Background Central memory T cells show superior persistence and antitumor immunity compared to effector memory and effector T cells. T effector cells respond quickly to tumors, but they are terminally differentiated and undergo apoptosis upon killing activity. T memory differentiate rapidly into T effector cells and maintain a pool of cells that can continuously differentiate thus sustaining a more lasting response. In adoptive cell therapy (ACT), T cells infused into patients may have a limited time of activity if they are terminally differentiated, and may rapidly undergo exhaustion and apoptosis. The development of new strategies based on novel agents able to generate memory T cells ex-vivo is important for a successful clinical application of ACT.We have studied the effect of a potent and selective HDAC6 inhibitor, ITF3756, on CD8 T cells differentiation during an in vitro induced exhaustion process.

Materials and Methods To induce exhaustion purified human CD8+ cells were stimulated twice with anti-CD3/CD28 beads (1:2) during 5 days, with or without ITF3756 1μM or 2μM added at all times of stimulation. At day 3 and 5 the expression of exhaustion, memory and effector T cells markers were analyzed by flow cytometry. Cells were also collected at day 5 for genes expression analysis. Expression of exhaustion, T phenotype, metabolic pathway and inflammatory cytokines were investigated by qPCR. Paired two-tailed t-tests was used to determine statistical significance between control versus treatment group at day 3 and 5 in 10 different donors. P-values ≤ 0.05 were considered significant.

Results ITF3756 1μM increased significantly the T central memory phenotype (CD45RO+CD62L+CCR7+) and decreased significantly the T effector phenotype (CD45RO+CD62L-CCR7-). The expression of CD62L in T central memory cells was significantly increased in agreement with the high expression of this marker in naïve and memory T cells. ITF3756 treatment decreased significantly the expression of exhaustion markers PD-1 and LAG-3. No effect was observed on TIM-3 expression. In agreement with the data obtained with protein analysis, treatment with ITF3756 reduced the mRNA level of Pd-1 and Lag-3. Gene expression of Tim-3 was also downmodulated, but this effect did not result in reduction of protein expression at the time of detection. ITF3756 reduced the expression of t-bet (Tbx21) driving T effector differentiation and increased genes related to T memory phenotype (Eomes, Lef-1 and albeit slightly, Tcf-7). T cell activation requires a metabolic reprogramming that supports highly proliferative phenotype and T effector differentiation. ITF3756 treatment decreased both Hif-1α and Glut-1 gene expression that are associated with TCR activation during the exhaustion process. T central memory cells produce less cytokines compared to T effector and effector memory cells. ITF3756 treatment decreased the genes expression of Il-2, Ifn-γ and Tnf-α. All these effects resulted dose dependent.

Conclusions The selective inhibitor of HDAC6 ITF3756 delays the terminal differentiation of CD8 T cells and increases the percentage of memory T cells with a reduced expression of exhaustion markers in vitro. These results are the basis to further explore the possible use of ITF3756 as a safe ex vivo treatment of CD8 T cells for adoptive cell transfer.

Disclosure Information C. Ripamonti: A. Employment (full or part-time); Significant; Italfarmaco SpA. C. Steinkuhler: A. Employment (full or part-time); Significant; Italfarmaco SpA. G. Fossati: A. Employment (full or part-time); Significant; Italfarmaco SpA.