Mol. Cells

No Time to Die, Born to Be Killers: Survival Is Accompanied by Exhaustion after an Endless Battle

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The immune system is well-equipped to eliminate foreign or dangerous entities, including naïve CD8+ T cells capable of differentiating into effector T cells (TEff or cytotoxic T lymphocytes) and memory T cells (TMem) (Yao et al., 2019). Persistent antigen interactions in chronic infections or cancer drive T cells toward a dysfunctional, named “exhausted” phenotype without antigen-independent self-renewal (Abdel-Hakeem et al., 2021; Khan et al., 2019; Yao et al., 2019). Exhausted T cells (TEx) cells characterize malignancies, such as hepatitis C virus or human immunodeficiency virus infection, and lung cancer (Khan et al., 2019).

TEx cells are a distinct subset with a unique transcriptional and epigenetic landscape from TEff and TMem (Abdel-Hakeem et al., 2021; Khan et al., 2019). Their epigenetic program differs similarly to epigenetic differences between distinct hematopoietic lineages (Beltra et al., 2020; Khan et al., 2019). Exhaustion is characterized by a reduced metabolic capacity and loss of cytokine production (Khan et al., 2019). This dysfunctional state induces a potential leakage of memory formation and T cell function, reduced proliferation, and impaired survival (Khan et al., 2019; Yao et al., 2019). TEx cells alter effector functions, express inhibitory receptors (IR), and reduce immunoreactivity. Programmed cell death protein-1 (PD-1), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin mucin-domain containing-3 (TIM-3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are examples of IR’s (Abdel-Hakeem et al., 2021; Khan et al., 2019; Kim et al., 2021; Yao et al., 2019). TEx cells are distinguished by their heterogeneity, allowing classification into four different subsets (Beltra et al., 2020).


While the progenitor and terminally exhausted TEx subtypes have already been described (Alfei et al., 2019; Khan et al., 2019; Kim et al., 2021; Yao et al., 2019), Beltra et al. (2020)recently identified four distinct TEx stages of exhaustion. As summarized in the figure, TEx cells can be classified as progenitor, intermediate, or terminally exhausted T cells based on the expression of Ly108 (or signaling lymphocyte activation molecule family member 6 [SLAMF6]), CD69, and Ki67. T cell factor-1 (TCF-1), thymocyte selection-associated high mobility group (HGM) box protein (Tox), T-box expressed in T cells (T-bet), and eomesodermin (Eomes) are transcription factors that determine TEx subsets. CD69 expression indicates a resident and anti-proliferative state, associated with Eomes and Tox but not with T-bet. These markers partially antagonize each other, Tox antagonizes T-bet, T-bet represses PD-1, and TCF-1+ cells are gradually lost when Ki67 positivity increases. As all TEx subsets express Tox, it is considered the master transcription factor of exhaustion (Abdel-Hakeem et al., 2021; Beltra et al., 2020).

Progenitor TEx 1 (TExprog1) express all previous markers but present low T-bet expression. Their Ki67 level is low, indicating a reduced proliferative potential. The key characteristics of this subset are quiescence, residency in the white pulp of the spleen, and inability to enter the bloodstream. However, the progenitor TEx 2 subset (TExprog2) expresses Ly108, but not CD69, and all other previously mentioned markers are only intermediately expressed. This subset proliferates, presents intermediate Ki67 expression levels, and circulates in the blood and red pulp of the spleen. Despite their anatomical, transcriptional, phenotypic, and functional differences, both progenitors share the potential to exchange through conversion and re-localization through the TCF-1 and Tox transcriptional checkpoints. The maintenance of progenitor cells depends on TCF-1, a key transcription factor of progenitor TEx (Abdel-Hakeem et al., 2021; Beltra et al., 2020). Under the epigenetic and transcriptional control of T-bet, the initial proliferation of the TExprog2 subset results in the appearance of the downstream intermediate TEx (TExint) subset, which is still considered irreversible.

