© 2016 Nature America, Inc. All rights reserved. of T of number the restore and IL-2R or IL-2 in deficiency with associated T rescue can Bim protein cursors T of differentiating survival the in be might IL-2 of role chief a that possibility the raised finding That Bcl2. molecule anti-apoptotic the of amounts increased by restored be can it sion, expres Foxp3 STAT5 in abolishes deficiency Although understood. T mature in expression IL-2R of importance the T and sion T extrathymic TGF- cytokine the with cooperation in IL-2, Correspondence should be addressed to J.D.F. ( North Chicago, Illinois, USA (A.K.K.), and Exploratory Biology, Juno Therapeutics,California, Seattle, USA. Washington, USA (J.D.F.). University, New York, New York, USA. New York, USA. New York, New York, USA. 1 of Foxp3 expression to induction the contribute can also signaling mus T of differentiation and Foxp3 of expression inducing the for indispensable are IL-2R, with associated kinases JAK of STAT5, downstream factor target a key transcription the and IL-2 (ref. IL-2 with interact collectively subunits three these fold when and IL-2R CD132) IL-2R to affinity (IL-2R 2 receptor interleukin of the expression abundant have cells Foxp3 to immune restrain responses self and foreign antigens (T cells T Regulatory from signaling via the T cell antigen receptor. 2R-dependent activation of the factor transcription STAT5 had an essential role in the suppressor function of T IL-2 was dispensable for the control of CD4 signaling in the suppressor function of T factor Foxp3, lineage–specification which has confounded experimental efforts to understand the role of IL-2R expression and T produced by activated T cells. This feature indicates a key role for a simple network based on the consumption of IL-2 by Regulatory T cells (T GasteigerGeorg Takatoshi Chinen function An essential role for the IL-2 receptor in T nature immunology nature Received 9 December 2015; accepted 27 July 2016; published online 5 September 2016; Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, New York, USA. reg While While the role of in IL-2R signaling the of induction Foxp3 expres cells in their suppressor function. However, congenital deficiency in IL-2R results in reduced expression of the T reg 5– 1 cells, but it does not prevent fatal autoimmunity fatal prevent not does it but cells, α 1 1 . IL-2R . 4 ; CD25) but are unable to produce IL-2. IL-2 binds with low with binds IL-2 IL-2. produce to unable are but CD25) ; . It has also been reported . that of Itablation the pro-apoptotic reported been has also reg 5 cell differentiation in the thymus is well established, established, well is thymus the in differentiation cell 8 reg Department of Pathology and Cell Biology, Columbia University, New York, New York, USA. Institute for Medical Microbiology and Hygiene, University of Mainz Medical Centre, Mainz, Germany. β cell differentiation cell and and α β or to heterodimers of the common common the of heterodimers to or (CD122), but receptor affinity increases ~1,000- increases but affinity receptor (CD122), 1 , reg 2 reg 3 1 γ Immunology Discovery, Biogen, Cambridge, Massachusetts, USA. , , c 8 2

are shared with the IL-15 receptor, whose whose receptor, IL-15 the with shared are cells) that express the transcription factor factor transcription the express that cells) cells), which have abundant expression of the interleukin 2 receptor (IL-2R), are reliant on IL-2 , , Yongqiang Feng , 10 reg aDV , , Arun K Kannan cells or their precursors from apoptosis apoptosis from precursors their or cells A 7 NCE ONLINE PUBLIC ONLINE NCE Nomis Foundation Laboratories for Immunobiology and Microbial Pathogenesis, The Salk Institute for Biological Studies, La Jolla, 1 3 . [email protected] reg cells. Using genetic gain- and approaches, we loss-of-function found that capture of 1 β + , , is also required for for required also is , 2 T cells but was important for limiting the activation of CD8 reg , , Jason D Fontenot 3 reg reg , 9 cells or their pre or their cells A cells in the thy the in cells , cells is not well well not is cells 10 TION , , Andrew G Levine 1 γ 5

. However, . -chain ( -chain α 1– -chain -chain 3 . . T 4 γ 1 reg 2 ). ). c - - - - . ;

) or A.Y.R. ( potential biological importance of this IL-2-based T IL-2-based this of importance biological potential of STAT5 activation T in of autologous inhibition the Foxp3 expression, T limitations. aforementioned the to due established firmly been not have function of IL-2R aspects other while of Foxp3, mainte nance or induction the on mainly focused have studies Previous triggering of JAK–STAT5, PI3K–Akt or to Ras–ERK signaling pathways. due function or proliferation maintenance, their support to cells T in differentiated signaling IL-2 and cell-intrinsic of suppression; source of IL-2; T a as serve that cells T self-reactive activated are which targets, their T peripheral T cells that which suggests IL-2R expression is T in dispensable peripheral rescue to reported IL-2R encoding transgene a of T the of T in expression Foxp3 and T of number the reduces thymus the of have removal that undergone mice adult in IL-2 of neutralization Antibody-mediated (T T cells of and T selection the differentiation on Bim and Bcl2 IL-2, in deficiency congenital of effect profound a 3 , reg 9 Despite their considerable reliance on IL-2 for the maintenance of on maintenance for the IL-2 reliance considerable their Despite & Alexander Y Rudensky cells has remained uncertain. Hypothetically, a role for IL-2R in in IL-2R for role a Hypothetically, uncertain. remained has cells 7 4 , 1 Herbert Irving Comprehensive Cancer Center, Columbia University, New York, doi:10.1038/ni.354 1 1 [email protected] , . . Thus, a role for in peripheral and IL-2R expression signaling reg 2 , Xiying , FanXiying eff cell lineage after differentiation after lineage cell cells) has confounded interpretation of that observation. observation. of that interpretation confounded has cells) reg 2 Immunology Program, Memorial Sloan Kettering Cancer Center, cells could be threefold: guidance for T reg 6 Department of Microbiology and Immunology, Columbia cell–mediated deprivation of cell–mediated IL-2 as a mechanism reg Il2rb cells are unable to produce IL-2. The reason for 0 10 1 ). −/− , These authors contributed equally to this work. 2 9 Present addresses: Biotechnology, AbbVie, reg , , Ulf Klein mice from lethal autoimmune disease, disease, autoimmune lethal from mice cells β exclusively in thymocytes has been been has thymocytes in exclusively 16 1 , , reg 2 1 7 . Thus, IL-2 supports stability stability supports IL-2 Thus, . cells and self-reactive effector effector and cells self-reactive reg 4 + – T cells, and that IL- 6 18 , , Ye Zheng , 1 cell 9 . However, expression However,. expression reg cells separable ticles e l c i rt A reg reg reg reg cells to sense cells and the the and cells cell–T 7 cell ,

reg eff cells cells cell cell reg reg  -

© 2016 Nature America, Inc. All rights reserved. recipients (data not shown). not (data recipients suppressive when transferred together with T u IL-2R ‘activated’ the CD103, CTLA-4, and ICOS of receptor expression costimulatory the the GITR, of upregulation their Despite signals. conditions the latter was sustained to some extent by lymphoidIL-2R-independent tissues, were dependent on IL-2, although CD62L the under inflammatoryCD62L the both that suggested tions in of T result the as upregulated was Foxp3, and CD25 T characteristic many of expression the ( intestines large and small CD62L which at sites at mice T of frequency in lower much the also because inflammation, severe of T of 1a Fig. ( CD44 and CD62L of expression their IL-2R of IL-2R molecules ‘signature’ Foxp3 ( IL-2R CD62L CD62L both of abundance the that found we CD62L for dispensable is but cells CD62L of maintenance the for required selectively is ( under-represented were random inactivation of the X chromosome, IL-2R 2R ( in lower both CD4 T peripheral in lacking was IL-2 to response in STAT5 of tion Il2rb in lower was germline with Foxp3 mice in observed that than milder somewhat albeit lymphoproliferation, and lesions inflammatory autoimmune fatal after Foxp3 was expressed. ( 2R we mice generated with T T in IL-2R for role a establish Todefinitively T for indispensable is IL-2R RESULTS T in signaling IL-2 and expression IL-2R for requirements differential the assessed STAT5, of we form active an of expression inducing simultaneously IL-2R we ablated T differentiated in pathways signaling downstream and 2R suppression in role (refs. of T in role state ‘unbound’ the T on maintain IL-2R to high-affinity needed is that IL-2 of suggested been repression has It unknown. remain also loop regulatory s e l c i rt A  Supplementary Fig. 1a Fig. Supplementary Foxp3 Fig. 1 Fig.

n able to control inflammation in the diseased mice and were not not were and mice diseased the in inflammation control to able β β Il2rb reg -sufficient -sufficient T by using Cre recombinase driven by the endogenous + fl/wt 20– β T cells and the expression of Foxp3 on a per-cell basis were were basis per-cell a on Foxp3 of expression the and cells T β Cre/wt deficiency cells were CD62L were cells Cre f than in its presence in healthy ). Although in diseased diseased in Although ). lo dfcet T -deficient fl/fl ). In healthy healthy In ). Foxp3 reg CD44 β 2 ) to delete delete to ) 4 sfiin ( -sufficient Foxp3 cell–mediated suppression by depriving T depriving by suppression cell–mediated lo ) ) T ); ); however, whether this mechanism has a non-redundant reg CD44 reg reg Cre Il2rb Il2rb hi cells ( cells reg cell homeostasis versus T versus homeostasis cell T cells had lower expression of Foxp3 T and the expression lower had cells Cre 3 α counterparts ( counterparts ( reg cells and IL-2R hi , , IL-2R Fig. 1a Fig. mice ( mice fl/fl fl/fl reg T Il2rb in vivo in lox cells was significantly lower in the absence of of absence the in lower significantly was cells Fig. 1 Fig. Il2rb reg cells from from cells Foxp3 Foxp3 Il2rb P-flanked P-flanked cell subset, including those residing in non- residing those including subset, cell ). In these mice, IL-2R mice, these In ). lo fl/fl reg reg β Fig. 1g Fig. – fl/fl CD44 Supplementary Fig. 1c Fig. Supplementary Il2rb and STAT5 in Foxp3-expressing cells. By cells. STAT5and Foxp3-expressing in e is unknown. To address the role of IL- of role the address To unknown. is c cell–specific conditional knockout of conditional IL- cell–specific cells, and unbound IL-2R serves a key key a serves IL-2R unbound and cells, α fl/wt Foxp3 Cre ). The frequency of Foxp3 of frequency The ). Supplementary Fig. 1b Fig. Supplementary ). The expression of IL-2R expression ). The Cre Foxp3 , CTLA-4, GITR and CD103 than that that than CD103 and GITR CTLA-4, , lo reg CD44 mice than in in than mice Foxp3 eihrl T peripheral fl/fl Fig. 1 Fig. Il2rb hi , cell function cell h β , this was probably a consequence consequence a probably was this , Cre Il2rb Foxp3 Il2rb ). It has been suggested that IL-2 IL-2 that suggested been has It ). -deficient -deficient T Cre/wt lo mice than in in than mice hi fl/fl Il2rb Cre/wt CD44 d cells were prevalent—i.e., the the prevalent—i.e., were cells hi alleles ( alleles Fig. 1i Fig. ), and tyrosine-phosphoryla and ), fl/fl Foxp3 CD44 Cre female mice, in which IL- which in mice, female reg reg fl/fl Foxp3 T ) mice developed systemic systemic mice developed cell markers, except for for except markers, cell hi eff cell suppressor activity. suppressor cell reg Foxp3 T Il2rb reg hi cells into lymphopenic , Cre lo reg reg j cell function function cell CD44 and and Il2rb T reg β cells than in their their in than cells cells regardless of of regardless cells β mice, the majority majority the mice, cells co-exist due to Cre -deficient ( -deficient reg -deficient -deficient T cells fl/wt ). These observa These ). Cre/wt Il2rb reg mice were still still were mice Il2rb cell subset and and subset cell Supplementary Supplementary fl/fl lo ). Accordingly, Accordingly, ). eff cell activation activation cell Foxp3 T β + hi ) in T in ) 2 reg cells of IL-2 IL-2 of cells cells among among cells fl/wt female mice and IL-2R 5 CD44 reg Foxp3 fl/fl . However, However, . cells was was cells cells and and cells Foxp3 Foxp3 Cre reg Il2rb reg in vivo in reg reg lo locus locus cells, cells, mice mice cells cells cells T cell cell fl/fl Cre Cre reg α - - ,

fatal disease ( T mice and the consequent expression of STAT5b-CA in IL-2R bination. Introduction of the CA is expressed only when the ( enhancers) galovirus CA driven by a CAG promoter (chicken the these mice in cells T peripheral in time overlower became transgene the of sion addition, both the activity of the proximal proliferation, which complicated the interpretation of those findings. In of this transgene early during thymopoiesis leads to leukemic lympho rescue from lymphoproliferative syndrome 2R mal promoter of the gene encoding the kinase Lck in the absence of IL- constitutively active form of STAT5b (STAT5b-CA) driven inby the the absenceproxi of IL-2R. A published study using a transgene encoding a gain-of-function form of STAT5b was able to restore T expressiona of whether determine to sought we IL-2R, ofthat versus T of dysfunction severe observed the to contributor key a as IL-2) of deprivation and consumption and signals, independent (i.e., detection of IL-2, transduction of STAT5-dependent and STAT5- sible to separate in loss of STAT5 from impairment observed in all IL-2R functions expression IL-2R T STAT5-deficient in decrease marked the However, STAT5 of activation that downstream of indicated IL-2R was continuously required for above T reported findings The T IL-2R-deficient in STAT5 signaling Restoring T for required was ing signal that IL-2R indicated results these studies, IL-2-neutralization IL-2R-deficient T harboring mice of that to similar way a in munity that downstream downstream of IL-2R in signaling T STAT5 of activation role the assess to sought next we pathways, ing induction Foxp3 to part in T IL-2 and IL-15, yet they were affected similarly. This was in contrast to in whereas in T differentiated in signaling IL-2 of loss the for sate compen effectively to unable was IL-15 that indicated also findings T in signaling IL-2R of role the to due not probably shown), (data onset delayed a with disease a aggressive in less resulted considerably knockout conditional with mice our of that as strain either in deficiency line IL-2R of ablation after observed that to similar severity and onset early an with disease a in Weabove. T that found in T ablation conditional a similar to that of from T tration seques its enables signaling, IL-2 facilitating to addition in (which, STAT5 impaired T Supplementary Fig. 2a reg reg reg lox To address that major caveat and to understand the role of STAT5of role the understand to and caveatTomajor that address Since IL-2R activates PI3K–Akt, MAPK, and JAK–STAT5 and signal MAPK, PI3K–Akt, activates IL-2R Since IL-2R of ablation whether question the raised findings Our β Il2ra Rosa26 cells rescued the mice from the systemic inflammation and early and inflammation systemic the from mice the rescued cells cell differentiation in the thymus, in which IL-15 can contribute contribute can IL-15 which in thymus, the in differentiation cell el ( cells showed restoration of T Stat5a P-flanked P-flanked fl/fl 9 aDV ‘gene-trap’ locus Foxp3 . Therefore, we generated a gene-targeted mouse strain using fl/fl upeetr Fg 1g Fig. Supplementary Il2rb Supplementary Fig. 2b Stat5b eff A Il2ra NCE ONLINE PUBLIC ONLINE NCE cells) would result in result T would cells) Il2rb Cre fl/fl reg reg mice, T mice, Foxp3 fl/fl allele (J.D.F., data not shown) and induced its its induced and shown) not data (J.D.F., allele cell function similarly to similarly ablation of cell function IL-2R 2 cells ( cells fl/fl 7 reg β is preceded by a a by preceded is Foxp3 reg ). In the resulting ( Foxp3 Il2ra cell fitness in a cell-intrinsic manner. cell-intrinsic a in fitness cell reg Supplementary Fig. 1d Fig. Supplementary cell–specific deficiency in IL-2R deficiency cell–specific Cre 1 reg 2 cells by means similar to those described described to those similar by means cells Supplementary Fig. 1g Fig. Supplementary 2 6 reg Rosa26 . in which a transgene encoding STAT5b- mice, they lacked signaling via both both via signaling lacked they mice, Cre or or cell differentiation in the thymus but not lox Cre cells lacked only signaling via IL-2, IL-2, via signaling only lacked cells mice were affected by fatal autoim fatal by affected were mice P sites undergo Cre-mediated recom Il2rb mice. Thus, we generated mice with reg Stat5bCA ). In these mice, the frequency and A – in mice on the same C57BL6/J C57BL6/J same the on mice in TION cells. We found that ablation of k β ). Thus, in agreement with with agreement in Thus, ). reg Rosa26 -actin promoter with cytome Lck lox cell deficiency and disease and disease deficiency cell allele into 9

