Circulating Extracellular Vesicles Induce Chimeric Antigen Receptor T Cell Dysfunction in Chronic Lymphocytic Leukemia (CLL)

Abstract

<h3>Introduction</h3> CD19 directed chimeric antigen receptor T cell (CART19) therapy has resulted in unprecedented clinical outcomes. However, its success in chronic lymphocytic leukemia (CLL) has been modest to date. An increasing body of evidence indicates that impaired CART cell fitness is the predominant mechanism of the relative dysfunction in CLL. The immunosuppressive microenvironment in CLL is well known and may be related to the abundance of circulating extracellular vesicles (EVs) bearing immunomodulatory properties. We hypothesized that CLL derived EVs contribute to CART cell dysfunction. <h3>Objectives</h3> We aimed to investigate the interaction between EVs from CLL patients and CART19 cells. <h3>Methods</h3> We characterized circulating EVs from platelet free plasma in untreated patients with CLL and normal controls using nanoscale flow cytometry. CLL derived EVs, CART19, and CLL B cells were imaged with super resolution microscopy. We stimulated CART19 cells with CD19⁺ JeKo1 cells at a 1:1 ratio in the presence of increasing concentrations of CLL derived EVs and measured inhibitory receptors by flow cytometry. To interrogate the transcriptome, we stimulated CART19 cells with irradiated JeKo1 cells in the presence of CLL derived EVs at ratios of 10:1 and 1:1 EV:CART19. Finally, immunocompromised mice were engrafted with the JeKo1 and randomized to treatment with untreated, CART19 cells, or CART19 cells co-cultured <i>ex vivo</i> with CLL derived EVs for six hours. <h3>Results</h3> Our findings indicate that CLL derived EVs impair normal donor CART19 antigen-specific proliferation (Fig 1A). Although there wasn't a difference in total EV particle count (Fig 1B), CLL derived EVs expressed significantly more PD-L1 than normal controls (Fig 1C). On super resolution microscopy, EVs were localized at the T cell-tumor junction (Fig 1D). Furthermore, CLL derived EVs were captured by T cells (Fig 1E). Having demonstrated this interaction, we sought to establish their functional impact on CART19 cells. There was a significant upregulation of inhibitory receptors on the T cells (Fig 1F), associated with a reduction in CART effector cytokines at higher concentrations of EVs (Fig 1G), suggesting a state of exhaustion. This was further supported by RNA sequencing, which indicated a significant upregulation of AP-1 and YY1 (Figs 1H), known T cell exhaustion pathways. Finally, to confirm the impact of CLL derived EVs on CART19 functions <i>in vivo</i>, treatment with CART19 cultured with EVs resulted in reduced anti-tumor activity compared to treatment with CART19 alone (Fig 1I). <h3>Conclusion</h3> Our results indicate that CLL derived EVs induce significant CART19 cell dysfunction <i>in vitro</i> and <i>in vivo</i>, by direct interaction with CART cells resulting in a downstream alteration of their exhaustion pathways. These studies illuminate a novel way that circulating and potentially systemic EVs can lead to CART cell dysfunction in CLL patients.