Boosted immune monitoring methods for determining antigen-specific t cell responses

Abstract

The present invention relates to a method for determining antigen-specific T cell responses by enhanced flow cytometry and enhanced ELISPOT techniques that simultaneously stain cytokines based on the use of specific combinations of monoclonal antibodies that identify specific T cell polarization profiles in their combinations. The method allows for the identification of Tfc CD8 + T cell responses that have not been identified so far.

Description

Technical Field

[0001] The present invention relates to methods for determining antigen-specific T-cell responses based on boosted flow cytometry and boosted ELISPOT techniques and the use of a combination of monoclonal antibodies directed against cytokines that define specific T-cell polarization profiles associated with disease states. Background Art

[0002] Despite 90 years of vaccination and 60 years of chemotherapy, tuberculosis (TB) remains the world's leading cause of death from infectious agents, surpassing human immunodeficiency virus / acquired immunodeficiency syndrome (HIV / AIDS) for the first time. The World Health Organization (WHO) estimates that there are approximately 10.4 million new cases and 1.8 million deaths from TB each year. One third of these new cases (approximately 3 million) remain unknown to the health system, and many do not receive adequate treatment.

[0003] Tuberculosis is an infectious bacterial disease caused by Mycobacterium tuberculosis (Mtb), which is transmitted from person to person by the respiratory route and most often affects the lungs, but can damage any tissue. One of the challenges of TB is that the pathogen persists in a latent state in many infected individuals for many years and can be reactivated to cause disease. The risk of progression to TB following infection is highest shortly after the initial infection, and the risk increases dramatically for people who are also infected with HIV / AIDS or other immune-compromising conditions.

[0004] TB disease requires multiple drug treatments for months. These long-term drug regimens are challenging for both patients and health care systems, especially in low- and middle-income countries (LMICs), where the burden of disease often far exceeds local resources. In some areas, the incidence of drug-resistant TB is increasing, requiring even longer treatments with more expensive and less tolerated drugs.

[0005] Great progress has been made in TB control over the past few decades. Despite progress, large gaps remain. While the Sustainable Development Goals (SDGs) seek to end the TB pandemic once and for all, declines in incidence have been disappointing. One in three TB patients remains “unknown to the health system”, many are not diagnosed and treated, and case detection and treatment success rates remain too low in high-burden countries. Unfortunately, global rates of multidrug-resistant (MDR) TB – defined as resistance to the two main TB drugs, isoniazid and rifampicin – are increasing with the emergence of extensively drug-resistant (XDR) TB, which is resistant to many second-line drugs, and strains resistant to all current drugs. These are now primarily the result of transmission rather than undertreatment.

[0006] If the ultimate goal of infectious disease control is to interrupt transmission, then reversing the TB trend will require early and accurate case detection, rapid initiation and adherence to effective treatment to prevent transmission, and, where possible, preventive treatment of latent TB.

[0007] Diagnosis in LMICs is primarily made by microscopic examination of stained smears of sputum from suspected patients; however, smear microscopy detects only 50-60% of all cases (smear positive). New diagnostic options have emerged in recent years, such as the Xpert MTB / RIF test, a DNA amplification test that provides rapid and sensitive detection of TB and rifampicin resistance, enabling diagnosis of TB in less than two hours and identification of MDR TB from sputum. However, this test is expensive and cannot be performed at the point of care. Zak D et al. (2016. The Lancet 387(10035):2312–22) developed a method to measure gene expression signatures in peripheral leukocytes that are elevated before latently infected healthy individuals progress to active TB.

[0008] Nonetheless, further TB diagnostic methods are needed to bring health care providers closer to TB control. A more sensitive diagnostic method that can differentiate between active and latent TB infection (ATBI and LTBI) would be desirable, thereby appropriately adjusting treatment and achieving such TB control.

[0009] Currently, approximately 38 million people are infected with HIV worldwide. See UNAIDS, http: / / www.unaids.org / ,November 2021. HIV infection is currently one of the major public health challenges in the world.

[0010] A minority of HIV-infected individuals (<1%, i.e., HIV controllers) naturally control viral replication in the absence of HAART, which may be due to innate and adaptive immune mechanisms (Jacobs E, et al., J. Virol. 2017; doi:10.1128 / jvi.02051-16, F, et al., J Acquir Immune Defic Syndr. 2015; doi:10.1097 / QAI.0000000500). A detailed understanding of this natural immune-mediated control is essential for developing effective immune strategies to prevent and treat HIV infection ( 2015, supra). By inducing potent cytotoxic CD8+ T cell responses (CTL or Tc 3Specific HLA class I genotypes have been shown to be associated with HIV disease progression to some extent, but this accounts for only 10-15% of the variability in the course of infection in untreated hosts (Jones R, et al., J. Clin. Invest. 2016; doi: 10.1172 / JCI80566, Fellay J, et al., Science 2007; 80, doi: 10.1126 / science.1143767). In addition, the relative contribution of different CTL specificities to overall viral control remains controversial because they are highly diverse in HIV controllers (Pereyra F, et al., J. Infect. Dis. 2008; doi: 10.1086 / 526786, Sáez-Cirión A, et al., J. Immunol. 2009; doi: 10.4049 / jimmunol.0803928). In addition to cellular immunity, humoral immune correlates of controlled HIV disease have also been explored. Specifically, broadly neutralizing antibodies (bnAb) passive immunization has shown convincing clinical efficacy, especially when structurally modified antibodies are used (Mendoza P, et al., Nature 2018; 561). These studies clearly show that neutralizing and non-neutralizing antibodies to HIV can trigger additional antiviral effector functions, involving Fc-mediated mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC) (Chen X, et al., Front. Immunol. 2018; doi: 10.3389 / fimmu.2018.02322). However, the ability of naturally induced neutralizing antibodies to control chronic viral replication appears to be limited, and broadly neutralizing antibody responses are only elicited in a minority of infected individuals; in addition, their presence is often associated with increased HIV replication and viral diversity (Overbaugh J, et al., Cold Spring Harb. Perspect. Med. 2012; doi: 10.1101 / cshperspect.a007039, Su B, et al., Front. Immunol. 2019; doi: 10.3389 / fimmu.2019.02968). In addition to affinity maturation that can produce bnAbs, the mechanisms that induce and maintain humoral responses with effector functions are not well understood.

[0011] While it is widely accepted that effective HIV control requires the induction of strong cellular and humoral adaptive responses, the nature of the most appropriate T cell populations to exert direct antiviral activity and support antibody production remains elusive. However, emerging data strongly suggest that this may be a question of specificity or polarization of the effector profile of such T helper (Th) cells driving this combined antiviral immunity. Pathogen-specific memory T cells can exist in different Th subsets that can have different or even divergent effector functions (Sallusto F, Annu. Rev. Immunol. 2016; 34: 317–334). While much is known about CD4 T cells and their differentiation and polarization into different Th subsets (e.g., Th1, Th2, Thx-like populations) and divergent effector function profiles, this classification of CD8+ T cells is less certain. Nevertheless, there are recent reports that CD8+ T cells also differentiate into distinct polarized subsets, characterized by effector functions in addition to their classical cytotoxic capacity, and are associated with different immune contexts, including those other than HIV infection (Sallusto, 2016, supra, Valentine K, et al., Front. Immunol. 2019; doi: 10.3389 / fimmu.2019.01322). For example, a CD8+ subtype with an abnormal Th2-like phenotype is associated with an effective immune response to Mycobacterium tuberculosis (Mtb).

[0012] Based on the broad spectrum of effector function profiles (defined primarily by cytokine production characteristics), T cell polarization is broadly divided into three major types: type 1 (CD4+Th1 and CD8+Tc1 subsets, characterized by secretion of IFN-γ, IL-2, and TNF-α); type 2 (CD4+Th2 and CD8+Tc2 subsets, mainly producing IL-4, IL-5, IL-10, and IL-13); and type 3 (CD4+Th17 and CD8+Tc17 subsets, characterized by IL-17 and IL-22 as cytokines) (Annunziato F, et al., J. Allergy Clin. Immunol. 2015; 135: 626–635). This T cell subset diversity is achieved by regulatory T cells (Treg, CD4+ or CD8+ Treg subsets, producing IL-10 and TGF-β), multiple less distinctive and rarer subsets (Th22 CD4+ or CD8+ subsets, defined by the production of TNF-α, IL-2 and IL-22; CD4+ and CD8+ Th9 characterized by the production of IL9 and other emerging subsets) (Chen J, et al., DNA Cell Biol. 2019; 38(10): 1040-1047, Jia L, et al., AdvExp Med Biol. 2014; 841: 209-230). One of the more recently identified additional T cell subsets is follicular T cells. Follicular T helper cells (Tfh) have been shown to play an important role in the development of humoral immunity (Joosten S, et al., Hum. Immunol. 2008; 69: 760–770). Activated naive CD4+ pre-Tfh cells are located at the edge of the T cell zone of secondary lymphoid organs, begin to express CXCR5, PD-1 and ICOS and migrate to the germinal center of the follicle (Song W, et al., Immunol. Rev. 2019; 288: 85–96). There, they interact with B cells and support their proliferation, isotype class switching and antibody affinity maturation through a series of somatic hypermutations. CD4 Tfh functionality is associated with IL-4 and IL-21 secretion, which is required for the establishment of antigen-specific, long-lived plasma cells and circulating memory B cells (Koutsakos M, et al., J. Immunol. 2019; 202: 360–367). Although this function was initially thought to be reserved for CD4+ T cells, increasing evidence suggests that CXCR5-expressing CD8+ T cells are also located within or near B cell follicles.These CD8+ T follicular cytotoxic cells (Tfcs) are reported to have a self-renewal and less exhausted profile, and maintain their cytotoxic functionality to eliminate infected cells while regulating antibody responses (Yu D, et al., Trends Immunol. 2018; 39: 965–979). These findings are consistent with previous observations that CD8 Tfcs are associated with B cell isotype switching and superior control of viral replication.

