T cells

Abstract

Tissue-resident memory T cells (T) are useful in therapies such as immunotherapy for cancer treatment. RM ) provides a method for producing. [Solution] A tissue-resident memory T cell (T) culture method comprising the steps of culturing lymphocytes in the presence of transforming growth factor beta (TGFβ) and / or co-culturing lymphocytes with regulatory T cells. RM This invention relates to a method for producing tissue-resident memory T cells (T) obtained by the method of the present invention. RM ) themselves, these T RM Compositions containing cells, and these T in therapies such as immunotherapy for the treatment of cancer RM The use of cells and compositions is also provided.

Description

[Technical Field]

[0001] The present invention relates to T cells, and more particularly to tissue-resident memory T cells (T RM A method for producing ) and tissue-resident memory T cells (T) obtained by the method of the present invention. RM ) themselves, these T RM Compositions containing cells, and these T in therapies such as immunotherapy for treating cancer RM Regarding the use of cells and compositions. [Background technology]

[0002] Immunotherapy using immune checkpoint inhibitors, such as those blocking antibodies against PD-1 or CTLA-4, has significantly improved cancer-free survival rates. Importantly, adoptive transfer of chimeric antigen receptor (CAR) T cells[1], common tumor detection delta-1 γδ T cells (DOT)[2,3], or MR-1-restricted T cells[4] has achieved very promising results. However, sustained responses are limited to some patients, and questions remain as to whether these approaches are sufficiently effective against solid tumors such as breast cancer. In particular, the success of T cell immunotherapy in solid tumors depends on delivering and activating tumor-specific lymphocytes with cytotoxic activity, such as CD8+ T cells, into the tumor tissue.

[0003] A breakthrough in tissue immunity allows for the deep penetration of tissue-resident memory CD8+T(T) RM The presence of T cells has been revealed [5, 6]. In recent years, T cells in tumors have been identified by the expression of the marker CD103. RM A strong correlation has been shown between the presence of cells and the patient's positive prognosis. RM In solid tumors such as breast cancer, lung cancer, ovarian cancer, and cervical cancer, T cells are more strongly correlated with extended overall survival and disease-free periods than the total CD8+ T cell count [7-15]. In fact, T RM The cells are directly linked to the enhanced cytotoxic T cell response in human solid tumors [16, 17]. [Overview of the project]

Means for Solving the Problem

[0004] Therefore, it is very important to explore an improved mechanism of immunotherapy by focusing on generating and delivering cytotoxic CD8+ T cells deep into tissues, which is an essential step change for the success of cell-based cancer immunotherapy against solid tumors.

[0005] The present inventors hypothesized that regulatory T (T REG ) cells can penetrate deeply into tissues and are important in the generation of T cells that are very effective against solid tumors. For the purpose of generating antitumor T cells with tissue penetration characteristics, the present inventors were able to elucidate and identify the factors necessary for the generation of T RM cells in vitro to enable the generation of T RM cells.

[0006] Therefore, in a first aspect of the present invention, there is provided a method for producing tissue-resident memory T cells (T RM ), which includes culturing lymphocytes in the presence of transforming growth factor beta (TGFβ) and / or co-culturing lymphocytes with regulatory T cells.

[0007] Advantageously, as described in the examples, the present inventors established the in vitro requirements necessary for the growth of tissue-penetrating T cells, that is, tissue-resident memory T cells (T RM ), and developed a protocol for generating T cells for use in cell therapy. The production of such cells results in the production of T RM cells with cytotoxic activity in diseased tissues such as tumor tissues and metastatic tumors, enabling the delivery and activation of disease-specific lymphocytes, thereby greatly expanding the T cell-based therapeutic toolkit. Furthermore, as described in the examples, the present inventors developed a new protocol for generating T REG cells for use in cell therapy without including T RM cells in the culture.

[0008] Preferably, this method is performed ex vivo or in vitro. Preferably, the method includes culturing lymphocytes in the presence of TGFβ. Preferably, in some embodiments, the method does not include culturing lymphocytes in the presence of regulatory T cells.

[0009] Preferably, TGFβ is physiologically active (i.e., activated). Preferably, TGFβ is mammalian. TGFβ may be rodent, dog, horse, or pig TGFβ. Rodents may be rats or mice. Most preferably, TGFβ is human TGFβ.

[0010] In one embodiment, TGFβ may be TGFβ1 represented by Genebank ID No: 7040, which is provided herein as Sequence ID No. 1: MPPSGLRLLPLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEW LSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS [Sequence ID 1] Therefore, preferably, TGFβ comprises or consists of the sequence substantially shown in SEQ ID NO: 1, or a fragment or variant thereof.

[0011] In one embodiment, TGFβ may be TGFβ2, represented by Genebank ID No: 7042, which is provided herein as Sequence ID No. 14: MHYCVLSAFLILHLVTVALSLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAV HEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCF RNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS [Sequence ID 14] Therefore, preferably, TGFβ comprises or consists of the sequence substantially shown in SEQ ID NO: 14, or a fragment or variant thereof.

[0012] In one embodiment, TGFβ may be TGFβ3, represented by Genebank ID No: 7043, which is provided herein as Sequence ID No. 16: MKMHLQRALVVLALLNFATVSLSLSTCTTLDFGHIKKKRV EAIRGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIH CPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKRALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS [Sequence ID 16] Therefore, preferably, TGFβ comprises or consists of the sequence substantially shown in SEQ ID NO: 16, or a fragment or variant thereof.

[0013] Preferably, TGFβ is present at a concentration between 0.01 ng / ml and 50 ng / ml. More preferably, TGFβ may be present at a concentration between 0.1 ng / ml and 20 ng / ml, or between 0.1 ng / ml and 10 ng / ml. Most preferably, TGFβ may be present at a concentration between 0.25 ng / ml and 5 ng / ml, more preferably between 0.5 ng / ml and 5 ng / ml.

[0014] Preferably, the lymphocytes are naive, effector, or memory CD8+ T lymphocytes. Preferably, the lymphocytes are naive or effector CD8+ T lymphocytes.

[0015] Preferably, the lymphocytes are naive CD8+ T lymphocytes. If the lymphocytes are human, naive CD8+ T lymphocytes are defined by the expression of differentiation cluster 45 isoform RA (CD45RA+), CC chemokine receptor type 7 (CCR7+), and / or differentiation cluster 27 (CD27+). Naive CD8+ T lymphocytes may also be characterized by the lack of expression of differentiation cluster 45 isoform RO (CD45RO-).

[0016] When lymphocytes are from mice, preferably rats, naive CD8+ T lymphocytes may be defined by the expression of differentiation cluster 67 isoform L (CD67L+), CC chemokine receptor type 7 (CCR7+), differentiation cluster 127 (CD127+), and / or differentiation cluster 27 (CD27+). Naive CD8+ T lymphocytes may be further defined by low levels of differentiation cluster 44 (CD44+).

[0017] Preferably, the lymphocytes are effector CD8+ T lymphocytes. If the lymphocytes are human, the effector CD8+ T lymphocytes may be characterized by the expression of differentiated cluster 45 isoform RA (CD45RA+) and / or differentiated cluster 45 isoform RO (CD45RO+). The effector CD8+ T lymphocytes may be further characterized by the absence of CC chemokine receptor 7 (CCR7-) expression.

[0018] When lymphocytes are from mice, preferably rats, effector CD8+ T lymphocytes can be characterized by high levels of expression of differentiation cluster 44 (CD44+) and / or lack of expression of differentiation cluster 62 ligand (CD62L).

[0019] Preferably, the lymphocytes are memory CD8+ lymphocytes. The memory CD8+ T lymphocytes may be central memory CD8+ T lymphocytes or effector memory CD8+ T lymphocytes. If the lymphocytes are human, the central memory CD8+ T lymphocytes are differentiated into cluster 45. Central memory CD8+ T lymphocytes may be characterized by the expression of the isoform RO (CD45RO+). They may also be characterized by the lack of expression of the differentiation cluster 45 isoform RA (CD45RA-), CC chemokine receptor type 7 (CCR7-), differentiation cluster 27 (CD27-), and / or differentiation cluster 62L (CD62L-).

[0020] If the lymphocytes are from mice, preferably from rats, central memory CD8+ T lymphocytes may be characterized by high levels of differentiation cluster 44 (CD44+) expression and / or differentiation cluster 62 ligand (CD62L).

[0021] If the lymphocytes are human, effector memory CD8+ T lymphocytes can be characterized by the expression of differentiation cluster 45 isoform RO (CD45RO+). Effector memory CD8+ T lymphocytes can further be characterized by the absence of expression of differentiation cluster 45 isoform RA (CD45RA-), CC chemokine receptor type 7 (CCR7-), differentiation cluster 27 (CD27-), and / or differentiation cluster 62L (CD62L-).

[0022] When the lymphocytes are from mice, preferably rats, effector memory CD8+ T lymphocytes can be characterized by high levels of expression of differentiation cluster 44 (CD44+) and / or absence of differentiation cluster 62 ligand (CD62L).

[0023] In one embodiment, CD45RA can be represented by Genebank ID No: 5788, which is provided herein as Sequence ID No. 18, as follows: QTYGDIEVDLKDTDKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLESAETEE VVDIFQVVKALRKARPGMVSTFEQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNGPASPALNQGS [Sequence ID 18] Therefore, preferably, CD45RA comprises or consists of the sequence substantially shown in Sequence ID No. 18, or a fragment or variant thereof.

[0024] In one embodiment, CD45RO can be represented by Genebank ID No: 5788, which is provided herein as Sequence ID No. 19, as follows: [Sequence ID 19] Therefore, preferably, CD45RO comprises or consists of the sequence substantially shown in Sequence ID No. 19, or a fragment or variant thereof.

[0025] In one embodiment, CCR7 can be represented by Genebank ID No: 1236, which is provided herein as Sequence ID No. 20, as follows: MDLGKPMKSVLVVALLVIFQVCLCQDEVTDDYIGDNTTVDYTLFESLCSKKDVRNFKAWFLPIMYSIICFVGLLGNGLVVLTYIYFKRLKTMTDTYLLNLAVADILFLLTLPFWAYSAAK SWVFGVHFCKLIFAIYKMSFFSGMLLLLCISIDRYVAIVQAVSAHRHRARVLLISKLSCVGIWILATVLSIPELLYSDLQRSSSEQAMRCSLITEHVEAFITIQVAQMVIGFLVPLLAMSFCYLVIIRT LLQARNFERNKAIKVIIAVVVVFIVFQLPYNGVVLAQTVANFNITSSTCELSKQLNIAYDVTYSLACVRCCVNPFLYAFIGVKFRNDLFKLFKDLGCLSQEQLRQWSSCRHIRRSSMSVEAETTTTFSP [Sequence ID 20] Therefore, preferably, CCR7 comprises or consists of the sequence substantially shown in Sequence ID No. 20, or a fragment or variant thereof.

[0026] In one embodiment, CD27 can be represented by Genebank ID No:939, which is provided herein as Sequence ID No. 21, as follows: MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTA RSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP [Sequence ID 21] Therefore, preferably, CD27 comprises or consists of the sequence substantially shown in Sequence ID No. 21, or a fragment or variant thereof.

[0027] In one embodiment, CD62L can be represented by Genebank ID No: 6402, which is provided herein as Sequence ID No. 22, as follows: MGCRRTREGPSKAMIFPWKCQSTQRDLWNIFKLWGWTMLCCDFLAHHGTDCWTYHYSEKPMNWQRARRFCRDNYTDLVAIQNKAEIEYLEKTLPFSRSYYWIGIRKIGGIWTWVGTNKSLTEEAENWGDGEPNNKKNKEDCVEIYIKRNKDAGKWNDDACHKLKAALCYTASCQPWSCSGHGECVEIINNYT CNCDVGYYGPQCQFVIQCEPLEAPELGTMDCTHPLGNFSFSSQCAFSCSEGTNLTGIEETTCGPFGNWSSPEPTCQVIQCEPLSAPDLGIMNCSHPLASFSFTSACTFICSEGTELIGKKKTICESGIWSNPSPICQKLDKSFSMIKEGDYNPLFIPVAVMVTAFSGLAFIIWLARRLKKGKKSKRSMNDPY [Sequence ID 22] Therefore, preferably, CD62L comprises or consists of the sequence substantially shown in Sequence ID No. 22, or a fragment or variant thereof.

[0028] Preferably, the lymphocytes are obtained from human or non-human animal tissue. Preferably, the non-human animal is a mammal. The non-human animal may be a rodent, dog, horse, or pig. The rodent may be a rat or mouse. Preferably, the lymphocytes are obtained from human tissue. The tissue may be selected from the group consisting of blood, spleen, lymph nodes, lungs, gastrointestinal tract, skin, prostatic mammary gland tissue, liver, bone marrow, and pancreas. Preferably, the tissue is blood or bone marrow.

[0029] This method may include obtaining lymphocytes from tissues obtained from humans or non-human animals. Lymphocytes may be obtained by any suitable method known in the art. Such methods include buffy coat or density gradient, fluorescence-activated cell sorting and / or magnetically activated cell sorting. These methods will be known to those skilled in the art.

[0030] Preferably, tissue-resident memory T cells (T) produced by the method of the present invention. RM ) are tissue-resident memory CD8+ T cells. Preferably, multiple tissue-resident memory T cells (T RM ) is produced using this method.

[0031] Tissue-resident memory CD8+ T cells can be characterized by the expression of differentiation cluster 8 (CD8), differentiation cluster 69 (CD69), zinc finger protein 683 (ZNF683 / HOBIT), aryl hydrocarbon receptor (AhR), and / or differentiation cluster 103 (CD103). Tissue-resident memory CD8+ (cytotoxic) T cells can be further characterized by the absence of killer cell lectin-like receptor subfamily G member (KLRG1) and / or emesodermine (Eomes).

[0032] Preferably, tissue-resident memory CD8+ (cytotoxic) T cells can be characterized by the expression of CD8, CD69, Hobit, AhR, and CD103. Preferably, tissue-resident memory CD8+ (cytotoxic) T cells can be characterized by the expression of CD8, CD69, Hobit, AhR, and CD103, and the absence of KLRG1 and Eomes expression.

[0033] In one embodiment, CD8 can be represented by Genebank ID No: 925, which is provided herein as Sequence ID No. 2, as follows: MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSA LSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV [Sequence ID 2] Therefore, preferably, CD8 comprises or consists of the sequence substantially shown in Sequence ID No. 2, or a fragment or variant thereof.

[0034] In one embodiment, CD69 can be represented by Genebank ID No:969, which is provided herein as Sequence ID No. 3, as follows: MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALSVGQYNCPGQYTFSMPSDSHVSSCSEDWVGYQRKCYFISTVKRSWTSAQNACSEHGATLAVIDSEKDMNFLKRYAGREEHWVGLKKEPGHPWKWSNGKEFNNWFNVTGSDKCVFLKNTEVSSMECEKNLYWICNKPYK [Sequence ID 3] Therefore, preferably, CD69 comprises or consists of the sequence substantially shown in Sequence ID No. 3, or a fragment or variant thereof.

[0035] In one embodiment, CD103 can be represented by Genebank ID No:3682, which is provided herein as Sequence ID No. 4, as follows: MWLFHTLLCIASLALLAAFNVDVARPWLTPKGGAPFVLSSLLHQDPSTNQTWLLVTSPRTKRTPGPLHRCSLVQDEILCHPVEHVPIPKGRHRGVTVVRSHHGVLICIQVLVRRPHSLSS ELTGTCSLLGPDLRPQAQANFFDLENLLDPDARVDTGDCYSNKEGGGEDDVNTARQRRALEKEEEEDKEEEEDEEEEEAGTEIAIILDGSGSIDPPDFQRAKDFISNMMRNFYEKCFECN FALVQYGGVIQTEFDLRDSQDVMASLARVQNITQVGSVTKTASAMQHVLDSIFTSSHGSRRKASKVMVVLTDGGIFEDPLNLTTVINSPKMQGVERFAIGVGEEFKSARTARELNLI ASDPDETHAFKVTNYMALDGLLSKLRYNIISMEGTVGDALHYQLAQIGFSAQILDERQVLLGAVGAFDWSGGALLYDTRSRRGRFLNQTAAAAADAEAAQYSYLGYAVAVLHKTCSL SYIAGAPRYKHHGAVFELQKEGREASFLPVLEGEQMGSYFGSELCPVDIDMDGSTDFLLVAAPFYHVHGEEGRVYVYRLSEQDGSFSLARILSGHPGFTNARFGFAMAAMGDLSQDK LTDVAIGAPLEGFGADDGASFGSVYIYNGHWDGLSASPSQRIRASTVAPGLQYFGMSMAGGFDISGDGLADITVGTLGQAVVFRSRPVVRLKVSMAFTPSALPIGFNGVVNVNRCFEI SSVTTASESGLREALLNFTLDVDVGKQRRRLQCSDVRSCLGCLREWSSGSQLCEDLLLMPTEGELCEEDCFSNASVKVSYQLQTPEGQTDHPQPILDRYTEPFAIFQLPYEKACKNK LFCVAELQLATTVSQQELVVGLTKELTLNINLTNSGEDSYMTSALNYPRNLQLKRMQKPPSPNIQCDDPQPVASVLIMNCRIGHPVLKRSSAHVSVVWQLEENAFPNRTADITVTVT NSNERRSLANETHTLQFRHGFVAVLSKPSIMYVNTGQGLSHHKEFLFHVHGENLFGAEYQLQICVPTKLRGLQVVAVKKLTRTQASTVCTWSQERACAYSSVQHVEEWHSVSCVIAS DKENVTVAAEISWDHSEELLKDVTELQILGEISFNKSLYEGLNAENHRTKITVVFLKDEKYHSLPIIIKGSVGGLLVLIVILVILFKCGFFKRKYQQLNLESIRKAQLKSENLLEEEN [Sequence ID 4] Therefore, preferably, CD103 comprises or consists of the sequence substantially shown in Sequence ID No. 4, or a fragment or variant thereof.

