Spontaneous Intestinal Tumorigenesis in Min Mice Requires ...downloads.hindawi.com/journals/jir/2015/860106.pdf · Tregs in the LP and investigated the role of the heterozygous Apc

Research ArticleSpontaneous Intestinal Tumorigenesis in 119860119901119888Min+ Mice RequiresAltered T Cell Development with IL-17A

Wook-Jin Chae and Alfred L M Bothwell

Department of Immunobiology Yale University School of Medicine New Haven CT 06520 USA

Correspondence should be addressed to Alfred L M Bothwell alfredbothwellyaleedu

Received 17 December 2014 Revised 6 May 2015 Accepted 6 May 2015

Academic Editor David E Gilham

Copyright copy 2015 W-J Chae and A L M Bothwell This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The control of inflammatory diseases requires functional regulatory T cells (Tregs) with significant Gata-3 expression Here weaddress the inhibitory role of Tregs on intestinal tumorigenesis in the 119860119901119888Min+ mouse model that resembles human familialadenomatous polyposis (FAP) 119860119901119888Min+ mice had a markedly increased frequency of Foxp3+ Tregs and yet decreased Gata-3 expression in the lamina propria To address the role of heterozygous Apc gene mutation in Tregs we generated Foxp3-CreApcflox+ mice Tregs from these mice effectively inhibited tumorigenesis comparable to wild type Tregs after adoptive transferinto 119860119901119888Min+ mice demonstrating that the heterozygous Apc gene mutation in Tregs does not induce the loss of control overtumor microenvironment Adoptive transfer of in vitro generated 119860119901119888Min+ iTregs (inducible Tregs) failed to inhibit intestinaltumorigenesis suggesting that naıve CD4 T cells generated from 119860119901119888Min+ mice thymus were impaired We also showed thatadoptively transferred IL-17A-deficient 119860119901119888Min+ Tregs inhibited tumor growth suggesting that IL-17A was critical to impair thetumor regression function of119860119901119888Min+ Tregs Taken together our results suggest that bothT cell development in a functional thymusand IL-17A control the ability of Treg to inhibit intestinal tumorigenesis in 119860119901119888Min+ mice

1 Introduction

The intrinsic connection between inflammation and cancerpromotion is well established [1] The main role of regu-latory T cells (Tregs) is to suppress inflammation [2] Thegeneration of peripheral Tregs is particularly important in theintestinal tract and about 20ndash30 of Tregs in the intestineare adaptively induced Tregs (iTregs) [3 4] Tregs expressa lineage specification marker Foxp3 and they use varioustypes of suppression mechanisms including the secretion ofimmunosuppressive cytokines (eg IL-10 and TGF-120573) andcell-cell contact [2 5] In addition to the expression of Foxp3a very recent study demonstrated that another Th2 typeCD4 T cell lineage marker Gata-3 is critical to maintainhomeostasis and suppressive function of Tregs in suppressingintestinal inflammation in vivo [6 7]

There is a high incidence of Tregs in tumor infiltratinglymphocyte (TIL) populations [8] In most human carcino-mas these Tregs have been considered as an obstacle for

effective antitumor immunity A variety of localized andmetastatic human carcinomas showed that the accumula-tion of Tregs in the tumor microenvironment is associatedwith unfavorable disease outcome [8] This has been alsodemonstrated in experimental animal models of cancer [9ndash12] In human colorectal cancer however a series of reportsargued that the high incidence of tumor infiltrating Tregs inpatient samples predicted improved survival or better diseaseoutcome [13] In line with this a previous study showed thatthe adoptive transfer of splenic wild type Tregs effectivelyregressed intestinal tumors in 119860119901119888Min+ mice [14]

The 119860119901119888Min+ mouse model is well established to investi-gate the mechanisms of early stages of spontaneous intestinaltumorigenesis It resembles human FAP (familial adenoma-tous polyposis) and many genetictherapeutic approacheswere tested in this model [15] 119860119901119888Min+ mice and humanFAP patients possess an inherited heterozygous germlinemutation in the Apc gene that results in a truncated form

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2015 Article ID 860106 11 pageshttpdxdoiorg1011552015860106

2 Journal of Immunology Research

of APC protein The Apc gene undergoes further loss ofheterozygosity (LOH) via the deletion of the wild type Apcallele and this results in activation of excessive Wnt signalingthat drives polyp formation in the intestinal tract [16] FAPpatients develop colon polyps early in life and all affectedindividuals develop cancer In contrast sporadic colon cancerpatients do not have a germline mutation in the Apc geneand generally develop disease much later in life However therole of Treg cells in the rapid onset of intestinal tumorigenesis119860119901119888Min+ mice has not been addressedIn the current study we characterized 119860119901119888Min+ mice

Tregs in the LP and investigated the role of the heterozygousApc gene mutation in Tregs in a spontaneous intestinaltumorigenesis model Tregs from IL-17A deficient 119860119901119888Min+

mice were used to assess the effect of the tumor environmentand functional thymus

2 Materials and Methods

21 Mice 119860119901119888Min+ mice were purchased from the JacksonLaboratory and have been bred and maintained in our SPFmouse facility in conventional housing condition All othermouse strains mentioned below were also housed in ourSPF mouse facility in conventional housing condition insuspended stainless wire-mesh cages Mouse health statuswas regularly monitored by Yale University InstitutionalAnimal Care and no infections were reported in animals usedin this study The animals were kept under normal lightdarkcycle In all experiments wild type (WT) littermates of119860119901119888

Min+ mice on the C57BL6 background were used IL-17A KO-119860119901119888Min+mice were generated previously in C57Bl6background [17] Foxp3-IRES-RFPmice (FIR) were providedby Richard Flavell (Yale University) and bred with 119860119901119888Min+

mice and C57BL6 mice to generate 119860119901119888Min+-Foxp3-IRES-RFP (119860119901119888Min+-FIR) mice in C57Bl6 background Foxp3-Cre transgenic mice were generously provided by AlexanderRudensky (Sloan-Kettering Cancer Center) and maintainedin our colonies for more than 6 generations in C57Bl6background 119860119901119888floxflox mice in C57Bl6 background wereobtained from the NCI and reported previously [18] Thisstrain was bred to Foxp3-Cre transgenic mice resulting intruncation of the Apc protein at amino acid 580 in Foxp3+cells All mouse protocols were approved by the Yale Uni-versity Institutional Animal Care and Use Committee inaccordance with the Association for Assessment and Accred-itation of Laboratory Animal Care International Genotypingconditions and primer sequences for mouse strains used inthis study are as follows119860119901119888

Min+ mice 94∘C 4min (94∘C 45 sec 64∘C 45 secminus03∘C 45 seccycle 72∘C 1min) 35 cycles 72∘C 5min

Forward primer 51015840-ttc cac ttt ggc ata agg c-31015840 Reverseprimer 51015840-ttc tga gaa aga cag aag tta-31015840

Foxp3-IRES-RFP mice 94∘C 3min (94∘C 30 sec 65∘C30 secminus03∘C45 seccycle 72∘C40 sec) 36 cycles 72∘C5minPrimer 1 51015840-caaaac caagaaaaggtgggc-31015840 Primer 2 51015840-ggaatg-ctcgtcaagaagacagg-31015840 Primer 3 51015840-cagtgctgttgctgtgtaagggtc-31015840

119860119901119888floxflox mice 94∘C 5min (94∘C 30 sec 58∘C 30 sec

72∘C 45 sec) 32 cycles 72∘C 5min Forward primer 51015840-gag aaaccc tgt ctc gaa aaa a-31015840 Reverse primer 51015840-agt gct gtt tct atgagt caa c-31015840

Foxp3-Cre transgenic mice 94∘C 3min (94∘C 30 sec52∘C 1min 72∘C 1min) 35 cycles 72∘C 2min Forwardprimer 51015840-agg atg tga ggg act acc tcc tgt a-31015840 Reverse primer51015840-tcc ttc act ctg att ctg gca att t-31015840

IL-17A KO mice 94∘C 2min (94∘C 15 sec 64∘C 30 sec72∘C 1min) 40 cycles 72∘C 5min 10min primer 1 51015840-actctt-catccacctcacacga-31015840 primer 2 51015840-gccatgatatagacgttgtggc-31015840primer 3 51015840-cagcatcagagactagaaggga-31015840 Primers 1 and 2 wereused to detect wild type allele and primers 1 and 3 were usedto detect mutant allele

22 Tumor Counts and Size Animals were examined formortality and clinical signs throughout the study All micesurviving to the end of studywere euthanized for determiningtumor size and number Following euthanasia the entiresmall intestinal tract from each mouse was immediatelyremoved and divided into 3 sections from the duodenumto the ileum The intestinal sections were opened anda dissecting microscope (magnification 20x) was used toidentify tumors in each section A calibrated reticle in aneyepiece of the dissecting scope was used to measure thediameter of each tumor at its maximum dimension Lesionsless than 05mm in diameter were not enumerated Largetumors (gt2mm) or small tumors (05ndash2mm) were counted