. . However, the expression promoter and the expres P-flanked STOP cassette cassette STOP P-flanked nature immunology nature Stat5bCA – f ). Of note, germ note, Of ). reg ) made it impos it made ) reg Il2rb reg reg mice, STAT5b- cells cells, because because cells, cell function. cell function reg eff fl/fl β cells. Our Our cells. cells. α -deficient resulted resulted Foxp3 β and Cre α ------

© 2016 Nature America, Inc. All rights reserved. by STAT5-dependent signaling but not by STAT5-independent STAT5-independent by not IL-2R Notably, these signaling. but signaling STAT5-dependent by IL-2R of expression the that IL-2R the of absence the despite from those in than mice of IL-2R 2R-sufficient number of T ( each in group per mice more or three with results, similar with experiments independent three of representative ( group per mice five than more with experiments two ( ( symbol Each ( in as mice of organs from (right) in Cre to fused (T staining Foxp3 intracellular female heterozygous ( (right). (MFI) intensity fluorescence mean as ( U/ml). (1,000 IL-2 STAT5 (pY-STAT5) in from ( CD28. co-receptor the to antibody and IL-4 100 bars, Scale margin). (left mice 1 Figure nature immunology nature IL-2R ± j g d a Foxp3 Foxp3 Spleen Thymus ) Expression of the markers IL-2R markers the of ) Expression s.e.m.). NS, not significant ( significant not NS, s.e.m.). Il2rb Il2rb

LNs Events (% of max) + 100 Il2rb cells (middle) and IL-17 and (middle) cells 20 40 60 80 /wt Cre Cre α 0 / expression remained unresponsive to IL-2 (

α Foxp3 10 10 10 10 IL-2R

fl/wt 0

was higher in T 10 0 3 2 4 5 2 YFP CD122 reg Foxp3 Skin b

Il2rb 10 0 5.69 5.95 1.91 3 β

Foxp3 10 , Foxp3 cells were comparable to or even surpassed those in IL- 2 c is indispensable for T for indispensable is Il2rb , 10 f 10 4 , 3 wt/wt h Cre 1.31 f – /wt Cre/wt 2.26 2.03 Il2rb

) Frequency of Foxp3 of ) Frequency 10 Il2rb Cre 10

j 5 and and 4 ) represents an individual mouse (among 3- to 5-week-old sex- and age-matched mice); small horizontal lines indicate the mean mean the indicate lines horizontal small mice); age-matched and sex- 5-week-old to 3- (among mouse individual an ) represents mice). ( mice). Foxp3 10 5 Pharynx 100

fl/wt fl/wt 20 40 60 80 Il2rb Il2rb reg 0 aDV Foxp3 Foxp3 8.05 6.51 reg cells from 1.8 + Cre Foxp3 P cells (right) among splenic CD4 splenic among (right) cells 0 fl/fl α cells) with (top right) or without (top left) expression of Cre (assessed as yellow fluorescent protein (YFP), which is which (YFP), protein fluorescent yellow as (assessed Cre of expression left) (top without or right) (top with cells)

h 10 fl/fl Il2rb A > 0.05); * > 0.05); g 2 mice ( ) Frequency of Foxp3 of ) Frequency n T on β . . ( Il2rb NCE ONLINE PUBLIC ONLINE NCE CD132 Foxp3 1.1 α Cre/wt -deficient T -deficient Foxp3 0.216 Cre Cre/wt 0.112

µ 10 β i /

(CD25), CTLA-4, GITR and CD103 by YFP by CD103 and GITR CTLA-4, (CD25), 3 ) Foxp3 expression in YFP in expression ) Foxp3 m. ( m. chain ( chain /wt and and reg

10 Lung d Foxp3

or or 4 reg Fig. 2 Cre cell function. ( function. cell ) Expression of the IL-2R subunits IL-2R subunits IL-2R the of ) Expression b Rosa26 Cre +

) LN cellularity of of cellularity ) LN 10 cells was controlled mainly mainly controlled was cells mice (key); gray shaded curve, isotype-matched control antibody. ( antibody. control isotype-matched curve, shaded gray (key); mice Il2rb Il2rb (T 5 P or < 0.05, ** < 0.05, Cr reg g h j Fig. 2 Fig. e a

) Flow cytometry of gated CD3 gated of cytometry ) Flow 2 + 100 ). Notably, the expression reg fl/fl fl/fl ) cells among LN CD3 LN among ) cells CD25 (MFI ×10 ) Foxp3 cells (%) Il2rb 20 40 60 80 10 Stat5bCA 0 1 2 3 4 0 0 5 Stomach cells with heightened heightened with cells Foxp3 Foxp3 Thymus Thymus a

a 0 *** wt/wt ), which suggested suggested which ),

NS 10 ) or three experiments with more than ten mice per group ( group per mice ten than more with experiments three ) or

+ 2 Il2rb Il2rb Fig. 2 cells among CD3 among cells A a P CD25 Cre Cre/wt Il2rb TION ) Histopathology of various tissues (above images) from from images) (above tissues various of ) Histopathology

< 0.01 and *** and < 0.01 10 3 Foxp3 / /wt Spleen Spleen T *** Foxp3 NS Il2rb reg Foxp3 10 b mice (above plots). Numbers adjacent to outlined areas indicate percent cells with with cells percent indicate areas outlined to adjacent Numbers plots). (above mice 4 fl/fl −

). Pancreas cells left unstimulated (US) or stimulated stimulated or (US) unstimulated left cells + Foxp3 10 Foxp3 Cre

Foxp3 5 Cr fl/wt Cre/wt LNs LNs *** NS e

mice, mice, Foxp3 + +

e − cells (left) and YFP and (left) cells CD4 Cre cells stimulated for 5 h with antibody to the invariant signaling protein CD3 protein signaling invariant the to antibody 5 h with for stimulated cells Events (% of max)

+ 100 + P

2 YFP cells (%) 20 40 60 80 CD4

Cre < 0.001 (two-tailed unpaired Student’s Student’s unpaired (two-tailed < 0.001 CTLA-4 (MFI ×10 ) 0 30 40 50 10 20 + Il2rb 0 2 4 6 8 0 cells (left) and Foxp3 expression by the CD3 the by expression Foxp3 and (left) cells Il2rb + and and Liver expression expression of STAT5b-CA ( Il2rb T of ability impaired ( disease fatal early sion of STAT5b-CA rescued similarly of IL-2R levels high reintroduced the that possibility the STAT5b-CAof sion raised T deficient CD4 Thymus Thymus + 0 /

+ 10 cells (left) and of YFP of and (left) cells

/wt 2 *** Foxp3 The observed restoration of the suppressor function of IL-2R of function suppressor the of restoration observed The pY-STAT5 Foxp3 β Il2rb + Foxp3 (CD122), (CD122), fl/fl 10

cells from the thymus, spleen and LNs (left margin) of healthy healthy of margin) (left LNs and spleen thymus, the from cells 3 Spleen Spleen Cre/wt Foxp3

+ 10 ***

** 4 fl/fl cells from organs of mice as in in as mice of organs from cells Cre/wt b reg

8 Foxp3 10 + 5 LN cells (×10 ) Il2rb Il2rb Foxp3

cells and rescue from the early fatal disease via expres via disease fatal early the from rescue and cells 0.0 0.5 1.0 1.5 Cre LNs LNs *** γ α ** c / /wt were responsible for these effects. However, expres effects. these for responsible were (CD132) and IL-2R and (CD132) mice and Il2rb Il2rb Il2rb Il2rb Cre Foxp3 Il2rb Foxp3 + Supplementary Fig. 2c Supplementary *** mice (key). ( (key). mice cells (right) from organs of mice as in in as mice of organs from (right) cells / /wt / /wt i

4 / 3 reg Foxp3 Foxp3 Cr GITR (MFI ×10 ) Foxp3 (MFI ×10 )

+ Foxp3 Foxp3 Cr Foxp3 10 11 12 (Cre-expressing) cells among Foxp3 among cells (Cre-expressing) e 0 1 2 3 e cells to capture and consume IL-2 in both both in IL-2 consume and capture to cells 8 9

Thymus c Thymus in vitro in Il2ra Cr Cr Cr Cr Fig. Fig. 2 Cre/wt + ** NS e e IFN- cells (%) YFP γ e e +IL-2 US +IL-2 US 10 15 e 0 5 Il2rb ) Intracellular tyrosine-phosphorylated tyrosine-phosphorylated ) Intracellular

fl/fl c – Spleen b Spleen d Foxp3 Il2rb ) Frequency of IFN- of ) Frequency for 20 min with recombinant mouse mouse recombinant with min 20 for c , , NS /wt Foxp3 c * * ), ), the ofreactivity CD4 e ** α t , , -test). Data are representative of representative are Data -test). f g Foxp3 (CD25) by CD4 by (CD25) f , fl/wt ) or are from one experiment experiment one from are ) or + h

cells + Il2ra – g Foxp3 cells (%) LNs LNs j , analyzed by flow cytometry. cytometry. flow by , analyzed NS 10 12 Foxp3 Cre ). ). + * Il2rb 0 2 4 6 8 Cre/wt CD4 + – fl/fl

mice was not ‘rescued’ via IL-4 cells (%) Il2rb Il2rb 10 15 h /wt

0 5 + ). ). Notably, the although *** Foxp3 Cre + 3 Foxp3 Foxp3 CD103 cells (%) Foxp3 (MFI ×10 ) / /wt 10 0 2 4 6 8 and and Foxp3 7 8 9 4 5 6 Foxp3 *** Il2rb Thymus Thymus γ Cr ticles e l c i rt A + + Cre Foxp3 e cells (left), (left), cells + ** Il2rb * * YFP / Cr

cells, presented presented cells, Cr e Foxp3 mice from the e

g Foxp3 (MFI) +

Il2rb + . Spleen Foxp3 Spleen 700 750 800 850 900 950 + + IL-17 cells (%) fl/fl

+ cells cells T cells was 0.0 0.1 0.2 0.3 0.4 0.5 cells cells * Cre/wt / Foxp3 Foxp3 + cells ***

LNs LNs *** *** ** e Cre

Cr β e



- - - © 2016 Nature America, Inc. All rights reserved. restrained restrained only marginally in these mice ( T cells, in particular that of CD8 activated of CD62L expansion population the however, contrast, In responses. IL-2 for was dispensable compete for T and capture to ability the that suggested results These fully controlled in these mice ( in + s.e.m. (mean each in group per ( three of representative ( T T of co-transfer without CD8 ( T of CD4 (STAT5b-CA expressing from sorted each) CD4 CD4 of ( anti-IL-2). (+ IL-2 to antibody neutralizing with or Ab) control (+ IgG antibody control the with birth) after 5–7 day from (starting CD44 of quantification in as mice 2-week-old of ( cells). (No cells without culture of medium in or IL-2, from sorted cells ( U/ml). (1,000 IL-2 mouse STAT5 T LN in tyrosine-phosphorylated Il2rb CD3 among s e l c i rt A  2f Fig. Figure 2 Figure a h e d a reg

, ) Quantification (top row) and frequency (bottom row) of IFN- of row) (bottom frequency and row) (top ) Quantification + hi 7 b + hi lo +

cells sorted from from sorted cells CD4 CD62L CD44 CD4 CD44 cells (×10 ) Foxp3 cells (%) reg , + + + e 0.0 1.0 0.5 10 15 20 wt/wt + hi 7 Foxp3 Foxp3 Foxp3 cells (%) 0 5

) indicate the mean ( mean the ) indicate CD4 CD44 cells (×10 ) cells (T cells 10 + 0.0 0.5 1.0 1.5 2.0 Cre , 20 40 60 80 Foxp3 h

0 0 ), ), Restoration of the suppressor activity of IL-2R-deficient T IL-2R-deficient of activity suppressor the of Restoration ). Although expansion of the CD8 the of expansion Although ). Cre Il2rb Il2rb − − − + Cre Cre Cre Cre T cells sorted either from from either sorted T cells CD8 or CD62L CD4 Il2rb * eff wt/wt − Il2rb Il2rb Il2rb Il2rb CD44 ) or with co-transfer of T of co-transfer with ) or fl/fl wt/wt + Foxp3 cells (left) and expression of CD122 and CD25 by CD3 by CD25 and CD122 of expression and (left) cells / / /wt /wt Foxp3 Foxp3 hi +anti-IL-2 +controlAb + Cre

+anti-IL-2 +controlAb CD122 (MFI) hi

CD44 + lo hi Foxp3 Rosa26 CD8 CD62L CD44 20 40 60 80 and CD8 and Cre 0

hi 6 reg + Il2rb Cre

a cells (×10 ) ) ) CD4 Cre activated CD3 activated Cre , a 0 1 2 3 cells, or with co-transfer of 2 × 10 of co-transfer with or cells, Il2rb d mice (+ non-T (+ mice (key). ( (key). / lo ± mice (key, left), together with T with together (key, left), mice , mice (Cre (Cre mice − c reg g s.e.m.). * s.e.m.). (CD4 T cells (1 × 10 (1 T cells ) Flow-cytometry-based bead-array analysis ( analysis bead-array ) Flow-cytometry-based , / Stat5bCA h NS + cell–mediated suppression of CD4 ) or two ( two ) or or CD8 or + Cre Fig. 2 Foxp3 + e Cre Cre Cre Cre Cre naive), CD8 naive), ) Frequency of naive (CD62L naive of ) Frequency Il2rb Foxp3 c 3 Il2rb Il2rb Il2rb Il2rb ; mean ; mean Il2rb

Il2rb CD25 (MFI ×10 ) P d + / − reg Rosa26 + 0 2 4 6 8 < 0.05, ** < 0.05, T reg b

CD44 + hi lo and CD4 Stat5bC

reg CD8 CD62L CD44 + hi hi / / /wt /wt / , cells obtained from mice as in in as mice from obtained cells

eff CD8 CD62L CD44

c + hi 7 cells sorted from from sorted cells Cre

Stat5bCA+controlAb 7 CD8 CD44 cells (×10 ) cells (%) Stat5bC

from Cre) or T or Cre) from fl/fl Stat5bCA +anti-IL-2 , Stat5bCA+anti-IL-2

cells among total CD4 total among cells cells ( 10 ) Stat5bCA +controlAb × e 6 20 40 60 80 Fig. Fig. 2 0.0 0.5 1.0 1.5 + , 0 0 1 2 3 4 5 Supplementary Supplementary Fig. 2d cells each) sorted from from sorted each) cells mice (+ Stat5b-CA (+ mice ± f ) and ) and hi Foxp3 hi ) independent experiments with similar results, with two or more ( more or two with results, similar with experiments ) independent s.e.m. in in s.e.m. wt A T cells in the LNs of T cell–deficient ( T cell–deficient of LNs the in T cells * + CD44 + Foxp3 Foxp3 A CD62L d P Rosa26 − b and < 0.01 and *** and < 0.01 and CD3 and pY-STAT5 MFI hi − Cre 200 400 600 800 d CD62L CD8 ) Quantification of various subsets of CD4 of subsets various of ) Quantification f 0 mice (open bars) or or bars) (open mice lo reg Supplementary Supplementary , g reg CD44 Stat5bCA , cells sorted from mice as in in as mice from sorted cells f h Foxp3 5 cells (2 × 10 (2 cells ).

