Immunomodulatory metabolites kye kintu ekikulu mu tumor microenvironment (TME), naye okuggyako ebitonotono, ebizikwatako okusinga tebimanyiddwa. Wano, twekenneenya ebizimba n’obutoffaali bwa T okuva mu bizimba n’amasasi g’abalwadde abalina kookolo w’omusaayi ow’omutindo ogwa waggulu (HGSC) okulaga metabolome y’ebitundu bino eby’enjawulo ebya TME. Ascites n’obutoffaali bw’ekizimba bulina enjawulo nnene mu metabolite. Bw’ogeraageranya n’obulwadde bwa ascites, obutoffaali bwa T obuyingira mu bizimba bugaggawalidde nnyo mu 1-methylnicotinamide (MNA). Wadde ng’omutindo gwa MNA mu butoffaali bwa T guli waggulu, okwolesebwa kwa nicotinamide N-methyltransferase (enziyiza ekola okukyusa ebibinja bya methyl okuva mu S-adenosylmethionine okudda mu nicotinamide) kukoma ku fibroblasts n’obutoffaali bw’ekizimba. Mu nkola, MNA ereetera obutoffaali bwa T okufulumya ekizimba ekitumbula ekizimba ekiyitibwa cytokine tumor necrosis factor alpha. N’olwekyo, TME-derived MNA eyamba mu kulungamya abaserikale b’omubiri mu butoffaali bwa T era ekiikirira ekiruubirirwa ekiyinza okukolebwa mu bujjanjabi bw’abaserikale b’omubiri okujjanjaba kookolo w’omuntu.
Ebirungo ebikyusa ebiriisa ebiva mu bizimba bisobola okuba n’ekikolwa eky’amaanyi eky’okuziyiza ku buziyiza bw’ekizimba, era obujulizi obusinga obungi bulaga nti era bisobola okukola ng’amaanyi amakulu agavuga obulwadde okukula (1). Ng’oggyeeko ekikolwa kya Warburg, emirimu egyakakolebwa gitandise okulaga embeera y’okukyusakyusa emmere y’obutoffaali bw’ekizimba n’enkolagana yaayo n’embeera y’abaserikale b’omubiri mu butonde obutono obw’ekizimba (TME). Okunoonyereza ku bikolwa by’ebibe n’obutoffaali bwa T obw’omuntu kulaga nti enkyukakyuka ya glutamine (2), enkyukakyuka ya oxidative (3) n’enkyukakyuka ya glucose (4) zisobola okukola nga zeetongodde ku bibinja by’obutoffaali bw’abaserikale b’omubiri eby’enjawulo. Ebirungo ebikyukakyuka ebiwerako mu makubo gano biziyiza omulimu gw’obutoffaali bwa T ogw’okulwanyisa ebizimba. Kikakasibwa nti okuziyiza kwa coenzyme tetrahydrobiopterin (BH4) kuyinza okwonoona okukula kw’obutoffaali bwa T, era okweyongera kwa BH4 mu mubiri kuyinza okutumbula okuddamu kw’abaserikale b’omubiri abalwanyisa ebizimba nga kutabaganyizibwa CD4 ne CD8. Okugatta ku ekyo, ekikolwa ekiziyiza obusimu obuziyiza endwadde ekya kynurenine kisobola okununulibwa nga kiweebwa BH4 ( 5 ). Mu isocitrate dehydrogenase (IDH) mutant glioblastoma, okufulumya enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) kuziyiza okukola kw’obutoffaali bwa T, okukula n’okusengejja obutoffaali Activity (6). Gye buvuddeko, kyalagibwa nti methylglyoxal, ekiva mu glycolysis, kikolebwa obutoffaali obuziyiza obw’ensibuko ya myeloid, era okutambuza obutoffaali bwa T obwa methylglyoxal kuyinza okuziyiza enkola y’obutoffaali bwa T obukola. Mu bujjanjabi, neutralization ya methylglyoxal esobola okuvvuunuka emirimu gy’obutoffaali obuziyiza myeloid-derived suppressor cells (MDSC) era synergistically okutumbula checkpoint blockade therapy mu mouse models (7). Okunoonyereza kuno okugatta kuggumiza omulimu omukulu ogw’ebirungo ebikyusa ebiriisa ebiva mu TME mu kulungamya enkola n’emirimu gy’obutoffaali bwa T.
Obutakola bulungi bwa T cell bubadde bumanyiddwa nnyo mu kookolo w’enkwaso (8). Kino kiva ku ngeri y’enkyukakyuka y’emmere esangibwa mu hypoxia n’emisuwa gy’ekizimba egitaali gya bulijjo (9), ekivaamu okukyusa glucose ne tryptophan okufuuka ebivaamu nga lactic acid ne kynurenine. Lactate esukkiridde ey’ebweru w’obutoffaali ekendeeza ku kukola interferon-γ (IFN-γ) era evuga okwawukana kw’ebibinja ebitono ebiziyiza myelosuppressive ( 10 , 11 ). Okulya tryptophan kiziyiza butereevu okukula kw’obutoffaali bwa T era kiziyiza obubonero obukwata obutoffaali bwa T ( 12-14 ). Wadde nga bino bye twetegereza, omulimu munene ogwetoolodde enkyukakyuka y’abaserikale b’omubiri gwakolebwa mu vitro T cell culture nga tukozesa optimized media, oba okukoma ku homologous mouse models in vivo, nga tewali n’emu ku zo eraga mu bujjuvu heterogeneity ya kookolo w’omuntu ne Physiological macro ne micro environment.
Ekintu ekitera okulabika ku kookolo w’enkwaso kwe kusaasaana mu lubuto n’okulabika ng’obulwadde bwa ascites. Okukuŋŋaanyizibwa kw’amazzi g’obutoffaali mu ascites kukwatagana n’obulwadde obugenda mu maaso n’okuteebereza obubi ( 15 ). Okusinziira ku lipoota, ekisenge kino eky’enjawulo tekirina mukka gwa mukka, kirimu emiwendo mingi egy’ekirungo ky’okukula kw’emisuwa (VEGF) ne indoleamine 2,3-dioxygenase (IDO), era nga kiyingizibwamu obutoffaali obulung’amya T n’obutoffaali obuziyiza myeloid (15-18). Embeera y’enkyukakyuka y’emmere mu mubiri (ascites) eyinza okuba ey’enjawulo ku y’ekizimba kyennyini, kale okuddamu okukola pulogulaamu y’obutoffaali bwa T mu kifo ky’omu lubuto tekitegeerekeka bulungi. Okugatta ku ekyo, enjawulo enkulu n’obutafaanagana wakati wa ascites n’ebirungo ebikyusa ebiriisa ebibeera mu mbeera y’ekizimba biyinza okulemesa okuyingira kw’obutoffaali bw’abaserikale b’omubiri n’okukola kwabwo ku bizimba, era okunoonyereza okulala kwetaagibwa.
Okusobola okugonjoola ebizibu bino, twakola enkola ya sensitive cell separation and liquid chromatography tandem mass spectrometry (LC-MS/MS) okunoonyereza ku bika by’obutoffaali eby’enjawulo (omuli CD4 + ne CD8 + T cells) awamu ne munda ne wakati w’ebizimba Its metabolites span cells in the same ascites and tumor environment of the patient. Tukozesa enkola eno nga tukwataganye ne high-dimensional flow cytometry ne single-cell RNA sequencing (scRNA-seq) okusobola okuwa ekifaananyi ekigonjoddwa ennyo eky’embeera y’enkyukakyuka y’emmere y’ebika bino ebikulu. Enkola eno yalaga okweyongera okw’amaanyi mu mutindo gwa 1-methylnicotinamide (MNA) mu butoffaali bwa T obuzimba, era okugezesa mu vitro kwalaga nti ekikolwa ky’okukyusa obusimu obuziyiza endwadde ekya MNA ku nkola y’obutoffaali bwa T kyali tekimanyiddwa. Okutwaliza awamu, enkola eno eraga enkolagana y’omubiri wakati w’ebizimba n’obutoffaali bw’abaserikale b’omubiri, era etuwa amagezi ag’enjawulo ku bikozesebwa mu kulungamya abaserikale b’omubiri, ebiyinza okuba eby’omugaso mu kujjanjaba T cell-based ovarian cancer immunotherapy Emikisa gy’obujjanjabi.
Twakozesa high-dimensional flow cytometry okugera omulundi gumu omuwendo gwa glucose uptake [2- (N- (7-nitrophenyl-2-oxa-1,3-diaza-4-yl) amino) -2-deoxyglucose (2-NBDG) n'emirimu mitochondrial [MitoTracker Deep Red (MT DR)] (7, 19, 20) bali ku mabbali typical obubonero obwawula obutoffaali bw’abaserikale b’omubiri n’obutoffaali bw’ekizimba (Table S2 and Figure S1A). Okwekenenya kuno kwalaga nti bw’ogeraageranya n’obutoffaali bwa T, obutoffaali bwa ascites n’obuzimba bulina emiwendo gya glucose egy’okutwala waggulu, naye nga bulina enjawulo entono mu mitochondrial activity. Omutindo gwa glucose ogwa wakati ogw’obutoffaali bw’ekizimba [CD45-EpCAM (EpCAM)+] gukubisaamu emirundi esatu oba ena okusinga ogw’obutoffaali bwa T, ate ogwa wakati ogwa glucose ogutwalibwa mu butoffaali bwa CD4 + T gukubisaamu emirundi 1.2 ogw’obutoffaali bwa CD8 + T, ekiraga nti Tumor infiltrating lymphocytes (TIL) zirina ebyetaago eby’enjawulo eby’okukyusakyusa ebirime ne mu TME y’emu (Figure 1A). Okwawukana ku ekyo, emirimu gya mitochondrial mu butoffaali bw’ekizimba gifaananako n’egya CD4 + T cells, era emirimu gya mitochondrial egy’ebika by’obutoffaali byombi giri waggulu okusinga egy’obutoffaali bwa CD8 + T (Figure 1B). Okutwaliza awamu, ebivuddemu bino biraga omutindo gw’enkyukakyuka mu mubiri. Enkola y’okukyusakyusa emmere y’obutoffaali bw’ekizimba eri waggulu okusinga ey’obutoffaali bwa CD4 + T, ate emirimu gy’okukyusakyusa emmere y’obutoffaali bwa CD4 + T giri waggulu okusinga egy’obutoffaali bwa CD8 + T. Wadde nga bino ebikosa mu bika by’obutoffaali, tewali njawulo nkyukakyuka mu mbeera y’okukyusakyusa emmere y’obutoffaali bwa CD4 + ne CD8 + T oba ebipimo byabwe ebikwatagana mu ascites bw’ogeraageranya n’ebizimba (Figure 1C). Okwawukana ku ekyo, mu kitundu ky’obutoffaali bwa CD45, ekitundu ky’obutoffaali bwa EpCAM+ mu kizimba kyeyongera bw’ogeraageranya n’obulwadde bwa ascites (Figure 1D). Twalaba n’enjawulo entegeerekeka mu nkyukakyuka y’emmere wakati w’ebitundu by’obutoffaali bwa EpCAM+ ne EpCAM-. Obutoffaali bwa EpCAM+ (tumor) bulina okutwala glucose okungi n’emirimu gya mitochondrial okusinga obutoffaali bwa EpCAM-, nga kino kisinga nnyo emirimu gy’okukyusakyusa emmere ya fibroblasts mu butoffaali bw’ekizimba mu TME (Ekifaananyi 1, E ne F).