[0036] In one embodiment, KLRG1 can be represented by Genebank ID No: 10219, which is provided herein as Sequence ID No. 5, as follows: MTDSVIYSMLELPTATQAQNDYGPQQKSSSSRPSCSCLVAIALGLLTAVLLSVLLYQWILCQGSNYSTCASCPSCPDRWMKYGNHCYYFSVEEKDWNSSLEFCLARDSHLLVITDNQEMSLLQVFLSEAFCWIGLRNNSGWRWEDGSPLNFSRISSNSFVQTCGAINKNGLQASSCEVPLHWVCKKCPFADQALF [Sequence ID 5] Therefore, preferably, KLRG1 comprises or consists of the sequence substantially shown in Sequence ID No. 5, or a fragment or variant thereof.

[0037] In one embodiment, Eomes may be represented by Genebank ID No: 8320, which is provided herein as Sequence ID No. 6, as follows: MQLGEQLLVSSVNLPGAHFYPLESARGGSGGSAGHLPSAAPSPQKLDLDKASKKFSGSLSCEAVSGEPAAASAGAPAAMLSDTDAGDAFASAAAVAKPGPPDGRKGSPCGEEELPSAAAAAAAAAAAAAATARYSMDSLSSERYYLQSPGPQGSELAAPCSLFPYQAAAGAPHGPVYPAP NGARYPYGSMLPPGGPAAAVCPPGRAQFGPGAGAGSGAGGSSGGGGGPGTYQYSQGAPLYGPYPGAAAAGSCGGLGGLGVPGSGFRAHVYLCNRPLWLKFHRHQTEMIITKQGRRMFPFLSFNINGLNPTAHYNVFVEVVLADPNHWRFQGGKWVTCGKADNNMQGNKMYVHPESPNTGS HWMRQEISFGKLKLTNNKGANNNNTQMIVLQSLHKYQPRLHIVEVTEDGVEDLNEPSKTQTFTFSETQFIAVTAYQNTDITQLKIDHNPFAKGFRDNYDSSHQIVPGGRYGVQSFFPEPFVNTLPQARYYNGERTVPQTNGLLSPQQSEEVANPPQRWLVTPV QQPGTNKLDISSYESEYTSSTLLPYGIKSLPLQTSHALGYYPDPTFPAMAGWGGRGSYQRKMAAGLPWTSRTSPTVFSEDQLSKEKVKEEIGSSWIETPPSIKSLDSNDSGVYTSACKRRRLSPSNSSNENSPSIKCEDINAEEYSKDTSKGMGGYYAFYTTP [Sequence ID 6] Therefore, preferably, Eomes comprises or consists of the sequence substantially shown in Sequence ID No. 6, or a fragment or variant thereof.

[0038] In one embodiment, Hobit can be represented by Genebank ID No: 257101, which is provided herein as Sequence ID No. 9, as follows. MKEESAAQLGCCHRPMALGGTGGSLSPSLDFQLFRGDQVFSACRPLPDMVDAHGPSCASWLCPLPLAPGRSALLACLQDLDLNLCTPQPAPLGTDLQGLQEDALSMKHEPPGLQASSTDDKKFTVKYPQNK DKLGKQPERAGEGAPCPAFSSHNSSSPPPLQNRKSPSPLAFCPCPPVNSISKELPFLLHAFYPGYPLLLPPPHLFTYGALPSDQCPHLLMLPQDPSYPTMAMPSLLMMVNELGHPSARWETLLPYPGAFQA SGQALPSQARNPGAGAAPTDSPGLERGGMASPAKRVPLSSQTGTAALPYPLKKKNGKILYECNICGKSFGQLSNLKVHLRVHSGERPFQCALCQKSFTQLAHLQKHHLVHTGERPHKCSIPWVPGRNHWKS FQAWREREVCHKRFSSSSNLKTHLRLHSGARPFQCSVCRSRFTQHIHLKLHHRLHAPQPCGLVHTQLPLASLACLAQWHQGALDLMAVASEKHMGYDIDEVKVSSTSQGKARAVSLSSAGTPLVMGQDQNN [Sequence ID 9] Therefore, preferably, Hobit comprises or consists of the sequence substantially shown in Sequence ID No. 9, or a fragment or variant thereof.

[0039] In one embodiment, Ahr can be represented by Genebank ID No: 196, which is provided herein as Sequence ID No. 10, as follows. MNSSSANITYASRKRRKPVQKTVKPIPAEGIKSNPSKRHRDRLNTELDRLASLLPFPQDVINKLDKLSVLRLSVSYLRAKSFFDVALKSSPTERNGGQDNCRAANFREGLNLQEGEFLLQALNGFVLVVTTDALVFYASSTIQDYLGFQQ SDVIHQSVYELIHTEDRAEFQRQLHWALNPSQCTESGQGIEEATGLPQTVVCYNPDQIPPENSPLMERCFICRLRCLLDNSSGFLAMNFQGKLKYLHGQKKKGKDGSILPPQLALFAIATPLQPPSILEIRTKNFIFRTKHKLDFTPIGC DAKGRIVLGYTEAELCTRGSGYQFIHAADMLYCAESHIRMIKTGESGMIVFRLLTKNNRWTWVQSNARLLYKNGRPDYIIVTQRPLTDEEGTEHLRKRNTKLPFMFTTGEAVLYEATNPFPAIMDPLPLRTKNGTSGKDSATTSTLSKDS LNPSSLLAAMMQQDESIYLYPASSTSSTAPFENNFFNESMNECRNWQDNTAPMGNDTILKHEQIDQPQDVNSFAGGHPGLFQDSKNSDLYSIMKNLGIDFEDIRHMQNEKFFRNDFSGEVDFRDIDLTDEILTYVQDSLSKSPFIPSDYQ QQQSLALNSSCMVQEHLHLEQQQQHHQKQVVVEPQQQLCQKMKHMQVNGMFENWNSNQFVPFNCPQQDPQQYNVFTDLHGISQEFPYKSEMDSMPYTQNFISCNQPVLPQHSKCTELDYPMGSF EPSPYPTTSSLEDFVTCLQLPENQKHGLNPQSAIITPQTCYAGAVSMYQCQPEPQHTHVGQMQYNPVLPGQQAFLNKFQNGVLNETYPAELNNINNTQTTTHLQPLHHPSEARPFPDLTSSGFL [Sequence ID 10] Therefore, preferably, Ahr comprises or consists of the sequence substantially shown in Sequence ID No. 10, or a fragment or variant thereof.

[0040] Preferably, the method involves culturing lymphocytes in the presence of interleukins 2, 4, 7, 12, 15 and / or 21 (IL-2, IL-4, IL-7, IL-12, IL-15 and / or IL-21).

[0041] Interleukins are preferably mammalian, and most preferably human interleukins. Preferably, this method includes culturing lymphocytes in the presence of interleukin-7 (IL-7).

[0042] Preferably, IL-7 is mammalian. Most preferably, IL-7 is human IL-7. In one embodiment, IL-7 may be represented by Genebank ID No: 3574, which is provided herein as Sequence ID No. 28: MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRH ICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH [Sequence ID 28] Therefore, preferably, IL-7 comprises or consists of the sequence substantially shown in Sequence ID No. 28, or a fragment or variant thereof.

[0043] Preferably, IL-7 may be present at a concentration between 0.1 ng / ml and 200 ng / ml. More preferably, IL-7 may be present at a concentration between 2 ng / ml and 100 ng / ml. Most preferably, IL-7 may be present at a concentration between 10 ng / ml and 50 ng / ml.

[0044] Preferably, this method further comprises culturing lymphocytes in the presence of interleukin-15 (IL-15). Preferably, IL-15 is mammalian. Most preferably, IL-15 is human IL-15. In one embodiment, IL-15 may be represented by Genebank ID No: 3600, which is provided herein as Sequence ID No. 7: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS [Sequence ID 7] Therefore, preferably, IL-15 comprises or consists of the sequence substantially shown in Sequence ID No. 7, or a fragment or variant thereof.

[0045] Preferably, IL-15 may be present at a concentration of 1 ng / ml to 100 ng / ml. More preferably, IL-15 may be present at a concentration of 5 ng / ml to 50 ng / ml. Most preferably, IL-15 may be present at a concentration of 10 ng / ml to 25 ng / ml.

[0046] Preferably, the method further comprises culturing in the presence of interleukin-33 (IL-33). Preferably, IL-33 is mammalian. Most preferably, IL-33 is human IL-33. In one embodiment, IL-33 can be represented by Genebank ID No: 90865, which is provided herein as Sequence ID No. 8: MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIKKEACYFRRETTKRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRALHDSSITGISPITEYLASLSTYNDQSIT FALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET [Sequence 8] Therefore, preferably, IL-33 comprises or consists of the sequence substantially shown in Sequence ID No. 8, or a fragment or variant thereof.

[0047] Preferably, IL-33 may be present at a concentration between 0.5 ng / ml and 100 ng / ml. More preferably, IL-33 may be present at a concentration between 2 ng / ml and 50 ng / ml. Most preferably, IL-33 may be present at a concentration between 10 ng / ml and 25 ng / ml.

[0048] Preferably, this method includes culturing lymphocytes in the presence of interleukin-2 (IL-2). Therefore, in one embodiment, the cultivation of naive CD8+ T lymphocytes in the presence of TGFβ is included in tissue-resident memory T cells (T RM A method for producing ) is provided.

[0049] Another embodiment provides a method for producing tissue-resident memory T cells, comprising culturing naive CD8+ T lymphocytes in the presence of TGFβ, IL-15, and IL-33.

[0050] Therefore, in one embodiment, a method is provided for producing tissue-resident memory CD8+ (cytotoxic) T cells, comprising culturing naive CD8+ T lymphocytes in the presence of TGFβ, IL-15, and IL-33.

[0051] Preferably, the method further comprises culturing in the presence of at least one interleukin-1 family member, such as IL-1α, IL-1β, and / or IL-18.

[0052] Preferably, the interleukin-1 family member is a mammal. Most preferably, the interleukin-1 family member is human. In one embodiment, IL-1α can be represented by Genebank ID No:3552, which is provided herein as Sequence ID No. 11, as follows: MAKVPDMFEDLKNCYSENEEDSSSIDHLSLNQKSFYHVSYGPLHEGCMDQSVSLSISETSKTSKLTFKESMVVVATNGKVLKKRRLSLSQSITDDDLEAIANDSEEEIIKPRSAPFSFLS NVKYNFMRIIKYEFILNDALNQSIIRANDQYLTAAALHNLDEAVKFDMGAYKSSKDDAKITVILRISKTQLYVTAQDEDQPVLLKEMPEIPKTITGSETNLLFFWETHGTKNYFTSVAHPNLFIATKQDYWVCLAGGPPSITDFQILENQA [Sequence ID 11] Therefore, preferably, IL-1α comprises or consists of the sequence substantially shown in Sequence ID No. 11, or a fragment or variant thereof.

[0053] Preferably, IL-1α may be present at a concentration of 0.1 ng / ml to 100 ng / ml. More preferably, IL-1α may be present at a concentration of 1 ng / ml to 50 ng / ml. Most preferably, IL-1α may be present at a concentration of 5 ng / ml to 20 ng / ml.

[0054] In one embodiment, IL-1β can be represented by Genebank ID No:3553, which is provided herein as Sequence ID No. 12, as follows: MAEVPELASEMMAYYSGNEDDLFFEADGPKQMKCSFQDLDLCPLDGGIQLRISDHHYSKGFRQAASVVVAMDKLRKMLVPCPQTFQENDLSTFFPFIFEEEPIFFDTWDNEAYVHDAPVRSLNCTLRDSQQKSL VMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSS [Sequence ID 12] Therefore, preferably, IL-1β comprises or consists substantially of the sequence shown in Sequence ID No. 12, or a fragment or variant thereof.

[0055] Preferably, IL-1β may be present at a concentration of 0.1 ng / ml to 100 ng / ml. More preferably, IL-1β may be present at a concentration of 1 ng / ml to 50 ng / ml. Most preferably, IL-1β may be present at a concentration of 5 ng / ml to 20 ng / ml.

[0056] In one embodiment, IL-18 can be represented by Genebank ID No:3606, which is provided herein as Sequence ID No. 13, as follows: MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED [Sequence ID 13] Therefore, preferably, IL-18 comprises or consists of the sequence substantially shown in Sequence ID No. 13, or a fragment or variant thereof.

[0057] Preferably, IL-18 may be present at a concentration between 0.1 ng / ml and 100 ng / ml. More preferably, IL-18 may be present at a concentration between 1 ng / ml and 50 ng / ml. Most preferably, IL-18 may be present at a concentration between 5 ng / ml and 20 ng / ml.

[0058] Lymphocytes can be cultured in a culture medium containing at least one aryl hydrocarbon receptor (AhR) ligand. The AhR ligand may be an agonist or an antagonist. Preferably, the AhR ligand is an agonist. Preferably, the AhR ligand is an antagonist.

[0059] AhR ligands may be selected from the group consisting of halogenated aromatic hydrocarbons, polycyclic aromatic hydrocarbons, dietary aryl hydrocarbons, heme metabolites, indigoids, StemRegenin1, and tryptophan metabolites.

[0060] The halogenated aromatic hydrocarbon may be tetrachlorodibenzo-p-dioxin (TCDD). The polycyclic aromatic hydrocarbon may be 3-methylcholanthrene. The tryptophan metabolite may be 6-formylindoro[3,2-b]carbazole (FICZ). The aryl hydrocarbon of food origin may be a flavone and / or an indole derivative. The indole derivative may be indole-3-carbinol (I3C) and / or its product diindolylmethane (DIM).

[0061] Lymphocytes can be cultured in a culture medium containing at least one lipid. Preferably, the lipid is cholesterol and / or medium-chain fatty acids (MCFAs). The MCFA may be oleic acid.

[0062] Lymphocytes can be further cultured with antigens. Specific types of antigens include T RM The choice of cells depends on their intended therapeutic application. For example, lymphocytes can be cultured together with tumor antigens.

[0063] Preferably, in some embodiments, the method includes culturing lymphocytes in the presence of regulatory T cells or type 1 regulatory T cells. Therefore, in another aspect of the present invention, tissue-resident memory T cells (T) include culturing lymphocytes in the presence of transforming growth factor beta (TGFβ) and / or co-culturing lymphocytes with type 1 regulatory T cells. RM A method for producing ) is provided.

[0064] Those skilled in the art will understand that “regulatory T cells” are T cells involved in peripheral immunity as a subset of CD4+ T cells. Preferably, regulatory T cells are characterized by the expression of the transcription factor forkheadbox P3 (Foxp3). In other embodiments, the method does not involve culturing lymphocytes in the presence of regulatory T cells.

[0065] Those skilled in the art will understand that “type 1 regulatory T cells” are a class of regulatory T cells involved in peripheral immunity as a subset of CD4+ T cells. Preferably, type 1 regulatory T cells are characterized by the expression of the transcription factor, forkhead box P3 (Foxp3), T-box transcription factor 21 (Tbet), and / or surface molecule CXC motif chemokine receptor 3 (CXCR3). In other embodiments, the method does not involve culturing lymphocytes in the presence of type 1 regulatory T cells.

[0066] In one embodiment, Foxp3 can be represented by Genebank ID No: 50943, which is provided herein as Sequence ID No. 23, as follows: MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFL KHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPE FLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP [Sequence ID 23] Therefore, preferably, Foxp3 comprises or consists of the sequence substantially shown in Sequence ID No. 23, or a fragment or variant thereof.

[0067] In one embodiment, Tbet can be represented by Genebank ID No:30009, which is provided herein as Sequence ID No. 24, as follows: MGIVEPGCGDMLTGTEPMPGSDEGRAPGADPQHRYFYPEPGAQDADERRGGGSLGSPYPGGALVPAPPSRFLGAYAYPPRPQAAGFPGAGESFPPPADAEGYQPGEGYAAPDPRAGLYPGPREDYALPAGLEV SGKLRVALNNHLLWSKFNQHQTEMIITKQGRRMFPFLSFTVAGLEPTSHYRMFVDVVLVDQHHWRYQSGKWVQCGKAEGSMPGNRLYVHPDSPNTGAHWMRQEVSFGKLKLTNNKGASNNVTQMIVLQSLHKYQ PRLHIVEVNDGEPEACNASNTHIFTFQETQFIAVTAYQNAEITQLKIDNNPFAKGFRENFESMYTSVDTSIPSPPGPNCQFLGGDHYSPLLPNQYPVPSRFYPDLPGQAKDVVPQAYWLGAPRDHSYEAEFRA VSMKPAFLPSAPGPTMSYYRGQEVLAPGAGWPVAPQYPPKMGPASWFRPMRTLPMEPGPGGSEGRGPEDQGPPLVWTEIAPIRPESSDSGLGEGDSKRRRVSPYPSSGDSSSPAGAPSPFDKEAEGQFYNYFPN [Sequence ID 24] Therefore, preferably, Tbet comprises or consists of the sequence substantially shown in Sequence ID No. 24, or a fragment or variant thereof.

[0068] In one embodiment, CXCR3 can be represented by Genebank ID No: 2833, which is provided herein as Sequence ID No. 25, as follows: MELRKYGPGRLAGTVIGGAAQSKSQTKSDSITKEFLPGLYTAPSSPFPPSQVSDHQVLNDAEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDRAFLPALYSLLFLLGLLGNGAVAAVLLSRRTALSSTDTFLLHLAVADTLLVLTLPLWAVDAAVQWVFGSGLCKVAGALFNINFYAGALLLACISFDRYLNIVHATQLY RRGPPARVTLTCLAVWGLCLLFALPDFIFLSAHHDERLNATHCQYNFPQVGRTALRVLQLVAGFLLPLLVMAYCYAHILAVLLVSRGQRRLRAMRLVVVVVVAFALCWTPYHLVVLVDILMDLGALARNCGRESRVDVAKSVTSGLGYMHCCLNPLLYAFVGVKFRERMWMLLLRLGCPNQRGLQRQPSSSRRDSSWSETSEASYSGL [Sequence ID 25] Therefore, preferably, CXCR3 comprises or consists of the sequence substantially shown in Sequence ID No. 25, or a fragment or variant thereof.

