Dɔlélenutsiŋutete ƒe lãmenugbagbeviwo nye nu vevi aɖe le tumor microenvironment (TME) me, gake negbe ʋɛ aɖewo ko la, womenya woƒe dzedzeme akpa gãtɔ o. Le afisia la, míedzro dɔdzẽ kple T lãmenugbagbeviwo me tso dɔnɔ siwo ŋu dɔdzẽ si woyɔna be high-grade serous carcinoma (HGSC) le ƒe dɔdzẽ kple ascites me be míaɖe TME ƒe akpa vovovo siawo ƒe metabolome afia. Vovototo gã aɖe le ascites kple tumor cells me le metabolite me. Ne wotsɔe sɔ kple ascites la, 1-methylnicotinamide (MNA) sɔ gbɔ ɖe T lãmenugbagbevi siwo gena ɖe dɔdzẽ me la me ŋutɔ. Togbɔ be MNA ƒe agbɔsɔsɔ le T lãmenugbagbeviwo me dzina ɖe edzi hã la, nicotinamide N-methyltransferase (enzyme si naa methyl ƒuƒoƒowo ƒe tɔtrɔ tso S-adenosylmethionine yi nicotinamide) ƒe ɖeɖefia le fibroblasts kple tumor cells me ko. Le dɔwɔwɔ gome la, MNA nana T lãmenugbagbeviwo dona cytokine si doa dɔdzẽ ɖe ŋgɔ si nye tumor necrosis factor alpha. Eyata MNA si wokpɔ tso TME me kpena ɖe T lãmenugbagbeviwo ƒe dɔlélenutsiŋutete ƒe ɖoɖowɔwɔ ŋu eye wòtsi tre ɖi na dɔlélenutsiŋutete ƒe dɔdamɔnu si ate ŋu anye taɖodzinu si woatsɔ ada amegbetɔ ƒe kansa.
Nusiwo wotsɔ ɖɔa li lãmenugbagbeviwoe siwo wokpɔna tso dɔdzẽ me ate ŋu akpɔ ŋusẽ gã aɖe ɖe dɔlélenutsiŋutete si tsia dɔdzẽ nu dzi, eye kpeɖodzi geɖe wu ɖee fia be woate ŋu anye ŋusẽ vevi aɖe si aʋã dɔlélea ƒe ŋgɔyiyi hã (1). Tsɔ kpe ɖe Warburg ƒe ŋusẽkpɔɖeamedzi ŋu la, wodze dɔ siwo wowɔ nyitsɔ laa gɔme be woatsɔ ade dzesi dɔdzẽ ƒe lãmenugbagbeviwo ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe nɔnɔme kple ƒomedodo si le wo kple dɔlélenutsiŋutete ƒe nɔnɔme si le dɔdzẽ ƒe nuto sue (TME) me. Numekuku siwo wowɔ le sisiblisi ƒe kpɔɖeŋuwo kple amegbetɔ ƒe T lãmenugbagbeviwo ŋu ɖee fia be glutamine ƒe asitɔtrɔ (2), oxidative metabolism (3) kple glucose metabolism (4) ate ŋu awɔ dɔ le wo ɖokui si ɖe dɔlélenutsiŋutete ƒe lãmenugbagbeviwo ƒe hatsotso sue vovovowo dzi. Nusiwo wotsɔna trɔa asi le nu ŋu geɖe siwo le mɔ siawo me xea mɔ na T lãmenugbagbeviwo ƒe dɔwɔwɔ si tsia tre ɖe dɔdzẽ ŋu. Woɖo kpe edzi be coenzyme tetrahydrobiopterin (BH4) ƒe mɔxexe ate ŋu agblẽ nu le T lãmenugbagbeviwo ƒe dzidziɖedzi ŋu, eye BH4 ƒe dzidziɖedzi le ŋutilã me ate ŋu ana dɔlélenutsiŋutete si tsia tre ɖe dɔdzẽ ŋu si CD4 kple CD8 doa mɔ na la nadzi ɖe edzi. Tsɔ kpe ɖe eŋu la, woate ŋu aɖe kynurenine ƒe dɔlélenutsiŋutete ƒe ŋusẽkpɔɖeamedzi to BH4 dodo me (5). Le isocitrate dehydrogenase (IDH) mutant glioblastoma me la, enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) ƒe dodo xea mɔ na T lãmenugbagbeviwo ƒe dɔwɔwɔ, woƒe dzidziɖedzi kple cytolysis Dɔwɔwɔ (6). Nyitsɔ laa la, woɖee fia be methylglyoxal, si nye glycolysis ƒe akpa aɖe, tso suppressor cells siwo tso myeloid gbɔ, eye T cell transfer of methylglyoxal ate ŋu axe mɔ na effector T cell ƒe dɔwɔwɔ. Le atikewɔwɔ me la, methylglyoxal ƒe dɔmawɔmawɔ nyuie ate ŋu aɖu myeloid-derived suppressor cells (MDSC) ƒe dɔwɔwɔ dzi eye wòana checkpoint blockade therapy nanyo ɖe edzi le sisiblisi ƒe kpɔɖeŋuwo me le nuwɔwɔ aduadu me (7). Numekuku siawo katã te gbe ɖe akpa vevi si TME-siwo me metabolite wɔna le T lãmenugbagbeviwo ƒe dɔwɔwɔ kple woƒe dɔwɔwɔ dzi kpɔkpɔ me dzi.
Woka nya ta le T lãmenugbagbeviwo ƒe dɔmawɔmawɔ nyuie ŋu le azigolo me kansa me (8). Esia ƒe akpa aɖe tso lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe nɔnɔme siwo le ya si mebɔ o kple dɔdzẽ ƒe ʋuka siwo mesɔ o me gbɔ (9), si wɔnɛ be sukli kple tryptophan trɔna zua nusiwo dona tso eme abe lactic acid kple kynurenine ene. Lactate si le lãmenugbagbeviwo godo fũ akpa ɖea interferon-γ (IFN-γ) ƒe wɔwɔ dzi kpɔtɔna eye wòʋãa myelosuppressive subgroups ƒe vovototodedeameme ( 10 , 11 ). Tryptophan zazã xea mɔ na T lãmenugbagbeviwo ƒe dzidziɖedzi tẽ eye wòxea mɔ na T lãmenugbagbeviwo ƒe xɔla ƒe dzesiwo (12-14). Togbɔ be wokpɔ nusiawo hã la, wowɔ dɔ geɖe tso dɔlélenutsiŋutete ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ŋu le in vitro T cell culture me to mɔnu nyuitɔ zazã me, alo seɖoƒe li na homologous mouse models in vivo, eye wo dometɔ aɖeke meɖe amegbetɔ ƒe kansa ƒe vovototodedeameme kple Physiological macro kple micro environment fia bliboe o.
Nusi bɔ ɖe azigolo me dɔdzẽ ŋue nye aɖuɖɔtoe me kaka kple ascites ƒe dzedzeme. Lãmenugbagbeviwo ƒe tsi ƒe ƒoƒo ƒu ɖe ascites me do ƒome kple dɔléle si de ŋgɔ kple ŋgɔdonya si mesɔ o (15). Nyatakakawo ɖee fia be akpa tɔxɛ sia nye esi me ya mesɔ gbɔ ɖo o, ʋukawo me lãmenugbagbeviwo ƒe tsitsi ƒe akpa (VEGF) kple indoleamine 2,3-dioxygenase (IDO) sɔ gbɔ ɖe eme, eye T-dziɖuɖu lãmenugbagbeviwo kple myeloid mɔxenu lãmenugbagbeviwo gena ɖe eme (15-18). Ascites ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe nɔnɔme ate ŋu ato vovo tso dɔdzẽa ŋutɔ tɔ gbɔ, eyata T lãmenugbagbeviwo ƒe ɖoɖo yeye wɔwɔ le akɔta ƒe teƒea me mekɔ o. Tsɔ kpe ɖe eŋu la, vovototo vevi siwo le ascites kple metabolites siwo le dɔdzẽ ƒe nɔnɔme me kple vovototodedeameme ate ŋu axe mɔ na dɔlélenutsiŋutete ƒe lãmenugbagbeviwo ƒe gege ɖe dɔdzẽwo me kple woƒe dɔwɔwɔ ɖe dɔdzẽwo dzi, eye numekuku bubuwo hiã.
Be míakpɔ kuxi siawo gbɔ la, míewɔ sensitive cell separation and liquid chromatography tandem mass spectrometry (LC-MS/MS) mɔnu aɖe be míasrɔ̃ nu tso lãmenugbagbevi ƒomevi vovovowo ŋu (siwo dome CD4 + kple CD8 + T cells hã le) kpakple le tumors me kple wo dome Eƒe metabolites span cells in the same ascites and tumor environment of the patient. Míezãa mɔnu sia kpe ɖe high-dimensional flow cytometry kple single-cell RNA sequencing (scRNA-seq) ŋu tsɔ naa ameha vevi siawo ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe nɔnɔme ƒe nɔnɔmetata si wokpɔ nyuie. Mɔnu sia ɖee fia be 1-methylnicotinamide (MNA) ƒe agbɔsɔsɔ dzi ɖe edzi ŋutɔ le tumor T cells me, eye dodokpɔ siwo wowɔ le vitro ɖee fia be womenya alesi MNA ƒe dɔlélenutsiŋutete ƒe dɔwɔwɔ ɖe T lãmenugbagbeviwo ƒe dɔwɔwɔ dzii tsã o. Le goawo katã me la, mɔnu sia ɖea lãmenugbagbeviwo ƒe dɔwɔwɔ ɖekae si le dɔdzẽwo kple dɔlélenutsiŋutete ƒe lãmenugbagbeviwo dome fiana, eye wònaa gɔmesese tɔxɛwo le dɔlélenutsiŋutete ƒe ɖoɖowɔwɔ ƒe lãmenugbagbeviwo ŋu, si ate ŋu aɖe vi na T lãmenugbagbeviwo dada na azigolo me dɔdzẽ ƒe dɔlélenutsiŋutete ƒe atikewɔwɔ Atikewɔwɔ ƒe mɔnukpɔkpɔwo.
Míezã sisi ƒe lãmenugbagbeviwo me dzodzro si ƒe didime lolo tsɔ bu sukli ƒe xɔxlɔ̃ ƒe agbɔsɔsɔme le ɣeyiɣi ɖeka me [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) kple mitochondrial ƒe dɔwɔwɔ [MitoTracker Deep Red (MT DR)] (7, 19, 20) le axadzi typical dzesi siwo dea vovototo dɔlélenutsiŋutete ƒe lãmenugbagbeviwo kple dɔdzẽ ƒe lãmenugbagbeviwo ƒe xexlẽme dome (Tabla S2 kple Nɔnɔmetata S1A). Kukuɖenuŋu sia ɖee fia be ne wotsɔe sɔ kple T lãmenugbagbeviwo la, sukli si xɔa ascites kple tumor cells sɔ gbɔ wu, gake vovototo sue aɖe koe le mitochondrial ƒe dɔwɔwɔ me. Sukli si woxɔna le tumor cells [CD45-EpCAM (EpCAM)+] me le mama dedie nu nye T lãmenugbagbeviwo tɔ zi gbɔ zi etɔ̃ va ɖo ene, eye sukli si CD4 + T cells xɔna le mama dedie nu nye CD8 + T cells ƒe teƒe 1.2, si fia be Tumor infiltrating lymphocytes (TIL) ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe nudidiwo to vovo le TME ɖeka ma ke gɔ̃ hã me (Nɔnɔmetata 1A). To vovo na ema la, mitochondrial ƒe dɔwɔwɔ le tumor cells me sɔ kple CD4 + T cells tɔ, eye mitochondrial ƒe dɔwɔwɔ le lãmenugbagbevi ƒomevi eveawo siaa me sɔ gbɔ wu CD8 + T cells tɔ (Nɔnɔmetata 1B). Le goawo katã me la, emetsonu siawo ɖea lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe agbɔsɔsɔme fiana. Dɔdzẽ ƒe lãmenugbagbeviwo ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ sɔ gbɔ wu CD4 + T lãmenugbagbeviwo tɔ, eye CD4 + T lãmenugbagbeviwo ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ sɔ gbɔ wu CD8 + T lãmenugbagbeviwo tɔ. Togbɔ be ŋusẽkpɔɖeamedzi siawo le lãmenugbagbevi ƒomevi vovovowo me hã la, vovototo aɖeke mele CD4 + kple CD8 + T lãmenugbagbeviwo ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe nɔnɔme alo woƒe agbɔsɔsɔme si sɔ le ascites me ne wotsɔe sɔ kple dɔdzẽ o (Nɔnɔmetata 1C). To vovo na ema la, le CD45-lãmenugbagbeviwo ƒe akpa suea me la, EpCAM+ lãmenugbagbeviwo ƒe agbɔsɔsɔ le dɔdzẽa me dzi ɖe edzi ne wotsɔe sɔ kple ascites (Nɔnɔmetata 1D). Míede dzesi vovototo si dze ƒã le lãmenugbagbeviwo ƒe dɔwɔwɔ me le EpCAM + kple EpCAM- lãmenugbagbeviwo ƒe akpawo dome hã. EpCAM+ (tumor) lãmenugbagbeviwo ƒe sukli xɔxlɔ̃ kple mitochondrial ƒe dɔwɔwɔ sɔ gbɔ wu EpCAM- lãmenugbagbeviwo, si lolo wu fibroblasts ƒe metabolic dɔwɔwɔ le tumor cells me le TME me sã (Nɔnɔmetata 1, E kple F).
