Ebirungo by‘omubiri ebirikuhindura omubiri n‘ekintu kikuru omu nkora y‘okukyebera ebizimba (TME), kwonka okwihaho bikye, okumanyisa kwabyo nikuguma kutamanyirwe munonga. Aha, tukashwijuma ebizimba n‘obutafaari bwa T kuruga omu bizimba n‘ebihaha by‘abarwaire abaine oburwaire bwa kansa y‘omutindo gw‘ahaiguru (HGSC) kubaasa kushoboorora metabolome y‘ebicweka ebi ebya TME ebitarikushushana. Ascites n‘obutafaari bw‘ebizimba bwine entaaniso mpango omu metabolite. Waagyeragyeranisa n‘okusheeshasheesha, obutafaari bwa T oburikutaaha omu bizimba nibutunga munonga 1-methylnicotinamide (MNA). N‘obu omutindo gwa MNA omu butafaari bwa T guraabe guri ahaiguru, okureebeka kwa nicotinamide N-methyltransferase (enzigye erikuhwera omu kuhindura guruupu za methyl kuruga omu S-adenosylmethionine kuza omu nicotinamide) nikugarukira aha butafaari bw‘ebizimba n‘obutafaari bw‘ebizimba. Omu muringo gw’okukora, MNA neereetera obutafaari bwa T kusheeshasheesha ekirungo ky’ebizimba ekirikureetaho okusheeshasheesha kw’ebizimba alpha. Nahabwekyo, MNA erikuruga omu TME neehwera omu kuhabura obutafaari bwa T kandi neejwekyera ekigyendererwa ekirikubaasa kukoresibwa omu kujanjaba endwara ya kansa y‘omuntu.
Ebirungo ebirikuruga omu bizimba nibibaasa kugira eki byakora kihango omu kuzibira obutafaari bw‘omubiri oburikurwanisa ebizimba, kandi obuhame oburikweyongyera nibworeka ngu nabyo nibibaasa kukora nk‘ekintu kikuru ekirikureetaho okutunguuka kw‘endwara (1). Okwongyera aha nkora ya Warburg, emirimo eyahererukireyo etandikire kushoboorora embeera y‘okuhindura eby‘okurya omu butafaari bw‘ebizimba n‘akakwate kaayo n‘embeera y‘obutafaari bw‘omubiri oburikurwanisa endwara (TME). Okucondooza aha bishushani by‘embeba n‘obutafaari bwa T bw‘omuntu kworekire ngu okuhindura kwa glutamine (2), okuhindura kwa oxidative (3) n‘okuhindura kwa shukaari (4) nibibaasa kukora byonka aha butafaari bw‘omubiri obutari bumwe na bumwe oburikurwanisa endwara. Ebirungo bingi ebiri omu miringo egi nibizibira obutafaari bwa T kukora gye omu kurwanisa ebizimba. Kikahamibwa ngu okuzibira ekirungo kya coenzyme tetrahydrobiopterin (BH4) nikibaasa kushiisha okukanyira kw‘obutafaari bwa T, kandi okweyongyera kwa BH4 omu mubiri nikubaasa kwongyera aha kugarukamu kw‘obutafaari bw‘omubiri oburikurwanisa ebizimba oburikuteebwaho CD4 na CD8. Okwongyera ahari ekyo, obutafaari bw‘omubiri oburikurwanisa endwara bwa kynurenine nibubaasa kucungurwa ahabw‘okuha BH4 (5). Omuri glioblastoma erikuhinduka omu isocitrate dehydrogenase (IDH), okushohoza kw‘eshagama y‘omubiri (R-2-HG) nikuzibira obutafaari bwa T kukora, okukanyira n‘okukora kw‘obutafaari bw‘omubiri (6). Enyimaho, kyorekwa ngu methylglyoxal, ekirugire omu glycolysis, nikikorwa obutafaari oburikuzibira obukomooko bwa myeloid, kandi obutafaari bwa T oburikutwara methylglyoxal nibubaasa kuzibira obutafaari bwa effector T kukora. Omu kujanjaba, okutaho obutafaari bwa methylglyoxal nikubaasa kusingura emirimo y‘obutafaari oburikuzibira obuhuta oburikuruga omu myeloid (MDSC) kandi omu muringo gw‘okukoragana kwongyera aha bujanjabi bw‘okuzibira okukyebera omu bishushani by‘embeba (7). Okucondooza oku kwona nikuhamya omurimo mukuru gw‘ebintu ebirikuruga omu TME omu kutebeekanisa emirimo n‘emirimo y‘obutafaari bwa T.
Okuremwa kukora gye kw‘obutafaari bwa T kugambirweho munonga omu ndwara ya kansa y‘omu nda (8). Eki nikireetwa ekicweka ky‘emitwarize y‘okuhindura eby‘okurya ebiri omu kubura oxygen n‘okutambura kw‘ebizimba okutari kwa buriijo (9), ekirikureetaho okuhindura shukaari na tryptophan kuba ebintu ebirikuruga omu mubiri nk‘ebiriisa bya lactic acid na kynurenine. Laktate y‘aheeru y‘obutafaari erikukirayo neekyendeeza aha kureetaho interferon-γ (IFN-γ) kandi neereetaho okutaanisa kw‘ebicweka by‘omubiri ebirikuremesa obutafaari bw‘omubiri (10, 11). Okurya tryptophan nikuzibira butoosha okukanyira kw‘obutafaari bwa T kandi nikuzibira obutafaari bwa T okumanyisa (12-14). N‘obu ebi biraabe byareebirwe, emirimo mingi eyehinguririize okuhindura kw‘obutafaari bw‘omubiri ekakorwa omu buhangwa bw‘obutafaari bwa T omu vitro barikukoresa eby‘okureeberaho ebihikire, nari kugarukira aha bishushani by‘embeba ebirikushushana omu vivo, tihaine na kimwe aharibyo ekirikworeka omu bwijwire obutashushana bw‘endwara za kansa z‘abantu n‘embeera y‘omubiri ey‘omubiri n‘ey‘omubiri.
Ekintu ekirikukirayo omu kookoro y‘omu nda n‘okujanjaara kw‘omu nda n‘okureebeka kw‘ebizimba by‘omubiri. Okuteerana kw‘amaizi g‘obutafaari omu nshaho y‘omubiri nikukwatagana n‘endwara erikutunguuka n‘okutambura kubi (15). Okurugirira ahari ripoota, ekicweka eki eky‘omutaano nikiba kitarimu oxygen, kiine ebiriisa bingi ebirikureetaho okukura kw‘emitsi (VEGF) na indoleamine 2,3-dioxygenase (IDO), kandi nikitaahamu obutafaari bwa T oburikutegyeka n‘obutafaari oburikuzibira myeloid (15-18). Embeera y‘okuhindura eby‘okurya omu nkora y‘okusheeshasheesha neebaasa kuba neetaanira kimwe n‘ey‘ekizimba kyonyini, n‘ahabw‘ekyo okugaruka kutebeekanisa obutafaari bwa T omu mwanya gw‘omubiri tikurikwetegyerezibwa gye. Okwongyera ahari ekyo, entaaniso nkuru n‘obutashushana ahagati y‘ebizimba n‘ebintu ebiri omu mubiri ebiri omu mwanya gw‘ebizimba nibibaasa kuremesa okutaaha kw‘obutafaari bw‘omubiri oburikurwanisa endwara n‘emirimo yaabyo aha bizimba, kandi okucondooza okurikukiraho nikwetengyesa.
Okubaasa kushoboorora ebizibu ebi, tukakora omuringo gw‘okwahukana kw‘obutafaari oburikwetegyerezibwa n‘okukyebera amaizi (LC-MS/MS) okwega aha miringo y‘obutafaari etari emwe n‘emwe (obariiremu obutafaari bwa CD4 + na CD8 + T) hamwe n‘omunda n‘ahagati y‘ebizimba Ebirungo byakyo nibijanjaaza obutafaari omu mwanya gumwe gw‘okusheeshasheesha n‘omurwaire w‘ebizimba. Nitukoresa omuringo ogu omu kukwatanisa n‘okupima okusheeshasheesha kw‘omubiri n‘okukuratana kwa RNA y‘obutafaari bumwe (scRNA-seq) kuha ekishushani ekishobooroirwe munonga eky‘embeera y‘okuhindura eby‘okurya omu bantu abakuru aba. Omuringo ogu gukashohoza okweyongyera kw’amaani omu rurengo rwa 1-methylnicotinamide (MNA) omu butafaari bwa T obw’ebizimba, kandi okucondooza okwakozirwe omu vitro kukareebeka ngu oku MNA erikuhindura obutafaari bw’omubiri oburikurwanisa endwara aha mirimo y’obutafaari bwa T kukaba kutamanyirwe ira. Omu kutwariza hamwe, omuringo ogu nigushuruura enkoragana y‘eby‘okurya ebiri ahagati y‘ebizimba n‘obutafaari bw‘omubiri oburikurwanisa endwara, kandi niguha okumanya kw‘omutaano aha bikwatiraine n‘obutafaari bw‘omubiri oburikurwanisa endwara, oburikubaasa kuba obw‘omugasho omu kujanjaba oburwaire bwa T cell-based ovarian cancer immunotherapy Emigisha y‘okujanjaba.
Twakoresa okupima okurikugyendera aha nkora y‘okusheeshasheesha kw‘omubiri (2-NBDG) n‘emirimo y‘omubiri [MitoTracker Deep Red, MT, DR01) omu bwire bumwe. obumanyiso bw‘omutaano oburikutaanisa obutafaari bw‘omubiri oburikurwanisa endwara n‘omubaro gw‘obutafaari bw‘ebizimba (Ekipande S2 n‘ekishushani S1A). Okweshwijuma oku kukooreka ngu waagyeragyeranisa n‘obutafaari bwa T, obutafaari bwa ascites n‘obutafaari bw‘ebizimba bwine omubaro gw‘ahaiguru gw‘okutwara shukaari, kwonka bwine entaaniso nkye omu mirimo ya mitochondrial. Omubaro gw‘okutwara shukaari omu butafaari bw‘ebizimba [CD45-EpCAM (EpCAM)+] nigukira emirundi eshatu kuhika ahari ena y‘obutafaari bwa T, kandi omubaro gw‘okutwara shukaari omu butafaari bwa CD4 + T nigukira emirundi 1.2 y‘obutafaari bwa CD8 + T, ekirikworeka ngu obutafaari bw‘ebizimba oburikutaahamu omu butafaari bw‘ebizimba (TIL) nibutunga obutafaari bw‘omubiri obutari bumwe na bumwe. Okutaana n‘ekyo, emirimo y‘obutafaari bw‘ebizimba neeshushana n‘ey‘obutafaari bwa CD4 + T, kandi emirimo y‘obutafaari bw‘emiringo yombi eri ahaiguru y‘obutafaari bwa CD8 + T (ekishushani 1B). Omu kutwariza hamwe, ebyarugamu ebi nibyoreka omutindo gw‘okuhindura eby‘okurya. Omurimo gw‘okuhindura eby‘okurya omu butafaari bw‘ebizimba nigukira ogw‘obutafaari bwa CD4 + T, kandi omurimo gw‘okuhindura eby‘okurya omu butafaari bwa CD4 + T nigukira ogw‘obutafaari bwa CD8 + T. N‘obu ebi biraabe biriho omu miringo y‘obutafaari, tihaine ntaaniso egumire omu mbeera y‘okuhindura eby‘okurya omu butafaari bwa CD4 + na CD8 + T nari emibaro yaabo omu nkora y‘okusheeshasheesha kugyeragyeranisa n‘ebizimba (ekishushani 1C). Okutaana n‘ekyo, omu kicweka kya CD45-cell, omubaro gw‘obutafaari bwa EpCAM+ omu kizimba gukeyongyera waagyeragyeranisa n‘okusheeshasheesha (ekishushani 1D). Kandi tukareeba entaaniso erikureebwa gye omu nkora y‘okuhindura eby‘okurya ahagati y‘ebicweka bya EpCAM+ na EpCAM- cell. Obutafaari bwa EpCAM+ (ebizimba) bwine okutwara shukaari nyingi n‘okukora kwa mitochondrial kukira obutafaari bwa EpCAM-, ekirikukirayo munonga emirimo y‘okuhindura eby‘okurya omu butafaari bw‘ebizimba omu TME (ekishushani 1, E na F).
