Systems and methods for incorporating engineered T cells

Abstract

Disclosed herein are methods, systems, and devices for modeling adaptive immune responses and developing and / or testing improved antibodies, vaccines, and other therapeutics. The adaptive immune response can be modeled using lymphoid tissue derived from a subject. The methods, systems, and devices disclosed herein can include engineered T cells.

Description

[Technical Field]

[0001] Cross-reference to other applications This application claims the benefit of U.S. Provisional Application No. 63 / 298,104, filed January 10, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Federal Funding Statement This invention was made with government support under Grant No. U19-AI057229 awarded by the National Institute of Allergy and Infectious Diseases and the Howard Hughes Medical Institute. The government has certain rights in this invention. [Background technology]

[0003] Antigen recognition by lymphocytes has been studied by immunologists since the discovery of antibodies and their specificities over a century ago, followed by the more recent discovery of T cells and their antigen receptors in the 1960s–1980s. B cells, responsible for generating neutralizing antibody responses, develop alongside the germinal centers (GCs) and extrafollicular regions of lymphoid organs. In response to antigen presentation by antigen-presenting cells (APCs), T follicular helper (TFH) cells and various hematopoietic and non-hematopoietic cells interact to deliver signals to GC B cells for survival, proliferation, antibody affinity maturation, class switch recombination, and differentiation. Almost all of these interactions have been elucidated through in vivo studies in inbred mouse strains. While these studies have yielded a wealth of important information, many mechanistic aspects remain obscured due to the lack of a system that recapitulates essential features of adaptive immunity, such as affinity maturation and class switching, as well as the effects of adjuvants.

[0004] Much of what is known about adaptive immunity comes from inbred mouse studies, using methods that are often difficult or impossible to confirm in humans. Vaccine responses in mice often do not fully predict responses to those same vaccines in humans. The present invention uses cells from lymphoid organs to develop functional organotypic systems that recapitulate key features of germinal center and adaptive immunity in vitro, including, but not limited to, antigen-specific antibody production, somatic hypermutation, affinity maturation, plasmablast differentiation, and class switch recombination.

[0005] Many methods for antibody production include immunizing non-human animals with antigens, generating hybridoma cell lines, and collecting and purifying antibodies. A challenge with these methods is the production of human glycosylation patterns in antibodies produced from non-human subjects. Non-human glycosylation patterns may elicit an immune response in humans.

[0006] The methods and systems disclosed herein utilize cells from human lymphoid organs to produce human antibodies in response to antigens. The immune system's regulatory systems, including regulatory T cells, prevent the production of antibodies against antigens produced by the body, such as self-antigens. The invention disclosed herein provides an advantageous system for producing high-affinity antibodies against therapeutic targets, including self-antigens. Summary of the Invention

[0007] Disclosed herein are methods, systems, and devices capable of producing human antibodies against antigens, including self-antigens. Disclosed herein are methods, systems, and devices capable of modeling adaptive immune responses. Disclosed herein are in vitro cell clusters comprising lymphoid cells, the in vitro cell clusters being derived from lymphoid tissue of a subject, the in vitro cell clusters comprising germinal centers configured to produce antibodies against an antigen, the germinal centers comprising genetically modified T cells. The antigen may be a self-antigen. The antigen may be a polysaccharide, lipid, nucleic acid, peptide, protein, or fragment thereof. The protein may be a viral protein, growth factor, cancer-associated protein, or autoimmune disease-associated protein. The antigen may be expressed by tissue of the subject. The antigen may be a vaccine or vaccine candidate.

[0008] The germinal center may contain genetically modified T cells. The genetically modified T cells may be regulatory T cells. The genetically modified T cells may be modified to knock down or knock out expression of a forkhead box transcription factor. The forkhead box transcription factor may be FoxP3. The genetically modified T cells may be modified to knock down or knock out expression of granzyme B (GZMB). The genetically modified T cells may be CD8 + The germinal center may be configured to undergo one or more of hypermutation maturation, affinity maturation, plasmablast differentiation, and class switching recombination. The cells may be configured to differentiate to form the in vitro cell clusters upon exposure to the antigen. The genetically modified CD8+ T cells may be modified to knock down or knock out expression of granzyme B. The in vitro cell clusters may further comprise one or more adjuvants. The one or more adjuvants may include aluminum hydroxide or imiquimod. The germinal center may be configured to undergo one or more of hypermutation maturation, affinity maturation, plasmablast differentiation, and class switching recombination. The cells may be configured to differentiate to form the in vitro cell clusters upon exposure to the antigen.

[0009] The antigen can be a peptide, a protein, or a fragment thereof. The protein can be a viral protein, a growth factor, a cancer-associated protein, or an autoimmune disease-associated protein.

[0010] Lymphoid cells can be derived from tonsil tissue, spleen tissue, adenoid tissue, thymus tissue, or lymph node tissue.

[0011] Germinal centers may include antigen-presenting cells (APCs) and T cells that at least partially surround the functional germinal center. APCs may include B cells. APCs may be professional antigen-presenting cells. APCs may include dendritic cells. Dendritic cells may be follicular dendritic cells. B cells may include CD38+ B cells. B cells may include CD27+ B cells. T cells may include CD8+ T cells. T cells may include CD4+ T cells. An antibody may have an affinity for the antigen of 1 nanomolar to 10 femtomolar. An antibody may have an affinity for the antigen of up to 1 femtomolar or 10 femtomolar.

[0012] Disclosed herein is a method for producing antibody from lymphoid organoid, comprising: placing lymphoid cells in culture medium to produce the lymphoid organoid, the lymphoid organoid comprises T cell and B cell, and the T cell is modified to have reduced B cell regulation compared with unmodified T cell; introducing antigen into the culture medium; incubating the lymphoid organoid with the antigen to produce the antibody; and isolating the antibody from the lymphoid organoid.Method can further comprise isolating the nucleic acid encoding the antibody from the lymphoid organoid.

[0013] The lymphoid cells can be obtained from a subject, and the antigen is expressed by tissue of the subject. The subject can be human. The T cells can be modified to reduce or eliminate expression of a forkhead box transcription factor. The forkhead box transcription factor can be FoxP3. The T cells can be modified to reduce or eliminate expression of granzyme B (GZMB). The T cells can be modified by in vitro programmed genome editing, for example, a CRISPR-based system.

[0014] The method can further comprise introducing a B cell activator into the culture medium.The method can further comprise introducing an adjuvant into the culture medium.The adjuvant can be aluminum hydroxide or imiquimod.The method can further comprise incubating the lymphoid organoid with the antigen and the adjuvant.The adjuvant can be introduced after the antigen.The incubation can be at least 48 hours.

[0015] The method may further include obtaining the lymphoid cells from the subject. The lymphoid cells may be obtained by biopsy. The lymphoid cells may be derived from tonsil tissue, spleen tissue, adenoid tissue, thymus tissue, or lymph node tissue. The antigen may be a polysaccharide, lipid, nucleic acid, peptide, protein, or fragment thereof. The protein may be a growth factor, a cancer-associated protein, or an autoimmune disease-associated protein. The antigen may be a vaccine or vaccine candidate. The method may further include introducing an immune system stimulator. The immune system stimulator may be a live attenuated influenza virus (LAIV). The LAIV may be introduced together with the antigen. The antibody may have an affinity for the antigen of 1 nanomolar to 10 femtomolar.

[0016] Disclosed herein is an in vitro cell cluster comprising lymphoid cells, the in vitro cell cluster being derived from a lymphoid tissue of a subject, the in vitro cell cluster comprising germinal centers configured to produce antibodies against an autoantigen, the germinal centers comprising genetically modified T cells. Disclosed herein is a method for producing antibodies from lymphoid organoids, the method comprising: placing lymphoid cells in a culture medium to produce lymphoid organoids, the lymphoid organoids comprising T cells and B cells, the T cells being modified to have reduced B cell regulation compared to unmodified T cells; introducing an autoantigen into the culture medium; incubating the lymphoid organoids with the autoantigen to produce antibodies; and isolating the antibodies from the lymphoid organoids.

[0017] Disclosed herein are in vitro cell clusters comprising lymphoid cells, the in vitro cell clusters comprising germinal centers and T cell aggregates, configured to maintain the germinal centers and T cell aggregates and cellular respiration for at least 24 hours. The function of the clusters can be modulated by one or more adjuvants, including aluminum hydroxide or imiquimod, or any of several adjuvants currently in use or under development. The germinal centers can be configured to perform one or more of hypermutation maturation, affinity maturation, plasmablast differentiation, class switching recombination, and antigen-specific antibody production. The cells can be configured to differentiate to form the in vitro cell clusters upon exposure to an antigen. The antigen can be a peptide, protein or fragment thereof, polysaccharide, lipid, nucleic acid, or other biomolecule. The protein can be a viral protein, growth factor, cancer-associated protein, or autoimmune disease-associated protein. The antigen can be a vaccine or vaccine candidate. The carbohydrate can be a bacterial coat protein or fragment thereof. Many natural protein antigens, such as influenza hemagglutinin, are glycosylated and can stimulate B cells or T cells that express protein / peptide-specific antibodies, or these specific lymphocytes can be stimulated by carbohydrates or glycosylated proteins or peptides in any combination. Lymphoid cells can be derived from tonsil, spleen, adenoid, thymus, or lymph node tissue. Germinal centers can contain antigen-presenting cells (APCs) and T cells that at least partially surround the functional germinal center. APCs can include B cells or dendritic cells. Dendritic cells can include follicular dendritic cells. B cells can include CD38+ B cells. B cells can include CD27+ B cells. T cells can include CD8+ or CD4+ T cells of the αβ type, or they can include γδ T cells.

[0018] Disclosed herein is an in vitro lymphoid culture system comprising a well containing a cell suspension of lymphoid cells, including T cells, B cells, and non-lymphoid cells found in lymphoid organoids and involved in these responses (Wagar et al., 2021), and culture medium, which provides nutrients and factors necessary for proper cell differentiation and the spatial organization of germinal centers and adjacent T cell aggregates. The culture medium may contain recombinant human B cell activating factor (BAFF) and IL-2. The culture medium may also contain one or more adjuvants and a vaccine or vaccine candidate, including, but not limited to, inactivated pathogen vaccines; attenuated live pathogen vaccines; messenger RNA (mRNA) vaccines; subunit, recombinant, polysaccharide, and conjugate vaccines; toxoid vaccines; and viral vector vaccine candidates. The one or more adjuvants may include aluminum hydroxide or imiquimod, or many other adjuvants currently in use or under development. Germinal centers may perform one or more of hypermutation maturation, affinity maturation, plasmablast differentiation, class switching recombination, and antigen-specific antibody production. Cells may be stimulated by introducing antigen into the culture medium. The antigen may be a protein or fragment thereof, or a carbohydrate, or a glycoprotein or fragment thereof. The protein may be a viral protein, a growth factor, a cancer-associated protein, or an autoimmune disease-associated protein. Lymphoid cells may be derived from one or more of tonsil tissue, spleen tissue, adenoid tissue, thymus tissue, or lymph node tissue. Germinal centers may contain antigen-presenting cells (APCs) and T cells outside the functional germinal center. APCs may include B cells or dendritic cells. Dendritic cells may include follicular dendritic cells. B cells may include CD38+ B cells. B cells may include CD27+ B cells. T cells may include CD8+ T cells or CD4+ T cells of the αβ type, and T cells may include γδ T cells. T cells can be genetically modified to knock down or knock out expression of forkhead box transcription factors.T cells can be genetically modified to knock down or knock out expression of granzyme B.

[0019] The spatial organization of the lymphoid tissue may be maintained for at least 4 days. The spatial organization of the lymphoid tissue may be maintained for at least 1 week. The spatial organization of the lymphoid tissue may be maintained for at least 2 weeks. The spatial organization of the lymphoid tissue may be maintained for at least 3 weeks.

[0020] Disclosed herein is a method for producing antibodies, in some embodiments, high-affinity antibodies, from lymphoid organoids, the method comprising: (a) disposing lymphoid cells in a culture medium to produce lymphoid organoids, the lymphoid organoids comprising a spatial organization of lymphoid tissue, the spatial organization comprising germinal centers and T cell aggregates; (b) introducing an antigen into the culture medium; and (c) incubating the lymphoid organoids with the antigen to produce the antibody, and isolating the antibody from the lymphoid organoids. Step (a) may further comprise introducing a B cell activator into the culture medium. Step (b) may further comprise introducing an adjuvant into the culture medium. The adjuvant may be aluminum hydroxide or imiquimod. Step (c) may further comprise incubating the lymphoid organoids with the antigen and the adjuvant. The adjuvant may be introduced after the antigen. The incubation can be for at least 48 hours. The method can further include obtaining the lymphoid cells from the subject. The lymphoid cells can be obtained by biopsy. The lymphoid cells can be derived from tonsil tissue, spleen tissue, adenoid tissue, thymus tissue, or lymph node tissue. The antigen can be a peptide, a protein, a protein encoded by an mRNA vaccine, or a fragment thereof. The protein can be a viral protein, a bacterial protein, a growth factor, a cancer-associated protein, or an autoimmune disease-associated protein. The virus can be a coronavirus. The coding sequence for the antibody can be identified and introduced into an expression vector for production of the selected antibody by conventional methods. The antibody produced in this manner can be a high-affinity antibody.

[0021] Disclosed herein is a method of screening a patient for an immune response to a therapy, comprising obtaining a sample containing lymphoid cells from the patient, placing the lymphoid cells in a system described herein, exposing the lymphoid cells to a therapy to stimulate antibody production, isolating the antibodies from the system, and assaying the antibodies. The sample may be selected from one or more of tonsil tissue, spleen tissue, adenoid tissue, thymus tissue, or lymph node tissue. The sample may be obtained by biopsy.

[0022] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be understood, 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 present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0023] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, it is intended that the present specification supersede and / or prevail over such conflicting material. [Brief explanation of the drawings]

[0024] The invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to scale. Conversely, the dimensions of the various features have been arbitrarily increased or reduced for clarity. The drawings include the following figures:

