Human spleen organoids to model human immune responses

Abstract

Methods and compositions are provided for modeling human adaptive immune responses in an in vitro organoid culture initiated from human spleen cells. The organoids comprise diverse functional immune cells, including B cells, T cells, and myeloid cells, e.g. dendritic cells. A suspension of spleen cells are grown in culture, for example within an air liquid interface transwell system. These diverse cells locally interact in a manner that allows for both recall and naïve immune responses. The cultures remain functional for extended periods of time, e.g. for up to 30 days, up to 45 days, up to 60 days, up to 90 days, or more.

Description

HUMAN SPLEEN ORGANOIDS TO MODEL HUMAN IMMUNE RESPONSESGOVERNMENT SUPPORT RESEARCH

[0001] This invention was made with Government support under contract AI057229 awarded by the National Institutes of Health. The Government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATION

[0002] Application claims benefit under 35 U.S.C. § 119(e) of provisional application 63 / 779,171 , filed March 27, 2025, which application is hereby incorporated by reference in its entirety.BACKGROUND

[0003] Much of what is known about adaptive immune responses is derived from mouse studies. In addition, new vaccines or drugs need to be tested in animal models before being moved into clinical trials, making this a long and expensive process, with no certainty of success. While mouse models have yielded a wealth of information, they are often poorly predictive of human immune responses. This indicates that there are significant differences between inbred mice and human beings. For example, a large survey of genes expressed in the immune cells of mice and humans found over 40 genes were found that were unique to humans, and were not in mice.

[0004] Further evidence of the discordance between humans and animal models is clear in the process of developing new vaccines, as multiple vaccines that have been optimized in animal models have failed to be efficacious in human trials, for example candidate vaccines in HIV, TB, and malaria. Therefore, there is an unmet need for human platforms to assess immune responses in vitro.

[0005] The present disclosure provides a scalable, fully human in vitro system that recapitulates all the components of human immune responses in vitro and permits detailed mechanistic analysis of underlying adaptive immunity and drug screening.SUMMARY

[0006] Methods and compositions are provided for modeling human adaptive immune responses in an in vitro organoid culture initiated from human spleen cells. The organoids comprise diverse functional immune cells, including B cells, T cells, and myeloid cells, e.g. dendritic cells. The diverse spleen cells locally interact with each other in a manner that allows for both recall and naive immune responses. The cultures remain functional for extended periods of time, e.g. for up to 30 days, up to 45 days, up to 60 days, up to 90 days, or more. The cultures are shown to be useful in assays that predict human immune responses.

[0007] In some embodiments, a human spleen organoid comprises one or more, two or more, three or more, four or more, five or more, immune cell types, and may, for example, comprise each of the immune cell types: naive B cells, follicular B cells, marginal zone B cells, atypical B cells, antibody secreting B cells (ASC), germinal center B cells (GBC), CD4+T cells, T follicular helper (Tfh) cells, regulatory T cells (Treg), CD8+T cells, mucosal-associated invariant T (MAIT) cells, type 3 innate lymphoid cells (ILC3), NK cells, eosinophils, monocyte / macrophages, dendritic cells and follicular dendritic cells.

[0008] A feature of the cultures is the ability to support complex immune responses by cells in the culture. In some embodiments, cells present in the organoids perform one or more of: hypermutation maturation, affinity maturation, plasmablast differentiation, class switching recombination, and antigen-specific antibody production, activation of naive B cells, activation of naive T cells, activation of memory B cells, activation of memory T cells, etc. In some embodiments, cells in the organoid are responsive to an antigen, e.g. a vaccine candidate, allergen, autoantigen, tumor-specific antigen, MHC protein, etc.

[0009] In an embodiment, spleen samples are obtained from a human donor. A plurality of organoids can be generated from a single spleen sample, allowing for multiple screenings. In some embodiments a panel of genetically distinct human spleen organoids is provided. A panel may comprise 5, 10, 15, 20, 25, 30 or more genetically distinct cells. In some embodiments a genetic difference comprises a targeted change in a gene of interest, e.g. a naturally occurring mutation, an introduced mutation, and the like. In some embodiments a genetic difference comprises a different ancestry, a different sex, a different region of origin, and the like. In some embodiments a cell population of interest, e.g. T cells, are targeted for a genetic change, e.g. by genome engineering. In some embodiments, cells are modified by, for example, CRISPR / cas engineering.

[0010] In some embodiments the organoid model is used to test candidate immunogen, adjuvant, immunomodulatory therapeutic, etc. for activity in a human immune system. Multiple agents can be tested to determine optimal efficacy, using a panel of organoid cultures, and can be tested against a plurality of genotypes. Various methods are useful in determining the effectiveness of an agent, for example using flow cytometry, live imaging, antibody titers, T cell activity, and the like. Parameters of interest include synthesis of antibodies, synthesis or cytokines and chemokines, T cell responsiveness, phenotypic changes in the immune cells, and the like. In some embodiments the immune response comprises activation of naive B cells. In some embodiments the immune response comprises activation of naive T cells.

[0011] In an embodiment, the efficacy of a biologic agent, e.g. a therapeutic antibody, cytokine, chemokine, hormone, etc., is tested by contacting a culture of the disclosure with an effective dose of the biologic agent, and the effect on the cells of the culture determined. The effect may be, for example, determining viability of a targeted cell population, change inphenotype or expression patterns in a targeted cell population, and the like. The biologic agent may be a known agent, for example where the response of an individual or population of interest is determined. Alternatively the biologic agent may be a candidate agent, for example where the response of atrial population of interest is determined.

[0012] In an embodiment, the immunogenicity of a biologic agent, e.g. a therapeutic antibody, cytokine, chemokine, hormone, etc., is tested by contacting a culture of the disclosure with an effective dose of the biologic agent, and the effect on the cells of the culture determined. The effect may be, for example, determining specificity, affinity and / or titer of antibodies, determining presence of T cells responsive to the biologic agent, and the like. In some embodiments the immune response comprises activation of naive B cells. In some embodiments the immune response comprises activation of naive T cells. The biologic agent may be a known agent, for example where the response of an individual or population of interest is determined. Alternatively the biologic agent may be a candidate agent, for example where the response of atrial population of interest is determined.

[0013] In an embodiment, the immunogenicity of an immunogen, e.g. a vaccine, tumor antigen, autoantigen, allergen, etc., is tested by contacting a culture of the disclosure with an effective dose of the immunogen, and the effect on the cells of the culture determined. A vaccine may be a protein vaccine, whole or fractionated pathogen, mRNA encoding an immunogen, carbohydrate immunogen, and the like. The effect may be, for example, determining the specificity, affinity and / or titer of antibodies for the immunogen, determining presence of T cells responsive to the immunogen, and the like. In some embodiments the immune response comprises activation of naive B cells. In some embodiments the immune response comprises activation of naive T cells. The immunogen may be a known agent, for example where the response of an individual or population of interest is determined. Alternatively the immunogen may be a candidate agent, for example where the response of a trial population of interest is determined.

[0014] In an embodiment, the effectiveness of an adjuvant, e.g. a vaccine adjuvant, is tested by contacting a culture of the disclosure with an effective dose of the adjuvant alone or in combination with an immunogen, and the effect on the cells of the culture determined. A adjuvant may be a protein, whole or fractionated pathogen, mRNA encoding an a protein of interest, carbohydrate, lipid, or other biomolecule. The effect may be, for example, determining the specificity, affinity and / or titer of antibodies, e.g. against an immunogen, determining presence of T cells subtypes, and the like. In some embodiments the immune response comprises activation of naive B cells. In some embodiments the immune response comprises activation of naive T cells. The adjuvant may be a known agent, for example where the response of an individual or population of interest is determined. Alternatively the adjuvantmay be a candidate agent, for example where the response of atrial population of interest is determined.

[0015] For example, it is shown herein that spleen organoids recapitulate key processes of germinal center responses to different vaccine modalities. Organoids not only develop memory responses but can also develop responses to antigens to which the spleen donor did not have prior exposure. Therefore, spleen organoids can be used to (1) test the effects of novel vaccine candidates and / or adjuvants and (2) investigate the underlying mechanisms that can help improve future vaccine development. By using spleen organoids derived from donors from different age groups, gender and ethnic distribution, the effects of these factors can be testing in a clinically relevant system.

