How to expand immune cells

Abstract

The present invention relates to a method for expanding immune cells, comprising co-culturing the immune cells with at least one epithelial organoid, wherein the epithelial organoid comprises more epithelial cells than mesenchymal cells. The immune cells obtained by this method are at least 5×10 3 Also provided are in vitro innate lymphoid cell (ILC) populations comprising 100 ILCs, pharmaceutical compositions and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to a method for expanding immune cells, comprising co-culturing the immune cells with at least one epithelial organoid, wherein the epithelial organoid comprises more epithelial cells than mesenchymal cells. The immune cells obtained by this method are at least 5×10 3 Also provided are in vitro innate lymphoid cell (ILC) populations comprising 100 ILCs, pharmaceutical compositions and uses thereof. [Background technology]

[0002] Innate lymphoid cells (ILCs) are tissue-resident cells abundant in mucosal membranes, mediating mucosal barrier integrity during homeostasis and providing a rapid, non-antigen-specific source of cytokines during infection. ILCs are currently divided into three groups containing five subtypes. The first group is T-bet + Eomes + Cytotoxic natural killer (NK) cells and T-bet + Eomes - The second group includes ILC1. + Gata3 + The third group includes ILC2s and RORγt + Lymphoid tissue inducer cells (LTi) and natural cytotoxicity receptors (NCRs) + / - These categories are recognized to be less strictly defined in humans than in mice, and there may be plasticity between groups.

[0003] The relative frequencies and phenotypes of these ILC subsets vary across tissues in both mice and humans. Understanding what drives this heterogeneity is of considerable interest, as relative ILC frequencies not only change during acute infection but can also become chronically dysregulated in inflammatory diseases and cancer, making them attractive targets for therapeutic manipulation. This is particularly true because ILCs engage in bidirectional interactions with epithelial cells. ILC1s promote epithelial cell proliferation via TGF-β1, ILC2s are activated and proliferate in response to tuft cell-derived IL-25, and ILC3s promote Lgr5 proliferation via IL-22. + It promotes the proliferation of intestinal stem cells. Finally, fetal LTi mediates the development of secondary lymphoid structures, but NK cells are circulating and not specifically enriched in the mucosa.

[0004] Mature ILCs are derived from common lymphoid progenitors (CLPs), which are ILC-restricted lineages in mice. - CD127 + Id2 + IL-7R + α4β7 + They differentiate into ILC progenitor cells (ILCPs). Sources of ILCPs have been identified in adult murine bone marrow, fetal liver, small intestine, and lung (Bando et al., 2015). ILC progenitors have also been identified in human bone marrow, tonsils, and fetal, pediatric, and adult intestines, although these are less well characterized than their murine counterparts (Elmentaite et al., 2021).

[0005] Common to both mice and humans is the low frequency of ILCs in vivo. This limited number of isolations may hinder attempts to further study ILCs in vitro or in vivo. Furthermore, the therapeutic potential of ILCs has yet to be realized due to the difficulty of obtaining numbers large enough for cell therapy.

[0006] In vitro attempts to generate ILCs have shown that CD34 +Hematopoietic cells have been shown to generate ILCs when cultured with feeder cells engineered to express Notch ligands, with or without the addition of IL-15 (Hernandez et al., 2021). However, these approaches do not efficiently promote the maturation of all subsets in parallel.

[0007] Regulatory ILC (ILCreg) populations have previously been described in mice and humans through their ability to produce IL-10 (Wang et al., 2017). However, the characteristics of this population are controversial, as other ILC subsets, such as ILC2s, can also produce IL-10 (Bando et al., 2019). In none of these cases has the population been described as expressing Foxp3, a characteristic transcription factor of regulatory T cells. The present invention seeks to address one or more of the above-mentioned problems. Summary of the Invention

[0008] The present invention provides a method for expanding immune cells, comprising: (a) co-culturing immune cells with at least one epithelial organoid, wherein the epithelial organoid comprises more epithelial cells than mesenchymal cells. The present invention also provides immune cells obtained by the method of the present invention. Also, the present invention provides an immune cell expansion method for at least about 5×10 3Also provided is an in vitro innate lymphoid cell (ILC) population, comprising 100 ILCs. The inventors have also identified a subset of human ILCs called human regulatory ILCs (or ILCregs). These will be defined in more detail below and can be used to suppress inflammation, particularly in the intestinal tract. The term "organoid" is a term known in the art and refers to multiple cells that self-organize in vitro to form complex structures. Organoids are 3D and resemble in vitro miniaturized versions of organs or parts of organs. Generally, organoids are 3D. More generally, co-culture is performed with at least one whole epithelial organoid. However, in some embodiments, culture is performed with a portion of at least one epithelial organoid. The portion may include a layer of cells that may contain all cell types contained in the complete organoid. It will be understood that in such embodiments, the portion may be obtained by mechanically or chemically cutting the organoid. In the context of the present invention, the term "epithelial organoid" refers to an organoid containing epithelial cells.

[0009] Advantageously, organoid can be viable and stable in vitro for a long period of time.For example, organoid can be viable and stable in vitro for at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months or at least 24 months.The long-term stability of organoid, together with its complex structure, provides an in vitro culture environment that is closely related to in vivo environment.This long-term stability also allows organoid to be studied for a long period of time that is more closely related to in vivo period.

[0010] The present inventors have found that co-culture of immune cells with at least one epithelial organoid, which comprises more epithelial cells than mesenchymal cells, can significantly expand immune cells.Before this finding, it was believed that mesenchymal cells were essential for ensuring the maintenance and expansion of immune cells.Therefore, the significant expansion obtained by the method of the present invention is completely unexpected.In addition, the present inventors have surprisingly found that co-culture of immune cell precursor cells with at least one epithelial organoid, which comprises more epithelial cells than mesenchymal cells, can differentiate immune cell precursor cells into a significant number of immune cells.The generation of such a significant number of immune cells can be particularly useful in cell therapy, where it is currently difficult to obtain a sufficient number of mature immune cells for therapeutic purposes or even in vitro studies.

[0011] As those skilled in the art will understand, mesenchymal cells are stromal cells. In the context of the present invention, mesenchymal cells are cells that have flexible adhesive properties under normal culture conditions and have a fibroblast-like morphology. Cultured mesenchymal cells can be CD90 and CD105 positive. Cultured mesenchymal cells can be CD90, CD105, and CD73 positive. Cultured mesenchymal cells can be CD73, CD90, CD105, CD44, CD106, and CD166 positive. Cultured mesenchymal cells can be CD11b, CD14, CD19, CD34, CD45, CD79a, and HLA-DR negative, and CD73, CD90, and CD105 positive. The cultured mesenchymal cells can be CD11b, CD14, CD19, CD34, CD45, CD79a, and HLA-DR negative, and CD73, CD90, CD105, CD44, CD106, and CD166 positive.

[0012] In the context of the present invention, when a cell is defined as being positive for a particular marker, such as CD45, it is understood that the cell contains a detectable level of the marker. On the other hand, when a cell is defined as being negative for a particular marker, it is understood that the cell contains an undetectable level of the marker. Methods for measuring the presence of markers / proteins / mRNA are known in the art and will be described in more detail below. In the context of the present invention, the terms "amount" and "level" are interchangeable.

[0013] The mesenchymal cells may include multipotent mesenchymal cells, which are capable of differentiating into multiple different cell types.

[0014] Preferably, the mesenchymal cells comprise or consist of fibroblasts.

[0015] In the context of the present invention, the term "expansion" refers to the generation or production of immune cells. Thus, in the context of the present invention, the term "expansion" includes the proliferation of immune cells and / or the differentiation of immune cells into differentiated immune cells. The term "expansion" does not relate to the activation of specific subclones, such as specific T cell clones.

[0016] In some embodiments, epithelial organoids comprise less than about 45% mesenchymal cells.In some embodiments, epithelial organoids comprise less than about 44% mesenchymal cells, less than about 43% mesenchymal cells, less than about 42% mesenchymal cells, less than about 41% mesenchymal cells, less than about 40% mesenchymal cells, less than about 39% mesenchymal cells, less than about 38% mesenchymal cells, less than about 37% mesenchymal cells, less than about 36% mesenchymal cells, less than about 35% mesenchymal cells, less than about 34% mesenchymal cells, less than about 33% mesenchymal cells, less than about 32% mesenchymal cells, less than about 31% mesenchymal cells, less than about 30% mesenchymal cells, less than about 29% mesenchymal cells, less than about 28% mesenchymal cells, less than about 27% mesenchymal cells, less than about 26% mesenchymal cells, less than about 25% mesenchymal cells, less than about 24% mesenchymal cells. cells, less than about 23% mesenchymal cells, less than about 22% mesenchymal cells, less than about 21% mesenchymal cells, less than about 20% mesenchymal cells, less than about 19% mesenchymal cells, less than about 18% mesenchymal cells, less than about 17% mesenchymal cells, less than about 16% mesenchymal cells, less than about 15% mesenchymal cells, less than about 14% mesenchymal cells, less than about 13% mesenchymal cells, less than about 12% mesenchymal cells, less than about 11% mesenchymal cells, less than about 10% mesenchymal cells, less than about 9% mesenchymal cells, less than about 8% mesenchymal cells, less than about 7% mesenchymal cells, less than about 6% mesenchymal cells, less than about 5% mesenchymal cells, less than about 4% mesenchymal cells, less than about 3% mesenchymal cells, less than about 2% mesenchymal cells, or less than about 1% mesenchymal cells.

[0017] In some embodiments, epithelial organoids comprise less than about 40% mesenchymal cells. In some embodiments, epithelial organoids comprise less than about 35% mesenchymal cells. In some embodiments, epithelial organoids comprise less than 30% mesenchymal cells. In some embodiments, epithelial organoids comprise less than 25% mesenchymal cells. In some embodiments, epithelial organoids comprise less than 24% mesenchymal cells. In some embodiments, epithelial organoids comprise less than 23% mesenchymal cells. In some embodiments, epithelial organoids comprise less than 22% mesenchymal cells. In some embodiments, epithelial organoids comprise less than 21% mesenchymal cells. In some embodiments, epithelial organoids comprise less than 20% mesenchymal cells. In some embodiments, epithelial organoids comprise less than 15% mesenchymal cells. In some embodiments, epithelial organoids comprise less than 10% mesenchymal cells.

[0018] In some embodiments, the epithelial organoids comprise between about 0.01% and about 45% mesenchymal cells. In some embodiments, the epithelial organoids comprise between about 0.01% and about 30% mesenchymal cells. In some embodiments, the epithelial organoids comprise between about 0.01% and about 25% mesenchymal cells. In some embodiments, the epithelial organoids comprise between about 0.01% and about 20% mesenchymal cells. In some embodiments, the epithelial organoids comprise between about 0.01% and about 15% mesenchymal cells. In some embodiments, the epithelial organoids comprise between about 0.01% and about 10% mesenchymal cells.

[0019] In some embodiments, the epithelial organoids comprise between about 0.1% and about 45% mesenchymal cells. In some embodiments, the epithelial organoids comprise between about 0.1% and about 30% mesenchymal cells. In some embodiments, the epithelial organoids comprise between about 0.1% and about 25% mesenchymal cells. In some embodiments, the epithelial organoids comprise between about 0.1% and about 20% mesenchymal cells.

[0020] In some embodiments, the epithelial organoids comprise between about 1% and about 45% mesenchymal cells. In some embodiments, the epithelial organoids comprise between about 1% and about 30% mesenchymal cells. In some embodiments, the epithelial organoids comprise between about 1% and about 25% mesenchymal cells. In some embodiments, the epithelial organoids comprise between about 1% and about 20% mesenchymal cells.

[0021] Epithelial organoids may not contain detectable levels of mesenchymal cells.Thus, in some embodiments, epithelial organoids contain undetectable levels of mesenchymal cells.

[0022] Various methods for detecting percentage or level are known to those skilled in the art.For example, the percentage or level of mesenchymal cells in organoid can be detected by flow cytometry.Other suitable detection methods can include, for example, fluorescent microscopy using confocal microscope.Other suitable methods will be known to those skilled in the art.Detecting percentage or level of mesenchymal cells can be carried out before step (a).Alternatively, detecting percentage or level of mesenchymal cells can be carried out during step (a) or after step (a).

[0023] Method can comprise, before step (a), depleting mesenchymal cells from epithelial organoid.Alternatively, the epithelial organoid of the present invention can be depleted of mesenchymal cells.It will be understood that " depleting " mesenchymal cells refers to removing mesenchymal cells from epithelial organoid.Therefore, " depleting " can refer to the epithelial organoid that has already been depleted of mesenchymal cells.

[0024] Prior to step (a), the method may include a step of depleting mesenchymal cells from the epithelial organoids immediately prior to step (a) and / or after full maturation of the epithelium.

[0025] Mesenchymal cell-depleted epithelial organoids contain, as defined above, less than about 45% mesenchymal cells, less than about 44% mesenchymal cells, less than about 43% mesenchymal cells, less than about 42% mesenchymal cells, less than about 41% mesenchymal cells, less than about 40% mesenchymal cells, less than about 39% mesenchymal cells, less than about 38% mesenchymal cells, less than about 37% mesenchymal cells, less than about 36% mesenchymal cells, less than about 35% mesenchymal cells, less than about 34% mesenchymal cells, less than about 33% mesenchymal cells, less than about 32% mesenchymal cells, less than about 31% mesenchymal cells, less than about 30% mesenchymal cells, less than about 29% mesenchymal cells, less than about 28% mesenchymal cells, less than about 27% mesenchymal cells, less than about 26% mesenchymal cells, less than about 25 ... The fibroblasts may comprise less than 24% mesenchymal cells, less than about 23% mesenchymal cells, less than about 22% mesenchymal cells, less than about 21% mesenchymal cells, less than about 20% mesenchymal cells, less than about 19% mesenchymal cells, less than about 18% mesenchymal cells, less than about 17% mesenchymal cells, less than about 16% mesenchymal cells, less than about 15% mesenchymal cells, less than about 14% mesenchymal cells, less than about 13% mesenchymal cells, less than about 12% mesenchymal cells, less than about 11% mesenchymal cells, less than about 10% mesenchymal cells, less than about 9% mesenchymal cells, less than about 8% mesenchymal cells, less than about 7% mesenchymal cells, less than about 6% mesenchymal cells, less than about 5% mesenchymal cells, less than about 4% mesenchymal cells, less than about 3% mesenchymal cells, less than about 2% mesenchymal cells, or less than about 1% mesenchymal cells.

[0026] Depletion comprises mechanical destruction of epithelial organoid.Advantageously, by mechanical destruction, mesenchymal fraction (if present) is separated from the epithelial structure of epithelial organoid.Then, can remove mesenchymal fraction, and leave the epithelial structure of organoid.In some embodiments, depletion comprises digestion of epithelial organoid, for example, digestion with collagenase.

[0027] The depletion may be repeated two or more times, for example, the depletion may be repeated three, four, five, or six times.

[0028] In some embodiments, depletion comprises repeating the mechanical destruction of epithelial organoid 3 times, 4 times or 5 times.In other embodiments, depletion comprises (i) the mechanical destruction of epithelial organoid, and (ii) the digestion of epithelial organoid with collagenase.Preferably, the digestion of epithelial organoid with collagenase is carried out after mechanical destruction.

[0029] Epithelial organoid can be primary organoid, or can be derived from stem cells.Stem cells can comprise or consist of induced pluripotent stem cells (iPSC) or adult stem cells.As those skilled in the art will understand, iPSC is pluripotent stem cells obtained by genetically reprogramming adult somatic cells into embryonic state.Primary organoid is understood to refer to organoid obtained from subject's biopsy sample.Subject's biopsy sample can be obtained during endoscopy.Subject's biopsy sample is preferably human biopsy sample.Subject's biopsy sample is more preferably human small intestine biopsy sample or human colon biopsy sample.Subject's biopsy sample is most preferably human small intestine biopsy sample.Organoid is preferably human small intestine biopsy-derived organoid or human colon biopsy-derived organoid.Most preferably organoid is human small intestine biopsy-derived organoid.The effectiveness of such organoid is shown in Example 10.

[0030] In embodiments involving iPSC-derived organoids, iPSCs may be obtained from the Human Induced Pluripotent Stem Cell Initiative (HipSci, https: / / www.hipsci.org).

[0031] Alternatively, in embodiments involving iPSC-derived organoids, the method may include a step prior to step (a) of generating iPSCs.

[0032] Producing iPSCs can include introducing a polynucleotide sequence encoding one or more of OCT3 / 4, SOX2, KLF4, and MYC into isolated primary cells. Preferably, the polynucleotide sequence encodes OCT3 / 4, SOX2, KLF4, and MYC. More preferably, the polynucleotide sequence encodes human OCT3 / 4, human SOX2, human KLF4, and human MYC. Introduction can include transduction or gene transfer, typically transduction. In some embodiments, a vector comprises the polynucleotide sequence. The vector can be a Sendai vector. After introduction, the cells can be cultured in iPS cell medium for at least 5 days, at least 10 days, at least 20 days, at least 30 days, or at least 40 days. Culturing can be performed on a feeder layer. The iPS cell medium can include improved DMEM.

[0033] The iPS cell culture medium may further comprise Knockout Serum Replacement (KSR), commercially available from Life Technologies. The iPS cell culture medium may comprise about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20% KSR. In some embodiments, the iPS cell culture medium comprises about 10% KSR.

[0034] In some embodiments, the iPS cell culture medium further comprises L-glutamine and / or fibroblast growth factor-2. The iPS cell culture medium may comprise L-glutamine at a concentration of at least about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. The iPS cell culture medium may comprise less than about 10 mM, less than about 7 mM, less than about 6 mM, or less than about 5 mM L-glutamine. In some embodiments, the iPS cell culture medium comprises about 1 mM to about 3 mM L-glutamine. In some embodiments, the iPS cell culture medium comprises about 2 mM L-glutamine.

[0035] The fibroblast growth factor-2 can be zebrafish fibroblast growth factor-2. In some embodiments, the fibroblast growth factor-2 is recombinant. The iPS cell culture medium can contain fibroblast growth factor-2 at a concentration of at least about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, or at least about 10 ng / ml. In some embodiments, the iPS cell culture medium contains less than about 20 ng / ml, less than about 10 ng / ml, or less than about 5 ng / ml of fibroblast growth factor-2. In some embodiments, the iPS cell culture medium contains about 4 ng / ml of fibroblast growth factor-2.

[0036] The iPS cell culture medium may further contain an antibiotic, such as penicillin / streptomycin. The iPS cell culture medium may contain about 0.1%, about 0.2%, about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5% penicillin / streptomycin. In some embodiments, the iPS cell culture medium contains about 1% penicillin / streptomycin.

[0037] The iPS cell culture medium may further comprise 2-mercaptoethanol. The iPS cell culture medium may comprise about 0.001%, about 0.002%, about 0.005%, about 0.007%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, or about 0.05% 2-mercaptoethanol. In some embodiments, the iPS cell culture medium comprises about 0.007% 2-mercaptoethanol.

[0038] Methods for obtaining primary epithelial organoids from a subject's biopsy are well known in the art. In embodiments involving primary epithelial organoids, the method may include a step prior to step (a) of obtaining primary epithelial organoids from a subject's biopsy. In embodiments involving primary epithelial organoids, the method may include a step prior to step (a) of obtaining primary epithelial organoids from a human biopsy. Obtaining primary epithelial organoids from a subject's biopsy may include culturing a suspension of the subject's biopsy (or a portion of the subject's biopsy) in a matrix and a basal medium. In the context of the present invention, a matrix is understood to refer to a cell culture matrix. Various cell culture matrices are commercially available. Suitable matrices may include, but are not necessarily limited to, Geltrex, Cultrex, Matrigel, collagen I hydrogel, collagen IV hydrogel, and other synthetic hydrogels derived from crosslinking functionalized polypeptides and / or polymers such as polyethylene glycol (PEG), as described, for example, in Jowett et al., 2021. In some embodiments, the matrix comprises or consists of Matrigel.

[0039] Alternatively, obtaining primary epithelial organoid from subject's biopsy sample can comprise culturing the suspension of subject's biopsy sample (or part of subject's biopsy sample) in hanging drop suspension.It will be understood that " hanging drop suspension " refers to floating in basal medium from the surface.A variety of hanging drop suspension culture modules are available to those skilled in the art.

[0040] In some embodiments, obtaining primary epithelial organoids from a subject's biopsy sample comprises culturing a suspension of the subject's biopsy sample (or a portion of the subject's biopsy sample) in Matrigel and basal medium.

[0041] The basal medium may include modified DMEM / F12. Other suitable basal media will be known to those skilled in the art.

[0042] The basal medium may further comprise L-glutamine, antibiotics, N2 supplement, B27 supplement, HEPES, and / or N-acetylcysteine.

[0043] The basal medium may contain L-glutamine at a concentration of at least about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. The basal medium may contain less than about 10 mM, less than about 7 mM, less than about 6 mM, or less than about 5 mM L-glutamine. In some embodiments, the basal medium contains about 1 mM to about 3 mM L-glutamine. In some embodiments, the basal medium contains about 2 mM L-glutamine.

[0044] The basal medium may contain HEPES at a concentration of at least about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 12 mM, about 15 mM, or at least about 20 mM. The basal medium may contain less than about 50 mM, less than about 40 mM, less than about 30 mM, less than about 20 mM, or less than about 10 mM HEPES. In some embodiments, the basal medium contains HEPES at a concentration of about 5 mM to about 15 mM. In some embodiments, the basal medium contains about 10 mM HEPES.

[0045] The basal medium may contain N-acetylcysteine at a concentration of at least about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. The basal medium may contain less than about 10 mM, less than about 7 mM, less than about 6 mM, or less than about 5 mM. In some embodiments, the basal medium contains about 0.5 mM to about 3 mM N-acetylcysteine. In some embodiments, the basal medium contains about 1 mM N-acetylcysteine.

[0046] Culturing the suspension of the subject's biopsy sample (or a portion of the subject's biopsy sample) can be carried out for a period of at least 120 hours, at least 168 hours, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks. In some embodiments, culturing the suspension of the subject's biopsy sample (or a portion of the subject's biopsy sample) is carried out for a period of 120 hours to 8 weeks.

[0047] Preferably, obtaining primary epithelial organoid from subject's biopsy sample comprises culturing the suspension of subject's biopsy sample (or part of subject's biopsy sample) in matrix and basal medium for at least 120 hours, at least 168 hours, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks or at least 8 weeks.In some embodiments, obtaining primary epithelial organoid from subject's biopsy sample comprises culturing the suspension of subject's biopsy sample (or part of subject's biopsy sample) in matrix and basal medium for 120 hours to 8 weeks.

[0048] Preferably, the basal medium comprises noggin. In some embodiments, the basal medium comprises R-spondin1. In some embodiments, the basal medium comprises EGF. In some embodiments, the basal medium comprises noggin, R-spondin1, and EGF. The basal medium may further comprise one or more of FGF10, CHIR, and a RhoK inhibitor.

[0049] More preferably, epithelial organoid is derived from iPSC or adult stem cell.Most preferably, epithelial organoid is derived from iPSC.Advantageously, organoid is derived from stem cell, so that stem cell can differentiate into multiple different cell types in vitro.This multiple different cell types can self-organize into organoid with complex 3D structure in vitro.

[0050] The method of generating epithelial organoid from iPSC or adult stem cell is known in the art.Therefore, in some embodiments, before step (a), method comprises the first step of generating organoid from iPSC or adult stem cell in vitro.Preferably, before step (a), method comprises the first step of generating organoid from iPSC in vitro.Generating organoid from iPSC in vitro can comprise culturing iPSC in organoid differentiation medium.In some embodiments, method comprises: (i) culturing the iPSCs in multiple different organoid differentiation media for a total period of at least about 48 hours to form organoid colonies; (ii) selecting organoid colonies, and optionally, (iii) culturing organoid colonies.

[0051] In some embodiments, the iPSCs are human iPSCs.

[0052] Organoid differentiation medium may include RPMI medium or E8 medium supplemented with supplements appropriate for differentiation.

[0053] For example, the medium suitable for generating intestinal organoid can comprise endoderm differentiation medium and midgut / hindgut differentiation medium.Example endoderm differentiation medium can comprise RPMI medium, B27 and activin A.Example midgut / hindgut differentiation medium can comprise RPMI medium, B27, FGF4 and CHIR.

[0054] Endoderm or midgut / hindgut differentiation medium may contain at least about 0.1% B27, at least about 0.2% B27, at least about 0.3% B27, at least about 0.4% B27, at least about 0.5% B27, at least about 0.6% B27, at least about 0.7% B27, at least about 0.8% B27, at least about 0.9% B27, at least about 1% B27, at least about 1.5% B27, at least about 2% B27, or at least about 3% B27. In some embodiments, endoderm differentiation medium contains less than about 10% B27, less than about 5% B27, or less than about 3% B27. In some embodiments, endoderm differentiation medium contains about 0.2% B27. In other embodiments, endoderm differentiation medium contains about 1% B27. In some embodiments, the midgut / hindgut differentiation medium comprises about 2% B27.

[0055] The endoderm differentiation medium may comprise at least about 0.5 μl / ml of activin A, at least about 1 μl / ml of activin A, at least about 2 μl / ml of activin A, at least about 3 μl / ml of activin A, or at least about 4 μl / ml of activin A. In some embodiments, the endoderm differentiation medium comprises less than about 5 μl / ml of activin A. In some embodiments, the endoderm differentiation medium comprises about 1 μl / ml of activin A.

[0056] The midgut / hindgut differentiation medium may comprise at least about 0.5 μl / ml of fibroblast growth factor 4 (FGF4), at least about 1 μl / ml of FGF4, at least about 2 μl / ml of FGF4, at least about 3 μl / ml of FGF4, or at least about 4 μl / ml of FGF4. In some embodiments, the midgut / hindgut differentiation medium comprises less than about 5 μl / ml of FGF4. In some embodiments, the midgut / hindgut differentiation medium comprises about 1 μl / ml of FGF4.

[0057] An example organoid generation method for generating human intestinal organoids is as follows. 1. Human iPSCs are cultured in E8 medium containing B27 and activin A for approximately 24 hours. 2. The E8 medium is replaced with endoderm differentiation medium, and the human iPSCs are cultured in endoderm differentiation medium for at least about 72 hours. 3. Replace the endoderm differentiation medium with midgut / hindgut differentiation medium and culture the human iPSCs in midgut / hindgut differentiation medium for at least about 72 hours.

[0058] Alternatively, method comprises the first step of generating organoid from adult stem cell in vitro, preferably from murine adult stem cell.Generating organoid from adult stem cell in vitro can comprise culturing adult stem cell in organoid differentiation medium for at least about 4 hours.In some embodiments, method comprises: (i) culturing adult stem cells in an organoid differentiation medium for at least about 4 hours to form organoid colonies; (ii) selecting organoid colonies, and optionally, (iii) culturing organoid colonies.

[0059] Organoid differentiation medium may include RPMI medium or E8 medium supplemented with supplements appropriate for differentiation.

[0060] For generating intestinal organoid from adult murine stem cells, organoid differentiation medium can comprise the basal medium as defined above.For example, organoid differentiation medium can comprise improved DMEM / F12, L-glutamine, antibiotics, N2 supplement, B27 supplement, R-spondin, EGF, Noggin, HEPES and / or N-acetylcysteine.Basic medium can further comprise one or more of FGF10, CHIR and RhoK inhibitor.

[0061] Other suitable methods for generating organoids will be known and available to those skilled in the art.

[0062] At least one epithelial organoid can be a plurality of epithelial organoids.For example, at least one epithelial organoid can be at least two epithelial organoids, at least three epithelial organoids, at least four epithelial organoids, at least five epithelial organoids, at least six epithelial organoids, at least seven epithelial organoids, at least eight epithelial organoids, at least nine epithelial organoids, at least ten epithelial organoids, at least twenty epithelial organoids, at least thirty epithelial organoids, at least forty epithelial organoids, at least fifty epithelial organoids, at least sixty epithelial organoids, at least seventy epithelial organoids, at least eighty epithelial organoids, at least ninety epithelial organoids, at least one hundred epithelial organoids, at least one fifty epithelial organoids, at least two hundred epithelial organoids or at least five hundred epithelial organoids.

[0063] In some embodiments, at least one epithelial organoid is less than 150 epithelial organoids, less than 100 epithelial organoids, less than 90 epithelial organoids, less than 80 epithelial organoids, less than 70 epithelial organoids, less than 60 epithelial organoids, less than 50 epithelial organoids, less than 40 epithelial organoids or less than 30 epithelial organoids.

[0064] In some embodiments, at least one epithelial organoid is 2 to 100 organoids.In some embodiments, at least one epithelial organoid is 25 to 100 epithelial organoids, preferably 25 to 50 epithelial organoids.

[0065] It should be understood that the number of organoids described above can be for one co-culture, for example, one co-culture well in tissue culture plate.Therefore, when using multiple co-culture wells and / or when the method is high-throughput method, the total number of organoids can be much more.For example, the total number of organoids can be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, at least 1000, at least 10000, at least 50,000, at least 100,000, at least 200,000, at least 300,000, at least 400,000, at least 500,000, at least 1,000,000 or at least 5,000,000.

