The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 181, 3137 -3147
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Related articles in The JI
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Radhakrishnan, S.
Right arrow Articles by Pease, L. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Radhakrishnan, S.
Right arrow Articles by Pease, L. R.

Reprogrammed FoxP3+ T Regulatory Cells Become IL-17+ Antigen-Specific Autoimmune Effectors In Vitro and In Vivo1

Suresh Radhakrishnan*, Rosalyn Cabrera*, Erin L. Schenk*, Pilar Nava-Parada*, Michael P. Bell*, Virginia P. Van Keulen*, Ronald J. Marler{dagger}, Sara J. Felts* and Larry R. Pease2,*

* Department of Immunology, College of Medicine, Mayo Clinic, Rochester, MN 55905; and {dagger} Department of Molecular Pharmacology and Experimental Therapeutics, College of Medicine, Mayo Clinic, Scottsdale, AZ 85259


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lymphocyte differentiation from naive CD4+ T cells into mature Th1, Th2, Th17, or T regulatory cell (Treg) phenotypes has been considered end stage in character. In this study, we demonstrate that dendritic cells (DCs) activated with a novel immune modulator B7-DC XAb (DCXAb) can reprogram Tregs into T effector cells. Down-regulation of FoxP3 expression after either in vitro or in vivo Treg-DCXAb interaction is Ag-specific, IL-6-dependent, and results in the functional reprogramming of the mature T cell phenotype. The reprogrammed Tregs cease to express IL-10 and TGFβ, fail to suppress T cell responses, and gain the ability to produce IFN-{gamma}, IL-17, and TNF-{alpha}. The ability of IL-6+ DCXAb and the inability of IL-6–/– DCXAb vaccines to protect animals from lethal melanoma suggest that exogenously modulated DC can reprogram host Tregs. In support of this hypothesis and as a test for Ag specificity, transfer of DCXAb into RIP-OVA mice causes a break in immune tolerance, inducing diabetes. Conversely, adoptive transfer of reprogrammed Tregs but not similarly treated CD25– T cells into naive RIP-OVA mice is also sufficient to cause autoimmune diabetes. Yet, treatment of normal mice with B7-DC XAb fails to elicit generalized autoimmunity. The finding that mature Tregs can be reprogrammed into competent effector cells provides new insights into the plasticity of T cell lineage, underscores the importance of DC-T cell interaction in balancing immunity with tolerance, points to Tregs as a reservoir of autoimmune effectors, and defines a new approach for breaking tolerance to self Ags as a strategy for cancer immunotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunotherapy aims to harness the immune system to impact the treatment of a wide variety of diseases. However, for any specific therapy to be successful, it is critical that it direct a specific immune attack on the disease while protecting the host from aggressive autoimmunity. This effect is particularly a challenge in the case of cancer, where tumor growth is driven by mutations or abnormal expression of normal cellular proteins. To the host immune system, these "tumor-associated Ags" are likely recognized as an extension of self, allowing the tumor protection via active immune tolerance.

T regulatory cells (Tregs)3 actively suppress both physiological and pathological immune responsiveness and are thus central players in the establishment of self-tolerance and the maintenance of immune homeostasis (1, 2, 3, 4). Natural Tregs originate in the thymus early in life as CD4+ T cells (5, 6), constitutively display the IL-2R {alpha}-chain (CD25), and characteristically express FoxP3, a master regulator of Treg functions (7, 8, 9, 10, 11). Tregs also arise in response to newly encountered T cell epitopes in the absence of costimulatory signals (12, 13, 14). Mutation of the FoxP3 gene results in Treg deficiency and autoimmune inflammatory disorders, referred to as Scurfy disease in mice and IPEX (immune dysregulation, polyendocrinopathy, enteropathy X-linked syndrome) in humans (15, 16, 17). Treg-mediated suppression of proliferation and secretion of cytokines by effector-type T cells involves cell-to-cell contact, engagement of the CTLA-4 receptor on T cells, killing of effector cells and APCs, as well as the secretion of IL-10 and TGFβ (4).

It is becoming increasingly clear that development of effective therapeutic approaches to manipulate Treg functions and release immune effector mechanisms from stringent regulatory constraints will be crucial to advancing immune-based treatments of autoimmunity or cancer (18, 19). However, many details are not clear, such as how Tregs communicate with other cell types like dendritic cells (DC), how the balance of tolerance and immunity is maintained, and what becomes of Tregs as immune tolerance shifts toward active immunity. We recently characterized a human B7-DC cross-linking Ab (B7-DC XAb) that binds to B7-DC/PD-L2 molecules on both murine and human DC and cross-links the molecules on the cell surface (20). B7-DC XAb-activated DCs (DCXAb) can redirect established Th2 cells toward a Th1 phenotype in an Ag-specific manner (21). In addition, administration of B7-DC XAb to mice prevents tumor growth (22) and allergic asthma (23). This therapeutic effect can be mimicked by adoptive transfer of DCXAb, suggesting that the Ab acts directly on DC, not by preventing B7-DC-PD-1 interaction (24, 25).

