MTHFD2 yog Lub Chaw Saib Xyuas Metabolic Tswj Kev Ua Haujlwm thiab Kev Tswj Xyuas T Cell Txoj hmoo thiab ua haujlwm

Graphical abstract

Cov kws sau ntawv:

Ayaka Sugiura, Gabriela Andrejeva, Kelsey Voss, ..., Ashutosh K. Mangalam, Joshua D. Rabinowitz, Jeffrey C. Rathmell

Ntawv sau:

jeff.rathmell@vumc.org

Hauv luv luv:

Nucleotide synthesis yuav tsum tau los txhawb T cell proliferation sai. Sugiura et al. qhia tias de novo purine metabolism taw qhia ncaj qha T cell sib txawv thiab ua haujlwm thiab txheeb xyuas MTHFD2 raws li qhov chaw kuaj mob metabolic thiab kho lub hom phiaj rau cov kab mob inflammatory.

Qhov tseem ceeb

• MTHFD2 yog qhov tseem ceeb rau kev qhib CD4 T hlwb kom tswj tau de novo purine synthesis

• Tsis txaus MTHFD2 txhawb nqa Treg cell-zoo li phenotypes thiab metabolism hauv Th17 hlwb d Inhibition ntawm MTHFD2 suppresses mTORC1 signaling thiab hloov histone methylation

• MTHFD2 tuaj yeem raug tsom los tiv thaiv o thiab autoimmunity hauv vivo

MTHFD2 yog lub chaw kuaj mob metabolic tswj kev tswj hwm thiab tswj T cell txoj hmoo thiab ua haujlwm

Ayaka Sugiura,¹Gabriela Andrejeva¹Kelsey Voss,¹Darren R. Heintzman,¹Xincheng Xu,⁵Matthew Z. Madden,¹Xiang Yee,¹Katherine L. Beier,¹Nowrin U. Chowdhury,²Melissa M. Hmab,²Arissa C. Young,¹Dalton L. Greenwood,¹Allison E. Sewell,¹Shailesh K. Shahi,³Samantha N. Freedman,³Alanna M. Cameron,⁴Patrik Foob,⁴Tim Bourne, ib.⁴Juan C. Garcia-Canaveras,⁵John Karijolich,¹Dawn C. Newcomb,²Ashutosh K. Mangalam,³Joshua D. Rabinowitz,⁵thiab Jeffrey C. Rathmell^1,6,*

¹Vanderbilt Center for Immunobiology, Department of Pathology, Microbiology, thiab Immunology, Vanderbilt University Medical Center, Nashville, TN 37232, USA

²Department of Medicine, Division of Hematology thiab Oncology, Vanderbilt University Medical Center, Nashville, TN 37232, USA

³Department of Pathology, University of Iowa, Iowa City, IA 52242, USA

⁴Sitryx Therapeutics Limited, Magdalen Center, Oxford Science Park, Oxford, UK

⁵Department of Chemistry, Ludwig Cancer Research Institute Princeton Branch, Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA6Cov neeg sib cuag

* Cov ntawv sau: jeff.rathmell@vumc.org

https://doi.org/10.1016/j.immuni.2021.10.011

TSEEM CEEB

Antigenic stimulation txhawb T cell metabolic reprogramming kom tau raws li biosynthetic, bioenergetic, thiab signaling xav tau. Peb pom tias ib-carbon (1C) metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) tswj de novo purine synthesis thiab signaling nyob rau hauv activated T hlwb los txhawb kev loj hlob thiab inflammatory cytokine ntau lawm. Nyob rau hauv pathogenic T tus pab-17 (Th17) hlwb, MTHFD2 tiv thaiv kev tsis txaus ntseeg ntawm kev hloov pauv ntawm FoxP3 nrog rau qhov tsis tsim nyog tau txais kev txhawb nqa. MTHFD2 tsis muaj peev xwm kuj txhawb kev tswj hwm T (Treg) cell sib txawv. Mechanistically, MTHFD2 inhibition coj mus rau depletion ntawm purine pas dej ua ke, tsub zuj zuj ntawm purine biosynthetic intermediates, thiab txo cov as-ham sensor mTORC1 signaling. MTHFD2 kuj tseem ceeb heev los tswj DNA thiab histone methylation hauv Th17 hlwb. Qhov tseem ceeb, MTHFD2 tsis txaus txo cov kab mob hnyav hauv ntau yam hauv vivo inflammatory kab mob qauv. MTHFD2 yog li qhov chaw kuaj mob metabolic kom sib xyaw purine metabolism nrog cov kab mob cuam tshuam ntawm tes taw qhia thiab yog lub hom phiaj kho mob hauv 1C txoj hauv kev.

Taw qhia

Kev ua kom muaj txiaj ntsig zoo ntawm kev hloov lub cev tiv thaiv kab mob yuav tsum muaj kev ua kom muaj zog ntawm CD4 ntxiv rau T hlwb kom muaj kev loj hlob ntawm tes thiab kev loj hlob sai. Qhov no suav nrog metabolic reprogramming, tsav los ntawm cov khoom noj khoom haus sensor mTORC1, los ntawm lub xeev catabolic so mus rau lub xeev kev loj hlob anabolic nrog nce biosynthetic, bioenergetic, thiab kev xav tau. Nyob ntawm cov cytokine milieu, CD4 ntxiv rau T hlwb sib txawv rau hauv effector nrog rau cov kev tswj hwm subsets uas muaj cov kev pab cuam metabolic sib txawv uas tsav lawv cov kev ua haujlwm (Bantug li al., 2018; Buck et al., 2015). Qhov sib npaug ntawm cov subsets no yog qhov tseem ceeb rau kev tiv thaiv ib puag ncig thaum tiv thaiv kab mob inflammatory thiab autoimmune. Piv txwv li, ntau yam sclerosis (MS) yog tus cwj pwm los ntawm kev nce interleukin -17 (IL- 17)-tsim T pab-17 (Th17) hlwb thiab txo qis lossis tsis muaj txiaj ntsig kev tswj hwm T (Treg) hlwb. (Dendrou et al., 2015). Targeting cov kev pab cuam metabolic tshwj xeeb ntawm cov T cell subsets muaj lwm txoj hauv kev rau immunotherapy.

Cell metabolism-raws li kev kho mob pib xyoo 1948 thaum cov tshuaj tiv thaiv folate chemotherapeutic tau pom tias muaj txiaj ntsig zoo rau cov menyuam yaus uas mob qog ntshav qog ntshav ntshav (Farber et al., 1948). Folate pab txhawb rau ib-carbon (1C) metabolism rau purine biosynthesis, tiam ntawm methyl pub, thiab kev saib xyuas ntawm cellular redox tshuav nyiaj li cas (Ducker thiab Rabinowitz, 2017; Yang thiab Vousden, 2016). Ntau cov tshuaj, suav nrog methotrexate (MTX), fluorouracil (5-FU), thiab mercaptopurine (6-MP), txij li ntawd los tau tsim los rau txoj hauv kev no. Kev loj hlob sai sai ntawm cov hlwb, suav nrog cov qog nqaij hlav qog noj ntshav thiab cov T hlwb ua haujlwm, sib koom cov kev vam khom ntawm cov txheej txheem no los ua ke DNA thiab RNA. MTX feem ntau siv los kho cov kab mob autoimmune xws li mob caj dab rheumatoid (RA) (Brown li al., 2016). Txawm li cas los xij, vim muaj kev nthuav dav dav ntawm cov enzymes tsom, cov tshuaj kho mob no cuam tshuam nrog ntau yam thiab muaj feem cuam tshuam loj heev. Kev txheeb xyuas cov hom phiaj metabolic enzyme uas xaiv qhov tseem ceeb hauv cov neeg ntawm tes ntawm kev txaus siab tuaj yeem ua rau kev txhim kho kev nyab xeeb thiab muaj txiaj ntsig zoo dua immunotherapy.

1C metabolism suav nrog cov folate thiab methionine cycles nrog kev hloov ntawm cov pa roj carbon ib leeg. Serine ua haujlwm raws li 1C pub dawb los qhib tetrahydrofolate (THF) mus rau 5, 10-methyleneTHF, nrog kev tsim cov glycine sib xyaw. 5, 10-methyleneTHF tuaj yeem oxidized siv NAD(P) los tsim cov purine precursor 10- formylTHF. Xwb, 10-formylTHF tuaj yeem ua ke los ntawm formate thiab THF hauv ATP-raws li. Hauv txoj kev cytosolic, 10-fomylTHF ntau lawm yog kho los ntawm methyleneTHF dehydrogenase 1 (MTHFD1). Kev poob ntawm MTHFD1 tshaib plab rau cytosolic 10-formylTHF, ablating purine biosynthetic muaj peev xwm, thiab kev hloov hauv MTHFD1 ua rau muaj kev tiv thaiv kab mob hnyav (SCID) (Field et al., 2015). Txoj kev mitochondrial cia siab rau mitochondrial MTHFD2 thiab MTHFD1-zoo li (MTHFD1L).

Daim duab 1. 1C metabolism thiab MTHFD2 yog upregulated nyob rau hauv activated CD4^ntxivT hlwb thiab hauv cov ntsiab lus ntawm EAE

(A) Kev hloov pauv ntawm nucleotide cov metabolism hauv hom hauv-vitro-differentiated CD4^ntxivT cell subsets piv nrog cov hlwb tsis zoo ntsuas los ntawm huab hwm coj spectrometry (n=3 bioreplicates).

(B) Kev ua haujlwm rau lub hom phiaj CRISPR kev tshuaj ntsuam xyuas hauv cov qauv hauv vivo mob ntsws.

(C) Kev hloov pauv hauv gRNA abundance los ntawm 1C metabolism-taim hauv vivo CRISPR kev tshuaj ntsuam hauv thawj CD4^ntxivT hlwb (kev txheeb xyuas txheeb cais ua los ntawm MAGECK, n=3 bioreplicates).

(D) mRNA qhia txog cov noob tau txheeb xyuas hauv (C) thaum lub sijhawm T cell txhim kho thiab ua kom ua haujlwm (cov ntaub ntawv los ntawm ImmGen RNA-seq data browser).

(E) Protein nthuav qhia ntawm cov noob tau txheeb xyuas hauv (C) hauv kev tsis ncaj ncees thiab qhib CD4^ntxivT hlwb (cov ntaub ntawv los ntawm Immunological Proteome Resource [ImmPRes]).

(F) mRNA qhia txog cov noob tau txheeb xyuas hauv (C) hauv cov ntshav tag nrho ntawm cov tib neeg uas muaj cov kab mob inflammatory uas cuam tshuam rau cov tib neeg tswj kev noj qab haus huv (Aune li al., 2017). RA-MTX, RA hauv kev kho MTX; SLE, systemic lupus erythematosus; MS-Tx naive, kho-naive MS thaum lub sijhawm kuaj mob; MS-tsim, MS nyob rau hauv kev kho mob thiab nyob rau hauv kev kho kab mob; n=3–8 tus neeg pub dawb.

(G) IHC qhia CD3 thiab MTHFD2 staining hauv cauda equina ntu ntawm tus txha caj qaum ntawm nas nrog cov tsos mob EAE ntawm 2003 (sab saum toj; nplai bar, 100 mm) thiab 4003 (hauv qab; nplai bar, 50 mm) magnification (cov ntaub ntawv yog tus sawv cev ntawm ob qhov kev sim siab ywj pheej).

