Madhav D. Sharma, De-Yan Hou, Babak Baban, Pandelakis A. Koni, Yukai He, Phillip R. Chandler, Bruce R. Blazar, Andrew L. Mellor, and David H.

Transcription

1 Immunity, Volume 33 Supplemental Information Reprogrammed Foxp3 + Regulatory T Cells Provide Essential Help to Support Cross-presentation and CD8 + T Cell Priming in Naive Mice Madhav D. Sharma, De-Yan Hou, Babak Baban, Pandelakis A. Koni, Yukai He, Phillip R. Chandler, Bruce R. Blazar, Andrew L. Mellor, and David H. Munn - 1 -

2 - 1 - Supplemental Figure S1

3 Supplemental Figure S1 (continued) Supplemental Figure S1 (related to Figure 1). Analysis of Foxp3 expression during Treg cell reprogramming. (A) Loss of detectable immunoreactive Foxp3 by intracellular FACS staining following vaccineinduced reprogramming of Treg cells in vivo. Consistent with previous reports by Rudensky and colleagues (Rubtsov et al., 2010), we found that Foxp3 expression was highly stable in Foxp3 GFP mice, and GFP fluorescence was not lost even after functional alteration (reprogramming) of Treg cells. Resting Foxp3 GFP mice were analyzed without treatment (left-hand plots), or after receiving adoptive transfer of OT-I cells followed by footpad immunization with OVA+CpG+IFA vaccine containing graded doses of CpG, as shown in Manuscript Figure 1E. Four days later, popliteal LNs were harvested. In all groups, cells were fixed, permeabilized and stained for Foxp3 using the ebioscience Foxp3 staining kit, with APC-conjugated anti-foxp3 antibody clone JFK-16 (ebioscience). The gated CD4 + GFP + Treg cell population is shown (a representative gate is shown in the first panel). Before vaccination, approximately 90% of GFP + cells expressed Foxp3 by intracellular FACS staining. After vaccination, the proportion of GFP + cells that stained with the Foxp3 antibody progressively decreased with increasing doses of CpG in - 2 -