[0016] In some embodiments an in-vitro spleen organoid culture is provided, comprising a well, the well comprising a cell-suspension of spleen-derived diverse immune cells, plus media; wherein said media provides nutrients and factors that are needed for proper cell differentiation and the spatial organization of germinal centers. In some embodiments, the media can comprise recombinant human B-cell activating factor (BAFF). In some embodiments the media is free of additional cytokines. In other embodiments the media may comprise IL-7 and IL-15 for stimulation of T cells. In some embodiments, the media can further comprise one or more adjuvants and an immunogen, e.g. a vaccine or vaccine candidate. In some embodiments, the one or more adjuvants can comprise a biologic agent of interest, for example antibodies, cytokines, or adjuvants in use or being developed, and the like. In some embodiments, the cells are modified by introduction of an antigen to said media. In some embodiments, the cells are manipulated with targeted genetic changes. In some embodiments the targeted genetic changes are cell-specific, e.g. T cells, B cells, etc. can be selectively modified. In some embodiments a population of spleen cells is depleted of granulocytes, e.g. neutrophils, prior to initiation of the culture. In other embodiments the population of spleen cells is not depleted of granulocytes. In some embodiment the culture comprises a candidate immunogen, adjuvant, immunomodulatory therapeutic, etc.

[0017] In some embodiments, a method is provided for generating a spleen organoid, the method comprising dissociating a spleen or fragment thereof into single cells, placing a suspension of the single cells in an air liquid interface transwell system, in a medium that allows long term maintenance and functionality of diverse immune cells and adaptive immune responses. In some embodiments the adaptive immune response comprises activation of naive B cells. In some embodiments the adaptive immune response comprises activation of naive T cells. In some embodiments a germinal center is formed. In some embodiments granulocytes are selectively removed from the spleen cells prior to initiation of the culture. In alternative embodiments granulocytes are not selectively removed. In some embodiments a subset of the spleen cells are genetically modified prior to initiation of the culture, e.g. bygenome editing. In some embodiments the methods further comprise contacting the spleen organoid with a candidate immunogen, adjuvant, immunomodulatory therapeutic, etc. In some embodiments the cultures remain functional for extended periods of time, e.g. for up to 30 days, up to 45 days, up to 60 days, up to 90 days, or more. Cultures may be maintained by replacing from about 25% to about 75%, e.g. around 50% of the medium at regular intervals, e.g. every 2 days, every 3 days, every 4 days, etc. In some embodiments, the suspension of spleen cells are grown within an air liquid interface transwell system. In other embodiments, the spleen cells are grown in a hydrogel matrix, suspension culture, and the like.

[0018] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0020] FIG. 1. Comparison of immunoglobulin synthesis at day 28 from lymph node and spleen organoids. The organoids were stimulated with live attenuated influenza vaccine (LAIV).

[0021] FIG. 2. Maintenance of spleen organoid cell type distribution over month-long cultures:granulocyte depletion protocol. Cultures were set up using the methods of Example 1. Comparison of cell type proportions in unstimulated day 0 splenocytes and day 28 spleen organoids identified by single cell RNA sequencing. Donors on day 0 and day 28 are unmatched. Each point is one donor with 5 donors per timepoint. Spleens were dissociated using the PBMC isolation kit to remove granulocytes.

[0022] FIGS. 3A-3B. Human spleen organoids present diverse cell composition and develop adaptive immune responses: non-depletion of granulocytes as disclosed in Example 2. (A) Single-cell RNAseq analysis of human spleen single-cells after tissue processing, showing diverse cell composition that replicates the cell composition expected from human spleen. Dissociation did not remove granulocytes using magnetic depletion. (B) Human spleen organoids treated with LAIV (live attenuated influenza vaccine) vs untreated organoids. Top: representative flow plots showing increase in antibody-secreting cell frequencies (PB) aftertreatment with LAIV. Bottom: increase in HA-specific B cells in spleen organoids treated with LAIV.

[0023] FIGS. 4A-4D show a time course (a) of stimulated spleen organoids from different donors (b), (c), (d), showing immunoglobulin synthesis after stimulation with live attenuated influenza vaccine (LAIV).

[0024] FIGS. 5A-5B. Naive responses: Spleen organoids demonstrate priming to novel immunogen in naive organoids. Human spleen organoids can develop naive responses to novel antigens. Here we modeled naive responses by treating human immune organoids with Yellow fever vaccine (YFV-17D). (A) Representative flow plots showing that spleen organoids generate YFV-specific CD8+ T cells at day 14 after treatment compared to untreated organoids. (B) YFV-specific antibodies in organoids supernatants at different time points after treatment in organoids treated with YFV-17D (orange bars) vs untreated organoids (grey bars). Increase in IgM specific antibodies at early time points (days 7 and 14) with subsequent increase in YFV-specific IgG antibodies at later time points (days 21 and 28).

[0025] FIGS. 6A-6B. Responses to different types of vaccine platforms: Human spleen organoids generate responses to mRNA vaccines. Human spleen organoids can generate responses to mRNA vaccines. Here, we treated human spleen organoids with Pfizer BioNTech mRNA COVID-19 vaccine (0.1 gg / mL) and analyzed the response at day 12 after treatment. (A) Representative flow plots of spleen organoids at day 12 after treatment with Pfizer mRNA vaccine vs untreated. Plots show B cell phenotype and highlight a significant increase in antibody-secreting cells (PB) and germinal center B cells (GC) in organoids treated with mRNA vaccine compared to untreated. (B) Spike-specific IgG measured in organoid supernatants. Results show a significant increase in spike-specific IgG in organoids treated with Pfizer mRNA vaccine vs untreated.

[0026] FIGS. 7A-7B. Testing effectiveness of commercial biologies in spleen organoids:Significant variation in Rituximab-mediated B cell depletion across donors. We tested the efficacy of rituximab in depletion of B cells in human spleen organoids across 8 donors. (A) B cell frequency of total live spleen organoid cells in unstimulated, live attenuated influenza vaccine (LAIV, 2x105FFU) stimulated, and rituximab (2 nM) stimulated spleen organoids. (B) B cell depletion across 8 donors. Depletion is determined by comparing B cell frequencies in rituximab-stimulated vs. unstimulated cultures: [(% B cell in unstim - % B cell in rituximab stimulated) / % B cell in unstim] * 100.

[0027] FIG. 8. Testing immunogenicity of commercial biologies: Human spleen organoids generate anti-drug antibodies to Pegloticase. Pegloticase is a recombinant enzyme therapeutic used for chronic refractory gout in patients failing standard therapy. It is also used for management of tumor lysis syndrome. We stimulated spleen organoids across 6 donors with 1 ig of pegloticase and measured anti-pegloticase antibodies 14 days after stimulation.

[0028] FIGS. 9A-9B. Testing the effectiveness of novel drugs: immunosuppressive effect of B-cell specific TGFbeta. Spleen organoids were used to test the immunosuppressive properties of a novel drug designed for selective ‘silencing’ of B cells (B-cell specific TGF beta). We treated spleen organoids with LAIV (live-attenuated influenza vaccine) with the drug of interest at different concentrations as indicated (TGM1-hCD19) or controls. B-cell specific TGF beta led to significant decrease in antibody secreting cells (A) and significant reduction of flu-specific IgG at days 4 and 7 after treatment (B). Results in Sun, Ogishi et al. Selective Immune Silencing by Targeted TGF- / 3 Agonists. bioRxiv 2026 Jan 20:2026.01.19.700410.

[0029] FIG. 10. Testing adjuvants to boost vaccine responses: human spleen organoids can be used to test the effects of different adjuvants on boosting responses to vaccines. The effects of different types of adjuvants in human spleen organoids were analyzed. Here we show the results of spleen organoids treated with RBD-nanoparticle vaccine (5 jxg) together with 3M-052 (0.1 [ig / mL; a TLR 7 / 8 agonist). Results show that 3M-052 boosts the antibody response to the RBD-nanoparticle vaccine.DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0031] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporatedherein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0033] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0034] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0035] The term "cell culture" or "culture" means the maintenance of cells in an artificial, in vitro environment. It is to be understood, however, that the term "cell culture" is a generic term and may be used to encompass the cultivation not only of individual cells, but also of tissues or organs.

