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The Journal of Immunology, 2008, 180, 1405 -1413
Copyright © 2008 by The American Association of Immunologists, Inc.
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Suppression of Mature Dendritic Cell Function by Regulatory T Cells In Vivo Is Abrogated by CD40 Licensing1

Jens Hänig and Manfred B. Lutz2

Department of Dermatology, University Hospital Erlangen, Erlangen; and Institute for Virology and Immunobiology, University of Wuerzburg, Wuerzburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The priming of CD4+ effector T cells (Teff) in vivo is induced by mature dendritic cells (DC) and controlled by CD4+CD25+Foxp3+ regulatory T cells (Treg). It remains unclear,however, how Teff priming vs Treg suppression are regulated during Ag presentation by DC in secondary lymphoid organs at the simultaneous presence of Teff and Treg. In this study, we used an peptide-specific DO11.10 TCR-transgenic adoptive transfer model to follow the Teff priming kinetics and the mechanisms of suppression by Treg. Treg activation was slower as compared with Teff and could not influence the early Teff expansion but limited the Teff response leading to lower Teff numbers in the memory phase. DC-Treg cell contacts remained unaltered during suppression by Treg and led to a down-regulation of the costimulatory molecules CD80, CD86, PD-L1, and PD-L2 but not MHC II, CD40, ICOS-L, or CD70 from the mature DC surface. This effect was observed only after DC maturation with TNF or LPS but not after additional CD40 licensing. Together, our data indicate that Treg suppression against nonself Ags in vivo occurs delayed due to the slower Treg response, is mediated to a large extent through DC modulation, but is controlled by the type of DC maturation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Adaptive CD4+ and CD8+ T cell responses to self and nonself Ags can be efficiently controlled by regulatory T cells of the CD4+CD25+Foxp3+ (Treg)3 subset (1, 2, 3, 4). Although many characteristics of Treg have been elucidated, the kinetics of regulation and suppressive mechanisms during Ag-presentation in vivo are not fully understood.

In vitro studies on the mechanisms of Treg suppression revealed that interaction of surface receptors such as CTLA-4 or membrane TGF-β can directly suppress Teff or delete them (5). Subsequent induction of IL-10 secreting regulatory T cells from the naive T cell pool by Treg has also been observed (6). The expansion of CD3/CD28 Ab-primed Teff cells can be inhibited by direct cell contact-dependent interactions of Treg with Teff, clearly indicating direct interaction between Treg on Teff (7). In addition, suppressive effects of human and murine Treg on DC have been observed in vitro and include the down-regulation of MHC II and costimulatory molecules or inhibition of cytokine production (8, 9, 10, 11). Whether DC are also targets of Treg suppression in vivo is presently unclear.

As already indicated by the in vitro suppressor assays, it became clear that the preactivation of Treg resulted in a stronger suppressive effect due to their requirement of both TCR activation and CD28 costimulation (12). Therefore, mature DC were more effective in the activation and homeostatic maintenance of Treg in vivo as compared with immature DC (13, 14, 15). Adjuvant-mediated immunizations after adoptive transfer of TCR-transgenic Treg demonstrated that their proliferative and suppressive activity occurred delayed as compared with the Teff response but an explanation for this effect was not provided (16). Others have used preactivated Treg for their adoptive transfer studies to ensure a suppressive effect. Under these conditions the transfer of CD25+ Treg could prevent CD25– Teff activation in this model of autoimmune diabetes, whereas simultaneous transfer of preactivated Teff and Teff aborted the suppressive effect (17). Similarly, TLR-mediated maturation or CD40 ligation of DC released them from the control by Treg (18). Stable associations between preactivated Teff and preactivated Treg were not observed and also the Teff clustering with DC was abrogated in the presence of activated Treg. However, both Teff and Treg interacted with DC, suggesting that Treg suppression in vivo may require DC (17). This was further supported by the finding that Treg could inhibit the contacts between Ag-specific Teff and peptide-pulsed DC (19).

Together, accumulating evidence suggests that Treg can directly regulate immunogenic DC functions and thereby convert them to suboptimal or even tolerogenic DC. How regulation of T cell responses occurs in a more physiological setting, where Ag-specific Treg and Teff are simultaneoulsy present in lymphoid organs together with the presenting DC, has not been investigated. We separated CD25– Teff and CD25+ Treg from OVA-specific TCR-transgenic DO11.10 mice, and adoptively transferred them together with OVA-loaded DC after three color fluorescence labeling into syngeneic mice to follow the kinetics of Teff cell priming and Treg suppression in the spleen. We demonstrate in vivo that Treg suppression against the nonself Ag OVA occurs late to allow the Teff cell response to develop but limits the magnitude of Teff expansion. Treg activation by TNF- or LPS-matured DC reversely in the down-regulation of the CD80, CD86, PD-L1, and PD-L2 costimulatory molecules from the DC surface. Such regulated DC are then only suboptimal CD4+ Teff cell stimulators. This indirect mechanism may cooperate with a direct suppressive effect of Treg on Teff. Treg effects on DC could not be observed on fully matured or "CD40-licensed" DC. These data indicate that the balanced outcome of Teff priming and Treg suppression at the simultaneous presence of both T cell types can still act on mature DC but is further controlled by further licensing signals.


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

Female 6- to 8-wk-old BALB/c mice were obtained from Charles River Laboratories or bred in our own facilities under SPF conditions, like the DO11.10 mice (provided by K. Murphy, Washington University School of Medicine, St. Louis, MO). All animal experiments were performed according the German animal protection law as well as after approval and under control of the local authorities.

Abs and FACS analyses

The directly conjugated mAbs CD4-allophycocyanin, CD11c-allophycocyanin, CD40-PE, CD69-PE, CD70-PE, CD80-PE, CD86-PE, MHC II-PE (M5/114), CD25-PE, and biotinylated PD-L1, PD-L2 detected with streptavidin-PE were obtained from BD Pharmingen. The KJ1–26 mAb specific for the DO11.10 TCR was obtained from Caltag Laboratories/Invitrogen. Cells stained for 30 min in the dark on ice as described (20). The murine Foxp3-PE staining kit was purchased from eBioscience and stained after fixation and permeabilization of the cells as indicated by the manufacturer. Cells were measured with a FACS Canto II (BD Biosciences) and analyzed with the FCS Express software (De Novo Software).

