Immune Regulation through Mitochondrion-dependent Dendritic Cell Death Induced by T Regulatory Cells

Abstract

Dendritic cells (DCs) harbor an active mitochondrion-dependent cell death pathway regulated by Bcl-2 family members and undergo rapid turnover in vivo. However, the functions for mitochondrion-dependent cell death of DCs in immune regulation remain to be elucidated. Here we show that DC-specific knockout of pro-apoptotic Bcl-2 family members, Bax and Bak, induced spontaneous T cell activation and autoimmunity in mice. In addition to a defect in spontaneous cell death, Bax^−/−Bak^−/− DCs were resistant to killing by CD4⁺FoxP3⁺ T regulatory (T_reg) cells compared to wild type DCs. T_reg cells inhibited the activation of T effector cells by wild type, but not Bax^−/−Bak^−/− DCs. Bax^−/−Bak^−/− DCs showed increased propensity for inducing autoantibodies. Moreover, the autoimmune potential of Bax^−/−Bak^−/− DCs was resistant to suppression by T_reg cells. Our data suggest that Bax and Bak not only mediate intrinsic spontaneous cell death in DCs, but also regulate DC killing triggered by T_reg cells. Bax- and Bak-dependent cell death mechanisms help to maintain DC homeostasis, and contribute to the regulation of T cell activation and the suppression of autoimmunity.

INTRODUCTION

During T cell development in the thymus, T cells that recognize self MHC molecules presenting autoantigens can survive by positive selection, while highly self-reactive T cells are subsequently deleted by negative selection (1–4). However, T cells that undergo successful maturation and eventually populate the peripheral lymphoid organs carry certain degree of autoreactivity. It is essential to keep these mature T cells in check to maintain peripheral tolerance. Programmed cell death of mature lymphocytes is a major mechanism for the maintenance of lymphocyte homeostasis and peripheral tolerance (5–7). In addition, natural T_reg cells that express FoxP3 have been established to play an essential role in the protection of immune tolerance (8–14).

DCs, the most efficient antigen presenting cells, are important regulators of both innate and adaptive immune responses (15–19). DCs may also play important roles in the maintenance of immune tolerance (20, 21). We have previously observed that DC-specific expression of the baculoviral caspase inhibitor, p35, leads to inhibition of Fas-mediated apoptosis in DCs and the development of systemic autoimmune symptoms (22). Consistently, knockout of Fas in DCs also induces the onset of autoimmunity in mice (23). Interestingly, interactions of antigen-pulsed DCs with the antigen-specific T cells may lead to accelerated loss of DCs in vivo (24). It is possible that Fas-dependent killing of DCs by activated T cells provides a negative feedback mechanism that helps to terminate the activation of lymphocytes by antigen-bearing DCs (25).

It has been recognized that DCs have a short lifespan since the original discovery of this cell type (26). The short half-life of DCs in vivo has been linked to an active mitochondrion-dependent cell death pathway regulated by Bcl-2 family members (27–29). The Bcl-2 family members are upstream regulators of mitochondrion-dependent apoptosis pathway (30, 31). They share the Bcl-2 homology (BH) domains and are divided into three subfamilies (30, 31), including pro-apoptotic Bax and Bak; the anti-apoptotic subfamily proteins, such as Bcl-2, Bcl-xL and Mcl-1; and the pro-apoptotic BH3-only subfamily, such as Bim, Bid and Bad. BH3-only proteins are the upstream sensors for different apoptosis signaling in specific cell types (32). BH3-only proteins initiate mitochondrion-dependent apoptosis by either inhibiting the anti-apoptotic molecules, or directly activating pro-apoptotic Bax or Bak to induce apoptosis (32, 33). While deficiency in either Bax or Bak has no salient phenotypes, knockout of both Bax and Bak abolishes mitochondrion-dependent apoptosis (34), indicating that Bax and Bak are essential for mitochondrion-dependent apoptosis but functionally redundant of each other. Both negative selection of developing T cells in the thymus and apoptosis of mature T cells are defective in Bax^−/−Bak^−/− mice (35), suggesting that Bax- and Bak-dependent mitochondrial apoptosis in T cells is involved in the regulation of lymphocyte homeostasis and immune tolerance.

The rapid turnover rates of DCs in vivo could be attributed to the active mitochondrion-dependent cell death pathway in DCs (27). Indeed, transgenic expression of anti-apoptotic Bcl-2 or deletion of pro-apoptotic Bim can inhibit spontaneous cell death in DCs (28, 29). In contrast, Fas-mediated signaling is not required for spontaneous cell death in DCs (29). It is therefore possible that mitochondrion-dependent and Fas-mediated cell death pathways regulate DC turnover at different phases of immune responses. However, the function for the active mitochondrion-dependent cell death pathway of DCs in immune regulation has not been well characterized. Furthermore, whether mitochondrion-dependent cell death in DCs plays a role in the regulation of immune tolerance is not known.

T_reg cells can interact with DCs to inhibit the activation of antigen-specific T cells in vivo (36–42). T_reg cells may down-regulate important co-stimulatory molecules on DCs (40, 43–48). Interestingly, one report has suggested that T_reg cells may cause the disappearance of DCs in the draining lymph nodes (38). Whether T_reg cells can induce cell death in DCs, and whether such interactions help to protect immune tolerance have not be determined. Using mice with deficiencies of Bax and Bak in DCs, we show that CD4⁺FoxP3⁺ T_reg cells efficiently induce mitochondrion-dependent cell death in DCs. Different from the killing of DCs by T effector cells through Fas, our current study suggests that T_reg cells exploit the active mitochondrion-dependent apoptosis pathway in DCs for immune regulation. Such interactions between T_reg cells and DCs potentially play a fundamental role in the regulation of initiation and expansion of antigen-specific immune responses, and in the protection of immune tolerance.

