Methods of determining whether a subject suffering from lupus nephritis (LN) will achieve a response with an induction therapy

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease in which circulating immune complexes can cause different types of glomerulonephritis, according to immune deposits and to the type of glomerular cell injury. Proliferative lesions represent the most severe form of lupus nephritis (LN) and often lead to kidney failure and death. Mucosal-associated invariant T (MAIT) are a subset of innate-like T cells that recognize microbial-derived ligands from the riboflavin synthesis pathway. Now the inventors analyzed the phenotype and function of peripheral blood MAIT cells by flow cytometry in 26 patients with LN and in a control group of 16 healthy volunteers. The inventors found that MAIT cell frequencies are markedly reduced in blood of LN patients. More particularly, among LN patients, baseline MAIT cell frequency, Ki-67 expression evaluating proliferative activity and granzyme B production measuring cytotoxicity represent promising prognostic factors of renal response one year after induction therapy. In conclusion, the inventors showed that baseline frequency and cytotoxic profile of MAIT cells may represent a promising prognostic factor of renal remission one year after induction therapy.

Description

[0001] METHODS OF DETERMINING WHETHER A SUBJECT SUFFERING FROM LUPUS NEPHRITIS (LN) WILL ACHIEVE A RESPONSE WITH AN INDUCTION THERAPY

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular nephrology and immunology.

[0004] BACKGROUND OF THE INVENTION:

[0005] The hallmark of systemic lupus erythematosus (SLE), a systemic autoimmune disease, is the presence of pathogenic autoantibodies, including high titers of double-stranded anti-DNA antibodies. Tissue deposition of autoantigen-autoantibody complexes (immune complexes) induces immune activation and causes inflammatory damage that affects various types of organs, mainly joints, skin, kidneys, and nervous system. About half of SLE patients develop kidney disease. The pathogenesis of lupus nephritis (LN) involves immune complexes renal deposition in addition to other injuries resulting in endothelial, podocytic, and tubulointerstitial lesions (1). LN displays diverse clinical presentations and outcomes. Multiple parameters are associated with complications and prognosis of LN, among them socio-demographic and economic factors, but also histopathological features and serological markers. In SLE patients, LN is linked to higher morbidity, characterized by evolution to chronic glomerular lesions leading to end stage renal disease (ESRD) requiring replacement therapy. Furthermore, LN remains the leading cause of mortality in SLE patients (2).

[0006] Therapeutic care of LN is mainly decided according to histopathologic characteristics determined on the renal biopsy at diagnosis. LN classification is established according to the type of glomerular injuries characterized by activity and chronicity indices. Whereas class I and II generally do not require any specific treatment, class III and IV proliferative LN, the most severe forms of the disease, require an early diagnostic in order to setup immunosuppressive treatment as soon as possible. Treatment of class V remains debated. Despite immunosuppressive medication guidelines, about 15% of patients progress to ESRD within 10 years after initial diagnosis. Furthermore, 40% of SLE patients with class III or class V LN will evolve into chronic kidney disease (3). For the most severe forms of LN, induction therapy and maintenance therapy aim to remission and prevention of relapses, respectively, which are correlated to good renal outcome. To date, there is no reliable biomarker to predict LN remission at initiation of induction therapy, and how to distinguish patients in whom remission will be obtained with standard therapy, from patients who will require more intensive induction therapy. Despite being routinely performed to perform histopathologic classification of LN, kidney biopsy is not a sufficient diagnosis tool as it fails to predict the renal response to standard immunosuppressive therapy. Renal biopsy is also a costly and invasive procedure with potential risks that cannot be repeated in the monitoring of LN. Current biomarkers, that are conventionally used for LN follow-up like albuminuria, urine protein / creatinine ratio (UPCR), eGFR, anti-dsDNA levels, and serum complement fractions have not been very effective prognostic tools in predicting response to induction immunosuppressive therapy and estimating the probability of renal relapse (1).

[0007] Innate-like T cells are characterized as a cell subset that differs from conventional T cells by expressing a restricted T-cell receptor (TCRs) repertoire. Among them, Mucosal-associated invariant T (MAIT) cells are relatively abundant in the liver, at mucosal sites and in the blood, where they represent 1-10% of whole T cell population in humans. MAIT cells express a semiinvariant TCR restricted to a non-polymorphic MHC -related molecule, called MR1 (4). Human MAIT cells express the TCR Va7.2-Ja33 chain associated with a limited number of TCRP chains (5). MAIT cells recognize non-peptide antigens including bacteria and fungi-derived riboflavin and folate (vitamins B2 and B9) precursors and byproducts (6)(7). MAIT cells highly express many NK-cell specific markers such as the C-type lectin receptor CD161, in which coexpression with TCR Va7.2 classically defines human MAIT cells (8)(9). MAIT cells also express inflammatory cytokine receptors such as IL-12R and IL-18Ra at steady state (10). Upon activation through TCR signaling (11), or cytokine stimulation (10), MAIT cells are able to respond as effector cells and are involved in mucosal immunity, but also in pathologic inflammation. This activation can induce release of Thl / Thl7 cytokines, such as IFN-y, TNF- a, and IL-17 as well as apoptosis induction by perforin and cytotoxic molecule granzyme B (GzB). MAIT cells may therefore fulfill important key functions in the early defense against microbial pathogens at mucosal barriers (12). In addition to their antimicrobial functions, MAIT cells could also participate in local immunity homeostasis by preserving epithelial and mucosal barrier integrity (13) (14) (15) (16). Indeed, loss of MAIT cells during infectious diseases like HIV infection (17) or autoimmune diseases like Type 1 diabetes (18) may damper the intestinal barrier integrity and promote microbial translocation with deleterious effects on immune reconstitution and promoting inflammation. As a result, and through their ability to respond to inflammatory cytokines, MAIT cells have been implicated in the pathogenesis of, chronic inflammatory diseases, autoimmune or metabolic diseases-(15). Increased quantities of MAIT cells were detected in tissue lesions of patients with type 1 diabetes (18), inflammatory bowel disease (19), obesity (20), psoriasis (21), multiple sclerosis (22), and rheumatoid arthritis (23).

[0008] In SLE patients, circulating MAIT cells frequencies were found to be lower than in controls (19). However, frequency of peripheral apoptotic MAIT cells was increased and remaining circulating cells had an impaired phenotype: they express activation marker CD69 (24), pro- apoptotic marker PD-1, exhibit deficient Ca2+ / calcineurin / NFATl signaling pathway (23), and are less responsive to stimuli. These studies suggested that MAIT cells could be lost in SLE due to activation-induced apoptosis in vivo. Although it was suggested that MAIT cells activation positively correlates with disease activity in SLE (24) (25), their role in LN remained undetermined.

