Nibii ni haa mɔ he waa lɛ ji nɔ titri ni haa mɔ he jɔ̃ɔ ehe (TME), shi kɛji akɛ ajie fioo komɛi kɛjɛ mli lɛ, aleee amɛhe saji babaoo. Yɛ biɛ lɛ, wɔpɛi tsɛŋemɔ helai kɛ T cells ni jɛ helatsɛmɛi ni yɔɔ high-grade serous carcinoma (HGSC) lɛ atsɛŋemɔ helai kɛ ascites lɛ amli lɛ amli koni wɔjie nɛkɛ TME fãi srɔtoi nɛɛ amli metabolome lɛ kpo. Ascites kɛ tumor mli wala yibii lɛ yɛ srɔtofeemɔi babaoo yɛ metabolite mli. Kɛ akɛto ascites he lɛ, T wala yibii ni boteɔ tsɛŋemɔ hela mli lɛ mli 1-methylnicotinamide (MNA) lɛ fa babaoo waa. Eyɛ mli akɛ MNA ni yɔɔ T wala yibii lɛ amli lɛ falɛ yaa hiɛ moŋ, shi nicotinamide N-methyltransferase (ni ji tsofa ko ni haa methyl kui lɛ jeɔ S-adenosylmethionine mli kɛyaa nicotinamide mli) lɛ kpojiemɔ lɛ ji fibroblasts kɛ tumor wala yibii lɛ pɛ. Yɛ nitsumɔ mli lɛ, MNA haa T wala yibii lɛ jieɔ tsofa ni haa mɔ he jɔ̃ɔ ehe lɛ kpo. No hewɔ lɛ, MNA ni jɛ TME mli lɛ yeɔ ebuaa ni gbɔmɔtso lɛ mli hewalɛ ni tsiɔ hela naa lɛ tsuɔ nii jogbaŋŋ, ni edamɔ shi kɛha tsofa ni tsiɔ hela naa lɛ ni baanyɛ atsu nii kɛtsa gbɔmɔtso lɛ kansa.
Nibii ni jɛɔ tsɛŋemɔ hela mli baa lɛ baanyɛ aná hewalɛ ni tsiɔ tsɛŋemɔ hela lɛ naa waa, ni odaseyelii babaoo tsɔɔ akɛ amɛbaanyɛ amɛtsu nii hu akɛ nɔ titri ni tsirɛɔ hela lɛ ni eyaa hiɛ (1). Kɛfata Warburg hewalɛ lɛ he lɛ, nitsumɔ ko ni afee nyɛsɛɛ nɛɛ eje shishi etsɔɔ bɔ ni tsɛŋemɔ hela mli wala yibii lɛ tsakeɔ amɛhe amɛhaa kɛ wekukpaa ni yɔɔ ekɛ tsɛŋemɔ hela mli wala yibii bibii ni tsiɔ hela naa lɛ ateŋ (TME). Nibii amlipɛimɔi ni afee yɛ ŋmeii kɛ gbɔmɔtso mli T wala yibii lɛ ahe lɛ etsɔɔ akɛ glutamine mli nibii ni tsakeɔ (2), oxidative mli nibii ni tsakeɔ (3) kɛ glucose mli nibii ni tsakeɔ (4) lɛ baanyɛ atsu nii amɛ diɛŋtsɛ amɛhe yɛ gbɔmɔtso lɛ mli wala yibii bibii srɔtoi anɔ. Nibii babaoo ni yɔɔ gbɛi nɛɛ anɔ lɛ tsiɔ T cells lɛ anitsumɔ ni teɔ shi woɔ hela lɛ naa. Atsɔɔ akɛ, kɛ́ atsĩ coenzyme tetrahydrobiopterin (BH4) lɛ naa lɛ, ebaanyɛ efite T cells lɛ ayibɔ, ni BH4 ni yaa hiɛ yɛ gbɔmɔtso lɛ mli lɛ baanyɛ aha gbɔmɔtso lɛ mli hewalɛ ni tsiɔ hela naa lɛ aya hiɛ ni CD4 kɛ CD8 lɛ tsuɔ he nii. Kɛfata he lɛ, abaanyɛ ajie kynurenine ni haa mɔ he waa lɛ kɛtsɔ BH4 (5) ni akɛhaa lɛ nɔ. Yɛ isocitrate dehydrogenase (IDH) mutant glioblastoma mli lɛ, enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) ni ajieɔ lɛ kpo lɛ tsiɔ T cell nitsumɔ, shweremɔ kɛ cytolysis nitsumɔ naa (6). Nyɛsɛɛ nɛɛ, atsɔɔ akɛ methylglyoxal, ni ji nɔ ko ni jɛɔ glycolysis mli baa lɛ, jɛɔ wala yibii ni tsiɔ nii anaa ni jɛ myeloid mli lɛ amli, ni T wala yibii ni akɛ methylglyoxal lɛ yaa lɛ baanyɛ atsĩ effector T wala yibii lɛ anitsumɔ naa. Yɛ helatsamɔ mli lɛ, methylglyoxal ni akɛtsuɔ nii lɛ baanyɛ aye myeloid-derived suppressor cells (MDSC) lɛ anitsumɔ nɔ kunim, ni ekɛ tsofafeemɔ ni tsiɔ nii anaa lɛ aya hiɛ yɛ ŋmɔtɔ mli (7). Nikasemɔi nɛɛ fɛɛ maa gbɛfaŋnɔ titri ni TME-ni jɛɔ mli baa lɛ tsuɔ yɛ T cell nitsumɔ kɛ nitsumɔ he gbɛjianɔtoo mli lɛ nɔ mi.
Abɔ T cell ni tsuuu nii jogbaŋŋ lɛ he amaniɛ babaoo yɛ fɔmɔ kotoku mli kansa mli (8). Enɛ fã ko jɛɔ nibii ni haa mɔ he jɔɔ lɛ ni yɔɔ hypoxia kɛ tumor vasculature ni ejaaa mli lɛ hewɔ (9), ni no haa glucose kɛ tryptophan tsakeɔ efeɔ nibii krokomɛi tamɔ lactic acid kɛ kynurenine. Nibii ni haa mɔ tsui nyɔɔ emli ni fa fe nine lɛ haa interferon-γ (IFN-γ) lɛ feemɔ baa shi, ni ehaa srɔtofeemɔ ni yɔɔ myelosuppressive kui bibii lɛ ateŋ lɛ yaa hiɛ (10, 11). 12-14 ) Tryptophan ni ayeɔ lɛ tsiɔ T cell lɛ shweremɔ naa tɛ̃ɛ, ni etsĩɔ T cell lɛ he okadifeemɔ naa (12-14). Yɛ nɛkɛ niiamlipɛimɔi nɛɛ fɛɛ sɛɛ lɛ, atsu nitsumɔi babaoo ni kɔɔ gbɔmɔtso mli nii ni tsiɔ hela naa lɛ he yɛ vitro T wala yibii akusum mli kɛtsɔ adafitswaa ni hi jogbaŋŋ nɔ, loo akɛto homologous mouse models yɛ vivo mli pɛ, ni eko kwraa ejieee heterogeneity ni yɔɔ gbɔmɔtso mli kansa kɛ gbɔmɔtsoŋ macro kɛ micro shihilɛ mli lɛ kpo kwraa.
Nɔ ni afɔɔ namɔ yɛ fɔmɔ kotoku mli kansa mli ji fɔmɔ kotoku lɛ mli gbɛfaŋnɔ kɛ ascites ni jeɔ kpo. Nibii ni haa mɔ he jɔ̃ɔ ehe yɛ ascites mli lɛ kɛ hela ni etee hiɛ waa kɛ hela ni baa lɛ he tsɔɔmɔ ni ehiii baa (15). Amaniɛbɔi tsɔɔ akɛ, nɛkɛ fã krɛdɛɛ nɛɛ mli kɔɔyɔɔ bɛ mli, eyɛ fãji ni haa lá mli fãji lɛ daraa (VEGF) kɛ indoleamine 2,3-dioxygenase (IDO) ni fa babaoo, ni T fãji ni kudɔɔ fãji kɛ fãji ni tsiɔ lá mli fãji anaa lɛ boteɔ mli (15-18). Ekolɛ bɔ ni ascites lɛ mli nibii ni haa mɔ he jɔɔ lɛ tsuɔ nii eha lɛ yɛ srɔto yɛ tsɛŋemɔ hela lɛ diɛŋtsɛ he, no hewɔ lɛ, bɔ ni T cells ni yɔɔ peritoneal space lɛ mli lɛ feɔ amɛnii amɛhaa lɛ mli kãaa shi faŋŋ. Kɛfata he lɛ, srɔtofeemɔi titri kɛ heterogeneity ni yɔɔ ascites kɛ metabolites ni yɔɔ tsɛŋemɔ hela lɛ mli lɛ ateŋ lɛ baanyɛ atsĩ gbɔmɔtso lɛ mli wala yibii ni tsiɔ helai anaa lɛ amlibotemɔ kɛ amɛnitsumɔ yɛ tsɛŋemɔ hela lɛ nɔ lɛ naa, ni ehe miihia ni atao nii amli babaoo.
Bɔni afee ni wɔtsu naagbai nɛɛ ahe nii lɛ, wɔfee gbɛ ni atsɔɔ nɔ akaseɔ wala yibii sɔrɔtoi (ni CD4 + kɛ CD8 + T wala yibii fata he) kɛ agbɛnɛ hu yɛ tsɛŋemɔ helai amli kɛ amɛteŋ. Wɔkɛ gbɛ nɛɛ tsuɔ nii kɛfataa ŋwɛi-dimensional flow cytometry kɛ single-cell RNA sequencing (scRNA-seq) he koni wɔkɛ mfoniri ni yɔɔ ŋwɛi ni kɔɔ nɛkɛ gbɔmɛi titrii nɛɛ amli nibii ni tsakeɔ amɛhe lɛ ahe lɛ aha. Gbɛ nɛɛ jie bɔ ni 1-methylnicotinamide (MNA) ni yɔɔ tumor T cells lɛ amli lɛ etee hiɛ waa lɛ kpo, ni shikpamɔi ni afee yɛ vitro mli lɛ tsɔɔ akɛ, aleee bɔ ni MNA lɛ tsuɔ nii ehaa yɛ T cells lɛ anitsumɔ nɔ tsutsu lɛ. Yɛ gbɛ ni waaa nɔ lɛ, gbɛ nɛɛ jieɔ bɔ ni tsɛŋemɔ helai kɛ gbɔmɔtso lɛ mli yibii ni tsiɔ hela naa lɛ tsuɔ nii amɛhaa lɛ kpo, ni ehaa wɔleɔ nibii ni tsiɔ hela naa lɛ ahe gbɛjianɔtoo krɛdɛɛ ko, ni baanyɛ aye abua kɛha T tsɛŋemɔ hela ni damɔ fɔmɔ kotoku mli kansa ni tsiɔ hela naa lɛ tsamɔ hegbɛi.
Wɔkɛ tsɔne ni akɛkwɛɔ lá mli nibii ni haa mɔ tsui nyɔɔ emli lɛ tsu nii koni wɔkɛna bɔ ni lá ni hoɔ yɛ gbɔmɔtso lɛ mli lɛ fa ha yɛ be kome mli. tsɔɔmɔi ni tsɔɔ srɔtofeemɔ ni yɔɔ gbɔmɔtso lɛ mli wala yibii ni tsiɔ hela naa lɛ kɛ tumor wala yibii ayibɔ teŋ (Table S2 kɛ Figure S1A). Nɛkɛ mlipɛimɔ nɛɛ tsɔɔ akɛ kɛ akɛto T cells lɛ ahe lɛ, ascites kɛ tumor cells lɛ yɛ glucose ni amɛheɔ babaoo, shi amɛyɛ srɔtofeemɔi bibii yɛ mitochondrial nitsumɔ mli. Nibii ni haa mɔ tsui nyɔɔ emli lɛ ateŋ nɔ ni fe fɛɛ ji CD45-EpCAM (EpCAM)+] ni ji T cells lɛ toi etɛ kɛyashi ejwɛ, ni glucose ni CD4 + T cells lɛ yeɔ lɛ ji CD8 + T cells lɛ toi 1.2, ni tsɔɔ akɛ tumor infiltrating lymphocytes (TIL) lɛ yɛ srɔtofeemɔi). Nɔ ni tamɔɔɔ enɛ lɛ, mitochondrial nitsumɔ ni yɔɔ tumor cells lɛ mli lɛ tamɔ nɔ ni yɔɔ CD4 + T cells lɛ mli lɛ, ni mitochondrial nitsumɔ ni yɔɔ cells henɔi enyɔ lɛ fɛɛ mli lɛ fa fe CD8 + T cells lɛ (Mfoniri 1B). Yɛ fɛɛ mli lɛ, nɛkɛ hetoi nɛɛ jieɔ bɔ ni gbɔmɔtso lɛ mli nibii ni haa mɔ he jɔɔ lɛ tsuɔ nii eha lɛ kpo. Nibii ni haa mɔ mli wala yibii lɛ tsuɔ nii lɛ fa fe CD4 + T wala yibii lɛ, ni CD4 + T wala yibii lɛ amli nibii ni tsakeɔ lɛ hu fa fe CD8 + T wala yibii lɛ. Yɛ nɛkɛ nibii nɛɛ ni baa yɛ wala yibii srɔtoi amli lɛ fɛɛ sɛɛ lɛ, srɔtofeemɔ ko bɛ CD4 + kɛ CD8 + T wala yibii lɛ amli loo amɛhe ni amɛyɔɔ yɛ ascites mli lɛ mli kɛ akɛto tumors he (Mfoniri 1C). Nɔ ni tamɔɔɔ enɛ lɛ, yɛ CD45-cell fã lɛ mli lɛ, EpCAM+ cells ni yɔɔ tsɛŋemɔ hela lɛ mli lɛ ayibɔ tee hiɛ kɛ akɛto ascites he (Mfoniri 1D). Wɔna srɔtofeemɔ ko ni yɔɔ faŋŋ yɛ EpCAM+ kɛ EpCAM- cell mli nibii lɛ ateŋ lɛ hu. EpCAM + (tumor) mli wala yibii lɛ yɛ glucose ni amɛheɔ kɛ mitochondrial nitsumɔ ni fa fe EpCAM- wala yibii lɛ, ni no fa kwraa fe fibroblasts ni yɔɔ tumor mli wala yibii lɛ amli lɛ anitsumɔ yɛ TME mli (Mfoniri 1, E kɛ F).
