Antigen-immune modulator (AIM) therapy platform for psoriasis and other autoimmune diseases

Abstract

A method for preparing an immune cell sample from a subject having an autoimmune disorder is provided, the method comprising obtaining a tissue sample from the subject and isolating a single immune cell in situ from the tissue sample using laser capture microdissection.

Description

[Background technology]

[0001] [Cross-references to related applications] This application claims priority to U.S. Provisional Patent Application No. 63 / 044,586, filed on 26 June 2020. The entire contents of the aforementioned application are incorporated herein by reference.

[0002] Autoimmune diseases are a global problem. For example, psoriasis is a chronic autoimmune disease that primarily affects the skin and often the joints, with an estimated global prevalence of 2-3%, or about 125 million people. Furthermore, the incidence of psoriasis is increasing. In retrospective cohorts of adults, the incidence of psoriasis was approximately 50.8 cases per 100,000 people between 1970 and 1974, and approximately 100.5 cases per 100,000 people between 1995 and 1999. Regardless of its prevalence, the identity of the autoinvasive immune cells and hypothetical target autoantigens useful for identifying and / or treating psoriasis remains unknown. [Overview of the Initiative]

[0003] A method is provided for preparing an immune cell sample from a subject with psoriasis, comprising the steps of: obtaining a psoriasis tissue sample; and isolating a single immune cell in situ from the psoriasis tissue sample using laser-captured microdissection. The isolated single immune cell may be an unmanipulated single immune cell. The technique may include one or more of laser-captured microdissection (e.g., immunoguided laser-captured microdissection), micromanipulation, vacuum pulse-assisted techniques, immunomagnetic cell separation, density gradient centrifugation, sedimentation, adhesion, aptamer, buoyancy-activated cell sorting, fluorescence-activated cell sorting (FACS), and microfluidics.

[0004] In some embodiments, the psoriasis tissue sample is approximately 5 μm to 15 μm thick. In some embodiments, the psoriasis tissue sample is obtained by horizontally cutting a biopsy specimen from a psoriasis lesion. In some embodiments, the psoriasis tissue sample is obtained by mixing biopsy specimens obtained from a psoriasis lesion in a microblender.

[0005] Some embodiments include (a) compressing a psoriasis tissue sample and (b) smearing the compressed sample onto a glass slide. In some embodiments, the compression is performed using a flat, round solid glass head.

[0006] In some embodiments, psoriasis tissue samples are obtained by vertically cutting a biopsy specimen taken from a psoriasis lesion.

[0007] Some embodiments include (a) freezing a psoriasis tissue sample and (b) sectioning the frozen sample. In some embodiments, freezing includes placing the specimen on dry ice. In some embodiments, freezing is performed using liquid nitrogen vapor or isopentane.

[0008] In some embodiments, the tissue sample is embedded in an embedding substrate before sectioning. In some embodiments, the embedding is performed before freezing the sample. In some embodiments, the embedding substrate is tragacanth or optimal cutting temperature (OTC) medium. In some embodiments, sectioning is performed using a microtome or a cryogenic bath.

[0009] In some embodiments, a psoriasis tissue sample is obtained by cutting a biopsy specimen taken from a psoriasis lesion, the method further comprising (a) flash freezing the sample, (b) crushing the frozen sample into fragments, (c) thawing the fragments, (d) vertically crushing one or more of the thawed fragments and horizontally pressing the thawed fragments, and then (e) smearing the fragments onto a glass slide.

[0010] Some embodiments include culturing isolated immune cells in a growth medium.

[0011] Some embodiments involve detecting T cell surface markers, transcription factors, cytokines, or combinations thereof associated with isolated immune cells. Some embodiments involve detecting T cell surface markers, transcription factors, cytokines, or combinations thereof associated with one or more cultured immune cells. In some embodiments, detection is performed via flow cytometry, deep sequencing, spectrophotometrics, or enzyme-linked immunosorbent assay.

[0012] Some embodiments further include performing an antigen challenge on cultured immune cells. In some embodiments, the antigen challenge involves contacting immune cells with an antigen and then detecting T cell surface markers, transcription factors, cytokines, or combinations thereof associated with one or more cultured or isolated immune cells (e.g., unmanipulated single immune cells). In some embodiments, the antigen is keratin, melanocyte ADAMTSL5, or antimicrobial peptide LL37. In some embodiments, the antigen is isolated from a skin tissue sample obtained from a subject.

[0013] Some embodiments include (a) homogenizing and fractionating a skin tissue sample; (b) performing an antigen challenge on the homogenized and fractionated skin tissue sample; (c) detecting a fraction that induces T cell stimulation based on the presence or absence of T cell surface markers, transcription factors, cytokines, newly synthesized DNA, ATP, or combinations thereof; (d) isolating polypeptides in the detected fraction by performing three-dimensional gel chromatography; (e) performing an antigen challenge on the isolated polypeptides; and (f) detecting isolated polypeptides that induce T cell stimulation based on the presence or absence of T cell surface markers, transcription factors, cytokines, newly synthesized DNA, ATP, or combinations thereof.

[0014] Some embodiments include detecting, in a peripheral blood sample obtained from a subject, the presence or absence of (i) one or more immune cells having detected T cell surface markers and / or transcription factors, and / or (ii) isolated polypeptides that induce T cell stimulation.

[0015] Some embodiments include synthesizing one or more polypeptides that induce T cell stimulation. Some embodiments include administering one or more polypeptides to a subject having psoriasis.

[0016] Furthermore, a method is provided for preparing immune cell samples from subjects with autoimmune disorders, comprising the steps of: obtaining a tissue sample from the subject; and isolating single immune cells in situ from the tissue sample using laser-captured microdissection or vacuum pulse-assisted techniques. A method is also provided for preparing immune cell samples from subjects with psoriasis, comprising the steps of: obtaining a psoriasis tissue sample; and isolating single immune cells in situ from the psoriasis tissue sample using techniques that do not substantially alter the cellular biochemical profile of the immune cells. The isolated single immune cells may be unmanipulated single immune cells. Techniques may include one or more of laser-captured microdissection (e.g., immunoguided laser-captured microdissection), micromanipulation, vacuum pulse-assisted techniques, immunomagnetic cell separation, density gradient centrifugation, sedimentation, adhesion, aptamer, buoyancy-activated cell sorting, fluorescence-activated cell sorting (FACS), and microfluidics.

[0017] In some aspects, autoimmune disorders include psoriasis, type 1 diabetes mellitus, rheumatoid arthritis, or multiple sclerosis.

[0018] In some embodiments, the tissue sample is approximately 5 μm to 15 μm thick. In some embodiments, the tissue sample is obtained by horizontally cutting a biopsy specimen from the affected tissue. In some embodiments, the tissue sample is obtained by mixing the biopsy specimen obtained from the affected tissue in a microblender. Some embodiments further include (a) compressing the tissue sample and (b) smearing the compressed sample onto a glass slide. In some embodiments, the compression is performed using a flat, round solid glass head. In some embodiments, the tissue sample is obtained by vertically cutting a biopsy specimen obtained from the affected tissue.

[0019] Some embodiments further include (a) freezing the tissue sample and (b) sectioning the frozen sample. In some embodiments, freezing includes placing the specimen on dry ice. In some embodiments, freezing is performed using liquid nitrogen vapor or isopentane. Some embodiments further include embedding the tissue sample in an embedding substrate before sectioning the sample. In some embodiments, embedding is performed before freezing the sample. In some embodiments, the embedding substrate is tragacanth or optimal cutting temperature (OTC) medium. In some embodiments, sectioning is performed using a microtome or a cryogenic bath.

[0020] In some embodiments, the tissue sample is obtained by cutting a biopsy specimen taken from the affected tissue, and the method further comprises (a) flash freezing the tissue sample, (b) crushing the frozen sample into fragments, (c) thawing the fragments, (d) vertically crushing one or more of the thawed fragments and horizontally pressing one or more of the thawed fragments, and then (e) smearing the fragments onto a glass slide.

[0021] Some embodiments further include culturing isolated immune cells in a growth medium.

[0022] Some embodiments further include detecting T cell surface markers, transcription factors, cytokines, newly synthesized DNA, ATP, or combinations thereof associated with isolated immune cells. Some embodiments further include detecting T cell surface markers, transcription factors, cytokines, or combinations thereof associated with one or more of the cultured immune cells. In some embodiments, detection is performed via flow cytometry, deep sequencing, spectrophotometrics, or enzyme-linked immunosorbent assay.