[0066] In some embodiments, the total number of organoids is less than 100,000,000 organoids. In some embodiments, the total number of organoids is 10 to 100,000,000 organoids. The total number of organoids can be 20 to 500,000 organoids. In some embodiments, the total number of organoids is 20 to 10,000 organoids, optionally 20 to 1,000 organoids. In some embodiments, the total number of organoids is 20 to 500 organoids. The total number of organoids can be 20 to 250 organoids.

[0067] Preferably, epithelial organoid is human or murine epithelial organoid.More preferably, epithelial organoid is human epithelial organoid.In the context of the present invention, it will be understood that the term " murine " includes rat and mouse.Therefore, in some embodiments, epithelial organoid is murine epithelial organoid, preferably mouse epithelial organoid.

[0068] Epithelial organoids can be human primary epithelial organoids, or can be derived from human iPSCs or human adult stem cells.Alternatively, epithelial organoids can be mouse primary epithelial organoids, or can be derived from mouse iPSCs, mouse embryonic stem cells, or mouse adult stem cells.When epithelial organoids are mouse primary epithelial organoids, the mice from which organoids are isolated can be germ-free or specific pathogen-free.The present inventors have found that organoids obtained from such germ-free mice or specific pathogen-free mice are suitable for the present invention.This is surprising, considering the known role of microorganisms in organ differentiation and maintenance, especially intestinal differentiation and maintenance.

[0069] In the embodiment that comprises a plurality of epithelial organoids, the plurality of epithelial organoids preferably comprises or consists of the epithelial organoids from the same species.For example, the plurality of epithelial organoids can comprise or consist of a plurality of human epithelial organoids.Alternatively, the plurality of epithelial organoids can comprise or consist of a plurality of murine epithelial organoids, for example, a plurality of mouse epithelial organoids.Alternatively, the plurality of epithelial organoids can comprise or consist of the epithelial organoids from at least two different species, for example, human and mouse.Optionally, the plurality of epithelial organoids comprises or consist of a plurality of murine and human epithelial organoids.

[0070] It should be understood that epithelial organoid is the in vitro miniaturized type of organ or its part, so any epithelial organoid can be used in the present invention.In particular, it can be the in vitro miniaturized type of any organ (or its part) that comprises epithelial cells.For example, epithelial organoid can be the epithelial organoid of skin, gastrointestinal tract, lung, thymus, thyroid gland, genital organ, bladder, kidney, pancreas or liver.In some embodiments, epithelial organoid is the epithelial organoid of skin, gastrointestinal tract, lung, thyroid gland, genital organ, bladder, kidney, pancreas or liver.In some embodiments, epithelial organoid is the epithelial organoid of lung, genital organ or gastrointestinal tract.In some embodiments, epithelial organoid is the epithelial organoid of lung or gastrointestinal tract.

[0071] Genital epithelial organoids can include fallopian tube epithelial organoids, ovarian epithelial organoids, prostate epithelial organoids, endometrial epithelial organoids, cervical epithelial organoids, vaginal epithelial organoids, and testicular epithelial organoids (these can also be called gonadal epithelial organoids).Cervical epithelial organoids can include endocervical canal epithelial organoids and / or ectocervix epithelial organoids.Genital epithelial organoids can be selected from fallopian tube epithelial organoids, ovarian epithelial organoids, prostate epithelial organoids, and endometrial epithelial organoids.

[0072] In the context of the present invention, the term "gastrointestinal tract" shall be understood to refer to oral mucosal organs, stomach, intestinal tract and anus.The term "oral mucosa" shall be understood to refer to oral mucosal organs such as salivary gland, pharynx, taste bud, tongue region and esophagus.Gastrointestinal epithelial organoid can therefore comprise gastric epithelial organoid, salivary gland epithelial organoid, taste bud epithelial organoid, tongue region epithelial organoid, esophageal epithelial organoid, pharyngeal epithelial organoid, intestinal epithelial organoid and anal epithelial organoid.In some embodiments, epithelial organoid is oral mucosal epithelial organoid or intestinal epithelial organoid.In some embodiments, epithelial organoid is oral mucosal epithelial organoid.Oral mucosal epithelial organoid can be selected from gastric epithelial organoid and esophageal epithelial organoid.

[0073] In some embodiments, epithelial organoid is intestinal epithelial organoid.It can be understood that intestinal organoid comprises small intestinal organoid, large intestinal organoid and rectal organoid.In some embodiments, intestinal epithelial organoid is small intestinal epithelial organoid.In other embodiments, intestinal epithelial organoid is large intestinal epithelial organoid.Large intestinal epithelial organoid can comprise or consist of colonic epithelial organoid.In some embodiments, intestinal epithelial organoid is rectal organoid.

[0074] Preferably, epithelial organoid is intestinal epithelial organoid or lung epithelial organoid.In some embodiments, epithelial organoid is intestinal epithelial organoid.In some embodiments, epithelial organoid is small intestinal epithelial organoid or lung epithelial organoid.Alternatively, epithelial organoid can be lung epithelial organoid.

[0075] Epithelial organoid can be epithelial cancer organoid.It is understood that epithelial cancer organoid refers to the organoid obtained from cancerous tumor biopsy sample.For example, epithelial cancer organoid can be epithelial skin cancer, intestinal cancer, lung cancer, thymus cancer, thyroid cancer, reproductive cancer, bladder cancer, kidney cancer, pancreatic cancer, oral mucosa cancer or liver cancer organoid.In some embodiments, epithelial cancer organoid is epithelial skin cancer, intestinal cancer, lung cancer, thyroid cancer, reproductive cancer, bladder cancer, kidney cancer, pancreatic cancer, oral mucosa cancer or liver cancer organoid.Epithelial cancer organoid can be epithelial head and neck cancer organoid.It is understood that epithelial head and neck cancer organoid is derived from head and neck cancer biopsy sample.

[0076] In some embodiments, epithelial organoid is the primary epithelial organoid of skin, intestine, lung, thymus, thyroid, reproductive organs, bladder, kidney, pancreas, oral mucosa or liver.In some embodiments, epithelial organoid is the primary epithelial organoid of skin, intestine, lung, thyroid, reproductive organs, bladder, kidney, pancreas, oral mucosa or liver.In some embodiments, epithelial organoid is the primary epithelial organoid of intestine, lung, reproductive organs or oral mucosa.As defined above, primary epithelial organoid is the organoid comprising epithelial cells obtained from subject's biopsy sample.Primary intestinal epithelial organoid can alternatively be called enteroid.Primary colonic epithelial organoid can alternatively be called colonoid.

[0077] The present inventors have surprisingly found that the immune cells obtained by the method of the present invention can be imprinted with the gene signature and phenotype specific to the immune cells in vivo in primary organ.This advantageously allows the generation of tissue-specific immune cells, which can be useful for treating certain diseases and / or can improve the homing ability to specific tissue when administered to subject.When epithelial organoid is intestinal organoid, the immune cells obtained can therefore have the gene signature and phenotype specific to intestinal tract.When epithelial organoid is lung organoid, the immune cells obtained can have the gene signature and phenotype specific to lung.Tissue-specific gene signature and phenotype will be described in more detail herein.

[0078] Preferably, the immune cells and at least one epithelial organoid are co-cultured in a medium that supports the culture of both the immune cells and the epithelial organoid. "Supporting the culture" is understood to mean allowing growth, maintenance, and differentiation as needed. Those skilled in the art can design such a medium. The medium preferably comprises a basal medium containing one or more of R-spondin, noggin, EGF, 2-mercaptoethanol, IL-2, and IL-7. Suitable basal media include, but are not necessarily limited to, modified DMEM / F12 and Essential 8 medium. Further suitable basal media are known to those skilled in the art. In some embodiments, the medium comprises a basal medium containing R-spondin, noggin, EGF, 2-mercaptoethanol, IL-2, and IL-7. In some embodiments, the medium comprises a basal medium containing R-spondin, noggin, EGF, 2-mercaptoethanol, IL-2, and IL-7. In some embodiments, the medium comprises a basal medium containing IL-2 and IL-7. In some embodiments, the medium comprises a basal medium containing IL-2, IL-7, and 2-mercaptoethanol. In some embodiments, the medium comprises a modified DMEM / F12 medium containing R-spondin, noggin, EGF, IL-2, and IL-7. In other embodiments, the medium comprises Essential 8 medium containing R-spondin, noggin, EGF, IL-2, and IL-7. In some embodiments, the basal medium further comprises IL-23. In some embodiments, the basal medium further comprises IL-15. In some embodiments, the basal medium further comprises IL-22. In some embodiments, the basal medium further comprises TGF-β.

[0079] In some embodiments, the basal medium comprises IL-2, IL-7, IL-15, and 2-mercaptoethanol. In some embodiments, the basal medium comprises R-spondin, noggin, EGF, IL-2, IL-7, IL-15, and β-mercaptoethanol. In some embodiments, the basal medium comprises a modified DMEM / F12 medium containing R-spondin, noggin, EGF, IL-2, IL-7, IL-15, and β-mercaptoethanol.

[0080] The basal medium may contain EGF at a concentration of at least about 1 ng / ml, at least about 10 ng / ml, at least about 20 ng / ml, at least about 25 ng / ml, at least about 30 ng / ml, at least about 35 ng / ml, at least about 40 ng / ml, at least about 45 ng / ml, at least about 50 ng / ml, at least about 55 ng / ml, at least about 60 ng / ml, at least about 65 ng / ml, at least about 70 ng / ml, at least about 75 ng / ml, at least about 80 ng / ml, at least about 85 ng / ml, at least about 90 ng / ml, at least about 95 ng / ml, or at least about 100 ng / ml. In some embodiments, the basal medium contains EGF at a concentration of at least about 10 ng / ml. In some embodiments, the basal medium contains EGF at a concentration of at least about 25 ng / ml, optionally at least about 50 ng / ml.

[0081] The basal medium may contain EGF at a concentration of less than about 1000 ng / ml, less than about 900 ng / ml, less than about 800 ng / ml, less than about 700 ng / ml, less than about 600 ng / ml, less than about 500 ng / ml, less than about 400 ng / ml, less than about 300 ng / ml, less than about 200 ng / ml, less than about 100 ng / ml, or less than about 50 ng / ml. In some embodiments, the basal medium contains EGF at a concentration of less than about 50 ng / ml.

[0082] In some embodiments, the basal medium contains EGF at a concentration of about 1 ng / ml to about 1000 ng / ml. The basal medium may contain EGF at a concentration of about 10 ng / ml to about 500 ng / ml, optionally about 10 ng / ml to about 100 ng / ml. In some embodiments, the basal medium contains EGF at a concentration of about 50 ng / ml.

[0083] The EGF can be recombinant, hi some embodiments, the EGF is murine or human.

[0084] The basal medium can comprise R-spondin at a concentration of at least about 100ng / ml, at least about 200ng / ml, at least about 300ng / ml, at least about 400ng / ml, at least about 500ng / ml, at least about 600ng / ml, at least about 700ng / ml, at least about 800ng / ml, at least about 900ng / ml, at least about 1μg / ml, at least about 2μg / ml, at least about 3μg / ml, at least about 4μg / ml, at least about 5μg / ml, at least about 6μg / ml, at least about 7μg / ml, at least about 8μg / ml, at least about 9μg / ml, at least about 10μg / ml, at least about 15μg / ml or at least about 20μg / ml.In some embodiments, the basal medium comprises R-spondin at a concentration of at least about 500ng / ml. In some embodiments, the basal medium comprises R-spondin at a concentration of at least about 700 ng / ml, optionally at least about 900 ng / ml.

[0085] The basal medium may comprise R-spondin at a concentration of less than about 25 μg / ml, less than about 24 μg / ml, less than about 23 μg / ml, less than about 22 μg / ml, or less than about 21 μg / ml. In some embodiments, the basal medium comprises R-spondin at a concentration of less than about 10 μg / ml.

[0086] In some embodiments, the basal medium comprises R-spondin at a concentration of about 100 ng / ml to about 20 μg / ml. The basal medium may comprise R-spondin at a concentration of about 200 ng / ml to about 10 μg / ml, optionally at a concentration of about 500 ng / ml to about 2 μg / ml. In some embodiments, the basal medium comprises R-spondin at a concentration of about 1 μg / ml.

[0087] R-spondin can be recombinant.In some embodiments, R-spondin is murine or human.In some embodiments, R-spondin is R-spondin-containing supernatant.Supernatant can be isolated from cell line that produces R-spondin, and various cell lines are available.

[0088] The basal medium may comprise at least about 1 ng / ml, at least about 2 ng / ml, at least about 3 ng / ml, at least about 4 ng / ml, at least about 5 ng / ml, at least about 6 ng / ml, at least about 7 ng / ml, at least about 8 ng / ml, at least about 9 ng / ml, at least about 10 ng / ml, at least about 11 ng / ml, at least about 12 ng / ml, at least about 13 ng / ml, at least about 14 ng / ml, at least about 15 ng / ml, at least about 16 ng / ml, at least about 17 ng / ml, at least about 18 ng / ml, at least about 19 ng / ml, at least about 20 ng / ml, at least about 21 ng / ml, at least about 22 ng / ml, at least about 23 ng / ml, at least about 24 ng / ml, at least about 25 ng / ml, at least about 26 ng / ml, In some embodiments, the basal medium may comprise Noggin at a concentration of at least about 20 ng / ml, at least about 27 ng / ml, at least about 28 ng / ml, at least about 29 ng / ml, at least about 30 ng / ml, at least about 31 ng / ml, at least about 32 ng / ml, at least about 33 ng / ml, at least about 34 ng / ml, at least about 35 ng / ml, at least about 36 ng / ml, at least about 37 ng / ml, at least about 38 ng / ml, at least about 39 ng / ml, at least about 40 ng / ml, at least about 41 ng / ml, at least about 42 ng / ml, at least about 43 ng / ml, at least about 44 ng / ml, at least about 45 ng / ml, at least about 46 ng / ml, at least about 47 ng / ml, at least about 48 ng / ml, at least about 49 ng / ml, or at least about 50 ng / ml. In some embodiments, the basal medium comprises noggin at a concentration of at least about 50 ng / ml, optionally at least about 70 ng / ml.

[0089] The basal medium may comprise Noggin at a concentration of less than about 1000 ng / ml, less than about 900 ng / ml, less than about 800 ng / ml, less than about 700 ng / ml, less than about 600 ng / ml, less than about 500 ng / ml, less than about 400 ng / ml, less than about 300 ng / ml, less than about 200 ng / ml, less than about 100 ng / ml, or less than about 50 ng / ml, hi some embodiments, the basal medium comprises Noggin at a concentration of less than about 200 ng / ml.

[0090] In some embodiments, the basal medium comprises Noggin at a concentration of about 1 ng / ml to about 1000 ng / ml. The basal medium may comprise Noggin at a concentration of about 10 ng / ml to about 500 ng / ml, optionally at a concentration of about 10 ng / ml to about 200 ng / ml. In some embodiments, the basal medium comprises Noggin at a concentration of about 50 ng / ml to about 200 ng / ml. In some embodiments, the basal medium comprises Noggin at a concentration of about 100 ng / ml.

[0091] The Noggin can be recombinant. In some embodiments, the Noggin is murine or human. In some embodiments, the Noggin is a Noggin-containing supernatant. The supernatant can be isolated from a cell line that produces Noggin, of which a variety of cell lines are available.

[0092] The basal medium may comprise at least about 1 ng / ml, at least about 2 ng / ml, at least about 3 ng / ml, at least about 4 ng / ml, at least about 5 ng / ml, at least about 6 ng / ml, at least about 7 ng / ml, at least about 8 ng / ml, at least about 9 ng / ml, at least about 10 ng / ml, at least about 11 ng / ml, at least about 12 ng / ml, at least about 13 ng / ml, at least about 14 ng / ml, at least about 15 ng / ml, at least about 16 ng / ml, at least about 17 ng / ml, at least about 18 ng / ml, at least about 19 ng / ml, at least about 20 ng / ml, at least about 21 ng / ml, at least about 22 ng / ml, at least about 23 ng / ml, at least about 24 ng / ml, at least about 25 ng / ml, at least about 26 ng / ml, In some embodiments, the basal medium may comprise IL-2 at a concentration of at least about 10 ng / ml, at least about 27 ng / ml, at least about 28 ng / ml, at least about 29 ng / ml, at least about 30 ng / ml, at least about 31 ng / ml, at least about 32 ng / ml, at least about 33 ng / ml, at least about 34 ng / ml, at least about 35 ng / ml, at least about 36 ng / ml, at least about 37 ng / ml, at least about 38 ng / ml, at least about 39 ng / ml, at least about 40 ng / ml, at least about 41 ng / ml, at least about 42 ng / ml, at least about 43 ng / ml, at least about 44 ng / ml, at least about 45 ng / ml, at least about 46 ng / ml, at least about 47 ng / ml, at least about 48 ng / ml, at least about 49 ng / ml, or at least about 50 ng / ml. In some embodiments, the basal medium comprises IL-2 at a concentration of at least about 10 ng / ml. In some embodiments, the basal medium comprises IL-2 at a concentration of at least about 15 ng / ml, optionally at least about 20 ng / ml.

[0093] The basal medium may comprise IL-2 at a concentration of less than about 1000 ng / ml, less than about 900 ng / ml, less than about 800 ng / ml, less than about 700 ng / ml, less than about 600 ng / ml, less than about 500 ng / ml, less than about 400 ng / ml, less than about 300 ng / ml, less than about 200 ng / ml, less than about 100 ng / ml, or less than about 50 ng / ml. In some embodiments, the basal medium comprises IL-2 at a concentration of less than about 50 ng / ml.

[0094] In some embodiments, the basal medium comprises IL-2 at a concentration of about 1 ng / ml to about 1000 ng / ml. The basal medium may comprise IL-2 at a concentration of about 10 ng / ml to about 500 ng / ml, optionally at a concentration of about 10 ng / ml to about 100 ng / ml. In some embodiments, the basal medium comprises IL-2 at a concentration of about 10 ng / ml to about 50 ng / ml. In some embodiments, the basal medium comprises IL-2 at a concentration of about 20 ng / ml.

[0095] Preferably, the IL-2 is recombinant. In some embodiments, the IL-2 is murine or human. More preferably, the IL-2 is recombinant human IL-2.

[0096] The basal medium may comprise at least about 1 ng / ml, at least about 2 ng / ml, at least about 3 ng / ml, at least about 4 ng / ml, at least about 5 ng / ml, at least about 6 ng / ml, at least about 7 ng / ml, at least about 8 ng / ml, at least about 9 ng / ml, at least about 10 ng / ml, at least about 11 ng / ml, at least about 12 ng / ml, at least about 13 ng / ml, at least about 14 ng / ml, at least about 15 ng / ml, at least about 16 ng / ml, at least about 17 ng / ml, at least about 18 ng / ml, at least about 19 ng / ml, at least about 20 ng / ml, at least about 21 ng / ml, at least about 22 ng / ml, at least about 23 ng / ml, at least about 24 ng / ml, at least about 25 ng / ml, at least about 26 ng / ml, In some embodiments, the basal medium may comprise IL-7 at a concentration of at least about 10 ng / ml, at least about 27 ng / ml, at least about 28 ng / ml, at least about 29 ng / ml, at least about 30 ng / ml, at least about 31 ng / ml, at least about 32 ng / ml, at least about 33 ng / ml, at least about 34 ng / ml, at least about 35 ng / ml, at least about 36 ng / ml, at least about 37 ng / ml, at least about 38 ng / ml, at least about 39 ng / ml, at least about 40 ng / ml, at least about 41 ng / ml, at least about 42 ng / ml, at least about 43 ng / ml, at least about 44 ng / ml, at least about 45 ng / ml, at least about 46 ng / ml, at least about 47 ng / ml, at least about 48 ng / ml, at least about 49 ng / ml, or at least about 50 ng / ml. In some embodiments, the basal medium comprises IL-7 at a concentration of at least about 10 ng / ml. In some embodiments, the basal medium comprises IL-7 at a concentration of at least about 15 ng / ml, optionally at least about 20 ng / ml.

[0097] The basal medium may comprise IL-7 at a concentration of less than about 1000 ng / ml, less than about 900 ng / ml, less than about 800 ng / ml, less than about 700 ng / ml, less than about 600 ng / ml, less than about 500 ng / ml, less than about 400 ng / ml, less than about 300 ng / ml, less than about 200 ng / ml, less than about 100 ng / ml, or less than about 50 ng / ml. In some embodiments, the basal medium comprises IL-7 at a concentration of less than about 50 ng / ml.

[0098] In some embodiments, the basal medium comprises IL-7 at a concentration of about 1 ng / ml to about 1000 ng / ml. The basal medium may comprise IL-7 at a concentration of about 10 ng / ml to about 500 ng / ml, optionally at a concentration of about 10 ng / ml to about 100 ng / ml. In some embodiments, the basal medium comprises IL-7 at a concentration of about 10 ng / ml to about 50 ng / ml. In some embodiments, the basal medium comprises IL-7 at a concentration of about 20 ng / ml.

[0099] Preferably, the IL-7 is recombinant. In some embodiments, the IL-7 is murine or human. More preferably, the IL-7 is recombinant murine IL-7.

[0100] Preferably, the basal medium contains R-spondin at a concentration of about 1 μg / ml, noggin at a concentration of about 100 ng / ml, EGF at a concentration of about 50 ng / ml, IL-2 at a concentration of about 20 ng / ml, and IL-7 at a concentration of about 20 ng / ml.

[0101] R-spondin can be recombinant.In some embodiments, R-spondin is murine or human.In some embodiments, R-spondin is R-spondin-containing supernatant.Supernatant can be isolated from cell line that produces R-spondin, and various cell lines are available.

[0102] In some embodiments, the basal medium comprises at least about 0.1 ng / ml, at least about 0.2 ng / ml, at least about 0.3 ng / ml, at least about 0.4 ng / ml, at least about 0.5 ng / ml, at least about 0.6 ng / ml, at least about 0.7 ng / ml, at least about 0.8 ng / ml, at least about 0.9 ng / ml, at least about 1 ng / ml, at least about 1.1 ng / ml, at least about 1.2 ng / ml, at least about 1.3 ng / ml, at least about The basal medium may contain IL-15 at a concentration of 1.4 ng / ml, at least about 1.5 ng / ml, at least about 1.6 ng / ml, at least about 1.7 ng / ml, at least about 1.8 ng / ml, at least about 1.9 ng / ml, at least about 2 ng / ml, at least about 3 ng / ml, at least about 4 ng / ml, at least about 5 ng / ml, at least about 6 ng / ml, at least about 7 ng / ml, at least about 8 ng / ml, at least about 9 ng / ml, or at least about 10 ng / ml. The basal medium may contain IL-15 at a concentration of less than about 20 ng / ml, less than about 15 ng / ml, less than about 12 ng / ml, less than about 10 ng / ml, or less than about 5 ng / ml. In some embodiments, the basal medium contains IL-15 at a concentration of about 0.1 ng / ml to about 5 ng / ml. In some embodiments, the basal medium contains IL-15 at a concentration of about 0.5 ng / ml to about 2 ng / ml. In some embodiments, the basal medium comprises IL-15 at a concentration of about 1 ng / ml. The IL-15 can be recombinant.

[0103] In some embodiments, the basal medium comprises 2-mercaptoethanol. The basal medium may comprise at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 11 mM, at least 12 mM, at least 13 mM, at least 14 mM, at least 15 mM, at least 16 mM, at least 17 mM, at least 18 mM, at least 19 mM, at least 20 mM, at least 21 mM, at least 22 mM, at least 23 mM, at least 24 mM, at least 25 mM, at least 26 mM, at least 27 mM, at least 28 mM, at least 29 mM, at least 30 mM, at least 31 mM, at least 32 mM, at least 33 mM, at least at least 34 mM, at least 35 mM, at least 36 mM, at least 37 mM, at least 38 mM, at least 39 mM, at least 40 mM, at least 41 mM, at least 42 mM, at least 43 mM, at least 44 mM, at least 45 mM, at least 46 mM, at least 47 mM, at least 48 mM, at least 49 mM, at least 50 mM, at least 51 mM, at least 52 mM, at least 53 mM, at least 54 mM, at least 55 mM, at least 56 mM, at least 57 mM, at least 58 mM, at least 59 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, or at least 100 mM 2-mercaptoethanol.

[0104] In some embodiments, the basal medium comprises no more than 500 mM, no more than 400 mM, no more than 300 mM, no more than 200 mM, no more than 100 mM, no more than 90 mM, no more than 80 mM, no more than 70 mM, or no more than 60 mM 2-mercaptoethanol.

[0105] In some embodiments, the basal medium comprises 1 mM to 500 mM 2-mercaptoethanol. In some embodiments, the basal medium comprises 10 mM to 100 mM 2-mercaptoethanol. Optionally, the basal medium comprises about 50 mM 2-mercaptoethanol.

[0106] Preferably, the basal medium contains R-spondin at a concentration of about 1 μg / ml, noggin at a concentration of about 100 ng / ml, EGF at a concentration of about 50 ng / ml, IL-2 at a concentration of about 20 ng / ml, IL-7 at a concentration of about 20 ng / ml, IL-15 at a concentration of about 1 ng / ml, and about 20 μm 2-mercaptoethanol.

[0107] In some embodiments, the medium / basal medium does not contain detectable levels of one or more of IL-23, IL-1β, IL-15, IL-12, IL-18, IL-25, and IL-33. In the context of the present invention, a medium / basal medium that does not contain detectable levels of a specific marker refers to the fact that the marker is not exogenously added or is not contained in the medium. Therefore, a medium / basal medium that does not contain detectable levels of markers refers to the medium at the beginning of the method, and does not exclude that detectable levels of markers are produced by immune cells and / or epithelial organoids in the medium during the method. In some embodiments, the medium / basal medium does not contain detectable levels of IL-15, IL-12, and IL-18. In some embodiments, the medium / basal medium does not contain detectable levels of IL-1β, IL-15, IL-12, IL-18, and IL-25. In some embodiments, the medium / basal medium does not contain detectable levels of IL-1β, IL-15, IL-12, IL-18, IL-25, and IL-33. In some embodiments, the medium / basal medium does not contain detectable levels of IL-23, IL-1β, IL-15, IL-12, IL-18, and IL-25. In some embodiments, the medium / basal medium does not contain detectable levels of IL-23, IL-1β, IL-15, IL-12, IL-18, IL-25, and IL-33.

[0108] Preferably, the medium is sterile or specific pathogen free. Most preferably, the medium is Medium A as disclosed in the Examples.

[0109] Preferably, immune cells and at least one epithelial organoid are co-cultured in matrix.Matrix can comprise or consist of Geltrex, Cultrex or Matrigel.Other commercially available matrices, particularly synthetic hydrogels, are known to those skilled in the art.In some embodiments, matrix comprises or consists of Matrigel.More preferably, immune cells and at least one epithelial organoid are co-cultured in matrix in medium as defined above, preferably in basal medium.

[0110] In some embodiments, immune cells and at least one epithelial organoid are co-cultured in transwell.Typically, in this embodiment, permeable insert separates cell population.Therefore, in this embodiment, immune cells can be preferably separated from at least one epithelial organoid by permeable insert.

[0111] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the medium is changed about every 24 hours to about every 72 hours.

[0112] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the medium is changed about every 24 hours. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the medium is changed about every 48 hours. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the medium is changed about every 72 hours.

[0113] Preferably, about 10% to about 70% of the medium is replaced every about 24 to about 72 hours, and more preferably, about 50% of the medium is replaced every about 24 to about 72 hours. This allows the conditioned medium to be maintained with the cells while new growth factors are added.

[0114] Preferably, immune cells and at least one epithelial organoid are co-cultured at a temperature of at least about 20 ℃, at least about 25 ℃, at least about 30 ℃ or at least about 35 ℃.More preferably, immune cells and at least one epithelial organoid are co-cultured at a temperature of about 37 ℃.

[0115] In some embodiments, immune cells and at least one epithelial organoid are co-cultured in at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, or at least about 20% CO2.Preferably, immune cells and at least one epithelial organoid are co-cultured in about 1% to about 10% CO2.More preferably, immune cells and at least one epithelial organoid are co-cultured in about 5% CO2.

[0116] In some embodiments, epithelial organoid comprises metabolic product, for example bacterial metabolic product such as succinic acid or butyric acid.For example, epithelial organoid can be injected with metabolic product, preferably succinic acid.

[0117] In some embodiments, the immune cells are human immune cells, equine immune cells, feline immune cells, canine immune cells, bovine immune cells, ovine immune cells, or murine immune cells. In some embodiments, the immune cells are human immune cells or murine immune cells. Optionally, the murine immune cells are mouse immune cells. In some embodiments, the immune cells are human immune cells.

[0118] Preferably, the immune cells are primary immune cells. More preferably, the immune cells are human primary cells. "Primary cells" are understood to refer to cells obtained from a subject. Primary cells are not immortalized cells from a cell line.

[0119] The primary immune cells can be autologous. Alternatively, the primary immune cells can be allogeneic. In some embodiments, the primary immune cells comprise a mixture of allogeneic and autologous immune cells.

