Targeting metabolism to reverse T‐cell exhaustion in chronic viral infections

CD8 T‐cells are an essential component of immune response to viral infections; however, in certain instances, such as chronic infections, they can reach an exhausted state, where T‐cells loose glycolytic activity together with cytotoxic capacity. Recent research has indicated that correcting this dysfunctional metabolic state could be key to reversing exhaustion and allowing for proper clearance of chronic viral infections. Here, we review current knowledge about T‐cell exhaustion during infection and discuss strategies targeting metabolism that hold promise for its reversal.

Summary

CD8 T‐cells are an essential component of the adaptive immune response accountable for the clearance of virus‐infected cells via cytotoxic effector functions. Maintaining a specific metabolic profile is necessary for these T‐cells to sustain their effector functions and clear pathogens. When CD8 T‐cells are activated via T‐cell receptor recognition of viral antigen, they transition from a naïve to an effector state and eventually to a memory phenotype, and their metabolic profiles shift as the cells differentiate to accomidate different metabolic demands. However, in the context of particular chronic viral infections (CVIs), CD8 T‐cells can become metabolically dysfunctional in a state known as T‐cell exhaustion. In this state, CD8 T‐cells exhibit reduced effector functions and are unable to properly control pathogens. Clearing these chronic infections becomes progressively difficult as increasing numbers of the effector T‐cells become exhausted. Hence, reversal of this dysfunctional metabolic phenotype is vital when considering potential treatments of these infections and offers the opportunity for novel strategies for the development of therapies against CVIs. In this review we explore research implicating alteration of the metabolic state as a means to reverse CD8 T‐cell exhaustion in CVIs. These findings indicate that strategies targeting dysfunctional CD8 T‐cell metabolism could prove to be a promising option for successfully treating CVIs.

Abbreviations

AKT

protein kinase B

AMP

adenosine monophosphate

AMPK

AMP‐activated protein kinase

APCs

antigen‐presenting cells

ATP

adenosine triphosphate

CTLA‐4

cytotoxic T‐lymphocyte‐associated protein‐4

CVIs

chronic viral infections

DCs

dendritic cells

FAO

fatty acid oxidation

FoxO1

forkhead box protein O1

GLS1

glutaminase 1

GLUT

glucose transporters

HBV

hepatitis B

HCV

hepatitis C

HIF1α

hypoxia‐inducible factor 1‐alpha

HIV

human immunodeficiency virus

IFNγ

interferon‐gamma

IL

interleukin

KHSV

Kaposi’s sarcoma‐associated herpesvirus

LAG‐3

lymphocyte‐activation gene‐3

LCMV

lymphocytic choriomeningitis virus

MHC‐I

major histocompatibility complex class I

miRNAs

micro‐RNAs

mTOR

mammalian target of rapamycin

NFAT

nuclear factor of activated T‐cells

OXPHOS

oxidative phosphorylation

p38 MAPK

p38 mitogen‐activated protein kinase

PEP

phosphoenolpyruvate

PD‐1

programmed death‐1

PD‐L1/2

programmed death ligand‐1/2

PI3K

phosphoinositol 3‐kinase

SHP2

Src homology region 2‐containing tyrosine phosphatase

SRC

spare respiratory capacity

T‐bet

T‐box transcription factor

TCA

tricarboxylic acid

TCR

T‐cell receptors

TIM‐3

T‐cell immunoglobulin mucin‐3

TNFα

tumour necrosis factor‐alpha

TOX

thymocyte selection‐associated high‐mobility group box protein

Introduction

T‐cell exhaustion is a depressed immune state in cytotoxic CD8 T‐cells that is observed in chronic viral infections (CVIs) caused by pathogens such as human immunodeficiency virus (HIV), hepatitis B and C (HBV and HCV) and, in mice, lymphocytic choriomeningitis virus (LCMV). ¹ , ² , ³ , ⁴ Although the factors that induce exhaustion are still being investigated, it is widely accepted that T‐cell exhaustion is due in part to dysfunction of normal glycolytic CD8 T‐cell metabolism. ⁵ In this exhausted state, CD8 T‐cells become unable to produce the factors necessary to induce apoptosis in pathogenic cells. ⁶ Thus, reversal of T‐cell exhaustion has emerged as a target for therapies against CVIs. Based on recent findings of the role of metabolism in T‐cell exhaustion, we explore metabolism‐related pathways that can be targeted therapeutically to reverse CD8 T‐cell exhaustion during CVIs.