Generation, Ag loading, and maturation of BM-DC

The method for generating BM-DC with GM-CSF has been described in detail before (20). In brief, BM cells obtained from hind limbs of mice were seeded at 2 x 106 per 100 mm petri dish and fed at days 0, 3, and 6 with fresh medium and GM-CSF from a transfected cell line (21). DC were used at day 7 or 8 for further treatment. Where indicated, DC were incubated with 10 µM OVA peptide327–339 (Sigma-Genosys) over night at 37°C/CO2 in the presence of the following maturation stimuli that were used at the concentrations 0.1 µg/ml LPS (E. coli 0127:B8; Sigma-Aldrich), 500 U/ml TNF-{alpha} (PeproTech) or 0.1 µg/ml LPS plus 5 µg/ml anti-CD40 (NA/LE; BD Pharmingen).

Cryosections and immunofluorescence

Lymph nodes and spleens were removed and immediately frozen in liquid nitrogen. The organs were embedded in Tissue-Tek O.C.T. Compound (Sakura) and stored at –80°C. The frozen organs were cut into 10 µm slices with a Leica CM3050 S Cryostat (Leica) and stored on glass slides at –20°C. The frozen sections were thawed and air dried for 10 min. For fixation 4% paraformaldehyde (Merck) was used for 20 min at room temperature. After washing with PBS and incubation with 0.1 M glycin for 15 min the sections were incubated for 15 min with a blocking solution of 2% BSA (PAA) and then embedded in Fluoromount mounting medium (Serva Electrophoresis). The Fluorescence analysis was performed with a Leica DMRD research microscope (Leica) connected with a CCD camera and analyzed with Openlab software (Improvision).

Separation of T cell subsets from DO11.10 mice

The spleens were removed from 6- to 8-wk-old DO11.10 mice and a single cell suspension was prepared by grinding the spleen between the rough ends of two slides. The cells were then filtered through with a 70 µm Falcon Cell Strainer (BD Bioscience) and the erythrocytes were lysed with 1.6% ammonium chloride solution at 37°C for 5 min. The splenocytes were then depleted from non-CD4+ T cells using the CD4 negative cell isolation kit (Dynal Biotech). The remaining CD4+ T cells were then further positively enriched by MACS technology (Miltenyi Biotec) with a CD25-PE mAb and anti-PE mAb MACS bead conjugate using a MiniMACS Cell Aeparator. The purities of the separated T cell subsets were throughout >90%. The Treg that were isolated by CD25PE-conjugated beads, injected, and re-analyzed by FACS had lost their CD25PE fluorescence as indicated by isotype control stainings and thus did not interfere with further PE surface stainings of the Treg (data not shown).

Fluorescence labeling of cells

For CFSE-labeling, the cells were adjusted to 2 x 107 cells/ml in PBS. The stock solution of CFSE (CFDA SE, Molecular Probes) was diluted in DMSO and PBS following the manufacturers protocol to a concentration of 5 µM and then mixed 1:1 with the cell suspension and incubated at room temperature for 10 min before washing with PBS and injection. For labeling with Cell Tracker Blue (CMAC; Molecular Probes/Invitrogen) or Cell Tracker Orange (CMTMR; Molecular Probes/Invitrogen) the cells were adjusted to a concentration of 1 x 107 cells/ml in serum-free HL-1 medium (BioWhittaker). The cells were incubated at 37°C in the dark for 45 min with 20 µM CMAC or 10 µM CMTMR and then washed. After incubation for another 30 min at 37°C in HL-1 medium the cells were suspended in PBS and injected.

Adoptive transfer experiments

Fluorescence-labeled immature of differentially matured DC (as indicated), Treg or Teff were all injected i.v. at the indicated cell numbers into syngeneic BALB/c mice of the same sex. In the experiments where DC and T cells were injected at the same day, the DC were applied first and the T cells 6 h later if not otherwise stated. Subsequent T cell injections were performed at the indicated time points after the last T cell injection. To analyze T cell responses ex vivo the spleens were removed at the indicated time points after the T cell injections.

Isolation of CFSE-labeled OVA-loaded DC from spleens

The splenic single cell suspensions were prepared as described above. Before erythrocyte lysis the spleens were treated with collagenase III (25 µg/ml; Sigma-Aldrich) and DNase I (100 µg/ml; Sigma-Aldrich) for 30 min. The cells were then stained with CD11c-allophycocyanin and the PE-conjugated mAbs as indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Adoptively transferred Teff and Treg form clusters with coinjected DC in the spleen

To study the interaction of OVA-peptide-loaded DC with Ag-specific CD4+ Teff cells in the presence or absence of Treg, we generated murine DC from bone marrow (BM) with GM-CSF, matured them with LPS, labeled them with CMAC (blue) and injected them i.v. into BALB/c mice. The isolated responder Teff and Treg cell populations from DO11-10 OVA-specific TCR transgenic mice were fluorescence-labeled with CFSE (green) or CMTMR (red) and Teff or Treg were injected individually or together 6 h later. After another 20 h, both individually injected Teff or Treg clustered around DC in the splenic T cell areas (Fig. 1, A and B), similarly as it has been described after s.c. injection of DC for the draining lymph nodes (22, 23). This clustering of both T cell types occurred also after concomitant injection (Fig. 1C) but not in the lymph nodes due to the lack of DC migration into these organs from the blood compartment, as expected (data not shown). DC injected without OVA do not induce clusters and immature DC were poorly interfering with Teff or Treg in vitro and in vivo (data not shown), consistent with previous reports (13, 14). This indicates that the clustering of both Teff and Treg subsets with peptide-loaded mature DC can occur in the spleen.


