Immunomodulatory metabolites nchintu chipati mubusena bwazilonda (TME), pesi kunze lyazimwi, zyina lyazyo tazizyibidwe pe. Aawa, twakavwuntauzya zilonda amaseelo aa T kuzwa kuzilonda a ascites zyabalwazi bajisi bulwazi bwakkansa ya serous carcinoma (HGSC) kutegwa tuzyibe metabolome yazibeela eezyi zya TME ziindene. Ascites amaselu aazilonda alijisi kwiimpana kupati kwa metabolite. Kweezyanisya a ascites, maselu aa T aanjila muzilonda alivwubide kapati mu 1-methylnicotinamide (MNA). Nikuba kuti mweelwe wa MNA mumaseelo aa T ulisumpukide, kutondezya kwa nicotinamide N-methyltransferase (insayimu iipa kuti tubunga twa methyl tuzwe kuzwa ku S-adenosylmethionine kuya ku nicotinamide) kuli buyo kuma fibroblasts amaseelo aajisi zilonda. Mukubeleka, MNA ilasungwaazya maselu aa T kuti agwisye cytokine isumpula zilonda zipa kuti zilonda zifwe. Aboobo, TME-yakazwa ku MNA ilagwasya mukulanganya malwazi aa T cells mpawo yiiminina nzila iikonzya kugwasya kusilika kkansa yabantu.
Ma metabolites azwa kuzilonda alakonzya kuba aanguzu zyakulesya malwazi aakulwana zilonda, alimwi bumboni bunji butondezya kuti alakonzya kubeleka mbuli nguzu zipati zyakusololela malwazi (1). Mukuyungizya ku Warburg effect, mulimu walino-lino watalika kuziba chiimo chakusanduka kwamaseelo aamubulowa akujatana kwawo abukkale bwakukwabilila mubili bwamubulowa (TME). Ziiyo zyakachitilwa mbeba amaseelo aa T aabantu zyakatondezya kuti kusanduka kwa glutamine (2), kusanduka kwa oxidative (3) antoomwe akusanduka kwa glucose (4) kulakonzya kubeleka kakunyina kweendelezya maseelo aakwabilila mubili. Zibeela zili mbozibede munzila eezyi zilalesya mulimo wa T cells wakulwana zilonda. Kwakasinizyigwa kuti kusinkililwa kwa coenzyme tetrahydrobiopterin (BH4) kulakonzya kunyonganya kuvwula kwamaseelo aa T, mpawo kuyungizigwa kwa BH4 mumubili kulakonzya kuyungizya nguzu zyakukwabilila mubili kulwana zilonda zyeendelezyegwa a CD4 a CD8. Mukuyungizya, nguzu zyakukwabilila mubili zya kynurenine zilakonzya kuvwunwa kwiinda mukubelesya BH4 (5). Mu isocitrate dehydrogenase (IDH) mutant glioblastoma, kuzwa kwa enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) kulesya T cell activation, proliferation and cytolysis Activity (6). Ino-ino, kwakatondezyegwa kuti methylglyoxal, chintu chizwa mu glycolysis, chipangwa amaseelo aazwa ku myeloid, mpawo T cell transfer ya methylglyoxal ilakonzya kulesya effector T cell. Mukusilikwa, kugwisya methylglyoxal kulakonzya kuzunda milimo ya myeloid-derived suppressor cells (MDSC) mpawo akuyungizya busilisi bwakulesya kulanga mu mbeba (7). Ziiyo eezyi antoomwe zikankaizya mulimo mupati wa TME-derived metabolites mukulanganya mulimo wa T cell.
Kutabeleka kabotu kwa T cell kwakaambwa kapati mu kkansa ya ovarian (8). Eezi zilachitika akaambo kazyintu zichitwa mumubili zili mukubula muuya mubotu antoomwe akubula nguzu kwamitsinga yazilonda (9), eezyo zipa kuti glucose a tryptophan zisanduke kuba zintu zimwi zilimbuli lactic acid a kynurenine. Kuvwula kwa lactate kunze lyamaseelo kulacesya kupanga kwa interferon-γ (IFN-γ) mpawo kupa kuti kube kwaandaana kwa tubunga twa myelosuppressive (10, 11). Kunywa musamu wa tryptophan kulesya kuvwula kwamaseelo aa T mpawo kulesya zitondezyo zya T cell (12-14). Nikuba kuti kuli eezyi zintu zyabonwa, mulimu mupati uujatikizya nzila zyakukwabilila mubili wakachitilwa mukukomezya maselu aa T kubelesya nzila ziluzi, naakuti wakabikkilwa buyo mbeba zikozyenye mumubili, kunyina akati kazeezi zitondezya kusiyana kwamalwazi aakkansa aabantu alimwi abusena bupati abuniini bwamubili.
Chimwi chintu chidumide ku kkansa yamumapunga nkumwaika kwamubili mpawo akuboneka kwa ascites. Kuyoboloka kwamaanzi aamaseelo mu ascites kulaswaangene abulwazi bupati akubula bulangilizi (15). Kweelana amalipooti, ​​eechi chibeela chilibedelede tachijisi muya mubotu pe, chili aama vascular endothelial growth factor (VEGF) antoomwe a indoleamine 2,3-dioxygenase (IDO), mpo chilinjidwe a T regulatory cells antoomwe a myeloid inhibitory cells (15-18). Busena bwakusanduka kwamubili bwa ascites bulakonzya kuba bwiindene abwachilonda, aboobo kuchincha kwamaseelo aa T muchibaka chamukati takulibonyi kabotu pe. Mukuyungizya, kwiimpana kupati akusiyana aakati ka ascites a metabolites ziliwo mubusena bwazilonda zilakonzya kulesya kunjila kwamaseelo aakwabilila mubili akubeleka kwawo kuzilonda, mpawo buvwuntauzi bumbi buyandika.
Kuti tumanizye mapenzi aaya, twakabamba nzila yakugwisya maselu aasalala antoomwe anzila yakupima bunji bwamaanzi (LC-MS/MS) kuti twiiye maseelo aasiyene (kubikkilizya a CD4 + a CD8 + T maselu) antoomwe mukati aakati kamatumbu. Tubelesya nzila eeyi antoomwe a high-dimensional flow cytometry antoomwe a single-cell RNA sequencing (scRNA-seq) kuti tupe chikozyano chilikabotu chabukkale bwabantu aaba. Eeyi nzila yakatondeezya kuyungizigwa kupati kwa 1-methylnicotinamide (MNA) mumaseelo aa T aajisi zilonda, mpawo mukusola kwa in vitro kwakatondezya kuti mulimo wa MNA wakukwabilila mubili kumaseelo aa T tiiwakazyibidwe pe. Muchiindi choonse, eeyi nzila ilayubununa kujatana aakati kamatumbu amaseelo aakwabilila mubili, mpawo ipa luzibo lulibedelede kujatikizya maseelo aakwabilila mubili, aakonzya kugwasya kusilika kkansa ya T cell-based ovarian immunotherapy.
Twakabelesya high-dimensional flow cytometry kuchitila kuti tukonzye kupima glucose uptake [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) antoomwe a mitochondrial activity [MitoTracker Deep Red (MT DR) (MT DR, 2017, 2019) zili kumbali zitondezyo zizibilikide zipa kwaandaanya maseelo aakwabilila mubili amaseelo aajisi zilonda (Table S2 a Figure S1A). Ooku kuvwuntauzya kwakatondezya kuti kweezyanisya amaseelo aa T, maseelo aa ascites alimwi amaseelo aajisi bulwazi bwa tumor alaa mweelwe mupati wa glucose, pele alijisi kwiindana kusyoonto mumilimo ya mitochondrial. Mweelwe waakati-kati wa glucose uunjila mumaseelo aacilonda [CD45-EpCAM (EpCAM)+] ulainda ziindi zyotatwe kusikila kuli zyone kwiinda wamaseelo aa T, mpawo mweelwe waakati-kati wa glucose uunjila mumaseelo aa CD4 + T ulainda ziindi zili 1.2 kwiinda wamaseelo aa CD8 + T, eezi zitondezya kuti ma Tumor infiltrating lymphocytes (TIL) ajisi ziyandikana zyaandeene mumubili nokuba muchifanikisyo chomwe cha TAME (Mufanikisyo 1). Mukwiimpana, mulimo wa mitochondrial mumaseelo aacilonda ulikozyenye a CD4 + T cells, alimwi mulimo wa mitochondrial wamaseelo oonse obile ulisumpukide kwiinda wa CD8 + T cells (Chitondeezyo 1B). Muchiindi choonse, eezyi mpindu zitondezya mweelwe wa metabolic. Ikubeleka kwamaseelo aamubulowa kulisumpukide kwiinda kwamaseelo aa CD4 + T, mpawo kubeleka kwamaseelo aa CD4 + T kulisumpukide kwiinda kwamaseelo aa CD8 + T. Nikuba kuti kuli zyintu eezyi zicitika akati kamisyobo yamaseelo, taakwe kwiimpana kuliko akati kamaseelo aa CD4 + a CD8 + T naa mweelwe wawo mu ascites kweelanisigwa azilonda (Chitondeezyo 1C). Mukwiimpana, mu CD45-cell fraction, mweelwe wa EpCAM+ cells mucilonda wakatanta kweelanisigwa a ascites (Chitondeezyo 1D). Twakabona akwalo kwiimpana kulibonya aakati ka EpCAM+ antoomwe a EpCAM- cell components. Maseelo aa EpCAM+ (chilonda) alijisi bunji bwakutambula glucose akubeleka kwa mitochondrial kwiinda maseelo aa EpCAM-, alo alisumpukide kwiinda mulimo wa metabolic wa fibroblasts mumaseelo aacilonda mu TME (Chitondeezyo 1, E a F).
