Rapid expansion of T cells: Effects of culture and cryopreservation and importance of short-term cell recovery.

BACKGROUND: Successful cell therapy relies on the identification and mass expansion of functional cells for infusion. Cryopreservation of cells is an inevitable step in most cell therapies which also entails consequences for the frozen cells. MATERIAL AND METHODS: This study assessed the impact of cryopreservation and the widely used protocol for rapid expansion of T lymphocytes. The effects on cell viability, immunocompetence and the impact on apoptotic and immunosuppressive marker expression were analyzed using validated assays. RESULTS AND CONCLUSION: Cryopreservation of lymphocytes during the rapid expansion protocol did not affect cell viability. Lymphocytes that underwent mass expansion or culture in high dose IL-2 were unable to respond to PHA stimulation by intracellular ATP production immediately after thawing (ATP = 16 ± 11 ng/ml). However, their reactivity to PHA was regained within 48 hours of recovery (ATP = 356 ± 61 ng/ml). Analysis of mRNA levels revealed downregulation of TGF-ß and IL-10 at all time points. Culture in high dose IL-2 led to upregulation of p73 and BCL-2 mRNA levels while FoxP3 expression was elevated after culture in IL-2 and artificial TCR stimuli. FoxP3 levels decreased after short-term recovery without IL-2 or stimulation. Antigen specificity, as determined by IFNγ secretion, was unaffected by cryopreservation but was completely lost after addition of high dose IL-2 and artificial TCR stimuli. In conclusion, allowing short-time recovery of mass expanded and cryopreserved cells before reinfusion could enhance the outcome of adoptive cell therapy as the cells regain immune competence and specificity.

AdCD40L immunogene therapy for bladder carcinoma--the first phase I/IIa trial.

PURPOSE: Immunotherapy with Bacillus Calmette-Guerin (BCG) instillation is recommended for high-risk, non-muscle invasive bladder cancer. Bacillus Calmette-Guerin is not effective in advanced tumors, and better alternatives are warranted. Immunostimulating gene therapy with adenoviral vectors expressing CD40 ligand (AdCD40L) has shown efficacy in tumor models. CD40 ligand stimulates systemic immunity and may be effective in local and invasive human disease. EXPERIMENTAL DESIGN: Patients with invasive bladder cancer scheduled for cystectomy or patients with T(a) tumors were enrolled in a phase I/IIa trial. Patients were treated with three cycles of intrabladder Clorpactin WCS-90 prewash, followed by AdCD40L instillation 1 week apart. Safety, gene transfer, immune effects, and antitumor responses were monitored. RESULTS: All eight recruited patients were treated as scheduled, and therapy was well tolerated. The main adverse effect was transient local pain during prewash. Postoperatively, urinary tract infections and one case of late septicemia with elevated potassium were reported. No adverse events were ascribed to vector therapy. Gene transfer was detected in biopsies, and bladders were heavily infiltrated with T cells. The effector marker IFN-gamma increased in biopsies, whereas levels of circulating T regulatory cells were reduced. Histologic evaluation indicated that AdCD40L therapy reduced the load of malignant cells. CONCLUSIONS: To our knowledge, this is the first report on immunogene therapy in bladder cancer and the first using AdCD40L in vivo. Local AdCD40L gene therapy was safe, boosted immune activation, and should be further evaluated as a single or an adjuvant therapy for urothelial malignancies.

CpG oligonucleotide therapy cures subcutaneous and orthotopic tumors and evokes protective immunity in murine bladder cancer.

Bacillus Calmette-Guerin (BCG) instillation is standard immunotherapy for superficial bladder carcinoma. However, many patients become refractory to BCG, giving impetus to the development of alternative therapies. CpG oligodeoxynucleotide (ODN) therapy has been shown to promote T(H)1-oriented antitumor responses in various tumor models. To investigate its therapeutic effect in bladder cancer, we used different CpG ODNs to treat C57BL/6 mice bearing the subcutaneous murine bladder tumor MB49. CpG type B ODN 1668 was superior at inhibiting tumor growth, leading to complete regression of large tumors. More importantly, CpG ODN 1668 also regressed orthotopically growing MB49 tumors for the first time. Rechallenge of CpG ODN-cured mice with MB49 showed that a majority of the mice were protected long term, demonstrating that CpG ODN therapy evokes a memory response. Adenoviral vectors (Ad) encoding CD40L, tumor necrosis factor-related activation-induced cytokine, lymphotactin, interleukin (IL) 2, and IL-15 were also investigated. AdCD40L and AdIL-15 transduction could abolish MB49 tumorigenicity, and these vectors were combined with CpG ODN 1668 to investigate any enhanced effects. No such effects were seen. All groups of mice treated with CpG ODNs, alone or in combination with adenoviral vector, exhibited increased serum concentrations of IL-12, indicative of a T(H)1 response. Our results show that CpG ODN therapy cures established subcutaneous and orthotopic bladder cancer via a T(H)1-mediated response and provides long-lasting protective immunity.