Revolutionizing tumor immunotherapy: unleashing the power of progenitor exhausted T cells

In exploring persistent infections and malignancies, a distinctive subgroup of CD8+ T cells, progenitor exhausted CD8+ T (Tpex) cells, has been identified. These Tpex cells are notable for their remarkable self-renewal and rapid proliferation abilities. Recent strides in immunotherapy have demonstrated that Tpex cells expand and differentiate into responsive exhausted CD8+ T cells, thus underscoring their critical role in the immunotherapeutic retort. Clinical examinations have further clarified a robust positive correlation between the proportional abundance of Tpex cells and enhanced clinical prognosis. Tpex cells have found noteworthy applications in the formulation of inventive immunotherapeutic approaches against tumors. This review describes the functions of Tpex cells in the tumor milieu, particularly their potential utility in tumor immunotherapy. Precisely directing Tpex cells may be essential to achieving successful outcomes in immunotherapy against tumors.


Introduction
The roots of harnessing the body's natural immune defenses to combat cancer can be traced to the visionary ideas of Wilhelm Busch 1 and Friedrich Fehleisen 2 .The "cancer immunological surveillance" hypothesis was transformative in advancing tumor immunotherapy 3 .Currently, immunotherapy is considered the fourth pillar in the cancer treatment arsenal, joining the other pillars of surgical intervention, radiotherapy, and chemotherapy.Immunotherapy focuses on addressing challenges faced by patients not amenable to surgical procedures or experiencing recurrence and metastasis post-treatment.Immunotherapies are notable for their goal of activating the patient's own immune system to pinpoint and eradicate tumor cells while minimizing the risk of adverse effects.Current immunotherapy strategies encompass a spectrum of techniques including immune checkpoint inhibitors (ICIs), adoptive cell therapy, cancer vaccines, and cytokine therapies.Despite its tremendous promise, tumor immunotherapy faces several challenges.One major hurdle in cancer treatment is CD8 + T cell exhaustion within the tumor microenvironment (TME), which plays a crucial role in tumor progression.In conditions of chronic infection and cancer, CD8 + T cells engage in a perpetual response to antigenic stimulation, and gradually lose their efficacy and succumb to a state termed "functional exhaustion" [4][5][6][7] .This state is marked by persistent elevation of inhibitory receptors, such as programmed cell death protein-1 (PD-1), lymphocyte-activation gene-3 (LAG-3), and T cell immunoglobulin and mucin domaincontaining protein-3 (TIM-3).Simultaneously, the secretion of effector molecules such as granzyme B (GzmB) and interferon-γ (IFN-γ) declines.T cell exhaustion is closely linked to the effectiveness of immunotherapy.Notably, even interventions such as PD-1 blockade in terminally exhausted CD8 + T (Tex term ) cells fall short of restoring the epigenetic state akin to that of memory or effector cells 8 .In clinical settings, disappointing effects of cancer vaccines have arisen from the exhausted state of tumorinfiltrating lymphocytes (TILs) 9 .
T cell differentiation is a multifaceted and intricate biological process important in modulating the effector functions of CD8 + T cells (Figure 1) 10 .The conceptualization of progenitor cells originated from investigations of the roles of T-bet and eomesodermin (Eomes) in T cell effector differentiation in mice with chronic infection 11 .Research on chronic infections and cancer has uncovered a fascinating subset of CD8 + T cells called Tpex cells.Distinguished by their expression of T cell factor-1 (TCF-1) and PD-1, these cells are critical for understanding of immune exhaustion [12][13][14][15][16][17][18][19] .Referred to as " progenitor exhausted" cells, they have the remarkable ability to transition into fully functional effector exhausted CD8 + T (Tex eff ) cells, which exhibit greater effector function and a weaker exhaustion state than Tex term cells.This finding may potentially represent a turning point in immunotherapeutic strategies.Functionally, Tpex cells display slightly diminished cytotoxicity and produce lower levels of GzmB than Tex eff cells.However, their primary value lies in their robust self-renewal and proliferative capacities-attributes essential for a sustained immune response.Their potential to differentiate into Tex eff cells underscores their importance in the context of immunotherapy, wherein adaptability is key.Essentially, Tpex cells are a unique subset of CD8 + T cells with "stem cell-like" properties that substantially influence the efficacy of immunotherapeutic interventions.This review examines the intricate relationship between Tpex cells and tumor immunotherapy, shedding light on their roles in modulating the immune response.