This intermediate subset expresses high T-bet, low Tox, and low Eomes but no Ly108, CD69, or TCF-1. Additionally, these cells express a high Ki67 level and are highly proliferative. TExint circulate in both the blood and the red pulp of the spleen. This subset is weakly cytotoxic and resembles circulating effector-like cells but with a distinct epigenetic makeup compared with TEff. Furthermore, these three subsets express CD44 with intermediate PD-1 levels.

The epigenetic and transcriptional checkpoint Tox regulates the final and irreversible transition. In contrast to TExint cells, this terminally exhausted TEx (TExterm) subset loses T-bet but gains Eomes expression and presents the highest Tox activity. These cells exit the cell cycle permanently, and CD69 expression is restored. TExterm is resident in peripheral tissues, blood, and the red pulp of the spleen. Considering their low Ki67 levels, the proliferation of these cells is unlikely. The high PD-1 expression and intermediate CD44 levels further underline their terminal exhaustion. To the best of our understanding, this TEx classification is preserved across species and presents the conserved core developmental mechanisms.


The corresponding epigenetic mechanisms were recently described (Abdel-Hakeem et al., 2021). Exhausted T cells were rescued from terminal exhaustion by an early antigen removal, with a four-week recovery after transferring them to healthy mice. These reinvigorated TEx cells were called recovered TEx (REC-TEx) because they switched from an exhausted to a memory-like phenotype. Nevertheless, the REC-TEx did not fully recover memory functions. Interestingly, Tox expression levels remained high after 500 days, potentially limiting reinvigoration. By eliminating the antigen, some transcriptional changes occurred compared to the originally transferred TEx cells. The gene expression profile associated with exhaustion, including Tox, Pdcd1 (encodes PD-1), and LAG-3, was decreased in REC-TEx, resulting in an intermediate transcriptional profile between TEx and TMem. In contrast, memory cell-specific genes, including IL-7 receptor (Il7r) and TCF-7 (encodes TCF-1), were upregulated. Tox remained high in REC-TEx, although TCF-7 was lower than in TMem. By comparing REC-TEx with different subtypes established by Beltra et al. (2020), Abdel-Hakeem et al. (2021) concluded that REC-TEx arises from TCF-1-expressing progenitor TEx. These recovered cells also expressed higher PD-1 but lower killer cell lectin-like receptor subfamily G member (KLGR)1 level, representing a more exhausted and less effector-like phenotype. Furthermore, upon rechallenge, the cytotoxicity potential of REC-TEx was inferior to TEff or TMem, presenting higher PD-1 and Tox expression levels. Compared with TEx, we observed only a slight improvement of effector functions. Overall, REC-TEx resembled TEx more than TMem. Investigating chromatin accessibility revealed consistent differences between REC-TEx and TMem open chromatin regions called “epigenetic scars” of exhaustion. Altogether, the recovery after antigen removal did not improve REC-TEx effector functions. This incomplete functional recovery reveals a lack of epigenetic plasticity and failure to reestablish the transcriptional programs of TEff and TMem. Nevertheless, these reinvigorated TEx cells could become a target for future immune checkpoint blockade (ICB) therapies that already targeted TEx without groundbreaking success.


Because ICB offered significant therapeutic improvement, a better characterization of the function and regulation of exhausted T cells is critical, as TEx cells are major cell types responding to anti-PD-1/PD-L1 biologics (Beltra et al., 2020). Among the different TEx subsets, only PD-1intCD44hi progenitor TEx responded efficiently to anti-PD-1/PD-L1 treatment compared to PD-1hiCD44int terminally exhausted TEx (Abdel-Hakeem et al., 2021; Beltra et al., 2020; Kim et al., 2021). After ICB, the circulatory subsets TExprog2 and T-bet high TExint expanded preferentially, and differentiation into different TEx subsets is most likely affected by anti-PD-1/PD-L1 blockade (Beltra et al., 2020). Hence, after PD-1/PD-L1 blockade, the chromatin landscape stabilized, and some characteristics of exhaustion were reversed. However, after ICB, the effector properties were only temporarily and partially restored, and the open chromatin landscape remained mostly unchanged (Abdel-Hakeem et al., 2021; Beltra et al., 2020). In REC-TEx, ICB with anti-PD-1/PD-L1 generated minor effects without causing significant proliferation (Abdel-Hakeem et al., 2021).

