene expression by the naive (Thy1.1-positive) T cells was also determined after 20 h of stimulation and was greatly inhibited by the presence of Treg cells (Fig. 1B), suggesting that suppression is occurring at early time points. This was confirmed by the experiment shown in Fig. 1C; efficient suppression of the proliferative response of naive HA-specific T cells only occurred when Treg cells were added at the time of priming (time zero), but not at later time points (5 or 17 h). Although we cannot formally exclude simple competition for MHC:peptide complexes as an explanation for the effects of Treg cells on activation and proliferation of naive T cells, the addition of naive T cells at the same ratio as Treg cells did not affect CD69 expression in parallel experiments (data not shown).


Figure 1
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  		FIGURE 1. Treg cells suppress early activation events. A, Treg cells suppress surface expression of CD69 on naive T cells stimulated with peptide. HA-specific naive CD4+ T cells were incubated with irradiated BALB/c splenocytes with or without HA peptide and HA-specific Treg cells. Surface expression of CD69 on naive T cells was measured after 20 h by flow cytometry. The graph shows the level of surface CD69 on naive T cells 20 h after stimulation with no peptide (unstimulated control; filled area), HA peptide (dashed line), or HA peptide in the presence of Treg cells (solid line). The results shown are representative of analyses of triplicate wells and repeated experiments. B, Treg cells suppress the expression of IL-2 by naive T cells. CFSE-stained HA-specific naive CD4+ T cells were incubated with bone marrow-derived B10.A x B10.D2 DC with or without HA peptide and HA-specific Treg cells. CFSEhigh cells were purified by FACS after 20 h, mRNA was extracted and reverse transcribed to cDNA, and real-time PCR was used to determine the relative gene expression level of IL-2. The graph shows the amount of IL-2 mRNA in each sample compared with the unstimulated sample and normalized to the amount of mRNA for a control gene (Pol2a) in the same sample. Analyses were performed in triplicate, using cells pooled from at least nine wells, and are representative of repeated experiments. C, Delayed addition of Treg cells reduces suppression of proliferation. CFSE-stained HA-specific naive Thy1.1+CD4+ T cells were incubated with ex vivo CD11c+ BALB/c DC with or without HA peptide. HA-specific Treg cells were added at a naive:Treg ratio of 1:4 after 0, 5, or 17 h of incubation. Naive T cell division, represented by the amount of CFSE present in each cell, was measured after 93 h by flow cytometry. The graphs are representative of analyses of triplicate wells and repeated experiments.

 
Actin polymerization and microtubule organizing center (MTOC) reorientation by naive T cells are not suppressed by Treg cells

The findings described above prompted us to look at very early activation events occurring in naive T cells that could be modified by the presence of Treg cells. In particular, we determined whether IS formation between the DC and the naive T cell could be a target of suppression.

The initial physical contact between the APC and the T cell triggers a few TCRs, which, in turn, induces actin polymerization, F-actin clustering in the contact region, and translocation of the MTOC to the junction (reviewed in Ref. 23). With the persistence of TCR signaling, surface receptors and signaling molecules in preassembled lipid rafts start accumulating in the center of the contact plane, and there is a selective recruitment of active PKC{theta} into the central SMAC (cSMAC). It has been previously described that without PKC{theta} recruitment, IL-2 production and cell proliferation were greatly impaired (Ref. 27 ; reviewed in Ref. 28). We thus addressed the question of whether F-actin polymerization, MTOC reorganization, and PKC{theta} recruitment to the immune synapse in naive T cells were normal in the presence of Treg cells.

For this, we produced cell conjugates using bone marrow-derived DC activated by LPS and loaded, or not, with the HA peptide, naive T cells from a single transgenic TCR-HA mouse, and Treg cells obtained from a double-transgenic TCR-HA x IG-HA mouse, as described in Materials and Methods. After 30 min of coincubation, cells were fixed and stained for F-actin, tubulin, and/or PKC{theta}, and cell conjugates were analyzed by confocal microscopy.