DCs also present Ag to the T regulatory compartment of the immune system. Recent studies have documented the down-regulation of FoxP3 in the Tregs (9, 26). However, the functions acquired by those converted cells have not been explored. Our experiments using DCXAb to induce antitumor immunity suggested to us that activated DC may modulate Treg functions. In testing this hypothesis, we found that DCXAb not only caused Tregs to down-regulate their suppressive functions, but also to adopt an effector phenotype that promotes Ag-specific autoimmunity in animal models.


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

C57BL/6J, BALB/CJ, DO11.10, OT-II, B6.129S2-IL6tm1Kopf/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-2d). 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-{gamma}-PE (XMG1.2) Ab were from BD Biosciences. Anti-mouse FoxP3-allophycocyanin (FJK-16s), anti-DO11.10 TCR-FITC (KJ1-26), anti-mouse TNF-{alpha}-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 DCXAb.

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.


Figure 1
View larger version (20K):
[in this window]
[in a new window]

  		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-{gamma} responses by ELISPOT following stimulation with p66-pulsed P815 cells ({square}), the rat Her-2/neu-positive parental tumor line TUBO ({blacksquare}), a mouse breast cancer cell line transfected with rat Her-2/neu, A2L2 (Figure 1), or the mock transfected breast cancer cell line 66.3neo (Figure 1). 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 x 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 x 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 x 106 Tregs were adoptively transferred into histocompatible mice along with 3 x 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-{gamma} 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 x 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 x 17 mm.

Induction of diabetes in mice

DC (2 x 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 {chi}2 distribution.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Treg depletion or DCXAb 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 DCXAb elicited a robust immunity against tumors expressing rat Her-2/neu. These data suggested that DCXAb 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. 1A. 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-{gamma} 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. 1B) and from XAb-treated (Fig. 1C) 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 DCXAb-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 DCXAb or DC treated with isotype control Ab (DCcntrl) for 48 h and assayed for FoxP3 expression. Only CD25+ T cells expressed FoxP3 mRNA, and when cultured with DCXAb, markedly down-regulated FoxP3 mRNA in comparison to Tregs cultured with Ag-pulsed DCcntrl (Fig. 2A). Down-regulation of FoxP3 expression was also observed at the protein level (Fig. 2B). Enriched CD4+ DO11.10 T cells (Tregs) or CD25–CD4+ D011.10 T cells (non-Tregs) were stimulated with OVA-pulsed DCXAb or DCcntrl for 48 h, then stained for intracellular FoxP3 and analyzed by flow cytometry. All non-Tregs were FoxP3–. Ag-pulsed DCXAb suppressed the expression of FoxP3 protein by CD4+ DO11.10 Tregs. Importantly, there was a requirement for contact between the DCXAb and the Tregs (Fig. 2C). 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. 2C, compare top right quadrants). DCXAb 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).


Figure 2
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 2. DCXAb 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 DCcntrl or DCXAb. 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 DCXAb 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 DCcntrl or DCXAb. 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. 3A, top histogram). As shown in Fig. 3A, recovered DO11.10 Tregs treated with Ag-pulsed DCcntrl retained FoxP3 expression, whereas DO11.10 Tregs recovered from culture receiving DCXAb no longer expressed FoxP3 (Fig. 3A, middle panels). Importantly, expression levels of FoxP3 were normal in the BALB/c CD4+CD25+ Tregs exposed to either DC (Fig. 3A, 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).


Figure 3
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 3. B7-DC XAb induces Ag specific down-regulation of FoxP3 in Tregs. A, In vitro experiment in which DC (1 x 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 x 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. 3B, 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 DCXAb 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 DCXAb. 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. 4A). As expected, Tregs activated with Ag-pulsed DCcntrl incorporated very little [3H]thymidine (Fig. 4A, left, open circles), whereas CD4+CD25– T effector cells responded to Ag and DC (Fig. 4A, left, filled circles). When Tregs were mixed in increasing numbers with CD4+CD25– effectors, the effector proliferation in response to Ag was suppressed (Fig. 4A, right, open circles). However, when B7-DC XAb was used to modulate the mixed culture, the T effectors responded vigorously (Fig. 4A, right, filled circles), thus no longer suppressed by the Tregs, and demonstrating that DCXAb can alter the function of Tregs.