[0069] Preferably, regulatory T cells (preferably type 1 regulatory T cells) express integrin alpha V beta 8 (αvβ8). Those skilled in the art will understand that αvβ8 is a dimer of integrin subunit (Itgβ8) and integrin subunit alpha V (Itgav). Therefore, preferably, regulatory T cells (preferably type 1 regulatory T cells) express Itgβ8 and Itgav.

[0070] In one embodiment, Itgβ8 can be represented by Genebank ID No:3696, which is provided herein as Sequence ID No. 26, as follows: MCGSALAFFTAAFVCLQNDRRGPASFLWAAWVFSLVLGLG QGEDNRCASSNAASCARCLALGPECGWCVQEDFISGGSRSERCDIVSNLISKGCSVDSIEYPSVHVIIPTENEINTQVTPGEVSIQLRPGAEANFMLKVHPLKKYPVDLYYLVDVSASMHNNIEKLNSVGNDLSRKMAFFSRDFRLGFGSYVDKTVSPYISIHPERIHNQCSDYNLDCMPPH GYIHVLSLTENITEFEKAVHRQKISGNIDTPEGGFDAMLQAAVCESHIGWRKEAKRLLLVMTDQTSHLALDSKLAGIVVPNDGNCHLKNNVYVKSTTMEHPSLGQLSEKLIDNNINVIFAVQGKQFHWYKDLLPLLPGTIAGEIESKAANLNNLVVEAYQKLISEVKVQVENQVQGIYFNIT AICPDGSRKPGMEGCRNVTSNDEVLFNVTVTMKKCDVTGGKNYAIIKPIGFNETAKIHIHRNCSCQCEDNRGPKGKCVDETFLDSKCFQCDENKCHFDEDQFSSESCKSHKDQPVCSGRGVCVCGKCSCHKIKLGKVYGKYCEKDDFSCPYHHGNLCAGHGECEAGRCQCFSGWEGDRCQCP SAAAQHCVNSKGQVCSGRGTCVCGRCECTDPRSIGRFCEHCPTCYTACKENWNCMQCLHPHNLSQAILDQCKTSCALMEQQHYVDQTSECFSSPSYLRIFFIIFIVTFLIGLLKVLIIRQVILQWNSNKIKSSSDYRVSASKKDKLILQSVCTRAVTYRREKPEEIKMDISKLNAHETFRCNF [Sequence ID 26] Therefore, preferably, Itgβ8 comprises or consists of the sequence substantially shown in SEQ ID NO: 26, or a fragment or variant thereof.

[0071] In one embodiment, Itgav may be represented by Genebank ID No:3685, which is provided herein as Sequence ID No. 27, as follows: MAFPPRRRLRLGPRGLPLLLSGLLLPLCRAFNLDVDSPAEYSGPEGSYFGFAVDFFVPSASSRMFLLVGAPKANTTQPGIVEGGQVLKCDWSSTRRCQPIEFDATGNRDYAKDDPLEFKSHQWFGASVRSKQDKILACAPLYHWRTEMKQEREPVGTCFLQDGTKTVEYAPCRSQDIDADGQGFCQGGFSIDFTKADRVLLGGPGSFYWQGQLISDQVAEIVSKYDPNVYSIKYNNQLATRTAQAIFDDSYLGYSVAVGDFNGDGIDDFVSGVPRAARTLGMVYIYDGKNMSSLYNFTGEQMAAYFGFSVAATDINGDDYADVFIGAPLFMDRGSDGKLQEVGQVSVSLQRASGDFQTTKLNGFEVFARFGSAIAPLGDLDQDGFNDIAIAAPYGGEDKKGIVYIFNGRSTGLNAVPSQILEGQWAARSMPPSFGYSMKGATDIDKNGYPDLIVGAFGVDRAILYRARPVITVNAGLEVYPSILNQDNKTCSLPGTALKVSCFNVRFCLKADGKGVLPRKLNFQVELLLDKLKQKGAIRRALFLYSRSPSHSKNMTISRGGLMQCEELIAYLRDESEFRDKLTPITIFMEYRLDYRTAADTTGLQPILNQFTPANISRQAHILLDCGEDNVCKPKLEVSVDSDQKKIYIGDDNPLTLIVKAQNQGEGAYEAELIVSIPLQADFIGVVRNNEALARLSCAFKTENQTRQVVCDLGNPMKAGTQLLAGLRFSVHQQSEMDTSVKFDLQIQSSNLFDKVSPVVSHKVDLAVLAAVEIRGVSSPDHVFLPIPNWEHKENPETEEDVGPVVQHIYELRNNGPSSFSKAMLHLQWPYKYNNNTLLYILHYDIDGPMNCTSDMEINPLRIKISSLQTTEKNDTVAGQGERDHLITKRDLALSEGDIHTLGCGVAQCLKIVCQVGRLDRGKSAILYVKSLLWTETFMNKENQNHSYSLKSSASFNVIEFPYKNLPIEDITNSTLVTTNVTWGIQPAPMPVPVWVIILA。 VLAGLLLLAVLVFVMYRMGFFKRVRPPQEEQEREQLQPHENGEGNSET [Sequence ID 27] Therefore, preferably, Itgav comprises or consists substantially of the sequence shown in Sequence ID No. 27, or a fragment or variant thereof.

[0072] In one embodiment, type 1 regulatory T cells (preferably type 1 regulatory T cells) may be activated by an anti-CD3 molecule, preferably such as an anti-CD3 antibody, and / or IL-2. αvβ8 expression can be enhanced in regulatory T cells (preferably type 1 regulatory T cells) by amphigerulin. Therefore, this method may further include contacting regulatory T cells (preferably type 1 regulatory T cells) with amphigerulin.

[0073] This method may further include culturing lymphocytes together with dendritic cells. Preferably, lymphocytes are 100E+03 cells / cm³ 2 From 2000E+03 cells / cm 2 They are cultured between [a certain number of cells]. More preferably, lymphocytes are cultured at 250E+03 cells / cm³. 2 From 1000E+03 cells / cm 2 They are cultured between [species name]. Most preferably, lymphocytes are cultured at 250E+03 cells / cm³. 2 From 1000E+03 cells / cm 2 It is cultured between them.

[0074] In one embodiment, this method involves extracting T from the culture. RM This further includes purifying the cells. Those skilled in the art will understand that any factors, such as cytokines, described herein may be mammalian. Mammals may be rodents, dogs, horses, or pigs. Rodents may be rats or mice. However, preferably, the factors described herein are human.

[0075] In the second aspect, tissue-resident memory T cells (T RM A method is provided for propagating a population of tissue-resident memory T cells, the method comprising culturing a population of tissue-resident memory T cells as defined in the first embodiment. Tissue-resident memory T cells may be as defined in the first embodiment.

[0076] The method according to the first or second embodiment may further include culturing tissue-resident memory T cells in the presence of IL-2, IL-4, IL-7, IL-12, IL-15 and / or IL-21.

[0077] Preferably, IL-2 is mammalian. Most preferably, IL-2 is human IL-2. In one embodiment, IL-2 can be represented by Genebank ID No: 3558, which is provided herein as Sequence ID No. 17: MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT [Sequence ID 17] Therefore, preferably, IL-2 comprises or consists of the sequence substantially shown in Sequence ID No. 17, or a fragment or variant thereof.

[0078] Preferably, IL-2 may be present at a concentration between 0.1 ng / ml and 200 ng / ml. More preferably, IL-2 may be present at a concentration between 2 ng / ml and 100 ng / ml. Most preferably, IL-2 may be present at a concentration between 10 ng / ml and 50 ng / ml.

[0079] Preferably, IL-21 is mammalian. Most preferably, IL-21 is human IL-21. In one embodiment, IL-21 is Genebank ID No: 5 It can be represented by 9067, which is provided herein as Sequence ID No. 15, as follows: MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS [Sequence ID 15] Therefore, preferably, IL-21 comprises or consists of the sequence substantially shown in Sequence ID No. 15, or a fragment or variant thereof.

[0080] Preferably, IL-21 may be present at a concentration between 0.1 ng / ml and 200 ng / ml. More preferably, IL-21 may be present at a concentration between 5 ng / ml and 100 ng / ml. Most preferably, IL-21 may be present at a concentration between 10 ng / ml and 50 ng / ml.

[0081] In a third embodiment, tissue-resident memory T cells are provided that can be obtained or obtained by the method according to the first embodiment. Tissue-resident memory T cells produced by the method of the present invention are particularly useful for therapeutic applications.

[0082] T cells have been shown to be a powerful tool for eradicating tumors. The presence of T cells, particularly tissue-resident memory T cells within tumor tissue, is known to correlate with the prognosis of positive cancers. 94 Several studies have shown that in solid tumors, T is identified by the expression of CD103. RM The presence of cells is particularly associated with high survival rates and an overall positive prognosis, even in advanced stages of cancer. 95、96 Furthermore, T RM The cells have been shown to significantly improve survival rates in cancer patients when used in combination with other known immunotherapies. 97 However, naturally occurring T RMThe number of T cells is usually small compared to other types of T cells for a significant therapeutic effect to be observed. RM Generating T cells for antitumor therapy in vitro with cellular attributes, including migratory and tissue-homing properties such as the expression of CD103, CD69, and CTLA-4, which would enable T cells to penetrate tumors, would be highly valuable as monotherapy or in combination therapy.

[0083] Therefore, preferably, the present invention T RM The cells express CD103. Preferably, the T of the present invention RM The cells express CD69. Preferably, the T of the present invention RM The cells express CTLA-4.

[0084] Accordingly, a fourth aspect of the present invention provides tissue-resident memory T cells according to the third aspect, and possibly a proliferated population thereof, for use in therapy. A fifth aspect of the present invention provides tissue-resident memory T cells, or optionally a proliferated population thereof, according to the third aspect, for use in T cell therapy.

[0085] Tissue-resident memory T cells (T RM It will be understood that the proliferated population of ) is particularly useful in treatment, especially in T-cell therapy. T-cell therapy may also be CAR-T cell therapy. T-cell therapy may be congenital T-cell therapy, such as gamma delta T cell, mucosa-associated invariant T cell, or natural killer T cell-based therapy.

[0086] A sixth aspect of the present invention provides tissue-resident memory T cells, optionally proliferated therein, according to the third aspect, for use in the prevention, treatment, or improvement of cancer or infectious diseases. A seventh aspect of the present invention provides a method for treating cancer or infection in a subject requiring such treatment, comprising administering, or having administered, a therapeutically effective amount of tissue-resident memory T cells, optionally a proliferated population thereof, according to the third aspect.

[0087] T RM The cells may be generated by in vitro culture of previously activated T cells in the presence of antigen-presenting cells, interleukin (IL)-15, and TGFβ. The addition of IL-2 or especially IL-7 is preferably used to stimulate the expression of differentiation cluster (CD)69, CD103, and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) in mouse models. RM This improves cell migration characteristics and cell retrieval from peripheral organs during adoptive migration.

[0088] It is understood that tissue-resident memory T cells produced according to the present invention may be used in monotherapy (i.e., (i) the use of tissue-resident memory T cells alone, or (ii) the use of a therapeutic composition containing tissue-resident memory T cells alone). Alternatively, tissue-resident memory T cells according to the present invention may be used as an adjunct to or in combination with known therapies for treating, improving, or preventing diseases, such as cancer.

[0089] The tissue-resident memory T cells according to the present invention can be combined into compositions having many different forms, particularly depending on how the composition is used. For example, the composition may be a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle solution, transdermal patch, liposome suspension, or any other suitable form that can be administered to a person or animal in need of treatment. It will be understood that the vehicle of the drug according to the present invention should be well acceptable to the subject to which it is administered.

[0090] The tissue-resident memory T cells of the present invention can be used in many ways. For example, oral administration may be required, in which case the drug may be contained in a composition that can be taken orally, for example, in the form of a tablet, capsule, or liquid. The antibiotic compositions and formulations of the present invention may be administered by inhalation (for example, intranasally). The compositions can also be formulated for topical use. For example, creams or ointments may be applied to the skin.

[0091] The tissue-resident memory T cell compositions and formulations according to the present invention may also be incorporated into sustained release or delayed release devices. Such devices can be inserted, for example, at specific tissue locations above or below the skin, and the agent can be released over a period of hours, days, weeks, or even months. The device can be positioned at least adjacent to the treatment site. Such devices can be particularly advantageous when long-term treatment with an agent used in accordance with the present invention is required and usually frequent administration (e.g., at least daily administration) is required.

[0092] In a preferred embodiment, the tissue-resident memory T cells according to the present invention can be administered to a subject by injection into the bloodstream or directly into the site requiring treatment. The injection can be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion), or intramuscular. Preferably, the tissue-resident memory T cells of the present invention are administered via peripheral blood. Preferably, the tissue-resident memory T cells of the present invention are administered intravenously.

[0093] The amount of tissue-resident memory T cells required is determined by their biological activity and bioavailability, which also depends on the mode of administration, the physicochemical properties of the tissue-resident memory T cells, and whether they are used as monotherapy or in combination therapy. The frequency of administration is also affected by the half-life of the tissue-resident memory T cells in the treated subject. The optimal dosage to be administered can be determined by those skilled in the art, and varies depending on the specific composition and formulation used, the strength of the pharmaceutical composition, the mode of administration, and the progression of the specific disease being treated. Dosage adjustments may be necessary due to additional factors that depend on the specific subject being treated, such as the subject's age, weight, gender, diet, time of administration, etc.

[0094] Generally, the T according to the present invention RMA daily dose of 0.001 μg / kg body weight to 10 mg / kg body weight of the cells or the preparation can be used according to the composition or preparation used. More preferably, the daily dose is between 0.01 μg / kg body weight and 1 mg / kg body weight, more preferably between 0.1 μg / kg and 100 μg / kg body weight, and most preferably between approximately 0.1 μg / kg and 10 μg / kg body weight. Generally, 10^5T RM of the cells of the present invention to 10^7T RM A daily dose between the cells can be used. Preferably, 10^5T RM of the cells of the present invention to 10^6T RM A daily dose between the cells can be used.

[0095] The composition or preparation can be administered before, during, or after the onset of the disease to be treated. The daily dose can be given as a single administration (e.g., a single injection per day). Alternatively, tissue-resident memory T cells may require more than two administrations per day. As an example, tissue-resident memory T cells are 10^5T RM of the cells of the present invention to 10^7T RM The cells can be administered as two (or more depending on the severity of the disease being treated) daily doses (assuming a body weight of 70 kg). The patient being treated can take the first dose upon waking up and then take the second dose in the evening (in the case of a two-dose regimen) or at intervals of 3 or 4 hours thereafter. Alternatively, a sustained-release device may be used to provide the patient with the optimal dose of the concentration according to the present invention without the need to administer repeated doses.

[0096] Using known procedures (e.g., in vivo experiments, clinical trials, etc.) conventionally employed in the pharmaceutical industry, specific formulations and precise treatment regimens (such as the daily dose of tissue-resident memory T cells, dosing frequency, etc.) according to the present invention can be formed.

[0097] In an eighth aspect, there is provided a pharmaceutical composition comprising tissue-resident memory T cells according to the third aspect, optionally a proliferating population thereof, and a pharmaceutically acceptable excipient. The present invention also provides, in its ninth aspect, a method for producing a pharmaceutical composition according to its eighth aspect, comprising combining a therapeutically effective amount of tissue-resident memory T cells according to its third aspect, optionally a proliferating population thereof, with a pharmaceutically acceptable excipient.

[0098] The "target" can be a vertebrate, mammal, or livestock. Therefore, the drug according to the present invention can be used to treat any mammal, such as livestock (e.g., horses), pets, and can also be used for other veterinary purposes. Most preferably, the target is human.

[0099] The "therapeutic effective dose" of tissue-resident memory T cells is any amount necessary to produce the desired effect when administered to the target. The amount of drug is approximately 10^5 T RM Approximately 10^7 T from the cell RM The amount may be cellular, and most preferably about 10^5 T RM Approximately 10^6 T from the cell RM Even a cellular amount is acceptable.

[0100] As used herein, “pharmaceutically acceptable vehicle” is any known compound or any combination of known compounds that is known to those skilled in the art to be useful in formulating pharmaceutical compositions, in particular formulations for T-cell-based therapies.

[0101] In one embodiment, the pharmaceutically acceptable vehicle may be solid, and the composition may be in the form of a powder or tablet. The solid pharmaceutically acceptable vehicle may be a flavoring agent, a lubricant, The composition may contain one or more substances that can also act as solubilizers, suspending agents, pigments, bulking agents, flow enhancers, compression aids, inert binders, sweeteners, preservatives, dyes, coating agents, or tablet disintegrants. The vehicle may also be an encapsulating material. In the case of a powder, the vehicle is a finely ground solid mixed with the finely ground activator according to the present invention. In the case of a tablet, the activator can be mixed with a vehicle having the required compressibility properties in an appropriate ratio and compressed to the desired shape and size. The powder and tablet preferably contain up to 99% of the activator. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low-melting-point waxes, and ion-exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel, and the composition may be in the form of a cream.

[0102] However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition may be in the form of a solution. Liquid vehicles are used in the preparation of solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The activators according to the present invention may be dissolved or suspended in water, organic solvents, mixtures of both, or pharmaceutically acceptable liquid vehicles such as pharmaceutically acceptable oils or fats. Liquid vehicles may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity modifiers, stabilizers, or osmotic pressure modifiers. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing the above additives, e.g., cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (monohydric and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the vehicle may also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid compositions for parenteral administration. Liquid vehicles for pressurized compositions may be halogenated hydrocarbons or other pharmaceutically acceptable propellants.

[0103] Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be administered, for example, by intramuscular, intrathecal, epidural, intraperitoneal, intravenous, and especially subcutaneous injection. The drug can also be prepared as a sterile solid composition that can be dissolved or suspended at the time of administration using sterile water, saline solution, or other suitable sterile injection medium.