(A kɛ B) Nibii ni haa mɔ tsui nyɔɔ emli (MFI) ni haa mɔ he jɔɔ lɛ (2-NBDG) (A) kɛ CD4 + T wala yibii lɛ anitsumɔ (MitoTracker dark red) (B) Najiaŋdamɔlɔi ahe mfonirii (abɛkugbɛ) kɛ saji ni ato he gbɛjianɔ (Ninejurɔgbɛ), CD8 + T wala yibii kɛ EpCAM + T wala yibii kɛ EpCAM + T wala yibii a. (C) CD4 + kɛ CD8 + wala yibii (ni ji CD3 + T wala yibii) ni yɔɔ ascites kɛ tumor mli lɛ mlijaa. (D) EpCAM + tsɛŋemɔ helai ni yɔɔ ascites kɛ tsɛŋemɔ hela mli lɛ mlijaa (CD45−). (E kɛ F) EpCAM + CD45-tsɛŋemɔ hela kɛ EpCAM-CD45-matrix glucose ni anáa (2-NBDG) (E) kɛ mitochondrial nitsumɔ (MitoTracker red red) (F) najiaŋdamɔlɔi ahe mfonirii (abɛkugbɛ) kɛ saji ni ato he gbɛjianɔ ( Ninejurɔgbɛ) Ascites kɛ tsɛŋemɔ hela mli wala yibii. (G) CD25, CD137 kɛ PD1 ni ajieɔ lɛ kpo lɛ he mfoniri kɛtsɔ tsɔne ni akɛkwɛɔ lá mli lɛ nɔ. (H kɛ I) CD25, CD137 kɛ PD1 kpojiemɔ yɛ CD4 + T wala yibii (H) kɛ CD8 + T wala yibii (I) nɔ. (J kɛ K) Naive, teŋgbɛ kaimɔ (Tcm), nitsumɔ (Teff) kɛ nitsumɔ kaimɔ (Tem) nibii ni damɔ CCR7 kɛ CD45RO kpojiemɔ nɔ. Mfoniri ni damɔ shi kɛha CD4 + T wala yibii (J) kɛ CD8 + T wala yibii (K) ni yɔɔ ascites kɛ tsɛŋemɔ helai amli lɛ (abɛkugbɛ) kɛ saji ni yɔɔ ŋwɛigbɛ (ninejurɔgbɛ) lɛ. P sɛɛnamɔi ni atsɔɔ kɛtsɔ t-kasemɔ ni ji enyɔ nɔ (* P <0.05, ** P <0.01 kɛ *** P <0.001). Line lɛ damɔ shi kɛha helatsɛmɛi ni kɛ amɛhe kpãa gbee (n = 6). FMO, la ni tsoɔ ni ajieɔ ekome kɛjɛɔ mli; MFI, la ni kpɛɔ yɛ teŋgbɛ.
Nibii amlipɛimɔ kroko ni tee nɔ lɛ jie srɔtofeemɔi krokomɛi ni he hiaa ni yɔɔ T cell phenotypic shihilɛ ni atsu he nii jogbaŋŋ lɛ teŋ lɛ kpo. Nibii ni tsuɔ nii (Mfoniri 1, G kɛyashi I) kɛ nibii ni tsuɔ nii (Mfoniri 1, J kɛ K) ni yɔɔ tsɛŋemɔ helai amli lɛ fɔɔ baa fe ascites (CD3 + T cells lɛ mlijaa). Nakai nɔŋŋ hu, kɛji akɛ akwɛ bɔ ni gbɔmɔtso lɛ mli nibii yɔɔ ha lɛ mli kɛtsɔ nitsumɔ he okadii (CD25 kɛ CD137) kɛ nibii ni haa mɔ he jɔ̃ɔ ehe [programmed cell death protein 1 (PD1)] lɛ nɔ lɛ, etsɔɔ akɛ, eyɛ mli akɛ nɛkɛ gbɔmɛi nɛɛ amli gbɔmɔtso lɛ mli nibii ni haa mɔ he waa lɛ ahe sui lɛ yɛ srɔtoi moŋ (Mfoniri S1, B kɛyashi E) , Shi srɔtofeemɔ ko ni he hiaa ko bɛ ni haa mɔ he jɔɔ lɛ mli yɛ naFigure S1 teŋ. F kɛyashi I). Akɛ tsɔne ni akɛkaseɔ nii lɛ tsuɔ nii koni akɛha tsɛŋemɔ helai ahe nibii ni yɔɔ gbɔmɔtso lɛ mli lɛ diɛŋtsɛ (21), ni ejie tsɛŋemɔ helai babaoo (CD45 + / CD3- / CD4 + / CD45RO +) ni yɔɔ helatsɛ lɛ ascites lɛ mli lɛ kpo ekoŋŋ (Mfoniri S2A ). Yɛ wala yibii sɔrɔtoi fɛɛ ni ayɔse lɛ ateŋ lɛ, nɛkɛ myeloid wala yibii ayibɔ nɛɛ jie glucose ni anáa kɛ mitochondrial nitsumɔ ni fa fe fɛɛ kpo (Mfoniri S2, B kɛyashi G). Nibii nɛɛ maa srɔtofeemɔi ni mli wa ni yɔɔ wala yibii srɔtoi ni anaa yɛ ascites kɛ tumors ni yɔɔ HGSC helatsɛmɛi amli lɛ ateŋ lɛ nɔ mi.
Naagba titri ni yɔɔ TIL lɛ mli nibii ni haa mɔ he jɔɔ lɛ shishinumɔ mli ji bɔ ni ehe hiaa ni ajie T cell nɔkwɛmɔnii ni he tse bɔ ni sa, ni hi jogbaŋŋ kɛ bɔ ni amɛfa ha lɛ kɛjɛ tsɛŋemɔ helai amli. Nibii amlipɛimɔi ni afee nyɛsɛɛ nɛɛ etsɔɔ akɛ, gbɛi ni atsɔɔ nɔ ajieɔ nibii ni yɔɔ gbɔmɔtso lɛ mli lɛ amli kɛ nibii ni haa mɔ tsui nyɔɔ emli ni damɔ flow cytometry nɔ lɛ baanyɛ aha tsakemɔi aba tsɛŋemɔ helai ni haa mɔ he jɔɔ lɛ amli (22-24). Bɔni afee ni wɔye naagba nɛɛ nɔ kunim lɛ, wɔkɛ bead enrichment gbɛ lɛ tsu nii jogbaŋŋ koni wɔkɛjie TIL kɛjɛ gbɔmɔ fɔmɔ kotoku mli kansa ni afee lɛ opireshɛn lɛ mli dani LC-MS/MS lɛ akwɛ mli (kwɛmɔ Nibii kɛ Gbɛi; Mfoniri 2A). Bɔni afee ni wɔkwɛ bɔ ni nɛkɛ gbɛjianɔtoo nɛɛ saa metabolite tsakemɔi ahe ehaa lɛ, wɔkɛ T cells ni mɛi ni kɛ amɛhe eha ni yɔɔ hewalɛ lɛ tsuɔ nii yɛ ŋwɛi ŋtayaa mligbalamɔ gbɛ ni yɔɔ ŋwɛi nɛɛ sɛɛ lɛ to wala yibii ni jeee ŋtayaa mligbalamɔ shi amɛhiɔ ais nɔ lɛ ahe. Nɛkɛ kpakpafeemɔ he niiamlipɛimɔ nɛɛ na akɛ tsakpaa ni nɔ kwɔ yɛ shihilɛi enyɔ nɛɛ ateŋ (r = 0.77), ni ŋaa gbɛ nɔ ni atsɔɔ nɔ atsuɔ nii ekoŋŋ yɛ kuu ni ji 86 metabolites lɛ mli lɛ yɛ sɛɛkuu ni nɔ kwɔ (Mfoniri 2B). No hewɔ lɛ, gbɛi nɛɛ baanyɛ afee metabolite mlipɛimɔ ni ja yɛ wala yibii ni miitsɔ wala yibii ahe nibii amli lɛ amli, ni no haa klɛŋklɛŋ gbɛjianɔtoo ni nɔ kwɔ ni akɛyooɔ metabolites pɔtɛɛ komɛi ni yɔɔ HGSC mli lɛ haa, ni no haa gbɔmɛi nuɔ wala yibii pɔtɛɛ komɛi ahe shishinumɔ ni mli kwɔ Sexual metabolism program lɛ he.
(A) Mfoniri ni tsɔɔ bɔ ni magnetic bead lɛ yaa hiɛ ehaa. Dani LC-MS/MS lɛ baapɛi mli lɛ, wala yibii lɛ baatsɔ magnetic bead enrichment mli shii etɛ ni nyiɛɔ sɛɛ loo amɛbaahi ais nɔ. (B) Nɔ ni nibii ni haa mɔ tsui nyɔɔ emli lɛ tsuɔ yɛ nibii ni haa mɔ he waa lɛ babaoo nɔ. Nibii ni asusuɔ he etɛ mlijaa kɛha nibii ni haa mɔ tsui nyɔɔ emli lɛ eko fɛɛ eko ± SE. Line ni yɔɔ ŋmɔlɔŋmɔlɔ lɛ damɔ shi kɛha wekukpaa ni ji 1:1. Intra-klas tsakpaa (ICC) ni ji susumɔi ni afɔɔ feemɔ ni atsɔɔ yɛ axis label lɛ mli lɛ. NAD, nikotinamiid adenin dinukleotaid. (C) Mfoniri ni tsɔɔ bɔ ni helatsɛ lɛ mli nibii ni haa mɔ he waa lɛ mlipɛimɔ yaa nɔ ehaa lɛ. Abuaa ascites loo tsɛŋemɔ helai anaa kɛjɛɔ helatsɛmɛi amli ni akɛtoɔ fɛ̃i mli. Akɛ flow cytometry tsu nii kɛpɛi nɔkwɛmɔnɔ fɛɛ nɔkwɛmɔnɔ lɛ fã bibioo ko mli, yɛ be mli ni nɔkwɛmɔnɔi ni eshwɛ lɛ tee CD4+, CD8+ kɛ CD45- cells lɛ ahe nibii ni akɛwoɔ mli lɛ mli shii etɛ. Akɛ LC-MS/MS tsu nii kɛpɛi nɛkɛ wala yibii nɛɛ amli. (D) Heat map ni tsɔɔ bɔ ni nibii ni wala yɔɔ mli lɛ fa ha lɛ. Dendrogram lɛ damɔ shi kɛha Ward Euclidean sɛɛjɛkɛmɔ ni yɔɔ nɔkwɛmɔnii lɛ ateŋ lɛ kuu. (E) Nibii ni he hiaa waa lɛ amlipɛimɔ (PCA) ni kɔɔ nɔkwɛmɔnɔ ni ji metabolite map lɛ he, ni tsɔɔ nɔkwɛmɔnɔ fɛɛ nɔkwɛmɔnɔ lɛ mli etɛ, nɔkwɛmɔnii ni jɛ helatsɛ kome mli lɛ ji nɔ ni akɛ line ko tsa. (F) PCA ni ji metabolite profile ni ji nɔkwɛmɔnɔ ni ato he gbɛjianɔ yɛ helatsɛ lɛ nɔ lɛ (ni ji, kɛtsɔ fã ko ni akɛtsuɔ nii); nɔkwɛmɔnɔ henɔ lɛ ji nɔ ni convex hull lɛ tsiɔ naa. PC1, ni ji nɔ titri ni yɔɔ mli 1; PC2, nɔ titri ni ji 2.
No sɛɛ lɛ, wɔkɛ nɛkɛ nibii ni haa mɔ tsui nyɔɔ emli nɛɛ tsu nii koni wɔkɛpɛi 99 metabolites ni yɔɔ CD4 +, CD8 + kɛ CD45-cell mli lɛ amli yɛ shishijee ascites kɛ tumors ni yɔɔ HGSC helatsɛmɛi enumɔ lɛ amli lɛ mli (Mfoniri 2C, Mfoniri S3A kɛ Table S3 kɛ S4). Mɛi ni yɔɔ miishɛɛ lɛ ayibɔ ji 2% kɛyashi 70% yɛ shishijee nɔkwɛmɔnɔ agbo ni ji wala yibii lɛ amli, ni wala yibii lɛ amlijaa lɛ yɛ srɔto kwraa yɛ helatsɛmɛi lɛ ateŋ. Beni ajara ŋmeii lɛ amli sɛɛ lɛ, miishɛɛ ni yɔɔ mli lɛ mlijaa (CD4+, CD8+ loo CD45-) lɛ feɔ nɔ ni fe 85% yɛ wala yibii fɛɛ ni yɔɔ nɔkwɛmɔnɔ lɛ mli lɛ mli kɛ aja mli. Nɛkɛ gbɛ ni atsɔɔ nɔ anáa nii babaoo nɛɛ haa wɔpɛiɔ wala yibii ayibɔ ni jɛɔ gbɔmɔtso lɛ mli fãji ni haa mɔ he jɔ̃ɔ ehe lɛ amli lɛ mli, ni eji nɔ ko ni anyɛɛɛ afee kɛjɛ nɔkwɛmɔnii wuji amli. Kɛtsɔ nɛkɛ gbɛjianɔtoo nɛɛ nɔ lɛ, wɔna akɛ l-kynurenine kɛ adenosine, nɛkɛ nibii enyɔ ni haa mɔ he waaa ni ajie lɛ kpo jogbaŋŋ nɛɛ tee hiɛ yɛ tsɛŋemɔ hela T wala yibii loo tsɛŋemɔ hela mli wala yibii amli (Mfoniri S3, B kɛ C). No hewɔ lɛ, nɔ ni jɛ mli ba nɛɛ tsɔɔ anɔkwayeli kɛ nyɛmɔ ni wɔ wala yibii amli gbalamɔ kɛ mass spectrometry ŋaalee lɛ yɔɔ ni ekɛtaoɔ nibii ni he hiaa ni haa mɔ he jɔɔ lɛ yɛ helatsɛ lɛ fãji amli.