. (C) Omugerageranyo gw’obutoffaali bwa CD4 + ne CD8 + (obw’obutoffaali bwa CD3 + T) mu ascites n’ekizimba. (D) Ekitundu ky’obutoffaali bw’ekizimba kya EpCAM + mu ascites n’ekizimba (CD45−). (E ne F) EpCAM + CD45-ekizimba ne EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) n’emirimu gya mitochondrial (MitoTracker emmyufu enzirugavu) (F) graphs ezikiikirira (ku kkono) ne tabulated data ( Ku ddyo) Ascites n’obutoffaali bw’ekizimba. (G) Grafu ezikiikirira CD25, CD137 ne PD1 okwolesebwa nga bakozesa flow cytometry. (H ne I) Okwolesebwa kwa CD25, CD137 ne PD1 ku butoffaali bwa CD4 + T (H) n’obutoffaali bwa CD8 + T (I). (J ne K) Naive, central memory (Tcm), effector (Teff) ne effector memory (Tem) phenotypes nga zeesigamiziddwa ku kwolesebwa kwa CCR7 ne CD45RO. Ebifaananyi ebikiikirira (ku kkono) n’ebikwata ku bipande (ku ddyo) eby’obutoffaali bwa CD4 + T (J) n’obutoffaali bwa CD8 + T (K) mu ascites n’ebizimba. Emiwendo gya P egyasalibwawo n’okugezesebwa kwa t-okugatta (*P<0.05, **P<0.01 ne ***P<0.001). Layini ekiikirira abalwadde abakwatagana (n = 6). FMO, okumasamasa okuggyako ekimu; MFI, amaanyi g’ekitangaala eky’omu makkati.
Okwongera okwekenneenya kwalaga enjawulo endala ez’amaanyi wakati w’embeera ya T cell phenotypic egonjoddwa ennyo. Okujjukira okukola (Ekifaananyi 1, G okutuuka ku I) n’okujjukira okukola (Ekifaananyi 1, J ne K) mu bizimba kutera nnyo okusinga ascites (ekitundu ky’obutoffaali bwa CD3 + T). Mu ngeri y’emu, okwekenneenya enkula y’omubiri (phenotype) nga tuyita mu kwolesebwa kw’obubonero bw’okukola (CD25 ne CD137) n’obubonero bw’okukendeera [programmed cell death protein 1 (PD1)] kyalaga nti wadde ng’engeri z’enkyukakyuka y’emmere y’ebika bino za njawulo (Ekifaananyi S1, B okutuuka ku E) , Naye tewali njawulo nnene mu nkyukakyuka y’emmere yalabibwa bulijjo wakati w’ebitundu ebitono ebitaliimu, ebikola oba eby’okujjukira (Ekifaananyi S1, F okutuuka ku I). Ebivuddemu bino byakakasibwa nga bakozesa enkola z’okuyiga ebyuma okugabanya obutoffaali mu ngeri ey’otoma (21), ekyayongera okulaga nti waliwo omuwendo omunene ogw’obutoffaali bw’obusimu bw’amagumba (CD45 + / CD3- / CD4 + / CD45RO +) mu ascites y’omulwadde (Figure S2A ). Mu bika by’obutoffaali byonna ebyazuulibwa, omuwendo guno ogw’obutoffaali bwa myeloid gwalaga okutwalibwa kwa glucose okusinga obungi n’emirimu gya mitochondrial (Figure S2, B to G). Ebivudde mu kunoonyereza kuno biraga enjawulo ey’amaanyi mu nkyukakyuka y’emmere wakati w’ebika by’obutoffaali ebingi ebisangibwa mu ascites n’ebizimba mu balwadde ba HGSC.
Okusoomoozebwa okukulu mu kutegeera engeri z’obutafaali obutambula mu TIL kwe kwetaaga okwawula sampuli z’obutoffaali bwa T obw’obulongoofu obumala, omutindo n’obungi okuva mu bizimba. Okunoonyereza okwakakolebwa kulaga nti enkola z’okusunsula n’okugaggawaza obululu nga zeesigamiziddwa ku flow cytometry ziyinza okuvaako enkyukakyuka mu cellular metabolite profiles ( 22-24 ). Okuvvuunuka ekizibu kino, twalongoosa enkola y’okugaggawaza obululu okwawula n’okwawula TIL okuva mu kookolo w’enkwaso y’omuntu eyalongoosebwa nga tetunnaba kwekenneenya nga tukozesa LC-MS/MS (laba Ebikozesebwa n’Enkola; Ekifaananyi 2A). Okusobola okwekenneenya okutwalira awamu enkola eno ku nkyukakyuka mu metabolite, twageraageranya metabolite profiles z’obutoffaali bwa T obukozesebwa abagaba obulamu oluvannyuma lw’omutendera ogwo waggulu ogw’okwawula obululu n’obutoffaali obutayawulwamu obululu naye nga busigala ku ice. Okwekenenya kuno okw’okulondoola omutindo kwazuula nti waliwo enkolagana ey’amaanyi wakati w’embeera zino ebbiri (r = 0.77), era okuddiŋŋana okw’ekikugu okw’ekibinja ky’ebirungo ebikyusa ebiriisa 86 kulina okuddiŋŋana okw’amaanyi (Ekifaananyi 2B). N’olwekyo, enkola zino zisobola okukola okwekenneenya okutuufu okw’ebirungo ebikyukakyuka mu butoffaali obugenda mu kugaggawala kw’ekika ky’obutoffaali, bwe kityo ne ziwa omukutu ogusooka ogw’obulungi obw’amaanyi okuzuula ebirungo ebikyukakyuka ebitongole mu HGSC, bwe kityo ne kisobozesa abantu okufuna okutegeera okw’amaanyi ku butonde bw’obutoffaali Enteekateeka y’okukyusakyusa eby’okwegatta.
(A) Ekifaananyi ekiraga okugaggawala kw’obululu bwa magineeti. Nga tebannaba kwekenneenya mu LC-MS/MS, obutoffaali bujja kuyita mu lawundi ssatu ez’omuddiring’anwa ez’okugaggawaza obululu bwa magineeti oba okusigala ku ice. (B) Enkola y’ekika ky’okugaggawaza ku bungi bw’ebirungo ebikyusa ebiriisa. Average y’ebipimo bisatu ku buli kika ky’okugaggawaza ± SE. Layini enzirugavu ekiikirira enkolagana ya 1:1. Enkolagana wakati w’ekibiina (ICC) ey’ebipimo ebiddiŋŋanwa eragiddwa mu kabonero k’ekisiki. NAD, ekirungo kya nicotinamide adenine dinucleotide. (C) Schematic diagram y’enkola y’emirimu gy’okwekenneenya metabolite y’omulwadde. Ascites oba ebizimba bikung’aanyizibwa okuva mu balwadde ne bikuumibwa mu cryopreserved. Ekitundu ekitono ekya buli sampuli kyakeberebwa mu nkola ya flow cytometry, ate sampuli ezisigaddewo zaayita mu lawundi ssatu ez’okugaggawaza obutoffaali bwa CD4+, CD8+ ne CD45-. Ebitundu by’obutoffaali bino byakeberebwa nga bakozesa LC-MS/MS. (D) Maapu y’ebbugumu ey’obungi bw’ebirungo ebikyusa ebiriisa ebituufu. Dendrogram ekiikirira okukuŋŋaanyizibwa kwa Ward okw’amabanga ga Euclidean wakati wa sampuli. (E) Principal component analysis (PCA) of sample metabolite map, okulaga ebikoppi bisatu ebya buli sampuli, sampuli okuva mu mulwadde omu ziyungibwa ne layini. (F) PCA y’enkula y’ebirungo ebikyukakyuka (metabolite profile) eya sampuli etegekeddwa ku mulwadde (kwe kugamba, nga tukozesa ekitundu ekitono); ekika kya sampuli kikoma olw’ekisenge ekiwanvu (convex hull). PC1, ekitundu ekikulu 1; PC2, ekitundu ekikulu 2.
Ekiddako, twakozesa enkola eno ey’okugaggawaza okwekenneenya ebirungo ebikyukakyuka 99 mu bitundutundu by’obutoffaali bwa CD4 +, CD8 + ne CD45 mu ascites n’ebizimba ebisookerwako eby’abalwadde mukaaga aba HGSC (Figure 2C, Figure S3A and Table S3 and S4). Omuwendo gw’obutoffaali ogufaayo gukola ebitundu 2% ku 70% ku sampuli ennene eyasooka ey’obutoffaali obulamu, era ekitundu ky’obutoffaali kyawukana nnyo wakati w’abalwadde. Oluvannyuma lw’okwawula obululu, ekitundu ekigaggawalidde eky’amagoba (CD4+, CD8+ oba CD45-) kikola ebitundu ebisukka mu 85% ku butoffaali bwonna obulamu mu sampuli ku kigero. Enkola eno ey’okugaggawaza etusobozesa okwekenneenya obungi bw’obutoffaali okuva mu nkyukakyuka y’ebitundu by’ekizimba ky’omuntu, ekitasoboka kukikola okuva mu sampuli ennene. Nga tukozesa enkola eno, twazuula nti l-kynurenine ne adenosine, bino ebibiri ebimanyiddwa obulungi immunosuppressive metabolites byali waggulu mu tumor T cells oba tumor cells (Figure S3, B ne C). N’olwekyo, ebivuddemu bino biraga obwesigwa n’obusobozi bwa tekinologiya waffe ow’okwawula obutoffaali n’okupima obutoffaali okuzuula ebirungo ebikulu mu biramu mu bitundu by’omulwadde.
