Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

‘Stem-like’ precursors are the fount to sustain persistent CD8+ T cell responses

Abstract

Virus-specific CD8+ T cells that differentiate in the context of resolved versus persisting infections exhibit divergent phenotypic and functional characteristics, which suggests that their differentiation trajectories are governed by distinct cellular dynamics, developmental pathways and molecular mechanisms. For acute infection, it is long known that antigen-specific T cell populations contain terminally differentiated effector T cells, known as short-lived effector T cells, and proliferation-competent and differentiation-competent memory precursor T cells. More recently, it was identified that a similar functional segregation occurs in chronic infections. A failure to generate proliferation-competent precursor cells in chronic infections and tumors results in the collapse of the T cell response. Thus, these precursor cells are major therapeutic and prophylactic targets of immune interventions. These observations suggest substantial commonality between T cell responses in acute and chronic infections but there are also critical differences. We are therefore reviewing the common features and peculiarities of precursor cells in acute infections, different types of persistent infection and cancer.

Main

The mammalian immune system rapidly clears most infections and forms functional memory T (Tmem) cells, which protect the host upon reencountering the same or a cross-reactive pathogen. Nonetheless, several viruses bypass this elimination and establish either a latent chronic infection, characterized by intermittent virus reactivation, or a chronic infection, in which virus particles are continuously produced. We commonly refer to the latter as ‘active chronic infections’ (Box 1). Latent infections are typically caused by herpesviruses such as cytomegalovirus (CMV), Epstein–Barr virus (EBV), or herpes simplex virus (HSV) and active chronic infections by the hepatitis B virus (HBV) and hepatitis C virus (HCV) or human immunodeficiency virus (HIV) in humans and certain lymphocytic choriomeningitis virus (LCMV) strains in mice. In active chronic infections, T cells often show the phenomenon of T cell exhaustion, referring to T cells with diminished effector functions compared to effector and memory T cells, which develop in resolved infections as reviewed elsewhere1,2,3,4,5,6,7. By contrast, pathogen-specific T cell populations in latent infections remain functional and can, for instance in CMV infections, considerably grow in size—a phenomenon known as memory inflation8.

Irrespective of these differences, there are core principles that universally apply to T cell responses across acute and chronic infections. These involve the effective recruitment, massive expansion of pathogen-specific naive or previously formed Tmem cells, and the formation of short-lived, terminally differentiated effector T (Teff) cells. Teff cells massively decline in numbers following the acute infection phase. For acute and latent infection, it is well established that this decline coincides with the appearance a subpopulation known as memory precursor T (Tmp) cells (see Box 1 for nomenclature details), which retain proliferative potential and progressively develop into Tmem cells that then can give rise to Teff cells, either by antigen reexposure following a secondary infection or following a reactivation of a latent infection9,10. Tmem cells are often further divided into: (1) central memory T (Tcm) cells that bear strong proliferative capacity, (2) effector memory T (Tem) cells, which exhibit limited survival but more immediate effector functions, and (3) stem-cell memory T (Tscm) cells—a population related to Tcm with superior re-expansion capacity (Box 1). In addition, there are tissue-confined T cells known as tissue-resident memory T (Trm) cells.

Reports about a limited survival and re-expansion potential of exhausted T cell populations spurred the assumption that proliferation-competent T cells with memory potential are not formed in active chronic infections. In contrast, we know now that they also contain a small subset of proliferation-competent precursors of exhausted T (Tpex) cells (Box 1) that express the transcription factor T cell factor 1 (TCF-1) encoded by Tcf7, which are preferentially found in secondary lymphoid organs and in the blood of mice11,12,13,14,15,16. These precursors are essential for the overall maintenance of the terminally differentiated (exhausted) T cell population (Tex cells; Box 1). Thus, the branching of activated and expanding T cells into terminally differentiated cells and into cells with proliferative potential is a universal core feature of the CD8+ T cell response in acute and chronic infections and we know that these cells are formed in acute and in chronic infection irrespective of antigen persistence or clearance (Figs. 1a–c and 2) and in the context of tumors. Given the importance of these proliferation-competent precursors (Tpex and Tmp cells), we summarize the current knowledge about particularities and molecular mechanisms that apply to these precursors in acute, latent and chronic infection and cancer.

Fig. 1: Illustration of the dynamics of antigen-specific T cell populations following reoccurring infections versus latent or active chronic infections.
figure 1

ac, White area under the curve indicates the total magnitude of the antigen-specific T cell response, while the red area under the curve indicates the fraction of proliferation-competent precursor cells. Yellow intervals illustrate the frequency of reactivation. The black line in b increases over time to indicate the rise in terminally differentiated effector cells during inflating T cell responses.

Fig. 2: The life cycle of precursor T cells in acute and chronic infection.
figure 2

In both acute and chronic infections, naive T cells differentiate during the T cell expansion phase into short-lived effector cells and proliferation-competent precursor cells. We refer to these latter cells in acute infections as Tmp cells and in active chronic infection as Tpex cells. a, Three possible scenarios for the generation of effector and memory T cells are: the bifurcation model according to which effector and memory T cells are formed independently of each other (left), the progressive differentiation model proposing that cells with fewer rounds of division form Tmp cells, while more excessively proliferating cells generate primarily Teff cells (middle), and the progression of memory T cells through an effector stage (right). b, Upon resolution of the acute infection, Tmp cells transition into Tmem cells, which themselves upon reinfection can differentiate again into Tmp cells and subsequently into secondary Tmem cells. c, The initial mixed short-lived Teff (blue) and Tex (red) cell population is generated independently of TCF-1-expressing Tpex cells, but this initial population becomes replaced at later time points by Tex cells derived from a self-maintaining Tpex population. Tpex cells are permanently exposed to antigen (albeit being considerably shielded against activation by the expression of co-inhibitory receptors), leading to their frequent (or permanent) activation, proliferation and differentiation into Tex cells. The latter follows a trajectory including Tpex2 and TexInt subpopulations. d, In case of an eventually occurring resolution of a chronic infection, or due to a loss of a particular epitope due to virus escape, Tpex cells likely transition into a resting population, which combines features of memory and of exhausted cells. These Tmem cells with signs of T cell exhaustion (Tmx) appear to have the capacity to regenerate Teff cells with an exhausted phenotype upon secondary stimulation (gray dashed arrows).

The dynamics of proliferation-competent precursors in different infections

Even though proliferation-competent T cells are an integral part of all T cell responses, their development is typically masked by the much larger terminally differentiated Teff cell population. For instance, our linear illustration of T cell response kinetics in acute infections, which includes expansion, contraction, Tmem cell formation and maintenance phase (Fig. 1a), does not reflect the dynamics nor do any of the three main models discuss how Tmp cells are formed. These models include: (1) a bifurcation model claiming that effector and memory T cell trajectories segregate at a certain time point following an asymmetric cell division (Fig. 2a); (2) a progressive differentiation model demonstrating that more extensively dividing cells become effector and less-stimulated cells become memory precursor cells (Tmp; Fig. 2a); and (3) a third and not mutually exclusive model showing that some T cells transition from an effector stage into Tmem cells (Fig. 2a). Experimental evidence exists that supports each of these models17,18,19,20,21,22, leaving the origin of Tmem cells unresolved. What is undisputed by all models is the existence of a trajectory, which connects the different types of proliferation-competent naive and memory T cells or, for instance, primary Tmem cells with secondary Tmem cells. Along this trajectory, cells transition from a resting state through a phase of rapid proliferation, followed by a partially activated Tmp cell state, before the cells return to quiescent Tmem cells, which only occasionally homeostatically proliferate. Upon reencountering antigen, Tmem cells again move through these differentiation phases. Because of this repeating nature, the dynamics of proliferation-competent cells can be viewed as a circular process (Fig. 2b).

In active chronic infections, the continuation of antigen exposure interrupts this circle and counteracts the return of proliferation-competent Tpex cell to the quiescent Tmem cell state. Instead, Tpex cells continuously proliferate to reproduce themselves and to generate Tex cells (Fig. 2c). Of note, bromodeoxyuridine (BrdU) incorporation studies revealed that proliferation occurs at a much slower pace than in the acute infection phase. This slower pace likely accounts for the contraction phase that is detectable even in active chronic infection after a certain time and despite abundant antigen being present23,24,25 (Fig. 1c). Another possible reason for this slowdown of proliferation is a reduced level of activation, owing to negative regulation by co-inhibitory receptors26.

In contrast, latent infections show a mixed phenotype that combines features of resolved and active chronic infections. Here, the intervals and the magnitude of pathogen reactivation determine the response pattern. With more frequent reactivation of the proliferation-competent precursors, a larger population of T cells with a Tem cell phenotype is induced27,28,29 in a process known as memory inflation (Fig. 1b). Of note, the proliferation-competent cells established in CMV infections do not exhibit signs of exhaustion in healthy individuals. Instead, they resemble Tcm-like cells established in resolved infection8. This observation indicates that the extent of (recurrent) antigen exposure is a critical determinant in shaping the phenotypic and functional profile of T cells in persisting infections25.