Figure 4
View larger version (46K):
[in this window]
[in a new window]

  		FIGURE 4. DCXAb 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 DCcntrl (•) and Tregs did not ({circ}). A mix of 3 x 105 CD4+CD25– T cells (non-Tregs) with an increasing number of CD25+ Tregs (right) in the presence of 0.5 x 106 OVA-pulsed DCcntrl ({circ}) inhibited proliferation but not if in the presence of OVA-pulsed DCXAb (•). 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-{gamma} and TNF-{alpha} or IFN-{gamma} 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-{gamma}, TNF-{alpha}, and IL-17 levels were significantly increased in cultures containing DCXAb and DO11.10 Tregs, whereas the level of the immunosuppressive cytokine IL-10 was low compared with Treg or DCcntrl 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-{gamma}, TNF-{alpha}, and IL-17 were up-regulated in the CD4 T cells cocultured with DCXAb, whereas IL-10 was down-regulated compared with controls (Fig. 4B). 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-{gamma} were also secreting TNF-{alpha} or IL-17 (Fig. 4C). TGFβ in the culture supernatants was also down-regulated in DCXAb-Treg cocultures but not in DCcntrl-Treg mix and was low in all non-Treg controls (Fig. 4D). Taken together, these studies indicate that when Tregs interact with DCXAb, 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-{gamma}, and TNF-{alpha}.


View this table:
[in this window]
[in a new window]

  		Table I. Multiplex cytokine analysis on Tregs stimulated with DCXAba

 
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 DCcntrl or DCXAb for 48 h before characterization. As shown in Fig. 5A, CFSE high Tregs cultured with DCcntrl were still FoxP3+ after 48 h. In contrast, CFSEhigh Tregs cultured with DCXAb 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.


Figure 5
View larger version (24K):
[in this window]
[in a new window]

  		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 DCcntrl or DCXAb. 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 DCcntrl (filled histograms) or DCXAb (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 DCcntrl (filled histograms) or DCXAb (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 DCXAb. First, FoxP3 and IL-17 levels were measured in the purified but unstimulated Tregs and non-Tregs (Fig. 5B). 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 x 106 each) were then labeled with CFSE and incubated with DC as in Fig. 5A. 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 DCXAb compared with DCcntrl (Fig. 5C, upper left). Yet, the non-Tregs remained FoxP3– and failed to express IL-17 (Fig. 5C, lower panels). In contrast, CFSE analysis of Tregs (CD25+) showed very little dilution upon stimulation with DCXAb (Fig. 5C, upper right). Intracellular staining showed that Tregs stimulated with DCcntrl remained FoxP3+ and IL-17–, whereas Tregs stimulated with DCXAb became FoxP3– and IL-17+ (Fig. 5C, 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 DCXAb.

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

We have previously shown that DCXAb 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. 6A, left panels). As observed previously, FoxP3 was down-regulated in Tregs cultured with WT DCXAb, but not if the DC were derived from IL-6 deficient mice (Fig. 6A, 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. 6B, 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.


Figure 6
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 6. IL-6 is crucial for DCXAb-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 x 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.


Figure 7
View larger version (20K):
[in this window]
[in a new window]

  		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 x 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.

 

View this table:
[in this window]
[in a new window]

  		Table II. B7-DC XAb-mediated tumor protection is IL-6 dependenta

 
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 DCXAb modulated tolerance by injecting RIP-OVAlow and RIP-OVAhigh mice with OVA-pulsed DCcntrl or DCXAb. 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. 8A, RIP-OVAhigh mice that received Ag-pulsed DCcntrl retained normal glucose levels, whereas mice that received Ag-pulsed DCXAb became diabetic within 10 days (glucose levels higher than 250 mg/dL). Similar phenotypes were observed using RIP-OVAlow hosts; RIP-OVAlow 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 DCXAb failed to induce diabetes in any of the mice.


Figure 8
View larger version (47K):
[in this window]
[in a new window]

  		FIGURE 8. Tregs reprogrammed by DCXAb become autoimmune effectors in immune diabetic model. OVA-pulsed DCXAb or DCcntrl (2 x 106) derived from WT or IL-6–/– mice were adoptively transferred (i.p.) into RIP-OVAlow or RIP-OVAhigh mice. Recipient mice were monitored regularly for hyperglycemia. A, Results show blood sugar profiles of individual RIP-OVAhigh 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-OVAlow mice (top row at low and high magnification) and from RIP-OVAhigh mice (bottom row at low and high magnification) are shown. C, Same as in A except RIP-OVAhigh mice received 2 x 106 CD4+CD25+ OT-II T cells pretreated with OVA-pulsed DCXAb or OVA-pulsed DCcntrl. In addition, some mice received 2.5 x 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. 8B). 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 DCXAb induced disease, in part, by down regulating Treg function (36).

We also approached the adoptive transfer in the opposite way, asking whether DCXAb-converted Tregs caused pathology in the RIP-OVA model. As shown in Fig. 8C, 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 DCcntrl 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 DCXAb (n = 8 mice). or alternatively, OVA-pulsed DCcntrl (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.