[0036] The term “culture system” is used herein to refer to the culture conditions in which explants are grown that promote prolonged tissue expansion with proliferation, multilineage differentiation and recapitulation of cellular and tissue ultrastructure.

[0037] “Gel substrate”, as used herein has the conventional meaning of a semi-solid extracellular matrix. Gel, e.g. a hydrogel, described here in includes without limitations, collagen gel, matrigel, extracellular matrix proteins, fibronectin, collagen in various combinations with one or more of laminin, entactin (nidogen), fibronectin, and heparin sulfate; human placental extracellular matrix.

[0038] An “air-liquid interface” is the interface to which the explant cells may be exposed to in the cultures described herein. The primary tissue may be mixed with a gel solution, e.g. a collagen gel, which is then poured over a layer of gel formed in a container with a lower semi- permeable support, e.g. a membrane. This container is placed in an outer container that contains the medium such that the gel containing the tissue in not submerged in the medium. The primary tissue is exposed to air from the top and to liquid medium from the bottom, see for example US Patent no. 9,464,275 herein specifically incorporated by reference.

[0039] By "contained' is meant a glass, plastic, or metal vessel that can provide an aseptic environment for culturing cells.

[0040] The term “sample” with reference to a patient encompasses solid tissue samples such as a biopsy specimen or cells derived therefrom and the progeny thereof. The term alsoencompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as diseased cells. The definition also includes samples that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc. The term “biological sample” encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like.

[0041] The term ‘‘explant’ is used herein to mean a piece of tumor tissue, and the immune and stromal cells present in that tissue; and the cells thereof originating from the tumor tissue that is cultured in vitro, for example according to the methods of the invention. The tissue from which the explant is derived is obtained from an individual.

[0042] The term “organoid’ is used herein to mean a 3-dimensional growth of tumor tissue in culture that retains characteristics of the tumor in vivo, e.g. recapitulation of cellular and tissue ultrastructure, immune cell interactions, etc.

[0043] As used herein, the term “immune cell” includes cells that are of hematopoietic origin and that play a role in the immune response. Immune cells include lymphocytes, such as B cells and T cells; natural killer cells; dendritic cells; myeloid cells, such as monocytes, myeloid cells, eosinophils, mast cells, basophils, and granulocytes. Immune cells of interest may include naive B cells, follicular B cells, marginal zone B cells, atypical B cells, antibody secreting B cells (ASC), germinal center B cells (GBC), CD4+T cells, T follicular helper (Tfh) cells, regulatory T cells (Treg), CD8+T cells, mucosal-associated invariant T (MAIT) cells, type 3 innate lymphoid cells (ILC3), NK cells, eosinophils, monocyte / macrophages, dendritic cells and follicular dendritic cells.

[0044] Methods are provided for the culture of small amounts of clinical specimens. Samples of interest include human tissue, e.g. solid tumor microbiopsy samples such as needle or fine needle aspirate. Samples may be taken at a single timepoint, or may be taken at multiple timepoints. Samples may be as small as 107cells, 106cells, 105cells, or less.

[0045] The phrase “mammalian cells’’ means cells originating from mammalian tissue.Typically, in the methods of the invention pieces of tissue are obtained surgically, e.g. biopsy, needle biopsy, etc. and minced to a size less than about 1 mm3, and may be less than about 0.5 mm3, or less than about 0.1 mm3. “Mammalian” used herein includes human, equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc. “Mammalian tissue cells” and “primary cells” have been used interchangeably.

[0046] “Ultrastructure’’ refers to the three-dimensional structure of a cell or tissue observed in vivo. For example, the ultrastructure of a cell may be its polarity or its morphology in vivo, while the ultrastructure of a tissue would be the arrangement of different cell types relative to one another within a tissue.

[0047] The term “candidate cells” refers to any type of cell that can be placed in co-culture with the tissue explants described herein. Candidate cells include without limitations, genetically engineered T cells including without limitation CAR-T cells, dendritic cells, phagocytic cells T cells, B cells, etc.

[0048] The term “candidate agent” means any oligonucleotide, polynucleotide, siRNA, shRNA, gene, gene product, peptide, antibody, small molecule or pharmacological compound that is introduced to an explant culture and the cells thereof as described herein to assay for its effect on the explants.

[0049] The term "contacting" refers to the placing of candidate cells or candidate agents into the explant culture as described herein. Contacting also encompasses co-culture of candidate cells with tissue explants for at least 1 hour, or more than 2 hrs or more than 4 hrs in culture medium prior to placing the tissue explants in a semi-permeable substrate. Alternatively, contacting refers to injection of candidate cells into the explant, e.g. into the lumen of an explant.

[0050] “Screening” refers to the process of either co-culturing candidate cells with or adding candidate agents to the explant culture described herein and assessing the effect of the candidate cells or candidate agents on the explant, including without limitation immune cells present in the explant. The effect may be assessed by assessing any convenient parameter, e g. phenotypic changes, protein expression, mRNA expression, etc.

[0051] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and / or being treated. In some embodiments, the mammal is a human. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals having a disease. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mice, rats, etc.

[0052] The term “diagnosis” is used herein to refer to the identification of a molecular or pathological state, disease or condition in a subject, individual, or patient.

[0053] The term “prognosis” is used herein to refer to the prediction of the likelihood of death or disease progression, including recurrence, spread, and drug resistance, in a subject, individual, or patient. The term “prediction” is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning, the likelihood of a subject, individual, or patient experiencing a particular event or clinical outcome. In one example, a physician may attempt to predict the likelihood that a patient will survive.

[0054] As used herein, the terms “treatment,” “treating,” and the like, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect on or in a subject, individual, or patient. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of effecting apartial or complete cure for a disease and / or symptoms of the disease. “Treatment,” as used herein, may include treatment of infection in a mammal, particularly in a human, and includes one or more of: (a) preventing disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease or its symptoms, i.e., causing regression of the disease or its symptoms. Treating may also refer to any indicia of success in the treatment or amelioration or prevention of a disease, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0055] As used herein, a "therapeutically effective amount" refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., to delay or minimize infection and the sequelae of infection. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Further, a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.

[0056] As used herein, the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0057] "In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of one or more therapeutic agents; or to the screening of two or more agents in a culture. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.

[0058] "Concomitant administration" means administration of one or more components at such time that the combination will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of components. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration.

[0059] The use of the term "in combination" does not restrict the order in which prophylactic and / or therapeutic agents are administered to a subject with a disorder. A first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject with a disorder.

[0060] As used herein, a "therapeutically effective amount" refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., to delay or minimize the growth and spread of cancer. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Further, a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.Culture methods

[0061] In an embodiment, a method is provided for generating a spleen, e.g. a human organoid, the method comprising dissociating a spleen or fragment thereof into single cells, placing a suspension of the single cells in an air liquid interface transwell system, with the cells suspended in media on a porous membrane, in a medium that allows long term maintenance and functionality of diverse immune cells and adaptive immune responses.

[0062] The general method of such a culture encompasses the following steps:(a) a spleen sample is obtained;(b) the spleen tissue cut into small pieces;(c) the pieces are subjected to a dissociation step for the creation of a single-cell suspension; (d) the single-cell suspension is optionally cryopreserved;(e) aliquots of cell suspension are transferred to a culture plate;(f) the cells are cultured exposed to a medium that allows long term maintenance and functionality of diverse immune cells and adaptive immune responses, optionally in an airliquid interface. An optional step depletes granulocytes from the single cell suspension.

[0063] The spleen tissue may comprise spleen of any species. In a primary embodiment, the spleen tissue is human spleen tissue. The spleen tissue may be obtained by any means. In one embodiment, the spleen tissue is obtained from a deceased subject such as an organ donor.

