Reprogrammed FoxP3⁺ T Regulatory Cells Become IL-17⁺ Antigen-Specific Autoimmune Effectors In Vitro and In Vivo

Mice and reagents

C57BL/6J, BALB/CJ, DO11.10, OT-II, B6.129S2-IL6^tm1Kopf/J, C57BL/6-Tg(Ins2-OV)59Wehi/WehiJ (high), and C57BL/6-Tg(Ins2-OV)307Wehi/WehiJ (low) mice and rat insulin promoter (RIP)-OVA (27) mice were purchased from The Jackson Laboratory. Female mice were used in diabetes experiments and in experiments using BALB-neuT mice (H-2^d). Otherwise, male mice were used. All experiments were conducted with Institutional Animal Care and Use Committee oversight. The mouse breast cancer cell line transfected with rat Her-2/neu (A2L2), a mock-transfected parental cell line (66.3neo), and a cloned cell line from established carcinoma that spontaneously arose in a BALB-neuT mouse (TUBO) (28, 29, 30) were gifts from E. Celis (Moffitt Cancer Center, Tampa, FL). Peptide p66 (TYVPANASL) derived from rat Her-2/neu was synthesized in Mayo Protein Core Facility. Hybridoma clone PC61 (anti-CD25) was a gift from W.-Z. Wei (Wayne State University, Detroit, MI). Anti-mouse CD4-PE (RM4-5) Ab and anti-mouse IFN-γ-PE (XMG1.2) Ab were from BD Biosciences. Anti-mouse FoxP3-allophycocyanin (FJK-16s), anti-DO11.10 TCR-FITC (KJ1-26), anti-mouse TNF-α-PE (MP6-XT22), anti-mouse IL-10-PE (JES5-16E3), and anti-mouse IL-17A-PE (clone eBio17B7) Abs were from eBioscience.

B7-DC XAb development

The human monoclonal IgM Abs sHIgM12 (B7-DC XAb) and sHIgM39 (isotype-matched control) arose from a screen to identify Abs that bound mouse DC using a pool of sera from patients with monoclonal gammopathies (20). The Abs were purified as described (20). Due to the B7-DC-dependence of its biologic properties, the requirement for the pentameric form, and the observed signals (S. Radhakrishnan, L. N. Arneson, J. L. Upshaw, C. L. Howe, S. J. Felts, M. Colonna, P. J. Leibson, M. Rodriguez, and L. R. Pease. TREM-2-mediated signaling induces antigen uptake and retention in mature dendritic cells. Submitted for publication.) in DC elicited by Ab binding (20, 21), we refer to this novel reagent as B7-DC cross-linking Ab (B7-DC XAb). DC treated with the B7-DC XAb reagent are referred to as DC^XAb.

Immunization protocol

Peptide and CpG immunization strategy (see Fig. 1⇓) and analysis of the ensuing immune response was conducted as previously described (28). For the in vivo Treg depletion experiments, 0.5 mg of anti-CD25 mAb (PC61) was injected i.p. on days −3, −2, and −1 before immunization with peptide on day 0. Other groups of mice were injected i.p. or s.c. with 10 μg of sHIgM39 control or B7-DC XAb on days −1, 0, and +1 relative to immunization. For all other experiments, mice received Ab, Ag, pretreated DC, or T cells i.v. as indicated.

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FIGURE 1.

Reversal of T cell tolerance in Her-2/neu model by Treg depletion or B7-DC XAb. A, Treatment regimen wherein mice that express rat Her-2/neu as self-Ag were immunized with a known immunogenic rat Her-2/neu peptide (p66) or control “irrelevant” peptide along with CpG adjuvant and concomitant depletion of Treg with the anti-CD25 Ab PC61 (top) or (bottom) in combination with B7-DC XAb treatment. B and C, Seven days after challenge with peptide, CD8⁺ T cells were isolated and assessed for IFN-γ responses by ELISPOT following stimulation with p66-pulsed P815 cells (□), the rat Her-2/neu-positive parental tumor line TUBO (▪), a mouse breast cancer cell line transfected with rat Her-2/neu, A2L2 (▧), or the mock transfected breast cancer cell line 66.3neo (▩). Data are representative of three or more independent experiments using at least n = 3 mice per group. All measurements were done in triplicate.

Isolation of Tregs and non-Tregs

Splenocytes were isolated from pooled spleens harvested from at least three mice in each treatment group. Tregs were isolated by positive selection using Mouse T Regulatory Isolation kit from Miltenyi Biotec (31), as per the manufacturer’s protocol. Briefly, splenocytes were incubated with anti-CD25 Ab coupled to magnetic beads for 15 min before binding to the MACS column. Unbound cells were washed three times with RPMI 1640 plus 10% FBS and used as non-Tregs. Adherent cells (Tregs) were eluted and washed before use. Flow cytometry analysis for FoxP3 expression showed the Treg population to be >95% pure.

