Ferritin heavy chain supports stability and function of the regulatory T cell lineage

A icle
Fe i in hea y chain suppo s s abili y and unc ion o
he egula o y T cell lineage
Qian Wu 1,2,15, Ana Ri a Ca los 1,3,15,FaouziB aza
1,15, Ma ie-Louise Be gman 1,JamilZKi oko 1,
Pa icia Bas os-Amado 1,EloyCuad ado
4, Rui Ma ins1, B una Sabino Oli ei a 1,Ve aCMa ins 1,
B endon P Scicluna 5, Jona han JM Land y 6,Fe isEJung 6, Temi ope W Ademolue1,
Mi ko Pei zsch 7, Jose Almeida-San os 1, Jessica Thompson1, Sil ia Ca doso1, Ped o Ven u a1,
Manon Slo 4, S ama ia Ron ogianni4,VanessaRibei o 3, Vi al Da Sil a Domingues 1, Inês A Cab al1,
Sebas ian Weis 8,9,10,Ma coG o h 11, C is ina Amenei o 12, Miguel Fidalgo 12, Fudi Wang 13,
Jocelyne Demengeo 1, De k Amsen4,14 &MiguelPSoa es 1✉
Abs ac
Regula o y T (TREG) cells de elop ia a p og am o ches a ed by he
ansc ip ion ac o o khead box p o ein P3 (FOXP3). Main enance o
he TREG cell lineage elies on sus ained FOXP3 ansc ip ion ia a
mechanism in ol ing deme hyla ion o cy osine-phospha e-guanine
(CpG)- ich elemen s a conse ed non-coding sequences (CNS) in
he FOXP3 locus. This cy osine deme hyla ion is ca alyzed by he
en–ele en ansloca ion (TET) amily o dioxygenases, and i in ol es
a edox eac ion ha uses i on (Fe) as an essen ial co ac o . He e, we
es ablish ha human and mouse TREG cells exp ess Fe- egula o y
genes, including ha encoding e i in hea y chain (FTH), a ela i ely
high le els compa ed o con en ional T helpe cells. We show ha
FTH exp ession in TREG cells is essen ial o immune homeos asis.
Mechanis ically, FTH suppo s TET-ca alyzed deme hyla ion o CpG-
ich sequences CNS1 and 2 in he FOXP3 locus, he eby p omo ing
FOXP3 ansc ip ion and TREG cell s abili y. This p ocess, which is
essen ial o TREG lineage s abili y and unc ion, limi s he se e i y o
au oimmune neu oinflamma ion and in ec ious diseases, and a o s
umo p og ession. These findings sugges ha he egula ion o
in acellula i on by FTH is a s able p ope y o TREG cells ha sup-
po s immune homeos asis and limi s he pa hological ou comes o
immune-media ed inflamma ion.
Keywo ds Regula o y T Cells; FOXP3; I on Me abolism; Fe i in Hea y
Chain; Ten–ele en T ansloca ion Enzymes
Subjec Ca ego ies Cance ; Ch oma in, T ansc ip ion & Genomics;
Immunology
h ps://doi.o g/10.1038/s44318-024-00064-x
Recei ed 26 Ap il 2023; Re ised 15 Feb ua y 2024;
Accep ed 20 Feb ua y 2024
Published online: 18 Ma ch 2024
In oduc ion
Iden ified and cha ac e ized (Pow ie and Mason, 1990;Sakaguchi
e al, 1982) o iginally on he basis o hei c i ical in ol emen in
main aining pe iphe al immune ole ance (Cou inho e al, 1993),
egula o y T (T
REG
) cells pa ake in di e en aspec s o immune
homeos asis (Campbell and Rudensky, 2020; Dikiy and Rudensky,
2023;Jose owicze al,2012; Pandu o e al, 2016). One o he main
unc ions o T
REG
cells, howe e , is mos likely o es ain he b ea h
o inna e and adap i e immune esponses agains commensal
mic obes o p e en immunopa hology (Belkaid, 2007;Demengeo
e al, 2006). This e olu iona ily conse ed ai was p obably co-
op ed h ough e olu ion o p e en pe iphe al sel - eac i e T and B
cells om elici ing au oimmune diseases (La aille e al, 1994;
Sakaguchi e al, 1995). As an e olu iona y ade-o (S ea ns and
Medzhi o , 2015), T
REG
cells a e pa hogenic, o example, when
limi ing immune-media ed inflamma o y esponses o pa hogens o
p omo e ch onic in ec ions (Belkaid, 2007; Demengeo e al, 2006)
o when es aining an i- umo immuni y, o p omo e cance
p og ession (Cu iel e al, 2004; Liu e al, 2016).
T
REG
cell de elopmen and unc ion a e con olled by he X-
ch omosome-encoded ansc ip ion ac o FOXP3 (Fon eno e al,
2003;Ho ie al,2003), oge he wi h auxilia y ansc ip ional
1Ins i u o Gulbenkian de Ciência, Oei as, Po ugal. 2In e na ional Ins i u es o Medicine, he Fou h A filia ed Hospi al o Zhejiang Uni e si y, School o Medicine, Yiwu, Zhejiang,
China. 3Depa amen o de Biologia Animal, Cen o de Ecologia, E olução e Al e ações Ambien ais, Faculdade de Ciências, Uni e sidade de Lisboa, Lisboa, Po ugal. 4Depa men
o Hema opoiesis and Depa men o Immunopa hology, Sanquin Resea ch and Lands eine Labo a o y, Ams e dam, The Ne he lands. 5Depa men o Applied Biomedical
Science, Facul y o Heal h Sciences, Ma e Dei Hospi al, and Cen e o Molecula Medicine and Biobanking, Uni e si y o Mal a, Msida, Mal a. 6Genomic Co e Facili y, Eu opean
Molecula Biology Labo a o y, Heidelbe g, Ge many. 7Ins i u e o Clinical Chemis y and Labo a o y Medicine, Uni e si y Clinic Ca l Gus a Ca us, TU D esden, D esden,
Ge many. 8Depa men o Anes hesiology and In ensi e Ca e Medicine, Jena Uni e si y Hospi al, F ied ich-Schille Uni e si y, Jena, Ge many. 9Ins i u e o In ec ious Disease
and In ec ion Con ol, Jena Uni e si y Hospi al, F ied ich-Schille Uni e si y, Jena, Ge many. 10Leibniz Ins i u e o Na u al P oduc Resea ch and In ec ion Biology, Hans-Knöll
Ins i u e-HKI, Jena, Ge many. 11Leibniz Ins i u e on Aging-F i z Lipmann Ins i u e, Jena, Ge many. 12Cen e o Resea ch in Molecula Medicine and Ch onic Diseases (CiMUS),
Uni e sidade de San iago de Compos ela-Heal h Resea ch Ins i u e (IDIS), San iago de Compos ela, Spain. 13The Second A filia ed Hospi al, School o Public Heal h, Zhejiang
Uni e si y School o Medicine, Hangzhou 310058, China. 14Depa men o Expe imen al Immunology, Ams e dam UMC, Uni e si y o Ams e dam, Ams e dam, The Ne he lands.
15These au ho s con ibu ed equally: Qian Wu, Ana Ri a Ca los, Faouzi B aza. ✉E-mail: [email p o ec ed]
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egula o s (Kanamo i e al, 2016). The ansc ip ional p og am
en o ced by FOXP3 specifies T
REG
cell lineage commi men in he
hymus and in he pe iphe y (Fon eno e al, 2003;Ho ie al,2003;
Lee e al, 2012), gene a ing hymic T
REG
( T
REG
) cells and
pe iphe ally de i ed T
REG
(pT
REG
) cells, espec i ely (Chen e al,
2003). Sus ained FOXP3 ansc ip ion main ains T
REG
cell lineage
s abili y (Williams and Rudensky, 2007), a oiding ansdi e en ia-
ion owa ds p o-inflamma o y T helpe (T
H
) cells (Ga in e al,
2007;Mo ikawae al,2014).
FOXP3 ansc ip ion is egula ed by di e en signal ansduc-
ion pa hways, emana ing om he T-cell ecep o (TCR),
in e leukin (IL-2) ecep o , and TGF-β ecep o (Benne e al,
2001; B unkow e al, 2001; Ho i and Sakaguchi, 2004), among
o he s. Sus ained FOXP3 ansc ip ion is en o ced epigene ically
(Ga in e al, 2007; Mo ikawa e al, 2014), in esponse o
en i onmen al cues (Chapman e al, 2020; Shi and Chi, 2019) ha
egula e di e en aspec s o T
REG
cell me abolism (E chega ay and
Mos osla sky, 2016). These epigene ic modifica ions include he
ela i e me hyla ion s a us o cy osine-phospha e-guanine (CpG)-
ich sequences in he FOXP3 conse ed non-coding sequences
(CNS) 1, 2, and 3 (Ohku a e al, 2012; Zheng e al, 2010), whe eby
cy osine me hyla ion ep esses while deme hyla ion sus ains
FOXP3 ansc ip ion (Ohku a e al, 2012; Zheng e al, 2010).
Cy osine me hyla ion is ca alyzed by DNA me hyl ans e ase
(DNMT) (Ohku a e al, 2012), while deme hyla ion is ca alyzed by
he en–ele en ansloca ion (TET) amily o dioxygenases (Wu and
Zhang, 2017;Yuee al,2016). Cy osine deme hyla ion consis s on
edox-based eac ions ha oxidize 5-me hylcy osine (5-mC) in o
5-hyd oxyme hylcy osine (5-hmC), 5- o mylcy osine (5- C) and
5-ca boxylcy osine (5caC) (Kohli and Zhang, 2013). TET dioxy-
genases ca alyze cy osine deme hyla ion a FOXP3 CNS1 and 2
(Ohku a e al, 2012), suppo ing T
REG
cell lineage s abili y
(Naka sukasa e al, 2019;WuandZhang,2017;Yuee al,2019;
Yue e al, 2016), and p e en ing T
REG
cell ansdi e en ia ing in o
inflamma o y e ec o T
H
cells, also e e ed as ex-T
REG
cells (Dua e
e al, 2009; Koma su e al, 2009;Zhoue al,2009).
TET dioxygenases use Fe as an essen ial co ac o and he
in e media e me aboli e α-ke oglu a a e as an obliga o y subs a e
(Huang and Rao, 2014;Pas o e al,2013). This TET eliance on Fe
a ailabili y en e ained he hypo hesis ha egula ion o cellula Fe
me abolism ac s ups eam o TET dioxygenases o modula e T
REG
cell lineage s abili y.
Se e al s udies ha e shown ha Fe me abolism impac s on
immuni y. Fo example, in acellula Fe a ailabili y and edox
ac i i y is essen ial o suppo B and T-cell de elopmen (Vanoaica
e al, 2014), ia a cy op o ec i e mechanism exe ed by he e i in
H chain (FTH) (Be be a e al, 2003;Phame al,2004), likely
in ol ing he mi ochond ia (Blankenhaus e al, 2019; Vanoaica
e al, 2014). Regula ion o cellula Fe con en and edox ac i i y also
modula e cy okine p oduc ion by e ec o T
H
cells, ia a mechanism
in ol ing he PolyC-RNA-Binding P o ein 1 (PCBP1) (Wang e al,
2018). Cellula Fe impo , ia he ans e in ecep o 1 (TFR1/
CD71), suppo s T
H
ype 1 (T
H
1) cell immuni y and i s egula ion
by induced T
REG
(iT
REG
) cells (Voss e al, 2023) as well as an ibody
esponses o accina ion (F os e al, 2021; Jiang e al, 2019)and
immuni y agains in ec ion by pa hogens such as Plasmodium, he
causa i e agen o mala ia (Wideman e al, 2023).
He e, we demons a e ha egula ion o Fe me abolism by
FTH ope a es ups eam o TET dioxygenases o en o ce cy osine
deme hyla ion a CpG- ich sequences in he CNS1 and 2 o he
FOXP3 locus, sus aining FOXP3 ansc ip ion, exp ession and T
REG
cell lineage iden i y. This cell-in insic p ope y o T
REG
cells is
essen ial o main ain immune homeos asis while exe ing a majo
impac on he ou come o immune-d i en inflamma ion.
Resul s
T
REG
cells exp ess ela i ely high le els o FTH
In a p e iously unbiased p o eomics analysis, we ound ha eshly
isola ed human nai e CD45RA+CD25hi T
REG
(nT
REG
) and memo y
CD45RA-CD25hi T
REG
(mT
REG
) cells exp essed ela i ely highe le els o
Fe- egula o y p o eins, including FTH and e i in L chain (FTL),
when compa ed o CD45RA+CD25-nai e con en ional (nTcon ) cells
o o CD45RA-CD25-ac i a ed/memo y (mTcon ) cells (Cuad ado
e al, 2018). The ela i ely highe exp ession o he FTH and FTL
componen s o he e i in complex was main ained upon expansion o
human CD4+CD127–CD25+T
REG
cells in i o, in compa ison o T
CONV
cells CD4+CD127+CD25–cells (Fig. 1A,B). Simila ly, mouse
CD4+Foxp3+T
REG
cells also exp essed ela i ely highe le els o FTH
p o ein, compa ed o CD4+Foxp3−
CD44lowCD62Lhigh nai e T
H
cells
(T
N
)o CD4
+Foxp3−
CD44highCD62Llow memo y T
H
cells (T
M
), as
de e mined by wes e n blo (Fig. 1C,D). This sugges s ha sus ained
and ele a ed le els o e i in exp ession a e a s able p ope y o
human and mouse T
REG
cells. O no e, mouse T
REG
cells exp ess
simila le els o F h mRNA, compa ed o T
N
cells, while
(CD4±Foxp3−
CD44highCD62L−
)T
M
cells exp ess ela i ely highe
le els o F h mRNA, compa ed o T
REG
cells (Appendix Fig. S1). This
sugges s ha he ela i ely highe le el o FTH p o ein exp ession in
T
REG
cells is en o ced pos - ansc ip ionally, simila o o he cell ypes
(Hen ze e al, 1987; Mey on-Hol z e al, 2004; Mucken hale e al,
2017; Rouaul e al, 1988).
We moni o ed FTH exp ession in induced T
REG
(iT
REG
) cells,
gene a ed om mouse T
N
cells ac i a ed in i o wi h an i-CD3/
CD28 mAb plus IL-2 and TGFβ(Chen e al, 2003) (Fig. 1E). To his
aim we used Foxp3GFP T
REG
cell epo e mice, in which a g een
fluo escen p o ein (GFP) humanized C e- ecombinase (GFP-
hC e) coding sequence is inse ed downs eam o he Foxp3 ATG
ansla ional s a codon in a bac e ial a ificial ch omosome (BAC)
ansgene ca ying he in ac Foxp3 p omo e (Foxp3-GFP-hC e;
e e ed he ein as Foxp3GFP)(Chene al,2003;Zhoue al,2008).
FTH p o ein exp ession was highe unde cul u e condi ions
con aining TGFβand en iched in CD4+GFP+iT
REG
cells, compa ed
o cul u e condi ions lacking TGFβand en iched in CD4+GFP-
T
CONV
cells (Fig. 1F). A simila end was obse ed o F h mRNA,
which was up egula ed in iT
REG
cells (Fig. 1G). The ela i e le el o
F h mRNA exp ession was simila in hymic, pe iphe al and iT
REG
cells (Appendix Fig. S2).
FTH exp ession in T
REG
cellsisessen ial omain ain
immune homeos asis
To de e mine he e ec o egula ion o in acellula Fe me abolism
by FTH on T
REG
cells, we in oduced an addi ional loxP-flanked F h
allele (F hfl/fl) (Da shan e al, 2009)in oFoxp3GFP mice (Zhou e al,
2008), dele ing F h specifically in T
REG
cells om Foxp3GFP-F hΔ/Δ s.
con ol Foxp3GFP mice (Fig. EV1A). F h dele ion was associa ed wi h
The EMBO Jou nal Qian Wu e al
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B
TCONV TREG
FTH
E-Ac in
0
0.02
0.04
0.06
0.08
0.1
FTH/ E-Ac in
A**
21
TCONV TREG
IJ
42
H
KDa
KDa
CD4 CD4
Foxp3GFP-F h∆/∆
11010
2103104105
101
102
103
104
105
106
12,4%
11010
2103104105
101
102
103
104
105
106
11010
2103104105
5,71%
11010
2103104105
2,65%13,3%
0
5
10
15
0
1
2
3
4
*** *** ***
11010
2103104105
101
102
103
104
105
106
11010
210310410
C
F h/H3
F h
H3
TNTREG TM
D
21
17
KDa
KDa
TNTREGTM
Spleen MLN
TREG cell
(CD4+GFP+
)
Thymus
TREG cell
(CD4+GFP+
)
TREG cell
(CD4+GFP+
)
EF
CD4
DCD3/28
IL-2
DCD3/28
IL-2 + TGFE
TN
T
CONV
G
iT
REG
F h
H3
21
15
1,15%
105
104
103
0
-103
105
104
103
0
-103
105
104
103
0
79,4%
DCD3/28
+IL-2
DCD3/28
+IL-2/TGFE0
0.2
0.4
0.6
0.8
F h/H3
P o ein
**
TCONV
iT
REG
0
0.2
0.3
0.1
F h/A bp0 mRNA
**
TCONV
iT
REG
KDa
TCONV
iT
REG
Foxp3GFP Foxp3GFP-F h∆/∆
Foxp3GFP
Foxp3GFP-F h∆/∆
Foxp3GFP
0
1
2
3
0
1
2
3
K
L
MLN
Foxp3-GFP
CD4
Foxp3GFP-F h∆/∆
Foxp3GFP
Foxp3-GFP
N p1
N p1
+
N p1
-
Nb . Foxp3-GFP+
Cells (x105)
N p1
+
N p1
-
Foxp3-GFP+ (%)
* *
0
1
2
3
2,15% 1,14%
*** NS
Foxp3-GFP+ (%)
Foxp3-GFP+ (%)
Nb . Foxp3-GFP+
Cells (x105)
Nb . Foxp3-GFP+
Cells (x106)
Foxp3-GFP+ (%)
Nb . Foxp3-GFP+
Cells (x105)
N p1+ (
TREG
)
N p1- (
pTREG
)
CD4+GFP+
cells
010
3104105010
3104105
0
20
40
60
80
100
0
1
2
3
4
** *** ***
82% 72,6%
0
103
104
Thymus
Spleen & MLN
E en s (%)
Foxp3GFP-F h∆/∆
Foxp3GFP
*** *** ***
103
010
4
Foxp3-GFP
Thymus
Spleen
MLN
0
1
2
3
4
Foxp3-GFP MFI (103)
Thymus
Spleen
MLN
TREG cell
CD4+GFP+
Foxp3-GFP
Foxp3-GFP
Foxp3-GFP
***
0.5
2
1.5
1
00
5
10
15
20
Qian Wu e al The EMBO Jou nal
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he accumula ion o in acellula labile Fe2+in T
REG
cells, isola ed
om he mesen e ic lymph nodes (MLN) o Foxp3GFP-F hΔ/Δ s.
con ol Foxp3GFP mice (Fig. EV1B).
