Characterisation of porcine T follicular helper cells and their response to vaccination and infection

自2000年被首次发现以来，T滤泡辅助细胞(TFH)的功能一直是一个密集和快速发展的研究主题。现在很明显，这些细胞在协调B细胞/抗体反应方面起着关键作用，它们已经成为改进靶向癌症免疫治疗和疫苗策略的重点。在人类和小鼠中，TFH的特征是表达CXC趋化因子受体5(CXCR5)、诱导性T细胞共刺激因子(ICOS)、程序性细胞死亡蛋白1(PD-1)、T细胞淋巴瘤6(Bcl-6)和白介素21(IL-21)。现在清楚的是，至少有五种表型存在于次级淋巴组织和循环中，每种表型在免疫反应的不同阶段普遍存在，每种表型都表达不同水平的原型Tfh表型标记。值得注意的是，在SARS-CoV-2和HIV感染中，循环Tfh的频率增加分别与疾病严重程度的降低和中和抗体反应的增强相关。对猪的Tfh细胞的研究有限，因为Tfh细胞既是主要的食物生产物种，也是重要的生物医学模型。因此，本项目旨在扩大我们对猪Tfh细胞表型和功能的了解。我们已经证明了几个TFH标记物的跨物种反应性，包括Bcl-6和ICOS，并与Roslin研究所合作，开发了一种用于标记CXCR5表达细胞的试剂。该项目的第一个目标是进一步开发和优化用于流式细胞术和共聚焦显微镜应用的染色面板，以识别和表征血液和次级淋巴器官中的猪Tfh。确定的TFH样细胞群将在功能分析中进行细胞分类和评估，包括评估共培养的同基因B细胞产生抗体的情况。分选的细胞将通过RNAseq转录分析进一步确定其特征。在资金允许的情况下，可以对淋巴器官切片进行空间转录分析，从而确定并说明每个细胞的空间位置。最后，所开发的工具将应用于疫苗接种和感染。例如，评估在感染猪繁殖与呼吸综合征病毒(PRRSV)期间循环和组织驻留的Tfh的频率和表型。TFH反应谱将与血清中的CXCL13、IL-21和IL-6水平、血液和组织中的病毒载量以及中和抗体反应进行比较，以确定是否存在关联。

Since they were first discovered in 2000, the function of T follicular helper (Tfh) cells has been a subject of intense and fast-moving research. It is now clear that these cells are critical in orchestrating the B cell/antibody response and they have become a focus in improving targeted cancer immunotherapy and vaccine strategies. In humans and mice, Tfh are characterised by expression of CXC chemokine receptor 5 (CXCR5), inducible T cell costimulatory (ICOS), programmed cell death protein 1 (PD-1), T-cell lymphoma 6 (Bcl-6) and interleukin 21 (IL-21). It is now clear that at least five phenotypes exist across secondary lymphoid tissues and in circulation, each prevalent at different stages of the immune response and each expressing different levels of the archetypal Tfh phenotypic markers. Notably, in both SARS-CoV-2 and HIV infection an increased frequency of circulating Tfh correlates with decreased disease severity and enhanced neutralising antibody responses, respectively.There are limited studies on Tfh cells in the pig, which is both a major food producing species and an important biomedical model. This project therefore intends to expand our knowledge of the phenotype and function of porcine Tfh cells. We have demonstrated cross-species reactivity of several Tfh markers, including Bcl-6 and ICOS, and in collaboration with the Roslin Institute, developed a reagent for labelling of CXCR5-expressing cells. The first aim of the project is to furtherdevelop and optimise staining panels for both flow cytometry and confocal microscopy applications to identify and characterise porcine Tfh in blood and secondary lymphoid organs. Defined Tfh-like populations will be cell sorted and assessed in functional assays involving assessment of antibody production from co-cultured syngeneic B cells. Sorted cells will be further characterised by RNAseq transcriptomic analysis. Funding permitting, a spatial transcriptomic analysis, whereby the spatial location of each cell is determined and accounted for, may be performed on lymphoid organ sections. Finally, the tools developed will be applied in the context of vaccination and infection. For example, assessing the frequency and phenotype of circulating and tissue resident Tfh during infection with the porcine reproductive and respiratory syndrome virus (PRRSV). Tfh response profiles will be compared to CXCL13, IL-21 and IL-6 levels in serum, virus loads in blood and tissues and neutralising antibody responses to determine whether associations exist.