Dmitry M. Gakamsky

Kinetic evidence for a ligand-binding-induced conformational transition in the T cell receptor.

Thermodynamics and kinetics of the interaction between T cell receptor specific for cytomegalovirus peptide (TCRCMV) and its specific ligand, pp65–HLA-A*0201 complex, were studied by surface plasmon resonance and stopped-flow methods. In the latter measurements, fluorescence resonance energy transfer (FRET) between fluorescently labeled reactants was used. Thermodynamic data derived from surface plasmon resonance measurements suggest that the complex formation is driven by both favorable enthalpy and entropy. Two reaction phases were resolved by the stopped-flow measurements. The rate constant of the first step was calculated to be close to the diffusion-controlled limit rate (3·105 to 106 M−1·s−1), whereas the second steps reaction rate was found to be concentration independent and relatively slow (2–4 s−1 at 25°C). These findings strongly suggest that the interactions between the TCR and its ligand, the peptide–MHC complex, proceed by a two-step mechanism, in which the second step is an induced-fit process, rate determining for antigen recognition by TCR.

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

The interactions between the TCR and peptides bound to class I MHC encoded molecules (pMHC) and a mechanism for CD8 cooperation in this process are reviewed. Observation of two TCR/CD8 populations with different lateral diffusion rate constants as well as two distinct association phases of class I MHC tetramers ((pMHC)4) with T‐cells suggest that the most efficient pMHC–T‐cell association route corresponds to a fast tetramer binding to a colocalized CD8/TCR population, which apparently resides within membrane rafts. Thus, ligand–cell association starts by pMHC binding to the CD8. This rather fast step promotes pMHC association with CD8–proximal TCRs and thereby enhances the overall association process. The model suggests that this raft‐associated CD8–TCR subpopulation is responsible for evoking T‐cell activation.

Efficient induction of cytotoxic CD8+ T cells against exogenous proteins: establishment and characterization of a T cell line specific for the membrane protein ActA of Listeria monocytogenes

The property of listeriolysin (LLO) to introduce soluble passenger proteins into the cytosol of antigen‐presenting cells allows the induction of CD8+ cytotoxic T cells against such antigens. To overcome the potential problem of presentation of the immunodominant epitope LLO91‐99 by H‐2Kd, a variant LLO92A was established in which Tyr 92 was replaced by Ala. Immunization of BALB/c mice with purified LLO92A failed to stimulate cytotoxic T cells specific for either the epitope LLO91‐99 or for any other LLO‐derived peptide. Injection of mixtures of purified LLO92A and soluble nucleoprotein (NP) of influenza virus into mice resulted in a strong cytotoxic T cell response exclusively directed against NP. The LLO92A variant was successfully used to generate, propagate and characterize a CD8+ T cell line specific for the membrane‐bound virulence factor ActA of Listeria monocytogenes. Interestingly, wild‐type ActA bound to the surface of live L. monocytogenes was not presented by MHC class I molecules to the CD8+ T cell line.

Photophysical analysis of class I major histocompatibility complex protein assembly using a xanthene-derivatized beta2-microglobulin.

Spectral changes and a sixfold increase in the emission intensity were observed in the fluorescence of a single xanthene probe (Texas red) attached to beta2m-microglobulin (beta2m) upon assembly of beta2m into a ternary complex with mouse H-2Kd heavy chain and influenza nuclear protein peptide. Dissociation of the labeled beta2m from the ternary complex restored the probes fluorescence and absorption spectra and reduced the emission intensity. Thus changes in xanthene probe fluorescence upon association/dissociation of the labeled beta2m molecule with/from the ternary complex provide a simple and convenient method for studying the assembly/dissociation mechanism of the class I major histocompatibility complex (MHC-I) encoded molecule. The photophysical changes in the probe can be accounted for by the oligomerization of free labeled beta2m molecules. The fluorescence at 610 nm is due to beta2m dimers, where the probes are significantly separated spatially so that their emission and excitation properties are close to those of xanthene monomers. Fluorescence around 630 nm is due to beta2m oligomers where xanthene probes interact. Minima in the steady-state excitation (550 nm) and emission (630 nm) anisotropy spectra correlate with the maxima of the high-order oligomer excitation and emission spectra, showing that their fluorescence is more depolarized. These photophysical features are explained by splitting of the first singlet excited state of interacting xanthene probes that can be modeled by exciton theory.