As shown in Fig. 2, naive HA-specific T cells showed actin polymerization at the contact site, as revealed by staining with phalloidin, when incubated with peptide-loaded DC. This polymerization was accompanied by MTOC reorientation to the junction, considered positive when strong staining for beta-tubulin was observed at the site of contact with the DC. As expected, a significantly higher percentage of T cells presented reorientation of the MTOC toward the contact site when the DC were loaded with peptide. Some MTOC reorientation toward the contact site was also observed upon incubation with unloaded DC (see histogram). This could be due to the fact that the DC used in this study are mature DC, and it has been reported that mature CD4+ T cell-DC synapses are formed in the absence of Ag and lead to several T cell responses: a local increase in tyrosine phosphorylation, small Ca2+ responses, weak proliferation, and long-term survival (29). When coincubated in the presence of Treg cells, no significant differences in the number of naive T cells presenting MTOC reorientation toward the contact site was observed, suggesting that suppression does not affect the formation of DC-naive T cell conjugates. Because MTOC reorientation requires triggering by a few TCRs, this also indirectly suggests that initial TCR signaling is not affected by Treg cells of the same Ag specificity.


Figure 2
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  		FIGURE 2. Treg cells do not significantly suppress actin polymerization or MTOC reorientation by naive T cells of the same specificity. Naive cells were incubated for 30 min with peptide-loaded or unloaded mature DC and with or without Treg cells that were previously stained with green cell tracker. Cell conjugates were adhered onto poly-L-lysine coverslips, fixed, and permeabilized as described in Materials and Methods. Blue staining represents beta-tubulin stained with anti-beta-tubulin Ab and revealed with goat anti-IgG mouse Alexa 633. Red staining represents F-actin stained with tetramethylrhodamine isothiocyanate-labeled phalloidin. Polarization of microtubules was considered positive when the MTOC was oriented toward the contact site with the DC. A total of 10–30 cell conjugates were analyzed per experiment. The histograms represent the mean and SD values of three independent experiments.

 
Treg cells inhibit PKC{theta} recruitment to the IS of the naive T cell

Because PKC{theta} recruitment to the contact site is considered to be the hallmark of a mature immune synapse, we next addressed its recruitment to the IS of the naive T cell in the presence of Treg cells of the same Ag specificity. First, it was important to confirm that the positive selection of T cells using the clonotypic mAb did not by itself induce PKC{theta} clustering. As shown in the first row and in the histogram of Fig. 3, only a small percentage of naive T cells that were in contact with unloaded DC recruited PKC{theta}, indicating that the purification protocol did not induce such recruitment. When incubated with peptide-loaded DC, the majority of naive HA-specific T cells recruited PKC{theta} to the contact site (Fig. 3, middle row). The majority of cell contacts between naive T cells and peptide-loaded DC corresponded to the slow dynamic contacts described by Friedl et al. (30), characterized by polarized and semicircular contacts between the T cell and the DC. When naive T cells were incubated with peptide-loaded DC in the presence of Treg cells, PKC{theta} recruitment by naive T cells was significantly reduced (lower row) in those conjugates containing the three cell types (DC, naive T cell, and Treg cell). Ten to 30 naive Treg-DC conjugates were analyzed at different planes on the z-axis for each experiment. An artificial color scale (pseudocolor) was assigned for PKC{theta}, as shown in the last column of Fig. 3, and polarization was considered positive when the color at the contact site was red. As shown in the histograms, 55% of naive T cells in such conjugates displayed a complete absence of PKC{theta} recruitment. Among the remaining 45%, PKC{theta} recruitment was considered positive according to our criteria, although in many of these cells, polarization was less marked. Fixing cells at later time points (60 or 120 min) after conjugation formation did not significantly alter the percentage of polarized cells (data not shown), suggesting that Treg cells inhibit PKC recruitment, not only delay their kinetics. Importantly, in cell conjugates on the same coverslip that contained only naive T cells and peptide-loaded DC, PKC{theta} recruitment was observed. These results suggest that Treg cells may require cell contact to suppress PKC{theta} recruitment by naive T cells.


Figure 3
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  		FIGURE 3. Treg cells significantly decrease PKC recruitment to the IS of the naive T cell if contacting the same DC. Cell conjugates were obtained, fixed, and permeabilized as described in Materials and Methods. Treg cells are stained in green. Blue staining represents anti-PKC{theta} Ab revealed with goat anti-IgG mouse Alexa 633, and red staining represents F-actin stained with tetramethylrhodamine isothiocyanate-labeled phalloidin. A pseudocolor was assigned to the PKC staining, and polarization was considered positive when the Ab concentration reached the maximum value in the scale (red). For each experiment, between 10 and 30 cell conjugates were analyzed at different planes. The histograms represent the mean and SD values for three independent experiments.