Surface molecular characterization of Tpex cells
TCF-1 and PD-1 are the quintessential signature of molecules adorning the surfaces of Tpex cells.Additional notable surface molecules include C-X-C chemokine receptor 5 (CXCR5), SLAMF6, and CD62L (Figure 2) 15,[20][21][22][23] .TCF-1, encoded by the Tcf7 gene, is a critical transcription factor downstream of the Notch signaling pathway.Its role extends beyond the thymic development of T cells and encompasses the formation of the memory phenotype 24 .TCF-1 primarily maintains the memory phenotype of Tpex cells through upregulation of key transcription factors, including inhibitor of DNA binding 3 (ID3), Eomes, c-Myb, and B cell lymphoma-2 protein (Bcl-2) 25,26 .The absence of TCF-1 mirrors the dysfunction observed in tumorspecific CD8 + T cells, which is similar to the unresponsiveness observed in patients undergoing ICI treatment in clinical scenarios 27 .SLAMF6, another crucial surface marker associated with precursor exhaustion, is co-expressed with TCF-1 in most Tpex cells 17,28 .Furthermore, within the TME in patients with non-small cell lung cancer (NSCLC), a distinct subset of CXCR5 + PD-1 + CD8 + T cells displays heightened TCF-1 expression 29 .Intriguingly, this phenomenon does not manifest uniformly across various tumor types, e.g., melanoma 17 .This divergence suggests a potential relationship between CXCR5 expression on Tpex cells and tumor types; therefore, further research is necessary to reveal these specific relationships.
The hallmark of CD8 + T cell exhaustion, C-X3-C motif chemokine receptor 1 (CX3CR1), has a critical role in the intricate dynamics of the TME.CX3CR1 + CD8 + T cells, characterized by their expression of CX3CR1, have emerged as key players in the anti-tumor defense within the TME (Figure 2).The downregulation of CX3CR1 is closely linked to the terminal exhaustion state of CD8 + T cells, and marks a shift in their functional capabilities.Single-cell RNA sequencing (scRNA-seq) has revealed that Tpex cells are precursors to SLAMF6 − CX3CR1 + CD8 + T cells.Investigations of chronic viral infections have elucidated the journey of TIM-3 − CX3CR1 + CD8 + T cells and traced their origin to Tpex cells.Notably, these cells can further differentiate into a subset characterized by TIM-3 + CX3CR1 + CD8 + T cells endowed with proliferative capabilities 30 .In the context of melanoma and breast cancer, a specific population of TCF-1 Low PD-1 + TIM-3 int CD8 + T cells are the primary responders to anti-Galectin-9 therapy 31 .Although Tpex cells might not have inherent anti-tumor capabilities, continuous differentiation of Tpex cells into functional Tex eff cells can mediate sustained and more robust anti-tumor response.Notably, in PD-1 blockade therapy, Tpex cells engage in asymmetric division, thereby maintaining the stability of the Tpex cell population while simultaneously generating a cohort of highly effective CD8 + T cells 32 .This intricate balance of differentiation highlights the nuanced yet critical role of Tpex cells in shaping anti-tumor immunity after immunotherapeutic interventions.