(A a B) Kuyuma kwa fluorescence (MFI) kwakutambula glucose (2-NBDG) (A) akubeleka kwa mitochondrial kwa CD4 + T cells (MitoTracker dark red) (B) Ma graph aayiminina (kulumwensyi) antoomwe a data iilembedwe (Kululyo), CD8 + T cells antoomwe a EpCAM + CD45-tumor cells mbuli zilonda. (C) Mweelwe wa CD4 + a CD8 + maselu (aa CD3 + T maselu) mu ascites amucilonda. (D) Mweelwe wa EpCAM + maselu aazilonda mu ascites a zilonda (CD45−). (E a F) EpCAM + CD45-tumor a EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) a mitochondrial activity (MitoTracker dark red) (F) representative graphs (lumwensyi) antoomwe a tabulated data ( Right) Ascites a tumor cells. (G) Ma graph aatondezya CD25, CD137 antoomwe a PD1 kwiinda mukubelesya nzila ya flow cytometry. (H a I) CD25, CD137 antoomwe a PD1 zitondezyo a CD4 + T maselu (H) a CD8 + T maselu (I). (J a K) Kutazyiba, kuyeeya kwaakati (Tcm), kugwasya (Teff) antoomwe akuyeeya kugwasya (Tem) kweendelana akutondezya kwa CCR7 a CD45RO. Zifwanikiso ziiminina (kulumwensyi) antoomwe a data iilembedwe (kululyo) ya CD4 + T maselu (J) antoomwe a CD8 + T maselu (K) mu ascites amuzilonda. P values ​​  yakajanwa a paired t-test (*P<0.05, **P<0.01 antoomwe a ***P<0.001). Muzila wiiminina balwazi bakozyenye (n = 6). FMO, kumweka kwakugwisya chimwi; MFI, nguzu zyamumuni zili akati-kati.
Kuyungizya kuvwuntauzya kwakatondeezya kwiimpana kupati aakati ka T cell phenotypic status. Ikubelesya (Chitondeezyo 1, G kusikila I) akuyeeya kwakuchita (Chitondeezyo 1, J a K) muzilonda zilavwula kwiinda ascites (mweelwe wamaseelo aa CD3 + T). Mbubwenya buyo, kulanga-langa chiimo chamuntu kwiinda mukutondezya zitondezyo zyakuchita (CD25 a CD137) antoomwe azitondezyo zyakumana [programmed cell death protein 1 (PD1)] kwakatondezya kuti nikuba kuti bube bwakusanduka bwabantu aaba buliindene (Chitondeezyo S1, B kusikila ku E), pesi taakwe kwiimpana kupati kwakabonwa aakati kakuyeeyelwa, naa kuchincha kwakuyeeyela (Mufanikisyo S1, F1) IME). Eezyi mpindu zyakasinizyigwa kwiinda mukubelesya nzila zyakwiiya zyaminchina kuti zipe maseelo (21), eezyo zyakayungizya kuyubununa mweelwe mupati wamaseelo aamubulowa (CD45 + / CD3- / CD4 + / CD45RO +) mu ascites yamulwazi (Chitondeezyo S2A ). Akati kamisyobo yoonse yamaseelo yakajanwa, eeyi maseelo aa myeloid yakatondezya kutambula glucose kupati akubeleka kwa mitochondrial (Chitondeezyo S2, B kusikila ku G). Eezyi mpindu zitondezya kwiimpana kupati kwakusanduka kwamaseelo aakati kamaseelo miingi aajanika mu ascites a tumors mubantu bachiswa HGSC.
Buyumu-yumu bupati mukumvwisisisya bube bwa TIL mbwakuyandika kwaandaanya zitondezyo zya T cell zisalala, zibotu amweelwe kuzwa kuzilonda. Ziiyo zyakachitika ino-ino zyakatondezya kuti nzila zyakusalazya akuyungizya nsinga zyeendelana a flow cytometry zilakonzya kuleta kuchincha kwamaseelo (22-24). Kuti tuzunde penzi eeli, twakabambulula nzila yakuyungizya nsinga kuti tukonzye kwaandaanya akwaandaanya TIL kuzwa ku kkansa yamumapunga yabantu yakagwisigwa kakutanaba kulanga-langa a LC-MS/MS (Langa Zibelesyo a Nzila; Cifwanikiso 2A). Kuti tubone mbuzigwasya nzila eeyi mukusanduka kwa metabolite, twakakozyanisya metabolite yamaseelo aa T akabelesyegwa abasikupa buumi bubotu nikwakainda ntaamu yakuandaanya nsinga zyaambwa atala aawa amaseelo aatakaandaanizyidwe pesi akachili aa aisi. Ooku kulanga-langa bubotu kwakajana kuti kuli kujatana kupati aakati kazeezi zibili (r = 0.77), mpawo kwiinduluka kwabusongo bwakabunga ka 86 metabolites kulijisi kwiinduluka kupati (Chitondeezyo 2B). Aboobo, nzila eezi zilakonzya kuchita buvwuntauzi buli kabotu bwamaseelo aali mukuyungizigwa kwamusyobo wamaseelo, aboobo kupa nzila yakusaanguna iisumpukide yakuzyiba maseelo amwi mu HGSC, aboobo kupa kuti bantu bamvwisisisye kapati pulogilamu yakucinca kwamaseelo.
(A) Mufanikisyo wakuvwuzya nsingo zyamagineti. Kakutanaba kuvwuntauzyigwa a LC-MS/MS, maselu ayooinda ziindi zyotatwe zitobelana zyakuvwuzya nsinga zyamagineti naakuti ayookkala aayizi. (B) Ikugwasya kwamusyobo wakuvwuzya kubunji bwazintu zipangwa mumubili. Mweelwe waakati-kati wazipimyo zyotatwe zyamusyobo umwi aumwi wakuvwuzya ± SE. Muzila wadota wiiminina chilongwe cha 1:1. Kuswaangana kwamukati-kati (ICC) kwakupima kwakaindulukwa kutondezyedwe aachikozyano cha axis. NAD, nicotinamide adenine dinucleotide. (C) Mufanikisyo wachikozyano chakubeleka kwakuvwuntauzya kwa metabolite yamulwazi. Ma ascites naakuti zilonda zilabwezegwa kuzwa kuli bachiswa mpawo zilabikkwa mumulilo. Kabeela kaniini ka sampulu imwi aimwi kakavwuntauzyigwa a flow cytometry, mpawo sampulu zyakasyeede zyakainda ziindi zyotatwe zyakuvwuzya CD4+, CD8+ antoomwe a CD45- maselu. Maselu aaya akavwuntauzyigwa kubelesya LC-MS/MS. (D) Maapu yakupya kwabunji bwazintu zipangwa mumubili. Dendrogram itondezya kubunganya kwa Ward kwa Euclidean misinzo aakati kazitondezyo. (E) Kuvwuntauzya kwazibeela zipati (PCA) zyamaapu ya metabolite, kutondezya ziindi zyotatwe zya musamu umwi aumwi, zitondezyo kuzwa kumulwazi omwe zilaswaanganizyigwa amuzila. (F) PCA ya metabolite profile ya sampulu yakabikkilwa kumulwazi (nkokuti, kubelesya cibeela ca redundancy); musyobo wachikozyano ulaa munyinza akaambo kachikozyano chiyumu. PC1, chibeela chipati 1; PC2, chibeela chipati 2.
Kuzwa waawo, twakabelesya nzila eeyi yakuyungizya kuti tuvwuntauzye 99 metabolites mu CD4 +, CD8 + antoomwe a CD45-cell fractions mu primary ascites a tumors yabantu bali lusele ba HGSC (Chitondeezyo 2C, Chitondeezyo S3A a Table S3 a S4). Mweelwe wabantu bayanda kugwasyigwa ulasika ku 2% kusika ku 70% ya maseelo mapati aakusaanguna, mpawo mweelwe wamaseelo ulaandeene akati kabantu bachiswa. Nikwakamana kwaandaanya nsinga, chibeela chakuyandisya (CD4+, CD8+ naa CD45-) chitola 85% yamaseelo woonse apona muchikozyano. Eeyi nzila yakuyungizya ilatuzumizya kulanga-langa mweelwe wamaseelo kuzwa kuzilonda zyabantu, eezyo zitakonzeki pe kuchitwa kuzwa kuzitondezyo zipati. Kubelesya nzila eeyi, twakajana kuti l-kynurenine a adenosine, aaya ma metabolites obile aajisi bube buli kabotu aakwabilila mubili kulwana malwazi akalisumpukide mumaseelo aa T naakuti maseelo aajisi bulwazi (Chitondeezyo S3, B a C). Aboobo, eezyi mpindu zitondezya kusyomeka akukonzya kwakwaandaanya maseelo anzila ya mass spectrometry kuti tujane zintu ziyandikana mumubili wamuntu uuchiswa.