[0013] For CD8+ and CD4+ T cells, an association between type 1 responses and reduced HIV viremia has been demonstrated. Although it was hypothesized many years ago, the importance of other T cell subsets that play alternative effector functions (not mediated by IFN-γ) in HIV pathogenesis remains understudied (Walker B, et al., Cold Spring Harb. Perspect. Med. 2012; doi: 10.1101 / cshperspect.a007054, Johnson S, et al., J. Virol. 2015; doi: 10.1128 / jvi.00438-15). Several aspects may be the cause of this gap in our understanding, including the small size of certain subsets in peripheral blood, their heterogeneity and plasticity, or the severe depletion of CD4+ T cells during acute HIV infection (Crotty S, et al., Cold Spring Harb. Perspect. Biol. 2018; 10: a032102). In the early 1990s, CD4+Th1 / Th2 balance was considered a hallmark of HIV disease progression, but this view was not unanimously recognized (Graziosi C, et al., Science 1994; 80: 265, Clerici M, et al., Immunol. Today 1993; 14). To date, although HIV-specific type 2 and type 3 responses remain poorly understood, the relevance of CD4+Th2 and Th17 in HIV disease control and vaccine response has begun to emerge (Sauce D, et al., Sci. Rep. 2016; doi: 10.1038 / srep28129). In contrast, HIV-specific CD8+Tc2 responses were associated with reduced cytotoxic activity, while retention of tissue-resident CD8+Tc17 during HIV infection was important for regulating immune activation (González S, et al., Front. Immunol. 2017; 8: 1–9, Perdomo-Celis F, et al., Front. Immunol. 2017; 8: 1–13). Regulatory CD4+T cells were associated with weaker HIV-specific cellular immune responses and were shown to inhibit HIV-induced immune hyperactivation, thereby limiting infection of conventional CD4+T cells (Hasenkrug K, et al., PLoS Pathog. 2018; 14: 1–22, Fenoglio D, et al., J. Allergy Clin. Immunol. 2018; 141: 2220-2233.e4).It has been reported that CD4+Tfh cells have different HIV antigen specificity compared to conventional CD4+Th1 cells and are required to help differentiate and mature HIV-specific B cell responses while supporting CTL activity (Locci M, et al., Immunity 2013; 39: 758–769, Martín-Gayo E, et al., JCI Insight 2017; 2: 1–17, Schultz B, et al., Immunity 2016; 44: 167–178). On the other hand, Tfh cells appear to be highly permissive for HIV entry and account for a large proportion of the latent viral reservoir, and the expansion of this cell subset is associated with disease progression (Aid M, et al., Front. Immunol. 2018; doi: 10.3389 / fimmu.2018.00895, Kohler S, et al., J. Immunol. 2016; doi: 10.4049 / jimmunol.1502174, Miles B, et al., Trends Microbiol. 2016; doi: 10.1016 / j.tim.2016.02.006, Lu J, et al., J. Infect. Public Health 2018; 11: 685–690).

[0014] Little is known about CD8+Tfc cells in HIV infection, and some studies have reported higher ex vivo cytotoxic activity compared to non-Tfc CD8+T cells (Petrovas C, et al., Sci. Transl. Med. 2017; doi: 10.1126 / scitranslmed.aag2285). This is consistent with the Tfc and Tc2-like phenotype CD8+T cells that mediate lower viral loads in chronic infection animal models (including LCMV-infected mice and SIV-infected macaques) (He R, et al., Nature 2016; doi: 10.1038 / nature19317, Mylvaganam G, et al., Proc. Natl. Acad. Sci. USA 2017; doi: 10.1073 / pnas.1621418114). These observations suggest that CD8+ Tfc with increased cytotoxic capacity and localization to lymph node follicles are critical for limiting the viral reservoir in CD4+ Tfh cells and provide additional help for the maturation of humoral immunity in follicles.

[0015] The diversity and functional polarization of the above-mentioned T cell repertoires are in stark contrast to the limited identification methods that can be used to date to identify, characterize and quantify T cell responses. Therefore, standard techniques do (i) miss these above-mentioned unconventional T cell differentiation repertoires (Guardo A, et al., AIDS 2015; 29, Ruíz-Riol M, et al., J. Infect. Dis. 2015; doi: 10.1093 / infdis / jiu534) and (ii) fail to provide further insight into disease states, which are reflected by the polarization of specific T cell responses. Therefore, there is an urgent need in the art for better tests, such as the novel flow cytometry and enhanced ELISPOT assays described herein, which can identify the broadest T cell polarization repertoire of antigen-specific T cells in a specific and sensitive manner. Summary of the invention

[0016] In a first aspect, the present invention relates to a method for determining a polarized T cell response specific for an antigen in a subject, comprising:

[0017] (i) contacting a biological sample comprising T cells from a subject with a composition comprising an antigen, and

[0018] (ii) determining the levels of various cytokines and / or chemokines produced by T cells in the sample by enhanced flow cytometry and / or enhanced ELISPOT,

[0019] wherein a combination of antibodies against multiple cytokines and / or chemokines is used, and the multiple cytokines and / or chemokines are associated with a specific T cell polarization profile,

[0020] The antigen is at least one antigen from Mycobacterium tuberculosis (M. tuberculosis).

[0021] In another aspect, the present invention relates to a kit comprising reagents for detecting one or more cytokines, including IFN-γ, IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17, IL-21, IL-22, TGF-β, CXCL-13 and TNF-α.

[0022] In another aspect, the present invention relates to use of the method of the first aspect for diagnosing tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1.The boosted flow assessment has the ability to capture more robust M.Tb infection compared to other assessments in ATBI (50% gold standard Quantiferon IGRA (QFN) vs 100% boosted (5μg protein stimulation) and 15% tuberculin skin test vs 100% LTBI (5μg protein stimulation)). Chi-square test was performed for inter-group comparisons. The percentage of patients with a positive response to the indicated test is indicated in the figure. P<0.05 was considered significant. P<0.0001=****, P<0.001=***, P<0.01=**.

[0024] Figure 2 .While ATBI produced mainly Th1-like and Th17-like profiles, LTBI produced a specific M.Tb response with Th2-like and Tfh effector functions. Chi-square test was performed for comparison between groups. The percentage of patients who responded positively to ESAT-6, CFP-10 or a combination of both proteins (5 μg stimulation) to enhance the specific T cell profile measured by flow is indicated in the figure. P<0.05 was considered significant. P<0.0001=****, P<0.001=***, P<0.01=**.

[0025] Figure 3 .Gating strategy for boosted flow screening. Gating on live CD3+ cells followed by gating on CD4 and CD8. Boosted flow channels for CD4+ cells (A) and CD8+ cells (B) are shown. Specific CD4 T cell (A) and CD8 T cell responses (B) in the presence of anti-CD3 / CD28 polyclonal stimulation (positive control) or in the absence of specific T cell stimulators (negative control).

[0026] Figure 4 Correlation between IL-4 / IL-21 responses measured by boost flow and ELISPOT on total PBMCs after HIV peptide pool-specific stimulation (non-parametric Spearman r test).

[0027] Figure 5 .HIV protein-specific polarized T cell responses. Magnitude of polarized responses in CD4+ T cells (A) and CD8+ T cells (B). Controllers are indicated by purple dots and non-controllers by light blue dots. Significant p-values ​​are highlighted in color, purple if the response is higher in controllers and light blue if the response is higher in non-controllers. The table below indicates the number of pools included in each protein screen, with the number of peptides in each pool in parentheses. Statistical significance was assessed using the non-parametric Mann-Whitney test (B).

[0028] Figure 6 Lack of significant correlation between HIV-specific CD8+Tfc-like responses and humoral responses for (a) neutralization and (b) ADCC. P indicates presence. A indicates absence.

[0029] Figure 7 In a cohort of 15 HIV+ controllers (C, purple) and 14 HIV+ noncontrollers (NC, light blue), boosted flow screening for specific T cell responses against the entire HIV proteome revealed a highly diverse set of polarized T cell responses in terms of (A) the number of responders, (B) the percentage of circulating HIV-specific T cells (top), and the size of the CD4 and CD8 T cell pools (bottom), respectively. (C) Inverse correlation between plasma viral load and measured CD8+ Tfc-like T cell responses to HIV in terms of breadth (left) and magnitude (right). (D) The ability of total PBMCs to simultaneously secrete the T follicle-associated cytokines IL-21 (left scatter plot), IL-4 (middle scatter plot), and IL-21 / IL-4 (right scatter plot) after antigen-nonspecific PHA stimulation, stratified by the presence (left bar) or absence (right bar) of HIV-specific circulating Tfc-like responses. The statistical significance of differences in responder frequencies was evaluated by the chi-square test, differences between group medians were evaluated by the nonparametric Mann-Whitney test, and the correlation between Tfc-like responses and plasma viral load was evaluated by Spearman correlation and its corresponding p-value. Statistical significance was set at p < 0.05.

[0030] Figure 8 .Comprehensive assessment of HIV protein-specific polarized T cell responses. Screening for HIV protein-specific T cell responses based on enhanced flow cytometry revealed antigen-dependent, distinct polarization profiles in both CD4+ and CD8+ T cells. Stacked bars of CD4+ (A) and CD8+ (B) T cells indicate the percentage of responders to each HIV-1 derived protein. Statistically significant (p<0.05) differences in protein-specific response frequencies between HIV+ controllers (C) and HIV+ non-controllers (NC) are indicated. Statistical significance was evaluated by chi-square test, and statistical significance was set at p<0.05.

[0031] Fig. 9.Memory phenotype of HIV-specific polarized T cell responses. The frequency of cells expressing the memory markers CD45RA and CCR7 was evaluated in both HIV-specific polarized CD4+ and CD8+ T cells. The pie chart shows the percentages of basal cells, Th1 / Tc1 cells, Th2 / Tc2 cells, and Tfh / Tfc-like cells classified as TCM (CD45RA-CCR7+), TEM (CD45RA-CCR7-), TEMRA (CD45RA+CCR7-), and TNaive (CD45RA+CCR7+). The statistical significance of the differences between the HIV-specific memory phenotypes in HIV+ controllers (C, left) and HIV+ non-controllers (NC, right) was evaluated by the non-parametric Mann-Whitney test, with statistical significance set at p>0.05, and only significant results are shown.

[0032] Figure 10. Association between HIV-specific CD8+Tfc-like responses and humoral responses. (A) Samples were stratified by the presence (n=17) or absence (n=13) of HIV-specific CD8+Tfc-like responses and the titers of IgA, IgG, and IgM and the ratios of IgA / IgM, IgG / IgM, and IgG / IgA were compared. (B) Correlation between anti-HIV-1NL4-3 (left) and HIV-1BaL (right) IgM titers and the size (left) and breadth (right) of the CD8+Tfc-like response. (C) Anti-HIV-1 NL4-3 (Left) and HIV-1 BaL (Right) Correlation between IgG titer and CD8+Tfc-like width. (D) Anti-HIV-1 NL4-3 (Left) and HIV-1 BaL (Right) Correlation between IgG / IgM ratio and CD8+Tfc-like width. (E) Anti-HIV-1 NL4-3 (Left) and HIV-1 BaL (Right) Correlation between the IgG / IgA ratio and the magnitude (left) and breadth (right) of CD8+Tfc-like responses. Statistical significance of comparisons was performed by the nonparametric Mann-Whitney test, while nonparametric Spearman r correlation was applied. DETAILED DESCRIPTION

[0033] Currently, there is no test that can distinguish between active and latent stages of TB. The inventors surprisingly found that enhanced flow cytometry outperformed prior art tests in both active and latent TB diagnosis: enhanced flow was significantly more powerful in detecting active M.Tb. infection compared to the gold standard Quantiferon IGRA (37% vs 100%) and tuberculin skin test (TST) (50% vs 100%). Using the present invention, different polarization profiles are evaluated simultaneously in the same sample without the need for additional parallel testing, and allows the differentiation of active and latent infection by the breakdown of T cell polarization profiles. This is particularly relevant and useful for children, the elderly and immunocompromised patients, such as patients coinfected with HIV-1 - where TB is a significant cause of death - or patients receiving immunosuppressive therapy. The ability of the claimed invention to discriminate between active and latent stages of TB disease is an important added value compared to the gold standard assays on the market and is very relevant for the adequacy of treatment to reduce multi-drug resistance which has become a worldwide challenge.

