Bortezomib Induces Selective Depletion of Activated T Lymphocytes and Modifies the Cytokine’s Production Pattern.

The NF-kB family has emerged as a key transducer of inflammatory signals involved in dendritic cell maturation and T lymphocyte activation. Accordingly, the diverse signaling pathways downstream of TCR/CD3 converge on several transcription factors including NFAT, AP-1 and NF-kB. We explored the ability of the proteosome inhibitor bortezomib, which prevents Nuclear Factor kB (NF-kB) activation, to block T-cell activation, proliferation and survival within alloreactive as compared to resting T-cells. For this purpose, T-cells were stimulated with PHA, aCD3/aCD28, allogeneic dendritic cells (APC) or through mixed lymphocyte cultures. NF-kB expression increased in activated T-lymphocytes as compared to resting T-cells. Interestingly, the higher the NF-kB expression, the more intense the proliferative blockade induced by bortezomib. This effect on proliferation was due to a selective induction of apoptosis among activated T-cells. Thus, after mixed lymphocyte reaction (MLR) cultures, alloreactive T-cells were 2 logs more sensitive to bortezomib-induced apoptosis than the resting T-cell counterpart as assessed by annexin / 7AAD staining. This effect of bortezomib was partially blocked by adding the caspase inhibitor Z-VAD to the culture. The addition of bortezomib not only decreased the proliferation and viability of activated T-lymphocytes but also the levels of IFN-g and IL-2, which were significantly decreased among activated T-cells cultured with bortezomib at doses ranging from 10 to 100 nM. Secondary mixed lymphocyte cultures demonstrated a selective loss of alloreactive T-cells while activation and proliferation against third party donor was partially preserved.

In conclusion, at concentrations reached in the clinical setting, bortezomib induces selective apoptosis and decreases Th1 response among alloreactive T-lymphocytes while it barely affects unstimulated T-cells. These results establish the basis for the clinical use of bortezomib in the management of GVHD