USE OF PD-1 AND CD38 PROTEIN AS MARKERS FOR ACTIVE AUTOIMMUNE DISEASE

Description

Technical field

[0001] The present invention relates to a method for predicting and / or prognosing active autoimmune hepatitis in a subject, comprising measuring the amounts of certain proteins in a biological sample of the subject, as well as to certain uses of these proteins.

[0002] In the description below, the references in brackets ([ ]) refer to the list of references presented at the end of the text. State of the art

[0003] Autoimmune hepatitis (AIH) is a chronic liver disease whose immunological mechanisms remain unknown. It is a rare disease with an incidence of 1.1 to 1.9 cases per 100,000 people per year in Europe, characterized by an attack of the liver parenchyma by cells of the lymphoid immune system (Liberal et al.: Cutting edge issues in autoimmune hepatitis. J. Autoimmun. (2016) 75, 6-19 ([1])).

[0004] The diagnosis of AIH is often a long one to make. It is based, as for other autoimmune liver diseases, on the elimination of other causes, on non-specific biological and biochemical blood tests (elevation of transaminases, absence of viral hepatitis, presence of autoantibodies and hypergammaglobulinemia) and on the histological reading of liver biopsy. In other cases of autoimmune liver disorder or not, the diagnosis is based on the symptoms, the presence or absence of autoantibodies and on the histological reading of the affected tissue. Monitoring of the response to treatments is generally based on the improvement of symptoms, the regulation of biological parameters and possibly on the histological reading of biopsy of the affected tissues.

[0005] The treatment combining corticosteroids and azathioprine, unchanged for more than 30 years (Manns et al.: Diagnosis and management of autoimmune hepatitis. Hepatol. Baltim. Md 51, 2010, 2193-2213 ([2])), aims to control inflammation to prevent progression to liver fibrosis. This non-selective immunosuppression is sufficient to induce complete remission in 70% of patients in the first year. European guidelines recommend attempting to stop azathioprine after 2 years of complete remission to limit the harmful effects of this in the long term (European Association for the Study of the Liver (2015). EASL Clinical Practice Guidelines: Autoimmune hepatitis. J. Hepatol. 63, 971-1004 ([3])); However, the risk of relapse is high and affects 60 to 80% of patients within six to twelve months of stopping treatment (Manns et al., 2010 ([2])).The state of complete clinical remission is currently based on biological and biochemical tests, in particular by normalization of transaminases and blood IgG, which do not predict the risk of relapse.

[0006] A better understanding / characterization of the immune response in AIH would be useful to predict the risk of relapse, but also to identify the immunological protagonists involved in the pathogenesis of this disease.

[0007] CD4 T lymphocytes (CD4 LT) and CD8 LT have been described as strongly involved in the attack of the liver parenchyma in AIH. They are widely represented in the portal and intralobular inflammatory infiltrate. However, the relationship between the immunological actors involved and the evolution of the disease is not yet known (Liberal et al., 2016 ([1])).

[0008] Recently, blood lymphocyte alterations have been highlighted at diagnosis, which persist during the remission phase under treatment (Renand et al., : Immune Alterations in Patients With Type 1 Autoimmune Hepatitis Persist Upon Standard Immunosuppressive Treatment. Hepatol. Commun. (2018) 2, 968-981 ([4])). However, some of these lymphocyte alterations were found in patients with dysmetabolic steatohepatitis (NASH), and thus seemed to reflect nonspecific hepatic inflammation (Böttcher et al.: MAIT cells are chronically activated in patients with autoimmune liver disease and promote pro-fibrogenic hepatic stellate cell activation. Hepatol. Baltim. Md, 2018 ([5]); Jeffery et al.: Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MRI. J. Hepatol., 2016, 64, 1118-1127 ([6]); Oo et al.: CXCR3-dependent recruitment and CCR6-mediated positioning of Th-17 cells in the inflamed liver. J.Hepatol. 57, 1044-1051, 2012 ([7])). Furthermore, cases of non-viral liver inflammation (such as NASH) with signs of autoimmunity (presence of blood autoantibodies in 10% of NASH patients) have been reported, but are not currently the subject of any specific treatment.