[0025] [Figure 1A]Adaptive immune responses in tonsillar organoids. Representative stereoscopic images from replicate cultures unprimed or primed with live attenuated influenza vaccine (LAIV) on day 5 show the workflow for tissue disruption and culture preparation. Bright areas indicate areas of high cell density. [Figure 1B] Figure 1 shows adaptive immune responses in tonsillar organoids. Cellular composition of immune cell types in freshly isolated, frozen, and revived tonsillar cells at day 0, unstimulated cultures at day 7, and cultures stimulated with LAIV at day 7 is shown. Frequencies were determined by flow cytometry, and plotted values ​​are mean ± sem (n = 5 donors). PB / PC, plasmablast / plasma cell; NK, natural killer. [Figure 1C] Figure 1 shows adaptive immune responses in tonsillar organoids. Plasmablast differentiation and specific antibody secretion in unstimulated and LAIV-stimulated organoids at day 7 in culture. P values ​​were calculated using a paired two-tailed Wilcoxon signed-rank test (n=15 donors). OD is optical density. [Figure 1D] Adaptive immune responses in tonsillar organoids. Antigen-specific antibodies and total IgG from unstimulated and LAIV-stimulated organoid cultures at day 7 are shown. Colors represent individual donors (n=6). [Figure 1E] Adaptive immune responses in tonsillar organoids. ASCs specific for influenza antigens from unstimulated and LAIV-stimulated cultures at day 7 are shown (n=5 donors). [Figure 1F] Adaptive immune responses in tonsillar organoids. Plasmablast frequency and antibody secretion from immune organoids derived from human lung-draining lymph nodes and spleens at day 14 (n=2 for each tissue). A / Cal is A / California H1N1 virus. [Figure 2A]Longitudinal tracking of immune organoids to reveal cellular and functional changes consistent with an adaptive response is shown. Figure 2A shows representative flow cytometry staining of B cell differentiation phenotypes in unstimulated and LAIV-stimulated cultures. Cells shown were pre-gated on total B cells (CD45+CD19+CD3-). [Figure 2B] Longitudinal tracking of immune organoids to reveal cellular and functional changes consistent with an adaptive response. Quantification of B cell phenotype over time in unstimulated and LAIV-stimulated organoids. [Figure 2C] Longitudinal tracking of immune organoids to reveal cellular and functional changes consistent with an adaptive response. Quantification of T cell phenotype over time in unstimulated and LAIV-stimulated organoids. [Figure 2D] Longitudinal tracking of immune organoids to reveal cellular and functional changes consistent with an adaptive response. Influenza-specific antibodies detected in culture supernatants. [Figure 2E] Longitudinal tracking of immune organoids to reveal cellular and functional changes consistent with an adaptive response is shown. Virus-neutralizing capacity of antibodies in culture supernatants against two of the four vaccine strains (LAIV2015-2016) represented by antigen challenge is shown. Full cellular and fluid data from n=3 donors (a-e) and longitudinal antibody detection data from an additional n=4 donors (d) are shown. [Figure 2F] Longitudinal tracking of immune organoids to reveal cellular and functional changes consistent with an adaptive response is shown. Influenza M1-specific CD8+ T cell responses in unstimulated and LAIV-stimulated tonsillar organoids from four HLA-A2+ donors are shown. Donor ages (years) are indicated in parentheses after the donor ID. [Figure 3A] Figure 1 shows the diversity and maturation of influenza responses. Confocal microscopy images of organoid cultures stimulated with LAIV on day 4 and the B cell region with light zone (LZ) and dark zone (DZ) organization are shown. [Figure 3B]Figure 1 shows the diversity and maturation of influenza responses. Single-cell RNA-seq of B cells from tonsillar cells on day 0 and organoid cultures on days 5 and 9 from a single donor is shown. Fold-change values ​​(at least 1.5-fold increase) of genes and antibodies (Abs) highly expressed in GCs compared to naive B cells are plotted from B cells on day 0 (d0) and B cells stimulated with LAIV on day 5 (d5). [Figure 3C] Figure 1 shows the diversity and maturation of influenza responses. Median fluorescence intensity (MFI) (top) and relative proportions of B cell subsets (bottom) of AID expression at the protein level on days 0, 4, and 7 of organoid culture. Box plots show median values ​​with hinges representing the first and third quartiles and whiskers representing maximum and minimum values ​​within 1.5 times the interquartile range of the hinge (n=4 donors). FMO is fluorescence minus 1. [Figure 3D] Figure 1 illustrates the diversity and maturation of influenza responses. Single-cell BCR sequencing of high-affinity A / California 2009 H1N1 HA-specific plasmablasts and GC B cells from one representative donor is shown. High-affinity B cells specific for HA are shown from organoid cultures stimulated with LAIV on day 0 (black dots) and day 7 (green, blue, and red dots representing IgM, IgA, and IgG isotypes, respectively). Clonal families are represented by open shading. [Figure 3E] This demonstrates the diversity and maturation of influenza responses. Organoid cultures pre-depleted of high-affinity A / California HA+ B cells and non-naive B cells generate new HA+ B cells. Effective depletion of HA+ B cells and non-naive populations on day 0 was confirmed by post-sort analysis, and new HA+ B cells were detectable on day 10. Data shown are representative plots from one of n=4 donors. [Figure 3F] This figure shows the diversity and maturation of influenza responses. Influenza-specific antibodies were detected in depleted organoid cultures from three of the four donors tested (n=4). The donor ages (years) are shown in parentheses after the donor ID. [Figure 4A] Figure 1 shows supported somatic hypermutation and antigen-directed affinity maturation in tonsillar organoids. BCR repertoire analysis workflow for cultures prepared from tonsillar cells depleted of high-affinity HA+ and total non-naive B cells (n=6 donors). [Figure 4B] Figure 1 shows somatic hypermutation and antigen-directed affinity maturation supported in tonsillar organoids. Influenza vaccine-specific and A / California 2009 H1N1 HA-specific antibodies after 7 days in unstimulated and LAIV-stimulated cultures. [Figure 4C] Figure 1 shows somatic hypermutation and antigen-directed affinity maturation supported in tonsillar organoids. Somatic hypermutation measured by the number of nucleotide mutations from the germline heavy chain BCR sequence. Data are mean ± sem, and significance was calculated using a two-tailed Welch t-test. [Figure 4D] Figure 1 shows somatic hypermutation and antigen-directed affinity maturation supported in tonsillar organoids. Clone size (measured by the number of RNA molecules per lineage) and diversity (measured by the number of unique RNA molecules per lineage) in A / California 2009H1N1 HA+ B cell lines compared to non-A / California HA+ lineages were measured in n=6 donors. Specificity for A / California HA was estimated from a pool of known day 0 HA+ BCRs. Box plots show median values ​​with hinges representing the first and third quartiles and whiskers representing maximum and minimum values ​​within 1.5 times the interquartile range of the hinge. Each overlaid point represents an individual lineage. The significance values ​​shown were calculated using a two-tailed Welch t-test. Donor age (years) is shown in parentheses after the donor ID. [Figure 4E]This figure shows the development of HA specificity in a BCR lineage from a single donor, with somatic hypermutation and antigen-directed affinity maturation supported in tonsillar organoids. The size of the nodes represents the number of RNA molecules detected, the distance between nodes is proportional to the edit distance (in nucleotides) between them, and the color reflects the edit distance (in amino acids) to the nearest known A / California HA-specific sequence, with light blue representing an exact match. The root sequence, major nodes, and a subset of major nodes with IgG class switches are highlighted. [Figure 5A] Depletion studies reveal the cell types required for plasmablast differentiation, specific antibody secretion, and antibody affinity in organoid cultures. The effect of cell subset depletion on plasmablast differentiation is shown. The fold change in plasmablast frequency is shown for sorted cells reconstituted with the depleted cell types. Sorting depletion experiments were performed on cells from n = 6 donors (naive and non-naive B cells), n = 2 donors (total B cells and plasmacytoid dendritic cells or pDCs), or n = 3 donors (myeloid cells + pDCs, regulatory T (Treg) cells, memory B cells, and plasmablasts). [Figure 5B] Depletion studies reveal the cell types required for plasmablast differentiation, specific antibody secretion, and antibody affinity in organoid cultures. The effect of cell subset depletion on specific antibody secretion is shown. The fold change in plasmablast frequency is shown for sorted cells reconstituted with the depleted cell types. Sorting depletion experiments were performed on cells from n = 6 donors (naive and non-naive B cells), n = 2 donors (total B cells and plasmacytoid dendritic cells or pDCs), or n = 3 donors (myeloid cells + pDCs, regulatory T (Treg) cells, memory B cells, and plasmablasts). [Figure 5C] Depletion studies reveal the cell types required for plasmablast differentiation, specific antibody secretion, and antibody affinity in organoid cultures. The effect of CD4+ depletion on plasmablast differentiation after 7 days in organoid cultures is shown (n=15 donors). Antibodies to influenza were not detected in unstimulated controls. Significance was determined using a paired two-tailed Wilcoxon signed-rank test. [Figure 5D] Depletion studies reveal the cell types required for plasmablast differentiation, specific antibody secretion, and antibody affinity in organoid cultures. The effect of CD4+ depletion on antibody production after 7 days in organoid cultures is shown (n=15 donors). Antibodies to influenza were not detected in unstimulated controls. Significance was determined using a paired two-tailed Wilcoxon signed rank test. [Figure 5E] Depletion studies reveal cell types required for plasmablast differentiation, specific antibody secretion, and antibody affinity in organoid cultures. The dependence of CD4+ cells on influenza-specific antibody secretion was associated with age. Intact or CD4+-depleted LAIV-stimulated cultures were stratified into ages ≤5 years (red) or >5 years (cyan). Box plots show median values ​​with hinges representing the first and third quartiles and whiskers representing maximum and minimum values ​​within 1.5 times the interquartile range of the hinge. Significance was calculated using an unpaired, two-tailed Mann-Whitney U test. [Figure 5F] Depletion studies reveal cell types required for plasmablast differentiation, specific antibody secretion, and antibody affinity in organoid cultures. Biolayer interferometry binding data from intact or CD4+-depleted cultures are shown. Dissociation rates, measured in kd (1 / s), are shown for four donors, with detailed binding traces from one representative donor. Data were collected from antibodies binding to full-length A / California 2009 H1N1 HA or the head domain alone. Stem-specific antibodies were detected in two donors, but only under CD4-containing conditions. [Figure 5-1] Depletion studies reveal cell types required for plasmablast differentiation, specific antibody secretion, and antibody affinity in organoid cultures. [Figure 5G]Depletion studies reveal the cell types required for plasmablast differentiation, specific antibody secretion, and antibody affinity in organoid cultures. The minimal cellular requirements for supporting influenza B cell responses are shown. Individual cell types were sorted and then recombined as indicated to test for plasmablast differentiation and antibody secretion 7 days after stimulation with LAIV (n = 7 donors). [Figure 5H] Depletion studies reveal the cell types required for plasmablast differentiation, specific antibody secretion, and antibody affinity in organoid cultures. The contribution of naive versus memory CD4+ T cells to B cell differentiation and influenza-specific antibody secretion under minimal cell conditions is shown. Naive B cells, APCs, and CD45- cells were sorted and combined with equal numbers of either naive (CD45RA+CD27+) or memory (all non-naive) CD4+ T cells in the presence of LAIV for 10 days (n = 5 donors). [Figure 6A] Figure 1 shows the ability to respond to non-influenza antigens and immunomodulation using adjuvants. Figure 2 shows plasmablast differentiation in tonsillar organoid cultures in response to MMR vaccine stimulation. Organoid cultures from seven donors were harvested on day 11 for flow cytometry analysis and antibody secretion. Significance values ​​were calculated using a paired two-tailed Wilcoxon signed-rank test. Donor ages are shown in parentheses. [Figure 6B] The ability to respond to non-influenza antigens and immunomodulation using adjuvants are shown. Plasmablast differentiation in tonsillar organoid cultures in response to MMR vaccine stimulation corresponds to MMR-specific IgG production. Organoid cultures from seven donors were harvested on day 11 for flow cytometry analysis and antibody secretion. Significance values ​​were calculated using a paired two-tailed Wilcoxon signed-rank test. Donor ages are shown in parentheses. [Figure 6C] Ability to respond to non-influenza antigens and immunomodulation with adjuvants are shown. PE-specific B cells were detected at low levels in unstimulated cultures (0.05% of total B cells) and increased with PE stimulation, but not with an irrelevant antigen (n=3 donors, day 11 post-stimulation). [Figure 6D] The ability to respond to non-influenza antigens and immunomodulation using adjuvants are shown. The frequency of PE-specific B cells was enhanced by stimulation in the presence of alum (n=4 donors, 10 days post-stimulation). A representative flow cytometry plot from one donor is shown. Individual points represent each donor. Treatment groups were tested for significance using a paired two-tailed t-test. [Figure 6E] This figure shows the ability to respond to non-influenza antigens and immunomodulation using adjuvants. Tonsillar organoid response to rabies vaccine, as indicated by pre-GC, GC, and plasmablast B cell phenotypes, and antigen-specific IgM after 14 days of culture. NP is nucleoprotein. P values ​​between treatment groups were determined using a paired two-tailed t-test (n = 10 donors). [Figure 6F] Lung-draining lymph node and spleen organoids were stimulated with rabies vaccine for 14 days, demonstrating their ability to respond to non-influenza antigens and immunomodulation using adjuvants. [Figure 6G-1] The ability to respond to non-influenza antigens and immunomodulation using adjuvants are shown. Tonsil organoids were stimulated with a SARS-CoV-2 vaccine candidate (n = 12 donors). Ad5 vectors containing either the spike protein (S), spike and nucleocapsid protein (SN), or the S1 spike subunit with nucleocapsid (S1N) were used for stimulation, and responses were measured after 14 days of culture. Box plots show median values ​​with hinges representing the first and third quartiles and whiskers representing maximum and minimum values ​​within 1.5 times the interquartile range of the hinge. Culture supernatants were collected and tested for antigen-specific IgM, IgG, and IgA. One donor was shown to be positive for multiple proteins. An unrelated adenoviral vector carrying norovirus VP1 was used as a control. The indicated P values ​​were calculated using a paired, two-tailed Wilcoxon signed-rank test (no adjustment for multiple comparisons). [Figure 6G-2]The ability to respond to non-influenza antigens and immunomodulation using adjuvants are shown. Tonsil organoids were stimulated with a SARS-CoV-2 vaccine candidate (n = 12 donors). Ad5 vectors containing either the spike protein (S), spike and nucleocapsid protein (SN), or the S1 spike subunit with nucleocapsid (S1N) were used for stimulation, and responses were measured after 14 days of culture. Box plots show median values ​​with hinges representing the first and third quartiles and whiskers representing maximum and minimum values ​​within 1.5 times the interquartile range of the hinge. Culture supernatants were collected and tested for antigen-specific IgM, IgG, and IgA. One donor was shown to be positive for multiple proteins. An unrelated adenoviral vector carrying norovirus VP1 was used as a control. The indicated P values ​​were calculated using a paired, two-tailed Wilcoxon signed-rank test (no adjustment for multiple comparisons). [Figure 7A-1] Figure 1 shows the characteristics of B cells from tonsillar organoid cultures. Flow cytometry gating schemes for representative unstimulated and LAIV-stimulated cultures are shown. [Figure 7A-2] Figure 1 shows the characteristics of B cells from tonsillar organoid cultures. Flow cytometry gating schemes for representative unstimulated and LAIV-stimulated cultures are shown. [Figure 7B] Figure 1 shows the characteristics of B cells from tonsillar organoid cultures. Representative ELISpot wells for detection of Ab-secreting cells with influenza vaccine specificity are shown. The number of detected spots is indicated in the corner of each well. [Figure 7C] Figure 1 shows the characteristics of B cells from tonsillar organoid cultures. A comparison of organoid cultures grown in standard flat-bottom wells versus transwells is shown. Data shown are from cultures stimulated with LAIV on day 7 (n=6 donors). The p-values ​​shown were determined using a paired Wilcoxon signed-rank two-tailed test. Box plots show the median with hinges representing the first and third quartiles and whiskers representing the maximum and minimum values ​​within 1.5 times the interquartile range of the hinge. [Figure 7D] Figure 1 shows the characteristics of B cells from tonsillar organoid cultures. Figure 2 shows the correlation between specific Ab secretion and plasmablast frequency in influenza-stimulated and unstimulated cultures on day 7. Figure 3 shows the detection of influenza-specific IgG antibodies by semi-quantitative indirect ELISA. Background optical density was corrected using unused culture medium as a control. Each donor (n=15) was tested under four conditions: no stimulation, stimulation with IIV, stimulation with LAIV, or stimulation with H1N1 WT virus. The frequency of plasmablasts (CD19+CD38+++CD27+) was determined by flow cytometry. [Figure 8A] Confocal imaging showing the distribution of different cell types and their interactions in tonsillar tissue and organoid cultures is shown. The overlay does not include DAPI staining for clarity. One fresh tonsillar tissue is shown stained with a panel of markers to define I and B cell regions and GC structures. [Figure 8B] Confocal imaging showing the distribution of different cell types and their interactions in tonsillar tissue and organoid cultures is shown. The overlay does not include DAPI staining for clarity. Unstimulated organoids on day 4 stained for I and B cell markers are shown. Representative examples from organoid cultures derived from three different donors are shown. [Figure 8C] Confocal imaging showing the distribution of different cell types and their interactions in tonsillar tissue and organoid cultures is shown. The overlay does not include DAPI staining for clarity. A day 4 LAIV organoid stained for germinal center markers is shown. Representative examples from organoid cultures derived from three different donors are shown. [Figure 8D]Confocal imaging showing the distribution of different cell types and their interactions in tonsillar tissue and organoid cultures is shown. The overlay does not include DAPI staining for clarity. B and l Unstimulated organoids on day 4 stained for cell distribution are shown. For organoid cultures, representative examples from organoid cultures derived from three different donors are shown. [Figure 8E] Confocal imaging showing the distribution of different cell types and their interactions in tonsillar tissue and organoid cultures is shown. The overlay does not include DAPI staining for clarity. A day 4 LAIV organoid stained for a marker of follicular helper cells is shown. Representative examples from organoid cultures derived from three different donors are shown. [Figure 9A] Quantification of organoid organization and function. CXCR4 and CD83 expression levels (mean intensity, left panel) and percent positivity (percentage of CD20+ B cells, right panel) were quantified in day 4 tonsillar organoids from a single donor. Two regions of LAIV-stimulated organoids (upper and lower GC, as also shown in Figure 3A) and a representative GC organization region from a non-stimulated organoid were used for calculation. [Figure 9B] Quantification of organoid organization and function is shown. Representative intracellular AID flow cytometry staining profiles are shown. Data shown are from day 4 LAIV-stimulated organoid cultures. Total B cells were subsetted based on CD38 and CD27 expression and shown as individual profiles (red) compared to the "no AID primary antibody" FMO control (gray). [Figure 10A]Figure 10A shows the sequencing of B cell receptors from HA-specific B cells. Figure 10A shows a phylogenetic tree from two donors (top). Clones on day 0 are indicated by black dots. Different isotypes (IgM, IgA, IgG) are indicated by colors (green, blue, and red, respectively). Oligoclonal populations and clonal families from single-cell data are represented by larger dots (open circles). Clonal families were defined as BCR sequences that use the same V and J genes and have at least 70% amino acid identity in the CDR3 regions of the heavy and light chains. [Figure 10B] Figure 1 shows sequencing of B cell receptors from HA-specific B cells. Figure 2 shows FACS staining and B cell phenotype of HA-specific B cells compared to the total B cell pool. [Figure 11] Figure 1 shows that tracking individual heavy chain BCR lineages in tonsillar organoids before and after LAIV stimulation shows evidence of isotype switching. Immunoglobulin heavy chain gene rearrangements of each isotype were sequenced from total memory B cells, germinal center B cells, and plasmablasts sorted from cultures of four donors on days 0 and 7. Heavy chain BCR sequences from lineages containing only IgM members on day 0 and only isotype-switched members on day 7 were compared for their somatic hypermutation levels. For each lineage, the average SHM was calculated for IgM members on day 0 and switched members on day 7, and the difference between these means was plotted. Lineages with increased mutations are shown in gray, and lineages with decreased mutations are shown in black. [Figure 12] This study demonstrates that depletion of existing HA-specific and non-naive B cells does not prevent the generation of new high-affinity HA+ B cells after organoid culture. B cells bearing high-affinity BCRs for A / California 2009 HA+ and all non-naive B cells were depleted by FACS, and depletion was confirmed by post-sort analysis. After 10 days of organoid culture, cells were harvested, re-stained for A / California HA+ B cells, and run on a flow cytometer. n = 4 donors were tested. [Figure 13A]Figure 1 shows the effect of cell depletion on influenza-specific antibodies and their affinity. Figure 2 shows plasmablast rescue and Ab responses to LAIV stimulation by supplementing pDC-depleted cultures with type I IFN. [Figure 13B] Figure 13B shows the effect of cell depletion on influenza-specific antibodies and their affinity. Figure 13B shows the frequency of CD4+ T cells (relative to live cells) in intact and CD4-depleted cultures on day 7. CD4+ cells were depleted by positive selection with magnetic particles. [Figure 13C] (b) Effect of cell depletion on influenza-specific antibodies and their affinity. Biolayer interferometry data showing Ab affinity to A / California 2009 H1N1 HA full-length, head-specific, and stem domains are shown. Colors correspond to patient samples in (b). [Figure 14-1] SARS-CoV2-specific Abs detected in organoid cultures stimulated with Ad5-vectored vaccine candidates are shown. Culture supernatants 14 days post-stimulation were tested for the presence of SARS-CoV2-specific IgM, IgG, and IgA in n=12 donors. Abs were detected using protein microarrays. Individual donors are represented by symbols. The signal intensities shown were background-subtracted based on unstimulated control cultures from the same donors. Box plots show median values ​​with hinges representing the first and third quartiles and whiskers representing maximum and minimum values ​​within 1.5 times the interquartile range of the hinge. [Figure 14-2] SARS-CoV2-specific Abs detected in organoid cultures stimulated with Ad5-vectored vaccine candidates are shown. Culture supernatants 14 days post-stimulation were tested for the presence of SARS-CoV2-specific IgM, IgG, and IgA in n=12 donors. Abs were detected using protein microarrays. Individual donors are represented by symbols. The signal intensities shown were background-subtracted based on unstimulated control cultures from the same donors. Box plots show median values ​​with hinges representing the first and third quartiles and whiskers representing maximum and minimum values ​​within 1.5 times the interquartile range of the hinge. [Figure 14-3] SARS-CoV2-specific Abs detected in organoid cultures stimulated with Ad5-vectored vaccine candidates are shown. Culture supernatants 14 days post-stimulation were tested for the presence of SARS-CoV2-specific IgM, IgG, and IgA in n=12 donors. Abs were detected using protein microarrays. Individual donors are represented by symbols. The signal intensities shown were background-subtracted based on unstimulated control cultures from the same donors. Box plots show median values ​​with hinges representing the first and third quartiles and whiskers representing maximum and minimum values ​​within 1.5 times the interquartile range of the hinge. [Figure 15] This figure shows PR3-specific autoantibodies produced by tonsillar organoids. T cell populations were depleted before stimulation with LAIV or LAIV with an autoantigen cocktail (LAIV+A). The results show that tonsillar organoids with depletion of CD4+CD25+ T cells produced large amounts of PR3-specific autoantibodies upon stimulation with LAIV+A, as measured by ELISA. [Figure 16A] Figure 1 shows the FACS results of FOXP3-KO in T cells using the CRISPR / Cas9 gene editing kit. [Figure 16B] Figure 1 shows the FACS results of FOXP3-KO in T cells using the CRISPR / Cas9 gene editing kit. [Figure 16C] Figure 1 shows the FACS results of FOXP3-KO in T cells using the CRISPR / Cas9 gene editing kit. [Figure 16D] Figure 1 shows the FACS results of FOXP3-KO in T cells using the CRISPR / Cas9 gene editing kit. [Figure 16E] Figure 1 shows the FACS results of FOXP3-KO in T cells using the CRISPR / Cas9 gene editing kit. [Figure 16F] Figure 1 shows the FACS results of FOXP3-KO in T cells using the CRISPR / Cas9 gene editing kit. [Figure 17]Figure 1 shows the production of specific autoantibodies against each autoantigen in FOXP3-KO T cells stimulated with LAIV+A. Four different autoantibodies were generated against each autoantigen from the autoantigen cocktail (proteinase 3 (PR3), double-stranded DNA (dsDNA), histone, and SNRNP70) and measured using ELISA. Knockout of FOXP3 alone in T cells is sufficient to enable the production of large amounts of specific autoantibodies. [Figure 18] 1 illustrates an example computer system configured to implement the methods provided herein. [Figure 19A] 1 shows the frequency of CD4+ and CD8+ regulatory T cells in human tonsils and PBMCs. Representative FACS plots showing the expression of CD25, FOXP3, and CXCR5 on CD4+ T cells from tonsils and PBMCs are shown. [Figure 19B] The frequencies of CD4+ and CD8+ regulatory T cells in human tonsils and PBMCs are shown. The percentages of FOXP3+ cells, Tfr (CXCR5+FOXP3+), CD25+FOXP3+ cells, and KIR+CD8+ T cells in CD4+ T cells from donors (N=7) using PBMC and tonsil samples are shown. Mean ± SEM is shown. ****p<0.0001. *p<0.05 (unpaired t-test). [Figure 20A] Figure 1 shows the phenotype of inflammatory T and B cells in FOXP3 and GZMB KO tonsillar organoids. The frequency of fold change in activated CD4 and CD8 T cells (CD27+CD38+) over controls in FOXP3 KO and GZMB KO tonsillar organoids was compared after 10 days of culture. Representative data from four independent experiments and mean ± SEM are shown. *p<0.05 (unpaired t-test). [Figure 20B] Figure 1 shows the phenotype of inflammatory T and B cells in FOXP3 and GZMB KO tonsillar organoids. The frequency of activated T cells in control, FOXP3 KO, and GZMB KO tonsillar organoids was compared after 10 days of culture. Representative data from three independent experiments and mean ± SEM are shown. **p<0.01 (paired t-test). [Figure 20C] Figure 1 shows the phenotype of inflammatory T and B cells in FOXP3 and GZMB KO tonsillar organoids. Representative FACS staining of B cell differentiation phenotypes in control, FOXP3 KO, and GZMB KO tonsillar organoids after 10 days of culture. Cells were pre-gated on total B cells (CD19+CD3-). [Figure 20D] Figure 1 shows the phenotype of inflammatory T and B cells in FOXP3 and GzmB KO tonsillar organoids. The frequencies of naive (CD38-CD27-), GC (CD38+CD27+), PB (CD38++CD27++), memory (CD38-CD27+), and pre-GC (CD38+CD27-) B cells were compared in control, FOXP3 KO, and GzmB KO tonsillar organoids after 10 days of culture. Representative data from four independent experiments are shown, with mean ± SEM. *p<0.05, **p<0.01 (paired t-test). [Figure 21A] Figure 1 shows that FOXP3 KO and GzmB KO tonsillar organoids developed differential autoimmune phenotypes after stimulation with LAIV and autoantigens. The fold change in OD values, representing the amount of autoantibodies specific for proteinase 3 (PR3), double-stranded DNA (dsDNA), small nuclear ribonucleoprotein 70 kDa (snRNP70), and core histones from control, FOXP3 KO, and GzmB KO tonsillar organoids, is shown. Data from three independent experiments are shown as mean ± SEM. *p<0.05, **p<0.01 (paired t-test). [Figure 21B] Figure 1 shows that FOXP3 KO and GZMB KO tonsillar organoids developed differential autoimmune phenotypes after stimulation with LAIV and autoantigen. Percentages of PB cells (CD27++CD38++ B cells), activated CD4+ and CD8+ T cells (CD38+CD27+) from control, FOXP3 KO, and GZMB KO tonsillar organoids after 10 days of culture are shown, derived from unstimulated (NS), LAIV-stimulated, and LAIV + autoantigen (LAIV+A)-stimulated control, FOXP3 KO, and GZMB KO tonsillar organoids. Data from three independent experiments are shown, with mean ± SEM. *p<0.05 (unpaired t-test). [Figure 22]When live attenuated influenza vaccine (LAIV) was used as an adjuvant in FoxP3 KO and granzyme B (GzmB) KO, the resulting bulk anti-influenza HA affinity was measured using interferometry. In most donors (n=9), FoxP3 KO significantly increased affinity, with up to an 18-fold decrease in off-rate (kd), while GzmB KO had a more modest effect in some donors. DETAILED DESCRIPTION OF THE INVENTION