(A na B) Obuhango bw‘okujwara (MFI) bw‘okutwara shukaari (2-NBDG) (A) n‘emirimo y‘obutafaari bwa CD4 + T (MitoTracker erikutukura munonga) (B) Ebishushani ebirikujwekyera (aha rubaju rwa bumosho) n‘ebihandiiko ebiteirwe omu nkora (aha rubaju rwa buryo), obutafaari bwa CD8 + T n‘obutafaari bwa EpCAM + T CD45-. (C) Omubaro gw‘obutafaari bwa CD4 + na CD8 + (obw‘obutafaari bwa CD3 + T) omu nshaho n‘omu bizimba. (D) Omubaro gw‘obutafaari bwa EpCAM + obw‘ebizimba omu nshaho n‘ebizimba (CD45−). (E na F) EpCAM + CD45-ekizimba n‘okutwara shukaari omu nkora ya EpCAM-CD45 (2-NBDG) (E) n‘emirimo y‘ebizimba (MitoTracker erikutukura munonga) (F) ebishushani ebirikujwekyera (aha rubaju rwa bumosho) n‘ebihandiiko ebihandiikirwe omu nkora ( Aha rubaju rwa buryo) Ascites n‘obutafaari bw‘ebizimba. (G) Ebishushani ebirikworeka CD25, CD137 na PD1 okurikworekwa omu kucondooza okurikukoresibwa okugyendera kw‘eshagama. (H na I) CD25, CD137 na PD1 okureebeka aha butafaari bwa CD4 + T (H) na CD8 + T (I). (J na K) Naive, okwijuka kwa rwagati (Tcm), okwijuka kw‘okukora (Teff) n‘okwijuka kw‘okukora (Tem) ebirikwegamira aha kushoboorora kwa CCR7 na CD45RO. Ebishushani ebirikujwekyera (aha rubaju rwa bumosho) n‘ebihandiiko by‘omu nkora (aha rubaju rwa buryo) eby‘obutafaari bwa CD4 + T (J) n‘obutafaari bwa CD8 + T (K) omu nshaho n‘ebizimba. Emihendo ya P erikumanyirwa aha kukyebera kwa t-okwa kabiri (*P<0.05, **P<0.01 na ***P<0.001). Omuringo nigujwekyera abarwaire abarikushushana (n = 6). FMO, okujwara ebirikwiragura okurugaho kimwe; MFI, obuhango bw‘okujwara omugati.
Okweyongyera kweshwijuma kukashohoza entaaniso ezindi z‘amaani ahagati y‘embeera y‘obutafaari bwa T obushobooroirwe munonga. Okwijuka okukozirwe (ekishushani 1, G kuhika I) n‘okwijuka okurikubaho (ekishushani 1, J na K) omu bizimba nibikira kubaho munonga kukira okusheeshasheesha (omubaro gw‘obutafaari bwa CD3 + T). Omu muringo nigwo gumwe, okushwijuma ebiri omu nkora y‘omubiri (phenotype) ahabw‘okumanyisa obumanyiso bw‘okukora (CD25 na CD137) n‘obumanyiso bw‘okukyendeeza [puroteyini y‘okufa kw‘obutafaari 1 (PD1)] kukooreka ngu n‘obu emitwarize y‘okuhindura eby‘okurya omu bantu aba eraabe neetaanira kimwe (ekishushani S1, B kuhika E) , Kwonka tihaine ntaaniso y‘amaani ey‘okuhindura eby‘okurya ahagati y‘okwijuka nari okuhinduka kw‘omubiri (Figure S1). F kuhika ahari I). Ebirugiremu ebi bikahamibwa barikukoresa emiringo y‘okwega eby‘ebyoma kuha obutafaari bw‘omubiri (21), ekyayongyeire kworeka okubaho kw‘obutafaari bw‘amagufa bwingi (CD45 + / CD3- / CD4 + / CD45RO +) omu nshaho y‘omurwaire (Ekishushani S2A ). Omu miringo yoona ey‘obutafaari eyamanyirwe, omubaro gw‘obutafaari obu ogwa myeloid gukooreka okutwara shukaari nyingi n‘emirimo y‘emibiri (ekishushani S2, B kuhika G). Ebirugiremu ebi nibyoreka entaaniso z‘amaani z‘okuhindura eby‘okurya ahagati y‘ebika by‘obutafaari bingi ebirikushangwa omu nshaho n‘ebizimba omu barwaire ba HGSC.
Ekizibu ekikuru omu kwetegyereza emitwarize ya metabonomic ya TIL n‘okwetenga kwetaanisa aha sampo z‘obutafaari bwa T obw‘obushemeire oburikumara, omutindo n‘obungi kuruga omu bizimba. Okucondooza okukozirwe juba kworekire ngu emiringo y‘okushoroora n‘okugaita oburungo erikwegamira aha flow cytometry neebaasa kureetera empindahinduka omu nkora y‘ebiriisa by‘omubiri (22-24). Okubaasa kusingura ekizibu eki, tukatunguura omuringo gw‘okugaita oburungo (bead enrichment method) kubaasa kutaanisa n‘okutaanisa TIL kuruga omu kookoro y‘omunda y‘omuntu eyashazirwe omu kushemezibwa tutakakyebeire LC-MS/MS (reeba Ebikoreso n‘emiringo; ekishushani 2A). Okubaasa kukyebera oku puroguraamu egi erikukwata aha mpinduka z‘eby‘okurya omu bwijwire, tukagyeragyeranisa ebirikukwata aha nkora y‘okuhindura eby‘okurya omu butafaari bwa T oburikukoresibwa abahi b‘obuhwezi abaine amagara marungi bwanyima y‘eidaara ry‘okwahukana kw‘obubare eri aharuguru n‘obutafaari obutataanisiibwe obubare kwonka bukaguma aha ice. Okushwijuma oku okw‘okurinda omutindo kukashanga ngu hariho akakwate kakuru ahagati y‘embeera ezi zombi (r = 0.77), kandi okugarukamu kw‘eby‘obuhangwa kw‘ekibiina ky‘ebicweka 86 eby‘omubiri kwine okugarukamu kw‘ahaiguru (ekishushani 2B). Nahabwekyo, emiringo egi neebaasa kukora okucondooza okuhikire okw‘ebiriisa omu butafaari oburikuraba omu kutunguuka kw‘omuringo gw‘obutafaari, n‘ahabw‘ekyo neeha omuhanda gw‘okubanza ogw‘okushoboorora ebiri omu HGSC, obwo erikubaasisa abantu kutunga okwetegyereza okurikukirayo aha nkora y‘ebiriisa omu nkora y‘okuteerana.
(A) Ekishushani ky’okutunguura eby’obugaiga bw’omubiri. Obutafaari butakakyebeirwe LC-MS/MS, nibwija kuraba omu nshonda ishatu ezirikukuratana z‘okugaita oburungo bw‘amashanyarazi nari kuguma aha ice. (B) Ekirikuruga omu muringo gw‘okugaita aha bwingi bw‘ebintu ebirikuhindura omubiri. Omubaro gw‘ebipimo bishatu ahari buri muringo gw‘okugaita ± SE. Omuringo gw’enzirugavu nigujwekyera omukago gwa 1:1. Enkoragana y‘omunda y‘ekibiina (ICC) y‘okugarukamu ebipimo ebirikworekwa omu kihandiiko ky‘omutindo. NAD, nikotiinamidi y’omubiri. (C) Ekishushani ky‘omuringo gw‘okukora kw‘okushwijuma ebiriisa by‘omurwaire. Ascites nari ebizimba nibitorotoorwaho kuruga omu barwaire kandi bibiikwa omu nju y‘obufuki. Ekicweka kikye kya buri sampo kikakyeberwa omu muringo gwa flow cytometry, obwo sampo ezisigaireho zikakora emirundi eshatu y’okugaita obutafaari bwa CD4+, CD8+ na CD45-. Ebicweka by’obutafaari ebi bikakyeberwa barikukoresa LC-MS/MS. (D) Maapu y‘okwotsya y‘obungi bw‘ebiriisa ebiri omu nkora. Dendrogram neejwekyera okuteerana kwa Ward kw’ebicweka bya Euclidean ahagati y’eby’okureeberaho. (E) Okushwijuma ebicweka ebikuru (PCA) bya maapu y‘eby‘okureeberaho by‘okuhindura ebiri omu mubiri, kworeka okugarukamu kashatu kwa buri sampo, eby‘okureeberaho kuruga omu murwaire omwe nibikwatanisibwa n‘omurongooti. (F) PCA y‘enkora ya metabolite y‘eky‘okureeberaho ekitebeekanisiibwe aha murwaire (nikwo kugira ngu, okukoresa okugaruka enyima kw‘ekicweka); ekika ky’eky’okureeberaho nikigarurwa enyima ahabw’ekicweka ky’omubiri ekirikushusha nk’ekibunu. PC1, ekicweka kikuru 1; PC2, ekicweka kikuru 2.
Ekindi, tukakoresa omuringo ogu gw‘okugaita okushwijuma ebiriisa 99 omu bicweka bya CD4 +, CD8 + na CD45-cell omu nkora y‘okubanza y‘okusheeshasheesha n‘ebizimba by‘abarwaire mukaaga ba HGSC (ekishushani 2C, ekishushani S3A n‘ekipande S3 na S4). Omubaro gw‘abantu abarikukunda nigukora 2% kuhika ahari 70% y‘eky‘okureeberaho kihango ky‘okubanza eky‘obutafaari obuhuriire, kandi omubaro gw‘obutafaari niguhinduka munonga ahagati y‘abarwaire. Bwanyima y‘okutaanisa obubare, ekicweka ky‘okutunga ekigaiga ky‘okukunda (CD4+, CD8+ nari CD45-) nikikora obutafaari bwona obuhuriire oburikurenga ahari 85% omu sampo omu mwanya. Omuringo ogu gw’okugaita nigutubaasisa kushwijuma obutafaari kuruga omu nkora y’enyama z’ebizimba by’omuntu, ekitarikubaasika kukora kuruga omu sampo mpango. Twakozesa enkora egi, tukashanga ngu l-kynurenine na adenosine, ebi bibiri ebiine obumanyiso burungi ebirikuzibira obutafaari bw‘omubiri kurwanisa endwara bikatunguuka omu butafaari bw‘ebizimba T nari obutafaari bw‘ebizimba (ekishushani S3, B na C). Nahabwekyo, ebyarugamu ebi nibyoreka obwesigwa n’obushoboorozi bw’okwahukana kw’obutafaari bwaitu n’okupima obuhango bw’ebintu (mass spectrometry technology) kutunga ebiriisa ebikuru omu by’amagara omu bicweka by’omubiri by’omurwaire.