4 the vaccine. CpG also drove progressively more reprogramming (seen as the population of CD40Lexpressing cells emerging in the Foxp3 GFP Treg cells). Even through Foxp3 became undetectable by conventional immunofluorescent antibody staining in many of the cells, they still retained fluorescence of the Foxp3 GFP fusion protein, as shown in the upper row of dot-plots. In some cases, a transient dualpositive population could be seen (CD40L + and staining with Foxp3 antibody, as in the group receiving 25 ug of CpG), but this was evanescent, and in most cases Foxp3 simply became undetectable by FACS staining. This loss of detectable immunoreactive Foxp3 by intracellular FACS staining was reproducibly observed in the reprogrammed Treg cells in over 15 experiments, using multiple different markers to define the reprogrammed population (IL-2, IL-17, TNFα or CD40L), and using multiple different commercial anti-foxp3 antibodies. (B) In vitro culture model, confirming loss of detectable Foxp3 by intracellular FACS staining after Treg cell reprogramming in vitro. Treg cells were induced to undergo reprogramming using an in vitro culture system (described in detail in ref. (Sharma et al., 2009)). In this system, Treg cells can be driven to undergo extensive reprogramming, with essentially complete loss of detectable Foxp3 by FACS staining. The same starting population of Treg cells can be induced to undergo reprogramming or not, depending on whether IDO is blocked or active during the co-cultures (similar to the in vivo model, as shown in manuscript Figure 4A). Resting Treg cells were isolated by FACS sorting from spleens of Foxp3 GFP knock-in mice, and activated in co-cultures comprising IDO-expressing pdcs isolated from tumor-draining LNs of B16F10 tumors, plus OT-I CD8 + T cells and SIINFEKL antigen peptide, plus a feeder layer of T cell-depleted spleen cells to maintain viability. Co-cultures were incubated for 2 days with or without 1MT, then harvested and stained for CD4 vs. intracellular Foxp3. (In these cultures, the sorted Treg cells were the only CD4 + cells present, so identification was unambiguous.) The dot-plots show the Treg cell gate (CD4+GFP+ cells) analyzed for GFP vs. immunoreactive Foxp3 by intracellular staining (similar to panel A). When IDO was blocked (i.e., Treg cells allowed to reprogram) essentially all Treg cells could be driven to lose detectable Foxp3 by intracellular FACS staining. Loss of detectable Foxp3 staining was observed with 2 different commercial antibodies (clones JFK-16 and NNRF-30, both from ebioscience, using the ebioscience fixation-permeabilization reagents according to the manufacturer s protocol). Loss of detectable Foxp3 was confirmed in >10 experiments, using both Foxp3 GFP Treg cells and WT B6 Treg cells. Despite this, however, the reprogrammed Treg cells remained clearly GFP positive, as shown. (Inset, immunoblot) Native Foxp3 remains detectable in Treg cells by immunoblot. Co-cultures were performed as shown in the previous panel, using Treg cells from wild-type C57BL/6 mice (sorted as CD4 + CD25 + cells). Cultures were incubated for 2 days, with and without 1MT, then the CD4 + population harvested, stained and sorted by FACS. (In these cultures, the original sorted CD4 + CD25 + cells were the only CD4 + cells present, so they could be unambiguously identified based on CD4 expression, irrespective of any change in CD25 or other markers). Sorted cells were resolved by SDS-PAGE gel electrophoresis, transferred to PVDF membranes, and Foxp3 detected by immunoblotting using polyclonal rabbit antibody (Abcam, Cat. # ab54501), diluted 1:2000. Blots were incubated overnight in primary antibody at 4 o C, then developed with secondary antibody and chemiluminescence detection as described (Hou et al., 2007). The unchanged Foxp3 band on immunoblot, despite the loss of detectable signal by intracellular staining on flow cytometry, suggested that Foxp3 was physically present, but became either immunologically masked (e.g., through modification or complex formation), or physically sequestered in an intracellular location that was not accessible to the usual intracellular staining protocol. Foxp3 is known to be subject to a number of posttranslational modifications, and it also can be bound in multi-unit macromolecular complexes, some of which are inactive as transcriptional repressors (Zhou et al., 2009b). (C) Characterization of F1(Foxp3-GFP-cre ROSA26-YFP) reporter mice. Spleen cells were harvested from F1(Foxp3-GFP-cre ROSA26-YFP) mice, stained for surface CD4, then sorted as live cells into GFP-YFP positive and negative fractions as shown above. For sorting, both GFP and YFP fluorescence were combined in the green channel (530/40 band-pass filter). After sorting, each - 3 -

5 population was then fixed, permeabilized and stained for intracellular Foxp3. (The GFP-cre and YFP fluorescent markers were sensitive to the fixation-permeabilization step that we used for this Foxp3 staining step, so staining was performed after sorting the live cells, as previously described by Bluestone and colleagues (Zhou et al., 2009a).) Foxp3 staining reagents were from BD-Pharmingen as follows: # (Fixation & Permeabilization reagents); # (anti-cd4 PerCP-Cy5.5); # (anti- Foxp3 Alexa Fluor-647). One of 3 experiments. (D) Resolution of GFP and YFP populations (gated CD4 + cells). F1(Foxp3-GFP-cre ROSA26-YFP) mice received OT-I adoptive-transfer and vaccination with OVA+CpG+IFA as in manuscript Figure 1D. After 4 days, cells from vaccine-draining LN were treated with low-dose PMA+ionomycin as described in Supplemental Methods, below, and stained for surface CD4-PerCP vs. intracellular IL-2-APC. Treg cell analysis was performed on a Becton Dickinson FACSCanto I flow cytometer. A special filter set was used (Omega Optical, Brattleboro, VT) comprising a 510/21 nm bandpass optical filter for egfp emission, and a 550/30 nm bandpass optical filter for eyfp emission and a 525 nm long-pass dichroic optical filter to split the two signals. These filters were used in place of the FITC and PE optical filters, respectively. The dichroic filter was placed in front of the eyfp bandpass filter. There was Treg cell reprogramming (upregulation of IL-2 expression) in the GFP-low, GFP-high and GFP-YFP dual-positive populations. (All FACS-plots show gated CD4 + cells.) (E) Intact host MyD88 and IL-6 pathways are required for normal vaccine-induced reprogramming of Foxp3 + Treg cells. Congenically-marked (Thy1.1 + ) Treg cells were sorted from Foxp3 GFP Thy1.1 donors and transferred into host mice with a disruption of either MyD88 or IL-6 genes (or into WT B6 hosts as controls). All mice then received OT-I adoptive-transfer and vaccination with OVA+CpG+IFA. Four days later, the transferred Treg cells in vaccine-draining LNs (identified as Thy1.1 +, as shown in the gating example) were analyzed for markers of re-programming (CD40L upregulation in this experiment). One of 3 similar experiments

