Understanding the regulation of residency and exhaustion in CD8-positive T cells
Keywords: Medicine.
Microbiology and Immunology.

CD8+ T cells are responsible for cell-based adaptive immunity and are required for protection against many pathogenic infections and malignant cellular transformation. To carry out their duty during the immune response, naïve CD8+ T cells (TN) can differentiate into many unique T cell states that have wide phenotypic variation and functional specialties. TN integrate both cell-intrinsic and external signals to determine their differentiation trajectory, which leads to the remodeling of the TN epigenome. In the context of acute antigenic stimulation, antigen-specific TN generate progeny of diverse potential, including short-lived, effector T cells (TEFF) and memory T cell precursors (TMP), which generate long-lived memory CD8+ T cells (TMEM). While each TEFF, TMP, and TMEM have distinct canonical roles in the immune response, there is phenotypic heterogeneity within each classification as well. Conversely, in the context of chronic antigen stimulation CD8+ T cells enter a hypofunctional state known as T cell exhaustion. Exhaustion is characterized by high co-inhibitory receptor expression, reduced cytokine expression, a distinct chromatin state, and reduced target cell killing. In this work, we sought to understand the transcriptional regulation and epigenetic state that underlies these complex and diverse CD8+ T cell states. We examined the single-cell chromatin landscape of CD8+ T cells over the course of in vivo acute viral infection and associated transcriptional changes to understand heterogeneity. We reveal an early bifurcation of memory precursors displaying distinct chromatin accessibility and define epigenetic trajectories that lead to a circulating (TCIRC) or tissue-resident memory T (TRM) cell fate. Further analysis of chromatin accessibility differences between TCIRC and TRM cell populations identified trans-regulatory elements (including ARID3A and ZFP384) as major regulators of memory T cell development in the liver. Moreover, despite being epigenetically heterogeneous within organs, TRM cells displayed a conserved epigenetic signature across tissues. We demonstrate that these cells exhibit tissue-specific signatures and identify transcription factors that regulate TRM cell populations in a site-specific manner. Last, we demonstrate that TRM cells and exhausted T (TEX) cells are distinct epigenetic lineages that are distinguishable early in their differentiation. Together, these findings show that TRM cell development is accompanied by dynamic alterations in chromatin accessibility that direct a unique transcriptional program resulting in a tissue-adapted and functionally distinct T cell state. We further sought to model and interrogate the regulation of primary human CD8+ T cell exhaustion. Here, we developed a model of chronic stimulation using sustained anti-CD3 antibody stimulation over the course of 10-14 days. T cells undergoing in vitro chronic stimulation displayed altered cell growth, increased expression of multiple checkpoint receptor molecules, decreased cytokine expression on re-stimulation, reduced target cell killing, and transcriptional heterogeneity reflective of tumor-infiltrating lymphocytes in human tumors. We leveraged this system to identify epigenetic and transcriptional regulators of many canonical phenotypes of T cell exhaustion, including increased checkpoint receptor expression and reduced effector cytokine expression. Ultimately, we unravel the network of factors that underlie T cells under chronic stimulation and identify novel factors that contribute to individual T cell phenotypes associated with exhaustion. Altogether, this work demonstrates how high-throughput genomic methods can be leveraged to understand novel facets of T cell differentiation and uncover regulators of T cell phenotype and fate.