Generation of bone marrow DCs

DCs were generated from the mouse bone marrow (32). Bone marrow cells were plated (1 × 10⁶/ml) in RPMI 10 containing 10 μg/ml murine GM-CSF and 1 ng/ml murine IL-4 PeproTech. The culture medium was refreshed on day 2 and pulsed with Ag (1 mg/ml), isotype control Ab, or B7-DC XAb (10 μg/ml) on day 6, followed by overnight incubation. Cells were washed on day 7 before use.

In vitro and in vivo activation of Tregs and non-Tregs

Bone marrow-derived wild-type (WT) or IL-6^−/− immature DCs (2 × 10⁶) were pulsed with Ag and treated with isotype control Ab or B7-DC XAb then used to stimulate naive DO11.10 Tregs or OT-II Tregs in culture (together in 24-well plates or separated into the bottom and top partitions of Boyden chambers) at a 1:1 ratio in vitro for 48 h. For in vivo studies, 1.5 × 10⁶ Tregs were adoptively transferred into histocompatible mice along with 3 × 10⁶ Ag-pulsed, Ab-treated bone marrow-derived DC. In experiments involving modulation of endogenous DC to activate Treg cells, CFSE-labeled OT-II Treg cells were transferred into mice along with isotype control Ab sHIgM39 or B7-DC XAb Ab (10 μg/ml) and OVA Ag (1 mg/ml). After 48 h, spleens were removed, pooled, and cells were prepared for analysis.

Suppression assays

DO11.10 Tregs, non-Tregs, or combinations of the two were stimulated with serially diluted Ag-pulsed, Ab-treated DC for 72 h. Proliferative responses were monitored by the addition of [³H]thymidine to the cultures for the last 18 h and measuring incorporation as previously described (20).

Cytokine responses

The frequency of CD8 T cells secreting IFN-γ was measured using ELISPOT (Mabtech) as described (28). Secretion of TGFβ1 into Treg:DC culture supernatants was measured using an ELISA kit from R&D Systems as per the manufacturer’s protocol. Multiplex cytokine assay was performed using a mouse cytokine panel and Bio-Plex Manager software version 4.0, according to the manufacturer’s protocol (Bio-Rad). For cytokine expression by intracellular staining, cells were permeabilized with CytoFix/CytoPerm kit (BD Biosciences) and incubated with the appropriate conjugated Ab at 4°C according to the manufacturer’s suggestions before analysis by flow cytometry as described (20, 21).

Analysis of FoxP3 expression

RNA was isolated using TRIzol reagent (Invitrogen). The 300 ng of RNA was used for quantitative RT-PCR (7900HT real-time PCR; Applied Biosystems) using FoxP3 forward 5′-CTACTTCAAGTACCACAATATGCGAC-3′, reverse 5′-CGTTGGCTCCTCTTCTTGCGAAACTC-3′ and actin control forward 5′-CGTCTGGACTTGGCTGGCCGGGACCT-3′, reverse 5′-AGTGGCCATCTCCTGCTCGAAGTCTA-3′. Intracellular staining for FoxP3 protein was performed as described for cytokine staining.

Tumor experiments

B16 tumor cells (5 × 10⁵) were injected s.c. into the flank of WT or IL-6^−/− mice along with 10 μg of control or B7-DC XAb Ab (i.v.) on days −1, 0, +1 as described (22). After 7 days, tumor draining lymph nodes were removed, and effector cells were prepared for in vitro cytotoxicity assays as previously described (33). Additional animals were monitored regularly for the development of tumors and euthanized if and when tumors measured 17 × 17 mm.

Induction of diabetes in mice

DC (2 × 10⁶) derived from WT or IL-6^−/− mice were pulsed with 100 μg of OVA and stimulated with 10 μg of control Ab or B7-DC XAb before injection into RIP-OVA mice. Mice were monitored regularly for blood sugar levels using a glucometer. Pancreata were excised, fixed in 2% formalin, and processed by Mayo Immunohistochemistry Core Facility for staining with anti-insulin Ab.

Toxicology studies

Eight-week-old male C57BL/6J mice, housed individually, received 300 μg of B7-DC XAb in 100 μl of PBS i.v. or 100 μl of PBS. Mice were observed and weighed periodically. Blood samples were collected on day 17 for hematology and chemistry analyses, including the number and percentage of white blood cells, lymphocytes, granulocytes, monocytes, and RBC as well as hematocrit, mean corpuscular volume, hemoglobin, mean corpuscular hemoglobin, mean corpuscular hemoglobin with concentration, red cell distribution width, mean platelet volume, with platelet counts and blood urea nitrogen, glucose, creatine L, alkaline phosphatase and alanine aminotransferase, and T-Pro measurements levels. Following the blood collection, the mice were euthanized and the heart, brain, spleen, kidney, liver, and lungs were analyzed for gross pathology and sectioned for histopathology in a blinded manner.

