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. 2008 Sep 1;181(5):3137-47.
doi: 10.4049/jimmunol.181.5.3137.

Reprogrammed FoxP3+ T regulatory cells become IL-17+ antigen-specific autoimmune effectors in vitro and in vivo

Suresh Radhakrishnan  1 , Rosalyn Cabrera, Erin L Schenk, Pilar Nava-Parada, Michael P Bell, Virginia P Van Keulen, Ronald J Marler, Sara J Felts, Larry R Pease
Affiliations

Reprogrammed FoxP3+ T regulatory cells become IL-17+ antigen-specific autoimmune effectors in vitro and in vivo

Suresh Radhakrishnan et al. J Immunol. .

Retraction in

Abstract

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 (DC(XAb)) can reprogram Tregs into T effector cells. Down-regulation of FoxP3 expression after either in vitro or in vivo Treg-DC(XAb) 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 TGFbeta, fail to suppress T cell responses, and gain the ability to produce IFN-gamma, IL-17, and TNF-alpha. The ability of IL-6(+) DC(XAb) and the inability of IL-6(-/-) DC(XAb) 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 DC(XAb) 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.

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Figures

FIGURE 1
FIGURE 1. Reversal of T cell tolerance in Her2/neu model by Treg depletion or B7-DC XAb
A, Treatment regimen wherein mice that express rat (r) Her2/neu as self antigen were immunized with a known immunogenic rHer2/neu peptide (p66) or control “irrelevant” peptide along with CpG adjuvant and (top) concomitant depletion of Treg with the anti-CD25 antibody PC61 or (bottom) in combination with B7-DC XAb treatment (s.c.=subcutaneous; i.p=intraperitoneal). Seven days after challenge with peptide, CD8+ T cells were isolated and assessed for IFNγ responses by ELISPOT (B, C) following stimulation with p66-pulsed P815 cells (open bars), the rHer2/neu+ parental tumor line TUBO (filled bars), a mouse breast cancer cell line transfected with rHer2neu, A2L2 (hatched bars), or the mock transfected breast cancer cell line, 66.3neo (dotted bars). Data are representative of 3 or more independent experiments using at least 3 mice per group. All measurements were done in triplicate.
FIGURE 2
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:T cell mixtures (1:1). The DC were pulsed with chicken ovalbumin (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 (upper panels) and CD4+CD25+ Tregs (lower panels) after co-incubation with OVA-pulsed DCcntrl or DCXAb. C, Expression of FoxP3 in Tregs co-cultured 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 3 or more independent experiments.
FIGURE 3
FIGURE 3. B7-DC XAb induces antigen 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 antigen and treated with control antibody or B7-DC XAb (10 μg/ml) and then used to stimulate a 1:1mixture 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 top panel). Data are representative of 3 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 OVA and 10 μg control antibody or B7-DC XAb. After 48 hours, CD4+ CD25+ Tregs were isolated from pooled spleens. KJ1-26 positive (i.e., transferred DO11.10) and negative (i.e., BALB/c host) cells were gated as indicated and analyzed for FoxP3 expression. Data are representative of 3 or more independent experiments using at least 3 mice per antibody treatment group.
FIGURE 4
FIGURE 4. DCXAb induce Tregs to lose suppressor activity and to express effector cell cytokines
A, T cell proliferation was measured by [3H] incorporation. DO11.10 CD4+CD25- nonTregs or CD4+CD25+ Tregs were incubated with OVA-pulsed and control antibody-treated DC (left panel). Non-Tregs proliferated when stimulated with DCcntrl (filled symbols) and Tregs did not (open symbols). Right panel: mixing 3×105 CD4+CD25- T cells (non-Tregs) with increasing numbers of CD25+ Tregs in the presence of 0.5×106 ovalbumin-pulsed DCcntrl (open symbols) inhibited proliferation but not if in the presence of ovalbumin-pulsed DCXAb (filled symbols). B, DO11.10 CD4+CD25+ Tregs were cultured with OVA-pulsed DC treated with control antibody or B7-DC XAb for 48 h. Cytokine profiles of CD4+ Tregs were characterized by intracellular staining and flow cytometry. C, Co-expression of effector cytokines IFNγ and TNFα or IFNγ and IL-17 in Tregs after conversion to CD4+FoxP3- cells. D, TFGβ1 levels from OVA-pulsed DC:Treg interactions were determined by ELISA using supernatants from 48 hr co-cultures. Measurements were done in triplicate. All data are representative of at least 3 independent experiments.
FIGURE 5
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 prior to stimulation with DC. C, Top panels: DO11.10 non-Tregs (left) or Tregs (right) were labeled with CFSE 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. Middle panels: analysis of FoxP3 and L-17 expression in recovered CFSE+ DO11.10 Tregs stimulated with OVA-pulsed DCcntrl (filled histograms) or DCXAb (open histograms). Bottom panels: analysis of FoxP3 and L-17 expression in recovered CFSE+ DO11.10 nonTregs. All data are representative of at least 3 independent experiments.
FIGURE 6
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 3 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 soluble OVA and 10 μg isotype control antibody or B7-DC XAb. FoxP3 levels were measured in recovered splenocytes 48 hours after adoptive transfer, antigen, and antibody treatments. FACS plots represent CFSE+ cells (i.e., transferred cells) on x-axis and FoxP3+ cells on y-axis. CFSE negative cells represent endogenous cells. All data are representative of 3 or more independent experiments using at least 3 mice per treatment group.
FIGURE 7
FIGURE 7. IL-6 is not required for the induction of anti tumor 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 intravenously on days -1, 0, and +1 with 10 μg control antibody (○, □) or B7-DC XAb (●, ■). 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 3 or more independent experiments using at least 3 mice per treatment group.
FIGURE 8
FIGURE 8. T regs reprogrammed by DCXAb become autoimmune effectors in immune diabetic model
Ovalbumin pulsed DCXAb or DCcntrl (2×106) derived from WT or IL-6-/- mice were adoptively transferred (i.p.) into RIP-Ovalo or RIP-Ovahi mice. Recipient mice were monitored regularly for hyperglycemia. A, Colored lines showing the blood sugar profiles of individual RIP-OVAhi 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-OVAlo mice (top rows at low and high magnification) and from RIP-OVAhi mice (bottom rows at low and high magnification) are shown. C, Same as in (A) except RIP-OVAhi mice received 2 × 106 CD4+CD25+ OT-II T cells pre-treated with OVA-pulsed DCXAb or OVA-pulsed DCcntrl. In addition, some mice received 2.5 × 105 naive OT-l 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 3 or more independent experiments using at least 3 mice per treatment group. See Table III for statistical comparisons of the treatment groups.
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