[0120] As those skilled in the art will understand, autologous cells are cells from the same subject, i.e., cells obtained from a subject and administered back to the same subject. Allogeneic cells are cells obtained from a subject different from the subject to which the cells are administered. The different subject is typically of the same species. Thus, allogeneic cells are genetically different from the subject to which they are administered.

[0121] Alternatively, the immune cells may comprise or consist of immortalized immune cells from a cell line.

[0122] In some embodiments, immune cell is primary immune cell, and epithelial organoid is primary epithelial organoid.In some embodiments, immune cell is primary immune cell, and epithelial organoid is derived from iPSC or adult stem cell.In some embodiments, immune cell is human primary immune cell, and epithelial organoid is human primary epithelial organoid.In some embodiments, immune cell is human primary immune cell, and epithelial organoid is derived from human iPSC or adult stem cell.

[0123] In some embodiments, immune cell is mouse primary immune cell, and epithelial organoid is human primary epithelial organoid.In some embodiments, immune cell is mouse primary immune cell, and epithelial organoid is derived from human iPSC or human adult stem cell.

[0124] In some embodiments, immune cell is human primary immune cell, and epithelial organoid is mouse primary epithelial organoid.Alternatively, immune cell can be human primary immune cell, and epithelial organoid can be derived from mouse iPSC or mouse adult stem cell.

[0125] In some embodiments, the immune cells are not mouse immune cells and the epithelial organoids are not mouse epithelial organoids.

[0126] Any immune cell is suitable for expansion in the methods of the present invention. However, the methods of the present invention are particularly useful for expanding immune cells that may be difficult to obtain in large numbers, or in numbers large enough for use in cell therapy. The methods of the present invention are also useful for generating and expanding immune cells from progenitor cells, such as hematopoietic stem cells and lymphoid progenitor cells. This advantageously allows for the generation of cells that may be difficult to obtain ex vivo, allowing for real-time studies of differentiation stages. Furthermore, the methods of the present invention can generate various immune cell types from a single progenitor cell type. This effectively makes the methods a "one-stop shop" for generating and expanding large numbers of different immune cells, reducing cost, complexity, and time. Thus, in some embodiments, the immune cells are hematopoietic stem cells and / or lymphoid progenitor cells.

[0127] As those skilled in the art will appreciate, hematopoietic stem cells are stem cells that can differentiate into myeloid and lymphoid cells. Myeloid cells include, but are not necessarily limited to, monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes. Lymphoid cells include T cells, B cells, and innate lymphoid cells (ILCs). Therefore, co-culturing hematopoietic stem cells eliminates the need for separate co-culturing of mature myeloid or lymphoid cells, since mature myeloid and lymphoid cells can all be generated in vitro from hematopoietic stem cells.

[0128] Lymphoid precursor cells may alternatively be referred to as lymphoid progenitor cells or thymocytes. As those skilled in the art will appreciate, lymphoid precursor cells arise from hematopoietic stem cells and are the precursor cell type to mature lymphoid cells. Thus, in some embodiments, immune cells comprise a mixture of hematopoietic stem cells and lymphoid precursor cells.

[0129] In some embodiments, immune cells are lymphocytes and / or bone marrow cells.In some embodiments, immune cells are bone marrow cells.For example, immune cells can be selected from one or more of the following cell types: monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes.In some embodiments, immune cells do not include macrophages.

[0130] In some embodiments, the immune cells are monocytes, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes, T cells, B cells, or innate lymphoid cells (ILCs). In some embodiments, the immune cells are neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes, T cells, B cells, or innate lymphoid cells (ILCs). In some embodiments, the immune cells are neutrophils, basophils, eosinophils, T cells, B cells, or innate lymphoid cells (ILCs).

[0131] In some embodiments, the immune cells are lymphoid cells. For example, the immune cells can be T cells, B cells, and / or innate lymphoid cells (ILCs). The immune cells can be human primary lymphoid cells, such as human primary T cells, B cells, and / or ILCs. In some embodiments, the immune cells comprise T cells and / or B cells. In some embodiments, the immune cells comprise T cells. The T cells can comprise or consist of αβ T cells. Alternatively, the T cells can comprise or consist of γδ T cells. The T cells can comprise or consist of natural killer T (NKT) cells. In some embodiments, the T cells are CD4 + In another embodiment, the T cells are CD8 + In some embodiments, the T cells are CD4 + T cells and CD8 + Contains T cells.

[0132] In other embodiments, the immune cells include or are ILCs. As those skilled in the art will appreciate, ILCs are the natural counterparts of T cells and can express cytokines at detectable levels. ILCs are typically found in mucosal barriers in vivo and can be identified by those skilled in the art.

[0133] In some embodiments, the immune cells comprise or consist of human or murine ILCs.

[0134] In the context of the present invention, ILCs can be classified into three groups: group 1, group 2, and group 3 ILCs. Group 1 ILCs may include NK cells and ILC1 cells. NK cells are preferably cytotoxic NK cells. Group 2 ILCs may include ILC2 cells, while group 3 may include ILC3 and lymphoid tissue inducer (LTi) cells. Group 2 and group 3 ILCs typically express CD127. Human regulatory ILCs (ILCreg) are a subset of ILCs identified by the present inventors and are defined in more detail below.

[0135] ILC subsets can be identified using a variety of methods: ILC subsets can be identified using flow cytometry and / or RNA sequencing to determine their expression profiles.

[0136] Murine Group 1 ILCs are Lineage - , NK1.1 + Murine Group 1 ILCs may include NKp46 + or NKp46 - In some embodiments, murine Group 1 ILCs may further comprise an expression profile of NKp46 + Murine group 1 ILCs express T-bet + In some embodiments, the Group 1 ILCs may further comprise an expression profile. -, RORγt - Klrg1 - CRTh2 - NK1.1 + NKp46 + In some embodiments, Group 1 ILCs comprise a Lineage - , Klrg1 - , NK1.1 + , NKp46 + It can further comprise expression profile.When a cell is positive for a certain marker, "expression profile" is understood to mean that the marker is detectable in or on the cell.When a cell is negative for a certain marker, it is understood to mean that the marker is not detectable in the cell.Expression can be determined by measuring the expression level of mRNA or protein.

[0137] Murine group 1 ILCs express CD49a + , CD49b + , CXCR6 + , CD200r1 + , and Ly49 receptor family + The expression profile may further include one or more of the following:

[0138] Human group 1 ILCs are - , RORγt - , CD127 + / - , CD56 + / - , CD161 + / - It may include an expression profile.

[0139] Human Group 1 ILCs preferably express detectable levels of one or more of the following genes: AOAH, CCL3, CCL4, CCL5, CD244, CD247, CST7, EOMES, FCGR3A, FGR, GNLY, GZMB, GZMK, IFNG, IKZF3, IL12RB2, ITGAX, ITGB2, KLRC1, KLRD1, NCAM1, NCR1, NKG7, PRF1, SAMD3, TBX21, TIGIT, and ZNF683. Human Group 1 ILCs preferably express detectable levels of all of these genes. Gene expression is typically measured by measuring messenger RNA (mRNA) expression, for example, using RNA sequencing or single-cell RNAseq (scRNAseq) as described in Example 8. As explained above, human Group 1 ILCs preferably include human NK cells and / or human ILC1 cells.

[0140] Murine Group 2 ILCs are Lineage - , CD127 + , RORγt - , GATA-3 + , Klrg1 + ILC2 + Murine group 2 ILCs may include a Lineage - , GATA-3 + , ST2 + ILC2 + Alternatively, murine group 2 ILCs may be expressed using the Lineage - , GATA-3 + , ICOS + ILC2 + It may include an expression profile.

[0141] Human group 2 ILCs are - , RORγt - , CD127 +, GATA-3 + , CRTH2 + , c-Kit + / - Alternatively, human group 2 ILCs may be identified by a Lineage - , GATA-3 + , CRTH2 + , c-Kit + / - , ST2 + It may include an expression profile.

[0142] Human Group 2 ILCs preferably express detectable levels of one or more of the following genes: ANXA1, BCL11B, CCR2, GATA3, HPGD, HPGDS, IL10RA, IL13, IL17RB, IL32, IL5, IL9R, KLRG1, LGALS1, MAF, MBOAT2, PPARG, PTGDR2, PTGER2, and TNFSF10, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Human Group 2 ILCs preferably express detectable levels of all of these genes. Gene expression is typically measured by measuring messenger RNA (mRNA) expression, for example, using RNA sequencing or single-cell RNAseq (scRNAseq) as described in Example 8. Murid Group 3 ILCs are Lineage - , CD127 + , RORγt - , NK1.1 + / - , NKp46 + / - , CCR6 + / , CD4 + / - It may include an expression profile.

[0143] Human group 3 ILCs are - , CD127 + , RORγt - , NKp44 + / - , c-Kit + / - , CCR6 + / - , HLA-DR + / - It may include an expression profile.

[0144] Human Group 3 ILCs preferably express detectable levels of one or more of the following genes, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22: AHR, BCL2A1, CCL20, CD81, CSF2, CXCL8, ID2, IKZF2, IL1R1, IL23R, IL4I1, IRF4, KIT, LIF, LTA4H, NCR1, PECAM1, RBPJ, RORC, TNFRSF25, TNFSF4, and TOX2. Human Group 3 ILCs preferably express detectable levels of all of these genes. Gene expression is typically measured by measuring messenger RNA (mRNA) expression, for example, using RNA sequencing or single-cell RNAseq (scRNAseq) as shown in Example 8.

[0145] As used herein with respect to mouse ILCs, "Lineage" refers to - " is CD3 - CD19 - Ly6G - As used herein with respect to human ILCs, "Lineage" refers to a specific expression profile. - " is CD3 - CD20 - CD14 - CD19 - It will be understood to refer to an expression profile.

[0146] The present invention also provides human regulatory ILCs (ILCregs) that express detectable levels of FOXP3. Preferably, human ILCregs further express one or more of the following genes at detectable levels: CCR5, CTLA4, FGGY, GATA3, GZMB, IL10, IL1R1, IL2RA, IL2RB, KAT2B, LGLS3, PIM1, PRDM1, RUNX1, SOX4, TNFRSF18, and TRAF1, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17. Preferably, human ILCregs express all of these genes at detectable levels. Gene expression is typically measured by measuring messenger RNA (mRNA) expression, for example, using RNA sequencing or single-cell RNA sequencing (scRNA sequencing). It is noteworthy that FOXP3, RUNX1, IL1R1, and CTLA4 are associated with human regulatory T cells (Tregs). Human ILCregs preferably further secrete detectable levels of IL-10, which can be measured using a standard cytokine release assay. Human ILCregs preferably express one or more, for example, two or three, of the following cell surface markers at detectable levels: CD25 (IL2RA), CD127, and CTLA4. Human ILCregs preferably express detectable levels of CD25 (IL2RA), CD127, CTLA4, CD25 and CD127, CD25 and CTLA4, CD127 and CTLA4, or CD25, CD127, and CTLA4. Surface marker expression can be measured using standard methods, such as flow cytometry.

[0147] The present invention also provides human regulatory ILCs (ILCregs) that express one or more, for example, two or three, of the following cell surface markers at detectable levels: CD25 (IL2RA), CD127, and CTLA4. Human ILCregs preferably express detectable levels of CD25 (IL2RA), CD127, CTLA4, CD25 and CD127, CD25 and CTLA4, CD127 and CTLA4, or CD25, CD127, and CTLA4. Surface marker expression can be measured using standard methods, such as flow cytometry. Human ILCregs preferably further express detectable levels of FOXP3. Human ILCregs preferably further express one or more of the following genes at detectable levels: CCR5, FGGY, GATA3, GZMB, IL10, IL1R1, IL2RB, KAT2B, LGLS3, PIM1, PRDM1, RUNX1, SOX4, TNFRSF18, and TRAF1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). Human ILCregs preferably express all of these genes at detectable levels. Gene expression is typically measured by measuring messenger RNA (mRNA) expression, for example, using RNA sequencing or single-cell RNA sequencing (scRNA sequencing). It is noteworthy that FOXP3, RUNX1, IL1R1, and CTLA4 are associated with human regulatory T cells (Tregs). Human ILCregs preferably further secrete detectable levels of IL-10. This can be measured using standard cytokine release assays.

[0148] In any of the above-described embodiments, human ILCregs preferably do not express one or more, such as 2, 3, 4, 5, or 6, of CD3, CD4, CD19, CD20, TCRαβ, and TCRγδ at detectable levels. Human ILCregs preferably do not express one or more, such as 2, 3, 4, or 5, of CD3, CD4, CD19, CD20, and TCRαβ at detectable levels. Human ILCregs preferably do not express detectable levels of one or more of these markers at the cell surface and / or RNA level. Human ILCregs preferably do not express detectable levels of CD3 and CD4. Human ILCregs preferably do not express detectable levels of either of these markers. Human ILCregs preferably do not express CD3 - , CD4 - , CD19 - , CD20 - , TCRαβ - , TCRγδ - The human ILCreg preferably comprises a CD3 - , CD4 - , CD19 - , CD20 - , TCRαβ - Includes expression profiles.

[0149] In some embodiments, the ILCs comprise one or more of Group 1, Group 2, and Group 3 ILCs. In some embodiments, the ILCs comprise at least Group 1 ILCs. For example, the ILCs may comprise NK cells and / or ILC1 cells. The NK cells may comprise a CD127 expression profile. The NK cells are preferably cytotoxic NK cells. ILC1 cells have a CD127 expression profile. + In some embodiments, the ILCs comprise or consist of NK cells. The NK cells are preferably cytotoxic NK cells. This is shown in Example 9.

[0150] In some embodiments, the ILCs comprise at least Group 2 ILCs. In some embodiments, the ILCs comprise at least Group 3 ILCs.

[0151] In some embodiments, the ILCs comprise one or more of Group 1 ILCs, Group 2 ILCs, Group 3 ILCs, and ILCregs. In some embodiments, the ILCs comprise at least Group 1 ILCs. In some embodiments, the ILCs comprise at least Group 2 ILCs. In some embodiments, the ILCs comprise at least Group 3 ILCs. In some embodiments, the ILCs comprise at least ILCregs. The ILCregs may be murine ILCregs as described in Wang et al. (2017). The ILCregs are preferably human ILCregs of the present invention. The present invention also provides a plurality or population of two or more human ILCregs of the present invention, as described in more detail below.

[0152] ILC1 cells express RORγt - Klrg1 - NK1.1 + / - NKp46 + Eomes - T-bet + The expression profile may include: Human ILC1 cells may express one or more of the above-listed genes at detectable levels.

[0153] NK cells are T-bet + Eomes + Alternatively, NK cells may express T-bet - Eomes + The expression profile of the NK cells may include a cytotoxic NK cell. The NK cells are preferably cytotoxic NK cells. Human NK cells preferably express one or more of the above-listed genes at detectable levels.

[0154] The present inventors unexpectedly found that a large number of different immune cell types can be expanded using the methods of the present invention. In other words, a large number of different immune cell types can be expanded under the same conditions. Thus, in some embodiments, the immune cells comprise a plurality of different immune cell subsets. Preferably, the immune cells comprise a plurality of different lymphoid cells, more preferably a plurality of different ILC groups. For example, the immune cells may comprise a plurality of Group 1 and Group 2 ILCs. In some embodiments, the immune cells comprise a plurality of Group 1 and Group 3 ILCs. Alternatively, the immune cells may comprise a plurality of Group 2 and Group 3 ILCs. In some embodiments, the immune cells comprise a plurality of Group 1 ILCs and ILCregs. In some embodiments, the immune cells comprise a plurality of ILCregs and Group 2 ILCs. In some embodiments, the immune cells comprise a plurality of ILCregs and Group 3 ILCs. In some embodiments, the immune cells comprise a plurality of Group 1 ILCs, Group 2 ILCs, and ILCregs. In some embodiments, the immune cells comprise a plurality of Group 1 ILCs, Group 3 ILCs, and ILCregs. In some embodiments, the immune cells comprise a plurality of Group 2 ILCs, Group 3 ILCs, and ILCregs. In some embodiments, the immune cells comprise a plurality of Group 1 ILCs, Group 2 ILCs, Group 3 ILCs, and ILCregs. The ILCregs may be murine ILCregs as described in Wang et al. (2017). The ILCregs are preferably human ILCregs of the present invention.

[0155] Without wishing to be bound by theory, the inventors believe that the ability to generate multiple different cell types more accurately represents the in vivo environment, thereby enabling the generation of immune cells more suitable for cell therapy. In some embodiments, the method generates a heterogeneous population of immune cells. A heterogeneous population of immune cells may comprise or consist of the same immune cell type (e.g., ILCs or T cells), but may have different expression profiles. For example, a heterogeneous population of immune cells may comprise a heterogeneous population of ILCs. The inventors believe that such heterogeneous populations more accurately represent in vivo populations, thereby increasing their usefulness in in vitro studies. Without wishing to be bound by theory, the inventors also believe that such heterogeneous populations more accurately represent in vivo populations, thereby improving viability and function. This may be particularly useful for therapeutic applications.

[0156] Alternatively, in other embodiments, the method generates a homogeneous population of immune cells.

[0157] In some embodiments, one or more of multiple different immune cell subsets are enriched over time. For example, in embodiments comprising multiple different ILC groups, preferably Group 1 ILCs, optionally ILC1s and / or NK cells, may be specifically enriched over time. In embodiments comprising multiple different ILC groups, Group 3 ILCs may be enriched over time. Alternatively, in embodiments comprising multiple different ILC groups, Group 2 ILCs may be enriched over time. In embodiments comprising multiple different ILC groups, Group 1 and Group 3 ILCs may be enriched over time. In embodiments comprising multiple different ILC groups, ILCregs may be enriched over time. ILCregs may be murine ILCregs, as described in Wang et al. (2017). ILCregs are preferably human ILCregs of the present invention.

[0158] In some embodiments, tissue-specific immune cells can be enriched over time.Tissue-specific immune cells are described herein.For example, when epithelial organoid is intestinal organoid, intestinal specific immune cells can be enriched over time.When epithelial organoid is lung organoid, lung specific immune cells can be enriched over time.

[0159] Advantageously, the method can generate a substantial number of immune cells from minimal starting material, e.g., from a negligible level of immune cells, such as only one immune cell. Thus, the method can also include a prior step of expanding the immune cells to generate a plurality of immune cells. Thus, the method can include: (ia) culturing the immune cells to generate immune cells; and The method may include (a) co-culturing immune cells with at least one epithelial organoid, wherein the epithelial organoid comprises more epithelial cells than mesenchymal cells.

[0160] In some embodiments, the method is for generating and expanding immune cells, the method comprising, prior to step (a): This involves co-culturing immune cell progenitor cells with epithelial organoids to expand the immune cell progenitor cells and differentiate them into immune cells.

[0161] As one skilled in the art will appreciate, an immune cell precursor is a progenitor cell of an immune cell. Thus, expansion and differentiation of an immune cell precursor results in the generation of an immune cell. In some embodiments, the immune cell precursor is a lymphocyte precursor cell as defined above. In some embodiments, the method includes a prior step of expanding the immune cell precursor to generate a plurality of immune cell precursors. Thus, in some embodiments, the method is for the generation and expansion of immune cells, the method comprising, prior to step (a), Culturing immune cell progenitor cells to generate immune cell progenitor cells; and This involves co-culturing immune cell progenitor cells with epithelial organoids to expand the immune cell progenitor cells and differentiate them into immune cells.

[0162] In some embodiments, the immune cell precursor cells are human immune cell precursor cells, equine immune cell precursor cells, feline immune cell precursor cells, canine immune cell precursor cells, bovine immune cell precursor cells, ovine immune cell precursor cells, or murine immune cell precursor cells. In some embodiments, the immune cell precursor cells are human immune cell precursor cells or murine immune cell precursor cells. Optionally, the murine immune cell precursor cells are mouse immune cell precursor cells. In some embodiments, the immune cell precursor cells are human immune cell precursor cells.

[0163] The immune cell progenitor cells may comprise or consist of murine or human ILC progenitor cells.

[0164] The murine immune cell progenitor cells may comprise or consist of murine ILC progenitor cells. In such embodiments, it will be understood that the resulting immune cells will be murine ILCs. In the context of the present invention, murine ILC progenitor cells are characterized by the expression of CD127 + , Lineage - In some embodiments, the murine ILC progenitor cells may comprise an expression profile of CD127 + , Lineage - α4β7 + In some embodiments, the murine ILC progenitor cells comprise a CD127 + , Lineage - , PD-1 + In some embodiments, the murine ILC progenitor cells comprise a CD127 + , Lineage - , PD-1 + , α4β7 + In some embodiments, the murine ILC progenitor cells comprise a CD127 + , Lineage - , α4β7 + , PD-1+ , Flt3 - , CD25 - , c-KIT + Expression profiles include: A variety of methods can be used to determine expression profiles, such as flow cytometry, fluorescence microscopy, RT-PCR, and RNA sequencing.

[0165] The human immune cell progenitor cells may comprise or consist of human ILC progenitor cells. In such embodiments, it will be understood that the resulting immune cells will be human ILCs. In the context of the present invention, human ILC progenitor cells are Lineage ILCs. - , CD127 + In some embodiments, the human ILC progenitor cells may comprise a Lineage Expression Profile. - , CD127 + , CD7 + / - , c-Kit + , CRTh2 - , KLRG1 - , CD56 - , NKp46 - In some embodiments, the human ILC progenitor cells comprise a Lineage Expression Profile. - , CD127 + , c-Kit + Human ILC progenitors are lineage-specific. - , CD34 + Alternatively, the human ILC progenitor cells may be expressed in a Lineage - , CD127 + , CD45RA + , CD62L - The expression profile may include a variety of methods for determining the expression profile, such as flow cytometry, fluorescence microscopy, RT-PCR, and RNA sequencing.

[0166] As used herein with respect to mouse ILC progenitors, "Lineage" refers to - " is CD3 - CD11b- TER-119 - Ly-G6 - CD5 - CD19 - and NK1.1 - As used herein with respect to human ILC progenitors, "Lineage" refers to a specific expression profile. - " is CD3 - , CD4 - , CD19 - , CD20 - , TCRαβ - , TCRγδ - It will be understood to refer to an expression profile.

[0167] Preferably, the immune cell precursor cells are primary immune cell precursor cells. More preferably, the immune cell precursor cells are human primary immune cell precursor cells. "Primary cells" are understood to refer to cells obtained from a subject. Primary cells are not immortalized cells from a cell line.

[0168] The primary immune cell precursor cells can be autologous. Alternatively, the primary immune cell precursor cells can be allogeneic. In some embodiments, the primary immune cell precursor cells comprise a mixture of allogeneic and autologous immune cell precursor cells.

[0169] Alternatively, the immune cell precursor cells may comprise or consist of immortalized immune cell precursor cells from a cell line.

[0170] In some embodiments, immune cell precursor cell is primary immune cell precursor cell, and epithelial organoid is primary epithelial organoid.In some embodiments, immune cell precursor cell is primary immune cell precursor cell, and epithelial organoid is derived from stem cell, preferably iPSC or adult stem cell.In some embodiments, immune cell precursor cell is human primary immune cell precursor cell, and epithelial organoid is human primary epithelial organoid.In some embodiments, immune cell precursor cell is human primary immune cell precursor cell, and epithelial organoid is derived from human iPSC or human adult stem cell.

[0171] In some embodiments, immune cell precursor is mouse primary immune cell precursor, and epithelial organoid is human primary epithelial organoid.In some embodiments, immune cell precursor is mouse primary immune cell precursor, and epithelial organoid is derived from human iPSC or human adult stem cell.

[0172] In some embodiments, immune cell progenitor cell is human primary immune cell progenitor cell, and epithelial organoid is mouse primary epithelial organoid.Alternatively, immune cell progenitor cell can be human primary immune cell progenitor cell, and epithelial organoid can be derived from mouse embryonic stem cell (ESC), mouse adult stem cell or mouse iPSC, and preferably derived from mouse ESC or mouse adult stem cell.

[0173] In some embodiments, the immune cell progenitor cells are not murine immune cell progenitor cells and the epithelial organoids are not murine epithelial organoids.

[0174] In the context of the present invention, primary immune cells or primary immune cell precursor cells are understood to be isolated primary immune cells or primary immune cell precursor cells, given the in vitro nature of the methods of the present invention. Primary immune cells or primary immune cell precursor cells are isolated from blood, bone marrow, fetal liver, tonsils, or the intestinal tract. In some embodiments, primary immune cells or primary immune cell precursor cells are isolated blood or bone marrow primary immune cells or primary immune cell precursor cells. In some embodiments, primary immune cells or primary immune cell precursor cells are isolated blood primary immune cells or primary immune cell precursor cells.

[0175] Advantageously, this method can be maintained for a long period of time, thereby can continuously and reliably produce a large number of immune cells.In some embodiments, immune cell and epithelial organoid are co-cultured for at least about 72 hours.In some embodiments, immune cell and epithelial organoid are co-cultured for at least about 96 hours, at least about 120 hours, at least about 144 hours, at least about 168 hours, at least about 192 hours, at least about 216 hours, at least about 240 hours, at least about 264 hours, at least about 288 hours, at least about 312 hours, at least about 336 hours, at least about 504 hours, at least about 672 hours or at least about 1008 hours.

[0176] In some embodiments, the immune cells and epithelial organoids are co-cultured for about 1440 hours, about 1008 hours, about 720 hours, about 672 hours, or about 504 hours or less.

[0177] In some embodiments, immune cells and epithelial organoids are co-cultured for about 96 hours to about 504 hours.Preferably, immune cells and epithelial organoids are co-cultured for about 120 hours to about 504 hours.More preferably, immune cells and epithelial organoids are co-cultured for about 168 hours to about 336 hours.

[0178] During the methods of the present invention, the immune cells preferably expand at a rate of at least 2-fold about every 24 hours. In some embodiments, the immune cells expand at a rate of at least 3-fold about every 24 hours.

[0179] The immune cells may expand at a rate of at least about 4-fold every 48 hours, optionally at a rate of at least about 6-fold every 48 hours.

[0180] Optionally, the immune cells expand at a rate of at least 256-fold about every 168 hours.

[0181] In some embodiments, the immune cells expand at a rate of at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 450-fold, at least 500-fold, at least 550-fold, at least 600-fold, at least 650-fold, at least 700-fold, at least 750-fold, at least 800-fold, at least 850-fold, or at least 900-fold per about 336 hours of co-culture.

[0182] In some embodiments, immune cells expand at a rate of 5000-fold or less, 2000-fold or less, 1000-fold or less, or 950-fold or less per about 336 hours of co-culture. For example, immune cells may expand at a rate of 400-fold to 900-fold per about 336 hours of co-culture. In some embodiments, immune cells expand at a rate of 500-fold to 1000-fold per about 336 hours of co-culture. Immune cells may expand at a rate of 500-fold to 800-fold per about 336 hours of co-culture. In some embodiments, immune cells expand at a rate of about 750-fold per about 336 hours of co-culture.

[0183] The present invention also provides immune cells obtained by the methods of the present invention. 3 Also provided is an in vitro innate lymphoid cell (ILC) population comprising 100 ILCs.

[0184] The present invention also provides an in vitro human ILCreg population comprising at least about two human ILCregs of the invention, preferably at least about 5, at least about 10, at least about 50, at least about 100, at least about 1000, or at least about 5 x 10 3 The human ILCreg of the present invention may be any of those defined above.

[0185] In all embodiments of the present invention, the in vitro population includes a cell population in a format suitable for administration to a subject. This may include a vial, bag, or needle containing the cells. The cells may be in aqueous solution or frozen form.

[0186] In some embodiments, the population is at least about 5 x 10 3 ILCs, at least about 1 x 10 4 ILCs, at least about 5 x 10 4 ILCs, at least about 1 x 10 5 ILCs, at least about 5 x 10 5 ILCs, at least about 1 x 10 6 ILCs, at least about 5 x 10 6 ILCs, at least about 1 x 10 7 ILCs, at least about 5 x 10 7 ILCs, at least about 1 x 10 8 ILCs, at least about 5 x 10 8 ILCs, at least about 1 x 10 9 ILCs, at least about 5 x 10 9 ILCs, at least about 1 x 10 10 ILCs, at least about 5 x 10 10 ILCs, at least about 1 x 10 11 ILCs, at least about 1 x 10 12 ILCs, at least about 1 x 10 13 ILCs, at least about 1 x 10 14 ILCs, at least about 1 x 10 15 ILCs, at least about 1x10 16 ILCs, at least about 1x10 17 ILCs, at least about 1x10 18 ILCs, at least about 1 x 10 19 ILCs, at least about 1 x 10 20 ILCs, at least about 1 x 10 25 ILCs, at least about 1 x 10 30 ILCs, at least about 1 x 10 35 ILCs, at least about 1 x 10 40ILCs, at least about 1 x 10 45 ILCs, at least about 1 x 10 50 ILCs, at least about 1 x 10 60 ILCs, at least about 1 x 10 70 ILCs, at least about 1 x 10 80 ILCs, at least about 1 x 10 90 ILCs, at least about 1 x 10 100 ILCs, at least about 1 x 10 150 ILCs, at least about 1 x 10 200 ILCs, at least about 1 x 10 250 ILCs, at least about 1 x 10 300 ILCs, at least about 1 x 10 350 ILCs, at least about 1 x 10 400 ILCs, at least about 1 x 10 450 ILCs, at least about 1 x 10 500 ILCs, at least about 1 x 10 550 ILCs, at least about 1 x 10 600 ILCs, at least about 1 x 10 650 ILCs, at least about 1 x 10 700 ILCs, at least about 1 x 10 750 ILCs, at least about 1 x 10 850 ILCs, at least about 1 x 10 950 ILCs, at least about 1 x 10 1000 ILCs, at least about 1 x 10 2000 ILCs, at least about 1 x 10 3000 ILCs, at least about 1 x 10 4000 ILCs, at least about 1 x 10 4000 ILCs, at least about 1 x 10 5000 ILCs, at least about 1 x 10 6000 ILCs, at least about 1 x 10 7000 ILCs, at least about 1 x 10 8000 ILCs, at least about 1 x 10 9000 ILCs, or at least about 1 x 10 10000In any of these embodiments, the ILCs may be ILCregs, murine ILCregs, or human ILCregs of the invention.