[0026] Disclosed herein are methods, systems, and devices that can mimic immune responses and functions, such as the production of high-affinity antibodies. The methods, systems, and devices may include immune cell structures that can model adaptive immune responses, including, for example, antigen-specific hypermutation, affinity maturation, and class switching of B cells. The immune cell structures may include in vitro cell clusters containing lymphoid cells. The lymphoid cells may be derived from human tonsil tissue. The lymphoid cells may be derived from human spleen tissue. The in vitro cell clusters may be contacted with cell culture medium in a transwell system, with cells suspended in medium on a porous membrane. The in vitro cell culture may be maintained at a temperature at or around 98°F. The in vitro cell clusters may be exposed to one or more adjuvants. The in vitro cell clusters may be exposed to one or more antigens. The antigen may be a protein, carbohydrate, glycoprotein, nucleic acid, mRNA encoding a protein, or a fragment thereof. The antigen may be a viral protein, a growth factor, a cancer-associated protein, or an autoimmune disease-associated protein.

[0027] The in vitro cell clusters can comprise the spatial organization of lymphoid tissue. The spatial organization can include germinal centers. The spatial organization can include aggregates of T cells and distribution of rare but essential non-lymphoid cells. The in vitro cell clusters can be configured to maintain spatial organization and cellular respiration for at least 24 hours.

[0028] The in vitro cell clusters can be configured to undergo one or more of hypermutation maturation, affinity maturation, plasmablast differentiation, class switching recombination, and antigen-specific antibody production. Cells can be configured to differentiate to form the in vitro cell clusters upon exposure to antigen. The germinal center can include antigen-presenting cells (APCs) and T cells at least partially surrounding the functional germinal center. The APCs can include B cells or dendritic cells. The dendritic cells can include follicular dendritic cells. The B cells can include CD38+ B cells. The B cells can include CD27+ B cells. The T cells can include CD8+ T cells. The T cells can include CD4+ T cells.

[0029] Before the present methods and compositions are described, it is to be understood that this invention is not limited to the particular methods or compositions 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, as the scope of the present invention will be limited only by the appended claims.

[0030] Where a range of values ​​is provided, it is understood that each intervening value, down to the tenth of the unit of the lower limit, between the upper and lower limit of that range is also specifically disclosed, unless the context clearly dictates otherwise. Each smaller range between any stated value or intervening value in a stated range and any other stated value 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, and each range in which either, neither, or both limits are included in the smaller range is also encompassed within the invention, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0031] Unless otherwise defined, 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 possible methods and materials are described below.

[0032] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, and a reference to "the peptide" includes a reference to one or more peptides and equivalents thereof known to those skilled in the art, such as polypeptides.

[0033] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should 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.

[0034] definition The term "subject," as used herein, generally refers to any animal or living organism. The animal may be a mammal, e.g., a human, a non-human primate, a rodent such as a mouse or rat, a dog, a cat, a pig, a sheep, a rabbit, etc. The animal may be a fish, a reptile, or other. The animal may be a newborn, infant, adolescent, or adult animal. The human may be an infant, a toddler, a child, a young adult, an adult, or an elderly person. The human may be about 1, 2, 5, 10, 20, 30, 40, 50, 60, 65, 70, 75, or over about 80 years of age.

[0035] Cell culture conditions The in vitro cell clusters can be contacted with a culture medium. The culture medium can include one or more components. The first component can be a medium component consisting of AIM V, IMDM, MEM, DMEM, RPMI 1640, Alpha Medium, or McCoy's Medium, or equivalent known culture medium components. The second component can be a serum component that can include human serum, fetal bovine serum, horse serum, or a serum-free replacement. The third component can be an activator that can include B-cell activating factor (BAFF). The fourth component can be an antibiotic to prevent microbial growth. The fifth component can be a basal medium supplement that can include non-essential amino acids, sodium pyruvate, an insulin / selenium / transferrin cocktail, growth factors, hormones, or cytokines. The culture medium can be supplemented with fetal bovine serum. The culture medium can be replaced every 1, 2, 3, or 4 days. The in vitro cell clusters can be contacted with cell culture medium in a transwell system, with cells suspended in the medium on a porous membrane.

[0036] antigen The cells can be configured to differentiate to form such in vitro cell clusters upon exposure to an antigen. The antigen can be any substance that binds to an antibody. The antigen can be derived from the environment or formed endogenously. The antigen can be a peptide, protein or fragment thereof, polysaccharide, lipid, nucleic acid, or other biomolecule. The protein can be a viral protein, bacterial protein, growth factor, cancer-associated protein, cancer-associated peptide, autoimmune disease-associated protein, autoimmune disease-associated peptide, or fragment thereof. The antigen can be an autoantigen.

[0037] The antigen may be a protein or a fragment thereof. The protein may be a viral protein, a growth factor, a cancer-associated protein, a bacterial antigen, a fungal antigen, or an autoimmune disease-associated protein. The viral protein may be derived from a virus. The viral protein may be derived from, for example, a helical virus, a polyhedral virus, a spherical virus, or a complex virus. The antigen may be a virus. The virus may be, for example, a coronavirus or influenza virus, adenovirus, megavirus, Epstein-Barr virus, adenovirus, coxsackievirus, megavirus, Nipah virus, Marburg virus, hepatitis C virus, influenza A virus, varicella-zoster virus, canine parvovirus, hepatitis B virus, rabies virus, monkeypox virus, human coronavirus HKU1, dengue virus, human immunodeficiency virus 1, severe acute respiratory syndrome-associated coronavirus, Middle East respiratory syndrome-associated coronavirus, feline immunodeficiency virus, feline leukemia virus, human metapneumovirus, parvovirus B19, human polyomavirus 2, Neisseria gonorrhoeae, or neisseria gonorrhoeae. The virus may be cocalicivirus, eastern equine encephalitis virus, BK virus, Hendra virus, Norwalk virus, Ross River virus, Variola virus, vaccinia virus, herpes simplex virus 1, Kaposi's sarcoma-associated herpesvirus, mumps virus, measles morbillivirus, SV40, human coronavirus 229E, koi herpesvirus 3, B virus, beta arterivirus suid1, Indiana vesiculovirus, hepatitis A virus, human herpesvirus 5, bacteriophage MS2, enterobacteriaceae phage T2, human coronavirus NL63, murine respirovirus, human herpesvirus 2, simian immunodeficiency virus, or equine alphaherpesvirus 1.

[0038] The antigen may be a vaccine or vaccine candidate. Vaccines known and used in the art include, but are not limited to, inactivated pathogen vaccines; attenuated live pathogen vaccines, messenger RNA (mRNA) vaccines; subunit, recombinant, polysaccharide, and conjugate vaccines; toxoid vaccines, and viral vector vaccines. Inactivated vaccines use killed versions of pathogens that cause diseases such as hepatitis A, influenza, rabies, etc. Live vaccines use attenuated forms of pathogens that cause diseases such as measles, mumps, rubella (MMR combination vaccine), rotavirus, smallpox, chickenpox, and yellow fever. mRNA vaccines encode pathogen proteins, such as SARS-CoV2, that elicit an immune response. Subunit, recombinant, polysaccharide, and conjugate vaccines use specific pathogen molecules, such as Hib (Haemophilus influenzae type b), hepatitis B, HPV (human papillomavirus), Bordetella pertussis, pneumococcal disease, meningococcal disease, and varicella-zoster virus. Toxoid vaccines use toxins produced by pathogens, such as diphtheria and tetanus. Viral vector vaccines use modified versions of different viruses as vectors to deliver sequences encoding pathogen proteins. Several different viruses have been used as vectors, including influenza, vesicular stomatitis virus (VSV), measles virus, and adenovirus. Viral vectors are currently being used for SARS-CoV2.

[0039] Adjuvants The immune response of immunological organoid model as described herein can be enhanced by using adjuvant or its derivative.Adjuvant can comprise, for example, aluminum salt, Freund's adjuvant, polyIC, polyICLC, MDP, MPL, CpG ODN, virosome, MF59, AS01, flagellin, R837 / R848, AS04, AS02, AS03, mineral adjuvant, such as aluminum hydroxide, phosphate adjuvant, calcium phosphate adjuvant, imiquimod, ISA51, or any combination thereof.

[0040] Lymphoid organoid can be incubated with antigen and adjuvant.Lymphoid organoid can be incubated with antigen before introducing adjuvant.Lymphoid organoid can be incubated with antigen 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, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or more hours with antigen and / or adjuvant. Incubating may be for at least 48 hours. Incubation with one or more antigens may increase the percentage 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 compared to before incubation.

[0041] Incubation with one or more antigens may increase the number of nucleotide variations from the germline heavy chain BCR sequence 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 compared to before incubation.

[0042] Incubation with one or more antigens may increase the percent of antibody-secreting B cells in the total population of 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 compared to before incubation.

[0043] cell type The immunological organ model may include in vitro cell clusters. The cell clusters may include lymphoid cells, such as B cells and T cells. The lymphoid cells may be derived from lymphoid organs. The lymphoid cells may be derived from tonsil tissue, spleen tissue, adenoid tissue, thymus tissue, or lymph node tissue. The lymphoid organs may include, for example, primary and secondary lymphoid organs. The lymphoid organs may include bone marrow, thymus, lymph nodes, spleen, tonsils, tissues of the mucosal layers of the body (e.g., the intestine, respiratory and urinary tract, and vaginal lining), small intestine (Peyer's patches), and appendix. The in vitro cell clusters may include germinal centers. The in vitro cell clusters may include T cell aggregates.

[0044] The method may further include obtaining the lymphoid cells from the subject. The lymphoid cells may be obtained by biopsy, swab, or aspiration. The lymphoid cells may be derived from tonsil tissue, spleen tissue, adenoid tissue, thymus tissue, or lymph node tissue. The lymphoid cells may be derived from a lymphoid organ. The lymphoid organ may include, for example, primary and secondary lymphoid organs. The lymphoid organ may include bone marrow, thymus, lymph nodes, spleen, tonsils, tissue of the mucosal lining of the body (e.g., intestine).

[0045] Lymphoid cells, such as T cells, can be modified to reduce or eliminate the expression of forkhead box transcription factors. The forkhead box transcription factor can be FoxP3. Lymphoid cells, such as T cells, can be modified to reduce or eliminate the expression of granzyme B (GZMB). Lymphoid cells can be modified by in vitro programmed genome editing, for example, a CRISPR-based system.

[0046] Cells may be separated from a mixture of cells by techniques that enrich for the desired cells, or may be manipulated and cultured without separation. A suitable solution may be used for dispersion or suspension. Such solutions are generally balanced salt solutions, such as normal saline, PBS, Hank's balanced salt solution, and the like, conveniently supplemented with fetal bovine serum or other naturally occurring factors, along with a low concentration, generally 5-25 mM, of an acceptable buffer. Convenient buffers include HEPES, phosphate buffer, lactate buffer, and the like.

[0047] Techniques for affinity separation may include magnetic separation using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents such as complement and cytotoxin cells linked to or used in conjunction with monoclonal antibodies, and "panning" using antibodies attached to a solid matrix, such as a plate, or other convenient techniques. Techniques that provide accurate separation include, but are not limited to, fluorescence-activated cell sorters, which may have various degrees of sophistication, such as multiple color channels, low-angle and obtuse-angle light scatter detection channels, impedance channels, etc. Cells may be selected against dead cells by using dyes that associate with dead cells (e.g., propidium iodide).