23 Adoptive Transfer Model and Treg Depletion For theisolation of119860119901119888Min+ Tregs and wild type Tregs spleens from6-7-week-old 119860119901119888Min+-Foxp3-IRES-RFP mice and their lit-termate controls (Foxp3-IRES-RFP) were prepared as singlecell suspensions After RBC (red blood cell) lysis cells werestained with CD16CD32 FcR (Fc Receptor) antibody (clone24G2) to block nonspecific binding and then subsequentlystained with anti-CD4 (clone RM4-5) antibody RFP+ cellswere sorted by FACSVantage and then transferred immedi-ately to 3-month-old119860119901119888Min+-Foxp3-IRES-RFPmice RFP+cells were subsequently sorted and then immediately trans-ferred into 3-month-old 119860119901119888Min+ mice For Apc mutantTregs (Apc HET Tregs) splenic CD4+YFP+ cells were iso-lated from 6-week-old Foxp3-Cre 119860119901119888flox+ mice and Foxp3-Cre transgenic mice CD4+CD45RBlowCD25-splenic Tregswere isolated from 6ndash8-week old IL-17A KO-119860119901119888Min+ miceFor each transfer experiment 4 times 105 cells were injected peranimal intravenously Mice were analyzed on week 16 Toperform cellular analysis mice were sacrificed and LP (lam-ina propria) cells were prepared For histological analysisthe terminal ileum from each mouse was dissected fixed in10 formalin and washed in PBS solution The specimenwas paraffin-embedded sectioned at 5 120583m and stained withhematoxylin and eosin (HampE)

24 Antibodies and Reagents Anti-mouse CD4 (clone RM4-5) anti-Helios (clone 22F6) anti-Foxp3 (clone FJK-16s)anti-ROR120574t (clone AFKJS-9) anti-GATA-3 (clone TWAJ)

Journal of Immunology Research 3

anti-Tbet (clone eBio4B10) anti-IL-17A (clone eBio 17B7)anti-IFN-120574 (clone XMG12) CD45RB (clone C36316A) RagIgG2a isotype control (clone eBR2a) anti-CD3 (clone 145-2C11) and anti-CD28 (clone 3751)were purchased fromeBio-science Human TGF-1205731 was purchased from RampD systemsDiphtheria toxin and collagenase type IV were purchasedfrom Sigma DNase I was purchased from Roche

25 Flow Cytometry and Cell Culture ForCD4 T cell differentiation naıve CD4 T cells(CD4+CD62LhiCD44loFoxp3(RFP)-) were isolated from6-7-week-old 119860119901119888Min+-Foxp3-IRES-RFP and littermatecontrol mice For inducible Treg transfer splenic naıve CD4+T cells from 6-week-old 119860119901119888Min+-Foxp3-IRES-RFP miceand their littermate controls (Foxp3-IRES-RFP) spleens wereactivated with anti-CD3 (25120583gmL) IL-2 (100UmL) and15 ngmL of TGF-120573 for 5 days For LPMC (lamina propriamononuclear cell) preparation 16-week-old 119860119901119888Min+ miceand Treg-transferred 119860119901119888Min+ mice were sacrificed andthe individual small intestines were processed as describedwith modification [19] Briefly small intestines were cutlongitudinally and fecal contents were removed Each smallintestine was cut into 5 cm long pieces and extensivelywashed with PBS Subsequently small intestine pieces wereincubated in shaking incubator with 5mM EDTA-PBSsolution To digest these pieces EDTA was removed andcollagenase andDNase I solution was added in 5 FBS RPMImedia for 30min at 37∘C After Percoll gradient separationcells were washed with ice-cold PBS twice and lymphocyteswere countedwith trypan blue exclusion Approximately 90of cell viability was obtained The yield of cells was presentedin Supplementary Figure 1 in Supplementary Materialavailable online at httpdxdoiorg1011552015860106For the stimulation of LPMC cells PMA (100 ngmL) andionomycin (1 120583M) were used Brefeldin A (1 120583gmL) wasadded for the 4 h of culture in a total 6 hr of activationAfter stimulation intracellular staining was performed asdescribed in the manufacturerrsquos protocol (eBioscience) AFACSCalibur (BD Biosciences) was used for flow cytometryand data were analyzed by FlowJo software (Treestar)

26 Real Time PCR Cells were harvested and washed withPBS and then suspended in Trizol cDNA was synthesizedwith the BDsprint cDNA synthesis kit (Clontech) and usedwith SYBR Green (Molecular Probes) Real-time PCR wasperformed using a Real time Bioanalyzer (BioRad) Primerswere synthesized by the Keck Biotechnology Institute (YaleUniversity) We calculated relative gene expression by theΔ119862119879 method [20] The mRNA expression was normalizedagainst HPRT Primer sequences are available upon requestsPCR reaction was done using the following cycle sequence95∘C 3min 95∘C 30 s 61∘C 20 s and 72∘C 45 s for 40 cycles

27 Statistical Significance Statistically significant differenceswere determined by two-tailed unpaired Studentrsquos 119905-test (119901 lt005 was taken as significant) or one-way ANOVA analysesfollowed by Bonferronirsquos post hoc test with Graph Pad Prismsoftware (GraphPad Software)

3 Results

31 Regulatory T Cells Are Selectively Expanded and Alteredin the Lamina Propria of 119860119901119888Min+ Mice We first decided tocharacterize the Treg cell compartment in119860119901119888Min+miceWeexamined the frequencies of Foxp3+ Treg among CD4 T cellsin 119860119901119888Min+ mice and their littermate controls In the spleenthere was no substantial enrichment of CD4+Foxp3+ cellsin 119860119901119888Min+ mice at week 16 (Figure 1(a)) The frequencies ofCD4T cells were reduced in119860119901119888Min+mice although the totalnumber of splenocytes was markedly increased (Figure 1(b))This reduced frequency of CD4 T cells was previous reportedand attributed to lymphodepletion [21] In mesenteric lymphnodes (MLNs) CD4 T cell and Foxp3+ cell populations werelargely unaltered (Figures 1(c) and 1(d)) Interestingly weshowed that there is an approximately 25-fold increase in thefrequency of Foxp3+ Treg in the LP of 119860119901119888Min+ versus wildtype C57BL6 mice (Figure 1(e)) suggesting that 119860119901119888Min+

Treg expansion is specific to the LP We also showed that119860119901119888Min+ mice had about 25 times more cells in the LP

(Figure 1(f) and Supplementary Figure 1) Taken togetherour results show that the accumulation of Foxp3+ Treg isspecifically found in the LP where intestinal tumorigenesisoccurs

32 Th17 Cells and Altered Foxp3+ Tregs Cells Are BothIncreased in the Lamina Propria of 119860119901119888Min+ Mice Thetumor microenvironment alters the effector phenotype ofCD4 T cells and the balance with regulatory T cells [822] We decided to characterize Th17 cells and Treg in theLP of 119860119901119888Min+ mice First we measured the expressionof the Th17 lineage transcription factor ROR120574t in the LPCD4 T cells from wild type and 119860119901119888Min+ mice As Th17cells are important to initiate intestinal tumorigenesis wehypothesized that the levels of ROR120574t+ and IL-17A+ CD4T cells in the LP of 119860119901119888Min+ mice could be substantiallyelevated Unexpectedly the frequency of ROR120574t+ CD4T cellswas decreased in the LP compared to control mice whilethat of Foxp3+ cells was increased (Figure 2(a)) We alsoexamined the expression of IL-17A in the LP as markersfor expression for Th17 cells (Figure 2(b)) Importantly thehighly expanded 119860119901119888Min+ Foxp3+ Treg in the LP did notcoexpress substantial levels of ROR120574t or IL-17A (Figures2(a) and 2(b)) Although the frequency of IL-17A+ CD4 Tcells was reduced by 30 the 25-fold increase of total LPcells resulted in only moderate increased of IL-17A+ CD4T cells (Figure 2(b) and Supplementary Figure 1) Thus thetumor microenvironment in 119860119901119888Min+ mice have markedlyincreased numbers of Foxp3+ Tregs but only moderatelyincreased IL-17A+ CD4 T cells in the LP suggesting that119860119901119888Min+ Foxp3+ Tregs are altered in the LPWe further examined possible alterations in 119860119901119888Min+

Foxp3+ Treg It has been reported that Gata-3 is a criticaltranscription factor needed to maintain Treg function in vivo[6] In addition it has been shown that Gata-3 is requiredfor IL-10 expression and IL-10 is critical for inhibitingintestinal polyposis [14 23] As endogenous 119860119901119888Min+ Treg


507

175

135

379

Wild type

CD4

Foxp

3

SPLApcMin+

(a)

Wild type

SPLns

Foxp

3+ ce

llsam

ong

CD4

T ce

lls (

)

30

15

0

ApcMin+

(b)

634

36

626

322

Wild type

Foxp

3

MLN

CD4

ApcMin+

(c)

Wild type

nsMLN

Foxp

3+ ce

lls

amon

g CD

4 T

cells

()

250

125

00

ApcMin+

(d)

CD4

252

483

21

194

Foxp

3

LPWild type ApcMin+

(e)

Foxp

3+ ce

lls

amon

g CD

4 T

cells

()

Wild type

LP

60

30

0

ApcMin+

lowastlowast

(f)

