Methods and assays for immune phenotyping

Abstract

Screening methods based on using an ELISpot assay to measure the distribution profile of spontaneous cytokine or chemokine maximum spot intensity or mean spot intensity, or the number of "low-intensity" spontaneous cytokine or chemokine producing cells as a clinically relevant metric of immune function, for diagnosing a patient as having a particular disorder based on one or more of these measurements, and for treating a patient for the particular disorder. In some embodiments, the cytokine is IFN-γ or TNF-α. Patients can optionally further be evaluated to determine whether they have an immunosuppressive immunological endotype or a hyperinflammatory endotype by quantitating cytokine and / or chemokine levels in the biological sample. Representative disorders include sepsis, autoimmune disease, autoimmunity, cancer, and lymphopenia.

Description

METHODS AND ASSAYS FOR IMMUNE PHENOTYPINGFIELDThe present disclosure generally relates to methods and compositions for immune phenotyping patients.BACKGROUNDThe enzyme-linked immunosorbent spot (ELISpot) assay is a type of assay that focuses on quantitatively measuring the frequency of cytokine secretion for a single cell. The ELISpot Assay is also a form of immunostaining since it is classified as a technique that uses antibodies to detect a protein analyte, with the word analyte referring to any biological or chemical substance being identified or measured.The FluoroSpot Assay is a variation of the ELISpot assay. The FluoroSpot Assay uses fluorescence in order to analyze multiple analytes, meaning it can detect the secretion of more than one type of protein.U.S. Publication No. 20220091133 by Hotchkiss et al. discloses ways to use the ELISpot assay to carry out immune phenotyping (evaluating adaptive and innate immune status) of a subject. A biological sample comprising whole blood, diluted whole blood or isolated peripheral blood mononuclear cells (PBMCs) is provided, and T cell interferon-gamma (FFN-y) and / or monocyte TNF-a production can be quantified using ELISpot on the biological sample. It can be determined that the subject has an immunosuppressive immunological endotype if T cell interferon-gamma (IFN-y) and / or monocyte TNF-a production is relatively low compared to a healthy subject (control), or a hyper-inflammatory endotype if it is relatively high compared to a healthy subject.By way of example, COVID-19-associated morbidity and mortality have been attributed to a pathologic host response. In some patients, i.e., those with a hyper-inflammatory endotype, a hyper-inflammatory ‘cytokine-storm’-mediated injury occurs, and an over-exuberant immune response can be mediated by excessive pro-inflammatory cytokines drives excessive lung injuryand a pro-coagulant state. In such patients, mortality and morbidity can result from inflammatory lung injury, disturbances in micro- and macro-circulation, and resultant respiratory failure or vascular coagulopathy. For such patients, appropriate treatment may involve anti-cytokine and / or anti-inflammatory therapies, such as anti-IL-6(R) antibodies, IL-1 receptor antagonists, and / or JAK-STAT inhibitors, such as Baricitinib, which was approved by the U.S. Food and Drug Administration for treating COVID- 19 patients.In other patients, failure of host protective immunity (i.e., an immunosuppressive immunological endotype) can result in unrestrained viral dissemination and organ injury. For these patients, morbidity and mortality can result from an ‘immunologic collapse’ of the host's protective system. This collapse of host protective immunity manifests itself as a failure to control unrestrained viral replication and dissemination with direct host cytotoxicity. In such patients, progressive and profound lymphopenia can be observed, and this correlates with increased secondary infections and death. Patients with this endotype often have a devastating loss of immune cells in their spleen and secondary lymphoid organs, and multiple lymphocyte subsets are lost, including CD4 T, CD8 T, and NK cells that play vital antiviral roles, and in B cells that are essential for making antibodies.In addition to COVID-19, sepsis patients may have either a hyper-inflammatory endotype or an immunosupressive immunological endotype.The methods disclosed in U.S. Publication No. 20220091133 allow one to evaluate immune function, and determine the appropriate endotype. This can be particularly useful in determining an appropriate treatment regimen. That is, a patient with a hyper-inflammatory endotype might be treated with an agent that lowers the immune response, such as a steroid or JAK inhibitor, and a patient with an immunosupressive immunological endotype might be treated with an agent, such as interleukin-7 (IL-7) or GM-CSF, that enhances the immune response.This type of assessment can be useful when evaluating patients with disorders that might be associated with either an immunosuppressive immunological endotype or a hyper-inflammatory endotype. Such patients include those who have, are suspected of having, or are at risk for developing sepsis, such as fungal wound sepsis, autoimmune disease, autoimmunity, or cancer, orwho have lymphopenia (<1100 cells / pL), or who have undergone organ transplantation, or who are in critical care.It can be important to identify which of these immune endotypes predominate, because the appropriate intervention is diametrically different depending upon whether the patient is suffering from hyper-inflammation or profound immunosuppression. For example, if administered to patients with profound immunosuppression, anti-IL-6(R) antibodies, IL-1 receptor antagonists and JAK-STAT inhibitors can further compromise the patient's ability to fight infection. Conversely, if administered to patients with hyper-inflammation, immune stimulants such as checkpoint inhibitors, IL-7, interferon-y, GM-CSF, and the like can exacerbate an already dysfunctional and robust inflammatory response, and worsen organ injury.The methods disclosed in U.S. Publication No. 20220091133 are useful for identifying a patient’s immune endotype. The methods of immune phenotyping (evaluating adaptive and / or innate immune status) in a subject include providing or having been provided a biological sample comprising whole blood, diluted whole blood or isolated peripheral blood mononuclear cells (PBMCs), and quantitating T cell interferon-gamma (FFN-y) and / or monocyte TNF-a production using ELISpot in the biological sample. If T cell interferon-gamma (ZFN- y) and / or monocyte TNF-a production are relatively low compared to a healthy subject, the subject can be diagnosed as having an immunosuppressive immunological endotype, and if they are relatively high, the subject can be diagnosed as having a hyper-inflammatory endotype. As the subject’s endotype may evolve over time, the assessment may be re-performed periodically, including to determine a subject’s response to therapy.While these methods can be useful in determining the immune endotype of patients, they are not absolutely predictive of the type of illness that is causing a patient to have an immunosuppressive immunological endotype or a hyper-inflammatory endotype, relative to a healthy subject. It would be useful to have additional methods for gleaning additional information from ELISpot assays that can help identify the type of disorder from which the patient is suffering. The present invention provides such methods.SUMMARYIn one embodiment, a highly sensitive, functional immunoassay, which is an enzyme- linked immunosorbent spot (ELISpot) assay, is disclosed. The assay analyzes biological samples, such as whole blood, diluted whole blood, or isolated peripheral blood mononuclear cells (PBMCs), and determines the mean spot intensity, the maximum spot intensity, and the number of low-intensity cells per unit volume present in a biological sample, as counted in a well in which an ELISpot assay is carried out.As used herein, the term “low-intensity” IFN-y producing cells is defined as those with a mean spot intensity of <45% of the maximum intensity measured in the well.As used herein, “spontaneous IFN-y” refers to the spontaneous production of IFN-y of cells in a biological sample, such as whole blood, diluted whole blood, or isolated peripheral blood mononuclear cells (PBMCs), applied to ELISpot plates pre-coated with IFN-y capture antibody, and, ideally, incubated at 37°C in a 5% CO2 incubator without stimulation. This is in contrast to IFN-y production elicited by administering CD3 / CD28 to the incubated cells.In some aspects of this embodiment, the screening methods described herein are based on the fact that the distribution profile of spontaneous IFN-y mean spot intensity produced in the ELISpot assay is a clinically relevant metric of immune function in humans.In other aspects of this embodiment, the screening methods described herein are based on the fact that the distribution profile of spontaneous IFN-y maximum spot intensity produced in the ELISpot assay is a clinically relevant metric of immune function in humans.In still other aspects of this embodiment, the screening methods described herein are based on the fact that the number of “low-intensity” spontaneous IFN-y producing cells measured in the ELISpot assay is a clinically relevant metric of immune function in humans.In another embodiment, a patient is diagnosed as having a particular type of disorder based, at least in part, on the spontaneous IFN-y mean spot intensity, spontaneous IFN-y maximum spotintensity, and / or number of low-intensity spots per unit volume present in a biological sample, as counted in the ELISpot assay plate.In one aspect of this embodiment, a comparison can be made to a control (i.e., the number of low-intensity spots per unit volume in a biological sample derived from a healthy subject), and / or based on a reference library that includes ranges of numbers of low-intensity spots per unit volume in a biological sample, which ranges are associated with a patient having a particular disorder. The library can include, for example, anywhere from two to a thousand or more of such ranges. Where ranges associated with more than one type of disorder overlap, additional clinically relevant information may be used to further identify the disorder, and appropriately diagnose the patient.In some aspects of this embodiment, the particular disorder may be associated with either an immunosuppressive immunological endotype or a hyper-inflammatory endotype, and a further assessment can be made of the patient’s immunological endotype. For example, an ELISpot assay, which can be the same ELISpot assay as used to diagnose the disorder, can be used to quantitate cytokine production by individual cells and / or chemokine levels, such as interferon-gamma (IFN- y) and / or monocyte TNF-a production, in the biological sample. If T cell interferon-gamma (IFN- y) and / or monocyte TNF-a production are relatively low compared to a healthy subject, the subject can be diagnosed as having an immunosuppressive immunological endotype, and if they are relatively high, the subject can be diagnosed as having a hyper-inflammatory endotype. As the subject’s immunological endotype may evolve over time, the assessment may be re-performed periodically, including to determine a subject’s response to therapy.In other embodiments, patients who have been diagnosed as having a particular disorder can be treated for this particular disorder using an appropriate treatment protocol. Where the disorder is one where the appropriate treatment depends on the subject’s immunological endotype, the appropriate treatment protocol may be determined by a) determining the particular disorder from which the patient is suffering, b) determining the patient’s immunological endotype, and c) selecting an appropriate treatment protocol for patients suffering from a given disorder, and having a given immunological endotype.For some patients, particularly those with a hyper-inflammatory endotype, appropriate treatment may involve anti-cytokine and / or anti-inflammatory therapies, such as anti-IL-6(R) antibodies, IL-1 receptor antagonists, and / or JAK-STAT inhibitors, such as Baricitinib, which was approved by the U.S. Food and Drug Administration for treating COVID-19 patients. For other patients, particularly those with an immunosuppressive immunological endotype, appropriate treatment may involve administration of an agent, such as interleukin-7 (IL-7) or GM-CSF, that enhances the immune response.Treatment of the patient with immune modulating agents (i.e., agents that reduce or enhance the immune response) may be beneficial, but it can also be important to treat the underlying disorder. For example, a patient with bacterial sepsis may be treated with one or more antibiotics, a patient with viral sepsis may be treated with one or more antivirals, and a patient with fungal sepsis may be treated with one or more antifungals.In some embodiments, the patient has, is suspected of having, or is at risk for developing sepsis, such as fungal wound sepsis, autoimmune disease, autoimmunity, or cancer, or has lymphopenia (<1100 cells / pL), or has undergone organ transplantation, or is in critical care.In one aspect of these embodiments, the patient has sepsis, including sepsis caused by bacteria, viruses, fungi and / or parasites, is suspected of having sepsis, or is at risk for developing sepsis. In some aspects of this embodiment, at the time of diagnosis, it is unclear whether the patient is a healthy patient, a sepsis patient, or a critically ill, non-sepsis patient. In still other aspects of this embodiment, at the time of diagnosis, it is unclear whether the patient is a sepsis patient, or a critically ill, non-sepsis patient. In either of these embodiments, the determination of the number of low-intensity spots per unit volume in a biological sample that are counted in the ELlSpot assay plate is used to differentiate sepsis patients from critically ill, non-sepsis patients, and, optionally, healthy patients.In another embodiment, the patient has, or is suspected of having, autoimmune disease, autoimmunity, or cancer, or has lymphopenia (<1100 cells / pL), or has undergone organ transplantation, or is in critical care. With these patients, data including the mean spot intensity, the maximum spot intensity, and / or the number of low-intensity cells per unit volume present in abiological sample is determined, using ELISpot conditions as described herein, for one or more of these disorders, based on patients known to have these disorders. Then, a biological sample is obtained from a patient, and the mean spot intensity, the maximum spot intensity, and / or the number of low-intensity cells per unit volume present in a biological sample is determined. The patient’s information is compared with the reference information, and a diagnosis can be made based on a comparison of the patient’s information to the closest-matching reference information.In still another embodiment, the patient has, or is suspected of having, autoimmune disease, autoimmunity, or cancer, or has lymphopenia (<1100 cells / pL), or has undergone organ transplantation, or is in critical care, and the methods allow one to identify and track immune dysfunction in any of these conditions, for example, to characterize the severity of the illness, to track progression of the disease over time, and / or to help with the selection of appropriate therapies.In any of these embodiments, certain of the information is based on a particular type of biological sample and sample size, for example, 5 pl of whole blood. If larger or smaller sample sizes are used, or the blood is diluted, then the values obtained would be expected to vary in a linear manner. As such, the methods described herein do not depend on following the precise steps in the working examples, but rather, extent to variations thereof using different biological samples, sample sizes, and dilutions.The methods described herein can be performed using any ELISpot assay conditions, for example after CD3 / CD28 stimulation, or following medical treatments, such as administration of dexamethasone or IL-7. While the examples describe results with IFN-y, the ELISpot assay can be performed using any appropriate cytokine or chemokine.In another embodiment, the methods are used to determine response to treatments, such as IL-7 or other immunomodulatory therapies such as corticosteroids (e.g. dexamethasone). In this embodiment, the methods can be used in connection with any condition where immunomodulatory agents are applied, including cancer, transplantation and autoimmune diseases. For example, cancer can be treated with immunomodulatory agents, and this technology can be used to determine the response to that treatment. Similarly, transplant patients can be treated withimmunosuppressive agents to prevent rejection. This technology can also be used as a way to track the efficacy of these treatments.DESCRIPTION OF THE DRAWINGSThose of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.FIGS. 1 A-D are representative ELISpot photomicrographs showing the results of adaptive immune suppression in COVID-19 patients. The representative ELISpot photomicrographs display IFN-y production following overnight stimulation with anti-CD3 / anti-CD28 antibodies for (1 A) healthy volunteers, (IB) CINS patients, and (1C) septic non-COVID-19 patients. (ID) shows three representative COVID- 19-positive samples.FIG. 2A-D are representative ELISpot mages showing unstimulated and stimulated ex vivo production of IFN-y and TNF-a in whole blood from septic patients using an ELISpot assay. FIG. (2A) shows representative ELISpot images depicting IFN-y production in media alone versus with CD3 / CD28 Ab. FIG. (2B) shows a graphic representation of n=15 septic patient responses between unstimulated and stimulated ex vivo cytokine production of IFN-y. FIG. (2C) shows representative ELISpot images depicting TNF-a production in media alone versus with LPS. FIG. (2D) is a graphic representation of n=15 septic patient responses between unstimulated and stimulated ex vivo cytokine production of TNF-a. Red lines represent mortalities.FIG. 3-A is a chart showing distributions of spontaneous IFN-y mean spot intensity in biological samples obtained from healthy patients (green), critically ill non-septic patients (blue) and septic patients (orange).FIG. 3-B is a chart showing distributions of spontaneous IFN-y maximum spot intensity in biological samples obtained from healthy persons (green), critically ill non-septic patients (blue) and septic patients (orange).FIG. 3-C is a chart showing distributions of spontaneous IFN-y spot size in biological samples obtained from healthy patients (green), critically ill non-septic patients (blue) and septic patients (orange).FIG. 3-D is a chart showing distributions of spontaneous IFN-y spot circularity in biological samples obtained from healthy patients (green), critically ill non-septic patients (blue) and septic patients (orange).FIG. 3-E is a chart showing distributions of spontaneous IFN-y total spot intensity in biological samples obtained from healthy patients (green), critically ill non-septic patients (blue) and septic patients (orange).FIG. 4-A is a chart showing distributions of CD3 / CD28 stimulated IFN-y mean spot intensity in biological samples obtained from healthy patients (green), critically ill non-septic patients (blue) and septic patients (orange).FIG. 4-B is a chart showing distributions of CD3 / CD28 stimulated IFN-y maximum spot intensity in biological samples obtained from healthy persons (green), critically ill non-septic patients (blue) and septic patients (orange).FIG. 4-C is a chart showing distributions of spot size in CD3 / CD28 stimulated biological samples obtained from healthy patients (green), critically ill non-septic patients (blue) and septic patients (orange).FIG. 4-D is a chart showing distributions of spot circularity in CD3 / CD28 stimulated biological samples obtained from healthy patients (green), critically ill non-septic patients (blue) and septic patients (orange).FIG. 4-E is a chart showing distributions of IFN-y total spot intensity in CD3 / CD28 stimulated biological samples obtained from healthy patients (green), critically ill non-septic patients (blue) and septic patients (orange).FIG. 5 is a chart showing the total number of “low intensity” spots in healthy patients (green) compared with the number of low intensity spots in patients with sepsis (orange), and non- septic critical illness (blue).FIG. 6 shows raw images from the ELISpot assay, and events identified following image analysis.DETAILED DESCRIPTIONEnzyme-linked immunosorbent spot (ELISpot) is a highly sensitive, functional immunoassay that measures the number of cytokine-secreting cells at the single-cell level, in response to ex vivo stimulation following an incubation period. The ELISpot assay has excellent dynamic range and may detect as few as one in 100,000 cytokine-secreting cells. Furthermore, ELISpot can test simultaneously the integrity and robustness of the two disparate arms of immunity, i.e., innate (blood monocytes and low-density granulocytes) and adaptive cellular immunity (blood lymphocytes) by focusing on the responses of individual cell populations to cellspecific agonists.In one embodiment, the present disclosure relates to an ELISpot assay that analyzes biological samples, such as whole blood, diluted whole blood, or isolated peripheral blood mononuclear cells (PBMCs), and determines the mean spot intensity, the maximum spot intensity, and / or the number of low-intensity cells per unit volume present in a biological sample, as counted in a well in which an ELISpot assay is carried out.The distribution profde of spontaneous IFN-y mean spot intensity, spontaneous IFN-y maximum spot intensity, and / or number of low-intensity spots per unit volume produced in the ELISpot assay is a clinically relevant metric of immune function in humans and can be used to diagnose patients as having a particular disorder. Patients can be subjected to appropriate treatments following diagnosis.In some embodiments, the assessment is re-performed periodically to determine a subject’s response to therapy. In other embodiments, the assessment is re-performed following exposure ofthe patient’s biological sample to agents that enhance (i.e., IL-7) or reduce (i.e., dexamethasone) the immune response.In some embodiments, the spontaneous mean spot intensity, maximum spot intensity, and / or number of low-intensity spots per unit volume of cytokines and / or chemokines other than IFN-y can be evaluated. For example, similar results can be obtained using TNF-a.A control sample or a reference sample as described herein can be a sample from a healthy subject. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample. Other iterations of ELISpot, such as FluoroSpot can be used.These embodiments are further described in detail below.DefinitionsDefinitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.As used herein, the term “low-intensity” IFN-y producing cells is defined as those with a mean spot intensity of <45% of the maximum intensity measured in the well.As used herein, “spontaneous IFN-y” refers to the spontaneous production of IFN-y of cells in a biological sample, such as whole blood, diluted whole blood, or isolated peripheral blood mononuclear cells (PBMCs), applied to ELISpot plates pre-coated with IFN-y capture antibody, and, ideally, incubated at 37°C in a 5% CO2 incubator. This is in contrast to IFN-y production elicited (stimulated) by administering CD3 / CD28 to the incubated cells.As a way to compare the two, spontaneous production and stimulated production were compared in FIGS. 2A-D, which show data (from looking at average values of intensity within particular wells) from ex vivo production of IFN-y and TNF-a in whole blood unstimulated with either anti-CD3 / CD28 or LPS for a series of septic patients.FIGS. 1A-D show adaptive immune suppression in COVID- 19 patients, with representative ELISpot photomicrographs displaying IFN-y production following overnight stimulation with anti-CD3 / anti-CD28 antibodies for (1A) healthy volunteers, (IB) CINS patients, and (1C) septic non-COVID-19 patients. FIG. (ID) shows three representative COVID-19- positive samples. The number of spots demonstrates the number of cytokine-producing T cells. Counts are presented as the corrected number of spots per thousand lymphocytes plated as fraction of the 2.5* 104PBMCS plated in each well. Note the reduction in IFN-y production in both septic and COVID-19 patients compared with CINS patients. Note also a degree of heterogeneity in IFN- y production in COVID-19 and septic patients. Each photomicrograph was captured with the same magnification, and each image is to scale. ELISpot assays were performed using the PBMC fraction from freshly drawn whole blood. Each condition was run in duplicate for control samples and triplicate for COVID-19 samples.FIGS. 2A-D show unstimulated and stimulated ex vivo production of IFN-y and TNF-a in whole blood from septic patients using an ELISpot assay. FIG. (2A) shows representative ELISpot images depicting IFN-y production in media alone versus with CD3 / CD28 Ab. FIG. (2B) shows a graphic representation of n=15 septic patient responses between unstimulated and stimulated ex vivo cytokine production of IFN-y. FIG. (2C) shows representative ELISpot images depicting TNF-a production in media alone versus with LPS. FIG. (2D) is a graphic representation of n=15 septic patient responses between unstimulated and stimulated ex vivo cytokine production of TNF- a. Red lines represent mortalities.Conventional ELISpot assays calculate and report the total number of spots, the average spot size, and the total well intensity. This approach focuses on total and average features, but does not assess the characteristics of the individual spots. In this particular example, there was essentially no production of IFN-y without stimulation by CD3 / CD28-activating Abs. This lack of cytokine production was true not only for whole blood ELISpot but also for ex vivo IFN-yproduction in PBMCs (data not shown). In contrast to IFN-y, septic patient samples produced spontaneous, unstimulated TNF-a (i.e., without