Statistical analyses

Normally distributed quantitative data are represented graphically and in tabular form with SEs of the mean. Qualitative data were analyzed using the χ² distribution.

Treg depletion or DC^XAb protocols break tolerance in a Her-2/neu mammary tumor model

Host Treg cells that normally function as helpful guardians of immune tolerance become detrimental suppressors of antitumor immune responses in the context of cancer (19). Depletion of host Tregs by systematically treating mice with CD25-specific Abs can allow antitumor responses to become effective. In earlier studies (28) we observed that DC^XAb elicited a robust immunity against tumors expressing rat Her-2/neu. These data suggested that DC^XAb had rendered Tregs in the NeuT mice functionally defective and thus had broken their tolerance of the Her-2/neu (self) Ag. As a test, we set up a direct comparison of the two immunomodulatory therapies. The treatment protocol is outlined in Fig. 1⇑A. BALB/c-NeuT (rat Her2neu transgenic) animals were treated with CD25-specific Abs or B7-DC XAb in the presence of CpG costimulation then challenged with an immunodominant Her-2/neu peptide determinant p66 (28). Generation of an immune reaction to the peptide challenge was determined by measuring in vitro IFN-γ production by CD8⁺ T cells isolated from the spleens of treated animals when challenged with various tumor cells.

CD8⁺ T cells from Treg-depleted (Fig. 1⇑B) and from XAb-treated (Fig. 1⇑C) animals were highly reactive to tumor cells that express Her-2/neu (TUBO or A2L2 tumor cell lines) and were much less reactive to either a non-Her-2/neu tumor (P815) or mock transfected cells (66.3). The similarities in the response patterns between mice treated with Treg-depleting Ab and mice treated with B7-DC XAb were consistent with our hypothesis that B7-DC XAb induces endogenous DC to modulate the function of endogenous Tregs. Because there is the possibility that this type of immune modulation might provide an Ag-specific method to affect self-tolerance, further characterization of this DC^XAb-Treg interaction seemed warranted.

Immunomodulated Tregs lose FoxP3 expression in an Ag-specific manner

To evaluate directly the hypothesis that treatment with B7-DC XAb down-regulates Treg function, we used a nontumor model (DO11.10) in which we could track reactive T cells with a specific TCR Ab (KJ-126). Treg function was initially monitored by the expression of the Treg-specific transcription factor FoxP3. Enriched CD25⁺ and CD25⁻ splenic T cells from DO11.10 mice were cocultured with OVA-pulsed DC^XAb or DC treated with isotype control Ab (DC^cntrl) for 48 h and assayed for FoxP3 expression. Only CD25⁺ T cells expressed FoxP3 mRNA, and when cultured with DC^XAb, markedly down-regulated FoxP3 mRNA in comparison to Tregs cultured with Ag-pulsed DC^cntrl (Fig. 2⇓A). Down-regulation of FoxP3 expression was also observed at the protein level (Fig. 2⇓B). Enriched CD4⁺ DO11.10 T cells (Tregs) or CD25⁻CD4⁺ D011.10 T cells (non-Tregs) were stimulated with OVA-pulsed DC^XAb or DC^cntrl for 48 h, then stained for intracellular FoxP3 and analyzed by flow cytometry. All non-Tregs were FoxP3⁻. Ag-pulsed DC^XAb suppressed the expression of FoxP3 protein by CD4⁺ DO11.10 Tregs. Importantly, there was a requirement for contact between the DC^XAb and the Tregs (Fig. 2⇓C). When DC and T cells were cocultured in Boyden chambers in which the two cell types were separated by a permeable membrane, FoxP3 expression in the Tregs remained unchanged (Fig. 2⇓C, compare top right quadrants). DC^XAb not pulsed with Ag were unable to suppress FoxP3 protein levels; time course analysis of the down-regulation of FoxP3 showed only a modest reduction at 36 h with complete down-regulation occurring by 48 h; and DC matured with CpG did not down-regulate FoxP3 expression by CD4⁺CD25⁺ DO11.10 Tregs (data not shown).

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FIGURE 2.

