Background: We have identified the expression of an Ig-superfamily protein, CD96, on a subset of conventional T cells and Foxp3+regulatory T (Treg) cells particularly in the CNS, which segregated with a distinct functional phenotype in CD96bright conventional T cells and Treg cells, respectively.
Hypothesis: CD96 is relevant for sensing niche specific environmental signals (via interaction with CD155) that then drive the functional specialization of conventional T cells and Treg cells in the CNS. This CD155/CD96 interaction constitutes a pathway that determines resolution vs perpetuation of T cell-mediated inflammation in the CNS.
Strategy: We will use inducible and conditional gene ablation approaches (for conventional T cells and Treg cells) to investigate the significance of CD96 for the fate of T cells in the CNS in the context of autoimmunity.
Summary
CD96 is an Ig-superfamily molecule that is expressed on autoreactive T cells in later stages of their activation and segregates with a reduced encephalitogenic capacity of myelin antigen specific T cells. A specific mode of IL-6 sensing by T cells (IL-6 cluster signalling) is able to efficiently downmodulate CD96 and rescue the encephalitogenic potential of T cells. In the CNS, a high expression of CD96 on conventional CD4+ T cells is associated with IL-10 production. Conversely, in the Foxp3+ Treg cell compartment, CD96high expressors produce more IFN-g than their CD96dimcounterparts. Moreover, CD96, which interacts with CD155 that is mainly expressed on myeloid cells (including microglia), is also present on NK cells. Therefore, we will use a conditional gene targeting approach to explore the function of CD96 that is specific to conventional T cells and Treg cells.
We have generated a floxed Cd96 allele that we have bred to CD4-Cre mice (T cell conditional ablation of CD96), to CD4-CreERT2 mice (tamoxifen-inducible CD4+ T cell conditional ablation of CD96), and to Foxp3-CreERT2 mice (tamoxifen-inducible Treg cell conditional ablation of CD96). With these genetic tools, we will investigate the role of CD96 in conventional T cells and Treg cells during experimental autoimmune encephalomyelitis (EAE). Here, we will explore CD96 as a determinant of the heterogeneity of T cells in the CNS (scRNAseq). We will analyse in depth the functional phenotype and its upstream regulators in conventional T cells and Treg cells that segregate with the expression of CD96 (RNAseq and ATACseq). This strategy will also allow us to make predictions on the major downstream signalling pathways of CD96 in conventional T cells vs Treg cells. The CreERT2 strains will then allow for a disease phase dependent genetic ablation of CD96 in later phases of EAE when immunoregulation in the target tissue is most relevant. Finally, we will target the ligand of CD96, i.e. CD155, specifically in the CNS through a Crispr/Cas9 mediated knock-out approach using an intracerebroventricular lentiviral approach.
We are confident that this work program will allow us to make definitive conclusions on the significance of CD96 as a checkpoint for the perpetuation vs resolution of T cell-driven immunopathology in the CNS.