Wɔ niiamlipɛimɔ lɛ hu jie metabolic mligbalamɔ ni mli wa ni yɔɔ helatsɛmɛi amli kɛ amɛteŋ lɛ kpo (Mfoniri 2D kɛ Mfoniri S4A). Yɛ gbɛ titri ko nɔ lɛ, kɛ akɛto helatsɛmɛi krokomɛi ahe lɛ, helatsɛ 70 lɛ jie bɔ ni gbɔmɔtso lɛ mli nibii tsakeɔ ehaa lɛ kpo (Mfoniri 2E kɛ Mfoniri S4B), ni tsɔɔ akɛ ekolɛ gbɔmɔtso lɛ mli nibii ni tsakeɔ lɛ amli srɔtofeemɔ ko ni he hiaa waa yɛ helatsɛmɛi lɛ ateŋ. E sa nɛ wa kai kaa ke a kɛ to hiɔtsɛmɛ kpahi a he (lita 1.2 kɛ ya si 2; Table S1) ɔ, ascites nɛ a bua naa ngɛ hiɔtsɛ 70 ɔ mi ɔ tsuo (80 ml) ɔ faaa. Nibii ni yɔɔ helatsɛmɛi ateŋ lɛ anɔkwɛmɔ yɛ fã titri lɛ mlipɛimɔ mli (akɛ nɔkwɛmɔ nɔ lɛ, kɛtsɔ fã ni ji redundancy mlipɛimɔ ni akɛtsuɔ nii lɛ nɔ) tsɔɔ tsakemɔi ni yaa nɔ yɛ wala yibii ahenɔi lɛ ateŋ, ni wala yibii henɔi lɛ kɛ/loo shihilɛ bibioo lɛ feɔ ekome yɛ faŋŋ mli taakɛ metabolite lɛ he saji lɛ tsɔɔ lɛ (Mfoniri 2F ). Nibii ni anáa kɛjɛɔ mli lɛ amlipɛimɔ lɛ ma nɛkɛ nibii nɛɛ anɔ mi ni ejie srɔtofeemɔi ni sa kadimɔ ni yɔɔ wala yibii ahenɔi kɛ shihilɛ bibii ni yɔɔ jɛmɛ lɛ ateŋ lɛ kpo. Ehe miihia ni wɔkadi akɛ srɔtofeemɔ ni fe fɛɛ ni ana ji MNA, ni bei pii lɛ, CD45- wala yibii kɛ CD4+ kɛ CD8+ wala yibii ni boteɔ tsɛŋemɔ hela lɛ mli lɛ fa babaoo (Mfoniri 3A). Yɛ CD4 + wala yibii lɛ agbɛfaŋ lɛ, nɛkɛ hewalɛ nɛɛ jeɔ kpo faŋŋ fe fɛɛ, ni etamɔ nɔ ni MNA ni yɔɔ CD8 + wala yibii lɛ amli lɛ hu naa shihilɛ ni yɔɔ jɛmɛ lɛ nɔ hewalɛ waa. Shi kɛlɛ, enɛ he ehiaaa, ejaakɛ helatsɛmɛi enumɔ lɛ ateŋ mɛi etɛ pɛ ji mɛi ni abaanyɛ akwɛ amɛ koni ana CD8+ ni yɔɔ amɛmli lɛ mli. Kɛfata MNA he lɛ, yɛ tsɛŋemɔ helai srɔtoi ni yɔɔ ascites kɛ tsɛŋemɔ helai amli lɛ amli lɛ, tsɛŋemɔ helai krokomɛi ni bɛ su kpakpa yɛ TIL mli lɛ hu yɛ srɔtoi ni yɔɔ nii (Mfoniri S3 kɛ S4). No hewɔ lɛ, saji nɛɛ jieɔ tsofai ni haa mɔ he waa lɛ ahe nitsumɔ ko ni hi jogbaŋŋ kpo kɛha niiamlitaomɔ kroko.
(A) MNA ni yɔɔ CD4+, CD8+ kɛ CD45- mli lɛ mli nibii ni jɛ ascites kɛ tumor mli lɛ mli nibii ni sa. Adeka lɛ mli mfoniri lɛ tsɔɔ teŋgbɛ (line), ŋwɛigbɛ ŋwɛigbɛ (frem hinge) kɛ data ŋwɛigbɛ, kɛyashi ŋwɛigbɛ ŋwɛigbɛ (frem whisker) lɛ shii 1.5. Taakɛ atsɔɔ mli yɛ Helatsɛ Nibii kɛ Gbɛi lɛ amli lɛ, okɛ helatsɛ lɛ limma sɛɛnamɔ lɛ atsu nii ni okɛna P sɛɛnamɔ (*P<0.05 kɛ **P<0.01). (B) MNA mli tsakemɔ he mfoniri (60). Nibii ni haa mɔ he jɔɔ lɛ: S-adenosyl-1-methionine; SAH, S-adenosin-1-homosistɛin; NA, tsofa ni haa mɔ tsui nyɔɔ emli; MNA, 1-mɛtilnikotinami; 2-PY, 1-mɛtil- 2-piridɔn-5-karboksamaid; 4-PY, 1-mɛtil-4-piridon-5-karboksamaid; NR, ni ji tsofa ni haa mɔ he jɔɔ lɛ; NMN, nikotinamiid ni ji tsofa kome. Nibii ni haa mɔ he jɔɔ lɛ (eŋɔli): NNMT, ni ji tsofa ni haa mɔ he jɔɔ lɛ; SIRT, nibii ni haa mɔ tsui nyɔɔ emli; NAMPT, ni ji tsofa ni haa mɔ he jɔɔ lɛ; AOX1, ni ji tsofa ni haa mɔ he jɔɔ lɛ 1; NRK, ni ji tsofa ni haa mɔ he jɔɔ lɛ; NMNAT, ni ji tsofa ni haa mɔ he jɔɔ lɛ; Pnp1, purin nukleosaid fɔsfɔrilɛs. (C) t-SNE ni ji scRNA-seq ni ji ascites (ŋmɔlɔ) kɛ tsɛŋemɔ hela (tsuru; n = helatsɛmɛi 3). (D) NNMT kpojiemɔ yɛ tsɛŋemɔ helai srɔtoi ni ayɔse kɛtsɔ scRNA-seq nɔ. (E) NNMT kɛ AOX1 kpojiemɔ yɛ SK-OV-3, gbɔmɔtso mli fɔmɔ kotoku (HEK) 293T, T wala yibii kɛ T wala yibii ni akɛ MNA tsuɔ he nii lɛ mli. Atsɔɔ wiemɔ ni afolɔ mli lɛ yɛ SK-OV-3 he. Atsɔɔ bɔ ni SEM jieɔ ehe kpo ehaa lɛ mli (n = 6 mɛi ni kɛ amɛhe haa ni yɔɔ hewalɛ). Ct values ​​ni fe 35 lɛ ji nɔ ni abuɔ akɛ anyɛɛɛ ana (UD). (F) SLC22A1 kɛ SLC22A2 ni ajieɔ lɛ kpo yɛ SK-OV-3, HEK293T, T tsɛŋemɔ helai kɛ T tsɛŋemɔ helai ni akɛ 8mM MNA tsuɔ he nii lɛ mli. Atsɔɔ wiemɔ ni afolɔ mli lɛ yɛ SK-OV-3 he. Atsɔɔ bɔ ni SEM jieɔ ehe kpo ehaa lɛ mli (n = 6 mɛi ni kɛ amɛhe haa ni yɔɔ hewalɛ). Ct values ​​ni fe 35 lɛ ji nɔ ni abuɔ akɛ anyɛɛɛ ana (UD). (G) Cell MNA ni yɔɔ T cells ni yɔɔ hewalɛ ni tsuɔ nii lɛ amli yɛ ŋmɛlɛtswai 72 ni akɛ MNA ewo mli lɛ sɛɛ. Atsɔɔ bɔ ni SEM jieɔ ehe kpo ehaa lɛ mli (n = 4 mɛi ni kɛ amɛhe haa ni yɔɔ hewalɛ).
MNA lɛ ji nɔ ni ajieɔ kɛjɛɔ S-adenosyl-1-methionine (SAM) mli kɛyaa nikotinamide (NA) mli kɛtsɔ nikotinamide N-methyltransferase (NNMT; Mfoniri 3B) lɛ nɔ. NNMT lɛ jieɔ ehe kpo fe nine yɛ adesai kansa srɔtoi amli ni ekɛ shweremɔ, tutuamɔ kɛ metastasis (25-27) yɔɔ tsakpaa. Kɛha MNA jɛɛhe ni yɔɔ T cells ni yɔɔ TME mli lɛ shishinumɔ jogbaŋŋ lɛ, wɔkɛ scRNA-seq tsu nii koni wɔkɛtsɔɔ bɔ ni NNMT lɛ jieɔ ehe kpo yɛ cells henɔi amli yɛ ascites kɛ tumors ni yɔɔ HGSC helatsɛmɛi etɛ amli lɛ mli (Table S5). 6,500 mlipɛimɔ tsɔɔ akɛ yɛ ascites kɛ tumor shihilɛi amli lɛ, NNMT kpojiemɔ lɛ ji fibroblast kɛ tumor cells ni asusuɔ akɛ amɛji lɛ pɛ (Mfoniri 3, C kɛ D). Ehe miihia ni wɔkadi akɛ NNMT kpojiemɔ ko ni yɔɔ faŋŋ bɛ gbɔmɛi akuu ko mli ni jieɔ PTPRC (CD45 +) kpo (Mfoniri 3D kɛ Mfoniri S5A), ni tsɔɔ akɛ akɛ MNA ni ana yɛ metabolite spectrum lɛ mli lɛ ewo T cells lɛ amli. (AOX1) lɛ kpojiemɔ lɛ tsakeɔ MNA lɛ efeɔ 1-methyl-2-pyridon-5-carboxamide (2-PYR) loo 1-methyl-4-pyridon-5-carboxamide (4- PYR); 3B) lɛ hu ji nɔ ni akɛtoɔ fibroblasts ni jieɔ COL1A1 kpo lɛ ayibɔ pɛ he (Mfoniri S5A), ni kɛ fɛɛ feɔ ekome lɛ, etsɔɔ akɛ T cells lɛ bɛ nyɛmɔ ni amɛyɔɔ ni amɛkɛtsakeɔ MNA nibii ni afɔɔ feemɔ lɛ. Akɛ nɛkɛ MNA-he tsɛŋemɔ helai nɛɛ ajiemɔ gbɛjianɔtoo lɛ tsu nii kɛtsɔ tsɛŋemɔ hela ni ji enyɔ ni yɔɔ ehe lɛ he saji ni ato kɛjɛ ascites ni jɛ HGSC helatsɛmɛi adɛŋ lɛ nɔ (Mfoniri S5B; n = 6) (16). Kɛfata he lɛ, qPCR mlipɛimɔ ni kɔɔ T cells ni yɔɔ hewalɛ ni akɛ MNA tsuɔ he nii lɛ he lɛ tsɔɔ akɛ, kɛ akɛto SK-OV-3 ovarian tumor cells ni kudɔɔ lɛ he lɛ, eshwɛ fioo ni NNMT loo AOX1 lɛ baaje kpo (Mfoniri 3E). Nɔ ni jɛ mli ba ni akpaaa gbɛ nɛɛ tsɔɔ akɛ MNA baanyɛ ajie kɛjɛ fibroblasts loo tumors amli kɛya T cells ni bɛŋkɛ TME lɛ mli.
Eyɛ mli akɛ mɛi ni miitao ni amɛye nɔ lɛ ateŋ mɛi komɛi ji weku ni ji bɔɔ nii ni kɛ nibii ni wala yɔɔ mli lɛ yaa 1 kɛyashi 3 (OCT1, OCT2 kɛ OCT3) ni tsɔne ni haa mɔ he jɔɔ lɛ 22 (SLC22) weku lɛ (SLC22A1, SLC22A2 kɛ SLC22A3) lɛ kɛwoɔ mli lɛ moŋ, shi mɛi ni baanyɛ atsɔ MNA lɛ nɔ lɛ atsɔɔɔ mli lolo (28) . QPCR ni ji mRNA ni jɛ T cells ni yɔɔ hewalɛ ni kɛ shika haa lɛ mli lɛ tsɔɔ SLC22A1 lɛ mlitsɔɔmɔ ni baa shi shi SLC22A2 lɛ mlitsɔɔmɔ ni anyɛɛɛ ana, ni ma nɔ mi akɛ abɔ he amaniɛ tsutsu yɛ woji amli (Mfoniri 3F) (29). Nɔ ni tamɔɔɔ enɛ lɛ, SK-OV-3 ovarian tumor cell line lɛ jieɔ tsɔnei enyɔ ni kɛ nibii yaa lɛ fɛɛ ayibɔ ni nɔ kwɔ kpo (Mfoniri 3F).
Bɔni afee ni aka bɔ ni T cells lɛ baanyɛ aŋɔ maŋsɛɛ MNA lɛ akwɛ lɛ, akwɛ T cells ni yɔɔ hewalɛ ni kɛ amɛhe haa lɛ anɔ ŋmɛlɛtswai 72 yɛ MNA ni falɛi srɔtoi yɔɔ mli lɛ mli. Kɛji akɛ exogenous MNA bɛ lɛ, anyɛɛɛ ana MNA mli nibii ni yɔɔ wala yibii amli lɛ (Mfoniri 3G). Shi, T cells ni akɛtsuɔ nii ni akɛ exogenous MNA tsuɔ he nii lɛ tsɔɔ akɛ MNA ni yɔɔ cells lɛ mli lɛ yaa hiɛ yɛ tsofa ni akɛhaa lɛ mli, kɛyashiɔ 6 mM MNA (Mfoniri 3G). Nɔ ni jɛ mli ba nɛɛ tsɔɔ akɛ yɛ bɔ ni tsɔne ni kɛ nibii yaa lɛ jieɔ ehe kpo ehaa lɛ faaa kɛ bɔ ni enzyme titri ni haa MNA lɛ tsakeɔ yɛ wala yibii lɛ amli lɛ bɛ lɛ fɛɛ sɛɛ lɛ, TIL baanyɛ aŋɔ MNA lɛ lolo.