[0064] The tissue may be cut by any means. Optionally, the tissue may be placed into decontamination media, for example for at least 30 minutes prior to cutting. In embodiments, scissors, scalpel, or other instrument is used to cut the spleen tissue. In some embodiments, the resulting pieces may be sized at about 2-10 grams, about 3-7 grams, or about 5 grams in mass.

[0065] Following the cutting step, the tissue is dissociated to create a single cell suspension.Any suitable method known in the art may be used to create the single cell suspension. Enzymes for the dissociation of tissue, for example, collagenases I and II, thermolysin, papainase, trypsin, dispase, etc. may be used.

[0066] Following dissociation, the cells may be filtered, centrifuged, and / or otherwise isolated.Lysis of red blood cells may be performed, for example using lysis buffers known in the art, for example, comprising NFLCI, sodium bicarbonate, and EDTA. For filtration, for example, a 120 micron filter may be used. The cells may be washed and spun one or more times and resuspended in a suspension buffer, for example, suspension medium comprising PBS, FBS, and EDTA. In one embodiment, granulocytes are removed, for example, by magnetic selection techniques.

[0067] The enriched cell suspension may be pelleted and resuspended in culture medium, for example RPMI 1640 medium.

[0068] Optionally, the cells are cryropreserved for storage. The cells may be frozen in any suitable cryopreservation medium, for example fetal bovine serum (FBS) with 10% DMSO. The cells may be frozen in aliquots, for example aliquots of 1-5 x 107cells per vial, for example, about 1.2 x 107cells per vial. The cells may be frozen, for example, at -80°C or in liquid nitrogen.

[0069] To initiate organoid cultures, cells are resuspended in medium at a concentration of around 5-10 x 10scells per well. This concentration is sufficient to provide cell diversity in theorganoid. The medium may comprise RPMI 1640 with HEPES + 10% FBS + 1x Penicillin / Streptomycin + 1x Non-Essential Amino Acids + 1x Sodium Pyruvate + 5ml Insulin / Transferrin / Selenium + 1ml Normocin, plus 1 y.g / ml BAFF.

[0070] The cells are placed in a transwell culture well. In some embodiments, the transwell culture well comprises: a first structure defining a first chamber for culture medium; a second structure defining a second chamber, the second chamber configured to hold cells therein; and a membrane located at an interface region between the first chamber and the second chamber to separate the first chamber from the second chamber. The membrane may comprise any suitable material, for example polycarbonate, polyester (PET), and collagen- coated polytetrafluoroethylene (PTFE). In some embodiments, the transwell wells are present in a 12 well plate, for example TRANSWELL™ plates by Corning.

[0071] In a primary embodiment, the organoids are cultured in an air-liquid culture system. In one embodiment medium is present in the first chamber of a transwell; cells are present in a second chamber of the transwell positioned above the first chamber; the first and second chamber are separated by a membrane; and the cells form a mass having an upper and lower surface. In air-liquid culture, the level of the culture medium liquid in the first chamber is adjusted such that the lower surface of the cells in the second chamber is in contact with liquid medium of the first chamber and the upper surface of the cell mass is not submerged, i.e. is in air.

[0072] The spleen organoids of the invention advantageously contain, and maintain for long periods of time, a diverse population of functional immune cells that recapitulate the cell populations in the spleen tissue from which each organoid was derived. In the organoid cultures of the invention, the human spleen organoid comprises one or more, two or more, three or more, four or more, five or more, distinct immune cell types, and may comprise each of naive B cells, follicular B cells, marginal zone B cells, atypical B cells, antibody secreting B cells (ASC), germinal center B cells (GBC), CD4+T cells, T follicular helper (Tfh) cells, regulatory T cells (Treg), CD8+T cells, mucosal-associated invariant T (MAIT) cells, type 3 innate lymphoid cells (ILC3), NK cells, eosinophils, monocyte / macrophages, dendritic cells and follicular dendritic cells.

[0073] Identification of these cell types can utilize known markers for phenotyping. For example and without limitation, cells may have one or more of the following features: naive B cells may be characterized as mature, unactivated lymphocytes that are CD19+, CD20+, CD22+, lgD+, lgM+, and CD27-. Follicular B cells may be characterized as expressing high levels of IgD and CD23 / Fc epsilon RII, and either high or low levels of IgM, and positive for CD22 / Siglec-2. Marginal zone B cells are identified by high expression of CD21 and surfaceIgM, low expression of CD23 and IgD, and positive for CD1 d, CD9, and CD27. Atypical B cells are distinguished from classical memory B cells by the upregulation of CD11c and T-bet and the downregulation of CD21 and CD27. Antibody secreting B cells are defined by the high expression of CD38 and CD138, alongside the loss of "pan-B cell" markers like CD19 and CD20. Germinal center B cells have high expression of the master transcription factor Bcl6, are CD19+, CD20+, CD95+. CD4+ T cells express CD4, and CD3, among other markers. T follicular helper cells express Bcl6, CXCR5, PD-1 ; IGOS; and SAP. Treg cells express CD4, CD25, and Foxp3. MAIT cells recognize MR1 tetramers loaded with the 5-OP-RU ligand, and also express CD161 , IL-18R, CD218a and CD26 (DPP4). ILC3 cells are primarily defined by the expression of the transcription factor RORyt, an ability to produce IL-17 and IL-22 and expression of CD127 and CD117. NK cells express CD56, CD16 and NKp46 and lack of CD3 expression. Eosinophils may express Siglec-8, CCR3 (CD193), CD125 (IL-5Ra), and CD11b. Monocytes may express CD11b (Mac-1); CD115 (CSF-1 R); HLA-DR; and CD64. Dendritic cells may express CD45, MHC-II (HLA-DR), and CD11c. Follicular dendritic cells express high levels of complement and Fc receptors and may express CD21 , CD35; CD23 (FCERII); CXCL13; and MFGE8.

[0074] In some embodiments, the organoids retain functional cells that perform one or more of: hypermutation maturation, affinity maturation, plasmablast differentiation, class switching recombination, and antigen-specific antibody production, activation of naive B cells, activation of naive T cells, activation of memory B cells, activation of memory T cells, etc. In some embodiments, the organoid is responsive to an antigen, e.g. a vaccine candidate, allergen, autoantigen, tumor-specific antigen, MHC protein, etc. In some embodiments the adaptive immune response comprises activation of naive B cells. In some embodiments the adaptive immune response comprises activation of naive T cells. In some embodiments a germinal center is formed.

[0075] The organoid culture can be initiated with a single cell suspension of spleen cells. The spleen cells may be obtained from a healthy adult, an adult with a known disease condition, etc., and are frequently obtained from a deceased adult. In some embodiments granulocytes are selectively removed from the spleen cells prior to initiation of the culture. Red blood cells may also be depleted prior to culture, e.g. by lysis, selection, etc.

[0076] An effective amount of cells in culture may refer to the number of splenocytes required to generate a diverse population of immune cells. A culture may be initiated with, for example, at least about 105cells in a single well, at least about 2 x 10scells, at least about 3 x 106cells, at least about 4 x 106cells, at least about 5 x 106cells, at least about 6 x 106cells, and up to about 107cells. The cells may be suspended in from about 0.5 to about 2 ml. of culture medium.

[0077] In some embodiments a subset of the spleen cells is genetically modified prior to initiation of the culture, e.g. by genome editing. In some embodiments the subset of cells are lymphocytes, e.g. CD4+ cells, CD8+ T cells, B cells, etc. The subset of cells may comprise one, two or more genetic modifications. The modifications may be introduced by genome engineering, e.g. CRISPR / cas methods. The modifications may be introduced by introduction of a gene of interest into the cells.

[0078] In some embodiments, the media can comprise one or more components. In some embodiments, the first component can comprise a media component comprised of AIM V, IMDM, MEM, DMEM, RPM1 1640, Alpha Medium or McCoy's Medium, or an equivalent known culture medium component. In some embodiments, the second can be a serum component which can comprise human serum, fetal bovine serum, or horse serum. In some embodiments, medium comprises an antibiotic to prevent microbial growth, basal media supplements which can comprise nonessential amino acids, sodium pyruvate, insulin / selenium / transferrin cocktail, growth factors, hormones, or cytokines. In some embodiments, the media is supplemented with fetal calf serum, he medium may comprise an effective amount of BAFF, e.g. from about 0.1 to about 10 |ig / ml BAFF, and may be around 1 p.g / ml BAFF. For stimulation of T cells, the medium may comprise an effective amount of one or both of IL-7 and IL-15, e.g. from about 0.1 to about 10 g / ml of each cytokine, and may be around 1 p.g / ml of each cytokine.

