Murine lupus autoantibodies identify distinct subsets of apoptotic bodies

The specific modification of autoantigens and their redistribution into blebs at the surface of apoptotic cells contribute to the induction of autoimmune responses. Blebs containing fragments of the apoptotic nucleus separate from the remainder of the cell to form membrane-bound sub-cellular particles (SCPs), otherwise known as apoptotic bodies. To determine whether apoptotic bodies containing nuclear antigens represent a defined subset of SCPs, we examined the heterogeneity of particles generated by Jurkat cells following synchronization of the cell cycle by serum withdrawal and inhibition of topoisomerase I by camptothecin. Particles were purified by filtration, incubated in the presence of antinucleosome or anti-cardiolipin autoantibodies, annexin V, and Sytox Orange and analyzed by flow cytometry and confocal microscopy. We demonstrate that nuclear autoantigens are associated with one clearly defined subset of SCPs that can be distinguished from other products of late apoptosis. Our experiments represent an important step towards characterizing the heterogeneity of SCPs that are generated in late apoptosis and identifying their contributions to tolerance and autoimmunity.