[0034] 1. Definition of General Terms and Expressions

[0035] As used herein, the term "AIDS" refers to the symptomatic phase of HIV infection and includes both acquired immunodeficiency syndrome (commonly referred to as AIDS) and "ARC" or AIDS-related complex (Adler M, et al., Brit. Med. J. 1987; 294: 1145-1147). The immunological and clinical manifestations of AIDS are well known in the art and include, for example, opportunistic infections and cancers resulting from immunodeficiency.

[0036] As used herein, the term "antigen" refers to a molecule that is recognized by the product of an adaptive immune response (specifically, by a specific T cell receptor (TCR) and a B cell receptor (BCR, or antibody). Antigens include, but are not limited to, cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide and non-peptide mimetics of polysaccharides and other molecules, small molecules, lipids, glycolipids, carbohydrates, viruses and viral extracts, and multicellular organisms such as parasites and allergens. The term antigen broadly includes any type of molecule that is recognized by the host's immune system, thereby including, but not limited to, pathogen-derived antigens, self-antigens, allergens, and toxins.

[0037] As used herein, the term "antiretroviral therapy" or "ART" refers to the administration of one or more antiretroviral drugs (i.e., HIV antiretroviral agents) to inhibit the replication of HIV. Typically, ART involves the administration of at least one antiretroviral agent (or often a mixture of antiretroviral agents) such as nucleoside reverse transcriptase inhibitors (e.g., zidovudine (AZT), lamivudine (3TC), and abacavir), non-nucleoside reverse transcriptase inhibitors (e.g., nevirapine and efavirenz), and protease inhibitors (e.g., indinavir, ritonavir, and lopinavir).

[0038] The term "antibody" as used herein refers to a complete immunoglobulin of any isotype or a fragment thereof that can compete with a complete antibody for specific binding to a target antigen, and includes, for example, chimeric, humanized, fully human, non-human and bispecific antibodies.Antibody is an antigen binding protein.Complete antibodies will generally include at least two full-length heavy chains and two full-length light chains, but may include fewer chains in some cases, such as antibodies naturally present in camelids, which may include only heavy chains.Antibodies may be derived from only a single source, or may be "chimeric", i.e., different parts of the antibody may be derived from two different antibodies, as further described below.Antigen binding proteins, antibodies or binding fragments may be produced in hybridomas by recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies.Unless otherwise indicated, the term "antibody" includes, in addition to antibodies including two full-length heavy chains and two full-length light chains, derivatives, variants, fragments and mutant proteins thereof, which may be connected to the cytokines of the present invention.

[0039] As used herein, the term "M.Tb treatment or therapy" refers to the treatment received by subjects suspected of or diagnosed with Mycobacterium tuberculosis (M.Tb) infection. For latent TB (LTBI), if there is a risk of developing active TB (ATBI), treatment is usually prescribed, and the most common treatment includes only two types of drugs. For active tuberculosis, antibiotics are prescribed for at least six to nine months. Especially for resistant strains, treatment will require the use of several drugs at the same time, and the most common drugs are isoniazid, rifampicin, ethambutol, pyrazinamide, and a combination of fluoroquinolones and other injectable antibiotics, such as amikacin or capreomycin, which are usually used for 20 to 30 months. Since some types of TB also develop resistance to these drugs, some additional drugs may be added to the treatment to combat resistance, such as bedaquiline or lindoxadiazole.

[0040] The term "multidrug resistance (MDR)" as used herein refers to the insensitivity or resistance of a microorganism to an administered antimicrobial drug that is structurally unrelated and has a different molecular target, despite being previously sensitive thereto.

[0041] As used herein, the term "biological sample comprising T cells" refers to a tissue or body fluid taken from a mammalian subject, and it includes T cells. In some embodiments, before the T cells are exposed to the antigen (one or more) of interest, the T cells are separated from the sample. The term "isolated" about T cells refers to preparing a cell population in the form of a cell population having at least 70%, 80%, 90%, 95%, 99% or 100% T cells. In some embodiments, the desired cell population is separated from other cell components, in some cases specifically excluding other cell types that may interfere with the study of the isolated cells. However, the "isolated" cell population can be incorporated into other cell types necessary for cell survival or the desired results provided by the present invention. For example, antigen presenting cells, such as monocytes (macrophages) or dendritic cells, may be present in the "isolated" cell population of T cells, or be added to the isolated T cell population to produce regulatory T cells. In some embodiments, these antigen presenting cells can be activated monocytes or dendritic cells. The cell population comprising T cells used in the method of the present invention can be isolated from a biological sample taken from a mammalian subject. Samples can be derived from a variety of sources, including but not limited to peripheral blood, leukapheresis blood products, apheresis blood products, bone marrow, thymus, tissue biopsy, tumor, lymph node tissue, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, liver, sites of immune damage (e.g., synovial fluid), pancreas, and cerebrospinal fluid.

[0042] As used herein, the term "enhanced flow cytometry" refers to an assay based on the detection of cells that secrete several cytokines simultaneously and detecting these cytokines in the same channel of a flow cytometer so that the detected signal is additive over the entire range of cytokines detected. Thus, "enhanced flow cytometry" results in increased sensitivity and allows coverage of a much larger set of effector functions than standard assays.

[0043] As used herein, the term "boosted ELISPOT" refers to an assay based on the detection of cells producing several cytokines by using several cytokine-specific antibodies that are labeled (for example, but not limited to enzymatic labels such as AP or fluorescent labels) to detect and quantify cells producing one or several of these cytokines. Thus, "boosted ELISPOT" allows the detection of a broader set of effector functions than the standard, usually single-function ELISPOT assay and provides higher sensitivity than the standard assay because the signals originating from the individual cytokines produced in a single cell all add up to form a spot.

[0044] The term "CD4+T cells" as used herein refers to T cells that present CD4 molecules (differentiation cluster 4 molecules) on their surface. The term refers to T helper cells, which coordinate the activation of macrophages and CD8+T cells (Th-1 cells), the production of antibodies caused by B cells (Th-2 cells), or have been shown to play an important role in autoimmune diseases (Th-17 cells). In addition, the term "CD4+T cells" also refers to other T cell populations expressing CD4, including but not limited to regulatory CD4+T cells (Treg), which account for about 10% of the total CD4+T cell population. Regulatory T cells play an important role in suppressing immune responses, preventing autoimmune diseases and oral tolerance.

[0045] The term "CD8+ T cells" as used herein refers to T cells that express CD8 glycoproteins on their surface, wherein the CD8 (differentiation cluster 8 molecule) glycoprotein is a transmembrane glycoprotein that acts as a co-receptor for the T cell receptor (TCR). CD8 binds to major histocompatibility complex (MHC) molecules, but is specific for class I MHC proteins. Exemplary CD8 T cells include cytotoxic memory CD8 T cells, regulatory CD8 T cells, cytotoxic effector CD8 T cells, and other cells that can be identified by the skilled person.

[0046] As used herein, the term "CMV" or "cytomegalovirus" refers to a genus of viruses in the order Herpesvirales, the family Herpesviridae, the subfamily Betaherpesvirinae. The genus CMV includes 11 species, including human beta herpesvirus 5 (HCMV, human cytomegalovirus, HHV-5 type) that infect humans. Diseases associated with HHV-5 include mononucleosis and pneumonia (ENA accession number GU980198).

[0047] As used herein, the terms "comprising" or "comprises" also disclose "consisting of" according to generally accepted patent practice.

[0048] As used herein, the term "controller" refers to an individual who is infected with HIV and exhibits (i) undetectable plasma HIV RNA or (ii) detectable but low levels of plasma HIV RNA (Deeks S, et al., Immunity 2007; 27: 406-416, Ferre A, et al., Blood 2009; 113(17): 3978-3989).

[0049] As used herein, the term "coronavirus" refers to any member of the Coronaviridae virus family. The Coronaviridae family includes single-stranded RNA viruses with a diameter of about 120 nanometers. The family is divided into two subfamilies: Letovirinae and Coronavirinae. The Coronavirus subfamily includes alphacoronaviruses (e.g., human coronavirus 229E (HCoV-229E), betacoronaviruses (e.g., human coronavirus HKU1, human coronavirus NL63 (HCoV-NL63, New Haven coronavirus), human coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome-related coronavirus (MERS-CoV or HCoV-EMC - the causative agent of MERS), severe acute respiratory syndrome coronavirus (SARS-CoV - the causative agent of SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the causative agent of COVID-19), deltacoronaviruses, and gammacoronaviruses. Coronaviruses can also infect non-human subjects such as, for example, cattle (e.g., bovine coronavirus (BCV), cats (e.g., feline coronavirus (FCoV), dogs (e.g., canine coronavirus (CCoV), pigs (e.g., porcine coronavirus HKU15, porcine epidemic diarrhea virus (PED or PEDV), rabbits (e.g., rabbit enteric coronavirus) and birds (e.g., infectious bronchitis virus (IBV), turkey coronavirus (TCV)). There are more than 40 species of coronaviruses.

[0050] As used herein, the term "cytokine" refers to a small protein secreted by a cell, which can change the behavior or properties of secretory cells or other cells. Cytokines bind to cytokine receptors and trigger intracellular behavior or properties, such as cell proliferation, death or differentiation. Exemplary cytokines include but are not limited to interleukins (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-1a, IL-1β and IL-1RA), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), oncostatin M, erythropoietin (EPO), leukemia inhibitory factor (LIF), interferon, TNF family members (e.g., TNF-α, TNF-β, LT-β). Cytokine levels and the cells that produce them can be determined with varying levels of sensitivity and specificity by several methods well known in the art such as ELISA, ELISPOT, Western blot, and flow cytometry.

[0051] As used herein, the term "chemokine" refers to a chemotactic cytokine secreted by cells and other cell types that induce directional movement of leukocytes. Exemplary chemokines include CC chemokines (CCL1-CCL28), CXC chemokines (CXCL1-CXCL17), C chemokines (XCL1 and XCL2), and CX3C chemokines such as CX3CL1. In the present invention, chemokine CXCL13 has a special relevance.

[0052] As used herein, the term "EBV" or "Epstein-Barr virus" refers to a double-stranded DNA virus member of the herpes virus family (Zanella M, et al., Clinical Microbiol Rev 2020; 33(4): e00027-20.0). EBV is most commonly transmitted through body fluids, mainly saliva. EBV can cause infectious mononucleosis, also known as mono or kissing disease virus, as well as other conditions, specifically several forms of cancer such as lymphoma and carcinoma.