T‐cell metabolism

Like almost all cells, CD8 T‐cells generate energy through glycolysis, oxidative phosphorylation (OXPHOS), and fatty acid oxidation (FAO). These processes metabolize glucose and fatty acids to generate energy by producing intermediates that enter the tricarboxylic acid (TCA) cycle that produces electron carriers that assist in adenosine triphosphate (ATP) production through the electron transport chain. In addition to these pathways, CD8 T‐cells also utilize alternate inputs to the TCA through the metabolism of glutamine and glutamate. Relying on each of these pathways has advantages that suit particular T‐cell subsets, and thus CD8 T‐cell metabolism is finely tuned to enable optimal immunological function. ⁷

When CD8 T‐cells are naïve, or have yet to encounter cognate antigens, survival through efficient energy production is essential, and therefore relies primarily on OXPHOS and FAO. OXPHOS produces the highest amount of ATP per molecule of glucose metabolized, and is achieved through the catabolism of glucose to pyruvate, which enters mitochondria and the TCA cycle. FAO also makes use of mitochondrial functions, breaking down fatty acids into the TCA input acetyl‐CoA. While these processes are highly efficient, they are unable to meet the bioenergetic demands of activated T‐cells. ⁸

During an acute viral infection, the metabolic requirements of CD8 T‐cells change from catabolic to anabolic as naïve T‐cells are activated through specific binding of viral antigens displayed by antigen‐presenting cells (APCs), including dendritic cells (DCs), to their T‐cell receptors (TCR). ⁹ This binding induces pyruvate dehydrogenase kinase activity, inducing a metabolic shift from OXPHOS to glycolysis. This shift is enhanced by ligation of costimulatory receptors, for example, CD28, which leads to phosphorylation of protein kinase B (AKT) through the phosphoinositol 3‐kinase (PI3K) pathway. This promotes activation of the mammalian target of rapamycin (mTOR) pathway, enhancing MYC and hypoxia‐inducible factor 1‐alpha (HIF1α) activity, which induce transcription of glucose transporters and glycolytic enzymes. ¹⁰ , ¹¹ TCR‐based activation also results in significant upregulation of glutaminolysis and the pentose phosphate pathway, which contribute to reactive oxygen species production. This upregulates MYC activity, further enhancing the glycolytic flux during T‐cell activation ¹² (Fig. 1).

Figure 1.

Chronic viral infections (CVIs) induce CD8 T‐cell exhaustion. In acute viral infections, normal levels of antigen (Ag) presented by antigen‐presenting cells (APCs), and of cytokines such as interleukin (IL)‐2 released by helper cells such as CD4 T‐cells, stimulate T‐cell receptors (TCRs) and co‐receptors such as CD28. Stimulation of these receptors enhances signalling to mammalian target of rapamycin (mTOR) via phosphoinositol 3‐kinase (PI3K) and protein kinase B (AKT), which upregulates hypoxia‐inducible factor 1‐alpha (HIF1α) and MYC, leading to an increase in glycolytic enzyme levels and glucose transporters (GLUT) expression. This shifts the cell from an oxidative phosphorylation (OXPHOS)‐ and fatty acid oxidation (FAO)‐ to a glycolytic‐based metabolism, allowing for the anabolic processes that define the activated effector state needed to clear an infection. Such processes include the production of cytotoxic factors such as cytokines, granzyme and perforin, and enhanced proliferation. In CVIs, on the other hand, an over‐abundance of antigen leads to upregulation of inhibitory receptors, such as programmed death‐1 (PD‐1), that block TCR activation, thus reducing the signalling required for the glycolytic metabolic phenotype that in turn is necessary for proper effector functions. This dysfunction is compounded by the upregulation of transcription factors that increase PD‐1 expression, the reduction of helper cell signalling for survival and proliferation (e.g. IL‐2), and enhancement of inhibitory signals. The combination of these events leads cells to metabolic dysfunction, reduced cytotoxic function and an exhausted phenotype.