 
Interestingly, we also observed that Treg cells alone did not recruit PKC{theta} when incubated with peptide-loaded DC (Fig. 3, lower row, green cells), although they did polymerize F-actin and reorient their MTOC toward the contact site with the APC (Fig. 2, lower row, green cells; our unpublished observations). The biological significance of this observation is currently being addressed, but it is in accordance with the absence of PKC{theta} recruitment among orally tolerized cells of known Ag specificity, as described recently (31).

Because the majority of mature immune synapses are formed within the first 30 min (23), we tested whether immune regulation in terms of proliferation could still be observed after mature IS had formed between naive T cells and Ag-loaded DC. For this, we tested the effect of adding Treg cells at earlier time periods than those described in Fig. 1 and under the same conditions and the same ratios as the confocal experiments described above. As shown in Fig. 4, 45% inhibition was achieved when adding the Treg cells at the same time, and such inhibition decreased if they were added after 30, 60, or 120 min. Such a percentage is similar to the inhibition of PKC recruitment observed in Fig. 2, which suggests that both phenomena are closely linked. However, these experiments do not exclude other mechanisms of regulation that may be acting after the IS has formed.


Figure 4
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  		FIGURE 4. Treg cells inhibit T cell proliferation when added before mature IS have formed. Thy1.1+ CFSE-stained naive HA-specific T cells were incubated with peptide-loaded DC in the presence or the absence of Treg cells at a naive:Treg ratio of 1:2. Treg cells were added at the same time (time 0) or 30, 60, or 120 min after contact between naive T cells and DC. Cell proliferation was determined by flow cytometry after 72 h. Shown is the percent inhibition of proliferation by naive T cells in the absence of regulatory T cells. This is one representative experiment of two performed.

 
PKC{theta} recruitment is not inhibited in T cells of different Ag specificity than the Treg cell

We have previously shown that these HA-specific Treg cells act in an Ag-specific manner both in vitro and in vivo; that is, they do not suppress proliferation or IL-2 secretion by naive CD4+ T cells specific for another Ag even if both Ags are presented by the same APC (26). As shown in Fig. 4, this was also true even if both types of naive cells (HA- and PCC-specific) were incubated in the same well with DC loaded with both peptides and in the presence of HA-specific Treg cells; only HA-specific naive T cells, not PCC-specific T cells, were inhibited in terms of proliferative response by HA-specific Treg cells. We thus sought to determine whether the inhibition of PKC{theta} recruitment observed in Fig. 3 was also Ag specific. For this, we performed cell conjugates with DC expressing both I-Ed and I-Ek and thus capable of presenting both peptides. These DC were incubated with naive PCC-specific T cells in the absence or the presence of HA-specific Treg cells (the latter stained in green). In parallel, we performed conjugates with naive HA-specific T cells in the absence or the presence of Treg cells, which are also HA specific. When mixing naive and Treg cells, only conjugates containing at least one naive T cell and one Treg cell were counted. The presence of Treg cells did not affect PKC{theta} recruitment among naive PCC-specific T cells, as shown in the representative images in the upper and middle rows of Fig. 6. For each experiment, we counted 20–30 conjugates containing at least one Treg cell and one naive T cell, and as shown in the histogram of Fig. 6, the percentage of naive PCC-specific T cells that recruited PKC{theta} in the absence or the presence of Treg cells was not altered (see histogram). As expected, in conjugates containing Treg and HA-specific naive T cells, the percentage of cells showing PKC{theta} polarization was decreased in presence of Treg cells, although for unknown reasons, such as the different nature of the DC, it was less efficient than that shown in Fig. 3 (see histogram). It should be pointed out that the greater inhibition of proliferation observed in Fig. 5 compared with the inhibition of PKC{theta} recruitment observed in the immune synapse experiments (Fig. 6) may be explained at least in part by the fact that the naive:Treg ratios used in such experiments were 1:4 vs 1:2, respectively. Finally, we also performed conjugates with both types of naive cells together with the HA-specific Treg cells. In this case, PCC-specific T cells were stained in red. In these experiments we did not obtain enough conjugates containing all three types of T cells to perform quantifications, but a representative image is shown (Fig. 6, bottom row). In this image, one can observe a conjugate containing one Treg cell (green), two PCC-specific naive T cells (red), and one HA-specific T cell (no color). In this conjugate, PKC{theta} recruitment was inhibited among the naive HA-specific, but not the PCC-specific, T cells. Overall, these results confirm that inhibition of PKC{theta} recruitment by naive T cells correlates with their suppression in terms of proliferation and IL-2 production.


