August 16, 2022

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Molecular perspective on T cell stemness and exhaustion

Molecular perspective on T cell stemness and exhaustion


Molecular perspective on T cell stemness and exhaustion

T cell stemness and exhaustion coexist as two key contrasting phenomena during chronic antigenic stimulation such as infection, transplantation, cancer, and autoimmunity .

T cell stemness describes the stem cell-like behavior of T cells, including self-renewal, pluripotency, and functional persistence.

T cell depletion refers to the gradual loss of effector function caused by chronic antigen exposure, and exhausted T cells ( T EX ) highly express multiple inhibitory receptors and exhibit severe defects in cell proliferation and cytokine production.


It is generally accepted that naive T cells and certain memory T cell subsets have stem cell-like properties.

In studying the exhausted differentiation of T cells in chronic infection and cancer, recent studies have highlighted the stemness of “exhausted precursor” T cells ( T PEX ) prior to final differentiation into T EX cells.

Clinically successful cancer therapy with checkpoint blockade appears to activate antitumor T PEX cells, but not T EX cells.


Therefore, understanding the transcriptional and epigenetic regulation of T cell stemness and exhaustion, and how systems immunology has been and will be used to define the molecular basis of T PEX to T EX cell transformation, will contribute to a better understanding of T cells dryness and depletion to develop new immunotherapy strategies.




Stemness of memory T cells

Adaptive immunity relies on the immune memory created after natural acute infection or vaccination.

The generation of long-lived antigen-specific memory lymphocytes is the cellular basis of immune memory, and T cell stemness includes the ability of T cells to self-renew and differentiate into multiple downstream cell types, which is called pluripotency.


Earlier studies of mouse memory T cells identified CD62L+CD44+ central memory T ( TCM ) and CD62L–CD44+ effector memory T ( TEM ) cells.

T CM cells are less terminally differentiated than T EM cells.

The progeny produced by a single naive T cell and a single T CM cell upon antigenic stimulation are very similar in size and diversity.

In addition, progeny from a single primary T CM cell contained secondary T CM cells.

When secondary T CM cells were individually transferred to a new host and exposed to antigen, they again gave rise to distinct progeny cells, including effector T ( TEFF ) , T CM and TEM cells .


There is a naive population of memory T cells that is even less differentiated than T CM cells. These cells are named memory stem T cells ( T SCM ).

T SCM cells share many similarities with naive T cells, however, they express Bcl-2, Sca-1, IL-2Rβ, CXCR3 and CD95 levels are elevated.


T SCM cells develop directly from naive T cells, IL-7 is essential for the development of human T SCM cells from naive T cells in vitro, and IL-15 supports their expansion.

Numerous studies support a progressive model of memory T cell differentiation, following naive T cells → T SCM → T CM → T EM .




Stemness of T PEX cells

PD -1/PD-L1 checkpoint blockade is effective in the treatment of different cancers.

Since TEX cells typically express high levels of PD-1, reversal of T cell depletion has been suggested as a potential mechanism for PD-1/PD-L1 blockade.


This view changed dramatically after the discovery of TCF1+PD-1+ T PEX cells in 2016 . We found that TCF1+PD-1+CD8+ T cell subsets resemble stem cells during chronic LCMV infection, undergo self-renewal and differentiate into terminal TCF1–PD-1+ T EX cells.

TCF1 is not only a marker for this cell subset, but also plays an important role in its production. More importantly, the proliferative burst following PD-1 blockade was almost exclusively derived from this subset of cells.


TCF1+PD-1+TILs exhibit stem cell-like functions as they mediate the proliferative response to immunotherapy, generating TCF1+PD-1+ and differentiated TCF1–PD-1+ cells.

A study that depleted tumor-specific TCF1+CD8+ T cells via the Tcf7 diphtheria toxin receptor ( DTR ) system found that depletion of TCF1+PD-1+TIL did limit immunotherapy response.

Therefore, immune checkpoint blockade may be dependent on the proliferation of stem cell-like TCF1+PD-1+TIL rather than the reversal of the T-cell exhaustion program.



Transcriptional regulation of T cell stemness

With the deepening of research, the transcription factors that regulate T cell stemness are gradually revealed. Transcription factor TCF1 is not only a marker of memory T cell stemness but also a key regulator of memory T cell stemness.

TCF1-deficient mice developed normal primary effector T cell responses, but at the peak of acute infection, they were largely devoid of CD8+ memory precursor cells.

Following acute infection, TCF1 deficiency severely impairs CD8+ T CM differentiation, longevity, and response to antigen. These findings suggest that TCF1 regulates the stemness and fate of CD8+ memory T cells.





TCF1 is also a marker and key regulator of TPEX cells during chronic antigen exposure. TCF1 deficiency leads to impaired production of TPEX cells. In addition, Chen et al. used scRNA-seq and lineage tracing to study T cell status early in chronic infection. They found that TCF1 inhibited the development of KLRG1 hi terminal effector T cells when TPEX cells were cultured. Therefore, although TCF1 promotes T PEX cell generation and maintains chronic responses, it may inhibit T cells from exerting direct effector functions.