Okwekenenya kwaffe era kwalaga okwawukana okw’amaanyi mu nkyukakyuka y’emmere ey’ebika by’obutoffaali munda ne wakati w’abalwadde (Ekifaananyi 2D n’Ekifaananyi S4A). Okusingira ddala, bw’ogeraageranya n’abalwadde abalala, omulwadde 70 yalaga engeri ez’enjawulo mu nkyukakyuka y’emmere (Figure 2E ne Figure S4B), ekiraga nti wayinza okubaawo obutafaanagana obw’amaanyi mu nkyukakyuka y’emmere wakati w’abalwadde. Kinajjukirwa nti bw’ogeraageranya n’abalwadde abalala (liita 1.2 ku 2; Omulongooti S1), omuwendo gwonna ogw’amasasi ogwakung’aanyizibwa mu mulwadde 70 (80 ml) gwali mutono. Okufuga obutafaanagana wakati w’abalwadde mu kiseera ky’okwekenneenya ebitundu ebikulu (okugeza, nga tukozesa okwekenneenya okutali kwa kitundu) kulaga enkyukakyuka ezitakyukakyuka wakati w’ebika by’obutoffaali, era ebika by’obutoffaali ne/oba microenvironment bikuŋŋaanyiziddwa bulungi okusinziira ku metabolite profile (Figure 2F ). Okwekenenya ebirungo ebikyukakyuka (single metabolites) kwassa essira ku bikolwa bino era ne kulaga enjawulo ennene wakati w’ebika by’obutoffaali n’obutonde obutonotono. Kinajjukirwa nti enjawulo esinga okusukkiridde okulabibwa ye MNA, etera okugaggawala mu butoffaali bwa CD45- ne mu butoffaali bwa CD4+ ne CD8+ obuyingira mu kizimba (Ekifaananyi 3A). Ku butoffaali bwa CD4 +, ekikolwa kino kisinga kweyoleka, era MNA mu butoffaali bwa CD8 + nayo erabika ng’ekosebwa nnyo obutonde. Wabula kino si kikulu, kubanga abalwadde basatu bokka ku mukaaga be basobola okwekenneenya obubonero bwa CD8+ mu kizimba. Ng’oggyeeko MNA, mu bika by’obutoffaali eby’enjawulo mu ascites n’ebizimba, ebirungo ebirala ebikyusa ebiriisa ebimanyiddwa obubi mu TIL nabyo biba bya njawulo (Figures S3 and S4). N’olwekyo, data zino ziraga ekibinja ekisuubiza eky’ebirungo ebikyusa obusimu obuziyiza endwadde okwongera okunoonyereza.
(A) Ebirimu ebya MNA ebya bulijjo mu butoffaali bwa CD4+, CD8+ ne CD45- okuva mu ascites n’ekizimba. Ploti ya box eraga wakati (layini), ebanga wakati wa quartile (frame hinge) n’obuwanvu bwa data, okutuuka ku mirundi 1.5 egy’obuwanvu wakati wa interquartile (frame whisker). Nga bwe kinyonyoddwa mu Patient Materials and Methods, kozesa omuwendo gwa limma gw’omulwadde okuzuula omuwendo gwa P (*P<0.05 ne **P<0.01). (B) Ekifaananyi ekiraga enkyukakyuka ya MNA (60). Ebirungo ebikyusa ebiriisa: S-adenosyl-1-methionine; SAH, S-adenosine-1-ekirungo kya homocysteine; NA, eddagala eriyitibwa nicotinamide; MNA, 1-methylnicotinamide, eddagala eriyitibwa eddagala eriyitibwa 1-methylnicotinamide; 2-PY, 1-metil- 2-piridoni-5-kaboksisaayidi; 4-PY, 1-metil-4-piridoni-5-kabokisaayidi; NR, ekirungo kya nicotinamide ribose; NMN, ekirungo kya nicotinamide mononucleotide. Enziyiza (eza kiragala): NNMT, nicotinamide N-methyltransferase; SIRT, ebiwuka ebiyitibwa sirtuins; NAMPT, ekirungo kya nikotinamide phosphoribosyl transferase; AOX1, ekirungo kya aldehyde oxidase 1; NRK, ekirungo kya nicotinamide riboside kinase; NMNAT, nicotinamide mono Nucleotide adenylate transferase, nga kino kirimu ekirungo kya ssennyiga omukambwe; Pnp1, purine nyukiliyosayidi phosphorylase. (C) t-SNE ya scRNA-seq ya ascites (enzirugavu) n’ekizimba (emmyufu; n = abalwadde 3). (D) Okwolesebwa kwa NNMT mu bungi bw’obutoffaali obw’enjawulo obuzuuliddwa nga tukozesa scRNA-seq. (E) Okwolesebwa kwa NNMT ne AOX1 mu SK-OV-3, ekibumba ky’omuntu (HEK) 293T, obutoffaali bwa T n’obutoffaali bwa T obujjanjabiddwa MNA. Okwolesebwa okuzingibwa kulagibwa okusinziira ku SK-OV-3. Enkola y’okwolesebwa ne SEM eragiddwa (n = 6 abagaba abalamu). Emiwendo gya Ct egisukka mu 35 gitwalibwa nga egitazuulibwa (UD). (F) Okwolesebwa kwa SLC22A1 ne SLC22A2 mu SK-OV-3, HEK293T, obutoffaali bwa T n’obutoffaali bwa T obujjanjabiddwa ne 8mM MNA. Okwolesebwa okuzingibwa kulagibwa okusinziira ku SK-OV-3. Enkola y’okwolesebwa ne SEM eragiddwa (n = 6 abagaba abalamu). Emiwendo gya Ct egisukka mu 35 gitwalibwa nga egitazuulibwa (UD). (G) Ebirimu MNA mu butoffaali mu butoffaali bwa T obugaba obulamu obukozesebwa oluvannyuma lw’essaawa 72 nga bufumbiddwa ne MNA. Enkola y’okwolesebwa ne SEM eragiddwa (n = 4 abagaba abalamu).
MNA ekolebwa nga ekyusa ekibinja kya methyl okuva mu S-adenosyl-1-methionine (SAM) okudda mu nicotinamide (NA) nga tuyita mu nicotinamide N-methyltransferase (NNMT; Ekifaananyi 3B). NNMT esukkiridde okulaga mu kookolo w’abantu ow’enjawulo era ekwatagana n’okusaasaana, okulumba n’okusaasaana (25-27). Okusobola okutegeera obulungi ensibuko ya MNA mu butoffaali bwa T mu TME, twakozesa scRNA-seq okulaga obubonero bw’okwolesebwa kwa NNMT mu bika by’obutoffaali mu ascites n’ebizimba by’abalwadde basatu aba HGSC (Table S5). Okwekenenya obutoffaali nga 6,500 kwalaga nti mu mbeera z’amasanda n’ebizimba, okwolesebwa kwa NNMT kwakoma ku bungi bw’obutoffaali bwa fibroblast n’ebizimba obuteeberezebwa (Figure 3, C and D). Kikulu okumanya nti tewali kwolesebwa kwa NNMT kweyoleka mu kibinja kyonna ekiraga PTPRC (CD45 +) (Ekifaananyi 3D ne Ekifaananyi S5A), ekiraga nti MNA ezuuliddwa mu spektrum y’ebirungo ebikyusa ebiriisa eyingiziddwa mu butoffaali bwa T. Okwolesebwa kwa aldehyde oxidase 1 (AOX1) kukyusa MNA mu 1-methyl-2-pyridone-5-carboxamide (2-PYR) oba 1-methyl-4-pyridone-5-carboxamide (4- PYR); Ekifaananyi 3B) era kikoma ku bungi bwa fibroblasts eziraga COL1A1 (Figure S5A), nga byonna awamu biraga nti obutoffaali bwa T tebulina busobozi bwa MNA metabolism eya bulijjo. Enkola y’okwolesebwa kw’obuzaale buno obukwatagana ne MNA yakakasibwa nga bakozesa data y’obutoffaali ey’okubiri eyetongodde okuva mu ascites okuva mu balwadde ba HGSC (Figure S5B; n = 6) (16). Okugatta ku ekyo, okwekenneenya quantitative polymerase chain reaction (qPCR) ku butoffaali bwa T obulamu obugaba obujjanjabiddwa ne MNA kwalaga nti bw’ogeraageranya n’obutoffaali bw’ekizimba ky’enkwaso obufuga SK-OV-3, NNMT oba AOX1 kumpi teyalabikira (Figure 3E). Ebivuddemu bino ebitali bisuubirwa biraga nti MNA eyinza okufulumizibwa okuva mu fibroblasts oba ebizimba mu butoffaali bwa T oburiraanye mu TME.
Newankubadde nga abesimbyewo mulimu famire ya organic cation transporters 1 okutuuka ku 3 (OCT1, OCT2 ne OCT3) encoded soluble carrier 22 (SLC22) famire (SLC22A1, SLC22A2 ne SLC22A3), abayinza okutambuza MNA tebakyategeezeddwa (28) . QPCR ya mRNA okuva mu butoffaali T obulamu obugabi yalaga emitendera emitono egy’okwolesebwa kwa SLC22A1 naye emiwendo egitazuulibwa gya SLC22A2, ekyakakasa nti kyali kyaloopebwa emabegako mu biwandiiko (Figure 3F) (29). Okwawukana ku ekyo, SK-OV-3 ovarian tumor cell line yalaga emitendera egya waggulu egy’ebitambuza byombi (Figure 3F).
Okusobola okugezesa obusobozi bw’obutoffaali bwa T okuba n’obusobozi okunyiga MNA ey’ebweru, obutoffaali bwa T obugaba obulamu bwakuzibwa okumala essaawa 72 nga waliwo ebirungo bya MNA eby’enjawulo. Mu butabeerawo MNA ya bweru, ebirimu mu butoffaali mu MNA tebisobola kuzuulibwa (Ekifaananyi 3G). Wabula, obutoffaali bwa T obukozesebwa obujjanjabiddwa ne MNA ow’ebweru bwalaga okweyongera okusinziira ku ddoozi mu MNA ebirimu mu butoffaali, okutuuka ku 6 mM MNA (Figure 3G). Ekivuddemu kino kiraga nti wadde nga waliwo omutindo omutono ogw’okwolesebwa kw’abatambuza n’obutaba na enzyme enkulu evunaanyizibwa ku nkyukakyuka ya MNA mu butoffaali, TIL ekyasobola okutwala MNA.