. (C) CD4 + kple CD8 + lãmenugbagbeviwo (siwo le CD3 + T lãmenugbagbeviwo me) ƒe xexlẽme le ascites kple tumor me. (D) EpCAM + tumor cells ƒe agbɔsɔsɔme le ascites kple tumor (CD45−) me. (E kple F) EpCAM + CD45-tumor kple EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) kple mitochondrial activity (MitoTracker dark red) (F) teƒenɔlawo ƒe nɔnɔmetatawo (miame) kple nyatakaka siwo woɖo ɖe kplɔ̃ dzi ( Nuɖusi) Ascites kple tumor cells. (G) CD25, CD137 kple PD1 ƒe ɖeɖefia to flow cytometry dzi ƒe nɔnɔmetata siwo le tsitre ɖi na. (H kple I) CD25, CD137 kple PD1 ƒe ɖeɖefia le CD4 + T lãmenugbagbeviwo (H) kple CD8 + T lãmenugbagbeviwo (I) dzi. (J kple K) Naive, central memory (Tcm), effector (Teff) kple effector memory (Tem) ƒe nɔnɔme siwo wotu ɖe CCR7 kple CD45RO ƒe ɖeɖefia dzi. CD4 + T lãmenugbagbeviwo (J) kple CD8 + T lãmenugbagbeviwo (K) ƒe nɔnɔmetata siwo le tsitre ɖi na (miame) kple kplɔ̃dzi nyatakakawo (ɖusime) le ascites kple tumors me. P ƒe asixɔxɔ siwo wokpɔ to eve eve t-dodokpɔ me (* P <0.05, **P <0.01 kple ***P <0.001). Fli la tsi tre ɖi na dɔnɔ siwo sɔ (n = 6). FMO, keklẽŋusẽ si me woɖea ɖeka le; MFI, fluorescence ƒe sesẽme le titina.
Kukuɖenuŋu bubuwo ɖe vovototo ɖedzesi bubuwo fia le T lãmenugbagbeviwo ƒe nɔnɔme si wokpɔ ta na ŋutɔ la dome. Activated (Nɔnɔmetata 1, G vaseɖe I) kple effector ŋkuɖodzinu (Nɔnɔmetata 1, J kple K) le dɔdzẽwo me bɔ wu ascites (CD3 + T lãmenugbagbeviwo ƒe agbɔsɔsɔme). Nenema ke, numekuku le phenotype ŋu to activation markers (CD25 kple CD137) kple depletion markers [programmed cell death protein 1 (PD1)] ƒe ɖeɖefia me ɖee fia be togbɔ be ameha siawo ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe nɔnɔmewo to vovo hã (Nɔnɔmetata S1, B vaseɖe E) , Gake womekpɔ vovototo gã aɖeke le lãmenugbagbeviwo ƒe dɔwɔwɔ me ɣesiaɣi le naive, effector alo memory subsets dome o (Nɔnɔmetata S1, F vaseɖe I). Woɖo kpe emetsonu siawo dzi to mɔ̃ɖaŋununya ƒe nusɔsrɔ̃mɔnu zazã me tsɔ de lãmenugbagbeviwo ƒe nɔnɔmewo asi na wo ɖokui (21), si gaɖe ƒumeŋuɖui me lãmenugbagbevi gbogbo aɖewo (CD45 + / CD3- / CD4 + / CD45RO +) ƒe anyinɔnɔ ɖe dɔnɔa ƒe ascites me fia (Nɔnɔmetata S2A ). Le lãmenugbagbevi ƒomevi siwo katã wode dzesii dome la, myeloid lãmenugbagbevi ƒe hatsotso sia ɖe sukli ƒe xɔxlɔ̃ kple mitochondrial ƒe dɔwɔwɔ fia wu (Nɔnɔmetata S2, B vaseɖe G). Nusiwo do tso eme siawo te gbe ɖe vovototo sesẽ si le lãmenugbagbevi ƒomevi vovovo siwo wokpɔna le ascites kple tumors me le HGSC dɔnɔwo me dome le lãmenugbagbeviwo ƒe dɔwɔwɔ me.
Kuxi vevitɔ si le TIL ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe nɔnɔmewo gɔmesese me enye alesi wòhiã be woaɖe T lãmenugbagbeviwo ƒe kpɔɖeŋu siwo ƒe dzadzɛnyenye, nyonyome kple agbɔsɔsɔ si sɔ la ɖe vovo tso dɔdzẽwo me. Numekuku siwo wowɔ nyitsɔ laa ɖee fia be vovototodedeameme kple bead enrichment mɔnu siwo wotu ɖe flow cytometry dzi ate ŋu ahe tɔtrɔ vɛ le lãmenugbagbeviwo ƒe metabolite profiles me ( 22-24 ). Be míaɖu kuxi sia dzi la, míewɔ bead enrichment mɔnua wònyo wu be míaɖe TIL ɖe vovo ahaɖee ɖe vovo tso amegbetɔ ƒe azigolo me kansa si woɖe le amekoko me hafi wodae kpɔ to LC-MS/MS dzi (kpɔ Nusiwo Wozã kple Mɔnuwo; Nɔnɔmetata 2A). Be míate ŋu akpɔ ŋusẽ si ɖoɖo sia kpɔna ɖe lãmenugbagbeviwo ƒe tɔtrɔ dzi bliboa la, míetsɔ T lãmenugbagbevi siwo nunala siwo le lãmesẽ me wɔ dɔ le bead mama ƒe afɔɖeɖe si le etame megbe ƒe metabolite profiles sɔ kple lãmenugbagbevi siwo womeɖe bead ma ɖe vovo o gake wotsi tsikpe dzi. Dzɔdzɔmeŋusẽdzikpɔkpɔ ƒe numekuku sia ɖee fia be kadodo gã aɖe le nɔnɔme eve siawo dome (r = 0.77), eye mɔ̃ɖaŋununya ƒe gbugbɔgawɔ ƒe ƒuƒoƒo si me metabolite 86 le la ƒe gbugbɔgawɔ ƒe ŋutete le gã (Nɔnɔmetata 2B). Eyata mɔnu siawo ate ŋu awɔ lãmenugbagbeviwo ƒe dɔwɔwɔ me dzodzro pɛpɛpɛ le lãmenugbagbevi siwo le lãmenugbagbevi ƒomevi ƒe dzidziɖedzi me, si ana woakpɔ mɔnu gbãtɔ si me woate ŋu ade dzesi lãmenugbagbeviwo ƒe tɔtrɔ tɔxɛwo le HGSC me, si ana amewo nase lãmenugbagbeviwo ƒe vovototodedeameme gɔme nyuie wu Gbɔdɔdɔ ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe ɖoɖo.
(A) Magnet bead ƒe ŋusẽdodo ƒe nɔnɔmetata si wowɔ ɖe ɖoɖo nu. Hafi woatsɔ LC-MS/MS adzro lãmenugbagbeviawo me la, woawɔ magnetic bead ƒe dzidziɖedzi zi etɔ̃ tsiã ɖe enu alo woanɔ tsikpe dzi. (B) Ŋusẽ si enrichment type kpɔna ɖe metabolites ƒe agbɔsɔsɔ dzi. Dzidzedze etɔ̃ ƒe mamã dedie na nusiwo wotsɔ doa ŋusẽe ƒomevi ɖesiaɖe ± SE. Fli ɣia tsi tre ɖi na ƒomedodo si nye 1:1. Intra-class correlation (ICC) si le dzidzenu siwo wogbugbɔ wɔ si woɖe fia le axis label me. NAD, si nye nicotinamide adenine dinucleotide, si nye atike si woyɔna be nicotinamide. (C) Dɔnɔwo ƒe metabolite numekuku ƒe dɔwɔwɔ ƒe ɖoɖowɔɖi ƒe nɔnɔmetata. Woƒoa ascites alo tumors nu ƒu tso dɔnɔwo gbɔ eye wodzraa wo ɖo le cryopreserved me. Wotsɔ flow cytometry da kpɔɖeŋu ɖesiaɖe ƒe akpa sue aɖe kpɔ, esime wowɔ kpɔɖeŋu susɔeawo ƒe dzidziɖedzi zi etɔ̃ na CD4+, CD8+ kple CD45- lãmenugbagbeviwo. Wozã LC-MS/MS tsɔ da lãmenugbagbevi sue siawo kpɔ. (D) Dzoxɔxɔ ƒe nɔnɔmetata si ɖe metabolite ƒe agbɔsɔsɔ si woɖo ɖe ɖoɖo nu fia. Dendrogram la tsi tre ɖi na Ward ƒe Euclidean didime siwo le kpɔɖeŋuwo dome ƒe ƒuƒoƒo. (E) Principal component analysis (PCA) of sample metabolite map, si ɖe kpɔɖeŋu ɖesiaɖe ƒe gbugbɔgawɔ etɔ̃ fia, wotsɔ fli do ƒome kple kpɔɖeŋu siwo tso dɔnɔ ɖeka me. (F) PCA si nye metabolite profile si le kpɔɖeŋua ƒe nɔnɔme si wowɔ ɖe dɔnɔa dzi (si fia be, wozã akpa aɖe ƒe redundancy); kpɔɖeŋu ƒomevi si woatsɔ awɔe la seɖoƒe li na le ʋua ƒe ʋuƒo si le ʋuʋu ɖi ta. PC1, akpa vevitɔ 1; PC2, akpa vevitɔ 2.
Eyome, míezã mɔnu sia si wotsɔ doa ŋusẽ ame tsɔ da lãmenugbagbeviwo ƒe dɔwɔwɔ 99 siwo le CD4 +, CD8 + kple CD45-lãmenugbagbeviwo ƒe akpa siwo le HGSC dɔnɔ ade ƒe ascites kple tumor gbãtɔwo me kpɔ (Nɔnɔmetata 2C, Nɔnɔmetata S3A kple Tabla S3 kple S4). Ameha si me wotsɔ ɖe le la nye 2% va ɖo 70% le lãmenugbagbevi gbagbewo ƒe kpɔɖeŋu gã gbãtɔ me, eye lãmenugbagbeviwo ƒe agbɔsɔsɔme toa vovo ŋutɔ le dɔnɔwo dome. Ne woma kuawo me vɔ la, ɖetsɔleme ƒe akpa si wodo ŋusẽe (CD4+, CD8+ alo CD45-) la wu lãmenugbagbevi gbagbe siwo katã le kpɔɖeŋua me ƒe 85% le mama dedie nu. Mɔnu sia si wotsɔ doa ŋusẽ lãmenugbagbeviwoe na míete ŋu bua lãmenugbagbeviwo ƒe xexlẽme ŋu tso amegbetɔ ƒe dɔdzẽ ƒe lãkusiwo ƒe asitɔtrɔ me, si manya wɔ tso kpɔɖeŋu gãwo me o. Míezã ɖoɖo sia tsɔ kpɔe be l-kynurenine kple adenosine, siwo nye dɔlélenutsiŋutete ƒe lãmenugbagbeviwo eve siawo siwo wonya nyuie la, wodo ɖe dzi le tumor T cells alo tumor cells me (Nɔnɔmetata S3, B kple C). Eyata nusiwo do tso eme siawo ɖe nuteƒewɔwɔ kple ŋutete si le míaƒe lãmenugbagbeviwo mama kple mass spectrometry mɔ̃ɖaŋununya ŋu be wòake ɖe nugbagbeviwo ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ veviwo ŋu le dɔnɔwo ƒe lãkusiwo me fia.
