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Cutting Through CD8 T Cell Heterogeneity with CUT&RUN

By Stuart P. Atkinson, Ph.D.

September 25, 2024

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Introduction: CD8 T Cell Heterogeneity – The Crux of the Problem

CD8 T cells (often called cytotoxic T lymphocytes) play critical roles in defending hosts against viral/bacterial infections and tumor immunity; however, deciphering the mechanisms controlling the loss of T cell function remains a challenge due to the sheer heterogeneity of cell types and states (Fan and Rudensky, Jameson and Masopust, and Kumar et al.). A more in-depth understanding of this process may encourage the development of strategies that prevent/reverse CD8 T cell dysfunction and, as such, treat tumors and chronic infections. In response, the application of a range of advanced techniques has supported the first descriptions of the transcriptomic and epigenetic states of isolated functional/dysfunctional CD8 T cell subsets; however, heterogeneity still represents a significant problem, and we understand little regarding the progression of these cells to dysfunction.

Researchers from the laboratories of Yuri Pritykin and Christina S. Leslie (Memorial Sloan Kettering Cancer Center) sought to cut through this heterogeneity by applying a uniform reprocessing and a statistically principled batch effect correction approach to epigenetic and transcriptomic data from independent studies of CD8 T cells across experimental mouse models of acute/chronic infection and tumors. They hoped that any success would support a direct comparison between genome-wide data from different studies and aid in defining similar steps present in the CD8 T cell dysfunction program across subtypes. Furthermore, they also applied single-cell epigenetic and transcriptomic analyses, and the cleavage under target and release under nuclease (CUT&RUN) chromatin profiling technique employed to define transcription factor binding profiles in low-cell number samples to their own model system to validate and explore their findings.

Their epigenetic and transcriptomic data, reported recently in Molecular Cell, suggest the existence of a "universal program" that describes the early commitment of CD8 T cells to a dysfunctional fate in response to antigen exposure that is controlled via the activity of state-specific transcription factors. They hope their findings will represent a valuable resource facilitating mechanistic and translational studies involving CD8 T cells (Prityki and van der Veeken et al.).

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Cutting through T Cell Heterogeneity: ATAC-seq and RNA-seq Data Identifies Dysfunctional CD8 T Cells

Prityki and van der Veeken et al. first evaluated transposase-accessible chromatin sequencing (ATAC-seq) datasets from previously reported studies of CD8 T cell dysfunction in mouse models of infection/cancer; interestingly, they discovered that the chromatin states of dysfunctional tumor-infiltrating T cells and T cells associated with chronic viral infections displayed a high degree of similarity, suggesting the presence of a universal program of CD8 T cell dysfunction. A similar analysis of RNA-sequencing (RNA-seq) datasets revealed consistent results, with differential expression between functional and dysfunctional cells significantly correlating with differential chromatin accessibility. Overall, these findings confirmed that dysfunctional CD8 T cells displayed epigenetic and transcriptomic similarities across mouse models of infection/cancer. Interestingly, genes associated with regulatory regions displaying the most substantial level of differential chromatin accessibility between functional and dysfunctional cells included those encoding for well-known markers of T cell activation, cytotoxicity, adhesion, apoptosis, key transcription factors, and cytokines/cytokine receptors.

Describing a Universal Program of CD8 T Cell Dysfunction

Subsequent comparisons suggested that T cells adopt a dysfunctional chromatin state in the early stages after antigen-mediated stimulation that correlated with alterations to gene expression profiles; furthermore, this analysis highlighted the existence of a universal program of T cell progression towards dysfunction - from progenitor to terminal dysfunction - across models. Their subsequent analysis identified common markers and transcription factors associated with T cell differentiation toward dysfunction; additionally, motif-based regression modeling of ATAC-seq data (associates transcription factors with functional states) suggested that the coordinated and hierarchical activity of a range of transcription factors (and not the activity of a single "master" transcription factor) established and maintained T cell functional states.