[0187] In some embodiments, the population is at least about 1 x 10 20 In some embodiments, the population comprises at least about 1 x 10 ILCs. 100 In some embodiments, the population comprises at least about 1 x 10 ILCs. 500 In some embodiments, the population comprises at least about 1 x 10 ILCs. 1000 In any of these embodiments, the ILCs may be ILCregs, murine ILCregs, or human ILCregs of the invention.

[0188] In some embodiments, the population is about 1 x 10 10000 In some embodiments, the population comprises about 1 x 10 ILCs or less. 500 In some embodiments, the population comprises about 5 x 10 ILCs or less. 4 ILC ~ approx. 1 × 10 10000 In some embodiments, the population comprises about 1 x 10 ILCs. 200 ILC ~ approx. 1 × 10 10000 In any of these embodiments, the ILCs may be ILCregs, murine ILCregs, or human ILCregs of the invention.

[0189] ILCs can be as defined above. For example, an in vitro ILC population can include or consist of human or murine ILCs. An in vitro ILC population can include one or more of group 1, group 2, and group 3 ILCs. An in vitro ILC population can include one or more of group 1 ILCs, group 2 ILCs, group 3 ILCs, and ILCregs. Murine and human group 1, group 2, and group 3 ILCs can be identified as described above. Mouse ILCregs can be identified as described in Wang et al. (2017). Human ILCregs can be identified as described above.

[0190] In some embodiments, the ILCs comprise at least Group 1 ILCs. Group 1 ILCs may comprise NK cells and / or ILC1 cells. The NK cells are preferably cytotoxic NK cells. This is shown in Example 9.

[0191] An in vitro ILC population may comprise a plurality of Group 1 and Group 2 ILCs. In some embodiments, an in vitro ILC population comprises a plurality of Group 1 and Group 3 ILCs. Alternatively, an in vitro ILC population may comprise a plurality of Group 2 and Group 3 ILCs. In some embodiments, an in vitro ILC population comprises a plurality of Group 1, Group 2, and Group 3 ILCs. In some embodiments, an in vitro ILC population comprises a plurality of Group 1 ILCs and ILCregs. In some embodiments, an in vitro ILC population comprises a plurality of ILCregs and Group 2 ILCs. In some embodiments, an in vitro ILC population comprises a plurality of ILCregs and Group 3 ILCs. In some embodiments, an in vitro ILC population comprises a plurality of Group 1 ILCs, Group 2 ILCs, and ILCregs. In some embodiments, an in vitro ILC population comprises a plurality of Group 1 ILCs, Group 3 ILCs, and ILCregs. In some embodiments, the in vitro ILC population comprises a plurality of Group 2 ILCs, Group 3 ILCs, and ILCregs. In some embodiments, the in vitro ILC population comprises a plurality of Group 1 ILCs, Group 2 ILCs, Group 3 ILCs, and ILCregs. The ILCregs may be murine ILCregs as described in Wang et al. (2017). The ILCregs are preferably human ILCregs of the present invention.

[0192] The in vitro ILC population can comprise at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% Group 1 ILCs.

[0193] An in vitro ILC population can comprise at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% NK cells. The NK cells are preferably cytotoxic NK cells.

[0194] The in vitro ILC population may comprise at least about 1%, at least about 5%, at least about 7%, or at least about 10% Group 2 ILCs. In some embodiments, the in vitro ILC population comprises no more than about 20% or no more than about 10% Group 2 ILCs.

[0195] The in vitro ILC population can comprise at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% Group 3 ILCs. In some embodiments, the in vitro ILC population comprises no more than about 90%, no more than about 80%, no more than about 75%, or no more than about 70% Group 3 ILCs.

[0196] An in vitro ILC population may comprise at least about 0.1%, at least about 0.2%, at least about 0.5%, at least about 0.8%, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% ILCregs, murine ILCregs, or human ILCregs of the present invention. The in vitro human ILCreg population of the present invention may comprise at least about 0.1%, at least about 0.2%, at least about 0.5%, at least about 0.8%, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the human ILCreg of the present invention.

[0197] Without wishing to be bound by theory, the inventors believe that ILC ratios may be dysregulated in some diseases, and therefore, certain tissue-specific ratios may be particularly useful in treating such diseases.

[0198] In some embodiments, the population comprises about 95% NK cells. This frequency of NK cells may be particularly beneficial for treating pulmonary diseases, as it may be associated with healthy human lung tissue. The NK cells are preferably cytotoxic NK cells. This is shown in Example 9.

[0199] In some embodiments, the population comprises about 60% Group 1 ILCs and about 20-40% Group 3 ILCs. In some embodiments, the population comprises about 60% Group 1 ILCs, about 35% Group 3 ILCs, and about 5% Group 2 ILCs.

[0200] In some embodiments, the population comprises about 90% Group 3 ILCs and about 10% Group 1 ILCs. In some embodiments, the population comprises an undetectable percentage of Group 2 ILCs. Such frequencies may be particularly beneficial for treating diseases of the colon or intestinal tract, as such frequencies may be associated with healthy human colon tissue.

[0201] In some embodiments, the population comprises approximately 60-70% Group 3 ILCs and approximately 20-30% NK cells. For example, the population may comprise approximately 66% Group 3 ILCs and approximately 27% NK cells. Such a population may be associated with healthy human tonsillar tissue. The NK cells are preferably cytotoxic NK cells. This is shown in Example 9.

[0202] In some embodiments, the population comprises about 40-50% Group 3 ILCs and about 30-50% Group ILCs. For example, the population may comprise about 48% Group 3 ILCs and about 40% Group ILCs. Such a population may be associated with healthy human tonsillar tissue.

[0203] In some embodiments, the in vitro ILC population is an in vitro NK cell population. The NK cells are preferably cytotoxic NK cells. This is shown in Example 9.

[0204] In some embodiments, the in vitro ILC population is a heterogeneous ILC population. In other embodiments, the in vitro ILC population is a homogeneous ILC population.

[0205] In some embodiments, at least 10% of the ILCs express detectable levels of one or more of NKp44, NKp46, CD56, c-KIT, ST2, CRTh2, Klrg1, CD122, CD127, T-bet, GATA3, ROR-yt, ID2, IL-10, IL-5, IL-13, IL-17a, IFN-γ, amphiregulin, granzyme B, perforin, or TGFβ. In some embodiments, the ILCs express detectable levels of one or more of IL-22, IL-5, IL-13, IL-17a, IFN-γ, amphiregulin, granzyme B, perforin, NKp44, T-bet, GATA3, ROR-yt, and HLA-DR. ILCs are preferably capable of antigen processing. This is shown in Example 7.

[0206] In some embodiments, at least 10% of the ILCs express detectable levels of HLA-DR. In some embodiments, the ILCs express detectable levels of HLA-DR. The ILCs are preferably capable of antigen processing. This is shown in Example 7.

[0207] In some embodiments, at least 10% of the ILCs express detectable levels of one or more receptors for IL-23, IL-1β, IL-15, IL-12, IL-18, IL-25, and IL-33.

[0208] In some embodiments, the in vitro ILC population comprises Group 1 ILCs. Group 1 ILCs may express detectable levels of IFN-γ. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the Group 1 ILCs express detectable levels of IFN-γ. In some embodiments, at least 30% of the Group 1 ILCs express detectable levels of IFN-γ.

[0209] The ILCs can express detectable levels of T-bet. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ILCs express detectable levels of T-bet.

[0210] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ILCs express detectable levels of GATA3. In some embodiments, at least 30% of the ILCs express detectable levels of GATA3. In some embodiments, at least 50% of the ILCs express detectable levels of GATA3. The ILCs may express detectable levels of GATA3.

[0211] At least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ILCs may express detectable levels of IL-13. In some embodiments, at least 50%, preferably at least 60%, of the ILCs express detectable levels of IL-13. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ILCs may express detectable levels of IL-5. In some embodiments, at least 40% of the ILCs express detectable levels of IL-5. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ILCs co-express detectable levels of IL-5 and IL-13. In some embodiments, at least 50% of the ILCs co-express detectable levels of IL-5 and IL-13.

[0212] ILCs may express detectable levels of IL-5 and / or IL-13. In some embodiments, ILCs co-express detectable levels of IL-5 and IL-13.

[0213] At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ILCs may express detectable levels of IL-22. In some embodiments, at least 30%, preferably at least 40%, of the ILCs express detectable levels of IL-22. The ILCs may express detectable levels of IL-22.

[0214] At least 1%, at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ILCs may express detectable levels of NKp44. In some embodiments, at least 10% of the ILCs express detectable levels of NKp44. In some embodiments, at least 15% of the ILCs express detectable levels of NKp44. In some embodiments, at least 20% of the ILCs express detectable levels of NKp44. In some embodiments, about 10% to about 30% of the ILCs, preferably about 15% to about 25% of the ILCs, express detectable levels of NKp44. The ILCs may express detectable levels of NKp44.

[0215] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ILCs express IL-22. In some embodiments, at least 20% of the ILCs express IL-22. In some embodiments, at least 50% of the ILCs express IL-22. In some embodiments, about 10% to about 70% of the ILCs express IL-22.

[0216] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ILCs express IL-17a. In some embodiments, at least 20% of the ILCs express IL-17a. In some embodiments, at least 50% of the ILCs express IL-17a. In some embodiments, about 10% to about 70% of the ILCs express IL-17a.

[0217] Preferably, at least 2% of the in vitro ILC population expresses detectable levels of NKp44. In some embodiments, at least 5% of the in vitro ILC population expresses detectable levels of NKp44. In some embodiments, at least 10% of the in vitro ILC population expresses detectable levels of NKp44. In some embodiments, at least 15% of the in vitro ILC population expresses detectable levels of NKp44. In some embodiments, at least 20% of the in vitro ILC population expresses detectable levels of NKp44. In some embodiments, at least 50% of the in vitro ILC population expresses detectable levels of NKp44. In some embodiments, the in vitro ILC population expresses detectable levels of NKp44.

[0218] In some embodiments, the in vitro ILC population comprises Group 2 ILCs. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the Group 2 ILCs express detectable levels of GATA3. In some embodiments, at least 30% of the Group 2 ILCs express detectable levels of GATA3. In some embodiments, at least 50% of the Group 2 ILCs express detectable levels of GATA3. Group 2 ILCs may express detectable levels of GATA3.

[0219] At least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ILCs in Group 2 may express detectable levels of IL-13. In some embodiments, at least 50%, preferably at least 60%, of the ILCs in Group 2 express detectable levels of IL-13. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ILCs in Group 2 may express detectable levels of IL-5. In some embodiments, at least 40% of the ILCs in Group 2 express detectable levels of IL-5. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ILCs in Group 2 co-express detectable levels of IL-5 and IL-13. In some embodiments, at least 50% of the ILCs in Group 2 co-express detectable levels of IL-5 and IL-13.

[0220] Group 2 ILCs may express detectable levels of IL-5 and / or IL-13. In some embodiments, Group 2 ILCs co-express detectable levels of IL-5 and IL-13.

[0221] At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ILCs in Group 2 may express detectable levels of IL-22. In some embodiments, at least 30%, preferably at least 40%, of the ILCs in Group 2 express detectable levels of IL-22. The ILCs in Group 2 may express detectable levels of IL-22.

[0222] The in vitro ILC population may comprise Group 3 ILCs. At least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the Group 3 ILCs may express detectable levels of NKp44. In some embodiments, at least 10% of the Group 3 ILCs express detectable levels of NKp44. In some embodiments, at least 15% of the Group 3 ILCs express detectable levels of NKp44. In some embodiments, at least 20% of the Group 3 ILCs express detectable levels of NKp44. In some embodiments, about 10% to about 30% of the Group 3 ILCs, preferably about 15% to about 25% of the ILCs, express detectable levels of NKp44. The Group 3 ILCs may express detectable levels of NKp44.

[0223] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ILCs in Group 3 express IL-22. In some embodiments, at least 20% of the ILCs in Group 3 express IL-22. In some embodiments, at least 50% of the ILCs in Group 3 express IL-22. In some embodiments, between about 10% and about 70% of the ILCs in Group 3 express IL-22.

[0224] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ILCs in Group 3 express IL-17a. In some embodiments, at least 20% of the ILCs in Group 3 express IL-17a. In some embodiments, at least 50% of the ILCs in Group 3 express IL-17a. In some embodiments, between about 10% and about 70% of the ILCs in Group 3 express IL-17a.

[0225] In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the ILCregs express or secrete detectable levels of IL-10. The ILCregs may be murine or human ILCregs of the invention.

[0226] In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the ILCregs express detectable levels of FOXP3. The ILCregs may be murine or human ILCregs of the present invention.

[0227] In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the ILCregs express detectable levels of CD25 (IL2RA). The ILCregs may be murine or human ILCregs of the invention.

[0228] In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the ILCregs express detectable levels of CD127. The ILCregs may be murine or human ILCregs of the present invention.

[0229] In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the ILCregs express detectable levels of CTLA4. The ILCregs may be murine ILCregs of the present invention or human ILCregs. At least 50% of the in vitro ILC population may co-express detectable levels of two or more cytokines. The ILCregs may be murine ILCregs as described in Wang et al. (2017). The ILCs may be human ILCregs of the present invention.

[0230] The in vitro ILC population can be a stimulated in vitro ILC population. Various stimulation methods are known to those skilled in the art. For example, the population can include an in vitro ILC population stimulated with PMA and ionomycin. Preferably, the stimulated in vitro ILC population expresses detectable levels of one or more cytokines as described above. In any of these embodiments, the ILCs can be ILCregs, murine ILCregs, or human ILCregs of the present invention.

[0231] In vitro ILC populations may have tissue-specific imprinting. "Tissue-specific imprinting" is understood to refer to gene signatures and phenotypes specific to immune cells of a specific organ in vivo. Thus, in some embodiments, in vitro ILC populations comprise or consist of tissue-specific ILC populations. Such tissue-specific ILCs may be useful for treating specific diseases and / or may have improved homing ability to specific tissues when administered to a subject. In some embodiments, in vitro ILC populations comprise or consist of ILCs specific to the skin, intestine, lung, thymus, thyroid, genitals, bladder, kidney, pancreas, oral mucosa, or liver. In some embodiments, in vitro ILC populations comprise or consist of ILCs specific to the skin, intestine, lung, thyroid, genitals, bladder, kidney, pancreas, oral mucosa, or liver. In some embodiments, in vitro ILC populations comprise or consist of ILCs specific to the intestine, lung, genitals, or oral mucosa. In any of these embodiments, the ILCs may be ILCregs, murine ILCregs, or human ILCregs of the invention.

[0232] Reproductive organ-specific ILCs may include fallopian tube-specific ILCs, ovarian-specific ILCs, prostate-specific ILCs, endometrial-specific ILCs, cervix-specific ILCs, vaginal-specific ILCs, and testis-specific ILCs. Reproductive organ-specific ILCs may be selected from fallopian tube-specific ILCs, ovarian-specific ILCs, prostate-specific ILCs, and endometrial-specific ILCs. In any of these embodiments, the ILCs may be ILCregs, murine ILCregs, or human ILCregs of the present invention.

[0233] Oral mucosa-specific ILCs can include salivary gland taste bud-specific ILCs, tongue-specific ILCs, and esophagus-specific ILCs. In some embodiments, oral mucosa-specific ILCs are esophagus-specific ILCs. In any of these embodiments, ILCs can be ILCregs, murine ILCregs, or ILCregs of the present invention.

[0234] In some embodiments, the in vitro ILC population comprises or consists of intestinal-specific ILCs and / or lung-specific ILCs. The intestinal-specific ILCs can be small intestine-specific ILCs and / or lung-specific ILCs. In any of these embodiments, the ILCs can be ILCregs, murine ILCregs, or ILCregs of the present invention.

[0235] Alternatively, the in vitro ILC population may comprise or consist of epithelial cancer-specific ILCs. In some embodiments, the in vitro ILC population may comprise or consist of skin cancer-specific ILCs, intestinal cancer-specific ILCs, lung cancer-specific ILCs, thymic cancer-specific ILCs, thyroid cancer-specific ILCs, reproductive cancer-specific ILCs, bladder cancer-specific ILCs, kidney cancer-specific ILCs, pancreatic cancer-specific ILCs, oral mucosal cancer-specific ILCs, or liver cancer-specific ILCs. The in vitro ILC population may comprise or consist of skin cancer-specific ILCs, intestinal cancer-specific ILCs, lung cancer-specific ILCs, thyroid cancer-specific ILCs, reproductive cancer-specific ILCs, bladder cancer-specific ILCs, kidney cancer-specific ILCs, pancreatic cancer-specific ILCs, oral mucosal cancer-specific ILCs, or liver cancer-specific ILCs. In any of these embodiments, the ILCs may be ILCregs, murine ILCregs, or ILCregs of the present invention.

[0236] In some embodiments, the ILCs are human ILCs, equine ILCs, feline ILCs, canine ILCs, bovine ILCs, ovine ILCs, or murine ILCs. In some embodiments, the ILCs are human ILCs or murine ILCs. Optionally, the murine ILCs are mouse ILCs. In some embodiments, the ILCs are human ILCs. The human ILCs can be ILCregs, murine ILCregs, or the human ILCregs of the present invention.

[0237] The in vitro ILC population may comprise or consist of small intestine-specific murine ILC1 cells. Small intestine-specific murine ILC1 cells are derived from Eomes- , CD127 + , CD49a + / - , CD49b - , TRAIL + / - , CD103 - , CD69 + , CD200r1 + , CXCR6 + / - , CD61 + The expression profile may include:

[0238] The in vitro ILC population may comprise or consist of liver-specific murine ILC1 cells. Liver-specific murine ILC1 cells are described by Eomes et al. - , CD127 + / - , CD49a + , CD49b - , TRAIL + , CD103 - , CD69 + , CD200r1 + , CXCR6 + , CD61 + The expression profile may include:

[0239] The in vitro ILC population may comprise or consist of oral mucosa-specific murine ILC1 cells. Oral mucosa-specific murine ILC1 cells are described by Eomes et al. + , CD127 - , CD49a + , CD49b + , TRAIL + , CD103 + / - , CD69 + , CD200r1 + , CXCR6 + , CD61 + The expression profile may include:

[0240] The in vitro ILC population can comprise or consist of small intestine-specific human ILC1 cells. Small intestine-specific human ILC1 cells express T-bet + , Eomes + / - , CD56 + / - , CD127 +, CD49a - , CD103 - , CD69 + / - , NKp44 + / - , CD16 - The small intestine-specific human ILC1 cells may express one or more particular genes, as described above, at detectable levels.

[0241] The in vitro ILC population can comprise or consist of lung-specific human ILC1 cells. Lung-specific human ILC1 cells express T-bet + / - , Eomes + / - , CD56 + / - , CD127 + , CD49a - , CD103 - , CD69 + / - , NKp44 + / - , CD16 - The lung-specific human ILC1 cells may express one or more particular genes, as described above, at detectable levels.

[0242] The in vitro ILC population can comprise or consist of liver-specific human ILC1 cells. Liver-specific human ILC1 cells express T-bet + , Eomes - , CD56 + / - , CD127 - , CD49a + , CD69 + , CD16 - The liver-specific human ILC1 cells may express one or more particular genes, as described above, at detectable levels.

[0243] The in vitro ILC population may comprise or consist of liver-specific murine ILC2 cells. Liver-specific murine ILC2 cells are designated ST2 + ,IL17RB + , KLRG1 + , CD69 + The expression profile may include:

[0244] The in vitro ILC population may comprise or consist of small intestine-specific murine ILC2 cells. Small intestine-specific murine ILC2 cells are known as ST2 + / - ,IL17RB + , IL18R1 - , KLRG1 + , CD69 + , NMUR1 + , VPAC1 / 2 + , CCR4 + , CCR8 + / - , MHCII + / - The expression profile may include:

[0245] The in vitro ILC population may comprise or consist of lung-specific murine ILC2 cells. Lung-specific murine ILC2 cells are designated ST2 + ,IL17RB + , IL18R1 + / - , KLRG1 + / - , CD69 + , NMUR1 + , VPAC1 / 2 + , CCR4 + , CCR8 + The expression profile may include:

[0246] The in vitro ILC population can comprise or consist of lung-specific human ILC2 cells. The lung-specific human ILC2 cells are CRTH2 + / - , KLRG1 + / - , CD117 + / - , CD49a + / - , ICOS + / - , CD69 + / - , NKp30 - , CD25 + / - , CCR6 + / - , CCR4 + / - The lung-specific human ILC2 cells may express one or more particular genes, as described above, at detectable levels.

[0247] The in vitro ILC population can comprise or consist of small intestine-specific human ILC2 cells. Small intestine-specific human ILC2 cells are CRTH2 + / - , KLRG1 + / - , CD117 + / - , CD49a + / - , ICOS + / - , CD69 + / - , NKp30 + / - , CD25 + / - , CCR6 + / - , CCR4 + / - , HLA-DR + / - The small intestine-specific human ILC2 cells may express one or more particular genes, as described above, at detectable levels.

[0248] The in vitro ILC population can comprise or consist of liver-specific human ILC2 cells. Liver-specific human ILC2 cells are CRTH2 cells. + / - , KLRG1 + / - , CD117 + / - , CD49a + / - , ICOS + / - , CD69 + / - , NKp30 + / - , CD25 + / - , CCR6 + / - , CCR4 + / - The liver-specific human ILC2 cells may express one or more particular genes, as described above, at detectable levels.

[0249] The in vitro ILC population can comprise or consist of small intestine-specific murine ILC3 cells. The small intestine-specific murine ILC3 cells express CXCR6 + / - , CCR7 + , CCR9 + , α4β7 + , MHCII + The expression profile may include:

[0250] The in vitro ILC population can comprise or consist of small intestine-specific human ILC3 cells. The small intestine-specific human ILC3 cells are NKp44 + / - , CD69 + / - , CCR7 - , ICOS + / - , CD39 + / - , CD45RA + / - , NRP1 + / - , CD25 + / - , HLA-DR + / - , IL1R1 - , IL23R + The small intestine-specific human ILC3 cells may express one or more specific genes, as described above, at detectable levels.

[0251] The in vitro ILC population can comprise or consist of lung-specific human ILC3 cells. The lung-specific human ILC3 cells are NKp44 + / - , CD69 + / - , CCR7 - , ICOS + / - , CD39 + / - , CD45RA + / - , NRP1 + / - , CD25 + , HLA-DR - The lung-specific human ILC3 cells may express one or more particular genes, as described above, at detectable levels.

[0252] Preferably, the ILCs are primary ILCs. More preferably, the ILCs are human primary ILCs. The primary ILCs can be autologous. Alternatively, the primary ILCs can be allogeneic. In some embodiments, the population comprises a mixture of allogeneic and autologous ILCs.

[0253] Preferably, the ILCregs are primary ILCregs. Primary ILCregs can be autologous. Alternatively, primary ILCregs can be allogeneic. In some embodiments, the population comprises a mixture of allogeneic and autologous ILCregs.

[0254] Preferably, the human ILCreg is a human primary ILCreg. The human primary ILCreg can be autologous. Alternatively, the human primary ILCreg can be allogeneic. In some embodiments, the population comprises a mixture of allogeneic and autologous ILCreg. The human ILCreg is preferably the human ILCreg of the present invention. Alternatively, the ILCs can comprise or consist of immortalized immune cells from a cell line. Alternatively, the ILCreg can comprise or consist of immortalized ILCreg or an ILCreg cell line.

[0255] In some embodiments, at least about 10% of the in vitro population comprises an exogenous polynucleotide. An "exogenous polynucleotide" refers to a polynucleotide introduced into ILCs or ILC precursor cells to genetically modify the ILCs or ILC precursor cells. Thus, in some embodiments, the in vitro ILC population is a genetically modified in vitro ILC population. Typically, the exogenous polynucleotide is recombinant. The exogenous polynucleotide typically encodes an exogenous polypeptide. It will be understood that the exogenous polypeptide may be a polypeptide that is endogenous to the ILCs but is expressed at a higher level in the cells after introducing the exogenous polynucleotide by genetic modification. Alternatively, the exogenous polypeptide may be a polypeptide that is not naturally expressed in the ILCs. In any of these exogenous polynucleotide and polypeptide embodiments, including those described below, the ILCs may be ILCregs, murine ILCregs, or human ILCregs of the present invention.

[0256] In some embodiments, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the in vitro population comprises exogenous polynucleotides.

[0257] In some embodiments, at least about 10% of the in vitro population expresses the exogenous polypeptide encoded by the exogenous polynucleotide at a detectable level, hi some embodiments, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the in vitro population expresses the exogenous polypeptide encoded by the exogenous polynucleotide at a detectable level.

[0258] The exogenous polypeptide can comprise a marker protein or an immunotherapeutic molecule. The marker protein can alternatively be called a reporter protein. Suitable marker proteins include, but are not limited to, GFP, MYC epitope tag, or FLAG epitope tag.

[0259] In some embodiments, exogenous polypeptide further comprises purification tag.As those skilled in the art will understand, purification tag can assist purification.Examples of purification tag include, but are not necessarily limited to, His tag, Arg tag, T7 tag, Strep tag, S tag, aptamer tag, V5 tag or AviTag.Various other tags are well known in the art.

[0260] The immunotherapeutic molecule can be any immunotherapeutic molecule that can further enhance the immunotherapeutic use of in vitro ILC populations. For example, the immunotherapeutic molecule can include an enzyme, an antibody, an antigen, a chimeric antigen receptor (CAR), an MHC class II cell receptor, a chemokine receptor, and / or a cytokine. The MHC class II cell receptor can preferably include HLA-DR.

[0261] In some embodiments, the immunotherapeutic molecule comprises or consists of a chimeric antigen receptor (CAR). A chimeric antigen receptor is an immune cell receptor that has been genetically engineered to confer the ability to target a specific antigen. Generally, a chimeric antigen receptor is specific to one or more cancer-associated antigens. Therefore, a chimeric antigen receptor is commonly used in the treatment of cancer.

[0262] A variety of CARs are known to those skilled in the art. In particular, a CAR can comprise or consist of a first-, second-, third-, or fourth-generation CAR.

[0263] First-generation CARs comprise or consist of a binding domain capable of specifically binding to an epitope in a target antigen, a transmembrane domain, and one or more intracellular signaling domains. The extracellular binding domain can comprise a single-chain variable fragment (scFv) from a monoclonal antibody. First-generation CARs typically comprise a CD3 zeta chain domain or a variant thereof as the intracellular signaling domain, which is the main signaling domain.

[0264] In addition to the components identified in first-generation CARs, second-generation CARs also contain costimulatory domains, such as CD28 and / or 4-1BB. The inclusion of an intracellular costimulatory domain enhances T cell proliferation, cytokine secretion, resistance to apoptosis, and persistence in vivo. The costimulatory domain of second-generation CARs is typically located in cis with and upstream of one or more intracellular signaling domains.

[0265] Third-generation CARs combine multiple costimulatory domains in cis position with one or more intracellular signaling domains to enhance T cell activity. For example, a third-generation CAR may contain a costimulatory domain derived from CD28 and 41BB together with an intracellular signaling domain derived from CD3ζ. Another third-generation CAR may contain a costimulatory domain derived from CD28 and OX40 together with an intracellular signaling domain derived from CD3ζ.

[0266] Fourth-generation CARs (also known as TRUCKs or armored CARs) combine the features of second-generation CARs with additional factors to enhance anti-tumor activity (e.g., cytokines, costimulatory ligands, chemokine receptors, or additional chimeric receptors of immune regulatory or cytokine receptors). The factors can be in the trans or cis position of the CAR, typically in the trans position of the CAR.

[0267] In some embodiments, the CAR is specific to a cancer antigen. The cancer antigen can be a solid tumor cancer antigen. "Specific" in the context of a CAR is understood to refer to the ability to specifically bind to a target antigen.

