2年目である本年度は①昨年実施したシングルセル解析のデータ解析、および②唾液腺障害と関連が報告されていない種類の自己抗体の詳細解析、の2つを進めた。
①に関しては、昨年度実施した解析データでクラスタリングを行い、複数のサブセットを同定し、その各細胞種のマーカー遺伝子/タンパク質となりそうな特異的に発現するタンパク質をリストアップし、免疫染色で確認した。漿液性腺房細胞、粘液性腺房細胞、導管細胞、上皮幹細胞の複数のマーカーの発現が蛍光免疫染色により確認された。また得られたリンパ球のBCR配列を解析し、そこから100種類以上の抗体のV領域を選定し、核酸合成を進めており、来年度に抗体として発現させ反応性を確認し、自己反応性の有無やそれによる遺伝子発現の特徴を検討する予定としている。
②に関して、筋炎、間質性肺炎、皮膚症状、関節症状の４病変が出現する事が知られる抗ARS抗体陽性の患者さんの肺胞洗浄液中のB細胞から抗体を作製し、抗ARS抗体が産生されている事を確認していたが、本研究内で、抗ARS抗体陽性の患者の唾液腺検体２例を偶発的に入手できた。そこから100種類の抗体を作製し解析した所、うち4割の抗体が抗ARS抗体もしくはそれに関連する自己抗体であった。これまで抗ARS抗体陽性患者で唾液腺障害が合併するという報告はなく、唾液腺で抗ARS抗体が産生されている事は想定外の結果であった。これらの抗体はsomatic hypermutationを蓄積して自己抗原への親和性を増しており、また抗原の様々な部位に反応していたことから、肺でも唾液腺でも同様に抗原に依存した自己抗体産生が行われていると考えられた。臨床的には抗ARS抗体症候群患者が口腔乾燥症状を訴えた場合、シェーグレン症候群の合併と判断されていたと思われ、今後どう疾患の口腔乾燥症状につき、唾液腺病変の精査と症例の蓄積を進める。
This year, the second year of the project, we proceeded with two studies: (1) data analysis of the single cell analysis conducted last year, and (2) detailed analysis of autoantibodies of types not reported to be associated with salivary gland disorders.
For (1), clustering was performed on the analysis data conducted last year to identify multiple cell subsets, and a list of specifically expressed proteins that are likely to be marker genes/proteins for each of these cell types was made. The candidates were confirmed by immunostaining. Expression of multiple markers in serous gland cells, mucous gland cells, ductal cells, and stem cells was confirmed by fluorescent immunostaining. The BCR sequences of the obtained lymphocytes were analyzed, and the V regions of more than 100 types of antibodies were selected from them, and nucleic acid synthesis is in progress. In the next year, we plans to produce antibodies and confirm their reactivity, and investigate the presence or absence of autoreactivity and the characteristics of gene expression.
For (2), we had produced antibodies from B cells in alveolar lavage fluid of patients positive for anti-ARS antibodies, which are known to cause four lesions: myositis, interstitial pneumonia, skin symptoms, and joint symptoms, and had confirmed that anti-ARS antibodies were produced in lungs. In this study, two salivary gland specimens from patients with anti-ARS antibody-positive disease were obtained by chance. We generated and analyzed 100 antibodies from them, and found that 40% of the antibodies were anti-ARS antibodies or related autoantibodies. The fact that anti-ARS antibodies are produced in the salivary glands was unexpected, since there have been no reports of salivary gland disorders in anti-ARS antibody-positive patients. These antibodies increased their affinity for self-antigens by accumulating somatic hypermutations, and reacted to various sites of antigens. These results suggest that antigen-dependent production of autoantibodies occurs in the lungs and salivary glands as well. Until now, clinically, when a patient with anti-ARS syndrome complained of xerostomia, it was considered to be a complication of Sjogren's syndrome. We will continue to examine antigen specificity of salivary glands B cells and accumulate cases of xerostomia in this disease.