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.

Class I major histocompatibility complex (MHC) heterodimer, composed of human leukocyte antigen (HLA)-A2 heavy chain and human beta(2)-microglobulin (beta(2)m), was produced by denaturation and gel filtration of the recombinant water-soluble HLA-A2/beta(2)m/peptide ternary complex in 8 M urea Tris-HCl buffer, followed by refolding of the separated chains without peptide. Peptide affinity and kinetics of the ternary complex formation and dissociation were investigated in real time by monitoring the fluorescence resonance energy transfer (FRET) from intrinsic HLA-A2 heavy-chain tryptophans to a dansyl fluorophore conjugated to the bound peptide. Peptide binding to the heterodimer was a second order process with rate constants linearly dependent upon temperature in Arrhenius coordinates over 0-20 degrees C. The binding rate constant of pRT6C-dansyl [ILKEPC(dansyl)HGV] at 37 degrees C evaluated by extrapolation of the Arrhenius plot was (2.0 +/- 0.5) x 10(6) M(-1) s(-1). Association of the heavy chain with beta(2)m was a first order process, apparently controlled by a conformational transition in the heavy chain. One of these conformations bound to beta(2)m to form the heavy chain/beta(2)m heterodimer whereas the second conformer oligomerized. Peptide dissociation from the ternary complex was a first-order reaction over the temperature range 20-37 degrees C, suggesting that the ternary complex also exists in two conformations. Taken together, the present data suggest that association of beta(2)m changes the HLA-A2 heavy-chain conformation thereby promoting peptide binding. Peptide dissociation from the ternary complex induces dissociation of the heavy-chain/beta(2)m heterodimer thereby causing oligomerization of the heavy chain. The lability of the HLA-A2/beta(2)m heterodimer and the strong tendency of the "free" heavy chain to oligomerize may provide an efficient mechanism for control of antigen presentation under physiological conditions by reducing the direct loading of HLA with exogenous peptide at the cell surface.

Thermodynamics and kinetics of interaction between a soluble class I MHC heterodimer composed of the H-2Kd heavy chain (H) and human beta 2microglobulin (beta 2m) with a dansylated peptide series based on residues 147-155 of influenza virus nucleoprotein sequence were studied by means of real-time fluorescence measurements. Peptide-heterodimer binding is a second-order process with specific rates practically independent of peptide structure (3-5 x 10(6) M-1 s-1). The ternary complex assembly involves a rate-limiting step of beta 2m association with H to yield an unstable heterodimer (tau < or = 5 s, 37 degrees C). Peptide binding provides a positive feedback enhancing H's affinity for beta 2m, thus stabilizing the ternary complex. The latter decays by either peptide or beta 2m dissociation. The first-order rate constants of peptide dissociation (0.5 x 10(-2))-(0.4 x 10(-3)) s-1, 37 degrees C) depend on their structures and are faster than that of beta 2m dissociation. The former process decreases the H affinity for beta 2m and induces their dissociation. This dissociation, in turn, drastically lowers H affinity for peptide. Thus, these three components produce a system which is stable as a trimer. This behavior is rationalized by the functional requirements of class I molecules: Peptide structure determines the ternary complex's lifetime, and peptide rebinding on the cell surface is rendered unlikely by the limited stability of the empty heterodimers and the very low peptide affinity of the heavy chains.