[0023] Some embodiments further include identifying a primary antigen that stimulates isolated immune cells. In some embodiments, the identifying step includes culturing the isolated immune cells to generate cultured immune cells and performing an antigen challenge on the cultured immune cells. In some embodiments, the antigen challenge includes contacting the immune cells with an antigen and then detecting a T cell surface marker, transcription factor, cytokine, or a combination thereof associated with one or more of the cultured immune cells or the isolated immune cells (e.g., an unmanipulated single immune cell). In some embodiments, the antigen is isolated from a tissue sample obtained from a subject.

[0024] Some embodiments further include (a) homogenizing and fractionating a tissue sample, (b) performing an antigen challenge on the homogenized and fractionated tissue sample, (c) detecting a fraction that induces T cell stimulation based on the presence or absence of a T cell surface marker, transcription factor, cytokine, newly synthesized DNA, ATP, or a combination thereof, (d) isolating a polypeptide in the detected fraction by performing three-dimensional gel chromatography, (e) performing an antigen challenge on the isolated polypeptide, and (f) detecting an isolated polypeptide that induces T cell stimulation based on the presence or absence of a T cell surface marker, transcription factor, cytokine, or a combination thereof.

[0025] Some embodiments further include detecting, in a peripheral blood sample obtained from a subject, the presence or absence of (i) one or more immune cells having a detected T cell surface marker and / or transcription factor, and / or (ii) an isolated polypeptide that induces T cell stimulation.

[0026] Some embodiments further include synthesizing one or more of the polypeptides that induce T cell stimulation.

[0027] Also provided is a method of treating a subject having an autoimmune disorder, the method comprising administering to the subject a therapeutically effective amount of the identified primary antigen.

[0028] Some embodiments further include culturing immune cells in a growth medium that induces the formation of regulatory immune cells. Some embodiments include a method for treating a subject having an autoimmune disorder, the method comprising administering a therapeutically effective amount of the induced regulatory immune cells to the subject. [Modes for carrying out the invention]

[0029] In this specification, the singular forms "a," "an," and "the" include both singular and plural forms unless otherwise clearly indicated by the context.

[0030] A description of a numerical range by its terminology includes all numbers and fractions contained within that range, as well as the terminology itself.

[0031] As used herein, the term “about” is intended to include variations of or from a specified value when referring to measurable values ​​such as parameters, quantities, time durations, and the like, such as variations within ±10% of the specified value, unless specifically identified to mean other variations, such as variations within +1 to 5%, ±1%, or ±0.1% of the specified value or from it. The value to which the “about” modifier refers is also understood to be specifically and preferably disclosed in itself.

[0032] The terms “quantity,” “amount,” and “level” are synonymous and are understood to be generally known in the art. These terms may particularly refer to the absolute quantification of a marker in a tested object (e.g., in or on cells, cell populations, tissues, or organs in a biological sample of interest), or the relative quantification of a marker in a tested object, i.e., relative to another value (e.g., relative to a reference value), or a range of values ​​indicating a baseline for the marker. For example, a baseline or reference value may be obtained based on the determination of the quantity, level, or amount of a marker in a subject (or in cells or cell populations from a subject) that has an autoimmune disorder or does not have one or more symptoms thereof. Such values ​​or ranges may be obtained as conventionally known. In some cases, quantity, amount, or level is a measured concentration. Amount can be quantified using well-known techniques, such as PCR, UV absorption, calorimetry, fluorescence-based measurement, diphenylamine reaction, and others.

[0033] This paper describes specific immunomodulatory platforms for diagnosing and / or treating autoimmune diseases such as psoriasis. While not theoretically bound, autoimmune disorders, including psoriasis, are thought to be caused by a loss of self-tolerance to specific autoantigens. These autoantigens trigger autoimmune processes that target organs specific to that particular autoantigen. For example, in type 1 diabetes mellitus, the autoantigen is located in the beta cells of the pancreas that produce insulin; in psoriasis, the autoantigen is located in the skin; and in autoimmune thyroiditis, the antigen is located in the thyroid gland.

[0034] Psoriasis is an autoimmune disease with multiple phenotypes, including plaque (also known as psoriasis vulgaris), guttate, intertriginous, pustular, and erythrodermic forms. Psoriasis has a strong genetic component located in the HLA region. One such locus is psoriasis susceptibility locus 1 (PSORS1). The strongest HLA association in psoriasis is located at HLA-C*O6 in the Caucasian population. The disease is characterized by remissions and acute exacerbations. The prevalence of psoriasis among first-degree relatives is high, i.e., about 4–19% higher than in the general population.

[0035] The pathophysiology of psoriasis is characterized by abnormal proliferation of corneal stromal cells and infiltration of immune cells in the epidermis and dermis. Although not constrained by theory, it is thought that abnormal recognition and presentation of hypothetical autoantigens triggers an autoinvasive T cell population, including Th1 and Th17 cells. The disease can be maintained and exacerbated by pro-inflammatory cytokines (e.g., TNF-alpha, IL-17, IL-22 and 23, CCL20). Epithelial damage can further accelerate skin destruction by triggering antimicrobial peptides such as CAMP, which then mediate plasmacytoid dendritic cell (pDC) activity.

[0036] This paper describes methods to replicate the process by which healthy individuals acquire self-tolerance to their body's immune system. For example, in healthy individuals, immune cells circulate in the infant's thymus from early life, where autoantigens from different organs are expressed. Some immune cells (specific to certain antigens) are eliminated. Some immune cells are not eliminated but remain suppressed by regulatory immune cells that are also specific to these autoantigens. Autoimmunity can occur when these regulatory cells lose control over the immune cells that have not been eliminated. Therefore, some embodiments involve introducing autoantigens as targets to stimulate regulatory cells to regain control and re-establish a healthy immune balance.

[0037] An "antigen" is a structural substance, such as a protein or polypeptide, that is recognized by the immune system and functions as a target for an immune response. A "self-antigen" or "autoantigen" is an antigen of biological origin that is not normally recognized by the immune system of that organism, but can become a target of immune attack and cause autoimmune diseases.

[0038] Against the above background, methods and compositions for identifying T cells and / or antigens in subjects having an autoimmune disorder are provided. In some aspects, the subject has been diagnosed with an autoimmune disorder. In some aspects, the subject is at risk of developing an autoimmune disorder. In some aspects, the autoimmune disorder is psoriasis. In some aspects, the autoimmune disorder is rheumatoid arthritis. In some aspects, the autoimmune disorder is type 1 diabetes mellitus. In some aspects, the autoimmune disorder is multiple sclerosis.

[0039] Methods and compositions for treating or preventing autoimmune disorders in subjects are also provided. Method for preparing tissue samples

[0040] A novel and inventive method is provided for preparing immune cell samples from subjects with autoimmune disorders such as psoriasis. Such a method can be used, for example, to identify immune cells present in the tissue of a subject.

[0041] Some embodiments involve obtaining tissue samples from a subject. The tissue may be any tissue affected by an autoimmune disease, such as skin, pancreatic tissue, cerebrospinal fluid, or synovial fluid. For example, the tissue of a subject with psoriasis may be skin. Exemplary psoriatic tissue includes psoriatic plaques or lesions, red, scaly skin with pustules, erythematous patches, red, shiny lesions, and / or scales. Tissue from a subject with multiple sclerosis may be cerebrospinal fluid (e.g., obtained from a cerebrospinal fluid aspiration). Tissue from a subject with type 1 diabetes mellitus may contain pancreatic cells. Tissue from a subject with rheumatoid arthritis may be synovial fluid.

[0042] In some embodiments, tissue samples are obtained during an autoimmune outbreak (e.g., an acute exacerbation of psoriasis). In some embodiments, tissue samples are obtained during remission or partial remission of an autoimmune disease (e.g., psoriasis). In some embodiments, tissue samples are isolated from serial biopsies obtained during the course of the symptoms and provide information, for example, about the dynamic changes in the autoimmune process and its self-regulatory recovery process.

[0043] Tissue samples can be obtained by any of the following methods. For example, tissue samples can be obtained directly from the subject or from a biopsy specimen taken from the subject. Exemplary types of biopsies include shave biopsies, punch biopsies, incision biopsies, and excision biopsies. In some embodiments, a biopsy is obtained by using a scalpel to remove part or all of the affected tissue from the subject. In some embodiments, individual cells (e.g., from cerebrospinal fluid or synovial fluid) can be collected from a solution, for example, by using a microscope with a micromanipulation device.

[0044] In some embodiments, the tissue sample is obtained by horizontally cutting (e.g., parallel to the skin layer) a biopsy specimen taken from the tissue of interest (e.g., a psoriatic lesion). In some embodiments, the tissue sample is obtained by vertically cutting a biopsy specimen taken from the tissue of interest. Such cutting can be performed by any known means, such as a scalpel. In some embodiments, the tissue sample is obtained by mixing the biopsy specimen taken from the tissue of interest in a microblender.

