Suppression of Mature Dendritic Cell Function by Regulatory T Cells In Vivo Is Abrogated by CD40 Licensing

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 × 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 peptide_327–339 (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-α (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 T_reg 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 T_reg (data not shown).

Fluorescence labeling of cells

For CFSE-labeling, the cells were adjusted to 2 × 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 × 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), T_reg or T_eff 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.

Adoptively transferred T_eff and T_reg form clusters with coinjected DC in the spleen

To study the interaction of OVA-peptide-loaded DC with Ag-specific CD4⁺ T_eff cells in the presence or absence of T_reg, 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 T_eff and T_reg cell populations from DO11-10 OVA-specific TCR transgenic mice were fluorescence-labeled with CFSE (green) or CMTMR (red) and T_eff or T_reg were injected individually or together 6 h later. After another 20 h, both individually injected T_eff or T_reg 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. 1⇓C) 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 T_eff or T_reg in vitro and in vivo (data not shown), consistent with previous reports (13, 14). This indicates that the clustering of both T_eff and T_reg subsets with peptide-loaded mature DC can occur in the spleen.

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FIGURE 1.

Similar DC clustering but slower activation and proliferation rate of T_reg in the spleen as compared with T_eff. A–C, OVA-loaded and LPS-matured BM-DC (1 × 10⁷, BALB/c) were labeled with CMAC (blue) and coinjected i.v. into syngeneic mice together with 8 × 10⁶ isolated and CFSE⁺ DO11.10 CD25⁻ T_eff (green) alone (A), 2 × 10⁶ CD25⁺ T_reg (red) alone (B), or CD25⁻ T_eff and CD25⁺ T_reg 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 × 10⁵ events were acquired. D, OVA-loaded immature or LPS-matured DC (2 × 10⁶) were injected i.v. into BALB/c mice and 6 h later 2 × 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 T_reg are reduced as compared with T_eff

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⁻ T_eff or CD25⁺ T_reg 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. 1⇑D, left), due to rearrangement of transgenic with endogenous TCR chains (24). As expected, immature DC did not stimulate both T_reg and T_eff populations (Fig. 1⇑D, 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 T_reg a shift into the CD25^high gate) and more pronounced for the CD25⁻ T_eff subset (Fig. 1⇑D, right). When the spleens were removed 4 days after injection and analyzed by FACS, the proliferation of CD4⁺KJ1–26⁺CD25⁺ T_reg was lower than of the CD4⁺KJ1–26⁺CD25⁻ T_eff but both populations remained in their Foxp3 positive or negative state, respectively (Fig. 1⇑E). Thus, the T_eff and T_reg populations, which are specific for a nonself peptide in this setting, differ in their activation and proliferation kinetics.

T_reg suppress T_eff only after delayed T_eff transfer

The observed contacts between T_eff 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 T_eff population in the absence of T_reg. How this could be influenced by T_reg was investigated by coinjections of both T cell subsets. OVA-loaded LPS-matured DC were injected together with T_reg followed by simultaneous or subsequent CFSE⁺ T_eff 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 T_eff vigorous CFSE dilution could be detected, independent of the time point of T_eff injection. When T_eff cells were coinjected simultaneously with T_reg at different ratios, the proliferation of T_eff remained unchanged. Suppression of the T_eff population was observed only when the T_eff were injected 10 or 24 h after the T_reg and the extent of suppression was dependent on the T_reg:T_eff ratio (Fig. 2⇓).

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FIGURE 2.

T_reg mediated suppression depends on their preactivation in vivo. OVA-loaded LPS-matured DC or unloaded DC (2 × 10⁶) together with 4 × 10⁵ (1:5 ratio) or 1.6 × 10⁶ (1:1 ratio) T_reg were injected i.v. into BALB/c mice. In addition, 1.6 × 10⁶ CFSE⁺ T_eff were injected either simultaneously, with a 10 h or 24 h delay with respect to the DC and T_reg 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 × 10⁶ events were acquired.

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

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

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FIGURE 3.