Ecological localization of Tpex cells in the TME
In the context of chronic viral infections, Tpex cells are found predominantly in peripheral lymphoid tissues, including the lymph nodes, bone marrow, and spleen 22,23,33,34 .However, the spatial distribution of Tpex cells within tumor tissues is highly complex.Connolly and colleagues 35 have identified a significant population of tumor-specific Tpex cells in the tumor-draining lymph nodes (TdLNs) of the mouse KP-NINJA lung cancer (LC) in situ tumor model; intriguingly, their abundance does not correlate with tumor progression.Similarly, both the mouse B16 melanoma tumor model and patients with head and neck squamous cell carcinoma (HNSCC) exhibit a higher proportion of Tpex cells within TdLNs compared to TME 17,36 .In patients with NSCLC and HNSCC, Tpex cells are localized primarily within the tertiary lymphoid structures (TLS) of the tumor and show minimal infiltration into the tumor parenchyma 37,38 .These observations suggest a potential key role of TLS in sustaining the functionality of Tpex cells.Furthermore, within the TME in renal cancer, these stem-like T cells, marked by positive TCF-1 expression, tend to concentrate in regions densely populated with APCs.Remarkably, these areas functionally mirror TLS, thus emphasizing the significance of such structures in shaping the dynamics of Tpex cells in the intricate landscape of the TME 16 .
Current discourse regarding the origin of Tpex cells revolves around 2 primary theories: one positing that Tpex cells undergo self-renewal and proliferation within tumors, and the other positing their continuous migration from TdLNs to tumor tissue.In the mouse KP-NINJA LC in situ tumor model, the differentiation level of tumor-infiltrating CD8 + T cells surpasses that in TdLNs.This continuous differentiation pattern from TdLNs to tumors suggests that tumor-infiltrating CD8 + T cells are likely to originate from the migration of CD8 + T cells in TdLNs 35 .Examination of patients with hepatocellular carcinoma (HCC) has revealed 2 distinct CD8 + T cell subgroups in TdLNs: tumor-specific memory T cells (Ttsm) and Tpex cells.In mouse models of B16 melanoma and Kras LSL-G12D/+ p53 fl/fl lung adenocarcinoma, Ttsm cells in TdLNs have been found to be critical for the response to PD-1/programmed deathligand-1 (PD-L1) immunotherapy.These cells undergo a linear differentiation trajectory from Ttsm cells to Tpex cells, and subsequently to tumor-infiltrating Tpex and Tex term cells during tumor control 39 .Additionally, a noteworthy increase in Tpex cells in TdLNs and their migration to tumors is observed after immunotherapy 28,40 .Recent research has further suggested that effector T cells under chronic antigen stimulation transform into Tex term cells, which exhibit long-term residence within tumors, and the intratumoral anti-tumor response is maintained by sustained recruitment of Tpex cells from peripheral sources.Over time, these Tpex cells migrate to lymph nodes through the lymphatic system 28 .Consequently, unraveling the intricate ecological positioning and origins of Tpex cells in the TME is imperative for formulating effective immunotherapy strategies.

Regulation of Tpex cells by CD4 + T cells
In LC research, the application of multiplex immunohistochemistry staining techniques has revealed intriguing patterns.Tpex cells tend to form clusters near CD4 + T cells 37 .This spatial arrangement hints at potential interactions between Tpex cells and antigen-specific CD4 + T cells.The abundant presence of CD4 + Th cells within the TME plays a critical role in activating CD8 + T cells (Figure 3).This activation is particularly facilitated by the secretion of interleukin (IL)-21, which engages the IL-21R signaling pathway in CD8 + T cells and consequently promotes differentiation of Tpex cells into effector cells 6,41 .Studies in the mouse B16F10 melanoma model have demonstrated that adoptive therapy with CD4 + T cells significantly amplifies the population of CX3CR1 + CD8 + T cells in the TME, thus effectively controlling tumor growth.In contrast, when the function of CD4 + T cells is compromised, the sustained accumulation of effector CX3CR1 + CD8 + T cells is hampered, as mediated primarily through the IL-21 signaling pathway 42 .Furthermore, in both GL261 (immunoresponsive) and CT2A (immunoresistant) glioblastoma multiforme (GBM) mouse models, effector CD4 + T cells have emerged as key contributors maintaining the Tpex cell state within CT2A tumors.This not only enhances the efficacy of αPD-1 therapy but also indicates the critical role of CD4 + T cells in orchestrating immunotherapeutic responses.Under CD40 agonist administration to compensate for the deficiency in CD4 + T cells in the CT2A TME, the proportion of Tpex cells is sustained.Combined with PD-1 blockade, this dual approach significantly decreases the tumor burden, thus underscoring the potential of this comprehensive strategy 43 .