Materials and Methods

Mice

Bak^−/−, Bax^flox, Bim^−/−, perforin^−/−, lpr, CD11c-cre transgenic, OVA-specific-OT1 or -OT2 transgenic mice were obtained from the Jackson Laboratory and maintained on the C57BL/6 background. Granzyme A^−/−granzyme B^−/− mice and wild type controls on the 129X1/SvJ background were also obtained from the Jackson Laboratory. FoxP3^GFP knock-in mice were provided by Dr. Alexander Rudensky (49). FoxP3^GFP mice were crossed with perforin^−/− mice to generate perforin^−/−FoxP3^GFP mice, and with OT2 mice to generate OT2-FoxP3^GFP mice. OT2 mice were also crossed with CD45.1 congenic mice. The mice were maintained in a specific-pathogen-free facility and used with the approval of the Institutional Animal Care and Use Committee at Baylor College of Medicine.

Flow cytometry, preparation of DCs, measurement of autoantibodies, cell death assays and histochemistry

Flow cytometry analyses of different cell types, preparation of bone marrow derived DCs (BMDCs) and splenic DCs, histochemistry, measurements of spontaneous cell death in DCs, DC turnover in vivo by bromodeoxyuridine (BrdU) labeling, and detection of autoantibodies in mice were preformed as described (22, 29).

Proliferation assays

Mice were immunized with ovalbumin (OVA; 50 μg/mouse) emulsified in complete Freud adjuvant (CFA) at the footpad. Ten days later, total cells (2×10⁵/well) from the draining popliteal lymph nodes were cultured in 96-well plates with various concentrations of OVA for 72 h. The cells were pulsed with 1 μCi/well [³H]-thymidine for the last 12 h and harvested to measure [³H]-thymidine incorporation.

CD4⁺FoxP3⁺ T_reg cells were sorted from FoxP3^GFP mice and expanded in vitro with anti-CD3-and anti-CD28-coated Dynabeads (Invitrogen; 10 μl beads/10⁶ cells) in the presence of 1000 U/ml IL-2 for 3 days. Wild type or Bax^−/−Bak^−/− DCs were either unpulsed or pulsed with OVA_323–339 peptide. DCs (10³/well) were mixed with freshly sorted CD4⁺CD25⁻ T cells from OT2 mice (10⁴/well) in 96-well U-bottom plates, in the presence of different numbers of expanded T_reg cells. The proliferation of OT2 T cells was measured by [³H]-thymidine incorporation 4 days later. Alternatively, CD4⁺CD25⁻ T_eff cells were sorted from wild type mice by flow cytometry. Different numbers of T_reg cells expanded as above were added to CD4⁺CD25⁻ T_eff cells (5×10⁴/well) at different ratios in the presence of 0.25 μg/ml soluble anti-CD3 (2C11; BD Biosciences) and irradiated (3000 rads) T cell- and DC-depleted syngeneic splenocytes (5×10⁴/well) in 96-well U-bottom plates. [³H]-thymidine incorporation was measured 3 days later.

CD4⁺ T cells from OT2 mice (CD45.1 × CD45.2) were either sorted and labeled with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen) at room temperature for 10 min. The CFSE-labeled OT2 T cells were injected into C57BL/6 mice retro-orbitally (2×10⁶/mouse). DCs (2×10⁵) with or without pulsing with OVA_323–339 peptide were injected intradermally at footpad 24 h later with or without T_reg cells. Four days later, popliteal lymph nodes were collected and CD4⁺CD45.1⁺ T cells were analyzed by flow cytometry to determine CFSE dilution.

Spontaneous cell death in DCs and Killing of DCs or other target cells by T_reg cells

CD11c⁺I-Ab⁺ DCs were sorted from the spleens of DC-DKO or control mice. DCs were cultured in RPMI complete medium for 0 or 24 h. Spontaneous cell death of DCs were quantitated as described (29). BMDCs or CD11c⁺CD11b⁺ splenic DCs from wild type, DC-DKO or Bim^−/− mice were labeled with 1 μM CFSE (Invitrogen) for 10 min at 37 °C. The killing of DCs was analyzed similar to the previously described protocol (50). T_reg cells were expanded as above. In some experiments, CD4⁺CD25^high T_reg cells were sorted from granzyme A^−/−granzyme B^−/− mice or wild type controls and expanded as above. T_reg cells were mixed with DCs (2×10⁴/well) at different ratios in 96-well U-bottomed plates for 6 h. the cells were incubated with 5 ng/ml 7-amino-actinomycin D (7-AAD; BD Biosciences) at room temperature for 10 min, followed by flow cytometry. Induction of cell death in DCs was quantified essentially as described (50) with the following formula: percentages of killing of DCs by T cells = 100% × (DC_control−DC_T)/DC_control, with DC_control and DC_T representing CFSE⁺7-AAD⁻ DCs in the absence or presence of T cells, respectively. In some experiments, 10 μg/ml blocking antibody to LAG3 (clone C9B7W; BD Biosciences), LFA-1 (clone M17/4; BD Biosciences) or I-A/I-E (clone M5/114.15.2; Biolegend), or rat IgG were added to the culture.

To measure mitochondrial membrane potential, DCs with or without incubation with T_reg cells above were incubated with 10 μM etramethylrhodamine ethyl ester (TMRE; Invitrogen) at 37 °C for 20 min, followed by flow cytometry. In some experiments, DCs were incubated with 10μg/ml agonist anti-I-A/I-E (clone 2G9; BD Bioscience) or control rat IgG for 6 h, followed with TMRE staining and flow cytometry.

Splenic CD11c⁺ DCs were purified with anti-CD11c-MACS beads (Miltenyi Biotec). Activated CD4⁺ T cells were generated by stimulating sorted CD4⁺CD25⁻ cells anti-CD3- and anti-CD28-coated Dynabeads in the presence of 100 U/ml IL-2 for 3 days. B cells were purified with anti-CD19 MACS beads and stimulated with 1 μg/ml LPS and 100 μM CpG for 48 h. T_reg-mediated killing of splenic DCs and activated T or B cells were measured as the killing of BMDCs above.