Okweshwijuma kwaitu kandi kukashohoza okwahukana kw‘amaani kw‘eby‘okurya omu miringo y‘obutafaari omunda n‘ahagati y‘abarwaire (ekishushani 2D n‘ekishushani S4A). Okukira munonga, waagyeragyeranisa n‘abarwaire abandi, omurwaire 70 akooreka emitwarize y‘okuhindura eby‘okurya etari emwe n‘emwe (ekishushani 2E n‘ekishushani S4B), ekirikworeka ngu nihabaasa kubaho obutashushana bw‘eby‘okurya bw‘amaani ahagati y‘abarwaire. Nikirungi kumanya ngu waagyeragyeranisa n‘abarwaire abandi (lita 1.2 kuhika 2; ekipande S1), omubaro gwona gw‘ebizimba ebyarundainwe omu murwaire 70 (miriyoni 80) gukaba guri mukye. Okutegyeka obutashushana ahagati y‘abarwaire omu bwire bw‘okushwijuma ebicweka ebikuru (eky‘okureeberaho, okukoresa okushwijuma kw‘ekicweka ky‘okugarukamu) nikworeka empindahinduka ezitarikuhinduka ahagati y‘ebika by‘obutafaari, kandi ebika by‘obutafaari na/nari embeera nkye nibiteeranirwa hamwe gye kurugiirira aha nkora ya metabolite (ekishushani 2F ). Okushwijuma ebiriisa bimwe bikahamya ebirugiremu ebi kandi byayoreka entaaniso mpango ahagati y‘ebika by‘obutafaari n‘embeera nkye. Nikirungi kumanya ngu entaaniso erikukirayo obuhango erikureebeka ni MNA, erikukira kuba erimu obutafaari bwa CD45- n‘obutafaari bwa CD4+ na CD8+ oburikutaaha omu kizimba (ekishushani 3A). Aha butafaari bwa CD4 +, eki nikikira kureebwa, kandi MNA omu butafaari bwa CD8 + nayo neeshusha ngu neekwatwaho munonga eby‘obuhangwa. Kwonka eki ti kikuru, ahabw‘okuba abarwaire bashatu bonka ahari mukaaga nibo barikubaasa kukyeberwa ahabw‘obubonero bwa CD8+ bw‘ebizimba. Oyihireho MNA, omu miringo etari emwe n‘emwe y‘obutafaari omu nshaho n‘ebizimba, ebindi bihingwa ebitarikumanyirwa gye omu TIL nabyo nibitunga obugaiga bw‘entaaniso (ebishushani S3 na S4). Nahabwekyo, ebihandiiko ebi nibyoreka ekicweka ky‘ebintu ebirikuraganisa ebirikukora aha nkora y‘okurwanisa endwara ahabw‘okucondooza okurikukiraho.
(A) Ebirimu bya MNA ebya buriijo omu butafaari bwa CD4+, CD8+ na CD45- kuruga omu nshaho n‘ebizimba. Puloti y‘akabokisi neeyoreka omubaro gw‘ahagati (omurongooti), omubaro gw‘ahagati y‘ebicweka bina (ekihingo kya fureemu) n‘omubaro gw‘ebihandiiko, kuhika aha mirundi 1.5 y‘omubaro gw‘ebicweka bina (frame whisker). Nk‘oku kyashoboororwa omu bihandiiko by‘omurwaire n‘emiringo, kozesa omuhendo gw‘omurwaire ogwa limma kubaasa kumanya omuhendo gwa P (*P<0.05 na **P<0.01). (B) Ekishushani ky‘okuhindura eby‘okurya bya MNA (60). Ebirungo ebiri omu mubiri: S-adenosili-1-metionini; SAH, S-ekirungo ky’omubiri-1-ekirungo ky’omubiri; NA, omubazi gw’okunywa; MNA, 1-omubazi gw’okukyebera; 2-PY, 1-omubiri- 2-piridoni-5-kaboksamidi; 4-PY, 1-omubiri-4-piridoni-5-kaboksamidi; NR, ebiriisa by’omubiri; NMN, ekirungo ky’omubiri ekirikureeta ebiriisa. Enzymes (eza kinyasi): NNMT, nikotinamidi N-ekiriisa ekirikuhindura ebiriisa; SIRT, ebicweka by’omubiri; NAMPT, ekirikuhindura ebiriisa omu mubiri; AOX1, ekirungo ky’omubiri 1; NRK, ekirikureetaho okusheeshasheesha kw’omubiri; NMNAT, nikotinamide emwe ey’okujanjaaza ebiriisa; Pnp1, purini y’omubiri. (C) t-SNE ya scRNA-seq y‘ebizimba (enzirugavu) n‘ekizimba (erangi erikutukura; n = abarwaire 3). (D) NNMT okureebeka omu butafaari obutari bumwe na bumwe obumanyirwe barikukoresa scRNA-seq. (E) Okworeka NNMT na AOX1 omu SK-OV-3, ensigo y‘omuntu ey‘omunda (HEK) 293T, obutafaari bwa T n‘obutafaari bwa T obujanjabirwe MNA. Enshoboorora ehindwirwemu neeyorekwa erikwingana na SK-OV-3. Enkora y‘okworeka na SEM neeyorekwa (n = 6 abahi b‘obuhwezi abaine amagara marungi). Emihendo ya Ct erikurenga 35 neetwarwa nk‘etarikubaasa kumanyirwa (UD). (F) Okworeka SLC22A1 na SLC22A2 omu SK-OV-3, HEK293T, obutafaari bwa T n‘obutafaari bwa T obujanjabirwe na 8mM MNA. Enshoboorora ehindwirwemu neeyorekwa erikwingana na SK-OV-3. Enkora y‘okworeka na SEM neeyorekwa (n = 6 abahi b‘obuhwezi abaine amagara marungi). Emihendo ya Ct erikurenga 35 neetwarwa nk‘etarikubaasa kumanyirwa (UD). (G) Ebirimu omu butafaari bwa MNA omu butafaari bwa T obuine amagara marungi obukozirwe bwanyima y‘eshaaha 72 z‘okubuteeka na MNA. Enkora y‘okworeka na SEM neeyorekwa (n = 4 abahi b‘obuhwezi abaine amagara marungi).
MNA neekorwa omu kuhindura ekibiina ky‘eby‘okurya kuruga omu S-adenosyl-1-methionine (SAM) kuza omu by‘okurya (NA) kurabira omu kuhindura eby‘okurya (NNMT; Ekishushani 3B). NNMT neeyorekwa munonga omu miringo etari emwe n‘emwe ya kansa z‘abantu kandi neekwatagana n‘okukanyira, okutaahirira n‘okujanjaara (25-27). Kubaasa kwetegyereza gye obukomooko bwa MNA omu butafaari bwa T omu TME, tukakoresa scRNA-seq kushoboorora oku NNMT erikworekwa omu miringo y‘obutafaari omu ascites n‘ebizimba by‘abarwaire bashatu ba HGSC (Ekipande S5). Okushwijuma obutafaari oburikuhika 6,500 kukooreka ngu omu mbeera z‘okusheeshasheesha n‘ebizimba, okureebeka kwa NNMT kukaba kuri aha butafaari oburikuteekateekwaho obw‘ebizimba n‘ebizimba (ekishushani 3, C na D). Nikirungi kumanya ngu tihaine NNMT erikureebwa omu bantu boona abarikworeka PTPRC (CD45 +) (ekishushani 3D n‘ekishushani S5A), ekirikworeka ngu MNA eyashangirwe omu nkora ya metabolite ekataaha omu butafaari bwa T. Okworeka kwa aldehyde oxidase 1 (AOX1) nikuhindura MNA kuba 1-metili-2-piridoni-5-kaboksamiidi (2-PYR) nari 1-metilii-4-piridoni-5-kaboksamiidi (4- PYR); Ekishushani 3B) nakyo nikigarukira aha mubaro gw‘ebicweka by‘omubiri ebirikworeka COL1A1 (ekishushani S5A), byona hamwe nibyoreka ngu obutafaari bwa T tiburikubaasa kuhindura eby‘okurya bya MNA eby‘obutoosha. Enkora y‘okworeka ebiriisa ebi ebiine akakwate na MNA ekahamibwa barikukoresa ebihandiiko bya kabiri eby‘obutafaari eby‘obwegyese kuruga omu ascites kuruga omu barwaire ba HGSC (Ekishushani S5B; n = 6) (16). Okwongyera ahari ekyo, okucondooza okwakozirwe aha nkora ya polymerase chain reaction (qPCR) aha butafaari bwa T obuine amagara marungi obujanjabirwe na MNA kukooreka ngu waagyeragyeranisa n‘obutafaari bw‘okukyebera SK-OV-3 obw‘ebizimba by‘omu nkari, NNMT nari AOX1 zikaba ziri haihi kutareebeka (ekishushani 3E). Ebirugire omu kucondooza oku ebitarikuteekateekwaho nibyoreka ngu MNA neebaasa kuruga omu fibroblasts nari ebizimba kuza omu butafaari bwa T oburi haihi omu TME.
N‘obu abarikwenda kwetsimbaho ​​baraabe barimu eka y‘ebintu ebirikutwara ebiriisa by‘omubiri 1 kuhika ahari 3 (OCT1, OCT2 na OCT3) ebiine enkodi z‘eka y‘ebintu ebirikutwara ebiriisa 22 (SLC22) (SLC22A1, SLC22A2 na SLC22A3), ebirikutwara ebintu ebirikubaasa kutwara MNA tibikashobooroirwe (28) . QPCR ya mRNA kuruga omu butafaari bwa T oburikuha obuhwezi oburimu amagara marungi ekooreka emiringo y‘okumanyisa SLC22A1 eri ahansi kwonka emiringo etarikureebwa eya SLC22A2, ekyahamize ngu ekaba egambirweho enyima omu bihandiiko (Ekishushani 3F) (29). Okutaana n‘ekyo, omubaro gw‘obutafaari bw‘ebizimba by‘omu nkari SK-OV-3 gukooreka emiringo y‘ahaiguru y‘ebirikutwara ebintu byombi (ekishushani 3F).
Okubaasa kugyezaho okubaasa kw’obutafaari bwa T kugira obushoboorozi bw’okunywa MNA ey’aheeru, obutafaari bwa T oburikuha obuhwezi oburimu amagara marungi bukakuzibwa kumara eshaaha 72 omu kubaho kw’ebicweka bya MNA ebitarikushushana. Omu kubura kwa MNA ey‘aheeru, ebiri omu butafaari bwa MNA tibirikubaasa kumanyirwa (ekishushani 3G). Kwonka obutafaari bwa T obukozirwe obujanjabirwe na MNA ey‘aheeru bukayoreka okweyongyera okurikurugiirira aha dose omu biri omu MNA omu butafaari, kuhika ahari 6 mM MNA (ekishushani 3G). Ekirugiremu eki nikyoreka ngu n‘obu omutindo gw‘okutwara ebintu guraabe guri ahansi kandi hatariho enzyme enkuru erikujunanizibwa n‘okuhindura MNA omunda y‘obutafaari, TIL neebaasa kuguma neetwara MNA.
