Induction of regular cytolytic T cell synapses by bispecific single-chain antibody constructs on MHC class I-negative tumor cells
Introduction
Among the various immune mechanisms the body can mount against tumor cells, cytotoxic T cell responses play a dominant role (Boon et al., 1994, Nagorsen et al., 2003). This is particularly evident from the growing number of T cell evasion mechanisms being discovered in late-stage tumor cells. Loss of MHC class I (Bubenik, 2003) and costimulatory molecules (Stopeck et al., 2000), overexpression by tumor cells of cytokines interfering with Th1 cell development (Gorelik and Flavell, 2001, Urosevic et al., 2001), of granzyme inhibitory serpins (Bladergroen et al., 2002), pro-apoptotic ligands (Dong et al., 2002, Lee and Ferguson, 2003), and a tryptophane-degrading enzyme (Uyttenhove et al., 2003) are examples of evasion mechanisms predominantly directed against cytotoxic T cells.
An elegant bypass for certain evasion mechanisms could be the direct recruitment of T cells to tumor cells via bispecific antibodies designed to bind a common T cell signaling molecule and, simultaneously, a surface molecule on tumor cells (Segal et al., 1999). Ideally, this will lead to a potent polyclonal T cell response against all tumor cells bearing the surface antigen, which should be uniquely expressed or accessible on tumor cells but not on normal cells. Countless efforts were made to design such bispecific antibodies but few met the requirements of target-dependent cytotoxicity, decent productivity, independence on costimulation, and necessary potency. One kind of bispecific antibodies are bispecific T cell engagers (BiTEs) (Baeuerle et al., 2003). BiTEs are small recombinant antibody constructs based on two tandemly arranged single-chain antibodies. They show extreme cytotoxic potency in a low picomolar range, work at very low effector:target (E:T) ratios, only activate T cells in the presence of target cells, can be produced in sufficient amounts and show high anti-tumor activity in animal models (Dreier et al., 2003, Dreier et al., 2002). The anti-CD3 domain of BiTEs recognizes the epsilon subunit of the CD3 complex. A BiTE molecule directed against the pan-B cell antigen CD19 and the CD3 complex (Loffler et al., 2003, Loffler et al., 2000) is in early clinical development for the treatment of B cell malignancies. The basis for the extreme potency of BiTEs is not fully understood. Complete protection of target cells from BiTE-induced T cell cytotoxicity by a calcium chelator suggested a role of perforin (Loffler et al., 2000).
Immunological synapses were first described for the interface between naive CD4 T cells and antigen-presenting cells (APCs) (for a review see (Dustin, 2003)), where they are required for T cell activation. Immunological synapses are composed of a ‘central supramolecular activation cluster’ (cSMAC) (Anton van der Merwe et al., 2000) containing essential signaling molecules. cSMACs are surrounded by a domain of heterotypic cell adhesion molecule interactions referred to as ‘peripheral supramolecular activation cluster’ (pSMAC). Cytolytic synapses are formed between cytotoxic T lymphocytes (CTLs) and target cells. They resemble immunological synapses made between APCs and CD4 T cells, but contain in their cSMAC in addition to the signaling domain a secretory domain for the focused release of cytotoxic molecules like perforin and granzymes (Stinchcombe et al., 2001).
Here we performed a confocal immunofluorescence microscopy analysis of cytolytic synapses formed between human cytotoxic CD8 T cells and human tumor cell lines to gain insight into the structural basis for BiTE-induced T cell cytotoxicity. By choosing a particular T cell/target cell system we could induce the formation of cytolytic synapses either by a T cell peptide in the correct T cell receptor/MHC class I context, or, by addition of a BiTE recognizing both CD3 and the pan-carcinoma antigen epithelial cell adhesion molecule Ep-CAM (Armstrong and Eck, 2003, Balzar et al., 1999). We also studied whether BiTEs can induce functional cytolytic synapses between human T cells and target cells lacking MHC class I.
Section snippets
Cell preparation and culture
The erbB2 peptide-specific CD8-positive T cell clone SuHie7 (Knabel et al., 2002) was cultivated in T cell medium (RPMI 1640, 7.5% human serum, 7.5% fetal calf serum, 1% non-essential amino acids, 1% penicillin/streptomycin, 1% glutamine, 1% sodium pyruvate, 200 U/ml IL2) and expanded by restimulation. Restimulations were performed with 1 × 105 SuHie7 T cells, 2.5 × 107 irradiated PBMC (5000 rad) and 5 × 106 irradiated EBV immortalized B cells (10,000 rad) as feeder cells in 25 ml T cell medium. IL-2 was
Target cell lysis induced by either T cell peptide antigen or Ep-CAM-specific BiTE
For our experiments, we selected the human CD8-positive T clone SuHie7, which has a T cell receptor with specificity for a peptide derived from the ErbB2 tyrosine kinase receptor and is restricted to MHC class I molecule HLA A 0201 (Knabel et al., 2002). The lytic activity of SuHie7 T cells was initially tested with the lymphoma line T2 as target. ErbB2 peptide-loaded (at 15 μg/ml) T2 cells were specifically lysed after 4 h to 50 and 70% at effector-to-target (E:T) ratios of 3:1 and 10:1,
Discussion
Immunological synapses have in recent years been recognized as key subcellular structures for specific T cell function and activation. There are essentially two types of T cell synapses: stimulatory synapses that form between naïve T cells and antigen-presenting cells, and lytic synapses that form between cytotoxic effector T cells and their target cells. Very recent studies have dissected in detail the structure/function relationships and kinetics of specific synapse function (Faroudi et al.,
Acknowledgements
We thank D. Zink and N. Sadoni for help in confocal laser microscopy and S. Zeman for critical reading of the manuscript. We are also very grateful to H. Bernhard for providing the SuHie7 T cell clone.
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