[0104] The agents and compositions of the present invention can be administered orally in the form of sterile solutions or suspensions containing other solutes or suspensions (e.g., saline or glucose sufficient to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monooleate, polysorbate 80 (its anhydrous copolymerized with oleic acid ester and ethylene oxide of sorbitol), etc. The agents used according to the present invention can also be administered orally in the form of liquid or solid compositions. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, as well as liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0105] The present invention will be understood to extend to any nucleic acid or peptide, or its variants, derivatives, or analogs, substantially comprising an amino acid sequence or nucleic acid sequence of any sequence referred herein, including its variants or fragments. The terms “substantially amino acid / nucleotide / peptide sequence,” “variant,” and “fragment” may be sequences having at least 40% sequence identity with any one of the amino acid / nucleotide / peptide sequences referred herein, for example, sequences identified as SEQ ID NOs. 1 to 28, etc.

[0106] More than 65%, more preferably more than 70%, of any of the referenced sequences, and More preferably, amino acid / polynucleotide / polypeptide sequences having more than 75% sequence identity, and even more preferably more than 80%, are also conceivable. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, and most preferably at least 99% identity with any of the sequences referred to herein.

[0107] Those skilled in the art will understand how to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences. To calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences, it is necessary to first prepare the alignment of the two sequences and then calculate the sequence identity value. The percentage identity of two sequences can take different values ​​depending on the following conditions: (i) the method used for sequence alignment, e.g., ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used in the alignment method, e.g., local alignment versus global alignment, the pair score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and the gap penalty, e.g., functional and constant.

[0108] When creating an alignment, there are various ways to calculate the percentage identity between two sequences. For example, the number of identities can be divided by: (i) the length of the shortest sequence; (ii) the length of the alignment; (iii) the average length of the sequences; (iv) the number of non-gap positions; or (v) the number of equivalent positions excluding overhangs. Furthermore, it will be understood that the percentage identity also depends strongly on length. Therefore, the shorter the pair of sequences, the higher the sequence identity that is expected to occur by chance.

[0109] Therefore, it will be understood that the precise alignment of protein or DNA sequences is a complex process. The general multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, pp. 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, pp. 4876-4882) is a preferred method for generating multiple alignments of protein or DNA according to the present invention. Suitable parameters for ClustalW are as follows: For DNA alignment: gap open penalty = 15.0, gap expansion penalty = 6.66, and matrix = identity. For protein alignment: gap open penalty = 10.0, gap expansion penalty = 0.2, and matrix = gonnet. For DNA and protein alignment: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will notice that it may be necessary to change these and other parameters for optimal sequence alignment.

[0110] Preferably, the percentage identity between two amino acid / polynucleotide / polypeptide sequences can be calculated from an alignment such as (N / T) × 100, where N is the number of positions in which the sequences share identical residues, and T is the total number of positions compared, either including gaps and including or excluding overhangs. Preferably, overhangs are included in the calculation. Therefore, the most preferred method for calculating the percentage identity between two sequences includes the steps of (i) preparing a sequence alignment using the ClustalW program with a suitable set of parameters, for example, as described above; and (ii) inserting the values ​​of N and T into the formula: Sequence Identity = (N / T) * 100.

[0111] Alternative methods for identifying similar sequences are known to those skilled in the art. For example, substantially similar nucleotide sequences are encoded by sequences that hybridize to a DNA sequence or its complement under stringent conditions. The inventors mean that stringent conditions involve hybridizing nucleotides to filter-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least one wash with 0.2x SSC / 0.1% SDS at about 20–65°C. Alternatively, substantially similar polypeptides may differ from sequences, for example, those shown in SEQ ID NOs. 1–28, which are amino acid sequences, by at least one, but fewer than 5, 10, 20, 50, or 100 amino acids.

[0112] Due to the degeneracy of the genetic code, it is evident that any nucleic acid sequence described herein can be altered or modified without substantially affecting the sequence of the protein it encodes, thereby providing functional variants thereof. Suitable nucleotide variants are those that have a sequence modified by the substitution of a different codon encoding the same amino acid in the sequence, resulting in a silent (synonymous) change. Other suitable variants have a homologous nucleotide sequence, but consist of all or part of the sequence, and are modified by the substitution of a different codon encoding an amino acid having a side chain with similar biophysical properties to the amino acid being substituted, resulting in a conservative change. For example, small nonpolar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Therefore, it is understood which amino acids can be replaced with those possessing similar biophysical properties, and those skilled in the art will know the nucleotide sequences encoding these amino acids.

[0113] All features described in this specification (including the appended claims, abstract, and drawings), and / or all steps of any method or process thus disclosed, may be combined in any combination with any of the above aspects, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0114] For a better understanding of the present invention, and to show how similar embodiments may be carried out, reference is made, by way of example, to the accompanying drawings:

Brief Description of the Drawings

[0115] [Figure 1] It is a diagram showing that Foxp3-dependent Tbx21 excision results in a decrease in the number of type 1 Treg cells. (a) Percentage of intestinal TRM or splenic CD8+ T cells stained for T-bet by flow cytometry analysis (n = 4 - 7). Ex vivo flow cytometry analysis of T cell populations in Foxp3WT and Foxp3ΔTbx21 mice in the indicated organs. (b, f) Percentage of Treg cells (CD4+Foxp3+) in the spleen, mesenteric lymph nodes (mLN), or lamina propria (LPL) expressing any of CXCR3, CCR6, or ST2 in (b) Foxp3WT or (f) Foxp3ΔTbx21 mice (n = 4 - 12). (c, d) Percentage of CXCR3 expressed in CD4+, CD8α+, and Treg cells in (c) the spleen or (d) the thymus (n = 5). (e) Representative flow cytometry plot of CXCR3 expression in splenic CD4+ T cells. (g - j) Percentage of Treg cells expressing (g) CD44, (h) Helios, (i) Nrpl1, or (j) KLRG1 in Foxp3WT (white circles) and Foxp3ΔTbx21 mice (black triangles) (n = 4 - 9). Bars represent the mean and error bars represent ±SEM. For statistical analysis, the Mann - Whitney U test, or multiple t-tests (g - j) were used. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. [Figure 2] This figure shows that Foxp3-dependent Tbx21 excision results in changes in the CD8 T cell population. (a, b) Flow cytometry analysis of the spleen CD8+ T cell population. (a) Percentages of naive (CD62LhiCD44lo), central memory (CD62LhiCD44hi), and effector memory (CD62L-CD44hi) CD8+ T cells in the spleen of Foxp3WT mice (white bars) n=8, Foxp3ΔTbx21 mice (black bars) n=4, and Foxp3ΔEomes mice (gray bars) (n=4). (b) Representative flow cytometry plots of CD62L and CD44 expression in spleen CD8+ T cells of specified mouse strains. (ce) Flow cytometry analysis of T cell populations, CD4+, Foxp3+, and CD8α+ T cells in the intestinal compartment; (c) Intraepithelial lymphocytes (IELs) of Foxp3WT (white symbols) or Foxp3ΔTbx21 (black symbols) mice; (d) Number of shown subpopulations of IELs in the same mice described in (c); and (e) Lamina propria (LPL) (n=8-9). (f, g) Ratios of CD4+Foxp3- and CD8+ T cells in shown organs of Foxp3WT and (f)Foxp3ΔTbx21 or (g)Foxp3ΔEomes (n=4-12). (h) Representative dot plots showing CD103 and CD69 expression of CD8+ T cells in IELs (upper panel) or LPL (lower panel) of shown mouse strains. (i) Cell counts of total CD8+ and CD8+CD103+ T cells in the jejunal LPL of Foxp3WT (white bars), Foxp3ΔTbx21 (black bars), and Foxp3ΔEomes (gray bars) mouse strains (n=5-11). (j) Ratio of total CD8+ T cells to CD8+CD103+ T cells observed in the LPL of the indicated mouse strains (n=6-13). Bars represent the mean, and error bars represent ±SEM. Multiple t-tests were used for statistical analysis. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3]This figure shows the reduced growth of TRM cells in the absence of type 1 Treg cells. (ad) Mucosal lamina propria lymphocytes were isolated from indicated small intestinal sections of Foxp3WT mice (white bars), Foxp3ΔTbx21 mice (black bars), and Foxp3ΔEomes mice (gray bars) and analyzed by flow cytometry. (a, b) Cells were gated with TCRβ+CD8α+ and KLRG1 expression was analyzed (n=4-8). (c, d) Representative flow cytometry plots showing (c) CD103 and KLRG1 expression, or (d) Eomes and KLRG1 expression, in the TCRβ+CD4-CD8α+ LPL of the indicated mouse strains. This figure shows representative plots (e) and cumulative data (fh) (n=7~11) showing the percentage of TCRβ+CD4-CD8α+ cells expressing KLRG1 in the liver and lungs of the indicated mouse strain (f), (g) the percentage of TRM cells in the liver (CD69+Eomes-KLRG1-) and (h) the percentage of TRM cells in the lungs (CD69+CD103+KLRG1-). Bars represent the mean, and error bars represent ±SEM. Mann-Whitney U tests or multiple t-tests (fh) were used for statistical analysis. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 4]This figure shows that decreased TRM cell growth leads to increased susceptibility to infection. (ab)TCRβ+CD8α+ mucosal lamina propria lymphocytes, Eomeshi, or Eomeslo from Foxp3ΔTbx21 mice were stimulated with 25 μg of anti-CD3 i.p. and then analyzed by flow cytometry for (a)PD-1 and (b)GrzmB expression 48 hours later (n=8-9). (ch)Foxp3WT (white symbols), (c, e, g)Foxp3ΔTbx21 (black symbols), or (d, f, h)Foxp3ΔEomes (gray symbols) mice were orally infected with 1000 E. vermiformis oocysts. (c, d)Cumulative number of oocysts collected from the feces of individual mice from day 5 to day 18. (e, f)Number of oocysts excreted per day per individual mouse. (g, h) Weight changes during the course of E. vermiformis infection (n=3-4 per experiment, 2 biological replicates). Bars represent the mean, and error bars represent ±SEM. Multiple t-tests or Wilcoxon tests (a, b) were used for statistical analysis. *P<0.05;**P<0.01;***P<0.001. [Figure 5]This figure shows that the recruitment of type 1 Treg cells determines the differentiation of TRM cells. (ab) Rag2-deficient mice were reconstituted in the bone marrow of Ctrl(CD45.1) or Foxp3ΔTbx21(CD45.2) mice. Contributions from each donor were evaluated for total CD8 T cells in the spleen and TRM(CD8+CD103+KLRG1-)LPL population; (a) representative dot plot, (b) summary of individual mice evaluated (n=8). (c) Description of the adoptive transfer model; Foxp3WT or Foxp3ΔTbx21 mice were intravenously administered C57Bl / 6 CD45.1+CD8α+ T cells one day prior to oral infection with E. vermiformis. After recovery from infection, 3-4 weeks post-inoculation, lamina propria lymphocytes (LPLs) were isolated from the small intestine and analyzed by flow cytometry. (d)TRM cell growth as the percentage of total CD8 T cells in LPL of CD45.1 host mice that were transfused with spleen Foxp3ΔTbx21-derived CD8 T cells as described in (c) and subsequently challenged with E. vermiformis (n=6). (e) Representative dot plots of lymphocytes pre-gated with CD45.1, followed by gating with TCRβ and CD8α, and analyzed for CD103 and Eomes expression in the indicated mouse strains. (fg) Cell counts of total CD8α+LPL and CD103+Eomes-CD8α+LPL obtained from the small intestine at (b) 3 weeks or (c) 9 weeks post-infection. Gating was performed as described in (a) (n=5-7). (h) Percentage of CD8α+CD45.1+ T cells that express CD103 after translocation of wild-type CD45.1+CD8α+ T cells and E. vermiformis challenge in the presence or absence of wild-type Treg cells in Foxp3ΔTbx21 mice. (i) Single-cell RNA sequencing analysis of Treg cell subtypes organized by type 1 (85 cells), type 2 (35 cells), type 3 (28 cells) or other undefined Treg cells. Bars represent the mean, and error bars represent ±SEM. Mann-Whitney U test was used for statistical analysis. **P<0.01;****P<0.0001, ns = not significant. [Figure 6]This figure shows that type 1 Treg cells promote TRM cell growth via TGFβ availability. (a, b) Foxp3WT or Foxp3ΔTbx21 mice were intravenously administered C57Bl / 6 CD45.1+CD8α+ T cells one day prior to oral infection with Yersinia pseudotuberculosis. Two to three weeks later, lamina propria lymphocytes (LPLs) were isolated from the small intestine and analyzed by flow cytometry. (a) Percentage of CD8α+CD45.1+ T cells recovered expressing CD103 (n=7-9). (b) Representative dot plots of lymphocytes pregated with CD45.1, followed by gating with TCRβ and CD8α, and analyzed for CD103 and Eomes expression in the indicated mouse strains. (cd) Ileal LPL Foxp3WT (white symbols) or Foxp3ΔTbx21 (black symbols) mice were analyzed for (c) the number of Tregs and (d) the number of Tregs expressing CXCR3 (n=4-9) in the steady state (circles) or 10 days after E. vermiformis (Ev) infection (squares). (e) C57BL / 6 mice were infected with or not infected with E. vermiformis; Cxcl10 mRNA levels exceeding HPrt were assessed in the ileum on day 10 (biological replicates of 2594, n=5-8). (fk) One day prior to oral infection with E. vermiformis, C57BL / 6 CD45.1+CD8α+ T cells were administered intravenously to Foxp3ΔTbx21 mice. Three weeks post-infection, LPL was isolated from the small intestine and CD103 expression was analyzed by flow cytometry. In addition to CD8CD45.1 cells, wild-type Treg cells or Treg cells deficient in (f)CXCR3, (h)IL-10, (i)IL-35, (j)integrin β8, or (k)TGFβ1 were co-transplanted. Mice administered with wild-type TREG cells were accumulated and used in the panel (f, hk). (g) One day prior to oral infection with E. vermiformis, the indicated mouse strain was intravenously administered CD45.1+CD8α+T cells. Ten days after infection, the small intestine was stained with CD45.1 (green), Dapi (blue), and Foxp3 (red). Representative immunohistochemical images (40x objective, 1.5x zoom) are shown starting from the region containing oocysts (n=4).White arrows indicate proximity between CD8 T cells and TREGs. Bars represent the mean, and error bars represent ±SEM. The Mann-Whitney U test was used for statistical analysis. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 7] This figure shows that Foxp3-dependent conditional deletion of Tbx21 results in a decrease in the number of type 1 Treg cells. Ex vivo flow cytometry analysis of cells; a) Representative plot showing Tbet and Eomes expression by intracellular staining of CD8 cells in a C57BL / 6 spleen or intestinal TRM population, b) Representative plot of CD4 T cells stained for Foxp3 and Tbet. ce) Number of Treg, CD4, and CD8 T cells in the thymus and spleen of Foxp3WT (white bars) and Foxp3ΔTbx21 (black bars) mice n=9. fh) Number of Treg, CD4 T cells, and CD8 T cells in Foxp3WT (white bars) and Foxp3ΔEomes (gray bars) mice; ij) Percentage of CXCR3 expression in CD4CD8α and Treg cells in i) spleen or j) thymus. Representative flow cytometry plots of CXCR3 expression in splenic CD4 T cells in flow cytometry analysis of the percentage of Treg cells expressing CXCR3, CCR6, or ST2 in the spleen, mesenteric lymph node mLN, and lamina propria mucosa (n≧4, white symbols Foxp3ΔWT, black symbols Foxp3ΔTbx2, error bars represent ±SEM; Mann-Whitney U test or multiple t-test hm for statistical analysis) were used. P<0.05, P<0.01, P<0.001, P<0.0001. [Figure 8]This figure shows that Tbet or Eomes deficiency in Treg cells is associated with changes in the T cell phenotype of the small intestine. Mucosal lamina propria lymphocytes (LPLs) were isolated from Foxp3WT, Foxp3ΔTbx21, and Foxp3ΔEomes mice and analyzed by flow cytometry. The cell counts in the LPL fractions of (a) duodenum (n=8-11), (b) jejunum (n=10-14), (c) ileum (n=9-13), and (d) colon (n=4-7) were determined by gating TCRβ+ cells followed by gating CD4+CD8α_YFP-(CD4+), CD4+CD8α_YFP+(Treg), and CD4-CD8α+ T cells (CD8+). g) Ratio of CD4+Foxp3-T cells to CD4+YFP+T cells in individual animals from ef)Foxp3WT (white circles) and e)Foxp3ΔTbx21 (black triangles) or (f)Foxp3ΔEomes (black diamonds) (n=8). g) Representative flow cytometry dot plots showing CD103 and CD69 staining of CD4+YFP- (upper panel) or CD8+ (lower panel) LPL cells of the indicated mouse strains. h) Total number of CD4+T cells and CD103+CD4+T cells in the lamina propria of Foxp3WT (white bars), Foxp3ΔTbx21 (black bars), and Foxp3ΔEomes (gray bars) mouse strains (n=5~11). i) Ratio of Foxp3-CD4+T cells / CD8α+CD103+T cells observed in LPL of the indicated mouse strains (n=6~13). Error bars represent ±SEM. The Mann-Whitney U test was used for statistical analysis. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 9]This figure shows that the absence of type 1 Treg leads to a decrease in TRM cells. Mucosal lamina propria lymphocytes were isolated from Foxp3WT, Foxp3ΔEomes, and Foxp3ΔTbx21 mice and examined by flow cytometry. a) Representative flow cytometry dot plots showing CD103 vs Eomes and KLRG1 vs CD69 in the indicated mouse strains. b) Representative flow cytometry dot plots showing KLRG1 vs Bcl2 protein expression. c) Representative flow cytometry dot plots showing CD103 vs TL tetramer (CD8α staining) (n=4). d) Representative flow cytometry dot plots and summary graphs showing in vivo staining of CD8α in blood and LPL compartments (n=5) *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 10] This figure shows that decreased TRM cell growth increases susceptibility to infection. Mucosal lamina propria lymphocytes (LPL) or splenic CD8 T cells were isolated from Foxp3WT and Foxp3ΔTbx21 mice 48 hours after intraperitoneal injection of anti-CD3ε antibody and examined by flow cytometry. ac)(a)Representative flow cytometry dot plot showing granzyme B vs. Eomes protein expression in PD1 or (b)LPL (n=4)b)Similar to a), but showing cumulative data from the spleen (n=6). (df) C57BL / 6 Rag2- / - mice were orally infected with 1000 E. vermiformis oocysts. (g) Daily fecal oocyst count per individual mouse for the indicated number of days, e, h) Cumulative oocyst count (days 6-17), f, i) Change in body weight during E. vermiformis infection (n=5-9, 2 biological replicates). Error bars represent ±SEM. For statistical analysis, Mann-Whitney U test (e, h), multiple t-tests (d, f, g, f), or Wilcoxon test (b) were used. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 11]Figure (ad) shows Tbet expression and TRM cell growth during immune activation. The T cells shown were evaluated by intracellular flow cytometry staining under steady state or 24 hours after in vivo anti-CD3 stimulation. a) Overview of Tbet expression in Foxp3WT (white symbols) and Foxp3ΔTbx21 (black symbols) spleen CD4 and CD8 T cells. b) Analysis of Tbet expression by intracellular staining of spleen CD8 naive T cells and memory T cells, as well as LPL-supplied TRM cells (n=11 12). Representative plots of spleen CD8 T cells from Foxp3WT and Foxp3ΔEomes mice stained for (c)Eomes or (d)Tbet 24 hours after activation (n=3 5). eh) CD8CD45.1 T cells were adopted into Foxp3WT (white symbols) and Foxp3ΔTbx21 (black symbols) mice, and the mice were challenged with E. vermiformis 1 day later. At the peak of infection, LPL cells were isolated on day 10 and CD45.1 was analyzed by flow cytometry for (e) the percentage of TRM cells (CD8+CD103+Eomes-) and (f) total CD8+CD45.1+ T cells (n=6-7). (g) Based on e), CD45.2+Foxp3ΔTbx21 mice were used as hosts and additional CD45.1+WtTreg cells were transfused, and representative flow cytometry plots of CD4+ T cells in LPL, endogenous (CD45.2+) and transfused (CD45.1+) CD4 and CXCR3-expressing Treg cells are shown. h) Summary of CXCR3 percentage of transfused Treg cells based on f and g). Mann-Whitney U tests were used for statistical analysis. [Figure 12]This figure shows the growth of TRM cells in selected mouse strains. a, b) CD4Foxp3 cells were isolated and counted from the lamina propria of the ileum of Foxp3WT and Foxp3ΔTbx21 mice in steady state (circles) or 10 days after E. vermiformis infection (squares) (n=5-8). c) The shown mouse strains were intravenously administered CD45.1+CD8α+ T cells 1 day prior to oral infection with Ev. 10 days post-infection, the small intestine was stained with CD45.1 (green), Dapi (blue), and Foxp3 (red). Representative immunohistochemical images (20x objective lens, 15x zoom, scale bar 50 μm), with the shown area magnified. Oocysts are characteristic unstained circular areas, such as those indicated by *. Areas without oocysts are shown for comparison (third panel). f) Relative expression levels of spleen CXCR3 or CXCR3+ Treg cells (n=4). LPLs were isolated from d, e) C57BL6 (control) or IL10-deficient mice, g, j) or C57BL6 mice or g, h) bone marrow chimeric mice generated from IL-35-deficient mice, g, i) Foxp3ΔItgβ8 mice, and g, j Foxp3Δtgfβ1 mice. A representative flow cytometry plot is shown (f), showing the cumulative percentage of CD103-expressing cells (hj) (n=7-10). Bars represent the mean. Statistical analysis was performed using the Mann-Whitney test, with ***P<0.001 and ****P<0.0001. [Figure 13] This figure shows the gating strategies for flow cytometry analysis. a) Representative flow cytometry plot from the spleen of C57BL / 6 Foxp3ΔTbx21 mice showing lymphocyte gating, doublet exclusion, and dead cell exclusion, followed by CD4 selection and Treg selection based on eYFP and tdRFP detection. b) Representative flow cytometry plot obtained from the LPL of C57BL / 6 Foxp3ΔTbx21 mice showing lymphocyte gating, doublet exclusion, and dead cell exclusion, followed by the inclusion of TCRβ+CD69+ and CD8α+. In the LPL, TRM is defined as CD103+. [Figure 14]This figure shows RFP detection in Foxp3ΔTbx21 mice. ac) Representative flow cytometry plots from the spleen of C57BL / 6 Foxp3ΔTbx21 mice; as shown in Figure 13a, eYFP and tdRFP detection in a) CD4 lifegate, b) CD4Foxp3YFP population, and c) CD8 population. Two representative plots from two individual mice are shown. [Figure 15] This diagram shows the experimental layout detailing the production and testing of TRM cells, from initial cell extraction from mice to selection of cell populations via a FACS machine. [Figure 16] This figure shows that bone marrow-derived dendritic cells (BMDCs) maintain CD69 expression in CD8 T cells. When effector CD8 T cells are cultured with anti-CD3 in the presence of IL-15 and TGFβ alone (circles) or BMDCs (squares), the CD69 marker, which is importantly expressed in TRM cells, is maintained in the presence of BMDCs but lost in the absence of BMDCs. [Figure 17] This figure shows that IL-7 induces CTLA-4 expression in TRMs. Two independent dual experiments show the difference in CTLA-4 expression when effector CD8 T cells were cultured in the absence of IL-7 (black) and with the addition of IL-7 (gray). [Figure 18] This figure shows that IL-2 also induces CTLA-4 expression in TRM. The data show CTLA4 expression when effector CD8 T cells were cultured in the absence of IL-7 and IL-2 (black), with IL-7 (light gray), and with IL-2 (dark gray). When cultured with IL-7 or IL-2, CD8 T cells express CTLA-4 at similar rates, but the expression level of CTLA-4 is, on average, more robust in cultures with IL-7 than in cultures with IL-2. [Figure 19] This figure shows the experimental layout detailing the production and testing of TRM cells, as well as in vivo challenge and ex vivo analysis performed on mouse organs after challenge. The inventors wanted to confirm whether the characteristics and phenotype of TRM cells produced in vitro correlated with in vivo seeding to organs. [Figure 20] This figure shows the expression profiles of the markers CD69, CD103, and CTLA-4 for various T cell cultures used in the in vivo challenge. The T cells used in the challenge were cultured under the following conditions or groups: Group 1: CD8+BMDC+aCD3+TGFb+IL-15=TRM; Group 2: CD8+BMDC+aCD3+TGFb+IL-15+IL-7=TRM+IL-7; Group 3: CD8+BMDC+aCD3+IL-15=Effector; and Group 4: CD8+BMDC+aCD3+TGFb+IL-15+IL-2=TRM+IL-2. [Figure 21] This figure shows the number of CD8+ T cells derived from the spleen of mice challenged over 40 days. Effector CD8 T cells were cultured under the conditions shown in Figure 20 and transferred to a complete C57BL6 / J host. The presence of transferred cells (CD45.1) in the spleen was assessed at indicated time points. The graph shows two pooled experiments. Experiment 1 (black symbols) did not include group 4, and analysis was performed only at time points 11-14. Experiment 2 (gray symbols) included group 4, and analysis was performed at several time points, showing only 2 of 6 mice from which cells were harvested at a later time point. The data show that a significant number of IL-7 cultured TRM cells (group 2) were found compared to TRM cells cultured without IL-7. [Figure 22] This figure shows the number of CD8+ T cells derived from the lamina propria of the intestinal mucosa of mice challenged over 40 days. Effector CD8 T cells were cultured under the conditions shown in Figure 20 and transferred to a complete C57BL6 / J host. At indicated time points, the presence of transferred cells (CD45.1) in the lamina propria of the intestinal mucosa was assessed. The graph shows two pooled experiments. Experiment 1 (black symbols) did not include group 4, and analysis was performed only at time points 11-14. Experiment 2 (gray symbols) included group 4, and analysis was performed at several time points, showing only 2 of 6 mice from which cells were harvested at later time points. The data show that a significant number of IL-7 cultured TRM cells (group 2) were found compared to TRM cells cultured without IL-7. [Figure 23] This figure shows the number of CD8+ T cells derived from the IEL compartments of the lungs and intestines of mice challenged over 32 days. Effector CD8 T cells were cultured under the conditions shown in Figure 20 and transferred into a complete C57BL6 / J host. At indicated time points, the presence of transferred cells (CD45.1) in the IEL compartments of the lungs and intestines was assessed. The graph shows experiments in both organs (Experiment 1 - black symbols, Experiment 2 - gray symbols) representing three groups, and the analysis was performed at several time points. The data show that IL-7 cultured TRM cells (Group 2) were found in significantly larger numbers compared to TRM cells cultured without IL-7. [Figure 24] This figure shows the comparative number of CD8+ T cells derived from the lamina propria of the intestinal mucosa and the spleen of mice challenged over a 40-day period. Effector CD8 T cells were cultured under the conditions shown in Figure 20 and transferred to a complete C57BL6 / J host. At indicated time, the presence of transferred cells (CD45.1) was assessed in the lamina propria of the spleen and intestinal mucosa. The data show that a significant number of IL-7 cultured TRM cells (Group 2) were found compared to TRM cells cultured without IL-7. [Modes for carrying out the invention]

