High-throughput discovery of novel transcriptional programs regulating T cell exhaustion - Summary
Successful control of chronic virus and cancer critically depends on our improved understanding of the cellular
and molecular mechanisms of T cell exhaustion. CD8 T cells responding to persistent antigens during chronic
infection and cancer gradually differentiate into a dysfunctional state, commonly known as T cell exhaustion.
Exhausted T cells acquire a unique and permanent epigenetic profile, which severely limits their functional
reinvigoration. Recent studies from us and others reveal that the “exhausted” T cell pool consists of multiple
phenotypically, functionally, and transcriptionally distinct subsets. Importantly, a TCF-1hi progenitor (TPRO)
subset functions as a population of resource cells that continuously replenishes the pool of terminally
exhausted T cells (TEXH) during chronic infection and cancer. Interestingly, we recently show that some TPRO
cells can break away from the path toward exhaustion and differentiate into a CX3CR1+ effector subset (TEFF)
with enhanced killing ability, which can be exploited as cellular therapies to control chronic viral infection and
cancer in animal models. Furthermore, recent research demonstrates that the formation of these distinct CD8
T cell subsets is governed by underlying transcriptional programs. For example, TCF-1, T-bet and TOX act as
subset-specific TFs to regulate the differentiation of TRPO, TEFF and TEXH, respectively. Perturbation of genetic
pathways can be exploited to skew CD8 T cell differentiation trajectory toward effector subset and limit T cell
exhaustion. Thus, the high-throughput discovery of novel transcription factors (TFs) that regulate the
phenotypic heterogeneity of CD8 T cells and rapidly validate their biological function in the animal models is
highly sought-after. Taking advantage of recently developed technologies in single cell sequencing and
CRISPR/Cas9 gene editing, we propose to screen a set of novel TFs selected computationally based on their
“regulon” activity in the respective subsets of CD8 T cells. This more targeted selection of candidate TFs
combined with the high-throughput screen by perturb-seq will greatly expedite the discovery of novel
transcriptional programs that regulate CD8 T cell differentiation and cell-fate choice. Furthermore, we will
validate the function of the top-hits TFs identified by Perturb-seq via employing both loss-of-function and gain-
of-function approaches to test the role of them in pathogen-specific CD8 T cells in vivo during chronic viral
infection. The successful completion of this high-throughput, rapid and cost-effective CRISPR screen and
functional validation of novel TFs in CD8 T cell differentiation will gain new knowledge in transcriptional
regulation of T cell exhaustion. It will also provide mechanistic insights into therapeutic designs in treating
cancer and infectious diseases.