[0268] Cancer antigens include Erbb1, Erbb3, Erbb4, Erbb2, mucins, PSMA, carcinoembryonic antigen (CEA), mesothelin, GD2, MUC1, folate receptors, NKG2D ligands, ligands bound by other NK receptors such as NKp30, NKp44, or NKp46, GPC3, CAIX, FAP, NY-ESO-1, gp100, PSCA, ROR1, PD-L1, PD-L2, EpCAM, EGFRvIII, CD19, CD20, and C. D22, GD3, CLL-1, ductal epithelial mucin, CA-125, GP36, TAG-72, glycosphingolipids, glioma-associated antigen, β-hCG, AFP (alpha-fetoprotein) and lectin-reactive AFP, thyroglobulin, receptor for advanced glycation end products (RAGE), TERT, telomerase, carboxylesterase, M-CSF, M-CSF receptor, PSA, tyrosinase, survivin, PCTA-1, melanoma-associated antigen (MAGE), e.g. A1, MAGE A2, MAGE A4, MAGE A8, CD22, IGF-1, IGF-2, IGF-1 receptor, MHC-related tumor peptide, 5T4, tumor stroma-associated antigen, WT1, MLANA, CA 19-9, epithelial tumor antigen (ETA), BCMA, cancer-testis antigens such as CTA, New York esophageal squamous cell carcinoma (NYESO), and glycoprotein 100 (GP100), preferentially expressed antigen in melanoma (PRAME), type IV collagen alpha 3 chain (COL6A3), MR1, CD1c, human epidermal growth factor receptor 2 (HER2), solute carrier family 3 member 2 (SLC3A2), and avb6 integrin.

[0269] In some embodiments, the cancer antigen is selected from NYESO, GP100, PRAME, COL6A3, MR1, CD1c, HER2, SLCA2, CD19, PSMA, AFP, CEA, CA-125, MUC1, ETA, tyrosinase, and MAGE. In some embodiments, the CAR is an anti-CD19, anti-SLC3A2, or anti-PSMA CAR.

[0270] In some embodiments, the CAR is an anti-CD19 or anti-PSMA CAR.

[0271] The MAGE may be selected from MAGE A1, MAGE A2, MAGE A4, or MAGE A8.

[0272] The CAR can be linked to a reporter protein, such as GFP, a MYC epitope flag, or a FLAG epitope tag. Other suitable reporter proteins will be known to those skilled in the art.

[0273] In some embodiments, the CAR comprises a second generation CAR.

[0274] Suitable CAR intracellular signaling domains can include any suitable signaling domain, including any region containing an immunoreceptor tyrosine-based activation motif (ITAM), as discussed, for example, by Love et al., Cold Spring Harbor Perspect. Biol 2010 2(6)1 a002485. In some embodiments, the signaling domain comprises the intracellular domain of the human CD3[zeta] chain, or a variant thereof, as described, for example, in U.S. Pat. No. 7,446,190.

[0275] Various costimulatory domains are known to operate on CAR cells. CARs can contain one or more of these domains. Suitable costimulatory domains include B7 / CD28 family members, such as B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-1, PD-L2, or PDCD6; or ILT / CD85 family proteins, such as LILRA3, LILRA4, LILRB1, LILRB2, LILRB3, or LILRB4; or tumor necrosis factor (TNF) superfamily members, such as 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, lymphotoxin-α, OX40, RELT, TACI, TL1A, TNF-α, or TNF RII; or a member of the SLAM family, such as 2B4, BLAME, CD2, CD2F-10, CD48, CD58, CD84, CD229, CRACC, NTB-A, or SLAM; or a member of the TIM family, such as TIM-1, TIM-3, or TIM-4; or other costimulatory molecules, such as CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP12, Dectin-1, DPPIV, EphB6, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, or TSLP R.

[0276] In some embodiments, the immunotherapeutic molecule comprises an MHC class II cell surface receptor. Preferably, the MHC class II cell surface receptor comprises or consists of HLA-DR. Without wishing to be bound by theory, exogenous expression of HLA-DR by ILCs may support immunomodulatory activity.

[0277] In some embodiments, the immunotherapeutic molecule comprises a cytokine. The cytokine may be an immunomodulatory cytokine, such as IL-10 or TGF-β. Expression of immunomodulatory cytokines or receptors in in vitro ILC populations may be particularly useful when ILCs are used to treat autoimmune diseases, such as inflammatory bowel disease (IBD), multiple sclerosis, or allergies. Additional autoimmune diseases and specific allergies are described below.

[0278] In other embodiments, the cytokine is a pro-inflammatory cytokine. Exemplary pro-inflammatory cytokines include, but are not necessarily limited to, IL-22, IL-17A, IL-5, IL-4, amphiregulin, IFN-γ, IL-2, IL-1, IL-18, TNF-α, and GM-CSF. Thus, in some embodiments, the cytokine comprises one or more of IL-22, IL-17A, IL-5, IL-4, amphiregulin, IFN-γ, IL-2, IL-1, IL-18, TNF-α, and GM-CSF. In some embodiments, the cytokine comprises one or more of IL-22, IL-17A, IL-5, IL-4, IFN-γ, TNF-α, and GM-CSF. Expression of such pro-inflammatory cytokines may be particularly useful when using ILCs to treat cancer.

[0279] A variety of chemokine receptors are known in the art. Example chemokine receptors include, but are not necessarily limited to, CXC chemokine receptors, CC chemokine receptors, XCR1, and CX3CR1.

[0280] In some embodiments, the chemokine receptor comprises a CC chemokine receptor. For example, the chemokine receptor may comprise one or more of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, and CCR11. In some embodiments, the chemokine receptor comprises or consists of CCR7 or CCR3. In some embodiments, the chemokine receptor comprises or consists of CCR7.

[0281] Without wishing to be bound by theory, the inventors believe that the expression of chemokine receptors in in vitro ILC populations may aid in tissue-specific therapeutic targeting of ILCs, which may further enhance the therapeutic efficacy of in vitro ILC populations.

[0282] Preferably, the vector contains an exogenous polynucleotide. The vector can be viral or non-viral. Various viral and non-viral vectors are known to those skilled in the art. Non-viral vectors include plasmids, episomal vectors, and human artificial chromosomes (see, for example, Harrington et al., 1997, Nat Genet. 15:345). For example, non-viral vectors useful for expressing exogenous polypeptides in mammalian (e.g., human) cells include pThioHis A, B, and C, pcDNA3.1 / His, pEBVHis A, B, and C, (Invitrogen, San Diego, Calif.), MPS V vector, and many other vectors known in the art for expressing other proteins. Useful viral vectors include retroviruses, adenoviruses, adeno-associated viruses, herpesvirus-based vectors, SV40-based vectors, papillomaviruses, HBP Epstein-Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV). See Brent et al., supra; Smith, 1995, Annu. Rev. Microbiol. 49:807; and Rosenfeld et al., 1992, Cell 68:143. In particular, retroviruses, lentiviruses, adenoviruses, or adeno-associated virus vectors are commonly used for expression in immune cells such as T cells. Examples of such vectors include the SFG retrovirus expression vector (see Riviere et al., 1995, Proc. Natl. Acad. Sci. (USA) 92:6733-6737).

[0283] In some embodiments, the vector is a retroviral or lentiviral vector.Optionally, the vector is an SFG retroviral vector.In some embodiments, the vector is a lentiviral vector.Lentiviral vectors include self-inactivating lentiviral vectors (so-called SIN vectors).

[0284] The choice of vector depends on the intended host cell in which the vector will be expressed. Expression vectors for mammalian host cells can contain expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, for example, Queen, et al., 1986, Immunol. Rev. 89:49-68), as well as necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or modifiable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPS V promoter, the tetracycline-inducible CMV promoter (e.g., the human immediate-early CMV promoter), the constitutive CMV promoter, the EF1α promoter, the phosphoglycerate kinase (PGK) promoter, and promoter-enhancer combinations known in the art.

[0285] The vector may further comprise a polynucleotide encoding a reporter gene. Suitable reporter genes include, but are not necessarily limited to, HNIS, hNET, and HSVtK.

[0286] Cultures of transformed organisms can be expanded under non-inducing conditions without biasing the in vitro ILC population toward sequences encoding expression products well tolerated. In addition to a promoter, other regulatory elements may also be necessary or desirable for efficient expression. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. Furthermore, the efficiency of expression can be improved by including enhancers appropriate for the cell system used (see, e.g., Scharf et al., 1994, Results Probl. Cell Differ. 20:125; and Bittner et al., 1987, Meth. Enzymol. 153:516). For example, to increase expression in mammalian host cells, SV40 enhancers or CMV enhancers can be used.

[0287] Genetic manipulation of immune cells such as ILCs can be performed according to standard cloning and expression techniques known in the art (e.g., as described in Sambrook, J., Fritsh, EF, and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Vectors can be introduced into an in vitro ILC population using such techniques. Introduction can include gene transfer or transduction into an in vitro ILC population.

[0288] The term "gene transfer" in its various forms is intended to encompass a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as, for example, electroporation, calcium-phosphate precipitation, DEAE-dextran transfer, etc.

[0289] As used herein, the term polynucleotide refers to a polymer that comprises two or more nucleotides.Preferably, polynucleotide comprises at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, or at least 100 nucleotides.Nucleotides can be of natural origin or artificial.

[0290] A nucleotide typically comprises a nucleobase, a sugar, and at least one linking group, such as a phosphate group, a 2'O-methyl group, a 2'methoxy-ethyl group, a phosphoramidate group, a methylphosphonate group, or a phosphorothioate group. The nucleobase is typically a heterocycle. Nucleobases include, but are not limited to, purines and pyrimidines, more specifically adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar and nucleobase together form a nucleoside. Preferred nucleosides include, but are not limited to, adenosine, guanosine, 5-methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. The nucleosides can be adenosine, guanosine, uridine, and cytidine.

[0291] Nucleotides are typically ribonucleotides or deoxyribonucleotides. Nucleotides can be deoxyribonucleotides. Nucleotides typically contain monophosphate, diphosphate, or triphosphate. The phosphate can be attached to the 5' or 3' side of the nucleotide.

[0292] Nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), and cytidine diphosphate. Cytidine triphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine deoxyguanosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP) Nucleotides include, but are not limited to, 5-methyl-2'-deoxycytidine monophosphate, 5-methyl-2'-deoxycytidine diphosphate, 5-methyl-2'-deoxycytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine diphosphate, and 5-hydroxymethyl-2'-deoxycytidine triphosphate. The nucleotide may be selected from AMP, UMP, GMP, CMP, dAMP, dTMP, dGMP, or dCMP. In some embodiments, the nucleotide is selected from dAMP, dTMP, dGMP, or dCMP.

[0293] Nucleotides may contain further modifications. In particular, suitable modified nucleotides include, but are not limited to, 2'-aminopyrimidines (e.g., 2'-aminocytidine and 2'-aminouridine), 2'-hydroxypurines (e.g., 2'-fluoropyrimidines (e.g., 2'-fluorocytidine and 2'-fluorouridine), hydroxylpyrimidines (e.g., 5'-α-P-boranouridine), 2'-O-methyl nucleotides (e.g., 2'-O-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytidine, and 2'-O-methyluridine), 4'-thiopyrimidines (e.g., 4'-thiouridine and 4'-thiocytidine), and nucleotides with nucleobase modifications (e.g., 5-pentynyl-2'-deoxyuridine, 5-(3-aminopropyl)-uridine, and 1,6-diaminohexyl-N-5-carbamoylmethyluridine).

[0294] One or more nucleotides in a polynucleotide may be modified, for example, with a label or tag. The label may be any suitable label that allows for detection of the nucleotide. Suitable labels include fluorescent molecules, radioisotopes, e.g. 125 I, 35 S, enzymes, antibodies, antigens, other polynucleotides, and ligands such as biotin.

[0295] The nucleotides in exogenous polynucleotides can be linked together in any way.Nucleotides can be linked by phosphate, 2'O-methyl, 2'methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate bond.Nucleotides are typically linked by their sugar and phosphate groups.Nucleotides can be linked through their nucleobases as pyrimidine dimers.

[0296] The exogenous polynucleotide may comprise deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Preferably, the exogenous polynucleotide comprises DNA. The exogenous polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid, or other synthetic polymers with nucleotide side chains.

[0297] Substitutions can be used to perform codon optimization and codon wobble, both of which are known to those skilled in the art. Thus, it will be understood that codon-optimized and codon-wobble exogenous polynucleotides are also contemplated. In some embodiments, the exogenous polynucleotide is codon-optimized for human expression.

[0298] Exogenous polynucleotide can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences.Direct chemical synthesis of polynucleotide can be obtained by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth.Enzymol.68:90; the phosphodiester method of Brown et al., 1979, Meth.Enzymol.68:109; the diethyl phosphoramidite method of Beaucage et al., 1981, Tetra.Lett.,22:1859; and the solid support method of U.S. Patent No. 4,458,066. Introducing mutations into polynucleotide sequences by PCR can be carried out as described, for example, in PCR Technology: Principles and Applications of DNA Amplification, H.A. Erlich (Ed.), Freeman Press, New York, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif., 1990; Mattila et al., 1991, Nucleic Acids Res. 19:967; and Eckert et al., 1991, PCR Methods and Methods, 1:17.

[0299] The present invention also provides pharmaceutical compositions comprising immune cells or in vitro populations obtained by the methods of the present invention, as defined above, and a pharmaceutically or physiologically acceptable diluent and / or carrier.The present invention also provides pharmaceutical compositions comprising human ILCregs of the present invention, or in vitro populations of human ILCregs of the present invention, and a pharmaceutically or physiologically acceptable diluent and / or carrier.

[0300] Carriers and / or diluents are generally selected to be appropriate for a given mode of administration and can include, for example, agents to modify, maintain, or preserve the composition's pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability. Typically, these carriers and / or diluents include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and / or buffered media.

[0301] Suitable additional agents for inclusion in pharmaceutical compositions include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids), bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), bulking agents, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., free serum albumin, gelatin, or immunoglobulins), colorants, flavorings, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium carbonate), and / or hydroxypropyl-β-cyclodextrin. sodium), preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants, or wetting agents (e.g., Pluronic®; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancers (e.g., sucrose or sorbitol), osmolality enhancers (e.g., alkali metal halides such as sodium chloride or potassium chloride, or mannitol, sorbitol), delivery vehicles, excipients, and / or pharmaceutical adjuvants.

[0302] The carrier and / or diluent may be a vehicle for parenteral administration, optionally for intravenous administration. Suitable parenteral vehicles include sodium chloride solution, Ringer's solution containing glucose, and lactated Ringer's solution containing glucose and sodium chloride. Suitable physiologically acceptable viscosity enhancers may be included, such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's solution containing glucose. In some cases, the pharmaceutical composition may contain agents to adjust the osmolarity of the composition, such as sugars, polyalcohols such as mannitol or sorbitol, or sodium chloride. For example, in many cases, it is desirable for the composition to be substantially isotonic. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present. The exact formulation will depend on the route of administration. Other relevant principles, methods, and ingredients for pharmaceutical formulations are well known (see, e.g., Allen, Loyd V. Ed, (2012) Remington's Pharmaceutical Sciences, 22nd Edition).

[0303] The pharmaceutical compositions of the present invention can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As those skilled in the art will appreciate, the route and / or mode of administration can vary depending on the desired results. The administration route for the pharmaceutical compositions of the present invention includes, for example, intravenous, intramuscular, intradermal, intraperitoneal, intrapleural, subcutaneous, intratumoral, spinal, or other parenteral routes of administration, such as by injection or infusion. As used herein, the term "parenteral administration" refers to a mode of administration other than enteral administration and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intratumoral, intrapleural, and intrasternal injection and infusion. In some embodiments, the pharmaceutical composition is administered intratumorally. In other embodiments, administration is intrapleural or intraperitoneal. When parenteral administration is considered, the pharmaceutical composition is usually in the form of a sterile, pyrogen-free, parenterally acceptable composition. A particularly suitable vehicle for parenteral injection is a properly preserved sterile, isotonic solution. The pharmaceutical composition can be in the form of a lyophilized product, such as a lyophilized cake.

[0304] Alternatively, the pharmaceutical compositions of the present invention can be administered by a non-parenteral route, such as a topical, epithelial, or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.

[0305] In some embodiments, the pharmaceutical composition is for subcutaneous administration.Typically, the pharmaceutical composition for subcutaneous administration comprises suitable stabilizers (for example, amino acids such as methionine, and / or sugars such as sucrose), buffers, and osmolality-increasing agents.Alternatively, the pharmaceutical composition can be for intravenous administration.

[0306] The present invention also provides kits comprising immune cells, in vitro populations, and / or pharmaceutical compositions obtained by the methods of the present invention, as defined above. The kits may further comprise instructions for use. In some embodiments, the immune cells, in vitro populations, and / or pharmaceutical compositions obtained by the methods of the present invention are provided in an aqueous solution, optionally in a buffer, and / or at a temperature of at least -20°C.

[0307] Also provided are methods of treating or preventing disease in a subject, the methods comprising administering to the subject the immune cells, in vitro populations, and / or pharmaceutical compositions of the invention.

[0308] In some embodiments, the immune cells are lymphoid cells. Preferably, the immune cells are T cells, B cells, and / or innate lymphoid cells (ILCs). More preferably, the immune cells are ILCs. The in vitro population may comprise Group 1 ILCs. In some embodiments, the in vitro population comprises ILC1 cells and / or NK cells. In some embodiments, the in vitro population comprises or consists of NK cells. The NK cells are preferably cytotoxic NK cells. This is shown in Example 9. The ILCs are preferably ILCregs, murine ILCregs, or human ILCregs of the present invention. The in vitro population preferably comprises ILCregs, murine ILCregs, or human ILCregs of the present invention. The in vitro population may be a heterogeneous population. Alternatively, the in vitro population may be a homogeneous population.

[0309] The method typically includes administering a therapeutically or prophylactically effective amount of the immune cells, in vitro populations, and / or pharmaceutical compositions of the present invention. A therapeutically effective amount is an amount that improves one or more symptoms, e.g., all symptoms, of a disease and / or eliminates one or more symptoms, e.g., all symptoms, of a disease. A therapeutically effective amount preferably cures a disease. A prophylactically effective amount is an amount that prevents the onset of a disease and / or prevents the onset of one or more symptoms, e.g., all symptoms, of a disease. A prophylactically effective amount preferably prevents a subject from developing a disease. Suitable amounts are described in more detail below.

[0310] The immune cells, in vitro populations, and / or pharmaceutical compositions of the present invention can be administered to subjects who exhibit disease symptoms. The immune cells, in vitro populations, and / or pharmaceutical compositions of the present invention can be administered to subjects who are asymptomatic, i.e., do not exhibit disease symptoms. The immune cells, in vitro populations, and / or pharmaceutical compositions of the present invention can be administered when the subject's disease state is unknown or when the patient is expected to be disease-free. The immune cells, in vitro populations, and / or pharmaceutical compositions of the present invention can be administered to subjects who are predisposed to developing disease, for example, who have a genetic predisposition.

[0311] The subject can be a mammal. Optionally, the subject is a human, horse, dog, or cat. In some embodiments, the subject is a human. Alternatively, the subject can be a horse.

[0312] A variety of diseases are suitable for treatment or prevention by administration of the immune cells, in vitro populations, and / or pharmaceutical compositions of the invention. Any disease that can be treated or prevented using immunotherapy is contemplated.

[0313] For example, the disease may be cancer, an infectious disease, an autoimmune disease, or an allergy. In some embodiments, the disease is cancer, an autoimmune disease, or an allergy. In some embodiments, the disease is cancer or an autoimmune disease. In other embodiments, the disease is an autoimmune disease or an allergy. Alternatively, the disease is an allergy or cancer. In some embodiments, the disease is cancer. In some embodiments, the disease is an autoimmune disease. In other embodiments, the disease is an allergy.

[0314] In some embodiments, the disease is an inflammatory disease. Such diseases are described in more detail below. In some embodiments, the disease comprises a chronic or acute inflammatory disease. A chronic or acute inflammatory disease may include a chronic or acute infectious disease.

[0315] Autoimmune diseases may include, but are not necessarily limited to, inflammatory bowel disease, eczema, rheumatoid arthritis, psoriasis, multiple sclerosis (MS), myasthenia gravis, type 1 diabetes, systemic lupus erythematosus (SLE or lupus), Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, Graves' disease, Hashimoto's thyroiditis, and vasculitis.

[0316] In some embodiments, the autoimmune disease is selected from inflammatory bowel disease, rheumatoid arthritis, psoriasis, multiple sclerosis (MS), type 1 diabetes, and systemic lupus erythematosus (SLE or lupus). Additional autoimmune diseases are described below. In some embodiments, the autoimmune disease comprises inflammatory bowel disease. Exemplary inflammatory bowel diseases include Crohn's disease and ulcerative colitis.

[0317] Cancers may include, but are not necessarily limited to, solid tumor cancers, soft tissue tumors, metastatic lesions, and hematological cancers. For example, cancer may be liver cancer, lung cancer, breast cancer, prostate cancer, lymphoid cancer, colon cancer, kidney cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, e.g., squamous cell carcinoma of the head and neck (SCCHN), cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney cancer, or or ureteral cancer, renal pelvic cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, myelodysplastic syndrome (MDS), chronic myeloid leukemia chronic phase (CMLCP), diffuse large B-cell lymphoma (DLBCL), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), hepatocellular carcinoma (HCC), gastrointestinal stromal tumor (GIST), non-small cell lung cancer (NSCLC), cutaneous melanoma, mucosal melanoma, cutaneous squamous cell carcinoma (CSCC), small cell lung cancer, lung squamous cell carcinoma, Merkel cell carcinoma, environmentally induced cancers including those induced by asbestos, and combinations of the above cancers. In some embodiments, the cancer is selected from the group described above.

[0318] The cancer can be a solid tumor cancer.

[0319] In some embodiments, the cancer is selected from the group consisting of head and / or neck cancer, ovarian cancer, malignant mesothelioma, breast cancer, pancreatic cancer, colorectal cancer, lung cancer, gastric cancer, bladder cancer, prostate cancer, esophageal cancer, endometrial cancer, hepatobiliary cancer, chronic or acute leukemia including acute myeloid leukemia, duodenal cancer, thyroid cancer, central nervous system cancer, or renal cell carcinoma.

[0320] In some embodiments, the cancer is selected from ovarian cancer, breast cancer, optionally triple-negative breast cancer, pancreatic cancer, chronic or acute leukemia, including acute myeloid leukemia, malignant mesothelioma, and combinations of the aforementioned cancers.

[0321] The subject may have been pre-treated with a chemotherapeutic agent. In some embodiments, the disease is cancer or an autoimmune disease and the subject has been pre-treated with a chemotherapeutic agent.

[0322] When the disease is cancer, administration of the immune cells, in vitro populations, and / or pharmaceutical compositions of the present invention to a subject may result in a reduction in tumor size of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100% compared to an untreated tumor.

[0323] Allergies may include, but are not necessarily limited to, allergic rhinitis (which may alternatively be called seasonal nasal allergies), dust mite allergies, animal allergies, food allergies, insect bite / sting allergies, drug allergies, latex allergies, mold allergies, allo-rejection reactions, and / or graft-versus-host disease.

[0324] Common food allergies include, but are not limited to, nut allergies, fruit allergies, shellfish allergies, milk protein allergies, egg allergies, and lactose allergies. Nut allergies can be peanut allergies. Fruit allergies can be strawberry, rhubarb, pineapple, apple, or pear allergies.

[0325] In some embodiments, the allergy is selected from allergic rhinitis, food allergy, allogeneic rejection, and graft-versus-host disease.

[0326] Also provided are methods for treating or preventing a disease in a subject, the methods comprising administering to the subject the human ILCregs of the present invention, the in vitro human ILCreg populations of the present invention, and / or the pharmaceutical compositions of the present invention containing the human ILCregs of the present invention. The above statements regarding the amount of cells and the subject equally apply to this method. The disease is preferably an inflammatory disease, such as an autoimmune disease, an infectious disease, or cancer. The inflammatory disease can be chronic or acute, as described above. The inflammatory disease can occur in cells of any of the tissues described above, including tissues of the skin, gastrointestinal tract, lung, thymus, thyroid, reproductive organs, bladder, kidney, pancreas, or liver. The inflammatory disease is a disease or infection involving damage and destruction of healthy living cells. Examples of inflammatory diseases include, but are not limited to, autoimmune diseases, allergies, asthma, celiac disease, nephritis, hepatitis, reperfusion injury, graft-versus-host disease (GvHD), graft rejection, and infectious diseases. An "infectious disease" is understood to be an infection caused by a pathogen. In some embodiments, the inflammatory disease comprises an autoimmune disease, an infectious disease, or a cancer.

[0327] The autoimmune disease may include rheumatoid arthritis, psoriasis, systemic lupus erythematosus (lupus), inflammatory bowel disease, multiple sclerosis, diabetes, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, aplastic anemia (AA), vasculitis, or a combination thereof. The autoimmune disease preferably includes inflammatory bowel disease. Exemplary inflammatory bowel diseases include Crohn's disease and ulcerative colitis.

[0328] The infectious disease can be an infection caused by any pathogen. The pathogen can be a bacterium, an archaea, a unicellular eukaryote such as an amoeba or a paramecium, a fungus, or a virus.

[0329] The bacteria can be gram-negative or gram-positive. The gram-positive bacteria are preferably from the genus Bacillus, Clostridium, Enterococcus, Mycobacterium, Staphylococcus, or Streptococcus. The gram-positive bacteria can be from the genus Pasteurella or Nocardia.

[0330] The Gram-negative bacteria are preferably of the genera Aggregatibacter, Bacteroides, Bartonella, Brucella, Campylobacter, Chylamidia, Enterbacter, Francisella, Haemophilus, Heliobacter, Klebsiella, Legionella, Moraxella, Neisseria, Porphyromonas, Pseudomonas, Salmonella, Serratia, Stenotrophomonas, Vibrio, or Yersinia. The Gram-negative bacteria may be of the genus Escherichia or Pseudomonas.

[0331] The bacteria may be of the genus Borrelia, Chlamydophila, Listeria, Mycoplasma, Proteus, or Treponema. The bacteria are preferably Aggregatibacter actinomycetemcomitans, Bacillus anthracis, Bacillus licheniformis, Bacteroides fragilis, Bartonella henselae, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Campylobacter jejuni, Chlamydia trachomatis, Chlamydophila pneumoniae, Clostridium difficile, Clostridium perfringens, Enterobacter aerogenes, Enterococcus faecalis, Enterococcus faecium, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Legionella pneumophila, Listeria monocytogenes, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycoplasma genitalium, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Porphyromonas gingivalis, Proteus mirabilis、Pseudomonas aeruginosa、Salmonella enterica、Serratia marcescens、Staphylococcus aureus、Staphylococcus epidermidis、Staphylococcus haemolyticus、Stenotrophomonas maltophilia、Streptococcus mutans、Streptococcuspyogenes, Streptococcus salivarius, Streptococcus sanguinis, Treponema pallidum, Vibrio cholera, Vibrio parahaemolyticus, or Yersinia enterocolitica.

[0332] Other specific examples of bacteria are Mycobacterium tuberculosis, Mycobacterium intracellulare, Mycobacterium kansaii, Mycobacterium gordonae, Streptococcus agalactiae, Streptococcus viridans group, Streptococcus faecalis, Streptococcus bovis, Streptococcus pneumoniae, Corynebacterium diptheriae, Erysipelothrix rhusiopathie, Clostridium tetani, Klebsiella pneumoniae, Pasteurella multocida, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pertenue, and Actinomyces israelii.

[0333] The microorganism is preferably a Mycobacterium species that can cause tuberculosis. The microorganism is preferably Mycobacterium tuberculosis (M. tuberculosis), Mycobacterium africanum (M. africanum), Mycobacterium orygis (M. orygis, which may also be called oryx bacilli), Mycobacterium bovis (M. bovis), Mycobacterium microti (M. microti), Mycobacterium canetti (M. canetti), Mycobacterium caprae (M. caprae), Mycobacterium pinnipedii (M. pinnipedii), Mycobacterium suricattae (M. suricattae), or Mycobacterium mungi (M. mungi).

[0334] The fungus is preferably of the genus Absidia, Acremonium, Aspergillus, Aureobasidium, Basidiobolus, Blastomyces, Blastoschizomyces, Candida, Cladosporium, Coccidioides, Cryptococcus, Cunninghamella, Curvularia, Debaryomyces, Exophiala, Exserohilum, Fonsecea, Fusarium, Geotrichum, Histoplasma, Issatchenkia, Kluyveromyces, Malezzesia, Mucor, Paracoccidioides, Paecilomyces, Penicillium, Pichia, Pneumocystis, Rhizomucor, Rhizopus, Rhodotorula, Saccharomyces, Scedosporium, Schizophyllum, Scopulariopsis, Sporothrix, Trichoderma, Trichophyton, or Trichosporon.