[0009] SUMMARY OF THE INVENTION:

[0010] The present invention is defined by the claims. In particular, the present invention relates to methods of determining whether a subject suffering from lupus nephritis (LN) will achieve a response with an induction therapy

[0011] DETAILED DESCRIPTION OF THE INVENTION:

[0012] Systemic lupus erythematosus (SLE) is an autoimmune disease in which circulating immune complexes can cause different types of glomerulonephritis, according to immune deposits and to the type of glomerular cell injury. Proliferative lesions represent the most severe form of lupus nephritis (LN) and often lead to kidney failure and death. Mucosal-associated invariant T (MAIT) are a subset of innate-like T cells that recognize microbial-derived ligands from the riboflavin synthesis pathway. Although abundant in peripheral blood, MAIT cells are enriched in mucosal and inflamed tissues. While previous studies have reported concordant results concerning lower MAIT cell frequencies in the blood of SLE patients, no information is known about MAIT cell activation status and LN severity. In the current study, the inventors analyzed the phenotype and function of peripheral blood MAIT cells by flow cytometry in 26 patients with LN and in a control group of 16 healthy volunteers. The inventors found that MAIT cell frequencies are markedly reduced in blood of LN patients. MAIT cells from SLE patients have an altered phenotype in terms of differentiation, activation and proliferation markers, notably in most severe forms of L Moreover, the frequency of MAIT cells secreting IL-17 as well as granzyme B upon in vitro restimulation with PMA / ionomycin were higher in LN patients. Among LN patients, baseline MAIT cell frequency, Ki-67 expression evaluating proliferative activity and granzyme B production measuring cytotoxicity are promising prognostic factors of renal response one year after induction therapy. In conclusion, the inventors report here that blood MAIT cells display an altered phenotype correlating with severe forms of lupus nephritis, but without association with systemic disease activity. Moreover, the inventors showed that baseline frequency and cytotoxic profile of MAIT cells may represent a promising prognostic factor of renal remission one year after induction therapy.

[0013] Accordingly the present invention relates to a method of determining whether a subject suffering from lupus nephritis (LN) will achieve a response with an induction therapy comprising quantifying the population of MAIT cells in a blood sample obtained from the patient before the induction wherein said level indicated whether the patient will achieve a response.

[0014] As used herein, the term “patient” is interchangeable with the term “individual” or “subject”, and refers to a mammal. Non-limiting examples of mammals include rodents (e.g., mice and rats), primates (e.g., lemurs, bushbabies, monkeys, apes, and humans), rabbits, dogs (e.g., companion dogs, service dogs, or work dogs such as police dogs, military dogs, race dogs, or show dogs), horses (such as race horses and work horses), cats (e.g., domesticated cats), livestock (such as pigs, bovines, donkeys, mules, bison, goats, camels, and sheep), and deer. In some embodiments, the mammal is a human. In some embodiments, the patient is a human infant. In some embodiments, the patient is a human child. In some embodiments, the patient is a human adult.

[0015] As used herein, the term “lupus nephritis” or “LN” has its general meaning in the art and refers to a medical condition that occurs as a complication of systemic lupus erythematosus (SLE), commonly known as lupus. SLE is an autoimmune disease where the body's immune system mistakenly attacks its own healthy tissues, leading to inflammation and damage in various organs, including the skin, joints, kidneys, heart, and lungs. Lupus nephritis specifically refers to the involvement of the kidneys in systemic lupus erythematosus. It is estimated that up to 50% of people with SLE will develop lupus nephritis at some point during the course of their disease. The condition occurs when the immune system's antibodies and immune complexes (a combination of antibodies and antigens) deposit in the kidneys, causing inflammation and affecting their normal function. The severity of lupus nephritis can vary widely, ranging from mild to severe. In some cases, it may cause only minor kidney dysfunction, while in others, it can lead to significant damage and eventually progress to chronic kidney disease or even kidney failure if left untreated. Symptoms of lupus nephritis may include: 1. Swelling (edema) in the legs, ankles, feet, or around the eyes, 2. Foamy or bubbly urine, 3. Blood in the urine (hematuria), 4. High blood pressure, 5. Increased frequency of urination, especially at night, and 6. Fatigue and weakness. Diagnosis of lupus nephritis involves a combination of clinical evaluation, blood tests, urine tests, and imaging studies (such as ultrasound or kidney biopsy) to assess the extent of kidney damage and determine the appropriate treatment. Treatment for lupus nephritis usually involves a combination of immunosuppressive medications to control the overactive immune response and reduce inflammation in the kidneys. The goal is to prevent further kidney damage, preserve kidney function, and improve the overall quality of life for individuals with this condition. The most widely used classification system for lupus nephritis is the International Society of Nephrology / Renal Pathology Society (ISN / RPS) classification, which was revised in 2003 and 2018. According to this system, lupus nephritis is divided into six classes (Class I to Class VI), based on specific histological findings observed in kidney biopsies:

[0016] 1. Class I: Minimal Mesangial Lupus Nephritis - In this stage, there is minimal or no detectable structural damage to the kidneys. Immune deposits are found only in the mesangial area of the glomeruli (tiny blood vessels in the kidneys).

[0017] 2. Class II: Mesangial Proliferative Lupus Nephritis - This stage shows an increase in the number of mesangial cells, and mild expansion of the mesangial area due to immune deposits.

[0018] 3. Class III: Focal Proliferative Lupus Nephritis - In this stage, there are active inflammatory changes in some, but not all, glomeruli. It is characterized by the presence of "active" lesions, including immune deposits, cellular proliferation, and inflammation.

[0019] 4. Class IV: Diffuse Proliferative Lupus Nephritis - This stage involves diffuse involvement of glomeruli with "active" lesions, as seen in Class III. However, in Class IV, the severity of the disease is more widespread, affecting a higher percentage of glomeruli.

[0020] 5. Class V: Membranous Lupus Nephritis - In this stage, there is thickening of the glomerular basement membrane due to immune complex deposition. It is often associated with the presence of nephrotic syndrome (a condition characterized by excessive protein loss in urine).

[0021] 6. Class VI: Advanced Sclerosing Lupus Nephritis - This stage represents the most severe form of lupus nephritis, with advanced scarring and irreversible damage to the kidneys. The ISN / RPS classification helps guide treatment decisions, as different classes of lupus nephritis may require varying approaches to immunosuppressive therapy.

[0022] In some embodiments, the patient suffers from LN of classes III, IV and V.

[0023] As used herein, the term “induction therapy” has its general meaning in the art and refers to the first phase of treatment. According the invention treatment for lupus nephritis usually involves a combination of immunosuppressive medications to control the overactive immune response and reduce inflammation in the kidneys. LN induction therapy is well known in the art, for example conventional treatment for LN induction therapy contained immunosuppressive drugs, specifically glucocorticoids (GC) combined with additional immunosuppressive measure such as cyclophosphamide (CPA), mycophenolate mofetil (MMF), and calcineurin inhibitors (CNI) including cyclosporine A (CSA) and tacrolimus (TAC) as described in Shin JI, et al. Induction and Maintenance Treatment of Lupus Nephritis: A Comprehensive Review ofMeta-Analyses. J Clin Med. 2022 Jan 11;11(2):343.

[0024] In particular embodiment, induction therapy is selected in the group consisting of : cyclophosphamide and azathioprine; methylprednisolone and cyclophosphamide; methylprednisolone and mycophenolate mofetil; methylprednisolone and mycophenolate mofetil and hydroxychloroquine; prednisone and hydroxychloroquine; prednisone and hydroxychloroquine and rituximab; prednisone and hydroxychloroquine and rituximab and mycophenolate mofetil; prednisone and mycophenolate mofetil and hydroxychloroquine; prednisone and rituximab; rituximab and cyclophosphamide.

[0025] The method of the present invention is particularly suitable for predicting the response at one year after the therapy.