[0053] As used herein, the term "ELISPOT" or "enzyme-linked immunospot" refers to a highly sensitive immunoassay that measures the frequency of cytokine-secreting cells at the single-cell level. In this assay, cells are cultured on a surface coated with a cytokine-specific capture antibody in the presence or absence of antigenic stimulation. Monoclonal or polyclonal antibodies specific for the selected analyte (e.g., cytokine) are usually pre-coated on a PVDF (polyvinylidene fluoride) backed microplate. The appropriately stimulated cells are pipetted into the wells and the microplates are placed in a humidified 37°C CO2 incubator for a specified time. During this incubation period, the fixed antibodies of the cells that are adjacent to the secretion of the analyte of interest bind to the secreted analyte. After washing away any cells and unbound material, a biotinylated polyclonal antibody specific for the selected analyte is added to the wells. After washing to remove any unbound biotinylated antibody, streptavidin conjugated to an enzyme (e.g., alkaline phosphatase) is added. The unbound enzyme is subsequently removed by washing and an enzyme-specific substrate solution (e.g., BCIP / NBT) is added. Colored precipitates (e.g., blue-black in the case of BCIP / NBT) form at the cytokine localization site and appear as spots, with each individual spot representing an individual analyte-secreting cell. Biotin-streptavidin enzymatic detection can replace fluorescent labels that allow direct detection of secreted analytes by a fluorescence detector. Spots can be counted with an automated ELISPOT reader system or manually with a stereomicroscope.

[0054] The term "HBV" or "hepatitis B virus" as used herein refers to a double-stranded DNA virus, a species of the genus Orthohepadnavirus, and a member of the family Hepadnaviridae. HBV disease is associated with acute and chronic hepatitis and can lead to the development of cirrhosis and hepatocellular carcinoma (Ryu W, Molecular Virology of Human Pathogenic Viruses (Academic Press, Cambridge, MA, USA, 2017, p. 247-260)).

[0055] As used herein, the term "HCV" or "hepatitis C virus" refers to a single-stranded, positive-sense RNA virus member of the genus Hepacivirus in the family Flaviviridae (Rosen H, et al., NEJM 2011; 364(25): 2429-2438). HCV can cause liver disease and cirrhosis. In some cases, people with cirrhosis may develop serious complications such as liver failure, liver cancer, or esophageal and gastric vascular dilation.

[0056] As used herein, the term "HIV" includes HIV-1, HIV-2 and SHIV. "HIV-1" means human immunodeficiency virus type 1. HIV-1 includes, but is not limited to, extracellular viral particles and forms of HIV-1 associated with cells infected by HIV-1. HIV-1 viruses may represent any known major subtype (A, B, C, D, E, F, G and H classes) or unrelated (minor, outlying) subtypes (O group) including laboratory strains and primary isolates. "HIV-2" means human immunodeficiency virus type 2. HIV-2 viruses include, but are not limited to, extracellular viral particles and forms of HIV-2 associated with cells infected by HIV-2.

[0057] The term "SIV" refers to Simian Immunodeficiency Virus, which is the primate HIV homologue virus that infects monkeys, chimpanzees and other non-human primates. SIV includes, but is not limited to, extracellular viral particles and forms of SIV associated with SIV-infected cells.

[0058] As used herein, the term "HIV exposure" refers to contact between an individual who is not infected with HIV and a person with HIV infection, or contact with body fluids from such an HIV-infected subject - wherein such fluids from the infected subject contact a mucous membrane, a wound or abrasion (e.g., a needle stick, unprotected sexual intercourse), or other surface in the tissue of an uninfected subject in such a way that the virus can be transferred from the infected person or the infected person's body fluids to the uninfected individual.

[0059] As used herein, the term "HIV infection" refers to indications of the presence of the HIV virus in an individual, including asymptomatic seropositivity, AIDS-related complex (ARC), and acquired immunodeficiency syndrome (AIDS).

[0060] As used herein, the term "non-controller" refers to an individual who is infected with HIV and who exhibits disease progression after initial infection, resulting in an increase in viral load, CD4 + Loss of T cells and subsequent decline of the immune system.

[0061] The term "kit" as used herein refers to a product comprising the different reagents necessary for implementing the method of the present invention, which is packaged to allow its transportation and storage. Suitable materials for packaging the components of the kit include crystals, plastics (e.g., polyethylene, polypropylene, polycarbonate), bottles, vials, paper or envelopes.

[0062] As used herein, the term "Mycobacterium tuberculosis" refers to a complex including at least nine species of the genus Mycobacterium, the family Mycobacteriaceae, and the order Actinomycetales (i.e., M. tuberculosis sensu stricto, M. africanum, M. canetti, M. bovis, M. caprae, M. microti, M. pinnipedii, M. mungi, and M. orygis), which are the cause of tuberculosis in humans and zoonoses.

[0063] As used herein, the term "pathogen" refers to a biological agent that causes a disease state (e.g., infection, cancer) in a host."Pathogens" include, but are not limited to, viruses, bacteria, archaea, fungi, protozoa, mycoplasmas, prions, and parasites.

[0064] As used herein, the term "PBMC" refers to peripheral blood mononuclear cells, including lymphocytes, monocytes, and macrophages. PBMCs can be isolated using methods known in the art, such as density gradient centrifugation.

[0065] As used herein, the term "subject" refers to an individual or animal, such as a human, non-human primate (e.g., chimpanzee and other ape and monkey species); farm animals, such as birds, fish, cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats, and guinea pigs. The term does not indicate a specific age or sex. The term "subject" encompasses embryos and fetuses. In some embodiments, the subject is a human.

[0066] As used herein, the term "T cell response" refers to an immune response in which T cells directly or indirectly mediate or otherwise contribute to an immune response in a mammal. T cell-mediated immune responses can be associated with cell-mediated effects (e.g., lymphokine-mediated effects), or even with B cell-related effects if B cells are stimulated, for example, by stimulation of lymphokines secreted by T cells. For MHC class I restricted CTLs, effector functions can be peptide pulses, peptide precursor pulses, or cracking of natural peptide-presenting target cells, secretion of cytokines (e.g., IFN-γ, TNF-α, IL-2), secretion of effector molecules (e.g., granzymes, perforins) or degranulation. For MHC class II restricted T helper cells, effector functions can be secretion or degranulation of peptide-induced cytokines (e.g., IFN-γ, TNF-α, IL-4, IL5, IL-10, IL-13, IL-2), but in some cases other effector functions such as cytotoxic activity may also be included. T cell responses can also be measured as the proliferation of T cells and can be observed in vivo as well as in vitro.

[0067] 2. Methods for measuring T cell responses

[0068] In a first aspect, the present invention relates to a method for determining a polarized T cell response specific for an antigen in a subject, comprising:

[0069] (i) contacting a biological sample comprising T cells from a subject with a composition comprising an antigen, and

[0070] (ii) determining the levels of various cytokines and / or chemokines produced by T cells in the sample by enhanced flow cytometry and / or enhanced ELISPOT,

[0071] wherein a combination of antibodies directed against multiple cytokines and / or chemokines is used, and the multiple cytokines and / or chemokines are associated with a specific T cell polarization profile,

[0072] The antigen is at least one antigen from Mycobacterium tuberculosis.

[0073] In a preferred embodiment, the antigen comprises at least one M.Tb protein, preferably ESAT-6, CFP-10 or a combination thereof.

[0074] In a preferred embodiment, the plurality of cytokines and / or chemokines include cytokines and / or chemokines associated with type 1 (Th1 / Tc1), type 2 (Th2 / Tc2), type 3 (Th17 / Tc17), regulatory T cells (Treg) and / or follicular T cells (Tfh / Tfc) polarization profiles and / or other minority emerging (emergent) polarization profiles (such as Th9 and Th22). Preferably, the plurality of cytokines and / or chemokines include cytokines and / or chemokines associated with type 1 (Th1 / Tc1) and / or type 2 (Th2 / Tc2) polarization profiles.

[0075] In a preferred embodiment, multiple cytokines of type 1 (Th1 / Tc1) include IFN-γ, IL-2, TNF-α, or a combination thereof. In a preferred embodiment, multiple cytokines of type 2 (Th2 / Tc2) include IL-4, IL-5, IL-10, IL-13, or a combination thereof. In a preferred embodiment, multiple cytokines of type 3 (Th17 / Tc17) include IL-17, IL-22, or a combination thereof. In a preferred embodiment, multiple cytokines of regulatory T cells (Treg) include IL-10 and TGF-β, or a combination thereof. In a preferred embodiment, multiple cytokines of follicular T cells (Tfh / Tfc) include IL-4, IL-21, CXCL-13, or a combination thereof. In a preferred embodiment, antibodies include monoclonal antibodies. In a preferred embodiment, the sample is a blood sample, preferably a peripheral blood sample. Preferably, the sample to be analyzed is a peripheral blood cell preparation.

[0076] In one aspect, the invention relates to a method for determining a polarized T cell response specific for an antigen in a subject, comprising:

[0077] (i) contacting a biological sample comprising T cells from a subject with a composition comprising an antigen, and

[0078] (ii) determining the levels of various cytokines produced by T cells in the sample by enhanced flow cytometry and / or enhanced ELISPOT,

[0079] A combination of antibodies against multiple cytokines was used, and multiple cytokines are associated with specific T cell polarization profiles.

[0080] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the sample containing T cells is a PBMC preparation.

[0081] In some embodiments, antigens include pathogen-derived antigens, autoantigens, allergens, toxins, or a combination thereof. In some embodiments, antigens include natural or synthetic antigens, or a combination thereof. In some embodiments, pathogens include HIV, HBV, HCV, coronavirus, CMV, EBV, Mycobacterium tuberculosis antigens, or a combination thereof. In some embodiments, autoantigens include chronic inflammatory demyelinating polyneuropathy, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, inflammatory bowel disease (IBD), multiple sclerosis (MS), myasthenia gravis, psoriasis, rheumatoid arthritis (RA), systemic lupus erythematosus, type 1 diabetes, vasculitis antigens, or a combination thereof.

[0082] In some embodiments, the multiple cytokines include cytokines associated with type 1 (Th1 / Tc1), type 2 (Th2 / Tc2), type 3 (Th17 / Tc17), regulatory T cells (Treg), or follicular T cells (Tfh / Tfc) polarization profiles. In some embodiments, multiple cytokines of type 1 (Th1 / Tc1) include IFN-γ, IL-2, TNF-α, or a combination thereof. In some embodiments, multiple cytokines of type 2 (Th2 / Tc2) include IL-4, IL-5, IL-10, IL-13, or a combination thereof. In some embodiments, multiple cytokines of type 3 (Th17 / Tc17) include IL-17, IL-22, or a combination thereof. In some embodiments, multiple cytokines of regulatory T cells (Treg) include IL-10 and TGF-β, or a combination thereof. In some embodiments, multiple cytokines of follicular T cells (Tfh / Tfc) include IL-4, IL-21, CXCL-13, or a combination thereof.