[0335] The fungi are preferably Aspergillus fumigatus, Aspergillus flavus, Aspergillus lentulus, Aspergillus terreus, Aspergillus nidulans, Aspergillus oryzae, Aspergillus niger, Candida albicans, Candida caribbica (Candida fermentati), Candida dubliniensis, Candida famata (Debaryomyces hansenii), Candida fukuyamaensis (Candida xestobii or Candida carpophila), Candida guilliermondii, Candida kefyr (Kluyveromyces marxianus), Candida krusei (Issatchenkia orientalis), Candida metapsilosis, Candida orthopsilosis, Candida parapsilosis, Candida parapsilosis, Candida pelliculosa, Candida psychrophila, Candida rugosa, Candida smithsonii, Candida tropicalis, Candida utilis, Coccidioides immitis, Cryptococcus bacillisporus, Cryptococcus gattii, Cryptococcus grubii, Cryptococcus neoformans, Debaryomyces coudertii, Debaryomyces maramus, Debaryomyces nepalensis, Debaryomyces prosopidis, Debaryomyces robertsiae, Debaryomyces udenii, Histoplasma capsulatum, Kluyveromyces lactis, Pichia cecembensis, Rhodotorula araucariae, Rhodotorula babjevae, Rhodotoruladairensis, Rhodotorula diobovatum, Rhodotorula glutinis, Rhodotorula kratochvilovae, Rhodotorula paludigenum, Rhodotorula sphaerocarpum, Rhodotorula toruloides, Rhodotorula mucliaginosa, Saccharomyces 'sensu stricto', Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces cariocanus, Saccharomyces kudiavzevii, Saccharomyces mikatae, Saccharomyces paradioxus, Saccharomyces pastorianus, Saccharomyces uvarum, Saccharomyces cerevisiae, or Tsuchiyaea wingfieldii.

[0336] The virus may be a virus from the Retroviridae family, such as a human deficiency virus, e.g., HIV-I (also called HTLV-III), HIV-II, LAC, IDLV-III / LAV, HIV-III, or other isolates, e.g., HIV-LP; a virus from the Picornaviridae family, such as poliovirus, hepatitis A, enterovirus, human coxsackievirus, rhinovirus, echovirus; a virus from the Caliciviridae family, such as viruses that cause gastroenteritis; a virus from the Togaviridae family, such as equine encephalitis virus and rubella virus; a virus from the Flaviviridae family, such as dengue virus, encephalitis virus, and yellow fever virus; a virus from the Coronaviridae family, such as a coronavirus (e.g., SARS-CoV or SARS-CoV-2 / COVID-19); a virus from the Rhabdoviridae family, such as vesicular stomatitis (vesicular stomatitis); stomata virus and rabies virus, Filoviridae, such as Ebola virus, Paramyxoviridae, such as parainfluenza virus, mumps virus, measles virus, and respiratory syncytial virus, Orthomyxoviridae, such as influenza virus, Bungaviridae, such as Hataan virus, Bungavirus, phleobovirus, and Nairovirus, Arenaviridae, such as hemorrhagic fever viruses, Reoviridae, such as reovirus, orbivirus, and rotavirus, Birnaviridae, Hepadnaviridae, such as hepatitis B virus, Parvoviridae, such as parvovirus, Papovaviridae, such as papillomavirus and polyomavirus, Adenoviridae, such as adenovirus, Herpesviridae, such as herpes simplex virus (HSV) I and II, varicella-zoster virus, and poxvirus, or Iridoviridae, such as African swine fever virus. The virus may be an unclassified virus, a causative agent of a spongiform encephalopathy, a pathogen of hepatitis delta, a pathogen of non-A, non-B hepatitis (class 1 enterally transmitted, class 2 parenterally transmitted, e.g., hepatitis C), Norwalk and related viruses, and astroviruses.

[0337] The cancer may be any of those described above.

[0338] By administering the human ILCregs of the present invention, including the populations or pharmaceutical compositions of the present invention, to a subject, symptoms may be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100% compared to an untreated subject.

[0339] In embodiments in which immune cells or in vitro populations are administered, the number of cells administered to a subject should take into consideration the route of administration, the disease being treated, the subject's weight, and / or the subject's age. Generally, about 1 x 10 6 ~Approx. 1×10 11 In some embodiments, about 1 x 10 immune cells are administered to the subject. 7 ~Approx. 1×10 10 of immune cells, or approximately 1 x 10 8 ~Approx. 1×10 9 of immune cells are administered to a subject. This also applies to embodiments of the invention involving human ILCregs.

[0340] The present invention also provides immune cells, in vitro populations, and / or pharmaceutical compositions of the present invention for use in any of the above-mentioned therapeutic methods. Accordingly, the present invention also provides immune cells, in vitro populations, and / or pharmaceutical compositions of the present invention for use in the treatment or prevention of disease, which may alternatively be referred to as for use in therapy. In particular, the present invention provides immune cells, in vitro populations, and / or pharmaceutical compositions of the present invention for use in the treatment or prevention of cancer, autoimmune disease, or allergy. Preferably, the present invention provides immune cells, in vitro populations, and / or pharmaceutical compositions of the present invention for use in the treatment or prevention of cancer or autoimmune disease.

[0341] The present invention also provides human ILCregs of the present invention, in vitro populations of human ILCregs of the present invention, and / or pharmaceutical compositions of the present invention comprising human ILCregs of the present invention for use in any of the above-mentioned therapeutic methods. Accordingly, the present invention also provides human ILCregs of the present invention, in vitro populations of human ILCregs of the present invention, and / or pharmaceutical compositions of the present invention comprising human ILCregs of the present invention for use in the treatment or prevention of disease, which may alternatively be referred to as for use in therapy. In particular, the present invention provides human ILCregs of the present invention, in vitro populations of human ILCregs of the present invention, and / or pharmaceutical compositions of the present invention comprising human ILCregs of the present invention for use in the treatment or prevention of inflammatory diseases, such as autoimmune diseases, infectious diseases, or cancer.

[0342] Also provided is the use of the immune cells, in vitro populations, and / or pharmaceutical compositions of the present invention for the manufacture of a medicament for the treatment or prevention of a disease. Optionally, the disease is cancer or an autoimmune disease. In some embodiments, the disease is cancer. Further provided is the use of the immune cells, in vitro populations, and / or pharmaceutical compositions of the present invention for therapy. Also provided is the use of the immune cells, in vitro populations, and / or pharmaceutical compositions of the present invention for the treatment or prevention of cancer. Also provided is the use of the immune cells, in vitro populations, and / or pharmaceutical compositions of the present invention for the treatment or prevention of an autoimmune disease.

[0343] The present invention also provides the use of the human ILCregs of the present invention, in vitro populations of human ILCregs of the present invention, and / or pharmaceutical compositions of the present invention comprising human ILCregs of the present invention for the manufacture of medicaments for the treatment or prevention of disease. The present invention also provides the use of the human ILCregs of the present invention, in vitro populations of human ILCregs of the present invention, and / or pharmaceutical compositions of the present invention comprising human ILCregs of the present invention for the treatment or prevention of disease. In both cases, the disease is preferably an inflammatory disease, such as an autoimmune disease, an infectious disease, or cancer. The present invention also provides the use of the human ILCregs of the present invention, in vitro populations of human ILCregs of the present invention, and / or pharmaceutical compositions of the present invention comprising human ILCregs of the present invention for therapy.

[0344] In this description and in the claims, the terms "comprise" and "containing," as well as variations of these terms, such as "comprising" and "including," mean "including but not limited to" and do not exclude other components, constituents, or steps. Furthermore, unless the context requires otherwise, the singular encompasses the plural, and where the indefinite article is used, the specification should be understood as contemplating the plural as well as the singular, unless the context requires otherwise.

[0345] Preferred features of each aspect of the invention may be as described with respect to any of the other aspects. It is expressly intended that within the scope of this application, the various aspects, embodiments, examples, and alternatives described in the preceding paragraphs, claims, and / or the following detailed description and drawings, in particular their individual features, may be taken independently or in any combination. That is, the features of all embodiments and / or any embodiment may be combined in any way and / or in any combination, unless such features are inconsistent.

[0346] One or more embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings. [Brief explanation of the drawings]

[0347] [Figure 1]Figure 1 shows that murine bone marrow-derived ILCPs produce group 1, group 2, and group 3 ILCs in coculture with epithelial-only SIOs. (a) Confocal sections (scale bar 50 μm) of apical ZO-1 (green) and cryptopancreatic Lyzozyme1 expression (magenta) within SIOs are shown. (b) A schematic diagram of the coculture system is shown, along with corresponding representative confocal images (scale bar 50 μm) of SIOs (EpCAM-magenta, nuclei-blue) in coculture with ILCPs (CD45.2-green) as 3D maximum intensity projections. (c) A schematic diagram of the experimental design is shown. (d) Representative flow plots of murine bone marrow-derived DAPI-, Lineage- (HSC cocktail (CD3, CD45R, CD11b, TER-119), Ly-G6, CD5, CD19, and NK1.1), CD127+, α4β7+, Flt3-, PD-1+ ILCPs, and CD25+ ILC2Ps, gated based on the PD-1 fluorescence minus one (FMO) control (overlaid in red). (e) Quantification of ILCP progenitor counts generated per animal (N = 15 animals). (f) Representative flow plots of DAPI-EpCAM+CD45- epithelial and EpCAM-CD45+ immune cell populations in 7-day cultures of PD-1+ ILCP-only, ILCP+SIO, and SIO-only cultures. Arrows indicate expression of the Lineage marker within the CD45+ population. (g) shows the fold change in CD45+Lineage- cells generated after culture relative to the number of seeded progenitor cells (N=15 animals in 5 experiments).(h) In cultures derived from ILCP alone or ILCP co-culture with SIO, putative group 1 (magenta; viable EpCAM-CD45+Lin-RORγt-, Klrg1-, NK1.1+, NKp46+), group 2 (green; viable EpCAM-CD45+Lin-RORγt-, NKp46-, Klrg1+, Sca-1+), NKp46+ group 3 (lavender; viable EpCAM-CD45+Lin-Klrg1-, RORγt+, NKp46+), and NKp46+ cells were identified. Contour plot overlays depicting NKp46, RORγt, NKp46, NK1.1, Klrg1, and Sca-1 expression in the Lineage populations from (f) pre-gated as 6-Group 3 (blue; viable EpCAM-CD45+Lin-Klrg1-, RORγt+, NKp46-), and the putative immature "other" population (gray; viable EpCAM-CD45+Lin-RORγt-, Klrg1-, NK1.1-, NKp46-). (i) Quantification of the pooled ILC group described in (h) (ILCPs from N=4 animals). Error bars represent SEM, p-values, unpaired Student's t-test. [Figure 2]Figure 2 shows that SIO cocultures support ILCPs and functionally mature ILCs in parallel. (a) Representative flow contour plots of ILCs generated from PD-1+ ILCP cocultures with or without SIO (color overlay from the PD-1+ SIO condition indicates the excluded population) or without SIO (NEG: black = ILCPs + SIO, magenta = ILCPs cultured without SIO). (b) Relative frequency of α4β7+PD-1+CD25+c-KIT+ cells among all Lineage- cells. (c) Relative frequency of RORγt-CCR6-NK1.1-NKp46-Klrg1-ST2- (NEG) cells within the Lineage- population, counts of NEG cells, and percent CD25+, c-KIT+, α4β7+, and PD-1+ cells within the negative population (parental frequency, SD). In (d), the frequencies and counts of ILC1s (viable EpCAM-CD45+Lin-RORγt-Klrg1-NK1.1+NKp46+Eomes-T-bet+) and NK cells (viable EpCAM-CD45+Lin-RORγt-NK1.1+NKp46+T-bet+Eomes+) described in (e) were obtained from PD-1+ ILCP cocultures with or without SIO and compared with putative ILC3s (viable EpCAM-CD45+Lin-RORγt+Klrg1-NK1.1-NKp46+ / -Eomes-T-bet+ / -) and ILC2s (viable EpCAM-CD45+Lin-RORγt-Klrg1+NK1.1-NKp46+ / -Eomes-T-bet-) (N=7 animals, 2 experiments). (f) shows the frequency of ILC1 and NK cells expressing IFN-γ (FMO overlaid in blue in e) and granzyme B after 4 hours of stimulation with PMA / ionomycin and IL-18, compared to ILC1 and NK cells isolated from primary small intestinal lamina propria as described in (g).In (h), the frequencies and counts of ILC3 (viable EpCAM-CD45+Lin-NK1.1+ / -NKp46+ / -, Klrg1-ST2-, RORγt+, CCR6-) and CCR6+ (viable EpCAM-CD45+Lin-, NK1.1+ / -NKp46+ / -, Klrg1-ST2-, RORγt+, CCR6+) LTi-like cells described in (i) were obtained from PD-1+ ILCP cocultures with or without SIO and compared with primary ILC3s (N=5 animals, 2 experiments). (j) Frequency of ILC3- and CCR6+ LTi-like cells expressing IL-22 (FMOs overlaid in blue in ei) and IL-17A (FMOs overlaid in magenta in i) after 4 hours of stimulation with PMA / ionomycin and IL-23 compared to ILC3 and LTi cells isolated from primary small intestinal lamina propria as described in (k). Error bars are SEM, p-values are unpaired Student's t-test. [Figure 3] In Figure 3, (a) shows a representative flow plot and corresponding quantification of IFN-γ expression in T-bet+ ILC1s (RORγt-NKp46+, NK1.1+, Klrg1-) after 4 hours of PMA / ionomycin stimulation (n = 8 mice in at least two experiments). (b) shows a representative flow plot overlaid with putative ILC1s, NK cells, ILC3s, and ILC2s obtained from cocultures of PD-1-ILCPs with or without SIO, and corresponding quantification. All error bars are SEM. [Figure 4]In Figure 4, (a) shows a representative flow plot overlaid with putative ILC2, ILC1, ILC3, and Lti-like cells after SIO coculture with PD-1+ ILCPs, PD-1- ILCPs, and CD25+ ILC2Ps, and corresponding quantification of counts and frequencies comprised by the seeded progenitor cells or resulting ILC populations (N = 3 mice). (b) shows a representative flow plot and corresponding quantification of IL-22 and IL-17A expression in ILCP-derived ROR-γt+ ILCs of Group 3 after 3-hour stimulation with PMA / ionomycin and IL-23 (N = 8 mice). All error bars are SEM. [Figure 5]Figure 5 shows that SIO provides a virtually sterile model for the imprinting, maturation, and expansion of intestinal-specific ILCs. (a) Representative flow plots of NKp46 expression in CD45+Lineage-(CD127+)PD-1+ILCP+SIO cocultures or RORγt+ILC3s derived from primary small intestinal lamina propria. (b) Frequency of NKp46+ILC3s (viable EpCAM-Lin-CD45+RORγt+) further quantified for ILCPs cultured without SIO or with GF-SIO (N = 3–5). (c) Circular representation of relative ILCs in group 1 (magenta; viable EpCAM-CD45+Lin-RORγt-, ST2+Klrg1-, NK1.1+, NKp46+), group 2 (green; viable EpCAM-CD45+Lin-RORγt-, NKp46-, ST2+Klrg1+, Sca-1+), NKp46+ group 3 (lavender; viable EpCAM-CD45+Lin-, ST2-, Klrg1-, RORγt+, NKp46+), and NKp46- group 3 (blue; ST2-, Klrg1-, RORγt+, NKp46-) in viable unstimulated co-cultures derived from SPF-SIO or GF-SIO compared with primary SPF ileum (without LTi-rich Peyer's patches). (d) Schematic diagram showing the transwell culture method. (e) Quantification of the relative frequencies of group 1, group 2, and group 3 ILCs obtained from PD-1+ILCP cocultures with SIOs or PD-1+ILCP cocultures with SIOs isolated by transwell (TW) inserts (N=3). (f) Counts of putative Lineage-RORγt-, NKp46-, Klrg1+, Sca-1+ ILC2s. (g) Geometric mean fluorescence intensity (GeoMFI) of Klrg1 in Lineage-ILCs after coculture of CD25+ ILC2s with SIOs, TW-isolated, or without SIOs (N=4). (h) Representative flow plots showing the expression of Gata3, IL-25R, IL-13, and IL-15 in ILC2P-derived and small intestinal lamina propria-derived ILC2s after 4 hours of stimulation with PMA / ionomycin, with FMO overlays in cyan and magenta, respectively, as quantified in (i)–(k) (error bars are SD, N=3).(l) Gene expression heatmap of genes of interest obtained from bulk RNA sequencing of EpCAM+CD45- intestinal epithelial cells after 7 days of co-culture with progenitor-derived lymphocytes, without immune cells but with IL-2 and IL-7, or in SIO basal medium (magenta = high, cyan = low, white = not detected). (m) Schematic diagram of metabolite microinjection method. (n) SIOs microinjected with 20 kDa FITC-dextran and 5 mM succinate retained the dye within the pseudolumen for at least 16 hours after injection, demonstrating the preservation of tight junctions in the presence of metabolites. (o) Representative confocal images of SIOs microinjected with PBS or 5 mM succinate in PBS stained for the tuft cell marker doublecortin-like kinase 1 (Dclk1) (green, scale bar 50 μm). (p) shows the expression of IL25 / IL17E normalized to the housekeeping gene Hprt1 in PBS- or succinate-injected SIOs (n=3 wells of SIOs per experiment). (q) shows the frequency of Klrg1+ ILC2s after co-culture of ILC2Ps with PBS- or 5 mM succinate-injected SIOs (ILC2Ps were divided between conditions from N=4 animals). Error bars are SEM, p-values are unpaired Student's t-test. [Figure 6]Figure 6 shows that coculture with intestinal and pulmonary organoids promotes tissue-specific ILC2 imprinting. (a) Representative brightfield and confocal images of murine primary distal pulmonary epithelial organoids (LO) with cystic or sacculus structures consisting of expanded EpCAM+ (green) budding areas are shown (scale bar 25 μm). (b) Fold-change expansion of viable EpCAM-CD45+ ILCPs after 7 days of coculture with LO or LO. (c) and (d) show circular graphs of the frequencies of viable EpCAM-CD45+ group 1 ILCs (magenta, viable, EpCAM-, CD45+, Lineage-, RORγt-, NKp46+, NK1.1+, Gata3-), ILC2s (green, gated as viable, EpCAM-, CD45+, Lineage-, RORγt-, NKp46-, NK1.1-, Gata3+), and ILC3s (blue, EpCAM-, CD45+, Lineage-, RORγt-, NKp46+ / -, NK1.1+ / -, Gata3-) obtained from 7-day coculture of PD-1+ / - ILCPs with SIO or LO, and the corresponding quantification of ILC2 frequencies (N=4). (e) Representative flow plots of Klrg1, ST2, ICOS, CD25, IL-13, and IL-5 expression in EpCAM-, CD45+, NKp46-, NK1.1-, and RORγt- putative ILC2s after coculture with 1-SIO or LO (IL-13 and IL-5 FMOs are overlaid in magenta and blue), and (f) GeoMFIs of IL-5 and IL-13 cytokine expression in ILC2s and IL-22 expression in RORγt+ ILC3s were quantified after 4 hours of unbiased PMA / ionomycin stimulation (N=4). (g) Schematic of the experimental design for gut-to-lung exchange to assess tissue-specific ILC2 phenotype development versus plasticity. (h) shows the frequency of Klrg1+, ST2+, and CD25+ ILC2s after 14 days of co-culture according to the experimental design shown in (g) (related to Figure (e)) (N=3-5). (i) shows the relative gene expression of Il33 in whole primary small intestinal and lung tissue (N=5 animals), and in (j) in epithelial-only SIO and LO (n=3 wells of organoids).(k) Frequency of Klrg1+, ST2+, and CD25+ ILC2s in cocultures switched from SIO to LO with and without a neutralizing dose of rmIL-33 blocking antibody (50 ng / ml, N=3). Error bars are SEM; p-values are unpaired Student's t-test. [Figure 7] Figure 7 shows representative flow plots overlaid with putative group 1, group 2, and group 3 ILCs after 7 days of co-culture with murine lung organoids (LO). [Figure 8]In Figure 8, (a) shows a Euclidean heatmap (average linkage) of genes of interest expressed in human intestinal organoids (G; N = 2) and lung organoids (L; N = 2) cultured for 24 or 72 hours with Cryptosporidium non-infected (NI) or infected (I), representative of N = 3 experiments. Values represent the [log(X + 1,2)] fold change (FC) relative to the housekeeping genes (HKG) ACTB, GAPDH, and HPRT1 (magenta: greater than HKG, cyan: less than HKG). Black boxes reveal significant differential expression of the gene of interest, IL33, in primary human small intestinal and lung epithelial organoids sequenced after 24 or 72 hours of culture with (infected - I) or without (infected - NI) microinjected Cryptosporidium infection (Benjamini, Krieger, and Yekultieli Two-stage linear step-up method, Q = 1%, p < 0.000001; q < 0.000001) (Accession number: GSE112991) (Heo and Dutta et al., 2018). Ingenuity pathway analysis (IPA) was used to annotate these datasets, isolating basally presented or secreted ligands and quality control genes (e.g., CDX2 in the intestinal epithelium). (b) shows a volcano plot of the data depicted in (a), revealing genes with statistically significant differential expression. (c) Frequency of Klrg1, CD25, and ST-positive cells in putative ILC2s isolated by FACS from 7-day SIO cocultures and reseeded for an additional 7 days in Matrigel with or without SIO, and without SIO but with rmIL-33 (N=2 mice). (d) Frequency of Klrg1, CD25, and ST-positive cells in putative ILC2s isolated by FACS from 7-day LO cocultures and reseeded for an additional 7 days in Matrigel replaced with LO or SIO culture (N=3). Error bars are SEM, unpaired Student's t-test. [Figure 9]Figure 9 shows that HIOs promote the proliferation and maturation of systemic human ILCPs. (a) shows the gating method for healthy PBMC-derived ILCPs, pre-gated on viable single CD45+ cells, with appropriate FMOs overlaid in blue and magenta. (b) shows a schematic diagram of HIO-ILCP coculture, demonstrating the presence of mesenchymal cells and CD45+ ILCPs. (c) shows an overlay of representative flow cytometry plots of EpCAM+ intestinal epithelial cells (E, green), double-negative mesenchymal cells (M, magenta), and CD45+ ILCs (blue) after 14 days of coculture, revealing Matrigel debris in gray. (d) Quantification of EpCAM-CD45+ Lineage-ILCs. (e) fold-change expansion of ILCs after 14 days of coculture relative to the number of ILCPs seeded on day 1 (N = 3–15 in approximately seven experiments). (f) shows counts of viable, EpCAM-, CD45+, Lineage-, RORγt+ ILCs after 14 days of coculture with mesenchymal-depleted HIOs or epithelial-depleted mesenchymal / fibroblasts expressing markers CCR6, NKp44, and / or T-bet (N=3).(g) shows the relative group 1 (surviving, CD45+Lin-CRTH2-c-kit-, and [CD127+, CD161+ in primary intestinal ILCs only] and [EpCAM-, RORγt-, GATA3- in MD-HIO and ED-HIO only], group 2 (surviving, CD45+Lin-CRTH2+c-kit+ / -, and [CD127+, CD161+ in primary intestinal ILCs only] and [EpCAM-, RORγt-, GATA3+ in MD-HIO and ED-HIO only]), and group 3 (surviving, CD45+ A circular blur of Lin-CRTH2-c-kit+NKp44+ / -, and [CD127+, CD161+ in primary intestinal ILCs only] and [EpCAM-, RORγt+, GATA3- in MD-HIO and ED-HIO only], and other Lineage-ILCs (e.g., undifferentiated progenitor cells, (viable, CD45+Lin-CRTH2-c-kit-, and [CD127+, CD161+ in primary intestinal ILCs only] and [EpCAM-, RORγt-, GATA3- in MD-HIO and ED-HIO only]) is shown (N=13, Kraemer Adapted from [Scientific Relations] et al., 2017, MD-HIO (N=13), ED-FB (N=7). (h) Representative images of CD45+ ILCPs co-cultured with mesenchyme-depleted E-cadherin+ HIO (scale bar 25 μm). Error bars represent SEM; p values are unpaired Student's t-test. [Figure 10]In Figure 10, (a) shows a representative confocal image of human intestinal organoids (HIOs; the apical actin ring is in magenta, the hindgut expression of the transcription factor CDX2 is in white, and the DAPI-stained nuclei are in cyan) in coculture with ILCPs (CD45, yellow arrows). Mesenchyme surrounds the complete epithelial-mesenchymal HIO structure (Comp. HIO) as CD45-CDX2-nuclei (blue arrows). The scale bar is 50 μm. (b) shows the count of CD45 immune cells after 14 days of coculture with or without complete epithelial-mesenchymal HIOs (day 1) seeded ILCPs. (c) Representative flow plots of GATA3 and IL-13 expression in CD45+Lineage-ROR-γt-CRTh2+ ILC2s stimulated with PMA / ionomycin for 4 hours on day 1 of pre-ILCP coculture and 14 days of coculture with complete HIOs (ILCPs from three donors). (d) Overlay of target gene expression in putative group 1 (magenta, viable, EpCAM-CD45+LIN-RORγtlowT-bet+) and group 3 (viable, EpCAM-CD45+LIN-, ROR-γt+) cells expressing CCR6 (orange), NKp44 (dark blue), and NKp44-free (light blue) after 4 hours of stimulation with PMA / ionomycin after 14 days of coculture with mesenchyme-depleted HIOs (MD-HIOs). (e) Overlay of putative Group 3, T-bet+, and Eomes+ Group 1 ILCs cocultured with complete HIOs with or without IL-18 stimulation, showing IFN-γ expression in PMA / ionomycin-stimulated cocultures, and corresponding quantification. (f) Representative flow plots of CD161 and RORγt expression in viable EpCAM-CD45+LIN- HIO-derived ILCs and corresponding quantification of CD161+RORγt+ cells. (g) Frequency of NKp44+ ILCs within the CD161+RORγt+ population. (h) Representative flow plots of IL-22 and IL-17A expression in complete HIO-derived ILCPs after 4 hours of stimulation with PMA / ionomycin, with or without further IL-23 stimulation, and corresponding quantification. Error bars represent SEM, unpaired Student's t-test, ILCPs from N=3 donors. [Figure 11] Figure 11 shows that human epithelial cells, but not mesenchymal cells, promote the proliferation and maturation of functional human ILCs. (a) shows the frequency of viable, EpCAM-, CD45+, Lineage-, RORγt+ ILCs expressing the markers CCR6, NKp44, and / or T-bet after 14 days of coculture. (b) shows the frequency of IL-22+ and IL-17A+ MD-HIO- and ED-FB-derived ILCs after 4 hours of stimulation with PMA / ionomycin. (c) shows a representative flow plot corresponding to (b) (FMO overlaid in blue and magenta). (d) shows the frequency of CD45+LIN-RORγt- ILCs expressing T-bet and / or Eomes. (e) Representative flow plots of CD56 and IFN-γ expression in T-bet+ and Eomes+ populations after 4 hours of stimulation with PMA / ionomycin (IFN-γ FMOs overlaid in magenta) and corresponding quantification. (f) Total counts of putative ILC2s after 14 days of coculture. (g) IL-5 and IL-13 expression in putative ILC2s after 4 hours of stimulation with PMA / ionomycin (FMOs overlaid in magenta and blue) and corresponding quantification. Error bars represent SEM, p-values, unpaired Student's t-test, ILCPs from N=3 donors. [Figure 12] Figure 12 shows representative flow plots of group 1-associated genes visualized by overlaying ROR-γt-T-bet+ (red), Eomes+ (purple), or "other" T-bet- (gray) ILCPs after 14 days of coculture with MD-HIO or ED-FB, and quantification of associated geometric mean fluorescence intensity after 4 h of stimulation with PMA / ionomycin. Error bars are SEM, unpaired Student's t-test, N=3 donors. [Figure 13]Figure 13 (a) shows the relative frequency of CD45+ Lineage- putative ILC2s (gating scheme in (b)) after 14 days of coculture with MD-HIOs or ED-FBs. (b) shows a representative flow plot overlaid with putative ILC2s (green) and other Klrg1- non-ILC2s (magenta) showing expression of group 2-associated genes after 4 hours of stimulation with PMA / ionomycin (ILCPs from N=3 donors). (c) shows a representative flow plot of putative ILC2s and IL-5 / IL-13 expression after 4 hours of stimulation with PMA-ionomycin after coculture of ILCPs with MD-HIOs, after separation of MD-HIOs from ILCPs using a transwell separator, or after culturing ILCPs in Matrigel without organoids (N=1 donor, n=2 technical replicates). (d) Counts of EpCAM-CD45+ immune cells and EpCAM-CD45- fibroblasts / mesenchymal cells in cocultures derived from 14-day MD-HIO cocultures, subsequently purified by FACS, and replated onto new MD-HIO or ED-FB. (e) Frequencies of IL-13+ and IL-5+ ILCs within putative ILC2s pre-matured in MD-HIO and replated onto MD-HIO or ED-FB for an additional 7 days. ILCPs from N=3 donors. All error bars are SEM, unpaired Student's t-test. [Figure 14]Figure 14 shows that transition of intestinal mature ILCs to HLOs recapitulates the tissue-specific human ILC2 phenotype. (a) Representative images of HIOs and human lung organoids (HLOs) showing E-cadherin+ epithelium (magenta), CD45+ ILCs (yellow), and nuclei (Hoechst, cyan) after 14 days of coculture (scale bar 50 μm). (b) Counts of EpCAM-, CD45+, LIN- ILCs and corresponding counts of EpCAM-CD45- fibroblasts / mesenchymal cells after 14 days of coculture with MD-HIOs or MD-HLOs. (c) Representative flow plots of RORγt, CCR6, IL-22, IL-17A, IFN-γ, and NKp44 after 14 days of coculture with MD-HIO or MD-HLO and 4 hours of PMA / ionomycin stimulation (pre-gated populations are shown in gray; representative of N = 3). (d) Expression of IL-5 and IL-13 in putative GATA3+ ILC2s after 14 days of coculture and 4 hours of PMA / ionomycin stimulation (FMOs overlaid in magenta and cyan), along with corresponding quantification. (e) Expression of CD25 and ST2 in putative GATA3+ ILC2s after 14 days of coculture with HIO or HLO, along with corresponding quantification. (f) Quantification of the relative frequencies of CD25+ and ST2+ putative ILC2s, and (g) the corresponding histogram overlay of ST2 MFI and ST2-PE in the same populations after 14 days of coculture with HIOs or HLOs, followed by HIO-to-HIO reseeding, HIO-to-HLO exchange, or HIO-to-HLO exchange with 50 ng / ml hIL-33 neutralizing antibody. All experiments were performed on ILCPs from N=3 donors, unpaired two-tailed Student's t-test, error bars are SEM. [Figure 15]Figure 15 shows HLA-DR expression in ILC3s generated from 13-day coculture of ILC progenitor cells (ILCPs) in mesenchyme-depleted human intestinal organoids (MD-HIO). The percentage of HLA-DR+ cells in the ILC3 population was pre-gated on viable, CD45+, Lin- (CD3-CD20-CD14-CD19-), ckit+ ILC3s. Red: ILC3s; Blue: Fluorescence Minus One (FMO) control. [Figure 16] Figure 16 shows antigen processing by ILCs via MHC-II, as indicated by hydrolysis of DQ-BSA, which leads to a fluorescent signal. (a) Shows the expression of dye-quenched bovine serum albumin (DQ-BSA) fluorescence in expanded ILCs after 4 hours of incubation with DQ-BSA at 37°C, on ice, or without DQ-BSA. ILCs were expanded from ILC progenitors derived from healthy adult PBMCs. (CD45+, Lin-, ILC1 are cKit-CRTH2-, CD56-, and CD161+; ILC2 are CRTH2+; ILC3 are cKit+, CRTH2-). (b) HLA-DR expression in expanded ILC populations after 4 hours of incubation with dye-quenched bovine serum albumin (CD45+, Lin-, ILC1 are cKit-CRTH2-, CD56-, and CD161+, ILC2 are CRTH2+, and ILC3 are cKit+, CRTH2-). (c) HLA-DR expression in expanded ILC populations after 4 hours of incubation with dye-quenched bovine serum albumin (gray dots are DQ-BSA-ILCs) (CD45+, Lin-, ILC1 are cKit-CRTH2-, CD56-, and CD161+, ILC2 are CRTH2+, and ILC3 are cKit+, CRTH2-). [Figure 17]Figure 17 shows single-cell RNA sequencing (scRNA sequencing) of organoid-generated ILCs. (A) shows a UMAP visualization of all cells (n=7,446) that passed quality control, color-coded based on the annotated ILC subset. (B) shows a dot plot showing the relative enrichment of selected previously described human ILC subset-specific genes used to annotate the cluster. While the gene expression characteristics of ILCregs in the human intestine have not yet been described, key genes used to distinguish these cells from other ILC subsets in mice (Wang et al., 2017) were found to be highly expressed in this cluster, including IL10, SOX4, and ID3, along with other genes characteristic of Tregs (e.g., FOXP3, CTLA4, and IL2RA). (C) shows a UMAP visualization of the enrichment scores of the human ILCP gene signature (Liu et al., 2021). The apparent clustering observed here within the ILC3 population indicates that a small proportion of ILCPs were captured and sequenced, even though these cells did not form their own specific cluster. (D) shows a UMAP visualization of the density of TBX21 and EOMES expression. Density mapping was used here because expression detection of these two transcription factors was weak. At the resolution used here, ILC1 and NK cells did not form specific clusters. However, the patterning of EOMES expression suggests that both cell types were successfully generated within this coculture. (E)-(F) show density plots and UMAP visualization of the enrichment scores of gene modules associated with human tissue-specific ILC populations (Mazzurana et al., 2021) in ILC3. The three modules examined here correspond to the circulating ILC module (mod11), the colonic ILC module (mod3), and the pulmonary ILC module (mod34). Mazzurana et al. did not identify any specific clusters of ILC1 or ILC2 in the colon, so only ILC3s were examined.Although ILC3s showed the greatest enrichment in circulating phenotypes, this was not uniform across ILC3 clusters (F); the clusters separated into cells with greater enrichment in circulating ILC signatures, including putative ILCP cells, and cells with more colonic (mod3) enrichment. Notably, these ILC3s showed the least enrichment in the lung ILC gene module, indicating that within intestinal cocultures, ILC3s can mature and differentiate toward a more intestinal tissue-specific phenotype. [Figure 18] Figure 18 shows that NK cells generated by co-culture of ILC progenitor cells (ILCP) with intestinal organoids are cytotoxic. The percentage (%) of dead target cells in CFSE-stained K562 cells is shown. CFSE-positive cells (target cells, T) were labeled with carboxyfluorescein succinimidyl ester (CFSE) before co-culture with CD56+ NK cells (effector cells, E) expanded from ILCP in human intestinal organoid co-cultures. These cells were co-cultured with IL-2 (20 ng / ml) at various effector-to-target (E:T) ratios ranging from 5:1, 2.5:1, to 1.25:1. Three control samples were prepared: target cells alone, effector cells, and a positive control for cell death (target cells treated with Tween 20). All experimental groups were stained with LIVE / DEAD fixable UV blue stain to measure cell viability. [Figure 19] Figure 19 shows the generation of ILCs in human small intestinal biopsy-derived organoids. After 15 days of culture with human small intestinal biopsy-derived organoids using 50% Intesticult (commercially available from StemCell Technologies) and 50% homemade organoid medium, the generation of differentiated ILCs (including ILC1, ILC2, ILC3, and NK cells) from ILC progenitor cells (ILCPs) is shown. (A) Cell counts at the beginning and end of culture are shown. (B) Visual representation (bright-field microscopy) of ILCs expanded in contact with human small intestinal organoids (dark areas). DETAILED DESCRIPTION OF THE INVENTION