Specific features of Tpex cells and their descendants

The identification of Tpex cells resolved the long-standing question of how short-lived, terminally differentiated Tex cells are maintained long term and how Tex cells could be reinvigorated via immune checkpoint blockade (ICB). In murine chronic LCMV infection, Tpex cells express TCF-1, CXCR5, Ly108, CD73, ID3 and low levels of CD127 and CD62L, while they lack expression of TIM-3 (refs. 13,14,30). TCF-1 is essential for the formation and function of this precursor population. Interestingly, its absence results in a normal primary T cell expansion (Fig. 2c), but T cells steadily decline over time14. This response resembles the pattern seen with TCF-1-deficient T cells in acute LCMV infection, where effector but not functional Tmp cells are formed without TCF-1 (refs. 31,32). Recently, Tpex cells were further divided into TCF-1+Ly108+CD69+ triple-positive Tpex1 cells, which then transition into further differentiated TCF-1+Ly108+CD69 double-positive Tpex2 cells. Likewise, Tpex2-derived terminally differentiated cells can be divided into CD69Ly108 (TexInt) cells that generate the most terminally differentiated CD69+Ly108 Tex cell population33 (Fig. 2a). Similarly, exhausted cell populations were also divided into TCF-1+Ly108+ precursors, Ly108CX3CR1 double-negative terminal cells, and into CD4-help-dependent CX3CR1+ cells34,35, or into TCF-1+CD101TIM3 precursors, CD101TIM3+CX3CR1+ transitional cells and CD101+TIM3+ double-positive terminal cells36. Despite the use of different markers, all studies depict similar differentiation concepts for exhausted T cell populations.

A key feature of Tpex cells is that they are stably committed to exhaustion and they transmit an exhausted phenotype to their progeny. Thus, even when Tpex cells from a chronic infection are experimentally transferred into new hosts experiencing an acute infection, their progeny retains an exhausted phenotype37. Similar observations were made following ICB. While the treatment boosts the overall T cell response and their effector capacity in chronic infection and tumors, the progeny of the reactivated Tpex cells still shows core features of exhausted T cells38. We know now that this stability is enforced by epigenetic imprints that, once established, cannot be overcome by reactivation of the Tpex cell or by checkpoint inhibition37,38,39,40. Several observations have indicated that a full commitment to T cell exhaustion requires time, such that an early transfer of virus-specific CD8+ T cells exposed to a chronic infection into a new setting of an acute infection prevents the acquisition of an exhausted phenotype41. Similar observations were made in the context of tumor-reactive T cells in mice40. Nonetheless, transcriptional and certain epigenetic differences between Tpex and Tmp cells can be detected as early as 5 days after infection42. Thus, the commitment toward T cell exhaustion begins early but requires time to stabilize.

This commitment to the generation of T cell exhaustion involves distinct metabolic states43,44 and epigenetic mechanisms, including the activity of methyltransferases such as Dnmt3a39, and also require continued high-level antigen exposure and strong T cell antigen receptor (TCR) stimulation that lead to NFAT-induced transcriptional networks. The transcription factors such as NR4A1, NR4A2 and TOX are essential for inducing or enforcing T cell exhaustion45,46,47,48,49,50,51. In addition, interleukin-2 receptor (IL-2R) signaling was shown to promote Tex cell differentiation at the expense of Tpex cell formation52, while IL-21R signaling at late time points prompted Tpex cell formation53. The transcription factor TOX is particularly interesting as its absence results in a T cell population with higher functional capacity and markedly reduced signs of exhaustion, including lower PD-1 expression. While the mechanisms by which TOX exerts this function remain unclear, it appears to be involved in establishing the exhausted phenotype in Tpex cells (discussed below)46. Nonetheless, it needs to be underlined that we are still far from fully understanding the molecular mechanisms that induce T cell exhaustion.

The functional similarities between Tpex and Tmp cells in chronic and acute infections, respectively, suggest that they are related populations. Although this notion holds true for certain aspects such as their ability to regenerate themselves and to generate more differentiated cells (stem-cell-like properties), there are also notable differences. Unlike Tmem cells, Tpex cells show markers and features of Tex cells including elevated levels of programmed death protein 1 (PD-1) and compromised tumor necrosis factor (TNF) and interferon-γ (IFNγ) secretion. Tpex cells also differ from Tmem cells in forming and surviving independently of CD4+ T cell help34,35, which is required to form or maintain Tmem cells in resolved infections10. By contrast, Tex cell differentiation and, in particular, the CX3CR1 subset strongly depends on CD4 help and the provision of interleukin (IL)-21 (refs. 34,35), while Teff cells in acute LCMV infections can be formed independently of CD4 help54. Thus, Tpex and Tex cells show divergent CD4 help requirements compared to Tmem and Teff cells in resolved infections.

While we clearly understand now that Tpex cells feed into the pool of CD8+ Tex cells in an antigen-dependent manner, it is less frequently considered that Tpex cells can survive following antigen withdrawal37,55. In fact, we consider that Tpex cells transition in the absence of antigen into cells with self-renewal capacity similar to that of conventional memory cells. In case of antigen reencounter, they then mount a recall response and produce terminally differentiated progeny (Fig. 2d)14,37. Related to this, it was recently identified that healthy humans harbor antigen-specific T cells that combine features of Tmem cells and exhaustion. These features include the expression of PD-1 and TIGIT inhibitory receptors and enhanced chromatin accessibility at exhaustion-related genomic sites, for instance, in the TOX, PDCD1 and TIGIT genes. These exhaustion traits are stable following activation and effector differentiation in vitro56 and are associated with enhanced response to immunosuppressive transforming growth factor-β (TGFβ) and reduced anti-leukemic activity when these cells are used as hosts for chimeric antigen receptor (CAR) expression in a humanized model of leukemia56. Intriguingly, the TCR clonal repertoire of these memory cells was largely non-overlapping with that of PD-1TIGIT Tmem cells. This finding implies that a specific set of antigens and/or stimulation conditions generates this specific cell population. Presently, we lack a name for these cells and we propose to refer to them as descendants of Tpex or Tmx cells (for Tmem cells with exhausted features). While more research is needed to understand their biology, we consider that these cells might originate from resolved active chronic infection (Fig. 2d), possibly from latently persisting pathogens, or from an acute infection with very strong T cell stimulation that induces T cell phenotypes seen otherwise in active chronic infection. In fact, different reports indicate that severe acute respiratory syndrome coronavirus 2 infection induces signs of T cell exhaustion57. This transition of Tpex cells into Tmx cells might at first glance appear as a rare scenario but these cells are readily found in humans. Of note, similar features can be seen in antigen-escape situations or pharmacologically resolved active human chronic infections, both of which support the formation of CD127-expressing cells featuring signs of T cell memory and exhaustion, simultaneously55.

Molecular determinants of precursor T cells

A large number of phenotypic markers, distinct transcriptional networks and specific epigenetic and metabolic profiles impact the differentiation of exhausted T cell populations1,3,7,58. Most of these features were established irrespective of the subsets of exhausted T cells. It is therefore important to identify how these mechanisms operate in Tpex cells. Here, we will mainly focus on a selection of molecules that were shown to affect the establishment or maintenance of the TCF-1+ Tpex cells.

PD-1, encoded by Pdcd1, was the first co-inhibitory receptor found to be constitutively expressed on exhausted T cells59. Its expression is regulated by the transcription factors NFAT and T-bet following T cell activation60,61. Pdcd1 deletion can result in lethal CD8+ T cell-mediated immunopathology59,62. PD-1 acts directly on Tpex cells, as adoptive transfer of LCMV-specific PD-1-deficient CD8+ T cells revealed compromised long-term maintenance combined with enhanced differentiation into Tex cells, suggesting that PD-1 is at least partly preserving Tpex cells63. This conclusion was confirmed by the demonstration that PD-1 stabilized the Tpex cell population64.

TCF-1 is a hallmark transcription factor for the formation and/or maintenance of Tpex cells2. It playshas a central role in the early bifurcation of terminal effector and Tpex cells by repressing the former and stabilizing the latter. This Tpex-stabilizing function relies on promoting EOMES and c-MYB expression, which antagonize terminal differentiation and potentially promote survival by supporting BCL-2 expression64. In addition, TCF-1 induces BCL-6 expression in virus-specific CD8+ T cells and this counteracts terminal differentiation by antagonizing type 1 interferon signaling16, while absence of TCF-1 impacts T cell maintenance without affecting the expression of exhaustion-related genes. Of note, it is important to mention that TCF-1 also plays a key role in other cells that bear strong proliferative potential such as naive, Tmem and Tmp cells.

ID3, a transcriptional regulator, is expressed in Tpex cells and sustains their survival by decreasing susceptibility to Fas–Fas ligand-mediated cell death65. Within Tpex cells, regulatory regions of the Id3 gene were more accessible than in Tex cells, whereas Id2 regulatory regions were more accessible in Tex cells42,66.

The transcription factors IRF4 and BATF are highly expressed in Tex cells in response to sustained TCR stimulation in a largely NFAT-dependent manner, promoting a transcriptional and metabolic profile of terminal exhaustion. This includes PD-1 expression, reduced glycolysis and oxidative phosphorylation and repression of TCF-1. Deletion of one Irf4 allele promoted memory-like T cell development in chronic LCMV infection67. Overexpression of BATF in murine CAR T cells early after activation results in preferential cooperation with IRF4 to promote effector cell differentiation at the expense of entering the exhaustion lineage68. Similar data were obtained by overexpressing the AP-1 family member c-Jun, a transcriptional partner of BATF and IRF4, in human CAR T cells69.

The transcription factor TOX (thymocyte selection-associated high-mobility group box protein) is induced by strong TCR stimulation in conjunction with calcineurin and NFATC2 signaling45,46,47. Multiple studies identified TOX as key factor for the establishment and maintenance of exhausted T cells in chronic viral infection or cancer, but not for the formation of Teff cells and Tmem cells in acute infection46,47,49. Mechanistically, TOX supports imprinting of an exhausted phenotype in Tpex cells, which is then passed on to their progeny. Absence of TOX results in the retention of an acute phenotype in Tpex cells, in a failure to maintain the Tpex cell population, and over time in a loss of the Tex cell population in chronic infection and in tumors46,48,70. This loss may involve direct regulation of TCF-1 (ref. 70), but there is no tight dependence as TCF-1 is expressed normally in TOX-deficient cells in acute infections46. Alternatively, reduced inhibitory receptor levels, such as PD-1, in the absence of TOX may lead to an overstimulation and terminal differentiation of Tpex into Tex cells. TOX expression seems to be particularly important for the instruction or epigenetic fixation of an ‘exhaustion program’, as delayed TOX inactivation (20 days after infection) did not show phenotypical or functional consequences46.

