Letter
1038/s41586-019-1215-2
Targeting the CBM complex causes Treg cells to
prime tumours for immune checkpoint therapy
Mauro Di Pilato1,2,6*, Edward Y. Kim1,2,6, Bruno L. Cadilha1, Jasper N. Prüßmann1,2, Mazen N. Nasrallah1,2, Davide Seruggia2,3,
Shariq M. Usmani1,2, Sandra Misale2,4, Valentina Zappulli5, Esteban Carrizosa1,2, Vinidhra Mani1,2, Matteo Ligorio2,4,
Ross D. Warner1, Benjamin D. Medoff1,2, Francesco Marangoni1,2, Alexandra-Chloe Villani1,2 & Thorsten R. Mempel1,2*
Solid tumours are infiltrated by effector T cells with the potential
to control or reject them, as well as by regulatory T (Treg) cells
that restrict the function of effector T cells and thereby promote
tumour growth1. The anti-tumour activity of effector T cells can
be therapeutically unleashed, and is now being exploited for the
treatment of some forms of human cancer. However, weak tumour-
associated inflammatory responses and the immune-suppressive
function of Treg cells remain major hurdles to broader effectiveness
of tumour immunotherapy2. Here we show that, after disruption of
the CARMA1–BCL10–MALT1 (CBM) signalosome complex, most
tumour-infiltrating Treg cells produce IFNγ, resulting in stunted
tumour growth. Notably, genetic deletion of both or even just
one allele of CARMA1 (also known as Card11) in only a fraction of
Treg cells—which avoided systemic autoimmunity—was sufficient
to produce this anti-tumour effect, showing that it is not the mere
loss of suppressive function but the gain of effector activity by
Treg cells that initiates tumour control. The production of IFNγ
by Treg cells was accompanied by activation of macrophages and
upregulation of class I molecules of the major histocompatibility
complex on tumour cells. However, tumour cells also upregulated
the expression of PD-L1, which indicates activation of adaptive
immune resistance3. Consequently, blockade of PD-1 together with
CARMA1 deletion caused rejection of tumours that otherwise do
not respond to anti-PD-1 monotherapy. This effect was reproduced
by pharmacological inhibition of the CBM protein MALT1. Our
results demonstrate that partial disruption of the CBM complex
and induction of IFNγ secretion in the preferentially self-reactive
Treg cell pool does not cause systemic autoimmunity but is sufficient
to prime the tumour environment for successful immune checkpoint
therapy.
Local exposure of tumour-infiltrating Treg cells to their cognate
antigens sustains their tumour-promoting immunosuppressive func-
tions4. We therefore explored which T-cell-receptor (TCR)-dependent
signalling pathways could be targeted to disable tumour-reactive Treg
cells. The scaffold protein CARMA1 nucleates assembly of the CBM
complex in T cells in response to TCR-dependent PKCθ activity and
promotes several functions, including activation of the AP-1, mTOR
and classical NF-κB pathways, as well as stabilization of mRNA5.
Constitutive genetic deletion of CARMA1, BCL10 or MALT1 abro-
gates thymic development of Treg cells6–10, but their role in mature
Treg cells is unknown.
When we conditionally deleted either one or both alleles of CARMA1
in mature Treg cells by crossing Foxp3YFP-cre to CARMA1flox/flox mice
(hereafter referred to as Fcre × C1f/+ or Fcre × C1f/f mice), levels of
CARMA1 protein were proportionally reduced in CD4+FOXP3+ Treg
cells from lymph nodes (LNs) (Extended Data Fig. 1a). Fcre × C1f/f, but
not Fcre × C1f/+ or C1+/+ control mice, stopped thriving at 17 days;
most died before 4 weeks of age after a T-helper-1 (TH1)-dominated
multiorgan inflammatory disease characterized by splenomegaly,
1
Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Boston, MA, USA. 2Harvard Medical School, Boston, MA, USA. 3Division of Hematology/Oncology, Boston
Children’s Hospital, Boston, MA, USA. 4Center for Cancer Research, Massachusetts General Hospital, Boston, MA, USA. 5Department of Comparative Biomedicine and Food Science, University of
Padua, Padova, Italy. 6These authors contributed equally: Mauro Di Pilato, Edward Y. Kim. *e-mail: mdipilato@mgh.harvard.edu; tmempel@mgh.harvard.edu
1 1 2 | N A T U RE | V O L 5 7 0 | 6 J U N E 2 0 1 9
https://doi.org/10.
lymphadenopathy, effector differentiation and inflammatory
cytokine secretion by conventional T (Tconv) cells, production of auto-
reactive IgG, and activation of the myeloid compartment (Fig. 1a,
Extended Data Figs. 1b–f, 2a–f). Hence, CARMA1 is essential for Treg
cells to maintain immune homeostasis, but a reduction in its expression
at least up to 50% is tolerated.
Absolute numbers of Treg cells were increased in LNs of Fcre × C1f/f
mice, concomitant with an increase in overall LN cellularity (Extended
Data Fig. 2g, h). However, the overall frequency of Treg cells among
CD4+ T cells did not vary with CARMA1 expression, whereas the pro-
portion of CD44highCD62L– effector Treg (eTreg) was strongly reduced
in its absence (Fig. 1b). Notably, CARMA1-deficient Treg cells, while
retaining expression of FOXP3, almost uniformly secreted IFNγ and—
at lower frequencies—secreted IL-4, IL-17 and TNF (Fig. 1c). Although
nearly all CARMA1-deficient Treg cells secreted the TH1-cytokine
IFNγ, far fewer—and only eTreg cells—expressed the TH1 lineage-defin-
ing transcription factor T-bet along with RORγt and, to a lesser degree,
GATA-3 (Fig. 1d and Extended Data Fig. 2i, j). Hence, complete—but
not partial—deletion of CARMA1 in Treg cells markedly dysregulates
their cytokine expression that (in the case of IFNγ) is dissociated
from expression of T-bet, and may contribute to the pathogenesis of
inflammatory disease. Indeed, Fcre × C1f/f mice died more rapidly than
Treg cell-deficient scurfy mice, but their lifespans were similar when
IFNγ was neutralized (Fig. 1e). Thus, under inflammatory conditions,
CARMA1-deficient Treg cells convert from an immunoregulatory cell
type into an IFNγ-secreting pathogenic cell type.
In heterozygous female Fcre/+ × C1f/f mice, random inactivation of
the X chromosome causes the yellow fluorescent protein–Cre recom-
binase (YFP–Cre) fusion protein to be expressed and CARMA1 to be
deleted in only half of the Treg cells, whereas the other half maintains
immune homeostasis (Extended Data Fig. 3a–c). Under such non-
inflammatory conditions, CARMA1-deficient Treg cells did not secrete
effector cytokines (Fig. 1f). However, in competition with CARMA1-
sufficient Treg cells for niche space, we observed a proportional decline
specifically in the frequency of eTreg cells—but not of central Treg (cTreg)
cells—that lacked one or both alleles of CARMA1 (Fig. 1g and Extended
Data Fig. 3e). The remaining YFP+ eTreg cells expressed less FOXP3 and
other proteins that are characteristically increased in expression during
the differentiation of cTreg cells into eTreg cells (Fig. 1h). They expressed
more of the pro-apoptotic protein BIM, but also more of the anti-
apoptotic protein BCL2, which probably reflects impaired differentia-
tion of eTreg cells, as control eTreg cells strongly downregulated both BCL2
and BIM relative to cTreg cells (Extended Data Fig. 3d–h). The in vitro
suppressive function of CARMA1-deficient Treg cells was reduced,
but not abrogated (Extended Data Fig. 4a, b), and they failed to per-
sist and did not suppress lymphopenia-induced expansion of effector
T cells after transfer into RAG1-deficient hosts (Extended Data Fig. 4c).
Failure to persist in vivo did not seem to result from accelerated apopto-
sis, because the previously described high apoptotic rate of eTreg cells11
 Letter RESEARCH

a b
was not further enhanced in the absence of CARMA1 (Extended Data 100 15 50 ****

Remaining animals (%)

Fig. 4d). Lack of CARMA1 did not lead to an increase in the formation C1+/+ Fcre × C1+/+ 40

Treg (% of CD4)
75 C1+/+
of FOXP3-negative ‘exTreg’ cells (Extended Data Fig. 4e). C1f/+ cTreg
10

eTreg (%)
C1f/f 30 C1f/+
50
On the basis of global gene expression analyses, CARMA1-deficient C1f/f

CD62L
FOXP3
20
5
eTreg cells were equally dissimilar to control eTreg cells as they were to 25
eTreg 10

control cTreg cells, whereas CARMA1-deficient cTreg cells were only 0

0 10 20 30
*
40 CD4 CD44
0 0

moderately dissimilar to control cTreg cells (Fig. 1i). In the latter, only

+
f/+

+
f/+
Days after birth

f/f

f/f
+/

+/
96 genes were differentially expressed, compared with 344 genes in IFNγ IL-4 IL-17 TNF
**** * ****
eTreg cells (Extended Data Fig. 5a and Supplementary Table 1). Based 100
**** 10 10 * ****
100
on differences between control cTreg and eTreg cells, we defined an ‘eTreg c C1+/+ C1f/+ C1f/f 80 8 8 80
C1+/+

signature’, which largely overlapped with a previously reported gene C1f/+

Cytokine+ (%)
5.1 0.3 7.3 0.4 0.3 20.6
60 6 6 60 C1f/f
set12,13. When examining these 689 genes, hemizygous CARMA1- 40 4 4 40

TNF
deletion had only a moderate effect, whereas homozygous deletion 20 2 2 20
induced major changes specifically in the eTreg cell gene expression 92.0 2.6 90.2 2.1 0 79.1
0 0 0 0
program (Fig. 1j and Extended Data Fig. 5b). Minor changes in the IFNγ

+
f/+

+
f/+

+
f/+

+
f/+
f/f

f/f

f/f

f/f
+/

+/

+/

+/
expression of the Bcl2 family of apoptotic regulator genes occurred T-bet GATA-3 RORγt
***
** **
during eTreg cell differentiation, but CARMA1-deficient eTreg cells 100 ** *** ***
d
did not deviate from this pattern apart from having less pronounced C1+/+ C1f/+ C1f/f 80 C1+/+
2.1 1.2 0.7 0.4 4.7 13.1
downregulation of BCL2 and BIM, confirming our observations on 60
C1f/+

TF+ (%)
C1f/f
protein expression (Extended Data Fig. 6). Thus, a complete—and even

T-bet
40

a partial—loss of CARMA1 expression impairs eTreg cell differentiation 20

2.5 97.5 1.4 80.0
and persistence, but does not induce the cells to become pathogenic or 94.2
RORγt
2.2
0

convert to exTreg cells under non-inflammatory conditions. However,

+
f/+

+
f/+

+
f/+
f/f

f/f

f/f
+/

+/

+/
in the context of incipient inflammation trigged by a global loss of the e f
IFNγ TNF
* **
suppressive function of Treg cells, CARMA1-deficient Treg cells secrete Remaining animals (%) 100 100 100
C1+/+ Fcre/+ × C1+/+ C1+/+
IFNγ, which further accelerates inflammatory disease. 80 80

Cytokine+ (%)
75 Scurfy C1f/+

Failed thymic development of Treg cells in the absence of CARMA1 50

C1f/f
C1f/f +
60 60 C1f/f

FOXP3
results from disabled NF-κB signalling and is restored through the 25
anti-IFNγ
40 40
20 20
expression of a constitutively active form of IκB kinase 2 (referred to *&# * * 0 0
as IKK2ca)12. Furthermore, the NF-κB proteins REL (also known as
0
0 10 20 30 40 YFP

+
f/+

+
f/+
f/f

f/f
+/

+/
Days after birth
c-Rel) and RELA (p65) have important roles in Treg cell function13–15,
g h FOXP3 CTLA-4 CD25 CD73 GITR
which suggests that failed activation of NF-κB may primarily account 8 30
****
*** ** 8 **** 20 **** 4 ** 20 **** 15 ****
** ** ** ** *** ****
for the effects of CARMA1-deletion in Treg cells. However, the expres-
YFP+ Treg (% of CD4)

6 6 15 3 15
sion of IKK2ca in Treg cells neither prolonged the lifespan of Fcre × C1f/f
MFI (×1,000)

MFI (×1,000)

MFI (×1,000)

MFI (×1,000)
MFI (×100)
20 10
eTreg (%)

mice, nor reduced the differentiation of effector Tconv cells (Extended 4 4 10 2 10

5
10
Data Fig. 7a, b). Restoring NF-κB activation did not restore differ- 2 2 5 1 5

entiation of eTreg cells, and did not limit the secretion of IFNγ, but 0 0 0 0 0 0 0

only of TNF (Extended Data Fig. 7c, d). Therefore, although NF-κB

f/+
f/+

f/+

f/+

f/+
+

+
+

+
f/+
+

f/f
f/f

f/f

f/f

f/f
f/+
+
f/f

+/

+/
+/

+/

+/
f/f
+/

+/

Il10

activation is evidently essential13,16, additional CBM-complex effector i j eTreg (689 eTreg signature genes)
Gzmb
Ccr2
1.0 Ltb4r1
functions are also crucial for maintaining Treg cell function. The rel- 0.5
Havcr2
Il1r2
Ccr5
evance of these additional functions will need to be investigated, but 0.0
Cxcr3
Tigit
Mmp9
an initial examination of CARMA1-deficient Treg cells from healthy Lag3
PC2

–0.5 Ebi3

Fcre/+ × C1f/f mice already revealed decreased expression and TCR-

Klrg1
–1.0 Itgae
Tnfrsf8
Ahr
induced phosphorylation of the AP-1 family protein JUN, as well as –1.5 Ccl1
Ccr4

changes to phosphorylation of FOXO1, possibly reflecting CARMA1- –2 –1 0 1 Icos

Prdm1
PC1 Pdcd1

and TBK1-driven regulation of AKT activity17 (Extended Data Fig. 7e, f). C1+/+ cTreg C1+/+ eTreg
Cd83
Itgb8
Ccr8
Considering the pro-inflammatory potential of CARMA1-deficient C1f/+ cTreg
C1f/f cTreg
C1f/+ eTreg
C1f/f eTreg
Cd44
Ctla4
Irf4
Treg cells, we examined their tumour response. Subcutaneous implan- +/+ f/+ f/f

tation of the poorly immunogenic BrafV600E × Pten−/− melanoma
D4M.3A18 into female Fcre/+ × C1f/f hosts amplified the effects of
CARMA1-deficiency on Treg cells, because the frequency not only
of eTreg cells but also of total Treg cells was reduced in tumours and
tumour-draining LNs (tdLNs) as a function of decreasing CARMA1
expression, accompanied also by a more pronounced reduction in
FOXP3 expression (Extended Data Fig. 8a–d). Notably, we observed
growth deceleration of D4M.3A melanoma, and of MC38 colon carci-
noma, when half of the Treg cells lacked one or both alleles of CARMA1
(Fig. 2a and Extended Data Fig. 8e). Because a mere loss of function
in only half of the Treg cells is not predicted to cause loss of tumour
tolerance19, this suggested active Treg-cell-mediated anti-tumour activ-
ity. Indeed, a large fraction of completely or even partially CARMA1-
deficient Treg cells secreted both TNF and IFNγ in situ, whereas these
effector cytokines were undetectable in tumour-infiltrating CD4+
and CD8+ Tconv cells (Fig. 2b, c). Importantly, no increase in cytokine
secretion by Treg cells was observed in tdLNs or in non-lymphoid tis-
sues, such as skin or lung (Fig. 2d and Extended Data Fig. 8f, g). IFNγ
expression in tumour tissue correlated with downregulation, but not
Fig. 1 | Loss of CARMA1 in Treg cells is fatal, but reduced expression is
sufficient to maintain immune tolerance. a, Survival of Fcre × C1+/+, C1f/+
and C1f/f mice (n = 8, 10 and 20 per group, respectively). b, Frequency of
Treg cells among CD4+ T cells and of eTreg cells among total Treg cells in
LNs. c, d, Expression of cytokines (c) and transcription factors (TF) (d) in
LN Treg cells after ex vivo stimulation. e, Survival of Fcre × C1f/f mice treated
with anti-IFNγ antibodies from day 14 of life, compared with Fcre × C1+/+
and scurfy mice. f, Cytokine expression of YFP+ Treg cells from LNs of nine-
week-old female heterozygous Fcre/+ × C1+/+, C1f/+ and C1f/f mice after ex
vivo stimulation. g, Frequency of YFP+ Treg cells among CD4+ T cells and
of YFP+ eTreg cells among total YFP+ Treg cells in LNs. h, Expression of
indicated proteins in YFP+ eTreg cells from 9-week-old mice. Data in b–h
represent two independent experiments with similar results. Data are mean
and individual replicates. In a and e, *P < 0.05 versus wild type (+/+),
&P < 0.05 versus scurfy, and #P < 0.05 versus anti-IFNγ (log-rank (Mantel–
Cox) test). In all other panels, *P < 0.05, **P < 0.01, ***P < 0.001,
****P < 0.0001 (one-way analysis of variance (ANOVA) with Tukey post
hoc test). i, j, Bulk RNA sequencing analysis of YFP+ cTreg and eTreg cells
from LNs of Fcre/+ × C1+/+, C1f/+ and C1f/f mice. i, Principal component
(PC) analysis of transcriptomes. j, Scaled expression of eTreg signature genes
by eTreg cells (defined by fold change > 2 and Padj < 0.01 between C1+/+
cTreg and C1+/+ eTreg cells). Selected eTreg cell genes are annotated.
6 J U N E 2 0 1 9 | V O L 5 7 0 | N A T U RE | 1 1 3
 RESEARCH Letter

a b c ****
80 * ** Tumour
C1+/+ C1f/+ C1f/f

IFNγ + TNF+ (%)

4 8 5 33 6 63 60

Treg (YFP+)
Fcre/+ × C1+/+ 40
Fcre/+ × C1f/f 1,250 Fcre/+ × C1f/+
D4M.3A Fcre/+ × C1f/f 20
1,000 79 9 52 10 16 14

Tumour size (mm3)

0
2 1 6 1 3 1

f/+

f/+

f/+
+

+
f/f

f/f

f/f
750

+/

+/

+/
CD8+ Tconv
Treg CD8+ CD4+

TNF
(YFP+) Tconv Tconv
500 *
implant.
Tumour

* 88 10 91 3 93 3 d 80 tdLN
250
* *

IFNγ + TNF+ (%)

1 0 8 0 11 1 60

CD4+ Tconv
0
0 0 5 10 15 18 40
Time (days) Tumour growth (days)
20
95 3 91 2 86 2
IFNγ 0

f/+

f/+

f/+
+

+
f/f

f/f

f/f
+/

+/

+/
Treg CD8+ CD4+
(YFP+) Tconv Tconv
e f
C57BL/6 Ifng–/– Ifng–/–
1,000 1,000
C1+/+ Treg i.v.
C1+/+ Treg i.v. C1+/+ Treg i.v. + anti-IFNγ
Tumour implant.
750 C1f/+ Treg i.v. 750

Tumour size (mm3)

C1f/+ Treg i.v.
Tumour size (mm3)

Treg i.v. Treg i.v. C1f/+ Treg i.v. + anti-IFNγ

(Fcre/+ × C1f/+) (Fcre/+ × C1f/+)
500 500
Treg i.v.

*
implant.
Tumour
Treg i.v.

250 * * 250
Anti-IFNγ
*

0 0
0 5 10 15 0 5 10 15 18
–1 0 –1 0 5 10 15
Tumour growth (days) Tumour growth (days)
Time (days) Time (days)

Fig. 2 | Reduced CARMA1 expression converts tumour-infiltrating injected intravenously (i.v.) into either C57BL/6 (e) or IFNγ-deficient (f)
Treg cells into IFNγ-secreting effector cells that dominantly control hosts, which were implanted with D4M.3A melanoma the next day, and
tumour growth. a, Female Fcre/+ × C1+/+, C1f/+ and C1f/f mice were tumour growth was recorded. Some IFNγ-deficient hosts were treated
implanted with D4M.3A melanoma, and tumour growth was recorded. with neutralizing anti-IFNγ antibody. Data represent two independent
b–d, In situ expression in tumour tissue (b, c) or tdLNs (d) of effector replicates with similar results. Data are mean and either individual
cytokines in YFP+ Treg cells lacking one or both alleles of CARMA1 as replicates (c, d) or s.e.m. (a, e, f). *P < 0.05, **P < 0.01, ****P < 0.0001
well as in CD4+ and CD8+ Tconv cells 18 days after tumour implantation. (two-way ANOVA with Bonferroni post hoc test in a, f; one-way ANOVA
e, f, One million YFP+ Treg cells from Fcre/+ × C1f/+ or C1+/+ mice were with Tukey post hoc test in c, d; two-tailed Student’s t-test in e).

loss of FOXP3 in both partially and fully CARMA1-deficient Treg cells C57BL/6 or Ifng–/– mice. In both hosts, Treg cells stunted tumour growth
(Extended Data Fig. 8h). Notably, destabilization of control Treg cells similarly, but not when IFNγ was neutralized, which indicates that
by IFNγ-producing Treg cells, as described in other settings20, did not Treg-cell-derived IFNγ is both necessary and sufficient for anti-tumour
occur because no increase in cytokine expression was detectable in effects (Fig. 2e, f and Extended Data Fig. 8j–m). Therefore, although
YFP− CARMA1-sufficient Treg cells in the same tumours (not shown). neither partially nor fully CARMA1-deficient Treg cells cause inflamma-
Neutralization of IFNγ fully restored tumour growth in Fcre/+ × C1f/f tion in healthy mice, both are selectively destabilized in tumour tissue
mice (Extended Data Fig. 8i), which suggests a crucial role for this and secrete IFNγ to decelerate tumour growth.
cytokine in Treg-cell-mediated anti-tumour immunity. However, IFNγ Expression of IKK2ca restored the frequencies of total Treg and
may also derive from other cellular sources after Treg cell destabilization. eTreg cells in tdLNs, but not in tumour tissue (Extended Data Fig. 9a).
To test the role of Treg-cell-produced IFNγ specifically, we transferred It did not restore FOXP3 expression, only partially reduced TNF
Treg cells with reduced expression of CARMA1 into tumour-bearing and IFNγ co-expression by tumour-infiltrating CARMA1-deficient

a b c 15 *
C1+/+
CD45+ C1f/f
MHC-II MFI (×104)

10
FcreERT2 × C1+/+
1,000 FcreERT2 × C1f/f
FcreERT2 × C1f/f
CD11B

5
Percentage
of max

750
Tumour size (mm3)

0
Tumour implant.

F4/80 MHC-II
f/f
+/

500 * *
Tamox. d C1+/+
6 10
*
C1f/f 8
MHC-I MFI (×103)

PD-L1 MFI (×103)

250 * 4
Cerulean (tumour)

FTY720 *
6
... 4
Percentage

0 2
of max

0 8 12 0 5 10 15 2
Time (days) Tumour growth (days)
0 0
CD45 MHC-I PD-L1
+

+
f/f

f/f
+/

+/

Fig. 3 | CARMA1-deleted Treg cells rapidly induce tumour inflammation expression on D4M.3A tumour cells (expressing blue-fluorescent
but also adaptive immune resistance. a, b, D4M.3A melanoma growth in H2B-Cerulean) (d) three days after the initiation of tamoxifen treatment.
FcreERT2 × C1+/+ and C1f/f mice treated with tamoxifen from days 8–12 as MFI, mean fluorescence intensity. Data represent two independent
well as with FTY720 starting the same day until the end of the experiment. replicates with similar results. Data are mean and individual replicates
Arrow in b indicates the start of treatment. c, d, MHC-II surface (c, d) or s.e.m. (b). *P < 0.05 (two-tailed Student’s t-test).
expression on F4/80+ tumour macrophages (c), and MHC-I and PD-L1

1 1 4 | N A T U RE | V O L 5 7 0 | 6 J U N E 2 0 1 9
 Letter RESEARCH

a FcreERT2 × C1f/f 800

C1+/+
C1+/+ + anti-PD-1 b destabilization of Treg cells was accompanied by pronounced induc-
Ƃ
C1f/f
C1f/f + anti-PD-1 CARMA1
tion of macrophage expression of cell-surface major histocompati-
bility complex class II (MHC-II) molecules, both after constitutive or

Tumour size (mm3)

600 * BCL10 MALT1
Tumour implant.

Mepazine

Anti-PD-1 400
Scaffold
function
Protease
function
MI-2 acute deletion of one or both alleles of CARMA1 in Treg cells (Fig. 3c
and Extended Data Fig. 9h). Furthermore, the expression of MHC-I
***
&& (TBK1) JNK IKK RELB Regnase-1 Glutamine
Tamox. 200 A20 Roquin-1/2 influx molecules on tumour cells increased, predictably sensitizing them to
... *#*& & #& #&
** * cytotoxic-T-lymphocyte (CTL)-mediated lysis (Fig. 3d). Although
(FOXO1) (AP-1) NF-κB mRNA mTORC1
0
0 9 13 0 5 10 15 20
stability
Treg cell-derived IFNγ thus caused widespread tumour inflammation, it
Time (days) Tumour growth (days)
also triggered tumour cell-expression of PD-L1, a ligand for the T cell
c Male C57BL/6 d Male Rag1–/– e f
C57BL/6
or Rag1–/– 1,000 1,500
inhibitory receptor PD-1, which suggests that concurrent induction
ƃ
Vehicle
Mepazine
Vehicle
Mepazine of adaptive immune resistance3 limited improved tumour control
MI-2 MHC-I PD-L1
resulting from enhanced anti-tumour immune effector functions
Tumour size (mm3)

750
*
Tumour implant.

* 1,000 100 15 * 3 8
(Fig. 3d).

IFNγ+ TNF+ Treg (%)

500 ** Treg (% CD4)
80 6
Considering the induction of PD-L1 on tumour cells, we hypoth-

MFI (×104)

MFI (×103)
10 2
60
500 4
MI-2 or *
mepazine 250
*
40
5 1
2
esized that antibody-mediated blockade of PD-1 may synergize with
...
0 0 0
20
0 0 0
the anti-tumour effects of IFNγ-secreting Treg cells. Indeed, treatment
0 10 0 5 10 15
Tumour growth (days)
0 5 10 15
Tumour growth (days)
Mep.: – + – + Mep.:– + – +
with anti-PD-1 antibody simultaneously with CARMA1 deletion in
Time (days)
Treg cells enabled much more rapid and consistent control of D4M.3A
g D4M.3A Vehicle h D4M.3A-SIINFEKL Vehicle
Male C57BL/6 1,500
Anti-PD-1
Mepazine 1,000 Anti-PD-1 melanoma than either treatment alone (Fig. 4a). Targeting the CBM
Mepazine
ƃ
Anti-PD-1
+ Mep. (0/5) Anti-PD-1 + Mep. complex in Treg cells may thus be highly effective at enhancing the
potency of immune checkpoint therapy (ICT) in patients with cancer.
Tumour size (mm3)
Tumour size (mm3)

750
Tumour implant.

1,000
Anti-PD-1
500
Mepazine
...
0 0
0 9
Time (days)
Fig. 4 | CARMA1 deletion in Treg cells and pharmacological inhibition of
MALT1 protease synergize with anti-PD-1 ICT. a, Female FcreERT2 × C1+/+
and C1f/f mice were implanted with D4M.3A melanoma, and treated with
tamoxifen starting on day 9 until the end of the experiment, as well as with
three doses of the anti-PD-1-antibody 29F.1A12 or isotype control, and
tumour growth was recorded. b, CBM complex effector pathways
and predicted effects (red arrows) of the MALT1 protease inhibitors
mepazine and MI-2. c, d, D4M.3A tumour growth in C57BL/6 (c) or
RAG1-deficient (d) hosts treated with MALT1 inhibitors (MI-2 or
mepazine). Dimethylsulfoxide (DMSO) was used as a vehicle control.
e, f, Effects of mepazine treatment for three days on intratumoral Treg cell
frequency and their in situ expression of effector cytokines (e), and on the
expression of MHC-I and PD-L1 on tumour cells (f). g, h, Synergistic control
of tumours by combined anti-PD-1 and mepazine treatment of poorly
immunogenic D4M.3A (g) and immunogenic D4M.3A-SIINFEKL (h)
tumours in male C57BL/6 hosts. Numbers in parentheses indicate fraction
of mice without relapse for more than 12 months after discontinuation of
treatment. Data in a, c–f represent two independent replicates with similar
results. Data are mean and individual replicates (e, f) or s.e.m. (a, c, d, g, h).
Arrows in tumour growth charts indicate the start of treatment. In a,
*P < 0.05 versus C1+/+, #P < 0.05 versus C1+/+ + anti-PD-1, and &P < 0.05
versus C1f/f. In g and h, *P < 0.05 versus vehicle (DMSO), #P < 0.05 versus
anti-PD-1, and &P < 0.05 versus and mepazine, respectively. In e, *P < 0.05
(two-way ANOVA with Bonferroni post hoc test in a, c, g, h; two-tailed
Student’s t-test in d–f).
Treg cells, and did not prevent their anti-tumour activity (Extended Data
Fig. 9b–d), which emphasizes the importance of CBM-complex effector
functions other than NF-κB activation16 in stabilizing tumour-reactive
Treg cells.
To examine whether CARMA1 deletion acutely destabilizes intra-
tumoral Treg cells, we generated Foxp3GFP-creERT2 × CARMA1f/f (here-
after termed FcreERT2 × C1f/f) mice and treated these with tamoxifen to
trigger Cre-mediated CARMA1 deletion when tumours were already
established (Fig. 3a and Extended Data Fig. 9e). To prevent subse-
quent recruitment of additional Treg cells from tdLNs, we concurrently
blocked lymphocyte tissue egress using the functional S1P receptor
antagonist FTY720, as previously described4. Within two days of
treatment, tumour growth decelerated (Fig. 3b). A similarly rapid, albeit
slightly less pronounced, growth effect as well as increased secretion
of effector cytokines by Treg cells resulted from deletion of CARMA1
in only half of the Treg cells (Extended Data Fig. 9f, g). Intratumoral
* Although pharmacological inhibitors of the scaffold protein
500 (0/5) (3/5)
* CARMA1 are, to our knowledge, currently not available, inhibi-
*#
# & 250 tors of MALT1 paracaspase are predicted to attenuate the majority
** *
#&
*& *&
* **&*& *& #
(5/5)
of CBM-complex-dependent effector pathways (Fig. 4b). Indeed,
0 5 10 15 20
Tumour growth (days)
0 5 10 15 20 25 30 35 40 45 similar to CARMA1-deficient mice, Treg cells are virtually absent in
Tumour growth (days)
mice that express mutant MALT1 proteins that lack paracaspase activity
(replicating continual and complete pharmacological inhibition)21–23.
We therefore tested the allosteric MALT1 inhibitor mepazine24,25 and
the catalytic site binder MI-226 for activity against solid tumours. Both
inhibitors produced a similar deceleration in melanoma growth to that
observed after deletion of CARMA1 in Treg cells (Fig. 4c), even when
CD8+ T cells were depleted (Extended Data Fig. 9i). Systemic inhibition
of MALT1 will also target cells other than Treg cells, including mela-
noma cells27. However, no effect on tumour growth occurred in RAG1-
deficient mice that lack lymphocytes (Fig. 4d). Inhibition of MALT1 did
not synergize with deletion of CARMA1 in Treg cells, which suggests
that the anti-tumour activity of MALT1 does not result from effects
other than attenuated CBM complex function in Treg cells (Extended
Data Fig. 9j). Because MALT1 inhibition is predicted to attenuate, and
not enhance lymphocyte effector functions28, we conclude that its effect
on tumour growth is probably mediated through destabilization of Treg
cells. Similar to Treg-cell-specific deletion of CARMA1, treatment with
mepazine caused a rapid, albeit less pronounced, induction of TNF
and IFNγ expression by tumour-infiltrating Treg cells (Fig. 4e). Short-
term in vitro treatment of Treg cells triggered only a minor reduction of
FOXP3, GITR and CTLA-4 expression (Extended Data Fig. 10a) and
did not induce IFNγ secretion (data not shown), which indicates that
the latter occurs only under the conditions of the tumour microenvi-
ronment. Accordingly, mepazine caused upregulation of the expression
of PD-L1 and MHC-I molecules on tumour cells in vivo (Fig. 4f) and
induction of Ifng and a wide range of IFNγ-regulated genes indicative
of both TH1 inflammation and adaptive immune resistance in tumour
tissue (Extended Data Fig. 10b). In contrast to constitutive deletion of
CARMA1, short-term inhibition of MALT1 did not reduce the fre-
quency of Treg cells, and the expression of Treg-cell-associated genes in
tumour tissue was not reduced (Fig. 4e and Extended Data Fig. 10c).
Nevertheless, in addition to overall enhanced infiltration of immune
cells, treatment with mepazine specifically increased the frequen-
cies of CTL and natural killer cells in tumour tissue (Extended Data
Fig. 10d–h).
A high tumour mutational load favours response to ICT in patients
with cancer29,30, and a low mutational burden remains a major chal-
lenge that limits the success of this form of immunotherapy to some
cancer types and to a minority of patients. Accordingly, D4M.3A
melanoma, which carries a negligible mutational load relative to the
C57BL/6J reference exome (D. E. Fisher, personal communication),

