The tumor necrosis factor alpha-induced protein 3(TNFAIP3, A20) imposes a brake on antitumoractivity of CD8 T cellsMarilyn Giordanoa,b,c, Romain Roncagallia,b,c, Pierre Bourdelya,b,c, Lionel Chassona,b,c, Michel Bufernea,b,c,Sho Yamasakid, Rudi Beyaerte,f, Geert van Looe,f, Nathalie Auphan-Anezina,b,c, Anne-Marie Schmitt-Verhulsta,b,c,and Grégory Verdeila,b,c,1

aCentre d’Immunologie de Marseille–Luminy, Aix–Marseille University (UM 2), Case 906, 13288 Marseille Cedex 9, France; bInstitut National de la Santé et de laRecherche Médicale, U1104, 13288 Marseille, France; cCentre National de la Recherche Scientifique, Unité Mixte de Recherche 7280, 13288 Marseille, France;dDivision of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan; eDepartment of Biomedical MolecularBiology, Ghent University, B-9052 Ghent, Belgium; and fInflammation Research Center, Vlaams Instituut voor Biotechnologie, B-9052 Ghent, Belgium

Edited by Michael J. Bevan, University of Washington, Seattle, WA, and approved June 24, 2014 (received for review April 6, 2014)

The transcription factor NF-κB is central to inflammatory signalingand activation of innate and adaptive immune responses. Activa-tion of the NF-κB pathway is tightly controlled by several negativefeedback mechanisms, including A20, an ubiquitin-modifying en-zyme encoded by the tnfaip3 gene. Mice with selective deletion ofA20 in myeloid, dendritic, or B cells recapitulate some human in-flammatory pathology. As we observed high expression of A20transcripts in dysfunctional CD8 T cells in an autochthonous mela-noma, we analyzed the role of A20 in regulation of CD8 T-cellfunctions, using mice in which A20 was selectively deleted in ma-ture conventional T cells. These mice developed lymphadenopathyand some organ infiltration by T cells but no splenomegaly and nodetectable pathology. A20-deleted CD8 T cells had increased sen-sitivity to antigen stimulation with production of large amounts ofIL-2 and IFNγ, correlated with sustained nuclear expression ofNF-κB components reticuloendotheliosis oncogene c-Rel and p65.Overexpression of A20 by retroviral transduction of CD8 T cellsdampened their intratumor accumulation and antitumor activity.In contrast, relief from the A20 brake in NF-κB activation in adop-tively transferred antitumor CD8 T cells led to improved control ofmelanoma growth. Tumor-infiltrating A20-deleted CD8 T cellshad enhanced production of IFNγ and TNFα and reduced ex-pression of the inhibitory receptor programmed cell death 1.As manipulation of A20 expression in CD8 T cells did not resultin pathologic manifestations in the mice, we propose it as a can-didate to be targeted to increase antitumor efficiency of adop-tive T-cell immunotherapy.

T-cell activation | tumor immunity | inflammation

Mechanisms controlling immune reactivity prevent excessiveinflammation and autoimmunity, but generally dampen

antitumor activity (1, 2). It is thus important to understand theconsequences of release from immune control mechanisms interms of increase in antitumor efficacy on the one hand and withrespect to the possibility of development of autoimmune pa-thologies on the other hand. The transcription factor NF-κB iscentral to inflammatory signaling, as well as to activation of in-nate and adaptive immune functions. Activation of the NF-κBpathway is regulated by ubiquitination and is tightly controlled byseveral feedback mechanisms (3). A20, an ubiquitin-modifyingenzyme encoded by the tnfaip3 gene, is one of the major inhib-itors of the canonical NF-κB signaling pathway (4). Genome-wide association studies (GWAS) have linked germ-line singlenucleotide polymorphisms of the TNFAIP3 gene with suscepti-bility to multiple human pathologies, including systemic lupuserythematosus (SLE) and psoriasis (5). For the latter autoim-mune diseases, causal mutations have been characterized thatcontrol either the level of expression or the function of A20.

When A20 is ubiquitously knocked out, mice are viable but de-velop severe multiorgan inflammation leading to premature death(6). Using mouse models expressing the recombinase Cre in specificcell types crossed to A20 flox/flox (A20fl/fl) mice, A20 deficiencyhas been well studied in B cells, myeloid cells, and dendritic cells(DCs) (7–12). With each cell type, specific deletion of A20 led tothe development of various degrees of autoimmune signs. SpecificA20 deletion in B cells led to the progressive development ofa SLE-type pathology (7, 9, 12), whereas mice with A20 deletion incells of myeloid origin developed spontaneous polyarthritis with theproduction of type II collagen autoantibodies. Mice with DC-spe-cific A20 deletion developed either features of SLE (10) or featuresof human inflammatory bowel disease (IBD) in independent studies(8). In both cases the lack of A20 in DCs induced aberrant acti-vation and proliferation of T cells. To our knowledge, no study ofA20 deficiency in primary T cells has been conducted, althoughthe involvement of A20 in T-cell receptor (TCR)-mediatedsignaling in cultured cells has been reported (13, 14).We observed a sustained high level expression of A20 tran-

scripts in dysfunctional CD8 T cells isolated from a progressingautochthonous melanoma in mice. This provided a strong in-centive to analyze the consequences of A20 deletion in mature

Significance

Mechanisms controlling immune reactivity prevent excessiveinflammation and autoimmunity, but generally dampen anti-tumor activity. The tumor necrosis factor alpha-induced protein3 gene encoding the A20 protein, a key molecule controllingNF-κB activation, has been linked to the development of mul-tiple inflammatory pathologies in humans, some of which arerecapitulated in mice with selective deletion of A20 in myeloid,dendritic, or B cells. Here, mice with selective deletion of A20 inmature conventional T cells presented no detectable pathol-ogy. CD8 T cells from these mice showed increased antigensensitivity with enhanced production of IL-2 and IFNγ. Impor-tantly, A20-deleted CD8 T cells possessed heightened antitumoractivity in vivo. Targeting this gene in adoptively transferredCD8 T cells could represent a promising mechanism to achievetumor rejection.