[0116] [Examples]

[0117] The inventors of the present invention, T REG T cells, which can penetrate deep into tissues, are highly effective against solid tumors. RM It was hypothesized that this is important in cell generation. The inventors of the present invention, T RM Determine the factors necessary for cell generation and T in vitro RMThe ultimate goal is to enable cell generation, adapt current culture protocols to generate antitumor T cells, provide them with tissue penetration properties to target both primary tumors and metastases that have migrated to tissues distant from the primary tumor, and ultimately provide vital whole-organ immune surveillance. The inventors also described in detail in Figure 15 how to generate T cells in culture medium. RM Cell generation is T REG The aim was to evaluate whether this was possible in the absence of cells. Furthermore, the inventors evaluated whether the cells produced in vitro maintained their therapeutic properties, particularly their ability to migrate and survive within tissues in vivo, as detailed in Figure 19.

[0118] material and method mouse The C57Bl / 6J and C57Bl / 6J CD45.1 mouse were purchased from Charles River in France. Tbx21 f / f (Tbx21 tm2Srnr ) and Eomes fl / fl (Eomes tm1Srnr ) was provided courtesy of Dr. Reiner 14、82 Foxp3 eYFP-Cre (Foxp3 tm4(YFP / icre)Ayr ) is Dr. Rudensky 83 Provided by Rosa26-tdRFP, Dr. Fehling 84 Rag2 - / - , IL15R - / - Provided courtesy of Jackson Labs. Mice were reared at the Instituto de Medicina Molecular in Lisbon, Portugal. Age- and sex-matched male and female mice aged 8–18 weeks were used. Animals were housed in IVC cages with free access to drinking water and food under temperature-controlled conditions with a 12-hour light / dark cycle. All mice were kept free of specific pathogens. Foxp3 eYFP-Cre All mice of the Rosa26-tdRFP strain have been genetically modified by PCR. Mice were identified and those with detected knockout alleles were discarded (~20%). The presence of appropriate Tbx21 was confirmed by blood typing of CD4 T cells expressing CXCR3. Furthermore, mice were counter-screened for inappropriate expression of RFP associated with eYFP (~10%) (Figures 13-14). Bone marrow chimeras were generated by intravenous injection of the resulting bone marrow cells following sublethal irradiation (450 rads) of Rag2-deficient mice. CXCR3 - / - (Cxcr3 tm1Dgen ) 85 It was raised at the German Cancer Research Center (DKFZ) in Heidelberg, Germany; IL-10 - / -86 It is raised at the Instituto Gulbenkian de Ciencia, Lisbon, and Ebi3 - / -87 The animals were reared at the Institute for Immunology, University Medical Center Mainz, Germany, while Itgb8f / f88 and Tgfb1f / f89, which were crossed with Foxp3yfp-Cre, were reared at the Immunology, Virology and Inflammation Department, Cancer Research Center of Lyon, France. All animal experiments complied with the regulations of the Direcao-Geral de Alimentacao e Veterinaria Portugal and local ethics review boards and guidelines.

[0119] Cell Isolation: Intestinal cells were isolated as described above. The intestines were washed with PBS to remove contents and opened longitudinally. After cutting into 1 cm sections, the IELs were incubated in PBS containing 20 mM Hepes, 100 U / ml penicillin, 100 μg / ml streptomycin, 1 mM pyruvate, 10% FCS, 100 μg / ml polymyxin B, and 10 mM EDTA for 30 minutes at 37°C with shaking to release the IELs. The IEL single-cell suspension was further purified using 37.5% isotonic Percoll. To isolate LPLs, the intestinal tissue was then digested in IMDM medium containing 0.5 mg / ml collagenase D (Roche) and 0.2 mg / ml DNase I (Roche) at 37°C for 25 minutes with shaking. Hepatic lymphocytes were separated by grinding the organ through a 70 μm filter, and the cells were subsequently purified with 37.5% isotonic Percoll. The lungs were shredded with scissors and digested in PBS containing 1 mg / ml collagenase D at 37°C for 30 minutes. A cell suspension containing lymphocytes was obtained after passing through a 50 μm cell strainer.

[0120] Adoptive cell transfer: CD8α + T cells and / or CD25 + Cells (Treg) were purified from single-cell suspensions of the spleen and lymph nodes. Briefly, cells were labeled with anti-CD8a-APC or anti-CD25-APC antibody and selected using anti-APC MACS microbeads according to the instructions. After counting, purity was determined by flow cytometry and the cell number was adjusted. To ensure broad TCR diversity in the transferred population, a minimum of 2 x 10⁶ cells was used. 6 CD8 T cells were used. Some recipient mice received 0.4–1x10⁶ cells. 6 An additional number of Treg cells were administered. Infection was induced one day after cell transfer (day 0).

[0121] Infection Challenge: Infecting animals with Eimeria vermiformis (Ev), as previously reported. 91Infection was induced. Briefly, oocysts were washed three times with deionized water, floated on sodium hypochlorite, and counted using a Fuchs-Rosenthal chamber. 500 oocysts of E. vermiformis were administered to mice by forced oral administration in 100 μl of water and analyzed after the infection was resolved (from week 3 p.i). To determine the infection load, animals were placed in individual cages and feces were collected daily until no oocysts were detected. The animals were as described above. 58 The animals were infected with Yersinia pseudotuberculosis (Yptb), which was kindly provided by Dr. T. Bergsbaken. The animals were administered by forced oral administration in 100 μl of water. 6 The patient was infected with Yptb. Tissue analysis was performed on days 15-19.

[0122] Flow cytometry: Agreed standards 92 Using the gating strategy shown accordingly Single-cell suspensions were prepared from the spleen, lymph nodes, intestines, lungs, and liver and stained with antibodies (see list) (Figure 13). In vivo staining was performed by intravenous injection of 3 μg of CD8a-APC antibody, with mice sacrificed 5 minutes later. TL-tetramers were provided courtesy of the NIH Tetramer Core Facility. Samples were run on a Fortessa X20 cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar).

[0123] Quantitative RT-PCR: RNA was isolated using the Qiagen RNeasy Mini kit, and cDNA was generated using the Applied Biosystems High Capacity RNA-to-cDNA kit. Amplification was performed using SYBR Select Master Mix (Applied Biosystems) and QuantiTect Primer Assays Mm_Cxcl10_1_SG, Mm_Tgfb1_1_SG, Mm_Itgb8_1_SG, and Mm_Hprt_1_SG (Qiagen).

[0124] Immunohistochemistry and microscopic observation: Intestinal tissues were rolled into "Swiss rolls", fixed with 10% formalin, rehydrated with 30% glucose, and frozen in OCT medium. The tissues were sectioned at 10 μm, and the sections were treated with 4% paraformaldehyde. Blocking was performed using 10% BSA, and the following antibodies were used for detection: CD45.1 (A20, Biolegend) and FOXP3 (FJK-16s, eBioscience). The slides were mounted with Fluoromount (Invitrogen) and imaged using a Zeiss LSM880 microscope. Analysis was performed using Fiji software.

[0125] scRNA-Seq analysis: The original data was 55 created and analyzed. From the first dataset, T REG cells were selected based on Foxp3, excluding Tmems, stressed cells, and low-quality cells. To analyze this subset, the R package Seurat 93 was used, following a similar approach to 55 . The "LogNormalize" method was used, and data normalization was performed using a magnification of 10 5 ; data scaling was performed using UMI based on a negative binomial model. The subtypes of T REG were defined using the following criteria: Type 1 (cells with raw counts assigned to the Tbx21, Stat1, and Cxcr3 genes); Type 2 (cells with raw counts assigned to the Gata3, Stat6, and Il1rl1 genes); Type 3 (cells with raw counts assigned to the Rorc, Stat3, and Ccr6 genes); Others (cells without raw counts assigned to the Tbx21, Gata3, and Rorc genes).

[0126] In vitro culture: Effector CD8 T cells were obtained from C57BL6 / J or CD45.1 C57BL6 / J mice that had been intraperitoneally injected with 25 μg of anti-CD3ε in advance. Cells were separated by AutoMACS bead selection and cultured at 200,000 cells per flat-bottom 96-well plate in IMDM medium. 100,000 BMDCs cultured via a standard protocol using GM-CSF were added under the indicated conditions. Cells were restimulated with 0.25 μg / ml of anti-CD3ε, 10 ng / ml of IL-15, and 0.5 ng / ml of TGFβ, and with 10 - 20 ng / ml of IL-2 or IL-7 when indicated. To test tissue homing, cells were expanded for 3 days prior to analysis or adoptive transfer into naive C57BL6 / J mice. T RM Cells were evaluated for the markers CD69, CD103, absence of KLRG-1, and CTLA-4 expression.

[0127] In vitro-differentiated cells were transferred into mice by intravenous injection. At the indicated time points, animals were sacrificed and lymphocytes (both IEL and LPL fractions) from the spleen, lung, and small intestine were isolated according to standard methods. Cell populations were analyzed by flow cytometry. Transferred cells were distinguished from endogenous cells by expression of the congenic marker CD45.1, and cell counts were performed using flow cytometry counting beads.

[0128] Example 1 Results FoxP3 + Deletion of Tbx21 in the cells results in a decrease in type 1 T REG cells.

