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.

The programmed death (PD)-1/PD-ligand 1 pathway regulates graft-versus-host-reactive CD8 T cells after liver transplantation.

Acute graft-versus-host disease (aGVHD) is a life-threatening complication after solid-organ transplantation, which is mediated by host-reactive donor T cells emigrating from the allograft. We report on two liver transplant recipients who developed an almost complete donor chimerism in peripheral blood and bone marrow-infiltrating T cells during aGVHD. By analyzing these T cells directly ex vivo, we found that they died by apoptosis over time without evidence of rejection by host T cells. The host-versus-donor reactivity was selectively impaired, as anti-third-party and antiviral T cells were still detectable in the host repertoire. These findings support the acquired donor-specific allotolerance concept previously established in animal transplantation studies. We also observed that the resolution of aGVHD was not accompanied by an expansion of circulating immunosuppressive CD4/CD25/FoxP3-positive T cells. In fact, graft-versus-host-reactive T cells were controlled by an alternative negative regulatory pathway, executed by the programmed death (PD)-1 receptor and its ligand PD-L1. We found high PD-1 expression on donor CD4 and CD8 T cells. In addition, blocking PD-L1 on host-derived cells significantly enhanced alloreactivity by CD8 T cells in vitro. We suggest the interference with the PD-1/PD-L1 pathway as a therapeutic strategy to control graft-versus-host-reactive T cells in allograft recipients.