(53b) Spatiotemporal Membrane Organization At the Single Molecular Level During T Cell Receptor Triggering

In the adaptive immune system, critical antigen-specific T cell responses are triggered by the specific recognition of an antigenic peptide-major histocompatibility complex (pMHC) by the T cell receptor (TCR). Despite extensive studies on this TCR engagement and subsequent activation of the T cell, the spatiotemporal dynamics between the molecules involved in the process are still poorly understood, especially at the single molecular level. In this study, simultaneous dual-color imaging of the molecules that have critical roles in T cell activation was performed at unprecedented high resolution. For this purpose, we employed dual-color photoactivated localization microscopy (dc-PALM) and direct stochastic optical reconstruction microscopy (dSTORM). T cell membrane organizations in both latent and activated states were examined in detail.

T cell blasts prepared from the lymph nodes of 5c.c7 TCR transgenic mice were examined under microscope 7 days after initial activation by 10 µM Moth cytochrome C (MCC, 88-103) peptide. As fluorescent labels for dc-PALM, either PSCFP2 or PAmCherry was genetically tagged onto the CD3ζ, the linker for activation of T cells (Lat), and the CD4 molecule, a pair of which was co-expressed in a cell. For dSTORM, Alexa488 and Alexa 568 were conjugated to the specific antibodies and used for the immunofluorescence staining. Lasers of 405 nm, 488 nm and 568 nm were used for the activation of both fluorescent proteins, the excitation of PSCFP2/Alexa488, and the excitation of PAmCherry/Alexa568, respectively. Both excitation lasers were used in the total internal reflection fluorescence microscopy setup. The localization of individual molecules, reconstruction of dual color images, and spatial analyses were performed using in-house software written in MATLAB.

One of the foremost interests of this study is on how the TCR, Lat, and CD4 molecules reside in the T cell membrane before and after T cell activation. On the poly-L-lysine coated glass slides, which were used as non-stimulatory surfaces, molecules reside in their own domains which are within the size of a couple of hundred nanometers, and these domains are well distributed over the cell membrane. On the stimulatory glass surfaces coated with I-E^k/MCC and B7.1, these protein domains concatenate to form bigger microclusters. But surprisingly the degree of co-localization of protein domains seems moderate at molecular level, and mainly occurs in the interface between the two microclusters. Detailed statistical spatial analyses were employed to characterize the behaviors of protein domains quantitatively. In the talk, discussions also will be made to connect these findings to the T cell receptor activation process or other membrane receptor behavior in general.