Omubaro gw‘ebintu ebirikuhinduka omu butafaari bwa T bw‘abarwaire n‘okucondooza okurikukwata aha kunywa MNA omu nkora ya vitro nikwongera aha kubaasa kw‘okugira ngu fibroblasts eziine akakwate na kansa (CAF) nizishohoza MNA kandi obutafaari bw‘ebizimba nibubaasa kutebeekanisa ebiri omu nkora n‘emirimo ya TIL. Okubaasa kumanya oku MNA erikukora aha butafaari bwa T, obutafaari bwa T oburikuha obuhwezi oburimu amagara marungi bukakorwa omu nkora ya vitro omu kubaho nari obutabaho bwa MNA, kandi okukanyira kwabwo n’okukora kwa cytokine bikakyeberwa. Bwanyima y‘ebiro 7 by‘okwongeraho MNA aha dozi y‘ahaiguru, omubaro gw‘okukanyisa abantu emirundi ebiri gukakyendeera omu muringo gw‘ahaiguru, obwo amaani garikuguma aha dozi zoona (ekishushani 4A). Okwongyera ahari ekyo, okujanjaba MNA ey‘aheeru y‘omubiri kukarugamu okweyongyera kw‘omubaro gw‘obutafaari bwa CD4 + na CD8 + T oburikworeka ekirikureetaho okusheeshasheesha kw‘ebizimba-α (TNFα; ekishushani 4B). Okutaana n‘ekyo, okukora kwa IFN-γ omu butafaari bw‘omubiri kukakyendeera munonga omu butafaari bwa CD4 + T, kwonka tihariho omu butafaari bwa CD8 + T, kandi hakaba hatariho mpindahinduka mpango omu butafaari bwa interleukin 2 (IL-2; Ekishushani 4, C na D). Nahabwekyo, okukyebera okurikukwatanisa n‘enzigye (ELISA) kw‘ebicweka by‘omubiri kuruga omu buhangwa bw‘obutafaari bwa T obujanjabirwe MNA kukooreka okweyongyera kw‘amaani omu TNFα, okukyendeera omu IFN-γ, kandi tihaine mpindahinduka omu IL-2 (ekishushani 4, E kuhika G). . Okukyendeera kwa IFN-γ nikworeka ngu MNA neebaasa kukora kihango omu kuzibira emirimo y‘okurwanisa ebizimba ey‘obutafaari bwa T. Okubaasa kugyeragyeranisa oku MNA erikukora aha butafaari bwa T oburikureetwaho obutwa bw‘omubiri, obutafaari bwa chimeric antigen receptor T (FRα-CAR-T) oburikuta omutima aha folate receptor α na CAR-T (GFP) oburikutegyekwa obutafaari bwa green fluorescent puroteyini (GFP) -CAR-T) nibukorwa obutafaari bw‘omubiri oburikwera oburikureeta eshagama ( mononunu PBMC). Obutafaari bwa CAR-T bukakuzibwa kumara eshaaha 24 omu maisho ga MNA, reero bwakuzibwa hamwe n’obutafaari bw’ebizimba by’omunda y’omuntu SK-OV-3 oburikworeka folate receptor α aha nkora y’okukora n’ekigyendererwa kya 10:1. Obujanjabi bwa MNA bukarugamu okukyendeera kw‘amaani omu mirimo y‘okwita obutafaari bwa FRα-CAR-T, ekyabaire nikishushana n‘obutafaari bwa FRα-CAR-T obwabaire bujanjabirwe na adenosine (ekishushani 4H).
(A) Omubaro gw‘obutafaari bwona oburikubaasa kubaho n‘okukanyisa kw‘abantu emirundi ebiri (PD) butunu kuruga omu buhangwa aha kiro kya 7. Ekishushani ky‘omubaro nikijwekyera omubaro gw‘ahagati + SEM gw‘abahi b‘obuhwezi mukaaga abaine amagara marungi. Nikijwekyera amakuru kuruga omu kucondooza okutarimu n = 3 okwetongoire. (B kuhika D) CD3/CD28 na IL-2 bikakoresibwa kureetaho obutafaari bwa T aha nkora zaabo eza MNA kumara ebiro 7. Obutafaari butakakyebeirwe, bukakyeberwa na PMA/ionomycin na GolgiStop kumara eshaaha 4. TNFα (B) okureebeka omu butafaari bwa T. Eky‘okureeberaho ekishushani (aha rubaju rwa bumosho) n‘ebihandiiko by‘omu nkora (aha rubaju rwa buryo) eby‘okureebeka kwa TNFα omu butafaari obuhuriire. IFN-γ (C) na IL-2 (D) okureebeka omu butafaari bwa T. Okworeka kwa cytokines kukapimwa omu kugyezaho okusheesheka kw‘eshagama. Ekishushani ky‘omubaro nikijwekyera omubaro gw‘ahagati (n = 6 abahi b‘obuhwezi abaine amagara marungi) + SEM. Koresa okucondooza kw‘omuringo gumwe kw‘okuhinduka n‘okugarukamu (*P<0.05 na **P<0.01) kubaasa kumanya omuhendo gwa P. Nikijwekyera amakuru kuruga omu kucondooza okutarimu n = 3 okwetongoire. (E kuhika G) CD3/CD28 na IL-2 bikakoresibwa kureetaho obutafaari bwa T aha nkora zaabo eza MNA kumara ebiro 7. Omubazi gukatorotoorwaho eshaaha 4 z‘okusiimura PMA/ionomycin zitakahikire n‘enyima. Ebicweka bya TNFα (E), IFN-γ (F) na IL-2 (G) bikapimwa omu muringo gwa ELISA. Ekishushani ky‘omubaro nikijwekyera omubaro gw‘ahagati (n = 5 abahi b‘obuhwezi abaine amagara marungi) + SEM. Omuhendo gwa P gumanyirwe barikukoresa okucondooza kw‘omuringo gumwe kw‘okuhinduka n‘okugarukamu okupima (*P<0.05). Omuringo gw’obutonyezi nigworeka ekigarukwamu ky’okumanyisa eky’okumanyisa. (H) Okukyebera okukyebera obutafaari. Obutafaari bwa FRα-CAR-T nari GFP-CAR-T bukahindurwa n‘ekiriisa kya adenosine (250μM) nari MNA (10 mM) kumara eshaaha 24, nari bukarekwa butaine bujanjabi (Ctrl). Ebicweka 10 ahari 100 eby’okwitwa kw’obutafaari bwa SK-OV-3 bikapimwa. Omuhendo gwa P ogurikumanyirwa aha kukyebera kwa Welch t (*P<0.5 na **P<0.01).
Okubaasa kutunga okwetegyereza kw‘omuringo gw‘okutegyeka okureebeka kwa TNFα okurikurugiirira ahari MNA, empindahinduka omu TNFα mRNA y‘obutafaari bwa T obujanjabirwe MNA zikakyeberwa (ekishushani 5A). Obutafaari bwa T obuine amagara marungi obujanjabirwe na MNA bukayoreka okweyongyera kw‘emirundi ebiri omu rurengo rw‘okuhandiika TNFα, ekirikworeka ngu MNA neerugiirira aha nkora y‘okuhandiika TNFα. Okucondooza aha nkora egi erikubaasa kutegyeka, enshonga ibiri ezimanyirwe ez‘okuhandiika ezirikutegyeka TNFα, nikwo kugira ngu activated T cell nuclear factor (NFAT) na puroteyini y‘omutaano 1 (Sp1), zikakyeberwa omu kugarukamu okukwatira hamwe kwa MNA aha nkora ya TNFα eri haihi ( 30 ). Omuhi w‘obuhwezi wa TNFα aine emyanya 6 emanyirwe ey‘okuteerana NFAT n‘emyanya 2 y‘okuteerana Sp1, erikuteerana aha mwanya gumwe [-55 base pairs (bp) kuruga aha 5‘cap] (30). Okukyebera okurikukwata aha nkora y‘okurwanisa endwara (ChIP) kukooreka ngu ku baajanjabirwe na MNA, okutaaha kwa Sp1 aha muhi w‘obuhwezi wa TNFα kukaba kweyongyera emirundi eshatu. Okutaho NFAT nakyo kikeyongyera kandi kyahika aha bukuru (ekishushani 5B). Ebihandiiko ebi nibyoreka ngu MNA neetebeekanisa okureebeka kwa TNFα kurabira omu kuhandiika kwa Sp1, kandi omu rulengo rukye okureebeka kwa NFAT.
(A) Kugyeragyeranisa n‘obutafaari bwa T obuhangirwe hatariho MNA, empindahinduka y‘emirundi y‘okureebeka kwa TNFα omu butafaari bwa T obujanjabirwe na MNA. Enkora y‘okworeka na SEM neeyorekwa (n = 5 abahi b‘obuhwezi abaine amagara marungi). Nikijwekyera amakuru kuruga omu kucondooza okutarimu n = 3 okwetongoire. (B) Omuhi w‘obuhwezi wa TNFα ow‘obutafaari bwa T obujanjabirwe nari obutaine 8 mM MNA bwanyima ya NFAT na Sp1 kugambwaho na (Ctrl) n‘okusiimura kwa PMA/ionomycin kumara eshaaha 4. Immunoglobulin G (IgG) na H3 zikakozesebwa nk‘okukyebera okubi n‘okurungi ahabw‘okukyendeeza aha nkora y‘okurwanisa endwara, nk‘oku biri. Okupima omubaro gwa ChIP kukooreka ngu okutaaha kwa Sp1 na NFAT aha TNFα promoter omu butafaari obujanjabirwe MNA kukeeyongyera emirundi mingi waagyeragyeranisa n’okukyebera. Nikijwekyera amakuru kuruga omu kucondooza okutarimu n = 3 okwetongoire. Omuhendo gwa P ogurikumanyirwa aha bigyezo bya t bingi (*** P <0.01). (C) Kugyeragyeranisa n‘okusheeshasheesha kwa HGSC, obutafaari bwa T (obutari bw‘obutwa) bukayoreka okweyongyera kw‘okureebeka kwa TNF omu kizimba. Erangi nizijwekyera abarwaire batari bamwe na bamwe. Obutafaari oburikworekwa bubaire nibutooranwa omu muringo gw‘okutooranamu kuhika ahari 300 kandi bwajitterwa kukyendeeza aha kuteera ebishushani ebirengire (** Padj = 0.0076). (D) Eky‘okureeberaho ekiteirweho ekya MNA ahabwa kansa y‘omu nda. MNA neekorwa omu butafaari bw‘ebizimba n‘obutafaari bw‘omubiri oburikwetwa fibroblasts omu TME kandi neetwarwa obutafaari bwa T. MNA neeyongyera aha kukwatira hamwe kwa Sp1 n’omuhi w’obuhwezi wa TNFα, ekirikureetaho okweyongyera kw’okuhandiika kwa TNFα n’okukora kwa TNFα cytokine. MNA nayo neereetera IFN-γ kukyendeera. Okuzibira obutafaari bwa T kukora nikireetaho okukyendeeza aha bushoboorozi bw‘okwita n‘okukura kw‘ebizimba omu bwangu.
Okurugirira ahari ripoota, TNFα eine ebirikurwanisa ebizimba n‘ebizimba ebirikwegamira aha maisho n‘enyima, kwonka eine omurimo ogumanyirwe gye omu kutunguura okukura n‘okujanjaara kwa kansa y‘omu nda (31-33). Okurugirira ahari ripoota, okuteerana kwa TNFα omu nyama z‘omubiri n‘ebizimba omu barwaire abaine ekookoro y‘omu nda nikukira okuri omu nyama z‘omubiri ezitari z‘akabi (34-36). Omu nkora, TNFα neebaasa kutebeekanisa okukora, okukora n‘okukanyira kw‘obutafaari bw‘eshagama oburikwera, n‘okuhindura aha nkora y‘obutafaari bwa kansa (37, 38). Okurugiirira aha byashangwa, okucondooza okurikukwata aha nkora y‘okumanyisa ebiri omu nkora y‘obuzaarwa (gene gene expression) kworekire ngu TNF ekaba neetunguuka munonga omu butafaari bwa T omu bicweka by‘ebizimba kugyeragyeranisa n‘okusheeshasheesha (ekishushani 5C). Okwongyezibwaho kw‘okureebeka kwa TNF kukaba nikureebeka omu butafaari bwa T bwonka oburimu obutafaari obutari bw‘obutwa (ekishushani S5A). Omu bugufu, ebihandiiko ebi nibishagika enteekateeka ngu MNA eine ebirugiremu bibiri ebirikuzibira obutafaari bw‘omubiri n‘okutunguura ebizimba omu HGSC.