Figure 1 Regulatory T cells are selectively expanded and altered in the lamina propria of 119860119901119888Min+ mice (andashd) Four-month-old 119860119901119888Min+

mice and their littermate controls (119899 = 3 for each group) were sacrificed Single cell suspensions from spleen mesenteric lymph nodes (MLN)and lamina propria (LP) were counted and analyzed by flow cytometry The percentage of Foxp3+ cells among CD4 T cells was presented inthe bar graph A representative of three independent experiments is shown Two-tailed 119905-test was performed lowastlowastlowast119901 lt 00005 lowastlowast119901 lt 0005lowast119901 lt 005 ns not significant

in the LP clearly failed to regress tumors we tested whetherthese expanded Treg stably express Gata-3 Interestinglythe percentage of the Gata-3-Foxp3+ 119860119901119888Min+ Treg pop-ulation markedly increased in the LP (882 versus 29)(Figure 2(c)) However the absolute number of Foxp3+Gata-3+ Tregs was also increased in 119860119901119888Min+ mice This suggeststhat the expansion of Gata-3-119860119901119888Min+ Tregwasmore favoredby the tumor microenvironment giving more nonfunctional

Treg It has been shown that Helios a member of the Ikarosfamily is expressed in more mature and proliferating CD4 Tcells and Treg as well as in thymic-derived Tregs [24ndash26] Wealso showed that the frequency of Helios+ Foxp3+ Treg wasmarkedly increased in 119860119901119888Min+ mice while Helios-Foxp3+Treg were very low (Figure 2(d)) Interestingly Helios+ cellswere not increased in theCD4 effector population suggestingthat the tumor microenvironment favors the activation and


239 067

144611

106 164

382496Foxp3

CD4 gated

Wild type

Wild type

30

15

0

ApcMin+

ApcMin+

ROR120574

tlowast

CD4+

ROR120574

t+ce

lls (

)

(a)

Wild type

Wild type

CD4+

IL-1

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cells

()

20

10

0

107 067

122764

69 169

39550

Foxp3 CD4 gated

IL-1

7A

ApcMin+

ApcMin+

lowastlowast

(b)

CD4

T ce

lls (

)

Wild type Wild type

Foxp3+ Gata-3minus Foxp3minus Gata-3+

969 264

29348

232 267

882413

Wild type

Foxp3CD4 gated

30

15

0

Gat

a-3

ApcMin+ ApcMin+

ApcMin+

lowast

lowastlowastlowast

(c)

512 642

80

40

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389

554

255

152

Wild type

Heli

os

Foxp3CD4 gated Wild type

CD4+

Heli

os+

Foxp

3+ ce

lls (

)

ApcMin+

ApcMin+

lowastlowastlowast

(d)

Figure 2Th17 cells and altered Foxp3+ Tregs cells are both increased in the lamina propria of119860119901119888Min+mice (andashd) Four-month-old119860119901119888Min+

mice and their littermate controls (119899 = 3 for each group)were sacrificed Single cell suspensions of LP cells were analyzed by flow cytometry LPcells were stimulated for 6 hrs to detect IL-17A A representative of three independent experiments is shown Two-tailed 119905-test was performedlowastlowastlowast119901 lt 00005 lowastlowast119901 lt 0005 lowast119901 lt 005 ns not significant


Tumornumber

76 30 39 87

Inhibition ()

65 55 0

Total

0

50

100

Tum

or n

umbe

rs (N

)lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastApcM

in+

Treg

WT

Treg

Apc H

ET T

reg

No

Treg

ldquonsrdquo

(a)

27 33 58 30

ldquonsrdquo 89 81 0

Tum

or n

umbe

rs (N

)

0

20

40

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

Large (gt2mm)

WT

Treg

Apc H

ET T

reg

No

Treg

ApcM

in+

Treg

(b)

49 265 326 57

54 43 0

Tum

or n

umbe

rs (N

)

0

40

80

lowastlowast

lowastlowast

Small (05ndash2mm)

WT

Treg

Apc H

ET T

reg

No

Treg

ApcM

in+

Treg

ldquonsrdquo

(c)

IL-23

c-myc

IL-6

Apc HET Treg Wild typeNo Treg Apc HET Treg Wild typeNo Treg


0030

0015

0000

004

002

000

012

006

000

10

05

00

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

IL-1120573

lowastlowast

(d)

Figure 3 Continued


Total

Tumor

Tum

or n

umbe

rs (N

)

number83 50

Inhibition ()

0 40

No Treg IL-17A KO

lowastlowast

120

60

0

ApcMin+ Treg

(e)

No Treg IL-17A KO

Tum

or n

umbe

rs (N

)29 13

0 55

lowastlowastlowast

40

20

0

Large (gt2mm)

ApcMin+ Treg

(f)

No Treg IL-17A KO

Tum

or n

umbe

rs (N

)

54 37

0 31

lowast80

40

0

Small (05ndash2mm)

ApcMin+ Treg

(g)

Figure 3 The heterozygous Apc gene mutation in Treg alone does not impair Treg-mediated tumor regression (andashc) 4 times 105 CD4+Foxp3+cells were isolated from 6-7-week-old119860119901119888Min+-FIR mice their wild type littermate controls (FIR mice) and Foxp3-Cre119860119901119888flox+ mice by cellsorting and then transferred to 3-month-old 119860119901119888Min+ mice Tumor numbers were measured 4 weeks after adoptive transfer Large tumors(gt20mm) and small tumors (05ndash20mm) were counted (d) Tumors (gt20mm) from each group of mice were excised and analyzed byreal time PCR Each group had more than 5 polyps analyzed mRNA expression was normalized against HPRT for their relative expressionlevel (HPRT = 1) (endashg) 4 times 105 Tregs from 6-7-week-old IL-17A deficient (KO)-119860119901119888Min+ mice were transferred to 3-month-old 119860119901119888Min+

mice (119899 = 6 per group) Tumor numbers were measured 4 weeks after adoptive transfer Data are shown as mean plusmn SEM For (andashc) one-wayANOVA analysis with Bonferronirsquos post hoc test was used For other analyses two-tailed 119905-tests were performed for all groups lowastlowastlowast119901 lt 00005lowastlowast119901 lt 0005 lowast119901 lt 005 ns not significant

proliferation of 119860119901119888Min+ Treg Taken together our resultsshow that 119860119901119888Min+ Foxp3+ Tregs undergo robust expansionin the intestinal tumor microenvironment and these Treghave altered Gata-3 expression that is important for Tregfunction in vivo

33 The Heterozygous Apc Gene Mutation in Treg Alone DoesNot Impair Treg-Mediated Control of Intestinal TumorigenesisNext we questioned whether the heterozygous mutation ofApc gene in Treg is responsible for their dysfunction inintestinal tumorigenesis It has been known that 119860119901119888Min+

mice display thymic atrophy and altered bone marrow sub-populations Transplantation of wild type bone marrow into119860119901119888Min+ mice does not correct these phenotypes thus the

developmental environment of immune cells is altered [21]It has been demonstrated that adoptive transfer of wild typeTreg could effectively inhibit intestinal tumorigenesis [14]

As the heterozygous mutation of the Apc gene does occurin all cell types in 119860119901119888Min+ mice we decided to generate aTreg-specific heterozygous Apc gene deletion using Foxp3-Cre119860119901119888flox+ (orApcHET)miceThis allowed us to assess therole of Treg-specificApc genemutation Tregs from this strainof mice express a truncated form of Apc protein having 580amino acids (ApcHET Treg) Unlike the complete deletion ofthe Apc gene in Foxp3-specific manner lymphocytes in ApcHETmice develop in amouse with a wild type hematopoietic

system (Supplementary Figure 2) We isolated splenic ApcHET Tregs based on YFP expression and transferred theminto 3 month-old 119860119901119888Min+ mice

Interestingly Apc HET Tregs showed a similar levelof inhibition of total tumor numbers as wild type splenicTregs (Figure 3(a)) Wild type Foxp3+ splenic Tregs potentlydecreased both large (Figure 3(b) 89) and small intesti-nal tumors (Figure 3(c) 54) The degree of inhibition bythese Apc HET Tregs was more evident in large tumors(Figure 4(b) 81) than small tumors (Figure 3(c) 46)Further analysis of excised intestinal polyps showed that theadoptive transfer of Apc HET Tregs reduced the levels ofproinflammatory mediators IL-6 IL-1120573 and IL-23 in thetumormicroenvironment compared with wild type intestinaltissue (Figure 3(d)) but the expression level of TNF was notdecreased (Supplementary Figure 3)The transfer ofApcHETTregs decreased the expression of c-myc in polyps indicatingthat the aberrant proliferation in the intestinal tract hasbeen inhibited (Figure 3(d)) Endogenous 119860119901119888Min+ Tregswere isolated from 6-7 week-old119860119901119888Min+ mice Importantly119860119901119888Min+ mice showed no readily visible tumors at this age

in our colonies Isolated 119860119901119888Min+ Tregs were adoptivelytransferred to 3-month-old 119860119901119888Min+ mice to compare theirinhibitory capacity withApcHETTreg Interestingly endoge-nous119860119901119888Min+ Tregs were defective in their ability to decreasetumor numbers (Figures 3(a) 3(b) and 3(c)) This suggests


that the direct effect of the heterozygous Apc gene mutationin Treg is minimal whenApcHETTreg cells develop in a wildtype thymic environment