(H thiab I) txheeb ze (H) txhais tau tias fluorescence siv (MFI) thiab (I) mRNA qhia ntawm MTHFD2 hauv CD4^ntxivT hlwb los ntawm tus po thiab tus txha caj qaum ntawm nas nrog cov tsos mob EAE thiab spleen ntawm cov nas tswj (txhais tau tias ± SD, ib-txoj kev ANOVA, cov ntaub ntawv yog tus sawv cev ntawm ob qho kev sim ywj pheej nrog 6 tag nrho cov khoom siv roj ntsha).

(J) MTHFD2 mRNA qhia hauv CD4 tsis txawv^ntxivT hlwb 0, 5, thiab 24 teev tom qab ua kom muaj cov tshuaj tiv thaiv CD3 thiab anti-CD28 cov tshuaj tiv thaiv (txhais tau tias ± SD, ib-txoj kev ANOVA, n=3 bioreplicates).

(K) Cov txheeb ze MTHFD2 MFI dhau 5 hnub tom qab ua kom ua haujlwm hauv CD4 ntxiv rau T cell subsets, normalized rau cov hlwb so (txhais tau tias ± SD, n=3 bioreplicates). Saib daim duab S1. Cov txiaj ntsig tseem ceeb ntawm cov ntawv sau npe (* p < 0.05,="" **="" p="">< 0="" 01,="" ***="" p="">< 0.001,="" ****="" p%="">

MTHFD2 yog ib qho ntawm cov noob caj noob ces ntau tshaj plaws hauv txhua qhov qog (Nilsson li al., 2014). Txawm hais tias muaj kev txhawb nqa dav dav thaum lub sijhawm embryogenesis, MTHFD2 tsis muaj qhov qhia me me hauv cov neeg laus feem ntau (Nilsson li al., 2014). Hauv kev kho mob qog noj ntshav (Zhu thiab Leung, 2020), MTHFD2 inhibition tuaj yeem ua rau muaj kev ntxhov siab oxidative (Ju li al., 2019; Wan li al., 2020), glycine dependency (Koufaris li al., 2016), thiab tsis txaus. purine synthesis (Ben-Sahra li al., 2016; Pikman li al., 2016). MTHFD2 tsis muaj peev xwm tseem tuaj yeem cuam tshuam mTORC1 kev ua haujlwm los ntawm ntau lub tswv yim, suav nrog los ntawm guanine depletion thiab tom qab inhibition ntawm mTORC1- ua kom GTPase Rheb (Emmanuel li al., 2017). MTHFD2 deficiency kuj ua rau tsub zuj zuj ntawm purine synthesis pathway intermediates, nrog rau cov adenosine monophosphate (AMP) analog 5-aminoimidazole carboxamide ribonucleotide (AICAR) (Ducker li al., 2016), uas tuaj yeem ua rau AMP-activated protein) kinase (AMP) inhibit mTORC1 (Su li al., 2019).

Vim tias 1C metabolism sib xyaw ua ke ntau yam khoom noj khoom haus thiab kho tau zoo, peb tau tshuaj xyuas lub luag haujlwm ntawm 1Cmetabolism hauv CD4.^ntxivT effector (Teff) thiab Treg cell subsets. Los ntawm kev tsis ncaj ncees nyob rau hauv vivo CRISPR-Cas9-raws li kev tshuaj ntsuam xyuas hauv thawj murine T hlwb, peb tau txheeb xyuas MTHFD2 raws li kev ntaus uas tseem raug kho tsis tu ncua rau cov tib neeg uas muaj kab mob inflammatory. Qhov tseem ceeb, MTHFD2 tsis muaj peev xwm cuam tshuam Teff cell proliferation thiab ua haujlwm. MTHFD2 tsis muaj peev xwm kuj txhawb nqa FoxP3 kev qhia tsis txaus ntseeg thiab kev ua kom muaj zog hauv Th17 hlwb thaum txhawb nqa Treg cell sib txawv. Cov teebmeem no tau cuam tshuam nrog kev txuam nrog AICAR, txo cov purine concentrations, downregulation ntawm mTORC1 signaling, nce mitochondrial metabolism, thiab hloov DNA thiab histone methylation. Hauv vivo, tsom MTHFD2 tiv thaiv ntau tus qauv kab mob. Cov ntaub ntawv no qhia tau hais tias MTHFD2 ua haujlwm raws li qhov chaw kuaj mob metabolic hauv Th17 thiab Treg hlwb thiab qhia txog lub peev xwm ntawm cov enzyme no ua lub hom phiaj rau kev tiv thaiv kev tiv thaiv kab mob.

TSEEM CEEB

1C metabolism thiab purine synthesis yog qhov sib txawv hauv CD4^ntxivT cell subsets

Peb xav tias nucleotide synthesis yuav txawv ntawm CD4^ntxivT cell subsets. CD4^ntxivT hlwb tau sib txawv hauv vitro rau hauv Th1, Th17, thiab Treg hlwb thiab sau tom qab 72 teev rau huab hwm coj spectrometry. Nucleotide ntau npaum li cas feem ntau tau nce siab hauv txhua qhov subsets piv nrog cov hlwb tsis zoo (Daim duab 1A). Tshwj xeeb tshaj yog, purine synthesis intermediates tau sib txawv ntau, nrog rau siab tshaj plaws ntawm glycinamide ribonucleotide (GAR), 1-(phosphoribosyl) imidazole carboxamide (SAICAR), thiab AICAR hauv Th17 hlwb (Daim duab S1A).

Txhawm rau kuaj qhov dependence ntawm CD4^ntxivT hlwb ntawm enzymes hauv 1C metabolism, peb tau ua ib qho hauv vivo CRISPR-Cas9-raws li kev tshuaj ntsuam hauv thawj CD4^ntxivT cells (Daim duab 1B). Ib daim ntawv qhia kev cai RNA (gRNA) lub tsev qiv ntawv tau tsim los rau lub hom phiaj enzymes hauv 1C metabolism, nrog rau tuberous sclerosis complex 2 (TSC2) raws li kev tswj zoo thiab kev tswj tsis zoo (NTCs). CD4^ntxivT hlwb raug rho tawm los ntawm ovalbumin (OVA) - tshwj xeeb T cell receptor (TCR) transgenic OT-II Cas9 ob-transgenic nas tau hloov nrog lub tsev qiv ntawv no thiab hloov mus rau hauv cov hlab ntsha hauv Rag1.^{__ /__}tus tswv. Cov nas tau txhaj tshuaj intranasal OVA txhawm rau ua rau mob ntsws. T hlwb rov qab los ntawm lub ntsws ntawm cov nas no tau ua ntu zus, thiab kev ua kom muaj zog lossis txo qis ntawm gRNAs tau tsim muaj txheeb ze rau cov khoom siv ntau zaus. TSC2 gRNA yog enriched, txhawb lub luag hauj lwm inhibitory ntawm no protein nyob rau hauv T hlwb. Nyob rau hauv sib piv, PPAT, AHCY, MAT2A thiab MTHFD1 gRNAs tau depleted tseem ceeb, thiab GART, DHFR, DNMT1, MTRR, MTR, SHMT2, thiab MTHFD2 gRNAs raug txo kom tsawg, qhia tias cov noob no pab txhawb rau T cell proliferation. hauv vivo (Figures 1C thiab S1B).

Peb tom ntej no tshuaj xyuas qhov kev qhia profile ntawm cov noob txheeb xyuas los ntawm qhov screen. Txawm hais tias txhua tus tau tswj hwm kev sib koom ua ke hauv kev tsim cov thymocytes, MTHFD2 mRNA thiab cov protein tau ua rau muaj zog tshaj plaws hauv cov hlwb T hlwb (Figures 1D thiab 1E). Ntxiv mus, nyob rau hauv ib tug cais RNA sequencing (RNA-seq) dataset los ntawm tag nrho cov ntshav ntawm cov tib neeg uas muaj ntau yam inflammatory thiab autoimmune kab mob (Aune li al., 2017), MTHFD2 tau overexpressed tsis tu ncua nyob rau hauv ntau yam mob, nrog rau cov kab mob ulcerative, Crohn tus kab mob. , kab mob celiac, RA, systemic lupus erythematosus (SLE), psoriasis, psoriatic mob caj dab, Sjo¨ gren's syndrome, thiab MS (Daim duab 1F). Qhov tseem ceeb, cov tib neeg uas tsis ntev los no tau kuaj pom MS tau nce siab MTHFD2 qhia txog kev noj qab haus huv los yog cov tib neeg uas muaj MS tab tom kho nrog kev tshem tawm cov kab mob (Daim duab S1C). Yog li, CD4^ntxivT cell proliferation thiab ciaj sia taus yog nyob ntawm tej yam 1C metabolism genes, thiab kev qhia ntawm cov noob no txawv los ntawm cov theem ntawm T cell txoj kev loj hlob, ua kom, thiab sib txawv raws li zoo raws li nyob rau hauv lub teeb ntawm pathological o.

Daim duab 2. MTHFD2 deficiency impairs CD4^ntxivT cell proliferation thiab ua haujlwm

(A–D) Proliferation ntsuas los ntawm (A) CellTrace Violet (CTV) dilution, (B) viability, (C) CD25 qhia, thiab (D) TF qhia hauv CD4^ntxivT cell subsets kho nrog MTHFD2i rau 72 h tom qab ua kom ua haujlwm (txhais tau tias ± SD, ib-txoj kev ANOVA, cov ntaub ntawv yog tus sawv cev ntawm peb qhov kev sim ywj pheej nrog 9 tag nrho cov khoom siv roj ntsha).

MTHFD2 muaj kev tswj hwm zoo hauv CNS-infiltrating CD4^ntxivT hlwb hauv kev sim autoimmune encephalomyelitis (EAE) thiab hauv CD4 activated^ntxivT cell hauv vitro

Kev cai ntawm MTHFD2 tsis tau piav qhia zoo hauv CD4 ntxiv rau T hlwb. Txhawm rau ntsuas qhov qhia ntawm MTHFD2 hauv cov kab mob inflammatory hauv vivo, CD4^ntxivT hlwb los ntawm myelin-tshwj xeeb TCR transgenic 2D2 nas tau qhib thiab hloov mus rau hauv Rag1^{__ /__}nas mus ntxias EAE. Tom qab cov nas pib nthuav tawm hindleg tuag tes tuag taw, ntu cauda equina ntawm tus txha caj qaum tau txheeb xyuas los ntawm immunohistochemistry (IHC). Cov nas uas tau txais 2D2 T hlwb pom tias muaj txiaj ntsig ntawm MTHFD2 thiab CD3^ntxivcov hlwb uas tsis nyob hauv kev tswj hwm (Daim duab 1G). Txhawm rau txiav txim ncaj qha seb CD4^ntxivT hlwb upregulated MTHFD2 nyob rau hauv qhov txhab mob, T hlwb los ntawm tus po thiab tus txha caj qaum ntawm nas raug rau ib tug myelin oligodendrocyte glycoprotein (MOG) thiab pertussis toxin (PTX)-vim tus qauv ntawm EAE tau sau rau kev soj ntsuam. CNS-infiltrating CD4^ntxivT hlwb overexpressed MTHFD2 piv nrog matched splenic CD4^ntxivT hlwb los ntawm kev tswj hwm thiab EAE nas, raws li ntsuas los ntawm flow cytometry (Daim duab 1H) thiab qRT-PCR (Daim duab 1I). Lub kinetics ntawm MTHFD2 qhia hauv CD4^ntxivT hlwb hauv vitro raug ntsuas tom ntej. Kev txhim kho muaj zog ntawm MTHFD2 mRNA tau kuaj pom tom qab 5 teev ntawm kev txhawb nqa thiab pib txo qis los ntawm 24 teev (Daim duab 1J), qhov sib txawv CD4^ntxivT cell subsets tau txais qhov kev qhia siab tshaj plaws ntawm MTHFD2 protein 48 teev tom qab ua haujlwm (Daim duab 1K). Cov ntaub ntawv no qhia tau hais tias MTHFD2 yog upregulated hauv CD4^ntxivT hlwb nrog kev ua kom muaj zog hauv vitro thiab ntawm qhov chaw ntawm qhov mob hauv vivo.