Regulation of Tpex cells by dendritic cells (DCs)
As guardians of immune activation, DCs not only serve as professional antigen-presenting cells but also are essential in sustaining the function of Tpex cells (Figure 3).Research on chronic viral infections has indicated DC1 cells postpone the differentiation of Tpex cells, thereby extending the window period of CD8 + T cell cytotoxic activity.Although DC1 cells might not be the primary drivers of Tpex cell proliferation, they carve out a crucial ecological niche and prevent the overstimulation of Tpex cells 44 .Magen et al. 45 have investigated the TME in patients with HCC responding favorably to PD-1 therapy and found collaborative effects involving migratory regulatory DCs (mregDCs), C-X-C chemokine ligand 13 (CXCL13) + CD4 + Th cells, and Tpex cells orchestrating the activation of T cell clones.This collaboration amplifies the proportion of tumor-specific CD8 + T cells, thereby illustrating a dynamic interplay that drives therapeutic success 45 .In the Kras LSL-G12D/+ -p53 fl/fl lung adenocarcinoma model in mice, tumor progression triggers a decline in the migration and activation functions of mature DC1 cells to TdLNs.However, after FLT3L/CD40 agonist therapy, the number and function of DC1 in TdLNs are significantly augmented, thus increasing the numbers of stem cell-like CD8 + T cells in TdLNs and enhances their anti-tumor effects 46 .Additionally, in the TME, mregDCs upregulate the expression of major histocompatibility complex-I molecules, and after ICI therapy, CD8 + T cells with high CD28 expression on their surfaces further promote the proliferation and differentiation of Tpex cells within the TME [47][48][49] .After αPD-L1 therapy, the number of DCs in TdLNs also increases and consequently enhances the functionality of CD8 + T cells, although the detailed mechanism remains to be elucidated 50 .

Regulation of Tpex cells by tumor-associated macrophages
Recent investigations have revealed a distinctive subset of immune cells within esophageal squamous cell carcinoma (ESCC), denoted CD8 + Tex-SPRY1.These cells demonstrate anti-tumor functionalities similar to those of Tpex cells and facilitate the polarization of tumor-associated macrophages (TAMs) toward the M1 phenotype, through the secretion of cytokines such as IFN-γ and tumor necrosis factor-α (TNF-α).Notably, M1-type macrophages reciprocally activate CD8 + Tex-SPRY1 cells, thereby amplifying their anti-tumor efficacy 51 .In the TME of HCC, the status of TAMs is critical in determining the efficacy of αPD-1 therapy.Whereas M2-type macrophages suppress tumor progression, their reprogramming into the M1 phenotype has been shown to bolster the immune response and consequently augment the effectiveness of PD-1 blockade therapy 52 .

Application of Tpex cells in tumor immunotherapy
The rapid advancement of immunotherapy seeking to eliminate tumor cells by leveraging the body's own immune system has led to newfound optimism regarding the potential to cure cancer in the future [53][54][55][56] .Encouragingly, immunotherapeutic approaches such as chimeric antigen receptor (CAR)-T and ICI have demonstrated substantial anti-tumor efficacy in certain patients with cancer.However, prolonged exposure to tumor antigens prompts activated CD8 + T cell re-entry into the "exhaustion state"; this crucial factor limits the sustained therapeutic effects of immunotherapy.Consequently, maintaining CD8 + T cells in the TME within the effector stage for an extended duration is imperative for enhancing the overall effectiveness of immunotherapy (Figure 4).