Ensengekera y’ebirungo ebikyusa ebiriisa mu butoffaali bwa T bw’abalwadde n’okugezesa okunyiga MNA mu kisenge (in vitro MNA absorption experiments) kwongera ku busobozi nti fibroblasts ezikwatagana ne kookolo (CAF) zifulumya MNA era obutoffaali bw’ekizimba buyinza okulungamya enkula n’enkola ya TIL. Okuzuula enkola ya MNA ku butoffaali bwa T, obutoffaali bwa T obugaba obulamu bwakolebwa mu vitro nga waliwo oba nga tewali MNA, era okukula kwabwo n’okukola kwa cytokine ne byekenneenyezebwa. Oluvannyuma lw’ennaku 7 nga bongerako MNA ku ddoozi esinga obunene, omuwendo gw’abantu ogw’okukubisaamu emirundi ebiri gwakendeera mu kigero, ate ng’amaanyi gakuumibwa ku ddoozi zonna (Ekifaananyi 4A). Okugatta ku ekyo, okujjanjaba MNA ow’ebweru kyavaamu okweyongera mu kitundu ky’obutoffaali bwa CD4 + ne CD8 + T obulaga tumor necrosis factor-α (TNFα; Ekifaananyi 4B). Okwawukana ku ekyo, okukola IFN-γ mu butoffaali kwakendeera nnyo mu butoffaali bwa CD4 + T, naye si mu butoffaali bwa CD8 + T, era tewaaliwo nkyukakyuka nnene mu interleukin 2 (IL-2; Ekifaananyi 4, C ne D). Nolwekyo, enzyme-linked immunosorbent assay (ELISA) of supernatants okuva mu bino MNA-treated T cell cultures yalaga okweyongera okunene mu TNFα, okukendeera mu IFN-γ, era tewali nkyukakyuka mu IL-2 (Figure 4, E to G). . Okukendeera kwa IFN-γ kulaga nti MNA eyinza okukola kinene mu kuziyiza emirimu gy’obutoffaali bwa T egy’okulwanyisa ebizimba. Okusobola okukoppa ekikolwa kya MNA ku butwa bwa T cell-mediated cytotoxicity, obutoffaali bwa chimeric antigen receptor T (FRα-CAR-T) obugenderera obutoffaali bwa folate receptor α ne CAR-T (GFP) obufugibwa obutoffaali bwa green fluorescent protein (GFP) -CAR-T) bukolebwa obutoffaali obulamu obw’omugabi obw’okumpi obw’omusaayi mononuclear cells (PBMC). Obutoffaali bwa CAR-T bwakuzibwa okumala essaawa 24 nga waliwo MNA, oluvannyuma ne bukuzibwa wamu n’obutoffaali bw’ekizimba ky’enkwaso obw’omuntu SK-OV-3 obulaga folate receptor α ku mugerageranyo gwa effector ku target ogwa 10:1. Obujjanjabi bwa MNA bwavaamu okukendeera okw’amaanyi mu kutta obutoffaali bwa FRα-CAR-T, nga bufaananako n’obutoffaali bwa FRα-CAR-T obwjjanjabwa ne adenosine (Figure 4H).
(A) Omuwendo gw’obutoffaali obulamu bwonna awamu n’okukubisaamu emirundi ebiri omuwendo gw’abantu (PD) butereevu okuva mu buwangwa ku lunaku 7. Grafu y’ebbaala ekiikirira wakati + SEM y’abawaayo omukaaga abalamu. Ekiikirira data okuva mu kugezesa okwetongodde waakiri n = 3. (B okutuuka ku D) CD3/CD28 ne IL-2 zaakozesebwa okukola obutoffaali bwa T ku bungi bwa MNA bwabwo okumala ennaku 7. Nga tebannaba kwekenneenya, obutoffaali bwasikirizibwa ne PMA/ionomycin ne GolgiStop okumala essaawa 4. Okwolesebwa kwa TNFα (B) mu butoffaali bwa T. Ekyokulabirako ekifaananyi (ku kkono) ne tabular data (ku ddyo) eby’okwolesebwa kwa TNFα mu butoffaali obulamu. Okwolesebwa kwa IFN-γ (C) ne IL-2 (D) mu butoffaali bwa T. Okwolesebwa kwa cytokines kwapimibwa mu nkola ya flow cytometry. Ebbaala graph ekiikirira wakati (n = 6 abagaba obuyambi abalamu) + SEM. Kozesa okwekenneenya okw’engeri emu okw’enjawulo n’ebipimo ebiddiŋŋanwa (*P<0.05 ne **P<0.01) okuzuula omuwendo gwa P. Ekiikirira data okuva mu kugezesa okwetongodde waakiri n = 3. (E to G) CD3/CD28 ne IL-2 zakozesebwa okukola obutoffaali bwa T ku bungi bwa MNA bwabwo okumala ennaku 7. Ekirungo kino kyakuŋŋaanyizibwa nga tebannaba n’oluvannyuma lw’essaawa 4 ez’okusikirizibwa kwa PMA/ionomycin. Ebirungo bya TNFα (E), IFN-γ (F) ne IL-2 (G) byapimibwa mu ngeri ya ELISA. Grafu y’ebbaala ekiikirira ekigero (n = 5 abagaba obuyambi abalamu) + SEM. Omuwendo gwa P ogwasalibwawo nga tukozesa okwekenneenya okw’engeri emu okw’enjawulo n’okupima okuddiŋŋana (*P<0.05). Layini eriko ennukuta eraga ekkomo ly’okuzuula ery’okuzuula. (H) Okukebera okusasika kw’obutoffaali. Obutoffaali bwa FRα-CAR-T oba GFP-CAR-T bwatereezebwa ne adenosine (250μM) oba MNA (10 mM) okumala essaawa 24, oba ne bulekebwa nga tebujjanjabiddwa (Ctrl). Ebitundu ku kikumi eby’okutta obutoffaali bwa SK-OV-3 byapimibwa. Omuwendo gwa P ogwasalibwawo n’okugezesebwa kwa Welch t (*P<0.5 ne **P<0.01).
Okusobola okufuna okutegeera okw’enkola ku MNA-dependent TNFα expression regulation, enkyukakyuka mu TNFα mRNA y’obutoffaali bwa T obujjanjabiddwa MNA zaali zeekenneenyezebwa (Figure 5A). Obutoffaali bwa T obulamu obugaba obujjanjabiddwa ne MNA bwalaga okweyongera kw’emirundi ebiri mu miwendo gy’okuwandiika TNFα, ekiraga nti MNA yeesigamye ku kulungamya okuwandiika kwa TNFα. Okunoonyereza ku nkola eno esoboka okulungamya, ensonga bbiri ezimanyiddwa transcription ezifuga TNFα, kwe kugamba activated T cell nuclear factor (NFAT) ne specific protein 1 (Sp1), zaali zeekenneenyezebwa mu kuddamu MNA okusiba ku proximal TNFα promoter ( 30 ). TNFα promoter erimu ebifo 6 ebizuuliddwa ebisiba NFAT n’ebifo 2 ebisiba Sp1, nga bikwatagana mu kifo kimu [-55 base pairs (bp) okuva ku 5’cap] (30). Chromatin immunoprecipitation (ChIP) yalaga nti bwe yajjanjabwa ne MNA, okusiba kwa Sp1 ku TNFα promoter kweyongera emirundi esatu. Okuyingiza NFAT nakyo kyeyongera era ne kisemberera obukulu (Ekifaananyi 5B). Ebiwandiiko bino biraga nti MNA etereeza okwolesebwa kwa TNFα okuyita mu kuwandiika kwa Sp1, ate ku kigero ekitono okwolesebwa kwa NFAT.
(A) Bw’ogeraageranya n’obutoffaali bwa T obukuziddwa awatali MNA, enkyukakyuka y’emirundi y’okwolesebwa kwa TNFα mu butoffaali bwa T obujjanjabiddwa ne MNA. Enkola y’okwolesebwa ne SEM eragiddwa (n = 5 abagaba abalamu). Ekiikirira data okuva mu kugezesa okwetongodde waakiri n = 3. (B) TNFα promoter y’obutoffaali bwa T obujjanjabiddwa ne oba nga tebulina 8 mM MNA oluvannyuma lwa NFAT ne Sp1 byagattibwa ne (Ctrl) ne PMA/ionomycin stimulation okumala essaawa 4. Immunoglobulin G (IgG) ne H3 zaakozesebwa nga negativu ne positive controls ku immunoprecipitation, okusinziira ku. Okugera obungi bwa ChIP kwalaga nti okusiba Sp1 ne NFAT ku TNFα promoter mu butoffaali obujjanjabiddwa MNA kweyongera emirundi egiwerako bw’ogeraageranya n’obufugibwa. Ekiikirira data okuva mu kugezesa okwetongodde waakiri n = 3. Omuwendo gwa P ogusaliddwawo n’okugezesebwa kwa t okungi (*** P <0.01). (C) Bw’ogeraageranya n’obusimu obuyitibwa ascites bwa HGSC, obutoffaali bwa T (obutali bwa butwa bwa cytotoxic) bwalaga okweyongera kw’okwolesebwa kwa TNF mu kizimba. Langi zino zikiikirira abalwadde ab’enjawulo. Obutoffaali obulagiddwa bubadde bwa sampuli mu ngeri ey’ekifuulannenge okutuuka ku 300 ne buwunyiriza okukomya okusika okusukkiridde (** Padj = 0.0076). (D) Ekiteeso ky’ekyokulabirako kya MNA ku kookolo w’enkwaso. MNA ekolebwa mu butoffaali bw’ekizimba ne fibroblasts mu TME era etwalibwa obutoffaali bwa T. MNA eyongera okusiba Sp1 ku TNFα promoter, ekivaako okweyongera kw’okuwandiika kwa TNFα n’okukola TNFα cytokine. MNA era ereeta okukendeera kwa IFN-γ. Okuziyiza okukola kw’obutoffaali bwa T kivaako okukendeeza ku busobozi bw’okutta n’okukula amangu kw’ekizimba.
