Abstract 703: Transient G1 cell cycle arrest with trilaciclib enhances the generation of polyfunctional CD4+ and CD8+ T cells
Authors: Ahn Sarah , Cao Subing , Russo Melissa A. , Yi John S. 
Published: April 2023

Abstract
When administered prior to chemotherapy, intravenous trilaciclib transiently arrests cyclin-dependent kinase 4/6-dependent cells in the G1 phase of the cell cycle, which has been shown to augment antitumor immunity. In an open-label, phase 2 trial in patients with metastatic triple-negative breast cancer (NCT02978716), administering trilaciclib prior to gemcitabine plus carboplatin improved overall survival, potentially through protection and direct activation of immune function. To determine the impact of trilaciclib on effector T-cell function, we conducted in vitro studies to assess the kinetics of cytotoxic function and generation of polyfunctional T cells.
Peripheral blood mononuclear cells (PBMCs) or naïve CD4+ and CD8+ T cells were purified from 6 healthy human donors and activated with CD2/3/28 beads. On days 0, 1, and 3 post activation, 100 nM trilaciclib was added to the cells. To visualize phenotypic and functional changes, T cells that had been activated for 3, 7, or 14 days with or without trilaciclib were restimulated with phorbol myristate acetate (PMA) and ionomycin. Dimension-reduction analysis of flow cytometric data was used to identify polyfunctional subpopulations of CD4+ and CD8+ T cells. T-cell clusters with polyfunctional profiles were identified based on the coproduction of IFNγ, TNFα, and IL-2 cytokines. Subsets of polyfunctional clusters were defined according to other immune phenotypic profiles, including T-cell exhaustion (PD-1 and LAG-3), degranulation (granzyme B), proliferation (Ki-67), and transcription factors (TOX, TCF-1, and T-bet).
For both CD4+ and CD8+ T cells, 3 polyfunctional subpopulations were identified based on differential expression of granzyme B, PD-1, and TOX. When CD4+ and CD8+ T cells were activated in the presence of trilaciclib and restimulated with PMA and ionomycin, there was an increase in the frequency of polyfunctional T-cell subsets producing granzyme B. This was compensated by a decrease in granzyme Blow polyfunctional cells, which was associated with increased PD-1high and TOXhigh expression. Clustering analysis of trilaciclib-treated PBMCs also revealed a reduction in proliferating CD8+ T cells with undetectable effector cytokine function based on a decrease in the frequency of CD8+ T cells expressing TOXhigh and Ki-67high.
Transient G1 cell cycle arrest in T cells may limit T-cell hyperactivation, which has been shown to induce immunopathological responses. The increased generation of polyfunctional CD8+ T cells is associated with enhanced effector T-cell responses and memory T-cell differentiation. In combination with previous data showing increased differentiation of naïve T cells into memory T cells upon exposure to trilaciclib, these data support a beneficial role for trilaciclib in enhancing antitumor T-cell responses.
Citation Format: Sarah Ahn, Subing Cao, Melissa A. Russo, John S. Yi. Transient G1 cell cycle arrest with trilaciclib enhances the generation of polyfunctional CD4+ and CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 703.