6 J U N E 2 0 1 9 | V O L 5 7 0 | N A T U RE | 1 1 5
 RESEARCH Letter
is completely resistant to anti-PD-1 monotherapy in male hosts
(Fig. 4g), in contrast to female hosts, in which Y-antigen-expressing
male D4M.3A tumours showed a partial response (Fig. 4a). Concurrent
MALT1 inhibition, however, synergized with anti-PD-1 treatment,
and arrested tumour growth even in male hosts (Fig. 4g). Anti-PD-1
treatment did not further increase Treg cell expression of IFNγ, indi-
cating that PD-1 did not restrict the pro-inflammatory function of
destabilized Treg cells (Extended Data Fig. 10i). Furthermore, when
we raised the immunogenicity of D4M.3A tumours by expressing the
chicken ovalbumin-derived SIINFEKL epitope as a surrogate muta-
tional neoantigen, we observed an initial response to anti-PD-1 mon-
otherapy, but 40% of tumours relapsed. A combination of anti-PD-1
antibodies with mepazine, however, produced accelerated rejection and
prevented relapse (Fig. 4h). Finally, to explore the effects of MALT1
inhibition on other cancer types, we treated mice implanted with
MC38 colon carcinoma. Although anti-PD-1 monotherapy had only
a moderate effect on late-stage tumours, combination with mepazine
enabled profound tumour control and relapse-free rejection in most
mice (Extended Data Fig. 10j). Hence, systemic inhibition of MALT1
inflames the tumour environment and renders poorly immunogenic
tumours responsive to anti-PD-1 therapy while enhancing responses
of immunogenic tumours and minimizing the frequency of relapse, a
common problem in clinical ICT31.
We propose that inhibition of MALT1 protease or of other CBM
complex functions could be a useful therapeutic strategy to elicit an
intratumoral TH1 autoimmune reaction mediated by locally destabi-
lized, preferentially self-reactive Treg cells. Pro-inflammatory effects
of destabilized Treg cells seem to outweigh any potential attenuation of
immune effector cell activities through MALT1 inhibition. Owing to
its selectivity for intratumoral Treg cells, this treatment may increase the
fraction of patients with cancer who respond to PD-1/PD-L1-targeted
ICT or other forms of immunotherapy, without inducing systemic
autoimmune toxicity.
Online content
Any methods, additional references, Nature Research reporting summaries, source
data, statements of data availability and associated accession codes are available at
https://doi.org/10.1038/s41586-019-1215-2.
Received: 15 December 2017; Accepted: 17 April 2019;
Published online 15 May 2019.
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Acknowledgements We thank the MGH Pathology Flow Cytometry Core and
N. Ali-Akbar for technical assistance. This study was funded by an EMBO
fellowship (ALTF534-2015) and a Marie Curie Global Fellowship (750973)
(M.D.P.), DFG Fellowships (PR 1652/1-1 to J.N.P and US 116/2-1 to S.M.U),
NIH T32 CA207021 (V.M.), a Sara Elizabeth O’Brien Fellowship (F.M.), and
Melanoma Research Alliance Senior Investigator Award MRA-348693, NIH
AI123349, and the Bob and Laura Reynolds MGH Research Scholar Award
(T.R.M.).
Reviewer information Nature thanks Shimon Sakaguchi and the other
anonymous reviewer(s) for their contribution to the peer review of this work.
Author contributions M.D.P. initiated, designed, performed and analysed the
experiments, and wrote the manuscript. E.Y.K. initiated the project, designed
and performed experiments, V.Z. performed histological analyses, S.M.U.
performed autoantibody assays, V.M. and F.M. performed Treg cell analyses in
lung and skin. F.M. performed in vitro Treg suppression assay. E.C. generated
tumour cell lines. M.N.N. and A.-C.V. performed RNA sequencing analyses,
B.D.M. provided genetic mouse models, D.S. designed and performed RT–qPCR
assay, B.L.C., S.M., J.N.P., R.D.W. and M.L. performed tumour growth studies and
survival studies, T.R.M. conceived the study, supervised the project, designed
experiments, and wrote the manuscript.
Competing interests M.D.P. and T.R.M. have filed a patent application (PCT/
US2018/067856) related to the use of MALT1 inhibitors. T.R.M. is a co-founder
of Monopteros Therapeutics. All other authors declare no competing interests.
Additional information
Extended data is available for this paper at https://doi.org/10.1038/s41586-
019-1215-2.
Supplementary information is available for this paper at https://doi.org/
10.1038/s41586-019-1215-2.
Reprints and permissions information is available at http://www.nature.com/
reprints.
Correspondence and requests for materials should be addressed to M.D.P. or
T.R.M.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
© The Author(s), under exclusive licence to Springer Nature Limited 2019

Methods
Mice. Foxp3YFP-cre (ref. 32), Foxp3GFP-creERT2 (ref. 33), ROSA26-stopf/f-YFP (ref. 34),
ROSA26-stopf/f-IKK2ca (ref. 35), Ifng−/−36, B6-scurfy (ref. 37) and C57BL/6/J mice
were purchased from Jackson Laboratories. R. J. Xavier and J. J. Moon provided
CARMA1f/f (ref. 38) and Rag1−/− mice, respectively. Animals were housed in
specific-pathogen-free facilities at the Massachusetts General Hospital (MGH)
and all experimental studies were approved and performed in accordance with
guidelines and regulations implemented by the MGH Institutional Animal Care
and Use Committee (IACUC). For survival studies, the age of mice at euthanasia
mandated by a moribund state of health was recorded in Kaplan–Meyer plots.
Tumour cell lines. The BrafV600E × Pten−/− melanoma cell line D4M.3A (ref. 18)
was provided by D. E. Fisher. For some experiments, D4M.3A cells were lentivirally
transduced to express a blue fluorescent histone H2B-Cerulean fusion protein
(D4M.3A-H2B-Cerulean), as previously described39, to facilitate detection by
flow cytometry. To generate D4M.3A-SIINFEKL tumours expressing the chicken
ovalbumin-derived H-2Kb-restricted SIINFEKL peptide, we transduced D4M.3A
cells with a VSV-G pseudotyped pHAGE-EF1α lentiviral vector engineered to
express a fusion of histone H2B and Cerulean separated by two copies of the
SIINFEKL minigene and its native flanking sequences in the ovalbumin protein to
facilitate processing for antigen presentation. The colon adenocarcinoma cell line
MC3840 was obtained from A. D. Luster. All tumour lines were grown in DMEM
with 10% fetal calf serum (FCS) and used for experiments when in exponential
growth phase.
Tumour growth studies and treatments. One million D4M.3A, D4M.3A-H2B-
Cerulean, D4M.3A-SIINFEKL or MC38 tumour cells were injected subcutane-
ously in 100 μl HBSS without Ca2+ into the flanks of mice. Wherever possible,
animals were randomized into treatment groups. Tumour volumes were meas-
ured every second to third day after the start of treatments and calculated as
V = (length × width2)/2.
Tamoxifen (1 mg per mouse in 100 μl of a 9:1 mixture of olive oil and ethanol)
was injected intraperitoneally daily as indicated. FTY720 (1 mg kg−1 bodyweight)
in 150 μl H2O was injected intraperitoneally every other day until the end of the
experiment. Anti-IFNγ antibody (500 μg per mouse; clone XMG1.2) was injected
intraperitoneally on day 14 after birth or on the day of tumour implantation
and then every other day thereafter until the end of the experiment. Anti-PD-1
(200 μg; clone 29F.1A12) or rat IgG2a isotype control (200 μg; clone 2A3) was
injected intraperitoneally three times in 100 μl PBS every other day at the indicated
time points. Anti-CD8α (150 μg; clone YTS169.4) was injected in 100 μl PBS every
other day from the indicated time point until the end of the experiment. Mepazine
(16 mg kg−1 bodyweight in 5% dimethylsulfoxide (DMSO)) or MI-2 (20 mg kg−1
in 5% DMSO in purfied H2O) was injected intraperitoneally daily starting at the
indicated time points until the end of the experiment, unless indicated otherwise.
For adoptive Treg cell transfer studies, CD4+ YFP+ Treg cells were purified to more
than 95% purity through magnetic-activated cell sorting (Miltenyi) from LNs and
spleen of Fcre × C1f/+ or C1+/+ mice and 106 cells per mouse were injected intrave-
nously into the tail vein the day before tumour implantation.
Preparation of single-cell suspensions, antibody staining and flow cytometry.
Heparinized peripheral blood collected through sub-mandibular vein puncture
was treated with ACK red blood cell lysis buffer. LNs and spleens were passed
through 40-μm cell strainers, followed by red blood cell lysis (spleens only).
Tumours and lung tissue were minced into small fragments and treated with
1.5 mg ml−1 collagenase IV and 50 U ml−1 DNase I for 30 min at 37 °C under
agitation. Skin tissue was digested in medium containing 2% FCS, 10 mM HEPES,
0.5 mg ml−1 hyaluronidase, 1.5 mg ml−1 collagenase IV, and 50 U ml−1 DNase I
for 45 min at 37 °C under agitation. Residual tissue fragments were mechanically
dissociated.
Cell surface proteins were stained for 20 min at 4 °C with the following anti-
bodies against: CD11b (M1/70), CD120b/TNFR2 (polyclonal Armenian hamster
IgG), CD274/PD-L1 (10F.9G2), CD357/GITR (DTA-1), CD4 (GK1.5), CD45
(30-F11), CD62L (MEL-14), CD73 (TY/11.8), CD8α (53-6.7), CD90.2 (30-H12),
F4/80 (BM8), H-2Kb (AF6-88.5), -I-A/I-E (M5/114.15.2), Ly-6C (HK1.4), Ly-6G
(1A8), CD45R/B220 (RA3-6B2), CD64 (FcγRI) (X54-5/7.1), CD11c (N418),
CD103 (2E7), NK-1.1 (PK136), CD335 (NKp46) (29A1.4), CD3 (17A2), CD19
(1D3/CD19), CD45RB (16A), and CD44 (IM7) (BioLegend), CD11c (HL3) and
CD25 (PC61.5) (eBIoscience).
Intracellular and nuclear proteins were stained for 60 min at room temperature
after permeabilization and fixation (Mouse regulatory T cell staining Kit; eBiosci-
ence) using antibodies against: CD152/CTLA-4-(UC10-4B9), TNF (MP6-XT22),
IL-4 (11B11), IL-17A (TC11-18H10.1), IFNγ (XMG1.2), T-bet (4B10), and
Ki67 (16A8) (BioLegend), BIM (C34C5), CARD11/CARMA1 (1D12) (Cell
Signaling), FOXP3 (FJK-16 s, eBioscience), GATA-3 (L50-823) and Ki67 (B56)
(BD Biosciences), RORγt (AFKJS-9) and GFP (rabbit polyclonal) (Invitrogen).
Polyclonal goat anti-rabbit Ig (H+L) secondary antibody (Life Technologies) was
used to reveal primary anti-CARMA1 staining.
Letter RESEARCH
Preceding antibody staining, dead cells were stained using the fixable viability
violet dye Zombie Red (Biolegend) for 15 min at room temperature, followed
by blocking of Fc receptors with TruStain FcX (Biolegend) for 20 min at 4 °C.
Cells were analysed on LSR II, LSRFortessa or LSRFortessa X-20 flow cytometers
(BD Biosciences), and data were analysed with FlowJo software v.9.9.5.
Phospho-protein analysis. LN single-cell suspensions were stained using the
fixable viability dye ZombieRed (Biolegend) for 15 min at room temperature, and
added for 30 min at 37 °C to tissue culture plates pre-coated overnight with anti-
CD3ε (clone 145-2C11) and anti-CD28 (clone 37.51) antibodies (at 10 μg ml−1
of each antibody), or to uncoated control plates. Samples were then fixed in 4%
paraformaldehyde (PFA) for 10 min at room temperature, and permeabilized for
20 min through dropwise addition of 1 ml ice-cold methanol. Cells were then
stained for CD90.2 (30-H12), CD4 (GK1.5), CD8α (53-6.7), CD44 (IM7)
(BioLegend), Foxp3 (FJK-16 s, eBioscience), pFoxo1 (Thr24)/Foxo3a (Thr32), p-c-
Jun (Ser73) (D47G9) (Cell Signaling) and GFP (rabbit polyclonal Ab) (Invitrogen).
Analysis of in situ and ex vivo stimulated cytokine secretion. To detect in situ
cytokine secretion, mice were slowly injected intravenously with 500 μg of brefel-
din A in 250 μl PBS 6 h before euthanization and intracellular cytokine staining.
To detect cytokine secretion in T cells upon ex vivo re-stimulation, single-cell
suspensions from tumours and LNs were resuspended in RPMI 1640 with
10% FCS and added to anti-CD3 (clone 145-2C11)/anti-CD28 (clone 37.51)
antibody-coated (overnight at 10 μg ml−1 antibody) tissue culture plates for
8 h at 37 °C in the presence of 1 μg ml−1 Golgiplug and Monensin (both from
Biolegend) and cells processed for intracellular cytokine staining.
Analysis of exTreg cells. CD4+ YFPbright cells were first purified by FACS from
LNs and spleens of Foxp3YFP-cre/+ × CARMA1f/f (or f/+ or +/+) × ROSA26YFP mice
and stained for FOXP3 expression for flow cytometry analysis, as described above.
In vivo and in vitro suppression. For in vivo suppression studies, 3 × 105 Miltenyi
(negative selection) enriched CD4+ and FACS sorted (>98% purity) CD45RBhigh
YFP− cells from LNs and spleens of Foxp3YFP-cre/cre mice were intravenously
injected into the tail vein of Rag1−/− mice with or without 1 × 105 Miltenyi (neg-
ative selection) enriched CD4+ and FACS sorted (>98% purity) YFPbright Treg
cells from LNs and spleens of Foxp3YFP-cre/+ × CARMA1f/f (or CARMA1f/+ or
CARMA1+/+) × ROSA26YFP mice.
For in vitro suppression studies, 1 × 104 FACS-sorted (>98% purity) CD4+
YFP− conventional T cells from LNs and spleens of Foxp3YFP-cre/cre mice were
labelled with 5 μM CellTrace Violet and stimulated with 250 ng ml−1 of anti-CD3
monoclonal antibody (145-2c11, Biolegend) in presence of 2.5 × 104 T-cell
depleted splenocytes and different concentrations (from 1:1 to 1:16) of Miltenyi
(negative selection) enriched CD4+ and FACS sorted (>98% purity) YFPbright
Treg cells from LNs and spleens of Foxp3YFP-cre/+ × CARMA1f/f (or CARMA1f/+
or CARMA1+/+) × ROSA26-stopf/f-YFP mice. CD4+ YFP– conventional T cell
proliferation was read out after 72 h, as previously described41. In brief, percentage
of suppression was scaled from 0 (proliferation of conventional T cell in absence
of Treg cells) to 100 (complete absence of proliferation).
In vitro apoptosis. Enriched CD4+ and FACS-purified (>99% purity) YFPbright
Treg cells from LNs of Foxp3YFP-cre/+ × CARMA1f/f (or CARMA1+/+) × ROSA26-
stopf/f-YFP mice were added for 6 and 18 h at 37 °C to tissue culture plates pre-
coated overnight with anti-CD3ε (clone 145-2C11) and anti-CD28 (clone 37.51)
antibodies (at 10 μg ml−1 of each antibody). Viability of CD44lowCD62L+ cTreg
and CD44highCD62L– eTreg cells was then read out by annexin V and Zombie Red
staining (Biolegend).
RNA sequencing studies: sample collection. CD4+ T cells from LNs and spleens
of Fcre/+ × C1+/+, C1 f/+ or C1 f/f mice were pre-enriched by immunomagnetic cell
sorting (Miltenyi negative selection) and then 5 × 103 YFP+CD4+CD44lowCD62L+
cTreg cells per animal and the same number of YFP+CD4+CD44highCD62L– eTreg
cells were sorted to >99% purity directly into 10 μl lysis buffer consisting of TCL
buffer (Qiagen) and 1% of β-mercaptoethanol. Samples were then flash-frozen
and kept at −80 °C before further processing following a modified version of the
Smart-Seq2 protocol11,12,14, as described below. A total of 18 samples was collected,
but 2 samples were discarded for technical reasons.
Reverse transcription. Samples were thawed on ice for 2 min, and then
centrifuged at 2,500 rpm at 4 °C for 1 min and the RNA concentration nor-
malized. RNA (1.9 μl per sample) was moved to a full-skirt 96-well plate
(Eppendorf ). Each sample was then mixed with 1 μl 10 μM RT primer
5′-AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTT
TTTTTTTVN-3′ (IDT), 1 μl 10 mM dNTP (Life Technologies/Thermo
Fisher Scientific), and 0.1 μl SUPERase·In RNase-Inhibitor (20 U μl−1, Life
Technologies/Thermo Fisher Scientific). Samples were denatured at 72 °C for
3 min using an Eppendorf Mastercycler and placed immediately on ice after-
wards. The Reverse Transcription Mix (7 μl) was subsequently added to every
well, consisting of: 2 μl 5× RT buffer (Thermo Fisher Scientific), 2 μl 5 M betaine
(Sigma-Aldrich), 0.9 μl 100 mM MgCl2 (Sigma-Aldrich), 1 μl 10 μM TSO
(5′-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′, Exiqon), 0.25 μl
 RESEARCH Letter
SUPERase·In RNase-Inhibitor (20 U μl−1, Life Technologies/Thermo Fisher
Scientific), 0.1 μl Maxima H Minus Reverse Transcriptase (200 U μl−1, Thermo
Fisher Scientific) and 0.75 μl nuclease-free water. Reverse transcription was carried
out by incubating the plate at 50 °C for 90 min, followed by heat inactivation at
85 °C for 5 min.
PCR pre-amplification and cDNA purification. PCR Mix (14 μl, consisting of
0.5 μl 10 μM PCR primer 5′-AAGCAGTGGTATCAACGCAGAGT-3′ (IDT),
12.5 μl 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems) and 1 μl nuclease-
free water) was added to each well for a final PCR reaction volume of 25 μl. The
reaction was carried out with an initial incubation at 98 °C for 3 min, followed by
16 cycles at 98 °C for 15 s, 67 °C for 20 s, and 72 °C for 6 min, and a final extension
at 72 °C for 5 min. PCR products were purified by mixing them with 20 μl (0.8×)
of Agencourt AMPureXP SPRI beads (Beckman-Coulter), followed by a 6-min
incubation period at room temperature. The plate was then placed onto a magnet
for 6 min before removing the supernatant. SPRI beads were washed twice with
100 μl of freshly prepared 70% ethanol, with care being taken to avoid loss of beads
during the washes. After removing all residual ethanol traces, SPRI beads were left
to dry at room temperature for 10 min. The beads were then resuspended in 20 μl of
TE buffer (Teknova) and incubated at room temperature for 5 min. The plate was
placed on the magnet for 5 min before transferring the supernatant containing the
amplified cDNA to a new 96-well plate. This cDNA SPRI clean-up procedure was
repeated a second time to remove all residual primer dimers. The concentration
of amplified cDNA was measured on the Synergy H1 Hybrid Microplate Reader
(BioTek) using the Qubit dsDNA High Sensitivity Assay Kit (Life Technologies/
Thermo Fisher Scientific). The cDNA size distribution of few selected wells was
assessed on a High-Sensitivity Bioanalyzer Chip (Agilent), and the expected size
distribution sharply peaked around 2 kb.
Sequencing library preparation. Library preparation was carried out using the
Nextera XT DNA Sample Kit (Illumina) with custom indexing adapters, allowing
the 18 libraries to be simultaneously generated in a 384-well PCR plate (Eppendorf).
For each library, the amplified cDNA was normalized to a 0.15–0.20 ng μl−1
concentration range. The tagmentation reaction consisted of mixing 0.625 μl of
normalized cDNA with 1.25 μl of Tagmentation DNA (TD) buffer and 0.625 μl of
Amplicon Tagment enzyme Mix (ATM). The 2.5-μl reaction was incubated at 55 °C
for 10 min and then immediately placed on ice upon completing this incubation
step. The reaction was quenched with 0.625 μl of Neutralize Tagment (NT) buffer
and incubated at room temperature for 10 min. The libraries were amplified by
adding 1.875 μl of Nexstera PCR Master (NPM) Mix, 0.625 μl of 10 μM i5 adaptor
5′-AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC-3′
(IDT), in which [i5] signifies the 8-bp i5 barcode sequence (see below for sequences),
and 0.625 μl of 10 μM i7 adaptor 5′CAAGCAGAAGACGGCATACGAGAT[i7]
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGG-3′ (IDT), in which
[i7] represents the reverse-complement of the 8-bp i7 barcode sequence (see below
for sequences used). The PCR was carried out at an initial incubation at 72 °C for
3 min, 95 °C for 30 s, followed by 12 cycles of (95 °C for 10 s, 55 °C for 30 s, 72 °C
for 1 min), and a final extension at 72 °C for 5 min. Following PCR amplification,
2.5 μl of each library were pooled together in a 1.5-ml Eppendorf tube. The pool
was mixed with 67.5 μl (0.9× ratio for 2.5 μl of 30 samples pooled together) of
Agencourt AMPureXP SPRI beads (Beckman-Coulter) and incubated at room
temperature for 5 min. The pool was then placed on a magnet (DynaMag-2, Life
Technologies) and incubated for 5 min. The supernatant was removed and the SPRI
beads were washed twice with 1 ml of freshly prepared 70% ethanol. After remov-
ing all residual ethanol traces, the SPRI beads were left to dry at room temperature
for 10 min. The beads were resuspended in 100 μl of nuclease-free water and
incubated at room temperature for 5 min. The tube was then placed back on the
magnet for 3 min before transferring the supernatant to a new 1.5-ml Eppendorf
tube. This SPRI clean-up procedure of the library was repeated a second time to
remove all residual primer dimers, using the same approach. The concentration of
the pooled libraries was measured using the Qubit dsDNA High Sensitivity Assay
Kit (Life Technologies/Thermo Fisher Scientific), and the library size distribution
measured on a High-Sensitivity Bioanalyzer Chip (Agilent), showing the expected
size distribution of 300–500 bp. The 18 pooled samples were sequenced as paired-
end on an Illumina NextSeq 500 instrument using the NextSeq 500/550 High
Output v2 kit (75 cycles).
i5 barcodes: AAGTAGAG, ACACGATC, TGTTCCGA. i7 barcodes:
GAATTGCT, GTCAAGTT, ATCCGACA, CAAGGCGA, AGTGTCTT,
GACCGAGA.
RNA sequencing analysis. Raw sequencing reads were demultiplexed and
converted to FASTQ files using Illumina bcl2fastq2 Illumina software (version
2.17.1.14). FASTQ sequencing reads were then aligned to mm10 reference genome
using the STAR aligner with default parameters42. RSEM (v.1.2.8) was used to
quantify gene expression level from aligned reads and generate count expression
matrices for each experimental condition43. We filtered out lowly expressed genes
with a count per million (CPM) < 0.5 in more than two conditions, leaving a total
of 14,168 genes for further analysis. The distribution of log2 normalized CPM data
was visualized to assess for coverage, and all conditions had similar distributions.
Gene expression analysis. Gene expression matrices were analysed using the
limma package in R44. The global topology of quantile normalized data was vis-
ualized using the multidimensional scaling (plotMDS) function in limma after
removing batch effects using the removebatchEffect function in limma with default
parameters taking into account design and batch matrices. Differential gene expres-
sion was performed using empirical Bayesian statistics (eBayes) function in limma
simultaneously correcting for batch using blocking terms for batch covariates.
Differentially expressed genes with log fold change greater than 1 and a P value
below cut-off were visualized using the heatmap.2 function in gplots. All P values
were corrected for multiple hypothesis testing using Benjamini–Hochberg correc-
tion. For R scripts used to perform the gene expression analyses see Supplementary
Methods. The same differential expression steps were used to re-analyse the gene
expression data from GEO accession GSE82008 to obtain the list of differentially
expressed genes between c-Rel and p65 knockout versus wild-type resting and
activated Treg cells. A list of 831 ‘eTreg signature’ genes from ref. 16 was obtained by
direct correspondence with the authors. Overlap between differentially expressed
genes, including the list of eTreg cell signatures from the current study and ref. 16,
was visualized using the vennDiagram function in limma.
Quantitative RT–PCR. For analysis of gene expression, RNA was isolated (AllPrep,
DNA/RNA Mini kit; Qiagen) from CD4+ GFP+ Treg cells sorted to >99% purity
from tdLNs and tumours, or from homogenized tumour tissue, and reverse
transcribed using iScript cDNA Synthesis Kit (Bio-RAD). Quantitative reverse
transcription (RT–PCR) was performed using iQ SYBR green supermix (Bio-
RAD) and primers: CARMA1 Fwd 5′-ACATGCTGAGCCGTTACATCA-3′,
CARMA1 Rev 5′-CCACATAGCCCCTTTGTCCC-3′, Ifng Fwd 5′-CGGCACAGT
CATTGAAAGCCTA-3′, Ifng Rev 5′-GTTG CTGATGGCC TGATTGTC-3′, Ctla4
Fwd 5′-GCTTCCTAGATTACCCCTTCTGC-3′, Ctla4 Rev 5′-CGGGCATGG
TTCTGGATCA-3′, CD25-Fwd 5′-CCACATTCAAAGCC CTCTCCTA-3′,
CD25-Rev 5′-GTTTTCCCACACTTCATCTTGC-3′, Foxp3 Fwd 5′-TTGG
CCAGCGCCA TCTT-3′, Foxp3 Rev 5′-TGCCTCCTCCAGAGAGAAGTG-3′,
GITR (also known as Tnfrsf18) Fwd 5′-AAGGTTCAGAACGGAAGTG-3′, GITR
Rev 5′-GGGTCTCCACAGTGGTACT-3′, CD73 (also known as Nt5e) Fwd
5′-CAA ATCCCACACAACCACTG-3′, CD73 Rev 5′-TGCTCACTTGGTCACA
GGAC-3′, Gzmb Fwd 5′-CATGTAGGGTCGAGAGTGGG-3′, Gzmb Rev
5′-CCTCCTGC TACTGCTGAC CT-3′, Pdl1 (also known as Cd274) Fwd
5′-TGCTGCATAATCAGCTACGG-3′, Pdl1 Rev 5′-GCTGGTCACATT
GAGAAGCA-3′, Socs1-Fwd 5′-ACAAGCTGCTACAACCAGG G-3′, Socs1
Rev 5′-ACT TCTGGCTGGAGACCTCA-3′, Tap1 Fwd 5′-GTGGCCGCAGTG
GGA CAAGAG-3′, Tap1 Rev 5′-AGGGCACTGGTGGCATCATC-3′, Stat1
Fwd 5′-TGGTGAAATTGCAAG AGCTG-3′, Stat1 Rev 5′-CAGACTTCCG
TTGGTGGATT-3′, Irf1 Fwd 5′-CAG AGGAAAG AGAGAAAGTCC-3′, Irf1
Rev 5′-CACACGGTGACAGTGCTGG, Cxcl10 Fwd 5′-CATC CTGCTGGGT
CTGAGTG-3′, Cxcl10-Rev 5′-ATTCTCACTGGCCCGTCATC, Nos2 Fwd
5′-CAAGAGAGTGCTGTTCCAGGT-3′ and Nos2 Rev 5′-GAGCACGCTGAGT
ACC TCATT-3′, Gapdh Fwd 5′-TGGTGAAGGTCGGTGAAC-3′ and Gapdh Rev
5′-CC ATGTAGTTGAGGTCAATGAAGG-3′. Results were expressed as 2−ΔCt
relative to the house keeping gene Gapdh.
Histology. Tissue samples obtained from all organs were fixed in 10% buffered
formalin for 48 h, trimmed and placed into microcassettes, and embedded in par-
affin wax. Sections of 5 μm were stained with haematoxylin and eosin according
to standard procedures.
Immunofluorescence. Kidney, liver, and stomach from a Rag1−/− mouse were
embedded in OCT and flash-frozen in cold methylbutane equilibrated on dry
ice. Sections of 10 μm were permeabilized with pre-cooled 90% methanol for
10 min at −20 °C, blocked in TruStain FcX (93, Biolegend) with 1% goat serum
and 0.25% BSA in PBS for 60 min, incubated with sera (1:100 dilution) from
Foxp3YFP-cre × CARMA1f/f (or CARMA1 f/+ or CARMA1+/+) mice for 120 min and
stained with anti-mouse IgG (H+L)-Alexa Fluor 647 (1:500) (A-21235, Thermo
Fisher) and DAPI (Sigma) for 120 min. Sections were mounted on coverslips in
Prolong (Thermo Fisher) and imaged with LSM 780 AxioObserver confocal micro-
scope (Carl Zeiss) using a 20× lens (Apochromat, 0.8 W).
Statistical analysis. A two-tailed Student’s t-test was used for comparisons between
two groups, and a two-way ANOVA with Bonferroni post hoc test (multiple
time points) or one-way ANOVA with Tukey post hoc test (single time points)
was used for comparisons across multiple groups, unless otherwise indicated. A
log-rank (Mantel–Cox) test was used to compare survival curves. All statistical
tests were performed with GraphPad Prism software, and P < 0.05 was considered
statistically significant. No statistical methods were used to predetermine sample
size. Investigators were not blinded to allocation during experiments and outcome
assessment.
Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this paper.

