The immune system is well-equipped to eliminate foreign or dangerous entities, including naïve CD8⁺ T cells capable of differentiating into effector T cells (T_Eff or cytotoxic T lymphocytes) and memory T cells (T_Mem) (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 (T_Ex) cells characterize malignancies, such as hepatitis C virus or human immunodeficiency virus infection, and lung cancer (Khan et al., 2019).

T_Ex cells are a distinct subset with a unique transcriptional and epigenetic landscape from T_Eff and T_Mem (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). T_Ex 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). T_Ex cells are distinguished by their heterogeneity, allowing classification into four different subsets (Beltra et al., 2020).

While the progenitor and terminally exhausted T_Ex 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 T_Ex stages of exhaustion. As summarized in the figure, T_Ex 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 T_Ex 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 T_Ex subsets express Tox, it is considered the master transcription factor of exhaustion (Abdel-Hakeem et al., 2021; Beltra et al., 2020).

Progenitor T_Ex 1 (T_Ex^prog1) 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 T_Ex 2 subset (T_Ex^prog2) 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 T_Ex (Abdel-Hakeem et al., 2021; Beltra et al., 2020). Under the epigenetic and transcriptional control of T-bet, the initial proliferation of the T_Ex^prog2 subset results in the appearance of the downstream intermediate T_Ex (T_Ex^int) 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. T_Ex^int 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 T_Eff. 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 T_Ex^int cells, this terminally exhausted T_Ex (T_Ex^term) 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. T_Ex^term 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 T_Ex 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 T_Ex cells were called recovered T_Ex (REC-T_Ex) because they switched from an exhausted to a memory-like phenotype. Nevertheless, the REC-T_Ex 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 T_Ex cells. The gene expression profile associated with exhaustion, including Tox, Pdcd1 (encodes PD-1), and LAG-3, was decreased in REC-T_Ex, resulting in an intermediate transcriptional profile between T_Ex and T_Mem. In contrast, memory cell-specific genes, including IL-7 receptor (Il7r) and TCF-7 (encodes TCF-1), were upregulated. Tox remained high in REC-T_Ex, although TCF-7 was lower than in T_Mem. By comparing REC-T_Ex with different subtypes established by Beltra et al. (2020), Abdel-Hakeem et al. (2021) concluded that REC-T_Ex arises from TCF-1-expressing progenitor T_Ex. 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-T_Ex was inferior to T_Eff or T_Mem, presenting higher PD-1 and Tox expression levels. Compared with T_Ex, we observed only a slight improvement of effector functions. Overall, REC-T_Ex resembled T_Ex more than T_Mem. Investigating chromatin accessibility revealed consistent differences between REC-T_Ex and T_Mem open chromatin regions called “epigenetic scars” of exhaustion. Altogether, the recovery after antigen removal did not improve REC-T_Ex effector functions. This incomplete functional recovery reveals a lack of epigenetic plasticity and failure to reestablish the transcriptional programs of T_Eff and T_Mem. Nevertheless, these reinvigorated T_Ex cells could become a target for future immune checkpoint blockade (ICB) therapies that already targeted T_Ex without groundbreaking success.

Because ICB offered significant therapeutic improvement, a better characterization of the function and regulation of exhausted T cells is critical, as T_Ex cells are major cell types responding to anti-PD-1/PD-L1 biologics (Beltra et al., 2020). Among the different T_Ex subsets, only PD-1^intCD44^hi progenitor T_Ex responded efficiently to anti-PD-1/PD-L1 treatment compared to PD-1^hiCD44^int terminally exhausted T_Ex (Abdel-Hakeem et al., 2021; Beltra et al., 2020; Kim et al., 2021). After ICB, the circulatory subsets T_Ex^prog2 and T-bet high T_Ex^int expanded preferentially, and differentiation into different T_Ex 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-T_Ex, 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 T_Ex 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-T_Ex 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 T_Ex^prog2 to T_Ex^int. T_Ex^int 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 T_Ex 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 T_Ex^term 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 T_Ex subsets and associated therapeutic approaches is quite promising. These findings may indicate a crucial target for future therapies to combat chronic diseases and cancer.

This work was supported by the research grants: Global Research Laboratory Program (2017K1A1A2004511 [Y.C.]).

The paper was written during an internship (S.D.) at the College of Pharmacy of the Seoul National University as a part of the master’s program “Molecular and Cellular Biology” of the University of Munich.

S.D. and Y.C. wrote the paper.

The authors have no potential conflicts of interest to disclose.