Kuvwuntauzya kwesu kwakatondeezya kwaandaana kupati kwamaseelo mukati aakati kabachiswa (Chitondeezyo 2D aChitondeezyo S4A). Mubwini, kweezyanisya abamwi bachiswa, bachiswa 70 bakatondezya bube bwakusanduka kwazyakulya bwiindene (Chitondeezyo 2E aChitondeezyo S4B), kutondezya kuti inga kwaba kusiyana kupati kwazyakulya akati kabachiswa. Nchintu chibotu kuzyiba kuti kweezyanisya abalwazi bamwi (1.2 kusikila kuli 2 malita; Chikozyano S1), mweelwe woonse wa ascites wakabungikwa kuli balwazi bali 70 (80 ml) wakali muniini. Kweendelezya kwakusiyana kwabachiswa muchiindi chakuvwuntauzya zibeela zipati (mbuli, kubelesya kuvwuntauzya kwakuvwula kwazibeela) kutondeezya kuchincha kutamani aakati kamisyobo yamaseelo, mpawo misyobo yamaseelo antoomwe/naakuti busena busyoonto bulabunganizigwa kweendelana a metabolite profile (Chitondeezyo 2F). Kuvwuntauzya kwa metabolites yomwe-yomwe kwakakankaizya zyintu eezyi mpawo kwakatondeezya kwiimpana kupati aakati kamaseelo aabusena. Nchintu chibotu kuzyiba kuti kwiimpana kupati kwakabonwa nkwa MNA, ooko kanjikanji kuvwubidwe mumaseelo aa CD45- alimwi amumaseelo aa CD4+ alimwi a CD8+ aayo aanjila mucilonda (Mufanikisyo 3A). Kumaseelo aa CD4 +, eechi chintu chilibonya loko, mpawo MNA mumaseelo aa CD8 + ayalo ilibonya kuti ilajatikizyigwa loko abusena. Nokuba boobo, eezi taziyandiki pe, nkaambo botatwe luzutu akati kabalwazi bali musanu ababili mbabakonzya kulingulwa kuti bazyibe zilonda zya CD8+. Mukuyungizya kuli MNA, mumaseelo aaindene-indene mu ascites amuzilonda, zimwi zintu zitazyibidwe kabotu mu TIL azyalo zilivwubide munzila ziindene (Mifanikisyo S3 a S4). Aboobo, data eeyi itondezya kuti kuli zyintu zikonzya kukwabilila mubili kuti zikonzye kuyanduulisisya.
(A) MNA iili kabotu mu CD4+, CD8+ antoomwe a CD45- maselu kuzwa ku ascites a zilonda. Mulongo wabbokesi utondeezya mweelwe waakati-kati (muzila), mweelwe waakati-kati (chikozyano chafulemu) antoomwe amweelwe wa data, kusikila ku 1.5 mweelwe waakati-kati (mweembo wafulemu). Mbuli mbokwaambidwe mubbuku lyazibelesyo zyabalwazi anzila zyakubelesya, kobelesya mweelwe wa limma wamuntu uuchiswa kuti ujane mweelwe wa P (*P<0.05 a **P<0.01). (B) Cifwanikiso cha MNA metabolism (60). Ma metabolites: S-adenosyl-1-methionine; SAH, S-adenosine-1-homocysteine; NA, nicotinamide; MNA, 1-methylnicotinamide; 2-PY, 1-methyl- 2-pyridone-5-carboxamide; 4-PY, 1-methyl-4-pyridone-5-carboxamide; NR, nicotinamide ribose; NMN, nicotinamide mononucleotide. Ma enzyme (asalala pyu): NNMT, nicotinamide N-methyltransferase; SIRT, sirtuins; NAMPT, nicotinamide phosphoribosyl transferase; AOX1, aldehyde oxidase 1; NRK, nicotinamide riboside kinase; NMNAT, nicotinamide mono Nucleotide adenylate transferase; Pnp1, purine nucleoside phosphorylase. (C) t-SNE ya scRNA-seq ya ascites (mubala wadota) acilonda (mubala uusalala pyu; n = 3 balwazi). (D) NNMT kutondezya mumaseelo aasiyene-siyene aakajanwa kubelesya scRNA-seq. (E) Kutondezya kwa NNMT antoomwe a AOX1 mu SK-OV-3, inswi zyamuntu (HEK) 293T, maseelo aa T antoomwe amaseelo aa T aasilikwa a MNA. Ikuvwungaila kwatondezyegwa kweendelana a SK-OV-3. Inzila yakutondezya a SEM itondezyedwe (n = 6 basikugwasizya bali kabotu). Ct values ​​ziinda ku 35 zilabonwa kuti tazikonzyi kubonwa (UD). (F) Kutondezya kwa SLC22A1 a SLC22A2 mu SK-OV-3, HEK293T, maseelo aa T alimwi amaseelo aa T aakasilikwa a 8mM MNA. Ikuvwungaila kwatondezyegwa kweendelana a SK-OV-3. Inzila yakutondezya a SEM itondezyedwe (n = 6 basikugwasizya bali kabotu). Ct values ​​ziinda ku 35 zilabonwa kuti tazikonzyi kubonwa (UD). (G) Maselu aa MNA aali mumaseelo aa T aajisi buumi bubotu aasangwa musule aamawoola aali 72 aakubikkwa a MNA. Inzila yakutondezya a SEM itondezyedwe (n = 4 basikugwasizya bajisi buumi bubotu).
MNA ilapangwa kwiinda mukugwisya kabunga ka methyl kuzwa ku S-adenosyl-1-methionine (SAM) kuya ku nicotinamide (NA) kwiinda mukubelesya nicotinamide N-methyltransferase (NNMT; Cifwanikiso 3B). NNMT ilajanika kapati mumalwazi aakkansa aasiyene-siyene aabantu alimwi ilaswaangene akuvwula, kunjila alimwi akuvwula (25-27). Kutegwa tumvwisisisye kabotu nkozizwa MNA mumaseelo aa T mu TME, twakabelesya scRNA-seq kuzyiba mboizwa NNMT mumaseelo aamisyobo ya ascites alimwi azilonda zyabalwazi botatwe ba HGSC (Table S5). Kuvwuntauzya kwamaseelo aasika ku 6,500 kwakatondeezya kuti mubusena bwa ascites amuzilonda, NNMT yakabikkilwa buyo ku fibroblast amaseelo aazilonda (Chitondeezyo 3, C a D). Nchintu chibotu kuzyiba kuti taakwe chitondezyo cha NNMT mulibamwi bantu batondezya PTPRC (CD45 +) (Chitondeezyo 3D aChitondeezyo S5A), eecho chitondezya kuti MNA yakajanwa mumulongo wa metabolite yakanjizigwa mumaseelo aa T. Kutondezya kwa aldehyde oxidase 1 (AOX1) kusanduka MNA kuba 1-methyl-2-pyridone-5-carboxamide (2-PYR) naa 1-methyl-4-pyridone-5-carboxamide (4- PYR); Cifwanikiso 3B) alimwi chili buyo kubantu ba fibroblasts batondezya COL1A1 (Cifwanikiso S5A), eezyo antoomwe zitondezya kuti maseelo aa T taakwe nguzu zyakuchita zintu zya MNA. Inzila yakutondezya ma gene aaya aajatikizya MNA yakasinizyigwa kubelesya maseelo aabili aatali aalikke kuzwa kulibachiswa ba HGSC (Chitondeezyo S5B; n = 6) (16). Mukuyungizya, kuvwuntauzya kwa quantitative polymerase chain reaction (qPCR) kwamaseelo aa T aajisi buumi bubotu aakasilikwa a MNA kwakatondezya kuti kweelanisigwa amaseelo aa SK-OV-3 aajisi bulwazi bwamumatumbu, NNMT naa AOX1 tiiyakatondezyegwa pe (Chitondeezyo 3E). Eezyi mpindu zitalangilwi zitondezya kuti MNA ilakonzya kuzwa kuma fibroblasts naakuti zilonda kuya kumaseelo aa T aali afwaafwi mu TME.
Nikuba kuti basikutola lubazu babikkilizya mukwasyi wa organic cation transporters 1 kusikila 3 (OCT1, OCT2 a OCT3) uulembedwe a soluble carrier 22 (SLC22) mukwasyi (SLC22A1, SLC22A2 a SLC22A3), basikweenzya ba MNA tabazyibidwe pe (28) . QPCR ya mRNA kuzwa kumaseelo aa T aajisi buumi bubotu yakatondezya zyeelelo zya SLC22A1 zisyoonto pesi zyeelelo zitabonwi zya SLC22A2, eezyo zyakasinizya kuti zyakali kwaambwa kale mumabbuku (Chitondeezyo 3F) (29). Mukwiimpana, SK-OV-3 ovarian tumor line line yakatondeezya mweelwe mupati wazyintu zyoonse zyobile (Chitondeezyo 3F).
Mukusola kulanga-langa naa maseelo aa T alakonzya kunywa MNA yakunze, maseelo aa T aatakwe buumi bubotu akakomezyegwa kwamawoola aali 72 kakuli MNA iindene-indene. Kakwiina MNA iizwa anze, zili mumaseelo aa MNA tazikonzyi kubonwa pe (Chitondeezyo 3G). Nokuba boobo, maselu aa T aabelesyegwa akasilikwa a MNA yakunze akatondezya kuyungizigwa kwa MNA mumaseelo, kusika ku 6 mM MNA (Chitondeezyo 3G). Eechi chakazwa chitondeezya kuti nikuba kuti zyintu zitola zintu zili munsi-munsi akubula enzyme mpati iilanganya MNA mukati kamaseelo, TIL ilakonzya kubweza MNA.