Nibii ni haa mɔ he jɔɔ lɛ ni yɔɔ helatsɛmɛi lɛ T cells lɛ amli kɛ in vitro MNA absorption shikpamɔi lɛ haa bɔ ni kansa he fibroblasts (CAF) lɛ jieɔ MNA kɛ tumor cells lɛ baanyɛ akudɔ TIL lɛ su kɛ enitsumɔ lɛ yaa hiɛ. Bɔni afee ni ana bɔ ni MNA lɛ tsuɔ nii ehaa yɛ T wala yibii lɛ anɔ lɛ, atsu T wala yibii ni yɔɔ hewalɛ ni kɛ amɛhe haa lɛ ahe nii yɛ vitro mli yɛ MNA ni yɔɔ loo bɛ mli lɛ mli, ni akwɛ bɔ ni amɛshwereɔ kɛ bɔ ni amɛfeɔ cytokine lɛ mli. Yɛ gbii 7 ni akɛ MNA fata he yɛ tsofa ni fe fɛɛ lɛ mli lɛ sɛɛ lɛ, gbɔmɛi ayibɔ ni feɔ shii enyɔ lɛ ba shi yɛ gbɛ ni sa nɔ, yɛ be mli ni hewalɛ hiɔ shi yɛ tsofai lɛ fɛɛ mli (Mfoniri 4A). Kɛfata he lɛ, MNA ni jɛɔ kponɔgbɛ lɛ tsamɔ ha CD4 + kɛ CD8 + T wala yibii ni jieɔ tumor necrosis factor-α (TNFα; Mfoniri 4B) lɛ kpo lɛ ayibɔ tee hiɛ. Nɔ ni tamɔɔɔ enɛ lɛ, IFN-γ ni ajieɔ yɛ wala yibii amli lɛ ba shi kwraa yɛ CD4 + T wala yibii lɛ amli, shi jeee yɛ CD8 + T wala yibii lɛ amli, ni tsakemɔ ko kwraa bɛ interleukin 2 (IL-2; Mfoniri 4, C kɛ D) lɛ mli. No hewɔ lɛ, enzyme-linked immunosorbent assay (ELISA) ni ji supernatants ni jɛ nɛkɛ MNA-ni akɛtsa T cell kusumii nɛɛ amli lɛ tsɔɔ akɛ TNFα lɛ etee hiɛ waa, IFN-γ lɛ eba shi, ni tsakemɔ ko bɛ IL-2 lɛ mli (Mfoniri 4, E kɛyashi G). . IFN-γ ni baa shi lɛ tsɔɔ akɛ MNA baanyɛ atsu gbɛfaŋnɔ ko he nii yɛ T cells lɛ anitsumɔ ni teɔ shi ewoɔ helai ni haa mɔ he jɔ̃ɔ ehe lɛ mli. Bɔni afee ni akase bɔ ni MNA lɛ tsuɔ nii ehaa yɛ T wala yibii lɛ anɔ lɛ, chimeric antigen receptor T (FRα-CAR-T) wala yibii ni tsɔɔ folate receptor α kɛ CAR-T (GFP) ni green fluorescent protein (GFP) -CAR-T) wala yibii lɛ kudɔɔ lɛ ji nɔ ni wala yibii ni yɔɔ hewalɛ ni haa mɔ he jɔɔ lɛ feɔ. Akɛ CAR-T wala yibii lɛ wo mli ŋmɛlɛtswai 24 yɛ MNA hiɛ, ni no sɛɛ lɛ akɛ gbɔmɔ SK-OV-3 fɔmɔ kotoku mli wala yibii ni jieɔ folate receptor α kpo lɛ wo mli yɛ 10:1 mli. MNA helatsamɔ ha FRα-CAR-T wala yibii lɛ agbemɔ nitsumɔ lɛ ba shi waa, ni eji nɔ ni tamɔ FRα-CAR-T wala yibii ni akɛ adenosine tsuɔ he nii lɛ (Mfoniri 4H).
(A) Total viable cell count kɛ gbɔmɛi ayibɔ ni yaa hiɛ shii enyɔ (PD) tɛ̃ɛ kɛjɛ kusum mli yɛ gbi 7. Bar graph lɛ damɔ shi kɛha sɛɛnamɔ + SEM ni ji mɛi enumɔ ni yɔɔ hewalɛ ni kɛ amɛhe haa lɛ. Edamɔ shi kɛha saji ni jɛ n = 3 shikpamɔi ni yɔɔ amɛ diɛŋtsɛ amɛhe. (B kɛyashi D) Akɛ CD3/CD28 kɛ IL-2 tsu nii koni akɛtsu T cells lɛ ahe nii yɛ amɛ MNA lɛ mli gbii 7 sɔŋŋ. Dani abaakwɛ mli lɛ, akɛ PMA/ionomycin kɛ GolgiStop tsirɛɔ wala yibii lɛ ŋmɛlɛtswai 4 sɔŋŋ. TNFα (B) kpojiemɔ yɛ T wala yibii lɛ amli. Nɔkwɛmɔnɔ mfoniri (abɛkugbɛ) kɛ tabular data (ninejurɔgbɛ) ni kɔɔ TNFα kpojiemɔ yɛ wala yibii amli. IFN-γ (C) kɛ IL-2 (D) ni ajieɔ lɛ kpo yɛ T wala yibii lɛ amli lɛ. Akɛ flow cytometry susu bɔ ni cytokines lɛ jieɔ amɛhe kpo amɛhaa lɛ he. Bar graph lɛ damɔ shi kɛha sɛɛnamɔ (n = 6 ni yɔɔ hewalɛ ni kɛ amɛhe haa) + SEM. Kɛ gbɛ kome nɔ ni akɛpɛiɔ srɔtofeemɔ kɛ susumɔi ni afɔɔ feemɔ (*P<0.05 kɛ **P<0.01) atsu nii ni okɛna P sɛɛnamɔ lɛ. Edamɔ shi kɛha saji ni jɛ n = 3 shikpamɔi ni yɔɔ amɛ diɛŋtsɛ amɛhe. (E kɛyashi G) Akɛ CD3/CD28 kɛ IL-2 tsu nii koni akɛtsu T cells lɛ ahe nii yɛ amɛ MNA lɛ mli gbii 7 sɔŋŋ. Akɛ tsɔne lɛ bua naa dani PMA/ionomycin stimulation ŋmɛlɛtswai 4 kɛ no sɛɛ. Akɛ ELISA susu TNFα (E), IFN-γ (F) kɛ IL-2 (G) lɛ mli nibii ni yɔɔ mli lɛ ahe. Bar graph lɛ damɔ shi kɛha sɛɛnamɔ (n = 5 ni yɔɔ hewalɛ ni kɛ amɛhe haa) + SEM. P sɛɛnamɔ ni atsɔɔ kɛtsɔ gbɛ kome nɔ nibii amlipɛimɔ ni kɔɔ srɔtofeemɔ kɛ susumɔi ni afɔɔ feemɔ he (* P<0.05). Line ni yɔɔ ŋwɛigbɛ lɛ tsɔɔ husu ni anaa yɛ nɔ ni ana lɛ he. (H) Cell lysis mlikwɛmɔ. Akɛ adenosine (250μM) loo MNA (10 mM) tsake FRα-CAR-T loo GFP-CAR-T wala yibii lɛ ŋmɛlɛtswai 24, loo ashi amɛ ni atsako amɛ (Ctrl). Asusu SK-OV-3 wala yibii ni agbeɔ lɛ mlijaa oha mlijaa lɛ he. P sɛɛnamɔ ni Welch t kaa lɛ tsɔɔ (* P <0.5 kɛ ** P <0.01).
Bɔni afee ni aná shishinumɔ ni kɔɔ MNA-ni damɔ TNFα kpojiemɔ he mla lɛ he lɛ, akwɛ tsakemɔi ni baa yɛ TNFα mRNA ni MNA-ni akɛtsaa T wala yibii lɛ mli lɛ mli (Mfoniri 5A). T cells ni yɔɔ hewalɛ ni akɛ MNA tsuɔ he nii lɛ jie TNFα niŋmaa lɛ mliyaa ni yaa hiɛ shii enyɔ kpo, ni tsɔɔ akɛ MNA lɛ damɔ TNFα niŋmaa he mla lɛ nɔ. Kɛha nɛkɛ gbɛjianɔtoo ni baanyɛ aba nɛɛ mlitaomɔ lɛ, akwɛ nibii enyɔ ni ale ni kɔɔ niŋmaa he ni kudɔɔ TNFα, ni ji T cell nuclear factor (NFAT) ni tsuɔ nii kɛ protein pɔtɛɛ 1 (Sp1), lɛ mli yɛ hetoo ni akɛhaa MNA ni kɛ TNFα ni bɛŋkɛɔ lɛ lɛ he lɛ haa ( 30 ). TNFα nɔkwɛlɔ lɛ hiɛ NFAT ni akɛfiɔ hei 6 ni ayɔse kɛ Sp1 ni akɛfiɔ hei 2, ni amɛkpeɔ yɛ he kome [-55 base pairs (bp) kɛjɛ 5’cap lɛ mli] (30). Chromatin immunoprecipitation (ChIP) tsɔɔ akɛ, kɛ́ akɛ MNA tsu nii lɛ, Sp1 ni kɛ TNFα ni tsiɔ mɔ naa lɛ kpãa gbee lɛ yaa hiɛ shii etɛ. NFAT ni akɛwo mli lɛ hu tee hiɛ ni ebɛŋkɛ hiamɔ he (Mfoniri 5B). Nɛkɛ saji nɛɛ tsɔɔ akɛ MNA kudɔɔ TNFα kpojiemɔ kɛtsɔɔ Sp1 niŋmaa nɔ, ni yɛ gbɛ bibioo nɔ lɛ, ekwɛɔ NFAT kpojiemɔ nɔ.
(A) Kɛ akɛto T cells ni akwɛɔ nɔ ni MNA bɛ mli lɛ he lɛ, tsakemɔ ni baa TNFα kpojiemɔ lɛ mli yɛ T cells ni akɛ MNA tsuɔ nii yɛ mli lɛ mli lɛ. Atsɔɔ bɔ ni ajieɔ SEM kpo ahaa lɛ mli (n = 5 mɛi ni kɛ amɛhe haa ni yɔɔ hewalɛ). Edamɔ shi kɛha saji ni jɛ n = 3 shikpamɔi ni yɔɔ amɛ diɛŋtsɛ amɛhe. (B) TNFα tsɔne ni tsiɔ T wala yibii ni akɛ 8 mM MNA tsuɔ nii loo akɛtsuuu nii yɛ NFAT kɛ Sp1 sɛɛ lɛ fata (Ctrl) kɛ PMA/ionomycin stimulation he ŋmɛlɛtswai 4 sɛɛ. Akɛ Immunoglobulin G (IgG) kɛ H3 tsu nii akɛ nɔ ni ehiii kɛ nɔ ni hi kɛha gbɔmɔtso lɛ mli hewalɛ ni tsiɔ hela naa lɛ, yɛ gbɛ kome nɔ. ChIP lɛ falɛ ni ato lɛ tsɔɔ akɛ Sp1 kɛ NFAT ni kɛ TNFα ni tsiɔ mɔ lɛ naa lɛ kpãa gbee yɛ MNA-ni akɛtsaa wala yibii lɛ amli lɛ tee hiɛ shii abɔ kɛ akɛto nɔ ni akudɔɔ lɛ he. Edamɔ shi kɛha saji ni jɛ n = 3 shikpamɔi ni yɔɔ amɛ diɛŋtsɛ amɛhe. P sɛɛnamɔ ni t-kaai babaoo tsɔɔ (*** P <0.01). (C) Kɛ́ akɛto HGSC lɛ ascites lɛ he lɛ, T cells (ni jeee cytotoxic) lɛ jieɔ TNF kpojiemɔ ni yaa hiɛ yɛ tsɛŋemɔ hela lɛ mli lɛ kpo. Kɔlai lɛ damɔ shi kɛha helatsɛmɛi srɔtoi. Akɛ tsɛŋemɔ helai ni ajie lɛ kpo lɛ ebɔi 300 ni ajie lɛ kpo koni akɛtsi nɔ ni fe nine lɛ naa (** Padj = 0.0076). (D) MNA nɔkwɛmɔnɔ ni atsɔɔ kɛha fɔmɔ kotoku mli kansa. Afeɔ MNA yɛ tsɛŋemɔ hela mli wala yibii kɛ fibroblasts ni yɔɔ TME mli lɛ amli, ni T wala yibii lɛ heɔ. MNA haa Sp1 ni kɛ TNFα sɛɛnyiɛlɔ lɛ kpãa gbee lɛ yaa hiɛ, ni no haa TNFα niŋmaa kɛ TNFα cytokine feemɔ yaa hiɛ. MNA lɛ hu haa IFN-γ lɛ baa shi. T cell lɛ nitsumɔ ni atsĩɔ naa lɛ haa nyɛmɔ ni akɛgbeɔ mɔ lɛ baa shi, ni ehaa tsɛŋemɔ hela lɛ daa oya.
Taakɛ amaniɛbɔi tsɔɔ lɛ, TNFα yɛ hiɛ kɛ sɛɛ-ni damɔ shi kɛha tsɛŋemɔ hela kɛ tsɛŋemɔ helai ahe hewalɛ, shi eyɛ gbɛfaŋnɔ ni ale jogbaŋŋ yɛ fɔmɔ kotoku mli kansa lɛ dalɛ kɛ esɛɛgbɛtsɔɔmɔ mli (31-33). Amaniɛbɔi tsɔɔ akɛ, TNFα ni yɔɔ ascites kɛ tumor fãji amli yɛ helatsɛmɛi ni yɔɔ ovarian kansa lɛ amli lɛ fa fe nɔ ni yɔɔ benign fãji amli lɛ (34-36). Yɛ gbɛ ni atsɔɔ nɔ atsuɔ nii lɛ gbɛfaŋ lɛ, TNFα baanyɛ akudɔ lá mli wala yibii yɛŋ lɛ nitsumɔ, amɛnitsumɔ kɛ amɛshweremɔ, ni etsake kansa mli wala yibii lɛ ahe mfoniri ( 37, 38 ). Nɔ ni kɛ nɛkɛ nibii nɛɛ kpãa gbee lɛ, srɔtofeemɔ ni yɔɔ tsɛŋemɔ helai amli lɛ mlipɛimɔ tsɔɔ akɛ TNF lɛ ji nɔ ni akwɛɔ nɔ jogbaŋŋ yɛ T tsɛŋemɔ helai ni yɔɔ tsɛŋemɔ hela mli lɛ amli kɛ akɛto ascites he (Mfoniri 5C). TNF kpojiemɔ lɛ mliyaa lɛ je kpo faŋŋ yɛ T cell ayibɔ ni bɛ cytotoxic phenotype lɛ pɛ mli (Mfoniri S5A). Yɛ kukufoo mli lɛ, saji nɛɛ fiɔ susumɔ ni ji akɛ MNA yɛ nibii ni tsiɔ helai anaa kɛ nibii ni haa mɔ he jɔ̃ɔ ehe yɛ HGSC mli lɛ sɛɛ.