[0026] The method is thus particularly suitable for discriminating responders from non-responders. As used herein, the term “responder” in the context of the present disclosure refers to a patient that will achieve a response, i.e., a subject who is under remission and, more particularly, a subject who does not suffer from lupus nephritis. A non-responder subject includes subjects for whom the disease does not show reduction or improvement after the treatment (e.g., the proteinuria remains stable or increases). As used herein, the term “MAIT cells” or “Mucosal-Associated Invariant T cells” refers to a population of T cells present in mammals, preferably humans, that display an invariant TCR alpha chain comprising Va7.2-Jot.33 (in humans), a CDR3 of constant length, and a limited number of V0 segments together with an activated phenotype (CD44) (see, e.g., Lantz and Bendelac. 1994. J. Exp Med. 180: 1097-106; Tilloy et al., J. Exp. Med., 1999, 1907-1921; Treiner et al. (2003) Nature 422: 164-169, the entire disclosures of each of which are herein incorporated by reference). MAIT cells are generally CD8+(expressing mostly the homodimeric form of CD8aa) or CD47CD8' (DN), and are restricted by the non-classical MHC class I molecule MR1. For the purposes of the present invention, any T cells that express the invariant Va7.2-Ja33 alpha TCR chain are considered to be MAIT cells. Typically, the alpha chain is associated with an invariant CDR3 and with either V02 or V013.

[0027] As used herein, the terms “expressing” or “+” and “not expressing” or are well known in the art and refer to the expression level of the phenotypic marker of interest, in that the expression level of the phenotypic marker corresponding to “+” is high or intermediate, also referred as The phenotypic marker corresponding to is a null expression level of the phenotypic marker or also refers to less than 10 % of a cell population expressing the said phenotypic marker.

[0028] In some embodiments, the method of the present invention comprises quantifying the population ofKi67+ MAIT cells.

[0029] As used herein the term "Ki67" has its general meaning in the art and refers to the marker of proliferation Ki-67 (Gene ID: 4288). Ki67 is also known as MKI67, KIA, MIB-, MIB-1, and PPP1R105.

[0030] In some embodiments, the method of the present invention comprises quantifying the population of GZB+ MAIT cells.

[0031] As used herein the term "GZB" has its general meaning in the art and refers to the granzyme B (Gene ID: 3002). GZB is also known as CCPI, CGL-1, CGL1, CSP-B, CSPB, CTLA1, CTSGL1, HLP, and SECT. In some embodiments, the quantification is absolute or relative. In some embodiments, when the quantification is relative, it consists in determining the frequency of the population in the general population of T cells (i.e. characterized by the expression of CD3) or the frequency of the population in the population of MAIT cells.

[0032] In some embodiments, high levels of MAIT cells (e.g. high frequency) indicate that the patient will achieve a response whereas low levels of MAIT cells (e.g. low frequency) indicated that the patient will not achieve a response.

[0033] In some embodiments, high levels of Ki67+ MAIT cells (e.g. high frequency) indicate that the patient will not achieve a response whereas low levels of Ki67+ MAIT cells (e.g. low frequency) indicate that the patient will achieve a response.

[0034] In some embodiments, high levels of GZB+ MAIT cells (e.g. high frequency) indicate that the patient will not achieve a response whereas low levels of GZB+ MAIT cells (e.g. low frequency) indicate that the patient will achieve a response.

[0035] As used herein, the term “high” refers to a measure that is significantly greater than normal, greater than a standard, such as a predetermined reference value or a subgroup measure, or that is relatively greater than another subgroup measure. For example, high levels of MAIT cells refers to a level of MAIT cells that is greater than a normal MAIT cells level. A normal MAIT cells level may be determined according to any method available to one skilled in the art. A high level of MAIT cells may also refer to a level equal to or greater than a predetermined reference value, such as a predetermined cutoff. A high level of MAIT cells may also refer to a level of MAIT cells wherein a high MAIT cells subgroup has relatively greater levels of MAIT cells than another subgroup. For example, without limitation, according to the present specification, two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a subgroup whose measure is high (i.e., higher than the median) and another subgroup whose measure is low. In some cases, a “high” level may comprise a range of levels that is very high and a range of levels that is “moderately high”, where moderately high is a level that is greater than normal but less than “very high”. As used herein, the term “low” refers to a level that is less than normal, or less than a standard, such as a predetermined reference value or a subgroup measure that is relatively less than another subgroup level. For example, a low level of MAIT cells means a level of MAIT cells that is less than a normal level in a particular set of samples of patients. A normal level of MAIT cells measure may be determined according to any method available to one skilled in the art. A low level of MAIT cells may also mean a level that is less than a predetermined reference value, such as a predetermined cutoff. A low level of MAIT cells may also mean a level wherein a low level MAIT cells subgroup is relatively lower than another subgroup. For example, without limitation, according to the present specification, two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a group whose measure is low (i.e., less than the median) with respect to another group whose measurement is high (i.e., greater than the median).

[0036] Typically, the predetermined reference value is a threshold or cutoff value. Typically, a "threshold value" or "cutoff value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based on the existing experimental and / or clinical conditions, as would be recognized by a person of ordinary skill in the art. For example, retrospective measurement in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the level of MAIT cells in a group of reference, one can use algorithmic analysis to statistically treat the levels determined in samples to be tested and thus obtain a classification standard having significance for sample classification. The full name of the ROC curve is the receiver operator characteristic curve, which is also known as the receiver operation characteristic curve. It is mainly used for clinical and biochemical diagnostic tests. The ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cutoff values (thresholds or critical values, boundary values between normal and abnormal diagnostic test results) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate, and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point with high sensitivity and specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result improves as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used to draw the ROC curve, such as MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

[0037] In some embodiments, the blood sample is a PBMC sample.

[0038] As used herein, the term “PBMC” or “peripheral blood mononuclear cells” or “unfractionated PBMC” refers to whole PBMC, i.e. to a population of white blood cells having a round nucleus, which has not been enriched for a given sub-population. Typically, these cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma. Additionally, PBMC can be extracted from whole blood using a hypotonic lysis which will preferentially lyse red blood cells. Such procedures are known to the expert in the art.

[0039] The quantification of the population of MAIT cells is determined by any method well known in the art and typically involves flow cytometry methods.

[0040] As used herein, the term "flow cytometric method" refers to a technique for counting cells of interest, by suspending them in a stream of fluid and passing them through an electronic detection apparatus. Flow cytometric methods allow simultaneous multiparametric analysis of the physical and / or chemical parameters of up to thousands of events per second, such as fluorescent parameters. Modern flow cytometric instruments usually have multiple lasers and fluorescence detectors. A common variation of flow cytometric techniques is to physically sort particles based on their properties, so as to purify or detect populations of interest, using "fluorescence-activated cell sorting". As used herein, "fluorescence-activated cell sorting" “or “FACS” refers to a flow cytometric method for sorting a heterogeneous mixture of cells from a biological sample into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell and provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.

[0041] Accordingly, FACS can be used with the methods described herein to isolate and detect the population of cells of the present invention. For example, fluorescence activated cell sorting (FACS) may be therefore used. It involves using a flow cytometer capable of simultaneous excitation and detection of multiple fluorophores, such as a BD Biosciences FACSCanto™ flow cytometer, used substantially according to the manufacturer's instructions. The cytometric systems may include a cytometric sample fluidic subsystem, as described below. In addition, the cytometric systems include a cytometer fluidically coupled to the cytometric sample fluidic subsystem. Systems of the present disclosure may include a number of additional components, such as data output devices, e.g., monitors, printers, and / or speakers, softwares (e.g. (Flowjo, Kaluza.... ), data input devices, e.g., interface ports, a mouse, a keyboard, etc., fluid handling components, power sources, etc.

[0042] More particularly, the blood sample is contacted with a panel of antibodies specific for the specific market of the population of cells of the interest.