Ikusiyana kwazintu zipangwa mumaseelo aa T aabalwazi alimwi akusunkwa kwakunywa MNA mu vitro kuyungizyila kuti ma fibroblasts aajatikizya kkansa (CAF) agwisye MNA alimwi maseelo aamubulowa alakonzya kweendelezya mboibede alimwi amulimo wa TIL. Kutegwa tuzyibe mulimo wa MNA kumaseelo aa T, maseelo aa T aajisi buumi bubotu akabelesyegwa mu vitro kakuli naa kakunyina MNA, alimwi kuvwula kwawo alimwi akupanga ma cytokine kwakalingulwa. Musule lyamazuba aali musanu ababili aakuyungizya MNA aamusamu mupati, mweelwe wabantu wakayungizya tubili wakaselemuka, mpawo manguzu akazumanana kumisela yoonse (Chitondeezyo 4A). Mukuyungizya, kusilikwa kwa exogenous MNA kwakapa kuti mweelwe wa CD4 + antoomwe a CD8 + T maselu aatondezya tumor necrosis factor-α (TNFα; Cifwanikiso 4B) uyungizigwe. Mukwiimpana, kupanga kwa IFN-γ mukati kamaseelo kwakaceya kapati mumaseelo aa CD4 + T, pele kutali mumaseelo aa CD8 + T, alimwi kwakanyina kucinca kupati mu interleukin 2 (IL-2; Cifwanikiso 4, C a D). Aboobo, enzyme-linked immunosorbent assay (ELISA) ya supernatants kuzwa kuli eezyi MNA-treated T cells yakatondezya kuyungizigwa kupati kwa TNFα, kuseluka kwa IFN-γ, akubula kucinca kwa IL-2 (Chitondeezyo 4, E kusikila ku G). . Ikucheya kwa IFN-γ kutondeezya kuti MNA ilakonzya kuchita mulimu wakulesya kulwana zilonda zyamaseelo aa T. Kuti tukonzye kweezyanisya mulimo wa MNA ku T cell-mediated cytotoxicity, chimeric antigen receptor T (FRα-CAR-T) cells iilanganya folate receptor α antoomwe a CAR-T (GFP) yeendelezyegwa a green fluorescent protein (GFP) -CAR-T) cells zipangwa a donor peripheral blood mononuclear cells (PBMC). Maseelo aa CAR-T akakomezyegwa kwamahola aali makumi obilwe atune (24) kakuli MNA, mpawo akakomezyegwa antoomwe amaseelo aa SK-OV-3 aajisi tuzunda twamumatumbu aatondezya folate receptor α a effector to target ratio ya 10:1. Busilisi bwa MNA bwakapa kuti kube kucheya kupati mukujaya kwamaseelo aa FRα-CAR-T, alo aakali kukozyanya amaseelo aa FRα-CAR-T aakasilikwa a adenosine (Chitondeezyo 4H).
(A) Mweelwe wamaseelo woonse aakonzya kupona akuvwula kwabantu (PD) kuzwa kukukomezya mubuzuba bwa 7. Igilafu itondezya mweelwe waakati-kati + SEM wabasikusanga bali lusele. Chiiminina data kuzwa ku n = 3 zyakusola zilikke. (B kusikila ku D) CD3/CD28 antoomwe a IL-2 zyakabelesegwa kuchita kuti maselu aa T abeleke mubunji bwawo bwa MNA kwamazuba aali 7. Kakutanaba kuvwuntauzya, maselu akakulwaizigwa a PMA/ionomycin a GolgiStop kwamahola one. TNFα (B) kutondezya mumaseelo aa T. Cifwanikiso chachikozyano (kulumwensyi) antoomwe a data iilembedwe (kululyo) ya TNFα mumaseelo apona. IFN-γ (C) antoomwe a IL-2 (D) zitondezyo mumaseelo aa T. Kutondezya kwama cytokines kwakapimwa a flow cytometry. Igilafu itondezya mweelwe waakati-kati (n = 6 basikusanga batakwe buumi bubotu) + SEM. Belesya nzila yomwe yakulanga-langa kusiyana akupima kwiinduluka-induluka (*P<0.05 a **P<0.01) kuti ujane mweelwe wa P. Chiiminina data kuzwa ku n = 3 zyakusola zilikke. (E kusikila ku G) CD3/CD28 antoomwe a IL-2 zyakabelesegwa kuchita kuti maselu aa T abeleke mubunji bwawo bwa MNA kwamazuba aali 7. Ibbazu lyakayobolwa kakutanaba akumana mawoola one aakusungwaazyigwa kwa PMA/ionomycin. Bunji bwa TNFα (E), IFN-γ (F) antoomwe a IL-2 (G) bwakapimwa a ELISA. Igilafu itondezya mweelwe waakati-kati (n = 5 basikusanga batakwe buumi bubotu) + SEM. Mpindu ya P yakajanwa kwiinda mukubelesya nzila yomwe yakulanga-langa kusiyana akupima kwiinduluka-induluka (*P<0.05). Muzila uujisi madoti utondeezya munyinza wakuzyiba kwakujana. (H) Kusunkwa kwakugwisigwa kwamaseelo. Maseelo aa FRα-CAR-T naa GFP-CAR-T akabambululwa a adenosine (250μM) naa MNA (10 mM) kwamahola aali makumi obilwe atune, naa kusiigwa kaatasilikwa (Ctrl). Mweelwe wakujaigwa kwamaseelo aa SK-OV-3 wakapimwa. P value yakajanwa a Welch t test (*P<0.5 a **P<0.01).
Kuti tujane kumvwisisisya kwa MNA-dependent TNFα expression regulation, kuchincha kwa TNFα mRNA ya MNA-treated T cells kwakali kulinguwe (Chitondeezyo 5A). Maseelo aa T aajisi buumi bubotu aakasilikwa a MNA akatondezya kuyungizigwa ziindi zyobile mu TNFα transcription levels, kutondezya kuti MNA iyeeme a TNFα transcription regulation. Kuvwuntauzya nzila eeyi iikonzya kweendelezya, zintu zyobile zizibinkene zyeendelezya TNFα, nkokuti activated T cell nuclear factor (NFAT) antoomwe a protein 1 (Sp1), zyakali kulinguwe mukwiingula MNA binding ku proximal TNFα promoter ( 30). I TNFα promoter ilijisi masena aali 6 aazyibidwe aa NFAT alimwi amasena aa Sp1 aali 2, aavwumbilidwe abusena bomwe [-55 base pairs (bp) kuzwa ku 5'cap] (30). Chromatin immunoprecipitation (ChIP) yakatondezya kuti nibakasilikwa a MNA, kujatana kwa Sp1 ku TNFα promoter kwakatanta ziindi zyotatwe. Kubikkilizyigwa kwa NFAT akwalo kwakayungizya akuswena kuyandikana (Chitondeezyo 5B). Eeyi data itondezya kuti MNA yeendelezya kutondezya kwa TNFα kwiinda mukulemba kwa Sp1, mpawo asyoonto kutondezya kwa NFAT.
(A) Kweezyanisya amaseelo aa T aakakomezyegwa kakunyina MNA, kucinca kwa TNFα mumaseelo aa T aakasilikwa a MNA. Inzila yakutondezya a SEM itondezyedwe (n = 5 basikugwasizya bali kabotu). Chiiminina data kuzwa ku n = 3 zyakusola zilikke. (B) TNFα promoter ya T cells yakasilikwa a 8 mM MNA naa pe NFAT a Sp1 zyakaswanizigwa a (Ctrl) antoomwe a PMA/ionomycin stimulation kwamahola one. Immunoglobulin G (IgG) antoomwe a H3 zyakabelesegwa mbuli nzila zitali kabotu azitali kabotu zyakukwabilila mubili, kweendelana. Kupima mweelwe wa ChIP kwakatondeezya kuti kuswaangana kwa Sp1 a NFAT ku TNFα promoter mumaseelo aakasilikwa a MNA kwakayungizyika ziindi zyiingi kweelanisigwa akweendelezya. Chiiminina data kuzwa ku n = 3 zyakusola zilikke. P value yakajanwa kwiinda mukubelesya t-tests zyiingi (*** P <0.01). (C) Kweezyanisya a ascites ya HGSC, maselu aa T (aatakwe ntenda) akatondezya kuyungizigwa kwa TNF muchilonda. Mibala yiiminina balwazi baindene. Maseelo aatondezyedwe akasalwa biyo kusika ku 300 mpawo akazungaanizyigwa kuchitila kuti kutabi kuvwuzya (** Padj = 0.0076). (D) Nzila yakayeeyelwa ya MNA ya kkansa yamumatumbu. MNA ilapangwa mumaseelo aazilonda antoomwe ama fibroblasts mu TME mpawo ilatolwa amaseelo aa T. MNA ilayungizya kujatana kwa Sp1 ku TNFα promoter, mpawo eezi zipa kuti TNFα iyungizye kulembwa antoomwe akupanga TNFα cytokine. MNA alimwi iletela kucheya kwa IFN-γ. Kulesyegwa kwakubeleka kwamaseelo aa T kuletela kucheya kwanguzu zyakujaya akufwambaanya kukomena kwacilonda.
Kweelana amalipooti, ​​TNFα ilaa nguzu zyakulwana zilonda azilonda, pesi ilaa mulimo uuzyibidwe mukusumpula kukomena akuvwula kwa kkansa yamumapunga (31-33). Kweelana amalipooti, ​​bunji bwa TNFα muzibeela zya ascites amuzilonda mubantu bachiswa kkansa yamumapunga mbupati kwiinda muzibeela zitakwe bulwazi (34-36). Kweelana anzila, TNFα ilakonzya kweendelezya kubeleka, kubeleka akuvwula kwamaseelo aagazi aatuba, akucinca mboziboneka maseelo aakkansa (37, 38). Kweendelana azintu eezyi zyajanwa, kuvwuntauzya kwakusiyana kwama gene kwakatondezya kuti TNF yakasumpulwa kapati mumaseelo aa T muzibeela zyacilonda kweelanisigwa a ascites (Chitondeezyo 5C). Kuyungizya kwakutondezya TNF kwakalibonya buyo mumaseelo aa T aatakwe cilengwa cakujaya maseelo (Chitondeezyo S5A). Muchi chifwiifwi, data eeyi ilagwasyilizya muzeezo wakuti MNA ilijisi nzila zyobile zyakukwabilila mubili akusumpula zilonda mu HGSC.