6 - 5 - Supplemental Figure S2

7 Supplemental Figure S2 (continued) C - 6 -

8 Supplemental Figure S2 (continued) D - 7 -

9 Supplemental Figure S2 (continued) E - 8 -

10 Supplemental Figure S2 (related to Figure 2). Help from reprogrammed Treg cells is needed in naive mice to support cross-presentation of a new antigen to CD8 + T cells; whereas conventional, non-treg CD4 + T cells can provide help when present at high frequency, or when pre-immunized with cognate antigen. (A) Activation of the CD8α + DC subset by reprogrammed Treg cells. Cross-presentation of antigens to CD8 + T cells is critically dependent on antigen presentation by the CD8α + subset of DCs (Hildner et al., 2008). To ask whether CD8α + DCs were activated by re-programmed Treg cells, Tcra -/- host mice received adoptive transfer of either sorted Foxp3 GFP Treg cells or sorted non-treg cells (CD4 + GFP ) as in manuscript Figure 2B. A third group received no CD4 + cells but were treated with FGK45 antibody (250 ug i.p. on the day of vaccination and 100 ug i.p. 2 days later) to artificially cross-link CD40. One day later all mice received OT-I and OVA+CpG+IFA vaccination; then after 4 days the draining LNs were analyzed for CD8α + DC activation (plots show gated CD11c + population). Results for CD80 expression is shown; similar results were obtained for CD86, not shown. Treg cells supported extensive activation of CD8α + DCs (comparable to induced CD40 cross-linking), whereas non-treg CD4 + cells did not support activation of DCs. Representative of 3 experiments. (B) Titratable helper activity of reprogrammed Treg cells in CD40L-deficient hosts. Experiments were performed using adoptive transfer into helper-deficient Cd40lg -/- hosts, as described in manuscript Figure 2D. Cd40lg -/- hosts received graded numbers of sorted wild-type Foxp3 GFP Treg cells as shown, or wild-type non-treg cells (CD4 + GFP ). Mice then received CFSE-labeled OT-I cells (congenically marked with Thy1.1) and were immunized with OVA+CpG+IFA vaccine. Proliferative response and granzyme B expression by OT-I cells were analyzed in vaccine-draining LNs on day 4. Representative of 3 similar experiments. (C) The conventional, non-treg CD4 + cell fraction acquires the ability to provide help for CD8 + T cells only after mice are pre-immunized with cognate antigen. Foxp3 GFP donor mice were immunized with OVA protein in CpG+IFA, followed by 2 booster immunizations. Tcra -/- recipient mice then received adoptive transfer of sorted Treg cells ( CD4 + GFP + ) or non-treg cells ( CD4 + GFP ) from the pre-immunized donors; control Tcra -/- mice received Treg cells or non-treg cells from naive Foxp3 GFP donors (not pre-immunized with OVA). Two days after transfer, all mice received CFSElabeled OT-I cells (CD8 + ) and immunization with OVA+CpG+IFA. Responses were analyzed in vaccine-draining LNs after 4 days. Upper dot-plots show CFSE dilution and granzyme B expression by gated OT-I cells. Lower dot-plots show expression of pro-inflammatory cytokines by the transferred CD4 + cells following activation with low-dose PMA+ionomycin in vitro. In naive donors, only the Treg cell fraction could upregulate pro-inflammatory cytokines and provide help for OT-I. In contrast, if the donors had been pre-immunized with OVA, then both the Treg cell and the non-treg cell fraction now contained cells capable of expressing pro-inflammatory cytokines after OVA vaccine, and both populations supplied functional helper activity. One of 3 identical experiments. (D) The non-treg CD4 + cell fraction of OVA-specific OT-II cells can provide cognate help for OT-I cells (but wild-type non-treg CD4 + cells cannot). OT-II mice (TCR-transgenic CD4 +, specific for a peptide of OVA presented on IA b ) (Barnden et al., 1998) were crossed to Foxp3 GFP mice to produce double-hemizygous (Foxp3 GFP+ OT-II + ) donors. Tcra -/- host mice received pre-adoptive transfer of sorted Treg cells (CD4 + GFP + ) or non-treg cells (CD4 + GFP ) from OT-II-Foxp3 GFP donors (range of transfer day -1 to day -7; timing was not critical). Control mice received either no CD4 + cells, or polyclonal Treg cells and non-treg cell fraction from Foxp3 GFP mice (without the OT-II transgene). Mice were rested for 1-7 days, then received CFSE-labeled OT-I cells (CD8 + ) and immunization with OVA+CpG+IFA. Responses were analyzed in draining LNs after 4 days. Plots show CFSE dilution (upper) and granzyme B expression by gated OT-I cells (lower). In the case of non-transgenic Foxp3 GFP donors (polyclonal CD4 + repertoire) only the Treg cell fraction could provide help for OT-I; while non-treg CD4 + cells - 9 -