The equency o hymic CD4+GFP+T
REG
cells was lowe in
Foxp3GFP-F hΔ/Δ s. con ol Foxp3GFP mice (Fig. 1H). This was no
associa ed, howe e , wi h a concomi an educ ion in he numbe o
CD4+GFP+T
REG
cells (Fig. 1H). In con as , he e was a ma ked
educ ion o bo h he equency and numbe s o T
REG
cells in he spleen
(Fig. 1I) and in he MLN (Fig. 1J) o Foxp3GFP-F hΔ/Δ s. con ol Foxp3GFP
mice. The equency and numbe o CD4+GFP+CXCR5+PD1+
ollicula T
REG
(FT
REG
) cells was also dec eased in he spleen o
Foxp3GFP-F hΔ/Δ s. con ol Foxp3GFP mice (Fig. EV1C). These obse a-
ions sugges ha egula ion o in acellula Fe by FTH is equi ed o
sus ain he numbe o ci cula ing T
REG
and FT
REG
cells in he pe iphe y,
wi hou in e e ing wi h hymic T
REG
cell ou pu .
We no iced ha he ela i e le el o GFP exp ession, epo ing on
Foxp3 ansc ip ion unde he con ol o an in ac Foxp3 locus (Chen
e al, 2003;Zhoue al,2008), was educed in hymic, splenic and MLN
T
REG
cells om Foxp3GFP-F hΔ/Δ s. con ol Foxp3GFP mice (Fig. 1K).
These obse a ions sugges ha FTH egula es Foxp3 ansc ip ion in
he hymus as well as in ci cula ing T
REG
cells, which is no su ficien
howe e , o in e e e wi h hymic T
REG
cell ou pu .
Thymic and pe iphe al T
REG
cell de elopmen gi e ise o T
REG
and iT
REG
cells, exp essing neu opilin1 (N p1) o no , espec i ely
(Weiss e al, 2012; Yada e al, 2012). The equency and numbe o
N p1+ T
REG
cells and N p1-iT
REG
cells was educed, o he same
ex en , as assessed in he MLN (Fig. 1L) o Foxp3GFP-F hΔ/Δ s. con ol
Foxp3GFP mice. This sugges s ha egula ion o Fe me abolism by
FTH is equi ed o he main enance o hymic-de i ed and
pe iphe ally induced T
REG
cells.
FTH es ains T
REG
cell ansdi e en ia ion in o
inflamma o y ex-T
REG
cells
The educ ion in T
REG
cells imposed by F h dele ion was associa ed
wi h an accumula ion o ac i a ed CD4+CD44highCD62Llow T
CONV
cells and CD8+CD44highCD62Llow cy o oxic T (T
C
)cells,in he
spleen (Fig. EV1D) and in MLN o Foxp3GFP-F hΔ/Δ s. con ol F hfl/fl
mice (Fig. EV1E). Concomi an ly, he e was an inc ease in he
equency o in e e on-γ(IFN γ)-exp essing ac i a ed CD4+T
H
cells and CD8+T
C
cells in he spleen (Fig. EV1F) and MLN
(Fig. EV1G). These obse a ions sugges ha egula ion o Fe
me abolism in T
REG
cells is essen ial o main ain immune home-
os asis, p e en ing he ac i a ion and accumula ion o inflamma-
o y CD4+T
H
cells and CD8+T cells.
Sec e ed e i in complexes can es ain human T-cell p oli e a-
ion in i o (G ay e al, 2001), en e aining he hypo hesis ha
e i in sec e ion suppo s he an ip oli e a i e unc ion o T
REG
cells. Howe e , T
REG
cells om Foxp3GFP-F hΔ/Δmice inhibi ed T-cell
p oli e a ion in i o, o a simila ex en as T
REG
cells om Foxp3GFP
(Fig. EV2A). This sugges s ha FTH is no essen ial o suppo he
an ip oli e a i e unc ion o T
REG
cells, consis en wi h Foxp3GFP-F hΔ/Δ
mice no de eloping o e au oimmune pa hologic lesions,
compa ed o con ol F hfl/flmice (Fig. EV2B).
To gain u he insigh in o he mechanism ia which FTH
modula es T
REG
cell unc ion in i o, we pe o med RNA
sequencing (RNAseq), o compa e he gene exp ession p ofiles o
CD4+CD25+GFP+T
REG
cells so ed om he lymph nodes o
Foxp3GFP-F hΔ/Δ s. con ol Foxp3GFP mice (Fig. 2A). T
REG
cells om
Foxp3GFP-F hΔ/Δmice up egula ed 1832 genes and down egula ed
1340 genes, compa ed o T
REG
cells om con ol Foxp3GFP mice
(Fig. 2B). F h dele ion was associa ed wi h dys egula ion o Foxp3-
dependen and -independen “T
REG
ansc ip ional signa u e”(Hill
e al, 2007), a ec ing a leas 194 genes in ol ed in T
REG
cell
unc ion and lineage main enance (Fig. 2C). Pa hway en ichmen
analysis (Fig. 2D,E) showed ha T
REG
cells om Foxp3GFP-F hΔ/Δmice
p esen ed T
H
1and ype2(T
H
2) ansc ip ional signa u es (Fig. 2D),
as illus a ed by he induc ion o he ansc ip ional mas e
egula o s o T
H
1andT
H
2 e ec o unc ions, Tbx21 (T-box
ansc ip ion ac o ; T-be ) and Ga a3 (GATA Binding P o ein 3)
as well as Runx3 (RUNX Family T ansc ip ion Fac o 3),
espec i ely (Fig. EV2C).
Consis en ly, he pe cen age o ac i a ed CD4+GFP+
CD44highCD62Llow T
REG
cellswashighe in hespleenandMLN
Figu e 1. T
REG
cell e i in exp ession is a s able p ope y o human and mouse T
REG
cells.
(A) FTH and β-Ac in p o ein exp ession, de ec ed by wes e n blo in whole-cell ex ac s om human T con en ional (CD4+CD45RA+CD127+CD25–;T
CONV
) and T
REG
(CD4+CD127–CD45RA+CD25hi) cells a e wo weeks o expansion wi h an i-CD3, an i-CD28 mAb and IL-2. (B) Rela i e quan ifica ion o FTH p o ein exp ession,
no malized o β-Ac in, de ec ed by wes e n blo as in (A). n=3 independen expe imen s. (C) FTH and his one H3 p o ein exp ession de ec ed by wes e n blo in whole-
cell ex ac s om so ed mouse nai e T cells (CD4+Foxp3-CD44lowCD62Lhigh;T
N
), (CD4+Foxp3+GFP+)T
REG
cells and memo y T cells (CD4+Foxp3−CD44highCD62Llow;T
M
),
by wes e n blo . (D) Rela i e quan ifica ion o FTH, no malized o his one H3, p o ein exp ession, de ec ed by wes e n blo as in (C). Da a we e no malized o FTH
exp ession in T
N
cells, pooled om ou independen expe imen s. (E) Schema ic ep esen a ion o he p o ocol used o he gene a ion o iT
REG
and ep esen a i e flow
cy ome y do plo s o mouse iT
REG
gene a ed om so ed nai e T cells (T
N
), s imula ed wi h an i-CD3 and an i-CD28 mAb plus IL-2 and TGFβ o 5 days. Con ol T
CONV
cells we e subjec ed o he same expe imen al condi ions, wi hou TGFβ.(F) FTH and his one H3 p o ein exp ession, de ec ed by wes e n blo in whole-cell ex ac s om
iT
REG
and T
CONV
gene a ed as depic ed in (E). Da a pooled om h ee independen expe imen s wi h simila end. (G) The ela i e le el o F h mRNA exp ession, quan ified
by qRT-PCR, using A bp0 as housekeeping gene. Da a pooled om h ee independen expe imen s, wi h simila end. (H–J) Schema ic ep esen a ion o he p o ocol used
( op panels), ep esen a i e flow cy ome y do plo s (middle panels) and co esponding quan ifica ion o pe cen age (%) in CD4+cells and cell numbe (Nb .) (bo om
panels) o li e (TCRβ+CD4+Foxp3+) GFP+T
REG
cells in (H) hymus, (I) spleen and (J) mesen e ic LN (MLN). (H) Da a om N=8 mice pe geno ype, pe o gan, om wo
independen expe imen s, wi h simila end. (I,J) Da a om N=12 mice pe geno ype, pe o gan, om h ee independen expe imen s, wi h simila end. (K) Schema ic
ep esen a ion o he expe imen al app oach ( op panel) used o moni o he exp ession o he GFP-hC e ansgene in he hymus, spleen, and MLN o (CD4+GFP+)T
REG
cells. Rep esen a i e flow cy ome y his og ams o GFP-hC e (bo om le panel). Rela i e quan ifica ion o GFP-hC e exp ession (bo om igh panel), ep esen ed as
mean fluo escence in ensi y (MFI). Da a om N=8 mice pe geno ype, pooled om wo independen expe imen s wi h simila end. (L) Schema ic ep esen a ion o he
expe imen al app oach (le panel) used, ep esen a i e flow cy ome y do plo s ( op panel) and co esponding quan ifica ion o pe cen age (%) (bo om le panel) and
cell numbe (Nb .) (bo om igh panel) o li e (TCRβ+CD4+GFP+) N p1+and N p1-T
REG
cells in MLN. Da a om N=8 mice pe geno ype, pooled om wo independen
expe imen s wi h simila end. Da a in o ma ion: Da a in (B,F,G) a e p esen ed as mean ± SD. Da a in (D) a e p esen ed as mean ± SEM. Ci cles in (H–L) co espond o
indi idual mice. P alues in (B,F–J) de e mined using unpai ed es wi h Welch’s co ec ion, in (D,H–J) using o dina y one-way ANOVA, and in (K,L) using Two-way
ANOVA wi h Sidak’s mul iple compa isons es . NS no significan (P> 0.05); *P< 0.05; **P< 0.01; ***P< 0.001. Sou ce da a a e a ailable online o his figu e.
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eIF4/p70S6
K
signaling
mTOR
signaling
EIF2
signaling
AB
0
2
4
6
8
10
C
F
T
GER
e u angislanoi pi csna llec
Fold Exp ession (log
2
)
-2 -1 0 1 2
0246
AHR signaling
In e e on signaling
Acu e phase esponse
ER s ess pa hway
BRCA1 in DNA damage esponse
DNA damage (14-3-3-σ signaling)
In lammasome pa hway
CHK in cell cycle checkpoin con ol
G1/S checkpoin egula ion
T
H
cell di e en ia ion
LXR/RXR ac i a ion
Cyclins and cell cycle egula ion
GADD45 signaling
T
H
2 pa hway
NRF2-oxida i e s ess esponse
Cell cycle con ol o ch om. eplic.
T
H
1 pa hway
Es ogen-media ed S-phase en y
G2/M DNA damage checkpoin
T
H
1 and T
H
2 ac i a ion
35
1
Ak 1
Nqo1
Dnajb11
Hmox1
Sod2
Mgs 1
Sqs m1
N e2l2
Ma k
Map2k3
Txn d1
Dnajc15
Sod1
Abcc1
Fkbp5
Bach1
Ube2e3
Ca
Pm 1
Gclm
Txn1
Vcp
P dx1
Pik3c2a
Gs
Gclc
Abcc4
Gs 2
Hacd3
Enc1
Ma
F l1
Ma g
Ma
R as2
Ac g2
Aox1
Map3k5
Oxida i e s ess
Foxp3
GFP
Foxp3
GFP-F h''
−1 0 1 2
PF
G
-3pxo
F
CD25
de
o
S
105
104
103
102
0
-102
105
104
103
0-103
lai inI
F h1
Mi 1
Top2a
Tigi Hmox1
Anxa2
Myo1
Myo6 His 1h1b
P une2
M 1
Slc25a23
S a6
Cdc42bpa
Hdac9
Nqo1
Ry 2
Cyp26b1
Tmem136
Cacna1i M 2
Gm13394
Mes
Clec2g
Pianp
Pls1
Pnlip
0
50
100
150
200
250
−8 −4 0048
Adjus ed p- alue (-log10)
Base mean
exp ession
250000
500000
750000
Log2 old change
Foxp3GFP-F hΔ/Δ s. Foxp3GFP
Foxp3
GFP
Foxp3
GFP-F h''
105
104
103
102
0
-102
Foxp3GFP
Foxp3GFP-F h''
So ing
Lymph nodes
CD25+
Foxp3+(GFP+)
RNAseq
D
P- alue (-log 10)
P- alue (-log 10)
E
8,12%
89,9%
Fold Exp ession (log2)
Qian Wu e al The EMBO Jou nal
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om Foxp3GFP-F hΔ/Δ s. con ol Foxp3GFP mice (Fig. EV2D). This was
associa ed wi h an inc ease in he equency o CD4+GFP+T
REG
cells exp essing IFNγin he spleen (Fig. EV2E). This sugges s ha
FTH is essen ial o p e en he ansdi e en ia ion o T
REG
cells in o
inflamma o y T
H
cells.
RNAseq analysis also e ealed ha F h dele ion in T
REG
cells led
o he induc ion o he canonical oxida i e s ess esponse
con olled by he ansc ip ion ac o nuclea ac o e y h oid-
de i ed 2-like 2 (NRF2) (Fig. 2D). This was associa ed wi h he
ac i a ion o o he canonical s ess esponses, including he cell
cycle and DNA damage, un olded p o ein, and hypoxic esponse
(Fig. 2D) as well as an o e all shu down o eIF2, mTOR and eIF4/
p70s6K signaling ansduc ion pa hways (Fig. 2E). Ac i a ion o he
oxida i e s ess esponse egula ed by NRF2 was cha ac e ized by
he induc ion o Nqo1 and Hmox1,amongse e alo he NRF2-
egula ed genes (Fig. 2F). These obse a ions sugges ha FTH is
essen ial o suppo a ansc ip ional p ofile ha main ains T
REG
cell
edox homeos asis, simila o desc ibed in o he cell ypes
(Blankenhaus e al, 2019;Vanoaicae al,2014).
FTH suppo s T
REG
cell lineage main enance
To es ablish whe he FTH en o ces T
REG
cell lineage main enance and
p e en s he ansdi e en ia ion o T
REG
cells in o inflamma o y T
H
cells, an addi ional Rosa26- andem dime ( d) Toma o-Flox-s op-Flox
allele was in oduced in o Foxp3GFP-F hΔ/Δmice, d i ing he exp ession
o dToma o ( dT) by C e-d i en excision o a Flox-s op-Flox casse e,
unde he con ol o Foxp3 egula o y egions (Fig. 3A). The esul ing
Foxp3GFP-F hΔ/Δ- dT mice allow dis inguishing CD4+GFP+ dT+T
REG
om
CD4+GFP- dT+ex-T
REG
cells ha a some poin in hei de elopmen al
his o y down egula ed Foxp3 (i.e., GFP-), while e aining TdT
exp ession (Fig. 3A). F h dele ion in T
REG
cells was associa ed wi h a
p og essi e educ ion in he equency o ci cula ing T
REG
cells om
Foxp3GFP-F hΔ/Δ- dT s. con ol Foxp3GFP- dT mice, as assessed om 2 o
24 weeks a e bi h (Figs. 3B and EV3A). Concomi an ly, he e was an
inc ease in he equency o ci cula ing ex-T
REG
cells (Figs. 3C
and EV3A), wi h 60% o ci cula ing T
REG
cells becoming ex-T
REG
cells
in Foxp3GFP-F hΔ/Δ- dT, 24 weeks a e bi h (Figs. 3D and EV3A). This
ela i e en ichmen in he p opo ion o ex-T
REG
cells sugges s ha F h
dele ion p omo es he con e sion o T
REG
cells in o ex-T
REG
cells.
We easoned ha i FTH p e en s T
REG
cells om ansdi e en ia ing
in o ex-T
REG
cells, hen F h dele ion in T
REG
cells should be associa ed
wi h an accumula ion o ex-T
REG
cells in he spleen and/o lymph nodes.
As expec ed, he pe cen age and numbe o T
REG
cells we e educed in
he spleen om Foxp3GFP-F hΔ/Δ- dT s. con ol Foxp3GFP- dT mice, as
assessed a 19–30 weeks a e bi h (Fig. 3E). The pe cen age and
numbe o splenic ex-T
REG
cells emained ela i ely s able (Fig. 3F), bu
he a io o ex-T
REG
o e dT+cells was highe in he spleen om
Foxp3GFP-F hΔ/Δ- dT s. con ol Foxp3GFP- dT mice (Fig. 3G), wi h o e 80% o
T
REG
cells becoming ex-T
REG
cells (Fig. 3G). F h was dele ed in ex-T
REG
cells om Foxp3GFP-F hΔ/Δ- dT mice, as de e mined by qRT-PCR
(Fig. EV3B), confi ming ha he ex-T
REG
cells in Foxp3GFP-F hΔ/Δ- dT mice
do o igina e om T
REG
cells, in which he F h allele was dele ed unde
he con ol o he Foxp3 p omo e .
We hen asked whe he he ansdi e en ia ion o T
REG
cells in o
ex-T
REG
cells was associa ed wi h he exp ession o p o-
inflamma o y cy okine, which is a ea u e o T
H
cells ac i a ion.
In s ong suppo o his hypo hesis, a la ge pe cen age o T
REG
and
ex-T
REG
cells in he LN and o a lesse ex en in he spleen om
Foxp3GFP-F hΔ/Δ- dT mice exp essed IFNγ,ascompa ed o helacko
IFNγexp ession in T
REG
and ex-T
REG
cells om con ol Foxp3GFP- dT
mice (Figs. 3H and EV3C). Mo eo e , a significan p opo ion o
T
REG
and ex-T
REG
cells in he LN (Fig. 3I) and spleen (Fig. EV3D)
om Foxp3GFP-F hΔ/Δ- dT mice co-exp essed he p oli e a ion ma ke s
Ki67 and CD71 (i.e., ans e in ecep o ), as compa ed o T
REG
and
ex-T
REG
cells om con ol Foxp3GFP- dT mice (Figs. 3IandEV3D).
This was no associa ed, howe e , wi h changes in he ela i e le els
o CD71 exp ession (Fig. EV3E). Taken oge he hese obse a ions
sugges ha FTH is essen ial o es ain T
REG
cells om
ansdi e en ia ing in o inflamma o y ex-T
REG
cells.
FTH suppo s T
REG
lineage main enance in a
cell-au onomous manne
To disen angle cell-au onomous om sys emic e ec s associa ed
wi h F h dele ion in T
REG
cells we used mixed bone ma ow (BM)
chime ic mice. B iefly, sub le hally i adia ed lymphogenic Rag2-
deficien (Rag2-/-) mice we e econs i u ed wi h BM cells om
CD45.2+Foxp3GFP-F hΔ/Δ- dT s. Foxp3GFP- dT mice (50%), plus congenic
BM cells (50%) om CD45.1+C57BL/6 mice (Fig. 4A). The
p opo ion o CD45.2+ s. CD45.1+T
REG
cells in LN (Fig. 4A–C)
was ma kedly educed in BM chime ic mice, when econs i u ed
wi h BM cells om Foxp3GFP-F hΔ/Δ- dT s. Foxp3GFP- dT mice. In
con as , he e we e no di e ences in he ela i e p opo ion o
CD45.2+ s. CD45.1+T
REG
cells in he hymus o hese BM chime ic
mice (Fig. EV3F,G). This sugges s ha FTH sus ains he numbe o
ci cula ing T
REG
cells ia a cell-au onomous mechanism ha does
no a ec T
REG
cell de elopmen in he hymus.
