Lub luag haujlwm rau BATF3 hauv Tu9 Qhov Sib Txawv Thiab T-cell-driven L Pathologies MuCOsal (Part 1)

Xav paub ntau ntxiv plz hu raudavid.wan@wecistanche.com

T helper 9 (T,9) hlwb yog qhov tseem ceeb rau kev txhim kho cov kab mob inflammatory thiab tsis haum. TH9 transcriptional network converges teeb liab los ntawm cytokines thiab antigen kev nthuav qhia tab sis tsis to taub tag. Ntawm no, peb tau txheeb xyuas TL1A, tus tswv cuab ntawm TNF superfamily, ua tus muaj zog inducer ntawm nas thiab tib neeg TH9 sib txawv.Mechanistically. } secretion nyob rau hauv Tμ9 thiab Ty9-TL1A-polarizing tej yam kev mob. Hauv vivo, siv tus qauv hloov pauv T-cell, peb tau pom tias TL1A txhawb nqa IL-9-nyob, T,9 cell-induced plab hnyuv thiab ntsws. Neutralizing IL-9 cov tshuaj tiv thaiv kab mob ua rau TL1A-tsav mucosal o. Batf3-7T_9-TL1A hlwb induced txo o thiabcytokine kev qhiahauv vivo piv rau WT hlwb. Peb cov txiaj ntsig tau pom tias TL1A txhawb nqa T_9 cell sib txawv thiab ua haujlwm thiab txhais lub luag haujlwm rau BATF3 hauv T-cell-driven mucosal o.

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Taw qhia

Cov subsets tshwj xeeb ntawm T pab (T-) hlwb ua lub luag haujlwm tseem ceeb hauv kev tswj cov ntaub so ntswg homeostasis thiab thaum lub sij hawm kev loj hlob ntawm cov kab mob inflammatory ntawm mucosal nto. TH9 hlwb tsis ntev los no tau txheeb pom tias yog ib qho kev ywj pheej T cell subset uas ua rau IL-9 tab sis kuj IL-10 thiab IL-21.12 Tμ9 hlwb tau cuam tshuam rau ntau yam kab mob, nrog rau kev ua xua ntawm lub ntsws. mob, sim autoimmunemob encephalomyelitis(EAE), colitis, 3- Tsis ntev los no, TH9 hlwb tau muaj kab mob cab kab mob thiab mob qog noj ntshav. tau tshaj tawm tias ua lub luag haujlwm hauv cov kab mob hauv plab hnyuv (IBD).-9Cov neeg mob plab hnyuv (UC) tau nce tus lej ntawm mucosal IL-9 ntxiv rau T hlwb thiab IL-9 receptor (IL -9R) yog upregulated ntawm txoj hnyuv epithelium. IL -9 deficiency inhibits kev loj hlob ntawm mob thiab ntevoxazolone-induced colitis, tus qauv uas ua raws li UC. Hauv cov neeg mob Crohn's disease (CD), qib siab -8-10 serum IL-9 cuam tshuam nrog cov kab mob hnyav.

Fig.1 TL1A txhim kho qhov sib txawv ntawm murine thiab tib neeg TH9 hlwb. Naive murine CD4 ntxiv rau T hlwb tau sib txawv raws li Ty0-los yog T9-polarizing tej yam kev mob nrog los yog tsis muaj TL1A rau 3 hnub, thiab ELJSA tsom xam ntawm ]L-9, IL{{9} }, thiab IL -13 secretion. b Naive human CD4 T cells (CD4'CD45RACD45ROCD25) raug cais tawm ntawm PBMCs ntawm cov neeg pub noj qab haus huv thiab sib txawv raws li TH9-polarizing tej yam kev mob nrog lossis tsis muaj TL1A rau 3 hnub. ELISA tsom xam ntawm tib neeg IL-9 tso zis ntawm 48 thiab 72 teev. Box-whisker zaj duab xis uas qhia txog IL-9 concentrations hauv kab lis kev cai supernatants rau TH9 thiab TH9-TL1A-kho hlwb. Cov thawv sawv cev rau qhov nruab nrab thiab 25 thiab 75 feem pua, thiab cov whiskers sawv cev qhov tsawg kawg nkaus thiab siab tshaj qhov tseem ceeb ntawm kev faib khoom. N=7-9. Cov ntaub ntawv sawv cev txhais tau tias ± SD ntawm ib qho kev sim ywj pheej ntawm plaub (kev sim ywj pheej. *p< 0.05,***p=""><0.005 determined="" by="" student's="">

TH9 hlwb sib txawv ntawm naive CD4 ntxiv rau T hlwb nyob rau hauv muaj TGF- 1 thiab IL-4. Ntau qhov kev hloov pauv hauv qab ntawm T-cell receptor (TCR), TGF- 1, thiab IL-4 receptors yog xav tau rau kev sib txawv ntawm TH9 hlwb suav nrog IRF4, STAT6, GATA3, PU.1, NF- kB, thiab yooj yim leucine zipper transcription factor ATF-zoo li (BATF.11-15 Txawm li cas los xij, cov kev pab cuam transcriptional thiab inflammatory triggers uas tsav qhov sib txawv ntawm Tμ9 hlwb tseem tsis tau to taub zoo thiab ib tug kab mob-txhais yam cuam tshuam nrog lL{{ 17}} kev qhia tsis tau txheeb xyuas.

Tsev neeg ntawm BATF transcription yam muaj xws liPAB, BATF2, thiabPAB 3thiab belongs rau AP-1 tsev neeg ntawm cov ntaub ntawv hloov pauv uas suav nrog JUN thiab FOS. Cov kev hloov pauv hauv BATF tsev neeg yog tsim los ntawm DNA-binding domain thiab leucine zipper motif tab sis tsis muaj kev hloov pauv thiab tau pib piav qhia tias yog tus tswj hwm tsis zoo ntawm AP-1 kev ua haujlwm." Txawm li cas los xij, cov kev tshawb fawb tsis ntev los no tau pom tias BATF tsev neeg. cuam ​​tshuam nrog IRF cov ntaub ntawv hloov pauv suav nrog IRF4 thiab IRF8, thiab khi rau AP-1-IRF cov khoom sib xyaw ua ke los tswj lawv cov hom phiaj hauv T hlwb thiab cov hlwb dendritic. BATF tau pom tias yuav tsum muaj rau kev loj hlob ntawm Ty9 hlwb ib yam nkaus. raws li TH2, TA17, thiab Try cells.15,19BATF3 tau piav qhia los tswj kev loj hlob ntawm CD8a ntxiv thiab CD103 ntxiv rau cov hlwb dendritic. "2' Hauv qhov sib piv rau BATF, qhov tsis muaj nuj nqis ntawm BATF3 hauv kev loj hlob ntawm Tu hlwb tsis tau ua. ua qauv qhia. Tsis tas li ntawd, cov stimuli extracellular uas tuaj yeem qhib txoj hauv kev BATF3 hauv CD4 ntxiv rau Tu hlwb tseem yuav tsum tau piav qhia.

Fig.2 TL1A upregulates BATF thiab BATF3 qhia thaum T,9 sib txawv. Transcriptional profileing ntawm TH9 thiab TH9-TL1A hlwb los ntawm RNA sequencing. Thaum tshav kub kub daim ntawv qhia qhia RNA-sequencing cov ntaub ntawv siv cov saum toj kawg nkaus 100 noob nrog cov loj tshaj plaws IQR (interquartile ntau). Lub dendrograms mus rau sab laug thiab saum toj ntawm daim ntawv qhia tshav kub sawv cev rau pawg ntawm cov noob (kab) thiab cov qauv (kab). b Naive CD4 ntxiv rau T hlwb tau sib txawv rau hauv TH subsets sib txawv thiab Tregs rau 3 hnub. Cov txheeb ze BATF mRNA qhia tau txheeb xyuas los ntawm PCR, c Naive CD4 ntxiv rau T hlwb tau sib txawv raws li TH0- lossis TH9-polarizing tej yam kev mob nrog lossis tsis muaj TL1A rau lub sijhawm qhia. PCR tsom xam ntawm tus txheeb ze BATF mRNA qhia, d Co-staining ntawm BATF thiab IRF4 (sab saum toj). Kev txheeb xyuas ntau ntawm cov feem pua ​​​​ntawm BATFIRF4 hlwb (hauv qab).e Co-staining ntawm BATF thiab IL-9. fg

TL1A. f Tus neeg sawv cev intracellular staining ntawm IL-9 thiab BATF ntawm 48h.g Zaus ntawmBATFfIL-9 ntxiv rau T cellssib txawv raws li Ty9 thiab T9-TL1A cov xwm txheej. Txhais tau tias yog qhia. Txhua lub cim sawv cev rau tus neeg pub dawb. N=4. h Cov kwv tij Batf3 mRNA qhia nyob rau hauv txawv murine Th subsets thiab Tregs tau soj ntsuam los ntawm qPCR.I kwv tij Batf3 mRNA qhia ntawm lub sij hawm cov ntsiab lus. j Tus sawv cev co-staining ntawm BATF3 thiab IL-9. k zaus ntawmBATF3IL-9 ntxiv rau T cellssib txawv raws li Ty9 thiab TH9-TL1A cov xwm txheej. Txhais tau tias yog qhia. Txhua lub cim sawv cev rau ib qho kev sim ntawm tus kheej. N=3. Im Naive tib neeg CD4 ntxiv rau T hlwb tau sib txawv raws li T,9-polarizing tej yam kev mob nrog lossis tsis muaj TL1A. Kuv Tus Neeg Sawv Cev intracellular staining ntawm IL-9 thiab BATF3 ntawm 48 h. m zaus ntawm BATF3L-9 ntxiv rau T hlwb sib txawv raws li TH9 thiab TH9-TL1A cov xwm txheej. Txhais tau tias yog qhia. Txhua lub cim sawv cev rau tus neeg pub dawb. N=4. Cov ntaub ntawv sawv cev txhais tau tias ± SD ntawm ib qho kev sim ywj pheej ntawm ob (a) lossis tsawg kawg peb (bd. i. k) kev sim ywj pheej.*n<0.05. n=""><0.01.***n <="" 0.005="" as="" determined="" by="" student's="">

Peb tsis ntev los no tau qhia tias TL1A, TNF tus tswv cuab superfamily uas ua lub luag haujlwm tseem ceeb hauv cov kab mob tiv thaiv kab mob xws li IBD, 25 induces secretion ntawm IL-9 hauv Tu17 hlwb. "Tsis tas li ntawd, kev tshaj tawm tsis ntev los no tau qhia tias TL1A, ntawm nws cov receptor DR3, txhawb nqa TA9 qhov sib txawv los ntawmIB -2-thiabSTAT5-nyob ntawm tab sis PU.1- thiab STAT6- ywj pheej mechanisms hauv kev ua xua rau lub ntsws o.2' Txawm li cas los xij, cov kev pab cuam transcriptional induced los ntawm TL1A thiab cov pathogenicity ntawm TL1A-induced TH9 hlwb nyob rau hauv plab hnyuv o thiab IBD tseem yuav. tau piav qhia.

Ntawm no, peb pom tias TL1A txhawb kev sib txawv ntawm tib neeg thiab murine TH9 hlwb ntawm txoj kev qhia tshiab. Peb txhais BATF3 raws li ib tug tshiab transcriptional regulator rau qhov sib txawv ntawm Tp9 hlwb. TL1A ua rau chromatin remodeling ntawm lI9 locus thiab nrhiav neeg ua hauj lwm ntawm cov pioneer transcription yam IRF4, thiab BATF, uas yog ib qho tseem ceeb Cheebtsam rau IL-9 transcriptional activation, thiab BATF3 rau conserved cheeb tsam nyob rau hauv lub 9 promotors. Tsis tas li ntawd, TL1A upregulates qhov kev qhia ntawm BATF thiab BATF3 nyob rau hauv ib tug STAT6-dependent yam.BATF- thiab Batf3-deficient T-9 thiab TL1A-induced TH9(T-9-TL1A ) cov hlwb puas lawm hauv lawv cov IL-9 ntau lawm.TH9-TL1Acov hlwb yog cov pro-inflammatory heev nyob rau hauv vivo thaum saws hloov mus rau hauv Ragi-7- nas raws li pom los ntawm cov mucosal mob hnyav nyob rau hauv cov hnyuv thiab lub ntsws, uas yog attenuated los ntawm kev kho mob nrog neutralizing IL-9 tshuaj tiv thaiv. Kev hloov pauv ntawm Batf3-/TH9-TL1A hlwb rau hauv Rag1-7 nas ua rau txo qis mucosal o thiab cytokine ntau lawm hauv kev sib piv nrog WT TH9-TL1A hlwb. Peb cov ntaub ntawv qhia txog lub luag haujlwm tshiab rau TL1A thiab BATF3 hauv kev tsim cov kab mob pathogenic effector T,9 hlwb thiab txheeb xyuas txoj kev taw qhia no raws li lub hom phiaj kho mob hauv T_9- tsav pathologies, suav nrog IBD thiab kab mob ntsws tsis haum.

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TL1A txhim kho murine thiab tib neeg Tμ9 ntawm tes sib txawv hauv vitro Txhawm rau txiav txim siab cov txiaj ntsig ntawm TL1A ntawm TH9 qhov sib txawv, peb tau txhawb nqa CD4 T hlwb hauv TH9 cov xwm txheej nrog lossis tsis muaj TL1A. TL1A tau txhim kho IL-9 zais cia thiab I/9 mRNA qhia (Fig. 1a, Ntxiv Daim duab 1a). TL1A ib leeg tsis tau ntxias IL-9 zais cia, qhia tias TL1A ua haujlwm sib koom ua ke nrog TGF- 1 thiab IL-4 los txhawb IL-9 ntau lawm. Kev zais cia thiab mRNA qhia ntawm T-9-sociated cytokines L-10 thiab IL-13 kuj tau txhim kho los ntawm TL1A (Fig.1a, Ntxiv daim duab 1a, b). Raws li cov ntaub ntawv tshaj tawm dhau los, kev sim lub sijhawm ntawm intracellular IL-9 staining qhia tawm ntu IL-9 qhia nrog qhov siab tshaj plaws induction nyob rau hauv T-9 cov xwm txheej nyob rau hnub 3 thiab poob rov qab mus rau qhov pib ntawm hnub 5(Cov Duab Ntxiv 1b).28,2TL1A ua rau muaj feem pua ​​​​siab dua ntawm IL-9- tsim cov hlwb thoob plaws lub sijhawm ntawm kev sim nrog kev hloov pauv hauv kinetics mus rau qhov ua ntej thiab muaj zog IL-9 induction uas txawm ntawm lub sijhawm ntxov tshaj qhov siab tshaj plaws ntawm IL-9 ntau lawm induced nyob rau hauv TH9 tej yam kev mob (Ntxiv daim duab 1b). Peb kuj tau pom qhov nce ntawm IL-9 ntxiv rau IL-10 ntxiv rau cov hlwb hauv lub xub ntiag ntawm TL1A (Cov duab ntxiv 1b). Peb tsis tau soj ntsuam ib qho kev hloov mus rau lwm yam Tu subsets (Cov duab ntxiv 1c). Kev txhim kho ntawm TH9 qhov sib txawv los ntawm TL1A tau ua tiav nyob ntawm nws cov receptor DR3 (Cov Duab Ntxiv 1d). Ua ke, cov ntaub ntawv no qhia tias TL1A koom ua ke nrog TGF- 1 thiab IL-4 hauv TA9 qhov sib txawv thiab IL-9 ntau lawm.

