IFN-Alpha and gm-CSF Can Reverse Immunomodulatory Effects of Imatinib

Presently, Imatinib (IM) is the first line therapy for chronic myeloid leukaemia (CML), but in most patients CML recurs after discontinuation of IM. In contrast, allogeneic stem cell transplantation (ASCT) is considered to be the only curative treatment for this malignancy curing by the immune effects of donor T-cells against CML progenitor cells. Recent reports have described that IM has inhibitory effects on both the function of T-cells and antigen presenting cells (APCs) like dendritic cells. In the present study we further characterized the immunomodulatory effects of IM on T-cells and APCs and analysed whether IFN-alpha (interferon alpha, IFN-a) alone or in combination with GM-CSF (granulocyte-macrophage colony-stimulating factor) can influence the negative IM effects in vitro. Patient derived CML cells were examined to stimulate allogeneic HLA-mismatched T-cells without and with Imatinib (1, 2 or 5 micro M). The activation profile (CD25) and the expression of the TCR-alpha (T-cell receptor) on the T-cells was determined by FACS after 5 days in presence of IM as well as the proliferative T-cell response which was evaluated using CFSE (Carboxy Fluoroscein Succinimidyl Ester). In [51Cr]-release assays the cytotoxicity of CD8+ T-cells was assessed. The cytokine profile of the culture supernatants was detected by CBA (Cytokine bead array, Beckton Dickinson, USA). We also characterized the antigen profile (HLA-DR, CD40, CD54, CD58, CD80 and CD86) of the CML cells as APCs over a 5 day culture with and without IM. The rescue of the CML cells was initiated by IFN-a or IFN-a/GM-CSF after 3 days incubation with IM. On day 6 the APC profile of the CML cells was obtained and cells were used as stimulators in terms of T-cell proliferation which was determined by CFSE. We could show that the proliferation of allogeneic T-cell was inhibited in the presence of IM in a dose-dependent manner. In addition, the T-cell activation marker CD25 and the TCR-alpha expression were significantly downregulated by different concentrations of IM. Moreover, IM impaired the cytotoxic function of allogeneic T-cells in a dose-dependent manner. Interestingly, after stimulation with CML-cells cytotoxic T-cell activity could only be induced in those cases that did not secrete IL-6. Down-regulation of the APC profile on CML-cells (adhesion/costimulatory molecules) by increasing concentrations of IM could be in part reversed by the addition of IFN-a and completely restored by the combination of IFN-a and GM-CSF. Although, the proportion of proliferating T-cells could not be increased further by the combination of IFN-a ± GM-CSF, the number of T-cells was increased in the presence of IFN-a + GM-CSF. In conclusion, the down-modulating effects of IM could be almost completely reversed by the addition of IFN-a and GM-CSF. It remains to be seen whether these findings can be successfully applied to the treatment of CML-patients.