Tumor cells use two proteolytic compartments to degrade incorrectly synthesized and other damaged proteins, proteasomes and lysosomes. The immune system then monitors the degradation products of these two systems when the peptides are presented via MHC class I and II, respectively. Recognition of MHC class II by Th1 CD4+ T cells and MHC class I by cytolytic CD8+ T cells will result in the destruction of infected and malignant cell. We have recently described that the nuclear antigen 1 of the oncogenic Epstein Barr virus (EBNA1) gains access to MHC class II presentation after autophagy. We now propose in this application to investigate in three aims if autophagic delivery of antigens constitutes a general pathway of endogenous MHC class II antigen processing. 1. Analysis of autophagy levels and overlap with MHC class II loading compartments in human tissues, during steady state and after immune activation by type I and II Interferons as well as viral infections. We will quantitatively assess the autophagy level in different human tissues with and without immune activation and address the overlap between autophagosomes and MHC class II loading vesicles. 2. Characteristics of viral and tumor antigens following an autophagic route to MHC class II presentation. We will investigate if other viral and tumor antigens, for which CD4+ T cell recognition after endogenous processing was demonstrated, follow the autophagic route for MHC class II presentation. We will construct deletion mutants of EBNA1 and analyze their capacity to access endogenous MHC class II antigen processing after autophagy. This analysis will reveal domains that target substrates for this degradation path. 3. Targeting of endogenous antigens for autophagic degradation and MHC class II presentation. We will target known CD4+ T cell antigens to autophagosomes for improved MHC class II presentation by extending their half-lifes, since long-lived proteins are primarily processed via autophagy. In addition, we will target for chaperone mediated autophagy with signal peptides and for macroautophagy with the LC3/Atg8 protein, which gets partially degraded in lysosomes after attachment to the inner autophagosome membrane. These studies will allow us to understand better how tumors can be detected by the immune system and how we can enhance immune responses to cancer. [unreadable] [unreadable] [unreadable]