ICI therapy
Tpex cells, a distinct subgroup among CD8 + T cells, exhibit more robust functionality than Tex term cells: they inhibit tumor growth more effectively and demonstrate enhanced responsiveness to PD-1 therapy.PD-1 blockade therapy has been shown to augment the proliferative capacity of Tpex cells and to lead to their differentiation into highly efficacious Tex eff cells 15,17,33,[57][58][59] .In patients with ESCC, NSCLC, and melanoma, a high proportion of Tpex cells is closely associated with sustained effectiveness and improved clinical outcomes of PD-1 blockade therapy 17,51,57,60 .scRNA-seq analysis of tumor samples from patients with NSCLC undergoing PD-1 inhibitor treatment has revealed a significant elevation in Tpex cell levels among those with a positive response to immunotherapy.This increase has been attributed to the expansion of pre-existing Tpex cells in the systemic circulation 61 .Furthermore, the CD8 + Tex-SPRY1 cell subset, identified by Liu et al. 51 in ESCC, exhibits antitumor activity comparable to that of Tpex cells; moreover, the expression level of CD8 + Tex-SPRY1 cells positively correlates with the efficacy of PD-1 inhibitor treatment.In a comparison of patients receiving solely radiotherapy vs. a combination of radiotherapy and cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor treatment, a significantly elevated proportion of the Tpex cell subset has been observed in the latter group and found to positively correlate with patient survival 62 .For patients with metastatic brain tumors and recurrent GBM undergoing ICI treatment (including αPD-1 or combined αPD-1 and αCTLA-4 therapy), Tpex cells are associated with prolonged survival 63 .Notably, ICI treatment of recurrent GBM promotes the differentiation of Tpex cells, whereas in metastatic brain tumors, ICI treatment promotes the differentiation of Tex term cells 64 .
In a mouse model of acute myeloid leukemia, the combination of a bromodomain and extra-terminal domain inhibitor with αPD-1 therapy promotes the proliferation of Tpex cells, thereby exhibiting synergistic effects 65 .Moreover, the heightened expression of lysine-specific demethylase 1 (LSD1) in various cancers contributes to tumor progression and influences the immune response.Targeting LSD1 in mouse models of MC38 colon carcinoma and B16F10 melanoma enhances the durable effects of αPD-1 therapy, primarily by fostering Tpex cell proliferation within the TME and transformation into more efficacious Tex eff cells.The positive effects on therapeutic outcomes are further underscored by the augmentation of the TCF-1 regulatory network associated with LSD1 depletion 66 .In a separate study, the engineering of particles, specifically R848@M2pep-MPsAFP has been found to successfully reprogram macrophages within the HCC TME, thereby resulting in phenotypic transformation, improved antigen-presenting capabilities, activation of Tpex cells, and significant enhancement of αPD-1 therapy efficacy 52 .
Chen et al. 47 have demonstrated that concurrent administration of high-mobility group nucleosome binding domain 1 (HMGN1) peptide with αPD-L1 significantly augments the population of Tpex cells within tumors, thereby enhancing the efficacy of αPD-L1 therapy.In investigations of HNSCC, αPD-L1 immunotherapy has been observed to diminish the proportion of Tpex cells in peripheral tissues.Nevertheless, Tpex cells located near DCs tend to show proliferation and differentiation into intermediate-exhausted CD8 + T cells (Tex-int).This phenomenon aligns with the increased presence of Tex-int clonotypes circulating in the peripheral blood 50 .
Recent research has demonstrated that the combination of αPD-1 and decitabine increases the proportion of Tpex cells and consequently augments anti-tumor efficacy 67 .However, Mariniello and colleagues 68 have proposed that the combination of chemotherapy with immunotherapy might influence the effectiveness of these drugs, particularly  regarding the Tpex cell subpopulation.Their findings suggest that PD-L1 inhibitors activate Tpex cells and facilitate their differentiation into effector cells, whereas chemotherapy drugs may impede this activation.Notably, the sequence of chemotherapy followed by immunotherapy has been demonstrated to be more efficacious than combination therapy 68 .Importantly, this observation awaits validation in mouse tumor models, and further in-depth mechanistic research is necessary to provide insights for developing clinical treatment strategies.

