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

CD4+FoxP3+调节性T细胞（Tregs）对于平衡对病原体的反应和耐受性至关重要。免疫控制过少会导致自身免疫，而过度控制可能会促进癌症的发展。Treg的功能是由共受体参与和下游信号通路决定的。具有免疫球蛋白和ITIM结构域的T细胞免疫受体（TIGIT）、CD226和CD96是一个相对较新的共受体家族，在效应T细胞和Tregs上均有表达，并与自身免疫和癌症有关，但其表达的作用尚不清楚。据报道，TIGIT+ Tregs具有优越的抑制功能，尽管不确定TIGIT+CD226+ Tregs是否如此，因为健康的离体Tregs往往是TIGIT+CD226-。CD226作用于CD8+ T细胞和NK细胞可能具有潜在的抗肿瘤作用，但其对Tregs的作用尚不清楚；最近的研究表明，在人和小鼠中，CD226阴性Tregs具有更强的抑制功能和增殖能力，但其他研究发现CD226+TIGIT- Tregs中抗炎IL-10的产生增加。大多数CD4+ T细胞表达CD96，但其功能尚不清楚。每种共受体与CD155结合的亲和力不同（TIGIT为3.15nm； CD96为37.6nm； CD226为119nm），因此可能存在调节下游信号传导的配体结合竞争。人们对这种共受体家族调节免疫功能的潜力越来越感兴趣，类似于共抑制性CTLA4-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可以调节胸腺细胞和CD8+ T细胞中的TCR信号。因此，TIGIT/CD226/CD96轴表达的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/ treg中的作用。光谱流式细胞术将用于分析转录因子和激活相关蛋白的表达和磷酸化流，以评估关键的信号蛋白。在初步的旋转工作中，我们发现在Tregs中，与CD4+ T细胞相比，TCR/CD28激活时pPKC0增加，但抗TIGIT降低了pPKC0，这表明TIGIT可能改变PKC0磷酸化的上游信号。在相同条件下评估下游蛋白（如JNK）的磷酸化，以确定哪些途径可能受到TIGIT参与的影响，这将是一项有趣的研究。此外，将评估CD226和CD96下游的潜在信号级联，以及这三种共受体如何相互作用/竞争信号级联。Phospho-flow结果可以通过western blot分析和细胞系模型中的特异性共受体突变来证实。此外，还将分析具有/不具有配体可用性的共受体敲除刺激的初级treg的rna测序和单细胞多组（RNA-seq, ATAC-seq）数据，以评估下游基因的激活和功能。这些数据将有助于进一步评估人类主要Treg亚群的功能。最终，这项工作将导致与传统T细胞亚群相比，鉴定TIGIT/CD226/CD96下游Treg亚群的信号体和功能结果。我们的发现可以进一步告诉我们Treg共受体/信号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，并潜在地强调未来的治疗途径。

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.