Kulemba kwa fluorescent kweendelanya a flow cytometry kwaba nzila mpati yakwiiya TIL metabolism. Ziiyo eezyi zyakatondezya kuti kweelanisigwa ama lymphocytes aamubulowa naakuti maselu aa T kuzwa kuzibeela zya lymphoid zyabili, murine amuntu TIL ilaa chiyandisyo chipati chakutambula glucose (4, 39) akusweekelwa kwakubeleka kwa mitochondrial (19, 40). Nikuba kuti twakabona mpindu zikozyania muchiiyo eechi, chintu chipati nkukozyanisya kusanduka kwamaseelo aamubulowa a TIL kuzwa kunyama yomwe yakakosolwa. Kweendelana amalipooti amwi aakainda, maselu aazilonda (CD45-EpCAM +) kuzwa ku ascites amatumbu ajisi glucose mpati kwiinda CD8 + antoomwe a CD4 + T maselu, zitondezya kuti glucose mpati iitola maselu aachilonda ilakonzya kweezyanisyigwa amaseelo aa T. Muzeezo wakuzundana kwa T cell. TME. Nokuba boobo, mulimo wamaseelo aacilonda ulisumpukide kwiinda wa CD8 + T cells, pele mulimo wa mitochondrial ulikozyenye a CD4 + T cells. Eezyi mpindu ziyumya mutwe wamakani uuzwa wakuti nzila ya oxidative metabolism ilayandika kapati kumaseelo aamubulowa (41, 42). Alimwi baamba kuti maseelo aa CD8 + T alakonzya kuciswa akaambo kakutabeleka kabotu kwiinda maseelo aa CD4 + T, naa kuti maseelo aa CD4 + T alakonzya kubelesya zintu zimbi zipa kaboni kunze lya glucose kubamba mulimo wa mitochondrial (43, 44). Chileelela kuzyibwa kuti tiitwakabona kwiimpana mukutambula glucose naakuti kubeleka kwa mitochondrial aakati ka CD4 + T effectors, T effector memory antoomwe a T central memory cells mu ascites. Mbubwenya buyo, kusiyana kwamaseelo aa CD8 + T mumatumbu takukwe makani akusanduka kwakutambula glucose, kutondezya kwiimpana kupati aakati kamaseelo aa T aakakomezyegwa mu vitro a TIL yabantu mu vivo (22). Eezyi zintu zyakabonwa zyakasinizyigwa akubelesya nzila yakupa maseelo aatakwe kampenda, eeyo yakayubununa kuti maseelo aa CD45 + / CD3- / CD4 + / CD45RO + ajisi glucose mpati alimwi akubeleka kwa mitochondrial kwiinda maseelo aamubulowa alivwulide pele ajisi maseelo aabeleka. Mweelwe ooyu ulakonzya kwiiminina mweelwe wamaseelo aa myeloid suppressor naakuti plasmacytoid dendritic cells aajanika mukuvwuntauzya kwa scRNA-seq. Nikuba kuti zyoonse zyobile zyakaambwa muzilonda zyamumapunga aabantu [45], zichili kuyandika Mulimo uumbi ngwakupandulula kabunga aaka ka myeloid.
Nikuba kuti nzila zya flow cytometry zilakonzya kusalazya kwiimpana kwa glucose a oxidative metabolism akati kamisyobo yamaseelo, zintu zipangwa a glucose naakuti zimwi zintu zipa kuti kube metabolism ya mitochondrial mu TME tazinazyibwa pe. Kupa kubako naa kutabako kwa metabolites ku TIL subset imwi kuyandika kusalazyigwa kwamaseelo kuzwa kunyama yakakosolwa. Aboobo, nzila yesu yakuyungizya maselu antoomwe a mass spectrometry ilakonzya kupa luzibo kujatikizya zintu zipangwa mumaseelo aa T alimwi amaseelo aajisi zilonda muzibeela zyabalwazi zyeendelana. Nikuba kuti eeyi nzila ilaampindu kwiinda kusalazya maseelo aabelesya mumuni, zimwi zibeela zya metabolite zilakonzya kujatikizyigwa akaambo kakukkalikana kwazyo alimwi/naakuti kufwambaana kwakucinca (22). Nikuliboobo, nzila yesu yakakonzya kujana zintu zyobile zizibinkene zikonzya kulesya malwazi, adenosine a kynurenine, nkaambo zilaandeene kapati aakati kamisyobo yasampulu.
Buvwuntauzi bwesu bwa metabolomic bwazilonda a TIL subtypes bupa luzibo lunji kujatikizya mulimo wa metabolites mu ovarian TME. Chakusanguna, kubelesya flow cytometry, twakajana kuti taakwe kwiimpana mumulimo wa mitochondrial aakati kamatumbu a CD4 + T cells. Nokuba boobo, buvwuntauzi bwa LC-MS/MS bwakayubununa kucinca kupati mubunji bwazintu zipangwa mumubili akati kabantu aaba, kutondezya kuti mamanino aajatikizya TIL metabolism alimwi amulimo wayo woonse wa metabolic ayandika kusandululwa kabotu. Chabili, MNA ngo metabolite uulaa lwiindano lupati aakati ka CD45-cells a T cells mu ascites, kutali zilonda. Aboobo, kwaandaanya antoomwe abusena bwacilonda kulakonzya kuba azintu ziindene-indene ku TIL metabolism, eezyo zitondezya kusiyana kwabusena bumwi. Chatatu, kutondezya kwa MNA-producing enzyme NNMT kuli buyo a CAF, eeyo iili maseelo aazilonda asyoonto, asi zyeelelo zya MNA zikonzya kubonwa zilabonwa mumaseelo aa T aazwa kuzilonda. Kuyungizya kwa NNMT mu ovarian CAF kulijisi mulimo uuzyibidwe wakusumpula kkansa, cibeela akaambo kakusumpula CAF metabolism, kunjila kwacilonda alimwi akuvwula kwa kkansa (27). Nikuba kuti mweelwe woonse wa TIL uli akati-akati, kutondezya kwa NNMT mu CAF kuliswaangene kapati a Cancer Genome Atlas (TCGA) mesenchymal subtype, eeyo iiswaangene akulangilwa kutali kabotu (27, 46, 47). Kumamanino, kutondezya kwa enzyme AOX1 iilanganya kunyonyooka kwa MNA akwalo kuli buyo kubantu ba CAF, eezi zitondezya kuti maselu aa T taakwe nguzu zyakusandula MNA. Eezyi mpindu zilagwasyilizya muzeezo wakuti nikuba kuti mulimu uumbi uyandika kuchitila kusinizya eezyi zyajanwa, mweelwe mupati wa MNA mumaseelo aa T ulakonzya kutondeezya kubako kwa immunosuppressive CAF microenvironment.
Twaambilizya mweelwe musyoonto wa MNA transporters antoomwe amweelwe uutabonwi wama protein aajatikizyidwe mu MNA metabolism, kubako kwa MNA mu T cells takulangilwi pe. Kunyina NNMT naa AOX1 zyakali kukonzya kujanwa kwiinda mukuvwuntauzya kwa scRNA-seq alimwi a qPCR iibikkilidwe maanu yabantu bobilo baliimpene. Eezyi mpindu zitondezya kuti MNA taipangwi amaseelo aa T, pesi ilagwisigwa kuzwa ku TME. Kusola kwa in vitro kutondezya kuti maselu aa T alayandisya kuyobolola MNA iizwa anze.
Ziiyo zyesu zya in vitro zyakatondezya kuti MNA iizwa anze ilasungwaazya TNFα mumaseelo aa T mpawo ilasumpula kuswaangana kwa Sp1 ku TNFα promoter. Nikuba kuti TNFα ilakonzya kulwana zilonda azyakulwana zilonda, mu kkansa yamumapunga, TNFα ilakonzya kusumpula kukomena kwa kkansa yamumatumbu (31-33). Kulesya TNFα mukukomezya maseelo aazilonda zyamumatumbu naa kugwisya citondezyo ca TNFα muzikozyanyo zya mbeba kulakonzya kugwasya TNFα-mediated inflammatory cytokine production akulesya kukomena kwacilonda (32, 35). Aboobo, muchiimo eechi, TME-yakazwa ku MNA ilakonzya kubeleka mbuli metabolite iikulwaizya kuzimba kwiinda munzila iiyeeme a TNFα kwiinda munzila ya autocrine loop, aboobo ikusumpula kujanika akumwaika kwa kkansa ya ovarian (31). Kwiinda mukukonzeka ooku, TNFα blockade ili mukwiiya mbuli musamu uukonzya kusilika kkansa yamumatumbu (37, 48, 49). Mukuyungizya, MNA ilanyonganya maseelo aa CAR-T kumaseelo aazilonda zyamumatumbu, kupa bumboni bumbi bwa MNA-mediated immune suppression. Kusanganya, eezyi mpindu zitondezya nzila oomo zilonda amaseelo aa CAF mozigwisya MNA mu TME iili kunze lyamaseelo. Kwiinda mu (i) TNF-yakapa kuti kkansa yamumatumbu ikomezyegwe antoomwe a (ii) MNA-yakapa kuti T cell ilesyegwe kubeleka, eezi zilakonzya kuba aampindu zyobile (Chitondeezyo 5D).