[0009] WO2006 / 096800 describes a method for diagnosing and monitoring the progression in response to therapy of a subject suffering from autoimmune hepatitis. In this method, T cells reactive with immunogenic peptides comprising T lymphocyte epitopes involved in autoimmune hepatitis are quantified.

[0010] Thus, there is a real need to identify a specific signature of hepatic autoimmunity, particularly to improve diagnosis or clinical and therapeutic management. Description of the invention

[0011] After extensive research, the inventors managed to solve this technical problem.

[0012] Surprisingly, the inventors have in fact highlighted a specific signature of autoimmune liver damage.

[0013] The inventors have in fact demonstrated, by flow cytometry on blood T lymphocytes (CD3+), that the detection of the markers PD-1 (Programmed cell death 1) and CD38, possibly in association with the markers CD3, CD4, CD8, CD45RA, CXCR5, CD127 and CD27, highlights a hepatic autoimmune signature. This autoimmune signature advantageously makes it possible to discriminate between patients with active autoimmune hepatitis (AIH) and patients with non-autoimmune hepatic inflammation (NASH).

[0014] The advantages of the invention are numerous.

[0015] For example, the invention can reduce diagnostic time by providing new information complementary to that conventionally obtained.

[0016] The invention also has the advantage of providing a means allowing better orientation and / or confirmation of the diagnosis, in particular in atypical cases (seronegative form, without autoantibodies) of AIH.

[0017] The invention can also make it possible to assess autoimmune activity in patients. Better patient care can therefore be advantageously envisaged, for example with an adjustment or not of the proposed treatment.

[0018] Advantageously, the invention is applicable to other hepatic and non-hepatic autoimmune disorders which face the same problem.

[0019] Furthermore, the invention advantageously makes it possible to highlight among patients with hepatic inflammation a subgroup of patients with associated hepatic autoimmunity. An improvement in their clinical and therapeutic management is therefore advantageously envisaged.

[0020] Advantageously, the invention allows a simplified evaluation of autoimmune activity in a patient, since it can be carried out by the detection of an autoimmune signature according to the invention in the patient's blood, for example by means of a simple blood test.

[0021] The invention also advantageously allows the activity of T lymphocytes, which are the main players in autoimmunity, to be measured in the blood. This method is less invasive for the patient than a biopsy and adds value to already known biological tests. In addition, directly measuring the activity of the protagonists of the pathology in the blood advantageously provides better sensitivity and possibly a complement in the case of atypical diagnosis and monitoring, such as in seronegative forms or those with low lymphocytic infiltrate of the tissue.

[0022] Thus, a first subject of the invention relates to a method for predicting and / or prognosing an active autoimmune pathology in a subject, comprising the detection of the co-expression of the PD-1 and CD38 proteins on the surface of T lymphocytes in a biological sample of the subject, according to the appended claims 1-9.

[0023] According to the invention, the active autoimmune pathology is autoimmune hepatitis. For example, the subject in whom the prediction and / or prognosis is sought may be a patient with hepatic inflammation.

[0024] The term "diagnosis" refers to the demonstration of autoimmunity in patients. This demonstration can advantageously make it possible to deduce or confirm that the patient is affected by AIH, or to demonstrate an autoimmune attack in the context of a predominantly non-autoimmune pathology. For example, the diagnosis can be made on atypical forms of AIH, for example a seronegative form, i.e. without autoantibodies, and / or with little or no lymphocytic infiltrate of the liver tissue. In another example, the diagnosis can be a discrimination of patients with autoimmune hepatitis from patients with non-alcoholic fatty liver disease (NASH), or even a demonstration of hepatic autoimmunity in patients with NASH.