CD4⁺FoxP3⁺ T_reg cells or CD4⁺FoxP3⁻ T_eff cells sorted from OT2-FoxP3^GFP mice were stimulated with OVA_323–339 peptide-pulsed DCs (T:DC=2:1) for 3 days. OT2 T_reg or T_eff cells were then isolated by removing DCs with anti-CD11c-MACS beads (Miltenyi Biotec) and used for killing of DCs with or without pulsing with OVA_323–339 peptide. Percentages of killing of DCs by T cells were measured as above.

Analyses of interaction between Treg cells and DCs

To detect interactions between T_reg cells and DCs in vivo, DCs and T_reg cells were labeled at 37 °C for 10 min with 10 μM 5-(and -6)-(((4-chloromethyl) benzoyl) amino) tetramethylrhodamine (CMTMR) and CFSE (Invitrogen), respectively. Wild type or DKO DCs (1×10⁶) were injected into the footpad of recipient mice. T_reg cells (0.5×10⁶) were also injected into the footpad of some recipient mice. Draining (popliteal) lymph nodes were harvested 24 h later and frozen sections were analyzed using a LSM 510 confocal microscope (Zeiss).

To determine the conjugate formation between DCs and T_reg cells in vitro, DCs and T_reg cells were labeled at 37 °C for 10 min with 0.3 μM CMTMR and CFSE, respectively. The cells were mixed at a ratio of 1:1 and centrifuged at 500 g for 5 min. The cells were incubated at 37 °C for 0, 1, 2 or 3 h in the absence or presence of 10 μg/ml anti-LAG3, anti-LFA-1 (BD Bioscience) or rat IgG. The cells were washed with PBS and analyzed by flow cytometry.

Adoptive transfer of DCs

To determine autoantibody production after adoptive transfer, wild type or Bax^−/−Bak^−/− BMDCs (10⁶ or 5×10⁶/mouse) were injected into 8-week-old C57BL/6 mice (6 mice/group) with or without T_reg cells (with T_reg:DC at 0.5:1) intraperitoneally essentially as described (29, 51). The mice were then injected with LPS (30 μg/mouse) intraperitoneally one day later. Sera were collected from the recipient mice 1 week after DC transfer. ANAs, anti-ssDNA and anti-dsDNA were measured as above. In parallel experiments, CFSE-labeled wild type or Bax^−/−Bak^−/−BMDCs (10⁶ or 5×10⁶/mouse) were injected into 8-week-old C57BL/6 mice (6 mice/group) with or without T_reg cells (with T_reg:DC at 0.5:1) at the footpad. Twenty four hours later, draining (popliteal) lymph nodes were collected. Total cell numbers were counted and percentages of CFSE⁺ DCs were determined by flow cytometry. Total CFSE⁺ DCs in the draining lymph nodes were calculated.

Western blot

To determine T_reg-mediated signaling in DCs, wild type or DKO DCs were incubated with T_reg cells at a ratio of 2:1 for 6 h at 37 °C. The cells were incubated with anti-CD11c-MACS beads (Miltenyi Biotec) to isolate DCs. DCs were then lysed for Western blot analyses. The following primary antibodies were used: polyclonal rabbit antibodies to caspase-8, caspase-3, Bcl-xL, Bad (Cell Signaling), Bax (Santa Cruz Biotechnology), Bcl-2, Bak (Upstate Biotechnology), Mcl-1 (Fitzgerald), Bim (Stressgen), Bid (Imgenex), Blk or Bmf (Biovision), or mouse monoclonal antibody to caspase-9 (MBL), Noxa (Imgenex) or XIAP (BD Bioscience). The blots were then probed with HRP-conjugated secondary antibodies and developed using the chemiluminescent method (Pierce). CD11c⁺CD11b⁺ DCs, CD3⁺ T cells or CD19⁺ B cells sorted from the spleen of DC-DKO and control mice were also used for Western blot analyses of Bax and Bak as above. The blots were also probed with anti-α-tubulin (Santa Cruz Biotechnology) to ensure equal loading.

Intracellular staining for cytokines

CD11c⁺ bone marrow–derived DCs (10⁶/ml) were cultured in the absence or presence of 1 μg/ml LPS (Sigma) or 0.5 μM phosphorothioate-stabilized CpG oligonucleotide (TCCATGACGTTCCTGATGCT) for 24 hours. Brefeldin A (1 μg/mL) and monensin (2 μM) were added during the last 6 hours to inhibit cytokine secretion. Cells were stained with FITC-anti-CD11c, followed by fixation and permeabilization with Cytofix/Cytoperm solution (BD Biosciences) and staining with PE-conjugated anti-IL-12p40/p70 (BD Biosciences), PE-conjugated IL-6 (BD Biosciences) or PE-conjugated rat IgG1 as isotype control. The cells were than analyzed by flow cytometry.

Statistical analysis

Data were presented as mean ± SD. P values were determined by two-tailed Student’s t-test using GraphPad Prism software version 4.0 for Macintosh. P<0.05 is considered to be statistically significant.

RESULTS

Accumulation of DCs due to deficiencies in Bax and Bak

To determine the functions of mitochondrion-dependent apoptosis in immune regulation, we generated double knockouts of Bax and Bak in DCs (DC-DKO) by crossing CD11c-cre mice with Bax^flox and Bak^−/− mice. Specific deletion of Bax in DCs were observed in DC-DKO mice (Supplemental Fig. 1A). Because Bax and Bak are functionally redundant of each other (34), deletion of Bak alone is not expected to affect apoptosis in other cell types. DC-DKO, but not Bak^−/− or DC-Bax^−/− mice, displayed enlargement of the spleen and lymph nodes (Supplemental Fig. 1B). Terminally differentiated DCs have low proliferative potentials, pulsing with bromodeoxyuridine (BrdU) in vivo can be used to measure the rate of DC turnover by the appearance of newly generated BrdU⁺ DCs (52). We found that DCs were labeled more slowly with BrdU in DC-DKO mice (Supplemental Fig. 1C), indicating a reduced DC turnover rate for DCs in these mice. Consistently, the percentages and total numbers of CD11c^highI-Ab⁺ or CD11c^highCD40⁺ conventional DCs were increased in DC-DKO mice (Fig. 1A and B). CD11c^lowPDCA-1⁺ plasmacytoid DCs increased to a lesser extent in DC-DKO mice (Fig. 1A and B). The percentages of T, B or NK cells were not elevated in DC-DKO mice (Supplemental Fig. 1D). DCs from DC-DKO, but not Bak^−/−, DC-Bax^−/−, Fas-deficient lpr, CD11c-cre or C57BL/6 control mice, showed defects in spontaneous cell death (Fig. 1C). However, splenic DCs from DC-DKO mice and wild type controls express similar levels of CD40, MHC-II, B7.1, B7.2 and ICAM-1 (Supplemental Fig. 1E), suggesting that deficiencies of Bax and Bak cause DC accumulation but not abnormal DC activation.