View this table:
[in this window]
[in a new window]

  		Table III. Reprogrammed Tregs cause autoimmunitya

 
Taken together, these studies indicate that Ag-pulsed DCXAb 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-{gamma}, and TNF-{alpha}. 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.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we demonstrate that Ag-pulsed DCXAb reprogram Tregs into functional effector cells and cause a break in tolerance in a number of Ag model systems. The reprogrammed Tregs down-regulated FoxP3 expression, secreted proinflammatory cytokines (IFN-{gamma}, IL-17, and TNF-{alpha}), and mounted potent responses against self-Ag in vivo. This phenotype conversion required DC-Treg contact, IL-6 secretion by the DC, and occurred in an Ag-specific manner. Surprisingly, this phenotypic conversion did not require cell division, though it took roughly 48 h to reach completion. Another striking finding was that reprogramming of Tregs did not cause a generalized autoimmunity, but rather, whether the model system used tumor Ags or neo-self-Ags expressed in the pancreas, a specific immune attack was elicited. This response raises the hypothesis that some pathogens can mimic the activation state of DC, which we have described using B7-DC XAb, to reprogram Tregs into autoimmune effectors.

Many factors contribute to lineage commitment of a naive T cell. Prevailing paradigms stipulate linear differentiation programs driving T cell lineage commitment, beginning with naive T cells that become Th1, Th2, Th17, or Tregs depending on the cytokine milieu the T cells encounter at the time of antigenic stimulation. In vitro studies have shown that the presence of IL-12 causes naive T cells to differentiate into Th1 cells; IL-4 drives naive T cells to become Th2 cells; TGFβ drives them to become Tregs, and the combination of IL-6 and TGFβ directs differentiation toward the Th17 lineage (37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49). In this study, we demonstrate an alternative pathway in which supposedly mature Tregs are reprogrammed. Although this pathway has been suggested by in vitro studies (50), we show that this reprogramming can take place in animal models using the endogenous Treg or DC. Together with our previous finding that committed Th2 cells can be reprogrammed to express Th1 cytokines during recall responses, identification of effector cells generated from Tregs provides new insights into the plasticity of T cell lineages.

T cell lineage specification is thought to be dependent on cell proliferation. Initial experiments reported by Bird and colleagues (51) demonstrated that expression of IL-2, IFN-{gamma}, and IL-4 tracks with T cell division. Adoptive transfer of labeled transgenic T cells into Rag–/– mice revealed a correlation between cell division and up-regulation of activation markers, with secretion of IFN-{gamma} and IL-2 (52). Moreover, blockade of T cell entry into early S phase of the cell cycle resulted in abrogation of the secretion of IFN-{gamma} and IL-10 (53). Finally, asymmetric cell division was characteristic of cells acquiring effector or memory phenotypes (54). The asymmetric fate of the daughter cells derived from first cell division following prolonged T cell interaction with APC suggests the possibility that the asymmetric nature of cell division may be mechanistically tied to lineage commitment. The possibility that T cells can acquire new traits without cell division suggested by our study will need to be addressed further. We have not yet determined the mechanism through which FoxP3 was down-regulated and whether reprogramming Tregs coincides with expression of T effector regulators such as T-bet, ROR{alpha}, or ROR{gamma} (55, 56, 57). Although we found that Treg modulation was IL-6-dependent, that signal may not be the defining character of DCXAb because TLR-activated DC also produced IL-6 (26), but did not reprogram mature Tregs in our experiments. Finally, we recovered reprogrammed cells from lymph nodes and spleens. More direct evidence of where reprogramming takes place remains to be determined.

In demonstrating the plasticity of Tregs, we have also described an approach for selectively depleting Tregs in an Ag-specific manner without promoting generalized autoimmunity. The ability to control Treg function is important for immune interventions in autoimmunity, cancer, and chronic viral infection. For example, during the course of tumor progression, changes in gene expression and the accumulation of mutations result in new antigenic profiles that shape T cell responses. Given that the appearance of new antigenic profiles in tissues is a normal process during the life of an individual, tolerance mechanisms are critical for restraining immune attack against emerging Ags. But because of the absence of overt danger signals in emerging cancers, the normal mechanisms of tolerance blunt potentially protective immune responses and interfere with efforts to use standard adjuvants to induce antitumor immunity. Strategies to eliminate Treg functions by blocking Treg effector mechanisms or by depleting Tregs from the immune repertoire have had mixed success (19), and the development of a generalized autoimmunity becomes increasing problematic.

An important aspect of this work is that the ability of T cells to be reprogrammed was revealed using DCs activated with the immune modulator, B7-DC XAb. How B7-DC XAb modulates DC function is only now emerging, but the biologic effects are significant. In short, matured DC regain their ability to take up Ag upon treatment with B7-DC XAb (58). A model now emerges in which DCXAb causes the up-regulation of Ag presentation of proteins reaching the draining lymph nodes, and the repolarization of Ag-specific Th2 effectors and Tregs into effectors with Th1 or Th17 characteristics. At the same time, there is a rapid mobilization of Ag-specific CD8 cytolytic cells that is also Ag-specific (33). The net change that results in the loss of down-modulatory effects of the Tregs and the alignment of the polarity of Th cells with the activated CTL can result in an effective antitumor response that can clear transplanted tumors (22, 59).