[0079] In some embodiments the cultures remain functional for extended periods of time, e.g.for up to 30 days, up to 45 days, up to 60 days, up to 90 days, or more. Cultures may be maintained by replacing from about 25% to about 75%, e.g. around 50% of the medium at regular intervals, e.g. every 2 days, every 3 days, every 4 days, etc. In some embodiments, the in-vitro cell culture can be held at a temperature at or around 37°C.

[0080] In some embodiments the methods further comprise contacting the spleen organoid with a candidate immunogen, adjuvant, immunomodulatory therapeutic, vaccine, biologic agent, etc. In some embodiments, an immunogen can be a protein, carbohydrate, glycoprotein or fragment thereof. In some embodiments, an immunogen can be a viral protein, a growth factor, a cancer related protein, or an auto-immune disease related protein.

[0081] In some embodiments, the in-vitro cell cluster can comprise a spatial organization of spleen tissue. In some embodiments, the spatial organization can comprise a germinal center. In some embodiments, the spatial organization can comprise an aggregate of T-cells. In some embodiments, the in-vitro cell cluster can be configured to perform one or more of: hypermutation maturation, affinity maturation, plasmablast differentiation, class switching recombination, and antigen-specific antibody production. In some embodiments, the cells can be configured to differentiate to form said in-vitro cell cluster upon exposure to an antigen. Insome embodiments, the germinal center can comprise antigen presenting cells (APCs) and T-cells at least partially surrounding said functional germinal center. In some embodiments, the APCs can comprise B-cells or dendritic cells. In some embodiments, the dendritic cells can comprise follicular dendritic cells. In some embodiments, the T-cells can comprise «p type T-cells. In some embodiments, the T-cells can comprise CD8+ T-cells. In some embodiments, the T-cells can comprise CD4+ T-cells. In some embodiments, the T-cells can comprise y5 T cells.

[0082] In some embodiments, an antigen or immunogen can be any substance that binds to an antibody. Antigens can be originated from the environment or formed inside the body. In some embodiments, the antigen can be a peptide, protein or fragment thereof, polysaccharides, lipids, nucleic acids, or other biomolecules. In some embodiments, the protein can be a viral protein, a bacterial protein, a growth factor, a cancer related protein, a cancer related peptide, an auto-immune disease related protein, an auto-immune disease related peptide, or fragment thereof.

[0083] In some embodiments, the antigen can be a protein or fragment thereof. In some embodiments, the protein can be a viral protein, a growth factor, a cancer related protein, a bacterial antigen, a fungal antigen, or an auto-immune disease related protein. In some embodiments, the viral protein can be derived from a virus. In some embodiments, the viral protein can be derived from, for example, a helical virus, a polyhedral virus, a spherical virus, or a complex virus. In some embodiments, the antigen can be a virus. In some embodiments, the virus can be, for example, a coronavirus or a flu virus.

[0084] Immune responses of the immunological organoid model as described herein can be altered by an immunomodulatory agent. The agent may be an adjuvant or derivative thereof. In some embodiments, the adjuvants can comprise, for example, aluminum salts, Freund’s adjuvant, Poly-IC, Poly-ICLC, MDP, MPL, CpG ODN, Virosome, MF59, AS01 , Flagellin, R837 / R848, AS04, AS02, AS03, mineral adjuvants such as aluminum hydroxide, phosphate adjuvants, calcium phosphate adjuvants, imiquimod, ISA51 , or any combination thereof. In other embodiments an adjuvant can be a cytokine. Other immunomodulatory agents include, for example, immunosuppressants such as rapamycin, corticosteroids like prednisone, calcineurin inhibitors like cyclosporine and tacrolimus, monoclonal antibodies like basiliximab, etc. Immunomodulatory agents may include immune checkpoint inhibitors such as nivolumab, pembrolizumab / MK-3475, pidilizumab and AMP-224 targeting PD-1 ; and BMS-935559, MEDI4736, MPDL3280A and MSB0010718C targeting PD-L1 and those targeting CTLA-4such as ipilimumab. Immunomodulatory agents may include agents that enhance a response, e.g. cytokines, chemokines, and the like.

[0085] In some embodiments, the spleen organoid is incubated with one or more of an immunogen, immunomodulatory agent, etc. In some embodiments, the spleen organoid can be incubated with the agent(s) for at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, or more. In some embodiments, the incubation can be for at least 48 hours. In some embodiments, the incubation with one or more antigens can modulate the percent of antigen specific B-cells by at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more as compared to before incubation.

[0086] Immunomodulatory agents of interest also include pathogen-associated molecular patterns (PAMPs). These microbial molecular markers may be composed of proteins, carbohydrates, lipids, nucleic acids and / or combinations thereof, and may be located internally or externally. Examples include the endotoxin lipopolysaccharide (LPS), single or doublestranded RNA, and the like.

[0001] Typically PAMP receptors (PRRs) are nonclonal, i.e. expressed on all cells of a given type, and germ-line encoded, or independent of immunologic memory. Once bound, PRRs tend to cluster, recruit other extracellular and intracellular proteins to the complex, and initiate signaling cascades that ultimately impact transcription. Further, PRRs are involved in activation of complement, coagulation, phagocytosis, inflammation, and apoptosis functions in response to pathogen detection. There are several types of PRRs including complement, glucan, mannose, scavenger, and toll-like receptors, each with specific PAMP ligands, expression patterns, signaling pathways, and anti-pathogen responses.

[0002] The Toll-like receptors are type I transmembrane (TM) PRRs that possess varying numbers of extracellular N-terminal leucine-rich repeat (LRR) motifs, followed by a cysteine- rich region, a TM domain, and an intracellular Toll / IL-1 R (TIR) motif. The LLR domain is important for ligand binding and associated signaling and is a common feature of PRRs. The TIR domain is important in protein-protein interactions and is typically associated with innate immunity. The TIR domain also unites a larger IL-1 R / TLR superfamily that is composed ofthree subgroups. The human TLR family is composed of at least 10 members, TLR1 through 10. Each TLR is specific in its expression patterns and PAMP sensitivities.

[0087] TLR agonists are currently under investigation as vaccine adjuvants in anticancer therapies for their ability to activate immune cells and promote inflammation. Examples of TLR agonists include pathogen-associated molecular patterns (PAMPs) and mimetics thereof. These microbial molecular markers may be composed of proteins, carbohydrates, lipids, nucleic acids and / or combinations thereof, and may be located internally or externally, as known in the art. Examples include, without limitation, lipopolysaccharide (LPS), zymosan, peptidoglycans, flagellin, synthetic TLR2 agonist Pam3cys, Pam3CSK4, MALP-2, triacylated lipoproteins, lipoteichoic acid, peptidoglycans, diacylated lipopeptides, and the like. The TLR2 ligand may include one or more of lipoteichoic acid (LTA), a synthetic tripalm itoylated lipopeptide (PAM3CSK4), zymosan, a lipoglycan such as lipoarabinomannan or lipomannan, a peptidoglycan, diacylated lipoprotein MALP-2, synthetic diacylated lipoprotein FSL-1 , heat shock protein HSP60, heat shock protein HSP70, heat shock protein HSP96 or high-mobility- group protein 1 (HMG-1).

[0088] The dose of TLR agonist that is effective in the methods of the invention is a dose that increases the efficiency of T cell response against antigens, relative to the same population in the absence of the TLR agonist. An effective dose may be up to about 10 pig / kg body weight, up to about 50 pg / kg, up to about 100 pg / kg, up to about 250 pg / kg, up to about 500 pg / kg, up to about 750 pg / kg, up to about 1 mg / kg, up to about 2.5 mg / kg, up to about 5 mg / kg, up to about 10 mg / kg or more.