Figure 6
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  		FIGURE 6. Treg cells do not inhibit PKC{theta} recruitment among naive T cells of different Ag specificity. HA- or PCC-specific naive T cells were coincubated with F1 (B10.A x B10.D2) DC loaded with equimolar concentrations of HA and PCC peptides and in the presence or the absence of HA-specific Treg cells, stained with cell tracker green, at a 1:2 ratio. Cell conjugates were fixed, permeabilized, and stained for PKC{theta}, shown in blue. In some experiments naive HA-specific T cells (not stained) and PCC-specific T cells (stained with cell tracker orange) were incubated together in the presence of Treg cells. The upper panel represents conjugates of PCC-specific cells with loaded DC. The middle panel represents conjugates of PCC-specific cells with loaded DC and Treg cells (green). The lower panel represents conjugates of PCC-specific (red) and HA-specific naive T cells with loaded DC in presence of Treg cells (green). Polarization for the naive T cells was evaluated as described above. For each experiment, between 20 and 30 cell conjugates containing both naive and Treg cells were analyzed on different planes. The histograms represent the mean and SD values for two independent experiments.

 

Figure 5
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  		FIGURE 5. Suppression of T cell proliferation by Treg cells of the same Ag specificity occurs even during coculture with proliferating T cells of a different specificity. CFSE-stained, HA-specific CD4+ T cells (left panel) or CFSE-stained, PCC-specific CD4+ T cells (right panel) were incubated, either together or separately, with irradiated B10.A x B10.D2 bone marrow-derived DC with or without HA and PCC peptides and with or without HA-specific Treg cells at a naive:Treg ratio of 1:4. Naive T cell division, represented by the amount of CFSE present in each cell, was measured after 86 h by flow cytometry. The graphs show the amount of CFSE present in naive T cells 86 h after stimulation with no peptide (unstimulated control; filled area), HA peptide (dashed line), or HA peptide in the presence of Treg cells (solid line). The results shown are representative of analyses of duplicate wells and repeated experiments.

 
CD25+ natural Treg cells also suppress PKC{theta} recruitment by CD25– naive T cells of the same Ag specificity

The Treg cells obtained from TCR-HA, IG-HA double-transgenic mice have certain features in common with CD25+ naturally occurring Treg cells, but may have a different origin and a different gene expression profile (A. D. Eaton, unpublished observations) and may well suppress using different mechanisms. Therefore, it was important to determine whether naturally occurring CD25+ Treg cells also suppressed PKC{theta} recruitment by naive CD25– T cells. To address this question, we used CD25+ and CD25– cells expressing the HA-specific TCR, obtained from single transgenic TCR-HA mice that were not deficient for RAG. Such cells were selected using the 6.5 clonotype-specific Ab and then sorted for high or no CD25 expression.

As expected (Fig. 7), a high percentage of CD25– cells (80%) recruited PKC{theta} to the IS when DC were preincubated with peptide, compared with the percentage of cells that recruited PKC{theta} when conjugated with unloaded DC. In the presence of CD25high cells of the same Ag specificity, the percentage of CD25– T cells showing PKC{theta} recruitment decreased to 50%, although these values did not reach statistical significance. This was again observed only when both naive and Treg cells were contacting the same DC. In almost half the conjugates observed, the CD25– cell appeared to redirect its IS toward the CD25+ T cell. Nevertheless, we could not exclude the presence of DC elongations between both T cells, and thus the CD25– cells were still counted as polarized T cells. Interestingly though, although HA-specific Treg cells from TCR-HA x IG-HA double-transgenic mice did not recruit PKC{theta} to their IS (Fig. 3, lower row, green cells), the CD25+ naturally occurring Treg cells did seem to recruit PKC{theta} upon TCR stimulation (Fig. 6, lower row, green cells). The reason for this is not known, but it could well reflect differences between these populations of Treg cells; we are currently addressing this issue.


