CD8 Kinetically Promotes Ligand Binding to the T-Cell Antigen Receptor

Abstract

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

Spatial coordination of CD8 and TCR molecules controls antigen recognition by CD8+ T‐cells

“…The fast constant has been assigned to their diffusion in disordered membrane domains whereas the slower one to that in the ordered membrane domains [29]. Independent confocal microscopy measurements have also suggested a distribution of both CD8 and TCR molecules between rafts and disordered membrane microdomains: A partial localization of these molecules within lipid raft domains has been observed by us [29] and by other studies [30][31][32][33][34].…”

“…This CD8 contribution is expected to increase sensitivity in proportion to the number of TCR-proximal CD8 molecules on the cell surface. This proportion between the raft and bulk resident CD8/TCR populations was found to depend of the time past after T-cell activation [29].…”

“…The flow cytometry measurements revealed that pMHC tetramers associate with T-cells in a bi-phasic time-course for all three different T-cell types examined (two CTL clones and one T-cell hybridoma) [29]. The fast phase rate constant was about an order of magnitude faster than that of the slow one.…”

“…The cholesterol depletion slowed down the fast phase binding rate by ca. 10-fold [29]. This suggested that the CD8 and TCR molecules involved in the fast tetramer association step probably reside in rafts.…”

“…The experimental resolution of two distinct CD8/TCR populations which interact with the pMHC ligands with about an order of magnitude different rates led us to suggest a mechanism controlling sensitivity of T-cells response to the TCR stimulus [29]. This was based on measurements of tetramer binding to CTL clones on different days after cell stimulation.…”