PI3K-δ Inhibition Influences T-Cell Populations and Anti-Tumoral Immune Function in Preclinical Models

Abstract

Chronic lymphocytic leukemia (CLL) is characterized by continuously activated B-cell receptor (BCR) signaling. Phosphoinositide 3-kinase delta (PI3K-δ) is an important mediator of BCR signaling, which controls cell survival and proliferation through activation of AKT. The PI3K-δ inhibitor idelalisib (IDELA) is a highly effective treatment option for relapsed/refractory CLL patients. In the frontline setting, IDELA in treatment-naïve patients was associated with frequent adverse effects (AEs), such as an increased infection rate, colitis, and pneumonitis. Therefore, understanding the impact of PI3K-δ inhibition on the immune cells in the CLL microenvironment is of importance to identify novel treatment strategies to overcome these AEs.Methods and Results: In this study, we specifically focused on the effect of PI3K-δ inhibition on T-cell function in CLL using the Eµ-TCL1 adoptive transfer (TCL1 AT) model. In brief, Eµ-TCL1 tumor cells were transferred intravenously into syngeneic littermates, followed by continuous treatment after reaching a tumor load of 50% in peripheral blood. GS-649443, a tool compound with more favorable murine pharmacokinetic properties than IDELA, was used for PI3K-δ inhibition in vivo . Tumor load, as well as the immune microenvironment, was analyzed after 2 weeks of continuous treatment of the drug.Since PI3K-δ signaling has been previously demonstrated to be important for CD4+ regulatory T-cell (Treg)-mediated suppression of antitumor immunity, we studied the impact of PI3K-δ inhibition on differentiation and activation of Tregs. GS-649443 treatment resulted in a dramatic reduction in numbers of Foxp3+ CD25+ CD4+ Tregs, as well as a decrease in Treg maturation and activation markers (CD103, GITR, KLRG1, and CD69) compared to vehicle treatment.We further assessed the impact of PI3K-δ inhibition on TCL1 AT-induced changes in CD8+ T-cell differentiation and subset distribution. Notably, treatment with GS-649443 resulted in a dose-dependent decrease in effector T-cell numbers, accompanied by a decrease in activation markers such as CD69 and CD137. In line with these findings, effector cell proliferation, as measured by Ki-67, significantly decreased in GS-649443-treated mice. In accordance with that, degranulation capacity, granzyme B, and IFNγ production by CD8+ T cells was substantially reduced by GS-649443 treatment. These results were further confirmed in a treatment study with PI-3065, another PI3K-δ-specific inhibitor.To investigate a direct impact of PI3K-δ inhibition on CD8+ T cells, we performed in vitro studies with GS-649443. For that purpose, we used splenocytes from Nur77-GFP mice, a reporter mouse model for T-cell receptor (TCR) activity, and stimulated these with aCD3 antibody after pre-treatment with GS-649443 or vehicle. PI3K-δ inhibition led to a profound reduction of TCR signaling, which was accompanied by decreased expression of activation markers like CD69, CD44, CD25, and CD137 on CD8+ T-cells. Furthermore, GS-649443 pre-treatment resulted in significant dose-dependent decrease in CD8+ T-cell proliferation rates, as measured by CFSE dilution. These findings were confirmed using aCD3-stimulated human peripheral blood mononuclear cells that were pretreated with IDELA in vitro, which resulted in an impairment of CD8+ T-cell activation measured by CD69 and CD25 expression, as well as proliferation measured by CFSE dilution.Conclusion: This study clearly shows that PI3K-δ inhibition abrogates the immunosuppressive activity of Tregs in an in vivo model of CLL. Additionally, PI3K-δ inhibition of CD8+ T cells leads to decreased activation and proliferation of these cells, which might contribute to the immune-mediated AEs observed with IDELA when used as frontline treatment. DisclosuresStilgenbauer:Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding.