Methods and assays for performing immunophenotyping

Abstract

Screening methods are provided that are based on using ELISpot assays to measure the distribution profile of maximum spot intensity or mean spot intensity of spontaneous cytokine or chemokine or the number of "low intensity" spontaneous cytokine or chemokine producing cells as a clinically relevant measure of immune function for diagnosing patients with a particular disorder based on one or more of these measurements and for treating the particular disorder in the patient. In some embodiments, the cytokine is IFN-gamma or TNF-alpha. Patients can optionally be further evaluated to determine whether they are of an immunosuppressed or hyperinflammatory immune phenotype by quantifying cytokine and / or chemokine levels in a biological sample. Representative disorders include sepsis, autoimmune disease, autoimmunity, cancer, and lymphopenia.

Description

Technical Field

[0001] This disclosure generally relates to methods and compositions for immunophenotyping patients. Background Technology

[0002] Enzyme-linked immunosorbent assay (ELISpot) is a type of assay focused on quantitatively measuring the frequency of cytokine secretion in individual cells. ELISpot assay is also a form of immunostaining because it is classified as a technique that uses antibodies to detect protein analytes; the term analyte refers to any biological or chemical substance being identified or measured.

[0003] The FluoroSpot assay is a variant of the ELISpot assay. The FluoroSpot assay uses fluorescence to analyze a variety of analytes, meaning it can detect the secretion of more than one type of protein.

[0004] Hotchkiss et al., U.S. Publication No. 20220091133, disclose a method for immunophenotyping (evaluating adaptive and innate immune status) of subjects using the ELISpot assay. Biological samples including whole blood, diluted whole blood, or isolated peripheral blood mononuclear cells (PBMCs) are provided, and the ELISpot can be used to quantify T-cell interferon-γ (IFN-γ) and / or monocyte TNF-α production in the biological samples. If T-cell interferon-γ (IFN-γ) and / or monocyte TNF-α production is relatively low compared to healthy subjects (controls), the subject can be identified as having an immunosuppressive immune endotype; or if it is relatively high compared to healthy subjects, the subject can be identified as having a hyperinflammatory endotype.

[0005] By way of example, COVID-19-related morbidity and death have been attributed to pathological host responses. In some patients (i.e., those with a hyperinflammatory endotype), hyperinflammatory “cytokine storm”-mediated damage occurs, and an overactive immune response may be mediated by excessive pro-inflammatory cytokines, driving excessive lung injury and a procoagulant state. In such patients, death and morbidity may result from inflammatory lung injury, microcirculatory and macrocirculatory disturbances, and the resulting respiratory failure or vasocoagulopathy. 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 is approved by the U.S. Food and Drug Administration for the treatment of COVID-19 patients).

[0006] In other patients, failure of host protective immunity (i.e., immunosuppressive endotype) can lead to uncontrolled viral transmission and organ damage. For these patients, morbidity and death may result from an “immune collapse” of the host’s protective system. This collapse manifests itself as an inability to control uncontrolled viral replication and spread, as well as direct host cytotoxicity. Progressive and severe lymphopenia is observed in such patients and is associated with increased secondary infections and mortality. Patients with this endotype typically exhibit devastating loss of immune cells in their spleen and secondary lymphoid organs, with loss of multiple lymphocyte subsets, including CD4 T, CD8 T, and NK cells, which play important antiviral roles, as well as B cells essential for antibody production.

[0007] In addition to COVID-19, sepsis patients may also have a hyperinflammatory endotype or an immunosuppressive endotype.

[0008] The method disclosed in U.S. Patent Publication No. 20220091133 allows for the evaluation of immune function and the identification of appropriate endotypes. This can be particularly useful in determining appropriate treatment regimens. That is, patients with a hyperinflammatory endotype can be treated with agents that reduce the immune response (such as steroids or JAK inhibitors), and patients with an immunosuppressive endotype can be treated with agents that enhance the immune response (such as interleukin-7 (IL-7) or GM-CSF).

[0009] This type of assessment can be useful when evaluating patients with disorders that may be associated with immunosuppressive intracellular or hyperinflammatory intracellular types. Such patients include those who have, are suspected of having, or are at risk of developing sepsis (such as fungal wound sepsis), autoimmune diseases, autoimmune or cancer, or have lymphopenia (≤ 1100 cells / μL), or have undergone organ transplantation, or are in intensive care.

[0010] Identifying which of these immune endogenous types is dominant may be important because appropriate interventions differ drastically depending on whether a patient has hyperinflammatory or severe immunosuppression. For example, in patients with severe immunosuppression, anti-IL-6(R) antibodies, IL-1 receptor antagonists, and JAK-STAT inhibitors may further impair their ability to fight infection. Conversely, in patients with hyperinflammatory, immunostimulants (such as checkpoint inhibitors, IL-7, interferon-γ, GM-CSF, etc.) may worsen an already dysregulated and intense inflammatory response and exacerbate organ damage.

[0011] The method disclosed in U.S. Publication No. 20220091133 can be used to identify a patient's immune endotype. Methods for immunophenotyping (evaluating adaptive and / or innate immune status) of subjects include providing or having provided biological samples including whole blood, diluted whole blood, or isolated peripheral blood mononuclear cells (PBMCs), and quantifying T-cell interferon-γ (IFN-γ) and / or monocyte TNF-α production in the biological samples using ELISpot. If T-cell interferon-γ (IFN-γ) and / or monocyte TNF-α production is relatively low compared to healthy subjects, the subject can be diagnosed with an immunosuppressive immune endotype, and if they are relatively high, the subject can be diagnosed with a hyperinflammatory endotype. Because a subject's endotype may evolve over time, periodic reassessments can be performed, including determining the subject's response to therapy.

[0012] While these methods can be used to determine a patient's immune endotype, they cannot absolutely predict the type of disease that will result in a patient having an immunosuppressive or hyperinflammatory immune endotype relative to healthy subjects. It would be useful if there were alternative methods to collect additional information from the ELISpot assay that could help identify the type of disorder a patient has. This invention provides such methods. Summary of the Invention

[0013] In one embodiment, a highly sensitive functional immunoassay is disclosed as an enzyme-linked immunosorbent assay (ELISpot). This assay analyzes biological samples, such as whole blood, diluted whole blood, or isolated peripheral blood mononuclear cells (PBMCs), and determines the average spot intensity, maximum spot intensity, and number of low-intensity cells per unit volume present in the biological sample, as counted in the wells used for the ELISpot assay.

[0014] As used herein, the term “low-intensity” IFN-γ producing cells are defined as cells with an average spot intensity less than 45% of the maximum intensity measured in the well.

[0015] As used herein, “spontaneous IFN-γ” refers to the spontaneous production of IFN-γ in cells from a biological sample (such as whole blood, diluted whole blood, or isolated peripheral blood mononuclear cells (PBMCs)) applied to an ELISpot plate pre-coated with an IFN-γ capture antibody and ideally incubated at 37°C in a 5% CO2 incubator without stimulation. This contrasts with IFN-γ production induced by administration of CD3 / CD28 to incubated cells.

[0016] In some aspects of this embodiment, the screening method described herein is based on the fact that the distribution profile of the average spot intensity of spontaneous IFN-γ generated in the ELISpot assay is a clinically relevant measure of human immune function.

[0017] In other aspects of this embodiment, the screening method described herein is based on the fact that the distribution profile of the spontaneous IFN-γ maximum spot intensity generated in the ELISpot assay is a clinically relevant measure of human immune function.

[0018] In other aspects of this embodiment, the screening method described herein is based on the fact that the number of “low-intensity” spontaneously producing IFN-γ cells measured in the ELISpot assay is a clinically relevant measure of human immune function.

[0019] In another embodiment, a patient is diagnosed with a specific type of disorder based at least in part on the spontaneous IFN-γ mean spot intensity, spontaneous IFN-γ maximum spot intensity, and / or the number of low-intensity spots per unit volume (as counted in an ELISpot assay plate) present in the biological sample.

[0020] In one aspect of this embodiment, comparisons can be made with controls (i.e., the number of low-intensity spots per unit volume in a biological sample derived from healthy subjects) and / or based on a reference library comprising ranges of low-intensity spots per unit volume in biological samples that are associated with patients having a specific disorder. The library may include any range from two to one thousand or more, for example. Where ranges overlap with those associated with more than one type of disorder, additional clinically relevant information can be used to further identify the disorder and make an appropriate diagnosis of the patient.

[0021] In some aspects of this embodiment, specific disorders may be associated with immunosuppressive endotypes or hyperinflammatory endotypes, and a patient's endotype can be further assessed. For example, an ELISpot assay (which may be the same ELISpot assay used to diagnose disorders) can be used to quantify cytokine production and / or chemokine levels, such as interferon-γ (IFN-γ) and / or monocyte TNF-α production, in individual cells of a biological sample. If T-cell interferon-γ (IFN-γ) and / or monocyte TNF-α production is relatively low compared to healthy subjects, the subject can be diagnosed with an immunosuppressive endotype, and if they are relatively high, the subject can be diagnosed with a hyperinflammatory endotype. Because a subject's endotype may evolve over time, periodic reassessments can be performed, including determining the subject's response to therapy.

[0022] In other embodiments, an appropriate treatment regimen can be used to treat the specific disorder in a patient who has been diagnosed with the specific disorder. Where the disorder is one in which appropriate treatment depends on the subject's immune endotype, the appropriate treatment regimen can be determined by: a) identifying the specific disorder the patient has, b) identifying the patient's immune endotype, and c) selecting an appropriate treatment regimen for a patient with a given disorder and a given immune endotype.

[0023] For some patients, particularly those with a hyperinflammatory intraepithelial type, 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 is approved by the U.S. Food and Drug Administration for the treatment of COVID-19 patients). For other patients, particularly those with an immunosuppressive intraepithelial type, appropriate treatment may involve the administration of agents that enhance the immune response, such as interleukin-7 (IL-7) or GM-CSF.