CD4^ntxivT cell subsets sib txawv xav tau MTHFD2 rau kev ua kom, kev loj hlob, ciaj sia taus, thiab cytokine ntau lawm

Peb tom ntej no sim lub luag haujlwm ntawm MTHFD2 hauv CD4 ntxiv rau T cell subset ua kom, sib txawv, kev loj hlob, thiab kev ua haujlwm. CD4^ntxivT hlwb tau qhib rau hauv vitro nyob rau hauv lub xub ntiag ntawm cytokines rau kev pom zoo Th1, Th17, thiab Treg cell sib txawv thiab lub tsheb los yog ib qho MTHFD2 inhibitor (MTHFD2i; DS18561882) (Kawai li al., 2019). Tom qab 72 teev, tag nrho cov subsets tau txo qis kev loj hlob (Daim duab 2A) thiab cov xov tooj ntawm cov neeg caij tsheb kauj vab (Figures S2A thiab S2B). Kev ua tau zoo tau raug txo qis hauv Th1 thiab Th17 hlwb (Daim duab 2B), thiab kev ua kom muaj zog, raws li ntsuas los ntawm CD25 qhia, raug txo qis hauv txhua qhov subsets (Daim duab 2C). Lineage-characterizing transcription factor (TF) qhia tau txo qis hauv Th1 hlwb (T-bet^ntxiv) tab sis tsis hloov pauv hauv Th17 hlwb (ROR t^ntxiv) thiab hloov me ntsis hauv Treg hlwb (FoxP3^ntxiv) (Daim duab 1D). Txhawm rau ntsuas Teff cell muaj nuj nqi, cov hlwb tau rov ua haujlwm nrog 12-myristate 13-acetate (PMA) thiab ionomycin. Tsawg dua Th1 hlwb qhia interferon-g (IFNg), thiab tsawg dua Th17 hlwb qhia IL-17 thaum sib txawv ntawm MTHFD2i (Daim duab 2E). Qhov no tsis yog vim qhov ua tsis tau zoo vim tias cov phenotypes tau khaws cia thaum cov tshuaj raug ncua mus txog 24 teev tom qab ua haujlwm, txawm hais tias kab hnub nyoog-tus cwj pwm TF qhia tsis hloov pauv hauv qhov chaw no (Figures S2C-S2F). MTHFD2 yog li yuav tsum tau rau qhov siab tshaj plaws CD4^ntxivT cell activation, proliferation, ciaj sia taus, thiab cytokine ntau lawm.

Txhawm rau txheeb xyuas cov txiaj ntsig tshuaj no, peb tsim Mthfd2^ib/flnas strain thiab hla nws nrog Cd 4- cre transgenic nas kom ua tiav cov txheej txheem genetic deletion. Raws li kev cia siab, qhib CD4^ntxivT cells los ntawm Mthfd2^ib/flCd4-cre plus (CD4^{ΔMthfd 2}) nas tau qis MTHFD2 qhia piv nrog cov hlwb los ntawm Mthfd2^ib/flCd4-cr^ntxiv(wild-type [WT]) littermates (Daim duab 2F). Ntawm qhov pib, CD4DMthfd2 nas muaj tsawg dua CD4^ntxivT hlwb hauv tus po piv nrog WT (Daim duab 2G). Thaum nyob rau hauv vitro ua kom thiab sib txawv, cov xov tooj ntawm tes thiab kev muaj peev xwm ntawm CD4^{ΔMthfd 2}Th17 hlwb thiab, rau qhov tsawg dua, Treg hlwb tau qis dua (Daim duab 2H). Tsis tas li ntawd, CD25 qhia tau txo qis hauv Th17 thiab Treg hlwb thiab qis dua hauv Th1 hlwb (Daim duab 2I). Thaum kawg, CD4^{ΔMthfd 2}Th1 thiab Th17 hlwb tau qis dua ntawm T-thawj koom ruam thiab ROR t, feem. FoxP3 hauv Treg hlwb, txawm li cas los xij, tsis hloov pauv (Daim duab 2J). Txawm hais tias muaj kev hloov pauv hauv TF kev qhia, Teff hlwb uas tau sib txawv tsim cov cytokines ib txwm muaj (Daim duab 2K). Qhov sib txawv ntawm cov txiaj ntsig ntawm cov tshuaj pharmacological thiab noob caj noob ces tuaj yeem raug ntaus nqi rau lub sijhawm thiab ua tiav ntawm kev ua haujlwm enzyme poob thaum lub sijhawm txhim kho thiab muaj peev xwm them rov qab cov txheej txheem.

MTHFD2i induces FoxP3 qhia hauv Th17 hlwb thiab txhim kho Treg cell sib txawv

Muab qhov kev cia siab ntawm Teff hlwb ntawm MTHFD2, peb tau sim MTHFD2i kev tswj hwm ntawm FoxP3 thiab Treg hlwb. Qhov tseem ceeb, MTHFD2i kev kho mob-vim tsis txaus ntseeg ntawm FoxP3 hauv Th1 thiab tseem ceeb dua hauv Th17 hlwb nyob rau hauv koob tshuaj (Daim duab 3A). Qhov no kuj tshwm sim thaum kho tau ncua mus txog 24 teev tom qab ua kom tiav (Daim duab 3B). Ib qho piv txwv zoo sib xws tau pom hauv CD4DMthfd2 Th17 hlwb (Daim duab 3C). Qhov kev txhawb nqa FoxP3 no ua haujlwm tau zoo vim tias MTHFD2i-kho Th17 hlwb tau txais kev muaj peev xwm txo qis thaum sib koom ua ke nrog CD8^ntxivT cells (Daim duab 3D). MTHFD2i tuaj yeem txhawb nqa FoxP3 thiab Treg cell-zoo li phenotypes hauv Th17 hlwb. MTHFD2i kuj tau txhim kho qhov sib txawv ntawm induced Treg hlwb. Treg hlwb tau sib txawv nrog ntau yam ntawm kev hloov pauv kev loj hlob b (TGF-b) cov concentrations nyob rau hauv lub xub ntiag ntawm MTHFD2i. Ntawm txhua qhov kev ntsuam xyuas tshwj tsis yog qhov siab tshaj plaws, FoxP3 qhia tau nce nrog kev kho MTHFD2i (Figures 3E thiab 3F). Qhov phenotype no kuj tau tswj xyuas nrog ncua kev kho MTHFD2i (Daim duab 3G).

Daim duab 3. MTHFD2 deficiency induces FoxP3 qhia thiab txhawb kev hloov mus rau oxidative phosphorylation

(A) FoxP3 qhia hauv MTHFD2i-kho Th1 thiab Th17 hlwb 72 h tom qab ua kom ua haujlwm (txhais tau tias ± SD, ib-txoj kev ANOVA, cov ntaub ntawv yog tus sawv cev ntawm peb qhov kev sim ywj pheej nrog 9 tag nrho cov khoom siv roj ntsha).

(B) FoxP3 qhia hauv Th1 thiab Th17 hlwb qhib rau 24 teev thiab tom qab ntawd kho nrog lub tsheb lossis 500 nM MTHFD2i rau 48 h (txhais tau tias ± SD, unpaired t test, cov ntaub ntawv yog tus sawv cev ntawm peb txoj kev sim ywj pheej nrog 9 tag nrho cov khoom siv roj ntsha).

(C) FoxP3 qhia hauv Th1 thiab Th17 hlwb los ntawm WT thiab CD4DMthfd2 littermates 72 h tom qab ua kom ua haujlwm (txhais tau tias ± SD, unpaired t test, cov ntaub ntawv yog tus sawv cev ntawm peb txoj kev sim ywj pheej nrog 9 tag nrho cov khoom siv roj ntsha).

(D) Kev Tshawb Fawb Kev Tshawb Fawb ntsuas qhov tsis txaus ntseeg muaj peev xwm ntawm MTHFD2i-kho Th17 hlwb. Pre-kho Th17 hlwb raug co-cultured nrog CTV-stained CD8 ntxiv rau T hlwb los ntsuas kev loj hlob thaum ua kom muaj anti-CD3 thiab anti-CD28 antibodies (txhais tau tias ± SD, ib-txoj kev ANOVA, n=3 lom replicates) .

(E) FoxP3 qhia hauv Treg hlwb sib txawv nrog ntau yam ntawm TGF-b concentrations thiab kho nrog MTHFD2i (txhais tau tias ± SD, unpaired t test, cov ntaub ntawv yog tus sawv cev ntawm peb txoj kev sim ywj pheej nrog 9 tag nrho cov khoom siv roj ntsha).

(F) Flow cytometry plots rau cov ntaub ntawv tabulated hauv (E).

(G) FoxP3 qhia hauv Treg hlwb qhib thiab sib txawv nrog tsawg concentrations TGF-b rau 24 h thiab tom qab ntawd kho nrog MTHFD2i rau 48h (txhais tau tias ± SD, unpaired t test, cov ntaub ntawv yog tus sawv cev ntawm peb txoj kev sim ywj pheej nrog 9 tag nrho cov khoom siv roj ntsha. ).

(H) Seahorse XF Cell Mito stress test ua rau MTHFD2i-kho Th17 thiab Treg hlwb (txhais tau tias ± SD, n=3 bioreplicates).(I) Basal ECAR, basal OCR, max OCR, thiab basal OCR/ECAR piv ntsuas hauv (H) (txhais tau tias ± SD, unpaired t test, cov ntaub ntawv yog tus sawv cev ntawm ob qho kev sim ywj pheej nrog 6 tag nrho cov khoom siv roj ntsha rov ua dua tshiab). ** p < 0 01, *** p < 0.001, **** p% 0.001).