Figure 1. Reduced spontaneous cell death of DCs, DC accumulation and spontaneous lymphocyte activation in DC-DKO mice.

(A) Splenocytes from 6-month-old DC-DKO and control mice were stained with antibodies to different markers. The cells were analyzed by flow cytometry of CD11c^highI-Ab⁺ or CD11c^highCD40⁺ conventional DCs and CD11c^lowPDCA-1⁺ pDCs. (B) CD11c^highI-Ab⁺ DCs in the spleen of 6-month-old wild type C57BL/6 (B6), CD11c-cre (DC-cre), Bak^−/−, DC-Bax^−/−, DC- Bax^−/−Bak^−/− (DC-DKO) and lpr mice. **P<0.01, n=5. (C) Splenic CD11c⁺CD11b⁺ DCs from DC-DKO, lpr or control mice were cultured in vitro for 0 or 24 h. Cell death after 24 h of culture was determined. **P<0.01.

Spontaneous T cell activation and systemic autoimmunity in DC-DKO mice

T cells from DC-DKO mice showed increased expression of an activation marker, CD69 (Supplemental Fig. 2A). In particular, more than half of CD4⁺ T cells were CD69⁺ (Supplemental Fig. 2A). CD69 was also increased on CD8⁺ T cells and CD19⁺ B cells in DC-DKO mice (Supplemental Fig. 2A). In addition, similar to lpr mice, T cells with the CD44⁺CD62L⁻ activated/memory phenotype were increased in DC-DKO mice compared to controls (Supplemental Fig. 2A). These data suggest that deficiencies of Bax and Bak in DCs lead to DC expansion and abnormal lymphocyte activation.

We determined whether deficiencies in mitochondrion-dependent cell death induce autoimmune responses in DC-DKO mice. DC expansion and enlargement of lymphocyte areas were observed in spleen sections of 6-month-old DC-DKO mice (Supplemental Fig. 1F). Severe perivascular lymphocyte infiltration was found in the liver, lung and kidney of DC-DKO mice, but not DC-Bax^−/−, Bak^−/− or wild type controls (Supplemental Fig. 1G). IgG deposits were also observed in the glomeruli of kidneys in DC-DKO mice (Supplemental Fig. 1H). We also detected autoantibodies, including anti-dsDNA, anti-ssDNA and antinuclear antibodies (ANAs) in the sera of 3- and 6-month-old DC-DKO mice, but not in controls by ELISA (Fig. 2A). Consistent with the production of ANAs, sera from DC-DKO mice showed nuclear staining of Hep2 cells (Fig. 2B). Together, these observations suggest that deficiencies of Bax and Bak in DCs lead to the development of systemic autoimmunity. Normal levels of CD4⁺FoxP3⁺ natural T_reg cells were detected in DC-DKO mice (Supplemental Fig. 2A). Moreover, natural T_reg cells from DC-DKO and control mice showed comparable activities in inhibiting the proliferation of T effector (T_eff) cells (Supplemental Fig. 2B), indicating that there is no intrinsic defect in natural T_reg cells in DC-DKO mice.

Figure 2. Systemic autoimmunity in DC-DKO mice.

(A) ELISA for autoantibodies in the sera of 3- or 6-month-old DC-cre mice as wild type (WT) control, DC-DKO, Bak^−/−, DC-Bax^−/−mice. *P<0.05, **P<0.01. (B) Hep2 cell slides were incubated with sera (1:640 dilution) from 6-month-old DC-DKO or control mice, followed by staining with FITC-anti-IgG. Scale bar: 30 μm. Data are representative results of 5 mice per group analyzed.

Increased immunogenicity of Bax^−/−Bak^−/− DCs

We found that cells from the draining lymph nodes of immunized DC-DKO mice responded better to antigen re-stimulation than those of wild type, Bak^−/− or DC-Bax^−/− mice (Fig. 3A). Consistently, Bax^−/−Bak^−/− DCs induced more robust proliferation of antigen-specific T cells than control DCs in vivo as assayed by CFSE dilution (Fig. 3B). This suggests that Bax^−/−Bak^−/−DCs are more efficient in priming antigen-specific T cells in vivo. However, Bax^−/−Bak^−/− DCs did not produce more IL-6 or IL-12 (Supplemental Fig. 2F), suggesting that Bax^−/−Bak^−/− DCs do not over-produce these cytokines to induce more activation of T_eff cells. Interestingly, the increase in the capacity for Bax^−/−Bak^−/− DCs to stimulate antigen-specific T_eff cells in vitro was detectable but less dramatic (Fig. 3C, D; Supplemental Fig. 2D, E). This is reminiscent of previous observations that cell death in DCs has a more profound effect on the immunogenicity of DCs in vivo than in vitro (53). Such differences between in vivo and in vitro observations could be due to the presence of other cell types that regulate the interactions between DCs and T cells in vivo.

Figure 3. Increased immunogenicity of Bax^−/−Bak^−/− DC.

