Killer artificial antigen-presenting cells: a novel strategy to delete specific T cells

Abstract

Several cell-based immunotherapy strategies have been developed to specifically modulate T cell-mediated immune responses. These methods frequently rely on the utilization of tolerogenic cell-based antigen-presenting cells (APCs). However, APCs are highly sensitive to cytotoxic T-cell responses, thus limiting their therapeutic capacity. Here, we describe a novel bead-based approach to modulate T-cell responses in an antigen-specific fashion. We have generated killer artificial APCs (kappaaAPCs) by coupling an apoptosis-inducing alpha-Fas (CD95) IgM mAb together with HLA-A2 Ig molecules onto beads. These kappaaAPCs deplete targeted antigen-specific T cells in a Fas/Fas ligand (FasL)-dependent fashion. T-cell depletion in cocultures is rapidly initiated (30 minutes), dependent on the amount of kappaaAPCs and independent of activation-induced cell death (AICD). kappaaAPCs represent a novel technology that can control T cell-mediated immune responses, and therefore has potential for use in treatment of autoimmune diseases and allograft rejection.

Figure 1

Composition and phenotypical characterization of κaAPC and control beads (CB). (A) Composition of a HLA-A₂ IgG1–based κaAPC and CB. (B) FACS analysis of IgM (α-Fas mAb; left panels) and IgG1 (HLA-A₂ IgG1; right panels) immobilized on CBs and κaAPCs. ■ represents the isotype control, whereas □ indicates α-Fas mAb (clone CH11) and HLA-A₂ IgG1.

Figure 2

Elimination of CMVpp65-specific CTLs by ^CMVpp65κaAPCs. (A) An example of CMVpp65-specific CTLs used throughout the experiments. CTLs were specifically stained with CMVpp65 tetramer (88.84%). Mart-1 tetramer (0.19%) served as control stain. (B) Elimination of (▓) CMVpp65- or (▒) Mart-1–specific CTLs from different donors in cocultures with CBs or κaAPCs loaded with CMVpp65 or Mart-1 peptide. Cocultures with unloaded CBs or κaAPCs served as negative controls. Numbers of viable cells (% viable cells = (% treated cells × 100%) /% untreated cells) after 48 hours of coculture are depicted (means ± SD) of 5 independent experiments.

Figure 3

Cell/bead contact does not lead to an activation of antigen-specific CTLs and subsequent AICD. Activation in CMVpp65-specific CTLs of a single donor cocultured with ^Mart-1CBs, ^CMVpp65CBs, ^Mart-1κaAPCs, or ^CMVpp65κaAPCs. CTL cultures stimulated with 7.5 ng/mL soluble CMVpp65 peptide served as positive control. Numbers of CD107a⁺ cells were determined 5 hours following initiation of cocultures. One representative experiment of 3 is shown.

Figure 4

Elimination of antigen-specific CTLs by κaAPCs is strongly ratio dependent. CTLs were cocultured with unloaded (circles), Mart-1–loaded (squares), and CMVpp65-loaded (triangles) CBs or κaAPCs. Closed symbols represents CTLs cocultured with CBs, whereas open symbols indicate CTLs cocultured with κaAPCs. Different bead-CTL ratios are indicated at the x-axis. Numbers of viable CTLs; (% viable cells = (% treated cells × 100%)/% untreated cells were determined by Annexin V and PI staining 48 hours following initiation of cocultures. One representative experiment of 3 is shown.

Figure 5

Elimination of CTLs occurs early during interaction with κaAPCs. CMVpp65-specific CTLs were cocultured with different loaded or unloaded bead preparations (x-axis). At indicated time points (30, 60, and 120 minutes following initiation of cocultures), beads were magnetically removed and CMVpp65-specific CTLs were further incubated. After 48 hours, numbers of viable CTLs, (% viable cells = (% treated cells × 100%)/% untreated cells were determined as described in the experimental protocols. CMVpp65-specific CTLs cocultured for 48 hours with the indicated beads served as positive control.

Figure 6

κaAPCs eliminate CTLs in an antigen-specific fashion. PKH26-labeled activated Fas⁺ effector-memory CTLs (■) and PKH67-labeled CMVpp65-specific CTLs (▒) from the same donor were mixed to a 1:1 ratio and cocultured with ^CMVpp65κaAPCs for 48 hours. Control cultures were treated with ^unloadedκaAPCs or 1 μg/mL of soluble α-Fas IgM (clone CH11). Minimal loss of viable cells was determined in untreated mixed CTL cultures (T_mix). Numbers of viable CTLs, (% viable cells = (% treated cells × 100%)/% untreated cells) for each CTL population of the T_mix are depicted. The illustrating graph is derived from 1 representative experiment out of 3 independent experiments.