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J. Biol. Chem., Vol. 278, Issue 7, 4500-4509, February 14, 2003
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From the Fluorescence-labeled soluble major
histocompatibility complex class I-peptide "tetramers" constitute a
powerful tool to detect and isolate antigen-specific
CD8+ T cells by flow cytometry. Conventional
"tetramers" are prepared by refolding of heavy and light chains
with a specific peptide, enzymatic biotinylation at an added C-terminal
biotinylation sequence, and "tetramerization" by reaction with
phycoerythrin- or allophycocyanin-labeled avidin derivatives. We show
here that such preparations are heterogeneous and describe a new
procedure that allows the preparation of homogeneous tetra- or
octameric major histocompatibility complex-peptide complexes. These
compounds were tested on T1 cytotoxic T lymphocytes (CTLs), which
recognize the Plasmodium berghei circumsporzoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid on
Lys259 in the context of H-2Kd. We report that
mutation of the CD8 binding site of Kd greatly impairs the
binding of tetrameric but not octameric or multimeric
Kd-PbCS(ABA) complexes to CTLs. This mutation
abolishes the ability of the octamer to elicit significant
phosphorylation of CD3, intracellular calcium mobilization, and CTL
degranulation. Remarkably, however, this octamer efficiently activates
CTLs for Fas (CD95)-dependent apoptosis.
Soluble Major Histocompatibility Complex-Peptide Octamers
with Impaired CD8 Binding Selectively Induce
Fas-dependent Apoptosis*,
,,, and
Ludwig Institute for Cancer Research,
Lausanne Branch, University of Lausanne and ¶ Institute for
Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland
*
This study was supported in part by grants from the Sandoz
Foundation, the Stanley Thomas Johnson Foundation, and the Swiss National Science Foundation (Grant 3100-061946.00).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains supplementary Figs. 1S and 2S.
§
Supported by a grant from the Giorgi-Cavalieri Foundation Swiss.
To whom correspondence should be addressed. Tel.:
41-21-692-5988; Fax: 41-21-653-4474; E-mail:
iluesche@eliot.unil.ch.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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