Cytokine therapy
IL-2, a critical cytokine in regulating T cell maturation and maintaining T cell function, has significant therapeutic promise in cancer immunotherapy [69][70][71][72][73] .Given the potential for widespread systemic toxicity and adverse effects associated with IL-2 therapy, current research on engineered IL-2-based drugs focuses primarily on modulating specific aspects of the immune response, through activating CD8 + T cells or natural killer cells while inhibiting regulatory T cells 71,74 .
In the context of treating chronic viral infections, the combined use of IL-2 and αPD-1 has an effectiveness approximately tenfold greater than that of αPD-1 alone.This synergistic therapy modulates Tpex cells and facilitates their transformation into more efficacious effector T cells 75 .PD1-IL2v, a novel bispecific antibody, simultaneously blocks PD-1 signaling on CD8 + T cells and activates IL-2R signaling.This design offers the advantage of selectively activating the IL-2 signal in PD-1 + CD8 + T cells with tumor antigen specificity.Studies in various mouse models of pancreatic cancer (PK5L1940, FC1242, and Panc02) have indicated that PD1-IL2v treatment significantly increases the population of stem-like Tpex cells in the TME 76,77 .
Several studies have explored the therapeutic effects of combining PD1-IL2v with various treatment modalities.In a mouse Panc02 pancreatic adenocarcinoma model, PD1-IL2v treatment leads to a substantially higher proportion of Tpex cells in the TME than single-agent therapy.These cells consequently differentiate into a CD8 + TIL subpopulation with enhanced functionality and lower exhaustion (TCF-1 − PD-1 + TIM-3 − GzmB + ) 77 .When combined with radiotherapy, PD1-IL2v further increases the frequency of Tpex cells and results in more durable anti-tumor responses 76 .Monotherapy with PD1-IL2v in mouse models of pancreatic neuroendocrine tumors and GBM has been found to lead to tumor regression.
Combining this therapy with αPD-L1 treatment exhibits stronger anti-tumor effects, with 90% of mice showing no relapse, and a significant increase in the proportion of Tpex cells and their descendant Tex cells in the TME 78 .Current research outcomes on IL-2 drugs underscore the essential role of the Tpex cell subpopulation in activating CD8 + T cell anti-tumor responses.
IL-15, another cytokine with substantial potential in cancer therapy, shares the β (CD122) and γ (CD132) chains with IL-2R [79][80][81][82] .This cytokine plays a crucial role in promoting the proliferation of CD8 + T cells, and its absence in the TME has been associated with impaired function of CD8 + T cells, tumor relapse, and diminished patient survival 83,84 .Previous studies have demonstrated that co-culturing CD8 + TILs derived from human renal cell carcinoma with IL-15 enhances the self-renewal capability of these cells 85 .
Combining IL-15 with αPD-1 not only amplifies the proliferation of CD28 − PD-1 + CD8 + TILs exhibiting a Tex term cell phenotype and CD28 + PD-1 + CD8 + TILs with Tpex cell characteristics, but also intensifies their anti-tumor effects.This action is crucial for the reactivation of CD8 + T cells post-PD-1 blockade 48,86 .The anti-tumor effect is attributed primarily to the cytotoxic activity of CD28 − PD-1 + CD8 + TILs, which differentiate predominantly from CD28 + PD-1 + CD8 + TILs showing similar characteristics to Tpex cells 87 .The development of pro-IL-15 by Guo 79 has addressed the toxicity associated with IL-15 while preserving its anti-tumor effects.In the mouse MC38 colon cancer model, pro-IL-15 promotes the proliferation of stem-like TCF-1 + TIM-3 − CD8 + TILs, controlling tumor growth through IFN-γ secretion, particularly when used in combination with PD-L1.This combination exhibits significantly better effects than PD-L1 monotherapy 79 .Moreover, CAR-T cells treated with IL-7 and IL-15 display a notable increase in the expression of memory genes such as Tcf7 and Sell, and show stronger reactivity to PD-1 blockade therapy and sustained anti-tumor effects 88 .This finding opens new avenues for enhancing the efficacy of CAR-T treatment in solid tumors.
Current research on IL-10 has revealed its intricate dual role in immune regulation: it promotes immunosuppressive phenomena and also is crucial in maintaining anti-tumor responses.Previous studies have established a correlation between the concentration of IL-10 in serum and the progression of chronic lymphocytic leukemia (CLL) 89,90 .However, recent reports have highlighted the essential role of IL-10R signaling in the maintenance and accumulation of PD-1 int TCF-1 + CD8 + T cells in CLL.Intriguingly, treatment with αIL-10R may exacerbate the progression of CLL 91 .
Another noteworthy study has observed that treatment with IL-10-Fc in a mouse B16F10 tumor model stimulates the proliferation of PD-1 + TIM-3 + CD8 + T cells through IL-10R, thereby promoting their sustained cytotoxic function.Importantly, this proliferative process is not contingent on the differentiation of PD-1 + TIM-3 − progenitor cells.Instead, it is achieved by enhancing the cells' anti-tumor action through metabolic reprogramming, particularly the upregulation of oxidative phosphorylation in T cells 92 .