The p opo ion o CD45.2+ s. CD45.1+Foxp3-CD3+CD4+T
H
cells and CD3+CD8+T
C
cells was indis inguishable in he LN
(Fig. 4A–C) o mixed BM chime as econs i u ed wi h CD45.2+
BM cells om Foxp3GFP-F hΔ/Δ- dT s. con ol Foxp3GFP- dT mice.
Mo eo e , he pe cen age and numbe o ac i a ed CD45.2+CD4+
CD44highCD62Llow T
CONV
cells and CD45.2+CD8+CD44highCD62Llow
T
C
cells in he LN (Fig. EV4A–C) we e also simila in hese BM
chime ic mice. This confi ms ha FTH sus ains he numbe o
Figu e 2. FTH exp ession in T
REG
cells p e en s ansdi e en ia ion in o inflamma o y ex-T
REG
cells.
(A) Schema ic ep esen a ion o expe imen al app oach (le panel) and ep esen a i e flow cy ome y do plo s o (CD4+CD25+GFP+)T
REG
cells om he lymph nodes
be o e and a e so ing. (B) Volcano plo ep esen a ion o RNA sequencing da a o genes o e exp essed ( ed) o unde -exp essed (blue) in (CD4+CD25+GFP+)T
REG
cells
so ed om Foxp3GFP-F hΔ/Δand con ol Foxp3GFP (N=5 pe geno ype) mice. P alues de e mined by Benjamini and Hochbe g adjus ed p obabili ies. (C) Hea map
ep esen a ion o T
REG
ansc ip ional signa u e genes di e en ially exp essed in T
REG
cells so ed om Foxp3GFP-F hΔ/Δ s. Foxp3GFP mice, as illus a ed in (A,B). (D,E) Pa hway
en ichmen analysis o gene ic p og ams o e exp essed (D) o unde -exp essed (E)inT
REG
cells so ed om Foxp3GFP-F hΔ/Δ s. Foxp3GFP mice, as illus a ed in (A,B). Da a
we e analyzed using g:SCS mul iple es ing co ec ion me hod wi h a significance h eshold o 0.05. (F) Hea map ep esen a ion o indi idual genes associa ed wi h
oxida i e s ess- esponsi e p og ams, di e en ially exp essed in T
REG
cells so ed om Foxp3GFP-F hΔ/Δ s. Foxp3GFP mice, as illus a ed in (A,B). Sou ce da a a e a ailable
online o his figu e.
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B
0
20
40
60
80
2 4 7 9 11151924
Time [Weeks]
0
2
4
6
8
10
2 4 7 9 11151924
Time [Weeks]
0
1
2
3
4
5
247911151924
Time [Weeks]
C
**** ****
H
D
GFP+TdT+
TREG Cells (%)
GFP-TdT+
ex-TREG Cells (%)
GFP-TdT+/TdT+
Cells (%)
*
E
% (GFP +TdT+)
TREG cells
% GFP -TdT +
ex-T REG cells
Nb . GFP+TdT+
TREG cells (x105)
F
Nb . GFP-TdT+
ex-T
REG
cells (x10
6
)
G
% GFP -TdT +/
TdT+ cells
0
2
4
6
0
2
4
6
0
5
10
15
20
25
0
0.5
1
15
2
2.5
** **
NS NS
0
20
40
60
80
100
IFNJ
E en s (%)
100
0
20
40
60
80
LN
100
0
20
40
60
80
Spleen
103104105
102103104105
102
LN Spleen
01041050104105
10³
10⁴
10⁵
106
0
107
10³
10⁴
10⁵
106
0
107
CD71
Ki67
7 28%
14,3% 23,5%
16,1%
TREG ex-TREG
(%)
Ki67+CD71+
ex-TREG
0
10
20
30
40
50
LN
*** ***
I
TREG ex-TREG
FMO
A
TREG cells
(GFP+ dT+)
ex-TREGcells
(GFP- dT+)
G p hC e
S op
LoxP LoxP
dT
Rosa26 locus
Foxp3 P omo e
F h locus
LoxPLoxP
F h P omo e E1
BAC
LN, Spleen
IFN
J+
TREG (GFP+ dT+)
Ex-TREG (GFP- dT+)
LN
Ki67
CD71
TREG (GFP+ dT+)
Ex-TREG (GFP- dT+)
Foxp3GFP-F h''- dT
Foxp3GFP- dT
TREG cells
(GFP+ dT+)
TREG cells
(GFP- dT+)
Blood Blood
Foxp3GFP-F h''- dT
Foxp3GFP- dT
Foxp3GFP-F h''- dT
Foxp3GFP- dT
Foxp3GFP-F h''- dT
Foxp3GFP- dT
TREG cells
(GFP+ dT+)
TREG cells
(GFP- dT+)
Spleen Spleen
Foxp3GFP-F h''- dT
Foxp3GFP- dT
Foxp3GFP-F h''- dT
Foxp3GFP- dT
Foxp3GFP-F h''- dT
Foxp3GFP- dT
Foxp3GFP-F h''- dT
Foxp3GFP- dT
Foxp3GFP-F h''- dT
Foxp3GFP- dT
****
0
20
40
60 **** **** ** *
IFNJ+(%)
TREG
ex-TREG
TREG
ex-TREG
TREG
Qian Wu e al The EMBO Jou nal
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ci cula ing T
REG
cells, ia a cell-au onomous mechanism ha
ac s i espec i ely o he sys emic inflamma o y esponse associa ed
wi h F h dele ion in T
REG
cells om Foxp3GFP-F hΔ/Δmice
(Fig. EV1D–G).
We hen asked whe he FTH es ains he ansdi e en ia ion o
T
REG
cells owa ds ex-T
REG
cells ia a cell-au onomous mechanism. In
suppo o his no ion, he e was a ma ked educ ion in he pe cen age
and numbe o CD45.2+CD3+CD4+GFP+ dT+T
REG
cells (Fig. 4D,E)
and CD45.2+CD3+CD4+GFP- dT+ex-T
REG
cells (Fig. 4D,F) in he LN
o mixed BM chime ic mice econs i u ed wi h BM cells om
Foxp3GFP-F hΔ/Δ- dT s. Foxp3GFP- dT mice. The a io o CD45.2+ex-T
REG
cells o e dT+cells was inc eased in he LN om chime ic mice
econs i u ed wi h BM cells om Foxp3GFP-F hΔ/Δ- dT s. Foxp3GFP- dT mice
(Fig. 4G). This sugges s ha FTH main ains pe iphe al T
REG
cell
lineage s abili y, ia a cell-au onomous mechanism, i espec i ely o
he sys emic inflamma o y esponse associa ed wi h F h dele ion in
T
REG
cells om Foxp3GFP-F hΔ/Δmice (Fig. EV1D–G).
FTH main ains T
REG
cell edox homeos asis ia a
cell-au onomous mechanism
We hen asked whe he FTH egula es gene exp ession in T
REG
cells,
i espec i e o he sys emic inflamma o y esponse associa ed wi h
F h dele ion in T
REG
cells om Foxp3GFP-F hΔ/Δmice (Fig. EV1D–G).
To es his hypo hesis, he gene exp ession p ofile o T
REG
cells
so ed om he LN o mixed BM chime as (Fig. 4H) was compa ed
o ha o T
REG
cells so ed om he LN o non-chime ic mice
(Fig. 2A). Analysis o RNAseq da a om BM chime ic mice showed
ha F h-dele ed T
REG
cells (CD45.2+GFP+ dT+) de eloping om he
BM o Foxp3GFP-F hΔ/Δ- dT mice, up egula ed 149 genes and down-
egula ed 96 genes, compa ed o F h-compe en T
REG
cells om
con ol Foxp3GFP- dT mice (Fig. 4I). In he same BM chime ic mice,
F h-dele ed ex-T
REG
cells (CD45.2+GFP- dT+) de eloping om he
BM o Foxp3GFP-F hΔ/Δ- dT mice up egula ed 90 genes and down-
egula ed 36 genes, compa ed o F h-compe en ex-T
REG
cells om
con ol Foxp3GFP- dT mice (Fig. 4J). The genes egula ed in T
REG
and
ex-T
REG
cells o igina ing om he BM o Foxp3GFP-F hΔ/Δ- dT s.
Foxp3GFP- dT mice in BM chime ic mice, we e associa ed wi h he
oxida i e s ess esponse egula ed by NRF2 (Fig. 4I,J). This
sugges s ha FTH exe s cell-au onomous con ol o T
REG
cell edox
homeos asis, i espec i e o he sys emic inflamma o y esponse
associa ed wi h F h dele ion in T
REG
cells om Foxp3GFP-F hΔ/Δmice
(Fig. EV1D–G).
The inflamma o y ansc ip ional signa u e o T
REG
cells om
Foxp3GFP-F hΔ/Δ s. Foxp3GFP mice (Fig. 2B–D) was no obse ed in T
REG
cells om BM chime ic mice, o igina ing om Foxp3GFP-F hΔ/Δ- dT s.
Foxp3GFP- dT (Fig. 4I,J). Among he 245 di e en ially exp essed genes in
F h-deficien (CD4+GFP+)T
REG
cells om BM chime ic mice (Fig. 4I,J),
134 ma ched hose di e en ially exp essed in F h-deficien T
REG
cells
om non-chime ic mice (Fig. 4K,L). Gene on ology analyzes o he
o e lapping genes, showed an en ichmen o pa hways ela ed o
oxida i e s ess esponse, comp ising se e al NRF2- egula ed genes
(Fig. 4K–M). In con as , F h-deficien (CD4+GFP+)T
REG
cells om
BM chime ic mice did no show an en ichmen o pa hways
associa ed wi h T
H
cell ac i a ion (Fig. 4K–M). This sugges s ha FTH
ac s in a cell-au onomous manne o suppo T
REG
cell edox
homeos asis and es ain T
REG
cell ansdi e en ia ion in o ex-T
REG
cells (Fig. 4). In con as , he inflamma o y p ofile associa ed wi h he
ansi ion o F h-dele ed T
REG
cells owa ds inflamma o y ex-T
REG
cells,
obse ed in Foxp3GFP-F hΔ/Δmice (Fig. 2)andFoxp3GFP-F hΔ/Δ- dT mice
(Fig. 2) equi es, in addi ion, he de elopmen o sys emic
inflamma ion.
FTH ac s in a cell-au onomous manne o suppo T
REG
cell homeos a ic expansion
We ook ad an age o he homeos a ic expansion o T
REG
cells, upon
adop i e ans e in o lymphopenic Rag2−/−mice (Dua e e al,
2009), o compa e he p oli e a i e capaci y o CD4+GFP+ dT+T
REG
cells om Foxp3GFP-F hΔ/Δ- dT s. con ol Foxp3GFP- dT mice. The
numbe o CD4+GFP+ dT+T
REG
cells eco e ed om he LN was
ma kedly educed when Rag2−/−mice ecei ed T
REG
cells om
Foxp3GFP-F hΔ/Δ- dT s. Foxp3GFP- dT mice (Fig. 5A–C). While he e we e
no di e ences in he numbe o CD4+GFP- dT+ex-T
REG
, he a io o
T
REG
o e dT+cells (GFP- dT+/TdT +)we ehighe inRag2−/−
mice ecei ing T
REG
cells om Foxp3GFP-F hΔ/Δ- dT s. Foxp3GFP- dT mice,
albei wi hou s a is ical significance (Fig. 5A–C). F h exp ession in
CD4+GFP+ dT+T
REG
cells used in he adop i e ans e was
confi med by qRT-PCR (Fig. EV4D,E).
We conside ed he possibili y o an inc ease in he a io o ex-T
REG
o T
REG
cells associa ed wi h F h dele ion in T
REG
cells eflec ing, o
some ex en , a T
REG
cell su i al de ec . The e o e, we asked whe he
FTH ac s in a cell-au onomous manne o suppo T
REG
iabili y and
p oli e a ion in i o. The equency and numbe o induced T
REG
(iT
REG
)cellsgene a ed omT
N
cells ac i a ed in i o wi h an i-CD3/
CD28 mAb plus IL-2 and TGFβ, we e indis inguishable ega dless o
Figu e 3. FTH en o ces T
REG
cell lineage s abili y.
(A) Schema ic ep esen a ion o Foxp3GFP-F hΔ/Δ- dT mice used o moni o he ansi ion o (CD4+GFP+ dT+)T
REG
cells in o (CD4+GFP- dT+)ex-T
REG
cells ha ep essed GFP
exp ession while e aining he exp ession o a dT ansgene. (B–D) Pe cen age o ci cula ing: (B)T
REG
and (C)ex-T
REG
cells in Foxp3GFP-F hΔ/Δ- dT and con ol Foxp3GFP- dT mice,
and (D) Pe cen age o ex-T
REG
cells among CD4+ dT+cells, calcula ed as he a io o CD4+GFP- dT+/CD4+ dT+cells in he same mice as (B,C). Da a om N=4–6 mice
pe geno ype was pooled om h ee independen expe imen s wi h a simila end. (E–G) Pe cen age and numbe o splenic (E)T
REG
cells, (F)ex-T
REG
cells and (G) ela i e
pe cen age o ex-T
REG
cells o e o al CD4+ dT+cells. Da a om N=4 mice pe geno ype, pooled om wo independen expe imen s wi h simila ends. (H)
Rep esen a i e flow cy ome y his og ams o IFNγexp ession by li e ac i a ed (CD4+GFP+ dT+)T
REG
and (CD4+GFP- dT+)ex-T
REG
cells in lymph nodes and spleen om
Foxp3GFP-F hΔ/Δ- dT and con ol Foxp3GFP- dT mice (le panels) and co esponding quan ifica ion o he pe cen age o IFNγexp essing (CD4+GFP+ dT+)T
REG
and
(CD4+GFP- dT+)ex-T
REG
cells ( igh panels). Exp ession o IFNγwas induced upon Pho bol-12-my is a e-13-ace a e (PMA) and Ionomycin e-ac i a ion in i o. Da a om
N=3 wells pe geno ype, in one expe imen , ep esen a i e o wo independen expe imen s wi h simila end. (I) Rep esen a i e flow cy ome y do plo s (le panels)
and co esponding quan ifica ion ( igh panel) o he pe cen age o (CD4+GFP+ dT+)T
REG
and (CD4+GFP- dT+)ex-T
REG
cells exp essing Ki67 and CD71 in he lymph nodes
Foxp3GFP-F hΔ/Δ- dT and con ol Foxp3GFP- dT mice. Da a om N=6 mice pe geno ype, pooled om wo independen expe imen s, wi h simila end. Da a in o ma ion: Da a in
(B–D) ep esen ed as mean ± SD. Ci cles co espond o mean alues. Da a in (E–I) ci cles co espond o indi idual mice and ed ba s o mean alues. (H,I) ep esen ed as
mean ± SD. P alues in (B–D,H,I) calcula ed using Two-way ANOVA analysis wi h Sidak’s mul iple compa isons es and in (E–G) wi h unpai ed es wi h Welch’s
co ec ion. NS no significan (P> 0.05); *P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001. Sou ce da a a e a ailable online o his figu e.
The EMBO Jou nal Qian Wu e al
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0
40
80
120
CD8
+
CD4
+
Foxp3
+
NS
CD45.2
CD45.1
0
10³
-10³
0
0
10³
-10³
0 0
35.1%
64,8%
45%
54,2%
44%
54,4%
32,2%
67,7%
35,3%
63,8%
86,9%
12,7%
Chime ism (%)
Foxp3GFP-F h''- dT
Foxp3GFP- dT
A
0
1
2
3
4
5
**
*** **
**
% GFP+TdT+
Nb .GFP-TdT+(x104)
Nb . GFP
+TdT
+(x104)
ex-TREG cells
% GFP-TdT+/TdT+
10
10³ 10
0
10³
10
10
-10³
-10³ 0 10
10³ 10-10³ 0
TdT
GFP
2,77% 13,2%
79,7% 4,34%
2,82% 3,43%
91,1% 2,68%
B
Foxp3
GFP-F h''- dT
Foxp3
GFP- dT
0
5
10
15
20
0
5
10
15
20
0
1
2
3
4
TREG cells Ra io
DE
0
10
20
30
40
50 *
GF
Lymph Nodes
Lymph Nodes
% GFP-TdT+
CD45.1
CD45.2
Foxp3
GFP-F h '' - d
T
Foxp3
GFP- dT
CD8
+
CD4
+
Foxp3
+
C
***
Rag2-/-
Lymph Nodes
CD45.1+
s.
CD45.2+
C57BL/6
50%
Foxp3
GFP-F h''- dT
Foxp3
GFP- dT
50%
BM
F h1
Hmox1
Nqo1
Vmn1 80
P une2
Fes
M 1 Gm5441
Ly6c2
Mcoln2Cxc 2
A p8b4
F h1−ps
Epd 1
M 2
Padi4
Cnga1
Mageh1
Npdc1
Amz1
Susd4
M gp b1
Pde4c
La ge2
Vmn1 68
0
20
40
60
−10 −5 0 5 10 15 20
Adjus ed p- alue (-log10)
Log2 old change
HI
TREG cells (GFP+ dT+)J
TREG cells (GFP+ dT+)
ex-TREG cells (GFP- dT+
)
Foxp3
GFP- dT
Foxp3
GFP-F h'' - dT
s.
Base mean
exp ession
50x105
10x105
15x105
KL M
Rag2-/-
Lymph nodes
C57BL/6
50%
BM
Foxp3
GFP-F h''- dT
Foxp3
GFP- dT
50%
CD45.2+
RNAseq
F h1
Hmox1
Nqo1
N n1
P pn5
Gm21909
Sema7a
Ly6d
Cc 6
M 1
Gm16086
Hi 3a
Gm29243
Gja10
Gm19951
I 172
F h1−ps
Sgcb
C ap54 M ap3l
N 3c2
Cd83
Smim10l2a
A hgap24
Plpp1
0
20
40
60
−10 −5 0 5 10 15 20
Log2 old change
Adjus ed p- alue (-log10)
ex-TREG cells (GFP- dT+)
Foxp3
GFP- dT
Foxp3
GFP-F h'' - dT
s.
Base mean
exp ession
50x105
10x105
15x105
Mixed BM chime ic/non-chime ic mice
TREG cells (GFP+)TREG cells (GFP+)TREG cells (GFP+)
C
hime i
c
111
134
Non−chime ic
3711
Up egula ed Down egula ed
300
100
20
10
0
10
20
100
150
-log10 (p- alue)
Mixed BM chime as
Non-chime ic
Bio.
p oc. KEGG Reac. TF WP
Cell edox
homeos asis
Cellula esponse o
chemical s imulus
Cell. esponse
o IFN-β
De oxi ica ion
De oxi ica ion
o ROS
BACH2
IRF1
IRF2
IRF
Fe op osis
Oxid. s ess
and edox
pa hway
Oxid. s ess
esponse
Response o
oxid. s ess
Response
o ROS
T ansc ip. ac i . by
NRF2 in esponse
o phy ochem.
