Jennifer Wu

"In vitro modeling of CD8+ T cell exhaustion uncovers novel transcriptional regulator Bhlhe40"

Jennifer E. Wu, Sasikanth Manne, Joanna T. Lee, Josephine R. Giles, Elizabeth Freilich, Hua Huang, Zeyu Chen, Ryan P. Staupe, Omar Khan, Junwei Shi, E. John Wherry

Persistent stimulation of the immune system during cancer or chronic infection leads to T cell exhaustion. Exhausted CD8 T cells are characterized by decreased effector function, lower proliferative potential, increased expression of inhibitory receptors (IRs), and unique transcriptional and epigenetic profiles. Exhaustion ultimately results in a stalemate in which T cells exert limited control over but cannot fully eradicate infection while minimizing damaging immune pathology. Most data about T cell exhaustion has been generating using mouse models of chronic viral infection which have limitations; for example, in vivo mouse models have low cellular yields that prohibit the use of screening and other high-throughput exploratory platforms. In contrast, an in vitro model allows for the directed study of discrete exhaustion-associated pathways in a robustly scalable manner.

We have developed an in vitro murine model of exhaustion that recreates multiple modules of the exhaustion phenotype: high IR expression, exhaustion-associated transcription factor (TF) signature, and decreased production of effector cytokines. Furthermore, this in vitro model recreates exhaustion-specific transcriptional and epigenetic signatures, which offers novel insight into and can be combined with new and exciting technology to creatively probe the biology of exhaustion. We have performed in vitro CRISPR screening of exhaustion-related TFs to investigate the contributions of specific genetic loci to initiation and maintenance of the exhaustion program. Integration of this analysis with transcriptional and epigenetic data from RNA- and ATAC-seq analysis has identified a novel TF Bhlhe40 that is important for regulating terminal differentiation of CD8 T cells. Thus, the development and application of this in vitro model of T cell exhaustion is a useful tool for both research and development, and will allow us to mechanistically interrogate the basic biology of exhaustion while scaling up for translational discovery-oriented assays.