TL1A kuj tseem txhim kho TH9 qhov sib txawv hauv qhov chaw tshwj xeeb antigen siv OVA-specific OT-ll hlwb (Cov duab ntxiv 1e, f). TL1A tsis cuam tshuam rau kev loj hlob ntawm tes thaum lub sij hawm TH9 sib txawv (Ntxiv daim duab 1g), raws li tau qhia rau lub cim xeeb CD4 ntxiv rau T hlwb. " Cov ntaub ntawv no qhia tias TL1A txhim kho TH9 qhov sib txawv yam tsis muaj kev cuam tshuam rau kev loj hlob. Tsis tas li ntawd, peb tau tshuaj xyuas IL-9 ntau lawm hauv cov hlwb uas txawv ntawm Ty9 thiab TH9-TL1A cov xwm txheej rau 3 hnub thiab rov ua dua nrog cov tshuaj tiv thaiv CD3s / CD28. Peb tau soj ntsuam IL-9 ntau lawm hauv TH9-TL1A hlwb piv rau TH9 hlwb tom qab kev txhawb siab thib ob uas tau txhim kho ntxiv nyob rau hauv lub xub ntiag ntawm TL1A (Cov duab ntxiv 1h, i). Cov ntaub ntawv no qhia tias TL1A Peb kuj tau txhim kho IL-9 ntau lawm los ntawm kev sib txawv ntawm TH9 hlwb. Tom ntej no, peb txiav txim siab qhov cuam tshuam ntawm TL1A ntawm kev sib txawv ntawm tib neeg Tu9 hlwb. Naive CD4 ntxiv rau T hlwb raug cais tawm ntawm PBMC los ntawm cov neeg pub noj qab haus huv thiab txhawb nqa nyob rau hauv Tu9 mob nyob rau hauv lub xub ntiag. Raws li peb cov ntaub ntawv los ntawm nas TH9 hlwb, qhov sib ntxiv ntawm TL1A tau txhim kho IL-9 tso tawm los ntawm tib neeg TH9 cell cultures (Fig. 1b).

TL1A induces qhov kev nthuav qhia ntawm transcription yam BATF thiab BATF3 Kom elucidate signaling pathways induced los ntawm TL1A, peb siv RNA sequencing los ntsuam xyuas cov transcriptomic profile nyob rau hauv TH9 thiab TH9-TL1A hlwb. TL1A tsis tau hloov pauv TH9 cov ntaub ntawv sau npe qhia tau hais tias TL1A tsis tau hloov pauv TH9 qhov sib txawv (cov ntaub ntawv tsis qhia). Txawm li cas los xij, 219 cov noob tau sib txawv hauv TH9-TL1A hlwb (Daim duab 2a, Cov Lus Qhia Ntxiv 1 thiab 2). Pathway enrichment analysis tau txheeb xyuas cov kev hloov pauv tseem ceeb hauv cov noob koom nrog hauv cytokine-cytokine receptor thiab JAK-STAT cov cim qhia txoj hauv kev hauv T,9-TL1A cov xwm txheej (Cov Lus Ntxiv 1 thiab 2, cov ntaub ntawv tsis qhia). BATF3 thiab ob peb BATF-tswj cov noob caj noob ces tau tswj hwm zoo raws li T,9-TL1A cov xwm txheej (Table 3) qhia tias cov ntaub ntawv hloov pauv BATF3 thiab BATF tuaj yeem koom nrog qhov sib txawv ntawm TH9-TL1A hlwb. BATF tau piav qhia tias yuav tsum tau txiav txim siab txog kev txhim kho ntawm Tp17 thiab TA9 hlwb.15,31-34 -txawm tias TL1A ua haujlwm rau BATF cov neeg hauv tsev neeg, peb thawj zaug ntsuas cov lus qhia ntawm BATF hauv cov subsets sib txawv thiab kuaj pom muaj zog BATF mRNA qhia hauv TH9 thiab TH2 hlwb (Fig. 2b). TL1A txuas ntxiv txhim kho kev qhia ntawm BATF raws li Tu9 cov xwm txheej, tshwj xeeb tshaj yog hnub 1 thaum TH9 cov xwm txheej ib leeg tsis txaus los ntxias BATF mRNA (Fig.2c). TL1A ib leeg ntxias BATF mRNA kom zoo ib yam li Tp9 tej yam kev mob (Fig. 2c). Intracellular staining rau BATF tau lees paub tias Ty9-TL1A stimulation tau nce qhov feem pua ​​​​ntawm BATF ntxiv rau cov hlwb (Fig. 2d). Tsis tas li ntawd, TL1A ib leeg tau nce qhov feem pua ​​​​ntawm BATF ntxiv rau cov hlwb thiab qhov tseem ceeb tshaj qhov feem pua ​​​​ntawm BATF * IRF4 ntxiv cov hlwb piv rau TH9 cov xwm txheej qhia tias TL1A tuaj yeem txhim kho qhov tsim ntawm IRF4-BATF kev koom tes complexes (Fig. 2d). TH9-TL1A stimulation kuj txhim kho qhov feem pua ​​​​ntawm BATF ntxiv rau IL-9 ntxiv rau cov hlwb (Fig. 2e).

Tsis tas li ntawd, tib neeg TH9 hlwb muaj qhov feem pua ​​​​siab dua ntawm IL-9- tsim cov hlwb thaum txhawb nqa nrog TL1A (Fig. 2f, g). Tshwj xeeb tshaj yog, feem ntau ntawm IL-9- tsim cov hlwb co-expressed BATF. Co-staining ntawm BATF thiab IRF4 tau pom tias qhov sib ntxiv ntawm TL1A nce qhov feem pua ​​​​ntawm BATF * IRF4 ntxiv rau cov hlwb thaum sib txawv ntawm tib neeg T-9 (cov ntaub ntawv tsis qhia), qhia tias kev txhim kho ntawm IL-9 ntau lawm ntawm BATF signaling txoj kev los ntawm TL1A yog kev txuag ntawm nas thiab tib neeg TH9 hlwb. Nyob rau hauv sib piv rau BATF, lub luag hauj lwm ntawm BATF3 nyob rau hauv qhov sib txawv ntawm TH hlwb yog tsawg dua txhais. Txhawm rau kom paub meej tias peb cov ntaub ntawv RNA-seq, peb tau txiav txim siab qhia theem ntawm Batf3 hauv T-9 hlwb thiab lwm yam TH subsets thiab pom tias Batf3 mRNA qhia tau siab tshaj plaws hauv T-9, T,1, thiab TH2 hlwb (Fig. 2h). TL1A tau txhim kho qhov kev qhia ntawm Batf3 raws li T-9 tej yam kev mob (Fig.2i). Intracellular staining rau BATF3 tau lees paub tias T,9-TL1A stimulation tau nce qhov feem pua ​​​​ntawm BATF3 ntxiv rau IL-9 ntxiv rau cov hlwb (Fig.2j, k).Nyob hauv tib neeg T9 hlwb, TL1A tau txhim kho qhov kev qhia ntawm BATF3. plus IL-9 plus cells(Fig.2l, m). Zoo ib yam li BATF, feem ntau ntawm IL-9- tsim cov hlwb co-expressed BATF3.

Fig,3 TL1A txhim kho kev khi ntawm BATE IRF4, thiab BATF3 rau //9 tus txhawb nqa thaum lub sij hawm Ty9 sib txawv, a, b Naive murine CD4 ntxiv rau T hlwb tau sib txawv hauv TH0- lossis Tμ9-polarizing tej yam kev mob nrog lossis tsis muaj TL1A rau 2 (BATF, IRF4, thiab AcH3), lossis 3 (BATF3) hnub. ChP kev soj ntsuam tau ua rau BATF, IRF4, BATF3, lossis AcH3 khi rau kev khaws cia tsis yog cov kab ke (CNS) 1 (a) thiab CNS0 (b) ntawm //9. Cov ntaub ntawv sawv cev txhais tau tias ± SD ntawm ib qho kev sim ywj pheej tawm ntawm ob qhov kev sim ywj pheej.*p<><>< 0.005="" as="" determined="" by="" student's="">

BATF thiab BATF3 yog qhov tseem ceeb ntawm kev hloov pauv hauv TL1A-induced T_9 qhov sib txawv

