University of Birmingham The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

Invariant natural killer T cells (iNKT cells) recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such ‘tonic’ activation of iNKT cells remain unclear. Here we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen presenting cells (APCs) modulates activation of iNKT cells. By using super-resolution microscopy, we demonstrate that CD1d molecules form nanoclusters at the cell surface of APCs, whose size and density is constrained by the actin cytoskeleton. Dual color single particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly, and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the TLR7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a novel mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen loaded CD1d nanoclusters. enigmatic. Here we demonstrate that iNKT cell activation is regulated by the lateral nanoscale organization of CD1d loaded with exogenous and endogenous antigens. Using a combination of advanced biophysical techniques we show that CD1d molecules organize in nanoclusters on the membrane of APCs. We further discover that the actin cytoskeleton prevents enhanced CD1d nanoclustering by hindering physical encountering between CD1d diffusing nanoclusters, reducing basal iNKT cell activation. As such, regulation of CD1d nanoclustering through the actin cytoskeleton a novel mechanism to fine-tune peripheral iNKT cell autoreactivity. nanoscale THP-1 emission depletion (STED) nanoscopy anti-CD1d42 hCD1d dorsal fixed THP-1 cells. artifacts to CytoD treatment on the dorsal cell membrane morphology affect the quantification of STED images, on WT-hCD1d and the TD-hCD1d SPT similar lateral diffusion of exogenous lipid-loaded ( α GalCer GalGalCer) TD-CD1d CytoD treated samples. custom-made algorithm Bayesian intensity distribution, data effective STED α -GalCer WT-hCD1d small nanoclusters on the surface of THP-1 cells median cluster size 3.8 median number hCD1d molecules/cluster nanocluster around 75 hCD1d/ µ m verify that CD1d nanoclustering is real and not resulting from the enhanced expression of hCD1d on transduced THP-1 cells, imaged hCD1d on blood-derived primary human CD14 + monocytes. Analysis of STED images the presence of hCD1d nanoclusters, albeit being smaller in size (49 nm as median cluster size) to THP-1 cells S3). These data demonstrate that hCD1d forms nanoclusters on the plasma of APCs. average of at least 30 pair correlations obtained over 3x3 µm 2 regions of interest.