Author contributions: R.R., N.A.-A., A.-M.S.-V., and G.V. designed research; M.G., R.R., P.B.,L.C., M.B., and G.V. performed research; S.Y., R.B., and G.v.L. contributed new reagents/analytic tools; M.G., R.R., P.B., A.-M.S.-V., and G.V. analyzed data; and N.A.-A., A.-M.S.-V.,and G.V. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: verdeil@ciml.univ-mrs.fr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1406259111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1406259111 PNAS | July 29, 2014 | vol. 111 | no. 30 | 11115–11120

IMMUNOLO

GYAND

INFLAMMATION

Dow

nloa

ded

by g

uest

on

Dec

embe

r 25

, 201

9

CD8 T cells on their differentiation in basal conditions and theircapacity to develop antitumor activity.

ResultsCharacteristics of Mice with Specific Deletion of A20 in Peripheral TCells. To achieve A20 deletion in primary T cells we crossedA20fl/fl mice (15) with mature T-Cre (maT-Cre) mice that spe-cifically express the Cre recombinase in 80% of the peripheralCD8 T cells, around 50% of peripheral CD4 T cells, and 25% ofregulatory T cells (maT-A20, Fig. S1). Recombination was lim-ited in thymocytes (Fig. S1 A and C) as well as in B cells (<1%),natural killer (NK) cells (<6%), CD11b+ myeloid cells (<3%),and CD11b+ CD11c+ DCs (<4%) (Fig. S1 B and D). These mice

developed lymphadenopathy as early as 6 wk after birth with anincrease in CD4 and CD8 T cells as well as B220+ cells in lymphnodes (LNs) (Fig. 1A). LN CD8 and CD4 T cells retained a naïvephenotype (Fig. 1D). Total splenocyte numbers were similar butsplenic CD4 and CD8 T-cell numbers were decreased in maT-A20 mice compared with littermates (Fig. 1B). Interestingly 35%of the remaining splenic T cells showed an effector-like pheno-type (Fig. 1D). In the liver, we observed an increased infiltrationof CD45+ cells, with no significant variation of major immunepopulations except an increase in the proportion of CD8 T cells(Fig. 1C). Most of these CD8 T cells had an effector-like phe-notype (Fig. 1D). Increased infiltration in the liver or the lung wasdetectable at 6, 15, or 30 wk of age, with a phenotype that did notevolve after 15 wk of age (Fig. 1E and Fig. S1E). Given the verylow recombination in myeloid populations, it seems unlikely thatthe activated T-cell phenotype observed in the maT-A20 miceresulted from the presence of A20-deleted non-T cells as pre-viously shown in mice with A20-deleted macrophages (16) or DCs(8, 10). A multiplex analysis of the serum of 15-wk-old miceshowed augmented levels of proinflammatory cytokines in-cluding IFNγ, TNFα, or IL-17. We also detected increased levelsof several chemokines in the serum from maT-A20 mice (CXCL2,CXCL10, CCL3, CCL4, and CCL5/Rantes, Fig. 1F). The latterthree chemokines may be produced directly by T cells (17),whereas the former two may be produced as the result of the in-direct activation of myeloid cells by T-cell–produced IFNγ orTNFα (18). In conclusion, in a model with a specific deletion ofA20 in peripheral T cells, we observed the development of somelymphadenopathy and infiltration of T cells in peripheral organs.Nevertheless, we did not detect any weight loss or any differencein survival when comparing maT-A20 mice to control littermates(Fig. S1 F and G).To further evaluate the in vivo consequence of A20 deletion in

CD8 T cells, we reconstituted Rag-2−/− mice with either CD8 Tcells alone or CD8 T cells plus CD4 T cells purified from maT-A20 mice LNs (Fig. S2). Results showed that transfer of A20-deleted CD8 T cells alone did not lead to organ infiltration (Fig.S2 B and C), whereas transfer of A20-deleted CD8 and CD4 Tcells resulted in a preferential expansion of splenic CD4 overCD8 T cells (Fig. S2A) and strong liver infiltration by T cells(Fig. S2 B and C). Relative to their representation in the spleen,CD8 T cells were highly enriched within the liver (Fig. S2 A andB). Altogether, these results suggested that the phenotype ob-served in the maT-A20 mice resulted from A20 deletion in bothconventional CD4 and CD8 T cells. A20-deleted CD8 T cellsalone did not cause autoimmune pathology.

A20 Deletion in CD8 T Cells Increases Their Cytokine ProductionThrough an Increased NF-κB Activation. To measure the effect ofA20 deletion on early T-cell activation, naïve CD8 T cells fromLNs of maT-A20 mice or littermates were stimulated for 2–120min with cross-linked anti-CD3 and anti-CD28 mAb. No differ-ence was detected in ZAP70, ERK, or protein kinase AKT phos-phorylation between the two samples (Fig. 2A). Antigen receptoractivation of the canonical NF-κB pathway requires phosphoryla-tion followed by degradation of the Inhibitor of NF-κB alpha(IκBα) to allow the translocation of active NF-κB components tothe nucleus. We observed a sustained decrease in the level of theIκBα protein in CD8 T cells from maT-A20 mice after stimulationwith anti-CD3/CD28 (at 60 and 120 min) compared with CD8 Tcells from WT mice (Fig. 2A and Fig. S3A).When stimulated with coated anti-CD3 and soluble anti-

CD28, CD8 T cells from maT-A20 mice produced a much higheramount of IL-2 and IFNγ compared with CD8 T cells from WTmice as early as 5 h poststimulation, with up to 100-fold moreIL-2 and 10-fold more IFNγ produced 18 h poststimulation (Fig.2B). At the latter time point, we detected a slightly increasedlevel of RelA/p65 and a clearly increased level of c-Rel protein inthe nucleus of CD8 T cells from maT-A20 mice that correlatedwith a marked decrease in IκBα in the cytoplasm of the cells (Fig.2C and Fig. S3B). Altogether, CD8 T cells from maT-A20 mice

A

Cell

num

ber(

106 )

CD8+ CD4+ B220+Lit KO

Litt.Tmat-A20

B

C

CD8+CD4+

B220+

CD11b+

Gr1-

CD11b+

Gr1+

NK1.1+ C

D3-

NK1.1+ C

D3+

% o

f CD

45+

cells

am

ong

tota

l liv

er c

ells

Lit KO

Liver

Litte

rmat

eTm

at-A

2015

wee

ks30

wee

ks

E

D

% C

D44

+ C

D62

L-

Lit KO Lit KO Lit KO

LN S Liv.

