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

自2000年首次发现以来，T滤泡辅助细胞（Tfh）的功能一直是激烈和快速发展的研究主题。现在清楚的是，这些细胞在协调B细胞/抗体应答中至关重要，并且它们已成为改进靶向癌症免疫疗法和疫苗策略的焦点。在人类和小鼠中，Tfh的特征在于表达CXC趋化因子受体5（CXCR 5）、诱导性T细胞共刺激（ICOS）、程序性细胞死亡蛋白1（PD-1）、T细胞淋巴瘤6（Bcl-6）和白细胞介素21（IL-21）。现在清楚的是，在次级淋巴组织和循环中存在至少五种表型，每种表型在免疫应答的不同阶段普遍存在，并且每种表型表达不同水平的原型Tfh表型标志物。值得注意的是，在SARS-CoV-2和HIV感染中，循环Tfh频率的增加分别与疾病严重程度的降低和中和抗体应答的增强相关。因此，本项目旨在扩大我们对猪Tfh细胞的表型和功能的了解。我们已经证明了几种Tfh标记物的跨物种反应性，包括Bcl-6和ICOS，并与罗斯林研究所合作，开发了一种用于标记CXCR 5表达细胞的试剂。该项目的第一个目的是进一步开发和优化流式细胞术和共聚焦显微镜应用的染色板，以识别和检测猪血液和次级淋巴器官中的Tfh。将对确定的Tfh样群体进行细胞分选，并在涉及评估来自共培养的同基因B细胞的抗体产生的功能测定中进行评估。分选的细胞将通过RNAseq转录组学分析进一步表征。如果资金允许，可以在淋巴器官切片上进行空间转录组学分析，从而确定和说明每个细胞的空间位置。最后，开发的工具将应用于疫苗接种和感染。例如，评估猪繁殖与呼吸综合征病毒（PRRSV）感染期间循环和组织驻留Tfh的频率和表型。将Tfh应答特征与血清中CXCL 13、IL-21和IL-6水平、血液和组织中病毒载量以及中和抗体应答进行比较，以确定是否存在相关性。

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.