(A) Draining lymph node cells from 6-week-old DC-cre mice as wild type (WT) control, DC-DKO, Bak^−/−, DC-Bax^−/− mice immunized with OVA (5 mice per group) were re-stimulated in vitro with OVA. Cell proliferation was measured by ³H-thymidine incorporation 4 days later. WT versus DKO: **P<0.01. (B) CD4⁺CD25⁻ OT2 T cells from OT2 transgenic mice crossed with CD45.1 congenic mice (CD45.1×CD45.2 OT2 F1) were sorted, labeled with CFSE and injected intravenously into recipient C57BL6 mice. Recipient mice (5 mice/group) were then immunized with bone marrow-derived DCs (BMDCs) from DC-DKO or control mice pulsed with OVA_323–339 peptide the next day. Proliferation of transferred CD4⁺CD45.1⁺ T cells by CFSE dilution in draining lymph nodes was analyzed 4 days later. The numbers of cell cycle (average ± SD) are shown. WT versus DKO: P<0.01, with 65% increased in cell cycles induced by DKO DCs. (C) BMDCs from DC-DKO or control mice with or without OVA_323–339 pulsing were incubated with CD4⁺CD25⁻ OT2 T cells. ³H-thymidine incorporation was measured on day 4. WT versus DKO: *P<0.05, **P<0.01. (D) CFSE-labeled OT2 T cells (5×10⁴/well) were incubated with BMDCs from DC-DKO or control mice pulsed with OVA_323–339 peptide (300 DCs/well) in 96-well plates. Cell proliferation was measured by CFSE dilution 4 days later. The numbers of cell cycle (average ± SD) are shown. WT versus DKO: P<0.05 (n=4), with 13.9% increase in cell cycles induced by DKO DCs. (E) BMDCs from DC-DKO or control mice pulsed with OVA_323–339 peptide were cultured with CD4⁺CD25⁻ T cells from OT2 mice, in the absence or presence of T_reg cells sorted and expanded from FoxP3^GFP mice. Proliferation of OT2 T cells was measured by ³H-thymidine incorporation 4 days later. Statistic comparison with control group containing no T_reg cells: **P<0.01 (n=3). (F) CD4⁺CD25⁻ OT2 T cells as in Fig. 3B were labeled with CFSE and injected intravenously into recipient C57BL6 mice. Recipient mice were then immunized at the footpad with BMDCs from DC-DKO or control mice pulsed with OVA_323–339 peptide together with T_reg cells stimulated with anti-CD3- anti-CD28-coated Dynabeads in the presence of IL-2 for 3 days (T_reg:DC: 0.3:1). The proliferation of CD45.1⁺ OT2 T cells in the draining popliteal lymph nodes was quantitated by CFSE dilution 4 days later. The numbers of cell cycle (average ± SD) are shown (n=4). Statistic comparison between groups with or without T_reg cells: P<0.01 (WT DC), not significant (DKO DC).

Deficiencies in Bax and Bak enable DCs to overcome the suppression by T_reg cells

Although natural FoxP3⁺ T_reg cells were not reduced or dysfunctional in DC-DKO mice (Supplemental Fig. 2A, Supplemental Fig. 2B), it remains possible that Bax^−/−Bak^−/− DCs might be refractory to suppression by T_reg cells to induce uncontrolled T cell activation. We investigated whether deficiencies of Bax and Bak might enable DCs to overcome the inhibitory effect of T_reg cells. We sorted and expanded CD4⁺FoxP3⁺ natural T_reg cells from FoxP3^GFP knock-in mice (49). To the culture of antigen-pulsed DCs and ovalbumin (OVA)-specific OT2 T_eff cells (with DCs:T_eff at a fixed ratio of 0.1:1), we added varied numbers of the expanded T_reg cells (with T_reg:T_eff ratios ranging from 0.01:1 to 1:1). When Foxp3⁺ T_reg cell numbers were low (T_reg:T_eff from 0.01:1 to 0.1:1), we detected significant suppression of antigen-specific T cell proliferation induced by wild type, Bax^−/−, and Bak^−/− but not Bax^−/−Bak^−/− DCs (Fig. 3E). When T_reg:T_eff ratios were higher (0.1:1 to 1:1), significant suppression of Bax^−/−Bak^−/− DC-induced proliferation of OT2 T_eff cells was detected (Fig. 3E). However, this could at least be partially attributed to a direct suppression of T_eff cells by T_reg cells at higher T_reg: T_eff ratios (Supplemental Fig. 2C).

Consistent with in vitro observations (Fig. 3E), CD4⁺FoxP3⁺ T_reg cells suppressed the proliferation of OT2 T_eff cells induced by wild type, Bax^−/− or Bak^−/− DCs in vivo as measured by CFSE dilution (Fig. 3F). In contrast, Bax^−/−Bak^−/− DC-induced T cell proliferation was less susceptible to inhibition by T_reg cells in vivo (Fig. 3F). This supports the conclusion that Bax^−/−Bak^−/− DCs are refractory to suppression by T_reg cells.

Induction of Bax- and Bak-dependent cell death in DCs by T_reg cells

We found that activated T_reg cells acquired cytotoxicity against DCs (Fig. 4A). We examined whether activated T_reg cells could directly induce cell death in DCs in a Bax- and Bak-dependent manner. We observed that DCs were more susceptible than activated B or T cells to killing by CD4⁺FoxP3⁺ T_reg cells (Fig. 4B). Wild type, but not Bax^−/−Bak^−/− DCs were sensitive to killing by T_reg cells (Fig. 4C; Supplemental Fig. 3A, B), suggesting that T_reg cells induce DC cell death in a Bax- and Bak-dependent manner. T_reg-mediated killing of DCs is not affected by treating DCs with LPS (Supplemental Fig. 3C), suggesting that T_reg-mediated killing of DCs is not affected by DC maturation. It has been shown that T_reg cells can negatively regulate DCs by restricting DC development (54), inhibiting the expression of co-stimulatory molecules on DCs or competing with T_eff cells in the interaction with DCs (36–38, 40). Apoptotic DCs may promote further induction of T_reg cells (55). Inducing Bax- and Bak-dependent cell death in DCs may provide another mechanism for DC regulation by T_reg cells.