+ + – hi control T control

+ CD4 Foxp3 CD44 T cells, was hi NS NS CD8 γ 6 hi CD44 + + cells (×10 ) 0 2 4 6 8 CD4 hi T CD44 + Il2rb Cre subset subset reg reg + or CD8 or + ** P T cell Foxp3 * mice (T mice *** Rosa26 ). Each symbol ( symbol Each ). cells in the presence of an active form of STAT5.of form ( active an of presence the in cells lo < 0.001 (two-tailed unpaired Student’s Student’s unpaired (two-tailed < 0.001 – + reg (CD8 lo fl/fl or CD8 or h NS ) T cells among CD3 among ) T cells a ). in vitro in cells sorted from from sorted cells + + Foxp3 and left unstimulated or stimulated stimulated or unstimulated left and CD4 5

− +

cells) sorted from mice as in in as mice from sorted cells) cells (bottom row) obtained from the LNs of mice as in in as mice of LNs the from obtained row) (bottom cells T h Stat5bCA eff + eff Rosa26

naive) or CD8 or naive) + + + + 6 status comparable to that of healthy controls, they gradually failed to failed gradually they to controls, that of comparable healthy status clinical improved substantially showed and death premature from Foxp3 after weeks 3 ( as birth early as mice these in expand to started gradually ( mice natal neo in controlled, perfectly not albeit controlled, well relatively was CD4 IFN- cells (%) CD4 IFN- cells ( 10 ) + – hi + T + + γ γ × 10 CD8 Foxp3 CD44 cells from from cells T cells in recipients 3 weeks after transfer of CD4 of transfer after 3 weeks recipients in T cells Foxp3 Cre 50

IL-2 capture assay) of residual human IL-2 in the medium of non-T of medium the in IL-2 human residual of assay) capture IL-2 6 2 3 0 1 0 0 cells (×10 ) 0 2 4 6 8 mice (Cre (Cre mice Stat5bCA +IL-2 Cre Stat5bCA US Cre Cre Cre Cre Cre reg Foxp3 Cre ) or ) or Supplementary Fig. 2i Supplementary STAT5b-CA STAT5b-CA STAT5b-CA Stat5bCA Tcrb Il2rb Il2rb Il2rb Il2rb Il2rb Il2rb a + , * cells (middle and right) from from right) and (middle cells b mice and a , ** / / / / wt/wt wt/wt Rosa26 e (key), assessed after culture for 2 h with recombinant human human recombinant 2 h with for culture after assessed (key), aDV −/− Cre +IL-2 US , * Tcrb Foxp3 f +IL-2 US Fig. 2 Fig. , + Tcrd Foxp3 mice and treated treated and mice + g Foxp3 Il2rb CD4 , – – – A h

−/− T T T NCE ONLINE PUBLIC ONLINE NCE ) represents an individual mouse; small horizontal lines lines horizontal small mouse; individual an ) represents NS ef ef ef Stat5bCA −/− f f f Cre Tcrd +STAT5b-C +STAT5b-C c + + fl/fl Rosa26 d Cre Foxp3 or CD8 or ) host mice 3 weeks after adoptive transfer of transfer adoptive after 3 weeks mice ) host −

* Uncaptured IL-2 (%) mice (+ Stat5b-CA (+ mice and and CD62L Stat5bCA). ( Stat5bCA). 100 mice (filled bars) and treated with TAT-Cre, with treated and bars) (filled mice 50 −/− 0 Foxp3 recipients 3 weeks after transfer of transfer after 3 weeks recipients − a + Cre Stat5bCA + Cre + Cre CD4 CD8 CD4 CD8 CD4 Supplementary Fig. 2f Fig. Supplementary and CD3 and Stat5bCA (key, right). ( (key, right). + hi A A cells among CD3 among cells + – CD44 + + + + +

T T naive memory naive naive naive

Cre ). Although ). both Although Il2r Il2r Il2r t in vitro in re re in vitro in -test). Data are from one experiment experiment one from are Data -test). * g g a mice (T mice a a a T b , wt/wt / / * b re Il2ra hi + ) Quantification of intracellular intracellular of ) Quantification

NS ) or three or more ( more or three ) or g T + + + + + 6 (CD8

re + T CD8 CD8 IFN-γ cells (%) CD8 IFN-γ cells (×10 ) Il2rb A 100 for 20 min with recombinant recombinant with min 20 for g re with TAT-Cre, without co-transfer TAT-Cre, with co-transfer without 10 50 TION g − 5 0 0 NS fl/fl STAT5b-CA STAT5b-CA STAT5b-CA a g T eff + ) Frequency of Foxp3 of ) Frequency ) Frequency of STAT5b-CA-of ) Frequency g + wt/wt reg

Foxp3 + + STAT5b-CA Foxp3 memory) T cells (1 × 10 (1 T cells memory) STAT5b-CA cells (%) 10 ) or STAT5b-CA-expressing STAT5b-CA-expressing ) or

50 + Foxp3 0 0 Foxp3 nature immunology nature + + + + + + + non-T No cells − Rosa26 + + + T T T T T T Cre cells (top row) and and row) (top cells

re re re re re re f T T T + ) Quantification ) Quantification ef ef ef g g g g g g Foxp3 a fromCre fromCre fromCre fromCre fromCreStat5bCA fromCre Cre mice were rescued mice were rescued CD4 f f f − +STAT5b-CA +STAT5b-CA ), this subset also also subset this ), treated for 2 weeks 2 weeks for treated

re cells from the LNs LNs the from cells Teff +STAT5b-CA Teff c g mice (Cre (Cre mice fromCre , + d + Stat5bCA , − T e and and reg , Il2rb Il2rb Il2ra Il2ra f , g ). ). + / / , / / h + – cells cells CD8

Teff +Treg

Stat5bCA

Stat5bCA Il2rb T T ) mice ) mice re re g g + + 6 reg Treg

fl/fl

-

© 2016 Nature America, Inc. All rights reserved. T CD4 of proliferation the on tion cell–mediated suppression. Thus, we tested the effect of STAT5 activa CD4 in not but of STAT5 activation that was CD8 in idea to that A corollary by T sumption CD4 were than IL-2 to sensitive more be to CD8 naive both Thus, CD4 naive while IL-2, to did not ( CD4 IL-2R activated of proportion small A IL-2R expressing cells in observed that than extent lesser a to albeit IL-2, to response STAT5 in activate to of IL-2R expression and subset. latter the of control in subset, although this mechanism seemed to be particularly prominent CD8 naive the both of tion activa and expansion population the suppressing in role redundant ( sion and of activation naive CD8 T cells was not restored, whereas suppression of the population expan CD8 memory suppress to ability their contrast, STAT5b-CA;in by CD4 of activation and expansion population the of suppression impaired of observations with Consistent recipients. penic CD4 of transfer adoptive by CD8 naive CD8 memory of expansion tion by T Foxp3 in subset CD8 of proliferation pronounced more and treatment. by this suppressed completely almost of CD8 or In marginally unaffected. contrast, the and activation proliferation ever, the activation of CD4 CD4 T the of production In control T of T mice starting from 5–7 d of age. As IL-2 supports the differentiation of Foxp3 accounted for the proliferation of CD8 T by IL-2 of consumption in impairment the if Todetermine T CD4 of by T consumption that IL-2 of age ( by 12 weeks approximately (LNs) and tissues in lymph nodes subsets expanded activated CD62L massively by accompanied disease to succumb to started and thrive nature immunology nature tein containing a membrane-permeable TAT peptide (trans-activating STAT5b-CA in these cells by treating them with recombinant Cre pro from cells T Fig. 2 Fig. reg reg reg Although Although the majority of activated CD8 CD8 the in reduction relative The Il2rb cells. For this purpose, we sorted CD4 we sorted For purpose, cells. this cells in the thymus, neutralization of IL-2 reduced the frequency frequency the of reduced IL-2 in neutralization thymus, the cells cells suppress CD8 suppress cells reg reg + + Rosa26 T cells was significantly reduced by neutralization of IL-2; how T cells by IL-2R-deficient T IL-2R-deficient by cells T α Cre Cre cells in all groups of mice and induced immunoactivation in in immunoactivation induced and mice of groups all in cells cells might selectively impair their suppression of suppression the popula their impair selectively might cells f Supplementary Fig. Supplementary 2i + + ). Thus, IL-2 consumption by ). Thus, IL-2 consumption T expression also responded to IL-2, but the majority of them them of majority the but IL-2, to responded also expression Il2rb fl/fl T cells observed in T observed cells T cells, was important for the restraint of CD8 of restraint the for important was cells, T Supplementary Fig. 3b Fig. Supplementary mice, we administered IL-2-neutralizing antibodies to mice, antibodies we those IL-2-neutralizing administered mice raised the possibility that loss of IL-2-consumption IL-2-consumption of loss that possibility the raised mice Rosa26 Foxp3 + T cells into the effector-cell pool. We tested this idea idea this tested We pool. effector-cell the into cells T Stat5bCA fl/wt Rosa26 reg + Foxp3 cells might have markedly affected their activation. activation. their affected have markedly might cells Cre Stat5bCA T cells might render the former resistant to T to resistant former the render might cells T Il2rb mice, which spontaneously developed disease, disease, developed spontaneously which mice, α H Stat5bCA 2 (T helper type 2) cytokines IL-4 and IL-13 by IL-13 and IL-4 cytokines 2) type helper (T 2 ( Cre Supplementary Fig. 3a Fig. Supplementary +

T cells and activated CD8 activated and cells T Il2rb fl/fl mice ( mice + + aDV + T cells did not ( not did cells T T cell responses via IL-2 depletion IL-2 via responses T cell hi and CD8 Foxp3 Foxp3 reg + Rosa26 CD44 T cell subset and memory CD8 memory and subset cell T + fl/fl cells, while dispensable for control the dispensable while cells, , A and CD8 and j ). ). These findings raised the possibility NCE ONLINE PUBLIC ONLINE NCE Fig. 2 Fig. Foxp3 + ). ). naive T CD8 + + Cre T cells was recovered only partially Cre or CD8 or T cells but not the recruitment of of recruitment the not but cells T hi reg Stat5bCA and CD62L mice and induced expression of expression induced and mice mice did not have detectable detectable have not did mice + + cells was completely ‘rescued’ ‘rescued’ completely was cells + e T cells was at best reduced only T cells in Cre reg CD62L and + α + T cells with undetectable undetectable with cells T cells seemed to have a seemed cells non- and and ( + cell subsets into lympho into subsets cell + + Il2rb T cells in T cells in the presence of presence the in cells T Supplementary Fig. 3b Fig. Supplementary Supplementary Fig. 3b Fig. Supplementary Foxp3 Supplementary Fig. 3a Fig. Supplementary + + CD62L T cells, and IL-2 con IL-2 and cells, T lo Rosa26 ), these cells were able able were cells these ), + Rosa26 fl/fl CD44 lo cells also responded responded also cells CD44 − Foxp3 Foxp3 and CD8 A + Il2rb TION T cells seemed seemed cells T hi hi Stat5bCA Stat5bCA CD44 hi T cell subset subset cell T + Cre Cre CD8 T cells. T fl/fl

mice, the the mice, mice was mice Foxp3 + + hi Il2ra + Foxp3 reg Il2rb T cells T cells + T cell cell T T cell cell T T cell cells cells fl/fl Cre fl/fl reg ). ). ). ). ). ). + − ------

T CD8 of expansion tion popula showed IL-2 to antibody and IL-2 of immunocomplexes of in experiments gain-of-function which IL-2 was provided in the form CD4 in activation T to resistance nounced pro conferred and cells of expansion population robust prompted CD8 STAT5 in that activation suggested observations These expression of STAT5b-CA, were well controlled by T CD4 activated of production cytokine and STAT5b-CAof suppression the with compared mild very was it observed, still was of some degree suppression of STAT5b-CA Foxp3 ( control either of presence the in expanded robustly tions to ( only 40–50% CD4 expressing STAT5b-CA STAT5b-CA transfer, whereas cell the expressed after 3 T weeks cells of the treated CD4 30% approximately in STAT5b-CAexpression induced initially Cre T without or with recipients lymphopenic into cells treated the transferred Weadoptively Cre). transcriptional activator from human immunodeficiency virus) (TAT- increase increase in number at the expense of T rapid their by followed was tamoxifen of administration single a by CD8 of control the for essential be to appeared suppression of mode this responses, T for dispensable was mice ( mice STAT5b-CA tamoxifen-treated STAT5b-CA in in higher was CD103 and Consistent KLRG1 state. marker activation the memory IL-7R, of expression a surface or possibilities, those state with activated an toward lation T the of biasing STAT5b-CA–imposed a of indicative CD62L of CD62L proportion higher a had and mice in STAT5b-CA tion had larger amounts of Foxp3, CD25, CTLA-4 and GITR than those Fig. 4d Rosa26 ERT2 (TCR) cells exhibited these a diverse use highly of the T cell antigen receptor Rosa26 ERT2) T tiated generated mice, with tamoxifen-inducible we expression of STAT5b-CA issue, in differen this address To work by uncoupling T of the aforementioned IL-2-dependent T ing offered a unique opportunity to explore the importance biological relieved T fl in both (counter-selection) recombination Cre-mediated escaped that cells T IL-2R-sufficient of expansion population STAT5b-CA-driven of and IL-2 to response in STAT5activation detectable of lack The T in STAT5 activation CD4 did than robustly Foxp3 eff cells cnrl mc ( mice (control) Cre Fig. 3 Fig. 1 Cre , Stat5bCA Stat5bCA β 7 e hi reg Rosa26 . Induction of STAT5b-CA expression in ~20–30% of T 2 ). In these mice, the proliferated STAT5b-CA -chain variable region similar to that in in that to similar region variable -chain ) T ) CD44 8 mice indicated that the expression of an active form of STAT5 reg cells (via the tamoxifen-sensitive estrogen receptor variant estrogen the (via tamoxifen-sensitive cells . Thus, while the ability to capture and compete for IL-2 IL-2 for compete and capture to ability the while Thus, . reg cells from on their dependence IL-2 This find signaling. d − ). Notably, in the LNs and Peyer’s patches, the number number the Peyer’s and patches, LNs the Notably, in ). T cells or STAT5b-CA or cells Foxp3 allele ( allele Stat5bCA − − lo reg T + CD8 el ( cells Fig. Fig. 2 T cells, which responded to excessive IL-2 more more IL-2 excessive to responded which cells, T reg cells in tamoxifen-treated tamoxifen-treated in cells + + T cells and CD8 T cells did not. Consistent with those findings, findings, those with Consistent not. did cells T cells in tamoxifen-treated Cre-ERT2 + reg Il2rb Supplementary Fig. 4a Fig. Supplementary T cells ( cells T reg upeetr Fg 4c Fig. Supplementary g + + cell function from IL-2 production by T reg Rosa26 T cells. T ). ). Notably, STAT5b-CA T cells and CD4 and cells T i. 3a Fig. cell–mediated suppression of CD4 of suppression cell–mediated reg cells boosts immunosuppression boosts cells fl/fl cell–mediated suppression, but STAT5 but suppression, cell–mediated mice remained healthy ( reg Foxp3 Fig. 2 Fig. + cells. Although treatment with TAT- with treatment Although cells. Stat5bCA T cells increased their frequency frequency their increased cells T – d Cre and and + + h T T more cells, than 95% of CD8 reg ). In contrast, the proliferation proliferation the contrast, In ). mice and reg Foxp3 reg Rosa26 cells with a non-recombined cells ( cells + cell–T upeetr Fg 4f Fig. Supplementary T + + T cells, regardless of the the of regardless cells, T CD8 reg Cre-ERT2 Rosa26 , Rosa26 b cells but not of CD4 of not but cells Stat5bCA + ). The experimental experimental The ). Fig. 2g Fig. ). It was notable that that notable was It ). eff Rosa26 hi CD8 + T by cells T cell regulatory net CD44 Rosa26 ticles e l c i rt A reg + Supplementary Supplementary mice than in in than mice T wt + wt cells ( T popula cell Foxp3 reg , Foxp3 reg Stat5bCA Foxp3 h hi cell popula ). Although Although ). wt cell popu cell cells than than cells + T Foxp3 Il2ra Fig. Fig. 2 + Cre–ERT2 Cre-ERT2 reg + Cre-ERT2 reg eff reg T cells cells T T cell cell T Il2ra cells cells cells. cells cells wt/wt Cre- h reg fl/ ), ), ). ).  + + ------