Okusinziira ku lipoota, TNFα erina ebikolwa ebiziyiza ebizimba n’ebizimba ebisinziira mu maaso n’emabega, naye erina omulimu ogumanyiddwa ennyo mu kutumbula okukula n’okusaasaana kwa kookolo w’enkwaso (31-33). Okusinziira ku lipoota, ekirungo kya TNFα mu bitundu by’omubiri ebiyitibwa ascites n’ebizimba mu balwadde abalina kookolo w’enkwaso kiri waggulu okusinga mu bitundu ebitali bya bulabe ( 34-36 ). Mu ngeri y’enkola, TNFα esobola okulungamya okukola, okukola n’okusaasaana kw’obutoffaali obweru, n’okukyusa enkula y’obutoffaali bwa kookolo ( 37 , 38 ). Okukwatagana n’ebizuuliddwa bino, okwekenneenya okwolesebwa kw’obuzaale obw’enjawulo kwalaga nti TNF yali etereezebwa nnyo mu butoffaali bwa T mu bitundu by’ekizimba bw’ogeraageranya n’obulwadde bwa ascites (Figure 5C). Okweyongera mu kwolesebwa kwa TNF kwalabika mu bungi bw’obutoffaali bwa T bwokka obulina phenotype etali ya butwa bwa cytotoxic (Figure S5A). Mu bufunze, data zino ziwagira endowooza nti MNA erina dual immunosuppressive ne tumor promoting effects mu HGSC.
Fluorescent labeling nga yesigamiziddwa ku flow cytometry efuuse enkola enkulu ey’okunoonyereza ku nkyukakyuka ya TIL. Okunoonyereza kuno kulaga nti bw’ogeraageranya n’obutoffaali bw’omusaayi obw’okumpi oba obutoffaali bwa T okuva mu bitundu by’omubiri eby’okubiri, TIL y’ensiri n’ey’omuntu erina omuze gwa waggulu ogw’okutwala glucose ( 4 , 39 ) n’okufiirwa mpolampola emirimu gya mitochondria ( 19 , 40 ). Wadde nga tulabye ebivuddemu ebifaanagana mu kunoonyereza kuno, enkulaakulana enkulu kwe kugeraageranya enkyukakyuka y’obutoffaali bw’ekizimba ne TIL okuva mu bitundu by’ekizimba bye bimu ebyasaliddwako. Okukwatagana n’ezimu ku lipoota zino ezaaliwo emabega, obutoffaali bw’ekizimba (CD45-EpCAM +) okuva mu ascites n’ebizimba bulina okutwalibwa kwa glucose okusinga obutoffaali bwa CD8 + ne CD4 + T, ekiwagira nti okutwala glucose omungi mu butoffaali bw’ekizimba kuyinza okugeraageranyizibwa ku butoffaali bwa T. Endowooza y’okuvuganya kw’obutoffaali bwa T. TME. Wabula emirimu gya mitochondrial egy’obutoffaali bw’ekizimba gisinga egy’obutoffaali bwa CD8 + T, naye emirimu gya mitochondrial gifaananako n’egy’obutoffaali bwa CD4 + T. Ebivuddemu bino binyweza omulamwa ogugenda guvaayo nti oxidative metabolism kikulu eri obutoffaali bw’ekizimba ( 41 , 42 ). Era bagamba nti obutoffaali bwa CD8 + T buyinza okuba nga businga okukwatibwa obuzibu mu kukola oxidative okusinga obutoffaali bwa CD4 + T, oba nti obutoffaali bwa CD4 + T buyinza okukozesa ensibuko za kaboni endala okuggyako glucose okukuuma emirimu gya mitochondrial ( 43 , 44 ). Kinaamanyibwa nti tetwalaba njawulo mu kutwala glucose oba mitochondrial activity wakati wa CD4 + T effectors, T effector memory ne T central memory cells mu ascites. Mu ngeri y’emu, embeera y’enjawulo y’obutoffaali bwa CD8 + T mu bizimba terina kakwate na nkyukakyuka mu kutwalibwa kwa glucose, ekiraga enjawulo nnene wakati w’obutoffaali bwa T obukuliddwa mu vitro ne TIL y’omuntu mu vivo ( 22 ). Ebintu bino bye twetegereza era byakakasibwa nga bakozesa enkola ya unbiased automatic cell population allocation, ekyayongera okulaga nti obutoffaali bwa CD45 + / CD3- / CD4 + / CD45RO + obulina glucose uptake esingako n’emirimu gya mitochondrial okusinga obutoffaali bw’ekizimba buyitiridde naye nga bulina Metabolic active cell population. Omuwendo guno guyinza okukiikirira ekibinja ekitono ekiteeberezebwa eky’obutoffaali obuziyiza myeloid oba obutoffaali bwa plasmacytoid dendritic obuzuuliddwa mu kwekenneenya kwa scRNA-seq. Newankubadde nga bino byombi bibadde bitegeezeddwa mu bizimba by’enkwaso z’omuntu [45], bikyetaaga Omulimu omulala kwe kunnyonnyola ekitundu kino ekya myeloid.
Newankubadde enkola ezesigamiziddwa ku flow cytometry zisobola okunnyonnyola enjawulo ez’awamu mu glucose ne oxidative metabolism wakati w’ebika by’obutoffaali, metabolites entuufu ezikolebwa glucose oba ensibuko endala za carbon ku mitochondrial metabolism mu TME tezinnaba kutegeerekeka. Okugabanya okubeerawo oba obutabaawo kwa metabolites ku TIL subset eweereddwa kyetaagisa okulongoosa omuwendo gw’obutoffaali okuva mu tissue esaliddwa. N’olwekyo, enkola yaffe ey’okugaggawaza obutoffaali nga egattibwa wamu n’okupima obutoffaali (mass spectrometry) esobola okuwa amagezi ku bikozesebwa ebigaggawaza mu ngeri ey’enjawulo mu butoffaali bwa T n’obungi bw’obutoffaali bw’ekizimba mu sampuli z’abalwadde ezikwatagana. Newankubadde enkola eno erina ebirungi ku fluorescence-activated cell sorting, ebimu ku metabolite libraries ziyinza okukosebwa olw’obutebenkevu obuzaaliranwa ne / oba amangu enkyukakyuka rate ( 22 ). Wadde kiri kityo, enkola yaffe yasobola okuzuula ebirungo bibiri ebimanyiddwa ebiziyiza obusimu obuziyiza endwadde, adenosine ne kynurenine, kubanga byawukana nnyo wakati w’ebika bya sampuli.
Okwekenenya kwaffe okwa metabolite ku bizimba n’ebika bya TIL kuwa amagezi amalala ku kifo ky’ebirungo ebikyusa ebiriisa mu TME y’enkwaso. Okusooka, nga tukozesa flow cytometry, twazuula nti tewali njawulo mu mitochondrial activity wakati w’ebizimba n’obutoffaali bwa CD4 + T. Wabula, okwekenneenya kwa LC-MS/MS kwalaga enkyukakyuka ez’amaanyi mu bungi bw’ebirungo ebikyusa ebiriisa mu bika bino, ekiraga nti ebimalirizibwa ku nkyukakyuka ya TIL n’emirimu gyayo egy’okukyusakyusa okutwalira awamu byetaaga okutaputa n’obwegendereza. Ekirala, MNA ye metabolite esinga enjawulo wakati wa CD45-cells ne T cells mu ascites, so si bizimba. Nolwekyo, okugabanya mu bitundu n’ekifo ky’ekizimba biyinza okuba n’ebikosa eby’enjawulo ku nkyukakyuka ya TIL, ekiraga obutafaanagana obuyinza okubaawo mu mbeera entonotono eweereddwa. Ekyokusatu, okwolesebwa kw’enziyiza ekola MNA NNMT okusinga kukoma ku CAF, nga buno butoffaali bwa kizimba ku kigero ekitono, naye emiwendo gya MNA egyazuulibwa gyeyolekera mu butoffaali bwa T obuva mu kizimba. Okwolesebwa okusukkiridde kwa NNMT mu CAF y’enkwaso kulina ekimanyiddwa ekitumbula kookolo, ekitundu kiva ku kutumbula CAF metabolism, okulumba ebizimba n’okusaasaana ( 27 ). Wadde ng’omutindo gwa TIL okutwalira awamu gwa kigero, okwolesebwa kwa NNMT mu CAF kukwatagana nnyo n’ekika kya Cancer Genome Atlas (TCGA) mesenchymal subtype, ekikwatagana n’okuteebereza okubi ( 27 , 46 , 47 ). N’ekisembayo, okwolesebwa kw’enziyiza AOX1 evunaanyizibwa ku kuvunda kwa MNA nakyo kikoma ku muwendo gwa CAF, ekiraga nti obutoffaali bwa T tebulina busobozi kukyusa MNA. Ebivuddemu bino biwagira endowooza nti wadde nga kyetaagisa okukola okwongera okukakasa kino ekizuuliddwa, emiwendo gya MNA egy’amaanyi mu butoffaali bwa T giyinza okulaga nti waliwo obutonde obutono obwa CAF obuziyiza obusimu obuziyiza endwadde.
Okusinziira ku mutindo omutono ogw’okwolesebwa kw’abatambuza MNA n’emitendera egitazuulibwa egya puloteyina enkulu ezikwatibwako mu nkyukakyuka ya MNA, okubeerawo kwa MNA mu butoffaali bwa T tekisuubirwa. Wadde NNMT oba AOX1 tezaasobola kuzuulibwa nga bakozesa scRNA-seq analysis ne targeted qPCR of two independent cohorts. Ebivuddemu bino biraga nti MNA tekolebwa butoffaali bwa T, wabula eyingizibwa okuva mu TME eyeetoolodde. Okugezesa mu vitro kulaga nti obutoffaali bwa T butera okukung’aanya MNA ey’ebweru.