[0045] In some embodiments, tissue samples obtained from biopsy specimens are further processed. In some embodiments, the further processing does not involve chopping or digesting the tissue samples. In some embodiments, the tissue samples may be compressed (e.g., manually or using a mechanically assisted process and a hard surface, using constant or variable pressure to flatten the tissue vertically), crushed (beaten or compressed so that the tissue separates into smaller, thinner pieces), pressed (moved through a given diameter to separate and / or thin the tissue), stretched, smeared, frozen, or any combination thereof.

[0046] In some embodiments, tissue samples are compressed manually or mechanically, for example, using a flat, round solid glass head, or using a mortar and pestle, or using a press or crusher. For example, the head may be rotated and pressed downwards and sideways to compress the sample mechanically. In some embodiments, the compressed sample may be smeared onto a glass slide and then used for microdissection of the sample.

[0047] In some embodiments, tissue samples are frozen using, for example, flash freezing or snap freezing techniques. Exemplary freezing techniques include exposure to dry ice, liquid nitrogen vapor, or isopentane. In some embodiments, tissue samples are cryoprotected before freezing (for example, by exposing the tissue samples to a cryoprotective agent such as 4% paraformaldehyde and / or sucrose). Preferably, the tissue samples are frozen without the development of ice crystals in the cells of the sample.

[0048] Some embodiments involve fragmenting a frozen tissue sample. Such fragmentation can be achieved, for example, by crushing the frozen tissue sample into fragments, either manually, via mechanical compression, or using a microblender. In some embodiments, the fragmented tissue sample is thawed and then further compressed or pressed (for example, by vertically crushing the thawed fragments and / or pressing them horizontally). In some embodiments, the fragmented tissue sample is smeared onto a glass slide.

[0049] In some embodiments, tissue samples are embedded in a substrate. The substrate may be rubber or adhesive. Exemplary substrates include poly[N-(2-hydroxypropyl)methacrylamide], 10% gelatin, carboxymethylcellulose, tragacanth, and combinations thereof. In some embodiments, the substrate is in an optimal cutting temperature (OCT) medium. Exemplary OCT mediums include, for example, a water-soluble mixture of glycol and resin that provides a matrix for sectioning tissue samples. See, for example, FISHER HEALTHCARE® TISSUE-PLUS® OCT Compound, or TISSUE-TEK® OCT Compound (SAKURA® Finetek). In some embodiments, frozen tissue samples are embedded in the substrate. In some embodiments, fresh tissue samples are embedded in the substrate. In some embodiments, fragmented tissue samples are embedded in the substrate.

[0050] Some embodiments involve sectioning (e.g., freeze-sectioning) or slicing the tissue sample. For example, the tissue sample may be sectioned using a blade or knife. In some embodiments, the tissue sample is sectioned using a microtome. In some embodiments, the tissue sample is sectioned using a cryobath.

[0051] The smeared or sectioned tissue sample may be of any thickness suitable for microdissection. For example, in some embodiments, the smeared or sectioned tissue sample is about 5 to 15 microns thick, such as about 8 to 10 microns thick. In some embodiments, the smeared or sectioned tissue sample is about 5 to 15 microns thick, such as 8 to 10 microns thick. In some embodiments, the smeared or sectioned tissue sample has a thickness of about 5 microns, about 6 microns, about 7 microns, about 8 microns, about 9 microns, about 10 microns, about 11 microns, about 12 microns, about 13 microns, about 14 microns, or about 15 microns. In some embodiments, the smeared or sectioned tissue sample has a thickness of 5 microns, 6 microns, 7 microns, 8 microns, 9 microns, 10 microns, 11 microns, 12 microns, 13 microns, 14 microns, or 15 microns. In some embodiments, the smeared or sectioned tissue sample contains a monolayer of cells. In some embodiments, a portion of the smeared or sectioned tissue sample contains a monolayer of cells.

[0052] In some embodiments, smeared or sectioned tissue samples are placed on a slide (e.g., a glass slide) or membrane for microdissection. Isolation of immune cells

[0053] Furthermore, methods for isolating one or more immune cells from a tissue sample are provided. The term “immune cells” refers to, but is not limited to, cells of the innate or adaptive immune system, including lymphocytes such as T cells and B cells (including any cell of the B cell lineage), naive cells, memory cells, antigen-presenting cells (APCs), dendritic cells, monocytes, macrophages, natural killer (NK) cells, mast cells, basophils, eosinophils or neutrophils, and any progenitor cells of such cells. In one preferred embodiment, the immune cells may be T cells. As used herein, the term “T cell” (i.e., T lymphocyte) is intended to include all cells in the T cell lineage, including thymocytes, immature T cells, mature T cells, and the like. The term “T cell” refers to CD4 + and / or CD8 + T cells, T helper (T h ) cells, for example, T h 1. T h 2 and T h 17 cells, T regulatory (T reg This may include ) cells, NKT cells (including variant and / or invariant cells), and gamma T cells.

[0054] In some embodiments, immune cells are isolated from tissues obtained during an autoimmune outbreak (e.g., acute exacerbation of psoriasis). In some embodiments, immune cells are isolated from tissues obtained during remission or partial remission of an autoimmune disease (e.g., psoriasis). In some embodiments, immune cells are isolated from serial biopsies obtained during the course of the disease, providing information, for example, on the dynamic changes in the autoimmune process and its self-regulatory restoration process.

[0055] In some embodiments, immune cells are isolated from smeared tissue samples. In some embodiments, immune cells are isolated from sectioned tissue samples. Smeared or sectioned tissue samples may be obtained according to the methods described above.

[0056] Immune cells can be isolated using microdissection techniques such as laser capture microdissection. Laser capture microdissection is described, for example, in Datta et al., "Laser capture microdissection: Big data from small samples," Histol Histopathol., 30(11): 1255-1269 (2015), which is incorporated herein by reference in its entirety. Briefly, laser capture microdissection allows for the isolation of cells of interest from tissue samples using an inverted light microscope (with or without a fluorescence module) equipped with a laser to facilitate cell visualization and acquisition. Some embodiments utilize infrared laser capture microdissection. Some embodiments utilize ultraviolet laser capture microdissection. In some specific embodiments, laser capture microdissection is immunoguided laser capture microdissection. Immunoguided laser capture microdissection allows for the use of immunophenotype to identify and isolate target cells from tissue samples, in addition to morphology and tissue location, by combining immunostaining with laser capture microdissection. This technique employs immunohistochemistry or immunofluorescence to guide the dissection process for isolating cells expressing specific molecular markers, and may be particularly useful when a particular cell population is not recognized by tissue staining.

[0057] After the cells of interest have been separated from the surrounding tissue, they may be extracted from a tissue sample. In some embodiments, the cells of interest are transferred from the slide using non-contact methods such as gravity (e.g., gravity-assisted microdissection), pressure catapult (e.g., laser pressure catapult), or laser-guided forward transfer. In some embodiments, the cells of interest are transferred from the slide by pressing an adhesive surface onto the cells and peeling them off the slide. In some embodiments, the cells are extracted using a thermal transfer process that attaches the cells of interest to a membrane without adhering to the surrounding tissue. In some embodiments, the cells are extracted using vacuum pulse-assisted techniques that can enable manual or automated single-cell isolation using microdissection instruments such as UnipicK®, UnipicK+®, and A-picK® (NeuroInDx, Inc.™).

[0058] In some embodiments, immune cells can be isolated by micromanipulation. Micromanipulation, a form of manual cell collection, is a cell isolation technique involving the use of an inverted microscope and ultrathin glass capillaries connected to an aspiration and release unit. The system moves through a motorized mechanical stage, allowing the operator to carefully select specific cells and apply aspiration via a micropipette to aspirate and isolate the cells. In certain embodiments, micromanipulation can be performed using vacuum pulse-assisted techniques that enable manual or automated single-cell isolation using microdissection instruments such as UnipicK®, UnipicK+®, and A-picK® (NeuroInDx, Inc.™).

[0059] In some embodiments, immune cells can be isolated using immunomagnetocellular separation. Immunomagnetocellular separation is a technique in which magnetic particles are used to isolate target cells from a heterogeneous mixture. To achieve this, the magnetic particles are bound to specific cell surface proteins on the target cells via antibodies, enzymes, lectins, or streptavidins. The sample is then placed in an electromagnetic field that pulls the magnetic particles, and labeled cells are carried along with the magnetic particles. Unlabeled cells remain in the tissue sample.