EGR2 (early growth response 2) is an anergy-associated transcription factor selectively expressed in CD8+ Tpex cells in chronic viral infections and tumors. Akin to TOX, EGR2 stabilizes the exhausted transcriptional state on the transcriptional and epigenetic levels71. In addition, EGR2 maintains an exhausted signature in the TCF-1 descendants of TCF-1+ precursors via epigenetic repression of AP-1 family transcription factors71.

EOMES and T-bet are key transcription factors involved in effector, memory and exhausted CD8+ T cell differentiation11,72,73. In Tex cells, an increased ratio of nuclear EOMES to T-bet is found compared to Tmem cells in mice and humans74,75, leading to weaker repression of Pdcd1 as opposed to memory cells with a higher ratio of T-bet to EOMES. Enforced nuclear localization of T-bet in Tex cells leads to effector-like differentiation, contrasting with the high EOMES/T-bet ratios that are indicative of terminally exhausted CD8+ T cells and which are responsible for shielding memory-like CD8+ T cells from differentiating into terminal Teff cells74.

BACH2 and BATF are transcription factors involved in opposing functions in the generation of early Tpex cells. BACH2 was shown to be upregulated in Tpex cells42,70. BACH2-deficient CD8+ T cells were impaired in the generation of early Tpex cells, whereas overexpression of BACH2 had the opposite effect70. Mechanistically, overexpression of BACH2 inhibited the function of IRF4 and AP-1 family members (such as BATF) that support proliferation and effector cell differentiation42 and instilled the differentiation of memory-like CD8+ T cells by repressing the expression of Prmd1, which encodes the transcription factor BLIMP-1 (ref. 70). During later phases of chronic LCMV infection, BATF and to some extent T-bet activity induced the differentiation of Tpex cells toward a CX3CR1+ effector-like population76, a transitory state that eventually feeds into Tex cells36.

BLIMP-1 is a zinc-finger-containing transcriptional repressor whose role in promoting terminal differentiation was initially described for B cells. In the chronic LCMV infection, Blimp-1 promoted the terminal differentiation of virus-specific CD8+ T cells and positively regulated expression of many co-inhibitory receptors. Conditional deletion of Prdm1 in CD8+ T cells promoted the formation of Tpex cells77.

Nuclear receptor subfamily 4 group A (NR4A) factors also have roles in CD8+ T cell fate decisions. In the setting of cancer-reactive CD8+ T cells, NR4A-deficient tumor-infiltrating CD8+ T cells exhibited lower TIM-3 expression as compared to their wild-type counterparts and showed increased functionality with respect to TNF and IFNγ expression after restimulation. NR4A-deficient tumor-infiltrating CD8+ T cells exhibited a gene expression profile characteristic of Teff cells, evidenced by low TCF-1 expression, suggesting that NR4A transcription factors serve to inhibit terminal differentiation and hence to maintain the Tpex cell population78.

Taken together, a number of regulatory pathways can promote or impair the generation and maintenance of the Tpex cell population, including cell surface receptors (PD-1) and transcription factors or regulators (TOX, TCF-1, ID3, EGR2, BACH2, BATF, IRF4, BATF, BLIMP-1, NR4As and ratios of EOMES to T-bet). Given the importance of Tpex cells to sustain CD8+ T cell responses in chronic infection and cancer, the discovery of additional regulatory pathways is warranted.

Precursors in exhausted human T cells

Chronic HCV infection in humans differs in many aspects from chronic LCMV infection in mice, including that HCV infections are organ specific rather than systemic. Nevertheless, both infections display key similarities that have, in a bidirectional fashion, nurtured our mechanistic understanding of T cell exhaustion14,37,46,55,79,80. For example, T cell responses in HCV also branch into TCF-1+CD127+Bcl-2+ antigen-specific CD8+ T cells alongside EOMEShiCD127CD38+ terminally differentiated cells14,55. As in chronic LCMV infection, the TCF-1+CD127+ population expands more than TCF-1CD127 cells upon antigen stimulation, suggesting a similar potential of TCF-1+ cells to maintain HCV-specific T cell populations. Comprehensive transcriptome analysis indicates that the TCF-1CD127 subset closely resembles LCMV-specific Tex cells, with conventional memory signatures being absent. Vice versa, TCF-1+CD127+ cells express several markers typically detected in Tpex cells in chronic LCMV infection37. At the same time, the expression of exhaustion-specific markers can be detected in TCF-1+CD127+ and TCF-1CD127cells as seen among LCMV-specific Tpex cells and Tex cells13,42,46,79,80,81,82. In addition, virus-specific CD8+ T cells with a phenotype similar to Tpex cells have also been identified in human chronic HBV83 and HBV/HDV infection84,85, underscoring similar precursor-mediated T cell maintenance mechanisms across different chronic human viral infections. Detailed unbiased single-cell RNA-based profiling even revealed a CD127int cell cluster that is situated between Tpex and Tex cells82, which further resembles the abovementioned diversity of exhausted T cells in chronically infected mice33. These developmental relationships are also underscored by TCR clones shared between these subclusters82. We therefore recommend to refer to HCV-specific and HBV-specific T cells in chronic infections that express hallmark features of T cell exhaustion also as Tpex cells and Tex cells, respectively.

HCV infection provided also unique insights regarding what happens with exhausted T cell populations upon infection resolution via direct-acting antivirals (DAAs). This treatment results in a loss of the TCF-1CD127PD-1+ terminally differentiated Tex cell population, as it was seen in mice following transfer of virus-specific CD8+ T cells from chronic infection into antigen-free hosts23,37. Nonetheless, the selective maintenance of quiescent cells with Tmem cell features such as CD127 and TCF-1 expression, secondary expansion capacity but also signs of T cell exhaustion is a typical outcome of DAA-resolved HCV infection55,79,80,81,82. As mentioned above, we suggest using the term Tmx for these Tpex-derived Tmem cells.

The retention of an exhausted signature among TCF-1+ cells in DAA-treated individuals mirrors the phenotypic stability and epigenetic enforcement of exhausted T cells seen in other instances2,37,38,39,40,79,80,81,82,86,87, including initial observations in mice37 and among tumor-specific T cells38,40,86. A series of recent publications refers to this epigenetic commitment in Tpex cells as the acquisition of molecular ‘scars’79,80,81,82. Interestingly, this ‘scaring’ or commitment is not observed among bona fide HCV-specific memory CD8+ T cells formed in HCV infections that were spontaneously and rapidly resolved by the immune system46, but it is evident among TCF-1+ cells specific for epitopes that were lost due to antigen escape. Noteworthy, viral escape occurs rather early during the first 6 months of HCV infection88, indicating that the epigenetic imprinting takes place in this time period. Of note, while variant-specific T cells still acquire features of T cell exhaustion, their molecular commitment is less prominent than that of Tpex cells specific for conserved epitopes79,80,82. This observation implies a progressive acquisition of features of T cell exhaustion as opposed to the relatively rapid fixation of T cell exhaustion in LCMV infection, whereby the systemic nature of the LCMV infection may be a factor speeding up this commitment. However, in line with recent reports of early onset of the commitment toward exhaustion in Tpex cells, it could also mean that the early infection period, during which very high antigen levels are present, has the highest potential to initiate the acquisition of a strong exhausted phenotype42,46. Of note, a related study of antiretroviral treatment in HIV infection suggests some differences, that is, while the treatment went along with increases in the TCF-1-expressing population, there was a prominent loss of PD-1 expression89. This finding implies that the induction of exhaustion and its epigenetic fixation depends on factors that are yet to be identified and not only on prolongated antigen exposure. Here, it needs to be considered that antiretroviral treatment was applied to the specific HIV study cohort >2 years after having an uncontrolled HIV infection. However, as this fixation occurs also in tumors38,40,86, it remains one of the key challenges in the field of immunology—how the underlying processes and mechanisms can be overcome.

Precursors in latent reactivating viral infections

The majority of the world population is infected with CMV90,91. It differs from other chronic infection as it perseveres mainly in form of vial latency with very limited expression of viral gene products during latency (reviewed in refs. 92,93,94). However, sporadic reactivation from latency can reinitiate lytic replication, which is in most cases well controlled by established potent CMV-specific immunity.

Human CMV (HCMV) and murine CMV (MCMV) infections trigger two divergent types of CD8+ T cell responses that differ in their kinetics, phenotypical composition and maintenance requirements. While CD8+ T cells specific for some CMV antigens follow the ‘classical’ expansion, contraction and establishment of stable memory kinetics, CD8+ T cells specific for certain CMV-derived epitopes follow an atypical kinetics with continued expansion and accumulation of functional, effector-like CMV-specific CD8+ T cells (termed ‘memory inflation’) in the circulation, secondary lymphoid organs and peripheral tissues8,95,96,97. Because of the latter, the CMV-specific CD8+ T cell population is dominated by inflationary CD8+ T cells during latency, which exhibit an effector memory-like phenotype (Tem-like CD62LCD127KLRG1+CD27lo). Inflationary CD8+ T cells exert potent effector functions and have, from a bioengineering standpoint, gained considerable interest for T cell-based vaccines against viral infections and tumors98,99,100,101,102,103,104,105. The inflationary CD8+ T cell response depends on sporadic reactivation events, on antigen processing via the constitutive proteasome106,107 and on antigen presentation by latently infected non-hematopoietic cells108,109.