© 2016 Nature America, Inc. All rights reserved. ished rather than being increased in the presence of STAT5b-CA T of presence the in increased being than rather ished as well as T gut the in (IgA) A T T to function suppressor enhanced ferred 5c Fig. ( tamoxifen of administration the after reduced were (DCs) cells dendritic and cells B by CD86 and CD80 CD4 by IL-4, prominently most cytokines, pro-inflammatory of production ( pool cell T and CD62L vivo in state of and of activation proliferative activity CD4 ( activity STAT5 active heightened expressing showed cells tion of STAT5b-CA ( CD8 of abundance The STAT5 activated tissues. in non-lymphoid distributed ‘preferentially’ ( mice STAT5b-CA of predominance the despite T of s e l c i rt A  also showed a tendency toward a decrease, but this was not statistically cytometry of splenic CD4 splenic of cytometry ( cytometry. flow by analyzed treatment, tamoxifen a single after age-matched and sex- of axes) (horizontal skin and lungs liver, (SILP), intestine CD3 3 Figure each ( each Student’s ( mean the indicate lines horizontal small mouse; individual an represents (B220 cells CD3 CD4 splenic by markers ( (right). GITR or (top) CTLA-4 of intensity fluorescence mean indicate row) (bottom areas outlined of margins along numbers CD62L left),

reg b a H + T

17 subset of helper T cells and class switching to immunoglobulin to immunoglobulin of T and switching 17 class helper cells subset 3 Foxp3 cell (%) cells ( cells reg Foxp3 (MFI ×10 ) + + 20 40 60 80 reg 0 0 1 2 3 4 CD8 CD4 Foxp3 10 10 10 10 a cells have been proposed to have cells of proposed promote responses been the systemic Supplementary Fig. 4b Fig. Supplementary cells did not increase after the administration of tamoxifen, tamoxifen, of administration the after increase not did cells 0 , 2 3 4 5 as well, as indicated by the decreased number of Ki67 of number decreased the by indicated as well, as ). These results indicated that expression of STAT5b-CA expression that con indicated results These ). Rosa Foxp Blood Blood d

CD25 + ** , Increased proliferative and suppressor activity of T of activity suppressor and proliferative Increased Supplementary Fig. 4i Supplementary + + 0 f t T cells and expression of the costimulatory molecules molecules costimulatory the of expression and cells T Foxp3 10 Foxp3 CD3 in Foxp3 of expression and (top) cells , -test). Data are from one experiment representative of two independent experiments with similar results, with five or more mice per group in group per mice more or five with results, similar with experiments independent two of representative experiment one from are Data -test). Fig. 3 Fig. 3 Stat5bC g + H Cre-ERT2 2 hi lo ) or are representative of three experiments with more than ten mice per group ( group per mice ten than more with experiments three of representative are ) or CD11c Fig. 3 Fig. 10 17 responses in secondary lymphoid organs 17 in responses were secondary dimin CD44 *** CD44 Sp Sp ** Cre-ERT2 3 A 10 Foxp3 − g cells (bottom) from mice as in in as mice from (bottom) cells 4 SILP SILP and and *** 10 e hi − 29 ** hi ) in the LNs of mice as in in as mice of LNs the ) in and and 5 cells (top right) or CD62L or right) (top cells Cre-ERT2 , 3 T 10 10 10 10 Supplementary Fig. 4h Supplementary 0 + 0 Liver Liver 2 3 4 5 . However, we found that serum and fecal IgA fecal and serum that found However, we . eff Supplementary Fig. 5c Fig. Supplementary *** Foxp3 + + Foxp3 Supplementary Fig. 5a Fig. Supplementary cells and cells much CD62L larger 0 Foxp3 Foxp3 10 Rosa 2 Lung Lung ** 10 * + Stat5bCA Cre-ERT2 T 3 + 10 T , reg + g ) and effectively suppressed the basal the basal suppressed ) and effectively Skin Skin 4 cells also increased after the induc the after increased also cells *** reg ); this suggested that T that suggested this ); cells from mice as in in as mice from cells 10 cells from mice as in in as mice from cells 5 c GITR KLRG-1 CD62L 10 10 10 10 10 10 10 10 10 10 10 10 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 Fig. 3 Fig. CTLA-4 ICOS CD4 1.4 71.8 reg 0 0 0 Foxp3 10 55.6 10 10 a + MFI 769 lo 2 1 4 , analyzed by flow cytometry. ( cytometry. flow by , analyzed T cells. 2 2 2 ). ). The ‘autonomous’ T CD44 23 a 10 10 10 f reg (numbers in plots as in in as plots in (numbers ). Serum IgM and IgE and IgM Serum ). Cre-ERT2 3 3 and and 3 , 10 10 10 b cells in the former former the in cells 4 + 24.7 4 4 ). Accordingly, the the Accordingly, ). in in vitro hi + .1 CD4 10 16 10 10 and CD8 cells (bottom); numbers in quadrants (middle row) indicate percent cells in each (throughout); (throughout); each in cells percent indicate row) (middle quadrants in numbers (bottom); cells MFI 1519 1 5 5 5 Supplementary Supplementary a hi 16.6 , analyzed by flow cytometry. ( cytometry. flow by , analyzed + 68.3 a reg Foxp3 CD44 Foxp3 52.5 MFI 1277 . Numbers adjacent to outlined areas (top row) indicate percent CD62L percent indicate row) (top areas outlined to adjacent . Numbers Rosa reg cells expressing an active form of STAT5.of form ( active an expressing cells suppressor suppressor 13.7 cells with with cells b Stat5bC Cre-ERT2 ) Expression of Foxp3 and CD25 by splenic CD4 splenic by CD25 and Foxp3 of ) Expression + + lo T cells 26.6 cells (bottom) obtained from the blood, spleen (Sp), lamina propria of the small small the of propria lamina (Sp), spleen blood, the from obtained (bottom) cells 8.5 6.5 + naive A cells cells

MFI 2424 8 3 reg d e + - - -

± ICOS (MFI)

Foxp CD8 Foxp CD4 c

3 s.e.m.). * s.e.m.). 150 200 250 300

, top). ( , top). CD25 (MFI ×10 ) 0 1 2 3 4 + + were very resistant to experimental autoimmune encephalomyelitis encephalomyelitis autoimmune experimental to resistant very were treatment, tamoxifen a after single months 2–3 at that found We autoimmunity. autoantigen-induced develop T ‘autonomous’ ment, colon-carcinoma modulating and settings physiological activity. suppressor T by suppressed potentially but promoted not to be seemed carcinogenesis of colonic However, stages early the formed. already are lesions pre-cancerous or tumors once tumors of mation by T These results were consistent with the idea that suppression of inflam in than mice in greater was size polyp average to yps similar or fewer than that of Apc intestine small the in polyps adenomatous multiple develop tion Apc T in gain a of effect an explored we carcinogenesis, colonic of promotion T of ablation acute upon T observed switching and switching class noglobulin T in increase the with significant ( g 3 3 ) ELISA of serum and fecal IgA in mice as in in as mice in IgA fecal and serum of ) ELISA – – Foxp3 In addition to restraining the basal immunological reactivity in in reactivity immunological basal the restraining to addition In to the linked have been responses immune intestinal altered Since *** Min Min CD62L 10 10 10 10 reg f 0 2 3 4 5 ) Expression of CD80 and CD86 on DCs (CD11c DCs on CD86 and CD80 of ) Expression cell suppressor function afforded by activated STAT5 in the the STAT5in activated by afforded function suppressor cell CD4 P Rosa26 model of colorectal cancer. Mice harboring the the cancer. harboring Mice of colorectal model Cre-ERT2 0 Foxp < 0.05, ** < 0.05, 10 44. 44.

16. CD69 (MFI) CD127 (MFI) 43. 4 2 100 150 200 250 100 150 200 250 e aDV 50 8 10 9 1 3 ) Flow cytometry of splenic CD3 splenic of cytometry ) Flow 6 reg 0 Supplementary Fig. Supplementary 5d Cre-ERT 3 b 10 cells in tumor microenvironments promotes the growth Stat5bCA Apc and and , A c 31. 8.97 4 *** , 10 NCE ONLINE PUBLIC ONLINE NCE *** e 2 2 5 ). Min P Rosa26 < 0.01 and *** and < 0.01

Foxp 2 + Rosa Foxp3 Foxp3

59. CTLA-4 (MFI ×10 ) KLRG-1 cells (%) 71. 4.14 20. reg 10 12 10 20 30 H 6 8 0 8 7 3 17 responses and in both T both in and responses 17 3 Stat5bC cells afforded superior protection against against protection superior afforded cells Cre-ERT a Stat5bCA ) Frequency of Foxp3 of ) Frequency *** Cre-ERT2 Cre-ERT2 *** 32. 5.47 A 2 1 Foxp3 + 2 + Apc T cells from mice as in in as mice from T cells GITR (MFI ×10 ) CD103 cells (%) Apc P 10 15 20 25 10 20 30 40 50 Rosa26 mice ( mice g mice developed a number of mice developed pol ). ). These results were in agreement 0 < 0.001 (two-tailed unpaired unpaired (two-tailed < 0.001 H A Min 1-type immunoglobulin class class immunoglobulin 1-type TION Cre-ERT2 Min 2 *** Serum IgA (µg/ml ×10 ) ** Rosa26 + 10 15 Foxp3 0 5 CD4 Stat5bCA

a Supplementary Fig. 5e Fig. Supplementary d reg . Each symbol ( symbol . Each ) Expression of various various of ) Expression f nature immunology nature reg mice (key) 3 months 3 months (key) mice + + cells with augmented augmented with cells CD80 (MFI) cells among among cells Foxp3 CD80 (MFI) cells 200 400 600 Stat5bCA Cre-ERT2 * 50 55 60 65 70 75 0 Foxp3 hi + MHCII CD44 3 − ** H ** 1 cells (top) and and (top) cells . 2-type immu 2-type Foxp3 Apc Fecal IgA (µg/ml) mice, but the Cre-ERT2 a 100 150 200 250 lo hi . . ( B cells 50 cells (top (top cells DCs

0 2 a ) and B ) and 2 CD86 (MFI ×10 ) c Min , CD86 (MFI ×10 ) 10 15 20 25 d ) Flow ) Flow 0 5 0 1 2 3 4 , Cre-ERT2 f muta , *** g mice ) ** * 3 2 ). ). - - - - - .

© 2016 Nature America, Inc. All rights reserved. above, as well as the diminished activation of the immune system system of Peyer’s patches and immune LNs the in the of activation diminished the as well as above, IL- of expression and T 2R capacity in proliferative activity their STAT5 with correlated studies, gain-of-function and loss- STAT5 T sustained potentiate how might of signaling question the address to sought we Next T in STAT5 for role activation A distinct activated on reliant are IL-2. of source a as cells T instead and STAT5 of activation omous T why for rationale a vide pro might progression cancer of modulation and agents infectious CA STAT5b- of presence the in reduced markedly were responses cell T ( treatment). modestly only tamoxifen suppressed single a after months Although 2–3 at (also mice in Foxp3 ( lower significantly was organs these into cells, CD4 IL-17-producing and neutrophils including cells, tory ( mice these of cord nal CD4 of ( adjuvant Freund’s complete in peptide glycoprotein myelin with by oligodendrocyte induced immunization CD3 by cytometry.flow analyzed ( ( of and sex- age-matched scores 4 Figure nature immunology nature (middle) or CD4 (middle) of CD8 for 5 h or re-stimulation after (left) re-stimulation and n hc fwr T fewer which in with experiments independent four from pooled ten with ( results, similar with experiments of two independent representative the ( mean indicate lines or of vaccinia-virus a three mixture (middle) or 5 after (left) h re-stimulation without by cytometry flow assessed virus, vaccinia replicating b d a

) Frequency of Foxp3 ) Frequency + +

Rosa2 EAE score α CD4 Foxp3 cells (%) 0 1 2 3 4 5 + + Rosa26 Foxp3 10 20 30 and Foxp3. However, the the However, Foxp3. and T CD4 0 Cre-ERT2 8 + reg T cells specific for the vaccinia virus peptide B8R (detected with H-2K with B8R (detected peptide virus for the vaccinia T specific cells +

6 Foxp3 Potent suppressor function of T function suppressor Potent cells ( cells + Cre-ERT2 Stat5bCA cells (left) and of IFN- (left) cells Stat5bCA UI Time afterimmunization(d) Listeria mice. Pathogen-specific responses were also diminished + + Fig. 4 Fig. Foxp3 cells was significantly greater in the brain and spi and brain the in greater significantly was cells Foxp3 Foxp3 Rosa reg - Inf monocytogenes cells were found than in in than found were cells e − + 15 Stat5bC cells (right), in sex- and age-matched in and sex- age-matched (right), cells cells among brain-infiltrating CD3 brain-infiltrating among cells ). Our observation of diminished responses to responses diminished of observation Our ). ± Cre-ERT2 Cre-ERT2 s.e.m.). * s.e.m.).

Fig. 4 Fig. A aDV c Foxp3 reg ) Quantification of brain-infiltrating cells in as cells mice in of brain-infiltrating ) Quantification + + Fig. 4 Fig.

Foxp3 CD4 IFN- cells (%) cells lack IL-2 production and auton and production IL-2 lack cells γ reg mice left uninfected (UI) or on day 8 after infection with with or (UI) on day 8 infection after uninfected left mice A 10 15 20 mice relative to those in γ b Cre-ERT2 0 5 + NCE ONLINE PUBLIC ONLINE NCE in vitro in cells’ suppressive ability. In genetic genetic In ability. suppressive cells’ ), and the infiltration of inflamma of infiltration the and ), cells (middle) or TNF (middle) cells P Rosa26 < ** 0.05, Cre-ERT2 21 d seii T -specific ), vaccinia-virus-specific CD8 vaccinia-virus-specific ), UI reg b cells expressing an active form of form an STAT5.active expressing cells ( suppression assays reported reported assays suppression

- + +

specific peptides (ISK-A33R-B5R). Each symbol ( symbol Each (ISK-A33R-B5R). peptides specific CD4 Foxp3 cells (%) and Stat5bCA Fig. 4a Fig. 60 20 40 0 reg n P = 11 (UI), in in vitro < and 0.01 *** Inf Rosa26 cells * H Foxp3 Fig. 4 Fig. Foxp3 – 1 responses were were responses 1 * c ). The frequency frequency The ). A with heat-killed heat-killed with + TION Stat5bCA

cells (right) among CD4 among (right) cells + + +

c CD4 TNF cells (%) Cre-ERT2 CD4 Cre-ERT2 Foxp3 ) than that of of that than ) n 10 15 0 5 = 15 (Inf, + + Foxp3

CD8 Foxp3 cells (%) + 10 20 30 40 50 cells (left) and CD3 (left) cells Foxp3 0 P reg Cre-ERT2 + < 0.001 (two-tailed unpaired Student’s unpaired < (two-tailed 0.001 UI mice, mice, T mice, mice, Cre-ERT2 cells cells H Cre-ERT2 17 17 Foxp3 * L. L. monocytogenes + - - - -

and a Inf – mice ( mice * Foxp3 and analyzed gene expression in these cells by high-throughput Rosa26 f TT, e otd aue T mature sorted we STAT5, activation of sustained by conferred function suppressor heightened T of STAT5b-CA activity suppressor enhanced the Thus, shown). not (data Foxp3 of expression high comparably despite recipients, penic T with together transferred when mice (control) CD25 of that than STAT5b-CA T 1.36-fold; Foxp3 Foxp3 cells from in Foxp3 of intensity fluorescence mean in difference CD25 the in noticeable particularly was STAT5b-CA in protein than pronounced STAT5b-CA by more caused one expression the Foxp3 in increase an to leads of T not after change Foxp3 binding does T of activity suppressor increased the for account could STAT5b-CA of presence the in expression Foxp3 of It that upregulation augmented. mild was also basis unlikely was also in T of increase observed immunosuppression enhanced Rosa26 the that suggested Cre-ERT2 b c –B8R tetramer (tet); left) and of left) IFN- (tet); tetramer –B8R reg reg c ; mean ; mean Rosa26

To gain insight into the potential mechanisms underlying the the underlying mechanisms potential the into insight gain To + 5