Figure 4. Susceptibility of DCs to killing by T_reg cells.

(A) Sorted CD4⁺FoxP3⁺ T_reg cells were cultured in vitro with anti-CD3- anti-CD28-coated Dynabeads in the presence of IL-2 for 0, 1, 2 or 3 days. BMDCs were labeled with CFSE and incubated with T_reg cells for 6 h. Killing of DCs by T_reg cells was determined as described in Materials and Methods. Day 3 versus day 0 at 0.3: and 1:1 T_reg: DC ratios: **P<0.01 (n=3). (B) CD4⁺FoxP3⁺ Treg cells were sorted and stimulated with anti-CD3- anti-CD28-coated Dynabeads in the presence of IL-2 for 3 days. Treg-mediated killing (n=5) of BMDCs, splenic DCs, or activated B or T cells were was determined as described in Materials and Methods. (C) CD4⁺FoxP3⁺ T_reg cells or CD4⁺FoxP3⁻T_eff cells sorted and expanded as in Fig. 3 were incubated with wild type or Bax^−/−Bak^−/− (DKO) BMDCs for 6 h, followed by staining with 7-AAD and analyses by flow cytometry. Loss of 7-AAD⁻ DCs was quantitated. WT versus DKO: **P<0.01 (n=3). (D) Killing of DCs by CD4⁺FoxP3⁺ T_reg cells as in (A) in the presence of anti-MHC-II or control IgG. **P<0.01 (n=3). (E) CD4⁺FoxP3⁺ T_reg cells or CD4⁺FoxP3⁻ T_eff cells were sorted from OT2/FoxP3^GFP mice and expanded with OVA_323–339 peptide-pulsed BMDCs for 3 days. OT2 T_reg or T_eff cells were then isolated by removing DCs with anti-CD11c-MACS beads (Miltenyi Biotec) and used for killing of BMDCs with or without pulsing with OVA_323–339 peptide. WT versus DKO: **P<0.01 (n=3).

Blocking MHC-II inhibited the killing of DCs by polyclonal T_reg cells (Fig. 4D). This suggests that the polyclonal T_reg cells are potentially autoreactive. By using OVA-specific OT2 FoxP3⁺ T_reg cells, we found that FoxP3⁺ T_reg cells killed antigen-pulsed, but not unpulsed DCs (Fig. 4E). Using FoxP3⁺ T_reg cells expressing a transgenic TCR specific for a foreign antigen may not recuperate the action of natural T_reg cells carrying TCRs that tend to be autoreactive. Nevertheless, our data obtained by using OT2 FoxP3⁺ T_reg cells suggest that recognition of antigens on DCs is important for killing by T_reg cells. In contrast to T_reg cells, polyclonal T_eff cells did not efficiently kill DCs in the absence of anti-CD3 (Fig. 4C), while OT2 T_eff cells showed killing of DCs in the presence of OVA antigen at a higher T:DC ratio (3:1; Fig. 4E).

Bax- and Bak-dependent clearance of DCs in the draining lymph nodes after adoptive transfer

We consistently detect more Bax^−/−Bak^−/− DCs in the draining lymph nodes after adoptive transfer (Fig. 5A). Moreover, co-transfer of T_reg cells led to the loss of wild type but not Bax^−/−Bak^−/− DCs in the draining lymph nodes (Fig. 5A). This is consistent with the conclusion that Bax and Bak regulate spontaneous cell death of DCs, as well as T_reg-dependent DC killing. T_reg cells can induce Bax- and Bak-dependent cell death in DCs. In addition, we also observed that T_reg cells formed conjugates with both wild type and Bax^−/−Bak^−/− DCs in the draining lymph nodes (Fig. 5A). Conjugate formation between T_reg cells and DCs was also detected in vitro (Supplemental Fig. 3D–H). Wild type and DKO DCs were similar in forming conjugates with T_reg cells after 1 hour of incubation in vitro (Supplemental Fig. 3E, H). Consistent with confocal microscopy analyses, more Bax^−/−Bak^−/− DCs were found in the draining lymphocytes than wild type DCs as determined by flow cytometry (Fig. 5B). Co-transfer of T_reg cells also promoted the clearance of wild type but not Bax^−/−Bak^−/− DCs (Fig. 5B). These data suggest that Bax and Bak do not affect conjugate formation between T_reg cells and DCs. Rather, Bax and Bak regulate spontaneous cell death of DCs, as well as T_reg-mediated clearance of DCs in vivo.

Figure 5. Clearance of DCs by Treg cells after adoptive transfer.

(A) Wild type and Bax^−/−Bak^−/− BMDCs were labeled with CMTMR (red) and injected subcutaneously into footpad. In some groups, CFSE (green)-labeled T_reg cells were also injected into footpad. Draining (popliteal) lymph nodes were harvested 24 h later. Frozen sections were prepared and analyzed by confocal microscopy. Scale bar: 20 μm. Quantification of DCs per focal section field (0.02 mm²) in the draining lymph nodes is shown (right panel). **P<0.01 (n=9). (B) CD11c⁺ WT or DKO BMDCs (10⁶ or 5×10⁶) labeled with CFSE were injected with or without T_reg cells (T_reg:DC=0.5:1) into syngenic C57BL/6 recipient mice at the footpad. Twenty-four hours later, the draining (popliteal) lymph nodes (LN) were harvested. CFSE⁺ DCs were analyzed by flow cytometry and total CFSE⁺ DCs in the draining LN were calculated. **P<0.01 (n=6).

