Analysis method and diagnosis method for CD8-positive t cell activation in patients with myasthenia gravis

Abstract

The present invention provides a specimen analysis method comprising a step for determining whether or not CD8-positive T cells are activated in a specimen obtained from a subject suffering from or potentially suffering from myasthenia gravis.

Description

Method for analyzing and diagnosing CD8-positive T cell activation in patients with myasthenia gravis

[0001] The present invention broadly relates to a method for analyzing a sample derived from a subject suffering from or suspected of suffering from myasthenia gravis.

[0002] Myasthenia gravis is an autoimmune disease caused by autoantibodies. A typical symptom is muscle weakness in the limbs, and one of the causes is the development of antibodies (autoantibodies) against components of the patient's own body. Neuromuscular transmission is inhibited by autoantibodies produced in the patient's body against molecules present in the postsynaptic membrane of the neuromuscular junction, such as acetylcholine receptors (AChRs) and muscle-specific tyrosine kinases (MuSKs). As a result, brain commands are not transmitted effectively from motor nerves to muscles, resulting in insufficient muscle contraction and ultimately muscle weakness.

[0003] Since the appearance of autoantibodies has been thought to be the main cause of myasthenia gravis, research into myasthenia gravis to date has mainly focused on CD4-positive T cells, which are involved in humoral immunity (Non-patent Documents 1-3).

[0004] Treatments for myasthenia gravis include steroids and immunosuppressants, but currently there is a shift to treatments such as molecular targeted drugs that target antibodies produced by B cells via CD4-positive T cells, the antibody-producing B cells themselves, cytokines that activate B cells, and complement that induces antibody-mediated cytotoxicity. Examples of antibodies used in these treatments include efgartigimod (anti-FcRn antibody), rituximab (anti-CD20 monoclonal antibody), satralizumab (IL-6 receptor neutralizing antibody), and ravulizumab (complement neutralizing antibody).

[0005] Nico Melzer et al., "Clinical features, pathogenesis, and treatment of myasthenia gravis: a supplement to the Guidelines of the German Neurological Society", J Neurol. 2016 Aug;263(8):1473-94. Anthony Behin et al., "New Pathways and Therapeutic Targets in Autoimmune Myasthenia Gravis", J Neuromuscul Dis. 2018; 5(3): 265-277.Fredrik Romi et al., "Pathophysiology and immunological profile of myasthenia gravis and its subgroups", Curr Opin Immunol. 2017 Dec;49:9-13.

[0006] Even with current drug and surgical therapies, myasthenia gravis cannot be cured completely, and many cases remain refractory. Therefore, there is a need to explore new targets for treating this condition.

[0007] The present inventors compared peripheral blood mononuclear cells (PBMCs) from patients with myasthenia gravis and healthy individuals using gene expression analysis and found that CD8+ T cells are activated in patients with myasthenia gravis. This suggests that CD8+ T cells involved in cellular immunity or their gene signatures may be novel therapeutic targets for myasthenia gravis.

[0008] That is, the present application encompasses the following inventions: [1] A method for analyzing a sample, comprising a step of determining whether or not CD8-positive T cells are activated in a sample derived from a subject who has or may have myasthenia gravis. [2] The method of [1], in which the presence or absence of activation is determined based on the level of expression of a signature gene of CD8-positive T cells or its expression product in the sample. [3] The method of [2], in which the signature gene is a gene related to the cytotoxicity and / or activation of CD8-positive T cells. [4] The signature genes were ABHD17A, AC004687.1, ACTB, ACTG1, ADGRG1, AL138963.3, ANXA2, ANXA6, AREG, ARHGDIB, ARPC1B, ARPC5L, B2M, BCL3, BIN2, BTG1, BTG2, BTN3A2, CALR, CAPZB, CCL4, CCL5, CCND3, CD27, CD3D, CD3E, CD5, CD52, CD6, CD63, CD69, CD7, CD74, CD8A, CD8B, CEBPD, CFL1, CIR1, CLIC1, CLIC3, CMC1, CORO1A, CRIP1, CST7, CTSW, CXCR3, C XCR4, CYBA, CYTIP, DDIT4, DDX3Y, DDX5, DNAJA1, DNAJB1, DUSP1, DUSP2, DUSP5, EEF1G, EFHD2, EIF1, EML4, EVL, FC GR3A, FCRL6, FGFBP2, FLNA, FOS, FTH1, GGNBP2, GIMAP4, GIMAP7, GSTP1, GZMA, GZMH, GZMK, HCST, HINT1, HIST1H4 C, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DRA, HLA-DRB1, HNRNPA2B1, HNRNPDL, HOPX, ID2, IFIT2, IFITM1, IFITM2, IL 32, IL7R, IRF1, ITGB2, ITGB7, JUN, JUNB, JUND, KIR2DL3, KLF2, KLF6, KLRB1, KLRC1, KLRD1, LCP1, LEF1, LGALS1, LINC02446, LMNA, LSP1, LTB, LY6E, MAP3K8, MT2A, MT-ATP6, MT-ATP8, MT-CO1, MT-CYB, MT-ND4L, MT-ND5, MT-ND6,MTRNR2L12, MYADM, MYL12A, MYL6, NCR3, NFKB1, NFKBIA, NKG7, NR4A2, OAZ1, PFN1 , PIK3R1, PMAIP1, PPDPF, PPP1CA, PPP1R15A, PRDM1, PRELID1, PRF1, PSME1, PSME 2, PTPRC, RAC2, RBM39, REL, RELB, RGS1, RORA, RPL10, RPL17, RPL34, RPL36, RPL3 7, RPL38, RPL39, RPL41, RPLP0, RPLP1, RPS10, RPS13, RPS14, RPS21, RPS26, RPS27 , RPS28, RPS29, RPS4Y1, S100A4, S100A6, S1PR1, SBDS, SH3BGRL3, SLC2A3, SNRPA1, SOCS3, SPOCK2, SPON2, SRGN, STK17A, TCF7, TENT5C, TLN1, TMSB10, TMSB4X, TNFAIP3, TRBC2, TRGV10, TRGV2, TSC22D3, TXNIP, TYROBP, UCP2, VIM, WHAMM, YPEL5, ZEB2, ZFP36, ZFP36L1 and ZFP36L2. [5] The method of any of [2] to [4], wherein the signature gene is one or more genes selected from the group consisting of: 1) genes expressed in early activated CD8-positive T cells, which are CXCR4, CD69, IFITM1, IL32, CCL4, and IFITM2, or 2) genes expressed in late activated CD8-positive T cells, which are IL32, IFITM2, CXCR4, IFITM1, CCL4, CCL5, ZFP36L2, CD74, JUNB, and TNFAIP3. [6] The method of any of [1] to [5], wherein the subject is suffering from myasthenia gravis and is undergoing treatment targeting CD8-positive T cells, a signature gene of the cells or an expression product thereof, a factor that activates CD8-positive T cells, or a molecule whose expression is enhanced in activated CD8-positive T cells. [7] The method according to [6], wherein the treatment is evaluated as being effective in treating the subject when activated CD8-positive T cells are reduced compared to before the treatment.[9] The method according to any one of [1] to [7], wherein a subject may be suffering from myasthenia gravis. [9] The method according to [8], wherein a subject is assessed as suffering from myasthenia gravis when the number of activated CD8-positive T cells is higher than that of a healthy subject, and a treatment targeting CD8-positive T cells, a signature gene of the cells or an expression product thereof, a factor that activates CD8-positive T cells, or a molecule whose expression is enhanced in activated CD8-positive T cells is effective.