[0048] The collected and optionally enriched cell population may be used immediately for genetic modification or may be frozen and stored at liquid nitrogen temperature, and then thawed and reused. Cells are typically stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0049] The lymphoid cell population can be genetically modified to include, for example, genetically modified T cells. The genetically modified T cells can be regulatory T cells. The genetically modified T cells can be modified to knock down or knock out expression of a forkhead box transcription factor. The forkhead box transcription factor can be FoxP3. The genetically modified T cells can be modified to knock down or knock out expression of granzyme B (GZMB). The genetically modified T cells can be CD8 + It may be a T cell.

[0050] Gene editing or genome editing is a type of genetic engineering in which DNA is inserted into, replaced with, or removed from a genome using nucleases. The nucleases may be artificially engineered. Alternatively, the nucleases may be naturally occurring. The nucleases create specific double-strand breaks (DSBs) at desired locations in the genome. The cell's endogenous repair mechanisms then repair the induced breaks through natural processes such as homologous recombination (HR) and non-homologous end joining (NHEJ). Nucleases include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPRs (e.g., CRISPR / Cas systems), engineered meganucleases, and engineered homing endonucleases. CRISPR nucleases include, but are not limited to, Cas nuclease, Cpf1 nuclease, C2c1 nuclease, C2c3 nuclease, and C2c3 nuclease.

[0051] In one embodiment, the nuclease constitutes a CRISPR / Cas system. The CRISPR (clustered regularly interspaced short palindromic repeats) locus, which encodes the RNA components of the system, and the Cas (CRISPR-associated) locus, which encodes the protein (Jansen et al., 2002. Mol. Microbiol. 43:1565-1575; Makarova et al., 2002. Nucleic Acids Res. 30:482-496; Makarova et al., 2006. Biol. Direct 1:7; Haft et al., 2005. PLoS Comput. Biol. 1:e60) constitute the genetic arrangement of the CRISPR / Cas nuclease system. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes and non-coding RNA elements that can program the specificity of CRISPR-mediated nucleic acid cleavage.

[0052] Type II CRISPR is one of the best-characterized systems and involves four sequential steps to create a double-strand break in target DNA. First, two non-coding RNAs, the pre-crRNA and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat region of the pre-crRNA and mediates pre-crRNA processing into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base pairing between the crRNA spacer and the target DNA protospacer adjacent to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of the target DNA, creating a double-strand break within the protospacer. The activity of the CRISPR / Cas system consists of three steps: (i) insertion of foreign DNA sequences into the CRISPR array to prevent future attacks in a process called "adaptation," (ii) expression of associated proteins and expression and processing of the array, followed by (iii) RNA-mediated interference with the foreign nucleic acid.

[0053] In certain embodiments, a Cas protein may be a "functional derivative" of a naturally occurring Cas protein. A "functional derivative" of a native sequence polypeptide is a compound that shares qualitative biological properties with the native sequence polypeptide. "Functional derivatives" include, but are not limited to, native sequence fragments and derivatives of native sequence polypeptides and their fragments, provided that they share a biological activity with the corresponding native sequence polypeptide. The biological activity contemplated herein is the ability of a functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" encompasses both amino acid sequence variants of a polypeptide, covalent modifications, and fusions thereof. Suitable derivatives of a Cas polypeptide or a fragment thereof include, but are not limited to, mutants, fusions, and covalent modifications of a Cas protein or a fragment thereof. Cas proteins, including, but not limited to, Cas proteins or fragments thereof and derivatives of Cas proteins or fragments thereof, may be obtained from cells, produced in vitro, or obtained by a combination of these two procedures. The cells may be cells that naturally produce a Cas protein, or cells that naturally produce a Cas protein and have been genetically engineered to produce the endogenous Cas protein at higher expression levels, or to produce a Cas protein from an exogenous, introduced nucleic acid encoding a Cas that is the same as or different from the endogenous Cas. In some cases, the cells do not naturally produce a Cas protein but are genetically engineered to produce a Cas protein.

[0054] The method also includes introducing a single guide RNA (sgRNA) into a lymphoid cell population. The guide RNA (sgRNA) comprises a nucleotide sequence complementary to a target chromosomal DNA. The sgRNA can be an engineered single-stranded guide RNA, for example, comprising a crRNA sequence (complementary to the target DNA sequence) and a consensus tracrRNA sequence, or as a crRNA-tracrRNA hybrid. The sgRNA can be introduced into a cell or organism as DNA (with an appropriate promoter), as in vitro transcribed RNA, or as synthetic RNA. The target DNA sequence can be a FoxP3 sequence. The target sequence can be a Granzyme B sequence.

[0055] Characteristics of germinal centers In vitro cell clusters can be configured to undergo one or more of somatic hypermutation, affinity maturation, plasmablast differentiation, class switching recombination, and antigen-specific antibody production. To develop antigen specificity, B cells in germinal centers undergo a process called somatic hypermutation, in which point mutations are introduced into the B cell receptor (BCR) gene sequence of both the heavy and light chains of antibody variable regions at a rate significantly higher than the background mutation rate observed in other genes. These mutated B cells differ from each other in their antigen specificity. To ensure high-affinity antibody production, mutated B cells are further selected based on the binding affinity of their receptors to antigens from follicular dendritic cells (FDCs), macrophages, or dendritic cells. B cells with negative antigen binding affinity undergo apoptosis, while B cells with positive binding affinity are selected. This process is called affinity maturation. These two events result in the generation of B cells whose BCRs bind specific antigens with high affinity. Selected B cells then differentiate into memory B cells, also known as antibody-secreting cells (ASCs), or plasmablasts. While plasmablasts produce large amounts of antibodies during the first wave before undergoing apoptosis within a few days, memory B cells provide longer-lasting immunity. Memory B cells can also secrete different classes of antibodies or immunoglobulins (Ig) through a process called class switching recombination. During this process, the DNA recombination process of the constant region of the antibody heavy chain changes while the variable region of the heavy chain remains the same. As a result, the antibody retains affinity for the same antigen but can interact with different effector molecules.

[0056] Germinal centers may contain antigen-presenting cells (APCs), e.g., "professional" antigen-presenting cells. Germinal centers may be at least partially surrounded by T cells. APCs may include B cells, macrophages, or dendritic cells. Dendritic cells may include follicular dendritic cells and pDCs. B cells may include, for example, CD3- B cells, CD45+ B cells, CD19+ B cells, CD38+ B cells, CD38- B cells, or CD27+ B cells. B cells may include transitional B cells, naive B cells, plasma B cells, memory B cells, pre-GC B cells, GC B cells, and plasmablasts. T cells may include, for example, helper T cells, follicular helper T cells, follicular regulatory T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, mucosal-associated invariant T cells, and gamma delta T cells. T cells may include, for example, CD8+ T cells and CD4+ T cells.

[0057] Germinal centers may include dark zones and light zones. The dark zones may contain about 5%, 10%, 15%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more CXCR4+ B cells compared to the light zones. The light zones may contain about 5%, 10%, 15%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more CD83+ B cells compared to the dark zones. At least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 30%, 40% or more of the cells in the dark zone may be CXCR4+ B cells. At least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 30%, 40%, or more of the cells in the bright zone may be CD83+ B cells. Germinal centers may contain at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 30%, or more of CD83+ B cells. Germinal centers may contain at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 30%, or more of CXCR4+ B cells. The bright zone may contain about 5%, 10%, 15%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more of CD86+ cells. CXCL12-expressing reticulum cells (CRCs) are stromal cells known to reside in the dark zone. The dark zone may contain approximately 5%, 10%, 15%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more CRCs.

[0058] antibody The term "antibody" as used herein is used broadly and specifically covers monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain-only antibodies, three-chain antibodies, single-chain Fvs, nanobodies, and the like, as well as antibody fragments, so long as they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170:4854-4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species; typically, human antibodies are produced by culture as described herein.

[0059] The term antibody can refer to a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or an immunologically active portion of any of these polypeptides, i.e., a polypeptide comprising an antigen-binding site that immunospecifically binds to an antigen of a target of interest or a portion thereof, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune disease. An antibody can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. Immunoglobulins can be derived from any species. In one aspect, the immunoglobulin is of human origin.

[0060] The term "variable" refers to the fact that certain portions of the variable domains differ significantly in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy- and light-chain variable domains each contain four FRs that largely adopt a beta-sheet configuration, connected by three hypervariable regions that form loops that connect, and in some cases form part of, the beta-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0061] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. A hypervariable region may comprise amino acid residues from the "complementarity determining regions" or "CDRs" and / or residues from the "hypervariable loops". "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0062] The variable region of interest contains three CDR sequences, which can be obtained from an available antibody with the desired specificity or from an antibody developed for this purpose. Those skilled in the art will understand that several definitions of CDRs are commonly used, including, but not limited to, the Kabat definition, which is based on sequence variability and is the most commonly used (see, "Zhao et al. A germline knowledge-based computational approach for determining antibody complementarity determining regions." Mol Immunol. 2010;47:694-700). The Chothia definition is based on the location of structural loop regions (Chothia et al. "Conformations of immunoglobulin hypervariable regions." Nature. 1989;342:877-883).Alternative CDR definitions include Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol.2001;309:657-670; Ofran et al. “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.” J Immunol.2008;181:6230-6235, Almagro “Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size:implications for the rational design of antibody repertoires.” J Mol Recognit.2004;17:132-143, and Padlanet al. “Identification of specificity-determining residues in antibodies.”Faseb J. 1995;9:133-139, each of which is specifically incorporated herein by reference.

[0063] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method.

[0064] As used herein, "intact antibody chain" includes a full-length variable region and a full-length constant region. An intact, "traditional" antibody includes an intact light chain and an intact heavy chain, and for secreted IgG, a light chain constant domain (CL) and heavy chain constant domains, CH1, hinge, CH2, and CH3. Other isotypes, such as IgM or IgA, may have different CH domains. The constant domains may be native-sequence constant domains (e.g., human native-sequence constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more "effector functions," which refer to biological activities attributable to the Fc constant region (native-sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include, but are not limited to, C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis (ADCP), and down-regulation of cell surface receptors. Constant region variants include those that alter effector profile, binding to Fc receptors, etc.

[0065] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes". There are five major classes of intact immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. Ig forms include hinge-modified or hingeless forms (Roux et al (1998) J. Immunol. 161:4083-4090, Lund et al (2000) Eur. J. Biochem. 267:7246-7256, US2005 / 0048572, US2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0066] "Affinity" refers to the strength of binding, with increased binding affinity correlating with a lower KD. In one embodiment, affinity is determined by surface plasmon resonance (SPR), for example, as used by the Biacore system. The affinity of one molecule for another can be determined by measuring the binding kinetics of the interaction, for example, the binding kinetics at 25°C. A "high affinity" antibody has a KD of 0.1 μM or better. D , 0.01 μM or better K D , 1 nM or better K D , 0.1 nM or better K D , 0.01 nM or better K D , 1 pM or better K D , 0.1 pM or better K D , 0.01 pM or better K D capable of binding to its cognate antigen.

[0067] cell survival The spatial organization of the lymphoid tissue may be maintained for at least about 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or more. The cell clusters may maintain cellular respiration for at least about 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or more. Cellular respiration may be measured, for example, by a resazurin reduction assay, a protease viability marker assay, an ATP assay, or a luciferase assay.

[0068] Autoantigens and autoantibodies Autoimmune diseases are conditions in which immune cells mistakenly attack the body's healthy cells. Some autoimmune diseases are caused by autoantibodies (or natural antibodies), which are antibodies produced against substances (e.g., expressed proteins) formed by an individual's own body. Autoantigens (or autoantigens) are substances (e.g., expressed proteins) that stimulate autoantibodies. These substances can be found in all cell types or can be highly specific to a particular cell type in a single tissue. Autoantigens can include proteins, nucleic acids, carbohydrates, lipids, and various combinations thereof. Most natural autoantibodies are polyreactive, meaning they bind to several unrelated antigens with moderate affinity, typically in the low nanomolar range. However, through somatic hypermutation and class switching, autoantibodies can be expressed, increasing antibody affinity (e.g., to the femtomolar range). Autoantigens can include, for example, Pr3, dsDNA, core histones, or SNRNP70.

[0069] The autoantibodies disclosed herein can have an affinity for an autoantigen of greater than 1 nanomolar, 0.1 nanomolar, 0.01 nanomolar, 0.001 nanomolar, 0.0001 nanomolar, 1000 femtomoles, 100 femtomoles, 10 femtomoles, or 1 femtomole. The autoantibodies disclosed herein can have an affinity for an autoantigen of 1 nanomolar to 10,000 femtomoles, 100,000 femtomoles to 100 femtomoles, 1,000 femtomoles to 10 femtomoles, or 100 femtomoles to 1 femtomole.

[0070] Transcription factor expression In some embodiments, organoid cells are genetically modified to knock down or knock out the expression of a transcription factor. In some embodiments, regulatory T cells are genetically modified to knock down or knock out the expression of a transcription factor. In some embodiments, T cells are genetically modified so that they do not regulate antibody production by B cells. In some embodiments, T cells are genetically modified to enable the production of autoantibodies by B cells. In some embodiments, T cells are genetically modified to enable the production of high-affinity antibodies by B cells. In some embodiments, the transcription factor is a member of the forkhead box family of transcription factors. The forkhead box family of transcription factors plays a role in regulating the expression of genes involved in cell growth, proliferation, differentiation, and longevity. The forkhead box P3 (FOXP3) gene encodes the FOXP3 transcription factor, which is important for the development and inhibitory function of regulatory T cells. FOXP3 is necessary for the effective maintenance of systemic tolerance and the prevention of autoimmune diseases. In some embodiments, T cells are modified to reduce or inactivate FOXP3 expression. Inactivation of the FOXP3 gene can include knockout (KO) or knockdown methods and can be permanent or transient. In some embodiments, FOXP3-knockout or knockdown T cells can be edited by any gene expression modification system known to those of skill in the art.

[0071] Granzyme B expression In some embodiments, organoid cells are genetically modified to knock down or knock out the expression of cytolytic protein.In some embodiments, the cells are T cells.In some embodiments, the cytolytic protein is granzyme B.In some embodiments, the T cells are CD8 + In some embodiments, the T cells are CD4 + T cells. Granzyme B (GZMB) is a serine protease known for its perforin-dependent proapoptotic function, which underlies the ability of cytotoxic immune cells, such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Granzyme B exerts perforin-dependent intracellular activities and extracellular perforin-independent functions, consisting of cleavage of multiple extracellular substrates, such as extracellular matrix (ECM) components, cytokines, cell receptors, angiogenesis, and coagulation proteins.

[0072] Gene editing Methods for knocking out or knocking down gene expression can include any gene editing system known in the art. Gene editing systems can include, for example, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic nucleotide sequences (CRISPR) / Cas nucleases, meganucleases, or primed editing. Gene editing systems can include, for example, siRNA, shRNA, DNA-based RNAi, antisense oligonucleotides, or CRISPR-mediated gene knockdown, CRISPR interference (CRISPRi) dCas9 without additional proteins, CRISPRi dCas9 in combination with other proteins, or Cas13 family enzymes. Gene editing systems can include, for example, microinjection, electroporation, lipofection, ultrasound, gene guns, viral delivery, and hydrodynamic application.

[0073] use Disclosed herein is a method for producing antibodies, for example, high-affinity antibodies, from lymphoid cell cultures, for example, lymphoid organoids, comprising placing lymphoid cells in a culture medium to produce cell cultures.Cell cultures can include specific spatial configurations of lymphoid tissues.Cell cultures can include, but are not limited to, one or more embryonic cells.Lymphoid cell cultures can include aggregates of T cells.Methods can include introducing antigens into culture medium, for example, to enhance the immune response of cell cultures to antigens.Methods can include incubating cell cultures with antigens to produce antibodies.The antibodies produced from cell cultures can be isolated from the cell cultures.

[0074] Genetic sequences encoding antibodies of interest, e.g., high affinity antibodies, antibodies specific for autoantigens, etc., can be identified, sequenced by any convenient method, and inserted into expression vectors for responsiveness analysis, structure-function analysis, production of selected antibodies, etc.

[0075] Coding sequences can be inserted into vectors for expression and / or integration. Many such vectors are available. Vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include, but are not limited to, viral vectors, plasmid vectors, and integrating vectors. Expression vectors can contain a promoter that is recognized by the host organism and is operably linked to the antibody coding sequence. Transcription in higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp in length, that act on a promoter to increase its transcription. Expression vectors for use in eukaryotic host cells also contain sequences necessary for the termination of transcription and stabilization of mRNA. Such sequences are commonly available from the 5' and, occasionally, 3' untranslated regions of eukaryotic or viral DNA or cDNA. Construction of suitable vectors containing one or more of the above components uses standard techniques. Suitable host cells for cloning constructs for expressing a selected antibody include the prokaryotes, yeast, or other eukaryotic cells described above. The expressed antibody can be isolated and purified from the host cells as known in the art.

[0076] The antibodies produced by the methods disclosed herein may be formulated with a pharmaceutically acceptable carrier (one or more organic or inorganic components, natural or synthetic, with which the subject agent is combined to facilitate its application. Suitable carriers include, but are not limited to, sterile saline, and other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those skilled in the art.

[0077] Antibodies can be formulated as compositions containing pharmaceutically acceptable excipients. The form depends on the intended mode of administration and application. Depending on the desired formulation, the composition may also contain, but is not limited to, pharmaceutically acceptable non-toxic carriers or diluents (defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration). Diluents are selected so that, when combined, they do not affect biological activity. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also contain, but is not limited to, other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers.

[0078] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are commercially available. Additionally, pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, tonicity adjusters, stabilizers, and wetting agents, are commercially available. Any compound useful in the methods and compositions of the present invention can be provided as a pharmaceutically acceptable base addition salt. "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological effectiveness and properties of the free acids, without being biologically or otherwise unsuitable. These salts are prepared by the addition of an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particular organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0079] The methods and systems disclosed herein can be useful for generating monoclonal antibodies for the testing and treatment of various diseases, including cancer, autoimmune diseases, and / or infectious processes, including viral infections, bacterial infections, and microbial infections, or combinations thereof. The cancer disease can be, for example, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, intestinal cancer, head cancer, neck cancer, breast cancer, gastric cancer, melanoma, glioblastoma, colorectal cancer, lung cancer, kidney cancer, or ovarian cancer. The autoimmune disease can be rheumatoid arthritis, Crohn's disease, celiac disease, pernicious anemia, autoimmune vasculitis, myasthenia gravis, Sjögren's syndrome, Graves' disease, Addison's disease, inflammatory bowel disease, systemic lupus erythematosus, type 1 diabetes, lupus, multiple sclerosis, or psoriasis.