Figure 1
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  		FIGURE 1. Similar DC clustering but slower activation and proliferation rate of Treg in the spleen as compared with Teff. A–C, OVA-loaded and LPS-matured BM-DC (1 x 107, BALB/c) were labeled with CMAC (blue) and coinjected i.v. into syngeneic mice together with 8 x 106 isolated and CFSE+ DO11.10 CD25– Teff (green) alone (A), 2 x 106 CD25+ Treg (red) alone (B), or CD25– Teff and CD25+ Treg together at a ratio of 5:1 (C) 6 h after the DC. Cyrosections of the spleen and the inguinal LN were prepared 20 h after T cell injection. The data are representative for two independent experiments with similar results. For each analysis 2 x 105 events were acquired. D, OVA-loaded immature or LPS-matured DC (2 x 106) were injected i.v. into BALB/c mice and 6 h later 2 x 106 CFSE+ CD4+ CD25– or CFSE+ CD4+ CD25+ T cell subsets isolated from DO11.10 mice were injected i.v. After another 20 h, the spleens were removed and the single cell suspensions stained for the indicated markers and analyzed by FACS. CD4+CD25+ or CD4+CD25– cells were further gated for their KJ1–26 expression to detect only OVA specific T cells (left, histograms). Solid lines represent marker staining and dotted lines the respective isotype controls. Dot plots show cells stained for the indicated markers and gated for CD4, KJ1–26, and CFSE. Numbers within rectangle or quadrant gates represent the relative percentages of the displayed cells. E, To measure the proliferation of CFSE+CD4+CD25– or CFSE+CD4+CD25+ T cells, mature OVA-loaded DC were injected as above and spleens removed after 4 days. CFSE dilution is shown gated on CD4+ cells and counterstained for KJ1–26 and Foxp3. The numbers above the histograms are the percentages of cells within the marked gates. All data are representative for at least 4 independent experiments with similar results.

 
Activation and proliferation of DO11.10 Treg are reduced as compared with Teff

For a better understanding of these interactions in the spleen at 26 h, we compared the separated activation of both T cell types by injecting mature OVA-loaded DC together with either CFSE-labeled CD25– Teff or CD25+ Treg both isolated from DO11.10 mice. As controls for nonstimulatory conditions also immature OVA-loaded DC were coinjected with the T cells. After 24h the spleen cells were removed and analyzed by FACS. T cell analysis was performed by gating on CD4+KJ1–26+ cells to detect only clonotypic, OVA-specific cells as only 81% of the CD4+CD25– and 61% of the CD4+CD25+ starting T cell population were KJ1–26+ (Fig. 1D, left), due to rearrangement of transgenic with endogenous TCR chains (24). As expected, immature DC did not stimulate both Treg and Teff populations (Fig. 1D, right), similar to immature or mature DC, which were not loaded with OVA Ag (data not shown). With mature OVA-loaded DC CD69 and CD25 were clearly up-regulated on both types of T cells (for Treg a shift into the CD25high gate) and more pronounced for the CD25– Teff subset (Fig. 1D, right). When the spleens were removed 4 days after injection and analyzed by FACS, the proliferation of CD4+KJ1–26+CD25+ Treg was lower than of the CD4+KJ1–26+CD25– Teff but both populations remained in their Foxp3 positive or negative state, respectively (Fig. 1E). Thus, the Teff and Treg populations, which are specific for a nonself peptide in this setting, differ in their activation and proliferation kinetics.

Treg suppress Teff only after delayed Teff transfer

The observed contacts between Teff and OVA-pulsed DC in the spleen induced T cell priming because we observed CD69 and CD25 up-regulation and proliferation upon transfer of the Teff population in the absence of Treg. How this could be influenced by Treg was investigated by coinjections of both T cell subsets. OVA-loaded LPS-matured DC were injected together with Treg followed by simultaneous or subsequent CFSE+ Teff injections. After 3 days, spleen cells were analyzed. We did not observe any more than basal T cell proliferation in the absence of OVA Ag-loading of the DC (Fig. 2). However, after transfer of OVA loaded DC together with Teff vigorous CFSE dilution could be detected, independent of the time point of Teff injection. When Teff cells were coinjected simultaneously with Treg at different ratios, the proliferation of Teff remained unchanged. Suppression of the Teff population was observed only when the Teff were injected 10 or 24 h after the Treg and the extent of suppression was dependent on the Treg:Teff ratio (Fig. 2).


Figure 2
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  		FIGURE 2. Treg mediated suppression depends on their preactivation in vivo. OVA-loaded LPS-matured DC or unloaded DC (2 x 106) together with 4 x 105 (1:5 ratio) or 1.6 x 106 (1:1 ratio) Treg were injected i.v. into BALB/c mice. In addition, 1.6 x 106 CFSE+ Teff were injected either simultaneously, with a 10 h or 24 h delay with respect to the DC and Treg at different ratios. Three days after the injections the spleens were stained with anti-clonotypic KJ1–26 and CD4 mAb and analyzed by FACS. The CFSE+ cells are shown within gates for KJ1–26+ and CD4+ cells. The data are representative for three independent experiments with similar results. For each analysis 1 x 106 events were acquired.

 
Treg control the total spleen cellularity and do not prevent but limit the Teff expansion leading to higher Teff numbers in the memory phase

Because Treg were activated with a delay or at reduced levels and needed prestimulation to suppress Teff cells in this setting, we wondered whether at simultaneous transfer with Teff regulation may occur at later stages of the Teff response. For this, Teff and the OVA-loaded DC populations were simultaneously injected i.v. with or without Treg. The expansion of the Teff was followed by counting the total number of spleen cells and the OVA-specific Teff by staining them with the anti-clonotypic Ab KJ1–26 together with a counterstaining for Foxp3 to exclude OVA-specific Treg from the population of expanding effectors. Within the first 2 days after injection the total spleen cellularity and the numbers of OVA-specific Teff increased at parallel levels, independent of the presence or absence of Treg (Fig. 3). At day 3, the maximum expansion in spleen size and Teff numberswere detected. Here a marked difference could be observed between spleens and OVA-specific Teff cellularities from mice that were injected or not with Treg, indicating that the Treg contributed first to the limitation of the total spleen cellularity, and second, the CD4+ effector T cell expansion. At days 5 and 8, the spleen cellularities remained high while the number of specific Teff declined as a result of the terminated T cell response in the presence of Treg (Fig. 3). However, the Teff levels in the absence of Treg did not reach the same low plateau as in their presence. The down-regulation of Teff numbers occurred to the same extent and with the same kinetics with or without Treg, indicating that this contraction phase of the Teff response is controlled by other mechanisms than Treg. Nevertheless, the absence of Treg led to the persistence of high Teff levels during the memory phase, similar to what could be detected at the maximum response in the presence of Treg. Together, Treg with specificity for a nonself Ag as in our model may be activated with a delay to allow a productive Teff response against a pathogen but then interfere to control overshooting and chronic immunity and thereby preventing immunopathology.