Taking CD8 T Cell Heterogeneity to the Single-cell Level

The authors then shifted gears, switching from bulk ATAC-seq to single-cell (sc)ATAC-seq analysis of mouse splenic CD8 T cells under conditions of acute/chronic infection; these assays helped to characterize the early divergence between CD8 T cell chromatin states, identify progenitor-like subpopulations, and lastly implicate cell-state-specific transcription factors. Subsequent scRNA-seq analysis then described a comprehensive atlas of CD8 T cell functional and dysfunctional states and confirmed the existence of progenitor/progenitor-like cell populations with similar transcriptional profiles. The scRNA-seq data also suggested that both chronic and acute infection gave rise to cells at similar functional, activation, and differentiation states (which accumulated at challenge-dependent proportions) and supported the existence of cell-state-specific expression of transcription factors that bulk ATAC and scATAC analyses had previously described.

Allele-specific Single-cell RNA-seq Identifies Cell-State-Specific Transcription Factor Activities

Interestingly, the team identified thousands of genes with significant allele-specific expression profiles in the progenitor-dysfunctional T cell scRNA-seq datasets (including genes involved in T cell activation and function) when seeking to complement observations of cell-state-specific transcription factor activities from bulk and scATAC-seq and RNA-seq data analyses. Integrating ATAC-seq data supported the identification of transcription factors that activated or repressed gene expression in progenitor-dysfunctional T cells; as an example, they revealed the binding of T cell factor 1 (TCF1) at promoters of expressed genes.

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CUT&RUN Enters the Party and Highlights the Importance of TCF1

As studies had previously linked TCF1 activity and progenitor-dysfunctional T cells (Im et al. and Utzschneider et al.), the team validated the TCF1-mediated activation of gene expression by CUT&RUN analysis in progenitor-dysfunctional T cells. Direct TCF1 binding sites displayed strong enrichment for the TCF1 binding motif and included many T cell activation genes and progenitor marker genes, while nearly all TCF1 targets displayed higher accessibility levels in progenitor-dysfunctional T cells than in terminally dysfunctional cells. Of note, allele-specific TCF1 binding significantly correlated with allele-specific expression of nearby genes in progenitor-dysfunctional T cells in chronic infections, consistent with a causal role for TCF1 in activation of gene expression. Additionally, allele-specific motif enrichment analysis in CUT&RUN-defined TCF1 binding sites revealed a range of TCF1 binding co-factors. Overall, the allele-specific analysis revealed cis-regulatory effects of transcription factor binding on gene expression in CD8 T cells that may have been overlooked in previous studies using standard approaches.

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The End of the Story for CD8 T Heterogeneity?

Overall, these fascinating findings suggest that tumor/viral antigen exposure can induce a universal program of CD8 T cell progression toward dysfunction, laying the groundwork to resolve fundamental questions regarding T cells; indeed, the authors hope their data will represent a valuable resource for fundamental and clinical research.

For more on how transcriptomic and epigenetic profiling can cut through the problem of CD8 cell heterogeneity (with a little help from CUT&RUN), see Molecular Cell, June 2021.

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About the author

Stuart P. Atkinson, Ph.D.

Stuart was born and grew up in the idyllic town of Lanark (Scotland). He later studied biochemistry at the University of Strathclyde in Glasgow (Scotland) before gaining his Ph.D. in medical oncology; his thesis described the epigenetic regulation of the telomerase gene promoters in cancer cells. Following Post-doctoral stays in Newcastle (England) and Valencia (Spain) where his varied research aims included the exploration of epigenetics in embryonic and induced pluripotent stem cells, Stuart moved into project management and scientific writing/editing where his current interests include polymer chemistry, cancer research, regenerative medicine, and epigenetics. While not glued to his laptop, Stuart enjoys exploring the Spanish mountains and coastlines (and everywhere in between) and the food and drink that it provides!

Contact Stuart on Twitter with any questions

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