[0080] In certain aspects, the methods and systems disclosed herein may be useful for determining a subject's progress in treatment. For example, such methods and systems may include screening a patient for an immune response to treatment. The subject may be healthy. The patient may test positive for a viral infection. The subject may test positive for an autoimmune disease. The subject may test positive for a fungal infection. The subject may test positive for a bacterial infection. The methods and systems may include screens that may be used prophylactically to identify vaccination in a patient. The methods and systems may include screens that may be used to identify immune responses within specific populations of individuals.

[0081] In certain aspects, the methods disclosed herein are useful for screening vaccine candidates. The method may include contacting lymphoid organoids with an effective dose of a candidate vaccine, including administering the candidate vaccine, which may include an adjuvant, and determining the antibody response produced by the lymphoid organoids. The antibody response may be measured by bulk specificity and affinity measurement. The antibody response may be measured by sequencing the mRNA encoding the antibody in the lymphoid organoid cells. The antibody response may be measured by isolating antibody clones and determining specificity and affinity. The response induced by the vaccine candidate may be compared with the response induced by a control antigen. The response induced by the vaccine candidate may be compared with the response induced by one or more different vaccine candidates. In some embodiments, the candidate vaccine or adjuvant is selected for development based on its ability to provide a specific and / or high-affinity antibody response.

[0082] This disclosure includes an in vitro system that supports one or more of antigen-specific somatic hypermutation, affinity maturation, and class switching of human B cells. Tonsils are a readily available but underutilized source of human lymphoid tissue, containing cell types involved in adaptive immunity, including those largely absent from peripheral blood. We used tonsillar organoids to characterize the human influenza response, extending findings from previous human and mouse studies. Pre-existing HA-specific B cells possessed 5–10 heavy chain nucleotide mutations (a 2–4% mutation rate), while organoids stimulated with LAIV prepared using naive-only B cells possessed 3–9 mutations, sufficient to generate high-affinity specific antibodies (Figure 4). These rates are consistent with the average 5% mutation rate measured in adults who received influenza vaccinations.

[0083] As seen in the current SARS-CoV-2 pandemic, the human immune response is highly variable, and a major goal of vaccine development is to confer as broad a protective immunity as possible. Another useful feature of the organoid system is its ability to assess the variability of immune responses. While most donor-derived organoids respond to LAIV, the magnitude and kinetics of their responses vary widely. In a study of 15 donors, only organoids prepared from one donor failed to respond to LAIV within 7 days (Figures 1A–1F). This individual was young, had not been vaccinated against influenza, and may have been naive to the flu, suggesting that organoids prepared from cells may require more time to mount a response. Longitudinal analysis (Figures 2A–2F) identified non-responders treated with daily inhaled corticosteroids, demonstrating the system's sensitivity to immunosuppressants. Further variability was observed using SARS-CoV-2 vaccine candidates, with some individuals failing to generate B and / or T cell responses.

[0084] Using depletion studies, we established the functional relevance of individual immune cell types to the influenza response. pDCs were critical for antibody responses but could be replaced with exogenous type I IFN. These data are consistent with previous studies finding that pDCs induce human blood B cell differentiation via type I IFN41, but contrast with influenza challenge studies in mice showing that pDCs are dispensable for the response, highlighting differences between mouse and human systems. We identified a role for pre-existing plasmablasts / plasma cells in controlling B cell differentiation (Figures 5A-5B). When plasmablasts were depleted at the initiation of culture, influenza-specific antibody production increased compared to wild-type controls. Because secondary lymphoid tissues are competitive niches for plasma cell survival outside the bone marrow, depletion of pre-existing plasma cells may promote the survival of antigen-specific plasmablasts. We demonstrated that T cell help plays an age-dependent role in the magnitude of influenza-specific antibody responses, indicating that CD4+ T cells play an additional role in affinity maturation and B cell selection (Figures 5A-5H). Memory CD4+ T cells from older children better maintained naive B cell responses, suggesting that T cell help is refined by in vivo exposure. These findings are consistent with previous mouse studies showing that memory T cells can contribute to GC responses, whereas naive B cells typically do not. Stem-specific antibodies were also detectable only in the presence of CD4+ T cells, suggesting that manipulating T cell help is a rational strategy to consider for future vaccine design.

[0085] Previous efforts to create artificial human lymphoid tissues relied on specialized bioreactors or complex in vitro differentiation strategies. Blood-derived mononuclear cells combined with monocyte-derived dendritic cells can stimulate IgM and cytokine secretion consistent with adaptive immune responses. It remains questionable whether in vitro-matured dendritic cells can recapitulate the conditions in lymphoid tissues that orchestrate T and B cell responses. More importantly, a major limitation of bioreactor systems is the lack of mature GCs, class switching, and affinity maturation, because many cells required for GC function may not be present in peripheral blood mononuclear cells. Recent studies have demonstrated that key aspects of the immune microenvironment can be captured in organoids, with several studies focused on conferring immune protection upon transplantation into mice or recapitulating characteristics of GC responses. These studies used engineered hydrogel scaffolds for B cells combined with engineered fibroblasts to reveal the transcriptional feedback loops involved in GC formation, but lacked antigen specificity. More recently, maleimide-functionalized hydrogels have been shown to support GC B cell activation and antigen-specific responses in mouse B cell organoid cultures. However, these methods do not incorporate autologous APCs and T cells, both of which are essential for refining adaptive immune responses in vivo. Due to the need for bioengineered fibroblast cell lines to provide the necessary survival signals, these techniques are difficult to translate to human cells due to the diversity of human leukocyte antigen genes and the generation of mixed leukocyte reactions. Previous techniques for creating in vitro models of human adaptive immunity have not been widely adopted due to their reliance on specialized equipment or difficult technical protocols, low throughput, lack of evidence for antigen-specific responses and affinity maturation, and / or the absence of cells known to be essential for key features of adaptive immunity. While humanized mice are increasingly used, they are expensive to procure and still have many limitations in terms of recapitulating human immunity.

[0086] LAIV has been shown to be less effective than inactivated formulations in adults, likely due to the presence of pre-existing antibodies at mucosal sites in non-naive individuals. Such conditions can be mimicked by introducing autologous serum or spiking influenza-specific antibodies at the initiation of organoid culture.

[0087] kit Kits may include, but are not limited to, one or more containers housing one or more of the components and instructions provided in this disclosure. Specifically, such kits may include, but are not limited to, one or more compositions described herein and instructions describing the intended uses and proper use and / or disposal of these compositions. Kits may contain components in concentrations or amounts appropriate for performing various experiments.

[0088] Machine Learning The methods and systems disclosed herein can utilize artificial intelligence / machine learning to generate optimal antibodies with high specificity and affinity. Artificial intelligence / machine learning can be used to predict antigens that may be specific to a particular type of cancer, autoimmune disease, or infectious disease. The predicted antigens can be used in antibody production from the methods and systems disclosed herein.

[0089] Computer Systems The present disclosure provides a computer system for implementing the methods provided herein. FIG. 18 illustrates an example of a computer system 1001. The computer system 1001 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 1005, which may be, but is not limited to, a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 1001 also includes, but is not limited to, memory or memory locations 1010 (e.g., random access memory, read-only memory, flash memory), electronic storage 1015 (e.g., hard disk), a communication interface 1020 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1025, such as cache, other memory, data storage, and / or electronic display adapters. The memory 1010, storage 1015, interface 1020, and peripheral devices 1025 communicate with the CPU 1005 via a communication bus (solid lines), such as a motherboard. The storage 1015 may be a data storage device (or data repository) for storing data. Computer system 1001 may be operatively coupled to a computer network (“network”) 1030 with the aid of communication interface 1020. Network 1030 may be the Internet, an internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. Network 1030, in some cases, is a telecommunications and / or data network. Network 1030 may include one or more computer servers, which may enable distributed computing such as, but not limited to, cloud computing. Network 1030, in some cases, with the aid of computer system 1001, may implement a peer-to-peer network, which may enable devices coupled to computer system 1001 to operate as clients or servers.

[0090] The CPU 1005 can execute sequences of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1010. The instructions may be directed to the CPU 1005, which may then program or otherwise configure the CPU 1005 to implement the methods of the present disclosure. Examples of operations performed by the CPU 1005 may include fetch, decode, execute, and write-back.

[0091] The CPU 1005 may be part of a circuit, such as an integrated circuit. One or more other components of the system 1001 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0092] Storage device 1015 may store files such as drivers, libraries, and saved programs. Storage device 1015 may store user data, such as user preferences and user programs. Computer system 1001 may, in some cases, include one or more additional data storage devices external to computer system 1001, such as located on a remote server that communicates with computer system 1001 via an intranet or the Internet.

[0093] Computer system 1001 may communicate with one or more remote computer systems via network 1030. For example, computer system 1001 may communicate with a user's remote computer system (e.g., a remote cloud server). Examples of remote computer systems include, but are not limited to, a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., an Apple iPad®, a Samsung Galaxy Tab®), a telephone, a smartphone (e.g., an Apple iPhone®, an Android-enabled device, a Blackberry®), or a personal digital assistant. A user may access computer system 1001 via network 1030.

[0094] The methods described herein may be implemented by machine (e.g., computer processor) executable code stored in an electronic storage location of the computer system 1001, such as, for example, memory 1010 or electronic storage 1015. The machine-executable or machine-readable code may be provided in the form of software. During use, the code may be executed by the processor 1005. In some cases, the code may be retrieved from storage 1015 and stored in memory 1010 for immediate access by the processor 1005. In some situations, the electronic storage 1015 may be excluded, and the machine-executable instructions are stored in memory 1010.

[0095] The code may be pre-compiled and configured for use on a machine having a processor adapted to execute the code, or may be compiled during run-time. The code may be supplied in a programming language that may be selected to allow the code to be executed in a pre-compiled or compiled fashion.

[0096] Aspects of the systems and methods provided herein, such as computer system 1001, may be embodied in programming. Various aspects of the technology may be considered "products" or "articles of manufacture," typically in the form of machine (or processor) executable code and / or associated data carried or embodied on some type of machine-readable medium. The machine-executable code may be stored in electronic storage, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. A "storage" type medium may include any or all of the tangible memory of a computer, processor, etc., or their associated modules, such as various semiconductor memories, tape drives, disk drives, etc., and may provide non-transitory storage for software programming at any time. All or portions of the software may sometimes be communicated via the Internet or various other telecommunications networks. Such communication may, for example, enable loading of the software from one computer or processor to another, such as from a management server or host computer to an application server computer platform. Thus, other types of media that may carry software elements include optical, electrical, and electromagnetic waves, such as those used over physical interfaces between local devices, over wired and optical landline networks, and over various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered software-bearing media. As used herein, unless limited to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0097] Thus, a machine-readable medium such as a computer-executable code may take many forms, including, but not limited to, a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include optical or magnetic disks, such as the storage devices in any computer, such as those used to implement a database, as shown in the drawings. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables, copper wire, and fiber optics, including the wires that comprise a bus within a computer system. Carrier wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer-readable media include, but are not limited to, for example, magnetic disks, floppy disks, hard disks, magnetic tape, any other magnetic media, CD-ROMs, DVDs, or DVD-ROMs, any other optical media, punch cards, paper tape, any other physical storage media with patterns of holes, RAM, ROM, PROMs, and EPROMs, FLASH-EPROMs, any other memory chips or cartridges, carrier waves that transport data or instructions, cables or links that transport such carrier waves, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer-readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0098] The computer system 1001 may include or be in communication with an electronic display 1035 that includes, for example, a user interface (UI) 1040 for providing an electronic output of the identified gene fusions. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0099] The methods and systems of the present disclosure may be implemented by one or more algorithms, which may be implemented by software when executed by the central processing unit 1005. [Example]

[0100] Example 1 - Preparation of immune organoids from tonsils and other lymphoid tissues Human tonsil cultures were developed using dissociated cells that were reaggregated in culture. Table 1 below shows the characteristics of the tissue donors.

[0101] [Table 1-1]

[0102] [Table 1-2]

[0103] [Table 1-3]

[0104] Whole tonsils from 150 consenting individuals undergoing surgery for obstructive sleep apnea, hypertrophy, or recurrent tonsillitis were collected in accordance with the Stanford University Institutional Review Board (IRB). Ethics approval was granted by the Stanford IRB (protocols 30837 and 47690). Written informed consent was obtained from adult participants and legal guardians of children aged 0–17 years. Written informed consent was also obtained from children aged 7 years and older. In this cohort, participants were children aged 2–17 years (n=57) and adults (n=3) undergoing surgery for obstructive sleep apnea and / or hypertrophy. Overall, tonsillar tissue was generally healthy. After surgery, whole tonsils were collected in saline and then immersed in an antibacterial bath of Ham's F12 medium (Gibco) containing Normocin (InvivoGen), penicillin, and streptomycin for 1 hour at 4°C for tissue decontamination. Tonsils were then briefly rinsed with PBS and processed for culture as required (see below).

[0105] Donor lung lymph nodes and spleens were provided to the University of Chicago by the Gift of Hope Organ and Tissue Donor Network. These tissues were determined to have IRB-exempt status by the University of Chicago IRB. Only de-identified demographic information was obtained.

[0106] For cryopreservation of tonsillar cells, tissue was cut into approximately 5 mm x 5 mm x 5 mm pieces and manually disrupted into a suspension by passing through a 100 μm strainer with a syringe plunger. Enzymatic dissociation was not required and did not improve the response to LAIV from cryopreserved cells. Tissue debris was reduced by Ficoll density gradient separation, but this step was not necessary for the development of tonsillar organoids. After washing with complete medium (RPMI containing Glutamax, 10% FBS, 1x non-essential amino acids, 1x sodium pyruvate, 1x penicillin-streptomycin, 1x Normocin (InvivoGen), and 1x insulin / selenium / transferrin cocktail (Gibco)), cells were counted and frozen in aliquots in FBS + 10% DMSO. Frozen cells were stored at -140°C until use.

[0107] For lung-draining lymph node and spleen samples, tissue was collected in saline or Hank's balanced salt solution, diced into small pieces, and forced through a nylon mesh (Nitex) to disrupt the tissue. Cells were isolated by Ficoll density gradient separation, washed, counted, and frozen in FBS + 10% DMSO. Frozen samples were stored in liquid nitrogen until use.

[0108] For culturing of cryopreserved cells, thaw an aliquot into complete medium, count, and for larger cultures, 6 x 10 cells per ml. 7 cells per ml for smaller cultures, 2 x 10 7Cells were resuspended to a concentration of 100 μl per well. Cells were seeded onto permeable (0.4 μm pore size) membranes (PTFE or polycarbonate membranes in standard 12-well plates with 24-well size, or 96-well polycarbonate membrane plates with single-well receiver trays, Corning or Millipore) with a lower chamber consisting of complete medium (1 ml for 12-well plates and 200 μl for 96-well plates) supplemented with 1 μg ml recombinant human B-cell activating factor (BAFF, BioLegend). Adding a small amount of BAFF improved total B-cell viability (and thereby increased overall cell recovery) but was not a requirement for plasmablast differentiation or antibody secretion.

[0109] Then, LAIV (1 μl per well, 1.6 × 10 per strain) was added. 4 ~1.6×10 5 equivalent to 10 fluorescent focus units per culture, FluMist Quadrivalent, Mediimmune), wild-type influenza virus (A / California / 07 / 2009 pandemic strain, a gift from H. Greenberg and X.-S. He), MMR vaccine (5 μl per culture, Merck), R-phycoerythrin (1 μg per culture, Thermo Fisher), rabies vaccine (10 μl per culture, Imovax, Sanofi Pasteur), or Ad5-vectored SARS-CoV-2 vaccine candidate (1 × 10 per culture). 8 Infectious units of 1000 mg / ml (Vaxart) were added directly to the cell-containing portion of the culture setup. For adjuvant testing, alum (0.01%, InvivoGen, sold as a 2% stock wet gel with a stock aluminum content of 9–11 mg / ml) or imiquimod (2.5 μg / ml, InvivoGen) were added directly to the cultures immediately after antigen addition. Cultures were incubated at 37°C in 5% CO2 with humidity, with the lower wells replenished with additional medium as needed. Hypoxic conditions (5% O2) were tested for B cell differentiation and antibody secretion, but there were no significant differences compared to cultures maintained at standard incubator oxygen levels (17–21%).

[0110] For the Ad5-vectored vaccine candidate, a recombinant adenovirus construct was generated using the publicly available SARS-CoV-2 DNA sequence (GenBank accession number MN908947.3). The spike and nucleocapsid protein sequences were synthesized, codon-optimized for expression in human cells, and cloned into the E1 region as previously described. The same vector backbone has previously been used in clinical trials of oral recombinant adenovirus tablets. The Ad-S adenovirus vector contains the spike protein under the control of the human cytomegalovirus (CMV) promoter. The Ad-SN vector contains the spike under the control of the CMV promoter and the nucleocapsid under the control of the human beta-actin promoter. The recombinant Ad-S1N vector uses a fusion sequence combining the S1 region of the SARS-CoV-2 spike gene (including the natural furin site between S1 and S2) and the full-length SARS-CoV-2 nucleocapsid gene. All vaccine candidates were purified by cesium chloride density centrifugation and provided in liquid form for cell culture experiments.

[0111] For organoid preparation, frozen single-cell suspensions from tonsil tissue were thawed and seeded at high density into wells of permeable membrane plates (e.g., transwells) along with the antigen of interest. After several days of culture, areas of reaggregation of clustered cells were visible, as can be seen in Figure 1A. The cellular composition of the reaggregated cultures was assessed after 7 days in the presence or absence of antigen, and the optimized conditions maintained the appropriate tonsillar cell composition, as can be seen in Figure 1B.