[0089] In order to develop antigen specificity, in the germinal center, B cells can undergo a process called somatic hypermutation where point mutations are introduced into B cell receptor (BCR) gene sequence of the antibody variable regions of both the heavy and light chains at a very high rate compared to the background mutation rates observed in other genes. These mutated B cells are different from each other in specificity of antigen. To ensure high affinity of antibody production, mutated B cell are further selected based on the binding affinity of their receptors to antigen from follicular dendritic cells (FDCs), macrophages, or dendritic cells. Those B cells that have a negative effect on antigen binding undergo apoptosis while those with positive binding affinity are selected. This process is called affinity maturation. These two events result in a generation of B cells whose BCRs bind to specific antigen with high affinity. Selected B cells then differentiate into memory B cells or plasmablasts, which are also known as antibody secreting cells (ASC). Plasmablasts produce a large amount of antibodies during the first wave before undergoing apoptosis within a few days while memory B cells provide longer immunity. Memory B cells can also secrete different class of antibodiesor immunoglobins (Ig) via a process called class switching recombination. During this process, a DNA recombination process of the constant region of the antibody heavy chain is changed while the variable region of the heavy chains remains the same. As a result, the antibody retains affinity for the same antigen but can interact with different effector molecules.

[0090] In some embodiments, the germinal center can comprise antigen presenting cells (APCs). In some embodiments, the germinal center can be at least partially surrounded by T- cells. In some embodiments, the APCs can comprise B-cells, macrophages, or dendritic cells. In some embodiments, the dendritic cells can comprise follicular dendritic cells, pDC. The B- cells can comprise, for example, CD3- B-cells, CD45+ B-cells, CD19+ B-cells, CD38+ B-cells, CD38- B-cells, or CD27+ B-cells. In some embodiments, the B-cells can comprise transitional B-cells, naive B-cells, plasma B-cells, memory B-cells, pre-GC B cells, GO B cells, plasmablasts. In some embodiments, the T-cells can comprise, for example, helper T-cells, follicular helper T-cells, follicular regulatory T-cells, cytotoxic T-cells, memory T-cells, regulator T-cells, natural killer T-cells, mucosal associated invariant T-cells, gamma delta T-cells. In some embodiments, the T-cells can comprise CD8+ T-cells, CD4+ T-cells.Screening Methods

[0091] Methods and culture systems are provided for screening candidate agents or cells for an activity of interest. In these methods, candidate agents or cells are screened fortheir effect on cells in the spleen organoid of the disclosure.

[0092] The effect of an agent or cells is determined by adding the agent or cells to the cells of the organoid as described herein, usually in conjunction with a control culture of cells lacking the agent or cells. The effect of the candidate agent or cell is then assessed by monitoring one or more output parameters. Parameters are quantifiable components of explants or the cells thereof, particularly components that can be accurately measured, in some instances in a high throughput system. For example, a parameter of the explant may be the growth, differentiation, survival, gene expression, proteome, phenotype with respect to markers etc. of the explant or the cells thereof, e.g. any cell component or cell product including cell surface determinant, receptor, protein or conformational or posttranslational modification thereof, lipid, carbohydrate, organic or inorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portion derived from such a cell component or combinations thereof. While most parameters will provide a quantitative readout, in some instances a semi-quantitative or qualitative result will be acceptable. Readouts may include a single determined value, or may include mean, median value or the variance, etc. Characteristically a range of parameter readout values will be obtained for each parameter from a multiplicity of the same assays. Variability is expected and a range of values for each of the set of test parameters will be obtained using standard statistical methods with a common statistical method used to provide single values.

[0093] Candidate agent or cells can be added to the cells within the intact organoid. In other embodiments, the organoids are dissociated, and candidate agent or cells is added to the dissociated cells. The cells may be freshly isolated, cultured, genetically altered as described above; or the like. The cells may be environmentally induced variants of clonal cultures: e.g. split into independent cultures and grown into organoids under distinct conditions, for example with or without pathogen; in the presence or absence of other cytokines or combinations thereof. The manner in which cells respond to an agent, particularly a pharmacologic agent, including the timing of responses, is an important reflection of the physiologic state of the cell.

[0094] Candidate agents of interest for screening include known and unknown compounds that encompass numerous chemical classes, primarily organic molecules, for example proteins, antibodies, cytokines, etc. genetic sequences, etc. An important aspect of the invention is to evaluate candidate agents to predict patient responsiveness to an immunogen.

[0095] In some cases, the output response is synthesis of antibodies. The term “antibody” or “antibody moiety” is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The specific or selective fit of a given structure and its specific epitope is sometimes referred to as a “lock and key” fit. The archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, other mammal, chicken, other avians, etc., are considered to be “antibodies.” Antibodies utilized in the present invention may be either polyclonal antibodies or monoclonal antibodies. Antibodies are typically provided in the media in which the cells are cultured.

[0096] Candidate agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

[0097] Candidate agents are screened for biological activity by adding the agent to at least one and usually a plurality of explant or cell samples, usually in conjunction with explants not contacted with the agent. The change in parameters in response to the test agent is measured,and the result evaluated by comparison to reference cultures, e.g. in the presence and absence of the agent, obtained with other agents, etc.

[0098] The agents are conveniently added in solution, or readily soluble form, to the medium of cells in culture. The agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to an otherwise static solution. In a flow-through system, two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the cells, followed by the second. In a single solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow-through method. Alternatively, the agents can be injected into the explant, e.g. into the lumen of the explant, and their effect compared to injection of controls.

[0099] Preferred agent formulations do not include additional components, such as preservatives, that may have a significant effect on the overall formulation. Thus preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g. water, ethanol, DMSO, etc. However, if a compound is liquid without a solvent, the formulation may consist essentially of the compound itself.

[0100] A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effective concentration of an agent typically uses a range of concentrations resulting from 1 :10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the growth rate.

[0101] In certain aspects, the methods and systems disclosed herein may be useful for determining a treatment course for a subject. For example, such methods and systems may involve screening a patient for an immune response to a treatment. In some embodiments, the subject can be healthy. In some embodiments, the patient can be positive for a viral infection. In some embodiments, the subject can be positive for an auto-immune disease. In some embodiments, the subject can be positive for a fungal infection. In some embodiments, the subject can be positive for a bacterial infection. In some embodiments, the methods and systems may involve a screen that can be used prophylactically to identify a vaccination for a patient. In some embodiments, the methods and systems may involve a screen that can be used to identify an immune response within a particular population of individuals.

[0102] In some embodiments, the methods and systems disclosed herein may be useful for generating monoclonal antibodies for testing and treatment of a variety of diseases, e.g., cancer, autoimmune disease, and / or infectious processes, including viral infection, bacterial infection, microbial infection, or a combination thereof. In some embodiments, the cancer disease can be, for example, chronic lymphocytic leukemia, Hodgkin’s lymphoma, NonHodgkin’s lymphoma, bowel cancer, head cancer, neck cancer, breast cancer, stomach cancer, melanoma, glioblastoma, colorectal cancer, lung cancer, kidney cancer, or ovarian cancer. In some embodiments, the autoimmune disease can be rheumatoid arthritis, Crohn’s disease, celiac disease, pernicious anemia, autoimmune vasculitis, myasthenia gravis, Sjogren’s syndrome, Graves’ disease, Addison’s disease, inflammatory bowel disease, systemic lupus erythematosus, Type 1 diabetes, Lupus, Multiple sclerosis, or psoriasis.

[0103] In another aspect, the present disclosure provides an in vitro system supporting one or more of hypermutation maturation, affinity maturation, plasmablast differentiation, class switching recombination, and antigen-specific antibody production, activation of naive B cells, activation of naive T cells, activation of memory B cells, activation of memory T cells, etc. In some embodiments, the organoid is responsive to an antigen, e.g. a vaccine candidate, allergen, autoantigen, tumor-specific antigen, MHO protein, etc.

[0104] A kit may include, but is not limited to, one or more containers housing one or more of the components provided in this disclosure and instructions for use. Specifically, such kits may include, but is not limited to, one or more compositions described herein, along with instructions describing the intended application and the proper use and / or disposition of these compositions. Kits may comprise the components in appropriate concentrations or quantities for running various experiments.