11 could not. In contrast, in the case of OVA-specific OT-II-Foxp3 GFP mice, both Treg cells and non-treg CD4 + cells could provide help. One of 3 experiments. (E) Antigen-experienced (CD62L lo CD44 hi ) non-treg CD4 + cells are unable to provide spontaneous help for CD8 + T cells, unless they have been pre-activated in vivo. The previous two figures showed that conventional, non-treg CD4 + cells were able to provide spontaneous help for priming CD8 + cells, but only under certain circumstances (e.g., when they were pre-immunized with cognate antigen, or were TCR-transgenic for cognate antigen). We hypothesized that one factor limiting their ability to provide spontaneous help might be the fact that most non-treg CD4 + cells are naive, and only a small subset are antigen-experienced. To test whether this antigen-experienced (memory-phenotype) subset of non-treg cells might be better able to mediated spontaneous help in our model, we sorted the CD62L lo subset of non-treg CD4 + cells from untreated (resting) F1(Foxp3-GFP-cre R26-YFP) donor mice. The majority of CD62L lo cells were also CD44 hi, as shown, consistent with a non-naive phenotype. As positive control cells, the Treg cell fraction (CD4 + GFP + ) was sorted from the same untreated (resting) donors. To assess the additional effect of nonspecific exposure to CpG adjuvant, some donor mice were extensively pretreated with an irrelevant vaccine (comprising a pure CD8 epitope of gp100 with 50 ug CpG in IFA). Each donor received 5 simultaneous doses of vaccine at widely spaced sites (total dose of 250 ug CpG), followed 3 days later by similar booster immunization at 5 separated sites (total dose of 250 ug CpG). One week after boosting, the CD62L lo non-treg CD4+ cells were sorted. Equal numbers ( ) of sorted cells from all 3 donor groups were adoptively transferred into Tcra -/- recipients. One day after transfer, all mice received CFSE-labeled OT-I cells (sorted CD8 + ) and were immunized with OVA+CpG+IFA vaccine. Responses were analyzed in vaccine-draining LNs after 4 days. The bottom row of dot-plots shows that adoptive transfer of resting Treg cells provided help for OT-I, as expected. In contrast, resting non-treg cells, even when sorted specifically for the CD62L lo antigen-experienced cells, failed to provide spontaneous help. However, when donor mice had been extensively pre-treated with CpG, then the CD62L lo non-treg CD4 + cells now became able to mediate spontaneous help. One of 3 similar experiments, using both Foxp3 GFP and F1(Foxp3-GFP-cre R26-YFP) donor mice

