(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 2017/147196 Al 31 August 2017 (31.08.2017) P O P C T (51) International Patent Classification: Kellie, E. [US/US]; 70 Lanark Road, Maiden, MA 02148 C12Q 1/68 (2006.01) (US). COLE, Michael, B. [US/US]; 233 1 Eunice Street, Berkeley, CA 94708 (US). YOSEF, Nir [IL/US]; 1520 (21) International Application Number: Laurel Ave., Richmond, CA 94805 (US). GAYO, En¬ PCT/US20 17/0 18963 rique, Martin [ES/US]; 115 Peterborough Street, Boston, (22) International Filing Date: MA 022 15 (US). OUYANG, Zhengyu [CN/US]; 15 Vas- 22 February 2017 (22.02.2017) sar Street, Medford, MA 02155 (US). YU, Xu [CN/US]; 6 Whittier Place, Apt. 16j, Boston, MA 02 114 (US). (25) Filing Language: English (74) Agents: KOWALSKI, Thomas, J. et al; Vedder Price English (26) Publication Language: P.C., 1633 Broadway, New York, NY 1001 9 (US). (30) Priority Data: (81) Designated States (unless otherwise indicated, for every 62/298,349 22 February 2016 (22.02.2016) US kind of national protection available): AE, AG, AL, AM, (71) Applicants: MASSACHUSETTS INSTITUTE OF AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, TECHNOLOGY [US/US]; 77 Massachusetts Ave., Cam BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, bridge, MA 02139 (US). THE REGENTS OF THE UNI¬ DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, VERSITY OF CALIFORNIA [US/US]; 1111 Franklin HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, Street, 12th Floor, Oakland, CA 94607 (US). THE GEN¬ KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, ERAL HOSPITAL CORPORATION [US/US]; 55 Fruit MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, Street, Boston, MA 021 14 (US). NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, (72) Inventors; and TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, (71) Applicants : SHALEK, Alexander, K. [US/US]; 2408 ZA, ZM, ZW. Massachusetts Ave., Lexington, MA 02421 (US). KOLB, [Continued on nextpage] (54) Title: METHODS FOR IDENTIFYING AND MODULATING IMMUNE PHENOTYPES (57) Abstract: The present invention provides tools and methods for the sy s J , tematic analysis of genetic interactions in immune cells. The present invention provides tools and methods for modulat ing immune cell phenotypes and com positions, combinatorial probing of cel lular circuits, for dissecting cellular cir cuitry, for delineating molecular path ways, and/or for identifying relevant tar gets for therapeutics development. o w o 2017/147196 Ai II II II I III IIII II I I II III Hill III II I II (84) Designated States (unless otherwise indicated, for every Published: Mnd of regional protection available): ARIPO (BW, GH, — with international search report (Art. 21(3)) GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, — before the expiration of the time limit for amending the TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, claims and to be republished in the event of receipt of DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, amendments (Rule 48.2(h)) 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). METHODS FOR IDENTIFYING AND MODULATING IMMUNE PHENOTYPES INCORPORATION BY REFERENCE [0001] This application claims benefit of and priority to U.S. provisional patent application 62/298,349 filed February 22, 20 6, incorporated herein by reference. [0002] All documents cited or referenced in the application cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. FEDERAL FUNDING LEGEND [0003] This invention was made with government support under Grant numbers AI078799, AI098484 and HL126554 awarded by the National institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION [0004] The present invention provides methods and tools for analyzing (epi)genetic and/or protein signatures of immune cells as well as (epi)genetic and/or protein signatures characteristic of immune cell phenotypes or immune cell behaviors or responses. The present invention relates to molecular profiling at the single ceil level as well as ceil population level. Applications include dissection of cell circuitry and delineation of functional or molecular pathways, as well as modulation of phenotypes, including modulation of immune cell population composition. The present invention is also relevant for therapeutics target discovery. BACKGROUND OF THE INVENTION [0005] Systems-level responses in the body represent the combined and coordinated behaviors of a highly diverse ensemble of cells. In the immune system, many specialized cells must work together