Next we questioned whether the exposure to tumor bur-den is important for119860119901119888Min+ Tregs for their tumor regressorfunction Previously it has been demonstrated that IL-17Ais a potent proinflammatory cytokine that drives intestinaltumorigenesis in 119860119901119888Min+ mice [17 27 28] We previouslyshowed that ablation of IL-17A in 119860119901119888Min+ mice achievedapproximately 90 inhibition of polyposis even at 20 weekssuggesting that the effect of IL-17A deficiency is long-lastingto inhibit the formation of a tumor microenvironment [17]We questioned whether Treg with the 119860119901119888Min+ mutationfrom a mouse with a greatly reduced number of intestinaltumors could regress tumors To this end we isolated Tregfrom IL-17A deficient-119860119901119888Min+ mice and transferred theminto 3-month-old 119860119901119888Min+ mice IL-17A deficient-119860119901119888Min+

mice have very few tumors by 6-7 weeks and thus Tregfrom this strain have not yet encountered a mature tumormicroenvironment

We observed that Tregs from IL-17A deficient 119860119901119888Min+

mice reduced tumors by 40 (Figure 3(e)) Large tumorswere more effectively inhibited (55) than small tumors(31) (Figures 3(f) and 3(g)) These data suggest that theexposure to tumor burden is critical to impair119860119901119888Min+ Tregsin the LP Taken together our results suggest that thymicT cell development and the exposure of 119860119901119888Min+ Tregs tothe tumor microenvironment are critical to impair theircapability for inhibiting intestinal tumorigenesis

34 Wild Type Inducible Treg Can Decrease Intestinal TumorMultiplicity It has been also reported that the maintenanceof adaptive Treg in the intestine is important to maintainhomeostasis [29 30] We tested whether 119860119901119888Min+ naıveCD4 T cells are altered in their ability to generate inducibleTregs (iTreg) Interestingly splenic 119860119901119888Min+ naıve CD4 Tcells were more readily induced to become iTregs than theirwild type counterparts (Figure 4(a)) There is approximatelya 4-fold increase in Foxp3+ cells in the presence of TGF-120573 (Figure 4(a)) This suggests the possibility that Treg couldbe induced more readily in the tumor microenvironmentTo test whether increased iTreg populations might correctintestinal tumorigenesis we adoptively transferred in vitrogenerated wild type iTreg and 119860119901119888Min+ iTreg Notably wildtype iTreg effectively reduced intestinal tumornumbers (69Figure 4(b)) while 119860119901119888Min+ iTregs showed only a partialinhibitory effect (26) Wild type iTregs reduced both largetumors (86 Figure 4(c)) as well as small tumors (61Figure 4(d)) This poor inhibitory capability of 119860119901119888Min+

iTreg wasmainly observed in small tumors (16 Figure 4(c))rather than large tumors (51 reduction Figure 4(d)) Thelimited capacity of 119860119901119888Min+ iTregs in terms of inhibitingtumor numbers suggests that these Tregs have been alteredby the intestinal tumor microenvironment This is alsoconsistent with our results above that the impaired T celldevelopment of 119860119901119888Min+ Treg affected their capacity to limittumor multiplicity and sizes of tumors (Figures 4(a)ndash4(c))

Hence both splenic119860119901119888Min+ nTreg and iTreg were function-ally impaired in 119860119901119888Min+ mice The expansion of Treg inthe LP was markedly inhibited by wild type iTreg transfersuggesting that wild type iTreg inhibited the formation oftumor microenvironment that induces Treg cell expansion(Figure 4(e)) Our results suggest that 119860119901119888Min+ naıve T cellshave an altered capacity for generating iTreg and are func-tionally impaired by the intestinal tumor microenvironment

4 Discussion

In this study we demonstrated the importance of T cell devel-opment and tumormicroenvironment in inhibiting intestinaltumormultiplicity byTregs in119860119901119888Min+mice Since the role ofregulatory T cells is to control inflammation yet they failedto inhibit tumor numbers the accumulation of endogenous119860119901119888Min+ Tregs in the LP raised a question on their role

in the intestinal tumorigenesis The marked reduction infrequency ofGata-3+Treg cells as well as the increase ofGata-3-Tregs suggests that tumor microenvironment favored theexpansion of these altered 119860119901119888Min+ Tregs

It should be noted that the Treg-specific heterozygousApc mutation did not alter the function of Tregs to limit inboth large and small sizes of tumors markedly suggesting thecritical role of thymic development in Treg-mediated controlof tumor multiplicity in 119860119901119888Min+ mice Unlike 119860119901119888Min+

mice Apc HET mice do not have a germline mutationin the Apc gene APC protein is a large protein and hasmultiple 120573-catenin binding sites and thus it might be possiblethat different length of mutant APC protein between ApcHET Tregs and 119860119901119888Min+ Tregs (580 and 850 amino acidsresp) might have resulted in differential results in our studyHowever recently it has been shown that canonical Wntsignaling negatively regulates suppressor function in colitismodel using 119860119901119888Min+ Tregs [31] Since shorter form of APCprotein would result in enhanced Wnt signaling in Tregswith fewer 120573-catenin binding sites Apc HET Tregs wouldhave more severe loss of suppressor function compared to119860119901119888Min+ Tregs Therefore it is likely that 119860119901119888Min+ Tregs

is primarily impaired by dysfunctional thymic developmentin 119860119901119888Min+ mice More future studies regarding the regu-latory function of APC protein in Tregs are warranted Wepreviously showed that the lack of IL-17A in 119860119901119888Min+ micecompletely restored thymic as well as splenic cellularity while119860119901119888Min+ mice underwent thymic atrophy and splenomegaly

[17] As 119860119901119888Min+ iTreg that were generated from naıve CD4T cells that did not encounter tumor microenvironment italso suggests that both nTregs and iTregs in119860119901119888Min+mice areimpaired in tumor inhibition function in an altered thymicdevelopment Of note 119860119901119888Min+ iTregs lost most of theirinhibitory effect on tumor multiplicity yet they still retaineda sizeable effect on inhibition of large tumors suggesting thatthe defect in 119860119901119888Min+ iTregs is mainly in controlling tumorinduction while their inhibitory capacity to control tumorgrowth in tumor microenvironment is conserved similar toIL-17A KO 119860119901119888Min+ Tregs The adoptive transfer of Treg


Wild type

0 0

126894

0 0

60

30

0581479

Foxp3

SSC

Foxp

3+ ce

lls (

)

Wild type ApcMin+

ApcMin+ lowastlowast

(a)

Total

Tumor number

83 26 61

Inhibition () 0 69 26

0

50

100

Tum

or n

umbe

rs (N

)

lowastlowastlowastlowastlowast

lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(b)

27 4 13

0 86 51

Large (gt2 mm)

Tum

or n

umbe

rs (N

)

0

20

40 lowastlowastlowast

lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(c)

56

0

22 48

61 16

Small (05ndash2 mm)

Tum

or n

umbe

rs (N

)

0

40

80 lowastlowastlowast lowastlowastlowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(d)

50

25

0

938 347

136423

60 111

137692

Foxp3

CD39

CD4+

Fox

p3+

CD39

+ ce

lls (

)

+ no Treg + WT iTreg

+ no Treg + WT iTreglowastlowast

ApcMin+ ApcMin+

ApcMin+ ApcMin+

(e)

Figure 4Wild type inducible Tregs can regress intestinal tumors in119860119901119888Min+mice (a) Splenic naıve CD4T cells from6-7-week-old119860119901119888Min+-FIR mice and their wild type littermate controls were isolated and differentiated into inducible Tregs (iTregs) for 120 hrs A representativeof three independent experiments is shown lowastlowast119901 lt 0005 (bndashd) Splenic naıve CD4 T cells from 6-7-week-old 119860119901119888Min+-FIR mice and theirwild type littermate controls were isolated and differentiated into inducible Tregs (iTregs) and sorted 4 times 105 iTregs were transferred to 4 times105 (119899 = 7 for WT iTreg 119899 = 6 for 119860119901119888Min+ iTreg) Tumor numbers were measured 4 weeks after adoptive transfer Data are shown as meanplusmn SEM For (bndashd) one-way ANOVA analysis with Bonferronirsquos post hoc test was used For other analyses two-tailed 119905-test was performedfor all groups lowastlowastlowast119901 lt 00005 lowastlowast119901 lt 0005 lowast119901 lt 005 ns not significant (e) LP cells from 119860119901119888Min+ mice that received WT iTregs werecompared with 119860119901119888Min+ mice (119899 = 3) and analyzed by flow cytometry


from IL-17A-deficient119860119901119888Min+mice and119860119901119888Min+ iTreg alsosuggests that the reduced tumor burden and normal thymicdevelopment of T cells are important to control intestinaltumor multiplicity Since the decrease of small tumor num-bers (31) was less potent than that of large tumors (55)it could be noted that the role of IL-17A from 119860119901119888Min+

Tregs is more important for large tumor formation in IL-17A-mediated tumor microenvironment Thus our results arguethat the control of tumor multiplicity by Tregs requires boththe absence of IL-17A and a functional thymus

While exogenous agents such as enterotoxigenic bacteriacan induce and expand Th17 cells in the LP [27] our resultssuggest that the marked expansion of Th17 cells may notbe required for spontaneous intestinal tumorigenesis buta moderate increase of Th17 cells is sufficient to inducetumorigenesis It is possible that the biological potency ofIL-17A is more important than Th17 cell expansion as IL-17A-deficient 119860119901119888Min+ mice showed marked reduction ofintestinal tumorigenesis [17] Previous reports have suggestedthat a sizable fraction of Treg may coexpress Foxp3 and IL-17 [32] However our direct analysis of cells from the LP in119860119901119888Min+ mice did not find such a high percentage of IL-

