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

Dmitry M. Gakamsky ^*, Immanuel F. Luescher , Aladdin Pramanik , Ronen B. Kopito , François Lemonnier ^¶, Horst Vogel ^||, Rudolf Rigler  and Israel Pecht ^*

^* Department of Immunology, and Department of Materials and Interfaces, Weizmann Institute of Science, 76100 Rehovot, Israel; Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, CH-1066 Epalinges, Switzerland; Department of Medical Biophysics, MBB, Karolinska Institute, S-17177 Stockholm, Sweden; ^¶ Laboratoire d'Immunité Cellulaire Anti-Virale, Department SIDA-Retrovirus, Institut Pasteur, 75724 Paris, Cédex 15, France; and ^|| Laboratory of Physical Chemistry of Polymers and Membranes, Department of Chemistry, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland

Correspondence: Address reprint requests to Dmitry M. Gakamsky, Tel.: 972-8-9342551; Fax: 972-8-9344141; E-mail: lidima{at}wisemail.weizmann.ac.il.

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 10⁵ 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 10³ M^–1 s^–1), and close to that of the association of soluble pMHC with CD8 ((1–2) x 10⁵ 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 10-fold after cells treatment with methyl-ß-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.

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