[0024] Treating patients with immunomodulators (i.e., agents that reduce or enhance the immune response) may be beneficial, but it is also important to be aware of potential barriers to treatment. For example, patients with bacterial sepsis may be treated with one or more antibiotics, patients with viral sepsis with one or more antiviral agents, and patients with fungal sepsis with one or more antifungal agents.

[0025] In some embodiments, the patient has, is suspected of having, or is at risk of developing sepsis (such as fungal wound sepsis), an autoimmune disease, an autoimmune disease or cancer, or has lymphopenia (≤ 1100 cells / μL), or has undergone an organ transplant, or is in intensive care.

[0026] 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 of developing sepsis. In some aspects of these embodiments, at the time of diagnosis, it is unclear whether the patient is healthy, has sepsis, or is critically ill but not sepsis-prone. In other aspects of these embodiments, at the time of diagnosis, it is unclear whether the patient has sepsis or is critically ill but not sepsis-prone. In any of these embodiments, the determination of the number of low-intensity spots per unit volume of biological sample counted in an ELISpot assay plate is used to distinguish between patients with sepsis and critically ill but not sepsis-prone, and optionally, healthy patients.

[0027] In another embodiment, the patient has or is suspected of having an autoimmune disease, autoimmune disease or cancer, or lymphopenia (≤ 1100 cells / μL), or has undergone organ transplantation, or is in intensive care. For these patients, based on patients known to have these disorders, data for one or more of these disorders are determined using the ELISpot criteria as described herein, including the mean blot intensity, maximum blot intensity, and / or the number of low-intensity cells per unit volume present in the biological sample. A biological sample is then obtained from the patient, and the mean blot intensity, maximum blot intensity, and / or the number of low-intensity cells per unit volume present in the biological sample are determined. The patient's information is compared with reference information, and a diagnosis can be made based on the comparison of the patient's information with the closest matching reference information.

[0028] In yet another embodiment, the patient has or is suspected of having an autoimmune disease, autoimmune disease or cancer, or lymphopenia (≤ 1100 cells / μL), or has undergone an organ transplant, or is in intensive care, and these methods allow for the identification and tracking of immune dysfunction in any of these conditions, for example, to characterize the severity of the disease, track the progression of the disease over time, and / or help in selecting appropriate therapies.

[0029] In any of these embodiments, some information is based on a specific type of biological sample and sample volume, such as 5 µl of whole blood. If larger or smaller sample volumes are used, or if the blood is diluted, the values ​​expected will vary linearly. Therefore, the methods described herein do not rely on following the precise steps in the working examples, but rather on the extent to which they vary with different biological samples, sample volumes, and dilutions.

[0030] The methods described herein can be performed, for example, after CD3 / CD28 stimulation or after medical treatment (such as administration of dexamethasone or IL-7) using any ELISpot assay conditions. While the examples describe results using IFN-γ, the ELISpot assay can be performed using any appropriate cytokine or chemokine.

[0031] In another embodiment, these methods are used to determine the response to treatments such as IL-7 or other immunomodulatory therapies such as corticosteroids (e.g., dexamethasone). In this embodiment, these methods can be used in conjunction with any condition for which immunomodulatory agents are administered, including cancer, transplantation, and autoimmune diseases. For example, cancer can be treated with immunomodulatory agents, and this technique can be used to determine the response to that treatment. Similarly, transplant patients can be treated with immunosuppressants to prevent rejection. This technique can also be used as a way to track the efficacy of these treatments. Attached Figure Description

[0032] Those skilled in the art will understand that the accompanying drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of this teaching in any way.

[0033] Figure 1A-D These are representative ELISpot micrographs illustrating the results of adaptive immunosuppression in COVID-19 patients. Representative ELISpot micrographs show IFN-γ production in (1A) healthy volunteers, (1B) CINS patients, and (1C) sepsis-free non-COVID-19 patients after overnight stimulation with anti-CD3 / anti-CD28 antibodies. (1D) shows three representative COVID-19 positive samples.

[0034] Figure 2A-D These are representative ELISpot images depicting unstimulated and stimulated in vitro production of IFN-γ and TNF-α from whole blood of sepsis patients, as measured using ELISpot. Figure (2A) shows a representative ELISpot image depicting IFN-γ production in culture medium alone compared to production with CD3 / CD28 Ab. Figure (2B) shows a graphical representation of the response in n = 15 sepsis patients between unstimulated and stimulated in vitro cytokine production of IFN-γ. Figure (2C) shows a representative ELISpot image depicting TNF-α production in culture medium alone compared to production with LPS. Figure (2D) is a graphical representation of the response in n = 15 sepsis patients between unstimulated and stimulated in vitro cytokine production of TNF-α. The red line represents death.

[0035] Figure 3A shows the distribution of the average spot intensity of spontaneous IFN-γ in biological samples obtained from healthy patients (green), critically ill non-septic patients (blue), and septic patients (orange).

[0036] Figure 3B shows the distribution of spontaneous IFN-γ maximum spot intensity in biological samples obtained from healthy individuals (green), critically ill non-septic patients (blue), and septic patients (orange).

[0037] Figure 3C shows the distribution of spontaneous IFN-γ spot sizes in biological samples obtained from healthy patients (green), critically ill non-septic patients (blue), and septic patients (orange).

[0038] Figure 3D shows the distribution of spontaneous IFN-γ spot circularity in biological samples obtained from healthy patients (green), critically ill non-septic patients (blue), and septic patients (orange).

[0039] Figure 3E is a graph showing the distribution of spontaneous total IFN-γ spot intensity in biological samples obtained from healthy patients (green), critically ill non-septic patients (blue), and septic patients (orange).

[0040] Figure 4A The figure shows the distribution of the average speckled intensity of CD3 / CD28-stimulated IFN-γ in biological samples obtained from healthy patients (green), critically ill non-septic patients (blue), and septic patients (orange).

[0041] Figure 4B The figure shows the distribution of maximum speckled intensity of CD3 / CD28-stimulated IFN-γ in biological samples obtained from healthy individuals (green), critically ill non-septic patients (blue), and septic patients (orange).

[0042] Figure 4C The figure shows the distribution of spot size in CD3 / CD28-stimulated biological samples obtained from healthy patients (green), critically ill non-septic patients (blue), and septic patients (orange).

[0043] Figure 4D The figure shows the distribution of spot roundness in CD3 / CD28-stimulated biological samples obtained from healthy patients (green), critically ill non-septic patients (blue), and septic patients (orange).

[0044] Figure 4E The figure shows the distribution of total IFN-γ spot intensity in CD3 / CD28-stimulated biological samples obtained from healthy patients (green), critically ill non-septic patients (blue), and septic patients (orange).

[0045] Figure 5 The graph shows a comparison of the total number of “low-intensity” spots in healthy patients (green) with the number of low-intensity spots in patients with sepsis (orange) and non-sepsis-related critical illness (blue).

[0046] Figure 6 The original image from the ELISpot determination is shown, along with the event identified after image analysis. Detailed Implementation

[0047] Enzyme-linked immunosorbent assay (ELISpot) is a highly sensitive functional immunoassay that measures the number of cytokine-secreting cells in response to in vitro stimulation after an incubation period at the single-cell level. ELISpot assays offer excellent dynamic range and can detect as few as one cell per 100,000 cytokine-secreting cells. Furthermore, ELISpot can simultaneously test the integrity and robustness of two distinct branches of immunity—namely, innate cellular immunity (blood monocytes and low-density granulocytes) and adaptive cellular immunity (blood lymphocytes)—by focusing on the response of individual cell populations to cell-specific agonists.

[0048] In one embodiment, this disclosure relates to an ELISpot assay that analyzes a biological sample, such as whole blood, diluted whole blood, or isolated peripheral blood mononuclear cells (PBMCs), and determines the average spot intensity, maximum spot intensity, and / or the number of low-intensity cells per unit volume present in the biological sample, as counted in the well in which the ELISpot assay is performed.

[0049] The distribution profile of the spontaneous IFN-γ mean spot intensity, the spontaneous IFN-γ maximum spot intensity, and / or the number of low-intensity spots per unit volume generated in an ELISpot assay are clinically relevant measures of human immune function and can be used to diagnose specific disorders in patients. Patients can receive appropriate treatment after diagnosis.

[0050] In some embodiments, the evaluation is periodically re-performed to determine the subject’s response to the therapy. In other embodiments, the evaluation is re-performed after the patient’s biological sample has been exposed to an agent that enhances (i.e., IL-7) or reduces (i.e., dexamethasone) the immune response.

[0051] 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-γ can be evaluated. For example, similar results can be obtained using TNF-α.

[0052] As described herein, control or reference samples can be samples from healthy subjects. Reference values ​​can be used instead of control or reference samples previously obtained from healthy subjects or a group of healthy subjects. Control or reference samples can also be samples or spiked samples with known amounts of a detectable compound. Other iterations of ELISpot, such as FluoroSpot, can be used.

[0053] These embodiments will be described in further detail below.

[0054] definition

[0055] The definitions and methods described herein are provided to better define this disclosure and to guide those skilled in the art in practicing it. Unless otherwise stated, the terminology should be understood by those skilled in the art based on its conventional usage.

[0056] As used herein, the term “low-intensity” IFN-γ producing cells are defined as cells with an average spot intensity less than 45% of the maximum intensity measured in the well.

[0057] As used herein, “spontaneous IFN-γ” refers to the spontaneous production of IFN-γ in cells from a biological sample (such as whole blood, diluted whole blood, or isolated peripheral blood mononuclear cells (PBMCs)) applied to an ELISpot plate pre-coated with an IFN-γ capture antibody and ideally incubated at 37°C in a 5% CO2 incubator. This contrasts with (stimulated) IFN-γ production induced by administration of CD3 / CD28 to incubated cells.

[0058] As a way to compare the two, Figure 2A-D The figures compare spontaneous and stimulated production, and show data on in vitro production of IFN-γ and TNF-α from whole blood of a series of sepsis patients without stimulation with anti-CD3 / CD28 or LPS (average values ​​of the intensity observed in specific wells).

[0059] Figure 1A-D Adaptive immunosuppression in COVID-19 patients is illustrated. Representative ELISpot micrographs show IFN-γ production in (1A) healthy volunteers, (1B) CINS patients, and (1C) sepsis-free non-COVID-19 patients after overnight stimulation with anti-CD3 / anti-CD28 antibodies. Figure (1D) shows three representative COVID-19 positive samples. The number of spots represents the number of cytokine-producing T cells. The count is presented as the corrected number of spots per thousand lymphocytes in the plating, as per a plating density of 2.5 × 10⁻⁶ in each well. 4 Fractions of PBMCs were used. Note that IFN-γ production was reduced in both sepsis and COVID-19 patients compared to CINS patients. It should also be noted that there is some heterogeneity in IFN-γ production between COVID-19 and sepsis patients. Each photomicrograph was captured at the same magnification, and each image is scaled. ELISpot assays were performed using PBMC fractions from freshly drawn whole blood. For control samples, each condition was run in duplicate, and for COVID-19 samples, each condition was run in triplicate.

[0060] Figure 2A-DThe figures show unstimulated and stimulated in vitro production of IFN-γ and TNF-α from whole blood of sepsis patients, as measured using ELISpot. Figure (2A) shows a representative ELISpot image depicting IFN-γ production in culture medium alone compared to production with CD3 / CD28 Ab. Figure (2B) shows a graphical representation of the response in n = 15 sepsis patients between unstimulated and stimulated in vitro cytokine production of IFN-γ. Figure (2C) shows a representative ELISpot image depicting TNF-α production in culture medium alone compared to production with LPS. Figure (2D) is a graphical representation of the response in n = 15 sepsis patients between unstimulated and stimulated in vitro cytokine production of TNF-α. The red line represents death.

[0061] Routine ELISpot assays calculate and report the total number of spots, mean spot size, and total pore intensity. This method focuses on total and mean characteristics but does not assess the characteristics of individual spots. In this particular instance, IFN-γ was essentially not produced without stimulation by CD3 / CD28 activating Abs. This lack of cytokine production is present not only in whole blood ELISpots but also in ex vivo IFN-γ production in PBMCs (data not shown). Sepsis patient samples produced spontaneous, unstimulated TNF-α (i.e., without LPS addition) in contrast to IFN-γ.

[0062] This data shows that different cytokines can be used to obtain different information; therefore, it may be useful to observe the results of different cytokines / chemokines for different disorders until a strong correlation can be observed, for example, between healthy patients and patients with a specific disorder or patients with different disorders that are typically difficult to distinguish using other methods. By finding the correct cytokine or chemokine for a given assessment, conditions for appropriate assays for diagnosing disorders and optionally for differentiating two or more disorders can be identified.

[0063] In some embodiments, the numbers representing the amount, properties (such as molecular weight, reaction conditions), etc., of components used to describe and claim certain embodiments of this disclosure should be understood to be modified by the term "about" in some cases. In some embodiments, the term "about" is used to indicate that the value includes the standard deviation of the average of the means or methods used to determine the value. In some embodiments, the numerical parameters listed in the written description and appended claims are approximations that vary depending on the desired properties sought to be obtained in a particular embodiment. In some embodiments, numerical parameters should be interpreted according to the number of significant figures reported and by applying common rounding techniques. While the ranges and parameters of numbers listing a broad range of some embodiments of this disclosure are approximations, the values ​​listed in particular instances are reported as precisely as possible. The numerical values ​​presented in some embodiments of this disclosure may contain some error that is necessarily caused by the standard deviation found in their respective test measurements. The description of ranges of values ​​herein is intended only as a shorthand method of individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into this specification as if it were individually referenced herein. The description of discrete values ​​should be understood to include the range between each value.

[0064] In some embodiments, the terms “a” and “an” and “the” as used in the context of describing particular embodiments (particularly in the context of some of the following claims), and similar references, may be interpreted to include both the singular and the plural, unless otherwise specifically stated. In some embodiments, the term “or” as used herein (including the claims) is used to mean “and / or”, unless it is explicitly stated that it refers only to alternatives or that the alternatives are mutually exclusive.

[0065] The terms “comprise,” “have,” and “include” are open-ended linking verbs. Any form or tense of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” is also open-ended. For example, any method that “comprises,” “has,” or “includes” one or more steps is not limited to having only those steps and may also include other steps not listed. Similarly, any composition or apparatus that “comprises,” “has,” or “includes” one or more features is not limited to having only those features and may include other features not listed.

[0066] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by the context. The use of any and all instances or exemplary language (e.g., “such”) provided with respect to certain embodiments herein is merely intended to better illustrate this disclosure and does not limit the scope of this disclosure as originally claimed. No language in this specification should be construed as indicating that any unclaimed element is necessary to practice this disclosure.

[0067] The grouping of alternative elements or embodiments disclosed herein should not be construed as limiting. Each member of a group may be mentioned and claimed individually or in any combination with other members of that group or other elements discovered herein. For convenience or patentability reasons, one or more members of a group may be included in or removed from the group. When any such inclusion or removal occurs, the specification herein is deemed to contain the group as modified, thereby fulfilling the written description of all Markush groups used in the appended claims.

[0068] 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 extent that each individual publication, patent, patent application or other reference is specifically and individually indicated as being incorporated herein by reference in its entirety for all purposes. References herein should not be construed as an admission that they are prior art to this disclosure.

[0069] I. ELISpot / FluoroSpot Assays as described herein may include capture antibodies (e.g., anti-cytokines) attached, coated, or immobilized on a surface or membrane. Cells stimulated by the antigen may secrete the antigen captured by the capture antibody. Detection antibodies may be added to bind to the antigen captured on the capture antibody. Chromogens (substances that can be readily converted into dyes or other coloring compounds) may be added.

[0070] Enzyme-linked immunosorbent assay (ELISpot) is a type of assay that focuses on quantitatively measuring the frequency of cytokine secretion in individual cells or cell populations. ELISpot assays are also a form of immunostaining because they are classified as techniques that use antibodies to detect analytes or proteins (such as biological or chemical substances being identified or measured). FluoroSpot assays are a variant of ELISpot assays. FluoroSpot assays use fluorescence to analyze multiple analytes, thus allowing the detection of secretion from more than one type of analyte or protein.

[0071] ELISpot mechanism Antibody coating (e.g., attachment, fixation, or coating): Throughout the ELISpot assay, different substances are added to and washed away from the wells. Wells exist on a laboratory plate, which has tiny dimples (dish / bowls) that can be filled with the substance to be tested; the amount of material in the wells varies, but typically ranges from 16 to 100. The first substance added to the wells can be a cytokine-specific monoclonal antibody. These antibodies can coat the well walls for future binding to the cytokine. Monoclonal antibodies are antibodies produced by a single-cell lineage and are only capable of binding to one protein epitope. Polyclonal antibodies, on the other hand, can bind to multiple epitopes of the same protein.

[0072] Cell incubation: The desired cells for observation and analysis are added to the wells. Each well may or may not contain a stimulus that activates cytokine secretion in the cells. During cell incubation, cells are allowed to respond to any present stimulus and secrete cytokines. Numerous procedures and methods are known in the art to ensure proper cell handling. To ensure high cell quality, if storage is longer than 3 hours, cells in blood samples may be gently agitated; blood samples may be diluted in PBS (phosphate-buffered saline) before storage; and blood samples may be granulocyte-free. Any cells that have been cryopreserved and thawed may be allowed to stand at 37°C (typical human body temperature) for one hour or longer. Several considerations can be included when incubating cells to ensure that cells do not experience sudden movement that could affect spot formation, or to ensure that the humidity of the incubator is high enough to avoid excessive evaporation and drying of the wells.

[0073] Cytokine capture: As the cells are surrounded by cytokine-specific monoclonal antibodies that coat the pore walls, cytokines already secreted by the incubated cells will begin to attach to the antibodies at specific epitopes.

[0074] Detection Antibodies: At this point, the wells can be flushed to remove cells and any other unwanted material. What remains are cytokine-specific monoclonal antibodies and any cytokines bound to the antibodies. Biotinylated cytokine-specific detection antibodies can then be added to the wells. These cytokine-specific detection antibodies will bind to any cytokines remaining in the wells, as the cytokine is still attached to the first set of antibodies used. Because the cytokines are attached to the first set of antibodies coating the wells, they are not washed away when the wells are flushed. Streptavidin-Enzyme Conjugate: A streptavidin-enzyme conjugate can be added to the wells to bind to the detection antibodies. The purpose of biotinylating the cytokine-specific detection antibodies added to the wells in the previous step is to allow the antibodies to 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. Substrate Addition: A substrate (e.g., a chromogenic substrate) can be added to the wells and catalyzed by the enzyme conjugate added in the previous step. This reaction forms an insoluble precipitate that forms spots in the wells. The substrate used in this step can depend on the type of enzyme used in the previous steps. If streptavidin-ALP (streptavidin and alkaline phosphatase conjugate) is used, using BCIP / NBT-plus (a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitrotetrazole blue chloride) as the substrate will produce more obvious and easier-to-analyze spots. If streptavidin-HRP (streptavidin and horseradish peroxidase conjugate) is used, using TMB (tetramethylbenzidine) as the substrate will produce better results.

[0075] Analysis: The resulting spots can then be read on an automated ELISpot reader or counted under a dissecting microscope and further used to calculate the frequency of cytokine secretion.

[0076] FluoroSpot's mechanism The FluoroSpot assay combines the sensitivity of ELISpot with the ability to simultaneously study the secretion of several analytes, enabling the study of cell populations with diverse functional profiles. 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 in a single well, while the conventional ELISpot assay can only analyze one analyte at a time. The FluoroSpot assay achieves this by using fluorescence instead of an enzyme-catalyzed reaction for detection. The procedure for the FluoroSpot assay is also similar, with some differences.