Míaƒe numekukua ɖee fia hã be lãmenugbagbevi ƒomeviwo ƒe lãmenugbagbeviwo ƒe mama sesẽ le dɔnɔwo me kple wo dome (Nɔnɔmetata 2D kple Nɔnɔmetata S4A). Vevietɔ ne wotsɔe sɔ kple dɔnɔ bubuwo la, dɔnɔ 70 ɖe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe nɔnɔme vovovowo fia (Nɔnɔmetata 2E kple Nɔnɔmetata S4B), si ɖee fia be lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe vovototo gã aɖe ate ŋu anɔ dɔnɔawo dome. Enyo be míade dzesii be ne wotsɔe sɔ kple dɔnɔ bubuwo (lita 1.2 va ɖo 2; Tabla S1) la, ascites siwo katã woƒo ƒu ɖe dɔnɔ 70 (ml 80) me la le sue wu. Dɔnɔwo dome vovototodedeameme dzi ɖuɖu le akpa veviwo me dzodzro me (le kpɔɖeŋu me, wozãa akpa aɖe ƒe redundancy numekuku) ɖea tɔtrɔ siwo nɔa anyi ɖaa le lãmenugbagbevi ƒomeviwo dome fiana, eye woƒo lãmenugbagbevi ƒomeviwo kple/alo nuto suewo nu ƒu kɔte le metabolite ƒe nɔnɔme nu (Nɔnɔmetata 2F ). Nusiwo wotsɔ ɖɔa li lãmenugbagbeviwoe ɖekaɖekawo me dzodzro te gbe ɖe ŋusẽkpɔɖeamedzi siawo dzi eye wòɖe vovototo gã aɖe fia le lãmenugbagbevi ƒomeviwo kple nuto suewo dome. Enyo be míade dzesii be vovototo si gbɔ eme wu si wokpɔe nye MNA, si zi geɖe la, CD45- lãmenugbagbeviwo kple CD4+ kple CD8+ lãmenugbagbevi siwo gena ɖe dɔdzẽa me la sɔ gbɔ ɖe eme (Nɔnɔmetata 3A). Le CD4 + lãmenugbagbeviwo gome la, ŋusẽkpɔɖeamedzi sia dzena wu, eye edze abe nutoa me kpɔa ŋusẽ ɖe MNA si le CD8 + lãmenugbagbeviwo me hã dzi vevie ene. Gake esia mele vevie o, elabena dɔnɔ adeawo dometɔ etɔ̃ koe woate ŋu ada akpɔ be tumor CD8+ ƒe dzesiwo le wo ŋu hã. Tsɔ kpe ɖe MNA ŋu la, le lãmenugbagbevi ƒomevi vovovo siwo le ascites kple tumors me la, metabolite bubu siwo ƒe dzesi mede nyuie le TIL me o hã sɔ gbɔ le mɔ vovovowo nu (Nɔnɔmetata S3 kple S4). Eyata nyatakaka siawo ɖe dɔlélenutsiŋutete ƒe asitɔtrɔ le lãmenugbagbeviwo ŋu ƒe hatsotso aɖe si ŋugbe wodo fia hena numekuku bubuwo wɔwɔ.
(A) MNA ƒe agbɔsɔsɔ si sɔ le CD4+, CD8+ kple CD45- lãmenugbagbeviwo me tso ascites kple tumor me. Aɖaka ƒe nɔnɔmetata la ɖe titina (fli), dometsotso si le eneawo dome (frame hinge) kple nyatakakawo ƒe didime fia, si ade enelia dome didime (frame whisker) ƒe teƒe 1.5. Abe alesi woɖe eme le Dɔnɔ ƒe Nuwo Kple Mɔnuwo me ene la, zã dɔnɔa ƒe limma ƒe asixɔxɔ nàtsɔ anya P ƒe asixɔxɔ (*P<0.05 kple **P<0.01). (B) MNA ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe nɔnɔmetata (60). Nusiwo wotsɔ ɖɔa li lãmenugbagbeviwoe: S-adenosyl-1-methionine; SAH, S-adenosine-1-homocysteine ​​si nye lãmenugbagbevi siwo woyɔna be S-adenosine-1-homocysteine; NA, si nye nicotinamide, si nye nicotinamide; MNA, si nye 1-metilnikotinamide; 2-PY, 1-methyl- 2-pyridone-5-carboxamide ƒe ƒuƒoƒo; 4-PY, 1-methyl-4-pyridone-5-carboxamide ƒe ƒuƒoƒo; NR, si nye nicotinamide si nye ribose; NMN, si nye nicotinamide si nye nucleotide ɖeka. Dɔlékuiwutikewo (amadede dzẽ): NNMT, nicotinamide N-methyltransferase; SIRT, sirtuins, si nye lãwo; NAMPT, si nye nicotinamide si woyɔna be phosphoribosyl transferase, si nye nikotinamide si woyɔna be phosphoribosyl transferase; AOX1, si nye aldehyde oxidase 1, si nye aldehyde oxidase 1; NRK, si nye nicotinamide riboside kinase, si nye nicotinamide si woyɔna be riboside kinase; NMNAT, nicotinamide mono Nucleotide adenylate transferase, si nye nusi wotsɔna ƒoa ƒui; Pnp1, si nye purine nucleoside phosphorylase, si nye ʋu si woyɔna be purine nucleoside phosphorylase. (C) t-SNE ƒe scRNA-seq ƒe ascites (grey) kple tumor (dzẽ; n = 3 dɔnɔwo). (D) NNMT ƒe ɖeɖefia le lãmenugbagbevi vovovo siwo wokpɔ to scRNA-seq zazã me. (E) NNMT kple AOX1 ƒe ɖeɖefia le SK-OV-3, amegbetɔ ƒe fugboe ƒe ayiku (HEK) 293T, T lãmenugbagbeviwo kple T lãmenugbagbevi siwo wotsɔ MNA da dɔe me. Woɖe nyagbɔgblɔ si woxatsa la fia ne wotsɔe sɔ kple SK-OV-3. Woɖe nyagbɔgblɔ ƒe ɖoɖo si wowɔ kple SEM fia (n = 6 nunala siwo le lãmesẽ me). Wobua Ct ƒe xexlẽme siwo wu 35 be womate ŋu ade dzesii o (UD). (F) SLC22A1 kple SLC22A2 ƒe ɖeɖefia le SK-OV-3, HEK293T, T lãmenugbagbeviwo kple T lãmenugbagbevi siwo wotsɔ 8mM MNA da dɔe me. Woɖe nyagbɔgblɔ si woxatsa la fia ne wotsɔe sɔ kple SK-OV-3. Woɖe nyagbɔgblɔ ƒe ɖoɖo si wowɔ kple SEM fia (n = 6 nunala siwo le lãmesẽ me). Wobua Ct ƒe xexlẽme siwo wu 35 be womate ŋu ade dzesii o (UD). (G) Lãmenugbagbeviwo ƒe MNA ƒe agbɔsɔsɔ le nunala T lãmenugbagbevi siwo le lãmesẽ me siwo wowɔ dɔ me le gaƒoƒo 72 ƒe ƒoƒo kple MNA megbe. Woɖe nyagbɔgblɔ ƒe ɖoɖo si wowɔ kple SEM fia (n = 4 nunala siwo le lãmesẽ me).
Wowɔa MNA to methyl ƒuƒoƒoa ƒe tɔtrɔ tso S-adenosyl-1-methionine (SAM) me yi nicotinamide (NA) me to nicotinamide N-methyltransferase (NNMT; Nɔnɔmetata 3B) dzi. NNMT dzena wògbɔa eme le amegbetɔ ƒe kansa vovovowo me eye wòdo ƒome kple dzidziɖedzi, amedzidzedze kple kaka ɖe teƒe bubuwo (25-27). Be míase MNA ƒe dzɔtsoƒe gɔme nyuie wu le T lãmenugbagbeviwo me le TME me la, míezã scRNA-seq tsɔ de dzesi NNMT ƒe ɖeɖefia le lãmenugbagbevi ƒomevi vovovowo me le HGSC dɔnɔ etɔ̃ ƒe ascites kple tumors me (Tabla S5). Lãmenugbagbevi siwo ade 6,500 me dzodzro ɖee fia be le ascites kple tumor ƒe nɔnɔmewo me la, NNMT ƒe ɖeɖefia seɖoƒe li na fibroblast kple tumor cell ƒe agbɔsɔsɔ si wosusu be (Nɔnɔmetata 3, C kple D). Edze be míade dzesii be NNMT ƒe ɖeɖefia si dze ƒã mele ameha aɖeke si ɖea PTPRC (CD45 +) fiana me o (Nɔnɔmetata 3D kple Nɔnɔmetata S5A), si ɖee fia be wotsɔ MNA si wokpɔ le metabolite spectrum me la de T lãmenugbagbeviwo me. Aldehyde oxidase 1 (AOX1) ƒe ɖeɖefia trɔa MNA wòzua 1-methyl-2-pyridone-5-carboxamide (2-PYR) alo 1-methyl-4-pyridone-5-carboxamide (4- PYR); Nutata 3B) hã se ɖe fibroblast siwo ɖea COL1A1 fiana ƒe xexlẽme ko (Nɔnɔmetata S5A), siwo katã ɖee fia be ŋutete mele T lãmenugbagbeviwo ŋu le MNA ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ si bɔ me o. Wozã lãmenugbagbeviwo ƒe nyatakaka evelia si le eɖokui si tso ascites tso HGSC dɔnɔwo gbɔ tsɔ ɖo kpe domenyiŋusẽfianu siawo siwo do ƒome kple MNA ƒe ɖeɖefia ƒe ɖoɖo dzi (Nɔnɔmetata S5B; n = 6) (16). Tsɔ kpe ɖe eŋu la, agbɔsɔsɔme polymerase kɔsɔkɔsɔ ƒe dɔwɔwɔ (qPCR) numekuku le nunala T lãmenugbagbevi siwo le lãmesẽ me siwo wotsɔ MNA da dɔe ŋu ɖee fia be ne wotsɔe sɔ kple SK-OV-3 azigolo me dɔdzẽ lãmenugbagbevi siwo dzi woɖu la, NNMT alo AOX1 meɖea NNMT alo AOX1 fiana kloe o (Nɔnɔmetata 3E). Nusiwo do tso eme siawo siwo womele mɔ kpɔm na o ɖee fia be MNA ate ŋu atso fibroblasts alo tumors me ayi T cells siwo te ɖe wo nɔewo ŋu me le TME me.
Togbɔ be ame siwo di be yewoaxɔ dɔa dometɔ aɖewoe nye organic cation transporters 1 va ɖo 3 (OCT1, OCT2 kple OCT3) ƒe ƒome si me soluble carrier 22 (SLC22) ƒomea (SLC22A1, SLC22A2 kple SLC22A3) ŋlɔ ɖe eme hã la, womeɖe MNA ƒe mɔzɔla siwo ate ŋu ava eme la gɔme haɖe o (28) . QPCR si mRNA tso nunala T lãmenugbagbevi siwo le lãmesẽ me ɖee fia be SLC22A1 ƒe ɖeɖefia ƒe agbɔsɔsɔ si le sue gake SLC22A2 ƒe agbɔsɔsɔ si womate ŋu ade dzesii o, si ɖo kpe edzi be wogblɔe va yi le agbalẽwo me (Nɔnɔmetata 3F) (29). To vovo na ema la, SK-OV-3 azigolo me dɔdzẽ ƒe lãmenugbagbevi ƒe hatsotso ɖe mɔzɔla eveawo siaa ƒe agbɔsɔsɔ gã aɖe fia (Nɔnɔmetata 3F).
Be woate ŋu ado ŋutete si le T lãmenugbagbeviwo si be woaxɔ duta MNA la kpɔ la, wode agble na T lãmenugbagbevi siwo le lãmesẽ me siwo wonana gaƒoƒo 72 le MNA ƒe agbɔsɔsɔ vovovowo ƒe anyinɔnɔ me. Ne MNA si tso egodo meli o la, womate ŋu ade dzesi MNA ƒe lãmenugbagbeviwo me o (Nɔnɔmetata 3G). Gake T lãmenugbagbevi siwo wowɔ dɔ siwo wotsɔ exogenous MNA da dɔe ɖee fia be MNA ƒe agbɔsɔsɔ si nɔ te ɖe agbɔsɔsɔme dzi le lãmenugbagbeviawo me, va ɖo 6 mM MNA (Nɔnɔmetata 3G). Nusi do tso eme sia ɖee fia be togbɔ be mɔzɔla ƒe ɖeɖefia le sue eye enzyme vevitɔ si kpɔa lãmenugbagbeviwo me MNA ƒe asitɔtrɔ dzi mele o hã la, TIL ate ŋu axɔ MNA kokoko.