12 Supplemental Figure S3 Supplemental Figure S3 (related to Figure 4). Treg cell reprogramming is not unique to CpG To ensure that our results were not unique to the CpG-1826 preparation, vaccines were compared using CpG-1826 (class B) and CpG-2395 (class C) (Vollmer et al., 2004). Tumor-bearing C57BL/6 mice were vaccinated with each preparation in combination with 1MT, using the protocol shown in manuscript Figure 4A. Four days later, TDLNs were analyzed for expression of IL-17, IL-2 and surface CD40L in gated CD4 + CD25 + Treg cell population (following in vitro stimulation with low-dose PMA+ionomycin). Both the class B and class C oligonucleotides drove extensive re-programming of Treg cells

13 Supplemental Figure S4

14 Supplemental Figure S4 (related to Figure 5). Rigorous depletion of Treg cells in tumor-bearing hosts enhances anti-tumor immune response, but abrogates the beneficial effect of 1MT. Foxp3-GFP-cre mice, bearing an EGFP-cre fusion protein under the Foxp3 promoter (the same parent strain as used in manuscript Figure 1D and in Supplemental Figure S1C), were mated with mice bearing a human diphtheria-toxin receptor (DTR) transgene behind a floxed STOP codon, under the ROSA26 promoter (R26-DTR mice) as described in Supplemental Methods. The F1 offspring, bearing DTR selectively on Treg cells, were treated with diphtheria toxin (DT), 1 ug/dose i.p., as described (Kim et al., 2007). Treatment with 4 doses of DT, as shown, resulted in >95% ablation of Treg cells, which is consistent with previous reports (Kim et al., 2007). This cre-lox system was chosen so that Treg cells could not escape ablation even if they had down-regulated their level of Foxp3 expression (since the crelox system ensured sustained expression of DTR in all cells that had ever been Foxp3 + ). (A) F1(Foxp3-GFP-cre R26-DTR) mice were implanted with B16F10 tumors, then treated with DT or vehicle control, beginning on day 5 as shown. Sub-groups received either 1MT in drinking water, or vehicle control. On day 7, all mice were treated with vaccine plus CFSE-labeled pmel-1, as in manuscript Figure 5A. TDLNs were harvested on day 11. Ungated plots show CD4 vs GFP fluorescence (from the EGFP-cre transgene, which marks Treg cells). Gated dot-plots show staining for IL-2 vs IL-17 (following low-dose PMA+ionomycin treatment) for the gated CD4 + GFP + (Treg cell) population, and the gated CD4 + GFP (non-treg cell) population. Depletion of Treg cells allowed the emergence of some activated non-treg CD4 + cells expressing IL-17 and IL-2, but the reprogrammed Treg cell population was lost. (In the ungated plots, the CFSE-labeled pmel-1 cells are also seen as the CD4 cells in the green channel.) One of 3 experiments. (B) Using the same experimental design as in the previous panel, on day 11 TDLN cells were harvested and stained for intracellular granzyme B expression. Plots show gated pmel-1 cells. The right-hand bar graph shows the size of dissected tumors (product of xy diameters) on day 11 for each treatment group. Group comparisons were by two-way ANOVA with Tukey s post hoc test. Pooled results of 3 experiments

15 Supplemental Figure S5 Supplemental Figure S5 (related to Figure 6). CD40L-sufficient Treg cells (but not non-treg CD4 + cells) restore the helper defect in Cd40lg -/- mice with established tumors. Cd40lg -/- host mice received adoptive transfer of CD40L-sufficient Foxp3 GFP Treg cells or CD4 + GFP non-treg cells. (Both transferred CD4 + populations were from Foxp3 GFP -Thy1.1 congenic donors to permit subsequent tracking of Treg cells.) All mice were implanted with B16F10 tumors, then received pmel-1 and gp100+cpg+ifa vaccine as shown. All mice were treated with 1MT. Four days later, pmel-1 response was analyzed in tumor-draining LN. Cytokine expression was analyzed in the transferred CD4 + cells following stimulation with low-dose PMA+ionomycin; surface CD40L expression was analyzed immediately ex vivo without any stimulation. Representative of 3 experiments