The shift to glycolysis provides additional energy for activated CD8 T‐cells, also known as effector T‐cells, to clear pathogens by inducing apoptosis in infected or damaged host cells. Effector T‐cells carry this out by producing inflammatory cytokines, such as interferon‐gamma (IFNγ) and tumour necrosis factor‐alpha (TNFα), and cytotoxic factors, like granzyme B and perforin. In addition to TCR activation, the effector response is promoted by helper cells, including macrophages and CD4 T‐cells, ¹³ , ¹⁴ through the production of cytokines, for example, interleukin (IL)‐2, that can enhance CD8 T‐cell proliferation by activating the mTOR pathway. ¹⁵ The increased proliferation in response to acute infection provided by IL‐2 signalling is necessary to produce sufficient numbers of antigen‐specific T‐cells to respond to a given pathogen, but is also very energetically demanding, with cell cycles as brief as 2 hr. ¹⁶ This exponential growth and need to produce massive quantities and varieties of cytotoxic factors requires enhanced use of resources and rapid energy production. ¹⁷ Therefore, as CD8 T‐cells shift from a naïve to an effector state, it is necessary that they also move from an OXPHOS‐ to aerobic glycolytic‐based metabolism. IL‐2 signalling can also alter other aspects of cellular metabolism that impact CD8 effector functions, upregulating autophagy‐related pathways, ¹⁸ which has been noted to be essential for CD8 T‐cell survival and memory formation during viral infection. ¹⁹ Additionally, certain CVIs such as HCV are able to modulate autophagy to help themselves to assist in viral replication. ²⁰ Other cytokines such as IL‐7, IL‐15 and IL‐10 also have the ability to modulate metabolic processes such as glycolysis, altering CD8 T‐cell activation and effector functions. Knockout of IL‐7 has been shown to reduce glycolysis, and IL‐10 and IL‐15 have been shown to reduce activation signalling that upregulates glycolysis. ²¹ , ²² , ²³ Combined, these factors lead to a peak of immune activation and pathogen clearance during an acute viral infection. ²⁴

Following successful clearance of an acute infection by effector CD8 T‐cells, a small fraction differentiate into memory T‐cells that are maintained in the absence of their specific antigens. These are resting cells, like naïve T‐cells, but are capable of responding to restimulation by the same antigen, triggering a secondary adaptive immune response far more rapid than the response of naïve cells. In order to respond in such a manner, memory T‐cells have enhanced mitochondrial spare respiratory capacity (SRC), and have been observed to have mitochondrial masses higher than those of effector CD8 T‐cells. ²⁵ , ²⁶ This enhanced SRC is a signature of increased reliance on OXPHOS. However, many studies have shown that memory T‐cells rely upon both FAO and OXPHOS, with drugs, metformin and rapamycin, known to enhance adenosine monophosphate (AMP)‐activated protein kinase (AMPK) activity and decrease mTOR activity, promoting the development of antigen‐specific memory CD8 T‐cells. ²⁷ , ²⁸ , ²⁹ , ³⁰ Together, the metabolic changes accompanying the shifts from naïve to effector to memory cells during T‐cell differentiation are the hallmark of the metabolic flux associated with the immune response to acute viral infections.

Chronic viral infections

While the adaptive immune response can successfully clear acute viral infections, particular infections including HIV, HBV, HCV and LCMV are capable of evading clearance, leading to chronic infections. ³¹ The long‐term nature of these infections often results in CD8 T‐cells reaching an exhausted state with dysfunctional metabolism, and a concomitant reduction in essential cytotoxic functions. This is largely due to the combination of continuous overstimulation, increased levels of immunosuppressive cytokines and receptors, and reduced numbers of helper cells. While the precise metabolic state of exhausted T‐cells is yet to be pinpointed, the factors known to induce exhaustion impact pathways essential to maintaining the glycolytic metabolic state necessary to T‐cell effector functions. This leads effector T‐cells to increase dependence on FAO and OXPHOS, which are unable to keep up with their energetic demands further dampening effector functions of the CD8 T‐cell response, rendering them unable to clear the CVIs. ³²