Nusiwo wotsɔna trɔa asi le nu ŋu ƒe vovototo si le dɔnɔwo ƒe T lãmenugbagbeviwo me kple dodokpɔ siwo wowɔna tsɔ xɔa MNA le vitro dzia alesi wòate ŋu adzɔe be fibroblast siwo do ƒome kple kansa (CAF) naɖe MNA ɖa eye dɔdzẽ ƒe lãmenugbagbeviwo ate ŋu aɖɔ TIL ƒe nɔnɔme kple dɔwɔwɔ ɖo. Be woanya ŋusẽ si MNA kpɔna ɖe T lãmenugbagbeviwo dzi la, wowɔ T lãmenugbagbevi siwo le lãmesẽ me siwo wonana la dɔ le vitro le MNA ƒe anyinɔnɔ alo manɔmanɔ me, eye woda woƒe dzidziɖedzi kple cytokine wɔwɔ kpɔ. Le ŋkeke 7 ƒe MNA tsɔtsɔ kpe ɖe eŋu le agbɔsɔsɔ si lolo wu me megbe la, amewo ƒe xexlẽme si dzi ɖe edzi zi gbɔ zi eve dzi ɖe kpɔtɔ vie, evɔ wolé ŋusẽ me ɖe asi le agbɔsɔsɔme ɖesiaɖe me (Nɔnɔmetata 4A). Tsɔ kpe ɖe eŋu la, atikewɔwɔ na exogenous MNA na CD4 + kple CD8 + T lãmenugbagbevi siwo ɖea tumor necrosis factor-α (TNFα; Nɔnɔmetata 4B) fiana ƒe agbɔsɔsɔ dzi ɖe edzi. To vovo na ema la, IFN-γ ƒe wɔwɔme le lãmenugbagbeviwo me dzi ɖe kpɔtɔ ŋutɔ le CD4 + T lãmenugbagbeviwo me, gake menye le CD8 + T lãmenugbagbeviwo me o, eye tɔtrɔ gã aɖeke meva le interleukin 2 me o (IL-2; Nɔnɔmetata 4, C kple D). Eyata enzyme-linked immunosorbent assay (ELISA) si wowɔ le tsi si le tsia dzi tso T cell culture siawo siwo wotsɔ MNA da dɔe me la ɖee fia be TNFα dzi ɖe edzi ŋutɔ, IFN-γ dzi ɖe kpɔtɔ, eye tɔtrɔ aɖeke meva le IL-2 me o (Nɔnɔmetata 4, E vaseɖe G). . IFN-γ ƒe ɖiɖi ɖee fia be MNA ate ŋu awɔ akpa aɖe le T lãmenugbagbeviwo ƒe dɔwɔwɔ si tsia tre ɖe dɔdzẽ ŋu nu. Be woate ŋu asrɔ̃ ŋusẽ si MNA kpɔna ɖe T lãmenugbagbeviwo ƒe aɖi si dona tso lãmenugbagbeviwo me dzi la, chimeric antigen receptor T (FRα-CAR-T) lãmenugbagbevi siwo tɔa ŋku folate receptor α kple CAR-T (GFP) siwo dzi green fluorescent protein (GFP) -CAR-T) lãmenugbagbeviwo kpɔa ŋusẽ ɖo la toa ʋumenugbagbevi siwo le lãmesẽ me siwo nye donor peripheral blood mononuclear cells (PBMC) dzi. Wode CAR-T lãmenugbagbeviwo gaƒoƒo 24 le MNA ƒe anyinɔnɔ me, eye emegbe wotsɔ amegbetɔ ƒe SK-OV-3 azigolo me dɔdzẽ lãmenugbagbevi siwo ɖea folate receptor α fiana le effector kple target ƒe xexlẽme si nye 10:1 me ɖekae. MNA dɔdamɔnu na FRα-CAR-T lãmenugbagbeviwo ƒe amewuwu ƒe dɔwɔwɔ dzi ɖe kpɔtɔ ŋutɔ, si sɔ kple FRα-CAR-T lãmenugbagbevi siwo wotsɔ adenosine da dɔe (Nɔnɔmetata 4H).
(A) Lãmenugbagbevi siwo ate ŋu anɔ agbe ƒe xexlẽme bliboa kple amewo ƒe xexlẽme ƒe dzidziɖedzi zi gbɔ zi eve (PD) tẽ tso dekɔnu me le ŋkeke 7. Bar graph la tsi tre ɖi na nunala ade siwo le lãmesẽ me ƒe mama dedie + SEM. Etsi tre ɖi na nyatakaka siwo tso dodokpɔ siwo wowɔ le wo ɖokui si n = 3 ya teti me. (B vaseɖe D) Wozã CD3/CD28 kple IL-2 tsɔ na T lãmenugbagbeviwo wɔ dɔ le woƒe MNA ƒe agbɔsɔsɔ me hena ŋkeke 7 sɔŋ. Hafi woada lãmenugbagbeviwo kpɔ la, wotsɔ PMA/ionomycin kple GolgiStop do ŋusẽ lãmenugbagbeviwo hena gaƒoƒo 4. TNFα (B) ƒe ɖeɖefia le T lãmenugbagbeviwo me. Kpɔɖeŋu nɔnɔmetata (miame) kple tabular data (ɖusime) le TNFα ƒe ɖeɖefia le lãmenugbagbevi gbagbewo me. IFN-γ (C) kple IL-2 (D) ƒe ɖeɖefia le T lãmenugbagbeviwo me. Wotsɔ flow cytometry dzidze cytokines ƒe ɖeɖefia. Bar graph la tsi tre ɖi na mama dedie (n = 6 nunala siwo le lãmesẽ me) + SEM. Zã mɔ ɖeka dzi numekuku le vovototo ŋu kple dzidzenu siwo wogbugbɔ dzidze (*P<0.05 kple **P<0.01) nàtsɔ anya P ƒe asixɔxɔ. Etsi tre ɖi na nyatakaka siwo tso dodokpɔ siwo wowɔ le wo ɖokui si n = 3 ya teti me. (E vaseɖe G) Wozã CD3/CD28 kple IL-2 tsɔ na T lãmenugbagbeviwo wɔ dɔ le woƒe MNA ƒe agbɔsɔsɔ me hena ŋkeke 7. Woƒo atike la nu ƒu do ŋgɔ na gaƒoƒo 4 ƒe PMA/ionomycin ƒe dzodzo kple emegbe. Wotsɔ ELISA dzidze TNFα (E), IFN-γ (F) kple IL-2 (G) ƒe agbɔsɔsɔme. Bar graph la tsi tre ɖi na mama dedie (n = 5 nunala siwo le lãmesẽ me) + SEM. P ƒe asixɔxɔ si wokpɔ to mɔ ɖeka dzi numekuku le vovototo ŋu kple dzidzedze siwo wogbugbɔ dzidze (*P<0.05) zazã me. Fli si dzi woŋlɔ nu ɖo la fia seɖoƒe si woate ŋu ade dzesii. (H) Lãmenugbagbeviwo ƒe ʋuʋu ƒe dodokpɔ. Wotsɔ adenosine (250μM) alo MNA (10 mM) trɔ asi le FRα-CAR-T alo GFP-CAR-T lãmenugbagbeviwo ŋu hena gaƒoƒo 24, alo wogblẽ wo ɖi womewɔ atike na wo o (Ctrl). Wodzidze SK-OV-3 lãmenugbagbeviwo ƒe amewuwu le alafa me. P ƒe asixɔxɔ si wokpɔ to Welch t dodokpɔ me (*P<0.5 kple **P<0.01).
Be woakpɔ mɔ̃ɖaŋununya gɔmesese le TNFα ƒe ɖeɖefia ƒe ɖoɖowɔwɔ si nɔa te ɖe MNA dzi ŋu la, woda tɔtrɔ siwo vaa TNFα mRNA me le T lãmenugbagbevi siwo wotsɔ MNA da dɔe me kpɔ (Nɔnɔmetata 5A). T lãmenugbagbevi siwo le lãmesẽ me siwo wotsɔ MNA da dɔe ɖee fia be TNFα ƒe nuŋɔŋlɔ ƒe agbɔsɔsɔ dzi ɖe edzi zi gbɔ zi eve, si ɖee fia be MNA nɔ te ɖe TNFα ƒe nuŋɔŋlɔ ƒe ɖoɖowɔwɔ dzi. Be woaku nu me le ɖoɖowɔwɔ ƒe mɔnu sia si ate ŋu anɔ anyi ŋu la, woda nuŋɔŋlɔ ƒe nu eve siwo wonya siwo kpɔa TNFα dzi, siwo nye activated T cell nuclear factor (NFAT) kple specific protein 1 (Sp1), kpɔ le MNA ƒe kadodo kple proximal TNFα promoter ( 30 ). TNFα ƒe ŋgɔdola la me NFAT ƒe kadodoƒe 6 siwo wode dzesii kple Sp1 ƒe kadodoƒe 2, siwo ƒoa xlã wo nɔewo le teƒe ɖeka [-55 base pairs (bp) tso 5’cap] (30). Chromatin immunoprecipitation (ChIP) ɖee fia be ne wotsɔ MNA da dɔe la, Sp1 ƒe kadodo kple TNFα ƒe ŋgɔdola dzina ɖe edzi zi gbɔ zi etɔ̃. NFAT ƒe dede eme hã dzi ɖe edzi eye wògogo vevienyenye (Nɔnɔmetata 5B). Nyatakaka siawo ɖee fia be MNA ɖɔa TNFα ƒe ɖeɖefia to Sp1 ƒe nuŋɔŋlɔ dzi, eye le mɔ sue aɖe nu la, NFAT ƒe ɖeɖefia dzi.
(A) Ne wotsɔe sɔ kple T lãmenugbagbevi siwo wode agble na MNA manɔmee la, TNFα ƒe ɖeɖefia ƒe tɔtrɔ zi gbɔ zi eve le T lãmenugbagbevi siwo wotsɔ MNA da dɔe me. Woɖe nyagbɔgblɔ ƒe ɖoɖo si wowɔ kple SEM fia (n = 5 nunala siwo le lãmesẽ me). Etsi tre ɖi na nyatakaka siwo tso dodokpɔ siwo wowɔ le wo ɖokui si n = 3 ya teti me. (B) Wotsɔ T lãmenugbagbeviwo ƒe TNFα ƒe ŋgɔdola si wotsɔ 8 mM MNA da dɔe alo wometsɔe da dɔe le NFAT kple Sp1 megbe o la ƒo ƒu kple (Ctrl) kple PMA/ionomycin ƒe dzodzo hena gaƒoƒo 4. Wozã dɔlélenutsiŋutete G (IgG) kple H3 abe dɔlélenutsiŋutete ƒe ʋuʋu ƒe mɔxexeɖedɔléle nu kple nyuitɔ ene. ChIP ƒe agbɔsɔsɔmedzidzenu ɖee fia be Sp1 kple NFAT ƒe kadodo ɖe TNFα ƒe ŋgɔdola ŋu le lãmenugbagbevi siwo wotsɔ MNA da dɔe me dzi ɖe edzi zi gbɔ zi geɖe ne wotsɔe sɔ kple esiwo dzi woɖu. Etsi tre ɖi na nyatakaka siwo tso dodokpɔ siwo wowɔ le wo ɖokui si n = 3 ya teti me. P ƒe asixɔxɔ si wokpɔ to t-dodokpɔ geɖe me (*** P <0.01). (C) Ne wotsɔe sɔ kple HGSC ƒe ascites la, T lãmenugbagbeviwo (siwo menye lãmenugbagbeviwo ƒe aɖi o) ɖee fia be TNF ƒe ɖeɖefia dzi ɖe edzi le dɔdzẽa me. Amadedeawo tsi tre ɖi na dɔnɔ vovovowo. Wotsɔ lãmenugbagbevi siwo woɖe fia la ƒe kpɔɖeŋu le vome va ɖo 300 eye woʋuʋu wo be woatsɔ aɖo seɖoƒe na wo ɖeɖe fũu akpa (** Padj = 0.0076). (D) MNA ƒe kpɔɖeŋu si wodo ɖa na azigolo me kansa. Wowɔa MNA le tumedɔlékuiwo me eye wowɔa fibroblasts le TME me eye T lãmenugbagbeviwo xɔae. MNA dzia Sp1 ƒe kadodo ɖe TNFα ƒe ŋgɔdola ŋu ɖe edzi, si wɔnɛ be TNFα ƒe nuŋɔŋlɔ kple TNFα cytokine wɔwɔ dzina ɖe edzi. MNA hã nana IFN-γ dzi ɖena kpɔtɔna. T lãmenugbagbeviwo ƒe dɔwɔwɔ nutsitsi nana be amewuwu ƒe ŋutete dzi ɖena kpɔtɔna eye dɔdzẽ ƒe tsitsi kabakaba.
Nyatakakawo ɖee fia be TNFα ƒe dɔwɔwɔ tsi tre ɖe dɔdzẽ kple dɔdzẽ ŋu si nɔa te ɖe ŋgɔgbe kple megbe dzi, gake ewɔa akpa si wonya nyuie le azigolo me dɔdzẽ ƒe tsitsi kple eƒe kaka ɖe teƒe bubuwo dodo ɖe ŋgɔ me (31-33). Nyatakakawo ɖee fia be TNFα ƒe agbɔsɔsɔ le ascites kple tumor tissues me le dɔnɔ siwo ŋu azigolo me kansa le me sɔ gbɔ wu esi le lãmeka siwo me dɔléle aɖeke mele o me ( 34-36 ). Le dɔwɔwɔ gome la, TNFα ate ŋu aɖɔ ʋumenugbagbevi ɣiwo ƒe dɔwɔwɔ, dɔwɔwɔ kple woƒe dzidziɖedzi ɖo, eye wòatrɔ kansa lãmenugbagbeviwo ƒe nɔnɔme ( 37 , 38 ). Le ɖekawɔwɔ me kple nusiwo ŋu woke ɖo siawo la, domenyiŋusẽfianu ƒe vovototodedeameme ƒe numekuku ɖee fia be TNF dzi ɖe edzi ŋutɔ le T lãmenugbagbeviwo me le dɔdzẽ ƒe lãkusiwo me ne wotsɔe sɔ kple ascites (Nɔnɔmetata 5C). TNF ƒe ɖeɖefia ƒe dzidziɖedzi dze le T lãmenugbagbevi siwo me lãmenugbagbeviwo ƒe aɖi mele o ƒe nɔnɔme me ko (Nɔnɔmetata S5A). Kpuie ko la, nyatakaka siawo ɖo kpe nukpɔsusu si nye be MNA ƒe dɔlélenutsiŋutete kple dɔdzẽ dodo ɖe ŋgɔ ƒe ŋusẽkpɔɖeamedzi eve le HGSC me dzi.