Adoptive cell therapy
CAR-T immunotherapy has shown considerable success in the treatment of hematologic malignancies, yet its efficacy in solid tumors remains limited [93][94][95][96][97][98] .This limitation is largely attributable to the tendency of CAR-T cells to enter a functionally diminished "exhausted" state 99,100 .Consequently, maintaining the anti-tumor effects of CAR-T therapy has emerged as a critical challenge in therapeutic development.The transcription factor B lymphocyte-induced maturation protein 1 (BLIMP1), encoded by PRDM1, has been identified as a key player in CD8 + T cell exhaustion 101,102 .Modulating the interaction between BLIMP1 and TCF-1 enhances the persistence and proliferative capacity of CAR-T cells, thereby offering new strategies for improving the treatment of solid tumors 103,104 .
In vivo studies have demonstrated that BLIMP1-depleted CD19-CAR-T cells exhibit stronger and more persistent anti-tumor effects in certain cancer models, albeit with a potential decrease in their immediate ability to release cytotoxic molecules, including GzmB and perforin 105,106 .In PC3-PSMA and NALM-6 B cell acute lymphoblastic leukemia (B-ALL) models, PRDM1 −/− NR4A3 −/− CAR-T cells have shown greater tumor control and a lower tendency toward the Tex term cell phenotype compared with traditional CAR-T cells 105 .The specific knockout of Regnase-1 in CAR-T cells designed for ALL research promotes the development of Tpex cells, decreases their exhaustion, and consequently enhances sustained anti-tumor action 107 .Recent studies have also identified that high expression of ETS1 in CD8 + T cells significantly inhibits the transformation of Tpex cells into Tex term cells-a suppression dependent on decreased BATF activity.Treating the hCD19-B16 melanoma model with ETS1deficient hCD19-CAR-T cells markedly enhances therapeutic efficacy 108 .

Cancer vaccine therapy
Therapeutic cancer vaccines are aimed at activating the immune system to combat tumors.Although most trials have shown modest effects, better therapeutic efficacy has been observed under specific conditions, such as strong T-cell responses or minimal immunosuppression 9 .scRNA-seq analysis of mouse B16 tumors has revealed 2 main CD8 + T cell populations in the TME post-vaccination: progenitorexhausted (TCF-1 + ) and transiently exhausted (CX3CR1 + GzmB + ).These findings suggest that cancer vaccine inoculation is conducive to establishing a persistent CD8 + T cell anti-tumor environment 15,109 .
Preclinical in vivo experiments in mice have indicated synergistic anti-tumor effects of vaccine therapy combined with ICIs.In melanoma, combining a vaccine with αPD-L1 achieves stronger tumor control than monotherapy with αPD-L1 109 .In the mouse MC38 colon cancer model, an adenovirus vector vaccine combined with αPD-1 immunotherapy effectively promotes the accumulation of Tpex cells in TdLNs.These cells then infiltrate tumors and differentiate into effector cells.Moreover, compared with monotherapy with αPD-1, the combination of the vaccine with αPD-1 exhibits stronger clonal capacity of these specific T cells and better anti-tumor effects 40 .
To enhance the precision of vaccine delivery in vivo, Baharom and colleagues 110,111 have developed a nanoparticle vaccine assembled from neoantigen peptides linked to Toll-like receptor 7/8 agonists.Treatment of MC38 tumor-bearing mice with this nanoparticle vaccine significantly increases the proportion of Tpex cells in the spleen, thus demonstrating robust anti-tumor activity 110,111 .Crucially, the injection method has been observed to influence the proportion of Tpex cells induced by this vaccine.Intravenous injection yields a greater proportion of Tpex cells (80%) than subcutaneous injection (35%).Notably, the intravenous vaccine injection not only produces a significantly higher percentage of Tpex cells but also demonstrates a more pronounced anti-tumor effect than subcutaneous injection 111 .