The large population of inflationary MCMV-specific CD8+ T cells is maintained at a remarkably stable level for the lifetime of the infected mouse. This pertains not only to the numbers of inflationary CD8+ T cells, but also to their overall antigen avidity110, at least over an observation period of 5 months. Considerably longer exposure periods tend to go along with decreased avidity29. A dynamic process is responsible for the apparent stable maintenance of the inflationary CD8+ T cell response, as the half-life of terminally differentiated inflationary T cells is 6–8 weeks in the circulation and 10–12 weeks in the lung111,112, implying that at least every 3 months 50% of the inflationary pool becomes replenished113. This replenishment depends on a small, lymph node (LN)-residing subset of the inflationary cells that exhibits a Tcm cell phenotype (CD62L+IL-7Rα+ and high proliferative potential)108,114. It is also conceivable that CD62LCX3CR1int cells contribute to the process of inflation115,116. Recently, the Tcm subpopulation of inflationary CD8+ T cells was shown to express TCF-1 (ref. 9) and depletion of TCF-1+ cells during established infection severely curtailed the pool of inflationary cells9. Moreover, there is large clonal overlap between inflationary TCF-1+ and TCF-1 cells of the same specificity, with highly abundant TCF-1+ clones also being highly abundant within the effector-like population9. Both document a critical role of TCF-1+ precursors for feeding the more differentiated Tem-like pool in inflationary, MCMV-targeting T cell responses. Whether in humans a proportion of HCMV-specific inflationary CD8+ T cells expresses TCF-1 remains to be formally shown117. A comparative analysis of the TCR repertoire in humans revealed only a partial overlap of HCMV-specific CD8+ Tcm (IL-7Rα+) and Tem (IL-7Rα) cells. However, as comparative clonal analyses in humans and in particular in tissues such as the spleen and lymph nodes (LNs) are difficult to perform, it is conceivable that under-sampling might explain this partial clonal overlap117. Nonetheless, HCMV-specific CD8+ T cells isolated from LNs have a higher proliferative capacity compared to cells isolated from the blood118, suggesting that also in humans secondary lymphoid organs host HCMV-specific memory cells with superior proliferation potential.

Role of tumor-specific precursor T cells

The identification of Tpex cells and their superior response to blocking the interaction of PD-1 and its ligand, PD-L1, in murine chronic infections had obvious implications for cancer treatment, and prompted a search for similar cells in human cancer. Initially, a hierarchy of differentiation among PD-1+CD8+ T cells in non-small cell lung cancer (NSCLC) was identified, where the CXCR5+TCF-1hiTIM-3 subpopulation was more functional and gave rise to differentiated CXCR5TCF-1loTIM-3+ Tex cells after ex vivo TCR stimulation119. Subsequent studies demonstrated that antigen-specific TCF-1hi Tpex cells are superior to TCF-1lo Tex cells in mediating regression of tumors upon PD-1 blockade in mice120,121. This difference appears to depend on genes typically expressed by murine Tmem cells or human Tscm cells122,123,124,125.

Nonetheless, it is still debated which T cell subset is preferentially targeted by ICB in humans. Increased baseline abundance of TCF-1+ CD8+ T cells in melanoma lesions predicted response to ICB targeting PD-1, CTLA-4 or both126. Accordingly, intratumoral CD8+ Tpex cells, but not terminally differentiated Tex cells, were recently shown, along with peripheral T cells, to be the major source of effectors infiltrating NSCLC after anti-PD-1 and chemotherapy127. In other studies, however, pretreatment abundance of intratumoral T cells expressing high levels of the Tex markers PD-1, TIM-3 or CXCL13 predicted long-term response to ICB in NSCLC128,129,130. These contrasting results could be explained by the different approaches used to define T cell subsets. Alternatively, the abundance of Tex cells, making up the majority of tumor-specific cells131,132,133,134,135, could simply reflect an ongoing immune response that relies on precursors originating from other sites (for more details, see ref. 136). Initial evidence challenging the concept that anti-PD-1 immunotherapy mainly targets tumor-resident T cells came from murine models, where regression of MC38 colon carcinomas was abrogated by blockade of sphingosine 1-phosphate receptor 1 (S1P1)-mediated T cell egress from secondary lymphoid tissues by the FTY720 inhibitor137. The ‘immunological’ response to anti-PD-1 ICB, that is, the appearance of Ki-67+ proliferating CD8+ T cells, is visible in the circulation of patients with melanoma as soon as 7 days following administration138. These proliferating cells are also clonally related to tumor-infiltrating CD8+ T cells139, possibly suggesting recruitment from secondary lymphoid organs. This hypothesis has been corroborated by recent data showing that T cell clones found in basal and squamous cell carcinoma lesions following anti-PD-1 ICB were, for the vast majority, not present before treatment, thus indicating clonal replacement140. These results were, in part, confirmed also in NSCLC127. In mice, tumor-draining LNs are a preferential reservoir and site of Tpex cell stimulation141,142. Moreover, Tpex cells are preferentially present in LNs compared to lung tumors or the adjacent peritumoral space56. Accordingly, administration of ICB via intratumoral, intradermal or intrapleural injection causing antibody accumulation in tumor-draining LNs, expanded the pool of intratumoral Tpex cells, identified in this case by Slamf6 expression143, and was associated with enhanced tumor regression compared to routes (that is, intravenous or intraperitoneal) resulting in systemic distribution in preclinical tumor models143,144.

It should be noted that the majority of the human studies mentioned so far analyzed polyclonal T cell subsets with limited information about their specificity. More recently, a comprehensive analysis of transcriptomes, TCR diversity and antitumor reactivity of several melanoma and NSCLC-specific CD8+ T cells, including those responding to neo-antigens, revealed that these cells display a phenotypic and gene expression profile distinct from bystander CD8+ T cells, in particular pertaining to expression of exhaustion-related markers and transcription factors134,135. Tumor-reactive clones were heterogeneous at the single-cell transcriptomic level, featuring mainly Tex cells but also Tpex cells expressing TCF-1 and proliferating/activated cells134. Among tumor-reactive CD8+ T cells, expression of IL7RA, TCF7 and GZMK, previously related to Tpex cells56,127,145, were associated with major tumor regression in three of six patients with NSCLC treated with neoadjuvant anti-PD-1 ICB135.

A major question in the field of cancer immunology and chronic infection is why cancer progresses and infections continue despite long-lived and potent Tpex cell responses being present in the body and at the site of the lesion. This paradox could be explained, at least in part, by the epigenetic stability of the exhausted T cell population38,39,40,86, including Tpex cells17,56. In fact, our understanding of epigenetic mechanisms that enforce an exhausted phenotype also urge conception of new approaches to improve functionality of T cells during immunotherapies. Using drugs capable of specifically modifying the exhaustion-enforcing epigenetic landscape would be warranted, but this approach is challenged by poor specificity so far. As it is established early after activation, preventing, rather than reverting exhaustion by pharmacological intervention at a yet uncommitted stage might be a promising strategy to improve functionality of adoptively transferred TCR- or CAR-engineered T cells. In this regard, culturing T cells with antioxidants124, glycogen synthase-3β (GSK-3β)146, AKT147 and MEK inhibitors148, among other molecules, preserved stemness and prevented terminal differentiation and expression of inhibitory receptors via downregulation of the mTOR signaling pathway and glycolytic metabolism. Similarly, overexpression of c-Jun, which is thought to compete with the formation of AP-1–IRF complexes involved in terminal differentiation and exhaustion, resulted in transcriptional rewiring in cultured CAR T cells, and enhanced stemness and functionality in xenogeneic tumor models69. A different strategy based on transient interruption of CAR-mediated signaling or inhibition of CAR proximal kinases during T cell expansion in vitro was also able to revert the chromatin accessibility landscape associated with exhaustion149. Collectively, these data support the notion that acquisition of terminal differentiation and exhaustion traits are major barriers to the successful outcome of ICB and adoptive T cell therapies.

Common signature genes of proliferation-competent cells

As outlined above, studies in LCMV infection have pioneered the identification of proliferation-competent Tmp cells in acute infections and Tpex cells in chronic infections. Subsequently, related populations were identified in different infections and tumors. While Tmp and Tpex cells differ in many critical aspects, they share the expression of a large number of genes. We extracted Tpex cell-defining genes from a representative single-cell sequencing dataset generated by Yao et al.49 (‘Data availability and analysis’) and have identified 117 genes that are upregulated in TCF-1+ compared to TCF-1 cells and 290 that are downregulated in TCF-1+ compared to TCF-1 cells. We illustrate the top 50 differentially expressed genes in Fig. 3a. These include those genes encoding adhesion molecules, negative regulators of apoptosis and promoters of cell expansion and survival and cell cycle regulators. In addition, we noticed differential expression of several genes, whose function still needs to be characterized in chronic infections and tumors. In line with previous reports, the TCF-1+ population shows higher expression levels of CCR7, while CCL5, CXCR6 and LGALS3 show higher expression levels in TCF-1 cells. Finally, TCF-1 cells show higher expression of GZMA, GZMB and GZMK than TCF-1+ cells. We propose that many of these genes represent a core signature of TCF-1+ cells across many conditions, as they were also enriched in TCF-1+ cells in CMV infection and in the tumor settings (Fig. 3b). This analysis strongly underlines that a common and robust molecular wiring orchestrates the differentiation between precursors (Tpex) and terminally differentiated (Tex) cells across very different conditions.