17A+Foxp3+ CD4 T cells This is may be due to differencesin the particular mouse housing facility the mouse strain orthe intestinal microenvironment since IL-17 production isvariable depending on gut microflora [33]

A series of recent findings in colon cancer implied thatthe role of accumulated Treg may be unique in that theyperform amore inhibitory role on cancer development [10 1213] On the other hand other studies reported controversialresults regarding the antitumorigenic role of Treg in sporadiccolorectal cancer patients [34 35] The heterogeneous natureof the human intestinal tumor microenvironment is likely tocontribute to this controversy for the role of Treg Thereforethe role of Tregmay primarily depend on the tumormicroen-vironment in sporadic colon cancer while in FAP patientsthere is also an altered T cell development issue that impairsthe function of Treg to regress intestinal tumors

As both FAP patients and 119860119901119888Min mice show typicallya high incidence of rapidly developing tumors our study isimportant to provide a potential clinical strategy for FAP inpatients particularly in the early stages of intestinal tumori-genesis where the role of Treg could be antitumorigenic Ourpositive findings using wild type iTreg may also be of useclinically since the number of natural Tregs in human bloodis low Taken together our results offer novel insight into themechanism of early and rapid onset of FAP by implicating alack of functional Treg cells as a major factor

5 Conclusion

Our work suggests that thymic T cell development may be animportant factor to consider in rapid intestinal tumorigenesisin119860119901119888Min+mice driven by IL-17A that resemble FAPpatientswho bear the germline Apc gene mutation unlike fromsporadic colorectal cancer (CRC)

Conflict of Interests

The authors claim no conflict of interests The authors do nothave competing financial conflict of interests

Acknowledgments

This work was supported by NIH 1R21AI107957-01 andNIH 1R01CA168670-01 (awarded to Alfred L M Bothwell)The authors thank Anna Fuller foundation and TrudeauPostdoctoral fellowship (awarded to Wook-Jin Chae) Theauthors thank Gouzel Tokmulina andThomas Taylor for cellsorting

References

[1] A Mantovani P Allavena A Sica and F Balkwill ldquoCancer-related inflammationrdquo Nature vol 454 no 7203 pp 436ndash4442008

[2] A Y Rudensky ldquoRegulatory T cells and Foxp3rdquo ImmunologicalReviews vol 241 no 1 pp 260ndash268 2011

[3] S K Lathrop N A Santacruz D Pham J Luo and C-S HsiehldquoAntigen-specific peripheral shaping of the natural regulatory Tcell populationrdquo Journal of Experimental Medicine vol 205 no13 pp 3105ndash3117 2008

[4] L J Thompson A C Valladao and S F Ziegler ldquoCuttingedge de novo induction of functional Foxp3+ regulatory CD4T cells in response to tissue-restricted self antigenrdquo Journal ofImmunology vol 186 no 8 pp 4551ndash4555 2011

[5] S Sakaguchi T Yamaguchi T Nomura and M Ono ldquoRegu-latory T cells and immune tolerancerdquo Cell vol 133 no 5 pp775ndash787 2008

[6] Y Wang M A Su and Y Y Wan ldquoAn essential role of thetranscription factor GATA-3 for the function of regulatory Tcellsrdquo Immunity vol 35 no 3 pp 337ndash348 2011

[7] E A Wohlfert J R Grainger N Bouladoux et al ldquoGATA3controls Foxp3+ regulatory T cell fate during inflammation inmicerdquo The Journal of Clinical Investigation vol 121 no 11 pp4503ndash4515 2011

[8] W Zou ldquoRegulatory T cells tumour immunity and immun-otherapyrdquo Nature Reviews Immunology vol 6 no 4 pp 295ndash307 2006

[9] W L Byrne K H G Mills J A Lederer and G C OrsquoSullivanldquoTargeting regulatory T cells in cancerrdquoCancer Research vol 71no 22 pp 6915ndash6920 2011

[10] P Salama M Phillips F Grieu et al ldquoTumor-infiltratingFOXP3+ T regulatory cells show strong prognostic significancein colorectal cancerrdquo Journal of Clinical Oncology vol 27 no 2pp 186ndash192 2009

[11] F Pages A Berger M Camus et al ldquoEffector memory T cellsearly metastasis and survival in colorectal cancerrdquo The NewEngland Journal of Medicine vol 353 no 25 pp 2654ndash26662005

[12] D M Frey R A Droeser C T Viehl et al ldquoHigh fre-quency of tumor-infiltrating FOXP3+ regulatory T cells predictsimproved survival in mismatch repair-proficient colorectalcancer patientsrdquo International Journal of Cancer vol 126 no 11pp 2635ndash2643 2010

[13] S Ladoire F Martin and F Ghiringhelli ldquoPrognostic role ofFOXP3+ regulatory T cells infiltrating human carcinomas the


paradox of colorectal cancerrdquo Cancer Immunology Immuno-therapy vol 60 no 7 pp 909ndash918 2011

[14] S E Erdman J J Sohn V P Rao et al ldquoCD4+CD25+regulatory lymphocytes induce regression of intestinal tumorsin ApcMin+ micerdquo Cancer Research vol 65 no 10 pp 3998ndash4004 2005

[15] K W Kinzler and B Vogelstein ldquoLessons from hereditarycolorectal cancerrdquo Cell vol 87 no 2 pp 159ndash170 1996

[16] I Nathke ldquoCytoskeleton out of the cupboard colon cancer andcytoskeletal changes induced by loss of APCrdquo Nature ReviewsCancer vol 6 no 12 pp 967ndash974 2006

[17] W-J Chae T F Gibson D Zelterman L Hao O Henegariuand A L M Bothwell ldquoAblation of IL-17A abrogates progres-sion of spontaneous intestinal tumorigenesisrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol107 no 12 pp 5540ndash5544 2010

[18] H Shibata K Toyama H Shioya et al ldquoRapid colorectaladenoma formation initiated by conditional targeting of theApc generdquo Science vol 278 no 5335 pp 120ndash133 1997

[19] B Weigmann I Tubbe D Seidel A Nicolaev C Becker andM F Neurath ldquoIsolation and subsequent analysis of murinelamina propria mononuclear cells from colonic tissuerdquo NatureProtocols vol 2 no 10 pp 2307ndash2311 2007

[20] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[21] P L Coletta A M Muller E A Jones et al ldquoLymphodepletionin the Apc119872119894119899+ mouse model of intestinal tumorigenesisrdquoBlood vol 103 pp 1050ndash1058 2004

[22] W Zou and N P Restifo ldquoT11986717 cells in tumour immunity andimmunotherapyrdquoNature Reviews Immunology vol 10 no 4 pp248ndash256 2010

[23] J Shoemaker M Saraiva and A OrsquoGarra ldquoGATA-3 directlyremodels the IL-10 locus independently of IL-4 inCD4+ T cellsrdquoThe Journal of Immunology vol 176 no 6 pp 3470ndash3479 2006

[24] T Akimova U H Beier L Wang M H Levine and W WHancock ldquoHelios expression is a marker of T cell activation andproliferationrdquo PLoS ONE vol 6 no 8 Article ID e24226 2011

[25] R A Gottschalk E Corse and J P Allison ldquoExpressionof Helios in peripherally induced Foxp3+ regulatory T cellsrdquoJournal of Immunology vol 188 no 3 pp 976ndash980 2012

[26] A M Thornton P E Korty D Q Tran et al ldquoExpressionof Helios an Ikaros transcription factor family member dif-ferentiates thymic-derived from peripherally induced Foxp3+T regulatory cellsrdquo Journal of Immunology vol 184 no 7 pp3433ndash3441 2010

[27] S Wu K-J Rhee E Albesiano et al ldquoA human coloniccommensal promotes colon tumorigenesis via activation of Thelper type 17 T cell responsesrdquo Nature Medicine vol 15 no 9pp 1016ndash1022 2009

[28] I Kryczek K Wu E Zhao et al ldquoIL-17+ regulatory T cells inthe microenvironments of chronic inflammation and cancerrdquoThe Journal of Immunology vol 186 no 7 pp 4388ndash4395 2011

[29] J L Round and S KMazmanian ldquoInducible Foxp3+ regulatoryT-cell development by a commensal bacterium of the intestinalmicrobiotardquo Proceedings of the National Academy of Sciences ofthe United States of America vol 107 no 27 pp 12204ndash122092010

[30] M J Barnes and F Powrie ldquoRegulatory T cells reinforceintestinal homeostasisrdquo Immunity vol 31 no 3 pp 401ndash4112009

[31] J van Loosdregt V Fleskens M Tiemessen et al ldquoCanonicalWnt signaling negatively modulates regulatory T cell functionrdquoImmunity vol 39 no 2 pp 298ndash310 2013

[32] E Gounaris N R Blatner K Dennis et al ldquoT-regulatorycells shift from a protective anti-inflammatory to a cancer-promoting proinflammatory phenotype in polyposisrdquo CancerResearch vol 69 no 13 pp 5490ndash5497 2009

[33] I I Ivanov R D L Frutos N Manel et al ldquoSpecific microbiotadirect the differentiation of IL-17-producingT-helper cells in themucosa of the small intestinerdquo Cell Host andMicrobe vol 4 no4 pp 337ndash349 2008

[34] S Le Gouvello S Bastuji-Garin N Aloulou et al ldquoHighprevalence of Foxp3 and IL17 in MMR-proficient colorectalcarcinomasrdquo Gut vol 57 no 6 pp 772ndash779 2008