[0060] In some embodiments, immune cells can be isolated using density gradient centrifugation (e.g., in a fluid tissue sample). Density gradient centrifugation takes advantage of the density variation of cells in a heterogeneous sample. The sample is layered at the top of a density gradient medium before centrifugation. During centrifugation, each cell type settles to its isodensity point, which is the location in the medium gradient where the density of the cells and the medium are equal.

[0061] In some embodiments, immune cells can be isolated using sedimentation. Sedimentation works on the basis that, due to gravity, larger and denser elements settle faster than smaller and less dense substances. The largest and densest elements in a sample, due to their high sedimentation rate, can be pelleted through initial low-load centrifugation. The supernatant can then be swirled again. As continuous centrifugation progresses, elements with lower sedimentation rates can be isolated. Leukocytes are generally separated from erythrocytes by dextran sedimentation.

[0062] In some embodiments, immune cells can be isolated using adhesion. The unique adhesion profiles of different cell types can be used to separate target cells from heterogeneous populations in fluid samples. Adherent cells can be isolated from cells in suspension by selectively enabling or inhibiting adhesion through the selection of suitable growth factors and cell culture plates. For example, macrophages are inherently adherent and are often isolated from peripheral blood and bone marrow by adhesion. Mononuclear cells can be cultured with serum and differentiation-inducing cocktails to promote the formation of an adherent monolayer of macrophages. Macrophages can be isolated after removing the supernatant containing unwanted cells. Alternatively, cells that spontaneously grow in suspension or have lost anchorage dependence can be isolated by culturing heterogeneous cell populations in plates designed for ultra-low adhesion. Since there is no surface to adhere to, adherent cells cannot survive, and target cells remain in suspension.

[0063] In some embodiments, immune cells can be isolated using aptamers. Aptamers are single-stranded RNA or DNA oligonucleotides that form structures capable of binding to highly specific targets. Through the systematic evolution of ligands by exponential enrichment (SELEX) technique, aptamers can be screened and synthesized to target any cell type. SELEX is an experimental procedure that allows for the extraction of oligomers with desired binding affinity to a given molecular target from an initially random pool of oligonucleotides. These aptamers have high affinity and specificity for their targets and can be labeled with fluorescent dyes or magnetic particles to facilitate cell separation. Aptamers are nonimmunogenic.

[0064] In some embodiments, immune cells can be isolated by buoyancy-activated cell sorting. Buoyancy-activated cell sorting is a cell separation technique that typically utilizes glass microbubbles labeled with antibodies specific to target cells. When the microbubbles are mixed into the sample, they bind to the target cells. Due to enhanced buoyancy, the microbubbles float to the surface, separating the target cells.

[0065] In some embodiments, immune cells can be isolated by fluorescence-activated cell sorting (FACS). FACS is a method for sorting heterogeneous mixtures of cells using flow cytometry and fluorescent probes. Antibodies tagged with fluorescent dye molecules bind to epitopes on specific antigens on target cells in a single-cell suspension. After tagging, flow cytometry concentrates the cell suspension into a homogeneous flow of single cells. This flow then passes through a set of lasers that excite the fluorescent dye molecules bound to the cells, resulting in light scattering and fluorescence emission. Based on the wavelengths generated by the laser excitation, the resulting photon signals are converted into a proportional number of electron pulses that assign charge to droplets formed around the cells. As each droplet falls between deflection plates, its charge causes it to either be deflected into a collection tube or fall into a waste chamber. In some implementations, FACS can be used to isolate single cells from enriched samples of isolated cells, including samples prepared by other means disclosed herein (e.g., immunomagnetic cell separation, density gradient centrifugation, sedimentation, adhesion, etc.).

[0066] In some aspects, immune cells can be isolated using microfluidics. Microfluidics manipulates fluids at a microscopic level to facilitate single-cell isolation. Microfluidics techniques are frequently built on microchips and are commonly known as "lab-on-a-chip" devices. These devices have several advantages, including smaller sample and reagent volumes required for use. Lab-on-a-chip devices are also portable and particularly useful as field-based diagnostic tools. Microfluidics methods can be divided into active and passive systems. Active microfluidics systems involve external forces, while passive microfluidics utilizes cell density and mass in combination with gravity. These methods can also be classified by the presence or absence of cell labeling. There are several different microfluidics methods used for cell isolation. These include, for example, acoustic phoresis, aqueous two-phase systems, biomimetic microfluidics, cell affinity chromatography, deterministic transverse displacement, electrophoretic sorting, field flow fractionation, gravity and sedimentation, magnetophoresis, microfiltration, and photosorting. In some implementations, microfluidics may be used to isolate single cells from a sample enriched with isolated cells, including samples prepared by other means disclosed herein (e.g., immunomagnetic cell separation, density gradient centrifugation, sedimentation, adhesion, etc.).

[0067] In some embodiments, the cells of interest are preferably isolated and extracted from a tissue sample in a manner that protects cell viability. In some embodiments, the cells of interest are preferably isolated and extracted from a tissue in a manner that does not alter, or substantially does not alter, the cell biochemical profile (e.g., proteome, genome, and / or transcriptome). For example, the cells of interest can be isolated and extracted from a tissue in a manner such that the cells remain unmanipulated (e.g., an unmanipulated single immune cell can be isolated and extracted from a tissue). The isolation and extraction methods can, for example, substantially protect cell surface receptors present on the cells. Some techniques for extracting cells from a tissue sample, such as those involving tissue digestants and / or embedding agents (e.g., paraffin or electron microscopy resin) that do not protect the cells within the tissue, can prevent the ability to profile individually isolated cells, can alter the cell biochemical profile of the cells within the tissue sample, and / or can kill the cells, preventing the ability to characterize an in vitro cell response to a stimulus associated with identifying an autoantigen as described elsewhere herein. In some embodiments, immune cells (e.g., T cells) can be isolated from a blood sample and challenged with an antigen identified from a tissue sample as described elsewhere herein. However, performing an antigen challenge on immune cells isolated and extracted from a relevant tissue sample (e.g., a tissue that experiences immune cell infiltration during an autoimmune reaction) can be advantageous in providing a higher concentration or proportion / frequency of immune cells specific to an autoantigen of interest.

[0068] In some embodiments, the cells of interest are cultured in a growth medium, for example, to generate colonies of cells. In some embodiments, the culture medium is a complete medium that provides IL-12 stimulation.

[0069] In some embodiments, the cells of interest are T reg cells, or T regCells are cultured in a growth medium that induces the formation of active cells. In some embodiments, cells of interest are cultured in a growth medium that induces Foxp3 expression. In some embodiments, cells are cultured in a growth medium containing one or more of TGF-β, IL-10, IL-4, and IL-35. Some embodiments are cultured by the methods described in Chen et al., J. Experimental Medicine, 198(12): 1875-1886 (2003); Cao et al., J. Am. Soc. Nephrol., 21: 933-942 (2010); or Collison et al., Nat. Immunol., 11(12): 1093-1101 (2010). reg This includes the conversion to cells, and each disclosure thereof is incorporated herein in whole. immune cell identification

[0070] Methods are also provided for characterizing isolated immune cells (e.g., single immune cells) or colonies of cells cultured in growth medium. Such characterization may include, but is not limited to, the analysis of the genomics, transcriptomics, proteomics, and / or metabolomics of immune cells. Some embodiments include determining the expression levels of surface markers, transcription factors, cytokines, signal transducers, or combinations thereof associated with immune cells. "Expression" refers to the process by which polynucleotides are transcribed from a DNA template (to mRNA or other RNA transcripts, etc.), and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Thus, "expression level" may refer to the amount of nucleic acid (e.g., mRNA) or protein in a sample.

[0071] In some embodiments, immune cells are T cells. Exemplary T cell surface markers include T cell receptors (TCRs), CD3, CD4, CD8, CXCR3, CCR4, CD25, CD127, FoxP3, CD19 / 20, CD196 (CCR6), CD197 (CCR7), CD62L, CD123, CD80 / 86, CD69, and CD45RO / RA. Exemplary T cell transcription factors include Aiolos, ATM, BATF, Bcl-6, Blimp-1, FOXP3, GATA3, Helios, Ikaros, IRF4, IRF7, Ki-67, NF-κB p65, p53, PCNA, SSRP1, STAT1, STAT4, T-bet, TCL1, Th-POK, and ZAP-70. Exemplary T cell cytokines include INF-γ, IL-17, TNF-α, IL-6, IL-4, IL-10, TGF-β, IL-12, IL-13, IL-2, and IL-23. Exemplary T cell signaling transducers include JAK1 / 3, PI3K, and others.