Figure 3
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  		FIGURE 3. Treg control the total spleen cellularity and do not prevent the Teff response but control the maximal expansion and the termination phase. OVA-loaded LPS-matured DC (1 x 107) and 1.6 x 107 Teff were injected i.v. into BALB/c mice with or without 4 x 106 Treg (ratio 5:1). A, At the indicated days after injection spleens were removed, erythrocytes lysed and the total white spleen cell numbers counted. B, FACS analysis of the spleen cells was performed after staining with CD4, KJ1–26, and Foxp3 mAb. CD4+KJ1–26+Foxp3+ Treg were gated of the analysis to follow only the CD4+KJ1–26+Foxp3– Teff expansion. Absolute Teff numbers were calculated from the total spleen cellularity and the percentage of Teff revealed by FACS analysis. Error bars represent the SDs from the means analyzing individual mice from three independent experiments. For each analysis 2 x 105 events were acquired.

 
Treg suppression is characterized by cluster disruption and reduced individual Teff-DC and Teff-Treg contacts

The data so far indicate that Treg suppressive activity occurs delayed in our experimental setting. However, how Teff and Treg interfere under regulatory and nonregulatory conditions as well as the mechanisms of suppression remain unclear. Therefore, we evaluated the cluster formation but also individual cell-cell contacts between the three adoptively transferred cell populations within the spleen under the simultaneous (nonregulatory) and delayed (regulatory) conditions. OVA-loaded mature DC were fluorescence-labeled in blue and Treg in red before transfer. Simultaneously or 24 h later, green-fluorescent Teff were injected. Spleens were removed 24 h after the Teff injections and cryopreserved before serial sectioning. Then we asked whether or not cell clustering could be observed between the three cell types. In contrast to the nonsuppressive simultaneous injections, the cluster formation was completely inhibited under the delayed condition where also Teff suppression was observed and only individual contacts remained (Fig. 4A). These findings from splenic interactions here are in agreement with other findings derived from DC-T cluster analysis within the lymph node (19).


Figure 4
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  		FIGURE 4. Treg inhibit clustering and individual contacts between DC-Teff and Treg-Teff. OVA-loaded LPS-matured DC (1 x 107) were injected i.v. into BALB/c mice. Four hours later, later 2 x 106 isolated CMTMR+ Treg derived from DO11.10 mice were injected i.v. into the same recipients. CFSE+ Teff (8 x 106) were injected either simultaneously or with a 24 h delay at a ratio of 5:1 (Teff:Tregs). A, Cryosections were prepared 24 h after the Teff injections to detect cell contacts and clustering by microscopical analysis. B, Teff-DC, Teff-Treg, and Treg-DC contacts were analyzed and quantified. Data shown are from at least two independent experiments where for each of it >20 cryosections that were analyzed by counting >3000 cell-cell contacts. Values of p were calculated by unpaired Student’s t test (*, p < 0.05; **, p < 0.01).

 
Further assessment of the individual contacts between the three adoptively transferred populations to each other revealed that the frequency of Treg-DC contacts remained stable under both suppressive (24 h delayed) and nonsuppressive (simultaneous) conditions. However, under suppressive conditions, a reduced frequency of contacts was observed between Teff-DC as well as Treg-Teff (Fig. 4B), correlating with the cluster disruption (Fig. 4A). Similar observations have been made in other systems and it had been discussed there whether this may indicate that in vivo the Teff suppression can be mediated also indirectly by Treg activity on the Ag-presenting DC (17).

Treg induce down-regulation of selected costimulatory molecules on DC

Next, we wanted to elucidate whether mature DC can be influenced by Treg in vivo. DC treated with TNF, LPS, or LPS plus anti-CD40 were analyzed by FACS for surface expression of MHC II, CD80, CD86, PD-L1, and PD-L2 before i.v. injection to analyze their maturation state. These data confirmed their mature surface phenotype as compared with untreated DC (Fig. 5). Then Teff alone or Treg plus Teff were coinjected according the simultaneous and the 24-h delayed conditions. After another 48 h, in vivo the CFSE+ OVA-loaded DC within the spleen were analyzed by FACS for expression of CD11c and a panel of costimulatory molecules. DC injected with Teff only or with simultaneous Treg and Teff transfer maintained their mature phenotype and expressed high levels of MHC II, CD80, CD86, CD40, PD-L1, and PD-L2 as well as moderate levels of ICOS-L and CD70 (Fig. 5 and data not shown). The latter two markers are absent on immature DC (data not shown). When the regulatory conditions of Teff injection (24 h delayed) were analyzed, a marked down-regulation could be observed for CD80, CD86, PD-L1, and PD-L2 on DC that were matured with TNF or LPS (Fig. 6 and data not shown). No changes could be observed for the expression of ICOS-L, CD70, CD40, and MHC II (data not shown). Interestingly, DC that had been matured with LPS plus anti-CD40 also did not show a modulation of any surface marker analyzed (Fig. 6B). The effect of Treg was also not correlated with increased cell death of DC because Annexin V or 7-AAD stainings of the DC after ex vivo isolation remained negative (data not shown). Thus, Treg activity in vivo, which leads to a block of Teff proliferation, may be mediated by the down-regulation of specific costimulatoy molecules from the DC surface, which in turn indirectly controls the Teff expansion.


Figure 5
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  		FIGURE 5. The BM-DC have a mature surface phenotype before injection. BM-DC were generated according our standard protocol and left untreated or were matured over night with the indicated stimuli. Cells were stained for CD11c and the indicated markers. Histograms shown represent events gated on CD11c+ cells. Straight lines show stainings of the indicated markers and dotted lines the respective isotype controls. For each analysis 2 x 104 events were acquired.