Higher viral loads cause CD8 T‐cell overstimulation via TCR signalling, inducing CD8 T‐cell exhaustion and, in certain cases, apoptosis. ³³ , ³⁴ In clinical HBV cases, T‐cell exhaustion has been observed to be sparked by overstimulation from the production of an excess of non‐infectious particles. Despite their lack of pathogenicity, these particles are advantageous to HBV in that they overwhelm TCRs. ³⁵ This constant stimulation induces an increasingly exhausted state characterized by decreasing cytokine production until the T‐cells reach an exhausted state expressing programmed death (PD)‐1, lymphocyte‐activation gene‐3 (LAG‐3), cytotoxic T‐lymphocyte‐associated protein‐4 (CTLA‐4) and T‐cell immunoglobulin mucin‐3 (TIM‐3) and, in severe cases, deleting through apoptosis. ³⁶ Similar findings have been observed in mouse models of chronic LCMV showing that, as antigen load increases, effector T‐cells become increasingly exhausted characterized by the increasing loss of IFNγ, TNFα and IL‐2 production until the T‐cells become fully exhausted and, at certain antigen loads, undergoing apoptosis. ³⁴ These findings show that sustained antigen presence drives T‐cell exhaustion, and have been recapitulated in a study examining T‐cell response in LCMV‐infected wild‐type mice and LCMV‐infected mice with knockout of major histocompatibility complex class I (MHC‐I). Results from this study showed that decreased antigen presentation through ablation of MHC‐I expression directly decreased the phenotypes of exhaustion in CD8 T‐cells compared with the T‐cell phenotype in wild‐type mice, but that direct interaction between antigen‐MHC complexes and cytotoxic T‐cells was necessary for clearance of CVIs. ³⁷ Hence, inhibition of MHC‐I function or expression are not feasible therapeutic strategies. Together, these findings show that viral antigen load is a driver of CD8 T‐cell exhaustion, and that it is unlikely to be overcome via direct targeting of antigen presentation. If antigen presentation could be more finely tuned through alteration of specific pathways, antigen overstimulation could be controlled without fully preventing APC‐mediated CD8 T‐cell activation. A potential avenue for this strategy is p38 mitogen‐activated protein kinase (p38 MAPK) activation by toll‐like receptors, which has been shown to control regulation of MHC expression. ³⁸ , ³⁹ However, such therapies would require further understanding of p38 MAPK pathways, as inhibitors of p38 have also been shown to increase antigen uptake for presentation by MHC‐I in a Japanese encephalitis model. ⁴⁰

While directly targeting antigen presentation could prove to be a difficult strategy for preventing or delaying exhaustion induced by CD8 T‐cell overstimulation, recent studies have identified another potential target: thymocyte selection‐associated high‐mobility group box protein (TOX), which is upregulated in exhausted CD8 T‐cells. Studies using HCV and LCMV showed that TOX is an early marker for, and is important for the induction of, exhaustion via reprogramming CD8 T‐cell epigenetic and transcriptional profiles. ⁴¹ Exhausted T‐cells have a distinct epigenetic profile essential to maintaining their exhausted phenotype. In the exhausted epigenetic state, the availability of chromatin regions related to inhibitory markers such as PD‐1 is enhanced, and there are widespread changes to transcription factor binding. ⁴² TOX plays a role in this process by reducing the transcriptional availability of genes related to CD8 effector differentiation via the binding and recruitment of proteins involved in repressive epigenetic events. ⁴³ Evidence of the impact of TOX expression can be observed in the link between TOX and T‐cell inhibitory marker expression, as a decrease in TOX levels was shown to lead to a correlated decline in PD‐1, CD39 and TIM‐3 expression, and enhanced effector functions. ⁴⁴ Additionally, knockout of TOX in mouse CD8 T‐cells during LCMV infection prevented development of CD8 T‐cell exhaustion, but TOX was not necessary for the formation of effector cells. ⁴⁵ These data indicate that TOX is a very promising therapeutic target for reversing T‐cell exhaustion. It would be interesting to determine whether ablating TOX expression is also able to shift the metabolism of exhausted T‐cells to glycolysis, determine if this in turn restores T‐cell effector functions, and study the link between the epigenetic and metabolic mechanisms that drive T‐cell exhaustion.