Fluorescent labeling si wotu ɖe flow cytometry dzi va zu mɔnu vevitɔ si dzi woato asrɔ̃ nu tso TIL ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ŋu. Numekuku siawo ɖee fia be ne wotsɔe sɔ kple ʋumenugbagbevi dzĩ siwo le gota alo T lãmenugbagbevi siwo tso ʋumenugbagbeviɣidɔ ƒe ŋutinu evelia me la, sisiblisi kple amegbetɔ ƒe TIL dina be yewoaxɔ sukli wu ( 4 , 39 ) eye mitochondria ƒe dɔwɔwɔ bu vivivi ( 19 , 40 ). Togbɔ be míekpɔ nu mawo tɔgbe le numekuku sia me hã la, nu vevitɔ si wowɔe nye be woatsɔ dɔdzẽ ƒe lãmenugbagbeviwo kple TIL ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ asɔ kple wo nɔewo tso dɔdzẽ ƒe lãkusi ɖeka ma ke si woɖe ɖa me. Le ɖekawɔwɔ me kple nyatakaka siawo dometɔ aɖewo la, dɔdzẽ (CD45-EpCAM +) lãmenugbagbevi siwo tso ascites kple dɔdzẽ me la xɔa sukli sɔ gbɔ wu CD8 + kple CD4 + T lãmenugbagbeviwo, si ɖo kpe edzi be woate ŋu atsɔ sukli si sɔ gbɔ ɖe dɔdzẽ ƒe lãmenugbagbeviwo me asɔ kple T lãmenugbagbeviwo. T lãmenugbagbeviwo ƒe hoʋiʋli ƒe nukpɔsusu. TME. Gake dɔdzẽ ƒe lãmenugbagbeviwo ƒe mitochondrial dɔwɔwɔ sɔ gbɔ wu CD8 + T lãmenugbagbeviwo tɔ, gake mitochondrial ƒe dɔwɔwɔ sɔ kple CD4 + T lãmenugbagbeviwo tɔ. Nusiwo do tso eme siawo do ŋusẽ tanya si le dodom be oxidative metabolism le vevie na tumor cells ( 41 , 42 ). Wogblɔ hã be CD8 + T lãmenugbagbeviwo ate ŋu axɔ oxidative dysfunction bɔbɔe wu CD4 + T lãmenugbagbeviwo, alo be CD4 + T lãmenugbagbeviwo ate ŋu azã carbon dzɔtsoƒe bubu siwo menye sukli o atsɔ alé mitochondrial ƒe dɔwɔwɔ me ɖe asi ( 43 , 44 ). Ele be míade dzesii be míekpɔ vovototo aɖeke le sukli ƒe xɔxlɔ̃ alo mitochondrial ƒe dɔwɔwɔ me le CD4 + T effectors, T effector memory kple T central memory cells dome le ascites me o. Nenema ke vovototodedeameme ƒe nɔnɔme si le CD8 + T lãmenugbagbeviwo me le dɔdzẽwo me meku ɖe tɔtrɔ siwo va le sukli ƒe xɔxlɔ̃ me ŋu o, si te gbe ɖe vovototo gã si le T lãmenugbagbevi siwo wode agble ɖe vitro kple amegbetɔ ƒe TIL le vivo dome dzi ( 22 ). Woɖo kpe ŋkuléleɖenuŋu siawo hã dzi to lãmenugbagbeviwo ƒe xexlẽme mama si me ameŋkumemakpɔmakpɔ le zazã me, si gaɖee fia be CD45 + / CD3- / CD4 + / CD45RO + lãmenugbagbevi siwo me sukli ƒe xɔxlɔ̃ kple mitochondrial ƒe dɔwɔwɔ sɔ gbɔ wu dɔdzẽ ƒe lãmenugbagbeviwo bɔ gake Metabolic active cell population le wo si. Ameha sia ate ŋu atsi tre ɖi na myeloid suppressor cells alo plasmacytoid dendritic cells ƒe hatsotso sue si wosusu be wokpɔ le scRNA-seq numekukua me. Togbɔ be wogblɔ nusiawo evea siaa le amegbetɔ ƒe azigolo me dɔdzẽwo me hã [45] hã la, wogahiã kokoko Dɔ bubue nye be woaɖɔ myeloid subpopulation sia.
Togbɔ be mɔnu siwo wotu ɖe flow cytometry dzi ate ŋu ana vovototo siwo le sukli kple oxidative metabolism me le lãmenugbagbevi ƒomeviwo dome le goawo katã me me nakɔ hã la, womekpɔ nya metabolite siwo tututu sukli alo carbon dzɔtsoƒe bubuwo wɔna hena mitochondrial metabolism le TME me haɖe o. Be woatsɔ metabolitewo ƒe anyinɔnɔ alo manɔmanɔ ade TIL ƒe hatsotso sue aɖe si wona me bia be woakɔ lãmenugbagbeviwo ƒe xexlẽme ŋu tso lãkusi si woɖe ɖa me. Eyata míaƒe lãmenugbagbeviwo dodo ɖe ŋgɔ ƒe mɔnu si wotsɔ kpe ɖe mass spectrometry ŋu ate ŋu ana gɔmesese nasu mía si le nusiwo wotsɔna trɔa asi le nu ŋu siwo ƒe xexlẽme to vovo le T lãmenugbagbeviwo kple dɔdzẽ ƒe lãmenugbagbeviwo ƒe xexlẽme me le dɔnɔwo ƒe kpɔɖeŋu siwo sɔ me. Togbɔ be viɖewo le mɔnu sia ŋu wu lãmenugbagbeviwo ƒe vovototodedeameme si wotsɔa keklẽŋusẽ wɔa dɔe hã la, ate ŋu akpɔ ŋusẽ ɖe lãmenugbagbeviwo ƒe agbalẽdzraɖoƒe aɖewo dzi le esi woli ke le dzɔdzɔme nu kple/alo woƒe tɔtrɔ kabakaba ta (22). Ke hã, míaƒe mɔnua te ŋu de dzesi dɔlélenutsiŋutete ƒe nugbagbevi eve siwo wonya, adenosine kple kynurenine, elabena woto vovo ŋutɔ le kpɔɖeŋu ƒomeviwo dome.
Míaƒe metabolite numekuku le tumors kple TIL subtypes ŋu naa gɔmesese geɖe wu le akpa si metabolites wɔna le ovarian TME me ŋu. Gbã la, míezã flow cytometry tsɔ kpɔe be vovototo aɖeke mele mitochondrial ƒe dɔwɔwɔ me le tumedɔlékuiwo kple CD4 + T lãmenugbagbeviwo dome o. Gake LC-MS/MS numekuku ɖe tɔtrɔ gãwo fia le lãmenugbagbeviwo ƒe agbɔsɔsɔ si le ameha siawo dome, si ɖee fia be nyataƒoƒo siwo ku ɖe TIL ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ kple eƒe lãmenugbagbeviwo ƒe dɔwɔwɔ bliboa ŋu hiã gɔmeɖeɖe nyuie. Evelia, MNA ye nye metabolite si me vovototo gãtɔ le CD45-cells kple T cells dome le ascites me, ke menye tumors o. Eyata, vovototodedeameme kple dɔdzẽ ƒe teƒe ate ŋu akpɔ ŋusẽ vovovowo ɖe TIL ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ dzi, si te gbe ɖe vovototodedeameme si ate ŋu anɔ nuto sue aɖe si wona me dzi. Etɔ̃lia, CAF, si nye dɔdzẽ ƒe lãmenugbagbeviwo le mɔ sue aɖe nu, koŋue na MNA-wɔla enzyme NNMT ƒe ɖeɖefia, gake wokpɔa MNA ƒe agbɔsɔsɔ si woate ŋu ade dzesii le T lãmenugbagbevi siwo wokpɔ tso dɔdzẽ me. NNMT ƒe ɖeɖefia fũ le azigolo me CAF me kpɔa ŋusẽ si wonya be edoa kansa ɖe ŋgɔ, eye eƒe akpa aɖe tso CAF ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ, dɔdzẽ ƒe amedzidzedze kple kaka ɖe teƒe bubuwo ƒe dodo ɖe ŋgɔ gbɔ ( 27 ). Togbɔ be TIL ƒe agbɔsɔsɔ bliboa le titina hã la, NNMT ƒe ɖeɖefia le CAF me do ƒome kplikplikpli kple Cancer Genome Atlas (TCGA) mesenchymal subtype, si do ƒome kple nyagblɔɖi gbegblẽ ( 27 , 46 , 47 ). Mlɔeba la, enzyme AOX1 si gbɔ MNA ƒe gbegblẽ tsona ƒe ɖeɖefia hã se ɖe CAF ƒe xexlẽme dzi, si ɖee fia be ŋutete mele T lãmenugbagbeviwo ŋu be woatrɔ MNA o. Nusiwo do tso eme siawo ɖo kpe susua dzi be togbɔ be ehiã be woawɔ dɔ bubuwo atsɔ aɖo kpe nusi ŋu woke ɖo sia dzi hã la, MNA ƒe agbɔsɔsɔ si sɔ gbɔ le T lãmenugbagbeviwo me ate ŋu afia be dɔlélenutsiŋutete ƒe CAF ƒe nuto sue aɖe li.
Ne míebu MNA mɔzɔlawo ƒe ɖeɖefia ƒe agbɔsɔsɔ si mede o kple protein vevi siwo kpɔa gome le MNA ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ me ƒe agbɔsɔsɔ si womate ŋu ade dzesii o ta la, MNA ƒe anyinɔnɔ le T lãmenugbagbeviwo me nye nusi womele mɔ kpɔm na o. Womete ŋu kpɔ NNMT alo AOX1 to scRNA-seq numekuku kple qPCR si woɖo taɖodzinu na le hatsotso eve siwo le wo ɖokui si me o. Nusiwo do tso eme siawo ɖee fia be menye T lãmenugbagbeviwoe wɔa MNA o, ke boŋ woxɔnɛ tso TME siwo ƒo xlãe me. Dodokpɔ siwo wowɔ le vitro ɖee fia be T lãmenugbagbeviwo dina be yewoaƒo MNA si tso egodo nu ƒu.
Míaƒe numekuku siwo míewɔ le vitro ɖee fia be exogenous MNA naa TNFα ƒe ɖeɖefia le T lãmenugbagbeviwo me eye wòdoa Sp1 ƒe kadodo ɖe TNFα ƒe ŋgɔdola ŋu ɖe ŋgɔ. Togbɔ be TNFα wɔa dɔ siwo tsia dɔdzẽ nu kple esiwo tsi tre ɖe dɔdzẽ ŋu siaa hã la, le azigolo me dɔdzẽ me la, TNFα ate ŋu ado azigolo me dɔdzẽ ƒe tsitsi ɖe ŋgɔ ( 31-33 ). TNFα ƒe akpaɖekedzimademade le azigolo me dɔdzẽ ƒe lãmenugbagbeviwo ƒe dekɔnu me alo TNFα ƒe dzesi ɖeɖeɖa le sisiblisi ƒe kpɔɖeŋuwo me ate ŋu ana TNFα-tome dɔdzẽ ƒe cytokine wɔwɔ nanyo ɖe edzi eye wòaxe mɔ ɖe dɔdzẽ ƒe tsitsi nu ( 32 , 35 ). Eyata le go sia me la, MNA si wokpɔ tso TME ate ŋu awɔ dɔ abe nusi doa alɔ dɔdzẽ ƒe lãmenugbagbeviwo ene to mɔnu si nɔa te ɖe TNFα dzi to autocrine loop dzi, si ana azigolo me dɔdzẽ ƒe dzɔdzɔ kple eƒe kaka nado ( 31 ). Wonɔ te ɖe alesi wòate ŋu adzɔe sia dzi le nu srɔ̃m tso TNFα ƒe mɔxexeɖedɔléle nu ŋu be enye atike si ate ŋu ada azigolo me kansa ( 37 , 48 , 49 ). Tsɔ kpe ɖe eŋu la, MNA gblẽa nu le CAR-T lãmenugbagbeviwo ƒe aɖi si le azigolo me dɔdzẽ ƒe lãmenugbagbeviwo ŋu me, si naa kpeɖodzi bubuwo be dɔlélenutsiŋutete ƒe nutete si MNA toa mɔ dzi. Le ƒuƒoƒo me la, emetsonu siawo ɖe kpɔɖeŋu aɖe si me dɔdzẽ kple CAF lãmenugbagbeviwo ɖea MNA ɖe gota le lãmenugbagbeviwo godo ƒe TME me. To (i) TNF-si na azigolo me kansa ƒe tsitsi ƒe ʋuʋu kple (ii) MNA-si na T cell cytotoxic activity inhibition me la, esia ate ŋu akpɔ ŋusẽ eve ɖe dɔdzẽ dzi (Nɔnɔmetata 5D).