[0077] Antibody Coating: Similar to ELISpot, cytokine-specific monoclonal capture antibodies are added to wells of a plate. For both assays, the plate is treated with ethanol to avoid contamination and data collection skew. For the FluoroSpot assay, a mixture of different types of capture antibodies is attached to the wells to detect multiple types of analytes. To obtain optimal results with ELISpot and FluoroSpot assays, proper plate coating techniques should be followed. The plate should be treated with ethanol, washed, and then coated with antibodies. The ethanol treatment method also varies depending on the type of plate used. For MSIP and IPFL plates, 15 μL of 35% ethanol can be added to all wells. Allow the ethanol to stand in the wells for one minute, then pour it out. For MAIPSWU plates, 50 μL of 70% ethanol can be added to all wells instead. Allow the ethanol to stand in the wells for two minutes, then pour it out. After treating the wells with ethanol, the wells can be washed with approximately 200 μL of sterile water. This washing process can be repeated a total of 5 times. Once the wells have been treated with ethanol and washed, cytokine-specific monoclonal capture antibodies can be added to each well.

[0078] Cell incubation: Cells or cell populations can be added to wells and incubated with or without stimuli that affect protein secretion.

[0079] Cytokine capture: Proteins / analytes secreted by incubated cells will bind to capture antibodies that are attached, immobilized, or coated onto the wells during the first step.

[0080] Detection antibodies: Similar to ELISpot, once the wells are flushed to remove cells and other substances not of interest for identification or measurement, biotinylated detection antibodies (which can be specific for one type of analyte to be quantified) can be added, followed by the addition of tagged detection antibodies for an optional second or third type of analyte under investigation.

[0081] Fluorophore-tagged conjugates: Instead of adding streptavidin-enzyme conjugates, FluoroSpot amplifies the detection of multiple analytes using fluorophore-tagged anti-tag antibodies and streptavidin-fluorophore conjugates. A fluorescence enhancer solution can also be added during this step to amplify the signal used later in the fluorescence color in the analysis wells. This fluorescence allows FluoroSpot to analyze and compare multiple analytes, unlike ELISpot.

[0082] Analysis: Because FluoroSpot relies on fluorescence rather than an enzymatic reaction, the step of adding a substrate to react with an enzyme (as required by ELISpot) is unnecessary. The final step in a FluoroSpot assay is the analysis of fluorophores under an automated fluorescence reader with individual filters for the different fluorophores being analyzed. These filters can be selected for specific wavelengths of the fluorophores for accurate measurements.

[0083] Because FluoroSpot assays identify and quantify the presence of multiple analytes, it is possible that the uptake of one analyte may affect the secretion of another; this is known as the capture effect. The effect of one analyte (e.g., cytokines) on another analyte can be positive or negative (the production of the second analyte can increase or decrease). To counteract the capture effect, co-stimulation can be used to avoid the reduction in analyte production. This involves adding a second antibody that stimulates the production of the same analyte to the well.

[0084] ELISpot and FluoroSpot assays can be used in many research fields: vaccine development, cancer, allergy, monocyte / macrophage / dendritic cell characterization, apolipoprotein analysis, and veterinary research. ELISpot can be used to study antigen-specific cytokine responses, antibody-specific secretory cells, tumor antigens, T cell granzyme B and perforin release, vaccine efficacy, epitope mapping, cytotoxic T cell activity, IL-4, IL-5, and IL-13 detection, vaccine-induced antibody responses, antigen-specific memory B cells, and more.

[0085] 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-γ, IL-2, TNF-α, IL-4, IL-5, and IL-13. The first three cytokines are produced by Th1 cells, while the latter three are produced by Th2 cells. Measuring T-cell responses through cytokine production also makes it possible to study vaccine efficacy. As another example, the T-cell FluoroSpot assay can monitor tumor-infiltrating lymphocytes. IFN-γ cytokines and granzyme B secretion can be analyzed to assess cytotoxic T-cell responses. Both are used in cancer research. As yet another example, the B-cell FluoroSpot assay can also observe vaccine efficacy by quantifying the secretion of IgG, IgA, and IgM before and after vaccination. This analysis of multiple immunoglobulins is made possible by the fluorescence method used in FluoroSpot.

[0086] II. Biological Samples Biological samples can include whole blood, diluted whole blood, peripheral blood, or isolated human peripheral blood mononuclear cells (PBMCs). PBMCs are a diverse mixture of highly specialized immune cells that play a crucial role in maintaining bodily health. Peripheral blood mononuclear cells (PBMCs) are any blood cells with a round nucleus, such as lymphocytes (e.g., T cells, B cells), monocytes, or macrophages. These blood cells are key components of the immune system in fighting infection and defending against invaders. The two main techniques for isolating peripheral blood mononuclear cells from whole peripheral blood are by using density gradient centrifugation or by apheresis. Blood contains several 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, including red blood cells (erythrocytes), white blood cells (leucocytes), and platelets. They exist in the circulating blood pool and do not remain in the lymphatic system, spleen, liver, or bone marrow.

[0087] In one embodiment, the biological sample is whole blood, diluted whole blood, or isolated PBMCs. In another aspect of this embodiment, the biological sample includes whole blood, which does not require isolated PBMCs. Whole blood ELISpots can simultaneously test the integrity and robustness of two distinct branches of immunity (i.e., innate cellular immunity (blood monocytes and low-density granulocytes) and adaptive cellular immunity (blood lymphocytes)) by focusing on the response of individual cell populations to cell-specific agonists. As shown in Example 1, T cell subsets are significantly reduced in COVID-19 patients. Additionally, stimulated blood mononuclear cells produce less than 40%-50% of the IFN-γ observed in patients with sepsis and CINS. And TNF-α, which is consistent with significantly impaired immune effector cell function.

[0088] III. Cytokines / Chemokines As described herein, the assays described herein are used to measure secreted cytokines, for example, to determine the immune status of a subject. As an example, cytokines are measured after ex vivo stimulation. Cytokines that can be measured to determine the immune status of a subject (cytokines related to cellular immunity) can be human cytokines, such as IFN-γ. Inflammatory cytokines may include interleukin-1 (IL-1), IL-12, and IL-18, tumor necrosis factor-α (TNF-α), interferon-γ (IFNγ), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Anti-inflammatory cytokines may include interleukin (IL)-1 receptor antagonists, IL-4, IL-6, IL-10, IL-11, and IL-13.

[0089]

[0090] The chemokines described in this article can be any of the chemokines listed below.

[0091]

[0092]

[0093] IV. Measurement Methods The assay methods described herein will be better understood by referring to the following description. Methods for determining maximum spot intensity, average spot intensity, and the total number of low-intensity cells per unit volume are discussed below.

[0094] In one embodiment, the ELISpot measurement is performed by acquiring a biological sample and conducting a routine ELISpot measurement (e.g., using conditions described by the manufacturer). In one aspect of this embodiment, to determine the average spot intensity, an automated ELISpot reader is used or the ELISpot image is analyzed using image analysis software such as ImageJ (https: / / imagej.nih.gov / ij / ).

[0095] Cytokine-producing cells are identified as “blobs.” In some embodiments, this identification is performed manually, and in others, it is performed using a computational algorithm. The amount of cytokines produced by each cell is correlated with the intensity of the blobs in the image. This can be measured as the average / mean intensity of the blobs or the maximum intensity of the blobs. In some embodiments, the blob intensity is normalized relative to the maximum intensity measured in the image. Individual blobs can be counted, and the number of cells with intensities below the maximum intensity can also be counted.

[0096] Methods for determining immunointentype and / or evaluating drug efficacy Immunophenotyping can be performed on patients before, after, or simultaneously with ELISpot assays, using information about specific spots to determine if a patient has a specific disorder. It can also be done by evaluating a patient's adaptive and / or innate immune status. For example, methods for immunophenotyping may involve providing or having provided biological samples including whole blood, diluted whole blood, or isolated peripheral blood mononuclear cells (PBMCs). ELISpot can then be used to quantify the production of T-cell interferon-γ (IFN-γ) and / or monocyte tissue necrosis factor-α (TNF-α) in the biological sample, and the levels of IFN-γ and / or TNF-α can be compared to those in healthy subjects.

[0097] If T-cell interferon-γ (IFN-γ) and / or monocyte TNF-α production is relatively low compared to healthy subjects, a patient can be diagnosed with an immunosuppressive endogenous type. In this context, relatively low can mean more than 20%, more than 30%, more than 40%, or more than 50% lower than the average healthy subject.

[0098] If T-cell interferon-γ (IFN-γ) and / or monocyte TNF-α production is relatively high compared to healthy subjects, a patient can be diagnosed with hyperimmune endogenous type. In this context, relatively high can mean more than 20%, more than 30%, more than 40%, or more than 50% higher than the average healthy subject.

[0099] On the other hand, once a patient has been diagnosed with a given condition and is being or has been treated with a therapeutic agent suitable for that condition, the efficacy of the treatment can be determined by measuring the subject's immune function. This may involve first determining whether the patient has an immunosuppressive endogenous or hyperimmune endogenous type, identifying appropriate treatment regimens for a) a specific condition and b) a specific endogenous type, treating the patient with the appropriate regimen, and re-measuring the patient's endogenous type and / or repeating the screening steps for identifying the patient with the given condition.

[0100] If the production of T-cell cytokines or monocyte cytokines is relatively low compared to the control, the subject can be defined as having an immunosuppressive endotype, or if it is relatively high compared to the control, the subject can be defined as having a hyperimmune endotype. The subject is given the drug and / or the subject's immune function in response to the drug is determined. In some embodiments, the T-cell cytokine is interferon-γ (IFN-γ). In some embodiments, the monocyte cytokine is selected from one or more of TNF-α, IL-2, IL-6, and / or IL-12. In some embodiments, the subject has sepsis, COVID-19, cancer, trauma, or an autoimmune disease; the subject is a critically ill non-septic (CINS) patient or a post-transplant patient; or the subject is an immunosuppressed patient or a pediatric patient.

