Electron tomography of the immunological synapse

The immunological synapse between T lymphocytes and Antigen presenting Cells is a critical interaction for the development of adaptive immune responses against microbial, viral and fungal pathogens. This interaction mainly involves the recognition by the T cell receptor of a specific antigenic peptide complexed with a molecule of the Major Histocompatibility Complex (MHCp).
TCR interaction with pMHC

In collaboration with the Dustin laboratory in the Skirball Institute, we are using correlations between TIRF microscopy and electron tomography to study this interaction between CD4+ T cells and MHCp molecules on a lipid bilayer supported by a glass coverslip. Elements of the antigen presenting cell are incorporated into the lipid bilayer using GPI anchors and T cells are introduced into a flow chamber. A grid deposited onto the coverslip allows identification of individual cells in both light and electron microscopes. Thus, we are able to correlate the distribution of individual molecules (e.g. TCR receptor or actin in the figure below) with the ultrastructure of the T-cell. The central area of TCR accumulation (red in the figure) is called the cSMAC and is where intracellular signalling by the TCR is turned off. Our goal is to understand the molecular mechanisms of this process.

T-cell TIRF

TIRF images reveal the distribution of TCR (red) and actin (green). The blue grid has been imposed on the cover slip to facilitate tracking of individual cells and later identification in the electron microscope.

T-cell correlative microscopy

The grey image is an en face section of the same cell shown above by TIRF. Such correlations have allowed us to characterize the vesicle secretion at the cSMAC.

 

Electron tomography of cross sectioned T cells reveals that these upon recognition of their cognate pMHC recptors, they polarize and develop a highly complex zone characterized by the accumulation of organelles in the vicinity of the contact interface. At the membrane, we were surprised to find that the cSMAC consists of an extracellular accumulation of vesicular structures, which are highly enriched in TCR. Currently, we are working on the elucidation of the molecular mechanisms responsible for generating these structures, which appear to involve elements of the ESCRT pathway. One working hypothesis generated by this work is that the HIV virus may have co-opted this pathway in order to facilitate transmission of the virus between cells of the immune system.

T-cell on lipid bilayer

Low magnification image of the T cell interacting with a lipid bilayer. To the right, a tomographic section is shown of the cSMAC region. Vesicles are shown in orange, the T cell plasma membrane in green, elements of the centriole in yelow, and the artificial lipid bilayer in cyan.
T-cell vesicles