2
1
4
6
7
5
3
−log10 (p alue)
Hmox1
Nqo1
Sqs m1
Sod1
Slc48a1
Txn d1
Txn1 P dx1 M 1
Ma g
F h1
F l1
Gclm
Gs
CD45.2+
CD45.1+
So ing
NS
NS
NS ***
Qian Wu e al The EMBO Jou nal
© The Au ho (s) The EMBO Jou nal Volume 43 | Issue 8 | Ap il 2024 | 1445 –1483 1453
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exp ession in T
REG
cells a o s umo p og ession. In suppo o his
hypo hesis, he ela i e g ow h o syngeneic B16 melanoma cells was
educed in Foxp3GFP-F hΔ/Δ s. con ol F hfl/flmice (Fig. 8H). This was
associa ed wi h lowe equency o umo -infil a ing T
REG
cells (Fig. 8I)
and highe equency o ac i a ed Foxp3-CD4+CD25+e ec o T
H
cells
(Fig. 8J). The equency and numbe o ac i a ed CD4+IFNγ+T
H
cells
isola ed om B16 melanomas was simila in Foxp3GFP-F hΔ/Δ s. con ol
Foxp3GFP mice (Fig. EV5F). In con as , he numbe o ac i a ed
CD8+IFNγ+T
C
cells was highe in B16 melanomas om Foxp3GFP-F hΔ/Δ
s. con ol Foxp3GFP mice (Fig. EV5F). This endency was also obse ed
o CD8+g anzymeB+T
C
cells, albei no s a is ically significan
(Fig. EV5G). This sugges s ha egula ion o Fe me abolism by FTH
in T
REG
cells suppo s umo p og ession, ia a mechanism ha hinde s
an i- umo immuni y.
Discussion
T
REG
cells espond o a ia ions in he ela i e le els o nu ien s,
i amins and me aboli es in hei en i onmen (Chapman e al,
2020; Shi and Chi, 2019), ia dedica ed anspo e - ecep o
coupled senso s ha modula e T
REG
cell unc ion and lineage
s abili y (Kempkes e al, 2019; Shi and Chi, 2019). In keeping wi h
Fe- egula o y genes being a co e p ope y o T
REG
cells (Cuad ado
e al, 2018), we ound ha he Fe- egula o y p o ein FTH is
essen ial o suppo T
REG
cell lineage main enance in i o (Figs. 1–4)
and suppo immune homeos asis (Figs. 2,EV1D–G, and EV2C).
FTH egula es T
REG
cell lineage s abili y (Figs. 3and 4A–G)
wi hou in e e ing wi h he an ip oli e a i e unc ion o T
REG
cells
(Fig. EV2A). Ins ead, FTH a ge s he in acellula pool o edox-
ac i e Fe2+(Fig. EV1B) o egula e T
REG
cell edox homeos asis
(Figs. 2Fand4K–M) in a manne ha con ols T
REG
cell: (i) ene gy
me abolism (Figs. 5F–IandEV5A–E), (ii) TET ac i i y (Fig. 6H),
(iii) cy osine deme hyla ion a CpG- ich sequences a CNS1 and
CNS2 in he FOXP3 locus (Fig. 6G), and (i ) FOXP3 ansc ip ion/
exp ession (Figs. 1K,L and 7F–H). As he la e is essen ial o
main ain he ansc ip ional p og am suppo ing T
REG
cell lineage
s abili y (Williams and Rudensky, 2007)(Naka sukasae al,2019;
Ohku a e al, 2012;Yuee al,2019)F h dele ion is associa ed wi h a
dec ease o T
REG
cells, including T
REG
cells and pT
REG
cells, wi hou
in e e ing wi h hymic T
REG
cell ou pu (Fig. 1H,L). These
obse a ions a e consis en wi h FTH ac ing ups eam o TET
me hylcy osine dioxygenases, o con ol edox-based cy osine
deme hyla ion in T
REG
cells (Fig. 6)(HuangandRao,2014;Pas o
e al, 2013).
Tha FTH a ge s he in acellula pool o edox-ac i e Fe2+ o
con ol TET enzyma ic ac i i y is sugges ed by he obse a ion ha
o e exp ession o a e oxidase deficien FTHmcomp omised TET
me hylcy osine dioxygenase ac i i y (Fig. 6H, I). Se e al non-
mu ually exclusi e mechanisms migh explain his obse a ion.
Fi s , FTH could ac “di ec ly” ia seques a ion o ca aly ic Fe2+
(Fig. EV1B), con olling he a ailabili y o his essen ial co ac o o
TET enzyma ic ac i i y (Kohli and Zhang, 2013;Pas o e al,2013).
Second, FTH could ac “indi ec ly” ia he egula ion o cellula
edox homeos asis (Figs. 2D,F and 4K–M), p e en ing ca aly ic
Fe2+ om ca alyzing oxida i e s ess, which comp omises TET
ac i i y (Niu e al, 2015). Mo eo e , his should es ain NRF2
ac i a ion (Figs. 2D,F and 4K–M) om ep essing Foxp3 exp ession
and impai T
REG
cell unc ion (Klemm e al, 2020).
Consis en wi h ou findings, in acellula Fe mobiliza ion ia
lysosome-media ed e i inophagy, was ecen ly shown o egula e
TET-d i en (de)me hyla ion o he pe oxisome p oli e a o -
ac i a ed ecep o γ(PPARγ) locus, he mas e egula o s o
adipocy e de elopmen (Suzuki e al, 2023). This sugges ha FTH
con ols Fe- esponsi e epigene ic p og ams defining di e en
cellula de elopmen al p og ams.
FTH egula es T
REG
cell mi ochond ial TCA cycle and OXPHOS
(Figs. 5F–I and EV5A–E), consis en wi h simila findings in o he cell
ypes (Blankenhaus e al, 2019; Oexle e al, 1999). While FTH does no
modula e he in acellula concen a ion o in e media e TCA cycle
me aboli es (Fig. EV5E), including α-ke oglu a a e (Fig. 5I), i
does appea o modula e he a e o isoci a e con e sion in o
α-ke oglu a a e, likely ac ing i espec i ely o α-ke oglu a a e gene a-
ion ia glu aminolysis. I is possible he e o e ha FTH egula es
he p oduc ion o his obliga o y subs a e o TET cy osine
dioxygenases (Kohli and Zhang, 2013;Pas o e al,2013) ia egula ion
Figu e 7. FTH is equi ed o sus ain Foxp3 ansc ip ion and exp ession.
(A) FTH p o ein de ec ed by wes e n blo in whole-cell ex ac s om HEK293T cells in ec ed wi h ecombinan len i i uses coding shRNAs a ge ing FTH (FTH429 and
FTH432) o con ol (C l.) ecombinan len i i uses non- a ge ing shRNA. (B) Mean fluo escence in ensi y (MFI) o FOXP3 exp ession, de ec ed by flow cy ome y in human
(CD4+CD45RA+CD25+)T
REG
cells in ec ed wi h he same ecombinan len i i uses as in (A). Da a om N=6 samples pe expe imen al g oup. (C) Schema ic
ep esen a ion o he expe imen al app oach (le panel) used o moni o GFP ansgene exp ession in he mesen e ic LN (MLN) o (CD4+GFP+N p1+) T
REG
cells and
(CD4+GFP+N p1−)pT
REG
cells. Rep esen a i e flow cy ome y his og am (middle panel) and quan ifica ion o ela i e GFP exp ession ( igh panel), shown as mean
fluo escence in ensi y (MFI). Da a om N=8 mice pe geno ype, pooled om wo independen expe imen s wi h simila end. (D) Schema ic ep esen a ion o he
expe imen al app oach (le panel) used o moni o Foxp3 exp ession by flow cy ome y in mouse spleen and MLN (CD4+Foxp3+)T
REG
cells. Rep esen a i e flow cy ome y
s aining o Foxp3 (middle panel). Rela i e quan ifica ion o Foxp3 exp ession ( igh panel), ep esen ed as mean o fluo escence in ensi y (MFI). Da a om N=9 mice pe
geno ype, pooled om wo o h ee independen expe imen s wi h simila end. (E) Schema ic ep esen a ion o he expe imen al app oach (le panel) used o moni o
Foxp3 exp ession in (CD45.2+CD4+GFP+ dT+)T
REG
cells isola ed om he spleen and LN o BM chime as. Rep esen a i e flow cy ome y o Foxp3 s aining (middle panel).
Rela i e quan ifica ion o Foxp3 exp ession ( igh panel), shown as mean fluo escence in ensi y (MFI). Da a in (E) om N=5–6 mice pe geno ype, ep esen a i e o wo
independen expe imen s wi h simila end. (F) Schema ic ep esen a ion o he expe imen al app oach (le panel) used o moni o GFP exp ession in he spleen and LN
o CD45.2+CD4+ dT+cells (T
REG
+ex-T
REG
) om BM chime as. Rep esen a i e flow cy ome y o GFP (F) s aining (middle panel). Rela i e quan ifica ion o GFP exp ession
( igh panel), ep esen ed as mean fluo escence in ensi y (MFI). Da a om N=10 mice pe geno ype, pooled om wo independen expe imen s wi h simila end. (G)
Schema ic ep esen a ion o he expe imen al app oach (le panel), whe e (CD4+ dT+) cells we e so ed om he spleen and LN o qRT-PCR (G, le panel). Rela i e
exp ession o Foxp3 ( igh panel). (H)G p, F h, and dT mRNA exp ession no malized o A bp0 o cells so ed as in (G). Da a in (G,H) om N=3–4 mice pe geno ype
om one expe imen . Da a in o ma ion: Da a in (B) a e p esen ed as mean ± SD, ci cles co espond o indi idual wells and ed ba s o mean alues. Da a in (C–H) a e
p esen ed as mean ± SD, ci cles co espond o indi idual mice and ed ba s o mean alues. P alues in (B) we e calcula ed using he Fiedman es wi h Dunn’s mul iple
compa ison es , in (C–H) using wo-way ANOVA wi h Sidak’s mul iple compa ison es . NS no significan (P> 0.05), *P< 0.05; **P< 0.01; ***P< 0.001;
****P< 0.0001. Sou ce da a a e a ailable online o his figu e.
The EMBO Jou nal Qian Wu e al
1460 The EMBO Jou nal Volume 43 | Issue 8 | Ap il 2024 | 1445 –1483 © The Au ho (s)
Downloaded om h ps://www.embop ess.o g on Janua y 15, 2025 om IP 193.144.79.182.

H
E
D
BC
G
F
F h
l/ l
Foxp3
GFP-F h''
F h
l/ l
Foxp3
GFP-F h''
J
I
0
40
80
67,7% 32,6%
***
Foxp3+ (%)
Foxp3
CD4
105
104
103
0
010
3104105010
3104105
10-2
10-3
10-2
1,32% 4,5%
0
2
4
6
8
Foxp3-CD25+ (%)
CD25
Foxp3
***
105
104
103
0
010
3104105010
3104105
F h
l/ l
Foxp3
GFP-F h''
F h
l/ l
Foxp3
GFP-F h''
0 5 10 15
0
25
50
75
100
Pe cen su i al
***
Days
0
1
2
3
4
5
6
7
iRBCs/PL (x106)
*
Foxp3
GFP
Foxp3
GFP-F h''
Days
9/9
1/9
**
CD4
010
3104105010
3104105
105
104
103
0
0
5
10
15
Foxp3-GFP (x10 )
0
2
4
1
3
NS
10% 4,86%
Foxp3
GFP
Foxp3
GFP-F h''
Tumo
Size
B16
Melanoma
Spleen
CD4+Foxp3+
Pcc
Pcc
CD4
IFNJ
0
10³
10
10
10
0
0
10
20
30
0
10
5
IFN
J
+CD4+ (
x10
6
)
IFN
J
+CD4+ (
%
)
NS *
12,6% 21,4%
CD4+
IFNJ
Pcc
105
104107
106
0
105
104107
106
Spleen
Foxp3
GFP
Foxp3
GFP-F h''
Foxp3
GFP
Foxp3
GFP-F h''
Foxp3
GFP
Foxp3
GFP-F h''
Su i al
Pa hogen
03 9 15612
Disease Se e i y
*
0 5 10 15 20 2530
0
0.5
1
1.5
2
2.5
3
Days
3.5
35
A
Disease Incidence (%)
EAE
MOG35-55
+CFA
0 5 101520253035
0
50
100
Days
25
25 *
Foxp3-GFP
Foxp3-GFP (%)
***
**
*
13 15 17 19
0
200
400
600
800
1000
Days
Tumo size (mm3)
IFNJ(%)
IL-17A+(%)
*
F h
l/ l
Foxp3
GFP-F h''
(IFNJ/IL-17)
CD4+
Spinal
Co d
MOG35-55
0
5
10
15
20
0
10
20
30
40
0
2
4
6
8
(%)
IFNJIL-17A+
*
16% 22%
IFNJ
IL-17A
18,7% 33,9%
105
104
103
0
010
3104105010
3104105
F h
l/ l
Foxp3
GFP-F h''
+CFA NS
1,4% 4%
F h
l/ l
Foxp3
GFP-F h''
** **
*
**
*
NS
NS
Qian Wu e al The EMBO Jou nal
© The Au ho (s) The EMBO Jou nal Volume 43 | Issue 8 | Ap il 2024 | 1445 –1483 1461
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o he TCA cycle. This in e p e a ion is consis en wi h o he signal
ansduc ion pa hways egula ing he TCA cycle, such as he one
igge ed by he p og ammed cell dea h 1 ligand 2 (PD-L2),
egula ing cy osine me hyla ion a CpG sequences in he T
REG
-specific
deme hyla ion egion, comp omising Foxp3 s abili y in T
REG
cells
(Hu ell e al, 2022).
The me abolic p og am o ches a ed by FOXP3, suppo s T
REG
cell an ip oli e a i e unc ion and lineage s abili y, ia a mechanism
ha elies on he supp ession o c-Myc, a ansc ip ion ac o ha
ep esses mi ochond ial OXPHOS and p omo es glycolysis
(Angelin e al, 2017). Impo an ly, c-Myc can ep ess FTH
ansc ip ion and ansla ion in o he cell ypes, he la e occu ing
ia a mechanism in ol ing he Fe- egula o y p o ein 2 (IRP2) (Wu
e al, 1999). This sugges s ha he FOXP3-d i en gene ic p og am
encompasses he induc ion o FTH ia a mechanism ha could
in ol e he ep ession o c-Myc. Mo eo e , he FTH p omo e
con ains a leas one Foxp3 DNA binding si e (Appendix
Figs. S3 and 4) and as such i is possible ha Foxp3 would egula e
FTH exp ession di ec ly. This would a gue o a posi i e eedback
loop in which FTH en o ces Foxp3 exp ession, ia he egula ion o
TET dioxygenases, and he la e en o ces he FTH exp ession
ansc ip ionally. This hypo hesis emains howe e o be es ed
expe imen ally.
While FTH p e en s he ansdi e en ia ion o T
REG
cells owa d
inflamma o y ex-T
REG
cells (Figs. 3and EV2C–E), ia a non-cell-
au onomous mechanism ha elies on sys emic inflamma ion
(Fig. EV1D–G), his is no associa ed wi h he accumula ion o
inflamma o y ex-T
REG
cells in Foxp3GFP-F hΔ/Δ- dT s. con ol Foxp3GFP- dT
mice (Fig. 3E–G). The same is ue in mixed BM chime ic mice
econs i u ed wi h BM cells om Foxp3GFP-F hΔ/Δ- dT s. con ol Foxp3GFP- dT
mice (Fig. 4A–G). One possible in e p e a ion is ha FTH is essen ial no
only o es ain T
REG
cells om ansdi e en ia ing in o inflamma o y ex-
T
REG
cells bu also o suppo he iabili y o highly p oli e a ing T
REG
and
ex-T
REG
cells. This is consis en wi h he lack o au oimmune lesions in
Foxp3GFP-F hΔ/Δmice(Fig.EV2B).Weno e,howe e , ha F h dele ion in
T
REG
cells does no comp omise he T
REG
cell hymic ou pu (Fig. 1H) no
he gene a ion and p oli e a ion o iT
REG
cells in i o (Fig. EV4F–J).
One canno exclude ha he highly me hyla ed s a us o he
Foxp3 locus om ex-T
REG
cells (Koma su e al, 2014; Miyao e al,
2012;Zhoue al,2009) oge he wi h he inc ease equency o ex-
T
REG
cells among CD45.2+CD4+ dT+cells (Fig. 4A–G), con ibu es
o he obse ed inc ease in he me hyla ion o he Foxp3 locus in
F h-dele ed T
REG
cells (Fig. 6A–G). This does no in alida e
howe e , ha F h dele ion ac s in a cell-au onomous manne o
dec ease Foxp3 ansc ip ion/exp ession (Figs. 1K,L and 7F–H),
he e o e inc easing he equency a which T
REG
cells ansdi e -
en ia e in o ex-T
REG
cells (Fig. 4A–G).
In con as wi h gene ic dele ion o Te 2 and Te 3 in T
REG
cells,
which p omo es he ansdi e en ia ion o T
REG
cells in o
inflamma o y ex-T
REG
cells and he de elopmen o au oimmuni y
(Naka sukasa e al, 2019;Ohku ae al,2012;Yuee al,2019), F h
dele ion in T
REG
cells is no associa ed wi h o e au oimmuni y
(Fig. EV2BG). This is consis en wi h FTH exe ing addi ional
e ec s, beyond he egula ion o TET dioxygenases, p e en ing
cellula s ess om comp omising he iabili y o he inflamma o y
ex-T
REG
cells ha would o he wise elici au oimmuni y.
F h dele ion in T
REG
cells led o an inc ease in EAE suscep ibili y
and se e i y (Fig. 8A–C), induced by an immuniza ion p o ocol
leading o low-g ade disease se e i y in con ol mice (Fig. 8C). This
was associa ed wi h a highe accumula ion o ac i a ed T
H
1and
T
H
17 cells as well as pa hogenic IFNγ+IL-17A+T
H
cells (Duhen
e al, 2013) in he cen al ne ous sys em (Fig. 8D), likely
o igina ing om au o- eac i e T
N
cells and/ om ex-T
REG
cells ha
los Foxp3 exp ession o become inflamma o y and p esumably
pa hogenic (Bailey-Buck ou e al, 2013). This is consis en wi h
egula ion o Fe me abolism modula ing he incidence and se e i y
o au oimmune condi ions, as demons a ed o sys emic lupus
e y hema osus (Gao e al, 2022a). Consis en wi h ou findings, his
was linked o modula ion o T
REG
(Gao e al, 2022a), T
H
17 (Teh
e al, 2021), and FT
H
(Gao e al, 2022b) cells and was associa ed
wi h egula ion o DNA deme hyla ion (Gao e al, 2022b; Teh e al,
2021). Howe e , whe he egula ion o Fe me abolism in T
REG
cells
a ec s sys emic lupus e y hema osus was, o he bes o ou
knowledge, no es ablished (Gao e al, 2022a).
F h dele ion in T
REG
cells inc eased suscep ibili y o mala ia
(Fig. 8E), consis en wi h dys egula ion o Fe me abolism p omo ing
mala ia le hali y (Fe ei a e al, 2008; Ramos e al, 2022; Ramos e al,
2019;Wue al,2023). This was associa ed wi h an inc ease in hos -
pa asi e bu den (Fig. 8E), in keeping wi h T
REG
cells being essen ial o
coun e he pa hogenesis o se e e p esen a ions o mala ia while
limi ing immune-d i en esis ance mechanisms d i ing pa asi e
clea ance (Hisaeda e al, 2004; Ku up e al, 2017; Wal he e al,
2005). We in e ha he p o ec i e e ec o T
REG
cells agains mala ia
ac s ia a mechanism ha is no associa ed wi h a educ ion o he
Figu e 8. FTH exp ession in T
REG
cells con ols he pa hologic ou come o expe imen al immune-d i en inflamma o y condi ions.
