CD4+ Regulatory T Cells Specific for the WT1 Antigen Are Present in Acute Myeloid Leukemia Patients: Implication for Immunotherapy.

Compelling evidences indicate a key role for regulatory T cells (T_regs) on the host response to cancer and recent studies indicated that the generation of effective WT1-specific cytotoxic T cells can be largely affected by the presence of T_regs. This is the first study to describe human T_regs generated specifically against the WT1 antigen which is overexpressed in several human leukemias and provide the mechanism by which these anti-WT1 T_regs inhibit the immune response in leukemia patients. We have generated T cell lines and clones that specifically recognized a WT1-84 peptide in an HLA DRB1*0402/TCR-Vb8-restricted manner. Importantly, they recognized HLADRB1* 04-matched fresh leukemic cells expressing the WT1 antigen. These clones exerted a Th2 cytokine profile, had a CD4⁺CD25⁺Foxp3⁺GITR⁺CD127⁻ T_regs phenotype, and significantly inhibited the proliferative activity of allogeneic T cells independently of cell-contact. Priming of allo-reactive T cells in the presence of T_regs strongly inhibited the expansion of NK; NK-T and CD8⁺ T cells, had an inhibitory effect on NK/NK-T cytotoxic activity but not on CD8⁺ T cells. Furthermore, priming of T cells with the WT1- 126 HLA-A0201-restricted peptide in the presence of T_regs strongly inhibited the induction of anti-WT1-126 CD8+ CTL responses as evidenced by both very low cytotoxic activity and IFN-g production. Moreover, these T_regs clones specifically produced Granzyme-B and selectively induced apoptosis in WT1-84 pulsed-autologous APCs but not in apoptoticresistant DR4-matched leukemic cells. Importantly, we have also detected anti-WT1-84 IL-5⁺/Granzyme-B⁺/Foxp3⁺ CD4⁺ T_regs in 5 out of 8 HLA-DR4⁺ AML patients. These findings suggest a critical role for anti-WT1 T_regs in the inhibition of T cell responses against leukemia. This study may have important implications for the clinical manipulation of T_regs such as immuno-targeting of TCR-Vb-8 with mAbs to eliminate anti-WT1 T_regs in leukemia patients in order to enhance GVL before vaccination with the WT1 antigen. On the other hand, leukemia patients with GVHD should be clinically-tried for vaccination with the current WT1-84 peptide or adoptively-treated with ex-vivo anti-WT1 T_reg cells to specifically enhance their frequency, which is known to be very low in these patients.