The B7-DC XAb Ab also binds and activates human DC (60). Accumulating studies demonstrate that just as in the laboratory mouse, this immune modulator rapidly activates immunity against human self-proteins (E. L. Schenk and L. R. Pease, unpublished observations). Important remaining questions include how effective this strategy will be in treating spontaneous tumors more characteristic of human cancers and indeed how effective this approach will be for treating human patients. As a first step toward clinical applications, a phase I trial is now in progress to evaluate B7-DC XAb as an immunotherapeutic immune modulator in advanced melanoma.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was funded by Grants R01 CA104996 and R01 HL077296 from the National Institutes of Health (to L.R.P.). Back

2 Address correspondence and reprint requests to Dr. Larry R. Pease, Department of Immunology, College of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail address: pease.larry{at}mayo.edu Back

3 Abbreviations used in this paper: Treg, T regulatory cell; DC, dendritic cell; RIP, rat insulin promoter; B7-DC XAb, B7-DC cross-linking Ab; DCXAb, B7-DC XAb-activated DC; DCcntrl, isotype control Ab-treated DC; WT, wild type. Back

Received for publication April 18, 2008. Accepted for publication June 27, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Sakaguchi, S.. 2004. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 22: 531-562. [Medline]
  2. Ziegler, S. F.. 2006. FoxP3: of mice and men. Annu. Rev. Immunol. 24: 209-226. [Medline]
  3. Zheng, Y., A. Y. Rudensky. 2007. Foxp3 in control of the regulatory T cell lineage. Nat. Immunol. 8: 457-462. [Medline]
  4. Miyara, M., S. Sakaguchi. 2007. Natural regulatory T cells: mechanisms of suppression. Trends Mol. Med. 13: 108-116. [Medline]
  5. Bonomo, A., P. J. Kehn, E. Payer, L. Rizzo, A. W. Cheever, E. M. Shevach. 1995. Pathogenesis of post-thymectomy autoimmunity: role of syngeneic MLR-reactive T cells. J. Immunol. 154: 6602-6611. [Abstract]
  6. Asano, M., M. Toda, N. Sakaguchi, S. Sakaguchi. 1996. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 184: 387-396. [Abstract/Free Full Text]
  7. Khattri, R., T. Cox, S. A. Yasayko, F. Ramsdell. 2003. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 4: 337-342. [Medline]
  8. Fontenot, J. D., M. A. Gavin, A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+ CD25+ regulatory T cells. Nat. Immunol. 4: 330-336. [Medline]
  9. Fontenot, J. D., J. P. Rasmussen, L. M. Williams, J. L. Dooley, A. G. Farr, A. Y. Rudensky. 2005. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22: 329-341. [Medline]
  10. Cabarrocas, J., C. Cassan, F. Magnusson, E. Piaggio, L. Mars, J. Derbinski, B. Kyewski, D. A. Gross, B. L. Salomon, K. Khazaie, et al 2006. Foxp3+ CD25+ regulatory T cells specific for a neo-self-antigen develop at the double-positive thymic stage. Proc. Natl. Acad. Sci. USA 103: 8453-8458. [Abstract/Free Full Text]
  11. Campbell, D. J., S. F. Ziegler. 2007. FoxP3 modifies the phenotypic and functional properties of regulatory T cells. Nat. Rev. Immunol. 7: 305-310. [Medline]
  12. Tang, Q., J. A. Bluestone. 2008. The Foxp3+ regulatory T cell: a jack of all trades, master of regulation. Nat. Immunol. 9: 239-244. [Medline]
  13. Wan, Y. Y., R. A. Flavell. 2007. "Yin-Yang" functions of transforming growth factor-β and T regulatory cells in immune regulation. Immunol. Rev. 220: 199-213. [Medline]
  14. Liston, A., A. Y. Rudensky. 2007. Thymic development and peripheral homeostasis of regulatory T cells. Curr. Opin. Immunol. 19: 176-185. [Medline]
  15. Bennett, C. L., J. Christie, F. Ramsdell, M. E. Brunkow, P. J. Ferguson, L. Whitesell, T. E. Kelly, F. T. Saulsbury, P. F. Chance, H. D. Ochs. 2001. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FoxP3. Nat. Genet. 27: 20-21. [Medline]
  16. Shevach, E. M.. 2002. CD4+ CD25+ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2: 389-400. [Medline]
  17. Gambineri, E., T. R. Torgerson, H. D. Ochs. 2003. Immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance (IPEX), a syndrome of systemic autoimmunity caused by mutations of FoxP3, a critical regulator of T-cell homeostasis. Curr. Opin. Rheumatol. 15: 430-435. [Medline]
  18. Pardoll, D.. 2003. Does the immune system see tumors as foreign or self?. Annu. Rev. Immunol. 21: 807-839. [Medline]
  19. Colombo, M. P., S. Piconese. 2007. Regulatory T-cell inhibition versus depletion: the right choice in cancer immunotherapy. Nat. Rev. Cancer 7: 880-887. [Medline]