Fluorescent ni akɛwoɔ nibii amli ni damɔ flow cytometry nɔ lɛ ebatsɔ gbɛ titri ni atsɔɔ nɔ akaseɔ TIL mli nibii ni tsakeɔ lɛ. Nibii nɛɛ etsɔɔ akɛ, kɛ́ akɛ lá mli lymphocytes loo T cells ni jɛ lymphoid fãi ni ji enyɔ lɛ amli lɛ to lɛ, murine kɛ gbɔmɔ TIL lɛ yɛ su ni nɔ kwɔ ni amɛkɛbaaŋɔ glucose (4, 39) kɛ mitochondrial nitsumɔ ni baa shi fiofio (19, 40). Eyɛ mli akɛ wɔna nɔ ni jɛ mli ba ni tamɔ nakai nɔŋŋ yɛ nikasemɔ nɛɛ mli moŋ, shi nɔ titri ni ba ji ni akɛ bɔ ni tsɛŋemɔ hela mli wala yibii lɛ kɛ TIL ni jɛ tsɛŋemɔ hela mli wala yibii ni ajie lɛ mli lɛ mli lɛ ato he. Nɔ ni kɛ amaniɛbɔi ni eho nɛɛ ekomɛi kpãa gbee lɛ, tsɛŋemɔ hela (CD45-EpCAM +) mli wala yibii ni jɛ ascites kɛ tsɛŋemɔ hela mli lɛ yɛ glucose ni fa fe CD8 + kɛ CD4 + T wala yibii lɛ, ni efiɔ sɛɛ akɛ abaanyɛ akɛ glucose ni fa babaoo ni tsɛŋemɔ hela mli wala yibii lɛ jieɔ lɛ ato T wala yibii lɛ ahe. T cell akaŋshii he susumɔ lɛ. TME. Shi kɛlɛ, mitochondrial nitsumɔ ni yɔɔ tsɛŋemɔ hela mli wala yibii lɛ amli lɛ fa fe CD8 + T wala yibii lɛ anɔ, shi mitochondrial nitsumɔ lɛ tamɔ nɔ ni CD4 + T wala yibii lɛ tsuɔ lɛ. Nibii ni jɛ mli ba nɛɛ maa saneyitso ni miiba lɛ nɔ mi akɛ, tsɔne ni haa mɔ he jɔɔ lɛ he hiaa waa kɛha tsɛŋemɔ hela mli wala yibii lɛ ( 41, 42 ). Amɛtsɔɔ hu akɛ CD8 + T wala yibii lɛ baanyɛ afee nɔ ni haa mɔ tsui nyɔɔ emli fe CD4 + T wala yibii lɛ, loo akɛ CD4 + T wala yibii lɛ baanyɛ amɛkɛ carbon jɛɛhei krokomɛi ni jeee glucose atsu nii koni amɛkɛkwɛ mitochondrial nitsumɔ lɛ nɔ (43, 44). E sa nɛ wa kai kaa wa na we slɔɔto ko ngɛ glucose nɛ a ngɔɔ loo mitochondrial ní tsumi ɔ mi ngɛ CD4 + T ní tsumi he ní tsumi ɔ, T ní tsumi he ní tsumi ɔ he kaimi kɛ T nɛ ngɛ ascites ɔ mi ɔ mi. Nakai nɔŋŋ hu, srɔtofeemɔ ni yɔɔ CD8 + T wala yibii ni yɔɔ tsɛŋemɔ helai amli lɛ mli lɛ bɛ nɔ ko kwraa ni ekɛ tsakemɔi ni baa yɛ glucose ni anáa lɛ mli lɛ yɔɔ, ni emaa srɔtofeemɔ ni he hiaa ni yɔɔ T wala yibii ni akwɛɔ yɛ vitro mli kɛ gbɔmɔ TIL ni yɔɔ vivo mli lɛ teŋ lɛ nɔ mi (22). Akɛ nɛkɛ nibii nɛɛ hu ma nɔ mi kɛtsɔ tsɔne ni haa mɔ tsui nyɔɔ emli lɛ nɔ, ni ejie lɛ kpo ekoŋŋ akɛ CD45 + / CD3- / CD4 + / CD45RO + wala yibii ni glucose ni fa babaoo kɛ mitochondrial nitsumɔ fe tumor wala yibii lɛ fa babaoo shi amɛyɛ Metabolic active wala yibii ayibɔ. Nɛkɛ yibɔ nɛɛ baanyɛ adamɔ shi aha myeloid ni tsiɔ wala yibii anaa loo plasmacytoid dendritic wala yibii ni ana yɛ scRNA-seq mlipɛimɔ lɛ mli lɛ. Eyɛ mli akɛ abɔ enɛɛmɛi enyɔ lɛ fɛɛ he amaniɛ yɛ gbɔmɔ fɔmɔ kotoku mli tsɛŋemɔ helai amli [45] moŋ, shi ehe miihia amɛ lolo Nitsumɔ kroko ji ni atsɔɔ nɛkɛ myeloid subpopulation nɛɛ mli.
Eyɛ mli akɛ gbɛi ni damɔ flow cytometry nɔ lɛ baanyɛ aha srɔtofeemɔi ni yɔɔ glucose kɛ oxidative metabolism ni yɔɔ wala yibii lɛ ateŋ lɛ mli aka shi faŋŋ moŋ, shi atsɔɔɔ glucose loo carbon jɛɛhei krokomɛi kɛhaa mitochondrial metabolism yɛ TME mli lɛ mli lolo. Ni akɛ metabolites ni yɔɔ loo bɛ lɛ baaha TIL kuu bibioo ko lɛ biɔ ni atsuu wala yibii lɛ ayibɔ kɛjɛ fãji ni ajie lɛ mli. No hewɔ lɛ, wɔ tsɛŋemɔ helai ahe gbɛ ni atsɔɔ nɔ akɛ mass spectrometry fataa he lɛ baanyɛ aha wɔle nibii ni haa mɔ he jɔɔ lɛ ni yɔɔ T tsɛŋemɔ helai kɛ tsɛŋemɔ helai ahe nibii amli lɛ ayibɔ ni kɛ amɛhe kpãa gbee lɛ mli. Eyɛ mli akɛ gbɛ nɛɛ yɛ sɛɛnamɔi fe fluorescence-activated cell sorting moŋ, shi metabolite wojiatoohei komɛi baanyɛ aná amɛnɔ hewalɛ yɛ fɔmɔ mli shihilɛ ni yɔɔ shiŋŋ kɛ/loo oyaiyeli ni amɛtsakeɔ lɛ hewɔ (22). Shi kɛlɛ, wɔ gbɛ lɛ nyɛ eyoo nibii enyɔ ni haa mɔ he waaa ni akpɛlɛɔ nɔ, ni ji adenosine kɛ kynurenine, ejaakɛ amɛsoro amɛhe waa yɛ nɔkwɛmɔnɔ henɔi lɛ ateŋ.
Wɔ metabolinomic mlipɛimɔ ni kɔɔ tsɛŋemɔ helai kɛ TIL subtypes lɛ he lɛ haa wɔleɔ nibii babaoo ni kɔɔ tsɛŋemɔ helai ni yɔɔ fɔmɔ kotoku lɛ mli lɛ ahe. Klɛŋklɛŋ lɛ, wɔkɛ flow cytometry tsu nii ni wɔkɛna akɛ srɔtofeemɔ ko bɛ mitochondrial nitsumɔ mli yɛ tsɛŋemɔ helai kɛ CD4 + T wala yibii lɛ ateŋ. Shi, LC-MS/MS mlipɛimɔ lɛ jie tsakemɔi ni sa kadimɔ yɛ nibii ni haa mɔ he jɔɔ lɛ babaoo ni yɔɔ nɛkɛ gbɔmɛi nɛɛ ateŋ lɛ kpo, ni etsɔɔ akɛ, naagbee wiemɔi ni kɔɔ TIL nibii ni tsakeɔ yɛ gbɔmɔtso lɛ mli lɛ kɛ e-metabolic nitsumɔ lɛ fɛɛ he lɛ biɔ ni atsɔɔ shishi jogbaŋŋ. Nɔ ni ji enyɔ lɛ, MNA ji tsofa ni haa mɔ he jɔɔ lɛ ni srɔtofeemɔ kpele yɔɔ CD45-cells kɛ T cells ni yɔɔ ascites mli lɛ ateŋ, shi jeee tumors. No hewɔ lɛ, fãi ni ajara mli kɛ he ni tsɛŋemɔ hela lɛ yɔɔ lɛ baanyɛ aná TIL mli nibii ni tsakeɔ lɛ anɔ hewalɛ srɔtoi, ni no maa bɔ ni ebaanyɛ eba akɛ eyɛ srɔto yɛ shihilɛ bibioo ko mli lɛ nɔ mi. Nɔ ni ji etɛ lɛ, MNA-ni feɔ enzyme NNMT lɛ kpojiemɔ lɛ ji CAF pɛ, ni ji tsɛŋemɔ hela mli wala yibii lɛ yɛ gbɛ bibioo nɔ, shi anaa MNA ni anyɛɔ ayɔseɔ lɛ yɛ T tsɛŋemɔ hela mli wala yibii ni jɛɔ tsɛŋemɔ hela mli lɛ amli. NNMT ni ajieɔ lɛ kpo fe nine yɛ ovarian CAF mli lɛ yɛ kansa-he-shweremɔ he hewalɛ ni ale, ni fã ko jɛɔ CAF metabolism, tumor invasion kɛ metastasis (27) lɛ hewɔ. Eyɛ mli akɛ TIL lɛ fɛɛ falɛ ji nɔ ni sa moŋ, shi NNMT kpojiemɔ yɛ CAF mli lɛ kɛ Cancer Genome Atlas (TCGA) mesenchymal subtype lɛ bɛŋkɛ kpaakpa, ni ekɛ sɛɛtsɔɔmɔ ni ehiii baa (27, 46, 47). Yɛ naagbee lɛ, enzyme AOX1 ni haa MNA fiteɔ lɛ kpojiemɔ lɛ hu ji CAF gbɔmɛi ayibɔ lɛ pɛ, ni tsɔɔ akɛ T cells lɛ bɛ nyɛmɔ ni amɛkɛbaatsake MNA lɛ. Nɔ ni jɛ mli ba nɛɛ fiɔ susumɔ ni ji akɛ, eyɛ mli akɛ nitsumɔ kroko he miihia koni akɛma nɔ ni ana nɛɛ nɔ mi moŋ, shi MNA ni fa babaoo yɛ T wala yibii lɛ amli lɛ baanyɛ atsɔɔ akɛ CAF shihilɛ bibioo ko ni tsiɔ hela naa lɛ yɛ.
Kɛ akɛ MNA tsɔnei lɛ akpojiemɔ ni baa shi kɛ protein titrii ni fata MNA mli lɛ ayibɔ ni anyɛɛɛ ana lɛ to he lɛ, MNA ni yɔɔ T wala yibii lɛ amli lɛ ji nɔ ni akpaaa gbɛ. NNMT loo AOX1 lɛ eko kwraa nyɛɛɛ ana kɛtsɔ scRNA-seq mlipɛimɔ kɛ qPCR ni akɛtoɔ kui enyɔ ni yɔɔ amɛ diɛŋtsɛ amɛhe lɛ anɔ. Nɔ ni jɛ mli ba nɛɛ tsɔɔ akɛ jeee T cells lɛ ji mɛi ni feɔ MNA lɛ, shi moŋ ejɛɔ TME ni ebɔle ehe lɛ mli. Shikpamɔi ni afee yɛ vitro mli lɛ tsɔɔ akɛ T wala yibii lɛ sumɔɔ ni amɛbua MNA ni jɛɔ kpo lɛ naa.
Wɔ vitro nikasemɔi lɛ etsɔɔ akɛ exogenous MNA lɛ haa TNFα jieɔ ehe kpo yɛ T cells lɛ amli ni ehaa Sp1 kɛ TNFα promoter lɛ kpãa gbee lɛ yaa hiɛ. Eyɛ mli akɛ TNFα yɛ nitsumɔi ni teɔ shi ewoɔ tsɛŋemɔ hela kɛ tsɛŋemɔ hela ni gbeɔ mɔ lɛ fɛɛ moŋ, shi yɛ fɔmɔ kotoku mli kansa mli lɛ, TNFα baanyɛ aha fɔmɔ kotoku mli kansa lɛ aya hiɛ (31-33). TNFα ni ajieɔ kɛjɛɔ fɔmɔ kotoku mli lɛ mli loo TNFα okadi ni ajieɔ kɛjɛɔ fɔmɔ kotoku mli lɛ baanyɛ aha TNFα-mediated inflammatory cytokine feemɔ aya hiɛ ni etsi fɔmɔ kotoku lɛ dalɛ naa (32, 35). No hewɔ lɛ, yɛ sane nɛɛ mli lɛ, TME-ni jɛ MNA mli lɛ baanyɛ atsu nii akɛ pro-inflammatory metabolite kɛtsɔ TNFα-ni damɔ gbɛ nɔ kɛtsɔ autocrine loop lɛ nɔ, ni no haa kansa ni baa fɔmɔ kotoku mli lɛ baa ni egbɛɔ eshwaa (31). Yɛ nɛkɛ gbɛ ni baanyɛ aba nɛɛ nɔ lɛ, akaseɔ TNFα blockade lɛ he nii akɛ eji tsofa ni baanyɛ atsa fɔmɔ kotoku mli kansa (37, 48, 49). Kɛfata he lɛ, MNA lɛ fiteɔ CAR-T wala yibii lɛ amli wala yibii lɛ kɛyaa fɔmɔ kotoku mli wala yibii lɛ amli, ni ekɛ odaseyeli kroko haa kɛha MNA-mediated immune suppression. Kɛ akɛto he lɛ, nɛkɛ hetoi nɛɛ tsɔɔ nɔkwɛmɔnɔ ko ni tsɛŋemɔ helai kɛ CAF wala yibii lɛ jieɔ MNA kɛyaa TME ni yɔɔ wala yibii krokomɛi amli lɛ mli. Kɛtsɔ (i) TNF-ni haa fɔmɔ kotoku mli kansa lɛ yaa hiɛ lɛ nɔ kɛ (ii) MNA-ni haa T cell cytotoxic nitsumɔ ni tsiɔ naa lɛ nɔ lɛ, enɛ baanyɛ aná tsɛŋemɔ hela ni ji enyɔ (Mfoniri 5D).