+ CD4 cells ( 10 )

cells was probably not due to the increase in Foxp3. in increase the to due not probably was cells Nevertheless, on IL-2. dependence their from were relieved cells × TCR 0 1 2 3 4 5 b + n Cre–ERT2 , analyzed by cytometry.flow , analyzed ( a CD8 ) ) or = 10 per group) at various times after immunization. immunization. after = times at 10 various per group) Stat5bCA Stat5bCA ) Experimental autoimmune encephalomyelitis (EAE) disease disease (EAE) encephalomyelitis autoimmune ) Experimental β ± Stat5bCA + s.e.m. in s.e.m. Fig. Fig. 3 Rosa26 L. L. monocytogenes Foxp3 n e + ** in the presence of DCs (middle and right). ( and right). of DCs (middle in the presence = 20 (Inf, cells (right) from mice as mice from in (right) cells * + reg in in vitro T

mice ( mice + + b Foxp3 Foxp3 CD8 B8R tet cells (%) – cells but that their suppressor activity on a per-cell per-cell a on activity suppressor their that but cells Foxp3 b reg − 10 15 20 25 e cells obtained from sex- and age-matched and sex- age-matched from obtained cells ), ), consistent that with STAT5b-CAthe observation 0 5 Stat5bCA ) represents an individual mouse; small horizontal horizontal small mouse; an individual ) represents cells exhibited more potent suppressor function function suppressor potent more exhibited cells

a + 5 hi CD8 cells ( 10 ) ) ) or ( four

stimulation with the vaccinia virus peptide B8R peptide virus the with vaccinia stimulation × Cre-ERT2 Foxp3 Cre-ERT2 10 15 Cre-ERT2 Rosa26 n 0 5 = 6): CD25 6): = ** + Foxp3 T (Inf), assessed by flow cytometry without without by cytometry flow assessed (Inf), t hi reg -test). Data are from one experiment are one Data from experiment -test). mice on day 8 after infection with non- with on mice day 8 infection after ** mice was not simply due to a numerical to a due numerical simply not was mice e mice with comparable expression of of expression comparable with mice Stat5bCA T ) mice per group ) in per ( mice group each * el ta i STAT5b-CA in than cells Cre-ERT2 reg

3 + +

3 CD8 IFN-γ cells (%) cells from from cells reg 10 d . The greater abundance of Foxp3 Foxp3 of abundance greater The . 0 2 4 6 8 ) Frequency of Foxp3 ) Frequency γ Foxp3 + hi cells from from cells + + 4

cells among CD8 among cells CD11b Gr1 cells (×10 ) cells, 1.06-fold; CD25 1.06-fold; cells, a 10 mice versus that in from those at day 22 after immunization, at day 22 immunization, after 0 2 4 6 8 +B8R reg Cre-ERT2 * lo reg T cells are activated, which which are activated, cells cells, as genome-wide genome-wide as cells, reg ** Rosa26 ) mice per group ) ( per mice group cell subset (average (average subset cell eff Foxp3

cells into lympho cells + + CD4 IFN-γ cells (%) s e l c i rt A e 0.0 0.2 0.4 0.6 ) Frequency ) Frequency + wt a + + 4 Foxp3 – + CD4 IL-17A cells ( 10 )

+ ISK-A33R-B5R × Foxp3 c cells among among cells Cre–ERT2 0 1 2 3 4 5 , Foxp3

e − ) ) or are T − cells cells * reg Cre-ERT2 lo Cre-ERT2 * cells, cells, + cells cells and and d T ). ). reg  + + -

© 2016 Nature America, Inc. All rights reserved. is proportional to the FDR-adjusted FDR-adjusted the to proportional is ( terms ontology STAT5b-CA ( perturbation). and enrichment both (reflecting STAT5b-CAin either expressed pathway the in genes STAT5b-CAin differentially expressed pathway the in genes (Overlap), margin right enrichment); of degree the to proportional is size circle pathway; the in differentially expressed genes of relationships inhibitory or activating on in STAT5b-CAin differentially expressed genes among enrichment showing margin) (left Genomes and Genes of Encyclopedia Kyoto the from function related with genes of expression the for enrichment significant most the with pathways the of analysis CD44 in manner a TCR-dependent downregulated (Act down) by inflammatory activation in T in activation inflammatory by down) (Act downregulated STAT5b-CAin expressed T in as cells in margin) (right ( (blue). downregulated or (red) upregulated genes total indicate plots in numbers genes; all of distribution the on based (adjusted T (Stat5bCA mice (log ( mouse. a single represents symbol Each 2 (PC2). component principal and 1 (PC1) component principal as presented variance, T (Control mice Foxp3 5 Figure T in expression gene identical, nearly were gene- the CD4 While naive of profiles (RNA-seq). expression cDNA for technologies sequencing s e l c i rt A  reg

Foxp3 cells versus STAT5b-CA-expressing T STAT5b-CA-expressing versus cells 2 c a receptors-ligand normalized read count) in T in count) read normalized TCR dependen

Cre-ERT2 PC2 (6.37%) –20 –10 Cell adhesio 10 20 Cytoskeleton

0 Cre-ERT2 Stat5bCA Control Cell surface RNA-seq analysis of T of analysis RNA-seq JAK-STAT P signaling −40 + value, value, T mice (Control T (Control mice reg T naive re −20 T Supplementary Table 1 Supplementary cells relative to their expression in in expression their to relative cells T PC1 (80.53% n g s t re reg reg ≤ g ) or naive T cells from from T cells naive ) or ); diagonal lines indicate change in expression of at least 1.5-fold (top line) or 0.67-fold (bottom line); colors indicate significant significant indicate colors line); (bottom 0.67-fold or line) (top 1.5-fold least at of expression in change indicate lines diagonal ); 0.05) upregulation (of at least 1.5-fold; red) or downregulation (of at least 1.5-fold; blue) of expression above a minimal threshold threshold a minimal above expression of blue) 1.5-fold; least at (of downregulation or red) 1.5-fold; least at (of upregulation 0.05) cells, presented as net pathway perturbation (status of pathway: activated (positive values) or inhibited (negative values)) based based values)) (negative inhibited or values) (positive activated pathway: of (status perturbation pathway net as presented cells,

Expression (log Control 2 0 −1 Stat5bCA Control +

T Stat5bCAT naive ) 4 0 1 0 reg a T (three replicates per cell subset (columns)), grouped by product function (left margin); margin); (left function product by grouped (columns)), subset cell per replicates (three reg naive cells (Expressed; change relative to that in in that to relative change (Expressed; cells Control T

), ), STAT5b-CA-expressing T T reg naive naive 0 2 reg ) cells expressing an active form of STAT5.of form ( active an expressing cells Stat5bCAT hi

cells from from cells reg P T + Irf Cxcl10 Cd200 Cxcr6 Tox Il1r2 Rhoc Vim Frmd5 Myo Drc Lgals1 Lama5 Itgb7 Selplg Cd47 Itgb3 Ptafr F2r Tnfsf8 Il9r Il1rl1 Klrg1 Socs2 Cish Actr3b b value of the enrichment and node size is proportional to gene-set size. gene-set to proportional is size node and enrichment the of value T cells from both groups of mice mice of groups both from cells T reg 8 Expression (log2) ); line thickness and color are proportional to the similarity coefficient between connected nodes; node color color node nodes; connected between coefficient similarity the to proportional are color and thickness line ); 1 T cells 6 reg reg Stat5bCA Treg –5 10 15 20 Rosa26 0 5 5.9 8.7 5.7 4.1 2.5 1.7 1.0 3.6 1.5 1.5 9.0 2.0 3.6 4.7 5.0 9.2 6.7 5.7 1.5 1.0 9.2 4.5 3.3 2.2 3.4 0.04 P cells. ( cells. –5 value Frmd5 342 3 × × × × × × × × × × × × × × × × × × × × × × × × × Gzmb Foxp3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 (right). Numbers in parentheses (key) indicate total genes in each group. ( group. each in genes total indicate (key) parentheses in Numbers (right). f Itgb3 0 ) Network analysis of gene-ontology term enrichment among genes significantly upregulated in upregulated significantly genes among enrichment term gene-ontology of analysis ) Network Expression (log –9 –1 –1 –5 –8 –6 –5 –1 –3 –4 –233 –2 –9 –5 –1 –2 –1 –2 –2 –1 –6 –1 –7 –7 –1 Il1rl1 Stat5bCA reg Myo6 d 5 7 0 6 2 5 3 0 2 6 6 8 4 3 control ) Empirical cumulative distribution function for the change in expression (log expression in change the for function distribution cumulative ) Empirical Klrg Foxp3 cells was markedly markedly was cells Cre-ERT2 + 5 e Crip1 T Cytokine-cytokine receptorinteraction 1 Lad1 reg Irf8 Foxp3 10 T Nr4a2 reg reg cells or or cells re Cre-ERT2 Lt g a Chemokine signalingpathway cells from from cells mice (Control T (Control mice cells Il1r JAK-STAT signalingpathway 2 1 ) Cd200 PI3K-Akt signalingpathway 2 5 Cre-ERT2 Stat ECM-receptor interaction 314 1 3 T 20 f Foxp3 3 reg (left) or the subsets of genes upregulated (TCR up) or downregulated (TCR down) in down) (TCR downregulated or up) (TCR upregulated genes of subsets the or (left) d Platelet activation cells; dashed outlines (added manually) indicate groups of similar gene- similar of groups indicate manually) (added outlines dashed cells; adhesion mice (Stat5bCA T (Stat5bCA mice

Rosa26 Cumulative fraction 0.0 0.2 0.4 0.6 0.8 1.0 Cell Regulation ofcell 0.01 Cre-ERT2 0.0 Foxp3 Regulation of proliferation cell motilit −4 reg Similarity coefficient a Act down(227): Act up(390): Expressed (10,589) ) Principal-component analysis of RNA-seq data sets of T of sets data RNA-seq of analysis ) Principal-component ) or STAT5b-CA-expressing T STAT5b-CA-expressing ) or populations or naive CD4 naive or populations 6a Fig. STAT5( of form active the by affected Stat5bCA (node color (line color) Cre-ERT2 T P Stat5bCA reg -value y −2 Expression (log + cells; far right, false-discovery-rate (FDR)-adjusted global global (FDR)-adjusted false-discovery-rate right, far cells; −3 T Foxp3 reg ). Among all genes expressed (~11,000) in either the T the either in (~11,000) expressed genes all Among ). T P Regulation ofresponse ) cells versus control control versus cells T reg <2.22 naive Immune response

re P aDV 2 0 g <2.22 cells) and for subsets of genes upregulated (Act up) or up) (Act upregulated genes of subsets for and cells) /control −2 Cre-ERT2 to woundin 1. ), assessed using the 15% of genes with the highest highest the with genes of 15% the using assessed ), 0 0 Net pathwayperturbation(z-score) A × 2 10 fold) NCE ONLINE PUBLIC ONLINE NCE

× 10 –1 T mice (Stat5bCA T (Stat5bCA mice −1 6 re –16 g g proces organismal multicellular Regulation of 4 reg Organ development 0 + s Foxp3 T cell populations analyzed, 342 genes genes 342 analyzed, populations cell T cells from from cells of biologicalproces + Positive regulation

T Cumulative fraction 0. 0. 0. 0. 0. 1. reg 0 2 4 6 8 0 cells relative to their expression expression their to relative cells 3 2 1 Signal transduction Cre-ERT2 −4 GPCR signalingpathway signaling pathway Cell surfacereceptor TCR down(16): TCR up(190): Expressed (10,589) reg proces multicellular organisma Negative regulationof P A values (far right), right), (far values Single-organis TION ), naive ), T from naive cells Rosa26 Stat5bCA c Fig. 5 Fig. ) Expression of selected genes genes selected of ) Expression s T proces e Expression (log −2 s ) Signaling-pathway-impact ) Signaling-pathway-impact reg

cells, divided by total total by divided cells, nature immunology nature

s P Stat5bCA a T <2.22×10 P 2 re b and and m =1.6×10 values) of all genes genes all of values) g ) Gene expression expression ) Gene 2 0 /control Overlap 26 /16 14 /74 20 /10 19 /81 27 /90 7 /20 l 2 Foxp3 fold) reg Supplementary Supplementary Foxp3 cells from from cells –16 2 9 –5 T re Foxp3 g

Cre-ERT2 0.002 0.051 0.023 0.00077 0.00067 0.00067 P

P value Cre-ERT2

value value

Cre-ERT2 1 4 reg

cell cell

© 2016 Nature America, Inc. All rights reserved. nature. A published study has shown that exposure of T of exposure that shown has study published A nature. T the STAT5b-CA reinforce that simply not did suggested STAT5b-CA in trend opposite an showed STAT5b-CA ( reorganization cytoskeletal and migration adhesion, cell in involved CA and T were upregulated and 314 genes were downregulated in STAT5b-CA (TCR TCR-sufficient of frequency the of summary Foxp3 percent indicate areas outlined to adjacent Numbers tamoxifen. with 2 weeks for CD4 of ( well. a single represents symbol each cytometry; flow by analyzed IU/ml), (100 IL-2 mouse recombinant of (right) presence or (left) absence the in axes) (horizontal ratios various at min 720 for together culture after cytometry flow by assessed Violet), CellTrace dye the CD11c and CFSE) dye the with labeled and mice age-matched and sex- from (obtained (key) ( manner. TCR-independent 6 Figure nature immunology nature T of depletion transient by induced inflammation mice per group in each. in group per mice ( three of representative experiment * for 2 weeks with tamoxifen, analyzed by flow cytometry. Each symbol ( × 10 (3 CD8 CD4 producing T in expression Foxp3 IL-2R GITR, Tcra in T cytokines the (activated) cells LNs and for of cells positive pro-inflammatory various sex- and age-matched b Fig. 5 Fig. LNs Splee P a reg

< 0.05, ** < 0.05, Conjugated cells (%) + cells relative to their expression in STAT5b-CA 10 20 30 fl/fl + Supplementary Fig. 6b Fig. Supplementary T 0 n Foxp3 Foxp3 4 0 + reg c 5 cells in the LNs and spleen of sex- and age-matched age-matched and sex- of spleen and LNs the in cells Foxp3

10 10 10 10 ). Several genes that were upregulated or downregulated in in downregulated or upregulated were that genes Several ). cells each) sorted from sex- and age-matched age-matched and sex- from sorted each) cells Augmented STAT5T in Augmented signaling cells encoded various cell-surface molecules and receptors receptors and molecules cell-surface various encoded cells 0 2 3 4 5 Foxp Tcra − 0 α TC T cells (5 × 10 (5 T cells

10 5.35 4.23 Cre-ERT2 2 (CD25) and KLRG1 in T in KLRG1 and (CD25) − P R /wt

T 10 + DC/T cel 3 < 0.01 and *** and < 0.01

3 Rosa Foxp Foxp β Cre-ERT2 and CD8 and No IL-2 13.2 10 15 reg 4 3 3

10 Stat5bCA

cells relative to their expression in naive T cells cells T naive in expression their to relative cells 5 Cre-ERT2 Cre-ERT2 and and l reg 1 8 4.11 6.25 Foxp Rosa Tcra cells (top row), expression of CTLA-4, IL-2R CTLA-4, of expression row), (top cells Foxp Tcra +

T non- 18.7 /wt 12.2 T cells (bottom row) in the LNs of of LNs the in row) (bottom T cells 3 Stat5bCA reg Cre-ERT2 a 5 aDV *** 3 ** ) ) cells each) together with T with together each) cells Cre-ERT2 T fl/fl 2 reg In vitro In ). The gene set upregulated in STAT5b- in upregulated set gene The ). P Rosa26 A < 0.001 (modified ANCOVA in Prism software ( software Prism in ANCOVA (modified < 0.001 10 20 30 40 0 NCE ONLINE PUBLIC ONLINE NCE + T 8 4 0 a Foxp3 cells (%) reg Tcra Tcra ) or two ( two ) or 10 15 20 25 formation of conjugates of of conjugates of formation 0 5 reg Stat5bCA

TCR /wt /wt cells from the LNs of those mice (bottom row), analyzed by flow cytometry. ( cytometry. flow by analyzed row), (bottom mice those of LNs the from cells reg β + Rosa Foxp LN DC/T cel cells increases the formation of conjugates of T of conjugates of formation the increases cells

TCR + IL-2 + S s 3 b Stat5bCA T Cre-ERT2 Foxp3 , β – d reg ) or four ( four ) or β TCR l + Foxp3 cells ( cells ) and TCR-deficient (TCR TCR-deficient ) and β + * NS Cre-ERT2 pleen