Figure 7
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  		FIGURE 7. CD25+ natural Treg cells also decrease PKC{theta} recruitment of CD25– T cells of the same Ag specificity. HA-specific, CD25+ and CD25– cells from a single TCR-HA transgenic mouse were sorted and incubated separately or together with peptide-loaded or unloaded DC at a 1:2:1 (CD25–:CD25+:DC) ratio. Cell conjugates were fixed and permeabilized as described in Materials and Methods. CD25+ cells were stained with cell tracker green. Blue staining represents PKC{theta}, and red staining represents F-actin. For each experiment, 20–30 cell conjugates were analyzed at different planes, and polarization was evaluated as described above. The histograms represent the mean and SD values for two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We show in this study that Treg cells must be present in the early stages of priming to exert their suppressive function on naive T cells of the same Ag specificity, and that they interfere with early activation events. In particular, they modify the type of IS that is established between the naive T cell and the Ag-loaded DC by inhibiting the recruitment of PKC{theta} to the contact site between the T cell and the peptide-loaded DC. This is true for Treg cells generated by encounter with self ligand in the thymus and periphery and seems to be true for CD25+ naturally occurring Treg cells, generated mainly in the thymus. It will be interesting to determine whether there is normal SMAC formation in those cells that are not recruiting PKC{theta} to the IS, although preliminary experiments with CD3{zeta} staining do not show effects on CD3 accumulation at the contact site between naive T cells and peptide-loaded DC in the presence of Treg cells, suggesting that a broad defective SMAC formation is unlikely (A. Sumoza-Toledo, unpublished observations).

It has been demonstrated that PKC{theta} is a central regulator of several pathways critical to T cell activation and ultimately affecting IL-2 production (reviewed in Ref. 32 , 33). It was shown that PKC{theta}-deficient T cells fail to mobilize the transcription factors AP1 and NF-{kappa}B, (34, 35), and one strain of mice also failed to mobilize NFAT (35). Furthermore, constitutively active PKC{theta} can stimulate an NF-{kappa}B reporter gene in T cells (36) through a recently proposed pathway involving interactions with Bcl10 and CARMA1 (also known as caspase recruitment domain (CARD) 11, Bimp3), which would, in turn, phosphorylate I{kappa}B kinase beta (37). Importantly, raft localization of PKC{theta} appears to be connected with its ability to exert its signaling functions, because the catalytic domain alone fails to activate transcription factors downstream of PKC{theta} (38). Thus, the results obtained in this study would explain many of the effects observed among suppressed T cells: the decrease in CD69 up-regulation, the drastic inhibition of IL-2 secretion, and the impaired proliferative response. In effect, it has been observed that PKC{theta}-deficient cells fail to up-regulate CD69, that activation and translocation of PKC{theta} to the IS is required for TCR-dependent proliferation, and that T cells with APC pulsed with suboptimal concentrations of Ag failed to cause recruitment of PKC{theta} (27). Furthermore, a recent study (31) indicates that orally tolerized T cells can form stable conjugates with APCs, but show defects in the translocation of TCR and PKC{theta} to the IS; this may account for the hyporesponsive state of these cells.

Although there is a relatively good correlation between the inhibition of proliferative responses and that of PKC{theta} recruitment by the naive cell under identical experimental conditions (Figs. 3 and 4), we certainly cannot exclude that there may be additional mechanisms of immune regulation in addition to the inhibition of PKC{theta} recruitment, because Treg cells added after establishment of mature IS still have a moderate effect on naive T cell proliferation (Figs. 1 and 4). The mechanisms by which PKC{theta} recruitment by the naive T cell is inhibited in the presence of Treg cells are unclear and need to be addressed by studying the recruitment of other signaling molecules, both upstream and downstream of PKC{theta}.