Okuhandiika ebiri omu mushana okurikurugiirira aha flow cytometry kihindukire omuringo mukuru gw‘okwega aha nkora ya TIL. Okucondooza oku kworekire ngu waagyeragyeranisa n‘obutafaari bw‘eshagama obw‘aheeru nari obutafaari bwa T kuruga omu bicweka by‘omubiri eby‘akabiri, TIL y‘omu nyamaishwa n‘ey‘omuntu eine omuze gw‘okutwara shukaari (4, 39) n‘okufeerwa mporampora emirimo y‘emibiri (19, 40). N‘obu turaabe twareebire ebyarugamu nk‘ebyo omu kucondooza oku, entunguuka enkuru n‘okugyeragyeranisa okuhindura kw‘obutafaari bw‘ebizimba na TIL kuruga omu nyama y‘ebizimba emwe eshazirweho. Okurugiirira ahari zimwe aha ripoota ezi ezabandize, obutafaari bw‘ebizimba (CD45-EpCAM +) kuruga omu nshaho n‘ebizimba bwine okutwara shukaari nyingi kukira obutafaari bwa CD8 + na CD4 + T, ekirikuhagira ngu okutwara shukaari nyingi kw‘obutafaari bw‘ebizimba nikubaasa kugyeragyeranisibwa n‘obutafaari bwa T. Enteekateeka y’okuhakanisa obutafaari bwa T. TME. Kwonka emirimo y‘obutafaari bw‘ebizimba neekira eya CD8 + T, kwonka emirimo y‘obutafaari bw‘ebizimba neeshushana n‘ey‘obutafaari bwa CD4 + T. Ebirugiremu ebi nibihamya omutwe ogurikurugayo ngu okuhindura eby‘okurya omu mubiri (oxidative metabolism) ni kikuru aha butafaari bw‘ebizimba (41, 42). Kandi nibagira ngu obutafaari bwa CD8 + T nibubaasa kukwatwaho munonga okutakora gye kw‘okwosya kukira obutafaari bwa CD4 + T, nari ngu obutafaari bwa CD4 + T nibubaasa kukoresa enshonga za kaboni ezindi ezitari shukaari kuguma nizikora gye (43, 44). Nikimanyisa ngu titukareeba ntaaniso yoona omu kutwara shukaari nari omu mirimo y‘emibiri ahagati y‘obutafaari bwa CD4 + T, okwijuka kwa T n‘obutafaari bwa T obw‘okwijuka obw‘ahagati omu kusheeshasheesha. Omu muringo nigwo gumwe, embeera y‘okutaanisa obutafaari bwa CD8 + T omu bizimba teine ​​kakwate n‘empindahinduka omu kutwara shukaari, ekirikworeka entaaniso mpango ahagati y‘obutafaari bwa T oburikuhinga omu vitro na TIL y‘omuntu omu vivo (22). Ebi bireebire bikahamibwa n‘okukoresa okugabanisa obutafaari obutarikuhakanisa, ekyayongyeire kworeka ngu obutafaari bwa CD45 + / CD3- / CD4 + / CD45RO + obuine okutwara shukaari nyingi n‘okukora kwa mitochondrial kukira obutafaari bw‘ebizimba nibukira kubaho kwonka bwine obutafaari oburikukora Metabolic. Omubaro ogu nigubaasa kujwekyera omubaro gw‘obutafaari oburikuteekateekwaho kuba obw‘okuzibira obutafaari bw‘omubiri nari obutafaari bwa plasmacytoid dendritic obumanyirwe omu kucondooza kwa scRNA-seq. N‘obu ebi byombi biraabe byagambirweho omu bizimba by‘omunda y‘abantu [45], nibyetenga Ebindi bikorwa n‘okushoboorora ekicweka eki eky‘omubiri eky‘omubiri (myeloid subpopulation).
N‘obu emiringo erikwegamira aha flow cytometry eraabe neebaasa kushoboorora entaaniso z‘omugasho omu nkora ya shukaari n‘okuhindura kw‘omubiri ahagati y‘ebika by‘obutafaari, ebiriisa ebihikire ebirikuruga omu shukaari nari ebindi bihingwa bya kaboni ahabw‘okuhindura kw‘omubiri omu TME tibikamanyirwe. Okuha okubaho nari obutabaho bw‘ebintu ebirikuhindura omubiri aha kicweka kya TIL ekihairwe nikyetengyesa okushemeza omubaro gw‘obutafaari kuruga omu nyama ezicwekirwe. Nahabwekyo, omuringo gwaitu gw‘okugaita obutafaari hamwe n‘okupima obuhango bw‘ebintu (mass spectrometry) nigubaasa kuha okumanya aha bikwatiraine n‘ebintu ebirikuhindura obutafaari ebirikugaita omu miringo etarikushushana omu butafaari bwa T n‘omubaro gw‘obutafaari bw‘ebizimba omu kuhikaana n‘eby‘okureeberaho by‘abarwaire. N‘obu omuringo ogu guraabe gwine ebirungi aha kushoroora obutafaari oburikukoresibwa fluorescence, amabiikiro g‘ebitabo agamwe ag‘ebiriisa nigabaasa kukwatwaho ahabw‘okugumaho kw‘obuhangwa n‘/nari omubaro gw‘okuhinduka kw‘ahonaaho (22). Nangwa n‘obu kiraabe kiri kityo, omuringo gwaitu gukabaasa kumanya ebiriisa bibiri ebirikumanywa ebirikuzibira obutafaari bw‘omubiri, adenosine na kynurenine, ahabw‘okuba nibitaana munonga ahagati y‘ebika by‘eky‘okureeberaho.
Okushwijuma kwaitu okwa metabonomic aha bizimba n‘ebicweka bya TIL nikuha okumanya kwingi aha murimo gw‘ebiriisa omu TME y‘omu nda. Eky‘okubanza, turikukoresa flow cytometry, tukashanga ngu tihaine ntaaniso omu mirimo y‘obutafaari bw‘omubiri ahagati y‘ebizimba n‘obutafaari bwa CD4 + T. Kwonka, okweshwijuma kwa LC-MS/MS kukashohoza empindahinduka z‘amaani omu bwingi bw‘ebiriisa omu bantu aba, ekirikworeka ngu ebirugire omu kucondooza aha nkora ya TIL n‘emirimo yaayo yoona ey‘okuhindura ebiriisa nibyetenga kushoboororwa gye. Ekyakabiri, MNA niyo metabolite erimu entaaniso mpango ahagati ya CD45-cells na T cells omu ascites, beitu bitari bizimba. Nahabwekyo, okutaho ebicweka n‘omwanya gw‘ebizimba nibibaasa kugira ebirugiremu bitarikushushana aha nkora ya TIL, ekirikworeka obutashushana oburikubaasa kubaho omu mbeera nkye erikuheebwa. Ekyakashatu, okureebeka kw‘enzigye erikukora MNA NNMT nikukira kuba ahari CAF, eri obutafaari bw‘ebizimba omu rulengo rukye, kwonka emihendo ya MNA erikureebeka neereebeka omu butafaari bwa T oburikuruga omu bizimba. Okweyongyera kwa NNMT omu CAF y‘omu nkari kwine ekimanyirwe ekirikureetaho ekookoro, ekicweka kimwe nikireetwa okutunguura okuhindura kwa CAF, okutaahirira kw‘ebizimba n‘okujanjaara (27). N‘obu omutindo gwa TIL gwona guraabe guri ahagati, okureebeka kwa NNMT omu CAF nikukwatagana munonga n‘omuringo gw‘okukyebera ogwa Cancer Genome Atlas (TCGA), ogurikukwatagana n‘okuteekateeka kubi (27, 46, 47). Eky’aha muheru, okureebeka kw’enzigye AOX1 erikujunanizibwa n’okusheenya MNA nayo neegarukira aha bantu ba CAF, ekirikworeka ngu obutafaari bwa T tiburikubaasa kuhindura MNA. Ebirugiremu ebi nibishagika ekiteekateeko ngu n‘obu haraabe hariho okukora ebindi ebirikwetengyesa kuhamya ekirugire omu kucondooza oku, emiringo y‘ahaiguru ya MNA omu butafaari bwa T neebaasa kworeka okubaho kw‘embeera y‘omubiri erikurwanisa endwara ya CAF.
Okweshushaniriza n‘omutindo gw‘okumanyisa ogw‘ahansi gw‘abatwazi ba MNA n‘omutindo ogutarikureebwa ogw‘ebirungo ebikuru ebirikwejumba omu kuhindura kwa MNA, okubaho kwa MNA omu butafaari bwa T tikurikuteekateekwaho. Tihariho NNMT nari AOX1 ezaabaire nizibaasa kumanyirwa aha kucondooza kwa scRNA-seq n‘okugyendera aha qPCR y‘ebicweka bibiri eby‘okwetongora. Ebirugiremu ebi nibyoreka ngu MNA terikukorwa obutafaari bwa T, kureka neenywa kuruga omu TME eyehinguririize. Okucondooza okwakozirwe omu vitro nikworeka ngu obutafaari bwa T nibukunda kurundaana MNA ey’aheeru.
Okucondooza kwaitu okwakozirwe omu vitro kworekire ngu exogenous MNA neereetaho okureebeka kwa TNFα omu butafaari bwa T kandi neeyongyera aha kukwatira hamwe kwa Sp1 n’omuhi w’obuhwezi wa TNFα. N‘obu TNFα eraabe eine emirimo y‘okurwanisa ebizimba n‘okurwanisa ebizimba, omu kookoro y‘omu nda, TNFα neebaasa kutunguura okukura kwa kookoro y‘omu nda (31-33). Okukyendeeza aha nkora ya TNFα omu buhangwa bw‘obutafaari bw‘ebizimba by‘omu nkari nari okumaraho akamanyiso ka TNFα omu bishushani by‘embeba nikibaasa kutunguura okukora kwa TNFα-mediated inflammatory cytokine n‘okuzibira okukura kw‘ebizimba (32, 35). Nahabwekyo, omu mbeera egi, MNA erikuruga omu TME neebaasa kukora nk‘ekintu ekirikureetaho okuzimba kurabira omu nkora erikwegamira ahari TNFα kurabira omu nkora ya autocrine loop, obwo erikutunguura okubaho n‘okujanjaara kwa kansa y‘omu nda (31). Okurugirira aha kubaasa oku, okuzibira TNFα nikukyeberwa nk‘ekintu ekirikubaasa kujanjaba ekookoro y‘omu nda (37, 48, 49). Okwongyera ahari ekyo, MNA neeshiisha obutwa bw‘obutafaari bwa CAR-T aha butafaari bw‘ebizimba by‘omu nkari, ekirikuha obuhame obundi obw‘okuzibira obutafaari bw‘omubiri oburikurwanisa endwara oburikureetwaho MNA. Omu kugaita, ebyarugamu ebi nibyoreka eky‘okureeberaho eki ebizimba n‘obutafaari bwa CAF birikusheesha MNA omu TME ey‘aheeru y‘obutafaari. Kurabira omu (i) okukura kwa kansa y‘omu nda okurikureetwaho TNF na (ii) okuzibira emirimo ya T cell cytotoxic erikureetwaho MNA, eki nikibaasa kugira ebizimba bibiri (ekishushani 5D).
Omu kuhendera, barikukoresa omugaati gw‘okutunguura obutafaari omu bwangu, okukuratana kw‘obutafaari bumwe n‘okucondooza aha nkora y‘okuhindura eby‘okurya, okucondooza oku kukashohoza entaaniso mpango z‘obutafaari bw‘omubiri ahagati y‘ebizimba n‘obutafaari bw‘omubiri oburikwetwa ascites omu barwaire ba HGSC. Okweshwijuma oku okuhikire kukooreka ngu hariho entaaniso omu kutwara shukaari n‘emirimo ya mitochondrial ahagati y‘obutafaari bwa T, kandi kukamanyisa MNA nk‘ekintu ekitari ky‘obutafaari ekirikutegyeka obutafaari bw‘omubiri. Ebihandiiko ebi biine akakwate n‘oku TME erikukora aha nkora y‘obutafaari bwa T omu ndwara za kansa z‘abantu. N‘obu okurwanagana okuhikire kw‘ebiriisa ahagati y‘obutafaari bwa T n‘obutafaari bwa kansa kwagambirweho, ebiriisa ebiri omu mubiri nibibaasa n‘okukora nk‘ebirikutegyeka ebitari butoosha kutunguura okukura kw‘ebizimba kandi nikibaasa kuzibira okugarukamu kw‘obutafaari bw‘omubiri oburikurwanisa endwara. Okushoboorora okurikukiraho aha murimo gw‘ebintu ebi ebirikutegyeka nikubaasa kwiguraho obukoryo obundi bw‘okutunguura okugarukamu kw‘obutafaari bw‘omubiri oburikurwanisa ebizimba.
Ebishushani by‘abarwaire n‘ebihandiiko by‘obujanjabi bikatunga kurabira omu kitongore ky‘okubiika enyama z‘ebizimba bya kansa eya BC ekyahamibwe ekitongore ky‘okubiika enyama ekya Canada. Okurugirira aha nkora eyaikiriziibwe akakiiko k‘emitwarize y‘okucondooza aha ndwara ya kansa aka BC na Yunivasite ya British Columbia (H07-00463), ebishushani by‘abarwaire boona n‘ebihandiiko by‘obujanjabi bikatunga okwikiriza okuhandiikirwe okumanyisiibwe nari bakarekura okwikiriza kwabo omu butongore. Sampuli ezi nizibiikwa omu BioBank erikuhamibwa (BRC-00290). Emitwarize y‘omurwaire omu bwijwire neeyorekwa omu bipande S1 na S5. Ahabw’okurinda omu bwire bw’obutiti, nibakoresa ekikwato ky’okushara (scalpel) kusheenya ekizimba ky’omurwaire omu muringo gw’ebyoma reero bakakihanika kurabira omu kicupuri kya maikoro 100 kubaasa kutunga obutafaari bumwe oburikuhamira. Ascites y‘omurwaire ekateebwa omu centrifuge aha 1500 rpm kumara edakiika 10 ahari 4°C kubaasa kusheenya obutafaari n‘okuihaho ebiri aha mutwe. Obutafaari oburugire omu bizimba n’omu nshaho z’omubiri bukabiikwa omu 50% y’eshagama y’omuntu etaine bwire bw’okwotsya (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) na 10% eya dimethyl sulfoxide. Ebi bihingwa ebibiikire eby’obutafaari bumwe bikafukiirwa kandi byakoresibwa omu kucondooza aha metabolomics n’okumanyisa metabolite nk’oku kirikushoboororwa ahaifo.
Omuringo oguhikire gurimu 0.22 μm ezishwijumirwe 50:50 eziyongyeirweho RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (eky‘okureeberaho ky‘okwotsya) eyongyeirwemu 10% y‘eshagama y‘omuntu AB etarikukora ahabw‘okwotsya (Sigma-Aldrich), 12.5 mM Hepes (eky‘okureeberaho ky‘okwotsya), 2 mM l-glutamine (eky‘okureeberaho ky‘okwotsya) S1. Omubazi gw’okukyebera obutwa (PenStrep) n’omubazi gwa 50 μMB-mercaptoethanol. AimV (Invitrogen) neeyongyerwamu Hepes 20 mM (eky’okureeberaho ky’eby’okurya) na 2 mM l-glutamine (eky’okureeberaho ky’eby’okurya). Ekicweka ky‘okukyebera ekirikukyebera okusheeshasheesha kw‘omubiri kikaba kirimu 0.22μm y‘omubazi gw‘okusheeshasheesha (PBS; Invitrogen) ogwongyeirwemu 3% y‘eshagama y‘omuntu eya AB etarikukora ahabw‘okwotsya (Sigma). Ekirungo ky‘okugaita obutafaari kirimu PBS eshenenekire 0.22μm kandi erimu 0.5% y‘eshagama y‘omuntu eya AB etarikukora ahabw‘okwotsya (Sigma-Aldrich).
Omu 37°C omu mwanya oguhikire, obutafaari bukateebwaho erangi 10 nM MT DR na 100 μM 2-NBDG kumara edakiika 30. Ekindi, obutafaari bukateebwaho erangi y’okugumaho eF506 ahari 4°C kumara edakiika 15. Garuka oshukye obutafaari omu FC Block (eBioscience) na Brilliant Stain Buffer (BD Biosciences), obite omu buffer y’okukyebera obutafaari bw’omubiri (okurugiirira aha biragiro by’omukozi), kandi obite omu nkungu kumara edakiika 10 aha butagasi bw’omu kishengye. Obutafaari bute erangi n‘ekicweka ky‘ebintu ebirikurwanisa endwara (Ekipande S2) omu nkora y‘okusiiga erangi ya flow cytometry aha 4°C kumara edakiika 20. Garuka ote obutafaari omu nkora y‘okukyebera obutafaari bw‘omubiri (Cytek Aurora; 3L-16V-14B-8R) otakakyebeire. Kozesa SpectroFlo na FlowJo V10 kucondooza aha makuru g’okubara obutafaari, kandi okozese GraphPad Prism 8 kuhanga amakuru. Omubaro gw‘okukyebera (MFI) gwa 2-NBDG na MT DR gukaba guri omu nkora ya log-normalized, reero bwanyima ekigyezo kya paired t kikakoresibwa omu kucondooza kw‘embariirira kushoboorora abarwaire abarikushushana. Ihaho abantu boona abaine ebintu ebitarikuhika 40 kuruga omu kucondooza; taasyamu omuhendo gwa MFI ogwa 1 ahabw‘omuhendo gwona ogutari murungi otakakozire okucondooza kw‘embariirira n‘okureeba amakuru.
Okubaasa kwongyera aha nkora y‘okutaho omuryango gw‘omu ngaro ey‘ekipande ky‘enkora ekiri aharuguru, tukakoresa okushoboorora okuhikire okurikukorwa omuti gw‘okuzibira ekishushani (FAUST) (21) kuha obutafaari omu bantu bwanyima y‘okumaraho obutafaari obufiire omu FlowJo. Nitutegyeka ebyarugamu omu ngaro kugaita abantu abarikureebeka nk‘abatarikugabwa kubi (okugaita PD1+ n‘obutafaari bwa PD1-tumor) n‘abantu abagumireho. Buri sampo erimu obutafaari oburikurenga ahari 2% omu mwanya, ahabw’omubaro gw’abantu 11.
Okukyebera okurikukoresibwa omu kutaanisa PBMC n’ebintu ebirikutaanisa obutafaari bw’eshagama (STEMCELL Technologies). Obutafaari bwa CD8 + T bukataanisibwa kuruga omu PBMC barikukoresa CD8 MicroBeads (Miltenyi) kandi bwagaziibwa omu nkora ehikire barikukoresa TransAct (Miltenyi) kumara wiiki 2 kurugiirira aha biragiro by‘omukozi. Obutafaari bukarekyererwa kwemerera kumara ebiro 5 omu mwanya oguhikire ogurimu IL-7 (10 ng/ml; PeproTech), reero bwagaruka bwagarurwamu amaani na TransAct. Aha kiro kya 7, kurugiirira aha biragiro by’omukozi, CD45 MicroBeads z’abantu (Miltenyi) zikakoresibwa kugaita obutafaari omu nshonda ishatu ezirikukuratana. Obutafaari bukateebwamu obutafaari bw‘okukyebera flow cytometry (nk‘oku kyashoboororwa ahaiguru), kandi obutafaari miriyooni emwe bukateebwaho obutafaari emirundi eshatu kukyeberwa LC-MS/MS. Sampuli zikaba zikozirwe omu nkora ya LC-MS/MS nk‘oku zirikushoboororwa ahaifo. Tukatebereza omuhendo gw’ebiriisa ebitariho n’omubaro gwa ion 1,000. Buri sampo neehindurwa omu buteeka n‘omubaro gw‘ayoni gwona (TIC), ehindurwa omu logarithm kandi ehindurwa omu buteeka omu MetaboAnalystR etakashwijumirwe.
Ekicweka ky‘obutafaari bumwe ekya buri murwaire kikasheesheka kandi kyasendwa kurabira omu kishwijuma kya 40 μm omu nkora ehikire (nk‘oku kyashoboororwa ahaiguru). Okurugirira aha nkora y‘omukozi, emirundi eshatu erikukuratana ey‘okutoorana gye kurabira omu kutaanisa obubare bwa magnetic barikukoresa MicroBeads (Miltenyi) ekakoresibwa kugaita eshampu z‘obutafaari bwa CD8+, CD4+ na CD45- (aha ice). Omu bugufu, obutafaari nibugaruka buteebwa omu buffer y’okugaita obutafaari (nk’oku kyashoboororwa ahaiguru) kandi bubarwa. Obutafaari bukateebwa omu nkora n’oburungo bwa CD8 bw’omuntu, oburungo bwa CD4 bw’omuntu nari oburungo bwa CD45 bw’omuntu (Miltenyi) aha 4°C kumara edakiika 15, reero bwayozibwa n’ekintu ekirikugaita obutafaari. Eky‘okureeberaho nikihingura omu nkora ya LS (Miltenyi), kandi obucweka oburungi n‘obubi niburundaanwa. Okubaasa kukyendeeza obwire n‘okutunguura eitembezo ry‘okugaruka omu butafaari, CD8-ekicweka nikiheza kikoresibwa omu rweteerane rwa kabiri rw‘okugaita CD4+, kandi CD4-ekicweka nikikoresibwa omu kugaita CD45 okurikukuratiraho. Reka omubazi aha barafu omu bwire bwona obu orikutaana.
Okutebeekanisa sampo z’okushwijuma metabolite, obutafaari bukanaabibwa omurundi gumwe n’omubazi gw’omunyugunyu ogurikwotsya nk’orubaare, kandi miriyoni 1 ya methanol 80% ekateebwa ahari buri sampo, bwanyima yaahindurwa kandi yaafumbirwa omu maizi ga nitrogen. Sampuli zikateebwa omu nkora eshatu z’okufuka-okufuka kandi zaateebwa omu nkora ya centrifuge aha 14,000 rpm kumara edakiika 15 ahari 4°C. Ekirungo ekirimu ebiriisa ebiri omu mubiri nikifuka kuhitsya obu kirikwoma. Ebirungo by’omubiri bikagaruka byashohozibwa omu 50 μl ya 0.03% formic acid, byateebwa omu vortex kubaasa kujwanzya, reero byateebwa omu centrifuge kwihaho ebisigarira.