[10] A marker for the treatment, prevention, or diagnosis of myasthenia gravis, comprising a signature gene of CD8-positive T cells or an expression product thereof.

[11] A pharmaceutical composition for the treatment or prevention of myasthenia gravis, comprising an active ingredient that targets CD8-positive T cells, a signature gene of the cells or an expression product thereof, a factor that activates CD8-positive T cells, or a molecule whose expression is enhanced in activated CD8-positive T cells.

[12] The pharmaceutical composition according to

[11] , wherein the CD8-positive T cells are early- or late-activated CD8-positive T cells.

[0009] Conventionally, there has been no method for evaluating myasthenia gravis using as an indicator whether or not CD8-positive T cells are activated. However, the present invention makes it possible to provide a novel method for analyzing myasthenia gravis and novel means for treatment and prevention that target CD8-positive T cells involved in cellular immunity.

[0010] Furthermore, activating factors of CD8-positive T cells activated in myasthenia gravis and factors whose expression is increased can serve as surrogate markers for determining the effectiveness of treatment when treatment targeting CD8-positive T cells is carried out.

[0011] A report by Wanlin Jin et al. presents the results of single-cell analysis of PBMCs derived from healthy individuals and patients with myasthenia gravis, and also mentions CD8+ T cells ("Single-cell RNA-Seq reveals transcriptional heterogeneity and immune subtypes associated with disease activity in human myasthenia gravis," Cell Discovery volume 7, Article number: 85 (2021)). However, the report only presents the results of combining analysis data of PBMCs from healthy individuals and patients with myasthenia gravis to characterize each cell population (e.g., Fig. 4a and 4b). This is not comparative data between healthy individuals and patients with myasthenia gravis, nor is it data demonstrating activation of CD8+ T cells in patients with myasthenia gravis.

[0012] Figure 1 shows the whole-cell clusters of samples from healthy individuals and myasthenia gravis patients subjected to single-cell RNA analysis. Figure 2 shows the general cell marker gene expression profiles used for annotation. Figure 3 shows violin plots visualizing the gene expression related to cytotoxicity in each cluster. Figure 4 shows the results of confirming the gene expression related to cell activation and cytotoxicity in each cluster.

[0013] Hereinafter, an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described, but the scope of the present invention should not be interpreted as being limited to the following embodiment.

[0014] (Analysis Method) In a first aspect, there is provided a method for analyzing a sample, the method comprising a step of determining whether CD8-positive T cells are activated in a sample derived from a subject who is suffering from or may be suffering from myasthenia gravis.

[0015] Myasthenia gravis (MG) is an autoimmune disease, and examples of its symptoms include ptosis, diplopia due to impaired eye movement, muscle weakness in the limbs, swallowing disorders, and dysarthria. In severe cases, hypoventilation occurs due to respiratory muscle paralysis. It is believed that myasthenia gravis is caused by the production of autoantibodies in patients against molecules present in the postsynaptic membrane of the neuromuscular junction, and that these antibodies inhibit neuromuscular transmission.

[0016] As used herein, the cause of myasthenia gravis is not limited to an autoimmune reaction, such as the production of antibodies against acetylcholine receptors (AChRs), but also includes cases caused by drugs such as immune checkpoint inhibitors, cases in which anti-Musk antibodies or anti-LRP4 antibodies are elevated, cases in which the titer of any autoantibody is not clearly elevated but the onset of myasthenia gravis is observed, and congenital cases in which AChRs are low from birth. Myasthenia gravis can be systemic or localized, for example, ocular myasthenia. The onset time and severity of symptoms are not limited, and myasthenia gravis includes all classifications, such as adult type I (ocular myasthenia), adult type II (systemic myasthenia), adult type III (acute severe symptoms), adult type IV (late severe myasthenia), adult type V (muscle atrophy), childhood onset, and cases involving thymoma.

[0017] Myasthenia gravis can be diagnosed by known methods, for example, based on the positivity of known markers such as AChR antibodies in a sample. The severity of myasthenia gravis can be classified using Class I to V in the clinical classification of the MGFA (MG Foundation of America). Class I or higher is diagnosed as myasthenia gravis.

[0018] In one embodiment, the myasthenia gravis is selected from the following groups as described in the Myasthenia Gravis / Lambert-Eaton Myasthenic Syndrome Clinical Practice Guidelines 2022: - Ocular MG (oMG) - AChR antibody-positive non-thymoma-type generalized MG - Early-onset MG (g-EOMG) (age of onset < 50 years) - Late-onset MG (g-EOMG) (age of onset > 50 years) - AChR antibody-positive thymoma-type generalized MG - Thymoma-associated MG (g-TAMG) - Systemic MG positive for pathogenic autoantibodies other than AChR antibodies - MuSK antibody-positive MG (g-MuSKMG) - Systemic MG with no detectable pathogenic autoantibodies - Antibody-negative MG (g-SNMG) (including LRP4 antibody-positive MG)

[0019] As used herein, "analysis" refers to any analysis related to myasthenia gravis, such as analysis that leads to elucidating the mechanism of action of myasthenia gravis (e.g., onset, progression, recurrence, etc.) and providing medically useful information regarding the diagnosis, treatment, prevention, prognosis, etc. of myasthenia gravis.

[0020] The analysis method may include, depending on the purpose, any steps necessary before or after the step of determining whether CD8-positive T cells are activated. These additional steps include, but are not limited to, a step of preparing a sample before analysis, a step of analyzing the sample from a different perspective, a step of further analyzing data regarding activation obtained in the analysis step, particularly data regarding differences from healthy subjects, and a step of providing information regarding the sample that is useful for diagnosis and treatment by physicians and others based on findings obtained by the analysis, such as a step of classifying the sample as a sample from a subject who has or may have myasthenia gravis.

