Introduction
In a functional immune system, the target and amplitude of immune responses are tightly controlled. Regulatory T cells (Tregs) are among the safeguards that prevent aberrant immune responses including those underlying autoimmunity, inflammatory diseases or allergy (1) . This critical function of Tregs is mediated by several suppressive mechanisms including production of anti-inflammatory cytokines (IL-10, TGF, IL-35, etc.), expression of co-inhibitory receptor signaling, and the disruption of effector cell growth and metabolism (2) . The expansion or enhancement of Tregs may be an effective means to enforce immune tolerance to transplanted grafts or restore homeostasis to patients suffering from autoimmune pathologies (3).
Introduction
Conversely, Treg depletion or inhibition approaches can be exploited to provoke effective antitumor immunity in the cancer setting. Therefore, a thorough understanding of the molecules and pathways important for the function of these cells can have broad therapeutic implications.
Introduction
The suppressive capabilities of Tregs are underpinned by a characteristic gene expression profile defined in large part by the transcription factor Foxp3. This forkhead/wing-helix family member works in concert with multiple co-regulator molecules (Eos, IRF-4, etc.) and a Tregspecific array of epigenetic modifications to shape the transcriptional landscape of Tregs (4) (5) (6) (7) (8) .
Introduction
This is typified by the stabilized expression of Treg-associated genes (e.g. Ctla4, Il2ra) and the silencing of genes associated with effector T cell lineages (e.g. Il2, Ifng). Stable expression of Foxp3 is not only characteristic of Tregs, it is also key for their identity and suppressive function.
Introduction
While the mechanisms controlling Foxp3 transcription are important in Treg induction and stability (9) , posttranslational regulation controls the stability and activity of the cellular Foxp3 protein pool and hence Tregs.. For example, multiple posttranslational modifications of Foxp3 have significant consequences for Treg function. Acetylation at specific lysine residues improves the stability of Foxp3 expression and enhances the association of Foxp3 with its target genes (10) (11) (12) . Phosphorylation of Foxp3 at distinct sites has been found to have positive and negative effects on Foxp3 activity and Treg functions (13) (14) .
Introduction
We and others have documented how ubiquitination affects the Foxp3 protein pool and the suppressive capacities of Tregs (15) (16) . Protein ubiquitination regulates diverse biological functions by virtue of both the modified target protein site and nature of the ubiquitin-to-ubiquitin linkages within the added polymer chains. For instance, the addition of ubiquitin monomers linked together at Lysine residue 48 (K48-linkage) often marks a target protein for degradation by the 26S proteasome (17) . Other types of ubiquitination, such as those involving K63-linked ubiquitin molecules, can be important for the activation of signaling molecules and protein trafficking events (18) (19) . Previously, we and others found that Foxp3 was subject to K48-linked polyubiquitination that mediated the transcription factor's degradation via the proteasome.
Introduction
Invoking or preventing this modification could effectively sabotage (15) or stabilize suppressive function (16) , respectively, in Tregs. However, the potentially diverse outcomes of Foxp3 ubiquitination and molecular pathways involved have been incompletely elucidated.
Introduction
Here we report a distinct pathway of K63-type Foxp3 ubiquitination capable of promoting Foxp3 nuclear localization, regulatory activity and Treg stability and function, mediated by the TNF receptor associated factor protein family member, TRAF6. Mice lacking TRAF6 specifically in Foxp3+ Tregs displayed compromised immune tolerance and robust immune activation at baseline compared to wild type controls (17) (18) (19) (20) (21) (22) (23) . But the mechanism of the regulation of TRAF6 to Foxp3 was not determined. We found such mice were resistant to aggressive melanomas accompanied by aberrant extranuclear accumulation of Foxp3 and robust anti-tumor immune responses. Furthermore, we show that the zinc finger and leucine zipper domains of Foxp3 and lysine residue 262 play a critical role in interacting with Traf6 and the process of K63-ubiquitination. Taken together, these results demonstrate a hitherto unknown, posttranslational mechanism controlling both the regulatory activity of Foxp3 and Treg suppressive potency and identify TRAF6 as a potential target for focused, tolerance-breaking immunotherapies.