Mukumanizya, kwiinda mukubelesya nzila yakufwambaana kuvwuzya maseelo, kutobelezya maseelo aamwi alimwi akulanga-langa zyakulya, ciiyo eeci cakayubununa kwiimpana kupati kwa immunometabolomic akati kamatumbu alimwi amaseelo aa ascites mubalwazi ba HGSC. Ooku kuvwuntauzya kwakatondezya kuti kuli kwiimpana mukutambula glucose akubeleka kwa mitochondrial aakati ka T cells, mpawo kwakazyibya kuti MNA nchintu chitali chamaseelo chilikwabilila mubili. Eeyi data ilaampindu aatala akuti TME ilajatikizya biyeni T cell metabolism mumalwazi aakkansa aabantu. Nikuba kuti kuzundana kwazyakulya aakati kamaseelo aa T amaseelo aakkansa kwakaambwa, ma metabolites alakonzya kubeleka mbuli kweendelezya kutaluleme kusumpula kuyaambele kwacilonda alimwi ambweni akulesya nzila zyakukwabilila mubili. Bupanduluzi bumbi bwamulimo wazyintu eezyi zyeendelezya zyakulya zilakonzya kujalula nzila zimbi zyakusumpula nguzu zyakukwabilila mubili kuzwa kuzilonda.
Zitondezyo zyabalwazi antoomwe a data yakusilikwa zyakajanwa kwiinda mu BC cancer tumor tissue repository yakazumizyigwa a Canadian Tissue Repository Network. Kweelana amulawu wakazumizigwa a BC Cancer Research Ethics Committee antoomwe a University of British Columbia (H07-00463), zitondezyo zyabalwazi boonse antoomwe a data yakusilikwa yakapegwa lugwalo lwakuzumizya naakuti bakaleka kuzumizya. Zitondezyo zilabikkwa mu BioBank (BRC-00290) iizumizyidwe. Zitondeezyo zyabalwazi zitondezyedwe muMatebulu S1 a S5. Mukukwabilila, chibelesyo chibelesyegwa kubola chilonda chamuntu uuchiswa mpawo chisunka kwiinda muchisefa cha 100-micron kuchitila kuti kube maseelo omwe. I ascites ya mulwazi yakabikkwa mu centrifuge a 1500 rpm kwamaminiti aali 10 a 4°C kuchitila kuti maselu agwisye maanzi. Maseelo aakajanwa kuzwa kucilonda a ascites akabikkwa mu 50% heat-inactivated human AB serum (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) antoomwe a 10% dimethyl sulfoxide. Eezyi zintu zyakakwabililwa zya selo yomwe zyakanyonyoonwa mpawo zyakabelesegwa mu metabolomics akuziba metabolite zyaambwa munsi.
Chiimo choonse chili a 0.22 μm yakasefwa 50:50 yakayungizigwa RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) yakayungizigwa a 10% heat-inactivated human AB serum (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) x1 Penicillin Streptomycin (PenStrep) solution (Thermo Fisher Scientific) antoomwe a 50 μMB-mercaptoethanol. AimV (Invitrogen) ilayungizigwa a 20 mM Hepes (Thermo Fisher Scientific) antoomwe a 2 mM l-glutamine (Thermo Fisher Scientific). I flow cytometer staining buffer yakajisi 0.22μm filtered phosphate buffered saline (PBS; Invitrogen) yakayungizigwa a 3% heat-inactivated AB human serum (Sigma). Ibbafa lyakuvwuzya maselu lipangidwe a 0.22μm ya PBS yakasefwa mpawo yakayungizigwa a 0.5% ya heat-inactivated human AB serum (Sigma-Aldrich).
Mu 37°C, maselu akabikkwa 10 nM MT DR antoomwe a 100 μM 2-NBDG kwamaminiti aali 30. Kuzwa waawo, maselu akabikkwa dayi eF506 a 4°C kwamaminiti ali kkumi amusanu. Bikka maselu mu FC Block (eBioscience) antoomwe a Brilliant Stain Buffer (BD Biosciences), kosanzya mu flow cytometer staining buffer (kweelana amalayilile aabasikupanga), mpawo kobikka mubusena bwakutontola kwamaminiti aali kkumi. Bika maselu ama antibodies (Table S2) mu flow cytometry staining buffer a 4°C kwamaminiti aali makumi abili. Bikka maselu mubbokesi lya flow cytometry staining (Cytek Aurora; 3L-16V-14B-8R configuration) katanavwuntauzyigwa. Belesya SpectroFlo antoomwe a FlowJo V10 kulanga-langa mweelwe wamaseelo, mpawo kobelesya GraphPad Prism 8 kupanga data. Imweelwe waakati-kati wamumuni (MFI) wa 2-NBDG antoomwe a MT DR wakaba a log-normalized, mpawo paired t test yakabelesegwa mukuvwuntauzya mweelwe wabantu bachiswa. Gwisya bantu boonse bajisi zintu zitasiki ku 40 kuzwa mukuvwuntauzya; lemba mweelwe wa MFI wa 1 kuli zyoonse zitali kabotu katanachita kuvwuntauzya kwamweelwe akulanga data.
Kuti tuyungizye nzila yakubelesya maanza yachibaka chili atala aawa, twakabelesya bupanduluzi buzulide bwamusamu wakukasya chiimo (FAUST) (21) kuti tupe maseelo kubantu boonse twamana kugwisya maseelo aafwide mu FlowJo. Tulabamba zyintu zizwa mumaanza kutegwa tusanganye bantu balibonya kuti tabakwe mulimo (kuswaanganya PD1+ amaseelo aa PD1-tumor) alimwi abantu bakazumanana. Isampulu imwi aimwi ilijisi mweelwe waakati-kati wamaseelo aayinda ku 2%, kubantu boonse antoomwe bali 11.
Ficoll gradient density centrifugation yakabelesegwa kwaandaanya PBMC kuzwa ku leukocyte separation products (STEMCELL Technologies). Maseelo aa CD8 + T akagwisigwa kuzwa ku PBMC kubelesya CD8 MicroBeads (Miltenyi) mpawo akayungizigwa munzila iizwide kubelesya TransAct (Miltenyi) kwamviki zyobile kweendelanya amalayilile aabasikupanga. Maseelo akazumizigwa kwiima kwamazuba aali 5 mubusena buzulide bujisi IL-7 (10 ng/ml; PeproTech), mpawo akuyungizigwa aTransAct. Mubuzuba bwa 7, kweendelanya amalailile aabasikupanga, ma CD45 MicroBeads (Miltenyi) aabantu akabelesegwa kuyungizya maselu muzibeela zyotatwe zitobelana. Maseelo akaandaanizyigwa kuti akabelesegwe munzila ya flow cytometry (mbuli mbokwaambidwe atala aawa), mpawo maseelo aali miliyoni yomwe akaandaanizyigwa ziindi zyotatwe kuti akabelesyegwe a LC-MS/MS. Isampulu zyakabelesegwa a LC-MS/MS mbuli mbokwaambidwe munsi. Twakayeeyela mweelwe wa metabolite uusweekede amweelwe wa ion wa 1,000. Isampulu imwi aimwi ilabambululwa amweelwe wa ion yoonse (TIC), yakasandulwa munzila ya logarithm mpawo yakabikkwa kabotu mu MetaboAnalystR kakutanaba kuvwuntauzyigwa.
Iseelo lyomwe lyamulwazi umwi awumwi lyakanyonyoonwa mpawo lyakasefwa kwiinda muchisefa cha 40 μm kuti libe aakati-kati koonse (mbuli mbokwaambidwe atala aawa). Kweelana amalailile aabasikupanga, ziindi zyotatwe zitobelana zyakusala kabotu kwiinda mukwaandaanya nsinga zyamagineti kubelesya MicroBeads (Miltenyi) zyakabelesegwa kuvwuzya zitondezyo zya CD8+, CD4+ antoomwe a CD45- maselu (aa aisi). Mubufwiifwi, maselu alabikkwa alimwi muchikwabilizyo chamaseelo (mbuli mbokwaambidwe atala aawa) mpawo abalwa. Maseelo akabikkwa antoomwe a CD8 yabantu, CD4 yabantu naakuti CD45 yabantu (Miltenyi) a 4°C kwamaminiti ali kkumi amusanu, mpawo akasanzyigwa a cell enrichment buffer. Isampulu yakainda mumulongo wa LS (Miltenyi), mpawo zibeela zibotu azibi zilayobolwa. Mukucheesya chiindi akuyungizya ntaamu yakubukulusya maselu, cibeela ca CD8 chibelesegwa muchiindi chabili chakuyungizya CD4+, mpawo cibeela cha CD4 chibelesegwa mukuyungizya CD45. Bikka musamu ooyu aachisani muchiindi choonse chakwaandaanya.
Kubamba zitondezyo kuti zibelesyegwe mukulanga-langa zintu zipangwa mumubili, maselu akasanzyigwa chiindi chomwe aamunyo uutontola mbuli aisi, mpawo 1 ml ya 80% methanol yakabikkwa kuli musamu umwi aumwi, mpawo yakazungaanizyigwa akubikkwa mu nitrogen. Izyakutondezya zyakabikkwa muziindi zyotatwe zyakutontola akutontola mpawo zyakabikkwa mumulilo a 14,000 rpm kwamaminiti ali kkumi amusanu a 4°C. Maanzi aali atalaa nyika aajisi zintu zipangwa mumubili alazwa mumuwo kusikila akayume. Ma metabolites akasandulwa alimwi mu 50 μl ya 0.03% ya formic acid, akuzungaanizyigwa kuti aswaangane, mpawo akuzungaanizyigwa kuti agwisye tombe.
