T cell exhaustion is a distinct cellular state that arises during prolonged exposure to chronic viral infections and cancer. Exhausted CD8 T cells (Tex) are characterized by reduced proliferative potential, heightened expression of inhibitory receptors (IRs), diminished production of effector cytokines, and distinct transcriptional and epigenetic profiles. This cellular exhaustion represents a significant impediment to effectively combating chronic diseases, and efforts to reverse this state have proven challenging. Consequently, there is a pressing need to uncover new therapeutic strategies to address T cell exhaustion effectively. Mouse models of chronic infection and cancer have played a pivotal role in advancing our understanding of CD8 T cell exhaustion. These models have not only helped establish the fundamental principles of Tex biology but have also led to the development of checkpoint pathway blockade, a groundbreaking therapeutic approach. However, these in vivo models have their limitations, particularly in terms of efficiency and cellular yield. They often generate limited numbers of Tex cells, making high-throughput experiments challenging due to the scale disparity between required and available cells. In vitro models offer a promising alternative for studying and interrogating Tex biology. They provide the advantage of scalability, enabling the generation of substantial cellular output for high-throughput assays. While in vitro models cannot fully replicate the complexities of in vivo Tex biology, they offer unique opportunities to dissect specific pathways and mechanisms. The ability to create reductionist in vitro models allows for the focused investigation of particular aspects of Tex biology, providing valuable insights. Sustained engagement of the T cell receptor (TCR) has been identified as a central driver of CD8 T cell exhaustion in vivo. Repeated or continuous TCR stimulation in vitro has successfully induced Tex-like features, including reduced proliferative potential, upregulated IR expression, and decreased effector function. These studies have demonstrated the utility of in vitro models in approximating Tex biology. However, the challenge lies in determining the extent to which these in vitro models accurately recapitulate the overall program of in vivo Tex biology.
In a new study published in the Journal Science Immunology, led by Professor John Wherry from the University of Pennsylvania, a significant leap has been made in our understanding of T cell exhaustion, a state that has long been a major challenge in the treatment of chronic viral infections and cancer. This study introduces an innovative in vitro model of T cell exhaustion, shedding light on the molecular mechanisms that drive this process and offering promising avenues for potential therapeutic interventions.
To address these challenges, the University of Pennsylvania research team developed an in vitro platform that models Tex through the chronic administration of a cognate peptide, simulating continuous antigenic stimulation. This in vitro model was then benchmarked against in vivo Tex generated during chronic lymphocytic choriomeningitis virus (LCMV) infection to assess the degree of similarity in phenotypic, functional, transcriptional, and epigenetic features. Leveraging this model, the team conducted CRISPR screening to identify the role of the transcription factor BHLHE40 in CD8 T cell exhaustion.
The authors found that chronic antigenic stimulation in vitro successfully induced Tex-like features, including reduced proliferative potential, high expression of IRs, and decreased effector cytokine production. The in vitro model closely mirrored in vivo Tex biology, validating its utility in studying Tex. They also conducted pooled CRISPR screening which identified a range of transcriptional regulators, some of which were known players in CD8 T cell biology, while others were newly identified as having a role in Tex. BHLHE40 emerged as a particularly intriguing regulator of Tex. It was highly ranked by Taiji analysis and was among the most robustly enriched leading-edge genes positively selected in CRISPR screening of both chronic stimulation in vitro and in vivo Tex.
According to the authors, BHLHE40 has been implicated in various aspects of T cell biology, including lineage commitment, maintenance of stemness in tumor-infiltrating lymphocytes, and the response to checkpoint blockade therapy. However, its role in Tex biology had remained unclear. The researchers demonstrated that BHLHE40 functions as a critical regulator of Tex differentiation. It influences the transition between progenitor Tex and intermediate and terminally differentiated subsets of Tex. When BHLHE40 activity was decreased, there was an accumulation of progenitor Tex at the expense of intermediate Tex, indicating that BHLHE40 plays a pivotal role in driving the differentiation of Tex cells towards a more effector-like state. Moreover, they reported that BHLHE40’s impact on Tex differentiation has significant implications for immunotherapy. PD-1 pathway blockade, a promising immunotherapeutic approach, was shown to be less effective in overriding the differentiation checkpoint controlled by BHLHE40. This suggests that BHLHE40 may be a key factor in determining the success of checkpoint blockade therapy by influencing the conversion of progenitor Tex into intermediate Tex.
In a nutshell, the study led by Professor John Wherry represents an milestone in our understanding of T cell exhaustion and offers new avenues for therapeutic intervention in chronic viral infections and cancer. The innovative in vitro model of Tex provides a valuable platform for studying and interrogating Tex biology, allowing for high-throughput experiments that were previously challenging in in vivo models. Additionally, the identification of BHLHE40 as a critical regulator of Tex differentiation sheds light on the molecular mechanisms underlying this complex cellular state.
Wu JE, Manne S, Ngiow SF, Baxter AE, Huang H, Freilich E, Clark ML, Lee JH, Chen Z, Khan O, Staupe RP, Huang YJ, Shi J, Giles JR, Wherry EJ. In vitro modeling of CD8+ T cell exhaustion enables CRISPR screening to reveal a role for BHLHE40. Sci Immunol. 2023;8(86):eade3369. doi: 10.1126/sciimmunol.ade3369.