Data availability
All datasets generated during the current study are available from the correspond-
ing authors upon reasonable request. RNA sequencing data have been deposited
at the Gene Expression Omnibus (GEO) under accession number GSE129480.
32. Rubtsov, Y. P. et al. Regulatory T cell-derived interleukin-10 limits inflammation
at environmental interfaces. Immunity 28, 546–558 (2008).
33. Rubtsov, Y. P. et al. Stability of the regulatory T cell lineage in vivo. Science 329,
1667–1671 (2010).
34. Srinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP
and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4 (2001).
35. Sasaki, Y. et al. Canonical NF-κB activity, dispensable for B cell development,
replaces BAFF-receptor signals and promotes B cell proliferation upon
activation. Immunity 24, 729–739 (2006).
36. Dalton, D. K. et al. Multiple defects of immune cell function in mice with
disrupted interferon-γ genes. Science 259, 1739–1742 (1993).
37. Godfrey, V. L., Wilkinson, J. E., Rinchik, E. M. & Russell, L. B. Fatal lymphoreticular
disease in the scurfy (sf) mouse requires T cells that mature in a sf thymic
Letter RESEARCH
environment: potential model for thymic education. Proc. Natl Acad. Sci. USA
88, 5528–5532 (1991).
38. Egawa, T. et al. Requirement for CARMA1 in antigen receptor-induced NF-κB
activation and lymphocyte proliferation. Curr. Biol. 13, 1252–1258 (2003).
39. Marangoni, F. et al. The transcription factor NFAT exhibits signal memory during
serial T cell interactions with antigen-presenting cells. Immunity 38, 237–249
(2013).
40. Spiess, P. J., Yang, J. C. & Rosenberg, S. A. In vivo antitumor activity of
tumor-infiltrating lymphocytes expanded in recombinant interleukin-2. J. Natl.
Cancer Inst. 79, 1067–1075 (1987).
41. Marangoni, F. et al. Tumor tolerance-promoting function of regulatory T cells is
optimized by CD28, but strictly dependent on calcineurin. J. Immunol. 200,
3647–3661 (2018).
42. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29,
15–21 (2013).
43. Li, B. & Dewey, C. N. RSEM: accurate transcript quantification from RNA-Seq
data with or without a reference genome. BMC Bioinformatics 12, 323 (2011).
44. Ritchie, M. E. et al. limma powers differential expression analyses for
RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).
RESEARCH Letter

a b 12 FCre x C1+/+
c Day 21 d Day 21
+ + + –
CD4 Foxp3 CD4 Foxp3 FCre x C1f/+

Body weight (gr)

10
FCre x C1+/+ (’C1+/+’) FCre x C1f/f
3593 3185
2880 2880 FCre x C1f/+ (’C1f/+’)
% of max

2076 3204 FCre x C1f/f (’C1f/f’) * * *

5

0
CARMA1 0 7 14 21 25
Days after birth

f/+
+
f/+
+

f/f
f/f

+/
+/
e Liver Skin Lung f Kidney Liver Stomach
C1+/+

C1+/+
C1f/+

C1f/+
C1f/f

C1f/f

100 µm

IgG (Serum autoantibody) DAPI

Extended Data Fig. 1 | Lymphoproliferative disease after Treg-cell-
specific deletion of CARMA1. a, CARMA1 protein in Treg cells and
CD4+ Tconv cells from LNs of Fcre × C1+/+, C1f/+ and C1f/f mice. b, Weight
curves (n = 5 per group). c, d, Appearance of 21-day-old mice (c), and
their spleens and LNs (d). e, Histological appearance of liver, skin and
lung at 21 days of age of indicated mice. Scale bars, 150 μm and 50 μm
(insets). f, Kidney, liver and stomach tissue sections of healthy C57BL/6
Rag1−/− mice were reacted with serum from 21-day-old mice of the
indicated genotypes, and self-tissue-reactive IgG revealed by anti-mouse
IgG staining (green). Nuclei were stained with DAPI (red). In b, *P < 0.05
versus C1+/+ and C1f/+ (two-way ANOVA with Bonferroni post hoc test).
 Letter RESEARCH

a CD45
+
CD11b+ CD11b+ CD11b+ CD11c– b c *
15.8 44.5 4.2 26.9 * **
*** ** hi
‘DC’ (CD11c MHC II )
hi
55.2 *** 100 ** **
50 – lo hi
C1+/+

‘Eos’ (Ly6G Ly6C SSC )

CD11b (% of CD45 )
+
hi
40 ‘Neu’ (Ly6G )
75 – hi

+
7.0 ‘Mo’ (Ly6G Ly6C )