[0083] In some embodiments, the plurality of cytokines includes cytokines associated with rarer subsets of the T cell polarization spectrum (eg, Th22, Th9, etc.).

[0084] In some embodiments, the methods of the present invention can be used to analyze different immune environments, including polarization of cellular immunity in infectious diseases (e.g., HIV, Mycobacterium tuberculosis, SARS-Cov2), autoimmune diseases (e.g., MS, RA), allergic and toxic reactions. In some embodiments, the methods of the present invention can be used to monitor the treatment of the subject's underlying disease, such as HIV infection or infection caused by other pathogens, cancer and / or autoimmune diseases.

[0085] 3. Kit

[0086] In the second aspect, the present invention relates to a kit comprising a reagent for detecting cytokines and / or chemokines according to the method of the first aspect. In a preferred embodiment, the kit comprises a reagent for detecting one or more cytokines associated with type 1 (Th1 / Tc1), type 2 (Th2 / Tc2), type 3 (Th17 / Tc17), regulatory T cells (Treg), Th22, Th9 or follicular T cells (Tfh) polarization spectrum. In some embodiments, the kit includes antibodies for detecting one or more cytokines selected from the following: IFN-γ, IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17, IL-21, IL-22, TGF-β, CXCL-13 and TNF-α.

[0087] On the other hand, the present invention relates to a kit comprising reagents for detecting one or more cytokines associated with type 1 (Th1 / Tc1), type 2 (Th2 / Tc2), type 3 (Th17 / Tc17), regulatory T cells (Treg), Th22, Th9 or follicular T cells (Tfh) polarization profiles. In some embodiments, the kit comprises antibodies for detecting one or more cytokines selected from the group consisting of IFN-γ, IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17, IL-21, IL-22, TGF-β, CXCL-13 and TNF-α. The components of the kit of the present invention may be optionally packaged in suitable containers and labeled for detecting antigens of the present invention (e.g., pathogen-derived antigens such as HIV, HBV, HCV, coronavirus, CMV, EBV, and Mycobacterium tuberculosis antigens; autoantigens such as chronic inflammatory demyelinating polyneuropathy, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, inflammatory bowel disease (IBD), multiple sclerosis (MS), myasthenia gravis, psoriasis, rheumatoid arthritis (RA), systemic lupus erythematosus, type 1 diabetes, and vasculitis antigens) and cancer antigens (e.g., p53-derived T cell antigens). The components of the kit may be stored in units or multi-dose containers as aqueous (preferably sterile) solutions or as lyophilized (preferably sterile) preparations for reconstitution. The container may be formed of a variety of materials such as glass or plastic and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). The kit may further include more containers that hold pharmaceutically acceptable carriers. They may also include other materials desirable from a commercial and user perspective, including, but not limited to, buffers, diluents, filters, needles, syringes or other active agents. The kit may contain instructions customarily included in commercial packages of diagnostic and therapeutic products, the instructions containing, for example, information about the indications, usage, dosage, manufacture, expiration date, administration, contraindications or warnings for the use of such diagnostic products.

[0088] 4. Purpose

[0089] In another aspect, the present invention relates to the use of the method of the first aspect or the kit of the second aspect for diagnosing tuberculosis. The method and kit of the present invention allow for the diagnosis of active tuberculosis or latent tuberculosis. Importantly, the present invention is particularly relevant for a population selected from children, the elderly, immunocompromised patients, subjects co-infected with HIV-1, subjects receiving immunosuppressive therapy, and any combination thereof.

[0090] When the present invention relates to HIV, preferably HIV-1, the present invention is described as follows: A method for determining a polarized T cell response specific for an antigen in a subject, comprising:

[0091] (i) contacting a biological sample comprising T cells from a subject with a composition comprising an antigen, and

[0092] (ii) determining the levels of various cytokines produced by T cells in the sample by enhanced flow cytometry and / or enhanced ELISpot,

[0093] Wherein use the combination of antibody for multiple cytokines, and multiple cytokines are relevant to specific T cell polarization spectrum, wherein the antigen is at least one antigen from HIV-1.Optionally, antigen may also include HBV, HCV, coronavirus, CMV, EBV, mycobacterium tuberculosis antigen or its combination.In the presence of other antigens, when antigen is autoantigen, the autoantigen may include chronic inflammatory demyelinating polyneuropathy, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, inflammatory bowel disease (IBD), multiple sclerosis (MS), myasthenia gravis, psoriasis, rheumatoid arthritis (RA), systemic lupus erythematosus, type 1 diabetes, vasculitis antigen or its combination.

[0094] In a preferred embodiment of the method, the multiple cytokines include cytokines associated with type 1 (Th1 / Tc1), type 2 (Th2 / Tc2), type 3 (Th17 / Tc17), regulatory T cells (Treg) or follicular T cells (Tfh / Tfc) polarization profiles or other minority emerging polarization profiles. Preferably, multiple cytokines of type 1 (Th1 / Tc1) include IFN-γ, IL-2, TNF-α, or a combination thereof. Preferably, multiple cytokines of type 2 (Th2 / Tc2) include IL-4, IL-5, IL-10, IL-13, or a combination thereof. Preferably, multiple cytokines of type 3 (Th17 / Tc17) include IL-17, IL-22, or a combination thereof. Preferably, multiple cytokines of regulatory T cells (Treg) include IL-10 and TGF-β, or a combination thereof. Preferably, multiple cytokines of follicular T cells (Tfh / Tfc) include IL-4, IL-21, CXCL-13, or a combination thereof. In a preferred embodiment, the antibody comprises a monoclonal antibody. Preferably, the sample to be analyzed is a peripheral blood sample, optionally a peripheral blood cell preparation. Another aspect is a kit comprising a reagent for detecting a cytokine as described above. In addition, another aspect is the use of the method or kit for diagnosing HIV diagnosis and immune monitoring.

[0095] All publications mentioned herein are incorporated by reference in their entirety.Having now summarized the invention, the invention will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention unless otherwise specified.

[0096] General Procedure

[0097] 1. Study Design

[0098] (a) TB: The aim of this study was to identify M.Tb-specific T cell responses in M.Tb-infected individuals with an alternative (non-unique IFN-γ-mediated) effector function profile that predicts M.Tb infection and the ability to differentiate between active and latent disease (ATBI and LTBI). Twenty-two M.Tb-infected individuals were included in the study. Ten participants were diagnosed with active TB infection (ATBI) based on microbiological culture determination and lung X-rays complemented by additional positive tests measured by bacillary visualization, PCR and / or TST, QFN, TB T-SPOT (see Table 1). Twelve participants were diagnosed with latent TB infection (LTBI) based on negative microbiological localization, X-ray, undetermined / negative microbiological culture, bacillary visualization and PCR, but positive TST, QFN and TB T-SPOT.

[0099] (b) HIV: One of the initial goals of the exemplary study was to identify HIV-specific T cell responses with alternative (non-IFN-γ mediated) effector function profiles associated with key aspects of the HIV humoral immune response. 44 HIV-infected, treatment-naive individuals (HIV+) and 8 HIV-seronegative controls (HIV-) were included in the study. 24 participants had a viral load (VL) <2000 copies / mL and were considered HIV controllers. They showed a median VL of 210 copies / mL (VL range: 25-1900 copies / mL) and a median CD4+T cell count of 786 cells / mm 3 (CD4+T cell count range: 364-1638 cells / mm 3 An additional 20 participants presented with VL > 2000 copies / mL (median 52435 copies / mL, range 5576-640803) and were considered non-controllers (median CD4+ T-cell count: 406 cells / mm 3 , range: 6-986 cells / mm 3 ). Anti-Env IgM, IgA and IgG titers of plasma samples of all individuals as well as plasma neutralization titers and ADCC activity were tested. Due to sample availability, 29 individuals (15 HIV controllers and 14 HIV non-controllers) were included in the enhanced flow analysis of cellular responses, and phenotypic characterization based on Th2 / Tc2 markers (CRTH2) and Tfh / Tfc markers (CXCR5+PD-1+ICOS+) was performed in 22 samples (8 HIV controllers and 14 HIV non-controllers) used in the enhanced flow analysis. The same 22 individuals were also tested by enhanced ELISPOT method with the same antigen used in the enhanced flow cytometry test. In vitro experiments were performed without blinding or randomization.

[0100] 2. Boosted Flow Screening for TB or HIV-Specific T Cell Responses

[0101] (a) TB: Cryopreserved peripheral blood mononuclear cells (PBMCs) from 10 ATBI and 12 LTBI individuals were thawed and stained. 5 × 10 PBMCs were cultured in R10 (RPMI medium (Gibco) supplemented with 2 mM L-glutamine (Gibco), 100 U / mL penicillin (Gibco), 100 μg / mL streptomycin (Gibco), and 10% heat-inactivated fetal bovine serum (FBS, Invitrogen) at 37 °C. 5PBMC / well for 16 h. PBMC were stimulated with ESAT-6, CFP-10, ESAT6+CFP-10 protein, and PPD protein as positive control in the presence of anti-CD49d and anti-CD28 (BD Biosciences) and GolgiStop (BD Biosciences). As negative control, medium was used instead of peptide pool. TM Cells were stained with Fixable Viability Stain575V, followed by extracellular staining for T cell lineage markers (anti-CD3 APC-Cy7, anti-CD4 BV786, anti-CD8-PerCP, anti-CCR7 BV711, Biolegend; anti-CD45RAFITC, BD Biosciences), B cell lineage markers (anti-CD19 PE-Dazzle594, Biolegend), and myeloid lineage markers (anti-CD14 A700, Biolegend). After a fixation and permeabilization step (Fix and Perm kit, Invitrogen), intracellular staining (ICS) was performed for type 1, type 2, type 3, Treg and T follicle-defining cytokines following the boosting strategy described in the art (Ruíz-Riol M, et al., J. Infect. Dis. 2015; doi: 10.1093 / infdis / jiu534; Romero-Martín, L et al. Front Immunol. 2022; 13: 928039). Antibodies against type 1-related cytokines were conjugated to BV605 (anti-IFN-α, anti-IL-2, anti-TNF-α, Biolegend), antibodies against type 2-related cytokines were conjugated to BV421 (anti-IL-4, anti-IL-10, anti-IL-13, Biolegend), antibodies against type 3-related cytokines were conjugated to PE-Cy7 (anti-IL-17-A, anti-IL-22, Biolegend), antibodies against Treg-related cytokines were conjugated to PE (anti-IL-10, anti-LAP, Biolegend), and antibodies against T follicle-related cytokines were conjugated to APC (anti-IL-4, anti-IL-21, Biolegend). Cells were classified as TCM (CD45RA-CCR7+), TEM (CD45RA-CCR7-), TEMRA (CD45RA+CCR7-), and TNaive (CD45RA+CCR7+).

