The Major Histocompatibility Complex (MHC) and the proteasome-ubiquitin pathway in T cell development

Abstract

The essential role of classical MHC molecules in T cell development as well as the proteasome role in antigen processing for MHC-mediated antigen presentation to T cells is well established. However, the contribution of nonclassical MHC molecules, the signals evoked by the MHC-TCR interactions, the signaling role of the proteasome-mediated proteolysis, and the regulation of the proteasome-ubiquitin proteolysis remain to be clarified. Here we address these more subtle but essential aspects of T cell development. We obtained mice deficient for MHC molecules encoded by the H-2K and H-2D genes. KbDb -/- mice have greatly reduced numbers of mature CD8+ T cells, indicating that selection of CD8+ T cells can not be compensated for by [beta]2m-associated molecules other than classical H-2K and D locus products. Spleen cells from KbDb -/- mice generate strong CD8+ MHC class I-specific responses after in vivo priming. Thus, a minor population of CD8+ T cells arises in the complete absence of classical MHC class I molecules. KbDb -/- animals also have self-tolerant natural killer (NK) cells that retain their cytotoxic potential. We utilize a fetal thymic organ culture (FTOC) system with a panel of proteasome inhibitors to implicate the proteasome in thymocyte apoptosis and negative selection. We find that proteasome inhibitors do not completely block but rather delay both dexamethasone- and antigen-triggered thymocyte apoptosis. We also show that proteasome activity is increased in apoptotic thymocytes,

(cont.) as visualized by active site labeling of proteasomal D subunits, indicating that proteasome functions as a positive regulator in thymocyte death cascade. We show that the deubiquitinating enzyme USP7 (HAUSP) is specifically and uniquely processed in apoptotic thymocytes. USP7 protein is highly expressed in thymus, spleen, and brain and is very similar in men and mice. Processing of USP7 does not occur in caspase 3-/- thymocytes but caspase 3 does not cleave USP7 directly. Our results suggest that thymocyte apoptosis leads to a modification of a deubiquitinating enzyme and may provide an additional link between the proteasome-ubiquitin pathway and the caspase cascade during programmed cell death.