Fig. 3: Gene expression signatures of TCF-1-positive, antigen-specific T cells.
figure 3

a, Total P14 T cells were obtained 4.5 and 7 days after infection with LCMV Armstrong or clone 13 by Yao et al.70 and analyzed by single-cell RNA sequencing. We reanalyzed these data and after clustering we identified the cluster expressing TCF-1 (Tcf7). We compared expression levels of genes in this cluster to the expression levels of the same genes in all other clusters. Depicted are the top genes that were either significantly upregulated or downregulated across both time points and the LCMV Armstrong versus clone-13 infection. We then compared the expression of these genes to the expression of the same genes in TCF-1+ and TCF-1 clusters in a similar LCMV dataset published by Chen et al.64, a dataset based on CMV-specific cells published by Highton et al.150, and two datasets of tumor-specific T cells published by Carmona et al.151 and Pauken et al.152. Shown are scaled expression levels (color intensity) and the percentage of expressing cells (circle size) for the top 50 upregulated (left) and downregulated (right) genes. Genes are grouped according to common biological functions. b, Gene signatures from a were tested for their ability to separate TCF-1+ and TCF-1 T cells in datasets derived from the publications indicated above. TF, transcription factor.

Conclusions, outlook and imminent questions

The central role of Tpex cells in sustaining antiviral and tumor-directed immunity underscores the importance of this population for immunotherapy. A better understanding of their biology is, in our opinion, a prerequisite to advance immunotherapy. Questions on how to augment the often-limited T cell response after immunotherapy are inevitably linked to the biology of the Tpex cell population, which we only partially understand so far. A particular challenge is to find solutions to potentiate the capacity of Tpex cells to give rise to Teff cells without compromising long-term persistence of Tpex cells. Indeed, the currently used checkpoint inhibition strategies are optimized for short-term effects, while the long-term consequences for Tpex cells and possibly negative effects on this population were insufficiently explored so far. The expression of signature genes of T cell exhaustion alongside classical Tmem cell traits also bears tremendous importance for immunotherapeutic interventions. It signals that T cell exhaustion-specific mechanisms and their epigenetic fixation should already be prevented at the level of the Tpex cell population. Presently, we lack any effective strategies to do so without compromising the Tpex cell population. Because of these and other aspects mentioned in this review, we see a major need to focus T cell exhaustion-related research directly at investigating the biology of Tpex cells.

Data availability and analysis

Raw read counts were obtained from NCBI under the accession numbers GSE119940 (ref. 49), GSE131535 (ref. 64), GSE128147 (ref. 150), GSE116390 (ref. 151) and GSE158520 (ref. 152). Only cells with more than 1,000 genes detected and with unique molecular identifier counts less than three standard deviations above the mean were kept for downstream analysis, with the exception of the dataset from Highton et al.150, where cells with more than 500 genes detected were kept. Contaminating cells were filtered out based on the cluster expression of the marker genes Cd14, Ly6d, H2-Aa, Cst3, Fcer1g, Fcgr3 and Lyz2. Raw counts were normalized for each sample separately using the R package sctransform (v0.3.2)153 (with glmGamPoi method). Downstream analysis was performed with the R package Seurat (v4.0.1)154. Principal-component analysis was calculated on the top 1,000 highly variable genes; k-nearest neighbor graph and uniform manifold approximation and projection were computed on the first 20 principal-component analysis dimensions. Clusters with a high percentage of cells expressing Tcf7 were labeled as the TCF-1+ subpopulation. In the dataset from Highton et al.150, cells with normalized expression higher than 4 were labeled as TCF+ cells. In the case of the dataset from Yao et al.70, anchors between replicates were identified on the top 1,000 highly variable genes and integration was performed on the first 20 dimensions. The integrated data were then used for cluster identification. For differential expression analysis instead, the data from all samples were merged and normalized using the R package sctransform (v0.3.2)153 (with glmGamPoi method). The TCF-1+ signatures were generated by contrasting the TCF-1+ versus the TCF-1 subpopulation using the function FindConservedMarkers with the Wilcoxon rank-sum test for differential expression followed by Bonferroni correction for multiple testing. The R package biomaRt (v2.46.3)155 was used to retrieve the Gene Ontology terms associated with each gene from the TCF+ signatures and they were grouped by common terms.

References

  1. McLane, L. M., Abdel-Hakeem, M. S. & Wherry, E. J. CD8+ T cell exhaustion during chronic viral infection and cancer. Annu Rev. Immunol. 37, 457–495 (2019).

  2. Blank, C. U. et al. Defining ‘T cell exhaustion’. Nat. Rev. Immunol. 19, 665–674 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Franco, F., Jaccard, A., Romero, P., Yu, Y. R. & Ho, P. C. Metabolic and epigenetic regulation of T cell exhaustion. Nat. Metab. 2, 1001–1012 (2020).

    CAS  PubMed  Google Scholar 

  4. Hashimoto, M. et al. CD8+ T cell exhaustion in chronic infection and cancer: opportunities for interventions. Annu. Rev. Med. 69, 301–318 (2018).

  5. Thommen, D. S. & Schumacher, T. N. T cell dysfunction in cancer. Cancer Cell 33, 547–562 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Philip, M. & Schietinger, A. CD8+ T cell differentiation and dysfunction in cancer. Nat. Rev. Immunol. https://doi.org/10.1038/s41577-021-00574-3 (2021).

  7. Kallies, A., Zehn, D. & Utzschneider, D. T. Precursor exhausted T cells: key to successful immunotherapy? Nat. Rev. Immunol. 20, 128–136 (2020).

    CAS  PubMed  Google Scholar 

  8. Klenerman, P. & Oxenius, A. T cell responses to cytomegalovirus. Nat. Rev. Immunol. 16, 367–377 (2016).

    CAS  PubMed  Google Scholar 

  9. Welten, S. P. M. et al. TCF-1+ cells are required to maintain the inflationary T cell pool upon MCMV infection. Nat. Commun. 11, 2295 (2020).

  10. Williams, M. A., Holmes, B. J., Sun, J. C. & Bevan, M. J. Developing and maintaining protective CD8+ memory T cells. Immunol. Rev. 211, 146–153 (2006).

  11. Paley, M. A. et al. Progenitor and terminal subsets of CD8+ T cells cooperate to contain chronic viral infection. Science 338, 1220–1225 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. He, R. et al. Follicular CXCR5-expressing CD8+ T cells curtail chronic viral infection. Nature 537, 412–428 (2016).

    CAS  PubMed  Google Scholar 

  13. Im, S. J. et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 537, 417–421 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Utzschneider, D. T. et al. T cell factor 1-expressing memory-like CD8+ T cells sustain the immune response to chronic viral infections. Immunity 45, 415–427 (2016). Together, the studies by He et al., Im et al. and Utzschneider et al. identified a subset of virus-specific CD8+ T cells, characterized by the expression of TCF-1 or CXCR5, that exhibits proliferative capacity and stem-cell traits such as self-renewal and the ability to differentiate into terminally differentiated cells. This population preferentially expanded after blockade of the PD-1 inhibitory pathway.

    CAS  PubMed  Google Scholar 

  15. Leong, Y. A. et al. CXCR5+ follicular cytotoxic T cells control viral infection in B cell follicles. Nat. Immunol. 17, 1187–1196 (2016).

    CAS  PubMed  Google Scholar 

  16. Wu, T. et al. The TCF-1–Bcl6 axis counteracts type I interferon to repress exhaustion and maintain T cell stemness. Sci. Immunol. https://doi.org/10.1126/sciimmunol.aai8593 (2016).

  17. Lugli, E., Galletti, G., Boi, S. K. & Youngblood, B. A. Stem, effector and hybrid states of memory CD8+ T Cells. Trends Immunol. 41, 17–28 (2020).

    CAS  PubMed  Google Scholar 

  18. Omilusik, K. D. & Goldrath, A. W. Remembering to remember: T cell memory maintenance and plasticity. Curr. Opin. Immunol. 58, 89–97 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Martin, M. D. & Badovinac, V. P. Defining memory CD8+ T cell. Front. Immunol. 9, 2692 (2018).

  20. Ahmed, R., Bevan, M. J., Reiner, S. L. & Fearon, D. T. The precursors of memory: models and controversies. Nat. Rev. Immunol. 9, 662–668 (2009).

    CAS  PubMed  Google Scholar 

  21. Zebley, C. C., Gottschalk, S. & Youngblood, B. Rewriting history: epigenetic reprogramming of CD8+ T cell differentiation to enhance immunotherapy. Trends Immunol. 41, 665–675 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Chang, J. T., Wherry, E. J. & Goldrath, A. W. Molecular regulation of effector and memory T cell differentiation. Nat. Immunol. 15, 1104–1115 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Wherry, E. J., Barber, D. L., Kaech, S. M., Blattman, J. N. & Ahmed, R. Antigen-independent memory CD8+ T cells do not develop during chronic viral infection. Proc. Natl Acad. Sci. USA 101, 16004–16009 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Wherry, E. J., Blattman, J. N., Murali-Krishna, K., van der Most, R. & Ahmed, R. Viral persistence alters CD8+ T cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J. Virol. 77, 4911–4927 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Utzschneider, D. T. et al. High antigen levels induce an exhausted phenotype in a chronic infection without impairing T cell expansion and survival. J. Exp. Med. 213, 1819–1834 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Sandu, I., Cerletti, D., Claassen, M. & Oxenius, A. Exhausted CD8+ T cells exhibit low and strongly inhibited TCR signaling during chronic LCMV infection. Nat. Commun. 11, 4454 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Welten, S. P. M., Sandu, I., Baumann, N. S. & Oxenius, A. Memory CD8+ T cell inflation vs tissue-resident memory T cells: same patrollers, same controllers? Immunol. Rev. 283, 161–175 (2018).