[0021] The sample may be any sample that can evaluate the activation of CD8-positive T cells. Examples of such samples include blood or its components, cell or tissue extracts, urine, cerebrospinal fluid, tracheal aspirate, bronchoalveolar lavage fluid, etc. from the subject from whom the sample is derived. Blood components include whole blood, blood cells, serum, plasma, etc. Peripheral blood or peripheral blood mononuclear cells (PBMCs) derived from peripheral blood are preferred as samples. PBMCs can be prepared using known methods such as density gradient centrifugation, immunomagnetic separation, and sedimentation.

[0022] The sample may include genomic DNA, RNA such as mRNA, protein, and / or a combination thereof collected from a subject. The sample preferably includes RNA to enable analysis of gene expression levels. The sample may also include cDNA generated using RNA as a template.

[0023] The sample is collected from a subject who is suffering from or may be suffering from myasthenia gravis. The number of times the sample is collected can be determined appropriately depending on the purpose of the analysis, and may be collected once for diagnosis or multiple times for purposes such as prognosis observation after treatment.

[0024] The subject from which the specimen is derived is not limited to humans, but may also be a non-human animal, particularly a mammal, that may develop myasthenia gravis, such as a rat, mouse, guinea pig, rabbit, sheep, horse, pig, cow, dog, cat, monkey, etc.

[0025] The subject may have been diagnosed with myasthenia gravis prior to the analysis method of the present invention by using known markers, such as anti-acetylcholine receptor (AChR) antibodies, anti-muscle-specific receptor tyrosine kinase (MuSK) antibodies and / or LDL receptor-related protein 4 (low-density lipoprotein receptor-related protein 4: Lrp4) antibodies, or by known tests, such as the edrophonium (Tensilon) test, ice pack test, and repetitive stimulation test.

[0026] After being diagnosed with myasthenia gravis, the subject may be further treated for myasthenia gravis. Such treatments include, in addition to known treatments such as anticholinesterase inhibitors, treatments that target CD8-positive T cells, signature genes of the cells or their expression products, factors that activate CD8-positive T cells, or molecules whose expression is increased in activated CD8-positive T cells.

[0027] As used herein, "CD8-positive T cells" refers to T cells or populations thereof that express CD8 on their surface. CD8-positive T cells are also referred to as cytotoxic T lymphocytes (CTLs), a term commonly used in the art.

[0028] Whether or not CD8-positive T cells in a sample are activated can be determined by comparison with CD8-positive T cells in a control, for example, a sample derived from a healthy subject, or by comparison with past CD8-positive T cells of the subject from whom the sample was derived. When gene changes are used as an indicator, the control includes a housekeeping gene.

[0029] In one embodiment, the method of analyzing a sample comprises determining that CD8-positive T cells are activated in the sample.

[0030] In CD8-positive T cells from patients with myasthenia gravis, the expression of certain genes or certain combinations of genes is significantly altered, and such changes can be used as indicators to determine whether or not the cells are activated.

[0031] As used herein, "activation" refers to any change in CD8-positive T cells derived from myasthenia gravis. Such a change may be an intrinsic change in CD8-positive T cells observed in myasthenia gravis patients, such as a change in the expression of genes or cell surface markers (e.g., CD25) strongly associated with myasthenia gravis, or a change in the proliferation of CD8-positive T cells. Multiple changes may be evaluated; for example, an expression profile of multiple genes in CD8-positive T cells may be used.

[0032] The change in CD8-positive T cells derived from myasthenia gravis is preferably at least a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 6- to 10-fold increase compared to a control.

[0033] The changes can be evaluated using known analytical methods such as transcriptome analysis, expression analysis, and expression variation analysis. The expression levels of nucleic acids and proteins in a sample can be measured qualitatively and / or quantitatively by microarray analysis, PCR (RT-PCR), immunohistochemistry, immunofluorescence, mass spectrometry, Northern blot, Western blot, or the like.

[0034] Single-cell RNA analysis is one method for analyzing expression variations. Single-cell analysis includes processes such as dimensionality reduction, clustering, and annotation. Clustering is a method for grouping cells based on similarity, allowing for the classification of cell populations with similar characteristics and gene expression patterns (e.g., populations of CD8-positive T cells and CD4-positive T cells). Furthermore, by plotting the expression levels of genes (markers) that are differentially expressed by cell type for each cluster, it is possible to estimate the function and importance of each gene.

[0035] In one embodiment, the presence or absence of activation is determined based on the level of expression of a signature gene of CD8-positive T cells or its expression product in a sample.

[0036] "Signature gene," as used herein in the context of CD8-positive T cells, refers to a gene specific to CD8-positive T cells or a gene that is statistically significantly expressed in CD8-positive T cells compared to other cells. The significance level may be, for example, a P value of 0.05 or less and a log2FC of greater than 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, or 0.95. Expression products of signature genes include polynucleotides such as mRNA, transcription products such as cDNA synthesized from mRNA, translation products such as proteins, and fragments thereof.

[0037] Signature genes of CD8-positive T cells can be identified by evaluating differential expression of genes in cells of healthy individuals and myasthenia gravis patients using known techniques such as single-cell analysis. For example, single-cell analysis can be used to evaluate the proportion of cells in which a gene is expressed, and a gene expressed in a greater proportion of CD8-positive T cells can be determined as a signature gene.

[0038] Signature genes of CD8-positive T cells are ABHD17A, AC004687.1, ACTB, ACTG1, ADGRG1, AL138963.3, ANXA2, ANXA6, AREG, ARHGDI B, ARPC1B, ARPC5L, B2M, BCL3, BIN2, BTG1, BTG2, BTN3A2, CALR, CAPZB, CCL4, CCL5, CCND3, CD27, CD3D, CD3E, CD5, CD52, CD6, CD63, CD69, CD7, CD74, CD8A, CD8B, CEBPD, CFL1, CIR1, CLIC1, CLIC3, CMC1, CORO1A, CRIP1, CST7, CTSW, CXCR3, CXCR4, CYBA, CYTIP, DDIT4, DD X3Y, DD X5, DNAJA1, DNAJB1, DUSP1, DUSP2, DUSP5, EEF1G, EFHD2, EIF1, EML4, EVL, FCGR3A, FCRL6, FGFBP2, FLNA, FOS, FTH1, GGNB P2, GIMAP4, GIMAP7, GSTP1, GZMA, GZMH, GZMK, HCST, HINT1, HIST1H4C, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DRA, HLA-DRB1, HNRNPA2B1, HNRNPDL, HOPX, ID2, IFIT2, IFITM1, IFITM2, IL32, IL7R, IRF1, ITGB2, ITGB7, JUN, JUNB, JUND, KIR2DL3, KLF2, KLF6, KLRB1, KLR C1, KLR D1, LCP!1, LEF1, LGALS1, LIN C02446, LMNA, LSP1, LTB, LY6E, MAP3K8, MT2A, MT-ATP6, MT-ATP8, MT-CO1, MT-CYB, MT-ND4L, MT-ND5, MT-ND6, MTRNR!2L12, MYADM, MYL12A, MYL6, NCR3, NFKB1, NFKBI A, NKG7, NR4A2, OAZ1, PFN1, PIK3R1, PMAIP1, PPDPF, PPP1CA, PPP1R15A, PRDM1, PRELID1, PRF1, PSME1, PSME2, PTPRC, RAC2, RBM39, REL, RELB, RGS1, RORA, RPL10, RPL17, RPL34, RPL36, RPL37, RPL38, RPL39, RPL41, RPLP0, RPLP1, RPS10, RPS13, RPS14It may be selected from the group consisting of RPS21, RPS26, RPS27, RPS28, RPS29, RPS4Y1, S100A4, S100A6, S1PR1, SBDS, SH3BGRL3, SLC2A3, SNRPA1, SOCS3, SPOCK2, SPON2, SRGN, STK17A, TCF7, TENT5C, TLN1, TMSB10, TMSB4X, TNFAIP3, TRBC2, TRGV10, TRGV2, TSC22D3, TXNIP, TYROBP, UCP2, VIM, WHAMM, YPEL5, ZEB2, ZFP36, ZFP36L1 and ZFP36L2.

[0039] In one embodiment, the signature genes of CD8-positive T cells are DUSP2, ZFP36, HLA-DRB1, MTRNR2L12, IL32, GZMA, ACTB, IFITM2, ZFP36L2, GZMH, FCGR3A, CXCR4, TXNIP, MT-ATP6, IFITM1, CD8B, PFN1, CD8A, HCST, CD74, LINC02446, S100A6, HLA-DPA1, HLA-DPB1, JUNB, LGALS1, CYBA, HOPX, ANXA6, HLA-DRA, CFL1, MT-ND4L, NKG7, CD63 , FGFBP2, FLNA, CD3D, CTSW, S100A4, TMSB4X, PRDM1, DDIT4, UCP2, AL138963 .3, ACTG1, ABHD17A, LSP1, CCND3, CCL4, CD52, ARPC1B, PPP1CA, CCL5, CORO1 A, CLIC3, HLA-C, TRGV2, TRBC2, LCP1, RAC2, DUSP1, EVL, B2M, SH3BGRL3, CLI C1, ITGB7, CRIP1, MYL6, ANXA2, CALR, OAZ1, PRF1, KIR2DL3, CD7, AREG, TRGV 10, PSME2, ​​CST7, FCRL6, SPON2, PSME1, KLRD1, MT-ND6, PTPRC, CD3E, MYL12A , CAPZB, TYROBP, PRELID1, TLN1, MT-CYB, GSTP1, KLRC1, ADGRG1, MT2A, ID2, NCR3, BIN2, HNRNPA2B1, TSC22D3, VIM, ARHGDIB, RPS14, DDX3Y, GGNBP2, SNR PA1, RPL34, REL, YPEL5, RPL39, DNAJA1, ZFP36L1, ARPC5L, RPL37, RPS27, HI and one or more selected from the group consisting of ST1H4C, RPLP0, MYADM, SBDS, RPS10, CIR1, SPOCK2, RELB, WHAMM, IRF1, S1PR1, STK17A, CD6, RBM39, PPP1R15A, RPS21, KLF6, EML4, LMNA, CD5, BCL3, RPS28, BTN3A2, IFIT2, JUN, RPL10, RPL17, GIMAP7, EIF1, EF1G, FTH1, MT-ATP8, RPS29, IL7R, PMAIP1, JUND, NFKBIA, RPS4Y1, and RPS26.

[0040] In one embodiment, the signature genes of CD8-positive T cells are DUSP2, MTRNR2L12, ZFP36, JUNB, MT-ATP6, CXCR4, DUSP1, GZMH, ZFP36L2, FGFBP2, NKG7, S100A4, IFITM2, HLA-DRB1, TXNIP, FCGR3A, CD8B, AL138963.3, TSC22D3, CD8A, SPON2, GZMA, I L32, KLRD1, CD74, HLA-DPA1, TRBC2, PFN1, S100A6, CFL1, CTSW, SH3BGRL3, HCST, CD69, CCND3, ANXA6, RPL41, IFITM1, FLNA, CCL4, HLA-DPB1, MT-ND5, ACTB, B2M, HOPX, DDIT4, EFHD2, DNAJB1, ABHD17A, CST7, TMSB10, MT-N D4L, PRDM1, FCRL6, HLA-C, LSP1, MT-CYB, ITGB2, LY6E, ZEB2, TMSB4X, CD52, CAPZB, ID2, MYL12A, LCP1, PPDPF , SRGN, MT2A, CD3D, BTG2, CMC1, RPL36, LEF1, RPL38, CD27, NFKB1, RPS13, RPL10, RPLP1, HINT1, RPL34, TCF7, R and one or more selected from the group consisting of PL37, HIST1H4C, RPS28, RPS27, EML4, CXCR3, NFKBIA, RPLP0, RPL39, RPS21, STK17A, S1PR1, GIMAP4, IL7R, BTN3A2, MT-ATP8, RPL17, FTH1, JUND, LTB, RPS29, JUN, EEF1G, GIMAP7, RPS4Y1, and RPS26.

[0041] In one embodiment, the signature genes of CD8-positive T cells are JUNB, DUSP2, ZFP36, CXCR4, ZFP36L2, CD69, FOS, DUSP1, TSC22D3, TXNIP, MTRNR2L12, DNAJB1, DDIT4, MT-ATP6, KLF2, SLC2A3, IFITM 1, BTG1, NR4A2, CD74, TMSB10, CCND3, AL138963.3, MT-ND4L, HLA-DRB1, IL32, CCL4, TNFAIP3, HLA-C, TENT5C, RGS1, DUSP5, CYTIP, BTG2, GZMH, MT-ND5, TMSB4X, MT-CYB, S OCS3, CD8B, PPP1R15A, PFN1, IFITM2, VIM, CFL1, MT-CO1, HLA-DPA1, GZMK, DDX5, CRI P1, PIK3R1, MAP3K8, B2M, AREG, HLA-DPB1, HNRNPDL, CMC1, RPL34, AC004687.1, RPL38 , RPL10, IL7R, HIST1H4C, RORA, RPL37, FTH1, RPL39, PRF1, GIMAP4, BTN3A2, S1PR1, RPS27, RPS21, CEBPD, GIMAP7, RPS29, KLRB1, RPS4Y1 and RPS26.

[0042] In one embodiment, the signature genes are ACTB, ACTG1, ADGRG1, AL138963.3, ANXA2, ANXA6, AREG, ARHGDIB, ARPC1B, B2M, BIN2, BTG1, BTG2, CALR, CAPZB, CCL4, CCL5, CCND3, CD3D, CD3E, CD52, CD63, CD69, CD7, CD74, CD8A, CD8B, CFL1, CLIC1, CLIC3, CMC1, CORO1A, CRIP1, CST7, CTSW, CXCR4, CYBA, CYTIP, DDIT4, DDX5, DNAJB1, DUSP1, DUSP2, DUSP5, EFHD2, EVL, FCGR3A, FCRL6, FGFBP2, FLNA, FOS, GS TP1, GZMA, GZMH, GZMK, HCST, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DRA, HLA-DRB1, HNRNPA2B1, HNRNPDL, HOPX, ID2, IFITM1, IFITM 2, IL32, ITGB2, ITGB7, JUNB, KIR2DL3, KLF2, KLRC1, KLRD1, LCP1, LGALS1, LINC02446, LSP1, LY6E, MAP3K8, MT2A, MT-ATP6, MT -CO1, MT-CYB, MT-ND4L, MT-ND5, MT-ND6, MTRNR2L12, MYL12A, MYL6, NCR3, NKG7, NR4A2, OAZ1, PFN1, PIK3R1, PPDPF, PPP1CA, PR DM1, PRELID1, PSME1, PSME2, ​​PTPRC, RAC2, RGS1, RPL41, S100A4, S100A6, SH3BGRL3, SLC2A3, SOCS3, SPON2, SRGN, TENT5C, TLN1, TMSB10, TMSB4X, TNFAIP3, TRBC2, TRGV10, TRGV2, TSC22D3, TXNIP, TYROBP, UCP2, VIM, ZEB2, ZFP36 and ZFP36L2.Signature genes may be multiple combinations of these genes, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 10, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 or 131 combination.

[0043] The signature gene is preferably one involved in the activation or cytotoxicity of CD8-positive T cells. Proteins encoded by genes involved in activation are generally known to increase in expression upon T cell activation. Among these activation proteins, those that have direct cytotoxicity are classified as cytotoxic proteins.

[0044] In one embodiment, the genes involved in activation are ZFP36, ZFP36L2, IL-32, IFITM2, CXCR4, IFITM1, CD74, CCL4, CCL5, JUNB, ITGB7, and the genes associated with cytotoxicity are GZMA, GZMH, NKG7, CTSW, PRF1.

[0045] In one embodiment, genes involved in activation are CXCR4, S100A4, IFITM2, IL-32, CD69, IFITM1, CCL4, JUNB, ZFP36L2, CD74, ITGB2, and genes associated with cytotoxicity are GZMH, NKG7, GZMA, CTSW.

[0046] In one embodiment, the genes involved in activation are CXCR4, CD69, IFITM1, IL-32, CCL4, IFITM2, JUNB, ZFP36L2, FOS, KLF2, CD74, and TNFAIP3, and the genes associated with cytotoxicity are GZMH and GZMK.

[0047] The signature gene may be a gene expressed in early activated CD8-positive T cells. CD69 is known to be a marker indicating the early activation of immune cells and the like (Stem Cells, Volume 12, Issue 5, 1994, Pages 456-465, doi: 10.1002 / stem.5530120502). As used herein, CD8-positive T cells expressing CD69 are considered to be early activated CD8-positive T cells, and CD8-positive T cells that have lost CD69 expression are considered to be late activated CD8-positive T cells.

[0048] In one embodiment, the late activated genes are DUSP2, ZFP36, HLA-DRB1, MTRNR2L12, IL32, GZMA, ACTB, IFITM2, ZFP36L2, GZMH, FCGR3A, CXCR4, TXNIP, MT-ATP6, IFITM1, CD8B, PFN1, CD8A, HCST, CD74, LINC02446, S100A6, HLA-DPAl, HLA-DPBl, JUNB, LGALS1, CYBA, HOPX, ANXA6, HLA-DRA, CFL1, MT-ND4L, NKG7, CD63, FGFB P2, FLNA, CD3D, CTSW, S100A4, TMSB4X, PRDM1, DDIT4, UCP2, AL138963.3, A CTG1, ABHD17A, LSP1, CCND3, CCL4, CD52, ARPC1B, PPP1CA, CCL5, CORO1A, CL IC3, HLA-C, TRGV2, TRBC2, LCP1, RAC2, DUSP1, EVL, B2M, SH3BGRL3, CLIC1, I TGB7, CRIP1, MYL6, ANXA2, CALR, OAZ1, PRF1, KIR2DL3, CD7, AREG, TRGV10, P SME2, ​​CST7, FCRL6, SPON2, PSME1, KLRD1, MT-ND6, PTPRC, CD3E, MYL12A, CA PZB, TYROBP, PRELID1, TLN1, MT-CYB, GSTP1, KLRC1, ADGRG1, MT2A, ID2, NCR 3, BIN2, HNRNPA2B1, TSC22D3, VIM, ARHGDIB, RPS14, DDX3Y, GGNBP2, SNRPA 1, RPL34, REL, YPEL5, RPL39, DNAJA1, ZFP36L1, ARPC5L, RPL37, RPS27, HIST and one or more selected from the group consisting of 1H4C, RPLP0, MYADM, SBDS, RPS10, CIR1, SPOCK2, RELB, WHAMM, IRF1, S1PR1, STK17A, CD6, RBM39, PPP1R15A, RPS21, KLF6, EML4, LMNA, CD5, BCL3, RPS28, BTN3A2, IFIT2, JUN, RPL10, RPL17, GIMAP7, EIF1, EF1G, FTH1, MT-ATP8, RPS29, IL7R, PMAIP1, JUND, NFKBIA, RPS4Y1, and RPS26.

[0049] In one embodiment, the genes that are activated at metaphase are DUSP2, MTRNR2L12, ZFP36, JUNB, MT-ATP6, CXCR4, DUSP1, GZMH, ZFP36L2, FGFBP2, NKG7, S100A4, IFITM2, HLA-DRB1, TXNIP, FCGR3A, CD8B, AL138963.3, TSC22D3, CD8A, SPON2, GZMA, IL32, K LRD1, CD74, HLA-DPA1, TRBC2, PFN1, S100A6, CFL1, CTSW, SH3BGRL3, HCST, CD69, CCND3, ANXA6, RPL41, IFIT M1, FLNA, CCL4, HLA-DPB1, MT-ND5, ACTB, B2M, HOPX, DDIT4, EFHD2, DNAJB1, ABHD17A, CST7, TMSB10, MT-ND4L , PRDM1, FCRL6, HLA-C, LSP1, MT-CYB, ITGB2, LY6E, ZEB2, TMSB4X, CD52, CAPZB, ID2, MYL12A, LCP1, PPDPF, S RGN, MT2A, CD3D, BTG2, CMC1, RPL36, LEF1, RPL38, CD27, NFKB1, RPS13, RPL10, RPLP1, HINT1, RPL34, TCF7, RP L37, HIST1H4C, RPS28, RPS27, EML4, CXCR3, NFKBIA, RPLP0, RPL39, RPS21, STK17A, S1PR1, GIMAP4, IL7R, BTN3A2, MT-ATP8, RPL17, FTH1, JUND, LTB, RPS29, JUN, EEF1G, GIMAP7, RPS4Y1 and RPS26.

[0050] In one embodiment, the early activating genes are JUNB, DUSP2, ZFP36, CXCR4, ZFP36L2, CD69, FOS, DUSP1, TSC22D3, TXNIP, MTRNR2L12, DNAJB1, DDIT4, MT-ATP6, KLF2, SLC2A3, IFITM1, BTG1 , NR4A2, CD74, TMSB10, CCND3, AL138963.3, MT-ND4L, HLA-DRB1, IL32, CCL4, TNFAI P3, HLA-C, TENT5C, RGS1, DUSP5, CYTIP, BTG2, GZMH, MT-ND5, TMSB4X, MT-CYB, SOCS3 , CD8B, PPP1R15A, PFN1, IFITM2, VIM, CFL1, MT-CO1, HLA-DPA1, GZMK, DDX5, CRIP1, PIK3R1, MAP3K8, B2M, AREG, HLA-DPB1, HNRNPDL, CMC1, RPL34, AC004687.1, RPL38, RPL10, IL7R, HIST1H4C, RORA, RPL37, FTH1, RPL39, PRF1, GIMAP4, BTN3A2, S1PR1, RPS27, RPS21, CEBPD, GIMAP7, RPS29, KLRB1, RPS4Y1 and RPS26.

[0051] In one embodiment, the signature genes are one or more selected from the group consisting of GZMA, GZMH, NKG7, CTSW, PRF1 and GZMK.

[0052] In one embodiment, the genes expressed in early activated CD8 positive T cells are one or more selected from the group consisting of CXCR4, CD69, IFITM1, IL32, CCL4, IFITM2, JUNB, ZFP36L2, FOS, KLF2, CD74 and TNFAIP3.

[0053] In one embodiment, the genes expressed in late activated CD8 positive T cells are one or more selected from the group consisting of IL32, IFITM2, CXCR4, IFITM1, CCL4, CCL5, ZFP36L2, CD74, JUNB, and TNFAIP3.

[0054] The analysis method can be used for the diagnosis and treatment of myasthenia gravis. Diagnosis includes determining the type of myasthenia gravis, determining the subsequent treatment plan, and predicting the prognosis.

[0055] In one embodiment, the analysis method includes a step of evaluating that the treatment is effective in treating the subject when activated CD8-positive T cells are reduced compared to before the treatment.

[0056] In one embodiment, the analysis method includes a step of assessing that, when the number of activated CD8-positive T cells is higher than in a healthy subject, the subject is affected with myasthenia gravis and that a treatment targeting CD8-positive T cells, a signature gene of the cells or an expression product thereof, or a factor that activates CD8-positive T cells, or a molecule whose expression is enhanced in activated CD8-positive T cells is effective.

[0057] The analytical method plays an auxiliary role in helping doctors and others diagnose myasthenia gravis. When used to diagnose myasthenia gravis, the analytical method can be performed in combination with known diagnostic methods. Such diagnostic methods include blood tests using anti-acetylcholine receptor (AChR) antibodies, anti-muscle-specific receptor tyrosine kinase (MuSK) antibodies, LDL receptor-related protein 4 (Lrp4) antibodies, etc. as indicators, as well as thyroid function tests, electromyography, etc.

[0058] For clinical evaluation of myasthenia gravis, the MGFA clinical classification for assessing severity, the QMG (quantitative MG) score, the MG-ADL (activities of daily living) scale for scoring the state of daily living, and the like are used.

[0059] Patients determined to have myasthenia gravis may be treated with a therapy targeting CD8-positive T cells, the signature gene or its expression product of the cells, or a factor that activates CD8-positive T cells, a therapy targeting a molecule whose expression is increased in activated CD8-positive T cells, or other known therapeutic methods. Examples of therapeutic methods for myasthenia gravis include methods using drugs such as anticholinesterase inhibitors such as mestinon and mytelase, corticosteroids, and immunosuppressants such as tacrolimus and cyclosporine, intravenous immunoglobulin injection, blood purification therapy, and extended thymectomy.

[0060] Treatment for myasthenia gravis is decided by a physician or other appropriate professional depending on the symptoms, type, severity, etc. of the disease. For example, in cases with thymoma, extended thymectomy is generally the first choice of treatment. In cases without thymoma, thymectomy is applicable only to patients who are anti-AChR antibody positive, who have systemic disease, and who have had the disease for less than five years. Thymectomy is not recommended for patients who are anti-MuSK antibody positive.

[0061] In the ocular muscle type, where weakness and fatigue of the eye muscles are limited, cholinesterase inhibitors may be used to monitor the progress, but in cases where this is ineffective, steroid drugs may be used. For rapid relief, steroid pulse therapy may be administered intermittently.

[0062] In the case of the systemic type, in which symptoms affect skeletal muscles throughout the body, including the limb muscles and trunk muscles, steroid therapy drugs and immunosuppressants are used in combination.

[0063] In refractory cases or acute exacerbations, blood purification therapy, high-dose immunoglobulin therapy, and steroid pulse therapy may be performed.

[0064] The therapeutic effect is evaluated according to the MGFA postintervention status index and classified into complete remission, pharmacological remission, minor symptoms, improvement, unchanged, worsening, related death, etc.

[0065] (Marker) In a second aspect, a marker for treating, preventing, or diagnosing myasthenia gravis, comprising a signature gene of CD8-positive T cells or an expression product thereof, is provided.

[0066] The type of marker is not particularly limited, and may be a diagnostic marker, a prognostic marker, a pharmacodynamic marker, a predictive marker, a monitoring marker, etc. Markers can also be used to classify a cell population containing CD8-positive T cells into specific subsets.

[0067] In one embodiment, the CD8-positive T cell signature gene or its expression product is a surrogate marker for assessing the efficacy of a therapy targeting CD8-positive T cells.

[0068] In one embodiment, the CD8-positive T cell signature gene or its expression product is a biomarker for selecting whether a therapy targeting CD8-positive T cells will be effective.

[0069] In one embodiment, the CD8+ T cell signature genes or their expression products are surrogate markers for assessing the efficacy of treatments targeting early and late CD8+ T cells.

[0070] In one embodiment, the CD8-positive T cell signature gene or its expression product is a biomarker for selecting whether a treatment targeting early or late CD8-positive T cells will be effective.

[0071] The above markers can be used in combination with known markers of myasthenia gravis, such as autoantibodies (AChR antibodies, MuSK antibodies, etc.).

[0072] (Pharmaceutical Composition) In a third embodiment, there is provided a pharmaceutical composition for treating or preventing myasthenia gravis, the pharmaceutical composition comprising an active ingredient that targets CD8-positive T cells, a signature gene of the cells or an expression product thereof, or a factor that activates CD8-positive T cells, or a molecule whose expression is enhanced in activated CD8-positive T cells.

[0073] In one embodiment, the CD8 positive T cells are early activated CD8 positive T cells.

[0074] In one embodiment, the CD8 positive T cells are late activated CD8 positive T cells.

[0075] The pharmaceutical composition may be combined with another active ingredient, as long as the function of the active ingredient is not impaired. Examples of such ingredients include components that target CD4-positive T cells, signature genes or expression products thereof, or factors that activate CD4-positive cells, or molecules whose expression is increased in activated CD8-positive T cells, and drugs known as therapeutic agents for myasthenia gravis, such as anticholinesterase inhibitors (e.g., mestinon and mytelase), corticosteroids, and immunosuppressants (e.g., tacrolimus and cyclosporine).

[0076] The pharmaceutical composition may contain pharmaceutically acceptable components such as buffers, stabilizers, preservatives, and other additives. Pharmaceutically acceptable components are well known to those skilled in the art, and can be appropriately selected and used by those skilled in the art within the scope of their ordinary skill in the art, for example, from components described in specifications such as the 18th Revised Japanese Pharmacopoeia, depending on the form of the formulation.

[0077] The present invention will be specifically explained below by showing examples, but the present invention is not limited to these examples.

[0078] Example 1. Identification of CD8+ T Cell Activation in Myasthenia Gravis Patients. Peripheral blood mononuclear cells (PBMCs) were collected from four patients with myasthenia gravis and six healthy individuals. All four patients had clinical signs of myasthenia gravis and had detectable AChR antibodies. Frozen PBMCs were quickly thawed in a 37°C water bath and washed three times with Mg / Ca-free 1x D-PBS containing 0.1% BSA. After resuspension in D-PBS / 0.1% BSA, remaining particles were removed using a 40 μm cell strainer. Cell count and viability were confirmed, and scRNA-seq sequencing libraries were prepared and analyzed according to the following procedure.

[0079] Approximately 10,000 cells were confirmed to have a viability of 80-90%. Gel beads-in-emulsions (GEMs) were generated by incorporating individual cells into a single oil emulsion using the Chromium Controller (10x Genomics) with gel beads containing reverse transcription reagents and barcoded oligonucleotides. Typically, 65% of the cells are incorporated into GEMs. The reverse transcription reaction allows cDNA to be labeled with a tag unique to each single-cell emulsion. cDNA preparation and library construction for single-cell gene expression were performed using the Chromium Next GEM Single Cell 5' Reagent Kits v2 (10x Genomics).

[0080] The resulting libraries were quantified using Qubit dsDNA Assay (ThermoFisher Scientific) and TapeStation D1000 ScreenTape (Agilent Technologies). They were then analyzed on a HiSeq platform (Illumina) using a 150-bp paired-end configuration, generating approximately 350 million paired-end reads per sample. These reads were further trimmed to 26 bp for Read 1 and 90 bp for Read 2.

[0081] For each sample, the resulting NGS data was processed using Cell Ranger (version 6.1.2). Reads were mapped to the human reference genome (GRCh 38), and UMIs were associated with gene and cell barcodes to generate expression profile matrices. These expression profile matrices were then analyzed using Seurat (version 4.1.1), a package for single-cell RNA sequencing data analysis in the statistical software R (version 4.1.3).

[0082] Prior to clustering, cells with low data quality were removed. Cells with fewer than 200 detected genes or mitochondrial gene expression levels exceeding 10% of the total were removed. T cell receptor genes, which were not important for the analysis, were also removed from the expression profile matrix. For the selected cells, UMI counts were normalized using the SCTransform function in Seurat, and genes with high variability were selected. To correct for batch differences between samples, the expression profile matrix was processed using the FindIntegrationAnchors and IntegrateData functions in Seurat. Furthermore, principal component analysis was performed using the RunPCA function in Seurat. Considering the contribution rate of principal components, we retained the first 30 principal components for subsequent analysis. Cell clustering was performed using the FindCluster function in Seurat. The cell type to which each cluster belonged was determined by comparing the expression levels of genes commonly known as cell type marker genes between clusters.

[0083] The resulting clusters were divided into cells derived from healthy individuals and those derived from myasthenia gravis patients, and expression profile analysis was performed on both groups to identify genes with significantly elevated expression levels in myasthenia gravis patient cells. The expression profile matrix was corrected using the PrepSCTFindMarkers function in Seurat, and expression profile analysis was performed using the FindMarker function. A significant increase in expression was defined as a Log2-Fold change of 0.25 and a p-value of 0.05 or less.

[0084] To visualize the results of clustering and annotation, the expression profiles were reduced in dimension using UMAP and plotted as shown in Figure 1. CD8-positive T cells were roughly divided into three clusters.

[0085] Figure 2 shows the expression levels of the marker genes used for annotation by cluster. The color of the circles in Figure 2 represents the logarithm of the average expression level, with the color closer to blue indicating a higher level. The size of the circle also indicates the proportion of cells in which the gene is expressed, with larger circles indicating that the gene is expressed in more cells.

[0086] The gene expression related to cytotoxicity was examined for CD8+ T cells divided into three clusters. The results are shown in Figure 3. The horizontal axis in the figure represents the logarithm of the average expression level. As a result, increased expression of GZMA, GZMH, NKG7, CTSW, PRF1, and GZMK was observed in patients with myasthenia gravis, indicating activation.

[0087] The results of evaluating CD8+ T cell activation are shown in the table below. In the table, p_val is the probability of significance, and avg_log2FC is the log2 of the expression variation ratio (MG / HC). Increased expression of GZMA, GZMH, NKG7, CTSW, PRF1, and GZMK was observed in patients with myasthenia gravis, indicating activation.

[0088] The table below shows genes whose expression was altered in CD8-positive T cells. In the table, p_val is the significance probability, and avg_log2FC is the log2 of the expression change ratio (MG / HC).

[0089]

[0090]

[0091] For each cell type, we examined the expression of genes related to cell activation and cytotoxicity. The results are shown in Figure 4. The horizontal axis represents the logarithm of the average expression level. As a result, we found that the expression of certain genes was elevated and activated in patients with myasthenia gravis. In other words, we found that these genes could be used as surrogate markers to evaluate the effectiveness of treatments targeting early and late CD8+ T cells, or as candidate biomarkers to screen for the effectiveness of treatments targeting early and late CD8+ T cells.

[0092] By checking the protein levels from the table above using techniques such as flow cytometry, various markers of CD8-positive T cells (activation markers, early activation markers, etc.) can be determined.

[0093] Currently, molecular targeted drugs for myasthenia gravis mainly target humoral immunity, but the above results suggest that CD8+ T cells, which are involved in cellular immunity, may be a new therapeutic target. Furthermore, since there are currently no surrogate markers for CD8+ T cell therapy, the above genes may be new markers that will be useful in the diagnosis and treatment of myasthenia gravis.

Claims

1. A method for analyzing a sample, comprising a step of determining whether CD8-positive T cells are activated in a sample from a subject suffering from or likely to suffer from myasthenia gravis.

2. The method according to claim 1, wherein the presence or absence of activation is determined based on the level of expression of a signature gene of CD8-positive T cells or its expression product in a sample.

3. The method according to claim 2, wherein the signature gene is a gene related to cytotoxicity and / or activation of CD8-positive T cells.

4. The signature genes are ABHD17A, AC004687.1, ACTB, ACTG1, ADGRG1, AL138963.3, ANXA2, ANXA6, AREG, ARHGDI B, ARPC1B, ARPC5L, B2M, BCL3, BIN2, BTG1, BTG2, BTN3A2, CALR, CAPZB, CCL4, CCL5, CCND3, CD27, CD3D, CD3E, CD5, CD52, CD6, CD63, CD69, CD7, CD74, CD8A, CD8B, CEBPD, CFL1, CIR1, CLIC1, CLIC3, CMC1, CORO1A, CRIP1, CST7, CTSW, CXCR3, CXCR4, CYBA, CYTIP, DDIT4, DD X3Y, DD X5, DNAJA1, DNAJB1, DUSP1, DUSP2, DUSP5, EEF1G, EFHD2, EIF1, EML4, EVL, FCG R3A, FCRL6, FG FBP2, FLNA, FOS, FTH1, GG NBP2, GIMAP4, GIMAP7, GSTP1, GZMA, GZMH, GZMK, HCST, HINT1, HIST1H4C, HLA - C, HLA - DPA1, HLA - DPB1, HLA - DRA, HLA - DRB1, HNRNPA2B1, HNRNPDL, HOPX, ID2, IFIT2, IFITM1, IFITM2, IL32, IL7R, IRF1, ITGB2, ITGB7, JUN, JUNB, JUND, KIR2DL3, KLF2, KLF6, KLRB1, KLR C1, KLRD1, LCP1, LEF1, LGALS1, LIN C02446, LMNA, LSP1, LTB, LY6E, MAP3K8, MT2A, MT - ATP6, MT - ATP8, MT - CO1, MT - CYB, MT - ND4L, MT - ND5, MT - ND6, MTRNR2L12, MYADM, MYL12A, MYL6, NCR3, NFKB1, NFKBIA, NKG7, NR4A2, OAZ1, PFN1, PIK3R1, PMAIP1, PPDPF, PPP1CA, PPP1R15A, PRDM1, PRELID1, PRF1, PSME1, PSME2, PTPRC, RAC2, RBM39, REL, RELB, RGS1, RORA, RPL10, RPL17, RPL34, RPL36, RPL37, RPL38, RPL39, RPL41, RPLP0, RPLP1, RPS10, RPS13, RPS14, RPS21,3. The method of claim 2, wherein the gene is one or more selected from the group consisting of RPS26, RPS27, RPS28, RPS29, RPS4Y1, S100A4, S100A6, S1PR1, SBDS, SH3BGRL3, SLC2A3, SNRPA1, SOCS3, SPOCK2, SPON2, SRGN, STK17A, TCF7, TENT5C, TLN1, TMSB10, TMSB4X, TNFAIP3, TRBC2, TRGV10, TRGV2, TSC22D3, TXNIP, TYROBP, UCP2, VIM, WHAMM, YPEL5, ZEB2, ZFP36, ZFP36L1 and ZFP36L2.

5. The method of claim 4, wherein the signature genes are one or more genes selected from the group consisting of: 1) genes expressed in early activated CD8-positive T cells, which are CXCR4, CD69, IFITM1, IL32, CCL4, and IFITM2; or 2) genes expressed in late activated CD8-positive T cells, which are IL32, IFITM2, CXCR4, IFITM1, CCL4, CCL5, ZFP36L2, CD74, JUNB, and TNFAIP3.

6. The method according to claim 1 or 2, wherein the subject is suffering from myasthenia gravis and is receiving treatment targeting CD8-positive T cells, a signature gene or its expression product of the cells, or a factor that activates CD8-positive T cells, or a molecule whose expression is enhanced in activated CD8-positive T cells.

7. The method according to claim 6, wherein the treatment is evaluated as being effective in treating the subject when activated CD8-positive T cells are decreased compared to before the treatment.

8. The method of claim 1 or 2, wherein the subject is suspected of suffering from myasthenia gravis.

9. The method according to claim 8, wherein, when the number of activated CD8-positive T cells is higher than that of a healthy subject, the subject is evaluated as suffering from myasthenia gravis and a treatment targeting CD8-positive T cells, a signature gene of the cells or an expression product thereof, or a factor that activates CD8-positive T cells, or a molecule whose expression is enhanced in activated CD8-positive T cells is evaluated as being effective.

10. A marker for the treatment, prevention, or diagnosis of myasthenia gravis, comprising a signature gene of CD8-positive T cells or its expression product.

11. A pharmaceutical composition for the treatment or prevention of myasthenia gravis, comprising an active ingredient that targets CD8-positive T cells, a signature gene of the cells or its expression product, or a factor that activates CD8-positive T cells, or a molecule whose expression is enhanced in activated CD8-positive T cells.

12. The pharmaceutical composition according to claim 11, wherein the CD8-positive T cells are early or late activated CD8-positive T cells.