[0112] Because much is already known about the characteristics of the human influenza response in vivo, influenza vaccines and viruses were used as model antigens. Stimulation with live attenuated influenza vaccine (LAIV) significantly increased B cell differentiation, leading to the development of more structural culture morphologies, suggesting additional activity in response to this immunogen. The ability of organoid cultures to support B cell maturation and function upon LAIV stimulation was then measured by staining the cultures, as can be seen in Figure 7A.

[0113] Flow cytometry. Organoids were collected from the top of the permeable membrane by rinsing the membrane with PBS. Cells were washed with FACS buffer (PBS + 0.1% BSA, 0.05% sodium azide, and 2 mM EDTA) and stained at 4°C with the following anti-human antibodies in the presence of Fc block and live / dead Aqua Zombie stain: Unless otherwise stated, all are from BioLegend: FITC CD138 (1 / 100), FITC CD116 (1 / 100), FITC CD21 (1 / 50), FITC or Ax488 CXCR5 (1 / 33), PerCP-Cy5.5 CD8 (1 / 100), PerCP-Cy5.5 CD33 (1 / 100), PE CD19 (1 / 100), PE CD56 (1 / 100), PE gamma-delta TCR (1 / 100), PE-Cy7 CD27 (1 / 100), PE-Cy7 CD123 (1 / 50), PE-Cy7 CD8 (1 / 100), APC CD38 (1 / 200), APC HLA-DR (1 / 100), APC CD27 (1 / 200), Ax700 CD45(1 / 100), Ax700 CD14(1 / 100), APC-Cy7 IgD(1 / 50), APC-Cy7 CD16(1 / 100), APC-Cy7 CD45RA(1 / 100), Pacific Blue HLA-DR(1 / 100), Pacific Blue PD-1(1 / 50), BV605 CD3 (1 / 100), BV650 CD4 (1 / 100), BV650 CD19 (1 / 100), BUV395 IgM (1 / 20, BD Biosciences), and BUV395 CD45RA (1 / 20, BD Biosciences).

[0114] For AID staining, after surface staining, cells were fixed and permeabilized (eBioscience) and intracellularly stained with biotinylated anti-AID antibody (1 / 100, clone mAID-2, eBioscience), followed by PE-streptavidin (eBioscience). A no-AID antibody control was used to identify positive signals. All analytical data were collected on a BD LSRII instrument and analyzed using FlowJo (TreeStar).

[0115] After 7 days, plasmablast frequencies were found to be significantly increased compared with unstimulated controls (Figure 1C, n = 15, P = 0.0002). Thirteen of the 15 donors tested, ranging in age from 2 to 14 years, produced influenza-specific IgG antibodies only in samples stimulated with LAIV (P < 0.0001). Of the two exceptions, one donor had a high response without stimulation, and the other was a true non-responder. The latter donor (aged 3 years) may have been naive to influenza infection and had not reported influenza vaccination. The production of specific antibodies was not a general effect of stimulation, as total (i.e., antigen-independent) IgG was reduced by LAIV (Figure 1D). Enzyme-linked immunosorbent spot (ELISpot) analysis was performed on day 7 cultures from five donors and found influenza-specific antibody-secreting cells (ASCs) ranging from 0.1 to 1.5% of total B cells (Figure 1E, representative ELISpot data in Figure 7B). In unstimulated tonsillar cultures, influenza-specific ASCs were rarely identified (less than 0.02% of total B cells in all donors). The organoid culture strategy in transwells was superior to flat-bottom tissue culture plates of similar surface area for growing tonsillar mononuclear cells (Figure 7C).

[0116] Antibody detection by ELISA. For influenza-specific antibody detection, ELISA plates (Costar) were coated with 0.1 μg of seasonally adapted Fluzone Quadrivalent inactivated influenza vaccine (based on the total HA content reported by the manufacturer, Sanofi) per well to serve as the capture antigen. For A / California HA antibody detection, recombinantly expressed soluble HA trimer was used as the capture antigen instead of the inactivated vaccine. Diluted (1:20 or 1:50) culture supernatant was added to the coated and blocked plates. A human pan-influenza monoclonal IgG antibody (H1N13-M, Alpha Diagnostics) was used as a standard to estimate specific antibody concentrations, allowing for quantitatively defined experiments. Bound antibodies were detected using horseradish peroxidase-conjugated anti-human secondary antibodies against either IgM / IgG / IgA (Sigma) or Fc-IgG alone (Bethyl, adsorbed against other isotypes). Plates were developed with TMB substrate solution (Thermo Scientific), quenched with sulfuric acid, and read at 450 nm. Neutralization experiments were performed by Monogram Biosciences using a pseudovirus neutralization assay containing HAs matching the vaccine antigen strains.

[0117] For detection of total IgG, culture supernatants were diluted 1:500 and assayed by ELISA (Thermo Scientific) according to the manufacturer's instructions. For detection of MMR-specific IgG, culture supernatants were tested by ELISA (Abcam) according to the manufacturer's instructions. Supernatants were diluted 1:2.5 using the provided sample diluent and serially diluted two-fold up to 1:40 to confirm signal specificity (data not shown). For measurement of rabies nucleoprotein-specific IgM and IgG, supernatants were diluted 1:5 using a specific IgM and IgG detection kit (Alpha Diagnostic).

[0118] Antibody detection by protein microarray. Organoid cultures stimulated with recombinant adenoviral vectors carrying SARS-CoV-2 sequences were tested for specific antibody production using a protein microarray. This technique has been previously described for the detection of influenza-specific antibodies and has recently been adapted to include commercially available SARS-CoV-2 proteins. SARS-CoV-2 proteins are commercially available from Sino Biological. Briefly, day 14 culture supernatants were diluted 1:1 with blocking buffer and incubated for 30 minutes. The diluted samples were then added to the microarray for overnight hybridization. The arrays were washed three times with Tris-buffered saline containing Tween 20 and then treated with Qdot-conjugated anti-IgM, IgG, or IgA secondary antibodies for two hours. After three additional washes, the arrays were dried and read. Signal intensity represents the relative amount of antibody bound to each protein spot.

[0119] We also tested whether the organoid culture strategy could support human lung-draining lymph node and spleen samples (Figure 1F). These organoids responded to both LAIV and wild-type A / California H1N1 influenza virus by producing influenza-specific antibodies at levels substantially above those of control cultures and comparable to those of tonsillar organoids.

[0120] Example 2: Longitudinal analysis of tonsillar cultures in response to LAIV Given the evidence of B cell maturation and antibody secretion in LAIV-stimulated organoid cultures, we performed longitudinal analyses to observe the differentiation process over time. The CD38 and CD27 expression patterns of B cells from LAIV-stimulated cultures clearly evolved (Figure 2A), showing an early transition to a pre-GC phenotype (CD38+CD27-), followed by plasmablast differentiation in two of three donors (Figure 2B). The frequency of plasmablasts typically correlated with the total amount of specific antibodies produced (Figure 7D). Changes in T cell phenotype were also observed, including the recovery of T cell (CXCR5+CD4+) cells during culture (in both unstimulated and LAIV-stimulated organoids) and transient CD4+ and CD8+ T cell activation at day 7 in LAIV-stimulated cultures (Figure 2C). Of the more than 50 donors tested, only organoids prepared from one donor (IMD012) failed to produce influenza-specific antibodies (Figure 2D). Four additional donors were used to confirm that influenza-specific antibodies were produced with the expected kinetics (between days 5 and 10). The ability of these cultures to produce neutralizing antibodies was measured. For two of the four LAIV strains tested, vaccine-stimulated culture supernatants exhibited neutralizing capacity, whereas unstimulated controls did not (Figure 2E).

[0121] T cell responses in HLA-A2+ donors were also evaluated using class I tetramers targeting the immunodominant influenza M1 specificity (Figure 2F). Three of the four donors tested showed a 2- to 10-fold expansion of M1-specific CD8+ T cells by day 7 after stimulation with LAIV. One non-responder had fewer M1-specific CD8+ T cells in its starting repertoire compared to the other donor. This may explain the poor CD8+ T cell response.

[0122] Tetramer staining. HLA-A2 tetramers were prepared as previously described using ultraviolet (UV)-sensitive peptide cleavage and exchange against the A2 immunodominant influenza M1 peptide (GILGFVFTL) or an irrelevant CMV pp65 peptide (NLVPMVATV) as a control. Tetramers were prepared from peptide-exchanged monomers by conjugation to PE-streptavidin and APC-streptavidin (eBioscience), respectively. For staining, organoids from HLA-A2 donors were harvested, washed with FACS buffer, and stained with tetramers (0.5 μg of monomer per test) in the presence of Fc block for 1 hour at 4°C. Lineage-determining antibodies were added during the final 30 minutes of tetramer staining, and samples were washed with FACS buffer. For analysis, influenza M1 tetramer-positive CD8+ T cells were identified as single-positive T cells for influenza tetramer (negative for CMV tetramer) and stained above the control without influenza tetramer staining.

[0123] Example 3: Spatial organization of tonsillar organoids During adaptive immune responses, peripheral lymphoid organs such as tonsils, lymph nodes, and spleens develop GCs. B cells aggregate in GC regions, whereas T cells are predominantly located in peripheral regions. In organoid cultures, GC-like structures containing distinct B- and T-cell-enriched aggregates were observed beginning approximately 48 hours after culture initiation in both LAIV-stimulated and unstimulated cultures. These progressed to well-defined clusters by days 4–7, particularly in LAIV-treated cultures (Figures 3A and 8). We analyzed embedded cryosections from three donors by fluorescence confocal microscopy and found evidence of the organization of bright and dark zones, as detected by the separation of B cells stained for CD83 and CXCR4, respectively. This is a hallmark of GCs (Figure 3A, representative images, Figures 8A–8E, and Figure 9A).

[0124] Immunofluorescence. Samples for immunofluorescence microscopy were prepared from frozen tonsillar cells stimulated with LAIV and harvested 4 or 7 days after stimulation. Permeable membrane inserts containing organoids were gently immersed in PBS, fixed with 4% paraformaldehyde in PBS for 30 minutes at 4°C, and washed three times with water. Cultures were kept at room temperature and incubated for 20 minutes with increasing concentrations of warmed (37°C) OCT compound (Fisher) diluted in PBS at 25%, 50%, and 75% (vol / vol), with a final incubation in pure OCT. Samples were snap-frozen on dry ice, the inserts removed with forceps, and the samples embedded in an additional layer of OCT and frozen. Embedded samples were sectioned at 25 μm and attached to poly-l-lysine-coated coverslips. Sections were dried in a dehumidified chamber for 4 minutes and permeabilized in acetone for 10 minutes at room temperature. Rehydration was performed in staining buffer (1% BSA, 1% normal goat serum, and 0.01% sodium azide in PBS) for 30 minutes. Sections were first stained with primary unconjugated antibodies for 3 hours at room temperature. Secondary antibody staining was performed for 1 hour at room temperature, followed by primary directly conjugated antibodies for 3 hours at room temperature.

[0125] Primary antibodies. Immunofluorescence staining was performed using the following antibodies at the indicated concentrations: BioLegend: CD3-BV421 (SK7, 1:25), CD4-AF594 (RPA-T4, 1:50), CD19-AF647 (SJ25C1, 1:25), CXCR4 (12G5, 1:100), CD21-FITC (Bu32, 1:100), PD-1-AF488 (NAT105, 1:100), BCL-6 (IG191E / A8, 1:50); Sigma-Aldrich: CXCR5 (polyclonal, 1:100), CD83 (polyclonal, 1:100); Thermo Scientific: Fisher:AID(ZA001, 1:100), CD8-AF488(AMC908, 1:100), CD20-eFluor615(L26, 1:100), CD20-eFluor660(L26, 1:100), BD Biosciences:Ki67-BV421(B56, 1:5), Fisher Science:IgD-AF488(IgD26, 1:10).

[0126] Secondary antibodies. The following secondary antibodies and their dilutions from Thermo Fisher Scientific were used: goat anti-mouse IgG (H+L) AF Plus555 (1:200), goat anti-rabbit IgG (H+L) AF Plus555 (1:200), and goat anti-rabbit IgG (H+L) AF Plus594 (1:200).

[0127] After collecting imaging data from the antibody markers, slides were stained with 1 μg ml-1 DAPI to visualize nuclear staining. Imaging was performed on an inverted Zeiss LSM880 confocal instrument using 25x magnification. Z-stacks of samples were acquired in 2 μm slices. DAPI was stimulated at 405 nm, FITC / Alexa Fluor 488 at 488 nm, Alexa Fluor 555 at 561 nm, and Alexa Fluor 594 at 594 nm. Images were processed in ImageJ (version 2.0.0). Tile stitching was performed using the grid / collection stitching tool, using positions from the files in the order defined by the image metadata. The fusion method used was linear blending with Computer Overlap and Ignore Calibration (all other parameters set to default). After despeckling, Z-stacks were overlaid using Z-Projection by Maximum Intensity, contrast was adjusted to better present structures, channels were stacked to RGB, and a scale bar was added.

[0128] To quantify the fraction of cells expressing CXCR4 and CD83 in the GC region, GCs were cut into two sections resembling bright and dark zone-like regions, as shown in the imaging images. CD20-, CD83-, and CXCR4-positive cells, in addition to double- and triple-positive cells, were manually counted (using the multipoint tool in ImageJ). The CD83+CXCR4-CD20+ and CD83-CXCR4+CD20+ cells in each region were calculated and expressed as a percentage of the total CD20+ population from the same region. To determine the average intensity of marker expression in the bright and dark zone regions, images were converted to RGB using "stack to RGB," and then a "color histogram," which displays a histogram of intensity values, was generated for each channel using ImageJ. The average of each channel was used to compare the average marker expression between different regions of comparable size.

[0129] ELISpot. ASCs were detected using an ELISpot protocol. Cultures either stimulated with LAIV for 7 days or left unstimulated were resuspended, counted, and then plated onto blocked 96-well PVDF membrane plates (Millipore) coated with inactivated influenza vaccine. Each sample was plated in triplicate at three 3-fold dilutions, and the total viable cell count was 2.22 x 10 per well at a 1:9 dilution. 4 ~1.07×10 5 A range of cells was obtained and used for enumeration of ASCs. Cells were incubated on these membranes and left undisturbed for 5 hours at 37°C. The plates were then washed and treated with horseradish peroxidase-conjugated anti-IgG / IgA / IgM secondary antibodies. After overnight incubation at 4°C, the plates were washed, developed with AEC substrate (BD), washed 20 times with water, dried, and the spots were counted. The frequency of ASCs among total B cells was determined by B cell flow cytometry data analysis and direct cell counting.

[0130] Example 4: Characterization of expression profiles Single-cell RNA-seq analysis was performed with DNA barcoding antibodies using the BD Rhapsody platform to characterize the expression profiles of GC phenotype B cells in organoid cultures. Dimensionality reduction (uniform manifold approximation and projection (UMAP)) was used to analyze GC B cells across different time points (Figure 3B). Overall, GC B cells (CD38+CD27+) from different days and time points clustered together, indicating that their expression profiles were similar. The top overexpressed genes in GC B cells (compared to naive B cells) on day 5 of LAIV-stimulated cultures were related to antibody secretion (immunoglobulin heavy chain, light chain, and J chain) and key B cell differentiation, proliferation, and B cell receptor (BCR) signaling genes (XBP1, POU2AF1, S100A10, and PCNA). These were also the top differentially expressed genes between direct ex vivo GC cells and naive B cells. These data demonstrate that the cultures are capable of functionally responding to antigen-specific stimulation, that light and dark zone phenotype B cells segregate into distinct regions (Figures 3A and 9A), and that B cells with the GC phenotype have similar transcriptional profiles at day 0 and day 5 of culture (Figure 3B), collectively supporting the conclusion that these cultures can be considered organoids.

[0131] Single-cell RNA-seq. Cells from either day 0 tonsils (processed, cryopreserved, and thawed) or organoids (days 5 and 9 after stimulation with LAIV or left unstimulated) were stained with a mixture of fluorophore-conjugated antibodies to allow sorting of CD45+CD19+CD3- B cells and sequence detection of DNA-tagged antibodies (CD20 clone L27, CD19 clone SJ25C1, CD71 clone L01.1, IgM clone G20-127, IgA clone A59, CD161 clone HP-3G10, CD27 clone L128, CD38 clone HIT2, IgD clone IA6-2, IgG clone G18-145, CD83 clone HB15E, CXCR4 clone 12G5, TCR Vg9 clone B3, CD3 UCHT1, CD4 clone SK3, CD8 clone RPA-T8, and CXCR5 clone RF8B2). A cocktail of DNA-tagged antibodies was used to enable manual gating of B cells (CD19+CD3-) and B cell subsets (CD27 and CD38) to identify GC B cells and naive B cells. Sorted cells were tagged with sample barcodes to enable pooling. Cells were loaded and captured from pooled samples using a BD Rhapsody pipeline, following the manufacturer's instructions for library preparation using a targeted human immune gene panel for amplification. Libraries were sequenced using the Illumina Novaseq platform, and the resulting data were processed using the Rhapsody analysis pipeline. Individual samples were debarcoded using SeqGeq (BD) software, and B cell subsets were gated based on DNA-barcoded antibodies for CD3, CD19, CD27, and CD38. Individual populations were then exported along with their gene expression profiles for analysis. UMAP dimensionality reduction was performed using the R package "umap" on gene (not DNA-tagged antibody) expression profiles. For fold change expression analysis, genes that were overexpressed by at least 1.5-fold in GC B cells were plotted.

[0132] Hemagglutinin-specific B cell diversity and maturation. We next examined whether activation-induced cytidine deaminase (AID), required for both somatic hypermutation and class switching, was expressed in these cultures. AID protein levels (Figure 3C, see representative staining in Figure 9B) were significantly increased in pre-GC and GC B cells in 4-day-old organoid cultures (dissemination shown in Figure 3C). BCRs were then single-cell sequenced from high-affinity A / California 2009 H1N1 hemagglutinin (HA)-specific plasmablasts (and other activated B cells) using an HA trimer construct for cultures stimulated with LAIV on days 0 and 7 to assess affinity maturation and isotype switching. As previously reported in peripheral blood plasmablasts from influenza-vaccinated volunteers, immunoglobulin heavy chain variable gene cluster (IGHV) 3 and IGHV 4 dominated the response (Figure 3D; Figures 10A and 10B for the complete data set). HA-specific BCRs from LAIV-stimulated organoids were highly diverse in both gene usage and isotype, with numerous oligoclonal expansions at day 7, but these clones were distinct from those detected in high-affinity HA+ B cells at day 0. Clonally related BCR families were also found in LAIV-stimulated organoids, with a clonal family containing a single amino acid change in one of two donors, demonstrating diversity within the HA-specific lineage. Using bulk BCR sequencing of total non-naive B cells, we found lineages that began as IgM isotypes and class-switched (primarily to IgG1 and IgA1) during organoid culture by day 7, usually with additional mutations (Figure 11).