Results
Traf6 is critical for enforcement of immune homeostasis by Tregs. TRAF6 plays a potentially significant role in Treg biology. In line with this notion, high levels of TRAF6 transcript were found in naïve CD4+ T cells differentiating into Tregs in vitro (induced or iTregs), but not those committing to other T helper lineages ( Fig.Fig. 1A ). Our previous study determined that miRNA-146b-5p increased TRAF6 expression in human tTregs (24) . We further found that nTregs freshly isolated from human peripheral blood expressed TRAF6 mRNA to a greater degree than their non-Treg CD4+ counterparts ( Fig.Fig. 1B) . This preferential expression of TRAF6 by multiple Treg subsets further implicates the E3 ligase as a key factor in the development and biology of these important suppressor cells.
Results
To further investigate the importance of TRAF6 for Treg identity and function, we explored the consequences of TRAF6-deficiency in vivo. Previously, global TRAF6 knockout was found to precipitate multi-organ autoimmunity (23) . Similarly, T cell-specific deletion of TRAF6 results in lymphoproliferative disease and systemic immune activation tied to T cells resistant to Treg suppression (25) . We set out to determine how much of this immune dysregulation stems from the absence of TRAF6 activity in Tregs by observing the consequences of Foxp3-driven TRAF6 Fig. 1C ). Increased cellularity was also noted in these lymphoid tissues in the absence of Treg-derived TRAF6 (Fig.Fig. 1D We next explored the effects of Treg-specific TRAF6 knockout on Treg differentiation. Prior work has shown that T cell-restricted TRAF6 deficiency does not impact in vitro, TGFβ-driven induction of Foxp3 expression (22) . Also, despite displaying stunted thymic Treg development, mice globally deficient in TRAF6 yield T cells more inclined to Foxp3 induction in vitro (23) . We found that in vitro activation of naïve CD4+ T cells from wild type and Traf6 fl/fl Foxp3Cre + mice in the presence of TGFβ and IL-2 resulted in similar Foxp3-induction, even with sub-optimal concentrations of TGFβ ( Fig. 1G and 1J ). However, in other experiments, addition of the proinflammatory, Th17-inducing cytokine, IL-6 to strongly Treg-inducing media (5 ng/ml TGFβ) resulted in disrupted iTreg skewing evidenced by low levels of Foxp3 induction. This was seen to an even greater degree when TRAF6 was deleted in the newly induced Tregs ( Fig. S2A and   2B (Fig. 3C) . Those lacking ectopic TRAF6 expression, however, showed only minor levels of Foxp3 polyubiquitination. Interestingly, expression of ubiquitin molecules possessing mutated K48 residues (K48R), which are incapable of supporting K48 polyubiquitination, still resulted in robust Foxp3 ubiquitination in this system. Constructs encoding ubiquitins having a lysine-toarginine (K-to-R) mutation at residue K63 (K63R), on the other hand, led to a marked reduction in Foxp3 ubiquitination as detected by anti-FLAG immunoblotting after Foxp3 pull-down. These findings demonstrate that TRAF6 plays an important role in facilitating Foxp3 ubiquitination, particularly in K63-type modifications, and they further suggest that such posttranslational modification may be prevalent under baseline conditions. This notion was confirmed in primary murine iTregs using a reciprocal endogenous TRAF6 and FoxP3 co-IP approach (Fig. 3D) .
Results
Importantly, we also found that the dysfunctional TRAF6-deficient Tregs displayed a relative dearth of K63-ubiquitinated Foxp3 species compared to normal Tregs ( Fig. 3E) , further implicating this modification of Foxp3 by TRAF6 as critical for optimal Treg function.
Results
The zinc finger and leucine zipper domains of Foxp3 and lysine residue 262 are necessary for interaction with Traf6 and ubiquitination. We then set out to further characterize the interaction between Foxp3 and TRAF6. To identify the TRAF6-interacting domain of Foxp3, we generated Foxp3 deletion mutants each lacking one or several of the recognized functional domains of the transcription factor ( Fig. S4A-C) . The ability of full-length Foxp3 and these deletion variants to interact with TRAF6 was determined by co-IP approaches.