Okunoonyereza kwaffe mu vitro kulaga nti MNA ey’ebweru ereeta okwolesebwa kwa TNFα mu butoffaali bwa T era n’eyongera okusiba Sp1 ku TNFα promoter. Wadde nga TNFα erina emirimu gyombi egy’okulwanyisa ebizimba n’okulwanyisa ebizimba, mu kookolo w’enkwaso, TNFα esobola okutumbula okukula kwa kookolo w’enkwaso ( 31-33 ). The neutralization of TNFα mu ovarian tumor cell culture oba okuggyawo TNFα signal mu mouse models kiyinza okulongoosa TNFα-mediated inflammatory cytokine production n’okuziyiza okukula kw’ekizimba ( 32 , 35 ). Nolwekyo, mu mbeera eno, TME-derived MNA esobola okukola nga pro-inflammatory metabolite okuyita mu TNFα-dependent enkola okuyita mu autocrine loop, bwe kityo okutumbula okubeerawo n’okusaasaana kwa kookolo w’enkwaso ( 31 ). Okusinziira ku kino ekisoboka, TNFα blockade esomesebwa ng’ekirungo ekiyinza okujjanjaba kookolo w’enkwaso ( 37 , 48 , 49 ). Okugatta ku ekyo, MNA eyonoona obutwa bw’obutoffaali bwa CAR-T eri obutoffaali bw’ekizimba ky’enkwaso, ekiwa obujulizi obulala ku kunyigirizibwa kw’abaserikale b’omubiri okutambulizibwa MNA. Okutwalira awamu, ebivuddemu bino biraga ekyokulabirako ebizimba n’obutoffaali bwa CAF mwe bifulumya MNA mu TME ey’ebweru w’obutoffaali. Okuyita mu (i) TNF-induced ovarian cancer growth stimulation ne (ii) MNA-induced T cell cytotoxic activity inhibition, kino kiyinza okuba n’ekizimba eky’emirundi ebiri (Figure 5D).
Mu kumaliriza, nga tukozesa omugatte gw’okugaggawaza obutoffaali obw’amangu, okulonda obutoffaali bumu n’okusengeka enkyukakyuka mu mubiri, okunoonyereza kuno kwalaga enjawulo ennene mu immunometabolomic wakati w’ebizimba n’obutoffaali bwa ascites mu balwadde ba HGSC. Okwekenenya kuno okujjuvu kwalaga nti waliwo enjawulo mu kutwala glucose n’emirimu gya mitochondrial wakati w’obutoffaali bwa T, era ne kuzuula MNA nga non-cell autonomous immune regulatory metabolite. Ebiwandiiko bino birina kye bikola ku ngeri TME gy’ekosaamu enkyukakyuka y’obutoffaali bwa T mu kookolo w’abantu. Wadde ng’okuvuganya obutereevu ku biriisa wakati w’obutoffaali bwa T n’obutoffaali bwa kookolo kubadde kutegeezeddwa, ebirungo ebikyusa ebiriisa nabyo bisobola okukola ng’ebifuga ebitali butereevu okutumbula okukula kw’ekizimba era nga kiyinzika okunyigiriza okuddamu kw’abaserikale b’omubiri okw’omunda. Okwongera okunnyonnyola omulimu gw’emirimu gy’ebirungo bino ebifuga ebikyukakyuka kiyinza okuggulawo enkola endala ez’okutumbula okuddamu kw’abaserikale b’omubiri abalwanyisa ebizimba.
Ebigezo by’abalwadde n’ebikwata ku bujjanjabi byafunibwa okuyita mu tterekero ly’ebitundu by’omubiri ebizimba kookolo wa BC eryakakasibwa ekitongole kya Canadian Tissue Repository Network. Okusinziira ku nkola eyayisibwa akakiiko akakwasisa empisa mu kunoonyereza ku kookolo aka BC ne Yunivasite y’e British Columbia (H07-00463), ebigezo by’abalwadde bonna n’ebikwata ku bujjanjabi bye baafunye okukkiriza mu buwandiike oba ne bava ku kukkiriza kwabwe mu butongole. Sampuli zino ziterekebwa mu BioBank ekakakasiddwa (BRC-00290). Ebikwata ku mulwadde mu bujjuvu biragiddwa mu bipande S1 ne S5. Ku ky’okukuuma mu cryopreservation, scalpel ekozesebwa okuvunda mu makanika sampuli y’ekizimba ky’omulwadde n’oluvannyuma n’akisika okuyita mu 100-micron filter okufuna obutoffaali bumu obuyimiriziddwa. Ascites y’omulwadde yafuumuulwa ku 1500 rpm okumala eddakiika 10 ku 4°C okusala obutoffaali n’okuggyamu amazzi agasukkulumye ku mazzi. Obutoffaali obwafunibwa okuva mu kizimba ne ascites bwakuumibwa mu cryopreserved mu 50% ebbugumu-inactivated human AB serum (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) ne 10% dimethyl sulfoxide. Ebisengejjo bino eby’obutoffaali obumu ebikuumiddwa byasaanuusibwa ne bikozesebwa mu metabolomics n’okusalawo metabolite ebyogeddwako wansi.
Ekirungo ekijjuvu kirimu 0.22 μm esengejeddwa 50:50 nga eyongezeddwamu RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) nga eyongezeddwamu ebitundu 10% eby’ebbugumu ly’omuntu AB serum (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) Fisher Scientific), 1 x Ekizigo kya Penicillin Streptomycin (PenStrep) (Thermo Fisher Scientific) ne 50 μMB-mercaptoethanol. AimV (Invitrogen) eyongerwako 20 mM Hepes (Thermo Fisher Scientific) ne 2 mM l-glutamine (Thermo Fisher Scientific). Ekiziyiza okusiiga amabala mu flow cytometer kyalimu 0.22μm filtered phosphate buffered saline (PBS; Invitrogen) nga eyongeddeko 3% heat-inactivated AB human serum (Sigma). Buffer y’okugaggawaza obutoffaali ekolebwa PBS esengejeddwa 0.22μm era n’eyongerwako 0.5% heat-inactivated human AB serum (Sigma-Aldrich).
Mu 37°C complete medium, obutoffaali bwasiigibwa ne 10 nM MT DR ne 100 μM 2-NBDG okumala eddakiika 30. Ekiddako, obutoffaali bwasiigibwa langi ya viability dye eF506 ku 4°C okumala eddakiika 15. Ddamu okuyimiriza obutoffaali mu FC Block (eBioscience) ne Brilliant Stain Buffer (BD Biosciences), bufukire mu flow cytometer staining buffer (okusinziira ku biragiro by’omukozi), era obufumbe okumala eddakiika 10 ku bbugumu erya bulijjo. Siiga obutoffaali n’ekibinja ky’obuziyiza (Table S2) mu flow cytometry staining buffer ku 4°C okumala eddakiika 20. Ddamu okuyimiriza obutoffaali mu flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) nga tonnaba kwekenneenya. Kozesa SpectroFlo ne FlowJo V10 okwekenneenya data y’omuwendo gw’obutoffaali, era kozesa GraphPad Prism 8 okukola data. Median fluorescence intensity (MFI) ya 2-NBDG ne MT DR ya log-normalized, n’oluvannyuma paired t test yakozesebwa okwekenneenya statistical okubala abalwadde abakwatagana. Ggyawo ebika byonna ebirina ebibaddewo ebitakka wansi wa 40 mu kwekenneenya; ssaamu omuwendo gwa MFI ogwa 1 ku muwendo gwonna omubi nga tonnaba kukola kwekenneenya kwa bibalo n’okulaba data.
Okusobola okugatta ku nkola ya manual gating ey’ekipande ky’enkola waggulu, twakozesa annotation enzijuvu n’omuti oguziyiza ekifaananyi (FAUST) (21) okugabanya obutoffaali mu bungi mu ngeri ey’otoma oluvannyuma lw’okumalawo obutoffaali obufu mu FlowJo. Tuddukanya mu ngalo ebifulumizibwa okugatta ebika ebirabika ng’ebigabanyizibwa obubi (nga tugatta PD1+ n’obutoffaali bw’ekizimba kya PD1) n’ebika ebikuumiddwa. Buli sampuli erimu obutoffaali obusukka mu bitundu 2% ku kigero, omugatte gw’ebika 11.
Ficoll gradient density centrifugation yakozesebwa okwawula PBMC okuva mu bikozesebwa ebyawula obutoffaali obutono (STEMCELL Technologies). Obutoffaali bwa CD8 + T bwawulwa okuva mu PBMC nga tukozesa CD8 MicroBeads (Miltenyi) ne bugaziwa mu kifo ekijjuvu nga tukozesa TransAct (Miltenyi) okumala wiiki 2 okusinziira ku biragiro by’omukozi. Obutoffaali bwakkirizibwa okuyimirira okumala ennaku 5 mu kifo ekijjuvu ekirimu IL-7 (10 ng/ml; PeproTech), oluvannyuma ne buddamu okusikirizibwa ne TransAct. Ku lunaku olw’omusanvu, okusinziira ku biragiro by’abakola, human CD45 MicroBeads (Miltenyi) zaakozesebwa okugaggawaza obutoffaali mu lawundi ssatu ez’omuddiring’anwa. Obutoffaali bwagabanyizibwamu okusobola okwekenneenya flow cytometry (nga bwe kyayogerwako waggulu), ate obutoffaali obukadde bumu bwagabanyizibwamu emirundi esatu okwekenneenya LC-MS/MS. Sampuli zalongoosebwa mu ngeri ya LC-MS/MS nga bwe kinyonyoddwa wansi. Twabalirira omuwendo gwa metabolite ogubula nga tulina ennamba ya ion 1,000. Buli sampuli efuulibwa eya bulijjo n’ennamba ya ion yonna (TIC), ekyusibwa mu ngeri ya logaritmu era n’efuuka eya bulijjo mu MetaboAnalystR nga tennaba kwekenneenyezebwa.
Ekisengejjo ky’obutoffaali obumu ekya buli mulwadde kyasaanuusibwa ne kisengejebwa okuyita mu ssengejjero lya 40 μm mu kifo ekijjuvu (nga bwe kyayogerwako waggulu). Okusinziira ku nkola y’omukozi, lawundi ssatu ez’omuddiring’anwa ez’okulonda okulungi nga tuyita mu kwawula obululu mu magineeti nga tukozesa MicroBeads (Miltenyi) zaakozesebwa okugaggawaza sampuli z’obutoffaali bwa CD8+, CD4+ ne CD45- (ku ice). Mu bufunze, obutoffaali buddamu okusibwa mu buffer y’okugaggawaza obutoffaali (nga bwe kyayogeddwa waggulu) ne bubalibwa. Obutoffaali bwafumbirwa n’obululu bwa CD8 obw’omuntu, obululu bwa CD4 obw’omuntu oba obululu bwa CD45 obw’omuntu (Miltenyi) ku 4°C okumala eddakiika 15, oluvannyuma ne bunaazibwa ne cell enrichment buffer. Sampuli eyisibwa mu kisenge kya LS (Miltenyi), era obutundutundu obulungi n’obubi ne bukuŋŋaanyizibwa. Okusobola okukendeeza ku bbanga n’okutumbula omutendera gw’okuzzaawo obutoffaali, olwo akatundu ka CD8 kakozesebwa mu lawundi eyookubiri ey’okugaggawaza CD4+, ate akatundu ka CD4 ne kakozesebwa mu kugaggawaza CD45 okuddako. Ekizimbulukusa kikuume ku ice mu nkola yonna ey’okwawula.
