Vaccination against infectious diseases is a major tool for prevention of morbidity and mortality for both veterans and active-duty soldiers. Effective vaccination, similar to the immune response to infection, requires activation of CD4+ helper T cells by professional antigen presenting cells expressing MHC class II-peptide complexes. Most vaccinations are delivered subcutaneously; thus, understanding the regulation of MHC class II- dependent immune responses in the skin will inform the rational design of vaccines. Surprisingly, we found that antigen processing and presentation by both lymphoid- resident and migratory DCs was required for clonal selection and expansion of CD4+ T cells following subcutaneous immunization. Early antigen presentation by lymphoid- resident DCs initiated activation and trapping of antigen-specific T cells in the draining LN, without inducing clonal expansion. Migratory DCs, however, interact with the CD4+ T cells retained in the LN to induce proliferation. Therefore, distinct DC subsets cooperate to alert and trap the appropriate cell and then license its expansion and differentiation. These preliminary results lead to the obvious question: why are two DC populations necessary to prime CD4+ T cells? In the current proposal, we will dissect the cellular and molecular mechanisms that underlie the requirement for two distinct populations of dendritic cells in the response to antigens delivered in the skin. We have three Specific Aims. In our first Aim, we will utilize large antigen-conjugated microspheres to limit antigen presentation to one population of DCs. This will test the hypothesis that antigen presentation restricted to migratory DCs cannot prime naove CD4+ T cells. In our second two Aims, we will examine two alternative explanations for the requirement for both lymphoid-resident and migratory DCs. First, immunofluorescence and molecular techniques will be utilized to dissect the localization of T cells following interaction with lymphoid-resident DCs. We will consider the hypothesis that naove T cells in the LN must change location to permit interaction with antigen-loaded migratory DCs. Finally, we will determine the biochemical setpoint and behavior of CD4+ T cells following priming by lymphoid-resident DCs. Does T cell activation occur in two distinct steps regulated by two different DC populations? Understanding the requirements for activation of CD4+ T cells will guide the development of more effective vaccinations against pathogens that cause significant disease in soldiers and veterans.