Inflammation-induced Id2 promotes plasticity in regulatory T cells

TH17 cells originating from regulatory T (Treg) cells upon loss of the Treg-specific transcription factor Foxp3 accumulate in sites of inflammation and aggravate autoimmune diseases. Whether an active mechanism drives the generation of these pathogenic ‘ex-Foxp3 TH17’ cells, remains unclear. Here we show that pro-inflammatory cytokines enhance the expression of transcription regulator Id2, which mediates cellular plasticity of Treg into ex-Foxp3 TH17 cells. Expression of Id2 in in vitro differentiated iTreg cells reduces the expression of Foxp3 by sequestration of the transcription activator E2A, leading to the induction of TH17-related cytokines. Treg-specific ectopic expression of Id2 in mice significantly reduces the Treg compartment and causes immune dysregulation. Cellular fate-mapping experiments reveal enhanced Treg plasticity compared to wild-type, resulting in exacerbated experimental autoimmune encephalomyelitis pathogenesis or enhanced anti-tumor immunity. Our findings suggest that controlling Id2 expression may provide a novel approach for effective Treg cell immunotherapies for both autoimmunity and cancer.


Supplementary Figure 1
Id2 is highly expressed in ex-Foxp3 TH17 cells.
a Microarray analysis (GEO accession code GSE48428) representing overlap of differentially expressed genes more in ex-Foxp3 TH17 cells and TH17 cells and less in iTreg cells than TH0 cells (2fold). b Gene Ontology (GO) analysis on 11 significant GO terms associated with 449 genes in which were normally suppressed by Foxp3 expression in iTreg cells while induced as absence of Foxp3 expression in ex-Foxp3 TH17 cells and TH17 cells. c Heat map of selected 19 target genes related to immune system and /or are involved in regulation of transcription from (b). d,e Validation of selected 19 target genes expression from two other independent studies (GEO accession code GSE80804;d, GSE60059;e).

Supplementary Figure 3
Effect of Id2 overexpression during TH17 and iTreg differentiation conditions. a A Schematic of retroviral transduction of naïve CD4 + T cells. Naïve CD4 + T cells that were activated in vitro were transduced with Empty-GFP RV or Id2-GFP RV on day 1 under each T helper (TH17 and iTreg) cell-differentiation conditions. The following day 2 after culture, GFP + cells were sorted and analyzed. b RT-qPCR analysis of T-bet, Gata3, Rorγt and Foxp3 mRNA expression between Empty RV and Id2 RV transduced T cells on day 3 post-infection during TH17 cell-differentiation conditions (n=3-5, per group). c Flow cytometry analysis of Rorγt expression in Empty RV or Id2 RV transduced (GFP + ) and nontransduced (GFP -) TH17 cells. d RT-qPCR analysis of the indicated cytokine encoding mRNA expression in Empty RV and Id2 RV transduced T cells after day 3 post-infection during iTreg cell-differentiation conditions. e Flow cytometry analysis of IL-17A, IL-17F and IL-22 from Empty RV or Id2 RV transduced CD4 + GFP + TH17 cells. NS=Non-Significant, * P < 0.05, ** P < 0.005 (Student's t-test). Data are representative two to three independent experiments (error bars, s.d.).

Supplementary Figure 4
Effect of Id2 overexpression concomitant to iTreg or TH17 differentiation.
a Naïve CD4 + T cells were sorted from wild-type C57BL/6 (B6) mice, and transduced with control vector (Empty RV) or vector encoding Id2 cDNA (Id2 RV) after 1 day activated by plate bound anti-CD3/28. Cells were then differentiated under iTreg or TH17 differentiation conditions. After 3 days cells were harvested and Id2 expression was measured by RT-qPCR. b,c Flow cytometry analysis of Foxp3 (b) expression and IL-17A, IL-17F and IL-22 (c) between Empty RV or Id2 RV transduced (GFP + ) and non-transduced (GFP -) iTreg cells. d, RT-qPCR analysis of mRNA expression for the indicated cytokines in Empty RV and Id2 RV transduced T cells sorted after 3 days post-infection followed by iTreg cell-differentiation. e,f Flow cytometry analysis of Rorγt (e) expression and IL-17A, IL-17F and IL-22 (f) in Empty RV or Id2 RV transduced (GFP + ) and non-transduced (GFP -) TH17 cells. g RT-qPCR analysis of the expressions of indicated cytokines in Empty RV and Id2 RV transduced T cells sorted after day 3 post-infection under TH17 cell-differentiation conditions. NS=Non-Significant, * P < 0.05, ** P < 0.005 (Student's t-test). Data are representative of two independent experiments (error bars, s.d.).

Supplementary Figure 6
Treg-specific ectopic expression of Id2 results in enhanced loss of Foxp3 expression from Treg cells, and not enhanced stability of promiscuous Foxp3 expressing naïve T cells.
a RT-qPCR analysis of Id2 mRNA expression given at different dose of IL-1β (upper) and IL-6 (lower) in iTreg cells. b Intracellular staining of IFN-γ, IL-17A and Foxp3 expression in W/O, IL-1β (20ng/ml), IL-6 (20ng/ml) and IL-1β+ IL-6 (20ng/ml) conditions for 2 days in iTreg cells. c ChIP-seq results for STAT3, IRF4 and BATF at the Id2 locus in TH0 and TH17 cells (GEO accession code GSM1004793). d Analysis of luciferase activity from six serial-deletion of Id2 promoterluciferase constructs transfected into HEK 293T cells with STAT3, IRF4 and BATF expressing vectors, respectively. (W/O; control). Luciferase activities were measured with the reporter activities normalized to renilla luciferase activity. ** P < 0.005 (Student's t-test). All data are representative three independent experiments with similar results (error bars, s.d.).

Supplementary Figure 8
Nucleotide sequences of the Foxp3 promoter and E2A binding sites.
a Sequences of Foxp3 promoter from -1741 to +1. Highly-putative E2A binding sites (score>10) are marked in blue, red and yellow (BS1, BS2 and BS3, respectively). Each ChIPprimers (F/R) were indicated, respectively. b Sequencing results of mutation of E2A binding sites (Mutants; BS1, BS2 and BS3) in the corresponding control (Foxp3-pGL4). The rectangle indicates the nucleotides comprising a major E2A binding sites (E-box site).

Supplementary Figure 9
Increased T cell infiltration and IL-17A expression in doxycycline-treated TetR-Id2 EmGFP mice.

Original western blot panels with gel makers for
Complete scanned gels for western blots shown in Figure 1c,1d and 1e. Western images are overlaid onto image containing MW markers. White dashed line identifies cropped region shown in respective figure.