[0043] Typically, the panel of antibodies comprises the following antibodies: anti-CD3 antibodies (e.g. OKT3 antibody), anti- Va7.2 antibodies (e.g. 3C10 antibody), anti-CD69 antibodies (e.g. FN50 antibody). The panel may further comprises anti-CD56 antibodies and anti-granzyme B antibodies (e.g. GB11 antibodies).

[0044] Such antibodies or antigen-binding fragments are available commercially from vendors such as R&D Systems, BD Biosciences, e- Biosciences, Biolegend, Proimmune and Miltenyi, or can be raised against these cell-surface markers by methods known to those skilled in the art. In some embodiments, an agent that specifically bind to a cell-surface marker, such as an antibody or antigen-binding fragment, is labelled with a tag to facilitate the isolation and detection of population of cells of the interest. As used herein, the terms "label" or "tag" refer to a composition capable of producing a detectable signal indicative of the presence of a target, such as, the presence of a specific cellsurface marker in a biological sample.

[0045] Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods to isolate and detect MAIT cell populations. Non-limiting examples of fluorescent labels or tags for labeling the agents such as antibodies for use in the methods of invention include Hydroxycoumarin, Succinimidyl ester, Aminocoumarin, Succinimidyl ester, Methoxycoumarin, Succinimidyl ester, Cascade Blue, Hydrazide, Pacific Blue, Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X, R-Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, PerCPeFluor 710, PE-CF594, Peri dinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY-FL, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), an APC-Cy7 conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, BV 785, BV711, BV421, BV605, BV510 or BV650.

[0046] The aforementioned assays may involve the binding of the antibodies to a solid support. The solid surface could be a microtitration plate coated with the antibodies. Alternatively, the solid surfaces may be beads, such as activated beads, magnetically responsive beads. Beads may be made of different materials, including but not limited to glass, plastic, polystyrene, and acrylic. In addition, the beads are preferably fluorescently labelled. In some embodiments, fluorescent beads are those contained in TruCount(TM) tubes, available from Becton Dickinson Biosciences, (San Jose, California). In some embodiments, PBMC were stained for detection of cytokines production after stimulation in RPMI medium supplemented with 10% fetal bovine serum with PMA and ionomycin at 25ng / mL and Ipg / mL, respectively, in the presence of brefeldin A at 10 pg / mL for 6 hours at 37°C. As being intra cellularly located, cytokine expression may be assessed by intracellular flow cytometry. Intracellular flow cytometry typically involves the permeabilization and fixation of the cells. Any convenient means of permeabilizing and fixing the cells may be used in practicing the methods. For example permeabilizing agent typically include saponin, methanol, Tween® 20, Triton X-100TM.

[0047] According to the present invention, the treatment consists of any method or drug that could be suitable for treating LN.

[0048] In some embodiments, the treatment is immunosuppressive. As used herein, the term “immunosuppressive treatment” refers to any substance capable of producing an immunosuppressive effect, e.g., the prevention or diminution of the immune response and, in particular, the prevention or diminution of the production of Ig. Immunosuppressive drugs include, without limitation, thiopurine drugs such as azathioprine (AZA) and metabolites thereof; nucleoside triphosphate inhibitors such as mycophenolic acid (Cellcept) and its derivative (Myfortic); derivatives thereof; prodrugs thereof; and combinations thereof. Other examples include but are not limited to 6-mercaptopurine ("6-MP"), cyclophosphamide, mycophenolate, prednisolone, sirolimus, dexamethasone, rapamycin, FK506, mizoribine, azathioprine and tacrolimus.

[0049] In some embodiments, the immunosuppressive drug is a calcineurin inhibitor. As used herein, the term “calcineurin inhibitor” has its general meaning in the art and refers to substances that block calcineurin (i.e., calcium / calmodulin-regulated protein phosphatase involved in intracellular signaling) dephosphorylation of appropriate substrates, by targeting calcineurin phosphatase (PP2B, PP3), a cellular enzyme that is involved in gene regulation. A calcineurin inhibitor of the present invention is typically an immunophilin-binding compound having calcineurin inhibitory activity. Immunophilin-binding calcineurin inhibitors are compounds forming calcineurin-inhibiting complexes with immunophilins, e.g., cyclophilin and macrophilin. Examples of cyclophilin-binding calcineurin inhibitors are cyclosporines or cyclosporine derivatives (hereinafter cyclosporines), and examples of macrophilin-binding calcineurin inhibitors are ascomycin (FR 520) and ascomycin derivatives (hereinafter ascomycins). A wide range of ascomycin derivatives are known, either naturally occurring among fungal species or are obtainable by manipulating fermentation procedures or by chemical derivatization. Ascomycin-type macrolides include ascomycin, tacrolimus (FK506), sirolimus, and pimecrolimus. Cyclosporine, originally extracted from the soil fungus Potypaciadium infilatum, has a cyclic 11 -amino acid structure and includes, e.g., Cyclosporines A through I, such as Cyclosporine A, B, C, D, and G. Voclosporin is a next-generation calcineurin inhibitor that is a more potent and less toxic semi -synthetic derivative of cyclosporine A. In some embodiments, the calcineurin inhibitor of the present invention is the trans-version of voclosporin, trans-ISA247 (Cas number 368455-04-3), which is described in, for example, US Patent Publication No.: 2006 / 0217309, which is hereby incorporated herein by reference. Further compositions of voclosporin are described, for example, in U.S. Pat. No. 7,060,672, which is hereby incorporated herein by reference. Tacrolimus (FK506) is another calcineurin inhibitor which is also a fungal product but has a macrolide lactone structure. Sirolimus (rapamycin) is a microbial product isolated from the actinomycete Streptomyces hygroscopicus. Sirolimus binds to an immunophilin (FK-binding protein 12, FKBP12), forming a complex which inhibits the mammalian target of the rapamycin (mTOR) pathway by directly binding the mTOR Complexl (mTORCl). Pimecrolimus is also a calcineurin inhibitor. Calcineurin inhibitors such as cyclosporine A, voclosporin, ascomycin, tacrolimus, pimecrolimus, an analogue thereof, or a pharmaceutically acceptable salt thereof, can be utilized in a mixed micellar composition of the present disclosure.

[0050] In some embodiments, the immunosuppressive drug is a corticosteroid. As used, the term “corticosteroids” has its general meaning in the art and refers to class of active ingredients having a hydrogenated cyclopentoperhydrophenanthrene ring system endowed with an antiinflammatory activity. Corticosteroid drugs typically include cortisone, cortisol, hydrocortisone (113,17-dihydroxy, 21-(phosphonooxy)-pregn-4-ene, 3,20-dione disodium), dihydroxy corti sone, dexamethasone (21-(acetyloxy)-9-fluoro-ip, 17-dihydroxy-16a-m- ethylpregna-l,4-diene-3, 20-dione), and highly derivatized steroid drugs such as beconase (beclomethasone dipropionate, which is 9-chloro-l l-P, 17,21, trihydroxy- 16P-methylpregna- 1,4 di ene-3, 20-dione 17,21 -dipropionate). Other examples of corticosteroids include flunisolide, prednisone, prednisolone, methylprednisolone, triamcinolone, deflazacort and betamethasone, cortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone propionate, fluocinonide, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide, clobetasol propionate, and dexamethasone.

[0051] In some embodiments, the immunosuppressive drug is a B cell depleting agent. As used herein, the term “B cell depleting agent” refers to any agent that is capable of triggering lymphodepletion of B cells. In some embodiments, the B cell depleting agent is an antibody having specificity for CD20. Examples of antibodies having specificity for CD20 include: “C2B8” which is now called “Rituximab” (“RITUXAN®”) (U.S. Pat. No. 5,736,137, expressly incorporated herein by reference), a chimaeric pan-B antibody targeting CD20; the yttrium-

[0090] -labeled 2B8 murine antibody designated “Y2B8” or “Ibritumomab Tiuxetan” ZEVALIN® (U.S. Pat. No. 5,736,137, expressly incorporated herein by reference), a murine IgGl kappa mAb covalently linked to MX-DTPA for chelating to yttrium-

[0090] ; murine IgG2a “BI,” also called “Tositumomab,” optionally labeled with radioactive 1311 to generate the “1311-B1” antibody (iodine 131 tositumomab, BEXXAR™) (U.S. Pat. No. 5,595,721, expressly incorporated herein by reference); murine monoclonal antibody “1F5” (Press et al. Blood 69 (2):584-591 (1987) and variants thereof including “framework patched” or humanized 1F5 (W003 / 002607, Leung, S.; ATCC deposit HB-96450); murine 2H7 and chimeric 2H7 antibody (U.S. Pat. No. 5,677,180, expressly incorporated herein by reference); humanized 2H7, also known as ocrelizumab (PRO-70769); Ofatumumab (Arzerra), a fully human IgGl against a novel epitope on CD20 huMax-CD20 (Genmab, Denmark; W02004 / 035607 (U.S. Ser. No. 10 / 687,799, expressly incorporated herein by reference)); AME-133 (ocaratuzumab; Applied Molecular Evolution), a a fully-humanized and optimized IgGl mAb against CD20; A20 antibody or variants thereof such as chimeric or humanized A20 antibody (cA20, hA20, respectively) (U.S. Ser. No. 10 / 366,709, expressly incorporated herein by reference, Immunomedics); and monoclonal antibodies L27, G28-2, 93-1B3, B-CI or NU-B2 available from the International Leukocyte Typing Workshop (Valentine et al, In: Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University Press (1987)). Further, suitable antibodies include e.g. antibody GAI 01 (obinutuzumab), a third generation humanized anti-CD20-antibody of Biogen Idec / Genentech / Roche. Moreover, BLX-301 of Biolex Therapeutics, a humanized anti CD20 with optimized glycosylation or Veltuzumab (hA20), a 2nd-generation humanized antibody specific for CD20 of Immunomedics or DXL625, derivatives of veltuzumab, such as the bispecific hexavalent antibodies of IBC Pharmaceuticals (Immunomedics) which are comprised of a divalent anti-CD20 IgG of veltuzumab and a pair of stabilized dimers of Fab derived from milatuzumab, an anti-CD20 mAb enhanced with InNexus' Dynamic Cross Linking technology, of Inexus Biotechnology both are humanized anti-CD20 antibodies are suitable. Further suitable antibodies are BM-ca (a humanized antibody specific for CD20 (Int J. Oncol. 2011 February; 38(2):335-44)), C2H7 (a chimeric antibody specific for CD20 (Mol Immunol. 2008 May; 45(10):2861-8)), PRO131921 (a third generation antibody specific for CD20 developed by Genentech), Reditux (a biosimilar version of rituximab developed by Dr Reddy's), PBO-326 (a biosimilar version of rituximab developed by Probiomed), a biosimilar version of rituximab developed by Zenotech, TL-011 (a biosimilar version of rituximab developed by Teva), CMAB304 (a biosimilar version of rituximab developed by Shanghai CP Guojian), GP-2013 (a biosimilar version of rituximab developed by Sandoz (Novartis)), SAIT- 101 (a biosimilar version of rituximab developed by Samsung BioLogics), a biosimilar version of rituximab developed by Intas Biopharmaceuticals, CT-P10), a biosimilar version of rituximab developed by Celltrion), a biosimilar version of rituximab developed by Biocad, Ublituximab (LFB-R603, a transgenically produced mAb targeting CD20 developed by GTC Biotherapeutics (LFB Biotechnologies)), PF-05280586 (presumed to be a biosimilar version of rituximab developed by Pfizer), Lymphomun (Bi-20, a trifunctional anti-CD20 and anti-CD3 antibody, developed by Trion Pharma), a biosimilar version of rituximab developed by Natco Pharma, a biosimilar version of rituximab developed by iBio, a biosimilar version of rituximab developed by Gedeon Richter / Stada, a biosimilar version of rituximab developed by Curaxys, a biosimilar version of rituximab developed by Coherus Biosciences / Daiichi Sankyo, a biosimilar version of rituximab developed by BioXpress, BT-D004 (a biosimilar version of rituximab developed by Protheon), AP-052 (a biosimilar version of rituximab developed by Aprogen), a biosimilar version of ofatumumab developed by BioXpress, MG-1106 (a biosimilar version of rituximab developed by Green Cross), IBI-301 (a humanized monoclonal antibody against CD20 developed by Innovent Biologies), BVX-20 (a humanized mAb against the CD20 developed by Vaccinex), 20-C2-2b (a bispecific mAb-IFNalpha that targets CD20 and human leukocyte antigen-DR (HLA-DR) developed by Immunomedics), MEDI-552 (developed by Medlmmune / AstraZeneca), the anti-CD20 / streptavidin conjugates developed by NeoRx (now Poniard Pharmaceuticals), the 2nd generation anti-CD20 human antibodies developed by Favrille (now MMRGlobal), TRU-015, an antibody specific for CD20 fragment developed by Trubion / Emergent BioSolutions, as well as other precloinical approaches by various companies and entities. All aforementioned publications, references, patents and patent applications are incorporated by reference in their entireties. All antibodies disclosed in therein may be used within the present invention.

[0052] FIGURES:

[0053] Figure 1. Baseline circulating MAIT cell phenotype as a prognosis marker of one-year renal response following induction therapy. (A) Baseline frequency of MAITs cells among CD3+ cells in LN patients with complete renal response (RC, n = 12) or partial response (PR) / no response (NR) (n = 9) at 1 year 15 after induction therapy. (B) Baseline frequency of MAITs cells expressing Ki-67 or (C) GzB among LN patients with complete renal response (RC) or partial response (PR) / no response) (NR) at 1 year after induction therapy. (D) Receiver operating characteristic (ROC) curve of the predictive value of GzB+ MAIT cell markers defining the outcome at 1 year after induction therapy. Data were analysed with Mann-Whitney U-tests. Horizontal lines are mean ± SD values.

[0054] EXAMPLE:

[0055] Methods:

[0056] Participants and clinical data

[0057] Patients with SLE having LN, and healthy individuals who did not report SLE or other inflammatory disease were recruited in Bichat Hospital between November 2017 and Mai 2019. Patients aged over 18-years old and presenting with a biopsy -proven LN were included in the study. Patients presented four or more criteria of the 1982 revised American College of Rheumatology (ACR) SLE classification. They did not have history of renal transplantation, surgery, cancer, urinary tract infection and systemic infection. Informed consent was obtained from all patients who participated in this study. Healthy individuals were also included as controls.

[0058] At diagnostic, demographic and laboratory data were collected: age, sex, serum albumin, blood creatinine, urine creatinine and 24-hour proteinuria. Systemic lupus activity was estimated according to the SLE Disease Activity Index (SELENA SLED Al) (26), a SLED Al score > 5 defining active SLE. LN class as well as activity and chronicity indexes according to the ISN / RPS 2003 classification were also collected. Class III and class IV LN with active lesions on kidney biopsy were defined as active class III or active class IV LN, respectively. Conversely, a non-active class III or class IV LN was defined by the absence of activity lesions on kidney biopsy. In addition, class I, class II and class V were classified as non-active LN.

[0059] The criteria for remission or absence of remission at 12 months were defined as follows, using estimated glomerular filtration rate (eGFR) and protein-to-creatinine ratio (UPCR). Complete remission was considered when UPCR < 0.5 g / g and eGFR > 60 ml / min / l,73m2, or if < 60 ml / min / l,73m2at baseline, and no decline (>20%) as compared to baseline. Partial renal response was defined as 50% improvement in UPCR compared to baseline and UPCR between 0.5 and 3 g / g and and eGFR > 60 ml / min / l,73m2, or if < 60 ml / min / l,73m2at baseline, no decline >20% compared to baseline. The absence of remission was defined as absence of complete or partial remission.

[0060] Cell isolation and activation

[0061] Peripheral blood mononuclear cells (PBMCs) isolation was performed on heparinized whole blood using Lymphosep (Biosera) according to the manufacturer’s instructions. Production of cytokines was measured after in vitro stimulation for 6 hours at 37°C. The latter was performed in RPMI medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) with PMA at 25 ng / ml and ionomycin at 1 pg / ml, in the presence of brefeldin A at 10 pg / ml (all from Sigma Aldrich).

[0062] Flow cytometric analysis

[0063] The list of Monoclonal antibodies (mAb) used in this study is presented in table SI. For cell surface staining, PBMC were incubated with appropriate mAb in PBS with 1 % BSA (FACS buffer) for 30 min on ice, followed by washes in FACS buffer prior to acquisition or subsequent intracellular or intranuclear staining. For intracellular staining, a fixation / permeabilization solution kit was used (Cytofix / Cytoperm, BD). For intranuclear staining, Foxp3 / transcription factor staining buffer set was used (eBiosciences). Data was acquired using a BD Biosciences LSRFortessa cytometer, and analyzed by FlowJo software (Tree Star). Dead cells were excluded by stringent FSC / SSC gating. Doublets were excluded by SSC-H / SSC-A and FSC- H / FSC-A gating.

[0064] Statistical Analyses

[0065] Mann-Whitney U or Unpaired t-test, when appropriate, were used for nonparametric tests. Spearman’s correlation test was used to perform Correlation analyses. Two-sided P values less than 0.05 were considered statistically significant. Statistical analyses were performed using GraphPad software (GraphPad Prism).

[0066] Study approval and Ethical Statement

[0067] The local Ethics Committee (Comite de protection des personnes Ile-de-France) approved the clinical investigations for both lupus and healthy subjects. All individuals provided written informed consent. This study was conducted in accordance with the Helsinki Declaration. Results:

[0068] Patients

[0069] All characteristics of studied population are shown in Table 1 including 26 LN patients with a mean age of 32 ± 11 years (88% were female). 13 patients (50%) were diagnosed as having an active LN (class III / IV + / -V). 21 patients were treated with an induction therapy in which 14 of them have either class III or a class IV + / -V LN with activity. 8 additional patients have pure LN class V with nephrotic syndrome.

[0070] Below are indicated the induction therapy received by the patients:

[0071] - Cyclophosphamide, azathioprine (n=l)

[0072] - Methylprednisolone, cyclophosphamide (n=6)

[0073] - Methylprednisolone, mycophenolate mofetil (n=3)

[0074] - Methylprednisolone, mycophenolate mofetil, hydroxychloroquine (n=l)

[0075] - Prednisone, hydroxychloroquine (n=l)

[0076] - Prednisone, hydroxychloroquine, rituximab (n=5)

[0077] - Prednisone, hydroxychloroquine, rituximab, mycophenolate mofetil (n=l)

[0078] - Prednisone, mycophenolate mofetil, hydroxychloroquine (n=l)

[0079] - Prednisone, rituximab (n=l)

[0080] - Rituximab, cyclophosphamide (n=l).

[0081] After 12 months of induction therapy, 12 patients (57%) achieved complete response, 3 (14%) partial response and the other 6 (29%) were nonresponsive (Table 2). 16 healthy volunteers (mean age of 42 ± 14 years) were also included into the control group. 10 healthy controls (63%) were women.

[0082] Conventional biomarkers are not related to renal response

[0083] As shown in Table 2, conventional biomarkers such as age, sex, renal SLE Disease Activity Index (SLED Al) score, renal activity / chronicity indexes, serum albumin level, eGFR or UPCR were not significantly different between patients with complete, partial or no response after induction therapy.

[0084] Circulating MAIT cell frequencies are markedly reduced in patients with lupus nephritis Frequency of circulating MAIT cells (identified as CD3+TCR76 TCR Va24 CD4 CD161hlVa7.2+cells) was first evaluated in the PBMC from patients and healthy controls (data not shown). The median MAIT cell frequency (defined as the percentage of MAIT cell among CD3+) was strongly decreased in LN patients as compared to controls (p = 0.0001). While 3.06% (0.19-11.5) of MAIT cells was observed in controls, within the range reported by others (19)(16)(23), only 0.25% (0.02-3.76) was seen in LN patients (data not shown). However, no significant differences in MAIT cell frequency was seen within different type of LN patients neither according to histologic classification (not shown) nor according to active (III or IV + / - V) and non-active (II and pure class V) status (data not shown).

[0085] Increased frequencies of CD127 CCR6’ MAIT cells in lupus nephritis patients.

[0086] As MAIT cells express tissue-homing chemokine receptors (9), we next evaluated the impact of LN disease activity on the remaining blood MAIT cell population. We found that virtually all blood MAIT cells expressed CD127 (IL7Ra) and CCR6 (CCL20R) in controls However, in patients with LN notably classes III and IV, we observed a lower frequency of MAIT cells expressing CD 127 (data not shown) and CCR6 (data not shown).

[0087] Remarkably, there was also a strong correlation in the expression of CCR6 and CD127 on MAITs in LN patients (p < 0.0001) (data not shown). The frequency of double negative CD127' CCR6 MAIT cells is increased among LN patients (data not shown). This phenotype is characteristic of short lived terminally differentiated effector memory T cells, displaying a complete exhaustion phenotype and loss of most cytokines and chemokines receptors (24). However, there was no difference in CD 127" or CCR6' MAIT cell frequency between active (III or IV + / -V) and non-active (II and pure class V) LN (data not shown).

[0088] We next wondered whether LN circulating MAIT cells display proliferation markers using Ki67 staining. Indeed, Ki67 expression was significantly increased on MAIT cells of patients presenting with proliferative LN (class III-IV) and class V (data not shown). Despite such proliferation profile, no significant differences were found between LN and controls concerning the expression of activation (CD25 and CD69) and differentiation (CD27) markers (data not shown). However, LN patients with SLEDAI > 6 (n = 23) displayed a higher percentage of circulating MAIT cells expressing CD25 (p = 0.0061) as well as a trend for a higher expression of CD69 (NS), in comparison with LN patients with low disease activity (n = 3) (data not shown). We therefore evaluated the expression of exhaustion markers such as PD-1 or TIM3, which mediate inhibition of TCR-induced activation and proliferation. While no differences were found for PD1 levels (data not shown), significantly lower expression of TIM-3 was noticed on MAIT cells in LN patients as compared to controls (data not shown) indicating an activated phenotype. There was no difference in the frequency of Ki67+ MAIT cells between active and non-active LN (data not shown).

[0089] Enhanced cytotoxic and Thl7 phenotype among MAIT cells in LN

[0090] To analyze a putative cytotoxic phenotype of MAIT cells in LN, we first studied the expression of CD56, a Natural Killer (NK) cell marker present on activated and / or cytotoxic MAIT cells. In LN MAIT cells, a positive correlation was indeed observed between the expression of CD56 and IL-7 receptor (CD127) suggesting a cytotoxic phenotype of these cells (data not shown). Moreover, while most of the MAIT population expressed the NK-associated cytotoxicity marker KLRG1 (Killer-cell Lectin-like Receptor Gl) in HC, there was a marked reduction in KLRG1 expression among LN patients (data not shown). The expression of KLRG1 marker was negatively correlated with CD27' phenotype of MAIT cells in LN (data not shown). However, we did not find any difference in the frequency of KLRG1+MAIT cells between active and non-active LN (data not shown).

[0091] We next wondered if the impaired phenotype of MAIT cells in LN patients was accompanied by functional changes. We therefore activated PBMC with PMA / ionomycin and measured MAIT intracellular production of multiple cytokines such IFN-y, TNF-a, IL- 17, IL-2, IL- 10, IL-4 and the cytotoxic effector molecule granzyme B (GzB). Restimulation with PMA and ionomycin, though nonspecific in contrast to antigen stimulation, measures the maximal capacity of lymphocytes to produce the cytokines. While production of TNF-a, IFN-y and IL- 10 was not altered between patients and controls (data not shown), significant increase was observed for levels of IL-2, IL-4 and IL-17, especially in classes III-IV (data not shown). However, there was no difference in the frequency of cytokine-producing MAIT cells between active and non-active LN (data not shown). Similarly, increased GzB production was seen in LN-derived MAIT cells as compared to healthy controls (data not shown). GzB+MAIT cells were mainly present in patients with LN of classes III, IV and V (data not shown). Unlike other cytotoxicity markers, GzB is only found in activated cytotoxic lymphocytes. Interestingly, in contrast to controls there was a negative correlation between GzB and CD56 expression on MAIT cells from LN patients (data not shown) indicating that circulating GzB+MAIT cells in LN express low levels of CD56. Nevertheless, no difference was noticeable in the frequency of GzB+ MAIT cells between active and non-active LN (data not shown). Baseline circulating MAIT cell frequency, Ki-67 expression and granzyme B production are new markers of renal response after induction therapy

[0092] We then assessed baseline circulating MAIT cell frequency and phenotype at time of diagnostic biopsy and before induction treatment initiation, as prognostic factors of the renal response observed one year after induction therapy initiation. Baseline MAIT cell frequencies were higher in LN patients who fulfilled complete response (median 0.31% of T cells) than in patients with partial response or non-response (median 0.08%, Figure 1A). Baseline frequency of Ki- 674- MAIT cells was significantly lower among LN patients with complete response than among those with partial response or non-response (p = 0.0347, Figure IB). There was no correlation between MAIT cell frequency, GzB production, and Ki-67 expression with clinical data such as serum albumin level, UPCR and eGFR (data not shown). Baseline frequency of cytotoxic (GzB4-) MAIT cells was significantly lower in LN patients who fulfilled complete response than among those with partial response or non-response (p = 0.0011, Figure 1C). The receiver operating characteristic (ROC) curve analysis was performed to compare the predictive value of blood MAIT cell frequency as well as Ki-674- and GzB4- MAIT cell proportions with one year outcome for LN patients. While ROC analysis of MAIT cell frequency did not reach significance, Ki-67 expression displayed a trend toward significance (Figure S10). Remarkably, ROC analysis of GzB production defined a predictive model for complete remission (Figure ID).

[0093] Discussion:

[0094] In this study, we evaluated the frequency, phenotype and function of peripheral MAIT cells in patients with active or non-active LN. MAIT cell frequency was found to be marked reduced among patients in comparison to HC, consistently with previous reports (23)(24). We furthermore observed that remaining circulating MAIT cells in LN patients displayed a modified phenotype with an increased proportion of CD127' CCR6' cells. One can hypothesized that depletion of peripheral blood MAIT cells during LN development is partly caused by modified expression of CCR6. Indeed, CCR6 is implicated in lymphocyte recruitment to inflamed tissues, and may be involved in T cell migration to kidneys during renal disease development (27)(28). In patients with LN, we hypothesize that MAIT cells may therefore partially disappear from the circulation due to recruitment to inflamed tissues and secondary lymphoid organs. Indeed, a recent study has identified MAIT cells (CD34-Va7.2TCR4-IL- 18RO.4-) in biopsies notably within the glomeruli and tubulointerstitium of patients with class III and IV LN (29). Interestingly the authors have shown that suppression of MAIT cell activation impaired autoantibody production and LN development in an animal model. Moreover, tissue infiltration by MAIT cells was also observed in other autoimmune diseases such as arthritis and diabetes (15) Nevertheless, detection and quantification of tissue MAIT cells is quite challenging because of the lack of specific surface markers. Indeed, CD161 and IL-18Ra, which are known to be specific markers of circulating MAIT cells among Va7.2+CD8aa+or CD8' CD4' T cells, are expressed by virtually all tissue-resident T cells. Until recently, the gold standard method for MAIT cells detection in human tissues, including the kidney, was the detection of MAIT cell-specific TCR transcripts (30)(31). In their recent study, Law et al. characterized MAIT cell phenotypes in healthy and pathologic renal tissue samples by flow cytometry (32). In both type of samples, kidney tissue-derived MAIT cells displayed a tissue-resident phenotype, characterized especially by CD 103 and CD69 expression. Among diseased biopsy samples, activation marker CD69 was significantly more expressed in those with fibrosis compared with nonfibrotic kidney tissues, suggesting that the local environment notably within fibrotic kidneys driven MAIT cells toward a more activated state. However, CD69 expression on MAIT cells was significantly increased on fibrotic as compared to nonfibrotic kidneys, suggesting that local environment within fibrotic kidneys drives MAIT cells toward a more activated state.

[0095] Another explanation for the decrease of circulating MAIT cells frequencies in LN patients could be an increased cell death induced by their activation (24). Indeed, there is a significant increase in CD69 and CD25 acute activation markers in LN patients with moderate to high activity (SLED Al score > 5) as compared to LN patients with no or mild activity. However, this should be confirmed by analyzing other chronic activation markers like CD38 or HLA-DR. Indeed, cytokine-driven activation can induce cell death which may be in part responsible for MAIT cell depletion in response to high levels of IL-12 and IL-18 leaving MAIT cell vulnerable to persistent activation (24)(33), a mechanism that has been proposed for invariant NKT cells, another subset of innate-like T cells (34).

[0096] A decrease of peripheral MAIT cells has also been described during HIV infection. MAIT cells recruitment to tissues has been proposed in response to microbial translocation following the loss of intestinal epithelial barrier integrity during infection (17) (35). Interestingly, intestinal microbial translocation occurs in both SLE and LN (36)(37). As MAIT cells recognize bacteria- derived ligands, this translocation may induce MAIT cell activation. This hypothesis should be investigated in future studies.

[0097] In adults, circulating MAIT cells have display an effector memory phenotype, characterized by expression of chemokine receptors CCR5 and CCR6, as well as high level of CD127. Our data show an increase of CCR6' CD127' double negative cells among remaining circulating MAIT cells in SLE patients with LN, which is the first report in the literature to our knowledge. As hypothesized above, it may be due to migration of double positive CCR6+CD127+cells toward inflamed tissue, or de novo differentiation toward terminally differentiated phenotype. CD127 forms the a-subunit of IL-7 receptor (IL-7Ra) that is associated to common y-chain (CD 132) to transmit its signal. IL-7 is the key cytokine for T-cell growth and development, as well as for regulation of naive and memory T-cell related homeostasis or long-term survival. IL-7Ra is indeed expressed by all naive T cells, as well as on memory T cells. On the other side, most CD8+effector T cells and FOXP3+regulatory T cells do not express IL-7a (38). In our study, fewer MAIT cells from class III-IV LN patients express CD127 compared with healthy individuals. Interestingly, loss of CD127 is also observed among CD8+T cells although not significant (not shown). Such phenomenon has been described in CD8+T cells from HIV- infected patients; it has been linked to the shedding or alternative splicing of CD 127 induced by IL-7 and mediated by STAT5 and MMP-9, and could be implicated in dysfunctional states of CD8+T cells that remain in the circulation after induction of efficient antiretroviral therapy (39)(40). Among papers analyzing the phenotype of blood MAIT cells in inflammatory diseases, a study described an increased CD127 expression in MS patients (41). This increase has been interpreted as a reflect of higher fluorescence mean intensity which may suggest an increase in the number of molecules at cell surface per MAIT cell, but not an increase in the frequency of CD127+MAIT cells. We and others reported previously rather a decrease of CD127-expressing circulating MAIT cells during severe SARS-Cov-2 infection (42), or chronic hepatitis delta virus infection (33). Contrary to the strong activation phenotype observed in these two papers, LN MAIT cells do not display higher expression of acute activation markers such as CD69 and CD25 in comparison to controls. Moreover, CD127 expression has been shown to be lost by effector T cells (43). Thus, we speculate that a subset of MAIT cells (CCR6‘ CD127' double negative cells) could be a result from reverting mechanism from effector memory to terminally differentiated memory T cells (TDEM) in the context of proliferative LN. Expansion of TDEM CD8+T cells (CD45RA+CCR7 CD27 CD28 ) producing high levels of proinflammatory cytokines and cytotoxic activity have been indeed reported in SLE with high disease activity and damage indexes (44) and have been shown to be associated with a higher risk of long-term kidney graft dysfunction (45)(46). Here, contrary to what has been noted in several studies, we do not report an increase in CD27' MAIT cells (data not shown). In HIV patients (35) and juvenile type I diabetes (47), the CD27 subset has been proposed to potentially represent a terminally differentiated or exhausted MAIT cell subset.

[0098] In healthy adults, most MAIT cells are noncycling cells with less than 1% Ki67+(9). Instead, contrary to controls, LN MAIT cells have a higher proliferating capacity (as defined by Ki-67 expression) in both class III-IV and class V. The systemic loss of MAIT cells may be attributable to increased turnover in LN patients. LN MAIT cells display a decreased expression of the inhibitory molecule TIM-3, together with low levels of KLRG1. TIM-3 is a marker of functional exhaustion of T cells. Low expression of this marker is compatible with rather activated than exhausted phenotype of MAITs in LN. But the functional tests are necessary to confirm this hypothesis. A tendency to low expression of TIM-3 on MAITs has been already described in patients with sarcoidosis where their sustained activation was associated with their decreased number in the peripheral blood (48). KLRG1 is an inhibitory receptor for NK cells, and its expression in T cells is associated to impaired ability to proliferate. In this study there was a trend to higher PD-1 expression on MAIT cells in LN patients comparing to HC but the difference did not reach significance. This data contrast to previous observations (19) that could be due to the different patient characteristics (LN versus SLE).

[0099] In this study, we did not analyze cytokines and GzB production after in vitro TCR specific 356 stimulation, since MAIT cells are usually exhausted in chronic inflammatory diseases (20) (24) and respond poorly to such activation. In contrast, restimulation with PMA / ionomycin allow stronger detection of effector molecules and showed increased IL-2, IL-4 and IL- 17 expression arguing in favor of a proinflammatory role of MAIT cells in LN notably in classes III-IV. Moreover, increased GzB production detected in CD561ow MAIT cells from classes III / IV LN patients suggests their deleterious cytotoxic effect on SLE renal tissues. Multivariable analysis of cytokines and GzB showed that monofunctional MAIT cells producing GzB were more frequent in classes II and III / IV than in class V (data not shown). The latter is a less inflammatory class of LN caused by glomerular membranous subepithelial deposition of immune complexes affecting podocytes without significant leukocyte infiltration in the renal tissue. Together, our findings show that MAIT cells in LN display activated and dysfunctional phenotype, which can become predictive factors for the achievement of 1 year remission. In the present study, patients were analyzed once at inclusion and it would be important to perform longitudinal studies after induction therapy in larger cohorts.

[0100] In conclusion, the current study described MAIT cells as a new prognostic factor for remission, complications, and relapse rate for LN patient care following induction therapy. High frequency of circulating MAIT cells may represent a favorable prognostic factor for complete remission, whereas high production of GzB is a risk factor for partial or unfavorable response to treatment in lupus nephritis. TABLES:

[0101] Table 1. Demographic, clinical and laboratory data of LN patients and healthy controls. LN, lupus nephritis; SD, standard deviation; IQR, Interquartile Range, SLEDAI, Systemic Lupus Erythematosus Disease Activity Index; ISN / RPS, International Society of Nephrology / Renal Pathology Society; GFR, Glomerular Filtration Rate; UPCR, Urine Protein Creatinine Ratio. Table 2. Conventional biomarkers of renal response. Complete renal response: UPCR < 0.5 g / g and eGFR > 60 ml / min, or if < 60 ml / min at baseline with no decline >20% compared to baseline; Partial renal response: 50% improvement in UPCR and UPCR between 0.5 and 3 g / g and stabilization (< 20% decrease) of eGFR. Values are given as median [interquartile range]. *Complete response vs. partial- or non-response among active LN only (n = 13, class III or IV ± V), Mann-Whitney test.

[0102] REFERENCES:

[0103] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method of determining whether a subject suffering from lupus nephritis (LN) will achieve a response with an induction therapy comprising quantifying the population of MAIT cells in a blood sample obtained from the patient before the induction wherein said level indicated whether the patient will achieve a response.

2. The method of claim 1 wherein the patient suffers from LN of classes III, IV and V.

3. The method according to claim 1 or 2 that comprises quantifying the population of Ki67+ MAIT cells.

4. The method according to claim 1 or 2 that comprises quantifying the population of GZB+ MAIT cells.

5. The method according to claim 1 or 2 wherein high levels of MAIT cells (e.g. high frequency) indicate that the patient will achieve a response whereas low levels of MAIT cells (e.g. low frequency) indicated that the patient will not achieve a response.

6. The method according to claim 3 wherein high levels of Ki67+ MAIT cells (e.g. high frequency) indicate that the patient will not achieve a response whereas low levels of Ki67+ MAIT cells (e.g. low frequency) indicate that the patient will achieve a response.

7. The method according to claim 4 wherein high levels of GZB+ MAIT cells (e.g. high frequency) indicate that the patient will not achieve a response whereas low levels of GZB+ MAIT cells (e.g. low frequency) indicate that the patient will achieve a response.

8. The method according to any one of claims 1 to 7 wherein the treatment is immunosuppressive.

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