Nanoscale Increases in CD2-CD48-mediated Intermembrane Spacing Decrease Adhesion and Reorganize the Immunological Synapse

Mengling Liu,P Anton Van Der Merwe,Martin K Wild,David L Stokes,Su-Yi Tseng,Oren Milstein,Yair Reisner,Michael L Dustin,Jaime Llodra,Andrea Nans,Toby Starr

doi:10.1074/jbc.m804756200

Abstract

The relationship between intermembrane spacing, adhesion efficiency, and lateral organization of adhesion receptors has not been established for any adhesion system. We have utilized the CD2 ligand CD48 with two (wild type CD48 (CD48-WT)), four (CD48-CD2), or five (CD48-CD22) Ig-like domains. CD48-WT was 10-fold more efficient in mediating adhesion than CD48-CD2 or CD48-CD22. Electron tomography of contact areas with planar bilayers demonstrated average intermembrane spacing of 12.8 nm with CD48-WT, 14.7 nm with CD48-CD2, and 15.6 nm with CD48-CD22. Both CD48-CD2 and CD48-CD22 chimeras segregated completely from CD48-WT in mixed contact areas. In contrast, CD48-CD2 and CD48-CD22 co-localized when mixed contacts were formed. Confocal imaging of immunological synapses formed between primary T lymphocytes and Chinese hamster ovary cells presenting major histocompatibility complex-peptide complexes, and different forms of CD48 demonstrated that CD48-CD2 and CD48-CD22 induce an eccentric CD2/T cell antigen receptor cluster. We propose that this reorganization of the immunological synapse sequesters the T cell antigen receptor in a location where it cannot interact with its ligand and dramatically reduces T cell sensitivity.

Highlights

Adhesion molecules that participate in T cell activation can be categorized into at least two groups: the integrin family adhesion molecules that include lymphocyte function-associated antigen (LFA)-1 on T cells, which interacts with intercellular adhesion molecule (ICAM)-1 on antigen-presenting cells, and the small immunoglobulin superfamily molecules like CD2 on T cells, which interacts with CD48 on the surface of antigen-presenting cells [3, 5]
Supported planar bilayers containing ICAM-1 and MHCp reconstitute formation of the immunological synapse (IS) [8]. This model IS is characterized by a bull’s eye-like configuration of a central supramolecular activation complex enriched in T cell antigen receptors (TCRs), a peripheral supramolecular activation complex enriched in LFA-1, and a distal supramolecular activation complex enriched in CD45 and F-actin [9]
We performed experiments with supported planar bilayers and imaging of T cell-APC IS in order to understand the basic rule of adhesion molecule segregation by size and how these relate to functional T cell activation

Summary

EXPERIMENTAL PROCEDURES

Cells and Constructs—CHO cell lines expressing recombinant CD48-WT, CD48-CD2, and CD48-CD22 were previously described [25]. 5-␮m glass beads, reacting these with saturating concentra- purified and fluorescently labeled CD48-WT, CD48-CD2, or tions of FITC-OX78, and analyzing fluorescence on a flow CD48-CD22 at different densities and compared the adhesion microfluorimeter using FITC standard beads (Bangs Laborato- of mouse T cell blasts by counting contact areas by interference ries) for calibration. T cells were allowed to interact with bilayers for 1 h at performed on a wide field fluorescence microscope with an 24 °C, which is sufficient time for adhesion by all forms to reach. Interference reflection microscopy revealed similar sized contact areas for CD48-WT and CD48-CD22 (Fig. 1B). These findings suggest that the CD48-WT is ϳ10-fold more efficient at mediating adhesion than either extended form of CD48. Similar results were obtained previously in cell-cell contacts with extended TCR-MHCp interactions [26]

Lateral Segregation Based on Differences in Intermembrane Distance

Findings

DISCUSSION