Le nyataƒoƒo me la, to lãmenugbagbeviwo ƒe dzidziɖedzi kabakaba, lãmenugbagbevi ɖeka ƒe ɖoɖowɔwɔ kple lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe nɔnɔmetatawo tsɔtsɔ ƒo ƒui zazã me la, numekuku sia ɖe dɔlélenutsiŋutete ƒe vovototo gã si le dɔdzẽ kple ascites lãmenugbagbeviwo dome le HGSC dɔnɔwo me fia. Kukuɖenuŋu blibo sia ɖee fia be vovototo le sukli ƒe xɔxlɔ̃ kple mitochondrial ƒe dɔwɔwɔ me le T lãmenugbagbeviwo dome, eye wòde dzesi MNA be enye non-cell autonomous immune regulatory metabolite. Nyatakaka siawo kpɔa ŋusẽ ɖe alesi TME kpɔa ŋusẽ ɖe T lãmenugbagbeviwo ƒe dɔwɔwɔ dzi le amegbetɔ ƒe kansa me dzi. Togbɔ be woka nya ta be hoʋiʋli tẽ le nunyiamewo ŋu le T lãmenugbagbeviwo kple kansa lãmenugbagbeviwo dome hã la, nusiwo woɖɔna ɖo ate ŋu awɔ dɔ abe ɖoɖowɔla siwo mele tẽ o ene hã be woado dɔdzẽ ƒe ŋgɔyiyi ɖe ŋgɔ eye ɖewohĩ woaxe mɔ ɖe dɔlélenutsiŋutete ƒe dɔwɔwɔ le lãmenugbagbeviwo me nu. Dɔ si ɖoɖowɔnu siawo wɔna le dɔwɔwɔ me ŋuti numeɖeɖe yi ŋgɔe ate ŋu aʋu mɔnu bubu siwo dzi woato ado dɔlélenutsiŋutete si tsi tre ɖe dɔdzẽ ŋu ɖe ŋgɔ.
Woxɔ dɔnɔwo ƒe kpɔɖeŋuwo kple dɔdamɔnu ŋuti nyatakakawo to BC kansa ƒe dɔdzẽ ƒe lãkusiwo ƒe nudzraɖoƒe si dzi Canada ƒe Lãmetsiwo Dzraɖoƒe ƒe Habɔbɔ da asi ɖo dzi. Le ɖoɖo si dzi BC Kansa Ŋuti Numekuku Agbenɔnɔ Ŋuti Kɔmiti kple British Columbia Yunivɛsiti (H07-00463) da asi ɖo nu la, dɔnɔawo katã ƒe dodokpɔwo kple dɔdamɔnu ŋuti nyatakakawo xɔ mɔɖeɖe si woŋlɔ ɖi alo woɖe asi le woƒe lɔlɔ̃nu faa ŋu le se nu. Wodzraa kpɔɖeŋuawo ɖo ɖe BioBank (BRC-00290) si ŋu woɖo kpee. Woɖe dɔnɔwo ƒe nɔnɔme tsitotsito fia le Tabla S1 kple S5 me. Le cryopreservation gome la, wozãa scalpel tsɔ gblẽa dɔnɔa ƒe dɔdzẽ ƒe kpɔɖeŋua to mɔ̃ dzi eye emegbe wotutuae to 100-micron filter me be woakpɔ lãmenugbagbevi ɖeka si wotsɔ ƒoe. Wotsɔ centrifuged dɔnɔa ƒe ascites le 1500 rpm hena miniti 10 le 4°C be woatsɔ aƒo lãmenugbagbeviawoe eye woaɖe tsi si le edzi la ɖa. Wodzra lãmenugbagbevi siwo wokpɔ tso dɔdzẽ kple ascites me ɖo le dzoxɔxɔ me le amegbetɔ ƒe AB serum 50% si wotsɔ dzoxɔxɔ mewɔa dɔ o (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) kple 10% dimethyl sulfoxide me. Wotsɔ lãmenugbagbevi ɖeka ƒe ʋuʋudedi siawo siwo wodzra ɖo la ƒu gbe eye wozã wo hena lãmenugbagbeviwo ƒe dɔwɔwɔ ŋuti numekuku kple lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe nyametsotso si ŋu woƒo nu tsoe le ete.
Atike bliboa nye 0.22 μm si woɖe 50:50 si wotsɔ kpe ɖe RPMI 1640: AimV ŋu. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) si wotsɔ amegbetɔ ƒe AB serum si dzoxɔxɔ mewɔa dɔ o 10% (Sigma-Aldrich), Hepes 12.5 mM (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) Fisher Scientific), 1 x kpe ɖe eŋu Penicillin Streptomycin (PenStrep) ƒe tsi (Thermo Fisher Scientific) kple 50 μMB-mercaptoethanol. Wotsɔa Hepes 20 mM (Thermo Fisher Scientific) kple 2 mM l-glutamine (Thermo Fisher Scientific) kpena ɖe AimV (Invitrogen) ŋu. Flow cytometer staining buffer la nye 0.22μm filtered phosphate buffered saline (PBS; Invitrogen) si wotsɔ 3% dzoxɔxɔ-si womewɔa dɔ o AB amegbetɔ ƒe ʋumenugbagbeviɣi (Sigma) kpe ɖe eŋu. PBS si woɖe tso 0.22μm ye nye lãmenugbagbeviwo ƒe dzidziɖedzi ƒe buffer la eye wotsɔ amegbetɔ ƒe AB serum si dzoxɔxɔ mewɔa dɔ o 0.5% (Sigma-Aldrich) kpena ɖe eŋu.
Le 37°C blibo me la, wotsɔ 10 nM MT DR kple 100 μM 2-NBDG de ama lãmenugbagbeviwo hena miniti 30. Eyome wotsɔ viability dye eF506 de ama lãmenugbagbeviawo le dzoxɔxɔ 4°C me hena miniti 15. Gbugbɔ tsɔ lãmenugbagbeviawo de FC Block (eBioscience) kple Brilliant Stain Buffer (BD Biosciences) me, tsɔ wo de flow cytometer staining buffer me (le atikewɔla ƒe mɔfiamewo nu), eye nàtsɔ wo ade xɔme ƒe dzoxɔxɔ me aɖabaƒoƒo 10. De ama lãmenugbagbeviawo kple dɔlékuiwutikewo ƒe hatsotso aɖe (Tabla S2) le flow cytometry staining buffer me le 4°C me hena miniti 20. Gbugbɔ tsɔ lãmenugbagbeviawo de flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) me hafi woado wo kpɔ. Zã SpectroFlo kple FlowJo V10 nàtsɔ adzro lãmenugbagbeviwo ƒe xexlẽme ŋuti nyatakakawo me, eye nàzã GraphPad Prism 8 atsɔ awɔ nyatakakaawo. Wowɔ 2-NBDG kple MT DR ƒe fluorescence intensity (MFI) si nye log-normalized, eye emegbe wozã paired t test hena akɔntabubu me dzodzro tsɔ bu akɔnta le dɔnɔ siwo sɔ ŋu. Ðe ameha siwo katã me nudzɔdzɔ siwo mede 40 o le ɖa le numekukua me; ŋlɔ MFI ƒe asixɔxɔ si nye 1 na asixɔxɔ manyomanyo ɖesiaɖe hafi nàwɔ akɔntabubu me dzodzro kple nyatakakawo ƒe nɔnɔmetatawo ɖeɖefia.
Be míatsɔ akpe ɖe asi agbonuwɔna ƒe mɔnu si le etame ƒe dɔwɔwɔ ƒe akpaa ŋu la, míezã nuŋlɔɖi bliboa to nɔnɔme mɔxexeɖeti (FAUST) (21) dzi tsɔ de lãmenugbagbeviwo asi na ameha la le eɖokui si le lãmenugbagbevi kukuwo ɖeɖeɖa le FlowJo me vɔ megbe. Míetsɔa asi kpɔa emetsonua dzi be míaƒo ameha siwo dze abe womema wo ɖe ɖoɖo nu o ene (siwo tsɔa PD1+ kple PD1-dɔdzẽ lãmenugbagbeviwo ƒoa ƒu) kple ameha siwo wolé ɖe te la nu ƒu ɖekae. Lãmenugbagbevi siwo wu 2% le kpɔɖeŋu ɖesiaɖe me le mama dedie nu, na lãmenugbagbevi 11 le wo katã me.
Wozã ficoll gradient density centrifugation tsɔ ɖe PBMC ɖa tso ʋumenugbagbevi dzĩwo mama ƒe nuwɔna (STEMCELL Technologies) gbɔ. Woɖe CD8 + T lãmenugbagbeviwo ɖe vovo tso PBMC me to CD8 MicroBeads (Miltenyi) zazã me eye wokeke wo ɖe enu le nu blibo me to TransAct (Miltenyi) zazã me hena kwasiɖa 2 le amesi wɔe ƒe mɔfiamewo nu. Wona lãmenugbagbeviawo tsi tre ŋkeke 5 le tsi blibo si me IL-7 (10 ng/ml; PeproTech) le me, eye emegbe wogatsɔ TransAct do ŋusẽ wo ake. Le ŋkeke 7 lia dzi la, le atikewɔla ƒe mɔfiamewo nu la, wozã amegbetɔ ƒe CD45 MicroBeads (Miltenyi) tsɔ do ŋusẽ lãmenugbagbeviwo le ɣeyiɣi etɔ̃ siwo kplɔ wo nɔewo ɖo me. Woma lãmenugbagbeviawo ɖe akpa vovovowo me hena flow cytometry numekuku (abe alesi míegblɔe le etame ene), eye woma lãmenugbagbevi miliɔn ɖeka ɖe akpa vovovowo me zi etɔ̃ hena LC-MS/MS numekuku. Wotsɔ LC-MS/MS trɔ asi le kpɔɖeŋuawo ŋu abe alesi woɖe eme le ete ene. Míebu akɔnta le metabolite ƒe asixɔxɔ si bu ŋu kple ion xexlẽme si nye 1,000. Wotsɔa ion xexlẽme bliboa (TIC) wɔa kpɔɖeŋu ɖesiaɖe wòsɔna ɖe ɖoɖo nu, wotrɔnɛ le logarithmically eye wowɔa eŋudɔ le eɖokui si le MetaboAnalystR me hafi wodzroa eme.
Wotsɔa lãmenugbagbevi ɖeka si wotsɔ tsia dɔnɔ ɖesiaɖe me la ƒua gbe eye woɖea wo to 40 μm ƒe tsilénu me yia nu blibo me (abe alesi míegblɔe le etame ene). Le adzɔnuwɔƒea ƒe ɖoɖo nu la, wozã tiatia nyui etɔ̃ siwo kplɔ wo nɔewo ɖo to magnetic bead separation to MicroBeads (Miltenyi) zazã me tsɔ do ŋusẽ kpɔɖeŋuawo na CD8+, CD4+ kple CD45- lãmenugbagbeviwo (le tsikpe dzi). Kpuie ko la, wogatsɔa lãmenugbagbeviawo dea lãmenugbagbeviwo ƒe dzidziɖedzi ƒe tsiléƒe (abe alesi míegblɔe le etame ene) eye woxlẽa wo. Wotsɔ amegbetɔ ƒe CD8-ku, amegbetɔ ƒe CD4-ku alo amegbetɔ ƒe CD45-ku (Miltenyi) de lãmenugbagbeviawo me le dzoxɔxɔ 4°C me hena miniti 15, eye emegbe woklɔ wo kple lãmenugbagbeviwo ƒe dzidziɖedzi ƒe nutrenu. Wotsɔa kpɔɖeŋua toa LS sɔti (Miltenyi) me, eye woƒoa akpa sue siwo nye nyui kple esiwo menyo o nu ƒu. Be woaɖe ɣeyiɣi didi si wòxɔna dzi akpɔtɔ eye woadzi lãmenugbagbeviwo ƒe hayahaya ƒe afɔɖeɖea ɖe edzi la, emegbe wozãa CD8-afã la hena CD4+ ƒe dzidziɖedzi evelia, eye wozãa CD4-afã la hena CD45 ƒe dzidziɖedzi si kplɔe ɖo. Na tsikpe la nanɔ tsikpe dzi le wo mama ƒe ɖoɖoa katã me.