0

20

40

60

***

***

Lit KO

% a

mon

g C

D45

+ ce

lls

CD8+ CD4+ B220+

Litt.Tmat-A20

0

20

40

60

80

100

**** ****

0

1

2

3

4

5

********

***

0

5

10

15

20 ****

Cell

num

ber(

106 )

0

100

200

300

F

0

20

40

60

80 ****

020406080

100 *

***

020406080

100****

****

Lit KO Lit KO Lit KO

LN S Liv.

0 5 10 15 20 25VEGF

G-CSFIFNg

TNFaIL-17IL-5IL-6

IL-10IL-18

CXCL10CCL2CCL3CCL4CCL5

CXCL2

Fold change

*******

******

******

***

% C

D44

+ C

D62

L- CD4+ CD8+

Cell

num

ber(

106 )

Cell

num

ber(

106 )

Fig. 1. T-cell–specific deletion of A20 in mice induces lymphadenopathyand infiltration of T cells in peripheral organs. (A–C) Total cell number (Left)or number of the indicated population (Right) per LN (A), spleen (B), orpercentage of CD45+ cells among total cells in the liver (Left) and percentageof the indicated population among CD45+ cells (Right) in the liver (C) ofmaT-A20 mice (KO, open circles) or WT littermates (Litt., black circles). Eachcircle represents the number of cells from 1 mouse. Results from 6-, 9-, or15-wk-old mice were pooled as we did not observe any significant variationbetween the various ages. Histograms represent pooled data from at least10 mice per condition. (D) Percentage of CD62L− CD44+ T cells was de-termined by flow cytometry in LN, spleen (S), or liver (Liv.) from Littermate(Litt.) or maT-A20 mice (KO). (E) Hematoxilin/eosin labeling was performedon livers from 15-wk-old maT-A20 mice or littermate controls. Representa-tive pictures from 6 individual mice are shown. (Scale bars, 100 μm.) (F) Foldincrease of the indicated cytokine/chemokine in serum of maT-A20 mice (n =12) versus littermate controls (n = 8) as measured by Luminex analysis.

11116 | www.pnas.org/cgi/doi/10.1073/pnas.1406259111 Giordano et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 25

, 201

9

have an increased capacity to produce IL-2 and IFNγ uponstimulation. These characteristics correlate with an increasedcytoplasmic degradation of IκBα, the inhibitor of NF-κB, anda higher level of c-Rel in the nucleus of these cells.

A20 Deletion in TCR Transgenic CD8 T Cells Increases Their Capacity toProduce Cytokines in Response to Low Doses of Peptide. To furtherstudy how A20 deletion affects CD8 T-cell activation and dif-ferentiation, we crossed maT-A20 mice with mice transgenic forthe P14 TCR specific for lymphocytic choriomeningitis virus(LCMV) GP33 associated with H-2Db (GP33/H-2Db) (P14-maT-A20) (19). In these mice, we did not detect increased in-filtration in the liver (Fig. S4A). CD8 T cells detected in the LNs,spleen, or liver kept a naïve phenotype (Fig. S4B) with a com-parable expression of the Vα and the Vβ chains of the P14 TCRas in WT P14 mice (Fig. S4C).After 24 h of in vitro stimulation with various amounts of

antigenic peptide (from 10−11 M to 10−8 M), the T cells fromWTor maT-A20 mice expressed similar levels of CD69 (Fig. S4D).At 48/72 h they both down-regulated CD62L expression at theirsurface to the same extent and expressed a high level of CD44similarly (Fig. 3A and Fig. S4E). The major differences betweenthe two cell types were the expression of CD25 and granzyme B(GzmB), both present at a higher level at 48 h and more strik-ingly at 72 h poststimulation in P14-maT-A20 CD8 T cells (Fig. 3A and C). Furthermore, P14-maT-A20 CD8 T cells producedfive times more IL-2 compared with WT P14 T cells and three tofour times more IFNγ, depending on the concentration of pep-tide (Fig. 3B). These results showed that A20 deletion in CD8 Tcells increased their capacity to produce large amounts of cyto-kines after stimulation with low amounts of peptide.

High Levels of A20 in Tumor-Specific CD8 T Cells Correlate with PoorIntratumor Accumulation. We previously developed a mousemodel of induced melanoma based on conditional deletion oftumor suppressor genes with concomitant expression of a natu-ral mouse tumor antigen (Tyr-iRas-P1A-transgenic Ink4a/Arfflox/flox or TiRP mice) (20). In this model, tumor-intrinsicfactors control the development of aggressive tumors and theirexpression of an inflammatory/ immunosuppressive program (21).Intratumor T cells expressed inhibitory receptors such as PD-1and showed a low level of GzmB and poor capacity to pro-duce IFN-γ upon restimulation (22, 23). We showed byquantitative RT-PCR that A20/tnfaip3 transcripts are over-expressed in CD8+ tumor infiltrating lymphocytes (TILs)sorted from TiRP melanomas compared with naïve or acti-vated T cells (Fig. 4A).To assess the consequences of a high level expression of A20 in

T cells on their capacity to accumulate in a tumor and to affecttumor progression, we used a system of adoptive transfer of TCRtransgenic CD8 T cells specific for the P1A35–43 epitope(TCRP1A) previously shown to induce regression of the P1A-expressing P511 mastocytoma (17). We cloned the cDNAencoding A20 into a retroviral vector also coding for GFP. Wenext activated and transduced tumor-specific TCRP1A CD8 Tcells that express luciferase as a transgene (TCRP1A-Luc) witha control retrovirus or with the retrovirus encoding A20 (Fig.S5A). Transduced CD8 T cells expressed a higher level of therelevant transcripts and of the A20 protein compared with mock-transduced T cells (Fig. S5 B and C). They were sorted on thebasis of their GFP expression (Fig. S5A) and transferred into

A

5 Litt. Tmat-A20

cytoplasm nucleusC

0 2 15 60 120 Littermate Tmat-A20

0 2 15 60 120

pERK1/2

ERK1/2

pZAP70

A20

IκBα

pS473 AKT

18 0 5 18 0 5 Litt. Tmat-A20

18 0 5 18 0

IL-2

(pg/

ml)