the addition of LPS).This data shows that different information can be obtained using different cytokines, so for different disorders, it may be useful to look at results for different cytokines / chemokines until a strong correlation can be seen between, for example, healthy patients and patients with the particular disorder, or patients with different disorders that are typically difficult to distinguish using other methodologies. By finding the right cytokine or chemokine for a given assessment, one can identify conditions for a suitable assay for diagnosing a disorder and, optionally, for distinguishing between two or more disorders.In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of areference herein shall not be construed as an admission that such is prior art to the present disclosure.I. ELISpot / FluoroSpotThe assay as described herein, can comprise a capture antibody (e.g., anti-cytokine) attached, coated, or immobilized on a surface or membrane. An antigen-stimulated cell can secrete antigens that are captured by the capture antibody. The detection antibody can be added to bind the antigen captured on the capture antibody. A chromogen, a substance which can be readily converted into a dye or other colored compound, can be added.An enzyme-linked immune absorbent spot (ELISpot) is a type of assay that focuses on quantitatively measuring the frequency of cytokine secretion for a single cell or a population of cells. The ELISpot Assay is also a form of immunostaining because it is classified as a technique that uses antibodies to detect an analyte or protein, such as a biological or chemical substance being identified or measured. The FluoroSpot Assay is a variation of the ELISpot assay. The FluoroSpot Assay uses fluorescence in order to analyze multiple analytes, thus it can detect the secretion of more than one type of analyte or protein.Mechanism of ELISpotAntibody coating (e.g., attached, immobilized, or coated): Throughout the ELISpot Assay technique, different substances are added to and washed away from wells. Wells are found on a laboratory plate with tiny dishes / bowls that can be filled with a substance to be examined; the amount of wells on a plate varies, but generally ranges from 16-100. The first substance added to the wells can be cytokine specific monoclonal antibodies. These antibodies can coat the walls of the wells for future binding to cytokine. The monoclonal antibodies are antibodies produced from a single cell lineage, and is only able to bind to one protein epitope. Polyclonal antibodies, on the other hand, are capable of binding to multiple epitopes of the same protein.Cell incubation: The desired cells being observed and analyzed are added to the wells. Each well can have the presence or absence of stimuli that activate the secretion of cytokine in cells. During cell incubation, the cells are allowed to react to any present stimuli and secrete cytokine.Many procedures and methods are known in the art to follow to ensure proper cell handling. To make sure that cells are of high quality, cells in blood samples can be lightly agitated if stored for longer than 3 hours, the blood samples can be diluted in PBS (phosphate buffered saline) before being stored, and the blood samples may be free of granulocytes. Any cells that have been cryopreserved and thawed can be allowed to rest for an hour or more at 37° C. (the typical temperature of the human body). When incubating cells, some considerations can include, ensuring that the cells do not experience sudden movements that could affect spot formation or ensuring the incubator's humidity is high enough to avoid excessive evaporation and drying out the wells.Cytokine capture: Since the cells are surrounded by cytokine-specific monoclonal antibodies that coat the walls of the wells, a cytokine that has been secreted by the incubated cells will start to attach to the antibodies at a specific epitope.Detection antibodies: At this point, the wells can be rinsed in order to get rid of the cells and any other undesirable substances. Remaining are the cytokine specific monoclonal antibodies and any cytokine that bonded to the antibodies. Biotinylated cytokine-specific detection antibodies can then be added to the well. These cytokine-specific detection antibodies will bind to any cytokine that is left in the well since the cytokine is still attached to the first set of antibodies used. Because the cytokine is attached to the first set of antibodies coating the wells, the cytokine is not washed away when the wells are rinsed. Streptavidin-enzyme conjugate: Streptavidin-enzyme conjugate can be added to the wells in order to bind with the detection antibodies. The purpose of biotinylating the cytokine-specific detection antibodies added to the wells in the previous step is so that the antibody can bind to the new streptavidin-enzyme conjugate. Biotinylation creates a strong affinity between the biotin on the cytokine-specific antibody and the streptavidin on the conjugate. Addition of substrate: A substrate (e.g., a chromogenic substrate) can be added to the wells, and is catalyzed by the enzyme conjugate added in the previous step. This reaction forms insoluble precipitate that forms spots in the wells. The substrate that is used in this step can depend on the type of enzyme used in the previous step. If streptavidin-ALP (streptavidin and alkaline phosphatase conjugate) is used, then using BCIP / NBT-plus (a mixture of 5-bromo-4-chloro-3- indolyl phosphate and nitroblue tetrazolium chloride) as a substrate can produce more distinct spots that are easier to analyze. If streptavidin-HRP (streptavidin and horseradish peroxidaseconjugate) is used, then using TMB (tetramethylbenzidine) as a substrate can produce better results.Analysis: The spots that are formed can then be read on an automated ELISpot reader, or counted under a dissection microscope, and further used to calculate the frequency of cytokine secretion.Mechanism of FluoroSpotThe FluoroSpot assay combines the sensitivity of ELISpot with the capacity to study secretion of several analytes simultaneously, enabling studies of cell populations with different functional profdes. The FluoroSpot assay is very similar to the ELISpot assay. The key difference is that the FluoroSpot assay can analyze the presence of multiple analytes on one plate of wells, whereas the conventional ELISpot assay can only analyze one analyte at a time. The FluoroSpot assay accomplishes this by using fluorescence rather than an enzymatic reaction for detection. The steps for a FluoroSpot assay are also similar, with a few differences.Antibody Coating: Similar to the ELISpot, cytokine-specific monoclonal capture antibodies are added to a plate with wells. For both assays, the plates are ethanol-treated to avoid contamination and skewed data collection. For the FluoroSpot assay, a mixture of different types of capture antibodies are attached to the wells in order to detect multiple types of analytes. In order to get optimal results with the ELISpot and the FluoroSpot assay, proper plate coating techniques should be followed. The plates should be treated with ethanol, washed, and then coated with antibodies. Ethanol treatment methods also vary depending on the type of plates that are used. For MSIP and IPFL plates, one can add 15 micro liters of 35% ethanol to all of the wells. Allow the ethanol to sit in the wells for one minute, and then pour it out. For MAIPSWU plates, one can instead add 50 micro liters of 70% ethanol to all of the wells. Allow the ethanol to sit in the wells for two minutes, and then pour it out. After the wells are treated with ethanol, the wells can be washed with about 200 micro liters of sterile water. This washing process can be repeated a total of 5 times. Once the wells have been treated with ethanol and washed, the cytokine-specific monoclonal capture antibodies can be added to each well.Cell Incubation: a cell or population of cells can be added to the wells and incubated in the presence or absence of stimuli that affect protein secretion.Cytokine Capture: Proteins / analytes that are secreted by the incubated cells will bind to the capture antibodies attached, immobilized, or coated to the wells during the first step.Detection Antibodies: Similar to the ELISpot, once the wells are rinsed to remove cells and other substances that are not of interest for identifying or measuring, a biotinylated detection antibody can be added (this can be specific for one type of analyte that will be quantified) and then tag-labeled detection antibodies are added for optional second or third types of analytes being studied.Fluorophore-labeled Conjugates: Instead of adding a streptavidin-enzyme conjugate, the detection of multiple analytes is amplified in the FluoroSpot with the use of fluorophore-labeled anti-tag antibody and streptavidin-fluorophore conjugate. A fluorescence enhancer solution can also be added during this step in order to enhance the signals later used when analyzing the fluorescence colors in the wells. This fluorescence makes it possible for the FluoroSpot to analyze and compare multiple analytes, unlike the ELISpot.Analysis: Because FluoroSpot relies on the use of fluorescence and not an enzymatic reaction, there is no need for a step that adds a substrate to react with enzymes (as needed for the ELISpot). The last step for the FluoroSpot assay is to analyze the fluorophores under an automated fluorescence reader that has separate filters for the different fluorophores being analyzed. These filters can be selected for the specific wavelengths of the fluorophores for accurate measurements.Since the FluoroSpot assay identifies and quantifies the presence of multiple analytes, it is possible that the absorption of one analyte can affect the secretion of another analyte; this is called capture effects. The affect an analyte (e.g., cytokine) has on another analyte could be positive or negative (the production of the second analyte can either increase or decrease). To counteract capture effects, it is possible to use co-stimulation in order to bypass the decreased production of an analyte. This is when a second antibody that stimulates the production of the same analyte is added to the wells.The ELISpot and FluoroSpot assays can be used in many research fields: vaccine development, cancer, allergies, monocytes / macrophages / dendritic cells characterization, apolipoproteins analysis, and veterinary research. With the ELISpot, antigen-specific cytokine responses, antibody specific secreting cells, tumor antigens, granzyme B and Perforin release by T cells, vaccine efficacy, epitope mapping, cytotoxic T cell activity, detection of IL -4, IL-5, and IL-13, vaccine-induced antibody responses, antigen-specific memory B cells, and more can be studied.As an example, the T cell ELISpot assay can be used to characterize T cell subsets. This is because the assay can detect the production of cytokines IFN-y, IL-2, TNF-alpha, IL-4, IL-5, and IL-13. The first three cytokines are produced by Thl cells, while the last three are produced by Th2 cells. Measuring T cell responses through cytokine production can also make it possible to study vaccine efficacy. As another example, with T cell FluoroSpot, tumor-infiltrating lymphocytes can be monitored. The IFN-y cytokine and granzyme B secretion can be analyzed in order to assess cytotoxic T cell responses. Both of these are used for cancer research. As yet another example, with B-cell FluoroSpot, vaccine efficacy can also be observed by quantifying the secretion of IgG, IgA, and IgM before and after vaccination. This analysis of multiple immunoglobulins is made possible because of the fluorescence method used in the FluoroSpot.II. Biological SamplesA biological sample can comprise or be whole blood, diluted whole blood, peripheral blood, or isolated human peripheral blood mononuclear cell (PBMC). PBMCs are a diverse mixture of highly specialized immune cells that play key roles in keeping our bodies healthy. A peripheral blood mononuclear cell (PBMC) is any blood cell having a round nucleus such as lymphocyte (e.g., T cells, B cells), monocyte, or a macrophage. These blood cells are a critical component in the immune system to fight infection and adapt to intruders. Two primary techniques that separate peripheral blood mononuclear cells from whole peripheral blood are through the use of a density gradient centrifugation process or by leukapheresis. Blood contains many types of cells: white blood cells (monocytes, lymphocytes, neutrophils, eosinophils, basophils, and macrophages), red blood cells (erythrocytes), and platelets. Peripheral blood cells are the cellular components of blood, comprising red blood cells (erythrocytes), white blood cells (leucocytes),and platelets, which are found within the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver, or bone marrow.In one embodiment, the biological sample is whole blood, diluted whole blood, or isolated PBMCs. In one aspect of this embodiment, the biological sample comprises whole blood, which does not require the isolation of PBMCs. Whole blood ELISpot can test simultaneously the integrity and robustness of the