  20. Radhakrishnan, S., L. T. Nguyen, B. Ciric, D. R. Ure, B. Zhou, K. Tamada, H. Dong, S. Y. Tseng, T. Shin, D. M. Pardoll, et al 2003. Naturally occurring human IgM antibody that binds B7-DC and potentiates T cell stimulation by dendritic cells. J. Immunol. 170: 1830-1838. [Abstract/Free Full Text]
  21. Radhakrishnan, S., K. R. Wiehagen, V. Pulko, V. Van Keulen, W. A. Faubion, K. L. Knutson, L. R. Pease. 2007. Induction of a Th1 response from Th2-polarized T cells by activated dendritic cells: dependence on TCR:peptide-MHC interaction, ICAM-1, IL-12, and IFN-{gamma}. J. Immunol. 178: 3583-3592. [Abstract/Free Full Text]
  22. Radhakrishnan, S., L. T. Nguyen, B. Ciric, D. Flies, V. P. Van Keulen, K. Tamada, L. Chen, M. Rodriguez, L. R. Pease. 2004. Immunotherapeutic potential of B7-DC (PD-L2) cross-linking antibody in conferring antitumor immunity. Cancer Res. 64: 4965-4972. [Abstract/Free Full Text]
  23. Radhakrishnan, S., K. Iijima, T. Kobayashi, M. Rodriguez, H. Kita, L. R. Pease. 2004. Blockade of allergic airway inflammation following systemic treatment with a B7-DC (PD-L2) cross-linking human antibody. J. Immunol. 173: 1360-1365. [Abstract/Free Full Text]
  24. Radhakrishnan, S., K. Iijima, T. Kobayashi, H. Kita, L. R. Pease. 2005. Dendritic cells activated by cross-linking B7-DC (PD-L2) block inflammatory airway disease. J. Allergy Clin. Immunol. 116: 668-674. [Medline]
  25. Van Keulen, V. P., B. Ciric, S. Radhakrishnan, K. L. Heckman, Y. Mitsunaga, K. Iijima, H. Kita, M. Rodriguez, L. R. Pease. 2006. Immunomodulation using the recombinant monoclonal human B7-DC cross-linking antibody rHIgM12. Clin. Exp. Immunol. 143: 314-321. [Medline]
  26. Pasare, C., R. Medzhitov. 2003. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 299: 1033-1036. [Abstract/Free Full Text]
  27. Kurts, C., W. R. Heath, F. R. Carbone, J. Allison, J. F. Miller, H. Kosaka. 1996. Constitutive class I-restricted exogenous presentation of self antigens in vivo. J. Exp. Med. 184: 923-930. [Abstract/Free Full Text]
  28. Nava-Parada, P., G. Forni, K. L. Knutson, L. R. Pease, E. Celis. 2007. Peptide vaccine given with a Toll-like receptor agonist is effective for the treatment and prevention of spontaneous breast tumors. Cancer Res. 67: 1326-1334. [Abstract/Free Full Text]
  29. Lucchini, F., M. G. Sacco, N. Hu, A. Villa, J. Brown, L. Cesano, L. Mangiarini, G. Rindi, S. Kindl, F. Sessa, et al 1992. Early and multifocal tumors in breast, salivary, harderian and epididymal tissues developed in MMTY-Neu transgenic mice. Cancer Lett. 64: 203-209. [Medline]
  30. Lachman, L. B., X. M. Rao, R. H. Kremer, B. Ozpolat, G. Kiriakova, J. E. Price. 2001. DNA vaccination against neu reduces breast cancer incidence and metastasis in mice. Cancer Gene Ther. 8: 259-268. [Medline]
  31. Fallarino, F., U. Grohmann, K.-W. Hwang, C. Orabona, C. Vacca, R. Bianchi, M. L. Belladonna, M. C. Fioretti, M. L. Alegre, P. Puccetti. 2003. Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 4: 1206-1212. [Medline]
  32. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, R. M. Steinman. 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176: 1693-1702. [Abstract/Free Full Text]
  33. Heckman, K. L., E. L. Schenk, S. Radhakrishnan, K. D. Pavelko, M. J. Hansen, L. R. Pease. 2007. Fast-tracked CTL: rapid induction of potent anti-tumor killer T cells in situ. Eur. J. Immunol. 37: 1827-1835. [Medline]
  34. Xystrakis, E., A. S. Dejean, I. Bernard, P. Druet, R. Liblau, D. Gonzalez-Dunia, A. Saoudi. 2004. Identification of a novel natural regulatory CD8 T-cell subset and analysis of its mechanism of regulation. Immunobiology 104: 3294-3301.
  35. Blocki, F. A., S. Radhakrishnan, V. P. Van Keulen, K. L. Heckman, B. Ciric, C. L. Howe, M. Rodriguez, E. Kwon, L. R. Pease. 2006. Induction of a gene expression program in dendritic cells with a cross-linking IgM antibody to the co-stimulatory molecule B7-DC. FASEB J. 20: 2408-2410. [Abstract/Free Full Text]
  36. Bettelli, E., Y. Carrier, W. Gao, T. Korn, T. B. Strom, M. Oukka, H. L. Weiner, V. K. Kuchroo. 2006. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441: 166-168. [Medline]
  37. Yamagiwa, S., J. D. Gray, S. Hashimoto, D. A. Horwitz. 2001. A role for TGF-β in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood. J. Immunol. 166: 7282-7289. [Abstract/Free Full Text]
  38. Fantini, M. C., C. Becker, G. Monteleone, F. Pallone, P. R. Galle, M. F. Neurath. 2004. Cutting edge: TGF-β induces a regulatory phenotype in CD4+CD25– T cells through Foxp3 induction and down-regulation of Smad7. J. Immunol. 172: 5149-5153. [Abstract/Free Full Text]
