CXCL9/10 producing cell clusters optimize Th1 cell positioning for interaction with antigen presenting cells

Abstract

Abstract Intracellular pathogen clearance is dependent on successful T cell mediated immunity. Th1 cells must localize to the site of infection to be activated into IFN-γ producing effector T cells. How Th1 cells find and interact with antigen presenting cells (APC) in infected tissues is poorly understood. Preliminary data using intravital imaging and the REX3 mouse, in which CXCL9 and CXCL10 producing cells express red and blue fluorescent proteins (RFP/BFP) respectively, suggest that CXCL9/10-producing cells form dense perivascular clusters that attract and arrest CXCR3+ Th1 cells. We hypothesize that these CXCL10+ cell clusters are enriched for APC that optimize Th1 cell activation and IFN-γ production. To determine the phenotype of the immune cells within the chemokine-producing clusters we crossed the REX3 mouse with a photoactivatable green fluorescent protein (PA-GFP) mouse. This will enable selective labeling of cells within chemokine-rich clusters prior to flow cytometry. Using precise multiphoton delivery of 830 nm light, we have successfully photoactivated the CXCL10+ clustered regions in the inflamed ear and have developed a flow cytometry panel to distinguish between macrophages, cDCs, and monocyte-derived cells that may be positioned within or outside these chemokine-clusters. Additionally, we will determine if these cell clusters enhance Th1 function by sorting photoactivated (in clusters) and non-photoactivated (outside clusters) Th1 cells from the inflamed ear and analyzing IFN-γ expression. Understanding how Th1 cells are positioned and activated at sites of inflammation will allow the field to boost responses to infectious agents and therapeutically attenuate Th1 responses in autoimmunity.