(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2018/191558 Al 18 October 2018 (18.10.2018) W !P O PCT (51) International Patent Classification: (72) Inventors; and C12N 5/071 (2010.01) C12Q 1/6813 (2018.01) (71) Applicants: RAJAGOPAL, Jayaraj [US/US]; C/o 55 C12N 5/079 (2010.01) C12Q 1/6881 (2018.01) Fruit Street, Boston, MA 021 14 (US). REGEV, Aviv C12Q 1/68 (2018.01) G01N 33/53 (2006.01) [US/US]; C/o 415 Main Street, Cambridge, MA 02142 (US). BITON, Moshe [US/US]; C/o 55 Fruit Street, (21) International Application Number: Boston, MA 021 14 (US). HABER, Adam [US/US]; CI PCT/US2018/027388 o 415 Main Street, Cambridge, MA 02142 (US). MON- (22) International Filing Date: TORO, Daniel [US/US]; C/o 55 Fruit Street, Boston, MA 12 April 2018 (12.04.2018) 021 14 (US). XAVIER, Ramnik [US/US]; C/o 55 Fruit Street, Boston, MA 021 14 (US). (25) Filing Language: English (74) Agent: SCHER, Michael, B.; Johnson, Marcou & Isaacs, (26) Publication Language: English LLC, P.O. Box 6 1, Hoschton, GA 30548 (US). (30) Priority Data: (81) Designated States (unless otherwise indicated, for every 62/484,746 12 April 2017 (12.04.2017) US kind of national protection available): AE, AG, AL, AM, (71) Applicants: THE BROAD INSTITUTE, INC. [US/US]; AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, 415 Main Street, Cambridge, MA 02142 (US). CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, MASSACHUSETTS INSTITUTE OF TECHNOLO¬ DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, GY [US/US]; 77 Massachusetts Avenue, Cambridge, MA HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, 02139 (US). THE GENERAL HOSPITAL CORPO¬ KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, RATION [US/US]; 55 Fruit Street, Boston, MA 021 14 MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (US). OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY,TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (54) Title: MODULATION OF EPITHELIAL CELL DIFFERENTIATION, MAINTENANCE AND/OR FUNCTION THROUGH T CELL ACTION, AND MARKERS AND METHODS OF USE THEREOF (57) Abstract: An atlas of intestinal epithelial cells, intestinal ep ithelial stem cells and intestinal immune cells identifies new cell populations, markers, networks, and responses to stimuli. Intestinal epithelial cell sub-types are also found in the trachea. Accordingly, disclosed are methods of modulating epithelial cell differentiation, maintenance and/or function, related methods for the treatment of disease, including IBD and asthma. Also disclosed are methods and kits for identifying cell types, their differentiation, homeostasis and activation. CD3r 3 ' 00 FIG. 1A 00 o [Continued on nextpage] WO 2018/191558 Al llll II II 11III II I I llll III I I 11III II I II (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Published: — with international search report (Art. 21(3)) — with sequence listing part of description (Rule 5.2(a)) MODULATION OF EPITHELIAL CELL DIFFERENTIATION, MAINTENANCE AND/OR FUNCTION THROUGH T CELL ACTION, AND MARKERS AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 62/484,746, filed April 12, 2017. The entire contents of the above-identified application is hereby fully incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under Grant Nos. OD020839, DKl 14784, DK043351 and DK097485 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD [0003] The subject matter disclosed herein is generally directed to compositions and methods for modulating, controlling or otherwise influencing epithelial cell differentiation, homeostasis and activation in the gut and respiratory system. This invention also relates generally to identifying and exploiting target genes and/or target gene products that modulate, control or otherwise influence epithelial cell differentiation, homeostasis and activation in a variety of therapeutic and/or diagnostic indications. This invention also relates generally to a gut and trachea atlas identifying novel cell types and markers for detecting, quantitating and isolating said cell types. BACKGROUND [0004] The functional balance between the epithelium and the constituents within the lumen plays a central role in both maintaining the normal mucosa and in disease. Intestinal epithelial cells (IECs) of the small intestinal epithelium comprise two major lineages - absorptive and secretory 1 - reflecting its dual roles to absorb nutrients and form a flexible barrier, monitoring and titrating responses to a variety of noxious substances or pathogens2. Enterocytes of the absorptive lineage comprise approximately 80% of the epithelium