    CAS  PubMed  Google Scholar 

  28. Grassmann, S. et al. Early emergence of T central memory precursors programs clonal dominance during chronic viral infection. Nat. Immunol. 21, 1563–1573 (2020).

    CAS  PubMed  Google Scholar 

  29. Schober, K. et al. Reverse TCR repertoire evolution toward dominant low-affinity clones during chronic CMV infection. Nat. Immunol. 21, 434–441 (2020).

    CAS  PubMed  Google Scholar 

  30. Toth, I. et al. Decreased frequency of CD73+CD8+ T cells of HIV-infected patients correlates with immune activation and T cell exhaustion. J. Leukoc. Biol. 94, 551–561 (2013).

    CAS  PubMed  Google Scholar 

  31. Jeannet, G. et al. Essential role of the Wnt pathway effector Tcf-1 for the establishment of functional CD8+ T cell memory. Proc. Natl Acad. Sci. USA 107, 9777–9782 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Zhou, X. et al. Differentiation and persistence of memory CD8+ T cells depend on T cell factor 1. Immunity 33, 229–240 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Beltra, J. C. et al. Developmental relationships of four exhausted CD8+ T cell subsets reveals underlying transcriptional and epigenetic landscape control mechanisms. Immunity 52, 825–841 (2020). In this report, the authors defined, based on transcriptional and epigenetic analyses, a CD8+ T cell differentiation landscape of exhausted CD8+ T cells during chronic viral infection, which involves four differentiation stages; these comprise two TCF-1+ progenitor subsets that gradually lose TCF-1 upon division and convert into a third intermediate population, which eventually differentiates into a fourth and terminally differentiated subset.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Kanev, K. et al. Proliferation-competent TCF-1+ CD8+ T cells in dysfunctional populations are CD4+ T cell help independent. Proc. Natl Acad. Sci. USA 116, 20070–20076 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Zander, R. et al. CD4+ T cell help is required for the formation of a cytolytic CD8+ T cell subset that protects against chronic infection and cancer. Immunity 51, 1028–1042 (2019). In this report, the authors demonstrate that the effector-like CX3CR1-expressing CD8+ T cell subset is required for viral control and that the differentiation of this population depends on CD4+ T cell provision of IL-21.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Hudson, W. H. et al. Proliferating transitory T cells with an effector-like transcriptional signature emerge from PD-1+ stem-like CD8+ T cells during chronic infection. Immunity 51, e1044 (2019).

    Google Scholar 

  37. Utzschneider, D. T. et al. T cells maintain an exhausted phenotype after antigen withdrawal and population reexpansion. Nat. Immunol. 14, 603–610 (2013). This study established that T cells undergo a stable and inheritable form of differentiation in chronic infection and that cells obtained from chronic infection retained their exhausted phenotype following transfer and re-expansion in acute infections.

    CAS  PubMed  Google Scholar 

  38. Pauken, K. E. et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 354, 1160–1165 (2016). This study demonstrated that Tex cells acquire a stable epigenetic signature that is maintained even after PD-L1 blockade, suggesting that limited plasticity of the epigenetic state may limit the long-term reprogramming of exhausted CD8+ T cells.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Ghoneim, H. E. et al. De novo epigenetic programs inhibit PD-1 blockade-mediated T cell rejuvenation. Cell 170, 142–157 (2017). This study showed that blocking de novo DNA methylation sustained effector functions in activated CD8+ T cells during persistent viral infection and in the context of cancer, demonstrating that de novo DNA methylation imprints T cell exhaustion and therefore limits T cell rejuvenation by inhibitory receptor blockade.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Philip, M. et al. Chromatin states define tumour-specific T cell dysfunction and reprogramming. Nature 545, 452–456 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Angelosanto, J. M., Blackburn, S. D., Crawford, A. & Wherry, E. J. Progressive loss of memory T cell potential and commitment to exhaustion during chronic viral infection. J. Virol. 86, 8161–8170 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Utzschneider, D. T. et al. Early precursor T cells establish and propagate T cell exhaustion in chronic infection. Nat. Immunol. 21, 1256–1266 (2020). This study established that TCF-1+ precursors with an exhausted phenotype can be detected 5 days after infection in chronic infections.

    CAS  PubMed  Google Scholar 

  43. Guo, Y. et al. Metabolic reprogramming of terminally exhausted CD8+ T cells by IL-10 enhances antitumor immunity. Nat. Immunol. 22, 746–756 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Gabriel, S. S. et al. Transforming growth factor-beta-regulated mTOR activity preserves cellular metabolism to maintain long-term T cell responses in chronic infection. Immunity 54, 1698–1714 (2021). In this report, the authors demonstrate that Tpex cells sustain mitochondrial fitness, while Tex cells deteriorate metabolically over time, with Tpex cells exhibiting TGFβ-mediated suppression of mTOR kinase signaling but retaining the ability to activate the mTOR pathway upon TCR signaling.

    CAS  PubMed  Google Scholar 

  45. Martinez, G. J. et al. The transcription factor NFAT promotes exhaustion of activated CD8+ T cells. Immunity 42, 265–278 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Alfei, F. et al. TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection. Nature 571, 265–269 (2019).

    CAS  PubMed  Google Scholar 

  47. Khan, O. et al. TOX transcriptionally and epigenetically programs CD8+ T cell exhaustion. Nature 571, 211–218 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Scott, A. C. et al. TOX is a critical regulator of tumour-specific T cell differentiation. Nature 571, 270–274 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Yao, C. et al. Single-cell RNA-seq reveals TOX as a key regulator of CD8+ T cell persistence in chronic infection. Nat. Immunol. 20, 890–901 (2019). All four studies have in parallel identified TOX as a key transcription factor for the generation or maintenance of exhausted T cell populations.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Seo, H. et al. TOX and TOX2 transcription factors cooperate with NR4A transcription factors to impose CD8+ T cell exhaustion. Proc. Natl Acad. Sci. USA 116, 12410–12415 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Scott-Browne, J. P. et al. Dynamic changes in chromatin accessibility occur in CD8+ T cells responding to viral infection. Immunity 45, 1327–1340 (2016). In this report, the authors perform genome-wide comparisons of chromatin accessibility and gene expression in virus-specific CD8+ T cells during chronic infection and report that exhausted CD8+ T cells exhibit distinct and stable changes in chromatin accessibility comprising consensus binding sites for NFAT and NR4A family members.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Beltra, J. C. et al. IL2Rβ-dependent signals drive terminal exhaustion and suppress memory development during chronic viral infection. Proc. Natl Acad. Sci. USA 113, E5444–E5453 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Snell, L. M. et al. CD8+ T cell priming in established chronic viral infection preferentially directs differentiation of memory-like cells for sustained. Immunity 49, 678–694 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Sun, J. C. & Bevan, M. J. Defective CD8+ T cell memory following acute infection without CD4+ T cell help. Science 300, 339–342 (2003).

  55. Wieland, D. et al. TCF-1+ hepatitis C virus-specific CD8+ T cells are maintained after cessation of chronic antigen stimulation. Nat. Commun. 8, 15050 (2017). The study found in humans that a TCF-1+CD127+PD-1+ HCV-specific CD8+ T cell subset has memory-like characteristics, including antigen-independent survival and recall proliferation.

    PubMed  PubMed Central  Google Scholar 

  56. Galletti, G. et al. Two subsets of stem-like CD8+ memory T cell progenitors with distinct fate commitments in humans. Nat. Immunol. 21, 1552–1562 (2020). This study identified that the human memory T cell pool includes a subpopulation that expresses signature genes of T cell exhaustion.

    PubMed  PubMed Central  Google Scholar 

  57. Kusnadi, A. et al. Severely ill COVID-19 patients display impaired exhaustion features in SARS-CoV-2-reactive CD8+ T cells. Sci. Immunol. 6, eabe4782 (2021).

    PubMed  PubMed Central  Google Scholar 

  58. Reina-Campos, M., Scharping, N. E. & Goldrath, A. W. CD8+ T cell metabolism in infection and cancer. Nat. Rev. Immunol. 21, 718–738 (2021).

    PubMed  PubMed Central  Google Scholar 

  59. Barber, D. L. et al. Restoring function in exhausted CD8+ T cells during chronic viral infection. Nature 439, 682–687 (2006).

    CAS  PubMed  Google Scholar 

  60. Oestreich, K. J., Yoon, H., Ahmed, R. & Boss, J. M. NFATc1 regulates PD-1 expression upon T cell activation. J. Immunol. 181, 4832–4839 (2008).

    CAS  PubMed  Google Scholar 

  61. Kao, C. et al. Transcription factor T-bet represses expression of the inhibitory receptor PD-1 and sustains virus-specific CD8+ T cell responses during chronic infection. Nat. Immunol. 12, 663–671 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Frebel, H. et al. Programmed death 1 protects from fatal circulatory failure during systemic virus infection of mice. J. Exp. Med. 209, 2485–2499 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Odorizzi, P. M., Pauken, K. E., Paley, M. A., Sharpe, A. & Wherry, E. J. Genetic absence of PD-1 promotes accumulation of terminally differentiated exhausted CD8+ T cells. J. Exp. Med. 212, 1125–1137 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Chen, Z. et al. TCF-1-centered transcriptional network drives an effector versus exhausted CD8+ T cell-fate decision. Immunity 51, 840–855 (2019). This report showed that PD-1 expression on virus-specific CD8+ T cells early during chronic viral infection supported TCF-1 expression, which stabilized the Tpex cell pool via promoting EOMES and c-MYB expression, being relevant for Bcl-2 expression and survival. These data identified PD-1 as a protector of the early established TCF-1+ memory-like subset during chronic viral infection.