Treg hlwb muaj cov mitochondrial metabolism ntau dua li Th17 hlwb (Michalek li al., 2011). Txhawm rau txiav txim siab qhov tshwm sim ntawm metabolic tshwm sim ntawm kev kho MTHFD2i, Th17 thiab Treg hlwb raug soj ntsuam los ntawm kev tshuaj ntsuam xyuas cov kab mob hauv lub cev. MTHFD2i tau nce qhov basal thiab siab tshaj plaws oxygen noj tus nqi (OCR) ntawm Th17 hlwb los qhia txog kev hloov mus rau mitochondrial respiration (Daim duab 3H). Hloov pauv, Th17 thiab Treg hlwb tau txo qis basal extracellular acidification tus nqi (ECAR) nrog kev kho MTHFD2i, tawm tswv yim txo glycolysis (Daim duab 3I). Qhov piv ntawm basal ntawm OCR rau ECAR tau nce qhov tseem ceeb hauv Th17 thiab Treg hlwb vim tias MTHFD2i hloov cov kev pabcuam metabolic los ntawm glycolysis mus rau oxidative phosphorylation. Cov ntaub ntawv no qhia tau tias MTHFD2i txhim kho FoxP3 qhia hauv Th17 hlwb thiab Treg hlwb sib txawv raws li TGF-b qis thiab hloov Th17 hlwb mus rau Treg cell zoo li metabolism.

MTHFD2 deficiency hauv tib neeg CD4 ntxiv rau T hlwb txo Th17 cell proliferation thiab viability thaum nce FOXP3 qhia thiab dampening mTORC1 kev ua.

Txhawm rau ntsuas qhov kev txhais lus ntawm qhov kev tshawb pom saum toj no rau tib neeg lub hlwb, CD4^ntxivT hlwb cais tawm ntawm cov ntshav peripheral ntawm cov neeg pub noj qab haus huv tau sib txawv hauv vitro rau hauv Th1, Th17, thiab Treg hlwb thiab kho nrog lub tsheb lossis MTHFD2i txij lub sijhawm ua haujlwm. Kev loj hlob tau txo qis hauv MTHFD2i-kho Th17 thiab Treg hlwb (Daim duab 4A), thiab Th17 hlwb kuj pom tau tias muaj apoptosis (Daim duab 4B). Txo kev loj hlob hauv Th17 hlwb tau rov ua dua nrog txoj hauv kev caj ces siv me me cuam tshuam RNA (siRNA) (Figures 4C thiab S2G). Zoo ib yam li murine hlwb, FOXP3 qhia tau siab dua hauv MTHFD2i-kho tib neeg Th17 hlwb thiab tau nce siab dua hauv MTHFD2i-kho tib neeg Treg hlwb (Figures 4D thiab S2H). Muab hais tias mTORC1-AMPK axis tseem ceeb heev rau Th17 thiab Treg cell sib txawv, phosphorylation ntawm mTORC1 lub hom phiaj ribosomal protein S6 (phospho-S6) kuj tau ntsuas. Qhov no pom tias txo qis mTORC1 kev ua hauv Th17 hlwb nrog MTHFD2i lossis siMTHFD2 (Daim duab 4E). Yog li, qhov poob ntawm kev loj hlob muaj peev xwm thiab induction ntawm FOXP3 qhia hauv Th17 thiab Treg hlwb tuaj yeem rov ua dua hauv tib neeg CD4.^ntxivT hlwb.

Daim duab 4. MTHFD2 deficiency impairs proliferation thiab induces FOXP3 qhia nyob rau hauv tib neeg Th17 hlwb

(A) Kev loj hlob ntsuas los ntawm CTV dilution hauv tib neeg CD4 ntxiv rau T cell subsets kho nrog lub tsheb lossis 2 mM MTHFD2i rau 72 h tom qab ua kom muaj cov tshuaj tiv thaiv CD3 thiab anti-CD28 cov tshuaj tiv thaiv (paired t test, cov ntaub ntawv yog tus sawv cev ntawm peb txoj kev sim ywj pheej nrog 5 -6 tus neeg pub noj qab haus huv).

(B) Apoptosis ntsuas los ntawm Annexin V hauv tib neeg CD4 ntxiv rau T hlwb kho nrog MTHFD2i rau 72 h (paired t test, cov ntaub ntawv yog sawv cev ntawm peb qhov kev sim ywj pheej nrog 5-6 tus neeg noj qab haus huv pub dawb).

(C) Ki-67 kev qhia hauv tib neeg CD4 ntxiv rau T hlwb hloov nrog NTC lossis siMTHFD2 (paired t test, cov ntaub ntawv yog tus sawv cev ntawm peb qhov kev sim ywj pheej nrog 4 tus neeg mob noj qab haus huv).

(D) FOXP3 qhia hauv tib neeg CD4 ntxiv rau T hlwb kho nrog MTHFD2i rau 72 h (paired t test, cov ntaub ntawv yog sawv cev ntawm peb txoj kev sim ywj pheej nrog 6-10 noj qab haus huv pub dawb).

(E) Hloov pauv hauv phospho-S6 qhia hauv tib neeg CD4 ntxiv rau T hlwb kho nrog MTHFD2i rau 72 h lossis hloov nrog siMTHFD2 (txhais tau tias ± SD, ib qho qauv t xeem, cov ntaub ntawv sawv cev ntawm peb qhov kev sim ywj pheej nrog 4-6 tus neeg noj qab haus huv pub dawb). Cov txiaj ntsig tseem ceeb tau sau npe (* p < 0="" {{10}}5,="" **="" p="">< 0.01,="" ***="" p="">< 0.001,="" ****="" p%="">

T hlwb cia siab rau MTHFD2 enzymatic muaj nuj nqi, thiab phenotypes tuaj yeem cawm tau los ntawm cov khoom

MTHFD2 yuav tsum muaj rau kev saib xyuas ntawm lub pas dej mitochondrial formate rau purine synthesis (Ma li al., 2017) tab sis kuj tseem tuaj yeem ua lub luag haujlwm tsis yog enzymatic. Txhawb nqa qhov tseem ceeb enzymatic kev ua ub no, kev kho MTHFD2i coj mus rau cov nyiaj them rov qab ntawm formate los ntawm nruab nrab hauv CD4 ntxiv rau T cell subsets (Daim duab 5A). 1C chav tsev siv rau formate synthesis yog muab los ntawm kev hloov ntawm serine rau glycine, thiab intracellular serine accumulated whereas glycine tau depleted nrog MTHFD2i kev kho mob nyob rau hauv tag nrho cov subsets (Daim duab 5B). Methionine concentrations kuj tau nce me ntsis, qhia tias inhibition ntawm lub voj voog methionine. Raws li qhov xav tau, muab cov kauj ruam hloov pauv serine-glycine yog nce ntawm MTHFD2, cov kev hloov pauv no tsis tau cawm los ntawm kev muab cov qauv hauv nruab nrab.

Daim duab 5. MTHFD2i teebmeem ntawm T cell subsets raug cawm los ntawm exogenous formate

(A) Formate uptake ntsuas los ntawm 1 H-magnetic resonance spectroscopy (MRS) hauv CD4 ntxiv rau T cell subsets kho nrog lub tsheb lossis 500 nM MTHFD2i rau 72 h tom qab ua kom ua haujlwm (txhais tau tias ± SD, unpaired t test, cov ntaub ntawv yog tus sawv cev ntawm ob qho kev sim ywj pheej nrog 6 tag nrho cov bioreplicates).

(B) Kev hloov pauv hauv serine, glycine, thiab methionine concentrations hauv CD4 ntxiv rau T hlwb kho nrog 500 nM MTHFD2i lossis 500 nM MTHFD2i ntxiv rau 1 mM formate rau 4-6 h piv nrog lub tsheb, ntsuas los ntawm huab hwm coj spectrometry (txhais tau tias ± SD, n { {8}} biological replicates).

(C) Nyob ntawm tes suav, muaj peev xwm, thiab CD25 qhia hauv MTHFD2i-kho CD4 ntxiv rau T hlwb cawm nrog 1 mM formate lossis 60 mM adenine thiab guanine purine tov rau 72 h tom qab ua kom (txhais tau tias ± SD, ib-txoj kev ANOVA, cov ntaub ntawv yog tus neeg sawv cev ntawm peb qhov kev sim ywj pheej nrog 9 tag nrho cov khoom siv lom neeg).

(D) Nyob ntawm tes suav, muaj peev xwm, thiab CD25 qhia hauv CD4 ntxiv rau T hlwb los ntawm WT lossis CD4DMthfd2 littermates cawm nrog formate lossis purines rau 72 teev tom qab ua kom (txhais tau tias ± SD, ib-txoj kev ANOVA, n=3 bioreplicates ).

Peb tom ntej no sim seb qhov ntxiv ntawm formate los yog adenine thiab guanine purine tov tuaj yeem cawm tsis muaj zog enzymatic thiab thim rov qab los ntawm MTHFD2 deficiency. Tseeb tiag, 1 mM formate rov qab muaj kev loj hlob, muaj peev xwm, thiab ua kom muaj zog hauv MTHFD2i-kho hlwb (Daim duab 5C). Purine supplementation cawm kev loj hlob ntawm txhua qhov subsets thiab ua kom muaj zog hauv Th1 thiab Th17 hlwb tab sis tsis muaj peev xwm nyob hauv txhua qhov subsets thiab ua kom muaj zog hauv Treg hlwb. Formate tseem cawm tau kev loj hlob, kev vam meej, thiab ua kom muaj zog hauv CD4^{ΔMthfd 2}T cells (Daim duab 5D). Kev puas tsuaj cytokine ntau lawm hauv MTHFD2i-kho Th1 thiab Th17 hlwb raug thim rov qab rau lub tsheb cov nyiaj nrog formate lossis purine ntxiv (Daim duab 5E). Qhov tseem ceeb, kev hloov pauv tsis zoo ntawm FoxP3 hauv Th17 hlwb tau txais kev cawmdim los ntawm purine ntxiv tab sis tsis formate, thaum formate thiab purine ntxiv txo FoxP3 qhia rau hauv paus hauv Treg hlwb sib txawv nrog tsawg concentrations ntawm TGF-b (Daim duab 5F). MTHFD2i phenotypes tshwm sim nyob ntawm MTHFD2 kev saib xyuas ntawm mitochondrial formate lossis purine pas dej.

MTHFD2 tswj mTORC1 kev ua haujlwm thiab DNA thiab histone methylation hauv CD4^ntxivT hlwb

Hauv cov qog nqaij hlav tsis txawj tuag, MTHFD2 tsis txaus tuaj yeem ua rau purine synthesis intermediates GAR, SAICAR, thiab AICAR, uas yog nce ntawm 10-formylTHF-mediated formylation cov kauj ruam (Ducker li al., 2016). CD4^ntxivT cell subsets raug coj mus kuaj rau 72 teev ua ntej raug MTHFD2i rau 4–6 teev. Lub sijhawm luv luv no tau raug xaiv kom tsis txhob muaj qhov cuam tshuam thib ob, suav nrog kev hloov pauv nyiaj. Transient MTHFD2i kev kho mob ua rau muaj kev sib txuam ntawm GAR, SAICAR, thiab AICAR hauv txhua qhov chaw (Daim duab 6A). Cov nyhuv no tau txais kev cawmdim los ntawm kev sib ntxiv ntawm 1 mM formate, txhawb lub luag haujlwm enzymatic ntawm MTHFD2. Nucleotides thiab nucleobases, suav nrog guanine, kuj tau depleted los ntawm MTHFD2i thiab cawm los ntawm formate hauv Th17 thiab Treg hlwb (Daim duab 6B).