Results
Molecules that contained intact Zinc-finger and Leucine zipper domains (full length Foxp3, N1, N2, and C3) readily pulled down TRAF6. Meanwhile those lacking these domains (N3, C1, and C2) failed to do so (Fig. S4D) . These revealed that the Zinc-finger and Leucine zipper domain of Foxp3 were indispensable for the interaction of TRAF6 with Foxp3.
Results
In order to identify potential target sites in the Foxp3 molecule subject to TRAF6-mediated ubiquitination, we also screened Foxp3 constructs with various K-to-R mutations for resistance to modification by this enzyme. As before prevalent ubiquitation was seen in the Foxp3 pool in the presence of TRAF6. As expected, we also found that mutating all twenty lysine residues We confirmed these results in primary murine CD4+ T cells by ectopically expressing either a normal Foxp3 construct or that encoding the K262R mutant (delivered by retroviral transduction), activating the recipient cells, and measuring IL-2 production by ELISA. (Fig. 5D, E) . Additionally, the dysfunction of TRAF6-deficient Tregs in our tumor experiments was found to be associated with this uncharacteristic distribution of were less effective suppressor cells (Fig. 6A, B) . We also explored the impact of K63 resistance on Treg function in vivo using a T cell-dependent mouse model of colitis. Here, adoptive transfer of naïve CD4+ T cells (Thy1.2+) into Rag2-/-mice induced gut inflammation and progressive wasting disease in recipients. Co-injection of normal Tregs along with the colitogenic T cells effectively prevented the development of disease. CD4+ Tregs transfected to express wild type Foxp3 were also largely protective in this model. K262R Foxp3 transductants, on the other hand, failed to prevent severe colitis and mice receiving these "engineered Tregs" resembled no-Treg controls in terms of weight loss. These trends were also seen in the degree of histopathology observed across these in the large bowel ( Fig. 6C-E (Fig. S7A) . Also the frequencies of these potentially inflammatory cells were consistently restrained in recipients of both isolated Tregs and lentiviral Foxp3 expressing cells ( Fig. S7B and C) . Reinforcing the notion that modification at K262 is important for stable Foxp3 expression, across the tissues involved, recipients of the K262R mutant saw marked loss of Foxp3 staining in the injected (Thy1.1+) "Treg" population. In contrast, most wild type Foxp3 transductants recovered in this experiment still expressed Foxp3
Results
( Fig. S7D-F) . These results further suggest that ubiquitination of Foxp3 at K262 is important for Treg function and stability. Additionally, since the overall numbers of K262R-carrying cells recovered from recipient mice were comparable to the wild type Foxp3 group (Fig. S7D) Recently Muto and colleagues reported on the consequences of Treg-specific TRAF6
Results
deficiency. In line with our own results they found Traf6 fl/fl Foxp3Cre + mice to be prone to dermatitis, lymphoproliferation and wide-spread immune pathologies (22) . Also, they found that
Results
Tregs from these mice were only modestly less suppressive in vitro than their wild type-derived counterparts, but phenotypically unstable and ineffectual suppressors in vivo (22, 37 we recently suggested positive relationship between TRAF6 expression and Treg function.
Results
We found that by antagonizing a specific microRNA (miR-146b), which targets TRAF6 transcript, they could enhance Foxp3 protein levels. Moreover, the in vitro and in vivo function of
Results
Tregs could be enhanced through a miR-146b antagomir (24) . Interestingly, while increased Additionally, it now appreciated that Foxp3 function is largely facilitated by a number of coregulator molecules such as Eos, IRF-4, GATA-3, and other members of the Foxp3
Results
"interactome" (4-6, 40). Posttranslational modification capable of influencing Foxp3 expression and activity are just now becoming clear (27) . Surprisingly, given the undisputed importance of In all, these findings reveal TRAF6 as a previously unappreciated posttranslational mediator of Foxp3 expression and activity in Tregs. They also provide insights into the molecular details of this distinct, non-proteolytic, and functionally enhancing brand of ubiquitination by TRAF6 including the specific modification site in the Foxp3 molecule.
Results
Importantly, this newly uncovered mode of control over Treg function may represent a target for future immunotherapies aiming to overcome immune suppression in cancer patients.
Materials and Methods
Mice. Traf6flox/flox mice on a C57BL/6 background were obtained from Dr. M Pasparakis (43).
Materials and Methods
Foxp3yfpCre transgenic mice (originally generated in the lab of Dr. Alexander Rudensky) and CD4Cre transgenic were obtained from the Jackson Laboratory. These mice were crossed to generate mice specifically lacking TRAF6 in their Tregs. Thy1.1+ and Thy1.2+ mice on a BALB/c background and CD45.1+ C57Bl/6 mice were purchased from Jackson laboratory. All mice were housed in a specific pathogen free facility in accordance with institutional guidelines.
Generation of mutant Foxp3 constructs. Single lysine and targeted residue mutant in Foxp3 constructs
were generated by Site-Directed Mutagenesis Kits (Thermo Fisher Scientific). Nonidet P-40, 0.5% Nadeoxycholate, 150 mM NaCl, 1 mM EDTA, with 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF and protease inhibitor (Sigma), followed by immunoprecipitation with the indicated antibodies, separation by SDS/PAGE, and analysis by Western blotting. Where applicable, band density indicating protein amount was quantified using Image J software.
Cell line transfections, Co

Retroviral transduction of primary T cells. Naïve T cells isolated from Wild type or Traf6fl/fl/CD4Cre+
mice were stimulated with plate-bound anti-CD3 (4μg ml-1) and soluble anti-CD28 (1μg ml-1) with 60U ml-1 human recombinant IL-2 for 16 h. Activated T cells were transduced with viral supernatants (carring empaty vector, Wt Foxp3 or Foxp3 mutant) supplemented with 60U ml-1 IL-2 and 8 μg ml-1 polybrene, followed by centrifugation for 1h at 2,500 rpm. Cells were cultured at 37ºC with 5% CO2 for an additional 48h and sorted out for ELISA analysis.
Retroviral transduction of primary T cells. Naïve T cells isolated from Wild type or Traf6fl/fl/CD4Cre+
T cell isolation and Treg suppression assays. Naïve CD4+ T cells from C57BL/6 mice were isolated from pooled lymph nodes and spleens by FACS as were congenically distinct Tregs (CD4+/CD25+) isolated from either wild type mice or the indicated conditional knockout strain prior to combination at the indicated ratios. Naïve T cells were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) and cocultured with equal numbers (2x10 iTregs were generated by activating naïve precursors in the presence of IL-2 (100u/ml) and the indicated concentrations of TGFβ.
Retroviral transduction of primary T cells. Naïve T cells isolated from Wild type or Traf6fl/fl/CD4Cre+
QRT-PCR and ELISA. RNA was isolated by a miniRNA extraction kit (QIAGEN). The cDNA archival kit (Applied Biosystem) was used per the manufacturer's instruction. Triplicate reactions were run using an ABI Prism 7500. mRNA levels were determined by comparative CT method and normalized to GAPDH expression. IL-2 production from the sorted T cell culture supernatants was measured by IL-2 Elisa kit.
Retroviral transduction of primary T cells. Naïve T cells isolated from Wild type or Traf6fl/fl/CD4Cre+
Immunofluorescence Microscopy. Cells were spun down on to microscope slides at 1800 rpm for 3 minutes at room temperature (RT). The slides were then fixed with 4% paraformaldehyde for 15 minutes at RT, then washed 3 times with PBS, incubated with 0.5% Triton-X100 for 5 minutes before washing again with PBS. The cells were then blocked with 1% BSA for 1 alyssa39@example.org. FITC-labeled anti-mouse Foxp3 antibodies were diluted 1:60 with PBS and incubated with the cells for 1 alyssa39@example.org. After washing the slides, DAPI was used as a nuclear counterstain. Images were obtained using an Eclipse E800 microscope equipped with a DS-Qi1Mc camera (Nikon) and NIS-Element AR 3.0 software. Leukocytes recovered from recipient lymph node, spleen and lamina propria were re-stimulated before surface staining for CD4, CD45.1 and CD45.2.
Retroviral transduction of primary T cells. Naïve T cells isolated from Wild type or Traf6fl/fl/CD4Cre+
Statistics. The significance of differences was determined by an unpaired student's t test, One-way ANOVA and Two-way ANOVA using Prism software (GraphPad). Differences were considered significant when P values were <0.05. 

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