Okwihaho ebiriisa nk’oku byashoboororwa ahaiguru. Hindurira ebiri aha mutwe gw’amaizi omu cupa y’amaizi g’okukyebera ebirikukora gye munonga ahabw’okucondooza aha metabolomics. Kozesa enkora y‘okujanjaba etari emwe n‘emwe kujanjaba buri sampo n‘omubaro gw‘obutafaari oburikushushana okuzibira ebirugire omu kujanjaba kw‘ekicweka. Twakora okucondooza kw‘omutindo gw‘ebintu ebirikuhindura omubiri omu nsi yoona ebyabaire bishohoziibwe enyima aha AB SCIEX QTRAP 5500 ebiri omu nkora y‘okupima obuhango bw‘ebintu (50). Okushwijuma kw’eby’obuhangwa n’okuteerana kw’ebicweka by’ahaiguru bikakorwa barikukoresa puroguraamu ya MultiQuant ey’omuringo 2.1 (Applied Biosystems SCIEX).
Okubara kwa ion kwa 1000 kukakoresibwa kuteekateeka aha muheendo gw‘ebiriisa ebibuzire, kandi TIC ya buri sampo ekakoresibwa kubara omwanya gw‘enshonga ogwa buriijo ogwa buri sampo eyashangirwe kushoboorora empindahinduka ezaareetsirweho okucondooza kw‘ebikwato kuruga omu kucondooza kwa sampo. Bwanyima ya TIC kuhindurwa omu buteeka, MetaboAnalystR(51) (paramita y‘okubanza) neekoresibwa omu kuhindura kwa logarithmic n‘okupima omurongooti gw‘obutoosha ogw‘okwemanyiza. Twakoresa PCA n‘ekipapura kya vegan R kukora okucondooza kw‘okucondooza aha ntaaniso z‘eby‘okurya ahagati y‘ebika by‘eky‘okureeberaho, kandi twakoresa okucondooza kw‘ekicweka ky‘okugarukamu kucondooza abarwaire. Koresa omuringo gwa Ward kwombeka maapu y‘okwotsya dendrogram okuteerana orugyendo rwa Euclidean ahagati y‘eby‘okureeberaho. Twakoresa limma (52) aha bwingi bw‘ebiriisa ebitebeekanisiibwe kubaasa kumanya ebiriisa ebiri omu nkora ebirikutaana omu muringo gwona gw‘obutafaari n‘embeera nkye. Kubaasa kworobya okushoboorora, nitukoresa omubaro gw‘obumanyiso bwa guruupu kushoboorora eky‘okureeberaho, kandi nituteekateeka aha miringo y‘obutafaari omu nkora y‘obuhangwa obukye nka buri guruupu (n = guruupu 6); aha kugyezibwa kw‘obukuru, tukakora ebipimo bishatu eby‘okugarukamu ahari buri metabolite Kubaasa kwetantara okugarukamu kw‘ebishuba, omurwaire akateebwamu nk‘ekizibu omu ntebeekanisa ya limma. Okubaasa kureeba entaaniso omu bihingwa ebiri omu mubiri ahagati y‘abarwaire batari bamwe na bamwe, tukahindura omu nkora ya limma erimu abarwaire omu muringo oguhamire. Nituha ripooti aha bukuru bw‘okutaana okwabandize kushoboororwa ahagati y‘omuringo gw‘obutafaari n‘embeera nkye ya Padj <0.05 (okutereeza kwa Benjamini-Hochberg).
Bwanyima y‘okugaita amaani barikukoresa ekikwato ky‘okuihaho obutafaari obufiire ekya Miltenyi (>80% viability), okukuratana kw‘obutafaari bumwe kukakorwa aha sampo z‘ebizimba n‘ebizimba byona ebiine amagara barikukoresa enkora y‘okumanyisa 10x 5′gene. Emirundi etaano erimu ebizimba ebirikushushana n‘ebizimba by‘omubiri bikakyeberwa, n‘obu okubaasa kugumaho okukye kuruga omu sampo emwe y‘ebizimba kwabaire kuremesa okugiteekamu. Okubaasa kutunga okutoorana abarwaire baingi, tukagaita eshampu za buri murwaire omu mihanda y‘ekikwato ky‘okukyebera 10x chromium, kandi twashwijuma emyanya y‘okusheeshasheesha n‘ebizimba omu muringo gw‘okwetaanisa. Bwanyima y’okukuratana [Ekicweka ky’omubiri ekya Quebec; omubaro gwa 73,488 na 41,378 okushoma buri butafaari ahabw‘ekizimba n‘okusheeshasheesha nk‘oku biri]], tukakoresa CellSNP na Vireo (53) (kurugiirira ahari CellSNP nk‘Omuntu owa buriijo SNP (VCF) erikuheebwa GRCh38 neeheebwa okumanya kw‘omuhi w‘obuhwezi. genotype y‘okumanya omurwaire the close's ku) (IBS), otabariiremu obutafaari obutaheebwayo n‘obutafaari obumanyirwe nk‘obw‘ebicweka bibiri (duplexes) n‘abahi b‘obuhwezi abarikuhikaana ahagati y‘eby‘okureeberaho by‘ebizimba n‘ebizimba (54 Aha musingi gw‘omurimo ogu, tukaguma n‘enshonga ishatu eziine obutafaari bwingi oburikujwekyera omu kizimba n‘ebizimba ahabw‘okushwijuma okuri ahansi y‘omugyera (BiCoran filtering step na)6. n‘obutafaari 6975 (obutafaari 2792 na 4183 kuruga omu bizimba n‘omu nshaho, nk‘oku biri) ahabw‘okushwijuma Nitukoresa igraph‘s (57) Louvain clustering y‘okugaaniira kw‘obutafaari bw‘omutaahi ori haihi (SNN) kurugiirira aha rugendo rwa Jaccard kuhika aha butafaari bw‘okuteerana kurabira omu kushoboorora kw‘omu ngaro. kandi ebirikureebeka n‘obutafaari bwa t-SNE nibushoboororwa ahabw‘okureebeka kwa CD8A na GZMA, otairemu ebicweka ebikye ebiine okureebeka kwa puroteyini ya ribosomal okukye Tukatunga amakuru agahandiikirwe ga Izar et al (16), obariiremu okutaho t-SNE yaabo, nibibaasa kutegyeka okureebeka kwa marNNMTs.
PBMC zikaba zitaanisiibwe n’ebintu ebirikutaanisa obutafaari bw’eshagama (STEMCELL Technologies) kurabira omu kucondooza kwa Ficoll okurikukoresibwa okutaana kw’obutafaari bw’eshagama. Obutafaari bwa CD3 + bukataanisibwa kuruga omu PBMC barikukoresa obubare bwa CD3 (Miltenyi). Omu kubaho nari obutabaho bwa MNA, obutafaari bwa CD3+ bukakora na CD3 ekwatsirwe aha mpapura (5 μg/ml), CD28 erikubaasa kusheesheka (3 μg/ml) na IL-2 (300 U/ml; Proleukin). Aha kiro ky’aha muheru ky’okujanjaaza, okubaasa kw’okujanjaara (Erangi y’okuhangaara erikubaasa kugumaho eFluor450, eBioscience) n’okukanyira (eBeads 123count, Thermo Fisher Scientific) bikakyeberwa omu muringo gw’okukyebera okusheeshasheesha kw’eshagama. Okushwijuma emirimo y‘ekikwato orikureetaho okusiimura obutafaari na PMA (20 ng/ml) na ionomycin (1μg/ml) na GolgiStop kumara eshaaha 4, kandi oreebere CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4) , BioLegend) na TNFore-PerCP (Mab11, BD). Okusiimura obutafaari bwa qPCR na ChIP na PMA (20 ng/ml) na ionomycin (1μg/ml) kumara eshaaha 4. Ekicweka kya ELISA ekyabaire kiri ahaiguru kikatorotoorwaho kitakatandikire n‘enyima y‘okusiimura na PMA (20 ng/ml) na ionomycin (1 μg/ml) kumara eshaaha 4.
Kuratirira ebiragiro by’abakozi kubaasa kutaanisa RNA orikukoresa ekikwato kya RNeasy Plus Mini Kit (QIAGEN). Kozesa ekikwato ky‘okusheenya (QIAGEN) kuhindura sampo kuba emwe. Kozesa ekikwato kya RNA kuhika ahari cDNA eky’obushoboorozi bw’ahaiguru (Thermo Fisher Scientific) kubaasa kuteerana DNA erikuhwera (cDNA). Kozesa TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) okupima obuhango bw‘okureebeka kw‘obuzaarwa (kurugiirira aha nkora y‘omukozi) n‘ebikwato ebi: Hs02786624_g1 [omubazi gw’okusheeshasheesha-3-fosifaati kuruga omu maizi (GAPDH)] na Hs01010726_m1 (SLC22A2). Sampuli zikaba zigyendesibwa aha nkora ya StepOnePlus ey‘obwire bwona eya PCR (Eby‘okureeberaho by‘Ebihangirwe) (Eby‘okureeberaho by‘Eby‘amagara) omu nkora ya MicroAmp ey‘okurahuka ey‘okureeba 96-well reaction plate (Eby‘okureeberaho by‘Eby‘amagara) n‘ekishushani ky‘okureeba ekya MicroAmp. Omuhendo gwona ogwa Ct ogurikurenga 35 nigutwarwa nk‘oguri ahaiguru y‘omutindo gw‘okukyebera kandi niguteebwaho akamanyiso nk‘ogutarikukyeberwa.
Kora ChIP nk‘oku kyashoboororwa enyima (58). Omubugufu, obutafaari bukajanjabwa na formaldehyde (omubaro gw’aha muheru 1.42%) kandi bwateebwa omu bwire bw’obutagatsi bw’omu kishengye kumara edakiika 10. Koresa ekirungo ky‘okuzimba ekiyongyeirweho (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl na 0.1% NP-40) aha barafu kumara edakiika 10, reero ogarukye ogite omu kirungo ky‘okuzimba nk‘oku kyashoboororwa (58 ). Sampuli bwanyima ekakyeberwa n‘enzindo ezi: enzindo 10 (20 1-second pulses) n‘obwire obutaine mpinduka bwa sekondi 40. Okukyebera obutafaari bw’omubiri oburikurwanisa endwara G (Tekinologiya y’okumanyisa obutafaari; 1μl), histone H3 (Tekinologiya y’okumanyisa obutafaari; 3μl), NFAT (Invitrogen; 3μl) na SP1 (Tekinologiya y’okumanyisa obutafaari; 3μl) n’eky’okureeberaho aha sha°CCn. Oteere oburungo bwa puroteyini A (Thermo Fisher Scientific) n’eky’okureeberaho aha 4°C orikugizunguza mpora mpora kumara eshaaha 1, reero okozese oburungo bwa chelex (Bio-Rad) kugaita DNA, kandi okozese puroteyini K (Thermo Fisher) ahabw’okugabura puroteyini. Omuhi w‘obuhwezi wa TNFα akashangwa na PCR: omu maisho, GGG TAT CCT TGA TGC TTG TGT; okutaana n‘ekyo, GTG CCA ACA EKITEISO GCC TTT ATA TG (ekintu ekiine 207-bp). Ebishushani ebi bikakorwa Image Lab (Bio-Rad) kandi byakyeberwa barikukoresa puroguraamu ya ImageJ.