(A) Schema ic ep esen a ion o he induc ion expe imen al au oimmune encephalomyeli is (EAE) in esponse o MOG
35-55
immuniza ion. (B) EAE incidence (pe cen age)
and (C) EAE se e i y in MOG
35-55
immunized mice. Da a om N=18–20 mice pe geno ype, pooled om h ee independen expe imen s, wi h simila end. (D)
Expe imen al app oach ( igh panel), ep esen a i e flow cy ome y do plo s (middle panel) and co esponding quan ifica ion (le panel) o he ela i e pe cen age o
ac i a ed T
H
1 (CD3+CD4+IFN-γ+), T
H
17 (CD3+CD4+IL-17A+); and double posi i e IFN-γ+IL-17A+T
H
cells in he spinal co d, 22 days a e MOG
35-55
immuniza ion. Da a
om N=3–4 mice pe geno ype. (E) Su i al (le panel) and numbe o ci cula ing Plasmodium chabaudi chabaudi (Pcc)-in ec ed ed blood cells (iRBC) pe µL o whole
blood (i.e., pa asi e bu den) ( igh panel). N=9 mice pe geno ype, pooled om wo independen expe imen s, wi h simila end. (F,G) Rep esen a i e flow cy ome y
do plo (le panels) and co esponding pe cen age and cell numbe s ( igh panels) o splenic (CD4+Foxp3-GFP+)T
REG
cells (F) and IFNγ+CD4+ac i a ed T
H
cells (G),
7 days a e Pcc in ec ion. Da a om N=8–9 mice pe geno ype, pooled om wo independen expe imen s, wi h simila end. (H) Rela i e umo (B16-F10-luc2) size,
13–19 days a e inocula ion (2 × 105cells). Da a om N=7–11 mice pe geno ype, pooled om 3 independen expe imen s, wi h simila end. (I,J) Rep esen a i e flow
cy ome y do plo s (le panels) and co esponding pe cen age ( igh panels) o li e umo -infil a ing (CD4+Foxp3+)T
REG
cells (I) and (CD4+Foxp3-CD25+) e ec o T
H
cells (J), 3 weeks a e umo inocula ion. N=7 mice pe geno ype, pooled om h ee independen expe imen s, wi h simila end). Da a in o ma ion: Ci cles in
(D,F,G,I,J) co espond o indi idual mice and ed ba s o mean alues. Da a in (C,H) a e p esen ed as mean ± SEM. Da a in (E, igh panel) a e p esen ed as mean ± SD. P
alues in (C), (E, igh panel), and (H) we e de e mined using Holm–Sidak me hod (mul iple es s), wi h alpha =0.05 unde he assump ion ha bo h geno ypes ha e
simila SEM, in (B,E) by log- ank (Man el–Cox) es , and in (D,F,G,I,J) by unpai ed es wi h Welch’s co ec ion. NS no significan (P> 0.05); *P< 0.05; **P< 0.01;
***P< 0.001. Sou ce da a a e a ailable online o his figu e.
The EMBO Jou nal Qian Wu e al
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hos -pa hogen bu den, a de ense s a egy known as disease ole ance
(Medzhi o e al, 2012; Soa es e al, 2017). P esumably,
he mechanism(s) ia which FTH ac s in T
REG
cells o es ablish
disease ole ance o mala ia is mul i ac o ial, es aining un e e ed
immune ac i a ion o p e en he pa hogenesis o se e e o ms o
mala ia.
Dys egula ion o Fe me abolism in F h-dele ed T
REG
cells was
associa ed wi h be e con ol o umo p og ession (Fig. 8H),
consis en wi h a ela i e educ ion o umo -infil a ing T
REG
cells
(Fig. 8I) and a mo e p onounced ac i a ion and/o infil a ion o
ac i a ed T e ec o cells (Fig. 8J), including CD8+IFNγ+T
C
cells
(Fig. EV5F). While he mechanism ia which FTH exp ession in
T
REG
cellsp omo es umo p og essionisno clea , hese
obse a ions a e consis en wi h he egula ion o Fe me abolism
in he umo mic oen i onmen impac ing on umo p og ession
(Alalu e al, 2020; Consonni e al, 2021).
In conclusion, egula ion o in acellula Fe me abolism by FTH
is essen ial o main ain T
REG
cell lineage and unc ion in i o,
eflec ing how in acellula ca aly ic Fe con ols he ac i i y o TET
dioxygenases ha sus ain FOXP3 ansc ip ion and suppo T
REG
cell lineage iden i y. Mo eo e , FTH migh egula e o he i on-
dependen mechanisms suppo ing T
REG
unc ion, o example, by
en o cing he exp ession o c-Ma in T
REG
cells (Zhu e al, 2023) ha
con ol immunological ole ance o he mic obio a (Xu e al, 2018).
We p opose ha a ge ing Fe me abolism pha macologically maybe
conside ed when manipula ing T
REG
cells o he apeu ic pu poses,
ei he o enhance T
REG
cell unc ion in he con ex o immune-
media ed inflamma o y diseases o o dampen T
REG
cell unc ion as
in he con ex o cance he apies.
Me hods
Reagen s and ools able
Reagen / esou ce
Re e ence o
sou ce
Iden ifie o ca alog
numbe
Expe imen al models
Human: HEK293T ATCC ATCC®CRL-3216™
Mouse: Tumo cells B16-F10-luc2
(B16)
Calipe LS B16-F10-luc2
Mouse: B6.C57BL/6 F hfl/flLukas Kuhn, ETH,
Swi ze land
(Da shan e al, 2009)
Mouse: B6129S-Tg(Foxp3 EGFP/
ic e)1aJbs/J backc ossed in o
B6.C57BL/6 backg ound
Jackson
Labo a o y
JAX s ock: 023161
Mouse: B6.Cg-G (ROSA)
26So m9(CAG- dToma o)Hze/J
Jackson
Labo a o y
JAX s ock: 007909
Mouse: RAG2 -/- (B6.129S6-
Rag2< m1Fwa>N12)
Taconic Taconic # RAGN12
Plasmodium chabaudi chabaudi
s ains: PcAS clone AJ4916
Reece &
Thompson, 2008
N/A
Blood samples om anonymized
heal hy male dono s we e
ob ained in acco dance wi h
guidelines es ablished by he
Sanquin Medical E hical
Commi ee.
This pape NA
Reagen / esou ce
Re e ence o
sou ce
Iden ifie o ca alog
numbe
Recombinan DNA
psPAX Addgene Ca #12260
pMD2.G Addgene Ca #12259
pCMV-FTH-3 ag3a This pape
pCMV-FTHmu -3 ag3a This pape
pCMV-3×FLAG-TET3(human)-
Neo
miaolingBio P45302
An ibodies
PE an i-human CD25 (Clone 2A3) BD Biosciences Ca #341011 (RRID:
AB_2783790)
PE-Cy7 an i-human CD45RA
(Clone HI100)
BD Biosciences Ca #560675 (RRID:
AB_1727498)
B illian Viole 421 An i-human
CD127 (Clone A019D5)
Biolegend Ca #351309 (RRID:
AB_10898326)
PE-Cy7 an i-Human FOXP3 (Clone
236A/E7)
eBioscience Ca #25-4777-42
(RRID: AB_2573450)
An i-Fe i in Hea y Chain Abcam ab65080
(RRID:AB_10564857)
CD45 APC-eFluo 780 eBioscience 30-F11, 47-0451-82
(RRID:AB_1548781)
TCR-βBV421 BioLegend H57-597, 109229
(RRID:AB_10933263)
TCR-βBV711 BioLegend H57-597, 109243
(RRID:AB_2629564)
CD4 PE-Cy7 eBioscience RM4-5, 25-0042-82
(RRID:AB_469578)
CD4 APC-eFluo 780 eBioscience GK1.5, 47-0041-82
(RRID:AB_11218896)
CD4 BV421 BioLegend GK1.5, 100438
(RRID:AB_10900241)
CD8 Pe cpCy5.5 eBioscience 53-6.7, 45-0081-82
(RRID:AB_1107004)
CD8 APC/Fi e 750 BioLegend 53-6.7, 100766
(RRID:AB_2572113)
CD44 eFluo 450 eBioscience IM7, 48-0441-82
(RRID:AB_1272246)
CD62L Pe-Cy7 eBioscience MEL-14, 25-0621-82
(RRID:AB_469633)
CD304 (NRP1) PE BioLegend 3E12, 145204
(RRID:AB_2561928)
PD1-PE eBioscience RMP1-30, 12-9981-82
(RRID:AB_466290)
CXCR5-bio in BD Biosciences 2G8, 551960 RRID:
AB_394301)
Alexa Fluo ®647 s ep a idin BioLegend 405237
CD25 PE-Cy7 BioLegend PC61, 102016
(RRID:AB_312865)
Foxp3 PE eBioscience FJK-16s, 12-5773-82
(RRID:AB_465936)
Foxp3 FITC eBioscience FJK-16s, 11-5773-82
(RRID:AB_465243)
Foxp3 eF450 eBioscience FJK-16s, 48-5773-82
(RRID:AB_1518812)
Qian Wu e al The EMBO Jou nal
© The Au ho (s) The EMBO Jou nal Volume 43 | Issue 8 | Ap il 2024 | 1445 –1483 1463
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Reagen / esou ce
Re e ence o
sou ce
Iden ifie o ca alog
numbe
CD71 BioLegend RI7217, 113811
(RRID:AB_2203383)
Ki67 eBioscience SolA15, 50-5698-82
(RRID:AB_2896285)
IL-17A eBioscience eBio17B7, 50-7177-82
(RRID:AB_11220280)
IFN-gamma eBioscience XMG1.2,12-7311-81
(RRID:AB_466192)
CD45RA BD Biosciences HI100 (RRID:
AB_1727498)
CD45.1 Pacific blue P oduced a IGC A20
CD45.2 AF647 P oduced a IGC 104.2
CD4 Mic oBeads, human Mil enyi Bio ec 130-045-101
(RRID:AB_2889919)
Thy1.1 P oduced a IGC 19E12
Thy1.2 P oduced a IGC 30H12
an i-CD3 mAb (Clone 1XE) Peliclus e M1654
(RRID:AB_10553652)
an i-CD28 mAb (Clone CD28.2) eBioscience 16-0289-85
(RRID:AB_468927)
HO-1 Enzo Li e
Sciences
ADI-SPA-896
(RRID:AB_10614948)
CD16/CD32 BioLegend 93, 101331
Oligonucleo ides and o he sequence-based eagen s
Human FTH1 shRNA: FTH429;
TRCN0000029429; a ge
sequence:
GCCTCGGGCTAATTTCCCATA
GPP Web Po al,
B oad Ins i u e
RHS3979-9596837
Human FTH1 shRNA: FTH432;
TRCN0000029432; a ge
sequence:
CCTGTCCATGTCTTACTACTT
GPP Web Po al,
B oad Ins i u e
RHS3979-9596840
P ime s o RT-qPCR, see Table 1See Table 1N/A
Chemicals, enzymes, and o he eagen s
TMRE-Mi ochond ial Memb ane
Po en ial Assay Ki
Abcam ab113852
Fe oFa Red™Go yo Chemical GC903-01
LIVE/DEAD™Fixable Aqua S ain The moFishe
Scien ific
L34957
eBioscience™Cell S imula ion
Cock ail
The moFishe
Scien ific
00-4970-93
P o ein T anspo Inhibi o
Cock ail
The moFishe
Scien ific
00-4980
CFSE The moFishe
Scien ific
C34554
Solid Phase Re e sible
Immobiliza ion (SPRI) beads
Beckman Coul e
So wa e Include e sion whe e applicable
ImageJ Schneide
e al, 2012
h ps://
imagej.nih.go /ij/
Flowjo BD Sciences Ve sion 10.8.1
R R Co e Team
2014, Vienna,
Aus ia
Ve sion 3.5.1
Reagen / esou ce
Re e ence o
sou ce
Iden ifie o ca alog
numbe
Fas QC me hod Bab aham
bioin o m.
Ve sion 0.11.5
Py hon (Linux/UNIX) Py hon Ve sion 2.7.12
T immoma ic Bolge e al, 2014 Ve sion 0.36
HISAT2 Kim e al, 2015 Ve sion 2.1.0
HTseq Ande s
e al, 2015
Ve sion 0.6.1p1
DESeq2 Lo e e al, 2014 Ve sion 1.26.0
O he
QIAamp DNA Mic o Ki QIAGEN 56304
NucleoSpin RNA XS Mache ey-Nagel 740902
ChIP DNA Clean & Concen a o
columns
Zymo Resea ch D5205
MagniSo ™Mouse CD4 T-cell
En ichmen Ki
The moFishe
Scien ific
8804-6821-74
NEBNex ®Enzyma ic Me hyl-seq
Ki
New England
Biolabs
E7120
Qubi HS dsDNA ki The moFishe
Scien ific
Q32851
High Sensi i i y DNA Bioanalyze
ki
Agilen 5067-4626
Seaho se XF Cell Mi o S ess Tes
Ki
Agilen
Technologies
103015-100
Animals
Mice we e b ed and main ained unde specific pa hogen- ee (SPF)
condi ions a he Ins i u o Gulbenkian de Ciência (IGC). All
expe imen al p o ocols we e app o ed by he E hics Commi ee o
he IGC, he “Ó gão Responsá el pelo Bem-es a dos Animais”
(ORBEA) (license numbe s A001-2017, A003-2017) and he
Po uguese Na ional En i y (Di ecção Ge al de Alimen ação e
Ve e iná ia) (no ifica ion numbe s 003722, 008830). Expe imen al
p ocedu es we e pe o med acco ding o he Po uguese (Po a ia
no. 1005/92, Dec e o-Lei no. 113/2013 and Dec e o-lei no.1/2019)
and Eu opean (Di ec i e 2010/63/EU) legisla ions, conce ning
housing, husband y, and animal wel a e. Foxp3GFP-F hΔ/Δmice we e
gene a ed by c ossing Foxp3GFP (i.e., B6129S-Tg(Foxp3 EGFP/ic e)
1aJbs/J) mice (Chen e al, 2003;Zhoue al,2008)wi hF hfl/flmice
(Da shan e al, 2009). Mouse p ogeny was geno yped o he
p esence o he C e allele. The Foxp3GFP mice exp ess a humanized
C e- ecombinase (GFP-hC e) om a Foxp3 ATG ansla ional s a
codon, inse ed in a bac e ial a ificial ch omosome (BAC)
ansgene (Chen e al, 2003;Zhoue al,2008). As C e exp ession
is no sex-dependen , his allows o F h dele ion in T
REG
cells om
male and emale Foxp3GFP-F hΔ/Δmice, using Foxp3GFP and F hfl/flmice
as con ols. Foxp3GFP-F hΔ/Δ- dT mice we e gene a ed by c ossing
Foxp3GFP-F hΔ/Δmice wi h Ai9 (RCL- dT) mice. Con ol Foxp3GFP- dT
mice we e gene a ed by c ossing Foxp3GFP mice wi h C57BL/6 Ai9
(RCL- dT) mice, simila o desc ibed abo e. P ogeny was geno yped
o he p esence o he humanized C e- ecombinase (G p-hC e)
allele. The gene ic backg ound o he mouse s ains used was
C57BL/6J, including Foxp3GFP mice, backc ossed in o C57BL/6/J
The EMBO Jou nal Qian Wu e al
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backg ound o o e 10 gene a ions. Rag2−/−mice used as
ecipien s o BM p ecu so cells we e in C57BL/6NTac backg ound.
Expe imen al au oimmune encephalomyeli is (EAE)
C57BL/6 mice we e immunized wi h he MOG
35–55
pep ide (s.c.;
200 µg), emulsified in Comple e F eund’s Adju an (CFA) con ain-
ing Mycobac e ium ube culosis (4 mg/mL; BD Diagnos ics). Mice
ecei ed 200 ng o Pe ussis oxin (i. .; Sigma-Ald ich) a he ime
o immuniza ion and 2 days he ea e . Clinical EAE se e i y sco es
we e e alua ed daily as ollows: 0, no mal; 1, limp ail; 2, pa ial
pa alysis o he hind limbs; 3, comple e pa alysis o he hind limbs;
4, hind-limb pa alysis and o elimb weakness; 5, mo ibund o
deceased, essen ially as desc ibed (Cho a e al, 2007).
Plasmodium in ec ion (mala ia)
Mice we e in ec ed by he inocula ion o blood isola ed om mice
in ec ed wi h a Plasmodium chabaudi chabaudi (Pcc) AS s ain [i.p.;
2×10
6in ec ed ed blood cells (iRBC) pe mouse]. Mice we e
moni o ed daily o pa asi emia, weigh , empe a u e, RBC
numbe , and su i al, essen ially as desc ibed (Seixas e al, 2009).
Tumo model
Tumo cells B16-F10-luc2 (B16) (Calipe LS) we e cul u ed a 37 °C
in RPMI 1640 (Li e Technologies) supplemen ed wi h 10% Fe al
Bo ine Se um (Biowes ), 1% penicillin–s ep omycin (Li e Tech-
nologies), 50 µg/mL Gen amicin (Li e Technologies), and 50 µM
2-Me cap oe hanol (Li e Technologies). A e ypsin (Li e Tech-
nologies) ea men , single-cell suspensions we e esuspended in
ice cold calcium- ee and magnesium- ee HBSS (Li e Technolo-
gies). Mice we e injec ed subcu aneously in he igh flank wi h
2×10
5B16 cells in 100 µL olume. Tumo size was measu ed wi h
a calipe e e y 2 o 3 days, om day 8 pos injec ion, and he
umo diame e (TD) was calcula ed as TD = (L +W)/2. Mice we e
sac ificed when TD ≥20 mm. By he end o each expe imen ,
umo clea ance (TD ≤5 mm) was confi med upon dissec ion.
Human pe iphe al blood mononuclea cells (PBMC)
Blood samples om anonymized heal hy male dono s we e ob ained
in acco dance wi h guidelines es ablished by he Sanquin Medical
E hical Commi ee. B iefly, PBMC was isola ed om esh bu y coa s
using Ficoll-Paque Plus (GEHeal hca e) g adien cen i uga ion. Nex ,
CD4+T cells we e isola ed using magne ic so ing wi h CD4
mic obeads (Mil enyi Bio ec) and iable cells we e sepa a ed using
flow cy ome ic so ing based on he exp ession o CD25, CD45RA,
and CD127 on a FACS A ia III (BD Biosciences).
Mouse leukocy e isola ion
Fo isola ion o leukocy es, spleen and lymph we e ha es ed,
dis up ed, passed h ough a cell s aine (70 μm) in PBS (3% FBS,
1mMEDTA),pelle ed(300×g,4°C,10min),andRBCwe elyzed
(5 mL RBC lysis bu e ; 5 min, RT). Lysis was s opped by adding
5 mL o medium, and cells we e passed h ough a 40-μmcell
s aine , cen i uged (300 × g, 4 °C, 10 min) and esuspended in PBS
con aining 3% FBS and 1 mM EDTA.