% of CD11b
– lo/– lo
30 ‘Mac’ (Ly6G Ly6C SSC )
50
40.1 2.9 29.9 20
14.7

+
25
55.6 10

MHC II
C1f/+

SSC
SSC

FSC 0 0

+
f/+

f/+
f/f

f/f
+/

+/
7.4

38.2 20.2 0.9 32.7

54.2
C1f/f

6.5

CD11b Ly6G CD11c Ly6C

d Mo Mac Neu Eos DC Mo Mac Neu Eos DC Mo Mac Neu Eos DC

**** **** **** **** **** * * ** **** *** **** ****
**** **** **** **** **** * * ** * **** *** *** ****
25 300 15
MHC I MFI (x1000)

PD-L1 MFI (x1000)

MHC II MFI (x100)

250
20
200 10
15
150
6
10
4 5
5
2
0 0 0

+
+

+
+

+
+

+
+

f/+
f/+

f/+

f/+

f/+
f/+

f/+

f/+
f/+

f/+

f/+
f/+

f/+

f/+

f/+

f/f

f/f
f/f

f/f

f/f

f/f
f/f

f/f

f/f
f/f

f/f

f/f
f/f

f/f

f/f

+/
+/

+/

+/

+/
+/

+/

+/
+/

+/

+/
+/

+/

+/

+/

Day 12 Day 21
e CD4+ CD8+ CD4+ CD8+
CD4+ Tconv CD8+ Tconv 60 **** *** ** **

%CD44hi CD62L–
+/+ f/+ f/f +/+ f/+ f/f
40
CD62L

CD62L

20

0
CD44 CD44

f/+

f/+
+

+
f/+

f/+
+

+
f/f

f/f
f/f

f/f
+/

+/
+/

+/
f ****
IFNγ
**** **
IL-4
*
IL-17
***
TNF
**
**** **** * * ** *
100 10 10 100

80 8 8 80
% cytokine+

60 6 6 60

40 4 4 40

20 2 2 20

0 0 0 0
+

+
f/+

f/+

f/+

f/+

f/+

f/+
+

+
f/+

f/+

f/f

f/f

f/f

f/f

f/f

f/f
f/f

f/f

+/

+/

+/

+/

+/

+/
+/

+/

CD4+ CD8
+
CD4
+
CD8
+
CD4
+
CD8
+
CD4
+
CD8
+

g 2.0 ***
*** h 1.25 **
**
FCre x C1+/+
#cells / LN (x107)

#Treg / LN (x105)

1.00
1.5 FCre x C1f/+
0.75 FCre x C1f/f
1.0
0.50
0.5 0.25

0 0
f/+
+
f/+
+

f/f
f/f

+/
+/

i j FCre xC1f/f
C1+/+ C1f/+ C1f/f C1+/+ C1f/+ C1f/f T-bet+ Treg GATA-3+ Treg RORγt+ Treg
3.4 0.7 1.1 0.2 13.6 5.0 2.1 1.1 1.1 0.4 10.3 4.3
RORγt

CD62L
T-bet

91.9 4.0 97.1 1.6 71.6 9.8 92.5 4.3 96.9 1.6 74.1 11.3
GATA-3 GATA-3 CD44

Extended Data Fig. 2 | Myeloid cell expansion and effector cytokine 21 days. f, Effector cytokine expression of Tconv cells from 21-day-old mice
secretion by Tconv and Treg cells after Treg-cell-specific deletion of after 8-h ex vivo stimulation on anti-CD3/CD28-coated plates. g, LN
CARMA1. a–c, Size of the CD11b+ splenic myeloid compartment cellularity. h, Absolute numbers of Treg cells in LNs. i, Co-expression of
and proportions of Ly6G+ neutrophils, CD11c+MHC IIhigh dendritic indicated transcription factors by Treg cells from LNs of indicated mice.
cells (DCs), Ly6Chigh monocytes, Lyc6GlowSSChigh eosinophils, and j, Expression of CD44 and CD62L by Fcre × C1f/f Treg cells expressing T-bet
Ly6ClowSSClow macrophages in Fcre × C1+/+, C1f/+ or C1f/f mice. (green dots), GATA-3 (blue dots), or RORγt (red dots), compared to
d, Expression of MHC-I, MHC-II and PD-L1 on splenic myeloid subsets. total C1f/f Treg cells (contour plots). *P < 0.05, **P < 0.01, ***P < 0.001,
e, Frequency of CD4+ and CD8+ Tconv cells with a CD44highCD62L− ***P < 0.0001 (one-way ANOVA with Tukey post hoc test).
effector memory phenotype in LNs of indicated mice at age 12 and
RESEARCH Letter

a Foxp3
YFP-Cre/+
x C1
f/f
Foxp3YFP-Cre +
YFP Treg
b CD4+ Tconv
+

CD8+ Tconv c
40 40
Foxp3+ (C1f/f is deleted) FCre/+
x C1 +/+
♀

% CD44hi CD62L–
Random
X-inactivation 30 FCre/+ x C1f/+ 30
– Cre/+
X-chr. 1 Foxp3YFP-Cre Foxp3 YFP-Cre
YFP Treg F x C1f/f
20 20
X-chr. 2 Foxp3+ Foxp3+ (maintain tolerance)

10 10

0 0
0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48

f/+
+
d

f/f
+/
F Cre/+
x C1 +/+
F Cre/+
x C1 f/+
F Cre/+
x C1 f/f Weeks after birth Weeks after birth

Foxp3 CTLA-4 CD25 CD73 GITR TNFR2 Ki67 Bim Bcl-2

cTreg

eTreg

e f Foxp3 CTLA-4 CD25 CD73 GITR

cTreg eTreg cTreg eTreg cTreg eTreg cTreg eTreg cTreg eTreg
**** **** ** **** * **** **** * ****
30 *** ** 8 * 8 20 4 ** 20 15
** ** * ** ** ** *** *** ****
#eTreg / LN (x103)

6 6 15 3 15

MFI (x1,000)
MFI (x1,000)

MFI (x1,000)

MFI (x1,000)
MFI (x100)

20 10
% eTreg

4 4 10 2 10

10 5
2 2 5 1 5

0 0 0 0 0 0 0
f/+

f/+

f/+
f/+

f/+
f/+

f/+

f/+

f/+

f/+

f/+
+

+
+

+
+

+
f/+
+

f/f

f/f

f/f
f/f

f/f
f/f

f/f

f/f

f/f

f/f

f/f
f/f

+/

+/

+/
+/

+/
+/

+/

+/

+/

+/

+/
+/

TNFR2 Ki67 Bim Bcl-2

cTreg eTreg cTreg eTreg cTreg eTreg cTreg eTreg
*** * ** *** 15 **
25 ** 20 *** 25 *** *
20 20
MFI (x1,000)

150
MFI (x1,000)
MFI (x100)

MFI (x100)

10
15 15
10
10 10
5
5
5 5

0 0 0 0
f/+

f/+
+

+
f/+

f/+

f/+

f/+

f/+

f/+
+

f/f

f/f
f/f

f/f

f/f

f/f

f/f

f/f

+/

+/
+/

+/

+/

+/

+/

+/

g h Foxp3 CTLA-4 CD25 CD73 GITR

cTreg eTreg cTreg eTreg cTreg eTreg cTreg eTreg cTreg eTreg
50 5 10 25 40 20 25
#eTreg / LN (x104)

40 4 8 20 20
30 15
MFI (x1,000)
MFI (x1,000)

MFI (x1,000)
MFI (x1,000)
MFI (x100)
% eTreg

30 3 6 15 15
20 10
20 2 4 10 10
10 5
10 1 2 5 5

0 0 0 0 0 0 0
f/+

f/+
f/+

+
+

f/+

f/+

f/+

f/+

f/+

f/+

f/+

f/+
+

+
f/+
+

f/f

f/f
f/f

f/f

f/f

f/f

f/f

f/f

f/f

f/f

f/f
f/f

+/

+/
+/

+/

+/

+/

+/

+/

+/

+/

+/
+/

TNFR2 Ki67 Bim Bcl-2

cTreg eTreg cTreg eTreg cTreg eTreg cTreg eTreg
25 20 25 15

20 20
MFI (x1,000)

15
MFI (x1,000)

MFI (x100)

10
MFI (x100)

15 15
10
10 10
5
5
5 5

0 0 0 0
f/+

f/+
+

+
f/+

f/+

f/+

f/+

f/+

f/+

f/f

f/f
+

+
f/f

f/f

f/f

f/f

+/

+/
f/f

f/f
+/

+/

+/

+/

+/

+/

Extended Data Fig. 3 | Role of CARMA1 in eTreg cell differentiation. proliferation marker Ki67, pro-apoptotic protein BIM and anti-apoptotic
a, Female heterozygous Fcre/+ × C1f/f mice express YFP–Cre and delete C1f/f protein BCL2 by YFP+ cTreg and eTreg cells (d, f) from nine-week-old
in half of the Treg cells owing to X-chomosomal location of the Foxp3YFP-cre Fcre/+ × C1+/+, C1f/+ or C1f/f mice. Note that some data on eTreg cells
allele and random X chromosome inactivation, whereas the other half of in e and f are also shown in Fig. 1g, h and shown here to facilitate the
the Treg cells remains functional. b, Frequency of CD4+ and CD8+ Tconv comparison to cTreg and YFP– Treg cells in g and h. g, h, Frequency and
cells with a CD44highCD62L– effector memory phenotype in peripheral absolute numbers of eTreg cells (g) and eTreg cell markers on YFP– cTreg
blood of ageing Fcre/+ × C1+/+, C1f/+ or C1f/f mice (n = 4 per group). and eTreg cells (h) from the same mice as shown in d and f. *P < 0.05,
c, Appearance of spleens and LNs of indicated mice at one year of age. **P < 0.01, ***P < 0.001, ***P < 0.0001 (one-way ANOVA with Tukey
d–f, Frequency and absolute numbers (e) of eTreg cells and expression post hoc test).
of FOXP3, indicated markers of eTreg cell differentiation, as well as
 Letter RESEARCH

a b *
**
**
****
***
**** **
100
C1+/+ Treg
+
CD4 ‘Tconv’ ‘Treg’ 80 C1f/+ Treg

% Suppression
C1f/f Treg
60
CD45RB

CD45RB
FACS 98.9 0.0 40

98.9
20
0.0

0
YFP YFP 1:16 1:8 1:4 1:2 1:1
Tconv : Treg ratio
c
No Treg C1+/+ Treg
3.7 Foxp3 0.0 1.9 12.0

Foxp3
Treg Tconv
Thy1

Thy1
y

Rag–/– 2.0 x 10
4
** * 5
1.2 x 10 * *
No Treg
99.2 87.1 1.5 x 104
C1+/+ Treg
4
8.0 x 10 C1f/+ Treg

cells / ml
CD4 YFP CD4 YFP

cells / ml
C1f/f Treg
f/+ f/f 1.0 x 104
+ C1 Treg C1 Treg
CD4 Tconv 4
2.6 14.1 4.9 4.9 4.0 x 10
+ 5.0 x 103

Treg (C1+/+, C1f/+, C1f/f)

Foxp3

Foxp3
Thy1

Thy1

0.0 0.0

84.3 93.1

CD4 YFP CD4 YFP

d cTreg eTreg
C1+/+ C1f/f C1+/+ C1f/f
3.0 0.2 3.1 0.1 25.3 3.8 23.2 2.1

C1+/+ AnnV+
0h C1+/+ AnnV+ / ZR+
C1f/f AnnV+
C1f/f AnnV+ / ZR+
96.8 0.0 96.8 0.0 70.6 0.2 74.7 0.0
cTreg eTreg
3.9 1.8 5.9 1.0 5.2 83.5 6.3 90.0
0h 6h 18 h 0h 6h 18 h
*** *
Annexin V

100 100

18 h
% apoptotic cells

75 75

94.4 0.0 93.1 0.0 11.0 0.4 3.8 0.0 50 50

5.0 6.1 4.4 5.4 2.8 21.4 4.4 21.7

25 25

18 h 0 0
αCD3/28 αCD3/28: – – + – + – – + – +

86.8 2.1 85.4 4.9 71.7 4.0 71.7 2.2

ZombieRed

e 100
1 year
80
% Foxp3–

60
6
Foxp3
CD4

23 40

20

0
YFP CD4
+
f/+
f/f
+/

Extended Data Fig. 4 | In vitro and in vivo suppression, apoptotic of various genotypes and Tconv cells were co-adoptively transferred into
rate and exTreg cell formation of CARMA1-deficient Treg cells. Rag-deficient hosts and their respective frequency in peripheral blood
a, CD4+CD45RBhighYFP– Tconv cells and CD4+CD45RBlowYFPbright was determined eight weeks later. d, CD4+YFP+ Treg cells of indicated
Treg cells were double-sorted to more than 98% purity from LNs and genotypes were cultured without exogenous IL-2 on anti-CD3/CD28-
spleens of Fcre × C1+/+ × ROSA26-stopf/f-YFP mice, which allow for clear coated or uncoated plates for 6 or 18 h and examined for reactivity with
differentiation of Cre-expressing Treg cells based on high expression annexin V and the viability dye ZombieRed. e, CD4+YFPbright cells were
of soluble enhanced yellow fluorescent protein (eYFP) in addition sorted from LNs of one-year-old and Fcre/+ × C1+/+ (or C1 f/+, C1 f/f) 
to the YFP–Cre fusion protein. b, YFPbright Treg from Fcre × C1+/+ or × ROSA26-stopf/f-YFP mice and subsequently stained for expression
Fcre/+ × C1f/+ or C1 f/f mice and CellTrace Violet-labelled Tconv cells from of FOXP3 protein to determine the frequency of FOXP3– exTreg cells.
Fcre × C1+/+ mice were co-cultured at indicated ratios for three days in *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA
the presence of anti-CD3 antibodies and T-cell-depleted splenocytes, and with Sidak post hoc test in b, c; two-tailed Student’s t-test in d).
suppression measured as reduction of Tconv cell proliferation. c, Treg cells
 RESEARCH Letter

a relative

cTreg (All 96 changed genes) row min row max

Hebp1
Gm14347
Rnf187
Nfil3
Ppm1d
Sft2d3
Bloc1s4
Mrs2
1810026J23Rik
Donson
Ccny
Lamp1
Glud1
1110008F13Rik
Dclk2
1300018J18Rik
Usp49
Bag1
Aggf1
Mrps30
Ccdc117
Tysnd1
Taf4b
Traf4
Ybx1
Sh2b3
Brap
Lmnb1
Pnrc1
Pim1
Cdk16
Wrnip1
Stt3b
E130309D02Rik
Rab24
Rfng
Ptk2b
Scaf4
Ubqln1
Ankrd13c
Tprgl
Ddi2
St8sia6
Kremen1
Sun2
Elavl1
Itsn2
Dbp
Mlxip
Stim2
Klhl21
Nol9
Tmem160
Gm13363
Patz1
Golph3
Herpud2
Mapk1
Isg20l2
Dtx1
Myo1e
Casp7
H2−Q8
Capn3
Tns1
Cd86
Ccr8
Nefh
Rangrf
Ncf1
Casp1
Dzip1
Mlh1
Atp9a
Myo1f
Usp54
Alms1
Cd83
Mgmt
Sdc4
Top2a
Hmgn3
Serpina3g
Prss12
Itgae
Tdgf1
Podnl1
2810029C07Rik
Fam160a1
Trip13
Pbk
Ccne2
Mxd3
Plekha8
Exo1
5830416I19Rik

C1+/+ C1f/+ C1f/f

eTreg (All 344 changed genes)