[0072] Surface markers, transcription factors, cytokines, and signal transducers can be identified by any preferred means. For example, some embodiments include using flow cytometry to identify surface markers, transcription factors, and / or cytokines. Some embodiments include using spectrophotometric methods. Some embodiments include using enzyme-linked immunosorbent assays. Some embodiments include using deep sequencing. Some embodiments include performing RNA-Seq or single-cell RNA-Seq. Some embodiments include performing single-cell genome (DNA) sequencing. Some embodiments include using tetramer or dextramer technology, e.g., DCODE® DEXTRAMER®, which can provide insights into antigen specificity, T cell receptor sequencing, and / or genomic profiling.

[0073] Some embodiments involve the identification of T cell receptors (TCRs) associated with isolated T cells. More specifically, TCRα and / or β chains may be characterized using antibodies and / or RT-PCR.

[0074] In some embodiments, immune cells are identified from an autoimmune outbreak (e.g., acute exacerbation of psoriasis). In some embodiments, immune cells are isolated from tissues obtained during remission or partial remission of an autoimmune disease (e.g., psoriasis). In some embodiments, immune cells are isolated from serial biopsies obtained during the course of the disease, providing information, for example, about the dynamic changes in the autoimmune process and its self-regulatory recovery process. For example, CD25hi+ / CD127 low, FoxP3+ population frequencies, and the ratio of naive and memory T cell populations are characterized in serial biopsies, providing such information about the dynamic changes in the autoimmune process and its self-regulatory recovery process. Antigen challenge and identification

[0075] Methods for performing antigen challenges on cultured immune cells are also provided. In some embodiments, cultured immune cells are brought into contact with an antigen, and the effect of exposing the immune cells to the antigen is determined by measuring, for example, the expression levels of surface markers associated with the immune cells, transcription factors, newly synthesized DNA (measured by, for example, the uptake of -3H-thymidine), ATP, cytokines, cellular signaling transducers, or combinations thereof. Antigen challenges are disclosed, for example, in Ridgway et al., "Following Antigen Challenge, T Cells Up-Regulate Cell Surface Expression of CD4 In Vitro and In Vivo," J. Immunol., 161: 714-720 (1998), and Brinke et al., "Monitoring T-Cell Responses in Translational Studies: Optimization of Dye-Based Proliferation Assay for Evaluation of Antigen-Specific Responses," Fontiers Immunol., 8: 1870 (2017), both of which are incorporated herein by reference in their entirety. Some embodiments include predicting T cell antigen specificity using a thorough analysis of TCRs, such as described in Fischer et al., "Predicting antigen-specificity of single T-cells based on TCR CDR3 regions," buiRxiv (Aug. 13, 2019), which is incorporated herein by reference in its entirety.

[0076] In some embodiments (e.g., in the case of psoriasis), the antigens used in the antigen challenge are corneal stromal cells (e.g., keratin), melanocytes (e.g., ADAMTSL5), antimicrobial peptides (e.g., LL37), any post-translational modification variant of the above, any fragment of the above, and / or any combination thereof. In some embodiments (e.g., in the case of multiple sclerosis), the antigens used in the antigen challenge are myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) peptide, alpha-beta-crystallin, S100 beta, DM20 isoform of proteolipidoprotein (PLP) myelin-associated antigen (MAG), myelin-associated oligodendrocyte basic protein (MOBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), any post-translational modification variant of the above, any fragment of the above, and / or any combination thereof. In some embodiments (for example, in the case of type 1 diabetes mellitus), the antigens used in the antigen challenge are preproinsulin, proinsulin, insulin A chain, insulin B chain, insulin C chain, GAD65, ICA512 / Ia2, HSP60, carboxypeptidase H, peripherin, ganglioside, any post-translational modification variant of the above, any fragment of the above, and / or any combination thereof. In some embodiments (for example, in the case of rheumatoid arthritis), the antigens used in the antigen challenge are collagen type II, proteoglycans, human chondrocyte glycoprotein 39, heat shock proteins (HSPs) (such as BiP), post-translational modification proteins (such as citrullinated filaggrin), ubiquitous proteins (such as glucose-6-phosphate isomerase), p205, N-acetylglucosamine-6-sulfate sulfatase (GNS), filamin (FLNA), any post-translational modification variant of the above, any fragment of the above, and / or any combination thereof.

[0077] In some embodiments, antigens are isolated from tissue samples obtained from subjects with autoimmune diseases. Such antigens may be isolated from tissues (e.g., psoriasis tissue) obtained from subjects, for example, via biopsy. In some embodiments, the tissue (e.g., psoriasis tissue) is etched and / or digested. In other embodiments, the tissue (e.g., psoriasis tissue) is not etched and / or digested.

[0078] Some embodiments include homogenizing the tissue. Some embodiments include fractionating the tissue. Some embodiments include performing an antigen challenge on the homogenized and fractionated tissue. Some embodiments include detecting the fraction that induces T cell stimulation in the antigen challenge. Some embodiments further include fractionating the tissue to identify the least reactive fraction. Some embodiments include isolating polypeptides (e.g., proteins) from one or more fractions that induce T cell stimulation. Such isolation may be performed using chromatographic techniques such as three-dimensional chromatography. Some embodiments include isolating polypeptides based on properties such as size, charge, and / or hydrophobicity. Some embodiments further include using mass spectrometry to isolate polypeptides, such as those described in Duong et al, "Review of Three-Dimensional Liquid Chromatography Platforms for Bottom-Up Proteomics," Int'l. J. Molecular Sci., 21(1524): 1-19 (2020), which is incorporated herein in whole by reference. Some embodiments include performing an antigen challenge on one or more isolated polypeptides. Some embodiments include identifying isolated polypeptides that induce T cell stimulation. Autoimmune detection / treatment

[0079] Methods for detecting T cells and / or antigens in subjects having or suspected of having an autoimmune disorder (such as psoriasis) are also provided. Some embodiments include detecting T cells and / or antigens in tissue (e.g., skin tissue) obtained from the subject. Some embodiments include detecting T cells and / or antigens in blood samples (e.g., peripheral blood samples) obtained from the subject. Some embodiments include identifying a subject having T cells and / or antigens as having an autoimmune disorder (e.g., psoriasis). Some embodiments include diagnosing a subject having an autoimmune disorder based on the presence of T cells and / or antigens. The antigen may be an autoantigen identified from a tissue sample according to any of the methods described elsewhere in this specification, and / or the T cell may be an antigen-specific T cell for an autoantigen identified from a tissue sample according to any one of the methods described elsewhere in this specification. Some embodiments include determining the stage of the autoimmune disorder (e.g., acute exacerbation, remission, partial remission, etc.) based on the presence of T cells and / or antigens. Some embodiments include determining the likelihood that the subject will respond favorably to treatment.

[0080] Methods for treating (including preventive treatment) subjects identified as having or being at risk of developing an autoimmune disorder are also provided. Treatment includes any kind of measure that benefits patients suffering from or at risk of developing an autoimmune disorder or its symptoms. Such benefits may include slowing, controlling, reversing, or halting the progression of one or more of the symptoms of the disorder and / or the disease process itself. Such treatments may include, but are not required, the complete elimination of all symptoms or a cure for the disorder.

[0081] Some embodiments involve administering a therapeutically effective amount of antigen, such as an antigen identified according to the methods described above, to a subject in need. In some embodiments, the administration of the antigen provides beneficial changes to regulatory immune cells and / or downregulates a pathological immune response. Some embodiments involve isolating or synthesizing the antigen for administration to a subject. In some embodiments, the antigen is administered using an adjuvant and / or a pharmaceutically acceptable vehicle. In various embodiments, the antigen may be administered according to either the method described in U.S. Patent Application Publication No. 2016 / 0361397, published December 15, 2016, by Orban et al., which is incorporated in whole herein by reference, or a general formulation.

[0082] In some aspects, T reg Cell, or T reg This includes administering to cells that have activity. In some embodiments, T reg Cell, or T reg Cells that have activity are T reg The cells of interest are produced by culturing cells of interest in a growth medium that induces the formation of active cells. In some embodiments, the administered cells express Foxp3. In some embodiments, the administered cells are produced by culturing cells of interest in a growth medium containing one or more of TGF-β, IL-10, IL-4, and IL-35.

[0083] The following examples are included to illustrate the compositions and methods described herein. These examples are not intended to limit the scope of the invention. Other embodiments will be apparent to those skilled in the art. example Example 1: Isolation of immune cells

[0084] Unprocessed, unmanipulated immune cells are isolated from target organ lesions. Instead of dissecting and digesting skin tissue to isolate immune cells, laser single-cell capture, or laser capture microdissection (LCM), is used. While not constrained by theory, such techniques are thought to allow access to unmodified immune cells in solid tissue samples for laser capture without degrading the extracellular matrix. For example, compared to skin biopsies, which are typically 6-8 mm thick, the tissue for performing LCM is approximately 5-15 microns thick.