[0105] In some embodiments, the methods and systems disclosed herein can utilize artificial intelligence / machine learning to generate optimal responses with high specificity and affinity. In some embodiments, the artificial intelligence / machine learning can be used to predict an antigen that might be specific to certain type of cancer, autoimmune disease or infection. In some embodiments, the predicted antigen can be used in antibody production from the methods and systems disclosed in this invention.EXPERIMENTALExample 1Dissociation of human spleen and preparation of single cell suspension for spleen organoid culture.

[0106] A human spleen sample is put into decontamination media for at least 30min at 4C, transferred to a sterile dish, and separated into approximately 5g pieces. Each piece is minced on a sterile plate in the medium. 1 ml of digestion medium (complete RPMI with 1 mg / ml collagenase, 200 U / ml DNAse) is added to the cells, then poured into a gentleMACS C-tube with total 10mL digestion media. The process is repeated for all spleen sections, then incubated while shaking at 37°C for 15-30 minutes. 100 ml of 500mM EDTA is added to 10ml solution in gentleMACs tube.

[0107] The suspension is then wet filtered, e.g. 120 micron filter, with suspension medium (PBS, 5% PBS, 5 mM EDTA) to provide a single cell suspension. The cell suspension is washed and spun at 300xg for 10 min. The supernatant is discarded and the pellet resuspended in ACK lysing buffer to lyse excess red blood cells. 30 ml of suspension media is added, and the cells spun down again. After removal of the supernatant, the pellet is resuspended in 40mL suspension media.

[0108] Granulocytes and RBS are moved with EasySep. Suspension medium is added to bring the volume to 20 ml. 50 jjJ / ml EasySep Isolation Cocktail is added to the sample, resuspend and incubate at room temperature for 5min. Suspension medium is added to the sample. 50 pl / mL of ORIGINAL volume) of RapidSpheres is added to the cells. The suspension is placed in an EasySep Magnet and incubated at room temperature for 10 minutes. The enriched cell suspension is transferred to a clean tube. The process is repeated 3 times. The final cell suspension is spun out at 400xg for 5 min. The supernatant is aspirated off, and the pellet resuspended in complete RPMI.

[0109] The cells are then frozen in fetal bovine serum (FBS) with 10% DMSO, typically in aliquots of 1 .2 x 107cells per vial. The cells are frozen at -80°C or in liquid nitrogen.

[0110] To initiate organoid cultures, cells are resuspended in medium at a concentration of around 5-10 x 106cells per well. This concentration is sufficient to provide cell diversity in the organoid. Medium is RPMI 1640 with HEPES + 10% FBS + 1x Penicillin / Streptomycin + 1x Non-Essential Amino Acids + 1x Sodium Pyruvate + 5ml Insulin / Transferrin / Selenium + 1 ml Normocin. 1 pg / ml BAFF is added to organoid media. The cells are placed in a transwell system, e.g. 1ml organoid media in each well of the prepared 12 well plate, outside the transwell, and 10Oul splenocyte suspension to inside of transwell.Example 2Dissociation of human spleen and preparation of single cell suspension for spleen organoid culture retaining granulocytes.

[0111] Spleen tissue is incubated in decontamination media for at least 30min at 4C, transferred to a sterile dish, and separated into approximately 5g pieces. Each piece is minced on a sterile plate in the medium. 1 ml of digestion medium (organoid media + benzonase) is added to the cells, then poured into a gentleMACS C-tube with total 10mL digestion media as in Example 1. The process is repeated for all spleen sections, then incubated while shaking at 37°C for 15-30 minutes. 100 ml of 500mM EDTA can be added to 10ml solution in gentleMACs tube. In the absence of gentleMACs the spleen is cut into very small pieces, and mashed through the 100 gm strainer.

[0112] The suspension is then wet-filtered through a 100 gm filter. The cell suspension is washed and spun out, and 5mL ACK lysing buffer added to the pellet and resuspended gently.30 ml of suspension media is added, and the cells spun down again. After removal of the supernatant, the pellet is resuspended in 30 mL suspension media.

[0113] The suspension is then overlaid on Ficoll and spun out at 1800rpm, 20 minutes. The interface is collected, and suspended in organoid media for counting. The cells are optionally aliquoted and frozen at that point, for example in fetal bovine serum (FBS) with 10% DMSO, typically in aliquots of 1 .2 x 107cells per vial, at -80°C or in liquid nitrogen.Media

[0114] Decontamination media is made with 500 ml hypothermosol (StemCell) or 500ml Ham’s F-12 (Gibco, Fisher Scientific # 31-765-092); 10 ml 100X pen / strep (ThermoFisher # 15240062); and 1 ml Normocin (FisherScientific # NC9273499).

[0115] Organoid media is made with 450 ml RPMI1640 with Glutamax (ThermoFisher # 61870127); 50 ml FBS (filtered); 5 ml 100X pen / strep (ThermoFisher # 15240062); 5ml non- essential aminoacids (ThermoFisher #11140050); 5 ml sodium pyruvate (ThermoFisher # 11360070); 1 ml normomicin (FisherScientific # NC9273499); and 5 ml insulin-transferrin- selenium supplement (ThermoFisher #41400045)

[0116] Spleen digestion media is made with organoid media + benzonase at 1 :1000 dilution.

[0117] Freezing media is made with FBS + 20% DMSO.Example 3Cell-type specific CRISPR-Cas9-mediated knock-out of multiple genes in human spleen organoids: T cells

[0118] Several protocols have been published for CRISPR-Cas9-mediated knock-out of genes in human T cells. Most protocols require stimulation with CD3 / 28 and supplementationwith high-dose IL-2 to permit adequate knock-out efficiency. This results in significant phenotypic changes and T cell differentiation incompatible with mechanistic studies in spleen organoids. As one example, IL-2 inhibits the T-follicular-helper cells and thus blunts CD4+ T cell dependent antibody responses to influenza vaccine in human spleen organoids. We required a protocol that enabled efficient knock-out without significant perturbation of existing T cell phenotypes. In our previous work on tonsil organoids, CRISPR-Cas9-mediated gene knock-outs were made possible by higher baseline T cell activation, permitting protein turnover within 1-2 days. However, T cells from human spleens are relatively quiescent and do not produce effective protein depletion using the protocols generated for T cells from tonsils. Thus, we have generated a minimal stimulation protocol as outlined below that permits efficient protein knock-down of multiple genes in T cells from human spleens.

[0119] In addition, the protocol below details how to perform cell-type specific knockouts such that only one cell type, in this case T cells, is genetically edited. This allows for mechanistic experiments testing the role of genes within one cell type on the entire organoid. This protocol is especially useful for hypotheses about cell-cell interactions, as the user can edit genes in one cell type and observe effects in another cell type.

[0120] At Day 0, T cells are isolated using a Miltenyi pan-T cell isolation kit and instructions from the manufacturer. The number of edited T cells is such that around 0.25 x 106cells are required for each condition. T cell medium is RPMI 1640 with HEPES + 10% FBS + 1x Penicillin / Streptomycin + 5ng / ml IL-7 + 5ng / ml IL-15. IL-7 and IL-15 are essential for low level stimulation of quiescent splenic T cells to induce protein turnover and efficient protein knockdown. Cells are resuspended at up to 5 x 106 / ml density and plated overnight.

[0121] The cells are divided for electroporation with the desired sgRNA. The final volume of Cas9-RNP is 17.5 J_LI for each knockout. HIFI-Cas9 is diluted into P3 buffer to 50 pM. Prepare Cas9 RNP by combining sgRNA and Cas9 to 40uM final concentration for each. If using multi- sgRNA kits (e.g. 3 sgRNA per gene), each gene’s 3sgRNA will total 40uM. Resuspend cells in 10Oul P3 buffer per cuvette, add 10Oul cells per cuvette into separate 1.5ml Eppendorf tubes, and add 17.5ul Cas9-RNP to appropriate 1.5ml Eppendorf tubes.