Be woadzra kpɔɖeŋuwo ɖo hena metabolite numekuku la, woklɔ lãmenugbagbeviawo zi ɖeka kple dze fafɛ si le abe tsikpe ene, eye wotsɔ methanol 80% ml 1 de kpɔɖeŋu ɖesiaɖe me, emegbe wotsɔ vortex ƒo wo eye wotsɔ wo ƒu gbe ɖe nitrogen si nye tsi me. Wotsɔ kpɔɖeŋuawo de tsikpe kple tsikpe ƒe tsatsam etɔ̃ me eye wotsɔ centrifuged le 14,000 rpm me aɖabaƒoƒo 15 le 4°C. Wotsɔa tsi si le edzi si me metabolitewo le la ƒua tsi vaseɖe esime wòƒu. Wogatsɔ metabolites la ƒo ɖe 50 μl 0.03% formic acid me, wotsɔ vortex ƒo wo be woatsaka, eye emegbe wotsɔ wo ƒo ɖe centrifuged me be woaɖe gbeɖuɖɔwo ɖa.
Ðe metabolites abe alesi míegblɔe le etame ene. Tsɔ tsi si le edzi la de tsi ƒe chromatography-tu si wɔa dɔ nyuie me hena metabolomics numekuku. Zã atikewɔwɔ ƒe ɖoɖo si wowɔ le vome nàtsɔ awɔ dɔ na kpɔɖeŋu ɖesiaɖe kple lãmenugbagbevi siwo ƒe xexlẽme sɔ be woaxe mɔ ɖe batch effects nu. Míewɔ xexeame katã ƒe metabolite siwo wota va yi le AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50) dzi ƒe nɔnɔme ŋuti numekuku. Wozã MultiQuant version 2.1 software (Applied Biosystems SCIEX) tsɔ wɔ chromatographic numekuku kple peak area integration.
Wozã ion xexlẽme si nye 1000 tsɔ bu akɔnta le metabolite ƒe asixɔxɔ si bu ŋu, eye wozã TIC si le kpɔɖeŋu ɖesiaɖe me tsɔ bu akɔnta le metabolite ɖesiaɖe si wokpɔ ƒe taƒoƒo ƒe teƒe si sɔ be woatsɔ aɖɔ tɔtrɔ siwo woto dɔwɔnu ƒe numekuku vɛ tso kpɔɖeŋu ƒe dɔwɔwɔ me ɖo. Ne wowɔ TIC wòsɔ ɖe ɖoɖo nu vɔ la, wozãa MetaboAnalystR(51) (default parameter) hena logarithmic ƒe tɔtrɔ kple automatic norm line scaling. Míezã PCA kple vegan R package tsɔ wɔ numekuku le metabolome ƒe vovototo siwo le kpɔɖeŋu ƒomeviwo dome ŋu, eye míezã akpa aɖe ƒe redundancy numekuku tsɔ da dɔnɔwo kpɔ. Zã Ward mɔnu nàtsɔ awɔ dzoxɔxɔ ƒe nɔnɔmetata dendrogram atsɔ aƒo Euclidean didime si le kpɔɖeŋuwo dome nu ƒu ɖe hatsotsowo me. Míezã limma (52) le metabolite agbɔsɔsɔme si woɖo ɖe ɖoɖo nu dzi tsɔ de dzesi metabolite gbogbo aɖewo le mɔ vovovowo nu le lãmenugbagbevi ƒomevi bliboa kple microenvironment me. Be numeɖeɖea nanɔ bɔbɔe la, míezãa ƒuƒoƒo ƒe mama dedie ƒe nɔnɔmetata tsɔ gblɔa kpɔɖeŋua, eye míebua lãmenugbagbevi ƒomevi siwo le nuto suea me abe ƒuƒoƒo ɖesiaɖe ene (n = ƒuƒoƒo 6); na vevienyenye dodokpɔa la, míewɔ dzidzenu etɔ̃ enuenu na metabolite ɖesiaɖe Be woaƒo asa na alakpa gbugbɔgawɔ la, wotsɔ dɔnɔa de limma ƒe ɖoɖoa me abe mɔxenu ene. Be míate ŋu akpɔ vovototo siwo le metabolites me le dɔnɔ vovovowo dome la, míetrɔ asi le limma model si me dɔnɔwo hã le ŋu le mɔ si woɖo ɖi nu. Míegblɔ vovototo si wogblɔ do ŋgɔ le lãmenugbagbevi ƒomevi kple microenvironment si le Padj <0.05 (Benjamini-Hochberg ɖɔɖɔɖo) dome ƒe vevienyenye.
Le ŋusẽ dodo ɖe ŋgɔ to Miltenyi Dead Cell Removal Kit (>80% viability) zazã me megbe la, wowɔ lãmenugbagbevi ɖeka ƒe transcriptome ƒe ɖoɖowɔwɔ ɖe ascites gbagbe siwo wofa tsikpe me kple tumor ƒe kpɔɖeŋuwo katã ŋu to 10x 5′gene expression protocol zazã me. Woda dɔlékui atɔ̃ siwo me dɔdzẽ kple ascites sɔ kple wo nɔewo le kpɔ, togbɔ be alesi dɔdzẽ ƒe kpɔɖeŋu ɖeka ƒe agbenɔnɔ mede o la xe mɔ na wo dede eme hã. Be míate ŋu akpɔ dɔnɔwo ƒe tiatia geɖe la, míeƒo dɔnɔ ɖesiaɖe ƒe kpɔɖeŋuwo nu ƒu le 10x chromium controller ƒe mɔwo dzi, eye míeda ascites kple tumor teƒewo kpɔ ɖe vovo. Le ɖoɖowɔwɔ vɔ megbe [Illumina HiSeq 4000 28×98 bp eve eve ƒe nuwuwu (PE), Quebec ƒe domenyiŋusẽfianu; le mama dedie nu la, nuxexlẽ 73,488 kple 41,378 le lãmenugbagbevi ɖesiaɖe me na tumedɔ kple ascites]], míezã CellSNP kple Vireo (53) (si wotu ɖe CellSNP dzi abe The common human SNP (VCF) provided by GRCh38 is assigned a donor identity. Míezãa SNPRelate tsɔ ƒoa nya ta le dɔnɔa ƒe domenyiŋusẽfianu ƒe nɔnɔme si te ɖe eŋu wu (IBS) ŋu, excluding unassigned cells and cells identified as duplexes and matching donors between ascites and tumor samples (54) Míenɔ te ɖe dɔ sia dzi lé nya etɔ̃ siwo me lãmenugbagbeviwo ƒe nɔnɔmetata gbogbo aɖewo le le dɔdzẽa me kple ascites hena anyime numekuku le esi míewɔ afɔɖeɖe si nye mass filtration le scater (55) kple scran (56) BioConductor packaging me vɔ la, esia na 697 cells (2792 and 4183 cells from tumor and ascites, respectively) for analysis. We use igraph's (57) Louvain clustering of shared nearest neighbor network (SNN) based on Jaccard distance to cluster cells by expression. The clusters were manually annotated to putative cell types based on marker gene expression and visualized with t-SNE. Cytotoxic T cells are si woɖe fia to CD8A kple GZMA ƒe ɖeɖefia me, si me subclusters siwo me ribosomal protein ƒe ɖeɖefia le sue mele o Míekpɔ Izar et al (16) ƒe nyatakaka siwo wota, siwo dome woƒe t-SNE embedding hã le, ate ŋu akpɔ nyagbɔgblɔ ƒe ƒoƒo ɖe wo nɔewo dzi le dɔlélenutsiŋutete ƒe lãmenugbagbeviwo ƒe dzesiwo kple NNMT ƒe ɖeɖefia dome.
Woɖe PBMC ɖa tso ʋumenugbagbevi dzĩwo mama ƒe nuwɔna (STEMCELL Technologies) me to Ficoll gradient density centrifugation me. Woɖe CD3 + lãmenugbagbeviwo ɖe vovo tso PBMC me to CD3 beads (Miltenyi) zazã me. Le MNA ƒe anyinɔnɔ alo manɔmanɔ me la, wotsɔ CD3 si wobla ɖe agba me (5μg/ml), CD28 si me wodea tsii (3μg/ml) kple IL-2 (300 U/ml; Proleukin) na CD3+ lãmenugbagbeviwo wɔa dɔ. Le kekeɖenudɔa ƒe ŋkeke mamlɛtɔ dzi la, wotsɔ flow cytometry da agbenɔnɔ (Fixable Viability Dye eFluor450, eBioscience) kple dzidziɖedzi (123count eBeads, Thermo Fisher Scientific) kpɔ. Da effector ƒe dɔwɔwɔ kpɔ to lãmenugbagbeviwo dodo ɖe ŋgɔ kple PMA (20 ng/ml) kple ionomycin (1μg/ml) kple GolgiStop hena gaƒoƒo 4, eye nàlé ŋku ɖe CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4) , BioLegend) kple TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD) ŋu. Do ŋusẽ qPCR kple ChIP lãmenugbagbeviwo kple PMA (20 ng/ml) kple ionomycin (1μg/ml) hena gaƒoƒo 4. Woƒo ELISA ƒe tsi si le edzi la nu ƒu do ŋgɔ na dzodzo kple emegbe kple PMA (20 ng/ml) kple ionomycin (1 μg/ml) hena gaƒoƒo 4.
Wɔ ɖe adzɔnuwɔƒea ƒe ɖoɖo dzi be nàɖe RNA ɖe vovo to RNeasy Plus Mini Kit (QIAGEN) zazã me. Zã QIAshredder (QIAGEN) nàtsɔ awɔ kpɔɖeŋua ɖekae. Zã RNA to cDNA kit si ƒe ŋutete lolo (Thermo Fisher Scientific) nàtsɔ awɔ DNA si kpe ɖe eŋu (cDNA). Zã TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) nàtsɔ adzidze domenyiŋusẽfianu ƒe ɖeɖefia (le amesi wɔe ƒe ɖoɖo nu) kple numekukumɔ̃ siwo gbɔna: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glyceraldehyde-3-phosphate si woɖe tso Haidrodzin (GAPDH)] kple Hs01010726_m1 (SLC22A2). Wowɔ kpɔɖeŋuawo le StepOnePlus ɣeyiɣi ŋutɔŋutɔ ƒe PCR ɖoɖo (Applied Biosystems) (Applied Biosystems) dzi le MicroAmp fast optical 96-well reaction plate (Applied Biosystems) me kple MicroAmp optical film. Wobua Ct ƒe asixɔxɔ ɖesiaɖe si wu 35 be ele dzeside ƒe dzidzenu dzi eye wode dzesii be womate ŋu ade dzesii o.
Wɔ ChIP abe alesi wogblɔe va yi ene (58). Kpuie ko la, wotsɔ formaldehyde (si ƒe agbɔsɔsɔme mamlɛtɔ nye 1.42%) da dɔ na lãmenugbagbeviawo eye wotsɔ wo de xɔme ƒe dzoxɔxɔ me aɖabaƒoƒo 10. Zã ʋuʋudedi si wotsɔ kpe ɖe eŋu (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl kple 0.1% NP-40) ɖe tsikpe dzi hena miniti 10, emegbe nàgatsɔe aƒu gbe ɖe dɔlélenutsiŋutete ƒe tsilénu me abe alesi wogblɔe ene (58 ). Emegbe wotsɔ tsatsam siwo gbɔna wɔ sonicated kpɔɖeŋua: tsatsam 10 (20 1-sɛkɛnd pulses) kple ɣeyiɣi si meʋãna o si nye sɛkɛnd 40. De ChIP-grade immunoglobulin G (Lãmenugbagbeviwo ƒe Dzesiwo Mɔ̃ɖaŋununya; 1μl), histone H3 (Lãmenugbagbeviwo ƒe Dzesiwo Mɔ̃ɖaŋununya; 3μl), NFAT (Invitrogen; 3μl) kple SP1 (Lãmenugbagbeviwo ƒe Dzesiwo Mɔ̃ɖaŋununya; 3μl) dɔlékuiwutikewo kple kpɔɖeŋua le 4°CC ʋuʋu me zã bliboa. Tsɔ protein A-ku (Thermo Fisher Scientific) kple kpɔɖeŋua de dzoxɔxɔ 4°C me eye nàʋuʋui blewu gaƒoƒo 1, emegbe nàzã chelex-ku (Bio-Rad) atsɔ ana DNA la nanyo ɖe edzi, eye nàzã proteinase K (Thermo Fisher) atsɔ awɔ protein ƒe nuɖuɖumeŋusẽ. Woto PCR dzi de dzesi TNFα ƒe ŋgɔdola: yi ŋgɔ, GGG TAT CCT TGA TGC TTG TGT; ke boŋ GTG CCA ACA ACT GCC TTT ATA TG (207-bp ƒe nuwɔna). Image Lab (Bio-Rad) ye wɔ nɔnɔmetataawo eye wozã ImageJ kɔmpiuta dɔwɔɖoɖo tsɔ bu woƒe agbɔsɔsɔme.