RelA/p65

cRel

laminB2

hours

β-actin

BIF

Nγ

(x10

IU/m

l)3

0 5 18 0 5 180

20004000

2500050000

hours post-activation

0 5 18 0 5 180

50100

10002000

hours post-activation

min

IκBα

Fig. 2. CD8 T cells from maT-A20 mice have increased NF-κB activation uponstimulation. Purified CD8 T cells from maT-A20 mice (maT-A20) or littermates(Litt.) were stimulated with cross-linked anti-CD3 and anti-CD28 (A) or withcoated anti-CD3 and soluble anti-CD28 (B and C) for the indicated time period.Cytoplasmic protein extracts (A) or cytoplasmic and nuclear protein extracts (C)were analyzed by Western blot for the indicated proteins. (B) Levels of IFNγand IL-2 were measured by ELISA in the in vitro cultures used in C.

A

C

B

2000

4000

6000

8000

10000Litt.maT-A20

IL-2

(pg/

ml)

0

500

1000 Litt.maT-A20

IFNγ

(IU/m

l)

GzmB CD250 102 103 104 105

0

20

40

60

80

100

% o

f Max

0 102 103 104 1050

20

40

60

80

100

% o

f Max

0 102 103 104 1050

20

40

60

80

100

% o

f Max

0 102 103 104 1050

20

40

60

80

100

% o

f Max

CD44 CD62L

48h 72h

[GP33]

0

5

10

15

20

MFI

Gzm

B (x

10 )3

MFI

CD

25 (x

10 )3

0

10

20

30

40

10-11

[GP33]10-10 10-9 10-8

50

10-11

[GP33]10-10 10-9 10-8 10-11

[GP33]10-10 10-9 10-8 10-11

[GP33]10-10 10-9 10-8

48h 72h

10-11 10-10 10-9 10-810-12 10-7

[GP33]10-11 10-10 10-9 10-810-12 10-7

0

Fig. 3. Increased cytokine production by CD8 T cells from maT-A20 mice.(A–C) CD8 T cells from LNs of P14-maT-A20 (gray line) or WT P14 littermate(black line) mice were left unstimulated (solid gray, A) or stimulated withGP33 peptide at a concentration ranging from 10−8 to 10−11 M and analyzedby flow cytometry for the indicated molecule. (A) Histograms represent thelevel of expression of the indicated molecules at 72 h postactivation (10−9 MGP33 peptide). (B) Supernatants from the P14-maT-A20 or WT P14 T cellsstimulation with the indicated concentration of GP33 peptide were tested intriplicates by ELISA for content of IL-2 (24 h) or IFNγ (48 h). (A and B) Resultsare representative of three independent experiments. (C) Mean fluores-cence intensity (MFI) of the indicated molecules was measured 48 or 72 hafter the initial stimulation of WT P14 (black line) or P14-maT-A20 (gray line)CD8 T cells with the indicated concentration of peptide.

Giordano et al. PNAS | July 29, 2014 | vol. 111 | no. 30 | 11117

IMMUNOLO

GYAND

INFLAMMATION

Dow

nloa

ded

by g

uest

on

Dec

embe

r 25

, 201

9

Rag−/−B10.D2 mice that had developed a solid tumor after in-jection of P511 mastocytoma 7 d before, as described (17). Wemeasured by bioluminescence the infiltration of the tumor byT cells. We detected a threefold lower luminescence intensitywhen using A20-transduced compared with mock-transducedTCRP1A-Luc T cells at day 7, the peak of infiltration of thetumor (Fig. 4B). This was associated with a similar decrease inCD8 T-cell number recovered from the tumors at that time (Fig. 4C and D), despite a similar proportion of TCRP1A T cells foundin the spleens of these animals (Fig. 4C). This dampened intra-tumor accumulation correlated with a delay in P511 regressionupon adoptive transfer of A20-transduced compared with mock-transduced TCRP1A T cells (Fig. 4E). In this transplanted tumormodel, tumor antigen-loss variants escape adoptive therapy byTCRP1A T cells leading to tumor regrowth after 20 d (17, 24).Regression of the P511 tumor, which remained incomplete uponadoptive transfer of A20-transduced TCRP1A T cells, was alsofollowed by a more robust tumor regrowth (Fig. 4E). The GFPlevel in mock- versus A20-transduced TCRP1A T cells was similarbefore adoptive transfer into the mice (Fig. S5A). As early as4 or 7 d after transfer, we observed a dramatic decrease in GFP

expression in A20-transduced TCRP1A T cells (Fig. S5 D and E),suggesting a negative effect of high level A20 expression on T-cellfitness and a selective expansion/survival of A20-low T cells. Todetermine whether this negative effect is linked to an early defectin survival after transfer or to a defect occurring after tumor-in-duced reactivation, we mixed mock-transduced GFP+ CD45.1with A20-transduced GFP+CD45.2 TCRP1A T cells at a 1:1 ratio.We then transferred these cells into Rag−/−B10.D2 mice that wereinjected or not with P511 mastocytomas 7 d earlier. In tumor-freemice, the ratio of CD45.1 to CD45.2 T cells remained identical 7 dposttransfer of the mixed TCRP1A T cells (Fig. 4F). This observationruled out any differential survival capacity during homeostasisafter adoptive transfer. However, in tumor-bearing mice, a 3.5ratio in favor of the mock-transduced TCRP1A T cells was ob-served both in the LNs and inside the tumor (Fig. 4F). This resultsuggests that A20-expressing cells developed a defective tumor-induced secondary response compared with control T cells andfailed to accumulate in tumor-bearing hosts. Maintenance ofa same ratio of CD45.1 to CD45.2 cells between LNs and TILsalso ruled out a potential defect in migration of A20-transducedT cells. In vitro, 48 h posttransduction, we did not detect anyincrease in annexin V labeling of either A20- or mock-trans-duced GFP+ CD8 T cells (Fig. S5F). We detected only a slightdelay in the proliferation of GFP+ A20-transduced CD8 T cells(Fig. S5G). Altogether, we show that high level expression ofA20 in CD8 T cells is detrimental for their antitumor efficiency.