DC^XAb induces down-regulation of FoxP3 in Tregs. A, FoxP3 mRNA was measured by RT-PCR in BALB/c DC (control), CD4⁺CD25⁻ non-Tregs, CD4⁺CD25⁺ Tregs isolated from DO11.10 TCR transgenic T cells, or DC to T cell mixes (1:1). The DC were pulsed with chicken OVA (1 mg/ml) and stimulated with isotype control or B7-DC XAb (10 μg/ml) for 48 h. Levels of RNA are shown relative to the amount detected in purified CD4⁺CD25⁺ splenic Tregs (sample 3). B, Intracellular FoxP3 protein level was measured by flow cytometry in CD4⁺CD25⁻ non-Tregs (top panels) and CD4⁺CD25⁺ Tregs (bottom panels) after coincubation with OVA-pulsed DC^cntrl or DC^XAb. C, Expression of FoxP3 in Tregs cocultured with OVA-pulsed DC as in B, except Boyden chambers were used to physically separate the T cells and the DC. Data are representative of three or more independent experiments.

We next sought to determine whether down-regulation of Treg function by DC^XAb was restricted only to Tregs specific for a given Ag presented. Therefore, we mixed OVA-specific DO11.10 Tregs with BALB/cJ Tregs that express a wide range of Ag specificities. To this mix we added OVA-pulsed DC^cntrl or DC^XAb. After 48 h, T cells were harvested from the cultures and analyzed for expression of FoxP3. BALB/c Tregs (KJ1-26⁻) and the OVA-specific DO11.10 Tregs (KJ1-26⁺) were distinguished using the KJ1-26 clonotypic marker (Fig. 3⇓A, top histogram). As shown in Fig. 3⇓A, recovered DO11.10 Tregs treated with Ag-pulsed DC^cntrl retained FoxP3 expression, whereas DO11.10 Tregs recovered from culture receiving DC^XAb no longer expressed FoxP3 (Fig. 3⇓A, middle panels). Importantly, expression levels of FoxP3 were normal in the BALB/c CD4⁺CD25⁺ Tregs exposed to either DC (Fig. 3⇓A, bottom panels), demonstrating that FoxP3 was down-regulated only in those Tregs specific for OVA Ag. We also detected CD4⁻ FoxP3⁺ cells, which may represent previously described CD8⁺ regulatory cells (34).

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FIGURE 3.

B7-DC XAb induces Ag specific down-regulation of FoxP3 in Tregs. A, In vitro experiment in which DC (1 × 10⁶) from BALB/c mice were pulsed with 1 mg/ml OVA Ag and treated with control Ab or B7-DC XAb (10 μg/ml) and then used to stimulate a 1:1 mix of DO11.10- and BALB/c-derived CD4⁺CD25⁺ Tregs. After 48 h, FoxP3 was measured by intracellular staining and flow cytometry in DO11.10 (KJ1-26⁺) or BALB/c (KJ1-26⁻) T cells (gated as shown in upper panel). Data are representative of three or more independent experiments. B, In vivo experiment in which DO11.10 Tregs (2 × 10⁶) were adoptively transferred (i.v.) into BALB/c mice along with 100 μg of OVA and 10 μg of control Ab or B7-DC XAb. After 48 h, CD4⁺CD25⁺ Tregs were isolated from pooled spleens. KJ1-26-positive (i.e., transferred DO11.10) and KJ1-26-negative (i.e., BALB/c host) cells were gated as indicated and analyzed for FoxP3 expression. Data are representative of three or more independent experiments using at least n = 3 mice per Ab treatment group.

We next asked whether endogenous DC could mediate the down-regulation of exogenous Tregs in vivo when activated by B7-DC XAb injection. BALB/cJ mice received adoptively transferred DO11.10 Tregs, OVA, and isotype control Ab or B7-DC XAb. Splenic T cells were recovered 48 h later and stained for expression of the DO11.10 clonotypic TCR (KJ1-26), CD4, and FoxP3. As shown in Fig. 3⇑B, coadministration of Ag with B7-DC XAb, but not with isotype control resulted in the down-regulation of FoxP3 expressed by the transferred DO11.10 T (KJ1-26⁺) cells, whereas the endogenous Tregs (KJ1-26⁻) retained FoxP3 expression. Thus, DC in the host animal can be activated by B7-DC XAb injection and, in turn, regulate FoxP3 expression by Tregs in vivo in an Ag-specific manner.

Down-regulation of FoxP3 following treatment with DC^XAb results in loss of suppressive activity by DO11.10 CD4⁺CD25⁺ T cells

FoxP3 expression is a marker for Treg phenotype, but it was important to establish whether there was any alteration in Treg function when FoxP3 was down-regulated by DC^XAb. Tregs were isolated from DO11.10 mice, stimulated in vitro with Ag-pulsed DC treated with activating or control Ab, then tested for their ability to suppress an Ag-specific response by effector T cells. Cells were incubated for 72 h and pulsed with [³H]thymidine during the last 18 h (Fig. 4⇓A). As expected, Tregs activated with Ag-pulsed DC^cntrl incorporated very little [³H]thymidine (Fig. 4⇓A, left, open circles), whereas CD4⁺CD25⁻ T effector cells responded to Ag and DC (Fig. 4⇓A, left, filled circles). When Tregs were mixed in increasing numbers with CD4⁺CD25⁻ effectors, the effector proliferation in response to Ag was suppressed (Fig. 4⇓A, right, open circles). However, when B7-DC XAb was used to modulate the mixed culture, the T effectors responded vigorously (Fig. 4⇓A, right, filled circles), thus no longer suppressed by the Tregs, and demonstrating that DC^XAb can alter the function of Tregs.

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FIGURE 4.

DC^XAb induce Tregs to lose suppressor activity and to express effector cell cytokines. A, T cell proliferation was measured by thymidine incorporation. DO11.10 CD4⁺CD25⁻ nonTregs or CD4⁺CD25⁺ Tregs were incubated with OVA-pulsed and control Ab-treated DC (left). Non-Tregs proliferated when stimulated with DC^cntrl (•) and Tregs did not (○). A mix of 3 × 10⁵ CD4⁺CD25⁻ T cells (non-Tregs) with an increasing number of CD25⁺ Tregs (right) in the presence of 0.5 × 10⁶ OVA-pulsed DC^cntrl (○) inhibited proliferation but not if in the presence of OVA-pulsed DC^XAb (•). B, DO11.10 CD4⁺CD25⁺ Tregs were cultured with OVA-pulsed DC treated with control Ab or B7-DC XAb for 48 h. Cytokine profiles of CD4⁺ Tregs were characterized by intracellular staining and flow cytometry. C, Coexpression of effector cytokines IFN-γ and TNF-α or IFN-γ and IL-17 in Tregs after conversion to CD4⁺ FoxP3⁻ cells. D, TGFβ1 levels from OVA-pulsed DC-Treg interactions were determined by ELISA using supernatants from 48 h cocultures. Measurements were done in triplicate. All data are representative of at least three independent experiments.

Changes in Treg suppressive activity correlated with changes in Treg cytokine production (Table I⇓). Using a bead-based cytokine assay, we found that IFN-γ, TNF-α, and IL-17 levels were significantly increased in cultures containing DC^XAb and DO11.10 Tregs, whereas the level of the immunosuppressive cytokine IL-10 was low compared with Treg or DC^cntrl cultures. To validate that these cytokines were emanating from the Tregs and not from other cell types in the cultures, DO11.10 Tregs were recovered and analyzed by intracellular flow cytometry. In concordance with the multiplex analysis, IFN-γ, TNF-α, and IL-17 were up-regulated in the CD4 T cells cocultured with DC^XAb, whereas IL-10 was down-regulated compared with controls (Fig. 4⇑B). The flow cytometry profiles suggest that the recovered T cells could be simultaneously producing all three cytokines. This pattern was confirmed in double-labeling experiments demonstrating that many cells secreting IFN-γ were also secreting TNF-α or IL-17 (Fig. 4⇑C). TGFβ in the culture supernatants was also down-regulated in DC^XAb-Treg cocultures but not in DC^cntrl-Treg mix and was low in all non-Treg controls (Fig. 4⇑D). Taken together, these studies indicate that when Tregs interact with DC^XAb, they lose the expression of FoxP3, production of TGFβ or IL-10, and the ability to suppress T effector cell responses and gain a T effector phenotype with the ability to produce the proinflammatory cytokines IL-17, IFN-γ, and TNF-α.

Modulation of Treg phenotype is not caused by the outgrowth of contaminating effector cells

We used enriched Treg populations for the experiments described. Thus, it was possible that the apparent modulation of the Treg phenotype was the consequence of the outgrowth of non-Treg effectors during the course of the experiments. To test for such proliferation, Tregs were labeled with CFSE and stimulated with Ag-pulsed, unlabeled DC^cntrl or DC^XAb for 48 h before characterization. As shown in Fig. 5⇓A, CFSE high Tregs cultured with DC^cntrl were still FoxP3⁺ after 48 h. In contrast, CFSE^high Tregs cultured with DC^XAb were FoxP3⁻. A similar number of T cells was recovered from each culture, indicating that the phenotype changes were not due to the death of FoxP3⁺ Tregs.

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FIGURE 5.

Decrease in FoxP3 comes from modulated Tregs and not due to effector cell outgrowth. A, DO11.10 Tregs were labeled with CFSE then incubated for 48 h in vitro with OVA-pulsed DC^cntrl or DC^XAb. Surface staining for CD4 was performed followed by intracellular staining for FoxP3. Flow cytometry scatter plot is shown for FoxP3 expression and CFSE content. B, DO11.10 non-Tregs (filled histograms) and Tregs (open histograms) were purified and analyzed for FoxP3 and IL-17 expression by intracellular staining before stimulation with DC. C, DO11.10 non-Tregs (left) or Tregs (right) were labeled with CFSE (upper panels) then incubated with OVA-pulsed DC^cntrl (filled histograms) or DC^XAb (open histograms). After 48 h, T cells were recovered and CFSE staining was measured by flow cytometry. Analysis of FoxP3 and IL-17 expression (middle panels) in recovered CFSE⁺ DO11.10 Tregs stimulated with OVA-pulsed DC^cntrl (filled histograms) or DC^XAb (open histograms). Analysis of FoxP3 and IL-17 expression in recovered CFSE⁺ DO11.10 non-Tregs (lower panels). All data are representative of at least three independent experiments.

The lack of significant CFSE dilution in cells displaying down-regulation of FoxP3 strongly suggested that the change in Treg phenotype was not due to the outgrowth of contaminating cell types. However, it was still possible that some small amount of non-Tregs in the Treg preparation were developing into effector cells. To evaluate this possibility, we followed the change in both Treg and effector cell markers in response to DC^XAb. First, FoxP3 and IL-17 levels were measured in the purified but unstimulated Tregs and non-Tregs (Fig. 5⇑B). The Tregs were 97% FoxP3⁺ and >99% IL-17⁻; the non-Tregs were 99% FoxP3⁻ and >99% IL-17⁻. The Treg or non-Tregs (2 × 10⁶ each) were then labeled with CFSE and incubated with DC as in Fig. 5⇑A. After 48 h, the total number of cells recovered (∼70% of the initial number plated) was the same for each group. CFSE analysis of those cells showed that some non-Tregs (CD25⁻ cells) did undergo at least one cell division in response to Ag-pulsed DC^XAb compared with DC^cntrl (Fig. 5⇑C, upper left). Yet, the non-Tregs remained FoxP3⁻ and failed to express IL-17 (Fig. 5⇑C, lower panels). In contrast, CFSE analysis of Tregs (CD25⁺) showed very little dilution upon stimulation with DC^XAb (Fig. 5⇑C, upper right). Intracellular staining showed that Tregs stimulated with DC^cntrl remained FoxP3⁺ and IL-17⁻, whereas Tregs stimulated with DC^XAb became FoxP3⁻ and IL-17⁺ (Fig. 5⇑C, middle panels). Because the preparation of non-Tregs did not develop into IL-17-producing cells, whereas the Treg preparation clearly produced IL-17 in the absence of significant CFSE dilution, we conclude that the induced effector phenotype was not due to outgrowth of CD25⁻ cells, but rather that Tregs were converted into IL-17 effectors by Ag-pulsed DC^XAb.

IL-6 is required for B7-DC XAb-induced down-regulation of FoxP3 in vitro and in vivo

We have previously shown that DC^XAb secrete IL-6 (35). IL-6 plays a dominant role in preventing newly activated T cells from differentiating into Tregs and favoring the development of the Th17 phenotype (26, 36). The importance of IL-6 expression in modulating the phenotype of established Tregs is less clear. Therefore, we evaluated the importance of IL-6 in mediating DC-Treg interactions using DC isolated from an IL-6-deficient subline of the C57BL/6 mouse lineage. We measured the down-regulation of FoxP3 expression in OVA specific OT-II CD4⁺CD25⁺ Tregs, an IL-6 WT subline of C57BL/6. FoxP3 expression was unaffected in Tregs cultured with WT or IL-6^−/− DC pulsed with OVA and treated with control Ab (Fig. 6⇓A, left panels). As observed previously, FoxP3 was down-regulated in Tregs cultured with WT DC^XAb, but not if the DC were derived from IL-6 deficient mice (Fig. 6⇓A, right panels). These results suggested that Treg modulation by B7-DC XAb required the ability of the DC to make IL-6. To test this IL-6-dependence further, we adoptively transferred CFSE-labeled OT-II Tregs into C57BL/6 WT or IL-6^−/− mice along with B7-DC XAb or control Ab. After 48 h, spleens were removed and recovered CFSE-labeled cells were analyzed for FoxP3 expression. As observed in vitro, FoxP3 was down-regulated in Tregs transferred into WT mice receiving Ag and B7-DC XAb, but not in Tregs transferred into IL-6^−/− mice (Fig. 6⇓B, compare right panels). The percentage of CFSE⁺ cells recovered from each treatment group in WT and knockout mice was comparable. Consistent with the data in Fig. 5⇑, FoxP3 was down-regulated in most of the CFSE-marked cells within 48 h. This down-regulation was IL-6-dependent not only using isolated DC in vitro, but also using endogenous DC in vivo.

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FIGURE 6.

IL-6 is crucial for DC^XAb-induced down-regulation of FoxP3 in vitro and in vivo. A, In vitro experiment in which FoxP3 expression was measured by intracellular staining and flow cytometry in CD4⁺CD25⁺ OT-II Tregs after 48 h incubation with OVA-pulsed DC generated from WT or IL-6^−/− mice. Data are representative of at least three independent experiments. B, In vivo experiment in which CFSE-labeled OT-II CD4⁺CD25⁺ Tregs (2 × 10⁶) were adoptively transferred into WT or IL-6^−/− mice along with 100 μg of soluble OVA and 10 μg of isotype control Ab or B7-DC XAb. FoxP3 levels were measured in recovered splenocytes 48 h after adoptive transfer and Ag and Ab treatments. FACS plots represent CFSE⁺ cells (i.e., transferred cells) on the x-axis and FoxP3⁺ cells on the y-axis. CFSE⁻ cells represent endogenous cells. All data are representative of three or more independent experiments using at least n = 3 mice per treatment group.

Ability to break tolerance to B16 tumor or islet-targeted OVA is IL-6-dependent

We reasoned that the modulation of Treg activity by B7-DC XAb or similar treatments would only have the potential to be clinically relevant if the converted T cells could break self-tolerance in model systems that had physiologic relevance. Our initial experiments in Her-2/neu mice suggested this effect was the case (Fig. 1⇑). So, we set out to extend those studies in the context of IL-6^−/− mice and test for specific responses toward tumor Ags. C57BL/6 (WT) and IL-6^−/− mice received B16 melanoma grafts and either B7-DC XAb or control Ab. After 7 days, T cells were isolated from draining lymph nodes and tested ex vivo for cytolytic activity toward B16 tumor or EL4 control cells. Tumor-reactive CTL were found in both groups of animals (Fig. 7⇓, left). Cells from IL-6^−/− mice were only slightly less reactive than WT mice. Despite the licensing of CTL in mice engrafted with tumor, only treatment with B7-DC XAb protected the C57BL/6 mice from otherwise lethal B16 melanoma grafts (Table II⇓); similar treatment of IL-6 knockout mice was not protective. All five WT animals treated with control Ab developed tumors and were sacrificed within 14 days, whereas all five B7-DC XAb-treated mice remained tumor-free for 6 mo (p < 0.05). In contrast, all of the seven IL-6^−/− mice treated with control Ab or with B7-DC XAb succumbed to the tumor grafts. Thus, immunomodulation by B7-DC XAb in this tumor model was also IL-6-dependent.

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FIGURE 7.

IL-6 is not required for the induction of antitumor CTL with B7-DC XAb. Left, WT mice (circles) or IL-6^−/− mice (squares) were engrafted with 0.5 × 10⁶ B16 melanoma tumor cells (s.c.) and treated i.v. on days −1, 0, and +1 with 10 μg of control Ab (open symbols) or B7-DC XAb (filled symbols). On day 7, T cells from draining lymph node cells were used as effectors to lyse ⁵¹Cr-labeled B16 tumor cell targets in vitro. Right, Lysis of ⁵¹Cr-labeled EL4 tumor-negative control targets. All data are representative of three or more independent experiments using at least n = 3 mice per treatment group.

We next addressed the functional relevance of modulating Tregs in another Ag-defined model. RIP-OVA mice express chicken OVA in their pancreas due to transcriptional control by the RIP. The mice treat the OVA Ag as a self-protein and develop normal pancreas morphology without evidence of inflammation (27). We first tested whether adoptive transfer of DC^XAb modulated tolerance by injecting RIP-OVA^low and RIP-OVA^high mice with OVA-pulsed DC^cntrl or DC^XAb. We also tested IL-6-dependence by using DC derived from WT or IL-6^−/− mice. Activated OVA-specific OT-I effector T cells were administered i.p. to RIP-OVA mice as a positive control (data not shown). All groups of animals were monitored periodically for their blood glucose levels.

As shown in Fig. 8⇓A, RIP-OVA^high mice that received Ag-pulsed DC^cntrl retained normal glucose levels, whereas mice that received Ag-pulsed DC^XAb became diabetic within 10 days (glucose levels higher than 250 mg/dL). Similar phenotypes were observed using RIP-OVA^low hosts; RIP-OVA^low mice that received OT-I T cells and OVA in CFA also became diabetic in that time frame (data not shown). Importantly, administration of IL-6-deficient DC^XAb failed to induce diabetes in any of the mice.

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FIGURE 8.

Tregs reprogrammed by DC^XAb become autoimmune effectors in immune diabetic model. OVA-pulsed DC^XAb or DC^cntrl (2 × 10⁶) derived from WT or IL-6^−/− mice were adoptively transferred (i.p.) into RIP-OVA^low or RIP-OVA^high mice. Recipient mice were monitored regularly for hyperglycemia. A, Results show blood sugar profiles of individual RIP-OVA^high mice. B, On day 15, the pancreata from mice in each group were isolated, fixed in formalin, and analyzed by histology for insulin. Staining patterns for the pancreata of RIP-OVA^low mice (top row at low and high magnification) and from RIP-OVA^high mice (bottom row at low and high magnification) are shown. C, Same as in A except RIP-OVA^high mice received 2 × 10⁶ CD4⁺CD25⁺ OT-II T cells pretreated with OVA-pulsed DC^XAb or OVA-pulsed DC^cntrl. In addition, some mice received 2.5 × 10⁵ naive OT-I cells along with the Tregs. No enhancement of islet destruction was observed by inclusion of the OVA-specific OT-I CD8⁺ T cells in the adoptive transfer experiments. All data are representative of three or more independent experiments using at least n = 3 mice per treatment group. See Table III⇓ for statistical comparisons of the treatment groups.

Blood glucose levels correlated with the integrity of the pancreas in each of the treatment groups. The number of islets was greatly reduced in the diabetic mice (Fig. 8⇑B). Furthermore, the immunohistochemical staining of insulin in the pancreas revealed only traces of insulin in mice that were diabetic. Mice that were nondiabetic had intact islets. We conclude from these studies that IL-6 expression by the adoptively transferred DC was required for the induction of diabetes in the RIP-OVA mice. The requirement for IL-6 is consistent with the interpretation that the Ag-pulsed DC^XAb induced disease, in part, by down regulating Treg function (36).

We also approached the adoptive transfer in the opposite way, asking whether DC^XAb-converted Tregs caused pathology in the RIP-OVA model. As shown in Fig. 8⇑C, when reprogrammed OT-II CD4⁺CD25⁺ Tregs were adoptively transferred into RIP-OVA mice with or without OT-I CD8 CTL precursors, hyperglycemia was evident by day 5 or 6, whereas blood glucose levels for all of the recipients of control Tregs was normal. Over the course of three experiments, 10 of 14 mice receiving reprogrammed OT-II Tregs developed diabetes, whereas none of the 10 recipients receiving Tregs treated with DC^cntrl developed the disease (Table III⇓; p < 0.001). To ensure that the pathogenesis was not caused by the outgrowth of contaminating CD4⁺CD25⁻ effector cells, mice were treated with enriched CD4⁺CD25⁻ OT-II cells that had been activated with OVA-pulsed DC^XAb (n = 8 mice). or alternatively, OVA-pulsed DC^cntrl (n = 7). None of these animals developed diabetes, demonstrating that any small number of CD4⁺CD25⁻ OT-II cells present in the population of enriched Tregs was not likely the source of effectors induced by DC activated with B7-DC XAb and that autoimmune effectors were derived from the Treg compartment and not from naive T cell precursors in this model of diabetes.

Taken together, these studies indicate that Ag-pulsed DC^XAb causes Tregs to down-regulate FoxP3 expression, stops Treg IL-10 and TGFβ production, and inhibits Treg ability to suppress CTL responses, whereas reprogramming the Tregs to produce effector cytokines IL-17, IFN-γ, and TNF-α. These reprogrammed Tregs effectively break tolerance, attacking tumors or causing autoimmune pathology, depending on the Ags used during reprogramming.

B7-DC XAb does not induce generalized autoimmunity

Because treatment of mice with B7-DC XAb rendered otherwise tolerant animals responsive to Ags associated with self-proteins in both the Her-2/neu and RIP-OVA transgenic models, we assessed whether the Ab might induce generalized autoimmunity when administered systemically to animals. Male C57BL/6J mice (n = 20), housed individually, were randomized and received i.v. either 300 μg of B7-DC XAb or PBS. The animals were scored over a 2-wk-period in a blinded manner for weight. Blood, heart, brain, spleen, kidney, liver, and lungs from each of the coded animals were examined for signs of abnormality by a board certified veterinary pathologist. During the course of the 2-wk observation period, one PBS-treated mouse died. There were no significant differences in body weight, blood chemistry, and cellularity, and no changes in organ morphology (including an absence of inflammation) among the surviving animals (data not shown). Moreover, B7-DC XAb treatment in other strains of mice (e.g., BALB/cJ) did not induce autoimmunity but protected mice from tumor (our unpublished observations). Therefore, treatment with B7-DC XAb does not induce a generalized autoimmunity.