Exploration of the lysis mechanisms of leukaemic blasts by chimaeric T-cells

Abstract

Adoptive transfer of specific cytotoxic T lymphocytes (CTL) and Cytokine Induced Killer Cells (CIK) following genetic engineering of T-cell receptor zeta hold promising perspective in immunotherapy. In the present work we focused on the mechanisms of anti-tumor action of effectors transduced with an anti-CD19 chimaeric receptor in the context of B-lineage acute lymphoblastic leukemia (B-ALL). Primary B-ALL blasts were efficiently killed by both z-CD19 CTL and z-CD19 CIK effectors. The use of death receptor mediated apoptosis of target cells was excluded since agonists molecules of Fas and TRAIL-receptors failed to induce cell death. Perforin/granzyme pathway was found to be the mechanism of chimaeric effectors mediated killing. Indeed, cytolytic effector molecules perforin as well as granzymes were highly expressed by CTL and CIK. CD19 specific stimulation of transduced effectors was associated with degranulation as attested by CD107 membrane expression and high IFN-gamma and TNF-alpha release. Moreover inhibitors of the perforin-based cytotoxic pathway, Ca(2+)-chelating agent EGTA and Concanamycin A, almost completely abrogated B-ALL blast killing. In conclusion we show that the cytolysis response of z-CD19 chimaeric effectors is predominantly mediated via perforin/granzyme pathway and is independent of death receptors signaling in primary B-ALL.

Figure 1

Killing of SupB15 (positive control) and tumor cells. The experiment is representative of 5 independent experiments. Similar results were produced with 3 donors of CTL and 2 donors of CIK.

Figure 2

Death receptors expressed by Jurkat, SupB15, or Raji cell lines and B-ALL blasts. (a) Percentages of cells expressing Fas. (b) Right histograms represent percentage of cells expressing TRAIL-R1 or -R2 agonists death receptors (DR4 or DR5) and left histogram decoy antagonist TRAIL-R3 or -R4 receptors (DcR1 or DcR2).

Figure 3

Apoptosis induced on Jurkat, SupB15, or Raji cell lines and B-ALL blasts by agonist antibody anti-Fas (clone CH11) or recombinant human TRAIL (killerTRAIL) measured after 4 hours and 18 hours exposition. Isotype control (Ig) is indicated.

Figure 4

z-CD19 chimaeric effectors express mediators of cytotoxicity. (a) CTL and CIK effectors were stained with antigranzyme A, -granzyme B or -perforin antibodies. Transfectant expressions were detected by a goat antihuman IgG (GAH) labeling on CD3⁺ cells. Percentages and MFI are indicated from CD3⁺ cells. (b) CTL and CIK effectors were cocultured in the presence of SupB15 CD19⁺ stimulating cells or Primary B-ALL blasts (AmMax, RoNir and SpSeb). Degranulation of effectors was analyzed by anti-CD107a and -CD107b antibodies gated on CD3⁺ cells. Similarly, IFN-γ and TNF-α specifically produced by responding effectors were analyzed on CD3⁺ gated cells. Similar results were produced with 2 donors of CTL and CIK.

Figure 5

Specific Lysis of z-CD19 chimaeric effectors is inhibited in the presence of perforin/granzyme inhibitors. Cytolytic activity of effectors was analyzed against SupB15 or Raji cell lines and B-ALL blasts (control) or in the presence of EGTA/MgCl₂ or Concanamycin A.