In the future, therapies specifically targeting selected exhaustion subtypes will play significant roles in immune cancer therapies, chronic, and autoimmune diseases. Following TEx recovery, the IL-7R-TCF-1 axis could target the poor accessibility of the chromatin regions. The upregulation of both CD127 and Il7r genes increased chromatin accessibility. In the TCF-1+ REC-TEx subset, this reinforced accessibility could create a survival mechanism with therapeutic potential for this restored cell subset. This suggests a combination of epigenetic and immunologic therapies to ensure durable and persistent memory formation and prevent widespread relapses (Abdel-Hakeem et al., 2021). In addition, targeting the TCF-1-T-bet axis is crucial for determining the progression of TExprog2 to TExint. TExint cells regain some effector-like functions and express high T-bet levels; by reinforcing this subtype, terminal exhaustion could be prevented. T-bet is a crucial transcription factor for different subsets because it regulates and balances different TEx subsets (Beltra et al., 2020). Even though exhausted cells remain rigid after therapy, Tox manipulation may have therapeutic value in the future (Khan et al., 2019).

CD4+ T-helper cells are another cell type to consider for future therapies, as Th17 immunity contributes to tumor-resident CD8+ T cell exhaustion. The depletion of CD4+ T cells promotes TExterm formation and increases PD-1 expression. Regardless of tumor type, injection route, or CD4 depletion, IL-17-producing cells cause terminal exhaustion within the tumor microenvironment. Inhibiting the PD-1/PD-L1 pathway increases the population of terminally exhausted CD8+ T cells in mice. Future investigation will show to what extent the inhibition of the retinoic acid receptor-related orphan nuclear receptor (Ror)γt pathway contributes to exhaustion caused by PD-1/PD-L1 ICB or CD4+ depletion. Recent discoveries point to novel anti-tumor immunity approaches by inhibiting IL-17 immunity or Rorγt pathways combined with other immunotherapies (Kim et al., 2021).

Overall, the research progress made by identifying different TEx subsets and associated therapeutic approaches is quite promising. These findings may indicate a crucial target for future therapies to combat chronic diseases and cancer.

Article information

Mol. Cells.Dec 31, 2021; 44(12): 879-882.
Published online 2021-12-24. doi:  10.14348/molcells.2021.0263
1BK21 Four Program, College of Pharmacy, Seoul National University, Seoul 08826, Korea
2Faculty of Biology, University of Munich, Planegg-Martinsried 82152, Germany
3Wide River Institute of Immunology, Seoul National University, Hongcheon 25159, Korea
*Correspondence: (SD); (YC)
Received November 11, 2021; Accepted November 25, 2021.
Articles from Mol. Cells are provided here courtesy of Mol. Cells


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Figure 1

From protective fighters to resting spectators: Changes within CD8+ T cells in chronic diseases and cancer. Naïve CD8+ T cells differentiate to effector T cells (TEff) and memory T cells (TMem) upon contact with foreign intruders during acute infections. However, chronic stimulation causes T cells to become exhausted and differentiate into progenitor exhausted T cells (TExprog1 and TExprog2), intermediate TEx (TExint), and irreversible terminally exhausted TEx (TExterm). Only progenitor TEx respond to PD-1/PD-L1 checkpoint inhibition. The attempt to reinvigorate TEx cells is only possible for TExprog, resulting in an intermediate phenotype, between TMem and TExprog, characterized by “epigenetic scars”. A high expression level is indicated by a blue bar, an intermediate expression by a green bar, and a low expression by a red bar.