Combining Established and Novel Immunomodulators: Synergistic Inhibition of Major T Cell Effector Functions by Dexamethasone and Dasatinib

Introduction. An increasing body of evidence suggests that in addition to their molecular targeted antitumor activity tyrosine kinase inhibitors (TKIs) exert immunomodulatory effects on T cells. Others (Blake et al 2008; Schade et al 2008) and we (Weichsel et al 2008) have recently shown that dasatinib (a multitargeting TKI) may lead to a complete inhibition of TCR dependent T cell effector functions via the SRC kinases LCK and FYN. Because TKIs act as competitive enzyme inhibitors with short half life, they permit better side effect control in the clinic. We investigated the impact of combining clinically relevant doses of dasatinib (1–100nM) with the established immunosuppressant dexamethasone (1–1000nM) on T cells.

Materials and Methods. Purified human CD3+ T cells from healthy blood donors were studied directly ex vivo. Functional outcomes assessed included cytokine production (IL-2), activation (CD69 upregulation), proliferation (CFSE dilution) and apoptosis/necrosis induction. EBV or CMV specific proliferation of antigen specific CD8+ T cells were evaluated applying tetramer technology. To distinguish between rapid non-genomic and genomic effects all assessments compared the impact of pre-treating T-cells with dexamethasone for 1 vs. 24h.

Results and Discussion. Complete inhibition of proliferation and activation occurred with dasatinib alone at levels of 50nM and above, whereas application of dexamethasone did not lead to a complete inhibition even at doses up to 1000nM. Dose-dependent inhibition of with the monoclonal CD3 antibody OKT3 induced T cell activation and proliferation was observed with the combination of dasatinib and dexamethasone. Strongest synergistic inhibitory effects of the drug combination were observed for OKT3 induced cytotoxic CD8+ T cell proliferation (mean±SEM given: OKT3 induced 63±5% proliferating T cells after 4 days incubation, OKT3 + 10nM dexamethasone 44±5%, OKT3 + 10nM dasatinib 39±10%, OKT3 + combination 16±4%; n=5; p<0.05). A significant inhibition of OKT3 induced up-regulation of the activation marker CD69 was demonstrated with the combination but not for dexamethasone alone (n=5). Our previously published data on dasatinib alone also showed inhibition of OKT3 induced up-regulation of CD69 expression, and this was potentiated in combination with dexamethasone. The pre-treatment time did not influence the dexamethasone effect except for increased reduction of IL-2 production after 24h vs. 1h pre-incubation. Overall, helper CD4+ T cells were more sensitive to the inhibitory effects of the drug combination regarding activation and proliferation than cytotoxic CD8+ T cells. Of note, synergistic effects occurred primarily in the different memory CD4 and CD8 T cell subsets but not in the naïve CD4 and CD8 T cells (e.g. for CD8+CD45RO+CD27+ memory T cells mean±SEM given for percentage of proliferating cells after 4 days: OKT3 92±1%, OKT3 + 100nM dexamethasone 77±2%, OKT3 + 10nM dasatinib 69±15%, and OKT3 + combination 27±11%; n=5; p<0.05). IL-2 production in purified T cells was significantly reduced (p<0.05) in a dose dependent nature for both dasatinib and dexamethasone compared to the OKT3 stimulated condition, either alone or in combination (n=5). Similarly, activation induced cell death (AICD) was significant reduced when the two drugs were combined, whereas no synergistic effects were observed regarding necrosis inhibition (n=5; p<0.05). In contrast, initial results suggested that dexamethasone did not inhibit clinically relevant EBV or CMV antigen specific CD8+ memory T cell proliferation when used alone and did not show synergistic effects with dasatinib (n=3). This may be due to a reduced sensitivity of the specific viral memory subsets towards dexamethasone.

Outlook and Conclusion. As viral reactivation especially with CMV is a major cause for morbidity after allogeneic stem cell transplantation, our results warrant further studies in vitro and in vivo. With an indication that each drug, when combined could be used at reduced dose, this research may then pave the way for synergistic uses of TKIs and glucocorticosteroids in treatment of graft versus host disease or autoimmune diseases potentially without increased risk of infections.