[35] G Betts E Jones S Junaid et al ldquoSuppression of tumour-specific CD4+ T cells by regulatory T cells is associated withprogression of human colorectal cancerrdquo Gut vol 61 pp 1163ndash1171 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


of APC protein The Apc gene undergoes further loss ofheterozygosity (LOH) via the deletion of the wild type Apcallele and this results in activation of excessive Wnt signalingthat drives polyp formation in the intestinal tract [16] FAPpatients develop colon polyps early in life and all affectedindividuals develop cancer In contrast sporadic colon cancerpatients do not have a germline mutation in the Apc geneand generally develop disease much later in life However therole of Treg cells in the rapid onset of intestinal tumorigenesis119860119901119888Min+ mice has not been addressedIn the current study we characterized 119860119901119888Min+ mice

Tregs in the LP and investigated the role of the heterozygousApc gene mutation in Tregs in a spontaneous intestinaltumorigenesis model Tregs from IL-17A deficient 119860119901119888Min+

mice were used to assess the effect of the tumor environmentand functional thymus

2 Materials and Methods

21 Mice 119860119901119888Min+ mice were purchased from the JacksonLaboratory and have been bred and maintained in our SPFmouse facility in conventional housing condition All othermouse strains mentioned below were also housed in ourSPF mouse facility in conventional housing condition insuspended stainless wire-mesh cages Mouse health statuswas regularly monitored by Yale University InstitutionalAnimal Care and no infections were reported in animals usedin this study The animals were kept under normal lightdarkcycle In all experiments wild type (WT) littermates of119860119901119888

Min+ mice on the C57BL6 background were used IL-17A KO-119860119901119888Min+mice were generated previously in C57Bl6background [17] Foxp3-IRES-RFPmice (FIR) were providedby Richard Flavell (Yale University) and bred with 119860119901119888Min+

mice and C57BL6 mice to generate 119860119901119888Min+-Foxp3-IRES-RFP (119860119901119888Min+-FIR) mice in C57Bl6 background Foxp3-Cre transgenic mice were generously provided by AlexanderRudensky (Sloan-Kettering Cancer Center) and maintainedin our colonies for more than 6 generations in C57Bl6background 119860119901119888floxflox mice in C57Bl6 background wereobtained from the NCI and reported previously [18] Thisstrain was bred to Foxp3-Cre transgenic mice resulting intruncation of the Apc protein at amino acid 580 in Foxp3+cells All mouse protocols were approved by the Yale Uni-versity Institutional Animal Care and Use Committee inaccordance with the Association for Assessment and Accred-itation of Laboratory Animal Care International Genotypingconditions and primer sequences for mouse strains used inthis study are as follows119860119901119888

Min+ mice 94∘C 4min (94∘C 45 sec 64∘C 45 secminus03∘C 45 seccycle 72∘C 1min) 35 cycles 72∘C 5min

Forward primer 51015840-ttc cac ttt ggc ata agg c-31015840 Reverseprimer 51015840-ttc tga gaa aga cag aag tta-31015840

Foxp3-IRES-RFP mice 94∘C 3min (94∘C 30 sec 65∘C30 secminus03∘C45 seccycle 72∘C40 sec) 36 cycles 72∘C5minPrimer 1 51015840-caaaac caagaaaaggtgggc-31015840 Primer 2 51015840-ggaatg-ctcgtcaagaagacagg-31015840 Primer 3 51015840-cagtgctgttgctgtgtaagggtc-31015840

119860119901119888floxflox mice 94∘C 5min (94∘C 30 sec 58∘C 30 sec

72∘C 45 sec) 32 cycles 72∘C 5min Forward primer 51015840-gag aaaccc tgt ctc gaa aaa a-31015840 Reverse primer 51015840-agt gct gtt tct atgagt caa c-31015840

Foxp3-Cre transgenic mice 94∘C 3min (94∘C 30 sec52∘C 1min 72∘C 1min) 35 cycles 72∘C 2min Forwardprimer 51015840-agg atg tga ggg act acc tcc tgt a-31015840 Reverse primer51015840-tcc ttc act ctg att ctg gca att t-31015840

IL-17A KO mice 94∘C 2min (94∘C 15 sec 64∘C 30 sec72∘C 1min) 40 cycles 72∘C 5min 10min primer 1 51015840-actctt-catccacctcacacga-31015840 primer 2 51015840-gccatgatatagacgttgtggc-31015840primer 3 51015840-cagcatcagagactagaaggga-31015840 Primers 1 and 2 wereused to detect wild type allele and primers 1 and 3 were usedto detect mutant allele

22 Tumor Counts and Size Animals were examined formortality and clinical signs throughout the study All micesurviving to the end of studywere euthanized for determiningtumor size and number Following euthanasia the entiresmall intestinal tract from each mouse was immediatelyremoved and divided into 3 sections from the duodenumto the ileum The intestinal sections were opened anda dissecting microscope (magnification 20x) was used toidentify tumors in each section A calibrated reticle in aneyepiece of the dissecting scope was used to measure thediameter of each tumor at its maximum dimension Lesionsless than 05mm in diameter were not enumerated Largetumors (gt2mm) or small tumors (05ndash2mm) were counted

23 Adoptive Transfer Model and Treg Depletion For theisolation of119860119901119888Min+ Tregs and wild type Tregs spleens from6-7-week-old 119860119901119888Min+-Foxp3-IRES-RFP mice and their lit-termate controls (Foxp3-IRES-RFP) were prepared as singlecell suspensions After RBC (red blood cell) lysis cells werestained with CD16CD32 FcR (Fc Receptor) antibody (clone24G2) to block nonspecific binding and then subsequentlystained with anti-CD4 (clone RM4-5) antibody RFP+ cellswere sorted by FACSVantage and then transferred immedi-ately to 3-month-old119860119901119888Min+-Foxp3-IRES-RFPmice RFP+cells were subsequently sorted and then immediately trans-ferred into 3-month-old 119860119901119888Min+ mice For Apc mutantTregs (Apc HET Tregs) splenic CD4+YFP+ cells were iso-lated from 6-week-old Foxp3-Cre 119860119901119888flox+ mice and Foxp3-Cre transgenic mice CD4+CD45RBlowCD25-splenic Tregswere isolated from 6ndash8-week old IL-17A KO-119860119901119888Min+ miceFor each transfer experiment 4 times 105 cells were injected peranimal intravenously Mice were analyzed on week 16 Toperform cellular analysis mice were sacrificed and LP (lam-ina propria) cells were prepared For histological analysisthe terminal ileum from each mouse was dissected fixed in10 formalin and washed in PBS solution The specimenwas paraffin-embedded sectioned at 5 120583m and stained withhematoxylin and eosin (HampE)

24 Antibodies and Reagents Anti-mouse CD4 (clone RM4-5) anti-Helios (clone 22F6) anti-Foxp3 (clone FJK-16s)anti-ROR120574t (clone AFKJS-9) anti-GATA-3 (clone TWAJ)






3 Results







507

175

135

379

Wild type

CD4

Foxp

3

SPLApcMin+

(a)

Wild type

SPLns

Foxp

3+ ce

llsam

ong

CD4

T ce

lls (

)

30

15

0

ApcMin+

(b)

634

36

626

322

Wild type

Foxp

3

MLN

CD4

ApcMin+

(c)

Wild type

nsMLN

Foxp

3+ ce

lls

amon

g CD

4 T

cells

()

250

125

00

ApcMin+

(d)

CD4

252

483

21

194

Foxp

3

LPWild type ApcMin+

(e)

Foxp

3+ ce

lls

amon

g CD

4 T

cells

()

Wild type

LP

60

30

0

ApcMin+

lowastlowast

(f)






239 067

144611

106 164

382496Foxp3

CD4 gated

Wild type

Wild type

30

15

0

ApcMin+

ApcMin+

ROR120574

tlowast

CD4+

ROR120574

t+ce

lls (

)

(a)

Wild type

Wild type

CD4+

IL-1

7A+

cells

()

20

10

0

107 067

122764

69 169

39550

Foxp3 CD4 gated

IL-1

7A

ApcMin+

ApcMin+

lowastlowast

(b)

CD4

T ce

lls (

)

Wild type Wild type


969 264

29348

232 267

882413

Wild type

Foxp3CD4 gated

30

15

0

Gat

a-3

ApcMin+ ApcMin+

ApcMin+

lowast

lowastlowastlowast

(c)

512 642

80

40

0448262

389

554

255

152

Wild type

Heli

os


CD4+

Heli

os+

Foxp

3+ ce

lls (

)

ApcMin+

ApcMin+

lowastlowastlowast

(d)




Tumornumber

76 30 39 87

Inhibition ()

65 55 0

Total

0

50

100

Tum

or n

umbe

rs (N

)lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastApcM

in+

Treg

WT

Treg

Apc H

ET T

reg

No

Treg

ldquonsrdquo

(a)

27 33 58 30


Tum

or n

umbe

rs (N

)

0

20

40

lowastlowastlowast


lowastlowastlowast

Large (gt2mm)

WT

Treg

Apc H

ET T

reg

No

Treg

ApcM

in+

Treg

(b)

49 265 326 57

54 43 0

Tum

or n

umbe

rs (N

)

0

40

80

lowastlowast

lowastlowast

Small (05ndash2mm)