two disparate arms of immunity, i.e., innate (blood monocytes and low-density granulocytes) and adaptive cellular immunity (blood lymphocytes) by focusing on the responses of individual cell populations to cell-specific agonists. As shown in Example 1, T cell subsets were profoundly reduced in COVID-19 patients. Additionally, stimulated blood mononuclear cells produced less than 40%-50% of the IFN-'- and TNF-a observed in septic and CINS patients, consistent with markedly impaired immune effector cell function.III. Cytokines / ChemokinesAs described herein, secreted cytokines are measured using the assays described herein, for example, to determine immune status of a subject. As an example, the cytokines are measured after ex vivo stimulation. Cytokines that can be measured to determine the immune status (cytokines associated with cellular immunity) of a subject can be human cytokine such as IFN-v, TNF-a, IL-ip, IL-6, or those listed below associated with cellular immunity. Inflammatory cytokines can include interleukin-1 (IL-1), IL-12, and IL-18, tumor necrosis factor alpha (TNF-a), interferon gamma (FFNy), and granulocyte-macrophage colony stimulating factor (GM-CSF). An anti-inflammatory cytokine can be interleukin (IL)-l receptor antagonist, IL-4, IL-6, IL- 10, IL-11, and IL-13.Cytokine ReceptorChromosome Molecular Receptor(s) Location(Da) andName Synonym(s) Amino Acids Weight2(s)FormInterleukinsIL- 1 -like hematopoietin-IL-la 271 2ql4 30606 CD121a, 2ql2,2ql2-CDwl21b q22IL- I catabolin 269 2ql4 20747 CD121a, 2ql2,2ql2-CDwl21b q22IL-IRA IL-1 receptor 177 2ql4.2 20055 CD121a 2ql2 antagonistIL- 18 interferon-Y 193 l lq22.2- 22326 IL- 2ql2 inducing q22.3 18Ra, P factorCommon g chain (CD 132)IL-2 T cell growth 153 4q26- 17628 CD25, 10pl5- factor q27 122, pl4,132 22ql3.1, Xql3.1IL-4 BSF-1 153 5q31.1 17492 CD 124, 16pl l.2-213al3, 12.1, X,132 Xql3.1IL-7 177 8ql2- 20186 CD 127, 5pl3, q 13 132 Xql3.1IL-9 T cell growth 144 5q31.1 15909 IL-9R, Xq28 or factor P40 CD132 Yql2, Xql3.1X,IL-13 P600 132 5q31.1 14319 CD213al,Xql3.1-213a2, q28,CD1243, 16pl l.2-132 12.1,Xql3.1IL-15 162 4q31 18086 IL-15Ra, 10pl4-CD122, pl4,132 22ql3.1,Xql3.1Common b chain (CD 131)Xp22.3IL-3 multipotential 152 5q31.1 17233 CD123, orCSF, MCGF CDwl31 Ypl l.3,22ql3.13p26-IL-5 BCDF-1 134 5q31.1 15238, CDwl25, p24, homodimer 131 22ql3.1Also relatedXp22.32GM-CSF CSF-2 144 5q31.1 16295 CD 116, orCDwl31 Ypl l.2,22ql3.1IL-6-likeIL-6 IPN- 2, 212 7p21 23718 CD126, lq21,BSF-2 130 5ql lIL- 11 AGIF 199 19ql3.3- 21429 IL-l lRa, 9pl3,13.4 CD130 5ql 1Also relatedG-CSF CSF-3 207 17qll.2- 21781 CD114 lp35- ql2 p34.3IL- 12 NK cell 219 / 328 3pl2- 24844 / 37169 CD212 19pl3.1, stimulatory pl3.2 / lp31.2 factor5q31.1- heterodimer q33.15pl3-LIF leukemia 202 22ql2.1- 22008 LIFR, pl2 inhibitory ql2.2 CD130 factorOSM oncostatin M 252 22ql2.1- 28484 OSMR, 5pl5.2- ql2.2 CD130 5pl2IL-10-likeIL-10 CSIF 178 lq31- 20517, CDw210 Hq23 q32 homodimerIL-20 176 2q32.2 20437 IL-20Ra, 0 ?OthersIL- 14 HMW-BCGF 498 1 54759 IL-14R ?IL- 16 LCF 631 15q24 66694, CD4 12pter- homotetramer pl2IL- 17 CTLA-8 155 2q31 17504, CDw217 22ql 1.1 homodimerInterferons21q22.1IFN-a 189 9p22 21781 CD11821q22.1IFN-P 187 9p21 22294 CD118IFN-y 166 12ql4 19348, CDwl l9 6q23- homodimer q24TNFCD 154 CD40L, 261 Xq26 29273, CD40 20ql2-TRAP homotrimer ql3.2LT-P 244 6p21.3 25390, LT R 12pl3 heterotrimerTNF -a cachectin 233 6p21.3 25644, CD 120a, b 12pl3.2, homotrimer lp36.3- p36.2TNF-P LT-a 205 6p21.3 22297, CD 120a, b 12pl3.2, heterotrimer lp36.3- p36.24-1BBL 254 19pl3.3 26624, CDwl37 lp36 trimer? (4- IBB) BCMA,APRIL T ALL-2 250 17pl3.1 27433, 16pl3.1, TACI trimer? 17pl l.2CD70 CD27L 193 19pl3 21146, CD27 12pl3 trimer?CD153 CD30L 234 9q33 26017, CD30 lp36 trimer?CD 178 FasL 281 lq23 31485, CD95 (Fas) 10q24.1trimer?G1TRL 177 lq23 20307, G1TR lp36.3 trimer? LTbR,LIGHT 240 16pl l.2 26351, 12pl3, HVEM trimer? lp36.3- p36.2OX40L 183 lq25 21050, 0X40 lp36 trimer?TALL-1 285 13q32-q34 31222, BCMA, 16pl3.1, trimer? TACI 17pl 1.2TRAILR1TRAIL Apo2L 281 3q26 32509, 8p214 trimer?TWEAK Apo3L 249 17pl3.3 27216, Apo3 lp36.2 trimer?RANK,TRANCE OPGL 317 13ql4 35478, 18q22.1,OPG trimer? 8q24TGF-PTGF-P 1 TGF-P 390 19ql3.1 44341, TGF-PR1 9q22 homodimerTGF-P2 414 lq41 47747, TGF-PR2 3p22 homodimerTGF-P3 412 14q24 47328, TGF-PR3 lp33- homodimer p32MiscellaneoushematopoietinsEpo erythropoietin 193 7q21 21306 EpoR 19pl3.3- pl3.2Tpo MGDF 353 3q26.3- 37822 TpoR lp34 q27Flt-3L 235 19ql3.1 26416 Flt-3 13ql2SCF stem cell 273 12q22 30898, CD117 4ql l- factor, c-kit homodimer ql2 ligandM-CSF CSF-1 554 lp21- 60119, CD115 5q33- p 13 homodimer q35MSP Macrophage 71 1 3p21 80379 CDwl36 3p21.3 stimulating factor, MST-1xList assembled using data from Gene Cards (World Wide Web URL: http: / / genome- www.stanford.edu / genecards). Note that some of the cytokines listed are not discussed in this chapter.2Data describes the unprocessed precursor.3Can be found in complexes.Chemokines as described herein can be any of those listed below.Amino Ligand Molecular Chemokine ReceptorName Synonym(s) Acids2Location Weight (Da)2Receptor(s) LocationC ChemokinesXCL1 lymphoactin a, 114 Iq21-q25 12517 XCR1 3p21SCM-la,ATACXCL2 lymphoactin b, 114 lq23 12567 XCR1 3p21 SCM-lb, ATACCC ChemokinesCCL1 1-309 96 17 10992 CCR8 3p22CCL2 MCP-1, 99 17ql l .2- 11025 CCR2 3p21MCAF ql2CCR1CCL3 MIP-la, 92 17ql l- 10085 3p21CCR5LD78a q21CCL4 MIP-ip,17ql 1- 10212 CCR5 3p21LAG-1, q23ACT-2CCR1CCL5 RANTES 91 17ql 1.2- 9990 3p21CCR3 ql2 CCR5CCR1CCL7 MCP-3 99 17ql 1.2- 11200 3p21CCR2 ql2 CCR3CCL8 MCP-2 99 17ql 1.2 11246 CCR3 3p21CCL1 1 eotaxin 97 17q21 .1 - 10732 CCR3 3p21 q21.2CCR2CCL13 MCP-4 98 17ql 1.2 10986 3p21CCR3CCL14 HCC-1 93 17ql 1.2 10678 CCR1 3p21CCR1CCL15 HCC-2, Lkn-1, 113 17ql 1.2 12248 3p21CCR3MIP-ld, MIP-5CCL16 HCC-4, LEC, 120 17ql l.2 13600 CCR1 3p21EMC, LCC-1CCL17 TARC 94 16ql3 10507 CCR4 3p24DC-CK1,CCL18 89 17ql l.2 9849 ?PARC,AMAC-a,MIP-4CCL19 MIP-3P, ELC, 98 9pl3 10993 CCR7 17ql2- exodus-3 q21.1MIP-3a,CCL20 96 2q33-q37 10762 CCR6 6q27LARC, exodus- 1CCL21 6Ckine, SLC, 134 9pl3 14646 CCR7 17ql2- exodus-2 q21.2CCL22 MDC, STCP-1 93 16ql3 10580 CCR4 3p22MPIF-1, M1P-CCL2312017ql l.2 13443 CCR1 3p213,CKb-8MPIF-2,CCL24 119 7ql l.23 13133 CCR3 3p21 eotaxin-2, CKb-6CCL25 TECK, MIP-4a 150 19pl3.2 16639 CCR9 3p21CCL26 eotaxin-3 94 7ql l.2 10648 CCR3 3p21Eskine,CCL27 112 9pl3 12618 CCR10 3p21CTACK,1ECCXC Chemokines ELR?GROa, MGSA-CXCL1 107 4q21 11301 CXCR1, 2q35 a+CXCR2GROb,CXCL2 107 4q21 11389 CXCR2 2q35MGSA-b,MIP-2a+GROg,CXCL3 107 4q21 11342 CXCR2 2q35MGSA-g,MIP-2b+CXCL4 PF4, oncostatin 101 4ql2-ql3 10845 ?CXCL5 ENA-78+ 114 4ql3-q21 11972 CXCR2 2q35CXCL6 GCP-2+ 114 4q21 11897 CXCR1, 2q35CXCR2NAP-CXCL7 375 4ql2-13 42823 CXCR2 2q352, PPBP+CXCL8 IL-8, NAP-1, 99 4ql2-13 11098 CXCR1, 2q35NAF,CXCR2 MDNCF+CXCL9125 4q21 14019 CXCR3 Xql3CXCL1098 4q21 10856 CXCR3 Xql3CXCL1194 4q21.2 10365 CXCR3 Xql3CXCL12 SDF-la / p- 93 lOql l. l 10666 CXCR4 2q21CXCL13 BLC, BCA-1- 109 4q21 12664 CXCR5 11CXCL14 BRAK- 99 5q31 11722 ?CX3C ChemokinesCX3CL1 fractalkine397 16ql3 42202 CX3CR1 3p211List based on terminology from Zlotnick and Yoshie, Immunity, 2000 Feb;12(2):121-7. doi: 10.1016 / sl074-7613(00)80165-x, with added annotation and data from Gene Cards (World Wide Web URL: http: / / genome-www.stanford.edu / genecards).2Data describes the unprocessed precursor.IV. Assay MethodsThe assay methods described herein will be better understood with reference to the following description. Methods for determining the maximum spot intensity, mean spot intensity, and total number of low-intensity cells per unit volume are discussed below.In one embodiment, an ELISpot assay is performed by taking a biological sample, performing a conventional ELISpot assay, for example, using the conditions described by the manufacturer. In one aspect of this embodiment, to determine the mean spot intensity, the ELISpot images are analyzed using an automated ELISpot plate reader or with image analysis software such as image j (https: / / imagej.nih.gov / ij / ).Cytokine producing cells are identified as “spots”. In some embodiments, this identification is done manually, and in other embodiments, with computational algorithms. The amount of cytokine produced by each cell is related to the intensity of the spot in the image. This can be measured as the average (mean) intensity of the spot or the maximum intensity of the spot. In some embodiments, spot intensity is normalized to the maximum intensity measured in the image. Individual spots can be counted, and the number of cells with an intensity lower than the maximum intensity can also be counted.Methods for Determining the Immune Endotype and / or Evaluating Drug EfficacyBefore, after or while performing the ELISpot assay, and using the information on specific spots to determine that a patient is suffering from a specific disorder, the patient can also be immune phenotyped by evaluating their adaptive and / or innate immune status. For example, amethod of immune phenotyping can involve providing, or having been provided, a biological sample comprising whole blood, diluted whole blood or isolated peripheral blood mononuclear cells (PBMCs). The T cell interferon-gamma (IFN-y) and / or monocyte tissue necrosys factor alpha (TNF-a) production can then be quantified using ELISpot on the biological sample, and the amount of IFN-y and / or TNF-a can be compared to that of a healthy subject.A patient can be diagnosed as having an immunosuppressive immunological endotype if the T cell interferon-gamma (IFN-y) and / or monocyte TNF-a production is relatively low compared to a healthy subject. In this context, relatively low can mean more than 20% lower, more than 30% lower, more than 40% lower, or more than 50% lower than an average healthy subject.A patient can be diagnosed as having a hyperimmune immunological endotype if the T cell interferon-gamma (IFN-y) and / or monocyte TNF-a production is relatively high compared to a healthy subject. In this context, relatively high can mean more than 20% higher, more than 30% higher, more than 40% higher, or more than 50% higher than an average healthy subject.In accordance with another aspect, once a patient has been diagnosed as having a given condition, and is being or has been treated with a therapeutic agent appropriate for that condition, the efficacy of the treatment can be determined by measuring the immune function in the subject. This can involve first determining whether a patient has an immosupressive or hyperimmune immune endotype, identifying an appropriate treatment regimen for a) the particular condition and b) the particular immune endotype, treating the patient with the appropriate treatment regimen, and re-measuring the patient’s immune endotype and / or re-performing the screening step that identified the patient as suffering from the given condition.A subject can be defined as having an immunosuppressive endotype if T cell cytokine or monocyte cytokine production is relatively low compared to a control, or a hyperimmune endotype if it is relatively high compared to a control. A drug to the subject and / or determining the immune function of the subject in response to the drug. In some embodiments, the T cell cytokine is interferon-gamma (IFN-y). In some embodiments, the monocyte cytokine is selected from one or more of TNF-a, IL-2, IL-6, and / or IL-12. In some embodiments, the subject has sepsis, COVID-19, cancer, trauma, or autoimmune disease; the subject is a critically ill non-septic (CINS) or posttransplant patient; or the subject is immunosuppressed or a pediatric patient.By way of example, once the patient has been diagnosed as having the disorder, and has been or is being treated, the same steps performed above for determining the patient’s immunological endotype can be performed. If the patient’s immunological endotype trends from hyperimmune or immunosuppressed towards a normal endotype, i.e., the amount which the patient’s T cell interferon-gamma (IFN-y) and / or monocyte TNF-a production was relatively high or relatively low, respectively, relative to that of a normal subject is reduced, then the treatment can be deemed to be working. If the trend is that the patient’s T cell interferon-gamma (IFN-y) and / or monocyte TNF-a production is even farther from normal levels, then the treatment can be deemed to not be working.An aspect of the present disclosure provides for a method of immune phenotyping a subject comprising: providing or having been provided a biological sample from the subject; optionally stimulating a T cell or monocyte cell or both to secrete a cytokine associated with cellular immunity; and / or quantitating at least one cytokine associated with cellular immunity using ELISpot assay or FluoroSpot assay in the biological sample. In some embodiments, the method further comprises determining that a subject has an immunosuppressive endotype if the cytokine associated with cellular immunity is a proinflammatory cytokine and / or proinflammatory cytokine production or secretion is decreased compared to a control. In some embodiments, the method further comprises determining that a subject has a hyper-inflammatory endotype if the cytokine associated with cellular immunity is a proinflammatory cytokine and / or the