− TCR reg Tcra Cre-ERT2 *** T A reg – TION β 12 reg cells leads to a to leads cells Fig. 5 Fig. cells with TCR ablation (TCR ablation TCR with cells c fl/fl ) independent experiments with similar results with three or more ( more or three with results similar with experiments ) independent mice (key) treated with tamoxifen for 2 weeks (top row), and expression of Foxp3, CTLA-4, CTLA-4, Foxp3, of expression and row), (top 2 weeks for tamoxifen with treated (key) mice cells ( c Tcrb Tcra

Foxp3 + hi

reg d reg Foxp3 CD4 CD44

c 3 6 Foxp3 −/− cell sig cell

), which which ), (MFI 10 ) cells ( 10 )

fl/wt × × cells to to cells Fig. Fig. 5 0 2 4 6 8 0 1 2 3 4 b

Tcrd + + 5 2 + Cre-ERT2 – α

Foxp3 CD4 IFN-γ cells (×10 ) CTLA-4 (MFI × 10 ) Foxp3 cells (%) d (CD25) and KLRG1 in T in KLRG1 and (CD25) 10 15 20 25 10 15 20 25 ) represents an individual mouse; small horizontal lines indicate the mean ( 0 5 0 2 4 6 8 0 5 Cre-ERT2 −/− b + -

* recipient mice 3 weeks after transfer of wild-type CD4 wild-type of transfer after 3 weeks mice recipient Cre-ERT2 * , , *** Tcra β they jointly regulated to potentiate the suppressor activity of T of by the activity affected set gene the that revealed However, analysis our cells. suppressor the potentiate to regulated jointly they expression whose genes of set overlapping largely a on acting were that the TCR- and STAT5-dependent signaling pathways in T pathways TCR- the that and STAT5-dependent signaling T STAT5 of form ( active settings matory the T by activated highly in found conferred those with correlated cells these in changes function of STAT5b-CA function suppressor marked change in their gene expression and a potent in increase their − non-T ) ) Foxp3 reg *** a **

* + hi fl/fl

) or two-tailed unpaired Student’s Student’s unpaired two-tailed ) or CTLA-4 CD8 CD44 cells to exert their suppressor function suppressor their to exert cells or or 2 6 Ros Foxp3 Rosa26 (MFl × 10 ) cells (×10 ) reg 10 15 Tcra 0 5 0 2 4 6 8 a cells or or cells + Stat5bC + β cells among CD4 among cells Cre-ERT TCR lo * *

fl/wt 3 CD3 + + 4 CD25 (MFI × 10 ) 3

Stat5bCA CD4 IL-4 cells (×10 )

reg Foxp3 (MFI 10 ) A

10 15 20 × + Foxp3 β 5 0 Rosa26 0 2 4 6 2 1 2 3 4 5 0 DCs (obtained from C57BL/6J mice and labeled with with labeled and mice C57BL/6J from (obtained DCs * − cells and DCs and potentiates suppressor function in a in function suppressor potentiates and DCs and cells reg lo cells (top left) or Foxp3 or left) (top cells Foxp3 ; ; TCR Fig. 5 Fig. cells (middle row) and quantification of cytokine- of quantification and row) (middle cells Cre-ERT ** * ** Foxp3 + + GlTR CD4 IFN-γ 3 Stat5bCA Cre-ERT2

3 4

− 6

(MFl 10 ) 2 ** . Consistent with the heightened suppressor suppressor heightened the with Consistent . × cells ( 10 )

T × d ** 0 1 2 * 0 1 2 3 + ). TCR signaling is required for the ability of ability for the is required signaling ). TCR reg Cre-ERT2 T + cells at left. ( left. at cells ** Tcra Tcra ) or TCR-sufficient T TCR-sufficient ) or reg Foxp3 or or cells, we found that the gene-expression Foxp3 / / ** * Ros Foxp3 Rosa26 or or

+ + 5 + + TC + TC + TC

Cre-ERT2 CD8 IFN-γ cells (×10 ) KLRG-1 cells (%) a 10 Foxp3 Stat5bC Cre-ERT2 4 6 8 0 1 2 3 0 2 t Cre-ERT -test ( -test R R R d + + + – –

) Frequency of T of ) Frequency CD25 CD4 IL-4

T T + T

Stat5bCA 2 5 A re re TCR re

c (MFl 10 ) cells ( 10 ) *** × × Foxp3 2 mice (above plots) treated treated plots) (above mice Cre–ERT 10 15 g g g * ) Frequency of CD44 of ) Frequency from from 0 2 4 6 8 from 0 5 b , , – Rosa26 β b 34 d *** *** Cre-ERT + ) Flow cytometry (left) (left) cytometry ) Flow * )). Data are from one one from are Data )). Tcra Tcra b Foxp3 , cells (top right). Right, Right, right). (top cells Foxp3 reg 3 , mice (key) treated treated (key) mice 5 c . Thus, it was possible . it Thus, possible was *** cells (TCR cells ) or five or more ( more or five ) or + / / ** 2 Foxp3 Cre-ERT Ros Foxp3 Stat5bCA reg Cre-ERT2 ticles e l c i rt A a reg cells in inflam in cells Stat5bC 2

Cre-ERT + + + − cells and and cells KLRG-1 CD8 IFN-γ and and

Foxp3 cells (%) 6 A cells (×10 ) 10 15 T + Foxp3 0 5 0 2 4 6 2 T reg hi ± reg ***

s.e.m.). cells cells reg Cre-ERT2 Cre-ERT )

d

cells cells

*** ) * 2 reg  -

,

© 2016 Nature America, Inc. All rights reserved. in y T by tion func suppressor of and phenotype acquisition the antigen-experienced for activated, an required is signaling TCR that shown has It been shown). not (data Foxp3 of induction the after immediately in function suppressor Tcra restore to failed expression STAT5b-CA ( ablation TCR undergone had CA T features of some Indeed, signals. TCR-dependent contemporaneous signal T of STAT5 activity suppressor the enhanced potentiated ing that suggested results these incomplete, ( recipients lymphopenic into STAT5b-CA firmed in experiments in which TCR-deficientflow-cytometry-sorted T in of T recovery treated gated in by part of expression the form active of STAT5 in tamoxifen- T cell activation and pro-inflammatory cytokine production was miti than in STAT5b-CA TCR-deficient not STAT5b-CA TCR-sufficient only of proportion the mice, and TCR-deficient T TCR-deficient and of form STAT5of in of TCR-sufficient ~50% active the observed was ( administration tamoxifen of weeks T TCR-deficient of proportion small a T of half a to up in TCR the of ablation in result theoretically can nation the of In heterozygous function activation suppressor in impaired from results results and that system mice immune these in efficient highly is inducible Cre-mediated ablation of analyzed we this, Rosa26 investigate To manner. TCR-independent a in T of function suppressor the STAT5potentiate of can vation in tamoxifen-treated the decreased expression of co-stimulatory molecules by DCs observed tions of STAT5b-CA of conjugates of T found that STAT5b-CA expression in T DCs tively active STAT5 in T DCs with interactions cells. target the with interactions their potentiate might 6c Fig. STAT5b-CA in expression among locomotion genes expressed differentiallycellular in STAT5b-CAand adhesion cell interactions, matrix and extracellular in cell-cell involved products encoding of sets gene over-representation revealed This enrichment. molecular-function activation of STAT5, we performed analysis of signaling pathways and activity. suppressor of T aspects distinct probably and of genes sets distinct largely T of activity suppressor the in role ble indispensa an served STAT5 pathway and pathway naling signaling ( manner TCR-dependent a in T in expressed that from distinct STAT5was of form active s e l c i rt A 1 0 The findings reported above raised the question of whether acti whether of question the raised above reported findings The Since intravital imaging of T T of aspects understand Tobetter

+ reg reg fl/fl reg T in vitro cells due to allelic exclusion at the the at exclusion allelic to due cells cells that had undergone ablation of the TCR was also con also was TCR the of ablation undergone had that cells el ta hd en bevd n C-ufcet STAT5b- TCR-sufficient in observed been had that cells ). This result suggested that in T in that suggested result This ). reg Rosa26 Stat5bCA Tcra Tcra el wr sil rsn i STAT5b-CA in present still were cells reg fl/fl fl/wt . . In agreement with the gene-set–enrichment analysis, we cells + reg T Rosa26 Stat5bCA Foxp3 cell suppressor function by the active form of form by STAT5active the function suppressor cell Foxp3 reg Tcra reg 3 4 el ad T and cells . Thus, our results suggested that activation of of activation that suggested results our Thus, . cells and DCs ( andcells DCs + Cre-ERT2 Rosa26 fl/wt reg T Stat5bCA Cre-ERT2 Foxp3 3 6 reg reg cells in in cells , we assessed the potential effect of constitu of effect potential the assessed we , Foxp3 cells on their ability to form conjugates with cells with DCs − Cre T Stat5bCA Foxp3 mice with a a with mice mice ( mice reg reg mice in which TCR deletion occurred occurred in TCR mice deletion which Cre-ERT2 eff i. 6 Fig. + Tcra cells ( cells i. 5 Fig. cells T i. 6c Fig. cells were adoptively transferred transferred adoptively were cells Tcra Cre-ERT2 reg Foxp3 Fig. 6 Fig. Fig. 6 fl/wt cells, was greater in these mice mice these in greater was cells, reg d Fig. 6 Fig. reg in vivo mice, Cre-mediated recombi Cre-mediated mice, (and thus the TCR) in T d reg Fig. 5e Fig. ). Although the ‘rescue’ was was ‘rescue’ the Although ). reg cell function potentiated by potentiated function cell reg cells promoted the formation . hs bt te C sig TCR the both Thus, ). Rosa26 , in vitro d a cells cells Cre-ERT2 cells in these mice after 2 after mice these in cells b mice ( cells, activation of STAT5 of activation cells, ). These enhanced interacenhanced ). These ). However, we note that that note we However, ). ). The marked increase in ). The increase marked Tcra Tcra reg + b has revealed their stable T ). Although expression expression Although ). , cells in the absence of absence the in cells f reg in vivo in and and Stat5bCA fl locus. We observed Weobserved locus. were consistent with Fig. 6 mice ( cells relative to their lee Tamoxifen- allele. + Supplementary Supplementary T by controlling controlling by c Foxp3 ). ). This partial reg Fig. 3 + cells that that cells T reg Cre-ERT2 reg f reg reg ). reg cells, cells, cells cells cells cells cells cell cell 3 4 ­ ------.

in vitro in and differentiation the linking of maintenance T nexus critical a as serve therefore sion expres Foxp3 of maintenance and induction the in involved is and Foxp3 of T maintenance and differentiation the linking in signaling IL-2R–STAT5 for role key a suggested results These deficiency. IL-2R from results that disease fatal the from mice STAT5rescued of form active an of expression T in STAT5deficiency that found we Furthermore, function. pressor T mature of tenance main in not the only signaling for role IL-2R essential cell-intrinsic, a demonstrate unequivocally to us enabled tools genetic new These of form STAT5. of an active expression induced with in combination of Il2rb deletion conditional the through issue this addressed function T by consumption IL-2 and in signaling IL-2R for T functional of absence the in disease autoimmune of T sion of IL-2R-sufficient T by consumption IL-2 whether vation T T wild-type and proliferation of effector T cells T of ability the for dispensable is IL-2R that deprivation 2 of T function suppressor the in role essential an serve to suggested been evidence indirect can also directly promote the suppressor ability of T of ability suppressor the promote directly also can signaling IL-2R whether of question the raised have findings Such function in the absence of STAT5 or IL-2R. Indeed, our analysis of of analysis our Indeed, IL-2R. STAT5 or of absence the T in of function loss severe the for account not could alone in Foxp3 expression decrease the Thus, is much more last severe. the T IL-2R-deficient STAT5- or of that resembling expression Foxp3 reduced exhibit do Although both T CNS2-deficient bind to CNS2 and the of Foxp3 expression during their extrathymic differentiation extrathymic their during expression Foxp3 of T mature of maintenance the in IL-2 the within elements of to IL-2 and regulatory as IL-2, antibody delineation well as genetic tion of IL-2 and provision of IL-2 in the form of immunocomplexes of in thymus the cells T of differentiation the and expression Foxp3 of induction the for required cytokine key a is IL-2 that demonstrated have subunits 2R IL- and IL-2 in deficiency germline with mice of analysis Published DISCUSSION the in IL-2R, signaling TCR permissive and TCR the of promotes T differentiation via signals, T of two require differentiation sequential these the for of ment reminiscent is observation That tion. T STAT5in mature function suppressor TCR-independent potentiated reg reg reg A chief limiting factor in efforts to experimentally assess a role role a assess experimentally to efforts in factor limiting chief A cells resulted in a similar loss of suppressor function and that that and function suppressor of loss similar a in resulted cells cell–mediated suppression that are independent of IL-2-depri of independent are that suppression cell–mediated el ta hd led udroe C-eedn matura TCR-dependent undergone already had that cells reg 3 Il2rb alleles and ablation of the expression of these genes in T in genes of these expression of the ablation and alleles 8 promoter and the intronic intronic the and promoter . Complexes of the transcription factors Runx and CBF and Runx factors transcription the of Complexes . cells by causing the death of activated CD4 of activated death the by causing cells 6 , study has proposed a role for IL-2 signaling on the basis of of basis the on signaling IL-2 for role a proposed has study 7 . However, the last studies left open the major question of of question major the open left studies last the However, . −/− n vivo in aDV reg mice from disease observed after adoptive transfer of of transfer adoptive after observed disease from mice 20– cells suggested the existence of major mechanisms of of mechanisms major of existence the suggested cells A NCE ONLINE PUBLIC ONLINE NCE 2 a be te ak f dqae eei tos We tools. genetic adequate of lack the been has 2 reg 4 reg 1 . On the other hand, other reports have suggested suggested have reports other hand, other the On . 5– . . In consumption addition, of IL-2 by T cells with their suppressor function. A published function. suppressor their with cells reg cells, the impairment of suppressor function in in function suppressor of impairment the cells, Foxp3 1 reg 1 reg Foxp3 cells in the thymus, where STAT5 signaling signaling STAT5 where thymus, the in cells . Furthermore, antibody-mediated neutraliza antibody-mediated . Furthermore, cells and their fitness but also in their sup their in also but fitness their and cells cells and their function. STAT5 the to function. binds their and cells eff 3 locus, have revealed an important role for an important have revealed locus, 7 cells, since IL-2 is a since probably main driver cells, . promoter and Foxp3 affect expression reg 8 , cell precursors that have experienced that cell precursors have experienced 3 9 reg reg . . Furthermore, the rescue of cells and CBF cells is essential for the suppres the for essential is cells Foxp3 A reg TION cells and in the stabilization stabilization the in and cells regulatory element CNS2 CNS2 element regulatory

nature immunology nature reg cells to suppress the the suppress to cells β + reg -deficient -deficient T T cells due to IL- due T cells reg cell suppressor suppressor cell reg reg cells in their their in cells cells and can can and cells cells. reg Il2ra cells has Il2ra reg reg 16 β , cells cells cells 28 also also and and , −/− 3 reg 4 8 0 ------. .

