Signalling and downstream effects of TIGIT/CD226/CD96 co-receptor family in human regulatory T cells

TIGIT/CD226/CD96 辅助受体家族在人类调节性 T 细胞中的信号传导和下游效应

• 批准号: 2720565
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2720565
• 关键词: Signalling; downstream; effects; TIGIT; CD226

CD4+FoxP3+ regulatory T cells (Tregs) are essential for balancing responses to pathogens and tolerance. Too little immune control can lead to autoimmunity and excessive control may promote cancer development. Treg functionality is determined by co-receptor engagement and downstream signalling pathways. T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), CD226 and CD96 are a relatively novel co-receptor family expressed on both effector T cells and Tregs and associated with autoimmunity and cancer, but the effect of their expression remains unclear. TIGIT+ Tregs reportedly have superior suppressive function, although it is uncertain whether this is true in TIGIT+CD226+ Tregs, since healthy ex vivo Tregs tend to be TIGIT+CD226-. CD226 on CD8+ T cells and NK cells may be co-stimulatory with potential antitumour effects, but its role on Tregs is less clear; recent work showed greater suppressive function and proliferation in CD226-negative Tregs in both humans and mice, but other work found increased anti-inflammatory IL-10 production in CD226+TIGIT- Tregs. Most CD4+ T cells express CD96, but its function remains unclear. Each co-receptor binds CD155 but with different affinity (TIGIT 3.15nm; CD96 37.6nm; CD226 119nm), thus there could be ligand-binding competition regulating downstream signalling. There is growing interest in this co-receptor family's potential to moderate immune function, similarly to how co-inhibitory CTLA4-Ig is used in treating arthritis: recombinant molecules or antibodies can dampen inflammation or activate regulatory mechanisms to alleviate disease. Publications suggest some of these co-receptors to modulate TCR signalling in thymocytes and CD8+ T cells. Therefore, altered expression of the TIGIT/CD226/CD96 axis may disrupt downstream stimulatory/inhibitory signalling.We will use anti-co-receptor antibodies, CRISPR knock-outs and lentiviral overexpression to investigate the roles of these co-receptors in T cells/Tregs. Spectral flow cytometry will be used to analyse transcription factor and activation-associated protein expression and phospho-flow to assess key signalling proteins. In preliminary rotation work we saw increased pPKC0 upon TCR/CD28 activation in Tregs versus CD4+ T cells, but this was reduced with anti-TIGIT, suggesting TIGIT may alter signalling upstream of PKC0 phosphorylation. It will be of interest to assess phosphorylation of downstream proteins (e.g. JNK) under the same conditions to determine which pathways could be affected by TIGIT engagement. Moreover, potential signalling cascades downstream of CD226 and CD96, and how the three co-receptors might interact/compete for signalling cascades will be assessed. Phospho-flow results may be confirmed by western blot analysis and specific co-receptor mutants in cell line models. RNA-sequencing and single-cell multiome (RNA-seq, ATAC-seq) data from primary Tregs with co-receptor knock-out stimulated with/without ligand availability will also be analysed to assess downstream gene activation and functionality. These data will lead to further functional assessment of primary human Treg subsets.Ultimately this work will lead to the identification of the signalosome and functional outcome of Treg subpopulations downstream of TIGIT/CD226/CD96 compared to conventional T cell subsets. Our findings could further inform us of Treg co-receptor/signalling alterations that can result in dysfunction. We may be able to translate this to autoimmune diseases which display reduced Treg function to further clarify possible disease pathogenesis and potentially highlight future therapeutic avenues.

CD4+ FOXP3+调节T细胞（Tregs）对于平衡对病原体和耐受性的反应至关重要。免疫控制太少会导致自身免疫性，过度控制可能会促进癌症的发展。 Treg功能是通过受体接合和下游信号通路确定的。具有免疫球蛋白和ITIM结构域（Tigit），CD226和CD96的T细胞免疫受体是一个相对新颖的共受体家族，在效应T细胞和Tregs上表达，并且与自身免疫和癌症有关，但其表达的效果仍然不清楚。据报道，Tigit+ Treg具有出色的抑制功能，尽管尚不确定在Tigit+ CD226+ Treg中是否正确，因为健康的离体Tregs倾向于Tigit+ CD226-。 CD8+ T细胞和NK细胞上的CD226可能与潜在的抗肿瘤作用共同刺激，但其在Tregs上的作用尚不清楚。最近的工作显示在人类和小鼠的CD226阴性Treg中表现出更大的抑制作用和增殖，但其他工作发现CD2226+Tigit-Tregs的抗炎IL-10产生增加了。大多数CD4+ T细胞表达CD96，但其功能尚不清楚。每个共受体结合CD155，但具有不同的亲和力（Tigit 3.15nm; CD96 37.6nm; CD226 119nm），因此可能会有调节下游信号的配体结合竞赛。与共同抑制性的CTLA4-Ig如何用于治疗关节炎的方式相似：重组分子或抗体可能会衰减炎症或激活调节机制以减轻调控机制以减轻疾病。出版物建议其中一些共受体，以调节胸腺细胞和CD8+ T细胞中的TCR信号传导。因此，TIGIT/CD226/CD96轴的表达改变可能会破坏下游刺激/抑制性信号。我们将使用抗CO受体抗体，CRISPR敲除和慢病毒过表达来研究这些共肽在T细胞中的作用Tregs。光谱流式细胞仪将用于分析转录因子和激活相关的蛋白质表达和磷酸流以评估关键信号蛋白。在初步旋转工作中，我们看到TREG与CD4+ T细胞的TCR/CD28激活后PPKC0增加了，但使用抗触发降低了PPKC0，这表明Tigit可能会改变PKC0磷酸化上游的信号传导。在相同条件下评估下游蛋白（例如JNK）的磷酸化以确定哪些途径可能受到Tigit参与的影响，这将是令人感兴趣的。此外，将评估CD226和CD96下游的潜在信号级联，以及如何评估三个共受体相互作用/竞争信号级联反应。可以通过细胞系模型中的蛋白质印迹分析和特定的共受体突变体证实磷酸流的结果。还将分析来自刺激/没有配体可用性的共受体敲除的RNA测序和单细胞多组（RNA-SEQ，ATAC-SEQ）数据，以评估下游基因激活和功能。这些数据将导致对原代人Treg子集进行进一步的功能评估。本工作将导致鉴定Tigit/CD226/CD96下游的Treg亚种群的信号体和功能结果，与常规T细胞亚群相比。我们的发现可以进一步告知我们Treg共受体/信号变化，这可能导致功能障碍。我们也许能够将其转化为自身免疫性疾病，这些疾病显示出降低的Treg功能，以进一步阐明可能的疾病发病机理，并可能突出未来的治疗途径。