16 Supplemental details of methods Reagents and 1MT For in vivo use, 1-methyl-D-tryptophan was supplied by NewLink Genetics Inc. (Ames, IA) and administered in drinking water at 2 mg/ml (or vehicle control) as described (Sharma et al., 2007). For in vitro studies (presented in the Supplemental Materials), both 1-methyl-D-tryptophan (Sigma catalog #452483) and 1-methyl-L-tryptophan (Sigma catalog #447439) were active as IDO inhibitors using tumor-draining LN pdcs. 1MT was dissolved in alkaline ph as described (Munn et al., 2005). CpG-1826 (phosphorothioate oligo 5 -TCCATGACGTTCCTGAGCTT-3 ) was synthesized based on the published sequence (Chu et al., 1997) by Tri-link Biotechnologies. Experiments shown in the manuscript used CpG-1826, but Treg cell reprogramming was a generalizable phenomenon, and similar results were seen with CpG-2395, and also with lentivirus-based vaccines in place of CpG, as described (Liu et al., 2009). Mouse strains TCR-transgenic OT-I mice (CD8 +, B6 background, recognizing the SIINFEKL peptide of ovalbumin on H2K b ) (Hogquist et al., 1994) were purchased from Jackson Laboratories (Bar Harbor, ME). GCN2-deficient Eif2ak4 -/- mice (Munn et al., 2005) (B6 background) were a generous gift from the laboratory of David Ron, New York University School of Medicine and maintained at our institution. Foxp3 GFP mice (Fontenot et al., 2005) were the gift of Alexander Rudensky and were inbred >5 generations on the B6 background. Pmel-1 mice (Overwijk et al., 2003), strain B6.Cg-Thy1 a /CyTg(TcraTcrb)8Rest/J, and Cd40lg -/- mice (Renshaw et al., 1994), strain B6.129S2- Cd40lg tm1imx /J, were obtained from Jackson Laboratories and bred in our colony. Tcra -/- (B6.129S2- Tcra tm1mom /J) and Il6 -/- (B6.129S2-Il6 tm1kopf /J) mice were obtained from Jackson Laboratories. MyD88- deficient mice (Myd88 -/- ), originally derived by Shizuo Akira and colleagues (Adachi et al., 1998), were obtained from Andrew Gewirtz at Emory University, with permission of Dr. Akira. OT-II mice (TCR-transgenic CD4 +, specific for a peptide of OVA presented on IA b ) (Barnden et al., 1998) were bred onto the Thy1.1 background (B6.PL-Thy1a/CyJ, Jackson) then crossed to Foxp3 GFP mice. Male F1 mice (double-hemizygous, Foxp3 GFP+ OT-II + ) were used as donors for all experiments. Mice transgenic for an EGFP-cre fusion protein under the Foxp3 promoter, derived by Bluestone and colleagues (Zhou et al., 2009a; Zhou et al., 2008) were obtained from Jackson Laboratories (NOD/ShiLt-Tg(Foxp3-EGFP/cre)1Jbs/J). These mice were crossed with reporter mice transgenic for YFP behind a floxed stop codon under the ROSA26 promoter (B6.129X1-Gt(ROSA)26Sor tm1(eyfp)cos /J, Jackson) (Srinivas et al., 2001). ROSA26-YFP mice were purchased inbred >6 generations on the B6 background, and were further inbred in our colony. The first-generation F1 (double-hemizygous) offspring were used for all experiments. The Foxp3-GFP-cre mice from Jackson were on the NOD background, but F1 offspring (NOD B6) are not diabetes-prone. Since the host F1 mice had one full K b haplotype from the ROSA-YFP mothers, they could present antigen to adoptively-transferred OT-I cells (also K b background), and were fully tolerant to the transferred OT-I (no host-versus-graft reaction) during the 4-day duration of the experiment. For DTR experiments, mice bearing a human diphtheria-toxin receptor (DTR) transgene behind a floxed STOP codon under the ROSA26 promoter (R26-DTR mice, (Buch et al., 2005)) were obtained from Jackson Laboratories (strain C57BL/6-Gt(ROSA)26Sor tm1(hbegf)awai /J). These mice were bred to the Foxp3-GFP-cre mice described above, to produce F1(Foxp3-GFP-cre R26-DTR) offspring expressing DTR selectively on Treg cells

