Understanding Immune Dysregulation in Tylosis with Oesophageal Cancer

泰勒病是一种罕见的局灶性非表皮炎性掌跖角化病，首次描述于18801年。随后，泰勒病被分为两个独立的组：A型，通常在7或8岁时出现角化过度，B型，在生命的第一年出现角化过度2。重要的是，B型被认为是良性的3，而A型与发生食管鳞状细胞癌（OSCC）的高终生风险相关，并被单独分类为食管癌的侵填体病（TOC[OMIM 148500]）4.TOC是一种常染色体显性遗传性状，具有完全显性，定位于染色体17 q255上的一个42.5kb区域，Blaydon等人在3个独立的家族中鉴定出与TOC相关的RHBDF 2基因内的错义突变。其他TOC家族已被鉴定出具有新的RHBDF 2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。功能TOC RHBDF 2突变导致免疫失调和上皮细胞发育不良以及角质形成细胞过度增殖。特别关注食管和皮肤作为临床相关的疾病部位。次要目的阐明TOC中免疫失调的基础机制，并阐明这是直接还是间接的TOC RHBDF 2突变的结果。结果和未来的工作TOC患者的外周血已经被很好地表征，并且已经观察到CD 8 +TEMRA细胞的扩增。然而，这种表型的功能相关性和机制基础还有待探索。在短期内，这项工作将在两个RHBDF 2小鼠模型中扩展：敲除和TOC功能获得敲入。使用流式细胞术，淋巴器官这些小鼠的胸腺、脾脏、淋巴结）进行完全免疫表型分析，以观察Rhom 2对免疫发育和外周免疫区室的影响，以及在人样品中观察到的CD 8 + TEMRA扩增是否在小鼠中复制。其次，计算分析已经鉴定了TOC发育异常食管活检中肥大细胞和M2巨噬细胞数量的升高，这是2型免疫应答的指示21。值得注意的是，在CIBERSORT和xCell输出之间存在一些患者差异;因此，这些发现需要验证。主要是，计划在TOC皮肤和卵巢组织切片库中对肥大细胞、M2巨噬细胞和其他2型相关免疫细胞（如Th 2细胞）进行免疫荧光染色。食管与皮肤作为TOC的临床相关部位，观察两者之间是否存在免疫表型差异将是一个有趣的研究课题。此外，将对RHBDF 2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.