Understanding Immune Dysregulation in Tylosis with Oesophageal Cancer

泰病是一种罕见的局灶性非表皮松解性掌足底角化病，于18801年首次被描述。随后，伤寒被分为两组；A型患者通常在7岁或8岁时出现角化过度，B型患者在出生后第一年出现角化过度。重要的是，B型被认为是良性的3，而A型与发展为食管鳞状细胞癌（OSCC）的高终生风险相关，并被单独归类为tylosis with oesophageal cancer (TOC[OMIM 148500])4。TOC是一种常染色体显性遗传性状，具有完全外显率，并定位于染色体17q255上42.5 kb的区域，Blaydon等人在3个不同的家族中发现了RHBDF2基因内与TOC相关的错义突变4。随后，其他TOC家族被鉴定出具有新的RHBDF2突变，这些突变仅聚集在几个氨基酸之间，并在表1中与尚未确定突变状态的家系一起描述。假设功能获得的TOC RHBDF2突变导致免疫失调和随后的上皮异常增生和角化细胞过度增生。主要目的：表征外周和组织内TOC的免疫表型，特别关注作为临床相关疾病部位的食道和皮肤。次要目的阐明TOC中免疫失调的机制，并揭示这是TOC RHBDF2突变的直接或间接后果。结果和未来的工作TOC患者的外周血已被很好地表征，CD8+TEMRA细胞的扩增已被观察到。然而，这种表型的功能相关性和机制基础尚未探索。在短期内，这项工作将在两种RHBDF2小鼠模型中扩展；敲除和TOC功能获得敲入。利用流式细胞术对这些小鼠的淋巴器官（胸腺、脾脏、淋巴结）进行充分免疫分型，观察firhom2对免疫发育和外周免疫室的影响，以及在人类样本中观察到的cd8 + TEMRA扩增是否在小鼠中复制。其次，计算分析发现TOC食管发育不良活检中肥大细胞和m2巨噬细胞数量升高，这表明存在2型免疫反应21。需要注意的是，CIBERSORT和xCell输出之间存在一些患者之间的差异；因此，这些发现需要验证。在TOC皮肤和食管组织切片中，主要计划对肥大细胞、M2巨噬细胞和其他2型相关免疫细胞（如Th2细胞）进行免疫荧光染色。作为TOC的临床相关部位，观察食管和皮肤之间是否存在免疫表型差异将是一件有趣的事情。此外，将对RHBDF2敲除和TOC敲入小鼠模型的皮肤和食管切片进行流式细胞术和免疫荧光染色。

Tylosis is a rare focal non-epidermolytic palmoplantar keratoderma, first described in 18801.Subsequently, tylosis has been stratified into two separate groups; type A which typicallypresents with hyperkeratosis by the ages of 7 or 8, and type B which presents withhyperkeratosis during the first year of life2. Importantly, type B is considered benign3, whereastype A is associated with a high lifetime risk of developing oesophageal squamous cellcarcinoma (OSCC) and is separately classified as tylosis with oesophageal cancer (TOC[OMIM 148500])4.TOC is inherited as an autosomal dominant trait with complete penetrance and followingmapping to a 42.5 kb region on chromosome 17q255, Blaydon et al. identified missensemutations within the RHBDF2 gene to be associated with TOC in 3 separate families4.Subsequently, other TOC families have been identified with novel RHBDF2 mutations thatcluster only a few amino acids apart and are described in Tab.1 alongside pedigrees with yetundefined mutational status.HypothesisThe gain-of-function TOC RHBDF2 mutation leads to immune dysregulation and subsequentepithelial dysplasia and keratinocyte hyperproliferation.Primary AimTo characterise the immune phenotype of TOC in the periphery and within the tissue, with aparticular focus on the oesophagus and skin as clinically relevant sites of disease.Secondary AimTo elucidate the mechanisms that underpin immune dysregulation in TOC and unravelwhether this is a direct or indirect consequence of the TOC RHBDF2 mutation.Results and Future WorkThe peripheral blood of TOC patients has been well characterised and an expansion of CD8+TEMRA cells has been observed. However, the functional relevance and mechanistic basis forthis phenotype is yet to be explored.In the immediate term, this work will be extended in two RHBDF2 mouse models; knock-outand TOC gain-of-function knock in. Using flow cytometry, the lymphoid organs (thymus,spleen, lymph nodes) of these mice will be fully immunophenotyped to observe the effects ofiRhom2 on immune development and the peripheral immune compartment, and whether theCD8+ TEMRA expansion observed in human samples is replicated in mice.Secondly, computational analyses have identified elevated numbers of mast cells and M2macrophages in TOC dysplastic oesophageal biopsies, which is indicative of a type 2 immuneresponse21. It's prudent to note that there were some patient by patient discrepancies betweenthe CIBERSORT and xCell outputs; therefore, these findings require validation.Predominantly, immunofluorescence staining for mast cells, M2 macrophages and other type2-related immune cells such as Th2 cells is planned in a bank of TOC skin and oesophagealtissue sections. As the clinically relevant sites of TOC, it will be interesting to observe whetherthere are immunophenotypic differences between the oesophagus and skin. Additionally, bothflow cytometry and immunofluorescence staining will be performed on both skin andoesophageal sections from both the RHBDF2 knock-out and TOC knock-in mouse models.