Ccdc164
Ampd1
Dapk1
Plaur
Ptger3
Dapl1
Adcy6
Ccdc78
Ggt1
Gm5346
Ovgp1
Zfp493
Slc30a4
Ptrf
Ntn5
Myo6
Tmod4
Lhfp
G0s2
Cnn3
Pla2g12a
Ly6c1
Homer3
1700028J19Rik
B020014A21Rik
Gm3435
Pde3b
Atf3
Serpinb8
Ggt5
Igfbp4
Dennd2c
Rnf187
Galntl6
Lypd6b
Lrp5
2610307P16Rik
Lcn6
Alyref
Lamp1
Ldhd
Igsf3
Faf1
Ada
Klf2
1110032F04Rik
Donson
Ccny
Emid1
Xkrx
Ppm1d
2610019F03Rik
Gm15708
Gpr146
Gpd2
Ncapg
Atp1b1
Col11a2
Treml2
Mmp11
Aven
Syn3
Sgk3
Gfod2
Sell
Prf1
Eif2ak3
Ccdc90a
Patz1
4933404O12Rik
Snx18
Itm2a
Tpx2
Igsf23
S1pr1
Asf1b
Birc5
Traf4
Trpc2
Ampd3
Satb1
1300018J18Rik
Itgb3
1700097N02Rik
C1galt1
Lmnb1
D030028A08Rik
Kremen1
Pole
A930005H10Rik
Itih5
Acss2
Rasgrp2
Mrs2
2810417H13Rik
Dusp2
Trmt10a
Sbk1
Cmya5
Stmn1
Glud1
Trat1
Cdc45
Mpi
Ecm1
Top2a
Stt3b
Prkd3
Smad4
Slc17a9
Lef1
Kbtbd11
Irf6
Gm11346
Fam49b
St8sia6
Tmem64
Pnrc1
Bcl9l
Aggf1
Mef2d
Bcl2
Rasa3
Pdlim1
Ybx1
Pisd−ps3
Klhdc5
Nfatc1
Pglyrp1
Zfp36l1
Nt5e
Rnf216
Prr13
Cd86
Pvt1
Cers4
Trpm4
Dnajc16
Ptpn11
Abi2
Ermp1
Gbp10
Tmem154
Galm
Cyp4v3
Ehd1
Gdpd5
Ikzf2
Il10ra
Dennd5a
Oas1a
Sh2d1a
Id2
Bcl2a1b
Myo1e
Sec14l1
Pop4
Wdfy1
H2−Q8
Itgb1
Man2a2
Gabarapl1
Tmem181c−ps
Reps1
Rilpl2
Cd101
Mlh1
Tbc1d4
Rogdi
Twsg1
AW112010
Acvr2a
Ahi1
Fam110a
Ahnak
Entpd1
Grn
Nrn1
Fam129a
Crmp1
Marcksl1
Slc45a4
Gpr155
Tiam1
Ptger2
Sdcbp2
Dst
Ptprv
Tmem38b
Dusp1
F2r
Furin
Gpr68
Ptpn13
Lclat1
Gprin3
Gadd45b
Gm3002
Fam109b
Ccdc92
Pear1
Efha2
Glrx
Itgb8
Creb3l2
H1f0
Tnfrsf1b
Ctla4
Pros1
E130308A19Rik
Casp1
Eea1
Tjp3
Matk
S100a11
Cyfip1
Icos
Atp9a
Naip2
Gpm6b
Stx11
Rora
Ncmap
Plscr1
Mmd
Cst7
Gm7609
Zcchc18
Angptl2
Alox8
Abhd4
Nav2
Irf5
I830012O16Rik
Osbpl3
Abcb1a
Setbp1
Dsel
Bmp7
Nr1d1
2900026A02Rik
Axl
Tmem140
Serpina3g
Chic1
Cd83
Ccr6
Adam8
Epcam
Cass4
Csprs
Cxcr6
Ebi3
Prdm1
Lrrfip2
Csf1
Pon3
Klf12
Amph
Spock2
Mx1
4932438H23Rik
Nanos1
Sdc4
Bcl2l2
Naip5
Tmem205
Cuedc1
Ccr2
Coro2b
Gfra4
Rsad2
Lag3
Rnase4
Gpr15
Ces2c
Hmga2
Pla2g2d
Tceal3
Npdc1
Naip6
Fnbp1l
Kcnk6
Plekhg3
Asb2
Megf8
Baiap3
Lingo4
Serpina3i
Krt18
Srxn1
Aebp1
Tigit
Mmp25
Lyl1
Adam33
Gm16894
Tmem243
Itgae
Tnip3
Hspb1
Il1r1
Itgb5
Efnb1
2210408F21Rik
Lilrb4
Ttn
Clip3
Itga1
Gm3020
Abcc3
Rhod
Ccl1
Ltb4r1
Sccpdh
Fgl2
Slc7a10
Col16a1
Hpn
Myo1d
E330021D16Rik
Matn2
Il23r
Enpp2
Cybb
Gm12169
Cnrip1
Mmp9
Ccr1
Gm3500
Plekhd1
Ces2d−ps
Miat
Klrg1
Areg
Sostdc1
Nr3c2
Havcr2
Nckap5
Ky
Gm5512
Noxa1
Amd2
Il10

C1+/+ C1f/+ C1f/f

relative

b eTreg (689 eTreg signature genes)

row min row max
Asb2
Amd2
Ces2c
Il10
Gzmb
Ptpn5
Arnt2
Ltb4r1
Ccr2
Havcr2
Lilrb4
Il23r
S100a4
5430421N21Rik
Il1r2
Tox2
Ccr5
Cxcr3
5830411N06Rik
Fam20a
Rorc
Ttc39c
Rapsn
Rin2
Tigit
Atp1a3
Cpne7
Mmp9
Gm12169
Serpina3f
Lag3
Adam12
Ebi3
Slc7a10
Nckap5
Mmp25
Serpina9
Sccpdh
Ccl20
Atp6v0d2
Hic1
Atp2b4
Hpn
Gp49a
Il1r1
Spock2
Il9r
Gcnt1
C030034L19Rik
Miat
Noxa1
Sostdc1
Il17re
Il21
Klrg1
Baiap3
Cpd
Ccr1
Plekhd1
Abcc3
Cysltr1
Ccr10
Cxcl10
S100a6
Gpm6b
Gstt3
Plcb4
Coro2b
Wnt10a
Naip6
Stom
Nav2
Fgl2
Angptl2
Serpina3g
Cxcr6
Fhdc1
Cacna1s
Ttn
Smtn
Chst2
Itgae
Tjp2
Axl
Rnase4
Vdr
Tnfrsf8
Dnahc7a
Klk1b27
Ky
Clip3
Cited4
Naprt1
Cela1
Maf
Ppap2a
Ankrd6
Anxa1
Enpp2
Lingo4
Ctsf
Plekhf1
Srxn1
Rab39b
Lgals3
Dkkl1
Cpe
Ttbk1
Col16a1
Gpr15
Ankrd29
Cybb
Ahr
Adam8
Igsf9b
Tmbim1
Ffar2
Matn2
Mpp2
1500009L16Rik
Serpinb1a
Nek2
Sema6d
Myo1f
Sntg2
Itga1
Cacna1d
Itgb5
Hdac9
Crmp1
Mnda
Eml6
Asns
Gbp1
Epcam
Cdc25c
Ccl1
Bcl2l15
Il18rap
Trpm6
Hip1
Slc2a6
Amph
Nfil3
Stxbp1
Ccr4
Plod2
Penk
Gpr25
Tnfsf14
Gna14
Dusp14
Podnl1
P2ry1
Selm
Nr3c2
Tnfsf13b
Osbpl3
Ang
Emp1
2010107G23Rik
Cntln
Wdr69
Tbx21
Itga2
Maged1
Tmem205
Adap1
Zcchc18
Zfp704
Kcnk6
4930558J18Rik
Fbn2
Plscr4
Ocln
Ndrg4
Glrx
Fam69b
Gal
Stfa3
Pik3ap1
Lima1
Bicd1
Bmp7
Ces2d−ps
Dnase1l3
Actg2
Ccr6
Myo3b
Npas4
Nr1d1
Repin1
Tg
Tmem2
Cst7
Fcrl5
Ctnnbip1
AI414108
6330403K07Rik
Dhrs3
Ptprv
H1f0
Tspan17
1700009P17Rik
Pon3
D930020B18Rik
Ccr3
Icos
Plxnb2
Npdc1
Rgs8
Gfra4
Prdm1
Wee1
Eea1
Ptpn3
Abhd4
Serpinc1
Stx11
March3
Fbxl21
St14
Dio3os
Tnfsf11
Bloc1s4
Naip5
Egln3
Timp2
Pdzk1ip1
Grhl1
Tshz2
Ahnak
S100a11
Atcay
Wdr35
Cav2
Itgb1
Fam160a1
Nucb2
Plscr1
Pdcd1
Bcl2a1d
Fam132a
Cxcr5
Arrdc4
Id2
Bhlhe40
Klf12
Lgalsl
Il17rb
Gpr68
Matk
Paqr4
Slc39a4
Csf1
Mmd
Casp4
Il1rl1
2810417H13Rik
Cd83
Dtl
Cyfip1
Setbp1
Mocos
Ccdc28b
Ccnb1
Itgb8
Furin
Gdpd5
Runx2
Ccna2
Sspo
Fgd6
Fam129a
Csprs
Nkg7
Il12rb1
Fut7
Dcxr
Cass4
Kif5c
Ccr8
2900026A02Rik
Rora
E130308A19Rik
Mki67
Anxa2
Ckb
Rgs16
Pmaip1
Racgap1
Scpep1
Gemin8
Twsg1
Rab11fip4
Cd38
Abcb1a
Pde4a
Irf5
Phactr2
Anxa4
Fbxw8
Rilpl2
Marcksl1
Gna15
Pear1
Naip2
Il18r1
Nrn1
Ptprs
Synpo
Nsmf
Slc43a1
Grn
Acot11
Gprin3
Tnfrsf1b
Ehd1
Fcrl1
Fignl1
Cd44
Gadd45b
Chn2
Cldn25
Pdcd1lg2
Ncmap
Nt5e
Cdk1
Ccdc92
Rnf43
F2rl2
Gpr155
Rrm2
Gucy1a3
Lclat1
Itfg3
Tk1
1110008F13Rik
Tspan3
Tbc1d4
AW112010
Ppfibp1
Tmem154
Cxx1c
Ctla4
Rxra
Sdcbp2
Acot7
Bcl2a1b
Prg4
Sdc4
Irf4
Inppl1
Lpar6
Kif15
Batf
Tiam1
Alox8
Ncapg2
Acvr2a
Mrps30
Ptger2
Atp9a
Casp3
Itgav
Adam19
F2r
Fam110a
Capg
Fuca2
Kif11
Lgals1
Acsbg1
Ptprj
Atf6
Hif1a
Malt1
Fam92a
Pard6g
Lztfl1
Sh3bgrl
Gns
Sh2d1a
Whsc1
Polr2m
Galm
Tpx2
Casp1
Slc4a8
Susd3
Atxn1
Kcnk5
Cdca8
Endod1
Abi2
Mapre3
Anp32b
Akr1e1
Prr13
Lrp10
Jazf1
Slc9a7
Ctsb
Tysnd1
Evi2a
Serpinb6a
Cks2
Efha1
Aqp3
Kcna3
Lmnb1
1810026J23Rik
Zfp808
Ubl3
Ctla2a
Slc27a1
Gpld1
Trps1
Neb
Gm3002
Cd82
Dst
Trerf1
Hivep3
Srgn
Nfatc1
Syt11
Gnaq
Rnf10
Uap1l1
Lipo1
Mrgpre
Il10ra
Plekhb2
Mrs2
Smpdl3a
Gabarapl1
Pros1
Uhrf1
Slc29a1
S1pr2
Hip1r
Xpa
Syne3
Ptpn13
Adam15
Pqlc3
AI504432
Ccdc50
Slc45a4
Lrrfip2
Ppm1m
Efhd2
Wrnip1
Lax1
Ahi1
Gng2
E130309D02Rik
Ptpn22
Top2a
Gsg2
Tpi1
Lpxn
Mdfic
Relb
Neu3
Ubash3b
Pias3
Nek7
Cd101
Vwa5a
Nr1d2
Sec14l1
Cyp4v3
Litaf
Mapre2
Cbx6
Sh2b3
Plbd2
Vmp1
Vps54
Myo1e
Nrp1
Tjp3
Arhgap31
Lpp
Plcxd2
Tex30
Heatr2
Pik3c2b
Agps
Pik3r5
Sidt1
Bcl9l
Gm20324
Sh3pxd2a
Stk38
Dnahc8
Atp1b3
Pydc3
5830415F09Rik
Trmt61a
A430078G23Rik
Mapk11
Snrpd3
Degs2
Afap1
Ppm1j
Ugcg
Rcn1
Fntb
Poc1b
Map3k8
Cdkn2d
Aim1
Atxn7l3
Serpini1
Actn1
Gsto1
D030028A08Rik
Smad4
H2−Ob
Pdlim1
Gemin6
Nedd4l
2900005J15Rik
Gadd45g
Mob3b
Ifngr2
Pydc4
Papd5
Ppp1r13b
Ahcyl2
Slc17a9
Cmya5
Cd55
Hid1
AI480653
Cenpj
Clcf1
Bach2
Klf7
2010003O02Rik
Lsm11
Yod1
Irf6
Txk
C030034I22Rik
Trat1
Rasa3
Gata1
Galnt10
Ets2
Itm2a
Bcl2
Gm11346
Oasl1
Maml3
Tacc2
Als2cl
Mmp11
Spice1
Gramd4
Gbp11
Adrb2
B130006D01Rik
Smim4
Ecm1
Lef1
Igsf23
C1galt1
Tlr12
Pdss1
Sfrp2
Csrnp2
Gsta4
Cyp2d22
Sema4a
Gpd2
Brpf3
Ptgir
Pigw
Zfp652
Itih5
Acss2
A930005H10Rik
Xkrx
B430306N03Rik
Mir17hg
Gfod2
Napepld
Pygm
Spon1
Prkd3
Rasgrp2
Sbk1
Slc25a23
Aven
Zfp345
Syn3
Gm14139
Galnt4
Nudt15
1110032F04Rik
Epb4.1l1
Treml2
Tmem120b
Vipr1
Ms4a4c
Itgb3
Gm14124
Tanc1
Ada
Dnahc17
Sell
Acvrl1
Satb1
Ldhd
Sgk3
Atp1b1
S1pr1
Col11a2
Trpm1
Sorcs2
Pde3b
Ms4a2
Pla2g12a
Card9
Plk2
1700025G04Rik
Klf2
Vmn2r96
Lypd6b
Gpr146
Selp
Ager
Prf1
Olfml3
Gm15708
2510049J12Rik
Gm13546
Myo6
Lrp5
Guca1b
Emid1
Clic3
Atp8b4
2610019F03Rik
Nup210l
Dennd2c
Unc13a
Igfbp4
9230115E21Rik
Ccdc78
Slc3a1
BC024582
Usp2
1110020A21Rik
Spred1
2610307P16Rik
5730422E09Rik
Jup
1700102P08Rik
Atp1a2
Comp
Lhfp
Cnn3
Cd177
Fabp3
Tspan9
Ly6c1
Ovgp1
Plaur
D830046C22Rik
Syk
Prkar1b
Syde1
Ampd1
Glt8d2
Adcy6
Ggt5
Homer3
G0s2
Tcf7l2
Ccdc164
Arhgap29
Ptger3
Dapl1
Dapk1

C1+/+ C1f/+ C1f/f

+
Extended Data Fig. 5 | Bulk RNA sequencing analysis of YFP cTreg differentially expressed (fold change > 2 and Padj < 0.05) between C1+/+
and eTreg cells from LNs of Fcre/+ × C1+/+, C1f/+ and C1f/f mice. and C1f/f mice. b, High-resolution, fully annotated heat map of eTreg
a, Scaled expression in cTreg cells (top) and eTreg cells (bottom) of genes signature genes as shown in Fig. 1j.
 Letter RESEARCH

a Anti-apoptotic Pro-apoptotic

10 * * * 8 * *
(log-transf./norm.)

8 cTreg
6
Expression

6 eTreg
4
4
2
2
0 0
-2 -2
Bcl2
Mcl1

Bcl1a1b
Bcl1a1d
Bcl2l2
Bcl2a1a
Bcl2l1 (Bcl-XL)

Bcap31

Bax

Bid
Bcl2l11 (Bim)

Bad

Pmaip1 (Noxa)

Bmf

Bik
Becn1
Bnip2

Bcl2l13

Bnip1

Bnip3
Bcl2l12
Bak1
Bbc3 (PUMA)

Bcl2l14

Hrk

Bcl2l15
b Anti-apoptotic (cTreg) Pro-apoptotic (cTreg) C1 ++
10 8 C1 f+
(log-transf./norm.)

8 C1 ff
6
Expression

6
4
4
2
2
0 0
-2 -2
Bcl2

Mcl1

Bcl2l1 (Bcl-XL)

Bcl1a1b

Bcl1a1d

Bcl2a1a
Bcl2l2

Bcl2l13

Bid

Bcl2l12

Bcl2l14

Bcl2l15
Bcap31

Bcl2l11 (Bim)

Bad

Bak1

Bmf

Bik
Becn1

Bnip2

Pmaip1

Hrk
Bax

Bnip1

Bnip3

Bbc3 (PUMA)
Anti-apoptotic (eTreg) Pro-apoptotic (eTreg) C1 ++
8 C1 f+
10
(log-transf./norm.)