Okuteekateeka sampuli okwekenneenya metabolite, obutoffaali bwanaazibwa omulundi gumu n’omunnyo omunnyogovu nga ice, era 1 ml ya 80% methanol yayongerwa mu buli sampuli, oluvannyuma ne vortexed ne snap frozen mu liquid nitrogen. Sampuli zino zakolebwako enzirukanya ssatu eza freeze-thaw ne zifuumuulwa ku 14,000 rpm okumala eddakiika 15 ku 4°C. Ekisengejjero ekiri waggulu ekirimu ebirungo ebikyusa ebiriisa kifuumuulwa okutuusa lwe kikala. Ebirungo ebikyusa ebiriisa byaddamu okusaanuusibwa mu 50 μl za 0.03% formic acid, ne bifuumuulwa okutabula, oluvannyuma ne bifuumuulwa okuggyawo ebisasiro.
Ggyako ebirungo ebikyusa ebiriisa nga bwe kyayogeddwa waggulu. Teeka amazzi agasukkulumye ku mazzi mu ccupa ya chromatography ey’amazzi ekola obulungi okunoonyereza ku metabolomics. Kozesa enkola y’obujjanjabi ey’ekifuulannenge okujjanjaba buli sampuli n’omuwendo gw’obutoffaali ogufaanagana okuziyiza ebikosa ebibinja. Twakola okwekenneenya okw’omutindo gw’ebirungo ebikyukakyuka mu nsi yonna ebyafulumizibwa emabegako ku AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Okwekenenya mu ngeri ya chromatographic n’okugatta ebitundu by’entikko byakolebwa nga tukozesa pulogulaamu ya MultiQuant version 2.1 (Applied Biosystems SCIEX).
Omuwendo gwa ion ogwa 1000 gwakozesebwa okubalirira omuwendo gw’ekirungo ekibulamu, era TIC ya buli sampuli yakozesebwa okubala ekitundu ky’entikko ekya bulijjo ekya buli kirungo ekizuuliddwa okutereeza enkyukakyuka ezayingizibwa okwekenneenya okw’ebikozesebwa okuva mu kukola sampuli. Oluvannyuma lwa TIC okufuuka eya bulijjo, MetaboAnalystR(51) (default parameter) ekozesebwa okukyusa logarithmic n’okugerageranya layini ya norm mu ngeri ey’otoma. Twakozesa PCA ne vegan R package okukola okwekenneenya okw’okunoonyereza ku njawulo za metabolome wakati w’ebika bya sampuli, era ne tukozesa okwekenneenya okutali kwa kitundu (partial redundancy analysis) okwekenneenya abalwadde. Kozesa enkola ya Ward okuzimba dendrogram ya maapu y’ebbugumu okukuŋŋaanya ebanga lya Euclidean wakati wa sampuli. Twakozesa limma (52) ku standardized metabolite obungi okuzuula differentially obungi metabolites okubuna ekika ky’obutoffaali kyonna n’obutonde obutono. Okusobola okwanguyiza ennyonyola, tukozesa ekigerageranyo ky’ekibinja ekitegeeza okulaga omuze, era ne tulowooza ku bika by’obutoffaali mu microenvironment nga buli kibinja (n = 6 ebibinja); ku lw’okugezesebwa kw’amakulu, twakola ebipimo bisatu ebiddiŋŋanwa ku buli metabolite Okusobola okwewala okukoppa okw’obulimba, omulwadde yassibwamu ng’ekiziyiza mu nteekateeka ya limma. Okusobola okukebera enjawulo mu metabolites wakati w’abalwadde ab’enjawulo, twatereeza limma model omuli abalwadde mu ngeri etakyukakyuka. Tutegeeza obukulu bw’enjawulo eyalagirwa nga tennabaawo wakati w’ekika ky’obutoffaali n’obutonde obutono obwa Padj <0.05 (Benjamini-Hochberg correction).
Oluvannyuma lw’okugaggawaza amaanyi nga tukozesa Miltenyi Dead Cell Removal Kit (>80% viability), ensengeka y’obutoffaali obumu (single-cell transcriptome sequencing) yakolebwa ku sampuli z’amasasi n’ebizimba byonna ebiramu ebifumbiddwa nga tukozesa enkola ya 10x 5′gene expression protocol. Emisango etaano egyali gikwatagana n’ebizimba n’obulwadde bwa ascites gyakeberebwa, wadde ng’obulamu obutono okuva mu sampuli y’ekizimba emu bwalemesa okugiyingizaamu. Okusobola okutuuka ku kulonda kw’abalwadde okungi, twagatta sampuli za buli mulwadde mu mirongooti gya 10x chromium controller, ne twekenneenya ebifo eby’okusannyalala n’ebizimba okwawukana. Oluvannyuma lw’okulonda [Illumina HiSeq 4000 28×98 bp paired end (PE), ensengekera y’obutonde bw’ensi mu Quebec; average of 73,488 and 41,378 reads per cell for tumor and ascites respectively]], twakozesa CellSNP ne Vireo (53) (okusinziira ku CellSNP nga The common human SNP (VCF) ewereddwa GRCh38 eweebwa endagamuntu y’omugabi.Tukozesa SNPRelate okuteebereza endagamuntu esinga okumpi (IBS) y’embeera y’ekikula ky’omulwadde (IBS), okuggyako obutoffaali obutaweebwa n’obutoffaali obuzuuliddwa nga duplexes n’okukwatagana abagaba wakati wa ascites ne sampuli z’ekizimba (54), twasigaza emisango esatu n’okukiikirira obutoffaali bungi mu kizimba ne ascites okwekenneenya wansi Oluvannyuma lw’okukola omutendera gw’okusengejja amasasi mu scater (55) ne scran (56) BioConductor packaging, kino kyavaamu 697 obutoffaali (2792 and 4183 cells from tumor and ascites, respectively) for analysis. We use igraph's (57) Louvain clustering of shared nearest neighbor network (SNN) based on Jaccard distance to cluster cells by expression. The clusters were manually annotated to putative cell types based on marker gene expression and visualized with t-SNE. Cytotoxic T cells are enyonyoddwa okwolesebwa kwa CD8A ne GZMA, okuggyako subclusters ezirina low ribosomal protein expression Twatuuka ku data efulumiziddwa eya Izar et al (16), omuli t-SNE embedding yaabwe, esobola okufuga okwolesebwa okukwatagana wakati w’obubonero bw’obutoffaali obuziyiza endwadde.
PBMC zaawulwa okuva mu bikozesebwa ebyawula obutoffaali obutono (STEMCELL Technologies) nga zikozesebwa Ficoll gradient density centrifugation. Obutoffaali bwa CD3 + bwawulwa okuva mu PBMC nga tukozesa obululu bwa CD3 (Miltenyi). Mu kubeerawo oba obutabaawo kwa MNA, obutoffaali bwa CD3+ bwakozesebwa ne CD3 esibiddwa ku pulati (5μg/ml), CD28 etabulwa (3μg/ml) ne IL-2 (300 U/ml; Proleukin). Ku lunaku olusembayo olw’okugaziya, obusobozi (Fixable Viability Dye eFluor450, eBioscience) n’okusaasaana (123count eBeads, Thermo Fisher Scientific) byakeberebwa nga bakozesa enkola ya flow cytometry. Weekenneenye enkola ya effector nga ositula obutoffaali ne PMA (20 ng/ml) ne ionomycin (1μg/ml) ne GolgiStop okumala essaawa 4, era olondoole CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4) , BioLegend) ne TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD). Simbula obutoffaali bwa qPCR ne ChIP ne PMA (20 ng/ml) ne ionomycin (1μg/ml) okumala essaawa 4. ELISA supernatant yakuŋŋaanyizibwa nga tebannaba n’oluvannyuma lw’okusikirizibwa ne PMA (20 ng/ml) ne ionomycin (1 μg/ml) okumala essaawa 4.
Goberera enkola y’omukozi okwawula RNA ng’okozesa RNeasy Plus Mini Kit (QIAGEN). Kozesa QIAshredder (QIAGEN) okugatta sampuli. Kozesa ekitabo kya RNA to cDNA eky’obusobozi obw’amaanyi (Thermo Fisher Scientific) okukola DNA ejjuliza (cDNA). Kozesa TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) okugera obungi bw’okwolesebwa kw’obuzaale (okusinziira ku nkola y’omukozi) n’ebikebera bino wammanga: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glyceraldehyde-3-phosphate okuva ku Haidrojeni (GAPDH)] ne Hs01010726_m1 (SLC22A2). Sampuli zino zaddukanyizibwa ku nkola ya StepOnePlus real-time PCR system (Applied Biosystems) (Applied Biosystems) mu MicroAmp fast optical 96-well reaction plate (Applied Biosystems) nga erina firimu ya MicroAmp ey’amaaso. Omuwendo gwonna ogwa Ct ogusukka 35 gutwalibwa okuba waggulu w’omusingi gw’okuzuula era guteekebwako akabonero nti teguzuulibwa.
Kola ChIP nga bwe kyayogerwako emabegako (58). Mu bufunze, obutoffaali bwalongoosebwa ne formaldehyde (final concentration 1.42%) ne bufumbirwa ku bbugumu erya bulijjo okumala eddakiika 10. Kozesa supplemented okuzimba buffer (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl ne 0.1% NP-40) ku ice okumala eddakiika 10, olwo resuspend mu immunoprecipitation buffer nga bwe kyayogerwako ( 58 ). Sampuli olwo ya sonicated n’enzirukanya zino wammanga: enzirukanya 10 (20 1-second pulses) n’obudde obutakyukakyuka bwa sikonda 40. Fumbira obutoffaali obuziyiza endwadde obwa ChIP-grade immunoglobulin G (Cell Signaling Technology; 1μl), histone H3 (Cell Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) ne SP1 (Cell Signaling Technology; 3μl) nga sampuli ku 4°CC ekankana ekiro kyonna. Fumbira obululu bwa puloteyina A (Thermo Fisher Scientific) ne sampuli ku 4°C ng’okankanya mpola okumala essaawa 1, olwo okozese obululu bwa chelex (Bio-Rad) okugaggawaza DNA, era okozese proteinase K (Thermo Fisher) okugaaya puloteyina. TNFα promoter yazuulibwa mu PCR: mu maaso, GGG TAT CCT TGA TGC TTG TGT; okwawukana ku ekyo, GTG CCA ACA ACT GCC TTT ATA TG (ekintu ekikolebwa mu 207-bp). Ebifaananyi byakolebwa ekitongole kya Image Lab (Bio-Rad) era ne bipimibwa mu bungi nga bakozesa pulogulaamu ya ImageJ.