 

Figure 6
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  		FIGURE 6. Treg downmodulate selected markers from the surface of TNF- and LPS-matured DC but not after LPS/CD40 maturation. A, CFSE+ OVA-loaded mature DC (1 x 107) and 2 x 106 DO11.10 derived Treg were injected i.v. into BALB/c mice. DO11.10 Teff (8 x 106) were injected simultaneously or with a 24 h delay (ratio 5:1, Teff:Treg). As a control, DC and Teff were also injected without Treg. Spleens were removed and analyzed 48h after injection of the DC. B, The data are representative for three independent experiments with similar results. Statistical evaluations were calculated by unpaired Student’s t test (*, p < 0.05). For each analysis 2 x 105 events were acquired.

 
Licensing of DC through CD40 aborts the suppressive activity of Treg

DC maturation can be performed with various classes of stimuli resulting in different qualities or maturation stages. DC licensing has been described to further enhance DC maturation, e.g., by LPS, to further improve their functional capacities, which are especially required for the priming of CD8+ T cells (25, 26). To investigate whether CD40 licensing could play a role on whether Treg suppression of DC may occur or not, DC were matured with the combination of LPS and CD40 mAb and tested in the same setting as for TNF- or LPS-matured DC. The data indicate that the addition of the CD40 stimulus on LPS-matured DC abrogates the down-regulation of surface markers by Treg (Fig. 6). This indicates that another quality of DC licensing or conditioning through CD40 may include the unresponsiveness to suppressive effects by Treg which then allows unimpaired T cell priming.

To test whether the inhibited down-regulation of the costimulatory molecules from the DC surface by LPS/CD40 treatment would also functionally abort the suppressive capacity of Treg on Teff proliferation, we directly compared OVA loaded DC matured with TNF, LPS, or LPS/CD40 under the 24-h delayed conditions in vivo. Although Teff proliferation was clearly suppressed when TNF or LPS mature DC were injected, the CFSE dilution was only marginally affected after injection of LPS/CD40 matured DC (Fig. 7). This indicates that Treg suppression of Teff proliferation in vivo is to a large extent mediated through DC and only a partial suppressive effect results from direct influence of Treg on Teff.


Figure 7
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  		FIGURE 7. CD40 licensing of DC largely blocks suppression of Teff proliferation by Treg. OVA-loaded and differentially matured DC (1 x 107) were injected i.v. into BALB/c mice alone or together with 2 x 106 Treg. In addition, 8 x 106 CFSE+ Teff were injected either simultaneously or with a 24 h delay at a 1:5 ratio (Treg:Teff). Three days after the injections the spleens were stained with anti-clonotypic KJ1–26 and CD4 mAb and analyzed by FACS. The CFSE+ cells are shown within gates for KJ1–26+ and CD4+ cells. The data are representative for two independent experiments with similar results. For each analysis 1 x 106 events were acquired.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Currently it is still under debate how regulation may occur in vivo, directly on Teff and/or through DC? In vitro data clearly indicate that Treg suppression can act directly on Teff (7, 27). Others (8, 10, 11, 28, 29, 30) reported inhibitory effects by Treg on murine and human DC in vitro but such a phenomenon has not been demonstrated in vivo so far. In addition, all of these reports used immature DC which were incubated with various maturation stimuli in the presence or absence of Treg, meaning that the Treg in these settings only prevented DC maturation.

In this study, we used an adoptive transfer system that allowed us to follow the effects of Treg on the Teff priming kinetics induced by mature DC in the spleen. The i.v. route of DC application excludes the secondary modification of the DC in vivo, whereas s.c. injected mature DC can be further influenced in their maturation state and induce bystander effects on endogenous DC after homing to the lymph node (31). Furthermore, the spleen does not require specific homing receptors for different stages of Teff such as central verus effector memory T cells. Therefore altered homing patterns of activated Teff subsets into the spleen in the presence or absence of Treg can be excluded as a mechanism to explain the lower numbers of Teff. Thus, the i.v. injection route and analysis of the spleen combines several advantages over previous approaches to follow the interaction of differentially matured DC with both CD4+ T cell types (Teff and Treg), undisturbed from secondary effects.

Our data reveal that Treg can act on TNF- and LPS-matured DC to induce the selective down-regulation of CD80/CD86 and PD-L1/PD-L2, but not MHC II, ICOS-L, CD40, and CD70, from the DC surface. This could point to a specific importance of these molecules not at preventing the Teff response but being decisive during later phases to limit the Teff expansion. Two ligands of the costimulatory molecule pairs on the T cell surface, CTLA-4 and PD-1, respectively, are well accepted to deliver negative signals into Teff and can work in an additive manner (32). Although CTLA-4 is expressed abundantly on resting Treg (7), PD-1 is located only intracellularly and up-regulated on activated Treg (33). Functionally, conflicting results exist for CTLA-4, as the effects mediated by Treg on human DC costimulatory molecule expression have been described as being both CTLA-4-dependent (28) and -independent (29). A functional role of PD-1 on Treg is not known yet.

Our data also show that the modulatory effect of Treg is not functional after a combined LPS+CD40 maturation, further extending our findings that the quality of DC maturation heavily influences T cell activation and differentiation (34). Endogenous inflammatory signals such as TNF, TLR ligands such as LPS, or CD40 ligation can result in DC maturation although the stage or quality of maturation are substantially different especially with respect to their cytokine production (35, 36). Although TNF-matured DC are rather semimature as they do not secrete cytokines and can even act tolerogenic in certain models, LPS will lead to full maturation including IL-12 production and Th type-1 priming (37). Signals through CD40 can also mature DC (38) leading to a so-called "conditioned" or "licensed" state that reflects a superior stimulation as achieved with TLR activation alone because only then it enables an effective priming and cross-priming of CD8+ T cells in vivo (25, 26, 39). The CD40L (CD154) is expressed on Treg (see below), activated platelets (40) and on CD4+ Th cells (41), The latter two could both serve as a licensing signal for DC. However, CD154 expressed on activated T cells may be encountered by the DC only at later time points after T cell activation in the lymph nodes, whereas CD154 on activated platelets may engage CD40 on DC already at the infection site simultaneously with a microbial TLR ligand. Although CD40 mediated maturation of DC is well established, evidences from our own work (37, 42) and in vivo experiments by others (43) suggest that CD40 Abs alone are insufficient for DC maturation and only the combination with a microbial stimulus such as through TLRs may be sufficient to reach the fully mature or licensed state. This is also supported by our data shown here. However, from the published data it is not entirely clear what renders the licensed state superior to a stage where DC have been matured through a TLR or CD40 alone. Although CD40 signals can enhance TLR-induced IL-12 production by DC, this quantitative effect may be insufficient to explain the qualitative effect on the priming of CD8+ T cells.