[0122] Cells are transferred with Cas9-RNP to electroporation cuvette. Electroporate cells using Lonza electroporator, then T cell media added. The cells are spun down and resuspended in T cell medium, then cultured overnight. Antibody staining and flow cytometry can be used to verify protein knock-down. If desired, perform genomic DNA extraction and PCR to verify DNA insertions / deletions at sgRNA binding sites.

[0123] To initiate organoid culture, thaw each splenocyte vial in 10ml of thawing media, wash 1x by pelleting cells via centrifugation, and resuspend in 10ml thawing media. Filter splenocytes through 10Oum cell strainer and rest for 1 hour at 37C in incubator. Deplete wildtype T cells from splenocyte suspension using miltenyi CD3 microbeads and instructions fromthe manufacturer. Reconstitute splenocyte suspension with edited T cells and wild-type non- T cells in 1.5ml. Ideal spleen organoid cultures contain ~4.5x 106non T cells and ~1.5x106T cells. Add relevant antigen to each tube and mix by gentle pipetting.

[0124] Prepare a 12 well plate for organoid culture by placing one 0.4um transwell in each well. Add 1ml organoid media (as in Example 1) in each well of the prepared 12 well plate, outside the transwell. Add 10Oul splenocyte suspension to inside of transwell. Be careful not to disturb the membrane. Culture at 37C.Example 4Screening Assays

[0125] As depicted in FIGS. 4-10, the spleen cultures generated by the methods of Example 1 or Example 2 are useful in screening assays. Media for all figures is RPMI 1640 with HEPES + 10% FBS + 1x Penicillin / Streptomycin + 1x Non-Essential Amino Acids + 1x Sodium Pyruvate + 5ml Insulin / Transferrin / Selenium + 1ml Normocin, plus 1 pg / ml BAFF. Organoids are fed every 3-4 days with the same media, but with BAFF concentration lowered to 0.5ug / ml BAFF. In all experiments unless otherwise noted, 1 microliter of LAIV is added per organoid. A standard human dose is 200 microliters which contains 1 o65-75fluorescence focus units (FFU) per strain. Using the average FFU of lO7, 1 microliter contains 50,000 FFU of each influenza virus strain for a total of 200,000 FFU across 4 strains

[0126] As shown in FIGS. 4A-4D and 5A-5B, the organoids are responsive to vaccines in a recall or naive immune response. In a recall response, FIG. 4A shows a time course from different donors showing immunoglobulin synthesis after stimulation with live attenuated influenza vaccine (LAIV).

[0127] To model a naive response (FIG. 5), organoids were stimulated with yellow fever vaccine (YFV-17D), to which the individuals were not expected to have been exposed. Representative flow plots showed that spleen organoids generate YFV-specific CD8+ T cells at day 14 after treatment compared to untreated organoids. The organoids produced IgM specific antibodies at early time points, and demonstrating class switching and affinity maturation then produced YFV-specific IgG antibodies at later time points (days 21 and 28).

[0128] The organoids are also responsive to mRNA vaccines, as shown in FIG. 6. The organoids were treated with an mRNA COVID-19 vaccine. The B cells were phenotyped 12 days after treatment, and show a significant increase in antibody-secreting cells (PB) and germinal center B cells (GC) compared to untreated controls. There is also an increase in spike-protein specific IgG in the organoid supernatants compared to a control.

[0129] Shown in FIGS. 7, 8 and 9, the spleen organoids are useful in determining the effectiveness and immunogenicity of biologic agents. Shown in FIG 7 are individual responses to depletion of B cells by rituximab, shown by measurement of the frequency of B cells in LAI V-stimulated, unstimulated, and Rituxan-treated organoids, for B cell frequency (A) and B cell depletion (B).

[0130] FIG. 8 demonstrates the power of the cultures in determining the immunogenicity of a biologic, where organoids were stimulated with a recombinant enzyme therapeutic, and the development of specific antibodies to the therapeutic measured after 14 days.

[0131] The effectiveness of novel drugs is shown in FIG. 9, which tested the immunosuppressive properties of a novel drug designed for selective ‘silencing’ of B cells (B- cell specific TGF beta). LAIV-stimulated cultures were treated with the drug, and a decrease in antibody secreting cells and specific antibody was measured.

[0132] In addition to the immunogenic components of vaccines, the cultures are useful for testing vaccine adjuvants, for example as shown in FIG. 10. A TLR 7 / 8 agonist was added to cultures in combination an RBD-nanoparticle vaccine. Results show that the adjuvant boosts the antibody response to the vaccine.

Claims

WHAT IS CLAIMED IS:1 . A method for producing a spleen organoid, the method comprising: dissociating a spleen or fragment thereof into single cells;placing the suspension of the single cells in a medium that allows long term maintenance and functionality of diverse immune cells and adaptive immune responses, wherein the diverse immune cells locally interact in a manner that allows for both recall and naive immune responses.

2. The method of claim 1, wherein the organoid model maintains functional splenocyte populations for up to 30 days, up to 45 days, up to 60 days, up to 90 days, or more.

3. The method of claim 1 or claim 2, wherein the adaptive immune response comprises activation of naive B cells.

4. The method of any of the preceding claims wherein the adaptive immune response comprises activation of naive T cells.

5. The method of any of the preceding claims wherein the adaptive immune response comprises activation of memory B or T cells.

6. The method of any of the preceding claims, wherein granulocytes are selectively removed from the spleen cells prior to initiation of the culture.

7. The method of any of the preceding claims wherein a subset of the spleen cells are genetically modified prior to initiation of the culture.

8. The method of claim 7, wherein the subset of cells comprises one or both of B cells and T cells.

9. The method of claim 7 or claim 8, wherein two or more genetic loci are modified.

10. The method of any of claims 7-9, wherein the genetic modification comprises genome editing.11 . The method of any of the preceding claims, wherein the medium comprises an effective amount of BAFF.

12. The method of any of the preceding claims, wherein the medium comprises an effective amount of one or both of IL-7 and IL-15.

13. The method of any of the preceding claims wherein the culture is maintained by replacing from about 25% to about 75%, e.g. around 50% of the medium at regular intervals.

14. The method of any of claims 1-6, further comprising the step of contacting the spleen organoid model with a candidate immunogen, biologic, vaccine, adjuvant, or immunomodulatory agent;and determining the effect of the agent on cells in the organoid model.

15. The method of claim 14, wherein the cells of the organoid are sorted or analyzed by flow cytometry or live imaging to determine the effect of the agent.

16. The method of claim 14 or 15, wherein effect of the agent is determined by antibody synthesis specific for an antigen of interest.

17. The method of claim 14 or 15, wherein effect of the agent is determined by T cell responsiveness specific for an antigen of interest.

18. The method of any of the previous claims, wherein the spleen organoid culture comprises one or more, two or more, three or more, four or more, five or more, and may comprise each of naive B cells, follicular B cells, marginal zone B cells, atypical B cells, antibody secreting B cells (ASC), germinal center B cells (GBC), CD4+T cells, T follicular helper (Tfh) cells, regulatory T cells (Treg), CD8+T cells, mucosal-associated invariant T (MAIT) cells, type 3 innate lymphoid cells (ILC3), NK cells, eosinophils, monocyte / macrophages, dendritic cells and follicular dendritic cells.

19. The method of any of claims 1-18, wherein the spleen cells are cultured in an air liquid interface transwell system.

20. The method of any of claims 1-18, wherein the spleen cells are cultured in a hydrogel matrix.

21. The method of any of claims 1-18, wherein the spleen cells are cultured in suspension media.-SO-22. An organoid model produced by the method of any of claims 1 -18.

23. A spleen organoid, comprising diverse immune cells that locally interact in a manner that allows for both recall and naive immune responses derived from a single spleen sample, wherein the diverse immune cells comprise five or more of naive B cells, follicular B cells, marginal zone B cells, atypical B cells, antibody secreting B cells (ASC), germinal center B cells (GBC), CD4+T cells, T follicular helper (Tfh) cells, regulatory T cells (Treg), CD8+T cells, mucosal-associated invariant T (MAIT) cells, type 3 innate lymphoid cells (ILC3), NK cells, eosinophils, monocyte / macrophages, dendritic cells and follicular dendritic cells.

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