[0102] On the LSR Fortessa BD instrument, approximately 10 5 cells and performed analysis using FlowJo 10.5.2. Figure 1). The percentage (i.e., quantity) of cytokine-producing cells detected in the unstimulated control was subtracted from the magnitude of antigen-stimulated cells. The threshold for a positive response was determined by the value of the most negative magnitude after subtracting the control in all individuals, as previously described (Roederer M, et al., Cytom. Part A 2011; doi: 10.1002 / cyto.a.21015). Multifunctional cytokine profiling was performed using Boolean gates in FlowJo 10.5.2, following the same strategy of subtracting the unstimulated cell signal and expressed using SPICE v6 (US National Institutes of Health, ImmunoTecnhnology Section, Vaccine Research Center, NIAID, NIH, Bethesda, MD, USA).

[0103] (b) HIV: Cryopreserved peripheral blood mononuclear cells (PBMCs) from 15 HIV+ controllers and 14 HIV+ non-controllers were thawed and stained. 5 × 10 cells / mL were cultured in R10 (RPMI medium (Gibco) supplemented with 2 mM L-glutamine (Gibco), 100 U / mL penicillin (Gibco), 100 μg / mL streptomycin (Gibco) and 10% heat-inactivated fetal bovine serum (FBS, Invitrogen) at 37°C. 5 PBMC / well for 16 h. PBMC were stimulated with a panel of 425 18-mer overlapping peptides (OLPs)—divided into 17 peptide pools (range: 21-27 OLPs / pool, each peptide at a final concentration of 10 μg / mL) covering the entire HIV proteome—or anti-CD3 / anti-CD28 beads (Gibco) as a positive control in the presence of anti-CD49d and anti-CD28 (BD Biosciences) and GolgiStop (BD Biosciences). As a negative control, medium was used instead of the peptide pool. TMCells were stained with Fixable Viability Stain 575V, followed by extracellular staining for T cell lineage markers (anti-CD3 APC-Cy7, anti-CD4 BV786, anti-CD8-PerCP, anti-CCR7BV711, Biolegend; anti-CD45RAFITC, BD Biosciences), B cell lineage markers (anti-CD19PE-Dazzle594, Biolegend), and myeloid lineage markers (anti-CD14 A700, Biolegend). After fixation and permeabilization steps (Fix and Perm kit, Invitrogen), intracellular staining (ICS) was performed for type 1, type 2, type 3, Treg and T follicle-defining cytokines following the boosting strategy described in the art (Ruíz-Riol M, et al., J. Infect. Dis. 2015; doi: 10.1093 / infdis / jiu534; Romero-Martín L, et al. Front Immunol. 2022; 13: 928039). Antibodies against type 1-related cytokines were conjugated to BV605 (anti-IFN-α, anti-IL-2, anti-TNF-α, Biolegend), antibodies against type 2-related cytokines were conjugated to BV421 (anti-IL-4, anti-IL-10, anti-IL-13, Biolegend), antibodies against type 3-related cytokines were conjugated to PE-Cy7 (anti-IL-17-A, anti-IL-22, Biolegend), antibodies against Treg-related cytokines were conjugated to PE (anti-IL-10, anti-LAP, Biolegend), and antibodies against T follicle-related cytokines were conjugated to APC (anti-IL-4, anti-IL-21, Biolegend). Cells were classified as TCM (CD45RA-CCR7+), TEM (CD45RA-CCR7-), TEMRA (CD45RA+CCR7-), and TNaive (CD45RA+CCR7+).

[0104] On the LSR Fortessa BD instrument, approximately 10 5 cells and performed analysis using FlowJo 10.5.2. Figure 3). The percentage of cytokine-producing cells detected in the unstimulated control was subtracted from the number of antigen-stimulated cells. The threshold of the positive response was determined by the value of the most negative number after subtracting the control in all individuals, as previously described (Roederer M, et al., Cytom. Part A 2011; doi: 10.1002 / cyto.a.21015). Multifunctional cytokine profile analysis was completed in FlowJo 10.5.2 using Boolean gates, following the same strategy of subtracting the unstimulated cell signal and using SPICE v6 representation (US National Institutes of Health, ImmunoTecnhnology Section, Vaccine Research Center, NIAID, NIH, Bethesda, MD, USA).

[0105] 3. Phenotypic characteristics of antigen-specific CD4+ Th2 and Tfh cells and CD8+ Tc2 and Tfc cells

[0106] Surface markers of Th2 / Tc2 (CRTH2) and Tfh / Tfc T cell polarization (CXCR5, ICOS, and PD-1) were characterized in cryopreserved PBMCs from 8 HIV+ controllers and 14 HIV+ non-controllers who had previously generated T cell responses in a boosted flow-wide proteome analysis. Cells were thawed and cultured at 5 × 10 5 PBMC / well plated for 16h. PBMCs were stimulated with peptide pools to show T cell activation as described above for initial screening or control stimulation. TM Cells were stained with Fixable Viability Stain 575V, followed by extracellular staining for T cell lineage markers (anti-CD3 APC-Cy7, anti-CD4 BV786, anti-CD8-PerCP, anti-CCR7 BV711, Biolegend; anti-CD45RAFITC, BD Biosciences), Th2 / Tc2 markers (anti-CRTH2 BV605, Biolegend) and Tfh / Tfc markers (anti-CXCR5PE, anti-ICOS PE-Dazzle594, anti-PD-1PE-Cy7, Biolegend). After fixation and permeabilization steps (Fix and Perm kit, Invitrogen), intracellular staining was performed using anti-IL-4BV421 and anti-IL-21APC (Biolegend).

[0107] On the LSR Fortessa BD instrument, approximately 10 5Cells and FlowJo 10.5.2 software was used for analysis. In addition, the CD3+ cell data set characterized by the negative control phenotype was down sampled to obtain 3000 events / fcs files. Unsupervised UMAP dimension reduction was performed using the default nearest neighbor setting of 15 and the minimum Euclidean distance of 0.5 on FlowJo 10.5.2. The number of clusters was determined by X-Shift (nearest neighbor number: 132, Euclidean distance metric) and FlowSOM and the expression level of the protein of interest in a total of 45 clusters in the CD3+ population was determined with it. The abundance of each cluster was determined in HIV+ controllers and non-controllers and the results of each cluster were expressed as log2 (% of CD3+ cells in C / % of CD3+ cells in NC).

[0108] 4. IL-4, IL-21 and IFN-γ enhanced ELISPOT

[0109] The Mabtech ELISPOT kit was used to count cells producing IFN-γ (capture clone 1-D1K, biotinylated clone 7-B6-1), IL-4 (capture clone IL-4I, biotinylated clone IL-4II), and IL-21 (capture clone MT216G, biotinylated clone MT21.3m) according to the manufacturer's instructions. An analysis similar to the enhanced flow technology was also performed to increase the detection sensitivity of the ELISPOT. For this purpose, the plate was coated with multiple capture antibodies to allow the detection of cells producing IL-4 and IL-21. A total of 2×10 cells from 8 HIV+ controllers and 14 HIV+ non-controllers were stimulated with the peptide pool at 37°C. 5 PBMC / well for 48h, these individuals reacted to the peptide pool in the initial screen, using R10 as a negative control, in triplicate, and PHA (25μg / mL) as a positive control. Spot forming cells (SFC) were counted using an automated Cellular Technology Limited (CTL) ELISPOT Reader Unit. The positive threshold was set to the highest of the following values: a) 50 SFC / 10 6 PBMC, b) mean SFC of negative control plus 3 times the standard deviation of negative control wells, or c) SFC of negative control wells / 10 6 3 times the PBMC average.

[0110] 5. Determination of anti-HIV Env IgM, IgA and IgG titers in plasma samples

[0111] HIV-1NL4-3 and HIV-1BaL infected MOLT cells (Blanco J, et al., J.Leukoc.Biol.2004; doi:10.1189 / jlb.0204100) were pre-incubated with plasma samples diluted 1 / 300 in 1% BSA (Miltenyi Biotec) PBS for 30 minutes at room temperature. After washing 3 times with PBS, the following secondary antibodies were added: PE-F (ab) 2 goat anti-human IgG (Fc specific); DyLight 649 goat anti-human IgA and DyLight 488-F (ab) 2 goat anti-human IgM (Fcm5 specific) (Jackson Immunoresearch) and incubated at room temperature for 15 minutes. After washing, the stained cells were washed twice with PBS and fixed with 1% formaldehyde (Sigma-Aldrich). Samples were acquired in a BD LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo 10.5.2 software. HIV-1NL4-3 and HIV-1BaL envelope protein-specific IgM, IgA, and IgG titers correlated with the levels of specific polarized, viral antigen-specific T cell subsets.

[0112] 6. Neutralization Assay

[0113] As previously described (Molinos-Albert L, et al., Sci. Rep. 2017; 7), a panel of 5 pseudoviruses, including HIV-1 strains classified as tiers 1A (NL4-3), 1B (BaL.01), 2 (398F1), TRO.11, and AC10, was used to determine the plasma neutralization capacity of 44 HIV+ individuals. Briefly, serial dilutions of heat-inactivated (56°C, 30 min) plasma samples were incubated with pseudoviruses in D10 (DMEM supplemented with 100 U / mL penicillin (Gibco), 100 g / mL streptomycin (Gibco), and 10% heat-inactivated FBS (Invitrogen). Thereafter, the serial dilutions were added to TZM-bl cells treated with DEAE-Dextran (Sigma) at 10 4Cells / well were plated and incubated at 37°C for 48 h. Then, 100 μL of supernatant was replaced with 100 μL of Britelite Luciferase Assay Substrate (Promega) and incubated at room temperature for 1.5 min. The cell-associated luciferase signal of each well was determined on an EnSight Multimode PlateReader (Perkin Elmer). The neutralizing activity of each serum dilution was calculated as the percentage inhibition of viral entry compared to positive and negative controls, untreated infected cells and uninfected cells, respectively. Using GraphPad Prism version 7, log 10 The transformed data were fitted with a sigmoidal dose-response curve to generate neutralization curves (percent inhibition of viral entry vs plasma dilution) and determine the IC 50 When the data were fitted to a sigmoidal curve (HIV-1NL4-3 and HIV-1BaL), the results were expressed as % neutralizer in each group, neutralization breadth (number of HIV-1 strains neutralized / number of HIV-1 strains tested*100) and logIC 50 (Reciprocal plasma dilution).

[0114] 7. Antibody-dependent cellular cytotoxicity (ADCC)

[0115] The ability of plasma from 44 HIV+ individuals to induce ADCC was determined by flow cytometry using a modified calcein-AM retention assay described previously (Gillissen M, et al., J. Immunol. Methods 2016; 434). HIV NL4-3 infected NKR-24 target cells were washed and incubated with 10 6 The cells were resuspended in 10 nM Calcein-AM (BioLegend) diluted in R10 at a concentration of 10 cells / mL for 30 min with intermittent shaking. The target cells were washed twice with R10 and 10 cells / mL were added in the presence or absence of serial dilutions of plasma from HIV+ individuals (1:1000 to 1:256.000). 4 Target cells were co-cultured with the NK effector cell line N6-ADR at an E:T ratio of 5:1 in R10 supplemented with 10 U / mL rh-IL-2 (Roche) for 4 hours, repeated twice. After incubation, cells were washed with PBS plus 1% heat-inactivated FBS (Invitrogen) and 3×10 4 Cells were counted and analyzed on FlowJo 10.5.2 software. Results were normalized to control conditions without human plasma and lysis was calculated as the mean of 100%-% Calcein-AM+ cells per replicate condition.

