We made use of the particular thin and extended form of LSEC that allows imaging of immune synapse formation at high resolution during the natural interaction of an APC with naive CD8 T cells. As naive CD8 T cells stop migrating after MHC I-restricted recognition of antigen on LSEC, we did not detect kinapse formation, as expected. Instead, we found that during priming of naive CD8 T cells by LSEC a multifocal immune synapse was Formoterol fumarate dihydrate formed, in which we observed both TCRb and CD11a clusters by confocal microscopy in the contact area. However, no overlap or spatial segregation into a c- or IMS2186 p-SMAC of TCRb and CD11a protein clusters were observed. Moreover, although PD-1 can be recruited into immune synapses and modifies proximal TCR signaling strength, PD-1 signaling in naive CD8 T cells undergoing priming by LSEC did neither affect immune synapse form nor the size or density of individual TCRb and CD11a clusters within the synapse. As PD-1 signaling is pivotal for development of the non-responsive phenotype in LSEC-primed T cells we conclude from these results that the dynamics of immune synapse formation does not contribute to the distinct programming of T cell differentiation by antigen-presenting LSEC. Naive CD8 T cells upregulate PD-1 after activation via the TCR. Although there was no detectable increase in PD-1 protein expression levels on the cell surface of CD8 T cells after 60 minutes of co-incubation with antigen-presenting LSEC, PD-1 mediated signaling controlled the production of IL-2 mRNA at this early time point after TCR stimulation. PD-1 protein expression levels then increased within 4 h until reaching a maximum at 24�C48 h and then remained stable for at least 4 days. As LSEC selectively upregulate co-inhibitory B7H1, but not costimulatory CD80 or CD86, during antigen-specific interaction with CD8 T cells, this implies that T cells continuously receive high levels of co-inhibitory, but insufficient co-stimulatory, signals during contact with antigen-presenting LSEC. Although LSEC do express other co-stimulatory molecules, like ICOSL and CD40, the presence of these molecules does not overcome B7H1-dependent inactivation of LSEC-stimulated CD8 T cells. For the development into fully functional effector T cells, naive T cells need to receive sustained TCR signaling for a distinct period of time. NaIve T cells that are given only a brief TCR stimulus, only transiently express CD25 and do not develop into effector T cells.