Daim duab 6. MTHFD2i tshwm sim hauv tsub zuj zuj ntawm purine synthesis intermediates dampened mTORC1 kev ua thiab hloov DNA thiab histone methylation

(A) Kev hloov pauv hauv GAR, SAICAR, thiab AICAR concentrations hauv CD4 ntxiv rau T hlwb kho nrog 500 nM MTHFD2i lossis 500 nM MTHFD2i ntxiv rau 1 mM formate rau 4-6 h txheeb ze rau lub tsheb, ntsuas los ntawm huab hwm coj spectrometry (txhais tau tias ± SD, ib- txoj kev ANOVA, n=3 bioreplicates).

(B) Hloov cov nucleotide metabolism hauv hom Th17 thiab Treg hlwb kho nrog MTHFD2i nrog lossis tsis muaj formate rau 4-6 h txheeb ze rau lub tsheb, ntsuas los ntawm huab hwm coj spectrometry (n=3 bioreplicates).

(C) Immunoblot ntawm phospho-S6, S6, Rheb, phospho-ACC, ACC, HIF-1a, thiab b-actin hauv CD4 ntxiv rau T hlwb kho nrog MTHFD2i nrog lossis tsis muaj formate rau 72 teev tom qab ua kom tiav (cov ntaub ntawv tus neeg sawv cev ntawm peb qhov kev sim ywj pheej nrog n=3 bioreplicates).

(D) Hloov hauv TCA voj voog metabolites hauv Th17 hlwb kho nrog MTHFD2i nrog lossis tsis muaj formate rau 6 h txheeb ze rau lub tsheb, ntsuas los ntawm huab hwm coj spectrometry (txhais tau tias ± SD, ib-txoj kev ANOVA, n=3 bioreplicates).

(E) Heatmaps ntawm H3K27me3 rau IgG tswj piv hauv ±10-kb thaj tsam nyob ib ncig ntawm cov neeg txhawb nqa ntawm UCSC paub cov noob hauv Th17 thiab Treg hlwb kho nrog MTHFD2i, ntsuas los ntawm CUT&RUN (cov ntaub ntawv sib sau los ntawm 3 cov khoom siv roj ntsha).

(F) Heatmap ntawm nruab nrab DNA methylation zaus ntawm Foxp3 locus los ntawm CpG site hauv Th1 thiab Th17 hlwb kho nrog tsheb lossis 500 nM MTHFD2i thiab hauv nTreg hlwb los ntawm WT lossis CD4DMthfd2 littermates (Mood's test, n=3 bioreplicates).

(G) Tabulation ntawm (F) los ntawm proximal promoter, distal promoter, thiab TSDR (paired t test). Saib cov duab S3 thiab S4. Cov txiaj ntsig tseem ceeb ntawm cov ntawv sau npe (* p < 0.05,="" **="" p="">< 0.01,="" ***="" p="">< 0.001,="" ****="" p%="">

AICAR thiab nucleotides yog tsim los tswj hwm ntawm mTORC1-AMPK axis (Emmanuel li al., 2017; Kim et al., 2016). Tseeb tiag, phospho-S6 qhia tau txo qis nrog kev kho MTHFD2i hauv txhua qhov subsets thiab tag nrho rov qab los ntawm kev sib ntxiv ntawm formate (Figures 6C thiab S3A). GTP-binding protein Rheb, lub luag haujlwm activator ntawm mTORC1, tuaj yeem nkag siab rau qhov muaj guanine intracellular (Emmanuel li al., 2017), thiab Rheb qhia tau dampened nrog MTHFD2i kev kho mob (Figures 6C thiab S3B). AICAR yog ib qho adenosine analog thiab AMPK activator (Rae thiab Mairs, 2019; Su li al., 2019). Txawm li cas los xij, 72 teev tom qab ua haujlwm, phosphorylation ntawm AMPK lossis nws lub hom phiaj, acetyl-coenzyme A(CoA) carboxylase (phospho-ACC) thiab Unc-51-zoo li autophagy activating kinase (phospho-ULK1), tsis hloov nrog kev kho MTHFD2i. (Daim duab 6C, S3C, thiab S3D). mTORC1 kev ua txhawb nqa Th17 thiab txo qis Treg cell sib txawv nyob rau hauv ib feem los ntawm induction ntawm HIF-1a-dependent glycolytic metabolism (Shi li al., 2011). Raws li qhov no, HIF-1 ib qho kev qhia tau txo qis hauv MTHFD2i-kho thiab CD4DMthfd2 Th17 hlwb (Figures 6C, S3E, thiab S3F). Cov kev hloov pauv hauv mTORC1 kev ua ub no tsis cuam tshuam nrog qhov tsis txaus ntawm TCR signaling, raws li ntsuas los ntawm Nur77 qhia nrog thiab tsis muaj kev rov ua dua (Daim duab S3G). Teeb liab transducer thiab activator ntawm transcription 3 (STAT3) kev ua, uas yog ib qho tseem ceeb rau Th17 cell txoj kev loj hlob, tsis hloov los yog nce me ntsis nrog kev kho MTHFD2i (Daim duab S3H). Kev ua haujlwm ntawm mTORC1 txo qis thiab hloov pauv ntawm glycolysis mus rau oxidative phosphorylation nrog kev kho MTHFD2i qhia txog kev hloov pauv ntawm cov txheeb ze ntawm tricarboxylic acid (TCA) cycle metabolites. Succinate thiab fumarate concentrations tau txo qis los ntawm MTHFD2i tab sis cawm tau los ntawm formate (Daim duab 6D).

TCA metabolites paub inhibitors ntawm DNA thiab histone demethylase (Su li al., 2016), thiab depletion ntawm succinate thiab fumarate pas dej ua ke yuav txhawb lub xeev hypomethylated. H3K27 tri-methylation (H3K27me3) thoob plaws genome tau ntsuas los ntawm CUT & RUN (cleavage nyob rau hauv lub hom phiaj thiab tso tawm siv nuclease) sequencing (Skene thiab Henikoff, 2017). Tseeb, MTHFD2i-kho Th17 hlwb tau txo H3K27me3 piv nrog lub tsheb (Daim duab 6E thiab S4A). DNA methylation ntawm Foxp3 locus kuj tau ntsuas los ntawm bisulfate sequencing los txiav txim seb qhov sib txawv methylation tau pab rau MTHFD2i induced FoxP3 upregulation. Txawm hais tias ob peb CpG loci sib txawv hauv methylation zaus ntawm lub tsheb thiab MTHFD2i cov xwm txheej, tsis muaj tus qauv pom hauv Treg cell-specific demethylated cheeb tsam (TSDR). Kuj tsis muaj qhov sib txawv ntawm nTreg hlwb (CD4 ntxiv rau CD25 ntxiv) cais tawm ntawm WT thiab CD4DMthfd2 nas (Daim duab 6F), txawm hais tias qhov purity ntawm txhua tus neeg muaj txiaj ntsig tsawg (Daim duab S4B). Txawm li cas los xij, muaj qhov txo qis hauv DNA methylation zaus dhau ntawm Foxp3 proximal proximal cheeb tsam hauv MTHFD2i-kho Th17 hlwb piv nrog lub tsheb (Daim duab 6G), qhia txog qhov ua tau zoo ntawm kev sau nrog MTHFD2i.

Ntxiv rau kev tswj hwm lub pas dej intracellular formate, MTHFD2 kev ua haujlwm txhawb nqa cellular redox tshuav nyiaj li cas los ntawm kev hloov pauv ntawm NAD^ntxivrau NADH thiab NADP^ntxivmus rau NADPH (Shin et al., 2017) thiab kev txhawb nqa ntawm txoj kev transsulfuration thiab tom qab glutathione synthesis. MTHFD2i-kho Th17 hlwb tau txo qis ntawm NAD^ntxiv, NADH, NADP^ntxiv, thiab NADPH, uas tau cawm nrog formate (Daim duab S4C). Txawm li cas los xij, NAD^ntxiv/ NADH thiab NADP^ntxiv/ NADPH piv tsis tau hloov pauv hauv cov xwm txheej. Qhov no, ua ke nrog qhov tsis muaj kev hloov pauv hauv cellular reactive oxygen hom (ROS) thiab mitochondrial superoxide (Daim duab S4D), qhia tias kev hloov pauv hauv lub xeev redox tsis zoo li yog qhov tseem ceeb rau MTHFD2i phenotype.

MTHFD2 deficiency hauv vivo txo ​​qhov mob ntawm ntau yam kab mob

Qhov ua tau zoo ntawm MTHFD2 raws li lub hom phiaj kho mob tau sim tom ntej hauv vivo T cell-dependent qeeb-hom hypersensitivity (DTH) qauv. Cov nas tau txhaj tshuaj tiv thaiv thiab sib tw ntawm pob ntseg nrog keyhole limpet hemocyanin (KLH) txhawm rau ua rau mob hauv zos thiab kho nrog lub qhov ncauj tsheb lossis MTHFD2i. Kev kho mob tsis pom muaj tshuaj lom vim tias tsiaj khaws lawv qhov hnyav (Daim duab S5A). Qhov tseem ceeb, pob ntseg tuab thiab qhov hnyav tau nce ntxiv hauv kev tswj hwm DTH nas tab sis tsis yog nyob rau hauv inhibitor-kho tsiaj, qhia txog kev tiv thaiv kev mob ntawm MTHFD2i kev kho mob (Figures 7A, 7B, thiab S5B). KLH-specific immunoglobulin G (IgG) kuj tau txo qis ntawm qhov koob tshuaj ntau tshaj ntawm inhibitor, qhia tias muaj peev xwm cuam tshuam rau B cell ua haujlwm (Daim duab 7C).

Daim duab 7. Targeting MTHFD2 hauv vivo txo ​​cov kab mob hnyav hauv DTH, EAE, thiab IBD qauv

(A thiab B) Pob ntseg (A) txheeb ze tuab thiab (B) qhov hnyav ntawm qhov nqaij ntuag 10 hnub tom qab KLH nrog kev txhaj tshuaj CFA thiab 3 hnub tom qab KLH kev sib tw los ntxias DTH (ib-txoj kev ANOVA). Cov nas raug kho ob zaug ib hnub nrog qhov ncauj 0, 100, lossis 300 mg / kg MTHFD2i (txhais tau tias ± SEM, n=8 bioreplicates).

(C) KLH-specific IgG concentrations nyob rau hnub 10 ntawm KLH-induced DTH (txhais tau tias ± SEM, Kruskal-Wallis test, n=8 biological replicates).

(D) Cov qhab nia nruab nrab ntawm kev kho mob nyob rau lub sijhawm hauv WT thiab CD4DMthfd2 littermates txhaj tshuaj tiv thaiv MOG nrog CFA thiab PTX kom ntxias EAE (txhais tau tias ± SEM, ntau qhov kev xeem Mann-Whitney, cov ntaub ntawv yog tus sawv cev ntawm ob qhov kev sim ywj pheej, txhua tus muaj 5 cov khoom siv roj ntsha).

(E) Cell suav thiab zaus ntawm CD4 ntxiv rau T hlwb hauv qaum qaum ntawm EAE nas ntawm cov kab mob siab tshaj plaws (txhais tau tias ± SEM, unpaired t test, cov ntaub ntawv yog tus sawv cev ntawm ob qhov kev sim ywj pheej nrog 9 tag nrho cov kab mob sib txawv).

(F) CD25 thiab CD44 qhia hauv CD4 ntxiv rau T hlwb los ntawm tus txha caj qaum ntawm EAE nas (txhais tau tias ± SEM, unpaired t test, n=4 biological replicates).

