In vitro stimulation and expansion of human tumour-reactive CD8+ cytotoxic T lymphocytes by anti-CD3/CD28/CD137 magnetic beads.

Adoptive immunotherapy with tumour-reactive CD8(+) cytotoxic T lymphocytes (CTLs) requires efficient in vitro approaches allowing the expansion of CTLs to large numbers prior infusion. Here, we investigated the antigen-independent activation and the expansion of human T cells in peripheral blood mononuclear cells (PBMCs) and in tumour-reactive CTLs using Dynabeads coated with monoclonal antibodies to CD3 and to the costimulatory molecules CD28 and CD137 (4-1BB). T cells in PBMCs showed an increased expansion rate of 15- to 17-fold during a 2-week culture period using antibody-conjugated beads with interleukin-2 (IL-2) added versus IL-2 alone. No significant difference between CD3/CD28 beads and CD3/CD28/CD137 beads was observed (P = 0.4). In contrast, expansion of tumour-reactive CD8(+) CTLs over 2 weeks was more efficient using CD3/CD28/CD137 beads (14.4-fold ± 1.2) compared with CD3/CD28 beads (10.6-fold ± 0.7) (P = 0.03) and matched well to the control arm using weekly stimulation with tumour cells. Although all modes of in vitro stimulation decreased the expression of central memory markers CD62L and CCR7 on CTLs, bead-activated cultures expressed consistently higher levels than tumour-stimulated cultures. CTLs analysed after bead-induced expansion versus weekly tumour stimulation showed equal IFN-γ production in ELISPOT assay. Furthermore, cytotoxicity assays demonstrated an either unchanged or slightly reduced capability of tumour cell lysis for antigen-independent stimulated CTLs versus those that maintained on weekly tumour stimulation, regardless of which type of beads was used. Our data suggest that the conjugation of anti-CD137 antibodies to conventional CD3/CD28 beads results in a minor but significant increase in the expansion capacity for tumour-reactive CD8(+) CTLs.

Surface expression of transglutaminase 2 by dendritic cells and its potential role for uptake and presentation of gluten peptides to T cells.

Celiac disease is a chronic small intestinal inflammation driven by gluten-reactive T cells of the intestinal mucosa. These T cells are HLA-DQ2 or -DQ8 restricted, and predominantly recognize gluten peptides that are deamidated by the enzyme transglutaminase 2 (TG2). Our recent results strongly suggest that duodenal CD11c(+) dendritic cells (DC) are directly involved in T cell activation in the celiac lesion. The aim of this study was to investigate whether surface-associated TG2 could be involved in receptor-mediated endocytosis of gluten peptides, a process that may contribute to the preferential recognition of deamidated peptides. We found that both monocyte-derived DC and local CD11c(+) DC in the duodenal mucosa expressed cell surface-associated TG2. As phenotypic characterization of CD11c(+) DC in the celiac lesion suggests that these cells may be derived from circulating monocytes, we used monocyte-derived DC in functional in vitro studies. Using a functional T cell assay, we obtained evidence that cell surface-associated TG2 is endocytosed by monocyte-derived DC. However, we were unable to obtain evidence for a role of surface TG2 in the loading and subsequent generation of deamidated gluten peptides in these cells.

Troybodies and pepbodies.

All antibodies (Abs) with effector function are produced in mammalian cells, whereas bacterial production is restricted to smaller targeting fragments (scFv and Fab) without effector functions. In this project, we isolated different peptides that bind one of several Ab effector molecules. We have developed bacterial expression vectors for direct cloning of these peptides as fusions to scFv and Fab, and have obtained targeting fragments that also have the ability to bind Ab effector molecules. Some of these fusions (pepbodies) may also initiate Ab effector functions. We have also genetically inserted T-cell epitopes into Abs with specificity for antigen-presenting cell (APC) surface molecules to target the Ab-T-cell epitope fusions (Troybodies) to APCs. The approach is to exchange loops in Ig constant domains with single copies of well-defined T-cell epitopes. We have shown that a number of such T-cell epitopes are loaded on to MHC class II on APCs and are presented to specific T-cells. An increase in T-cell activation of up to four orders of magnitude is achieved compared with synthetic peptide. Our current goal is to identify all the loops in all Ig constant domains that may be loaded with T-cell epitopes to produce a multi-vaccine.