Woƒo lãmenugbagbeviwo ƒe dekɔnu ƒe tsi si le edzi la nu ƒu abe alesi míegblɔe le etame ene. Wowɔ nyametsotsoa le amesi wɔe ƒe ɖoɖowo nu le amegbetɔ ƒe TNFα ELISA kit (Invitrogen), amegbetɔ ƒe IL-2 ELISA kit (Invitrogen) kple amegbetɔ ƒe IFN-γ ELISA kit (Abcam) nu. Le amesi wɔe ƒe ɖoɖo nu la, woɖe tsi si le edzi la 1:100 be woatsɔ ade dzesi TNFα kple IL-2, eye wotsɔ 1:3 de dzesi IFN-γ. Zã EnVision 2104 Multilabel Reader (PerkinElmer) nàtsɔ adzidze eƒe xɔxlɔ̃ le 450 nm.
Woɖe PBMC ɖa tso ʋumenugbagbevi dzĩwo mama ƒe nuwɔna (STEMCELL Technologies) me to Ficoll gradient density centrifugation me. Woɖe CD3 + lãmenugbagbeviwo ɖe vovo tso PBMC me to CD3 beads (Miltenyi) zazã me. Le MNA ƒe anyinɔnɔ alo manɔmanɔ me la, wotsɔ CD3 si wobla ɖe agba me (5μg/ml), CD28 si me wodea tsii (3μg/ml) kple IL-2 (300 U/ml; Proleukin) wɔ CD3+ lãmenugbagbeviwo ŋudɔ hena ŋkeke 3. Le ŋkeke 3 megbe la, woƒo lãmenugbagbeviawo nu ƒu eye woklɔ wo kple 0.9% saline, eye wotsɔ pellet la ƒu gbe enumake. Wowɔ lãmenugbagbeviwo ƒe xexlẽme to flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) me to 123count eBeads zazã me.
Ðe metabolites abe alesi míegblɔe le etame ene. Wogbugbɔ wɔ nusi woɖe tso eme ƒuƒu la le agbɔsɔsɔ si nye lãmenugbagbeviwo ƒe sɔsɔme 4000/μl me. Dzro kpɔɖeŋua me to reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) kple CORTECS T3 sɔti (2.1×150 mm, nusuewo ƒe lolome 1.6-μm, ʋuʋudedi ƒe lolome 120-Å; #186008500, Waters). Polar mass spectrometer (6470, Agilent), si me electrospray ionization wɔa dɔ le nɔnɔme nyui me. Ʋuʋu ƒe akpa A nye 0.1% formic acid (le H2O me), ʋuʋu ƒe akpa B nye 90% acetonitrile, 0.1% formic acid. LC ƒe ʋuʋudedi nye miniti 0 va ɖo 2 na 100% A, miniti 2 va ɖo 7.1 na 99% B, kple miniti 7.1 va ɖo 8 na 99% B. Emegbe nàgbugbɔ ada sɔti la kple mobile phase A le sisi ƒe agbɔsɔsɔme si nye 0.6 ml/min hena miniti 3. . Sisi ƒe agbɔsɔsɔme nye 0.4ml/min, eye wodoa dzo na sɔtixɔa va ɖoa 50°C. Zã MNA ƒe atike dzadzɛ ƒe dzidzenu (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) nàtsɔ aɖo ɣeyiɣi si woatsɔ alé nu ɖe ​​te (RT) kple tɔtrɔ (RT = miniti 0.882, tɔtrɔ 1 = 137→94.1, tɔtrɔ 2 = 137→92 , Tɔtrɔ 3 = 137→78). Ne tɔtrɔ etɔ̃awo katã dzɔ le ɣeyiɣi si sɔ me la, wozãa tɔtrɔ 1 tsɔ bua agbɔsɔsɔme be woakpɔ egbɔ be wowɔe tẽ. Wowɔ MNA (Toronto Research Chemical Company) ƒe dzidzenu ƒe ʋuʋudedi to stock solution (1 mg/ml) ƒe tsidede ɖe wo nɔewo yome ade me be woakpɔ dzidzenu siwo nye 0.1, 1.0, 10 kple 100 ng/ml kple 1.0 kple 10μg/ml le tsi me. Seɖoƒe si woate ŋu akpɔe nye 1 ng/ml, eye flidzidzedze ƒe ŋuɖoɖo le 10 ng/ml kple 10μg/ml dome. Wozãa kpɔɖeŋu kple dzidzenu ƒe microlitre eve ƒe atike ɖesiaɖe hena LC/MS numekuku, eye wowɔa dodokpɔ ƒe nyonyomedzikpɔkpɔ ƒe kpɔɖeŋu si wotsaka le dodo enyi ɖesiaɖe me be woakpɔ egbɔ be numekuku ƒe mɔ̃a li ke. Lãmenugbagbevi siwo katã wotsɔ MNA da dɔe ƒe MNA ƒe ŋuɖoɖowo nɔ dodokpɔa ƒe fli me. Wozã MassHunter agbɔsɔsɔme me dzodzro ƒe kɔmpiuta dɔwɔɖoɖo (v9.0, Agilent) tsɔ wɔ nyatakakawo me dzodzro.
Woɖe dzidzime evelia ƒe αFR-CAR wɔwɔme tso Song et al. (59) . Kpuie ko la, nusiwo gbɔna le xɔtuɖoɖoa me: CD8a kplɔla ƒe ɖoɖo, amegbetɔ ƒe αFR-koŋ ƒe kɔsɔkɔsɔ ɖeka ƒe tɔtrɔ ƒe kakɛ, CD8a hinge kple transmembrane nuto, CD27 lãmenugbagbeviwo me domenyinyi kple CD3z lãmenugbagbeviwo me domenyinyi. Wotsɔ GenScript wɔ CAR ƒe ɖoɖo bliboa, eye emegbe wowɔ eƒe nɔnɔmetata ɖe dzidzime evelia ƒe lentiviral expression vector si le GFP expression cassette si wozã tsɔ da dɔlékui ƒe dɔwɔwɔ nyuie kpɔ la ƒe dzigbe gome.
Wowɔa lentivirus to HEK293T lãmenugbagbeviwo ƒe dɔlékuiwo dodo me [American Type Culture Collection (ATCC); wodoe le Dulbecco ƒe Eagle medium si wogbugbɔ trɔ si me fugboe ƒe nyitsu ƒe ʋumenugbagbeviɣi (FBS) 10% kple PenStrep 1% le me, eye wozã CAR-GFP vector kple The packaging plasmids (psPAX2 kple pMD2.G, Addgene) zã lipofection amine (Sigma-Aldrich). Woƒo tsi si le tsia dzi si me dɔlékui le nu ƒu le gaƒoƒo 48 kple 72 le dɔlékuiwo dodo vɔ megbe, woɖee, eye wotsɔ ultracentrifugation ƒoe. Dzra dɔlékui ƒe tsi si le tsia dzi si me wode tsii ɖo ɖe -80°C vaseɖe esime wòatrɔe.
Woɖea PBMC ɖa tso nunala siwo le lãmesẽ me ƒe ʋumenugbagbevi dzĩwo mama (STEMCELL Technologies) gbɔ to Ficoll gradient density centrifugation me. Zã positive selection CD8 microbeads (Miltenyi) nàtsɔ aɖe CD8+ lãmenugbagbeviwo ɖe vovo tso PBMC me. Do ŋusẽ T lãmenugbagbeviwo kple TransAct (Miltenyi) kple le TexMACS medium [Miltenyi; wotsɔ amegbetɔ ƒe ʋumenugbagbeviɣi si me dzoxɔxɔ mewɔa dɔ o ƒe 3%, PenStrep 1% kple IL-2 (300 U/ml) kpe ɖe eŋu]. Gaƒoƒo blaeve vɔ ene le dzodzo megbe la, wotsɔ lentivirus (10 μl concentrated virus supernatant le lãmenugbagbevi 106 ɖesiaɖe me) trɔ T lãmenugbagbeviwo. Ŋkeke 1 va ɖo 3 le lãmenugbagbeviawo ƒe tɔtrɔ le Cytek Aurora (le FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+) dzi megbe la, da lãmenugbagbeviawo ƒe GFP ɖeɖefia kpɔ be woaɖe tɔtrɔ ƒe ŋutete si nye 30% ya teti afia .
Wode CAR-T lãmenugbagbeviwo gaƒoƒo 24 le Immunocult (STEMCELL Technologies; wotsɔ 1% PenStrep kpe ɖe eŋu) le nɔnɔme siwo gbɔna me: womewɔ atike na wo o, wotsɔ 250 μM adenosine alo 10 mM MNA da dɔe. Le atikewɔwɔ do ŋgɔ megbe la, woklɔ CAR-T lãmenugbagbeviwo kple PBS eye wotsɔ wo ƒo ƒu kple SK-OV-3 lãmenugbagbevi 20,000 [ATCC; le McCoy 5A medium (Sigma-Aldrich) si wotsɔ 10% FBS kple 1% PenStrep kpe ɖe eŋu le 10 me: Wodo effector kple target ratio si nye 1 ɖe ŋgɔ zi etɔ̃ le Immunocult medium si wotsɔ kpe ɖe eŋu me. Wozã SK-OV-3 lãmenugbagbeviwo kple SK-OV-3 lãmenugbagbevi siwo wotsɔ digitalis saponin (0.5mg/ml; Sigma-Aldrich) lã abe lãmenugbagbevi siwo mesɔ o kple esiwo nyo ene. Le gaƒoƒo 24 ƒe agbledede ɖekae megbe la, woƒo tsi si le edzi la nu ƒu eye wodzidze lactate dehydrogenase (LDH) le atikewɔla ƒe mɔfiamewo nu (LDH Glo Cytotoxicity Assay Kit, Promega). Wotsɔ LDH ƒe tsi si le edzi la ƒo ɖe LDH buffer me 1:50. Wozã mɔnu si gbɔna tsɔ dzidze amewuwu ƒe alafa memamã: amewuwu ƒe alafa memamã = ɖɔɖɔɖo ƒe alafa memamã / amewuwu ƒe agbɔsɔsɔme si sɔ gbɔ wu x 100%, afisi ɖɔɖɔɖo ƒe alafa memamã = co-culture-T cells ɖeɖeko, kple amewuwu ƒe agbɔsɔsɔme si sɔ gbɔ wu = positive control-negative control.
Abe alesi woɖe eme le nuŋɔŋlɔa alo nusiwo wotsɔ wɔe kple mɔnuwo me ene la, zã GraphPad Prism 8, Microsoft Excel alo R v3.6.0 hena akɔntabubu me dzodzro. Ne woxɔ kpɔɖeŋu geɖe tso dɔnɔ ɖeka gbɔ (abe ascites kple tumor ene) la, míezãa paired t test alo tsɔa dɔnɔa dea eme abe random effect ene le linear alo generalized model me abe alesi wòsɔ ene. Le metabolomics numekuku gome la, wowɔa vevienyenye dodokpɔa zi etɔ̃.
Ne èdi kpeɖeŋutɔ nyati sia la, taflatse kpɔ http://advances.sciencemag.org/cgi/content/full/7/4/eabe1174/DC1
Esia nye nyati si woateŋu akpɔ le mɔ gbadza nu si woma le Creative Commons Attribution-Non-Commercial License ƒe sewo nu, si ɖe mɔ ɖe eŋudɔwɔwɔ, mama kple egbugbɔgawɔ le mɔnu ɖesiaɖe me, nenye be zazã mamlɛtɔ menye asitsatsa ƒe viɖe ta o eye gɔmeɖoanyia nye be dɔ gbãtɔa sɔ. Ɖeɖe fiã.
De dzesii: Ðeko míebia tso asiwò be nàtsɔ wò e-mail adrɛs ana ale be amesi nèkafu na axaa nanya be yedi be yeakpɔ e-mail la eye be menye nyatakaka gbegblẽe wònye o. Mímalé e-mail adrɛs aɖeke ɖe mɔ̃ dzi o.
Wozãa biabia sia tsɔ doa amedzro kpɔ nenye be amedzroe nènye eye woxea mɔ na nyatakaka gbegblẽwo ɖoɖo ɖe amewo le wo ɖokui si.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellardini (Ralph J. DeBerardinis), Russell G. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T. 1999 ƒe ɣleti gbãtɔ me.
MNA kpena ɖe T lãmenugbagbeviwo ƒe dɔlélenutsiŋutete ŋu eye wòtsi tre ɖi na dɔlélenutsiŋutete ƒe dɔdamɔnu si ate ŋu anye taɖodzinu si woatsɔ ada amegbetɔ ƒe kansa.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellardini (Ralph J. DeBerardinis), Russell G. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T. 1999 ƒe ɣleti gbãtɔ me.
MNA kpena ɖe T lãmenugbagbeviwo ƒe dɔlélenutsiŋutete ŋu eye wòtsi tre ɖi na dɔlélenutsiŋutete ƒe dɔdamɔnu si ate ŋu anye taɖodzinu si woatsɔ ada amegbetɔ ƒe kansa.
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