[0348] material and method animal All animals were terminated by cervical dislocation according to standard ethical procedures performed by trained personnel. Death was confirmed by femoral artery slicing or decapitation (depending on the protocol in place) before organ and tissue collection. Animals were maintained under specific pathogen-free conditions (unless otherwise stated) in the licensed Charles River and King's College London animal units in accordance with the UK Animals (Scientific Procedures) Act 1986 (UK Home Office Project License (PPL: 70 / 7869 until September 2018; P9720273E from September 2018).

[0349] Isolation of murine organoids Murine epithelial small intestine (ileum) was isolated from 6-8 week-old female C57BL / 6 mice according to a recognized protocol (Sato et al., 2011, incorporated herein by reference). Small intestinal epithelial organoids (SIOs) were cultured in 3D Matrigel bubbles and passaged every 5-7 days using a curved pipette tip to disrupt crypts. SIOs were cultured in basal medium supplemented with R-spondin 1 (50 μl supernatant / ml or 1 mg / ml from an R-spondin-producing cell line), noggin (50 μl supernatant / ml or 100 μg / ml), and rm-EGF (50 μg / ml).

[0350] Murine lung organoids were isolated from the distal lung tip according to a recognized protocol (McQualter et al., 2010, incorporated herein by reference). The tissue was cut into 5-20 mm pieces. 2The tissue was cut into small pieces, rinsed in PBS, and then digested with 1.5 mg / ml Dispase II, 0.5 mg / ml Collagenase, and 10 μg / ml DNAse in 10 ml of PBS containing 2% FCS for 1 h at 37°C on a shaker set at 100-250 rpm, vortexing for 15 s every 20 min. The sample was then allowed to settle, and the cloudy fraction, enriched for fibroblasts and immune cells, was discarded. The remaining large chunks were then incubated in EDTA and HEPES for an additional 1 h, then resuspended in Matrigel and cultured for 4 days in basal medium containing R-spondin, Noggin, EGF, FGF10 (500 ng / ml), 5 μM CHIR, and 1 μM RhoK inhibitor. Once luminal structures formed, the RhoK inhibitor was removed, and the cultures were expanded for a minimum of 3 weeks to enrich for alveolar base gland stem cells. These heterogeneous organoid cultures were then FACS purified and analyzed for viable, CD45 - , EpCAM high Epithelial cells were isolated and expanded as epithelial-only structures in media containing expansion medium with a Rho-K inhibitor for 4 weeks.

[0351] Human Organoid Differentiation KUTE-4 (Leha et al., 2016) and FS13B human iPSCs were previously generated using recognized protocols (Kilpinen et al., 2017; Yusa et al., 2011, both incorporated by reference). Human iPS cells were maintained on vitronectin (StemCell Technologies) in E8 medium and passaged as disrupted clusters every 4–6 days using Versene® (GIBCO).

[0352] HIOs were obtained according to a previously published protocol (McCracken et al., 2011, incorporated by reference), substituting CHIR99021 for recombinant Wnt3a. HIOs were further matured by adding 20 ng / ml IL-2 to the expansion medium. HIOs were passaged and replated in Matrigel as whole structures every 10–14 days. Organoids were allowed to mature for 4–8 weeks before use in experiments.

[0353] Isolation of murine ChILPs and ILCs ILCPs were isolated from the bone marrow (BM) of femurs and tibias of 6-8 week-old C57BL / 6 female murines according to a recognized protocol (Gronke et al., 2017, incorporated herein by reference). Soft tissue was physically removed from the bones, which were then sterilized in 70% ethanol for 2 minutes and rinsed in ice-cold PBS. The ends of the bones were cut off with dissecting scissors, and the midsection was then washed with PBS using a 27-gauge needle. The bone marrow was pulverized, transferred to a 50 ml Falcon® tube, and centrifuged at 1500 RPM / 500 x G for 3 minutes. After removing the supernatant, red blood cells were depleted using 2 ml of standard ACK lysis buffer at room temperature for 2 minutes. The reaction was quenched with 30 ml of PBS and centrifuged at 500 x G for 3 minutes. The remaining pellet was resuspended in PBS supplemented with 2% FCS, 0.1 M EDTA, and 1 mM HEPES (FACS buffer) and filtered through a 40 μm sterile mesh into a flow tube. Fc receptors were blocked with anti-CD16 / CD32 (2.4G2) for 10 min at 4°C. Five million cells in 100 μl of FACS buffer were stained with primary conjugated antibodies for 30 min at 4°C in the dark, rinsed in FACS buffer, centrifuged, and resuspended in 200–300 μl of FACS buffer containing 0.1 μg / ml DAPI (4',6-diamidino-2-phenylindole; Sigma) to exclude dead cells. Unstained cells and UltraComp beads were used as unstained and single-color controls to calculate compensation values. Fluorescence minus one (FMO) was used for CD127 (IL7Rα) and Lineage + DAPI ChiLPs were isolated by fluorescence-activated cell sorting (FACS) on an ARIA-III (BD Biosciences) using DIVA software, with the assistance of Guy's, King's and St. Thomas' (GSST) Biomedical Research Council (BRC) flow core staff.

[0354] Isolation of human ILCPs Systemic ILCPs were isolated from white blood cell cones (NHS-BT) according to recognized protocols (Lim et al., 2017a, incorporated herein by reference). Briefly, lymphocytes were purified from the cones using FICOLL density gradient separation. A small aliquot from each cone was phenotyped using a hILCP panel to assess ILCP frequency and antibody titers, and the remaining cones were divided into 20–30 cryovials of approximately equal hILCP frequency (10% DMSO in fetal bovine serum, added dropwise, frozen in Mr. Frosty isopropanol containers, and stored in liquid nitrogen). This allowed experiments to be performed from the same three donors, reducing variability, and allowing for the addition of sufficient lineage antibodies to exclude non-ILC subtypes. On day 1 of the experiment, frozen vials were thawed and rested in medium containing 10% FCS and Fc block for 1 hour, rinsed with PBS for fixable Live / Dead staining, and stained and sorted on a FACSARIA-III (BD biosciences).

[0355] ILCP co-culture with organoids On day 1, murine SIO / LO were mechanically disrupted to whole crypts, and HIO / HLO were digested with 0.1 mg / ml collagenase at 37°C for 15 minutes, followed by mechanical disruption until a cloudy mesenchymal-enriched fraction appeared. Epithelial structures were allowed to settle to the bottom of a 15 ml Falcon tube, and the mesenchymal-enriched fraction was removed. This step was repeated 3–5 times or until the epithelial-enriched fraction became clear. Approximately 50–100 murine and 25–50 human organoid structures were transferred to a 1.5 ml Eppendorf tube, and then approximately 500–1000 ILCPs were added to these tubes and centrifuged at 300 g for 5 minutes. After carefully removing the supernatant, the resulting cell mixture was resuspended in 30 μl of ice-cold Matrigel, carefully avoiding the formation of bubbles, and the combined cell mixture was pipetted into the center of a pre-warmed tissue culture-treated well and allowed to solidify at 37° C. for at least 15 minutes. Then, pre-warmed basal medium supplemented with R-spondin, noggin, EGF, and 50 mM 2-mercaptoethanol (R&D), 20 ng / ml rhIL-2 (Sigma), and 20 ng / ml rmIL-7 (R&D) was carefully added to each well. This medium may be referred to as Medium A herein.

[0356] Half-medium changes (removing 50% of the medium and replenishing 60% of the remaining volume) were performed every 1–3 days, leaving the conditioned medium in the wells while adding fresh 2x growth factors to ensure organoid and ILC viability without inhibiting ILC-epithelial interactions. To maintain consistency between conditions, no small molecules, FGF7, or FGF10 were added to lung organoid cultures.

[0357] For transwell experiments, permeable inserts separated the organoid fraction (top) and the ILCP fraction (bottom), both of which were resuspended in 25 μl of Matrigel (Falcon 24-well (Corning), 1 cm 2 1.6 x 10 per 6 hole).

[0358] In organ-to-organ exchange experiments, ILCP+ organoid cocultures were detached with TryPLE on day 7, and the entire viable EpCAM-, CD45+, Lineage- population was isolated by FACS from half of the culture, while the other half was used for flow cytometry analysis. This prevented epitope blocking in the secondary analysis on day 14. The organoid-matured ILC population obtained by FACS was then replated into the same or opposite organoid culture in fresh Matrigel, and the protocol was resumed as on day 1.

[0359] For routine analysis, day 7 or day 14 (replacement) cocultures were either fixed in 4% PFA for immunocytochemistry, rinsed with PBS, and detached with TrypLE (Gibco) and DNAse (because epithelial cells may be dead) for 20 min to obtain single-cell suspensions for flow spectrometry, or individual populations were purified by FACS for RT-qPCR. All suspensions containing single epithelial cells were maintained in 2% FCS containing 0.1 mM EDTA and 1 mM HEPES to prevent cell clumping.

[0360] Co-culture analysis Flow cytometry Flow cytometry data were acquired on a Fortessa II (BD Biosciences) using DIVA software and analyzed using FlowJo 10.4.1.

[0361] RT-qPCR RNA was extracted using the RNAeasy Micro Kit (Qiagen), and 10 μl / ml β-ME was added to the RLT lysis buffer to mitigate degradation in RNAse-rich epithelial tissues. cDNA reverse transcription was performed using 0.5 μl of random primers and 0.5 μl of oligo-dTTT primer per 10 μl reaction using the RevertAid Synthesis Kit (ThermoFisher) according to the manufacturer's protocol. RTqPCR was performed using SYBR (Applied Biosciences) or TAQ-FAM probes (ThermoFisher) and TAQ enzyme (Applied Biosciences) with primers ordered from Invitrogen. The PCR was performed on a BioRad real-time CFX384 Touch using CFX Maestro software, and data were processed and normalized in Microsoft Excel.

[0362] Confocal and live imaging Live imaging was performed overnight in co-cultures 1 day after seeding for Cell Trace far-red experiments in which ILCPs were labeled prior to co-culture, or for Rorc eGFP The ILCP culture was performed on day 4 in animals. Images were taken using a NIKON A1R inverted confocal microscope with incubation capabilities and phenol-free medium. To preserve relative ILC-organoid localization, the cocultures were then fixed in 4% PFA at room temperature for 5–15 minutes within a 3D Matrigel bubble. Samples were either stained as whole organoids or cryopreserved in 30% glucose, embedded in OCT overnight at 4°C, the Matrigel dried, and then frozen for cryosectioning. Samples were permeabilized with 0.05% Triton-X, stained with primary antibodies overnight at 4°C, and stained with secondary antibodies and Hoechst for 1 hour at room temperature (RT). Images were acquired using LAX software on a Leica SP8 confocal microscope, and processed using FIJI (ImageJ).

[0363] RNA sequencing of murine SIO Lymphoid progenitor cells (Lin-Cd127+α4β7-Ftl3+) were harvested from BM, sorted as described above, and cultured with SIO for 7 days in the presence of 50 mM β-mercaptoethanol (R&D), 20 ng / ml rhIL-2 (Sigma), 20 ng / ml rmIL-7 (R&D), and 20 ng / ml Flt3-ligand (R&D). + Cd45 - The cells were sorted into lysis buffer, and RNA was extracted as described above. Libraries were prepared using the SMARTer Stranded Total RNA Seq kit (pico input mammalian) and sequenced on a HiSeq2500 at King's College London Genome Centre, with basic alignment and quality control. Normalized counts were expressed as (logX + 1,2) in Excel 16.16.20 and as heatmaps in GraphPad Prism 8.2.1.

[0364] Quantitative and statistical analysis Data were analyzed using Microsoft® Office 365 Excel 16.16.20 and GraphPad Prism 8.2.1. Meta-analysis of published RNA sequencing data was performed by normalizing raw FPKM values to the geometric mean values of the housekeeping genes Actb / ACTB, Hprt1 / HPRT1, and Gapdh / GAPDH, and then analyzing the (logX + 1,2) of these values using a multiple row t-test. Log-q values were visualized as volcano plots in GraphPad Prism 8. Heatmaps were generated using heatmapper.ca / expression / , and clustering was applied to rows and columns using centroid linkage and Euclidean distance measures (clustering dendrograms were applied to columns).

[0365] The reagents and models used are shown. [Table 1] Table 1 shows details of the experimental models and subjects.

[0366] [Table 2] JPEG2025525351000003.jpg226160JPEG2025525351000004.jpg227160JPEG2025525351000005.jpg27160Table 2 shows the antibodies.

[0367] [Table 3] JPEG2025525351000007.jpg212131Table 3 shows cell culture reagents.

[0368] [Table 4] Table 4 shows the kits used. [Example]

[0369] Example 1: Small intestinal organoids (SIOs) promote the development of ILCs from ILCPs Murine small intestinal epithelial organoids (SIOs) express Lgr5 + and Lyzozyme + The SIO consists of stem cell crypts (Fig. 1a), which bud into the surrounding extracellular matrix and differentiate into absorptive and secretory cells toward the center of the SIO. The SIO maintains polarity in vitro and expresses ZO-1. + Tight junctions form a pseudo-lumen at the apical surface, enclosed by apical tubular structures (Fig. 1b). To assess the ability of the basal epithelium to provide a niche for innate lymphoid cells (ILCs), CD127 + , Lineage - , Flt3 - , α4β7 + , CD25 - , PD-1 +ILC progenitor cells (ILCPs) were harvested from adult murine bone marrow, and approximately 500–1000 ILCPs were resuspended and replated into 3D Matrigel bubbles with or without 50–100 intact SIO structures (Figure 1c–e).

[0370] Serially passaged primary SIO cultures consistently and reproducibly lack non-epithelial cells, such as immune or mesenchymal cells (Sato et al., 2009). However, as an additional precaution, to ensure that mature ILCs were derived from ILCPs and not carried over from primary tissue, SIOs were also plated alone, but lineages were not observed in SIO-only cultures. - No immune cells were observed. Cocultures were supplemented with EGF (epidermal growth factor), R-spondin 1, and noggin to support epithelial crypts, and IL-2 and IL-7 to promote ILCP survival. ILCPs migrated freely within the Matrigel bubble, where they were exposed to basally presented or secreted epithelial ligands. After 7 days, CD45 + , Lineage - Immune cells, EpCAM + The epithelial cells were then exfoliated using TryPLE for downstream analysis (Fig. 1f). + ILCPs expanded significantly in SIO coculture (20.33 fold change (FC) from seeded cells), and under the same culture conditions, CD45 + , Lineage - The presence of epithelial cells was important for this expansion, as immune cells were reduced by 0.56FC (Fig. 1g). + It allowed the proliferation of ILC2P (2.3FC) but not the remaining CD25 - , PD-1 - The ILC population did not expand in the presence of SIO (0.8FC).

[0371] To assess whether SIO promotes not only expansion but also maturation (or differentiation), we next transfected ILCP into RORγt reporter mice (Rorc GFP / + ) and obtained by isolation.- ILCPs significantly upregulated the expression of this group 3 transcription factor when cultured with SIOs (Fig. 1h, 1i). SIOs also upregulated the expression of type 1 (Klrg1) transcription factors. - , NKp46 + , NK1.1 + ) and Group 2 ILCs (RORγt - , Klrg1 + This promoted the maturation of populations expressing extracellular markers associated with epithelial cell types (ILC2s). This did not significantly occur in ILCPs cultured in Matrigel with IL-2 and IL-7 alone, suggesting that epithelial cell differentiation can be promoted with or without the addition of subset-specific cytokines (e.g., IL-23, IL-1β, IL-15, IL-12, IL-18, IL-25, and / or IL-33) that are consistently used in other recognized in vitro protocols.

[0372] ILCPs cultured with or without SIO retained lymphocyte populations that did not express these maturation markers. To assess the identity of these cells and address whether their differentiation was actively promoted or resulted from suppression of progenitor stemness, additional markers associated with mature ILC subsets were pooled within a flow cytometry panel (Figure 2a). The remaining negative population (NEG) broadly expressed the ILCP marker c-KIT, and c-KIT expression with or without SIO was significantly reduced. + and CD25 + There was no significant difference in the relative proportion of NEG cells in the SIO coculture (Figure 2a-c). However, the absolute number of NEG cells was significantly expanded in the SIO coculture (Figure 2c), and this population upregulated the gut-homing integrin α4β7 and the ILC marker PD-1. This suggests that the adult mucosa has the capacity to actively maintain a heterogeneous population of ILC progenitors.

[0373] Since maturation is not promoted by suppressing ILCP stemness, we next examined the functional maturation of putative ILC groups using a group-specific intracellular flow cytometry panel. ILCPs cultured with SIO expressed significantly more RORγt cells than ILCPs cultured without SIO. - Klrg1 - NK1.1 + NKp46 + Eomes - T-bet + T-bet produced ILC1s (Fig. 2d, e). + Eomes + A small but significant population of NK cells was also present in the cocultures. There was no significant difference in the absolute number of NK cells between the culture conditions. In fact, the relative proportion of NK cells was significantly reduced by SIO (Figure 2d). When stimulated with nonspecific PMA and ionomycin, ILC1s in these cocultures demonstrated the ability to express IFN-γ, even without the addition of IL-12, IL-15, or IL-18. Furthermore, upon IL-18 stimulation, ILC1s and NK cells expressed IFN-γ and granzyme B at higher rates in the cocultures than comparable group 1 cells isolated from primary small intestinal lamina propria, suggesting that the precursor-derived group 1 ILCs were functionally mature (Figure 2e, f).

[0374] Next, RORγt + Population identity was assessed. Only in SIO cultures were CCR6 + RORγt + We observed a small but significant increase in cell numbers, and all ILC subtypes, including LTi-like cells (Klose et al., 2014a), expressed PD-1 in appropriate culture conditions. + After 7 days of co-culture, unbiased stimulation with PMA / ionomycin for 3 hours resulted in approximately 5% RORγt proliferation, suggesting that the cells maintain a shared lineage potential with ILCPs (Fig. 2h, i). +Approximately 2.5% of SIO-derived ILC3s expressed IL-22, and approximately 2.5% expressed IL-17A (but not IL-23, Figure 4a). These cells further responded strongly to subset-specific IL-23 stimulation, with SIO-derived ILC3s secreting IL-22 at rates comparable to primary ILC3s. Unlike unbiased PMA / ionomycin, this stimulation also induced some RORγt and moderate IL-22 expression in ILCPs cultured without SIO, although the absolute number and proportion of mature ILC3s were significantly reduced relative to ILCPs cultured with SIO (Figure 2i). Both SIO and primary ILC3s expressed low levels of IL-17A, which may explain the CCR6 expression in both SIO and primary SI-ILCs. + The number of ILC3s and LTi-like cells was significantly higher in the presence of SIO than in the presence of PD-1. - They were reduced in cocultures but not without SIO (Fig. 4b). Finally, ILC2 progenitors, which predominantly give rise to putative ILC2s, produced only minor numbers of ILC3s and no LTi-like cells.

[0375] Example 2: SIO recapitulates ILC subset proportions characteristic of the small intestine Cytokine production in SIO-derived ILC3s was comparable to that of primary ILC3s, but approximately 3% of this RORγt + Only a small proportion of the population expresses the natural cytotoxicity receptor (NCR) NKp46, with approximately 18% of NKp46CR in primary SI (excluding Peyer's patches). + The proportion of this population was significantly lower than that of ILC3s (Fig. 5a). NKp46 expression in ILCs was significantly higher than that in adult NKp46s. +It has previously been reported that ILC3 maturation is partially regulated by the microbial context, which can affect the expression of epithelial Notch ligands (DLL1, JAG1, JAG2), which may be essential, but not sufficient, for the final maturation of ILC3s. Intestinal organoids can maintain the epigenetic signature of the donor tissue from which they were derived. However, it remains somewhat controversial whether serially passaged organoids resemble germ-free (GF) epithelium in vitro or whether they retain the epigenetic signature of previous specific pathogen-free (SPF) microbial exposure. To address this question, SIOs were further derived from GF animals and cultured in Rorc. eGFP Both SPF and GF SIOs were cultured with ILCP. - There was no significant difference in their ability to generate ILC3s and promote ILC maturation, suggesting that SPF-derived SIOs essentially provide an epithelial model of GF (Figure 5b).

[0376] It is noteworthy that although NKp46 expression differed within primary ILC3s and SIO-derived ILC3s, the overall distribution of mature ILC groups generated from unstimulated SIO cultures was similar to the global tissue-specific ratios of ILC subsets observed in the homeostatic primary small intestine, which was greatly enriched for group 3 ILCs during homeostasis (Figure 5c). To assess whether ILCPs require contact with the epithelium to induce this intestinal-specific group distribution, ILCPs were isolated from SIOs using transwell inserts (TW). This did not affect group 1 and group 3 ILCs but significantly reduced the number of putative ILC2s (Figure 5d, Figure 5e). These experiments were performed using CD25 + Repeated with ILC2Ps, as their relative abundance in the murine bone marrow (Fig. 1d) allowed for isolation into more experimental conditions from the same animal. - Klrg1 +This cell loss was recapitulated in ILC2Ps, and the frequency (Figure 5f) and expression of Klrg1 (Figure 5g) of this population were significantly reduced when cultured either without SIO or with SIO isolated by transwell inserts. To further explore the necessary and potentially tissue-specific interactions between intestinal epithelial cells and ILC2s, we next compared the maturation of ILC2P-derived ILC2s with primary ILC2s, as previously shown for other ILC subtypes. Cocultured ILC2s and primary tissue-derived ILC2s showed no difference in expression of the type 2 transcription factor Gata3 (Figure 5h, Figure 5i). Upon stimulation, approximately 80% of ILC2s expressed IL-5, and nearly all ILC2s expressed IL-13, showing no significant differences in cytokine expression compared to primary ILC2s (Figure 5j).

[0377] Just as NKp46 expression was reduced in SIO-derived ILC3s, IL-25R expression, characteristic of intestinal ILC2s, was significantly reduced in cocultures relative to primary intestinal ILC2s (Fig. 5k). To assess the presence or absence of epithelial stimuli to promote this maturation, we examined the expression of EpCAM in cocultures of unstimulated common ILC progenitors with SIOs, and in SIOs cultured without lymphocytes in medium with or without IL-2 and IL-7. + CD45 - The epithelial fraction was characterized by bulk RNA sequencing (Figure 5l). Targets associated with ILC maturation included genes for the Notch ligands Dll1, Dll4, Jag1, and Jag2, the cytokines IL-15 and IL-18, and the expression of basal secretory growth factors of the Bmp, Wnt, Fgf, and Tgf families. Neither these genes of interest nor transcriptional signatures related to small molecule biosynthesis (including the gene for the ILC3-associated retinoic acid (RA) synthase aldh1a1) were significantly differentially expressed between culture conditions. Without wishing to be bound by theory, this may suggest that epithelial cells have a steady-state ability to promote ILC maturation that is independent of and / or precedes the presence of immune cells.

