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

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.