Txhawm rau txhawm rau txheeb xyuas lub luag haujlwm ntawm BATF thiab BATF3, peb tau ua qhov kev ntsuam xyuas chromatin immunoprecipitation (ChIP) los txiav txim siab kev khi ntawm ob lub Il9 khaws cia tsis yog cov kab ke (Il9 CNS1, CNSO).5 TL1A ib leeg ua rau muaj kev cuam tshuam ntau dua ntawm BATF rau II9 CNS1 piv rau cov xwm txheej. thiab TH9-TL1A ntxiv txhim kho BATF khi (Fig.3a). Ntawm qhov siab tshaj I/9 CNS0, TL1A kuj tau txhim kho BATF khi rau hauv T,9 cov xwm txheej thaum TL1A ib leeg tsis muaj txiaj ntsig (Fig.3b). TL1A txhim kho IRF4 khi nyob rau hauv Ty9 tej yam kev mob ntawm CNS1 thiab CNS0(Fig.3a, b). Interestingly, kev khi ntawm acetylated histone 3 (AcH3) ntawm CNS1 thiab CNSO uas tau piav qhia yav dhau los los pab txhawb kev hloov kho chromatin ntawm Il9 locus thaum lub sij hawm TH9 sib txawv kuj tau txhim kho nyob rau hauv Ty9-TL1A tej yam kev mob (Fig.3a,b). BATF3 binding rau CNS1 tau upregulated nyob rau hauv TH9 hlwb thiab cov binding tau ntxiv txhim kho nyob rau hauv TH9-TL1A tej yam kev mob thaum BATF3 tsis khi rau CNS0 (Fig.3a, cov ntaub ntawv tsis qhia). Cov ntaub ntawv no qhia tias TL1A txhim kho BATF, BATF3, thiab IFR4 khi rau //9 tus txhawb nqa raws li TH9 cov xwm txheej kom ntxias ll9 qhia. Txhawm rau paub meej lub luag haujlwm ntawm BATF thiab BATF3 hauv TL1A-dependent thiab Batf3-7-T hlwb. IL-9, IL- Ty9 qhov sib txawv, peb siv Batf7- ib 10, thiab IL-13 ntau lawm tau ua rau muaj kev cuam tshuam loj heev hauv BATF-/-cells nyob rau hauv Tμ9 tej yam kev mob (Fig. 4 a-d). Txawm li cas los xij, TL1A stimulation yam tsawg kawg ib nrab overcame qhov yuav tsum tau rau BATF rau IL-9 thiab IL-13 ntau lawm thaum Ty9 sib txawv (Fig.4a, b, d). Thaum tsis muaj BATF3, qhov induction ntawm IL-9 tau txo qis, tshwj xeeb tshaj yog nyob rau hauv Tμ9-TL1A tej yam kev mob (Fig.4e,f, Supplementary Figure 2a).Thiab rau BATF, BATF3 tsis tas yuav tsum tau. rau -10 lossis IL-13 kev qhia (Fig.4g, h, Ntxiv daim duab 2b, c).Kom txhais cov kev taw qhia uas ua rau BATF thiab BATF3 upregulation nyob rau hauv lub xub ntiag ntawm TL1A, peb siv Stat { {79}}thiab p50-/cells. p50-/TH9 hlwb tau txo qis feem pua ​​​​ntawm BATF ntxiv rau IRF4t cov hlwb thiab qhov nce ntawm feem pua ​​​​ntawm BATF ntxiv rau IRF4 ntxiv cov hlwb hauv qab TL1A stimulation yuav luag tag (Cov duab ntxiv 3a, b). Upregulation ntawm BATF thiab Batf3 mRNA nyob rau hauv T.9 tej yam kev mob yog p50- ywj siab, thaum txhim kho BATF thiab Batf3 qhia nyob rau hauv TH9-TL1A tej yam kev mob yog kiag li p50-dependent (Ntxiv daim duab 3c , d). Stat6-/T9 hlwb tau txo cov feem pua ​​​​ntawm BATFIRF4 ntxiv rau cov hlwb thiab lawv qhov nce hauv Tμ9-TL1A stimulation tau raug tshem tawm tag nrho (Cov duab ntxiv 4a, b). Ib yam li ntawd, kev tswj hwm ntawm BATF thiab Batf3 mRNA yog nyob ntawm STAT6 nyob rau hauv TH9 thiab TH9-TL1A tej yam kev mob (Cov duab ntxiv 4c, d). Peb pom tsis muaj qhov cuam tshuam tseem ceeb ntawm BATF lossis BATF3 qhov tsis txaus ntawm qhov kev qhia ntawm Batf3 thiab BATF, feem, qhia tias tsis muaj kev them nyiaj ntawm ob qhov kev hloov pauv (Cov duab ntxiv 5). Ua ke, cov ntaub ntawv no qhia tias BATF thiab BATF3 ua lub luag haujlwm tseem ceeb thiab sib txawv thaum lub sij hawm T_9 sib txawv ntawm TL1A thiab qhov nce BATF thiab BATF3 qhia nyob rau hauv TL1A stimulation yog nyob ntawm STAT6 thiab canonical NF-KB txoj hauv kev. Tom ntej no, peb ua RNA sequencing ntawm WT thiab Batf3-/-Tμ{119}}TL1A hlwb. Tag nrho, 139 cov noob tau sib txawv hauv WT piv rau Batf3-7Ty9-TL1A hlwb (Fig.4i, Cov Lus Ntxiv 4). Gene ontology pathway enrichment tsom xam pom cov kev hloov pauv tseem ceeb hauv cov noob koom nrog hauv kev tswj hwm ntawm kev hloov pauv, cell proliferation, thiab intracellular signal transduction (Cov Lus Ntxiv 5 thiab 6). Cov txiaj ntsig no qhia txog lub luag haujlwm rau BATF3 hauv kev sib txawv ntawm Ty9-TL1A hlwb. T-9-TL1A hlwb yog cov kab mob loj heev hauv vivo Los txiav txim siab cov kab mob muaj peev xwm ntawm TH9-TL1A hlwb hauv vivo, peb tau txais hloov pauv ex vivo-differentiated CD45.1 ntxiv rau Tμ9 lossis TA9-TL1A cells rau hauv Rag1-'-mice. Cov nas uas tau txais Ty9 hlwb tsis tsim qhov hnyav hnyav (Fig. 5a). Txawm li cas los xij, cov nas uas tau txais TH9-TL1A hlwb poob phaus thiab tsim cov hnyuv loj dua, tshwj xeeb tshaj yog nyob rau hauv cov hnyuv me (Fig. 5a-c). Peb kuj tau pom cov mob ntsws loj heev, uas yog tus cwj pwm los ntawm peribronchial thiab perivascular cellular infiltration ntawm inflammatory cells hauv TA{150}}TL1A cov neeg tau txais. Staining nrog Alcian Blue periodic acid-Schiff (AB-PAS) pom tau tias hyperproliferation ntawm mucin-tsim hlwb nyob rau hauv lub pa epithelium ntawm Ty9-TL1A cov neeg tau txais (Cov duab ntxiv 6a, b). Peb tau pom cov cellularity ntau dua hauv spleens thiab lamina propria mononuclear hlwb (LPMC), thiab cov feem pua ​​​​ntawm cov splenic, MLN, thiab LPMC CD4 ntxiv rau cov hlwb hauv TH9-TL1A cov neeg tau txais (Fig. 5d, e) qhia tias Ty 9-TL1A hlwb muaj peev xwm loj hlob ntau dua hauv vivo. Ki67 staining paub tseeb tias cov hlwb los ntawm MLN, spleen thiab LPMC los ntawm TH9-TL1A cov neeg tau txais kev loj hlob ntau dua txawm tias 6 lub lis piam tom qab hloov T-cell (Fig. 5f). Kev tso tawm ntau dua ntawm IL{170}} thiab IL{171}} hauv cov tshuaj tiv thaiv CD3 / anti-CD28-restimulated MLN hlwb los ntawm TH9-TL1A cov neeg tau txais kev pom zoo (Fig. 5g), thiab ntau dua tso tawm ntawm IL{179}} thiab IL{180}} hauv anti-CD3/anti-CD{184}}restimulated LPMC los ntawm Ty9-TL1A cov neeg tau txais (Daim duab 5h). Peb tau pom ntau dua tus naj npawb ntawm splenic thiab MLN IL{188}} ntxiv rau , IL-13 ntxiv , thiab IL-17 ntxiv cov hlwb thiab ntau dua tus naj npawb ntawm MLN, splenic, thiab LPMC CCR6 ntxiv thiab CD103 ntxiv rau T hlwb hauv TH9-TL1A cov neeg tau txais kev sib raug zoo nrog lub luag haujlwm ntawm cov hlwb ntawm cov mucosal nto (Fig.5i,j). IL-9 yuav tsum tau rau hauv vivo o uas tau tsav los ntawm T#9-TL1A hlwb Los tshuaj xyuas seb puas nce IL{199}} ntau lawm los ntawm TH9-TL1A hlwb pab rau plab hnyuv thiab mob ntsws, peb tau kho cov nas uas tau txais TH9-TL1A hlwb nrog neutralizing IL-9 lossis tswj cov tshuaj tiv thaiv rau lub sijhawm ntawm kev sim hloov T-cell. Kev tshuaj xyuas histopathological pom tau tias txo cov plab hnyuv loj hauv kev tiv thaiv lL-9- kho nas piv rau isotype tswj (Fig. 6a, b). Peb kuj tau pom qhov txo qis hauv lub ntsws nrog txo qis peribronchial infiltrates thiab txo qis hauv cov qog ua kua hauv cov tshuaj tiv thaiv lL-9-kho nas (Cov duab ntxiv 6c). Tag nrho cov xov tooj ntawm tes thiab tag nrho CD4tCD45.1 ntxiv rau T hlwb hauv MLN tau txo qis hauv cov tshuaj tiv thaiv IL-9- kho nas piv rau isotype tswj (Fig. 6c). Anti-IL-9 kev kho mob kuj txo qis kev tsim tawm ntawm IL-13 thiab IL-17 hauv MLN thiab spleens, thaum IFN-y ntau lawm tsis hloov pauv (Daim duab 6d, cov ntaub ntawv tsis qhia). Ua ke, cov ntaub ntawv no qhia tau hais tias T-9-TL1A hlwb yog cov muaj zog effector hlwb thiab ua rau cov hnyuv thiab lub ntsws o nyob rau hauv vivo thiab hais tias cov kab mob tshwm sim yog IL-9- nyob ntawm.