to defend against myriad pathogenic threats, maintain long-term memory, and establish tolerance (Germain 2012). Moreover, the interplay between these cells must establish cheeks and balances to protect against autoimmunity or immunodeficiency (Littman and Rudensky 2010, Yosef, Shalek et al. 2013). Measuring these phenomena in bulk, however, blends and potentially masks the unique contributions of individual cells, particularly when their behaviors are heterogeneous or driven by rare cell types/states. [0006] To overcome this issue, to date, analyses of immune cells have primarily relied on first dividing the system into distinct subpopulations from the "top-down," typically based on the expression of cellular markers, and subsequently characterizing each bi independently. This strategy has cataloged the major cell types of the mammalian immune system, established more nuanced functional divisions (Shay and Kang 2013), and uncovered that balanced composition is essential for proper function. Illustratively, overproduction of a subset of T helper cells (pro- inflammatory Thl7) (Yosef, Shalek et al. 2013), or an imbalance in the relative proportions of DC subtypes(Nakahara, Uchi et al. 2010), can lead to autoimmune disease; similarly, in cancer, the density and diversity of tumor-infiltrating lymphocytes (TILs) has been shown to be predictive of tumor recurrence and clinical outcome (Galon, Costes et al. 2006). Yet, while informative, these "top-down" approaches depend on pre-selected markers, biasing experimental design. Recent work has shown that even seemingly identical cells can exhibit significant and functionally important heterogeneities (S. C . Bendall et al, Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum. Science 332, 677-678 (201 1; A . A . Cohen et al, Dynamic Proteomics of Individual Cancer Cells in Response to a Drug. Science 322, 151 1-1516 (2008), A . Shalek et al., Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells. Nature 498, 236- 240 (2013); A . K . Shalek et al., Single-cell RNA-seq reveals dynamic paracrine control of cellular variation. Nature 510, 363-369 (2014); N . Yosef et al, Dynamic regulatory network controlling TH17 cell differentiation. Nature 496, 461-468 (2013); O . Feinerman et al.. Single- cell quantification of IL-2 response by effector and regulatory T cells reveals critical plasticity in immune response. Molecular Systems Biology 6, 1-16 (2010)). Moreover, recent molecular studies have shown that even "identical" cells can substantially differ in gene expression, protein levels and phenotypic output (Cohen, Geva-Zatorsky et al. 2008, Raj and Van Oudenaarden 2009, Feinerman, Jentsch et al. 2010, Sharma, Lee et al. 2010, S. C . Bendall et al. 20 , Dalerba, Kalisky et al. 201 1), with important functional consequences (Cohen, Geva-Zatorsky et al. 2008, Feinerman, Jentsch et al. 2010, Sharma, Lee et al. 2010), highlighting the shortcomings of "top- down" schemes. [0007] A complementary approach is to examine a system from the "bottom-up," profiling its component cells individually. For example, in order to uncover salient variables in an immune response, one approach is to explore deviations that funda e ta y alter human immunity (J. L . Casanova, Human genetic basis of interindividual variability in the course of infection. Proceedings of the National Academy of Sciences of the United States of America 112, E71 18- 7127 (2015)). Until recently, single-cell-based approaches, such as fluorescence activated cell sorting (FACS) or immunofluorescence, had been technically limited to probing a few pre selected RNAs or proteins (Cohen, Geva-Zatorsky et al. 2008, Raj and Van Oudenaarden 2009, Sharma, Lee et al. 2010, Bendall, Simonds et al. 201 1, Dalerba, Kali sky et al. 20 ), hindering Applicants' ability to uncover novel factors. The recent emergence of single cell genomic approaches, and especially single-cell RNA-Seq (scRNA-seq), opens a new path for unbiased molecular profiling of individual immune cells from which Applicants can identify cell states and their associated signatures. [0008] For example, analysis of T-cell lines from persons resistant to HIV-1 infection linked genetic variation in CCR5 to reduced risk (R. Liu et al, Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86, 367- 377 (1996)). Similarly, studies of elite controllers (ECs) - a rare (-0.5%) subset of HIV-1 infected individuals whose immune systems naturally suppress viral replication without combination antiretroviral therapy (cART) (J.