A20 Deletion in CD8 T Cells Increases Their Capacity to EliminateTumor Burden. Given the high level of A20 in nonresponsiveTILs and the increased capacity for CD8 T cells lacking A20 todevelop effector functions, we further tested the capacity toeliminate tumor cells by naïve tumor-specific CD8 T cells frommaT-A20 compared with WT mice. For this, we used GP33/H-2Db-specific CD8 T cells from P14 mice, which are inefficientat preventing the growth of B16F10 melanoma cells expressingthe LCMV GP33 epitope (B16-GP33) (25). We injected WTC57BL/6 mice with B16-GP33 melanoma cells and the followingday we transferred purified naïve P14 T cells from WT or maT-A20 mice. Tumor growth was significantly decreased in thepresence of P14-maT-A20 CD8 T cells compared with WT P14CD8 T cells (Fig. S6A). In another protocol, we first injected 106

B16-GP33 cells in C57BL/6 CD45.1 mice and waited for theformation of a solid mass (at day 7) before transferring 6–8 106in vitro preactivated CD45.2 P14-maT-A20 or WT P14 CD8T cells. We observed a reduction of the tumor mass with bothcell types (Fig. 5A) but only P14-maT-A20 CD8 T cells clearedthe tumor burden (in 75% of the mice, Fig. 5B). At day 7 afterT-cell transfer, we measured a threefold increase of IFNγ andTNFα in the serum of the mice that received P14-maT-A20compared with WT P14 CD8 T cells (Fig. 5C). The analysis ofthe transferred CD45.2 CD8 T cells revealed similar numbers ofcells in the LNs and slightly increased P14-maT-A20 comparedwith WT P14 CD8 TILs (Fig. 5D). When tested ex vivo for theircapacity to produce IFNγ in response to stimulation by theircognate peptide GP33, P14-maT-A20 CD8 T cells from LNsresponded slightly better than the WT P14 counterparts. Thedifference was much increased in favor of the P14-maT-A20 cellswhen TILs were compared (Fig. 5E for MFI; Fig. S6B for per-centage of positive cells). A similar trend was observed whenTNFα producing CD8 T cells were analyzed (Fig. 5E and Fig.S6B). The expression of GzmB was higher in TILs than in LNs forboth WT P14 and P14-maT-A20 CD8 T cells (Fig. 5E and Fig.S6B) without a significant difference between the two groups.Because high expression of the inhibitory receptor PD-1 has beenassociated with dysfunction of intratumor T cells, we compared itslevel of expression on the two types of transferred T cells. Theexpression of PD-1 was about threefold lower on P14-maT-A20than on WT P14 TILs (Fig. 5E). Interestingly, this PD-1 level ofthe adoptively transferred P14-maT-A20 CD8 T cells was alsolower than that of endogenous CD8 T cells (CD45.1) present inthe same tumor (Fig. S6C), indicating that low level expression of

A

C

N ActTILs

0

2

4

6

rela

tive

units

100 101 102 103 104100

101

102

103

104

7.74

100 101 102 103 104100

101

102

103

104

1.82

100 101 102 103 104100

101

102

103

104

10.6

100 101 102 103 104100

101

102

103

104

9.91Spl

een

TILs

CD

45

CD8

Mock-GFP A20-GFP

F

*

DBMock-GFPA20-GFP

* *

0 5 10 150

20

40

60

80

Days post-transfer

phot

onsx

10 /

mm

3

2

E

**

**

0 10 20 300

50

100

150 Mock-GFP+A20-GFP+No T cells

Days post-transfer

mm

2

CD8+ GFP+

MockA20

Mock A20

Cel

l num

ber (

x10

)

******

0

2

4

6

8

10

3

LN

020406080

100

LN TILs% o

f CD

45.1

or .

2 am

ong

CD

8 T

cells

MockA20

MockA20MockA20

***

with tumor

notumor

Fig. 4. Inefficient tumor infiltration/elimination by CD8 T cells expressinghigh level A20. (A) CD8 T cells were sorted from melanomas that developedin TiRP mice (three independent samples). RNA levels of A20 from these cells(TILs) were compared with those from naïve CD8 T cells (N) and from in vitroactivated CD8 T cells (Act) by quantitative RT-PCR. (B–F) The 5 × 104 sortedGFP+ TCRP1A-Luc CD8 T cells transduced with mock or A20 encoding ret-roviral constructs were injected in Rag−/−B10.D2 mice, injected with 106 P511mastocytoma 7 d earlier. (B) P511 infiltration by TCRP1A-Luc T cells wasmonitored by bioluminescence. (C) At day 7, splenocytes and TILs were an-alyzed by flow cytometry for the indicated markers. Percentages of CD45+

CD8+ T cells are indicated on the dot plots. (D) TCRP1A CD8 T-cell numbers inTILs from three pooled experiments are shown. (E) Tumor growth wasmonitored every 2–3 d and pooled results from two independent experi-ments (10 mice per condition) are shown. (F) After sorting, CD45.1 mock-transduced and CD45.2 A20-transduced TCRP1A T cells were mixed at a 1:1 ratioand transferred in Rag−/−B10.D2 mice (hatched bars) or Rag−/−B10.D2 miceinjected with 106 P511mastocytoma 7 d earlier (empty white or gray bars). Meanpercentage CD45.1+ or CD45.2+ among CD8 T cells at day 6 posttransfer in theLNs or in TILs (two experiments with 6 mice per condition) are shown.

11118 | www.pnas.org/cgi/doi/10.1073/pnas.1406259111 Giordano et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 25

, 201

9

PD-1 is a property intrinsic to the P14-maT-A20 CD8 T cells and isnot related to differences in the tumor microenvironment. We alsoobserved that endogenous NK cells (CD45.1) present within thetumors of mice that received P14-maT-A20 CD8 T cells had morethan twofold higher expression of GzmB than those from mice thatreceived WT P14 CD8 T cells (Fig. S6D). This property of “by-stander activation” may further contribute to antitumor activity.In conclusion P14 T cells deleted for A20 expression showed an

improved capacity to eliminate tumor cells. This improvement wasassociated with their enhanced capacity to produce IFNγ and TNFαand their moderate expression of the inhibitory receptor PD-1. Inaddition, presence of these T cells led to bystander activation of NKcells within the tumor microenvironment. The latter property mayfurther contribute to contain the escape from T-cell therapy of tu-mor antigen- or MHC-loss variants (17).

