Development of a seven-color immunofluorescent panel to identify a rare B cell population (IgM +IgD lo) and its effect on T regulatory cell (Treg) proliferation

Abstract Multiple sclerosis (MS) is caused by a T cell orchestrated immune response to myelin sheath antigens of the central nervous system. Autoimmune responses can be suppressed CD4 +Foxp3 +T regulatory cells (Treg); therefore, therapeutic strategy increase endogenous Treg numbers. Our studies using mouse model MS, experimental autoimmune encephalomyelitis, have identified unique subset IgM +IgD low/−B (BD L) that induce proliferation in glucocorticoid-Induced TNFR-related (GITR:GITRL)-dependent manner. However, where BDLlocalize with spleen unknown and imaging challenging due rarity both populations. Because remain within zone, we hypothesize BDL-Treg interactions take place T-B boundary. To visualize localize BD L-Treg-interactions spleen, seven-color microscopy panel was developed identify splenic architecture Nikon Inverted Eclipse Ti2 microscope SPECTRA Light Engine filter cube. Fluorescent conjugated antibodies marginal zone (MAdCAM-1), L(IgM, IgD), (CD4, Foxp3), (Ki-67), chemokine receptors (used for lymphocyte localization spleen) were selected. select fluorescent low background spectral overlap thereby ensuring detection real signal, cytospin slides sorted CD19 +B or single-stained controls utilized. This method applied variety experiments will beneficial other applications. By completing this project, hope better understand L-Treg develop L-based numbers reduce autoimmunity. MCW Center Immunology GRIT Award