Autoimmune potential of Bax^−/−Bak^−/− DCs in adoptive transfer

To directly test whether Bax^−/−Bak^−/− DCs can induce autoimmune responses, we performed adoptive transfer of wild type and Bax^−/−Bak^−/− DCs with or without T_reg cells. It has been shown that transfer of excessive activated DCs can trigger the production of autoantibodies in recipient mice (51, 56). Adoptive transfer of Bax^−/−Bak^−/− DCs at a low dose (1×10⁶ cells/mouse) induced the production of anti-dsDNA, anti-ssDNA and ANAs, while a higher dose (5×10⁶ cells/mouse) triggered more autoantibody production (Fig. 6). Co-transfer of T_reg cells did not significantly suppress autoantibody production induced by Bax^−/−Bak^−/− DCs (Fig. 6). In contrast, wild type DCs induced detectable levels of autoantibody production only at the high dose (5×10⁶ cells/mouse), which was efficiently suppressed by co-transfer of T_reg cells (Fig. 6). These data provide direct evidence to support the conclusion that Bax^−/−Bak^−/− DCs have the propensity for triggering autoantibody production. Moreover, Bax^−/−Bak^−/−, but not wild type DCs, are resistant to suppression by T_reg cells in the induction of autoimmune responses.

Figure 6. Suppression of DC-induced autoantibody production by Treg cells after adoptive transfer.

CD11c⁺ WT or DKO BMDCs (10⁶ or 5×10⁶) were injected with or without T_reg cells (T_reg:DC=0.5:1) into syngenic C57BL/6 recipient mice intraperitoneally (6 mice/group), followed by injection of LPS intraperitoneally (30 μg/mouse) 24 hours later. Sera were collected from recipient mice 1 week later for quantitation of ANA, anti-ssDNA, and anti-dsDNA by ELISA. **P<0.01 (n=6).

Molecules involved in the killing of DCs by activated Treg cells

We next investigated apoptosis signaling in DCs induced by T_reg cells. After incubation with T_reg cells, wild type DCs lost mitochondrial membrane potential (ΔΨm), while DKO DCs were relatively resistant to the loss of ΔΨm (Fig. 7A). Consistent with the activation of mitochondrion-dependent cell death in wild type DCs, T_reg cells induced the activation of caspase-9 and caspase-3 in wild type, but not Bax^−/−Bak^−/− DCs (Fig. 7B). It has been shown that cleavage of Bid into active tBid by caspases can trigger mitochondrial apoptosis (57). Caspase-dependent cleavage may inactivate anti-apoptotic Bcl-2 family proteins to promote mitochondrion-dependent cell death (58). However, we did not find processing of Bcl-2 family proteins in DCs after incubation with T_reg cells (Supplemental Fig. 4A). T_reg cells also did not change the expression of Bcl-2 family proteins in DCs (Fig. 7B, Supplemental Fig. 4A). Interestingly, Bim^−/− DCs showed resistance to killing by T_reg cells compared to wild type DCs, while deletion of either Bax or Bak in DCs did not affect their killing by T_reg cells (Fig. 7C). This suggests that Bim serves as a mediator to activate Bax and Bak in DCs after encountering T_reg cells. Although the precise mechanism for the activation of Bim is not resolved, Bim potentially transmits cell death signaling by sequestering anti-apoptotic molecules or directly activating Bax and Bak (59, 60).

Figure 7. Induction of mitochondrion-dependent cell death in DCs by T_reg cells.

(A) Wild type or DKO BMDCs with or without incubation with T_reg cells for 6 h were labeled with TMRE, followed by flow cytometry. Percentages of TMRE⁺ cells (mean ± SD): wild type, 89.4 ± 2.1 (no T_reg), 44.9 ± 5.0 (with T_reg), P=0.0001; DKO, 91.2 ± 1.5 (no T_reg), 83.2 ± 5.8 (with T_reg), P=0.081 (n=3). (B) Wild type or DKO BMDCs with or without incubation with T_reg cells for 6 h were lysed for Western blotting of caspase-8 (Casp8), Casp9, Casp3, Bax or Bak. (C) T_reg cells were incubated with wild type, Bak^−/−, Bax^−/−, Bim^−/− or DKO BMDCs, followed by analysis of killing of DCs as in Fig. 4. Bim^−/− or DKO versus WT DCs: *P<0.05, **P<0.01 (n=3). (D) T_reg cells were incubated with wild type BMDCs for 6 h in the presence of various antibodies of control IgG, followed by quantification of killing of DCs as in Fig. 6A. Antibody treatments versus IgG control: *P<0.05; **P<0.01 (n=3). (E) CD4⁺FoxP3⁺ T_reg cells with or without stimulation with anti-CD3/anti-CD28 Dynal beads plus IL-2 for 3 days were stained with PE-conjugated anti-LAG3 (solid line) or an isotype control (dashed line), followed by analyses by flow cytometry. (F) WT or DKO DCs were incubated with anti-I-A^b or control IgG for 6 h, followed by incubation with TMRE. The cells were analyzed by flow cytometry and the loss of TMRE⁺ cells were calculated. **P<0.01 (n=3).

We then investigated which effector molecules in T_reg cells that could induce cell death in DCs. The killing of DCs by T_eff cells involves Fas and perforin (22, 61, 62), but does not appear to require Bax and Bak in DCs (Supplemental Fig. 4B and C), suggesting that T_reg and T_eff cells use different mechanism to kill DCs. Interestingly, T_reg cells in tumor tissues can kill DCs in a perforin/granzyme-dependent manner (41), while T_reg cell-induced cell death in T_eff cells is independent of Fas or perforin (63). We found that T_reg-mediated killing of DCs did not involve Fas but required prior activation of T_reg cells (Fig. 4A, Supplemental Fig. 4D). Also, T_reg cells did not induce proteolytic activation of caspase-8 in DCs (Fig. 7B), consistent with the possibility that T_reg cells do not engage Fas on DCs to activate caspase-8. In addition, deficiencies in perforin or granzymes A/B in T_reg cells did not affect the killing of DCs (Supplemental Fig. 4E), suggesting that these molecules are not required for the killing of DCs by natural T_reg cells.

