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.

CD 4 + FoxP 3+调节性T细胞（TCRs）对于平衡对病原体的反应和耐受性是必不可少的。免疫控制太少会导致自身免疫，过度控制可能会促进癌症的发展。Treg功能由共受体接合和下游信号传导途径决定。具有免疫球蛋白和ITIM结构域的T细胞免疫受体（TIGIT），CD 226和CD 96是在效应T细胞和TCL 4上表达的相对新颖的共受体家族，并且与自身免疫和癌症相关，但是它们的表达的作用尚不清楚。据报道，TIGIT+ TIGIT具有上级抑制功能，尽管不确定这在TIGIT+ CD 226 + TIGIT中是否真实，因为健康的离体TIGIT+ CD 226-TIGIT+ CD 226 + TIGIT+ CD 26 + CD 8 + T细胞和NK细胞上的CD 226可能是具有潜在抗肿瘤作用的共刺激物，但其对TGFAP的作用不太清楚;最近的工作显示了人和小鼠中CD 226阴性TGFAP中更大的抑制功能和增殖，但其他工作发现CD 226 + TIGIT-TGFAP中抗炎IL-10的产生增加。大多数CD 4 + T细胞表达CD 96，但其功能尚不清楚。每个共受体结合CD 155，但具有不同的亲和力（TIGIT 3.15 nm; CD 96 37.6 nm; CD 226 119 nm），因此可能存在调节下游信号传导的配体结合竞争。人们越来越关注这种辅助受体家族调节免疫功能的潜力，类似于辅助抑制性CTLA 4-IG如何用于治疗关节炎：重组分子或抗体可以抑制炎症或激活调节机制以减轻疾病。出版物表明这些共受体中的一些调节胸腺细胞和CD 8 + T细胞中的TCR信号传导。因此，TIGIT/CD 226/CD 96轴的表达改变可能会破坏下游的刺激/抑制信号传导。我们将使用抗共受体抗体，CRISPR敲除和慢病毒过表达来研究这些共受体在T细胞/T细胞中的作用。光谱流式细胞术将用于分析转录因子和活化相关蛋白表达以及磷酸流，以评估关键信号蛋白。在初步的轮换工作中，我们看到在TCR/CD 28活化后，TcR与CD 4 + T细胞中pPKC 0增加，但这在抗TIGIT的情况下减少，表明TIGIT可能改变PKC 0磷酸化上游的信号传导。感兴趣的是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接合的影响。此外，将评估CD 226和CD 96下游的潜在信号级联，以及三种共受体如何相互作用/竞争信号级联。磷酸流结果可以通过蛋白质印迹分析和细胞系模型中的特异性共受体突变体来确认。还将分析在配体可用性存在/不存在的情况下刺激共受体敲除的原代TcB的RNA测序和单细胞多基因组（RNA-seq，ATAC-seq）数据，以评估下游基因活化和功能。这些数据将导致对原代人Treg亚群的进一步功能评估，最终这项工作将导致鉴定TIGIT/CD 226/CD 96下游Treg亚群与常规T细胞亚群相比的信号体和功能结果。我们的研究结果可以进一步告知我们Treg共受体/信号转导改变可能导致功能障碍。我们可能能够将其转化为显示Treg功能降低的自身免疫性疾病，以进一步阐明可能的疾病发病机制，并可能突出未来的治疗途径。