Fig.4 Cov teebmeem ntawm BATF thiab BATF3 tsis txaus ntawm TH9 qhov sib txawv. ad Naive CD4 T hlwb los ntawm Batf7 lossis WT nas tau sib txawv raws li TH0- lossis TH9-polarizing tej yam kev mob nrog lossis tsis muaj TL1A rau 3 hnub. Intracellular staining ntawm IL-9 thiab IL-10.b ELISA tsom xam ntawm IL-9 production.c ELISA tsom xam ntawm IL-10 production.d ELISA tsom xam ntawm IL{{14} } ntau lawm. eh Naive CD4 ntxiv rau T hlwb los ntawm Batf3-7- lossis WT nas tau sib txawv raws li T, 9-polarizing tej yam kev mob nrog lossis tsis muaj TL1A rau 3 hnub. e Intracellular staining ntawm IL-9 thiab IL-10.f ELISA tsom xam ntawm IL-9 ntau lawm. g ELSA tsom xam ntawm IL-10 ntau lawm. h ELISA tsom xam ntawm IL-13 ntau lawm. Kuv Transcriptional profileing ntawm WT thiab Batf3′TH9-TL1A hlwb los ntawm RNA sequencing. Daim ntawv qhia tshav kub uas qhia txog RNA-sequencing cov ntaub ntawv ntawm cov noob sib txawv qhia nrog p<0.01. the="" dendrograms="" to="" the="" left="" and="" above="" the="" heat="" map="" represent="" the="" clustering="" of="" genes="" (rows)="" and="" samples="" (columns).="" data="" represent="" means±="" sd="" of="" one="" independent="" experiment="" out="" of="" two="" (i)="" or="" three="" (a-h)="" independent=""><0.05,**p <="" 0.01,="" ***p=""><0.005 determined="" by="" student's="">