Molecular perspective on T cell stemness and exhaustion



Transcription factor BACH2 is required for memory differentiation of CD8+ T cells.

We found that BACH2 binds to enhancers of TCR-driven genes, reducing the availability of AP-1 loci to Jun family transcription factors, a process that limits terminal effector cell differentiation.

Thus, effector T cells downregulate BACH2 expression to promote effector fate differentiation, in contrast, BACH2 expression is able to generate long-lived memory cells.


BACH2 is an important regulator of TPEX cells during chronic antigen exposure .

Studies have shown that BACH2 deficiency impairs the generation of TCF1+CD8+ T PEX cells, whereas BACH2 overexpression enhances their generation in the early stages of chronic infection.

Thus, BACH2 actively promotes the generation of stem cell-like CD8+ T PEX lineages and protects T PEX cells from terminal exhaustion.


Id3 is a member of the Id protein family that negatively regulates DNA-binding E proteins and is essential for the formation of CD8+ memory T cells. Id3 is highly expressed in TCF1+TIM3- T PEX cells.

When TPEX cells finally differentiated into TIM3 + T cells, Id3 and TCF1 were gradually lost. Therefore, both Id3 and TCF1 are transcriptional markers of TPEX cells . Whether Id3 regulates T PEX cell state is unclear.


The transcription factor c-Myb is another key regulator of CD8+ T cell stemness. After antitumor vaccination, Myb-deficient CD8+ T cells are prone to terminal differentiation and produce fewer stem-like T CM cells than Myb-sufficient T cells.

Conversely, c-Myb overexpression promoted CD8+ T cell memory and recall responses, which elicited therapeutic antitumor immunity.

Systems immunology approaches further showed that in CD8+ T cells, c-Myb promoted T SCM cell formation, inhibited terminal differentiation, and promoted the persistence of TCF1+ T PEX cells.




Epigenetic regulation of memory T cell stemness

Upon acute infection, T cells undergo significant epigenetic reorganization to achieve multiple functions.


DNA methylation normally suppresses gene expression in cells.

We found that CD8+ memory precursors initially acquire a Dnmt3a-dependent de novo DNA methylation program to repress the expression of certain naive-associated genes and to demethylate ( permit expression ) at sites of effector-associated genes.

To develop into long-lived memory T cells, these memory precursor cells scavenge the de novo methylation program at the original associated gene locus to allow their re-expression, while key effector genes remain demethylated.

Therefore, during memory cell development, T cells may lose and re-express some stem cell/naive-related genes.

Fully developed memory T cells express naive associated genes and exert effector functions upon reinfection.


Methylation of histone H3 lysines 9 ( H3K9 ) and 27 ( H3K27 ) is highly correlated with transcriptional repression. SUV39H1 is a histone methyltransferase that methylates H3K9.

We found that SUV39H1-depleted CD8+ T cells were unable to silence stem/memory genes during terminal effector cell differentiation and increased long-term memory reprogramming capacity.


Polyclonal repressive complex 2 ( PRC2 ) silences gene expression through methylation of H3K27. EZH2 is the catalytic subunit of PRC2. EZH2 deficiency impairs terminal effector cell differentiation.



T cell exhaustion

Nearly three decades ago, Moskophidis et al. infected mice with LCMV-D, and the presence of large amounts of LCMV-D produced a high antigen load that forced the depletion of specific antiviral CD8+ cytotoxic T cells.

Due to the technical limitations of the time, the research uncovered phenomena that have not yet been explained by molecular mechanisms.


Today, systems immunology approaches allow us to go beyond traditional cell surface marker or cytokine expression, allowing us to observe the transcriptional, epigenetic, and metabolic programming that underpin various T cell states.

In 2007, Wherry et al. used microarray analysis to investigate the molecular features of CD8+ T cell exhaustion following chronic LCMV infection. CD8+ T EX cells were found to overexpress PD-1 and other inhibitory receptors, express a unique set of transcription factors, and have severe metabolic and bioenergetic deficits.

These findings reveal T EX for the first timeHow cells gradually lose effector function at the molecular level.


ScRNA-seq technology opens up new fields for studying T cell biology.

Many initial scRNA-seq studies examined T cell status in different types of cancer, CD8+ T EXCells are often preferentially enriched in the tumor microenvironment.

Since then, T-cell depletion has become an accepted term to describe T-cell responses to chronic infections and cancer.




Transcriptional regulation of T cell exhaustion

T cell exhaustion occurs during chronic antigen stimulation, and high antigen load leads to T cell exhaustion.

A high antigen load not only maintains chronic stimulation of antigen, but also provides repeated antigen stimulation through TCR.


TCR-induced signals activate the NFAT transcription factor family, and activated NFAT proteins in turn interact with other transcription factors, such as the AP-1 family , to control T cell activation and effector cell differentiation.