[0085] Tissue or unmodified cells are obtained from sites affected by autoimmune diseases (e.g., affected tissues such as synovial fluid or biopsy in rheumatoid arthritis, cerebrospinal fluid or brain biopsy in multiple sclerosis, pancreatic biopsy in type 1 diabetes mellitus, or skin biopsy in psoriasis). Isolated cells are placed on glass slides, or alternatively, tissues are prepared according to one of the techniques described below.

[0086] SAS method (disintegration and smearing): Using a sharp scalpel for microdissection, a fresh tissue sample is cut horizontally (i.e., parallel to the skin layer) from the psoriatic lesion as thinly as possible (intact portions of the dermis and adipose tissue are discarded). Alternatively, a microblender may be used to break down the psoriatic lesion into small pieces. The sample is then disintegrated using a flat, round solid glass head, by rotating the head and pressing downwards and sideways, further mechanically compressing the sample on a hard surface (each thin section of sample is disintegrated separately). Using a glass slide with a sharp edge, each disintegrated section is smeared onto the glass slide.

[0087] FAST Method #1: (Flash Freezing and Slicing, Thawing): Using a sharp scalpel for microdissection, a fresh tissue sample is cut vertically (i.e., across the skin layer) from the psoriatic lesion as thinly as possible (intact tissue, i.e., fat, etc., is cut). The slices are placed on dry ice and can be stored in a sterile container at -80°C or can be used immediately. Although not constrained by theory, it is thought that the cells are frozen without the development of ice crystals in the cells. The slices are then embedded (e.g., in a tragacanth), and a microtome / crisp is used to slice the specimen to the desired thickness (e.g., 8-10 microns).

[0088] FAST Method #2: (Flash Freezing and Slicing, Thawing): Using a sharp scalpel for microdissection, a fresh tissue sample is cut vertically (i.e., across the skin layer) from the psoriatic lesion as thinly as possible. The specimen is flash-frozen (e.g., using dry ice, or liquid nitrogen vapor (not submerged), or isopentane). Although not constrained by theory, it is believed that the cells are frozen without the development of ice crystals in the cells. Alternatively, a fresh specimen is used without freezing. In either case, OCT (Optimal Cutting Temperature) medium (containing glycerol and resin) is used to embed the specimen, and a microtome / cryotub is used to slice the specimen to the desired thickness (e.g., 8-10 microns).

[0089] FAST and SAS Combo Method: A sharp scalpel is used for microdissection to cut fresh tissue samples from psoriatic lesions as small pieces. The specimens are flash-frozen (e.g., using dry ice, or liquid nitrogen vapor (not submerged), or isopentane, or dimethyl sulfoxide (DMSO)). Although not constrained by theory, it is believed that the cells are frozen without the development of ice crystals in the cells. The frozen specimens are fragmented into the smallest possible fragments. The fragments are then thawed, and each fragment is again fragmented vertically and pressed horizontally. The samples are then smeared onto glass slides. Example 2: Identification and characterization of infiltrating immune cells in psoriasis plaques or other autoimmune disease target organs.

[0090] Clones are propagated from isolated single immune cells and characterized by flow cytometry to provide the full spectrum of T cells in lesions, elucidating, for example, the role of CD4+ T cells in psoriasis. Furthermore, the following surface markers, namely CD8, CD4, CD3, CD25, CD127, Foxp3, CD19 / 20, CD45RO / RA, CD62L, CD123, and CD80 / 86, are characterized. Serial biopsies (e.g., during partial remission and / or acute exacerbation) are monitored, and the T cell spectrum is characterized in the serial biopsies. The frequency of CD25hi+ / CD127low and FoxP3+ populations, as well as the ratio of naive to memory T cell populations, are also characterized in the serial biopsies to provide information on the dynamic changes in the autoimmune process and its self-regulatory recovery process. Additionally, or alternatively, genetic analysis (e.g., thorough TCR analysis), immunophenotyping (e.g., using cell surface markers), and / or proteomics and eicosanoid analysis (e.g., leukotrienes (LTB4, etc.)) are performed on immune cells. Example 3: Target antigen identification and testing

[0091] T cell clones from psoriatic lesions are used to "fish out" their corresponding autoantigens in cell culture tests. The antigenic response to T cells is characterized along with the T cell cytokine profile (e.g., INF-gamma, IL-17, TNF-α, IL-6, IL-4, IL-10, TGF-beta, and IL-13).

[0092] In cases of psoriasis, antigen challenges are performed using one or more of three virtual antigens: keratin, melanocyte ADAMTSL5, and the antimicrobial peptide LL37. Additional antigens are identified using relevant patient-derived skin tissue. More specifically, the skin tissue is homogenized and fractionated, and fractions that induce significant T-cell stimuli are further fractionated. The least reactive fraction is separated by 3D gel chromatography, and T-cell clones are tested against isolated proteins / peptides to identify antigens that induce a T-cell response. Identified antigens are cloned and sequenced.

[0093] In cases of type 1 diabetes mellitus, an antigen challenge is performed using one or more of the following: preproinsulin, proinsulin, insulin A chain, insulin B chain, insulin C chain, GAD65, ICA512 / Ia2, HSP60, carboxypeptidase H, peripherin, ganglioside, and immunoactive fragments, or any variant of the above (including any post-translational modified derivatives). Additional antigens are identified using relevant patient-derived pancreatic tissue.

[0094] In cases of rheumatoid arthritis, an antigen challenge is performed using collagen type II, proteoglycans, human chondrocyte glycoprotein 39, heat shock proteins (HSPs), post-translational modified proteins (such as citrullinated filaggrin), ubiquitous proteins (such as glucose-6-phosphate isomerase or p205), HSPs secreted during stress (such as BiP), N-acetylglucosamine-6-sulfatase (GNS), filamin (FLNA), and post-translational modified variants, and / or one or more of any of the above fragments. Additional antigens are identified using the affected joints of the patient themselves.

[0095] In cases of multiple sclerosis, antigen challenges are performed using one or more of the following: myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) peptide, alpha-beta-crystallin, S100 beta, DM20 isoform of proteolipidoprotein (PLP), myelin-associated antigen (MAG), myelin-associated oligodendrocyte basic protein (MOBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), and post-translational modification variants, and / or any of the above fragments. Additional antigens are identified using the affected cerebrospinal fluid or brain tissue of the relevant patient. Example 4: Identification of peripheral blood immune cell responses to antigens

[0096] Peripheral blood samples are collected from the subject around tissue samples taken at the same time (e.g., skin biopsy, synovial fluid or biopsy, cerebrospinal fluid or brain biopsy, pancreatic biopsy), and findings from the tissue samples are correlated with the patient's own blood samples. More specifically, the blood samples are characterized to determine the presence or absence of T cells and / or their associated antigens identified from the target organ (e.g., skin biopsy). Given that the presence of such cells and / or antigens in the target organ is confirmed, such elements may be identifiable in the blood, even if they are present at a low frequency.

[0097] Given the heterogeneity associated with autoimmune disorders (i.e., the possibility of different groups of patients having different autoantigens that respond to autoimmunity), blood tests may be used to develop personalized immunomodulatory therapies based on autoantigens associated with the pathological response. Blood tests may also be used as a diagnostic tool in subjects at risk or in subjects in the early stages of the disease (e.g., early or atypical disease symptoms). Example 5: Autoimmune cell biomarkers in psoriasis patients to validate markers and measure the level of activity and / or progression of autoimmune destruction over time.

[0098] Disease-specific and / or antigen-specific memory T cells may be used to assess the level of autoimmune activity or disease progression. While not constrained by theory, it is thought that the overall increase in memory T cells is due to the autoimmune process activating naive T cells, which then become disease-specific T cells, thus increasing the memory T cell pool. Therefore, the number of naive cells relative to newly identified disease-specific memory cells may be used to determine disease progression, providing a much stronger signal with increased accuracy, sensitivity, and specificity compared to using the entire memory cell pool in the formula, especially in the early stages of the autoimmune process. For example, to determine that the marker can predict the severity of psoriasis and its impending acute exacerbation, an autoimmune cell marker for diagnosing the presence of autoimmunity, disclosed in Orban's U.S. Patent Application Publication No. 2019 / 0137483, published on 9 May 2019, which is incorporated herein by reference in its entirety, is tested. More specifically, flow cytometry analysis is performed on blood samples from the subject to identify phenotypic markers. Using fluorescence-activated cell sorting (FACS), CD4+ cells are detected using anti-CD4 antibodies labeled with fluorescent dye molecules. For the co-detection of CD45RO, specific anti-CD45RO antibodies with different fluorescent dye molecules are also used. (Fluorescently labeled anti-CD4 and anti-CD45RO antibodies are commercially available from various sources, such as BD Bioscience in San Jose, California.) Each fluorescent dye molecule has characteristic peak excitation and emission wavelengths, and therefore, they can be distinguished by using fluorescence-activated cell sorting instruments such as the Becton-Dickinson FACSCALIBUR® or FACSARIA® systems. Against the background of single-cell analysis, the number of naive cells relative to identified memory cells may change over time (e.g., when cells are detected at different points in time). Example 6: Autoimmune cell biomarkers in patients with autoimmune disorders to validate markers and measure the level of activity and / or progression of autoimmune destruction over time.