WT

Treg

Apc H

ET T

reg

No

Treg

ApcM

in+

Treg

ldquonsrdquo

(c)

IL-23

c-myc

IL-6



0030

0015

0000

004

002

000

012

006

000

10

05

00

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

IL-1120573

lowastlowast

(d)

Figure 3 Continued


Total

Tumor

Tum

or n

umbe

rs (N

)

number83 50

Inhibition ()

0 40

No Treg IL-17A KO

lowastlowast

120

60

0

ApcMin+ Treg

(e)

No Treg IL-17A KO

Tum

or n

umbe

rs (N

)29 13

0 55

lowastlowastlowast

40

20

0

Large (gt2mm)

ApcMin+ Treg

(f)

No Treg IL-17A KO

Tum

or n

umbe

rs (N

)

54 37

0 31

lowast80

40

0

Small (05ndash2mm)

ApcMin+ Treg

(g)




















4 Discussion








Wild type

0 0

126894

0 0

60

30

0581479

Foxp3

SSC

Foxp

3+ ce

lls (

)

Wild type ApcMin+


(a)

Total

Tumor number

83 26 61


0

50

100

Tum

or n

umbe

rs (N

)


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(b)

27 4 13

0 86 51

Large (gt2 mm)

Tum

or n

umbe

rs (N

)

0

20


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(c)

56

0

22 48

61 16

Small (05ndash2 mm)

Tum

or n

umbe

rs (N

)

0

40


ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(d)

50

25

0

938 347

136423

60 111

137692

Foxp3

CD39

CD4+

Fox

p3+

CD39

+ ce

lls (

)



ApcMin+ ApcMin+

ApcMin+ ApcMin+

(e)









5 Conclusion




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results







507

175

135

379

Wild type

CD4

Foxp

3

SPLApcMin+

(a)

Wild type

SPLns

Foxp

3+ ce

llsam

ong

CD4

T ce

lls (

)

30

15

0

ApcMin+

(b)

634

36

626

322

Wild type

Foxp

3

MLN

CD4

ApcMin+

(c)

Wild type

nsMLN

Foxp

3+ ce

lls

amon

g CD

4 T

cells

()

250

125

00

ApcMin+

(d)

CD4

252

483

21

194

Foxp

3

LPWild type ApcMin+

(e)

Foxp

3+ ce

lls

amon

g CD

4 T

cells

()

Wild type

LP

60

30

0

ApcMin+

lowastlowast

(f)






239 067

144611

106 164

382496Foxp3

CD4 gated

Wild type

Wild type

30

15

0

ApcMin+

ApcMin+

ROR120574

tlowast

CD4+

ROR120574

t+ce

lls (

)

(a)

Wild type

Wild type

CD4+

IL-1

7A+

cells

()

20

10

0

107 067

122764

69 169

39550

Foxp3 CD4 gated

IL-1

7A

ApcMin+

ApcMin+

lowastlowast

(b)

CD4

T ce

lls (

)

Wild type Wild type


969 264

29348

232 267

882413

Wild type

Foxp3CD4 gated

30

15

0

Gat

a-3

ApcMin+ ApcMin+

ApcMin+

lowast

lowastlowastlowast

(c)

512 642

80

40

0448262

389

554

255

152

Wild type

Heli

os


CD4+

Heli

os+

Foxp

3+ ce

lls (

)

ApcMin+

ApcMin+

lowastlowastlowast

(d)




Tumornumber

76 30 39 87

Inhibition ()

65 55 0

Total

0

50

100

Tum

or n

umbe

rs (N

)lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastApcM

in+

Treg

WT

Treg

Apc H

ET T

reg

No

Treg

ldquonsrdquo

(a)

27 33 58 30


Tum

or n

umbe

rs (N

)

0

20

40

lowastlowastlowast


lowastlowastlowast

Large (gt2mm)

WT

Treg

Apc H

ET T

reg

No

Treg

ApcM

in+

Treg

(b)

49 265 326 57

54 43 0

Tum

or n

umbe

rs (N

)

0

40

80

lowastlowast

lowastlowast

Small (05ndash2mm)

WT

Treg

Apc H

ET T

reg

No

Treg

ApcM

in+

Treg

ldquonsrdquo

(c)

IL-23

c-myc

IL-6



0030

0015

0000

004

002

000

012

006

000

10

05

00

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

IL-1120573

lowastlowast

(d)

Figure 3 Continued


Total

Tumor

Tum

or n

umbe

rs (N

)

number83 50

Inhibition ()

0 40

No Treg IL-17A KO

lowastlowast

120

60

0

ApcMin+ Treg

(e)

No Treg IL-17A KO

Tum

or n

umbe

rs (N

)29 13

0 55

lowastlowastlowast

40

20

0

Large (gt2mm)

ApcMin+ Treg

(f)

No Treg IL-17A KO

Tum

or n

umbe

rs (N

)

54 37

0 31

lowast80

40

0

Small (05ndash2mm)

ApcMin+ Treg

(g)




















4 Discussion








Wild type

0 0

126894

0 0

60

30

0581479

Foxp3

SSC

Foxp

3+ ce

lls (

)

Wild type ApcMin+


(a)

Total

Tumor number

83 26 61


0

50

100

Tum

or n

umbe

rs (N

)


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(b)

27 4 13

0 86 51

Large (gt2 mm)

Tum

or n

umbe

rs (N

)

0

20


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(c)

56

0

22 48

61 16

Small (05ndash2 mm)

Tum

or n

umbe

rs (N

)

0

40


ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(d)

50

25

0

938 347

136423

60 111

137692

Foxp3

CD39

CD4+

Fox

p3+

CD39

+ ce

lls (

)



ApcMin+ ApcMin+

ApcMin+ ApcMin+

(e)









5 Conclusion




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















507

175

135

379

Wild type

CD4

Foxp

3

SPLApcMin+

(a)

Wild type

SPLns

Foxp

3+ ce

llsam

ong

CD4

T ce

lls (

)

30

15

0

ApcMin+

(b)

634

36

626

322

Wild type

Foxp

3

MLN

CD4

ApcMin+

(c)

Wild type

nsMLN

Foxp

3+ ce

lls

amon

g CD

4 T

cells

()

250

125

00

ApcMin+

(d)

CD4

252

483

21

194

Foxp

3

LPWild type ApcMin+

(e)

Foxp

3+ ce

lls

amon

g CD

4 T

cells

()

Wild type

LP

60

30

0

ApcMin+

lowastlowast

(f)






239 067

144611

106 164

382496Foxp3

CD4 gated

Wild type

Wild type

30

15

0

ApcMin+

ApcMin+

ROR120574

tlowast

CD4+

ROR120574

t+ce

lls (

)

(a)

Wild type

Wild type

CD4+

IL-1

7A+

cells

()

20

10

0

107 067

122764

69 169

39550

Foxp3 CD4 gated

IL-1

7A

ApcMin+

ApcMin+

lowastlowast

(b)

CD4

T ce

lls (

)

Wild type Wild type


969 264

29348

232 267

882413

Wild type

Foxp3CD4 gated

30

15

0

Gat

a-3

ApcMin+ ApcMin+

ApcMin+

lowast

lowastlowastlowast

(c)

512 642

80

40

0448262

389

554

255

152

Wild type

Heli

os


CD4+

Heli

os+

Foxp

3+ ce

lls (

)

ApcMin+

ApcMin+

lowastlowastlowast

(d)




Tumornumber

76 30 39 87

Inhibition ()

65 55 0

Total

0

50

100

Tum

or n

umbe

rs (N

)lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastApcM

in+

Treg

WT

Treg

Apc H

ET T

reg

No

Treg

ldquonsrdquo

(a)

27 33 58 30


Tum

or n

umbe

rs (N

)

0

20

40

lowastlowastlowast


lowastlowastlowast

Large (gt2mm)

WT

Treg

Apc H

ET T

reg

No

Treg

ApcM

in+

Treg

(b)

49 265 326 57

54 43 0

Tum

or n

umbe

rs (N

)

0

40

80

lowastlowast

lowastlowast

Small (05ndash2mm)

WT

Treg

Apc H

ET T

reg

No

Treg

ApcM

in+

Treg

ldquonsrdquo

(c)

IL-23

c-myc

IL-6



0030

0015

0000

004

002

000

012

006

000

10

05

00

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

IL-1120573

lowastlowast

(d)

Figure 3 Continued


Total

Tumor

Tum

or n

umbe

rs (N

)

number83 50

Inhibition ()

0 40

No Treg IL-17A KO

lowastlowast

120

60

0

ApcMin+ Treg

(e)

No Treg IL-17A KO

Tum

or n

umbe

rs (N

)29 13

0 55

lowastlowastlowast

40

20

0

Large (gt2mm)

ApcMin+ Treg

(f)

No Treg IL-17A KO

Tum

or n

umbe

rs (N

)

54 37

0 31

lowast80

40

0

Small (05ndash2mm)

ApcMin+ Treg

(g)




















4 Discussion








Wild type

0 0

126894

0 0

60

30

0581479

Foxp3

SSC

Foxp

3+ ce

lls (

)

Wild type ApcMin+


(a)

Total

Tumor number

83 26 61


0

50

100

Tum

or n

umbe

rs (N

)


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(b)

27 4 13

0 86 51

Large (gt2 mm)

Tum

or n

umbe

rs (N

)