proinflammatory cytokine production or secretion is increased compared to a control. In some embodiments, the method further comprises determining if the subject has immunosuppressive endotype if immune cells amount is reduced compared to a control or hyper-inflammatory endotype if cytokine production is increased compared to a control. In some embodiments, the method further comprises detecting a level of innate immunity comprising detecting a level of blood monocytes or detecting a level low-density granulocytes or detecting a level of monocyte function or low- density granulocyte function. In some embodiments, the method further comprises detecting a level of adaptive cellular immunity comprising detecting a level of blood lymphocytes or blood lymphocytes function. In some embodiments, the subject has an immunosuppressive endotype ifan amount of CD4+ and / or CD8+ T cells is reduced compared to a control, has reduced responsiveness of the T cells to T cell receptor activation, or both. In some embodiments, the cytokine associated with cellular immunity is a proinflammatory cytokine selected from the group consisting of T cell interferon-gamma (IFN-y), monocyte tumor necrosis factor alpha (TNF-a), IL- ip, or combinations thereof. In some embodiments, the cytokine associated with cellular immunity is selected from IFN- , TNF-a, IL-ip, IL-6, IL-7, IL-8, IL-10, IL-12, MCP-1, IL-IRA, or any combination thereof; or EGF, Eotaxin, FGF-basic, G-CSF, GM-CSF, HGF, IFN-a, IFN-y, IL-ip, IL-la, IL-IRA, IL-2, IL-2R, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40 / p70) IL- 13, IL-15, IL-17A, IL-17F, IL-22, IP-10, MCP-1, MIG, MIP-la, MIP-10, RANTES, TNF-a, VEGF, or any combination thereof. In some embodiments, quantitating cytokines associated with cellular immunity comprises: detecting an amount of cytokine-producing immune effector cells; or detecting an amount of cytokine produced on a cell. In some embodiments, quantitating cytokines associated with cellular immunity is measured in units of response per volume of blood. In some embodiments, the biological sample comprises: whole blood; diluted whole blood; circulating peripheral blood; whole blood diluted in about a 1 : 1 ratio with PBS; T cells, monocytes, and / or B cells; or plasma, leukocytes, red blood cells (RBCs), white blood cells (WBCs), platelets, cytokines, chemokines, or combinations thereof. In some embodiments, the biological sample does not comprise isolated peripheral blood mononuclear cells (PBMCs). In some embodiments, the method further comprises evaluating adaptive and / or innate immune status; evaluating monocyte or leukocyte function; evaluating progression of immune dysfunction in a subject; evaluating an effect of an immune therapy to restore innate and / or adaptive immunity in an immunosuppressed patient, optionally an immuno-adjuvant therapy to enhance host immunity; identifying optimal immune therapy for use in a subject; or improving immune function in a subject. In some embodiments, the subject has, is suspected of having, or is at risk for developing sepsis, autoimmune disease, autoimmunity, or cancer; the subject has Fungal Wound Sepsis; the subject has lymphopenia (<1100 cells / pL); the subject has undergone organ transplantation; and / or the subject is in critical care. In some embodiments, the method further comprises measuring ex vivo cytokine production as a response to external stimuli. In some embodiments, the subject is septic or is determined to be at risk for premature death if: an amount of proinflammatory cytokine producing immune effector cells are decreased compared to a control; or an amount of proinflammatory cytokine produced per cell measured by spot intensity are decreased comparedto a control. In some embodiments, if the subject does not have an immunosuppressive endotype or the subject has a hyper-inflammatory endotype, the subject is administered a drug, like dexamethasone, that blocks proinflammatory cytokines or inhibits an inflammatory signaling cascade; if the subject has an immunosuppressive endotype, then the subject is administered IL-7 or a similar immune-enhancing treatment to restore disease-induced T cell exhaustion. If the subject has sepsis and / or has an immunosuppressive endotype, a drug restoring immunity is administered to the subject; if the subject is septic and / or immunosuppressed, then the subject is not administered corticosteroid therapy, optionally dexamethasone; the subject has sepsis and / or has the immunosuppressive endotype, the subject is at high risk for death. If the subject has the immunosuppressive endotype, the subject is treated with immuno-modulatory drug therapies or immune adjuvants that enhance host immunity; if the subject has an immunosuppressive endotype, then the subject is administered checkpoint inhibitors and / or common y-chain cytokines that stimulate CD4 and / or CD8 T cells, optionally IL-17, if the subject has a hyper-inflammatory endotype or does not have an immunosuppressive endotype, the subject is treated with drugs to inhibit a host inflammatory response; if cytokine production in the subject is high, the subject is not treated with immunostimulant therapy; or if cytokine production in the subject is high, the subject is treated with anti-cytokine therapy or drugs to negatively modulate an inflammatory response.In some embodiments, the method further comprises detecting an immunosuppressive endotype or a hyper-inflammatory endotype during progression of a disease, disorder, or condition or during treatment of a disease, disorder, or condition. In some embodiments, the method further comprises administering a drug to a subject in need thereof and / or determining immune function or leukocyte function of the subject in response to the drug, optionally, during a course of immune therapy. In some embodiments, the subject has sepsis, COVID- 19, cancer, trauma, or autoimmune disease; the subject is a critically ill nonseptic (CINS) or post-transplant patient; or the subject is immunosuppressed or a pediatric patient. In some embodiments, the biological sample is placed in fluid contact with a test therapeutic agent, optionally cytokines / chemokines, IL6, anti-PD-1, anti-PD-Ll, GM512, CSF, IL-7. In some embodiments, the assay comprises a well pre-coated with a treatment directed at detecting one or more cytokines or chemokines.V. Diagnostic MethodsUsing the methods described herein, one can evaluate a biological sample, such as whole blood, diluted whole blood, or isolated PBMC cells from a patient, and determine what disorder(s) they may have. As discussed elsewhere herein, one can determine whether a patient is healthy, has sepsis, or has CINS, based on an evaluation of the mean spot intensity, the maximum spot intensity, and / or the number of low-intensity cells per unit volume present in a biological sample, as counted in a well in which an ELISpot assay is carried out.Information on mean spot intensity, maximum spot intensity and / or number of low intensity cells per unit volume can also be determined for other disorders, such as Covid-19 or other respiratory disorders, cancer, immunosuppressive disorders, and the like. The variance of mean spot intensity, maximum spot intensity and / or number of low intensity cells per unit volume between healthy subjects and patients known to have these specific disorders can be determined, and this information can be stored in a reference library. The library can have anywhere from 2 to 100 or more values for one or more of mean spot intensity, maximum spot intensity and / or number of low intensity cells per unit volume associated with various disorders. Then, once a patient has been screened using the ELISpot analysis, the results can be compared with the reference library, and a best-fit analysis can identify the particular disorder the patient likely has.VL Therapeutic MethodsOnce a patient has been diagnosed as having a particular disorder, a treating physician can consider an appropriate treatment regimen. In some case, the particular treatment regimen will depend on the patient’s particular immune endotype. Accordingly, in addition to treating the underlying disorder, such as sepsis, cancer, and the like, one can also modulate the immune system depending on whether the patient has a hyperimmune endotype or an immunosuppressive endotype. Representative treatments for various disorders, and also for increasing or decreasing the immune response, are discussed below.Antimicrobial CompoundsMicrobial infections, such as bacterial, viral, or fungal infections, can be treated with conventional antimicrobial compounds.Examples of conventional antibiotic agents include, but are not limited to, amikacin, tobramycin, gentamicin, piperacillin, mezlocillin, ticarcillin, imipenem, ciprofloxacin, ceftazidime, aztreonam, ticarcillin-clavulanate, dicloxacillin, amoxicillin, trimethoprimsulfamethoxazole, cephalexin, piperacillin-tazobactam, linezolid, daptomycin, vancomycin, metronidazole, clindamycin, colistin, tetracycline, levofloxacin, amoxicillin and clavulanic acid (Augmentin®), cioxacillin, dicloxacillin, cefdinir, cefprozil, cefaclor, cefuroxime, erythromycin / sulfisoxazole, erythromycin, clarithromycin, azithromycin, doxycycline, minocycline, tigecycline, imipenem, meropenem, colistimethate / Colistin®, methicillin, oxacillin, nafcillin, carbenicillin, azlocillin, piperacillin and tazobactam (Zosyn®), cefepime, ethambutol, rifampin, and meropenem.These antibiotics can also be combined with compounds that bind to or adsorb bacterial toxins, which can be particularly useful where bacterial toxins result in tissue damage. By way of example, Pseudomonas aeruginosa produces a variety of toxins that lead to cell lysis and tissue damage in the host. Type II toxins include Exotoxin U (Exo U), which degrades the plasma membrane of eukaryotic cells, leading to lysis, phospholipase C (PLC), which damages cellular phospholipids causing tissue damage and stimulates inflammation, alkaline protease, which leads to tissue damage, cytotoxin, which damages cell membranes of leukocytes and causes microvascular damage, elastase, which destroys elastin, a protein that is a component of lung tissue, and pyocyanin, a green to blue water-soluble pigment that catalyzes the formation of tissuedamaging toxic oxygen radicals, impairs ciliary function, and stimulates inflammation. Examples of compounds that bind these toxins include polyphenols and polyanionic polymers.Antifungals can also be co-administered, where the microbe is a fungus. Representative antifungal agents which can be used include fluconazole, posaconazole, viroconazole, itraconazole, echinocandin, amphotericin, and flucytosine. The choice of an appropriate antifungal agent can be made by a treating physician, and the following is a summary of fungal pulmonary infections and their treatments.Histoplasmosis is caused by the fungus Histoplasma capsulatum, and conventional treatment includes Itraconazole mild and chronic pulmonary disease, and Amphotericin B (AmB) with itraconazole for moderate-to-severe histoplasmosis.Blastomycosis is caused by Blastomyces dermatitidis, and conventional treatment includes itraconazole for mild-to-moderate disease and liposomal AmB (L-AmB) followed by itraconazole for life-threatening pulmonary infections.Sporotrichosis is caused by Sporothrix schenckii, and conventional treatment for mild-to- moderate pulmonary disease requires itraconazole, whereas AmB followed by itraconazole is recommended for severe disease.Coccidioidomycosis is caused by Coccidioides immitis and Coccidioides posadasii. Immunocompetent infected hosts may not require treatment, but immunocompromised patients are treated with fluconazole or itraconazole, and, in serious cases with AmB, followed by an azole.Opportunistic fungal infections primarily cause infections in patients who tend to be immunocompromised through a congenital or acquired disease process. Representative opportunistic infections are discussed below.Aspergillosis is caused by Aspergilli, and the associated disorders include invasive pulmonary aspergillosis (IPA), chronic necrotizing aspergillosis, Aspergilloma, and allergic bronchopulmonary aspergillosis. Conventional treatments for IPA include voriconazole, lipid- based AmB formulations, echinocandins, and posaconazole.Cryptococcosis is an opportunistic infection seen in immunocompromised individuals, including HIV or AIDS patients and organ-transplant recipients. Conventional treatments include AmB, with or without flucytosine, followed by oral fluconazole. For immunosuppressed or immunocompetent patients exhibiting mild-to-moderate symptoms, fluconazole therapy is recommended.Candidiasis can be caused when lung parenchyma become colonized with Candida species. Many critically ill patients are empirically treated with broad-spectrum antibiotics. Further clinical deterioration and lack of improvement in these cases suggest the initiation of empiric antifungal therapy. Triazole antifungals and echinocandins exhibit excellent lung penetration, so, in addition to AmB formulations, can be used to treat pulmonary candidiasis.Mucormycosis often occurs in patients with diabetes