Cell so ing
Mice we e sac ificed, and LN (i.e., inguinal, b achial, axilla y,
mandibula , supe ficial ce ical, mesen e ic, panc ea ic, enal, and
lumba ) o spleen we e collec ed, and leukocy es isola ed as desc ibed
abo e. The nega i e ac ion om CD4+T cells en ichmen
(MagniSo ™Mouse CD4 T-cell En ichmen Ki ) was eco e ed,
cen i uged, and s ained wi h he ollowing an ibody panel: an i-
CD11b A647, an i-B220 A647, an i-CD8 A647, an i-CD4 Pe CPCy5.5,
an i-CD62L Pe-Cy7, and an i-CD44 eF450. Foxp3+cells we e so ed
based on endogenous Foxp3EGFP/ic e exp ession (FACS A ia; BD
Biosciences). When indica ed, nai e T cells (CD11b/B220/
CD8-CD4+Foxp3-CD44lowCD62Lhigh), memo y/ac i a ed T cells
(CD11b/B220/CD8-CD4+Foxp3-CD44 highCD62Llow)andT
REG
cells
(CD11b/B220/CD8-CD4+Foxp3+) we e so ed and eco e ed. A
simila p ocedu e was ollowed o so ing ex-T
REG
cells, based on
endogenous exp ession o Toma o wi hin GFP+(CD4+GFP+TdT+)
and GFP-(CD4+GFP−
TdT+)cellpopula ions.
Immunopheno yping
Cells isola ed as desc ibed in “Leukocy e isola ion”we e s ained o
flow cy ome y analysis. Fo su ace s aining, cells we e incuba ed
wi h Fc block oge he wi h LIVE/DEAD™Fixable Aqua S ain in
PBS, ollowed by incuba ion wi h an ibodies agains he ollowing
su ace ma ke s: CD8, CD4, CD62L, CD44, CD25, TCRβ,CD11b,
and CD304 (N p1). In acellula Foxp3 s aining was pe o med
using Foxp3/T ansc ip ion Fac o S aining Bu e Se . B iefly, upon
su ace s aining, cells we e fixed, washed wi h pe meabiliza ion
bu e , and incuba ed wi h an i-Foxp3 an ibody in pe meabiliza ion
bu e . Fo T
REG
cells (CD4+GFP+ dT+)andex-T
REG
cells (CD4+
GFP- dT+) s aining, cells we e fixedandincuba edwi hFcblock,
ollowed by incuba ion wi h an ibodies di ec ed agains he
ollowing su ace ma ke s: CD4, CD62L, CD44, CD3, TCRβ.T
REG
cells and ex-T
REG
cells we e dis inguished based on endogenous
Foxp3EGFP/ic e and Toma o exp ession. Follicula T cells we e fixed
and incuba ed wi h Fc block, ollowed by incuba ion wi h
an ibodies agains su ace ma ke s: CD4, CD3, TCRβ,CXCR5,
and PD1. Foxp3+cells we e selec ed based on endogenous
Foxp3EGFP/ic e exp ession. Fo T
REG
cells lineage main enance analysis
compa ing T
REG
cells (CD4+GFP+ dT+)andex-T
REG
cells (CD4+
GFP- dT+), cells we e incuba ed wi h Fc block oge he wi h LIVE/
DEAD™Fixable Aqua S ain in PBS, ollowed by incuba ion wi h
an ibodies agains su ace ma ke s: CD4, CD3, CD71, and fixa ion
wi h in acellula s aining o he p oli e a ion ma ke Ki67. Cell
acquisi ion was pe o med using a CYTEK Au o a (Cy ek
Biosciences) flow cy ome e , and da a was analyzed using FlowJo
so wa e Ve sion 10.8.1.
Cy okine s aining
Cells we e isola ed as desc ibed in “Leukocy e isola ion“and
we e s imula ed using Cell S imula ion Cock ail oge he wi h
P o ein T anspo Inhibi o Cock ail (4 h; 37 °C) in comple e
RPMI (10% FBS, 100 U/mL Penicillin and 100 µg/mL S ep omy-
cin). Fo su ace s aining, cells we e incuba ed wi h Fc block
oge he wi h LIVE/DEAD™Fixable Aqua S ain, ollowed by
incuba ion wi h an ibodies di ec ed agains he ollowing
su ace ma ke s: CD8, CD4, and TCRβ.In acellula Foxp3,IFNγ,
Qian Wu e al The EMBO Jou nal
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and IL-17 s aining we e pe o med using Foxp3/T ansc ip ion
Fac o S aining Bu e Se . B iefly, we e fixed upon su ace s aining
cells, washed wi h pe meabiliza ion bu e , and incuba ed wi h
an i-Foxp3, an i-IFNγ, and an i-IL-17 an ibodies in pe meabiliza-
ion bu e . Cell acquisi ion was pe o med using BD LSRFo essa
X-20 (BD Biosciences) flow cy ome e . Al e na i ely, cells we e
incuba ed wi h Fc block oge he wi h LIVE/DEAD™Fixable
Yellow S ain, ollowed by incuba ion wi h an ibodies agains he
ollowing su ace ma ke s: CD8, CD4 and TCRβ. In acellula
Foxp3, IFNγ, IL-17 and IL10 s aining we e pe o med using
Foxp3/T ansc ip ion Fac o S aining Bu e Se . B iefly, upon
su ace s aining cells we e fixed, washed wi h pe meabiliza ion
bu e , and incuba ed wi h an i-Foxp3, an i-IFNγ, an i-IL-17 and
an i-IL-10 an ibodies in pe meabiliza ion bu e . Cell acquisi ion
was pe o med using a CYTEK Au o a (Cy ek Biosciences)
flow cy ome e . Da a we e analyzed wi h FlowJo so wa e Ve sion
10.8.1.
Leukocy e s aining wi h fluo escen p obes
Cells we e isola ed as desc ibed in “Leukocy es isola ion”. To e alua e
mi ochond ial memb ane po en ial, cells we e incuba ed wi h
( e ame hyl hodamine, e hyl es e ) TMRE-Mi ochond ial Memb ane
Po en ial p obe (20 nM) in RPMI wi hou se um (20 min 37 °C).
Con ol samples we e p e-incuba ed wi h he ionopho e uncouple o
oxida i e phospho yla ion FCCP (ca bonyl cyanide 4-( ifluo o-
me hoxy) phenylhyd azone; 20 μM, 10 min, 37 °C), o elimina e
mi ochond ial memb ane po en ial and posi i e TMRE s aining. To
de ec in acellula labile Fe2+, cells we e incuba ed wi h Fe oFa Red
(5 μM; 1 h 37 °C) in RPMI wi hou se um. A e incuba ion wi h he
fluo escen p obes, cells we e s ained wi h Fc block oge he wi h
LIVE/DEAD™Fixable Aqua S ain, ollowed by incuba ion an ibodies
agains he ollowing su ace ma ke s: CD4, CD44, CD62L, and TCRβ.
Foxp3+cells we e selec ed based on endogenous Foxp3GFP exp ession.
Cell acquisi ion was pe o med using BD LSRFo essa X-20 (BD
Biosciences) flow cy ome e and da a we e analyzed using FlowJo
so wa e Ve sion 10.8.1.
Bone ma ow ansplan s
Bone ma ow (BM) cells we e ha es ed by flushing he emu s and
ibias o dono mice, and T cells we e deple ed by an ibody-
media ed complemen killing. The abbi complemen was
p epa ed esh, ia incuba ion on ice (30 min), cen i uga ion
(300 × g,10min,4°C)andfil e ing (0.22 µm). BM cell suspensions
(1 × 107/mL in PBS) we e incuba ed wi h an an i-Thy1.2 mAb
(0.5 µg/mL) and mixed gen ly (e e y 15 min) wi h abbi comple-
men (LowTox-M, CL3051, CEDARLANE) a a a io o 100 µL pe
mL o BM cell suspension (37 °C, 1 h). Complemen ac i i y was
neu alized (FBS, 200 µL/mL), cell suspensions we e fil e ed (70-μm
mesh, cell s aine ), washed (2x in PBS, 2% FBS and 1× in PBS
wi hou se um), and cell numbe s adjus ed (108/mL) in PBS. BM
cells om C57BL/6 CD45.1+mice we e mixed wi h BM cells om
congenic C57BL/6 CD45.2+Foxp3GFP-F hΔ/Δ- dT o con ol Foxp3GFP- dT
mice a a 1:1 a io and injec ed (i. .; ail ein, 200 µL) in o congenic
C57BL/6- ecipien Rag2-deficien (Rag2−/−) emale mice, 2 h a e
i adia ion (600 Gys). Hema opoie ic chime ism was moni o ed by
immunopheno yping, 6 weeks a e bone ma ow econs i u ion
and he ea e .
T
REG
cell in i o homeos a ic expansion
LN (i.e., supe ficial, ce ical, axilla y, b achial, mesen e ic, inguinal,
lumba , enal, caudal, and popli eal) and spleen we e collec ed om
Foxp3GFP- dT and Foxp3GFP-F hΔ/Δ- dT mice and gen ly dis up ed in 70-
μm mesh issue o isola e leukocy es. Cell suspensions we e washed
in cold PBS, ed blood cells we e lyzed (i.e., ammonium chlo ide),
passed h ough 40-μm cell s aine and CD4+GFP+TdT+T
REG
cells
we e so ed upon su ace ma ke s aining, as desc ibed in “Cell
so ing”.To es T
REG
cell s abili y in i o, CD4+GFP+TdT+T
REG
cells om Foxp3GFP- dT and Foxp3GFP-F hΔ/Δ- dT mice we e adop i ely
ans e ed (i. ., 1 × 105cells) o Rag2-/- mice. LN and spleen we e
collec ed six weeks la e , dis up ed, and RBC was lysed. The
numbe o CD4+GFP+TdT+T
REG
cells and CD4+GFP-TdT+ex-T
REG
cells was quan ified by flow cy ome y, as desc ibed in “Immuno-
pheno yping”. An addi ional aliquo o spleen cells was used o so
CD4+GFP+TdT+T
REG
cells, as desc ibed in “Cell so ing”.These
we e used o ex ac mRNA and moni o F h mRNA exp ession in
he CD4+GFP+TdT+T
REG
cells used o he adop i e ans e .
T
REG
cell p oli e a ion supp ession assay
Nai e T cells we e so ed, as desc ibed in “Cell so ing”, washed
wi h PBS, and incuba ed wi h Cell T ace Viole (CTV) (The mo-
fishe ; 1/1000 in PBS wi hou se um) a RT in he da k o 15 min.
S aining was s opped by adding fi e olumes o comple e media
(con aining 10% FBS). So -pu ified T
REG
cells we e pla ed and
se ially wo old dilu ed, s a ing a 2.5 × 104cells/well in ound-
bo om 96-well pla es wi h 1 µg/mL soluble an i-CD3 mAb and
5×10
4i adia ed splenocy es. CTV-labeled T
N
cells we e pla ed
(2.5 × 104cells/well), esul ing in T
REG
:T
NAIVE
a io anging om 1:1
o 1:64. Cul u es we e se in iplica es in a final olume o 200 µL.
On day 3 o cul u e, CTV in ensi y was measu ed in esponde
Tcellsdefined as Thy1.1+Thy1.2-TCRb+CD4+, li e lymphocy es.
In i o induc ion o T
REG
cells and flow cy ome y analysis
Nai e T cells so ed, as desc ibed in “Cell so ing”, we e cul u ed o
5 days on a maxiso b 96-well pla e p e-coa ed wi h an i-CD3 mAb
(1 µg/mL; 100 µL/well in PBS) in RPMI comple e media (10% FBS,
100 U/mL Penicillin and 100 µg/mL S ep omycin), supplemen ed
wi h an i-CD28 mAb (1 µg/mL), mouse ecombinan IL-2 (20 ng/mL)
and TGFβ(5 ng/mL) (iT
REG
cell di e en ia ion medium). Al e na i ely,
nai e T cells we e cul u ed in he same media, wi hou TGFβ
(con en ional T-cell di e en ia ion medium). Fo cell su ace s aining,
cells we e incuba ed wi h Fc block oge he wi h LIVE/DEAD™Fixable
Aqua S ain, ollowed by incuba ion wi h an i-CD4 an ibody. Fo
in acellula Foxp3 s aining cells we e fixed a e su ace s aining,
washed wi h pe meabiliza ion bu e , and incuba ed wi h an i-Foxp3
an ibody in pe meabiliza ion bu e , acco ding o he Foxp3/
T ansc ip ion Fac o S aining Bu e Se . Cells we e acqui ed in a
BD LSRFo essa X-20 (BD Biosciences) flow cy ome e and analyzed
using FlowJo so wa e Ve sion 10.8.1. Fo analysis a day 12 a e T
REG
induc ion, induced iT
REG
cells we e e-pla ed a day 5 in RPMI media
supplemen ed wi h IL-2 (100 ng/mL) wi h o wi hou Fe sul a e
(20 µM) and cul u ed o 7 days. To de e mine cell p oli e a ion, nai e
T cells (5 × 106/mL) we e s ained wi h CFSE (5 µM, 20 min RT),
washed wi h comple e medium o s op he eac ion and e-cul u ed in
T
REG
o con en ional T cells medium.
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Len i i al ansduc ion
Len i i us was p oduced by ans ec ing confluen human
HEK293T cells wi h packaging (psPAX2) and en elope plasmids
(pMD2.G) wi h pLKO.1. HEK293T cells we e cul u ed in DMEM
wi h HEPES (Li e Technologies) supplemen ed wi h 10% e al cal
se um and 1% penicillin/s ep omycin. Polye hylenimine (Poly-
sciences, Hi schbe g an de Be gs asse, Ge many) was used as a
ans ec ion eagen . A e 24 h, he cul u es we e e eshed wi h
medium wi h 2% FCS and 24 h la e , len i i al pa icles we e
concen a ed and pu ified by ul acen i uga ion a 50,000 × g,
2.5h,8°C.Nai eT
CONV
(CD4+, CD127+,CD25
−, CD45RA+)and
nai e T
REG
(CD4+, CD127-,CD25
+,CD45RA
+) cells we e isola ed
by FACS so ing (FACS A ia III, BD Biosciences) as desc ibed
abo e. The cells we e hen cul u ed in p esence o 0.1 µg/mL o
an i-CD3 mAb (M1654, clone 1XE, PeliClus e ) and an i-CD28
mAb (16-0289-85, clone CD28.2, eBioscience) o 5 days in IMDM
con aining 10% FCS and 300 U/mL IL-2 and es imula ed one day
p io o ansduc ion. Cells we e hen in ec ed in Re oNec in®
(Clon ech) coa ed pla es o 24 h. A e ha , he cells we e
ans e ed in o issue cul u e- ea ed pla es wi h medium con ain-
ing 100 U/mL IL-2 and pu omycin. A e 5 days, he cells we e
di ec ly used o FACS analysis o lysed o wes e n blo assays.
Wes e n blo
Human T con en ional (CD4+CD45RA+CD127+CD25–), T
REG
(CD4+CD127–CD45RA+CD25hi) cells o HEK293T we e washed
(2× in PBS) and di ec ly lysed in RIPA bu e . Cell lysa es
con aining equal amoun s o p o ein we e boiled in a sample bu e
p io o gel elec opho esis. SDS-PAGE gel elec opho esis was
pe o med using he NuPAGE elec opho esis sys em (No ex, Li e
Technologies). P o eins we e ans e ed using he iBlo sys em
(The mo Scien ific) and analyzed using he co esponding
an ibodies. ECL signals on Wes e n blo s we e de eloped using
he Pie ce ECL subs a e ki (Pie ce) ollowed by au o adiog aphic
de ec ion on film (Fuji Medical). So ed cells mouse T
REG
,T
M
,and
T
N
cells we e di ec ly lysed in 2× SDS-page sample bu e (20%
glyce ol, 4% SDS, 100 mM T is pH 6.8, 0.002% b omophenol blue,
100 mM di hio h ei ol). Samples we e hen sonica ed o ea ed
wi h Benzonase o deg ade DNA, hea ed (10 min; 70 °C), and
cen i uged. The supe na an was collec ed, and he p o ein was
quan ified using NanoD op™1000. An i-FTH1 (1:1000), an i-
His one H3 (1:1000) we e de ec ed using pe oxidase-conjuga ed
seconda y an ibodies (1 h; RT) and de eloped wi h Supe Signal™
Wes Pico PLUS Chemiluminescen Subs a e (The moFishe
Scien ific). ECL signal was de eloped using Pie ce ECL subs a e
ki ollowed by au o adiog aphic de ec ion on film (Fuji Medical).
Al e na i ely, wes e n blo s we e de eloped using Ame sham
Image 680 (GEHeal hca e), equipped wi h a Pel ie -cooled
Fujifilm Supe CCD. Densi ome y analysis was pe o med wi h
ImageJ using images wi hou sa u a ed pixels.
qRT-PCR
RNA was isola ed om cells using NucleoSpin RNA XS ki
(Mache ey-Nagel) acco ding o he manu ac u e ’s ins uc ions.
cDNA was ansc ibed om o al RNA wi h ansc ip o fi s
s and cDNA syn hesis ki (Roche) o Xpe cDNA Syn hesis Ki
(GRiSP). Quan i a i e eal- ime PCR (qRT-PCR) was pe o med
using 1 μg cDNA and SYBR G een Mas e Mix (Applied
Biosys ems, Fos e Ci y, CA, USA) in duplica e on a 7500 Fas
Real-Time PCR Sys em (Applied Biosys ems) unde he ollowing
condi ions: 95 °C/10 min, 40 cycles/95 °C/15 s, annealing a 60 °C/
30 s, and elonga ion 72 °C/30 s. P ime s lis ed in Table 1we e
designed using P ime Blas (Ye e al, 2012).
Se ology
Mice we e eu hanized using CO
2
inhala ion. Whole blood was
collec ed by ca diac punc u e and ans e ed in o an EDTA o
hemog am analyses o hepa in ubes o se ology (I on, T ans e -
in, and T ans e in sa u a ion). Analysis was pe o med by
DNA ech (Lisbon).
His ology
O gans we e ha es ed, fixed (10% o malin), embedded in
pa a fin, sec ioned (3-μm- hick sec ions), and s ained wi h
Hema oxylin and Eosin (H&E). Whole sec ions we e analyzed,
and images acqui ed wi h a Leica DMLB2 mic oscope (Leica) and
NanoZoome -SQ Digi al slide scanne (Hamama su).
m DNA/nDNA qPCR
To alisola edDNAwasused ope o m hequan ifica ion o
mi ochond ial DNA (m DNA) in compa ison o nuclea DNA
(nDNA) using a qRT-PCR-based me hod (Qui os e al, 2017).
B iefly, qRT-PCR was pe o med o m using 20 ng o DNA and
SYBR G een Mas e Mix (Bio-Rad), in duplica e on a 7500 Fas
Real-Time PCR Sys em (Applied Biosys ems), unde he ollowing
condi ions: 50 °C/2 min and 95 °C/5 min (Hold s age), 45 cycles/
95 °C/10 s, annealing a 60 °C/30 s, and elonga ion 72 °C/20 s,
ollowed by mel ing cu e: 95 °C/15 s, 60 °C/1 min, and g adual
inc ease in empe a u e up o 95 °C. P ime s o NADH-
ubiquinone oxido educ ase chain 1 encoded by he mi ochond ial
gene MT-Nd1 (Nd1) and o he nuclea -encoded hexokinase 2 gene
(Hk2) (Qui os e al, 2017) a e lis ed in Table 1.Mi ochond ia
numbe pe cell was calcula ed acco ding o he a io o mRNA
exp ession o he single copy mi ochond ial gene Nd1 and he
single copy nuclea gene Hk2.