T_reg control the total spleen cellularity and do not prevent the T_eff response but control the maximal expansion and the termination phase. OVA-loaded LPS-matured DC (1 × 10⁷) and 1.6 × 10⁷ T_eff were injected i.v. into BALB/c mice with or without 4 × 10⁶ T_reg (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⁺ T_reg were gated of the analysis to follow only the CD4⁺KJ1–26⁺Foxp3⁻ T_eff expansion. Absolute T_eff numbers were calculated from the total spleen cellularity and the percentage of T_eff revealed by FACS analysis. Error bars represent the SDs from the means analyzing individual mice from three independent experiments. For each analysis 2 × 10⁵ events were acquired.

T_reg suppression is characterized by cluster disruption and reduced individual T_eff-DC and T_eff-T_reg contacts

The data so far indicate that T_reg suppressive activity occurs delayed in our experimental setting. However, how T_eff and T_reg 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 T_reg in red before transfer. Simultaneously or 24 h later, green-fluorescent T_eff were injected. Spleens were removed 24 h after the T_eff 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 T_eff suppression was observed and only individual contacts remained (Fig. 4⇓A). These findings from splenic interactions here are in agreement with other findings derived from DC-T cluster analysis within the lymph node (19).

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FIGURE 4.

T_reg inhibit clustering and individual contacts between DC-T_eff and T_reg-T_eff. OVA-loaded LPS-matured DC (1 × 10⁷) were injected i.v. into BALB/c mice. Four hours later, later 2 × 10⁶ isolated CMTMR⁺ T_reg derived from DO11.10 mice were injected i.v. into the same recipients. CFSE⁺ T_eff (8 × 10⁶) were injected either simultaneously or with a 24 h delay at a ratio of 5:1 (T_eff:T_regs). A, Cryosections were prepared 24 h after the T_eff injections to detect cell contacts and clustering by microscopical analysis. B, T_eff-DC, T_eff-T_reg, and T_reg-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 T_reg-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 T_eff-DC as well as T_reg-T_eff (Fig. 4⇑B), correlating with the cluster disruption (Fig. 4⇑A). Similar observations have been made in other systems and it had been discussed there whether this may indicate that in vivo the T_eff suppression can be mediated also indirectly by T_reg activity on the Ag-presenting DC (17).

T_reg induce down-regulation of selected costimulatory molecules on DC

Next, we wanted to elucidate whether mature DC can be influenced by T_reg 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 T_eff alone or T_reg plus T_eff 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 T_eff only or with simultaneous T_reg and T_eff 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 T_eff 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. 6⇓B). The effect of T_reg 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, T_reg activity in vivo, which leads to a block of T_eff proliferation, may be mediated by the down-regulation of specific costimulatoy molecules from the DC surface, which in turn indirectly controls the T_eff expansion.

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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 × 10⁴ events were acquired.

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FIGURE 6.

T_reg 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 × 10⁷) and 2 × 10⁶ DO11.10 derived T_reg were injected i.v. into BALB/c mice. DO11.10 T_eff (8 × 10⁶) were injected simultaneously or with a 24 h delay (ratio 5:1, T_eff:T_reg). As a control, DC and T_eff were also injected without T_reg. 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 × 10⁵ events were acquired.

Licensing of DC through CD40 aborts the suppressive activity of T_reg

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 T_reg 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 T_reg (Fig. 6⇑). This indicates that another quality of DC licensing or conditioning through CD40 may include the unresponsiveness to suppressive effects by T_reg 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 T_reg on T_eff proliferation, we directly compared OVA loaded DC matured with TNF, LPS, or LPS/CD40 under the 24-h delayed conditions in vivo. Although T_eff 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 T_reg suppression of T_eff proliferation in vivo is to a large extent mediated through DC and only a partial suppressive effect results from direct influence of T_reg on T_eff.

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FIGURE 7.

CD40 licensing of DC largely blocks suppression of T_eff proliferation by T_reg. OVA-loaded and differentially matured DC (1 × 10⁷) were injected i.v. into BALB/c mice alone or together with 2 × 10⁶ T_reg. In addition, 8 × 10⁶ CFSE⁺ T_eff were injected either simultaneously or with a 24 h delay at a 1:5 ratio (T_reg:T_eff). 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 × 10⁶ events were acquired.