17 FACS and intracellular cytokine staining with low-dose PMA Treg cells were fragile during staining, and we found that it was important to disaggregate LNs rapidly (by passing once through a 40 um mesh) and stain quickly using short incubation times. Our intracellular cytokine staining method was modified from the method of (Bettelli et al., 2006). In preliminary validation studies, we found that the usual commercial PMA+ionomycin reagents caused artifactual activation of large numbers of resting CD4 + T cells, which obscured the specific effect of vaccination on Treg cells. This nonspecific background could be minimized by using a lower concentration of PMA+ionomycin; under these conditions the high-responsive Treg cells continued to respond robustly, while the non-specific background was substantially reduced. When using a commercial PMA+ionomycin kit (BD-Pharmingen, Cat. #550583), titration studies showed that optimal background reduction was obtained by using one-tenth (1/10) the recommended concentration of the PMA+ionomycin reagent, supplemented with additional GolgiPlug reagent used at the recommended full-strength concentration. In other studies, cells were incubated for 4 hrs with 100 ng/ml PMA (Sigma, P8139) um ionomycin (Sigma, I0634) in the presence of brefeldin A (GolgiPlug, BD Bioscience). In all cases, concentrations were determined empirically for each reagent and supplier by preliminary validation studies, based on their ability to give good activation of Treg cells with low non-specific activation of other, resting CD4 + cells. In all cases, we found that nonspecific background was much lower when using the fixation-permeabilization reagent from ebioscience (Cat. # ), and this was used throughout. All cells were acquired on a BD FacsCalibur or FacsCanto with doublet discrimination. (Cells in Supplemental Fig. S2 were acquired as described.) Antibodies were obtained from BD-Pharmingen as follows: phospho-stat5 (clone 47) used with the manufacturer s reagents according to Phos-flow Protocol #3; CD69 (H1.2F3); CD4 (GK1.5); CD8α (53-6.7); CD80 (16.10A1); CD86 (GL1). Antibodies from ebioscience were as follows: anti-il-2 (JES6-5H4); anti-tnfα (MP6-XT22); anti-il-17a (17B7); anti-cd40l (MR1); anti-granzyme B (16G6); anti-foxp3 (clones JFK-16 and NNRF-30). Antibody against CXCR3 was from R&D Systems (220803). References Adachi, O., Kawai, T., Takeda, K., Matsumoto, M., Tsutsui, H., Sakagami, M., Nakanishi, K., and Akira, S. (1998). Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9, Barnden, M.J., Allison, J., Heath, W.R., and Carbone, F.R. (1998). Defective TCR expression in transgenic mice constructed using cdna-based alpha- and beta-chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76, Bettelli, E., Carrier, Y., Gao, W., Korn, T., Strom, T.B., Oukka, M., Weiner, H.L., and Kuchroo, V.K. (2006). Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441, Buch, T., Heppner, F.L., Tertilt, C., Heinen, T.J., Kremer, M., Wunderlich, F.T., Jung, S., and Waisman, A. (2005). A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration. Nat Methods 2, Chu, R.S., Targoni, O.S., Krieg, A.M., Lehmann, P.V., and Harding, C.V. (1997). CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Th1) immunity. J. Exp. Med. 186, Fontenot, J.D., Rasmussen, J.P., Williams, L.M., Dooley, J.L., Farr, A.G., and Rudensky, A.Y. (2005). Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22,