We observed that blocking LFA-1 inhibited T_reg-mediated killing of DCs (Fig. 7D). The conjugate formation between T_reg cells and DCs was inhibited by blocking LFA-1 (Supplemental Fig. 4G, H), suggesting that cell adhesion promotes the killing of DCs by T_reg cells. Molecules highly expressed by T_reg cells have been suggested to be critical for T_reg functions (11, 40, 64, 65). We observed that inhibition of LAG3 partially suppressed the killing of DCs by T_reg cells (Fig. 7D), while neutralization antibodies to soluble factors, including IL-10 and TGF-β, have no effect (data not shown). LAG3 is a potent ligand for MHC II molecules and can trigger negative signaling in DCs to suppress maturation and immunostimulatory capacity of DCs (66). On un-stimulated T_reg cells, the expression of LAG3 was low (Fig. 7E). T_reg cells acquired cytotoxicity against DCs after activation (Fig. 4A), while LAG3 was also significantly up-regulated on activated T_reg cells (Fig. 7E). Increased LAG3 on activated T_reg cells may enable T_reg cells to trigger cell death in DCs by engaging MHC-II. Indeed, we observed that engagement of MHC-II on DCs with an agonist antibody triggered the loss of ΔΨm in wild type, but not Bax^−/−Bak^−/− DCs (Fig. 7F). Engagement of MHC-II or other molecules on DCs by activated T_reg cells may transmit signals into DCs through BH3-only molecules, such as Bim, to induce the activation of Bax and Bak, leading to mitochondrial disruption and cell death.

DISCUSSION

DCs harbor an active mitochondrion-dependent apoptosis pathway regulated by Bcl-2 family members (27–29). In this study, we determined the functions of mitochondrion-dependent cell death in DCs in immune regulation using mice with DC-specific knockout of Bax and Bak. Deficiencies of Bax and Bak in DCs resulted in DC expansion, spontaneous T cell activation and development of systemic autoimmunity. In addition to regulating spontaneous cell death by Bax and Bak, our data suggest another level of regulation of T_reg-induced cell death in DCs through Bax and Bak. Expression of LAG3 may enable T_reg cells to trigger mitochondrion-dependent cell death in DCs. Adoptive transfer experiments provide direct evidence to show that Bax- and Bak-deficient DCs have increased propensity for inducing the production of autoantibodies. Moreover, T_reg cells inhibited wild type, but not Bax- and Bak-deficient DCs in the induction of autoantibodies after adoptive transfer. Our data suggest that Bax- and Bak-dependent pathway is involved in both spontaneous cell death and T_reg-mediated killing of DCs, and both of these mechanisms are important for maintaining DC homeostasis and preventing autoimmunity.

Bax- and Bak-dependent spontaneous cell death in DCs may occur throughout the courses of immune responses. In contrast, only pre-activated T_reg cells expressed higher levels of LAG3 (Fig. 7E) and showed the capacity to kill DCs (Fig. 4A), suggesting that Bax- and Bak-dependent killing of DCs may happen only after T_reg cells are activated. We and others have previously demonstrated that DCs are susceptible to Fas-mediated apoptosis (22, 23). Fas-dependent killing of DCs may take place when activated T cells that express high levels of Fas ligand are present. This potentially provide a negative feedback mechanism for the suppression of DC-dependent activation of T cells at late stages of immune responses, possibly after significant T cell activation induced by DCs has taken place. It has been reported that Treg cells can kill autologous CD8⁺ T cells or LPS-induced monocytes through FasL/Fas interactions (67, 68). Other studies have shown that the suppressive effect of T_reg cells is not inhibited by neutralization of FasL or using FasL-deficient Treg cells (69, 70). We also observed that the killing of DCs by Treg cells was independent of Fas (Supplemental Fig. 4). Such differences in the involvement of FasL/Fas interactions could be due to different target cells used in these studies.

Fas-mediated killing is usually dependent on activated T cells that express high levels of FasL. Spontaneous cell death in DCs through mitochondrion-dependent intrinsic apoptosis pathway is regulated by Bcl-2 family members (28, 71). Different from Fas-dependent cell death, Bax- and Bak-dependent spontaneous cell death in DCs may have a more broad influence at the initiation, expansion and contraction phases of immune responses. On the other hand, killing of DCs by activated T_reg cells through Bax and Bak may only function at the contraction phase of immune responses.

Recognition of antigens by TCRs on T_reg cells and LFA-1-dependent T_reg-DC conjugate formation may be critical for T_reg cells to kill DCs. Interestingly, activated T_reg cells expressed elevated LAG3 and acquired killing activities towards DCs, and blocking of LAG3 partially inhibited the killing of DCs by T_reg cells (Fig. 7). It has been shown that LAG3 can trigger negative signaling to inhibit the maturation and immunostimulatory capacity of DCs (66). Interestingly, crosslinking of MHC-II triggered the loss of ΔΨm in DCs in a Bax- and Bak-dependent manner (Fig. 7H). Our data suggest that LAG3 on activated T_reg cells can engage MHC-II on DCs to induce mitochondrial disruption and cell death signaling in DCs.

Certain DC subsets can induce the generation of T_reg cells in vitro and in vivo (20, 72–77). On the other hand, T_reg cells also restrict the development of DCs in vivo (10, 54). Our data suggest that T_reg cells can restrict DC expansion through Bax- and Bak-dependent killing, and deficiency in the killing of DCs by T_reg cells contributes to DC accumulation and induction of autoimmunity in DC-Bax^−/−Bak^−/− mice. Therefore, dynamic interplays between DCs and T_reg cells are important for the maintenance of a balanced immune system. Induction of mitochondrion-dependent cell death in DCs by T_reg cells may serve as one important mechanism for immune regulation. Promoting the interactions between T_reg cells and DCs may provide effective avenues to prevent and treat autoimmune diseases, whereas inhibiting the killing of DCs by T_reg cells may help to boost immune responses to infections and cancer.

Abbreviations used

7-AAD

7-amino-actinomycin D

ANA

antinuclear antibodies

BMDCs

bone marrow-derived DCs

CMTMR

5-(and -6)-(((4-chloromethyl) benzoyl) amino) tetramethylrhodamine

TMRE

etramethylrhodamine ethyl ester

T_eff

T effector

T_reg

T regulatory

ΔΨm

mitochondrial membrane potential