Yɛ naagbee lɛ, kɛtsɔ tsɛŋemɔ helai ni yaa hiɛ oya, tsɛŋemɔ hela kome mli nibii ni ato he gbɛjianɔ kɛ nibii ni haa mɔ he waa lɛ ahe saji ni akɛtsuɔ nii lɛ nɔ lɛ, nikasemɔ nɛɛ jie srɔtofeemɔi wuji ni yɔɔ tsɛŋemɔ helai kɛ ascites tsɛŋemɔ helai ni yɔɔ HGSC helatsɛmɛi lɛ ateŋ lɛ kpo. Nɛkɛ mlipɛimɔ ni mli kwɔ nɛɛ tsɔɔ akɛ srɔtofeemɔi yɛ glucose ni anáa kɛ mitochondrial nitsumɔ mli yɛ T wala yibii lɛ ateŋ, ni etsɔɔ MNA akɛ eji nɔ ko ni jeee wala yibii ni yeɔ amɛhe nɔ ni haa gbɔmɔtso lɛ mli hewalɛ ni tsiɔ helai anaa lɛ tsuɔ nii. Nɛkɛ saji nɛɛ yɛ hewalɛ ko yɛ bɔ ni TME lɛ saa T cell metabolism he yɛ gbɔmɔtso mli kansa helai amli lɛ nɔ. Eyɛ mli akɛ abɔ akaŋshii ni yɔɔ T wala yibii kɛ kansa wala yibii ateŋ lɛ he amaniɛ moŋ, shi nibii ni tsakeɔ wala yibii lɛ hu baanyɛ atsu nii akɛ nɔ ni haa hela lɛ yaa hiɛ tɛ̃ɛ ni ekolɛ ebaatsi gbɔmɔtso lɛ mli nii ni tsiɔ hela naa lɛ anaa. Nɛkɛ nibii nɛɛ anitsumɔ mlitsɔɔmɔ kroko baanyɛ agbele gbɛi krokomɛi ni atsɔɔ nɔ ahaa gbɔmɔtso lɛ mli nii ni gbeɔ helai lɛ ahetoo lɛ yaa hiɛ.
Akɛ helatsɛmɛi ahe nɔkwɛmɔnii kɛ helatsamɔhe saji tsu nii kɛtsɔ BC kansa hela lɛ mli fãji ni akɛtoɔ he ni Canada fãji ni akɛtoɔ he lɛ he odaseyeli lɛ nɔ. Taakɛ gbɛjianɔtoo ni BC Kansa He Nibii Amli Taomɔ He Saji Ahe Ajinafoi Akuu lɛ kɛ University of British Columbia (H07-00463) lɛ kpɛlɛ nɔ lɛ tsɔɔ lɛ, helatsɛmɛi lɛ fɛɛ akwɛmɔi kɛ helatsamɔhe saji lɛ ná hegbɛ ni aŋmala ashwie shi loo amɛjie amɛkpɛlɛmɔ lɛ kɛjɛ mli yɛ gbɛ ni sa nɔ. Akɛ nɔkwɛmɔnii lɛ toɔ BioBank (BRC-00290) ni awo he odase lɛ mli. Atsɔɔ helatsɛ lɛ su fitsofitso yɛ Tables S1 kɛ S5. Kɛha cryopreservation lɛ, akɛ scalpel tsuɔ nii koni akɛtsɔ tsɔne nɔ afite helatsɛ lɛ fɔmɔ kotoku lɛ mli nɔkwɛmɔnɔ lɛ, ni no sɛɛ lɛ atsɔ 100-micron filter mli koni ana wala yibii kome ni akɛwoɔ mli lɛ. Akɛ helatsɛ lɛ ascites lɛ wo centrifuge mli yɛ 1500 rpm mli minitii 10 yɛ 4°C koni akɛfee pellet ni akɛjie nɔ ni yɔɔ nɔ lɛ. Akɛ wala yibii ni anáa kɛjɛɔ tsɛŋemɔ hela kɛ tsɛŋemɔ hela mli lɛ to gbɔmɔtso mli wala yibii ni hulutsoo bɛ mli lɛ mli 50% (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) kɛ 10% dimethyl sulfoxide mli. Akɛ nɛkɛ wala yibii kome ni ato he gbɛjianɔ nɛɛ shwie shi ni akɛtsu nii kɛha metabolomics kɛ metabolite yiŋkpɛɛ ni atsɔɔ mli yɛ shishigbɛ nɛɛ.
1640: AimV ni akɛfata he lɛ ji 0.22 μm ni atsɔ mli 50:50 ni akɛfata he lɛ. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) ni akɛ 10% ni haa mɔ he jɔɔ lɛ fataa he (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) Streptomycin (PenStrep) tsabaa (Thermo Fisher jeŋ nilee) kɛ 50 μMB-mercaptoethanol. Akɛ Hepes 20 mM (Tɛrmo Fisher Jeŋ Nilee) kɛ 2 mM l-glutamine (Tɛrmo Fisher Jeŋ Nilee) fataa AimV (Invitrogen) he. Nɔ ni yɔɔ tsɔne ni akɛkwɛɔ nibii amli lɛ mli ji 0.22μm ni akɛ tsofa ni ji phosphate ni ajie lɛ kpo (PBS; Invitrogen) ni akɛ 3% ni ji AB gbɔmɔtso mli lá ni haa mɔ he jɔɔ lɛ (Sigma) fata he. 0.22μm PBS ni atsɔ mli lɛ ji nɔ ni yɔɔ tsɛŋemɔ hela lɛ mli, ni akɛ 0.5% ni haa mɔ he jɔɔ lɛ AB serum (Sigma-Aldrich) fata he.
Yɛ 37°C mli lɛ, akɛ 10 nM MT DR kɛ 100 μM 2-NBDG wo wala yibii lɛ amli minitii 30. No sɛɛ lɛ, akɛ viability dye eF506 wo wala yibii lɛ amli yɛ 4°C mli minitii 15 sɔŋŋ. Akɛ FC Block (eBioscience) kɛ Brilliant Stain Buffer (BD Biosciences) lɛ mli tsɛŋemɔ helai lɛ awo mli ekoŋŋ, ni akɛwo flow cytometer staining buffer mli (tamɔ bɔ ni mɔ ni fee lɛ tsɔɔ lɛ naa), ni akɛwo mli minitii 10 yɛ shĩa mli latsaa mli. Akɛ tsofai ni gbeɔ helai (Table S2) lɛ awo tsɛŋemɔ helai ni haa mɔ he jɔɔ lɛ amli yɛ 4°C mli minitii 20. Akɛ tsɔne ni haa mɔ he jɔɔ lɛ (Cytek Aurora; 3L-16V-14B-8R gbɛjianɔtoo) lɛ woɔ wala yibii lɛ amli ekoŋŋ dani akwɛ mli. Kɛ SpectroFlo kɛ FlowJo V10 atsu nii ni okɛpɛi tsɛŋemɔ helai ahe saji amli, ni okɛ GraphPad Prism 8 afee saji lɛ. 2-NBDG kɛ MT DR lɛ mli la ni tsoɔ waa lɛ mli hewalɛ (MFI) lɛ ji log-normalized, ni no sɛɛ lɛ akɛ paired t test tsu nii kɛha akɔntaabuu mlipɛimɔ koni akɛbu helatsɛmɛi ni kɛ amɛhe kpãa gbee lɛ ahe akɔntaa. Jiemɔ gbɔmɛi ayibɔ fɛɛ ni nibii ni tee nɔ lɛ shɛɛɛ 40 kɛjɛ mlipɛimɔ lɛ mli; kɛ MFI sɛɛnamɔ ni ji 1 awo mli kɛha sɛɛnamɔi ni ehiii fɛɛ dani ofee akɔntaabuu mlipɛimɔ kɛ saji amlipɛimɔ.
Kɛha gbɛjianɔtoo ni yɔɔ ŋwɛi nɛɛ mli lɛ, wɔkɛ niŋmaa ni yɔɔ shape restriction tree (FAUST) (21) lɛ mli lɛ fɛɛ tsu nii koni wɔkɛ tsɛŋemɔ helai aha gbɔmɛi ayibɔ lɛ yɛ be mli ni ajie tsɛŋemɔ helai ni egboi yɛ FlowJo lɛ mli lɛ sɛɛ. Wɔkɛ wɔdɛŋ kwɛɔ nɔ ni jɛɔ mli baa lɛ nɔ koni wɔkɛ gbɔmɛi ayibɔ ni etamɔ nɔ ni ajieee amɛ kpo yɛ gbɛ ni ejaaa nɔ lɛ afata he (ni akɛ PD1+ kɛ PD1-tumor cells feɔ ekome) kɛ gbɔmɛi ayibɔ ni ahiɛ mli lɛ. Nɔkwɛmɔnɔ fɛɛ nɔkwɛmɔnɔ lɛ hiɛ wala yibii ni fa fe 2%, kɛha gbɔmɛi ayibɔi 11 fɛɛ.
Akɛ Ficoll gradient density centrifugation tsu nii koni akɛgbala PBMC kɛjɛ lá mli wala yibii amligbalamɔ nibii amli (STEMCELL Technologies). Akɛ CD8 + T cells lɛ je PBMC mli kɛtsɔ CD8 MicroBeads (Miltenyi) nɔ ni agbɛ ashwa yɛ gbɛ ni yeɔ emuu nɔ kɛtsɔ TransAct (Miltenyi) nɔ otsii enyɔ taakɛ mɔ ni fee lɛ tsɔɔ lɛ. Aŋmɛ gbɛ ni wala yibii lɛ damɔ shi gbii enumɔ yɛ tsɔne ni mli eyi obɔ ni IL-7 (10 ng/ml; PeproTech) yɔɔ mli lɛ mli, ni no sɛɛ lɛ akɛ TransAct lɛ tsirɛ amɛ ekoŋŋ. Yɛ gbi ni ji kpawo lɛ nɔ lɛ, taakɛ mɔ ni fee lɛ tsɔɔ lɛ, akɛ adesa CD45 MicroBeads (Miltenyi) tsu nii koni akɛha wala yibii lɛ aya hiɛ yɛ shii etɛ ni nyiɛ sɛɛ lɛ amli. Akɛ wala yibii lɛ wo mli kɛha flow cytometry mlipɛimɔ (tamɔ bɔ ni atsɔɔ mli yɛ yiteŋgbɛ lɛ), ni ajie wala yibii akpekpe kome mli shii etɛ kɛha LC-MS/MS mlipɛimɔ. Akɛ LC-MS/MS tsu nɔkwɛmɔnii lɛ ahe nii taakɛ atsɔɔ mli yɛ shishigbɛ lɛ. Wɔbu metabolite ni elaaje lɛ jara kɛ ion yibɔ ni ji 1,000 he akɔntaa. Nɔkwɛmɔnɔ fɛɛ nɔkwɛmɔnɔ lɛ, akɛ ion yibɔ fɛɛ (TIC) feɔ nɔ ni sa, ni atsakeɔ yɛ logarithm gbɛ nɔ ni ekɛtoɔ ehe yɛ MetaboAnalystR mli dani akwɛɔ mli.
Akɛ helatsɛ fɛɛ helatsɛ mli wala yibii kome ni akɛwoɔ mli lɛ shwie shi ni atsɔ 40 μm filter nɔ atsɔ mli kɛtee mli ni eye emuu (tamɔ bɔ ni atsɔɔ mli yɛ yiteŋgbɛ lɛ). Taakɛ nifeelɔi lɛ agbɛjianɔtoo lɛ tsɔɔ lɛ, akɛ halamɔ kpakpa ni nyiɛɔ sɛɛ etɛ kɛtsɔ magnetic bead mligbalamɔ ni akɛ MicroBeads (Miltenyi) tsuɔ nii lɛ nɔ lɛ tsu nii koni akɛha nɔkwɛmɔnii ni akɛhaa CD8+, CD4+ kɛ CD45- cells (ni yɔɔ ais nɔ lɛ) lɛ aya hiɛ. Yɛ kukufoo mli lɛ, ajieɔ wala yibii lɛ awoɔ wala yibii ni haa mɔ he waa lɛ mli ekoŋŋ (tamɔ bɔ ni atsɔɔ mli yɛ yiteŋgbɛ lɛ) ni akaneɔ amɛ. Akɛ gbɔmɔ CD8 ŋmeii, gbɔmɔ CD4 ŋmeii loo gbɔmɔ CD45 ŋmeii (Miltenyi) wo wala yibii lɛ amli yɛ 4°C mli minitii 15, ni no sɛɛ lɛ akɛ wala yibii lɛ ahe nibii ni haa mɔ tsui nyɔɔ emli lɛ tsuɔ he nii. Akɛ nɔkwɛmɔnɔ lɛ tsɔɔ LS ŋwɛitsu (Miltenyi) lɛ mli, ni abuaa fãi ni hi kɛ fãi ni ehiii lɛ anaa. Bɔni afee ni be ni akɛtsuɔ nii lɛ aba shi ni aha tsɛŋemɔ hela lɛ aya hiɛ babaoo lɛ, no sɛɛ lɛ akɛ CD8-fã lɛ tsuɔ nii kɛhaa CD4+ shweremɔ ni ji enyɔ lɛ, ni akɛ CD4-fã lɛ tsuɔ nii kɛhaa CD45-shweremɔ ni nyiɛ sɛɛ lɛ. Ha tsofa lɛ ahi ais nɔ yɛ mligbalamɔ be lɛ fɛɛ mli.