C1 ff
8 6
Expression

6 4
4
2
2
0
0
-2 -2
Bid
Bcap31

Bad

Bik

Bmf
Becn1

Bnip2

Bax

Bcl2l13

Bnip1

Pmaip1

Bcl2l12

Bnip3

Bcl2l14

Hrk
Bak1

Bcl2l15

Bbc3 (PUMA)
Bcl2l11 (Bim)
Bcl2

Mcl1

Bcl2l1 (Bcl-XL)

Bcl1a1b

Bcl1a1d

Bcl2l2

Bcl2a1a

Extended Data Fig. 6 | Expression of apoptotic regulator genes. Fcre/+ × C1+/+ mice, based on RNA sequencing analyses. b, Comparison of
a, Comparison of normalized, log-transformed mRNA expression levels expression of the same genes in cTreg (top) and eTreg (bottom) cells of the
of anti- and pro-apoptotic Bcl2 family genes in YFP+ cTreg and eTreg from indicated genotypes. *Padj < 0.05.
RESEARCH Letter

a b c d IFNγ TNF
*** **** ****
CD4+ CD8+ **** ****
**** **** *** *** **** **** ****
* ****
75 **** **** 20 *** 60 *** 100
100 **** ****
****
****
% Remaining animals

C1+/+ C1+/+ C1+/+ 80 C1+/+

%CD44hi CD62L–

Treg (% of CD4)
75 C1+/+ x IKK2ca 15 C1+/+ x IKK2ca C1+/+ x IKK2ca

% cytokine+
C1+/+ x IKK2ca 50 40
C1f/f C1f/f C1f/f

% eTreg
C1f/f 60
50 C1f/f x IKK2ca C1f/f x IKK2ca 10 C1f/f x IKK2ca C1f/f x IKK2ca
40
25 20
25 5 20

0 ** 0 0 0 0
0 10 20 30 40
Days after birth
e f p-JunS73 p-Foxo1/3aT24/T32
*** *
3500 ** 800 *
C1+/+ C1f/f C1+/+ baseline
3000
Baseline αCD3/CD28 Baseline αCD3/CD28 C1+/+ αCD3/CD28
600
2500 C1f/f baseline
C1f/f αCD3/CD28

MFI
2000

MFI
400
1500
1000 200
500
0 0
p-JunS73 p-Foxo1/3aT24/T32

Extended Data Fig. 7 | Restoring NF-κB activation in CARMA1-
deficient Treg cells. a, Survival of Fcre × C1+/+ (or C1f/f) × ROSA26-
stopf/f-IKK2ca mice that express one allele of IKK2ca after expression of
Foxp3cre. b–d, Frequency of CD4+ and CD8+ Tconv cells with a CD44high
CD62L– effector memory phenotype in LNs (b), frequency of Treg among
CD4+ T cells and of eTreg cells among total Treg cells in LNs (c), and
effector cytokine expression of Tconv cells after 8-h ex vivo stimulation
on anti-CD3/CD28-coated plates (d) in indicated mice. e, f, Expression
of indicated phospho-proteins by YFP+ Treg cells from Fcre/+ × C1+/+
or × C1f/f mice at baseline and after 30 min. Anti-CD3/CD28 in vitro
stimulation. Grey solid histograms show unstained control cells. Tconv cells
were used as internal controls, and showed no differences (not shown).
*P < 0.05, ***P < 0.001, ****P < 0.0001 (log-rank (Mantel–Cox) test in a;
one-way ANOVA with Tukey post hoc test in b–d, f).
 Letter RESEARCH

a tdLN tdLN TU b tdLN tdLN TU c tdLN tdLN TU d tdLN TU

(total Treg) (eTreg) (total Treg) (eTreg) (total Treg) (eTreg)
**** **** *** ** ** *

#YFP+ eTreg / tdLN (x104)

8 2.0 20 2.0 * * * **** **** *

#YFP+ Treg / tdLN (x105)

** * **** * ** 5 1.2 105 1.5 * ** 1.5 **** 1.5 2.5 4
YFP+ Treg (% of CD4)

8.0 104

#cells / tdLN (x107)

#YFP+ Tregs / TU

Foxp3 MFI (norm.)

#cells / TU (x107)
6 1.5 15 1.5 4 4.0 104 2.0
3
3.0 103 1.0 1.0 1.0
3 1.5
4 1.0 10 1.0 2
2 1.0
1.5 103 0.5 0.5 0.5
2 0.5 5 0.5 1
1 0.5

0 0.0 0 0.0 0 0.0 0.0 0.0 0.0 0.0 0

+

+
f/+

f/+
+
f/+

+
+

f/+

+
f/+

f/+
f/+
+

+
f/f

f/f

f/+

f/+

f/+
f/f

f/f
f/+
f/f
+/

+/

f/f
f/f
+/

f/f

f/f

f/f
+/
+/

+/
+/

f/f
+/

+/

+/
+/
e f Lung
80

MC38 60
400

% IFNγ+
20.6
40

Foxp3

Foxp3
CD45

CD45
FCre/+ x C1+/+
300 FCre/+ x C1f/+
Tumor size (mm3)

20

* 0
200 Thy1 (i.v.) CD4 YFP IFNγ

+
f/+
f/f
+/
* g Skin
80
*
100
60

% IFNγ+
14.7
Foxp3 40

Foxp3
CD45

CD45

0
0 5 10 15 19
Days of TU growth 20

0
Thy1 (i.v.) CD4 YFP IFNγ

+
f/+
f/f
+/
h +/+ f/+ f/f
i C1+/+
C1 C1 C1
FCre/+ x C1f/f C1f/f
1.2 ** 1000
C1+/+/α-IFNγ
+/+ f/+ f/f
C1 C1 C1 C1f/f/α-IFNγ
Foxp3 MFI (norm.)

1.0 –
/ IFNγ
+
0.8 / / IFNγ 750

3
TU size (mm )
0.6
Foxp3

TU implant.
p

0.4
500
0.2
n.d.
IFNγ 0.0
α-IFNγ 250 *

0
0 5 10 15 0 5 10 15
Time (Days) Days of TU growth

j tdLN k TU l m
C1+/+ –> Ifng KO C1+/+ –> Ifng KO
12 3 IFNγ TNF TNF + IFNγ
**** ****
2.0 60 60 60 25
YFP Treg (% of CD4)

YFP Treg (% of CD4)

0.2
Foxp3

TNF
p
CD4

20
YFP (% of CD4)

1.5
+
% cytokine

40 40 40
15
79 6 1.0
0.1 10
YFP YFP IFNγ 20 20 20
C1f/+–> Ifng KO C1f/+–> Ifng KO 0.5
+

5
6 22
0.0 0 0 0 0
0.0
+

+
Foxp3

f/+

f/+

f/+

f/+
+
f/+

+/

+/

+/

+/
+/
TNF
CD4

+
f/+
+/

45 27
YFP YFP IFNγ

Extended Data Fig. 8 | Role of IFNγ secretion by FOXP3int unstable Treg Gates for IFNγ+ cells drawn based on fluorescence-minus-one (FMO)
cells selectively in tumour tissue. a–c, Frequencies of total YFP+ Treg cells controls. h, Normalized FOXP3 expression in IFNγ+ and IFNγ– Treg cells
and of YFP+ eTreg cells among CD4+ T cells (a), absolute YFP+ Treg and from tumour tissue. n.d., not detectable. i, Tumour growth in indicated
eTreg cell numbers (b), and normalized FOXP3 expression in YFP+ Treg mice implanted with D4M.3A melanoma and19 treated with or without
and eTreg (c) in tdLN and tumour tissue of 18-day-old D4M.3A tumours neutralizing anti-IFNγ antibody. j, Frequency of adoptively transferred,
in female heterozygous Fcre/+ × C1+/+, C1 f/+ or C1 f/f mice. d, tdLN and YFP+ Treg cells of indicated genotypes in tdLNs of Ifng−/− hosts at day 18 of
tumour cellularities. e, Growth of MC38 tumours in female heterozygous tumour growth. k–m, Frequency (k, l) and effector cytokine expression (m)
Fcre/+ × C1+/+ or C1 f/+ mice. f, g, Tumour-bearing mice were treated of adoptively transferred, YFP+ Treg cells in tumours in Ifng−/− hosts.
with brefeldin A for 5 h, injected intravenously with 3 μg of anti-THY1.2 *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way ANOVA
monoclonal antibodies, and 3 min later collected for direct ex vivo analysis with Tukey post hoc test in a–d, f, g; two-tailed Student’s t-test in e, h, j, l, m;
of IFNγ expression in extravascular YFP+ Treg cells in the lung (f) and skin (g). two-way ANOVA with Bonferroni post hoc test in i).
 RESEARCH Letter

a tdLN tdLN TU b tdLN tdLN TU

Cre/+ f/f
(total Treg) (eTreg) (total Treg) (eTreg)
F x C1 x IKK2ca
*** **** ****
** *** **** **** ****
**** ***
6 2.0 * * 20 *** 1.5 **** ****
C1+/+ ** * C1+/+
YFP+ Treg (% of CD4) C1+/+ x IKK2ca C1+/+ x IKK2ca

Foxp3 MFI (norm.)

TU implant.

1.5 15
4 C1f/f 1.0
C1f/f
C1f/f x IKK2ca C1f/f x IKK2ca
1.0 10

2 0.5
0.5 5

0 0.0 0 0.0
0
Time (Days)

c d 750
FCre/+ x C1+/+
**** FCre/+ x C1+/+ x IKK2ca
**** FCre/+ x C1f/f
****
****
Tumor size (mm3)

100 *** FCre/+ x C1f/f x IKK2ca

C1+/+ 500
C1+/+ x IKK2ca
80
C1f/f
% IFNγ+ TNF+

60 C1f/f x IKK2ca *&

250 *
40 * &
*& *
*&
20

0 0
0 5 10 15 20
Days of TU growth

f FCreERT2/+ x C1+/+ g
e 1000 FCreERT2/+ x C1f/f
*
FCreERT2 x C1f/f 80 *

tdLN TU 750 FCreERT2/+ x C1+/+ FCreERT2/+ x C1f/f FCreERT2 x C1f/f

% IFNγ+ TNF+
60
FCreERT2 x C1+/+ 12.3 26.4 10.4 61.6 14.9 62.8
TU size (mm3)

0.04 0.015
FCreERT2 x C1f/f
40
Rel. mRNA (2- CT )

TNF

0.03 500 *
0.010
20
0.02 *# *#
250 * *# 54.7 6.5 20.4 7.6 17.4 5.0
0.005 0
0.01 * IFN-γ

e/ /+
Cr x f/f
/f
xf
Cr x +
+
e
n.d.

+
e/
0.00 0.000 0

Cr
0 5 10 15 20
Days of TU growth

h FCre/+ x C1f/f
i C57BL/6 1250 j 600
D4M.3A
Veh. FCre/+ x C1+/+
♂ Mep. FCre/+ x C1+/+ + Mep
Day 21 1000 FCre/+ x C1f/f
*
** FCre/+ x C1f/f + Mep.
Tumor size (mm3)

Tumor size (mm3)

25 ** F Cre/+
x C1 +/+
400
MHC II MFI (x 1,000)

FCre/+ x C1f/+ 750

20
TU implant.

FCre/+ x C1f/f
TU implant.

α-CD8 *
15 500 *##
... 200
** *
10
Mepazine 250
5
...
0 0 0
0 0 9 0 5 10 15 17 0 5 10 15 20
+
f/+
f/f
+/

Time (Days) Time (Days) Days of TU growth Days of TU growth

Extended Data Fig. 9 | Tumour response of CARMA1-deficient Treg days after CARMA1 deletion in half or all Treg cells. h, MHC-II expression
cells after restoration of NF-κB activation. a–d, D4M.3A melanoma on tumour-associated macrophages in D4M.3A-implanted Fcre/+ × C1+/+,
cells were implanted into Fcre/+ × C1+/+ (or C1 f/f) × ROSA26-stopf/f-IKK2ca C1f/+ or C1f/f mice. i, D4M.3A tumour growth in mice treated with
mice to record frequencies of YFP+ Treg and eTreg cells among CD4+ T cells depleting anti-CD8 antibody from day 8 and treated with mepazine or
(a) and their normalized FOXP3 expression (b) in tdLNs and tumour vehicle from day 9. j, D4M.3A tumour growth in Fcre/+ × C1+/+ or C1f/f
tissue, effector cytokine expression by tumour-infiltrating Treg cells (c), mice treated with mepazine or vehicle starting on day 9. Data are mean
and tumour growth (d). e, YFP+ Treg cells were sorted from D4M.3A and individual replicates or s.e.m. In a–c, g–i, P < 0.05, **P < 0.01,
melanoma tissue and tdLNs after five days of treatment of FcreERT2 × C1+/+ ***P < 0.001, ****P < 0.0001. In d, *P < 0.05 versus C1+/+, &P < 0.05
and C1f/f with tamoxifen and analysed for CARMA1 expression by versus C1+/+ + IKK2ca. In f, *P < 0.05 versus FcreERT2 × C1+/+, #P < 0.05
RT–qPCR. f, Tumour growth in female FcreERT2 or FcreERT2/+ × C1+/+ versus FcreERT2/+ × C1f/f. In j, *P < 0.05 versus C1+/+, #P < 0.05 versus
and C1 f/f mice, in which CARMA1 was deleted in all (FcreERT2) or half C1+/+ + mepazine (one-way ANOVA with Tukey post hoc test in a–c,
(FcreERT2/+) of Treg cells. Arrow indicates tamoxifen treatment start. g, h; two-way ANOVA with Bonferroni post hoc test in d, f, j: two-tailed
g, In situ expression in tumour tissue of effector cytokines by YFP+ Treg five Student’s t-test in i).
 Letter RESEARCH

a * **
8h
** ***
24h
***
8 8 3 20 12 4 10 3 100 100

GITR MFI (x10,000)

CTLA4 MFI (x1,000)

Foxp3 MFI (x1,000)

CD25 MFI (x1,000)

CTLA-4 MFI (x100)
Foxp3 MFI (x1,000)

CD25 MFI (x1,000)

8 80 80

GITR MFI (x1,000)

6 6 15 3

% live cells
2 8 2

% eTreg
6 60 60
4 4 10 2
4 40 40
1 4 1
2 2 5 1
2 20 20

0 0 0 0 0 0 0 0 0 0
Mep.: – + – + – + – + – + – + – + – + Mep.: – + – + – + – + – + – +
αCD3/28.: – + – + – + – +

b Ifng Cd274 Socs1 Tap1 Stat1 Irf1 Cxcl10 Nos2

1.5 10-3 * 1.5 x 10
-2
*** 1 x 10-2 * 5 x 10
-2 * 1 x 10
-1
4 x 10
-2 * 8 x 10
-2
* 5 x 10
-3 *
Rel. mRNA (2- CT )

8 x 10-3 4 x 10
-2
-2 -2 4 x 10
-3
3 x 10 6 x 10
-2
1.0 10-3 1.0 x 10
6 x 10-3 3 x 10
-2
3 x 10-3
5 x 10-2 2 x 10-2 4 x 10
-2
-3 -2 -3
-3
4 x 10 2 x 10 2 x 10
5.0 10-4 5.0 x 10 -2
2 x 10-3 -2 1 x 10-2 2 x 10 -3
1 x 10 1 x 10

0.0 0 0 0 0 0 0 0
Mep.: – + – + – + – + – + – + – + – +

c Foxp3 CTLA4 CD25 CD73 GITR

5 10-4 2.0 10-3 5 10-5 1 10-2 1.5 10-3
Rel. mRNA (2- CT )

4 10-4 4 10-5 8 10-3

1.5 10-3
1.0 10-3
3 10 -4
3 10 -5
6 10-3
1.0 10-3
2 10-4 2 10-5 4 10-3
5.0 10-4
5.0 10-4
1 10 -4
1 10 -5
2 10-3

0 0.0 0 0 0.0
Mep.: – + – + – + – + – +

d Neu
4.9
Mo Mac DC DC1 vs DC2
Thy.1/B220/Ly6G

17.0 64.4
Cerulean (TU)

82.5
21.8
CD11b
MHC II
CD64
Ly6G

10.3
14.7

CD45 CD11b Ly6C F4/80 CD11c CD103

CD8 CD4 B NK/NK T NK vs NK T
5.8
6.4 80.0
5.5 19.8
NKp46

NK1.1

7.5
B220
Thy1

Thy1

CD8 CD4 CD19 NK1.1 CD3

e f Mo Mac Neu DC1 DC2 CD8 CD4 B NK NK T g CD8 CD4 h Mac

** * * *
15 8 0.20 1.5 60 60 25
CD45 (% of live cells)

MHC II MFI (x1000)

20
6 0.15
% of live cells

10 1.0 40 40
hi

15
%Ki67

4 0.10
10
5 0.5 20 20
+

2 0.05
5

0 0 0.00 0.0 0 0 0
Mep.: – + Mep.: – + – + – + – + – + – + – + – + – + Mep.: – + – + Mep.: – +

i j MC38
1250
Vehicle
20 Vehicle α-PD-1
* Mepazine
Mepazine 1000
Vehicle Mepazine αPD-1 αPD-1 + Mepazine α-PD-1 + Mep.
Tumor size (mm3)
% IFNγ+ TNF+

15 α-PD-1
0.0 0.9 2.3 9.1 1.2 3.9 0.6 3.8
α-PD-1 + Mep. 750
10
TNF

500
5 (0/6)
97.9 1.2 85.4 3.2 92.8 2.1 93.9 1.8 0 250 *&
IFN-γ *
* *& *#& *#& (4/6)
0
0 5 10 15 20 25
Days of TU growth

Extended Data Fig. 10 | See next page for caption.