© 2016 Nature America, Inc. All rights reserved. on Note: Any Supplementary Information and Source Data files are available in the codes. Accession the t of in version available are references associated any and Methods M cells. T pressive ducks of IL-2R conservation of evolutionary and importance chicken in activity suppressor CD4 a although that thy T differentiated of function driven activation of STAT5 in supporting and boosting the suppressor and transplantation. organ disorders inflammatory and autoimmune of variety a for pies T T of of design optimal the for modification promise hold might such that in suggest would autoimmunity presence their organ-specific of severity reduced significantly T of activity sor suppres enhanced observed The cancer. of forms some in therapy targeting. specific for suppressor of T the function for booster a is IL-2 while Thus, IL-2. of source lar T T CD4 of suppression the of cue’ function suppressor their of ‘licensing’ enables consumption. IL-2 through CD8 of control the for mechanism distinct a for need the for explanation likely a provide features ing T into T cells IFN- and IL-4 as such cytokines of effector to production the in contrast TCR engagement, CD4 naive both of vation acti upon is produced IL-2 Furthermore, stimulation. IL-2-induced CD8 naive both of sensitivity exquisite observed the given sense makes finding unexpected ingly CD8 of suppression the for sable T by IL-2 of consumption IL-2R-dependent CD4 T the of T expression peripheral in transgene STAT5b-CA-encoding reduced and cells B and cells T pre-leukemic of expansion population massive the to due problematic is result last in the thymus cell differentiation in disease E and promoter proximal the by driven STAT5b-CA encoding transgene a that finding published the with odds at be to appear might results Such of T of of an form active of STAT5 ‘rescues’ the nearly partially complete loss to bind to DCs and suppress their activation. Furthermore, expression T of ability STAT5 of heightened a form to pointed active the of expression by conferred features functional and expression gene nature immunology nature reg reg reg line version of the pape ethods Our findings have highlighted a central role for IL-2R-signaling- for role central a highlighted have findings Our of means potent a as emerging are cells T modified Genetically It has been suggested that sensing of local IL-2 production by T T depriving that demonstrated have studies Our cells was fully dispensable for was cells the dispensable suppression fully of IL-2R-sufficient cells expressing an form expressing cells active of STAT5 that activated suggested cells can suppress autoimmunity without identifying the cellu the identifying without autoimmunity suppress can cells reg + + T cells, even though IL-2R signaling was required. However, required. was signaling IL-2R though even cells, T T cell subsets with high expression of IL-2R of expression high with subsets cell T cell suppressor function in the absence of TCR signaling TCR of absence the in function suppressor cell Il2rb he pape he eff reg cells on cells a much scale time longer −/− cells, it might not have an indispensable role as role a cue not have it an might indispensable cells, µ reg GEO: microarray data, data, microarray GEO: enhancer fails to curtail fatal lymphoproliferative lymphoproliferative fatal curtail to fails enhancer mice despite restoring Foxp3 expression and T and expression Foxp3 restoring despite mice r cells expressing an active form of STAT5 and and STAT5 of form active an expressing cells Foxp3 . r . γ

, which requires the differentiation of naive naive of differentiation the requires which , + aDV ortholog has not been identified in birds, birds, in identified been not has ortholog T cells and CD8 and cells T reg + A T cells and activated CD8 activated and cells T cells. In this context, it is notewor is it context, this In cells. + NCE ONLINE PUBLIC ONLINE NCE T cell responses by IL-2R-deficient IL-2R-deficient by responses cell T 9 + . . However, of the interpretation the T cell responses. The last, seem last, The responses. cell T + T cell responses by T by responses cell T GSE8455 + 42 T cells within hours of of hours within cells T reg , 4 2 3 cells was indispen was cells 4 reg 1 . This suggests the the suggests This . reg 1 . However, the ‘res However, the . . . Such distinguish α cell–based thera cell–based 3 eff cells. A function in sup function . TION α cells of IL-2 by IL-2 of cells have have +

T cells to to cells T reg reg in vitro in reg reg online online cells cells cells cells cells cells 34 cells Lck , 3 reg 5 ------.

reprints/ R version of t The authors declare competing interests:financial details are available in the the Y.F. conducted experiments; U.K. generated the and wrote and edited the manuscript.; T.C., A.K.K., A.G.L., X.F., Y.Z., G.G. and T.C., J.D.F. and A.Y.R. conceived of the project, the designed experiments Cancer Research (A.Y.R.), and the Howard Hughes Medical Institute (A.Y.R.). Prevention Initiative funded by the Conrad N. Hilton Foundation and Ludwig at Memorial Sloan Kettering Cancer Center (A.Y.R.), Hilton-Ludwig Cancer AI034206 to A.Y.R.), DFG Emmy Noether programme (G.G.), the Ludwig Center CA008748 to Memorial Sloan Kettering Cancer Center Core Facilities; and R37 MD-PhD Program, for A.G.L. and X.F.), the US National Institutes of Health (P30 to (T32GM07739 the Weill KetteringCornell/Rockefeller/Sloan Tri-Institutional (T.C.), the US National Institutes of Health Medical Scientist Training Program Circulation (T.C.), Lucille Castori Center for Microbes, Inflammation & Cancer Strategic Young Researcher VisitsOverseas Program for Accelerating Brain Stat5a-Stat5b construct; and L. Henninghausen (US National Institutes of Health) for (pMX-STAT5B(1*6)); K. Rajewsky (Max Delbrück Center) for the We thank T. Kitamura (The University of Tokyo) for the mSTAT5b-CA vector 18. 17. 16. 15. 14. 5. 4. 3. 2. 1. 13. 12. 11. 10. 9. 8. 7. 6. C AUTH A eprints and permissions information is available online at at online available is information permissions and eprints ck OM

Il2ra Komatsu, N. Komatsu, Y.P.Rubtsov, maintenance Homeostatic S. Sakaguchi, & T. Takahashi, S., Hori, R., Setoguchi, L. Barron, S. Malin, Furtado, G.C., Curotto de Lafaille, M.A., Kutchukhidze, N. & Lafaille, J.J. Interleukin Waldmann,T.A.receptor. interleukin-2 multi-subunit The development the programs Foxp3 A.Y. Rudensky, & M.A. Gavin, J.D., Fontenot, by development cell T regulatory of Control S. Sakaguchi, & T. Nomura, S., Hori, Immunologic M. Toda, & M. Itoh, M., Asano, N., Sakaguchi, S., Sakaguchi, Chen, W. Chen, K.B. Vang, IL-2. on depends crucially immunity, not Tolerance, A.L. Bayer, & T.R. Malek, Z. Yao, receptor IL-2 Farrar,M.A. & Blazar,B.R. Vogtenhuber,C., Yang,J., M.A., Burchill, for function A A.Y. Rudensky, & M.A. Gavin, J.P., Rasmussen, J.D., Fontenot, Malek, T.R., Yu, A., Vincek, V., Scibelli, P. & Kong, L. CD4 regulatory T cells prevent Almeida, A.R., Legrand, N., Papiernik, M. & Freitas, A.A. Homeostasis of peripheral Acad. Sci. USA Sci. Acad. T-cellplasticity.retaining population minor uncommitted an and lineage (2010). 1667–1671 neutralization. IL-2 (2005). by disease autoimmune of of natural Foxp3 cells. T regulatory functional development. cell (2010). pro-B during recombination 198 TGF- by cells T regulatory CD4 for required is signaling 2 (1989). 875–911 (2003). CD4 of function and Foxp3. various factor transcription the causes self-tolerance of mechanism single diseases. a autoimmune of Breakdown receptor IL-2 (CD25). expressing cells T activated by maintained self-tolerance 3285–3290 (2008). 3285–3290 CD4 govern redundantly Immunol. Rev. Nat. 109 cells. T β (2005). cells. T regulatory Foxp3-expressing in 2 interleukin IL-2. of function IL-2R in autoimmunity lethal CD4 controls CD4 (2002). 851–857 -dependent STAT5regulatory -dependent Foxp3+ of development the for required is activation now P O E , 1875–1886 (2003). 1875–1886 , , 4368–4375 (2007). 4368–4375 , + index.htm fl TI cls I-R lh ad L2 hp a ouain f euaoy el that cells regulatory of population a shape IL-2 and alpha IL-2R cells: T R R C allele. he pape et al. et N l J. Immunol. J. e ON et al. et G G FI et al. et fl dg mice. Supported by the Japan for Society the Promotion of Science, t al. et t al. et + Nonredundant roles for Stat5a/b in directly regulating Foxp3. regulating directly in Stat5a/b for roles Nonredundant et al. et TRIBUTI et al. et T cell numbers. cell T N men Role of STAT5 in controlling cell survival and immunoglobulin gene immunoglobulin and STAT5survival of cell Role controlling in l Conversion of peripheral CD4 peripheral of Conversion .

r + 106 . A CD25 utn eg: ehnss f L2dpnet aneac of maintenance IL-2-dependent of mechanisms edge: Cutting Immunity Heterogeneity of natural Foxp3 natural of Heterogeneity N L2 -, n -5 bt o tyi srml lymphopoeitin, stromal thymic not but -15, and -7, IL-2, Stability of the regulatory T cell lineage in vivo. in lineage cell T regulatory the of Stability

CIAL CIAL I 4 , 1903–1908 (2009). 1903–1908 , t 178 , 665–674 (2004). 665–674 , s + J. Immunol. J. + CD25 ONS + CD4 Foxp3 , 280–290 (2007). 280–290 , β

nuto o tasrpin atr Foxp3. factor transcription of induction N 17 + + J. Immunol. J. β T euaoy cells. T regulatory regulatory T cells by interleukin (IL)-2 and induction , 167–178 (2002). 167–178 , dfcet ie Ipiain fr thenonredundant for Implications mice. -deficient + E Science J. Immunol. J. regulatory T cell development. cell T regulatory + R euaoy cl function. cell T regulatory ES

155 T S

, 1151–1164 (1995). 1151–1164 , 299

185

, 1057–1061 (2003). 1057–1061 , 169 + Il2rb CD25 , 6426–6430 (2010). 6426–6430 , , 4850–4860 (2002). 4850–4860 , + fl T cells: a committed regulatory committed a cells: T a. Immunol. Nat. . x. Med. Exp. J. allele; andallele; J.D.F. generated − Nat. Immunol. Nat. a. Immunol. Nat. naive T cells to CD4 to cells T naive Annu. Rev.Annu. Biochem. http://www. ticles e l c i rt A . x. Med. Exp. J. J. Immunol. J. Rosa26

201

6

11 , 1142–1151 , Science 4 . x. Med. Exp. J. nature.com/

171–179 , 330–336 , 723–735 , Proc. Natl. Proc.

α + -chains online CD25

Blood 329 181 196

58 11

+ , , , ,

© 2016 Nature America, Inc. All rights reserved. 30. 29. 28. 27. 26. 25. 24. 23. 22. 21. 20. 19. s e l c i rt A 12

Cong, Y., Feng, T., Fujihashi, K., Schoeb, T.R. & Elson, C.O. A dominant, coordinated Y. Selective Chen, J. Sprent, & C.D. Surh, M.P., Rubinstein, M., Kovar, O., Boyman, M. Onishi, Zambrowicz,B.P. K.S. Smigiel, T. Yamaguchi, D. Busse, Barthlott,T. IL-2 edge: Cutting E.M. Shevach, & C.A. Piccirillo, E.E., Donovan, A.M., Thornton, CD4 M.J. Lenardo, & J. Reed, S., Ishihara, P.,L., Pandiyan, Zheng, Foxp3 of plasticity phenotypic and stability Lineage S. Hori, exploit it for the inductiontheexploitforIL-10production. itof CD4 of activation function. vitro in the for required critically is cells. T CD4 effector of apoptosis deprivation-mediated cytokine induce cells T regulatory USA microbiota. intestinal the to response cell-IgA regulatory T interleukin-2. of regulation through vivo 311 complexes. immune antibody-cytokine with subsets cell T of stimulation proliferation. cell promotes that mutant hematopoieticand cells. strainwidespreadtrapgeneleadsto expression of (2014). subsets. cell T regulatory distinct homeostatically USA Sci. expression. IL-2 and CTLA-4 controlling by cells T conventional USA microenvironments. cellular in lymphocytes T regulatory and Immunol. Rev. Immunol. , 1924–1927 (2006). 1924–1927 ,

106 107 Nat. Immunol. Nat.

et al. et , 19256–19261 (2009). 19256–19261 , (2010). 3058–3063 , J. Immunol. J. 110 t al. et et al. et et al. et , E2116–E2125 (2013). E2116–E2125 , Foxp3

259 t al. et Identification and characterization of a constitutively active STAT5 active constitutively a of characterization and Identification t al. et optn febc los hp I- sgaig ewe helper between signaling IL-2 shape loops feedback Competing etal. CD25 , 159–172 (2014). 159–172 , +

osrcin f efrcgiig euaoy cls from cells T regulatory self-recognizing of Construction Disruption ofoverlapping transcripts inthe ROSA beta geo26 C7 rvds oaie acs t I- ad defines and IL-2 to access localized provides CCR7 regulatory T cells promote T helper 17 cell development in development cell 17 helper T promote cells T regulatory 172

+ 8 Proc.Natl.Acad.USASci. CD4 , 1353–1362 (2007). 1353–1362 , , 6519–6523 (2004). 6519–6523 , + T cellscompeteTnaivewithCD4 Mol. Cell. Biol. Cell. Mol. Immunity Int. Immunol.Int. β

-galactosidasemouseembryosin

34 94 . x. Med. Exp. J. , 409–421 (2011). 409–421 , , 3789–3794, (1997).

+ 18 CD25 , 3871–3879 (1998). 3871–3879 ,

rc Nt. cd Sci. Acad. Natl. Proc. Proc. Natl. Acad. Sci. Acad. Natl. Proc. 17 + + T cells for IL-2 andIL-2cellsfor T + euaoy cells. T regulatory , 279–288(2005)., cl suppressor cell T Proc. Natl. Acad. Natl. Proc.

211 + CD25 121–136 , + Science Foxp3 + +

43. 42. 41. 40. 39. 38. 37. 36. 35. 34. 33. 32. 31.

Andersen, K.G., Nissen, J.K. & Betz, A.G. Comparative genomics reveals key gain- key reveals genomics Comparative A.G. Betz, & J.K. Nissen, K.G., Andersen, chicken of properties cell T Regulatory R.K. Selvaraj, & R. Shanmugasundaram, CD4 by secretion IL-2 N.J. Singh, & R.H. Schwartz, D., Bruniquel, D.K., Sojka, D. Rudra, D.Q. Tran, Feng, Y. Lio, C.W. & Hsieh, C.S. A two-step process for thymic regulatory T cell development. C.E. Tadokoro, J.C. Vahl, the for requirement Continuous A.Y. Rudensky, & W. Jin, A., Arvey, A.G., Levine, A. Arvey, L.K. Su, Kim,J.M., Rasmussen, J.P. Rudensky,& A.Y. Regulatory cellsT prevent catastrophic 113 (2012). 113 evolution. cell T regulatory during Foxp3 in events of-function CD4 competition. TCR-specific by influenced and transient, Immunol. J. rapid, is vivo in cells T (2009). cells. T regulatory in Foxp3 factor transcription (2009). human FOXP3 locus. Foxp3 the in Immunity vivo. in cells dendritic and cells T pool. cell T function. cell T regulatory in TCR cells. T regulatory (2014). in Foxp3 factor transcription gene. APC the of homolog mice. of lifespan(2007). the throughout autoimmunity + CD25 et al. aDV t al. et et al. et 28 t al. et + et al. et cells.

Immunity 172 , 100–111 (2008). 100–111 , Control of the inheritance of regulatory T cell identity by a cis element t al. et + A utpe netnl epai cue b a uain n h murine the in mutation a by caused neoplasia intestinal Multiple regulatory T cell suppressor function. Continuous T cell receptor signals maintain a functional regulatory functional a maintain signals receptor cell T Continuous t al. et Analysis of adhesion molecules, target cells, and role of IL-2 in IL-2 of role and cells, target molecules, adhesion of Analysis NCE ONLINE PUBLIC ONLINE NCE Inflammation-induced repression of chromatin bound bythe bound of chromatin repression Inflammation-induced , 6136–6143 (2004). 6136–6143 , J. Immunol. J. Cell uxCFea opee cnrl xrsin f the of expression control complexes Runx-CBFbeta

euaoy cls nii sal cnat bten CD4 between contacts stable inhibit cells T Regulatory 41

158 , 722–736 (2014). 722–736 , Science , 749–763 (2014). 749–763 ,

186 Nat. Immunol. Nat.

J. Exp. Med. Exp. J. 256 , 1997–2002 (2011). 1997–2002 , , 668–670 (1992). 668–670 , A TION

203

a. Immunol. Nat. 15 a. Immunol. Nat. nature immunology nature a. Immunol. Nat. J. Immunol. , 1070–1078 (2014). 1070–1078 , , 505–511 (2006). 505–511 , Front. Immunol. Front.