Although we cannot conclude that direct naive cell to Treg cell contact is required, at least in these experiments, contacting the same APC was essential for the inhibition of PKC{theta} recruitment. It is known that PKC{theta} translocation is dependent on its activation. Activation of PKC{theta} seems to depend on signals generated by the TCR, such as SLP-76 and Zap70, as well as on signals generated by TCR and CD28 activation, such as Vav (39). In fact, a nonconventional translocation pathway dependent on Vav and PI3K has been proposed, and it has been shown that although TCR ligation alone induces weak PKC{theta} raft association, CD28 costimulation allows concentration of PKC{theta} to the cSMAC, resulting in strong association of PKC{theta} with lipid rafts (38). Thus, one possibility is that the CTLA4 expressed in high levels by the Treg cells used in this study (24) could be sequestering CD80 and CD86 on the DC and inhibiting CD28-dependent costimulation on the naive T cell and PKC{theta} recruitment to the cSMAC. Another possibility could be simply the sequestration of MHC:peptide complexes of the DC by the Treg cell, inhibiting PKC{theta} activation signals through the TCR. Either of these possibilities would explain why regulation can be broken under certain conditions by high antigenic doses (26), but only the latter would explain the Ag-specific effect on PKC{theta} recruitment and proliferation that we observed in our model. In any case, these possibilities need to be addressed by additional experimentation. Clearly, the behaviors of molecules upstream (CD28) and downstream (CARMA-1 and I{kappa}B kinase beta) of PKC{theta} in this system need to be addressed.

Importantly, we compared CD25+ naturally occurring HA-specific Treg cells with the HA-specific Treg cells used in this study. Interestingly, although they are surely different in terms of origin, gene expression, and mechanisms of action (6), they also seem to have an inhibitory effect on PKC{theta} recruitment by naive CD25– T cells of the same Ag specificity and do so only when contacting the same DC. The fact that they inhibit less efficiently than HA-specific Treg cells from double-transgenic mice may be due to the heterogeneity among CD25highCD4 T cells and the fact that all of them may not be regulatory. Interestingly, we found that CD25+ T cells do not seem to have defects in their own capacity to recruit PKC{theta} upon antigenic stimulation. This contrasts with Treg cells obtained from double-transgenic mice (this study) and with orally tolerized, Ag-specific T cells (31). These results suggest that the inability to recruit PKC{theta} by the Treg cell may not be directly linked to its ability to suppress PKC{theta} recruitment and activation in the naive T cell.

Overall, the results obtained in this study suggest that although Treg cells may be acting in different manners and via different molecules, inhibition of PKC{theta} recruitment by the effector T cell may be a common feature that leads to the absence of NF-{kappa}B activation and the block of IL-2 secretion that are characteristic of immune suppression.


   Acknowledgments
 
We thank Gerardo Arrellin (Mexico) and Sandrine Léaument (Paris, France) for excellent animal breeding and care. We are grateful to Claire Hivroz and Gloria Soldevila for valuable advice and critical reading of the manuscript. We are grateful to Norma Moreno for help with the confocal microscopy, and Carlos Castellanos for help with cell sorting.


   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 funded by Consejo Nacional de Ciencia y Technologia Grant 44212, Juvenile Diabetes Foundation Grant GRT2003-1335, and Institut National de la Santé et de la Recherche Médicale. A.D.E. was funded by Institut National de la Santé et de la Recherche Médicale and the Juvenile Diabetes Foundation. A.S.-T. was funded by a fellowship from the Universidad Nacional Autónoma de México, and A.S. was funded by Institut National de la Santé et de la Recherche Médicale and Consejo Nacional de Ciencia y Technologia. Back

2 Address correspondence and reprint requests to Dr. Adelaida Sarukhan, Department of Immunology, Institute of Biomedical Investigation, UNAM, Mexico City, Mexico E-mail address: sarukhan{at}biomedicas.unam.mx Back

3 Abbreviations used in this paper: Treg, T regulatory; cSMAC, central SMAC; DC, dendritic cell; FoxP3, Forkhead/winged helix transcription factor 3; HA, hemagglutinin; IS, immunological synapse; MTOC, microtubule organizing center; PCC, pigeon cytochrome c; PKC{theta}, protein kinase C{theta}; SMAC, supramolecular activation cluster. Back