Kɛha nɔkwɛmɔnii ahe saamɔ kɛha metabolite mlipɛimɔ lɛ, akɛ ŋmei ni mli ejɔ tamɔ ais tsuɔ nii shikome, ni akɛ 80% methanol 1 ml fata nɔkwɛmɔnii lɛ eko fɛɛ eko he, no sɛɛ lɛ, atsɔ vortex nɔ ni akɛ snap frozen wo nitrogen ni ji nu mli. Akɛ nɔkwɛmɔnii lɛ wo fɛ̃i-fɛ̃i shii etɛ mli ni akɛwo 14,000 rpm mli minitii 15 yɛ 4°C. Akɛ nu ni yɔɔ ŋwɛi ni metabolites lɛ yɔɔ mli lɛ shwieɔ shi kɛyashiɔ be mli ni egbiɔ. Akɛ nibii ni tsakeɔ lɛ wo 50 μl ni ji 0.03% formic acid mli ekoŋŋ, ni atsɔ mli koni akɛfutu, ni no sɛɛ lɛ akɛwo tsɔne mli koni ajie sũ ni efite lɛ.
Jiemɔ nibii ni haa mɔ he jɔɔ lɛ tamɔ bɔ ni atsɔɔ mli yɛ yiteŋgbɛ lɛ. Tsɔɔmɔ nu ni hoɔ lɛ kɛya nu ni tsuɔ nii jogbaŋŋ ni akɛkwɛɔ nibii amli lɛ mli kɛha metabolomics niiamlitaomɔ. Kɛ random treatment protocol atsu nii ni okɛtsa nɔkwɛmɔnɔ fɛɛ nɔkwɛmɔnɔ kɛ tsɛŋemɔ helai ayibɔ ni tamɔ nakai nɔŋŋ koni akɛtsi batch nibii ni baa lɛ anaa. 10:10, 11. 10 Wɔfee jeŋ muu fɛɛ nibii ni tsakeɔ nibii ni yɔɔ gbɔmɔtso lɛ mli lɛ ahe niiamlipɛimɔ ko ni akala tsutsu yɛ AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50) lɛ nɔ. Akɛ MultiQuant 2.1 tsɔne ni akɛtsuɔ nii (Applied Biosystems SCIEX) lɛ tsu nii kɛpɛi ŋwɛi ŋta kɛ ŋwɛi ŋta ni akɛwoɔ mli lɛ mli.
Akɛ ion count ni ji 1000 tsu nii ni akɛbu metabolite ni elaaje lɛ he akɔntaa, ni akɛ TIC ni yɔɔ nɔkwɛmɔnɔ fɛɛ nɔkwɛmɔnɔ mli lɛ tsu nii ni akɛbu normalized peak area ni yɔɔ metabolite fɛɛ metabolite ni ana lɛ mli lɛ he akɔntaa koni akɛsaa tsakemɔi ni tsɔne mlipɛimɔ ni jɛ nɔkwɛmɔnɔ nitsumɔ mli lɛ kɛba lɛ. Yɛ be mli ni TIC lɛ ji nɔ ni sa lɛ sɛɛ lɛ, akɛ MetaboAnalystR(51) (paramita ni yɔɔ tsutsu lɛ) tsuɔ nii kɛhaa logarithmic tsakemɔ kɛ tsɔne ni haa mɔ tsui nyɔɔ emli lɛ he sɛkɛli. Wɔkɛ PCA kɛ vegan R paketi tsu nii koni wɔkɛfee niiamlipɛimɔ ni kɔɔ metabolome srɔtofeemɔi ni yɔɔ nɔkwɛmɔnɔ henɔi lɛ ateŋ lɛ he, ni wɔkɛ fã ko ni ji redundancy mlipɛimɔ tsu nii kɛpɛi helatsɛmɛi amli. Kɛ Ward gbɛ lɛ afee heat map dendrogram ni okɛbua Euclidean sɛɛjɛkɛmɔ ni yɔɔ nɔkwɛmɔnii lɛ ateŋ lɛ naa. Wɔkɛ limma (52) tsu nii yɛ metabolite babaoo ni ato he gbɛjianɔ lɛ nɔ koni wɔkɛyoo metabolites babaoo ni yɔɔ srɔtoi yɛ wala yibii lɛ fɛɛ kɛ shihilɛ mli nibii bibii lɛ amli. Bɔni afee ni shishitsɔɔmɔ lɛ afee mlɛo lɛ, wɔkɛ kuu lɛ mli shishitoo mla lɛ tsuɔ nii kɛtsɔɔ nɔkwɛmɔnɔ lɛ mli, ni wɔsusuɔ wala yibii henɔi ni yɔɔ shihilɛ bibioo lɛ mli lɛ ahe akɛ kuu fɛɛ kuu (n = kui 6); kɛha shishinumɔ he kaa lɛ, wɔfee susumɔi ni afɔɔ feemɔ shii etɛ kɛha metabolite lɛ eko fɛɛ eko Bɔni afee ni wɔtsi wɔhe kɛjɛ apasa sɛɛkuu he lɛ, akɛ helatsɛ lɛ fata he akɛ gbɛtsii nii ko yɛ limma lɛ he gbɛjianɔtoo lɛ mli. Bɔni afee ni wɔkwɛ srɔtofeemɔi ni yɔɔ metabolites ni yɔɔ helatsɛmɛi srɔtoi ateŋ lɛ mli lɛ, wɔtsake limma model ni helatsɛmɛi fata he lɛ yɛ gbɛ ni ato he gbɛjianɔ nɔ. Wɔbɔɔ shishinumɔ ni yɔɔ srɔtofeemɔ ni atsɔɔ mli momo lɛ he amaniɛ yɛ tsɛŋemɔ hela lɛ henɔ lɛ kɛ Padj <0.05 (Benjamini-Hochberg tsakemɔ) lɛ teŋ lɛ he amaniɛ.
Yɛ hewalɛ ni akɛ Miltenyi Dead Cell Removal Kit (>80% viability) tsu nii lɛ sɛɛ lɛ, afee tsɛŋemɔ hela kome mli nibii ni aŋmala ashwie shi lɛ ahe mfoniri yɛ 10x 5′gene kpojiemɔ gbɛjianɔtoo lɛ nɔ. Akwɛ helai enumɔ ni yɔɔ tsɛŋemɔ helai kɛ ascites ni kɛ amɛhe kpãa gbee lɛ amli, eyɛ mli akɛ bɔ ni tsɛŋemɔ hela kome mli tsɛŋemɔ hela lɛ mli tsɛŋemɔ hela lɛ he nɔkwɛmɔnɔ ni baa shi lɛ tsi enaa akɛ akɛwo mli moŋ. Bɔni afee ni wɔnine ashɛ helatsɛmɛi babaoo ni wɔhala lɛ anɔ lɛ, wɔkɛ helatsɛ fɛɛ helatsɛ nɔkwɛmɔnii lɛ fata he yɛ 10x chromium controller lɛ gbɛi lɛ anɔ, ni wɔpɛi ascites kɛ tumor hei lɛ amli srɔtoi. Beni ato amɛhe gbɛjianɔ sɛɛ lɛ, [Illumina HiSeq 4000 28×98 bp ni ji enyɔ naagbee (PE), Quebec tsɛŋemɔ hela lɛ; 73,488 kɛ 41,378 kaneɔ yɛ wala yibii fɛɛ mli kɛha tsɛŋemɔ hela kɛ ascites]], wɔkɛ CellSNP kɛ Vireo tsu nii (53) (ni damɔ CellSNP nɔ akɛ Adesa SNP (VCF) ni afɔɔ namɔ ni GRCh38 kɛhaa lɛ ji nɔ ni akɛhaa mɔ ni kɛ ehe haa lɛ he okadi (wɔkɛ SNPRBSI helatsɛ lɛ he okadi ni ji close's lɛ tsuɔ nii) (IBS), ni tsɛŋemɔ helai ni akɛhako amɛ kɛ tsɛŋemɔ helai ni ayɔse akɛ amɛji duplexes kɛ mɛi ni kɛ amɛhe kpãa gbee yɛ ascites kɛ tsɛŋemɔ helai ahe nɔkwɛmɔnii lɛ ateŋ lɛ fataaa he (54) Yɛ nitsumɔ nɛɛ nɔ lɛ, wɔhiɛ saji etɛ ni tsɛŋemɔ helai babaoo damɔ shi kɛha tsɛŋemɔ hela lɛ mli kɛha shishigbɛ mlipɛimɔ (BiCoran filtration step yɛ) 6975 tsɛŋemɔ helai (2792 kɛ 4183 tsɛŋemɔ helai kɛjɛ tsɛŋemɔ hela kɛ ascites, yɛ gbɛ kome nɔ) kɛha mlipɛimɔ ni akɛ t-SNE kwɛɔ lɛ ji nɔ ni atsɔɔ mli kɛtsɔ CD8A kɛ GZMA lɛ kpojiemɔ nɔ, ni subclusters ni yɔɔ ribosomal protein kpojiemɔ ni baa shi lɛ fataaa he Wɔnine shɛ saji ni akala ni Izar kɛ ekrokomɛi (16) lɛ jieɔ lɛ kpo lɛ nɔ, ni amɛ t-SNE ni akɛwoɔ mli lɛ fata he lɛ, baanyɛ akudɔ kpojiemɔ ni tsɔɔ marNN cell lɛ kpojiemɔ lɛ.
Akɛ Ficoll tsɔne ni akɛjieɔ lá mli wala yibii lɛ mligbalamɔ nibii (STEMCELL Technologies) lɛ gbala PBMC mli. Akɛ CD3 + tsɛŋemɔ helai (Miltenyi) lɛ tsu nii ni akɛjie CD3 + tsɛŋemɔ helai kɛjɛ PBMC mli. Yɛ MNA ni yɔɔ loo bɛ lɛ mli lɛ, akɛ CD3+ ni akɛwoɔ plɛte mli lɛ tsuɔ nii kɛ CD3 (5μg/ml), CD28 ni mli tse (3μg/ml) kɛ IL-2 (300 U/ml; Proleukin) lɛ. Yɛ naagbee gbi ni akɛgbɛɔ eshwaa lɛ nɔ lɛ, akwɛ bɔ ni ebaanyɛ ehi shi eha lɛ mli kɛtsɔ flow cytometry nɔ. ng/ml kɛ ionomycin (1μg/ml) kɛ GolgiStop ŋmɛlɛtswai ejwɛ, ni okwɛ CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4), BioLegend) kɛ TNForethicein (MAb 11, BD). ng/ml kɛ ionomycin (1μg/ml) tsirɛɔ qPCR kɛ ChIP wala yibii lɛ ŋmɛlɛtswai ejwɛ. Akɛ ELISA mli nu ni yɔɔ ŋwɛi lɛ bua naa dani akɛ PMA (20 ng/ml) kɛ ionomycin (1 μg/ml) woɔ mɔ mli ŋmɛlɛtswai 4 kɛ sɛɛ mli.
Nyiɛmɔ mɔ ni fee lɛ gbɛjianɔtoo lɛ sɛɛ koni okɛ RNeasy Plus Mini Kit (QIAGEN) lɛ atsu nii ni okɛjie RNA lɛ kɛjɛ mli. Kɛ QIAGEN (QIAGEN) afee nɔkwɛmɔnɔ lɛ ekome. Kɛ RNA ni he wa waa kɛ cDNA kit (Thermo Fisher Scientific) atsu nii ni okɛfee DNA ni yeɔ ebuaa (cDNA) lɛ. Kɛ TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) atsu nii ni okɛtsɔɔ bɔ ni wala yibii lɛ jieɔ amɛhe kpo amɛhaa (tamɔ bɔ ni mɔ ni fee lɛ tsɔɔ lɛ) kɛ nibii ni nyiɛ sɛɛ nɛɛ: Hs02786624_g1 [glyceraldehyde-3-phosphate ni jɛɔ nu mli (GAPDH)] kɛ Hs01010726_m1 (SLC22A2). Akɛ nɔkwɛmɔnii lɛ tsu nii yɛ StepOnePlus be-mli PCR gbɛjianɔtoo lɛ nɔ (nibii ni wala yɔɔ mli ni akɛtsuɔ nii) (nibii ni wala yɔɔ mli ni akɛtsuɔ nii) yɛ MicroAmp oyaiyeli ni haa hulu kpɛɔ 96-well reaction plate (Nibii ni wala yɔɔ mli ni akɛtsuɔ nii) kɛ MicroAmp ŋmɛlɛtswaa sini. Abuɔ Ct sɛɛnamɔ fɛɛ sɛɛnamɔ ni fe 35 lɛ akɛ eshɛɔ nɔ ni anaa lɛ he, ni akadiɔ lɛ akɛ anyɛɛɛ ana.
Feemɔ ChIP taakɛ atsɔɔ mli kɛtsɔ hiɛ lɛ (58). Yɛ kukufoo mli lɛ, akɛ formaldehyde (naagbee 1.42%) tsu wala yibii lɛ ahe nii, ni akɛwo ŋwɛi fɛ̃i mli minitii 10. mM Hepes, 1.5 mM MgCl2, 10 mM KCl kɛ 0.1% NP-40) yɛ ais nɔ minitii 10, no sɛɛ lɛ, okɛwo tsofa ni haa mɔ he waa lɛ mli ekoŋŋ taakɛ atsɔɔ mli lɛ (58 ). No sɛɛ lɛ, akɛ sɛɛkuu-kɛsaramɔi ni nyiɛ sɛɛ nɛɛ tsu nɔkwɛmɔnɔ lɛ he nii: sɛɛkuu-kɛsaramɔi 10 (20 1-second pulses) kɛ be ni bɛ tsakemɔ ni ji sɛkɛndii 40. Akɛ ChIP-grade immunoglobulin G (Tɛknɔlɔji ni haa mɔ he waaa; 1μl), histone H3 (Tɛknɔlɔji ni akɛtsɔɔ wala yibii ahe okadi; ​​3μl), NFAT (Invitrogen; 3μl) kɛ SP1 (Tɛknɔlɔji ni akɛtsɔɔ wala yibii amli; 3μl) nibii ni gbeɔ hela lɛ awo mli yɛ nɔkwɛmɔnɔ ni ji sha°4night nɔ. Akɛ protein A ŋmeii (Thermo Fisher Scientific) lɛ awo nɔkwɛmɔnɔ lɛ mli yɛ 4°C kɛ ŋmɔlɔ blɛoo ŋmɛlɛtswaa kome, no sɛɛ lɛ akɛ chelex ŋmeii (Bio-Rad) atsu nii ni akɛha DNA lɛ aya hiɛ, ni akɛ proteinase K (Thermo Fisher) atsu nii kɛha protein ni shwieɔ shi lɛ. TNFα sɛɛnyiɛlɔ lɛ ji nɔ ni ana kɛtsɔ PCR nɔ: hiɛ, GGG TAT CCT TGA TGC TTG TGT; nɔ ni tamɔɔɔ enɛ lɛ, GTG CCA ACA NYƆŊMƆ GCC TTT ATA TG (207-bp nibii). Image Lab (Bio-Rad) ji mɔ ni fee mfonirii lɛ ni akɛ ImageJ tsɔne lɛ tsu nii kɛsusu bɔ ni amɛyi fa ha lɛ he.