and are specialized for digestion and transport of nutrients3. The secretory lineage comprises five further terminally differentiated types of IECs: goblet, Paneth, enteroendocrine, tuft and microfold (M) cells4 6 - each with distinct and specialized sensory and effector functions. [0005] The epithelium is organized in a repeating structure of villi, which project toward the lumen, and nearby crypts (Fig. la). The crypts of the small intestine are the proliferative part of the epithelium, in which intestinal stem cells (ISCs) and progenitors, termed transit- amplifying cells (TAs), reside6 7. In contrast, only fully differentiated cells are found on the villi2 7. The crypt also contains Paneth cells, which secrete anti-microbial peptides (AMPs), such as defensins and lysozyme, into the lumen to keep the microbiota in check8 9. The highly proliferative TA cells migrate along the crypt-villus axis and differentiate into functionally distinct epithelial cell types that subsequently reach the tip of the villus, where mature cells undergo apoptosis and shed to the lumen 1. [0006] Epithelial tissue turns over rapidly (~5 days)8, allowing it to dynamically shift its composition in response to stress or pathogens. For example, parasitic infection typically induces hyperplasia of goblet cells, which produce and secrete mucins to prevent pathogen attachment, strengthening the epithelial barrier and facilitating parasite expulsion 10 . Rare (0.5-1%) enteroendocrine cells (EECs) secrete over 20 individual hormones and are key mediators of intestinal response to nutrients 11 12 by directly detecting fluctuations in luminal nutrient concentrations via G-protein-coupled receptors (GPCRs) 11. Finally, IECs communicate with immune cells to initiate either inflammatory responses or tolerance in response to lumen signals2 13 . Tuft cells5, a rare IEC population, promote type-2 immunity in response to intestinal parasites by expressing interleukin-25 (1125), which in turn mediates the recruitment of group 2 of innate lymphoid cells (ILC2s) that initiate the expansion of T- helper type 2 (Th2) cells upon parasite infection 14 16. M cells, which reside exclusively in follicle-associated epithelia (FAE) 17, play an important role in immune sensing by transporting luminal content to immune cells found directly below them 18 in Peyer's patches, gut associated lymphoid follicles. Disruption in any of the major innate immune sensors and proximity effector functions of IECs may result in increased antigenic load through weakening of the epithelial barrier, and may lead to the onset of acute or chronic inflammation. [0007] Despite this extensive knowledge, given the complexity of the epithelial cellular ecosystem, many questions remain open. First, do we know all the discrete epithelial cell types of the gut, or are there additional types, or new sub-types that have eluded previous studies. Second, what are the molecular characteristics of each type. For example, mapping the GPCRs and hormones expressed by EECs has important therapeutic applications; charting known and new specific cell surface markers would provide handles for specific cell isolation, and help assess the validity of legacy ones; and finding differentially expressed transcription factors (TFs) will open the way to study the molecular processes that accompany the differentiation of IECs, such as tuft or enteroendocrine cells. Third, we still know little about the response of individual cell populations to pathogenic insult, both in terms of changes in cellular proportions and cell-intrinsic responses. [0008] A systematic atlas of single-cell RNA profiles can help address these questions, as the gene-expression program of a given cell closely reflects both its identity and function 19 20 . Most previous studies have examined the gene-expression profiles of IECs, but relied on known markers to purify cell populations6 15'2 1 22, which may isolate either a mixed population if marker expression is more promiscuous than assumed, or a subset of a larger group if overly specific. They may further fail to detect rare cellular populations or intermediate, transient states on a continuum. A recent study23 attempted to overcome these limitations using single-cell RNAseq (scRNA-seq), but analyzed only several hundred single cells, which may be insufficient to address the diversity of IECs, especially for subtypes that occur at a frequency of less than 0.1% '12 . Additional, studies53'30'145 also attempted to overcome these limitations using single-cell RNAseq (scRNA-seq).