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Menner, A. J. et al. Id3 controls cell death of 2B4+ virus-specific CD8+ T cells in chronic viral infection. J. Immunol. 195, 2103–2114 (2015).

    CAS  PubMed  Google Scholar 

  66. Jadhav, R. R. et al. Epigenetic signature of PD-1+ TCF-1+ CD8+ T cells that act as resource cells during chronic viral infection and respond to PD-1 blockade. Proc. Natl Acad. Sci. USA 116, 14113–14118 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Man, K. et al. Transcription factor IRF4 promotes CD8+ T cell exhaustion and limits the development of memory-like T cells during chronic infection. Immunity 47, 1129–1141 (2017).

    CAS  PubMed  Google Scholar 

  68. Seo, H. et al. BATF and IRF4 cooperate to counter exhaustion in tumor-infiltrating CAR T cells. Nat. Immunol. https://doi.org/10.1038/s41590-021-00964-8 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  69. Lynn, R. C. et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 576, 293–300 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Yao, C. et al. BACH2 enforces the transcriptional and epigenetic programs of stem-like CD8+ T cells. Nat. Immunol. 22, 370–380 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Wagle, M. V. et al. Antigen-driven EGR2 expression is required for exhausted CD8+ T cell stability and maintenance. Nat. Commun. 12, 2782 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Pearce, E. L. et al. Control of effector CD8+ T cell function by the transcription factor Eomesodermin. Science 302, 1041–1043 (2003).

    CAS  PubMed  Google Scholar 

  73. Intlekofer, A. M. et al. Effector and memory CD8+ T cell fate coupled by T-bet and Eomesodermin. Nat. Immunol. 6, 1236–1244 (2005).

    CAS  PubMed  Google Scholar 

  74. McLane, L. M. et al. Role of nuclear localization in the regulation and function of T-bet and Eomes in exhausted CD8+ T cells. Cell Rep. 35, 109120 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Buggert, M. et al. T-bet and Eomes are differentially linked to the exhausted phenotype of CD8+ T cells in HIV infection. PLoS Pathog. 10, e1004251 (2014).

    PubMed  PubMed Central  Google Scholar 

  76. Chen, Y. et al. BATF regulates progenitor to cytolytic effector CD8+ T cell transition during chronic viral infection. Nat. Immunol. https://doi.org/10.1038/s41590-021-00965-7 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  77. Shin, H. et al. A role for the transcriptional repressor Blimp-1 in CD8+ T cell exhaustion during chronic viral infection. Immunity 31, 309–320 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Chen, J. et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature 567, 530–534 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Yates, K. B. et al. Epigenetic scars of CD8+ T cell exhaustion persist after cure of chronic infection in humans. Nat. Immunol. 22, 1020–1029 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Tonnerre, P. et al. Differentiation of exhausted CD8+ T cells after termination of chronic antigen stimulation stops short of achieving functional T cell memory. Nat. Immunol. 22, 1030–1041 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Abdel-Hakeem, M. S. et al. Epigenetic scarring of exhausted T cells hinders memory differentiation upon eliminating chronic antigenic stimulation. Nat. Immunol. 22, 1008–1019 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Hensel, N. et al. Memory-like HCV-specific CD8+ T cells retain a molecular scar after cure of chronic HCV infection. Nat. Immunol. 22, 229–239 (2021). The studies by Tonnerre et al., Abdel-Hakeem et al. and Hensel et al. identified that the molecular signature of T cell exhaustion is maintained in T cells in mice and human HCV-specific CD8+ T cells even after the cessation of chronic antigen stimulation.

    CAS  PubMed  Google Scholar 

  83. Schuch, A. et al. Phenotypic and functional differences of HBV core-specific versus HBV polymerase-specific CD8+ T cells in chronically HBV-infected patients with low viral load. Gut 68, 905–915 (2019).

    CAS  PubMed  Google Scholar 

  84. Kefalakes, H. et al. Hepatitis D virus-specific CD8+ T cells have a memory-like phenotype associated with viral immune escape in patients with chronic hepatitis D virus infection. Gastroenterology 156, 1805–1819 (2019).

    CAS  PubMed  Google Scholar 

  85. Karimzadeh, H. et al. Mutations in hepatitis D virus allow it to escape detection by CD8+ T cells and evolve at the population level. Gastroenterology 156, 1820–1833 (2019).

    CAS  PubMed  Google Scholar 

  86. Sen, D. R. et al. The epigenetic landscape of T cell exhaustion. Science 354, 1165–1169 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. Charmoy, M., Wyss, T., Delorenzi, M. & Held, W. PD-1+ TCF-1+ CD8+ T cells from established chronic infection can form memory while retaining a stableimprint of persistent antigen exposure. Cell Rep. 36, 109672 (2021).

    CAS  PubMed  Google Scholar 

  88. Thimme, R. T cell immunity to hepatitis C virus: lessons for a prophylactic vaccine. J. Hepatol. 74, 220–229 (2021).

    CAS  PubMed  Google Scholar 

  89. Rutishauser, R. L. et al. TCF-1 regulates HIV-specific CD8+ T cell expansion capacity. JCI Insight https://doi.org/10.1172/jci.insight.136648 (2021).

  90. Numazaki, Y., Yano, N., Morizuka, T., Takai, S. & Ishida, N. Primary infection with human cytomegalovirus: virus isolation from healthy infants and pregnant women. Am. J. Epidemiol. 91, 410–417 (1970).

    CAS  PubMed  Google Scholar 

  91. Ho, M. Epidemiology of cytomegalovirus infections. Rev. Infect. Dis. 12, S701–S710 (1990).

    PubMed  Google Scholar 

  92. Reeves, M. & Sinclair, J. Regulation of human cytomegalovirus transcription in latency: beyond the major immediate-early promoter. Viruses 5, 1395–1413 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Poole, E. & Sinclair, J. Sleepless latency of human cytomegalovirus. Med. Microbiol. Immunol. 204, 421–429 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Schwartz, M. & Stern-Ginossar, N. The transcriptome of latent human cytomegalovirus. J. Virol. https://doi.org/10.1128/JVI.00047-19 (2019).

  95. Holtappels, R., Thomas, D., Podlech, J. & Reddehase, M. J. Two antigenic peptides from genes m123 and m164 of murine cytomegalovirus quantitatively dominate CD8+ T cell memory in the H-2d haplotype. J. Virol. 76, 151–164 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  96. Karrer, U. et al. Memory inflation: continuous accumulation of antiviral CD8+ T cells over time. J. Immunol. 170, 2022–2029 (2003).

    CAS  PubMed  Google Scholar 

  97. O’Hara, G. A., Welten, S. P., Klenerman, P. & Arens, R. Memory T cell inflation: understanding cause and effect. Trends Immunol. 33, 84–90 (2012).

    PubMed  Google Scholar 

  98. Beyranvand Nejad, E. et al. Demarcated thresholds of tumor-specific CD8+ T cells elicited by MCMV-based vaccine vectors provide robust correlates of protection. J. Immunother. Cancer 7, 25 (2019).

    PubMed  PubMed Central  Google Scholar 

  99. Borkner, L. et al. Immune protection by a cytomegalovirus vaccine vector expressing a single low-avidity epitope. J. Immunol. 199, 1737–1747 (2017).

    CAS  PubMed  Google Scholar 

  100. Hansen, S. G. et al. Profound early control of highly pathogenic SIV by an effector memory T cell vaccine. Nature 473, 523–527 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  101. Hansen, S. G. et al. Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge. Nat. Med. 15, 293–299 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. Karrer, U. et al. Expansion of protective CD8+ T cell responses driven by recombinant cytomegaloviruses. J. Virol. 78, 2255–2264 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  103. Klyushnenkova, E. N. et al. A cytomegalovirus-based vaccine expressing a single tumor-specific CD8+ T cell epitope delays tumor growth in a murine model of prostate cancer. J. Immunother. 35, 390–399 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  104. Qiu, Z. et al. Cytomegalovirus-based vaccine expressing a modified tumor antigen induces potent tumor-specific CD8+ T cell response and protects mice from melanoma. Cancer Immunol. Res. 3, 536–546 (2015).

    CAS  PubMed  Google Scholar 

  105. Snyder, C. M., Cho, K. S., Bonnett, E. L., Allan, J. E. & Hill, A. B. Sustained CD8+ T cell memory inflation after infection with a single-cycle cytomegalovirus. PLoS Pathog. 7, e1002295 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. Dekhtiarenko, I. et al. Peptide processing is critical for T cell memory inflation and may be optimized to improve immune protection by CMV-based vaccine vectors. PLoS Pathog. 12, e1006072 (2016).

    PubMed  PubMed Central  Google Scholar 

  107. Hutchinson, S. et al. A dominant role for the immunoproteasome in CD8+ T cell responses to murine cytomegalovirus. PLoS ONE 6, e14646 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  108. Torti, N., Walton, S. M., Brocker, T., Rulicke, T. & Oxenius, A. Non-hematopoietic cells in lymph nodes drive memory CD8+ T cell inflation during murine cytomegalovirus infection. PLoS Pathog. 7, e1002313 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  109. Seckert, C. K. et al. Antigen-presenting cells of haematopoietic origin prime cytomegalovirus-specific CD8+ T cells but are not sufficient for driving memory inflation during viral latency. J. Gen. Virol. 92, 1994–2005 (2011).

    CAS  PubMed  Google Scholar 

  110. Baumann, N. S. et al. Early primed KLRG1 CMV-specific T cells determine the size of the inflationary T cell pool. PLoS Pathog. 15, e1007785 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  111. Baumann, N. S. et al. Tissue maintenance of CMV-specific inflationary memory T cells by IL-15. PLoS Pathog. 14, e1006993 (2018).