[0133] B cell receptor sequencing. For isotype switching analysis, tonsillar cells were collected from organoids at day 0 or day 7, washed with FACS buffer, stained with a cocktail of lineage-defining antibodies as described above in the presence of Fc block, and then bulk-sorted using a FACS Fusion or Aria II instrument. Bulk sequencing of immunoglobulin heavy chain gene rearrangements for isotype switching analysis was performed as previously reported. Briefly, RNA was isolated from sorted (memory CD38-CD27+, GC CD38+CD27+, and plasmablast CD38+++CD27+) cells using Trizol (Thermo Fisher Scientific) and reverse-transcribed into cDNA using Superscript II (Life Technologies) primed with random hexamer primers. Amplicons from IgM, IgD, IgG, IgA, and IgE were PCR amplified in separate reactions using IGHV framework region 1 primers and isotype primers for the first constant region exon, and modified to include a partial Illumina linker sequence, sample barcode sequence, and randomized nucleotides to ensure sequence diversity during the initial cycles of sequencing. A second PCR was performed to complete the Illumina linker sequence, followed by amplicon quantification, pooling, gel extraction (Qiagen), and sequencing on an Illumina MiSeq instrument using a kit for 600 cycles with 2 × 300 paired-end reads. Bulk BCR heavy chain sequences were analyzed using an in-house developed pipeline based on IgBLAST for V, D, and J gene segment alignment and CDR-H3 analysis. Clonally related sequences in the bulk sequencing data were identified based on shared use of IGHV and IGHJ genes, equal CDR-H3 lengths, and monophyletic clustering of CDR-H3 nucleotide sequences at a 90% identity threshold.

[0134] For analysis of somatic hypermutation and A / California 2009 HA-specific B cells, tonsillar cells were collected from organoids on day 0 or day 7, washed with FACS buffer, and then treated with 2 μg (4 μg ml) per sample of biotinylated recombinant A / California influenza HA1 hemagglutinin (Y98F mutant, a gift from B. Graham and the Vaccine Research Center) in the presence of Fc block, then washed and stained with 0.2 μg ml of fluorescently labeled streptavidin and a cocktail of lineage-determining antibodies.

[0135] For single-cell sorting, GCs were sorted for HA+ B cell or plasmablast phenotype (CD38+CD27+ or CD38+++CD27+, respectively). Single-cell antibody sequencing was performed as previously described. After single-cell sorting into 96-well plates, cDNA was labeled with well-specific barcode oligonucleotides and pooled per plate, followed by gene-specific PCR and library preparation using previously reported primer sequences. Libraries were sequenced using 2x300 paired-end sequencing on an Illumina MiSeq. Sequence analysis was performed as previously described. Briefly, fastq generation and plate demultiplexing were completed using the built-in MiSeq Generate FASTQ workflow. After quality filtering, paired reads were stitched and separated by well ID, and consensus sequences were determined by clustering well ID reads into operational taxonomic units. Consensus operational taxonomic unit sequences were analyzed using IMGT HighV-QUEST version 1.5.7.1. For single-cell data, clonal families were defined by the use of the same V and J genes for both heavy and light chains and at least 70% amino acid identity at the CDR3 locus. A caveat to sequencing single-cell HA-specific B cells is that the most vigorously responding B cells may lose surface immunoglobulin expression as they convert to antibody secretion, and therefore, the best ASCs may not have been captured during sorting of HA-specific B cells.

[0136] Bulk IgH sequencing and analysis were performed using MIDCIRS as previously described. Sequencing was performed on an Illumina MiSeq using the v3, 600-cycle kit. mRNA molecules were tagged with 12N randomized molecular identifiers (MIDs) during reverse transcription. Reads with the same MID were grouped together and then further clustered into subgroups based on 85% sequence similarity to separate distinct mRNA molecules tagged with the same MID. Consensus sequences were then formed from MID subgroups to average PCR and sequencing errors and reduce amplification and sequencing bias. Clonal lineages were defined using monophyletic clustering of consensus sequences using the same criteria as above. Clonal lineage size refers to the total number of consensus sequences or mRNA molecules comprising the lineage, and lineage diversity refers to the number of unique consensus sequences within the lineage.

[0137] To estimate the HA specificity of unsorted total B cells from day 7 tonsillar cultures, the sequences of all FACS-sorted HA-specific B cells were first grouped together (n = 20,977). All nucleotide sequences were then translated into amino acid sequences, and each day 7 sequence was aligned to the pool of HA+ sequences to find the minimum distance to the nearest HA-specific sequence. If any sequence within a clonal lineage was found to either perfectly match or differ by a single substitution from a known HA-specific sequence, the lineage was labeled "HA putative." Clonal lineages were then divided into HA putative and non-A / California HA for further analysis.

[0138] Example 5: Affinity maturation in tonsillar organoids Affinity maturation is another key function of GCs. To assess this, we performed depletion experiments using FACS to eliminate pre-existing high-affinity HA+ B cells and any non-naive B cells from the tonsillar cell pool. Cultures were then prepared from the depleted cells, stimulated with LAIV, and stained again on day 10 to assess the development of new high-affinity HA+ B cells (Figure 3E and Figure 12). In three of the four donors tested, HA+ B cells reappeared in the depleted cultures, and influenza-specific secreted antibodies were detectable (Figure 3F), demonstrating that affinity maturation can be directly observed in this system.

[0139] Cell depletion experiments. Thawed tonsillar cells were stained and bulk sorted into culture medium using a FACS Aria II or Fusion (BD). Sorting experiments involved separating individual cell subsets using the following markers: myeloid cells and DCs (CD45+CD3-CD19-HLA-DR+, CD116+, or CD33+), pDCs (CD45+CD3-CD19-HLA-DR+CD123+), Treg cells (CD45+CD3+CD19-CD4+CD25+CD1271), total B cells (CD45+CD3-CD19+), non-naive B cells (CD38+ and / or CD27+IgM- and / or IgD-), naive B cells (CD38-CD27-IgM+IgD+), pre-GC B cells (CD38+CD27-), GC B cells (CD38+CD27+), memory B cells (CD38-CD27+), and plasmablasts / plasmocytes (CD38+++CD27+). Cell types were depleted from day 0 tonsillar cells by FACS and cultured with LAIV for 7 days. As a control, depleted cultures were reconstituted with the originally sorted cell types and seeded at the same cell density as the depleted cultures. For depletion of HA-specific B cells, tonsillar samples were stained as described in "BCR Sequencing," and HA-specific B cells were defined as CD45+CD19+CD3-HA+. Post-sorting analysis was used to ensure the purity of the depletion.

[0140] Statistical Analysis. All statistical analyses were performed in R. A paired Wilcoxon signed-rank test (two-tailed) was used to compare paired samples (plasmablast frequency and antibody secretion in unstimulated vs. LAIV-stimulated cultures, standard vs. transwell responses, unstimulated vs. MMR-stimulated cultures, somatic hypermutation analysis, and studies of the effect of CD4+ cell depletion on plasmablast differentiation and specific antibody secretion). An unpaired Mann-Whitney U test (two-tailed) was used to analyze the effect of CD4 depletion in younger vs. older children. A Welch t-test (two-tailed) was used to compare somatic hypermutation levels between culture conditions and the clonal size and diversity of putative HA-specific and non-specific B-cell lineages. A two-tailed, paired t-test was used to compare the effect of PE stimulation with and without adjuvant on the frequency of PE+ B cells.

[0141] Next, we performed IgH sequencing to analyze the BCR repertoire in naive versus affinity-matured B cells in response to LAIV stimulation. Here, we prepared cultures in which naive HA- B cells were the sole B cell source (Figure 4A). Day 0 HA+ B cells were used as a reference for high-affinity BCRs to estimate HA specificity. On day 7, organoid cultures were harvested for bulk BCR repertoire sequencing (Figure 4A). The repertoire of these cultures was compared with both in vivo high-affinity HA+ B cells from day 0 tonsillar cells depleted by FACS and naive HA- B cells that entered the culture preparation. Of the six donors tested, five secreted antibodies specific to the vaccine components, and four had detectable antibodies specific to the A / California 2009 H1N1 HA protein at day 7 after stimulation (Figure 4B), indicating that HA-specific antibodies could be derived from naive B cells.

[0142] Somatic hypermutation significantly increased in cultures stimulated with LAIV on day 7 compared with unstimulated control cultures, and was particularly enhanced when A / California 2009 H1N1 HA antibodies were detected (IMD006, 013, 014, and 102, Figure 4C). The development of BCR sequences likely specific (within one amino acid) to influenza strain A / California H1N1 continued. In two of the six donors tested (IMD006 and IMD014), B cell clones with predicted HA specificity for this strain were also greater than those without known HA specificity, and this depended on vaccine stimulation (Figure 4D). In these same donors, mean diversity also increased, with more unique molecules per clonal lineage in HA-specific B cells from LAIV-stimulated cultures (Figure 4D). Only in vaccine-stimulated cultures were other expanded and diverse lineages found, likely responding to other antigens or strains. Direct evidence for BCR sequence evolution toward increased HA affinity was found (Figure 4E). Here, the root sequence, which harbored four nucleotide changes from the germline heavy chain sequence, acquired an additional 10 mutations to achieve precise amino acid matching with the known high-affinity HA specificity. Taken together, these data support antigen-driven somatic hypermutation, affinity maturation, and class switching in tonsillar organoid cultures.

[0143] Example 6: Contribution of individual cell types to influenza responses A major advantage of in vitro systems is their ability to define essential components. Therefore, we depleted APC, T cell, and B cell subsets and compared plasmablast differentiation to wild-type organoids (Figures 5B and 5D). Despite their low initiation frequency (0.1–0.5% of viable cells), depleting myeloid and plasmacytoid dendritic cell (pDC) populations completely blocked plasmablast differentiation (Figure 5A), and pDC depletion alone was sufficient to dramatically reduce antibody responses to LAIV (Figure 5B). The effects of pDC depletion were rescued by adding type I interferon (IFN), indicating that type I IFN production is a primary pDC function (Figure 13A).

[0144] As expected, depleting CD4+ cells (Figure 13B) significantly reduced plasmablast differentiation, although some influenza-specific antibodies were still detectable (Figures 5C and 5D). The effect of donor age was analyzed, and organoids depleted of CD4+ T cells from younger children were found to be almost completely unable to secrete influenza-specific antibodies (Figure 5D), in contrast to older donors who were able to mount an antibody response.

[0145] Antibody affinity was characterized against A / California / 07 / 2009 H1N1 HA in CD4+-depleted cultures. The dissociation rates of antibodies generated in the absence of CD4+ cells were 4-10 times higher than wild-type controls, even when the amount of specific antibody was comparable between intact and CD4+-depleted cultures (Figure 5F and Figure 13C). Thus, CD4+ T cells are important for both the magnitude and affinity of the antibody response.

[0146] We determined that a minimum of three to four cell types were required to consistently achieve detectable plasmablast differentiation and naive antibody responses to LAIV: naive B cells, APCs, CD4+ T cells, and, in some donors, a mixed population of CD45- stromal cells (Figure 5G). Notably, naive CD4+ T cells were comparable to or superior to memory CD4+ T cells in their ability to help B cells differentiate into plasmablasts and stimulate influenza-specific antibodies (Figure 5H).

[0147] Example 7: Responses to other antigens and the effect of adjuvants We next investigated whether tonsillar organoids could also respond to non-influenza memory antigens. We stimulated tonsillar organoids with the measles, mumps, and rubella (MMR) vaccine, recommended for administration at 12 months of age, and assessed plasmablast formation and antigen-specific IgG responses. These cultures significantly increased plasmablasts compared to controls (Figure 6A), and six of seven donors produced measles-specific IgG, whereas mumps and rubella responses were weaker and were only detected in three of the older children tested (Figure 6B).

[0148] Affinity binding experiments. Binding affinities of the indicated antigens to antibody-containing supernatant samples were determined by biolayer interferometry using an Octet QK instrument (Pall ForteBio). For analysis of the influence of T cells on antibody responses, antibodies were purified from culture supernatants using a Protein G affinity column (GE Healthcare Life Sciences). Full-length, head, and stem domain H1 CA / 09HA antigens were purified using a Ni-NTA affinity column followed by size-exclusion cleanup, as previously described. Purified antigens were captured on an anti-pentaHis (HIS1K) biosensor chip in PBS-T (PBS containing 0.05% Tween 20, pH 7.4). Ligand-bound sensors were immersed in control wells or purified antibody (200–500 nM). Similar antibody concentrations were used for all evaluated conditions from individual donors. Ligand-free sensors immersed in analyte served as controls for nonspecific binding. Traces were processed using ForteBio data analysis software (v8.0). Data were globally fitted to a simple 1:1 Langmuir interaction model to obtain kinetic parameters. Each binding interaction was repeated at least three times.

[0149] To determine whether tonsillar organoids could serve as a platform for priming antigen-specific adaptive responses, cells were first stimulated with the naive antigen phycoerythrin (PE), with and without adjuvant. Here, a substantial (10-fold) increase in PE+ B cells was observed in PE-stimulated cultures compared with unstimulated or irrelevant antigen-stimulated cultures (Figure 6C). The frequency of PE+ B cells was also enhanced by alum hydrogel but not by imiquimod (Figure 6D), indicating that at least some naive responses can be elicited and modulated by an appropriate adjuvant. We then tested the ability of tonsillar, lung-draining lymph node, and spleen cultures to respond to a T cell-dependent rabies vaccine. The donors had not previously received this vaccine. Tonsillar B cells were more differentiated in vaccine-primed cultures after 14 days of organoid culture, with four of ten donors having detectable increases in rabies nucleoprotein-specific IgM (Figure 6E). Lung-draining lymph node and splenic organoids also showed modest rabies-specific antibody production, with some specific IgM (but not IgG) production, consistent with a priming response (Figure 6F).

[0150] Example 8: Testing of vaccine candidates on tonsillar organoids We investigated the ability of tonsillar organoids to respond to a series of vaccine candidates developed in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These vaccine candidates use replication-deficient adenovirus type 5 (Ad5) encoding either the full-length viral spike protein, spike and nucleocapsid proteins, or the S1 spike subunit with nucleocapsid within the Ad5 E1 region. All tonsillar tissue samples were collected before the SARS-CoV-2 pandemic and were therefore naive to these antigens. At day 14 post-stimulation, plasmablast differentiation and significant CD8 T cell activation were observed in a subset of donors compared with unstimulated controls (Figure 6G). Using protein microarrays to detect antibody specificity against SARS-CoV-2, IgG and IgA antibodies specific for several spike and nucleocapsid proteins were present in some donors. One particularly robust responder, IMD163, is shown in Figure 6G (data from all donors is available in Figure 14), demonstrating that immune organoid cultures can be used to analyze and compare vaccine candidates against new and previously encountered antigens.

[0151] Example 9: Autoantibodies generated from FOXP3-KO T cells in tonsillar organoids To identify which T cell population plays a major role in antibody production, specific T cell populations were depleted from tonsillar organoids and then stimulated with either LAIV or LAIV with an autoantigen cocktail (LAIV+A). The autoantigen cocktail included proteinase 3 (PR3), double-stranded DNA (dsDNA), histones, and SNRNP70, which are autoantigens commonly found in patients with autoimmune diseases (e.g., lupus). The secretion of autoantibodies specific to the PR3 autoantigen was then assessed by ELISA (Figure 15). Figure 15 shows that CD4+CD25+ T cells, a subset of regulatory T cells, play a suppressive role in autoantibody production. Depletion of CD4+CD25+ T cells enabled tonsillar organoids to produce large amounts of PR3-specific autoantibodies compared to other conditions and other cell depletions.

[0152] The immune system carefully regulates T and B cell responses to self-antigens while focusing responses to foreign antigens. In this way, the body avoids most self-reactivity, but this can go awry in individuals with autoimmune diseases such as lupus erythematosus, multiple sclerosis, and rheumatoid arthritis, as well as dozens of other diseases in which the immune system attacks specific organs or tissues. A key factor in avoiding self-reactivity is T cells expressing the transcription factor FoxP3, known as regulatory T cells. Because of their crucial role in preventing self-reactivity, for example, mice and humans lacking FoxP3 die in childhood from massive inflammation resulting from the lack of these T cells.