Recently, it has been shown (10) that CD40 licensing also relieves human DC from Treg activity in vitro in a preventive setting. We can extend on this by showing CD40 licensing also in vivo. Others have reported that in vitro immature murine BM-DC and splenic DC were prevented from up-regulation of costimulatory molecules but when the DC were matured with LPS before adding the Treg, there was no effect on the expression level of DC surface markers (18). In contrast, we still observe modulatory effects on mature DC after stimulation with TNF or LPS in vivo. These conflicting results may indicate that the in vivo requirements for regulation may be different from in vitro cultures. The reasons for this remain to be investigated.

It is of note that CD154 (CD40L) is also expressed at constitutively low levels by Treg, up-regulated after activation and required for their functionality (44, 45). In light of this our results may indicate that there are either quantitative differences in CD40L expression between Treg and activated platelets or an early CD40 signal through platelets may desensitize DC for further CD40 signaling by Treg in the lymph node or the signals received by the DC are qualitatively different. The latter possibility could be due to the differential expression of the five splice variants for CD40 existing on immature or mature DC (46). In this scenario, the CD40 isoforms triggered on immature DC contacting platelets at the site of microbial encounter would be different from the ones triggered by DC, which had matured and migrated into the T cell areas of the lymph node. In fact, three isoforms on mature DC have been shown to rather disable maturation signaling and thereby impair their IL-12 production (46).

Our kinetic analysis revealed that the total splenic cellularity as well as the number of OVA-specific Teff is reduced only at later stages of the T cell response but not in the initial T cell expansion phase. Our data are similar to analyses of Teff cell expansion controlled by Treg in the draining lymph nodes after immunization with Freund’s adjuvant (16) or results about the role of IL-2 as a major mediator to allow Teff expansion before Treg activation and thereby controlling the Teff response (47). As both systems used nonself Ags in their stimulation of T cells, this may indicate that in such settings the induction of immunity will be mediated by slower activation of nonself specific Treg to allow pathogen defense by Teff. At the maximum of the response Treg may contribute to limit Teff expansion to avoid immunopathology. However, in the termination phase of the Teff response, Teff contraction occurs with a similar kinetics and quantity in the presence or absence of Treg. When the Treg-dependent limitation is lacking and the subsequent Treg-independent contraction has occured, during the following memory phase the cellularity of Teff remains at high levels, similar as they appear at the maximum response in the presence of Treg. The persistence of high Teff numbers can obviously not be further down-regulated by the Treg, probably due to the lack of Ag and thereby Treg activation at this time point.

A possible mechanism for the reduced contacts between Teff-DC and Teff-Treg we observed in the experiments under delayed conditions may also depend on simple competition for the access of the delayed injected Teff to the respective cells. Also the ratio of Teff:Treg, that had been chosen 5:1 in most of our settings, certainly can influence the extent of competition and thus regulation. However, we found many small clusters and two cell interactions in the sections, which would argue against competition. Also others (23) have excluded competition as a mechanism for reduced T cell to DC contacts within murine lymph nodes. Furthermore, it remains to be analyzed whether T cell specificities other than for OVA, due to differential selection of Treg and Teff in the thymus, could influence the kinetics of Treg activity on different types of Teff that may express low vs high affinity TCRs for the same peptide but also in a bystander fashion.

Recently, it has been shown (19) that Treg form stable contacts with DC in vivo, which we also found in our study and this may indicate effects of Treg on DC in vivo. This was then shown in a system where Treg-DC contacts led to a complete loss of significant interactions between Teff with DC, indicating that such direct regulation of Teff may be less important in vivo (17). In contrast, we still found substantial individual interactions between these two cell types at a level of ~50% compared with what we observed in the absence of regulation. This may indicate that in our system Treg may act though both pathways, directly on Teff as well as directly on DC. The latter mechanism may then indirectly result in an impaired Teff activation by the DC and together with the direct suppression by Treg resulting in a potentiated regulatory effect.

In conclusion, our data contribute to the questions why and how Treg can act on mature DC in vivo, but also how DC can evade this Treg control through CD40 licensing. Both phenomena may combine with other mechanisms that control desired immunity against pathogen vs an exacerbated immunopathological reponse.


   Acknowledgments
 
We thank Ken Murphy for providing the DO11.10 mice, as well as Gerold Schuler and Thomas Hünig for their support of our work. We are greatful to K. Khazaie and T. Hünig for reading the manuscript and providing critical comments.


   Disclosures
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 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 Deutsche Forschungsgemeinschaft through the Research Training Group GK 592, the Collaborative Research Center SFB 643 in Erlangen, and the Institute of Virology and Immunobiology in Wuerzburg. Back

2 Address correspondence and reprint requests to Dr. Manfred B. Lutz, University of Wuerzburg, Versbacherstrasse 7, Wuerzburg 97987, Germany. E-mail address: m.lutz{at}vim.uni-wuerzburg.de Back

3 Abbreviations used in this paper: Treg, CD25+ regulatory T cell; BM, bone marrow; DC, dendritic cell; Teff, CD25– effector T cells; CMAC, Cell Tracker Blue; CMTMR, Cell Tracker Orange. Back

