Is differential T cell receptor patterning at the immune synapse mediated by glycosylation of the viral ligand UL18 and the inhibitory receptor ILT2?

Abstract

BackgroundThe T cell immune synapse is important for communication between B cells and T cells. The ILT2 receptor is expressed on T cells and binds MHC I. ILT2 also binds the highly glycosylated human cytomegalovirus MHC I-homologue UL18 at 1000 times greater affinity than native MHC I ligand. The higher affinity viral ligand results in repatterning at the immune synapse and can induce differential signalling, thus enhancing the inhibitory signals of T cells aiding viral immune evasion. We aimed to understand the mechanism by which the viral ligand UL18 interacts with the ILT2 receptor at the immune synapse to accentuate viral immune evasion. MethodsConfocal microscopy was used to characterise the patterning at the immune synapse in Jurkat T cells and superantigen pulsed Raji B cells. Overexpression of high-affinity ILT2 in the model T cells facilitated analysis of the role of affinity in the repatterning at the T cell synapse. The contribution of glycosylation was analysed by treatment of UL18-overexpressing B cells with deglycosylation reagents tunicamycin and endo H. Western blotting was used to analyse signalling at the immune synapse. FindingsThe T cell receptor (TCR) naturally clustered at the centre of the immune synapse in response to antigen. However, engaging ILT2 with the native MHC I ligand resulted in ILT2 accumulation at the centre of the immune synapse and exclusion of TCRs to the synapse periphery. Furthermore, the heavily glycosylated viral ligand UL18 altered this topography and instead co-clustered ILT2 and the TCR at the centre of the immume synapse. Deglycosylation of UL18 resulted in the separation of the ILT2 and TCR receptors once again. Analysis of signalling events showed that after superantigen stimulation the phosphorylation of Vav1 was attenuated in ILT2 and TCR clusters and that this effect was further enhanced in ILT2 and UL18 ligand binding. InterpretationOur findings show that ILT2 interaction with native versus viral ligands induces differential TCR clustering at the immune synapse, and that the viral ligand UL18 results in the co-clustering of ILT2 and TCR, possibly promoting inhibitory signalling. UL18 glycosylation is essential for this co-clustering. The proximity of the inhibitory receptor enhances the reduction of the antigen-induced Vav1 signalling. Re-patterning of the immune synapse by glycosylated UL18 might enhance inhibitory signals in immune evasion. FundingWellcome Trust.