[0099] Disease-specific and / or antigen-specific memory T cells may be used to assess the level of autoimmune activity or disease progression. While not constrained by theory, it is thought that the overall increase in memory T cells is due to the autoimmune process activating naive T cells, which then become disease-specific T cells, thus increasing the memory T cell pool. Therefore, the number of naive cells relative to newly identified disease-specific memory cells may be used to determine disease progression, providing a much stronger signal with increased accuracy, sensitivity, and specificity compared to using the entire memory cell pool in the formula, especially in the early stages of the autoimmune process. For example, to determine that the marker can predict the severity of an autoimmune disorder (such as rheumatoid arthritis, multiple sclerosis, or type 1 diabetes mellitus) and its impending acute exacerbation, an autoimmune cell marker for diagnosing the presence of autoimmunity, disclosed in Orban's U.S. Patent Application Publication No. 2019 / 0137483, published on 9 May 2019, which is incorporated herein by reference in its entirety, is tested. More specifically, flow cytometry analysis is performed on blood samples from subjects to identify phenotypic markers. Using fluorescence-activated cell sorting (FACS), CD4+ cells are detected using anti-CD4 antibodies labeled with fluorescent dye molecules. For the co-detection of CD45RO, specific anti-CD45RO antibodies with different fluorescent dye molecules are also used. (Fluorescently labeled anti-CD4 and anti-CD45RO antibodies are commercially available from various sources, such as BD Bioscience in San Jose, California.) Each fluorescent dye molecule has characteristic peak excitation and emission wavelengths, and therefore, they can be distinguished by using fluorescence-activated cell sorting instruments such as the Becton-Dickinson FACSCALIBUR® or FACSARIA® systems. Against the background of single-cell analysis, the number of naive cells against identified memory cells may change over time (e.g., when cells are detected at different points in time). Example 7: Synthesis of a hypothetical antigen

[0100] The identified primary antigens are synthesized under GMP conditions. The synthesized antigens are used in preclinical and human clinical trials. Antigens that influence pathological immune responses form the basis of drug development. Some antigens may be more effective in specific patient populations. Example 8: Preclinical animal testing

[0101] Preclinical animal studies are performed using one of the available animal models known for the autoimmune disease of interest. For example, such an animal model for psoriasis is disclosed in Bochenska et al, Int. J. Mol. Sci., 2017, 18, 2514, which is incorporated herein in whole by reference. Two preferred animal models for candidate antigen testing are (a) SCID / SCID mice with psoriasis skin grafts and (b) induced psoriasis in Balb / c mice using 5% imiquimod applied to the skin at a dose of 62.5 mg / day. In the model of induced psoriasis, phenotypic psoriasis plaques develop somewhat rapidly (5-6 days, on the underside of the skin) and show high similarity to the human disease itself.

[0102] To test candidate drugs in animal models, toxicological protocols are employed, and the effects of the drugs on skin lesions are evaluated using scoring methods used in human trials (Psoriasis Area and Severity Index (PASI) score (PASI90 indicates a 90% or greater improvement from the baseline PASI score) and static Physician's Global Assessment (sPGA)). Animal studies are initiated with a dose equivalent to the human dose of 2 mg in mice. Example 9: Human Test

[0103] Currently, immunosuppressants are being tested and used for the treatment of psoriasis or other autoimmune diseases, and all have serious potential side effects. The antigen-based approach described herein is non-immunosuppressive, and its potential side effects are minimal to moderate.

[0104] A Phase 1 clinical trial will be conducted using the National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE) to test the safety of the drug and monitor adverse events. The clinical trial is a double-blind, placebo-controlled, four-group trial (20 patients in each group). Three groups will receive the active drug (with increasing doses), and one group will receive a placebo. The initial dose will be estimated from preclinical studies. The following references are incorporated herein to the extent that a person skilled in the art can carry out the methods described above. [References] 1 Greb JE et al., Psoriasis. Nat Rev Dis Primers 2:16082 (2016). 2 Icen M. et al., Trends in incidence of adult-onset psoriasis over three decades: a population-based study. J Am Acad Dermatol 60(3):394-401 (2009). 3 Raphael I. et al., T cell subsets and their signature cytokines in autoimmune and inflammatory diseases. Cytokine 74(1):5-17 (2015). 4 Lande R. et al., Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus. Sci Transl Med 3(73): 73ra19 (2011). 5 Lande R. et al., Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 449:564-569 (2007). 6 Nederstigt C. et al., Incidence and prevalence of thyroid dysfunction in type 1 diabetes. J Diabetes Complications 30(3):420-425 (2016). 7 Bakker S. F. et al., Screening for coeliac disease in adult patients with type 1 diabetes mellitus: myths, facts and controversy. Diabetol Metab Syndr 8:51 (2016). 8 Valdimarsson H. et al., Psoriasis--as an autoimmune disease caused by molecular mimicry. Trends Immunol 30(10):494-501 (2009). 9 Prinz J. C. Melanocytes: Target Cells of an HLA-C*06:02-Restricted Autoimmune Response in Psoriasis. J Invest Dermatol 137(10):2053-2058 (2017). 10 Lande R. et al., The antimicrobial peptide LL37 is a T-cell autoantigen in psoriasis. Nature Communications 5:5621 (2014).1 11. Kim S. M. et al., Analysis of the paired TCR alpha- and beta-chains of single human T cells. PLoS ONE 7(5): e37338 (2012). 12 Bochenska K. et al., Models in the Research Process of Psoriasis. Int J Mol Sci 18(12) 2514 (2017). 13 Paul C. et al., Ixekizumab provides superior efficacy compared with ustekinumab over 52 weeks of treatment: Results from IXORA-S, a phase 3 study. J Am Acad Dermatol 80(1):70-79 (2019)

Claims

1. A method for preparing an immune cell sample from a subject with psoriasis, The step of obtaining a psoriasis tissue sample; and, The step of isolating a single immune cell in situ from the psoriasis tissue sample using laser capture microdissection. Equipped with, The psoriasis tissue sample is obtained by mixing biopsy specimens taken from psoriasis lesions in a microblender. method.

2. A method for preparing an immune cell sample from a subject with psoriasis, The step of obtaining a psoriasis tissue sample; and, Laser capture microdissection, and, The step of isolating a single viable immune cell in situ from the psoriasis tissue sample using a technique comprising one or more of the following: immunoguided laser capture microdissection; micromanipulation; vacuum pulse-assisted techniques; immunomagnetic cell separation; density gradient centrifugation; sedimentation; adhesion; aptamer; buoyancy-activated cell sorting; fluorescence-activated cell sorting (FACS); and microfluidics. Equipped with, The psoriasis tissue sample is obtained by mixing biopsy specimens taken from psoriasis lesions in a microblender. method.

3. The method according to either claim 1 or 2, wherein the psoriasis tissue sample has a thickness of 5 μm to 15 μm.

4. The method according to any one of claims 1 to 3, wherein the psoriasis tissue sample is obtained by horizontally cutting a biopsy specimen from a psoriasis lesion.

5. The aforementioned method further, (a) the step of compressing the psoriasis tissue sample; and (b) The step of spreading the compressed sample onto a glass slide. The method according to claim 1 or 2, comprising:

6. The method according to claim 5, wherein the compression step is performed using a flat, round solid glass head.

7. The method according to any one of claims 1 to 3, wherein the psoriasis tissue sample is obtained by vertically cutting a biopsy specimen obtained from a psoriasis lesion.

8. The aforementioned method further, (a) the step of freezing the psoriasis tissue sample; and (b) Sectioning the frozen sample. The method according to any one of claims 1 to 7, comprising:

9. The method according to claim 8, wherein the freezing step includes the step of placing the psoriasis tissue sample on dry ice.

10. The method according to claim 8, wherein the freezing step is performed using liquid nitrogen vapor or isopentane.

11. The method according to any one of claims 8 to 10, further comprising the step of embedding the psoriasis tissue sample in an embedding matrix before sectioning the sample.