mellitus, organ or hematopoietic stem cell transplant, neutropenia, or malignancy. Pulmonary mucormycosis is primarily observed in patients with a predisposing condition of neutropenia or corticosteroid use. Due to fungal adherence to and damage of endothelial cells, fungal angioinvasion, vessel thrombosis, and successive tissue necrosis, conventional antifungal agents have a difficult time penetrating throughthe lung tissue. For this reason, conventional treatment includes debridement of necrotic tissue and antifungal therapy, using AmB formulations, posaconazole, and iron chelation therapy.Pneumocystis jirovecii Pneumonia (PCP) occurs in patients with HIV / AIDS, hematologic and solid malignancies, organ transplant, and diseases requiring immunosuppressive agents. PCP is extremely resistant to common antifungal therapy, including AmB formulations and triazole antifungals, but can be treated with Trimethoprim / sulfamethoxazole. Second-line agents primaquine plus clindamycin, atovaquone, IV pentamidine, or dapsone.The antifungal agents identified herein can be co-administered with the phototherapy approaches described herein. However, the use of phototherapy can lessen the duration of, and / or increase the efficacy of, such antifungal treatments.Antiviral treatments for viral disorders are well known. Examples include nucleotide reverse transcriptase inhibitors, non-nucleotide reverse transcriptase inhibitors, protease inhibitors, entry inhibitors, and the like. When a patient has a viral pulmonary infection, conventional antiviral agents used for such viruses can be administered. The selection of antivirals typically depends on the viral infection being treated. Influenza virus is typically treated with oseltamivir (Tamiflu), zanamivir (Relenza), or peramivir (Rapivab), and RSV with ribavirin (Virazol). Coronavirus is also being treated with Tamiflu, ribavirin, certain anti-HIV compounds, and certain interferons, including Betaferon, Alferon, Multiferon, and Wellferon.Combination Therapy for Particular Use in Treating Covid- 19 InfectionsThere are various compounds useful for treating Covid- 19 infections, or other respiratory infections with similar pathology, which can be administered by themselves, or in combination with antiviral compounds.Examples include fusion inhibitors, entry inhibitors, protease inhibitors, polymerase inhibitors, antiviral nucleosides, such as remdesivir, GS-441524, N4-hydroxycytidine, and other compounds disclosed in U.S. Patent No. 9,809,616, and their prodrugs, viral entry inhibitors, viral maturation inhibitors, JAK inhibitors, angiotensin-converting enzyme 2 (ACE2) inhibitors, SARS-CoV- specific human monoclonal antibodies, including CR3022, and agents of distinct or unknown mechanism.Umifenovir (also known as Arbidol) is a representative fusion inhibitor.Representative entry inhibitors include Camostat, luteolin, MDL28170, SSAA09E2, SSAA09E1 (which acts as a cathepsin L inhibitor), SSAA09E3, and tetra-O-galloyl-P-D-glucose (TGG).Additionally, when a patient has a hyperimmune endotype, one can administer compounds which inhibit the cytokine storm, such as dexamethasone. Further, when a patient has, is suspected of having, or is at risk of developing blood clots, anti-coagulants and / or platelet aggregation inhibitors that address blood clots, or compounds which chelate iron ions released from hemoglobin by viruses such as COVID- 19.Representative ACE-2 inhibitors include sulfhydryl-containing agents, such as alacepril, captopril (capoten), and zefnopril, dicarboxylate-containing agents, such as enalapril (vasotec), ramipril (altace), quinapril (accupril), perindopril (coversyl), lisinopril (listril), benazepril (lotensin), imidapril (tanatril), trandolapril (mavik), and cilazapril (inhibace), and phosphonate- containing agents, such as fosinopril (fositen / monopril).Compounds for Inhibiting the Cytokine StormThroughout its activation, the inflammatory response must be regulated to prevent a damaging systemic inflammation, also known as a “cytokine storm.” A number of cytokines with anti-inflammatory properties are responsible for this, such as IL- 10 and transforming growth factor P (TGF-P). Each cytokine acts on a different part of the inflammatory response. For example, products of the Th2 immune response suppress the Thl immune response and vice versa.By resolving inflammation, one can minimize collateral damage to surrounding cells, with little or no long-term damage to the patient. Accordingly, in addition to using the compounds described herein to inhibit the viral infection, one or more compounds which inhibit the cytokine storm can be co-administered.Compounds for Treating or Preventing Blood ClotsViruses that cause respiratory infections, including Coronaviruses such as Covid- 19, can be associated with pulmonary blood clots, and blood clots that can also do damage to the heart.The compounds described herein can be co-administered with compounds that inhibit blood clot formation, such as blood thinners, or compounds that break up existing blood clots, suchas tissue plasminogen activator (TP A), Integrilin (eptifibatide), abciximab (ReoPro) or tirofiban (Aggrastat).Blood thinners prevent blood clots from forming, and keep existing blood clots from getting larger. There are two main types of blood thinners. Anticoagulants, such as heparin or warfarin (also called Coumadin), slow down biological processes for producing clots, and antiplatelet aggregation drugs, such as Plavix, aspirin, prevent blood cells called platelets from clumping together to form a clot.By way of example, Integrilin® is typically administered at a dosage of 180 mcg / kg intravenous bolus administered as soon as possible following diagnosis, with 2 mcg / kg / min continuous infusion (following the initial bolus) for up to 96 hours of therapy.Representative platelet aggregation inhibitors include glycoprotein IIB / IIIA inhibitors, phosphodiesterase inhibitors, adenosine reuptake inhibitors, and adenosine diphosphate (ADP) receptor inhibitors. These can optionally be administered in combination with an anticoagulant.Representative anti-coagulants include coumarins (vitamin K antagonists), heparin and derivatives thereof, including unfractionated heparin (UFH), low molecular weight heparin (LMWH), and ultra-low-molecular weight heparin (ULMWH), synthetic pentasaccharide inhibitors of factor Xa, including Fondaparinux, Idraparinux, and Idrabiotaparinux, directly acting oral anticoagulants (DAOCs), such as dabigatran, rivaroxaban, apixaban, edoxaban and betrixaban, and antithrombin protein therapeutics / thrombin inhibitors, such as bivalent drugs hirudin, lepirudin, and bivalirudin and monovalent argatroban.Representative platelet aggregation inhibitors include pravastatin, Plavix (clopidogrel bisulfate), Pletal (cilostazol), Effient (prasugrel), Aggrenox (aspirin and dipyridamole), Brilinta (ticagrelor), caplacizumab, Kengreal (cangrelor), Persantine (dipyridamole), Ticlid (ticlopidine), Yosprala (aspirin and omeprazole).Antiviral AgentsRepresentative antiviral agents include lopinavir, ritonavir, niclosamide, promazine, PNU, UC2, cinanserin (SQ 10,643), Calmidazolium (C3930), tannic acid, 3-isotheaflavin-3-gallate, theaflavin-3,3’ -digallate, glycyrrhizin, S-nitroso-N-acetylpenicillamine, nelfinavir, niclosamide, chloroquine, hydroxychloroquine, 5-benzyloxygramine, ribavirin, Interferons, such as Interferon (IFN)-a, IFN-P, and pegylated versions thereof, as well as combinations of these compounds withribavirin, chlorpromazine hydrochloride, triflupromazine hydrochloride, gemcitabine, imatinib mesylate, dasatinib, and imatinib.ImmunosuppressantsImmunosuppressants are drugs that work to broadly dampen the immune response. Some examples of traditional immunosuppressants include anti-IL-6(R) antibodies, IL-1 receptor antagonists, methotrexate, sulfasalazine, cyclosporine, azathioprine, leflunomide, hydroxychloroquine, JAK-STAT inhibitors, such as Baricitinib, Tofacitinib, and Jakafi, and steroids such as dexamethasone, prednisone, prednisolone, methyl prednisolone and cortisone. Representative examples of biologic drugs are abatacept (Orencia), adalimumab (Humira), dupilumab (Dupixent), etanercept (Enbrel), infliximab (Remicade), mepolizumab (Nucala), omalizumab (Xolair), rituximab (Rituxan), secukinumab (Cosentyx), tocilizumab (Actemra), and ustekinumab (Stelara).Disease-modifying therapies for MSMS is treated with disease-modifying therapies (DMTs). These drugs help to lower levels of inflammation, thereby lowering the likelihood of relapses and preventing additional damage to myelin and the nerves under it. Examples include cladribine (Mavenclad), fingolimod (Gilenya), glatiramer acetate (Copaxone, Glatopa), interferon-beta la (Avonex, Rebif), interferon-beta lb (Betaseron, Extavia), natalizumab (Tysabri), ocrelizumab (Ocrevus), ofatumumab (Kesimpta), and siponimod (Mayzent).Cancer TreatmentsExemplary cancer treatments include radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, and angiogenesis inhibitors.Radiation therapyRadiation therapy (also called radiotherapy, X-ray therapy, or irradiation) is the use of ionizing radiation to kill cancer cells and shrink tumors by damaging their DNA (the molecules inside cells that carry genetic information and pass it from one generation to the next), making itimpossible for these cells to continue to grow and divide. Radiation therapy can either damage DNA directly or create charged particles (free radicals) within the cells that can in turn damage the DNA. Radiation therapy can be administered externally via external beam radiotherapy (EBRT) or internally via brachytherapy. The effects of radiation therapy are localised and confined to the region being treated. Although radiation damages both cancer cells and normal cells, most normal cells can recover from the effects of radiation and function properly. The goal of radiation therapy is to damage as many cancer cells as possible, while limiting harm to nearby healthy tissue. Hence, it is given in many fractions, allowing healthy tissue to recover between fractions.Radiation therapy may be used to treat almost every type of solid tumor, including cancers of the brain, breast, cervix, larynx, liver, lung, pancreas, prostate, skin, stomach, uterus, or soft tissue sarcomas. Radiation is also used to treat leukemia and lymphoma. Radiation dose to each site depends on a number of factors, including the radio sensitivity of each cancer type and whether there are tissues and organs nearby that may be damaged by radiation. Thus, as with every form of treatment, radiation therapy is not without its side effects.ChemotherapyChemotherapy is the treatment of cancer with drugs ("anticancer drugs") that can destroy cancer cells. Chemotherapy can be given in a variety of ways such as injections into the muscles, skin, artery, or vein, or it could even be taken by mouth in the form of a pill. In current usage, the term "chemotherapy" usually refers to cytotoxic drugs which affect rapidly dividing cells in general, in contrast with targeted therapy. Chemotherapy drugs interfere with cell division in various possible ways, e.g. with the duplication of DNA or the separation of newly formed chromosomes. Most forms of chemotherapy target all rapidly dividing cells and are not specific to cancer cells, although some degree of specificity may come from the inability of many cancer cells to repair DNA damage, while normal cells generally can. Hence, chemotherapy has the potential to harm healthy tissue, especially those tissues that have a high replacement rate (e.g. intestinal lining). These cells usually repair themselves after chemotherapy.Because some drugs work better together than alone, two or more drugs are often given at the same time. This is called "combination chemotherapy"; most chemotherapy regimens are given in a combination.Targeted therapiesTargeted therapy constitutes the use of agents specific for the deregulated proteins of cancer cells. Small molecule targeted therapy drugs are generally inhibitors of enzymatic domains on mutated, overexpressed, or otherwise critical proteins within the cancer cell. Prominent examples are the tyrosine kinase inhibitors imatinib (Gleevec / Glivec) and gefitinib (Iressa).Monoclonal antibody therapy is another strategy in which the therapeutic agent is an antibody which specifically binds to a protein on the surface of the cancer cells. Examples include the anti-HER2 / neu antibody trastuzumab (Herceptin) used in breast cancer, and the anti-CD20 antibody rituximab, used in a variety of B-cell malignancies.Targeted therapy can also involve small peptides as "homing devices" which can bind to cell surface receptors or affected extracellular matrix surrounding the tumor. Radionuclides which are attached to these peptides (e.g. RGDs) eventually kill the cancer cell if the nuclide decays in the vicinity of the cell. Especially oligo- or multimers of these binding motifs are of great interest, since this can lead to enhanced tumor specificity and avidity.Photodynamic therapy (PDT) is a ternary treatment for cancer involving a photosensitizer, tissue oxygen, and light (often using lasers