(G thiab H) Cell suav ntawm (G) T-bet ntxiv , RORgt ntxiv , FoxP3 ntxiv , FoxP3 ntxiv / T-bet ntxiv piv, FoxP3 ntxiv / RORgt ntxiv piv thiab (H) IL-17 ntxiv, IFNg ntxiv , thiab IL-17 ntxiv rau IFNg ntxiv rau CD4 ntxiv rau T hlwb los ntawm tus txha caj qaum ntawm EAE nas (txhais tau tias ± SEM, unpaired t test, cov ntaub ntawv yog tus sawv cev ntawm ob qho kev sim ywj pheej nrog 9 tag nrho cov khoom siv roj ntsha).

(I) H&E, anti-CD3 IHC, thiab Luxol Fast Blue (LFB) staining rau myelin nyob rau hauv tus txha caj qaum ntawm WT nas uas tsis muaj kev txhaj tshuaj thiab WT thiab CD4DMthfd2 littermates nrog EAE ntawm cov kab mob siab tshaj (2003 magnification, tus neeg sawv cev ntawm 5 bioreplicates ).

(J) Hloov lub cev hnyav dhau lub sijhawm ntawm Rag1^__/__nas txhaj ip nrog WT los yog CD4DMthfd2 naive CD4 ntxiv rau T hlwb kom induce IBD colitis (txhais tau tias ± SEM, ntau t xeem, n=8 lom replicates).

(K thiab L) Cell suav ntawm (K) tag nrho CD4 ntxiv rau T hlwb thiab (L) T-bet ntxiv , RORgt ntxiv , thiab FoxP3 ntxiv rau CD4 ntxiv rau T hlwb los ntawm MLNs ntawm IBD nas (txhais tau tias ± SEM, unpaired t test n { {4}} biological replicates). Saib cov duab S5 thiab S6. Cov txiaj ntsig tseem ceeb tau sau npe (* p < 0="" 05,="" **="" p="">< 0="" 01,="" ***="" p="">< 0.001,="" ****="" p%="">

Ib yam li ntawd, dual MTHFD1/2 inhibitor LY345899 (Gustafsson li al., 2017) tau ua haujlwm tau zoo hauv cov kab mob ameliorating hauv EAE qauv. EAE raug ntxias nrog MOG thiab PTX, thiab nas tau kho txhua hnub nrog kev txhaj tshuaj intraperitoneal (ip) ntawm DMSO lossis LY345899. Inhibitor kev kho mob tau ua rau txo qis tus kab mob hnyav thiab cov qhab nia kho mob sib piv nrog lub tsheb (Figures S5C thiab S5D). Cov nas tau txi rau hnub 26, thiab cov hlwb txha caj qaum-infiltrating tau sau rau kev tiv thaiv kab mob. Qhov tseem ceeb, qhov tseem ceeb tsawg dua CD45^ntxiv, cd4 ua^ntxiv,, ua cd8^ntxivcov hlwb infiltrated tus txha caj qaum ntawm LY345899-kho nas (Figures S5E–S5G). Tsis muaj kev hloov pauv hauv zaus ntawm ROR t^ntxivlos yog FoxP3^ntxivcell ntawm infiltrating CD4^ntxivT cells (Daim duab S5H). Txawm li cas los xij, muaj qhov txo qis ntawm IFNg ntxiv rau IL-17 ntxiv cov hlwb thiab nce hauv IL-17 ntxiv rau cov hlwb (Daim duab S5I), qhia txog qhov muaj peev xwm hloov mus rau qhov tsis tshua muaj kab mob phenotype.

Txhawm rau kuaj ncaj qha T cell-intrinsic dependence ntawm MTHFD2 nyob rau hauv vivo, WT thiab CD4DMthfd2 littermates tau raug rau peb yam qauv: EAE, inflammatory plob tsis so tswj kab mob (IBD), thiab kab mob ua xua. EAE tau rov ua dua nrog MOG thiab PTX, thiab, zoo ib yam li qhov kev sim inhibitor, CD4DMthfd2 nas tau txo qis tus kab mob hnyav dhau 26 hnub (Daim duab 7D). Ib pawg sib cais tau euthanized ntawm cov kab mob siab tshaj plaws nyob rau hnub 15 tom qab induction rau T cell phenotyping (Figures S6A thiab S6B). Txawm hais tias CD4^ntxivlub cd8^ntxivT cell suav tsis tau hloov pauv hauv tus po (Daim duab S6C), CD4 ntxiv rau T cell infiltration rau hauv tus txha caj qaum tau poob qis (Daim duab 7E). Spinal cord-infiltrating CD4 plus T cells in CD4DMthfd2 nas muaj CD25 qhia zoo ib yam li WT tab sis txo qis ntawm CD44, taw qhia rau qee qhov kev ua kom tsis zoo (Daim duab 7F). Tus lej ntawm T-bet^ntxiv, ROR t plus , thiab FoxP3 ntxiv cov hlwb (Daim duab 7G), nrog rau IL-17 ntxiv thiab IFNg ntxiv cov hlwb (Daim duab 7H), kuj tau txo qis hauv tus txha caj qaum ntawm CD4DMthfd2 nas txheeb ze rau WT. Qhov tseem ceeb, qhov sib piv ntawm FoxP3 ntxiv rau T-thawj ntxiv thiab RORgt ntxiv cov hlwb tau nce siab hauv CD4DMthfd2 nas piv nrog WT ntawm tus kab mob siab tshaj plaws, tsom rau qhov kev tshawb pom hauv vitro ntawm kev nthuav qhia FoxP3 ntxiv nrog MTHFD2 tsis txaus (Daim duab 7G). Txawm hais tias qhov zaus ntawm TF nthuav qhia cov hlwb ntawm cov hlwb infiltrating tag nrho cov kev sib tw siab dua, qhov zaus ntawm cytokine qhia tsis hloov pauv (Figures S6D thiab S6E). Histology ntawm tus txha caj qaum ntawm cov nas tsis muaj EAE thiab WT thiab CD4^{ΔMthfd 2}nas nrog EAE pom tau tias muaj zog cellularity thiab CD3 zoo nyob rau hauv tus txha caj qaum hauv WT nas uas tau txuam nrog demyelination, raws li qhia los ntawm Luxol Fast Blue staining (Daim duab 7I). Cov kev hloov no tsis muaj nyob rau hauv tsis muaj-EAE tswj thiab CD4^{ΔMthfd 2}nas.

Lub luag haujlwm ntawm MTHFD2 tau sim tom ntej hauv IBD thiab cov qauv kab mob ua xua. WT thiab CD4DMthfd2 naive CD4 ntxiv rau T hlwb raug xa mus rau Rag1 / cov neeg tau txais los pib IBD. Txawm hais tias cov nas uas tau txais WT hlwb pib poob phaus raws li IBD zuj zus, cov neeg tau txais CD4DMthfd2 T hlwb txuas ntxiv nce qhov hnyav (Daim duab 7J). Tsawg tsawg tus lej thiab zaus ntawm CD4DMthfd2 T hlwb tau pom nyob rau hauv spleens (Daim duab S6F) thiab mesenteric lymph nodes (MLNs) ntws cov nyuv (Figures 7K thiab S6G). Tus lej ntawm T-thawj ntxiv , ROR t ntxiv , thiab FoxP3 ntxiv rau CD4DMthfd2 T hlwb kuj tau txo qis hauv MLNs, tab sis qhov zaus ntawm FoxP3 ntxiv cov hlwb tau nce (Figures 7L thiab S6H). Txawm li cas los xij, tsis muaj qhov sib txawv hauv FoxP3 / T-thawj koom ruam thiab FoxP3 / RORgt ntawm tes suav qhov sib piv hauv MLNs ntawm lub sijhawm kawg no (Daim duab S6I). Zoo ib yam li kev tshawb pom ntawm MTHFD2 raws li kev sib tw me me hauv vivo airway o CRISPR screen (Daim duab 1C), CD4DMthfd2 nas raug rau Alternaria-induced allergic airway kab mob muaj ib qho kev txo qis neutrophil abundance nyob rau hauv lub bronchioalveolar lavage kua (BALF), txawm tias tsis muaj. Kev hloov pauv tau pom hauv BALF lymphocyte, eosinophil, thiab macrophage suav (Daim duab S7A).

Thaum kawg, cov txiaj ntsig ntawm MTHFD2i tau sim ntawm kev tiv thaiv kab mob. OVA tshwj xeeb OT-II CD45.2^ntxivCD4^ntxivT hlwb tau hloov mus rau hauv CD45.1^ntxivnas, uas tau txhaj tshuaj subcutaneously nrog ib qho emulsion ntawm OVA nrog ua tiav Freund's adjuvant (CFA) thiab kho txhua hnub nrog lub qhov ncauj tsheb lossis MTHFD2i. Lub cev hnyav tsis hloov pauv raws li txhua yam kev mob (Daim duab S7B). Kev nthuav dav ntawm qhov hloov pauv OT-II CD45.2^ntxivcov hlwb tau qeeb me ntsis hauv kev txhaj tshuaj MTHFD2i-kho nas tab sis mus txog cov lej tsheb los ntawm hnub 7 (Figures S7C–S7E). Txawm li cas los xij, qhov zaus ntawm IFNg ntxiv cov hlwb raug txo qis hauv cov kua dej LNs (Daim duab S7F). Cov ntaub ntawv no qhia tau hais tias nyob rau hauv vivo MTHFD2i kev kho mob tso cai rau antigen tshwj xeeb T cell expansion tom qab mob stimulation, tab sis nrog dampened inflammatory cytokine ntau lawm.

Kev sib tham

T cell activation, sib txawv, thiab muaj nuj nqi yuav tsum tsim nyog metabolic reprogramming kom ua tau raws li lub hlwb xav tau ntau zog, biosynthetic tsev blocks thiab signaling molecules. Hauv txoj kev tshawb no, peb tau tshawb xyuas qhov ua haujlwm ntawm 1C metabolism hauv thawj CD4 ntxiv rau T hlwb. Siv kev sib xyaw ua ke ntawm vivo thawj T cell CRISPR-raws li kev tshuaj ntsuam xyuas thiab cov ntaub ntawv qhia cov noob, MTHFD2 tau txheeb xyuas tias yog lub hom phiaj rau kev kho mob tiv thaiv kab mob. MTHFD2 inhibition lossis deficiency feem ntau ua rau txo qis CD4 ntxiv rau T cell proliferation. Qhov tseem ceeb, qhov no tau cuam tshuam nrog induction ntawm FoxP3 kev qhia thiab kev ua haujlwm tsis zoo hauv Th17 hlwb nrog rau kev txhim kho Treg cell sib txawv. Cov ntaub ntawv no qhia tias MTHFD2 tuaj yeem ua haujlwm raws li qhov chaw kuaj mob metabolic hauv Th17-Treg cell axis, nrog MTHFD2i kev kho mob sewing qhov sib npaug los ntawm cov kab mob mus rau ntau yam tshuaj tiv thaiv phenotype. Tseeb tiag, MTHFD2 inhibition lossis genetic deficiency ameliorated kab mob hnyav hauv ntau yam hauv vivo qauv ntawm autoimmunity thiab hypersensitivity.