182 10

15 1170–1177 , , 2929–2938 8 191–197 , 580–587 ,

3 + + ,

© 2016 Nature America, Inc. All rights reserved. CD4 ( anti-B220 and anti-CD8 with stained were cells The enriched. were cells T and panning by cells accessary and cells B of In vitro T sorted into injected ( T cell–deficient and PBS, in resuspended FCS, 10% containing RPMI with washed 50 (Millipore; nase a TAT-Crecontaining medium RPMI in 2 recombi ml of serum-free pended of LNs the and spleens non-T in cells. T of TAT-Cre treatment 200 BioXcell), 2A3; IgG2a, (rat antibody control isotype-matched or (BioXcell), and S4B6-1 JES6-1 antibodies, monoclonal anti-IL-2 of two different cocktail vivo In cytometry. by flow detected were cells T cytokine-producing and A, vaccinia- various with (1 peptides antigenic virus-derived re-stimulated were Splenocytes 8. day on analyzed 10 × intrave (5 given virus were non-replicating of mice injection nous infection, virus vaccinia For cytometry. detected were flow cells by T cytokine-producing and A, brefeldin of presence the cells from T obtained splenic with co-cultured then were cells The analysis. the before h 6 plate U-bottom 96-well 10 × (2 a CD11c of using wells in sorted cultured were mice (Miltenyi) B6 microbeads unchallenged from DCs splenic responses, and analyzed on day 8. For the detection of monocytogenes L. Listeria sorter. cell FACSAria BD II a using expression GFP or YFP on Foxp3 of sorting Cell Biosciences). (BD anti-pY-STAT5antibody with stained and PFA 4% methanol, 90% with by followed permeabilized and fixed min, 20 for rmIL-2 without or with stimulated were cells STAT5staining, phosphorylated intracellular For kit. Permeabilization Fixation eBioscience with and stained harvested monensin, (5 to and CD3 CD28 antibodies software FlowJo (TreeStar). using For intracellular cytokine analyzed staining, cells were were stimulated data for 5 h with cytometry Flow cytometer. flow ( BioLegend or Bioscience Tonbo Biosciences, BD eBioscience, from purchased antibodies and cell sorting. Flow cytometry and tamoxifen of gavage previously described single as assessed a after months 2–3 challenged were mice experiments, infection and encephalomyelitis autoimmune experimental In 100 of gavage mg/ml. oral 40 given were of Mice concentration a at oil tamoxifen olive in treatment, dissolved was tamoxifen (Sigma-Aldrich) For non-survivors. as counted were and lethargic found were they once euthanized were mice unhealthy daily; tored moni were mice analysis, For survival guidelines. institutional the with ance accord in performed were and animal Center Cancer Kettering All Sloan Memorial at Center. committee use Cancer and care animal Kettering institutional by approved were Sloan experiments Memorial at facility animal pathogen-free specific the in housed and bred background, (B6) C57BL/6J a ously alleles are shown in The targeting strategies to generate L. Henninghausen. Il2ra Mice. METHODS ONLINE doi: of wells among eight were divided cells sorted The population. CD8-negative 10.1038/ni.3540 µ + fl 3 g each, twice a week, starting from 5–7 d after birth. after d 5–7 from starting week, a twice each, g T

ie ee eeae b J.D.F. by generated were mice 4 . The experimental mice were either generated on or backcrossed ontoon were generated mice or either backcrossed . The experimental Foxp3 4 IL-2 neutralization. IL-2 IL-2 capture assay. capture IL-2 reg and vaccinia infection. vaccinia and cells/well) with heat-killed heat-killed with cells/well) reg reg cells were sorted on the basis of GFP (YFP) expression alone in in alone expression (YFP) GFP of basis the on sorted were cells cells for for cells cells, 1 × 10 × 1 cells, Cre L. monocytogenes L. upeetr Tbe 2 Table Supplementary and and (LM10403S; 2000 cells/mouse) into the tail vein on day 0 day on vein tail the into cells/mouse) 2000 (LM10403S; Supplementary Figure 7 Apc in vivo in µ g/ml) and incubated at 37 °C for 45 min. The cells were were cells The min. 45 for °C 37 at incubated and g/ml) Tcrb Foxp3 Foxp3 Min 7 CD4 −/− suppression assay. suppression mice were from purchased the Jackson Laboratory. Pooled cells from LNs and spleens were depleted depleted were LNs spleens and from cells Pooled Tcrd Cre–ERT2 Cre Mice were given intraperitoneal injection of a of injection intraperitoneal given were Mice + For the induction of STAT5b-CA expression STAT5b-CAexpression of induction the For and µ Foxp3 3 –infected mice (1 × 10 × (1 mice –infected −/− 8 Cells were stained with fluorescence-tagged Mice were given intravenous injection of of injection intravenous given were Mice g/ml each) in the presence of brefeldin A of or brefeldin in presence the each) g/ml . Il2rb µ ) ) mice together with or without separately + µ L. monocytogenes L. Rosa26 ie ee ecie previously described were mice and Foxp3 and g/ml) for g/ml) 5 h of in the presence brefeldin l of tamoxifen emulsion per treatment. treatment. per emulsion tamoxifen of l − fl or CD8 or (generated by UK) and ) and analyzed using a BD LSR II II LSR BD a using analyzed and ) Stat5a/b L. L. monocytogenes Stat5bCA . Tcra Supplementary Table 2 Table Supplementary + 7 − PFU/mouse) on day 0 and and 0 day on PFU/mouse) Foxp3 fl cells was performed based based performed was cells fl mice were described previ mice were provided by by provided were mice Foxp3 (2 × 10 × (2 5 − cells/well) for 5 h in in h 5 for cells/well) T cells sorted from from sorted cells T Cr e –specific –specific immune mice were resus mice 7 cells/well) for for cells/well) Rosa26 Stat5bCA ), and and ), 17 , 4 4 ------.

rescence intensity of CellTrace Violet of the gated cells. gated the of Violet CellTrace of intensity rescence and Foxp3 T and cells T responder of proliferation Cell 1 of presence 1 × 10 to genomic features using HTSeq v0.6.1p1. The overall read alignment alignment read overall The v0.6.1p1. HTSeq using features genomic counted being to before v0.1.19 SAMtools with sorted were alignments settings. Read default with v2.2.2 Bowtie2 implementing v2.0.11 TopHat2 using GRCm38) assembly (Ensembl genome mouse to the aligned contamination then were reads adaptor and reads low-quality remove to settings standard with v0.32 Trimmomatic using trimmed were reads Raw effects. batch avoid to lane same the on sequenced and time same a at processed were samples All sample. per pairs read 50-bp of × to 27.5 10 an depth 2500 average HiSeq on Illumina the were sequenced Samples protocols. manufacturer’s following preparation library paired-end TruSeq and Illumina selection for Totaland poly(A) used RNA extracted was group. experimental each for analysis RNA-seq to subjected were replicates same the in group into were biological three replicate; pooled one experimental mice biological individual three from gener isolated were subsets samples cell T 12 of Spleen ated. total a and sorter, cell II FACSAria BD a using CD4 CD4 Splenic isolation. months 4 before gavage oral by tamoxifen of mg) (4 dose single a received group, and CA) sequencing. RNA positive. was two-tailed unpaired Student’s with software using from Prism the study.were performed analyses Statistical after or preparation after vitality cell 70% tal anomalies were excluded from the study. Cell samples that showed less than and after data analysis was completed. Wild-type mice with suspected congeni experiment a in single included size of sample for adjustment except of mice, genotypes to the were blinded number, and researchers identification unique experiments. animal for analysis Statistical by ELISA. measured IgA was centrifugation, and titrated, after collected were Supernatants Roche). mini; (Complete cocktail inhibitor protease DTT, and mM EDTA,1 1mM NP-40, 0.5% NaCl, (7 buffer extraction in dissolved and collected were pellets fecal fresh IgA, fecal of For measurement streptavidin. HRP-conjugated and biotinylated Biosciences) using BD (R35-118, performed anti-IgE was ELISA IgE Biotech). (Southern System IgG2b, IgG2c, IgG3 and IgA were by measured using ELISA SBA Clonotyping Measurement of serum and fecal immunoglobulin. naive CD4 10 × naive 4 Violet. CellTrace with stained and cytometry flow by purified were In vitro CTV (% of T Frequencies of IU/ml). (100 rmIL-2 absence 10 × 1 to or non-T CFSE. DCs were stained with CellTrace Violet (Molecular Probes). 1 × 10 T cells. of melanoma B16 Flt3L-secreting injection given mice B6 from sorting cell magnetic-activated by isolated CD11c Splenic assay. capture IL-2 the in as manner same the in In vitro Biosciences). (BD instructions manufacturer’s the to according Set Flex Sensitivity Enhanced IL-2 Human and Array Bead Cytometric BD the with assessed was medium the from of IL-2 Depletion for 2 h at °C. 37 by incubation followed U/ml), 12 to (0.016 IL-2 human recombinant of doses increasing without or with FCS) 10 × (2 plate V-bottomed 96-well a µ Read alignment and processing followed a method previously described previously a method followed and processing alignment Read g/ml anti-CD3 (145-2C11, Bioxcell) in a 96-well round-bottom plate for 80 h. + Foxp3(YFP/GFP) T cell–DC conjugation assay.T cell–DC suppression assay.suppression + 5 reg CFSE ) in a 96-well round-bottomed plate for 720 min in the presence or presence the in min 720 for plate round-bottomed 96-well a in ) Foxp3 + + cells were cultured together with graded numbers of numbers (1 DCs × graded with 10 together were cells cultured ) was determined by flow cytometry based on of the based dilution fluo by cytometry ) flow was determined T cells were cultured with graded numbers of T of numbers graded with cultured were cells T P values of <0.05 were considered significant. considered were <0.05 of values + /CFSE Cre–ERT2 5 Male 8-week-old 8-week-old Male irradiated, T cell–depleted, CFSE-stained splenocytes and splenocytes CFSE-stained T cell–depleted, irradiated, µ l per mg pellet) containing 50 mM Tris-HCl, 150 mM mM 150 Tris-HCl, mM 50 containing pellet) mg per l + ) were analyzed by flow cytometry. by flow analyzed were ) − (control) mice, nine mice for each experimental experimental each for mice nine mice, (control) CD62L Naive CD4 t -test. Welch’s correction was applied when F-test + hi Foxp3(YFP/GFP) CD44 5 cells/well) in 25 25 in cells/well) Rosa26 T reg + T cells (responder cells) and cells) T T (responder cells reg lo and non-T naive T cells were double sorted sorted double were cells T naive cells and non-T in vitro in reg Stat5bCA Each mouse was tagged with a with tagged was mouse Each cells (live CFSE (live cells reg stimulation were excluded excluded were stimulation nature immunology nature cells conjugated with DCs DCs with conjugated cells + reg Serum IgM, IgG1, IgG2a, GITR Foxp3 µ cells were stained with were stained cells l RPMI medium (10% (10% medium RPMI l reg hi Cre-ERT2 CD25 4 cells were sorted 6 . The trimmed trimmed The . reg − CD4 cells in the the in cells + hi (STAT5b- DCs were were DCs T + reg Foxp3 reg 4 and and cells T 4 reg 5 − 6 4 4 - - - .

© 2016 Nature America, Inc. All rights reserved. Bonferroni correction. Bonferroni accumulations. perturbation net of Global distribution null the of the s.d. and and mean accumulation, perturbation net observed the using calculated was perturbation pathway net The 2015. October 5 on accessed pathways tion of 90 as one were and analyzed for in perturba changes set expression, enrichment name same the of T of depletion transient from recovering change; (twofold upregulated CD44 lated’ genes were upregulated (at least 1.75-fold; to inTCR-sufficientexpression their CD44 regulated (a change in expression of at least 0.57-fold) in TCR-deficient relative FDR-adjusted a and respectively, 0.67-fold, or 1.5-fold least at of expression in change a with genes expressed as defined were cells genes in (314) STAT5b-CA T genes were 39,179 called as of present. Genes significantly upregulated10,589 (342) or downregulatedreads, normalized ~60 of threshold this Using sion. expres for threshold the be to chosen was peaks two the between minimum genes marker known to be of expressed or not expressed in T examination by supported was genes ‘non-expressed’ and ‘expressed’ to corresponded this that assumption The bimodal. was genes all was RNA-seq. of analysis expression Bioinformatics gene Differential 3.1.0) 74.5%. (version R in was 1.6.3 DESeq2 using samples analyzed all across rate nature immunology nature Signaling pathway impact analysis was performed using the R package package R the using performed was analysis impact pathway Signaling ‘TCR-upregulated’ (i.e., TCR-dependent) genes were defined as genes down hi T P values were calculated using the normal inversion method with with method inversion normal the using calculated were values reg Mus Mus musculus cells ( cells 4 GSE6107 8 . Genes significantly up- and downregulated, and their their and downregulated, and up- significantly Genes . KEGG (Kyoto Encyclopedia of (Kyotoand KEGG Genes Genomes) Encyclopedia 7 reg ) 3 P cells relative to expression in their control T 4 adj . ‘Activation-upregulated’ genes were genes genes were genes ‘Activation-upregulated’ .

≤ 0.01) in T in 0.01) The distribution of read counts across across counts read of distribution The reg hi cells ( cells reg T cells and naive T cells. The local reg reg P cells, while ‘TCR-downregu while cells, adj GSE5575 cells from from cells 4

≤ 7 . 0.001) in TCR-deficient P value of of value 3 ) 3 3 Foxp3 . ≤ 0.05. DTR z -score -score mice mice reg - - - -

49. 48. 47. 46. 45. 44. circled. manually were terms GO similar of Groups iterations. 500 and settings default with Cytoscape in Layout’ Force-Directed ‘Prefuse the default using was The visualized network genes. ten than fewer with of sets gene and exclusion of 0.005, cutoff adjusted of cutoff 0.2, a a coefficient Jaccard similarity using generated was EnrichmentMap An results. the visualize and terms GO (v2.0.1) EnrichmentMap into imported on downloaded ( were 2015 used October files 25 annotation and ontology GO the and set, FDR-adjusted of cutoff significance a P applying and test hypergeometric (v3.0.3) BiNGO using calculated was 50. value of of value Biological-process (BP) gene-ontology (GO) term over-representation over-representation term (GO) gene-ontology (BP) Biological-process

ar, . Hyas K & upr M BNO a yocp pui t assess to plugin Cytoscape a BiNGO: M. Kuiper, & K. Heymans, S., Maere, and change fold A.L. Tarca, of estimation Moderated S. Anders, & W. Huber, M.I., Love, Illumina for trimmer flexible Trimmomatic:a B. Usadel, & M. Lohse, A.M., Bolger, S. Anders, Y.P.Rubtsov, eio D, seln R, tee, . Eii A & ae, .. nihet map: Enrichment G.D. Bader, & A. Emili, O., Stueker, R., Isserlin, D., Merico, overrepresentation of gene ontology categories in biological networks. (2009). 75–82 PLoS One PLoS interpretation. and visualization enrichment gene-set for method network-based a 21 dispersion for RNA-seq data with DESeq2. with data RNA-seq for dispersion data. sequence Bioconductor. and R using data interfaces. environmental , 3448–3449 (2005). 3448–3449 , ≤ 0.05. The 10,589 expressed genes were entered as the reference reference the as entered were genes expressed 10,589 The 0.05.

5 t al. et t al. et , e13984 (2010). e13984 , et al. et Bioinformatics on-ae dfeeta epeso aayi o RA sequencing RNA of analysis expression differential Count-based nvl inln ptwy mat analysis. impact pathway signaling novel A Supplementary Table 1 Table Supplementary Regulatory T cell-derived interleukin-10 limits inflammation at inflammation limits interleukin-10 cell-derived T Regulatory Immunity

30 Nat. Protoc. Nat. , 2114–2120 (2014). 2114–2120 ,

28 4 , 546–558 (2008). 546–558 , 9 in Cytoscape v3.2.1, employing the the employing v3.2.1, Cytoscape in 5 0 in Cytoscape to cluster redundant redundant cluster to Cytoscape in Genome Biol. Genome

). The output from BiNGO was was BiNGO from output The ). 8 P , 1765–1786 (2013). 1765–1786 , -value cutoff of cutoff -value an 0.001, FDR-

15 doi: , 550 (2014). 550 , Bioinformatics 10.1038/ni.3540 Bioinformatics

25 ,