Received for publication June 26, 2007. Accepted for publication November 19, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Sakaguchi, S., M. Ono, R. Setoguchi, H. Yagi, S. Hori, Z. Fehervari, J. Shimizu, T. Takahashi, T. Nomura. 2006. Foxp3+CD25+CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol. Rev. 212: 8-27. [Medline]
  2. Shevach, E. M., R. A. DiPaolo, J. Andersson, D. M. Zhao, G. L. Stephens, A. M. Thornton. 2006. The lifestyle of naturally occurring CD4+CD25+Foxp3+ regulatory T cells. Immunol. Rev. 212: 60-73. [Medline]
  3. Mempel, T. R., M. J. Pittet, K. Khazaie, W. Weninger, R. Weissleder, H. von Boehmer, U. H. von Andrian. 2006. Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation. Immunity 25: 129-141. [Medline]
  4. Belkaid, Y., B. T. Rouse. 2005. Natural regulatory T cells in infectious disease. Nat. Immunol. 6: 353-360. [Medline]
  5. von Boehmer, H.. 2005. Mechanisms of suppression by suppressor T cells. Nat. Immunol. 6: 338-344. [Medline]
  6. Dieckmann, D., C. H. Bruett, H. Plöttner, M. B. Lutz, G. Schuler. 2002. Human CD4+CD25+ regulatory, contact-dependent T cells induce interleukin-10 producing, contact-independent type-1 regulatory T cells. J. Exp. Med. 196: 247-253. [Abstract/Free Full Text]
  7. Dieckmann, D., H. Plottner, S. Berchtold, T. Berger, G. Schuler. 2001. Ex vivo isolation and characterization of CD4+CD25+ T cells with regulatory properties from human blood. J. Exp. Med. 193: 1303-1310. [Abstract/Free Full Text]
  8. Cederbom, L., H. Hall, F. Ivars. 2000. CD4+CD25+ regulatory T cells down-regulate costimulatory molecules on antigen-presenting cells. Eur. J. Immunol. 30: 1538-1543. [Medline]
  9. Sato, K., S. Tateishi, K. Kubo, T. Mimura, K. Yamamoto, H. Kanda. 2005. Downregulation of IL-12 and a novel negative feedback system mediated by CD25+CD4+ T cells. Biochem. Biophys. Res. Commun. 330: 226-232. [Medline]
  10. Misra, N., J. Bayry, S. Lacroix-Desmazes, M. D. Kazatchkine, S. V. Kaveri. 2004. Cutting edge: human CD4+CD25+ T cells restrain the maturation and antigen-presenting function of dendritic cells. J. Immunol. 172: 4676-4680. [Abstract/Free Full Text]
  11. Veldhoen, M., H. Moncrieffe, R. J. Hocking, C. J. Atkins, B. Stockinger. 2006. Modulation of dendritic cell function by naive and regulatory CD4+ T cells. J. Immunol. 176: 6202-6210. [Abstract/Free Full Text]
  12. Salomon, B., D. J. Lenschow, L. Rhee, N. Ashourian, B. Singh, A. Sharpe, J. A. Bluestone. 2000. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12: 431-440. [Medline]
  13. Oldenhove, G., M. de Heusch, G. Urbain-Vansanten, J. Urbain, C. Maliszewski, O. Leo, M. Moser. 2003. CD4+ CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo. J. Exp. Med. 198: 259-266. [Abstract/Free Full Text]
  14. Yamazaki, S., T. Iyoda, K. Tarbell, K. Olson, K. Velinzon, K. Inaba, R. M. Steinman. 2003. Direct expansion of functional CD25+CD4+ regulatory T cells by antigen-processing dendritic cells. J. Exp. Med. 198: 235-247. [Abstract/Free Full Text]
  15. Brinster, C., E. M. Shevach. 2005. Bone marrow-derived dendritic cells reverse the anergic state of CD4+CD25+ T cells without reversing their suppressive function. J. Immunol. 175: 7332-7340. [Abstract/Free Full Text]
  16. 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]
  17. Tang, Q., J. Y. Adams, A. J. Tooley, M. Bi, B. T. Fife, P. Serra, P. Santamaria, R. M. Locksley, M. F. Krummel, J. A. Bluestone. 2006. Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice. Nat. Immunol. 7: 83-92. [Medline]
  18. Serra, P., A. Amrani, J. Yamanouchi, B. Han, S. Thiessen, T. Utsugi, J. Verdaguer, P. Santamaria. 2003. CD40 ligation releases immature dendritic cells from the control of regulatory CD4+CD25+ T cells. Immunity 19: 877-889. [Medline]
  19. Tadokoro, C. E., G. Shakhar, S. Shen, Y. Ding, A. C. Lino, A. Maraver, J. J. Lafaille, M. L. Dustin. 2006. Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo. J. Exp. Med. 203: 505-511. [Abstract/Free Full Text]
  20. Lutz, M. B., N. Kukutsch, A. L. Ogilvie, S. Rössner, F. Koch, N. Romani, G. Schuler. 1999. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223: 77-92. [Medline]
  21. Zal, T., A. Volkmann, B. Stockinger. 1994. Mechanisms of tolerance induction in major histocompatibility complex class II-restricted T cells specific for a blood-borne self-antigen. J. Exp. Med. 180: 2089-2099. [Abstract/Free Full Text]
  22. Ingulli, E., A. Mondino, A. Khoruts, M. K. Jenkins. 1997. In vivo detection of dendritic cell antigen presentation to CD4+ T cells. J. Exp. Med. 185: 2133-2141. [Abstract/Free Full Text]
  23. Hommel, M., B. Kyewski. 2003. Dynamic changes during the immune response in T cell-antigen-presenting cell clusters isolated from lymph nodes. J. Exp. Med. 197: 269-280. [Abstract/Free Full Text]
  24. Kawahata, K., Y. Misaki, M. Yamauchi, S. Tsunekawa, K. Setoguchi, J. Miyazaki, K. Yamamoto. 2002. Generation of CD4+CD25+ regulatory T cells from autoreactive T cells simultaneously with their negative selection in the thymus and from nonautoreactive T cells by endogenous TCR expression. J. Immunol. 168: 4399-4405. [Abstract/Free Full Text]
  25. Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393: 478-480. [Medline]