A mechanism through which CD8 T‐cells responding to CVIs protect themselves from the high viral antigen loads accompanying such infections is expression of inhibitory receptors including PD‐1. The upregulation of PD‐1 expression has been seen in diverse CVIs, including HIV, HCV and LCMV, and is frequently used as a marker for identifying exhausted CD8 T‐cells. ⁴⁶ PD‐1 expression on HIV‐specific CD8 T‐cells has been associated with decreased cytokine production and proliferative capacity. ⁴⁷ PD‐1 interacts with programmed death ligand (PD‐L)1 and 2 to provide negative regulatory signalling to CD8 T‐cells through the protein tyrosine phosphatase Src homology region 2‐containing tyrosine phosphatase (SHP2), and this signalling inhibits RAS and PI3K signalling from TCR and CD28 stimulation ⁴⁸ , ⁴⁹ (Fig. 1). While this process protects CD8 T‐cells from overstimulation by antigen, it also reduces signalling to transcription factors that are key to a proper immune response. ⁵⁰ Additionally, blocking this pathway reduces mTOR signalling from PI3K, which decreases glycolytic capacity, resulting in metabolic dysfunction, and with insufficient production of energy to meet the anabolic demands of an effector CD8 T‐cell, a state of exhaustion is induced. ⁵¹ The mechanisms controlling PD‐1 signalling are still being explored; however, it has been shown that PD‐1 expression is increased by persistent TCR stimulation, and that transcription factors including T‐box transcription factor (T‐bet) and forkhead box protein O1 (FoxO1) regulate PD‐1 expression. ⁵² T‐bet is responsible for repression of PD‐1 expression, and is downregulated in cases of persistent stimulation. ⁵⁰ This leads to a further increase in PD‐1 expression, as this causes more severe T‐cell dysfunction; therefore, targeting such transcription factors may help to reverse T‐cell exhaustion. ⁵⁰ FoxO1 plays a role opposite to that of T‐bet, upregulating expression of PD‐1 and eomesodermin, the latter of which is a transcription factor that promotes exhaustion during CVIs. ⁵² While PD‐1 does play an important role in preventing overstimulation‐induced apoptosis of exhausted CD8 T‐cells, the outcome of reduced TCR and costimulatory signalling is the decline of key effector functions including proliferation and cytokine production due to metabolic dysfunction related to reduction in glycolysis.

Other inhibitory receptors, including LAG‐3, also stifle the CD8 T‐cell response and are indicators of exhaustion. ⁵³ LAG‐3 expression is associated with decreased virus‐specific CD8 T‐cell responses in LCMV infection, ⁵⁴ and blockade of LAG‐3 has been shown to restore activity in chronic HBV cases. ⁵⁵ Furthermore, co‐treatment with blockade of PD‐1/PD‐L1 interaction and LAG‐3 proved more effective than either treatment alone in an LCMV model. ⁵⁶ Together, these studies suggest that blockade treatment of inhibitory receptors can shift T‐cell metabolism toward a glycolytic state.

In addition to inhibitory receptors, certain cytokines, including IL‐10, can also induce exhaustion in CD8 T‐cells. ⁵⁷ IL‐10 can be produced by diverse cell types, and has the ability to decrease proliferation and cytokine production in CD8 T‐cells. ⁵⁸ In the context of CVIs, IL‐10 has been shown to be an indicator for persistence of HIV and HCV. ⁵⁹ , ⁶⁰ Treatments targeted at IL‐10 also show promise, as removal of IL‐10/IL‐10 receptor (IL‐10R) interaction via antibody blockade, and knockout of IL‐10 signalling in LCMV models, have been shown to enhance clearance of CVIs. ⁶¹ , ⁶² Blockade of the IL‐10/IL‐10R interaction holds promise and a means to reverse exhaustion.

Chronic viral infections are also able to indirectly induce CD8 T‐cell exhaustion and metabolic dysfunction by altering helper cell function (Fig. 1). CD4 T‐cells promote CD8 T‐cell responses to CVIs by secreting cytokines, including IL‐2, that induce high levels of mTOR signalling in CD8 T‐cells. This process is essential for differentiation of CD8 T‐cells from naïve to effector, and for maintaining effector functions. Studies have shown that in chronic infections including LCMV and HCV, CD4 T‐cell capacity to both proliferate and secrete IL‐2 is diminished. ¹⁵ , ⁶³ , ⁶⁴ , ⁶⁵ Other helper cells including DCs have also displayed functional impairments in the context of CVIs. DCs isolated from patients with chronic infections including HIV, HBV and HCV exhibit diminished ability to produce cytokines and induce T‐cell activation. ⁶⁶ , ⁶⁷ Such reductions in helper‐cell functions impair the CD8 T‐cell anti‐viral response and drive exhaustion. Hence, therapies aimed at enhancing the function of these helper cells could potentially improve the anti‐viral function of CD8 T‐cells.