BATF3 pab txhawb rau hauv vivo o uas tau tsav los ntawm TH9-TL1A hlwb Los txiav txim seb BATF3 puas ua lub luag haujlwm hauv Tu9-tsav pathologies hauv vivo, peb tau txais kev hloov pauv ex vivo-differentiated WT lossis Batf3- /-TA9-TL1A rau Rag1-/-mice. Cov nas uas tau txais Batf3-'- TA9-TL1A cov hlwb tsim cov qog pulmonary tsawg dua (Fig. 7a, b, Ntxiv Daim duab 6e) thiab ib qho kev hloov mus rau kev txo cov hnyuv (Histoscore: 3.6 vs. 1.2)( Ib. 7a). Peb tau pom tias txo qis cellularity hauv ntsws thiab LPMC, thiab qis feem pua ​​​​ntawm MLN thiab ntsws CD4 ntxiv rau T hlwb hauv Batf37′-TL1A cov neeg tau txais piv rau WT TH9-TL1A cov neeg tau txais (Daim duab 7c, Ntxiv daim duab 6d). Qhov tseem ceeb, qis feem pua ​​​​ntawm IL-13 ntxiv thiab IL-17 ntxiv rau cov hlwb hauv spleens, MLN, thiab ntsws los ntawm Batf3-'-Ty9-TL1A cov neeg tau txais (Fig. 7d ib). Ua ke, peb cov ntaub ntawv qhia tias BATF3 ua lub luag haujlwm hauv IL-9 ntau lawm thiab T#9 kev loj hlob hauv vitro thiab hauv Tp9-TL1A-tsav mucosal o hauv vivo.

Fig.6 IL-9 yuav tsum tau rau hauv vivo mucosal o uas tau tsav los ntawm Ty9-TL1A hlwb. T,9 lossis Ty9-TL1A hlwb raug txhaj rau Rgg1-7 nas, thiab nas tau kho peb zaug hauv ib lub lis piam nrog cov tshuaj tiv thaiv IL-9 tshuaj tiv thaiv lossis isotype tswj, Tus Neeg Sawv Cev H&E stainings ntawm lub duodenum, Scale bar; 200 μm. b Histology cov qhab nia rau txoj hnyuv.c Tag nrho MLN cell suav (sab laug), zaus ntawm CD45.1 ntxiv rau cov hlwb (nruab nrab), thiab tag nrho cov cell suav ntawm CD45.1 ntxiv rau cov hlwb (txoj cai).d ELISA tsom xam ntawm IL{{15} }, IL-17, thiab IL-13 ntau lawm los ntawm MLN tom qab ex vivo restimulation nrog anti-CD3 thiab anti-CD28. Cov ntaub ntawv sawv cev txhais tau tias ± SD ntawm ib qho kev sim ywj pheej tawm ntawm ob qhov kev sim ywj pheej. n=4-5/pab.*p<><0.01, as="" determined="" by="" student's="" t-test="" (b)="" or="" mann-whitney="" u="" test="" (c,="">