[0129] T RM cells express T-bet but not Eomes (Figure 1a, Figure 7a) 37 . T REG cells express lineage-related chemokine receptors in various tissues with immune type 1, 2, and 3 characteristics (Figure 1b). T-bet or Eomes-expressing T REG cells, T RMTo test whether it affects cells, the inventors of Foxp3 eYFP-Cre Tbx21 fl / fl Rosa26 tdRFP / tdRFP and Foxp3 eYFP-Cre Eomes fl / fl Rosa26 tdRFP / tdRFP Mouse strains (each Foxp3) ΔTbx21 and Foxp3 ΔEomes (called) and the control Foxp3 eYFP-Cre Rosa26 tdRFP / tdRFP Strain (Foxp3 WT (Method, Figure 14) was used. T-bet and Eomes were compared using T-bet. 38 It activates the transcription of genes important for type 1 immune responses, such as the chemokine receptor CXCR3, which is transactivated by T. REG Consistent with enhanced type 1 inflammation in the absence of T-bet in cells. 35、39 (Figure 7b) The spleen, not the thymus, is shown in Foxp3. ΔTbx21 In mice, CXCR3 + CD4 + and CXCR3 + CD8 + A proportional increase in T cells was observed (Figure 1c-d). CD4 in the thymus or spleen. + Foxp3 - or T REG (CD4 + Foxp3 + The number of cells was similar (Figure 7c-h), but the CD8 in the spleen + T cells are Foxp3 ΔTbx21 The animal population showed an increasing trend (Figure 7e). 29、35、39 No signs of autoimmunity were observed in mice up to 3 months of age.

[0130] The inventors have developed Foxp3-specific targeting, CD4 + CXCR3 + Foxp3 + Instead, CD4 + CXCR3 + Foxp3 - T cells, Foxp3 ΔTbx21 It was confirmed to be present in mice (Figure 1c-e), but Foxp3 ΔEomesCXCR3 in the peripheral lymphoid organs of mice + T REG An increase in the proportion and number of cells was observed (Figure 7i-k). REG Excision of T-bet in cells is associated with type 3 T REG As the number of cells increases, REG This resulted in a change in the distribution, not the number of subsets (Figure 1f, Figure 7l-n). LPL's T REG The cell population exhibited a more activated phenotype and expressed higher levels of CD44 compared to those present in secondary lymphoid organs (SLOs) (Figure 1g). Neuropilin-1 (Nrpl-1) and the transcription factor Helios were primarily used in SLO T REG It was present in cells, but decreased in the intestines. REG The cells are Foxp3 WT Compared to the control mouse, Foxp3 ΔTbx21 The same phenotype was observed in (Figure 1h-i). 29 Effector T cells and T EM The co-inhibitory receptor killer cell lectin-like receptor G1 (KLRG1) expressed in cells 40、41 Foxp3 WT Compared to the control mouse, Foxp3 ΔTbx21 T from REG It increases in cells (Figure 1j). In summary, these data suggest that T-bet expression T REG In the absence of cells, T REG The number of cells and their phenotypes remain similar, REG This indicates that the proportion of the subset is changing.

[0131] T REG Excision of Tbx21 or Eomes in cells alters the distribution of CD8 T cells. Foxp3 ΔTbx21 Mouse spleen CD8 + The T cell compartment is an effector (T eff ) / T EM This shows an increase in T cells (Figure 2a-b). However, Foxp3 ΔTbx21 The intraepithelial fraction of mouse intestines contains Foxp3 WTCompared to the control, a decrease in CD4 and CD8 T cells is observed (Figure 2c). Within the CD8IEL population, induced CD8αβ + Foxp3 ΔTbx21 A significant decrease is observed in mice, but natural CD8αα + Not observed within the IEL population (Figure 2d). Foxp3 ΔTbx21 The lamina propria (LP) compartment of the mouse mucosa is Foxp3 WT Compared to the control, CD4 + It shows a decrease in T cells, CD8 + No decrease in T cells was observed (Figure 2e). This difference was evident in all intestinal sections except the colon (Figure 8a-d), and CXCR3 and T REG T-bet expression in cells is random. 42 .

[0132] CD4 in the LPL compartment + Foxp3 - T cells and T REG Despite changes in cell number and regardless of Foxp3-dependent excision of Tbx21 or Eomes, CD4 + T cells and T REG The cell proportions remain stable (Figure 8e-f). However, Foxp3 ΔTbx21 The animal T cell population shows a significant proportional bias toward CD8 T cells (Figure 2f), but Foxp3 ΔEomes In animals, the opposite is observed, especially in the proximal intestine (Figure 2g). These data are T REG If T-bet or Eomes are not present in the cells, CD4 + Foxp3 + and CD4 + Foxp3 - The proportional distribution among T cells does not change, but CD8 in the small intestine + This indicates a significant impact on the proportion of T cell subsets.

[0133] Tbx21 + and Eomes + T REG The cells influence the CD8 T cell memory compartment. T-bet is sufficientREG T cells in the intestine in the absence of cells RM Cellular reduction and circulatory effector / T EM The increase in the percentage of cells is T RM This suggests a potential role for these cells in cell generation or maintenance. Despite the decrease in IEL count (Figure 2c-d), all CD8 + IEL is T RM The cell markers CD103 and CD69 are expressed (Figure 2h). Foxp3 ΔTbx21 Foxp3 ΔEomes and Foxp3 WT The mouse LPL compartment expresses high levels of CD69, and about half of it co-expresses CD44 with CD103. + Foxp3 - Similarities were observed with respect to T cells (Figure 8g). The inventors of this invention developed CD4T RM Although no differences in cellular phenotypes were observed, Foxp3 ΔTbx21 Animals, CD4 + Foxp3 - T cells and CD4 + CD103 + A general trend was observed in the decrease in cell number (Figure 8h).

[0134] Foxp3 WT and Foxp3 ΔEomes In the animal LPL compartment, most CD8 T cells are T RM It expresses markers CD69 and CD103 (Figure 2h-i). In contrast, Foxp3 ΔTbx21 In animals, more than half of CD8+ T cells do not express CD103 (Figure 2h-i). Therefore, even if the total number of CD8+ T cells is similar, T-bet expression T REG In the absence of cells, CD8+T RM The number of cells decreases in the intestines of these animals, RM The proportion of effectors contributing to cells is high (Figure 2i-j), and in all three mouse strains, CD4T cells and CD8T cells were present. RM The cell ratio remained constant (Figure 8i). These data suggest that the gut immune network is fine-tuned, and Foxp3 ΔTbx21The increase in the CD8+ T cell ratio observed in animals is T RM This suggests that the problem is caused by the accumulation of CD8+ effector T cells that do not develop into proper cells.

[0135] Tbx21 + T REG Cells are T in multiple tissues RM It affects cell growth. In the event of a skin infection, KLRG1 + CD103 - CD8 + These effector T cells have been reported in the early dermis, not in the later stages or the epidermis. 19 . Foxp3 in a steady state ΔTbx21 CD103 observed in the small intestine of animals - CD8 + Consistent with the T cell population, KLRG1, representing approximately 20% of the total CD8 T cell population, was found in all sections of the small intestine. + CD8 + A significant population of T cells was observed (Figure 3a). In contrast, increased CXCR3 + T REG Foxp3 that harbors cells ΔEomes The animal (Figure 7h) has KLRG1 in its proximal intestine. + CD8 + Decreased number of T cells This was shown (Figure 3b).

[0136] Co-staining with CD103 revealed that the KLRG1 protein is expressed in a mutually exclusive manner with CD103. 19、26、43 , mainly Foxp3 ΔTbx21 It was confirmed to be present in mice (Figure 3c). Foxp3 ΔTbx21 CD103 present in animals - KLRG1 + CD8 T cells expressed high levels of Eomes, but Foxp3 WT It is rarely seen in Foxp3 ΔEomes The incidence was even lower in animals (Figure 3d, Figure 9a). This corresponds to the effector state of Foxp3 ΔTbx21 KLRG1 in animals + Eomes +CD8 T cells have low expression levels of the survival-promoting protein Bcl-2, RM Upregulated during cell maturation 44、45 (Figure 9b). Furthermore, it is expressed together with the CD8αβ heterodimer, and epithelial memory CD8 T cells 46 The proportion of cells expressing the characteristic CD8αα homodimer is Foxp3 WT Compared to the control, Foxp3 ΔTbx21 The number of cells decreased in animals (Figure 9c). Finally, in vivo staining confirmed that the majority of cells isolated from the LPL compartment were not recently circulating (Figure 9d, e). In summary, while we do not wish to be bound to any particular theory, these data suggest that T REG If T-bet is not expressed in the cell, CD8 + Effector T cells accumulate in the intestinal barrier, RM This indicates that it does not progress towards the cells.

[0137] T RM The presence of cells has been reported in many tissues. 4 The results were consistent with those in the gut, and Foxp3 ΔTbx21 The liver and lungs of mice are Foxp3 ΔEomes and Foxp3 WT Compared to animals, the proportion of effector CD8 T cells expressing high levels of KLRG1 and Eomes was increased (Figure 3e-f). CD103 expression was associated with T cells in the liver. RM Since CD8 is not considered a sufficient marker for cells, the inventors have identified CD8 that is negative for KLRG1 and Eomes. + CD69 + The proportion of cells was evaluated. Foxp3 ΔTbx21 The mouse is Foxp3 ΔEomes and Foxp3 ΔWT Compared to mice, less T in the evaluated non-lymphatic tissues. RM The cells contained (Figure 3g-h). While we do not wish to be bound by any particular theory, these data suggest that type 1 T REG Cells in multiple tissues RM This suggests that it is important in cell generation.

[0138] T RM When cellular compartments are damaged, protection against pathogen invasion is reduced. The inventors of the present invention, T RM Because bystander-mediated activation of cells is an important defense mechanism that limits the entry of pathogens, Foxp3 ΔTbx21 T in mice RM We hypothesized that a decrease in cell count could reduce protection against new infections. 47、48、49 Two days later, the acute response of intestinal CD8+ T cells was tested by administering an anti-CD3 antibody that evaluated the LPL T cell response. Eomes + CD8 T cells are Eomes - Compared to CD8 T cells, the activity profile was reduced, PD-1 expression was increased, and granzyme B was decreased (Figures 4a, b, 10a-c). 50 .

[0139] Next, the inventors challenged mice with Eimeria vermiformis (Ev), an intracellular protozoan parasite that infects the epithelial cells of the mouse small intestine. In this infection model, lymphocytes reduced the burden on the parasite (Figure 10d-f), and CD8+ T cells and IFNγ played important roles in clearance. 10、51、52 Type 1 T cells are expected to lead to enhanced T-cell immunity. REG Although cells do not exist 53 In fact, Foxp3 ΔTbx21 The mouse is Foxp3 WT and Foxp3 ΔEomes Compared to animals, the suppression of Ev infection was impaired (Figure 4c-f). This is in contrast to mice lacking lymphocytes (Figure 10d-e), T RM IL-15Rα-deficient mice, which are necessary for cell survival. 26 Similarly, Foxp3 has a higher concentration of effector cells but exhibits an exhausted phenotype. ΔTbx21 The mouse is Foxp3 WT Foxp3 ΔEomes , and Rag2 - / - Compared to other animals, their body weight decreased (Figure 4g-h, Figure 10f). These data are from TRM Cells of the liver apicomplex parasite malaria parasite 49 and skin viruses 26 To provide protection against It is consistent with being there.

[0140] 1 type T REG Cells are T RM To promote growth. Foxp3 ΔTbx21 Or Foxp3 ΔEomes T cells from mice are analyzed in steady state, activated state, or T state. RM During cell establishment, regarding the expression of Tbet or Eomes, Foxp3 WT Indistinguishable from the control (Figure 11a-d). Foxp3 ΔTbx21 Accumulation of effector T cells and T cells in animal tissues RM Cell reduction (Foxp3 WT To assess whether (compared to) is specific to CD8 T cells, we used CD45.1 controls and Foxp3 ΔTbx21 A mixed bone marrow chimera with a mouse was generated. RM The cells showed similar contributions from both donors (Figure 5a-b). Furthermore, Foxp3 ΔTbx21 CD8 T cells supplied from mice (CD45.2) were transferred to control CD45.1 animals. After 1 day, the mice were challenged with Ev, and the challenge was resolved after 2 weeks. 54 CD8 CD45.1 CD103 + T RM Cell growth occurs one week after parasite elimination, T eff The cell count was evaluated when it decreased (Figure 5c). The transferred CD45.2 + Within the group, T RM Cells were generated with high efficiency (Figure 5d). In summary, these results indicate that T RM Foxp3 inhibits cell regeneration. ΔTbx21 This suggests an exogenous defect in mouse CD8 T cells. To confirm this, CD45.1 + CD8 + T cells (CD8 CD45.1 ) to CD45.2 + Foxp3 ΔTbx21Or Foxp3 WT It was introduced into animals. At the peak of infection (day 10), T RM Cells and effector T cells are present in the LPL (Figure 11e, f). Foxp3 WT In the host, the introduced CD8 CD45.1 Most cells exhibit characteristic T103 expression with low Eomes levels. RM The cell profile is shown (Figure 5e). In contrast, Foxp3 ΔTbx21 CD8 transferred to the host CD45.1 T cells express Eomes and lack CD103; the majority of cells exhibit an effector phenotype; partial T RM Cell formation was observed (Figure 5e). Transplanted CD8 CD45.1 The cumulative number of T cells is Foxp3 WT Foxp3 compared to the host ΔTbx21 They are similar in host, and in the former, T RM The number of cells was reduced (Figure 5f). Analysis of mice 9 weeks after Ev infection showed that Foxp3 ΔTbx21 In the entire animal population, detectable imported CD8 CD45.1 The number of T cells was shown to have decreased (Figure 5g). These data suggest that Foxp3 ΔTbx21 CD8 observed in animals + T RM Disorders in cell differentiation, CD8 + It is confirmed to be exogenous for the T cell population. The inventor's transfer system allows T REG The cell, CD8 CD45.1 T cells and T REG WT Cellular Foxp3 ΔTbx21 Through simultaneous introduction of animals RM It became possible to test the hypothesis that it promotes cell growth (Figure 11g). The hypothesis was supported by Foxp3. ΔTbx21 T in animals RM Cell generation is controlled by T REG In the presence of cells, Foxp3 WT The levels recovered to those observed in the control group (Figure 5h).

[0141] T-bet-expressing T REG Cells, TRM To understand whether it has certain functional attributes that can explain its role in growth, the inventors have recently published a single T REG Using a set of cell sequencing data 55 Previous report 42、56 In line with this, although a small trend may exist, the inventors define T by the presence of characteristic lineage transcription factors Tbx21, Gata3, or Rorc. REG Across cellular subsets, TGFβ, CD25, LAG3, or CTLA-4 are present. REG No significant differences in cellular effector molecules were found (Figure 5i). Furthermore, T-bet-deficient T REG The cells were controlled by T REG It has been reported that it exhibits inhibitory activity similar to that of cells. 35、57 .

[0142] T RM The growth of T REG By relying on the mobilization of these factors, TGFβ is made locally bioavailable. The inventors' observations relied on the presence of microorganisms under specific pathogen-free conditions and the intracellular intestinal parasite Ev to induce a highly localized response. We have identified our CD8 CD45.1 Using a T cell transfer system (Figure 5c) to treat bacterial Yersinia pseudotuberculosis (Y Challenge the mouse in ptb, T RM They reported that it induces cells. 58、59 Consistent with the results obtained using Ev, the Yptb challenge is Foxp3 ΔTbx21 Foxp3 has decreased significantly in animals. WT Efficient T in animals RM This resulted in cell growth (Figure 6a, b).

[0143] CD8 CD45.1 T cell Foxp3 ΔTbx21 Using a mouse transfer model, the inventors found that T RM To promote cell growth, T REG We were able to investigate the contribution made by cells. In the absence of T-bet, T REGThe cells had similar levels of CD103, CCR6, and P-selectin. 35 It expresses CXCR3, which is important for the localization of T cells to infected areas in non-lymphoid tissues, but it cannot express CXCR3. 7、35、39、60 The local inflammatory environment is T RM Progenitor cells 58、61 , and T REG Controls cell recruitment (Figure 6c). Importantly, T cells recruited during Ev infection. REG The cells exhibit a type 1 phenotype, primarily expressing CXCR3 (Figure 6d, Figure 11g-h). Foxp3, in which CXCR3 expression is not T-bet dependent. ΔTbx21 Effectors and T in the colon of mice RM Considering the lack of change in the cells, the inventors of the present invention, T REG T-bet expression in a subpopulation of cells promotes the recruitment of these cells to the infection site, and T RM We hypothesized that it would be placed in close proximity to progenitor cells. Upon confirmation of this, Foxp3-dependent excision of Tbx21 revealed that Tbx21 cells were present during Ev infection. REG This resulted in decreased cell recruitment (Figure 6c), which was mainly due to cells expressing CXCR3 (Figure 6d). Type 1 T REG Consistent with cell recruitment, the inventors found that the expression of the CXCR3 ligand, CXCL10, increased in intestinal tissue during Ev infection (Figure 6e). The number of CD4 T cells was increased by Foxp3 ΔTbx21 The LPL compartment in mice decreases in a steady state (Figure 1e), T RM It may play an additional role in cell generation. However, Ev infection resulted in robust recruitment of CD4 T cells into the LPL compartment with a dominant T helper 1 phenotype (Figure 12a, b). Using the transfer system (Figure 5a), CD8 CD45.1 When cells are transferred simultaneously, CXCR3-deficient T REG Cells with sufficient CXCR3 compared to controls, T RM It failed to support efficient cell growth. (Figure 6f)

[0144] CD8 + and CD4 +T cell aggregates, though not accompanied by B cells, are commonly observed in areas of microbial invasion, often alongside other immune cells such as macrophages and dendritic cells. 58、62、63、64、65 CD4 + and CD8 + Interactions between T cells are not necessary for the maintenance of T cells. 59 , T RM It may constitute different microenvironments that could support differentiation. Accordingly, the inventors of Foxp3 WT Mouse Foxp3 expression T REG Implanted CD8 in close proximity to the cell CD45.1 T cells were frequently observed, despite similar CD8 T cell infiltration, Foxp3 ΔTbx21 This phenomenon was not easily observed in animals (see Figures 6g and 12c).