  26. Ridge, J. P., F. Di Rosa, P. Matzinger. 1998. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 393: 474-478. [Medline]
  27. Thornton, A. M., E. M. Shevach. 2000. Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J. Immunol. 164: 183-190. [Abstract/Free Full Text]
  28. Oderup, C., L. Cederbom, A. Makowska, C. M. Cilio, F. Ivars. 2006. Cytotoxic T lymphocyte antigen-4-dependent down-modulation of costimulatory molecules on dendritic cells in CD4+CD25+ regulatory T-cell-mediated suppression. Immunology 118: 240-249. [Medline]
  29. Houot, R., I. Perrot, E. Garcia, I. Durand, S. Lebecque. 2006. Human CD4+CD25high regulatory T cells modulate myeloid but not plasmacytoid dendritic cells activation. J. Immunol. 176: 5293-5298. [Abstract/Free Full Text]
  30. Bayry, J., F. Triebel, S. V. Kaveri, D. F. Tough. 2007. Human dendritic cells acquire a semimature phenotype and lymph node homing potential through interaction with CD4+CD25+ regulatory T cells. J. Immunol. 178: 4184-4193. [Abstract/Free Full Text]
  31. Voigtlander, C., S. Rossner, E. Cierpka, G. Theiner, C. Wiethe, M. Menges, G. Schuler, M. B. Lutz. 2006. Dendritic cells matured with TNF can be further activated in vitro and after subcutaneous injection in vivo which converts their tolerogenicity into immunogenicity. J. Immunother. 29: 407-415. [Medline]
  32. Probst, H. C., K. McCoy, T. Okazaki, T. Honjo, M. van den Broek. 2005. Resting dendritic cells induce peripheral CD8+ T cell tolerance through PD-1 and CTLA-4. Nat. Immunol. 6: 280-286. [Medline]
  33. Raimondi, G., W. J. Shufesky, D. Tokita, A. E. Morelli, A. W. Thomson. 2006. Regulated compartmentalization of programmed cell death-1 discriminates CD4+CD25+ resting regulatory T cells from activated T cells. J. Immunol. 176: 2808-2816. [Abstract/Free Full Text]
  34. Lutz, M. B.. 2006. Differentiation stages and subsets of tolerogenic dendritic cells. M. B. Lutz, and N. Romani, and A. Steinkasserer, eds. Handbook of Dendritic Cells: Biology, Diseases, and Therapy 517-543. VCH-Wiley, Weinheim.
  35. Morelli, A. E., A. F. Zahorchak, A. T. Larregina, B. L. Colvin, A. J. Logar, T. Takayama, L. D. Falo, A. W. Thomson. 2001. Cytokine production by mouse myeloid dendritic cells in relation to differentiation and terminal maturation induced by lipopolysaccharide or CD40 ligation. Blood 98: 1512-1523. [Abstract/Free Full Text]
  36. Lutz, M. B., G. Schuler. 2002. Immature, semi-mature, and fully mature dendritic cells: which signals induce tolerance or immunity?. Trends Immunol. 23: 445-449. [Medline]
  37. Menges, M., S. Rossner, C. Voigtlander, H. Schindler, N. A. Kukutsch, C. Bogdan, K. Erb, G. Schuler, M. B. Lutz. 2002. Repetitive injections of dendritic cells matured with tumor necrosis factor {alpha} induce antigen-specific protection of mice from autoimmunity. J. Exp. Med. 195: 15-21. [Medline]
  38. Caux, C., C. Massacrier, B. Vanbervliet, B. Dubois, C. Van Kooten, I. Durand, J. Banchereau. 1994. Activation of human dendritic cells through CD40 cross-linking. J. Exp. Med. 180: 1263-1272. [Abstract/Free Full Text]
  39. Fujii, S., K. Liu, C. Smith, A. J. Bonito, R. M. Steinman. 2004. The linkage of innate to adaptive immunity via maturing dendritic cells in vivo requires CD40 ligation in addition to antigen presentation and CD80/86 costimulation. J. Exp. Med. 199: 1607-1618. [Abstract/Free Full Text]
  40. Henn, V., J. R. Slupsky, M. Grafe, I. Anagnostopoulos, R. Forster, G. Muller-Berghaus, R. A. Kroczek. 1998. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature 391: 591-594. [Medline]
  41. Roy, M., T. Waldschmidt, A. Aruffo, J. Ledbetter, R. Noelle. 1993. The regulation of the expression of gp39, the CD40 ligand, on normal and cloned CD4+ T cells. J. Immunol. 151: 2497-2510. [Abstract]
  42. Theiner, G., S. Rössner, A. Dalpke, K. Bode, T. Berger, A. Gessner, M. B. Lutz. 2008. TLR9 cooperates with TLR4 to increase IL-12 release by murine dendritic cells. Mol. Immunol. 45: 244-252. [Medline]
  43. Schulz, O., A. D. Edwards, M. Schito, J. Aliberti, S. Manickasingham, A. Sher, C. Reis e Sousa. 2000. CD40 triggering of heterodimeric IL-12 p70 production by dendritic cells in vivo requires a microbial priming signal. Immunity 13: 453-462. [Medline]
  44. Kumanogoh, A., X. Wang, I. Lee, C. Watanabe, M. Kamanaka, W. Shi, K. Yoshida, T. Sato, S. Habu, M. Itoh, et al 2001. Increased T cell autoreactivity in the absence of CD40-CD40 ligand interactions: a role of CD40 in regulatory T cell development. J. Immunol. 166: 353-360. [Abstract/Free Full Text]
  45. Guiducci, C., B. Valzasina, H. Dislich, M. P. Colombo. 2005. CD40/CD40L interaction regulates CD4+CD25+ T reg homeostasis through dendritic cell-produced IL-2. Eur. J. Immunol. 35: 557-567. [Medline]
  46. Tone, M., Y. Tone, P. J. Fairchild, M. Wykes, H. Waldmann. 2001. Regulation of CD40 function by its isoforms generated through alternative splicing. Proc. Natl. Acad. Sci. USA 98: 1751-1756. [Abstract/Free Full Text]
  47. Knoechel, B., J. Lohr, E. Kahn, J. A. Bluestone, A. K. Abbas. 2005. Sequential development of interleukin 2-dependent effector and regulatory T cells in response to endogenous systemic antigen. J. Exp. Med. 202: 1375-1386. [Abstract/Free Full Text]



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