1C metabolism muaj xws li serine-glycine metabolism, lub voj voog folate, thiab lub voj voog methionine thiab yog lub hauv paus rau ntau yam txheej txheem, suav nrog de novo purine synthesis, methyl pub dawb tiam, thiab kev cai redox (Yang thiab Vousden, 2016). 1C metabolism tau koom nrog TCR stimulation (Tan li al., 2017) thiab yuav tsum tau txhawb T cell expansion (Ma li al., 2017). Lub voj voog folate yog compartmentalized rau hauv cytosolic thiab mitochondrial Cheebtsam. Hauv cytosol, MTHFD1 interconverts 5, 10-methyleneTHF, 10-formylTHF, thiab formate. Hauv mitochondria, tib qhov kev xav tau ua tiav los ntawm MTHFD2 thiab MTHFD1L. Cov txiaj ntsig nruab nrab no yog siv los txhawb cov kauj ruam formylation hauv de novo purine synthesis. MTHFD1 thiab MTHFD2 tuaj yeem hloov kho lub xeev redox los ntawm NAD(H) thiab NADP(H) tiam (Ducker thiab Rabinowitz, 2017). Txawm hais tias qee lub xovtooj ntawm tes qhia tau yooj yim hloov mus rau cytosolic qhov chaw nyob rau hauv qhov chaw ntawm mitochondrial txoj kev ua haujlwm tsis zoo (Ducker li al., 2016), T hlwb tau raug npaj ua ntej los nyob ntawm feem ntau ntawm txoj kev mitochondrial los muab 1C units thiab txo hom (Ron- Harel et al., 2016). T hlwb kuj tau pom tias vam khom serine, thiab hauv vivo lub cev tiv thaiv kab mob tuaj yeem hloov kho los ntawm kev noj zaub mov serine. Cov teebmeem ntawm serine tshaib plab yog kho los ntawm kev txwv purine biosynthesis txawm tias nyob rau hauv lub xub ntiag ntawm txoj kev khaws cia tsis zoo thiab tuaj yeem hla dhau los ntawm kev muab glycine thiab formate (Ma li al., 2017). Peb cov ntaub ntawv ntxiv rau cov kev tshawb pom no thiab taw qhia rau MTHFD2 ua tus tswj hwm tseem ceeb uas cuam tshuam rau CD4^ntxivT cell proliferation thiab sib txawv.

Echanicoside

Txawm hais tias 1C metabolism ua lub luag haujlwm dav hauv cell metabolism, peb pom tias qee qhov haujlwm tau txawv los xaiv T cell subsets. Txhua tus tib neeg thiab nas T hlwb loj hlob mus rau qhov tsawg dua nrog MTHFD2 tsis txaus, qhia txog kev sib koom ua haujlwm rau MTHFD2 los txhawb T cell loj hlob thiab faib. Cov teebmeem ntawm MTHFD2 deficiency ntawm T cell sib txawv thiab effector muaj nuj nqi, txawm li cas los xij, sib txawv hauv txhua qhov kev sim. Th1 hlwb tau pom tias muaj kev cuam tshuam tsis zoo nrog kev txo qis ntawm T-bet thiab txo qis cytokine ntau lawm. Th17 hlwb kuj tau hloov qhov sib txawv thiab txo cov cytokine ntau lawm. Txawm hais tias ROR t tsis tau hloov pauv, MTHFD2- tsis txaus Th17 hlwb tau tswj hwm Treg cell TF FoxP3 thiab tau txais lub peev xwm los txwv kev loj hlob ntawm CD8 ntxiv rau T hlwb. Treg hlwb nthuav tawm txhim kho qhov sib txawv hauv qhov qis TGF-b. Nws yog tam sim no tsim tau zoo tias txhua qhov ntawm cov subsets no tuaj yeem muaj cov kev xav tau ntawm metabolic (Bantug li al., 2018; Buck li al., 2015). Peb cov ntaub ntawv qhia tias MTHFD2 kuj tseem xaiv tau rau Teff hlwb thaum txhawb Treg cell txoj hmoo thiab ua haujlwm. Nws yog ib qho tseem ceeb tias cov txiaj ntsig no txawv ntawm GLUT1, GLS, lossis ASCT2 qhov tsis zoo uas cuam tshuam lossis hloov Teff cell sib txawv thaum muaj kev cuam tshuam ncaj qha los txhawb Treg cell sib txawv lossis hloov pauv ntawm Th17 rau Treg hlwb (Johnson li al., 2018; Macintyre thiab al. ., 2014; Nakaya et al., 2014).

Cov kab mob T cell sib txawv no tshwm sim los ntawm kev ua haujlwm enzymatic ntawm MTHFD2 vim tias cov teebmeem ntawm MTHFD2 tsis muaj peev xwm cawm tau nrog formate lossis purine nucleobases. Txawm hais tias peb pom tsis muaj qhov cuam tshuam tseem ceeb ntawm kev hloov pauv redox tshuav nyiaj li cas nrog MTHFD2 tsis txaus, hloov pauv de novo purine synthesis zoo li tseem ceeb. MTHFD2i kev kho mob ua rau txo qis purine concentrations, tshwj xeeb tshaj yog guanine thaum inducing tsub zuj zuj ntawm purine synthesis intermediates GAR, SAICAR, thiab AICAR. Cov intermediates no yuav tsum tau formmylation rau biosynthesis ntxiv, thiab cov teebmeem raug cawm thaum MTHFD2- muab cov metabolite formate ntxiv rau nruab nrab, txhawb nqa lub hom phiaj biochemical rau MTHFD2i. De novo nucleotide synthesis los txhawb DNA thiab RNA synthesis yog qhov tseem ceeb rau T cell proliferation (Que´ me´ neur et al., 2003, 2004). Yog li, ib feem ntawm lub tshuab uas MTHFD2i impairs T cell expansion zoo li tsis txaus tiam ntawm nucleotides.

Flavonoid

Kev ua tsis tau zoo ntawm nucleotides tuaj yeem cuam tshuam cov nyhuv T hlwb los ntawm ntau lub tshuab. AMPK thiab mTORC1 yog cov tsav tsheb loj ntawm metabolic reprogramming nrog feem ntau tawm tsam hauv Teff thiab Treg hlwb (Bantug li al., 2018; Buck li al., 2015). Peb qhia tau hais tias kev kho MTHFD2i hauv T hlwb tau ua rau muaj mob hnyav ntawm AICAR, cov metabolite tseem ceeb xws li adenosine analog thiab AMPK activator (Rae thiab Mairs, 2019; Su li al., 2019). Txawm li cas los xij, tsis muaj pov thawj ntawm kev nce AMPK cov haujlwm ntawm lub sijhawm xaiv. Qhov no tuaj yeem cuam tshuam txog kev cai ntxiv ntawm AMPK, tab sis AICAR tseem tuaj yeem muaj cov teebmeem AMPK-kev ywj pheej (Dembitz li al., 2019). mTORC1 kuj tseem nkag siab rau purine concentrations vim tias qhov tsis txaus guanine txo qis kev ua haujlwm ntawm mTORC1 activator Rheb (Emmanuel li al., 2017; Hoxhaj li al., 2017). AICAR tsub zuj zuj thiab txo qis hauv mTORC1 kev ua haujlwm tuaj yeem ua rau muaj kev cuam tshuam tsis zoo ntawm MTHFD2i-kho Th1 thiab Th17 hlwb thiab txhim kho tiam ntawm Treg hlwb. Kev txwv tsis pub ntawm mTORC1 teeb liab kuj tseem tuaj yeem ua rau muaj kev hloov pauv hauv cov txheej txheem metabolic los ntawm glycolysis mus rau mitochondrial ua pa thiab hloov TCA metabolite ntau hauv MTHFD2i-kho Th17 hlwb. Txoj hauv kev mTORC1 ua lub luag haujlwm tseem ceeb hauv kev txhawb nqa cov metabolism hauv anabolic thiab tuaj yeem tsav kev sib sau ntawm TF ATF4, uas, dhau los, tuaj yeem ua rau MTHFD2 qhia (Ben-Sahra li al., 2016).

Metabolic flux yog khi rau kev tswj hwm epigenetic vim tias cov metabolites ua rau muaj ntau yam kev hloov pauv hauv epigenetic (Sharma thiab Rando, 2017), suav nrog 1C metabolism los tsim cov methyl universal pub S-adenosyl methionine (SAM) rau cov protein thiab DNA methylation. Cov kev sim carbon tracing hauv activated T hlwb, txawm li cas los xij, qhia tias 1C units tau muab los ntawm cov piam thaj los ntawm cov piam thaj thiab exogenous serine tsis muaj txiaj ntsig rau lub voj voog methionine thiab methylation (Ma li al., 2017). Metabolic flux kuj tseem tuaj yeem cuam tshuam rau cov qauv methylation los ntawm kev hloov pauv hauv ntau ntawm TCA metabolites, suav nrog alpha-ketoglutarate, fumarate, thiab succinate. Tshwj xeeb, fumarate thiab succinate inhibit DNA thiab histone demethylase (Su li al., 2016). Raws li kev depletion ntawm cov metabolites hauv MTHFD2i kho Th17 hlwb, H3K27me3 raug txo kom dav thoob plaws genome, thiab DNA methylation tau txo qis tshwj xeeb ntawm Foxp3 proximal proximal, uas tej zaum yuav ua rau muaj kev hloov pauv phenotype. Th17 cell transdifferentiation phenotype tsis tau txais kev cawmdim los ntawm formate supplementation, qhia tias txoj hauv kev ntxiv yuav pab txhawb rau FoxP3 kev tswj hwm, xws li los ntawm kev ua haujlwm ntawm lwm yam nucleotide-concentration-sensing mechanisms.

Acteoside (Verbascoside)

MTHFD2 kuj tau tshaj tawm los ua lub luag haujlwm uas tsis yog-enzymatic uas tuaj yeem ua rau muaj kev cuam tshuam ntawm cov enzyme no. Hauv cov kab mob qog noj ntshav, MTHFD2 tuaj yeem muaj cov haujlwm nuclear, kev sib koom ua ke nrog DNA replication qhov chaw los txhawb kev loj hlob ntawm tes (Gustafsson Sheppard li al., 2015). Hauv murine qia hlwb, MTHFD2 tau pom tias yuav hloov kho DNA kom muaj kev ruaj ntseg genomic (Yue li al., 2020). Hauv lub raum cell carcinoma, MTHFD2 tau pom tias yog ib qho tseem ceeb rau metabolic reprogram ming ntawm mRNA methylation (Green li al., 2019). Txawm hais tias cov luag haujlwm no rau MTHFD2 tsis yog sib koom nrog peb qhov kev tshawb pom, lub peev xwm ntawm formate los cawm ntau qhov phenotypes ntawm MTHFD2 deficiency hauv T hlwb qhia tias MTHFD2 enzymatic kev ua si yog thawj tsav tsheb ntawm T cell proliferation thiab txoj hmoo.