[0025] For the purposes of the present invention, the term "prediction" means the anticipation of the progression of the disease in a subject during monitoring over time. This may in particular be a prediction of a risk, for example the risk of developing an autoimmune pathology in a subject, or the risk of relapse of a patient suffering from an autoimmune pathology, in particular following the cessation or adjustment of their treatment, or an immune-related adverse event (IRAE), for example during the use of antibodies targeting checkpoint inhibitors in the context of anti-cancer immunotherapy.

[0026] For the purposes of the present invention, the term "prognosis" means the prediction of the chances of survival of a patient suffering from an active autoimmune pathology in a subject. For example, the prognostic method of the invention can make it possible to evaluate the survival rate, this rate indicating the percentage of people having participated in a study and living for a given period, generally 5 to 10 years after the diagnosis of an active autoimmune pathology. Generally, a good prognosis, for the purposes of the invention, is associated with a median survival greater than 5 years, while a poor prognosis is associated with a median survival less than 5 years.

[0027] For the purposes of the present invention, the term "detection of protein co-expression" means any detection of the joint expression of the PD-1 and CD38 proteins on the surface of each T lymphocyte present in the biological sample. It is therefore the frequency of joint expression of the PD-1 and CD38 proteins on the surface of each T lymphocyte present in the biological sample that is measured. The detection may be, for example, a comparison between the level of the frequency of joint expression of these proteins in a biological sample from a subject, and a sample or a reference value (also called "threshold value" or "normal value") allowing a diagnosis, a prediction and / or a proven prognosis of the active autoimmune pathology. Indeed, the reference value represents the frequency of joint expression of these proteins in the population not presenting the pathology sought.The reference value can be defined on the basis of measurements obtained on a sample of healthy subjects, i.e. subjects not presenting the pathology sought. This may be, for example, subjects who do not have viral hepatitis, nor any sign of autoimmunity in the liver and / or any chronic condition other than, possibly, hepatic inflammation due to a dysmetabolic disorder (also called "fatty liver" or "soda disease"). Thus, it is understood that the "healthy subjects" used to define the reference value may be patients suffering from NASH (or not suffering from NASH), but not presenting viral hepatitis, nor any sign of autoimmunity in the liver and / or any other chronic condition other than hepatic inflammation due to a dysmetabolic disorder. The reference value can be defined, for example, according to the protocol described in example 2 below.The reference value may be established on a significant number of healthy subjects, for example at least 100 healthy subjects. Advantageously, the threshold value may be defined as greater than a frequency of LTCD3+ jointly expressing PD1 and CD38 of 2.77 percent. Thus, a frequency greater than 2.77 percent will be characteristic of hepatic autoimmunity. Detection may be carried out by any method known to those skilled in the art. This may be, for example, a method chosen from flow or mass cytometry, an immunological test technology, such as direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA, multiplex ELISA, a radioimmunoassay (RIA) or an ELISPOT technology, a mass spectrometry analysis method, a chromatography method, a qPCR method and a combination of at least two of these methods.Thus, in one example described, the detection of an autoimmune signature can be carried out in the blood, for example whole blood, of the patient by a simple blood sample followed by a simplified test consisting of incubating the blood cells with a mixture of specific fluorescent antibodies, followed by analysis on a flow cytometer.

[0028] PD-1 and CD38 proteins can be detected on the surface of any cell known to express them on its surface. This could be, for example, CD3 T cells and / or CD8 T cells and / or CD4 T cells.

[0029] The biological sample used may be any biological sample commonly used for biological analyses. It may be, for example, a blood sample, for example a blood fraction, or alternatively a whole blood sample, or a biopsy fraction, for example liver, of tissue. In the method of the invention, the biological sample is a blood sample.