[0101] By way of example, once a patient has been diagnosed with the disorder and is receiving or is receiving treatment, the same steps described above for determining the patient's immune endotype can be performed. If the patient's immune endotype tends to change from hyperimmune or immunosuppressed to normal, i.e., the patient's T-cell interferon-γ (IFN-γ) and / or monocyte TNF-α production is reduced relative to relatively high or relatively low levels in normal subjects, then treatment can be considered effective. If the trend is that the patient's T-cell interferon-γ (IFN-γ) and / or monocyte TNF-α production moves even further away from normal levels, then treatment can be considered ineffective.

[0102] One aspect of this disclosure provides a method for immunophenotyping a subject, the method comprising: providing or having provided a biological sample from the subject; optionally stimulating T cells or monocytes or both to secrete cytokines associated with cell-mediated immunity; and / or quantifying at least one cytokine associated with cell-mediated immunity in the biological sample using an ELISpot assay or a FluoroSpot assay. In some embodiments, the method further comprises determining that the subject has an immunosuppressive endotype if the cytokine associated with cell-mediated immunity is a pro-inflammatory cytokine and / or the production or secretion of pro-inflammatory cytokines is reduced compared to a control. In some embodiments, the method further comprises determining that the subject has a hyperinflammatory endotype if the cytokine associated with cell-mediated immunity is a pro-inflammatory cytokine and / or the production or secretion of pro-inflammatory cytokines is increased compared to a control. In some embodiments, the method further comprises determining whether the subject has an immunosuppressive endotype if the number of immune cells is reduced compared to a control, or determining whether the subject has a hyperinflammatory endotype if cytokine production is increased compared to a control. In some embodiments, the method further includes detecting innate immune levels, which includes detecting the level of blood monocytes or low-density granulocytes or detecting the level of monocyte function or low-density granulocyte function. In some embodiments, the method further includes detecting adaptive cellular immune levels, which includes detecting the level of blood lymphocytes or blood lymphocyte function. In some embodiments, if the amount of CD4+ and / or CD8+ T cells is reduced compared to a control, the responsiveness of T cells to T cell receptor activation is reduced, or both, the subject has an immunosuppressive endotype. In some embodiments, the cytokines associated with cellular immunity are pro-inflammatory cytokines selected from the group consisting of: T-cell interferon-γ (IFN-γ), monocyte tumor necrosis factor-α (TNF-α), IL-1β, or combinations thereof. In some embodiments, the cytokines associated with cellular immunity are selected from IFN-α, TNF-α, IL-1β, IL-6, IL-7, IL-8, IL-10, IL-12, MCP-1, IL-1RA, or any combination thereof; or EGF, eosinophil chemokine, basic FGF, G-CSF, GM-CSF, HGF, IFN-α, IFN-γ, IL-1β, IL-1α, IL-1RA, 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-1α, MIP-1β, RANTES, TNF-α, VEGF, or any combination thereof.In some embodiments, quantifying cytokines associated with cellular immunity includes: detecting the amount of cytokines produced by immune effector cells; or detecting the amount of cytokines produced on cells. In some embodiments, quantification of cytokines associated with cellular immunity is measured in units of reaction / volume of blood. In some embodiments, the biological sample includes: whole blood; diluted whole blood; circulating peripheral blood; whole blood diluted with PBS at a ratio of about 1:1; T cells, monocytes, and / or B cells; or plasma, leukocytes, erythrocytes (RBCs), white blood cells (WBCs), platelets, cytokines, chemokines, or combinations thereof. In some embodiments, the biological sample does not include isolated peripheral blood mononuclear cells (PBMCs). In some embodiments, the method further includes evaluating adaptive and / or innate immune status; evaluating monocyte or leukocyte function; evaluating the progression of immune dysfunction in the subject; evaluating the effectiveness of immunotherapy in restoring innate and / or adaptive immunity in immunosuppressed patients, optionally with the effect of adjuvant immunotherapy in enhancing host immunity; identifying the optimal immunotherapy for use in the subject; or improving the subject's immune function. In some embodiments, the subject has, is suspected of having, or is at risk of developing sepsis, an autoimmune disease, autoimmune disease, or cancer; the subject has fungal wound sepsis; the subject has lymphopenia (≤ 1100 cells / μL); the subject has undergone organ transplantation; and / or the subject is in intensive care. In some embodiments, the method further includes measuring the production of ex vivo cytokines as a response to external stimuli. In some embodiments, a subject has sepsis or is identified as being at risk of premature death if: the number of pro-inflammatory cytokine-producing immune effector cells is reduced compared to a control; or the amount of pro-inflammatory cytokines produced per cell, measured by speckle intensity, is reduced compared to a control. In some embodiments, if the subject does not have an immunosuppressive endotype or the subject has a hyperinflammatory endotype, the subject is administered a drug that blocks pro-inflammatory cytokines or inhibits the inflammatory signaling cascade, such as dexamethasone; if the subject has an immunosuppressive endotype, the subject is administered IL-7 or a similar immunostimulatory therapy to restore disease-induced T cell exhaustion. If a subject has sepsis and / or has an immunosuppressive endogenous form, the subject is given an immune-restoring drug; if a subject has sepsis and / or is immunosuppressed, corticosteroid therapy is not given, and dexamethasone is optional; if a subject has sepsis and / or has an immunosuppressive endogenous form, the subject is at high risk of death.If the subject has an immunosuppressive endotype, treat the subject with immunomodulatory drugs or immune adjuvants that enhance host immunity; if the subject has an immunosuppressive endotype, administer checkpoint inhibitors and / or common γ-chain cytokines (optionally IL-17) that stimulate CD4 and / or CD8 T cells; if the subject has a hyperinflammatory endotype or does not have an immunosuppressive endotype, treat the subject with drugs that suppress the host inflammatory response; if the subject has high cytokine production, do not treat the subject with immunostimulatory therapy; or if the subject has high cytokine production, treat the subject with anticytokine therapy or drugs that negatively regulate the inflammatory response.

[0103] In some embodiments, the method further includes detecting an immunosuppressive endotype or a hyperinflammatory endotype during the progression of a disease, disorder, or condition, or during treatment of a disease, disorder, or condition. In some embodiments, the method further includes optionally administering the drug to a subject in need during an immunotherapy procedure and / or determining the subject's immune function or leukocyte function in response to the drug. In some embodiments, the subject has sepsis, COVID-19, cancer, trauma, or an autoimmune disease; the subject is a critically ill non-septic (CINS) patient or a post-transplant patient; or the subject is an immunosuppressed patient or a pediatric patient. In some embodiments, the biological sample is placed in fluid contact with a test therapeutic agent (optionally a cytokine / chemokine, IL6, anti-PD-1, anti-PD-L1, GM512, CSF, IL-7). In some embodiments, the assay includes wells pre-coated with a treatment for detecting one or more cytokines or chemokines.

[0104] V. Diagnostic methods Using the methods described herein, biological samples (such as whole blood from a patient, diluted whole blood, or isolated PBMC cells) can be evaluated, and it can be determined what kind or which disorders they may have. As discussed elsewhere in this document, it is possible to determine whether a patient is healthy, has sepsis, or has CINS based on evaluations of the average spot intensity, maximum spot intensity, and / or the number of low-intensity cells per unit volume (such as counts in wells used for ELISpot assays).

[0105] Information regarding mean speckle intensity, maximum speckle intensity, and / or the 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, etc.). The variability in mean speckle intensity, maximum speckle intensity, and / or the number of low-intensity cells per unit volume can be determined between healthy subjects and patients known to have these specific disorders, and this information can be stored in a reference library. The library can have any values ​​from 2 to 100 or more for one or more of the mean speckle intensity, maximum speckle intensity, and / or the number of low-intensity cells per unit volume associated with various disorders. Patients can then be screened using ELISpot analysis, and the results can be compared to the reference library, with best-fit analysis identifying the specific disorders a patient may have.

[0106] VI. Treatment methods Once a patient has been diagnosed with a specific disorder, the treating physician can consider an appropriate treatment plan. In some cases, the specific treatment plan will depend on the patient's specific immune endotype. Therefore, in addition to treating the underlying disorder (such as sepsis, cancer, etc.), the immune system can be modulated based on whether the patient has a hyperimmune endotype or an immunosuppressive endotype. Representative treatments for various disorders and representative treatments for increasing or decreasing the immune response are discussed below.

[0107] Antimicrobial compounds Microbial infections (such as bacterial, viral, or fungal infections) can be treated with conventional antimicrobial compounds.

[0108] Examples of commonly used antibiotics include, but are not limited to, amikacin, tobramycin, gentamicin, piperacillin, meropenem, ticarcillin, imipenem, ciprofloxacin, ceftazidime, aztreonam, ticarcillin-clavulanic acid, dicloxacillin, amoxicillin, trimethoprim-sulfamethoxazole, cephalexin, piperacillin-tazobactam, linezolid, daptomycin, vancomycin, metronidazole, clindamycin, colistin, tetracycline, levofloxacin, amoxicillin, and clavulanic acid (Augmenti). (n®), cloxacillin, dicloxacillin, cefdinir, cefprozil, cefaclor, cefuroxime, erythromycin / sulfamethoxazole, erythromycin, clarithromycin, azithromycin, doxycycline, minocycline, tigecycline, imipenem, meropenem, colistin mesylate / Colistin®, methicillin, oxacillin, ethoxynaphthylpenicillin, carbenicillin, azoxycillin, piperacillin and tazobactam (Zosyn®), cefepime, ethambutol, rifampin and meropenem.

[0109] These antibiotics can also be combined with compounds that bind to or adsorb bacterial toxins, which can be particularly useful in cases where bacterial toxins cause tissue damage. By way of example, *Pseudomonas aeruginosa* produces a variety of toxins that cause 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 stimulating inflammation; alkaline protease, which causes tissue damage; cytotoxin, which damages the cell membrane 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), which catalyzes the formation of tissue-damaging toxic oxygen free radicals, impairing ciliary function and stimulating inflammation. Examples of compounds that bind to these toxins include polyphenols and polyanionic polymers.