12. The method according to claim 11, wherein the embedding step is performed before freezing the sample.

13. The method according to claim 11 or 12, wherein the embedding substrate is tragacanth or an optimal cutting temperature (OTC) medium.

14. The method according to any one of claims 8 to 13, wherein the sectioning is performed using a microtome or a cryogenic bath.

15. The psoriasis tissue sample is obtained by cutting a biopsy specimen taken from a psoriasis lesion, and the method further, (a) The step of flash-freezing the psoriasis tissue sample; (b) The step of crushing the flash-frozen sample into fragments; (c) The step of thawing the aforementioned fragment; (d) vertically crushing one or more of the thawed fragments and pressing the one or more thawed fragments horizontally; and then (e) The step of spreading the fragment onto a glass slide. The method according to any one of claims 1 to 3, comprising:

16. The method according to any one of claims 1 to 15, further comprising the step of culturing the isolated immune cells in a growth medium.

17. The method according to any one of claims 1 to 15, further comprising the step of detecting and analyzing T cell surface markers, TCRs, single-cell genome (DNA) sequencing, transcription factors, cytokines, newly synthesized DNA, ATP, or combinations thereof from the isolated immune cells.

18. The method according to claim 16, further comprising the step of detecting and analyzing one or more of the cultured immune cells for T cell surface markers, TCRs, single-cell genome (DNA) sequencing, transcription factors, cytokines, or combinations thereof.

19. The method according to claim 17 or 18, wherein the detection step is performed via flow cytometry, deep sequencing, spectrophotometric analysis, or enzyme-linked immunosorbent assay.

20. The method according to any one of claims 16, 18, and 19, further comprising the step of performing an antigen challenge on the cultured immune cells.

21. The method according to claim 20, wherein the antigen challenge comprises the steps of contacting the immune cells with an antigen, and then detecting and analyzing one or more of the cultured or isolated immune cells for T cell surface markers, TCRs, single-cell genome (DNA) sequencing, newly synthesized DNA, ATP, transcription factors, cytokines, or combinations thereof.

22. The method according to claim 20 or 21, wherein the antigen is keratin, ADAMTSL5, or LL37.

23. The method according to any one of claims 20 to 22, wherein the antigen is isolated from a skin tissue sample obtained from the subject.

24. (a) A step of homogenizing and fractionating the skin tissue sample, (b) The step of performing the antigen challenge using the homogenized and fractionated skin tissue sample, (c) Steps to detect fractions that induce T cell stimulation, (d) A step of isolating the polypeptide in the detected fraction by performing three-dimensional gel chromatography. (e) The step of performing an antigen challenge using the isolated polypeptide, and (f) The step of detecting the isolated polypeptide that induces T cell stimulation. The method according to claim 23, further comprising the above.

25. The method according to claim 24, further comprising the step of detecting in a peripheral blood sample obtained from the subject: (i) the presence or absence of one or more immune cells having the detected T cell surface marker and / or transcription factor, and / or (ii) the presence or absence of isolated polypeptides that induce T cell stimulation.

26. The method according to claim 24, further comprising the step of synthesizing one or more polypeptides that induce T cell stimulation.

27. A method for preparing an immune cell sample from a subject with an autoimmune disorder, The step of obtaining tissue samples affected by the autoimmune disorder from the subject; and The step of isolating a single immune cell in situ from the tissue sample using laser capture microdissection or vacuum pulse-assisted technology. Equipped with, The aforementioned tissue sample is obtained by mixing biopsy specimens obtained from the affected tissue in a microblender. method.

28. A method for preparing an immune cell sample from a subject with an autoimmune disorder, The step of obtaining tissue samples affected by the aforementioned autoimmune disorder; and Laser capture microdissection, and, A step of isolating a single viable immune cell in situ from a tissue sample using a technique comprising one or more of the following: immunoguided laser capture microdissection; micromanipulation; vacuum pulse-assisted techniques; immunomagnetic cell separation; density gradient centrifugation; sedimentation; adhesion; aptamer; buoyancy-activated cell sorting; fluorescence-activated cell sorting (FACS); and microfluidics. Equipped with, The aforementioned tissue sample is obtained by mixing biopsy specimens obtained from the affected tissue in a microblender. method.

29. The method according to any one of claims 27 or 28, wherein the autoimmune disorder is psoriasis, type 1 diabetes mellitus, rheumatoid arthritis, or multiple sclerosis.

30. The method according to any one of claims 27 to 29, wherein the tissue sample has a thickness of 5 μm to 15 μm.

31. The method according to any one of claims 27 to 30, wherein the tissue sample is obtained by horizontally cutting a biopsy specimen from the tissue affected by the autoimmune disorder.

32. The aforementioned method further, (a) The step of compressing the tissue sample, and (b) The step of spreading the compressed sample onto a glass slide. The method according to claim 27 or 28, comprising:

33. The method according to claim 32, wherein the compression step is performed using a flat, round solid glass head.

34. The method according to any one of claims 27 to 30, wherein the tissue sample is obtained by vertically cutting a biopsy specimen obtained from the affected tissue.

35. The aforementioned method further, (a) The step of freezing the tissue sample, (b) Sectioning the frozen sample. The method according to any one of claims 27 to 34, comprising:

36. The method according to claim 35, wherein the freezing step includes the step of placing the biopsy specimen on dry ice.

37. The method according to claim 35, wherein the freezing step is performed using liquid nitrogen vapor or isopentane.

38. The method according to any one of claims 35 to 37, further comprising the step of embedding the tissue sample in an embedding matrix before sectioning the sample.

39. The method according to claim 38, wherein the embedding step is performed before freezing the sample.

40. The method according to claim 38 or 39, wherein the embedding substrate is tragacanth or an optimal cutting temperature (OTC) medium.

41. The method according to any one of claims 35 to 40, wherein the sectioning is performed using a microtome or a cryogenic bath.

42. The tissue sample is obtained by cutting a biopsy specimen taken from the affected tissue, and the method further, (a) Step of flash freezing the tissue sample, (b) The step of crushing the flash-frozen sample into fragments, (c) The step of thawing the fragment, (d) The step of vertically crushing one or more of the thawed fragments and horizontally pressing the one or more thawed fragments, and then (e) The step of spreading the fragment onto a glass slide. The method according to any one of claims 27 to 30, comprising:

43. The method according to any one of claims 27 to 42, further comprising the step of culturing the isolated immune cells in a growth medium.

44. The method according to any one of claims 27 to 42, further comprising the step of detecting and analyzing T cell surface markers, TCRs, single-cell genome (DNA) sequencing, transcription factors, cytokines, newly synthesized DNA, ATP, or combinations thereof from the isolated immune cells.

45. The method according to claim 43, further comprising the step of detecting and analyzing one or more of the cultured immune cells for T cell surface markers, TCRs, single-cell genome (DNA) sequencing, transcription factors, cytokines, newly synthesized DNA, ATP, or combinations thereof.

46. The method according to claim 44 or 45, wherein the detection step is performed via flow cytometry, deep sequencing, spectrophotometric analysis, or enzyme-linked immunosorbent assay.

47. The method according to any one of claims 27 to 46, further comprising the step of identifying a primary antigen that stimulates the isolated immune cells.

48. The method according to claim 47, wherein the identifying step includes culturing the isolated immune cells to produce cultured immune cells, and performing an antigen challenge on the cultured immune cells.

49. The method according to claim 48, wherein the antigen challenge comprises the steps of contacting the immune cells with an antigen, and then detecting and analyzing one or more of the cultured immune cells or isolated immune cells for T cell surface markers, TCRs, single-cell genome (DNA) sequencing, newly synthesized DNA, ATP, transcription factors, cytokines, or combinations thereof.

50. The method according to claim 49, wherein the antigen is isolated from a tissue sample obtained from the subject.

51. (a) A step of homogenizing and fractionating the tissue sample, (b) The step of performing the antigen challenge using the homogenized and fractionated tissue samples, (c) Steps to detect fractions that induce T cell stimulation, (d) A step of isolating the polypeptide in the detected fraction by performing three-dimensional gel chromatography. (e) The step of performing an antigen challenge using the isolated polypeptide, and (f) The step of detecting the isolated polypeptide that induces T cell stimulation. The method according to claim 50, further comprising:

52. The method according to claim 51, further comprising the step of detecting in a peripheral blood sample obtained from the subject: (i) the presence or absence of one or more immune cells having the detected T cell surface marker and / or transcription factor, and / or (ii) the presence or absence of isolated polypeptides that induce T cell stimulation.

53. The method according to claim 51, further comprising the step of synthesizing one or more polypeptides that induce T cell stimulation.