18 Hildner, K., Edelson, B.T., Purtha, W.E., Diamond, M., Matsushita, H., Kohyama, M., Calderon, B., Schraml, B.U., Unanue, E.R., Diamond, M.S., et al. (2008). Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity. Science 322, Hogquist, K.A., Jameson, S.C., Heath, W.R., Howard, J.L., Bevan, M.J., and Carbone, F.R. (1994). T cell receptor antagonist peptides induce positive selection. Cell 76, Hou, D.Y., Muller, A.J., Sharma, M.D., Duhadaway, J.B., Banerjee, T., Johnson, M., Mellor, A.L., Prendergast, G.C., and Munn, D.H. (2007). Inhibition of IDO in dendritic cells by stereoisomers of 1-methyl-tryptophan correlates with anti-tumor responses. Cancer Res. 67, Kim, J.M., Rasmussen, J.P., and Rudensky, A.Y. (2007). Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat Immunol 8, Liu, Y., Peng, Y., Mi, M., Guevara-Patino, J., Munn, D.H., Fu, N., and He, Y. (2009). Lentivector immunization stimulates potent CD8 T cell responses against melanoma self-antigen tyrosinaserelated protein 1 and generates antitumor immunity in mice. J Immunol 182, Munn, D.H., Sharma, M.D., Baban, B., Harding, H.P., Zhang, Y., Ron, D., and Mellor, A.L. (2005). GCN2 kinase in T cells mediates proliferative arrest and anergy induction in response to indoleamine 2,3-dioxygenase. Immunity 22, Overwijk, W.W., Theoret, M.R., Finkelstein, S.E., Surman, D.R., de Jong, L.A., Vyth-Dreese, F.A., Dellemijn, T.A., Antony, P.A., Spiess, P.J., Palmer, D.C., et al. (2003). Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells. J. Exp. Med. 198, Renshaw, B.R., Fanslow, W.C., 3rd, Armitage, R.J., Campbell, K.A., Liggitt, D., Wright, B., Davison, B.L., and Maliszewski, C.R. (1994). Humoral immune responses in CD40 ligand-deficient mice. J Exp Med 180, Rubtsov, Y.P., Niec, R.E., Josefowicz, S., Li, L., Darce, J., Mathis, D., Benoist, C., and Rudensky, A.Y. (2010). Stability of the regulatory T cell lineage in vivo. Science 329, Sharma, M.D., Baban, B., Chandler, P., Hou, D.Y., Singh, N., Yagita, H., Azuma, M., Blazar, B.R., Mellor, A.L., and Munn, D.H. (2007). Plasmacytoid dendritic cells from mouse tumor-draining lymph nodes directly activate mature Tregs via indoleamine 2,3-dioxygenase. J. Clin. Invest. 117, Sharma, M.D., Hou, D.Y., Liu, Y., Koni, P.A., Metz, R., Chandler, P., Mellor, A.L., He, Y., and Munn, D.H. (2009). Indoleamine 2,3-dioxygenase controls conversion of Foxp3+ Tregs to TH17-like cells in tumor-draining lymph nodes. Blood 113, Srinivas, S., Watanabe, T., Lin, C.S., William, C.M., Tanabe, Y., Jessell, T.M., and Costantini, F. (2001). Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 1, 4. Vollmer, J., Weeratna, R., Payette, P., Jurk, M., Schetter, C., Laucht, M., Wader, T., Tluk, S., Liu, M., Davis, H.L., and Krieg, A.M. (2004). Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. Eur. J. Immunol. 34, Zhou, X., Bailey-Bucktrout, S.L., Jeker, L.T., Penaranda, C., Martinez-Llordella, M., Ashby, M., Nakayama, M., Rosenthal, W., and Bluestone, J.A. (2009a). Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nat. Immunol. 10, Zhou, X., Jeker, L.T., Fife, B.T., Zhu, S., Anderson, M.S., McManus, M.T., and Bluestone, J.A. (2008). Selective mirna disruption in T reg cells leads to uncontrolled autoimmunity. J Exp Med 205, Zhou, Z., Song, X., Berezov, A., Li, B., and Greene, M.I. (2009b). Structural aspects of the FOXP3-17 -

19 regulatory complex as an immunopharmacological target. International immunopharmacology 9,