[0013] ). PDT can be used as treatment for basal cell carcinoma (BCC) or lung cancer; PDT can also be useful in removing traces of malignant tissue after surgical removal of large tumors.Targeted therapies under pre-clinical development as potential cancer treatments include morpholino splice switching oligonucleotides, which induce ERG exon skipping in prostate cancer models, multitargeted kinase inhibitors that inhibit the PI3K with other pathways including MEK and PIM, and inhibitors of NF-KB in models of chemotherapy resistance.Hormonal therapyThe growth of some cancers can be inhibited by providing or blocking certain hormones. Common examples of hormone-sensitive tumors include certain types of breast and prostate cancers. Blocking estrogen or testosterone is often an important additional treatment. In certain cancers, administration of hormone agonists, such as progestogens may be therapeutically beneficial.Angiogenesis inhibitorsAngiogenesis inhibitors prevent the extensive growth of blood vessels (angiogenesis) that tumors require to survive. Some, such as bevacizumab, have been approved and are in clinical use. One of the main problems with anti-angiogenesis drugs is that many factors stimulate blood vessel growth in cells normal or cancerous. Anti-angiogenesis drugs only target one factor, so the other factors continue to stimulate blood vessel growth. Other problems include route of administration, maintenance of stability and activity and targeting at the tumor vasculature.Cancer ImmunotherapyA patient suffering from cancer can be treated with various types of immunotherapy, examples of which are listed below:Immune checkpoint inhibitors function by turning off signals that prevent immune cells from responding to cancer cells.Cytokines are small proteins that are involved in immune system signaling.Immunomodulating agents are groups of drugs such as thalidomide and lenalidomide that target immune pathways, and are typically used for cancers such as multiple myeloma.Chimeric antigen receptor (CAR) T-cell therapy extracts immune cells called T cells from the patient’s blood. These cells are then modified in a lab so that they specifically respond to the patient’s cancer.Cancer vaccines stimulate the immune system to respond to cancer. Representative cancers that can be treated using cancer vaccines include melanoma, prostate cancer, and certain lung cancers.Immunostimulatory AgentsImmunostimulants, also known as immunostimulators, are substances (drugs and nutrients) that stimulate the immune system by inducing activation or increasing activity of any of its components. One notable example is the granulocyte macrophage colony-stimulating factor (GM-CSF). When it is appropriate to enhance the immune response, immunostimulatory agents such as IL-7, GM-CSF, prolactin, growth hormone, checkpoint inhibitors, intcrfcron-y, or vitamin D can be used. Deoxycholic acid stimulates macrophages, and imiquimod and resiquimod activate immune cells through the toll-like receptor 7, and these agents can also be used.Evaluation of Drug EfficacyYet another aspect of the present disclosure provides for an ELISpot or FluorSpot assay comprising wells, wherein the wells are precoated with one or more test therapeutic agents or one or more cytokine or chemokine detecting agents. In some embodiments, the one or more test therapeutic agents are tocilizumab, haptoglobin, hemopexin, ox40, IL7, or steroids. In some embodiments, the method further comprises a biological sample in fluid contact with the precoated wells, wherein the biological sample comprises whole blood, diluted whole blood, or isolated immune cells. In some embodiments, the biological sample is obtained from a subject having or suspected of having sepsis, COVID-19, cancer, trauma, or autoimmune disease; a critically ill nonseptic (CINS) subject or post-transplant patient; or an immunosuppressed or a pediatric patient. In some embodiments, the assay produces accelerated results compared to conventional PBMC assays.KitsYet another aspect of the present disclosure provides for a kit comprising an ELISpot or FluoroSpot assay comprising test agent-coated wells or wells coated with cytokine or chemokine detecting agents; and / or optionally a biological sample comprising whole blood, diluted whole blood, or PBMCs.Other objects and features will be in part apparent and in part pointed out hereinafter.Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.EXAMPLESThe following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Example 1: A Population of Low-Intensity IFN-y Producing T Cells in Patients with SepsisThe ELISpot immune function assay has been used to identify immunosuppression in critically ill patients. The ELISpot assay has typically reported metrics of cytokine production, including the number of “spot forming units”, the average “Spot Size”, and the total intensity of signal in the well.Underlying these aggregate features are high resolution data which include the characteristics of individual spots in each well, including the mean intensity, maximum intensity and size of each individual spot. FIG. 6 shows raw images from the ELISpot assay, and the events that are identified (algorithmically) following image analysis, where individual event (“spot”) metrics are measured. The results are also tabulated in the table below.The currently available technology calculates and reports the total number of spots, the average spot size, and the total well intensity. This approach focuses on total and average features, but does not assess the characteristics of the individual spots.The present inventor hypothesized that a high resolution analysis of individual spot metrics would provide additional biologically relevant information regarding the state of the immune system. The instant example demonstrates the utility of information gleaned from looking at individual spot metrics.Methods:Whole blood was collected on 1, 4 and 7 days after diagnosis from critically-ill non-septic (CINS) subjects (n=20) and subjects with sepsis (n=39). Samples were also collected from healthy donors (n=20). 5 pl of whole blood was applied to ELISpot plates pre-coated with IFN-y capture antibody and incubated at 37°C in a 5% CO2 incubator. After 22 hours, the plates were washed and developed with secondary antibody against IFN-y. The plates were imaged on a CTL S6 entry M2 analyzer, and spot features recorded in .fcs files using following manufacturer’s procedures. The .fcs files were analyzed using the OMIQ platform (www.omiq.ai).The mean intensity, maximum intensity, size, circularity and total intensity of each spot were extracted and exported to the SPSS and GraphPad Prism. Spot frequency was compared by Kruskal-Wallis Test followed by Dunn’s Post Test.The Distribution Profile of Spontaneous IFN-y Mean Spot Intensity Produced in the ELISpot Assay is a Clinically Relevant Metric of Immune Function in HumansHigh-resolution analysis of spontaneous IFN-y production was performed in a cohort of healthy subject, subjects with sepsis and subjects with non-septic critical illness. Non-septic critical illness and sepsis are presented as examples of disparate states of immune function. We found that healthy persons, critically ill non-septic patients and septic patients had characteristic distributions of spontaneous IFN-y mean spot intensity (Figure 3-A). Blood from healthy controls (green tracing) produce a single sharp peak of high-intensity IFN-y production at 50% of the maximum intensity detected in the assay well. Blood from septic patients (orange tracing) produces a broader peak of lower intensity of 35% of the maximum intensity in the well. Bloodfrom non-septic critically ill patients (blue tracing) produces a bimodal distribution with peaks at -45% of maximum intensity and a second peak at approximately 32% of peak well intensity. These results demonstrate that the distribution profde of maximum spontaneous interferon spot intensity produced in the ELISpot assay can be used as a clinically relevant metric of immune function in humans.The Distribution Profile of Spontaneous IFN-y Maximum Spot Intensity Produced in the ELISpot Assay is Clinically Relevant Metric of Immune Function in HumansThe data show that healthy persons, critically ill non-septic patients and septic patients had characteristic distributions of spontaneous IFN-y maximum spot intensity (FIG. 3-B). Blood from healthy controls (green tracing) produce a single sharp peak of high-intensity IFN-y production at 50% of the maximum intensity detected in the assay well. Blood from septic patients (orange tracing) produces a broader peak of lower intensity of 35% of the maximum intensity in the well. Blood from non-septic critically ill patients (blue tracing) produces a bimodal distribution with peaks at -45% of maximum intensity and a second peak at approximately 32% of peak well intensity. These results demonstrate that the distribution profde of maximum spontaneous interferon spot intensity produced in the ELISpot assay can be used as a clinically relevant metric of immune function in humans.Figures 3-C through 3-E show distributions of spot size, spot circularity, and IFN-y total spot intensity in biological samples obtained from healthy patients (green), critically ill non-septic patients (blue) and septic patients (orange). As shown in these figures, the results do not vary significantly between these patient populations, at least with respect to healthy patients, patients with sepsis, and critically ill, non-septic patients.The Distribution Profile of CD3 / CD28-Stimulated IFN-y Production Maximum Spot Intensity Produced in the ELISpot AssayThe protocol for spontaneous IFN-y production was repeated, but with CD3 / CD28 stimulation by CD3 / CD28-activating antibodies (Abs). The data is shown in FIGS. 4A-E, withFIG. 4A showing the mean spot intensity, Figure 4B showing the maximum spot intensity, and FIGS 4C-E showing distributions of spot size, spot circularity, and IFN-y total spot intensity in CD3 / CD28 stimulated biological samples obtained from healthy patients (green), critically ill non- septic patients (blue) and septic patients (orange).Comparing FIGS. 3A / 3B (spontaneous IFN-y production) with FIGS. 4A / 4B (stimulated IFN-y production), the data show that there are differences in the mean intensity and maximum intensity between spontaneous and stimulated IFN-y production. Looking at FIGS. 3A / 3B, there are profound differences between the mean and maximum intensity associated with healthy, septic and CINS patients, whereas, as shown in FIGS. 4A / 4B, there are significantly less differences between healthy, septic and CINS patients.SummarySpontaneous or CD3 / CD28 stimulated IFN-y production was measured by ELISpot. Data from Days 1, 4, and 7 were pooled, spot level data including mean intensity, maximum intensity, spot size, circularity and total intensity were extracted, and the distribution of each metric compared.Sepsis was associated with a population of mean-intensity spontaneous IFN-y producing cells, as compared to healthy controls. NSCI subjects had a heterogeneous phenotype.The number of lower intensity spots were compared by Kruskal-Wallis Test followed by Dunn’s Post Test. **p<0.01The results are summarized in Figure 5, which shows the number of low intensity spots in sepsis, CINS and healthy patients.The number of “Low-Intensity” Spontaneous IFN-y producing cells measured in the ELISpot Assay is a Clinically Relevant Metric of Immune Function in HumansThe ELISpot assay can effectively measure IFN-y production by T cells in the whole blood of critically ill patients. The ELISpot detection algorithm can identify IFN-y producing features across wide ranges of both spot size and spot intensity.Conventional ELISpot analysis incorporating spot number, spot size and total well intensity poorly discriminates septic from critically-ill non-septic patients.High resolution analysis of the distribution of spot intensity identifies a novel population of low-intensity IFN-y producing T cells in septic patients, which are absent in healthy controls.The number of low-intensity IFN-y producing cells tightly discriminates septic and non- septic critically ill patients, demonstrating that high resolution analysis of ELISpot data using single cell analysis approaches is a powerful tool to extract additional information from the ELISpot assay.The term “low-intensity” IFN-y producing cells is defined as those with a mean spot intensity of <45% of the maximum intensity measured in the well (see, for example, FIGS. 3 A and 4A). The total number of “low intensity” spots was enumerated and compared with the number of low intensity spots in patients with sepsis, and non-septic critical illness (FIG. 5). Whole blood samples from healthy patients, subjected to EliSpot analysis, produced on average 1.3 low- intensity spots, whereas blood samples from CINS patients produced on average 4.3 spots and blood samples from sepsis patients produced 18.5 spots. These resulted in statistically significant differences between the cohorts.The number of low-intensity IFN-y producing cells therefore tightly discriminates septic and non-septic critically-ill patients, demonstrating that the number of low intensity spontaneous IFN-y producing cells can be used as a clinically relevant metric of immune function in humans.Conclusions:The ELISpot assay can effectively measure IFN-y production by T cells in the whole blood of critically ill patients. The ELISpot spot detection algorithm can identify IFN-y producing features across wide ranges of both spot size and spot intensity. High resolution analysis of the distribution of spot intensity identifies a novel population of low-intensity IFN-y producing T cells in septic patients which are absent in healthy controls. The number of low-intensity IFN-y producing cells tightly discriminates septic and non-septic critically-ill patients, demonstrating that high resolution analysis of ELISpot data using single cell analysis approaches is a powerful tool to extract additional information from the ELISpot assay.The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will becomeapparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

