Molecular, Epigenetic, and Phenotypic Repolarization of T Lymphocytes From Chronic Lymphocytic Leukemia Patients Using 5-Aza-2′-Deoxycytidine

Abstract 4651

The improper polarization of helper and cytotoxic T lymphocytes is a trademark immunosuppressive strategy employed by chronic lymphocytic leukemia (CLL). Mounting data implicates chromatin regulation, namely promoter methylation, in the plasticity of naïve human T cells. Very recent in-vitro evidence indicates that this plasticity may be phenotypically replicated by using methylation inhibitors which are approved for clinical use in certain types of cancer, however the longevity and therapeutic potential of repolarized cells remains to be elucidated. These results beg the question: can the ineffective polarization of T lymphocytes in the context of CLL be effectively modulated using methylation inhibitors in a sustainable therapeutic fashion? To answer this question our laboratory has focused on repolarizing helper and cytotoxic T lymphocytes using 5-aza-2′-deoxycytidine (5A2) tracking changes in well characterized molecular and epigenetic signaling pathways involved in effective polarization. Moreover, we sought to investigate the consequences of methylation inhibitor treatment on lymphocyte survival, activation intensity, and naïve cell polarization. Our studies relied upon intracellular staining, methylation specific PCR, western blot, and phosflow anlaysis. The data indicates that 5A2 treatment can repolarize Th2 cells to effectively secrete interferon gamma, signal via T-bet, and achieve demethylation of critical Th1 specific promoters. We also show that this repolarization can be maintained. Moreover, we demonstrate that 5A2 can force Th1 polarization of naïve T cells despite a strong IL-4 stimuli and a lack of IL-12. Finally, we have identified a cytotoxic threshold for 5A2 treatment which is above the concentration necessary to achieve effective and sustained molecular and epigenetic repolarization. All in all our data seeks to define a modality in which improper or ineffective T cell polarization can be altered ex post facto and continued immunotherapeutic intervention may benefit from the naïve polarization of anti-cancer responses.