Ekisengejjo ky’obutoffaali (cell culture supernatant) kyakuŋŋaanyizibwa nga bwe kyayogerwako waggulu. Okusalawo kwakolebwa okusinziira ku nkola z’omukozi w’ekintu kya human TNFα ELISA kit (Invitrogen), human IL-2 ELISA kit (Invitrogen) ne human IFN-γ ELISA kit (Abcam). Okusinziira ku nkola y’omukozi, amazzi agasukkulumye ku mazzi gafukibwa 1:100 okuzuula TNFα ne IL-2, ne 1:3 okuzuula IFN-γ. Kozesa EnVision 2104 Multilabel Reader (PerkinElmer) okupima okunyiga ku 450 nm.
PBMC zaawulwa okuva mu bikozesebwa ebyawula obutoffaali obutono (STEMCELL Technologies) nga zikozesebwa Ficoll gradient density centrifugation. Obutoffaali bwa CD3 + bwawulwa okuva mu PBMC nga tukozesa obululu bwa CD3 (Miltenyi). Mu kubeerawo oba obutabaawo kwa MNA, obutoffaali bwa CD3+ bwakozesebwa ne CD3 (5μg/ml), CD28 etabulwa (3μg/ml) ne IL-2 (300 U/ml; Proleukin) okumala ennaku 3. Oluvannyuma lw’ennaku 3, obutoffaali bwakuŋŋaanyizibwa ne bunaazibwa n’omunnyo 0.9%, era ekikuta ne kifuulibwa mu bbugumu amangu. Okubala obutoffaali kwakolebwa mu ngeri ya flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) nga tukozesa 123count eBeads.
Ggyako ebirungo ebikyusa ebiriisa nga bwe kyayogeddwa waggulu. Ekirungo ekikalu kyaddamu okukolebwa ku kigero kya 4000 cell equivalents/μl. Yeekenneenya sampuli nga okozesa enkola ya reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) ne CORTECS T3 column (2.1×150 mm, obutundutundu 1.6-μm, obutuli obunene 120-Å; #186008500, Waters). Polar mass spectrometer (6470, Agilent), nga mu kino electrospray ionization ekola mu mbeera ya positive. Omutendera ogutambula A guli 0.1% asidi wa formic (mu H2O), omutendera ogutambula B guli 90% acetonitrile, 0.1% asidi wa formic. LC gradient eri eddakiika 0 ku 2 ku 100% A, eddakiika 2 ku 7.1 ku 99% B, n’eddakiika 7.1 ku 8 ku 99% B. Oluvannyuma ddamu okutebenkeza empagi ne mobile phase A ku sipiidi y’okukulukuta kwa 0.6 ml/min okumala eddakiika 3. . Omuwendo gw’amazzi agakulukuta guli 0.4ml/min, era ekisenge ky’empagi kibuguma okutuuka ku 50°C. Kozesa omutindo gw’eddagala omulongoofu ogwa MNA (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) okuteekawo obudde bw’okusigala (RT) n’enkyukakyuka (RT = eddakiika 0.882, enkyukakyuka 1 = 137→94.1, enkyukakyuka 2 = 137→92 , Okukyusa 3 = 137→78). Enkyukakyuka zonsatule bwe zibaawo mu kiseera ekituufu eky’okusigala, enkyukakyuka 1 ekozesebwa okupima obungi okukakasa nti enjawulo. Standard curve ya MNA (Toronto Research Chemical Company) yakolebwa nga serial dilutions mukaaga eza stock solution (1 mg/ml) okufuna omutindo gwa 0.1, 1.0, 10 ne 100 ng/ml ne 1.0 ne 10μg/ml respectively liquid. Ekkomo ly’okuzuula liri 1 ng/ml, ate okuddamu okw’ennyiriri kuli wakati wa 10 ng/ml ne 10μg/ml. Buli mpiso ya microliters bbiri eza sampuli ne standard ekozesebwa okwekenneenya LC/MS, era sampuli etabuddwamu ey’okulondoola omutindo eddukanyizibwa buli mpiso munaana okukakasa nti ekifo eky’okwekenneenya kinywevu. Eby’okuddamu bya MNA ebya sampuli z’obutoffaali zonna ezajjanjabwa MNA byali mu bbanga lya layini ery’okukebera. Okwekenenya amawulire kwakolebwa nga tukozesa pulogulaamu ya MassHunter okwekenneenya obungi (v9.0, Agilent).
Omulembe ogwokubiri αFR-CAR construct yaggyibwa mu Song et al. (59) (59). Mu bufunze, ekizimbisibwa kirimu bino wammanga: CD8a leader sequence, human αFR-specific single-chain variable fragment, CD8a hinge ne transmembrane region, CD27 intracellular domain ne CD3z intracellular domain. Omutendera gwa CAR omujjuvu gwasengekebwa GenScript, oluvannyuma ne gukolebwa mu clone mu lentiviral expression vector ey’omulembe ogwokubiri waggulu wa GFP expression cassette ekozesebwa okwekenneenya obulungi bw’okukyusa.
Lentivirus ekolebwa nga ekyusa obutoffaali bwa HEK293T [American Type Culture Collection (ATCC); ekuliddwa mu Dulbecco’s modified Eagle medium erimu 10% fetal bovine serum (FBS) ne 1% PenStrep, era nga ekozesebwa CAR-GFP vector ne The packaging plasmids (psPAX2 ne pMD2.G, Addgene) zikozesa lipofection amine (Sigma-Aldrich). Ekisengejjero ekirimu akawuka kyakuŋŋaanyizibwa oluvannyuma lw’essaawa 48 ne 72 nga kikyusiddwa, ne kisengejebwa, ne kisengejebwa nga bakozesa enkola ya ultracentrifugation. Teeka amazzi g’akawuka agasukkiridde ku -80°C okutuusa nga gakyusiddwa.
PBMC zaawulwa okuva mu bintu ebiramu eby’okwawula obutoffaali obukola omusaayi (STEMCELL Technologies) nga zikozesebwa Ficoll gradient density centrifugation. Kozesa positive selection CD8 microbeads (Miltenyi) okwawula obutoffaali bwa CD8+ okuva mu PBMC. Okusitula obutoffaali bwa T ne TransAct (Miltenyi) ne mu TexMACS medium [Miltenyi; nga kwongerako ebitundu 3% ku serum y’omuntu etaliimu bbugumu, 1% PenStrep ne IL-2 (300 U/ml)]. Oluvannyuma lw’essaawa 24 oluvannyuma lw’okusikirizibwa, obutoffaali bwa T bwakyusibwa ne lentivirus (10 μl concentrated virus supernatant per 106 cells). Ennaku 1 ku 3 oluvannyuma lw’okukyusa ku Cytek Aurora (ku FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), okwekenneenya GFP okwolesebwa kw’obutoffaali okulaga obulungi bw’okukyusa waakiri 30% .
Obutoffaali bwa CAR-T bwakuzibwa okumala essaawa 24 mu Immunocult (STEMCELL Technologies; bwayongerwako 1% PenStrep) mu mbeera zino wammanga: obutajjanjabiddwa, bwajjanjabwa ne 250 μM adenosine oba 10 mM MNA. Oluvannyuma lw’okusooka okulongoosebwa, obutoffaali bwa CAR-T bwanaazibwa ne PBS ne bugattibwa n’obutoffaali bwa SK-OV-3 20,000 [ATCC; mu McCoy 5A medium (Sigma-Aldrich) nga eyongezeddwamu 10% FBS ne 1% PenStrep ku 10: Omugerageranyo gwa effector ku target ogwa 1 gwayongezebwako emirundi esatu mu Immunocult medium eyongezeddwamu. Obutoffaali bwa SK-OV-3 n’obutoffaali bwa SK-OV-3 obwa lysed ne digitalis saponin (0.5mg/ml; Sigma-Aldrich) bwakozesebwa nga negativu ne positive controls, respectively. Oluvannyuma lw’essaawa 24 nga balima wamu, amazzi agasukkulumye ku mazzi gaakuŋŋaanyizibwa era ekirungo kya lactate dehydrogenase (LDH) ne kipimibwa okusinziira ku biragiro by’omukozi (LDH Glo Cytotoxicity Assay Kit, Promega). LDH supernatant yafukibwa 1:50 mu LDH buffer. Ebitundu ku kikumi eby’okutta byapimibwa nga tukozesa enkola eno wammanga: ebitundu ku kikumi eby’okutta = ebitundu ku kikumi eby’okutereeza / omuwendo gw’okutta ogusinga obunene x 100%, nga ebitundu ku kikumi eby’okutereeza = obutoffaali bwa co-culture-T bwokka, n’omuwendo ogusinga obunene ogw’okutta = positive control-negative control.
Nga bwe kinyonyoddwa mu kiwandiiko oba ebikozesebwa n’enkola, kozesa GraphPad Prism 8, Microsoft Excel oba R v3.6.0 okwekenneenya ebibalo. Singa sampuli eziwera zikunganyizibwa okuva mu mulwadde omu (nga ascites n’ekizimba), tukozesa paired t test oba tussaamu omulwadde nga random effect mu linear oba generalized model nga bwe kisaanidde. Ku kwekenneenya metabolomics, okugezesebwa kw’obukulu kukolebwa mu mirundi esatu.
Okumanya ebikozesebwa eby’okugatta ku kiwandiiko kino, laba 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 (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellardini (Ralph J. Deberardini), Russell G. Jones (Russell). G. Jones), Phineas T. Hamilton (Ekibiina ky’Oluganda T.
MNA eyamba mu kuziyiza abaserikale b’omubiri mu butoffaali bwa T era ekiikirira ekigendererwa ekiyinza okuweebwa obujjanjabi bw’abaserikale b’omubiri okujjanjaba kookolo w’omuntu.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellardini (Ralph J. Deberardini), Russell G. Jones (Russell). G. Jones), Phineas T. Hamilton (Ekibiina ky’Oluganda T.
MNA eyamba mu kuziyiza abaserikale b’omubiri mu butoffaali bwa T era ekiikirira ekigendererwa ekiyinza okuweebwa obujjanjabi bw’abaserikale b’omubiri okujjanjaba kookolo w’omuntu.
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