[0116] 8. Statistical Analysis

[0117] GraphPad Prism version 7 for Mac OS X (La Jolla, CA, USA) was used for statistical analysis. To compare medians between groups, the nonparametric Mann-Whitney (MW) test was performed. 2 The analysis detected the difference between the frequency of T cells with a specific secretion cytokine pattern and the number of samples with neutralizing activity. The correlation between the two measurements was analyzed using the non-parametric Spearman r test, and the relevant p value was also calculated. The statistical significance criterion was set at p < 0.05.

[0118] Example 1: Enhanced flow screening identifies a greater number of positive M.Tb-specific T cell responses with selectable cytokine polarization than other gold standard assays

[0119] An exhaustive screening of cellular responses to M.Tb was performed by boosting flow to assess the presence of M.Tb-specific T cells with an alternative (non-IFN-γ-based, i.e., Th1) cytokine polarization profile. Total PBMCs from 10 ATBI and 12 LTBI cohorts were stimulated with different M. tuberculosis proteins commonly tested in other standard assays on the market. The signals observed in the flow cytometry data are expressed as the number of responders (% of individuals that elicited a specific polarization response). Figure 1 As shown, boosted flow had a greater ability to capture M.Tb infection compared to the other assays evaluated (gold standard Quantiferon IGRA 37% vs boosted flow 100% in ATBI and tuberculin skin test 50% vs boosted flow 100% in LTBI).

[0120] Example 2: Boosted Flow Screening Identification of M.Tb-Specific T Cell Responses with Alternative Cytokine Polarization Associated with ATBI and LTBI

[0121] Although T-SPOT detects active and latent TB infection at rates similar to boosted flow, T-SPOT cannot distinguish between the two infections. The analysis of M.Tb-specific T cell responses by T cell polarization profiles enables the discrimination of ATBI from LTBI infection, which is not possible with conventional assessments. In addition, different polarization profiles can be assessed simultaneously in the same sample without the need for additional parallel testing. Figure 2 As shown, while ATBI primarily generates a Th1-like profile, LTBI generates a specific M.Tb response with Th2-like, Tfh, and Th17 effector functions.

[0122] Example 3: Enhanced Flow Screening Identification of HIV-Specific T Cell Responses with Selectable Cytokine Polarizations Associated with Viral Control

[0123] An exhaustive screen of cellular responses against HIV was performed by flow boosting to assess the presence of HIV-specific T cells with alternative (non-IFN-γ-dominant, i.e., Th1) cytokine polarization profiles. Total PBMCs from a cohort of 15 HIV+ controllers (C) and 14 HIV+ non-controllers (NC) were stimulated with a panel of 17 peptide pools covering the entire HIV proteome. The signals observed in the flow cytometry data were expressed as the number of responders (% of individuals that elicited a specific polarized response), as the breadth of the response (number of reactive peptide pools), and as the size of the response (% of positive cells). Although anti-HIV responses of type 1 CD4+ Th1-like cells and CD8+ Tc1-like cells were the most commonly detected responses, 86% of the 30 individuals tested (n=24, including 13 controllers and 11 non-controllers) also exhibited non-type 1-like T cell responses. See Figure 7 A. Among them, 5 individuals (2 controllers and 3 non-controllers) only elicited responses with alternative polarization but not type 1 CD4+ or CD8+ T cell responses. The main non-type 1 responses were type 2 in CD4+ (percentage of responders: CD4+ Th2-like cells 50%, CD8+ Tc2-like cells 47%) and follicular-like in CD8+ T cells (percentage of responders: CD4+ Tfh-like cells 44%, CD8+ Tfc-like cells 53.5%). Type 3 polarized responses (percentage of responders: CD4+ Th17-like cells 16%, CD8+ Tc17-like cells 13%) and cells with regulatory functions (percentage of responders: CD4+ Treg-like 24%, CD8+ Treg-like 20%) were also detected. See Figure 7 In conclusion, natural control of HIV infection was associated with increased anti-HIV CD4+Tfh-like, CD8+Tc2-like, and Tfc-like responses, neither of which was detected by standard intracellular cytokine staining.

[0124] These findings are further supported by the increased number (p = 0.0268) and breadth (p = 0.0582) of HIV-specific CD4+ Tfh-like T cells in HIV+ controllers compared with non-controllers ( Figure 7 B) CD8+ T cell-mediated responses with Tc2- and Tfc-like profiles were also more frequent in HIV+ controllers (χ 2 p = 0.0402 and 0.0015) ( Figure 7 B), HIV+ controllers also showed significantly greater magnitude and breadth of CD8+Tfc-like responses than non-controllers (size, p=0.0005; breadth p=0.0002) ( Figure 7B). The inverse correlation between the breadth and magnitude of HIV-specific CD8+ Tfc-like responses and plasma viral load further supports the predominance of these CD8 T cell responses in HIV controllers ( Figure 7 C).

[0125] Since there were distinct CD8+Tfc-like responses between HIV controllers and non-controllers, the possibility that the acquisition of the anti-HIV CD8+Tfc-like phenotype was associated with a higher ability of PBMCs to secrete T cell follicle-associated cytokines (IL-4, IL-21) was explored. Total PBMCs (8 HIV+ controllers and 14 HIV+ non-controllers from the boosted flow study) were stimulated with a pool of peptides found to trigger Tfc-like responses in the initial boosted flow cytometry screen and cytokine secretion of IFN-γ, IL-4, or IL-21 was assessed by single cytokine or boosted ELISPOT assays alone or in combination (IL-4 and IL-21). Although only 54% of the tested peptide pools induced IL-21 SFCs and only 18% induced IL-4 SFCs, combined detection of IL-4 and IL-21 increased the sensitivity of the ELISPOT assay and detected responses against 72% of the peptide pools, which is consistent with the boosted flow analysis results. Some responses that would otherwise be missed were detected ( Figure 4 Interestingly, individuals who were shown to harbor HIV-specific CD8+ Tfc-like responses displayed a significantly higher innate ability to secrete IL-21, but not IL-4, in response to nonspecific stimulation (PHA, Figure 7 D).

[0126] It has been shown that T cells responding to different HIV proteins exert different antiviral activities. This may be related to antigen presentation, viral sequence variability, but may also be related to differences in maturation and effector spectrum polarization in response to different viral proteins (Shiner E, et al., PLoS One 2014; doi:10.1371 / journal.pone.0100175.45). In fact, analysis of T cell responders stratified by HIV-derived proteins revealed significant differences in the polarization profiles of reactive T cell populations. See Figure 8 Furthermore, these alterations were distinct in HIV controllers and non-controllers, thus directly linking viral protein specificity to specific polarization of T cell effector function and natural viral control in vivo. Specifically, and consistent with previous studies, CD4+ T cell responses to envelope and CD4+ T cell responses with a Th1-like profile were more frequent in HIV+ non-controllers compared with controllers (χ 2 p = 0.0536), while more CD4+Tfh-like, Env-specific responders were observed in HIV+ controllers (χ 2p=0.0772). HIV+ non-controllers also triggered more obvious Pol-specific Treg-like responses. Figure 8 A. In the context of CD8+ T cells, a statistically significant difference in the frequency of type 1 responses to Gag-reactive T cell responses was observed in HIV controllers (χ 2 p = 0.0079), which is also consistent with previous studies using a single cytokine (IFN-γ) based ELISPOT assay ( R, et al., J. Virol. 2006; 80). 2 Controls also showed a significantly higher number of Tc2-like responders after Pol and Env stimulation (χ 2 Controllers also showed a significantly greater number of Tc2-like responders after 4-6 months of follow-up (p = 0.0271 and 0.0374). Figure 8 B. When comparing the magnitude of the response, CD8+Tc1-like Gag specificity (MW p=0.0169) and Tfc-like Pol specificity (MW p=0.0245) were greater in controllers compared to non-controllers. Of note, accessory proteins (Rev, Vpr, Tat and Vpu) elicited alternative effector functions rather than type 1 responses.

[0127] Multifunctional analysis performed using SPICE v6 software revealed that the majority of T cell responses had unique T cell polarization profiles (i.e., did not show mixed polarization profiles), which is consistent with responses maturing into distinct polarization profiles and strongly supports the specificity of the boosted flow analysis used. These results suggest that alternative HIV-specific T cell profile analysis beyond conventional antiviral type 1 responses can identify new T cell responses with protein-specific polarizations associated with viral control. These data also demonstrate that CD8+Tfc-like cells may play a particularly important role in this potent anti-HIV immune response and extend previous observations to hitherto unrecognized T cell populations driving HIV control in vivo.

[0128] Example 4: Circulating type 2 and follicular T cells display distinct memory profiles in HIV+ controllers and non-controllers

[0129] To determine whether polarization of anti-HIV T cell responses and apparent control of viral control in vivo is associated with the induction of specific T cell differentiation and maturation, we determined CCR7 and CD45RA expression on these HIV-specific CD4+ and CD8+ type 1, type 2, and follicular T cells. No statistically significant differences were observed when comparing unstimulated total CD4+ and CD8+ basal memory phenotypes in HIV+ controllers to non-controllers (median percentages of CD4 populations: TCM 18%, TEM 34.5%, TEMRA 7.5%, and TNaive 40%; CD8+ T cells: TCM 3%, TEM 37.8%, TEMRA 29.9%, and TNaive 29.3%). See Fig. 9 In contrast, HIV-specific T cells showed significant differences in memory subsets among CD4+ Th2-like and Tfh-like cells, but not among Th1-polarized cells, with a higher proportion of Th2-like and Tfh-like cells displaying TCM and TEM phenotypes. Notably, levels of CD4+ TEMs that contribute to Th2-like polarization were significantly higher in HIV+ controllers (χ 2 p = 0.007), while TNaive was more frequent in non-controllers (χ 2 =0.0247). Although the difference in memory subset distribution was less pronounced for CD8+ T cells, a significantly higher frequency of TEM Tc2 was observed in HIV-specific CD8+ cells when comparing controllers to non-controllers (χ 2 =0.0247) and a reduction in the T naive Tc2 population. Thus, enhanced flow cytometry spanning profiles that have not previously been evaluated by this method identified new T cell populations directly associated with viral control, which has important implications for preventive HIV vaccine development and optimization and monitoring of therapeutic (vaccine-based) interventions.

[0130] Example 5: Expression of cell surface polarization markers in individuals displaying CD8+Tc2 and Tfc-like responses

[0131] To further validate that the CD4+ and CD8+ type 2 and follicular HIV-specific T cells identified by our enhanced flow cytometry approach expressed markers indicative of such polarized T cell populations, we determined CRTH2 expression for Th2 / Tc2 or CXCR5, PD-1, and ICOS expression for Tfh / Tfc.

[0132] A total of 45 clusters based on the expression of CD4, CD8, CCR7, CD45RA, CRTH2, CXCR5, PD-1, and ICOS were obtained by unsupervised analysis with X-Shift, and heat maps were performed based on the expression level (MFI) of each marker. The contribution of each cluster within the CD3+ population was used to compare HIV controller vs non-controller status, expressed as fold change. Clusters with ±2-fold changes in contribution (%) to the CD3+ population were included in this analysis. Three different clusters in the two groups—one corresponding to CD8+T cells and two corresponding to CD4+—were represented differently. Among them, one corresponded to CD4+Tfh cells expressing ICOS, CXCR5, and PD-1. The other two clusters (one CD4+, one CD8+) expressed high levels of CXCR5, but lacked expression of PD-1 and ICOS. Manual supervised gating was used to analyze the potential relationship between the presence of circulating CRTH2+Th2 / Tc2 and circulating CXCR5+Tfh / Tfc populations and HIV control. These analyses did not reveal any differences in the levels of CD4+Th2 cells expressing CRTH2 or CD4+Tfh cells expressing CXCR5, PD-1, and ICOS. Among CD8+T cells, no differences were found in CRTH2-expressing Th2 cells, but CD8+CXCR5+PD-1+ICOS+T cells were significantly elevated in HIV+ controllers and also in individuals who were able to elicit HIV-specific CD8+Tfc-like cytokine responses - observed by enhanced flow, further validating the applicability, sensitivity, and specificity of the enhanced flow cytometry approach to discriminate between different disease controls in HIV infection and highlighting the potential of this approach to identify novel T cell responses involved in viral control in vivo.

[0133] Example 6: Higher levels of CD8+Tfc-like cells in HIV+ controllers are associated with maturation of humoral immune responses and antibody effector functions

[0134] CD4+Tfh-like cells have been shown to be required for the induction of effective humoral immunity. Therefore, we evaluated whether virus-specific CD4+ cells, and in particular CD8+Tfc-like cells, are associated with antibody isotype switching, neutralization capacity, and ADCC effector function during HIV infection. To this end, plasma titers of HIV-1BaL and HIV-1NL4-3 Env-specific IgA, IgM, and IgG responses were analyzed in 24 HIV+ controllers (C), 20 HIV+ non-controllers (NC), and 8 HIV-, of which 15 HIV+ controllers and 14 HIV+ non-controllers were also analyzed in parallel by enhanced flow cytometry. Uncontrolled HIV infection was associated with increased median IgA (p=0.0515) and IgM titers (p=0.0009), while HIV+ controllers had higher IgG titers in their plasma (p=0.0182). Consistent with this finding, IgG / IgM and IgG / IgA ratios—reflecting isotype class switching from I to G—are elevated in HIV controllers.

[0135] Neutralization capacity and ADCC activity were determined in the same group of HIV controllers and non-controllers. Neutralization capacity was assessed against a panel of 5 HIV-1 pseudoviruses, including strains from tiers 1A (NL4-3), 1B (BaL.01), and 2 (398F1, TRO11, and AC10). Among HIV+ controllers, the proportion of individuals able to neutralize HIV-1NL4-3 was significantly higher (76%) than in HIV non-controllers (37%, χ 2 p = 0.0137). A similar trend was observed for the neutralization of HIV-1BaL.01 (68% C vs 42% NC, χ 2 p = 0.0657). Controllers also had a significantly greater breadth of neutralization compared with non-controllers (median: 40% vs. 20%, respectively; p = 0.0111). In controllers, titers of HIV-1 NL4-3 strain-specific nAbs were also significantly elevated (p = 0.0059), and a neutralization trend toward HIV-1 BaL was also observed (p = 0.0819). In addition to neutralization, more potent ADCC activity was observed in the plasma of controllers compared with non-controllers, at least at the lowest dilution tested (1:10 3 , 1:10 3.6 , 1:10 4.2 ADCC ability (1:10) of HIV-1NL4-3 (r=0.4562, p=0.0019) and HIV-1BaL (r=0.3461, p=0.0214) was also observed. 3.6 dilution) and neutralization IC 50There is a significant positive correlation between. These associations are driven by the IgG levels in plasma samples, because the Env-specific IgG (but not other isotypes) titers of HIV-1NL4-3 (Spearman r rho=0.5919, p value <0.0001) and HIV-1BaL (Spearman r rho=0.6423, p value <0.0001) are found to be positively correlated with logIC50. Consistent with this, the ratio of IgG / IgM is also related to the neutralization of HIV-1NL4-3 (r=0.5573, p<0.0001) and HIV-1BaL (r=0.3028, p=0.0458). A similar relationship between the ratio of IgG / IgM and ADCC ability is observed. Not only was the IgG / IgM ratio of HIV-1NL4-3 Env (r=0.2770, p=0.0687) the same HIV strain as expressed by NKR-24 ADCC target cells, but the IgG / IgM ratio of HIV-1BaL Env was also the same (r=0.3799, p=0.0110).

[0136] To further explore the possible relationship between humoral immune response and HIV control, antibody titers and functional responses were correlated with three clinical parameters (CD4 count, CD4 / CD8 ratio, and plasma viral load). Anti-HIV-1BaL IgM titers showed a negative correlation with CD4 count (Spearman r rho = -0.6352, p < 0.0001), CD4 / CD8 ratio (Spearman rrho = -0.5628, p < 0.0001), and a positive correlation with plasma viral load (Spearman r rho = 0.4177, p = 0.0048), which is consistent with the increased IgM levels observed in HIV non-controllers. In addition, plasma viral load was also negatively correlated with the IgG / IgM ratio (r=-0.3094, p=0.0410), the neutralization ability of HIV-1NL4-3 (r=-0.3989, p=0.0073) and ADCC activity (r=-0.4863, p=0.0008).

[0137] Example 7: HIV-specific CD8+Tfc-like responses are associated with antibody isotype class switching and humoral function

[0138] The above experimental data showed that HIV-specific CD8+Tfc-like responses were elevated in HIV controllers and that isotype ratios and IgG titers were also associated with HIV control. Given these results, we examined whether the presence of CD8+Tfc-like cells was associated with beneficial markers of humoral immune responses.

[0139] Plasma from individuals with detectable CD8+Tfc-like cells showed that the titer of IgG to HIV NL4-3 Env increased (p=0.0037), while the titer of IgM HIV-1BaL Env decreased (p=0.0118). This resulted in a significantly higher IgG / IgM ratio for both viral isolates (HIV-1NL4-3 MW p=0.0215, HIV-1BaL MW p=0.0123), and reflected an increase in antibody isotype class switching compared to individuals with undetectable CD8+Tfc-like responses. When measuring the balance of IgG and IgA levels, the results showed that when CD8+Tfc-like responses occurred, there was a clear bias towards IgG-mediated Ab activity (HIV-1NL4-3MW p=0.0059, HIV-1BaL MW p=0.0037). See Figure 10.

[0140] Finally, the association between CD8+Tfc-like responses, isotype switching, and antibody-mediated effector functions was analyzed by correlating CD8+Tfc-like size and width with antibody titers, neutralization capacity, and ADCC activity. The size and width of the CD8+Tfc-like response were negatively correlated with anti-HIV-1BaL IgM (size: r = -0.5764, p value = 0.0011; width: r = -0.5561, p value = -0.0017, Fig. 10B ), while compared with anti-HIV-1NL4-3 IgG of both strains (width: r=0.4729, p value=0.0096, Fig. 10C ) and the IgG / IgM ratio were positively correlated (HIV-1NL4-3: r = 0.3849, p value = 0.0393; HIV-1BaL: r = 0.4252, p value = 0.0215, Fig. 10D ). Interestingly, the IgG / IgA ratio also correlated positively with Tfc-like size and breadth, suggesting that conversion to IgG over IgA isotypes is preferential in individuals that elicit this alternative Tfc-like CD8+ effector function. Fig.10E .

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Claims

1. A method for determining a polarized T cell response specific for an antigen in a subject, comprising: (i) contacting a biological sample comprising T cells from a subject with a composition comprising the antigen, and (ii) determining the levels of a plurality of cytokines and / or chemokines produced by the T cells in the sample by enhanced flow cytometry and / or enhanced ELISPOT, Wherein a combination of antibodies against said plurality of cytokines and / or chemokines is used and said plurality of cytokines and / or chemokines are associated with a specific T cell polarization profile, wherein said antigen is at least one antigen from Mycobacterium tuberculosis (M. tuberculosis).

2. The method according to claim 1, wherein the antigen comprises at least one M.tb protein, preferably ESAT-6, CFP-10 or a combination thereof.

3. The method according to any one of claims 1 or 2, wherein the multiple cytokines and / or chemokines include cytokines and / or chemokines associated with type 1 (Th1 / Tc1), type 2 (Th2 / Tc2), type 3 (Th17 / Tc17), regulatory T cells (Treg) and / or follicular T cells (Tfh / Tfc) polarization spectra and / or other minority emerging polarization spectra. 4 . The method of claim 3 , wherein the plurality of cytokines and / or chemokines comprises cytokines and / or chemokines associated with type 1 (Th1 / Tc1) and / or type 2 (Th2 / Tc2) polarization profiles.

5. The method according to any one of claims 1 to 4, wherein the type 1 (Th1 / Tc1) multiple cytokines include IFN-γ, IL-2, TNF-α or a combination thereof.

6. The method according to any one of claims 1 to 5, wherein the type 2 (Th2 / Tc2) multiple cytokines include IL-4, IL-5, IL-10, IL-13 or a combination thereof.

7. The method according to any one of claims 1 to 6, wherein the type 3 (Th17 / Tc17) multiple cytokines include IL-17, IL-22 or a combination thereof.

8. The method according to any one of claims 1 to 7, wherein the regulatory T cell (Treg) multiple cytokines include IL-10 and TGF-β or a combination thereof.

9. The method according to any one of claims 1 to 8, wherein the follicular T cell (Tfh / Tfc) multiple cytokines include IL-4, IL-21, CXCL-13 or a combination thereof.

10. The method of any one of the preceding claims, wherein the antibody comprises a monoclonal antibody.

11. The method according to any one of the preceding claims, wherein the sample to be analyzed is a peripheral blood cell preparation.

12. A kit comprising reagents for detecting a cytokine and / or chemokine according to any one of the preceding claims.

13. Use of the method according to claims 1 to 11 or the kit according to claim 12 for diagnosing tuberculosis.

14. The use according to claim 13, for diagnosing active tuberculosis or latent tuberculosis.

15. The use according to any one of claims 13 or 14, wherein the subject belongs to a group selected from children, elderly people, immunocompromised patients, subjects co-infected with HIV-1, subjects receiving immunosuppressive therapy, and any combination thereof.

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