Received for publication October 12, 2005. Accepted for publication February 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor {alpha}-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155: 1151-1164. [Abstract]
  2. Baecher-Allan, C., J. A. Brown, G. J. Freeman, D. A. Hafler. 2001. CD4+CD25high regulatory cells in human peripheral blood. J. Immunol. 167: 1245-1253. [Abstract/Free Full Text]
  3. Levings, M. K., R. Sangregorio, M. G. Roncarolo. 2001. Human CD25+CD4+ T regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function. J. Exp. Med. 193: 1295-1302. [Abstract/Free Full Text]
  4. Sakaguchi, S.. 2000. Regulatory T cells: key controllers of immunologic self-tolerance. Cell 101: 455-458. [Medline]
  5. Jordan, M. S., A. Boesteanu, A. J. Reed, A. L. Petrone, A. E. Holenbeck, M. A. Lerman, A. Naji, A. J. Caton. 2001. Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2: 301-306. [Medline]
  6. Apostolou, I., A. Sarukhan, L. Klein, H. von Boehmer. 2002. Origin of regulatory T cells with known specificity for antigen. Nat. Immunol. 3: 756-763. [Medline]
  7. Khattri, R., T. Cox, S. A. Yasayko, F. Ramsdell. 2003. An essential role for scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 4: 337-342. [Medline]
  8. Hori, S., T. Nomura, S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299: 1057-1061. [Abstract/Free Full Text]
  9. Fontenot, J. D., M. A. Gavin, A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4: 330-336. [Medline]
  10. Apostolou, I., H. von Boehmer. 2004. In vivo instruction of suppressor commitment in naive T cells. J. Exp. Med. 199: 1401-1408. [Abstract/Free Full Text]
  11. Chen, M. L., M. J. Pittet, L. Gorelik, R. A. Flavell, R. Weissleder, H. von Boehmer, K. Khazaie. 2005. Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-beta signals in vivo. Proc. Natl. Acad. Sci. USA 102: 419-424. [Abstract/Free Full Text]
  12. Green, E. A., L. Gorelik, C. M. McGregor, E. H. Tran, R. A. Flavell. 2003. CD4+CD25+ T regulatory cells control anti-islet CD8+ T cells through TGF-beta-TGF-beta receptor interactions in type 1 diabetes. Proc. Natl. Acad. Sci. USA 100: 10878-10883. [Abstract/Free Full Text]
  13. Asseman, C., S. Mauze, M. W. Leach, R. L. Coffman, F. Powrie. 1999. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190: 995-1004. [Abstract/Free Full Text]
  14. Suri-Payer, E., A. Z. Amar, A. M. Thornton, E. M. Shevach. 1998. CD4+CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells. J. Immunol. 160: 1212-1218. [Abstract/Free Full Text]
  15. Gondek, D. C., L. F. Lu, S. A. Quezada, S. Sakaguchi, R. J. Noelle. 2005. Cutting edge: contact-mediated suppression by CD4+CD25+ regulatory cells involves a granzyme B-dependent, perforin-independent mechanism. J. Immunol. 174: 1783-1786. [Abstract/Free Full Text]
  16. Taylor, P. A., C. J. Lees, S. Fournier, J. P. Allison, A. H. Sharpe, B. R. Blazar. 2004. B7 expression on T cells down-regulates immune responses through CTLA-4 ligation via T-T interactions. J. Immunol. 172: 34-39. [Abstract/Free Full Text]
  17. Fontenot, J. D., J. P. Rasmussen, L. M. Williams, J. L. Dooley, A. G. Farr, A. Y. Rudensky. 2005. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22: 329-341. [Medline]
  18. Belghith, M., J. A. Bluestone, S. Barriot, J. Megret, J. F. Bach, L. Chatenoud. 2003. TGF-beta-dependent mechanisms mediate restoration of self-tolerance induced by antibodies to CD3 in overt autoimmune diabetes. Nat. Med. 9: 1202-1208. [Medline]
  19. Vieira, P. L., J. R. Christensen, S. Minaee, E. J. O’Neill, F. J. Barrat, A. Boonstra, T. Barthlott, B. Stockinger, D. C. Wraith, A. O’Garra. 2004. IL-10-secreting regulatory T cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4+CD25+ regulatory T cells. J. Immunol. 172: 5986-5993. [Abstract/Free Full Text]
  20. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. de Vries, M. G. Roncarolo. 1997. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389: 737-742. [Medline]
