The adaptive immune system protects us from nocuous microbes, yet excessive immune reactions can lead to the development of autoimmunity. The initiation of adaptive immune responses takes place in lymphoid organs, including peripheral lymph nodes (PLN). Inside PLN, T cells become activated through the molecular interactions of the T cell receptor with cognate peptide-MHC (pMHC) molecules and costimulatory molecules on dendritic cells (DC). TCR-mediated signals lead to the activation of the phosphoinositol-3-kinase (PI3K) pathway via the PI3K? isoform, which is required for full T cell activation in vitro.However, little is known about the role of the PI3K? during primary and secondary in vivo T cell activation. Here, we will use twophoton microscopy (2PM) to observe the interactions of antigen-specific control and PI3K?-mutant CD4+ T cells and pMHC-loaded DC deep inside PLN of live, anesthetized mice. Comparing the dynamic interaction parameters obtained in 2PM imaging with the flow cytometric analysis of the activation status of control and PI3K?-mutant T cells, we will delineate the precise contribution of the PI3K? for primary T cell activation in vivo.We will furthermore perform an analysis of the secondary activation of control and PI3K?-mutant effector CD4+ T cells in a mouse model of antigen-induced arthritis. Therefore, we will establish an intravital knee-joint 2PM model to follow the dynamic behavior of control and PI3K?-mutant effector T cells with antigen-presenting macrophages in the inflamed tissue. We will combine imaging data with disease scoring, flow cytometry and the use of a PI3K?-specific pharmacological inhibitor for a comprehensive analysis of PI3K? function at sites of inflammation.In summary, we propose to perform an in-depth analysis of the role of PI3K? during adaptive immune response initiation and maintenance. This is also clinically relevant since PI3K? inhibitors are in development for immunomodulation.