  39. Chen, W., W. Jin, N. Hardegen, K. J. Lei, L. Li, N. Marinos, G. McGrady, S. M. Wahl. 2003. Conversion of peripheral CD4+CD25– naïve T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3. J. Exp. Med. 198: 1875-1886. [Abstract/Free Full Text]
  40. Harrington, L. E., R. D. Hatton, P. R. Mangan, H. Turner, T. L. Murphy, K. M. Murphy, C. T. Weaver. 2005. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat. Immunol. 6: 1123-1132. [Medline]
  41. Park, H., Z. Li, X. O. Yang, S. H. Chang, R. Nurieva, Y. H. Wang, Y. Wang, L. Hood, Z. Zhu, Q. Tian, C. Dong. 2005. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat. Immunol. 6: 1133-1141. [Medline]
  42. Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, B. Stockinger. 2006. TGFβ in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing cells. Immunity 24: 179-189. [Medline]
  43. Mangan, P. R., L. E. Harrington, D. B. O'Quinn, W. W. Helms, D. C. Bullard, C. O. Elson, R. D. Hatton, S. M. Wahl, T. R. Schoeb, C. T. Weaver. 2006. Transforming growth factor-β induces development of the TH17 lineage. Nature 441: 231-234. [Medline]
  44. Hori, S., T. Nomura, S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299: 1057-1061. [Abstract/Free Full Text]
  45. Sugimoto, N., T. Oida, K. Hirota, K. Nakamura, T. Nomura, T. Uchiyama, S. Sakaguchi. 2006. Foxp3-dependent and -independent molecules specific for CD25+CD4+ natural regulatory T cells revealed by DNA microarray analysis. Int. Immunol. 18: 1197-1209. [Abstract/Free Full Text]
  46. Weaver, C. T., L. E. Harrington, P. R. Mangan, M. Gavrieli, K. M. Murphy. 2006. Th17: an effector CD4 T cell lineage with regulatory T cell ties. Immunity 24: 677-688. [Medline]
  47. Weaver, C. T., R. D. Hatton, P. R. Mangan, L. E. Harrington. 2007. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu. Rev. Immunol. 25: 821-852. [Medline]
  48. Stockinger, B., M. Veldhoen. 2007. Differentiation and function of Th17 T cells. Curr. Opin. Immunol. 19: 281-286. [Medline]
  49. Reiner, S. L.. 2007. Development in motion: helper T cells at work. Cell 129: 33-36. [Medline]
  50. Xu, L., A. Kitani, I. Fuss, W. Strober. 2007. Cutting edge: regulatory T cells induce CD4+CD25–Foxp3– T cells or are self-induced to become Th17 cells in the absence of exogenous TGF-β. J. Immunol. 178: 6725-6729. [Abstract/Free Full Text]
  51. Bird, J. J., D. R. Brown, A. C. Mullen, N. H. Moskowitz, M. A. Mahowald, J. R. Sider, T. F. Gajewski, C. R. Wang, S. L. Reiner. 1998. Helper T cell differentiation is controlled by the cell cycle. Immunity 9: 229-237. [Medline]
  52. Gudmundsdottir, H., A. D. Wells, L. A. Turka. 1999. Dynamics and requirements of T cell clonal expansion in vivo at the single-cell level: effector function is linked to proliferative capacity. J. Immunol. 162: 5212-5223. [Abstract/Free Full Text]
  53. Richter, A., M. Lohning, A. Radbruch. 1999. Instruction for cytokine expression in T helper lymphocytes in relation to proliferation and cell cycle progression. J. Exp. Med. 190: 1439-1450. [Abstract/Free Full Text]
  54. Chang, J. T., V. R. Palanivel, I. Kinjyo, F. Schambach, A. M. Intlekofer, A. Banerjee, S. A. Longworth, K. E. Vinup, P. Mrass, J. Oliaro, et al 2007. Asymmetric T lymphocyte division in the initiation of adaptive immune responses. Science 315: 1687-1691. [Abstract/Free Full Text]
  55. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, L. H. Glimcher. 2000. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100: 655-669. [Medline]
  56. Yang, X. O., B. P. Pappu, R. Nurieva, A. Akimzhanov, H. S. Kang, Y. Chung, L. Ma, B. Shah, A. D. Panopoulos, K. S. Schluns, et al 2008. T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR{alpha} and ROR{gamma}. Immunity 28: 29-39. [Medline]
  57. Ivanov, I. I., B. S. McKenzie, L. Zhou, C. E. Tadokoro, A. Lepelley, J. J. Lafaille, D. J. Cua, D. R. Littman. 2006. The orphan nuclear receptor ROR{gamma}t directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126: 1121-1133. [Medline]
  58. Radhakrishnan, S., E. Celis, L. R. Pease. 2005. B7-DC cross-linking restores antigen uptake and augments antigen-presenting cell function by matured dendritic cells. Proc. Natl. Acad. Sci. USA 102: 11438-11443. [Abstract/Free Full Text]
  59. Pavelko, K. D., K. L. Heckman, M. J. Hansen, L. R. Pease. 2008. An effective vaccine strategy protective against antigenically distinct tumor variants. Cancer Res. 68: 2471-2478. [Abstract/Free Full Text]
  60. Radhakrishnan, S., L. T. Nguyen, B. Ciric, V. P. Van Keulen, L. R. Pease. 2007. B7-DC/PD-L2 cross-linking induces NF-{kappa}B-dependent protection of dendritic cells from cell death. J. Immunol. 178: 1426-1432. [Abstract/Free Full Text]