DiscussionWe show that deletion of A20 in peripheral CD8 and CD4conventional T cells, under conditions that did not affect themajority of regulatory T cells, induced early enlargement oflymph nodes and lymphocytic infiltration of primary organs.Surprisingly, this phenotype observed in 6-wk-old mice did notworsen with age. In maT-A20 mice crossed with P14 TCR

transgenic mice, infiltration of the lung or liver with CD8 T cellswas abrogated compared with the phenotype we observed inmaT-A20 mice with a full TCR repertoire. In addition to therestricted TCR repertoire expressed by the P14 CD8 T cells, thedevelopment of CD4 T cells is greatly diminished in P14 trans-genic mice (19). The paucity of A20-deleted CD4 T cells in thesemice may be the reason for the absence of lymphocytic in-filtration in peripheral organs as our data on reconstitution ofRag-2−/− mice indicated that A20-deleted CD8 T cells alonefailed to reproduce the organ infiltration that was observed uponcoinjection of A20-deleted CD4 T cells. The control of the in-filtration over time in WT A20 mice could depend on theestablishment of mechanisms of peripheral tolerance exertedextrinsically, such as by T regulatory cells or intrinsically by in-duction of deletion or anergy in the CD8 T cells (26). In vitroA20-deficient CD8 T cells had an increased capacity to produceIFNγ and IL-2 in response to limited amounts of antigen. Theseresults are consistent with previous observations using Jurkatcells transfected with siRNA targeting A20 (14).NF-κB signaling is tightly controlled by ubiquitination, and

A20, through its deubiquinating activity, is one of the proteinsthat affects this process. The CARMA1, BCL10, and MALT1signalosome bridges TCR signaling to the IκB kinase (IKK)complex activating the canonical NF-κB pathway, which involvesmainly NF-κB heterodimers consisting of RelA, c-Rel, and p50(27). Activation of this pathway involves the ubiquitin-dependentproteasomal degradation of the IκB inhibitory proteins upontheir phosphorylation at the IKK complex. Release from the IκBproteins leads to nuclear translocation of the NF-κB dimers andtheir DNA binding. In studies performed in the Jurkat line, A20has been shown to control the ubiquitination of MALT1, therebypreventing the interaction between the ubiquitinated MALT1protein and the IKK complex, acting as a negative regulator ofthe NF-κB pathway (14). In our study, we observed an increasein nuclear translocation of c-Rel compared with RelA NF-κBcomponents in the response to CD3/CD28 engagement of A20-deleted versus WT CD8 T cells. Whether the relief from A20control of MALT1 activity favors the activation of c-Rel overRelA remains to be determined. This is suggested to occur in Bcells, where MALT1 directs BCR-induced canonical NF-κBsignaling selectively to the c-Rel subunit (28). Interestingly, forCD8 T-cell activation, c-Rel deficiency has been reported tophenocopy deficiency of PKCθ thought to activate the CARMA1,BCL10, and MALT1 signalosome, but not BCL10 deficiency(29). Irrespective of the detailed mechanism of increase inNF-κB activity, we show that A20 deletion in CD8 T cells greatlyenhances their capacity to produce IL-2 and IFNγ. This is co-herent with the described defects in IL-2 production by T cellslacking c-Rel (30) and c-Rel requirement for chromatin re-modeling across the IL-2 gene promoter (31). It is also consistentwith the observed strict dependency of IFNγ gene transcriptionupon binding of NF-κB to cis-regulatory elements of the gene forboth TCR- and cytokine (IL-12/IL-18)-induced signaling in CD8T cells (32).We have observed that dysfunctional CD8 T cells isolated

from a progressing autochthonous melanoma in mice overex-pressed A20 transcripts compared with naïve or acutely activatedCD8 T cells. We demonstrated that overexpression of A20 byretroviral transduction of tumor-specific CD8 T cells led toa drastic inhibition of their intratumor accumulation. We showthat this effect is not related to the homing capacity of the T cellsand that it requires restimulation induced by the tumor. Becauseof a rapid loss of the T cells expressing high levels of A20, wewere not able to determine whether it was related to a defect inactivation, proliferation, or survival.Importantly, relief from the A20 brake in NF-κB activation

in adoptively transferred antitumor CD8 T cells led to in-creased efficiency in induction of regression of the B16 mel-anoma with concomitant detection of high levels of IFNγ andTNFα in the serum of the mice. In addition to a small increase(1.6-fold) in the number of A20-deleted compared with WT

C D

P14 Litt. (n=13)P14 maT-A20 (n=7)

No P14 (n=6)

* ** *

**

Days

0

50

100

150

200

0 10 3020

mm

2

IFNγ

in m

ice

sera

(IU

/ml)

0.0

0.1

0.2

0.3

0.4 ***

0

50

100

150 *

TNFα

in m

ice

sera

(pg/

ml)

BP14 Litt. (n=13)P14 maT-A20 (n=7)

p=0.0004

No P14 (n=6)

0

50

% o

f tum

or fr

ee m

ice

100

0 10 20 30

A

0

500

1000

1500

***CD8+ NK1.1+

E

MFI

Gzm

B

MFI

PD

-1

0

1000

2000

3000

4000

5000***

LN T0

100

200

300

400

500 * ****

0

50

100

150

200

250

*****

MFI

IFNγ

MFI

TN

Fα

LN T LN T LN T LN T

6

0.00

0.01

0.02

0.03

0.04

0

20

40

60 *

notumor

B16-GP33

LN Tumor

B16-GP33

P14

T c

ells

(x10

)

% P

14 T

cel

ls (a

mon

g C

D8+

)

Days

Fig. 5. P14 CD8 T cells from maT-A20 mice have increased antitumor po-tential. B16-GP33 cells (106) were injected s.c. in CD45.1 C57BL/6 mice and invitro preactivated WT CD45.2 P14 or CD45.2 P14-maT-A20 CD8 T cells (6–8 ×106 cells) were transferred 7 d later. (A and B) Tumor growth was monitoredwith a caliper every 2–3 d. (C) At day 7 after transfer, levels of IFNγ and TNFαin mice sera were measured by ELISA. (D) On the same day, the number ofCD45.2+ P14 T cells present in LNs and their proportion in TILs among totalCD8 T cells was determined; number of CD45.2+ P14 T cells present in LNs ofmice that received no tumor (no tumor) is also shown. (E) MFI of the in-dicated molecules was determined by flow cytometry on CD45.2+ P14 T cellsand on endogenous CD45.1+ NK1.1+ cells (for GzmB only) present in the LNsor in TILs (T) directly ex vivo (PD-1 and GzmB) or after 4 h restimulation withGP33 peptide (IFNγ and TNFα). (A–E) Results are representative of two in-dependent experiments. Numbers of mice are indicated in A and B and sixmice per condition were used for C–E.

