Regulatory T cell (Treg) therapy has the potential to induce transplantation tolerance so that immunosuppression and associated morbidity can be minimized. Alloantigen-reactive Tregs (arTregs) are more effective at preventing graft rejection than polyclonally expanded Tregs (PolyTregs) in murine models. We have developed a manufacturing process to expand human arTregs in short-term cultures using good manufacturing practice-compliant reagents. This process uses CD40L-activated allogeneic B cells to selectively expand arTregs followed by polyclonal restimulation to increase yield. Tregs expanded 100- to 1600-fold were highly alloantigen reactive and expressed the phenotype of stable Tregs. The alloantigen-expanded Tregs had a diverse TCR repertoire. They were more potent than PolyTregs in vitro and more effective at controlling allograft injuries in vivo in a humanized mouse model.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in certain tumor cells. In addition, TRAIL and chemotherapy can act cooperatively, possibly as a result of chemotherapy-induced increases in expression of a TRAIL receptor, DR5. We used cell lines derived from a highly chemoresistant tumor, malignant mesothelioma, to learn whether TRAIL was effective alone or together with chemotherapy and whether cooperativity depended on increases in DR5 expression. TRAIL (codons 95-285) was expressed in a bacterial expression vector and purified by nickel affinity chromatography. TRAIL alone (25 to 500 ng/ml) had little effect on mesothelioma cells. TRAIL plus chemotherapy (doxorubicin, cis-platinum, etoposide, or gemcitabine) acted cooperatively to induce apoptosis in mesothelioma cells (M28, REN, VAMT, and MS-1). For example, in M28 cells treated for 18 h, apoptosis from TRAIL (100 ng/ml) plus doxorubicin (0.6 microg/ml; 71 +/- 11%) greatly exceeded that from TRAIL alone (21 +/- 8%) or from doxorubicin alone (6 +/- 2%) (means +/- standard deviation; P < 0.03). Mesothelioma cells treated with chemotherapy showed no change in DR5 protein by Western analysis or by immunocytochemistry. TRAIL plus chemotherapy was associated with an increase in mitochondrial cytochrome c release and mitochondrial depolarization. We conclude that TRAIL and chemotherapy act cooperatively to kill mesothelioma cell lines, not by increases in DR5 receptor but in association with mitochondrial amplification of apoptotic signals.