Increased Sensitivity of Antigen-Experienced T Cells through the Enrichment of Oligomeric T Cell Receptor Complexes

Abstract

Although memory T cells respond more vigorously to stimulation and they are more sensitive to low doses of antigen than naive T cells, the molecular basis of this increased sensitivity remains unclear. We have previously shown that the T cell receptor (TCR) exists as different-sized oligomers on the surface of resting T cells and that large oligomers are preferentially activated in response to low antigen doses. Through biochemistry and electron microscopy, we now showed that previously stimulated and memory T cells have more and larger TCR oligomers at the cell surface than their naive counterparts. Reconstitution of cells and mice with a point mutant of the CD3ζ subunit, which impairs TCR oligomer formation, demonstrated that the increased size of TCR oligomers was directly responsible for the increased sensitivity of antigen-experienced T cells. Thus, we propose that an "avidity maturation" mechanism underlies T cell antigenic memory.

Cognate Peptide–MHC Complexes Are Expressed as Tightly Apposed Nanoclusters in Virus-Infected Cells To Allow TCR Crosslinking

“…It may even be less, considering on the one hand that each 25D1.16 mAb has two binding sites for the K b OVAp complex, and on the other hand that steric hindrance between the protein A-gold conjugates and a labeling efficiency of ,100% might not allow detection of all K b OVAp complexes within a cluster. These distances are in the range of the estimated diameter of the TCR complex (35) and spacing of individual TCR complexes within TCR nanoclusters (7,8). The cognate pMHC clusters could therefore facilitate multivalent interactions with TCR nanoclusters that in turn could give rise to the enhanced T cell response we observed.…”

“…However, the pMHC-TCR interaction is of low affinity (4-6), making it difficult to explain how T cells achieve high sensitivity. We previously showed that the TCR was organized in nanoclusters prior to Ag engagement and that this clustering endowed T cells with higher sensitivity (7,8). Furthermore, it has long been recognized that experimental TCR ligands, such as TCR-specific Abs and soluble pMHC complexes, need to be at least bivalent to be capable of stimulating T cells (9,10).…”

“…In the case of labeling with the 25D1.16 mAb (mouse IgG1), a bridging hamster anti-mouse IgG was used to overcome the low affinity of protein A for murine IgG1 Abs. Replica generation was done as previously described (7,8). Replicas were analyzed on a JEOL 1010 transmission electron microscope operating at 80 kV, and images were acquired with a TemCAM F416 camera operated by EM-MENU software (Tietz Video and Image Processing Systems).…”

“…We used our previously established approach of cell surface replica generation of fixed cells labeled with primary Abs and colloidal gold-conjugated secondary reagents in combination with transmission electron microscopy (7,8). This approach permits molecular resolution of the cell surface localization of endogenous proteins and, in contrast to most high-resolution light microscopy techniques, visualizes the cell surface membrane opposite to the one in contact with the experimental support.…”

The coreceptor CD4 is expressed in distinct nanoclusters and does not colocalize with T-cell receptor and active protein tyrosine kinase p56lck

“…The molecular assembly and clusters such as supramolecular activation clusters (SMACs) (15) of immunological synapse (IS) (14), microclusters (16)(17)(18)(19)(20), and their roles in T-cell signaling have been widely studied. More recently, the presence and unique roles of smaller-sized protein clusters, termed "nanoclusters" or "protein islands," of TCR-CD3 complex (21)(22)(23)(24), linker for activation of T cell (LAT) (21,22,24,25), Lck (26), and other signaling molecules (24) were revealed by electron microscopy and the newly available superresolution fluorescence microscopy.…”