Giordano et al. PNAS | July 29, 2014 | vol. 111 | no. 30 | 11119

IMMUNOLO

GYAND

INFLAMMATION

Dow

nloa

ded

by g

uest

on

Dec

embe

r 25

, 201

9

CD8 T cells infiltrating the tumors, the A20-deleted CD8TILs produced higher amounts of IFNγ and TNFα ex vivo.Intratumor IFNγ has been shown to have beneficial antitumoreffects, including the activation of tumor-associated macro-phages toward a tumoricidal rather than tumor-promoting pro-gram (33) as well as angiostatic effects (34). In synergy withTNFα, IFNγ further mediates the destruction of the tumorstroma (35). Whereas injection of these cytokines may produceunwanted toxicity, tumor antigen-specific T cells localize to thetumors and release cytokines without evidence for systemictoxicity. In contrast to the beneficial antitumor effects of IFNγ,this cytokine has also been shown to contribute to the adaptiveimmune resistance of tumor cells (1). This is thought to occur byits capacity to increase the expression on tumor cells of the li-gand PDL-1 of the inhibitory receptor PD-1. PD-1 is transientlyexpressed upon T-cell activation, but its sustained expression onCD8 T cells contributes to the generation of dysfunctional CD8T cells in numerous settings involving persistent antigen, such aschronic viral infections and cancer. Engagement of T-cell–expressedPD-1 by tumor-expressed PDL-1 has been shown to inhibit CD8T-cell effector functions (36). The intrinsic characteristic of mod-erate levels of expression of PD-1 on A20-deleted CD8 TILs ob-served in our study may thus contribute to the enhanced antitumorefficacy of these cells upon adoptive immunotherapy.This work contributes to the identification of a major pathway

affected in dysfunctional CD8 T cells within tumors, which, in thecase studied here, could be reversed without apparent pathologicconsequences. Thus, A20 may be an interesting candidate to betargeted in the context of adoptive T-cell immunotherapy.

Materials and MethodsMice and Cell Lines. TiRP-10B; Ink4a/Arf flox/flox mice, “TiRP mice,”were kepton a B10.D2 background and treated with 4OH-tamoxifen as described

(20, 23). A20flox/flox mice (15) were crossed on maT-Cre mice (37) (Fig. S1).Mice heterozygous for the H-2Ld/P1A35–43-specific TCR-transgene (TCRP1A)(17) and luciferase expressing TCRP1A mice (TCRP1A-Luc) (24) were kept onthe Rag-1−/−B10.D2 background. P14 TCRmice (19), Rag-1−/−B10.D2, and C57BL/6(CD45.2) as well as congenic C57BL/6 (CD45.1) mice were also used. All micewere bred in the Centre d’Immunologie de Marseille–Luminy animal facility.Animal experiments were approved by the regional Provence-Alpes-Côted’Azur committee on ethics for animal experimentation and respectedFrench and European directives. B16F10 melanoma cells expressing theglycoprotein epitope amino acids 33–41 (B16GP33) were a kind gift from H.Pircher (University of Freiburg, Freiburg, Germany). P511 mastocytoma cellsexpressing the P1A gene were used as described (17).

CD8 T-Cell Activation and Retroviral Infections. FL cDNA vector coding for A20(clone ID 6838303 from Thermo Scientific ABgene) was cloned into the retroviralvector pMX-IRES-GFP after PCR amplification using PFUultra HF (Agilent) usingthe following primers GGAAGATCTCCCCAAGAGGCCTTGT/TTGCTGGACCTGT-CAAT (A20) and TCGGAAATATACCA AAGC/TTTTCCTTTTGCGGCCGCTTCTTTG-ACGTGCTTGG (NR4A2). Retroviral particles were produced as described (38).TCRP1A T cells were activated with 10−7 M of the P1A35–43 (LPYLGWLVF) peptide,retrovirally transduced after 20 h as described (38) and further cultured for an-other 48 h.

Additional materials and methods can be found in SI Materials andMethods.

ACKNOWLEDGMENTS.We thank Y. Hamon for technical advice, L. Lesermanfor suggestions on the manuscript, T. Lawrence for his support, and theCentre d’Immunologie de Marseille–Luminy imaging and animal facilities forassistance. This work was supported by funding from Institut National de laSanté et de la Recherche and Centre National de la Recherche Scientifiqueand by grants from the Agence Nationale de la Recherche (ANR Retour Post-Doctorants to G.V.) and the Association pour la Recherche sur le Cancer(to A.-M.S.-V.).

1. Pardoll DM (2012) The blockade of immune checkpoints in cancer immunotherapy.Nat Rev Cancer 12(4):252–264.

2. Quezada SA, Peggs KS, Simpson TR, Allison JP (2011) Shifting the equilibrium incancer immunoediting: From tumor tolerance to eradication. Immunol Rev 241(1):104–118.

3. Oeckinghaus A, Hayden MS, Ghosh S (2011) Crosstalk in NF-κB signaling pathways.Nat Immunol 12(8):695–708.

4. Catrysse L, Vereecke L, Beyaert R, van Loo G (2014) A20 in inflammation and auto-immunity. Trends Immunol 35(1):22–31.

5. Ma A, Malynn BA (2012) A20: Linking a complex regulator of ubiquitylation to im-munity and human disease. Nat Rev Immunol 12(11):774–785.

6. Lee EG, et al. (2000) Failure to regulate TNF-induced NF-kappaB and cell death re-sponses in A20-deficient mice. Science 289(5488):2350–2354.