Claims1. A method of determining the mean spot intensity, the maximum spot intensity, and / or the number of low-intensity cells per unit volume present in a biological sample from a subject, comprising: a. providing or having been provided a biological sample from the subject; b. placing the sample in a well pre-coated with an agent that stimulates a T cell or monocyte cell or both to secrete a cytokine or chemokine associated with cellular immunity, and c. quantitating the mean spot intensity, the maximum spot intensity, and / or the number of low-intensity cells per unit volume present in a biological sample using ELISpot assay or FluoroSpot assay, wherein the ELISpot assay or FluoroSpot assay detects spots associated with cells that spontaneously secrete a cytokine or chemokine, and those cells have a mean spot intensity, a maximum spot intensity and individual intensity that can be measured, wherein the term “low-intensity” cytokine or chemokine producing cells is defined as those with a mean spot intensity of <45% of the maximum intensity measured in the well, and the term “spontaneously secrete” refers to the spontaneous production of the cytokine or chemokine by cells in the biological sample after they are applied to ELISpot plates pre-coated with a capture antibody specific for the cytokine or chemokine.

2. The method of Claim 1, wherein the cytokine is IFN-y or TNF-a.

3. The method of Claim 1, wherein the biological sample is whole blood, diluted whole blood, or isolated PBMC cells.

4. The method of Claim 1, wherein the number of low-intensity cells per unit volume is determined.

5. The method of Claim 1, further comprising comparing the mean spot intensity, the maximum spot intensity, and / or the number of low-intensity cells per unit volume present in a biological sample from a subject to one or more reference controls.

6. The method of Claim 5, wherein the one or more reference controls comprise one or more of a healthy patient, a sepsis patient, or a critically ill non-sepsis patient.

7. The method of Claim 6, wherein: a) when one or more of the mean spot intensity, the maximum spot intensity, and / or the number of low-intensity cells per unit volume present in the biological sample is similar to the mean spot intensity, the maximum spot intensity, and / or the number of low-intensity cells per unit volume present in a reference control associated with sepsis, the patient is identified as having sepsis, b) when one or more of the mean spot intensity, the maximum spot intensity, and / or the number of low-intensity cells per unit volume present in the biological sample is similar to the mean spot intensity, the maximum spot intensity, and / or the number of low-intensity cells per unit volume present in a reference control associated with a critically ill non-sepsis patient, the patient is identified as being a critically ill non-sepsis patient, and c) when one or more of the mean spot intensity, the maximum spot intensity, and / or the number of low-intensity cells per unit volume present in the biological sample is similar to the mean spot intensity, the maximum spot intensity, and / or the number of low-intensity cells per unit volume present in a reference control associated with a healthy patient, the patient is identified as being a healthy patient.

8. The method of Claim 7, further comprising treating the patient for sepsis if the patient has been identified as having sepsis, and not treating the patient for sepsis if the patient is not identified as having sepsis.

9. The method of Claim 7, further comprising determining whether the patient has an immunosuppressive immunological endotype or a hyper-inflammatory endotype by evaluating the amount of the cytokine associated with cellular immunity, and determining whether this amount is decreased relative to a control, wherein the control is obtained from a healthy patient, wherein if the amount of the cytokine is decreased relative to a control, the patient has an immunosuppressive immunological endotype, and if the amount is increased relative to a control, the patient has a hyper-inflammatory endotype.

10. The method of Claim 9, further comprising treating the patient with an agent that enhances the immune response if the patient is identified as having an immunosuppressive immunological endotype, or treating the patient with an agent that suppresses the immune response if the patient is identified as having a hyper-inflammatory endotype.

11. The method of Claim 10, wherein if the patient has an immunosuppressive immunological endotype, the patient is treated with an immunostimulatory agent.

12. The method of Claim 11, wherein the immunostimulatory agent is IL-7.

13. The method of Claim 10, wherein if the patient has an hyper-inflammatory endotype the patient is treated with an immunomodulatory agent.

14. The method of Claim 11, wherein the immunomodulatory agent is dexamethasone.

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