MTHFD2 tau raug suav hais tias yog lub hom phiaj siv tshuaj tiv thaiv kab mob. Nws kuj tseem yuav yog lub hom phiaj tiv thaiv kab mob thiab muaj kev cuam tshuam tsawg dua piv nrog tam sim no muaj cov tshuaj tiv thaiv folates, muab cov lus qhia qis hauv cov neeg laus feem ntau. Piv txwv li, ib lub hom phiaj ntawm MTX, DHFR, tau nthuav dav dav hauv cov neeg laus cov ntaub so ntswg (Nilsson li al., 2014). Yog li ntawd, MTX tuaj yeem cuam tshuam nrog ntau yam kev phiv, suav nrog kev mob plab hnyuv. Ntxiv mus, cov txheej txheem ntawm kev ua ntawm MTX tseem tsis to taub txawm hais tias nws cov keeb kwm dav dav (Cronstein thiab Aune, 2020). Kev tswj hwm kev qhia ntawm MTHFD2 thiab muaj peev xwm rov ua dua nrog txoj hauv kev cytosolic MTHFD1 tuaj yeem ua rau muaj kev xaiv xaiv ntawm cov cell tshwj xeeb ntawm MTHFD2. Peb qhov kev tshawb pom tau txheeb xyuas MTHFD2 yog qhov tseem ceeb hauv kev kuaj mob metabolic hauv CD4^ntxivT hlwb. Txawm hais tias muaj kev sib tshooj dav dav ntawm kab mob qog noj ntshav thiab cov kab mob biology ntawm kev loj hlob sai T hlwb, peb pom tias CD4 ntxiv rau T cell subsets qhia qhov kev xav tau ntxiv los ntawm kev hloov pauv ntawm tes sib txawv thiab ua haujlwm. Nws zoo li tias kev tsom mus rau MTHFD2 hauv kev kho mob qog noj ntshav tuaj yeem txwv kev tiv thaiv qog noj ntshav. Txawm li cas los xij, kev teeb tsa xws li mob qog nqaij hlav hauv plab, qhov twg Th17 cell-mediated o ua rau muaj kab mob, yuav ua tau zoo rau MTHFD2is. T cell rhiab heev rau MTHFD2is tuaj yeem yog ib qho kev tiv thaiv kab mob hauv cov chaw ntawm CD4.^ntxivT cell-driven o dhau mob qog noj ntshav thiab ua rau muaj kev cuam tshuam tsawg dua li cov tshuaj kho tam sim no.

Kev txwv kev kawm

MTHFD2i siv hauv txoj kev tshawb no muaj IC50 tus nqi ntawm 0.0063 mMfor MTHFD2 thiab 0.57 mM rau MTHFD1; Yog li ntawd, cov teebmeem me me ntawm MTHFD2 thiab MTHFD1 dual inhibition tsis tuaj yeem raug cais tawm. Muab hais tias lub aberrant FoxP3 upregulation nyob rau hauvTh17 hlwb tsis tau cawm los ntawm kev muab formate tab sis mTORC1activity yog, lub mechanism ntawm lub hauv paus ntawm no phenotype yuav tsum tau soj ntsuam ntxiv. Tsis tas li ntawd, tag nrho cov qauv hauv vivo tau ua nrog MTHFD2i kev kho mob lossis caj ces ablation los ntawm lub sijhawm muaj kab mob induction, thiab T cell phenotyping tau ua nyob rau tib lub sijhawm. Txhawm rau ua kom zoo dua qhov cuam tshuam ntawm MTHFD2 qhov tsis txaus ntawm cov kab mob pathogenesis, cov kev tshawb fawb ntxiv yuav tsum suav nrog kev sim sijhawm nrog cov cim ntawm T cell kom paub tseeb tias CD4 ntxiv rau T cell subsets cuam tshuam qhov sib txawv hauv vivo. Thaum kawg, txhawm rau txhim kho qhov cuam tshuam ntawm txoj kev tshawb fawb no rau kev siv tshuaj kho mob, qhov ua tau zoo ntawm kev kho MTHFD2i pib tom qab pib kab mob yuav tsum tau sim.

STAR★ txoj kev

Cov txheej txheem ntxaws ntxaws tau muab rau hauv online version ntawm daim ntawv no thiab suav nrog cov hauv qab no:

• KHOOM PLIG TABLE

• RESOURCE AVAILABILITY

• B Coj kev tiv tauj

• B Cov ntaub ntawv muaj

• B Cov ntaub ntawv thiab cov lej muaj

  
  

• EXPERIMENTAL MODEL THIAB SUBJECT DETAILS

• B Mice B Human T cells

• B Cell Kab

  
  

• METHOD DETAILS

• B In vitro nas CD4^ntxivT cell activation thiab sib txawv

• B Th17 cell suppression assay

• B In vitro human CD4^ntxivT cell kab lis kev cai

• B CRISPR tshuaj ntsuam B Mass spectrometry metabolomics

• B Proton nuclear sib nqus resonance spectroscopy (1H-MRS)

• IB Immunoblotting

• B CUT&RUN B EpigenDx Targeted NextGen Bisulfifiite Sequencing

• B hauv vivo EAE qauv rau IHC thiab ntws cytometry

• B In vivo LY345899 kev kho mob hauv EAE qauv

• B Hauv vivo DS18561882 kev kho mob hauv DTH qauv B Hauv vivo IBD qauv nrog WT thiab CD4^{ΔMthfd 2}cov phooj ywg

• B nyob rau hauv vivo tus kab mob ua xua kab mob nrog WT thiab CD4^{ΔMthfd 2}cov phooj ywg

• B In vivo DS18561882 kev kho mob hauv OVA kev txhaj tshuaj qauv

  
  

• QUANTIFICATION THIAB STATISTICAL analysis

NYEEM NTXIV

Cov ntaub ntawv ntxiv tuaj yeem pom hauv online ntawm https://doi.org/10.1016/j. immuni.2021.10.01.

TXOJ CAI

Peb ua tsaug rau cov tswv cuab ntawm Rathell lab rau kev pab txhawb rau txoj haujlwm no. Peb ua tsaug Thomas Aune rau kev muab cov ntaub ntawv RNA-seq thiab J. Cools (VIB) rau kev muab pMx-U6-gRNA-GFP tsim. Peb ua tsaug Max R. Van Belkum rau tsim MTHFD2 qPCR primers. Peb ua tsaug Nello Mainolfifi, Vipin Suri, Adam Friedman, thiab Mark Manfredi los ntawm Raze Therapeutics, Inc. (Boston, MA) rau kev muab Raze 1459 compound, uas tau siv los txheeb xyuas qhov kev tshawb pom (cov ntaub ntawv tsis qhia). Cov duab kos tau tsim nrog BioRender. Peb lees paub Translational Pathology Shared Resource, txhawb nqa los ntawm NCI/NIH Cancer Center txhawb nqa nyiaj 5P30 CA68485-19 thiab sib koom cov cuab yeej siv nyiaj S10 OD023475-01A1, rau Leica Bond RX. Txoj haujlwm no tau txais kev txhawb nqa los ntawm William E. Paul Distinguished Innovator Award rau Lupus Research Alliance (rau JCR), R01s DK105550 (rau JCR), HL136664 (rau JCR thiab DCN), CA217987 (rau JCR), AI153167 (rau JCR), AI137075 (rau AKM), T32 DK101003 (KV), thiab T32 GM007347 (rau AS).

AUTHOR CONTRIBUTIONS

AS, GA, thiab JCR tsim cov kev tshawb fawb. AS, GA, KV, DRH, XX, MZM, XY, KLB, NC, MMW, ACY, DLG, AES, SKS, SNF, AMC, PF, TB, and JCG-C. ua qhov kev tshawb fawb. AS, GA, XY, JK, DCN, AKM, JDR, thiab JCR tau txheeb xyuas cov ntaub ntawv. AS thiab JCR tau sau daim ntawv nrog kev koom tes los ntawm lwm tus kws sau ntawv.

DECLARATION OF INTERESTS

JCR yog tus tsim, pawg thawj coj saib xyuas kev tshawb fawb, thiab cov tswv cuab ntawm Sitryx Therapeutics; ib tug tswv cuab ntawm pawg thawj coj saib xyuas kev tshawb fawb thiab cov tswv cuab ntawm Caribou Biosciences; ib tug tswv cuab ntawm pawg thawj coj saib xyuas kev tshawb fawb ntawm NirogyTherapeutics; tau sab laj rau Merck, Pfizer, thiab Mitobridge hauv 3 xyoo dhau los; thiab tau txais kev pab tshawb fawb los ntawm Incyte Corp., CalitheraBiosciences, thiab Tempest Therapeutics. JDR yog tus tsim thiab koom nrog hauv Raze Therapeutics, Toran, Serien Therapeutics, thiab Farber Partners thiab tus kws pab tswv yim thiab cov tswv cuab hauv Agios Pharmaceuticals, Kadmon Pharmaceuticals, Bantam Pharmaceuticals, Colorado Research Partners, RafaelHoldings, Barer Institute, thiab LEAF Pharmaceuticals nws tau txais kev sab laj tus nqi thiab kev tshawb fawb nyiaj txiag los ntawm Pfizer thiab Rafael thiab yog tus tsim ntawm patents tuav los ntawm Princeton University. AMC, PF, thiab TB yog cov neeg ua haujlwm ntawm Sitryx Therapeutics.

NYIAJ THIAB DIVERSITY

Peb tau ua haujlwm los xyuas kom muaj kev sib deev sib luag hauv kev xaiv cov yam tsis yog tib neeg. Ib lossis ntau tus kws sau ntawv ntawm daim ntawv no tus kheej qhia tias yog ib haiv neeg tsawg tsawg hauv kev tshawb fawb. Thaum hais txog cov ntaub ntawv hais txog kev tshawb fawb muaj feem xyuam rau txoj haujlwm no, peb kuj tau ua haujlwm txhawb nqa kev sib npaug ntawm poj niam txiv neej hauv peb cov npe siv. Cov neeg sau npe ntawm daim ntawv no suav nrog cov neeg koom nrog los ntawm qhov chaw uas tau tshawb fawb uas tau koom nrog hauv kev sau cov ntaub ntawv, tsim, tshuaj xyuas, thiab / lossis kev txhais cov haujlwm.

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STAR★ txoj kev

KHOOM PLIG TABLE

RESOURCE AVAILABILITY

Coj kev sib cuag

Cov ntaub ntawv ntxiv thiab kev thov rau cov peev txheej thiab cov tshuaj reagents yuav tsum raug coj mus rau thiab yuav ua tiav thaum ua tiav ntawm kev tsim nyog MTA los ntawm Tus Thawj Coj Hu, Dr. Jeffrey Rathmell (jeff.rathmell@vumc.org).

Cov ntaub ntawv muaj

Plasmids thiab nas kab tsim nyob rau hauv txoj kev tshawb no yuav ua kom tiav thaum ua tiav ntawm MTA raws li kev thov.

Cov ntaub ntawv thiab code muaj

CUT&RUN sequencing cov ntaub ntawv tau muab tso rau ntawm GEO thiab tau tshaj tawm rau pej xeem txij li hnub tshaj tawm (GSE180356). Microscopy cov ntaub ntawv thiab thawj cov duab western blot qhia nyob rau hauv daim ntawv no yuav muab sib koom los ntawm cov lead tiv tauj raws li kev thov. Cov ntaub ntawv ntxiv uas yuav tsum tau ua kom rov ua dua cov ntaub ntawv qhia hauv daim ntawv no yog muaj los ntawm tus thawj coj hu thaum thov.

Nyem rau Cistanches rau kev tiv thaiv