Ekicweka ky’obuhangwa bw’obutafaari kikatorotoorwaho nk’oku kyashoboororwa ahaiguru. Okucondooza kukakorwa kurugiirira aha nkora z‘abakozi b‘ekikwato ky‘okukyebera TNFα y‘omuntu (Invitrogen), ekikwato ky‘okukyebera IL-2 ky‘omuntu (Invitrogen) n‘ekikwato ky‘okukyebera IFN-γ y‘omuntu (Abcam). Okurugirira aha nkora y’abakozire, amaizi g’ahaiguru gakakyeberwa 1:100 kubaasa kureeba TNFα na IL-2, kandi 1:3 kubaasa kumanya IFN-γ. Koresa ekikwato ky‘okushoma ebiri omu nkora (PerkinElmer) kupima okunywanisa aha 450 nm.
PBMC zikaba zitaanisiibwe n’ebintu ebirikutaanisa obutafaari bw’eshagama (STEMCELL Technologies) kurabira omu kucondooza kwa Ficoll okurikukoresibwa okutaana kw’obutafaari bw’eshagama. Obutafaari bwa CD3 + bukataanisibwa kuruga omu PBMC barikukoresa obubare bwa CD3 (Miltenyi). Omu kubaho nari obutabaho bwa MNA, obutafaari bwa CD3+ bukakora na CD3 ekwatsirwe aha mpapura (5 μg/ml), CD28 erikushohora (3 μg/ml) na IL-2 (300 U/ml; Proleukin) kumara ebiro 3. Bwanyima y’ebiro 3, obutafaari bukatorotoorwamu kandi bwanaabibwa n’omubazi gw’omunyugunyu 0.9%, kandi pellet ekakyeberwa (snap frozen). Okubara obutafaari kukakorwa omu muringo gw’okupima okutambura kw’obutafaari (Cytek Aurora; 3L-16V-14B-8R) barikukoresa eBeads z’okubara 123.
Okwihaho ebiriisa nk’oku byashoboororwa ahaiguru. Ekirungo ekyo ekyomire kikagaruka kyateebwamu aha nkora y’obutafaari 4000 oburikwingana/μl. Okushwijuma eky‘okureeberaho orikukoresa enkora y‘okugaruka enyima (1290 Infinity II, Agilent Technologies, Santa Clara, CA) n‘enyomyo ya CORTECS T3 (2.1×150 mm, obuhango bw‘ebicweka 1.6-μm, obuhango bw‘ebiina 120-Å; #186008500, #186008500). Ekipimo ky’okupima obuhango bw’enyonyoozi (6470, Agilent), omu kukyebera kw’amashanyarazi kurikukora omu muringo murungi. Omuringo gw’okugyenderamu A ni 0.1% asidi ya formiki (omu H2O), omuringo gw’okugyenderamu B ni 90% asetonitrile, 0.1% asidi ya formiki. Omubaro gwa LC ni 0 kuhika ahari 2 edakiika ahari 100% A, edakiika 2 kuhika ahari 7.1 ahari 99% B, n‘edakiika 7.1 kuhika ahari 8 ahari 99% B. Reero ogarukye ogyeragyeranise enyomyo n‘omuringo gw‘okugyenderamu A aha nkora y‘okusheesheka ya 0.6 ml/min kumara edakiika 3. . Omubaro gw’okusheeshasheesha ni 0.4ml/min, kandi ekishengye ky’enyomyo nikiheebwa omuriro kuhika ahari 50°C. Koresa omutindo gw‘eby‘obuhangwa ogwa MNA ogushemeire (M320995, kampuni y‘okucondooza ey‘eby‘obuhangwa eya Toronto, North York, Ontario, Canada) kutaho obwire bw‘okurinda (RT) n‘okuhindura (RT = edakiika 0.882, okuhindura 1 = 137→94.1, okuhindura 2 = 137, okuhindura→372). Enhinduka zoona ishatu ku zirikubaho omu bwire bw‘okurinda obuhikire, enhinduka 1 neekoresibwa omu kupima omubaro kureeba ngu hariho obumanyiso. Omutindo gw‘okugyeragyeranisa ogwa MNA (Kampuni y‘okucondooza ey‘eby‘emibazi eya Toronto) gukakorwaho okukyendeeza aha nkora mukaaga z‘omubaro gw‘ebintu (1 mg/ml) kutunga omutindo gwa 0.1, 1.0, 10 na 100 ng/ml na 1.0 na 10μg/ml nk‘amaizi. Ekipimo ky‘okukyebera ni 1 ng/ml, kandi okugarukamu kw‘omuringo gumwe kuri ahagati ya 10 ng/ml na 10μg/ml. Buri nshaho ya maikorolita ibiri z‘eky‘okureeberaho n‘omutindo neekoresibwa omu kucondooza kwa LC/MS, kandi sampo y‘okurinda omutindo etaburiine neegyendesibwa buri nshaho munaana kureeba ngu enkora y‘okushwijuma yaagumaho. Ebigarukwamu bya MNA eby‘eby‘okureeberaho by‘obutafaari byona ebyabaire bijanjabirwe MNA bikaba biri omu nkora y‘okukyebera. Okushwijuma ebihandiiko kukakorwa barikukoresa puroguraamu ya MassHunter ey‘okushwijuma omubaro (v9.0, Agilent).
Omurembe gwa kabiri ogw‘okwombeka αFR-CAR gukatwarwa kuruga omu Song et al. (59). Omu bugufu, ekyombeko eki kirimu ebi: CD8a omwebembezi orikukuratana, ekicweka ky‘omuntu αFR-specific ekirikuhinduka eky‘omurunga gumwe, CD8a hinge na transmembrane region, CD27 omunda y‘obutafaari n‘omunda ya CD3z omunda y‘obutafaari. Omukuratana gwa CAR gwona gukakorwa GenScript, reero bwanyima gwakorwa omu nkora y’okumanyisa lentiviral ey’omurembe gwa kabiri aha mutwe gwa kaseti y’okumanyisa GFP eyakozesiibwe kukyebera oburungi bw’okuhindura.
Lentivirus neekorwaho okuhindura obutafaari bwa HEK293T [Ekitongore ky’obuhangwa bw’omuringo gw’America (ATCC); okukura omu nkora ya Dulbecco ehindwirwemu eya Eagle erimu 10% y’eshagama y’ente y’omunda (FBS) na 1% ya PenStrep, kandi ekakoresa CAR-GFP vector na Plasmids z’okupakira (psPAX2 na pMD2.G, Addgene) nizikoresa omubazi gw’okusheeshasheesha (Sigma-Aldrich). Ekirungo ekirimu akakoko aka kikatorotoorwamu bwanyima y’eshaaha 48 na 72 bwanyima y’okukihindura, kyasendwa, kandi kyateebwa omu nkora ya ultracentrifugation. Biika ebiriisa bya virus ebirimu ebiriisa bingi ahari -80°C kuhitsya obu birahindurwe.
PBMC nizitaanisibwa n‘ebintu by‘okwahukana kw‘eshagama y‘abahi b‘obuhwezi abaine amagara marungi (STEMCELL Technologies) kurabira omu kucondooza kwa Ficoll. Koresa obutafaari bwa CD8 obukye oburikutoorana gye (Miltenyi) kutaanisa obutafaari bwa CD8+ kuruga omu PBMC. Okusiimura obutafaari bwa T na TransAct (Miltenyi) kandi omu nkora ya TexMACS [Miltenyi; eyongyeirwemu 3% y‘eshagama y‘omuntu etarikukora ahabw‘okwotsya, 1% PenStrep na IL-2 (300 U/ml)]. Eshaaha makumi abiri na ina bwanyima y‘okusiimura, obutafaari bwa T bukahindurwa na lentivirus (10 μl y‘obukooko oburikwegamira aha butafaari 106). Ebiro 1 kuhika ahari 3 bwanyima y’okuhindura aha Cytek Aurora (aha FSC (Okujanjaara kw’omumaisho)/SSC (Okujanjaara kw’aha rubaju), Singlet, GFP+), shwijuma oku GFP erikworeka obutafaari kworeka obushoboorozi bw’okuhindura haakiri 30% .
Obutafaari bwa CAR-T bukakuzibwa kumara eshaaha 24 omu Immunocult (STEMCELL Technologies; obuyongyeirwemu 1% PenStrep) omu mbeera ezi: butajanjabirwe, bujanjabirwe na 250 μM adenosine nari 10 mM MNA. Bwanyima y‘okubanza kujanjaba, obutafaari bwa CAR-T bukanaabibwa na PBS kandi bwagambwaho n‘obutafaari 20,000 bwa SK-OV-3 [ATCC; omu nkora ya McCoy 5A (Sigma-Aldrich) eyongyeirwemu 10% FBS na 1% PenStrep ahari 10: Omubaro gw‘okukora n‘ekigyendererwa kya 1 gukagarurwamu amaani omu mirundi eshatu omu nkora ya Immunocult eyongyeirwemu. Obutafaari bwa SK-OV-3 n‘obutafaari bwa SK-OV-3 obucwekirwe n‘obutafaari bwa digitalis (0.5mg/ml; Sigma-Aldrich) bukakoresibwa nk‘okukyebera okubi n‘okukyebera okurungi, nk‘oku biri. Bwanyima y’okuhinga hamwe kumara eshaaha 24, amaizi g’omu itaka gakatorotoorwaho kandi amaizi g’omubiri gakapimwa kurugiirira aha biragiro by’omukozi (LDH Glo Cytotoxicity Assay Kit, Promega). Ekicweka kya LDH ekyabaire kiri ahaiguru kikaba kikyebeirwe 1:50 omu buffer ya LDH. Ebicweka by‘okwitwa bikapimwa barikukoresa enkora egi: ebicweka by‘okwitwa = ebicweka by‘okutereeza / omubaro gw‘okwitwa ogurikukirayo x 100%, ahu ebicweka by‘okutereeza = obutafaari bwa co-culture-T bwonka, n‘omubaro gw‘okwitwa ogurikukirayo = okutegyeka okurungi-okutegyeka kubi.
Nkoku kyashoboororwa omu bihandiiko nari ebikwato n‘emiringo, kozesa GraphPad Prism 8, Microsoft Excel nari R v3.6.0 ahabw‘okushwijuma eby‘embariirira. Sampuli nyingi zaaba nizitorotoorwaho kuruga omu murwaire omwe (nk‘okusheeshasheesha n‘ekizimba), nitukoresa ekigyezo kya paired t nari nitutaho omurwaire nk‘ekirikurugamu omu muringo gw‘okutooranamu omu nkora y‘omuringo gumwe nari ey‘omugasho nk‘oku kirikubaasika. Aha kweshwijuma kwa metabolomics, okukyebera obukuru nikukorwa omu miringo eshatu.
Okumanya ebihandiiko by’okwongeraho aha kihandiiko eki, reeba aha http://advances.sciencemag.org/cgi/ebirimu/omu bwijwire/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). (Russell G. Jones), Fineas T.
MNA neehwera omu kuzibira obutafaari bw‘omubiri oburikurwanisa endwara bwa T kandi neejwekyera ekigyendererwa ky‘obutafaari bw‘omubiri oburikubaasa kujanjaba endwara ya kansa y‘omuntu.
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). (Russell G. Jones), Fineas T.
MNA neehwera omu kuzibira obutafaari bw‘omubiri oburikurwanisa endwara bwa T kandi neejwekyera ekigyendererwa ky‘obutafaari bw‘omubiri oburikubaasa kujanjaba endwara ya kansa y‘omuntu.
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