RESEARCH Letter
Extended Data Fig. 10 | Mepazine effects on the tumour
microenvironment. a, YFP+ Treg cells were sorted from Fcre × C1+/+ mice
and treated with 10 μM mepazine or vehicle for 8 or 24 h with or without
concurrent anti-CD3/28 monoclonal antibody TCR stimulation (8-h time
point only). Expression of FOXP3, markers of eTreg cell differentiation,
cell viability, and frequency of eTreg cells were recorded. b, c, RT–qPCR
analysis of expression of Ifng and genes of adaptive immune resistance
(the PD-L1 genes Cd274 and Socs1), antigen presentation (Tap1), IFNγ
signalling (Stat1 and Irf1), T-cell recruitment (Cxcl10), M1 macrophage-
activation (Nos2) (b) and of Foxp3 and various Treg-cell-associated genes (c)
in whole tumour tissue lysate after three days of treatment with mepazine
or vehicle control. d–h, Composition of the tumour tissue immune
infiltrate and frequencies of CD45+ cells (e) and of various immune cell
subsets (f) as well Ki67 expression by Tconv cells (g) and MHC-II expression
by macrophages (h) after three days of treatment with mepazine or vehicle
control. i, Effector cytokine co-expression by tumour-infiltrating Treg cells
after 12 days of treatment with mepazine and anti-PD-1 antibody.
j, Synergistic tumour control of MC38 colon carcinoma through
anti-PD-1 and mepazine combination treatment in female C57BL/6 hosts.
Numbers in parentheses indicate fraction of mice without relapse for more
than 12 months after discontinuation of treatment. In j, *P < 0.05 versus
vehicle, #P < 0.05 versus anti-PD-1, and &P < 0.05 versus mepazine. In
all other panels, *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student’s
t-test in a–c, e–h; one-way ANOVA with Tukey post hoc test in i; two-way
ANOVA with Bonferroni post hoc test in j).
 nature research | reporting summary
Corresponding author(s): Thorsten R. Mempel, Mauro Di Pilato
Last updated by author(s): Apr 1, 2019

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Data exclusions 2 samples were excluded from RNA sequencing analysis due to contamination of the sorted cell population with irrelevant immune cells, as
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Antibodies
Antibodies used Antibody-Dye (Clone), Catalog-Number, Lot-Number, Supplier;
Anti-GFP-AlexaFluor488 (rabbit polyclonal), A21311, 1891008, Invitrogen;
Anti-Human/Mouse CARD11/CARMA1 (1D12), 04/2017, 5, Cell Signaling Technologies;
Anti-Human/Mouse/Rat Bim-PE (C34C5), 11/2017, 7, Cell Signaling Technologies;
Anti-Human/Mouse Phospho-c-JUN (Ser73) (D47G9) 11/2018, 3, Cell Signaling Technologies;
Anti-Mouse Phospho-FoxO1 (Thr24)/FoxO3a (Thr32), 11/2018, 7, Cell Signaling Technologies;
Anti-Mouse CD11b-PE/Cy7 (M1/70), 101216, B203625, BioLegend;
Anti-Mouse CD11b-FITC, (M1/70), 101215, B246149, BioLegend;
Anti-Mouse CD120b-Biotin (TR75-89), 113403, B200975, BioLegend;
Anti-Mouse CD152-APC (UC10-4B9), 106310, B230773, BioLegend;
Anti-Mouse CD152-BrilliantViolet605 (UC10-4B9), 106323, B233636, BioLegend;
Anti-Mouse CD274-PE (10F.9G2), 124307, B223286, BioLegend;
October 2018

Anti-Mouse CD279 (29F.1A12), BE0273, 667617A2, BioXCell;

Anti-Mouse CD357-APC (DTA-1), 126312, B218262, BioLegend;
Anti-Mouse CD4-APC/Cy7 (GK1.5), 100414, B237980, BioLegend;
Anti-Mouse CD45-AlexaFluor700 (30-F11), 103128, B241345, BioLegend;
Anti-Mouse CD45- PerCP/Cy5.5 (30-F11), 103132, B249564, BioLegend
Anti-Mouse CD62L-BrilliantViolet421 (MEL-14), 104435, B238233, BioLegend;
Anti-Mouse CD62L-BrilliantViolet510 (MEL-14), 104441, B213054, BioLegend;
Anti-Mouse CD62L-PE/Cy7 (MEL-14), 104418, B209901, BioLegend;
Anti-Mouse CD73-AlexaFluor647 (TY/11.8), 127208, B228746, BioLegend;

2

Validation
Eukaryotic cell lines
Policy information about cell lines
Cell line source(s)
Authentication
Mycoplasma contamination
Commonly misidentified lines
(See ICLAC register)
Anti-Mouse CD8a-BrilliantViolet510 (53-6.7), 100751, B237067, BioLegend;
Anti-Mouse CD8a-PerCP/Cy5.5 (53-6.7), 100733, B249621, BioLegend
nature research | reporting summary
Anti-Mouse CD90.2-AlexaFluor700 (30-H12), 105320, B207170, BioLegend;
Anti-Mouse CD90.2-BrilliantViolet421 (30-H12), 105341, B240026, BioLegend;
Anti-Mouse CD90.2-PE/Cy7 (30-H12), 105325, B244113, BioLegend;
Anti-Mouse CD90.2-APC (30-H12), 105311, B244113, BioLegend;
Anti-Mouse CD90.2-Pacific Blue (53-2.1), 104306, B245912, BioLegend;
Anti-Mouse F4/80-APC (BM8), 123116, B241397, BioLegend;
Anti-Mouse F4/80-BrilliantViolet510 (BM8), 123135, B254669, Biolegned;
Anti-Mouse H-2Kb-PerCP/Cy5.5 (AF6-88.5), 116516, B188334, BioLegend;
Anti-Mouse I-A/I-E-BrilliantViolet605 (M5/114.15.2), 107639, B219482, BioLegend;
Anti-Mouse IFN-gamma (XMG1.2), BE0055, 630017J1, BioXCell;
Anti-Mouse IFN-gamma-AlexaFluor700 (XMG1.2), 505824, B224155, BioLegend;
Anti-Mouse IFN-gamma-PE (XMG1.2), 505808, B213720, BioLegend;
Anti-Mouse IFN-gamma-PE/Cy7 (XMG1.2), 505826, B217032, BioLegend;
Anti-Mouse IL-17A-PE/Cy7 (TC11-18H10.1), 506921, B236124, BioLegend;
Anti-Mouse IL-4-PerCP/Cy5.5 (11B11), 504123, B204723, BioLegend;
Anti-Mouse Ki67-BrilliantViolet605 (16A8), 652413, B223900, BioLegend;
Anti-Mouse Ki67-FITC (B56), 556026, 6312779, BD Biosciences;
Anti-Mouse Ly-6C-PerCP/Cy5.5 (HK1.4), 128012, B229409, BioLegend;
Anti-Mouse Ly-6C-APC/Cy7 (HK1.4), 128025, B2478350, Biolegend
Anti-Mouse TNF-AlexaFluor647 (MP6-XT22), 506314, B232130, BioLegend;
Anti-Mouse TNF-BrilliantViolet605 (MP6-XT22), 506329, B238894, BioLegend;
Anti-Mouse TruStain fcX (93), 101320, B234518, BioLegend;
Anti-Mouse/Human CD44-AlexaFluor700 (IM7), 103026, B244378, BioLegend;
Anti-Mouse/Human CD44-BrilliantViolet605 (IM7), 103047, B236770, BioLegend;
Anti-Mouse/Human CD44-PerCP/Cy5.5 (IM7), 103032, B213817, BioLegend;
Anti-Mouse/Rat Foxp3-AlexaFluor700 (FJK-16s), 56-5773-82, 4307313, eBioscience;
Anti-Mouse/Rat FoxP3-PE (FJK-16s), 12-5773-82, 4307350, eBioscience;
Anti-Mouse/Rat FoxP3-PE-Cy7 (FJK-16s), 25-5773-80, 4307350, eBioscience;
Anti-Rabbit Ig (H+L) goat-AlexaFluor488 (Polyclonal), A11008, 1583138, Life Technologies;
Anti-Rabbit Ig (H+L) goat-AlexaFluor405 (Polyclonal), A31556, 1984053, Life Technologies;
Rat IgG2a isotype control (2A3), BE0089, 654817O1, BioXCell;
Streptavidin-AlexaFluor700, S21383, 1800816, Invitrogen;
Streptavidin-PE/Cy7, 405206, B207311, BioLegend;
Zombie Red Fixable Viability Kit, 423110, 223441, BioLegend.
Anti-Mouse Tbet-BrilliantViolet 421 (4B10), 644815, B210885, BioLegend;
Anti-Mouse GATA3-PE-CF594 (L50-823), 563510, 7202530, BD Biosciences;
Anti-Human/Mouse ROR gamma (t)-PE (AFKJS-9), 12-6988-80, 4330086, Invitrogen;
Anti-mouse Ly-6G-APC (1A8), 127613, B259669, Biolegend;
Anti-mouse/human CD45R/B220-APC (RA3-6B2), 103212, B172317, BioLegend;
Anti-mouse Bcl-2 (BCL/10C4), 633510, B253631, Biolegend;
Anti-mouse CD64 (FcγRI)-PE (X54-5/7.1), 139303, B245515, Biolegend;
Anti-mouse CD11c-FITC (N418), 117305, B244373, BioLegend;
Anti-Mouse CD11c-PE/Cy7 (HL3), 561022, 7223543, eBioscience;
Anti-mouse CD103-PerCP/Cy5.5 (2E7), 121416, B252750, BioLegend;
Anti-mouse NK-1.1-PE/Cy7 (PK136), 108713, B249073, BioLegend;
Anti-mouse CD335 (NKp46)-APC (29A1.4), 137607, B252577, BioLegend;
Anti-mouse CD3-PerCP/Cy5.5 (17A2), 100217, B260626, BioLegend;
Anti-mouse CD25-PE-Cy7 (PC61.5), 25-0251-81, 4289561, eBioscience;
Anti-mouse CD19-PerCP/Cy5.5 (1D3/CD19) 152405, B255188 Biolegend;
Anti-mouse CD45RB-PE (16A), 553101, 7348606, BD Biosciences;
Anti-Mouse IgG (H+L)-Alexa Fluor647, A-21235,1764240, Thermo Fisher.
All antibodies are commercially available and validated by previous studies done by others or our laboratory.
The BRAF^V600E x PTEN^null melanoma cell line, D4M.3A, was provided by David E. Fischer.
D4M.3A-H2B-Cerulean and D4M.3A-SIINFEKL-H2B-Cerulean were generated by the Mempel Laboratory through lentiviral
transduction.
The colon adenocarcinoma cell line, MC38, was provided by Andrew D. Luster.
October 2018
D4M.3A cells were exome sequenced and determined to be of mouse origin.
All cell lines were regularly tested for mycoplasma contamination.
No cell line used is listed in the database of commonly misidentified cell lines maintained by ICLAC.
3
Animals and other organisms

nature research | reporting summary

Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research
Laboratory animals 6 to 8 weeks old Foxp3^YFP-Cre, Foxp3^GFP-Cre-ERT2, Rosa26-STOP f/f-YFP, Rosa26-STOP f/f-IKK2ca, Ifng KO and C57BL/6J mice
were purchased from Jackson Laboratories.
Ramnik J. Xavier and James J. Moon (MGH) provided CARMA1^f/f and Rag1 KO mice, respectively.
CARMA1^f/f mice were bred with Rosa26-STOP f/f-YFP, Rosa26-STOP f/f-IKK2ca and either Foxp3^YFP-Cre, Foxp3^GFP-Cre-ERT2,
Rosa26^YFP.

Wild animals No wild animals were employed in the study.

Field-collected samples No field-collected samples were used in this study.

Ethics oversight All mouse experiments were done with the approval of Institutional Animal Care and Use Committee.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots
Confirm that:
The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Heparinized peripheral blood collected through sub-mandibular vein puncture was treated with ACK red blood cell lysis buffer.
LNs and spleens were passed through 40 um cell strainers, followed by red blood cell lysis (spleens only). Tumors and lungs were
minced into small fragments and treated with 1.5 mg/ml collagenase IV and 50U/ml DNAse I for 30 min. at 37oC under agitation.
Skin tissue was digested in medium containing 2% FCS, 10mM Hepes, 0.5 mg/ml hyaluronidase, 1.5 mg/ml collagenase IV, and 50
U/ml DNAse I for 45 min. at 37oC under agitation. Residual tissue fragments were mechanically dissociated.
Cell surface proteins were stained for 20 minutes at 4oC.
Intracellular and nuclear proteins were stained for 60 minutes at room temperature after permeabilization and fixation (Mouse
regulatory T cell staining Kit; eBioscience).
Preceding antibody staining, dead cells where stained using the fixable viability violet dye Zombie Red (Biolegend) for 15 minutes
at room temperature, followed by blocking of Fc receptors with TruStain fcX (Biolegend) for 20 minutes at 4oC.

Instrument Cells were analyzed on LSR II, LSRFortessa or LSRFortessa X-20 flow cytometers (BD Biosciences).

Software BD FACSDiva was used to acquire the data. Data were analyzed with FlowJo software version 9.9.5 and 10.5.3.

Cell population abundance
10,000 CD4-pos. GFP-pos. cells from either lymph nodes or tumors of Foxp3^CreERT2 x CARMA1 +/+ or fl/fl mice were purified
to >99% purity for RT-PCR analysis.
100,000 CD4-pos. YFP-pos. cells from lymph nodes of Foxp3^Cre/+ x CARMA1 +/+, or fl/fl x R26^YFP mice were purified to >99%
purity for the in vitro apoptosis assay.
10,000 CD4-pos. YFP-pos. cells from lymph nodes of Foxp3^Cre/+ x CARMA1 +/+, fl/+, or fl/fl x R26^YFP mice were purified to
>99% purity for Foxp3 staining.
5,000 CD4-pos. YFP-pos. CD44-low CD62L-pos cTreg/animal and the same number of CD4-pos. YFP-pos. CD44-high CD62-Lneg
eTreg/animal from lymph nodes of Foxp3^Cre/+ x CARMA1 +/+, fl/+, or fl/fl x R26^YFP mice were purified to >99% purity for
RNA-sequencing studies.
6,000,000 CD4-pos. CD45Rb-high YFP-neg. cells from lymph nodes of Foxp3^Cre/Cre x CARMA1 +/+ and 600,000 CD4-pos. YFP-
pos. cells from lymph nodes of of Foxp3^Cre/+ x CARMA1 +/+, fl/+, or fl/fl x R26^YFP mice were purified to >98% purity for in
vivo suppression study.
2,000,000 CD4-pos. YFP-neg. cells cells from lymph nodes of Foxp3^Cre/Cre x CARMA1 +/+ and 200,000 CD4-pos. YFP-pos. cells
cells from lymph nodes of of Foxp3^Cre/+ x CARMA1 +/+, fl/+, or fl/fl x R26^YFP mice were purified to >98% purity for in vitro
suppression study.
October 2018

CD4-pos. YFP-pos. cells from lymph nodes of of Foxp3^Cre/+ x CARMA1 +/+, fl/+ x R26^YFP mice were purified to >95% purity
through magnetic-activated cell sorting (Miltenyi) purity and 1,000,000 cells/mouse injected
for adoptive Treg cell transfer studies.

Gating strategy Lymph node Treg analysis was gated on Zombie Red-negative, singlets, CD4-pos. CD8-neg./Foxp3-pos. events.
Tumor Treg analysis was gated on Zombie Red-neg., CD45-pos. CD90-pos., singlets, CD4-pos. CD8-neg./Foxp3-pos. events.
Tumor macrophage analysis was gated on Zombie Red-neg., CD45-pos., singlets, F4/80-pos. CD11b-pos. events.
Tumor cell analysis was gated on Zombie Red-neg., CD45-neg., singlets, Cerulean-pos. events.

4
 Splenic myeloid compartment was gated on CD11b-pos. and proportions of Ly6G-pos. neutrophils, CD11c-pos. MHC II-high DCs,
Ly6C-high monocytes, Lyc6G-low SSC-high eosinophils, and Ly6C-low SSC-low macrophages were defined.

nature research | reporting summary

Tumor tissue immune infiltrates were gated on CD45pos. cells and proportion of CD90-pos. CD8-pos T cells, CD90-pos. CD4-pos
T cells,
B220-pos. CD19-pos. B cells, Nkp46-pos. NK1.1-pos CD3-pos./neg. NK/NKT cells, Ly6G-pos. CD11b-pos. Neutrophils, CD90-neg
B220-neg Ly6G-neg Ly6C-pos Monocytes and Ly6C-neg CD64-pos. F4/80 pos. Macrophages, and CD64-neg. F4/80 neg. MHC II
high CD11c pos. Dendritic cells were defined.
For analysis of skin and lung Tregs, we excluded cells stained in vivo by i.v.-injected Thy1 Ab, then gated on CD45+ CD4+, Foxp3-
YFP+ cells.

Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.

October 2018