Seaho se assays
Oxygen consump ion a e (OCR) and ex acellula acidifica ion
a e (ECAR) we e measu ed usingaSeaho seXFe96analyze
(Agilen Tech.) and he Seaho se XF Cell Mi o S ess Tes Ki
acco ding o ins uc ions om he manu ac u e . Specifically so ed
TREG and TCONV cells we e pla ed on poly-L lysine-coa ed
Seaho se XF96 pla es (15 × 103cells/well) XF medium (10 mM
glucose, 1 mM sodium py u a e, 2 mM L-glu amine, pH 7.4), and
cen i uged (400 × g, o 5 min) o p omo e cell adhesion. Cells
we e incuba ed in a non-CO
2
incuba o (37 °C; 1 h) p io o he
assay. The analyze was p og ammed o calib a e and equalize
samples, ollowed by h ee baseline measu emen s (3 min each) and
mixing (2 min) be ween measu emen s p io o inhibi o injec ion.
The inhibi o s we e injec ed in he ollowing o de : Oligomycin
(1 μM); FCCP (2 μM); An imycin A/Ro enone (1 μM); and h ee
Qian Wu e al The EMBO Jou nal
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measu emen s (3 min each) we e made ollowing each injec ion
wi h 2 min o mixing be ween measu emen s.
RNA sequencing
RNA was ex ac ed om (CD4 +GFP +)T
REG
cells so ed om he
LN o Foxp3GFP-F hΔ/Δ s. con ol Foxp3GFP mice (see Fig. 2) o om
(CD45.2 +CD3 +CD4 +GFP+ dT +)T
REG
and (CD45.2 +CD3 +
CD4 +GFP- dT +)ex-T
REG
cells so ed om LN o BM chime ic
mice (see Fig. 4). RNA sequencing da a om non-chime ic mice was
analyzed as ollows: RNA sequencing was pe o med using Illumina’s
nex -gene a ion sequencing (Ben ley e al, 2008). Quali y check and
quan ifica ion o o al RNA was done using he Agilen Bioanalyze
2100 in combina ion wi h he RNA 6000 pico ki (Agilen
Technologies). Lib a y p epa a ion was done using SMARTe
S anded To al RNA-Seq Ki 2-Pico Inpu Mammalian (Taka a)
ollowing he manu ac u e ’s desc ip ion. Lib a y quan ifica ion and
quali y check was done using he Agilen Bioanalyze 2100 in
combina ion wi h he DNA 7500 ki . Lib a ies we e sequenced on a
HiSeq2500 unning in 51cycle/single-end/ apid mode. All lib a ies
we e pooled and sequenced in wo lanes. Sequence in o ma ion was
ex ac ed in Fas Q o ma using Illumina’s bcl2 as q .2.19.1.403.
Sequencing esul ed in a ound 29mio eads pe sample. RNA
sequencing da a om cells ex ac ed om mixed BM chime ic mice
was analyzed as ollows: RNA was ex ac ed and quali y assessed using
Agilen Bioanalyze 2100 using he RNA 6000 pico ki (Agilen
Technologies). Full-leng h cDNAs and sequencing lib a ies we e
gene a ed ollowing he SMART-Seq2 p o ocol62. Lib a y p epa a ion,
including cDNA “ agmen a ion”, PCR-media ed adap o addi ion and
lib a y amplifica ion was pe o med using he Nex e a lib a y
p epa a ion p o ocol (Nex e a XT DNA Lib a y P epa a ion ki ,
Illumina). Lib a ies we e sequenced (Nex Seq 500, Illumina) using
High Ou pu ki 2.5 (75 cycles). Sequencing da a was ex ac ed in
Fas Q o ma , using Illumina’s bcl2 as q .2.19.1.403, p oducing
30.14 × 106 eads pe sample on a e age. Lib a y p epa a ion and
nex -gene a ion sequencing we e pe o med a he IGC Genomics
Uni . Fas q eads we e aligned agains he mouse e e ence genome
GRCm39 using he GENCODE M27 anno a ion o ex ac splice
junc ion in o ma ion (STAR; .2.5.2a)64. Read summa iza ion was
pe o med by assigning uniquely mapped eads o genomic ea u es
using Fea u eCoun s (sub ead package .1.5.0-p1). Gene exp ession
ables we e impo ed in o he R en i onmen ( .4.1.0) o pe o m
di e en ial gene exp ession, unc ional en ichmen analyses, and da a
isualiza ion (R Co e-Team, 2021). Di e en ial gene exp ession
analysis was pe o med using he DESeq2 R package ( .1.32). Gene
exp ession was modeled by geno ype. Genes no exp essed o wi h
ewe han 10 coun s ac oss he samples we e emo ed. We
subsequen ly an he unc ion DESeq o es ima e he size ac o s (by
es ima eSizeFac o s), dispe sion (by es ima eDispe sions) and fi a
binomial GLM fi ing o βicoe ficien and Wald s a is ics (by
nbinomWaldTes ). Pai wise compa isons we e pe o med wi h he
unc ion esul s (alpha = 0.05), and he log
2
old change o each
pai wise compa ison was sh unken wi h he unc ion l cSh ink using
he algo i hm ash ( .2.2-47)65. Di e en ially exp essed genes we e
conside ed as genes wi h an adjus ed P alue < 0.05 and an absolu e
log
2
old change >0. No malized gene exp ession coun s we e ob ained
wi h he unc ion coun s using he op ion no malized = TRUE.
Regula ized log- ans o med gene exp ession coun s we e ob ained
wi h log, using he op ion blind = TRUE. Ensembl gene ids we e
con e ed in o gene symbols om Ensembl ( .104 - May 2021—
h ps://may2021.a chi e.ensembl.o g) by using he mouse e e ence
(GRCm39) da abase wi h biomaR R package ( .2.48.2). All plo s we e
c ea ed using he ggplo 2 R package ( .3.3.5). Hea maps we e c ea ed
wi h pHea map ( .1.0.12), using Euclidean dis ance and Wa d.D2
me hods o clus e ing es ima ion. Fo hie a chical clus e ing, gene
exp ession coun s we e scaled (Z-sco e) wi h he unc ion scale.
Func ional en ichmen analysis was pe o med using he gp ofile 2 R
package ( .0.2.1). En ichmen was pe o med wi h he unc ion gos
based on he lis o up- o down egula ed genes, be ween each pai wise
compa ison, agains anno a ed genes (domain_scope = “anno a ed”)
Table 1. RT-qPCR p ime s.
Oligonucleo ides Sequences Re e ence
A bp0 Fwd 5′-CTTTGGGCATCACCACGAA-3′Blankenhaus e al, 2019
A bp0 Re 5′-GCTGGCTCCCACCTTGTCT-3′Blankenhaus e al, 2019
F h Fwd 5′-CCATCAACCGCCAGATCAAC-3′Blankenhaus e al, 2019
F h Re 5′-GCCACATCATCTCGGTCAAA-3′Blankenhaus e al, 2019
F l Fwd 5’-AAGATGGGCAACCATCTGAC-3’This wo k
F l Re 5’-GCCTCCTAGTCGTGCTTGAG-3’This wo k
Hk2 Fwd 5′-GCCAGCCTCTCCTGATTTTAGTGT-3′Qui os e al, 2017
Hk2 Re 5′-GGGAACACAAAAGACCTCTTCTGG-3′Qui os e al, 2017
Nd1 Fwd 5′-CTAGCAGAAACAAACCGGGC-3′Qui os e al, 2017
Nd1 Re 5′-CCGGCTGCGTATTCTACGTT-3′Qui os e al, 2017
GFP Fwd 5′-CGACGTAAACGGCCACAAGTTCAG-3′Liu e al, 2012
GFP Re 5′-CCGTAGGTCAGGGTGGTCACGAG-3′Liu e al, 2012
Td Fwd 5′-GCCGACATCCCCGATTACAAGA-3′Wiene e al, 2015
Td Re 5′-CGATGGTGTAGTCCTCGTTGTGG-3′Wiene e al, 2015
Foxp3 Fwd 5′-GGCCCTTCTCCAGGACAGA-3′Fon eno e al, 2003
Foxp3 Re 5′-GCTGATCATGGCTGGGTTGT-3′Fon eno e al, 2003
The EMBO Jou nal Qian Wu e al
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o he o ganism Mus musculus (o ganism = “mmusculus”). Gene lis s
we e so ed based on adjus ed p alue (o de ed_que y = TRUE) o
gene a e GSEA (Gene Se En ichmen Analysis) s yle p alues. Only
s a is ically significan (use _ h eshold=0.05) en iched unc ions a e
e u ned (significan =TRUE) a e mul iple es ing co ec ions wi h
he de aul me hod g:SCS (co ec ion_me hod = “analy ical”).
Gp ofile 2 que ies we e un agains he de aul unc ional da abases
o mouse which include Gene On ology (GO:MF, GO:BP, GO:CC),
KEGG (KEGG), Reac ome (REAC), TRANSFAC (TF), miRTa Base
(MIRNA), Human pheno ype on ology (HP), WikiPa hways (WP),
and CORUM (CORUM). Gp ofile 2 was pe o med using da abase
e sions Ensembl 104, and Ensembl gene 51 (da abase upda ed on 07/
05/2021).
RNA-sequencing da a analysis
Sequence ead quali y was assessed by means o he Fas QC me hod
( 0.11.5; h p://www.bioin o ma ics.bab aham.ac.uk/p ojec s/ as qc/).
T immoma ic e sion 0.36 was used o im Illumina adap e s and
poo -quali y bases ( immoma ic pa ame e s: leading=3, ailing=3,
sliding window=4:15, minimum leng h=40). The emaining high-
quali y eads we e used o align agains he Genome Re e ence
Conso ium mouse genome build 38 (GRCm38). Mapping was
pe o med by HISAT2 e sion 2.1.0 wi h pa ame e s as de aul . Coun
da a we e gene a ed by means o he HTSeq me hod and analyzed using
he DESeq2 me hod in he R s a is ical compu ing en i onmen (R Co e
Team 2014. R: A language and en i onmen o s a is ical compu ing. R
Founda ion o S a is ical Compu ing, Vienna, Aus ia). S a is ically
significan di e ences we e defined by Benjamini & Hochbe g adjus ed
p obabili ies <0.05. Canonical signaling pa hways and bio unc ions we e
gene a ed by Ingenui y Pa hway Analysis (IPA; QIAGEN) speci ying
mouse species and ingenui y da abase as e e ence. Benjamini &
Hochbe g adjus ed p obabili ies <0.05 dema ca ed significance. Func-
ional en ichmen analysis was pe o med wi h gP ofile (Kolbe g e al,
2023). Da a we e analyzed using g:SCS mul iple es ing co ec ion
me hod wi h a significance h eshold o 0.05.
Ta ge ed me abolomics
Ta ge ed me abolomics analyses o cell ex ac s o in e media es o he
ica boxylic acid cycle (TCA cycle; ci a e, isoci a e, α-ke oglu a a e,
succina e, uma a e, mala e, cis-aconi a e) and addi ional me aboli es
closely linked o he TCA cycle (py u a e, lac a e, DL-2-hyd oxyglu-
a a e, i acona e, and he amino acids aspa a e, glu ama e, glu amine)
we e pe o med by liquid ch oma og aphy- andem mass spec ome y
(LC-MS/MS), as desc ibed elsewhe e (Rich e e al, 2019). Fo
me aboli e quan ifica ion, a ios o analy e peak a eas o peak a eas
o espec i e s able iso ope labeled in e nal s anda ds de e mined in cell
ex ac s we e compa ed o hose in calib a o s.
Genome-wide me hyl-sequencing (EM-seq)
The ini ial genomic DNA (gDNA) was quan ified using he In i ogen
Qubi 4 as pe he manu ac u e ’s p o ocol (1 µL in 199 µL o Qubi
wo king solu ion). Fo lib a y p epa a ion, NEB enzyma ic Me hy-Seq
ki was used, ollowing he manu ac u e ’s p o ocol o la ge inse
lib a ies. Based on he quali y assessmen , samples we e s anda dized
o 10 ng o DNA in a olume o 50 µL, including a spike-in o pUC
and Lambda DNA p o ided wi h he ki , as a con ol o me hyla ion
e ficiency as pe he manu ac u e ’s p o ocol. Samples we e agmen-
ed using he Co a is S2 sys em, wi h se ings o achie e an a e age
agmen size o 350-400 bp and indi idual ba coded du ing he PCR,
using eigh PCR cycles as pe he manu ac u e ’s p o ocol. Lib a ies
we e quan ified using he Qubi HS DNA assay as pe he
manu ac u e ’s p o ocol (1 µL o sample in 199 µL o Qubi wo king
solu ion). The quali y and mola i y o he lib a ies we e assessed using
Agilen Bioanalyze wi h he DNA HS Assay ki as pe he
manu ac u e ’s p o ocol. Mola i y was used o equimola ly combine
indi idual lib a ies in o one pool, loaded and sequenced on an
Illumina Nex Seq 2000 pla o m (Illumina, San Diego, CA, USA)
using a P3 300 cycle ki and a ead-leng h o 155 bp pai ed-end eads.
Raw sequencing eads we e deposi ed o ENA unde he accession
numbe : Sequencing eads we e p ocessed by he me hylseq ( 1.6.1)
(h ps://zenodo.o g/ eco d/2555454/expo /xd) n -co e (Ewels e al,
2020). De aul pa ame e s we e used wi h BWA-me h as he aligne ,
GRCm38 as he e e ence genome. The --em-seq and --me hyl_ki
op ions we e also used o downs eam analysis. R me hylki package
( 1.22.0) (Akalin e al, 2012) was used o load he me hyla ion calls,
calcula e he me hyla ion a e, and gene a e plo s p esen ed in his
pape . Sliding Linea Model (SLIM) mul iple es ing co ec ion (Wang
e al, 2011) was used o ex ac he significan me hyla ed si es.
TET ac i i y assay
TET enzyma ic ac i i y was moni o ed in human HEK293T cells,
ansien ly ans ec ed wi h human TET3 (pCMV-3×FLAG-TET3(hu-
man)-Neo; MiaolingBio P45302) plus human FTH (pCMV-FTH-
3Tag3a) o FTH mu an (FTHmu ;pCMV-FTH
mu -3Tag3a; Glu 62 and
His 65 and Lys86 we e mu a ed as Lys, Gly, Gln, espec i ely, o
achie e ully abla ion o e oxidase ac i i y) lacking e oxidase mu an
FTH (FTHmu ), simila o p e iously desc ibed (B oxmeye e al, 1991),
ollowed by nuclea p o ein ex ac ion and ELISA-based TET ac i i y
measu emen . B iefly, HEK293T cells, a 60% confluency in six-well
pla es, we e ansien ly ans ec ed wi h he human TET3 (800 ng/well)
oge he wi h FTH (200 ng/well), FTHmu (200 ng/well) cDNA exp es-
sing ec o s o emp y ec o (pCMV-3Tag3a; 200 ng/well) using
ans ec ion eagen (YEASEN, 40802ES03). Nuclea p o eins we e
ex ac ed 48 h a e ans ec ion, using a comme cial ki (EpiQuik, OP-
0002-1) and quan ified (BCA assay; Meilunbio MA0082-2). TET
ac i i y was measu ed by ELISA-based he manu ac u e ’s ins uc ions
(EpiQuik, P-3087). Exp ession o flag- agged TET3, FTH and FTHmu
was moni o ed by wes e n blo . B iefly, cy osol and nuclea ex ac s
we e loaded on a 4–20% g adien SDS-PAGE p ecas -Gel (ACE
Bio echnology, ET15420LGel) and p o eins we e ans e ed on PVDF
memb anes. These we e blocked (5% skimmed milk in TBST; 1 h) and
incuba ed (4 °C; o e nigh ) wi h an HRP-labeled an i-FLAG-HRP
an ibody (Sigma, A8592). Memb anes we e washed (3 × 10 min; TBST)
and HRP ac i i y was de ec ed by ECL (The mo Scien ific, 32106).
Images we e cap u e by Molecula Image ®ChemiDocTM XRS+
wi h Image LabTM So wa e (BIO-RAD). Memb anes we e u he
incuba ed (1.5 h, RT) wi h an i-Lamin A/C (P o ein ech, 10298-1-AP)
and an i-GAPDH (Abclonal, AC033) an ibodies, used as e e ence
cy osolic and nuclea p o eins, espec i ely. Memb anes we e washed
(3 × 10 min; TBST), incuba ed wi h goa an i- abbi (Abclonal, AS014)
and goa an i-mouse (Abclonal, AS003), seconda y an ibodies,
espec i ely, washed (3 × 10min in TBST), and HRP signal was
de ec ed by ECL o blo e e ences p o eins o cy osol and nuclea
ac ions, espec i ely, as abo e.
Qian Wu e al The EMBO Jou nal
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A
B
TH cell ac i a ion
Fold Exp ession (log2)
−1 0 1 2
Icosl
Il12 b1
Il4
Tbx21
Cd247
Ga a3
Il17 b
Il10
Il12 b2
I 1
Psen1
H
a c 2
Cd4
J
ak2
Icos
Il1
l1
Il18 1
S a 1
N
a c2
Pik3c2a
Cd40lg
Il10
a
N
a c1
C
l 2
Cc 3
Icam1
Cc 8
Cc 4
Cxc 6
Cxc 3
I ng
Ma
N il3
Il2 b
Runx3
Cd80
Cc 5
Foxp3GFP-F h''
0
20
40
60
80
CD4
+
Foxp3
+
CD44
high
CD62L
low
(%)
* *
Foxp3
10
5
10
4
10
3
0
-10
3
10
5
10
4
10
3
010
5
10
4
10
3
0
1,52% 5,05%
0
2
4
6
8
IFN- J
+
Foxp3
+
(
%
)
***
CD4+
Foxp3+
(GFP+)
RNAseq
C
Spleen
CD4+
Foxp3 +
IFN J
+
IFNJ
Foxp3GFP-F h''
E
Foxp3 GFP Foxp3 GFP-F h''
SpleenMLN
MLN & Spleen
CD4+Foxp3
CD44
+
CD62L
+
Foxp3GFP-F h''
F h
l/ l
F h
l/ l
F h
l/ l
F h
l/ l
Foxp3
GFP-F h''
Li e Lung Kidney Colon Panc eas
1.25x
1.25x
1.25x
1.25x
1.25x
10x
10x
10x
10x
D
103104105
Cell T acke Viole
8421
0
50
100
150
Inhibi ion o
TN P oli e a ion (%)
TN pe TREG
NS
NS
8/1
4/1
2/1
1/1
8/1
4/1
2/1
1/1
TREG/TN
NS
NS
TCONV cell di ision
TREG/TN
Foxp3GFP-F h''
Foxp3GFP
MLN
T
REG
(GFP+)
So ing
DCD3/28
+IL-2
Foxp3GFP
Foxp3GFP-F h''
T
CONV
cell
p oli e a ion
So ing
The EMBO Jou nal Qian Wu e al
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Figu e EV2. FTH exp ession in T
REG
cells p e en s sys emic cellula inflamma ion.
