Pharmacologic Inhibition of Src Family Kinases Differentially Affects T Cell Cytokine Production and Proliferation: Implications for Novel Immunomodulatory Therapies.

It is well-recognized that the T cell receptor (TCR) signaling pathway, catalyzed by the Src family kinase (SFK) Lck, is the essential first step in T cell immune responses. The long-held goal of specifically targeting SFK activity for immunomodulatory therapy is becoming more of a reality as SFK inhibitors are entering into clinical use. In particular, dasatinib is an oral small molecule inhibitor of Abl and SFK, including Lck. Given the central importance of Lck in transmitting signals from the TCR signaling complex, and the potent ability of dasatinib to inhibit Lck activity, we hypothesized this agent could provide a novel route of immunomodulation via targeted inhibition of antigen-induced signaling, particularly in combination with currently available immunomodulatory agents. Using intracellular phospho-flow cytometry and western blotting, we show that low nanomolar concentrations of dasatinib potently inhibit TCR induced global protein tyrosine phosphoryation, including the key signaling elements CD3, ZAP-70, LAT, and PLC. In addition, the second messenger pathway Ras/Raf/MEK/ERK is potently blocked, whereas the PI3K-AKT pathway is minimally affected. Furthermore, induction of many pro-inflammatory cytokines, including TNF-α, IFN-γ, IL-2, GM-CSF, IL-17, IP-10, RANTES, and IL-6 (measured by cytokine antibody array) was significantly inhibited when the TCR was triggered in the presence of dasatinib. T cell proliferation, assessed by flow cytometric CFSE dilution analysis, was inhibited in a dose-dependent manner with dasatinib, showing complete inhibtion at 10 nM. Cell cycle analysis based on DNA content by PI staining revealed that dasatinib-treated T cells failed to enter the cell cycle. Considering that the PI3K-AKT pathway was only minimally affected by dasatinib, we examined the ability to achieve enhanced inhibition by using sub-optimal doses of dasatinib and rapamycin that individually produce mild-moderate inhibition of proliferation (20% and 40% inhibition, respectively). When dasatinib and rapamycin are combined at these lower doses, there was a consistently enhanced inhibitory effect on T cell proliferation (80–95% inhibition). Concurrent CD28 stimulation failed to overcome the inhibitory effects of SFK inhibition when the inhibitor was present throughout the stimulation. Interestingly, when SFK inhibition was delayed for at least 24 hours after the initiation of T cell activation with concurrent CD28 stimulation, we observed a divergent effect with regard to cytokine production and proliferation. Pro-inflammatory cytokine production was significantly decreased even if SFK activity was inhibited 48 hours after stimulating T cells, suggesting that ongoing TCR-dependent SFK activity is essential for cytokine production. However, late inhibition of SFK activity with dasatinib increased proliferation, with greater numbers of T cells achieving more rounds of cell division. Thus, while necessary for cytokine production, once the cells have entered the cell cycle, SFK activity regulates negative feedback in CD28-mediated proliferation. In conclusion, targeted inhibition of SFK activity is a promising approach for novel immunomodulatory therapy, particularly in combination with other signaling pathway inhibitors, but timing of SFK inhibition in relation to T cell activation could have an important impact on the immunomodulatory effect.