Conclusions
The discovery of Tpex cells has offered a fresh perspective for understanding the intricacies of CD8 + T cell exhaustion and has shed light on their essential role in cancer immunotherapy.These findings not only underscore the dynamic nature of CD8 + T cell exhaustion but also reveal a previously unknown complexity.Tpex cells significantly shape the response to immunotherapy, thereby emphasizing the crucial role of augmenting Tpex cell proportions within the TME to increase therapeutic efficacy.Currently, as the principal cells responsive to immunotherapy, Tpex cells are regulated primarily by immunotherapy, as discussed above.This approach correlates with improved response rates and extended patient survival after immunotherapy.Unfortunately, no specific drugs or compounds have been designed to specifically regulate the proliferation and renewal of Tpex cells.Consequently, future endeavors in drug development and delivery strategies targeting Tpex cells will be crucial avenues for augmenting the efficacy of immunotherapy.Furthermore, a thorough exploration of the molecular mechanisms governing Tpex cell differentiation and homeostasis will be essential for advancing novel immunotherapies.Special consideration should be given to the critical role of TCF-1 in regulating the delicate balance between T cell exhaustion and functionality, particularly its effects on human tumors.Additionally, elucidating the interactions between Tpex cells and other key T cell subsets, such as tissue-resident memory T cells crucial for tumor control, will be necessary to formulate effective immunotherapeutic strategies.Addressing these fundamental biological questions is poised to propel advancements in cancer immunotherapy.

Figure 1
Figure 1 Differentiation process of CD8 + T cells in the TME.(A) In the TME, sustained antigenic stimulation prompts the differentiation of naive CD8 + T cells into Tpex cells.In the absence of CD4 + T cells and antigen-presenting cells (APCs), Tpex cells undergo direct differentiation into terminally exhausted dysfunctional CD8 + T cells.This process is concomitant with an upregulation in the expression of surface molecules such as PD-1, TIM-3, LAG-3, and CD39, along with the transcription factors Tox and Eomes.(B) When sufficient CD4 + T cells and APCs are present, Tpex cells initially differentiate into Tex eff cells with augmented effector functions.These cells exhibit heightened secretion of GzmB and IFN-γ and exert potent anti-tumor effects.However, prolonged antigenic stimulation leads to the eventual differentiation of Tex eff cells into Tex term cells.

Figure 2
Figure 2 Surface molecules characterized during the differentiation of CD8 + T cell subpopulations.Differences in the expression of surface molecules, transcription factors, and effector molecules during CD8 + T cell exhaustion in Tpex cells, Tex eff cells, and Tex term cells.Int, intermediate.

Figure 3
Figure 3 Interactions between immune cells and Tpex cells in the TME.(A) DCs in TdLNs create a microenvironment conducive to the survival and development of Tpex cells.After ICI treatment, Tpex cells in TdLNs proliferate and migrate to tumor tissue, and differentiate into Tex eff cells with functional efficacy.(B) CD4 + Th cells maintain the effector functions of Tex cells, and promote the self-renewal and differentiation of Tpex cells into Tex eff cells through secretion of the cytokine IL-21.(C) IL-10 or TGF-β, secreted by M2 cells, suppresses the function of Tpex cells.In contrast, DCs and M1-type macrophages within the TME synergistically and collaboratively activate Tpex cells, thereby enhancing their anti-tumor effects.(D) Tpex cells in the TME can also enter TdLNs through the lymphatic system.

Figure 4
Figure 4 Tumor immunotherapy targeting Tpex.ICI therapy: Tpex cells are the primary population responsive to PD-1/PD-L1 treatment and can further differentiate into more Tex eff cells after ICI therapy.Cancer vaccine therapy: cancer vaccines augment their anti-tumor efficacy by bolstering the interaction between antigen-presenting cells and Tex eff cells.Tpex cells in the TME sustain this prolonged effector function.Cytokine therapy: cytokines such as IL-2 and IL-15 facilitate the generation of Tpex cells, thereby ensuring an ample supply of Tex eff cells within the TME to eliminate tumor cells.CAR-T therapy: CAR-T cells subjected to gene editing technology can sustain an enduring effector state while circumventing rapid differentiation into a terminal exhaustion state.Moreover, the concurrent application of immunotherapy can activate Tpex cells within the TME, thus sustaining their anti-tumor functions and facilitating prolonged therapeutic effects.