[0378] SIO expression of the tuft cell-derived IL-25R / IL-17RB ligand Il25 was low or absent in all conditions. It has been reported that in adult animals, the expression of epithelial-derived cytokines can be regulated by luminal metabolites, for example, succinate contributes to IL-25 / IL-17E expression in tuft cells. The absence of IL-25 in SIO provided an optimal template for assessing the ability of these essentially GF organoids to recapitulate this well-recognized succinate-tuft-ILC2 circuit. We took advantage of the organoid's closed compartmental nature, which allows components to be microinjected into the simulated lumen (Figure 5m). The epithelial integrity of this compartment was demonstrated by the retention of the 204 kDa molecule fluorescein isothiocyanate-dextran (FITC) (Figure 5n). Succinate injection significantly upregulated Dclk1 in SIO. + The number of tuft cells increased (Fig. 5o), which correlated with increased expression of Il17e / Il25 (Fig. 5p). Furthermore, ILC precursors co-cultured with succinate-injected SIOs expressed significantly more Klrg1 + These results suggest that SIO-derived ILC2s retain the ability to proliferate in response to epithelial-derived IL-25, despite the low level of expression of IL-25R (Fig. 5q).

[0379] Example 3: Epithelial identity promotes tissue-specific ILC2 phenotypes While ILC3s are the dominant population in the adult murine small intestine, ILC2s are relatively more abundant in the postnatal murine lung (Dutton et al., 2018; Saluzzo et al., 2017). To assess whether epithelial identity alone is sufficient to induce the development of this "expected" ILC2 imprinting from bone marrow ILC progenitors, we analyzed adult murine EpCAM (EpCAM)-derived ILCs. +Murine lung organoids (LOs) were obtained from enriched primary lung tip epithelial cells, producing a mixture of "cystic" round organoids and "saccular" organoids consisting of small budding structures (Figure 6a). Cystic lung organoids consist of airway epithelial cells enriched in club cells, goblet cells, and ciliated cells, whereas saccular structures contain surfactant-rich alveolar AEC1 and AEC2 cells that enable gas exchange (McQualter et al., 2010). Thus, like SIOs, LOs provide a rich snapshot of many cell types in the complex lung epithelium. Also, like SIOs, LOs supported the expansion of putative group 1, group 2, and group 3 ILCs (Figure 6b) from common helper-like ILC progenitors (Figure 6c, Figure 7). Notably, EpCAM-mediated signaling was observed in lung organoid cocultures. - , CD45 + , Lineage - , RORγt - , NKp46 - , NK1.1 - , Gata3 + The relative frequency of ILC2s was significantly increased. Indeed, these ILC2s recapitulated the tissue-specific characteristics that distinguish murine intestinal and pulmonary ILC2s in vivo (GSE117568, (Ricardo-Gonzalez et al., 2018a)) (Figure 6d). Despite the absence of other immune cells, stromal cells, or tissue-specific microbiota, epithelial-only SIO cocultures harbored a higher proportion of Klrg1 cells than LO. + LO-derived ILC2s expressed higher levels of the IL-33 receptor ST2 (Fig. 6e). Finally, ILC3s from these cocultures did not differ significantly in IL-22 expression, whereas intestinal ILC2s were observed to express higher amounts of type 2 cytokines, and IL-13 was significantly enriched in SIO-derived ILC2s, as predicted by a meta-analysis of RNA expression in murine intestinal and pulmonary primary ILC2s (Fig. 6f).

[0380] These tissue-specific ILC2 patterns are relevant in murine pulmonary helminth infections.high “Pro-inflammatory” iILC2s (Huang et al., 2015) expand in response to IL-25 and subsequently migrate from the gut to the lungs to clear infection (Huang et al., 2018). Indeed, Klrg1 low Only adoptive transfer of these iILC2-enriched intestinal ILCs, but not "natural" nILC2-enriched lung ILCs or bone marrow ILCPs, results in normal pathogen clearance. The ultimate fate of intestinal iILC2s in the lung is unknown, which may be due to the Klrg1 high This is because ILC2s are no longer detectable in the lung after recovery from infection. To address these phenomena in a reductionist organoid system, ILC populations were isolated from the SIO by FACS after 7 days of co-culture and then reseeded into new LOs, virtually recapitulating the post-migration kinetics of this population as it reached the lung (Figure 6g). After an additional 7 days of co-culture, the previous Klrg1 high We observed a significant downregulation of Klrg1 in SIO-derived ILC2s (Fig. 6h), which suggests that Klrg1 low These results suggest that SIO-ILC2s may become less distinguishable from lung / nILC2s. Conversely, ST2 and CD25 were significantly upregulated by SIO-ILC2s upon transfer to LO. Interestingly, ST2 upregulation was significantly higher in SIO-LO-exchanged ILCs compared with LO-derived ILCs reseeded in LO. This suggests that ILC2s adopt imprinting of their gut origin. This may make them more receptive to lung stimuli than nILC2s, potentially explaining their unique ability to clear pulmonary helminth infections.

[0381] To understand how intestinal and lung epithelia differed in expression of previously examined target genes (Figure 5l), we performed a meta-analysis of gene expression in published equivalent human adult primary intestinal and lung epithelial-only organoids (GSE112991) (Heo et al., 2018) (Figure 8a, Figure 8b). In this dataset, lung and intestinal epithelial cells clustered separately, recapitulating the expected tissue-specific expression of intestinal antimicrobial peptide (e.g., LYZ, REG4) and lung surfactant protein (e.g., SCGB1A1) transcripts. As in murine organoids, IL17E / IL25 were not detected in either intestinal or lung human organoids, and as in SIOs cultured with or without lymphocytes and immune cytokines, the majority of basal-presenting or basally-secreting genes of interest were not significantly differentially regulated between the two epithelia. However, expression of the ST2 ligand IL-33 was significantly enriched in lung epithelial cells (Figure 8b). This expression difference was conserved in primary murine small intestine and lung tissue (Figure 6i) as well as in the SIO and LO cultures used in these experiments (Figure 6j). To assess the role of this tissue-specific difference in mediating the phenotypic transition of gut-derived ILC2s, a neutralizing dose of murine anti-IL33 antibody was added to the cocultures exchanged from gut to lung. IL-33 inhibition did not affect the downregulation of Klrg1 but significantly suppressed the upregulation of ST2 and CD25 (Figure 6k). To confirm that these changes were actively induced by IL-33-containing lung epithelial cells and not simply due to the loss of some gut-specific inhibitory stimulus, SIO-derived ILCs were further replated on Matrigel without SIO or LO, but with or without recombinant murine IL-33 (Figure 8c). Loss of contact with SIO was not sufficient to recapitulate the SIO-to-LO phenotype, suggesting that ST2 and CD25 expression is not suppressed by the gut environment. Instead, recombinant IL-33 alone was sufficient to induce high levels of ST2 and CD25 expression in SIO-mature ILCs (Fig. 8c).Finally, when LO-mature ILCs were exchanged into SIO, no significant differences in the expression of CD25, ST2, or Klrg1 were observed (Fig. 8d).

[0382] Example 4: Human intestinal organoids promote human systemic ILCP maturation Translating experimental approaches used in mice to human distal tissues is challenging, especially when studying rare populations such as ILCs. This makes the potential of in vitro human mucosal organoid systems particularly attractive. However, human bone marrow ILC progenitors are difficult to access. Therefore, to translate the previously described murine ILCP-organoid system, readily available systemic ILCPs were isolated from peripheral blood mononuclear cells (PBMCs) instead of bone marrow, while human intestinal organoids (HIOs) were derived from healthy KUTE-4 human induced pluripotent stem cells (McCracken et al., 2011). The structure of these ILCPs is well-recognized and they can be matured by adding IL-2.

[0383] Heterogeneous systemic ILCPs (Figure 9a) were cocultured with these hiPSC-derived epithelial-mesenchymal HIOs (Figure 9b). Coculture with HIOs significantly expanded the ILCPs (approximately 20-fold) relative to the number of cells seeded without HIOs and compared to the number of ILCPs seeded on day 1 (d1) (Figures 10a and 10b). As in murine cocultures, human ILCPs developed as putative group 2 (Figure 10c), group 3 (Figure 10d), and group 1 ILCs (Figure 10e) when cultured with whole HIOs supplemented with IL-2 and IL-7. Previous in vitro systems have relied on the addition of IL-1β + IL-23 for robust development of ILC3s, IL-25 + IL-33 for ILC2s, and IL-12 + IL-18 for ILC1s (Lim et al., 2017b), or CD34 +Although maturation from hematopoietic stem cells required the addition of IL-15 and differentially expressed Notch ligands (Hernandez et al., 2021), fold expansion and development occurred without the addition of IL-1β or subset-specific cytokines. Instead, HIOs expressed RORγt alone. + It significantly supported ILC maturation (Fig. 10f), which significantly upregulated NKp44 (Fig. 10g) and c-KIT between the periphery and intestine, as observed by Lim et al. + These NKp44 + / - CCR6 + / - Group 3 RORγt + ILCs produced low levels of IL-22 and IL-17A upon nonspecific PMA / ionomycin stimulation and significantly responded to a further 4 h of stimulation with IL-23 (Fig. 10h), whereas putative group 1 ILCs significantly upregulated the expression of T-bet and IFN-γ secretion in response to 4 h of stimulation with IL-18 (Fig. 10e).

[0384] Example 5: Epithelial cells, but not mesenchymal cells, promote robust ILC expansion and maturation These data suggest that the microenvironment of human intestinal organoids stimulates the baseline maturation of ILC subsets from ILCPs. However, unlike murine primary epithelium-only organoids, hiPSC-derived hindgut organoids co-occur with a rich and complex native mesenchyme. These cells not only contribute to epithelial cell maturation (Stallmach et al., 1989), but are also known to be a source of IL-7, an ILC survival factor and CD127 ligand (Xu et al., 2015). To assess whether epithelial or mesenchymal cells predominantly drive ILC maturation, we digested the matrix of the HIO structure with collagenase, and isolated a single-cell suspension of mesenchyme from the 3D epithelial structure by continuous gravity gradient separation to obtain a mesenchyme-depleted HIO fraction (MD-HIO) and an epithelial-depleted fibroblast-enriched fraction (ED-FB) (Figure 9c). These fractions were cultured with ILCPs for 14 days, and both fractions were able to sustain ILC survival and expansion (Figure 9c). However, unexpectedly, the mesenchyme-depleted epithelial fraction lost EpCAM after culture. - , CD45 + , Lineage - This resulted in a dramatic increase in cells (Figure 9d) and significantly increased the fold-change expansion of ILCPs seeded on epithelial-depleted mesenchyme or on Matrigel alone (Figure 9e). Furthermore, while fully epithelial-mesenchymal HIOs induced NKp44 expression in approximately 2–6% of group 3 ILCs (Figure 10g), this upregulation was significantly greater (15–22%) in mesenchymal-depleted cocultures (Figure 9f, Figure 11a). Indeed, the epithelial-enriched MD-HIO fraction promoted a pattern of ILC subset maturation that more closely resembled the distribution of mature ILCs in the healthy human intestine (Kraemer et al., 2017) than the epithelial-depleted mesenchymal fraction (Figure 9g). Like NKp46 expression in a murine model of GF (Figure 5a), NKp44 - NKp44 against putative ILC3s + The relative proportion of putative ILC3s was significantly higher than that of NCRs in GF-environmentally controlled MD-HIOs (Fig. 9h), where ILCPs were free to interact with the epithelium. -Maturation was biased towards the group (Fig. 9f, Fig. 9g).

[0385] The current gold standard for studying murine and human ILC maturation in vitro is based on modified murine bone marrow stromal cells (Nakano, 1996). Therefore, the relatively small expansion rate of ILCPs induced by hiPSC-derived intestinal mesenchyme was unexpected. We hypothesized that the increased cell number in mesenchyme-depleted HIOs may represent the proliferation of poorly differentiated, immature ILCs, while stromal cells may promote the production of smaller, but more mature, ILCs. To test this, ILCs derived from cocultures of MD-HIOs and ED-FBs were unbiasedly stimulated with PMA / ionomycin. We first assessed the maturation of putative group 3 ILCs. Here, RORγt + NKp44 in ILCs + Only the frequency of cells significantly expanded in MD-HIOs, and CCR6 + , NKp44 - , and T-bet + The relative frequency of ILCs was constant in both conditions (Fig. 11a). However, CCR6 + and NKp44 + / - ILCs express significantly higher amounts of IL-22 and CCR6 than corresponding populations derived from epithelial-depleted mesenchymal fractions. + and NKp44 - ILCs expressed significantly more IL-17A than the corresponding population from the epithelial-depleted mesenchymal fraction (Fig. 11b). Indeed, IL-22 + The frequency of ILCs was equal to or greater in the unbiased PMA / ionomycin-stimulated mesenchymal-depleted fraction than in the IL-22-induced IL-23-stimulated complete HIO cocultures (Fig. 10h). + / - , T-bet + and Eomes + Not only was the proportion of ILCs (Figure ​(Figure12),12) significantly reduced in the epithelial-depleted fraction (Figure11d), but also, upon stimulation with PMA / ionomycin, no IFN-γ expression was observed in either population in the absence of epithelial cells (Figure11e). Similar to ILCs in group 3, T-bet expression was also observed in the epithelial-depleted fraction.+ Percentage of ILCs and IFN-γ + The number of cells was greater in the absence of mesenchyme than in the absence of epithelium (Fig. 10e).

[0386] Unlike group 1 and 3 ILCs, the overall count (Figure 11f) and relative frequency (Figure 13a) of putative group 2 ILCs (Figure 13b) were not significantly affected by mesenchyme depletion. Nevertheless, both IL-5 and IL-13 expression significantly and strongly increased in the mesenchyme-depleted HIO fraction upon unbiased stimulation with PMA / ionomycin (Figure 11g). The lack of difference in ILC2 numbers induced by epithelial depletion contrasts with findings in murine ILC2P transwell cultures, where ILC2 numbers were significantly reduced upon physical separation from epithelial cells (Figures 9d-f). To assess whether contact-dependent mechanisms are conserved in the maturation of human ILCP-derived ILC2s, ILCPs were also isolated from mesenchyme-depleted HIOs (Figure 13c). Although ILC2 production remained greater in transwell-isolated cocultures than in ILCPs cultured without organoids, both overall cell count and relative ILC2 frequency were observed to be reduced in transwell cultures, and these ILC2s did not express IL-5 or IL-13 (Figure 13c). Finally, we sought to assess whether the lack of cytokine expression in epithelial-depleted cocultures was due to the inability of ILC2s to mature without epithelial cells, or whether this was the result of active suppression of ILC2 cytokines by the mesenchyme, via secretion of inhibitory factors or competitive depletion of required ligands. To test this, we first allowed ILCPs to mature normally in MD-HIOs, and then cultured them in the presence of the resulting EpCAM-derived ILCPs. - CD45 + Lineage -ILCs were purified by FACS and replated in MD-HIO or switched to ED-FB (Fig. 13d). ILCs replated without epithelial cells were observed to have a reduced proliferation rate and no difference in IL-13 expression (Fig. 13e). However, the presence of more mesenchyme significantly reduced IL-5 expression by ILC2 (Fig. 13e). This suggests that IL-13 + These findings suggest that stable development of ILC2s can be promoted by contact with the intestinal epithelium, whereas ILC2 proliferation and IL-5 expression may be actively suppressed by the intestinal mesenchyme or may require constant exposure to the epithelium.

[0387] Example 6: Human mucosal epithelial identity contributes to tissue-specific ILC maturation To assess whether any tissue-specific ILC phenotypes could be captured in these human organoid systems (Figure 14a), ILCPs were cultured with either hiPSC-derived small intestine or hiPSC-derived lung organoids. ILCPs expanded in both cocultures (Figure 14b), and the absolute number of fibroblasts after mesenchymal depletion was well matched between conditions. HLOs showed comparable ability to produce mature group 1 and group 3 cells expressing IL-22, IL-17A, and IFN-γ (Figure 14c). Both HIOs and HLOs expressed GATA3, which expressed IL-5 and IL-13. + Like ED-FB, HLO produced IL-5 ILCs (Fig. 14d). + Similarly, the recently described c-KIT + , CRTh2 low , IL-17A +A further ILC2 population was present in HIO but not in HLO cultures, suggesting that, like the murine system, the human intestinal microenvironment may also support group 3 maturation by inducing ILC2-to-ILC3 plasticity in mature MD-HIO-derived ILC2s. However, unlike the murine system, coculture with HIO or HLO alone was not sufficient to induce statistically significant differences in the frequency of CD25 and ST2 ILCs within this putative GATA3+ ILC2 population (Figure 14e).

[0388] Finally, we assessed the ability of human organoids to recapitulate the gut-lung transition approach introduced in murine co-cultures (Figure 6e). As performed in the murine system, ILCPs were matured in MD-HIO for 2 weeks, then FACS-purified and replated in MD-HIO, MD-HLO, or MD-HLO with neutralized human IL-33. HiPSC-derived lung organoids express CD25 + The coculture of MD-HIO-derived ILC2s did not significantly affect the frequency of ST2-positive ILC2s (Fig. 14f). However, the expected increase in ST2 expression in lung ILC2s, which was not captured by HIO and HLO coculture alone, was significantly observed when MD-HIO-derived ILC2s were transferred into lung cultures (Fig. 14g). This effect was suppressed by the addition of neutralizing IL-33.

[0389] Example 7: Antigen processing of ILCs of the invention The ability of the ILCs of the present invention to process antigens via MHC-II was tested by hydrolysis of DQ-BSA, which leads to a fluorescent signal. A positive result is shown in Figure 16.

[0390] Example 8: Single-cell RNAseq (scRNAseq) in organoid-generated ILCs CD45 cytosol from the co-cultures via fluorescence-activated cell sorting (FACS) + Lin -We isolated five "superclusters" of cells that corresponded to the cytotoxic ILC1 population, two specific ILC2 clusters (ILC2a and ILC2b), an ILC3 cluster, and a small cluster of cells with significantly upregulated expression of genes characteristic of regulatory T cells (Treg) and regulatory ILC (ILCreg) populations previously described in mice (Wang et al., 2017).

[0391] Helper-like ILC cell types (ILC1-3) were identified by determining the expression patterns of key genes previously described to distinguish different ILC family members in single-cell RNA sequencing studies using human samples (Bjoerklund et al., 2016; Cella et al., 2019; Liu et al., 2021; Mazzurana et al., 2021). Significant enrichment of these genes in each cluster was determined by unbiased differential gene expression analysis comparing gene expression within each cluster to that of all other clusters (Figure 17B). Thus, the ILC1 population was identified by the enrichment of key cytokines and chemokines, including IFNG and CCL3, as well as the relatively high expression of genes such as KLRC1, PRF1, and GNLY, which have previously been shown to characterize the cytotoxic population of ILC1. Increased expression of the transcription factor GATA-3 (encoded via GATA3) and the cytokine signaling genes IL33 and IL17RB, along with upregulation of additional ILC2-associated genes such as HPGDS and KLRG1, were used to identify the two ILC2 populations. Finally, ILC3s showed significantly greater enrichment of genes encoding their signature transcription factors (e.g., RORC), cell surface markers (e.g., KIT), and cytokine signaling pathways (e.g., IL23RA). These genes were found to be coupled with the expression of markers previously shown to be highly expressed in both ILC3s and ILC1s, including NCR1 and PECAM1, further confirming the identity of both clusters.

[0392] However, annotation of ILCreg clusters has primarily been performed using markers identified in mouse studies of ILCregs. This is because, although an IL-10-expressing ILC population has been identified in the human intestine, this population has not yet been extensively characterized in human patients (Wang et al., 2017). This included genes encoding its main cytokine, IL-10 (IL10), the cell surface marker CD25 (IL2RA), and the transcription factors ID3 and SOX4. Interestingly, ILCreg clusters also expressed genes associated with human T regulatory (Treg) cells, including FOXP3, RUNX1, IL1R1, and CTLA4. Importantly, the T cell markers CD4 and CD3 were included within the lineage gating panel when sorting ILCs from cocultures, and their absence was further confirmed at the RNA level. Therefore, these cells were considered to be human ILCs equivalent to Tregs.

[0393] Using cell-specific enrichment scores for the human ILCP gene signature (Liu et al., 2021), we identified a small population of these progenitor cells within the larger ILC3 supercluster (Figure 17C). Furthermore, the cytotoxic ILC1 supercluster displayed a specific expression pattern of the transcription factors TBX21 and EOMES, suggesting that it contained both ILC1 and NK-like cells (Figure 17D). Overall, we found cells corresponding to the major ILC cell types expected to be observed in the coculture model, along with a putative population of human ILCregs.

[0394] To determine whether ILCs exhibit intestinal tissue-specific phenotypes, we created cell-specific enrichment scores for three gene modules identified by Mazzurana et al. (2021) as associated with blood / tonsillar circulating ILCs (mod11), colonic ILCs (mod3), or lung ILCs (mod34) (Mazzurana et al., 2021). Because Mazzurana et al. did not identify specific clusters of ILC1 or ILC2 in the colon, we considered only ILC3s here. We found that the ILC3 supercluster showed the greatest enrichment in the circulating gene module (Figure 17E), followed by the colonic gene module, with minimal cells showing enrichment in lung ILC-associated genes. Given that we observed a putative ILCP population within ILC3s, we subsequently overlaid the enrichment scores onto the UMAP to determine any heterogeneity in the expression patterns of these gene modules (Figure 17F). This revealed that ILC superclusters could clearly differentiate into cells displaying a more circulating phenotype compared with cells expressing a gene signature more closely resembling colonic ILC3s. As expected, the number of cells enriched for the lung module was minimal. Together, these data suggest that a substantial proportion of ILC3s in this co-culture model differentiate from a circulating phenotype and acquire a colonic gene expression signature.

[0395] Experimental Overview Circulating innate lymphoid progenitors (ILCPs; CD45+Lin-CD56-CD127+CRTh2-cKIT+NKp46-KLRG1-) were isolated from the blood of age-matched male and female donors (n=1) and placed in coculture with iPSC-derived human intestinal organoids for 14 days. After this 14-day period, four populations of ILCs were isolated from the cocultures using FACS: ILC2 (CD45+Lin-CRTh2+), ILC3 (CD45+Lin-CRTh2+cKIT+), ILC1 / NK cells (CD45+Lin-CRTh2-cKIT-CD56+ / -CD161+), and "other" ILCs (CD45+Lin-CRTh2-cKIT-CD56+ / -CD161-). Each population was then pooled at a 1:1 ratio of male to female cells, resulting in a final total percentage of each ILC population of 25, 15, 35, and 15%, respectively. These percentages were determined based on previous knowledge of the heterogeneity of each ILC subset, with ILCs exhibiting greater heterogeneity contributing a greater percentage of the final cell count. This cell pool was then sequenced as a single sample using 10x sequencing.

[0396] Processing and analysis of scRNA-seq data Barcoding, cDNA synthesis, and library preparation were performed using the Chromium Next GEM Single Cell 3' Library & Gel Beads Kit v3.1 according to the manufacturer's instructions. Libraries were sequenced by Novogene using NovaSeq with a target of 20,000 reads per cell. Preprocessing of single-cell RNA sequencing fastq files was performed using Cell Ranger v7.0.1 (10X Genomics), and reads were aligned to the GRCh38 reference genome. Further data preprocessing was performed in R (v4.2.2) using the Seurat package (v4.3.0). Cells with fewer than 1,000 genes and more than 10% of transcripts derived from mitochondrial genes were removed, as they were considered lysed / apoptotic. Additionally, cells with more than 7,500 genes or more than 40,000 UMIs were excluded, as they were presumed to contain doublets. The filtered dataset was then normalized using the Seurat SCTransform function (v2) to remove the effects of the cell cycle. The top 30 principal components were then used as input for Louvain clustering via the FindClusters() function in Seurat at a resolution of 0.2. UMAP was used to visualize the clusters. Differentially expressed genes were determined using the Wilcox test in the FindAllMarkers() function in Seurat. Genes with a log fold change of ≥ 2.5 and a false discovery rate of P < 0.05 were considered significantly differentially expressed. To visualize low-expression genes (TBX21 and EOMES), the plot_density function in the R package Nebulosa (v1.8.0) was used. The R package AUCell (v1.6.1) was used to determine the enrichment of various ILC gene signatures, and the resulting scores were plotted for visualization.

[0397] The results are shown in Figure 17 and Table 5 below. Table 5 shows the results of scRNAseq in organoid-generated ILCs. [Table 5]

[0398] Example 9: NK cells generated by co-culture of ILC progenitor cells (ILCP) with intestinal organoids are cytotoxic The cytotoxicity of NK cells generated by co-culture of ILC progenitor cells (ILCP) with intestinal organoids was tested using standard methods, and positive results are shown in Figure 18.

[0399] Example 10: Generation of ILCs in human small intestinal biopsy-derived organoids Differentiated ILCs (including ILC1, ILC2, ILC3, and NK cells) were generated from ILC progenitor cells (ILCPs) after 15 days of culture with human small intestinal biopsy-derived organoids. The results are shown in Figure 19.

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Claims

1. A method for expanding immune cells, (a) A method comprising the step of co-culturing the immune cells with at least one epithelial organoid, wherein the epithelial organoid comprises more epithelial cells than mesenchymal cells.

2. The method according to claim 1, wherein the epithelial organoid contains less than 45% mesenchymal cells.

3. The method according to claim 1, wherein the epithelial organoid contains less than 30% mesenchymal cells.

4. The method according to claim 1, wherein the epithelial organoid does not contain detectable levels of mesenchymal cells.

5. The method according to claim 1, further comprising the step of depleting mesenchymal cells from the epithelial organoid before step (a).

6. The method according to claim 1, wherein the epithelial organoids are subjected to depletion of mesenchymal cells.

7. The method according to claim 1, wherein the epithelial organoid is human or murine epithelial organoid.

8. The method according to claim 1, wherein the epithelial organoid is derived from induced pluripotent stem cells (iPSCs).

9. The method according to claim 1, wherein the epithelial organoid is a primary epithelial organoid.

10. The method according to claim 1, wherein the epithelial organoid is an organoid of the skin, intestines, lungs, thymus, thyroid gland, reproductive organs, bladder, kidneys, pancreas, oral mucosa, or liver.

11. The method according to claim 1, wherein the epithelial organoid is an intestinal epithelial organoid or a lung epithelial organoid.

12. The method according to claim 1, wherein the immune cells are hematopoietic stem cells and / or lymphocyte progenitor cells.

13. The method according to claim 1, wherein the immune cells are lymphocytes and / or bone marrow cells.

14. The method according to claim 13, wherein the immune cells are T cells, B cells, and / or innate lymphoid cells (ILCs).

15. The method according to claim 14, wherein the immune cells are ILCs.

16. The method according to claim 14 or 15, wherein the ILC comprises one or more ILCs of group 1, group 2, and group 3, or one or more ILCs of group 1, group 2, group 3, and a controllable ILC (ILCreg).

17. The method according to claim 1, wherein the immune cells include a plurality of different immune cell subsets, and optionally ILCs from a plurality of different ILC groups.

18. The method according to claim 17, wherein one or more of the aforementioned multiple different immune cell subsets are enriched over time.

19. The method described above is for the production and expansion of immune cells, and the method includes, before step (a), The method according to claim 1, comprising co-culturing immune cell progenitor cells with at least one epithelial organoid to expand the immune cell progenitor cells and differentiate the immune cell progenitor cells into immune cells.

20. The method according to claim 19, wherein the immune cell progenitor is a lymphocyte progenitor.

21. The method according to claim 19 or 20, wherein the immune cell progenitor cells include isolated ILC progenitor cells, and the immune cells are ILCs.

22. The method according to claim 1, wherein the immune cells are primary immune cells.

23. The method according to claim 1, wherein the immune cells are human immune cells.

24. The method according to claim 1, wherein the immune cells and the at least one epithelial organoid are co-cultured for at least about 72 hours.

25. The method according to claim 1, wherein the immune cells expand at a rate of at least twice their original size every 24 hours.

26. Immune cells obtained by the method described in claim 1.

27. At least 5 x 10 ３ An in vitro population of innate lymphoid cells (ILCs), including one individual ILC.

28. The in vitro ILC group according to claim 27, wherein the ILC comprises one or more ILCs from group 1, group 2, and group 3.

29. The in vitro ILC population according to claim 28, wherein the ILC comprises ILC1 cells and / or NK cells.

30. The in vitro ILC population according to claim 27, wherein at least 10% of the ILC population express a detectable level of NKp44.

31. The in vitro ILC population according to claim 27, wherein the population comprises ILCs from a plurality of different ILC groups.

32. The in vitro ILC population according to claim 27, wherein at least 10% of the in vitro population comprises exogenous polynucleotides.

33. A pharmaceutical composition comprising an immune cell population according to claim 26, or an in vitro population according to any one of claims 27 to 32, and a pharmaceutically or physiologically acceptable diluent and / or carrier.

34. A population of immune cells according to claim 26, or an in vitro population according to any one of claims 27 to 32, for use in the treatment or prevention of disease.

35. The pharmaceutical composition according to claim 33 for use in the treatment or prevention of disease.

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