    PubMed  PubMed Central  Google Scholar 

  112. Snyder, C. M. et al. Memory inflation during chronic viral infection is maintained by continuous production of short-lived, functional T cells. Immunity 29, 650–659 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. Welten, S. P. M., Baumann, N. S. & Oxenius, A. Fuel and brake of memory T cell inflation. Med. Microbiol. Immunol. https://doi.org/10.1007/s00430-019-00587-9 (2019).

    Article  PubMed  Google Scholar 

  114. Quinn, M. et al. Memory T cells specific for murine cytomegalovirus re-emerge after multiple challenges and recapitulate immunity in various adoptive transfer scenarios. J. Immunol. 194, 1726–1736 (2015).

    CAS  PubMed  Google Scholar 

  115. Bottcher, J. P. et al. Functional classification of memory CD8+ T cells by CX3CR1 expression. Nat. Commun. 6, 8306 (2015).

    PubMed  Google Scholar 

  116. Klenerman, P. The (gradual) rise of memory inflation. Immunol. Rev. 283, 99–112 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  117. Remmerswaal, E. B. et al. Clonal evolution of CD8+ T cell responses against latent viruses: relationship among phenotype, localization and function. J. Virol. 89, 568–580 (2015).

    PubMed  Google Scholar 

  118. Miron, M. et al. Human lymph nodes maintain TCF-1hi memory T cells with high functional potential and clonal diversity throughout Life. J. Immunol. 201, 2132–2140 (2018).

    CAS  PubMed  Google Scholar 

  119. Brummelman, J. et al. High-dimensional single cell analysis identifies stem-like cytotoxic CD8+ T cells infiltrating human tumors. J. Exp. Med. 215, 2520–2535 (2018). This study demonstrates that TCF-1+ stem-like T cells are also found among tumor-infiltrating T cells in humans.

    CAS  PubMed  PubMed Central  Google Scholar 

  120. Miller, B. C. et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol. 20, 326–336 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  121. Siddiqui, I. et al. Intratumoral TCF-1+PD-1+CD8+ T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. Immunity 50, 195–211 (2019).

    CAS  PubMed  Google Scholar 

  122. Gattinoni, L. et al. Wnt signaling arrests effector T cell differentiation and generates CD8+ memory stem cells. Nat. Med. 15, 808–813 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  123. Gattinoni, L. et al. A human memory T cell subset with stem cell-like properties. Nat. Med. 17, 1290–1297 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. Pilipow, K. et al. Antioxidant metabolism regulates CD8+ T memory stem cell formation and antitumor immunity. JCI Insight https://doi.org/10.1172/jci.insight.122299 (2018).

  125. Fraietta, J. A. et al. Determinants of response and resistance to CD19 chimeric antigen receptor T cell therapy of chronic lymphocytic leukemia. Nat. Med. 24, 563–571 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  126. Sade-Feldman, M. et al. Defining T cell states associated with response to checkpoint immunotherapy in melanoma. Cell 175, 998–1013 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  127. Liu, B. et al. Temporal single-cell tracing reveals clonal revival and expansion of precursor exhausted T cells during anti-PD-1 therapy in lung cancer. Nat. Cancer 3, 108–121 (2022).

    CAS  PubMed  Google Scholar 

  128. Thommen, D. S. et al. A transcriptionally and functionally distinct PD-1+ CD8+ T cell pool with predictive potential in non-small-cell lung cancer treated with PD-1 blockade. Nat. Med. 24, 994–1004 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  129. Clarke, J. et al. Single-cell transcriptomic analysis of tissue-resident memory T cells in human lung cancer. J. Exp. Med. 216, 2128–2149 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  130. Zhang, Y. et al. Single-cell analyses reveal key immune cell subsets associated with response to PD-L1 blockade in triple-negative breast cancer. Cancer Cell 39, 1578–1593 (2021).

    CAS  PubMed  Google Scholar 

  131. Duhen, T. et al. Coexpression of CD39 and CD103 identifies tumor-reactive CD8+ T cells in human solid tumors. Nat. Commun. 9, 2724 (2018).

    PubMed  PubMed Central  Google Scholar 

  132. Li, H. et al. Dysfunctional CD8+ T cells form a proliferative, dynamically regulated compartment within human melanoma. Cell 176, 775–789 (2019).

    CAS  PubMed  Google Scholar 

  133. Simoni, Y. et al. Bystander CD8+ T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature 557, 575–579 (2018).

    CAS  PubMed  Google Scholar 

  134. Oliveira, G. et al. Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma. Nature https://doi.org/10.1038/s41586-021-03704-y (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  135. Caushi, J. X. et al. Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers. Nature https://doi.org/10.1038/s41586-021-03752-4 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  136. Yost, K. E., Chang, H. Y. & Satpathy, A. T. Recruiting T cells in cancer immunotherapy. Science 372, 130–131 (2021).

    CAS  PubMed  Google Scholar 

  137. Spitzer, M. H. et al. Systemic immunity is required for effective cancer immunotherapy. Cell 168, 487–502 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  138. Huang, A. C. et al. A single dose of neoadjuvant PD-1 blockade predicts clinical outcomes in resectable melanoma. Nat. Med. 25, 454–461 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  139. Huang, A. C. et al. T cell invigoration to tumour burden ratio associated with anti-PD-1 response. Nature 545, 60–65 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  140. Yost, K. E. et al. Clonal replacement of tumor-specific T cells following PD-1 blockade. Nat. Med. 25, 1251–1259 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  141. Connolly, K. A. et al. A reservoir of stem-like CD8+ T cells in the tumor-draining lymph node preserves the ongoing antitumor immune response. Sci. Immunol. 6, eabg7836 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  142. Schenkel, J. M. et al. Conventional type I dendric cells maintain a reservoir of proliferative tumor-antigen specific TCF-1+ CD8+ T cells in tumor-draining lymph nodes. Immunity https://doi.org/10.1016/j.immuni.2021.08.026 (2021).

    Article  PubMed  Google Scholar 

  143. Dammeijer, F. et al. The PD-1/PD-L1-checkpoint restrains T cell Immunity in tumor-draining lymph nodes. Cancer Cell 38, 685–700 (2020).

    CAS  PubMed  Google Scholar 

  144. Francis, D. M. et al. Blockade of immune checkpoints in lymph nodes through locoregional delivery augments cancer immunotherapy. Sci. Transl. Med. https://doi.org/10.1126/scitranslmed.aay3575 (2020).

  145. van der Leun, A. M., Thommen, D. S. & Schumacher, T. N. CD8+ T cell states in human cancer: insights from single-cell analysis. Nat. Rev. Cancer 20, 218–232 (2020).

    PubMed  PubMed Central  Google Scholar 

  146. Sabatino, M. et al. Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B cell malignancies. Blood 128, 519–528 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  147. van der Waart, A. B. et al. Inhibition of Akt signaling promotes the generation of superior tumor-reactive T cells for adoptive immunotherapy. Blood 124, 3490–3500 (2014).

    PubMed  PubMed Central  Google Scholar 

  148. Verma, V. et al. MEK inhibition reprograms CD8+ T lymphocytes into memory stem cells with potent antitumor effects. Nat. Immunol. 22, 53–66 (2021).

    CAS  PubMed  Google Scholar 

  149. Weber, E. W. et al. Transient rest restores functionality in exhausted CAR T cells through epigenetic remodeling. Science https://doi.org/10.1126/science.aba1786 (2021).

  150. Highton, A. J. et al. Single-cell transcriptome analysis of CD8+ T cell memory inflation. Wellcome Open Res. 4, 78 (2019).

    PubMed  PubMed Central  Google Scholar 

  151. Carmona, S. J., Siddiqui, I., Bilous, M., Held, W. & Gfeller, D. Deciphering the transcriptomic landscape of tumor-infiltrating CD8 lymphocytes in B16 melanoma tumors with single-cell RNA-seq. Oncoimmunology 9, 1737369 (2020).

    PubMed  PubMed Central  Google Scholar 

  152. Pauken, K. E. et al. Single-cell analyses identify circulating anti-tumor CD8+ T cells and markers for their enrichment. J. Exp. Med. https://doi.org/10.1084/jem.20200920 (2021).

  153. Hafemeister, C. & Satija, R. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol. 20, 296 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  154. Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184, 3573–3587 2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  155. Durinck, S., Spellman, P. T., Birney, E. & Huber, W. Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. Nat. Protoc. 4, 1184–1191 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

D.Z. is supported by a European Research Council consolidator grant (ToCCaTa) and grants from the German Research Foundation (SFB1054 and SFB1371). A.O. is supported by the Swiss National Science Foundation (grant no. IZHRZ0_180552 and grant no. 310030B_185374). E.L. is a CRI Lloyd J. Old STAR (CRI award 3914) and is supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC IG 20676 and AIRC 5×1000 UniCanVax 22757). R.T. is supported by CRC/TRR 179-Project 01 and CRC 1160-Project A02.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Dietmar Zehn, Robert Thimme, Enrico Lugli or Annette Oxenius.

Ethics declarations

Competing interests

D.Z. has a consulting agreement and research collaboration agreement with Pieris Pharmaceuticals related to manipulation of Tpex cells. E.L. receives research grants from Bristol Myers Squibb and is inventor on a patent describing methods for the generation and isolation of Tscm cells.

Peer review

Peer review information

Nature Immunology thanks Axel Kallies and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: L. A. Dempsey, in collaboration with the Nature Immunology team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zehn, D., Thimme, R., Lugli, E. et al. ‘Stem-like’ precursors are the fount to sustain persistent CD8+ T cell responses. Nat Immunol 23, 836–847 (2022). https://doi.org/10.1038/s41590-022-01219-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41590-022-01219-w

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing