CD4+ T cells recognize peptides (epitopes) presented in the context of MHC class II molecules (MHC class II) and play a key role in host defense by guiding and potentiating effector responses. In the classical model, such peptides are derived from extracellular (""""""""exogenous"""""""") antigens that are unfolded, digested and loaded onto MHC class II in the late endosomal compartment with the assistance of a molecule termed H2-M. The """"""""Site 1"""""""" (S1) epitope within the influenza A/PR/8/34 hemagglutinin (HA) molecule fits this description. Despite investigation for several decades, key aspects of the classical pathway remain unclear. For example, we have shown that the presentation of S1 requires a reductase activity in the late endosome but the specific reductase is not yet known. We have identified two additional pathways that are naturally utilized for the presentation of influenza antigens. First is a recycling pathway in which class II molecules are loaded in the early endosome without participation of H2-M. The """"""""Site 3"""""""" (S3) epitope within HA is presented from exogenously provided virus via this pathway. Second is an endogenous proteasome-dependent presentation pathway. Both S3 and NA79 (a neuraminidase [NA]-derived epitope) are presented from biosynthesized HA and NA via a pathway that requires proteasome and TAP (transporter of antigenic peptide) activities, both generally associated with MHC class I- but not class II-restricted presentation. The recycling and endogenous pathways are poorly understood although our results suggest that both could be just as important as the classical pathway in host defense. Our goal is to gain an understanding of all three pathways at a depth that will drive vaccine design to a less empirical process. This is a pressing need even without bioterrorism and avian influenza. To do this, we propose two discovery-based approaches for identification of critical cellular components that are specific to each of these pathways. First, utilizing epitope-specific reagents that are in hand and to be developed we will isolate antigen-presenting cell mutants that are selectively defective in presenting S1, S3 and/or NA79. Using standard genetic approaches we will then determine the basis for any defect. Second, we will interrogate small molecule libraries for compounds that selectively inhibit presentation and then identify the targets of any positive hit compounds using genetic and/or biochemical approaches. We anticipate that the execution of these complementary approaches will lead to the discovery of key processing components that would otherwise remain hidden and that will help pave the way for more rational vaccine design. ? ? ?