Abua tsɛŋemɔ helai ahe nibii ni yɔɔ ŋwɛi lɛ anaa taakɛ atsɔɔ mli yɛ yiteŋgbɛ lɛ. Akɛ yiŋkpɛɛ lɛ tsu nii yɛ gbɛ ni mɛi ni fee lɛ tsɔɔ nɔ amɛfeɔ gbɔmɔtso lɛ TNFα ELISA kit (Invitrogen), gbɔmɔtso IL-2 ELISA kit (Invitrogen) kɛ gbɔmɔtso IFN-γ ELISA kit (Abcam) lɛ nɔ. Taakɛ mɔ ni fee lɛ gbɛjianɔtoo lɛ tsɔɔ lɛ, akɛ nu ni hoɔ yɛ nu lɛ mli lɛ woɔ mli 1:100 koni ana TNFα kɛ IL-2, kɛ 1:3 koni ana IFN-γ. 2104 Multilabel Kanemɔ (PerkinElmer) lɛ atsu nii ni okɛsusu bɔ ni hulutsoo be lɛ shɛɔ 450 nm lɛ he.
Akɛ Ficoll tsɔne ni akɛjieɔ lá mli wala yibii lɛ mligbalamɔ nibii (STEMCELL Technologies) lɛ gbala PBMC mli. Akɛ CD3 + tsɛŋemɔ helai (Miltenyi) lɛ tsu nii ni akɛjie CD3 + tsɛŋemɔ helai kɛjɛ PBMC mli. Yɛ MNA ni yɔɔ loo ebɛ mli lɛ mli lɛ, akɛ CD3+ ni akɛwoɔ plɛte mli lɛ tsuɔ nii kɛ CD3 (5μg/ml), CD28 ni mli tse (3μg/ml) kɛ IL-2 (300 U/ml; Proleukin) gbii 3 sɔŋŋ. Yɛ gbii 3 sɛɛ lɛ, abua tsɛŋemɔ helai lɛ anaa ni akɛ 0.9% saline tsu he, ni akɛ pellet lɛ wo fɛ̃i mli oya. Akɛ 123count eBeads tsu nii kɛkane wala yibii lɛ kɛtsɔ tsɔne ni akɛkwɛɔ wala yibii lɛ anɔ lɛ nɔ.
Jiemɔ nibii ni haa mɔ he jɔɔ lɛ tamɔ bɔ ni atsɔɔ mli yɛ yiteŋgbɛ lɛ. Akɛ nu ni egbi lɛ fee ekoŋŋ yɛ 4000 cell equivalents/μl mli. 1290 Infinity II, Agilent Technologies, Santa Clara, CA) kɛ CORTECS T3 ŋwɛi ŋta (2.1 × 150 mm, fãji bibii lɛ dalɛ 1.6-μm, ŋtayaa lɛ dalɛ 120-Å; #186008500). Polar mass spektromita (6470, Agilent), ni tsɔne ni akɛshwieɔ nu mli lɛ tsuɔ nii yɛ gbɛ ni hi nɔ. Mobil fã A ji 0.1% fɔmik asid (yɛ H2O mli), mobile fã B ji 90% asetonitrile, 0.1% fɔmik asid. LC gradient lɛ ji minitii 0 kɛyashi 2 kɛha 100% A, minitii 2 kɛyashi 7.1 kɛha 99% B, kɛ minitii 7.1 kɛyashi 8 kɛha 99% B. No sɛɛ lɛ, okɛ mobile phase A lɛ afee ekome ekoŋŋ yɛ 0.6 ml/min ni hoɔ oya lɛ mli kɛha minitii 3. . Nibii ni hoɔ lɛ falɛ ji 0.4ml/min, ni akɛwoɔ ŋwɛitsu lɛ mli la kɛyashɛɔ 50°C. Kɛ MNA tsofafeemɔ he mla krɔŋŋ (M320995, Toronto Nibii Amlipɛimɔ Tsofafeemɔ Nitsumɔhe, North York, Ontario, Canada) lɛ atsu nii ni okɛto be ni akɛhiɛɔ nii amli (RT) kɛ tsakemɔ (RT = minitii 0.882, tsakemɔ 1 = 137→94.1, tsakemɔ 2 = 137, tsakemɔ→372) lɛ he gbɛjianɔ. Kɛji tsakemɔi etɛ lɛ fɛɛ ba yɛ be ni sa ni akɛhiɛɔ mli lɛ mli lɛ, akɛ tsakemɔ 1 lɛ tsuɔ nii kɛhaa yibɔ ni akɛbaakwɛ koni ana nɔ ni yɔɔ pɔtɛɛ. 1.1, 1.0, 10 kɛ 100 ng/ml kɛ 1.0 kɛ 10μg/ml ni ji nu. ng/ml ji nɔ ni anaa, ni linear hetoo lɛ yɛ 10 ng/ml kɛ 10μg/ml teŋ. Akɛ nɔkwɛmɔnɔ kɛ shishitoo mla ni ji microliters enyɔ ni akɛwoɔ mɔ mli lɛ eko fɛɛ eko tsuɔ nii kɛpɛiɔ LC/MS mli, ni akɛ nɔkwɛmɔnɔ ni akɛfutuɔ nibii ni hi jogbaŋŋ lɛ tsuɔ nii daa ekpaa mli koni ana nɔmimaa akɛ mlipɛimɔ gbɛjianɔtoo lɛ damɔ shi shiŋŋ. MNA hetooi ni jɛ MNA-ni akɛtsaa wala yibii lɛ fɛɛ mli lɛ yɛ linear range ni yɔɔ assay lɛ mli. Akɛ MassHunter nibii amlipɛimɔ tsɔne (v9.0, Agilent) tsu nii kɛpɛi saji amli.
Ajie yinɔ ni ji enyɔ αFR-CAR tsu lɛ kɛjɛ Song kɛ ekrokomɛi adɛŋ. (59). Yɛ kukufoo mli lɛ, nibii ni nyiɛ sɛɛ nɛɛ yɔɔ tsu lɛ mli: CD8a hiɛnyiɛlɔ sɛɛkuu, gbɔmɔ αFR-pɔtɛɛ ni yɔɔ ŋwɛi ŋta kome mli lɛ mli fã, CD8a ŋta kɛ transmembrane kpokpaa, CD27 ŋta ni yɔɔ wala yibii amli kɛ CD3z ŋta ni yɔɔ wala yibii amli. Akɛ GenScript fee CAR lɛ fɛɛ, ni no sɛɛ lɛ afee lɛ tamɔ lentiviral kpojiemɔ vector ni yɔɔ yinɔ ni ji enyɔ lɛ mli yɛ GFP kpojiemɔ kasɛt ni akɛtsuɔ nii koni akɛpɛi bɔ ni etsɔɔ mli jogbaŋŋ lɛ mli.
Akɛ HEK293T wala yibii [Amerikabii Akusum Nifeemɔi Ahe Nitsumɔhe (ATCC); 10% fɔmɔ kotoku mli lá (FBS) kɛ 1% PenStrep yɔɔ mli, ni akɛ CAR-GFP vector kɛ Plasmids ni akɛwoɔ mli lɛ (psPAX2 kɛ pMD2.G, Addgene) kɛ lipofection amine (Sigma-Aldrich) tsu nii. Abua nu ni yɔɔ mli lɛ naa yɛ ŋmɛlɛtswai 48 kɛ 72 sɛɛ beni ajie hela lɛ mli lɛ, ajie mli, ni akɛ ultracentrifugation tsu nii ni akɛwo mli. Kɛ hela ni haa mɔ he jɔ̃ɔ ehe lɛ ato -80°C kɛyashi be mli ni abaajie lɛ kpo.
Akɛ Ficoll tsɔne ni akɛjieɔ lá mli wala yibii ni haa mɔ he waa lɛ mligbalamɔ nibii (STEMCELL Technologies) lɛ mli gbalaa PBMC kɛjɛɔ mli. Kɛ CD8 nibii bibii (Miltenyi) ni ji halamɔ kpakpa lɛ atsu nii ni okɛjie CD8+ wala yibii kɛjɛ PBMC mli. Akɛ TransAct (Miltenyi) kɛ TexMACS mli [Miltenyi; ni akɛ 3% ni hulutsoo bɛ mli lɛ fata he, 1% PenStrep kɛ IL-2 (300 U/ml)]. Beni ajie ŋmɛlɛtswai nyɔŋmai enyɔ kɛ ejwɛ sɛɛ lɛ, akɛ lentivirus (10 μl ni mli eyi obɔ kɛ virus supernatant yɛ wala yibii 106 mli) tsake T wala yibii lɛ. 1 kɛyashi 3 gbii sɛɛ beni atsake yɛ Cytek Aurora nɔ (yɛ FSC (Hiɛyaa Scatter)/SSC (Side Scatter), Singlet, GFP+), kwɛmɔ GFP kpojiemɔ ni yɔɔ wala yibii lɛ amli lɛ mli koni otsɔɔ tsakemɔ nitsumɔ ni kɛ hoo lɛ 30% .
Akɛ ŋmɛlɛtswai 24 wo Immunocult (STEMCELL Technologies; ni akɛ 1% PenStrep fata he) mli yɛ shihilɛi ni nyiɛ sɛɛ nɛɛ amli: atsaaa, akɛ 250 μM adenosine loo 10 mM MNA tsu nii. Beni atsu CAR-T wala yibii lɛ ahe nii dani agbe naa lɛ, akɛ PBS tsu nii ni akɛ SK-OV-3 wala yibii 20,000 [ATCC; 10% FBS kɛ 1% PenStrep fata he yɛ McCoy 5A mli yɛ McCoy 5A mli (Sigma-Aldrich) ni akɛfata he yɛ 10: Akɛ nɔ ni tsuɔ nii kɛyaa otii lɛ mlijaa 1 lɛ tee hiɛ yɛ shii etɛ mli yɛ Immunocult mli ni akɛfata he lɛ mli. Akɛ SK-OV-3 tsɛŋemɔ helai kɛ SK-OV-3 tsɛŋemɔ helai ni akɛ digitalis saponin (0.5mg/ml; Sigma-Aldrich) shwie nɔ lɛ tsu nii akɛ nɔ ni ehiii kɛ nɔ ni hi, yɛ gbɛ kome nɔ. Yɛ ŋmɛlɛtswai 24 ni akɛfee ekome kɛtsu nii lɛ sɛɛ lɛ, abua nu ni hoɔ lɛ naa ni asusu lactate dehydrogenase (LDH) lɛ he yɛ bɔ ni mɔ ni fee lɛ tsɔɔ lɛ naa (LDH Glo Cytotoxicity Assay Kit, Promega). Akɛ LDH ni yɔɔ ŋwɛi lɛ wo LDH buffer mli 1:50. Akɛ fɔmula ni nyiɛ sɛɛ nɛɛ susu gbɔmɔgbee mlijaa oha mlijaa lɛ he: gbɔmɔgbee mlijaa oha mlijaa = jajemɔ mlijaa oha mlijaa / gbɔmɔgbee ni fa fe fɛɛ x 100%, he ni jajemɔ mlijaa oha mlijaa = co-culture-T cells pɛ, kɛ gbɔmɔgbee mlijaa oha mlijaa = positive control-negative control.
Taakɛ atsɔɔ mli yɛ niŋmaa loo nibii kɛ gbɛi lɛ amli lɛ, kɛ GraphPad Prism 8, Microsoft Excel loo R v3.6.0 atsu nii kɛha akɔntaabuu mlipɛimɔ. Kɛji akɛ abua nɔkwɛmɔnii babaoo anaa kɛjɛ helatsɛ kome mli (tamɔ ascites kɛ tumor), wɔkɛ paired t test tsuɔ nii loo wɔkɛ helatsɛ lɛ fataa he akɛ nɔ ko ni baa trukaa yɛ linear loo generalized model mli bɔ ni sa. Kɛha metabolomics mlipɛimɔ lɛ, afeɔ hiɛdɔɔ kaa lɛ yɛ shii etɛ mli.
Kɛji ootao nibii krokomɛi ni fata he kɛha niŋmaa nɛɛ, ofainɛ kwɛmɔ http://advances.sciencemag.org/cgi/content/full/7/4/eabe1174/DC1.
Enɛ ji niŋmaa ko ni anyɛɔ ashɛɔ he ni ajaa yɛ Creative Commons Attribution-Non-Commercial License lɛ mlai lɛ ashishi, ni ŋmɛɔ gbɛ akɛ akɛtsuɔ nii, ajaa ni afee ekoŋŋ yɛ gbɛ fɛɛ gbɛ nɔ, kɛji akɛ naagbee nitsumɔ lɛ jeee kɛha jarayeli sɛɛnamɔ ni nɔ ni yɔɔ mli ji akɛ shishijee nitsumɔ lɛ ja. Kaimɔ nii.
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Marisa K. Kilgour (Marisa K. Kilgour), Sara MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Jones (Brad H. Nelson), Ralph J. De Bell (Rusel G. Jɔɔns), Finias T. Hamiltɔn (Finias T.
MNA yeɔ ebuaa ni gbɔmɔtso lɛ mli nii ni tsiɔ hela naa lɛ tsiɔ T wala yibii lɛ anaa, ni edamɔ shi kɛha gbɔmɔtso lɛ mli nii ni tsiɔ hela naa lɛ ni abaanyɛ atsu he nii kɛtsa gbɔmɔtso lɛ kansa.
Marisa K. Kilgour (Marisa K. Kilgour), Sara MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Jones (Brad H. Nelson), Ralph J. De Bell (Rusel G. Jɔɔns), Finias T. Hamiltɔn (Finias T.
MNA yeɔ ebuaa ni gbɔmɔtso lɛ mli nii ni tsiɔ hela naa lɛ tsiɔ T wala yibii lɛ anaa, ni edamɔ shi kɛha gbɔmɔtso lɛ mli nii ni tsiɔ hela naa lɛ ni abaanyɛ atsu he nii kɛtsa gbɔmɔtso lɛ kansa.
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