0

20


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(c)

56

0

22 48

61 16

Small (05ndash2 mm)

Tum

or n

umbe

rs (N

)

0

40


ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(d)

50

25

0

938 347

136423

60 111

137692

Foxp3

CD39

CD4+

Fox

p3+

CD39

+ ce

lls (

)



ApcMin+ ApcMin+

ApcMin+ ApcMin+

(e)









5 Conclusion




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















239 067

144611

106 164

382496Foxp3

CD4 gated

Wild type

Wild type

30

15

0

ApcMin+

ApcMin+

ROR120574

tlowast

CD4+

ROR120574

t+ce

lls (

)

(a)

Wild type

Wild type

CD4+

IL-1

7A+

cells

()

20

10

0

107 067

122764

69 169

39550

Foxp3 CD4 gated

IL-1

7A

ApcMin+

ApcMin+

lowastlowast

(b)

CD4

T ce

lls (

)

Wild type Wild type


969 264

29348

232 267

882413

Wild type

Foxp3CD4 gated

30

15

0

Gat

a-3

ApcMin+ ApcMin+

ApcMin+

lowast

lowastlowastlowast

(c)

512 642

80

40

0448262

389

554

255

152

Wild type

Heli

os


CD4+

Heli

os+

Foxp

3+ ce

lls (

)

ApcMin+

ApcMin+

lowastlowastlowast

(d)




Tumornumber

76 30 39 87

Inhibition ()

65 55 0

Total

0

50

100

Tum

or n

umbe

rs (N

)lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastApcM

in+

Treg

WT

Treg

Apc H

ET T

reg

No

Treg

ldquonsrdquo

(a)

27 33 58 30


Tum

or n

umbe

rs (N

)

0

20

40

lowastlowastlowast


lowastlowastlowast

Large (gt2mm)

WT

Treg

Apc H

ET T

reg

No

Treg

ApcM

in+

Treg

(b)

49 265 326 57

54 43 0

Tum

or n

umbe

rs (N

)

0

40

80

lowastlowast

lowastlowast

Small (05ndash2mm)

WT

Treg

Apc H

ET T

reg

No

Treg

ApcM

in+

Treg

ldquonsrdquo

(c)

IL-23

c-myc

IL-6



0030

0015

0000

004

002

000

012

006

000

10

05

00

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

IL-1120573

lowastlowast

(d)

Figure 3 Continued


Total

Tumor

Tum

or n

umbe

rs (N

)

number83 50

Inhibition ()

0 40

No Treg IL-17A KO

lowastlowast

120

60

0

ApcMin+ Treg

(e)

No Treg IL-17A KO

Tum

or n

umbe

rs (N

)29 13

0 55

lowastlowastlowast

40

20

0

Large (gt2mm)

ApcMin+ Treg

(f)

No Treg IL-17A KO

Tum

or n

umbe

rs (N

)

54 37

0 31

lowast80

40

0

Small (05ndash2mm)

ApcMin+ Treg

(g)




















4 Discussion








Wild type

0 0

126894

0 0

60

30

0581479

Foxp3

SSC

Foxp

3+ ce

lls (

)

Wild type ApcMin+


(a)

Total

Tumor number

83 26 61


0

50

100

Tum

or n

umbe

rs (N

)


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(b)

27 4 13

0 86 51

Large (gt2 mm)

Tum

or n

umbe

rs (N

)

0

20


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(c)

56

0

22 48

61 16

Small (05ndash2 mm)

Tum

or n

umbe

rs (N

)

0

40


ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(d)

50

25

0

938 347

136423

60 111

137692

Foxp3

CD39

CD4+

Fox

p3+

CD39

+ ce

lls (

)



ApcMin+ ApcMin+

ApcMin+ ApcMin+

(e)









5 Conclusion




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Tumornumber

76 30 39 87

Inhibition ()

65 55 0

Total

0

50

100

Tum

or n

umbe

rs (N

)lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastApcM

in+

Treg

WT

Treg

Apc H

ET T

reg

No

Treg

ldquonsrdquo

(a)

27 33 58 30


Tum

or n

umbe

rs (N

)

0

20

40

lowastlowastlowast


lowastlowastlowast

Large (gt2mm)

WT

Treg

Apc H

ET T

reg

No

Treg

ApcM

in+

Treg

(b)

49 265 326 57

54 43 0

Tum

or n

umbe

rs (N

)

0

40

80

lowastlowast

lowastlowast

Small (05ndash2mm)

WT

Treg

Apc H

ET T

reg

No

Treg

ApcM

in+

Treg

ldquonsrdquo

(c)

IL-23

c-myc

IL-6



0030

0015

0000

004

002

000

012

006

000

10

05

00

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

Relat

ive e

xpre

ssio

n le

vel

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

IL-1120573

lowastlowast

(d)

Figure 3 Continued


Total

Tumor

Tum

or n

umbe

rs (N

)

number83 50

Inhibition ()

0 40

No Treg IL-17A KO

lowastlowast

120

60

0

ApcMin+ Treg

(e)

No Treg IL-17A KO

Tum

or n

umbe

rs (N

)29 13

0 55

lowastlowastlowast

40

20

0

Large (gt2mm)

ApcMin+ Treg

(f)

No Treg IL-17A KO

Tum

or n

umbe

rs (N

)

54 37

0 31

lowast80

40

0

Small (05ndash2mm)

ApcMin+ Treg

(g)




















4 Discussion








Wild type

0 0

126894

0 0

60

30

0581479

Foxp3

SSC

Foxp

3+ ce

lls (

)

Wild type ApcMin+


(a)

Total

Tumor number

83 26 61


0

50

100

Tum

or n

umbe

rs (N

)


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(b)

27 4 13

0 86 51

Large (gt2 mm)

Tum

or n

umbe

rs (N

)

0

20


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(c)

56

0

22 48

61 16

Small (05ndash2 mm)

Tum

or n

umbe

rs (N

)

0

40


ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(d)

50

25

0

938 347

136423

60 111

137692

Foxp3

CD39

CD4+

Fox

p3+

CD39

+ ce

lls (

)



ApcMin+ ApcMin+

ApcMin+ ApcMin+

(e)









5 Conclusion




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Total

Tumor

Tum

or n

umbe

rs (N

)

number83 50

Inhibition ()

0 40

No Treg IL-17A KO

lowastlowast

120

60

0

ApcMin+ Treg

(e)

No Treg IL-17A KO

Tum

or n

umbe

rs (N

)29 13

0 55

lowastlowastlowast

40

20

0

Large (gt2mm)

ApcMin+ Treg

(f)

No Treg IL-17A KO

Tum

or n

umbe

rs (N

)

54 37

0 31

lowast80

40

0

Small (05ndash2mm)

ApcMin+ Treg

(g)




















4 Discussion








Wild type

0 0

126894

0 0

60

30

0581479

Foxp3

SSC

Foxp

3+ ce

lls (

)

Wild type ApcMin+


(a)

Total

Tumor number

83 26 61


0

50

100

Tum

or n

umbe

rs (N

)


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(b)

27 4 13

0 86 51

Large (gt2 mm)

Tum

or n

umbe

rs (N

)

0

20


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(c)

56

0

22 48

61 16

Small (05ndash2 mm)

Tum

or n

umbe

rs (N

)

0

40


ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(d)

50

25

0

938 347

136423

60 111

137692

Foxp3

CD39

CD4+

Fox

p3+

CD39

+ ce

lls (

)



ApcMin+ ApcMin+

ApcMin+ ApcMin+

(e)









5 Conclusion




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























4 Discussion








Wild type

0 0

126894

0 0

60

30

0581479

Foxp3

SSC

Foxp

3+ ce

lls (

)

Wild type ApcMin+


(a)

Total

Tumor number

83 26 61


0

50

100

Tum

or n

umbe

rs (N

)


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(b)

27 4 13

0 86 51

Large (gt2 mm)

Tum

or n

umbe

rs (N

)

0

20


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(c)

56

0

22 48

61 16

Small (05ndash2 mm)

Tum

or n

umbe

rs (N

)

0

40


ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(d)

50

25

0

938 347

136423

60 111

137692

Foxp3

CD39

CD4+

Fox

p3+

CD39

+ ce

lls (

)



ApcMin+ ApcMin+

ApcMin+ ApcMin+

(e)









5 Conclusion




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Wild type

0 0

126894

0 0

60

30

0581479

Foxp3

SSC

Foxp

3+ ce

lls (

)

Wild type ApcMin+


(a)

Total

Tumor number

83 26 61


0

50

100

Tum

or n

umbe

rs (N

)


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(b)

27 4 13

0 86 51

Large (gt2 mm)

Tum

or n

umbe

rs (N

)

0

20


lowast

ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(c)

56

0

22 48

61 16

Small (05ndash2 mm)

Tum

or n

umbe

rs (N

)

0

40


ApcM

in+

iTre

g

WT

iTre

g

No

Treg

(d)

50

25

0

938 347

136423

60 111

137692

Foxp3

CD39

CD4+

Fox

p3+

CD39

+ ce

lls (

)



ApcMin+ ApcMin+

ApcMin+ ApcMin+

(e)









5 Conclusion




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























5 Conclusion




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Spontaneous Intestinal Tumorigenesis in Min Mice Requires ...downloads.hindawi.com/journals/jir/2015/860106.pdf · Tregs in the LP and investigated the role of the heterozygous Apc