In vitro co-stimulation with anti-CD28 synergizes with IL-12 in the generation of T cell immune responses to leukaemic cells; a strategy for ex-vivo generation of CTL for immunotherapy

Abstract

The existence of an immune based graft-versus-leukaemia (GvL) effect highlighted the prospect of managing relapsed leukaemias with T cell-based adoptive immunotherapy. Thus, various strategies have been explored for the in vitro expansion of acute myeloid leukaemia (AML)-specific T cells. In a popular approach, AML blasts have been genetically modified to express co-stimulatory molecules essential for effective T cell priming. One such tactic has been the modification of AML cells to express the B7/CD80 co-stimulatory molecule that binds to CD28 on T cells initiating events that culminate in enhanced cytokine production, proliferation and development of effector functions by T cells. The success of these strategies has been limited by difficulties in attaining sufficient transduction efficiencies and associated high levels of CD80 expression. We demonstrate that these problems can be circumvented by using anti-CD28 monoclonal antibody. Furthermore, we show that the synergistic relationship between CD80/CD28 pathway and interleukin 12 cytokine (IL-12), documented in the generation of cytotoxic T lymphocytes (CTL) for solid tumours, also applies to AML. CD28/IL-12 synergy facilitated the proliferation of allogeneic T cells in response to stimulation with primary AML blasts. The synergy also favoured generation of a Th1-type immune response, evidenced by gamma interferon (IFN-gamma) secretion and facilitated naive and memory T cell proliferation. Unlike some methods of in vitro T cell expansion, use of CD28/IL-12 synergy left T cells in the physiologically appropriate CD45RA-/CCR7- subsets known to be associated with immediate cytotoxic functions.

Fig. 1

Anti-CD28/IL-12 synergy drives the proliferation of allogeneic T cells to primary AML blasts. (a) Proliferation, as determined by [³H] thymidine uptake, of allogeneic T cells (2 × 10⁵ cells/well) stimulated with primary AML blasts (2 × 10⁴cells/well) in the presence of anti-CD28 antibody , IL-12 cytokine (▪) or anti-CD28 in combination with IL-12 cytokine (□). Fold increase was calculated relative to the proliferation of T cells stimulated with AML blasts alone. (b) The effect of anti-CD28 on allogeneic T cell response to AML blasts plus IL-12.

Fig. 2

Blastogenesis of T cells in response to (a) AML blasts alone; (b) AML blasts plus anti-CD28; (c) AML blasts plus IL-12; (d) AML blasts plus anti-CD28 and IL-12. The data are presented as flow cytometric dot plots of forward angle light scatter (indicative of cell size) versus CD3 expression. The percentages represent the CD3⁺ blast cells as a proportion of total CD3⁺ cells. This is representative of five experiments.

Fig. 3

Two cases of autologous T cell proliferation to AML blasts. AML 55 (▪) AML 65 (□).

Fig. 4

Anti-CD28/IL-12 synergy stimulates T cells to produce IFN-γ in response to primary AML blasts. IFN-γ production by CD3⁺ selected T cells, stimulated with AML blasts for 5 days in the presence of anti-CD28 alone, IL-12 alone or the two agents in combination. Percentage of cytokine-producing cells determined using MACs IFN-γ secretion assay and measured by flow cytometry.

Fig. 5

Intracellular perforin expression in CD3⁺/CD8⁺ T cells after stimulation with anti-CD28, IL-12 or a combination of both.

Fig. 6

Differential proliferative responses of CD45RA versus CD45RO T cells to allogeneic AML blasts: non-sorted T cells; (b) CD45RO⁺ T cells; (c) CD45RA⁺ T cells. Three separate AML blasts were tested, represented by the variously shaded bars.

Fig. 7

The CD45RA⁻/CCR7⁻ T cell subset proliferates following stimulation with AML blasts using the anti-CD28/IL-12 synergy. Expression of CD45RA and CCR7 by sorted CD3⁺ allogeneic T cells, stimulated for 5 days with AML blasts and anti-CD28/IL-12 (5 µg/ml anti-CD28) or PHA only (4 µg/ml).