  21. Klein, L., K. Khazaie, H. von Boehmer. 2003. In vivo dynamics of antigen-specific regulatory T cells not predicted from behavior in vitro. Proc. Natl. Acad. Sci. USA 100: 8886-8891. [Abstract/Free Full Text]
  22. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, M. L. Dustin. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285: 221-227. [Abstract/Free Full Text]
  23. Kupfer, A., H. Kupfer. 2003. Imaging immune cell interactions and functions: SMACs and the immunological synapse. Semin. Immunol. 15: 295-300. [Medline]
  24. Lechner, O., J. Lauber, A. Franzke, A. Sarukhan, H. von Boehmer, J. Buer. 2001. Fingerprints of anergic T cells. Curr. Biol. 11: 587-595. [Medline]
  25. Jooss, K., B. Gjata, O. Danos, H. von Boehmer, A. Sarukhan. 2001. Regulatory function of in vivo anergized CD4+ T cells. Proc. Natl. Acad. Sci. USA 98: 8738-8743. [Abstract/Free Full Text]
  26. Tanchot, C., F. Vasseur, C. Pontoux, C. Garcia, A. Sarukhan. 2004. Immune regulation by self-reactive T cells is antigen specific. J. Immunol. 172: 4285-4291. [Abstract/Free Full Text]
  27. Monks, C. R., H. Kupfer, I. Tamir, A. Barlow, A. Kupfer. 1997. Selective modulation of protein kinase C-{theta} during T-cell activation. Nature 385: 83-86. [Medline]
  28. Altman, A., M. Villalba. 2003. Protein kinase C-{theta} (PKC{theta}): it’s all about location, location, location. Immunol. Rev. 192: 53-63. [Medline]
  29. Revy, P., M. Sospedra, B. Barbour, A. Trautmann. 2001. Functional antigen-independent synapses formed between T cells and dendritic cells. Nat. Immunol. 2: 925-931. [Medline]
  30. Friedl, P., J. Storim. 2004. Diversity in immune-cell interactions: states and functions of the immunological synapse. Trends Cell Biol. 14: 557-567. [Medline]
  31. Ise, W., K. Nakamura, N. Shimizu, H. Goto, K. Fujimoto, S. Kaminogawa, S. Hachimura. 2005. Orally tolerized T cells can form conjugates with APCs but are defective in immunological synapse formation. J. Immunol. 175: 829-838. [Abstract/Free Full Text]
  32. Isakov, N., A. Altman. 2002. Protein kinase C{theta} in T cell activation. Annu. Rev. Immunol. 20: 761-794. [Medline]
  33. Sedwick, C. E., A. Altman. 2004. Perspectives on PKC{theta} in T cell activation. Mol. Immunol. 41: 675-686. [Medline]
  34. Sun, Z., C. W. Arendt, W. Ellmeier, E. M. Schaeffer, M. J. Sunshine, L. Gandhi, J. Annes, D. Petrzilka, A. Kupfer, P. L. Schwartzberg, et al 2000. PKC-{theta} is required for TCR-induced NF-{kappa}B activation in mature but not immature T lymphocytes. Nature 404: 402-407. [Medline]
  35. Pfeifhofer, C., K. Kofler, T. Gruber, N. G. Tabrizi, C. Lutz, K. Maly, M. Leitges, G. Baier. 2003. Protein kinase C{theta} affects Ca2+ mobilization and NFAT cell activation in primary mouse T cells. J. Exp. Med. 197: 1525-1535. [Abstract/Free Full Text]
  36. Coudronniere, N., M. Villalba, N. Englund, A. Altman. 2000. NF-{kappa}B activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-{theta}. Proc. Natl. Acad. Sci. USA 97: 3394-3399. [Abstract/Free Full Text]
  37. Wang, D., Y. You, S. M. Case, L. M. McAllister-Lucas, L. Wang, P. S. DiStefano, G. Nunez, J. Bertin, X. Lin. 2002. A requirement for CARMA1 in TCR-induced NF-{kappa}B activation. Nat. Immunol. 3: 830-835. [Medline]
  38. Bi, K., Y. Tanaka, N. Coudronniere, K. Sugie, S. Hong, M. J. van Stipdonk, A. Altman. 2001. Antigen-induced translocation of PKC-{theta} to membrane rafts is required for T cell activation. Nat. Immunol. 2: 556-563. [Medline]
  39. Villalba, M., K. Bi, J. Hu, Y. Altman, P. Bushway, E. Reits, J. Neefjes, G. Baier, R. T. Abraham, A. Altman. 2002. Translocation of PKC{theta} in T cells is mediated by a nonconventional, PI3-K- and Vav-dependent pathway, but does not absolutely require phospholipase C. J. Cell Biol. 157: 253-263. [Abstract/Free Full Text]



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