1058 - T-cell Transcriptional and Mitochondrial Fitness Reprogramming in CLL Using PI3Kδ Inhibitors

T-cell dysfunction associated with chronic lymphocytic leukemia (CLL) presents a major barrier for successful adoptive cell therapy in patients. This problem is attributed to the phenotypic skewness of CLL T cells towards terminal differentiation phenotypes that, in most cases, cannot tolerate the ex vivo stimulation and expansion protocols. Importantly, a handful of recent reports have demonstrated metabolic defects in T cells from CLL patients which represent a new facet for the T-cell dysfunction problem in this disease. Nevertheless, the field is still lacking sufficient studies to validate these metabolic abnormalities, in order to correlate with the differentiation and transcriptional changes of T cells in the preclinical Eµ-TCL1 murine model.

PI3K signaling pathway is a main regulator of T-cell differentiation and metabolic programming following antigen recognition. Previous evidence has shown that T cells can maintain proliferative capacity following PI3K/AKT inhibition resulting in durable T cell phenotypes with enhanced functional capacity. Despite these observations, specific application of this knowledge in CLL is still required to improve adoptive cell therapies. In this study, 1) we utilized the Eµ-TCL1 preclinical model for uncovering the potential correlation between the mitochondrial/metabolic defects and the exhausted-like features of T cells in CLL and 2) we investigated the potential benefits of ex vivo priming of Eµ-TCL1 T cells. By using a PI3Kδ inhibitor, Idelalisib, we asked to improve the metabolic fitness and T-cell transcriptional reprogramming (more naïve/less-differentiated T-cell phenotypes with high persistence and effective cytotoxicity).

In this study, CLL was induced in mice via adoptive transfer (AT) of leukemic splenocytes from transgenic Eµ-TCL1 mice into wild type (WT) recipients. Multiparameter flow cytometry and transmission electron microscopy (TEM) analyses revealed the accumulation of small-sized depolarized mitochondria with significant changes in reactive oxygen species (ROS) levels in AT Eµ-TCL1 CD8+ T cells. These abnormalities were associated with the expansion of specific Eµ-TCL1 CD8+ T-cell populations with high EOMES and low TCF-1 expression, and a significant increase in effector phenotypes highly expressing immune suppressive markers such as PD-1 and Tim-3. Upon ex vivo activation of AT Eµ-TCL1 CD8+ T cells for 5 to 6 days using CD3/CD28 dynabeads and 60IU/ml hIL-2, we found significant loss in mitochondrial spare respiratory capacity (SRC), using mitochondrial stress test, compared to activated WT controls. Nanostring gene expression analysis comparing metabolic gene expression profiles of AT Eµ-TCL1 versus WT CD8+ T cells revealed upregulation of glycolysis, mitochondrial respiration, T-cell receptor and costimulatory pathways. On the other hand, there was a downregulation in autophagy, AMPK and amino acid transporter pathways.

For ex vivo reprogramming experiments, AT Eµ-TCL1 CD8+ T cells were primed with 5µM dose of idelalisib (CAL-101) and activated using CD3/CD28 dynabeads and 60IU/ml hIL-2 for 5 to 7 days. Idelalisib-treated Eµ-TCL1 T-cell cultures showed comparable expansion to vehicle-treated cells. Flow cytometric analysis showed significant increase in central memory CD8+ T-cell populations with marked CD62L expression intensity in idelalisib-treated cultures compared to more effector and terminally differentiated phenotypes in vehicle-treated counterparts. Interestingly, the idelalisib-induced effect was associated with a shift in the transcriptional signature of Eµ-TCL1 CD8+ T cells into significantly less EOMES and TOX expression and a significant upregulation of TCF-1. This indeed was accompanied by downregulation of immune checkpoint markers such as Tim-3 and KLRG-1. From a metabolic perspective, idelalisib treatment maintained high expression of PGC-1α in Eµ-TCL1 CD8+ T cells and resulted in an increase in oxygen consumption rate and mitochondrial SRC compared to the more terminally differentiated vehicle-treated cells. This increase in mitochondrial capacity was also demonstrated by the increase in mitochondrial membrane potential and ROS production.

Finally, to demonstrate the functional advantage of ex vivo CLL T-cell reprogramming, we performed a cytotoxicity study of idelalisib-treated cells against autologous Eµ-TCL1 CLL cells. Compared to vehicle controls, PI3K inhibition in Eµ-TCL1 T cells resulted in a significantly higher killing capacity at various effector: target ratios.

In conclusion, we characterized for the first time the mitochondrial defects of CD8+ T cells from the Eµ-TCL1 murine model and outlined the correlation of these defects with the T-cell transcriptional changes in this disease. Additionally, we demonstrated successful application of ex vivo PI3K inhibition on Eµ-TCL1 T cells for improving adoptive cell therapy product quality and effectiveness which can be translated into CLL patient studies.