(A) Schema ic ep esen a ion o expe imen al app oach (le panel) o (CD4+CD25+GFP+)T
REG
cell so ing om he mesen e ic lymph nodes (MLN) and cocul u e wi h
con en ional ac i a ed T cells (αCD3/28 +IL-2) o e alua e supp essi e unc ion o T
REG
cells. Rep esen a i e flow cy ome y p oli e a ion his og ams (Cell T ace Viole )
o in i o supp ession assay o mouse T
N
by di e en a ios o T
REG
cells (middle panel). Inhibi ion o T
N
cell p oli e a ion quan ified as pe cen age o undi ided cells ( igh
panel). Da a om 1 ou o 3 ep esen a i e expe imen s, wi h simila end. (B) Rep esen a i e images o H&E-s ained li e , lung, kidney, colon, and panc eas om N=3–4
mice pe geno ype a 27–31 weeks a e bi h. (C) Schema ic ep esen a ion o he expe imen al app oach (le panel) used o gene a e he Hea map ( igh panel) o
indi idual genes associa ed wi h T
H
e ec o unc ion p og ams, di e en ially exp essed in (CD4+GFP+)T
REG
cells so ed om Foxp3GFP-F hΔ/Δ s. Foxp3GFP mice (same
expe imen as Fig. 2A,B). (D) Schema ic ep esen a ion o he expe imen al app oach used (le panel) o e alua e he pe cen age ( igh panel) o
(CD4+Foxp3+CD44highCD62Llow) ac i a ed T
REG
cells in he MLN and spleen. Da a om N=7–8 mice pe geno ype, pooled om h ee independen expe imen s, wi h
simila end. (E) Schema ic ep esen a ion o he expe imen al app oach used ( op panel), ep esen a i e flow cy ome y do plo s (bo om le panel) and pe cen age
(bo om igh panel) o splenic (CD4+Foxp3+) IFNγ-sec e ing T
REG
. Da a om N=5 mice pe geno ype, pooled om wo independen expe imen s, wi h simila end. Da a
in o ma ion: Da a in (A,D,E) ep esen ed as mean ± SD. Ci cles in (A) ep esen indi idual wells, and ed ba s a e mean alues. Ci cles in (D,E) ep esen indi idual mice,
and ed ba s a e mean alues. P alues in (A,D) calcula ed using Two-way ANOVA wi h Sidak’s mul iple compa ison es and in (E) using unpai ed es wi h Welch’s
co ec ion. NS no significan (P> 0.05), *P< 0.05; ***P< 0.001. Sou ce da a a e a ailable online o his figu e.
Qian Wu e al The EMBO Jou nal
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B
01010 01010
10³
10
10
10
0
10
10³
10
10
10
0
10
CD71
Ki67
TREG ex-TREG
0
20
40
60
80
**
Lo em
21,1%
47,4% 59,6%
32,1%
Foxp3
GFP-F h''- d
T
Foxp3
GFP- dT
*
F h/A bp 0 mRNA
C D
**
0
0.1
0.2
0.3
0.4
0.5
(%)
Ki67 +CD71 +
4
8
12
IFNJMFI (x103)
0
LN Spleen
*** *** * *
TREG
A
dT
GFP
Week 7Week 9Week 11 Week 15 Week 24
6,89%
1,83%
5,83%
2,19%
7,15%
1,77%
6,08%
1,79%
5,26%
1,42%
2,70%
2,77%
5,83%
2,17%
3,48%
3,48%
4,30%
3,56%
2,77%
2,64%
3,04% 3,25%
Week 2Week 4
5,39%
1,2%
4,27%
1,65%
5,01%
1,37%
4,95%
1,61%
Foxp3
GFP-F h''- dT
Foxp3
GFP- dT
Week 19
105
104
103
0
010
3104105010
3104105010
3104105010
3104105010
3104
105
010
3104105
010
3104105
010
3104105
102
105
104
103
0
102
Spleen
1
2
3
CD71 M FI (x104)
0
LN Spleen
EF
Thymus
CD4+CD8-CD8+CD4-
Foxp3+
CD45.2
CD45.1
0
10³
10
10
-10³
10
-10
-10 010 10 -10 010 10 -10 010 10 -10 010 10 -10 010 10
83,7%
16,2%
52,9%
46,6%
42,4%
55,6%
52,9%
47,1%
77,8%
21,9%
34,2%
64,5%
38,3%
61,2%
34,3%
64,7%
35,1%
63,5%
48,8%
51,2%
0
10³
10
10
-10³
10
-10
CD4+CD8+
CD4-CD8-
0
40
80
120
CD45.1
CD45.2
Foxp3
GFP-F h''- dT
Foxp3
GFP- dT
Chime ism (%)
G
Thymus
CD4+CD8-CD8+CD4-
Foxp3
+
CD4+CD8+
CD4-CD8-
NS NS NS NS
ex-TREG
TREG
ex-TREG
TREG
ex-TREG
TREG
ex-TREG
TREG
ex-TREG
TREG
ex-TREG
Foxp3
GFP-F h''- dT
Foxp3
GFP- dT
Foxp3
GFP-F h''- dT
Foxp3
GFP- dT
3,12% 3,5%
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
Foxp3
GFP-F h''- dT
Foxp3
GFP- dT
LN
The EMBO Jou nal Qian Wu e al
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Figu e EV3. FTH exp ession in T
REG
cells p e en s T
REG
ansdi e en ia ion in o inflamma o y T
REG
cells.
(A) Rep esen a i e flow cy ome y do plo s o GFP and dT exp ession in ci cula ing CD4+cells (same expe imen as Fig. 3A–D). Numbe s in quad an s co espond o
pe cen ages o posi i e cells a he indica ed weeks a e bi h. (B) Rela i e quan ifica ion o F h mRNA, by qRT-PCR, no malized o A bp0 mRNA, in (CD4+GFP+ dT+)T
REG
and (CD4+GFP- dT+)ex-T
REG
cells so ed om he lymph nodes (LN). Da a om N=5–7 mice pe geno ype, pooled om wo expe imen s wi h simila end. (C) Mean
fluo escence in ensi y (MFI) o IFNγin ac i a ed (CD4+GFP+ dT+)T
REG
cells and (CD4+GFP- dT+)ex-T
REG
om he lymph nodes (LN) and spleen in he same expe imen as
(Fig. 3H). Da a om N=3 wells pe geno ype in one expe imen , ep esen a i e o 2 independen expe imen s wi h simila end. (D) Rep esen a i e flow cy ome y do
plo s (le panel) and co esponding pe cen age o Ki67+CD71+( igh panel) among splenic (CD4+GFP+TdT+)T
REG
cells and (CD4+GFP-TdT+)ex-T
REG
cells. Da a om
N=6 mice pe geno ype, pooled om wo independen expe imen s, wi h simila end. (E) Mean fluo escence in ensi y (MFI) o CD71 exp ession in Ki67+T
REG
and ex-T
REG
cells om he lymph nodes (LN) and spleen, om he same expe imen s as in (D). (F) Rep esen a i e flow cy ome y do plo s and (G) co esponding pe cen ages o
CD45.1+and CD45.2+double nega i e (DN), double posi i e (DP) hymocy es, T
H
cells, cy o oxic T cells and (CD4+Foxp3+)T
REG
cells in he hymus om he same BM
chime ic mice illus a ed in (Fig. 4A–G). Da a om N=11–12 mice pe geno ype, pooled om wo independen expe imen s wi h simila end. Da a in o ma ion: Da a in
(B–E,G) a e p esen ed as mean ± SD, ci cles in (B,D,E) co espond o indi idual mice o indi idual wells (C) and ed ba s a e mean alues. P alues in Panel (B–E,G) we e
calcula ed using wo-way ANOVA wi h Sidak’s mul iple compa ison es . NS no significan (P> 0.05), *P< 0.05; **P< 0.01; ***P< 0.001. Sou ce da a a e a ailable online
o his figu e.
Qian Wu e al The EMBO Jou nal
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17,3% 5,71%
8,02% 1,71%
A
Rag2-/-
CD45.2+
C57BL/6
50%
Foxp3GFP-F h''- dT
Foxp3GFP- dT
50%
Lymph Nodes
Foxp3
GFP-F h''- dT
Foxp3
GFP- dT
4DC(L26DC
+
)
8DC(L26DC
+
)
BC
DE
BM
CD44
103104105
102106103104105
102106
-103
105
104
103
0
-103
105
104
103
0
0
0.05
0.1
0.15
F h/A bp0 mRNA
***
dT
GFP
0-10³ 10³ 10 10 0-10³ 10³ 10 10
-10²
0
10²
10³
Pos -
So ing
P e-
So ing
TREG cells
(GFP+ dT+)
So ing
TREG cells
(GFP+ dT+)
98,8%16,3%
Foxp3
GFP-F h''- dT
Foxp3
GFP- dT
Foxp3
GFP-F h''
Foxp3
GFP
F
0
25
50
75
100
T
N
DCD3/28
+IL-2+TGFE
CD3/28
+IL-2
Foxp3+Cells (%)
8,5%
96,8%
8,2%
CD4
Foxp3
DCD3/28
IL-2
DCD3/28
IL-2 + TGF E
010
3104105010
3104105
-103
105
104
103
0
-103
105
104
103
0
96,9%
TCONV
iT
REG
5d
NS
NS
Foxp3
GFP
Foxp3
GFP-F h''
Foxp3
GFP
Foxp3
GFP-F h''
GH
I
103
104
105
106
107
Foxp3+Cells (Nb .)
Foxp3
GFP
Foxp3
GFPF h''
12345678
0
10
20
30
Nb . o cell di isions
TREG (%)
0
20
40
60
80
100
0103
0
20
40
60
80
100
104105
1
2
3
4
5
6
7
8
1
2
3
4
6
7
8
5
Foxp3
GFP
Foxp3
GFPF h''
TCONV
iT
REG
J
NS
NS
E en s (%)
CFSE
Cells
0
10
20
30
40
50
0
0.5
1
1.5
2
CD44
high
CD62L
low
(%)
CD8
+
CD4
+
CD44
high
CD62L
low
(x10
6
NS NS NS NS
)
CD45.2+
CD8
+
CD4
+
Foxp3
GFP-F h''- dT
Foxp3
GFP- dT
Foxp3
GFP-F h''- dT
Foxp3
GFP- dT
Foxp3
GFP
Foxp3
GFPF h''
iTREG
cells
iTCONV
cells
The EMBO Jou nal Qian Wu e al
EV7 The EMBO Jou nal Volume 43 | Issue 8 | Ap il 2024 | 1445 –1483 © The Au ho (s)
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Figu e EV4. FTH ac s in a non-cell-au onomous manne o p e en T
REG
cells om ansdi e en ia ing in o inflamma o y T
REG
cells.
(A) Schema ic ep esen a ion o he expe imen al app oach used o flow cy ome y analysis om he lymph nodes o BM chime ic mice (same expe imen as Fig. 4A).
(B,C) Rep esen a i e flow cy ome y do plo s (B), quan ifica ion o pe cen age (le panel) and numbe ( igh panel) (C) o li e ac i a ed
(CD45.2+CD4+CD44highCD62Llow) and (CD45.2+CD8+CD44highCD62Llow) cells in he lymph nodes o BM chime ic mice om (A). Da a in (C) om n=11–12 mice pe
geno ype, pooled om 2 independen expe imen s, wi h simila end. (D) Schema ic ep esen a ion o cell so ing o adop i e ans e s in he expe imen illus a ed in
Fig. 5A. (E) Rep esen a i e flow cy ome y do plo s o (CD4+GFP+ dT+)T
REG
cells and ela i e le el o F h mRNA exp ession in (CD4+GFP+ dT+)T
REG
cells ( igh panel)
used o adop i e ans e s in he expe imen illus a ed in Fig. 5A. (F) Schema ic ep esen a ion o expe imen al app oach used o in i o gene a ion o induced T
REG
(iT
REG
) cells and con en ional T
H
(T
CONV
) cells om so ed nai e T
H
(T
N
) cells, ac i a ed wi h an i-CD3 and an i-CD28 mAb plus IL-2 and TGFβ.(G) Rep esen a i e flow
cy ome y do plo s o iT
REG
and T
CONV
cells, gene a ed in (F). (H) Pe cen age (%) and (I) Numbe (Nb .) o Foxp3+T
CONV
and iT
REG
cells, gene a ed as desc ibed in (F).
N=2–5 independen expe imen s wi h simila end. Each expe imen co esponds o he a e age o di e en wells. (J) Rep esen a i e flow cy ome y ca boxyfluo escein
succinimidyl es e (CFSE) s aining (le panel) and quan ifica ion o pe cen age (%) ( igh panel) o p oli e a ing (CD4+Foxp3+)iT
REG
cells, gene a ed as desc ibed in (F).
Da a om 3 o 6 echnical eplica es in 1 ou o 3 independen expe imen s, wi h simila end. Da a in o ma ion: Da a in (C,E) a e p esen ed as mean ± SD, ci cles
co espond o indi idual mice and ed ba s a e mean alues. Ci cles in (H–J) co espond o indi idual wells and ed ba s a e mean alues. P alues in panels (C,H,I) we e
calcula ed using wo-way ANOVA wi h Sidak’s mul iple compa ison es . P alues in (E) we e calcula ed using Mann–Whi ney es . NS no significan , ***P< 0.001. Sou ce
da a a e a ailable online o his figu e.
Qian Wu e al The EMBO Jou nal
© The Au ho (s) The EMBO Jou nal Volume 43 | Issue 8 | Ap il 2024 | 1445 –1483 EV8
Downloaded om h ps://www.embop ess.o g on Janua y 15, 2025 om IP 193.144.79.182.

ABC
Foxp3GFP
FCCPOligo. A/A+Ro .
OCR
(pmol/min/106cells)
0 20406080100
0
200
400
600
800
-100
NS
FCCPOligo. A/A+Ro .
OCR
(pmol/min/106cells)
0 20406080100
0
200
400
600
800
-100
Foxp3GFP-F h''
REG
CONV
T
T
REG
CONV
T
T
Time (minu es) Time (minu es)
OCR
(pmol/min/106cells)
****
TCONV
TREG
TCONV
TREG
Foxp3GFP
REG
CONV
T
T
OCR
(pmol/min/106cells)
Foxp3GFP-F h''
REG
CONV
T
T
D
Me aboli e Concen a ion
[pmol/103 cells]
Foxp3GFP-F h'''
Foxp3GFP
Ci a e
Fuma a e
Isoci a e
D-ke oglu a a e
D,L-2
hyd oxyglu a a e
Succina e
Cis-aconi a e
Py u a e
Mala e
Aspa a e
Glu ama e
Glu amine
I aconic acid
Lac a e
0
100
200
300
400
0.001
0.01
0.1
1
10
100 **
T age ed
Me abolomics
Spleen
TREG cells
(GFP+)
So ing
E
IFN
105
104
103
0
105
104
103
0
NS
CD4
+
CD8
+
0
20
40
60
80
NS
23,3%
105
104
103
0
105
104
103
0
28,8%
105
104
103
0
G anzyme B
CD8
53,9%
54,9%
105
104
103
0105
104
103
0
CD4
CD8
0
2
4
6
pe umo (x10
5
)
CD4
+
CD8
+
NS *
15,0% 26,6%
0
20
40
60
NS
0
2.5
5
7.5
10
pe umo (x10
5
)
NS
Tumo -in il a ing
CD8+G zmB+ cells
B16
Melanoma
G
B16
Melanoma
F
Foxp3GFP-F h''
Foxp3GFP
Foxp3GFP-F h''
Foxp3GFP
CD8+G zmB+ cells (%)
Nb . CD8+G zmB+ cells
0
200
400
600
-100 0
100
200
300
400
The EMBO Jou nal Qian Wu e al
EV9 The EMBO Jou nal Volume 43 | Issue 8 | Ap il 2024 | 1445 –1483 © The Au ho (s)
Downloaded om h ps://www.embop ess.o g on Janua y 15, 2025 om IP 193.144.79.182.
Figu e EV5. FTH egula es mi ochond ial ene gy me abolism and CPG me hyla ion in T
REG
cells.
(A) Oxygen consump ion a e (OCR) in li e splenic (CD4+GFP+)T
REG
cells and (CD4+GFP-)T
CONV
cells om Foxp3GFP mice. (B) Quan ifica ion o spa e espi a o y
capaci y, om da a ep esen ed in (A). (C) Oxygen consump ion a e (OCR) in li e splenic (CD4+GFP+)T
REG
cells and (CD4+GFP-)T
CONV
cells om Foxp3GFP-F hΔ/Δmice.
(D) Quan ifica ion o spa e espi a o y capaci y, om da a ep esen ed in (C). Da a in (A–D) pooled om N=3 mice pe geno ype, ep esen ed as mean ± SD. N=3–5
echnical eplica es in 1 ou o 3 independen expe imen s, wi h simila end. Oligomycin (Oligo.), ca bonilcyanide p- iflou ome hoxyphenylhyd azone (FCCP), An imycin
A/Ro enone (A/A+Ro .). (E) Schema ic ep esen a ion o so ing o splenic (CD4+GFP+)T
REG
cells used o a ge ed me abolomics (le panel). Quan ifica ion o
in e media e me aboli es om a ge ed me abolomics analyzes o splenic T
REG
cells ( igh panel). Da a om N=3–4 mice pe geno ype in one expe imen ep esen a i e
o 3 independen expe imen s wi h simila end. (F) Schema ic ep esen a ion o he expe imen al app oach used o flow cy ome y analysis o umo -infil a ing cells
(le panel), ep esen a i e flow cy ome y do plo s (middle panel) and co esponding pe cen age and numbe ( igh panel) o li e umo -infil a ing (CD4+IFNγ+)T
H
cells
(CD8+IFNγ+)T
C
cells, 3 weeks a e umo inocula ion (2 × 105B16 cells). Da a om N=6 mice pe geno ype, pooled om wo independen expe imen s, wi h simila
end. (G) Schema ic ep esen a ion o he expe imen al app oach used o flow cy ome y analysis o umo -infil a ing cells (le panel), ep esen a i e flow cy ome y
do plo s (middle panel) and co esponding pe cen age and numbe ( igh panels) o li e umo -infil a ing (CD8+G zmB+) T cells, 3 weeks a e umo inocula ion (2 × 105
B16 cells). Da a om N=6 mice pe geno ype, pooled om wo independen expe imen s, wi h simila end. Da a in o ma ion: Ci cles and iangles in (A,C) co espond
o mean alues, ci cles, and iangles in (B,D) co espond o indi idual wells, and ci cles in (E–G) co espond o indi idual mice, and ed ba s a e mean alues. P alues in
(A,C) calcula ed using wo-way ANOVA wi h Bon e oni’s(A,C) o Sidak´s (E,F) mul iple compa isons es , in (B,D,G) using unpai ed es wi h Welch’s co ec ion. NS,
no significan (P> 0.05); *P< 0.05; **P< 0.01; ****P< 0.0001. Sou ce da a a e a ailable online o his figu e.
Qian Wu e al The EMBO Jou nal
© The Au ho (s) The EMBO Jou nal Volume 43 | Issue 8 | Ap il 2024 | 1445 –1483 EV10
Downloaded om h ps://www.embop ess.o g on Janua y 15, 2025 om IP 193.144.79.182.