Identifying the Role of Endoplasmic Reticulum Stress in CLL T-Cell Dysfunction
Authors: Gamal Wael , Mediavilla Varela Melanie , Uriepero Palma Angimar , Ammad Ud Din Mohammad , Chavez Julio C. , Sahakian Eva , Rodriguez Paulo C. , Pinilla-Ibarz Javier 
Published: December 2024

Chronic lymphocytic leukemia (CLL) presents a significant clinical challenge due to the increasing incidence of resistance to targeted therapies. Despite the potential of adoptive T-cell therapy (ACT), its efficacy in CLL has been limited, mainly due to CLL-induced T-cell dysfunction and a skewed differentiation toward terminal subsets. The underlying mechanisms of these T-cell alterations remain poorly understood. Given that T-cell activation and differentiation are metabolically intensive processes, previous research (van Bruggen et al, Blood 2019) has highlighted aberrant mitochondrial properties in CLL T cells. However, till now, no focus has been given to the involvement of the endoplasmic reticulum (ER) signaling pathways in T-cell dysfunction in CLL. This is particularly important since there is a close relationship between the mitochondria and ER in maintaining cell homeostasis under stress. Moreover, evidence (Hurst et al., Cancer Immunology Research 2019) suggests that chronic ER stress significantly contributes to T-cell mitochondrial dysfunction in solid tumor models. Using the Eμ-TCL1 murine model, our study aims to elucidate the association between T-cell differentiation, metabolism, and ER signaling disturbance in CLL. In the objective of identifying new drivers of T-cell dysfunction, we aim to determine the role of C/EBP homologous protein (CHOP), a downstream sensor of ER stress, utilizing Ddit3flox/flox CD4-Cre (Ddit3T cell-KO) mice in CLL adoptive transfer experiments.

Splenocytes from transgenic Eμ-TCL1 mice were adoptively transferred (AT) into wild-type syngeneic C57BL/6 mice. Splenic T cells were evaluated for alterations in the expression of ER unfolded protein response (UPR) targets at the protein and gene levels. Moreover, T cells' metabolic and differentiation trajectory changes were assessed on the transcriptional and epigenetic levels at various disease stages. Ddit3T cell-KO mice were AT with leukemic Eμ-TCL1 splenocytes and were monitored for peripheral leukemic burden and survival.

Eμ-TCL1 T cells showed significant upregulation of UPR targets, including PERK, phospho-PERK, XBP1s, and the downstream target CHOP. Moreover, there were substantial changes in T-cell ER mass measured by the ER tracker green probe at different disease stages. The ER stress response disturbance was associated with depolarized dysfunctional mitochondria accumulation, elevated reactive oxygen species (ROS) levels, and reduced spare respiratory capacity post-T-cell receptor (TCR) activation. Moreover, these metabolic changes were associated with terminal differentiation and an exhausted-like T-cell phenotype characterized by a significant increase in TOX, Eomes, and PD-1 and a marked reduction in the self-renewal factor TCF-1. AT of Eμ-TCL1 splenocytes into Ddit3T cell-KO mice showed slower disease progression and improved survival compared to control mice.

Our research has uncovered for the first time the upregulation of the ER stress response activity in CLL T cells that goes hand in hand with mitochondrial disturbance and T-cell exhaustion. Additionally, we highlight the specific role of CHOP in T cells in controlling CLL progression in the murine model. These findings will enable us to identify novel targets that can be investigated for rejuvenating CLL T-cell activity to enhance the efficacy and persistence of ACT products for CLL.