[0110] Antifungal agents can also be used concurrently when the microorganism is a fungus. Representative antifungal agents that can be used include fluconazole, posaconazole, voriconazole, itraconazole, echinococcin, amphotericin B, and flucytosine. The appropriate antifungal agent can be selected by the treating physician, and the following is a summary of fungal lung infections and their treatment.

[0111] Histoplasmosis is caused by the fungus Histoplasma capsulatum, and routine treatment includes itraconazole for mild and chronic lung disease, and amphotericin B (AmB) in combination with itraconazole for moderate to severe histoplasmosis.

[0112] Blastomycosis is caused by Blastomyces dermatitidis, and routine treatment includes itraconazole for mild to moderate cases, liposomal AmB (L-AmB), followed by itraconazole for life-threatening lung infections.

[0113] Sporothrix schenckii is caused by the fungus Sporothrix schenckii. Itraconazole is routinely used for the treatment of mild to moderate lung disease, while AmB is recommended for severe cases, followed by itraconazole.

[0114] Coccidioidomycosis is caused by *Coccidioides immitis* and *Coccidioides posadasii*. Immunocompromised hosts may not require treatment, but immunocompromised patients are treated with fluconazole or itraconazole, and in severe cases, with AmB followed by azole.

[0115] Opportunistic fungal infections primarily occur in patients with compromised immune systems due to congenital or acquired disease processes. Representative opportunistic infections are discussed below.

[0116] Aspergillosis is caused by Aspergillus, and related disorders include invasive pulmonary aspergillosis (IPA), chronic necrotizing aspergillosis, aspergilloma, and allergic bronchopulmonary aspergillosis. Routine treatment for IPA includes voriconazole, lipid-based AmB formulations, echinococcin, and posaconazole.

[0117] Cryptococcosis is an opportunistic infection observed in immunocompromised individuals, including those with HIV or AIDS and organ transplant recipients. Standard treatment includes AmB (with or without flucytosine), followed by oral fluconazole. Fluconazole therapy is recommended for immunosuppressed or immunocompetent patients presenting with mild to moderate symptoms.

[0118] Candidiasis can occur when the lung parenchyma is colonized by 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 empirical antifungal therapy. Triazole antifungals and echinococcin exhibit excellent lung penetration and are therefore used in addition to AmB formulations for the treatment of pulmonary candidiasis.

[0119] Mucormycosis typically occurs in patients with diabetes, organ or hematopoietic stem cell transplantation, neutropenia, or malignant tumors. Pulmonary mucormycosis is primarily observed in patients with neutropenia or predisposing conditions requiring corticosteroids. Conventional antifungal agents struggle to penetrate lung tissue due to fungal adhesion to and damage to endothelial cells, fungal vascular invasion, vascular thrombosis, and subsequent tissue necrosis. For this reason, standard treatment includes debridement of necrotic tissue and antifungal therapy (using AmB formulations, posaconazole) and iron chelation therapy.

[0120] Pneumocystis jirovecii pneumonia (PCP) occurs in patients with HIV / AIDS, hematologic and solid malignancies, organ transplant recipients, and those requiring immunosuppressants. PCP is highly resistant to common antifungal therapies, including AmB formulations and triazole antifungals, but can be treated with trimethoprim / sulfamethoxazole. Second-line agents include primaquine plus clindamycin, atovaquinone, intravenous pentamicillin, or dapsone.

[0121] The antifungal agents identified in this article can be administered in conjunction with the phototherapy methods described herein. However, the use of phototherapy can shorten the duration of such antifungal treatment and / or increase its efficacy.

[0122] Antiviral treatment for viral disorders is well-known. Examples include nucleotide reverse transcriptase inhibitors, non-nucleotide reverse transcriptase inhibitors, protease inhibitors, entry inhibitors, etc. When a patient has a viral lung infection, conventional antiviral agents used for that type of virus can be administered. The choice of antiviral agent typically depends on the viral infection being treated. Influenza virus is typically treated with oseltamivir (Tamiflu), zanamivir (Relenza), or peramivir (Rapivab), and RSV is treated with ribavirin (Virazol). Coronaviruses are also treated with Tamiflu, ribavirin, certain anti-HIV compounds, and certain interferons (including betaferon, alferon, multiferon, and wellferon).

[0123] Combination therapy used specifically for the treatment of Covid-19 infection Various compounds exist that can be used to treat Covid-19 infection or other respiratory infections with similar pathology, and can be administered alone or in combination with antiviral compounds.

[0124] Examples include fusion inhibitors, entry inhibitors, protease inhibitors, polymerase inhibitors, antiviral nucleosides (such as remdesivir, GS-441524, N4-hydroxycytidine, and other compounds and their prodrugs disclosed in U.S. Patent No. 9,809,616), viral entry inhibitors, viral maturation inhibitors, JAK inhibitors, angiotensin-converting enzyme 2 (ACE2) inhibitors, SARS-CoV-specific human monoclonal antibodies (including CR3022), and agents with different or unknown mechanisms.

[0125] Umifenovir (also known as Arbidol) is a representative fusion inhibitor.

[0126] Representative entry inhibitors include carmostatin, luteolin, MDL28170, SSAA09E2, SSAA09E1 (which acts as a cathepsin L inhibitor), SSAA09E3, and tetra-O-galloyl-β-D-glucose (TGG).

[0127] Additionally, when patients have hyperimmune endotype, compounds that suppress cytokine storms, such as dexamethasone, can be administered. Furthermore, when patients have, are suspected of having, or are at risk of developing blood clots, anticoagulants and / or platelet aggregation inhibitors that resolve blood clots, or compounds that chelate iron ions released from hemoglobin by viruses (such as COVID-19) can be provided.

[0128] Representative ACE-2 inhibitors include thiol-containing agents such as alapril, captopril, and zolfenpril; dicarboxylate-containing agents such as enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, sinepril, and inhibace; and phosphonate-containing agents such as fositen / monopril.

[0129] Compounds used to suppress cytokine storm Throughout activation, the inflammatory response must be modulated to prevent destructive systemic inflammation, also known as a "cytokine storm." Many cytokines with anti-inflammatory properties contribute to this, such as IL-10 and transforming growth factor β (TGF-β). Each cytokine acts on a different part of the inflammatory response. For example, products of the Th2 immune response suppress the Th1 immune response, and vice versa.

[0130] By reducing inflammation, collateral damage to surrounding cells can be minimized with little or no long-term harm to the patient. Therefore, in addition to using the compounds described herein to suppress viral infection, one or more compounds that inhibit cytokine storms can be administered in combination.

[0131] Compounds used to treat or prevent blood clots Viruses that cause respiratory infections, including coronaviruses such as Covid-19, may be associated with blood clots in the lungs and blood clots that may also damage the heart.

[0132] The compounds described herein may be used in combination with compounds that inhibit blood clot formation (such as blood thinners) or compounds that break down existing blood clots (such as tissue plasminogen activator (TPA), integrilin (epitibatide), abciximab (ReoPro), or tirofiban (Aggrastat)).

[0133] Blood thinners prevent blood clots from forming and prevent existing clots from growing larger. There are two main types of blood thinners. Anticoagulants (such as heparin or warfarin, also known as coumarin) slow down the biological process that produces clots, while antiplatelet aggregation drugs (such as clopidogrel or aspirin) prevent blood cells called platelets from clumping together to form a clot.

[0134] By way of example, Integrilin® is typically administered as soon as possible after diagnosis with an intravenous bolus of 180 mcg / kg, and followed by a continuous infusion of 2 mcg / kg / min (after the initial bolus) for up to 96 hours.

[0135] Representative platelet aggregation inhibitors include glycoprotein IIB / IIIA inhibitors, phosphodiesterase inhibitors, adenosine reuptake inhibitors, and adenosine diphosphate (ADP) receptor inhibitors. These can optionally be used in combination with anticoagulants.

[0136] Representative anticoagulants include coumarin (vitamin K antagonists), heparin and its derivatives (including unclassified heparin (UFH), low molecular weight heparin (LMWH), and ultra-low molecular weight heparin (ULMWH)), factor Xa synthesis pentasaccharide inhibitors (including fondaparinux, idraparinux, and idrabiotaparinux), direct-acting oral anticoagulants (DAOCs) (such as dabigatran, rivaroxaban, apixaban, edoxaban, and betrixaban), and antithrombin protein therapy agents / thrombin inhibitors (such as the bivalent drugs hirudin, lepirudin, and bivalirudin, and the monovalent drug argatroban).

[0137] Representative platelet aggregation inhibitors include pravastatin, clopidogrel (Plavix), cilostazol (Pletal), prasugrel (Effient), aggrenox (aspirin and dipyridamole), ticagrelor (Brilinta), capsulbizumab, cangrelor (Kengreal), dipyridamole (Persantine), ticlopidine (Ticlid), and omeprala (aspirin and omeprazole).

[0138] antiviral agents Representative antiviral agents include lopinavir, ritonavir, niclosamide, promazine, PNU, UC2, sinacerin (SQ 10,643), carmidazolium (C3930), tannic acid, 3-isotheaflavin-3-gallate, theaflavin-3,3'-digallate, glycyrrhizin, S-nitroso-N-acetylpenicillamine, nelfinavir, niclosamide, chloroquine, hydroxychloroquine, 5-benzyloxyarbutin, ribavirin, interferons (such as interferon (IFN)-α, IFN-β and their PEGylated forms), and combinations of these compounds with ribavirin, chlorpromazine hydrochloride, trifluoropromazine hydrochloride, gemcitabine, imatinib mesylate, dasatinib, and imatinib.

[0139] Immunosuppressants Immunosuppressants are drugs that broadly weaken 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, methylprednisolone, and cortisone). Representative examples of biologics include abatacept (Orencia), adalimumab (Humira), dupilumab (Dupixent), etanercept (Enbrel), infliximab (Remicade), mepolilimab (Nucala), omaliximab (Xolair), rituximab (Rituxan), secukinumab (Cosentyx), tocilizumab (Actemra), and uterotonicumab (Stelara).

[0140] Disease-modifying therapy for MS MS is treated with disease-modifying therapies (DMTs). These medications help reduce inflammation levels, thereby reducing the likelihood of relapse and preventing further damage to myelin and its underlying nerves. Examples include cladribine (Mavenclad), fingolimod (Gilenya), glatiramer acetate (Copaxone, Glatopa), interferon-β1a (Avonex, Rebif), interferon-β1b (Betaseron, Extavia), natezumab (Tysabri), osprezumab (Ocrevus), oframumab (Kesimpta), and sinimod (Mayzent).

[0141] Cancer treatment Exemplary cancer treatments include radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormone therapy, and angiogenesis inhibitors.

[0142] Radiation therapy Radiation therapy (also known as radiotherapy, X-ray therapy, or irradiation) uses ionizing radiation to kill cancer cells and shrink tumors by damaging their DNA (the molecules within cells that carry genetic information and pass it down from generation to generation), making these cells unable to continue growing and dividing. Radiation therapy can directly damage DNA or create charged particles (free radicals) within cells, which in turn can damage DNA. Radiation therapy can be administered externally via external beam radiation therapy (EBRT) or internally via brachytherapy. The effects of radiation therapy are localized and limited to the area being treated. Although radiation damages both cancer cells and normal cells, most normal cells can recover from the effects of radiation and function normally. The goal of radiation therapy is to damage as many cancer cells as possible while limiting damage to nearby healthy tissue. Therefore, it is administered in many fractions, allowing healthy tissue to recover between fractions.

[0143] Radiation therapy can be used to treat almost every type of solid tumor, including brain cancer, breast cancer, cervical cancer, laryngeal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, or soft tissue sarcoma. Radiation is also used to treat leukemia and lymphoma. The radiation dose at each site depends on many factors, including the radiosensitivity of each type of cancer and the presence of nearby tissues and organs that could be damaged by radiation. Therefore, as with every form of treatment, radiation therapy is not without side effects.

[0144] Chemotherapy Chemotherapy treats cancer with drugs that can destroy cancer cells (“anti-cancer drugs”). Chemotherapy can be administered in various ways, such as by injection into muscles, skin, arteries, or veins, or it can even be taken orally in the form of pills. In current use, the term “chemotherapy” generally refers to cytotoxic drugs that affect rapidly dividing cells in general, in contrast to targeted therapy. Chemotherapy drugs interfere with cell division in a variety of possible ways, such as interfering with DNA replication or the separation of newly formed chromosomes. Most forms of chemotherapy target all rapidly dividing cells and are not specifically targeted at cancer cells, although some degree of specificity may stem from the fact that many cancer cells cannot repair DNA damage that normal cells often can. Therefore, chemotherapy has the potential to harm healthy tissues, especially those with high replacement rates (e.g., the intestinal lining). These cells typically repair themselves after chemotherapy.

[0145] Because some drugs are more effective when used in combination than when used alone, two or more drugs are often administered simultaneously. This is known as "combination chemotherapy"; most chemotherapy regimens are given in combination.

[0146] Targeted therapy Targeted therapy involves the use of agents that are specific to dysregulated proteins in cancer cells. Small molecule targeted therapies are typically inhibitors of enzyme domains on mutated, overexpressed, or other key proteins within cancer cells. Prominent examples are the tyrosine kinase inhibitors imatinib (Gleevec / Glivec) and gefitinib (Iressa).

[0147] Monoclonal antibody therapy is another strategy in which the therapeutic agent is an antibody that specifically binds to proteins on the surface of cancer cells. Examples include trastuzumab (Herceptin), an anti-HER2 / neu antibody used for breast cancer, and rituximab, an anti-CD20 antibody used for various B-cell malignancies.

[0148] Targeted therapies can also include small peptides that act as “homing devices”, binding to cell surface receptors or the affected extracellular matrix surrounding the tumor. If the radionuclide decays near the cell, the radionuclide attached to these peptides (e.g., RGD) eventually kills the cancer cells. Oligomers or multimers of these binding motifs, in particular, have attracted considerable interest because they can lead to enhanced tumor specificity and affinity.

[0149] Photodynamic therapy (PDT) is a ternary treatment for cancer, which includes photosensitizers, tissue oxygenation, and light (usually using lasers).

[13] PDT can be used to treat basal cell carcinoma (BCC) or lung cancer; PDT can also be used to remove small amounts of malignant tissue after surgical removal of large tumors.

[0150] Targeted therapies in preclinical development as potential cancer treatments include morpholino-splicing oligonucleotides (which induce ERG exon skipping in prostate cancer models), multi-target kinase inhibitors that inhibit PI3K and other pathways (including MEK and PIM), and NF-κB inhibitors in chemotherapy resistance models.

[0151] Hormone therapy The growth of some cancers can be suppressed by providing or blocking certain hormones. Common examples of hormone-sensitive tumors include certain types of breast cancer and prostate cancer. Blocking estrogen or testosterone is often an important additional treatment. In some cancers, the administration of hormone agonists (such as progestins) may be beneficial in treatment.

[0152] Angiogenesis inhibitors Angiogenesis inhibitors prevent the extensive growth of blood vessels (angiogenesis) necessary for tumor survival. Some drugs, such as bevacizumab, have been approved and are in clinical use. One of the main problems with anti-angiogenic drugs is that many factors stimulate blood vessel growth in both normal and cancerous cells. Anti-angiogenic drugs target only one factor, so other factors continue to stimulate blood vessel growth. Other issues include the route of administration, stability and maintenance of activity, and targeting within the tumor vascular system.

[0153] Cancer immunotherapy Patients with cancer can be treated with various types of immunotherapy, examples of which are listed below: Immune checkpoint inhibitors work by shutting down signals that prevent immune cells from responding to cancer cells.

[0154] Cytokines are small proteins that participate in the signal transduction of the immune system.

[0155] Immunomodulators are a group of drugs that target immune pathways (such as thalidomide and lenalidomide) and are typically used in cancers such as multiple myeloma.

[0156] Chimeric antigen receptor (CAR) T-cell therapy involves extracting immune cells called T cells from a patient's blood. These cells are then modified in the laboratory to make them specifically respond to the patient's cancer.

[0157] Cancer vaccines stimulate the immune system to respond to cancer. Representative cancers that can be treated with cancer vaccines include melanoma, prostate cancer, and some types of lung cancer.

[0158] Immunostimulants Immunostimulants (also known as immunostimulators) are substances (drugs and nutrients) that stimulate the immune system by inducing activation or increasing the activity of any component of the immune system. A notable example is granulocyte-macrophage colony-stimulating factor (GM-CSF). Immunostimulatory agents such as IL-7, GM-CSF, prolactin, growth hormone, checkpoint inhibitors, interferon-γ, or vitamin D can be used when appropriate to enhance the immune response. Deoxycholic acid stimulates macrophages, and imiquimod and requimod activate immune cells through Toll-like receptor 7, and these agents can also be used.

[0159] Evaluation of drug efficacy Another aspect of this disclosure provides an ELISpot or FluorSpot assay comprising wells pre-coated with one or more assay therapeutics or one or more cytokine or chemokine detection agents. In some embodiments, the one or more assay therapeutics are tocilizumab, haptoglobin, hemoglobin-binding protein, ox40, IL7, or steroids. In some embodiments, the method further comprises fluidly contacting a biological sample with the pre-coated 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 who has or is suspected of having sepsis, COVID-19, cancer, trauma, or an autoimmune disease; a critically ill non-sepsis (CINS) subject or a post-transplant patient; or an immunosuppressed patient or a pediatric patient. In some embodiments, this assay produces accelerated results compared to conventional PBMC assays.

[0160] Reagent test kit Another aspect of this disclosure provides a kit comprising an ELISpot or FluoroSpot assay, the assay comprising wells coated with a test agent or wells coated with a cytokine or chemokine detection agent; and / or optionally comprising biological samples including whole blood, diluted whole blood, or PBMCs.

[0161] Other purposes and features will be partially clear and will be partially indicated below.

[0162] Having described this disclosure in detail, it will be clear that modifications, variations, and equivalent embodiments are possible without departing from the scope of this disclosure as defined in the appended claims. Furthermore, it should be understood that all examples in this disclosure are provided as non-limiting examples.

[0163] Example The following non-limiting examples are provided to further illustrate this disclosure. Those skilled in the art will understand that the techniques disclosed in the following examples represent methods that the inventors have found to work well in the practice of this disclosure, and can therefore be considered as examples of patterns for use in their practice. However, those skilled in the art will understand from this disclosure that many changes can be made to the specific embodiments disclosed and similar or analogous results can still be obtained without departing from the spirit and scope of this disclosure.

[0164] Example 1: Low-intensity IFN-γ producing T cell population in patients with sepsis The ELISpot immunoassay has been used to identify immunosuppression in critically ill patients. The ELISpot assay typically reports measures of cytokine production, including the number of “spot-forming units” in the wells, the average “spot size”, and the total signal intensity.

[0165] The basis for these comprehensive features is high-resolution data, which includes features of individual spots in each well, including average intensity, maximum intensity, and the size of each individual spot. Figure 6 The original images from the ELISpot determination are shown, along with the events identified (by an algorithm) after image analysis, in which individual event (“blob”) metrics were measured. The results are also tabulated in the table below.

[0166]

[0167] Current available techniques calculate and report the total number of spots, average spot size, and total pore intensity. This method focuses on total and average characteristics but does not evaluate the characteristics of individual spots.

[0168] The inventors hypothesize that high-resolution analysis of individual blot measures will provide additional biologically relevant information about the state of the immune system. This example demonstrates the practicality of information gathered from observing individual blot measures.