[0145] T RM The requirements for CXCR3 to promote growth are T REG This suggests that they provide short-range action or cell-binding effector molecules. Type 1 cytokines like IL-12 can maintain high levels of T-bet and Eomes, therefore T RM It inhibits cell differentiation and CD103 expression. IL12Rβ2-deficient CD8+ T cells have an increased proportion of cells expressing CD103, and T cell clusters have high TGFβ transcript levels. 59 IL-10 may reduce IL-12 expression and dendritic cell maturation. 66 However, IL-10 deficiency T REG Cells are T RM It can help cells grow efficiently (Figure 6h), and IL-10-deficient animals are T RM No reduction in cellular compartments was observed (Figure 12d, e). Furthermore, EBI3-deficient T cells, which are unable to produce IL-35, were also found to be inactive. REG Cells are T RM It similarly promotes cell generation (Figure 6i), and in EBI3-deficient bone marrow chimeric animals, T RM Cellular compartments did not decrease (Figure 12g, h).

[0146] Many cells, especially those in the mucosal barrier, can produce TGFβ. 67 However, TGFβ is produced as an inactive precursor and requires cleavage from latent-related peptides. -β possesses potent cellular regulatory activity and acts on numerous immune and non-immune cell types; therefore, its availability is strictly regulated in the local microenvironment. Recently, T REG Cells can activate TGFβ via integrin αvβ8, and this protein is activated / effector T REG It has been shown that it is upregulated in cells, thereby reducing locally bioactive TGFβ. 68 The present inventors have identified a CXCR3-expressing T2 that does not exhibit differential expression of TGFβ1 or Itgb8 under steady state conditions. REG Specific recruitment of effector CD8 T cells to specific sites (Figure 5f, Figure 12f) 42 However, it was assumed that this would enable the local release of TGFβ. Foxp3 ΔTbx21 Similar to the mouse, Foxp3 ΔItgβ8 Bone marrow chimeras generated from mice are T RM This showed a decrease in the number of cells and an increase in KLRG1-expressing effector CD8+ T cells (Figure 12g, i). Importantly, T REG If only cells lack Itgβ8, RM There was an inefficient promotion of cell generation (Figure 6j). REG To understand whether the derived TGFβ plays a deterministic role, the inventors of Foxp3 ΔTgfβ1 I used a mouse. Foxp3 ΔItgβ8 Matching the mouse, T REG In the absence of TGFβ1 from cells, there is an increase in effector T cells and T RM This only resulted in a decrease in the proportion of cells (Figure 12g, j). In adoptive transfer, the supply of TGFβ1 is insufficient. REG The cells are Foxp3 ΔTbx21 Mouse T RM We were unable to rescue the cells from growth (Figure 6k). In summary, we do not want to be bound by any particular theory, but the data suggests that T REGCells are recruited via T-bet-inducible expression of CXCR3, generating TGFβ1, which enables local bioavailability via αvβ8 integrin expression, and T in inflammatory tissues. RM This indicates that it promotes cell growth.

[0147] Consideration Induction of long-term cellular immunity in non-lymphoid tissues is crucial for preventing reinfection and is a primary objective of vaccine design. Our data show that CD8+ T cells, acting as effector cells or memory cell precursors, hom into tissues and then, upon receiving a local cue, T cells... RM Supports a model of cell differentiation. 69 T cell activation in SLO induces the expression of a wide variety of tissue-homing receptors that guide activated T cells to non-lymphoid tissues, enabling effector T cells to examine most peripheral tissues. 69、70 . T RM The unique cellular profile is due to factors in the tissue microenvironment affecting the T cells of effector cells. RM This suggests that it directs the differentiation of cells.

[0148] The inventors' data are based on local infection models and polyclonal TCR repertoires. Inflammation-mediated transport and alloantigens in the local microenvironment are optimal for T RM Local infection models have shown that it is necessary for cell growth but not for maintenance. 61、71、72 This is in contrast to observations using systemic viral infections where the IEL count remains stable. 69 The inventor's observations suggest that previously reported small intestinal infections 58 This is consistent with the findings, suggesting that this is a characteristic of local inflammation. T cells from recruited effector T cells or memory progenitor T cells. RM Cell differentiation may require local cues such as encounters with cytokines or secondary antigens. The inventor's data supports this model, and T REGThis model is extended by the need to recruit cells to the site of inflammation and their ability to increase the levels of bioactive TGFβ, which promotes the growth from effector to memory. REG When cells are depleted, the T cells in the central nervous system... RM The number of cells decreases due to viral infection. 73 , T RM T in cell growth or maintenance REG The role of cells was suggested. The inventors of this invention have identified type 1 T REG Local cell recruitment is important, and in this process, Itgβ8 expression is crucial. RM This observation is extended by demonstrating that the growth of [TGFβ] is highly dependent on locally supplied TGFβ and its bioavailability. 23、26、59 Local antigens can retain CD8+ T cells in the tissue, forming stable contact with infected cells, and in this process, CD69 is reexpressed, which requires CXCR3 expression. 74 In non-inflammatory tissues, the inventors found that CD4 and T REG Although a decrease in cells has been reported, CD4 T cells are recruited during inflammation. REG Cell replenishment is T RM This is sufficient to improve cell growth, but this is C This does not preclude the supportive role of D4 T cells. The inventors have identified a potential role for human type 1 T cells. REG CXCR3 expression, which also identifies cells 75 , T REG This demonstrates that cells are required to mobilize to inflammatory sites, providing rationale for the need for the upstream transcription factor T-bet. In the absence of CXCR3, T REG Cell and tissue recruitment is limited, leading to improvements in type 1 immunology and immunopathology. 42、76 . T REG The absence of cells can enhance the immune response and even lead to sterile immunity, but subsequent loss of immunity has been reported. 53 The inventors here described the Type 1 T REG Cells provide TGFβ, and by expressing αvβ8 integrin, it becomes locally available, RM This study demonstrates that it promotes cell growth, supports lifelong immune surveillance, and enhances tissue protection against invading microorganisms.

[0149] The inventors of this invention have developed a T-bet-expressing T REG In several tissues evaluated in the absence of cells, T RM A decrease in differentiation was observed. This is because the specific tissue microenvironment is T RM Cell growth or type 1 T REG It emphasizes that it does not play a significant role in cell recruitment. Nevertheless, certain differences in tissues are T RM This may alter the growth or the amplitude of their phenotypes. RM While cells are known to primarily produce IFNγ in the gut, it has been shown that they can produce IL-17 in the epidermis after microbial challenge. Furthermore, additional tissue damage can lead to epidermal T RM It can alter cellular function and contribute to wound healing. 77、78 The colon is type 1 T REG In the absence of cells, the inventors of T RM Alternatively, it was a notable exception in which no changes were found in effector cells, nor were there any changes in the proportion of T cell subsets. REG Cells express CXCR3 and require T-bet to be recruited to the site of inflammation, but T-bet does not appear to regulate CXCR3 expression in the colon. 42 The isolation of T-bet and CXCR3 in the colon suggests alternative immunomodulation in the organ with the highest microbial content.

[0150] TGFβ strongly promotes CD103 expression in CD8+ T cells in vitro and in vivo. 22 It has been shown to reduce KLRG1 expression. 43 Furthermore, T RM The importance of reducing inflammation for cell growth is suggested by the decrease in CD103 expression during chronic infections. 23、79 Furthermore, TGFβRII-deficient CD8 T cells are T RM Unable to become a cell, or T RM Do not remain in cells 19、58 CD103 + T RM In addition to cells, CD103- T RM Cell populations have been reported. 22、58、62 The stability of this population may depend on the tissue type and the persistence of the antigen. In the inventor's model in control mice, the steady state under specific pathogen-free conditions, as well as the state after Ev challenge, showed that CD103 - T cells were a minority group. Instead, Foxp3 ΔTbx21 CD103 observed in mice - The T cells expressed CD103 and KLRG1, which is characteristic of transitional T cells that switch off Eomes, as well as high levels of Eomes. 80 While we do not wish to be bound by any particular theory, the inventors' data is T RM Type 1 T cells in cell growth REG While revealing the important roles of cells, smaller T RM The continued potential for cell populations suggests that other cells may make further contributions to TGFβ release. RM Alternative sources of bioactive TGFβ, including stromal epithelial cells crucial for cell maintenance, have been reported. 81 .

[0151] T REG T cells are thought to be important in weakening excessive immune responses and thereby preventing autoimmunity and immunopathology, and in reducing the amplitude of infection and vaccination responses as measured in the blood. However, our data highlight their crucial role in efficiently generating otherwise depleted tissue-resident memory T cells from effectors or memory precursors. REG The cells ensure that a critical number of T cells are available for immune surveillance within the tissue, preventing or mitigating reinfection and reducing the pathogen burden from new infections.

[0152] Example 2 result As detailed in Figure 15, the inventors have added T to the culture medium. REG In the absence of cells, T RMWe evaluated whether cell generation was possible.

[0153] Referring to Figure 16, the addition of TGFβ to a culture containing IL-15, antigen-presenting cells (BMDCs), and previously activated CD8 T cells results in additional T REG In the absence of cells, continuous expression of CD69 and CD103, etc. RM It has been shown to be sufficient to establish functionality.

[0154] Referring to Figure 17, the combination of IL-15, TGFβ, antigen-presenting cells, and previously activated CD8 T cells with IL-7 added resulted in the generation of T cells based on their CTLA-4 expression profiles. RM It has been shown that it can enhance the migratory ability of cells. (Front. Immunol., November 27, 2018; Brunner-Weinzierl and Rudd; Kieke et al., PLOS One, 27 / 5 / 09) Referring to Figure 18, the combination of IL-15, TGFβ, antigen-presenting cells, and previously activated CD8 T cells with IL-2 added resulted in T cells generated based on their CTLA-4 expression profiles. RM While it has been shown that it can enhance the migratory capacity of cells, this effect is not as consistent as that of IL-7 addition in terms of the amount per cell or biological repeats.

[0155] Furthermore, the inventors evaluated whether the cells produced in vitro retained their therapeutic properties, particularly their ability to migrate and survive in vivo within tissues, as detailed in Figure 19.

[0156] The inventors replicated in vivo conditions in an in vitro setup consisting of effector CD8+ T cells and bone marrow-derived dendritic cells (BMDCs). The T cells were stimulated in vitro and could be proliferated in a manner similar to that used to produce large quantities of cells for T cell therapy. The inventors found that the addition of bioactive TGFβ resulted in T RM T cells in the growth of T cells, which are similar to other cells. REGThis demonstrates that the cell's role can be replaced, and the expression of markers and tissue-retaining factors CD69 and CD103 can be continued (Figure 20). CD69 is normally an activation marker that is transiently expressed during T cell activation. CD69 is a type C lectin and is used in non-lymphoid tissues, including solid tumors. RM Most likely involved in cell retention 98 CD69 forms a complex with sphingosine-1-phosphate (S1P)1, thereby inhibiting its binding to the S1P receptor, which triggers the release of T cells outside the tissue. Furthermore, data show that the addition of IL-7 and IL-2 induces potent CTLA-4 expression (Figures 17 and 18), which is associated with enhanced T cell migration. 99、100、101 CTLA-4 expression appears to be enhanced with the addition of IL-2, but replication experiments showed that the addition of IL-7 was more consistent than that of IL-2. Furthermore, the addition of IL-7 was shown to enhance CTLA-4 expression in individual cells.

[0157] Data obtained from transferring T cells generated and proliferated in vitro into the tissue in vivo show that effector cells generated in the absence of TGFβ are not found in substantial numbers in the tissue (Figures 21, 22, 23, and 24). RM The cells, especially when stimulated with IL-7, were readily detectable 40 days after transplantation in all the organs tested, including the lungs, liver, lamina propria, and small intestinal intraepithelial compartment (Figures 21, 22, 23, and 24).

[0158] summary In summary, the inventors have developed a method for deeply penetrating tumors (and especially solid tumors) to treat tumors or T cells (T) that can contribute to the stepwise evolution of T cell therapy for infectious diseases RMWe have developed a highly novel and innovative protocol for generating T cells (known as T cells). Our protocol results in cells generated and proliferated in vitro with migratory and tissue-penetrating cell phenotypes based on the expression of CTLA-4, CD69, and CD103. Matching their phenotypes and in contrast to effector T cells, the generated cells are readily found in various lymphoid and non-lymphoid tissues at least 40 days after adoptive transfer to a complete mouse host. Our research will represent a significant advance for the effective treatment of organ infections and for cancer patients suffering from solid tumors that are far more difficult to treat. However, we believe that T RM We believe that the ability of cells to penetrate tissues can provide crucial organ-wide immune surveillance not only for targeting infections or primary tumors, but also against metastases that have migrated to tissues far from the primary tumor, which are often difficult to access.