Gwisya zintu zipangwa mumubili mbuli mbokwaambidwe atala aawa. Bweza maanzi aali atalaa nyika akubika mubbodela lya chromatography lyamaanzi libeleka obotu kuti livwuntauzye metabolomics. Belesya nzila yakusilisya iitayeeyelwi kusilika sampulu imwi aimwi amweelwe wamaseelo uukozyenye kuchitila kutabilila ntenda zyakuvwula. Twakachita bweendelezi bwakulanga-langa zintu zipangwa munyika yoonse zyakasimbwa musule a AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Kuvwuntauzya kwa chromatographic akuswaanganya busena bwaatala kwakachitwa kubelesya MultiQuant version 2.1 software (Applied Biosystems SCIEX).
Mweelwe wa ion wa 1000 wakabelesegwa kweezyeezya mpindu ya metabolite yakasweeka, mpawo TIC ya sampulu imwi aimwi yakabelesegwa kubala busena bwa metabolite imwi aimwi yakajanwa kuchitila kululamika kucinca kwakaletwa akubelesya zibelesyo kuzwa mukubamba sampulu. Nikwakainda TIC, MetaboAnalystR(51) (default parameter) yakabelesegwa kusanduka kwa logarithmic antoomwe a automatic norm line scaling. Twakabelesya PCA antoomwe a vegan R package kuchita buvwuntauzi bwakuvwuntauzya kwiimpana kwa metabolome aakati kamisyobo yasampulu, mpawo twakabelesya buvwuntauzi bwakuvwula kwazibeela kuvwuntauzya balwazi. Belesya nzila ya Ward kupanga maapu ya heat dendrogram kuti ubunganye musinzo wa Euclidean aakati kazitondezyo. Twakabelesya limma (52) aatala akuvwula kwa metabolite kuti tuzyibe bunji bwa metabolite bwiindene-indene mumusyobo woonse wamaseelo amubusena busyoonto. Kutegwa bupanduluzi bube buubauba, twabelesya mweelwe waakati-kati wabantu kuti tubone nzila, mpawo tulangisisya misyobo yamaseelo aali mubusena busyoonto mbuli kabunga kamwi akamwi (n = tubunga tuli 6); mukusunka bupati, twakachita zipimyo zyotatwe zyakwiinduluka ku metabolite imwi aimwi Kutegwa kutabi kwiinduluka kwakubeja, mulwazi wakabikkilizyigwa mbuli chintu chisinkila mubupange bwa limma. Kuti tubone kwiimpana kwama metabolites aakati kabalwazi baindene, twakachincha nzila ya limma kubikkilizya abalwazi munzila iitacinci. Twaamba mbociyandika kwiimpana kwakaambwa kale aakati kamusyobo wamaseelo abusena busyoonto bwa Padj <0.05 (kululamika kwa Benjamini-Hochberg).
Musule akuyungizigwa kwanguzu kubelesya Miltenyi Dead Cell Removal Kit (>80% viability), kutobelezya kwa selo lyomwe kwakachitwa kuzibeela zyoonse zya ascites a zilonda kubelesya nzila ya 10x 5′gene expression protocol. Milandu yosanwe yakajisi zilonda zyeelene a ascites yakavwuntauzyigwa, nikuba kuti buumi busyoonto kuzwa kuchilonda chomwe kwakakasya kubikkilizyigwa. Kuti tujane kusala kwabalwazi biingi, twakaswaanganya zitondezyo zyamulwazi umwi awumwi munzila zya 10x chromium controller, mpawo twakalanga-langa busena bwa ascites a zilonda zyoonse zyobile. Musule akubamba [Illumina HiSeq 4000 28×98 bp paired end (PE), Quebec genome; mweelwe waakati-kati wa 73,488 a 41,378 zyakubala kuli selo lyomwe lyacilonda a ascites]], twakabelesya CellSNP a Vireo (53) (kweendelana a CellSNP mbuli I common human SNP (VCF) yakapegwa a GRCh38 ilapegwa zyina lyasikusanga. Tubelesya SNPRelate kuzyiba zyina lyaafwaafwi (IBS) musyobo wabachiswa (IBS), kutabikkilizya maselu aatabikkidwe antoomwe amaselu aazyibidwe kuti ngama duplexes alimwi abasikusanga bakozyenye aakati ka ascites amasampuli aazilonda (54 Aawo amulimu ooyu, twakabamba zibeela zyotatwe zyamaselu miingi muchilonda amuma ascites kuti tukonzye kulanga-langa. (2792 antoomwe a 4183 maselu kuzwa kuchilonda a ascites, kweendelana) mukulanga-langa twabelesya igraph's (57) Louvain clustering of shared nearest neighbor network (SNN) kweendelanya a Jaccard distance ku cluster cells kwiinda mukutondezya. maseelo alapandululwa kwiinda mukutondezya CD8A a GZMA, kakutalangwi tubunga tusyoonto tujisi ma protein masyoonto. Twakajana data yakamwaigwa ya Izar et al.
PBMC zyakaandaanizyigwa kuzwa kuzintu zyaandaanya maseelo aagazi (STEMCELL Technologies) a Ficoll gradient density centrifugation. Maseelo aa CD3 + akagwisigwa kuzwa ku PBMC kubelesya CD3 beads (Miltenyi). Mukubako naa kutabako kwa MNA, maseelo aa CD3+ akali kubelesegwa a CD3 (5μg/ml), CD28 (3μg/ml) a IL-2 (300 U/ml; Proleukin). Mubuzuba bwamamanino bwakuyungizya, buumi (Fixable Viability Dye eFluor450, eBioscience) akuvwula (123count eBeads, Thermo Fisher Scientific) zyakali kulinguwe kwiinda mukubelesya nzila ya flow cytometry. Langa mulimo wa effector kwiinda mukusungwaazya maselu a PMA (20 ng/ml) antoomwe a ionomycin (1μg/ml) a GolgiStop kwamahola one, mpawo ulangisisye CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4) , BioLegend) antoomwe a TNFα-fluoresce isothiyanate (TCb1) BD). Amusungwaazye qPCR antoomwe a ChIP maselu a PMA (20 ng/ml) antoomwe a ionomycin (1μg/ml) kwamawoola one. Maanzi aa ELISA akabungikwa kakutanaba a PMA (20 ng/ml) antoomwe a ionomycin (1 μg/ml) kwamawoola one.
Tobela nzila yasikupanga kuti ukonzye kwaandaanya RNA kobelesya RNeasy Plus Mini Kit (QIAGEN). Belesya QIAshredder (QIAGEN) kuchita kuti musamu ube omwe. Belesya RNA iijisi nguzu kapati kuya ku cDNA kit (Thermo Fisher Scientific) kupanga DNA (cDNA). Belesya TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) kuti ubone mweelwe wamajini (kweendelana amalailile aabasikupanga) aama probe aaya: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427555m15S (AX1), Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] a Hs01010726_m1 (SLC22A2). Isampulu zyakabelesegwa a StepOnePlus real-time PCR system (Applied Biosystems) (Applied Biosystems) mu MicroAmp fast optical 96-well reaction plate (Applied Biosystems) antoomwe a MicroAmp optical film. Kufumbwa mweelwe wa Ct wiinda ku 35 ulabonwa kuti uli atala aachiimo chakuzyiba mpawo ulabikkwa kuti taukonzyi kuzyibwa.
Chita ChIP mbuli mbokwaambidwe musule (58). Mubufwiifwi, maselu akasilikwa a formaldehyde (kumamanino 1.42%) mpawo akabikkwa mubusena bwakupya kwamaminiti aali kkumi. Belesya musamu wakuzimba uuyungizyidwe (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl a 0.1% NP-40) aa aisi kwamaminiti aali kkumi, mpawo ubwezelezye mu immunoprecipitation buffer mbuli mbokwaambidwe (58 ). Isampulu mpawo yakabikkwa mumumuni kwiinda mukubelesya nzila zitobela: ziindi zili kkumi (20 1-second pulses) antoomwe achiindi chamasekondi aali makumi aane. Bikka ChIP-grade immunoglobulin G (Cell Signaling Technology; 1μl), histone H3 (Cell Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) antoomwe a SP1 (Cell Signaling Technology; 3μl) antibodies antoomwe asampulu a 4°CC kuzungaanya masiku woonse. Bikka mapulotini A (Thermo Fisher Scientific) antoomwe asampulu a 4°C akuzungaanya asyoonto-syoonto kwawoola lyomwe, mpawo kobelesya mapulotini aa chelex (Bio-Rad) kuvwuzya DNA, mpawo kobelesya mapulotini K (Thermo Fisher) kugaya mapulotini. TNFα promoter yakajanwa aPCR: kuya kunembo, GGG TAT CCT TGA TGC TTG TGT; mukwiimpana, GTG CCA ACA ACT GCC TTT ATA TG (207-bp chipangwa). Zifwanikiso zyakabambwa a Image Lab (Bio-Rad) mpawo zyakapimwa kubelesya pulogilamu ya ImageJ.
Maanzi aakukomezya maseelo akabungikwa mbuli mbokwaambidwe atala aawa. Kusala kwakachitwa kweendelana anzila zyabasikupanga zya human TNFα ELISA kit (Invitrogen), human IL-2 ELISA kit (Invitrogen) antoomwe a human IFN-γ ELISA kit (Abcam). Kweelana amalailile aabasikupanga, maanzi aakasyaala akasanzyigwa 1:100 kuti abone TNFα a IL-2, antoomwe a 1:3 kuti abone IFN-γ. Belesya EnVision 2104 Multilabel Reader (PerkinElmer) kupima kujatana a 450 nm.
PBMC zyakaandaanizyigwa kuzwa kuzintu zyaandaanya maseelo aagazi (STEMCELL Technologies) a Ficoll gradient density centrifugation. Maseelo aa CD3 + akagwisigwa kuzwa ku PBMC kubelesya CD3 beads (Miltenyi). Mukubako naa kutabako kwa MNA, maseelo aa CD3+ akali kubelesegwa a CD3 (5μg/ml), CD28 (3μg/ml) a IL-2 (300 U/ml; Proleukin) kwamazuba otatwe. Nikwakainda mazuba otatwe, maselu akabungikwa akusanzyigwa a 0.9% saline, mpawo pellet yakabikkwa mumuwo. Kubala kwamaseelo kwakachitwa a flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) kubelesya 123count eBeads.
Gwisya zintu zipangwa mumubili mbuli mbokwaambidwe atala aawa. Ikugwisigwa kwakayuminina kwakabambululwa abulemu bwa 4000 cell equivalents/μl. Langa musamu kwiinda mukubelesya nzila yakujokela musule (1290 Infinity II, Agilent Technologies, Santa Clara, CA) antoomwe a CORTECS T3 column (2.1×150 mm, bupati bwazibeela 1.6-μm, bupati bwamapopwe 120-Å; #186008500, Waters). Polar mass spectrometer (6470, Agilent), oomo electrospray ionization moibeleka munzila iili kabotu. Chibeela chakweendeenda A chili a 0.1% asidi yafoomu (mu H2O), cibeela chakweendeenda B chili a 90% acetonitrile, 0.1% asidi yafoomu. I LC gradient ili 0 kusikila ku 2 maminiti ku 100% A, 2 kusikila ku 7.1 maminiti ku 99% B, antoomwe a 7.1 kusikila ku 8 maminiti ku 99% B. Mpawo ubike antoomwe kkolumini a mobile phase A amweelwe wa 0.6 ml/min kwamaminiti otatwe. . Mweelwe wakuyoya ngu 0.4ml/min, mpawo ŋanda yamulongo ilapya kusika ku 50°C. Belesya MNA's pure chemical standard (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) kuti ujane chiindi chakubamba (RT) akusanduka (RT = 0.882 maminiti, kusanduka 1 = 137→94.1, kusanduka 2 = 137→92 , Kusanduka = ​​13→77). Eelyo kucinca koonse kutatu nokwachitika muchiindi cheelede, kucinca 1 kubelesegwa mukupima kuchitila kuti kube kusalazya. Mulongo wa MNA (Toronto Research Chemical Company) wakapangwa akusanzya ziindi zili musanu azibili (1 mg/ml) kuti kujane zyeelelo zya 0.1, 1.0, 10 a 100 ng/ml antoomwe a 1.0 a 10 μg/ml mbuli maanzi. Mugaano wakuzyiba ngu 1 ng/ml, mpawo nsandulo iili akati ka 10 ng/ml a 10μg/ml. Kunjizya kuli koonse kwama microlita obile aamusamu abulemu kubelesyegwa mukulanga-langa kwa LC/MS, mpawo musamu uusanganizidwe wakulanga-langa bubotu ulabikkwa muzibeela zili musanu atutatu kuchitila kuti nzila yakulanga-langa itazungaani. Bwiinguzi bwa MNA bwamaselu woonse aakasilikwa a MNA bwakali mukati kachiimo cheenzu cha assay. Kuvwuntauzya data kwakachitwa kubelesya pulogilamu ya MassHunter (v9.0, Agilent).
Izyalani lyabili lya αFR-CAR lyakazwa kuli Song abamwi. (59). Mubufwiifwi, chipangwa eechi chili azintu zitobela: CD8a musololi, cibeela chamuntu αFR-specific single-chain variable fragment, CD8a hinge antoomwe a transmembrane region, CD27 intracellular domain antoomwe a CD3z intracellular domain. Mulongo wa CAR woonse wakapangwa a GenScript, mpawo wakabikkwa mumulongo wabili wa lentiviral expression vector kujulu lya GFP expression cassette yakabelesegwa kulanga-langa bubotu bwa transduction.
Lentivirus ilapangwa kwiinda mukunjizya maselu aa HEK293T [American Type Culture Collection (ATCC); yakakomezyegwa mu Dulbecco's modified Eagle medium iijisi 10% fetal bovine serum (FBS) antoomwe a 1% PenStrep, mpawo yakabelesya CAR-GFP vector antoomwe a The packaging plasmids (psPAX2 a pMD2.G, Addgene) yakabelesya lipofection amine (Sigma-Aldrich). Maanzi aajisi kazunda akabungikwa 48 a 72 mahola kuzwa nikwakabikkwa, akasefwa, mpawo akabikkwa antoomwe a ultracentrifugation. Bika ma virus aayungizyidwe a -80°C mane akasandulwe.
PBMC zilaandaanizyigwa kuzwa kuzintu zipa kuti bulowa bube kabotu (STEMCELL Technologies) kwiinda mukubelesya Ficoll gradient density centrifugation. Belesya CD8 microbeads (Miltenyi) yakusala kabotu kuti ugwisye CD8+ maselu kuzwa ku PBMC. Amusungwaazye maseelo aa T a TransAct (Miltenyi) amu TexMACS medium [Miltenyi; yakayungizigwa a 3% yabulowa bwamuntu butakwe mulilo, 1% ya PenStrep a IL-2 (300 U/ml)]. Nokwakainda mawoola aali makumi obile aane kuzwa kukusungwaazyigwa, maselu aa T akasandulwa a lentivirus (10 μl ya virus supernatant kuli 106 maselu). 1 kusikila kuli 3 mazuba kuzwa nikwakasandulwa a Cytek Aurora (a FSC (Kumwaika Kunembo)/SSC (Kumwaika Kumbali), Singlet, GFP+), langa GFP kutondezya kwamaseelo kutondezya kugwasya kwakusanduka kwa 30%.
Maseelo aa CAR-T akakomezyegwa kwamahola aali makumi obilwe atune (24) mu Immunocult (STEMCELL Technologies; yakayungizigwa a 1% PenStrep) muchiimo eechi: tiiyakasilidwe, yakasilikwa a 250 μM adenosine naa 10 mM MNA. Musule akusilikwa, maselu aa CAR-T akasanzyigwa a PBS mpawo akasanganyigwa a 20,000 maselu aa SK-OV-3 [ATCC; mu McCoy 5A medium (Sigma-Aldrich) yakayungizigwa a 10% FBS antoomwe a 1% PenStrep a 10: Mweelwe wa 1 wakayungizigwa ziindi zyotatwe mu Immunocult medium. Maseelo aa SK-OV-3 antoomwe amaseelo aa SK-OV-3 aakakosolwa a digitalis saponin (0.5mg/ml; Sigma-Aldrich) akabelesegwa mbuli zyintu zitali kabotu azitali kabotu, kweendelana. Musule amawoola aali makumi obilwe atune (24) aakulima antoomwe, maanzi aakasyaala akabungikwa mpawo lactate dehydrogenase (LDH) yakapimwa kweendelana amalayilile aabasikupanga (LDH Glo Cytotoxicity Assay Kit, Promega). I LDH supernatant yakasanzyigwa 1:50 mu LDH buffer. Mweelwe wakujaya wakapimwa kubelesya nzila eeyi: mweelwe wakujaya = mweelwe wakululamika / mweelwe mupati wakujaya x 100%, aawo mweelwe wakululamika = co-culture-T cells luzutu, mpawo mweelwe mupati wakujaya = kweendelezya kubotu-kweendelezya kutali kabotu.
Mbuli mbokwaambidwe mumalembe naakuti zibelesyo anzila, kobelesya GraphPad Prism 8, Microsoft Excel naakuti R v3.6.0 mukuvwuntauzya. Kuti zitondezyo zyiingi zyabwezegwa kuzwa kumulwazi omwe (mbuli ascites a tumor), tulabelesya paired t test naakuti tubikkilizye mulwazi mbuli chintu chitalibonyi muchikozyano cha linear naakuti generalized model mbuli mbukweelede. Mukuvwuntauzya kwa metabolomics, musunko wakuyandika ulachitwa ziindi zyotatwe.
Kuti ujane zimwi zilembedwe muchibalo eechi, langa http://advances.sciencemag.org/cgi/content/full/7/4/eabe1174/DC1
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Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. Dephrini (R.R.R. Bellnis), Dephardnis Deph. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T.
MNA ilagwasya mukulesya malwazi aa T cells mpawo yiiminina nzila iikonzya kubelesyegwa kusilika kkansa yabantu.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. Dephrini (R.R.R. Bellnis), Dephardnis Deph. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T.
MNA ilagwasya mukulesya malwazi aa T cells mpawo yiiminina nzila iikonzya kubelesyegwa kusilika kkansa yabantu.
©2021 Kabunga ka Amelika kakusumpula sayensi. zyeelelo zyoonse zilikwabilidwe. AAAS mweenzinyina wa HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef a COUNTER. ScienceAdvances ISSN 2375-2548.