Monitoring Markers of T Cell Exhaustion

T cells follow a distinct process of activation, proliferation, and differentiation. However, in settings of chronic infection, autoimmunity, and cancer, they can subsequently become functionally and phenotypically exhausted. T cell exhaustion (T_ex) is a blanket term that covers a compromised function state exhibited by CD8⁺ T lymphocytes. Cancer immunotherapeutic approaches such as targeted antibodies, adoptive cell therapy (CAR-T), and cancer vaccines are dependent on an effective host immune response to attack and kill tumor cells, so T cell exhaustion is recognized as a significant threat to favorable treatment responses. A common feature of chronic viral infection and cancer is that both are prolonged diseases characterized by a persistence of antigen. While CD8⁺ T lymphocytes become exhausted during chronic viral infection, CD8⁺ tumor-infiltrating lymphocytes (TILs) are similarly hyporesponsive when confronted with the progressively expanding tumor.

Mechanisms and markers of T cell exhaustion

Immune Checkpoint Receptors

The onset of T cell exhaustion coincides with the surface expression of co-inhibitory receptors, which control CD8⁺ T cell function. Such immune checkpoints, which include programmed cell death protein (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and lymphocyte-activation gene 3 (LAG-3) are believed to have evolved to constrain T cell activation, thereby preventing excessive adverse inflammatory and autoimmune events. Immune checkpoints also appear to function throughout exhaustion as blocking interactions between PD-1 and its ligand (PD-L1) can restore the function and survival of CD8⁺T_ex. This observation was pivotal in opening the way for rapid clinical development of agents targeting immune checkpoints—immune checkpoint inhibitors (ICI)—that have gone on to demonstrate durable responses in limited sets of cancer patients.

Although CD8⁺T_ex was originally thought to be a uniform population that progressively loses effector function in response to persistent antigen, single-cell analysis has now revealed it to be composed of multiple interconnected subpopulations. The heterogeneity within the CD8⁺T_ex lineage is composed of immune checkpoint blockade (ICB) permissive and refractory subsets termed stem-like and terminally differentiated cells, respectively. These populations occupy distinct peripheral and intratumoral niches and are characterized by transcriptional processes that drive transitions between cell states. For example, PD-1⁺CD8⁺ T_ex can be further separated into PD-1^lo (low level expressors of PD-1) and PD-1^hi (high level expression of PD-1) subsets. A hypothesis stemming from this proposes that PD-1^lo cells differentiate into the PD-1^hi subset as CD8⁺ T cells exhaust.

TCF-1: T Cell factor 1

Evidence has shown that T cell responses are sustained by a small subset of CD8⁺ T cells with memory characteristics, equating to TCF-1⁺ T cells with exhausted phenotype. Similar to memory or stem-like T cells, chronically stimulated TCF-1⁺ T cells are able to both survive long-term and self-renew, and also retain proliferative potential. However, unlike conventional memory T cells, these TCF-1⁺ T cells can express inhibitory receptors such as PD-1 and constantly replenish the pool of terminally differentiated exhausted effector T cells.

TOX: Thymocyte selection-associated HMG Box

TOX is a member of a family of transcriptional factors that contain the highly conserved high mobility group box (HMG-box) region. Its expression is upregulated in dysfunctional T cells, driven by chronic T cell receptor stimulation and activation of transcription factors known as nuclear factor of activated T cells (NFATs). A strong and growing body of evidence has shown that TOX is involved in maintaining tumors and promoting T cell exhaustion.

Additionally, a range of other biomarkers have been identified and are under increased scrutiny to understand their role and contribution to the generation, or maintenance, of the exhausted T cell state.

Monitoring T cell dysfunction

In recent years, various innovative technologies have emerged that align themselves well with the challenge of profiling immune infiltrates. Specifically, given the complexity and heterogeneity of the functional states within T cell populations, technologies able to assess T cell dysfunction at the single-cell level have been most heavily exploited.

Mass cytometry

Mass cytometry, or cytometry by time-of-flight (CyTOF), is a valuable tool in the high-dimensional analysis of single cells. During CyTOF, antibodies are labeled with metallic isotopes (lanthanides) and used to detect cellular targets, prior to analysis using high-throughput spectrometry. The CyTOF technology brings the benefit of profiling a large number (up to 50) of cellular parameters in parallel, which, with traditional fluorescence-based approaches, would need multiple overlapping panels. Importantly, the use of heavy metal isotopes reduces the background compared to using fluorescent antibodies, while the detection overlap between the different heavy metals is very low. Currently the costs of CyTOF are high due to expensive reagents (metal-tagged antibodies and conjugation kits), and the data collection rate is slow compared to flow cytometry. Yet, the technology has allowed for detailed characterization of patient immune infiltrates to define the immune repertoire present and determine the status of key T_ex markers.

Single-cell RNA sequencing

Single-cell RNA sequencing (scRNA-seq) is a technique used to study the transcriptomes of individual cells and is now considered the gold standard for defining cell states and phenotypes. Due to the small amount of material available, it is not possible to obtain a complete expression profile from each cell, but patterns of gene expression can be derived through clustering analysis. scRNA-seq of immune cells derived from tumor tissue has been used to identify transcriptional signatures associated with T cell activation, cytotoxicity, and exhaustion.

Conclusions and future perspectives

The fundamental changes in metabolism, regulation, epigenetic control, and reversibility of T cell exhaustion need further elucidation to enable development of new therapeutic approaches and support the use of existing therapies in rational combinations. Improved understanding may also allow us to ask—and potentially address—whether T cell exhaustion can be prevented in response to immune checkpoint inhibition.

The complexity of the underlying biology and influence outside of the immediate tumor microenvironment (TME) highlights the need to go further and better understand the wider processes governing T cell exhaustion and the response to immunotherapeutics and immune checkpoint inhibitors (ICI). Following recent studies this now also extends to the gut microbiome and how its composition can influence or predict the patient response to ICI.

Ultimately the hope is that our emerging understanding of T cell exhaustion can be further refined and utilized to develop personalized strategies to restore effective antitumor immunity.

References

Dolina JS, Van Braeckel-Budimir N, Thomas GD, Salek-Ardakani S. CD8+ T Cell Exhaustion in Cancer. Front Immunol. 2021 Jul 20;12:715234.

Budimir N, Thomas GD, Dolina JS, Salek-Ardakani S. Reversing T-cell Exhaustion in Cancer: Lessons Learned from PD-1/PD-L1 Immune Checkpoint Blockade. Cancer Immunol Res. 2022 Feb;10(2):146-153.

McLane LM, Abdel-Hakeem MS, Wherry EJ. CD8 T Cell Exhaustion During Chronic Viral Infection and Cancer. Annu Rev Immunol. 2019 Apr 26;37:457-495.

Verdon DJ, Mulazzani M, Jenkins MR. Cellular and Molecular Mechanisms of CD8+ T Cell Differentiation, Dysfunction and Exhaustion. Int J Mol Sci. 2020 Oct 5;21(19):7357.

Thommen DS, Schumacher TN. T Cell Dysfunction in Cancer.  Cancer Cell. 2018 Apr 9;33(4):547-562.