[0159] References 1. Stary, G. et al. VACCINES. A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells. Science 348, aaa8205 (2015). 2. Masopust, D., Vezys, V., Marzo, AL & Lefrancois, L. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291, 2413-2417. (2001). 3. Reinhardt, RL, Khoruts, A., Merica, R., Zell, T. & Jenkins, MK Visualizing the generation of memory CD4 T cells in the whole body. Nature 410, 101-105. (2001). 4. Mueller, S.N. & Mackay, L.K. Tissue-resident memory T cells: local specialists in immune defence. Nature reviews. Immunology 16, 79-89 (2016). 5. Konjar, S., Ferreira, C., Blankenhaus, B. & Veldhoen, M. Intestinal Barrier Interactions with Specialized CD8 T Cells. Front Immunol 8, 1281 (2017). 6. Ariotti, S. et al. Tissue-resident memory CD8+ T cells continuously patrol skin epithelia to quickly recognize local antigen. Proceedings of the National Academy of Sciences of the United States of America 109, 19739-19744 (2012). 7. Schenkel, J.M., Fraser, K.A., Vezys, V. & Masopust, D. Sensing and alarm function of resident memory CD8(+) T cells. Nature immunology 14, 509-513 (2013). 8. Slutter, B., Pewe, L.L., Kaech, S.M. & Harty, J.T. Lung airway-surveilling CXCR3(hi) memory CD8(+) T cells are critical for protection against influenza A virus. Immunity 39, 939-948 (2013). 9. Wu, T. et al. Lung-resident memory CD8 T cells (TRM) are indispensable for optimal cross-protection against pulmonary virus infection. J Leukoc Biol 95, 215-224 (2014). 10. Konjar, S. et al. Mitochondria maintain controlled activation state of epithelial-resident T lymphocytes. Sci Immunol 3, eaan2543 (2018). 11. Intlekofer, A.M. et al. Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. Nature immunology 6, 1236-1244 (2005). 12. Pearce, E.L. et al. Control of effector CD8+ T cell function by the transcription factor Eomesodermin. Science 302, 1041-1043 (2003). 13. Banerjee, A. et al. Cutting edge: The transcription factor eomesodermin enables CD8+ T cells to compete for the memory cell niche. Journal of immunology 185, 4988-4992 (2010). 14. Intlekofer, A.M. et al. Requirement for T-bet in the aberrant differentiation of unhelped memory CD8+ T cells. The Journal of experimental medicine 204, 2015-2021 (2007). 15. Joshi, N.S. et al. Inflammation directs memory precursor and short-lived effector CD8(+) T cell fates via the graded expression of T-bet transcription facto r. Immunity 27, 281-295 (2007). 16. Pipkin, M.E. et al. Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells. Immunity 32, 79-90 (2010). 17. Joshi, N.S. et al. Increased numbers of preexisting memory CD8 T cells and decreased T-bet expression can restrain terminal differentiation of secondary effector and memory CD8 T cells. Journal of immunology 187, 4068-4076 (2011). 18. Kaech, S.M. & Ahmed, R. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells. Nature immunology 2, 415-422. (2001). 19. Mackay, L.K. et al. The developmental pathway for CD103(+)CD8+ tissue-resident memory T cells of skin. Nature immunology 14, 1294-1301 (2013). 20. Wakim, L.M. et al. The molecular signature of tissue resident memory CD8 T cells isolated from the brain. Journal of immunology 189, 3462-3471 (2012). 21. Nakayamada, S. et al. Early Th1 cell differentiation is marked by a Tfh cell-like transition. Immunity 35, 919-931 (2011). 22. Casey, K.A. et al. Antigen-independent differentiation and maintenance of effector-like resident memory T cells in tissues. Journal of immunology 188, 4866-4875 (2012). 23. Zhang, N. & Bevan, M.J. Transforming growth factor-beta signaling controls the formation and maintenance of gut-resident memory T cells by regulating migration and retention. Immunity 39, 687-696 (2013). 24. Sheridan, B.S. et al. Oral infection drives a distinct population of intestinal resident memory CD8(+) T cells with enhanced protective function. Immunity 40, 747-757 (2014). 25. Laidlaw, B.J. et al. CD4+ T cell help guides formation of CD103+ lung-resident memory CD8+ T cells during influenza viral infection. Immunity 41, 633-645 (2014). 26. Mackay, L.K. et al. T-box Transcription Factors Combine with the Cytokines TGF-beta and IL-15 to Control Tissue-Resident Memory T Cell Fate. Immunity 43, 1101-1111 (2015). 27. Laidlaw, B.J., Craft, J.E. & Kaech, S.M. The multifaceted role of CD4(+) T cells in CD8(+) T cell memory. Nature reviews. Immunology 16, 102-111 (2016). 28. Feau, S., Arens, R., Togher, S. & Schoenberger, S.P. Autocrine IL-2 is required for secondary population expansion of CD8(+) memory T cells. Nature immunology 12, 908-913 (2011). 29. Yu, F., Sharma, S., Edwards, J., Feigenbaum, L. & Zhu, J. Dynamic expression of transcription factors T-bet and GATA-3 by regulatory T cells maintains immunotolerance. Nature immunology 16, 197-206 (2015). 30. McNally, A., Hill, G.R., Sparwasser, T., Thomas, R. & Steptoe, R.J. CD4+CD25+ regulatory T cells control CD8+ T-cell effector differentiation by modulating IL-2 homeostasis. Proceedings of the National Academy of Sciences of the United States of America 108, 7529-7534 (2011). 31. de Goer de Herve, M.G., Jaafoura, S., Vallee, M. & Taoufik, Y. FoxP3(+) regulatory CD4 T cells control the generation of functional CD8 memory. Nature communications 3, 986 (2012). 32. Pace, L. et al. Regulatory T cells increase the avidity of primary CD8+ T cell responses and promote memory. Science 338, 532-536 (2012). 33. Laidlaw, B.J. et al. Production of IL-10 by CD4(+) regulatory T cells during the resolution of infection promotes the maturation of memory CD8(+) T cells. N ature immunology 16, 871-879 (2015). 34. Kalia, V., Penny, L.A., Yuzefpolskiy, Y., Baumann, F.M. & Sarkar, S. Quiescence of Memory CD8(+) T Cells Is Mediated by Regulatory T Cells through Inhibitory Receptor CTLA-4. Immunity 42, 1116-1129 (2015). 35. Koch, M.A. et al. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nature immunology 10, 595-602 (2009). 36. Hall, A.O. et al. The cytokines interleukin 27 and interferon-gamma promote distinct Treg cell populations required to limit infection-induced pathology. Immunity 37, 511-523 (2012). 37. Reis, B.S., Hoytema van Konijnenburg, D.P., Grivennikov, S.I. & Mucida, D. Transcription factor T-bet regulates intraepithelial lymphocyte functional maturation. Immunity 41, 244-256 (2014). 38. Lord, G.M. et al. T-bet is required for optimal proinflammatory CD4+ T-cell trafficking. Blood 106, 3432-3439 (2005). 39. Levine, A.G. et al. Stability and function of regulatory T cells expressing the transcription factor T-bet. Nature 546, 421-425 (2017). 40. Obar, J.J. & Lefrancois, L. Memory CD8+ T cell differentiation. Ann N Y Acad Sci 1183, 251-266 (2010). 41. Omilusik, K.D. et al. Sustained Id2 regulation of E proteins is required for terminal differentiation of effector CD8(+) T cells. The Journal of experimental medicine 215, 773-783 (2018). 42. Tan, T.G., Mathis, D. & Benoist, C. Singular role for T-BET+CXCR3+ regulatory T cells in protection from autoimmune diabetes. Proceedings of the National Academy of Sciences of the United States of America 113, 14103-14108 (2016). 43. Schwartzkopff, S. et al. TGF-beta downregulates KLRG1 expression in mouse and human CD8(+) T cells. European journal of immunology 45, 2212-2217 (2015). 44. Janssen, E.M. et al. CD4+ T-cell help controls CD8+ T-cell memory via TRAIL-mediated activation-induced cell death. Nature 434, 88-93 (2005). 45. Oh, S. et al. IL-15 as a mediator of CD4+ help for CD8+ T cell longevity and avoidance of TRAIL-mediated apoptosis. Proceedings of the National Academy of Sciences of the United States of America 105, 5201-5206 (2008). 46. Madakamutil, L.T. et al. CD8alphaalpha-mediated survival and differentiation of CD8 memory T cell precursors. Science 304, 590-593 (2004). 47. Lertmemongkolchai, G., Cai, G., Hunter, C.A. & Bancroft, G.J. Bystander activation of CD8+ T cells contributes to the rapid production of IFN-gamma in response to bacterial pathogens. Journal of immunology 166, 1097-1105 (2001). 48. Chu, T. et al. Bystander-activated memory CD8 T cells control early pathogen load in an innate-like, NKG2D-dependent manner. Cell reports 3, 701-708 (2013).49. Fernandez-Ruiz, D. et al. Liver-Resident Memory CD8(+) T Cells Form a Front-Line Defense against Malaria Liver-Stage Infection. Immunity 45, 889-902 (2016).50. Herndler-Brandstetter, D. et al. KLRG1(+) Effector CD8(+) T Cells Lose KLRG1, Differentiate into All Memory T Cell Lineages, and Convey Enhanced Protective Immunity. Immunity 48, 716-729 e718 (2018). 51. Rose, M.E., Joysey, H.S., Hesketh, P., Grencis, R.K. & Wakelin, D. Mediation of immunity to Eimeria vermiformis in mice by L3T4+ T cells. Infection and immunity 56, 1760-1765 (1988). 52. Rose, M.E., Wakelin, D. & Hesketh, P. Gamma interferon controls Eimeria vermiformis primary infection in BALB / c mice. Infection and immunity 57, 1599-1603 (1989). 53. Belkaid, Y., Piccirillo, C.A., Mendez, S., Shevach, E.M. & Sacks, D.L. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420, 502-507 (2002). 54. Ramsburg, E., Tigelaar, R., Craft, J. & Hayday, A. Age-dependent requirement for gammadelta T cells in the primary but not secondary protective immune response against an intestinal parasite. The Journal of experimental medicine 198, 1403-1414 (2003). 55. Miragaia, R.J. et al. Single-Cell Transcriptomics of Regulatory T Cells Reveals Trajectories of Tissue Adaptation. Immunity 50, 493-504 e497 (2019). 56. Zemmour, D. et al. Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR. Nature immunology 19, 291-301 (2018). 57. Xiong, Y., Ahmad, S., Iwami, D., Brinkman, C.C. & Bromberg, J.S. T-bet Regulates Natural Regulatory T Cell Afferent Lymphatic Migration and Suppressive Function. Journal of immunology 196, 2526-2540 (2016). 58. Bergsbaken, T. & Bevan, M.J. Proinflammatory microenvironments within the intestine regulate the differentiation of tissue-resident CD8(+) T cells responding to infection. Nature immunology 16, 406-414 (2015). 59. Bergsbaken, T., Bevan, M.J. & Fink, P.J. Local Inflammatory Cues Regulate Differentiation and Persistence of CD8(+) Tissue-Resident Memory T Cells. Cell reports 19, 114-124 (2017). 60. Harris, T.H. et al. Generalized Levy walks and the role of chemokines in migration of effector CD8+ T cells. Nature 486, 545-548 (2012). 61. Khan, T.N., Mooster, J.L., Kilgore, A.M., Osborn, J.F. & Nolz, J.C. Local antigen in nonlymphoid tissue promotes resident memory CD8+ T cell formation during viral infection. The Journal of experimental medicine 213, 951-966 (2016). 62. Wakim, L.M., Woodward-Davis, A. & Bevan, M.J. Memory T cells persisting within the brain after local infection show functional adaptations to their tissue of residence. Proceedings of the National Academy of Sciences of the United States of America 107, 17872-17879 (2010). 63. Iijima, N. & Iwasaki, A. T cell memory. A local macrophage chemokine network sustains protective tissue-resident memory CD4 T cells. Science 346, 93-98 (2014). 64. Natsuaki, Y. et al. Perivascular leukocyte clusters are essential for efficient activation of effector T cells in the skin. Nature immunology 15, 1064-1069 (2014). 65. Collins, N. et al. Skin CD4(+) memory T cells exhibit combined cluster-mediated retention and equilibration with the circulation. Nature communications 7, 11514 (2016). 66. Corinti, S., Albanesi, C., la Sala, A., Pastore, S. & Girolomoni, G. Regulatory activity of autocrine IL-10 on dendritic cell functions. Journal of immunology 166, 4312-4318 (2001). 67. Li, M.O., Wan, Y.Y., Sanjabi, S., Robertson, A.K. & Flavell, R.A. Transforming growth factor-beta regulation of immune responses. Annual review of immunology 24, 99-146 (2006). 68. Worthington, J.J. et al. Integrin alphavbeta8-Mediated TGF-beta Activation by Effector Regulatory T Cells Is Essential for Suppression of T-Cell-Mediated Inflammation. Immunity 42, 903-915 (2015). 69. Masopust, D. et al. Dynamic T cell migration program provides resident memory within intestinal epithelium. The Journal of experimental medicine 207, 553-564 (2010). 70. Butcher, E.C. & Picker, L.J. Lymphocyte homing and homeostasis. Science 272, 60-66. (1996). 71. Gebhardt, T. et al. Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus. Nature immunology 10, 524-530 (2009). 72. Mackay, L.K. et al. Long-lived epithelial immunity by tissue-resident memory T (TRM) cells in the absence of persisting local antigen presentation. Proceedings of the National Academy of Sciences of the United States of America 109, 7037-7042 (2012). 73. Graham, J.B., Da Costa, A. & Lund, J.M. Regulatory T cells shape the resident memory T cell response to virus infection in the tissues. Journal of immunology 192, 683-690 (2014). 74. Hickman, H.D. et al. CXCR3 chemokine receptor enables local CD8(+) T cell migration for the destruction of virus-infected cells. Immunity 42, 524-537 (2015). 75. Duhen, T., Duhen, R., Lanzavecchia, A., Sallusto, F. & Campbell, D.J. Functionally distinct subsets of human FOXP3+ Treg cells that phenotypically mirror effector Th cells. Blood 119, 4430-4440 (2012). 76. Paust, H.J. et al. CXCR3+ Regulatory T Cells Control TH1 Responses in Crescentic GN. J Am Soc Nephrol 27, 1933-1942 (2016). 77. Harrison, O.J. et al. Commensal-specific T cell plasticity promotes rapid tissue adaptation to injury. Science 363 (2019). 78. Ariotti, S. & Veldhoen, M. Immunology: Skin T Cells Switch Identity to Protect and Heal. Curr Biol 29, R220-R223 (2019). 79. Beura, L.K. et al. Lymphocytic choriomeningitis virus persistence promotes effector-like memory differentiation and enhances mucosal T cell distribution. J Leukoc Biol 97, 217-225 (2015). 80. Mueller, S.N., Gebhardt, T., Carbone, F.R. & Heath, W.R. Memory T cell subsets, migration patterns, and tissue residence. Annual review of immunology 31, 137-161 (2013). 81. Mohammed, J. et al. Stromal cells control the epithelial residence of DCs and memory T cells by regulated activation of TGF-beta. Nature immunology 17, 414-421 (2016). 82. Intlekofer, A.M. et al. Anomalous type 17 response to viral infection by CD8+ T cells lacking T-bet and eomesodermin. Science 321, 408-411 (2008). 83. Rubtsov, Y.P. et al. Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces. Immunity 28, 546-558 (2008). 84. Luche, H., Weber, O., Nageswara Rao, T., Blum, C. & Fehling, H.J. Faithful activation of an extra-bright red fluorescent protein in "knock-in" Cre-reporter mice ideally suited for lineage tracing studies. European journal of immunology 37, 43-53 (2007). 85. Hancock, W.W. et al. Requirement of the chemokine receptor CXCR3 for acute allograft rejection. The Journal of experimental medicine 192, 1515-1520 (2000). 86. Kuhn, R., Lohler, J., Rennick, D., Rajewsky, K. & Muller, W. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75, 263-274 (1993). 87. Nieuwenhuis, E.E. et al. Disruption of T helper 2-immune responses in Epstein-Barr virus-induced gene 3-deficient mice. Proceedings of the National Academy of Sciences of the United States of America 99, 16951-16956 (2002). 88. Travis, M.A. et al. Loss of integrin alpha(v)beta8 on dendritic cells causes autoimmunity and colitis in mice. Nature 449, 361-365 (2007). 89. Azhar, M. et al. Generation of mice with a conditional allele for transforming growth factor beta 1 gene. Genesis 47, 423-431 (2009). 90. Li, Y. et al. Exogenous stimuli maintain intraepithelial lymphocytes via aryl hydrocarbon receptor activation. Cell 147, 629-640 (2011). 91. Figueiredo-Campos, P., Ferreira, C., Blankenhaus, B. & Veldhoen, M. Eimeria vermiformis Infection Model of Murine Small Intestine. Bio-Protocol 8 (2018). 92. Cossarizza, A. et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). Eur J Immunol 49, 1457-1973 (2019). 93. Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 36, 411-420 (2018). 94. Savas, P., Virassamy, B., Ye, C. et al. Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis. Nat Med 24, 986-993 (2018). 95. Zhi-Qiang Wang, Katy Milne, Heather Derocher, John R. Webb, Brad H. Nelson and Peter H. Watson. CD103 and Intratumoral Immune Response in Breast Cancer. Clin Cancer Res (22) (24) 6290-6297 (2018). 96. John R. Webb, Katy Milne, Peter Watson, Ronald J. deLeeuw and Brad H. Nelson. Tumor-Infiltrating Lymphocytes Expressing the Tissue Resident Memory Marker CD103 Are Associated with Increased Survival in High-Grade Serous Ovarian Cancer. Clin Cancer Res (20) (2) 434-444 (2014). 97. Jarem Edwards et al. CD103 + Tumor-Resident CD8 + T Cells Are Associated with Improved Survival in Immunotherapy-Naive Melanoma Patients and Expand Significantly During Anti-PD-1 Treatment. Clin Cancer Res (24) (13) 3036-3045 (2018). 98. Mami-Chouaib F, Blanc C, Corgnac S, et al. Resident memory T cells, critical components in tumor immunology. Journal for ImmunoTherapy of Cancer 6:87 2018. 99. Biliana Lozanoska-Ochser, Nigel J. Klein, Guo C. Huang, Raymond A. Alvarez and Mark Peakman. Expression of CD86 on Human Islet Endothelial Cells Facilitates T Cell Adhesion and Migration. J Immunol. 181 (9) 6109-6116 (2008), 100. Knieke K, Hoff H, Maszyna F, Kolar P, Schrage A, Hamann A, et al. (2009) CD152 (CTLA-4) Determines CD4 T Cell Migration In Vitro and In Vivo. PLoS ONE 4(5): e5702. 101. Brunner-Weinzierl Monika C., Rudd Christopher E. CTLA-4 and PD-1 Control of T-Cell Motility and Migration: Implications for Tumor Immunotherapy. JOURNAL=Frontiers in Immunology

Claims

1. A tissue-resident memory T cell (T) cell culture method comprising the steps of culturing lymphocytes in the presence of transforming growth factor beta (TGFβ) and / or co-culturing lymphocytes with regulatory T cells. ＲＭ A method for producing ).

2. The method according to claim 1, wherein lymphocytes are cultured in the presence of TGFβ, and preferably the lymphocytes are not co-cultured with regulatory T cells.

3. The method according to claim 1 or 2, wherein the lymphocytes are naive, effector, or memory CD8+ T lymphocytes.

4. The method according to any one of claims 1 to 3, wherein TGFβ is present at a concentration between 0.01 ng / ml and 50 ng / ml.

5. The method according to any one of claims 1 to 4, wherein the lymphocytes are obtained from human or non-human animal tissue, and optionally the tissue may be selected from the group consisting of blood, spleen, lymph nodes, lungs, gastrointestinal tract, skin, prostatic mammary gland tissue, liver, bone marrow, and pancreas.

6. T ＲＭ The method according to any one of claims 1 to 5, characterized by the expression of differentiation cluster 8 (CD8), differentiation cluster 69 (CD69), Hobit, aryl hydrocarbon receptor (AhR), and / or differentiation cluster 103 (CD103).

7. T ＲＭ The method according to any one of claims 1 to 6, characterized by the absence of expression of killer cell lectin-like receptor subfamily G member (KLRG1) and / or eomesodermine (Eomes).

8. The method according to any one of claims 1 to 7, comprising the step of culturing lymphocytes in the presence of IL-2, IL-4, IL-7, IL-12, IL-15 and / or IL-21.

9. The method according to any one of claims 1 to 8, further comprising the step of culturing lymphocytes in the presence of interleukin 15 (IL-15).

10. The method according to any one of claims 1 to 9, further comprising the step of culturing lymphocytes in the presence of interleukin 33 (IL-33).

11. The method according to any one of claims 1 to 10, comprising the step of culturing lymphocytes in the presence of interleukin 7 (IL-7).

12. The method according to any one of claims 1 to 11, comprising the step of culturing lymphocytes in the presence of interleukin-2 (IL-2).

13. The method according to any one of claims 1 to 12, further comprising the step of culturing lymphocytes in the presence of at least one interleukin 1 family member.

14. The method according to claim 13, wherein at least one interleukin-1 family member is IL-1a, IL-1b and / or IL-18.

15. The method according to any one of claims 1 to 14, wherein lymphocytes are cultured in a medium containing at least one aryl hydrocarbon receptor (AhR) ligand.

16. The method according to claim 15, wherein the AhR ligand is selected from halogenated aromatic hydrocarbons, polycyclic aromatic hydrocarbons, dietary aryl hydrocarbons, heme metabolites, indigoids, StemRegenin 1, and tryptophan metabolites.

17. The method according to any one of claims 1 to 16, wherein lymphocytes are cultured in a medium containing at least one lipid.

18. The method according to any one of claims 1 to 17, wherein lymphocytes are cultured in a culture medium containing an antigen, and optionally the antigen is a tumor antigen.

19. The method according to any one of claims 1 to 18, wherein regulatory T cells are characterized by or absent by the expression of Foxp3.

20. The method according to any one of claims 1 to 19, further comprising the step of culturing lymphocytes together with dendritic cells.

21. Tissue-resident memory T cells (T ＲＭ The method according to any one of claims 1 to 20, further comprising the step of propagating a population of )

22. In the presence of IL-2, IL-4, IL-7, IL-12, IL-15 and / or IL-21, T ＲＭ The method according to claim 21, further comprising the step of culturing cells.

23. Tissue-resident memory T cells (T) obtained or obtainable by the method described in any one of claims 1 to 22 ＲＭ ).

24. Tissue-resident memory T cells (T) according to claim 23 for use in treatment ＲＭ ), and their population, chosen at will.

25. Tissue-resident memory T cells (T) according to claim 23 for use in T cell therapy ＲＭ ), and their population, chosen at will.

26. Tissue-resident memory T cells according to claim 23, for use in the prevention, treatment, or improvement of cancer or infectious diseases.

27. A pharmaceutical composition comprising tissue-resident memory T cells as described in claim 23, optionally a proliferating population thereof, and a pharmaceutically acceptable excipient.

28. A method for producing the pharmaceutical composition according to claim 27, comprising the step of combining a therapeutically effective amount of tissue-resident memory T cells according to claim 23, optionally a proliferating population thereof, with a pharmaceutically acceptable excipient.

Images