CD8+ T cells lacking NFAT fail to express exhaustion-related inhibitory receptors.


TOX is a secondary transcription factor induced by NFAT initiation.

TOX may not be involved in the differentiation of effector and memory T cell status upon acute infection. Conversely, in chronic infection and cancer, TOX is a key driver of the CD8+ T cell depletion program.

We found that TOX expression is critical for the formation and maintenance of TCF1+ T PEX cells and subsequent terminal T EX cell differentiation.

Without TOX, T PEX cell formation is impaired and T EX cells fail to form. Tox-deficient CD8+ cells do not upregulate genes for inhibitory receptors such as Pdcd1, Entpd1, Havcr2, Cd244 and Tigit.


NR4As ( NR4A1, NR4A2, and NR4A3 ) are also secondary transcription factors that initiate NFAT induction.

In chronic viral infections or tumors, CD8+ T cells express high levels of NR4As, which play an important role in the T cell dysfunction/exhaustion program.

Knockout of all three NR4As in CAR-T cells promoted tumor regression and prolonged survival in tumor-bearing mice.


BATF is a member of the AP-1 family of transcription factors. It dimers with JunB and acts as a repressor of AP-1 activity in activated T cells.

It has been suggested that BATF is one of the depletion drivers, and elevated BATF expression correlates with CD8+ T cell depletion upon HIV infection. Silencing BATF in T cells rescues HIV-specific T cell function.


BATF, BACH2 and NR4A1 all have inhibitory effects on AP-1 activity. BACH2 represses AP-1-dependent effector genes to maintain TPEX cells , whereas BATF and NR4A1 promote terminal depletion . In conclusion, the TCR-NFAT-TOX/NR4A axis drives the depletion program of CD8+ T cells.




Epigenetic signatures of T cell exhaustion

The epigenetic profile of exhausted T cells is markedly different from that of effector and memory T cells. Sen et al. used ATAC-seq to define chromatin accessible regions ( CHAR ) in CD8+ T cells.

T EX cells showed higher peak intensities of ChARs near depletion-related genes ( eg, Pdcd1, Havcr2, Batf ) compared to effector/memory T cells .


Furthermore, we found that after LCMV-Cl13 infection, antigen-specific T cells acquired exhaustion-related epigenetic signatures distinct from effector and memory T cells.

While PD-L1 blockade reactivated T cell responses to LCMV-Cl13, it largely failed to remodel exhaustion-related epigenetic signatures.

It is therefore concluded that the epigenetic stability of TEX cells limits the persistence of PD-L1 blockade effects.


Another study showed that depleted CAR-T cells can undergo epigenetic remodeling after a brief rest in CAR-T cells induced by a drug-regulated degron system.

Four-day induction resulted in an epigenetic profile of depleted CAR-T cells similar to non-depleted control CAR-T cells.

Importantly, a brief rest restores the antitumor function of depleted CAR-T cells.

In conclusion, T EX cells have epigenetic features distinct from effector and memory T cells. The stability of depletion-related epigenetic states may differ in exhausted CAR-T cells from T EX cells.




Influence of tissue environment and metabolism

Anatomical location and accessibility to antigen determine T cell status.

For example, in chronic viral infection studies, more terminally differentiated T EX cells were present in the red pulp of the spleen, which is the main site of LCMV infection.

The less differentiated T PEX cells were mainly located in the T cell region of the white pulp.


Does the environment dictate T cell status through ligand binding, nutrient availability, and local cytokine production? Does antigen abundance due to proximity lead to excessive TCR stimulation leading to end-effector differentiation or depletion? These questions remain to be answered.

However, it is undeniable that environmental factors play an extremely important role in determining T cell status.

The tissue environment may be an attractive target for improving or inhibiting T cell function.


For example, in the tumor microenvironment, elevated extracellular potassium caused by cancer cell necrosis inhibits TCR-driven Akt-mTOR phosphorylation and TIL effector function.

This inhibitory effect depends on the activity of the serine/threonine phosphatase PP2A. Other local immune cells also alter the tumor microenvironment, thereby altering T cell responses.

For example, tumor-associated macrophages can secrete endogenous glucocorticoids, which downregulate effector function, leading to T cell dysfunction and poor response to checkpoint blockade.

M2-like suppressor macrophages in the tumor microenvironment form bidirectional inhibitory interactions with depleted EOMES hi CD8+ T cells, leading to worse clinical outcomes.





Over the past few decades, tremendous progress has been made in our understanding of the transcriptional, epigenetic and metabolic regulation of T cell stemness and exhaustion.

These exciting new discoveries about various T cell states and their regulators may provide insight into how to find the entry point for immunotherapy.

As more mechanisms are revealed, finding ways to modulate the fate and function of T PEX and T EX cells will potentially revolutionize the current landscape of tumor immunotherapy.






1. Schrödinger’s T Cells: Molecular Insights Into Stemness and Exhaustion. Front Immunol. 2021; 12: 725618.

Molecular perspective on T cell stemness and exhaustion

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