7. Chu Y, et al. (2011) B cells lacking the tumor suppressor TNFAIP3/A20 display impaireddifferentiation and hyperactivation and cause inflammation and autoimmunity inaged mice. Blood 117(7):2227–2236.

8. Hammer GE, et al. (2011) Expression of A20 by dendritic cells preserves immune ho-meostasis and prevents colitis and spondyloarthritis. Nat Immunol 12(12):1184–1193.

9. Hövelmeyer N, et al. (2011) A20 deficiency in B cells enhances B-cell proliferation andresults in the development of autoantibodies. Eur J Immunol 41(3):595–601.

10. Kool M, et al. (2011) The ubiquitin-editing protein A20 prevents dendritic cell activation,recognition of apoptotic cells, and systemic autoimmunity. Immunity 35(1):82–96.

11. Matmati M, et al. (2011) A20 (TNFAIP3) deficiency in myeloid cells triggers erosivepolyarthritis resembling rheumatoid arthritis. Nat Genet 43(9):908–912.

12. Tavares RM, et al. (2010) The ubiquitin modifying enzyme A20 restricts B cell survivaland prevents autoimmunity. Immunity 33(2):181–191.

13. Coornaert B, et al. (2008) T cell antigen receptor stimulation induces MALT1 para-caspase-mediated cleavage of the NF-kappaB inhibitor A20. Nat Immunol 9(3):263–271.

14. Düwel M, et al. (2009) A20 negatively regulates T cell receptor signaling to NF-kappaBby cleaving Malt1 ubiquitin chains. J Immunol 182(12):7718–7728.

15. Vereecke L, et al. (2010) Enterocyte-specific A20 deficiency sensitizes to tumor ne-crosis factor-induced toxicity and experimental colitis. J Exp Med 207(7):1513–1523.

16. Wang L, et al. (2012) A20 controls macrophage to elicit potent cytotoxic CD4(+) T cellresponse. PLoS ONE 7(11):e48930.

17. Shanker A, et al. (2007) CD8 T cell help for innate antitumor immunity. J Immunol179(10):6651–6662.

18. Griffin GK, et al. (2012) IL-17 and TNF-α sustain neutrophil recruitment during inflammationthrough synergistic effects on endothelial activation. J Immunol 188(12):6287–6299.

19. Pircher H, Bürki K, Lang R, Hengartner H, Zinkernagel RM (1989) Tolerance induction indouble specific T-cell receptor transgenicmice varies with antigen.Nature 342(6249):559–561.

20. Huijbers IJ, et al. (2006) An inducible mouse model of melanoma expressing a definedtumor antigen. Cancer Res 66(6):3278–3286.

21. Wehbe M, et al. (2012) Epithelial-mesenchymal-transition-like and TGFβ pathwaysassociated with autochthonous inflammatory melanoma development in mice. PLoSONE 7(11):e49419.

22. Auphan-Anezin N, et al. (2013) Immunosuppression in inflammatory melanoma: Can itbe resisted by adoptively transferred T cells? Pigment Cell Melanoma Res 26(2):167–175.

23. Soudja SM, et al. (2010) Tumor-initiated inflammation overrides protective adaptiveimmunity in an induced melanoma model in mice. Cancer Res 70(9):3515–3525.

24. Grange M, et al. (2012) Activated STAT5 promotes long-lived cytotoxic CD8+ T cellsthat induce regression of autochthonous melanoma. Cancer Res 72(1):76–87.

25. Prévost-Blondel A, et al. (1998) Tumor-infiltrating lymphocytes exhibiting high ex vivocytolytic activity fail to prevent murine melanoma tumor growth in vivo. J Immunol161(5):2187–2194.

26. Redmond WL, Sherman LA (2005) Peripheral tolerance of CD8 T lymphocytes. Im-munity 22(3):275–284.

27. Paul S, Schaefer BC (2013) A new look at T cell receptor signaling to nuclear factor-κB.Trends Immunol 34(6):269–281.

28. Ferch U, et al. (2007) MALT1 directs B cell receptor-induced canonical nuclear factor-kappaB signaling selectively to the c-Rel subunit. Nat Immunol 8(9):984–991.

29. Deenick EK, et al. (2010) c-Rel phenocopies PKCtheta but not Bcl-10 in regulating CD8+T-cell activation versus tolerance. Eur J Immunol 40(3):867–877.

30. Köntgen F, et al. (1995) Mice lacking the c-rel proto-oncogene exhibit defects inlymphocyte proliferation, humoral immunity, and interleukin-2 expression. GenesDev 9(16):1965–1977.

31. Rao S, Gerondakis S, Woltring D, Shannon MF (2003) c-Rel is required for chromatinremodeling across the IL-2 gene promoter. J Immunol 170(7):3724–3731.

32. Balasubramani A, et al. (2010) Modular utilization of distal cis-regulatory elementscontrols Ifng gene expression in T cells activated by distinct stimuli. Immunity 33(1):35–47.

33. Biswas SK, Mantovani A (2010) Macrophage plasticity and interaction with lympho-cyte subsets: Cancer as a paradigm. Nat Immunol 11(10):889–896.

34. Qin Z, et al. (2003) A critical requirement of interferon gamma-mediated angiostasisfor tumor rejection by CD8+ T cells. Cancer Res 63(14):4095–4100.

35. Zhang B, Karrison T, Rowley DA, Schreiber H (2008) IFN-gamma- and TNF-dependentbystander eradication of antigen-loss variants in established mouse cancers. J ClinInvest 118(4):1398–1404.

36. Iwai Y, et al. (2002) Involvement of PD-L1 on tumor cells in the escape from hostimmune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad SciUSA 99(19):12293–12297.

37. Roncagalli R, et al. (2014) Quantitative proteomics analysis of signalosome dynamicsin primary T cells identifies the surface receptor CD6 as a Lat adaptor-independentTCR signaling hub. Nat Immunol 15(4):384–392.

38. Verdeil G, Puthier D, Nguyen C, Schmitt-Verhulst AM, Auphan-Anezin N (2006)STAT5-mediated signals sustain a TCR-initiated gene expression program towarddifferentiation of CD8 T cell effectors. J Immunol 176(8):4834–4842.

11120 | www.pnas.org/cgi/doi/10.1073/pnas.1406259111 Giordano et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 25

, 201

9