Related articles in The JI:

IN THIS ISSUE

The JI 2008 181: 2939-2940. [Full Text]  



This article has been cited by other articles:


Home page
 J. Immunol.Home page
J. M. Fletcher, R. Lonergan, L. Costelloe, K. Kinsella, B. Moran, C. O'Farrelly, N. Tubridy, and K. H. G. Mills
CD39+Foxp3+ Regulatory T Cells Suppress Pathogenic Th17 Cells and Are Impaired in Multiple Sclerosis
J. Immunol., December 1, 2009; 183(11): 7602 - 7610.
[Abstract] [Full Text] [PDF]

Home page
 J Mol Cell BiolHome page
Y. Y. Wan and R. A. Flavell
How Diverse--CD4 Effector T Cells and their Functions
J Mol Cell Biol, October 1, 2009; 1(1): 20 - 36.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
B. Baban, P. R. Chandler, M. D. Sharma, J. Pihkala, P. A. Koni, D. H. Munn, and A. L. Mellor
IDO Activates Regulatory T Cells and Blocks Their Conversion into Th17-Like T Cells
J. Immunol., August 15, 2009; 183(4): 2475 - 2483.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
X. Chen, R. Das, R. Komorowski, A. Beres, M. J. Hessner, M. Mihara, and W. R. Drobyski
Blockade of interleukin-6 signaling augments regulatory T-cell reconstitution and attenuates the severity of graft-versus-host disease
Blood, July 23, 2009; 114(4): 891 - 900.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
X. Yuan, M. J. Ansari, F. D'Addio, J. Paez-Cortez, I. Schmitt, M. Donnarumma, O. Boenisch, X. Zhao, J. Popoola, M. R. Clarkson, et al.
Targeting Tim-1 to overcome resistance to transplantation tolerance mediated by CD8 T17 cells
PNAS, June 30, 2009; 106(26): 10734 - 10739.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. D. Sharma, D.-Y. Hou, Y. Liu, P. A. Koni, R. Metz, P. Chandler, A. L. Mellor, Y. He, and D. H. Munn
Indoleamine 2,3-dioxygenase controls conversion of Foxp3+ Tregs to TH17-like cells in tumor-draining lymph nodes
Blood, June 11, 2009; 113(24): 6102 - 6111.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
G. Beriou, C. M. Costantino, C. W. Ashley, L. Yang, V. K. Kuchroo, C. Baecher-Allan, and D. A. Hafler
IL-17-producing human peripheral regulatory T cells retain suppressive function
Blood, April 30, 2009; 113(18): 4240 - 4249.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
X. Yuan, J. Paez-Cortez, I. Schmitt-Knosalla, F. D'Addio, B. Mfarrej, M. Donnarumma, A. Habicht, M. R. Clarkson, J. Iacomini, L. H. Glimcher, et al.
A novel role of CD4 Th17 cells in mediating cardiac allograft rejection and vasculopathy
J. Exp. Med., December 22, 2008; 205(13): 3133 - 3144.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
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 Myeloid Dendritic Cells
J. Immunol., December 1, 2008; 181(11): 7863 - 7872.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Related articles in The JI
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Radhakrishnan, S.
Right arrow Articles by Pease, L. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Radhakrishnan, S.
Right arrow Articles by Pease, L. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS