Septins are cytoskeletal proteins with filament forming capabilities, which have multiple roles during cell division, cellular polarization, morphogenesis, and membrane trafficking. Autoantibodies against septin-5 are associated with non-paraneoplastic cerebellar ataxia, and autoantibodies against septin-7 with encephalopathy with prominent neuropsychiatric features. Here, we report on newly identified autoantibodies against septin-3 in patients with paraneoplastic cerebellar ataxia. We also propose a strategy for anti-septin autoantibody determination. Sera from three patients producing similar immunofluorescence staining patterns on cerebellar and hippocampal sections were subjected to immunoprecipitation followed by mass spectrometry. The identified candidate antigens, all of which were septins, were expressed recombinantly in HEK293 cells either individually, as complexes, or combinations missing individual septins, for use in recombinant cell-based indirect immunofluorescence assays (RC-IIFA). Specificity for septin-3 was further confirmed by tissue IIFA neutralization experiments. Finally, tumor tissue sections were analyzed immunohistochemically for septin-3 expression. Immunoprecipitation with rat cerebellum lysate revealed septin-3, -5, -6, -7, and -11 as candidate target antigens. Sera of all three patients reacted with recombinant cells co-expressing septin-3/5/6/7/11, while none of 149 healthy control sera was similarly reactive. In RC-IIFAs the patient sera recognized only cells expressing septin-3, individually and in complexes. Incubation of patient sera with five different septin combinations, each missing one of the five septins, confirmed the autoantibodies’ specificity for septin-3. The tissue IIFA reactivity of patient serum was abolished by pre-incubation with HEK293 cell lysates overexpressing the septin-3/5/6/7/11 complex or septin-3 alone, but not with HEK293 cell lysates overexpressing septin-5 as control. All three patients had cancers (2 × melanoma, 1 × small cell lung cancer), presented with progressive cerebellar syndromes, and responded poorly to immunotherapy. Expression of septin-3 was demonstrated in resected tumor tissue available from one patient. Septin-3 is a novel autoantibody target in patients with paraneoplastic cerebellar syndromes. Based on our findings, RC-IIFA with HEK293 cells expressing the septin-3/5/6/7/11 complex may serve as a screening tool to investigate anti-septin autoantibodies in serological samples with a characteristic staining pattern on neuronal tissue sections. Autoantibodies against individual septins can then be confirmed by RC-IIFA expressing single septins.

中文翻译：

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副肿瘤性小脑性共济失调患者的 Septin-3 自身免疫

Septins 是具有细丝形成能力的细胞骨架蛋白，在细胞分裂、细胞极化、形态发生和膜运输过程中具有多种作用。针对 septin-5 的自身抗体与非副肿瘤性小脑性共济失调相关，针对 septin-7 的自身抗体与具有显着神经精神特征的脑病相关。在这里，我们报告了在副肿瘤性小脑性共济失调患者中新发现的针对 septin-3 的自身抗体。我们还提出了一种抗败血症自身抗体测定策略。来自三名患者的血清在小脑和海马切片上产生相似的免疫荧光染色模式，进行免疫沉淀，然后进行质谱分析。鉴定出的候选抗原都是败血症，在 HEK293 细胞中单独、作为复合物或缺少单个败血症的组合表达，用于基于重组细胞的间接免疫荧光测定 (RC-IIFA)。组织 IIFA 中和实验进一步证实了 septin-3 的特异性。最后，对肿瘤组织切片进行免疫组织化学分析以了解 septin-3 的表达。用大鼠小脑裂解物进行的免疫沉淀揭示了 septin-3、-5、-6、-7 和 -11 作为候选靶抗原。所有三名患者的血清都与共表达 septin-3/5/6/7/11 的重组细胞发生反应，而 149 名健康对照血清中没有一人具有类似的反应性。在 RC-IIFA 中，患者血清仅识别单独和复合表达 septin-3 的细胞。用五种不同的败血症组合孵育患者血清，每个缺失五个败血症中的一个，证实了自身抗体对 septin-3 的特异性。患者血清的组织 IIFA 反应性通过与过表达 septin-3/5/6/7/11 复合物的 HEK293 细胞裂解物或单独的 septin-3 预孵育而被消除，但与作为对照的过表达 septin-5 的 HEK293 细胞裂解物没有。这三名患者都患有癌症（2 例黑色素瘤，1 例小细胞肺癌），表现为进行性小脑综合征，并且对免疫疗法反应不佳。septin-3 的表达在一名患者的切除肿瘤组织中得到证实。Septin-3 是副肿瘤性小脑综合征患者的新型自身抗体靶点。根据我们的发现，带有表达 septin-3/5/6/7/11 复合物的 HEK293 细胞的 RC-IIFA 可以作为一种筛选工具来研究在神经元组织切片上具有特征性染色模式的血清学样本中的抗败血症自身抗体。然后可以通过表达单个败血症的 RC-IIFA 来确认针对单个败血症的自身抗体。