[0169] method: Whole blood was collected from critically ill non-septic (CINS) subjects (n = 20) and subjects with sepsis (n = 39) at 1, 4, and 7 days post-diagnosis. Samples were also collected from healthy donors (n = 20). 5 μl of whole blood was applied to an ELISpot plate pre-coated with IFN-γ capture antibody and incubated at 37°C in a 5% CO2 incubator. After 22 hours, the plate was washed and visualized with a secondary antibody against IFN-γ. The plate was imaged on a CTL S6 entry M2 analyzer, and the spot characteristics were recorded in a .fcs file using a procedure from the following manufacturer. The .fcs file was analyzed using the OMIQ platform (www.omiq.ai).

[0170] The average intensity, maximum intensity, size, roundness, and total intensity of each spot were extracted and exported to SPSS and GraphPad Prism. Spot frequencies were compared using the Kruskal-Wallis test, followed by the Dunn post-hoc test.

[0171] The distribution profile of the mean intensity of spontaneous IFN-γ spots generated in an ELISpot assay is a clinically relevant measure of human immune function. High-resolution analysis of spontaneous IFN-γ production was performed in cohorts of healthy subjects, subjects with sepsis, and subjects with non-sepsis-related critical illness. Non-sepsis-related critical illness and sepsis are presented as examples of different states of immune function. We found characteristic distributions of mean spot intensity of spontaneous IFN-γ in healthy individuals, critically ill non-sepsis patients, and sepsis patients (Figure 3A). Blood from healthy controls (green trace) produced a single spike of high-intensity IFN-γ production at 50% of the maximum intensity detected in the assay well. Blood from sepsis patients (orange trace) produced a broader peak with a lower intensity (35% of the maximum intensity in the well). Blood from non-sepsis-related critically ill patients (blue trace) produced a bimodal distribution with a peak at approximately 45% of the maximum intensity and a second peak at approximately 32% of the peak well intensity. These results demonstrate that the distribution profile of the maximum spontaneous interferon spot intensity produced in the ELISpot assay can be used as a clinically relevant measure of human immune function.

[0172] The distribution profile of the maximum spontaneous IFN-γ spot intensity generated in the ELISpot assay is a clinically relevant measure of human immune function. Data showed that healthy individuals, critically ill non-septic patients, and septic patients exhibited characteristic distributions of spontaneous IFN-γ maximum spot intensity (Figure 3B). Blood from healthy controls (green trace) produced a single spike of high-intensity IFN-γ production at 50% of the maximum intensity detected in the well. Blood from septic patients (orange trace) produced a broader peak with lower intensity (35% of the maximum intensity in the well). Blood from critically ill non-septic patients (blue trace) produced a bimodal distribution with a peak at approximately 45% of the maximum intensity and a second peak at approximately 32% of the peak intensity in the well. These results demonstrate that the distribution profile of the maximum spontaneous interferon spot intensity produced in the ELISpot assay can be used as a clinically relevant measure of human immune function.

[0173] Figures 3C through 3E show the distribution of spot size, spot roundness, and total IFN-γ spot intensity in biological samples obtained from healthy patients (green), critically ill non-septic patients (blue), and septic patients (orange). As these figures indicate, the results did not vary significantly among these patient groups (at least regarding healthy patients, patients with sepsis, and critically ill non-septic patients).

[0174] Distribution of maximum spot intensity of CD3 / CD28-stimulated IFN-γ generated in ELISpot assays The protocol for spontaneous IFN-γ generation was repeated, but CD3 / CD28 stimulation was performed using a CD3 / CD28 activating antibody (Ab). Data are shown in... Figure 4A In -E, Figure 4A The average spot intensity is shown in CD3 / CD28-stimulated biological samples obtained from healthy patients (green), critically ill non-septic patients (blue), and septic patients (orange). Figure 4B The maximum spot intensity in these biological samples is shown, and Figure 4C - E shows the distribution of spot size, spot roundness, and total IFN-γ spot intensity in these biological samples.

[0175] Compare Figure 3A / Figure 3B (spontaneous IFN-γ generation) with Figure 4A / Figure 4B (Stimulated IFN-γ production) Data showed differences in mean and maximum intensity between spontaneous and stimulated IFN-γ production. Observing Figures 3A / 3B, significant differences were found between mean and maximum intensity associated with healthy patients, sepsis patients, and CINS patients. Figure 4A / Figure 4B As shown, the differences between healthy patients, sepsis patients, and CINS patients were significantly smaller.

[0176] Overview Spontaneous IFN-γ production or IFN-γ production stimulated by CD3 / CD28 was measured using ELISpot. Data from days 1, 4, and 7 were pooled, and speckle level data (including mean intensity, maximum intensity, speckle size, roundness, and total intensity) were extracted, and the distribution of each metric was compared.

[0177] Compared to healthy controls, sepsis was associated with a population of spontaneously producing IFN-γ cells of average intensity. NSCI subjects exhibited a heterogeneous phenotype.

[0178] The number of lower intensity spots was compared using the Kruskal-Wallis test, followed by the Dunn post-hoc test. p<0.01 The results are summarized in Figure 5 The figure shows the number of low-intensity spots in patients with sepsis, CINS, and healthy patients.

[0179] The number of “low-intensity” spontaneous IFN-γ producing cells measured in the ELISpot assay is a clinically relevant measure of human immune function. The ELISpot assay is an effective measure of IFN-γ production by T cells in the whole blood of critically ill patients. The ELISpot detection algorithm can identify IFN-γ production characteristics across a wide range of spot sizes and intensities.

[0180] Conventional ELISpot analysis, which includes the number of spots, spot size, and total pore intensity, is insufficient to distinguish between sepsis patients and critically ill non-septic patients.

[0181] High-resolution analysis of speckle intensity distribution identified a novel population of low-intensity IFN-γ-producing T cells in sepsis patients that were absent in healthy controls.

[0182] The number of low-intensity IFN-γ producing cells strictly distinguishes between sepsis patients and non-septic critically ill patients, demonstrating that high-resolution analysis of ELISpot data using single-cell analysis methods is a powerful tool for extracting additional information from ELISpot assays.

[0183] The term "low-intensity" IFN-γ producing cells is defined as cells with an average spot intensity < 45% of the maximum intensity measured in the well (see, for example, Figure 3A and...). Figure 4A Enumerate the total number of "low-intensity" spots and compare it to the number of low-intensity spots in critically ill patients with and without sepsis. Figure 5 Whole blood samples from healthy patients analyzed by EliSpot produced an average of 1.3 low-intensity spots, while blood samples from CINS patients produced an average of 4.3 spots, and blood samples from sepsis patients produced 18.5 spots. These differences resulted in statistically significant differences between cohorts.

[0184] Therefore, the number of low-intensity IFN-γ producing cells strictly distinguishes between sepsis patients and critically ill non-septic patients, demonstrating that the number of low-intensity spontaneous IFN-γ producing cells can be used as a clinically relevant measure of human immune function.

[0185] in conclusion: The ELISpot assay is an effective measure of IFN-γ production by T cells in whole blood from critically ill patients. The ELISpot speckle detection algorithm can identify IFN-γ production characteristics across a wide range of speckle sizes and intensities. High-resolution analysis of speckle intensity distribution identified a novel population of low-intensity IFN-γ-producing T cells in sepsis patients, cells absent in healthy controls. The number of low-intensity IFN-γ-producing cells strictly distinguishes between sepsis patients and non-sepsis patients, demonstrating that high-resolution analysis of ELISpot data using single-cell analysis methods is a powerful tool for extracting additional information from ELISpot assays.

[0186] The scope of this invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention, in addition to those described, will become apparent to those skilled in the art based on the above description and drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A method for determining the average spot intensity, maximum spot intensity, and / or the number of low-intensity cells per unit volume present in a biological sample from a subject, the method comprising: a. A biological sample from the subject has been provided or has already been provided; b. Place the sample in wells pre-coated with a drug that stimulates T cells or monocytes, or both, to secrete cytokines or chemokines associated with cellular immunity. c. Use ELISpot or FluoroSpot assays to quantify the average spot intensity, maximum spot intensity, and / or the number of low-intensity cells per unit volume in biological samples. The ELISpot assay or FluoroSpot assay detects spots associated with cells that spontaneously secrete cytokines or chemokines, and those cells have measurable mean spot intensity, maximum spot intensity, and individual spot intensity. The term "low-intensity" cytokine or chemokine-producing cells are defined as cells with an average spot intensity less than 45% of the maximum intensity measured in the well, and the term "spontaneous secretion" refers to cells in the biological sample spontaneously producing the cytokine or chemokine after the biological sample has been applied to an ELISpot plate pre-coated with a capture antibody specific to the cytokine or chemokine.

2. The method of claim 1, wherein the cytokine is IFN-γ or TNF-α.

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 average spot intensity, maximum spot intensity, and / or the number of low-intensity cells per unit volume present in a biological sample from the subject with one or more reference controls.

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

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

8. The method of claim 7, further comprising treating the patient for sepsis if the patient has been diagnosed with sepsis, and not treating the patient for sepsis if the patient has not been diagnosed with sepsis.

9. The method of claim 7, further comprising determining whether the patient has an immunosuppressive endogenous or hyperinflammatory endogenous form by evaluating the amount of cytokines associated with cellular immunity, and determining whether the amount is reduced relative to a control obtained from a healthy patient, wherein if the amount of the cytokine is reduced relative to the control, the patient has an immunosuppressive endogenous form, and if the amount is increased relative to the control, the patient has a hyperinflammatory endogenous form.

10. The method of claim 9, further comprising treating the patient with an immune-enhancing agent if the patient is identified as having an immunosuppressive endotype, or treating the patient with an immune-suppressing agent if the patient is identified as having a hyperinflammatory endotype.

11. The method of claim 10, wherein if the patient has an immunosuppressive endogenous immune type, the patient is treated with an immunostimulatory agent.

12. The method of claim 11, wherein the immunostimulant is IL-7.

13. The method of claim 10, wherein if the patient has a hyperinflammatory intraepithelial type, the patient is treated with an immunomodulatory agent.

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

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