[0153] To investigate the function of FOXP3 in T cells, T cells were first isolated from tonsillar organoids using a pan T cell isolation kit (Miltenyi Biotec) according to the manufacturer's protocol. Next, FOXP3 knockout was performed using a CRISPR / Cas9 gene editing kit (Lonza). Lonza P3 electroporation buffer was allowed to reach room temperature. Tonsillar medium was warmed to 37°C, and several culture plates containing medium were also preheated. HIFI-Cas9 was diluted to 40 μM in Lonza P3 electroporation buffer. Ribonucleoprotein (RNP) complexes were prepared by slowly mixing 40 μM FOXP3 gRNA and 40 μM HIFI-Cas9 in a 1:1 volume ratio to generate 20 μM Cas9-RNP (gRNA-Cas9 complex). The RNP complexes were incubated at 37°C for 15 minutes. When experiments were performed with 100,000 to 1 million cells, 16-well strips were prepared by resuspending 3.5 μL of RNP complex per well in 20 μL of Lonza P3 electroporation buffer per well. Next, 21 μL of the mixture was transferred to the electroporation well, and the 4D Nucleofector System (Lonza) was used for electroporation of unstimulated primary T cells using the manufacturer's electroporation protocol. Immediately after electroporation, 80 μL of prewarmed tonsillar medium was added to the 16-well strip, and the cells were incubated in the electroporation plate at 37°C in a 5% CO2 incubator for 30 minutes before transferring the cells to a preheated culture plate. When experiments were performed with 1 million to 20 million cells, cuvettes were prepared by resuspending 17.5 μL of RNP complex per well in 100 μL of Lonza P3 electroporation buffer per well. Next, 115 μL of the mixture was transferred to an electroporation cuvette and the 4D Nucleofector System (Lonza) was run on unstimulated primary T cells using the manufacturer's electroporation protocol.Immediately after electroporation, 900 μL of warmed tonsillar medium was added to the cuvette, and the cells were incubated in the electroporation plate at 37°C in a 5% CO2 incubator for 30 minutes before being transferred to a preheated culture plate. The results of FOXP3 KO in T cells were confirmed using FACS (data from Figures 16A-16F). These KO cells were cultured and stimulated with either LAIV or LAIV with an autoantigen cocktail (LAIV+A). The secretion of autoantibodies against each autoantigen in the cocktail was measured using ELISA (data from Figure 17). The results show that upon stimulation with LAIV+A, B cells interacting with FOXP3-KO T cells secrete large amounts of autoantibodies specific to each autoantigen, including PR3, dsDNA, histones, and SNRNP70.

[0154] The results demonstrate that CRISPR-mediated elimination of FOXP3 gene expression in T cells in the tonsillar organoids of the present invention enables B cells to produce and secrete autoantibodies that can mature. This does not occur when the FOXP3 gene is intact and functional. These results suggest that simple elimination of the FOXP3 gene makes it possible to utilize the immune organoid system to produce fully human antibodies against any antigen, self or non-self. There is a natural limit to antibody affinity, typically in the low nanomolar range (1-10 nM). However, individuals with defects in the AIRE gene, an autoimmune regulatory gene, can exhibit antibody affinity in the femtomolar range, much higher than normal. This suggests that the affinity of antibodies generated by the present invention is much higher than that of normal antibodies. The results herein demonstrate that this is also a mode of suppression by FoxP3-mediated T cell regulation.

[0155] Example 10. Identification of tonsillar and circulating CD4+ and CD8+ regulatory T cell subsets in donor-matched samples FOXP3+CD4+ regulatory T (Treg) cells and CD8+KIR+ T cells are important for maintaining immune tolerance. To determine the percentages of the two Treg subsets in tonsils and to examine whether their percentages in tonsils are similar to those in blood, the two regulatory T cell subsets were compared in matched peripheral blood and tonsil samples from seven adult donors.

[0156] Human studies Ethics approval was granted by the Stanford University IRB. For child volunteers (IRB protocol 30837), written informed consent was obtained from the legal guardians of children aged 0–17 years and from children aged 7 years and older. For adult volunteers (IRB protocol 60741), written informed consent was obtained and 20 ml of blood was collected from each patient. Subjects taking systemic immunomodulatory medications, having a history of immunosuppressive or autoimmune disease, or having a serious active infection at the time of the procedure were excluded.

[0157] Sample collection and processing Tonsil samples were collected and processed as previously described (PMID: 33432170). Briefly, tonsils were surgically removed for various clinical conditions (e.g., ENT patients undergoing tonsillectomy for sleep apnea and / or cardiothoracic patients undergoing thymectomy). All surgical procedures were performed in accordance with the Stanford University Institutional Review Board (IRB). Whole tonsils were collected in saline after surgery and decontaminated by immersion in an antibacterial bath of Ham's F12 medium (Gibco) containing Normocin (InvivoGen), penicillin, and streptomycin for 1 hour at 4°C. Tonsil tissue was cut into small pieces (approximately 5 mm thick) using a scalpel and scissors and manually disrupted into a single-cell suspension by processing through a 100 μm strainer using a syringe plunger. After washing with complete medium (RPMI containing Glutamax, 10% FBS, 1x non-essential amino acids, 1x sodium pyruvate, 1x penicillin-streptomycin, 1x Normocin (InvivoGen), and 1x insulin / selenium / transferrin cocktail (Gibco)), cells were frozen in aliquots in FBS + 10% DMSO and stored at −140°C until use.

[0158] Flow cytometry Cultured organoids were resuspended by rinsing the membrane with medium and collected from the transwell. Cells were washed with FACS buffer (PBS + 0.1% BSA, 0.05% sodium azide, and 2 mM EDTA) and stained with Fc block (1 / 20), live / dead Aqua Zombie stain (1 / 100), and anti-human antibody at 4°C. For FOXP3 staining, after surface staining, cells were fixed and permeabilized (eBioscience) and intracellularly stained with anti-FOXP3. All analytical data were collected on a BD LSRII instrument and analyzed using FlowJo (TreeStar).

[0159] Multiparameter flow cytometry-based characterization of CD4+ and CD8+ T cell populations in peripheral blood mononuclear cells (PBMCs) and tonsil-derived mononuclear cells was performed (Figure 19A). The frequencies of total CD4+ Tregs and CD4+ Tregs expressing CXCR5 were significantly higher in tonsils compared with blood (Figure 19A). Circulating CD4+ Tregs typically express high surface levels of CD25, which serves as an additional marker. However, nearly half of the tonsil CD4+ Treg cell population expressed low levels of CD25, and the percentage of CD25 low FOXP3+ CD4+ T cells was significantly higher compared with blood (Figure 19B). The percentage of CD8+ T cells expressing KIR was comparable between tonsils and blood.

[0160] As shown in Figure 19A, CD4 + T cells were analyzed by FACS for CD25, FOXP3, and CXCR5 expression. CD4 T cells from donors (N=7) in PBMC and tonsil samples were analyzed. + FOXP3 in T cells + cells, Tfr(CXCR5 + FOXP3 + ), CD25+FOXP3 + The percentages of KIR+CD8+ T cells are shown in Figure 19B.

[0161] Example 11: Inflammatory and autoimmune cells in lymphoid cell cultures with T cells genetically modified to lack FoxP3 or Granzyme B (GZMB) expression Next, knockout experiments were performed to investigate the contribution of ablation of the FOXP3 and GZMB genes in T cells to the phenotype of whole tonsillar organoids.

[0162] Cas9 ribonucleoprotein (RNP) assembly and electroporation Cas9 RNP was prepared immediately prior to the experiment by incubating 20 μM Cas9 with 20 μM sgRNA at a 1:1 ratio for 15 minutes at 37°C to a final concentration of 10 μM. T cells were electroporated using a Neon transfection kit and device (Invitrogen) according to the manufacturer's instructions. Briefly, T cells were gently resuspended at 2 million cells per 20 μl in P3 buffer (Lonza Bioscience) with supplements. The Cas9 RNP and T cells were then gently mixed in P3 buffer. This mixture was then transferred to a 4D-Nucleofector cuvette (Lonza Bioscience) and pulsed with code EH105. After electroporation, the 4D-Nucleofector cuvette was placed in a tissue culture incubator at 37°C for 30 minutes to allow cells to recover. After recovery, the cells were ready for culture.

[0163] cell culture For culturing of cryopreserved cells, thaw an aliquot into complete medium, count, and for larger cultures, 6 x 10 cells per ml. 7 cells per ml for smaller cultures, 2 x 10 7 Cells were resuspended to a concentration of 100 μl per well onto permeable (0.4 μm pore size) membranes (PTFE or polycarbonate membranes in 24-well sizes in standard 12-well plates, or 96-well polycarbonate membrane plates with single-well receiver trays, Corning or Millipore), with the lower chamber consisting of complete medium (1 ml for 12-well plates, 200 μl for 96-well plates) supplemented with 1 μg ml recombinant human B cell activating factor (BAFF, BioLegend) and 1 ng / ml IL-21.

[0164] Figure 20A shows the activation of CD4 and CD8 T cells (CD27 + CD38+ Figure 20B shows the fold change frequency of activated T in control, FOXP3 KO, and GZMB KO tonsillar organoids after 10 days of culture. FH Representative FACS staining of B cell differentiation phenotypes in control, FOXP3 KO, and GZMB KO tonsillar organoids after 10 days of culture is shown in Figure 20C.

[0165] After 10 days of culture, FOXP3 and GZMB KO tonsillar organoids significantly increased expression of the activation marker CD38 and the costimulatory molecule CD27 (Figure 20A), as well as CD38+ T follicular helper (T FH ) cells (Figure 20B). In addition, KO tonsillar organoids showed B cell activation as a decreased percentage of naive B cells and an increased percentage of germinal center B cells and plasmablast B cells compared to controls (Figures 20C and 20D). Note that compared to FOXP3 KO tonsillar organoids, GZMB KO tonsillar organoids showed significantly higher levels of inflammation accompanied by greater T cell and B cell activation (Figure 20D). However, increased autoantibody production was not detected in FOXP3 or GZMB KO amygdala organoids compared to controls.

[0166] Example 12: FOXP3 KO and GZMB KO tonsillar organoids exhibited differential autoimmune phenotypes after LAIV and autoantigen stimulation. Viral infection has been implicated as a major factor in the initiation of autoimmune disease, and mouse models also developed an autoimmune phenotype upon viral infection. To determine whether autoimmune responses in germinal centers could be induced by viral antigens, control, FOXP3 KO, or GZMB KO tonsillar organoids were either left unstimulated (NS) or stimulated with live attenuated influenza virus (LAIV) after seeding in transwells. In addition, LAIV and an autoantigen cocktail (LAIV+A) were tested to determine whether this could further increase autoantibodies. The autoantigen cocktail includes proteinase 3 (PR3), core histones, double-stranded DNA (dsDNA), and small nuclear ribonucleoproteins (snRNPs), which are relatively common autoantigens targeted by autoantibodies found in patients with autoimmune disease and viral infection.

[0167] cell culture Cell culture was performed as described in the previous example. LAIV (1 μl per well, 1.6 × 10 per strain) was then added. 4 ~1.6×10 5 FluMist Quadrivalent (Medimmune, equivalent to 1000 fluorescent focus units) was added directly to the cell-containing portion of the culture setup. Cultures were incubated at 37°C in 5% CO2 with humidity, and the lower wells were replenished with additional medium as needed.

[0168] Antibody detection by ELISA For detection of influenza-specific antibodies, ELISA plates (Costar) were coated with 0.1 μg per well of 2021-2022 Fluzone Quadrivalent Inactivated Influenza Vaccine (Sanofi). For detection of autoantigen-specific antibodies, ELISA plates (Costar) were coated with 0.1 μg per well of proteinase 3 (PR3), small nuclear ribonucleoprotein (SnRNP), core histone, and dsDNA as capture antigens. The plates were coated with the capture antigen overnight and then blocked with blocking reagent for 2 hours. Cell supernatant from tonsil cultures was then added to the coated and blocked plates. After washing with wash buffer, a horseradish peroxidase-conjugated anti-human secondary antibody against IgM / IgG / IgA (Sigma) was added to the plates for 1 hour, followed by the addition of TMB substrate solution (Thermo Scientific). The reaction was stopped by adding sulfuric acid, and the plates were read at 450 nm.

[0169] As shown in Figures 21A and 21B, FOXP3 KO and GZMB KO tonsillar organoids developed differential autoimmune phenotypes after stimulation with LAIV and autoantigens. Figure 21A shows the fold change in the amount of autoantibodies specific for proteinase 3 (PR3), double-stranded DNA (dsDNA), small nuclear ribonucleoprotein 70 kDa (snRNP70), and core histones from control, FOXP3 KO, and GzmB KO tonsillar organoids. Figure 21B shows the fold change in the amount of autoantibodies specific for PB cells (CD27) from control, FOXP3 KO, and GzmB KO tonsillar organoids after 10 days of culture, with no stimulation (NS), stimulation with LAIV, and stimulation with LAIV plus autoantigen (LAIV+A). ++ CD38 ++ B cells), activated CD4 + and CD8 + T cells (CD38 + CD27 +) are shown. It is important to note that tonsillar organoids depleted of GZMB KO or CD8+KIR+ T cells produced some autoantibodies upon stimulation, but had significantly higher percentages of activated T cells and plasmablasts than FOXP3 KO and controls (Figure 21B).

[0170] Example 13: FOXP3 KO tonsillar organoids stimulated with LAIV produced high affinity HA antibodies. Because FOXP3 is important in regulating autoantibody responses, we investigated whether FOXP3 might also affect antigen-specific antibody responses, including antibody quality, by measuring the binding affinity of antigen-specific antibodies produced from FOXP3 KO tonsillar organoids stimulated with LAIV. We focused on antibodies against hemagglutinin (H1), the primary antibody target in vaccinated individuals.

[0171] Measurement of binding affinity The binding affinity of full-length H1 California / 04 / 2009 influenza hemagglutinin (CA / 09HA) to antibodies secreted into the culture medium of the indicated human tonsillar organoids was measured using biolayer interferometry (BLI) with an Octet QK instrument (Pall ForteBio, CA, USA). Antigen (H1 CA / 09HA) diluted in PBST (PBS containing 0.05% Tween 20, pH 7.4) was captured using a Ni-NTA biosensor. The ligand-bound biosensor was immersed in serially diluted culture supernatant. Both association and dissociation were monitored for 1 h. Double referencing was performed using an unliganded biosensor and an unrelated E. coli maltose-binding protein (MBP). The dissociation rate constant (k d ) was determined by global fitting of exponential decay kinetics. Each binding interaction was performed in duplicate.

[0172] Live attenuated influenza vaccine (LAIV) was used as an adjuvant in FoxP3 and granzyme B KO, allowing measurement of the resulting bulk anti-influenza HA affinity using interferometry. As shown in Figure 22, in most donors (n=9), there was a significant increase in affinity with FoxP3 KO, with a significant decrease in off-rate (k d ) was reduced by up to 18-fold, and in some donors, the effect of GzmB KO was more modest.

[0173] The dissociation rate constant (Kd) of antibodies against H1 CA / 09 HA in the FOXP3 KO condition was on average 4-fold lower than that in the donor-matched wild-type condition. The results in Figure 22 demonstrate that high-affinity HA antibodies can be generated by defeating the regulatory checkpoint by knocking out FOXP3, suggesting an efficient strategy for manipulating the binding affinity of antigen-specific antibodies using a genetically modified tonsillar system.

[0174] Overall, these results demonstrate that FOXP3 KO tonsillar T cells produced autoantibodies when stimulated with a panel of classical autoantigens, whereas GZMB KO cells exhibited a significant increase in autoreactive CD8+ T cells and plasmablasts but only low levels of autoantibodies. CD8+ and CD4+ Tregs have distinct and complementary roles in controlling cellular and humoral responses and preventing autoimmunity. Importantly, this genetically modified tonsillar system enables in vitro modeling of autoreactive immune cell responses, which remains a key obstacle for understanding the mechanisms of autoimmunity in humans. These results also demonstrate increased autoantibody production in FOXP3 KO cells with defective CD4+ Tregs and increased follicular helper T cells and autoreactive CD8+ T cells in GZMB KO cells with defective CD8+ Tregs.

[0175] While embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. An in vitro cell cluster comprising lymphoid cells, comprising: the in vitro cell clusters are derived from lymphoid tissue of a subject, the in vitro cell clusters comprise germinal centers adapted to produce antibodies against an antigen, and the germinal centers comprise genetically modified T cells. In vitro cell clusters.

2. The in vitro cell cluster of claim 1 , wherein the antigen is an autoantigen.

3. 2. The in vitro cell cluster of claim 1, wherein the antigen is selected from the group consisting of a polysaccharide, a lipid, a nucleic acid, a peptide, a protein, or a fragment thereof, and any combination thereof.

4. The in vitro cell cluster of claim 3 , wherein the protein is a viral protein, a bacterial protein, a growth factor, a cancer-associated protein, or an autoimmune disease-associated protein.

5. The in vitro cell cluster of claim 1 , wherein the antigen is expressed by a tissue of the subject.

6. The in vitro cell cluster of claim 1 , wherein the antigen is a vaccine or vaccine candidate.

7. 2. The in vitro cell cluster of claim 1, wherein the genetically modified T cells comprise regulatory T cells.

8. 2. The in vitro cell cluster of claim 1, wherein the genetically modified T cells are modified to knock down or knock out expression of a forkhead box transcription factor.

9. 9. The in vitro cell cluster of claim 8, wherein the forkhead box transcription factor is FoxP3.

10. The genetically modified T cells are CD8 ＋ The in vitro cell cluster of claim 1 , comprising T cells. Claim 11. The CD8 ＋ The in vitro cell cluster of claim 10, wherein the T cells are modified to knock down or knock out expression of granzyme B.

12. The in vitro cell cluster of claim 1 further comprising one or more adjuvants.

13. 13. The in vitro cell cluster of claim 12, wherein the one or more adjuvants comprise aluminum hydroxide or imiquimod.

14. 2. The in vitro cell cluster of claim 1, wherein the germinal centers are configured to perform one or more of hypermutation maturation, affinity maturation, plasmablast differentiation, and class switching recombination.

15. The in vitro cell cluster of claim 1 , wherein the lymphoid cells are derived from tonsil tissue, spleen tissue, adenoid tissue, thymus tissue, or lymph node tissue.

16. The in vitro cell cluster of claim 1 , wherein the germinal center comprises antigen-presenting cells (APCs) and T cells that at least partially surround the germinal center.

17. 17. The in vitro cell cluster of claim 16, wherein the APC comprises B cells, dendritic cells, or both.

18. 2. The in vitro cell cluster of claim 1, comprising CD38+ B cells, CD27+ B cells, CD8+ T cells, CD4+ T cells, or a combination thereof.

19. The in vitro cell cluster of claim 1 , wherein the lymphoid cells are human cells.

20. 1. A method for producing antibodies from lymphoid organoids, comprising: (a) disposing lymphoid cells in a culture medium to produce lymphoid organoids, wherein the lymphoid organoids comprise T cells and B cells, and the T cells are modified to have reduced control of B cells compared to unmodified T cells; (b) introducing an antigen into the medium; (c) incubating the lymphoid organoids with the antigen to produce the antibody; (d) isolating the antibody or a nucleic acid encoding the antibody from the lymphoid organoid. A method comprising:

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