[0030] The method of the invention comprises the steps of: (i) detecting the co-expression of PD-1 and CD38, on the surface of T lymphocytes, in said blood sample and determining the frequency of co-expression of PD-1 and CD38; (ii) comparing the frequency of co-expression of PD-1 and CD38 determined in (i) with a reference value of the frequency of co-expression of PD-1 and CD38, (iii) investigating the presence or absence of a deviation of the frequency of co-expression of PD-1 and CD38 determined in (ii) from the reference value; (iv) attributing the presence or absence of deviation to a prediction and / or prognosis of active autoimmune hepatitis in the subject.

[0031] A frequency of co-expression of PD-1 and CD38, in said sample, higher than a reference value indicates that the subject presents a risk of relapse of the active autoimmune pathology.

[0032] In addition to the PD-1 and CD38 proteins, it is possible, according to the invention, to detect the co-expression of PD-1 and CD38 with at least one other biological marker chosen from the group consisting of CD3, CD4, CD8, CD45RA, CXCR5, CD127 and CD27. It may be one marker, or two markers, or three markers, or four markers, or five markers, or six markers, or seven markers. This detection of at least one other marker may be carried out at the same time as the detection of the co-expression of PD-1 and CD38, or subsequently to the step of detecting the co-expression of PD-1 and CD38. For example, co-expression of the combinations of biological markers selected from the combinations PD-1 / CD38 / CD3, PD-1 / CD38 / CD3 / CD4 / CD8 / CD45RA, PD-1 / CD38 / CD3 / CD4 / CD8 / CD45RA / CD127, PD-1 / CD38 / CD3 / CD4 / CD8 / CD45RA / CXCR5 / CD127 and PD-1 / CD38 / CD3 / CD4 / CD8 / CD45RA / CXCR5 / CD127 / CD27 can be detected.Advantageously, the analysis of the co-expression of PD-1 and CD38 on the surface of CD3 (CD3+) LTs can be carried out in association with the detection of the frequency of lymphocyte populations defined by the phenotypes below: . a) CD45RA (study of the CD3+CD45RA-PD-1+CD38+ subpopulation); b) CD45RA, CD4 and CD8 (study of the CD3+CD4+CD8-CD45RA-PD-1+CD38+ and CD3+CD4-CD8+CD45RA-PD-1+CD38+ subpopulations); c) CD45RA, CD4, CD8 and CD127 (study of the CD3+CD4+CD8-CD45RA-CD127-PD-1+CD38+ and CD3+CD4-CD8+CD45RA-CD127-PD-1+CD38+ subpopulations); d) CD45RA, CD4, CD8, CD127 and CXCR5 (study of the CD3+CD4+CD8-CD45RA-CD127-CXCR5-PD-1+CD38+ and CD3+CD4-CD8+CD45RA-CD127- CXCR5-PD-1+CD38+ subpopulations); and e) CD45RA, CD4, CD8, CD127, CXCR5 and CD27 (study of the CD3+CD4+CD8-CD45RA-CD127-CXCR5-CD27+PD-1+CD38+ and CD3+CD4-CD8+CD45RA-CD127- CXCR5-CD27+PD-1+CD38+ subpopulations).

[0033] Advantageously, the associations of the cited markers can make it possible to highlight CD4 and CD8 T lymphocyte populations which are significantly increased in patients with an active autoimmune pathology, in particular active AIH, in comparison with a reference value, or for example in patients with NASH when it is a comparison with AIH.

[0034] Another object of the invention relates to the use in vitro of the PD-1 (Programmed cell death 1) and CD38 protein pool as biological markers co-expressed on the surface of T lymphocytes, measured in a blood sample taken from a subject for the prediction and / or prognosis of active autoimmune hepatitis in a subject.

[0035] Another disclosed (unclaimed) subject matter relates to a device for detecting co-expression of PD-1 and CD38 on the surface of T lymphocytes in a sample taken from a subject, comprising: (i) optionally means for obtaining a sample taken from the subject, and (ii) means for detecting the co-expression of PD-1 and CD38 on the surface of T lymphocytes in said sample, and (iii) means for determining the frequency of co-expression of PD-1 and CD38 in the sample. Advantageously, said means for determining the frequency of co-expression may make it possible to indicate whether this frequency, in said sample, is higher or lower than a reference value and / or whether this frequency in said sample differs or not from a reference value, said reference value representing a diagnosis, a prediction and / or a prognosis of an active autoimmune pathology in the subject.

[0036] Other advantages may still become apparent to those skilled in the art upon reading the examples below, illustrated by the attached figures, given for illustrative purposes. Brief description of the figures

[0037] [ Fig. 1 ] represents: A) representation of the unsupervised analysis strategy (FlowSOM) of flow cytometry data in 12 AIH patients and 12 NASH patients. B) Heatmap representing the expression of surface markers of the 4 lymphocyte populations (nodes) showing a significant increase (p<0.05) in AIH. Expression intensity ranging from striped (high expression) to white (low expression). [ Fig. 2 ] represents: A and D) Representation of flow cytometry data of PD-1 and CD38 markers on CD3+CD4+CD45RA-CXCR5-CD127-CD27+ populations (A) and CD3+CD8+CD45RA-CXCR5-CD127-CD27+ (D). B) and E) graphs representing the percentage of PD-1+CD38+ cells per CD3+CD4+CD45RA·CXCR5-CD127-CD27+ cells (B) and per CD3+CD8+CD45RA-CXCR5-CD127-CD27+ cells (E) in 32 AIH patients and 18 NASH patients. C and F) Graphs representing the percentage of CD4+CD45RACXCR5-CD127-CD27+PD-1 +CD38+ cells (C) and CD8+CD45RA-CXCR5-CD127-CD27+PD1+CD38+ cells (F) per CD3+ cells. P values ​​were determined using the Mann-Whitney statistical test. Fig. 3 ] represents the ROC curves based on the percentage of PD-1+CD38+ cells of LTCD4 and LTCD3 in the parent population identified with the markers annotated above the ROC curve. In italics the cutoff value is indicated in black with in parentheses the sensitivity and specificity values. The AUC values ​​are indicated on each graph. [ Fig. 4 ] represents the ROC curves based on the percentage of PD-1+CD38+ cells of LTCD8 and LTCD3 in the parent population identified with the markers annotated above the ROC curve. In italics the cutoff value is indicated in black with the sensitivity and specificity values ​​in parentheses. The AUC values ​​are indicated on each graph. [ Fig. 5 ] is a representation of the flow cytometry data values ​​of PD-1 and CD38 markers in HAI patients in complete remission under treatment. A) Heatmap representing flow cytometry data values ​​of PD-1 and CD38 markers in HAI patients in complete remission under treatment. In gray, data above the threshold values ​​identified previously. B) graphs representing the percentage of patients in complete remission under treatment with PD-1 and CD38 marker values ​​above the threshold values ​​determined previously ( figures 3 And 4 ). [ Fig.6 ] is a representation of the analysis data between the simple measurement of PD-1 on the surface of T cells versus the measurement of co-expression of PD-1 with CD38 on the surface of T cells. Comparison of the percentage of CD3+PD-1+CD38+ (A), CD3+PD-1+ (B), CD3+CD4+PD-1+CD38+ (C), CD3+CD4+PD-1+ (D), CD3+CD8+PD-1+CD38+ (E) and CD3+CD8+PD-1+ (F) cells of CD3+ between patients with AIH and patients with NASH. The p values ​​were determined using the Mann-Whitney statistical test. ROC curves (sensitivity / specificity in %) based on the percentage of CD3+PD-1+CD38+ (G), CD3+PD-1+ (H), CD3+CD4+PD-1+CD38+ (I), CD3+CD4+PD-1+ (J), CD3+CD8+PD-1+CD38+ (K) and CD3+CD8+PD-1+ (L) cells of CD3+ between patients with AIH and patients with NASH are shown. AUC values ​​are indicated on each graph. Examples Example 1: Identification of differential lymphocyte populations between patients with AIH and patients with NASH

[0038] With the aim of identifying a specific signature of hepatic autoimmunity, we performed multiparametric flow cytometry data analyses by unsupervised statistical methods (FlowSOM) between 12 HAI patients and 12 NASH patients, which represent the best choice as a control population to answer our question.

[0039] This unsupervised approach made it possible to establish a flow cytometry identification strategy, using a total of 13 parameters including 10 cell surface markers (CD3, CD4, CD8, CD45RA, CXCR5, CCR6, CD27, CD127, PD-1 and CD38) and 1 viability marker, of two cell populations (LT CD4 and CD8) which are characterized by the expression of PD-1 and CD38 ( Figure 1 ).

[0040] In a cohort of 32 active HAI patients and 18 NASH patients, both LT CD4 and LT CD8 cell populations expressing PD-1 and CD38 are significantly increased in the blood of HAI patients compared to NASH patients ( Figure 2 ). This strategy makes it easy to discriminate between hepatic autoimmunity (HAI) and hepatic inflammation (ROC curve; Figures 3 And 4 ).

[0041] In comparison, the sole measurement of PD-1 expression on the surface of T lymphocytes (CD3+, CD3+CD4+ and CD3+CD8+) is much less sensitive to discriminate between hepatic autoimmunity (HAI) and hepatic inflammation (NASH) than the measurement of the co-expression of PD-1 and CD38 on the surface of T lymphocytes ( Figure 6 . ROC curve). Indeed, the analysis of the difference in the frequency of T lymphocytes expressing PD-1 between active HAI patients and NASH patients is much less significant than the results obtained with the measurement of the co-expression of PD-1 and CD38 ( Figure 6 ).

[0042] These results demonstrate the interest in using these markers, particularly in flow cytometry, as potential biomarkers of hepatic autoimmunity. Example 2: Determination of biomarker threshold values

[0043] In order to propose the most sensitive threshold values ​​possible, we analyzed the generated results in various ways ( Figures 3 And 4 ). The principle was to carry out the analysis of the percentage of expression of PD-1 and CD38 by modifying the number of associated markers, in particular with a view to simplifying subsequent use.

[0044] On the LT CD4 population ( Figure 3 ) and LT CD8 ( Figure 4 ), and on the total LT CD3 population ( Figures 3 And 4), the percentage of PD-1 and CD38 expression (in the parent cell population using a gating strategy for flow cytometry analysis) was analyzed in the CD45RA- (6m); CD45RA-CD127- (7m); CD45RA-CD127-CXCR5- (8m) and CD45RA-CD127-CXCR5-CD27+ (9m) subpopulations ( Figures 3 And 4 ). The analysis was performed using ROC curves. In all analysis conditions we obtained areas under the curve (AUC) greater than 0.85, which demonstrates the strong potential of these markers as biomarkers of hepatic autoimmunity.

[0045] Threshold values ​​for the autoimmune signature could thus be defined with sensitivities ranging from 77% to 100% and specificities ranging from 78% to 87%.

[0046] In a small group of HAI patients in complete remission under treatment (pilot study) we observed that approximately 50% of HAI patients in complete remission under treatment (n=19) maintain a high level of these biomarkers, while it is normalized in the others ( Figure 5 ). This suggests a possible association between the persistence of these blood biomarkers and the patient's outcome (risk of relapse).

[0047] All of these results demonstrate that it is possible to define an autoimmune lymphocyte signature in the blood of patients which is discriminatory of non-specific inflammation. Example 3: Search for combinations of biomarkers of interest

[0048] The study focuses on 1) clinical interpretation of cytometry data; 2) validation of the data in a larger number of patients and in other autoimmune pathologies; and 3) simplification and development of a prototype test on whole blood.

[0049] Regarding the simplification and development of a prototype test on whole blood, different grouping associations of the previously mentioned markers are tested, in order to group the negative markers together to create exclusion "windows" and thus facilitate the reading of the test.

[0050] For example, for the 9m panel, an initial selection of CD3+CD45RA-CXCR5-CD127- T cells is performed, with CD45RA, CXCR5 and CD127 grouped in the same reading channel to facilitate their exclusion. It is thus possible to move from 9 reading channels in flow cytometry to 6 or 7 channels.

[0051] This therefore makes it possible to find the best possible combinations to offer a simple and as sensitive test as possible.

[0052] A sensitivity study is further performed on the analysis of the co-expression of PD-1 and CD38 on the surface of CD3 (CD3+) LT in association with the marker(s): a) CD45RA (study of the CD3+CD45RA-PD-1+CD38+ subpopulation); b) CD45RA, CD4 and CD8 (study of the CD3+CD4+CD8-CD45RA-PD-1+CD38+ and CD3+CD4-CD8+CD45RA-PD-1+CD38+ subpopulations); c) CD45RA, CD4, CD8 and CD127 (study of the CD3+CD4+CD8-CD45RA-CD127-PD-1+CD38+ and CD3+CD4-CD8+CD45RA-CD127-PD-1+CD38+ subpopulations); d) CD45RA, CD4, CD8, CD127 and CXCR5 (study of the CD3+CD4+CD8-CD45RA-CD127-CXCR5-PD-1+CD38+ and CD3+CD4-CD8+CD45RA-CD127- CXCR5-PD-1+CD38+ subpopulations); and e) CD45RA, CD4, CD8, CD127, CXCR5 and CD27 (study of the CD3+CD4+CD8-CD45RA-CD127-CXCR5-CD27+PD-1+CD38+ and CD3+CD4-CD8+CD45RA-CD127- CXCR5-CD27+PD-1+CD38+ subpopulations).

[0053] This study aims to identify the most sensitive marker association associated with the co-expression of PD-1 and CD38 to measure autoimmunity activity in patients with autoimmune hepatitis. List of references

[0054] 1. Liberal , R. , Krawitt , EL , Vierling , JM , Manns , MP , Mieli-Vergani , G. , and Vergani , D. (2016). Cutting edge issues in autoimmune hepatitis. J. Autoimmun. 75, 6-1 [ PubMed ] 2. Manns, MP, Czaja, AJ, Gorham, JD, Krawitt, EI, Mieli-Vergani, G, Vergani, D, Vierling, JM, and American Association for the Study of Liver Diseases. Diagnosis and management of autoimmune hepatitis. Hepatol. Baltim. Md 51, 2193–2 3. European Association for the Study of the Liver (2015). EASL Clinical Practice Guidelines: Autoimmune hepatitis. J. Hepatol. 63, 971–1004. 4. A. Renand, S. Habes, J.-F. Mosnier, H. Aublé, J.-P. Judor, N. Vince, P. Hulin, S. Nedellec, S. Metairie, 1. Archambeaud, et al. (2018). Immune Alterations in Patients With Type 1 Autoimmune Hepatitis Persist Upon Standard Immunosuppressive Treatment. Hepatol. Commun. 2, 968-9 5. Böttcher , K , Rombouts , K , Saffioti , F , Roccarina , D , Rosselli , M , Hall , A , Luong , T , Tsochatzis , EA , Thorburn , D ., and Pinzani, M. (2018). MAIT cells are chronically activated in patients with autoimmune liver disease and promote pro-fibrogenic hepatic stellate cell activation. Hepatol. Baltim. Md. 6. Jeffery, H.C., van Wilgenburg, B., Kurioka, A., Parekh, K., Stirling, K., Roberts, S., Dutton, E.E., Hunter, S., Geh, D., Braitch, M.K., et al. (2016). Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MRI. J. Hepatol. 64, 1118-1127. 7. Oo, Y.H., Banz, V., Kavanagh, O., Liaskou, E., Withers, D.R., Humphreys, E., Reynolds, G.M., Lee-Turner, L., Kalia, N., Hubscher, S.G., et al. (2012). CXCR3-dependent recruitment and CCR6-mediated positioning of Th-17 cells in the inflamed liver. J. Hepatol. 57, 1044-1051.

Claims

1. Method of predicting and / or prognosing active autoimmune hepatitis in a subject, comprising the following steps : (i) detect the co-expression of PD-1 (Programmed cell death 1) and CD38 on the surface of T lymphocytes, in a blood sample of the subject, and determine the frequency of co-expression of PD-1 and CD38; (ii) compare the frequency of co-expression of PD-1 and CD38 determined in (i) with a reference value for the frequency of co-expression of PD-1 and CD38; (iii) search for presence or absence of a deviation of the frequency of co-expression of PD-1 and CD38 determined in (ii) from the reference value ; and (iv) attribute the presence or absence of a deviation to prediction and / or a prognosis of active autoimmune hepatitis in the subject, wherein a frequency of co-expression of PD-1 and CD38 in said sample greater than a reference value indicates that the subject is at risk of relapse of active autoimmune hepatitis.

2. Method according to claim 1, wherein said blood sample is a blood fraction or a whole blood sample.

3. Method according to claim 1 or 2, wherein PD-1 and CD38 proteins are detected on the surface of CD3 and / or CD8 and / or CD4 T lymphocytes.

4. Method according to any one of the preceding claims, wherein the expression of at least one other biological marker selected from the group consisting of CD3, CD4, CD8, CD45RA, CXCR5, CD127 and CD27 is detected.

5. Method according to claim 4, wherein is detected the co-expression of associations of biological markers chosen from the associations PD-1 / CD38 / CD3, PD-1 / CD38 / CD3 / CD4 / CD8 / CD45RA, PD-1 / CD38 / CD3 / CD4 / CD8 / CD45RA / CD127, PD-1 / CD38 / CD3 / CD4 / CD8 / CD45RA / CXCR5 / CD127 et PD-1 / CD38 / CD3 / CD4 / CD8 / CD45RA / CXCR5 / CD127 / CD27.

6. Method according to claim 5, wherein is detected the frequency of lymphocytes populations defined by the phenotypes below: a) CD3+CD45RA-PD-1+CD38+ ; b) CD3+CD4+CD8-CD45RA-PD-1+CD38+ et / ou CD3+CD4-CD8+CD45RA-PD-1+CD38+ ; c) CD3+CD4+CD8-CD45RA-CD127-PD-1+CD38+ et / ou CD3+CD4-CD8+CD45RA-CD127-PD-1+CD38+ ; d) CD3+CD4+CD8-CD45RA-CD127-CXCR5-PD-1+CD38+ et / ou CD3+CD4-CD8+CD45RA-CD127 - CXCR5-PD-1+CD38+ ; et e) CD3+CD4+CD8-CD45RA-CD127 -CXCR5-CD27 + PD-1 +CD38+ et / ou CD3+CD4-CD8+CD45RA-CD127- CXCR5-CD27+PD-1+CD38+.

7. Method according to any one of the preceding claims, wherein the detection is performed by a method selected from flow or mass cytometry, an immunoassay technology, such as direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA, multiplex ELISA, radioimmunoassay (RIA) or ELISPOT technology, a mass spectrometry analysis method, a chromatography method, a qPCR method and a combination of at least two of these methods.

8. Method according to any one of the preceding claims, wherein said prediction is a prediction of the risk of relapse following discontinuation or adjustment of a treatment.

9. Method according to any one of the preceding claims, wherein said autoimmune hepatitis is an atypical form of autoimmune hepatitis, in particular a seronegative form and / or with little or no lymphocytic infiltrate of the liver tissue.

10. In vitro use of the PD-1 (Programmed cell death 1) and CD38 protein pool as biological markers co-expressed on the surface of T lymphocytes, measured in a blood sample taken from a subject, for the prediction and / or prognosis of active autoimmune hepatitis in a subject.