Soluble MHC-peptide (pMHC) complexes, commonly referred to as tetramers, are widely used to enumerate and to isolate Ag-specific CD8(+) CTL. It has been noted that such complexes, as well as microsphere- or cell-associated pMHC molecules compromise the functional integrity of CTL, e.g., by inducing apoptosis of CTL, which limits their usefulness for T cell sorting or cloning. By testing well-defined soluble pMHC complexes containing linkers of different length and valence, we find that complexes comprising short linkers (i.e., short pMHC-pMHC distances), but not those containing long linkers, induce rapid death of CTL. This cell death relies on CTL activation, the coreceptor CD8 and cytoskeleton integrity, but is not dependent on death receptors (i.e., Fas, TNFR1, and TRAILR2) or caspases. Within minutes of CTL exposure to pMHC complexes, reactive oxygen species emerged and mitochondrial membrane depolarized, which is reminiscent of caspase-independent T cell death. The morphological changes induced during this rapid CTL death are characteristic of programmed necrosis and not apoptosis. Thus, soluble pMHC complexes containing long linkers are recommended to prevent T cell death, whereas those containing short linkers can be used to eliminate Ag-specific CTL.

CD8؉ cytotoxic T lymphocyte (CTL) can recognize and kill target cells that express only a few cognate major histocompatibility complex class I-peptide (pMHC) complexes. To better understand the molecular basis of this sensitive recognition process, we studied dimeric pMHC complexes containing linkers of different lengths. Although dimers containing short (10 -30-Å) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers containing long linkers (>80 Å) did not. Based on this and on fluorescence resonance energy transfer experiments, we describe a dimeric binding mode in which two T cell receptors engage in an antiparallel fashion two pMHC complexes facing each other with their constant domains. This binding mode allows integration of diverse low affinity interactions, which increases the overall binding and, hence, the sensitivity of antigen recognition. In proof of this, we demonstrated that pMHC dimers containing one agonist and one null ligand efficiently activate CTL, corroborating the importance of endogenous pMHC complexes in antigen recognition.A hallmark of CD8ϩ CTLs 1 is their extraordinary sensitivity in recognizing and killing target cells that express only very few cognate pMHC complexes (1, 2). Although soluble TCR and CD8 have been shown to bind pMHC complexes typically with low affinities, fast dissociation kinetics, and in an independent manner (3, 4), the molecular basis of this highly sensitive recognition process is not clear.For several hormone, cytokine, and chemokine receptors, it has been shown that receptor dimerization is a means to strengthen receptor ligand binding and to promote receptor signaling (5, 6). Dimerization and aggregation, pMHC driven or not, have also been proposed for TCR (7-10); however, the evidences provided never gained general acceptance, leaving this issue open (11-13). In addition, none of these studies provided a plausible structural explanation for TCR dimerization. In view of the wealth of structural information on pMHC, TCR, CD8, pMHC-TCR, and pMHC-CD8 complexes, there should be a structural explanation for TCR dimerization, if it indeed exists. The crystal structure of TCR has uncovered three features that are unique to TCR, i.e. are not found in other immunoglobulin (Ig) super-family members. 1) In the V␣ domain, the CЈ strand forms hydrogen bonds with the D strand and not with the CЉ strand (14). 2) C␣ has only 12-18% sequence homology with other Ig-constant domains, and 12-15 residues are missing, resulting in a less ordered structure and a flatter outer surface as compared with C␤ (15). 3) C␤ has a prominent, surface-exposed FG loop (15). Because of these structural features, TCR␣ chains are more likely to dimerize than TCR␤ chains (14).Because TCR are naturally membrane-integrated and associated with CD3 units, studies on TCR dimerization/aggregation should be performed on cells (11). The CD3⑀␥␦ chains each contain an extracellular Ig domain and a cytoplasmic tail harboring one immunotyrosine...

CD8؉ cytotoxic T lymphocytes (CTL) can recognize and kill target cells expressing only a few cognate major histocompatibility complex (