Virus‐induced alteration of the metabolism of infected cells may play a role in CD8 T‐cell exhaustion. The metabolism of both infected host cells and uninfected neighbouring cells has been shown to be altered by particular viruses. ⁶⁸ Kaposi’s sarcoma‐associated herpesvirus (KHSV), for example, alters infected host cell glucose metabolism by upregulating HIF1α, reducing mitochondrial capacity and releasing micro‐RNAs (miRNAs) into the microenvironment. ⁶⁹ , ⁷⁰ , ⁷¹ Particular secreted miRNAs have been shown to reprogramme metabolism in cells, and other miRNAs secreted by KHSV have been implicated in alteration of glycolysis through upregulation of HIF1α. ⁷² , ⁷³ HBV has also been shown to impact infected host cell metabolism, by upregulating expression of glucose transporters (GLUT) through mTOR signalling, thereby enhancing glycolysis. ⁷⁴ , ⁷⁵ It is highly likely that alterations to the infected cell’s metabolism could impact immune function by modifying the microenvironment, reducing the levels of available nutrients, including glucose, or by producing miRNAs that induce changes to CD8 T‐cell function. This lack of available substrate could be a limiting factor even if normal glycolytic metabolism could be restored, and requires further understanding for such therapies to be successful. An additional metabolic aspect that could play a role in T‐cell exhaustion is whole‐body metabolism and nutrition. Studies have identified leptin as playing a key role in inducing CD8 effector differentiation, ⁷⁶ , ⁷⁷ and additional inquiry should be made to examine its role in T‐cell exhaustion. In sum, CVIs lead to exhausted phenotypes and metabolic dysfunction in CD8 T‐cells through diverse mechanisms, and therefore multifaceted approaches will be required to reverse exhaustion such that infections are successfully treated.

Metabolic targets for therapies

Multiple potential therapeutic approaches can be aimed at rescuing CD8 T‐cell exhaustion by reversing metabolic dysfunction through restoration of normal immune function (Fig. 2). Antibody blockade of inhibitory receptors, including PD‐1, LAG‐3 and IL‐10R, have shown promise in reinvigorating T‐cells. These blockades have the ability to help reverse metabolic dysfunction by restoring TCR signalling, which stimulates glycolysis‐enhancing pathways. Blockade of PD‐1 has proved to be an effective immunotherapy in many cancer models of CD8 T‐cell exhaustion, ⁷⁸ , ⁷⁹ and has shown promising results in the treatment of CVIs. Blockade of PD‐1/PD‐L1 interaction in HIV, HBV, HCV and LCMV have all shown enhancement of T‐cell effector capacities. ⁸⁰ , ⁸¹ , ⁸² , ⁸³ , ⁸⁴ Blockade of PD‐1 has also been shown to have enhanced therapeutic efficacy when combined with other antibodies including LAG‐3. This combination displayed an increased ability to rescue CD8 T‐cells from an exhausted state in LCMV. ⁵⁶ While these antibody therapies do not directly target metabolic processes, an important component of their efficacy is that by reducing inhibitory‐pathway activity, they help to repair metabolic dysfunction. PD‐1 inhibits glucose transport and utilization by downregulating mTOR activity, and thus inhibition of PD‐1 signalling increases glucose availability to T‐cells. ⁸⁵ , ⁸⁶ The importance of restoring mTOR activity has also been demonstrated in a study showing that rapamycin, an mTOR inhibitor, can reverse the positive effects of PD‐L1 blockade in an LCMV model of exhaustion. ⁵² Together, these findings show that a key outcome of PD‐1 blockade and reversal of CD8 T‐cell exhaustion is the return of a glycolytic metabolism.

Figure 2.

Potential therapeutic targets to reverse CD8 T‐cell exhaustion. (a) Therapies blocking or downregulating inhibitory receptors via antibody blockade or modulating transcription factor expression, enhancing T‐cell receptor (TCR) pathway signals, or directly targeting metabolism by enhancing substrate availability, could reverse T‐cell exhaustion by enhancing glycolysis in CD8 T‐cells. (b) Potential strategies for reinvigorating exhausted CD8 T‐cells to their effector state with high glycolytic metabolism via currently available methods could hold the key to reversing T‐cell exhaustion and clearing chronic viral infections (CVIs) more efficiently. PHD, prolyl hydroxylases; TCR, T‐cell receptor; VHL, Von Hippel‐Lindau protein.

Another strategy for antibody‐based therapies aimed at reversing CD8 T‐cell exhaustion is to enhance the function of cells that augment CD8 T‐cell effector functions, including CD4 T‐cells and DCs. One way to achieve this is through IL‐10 neutralization, as IL‐10 inhibits CD4 helper and DC functions, and exhibits increased expression during CVI. ²¹ , ⁶² , ⁸⁷ , ⁸⁸ , ⁸⁹ Reduction of IL‐10 signalling has been shown to be effective in enhancing the immune response and clearance of LCMV, ⁶¹ , ⁶² , ⁸⁸ and treatment of HIV with a combined IL‐10 and PD‐1 blockade resulted in a 10‐fold increase in IFNγ production compared with treatment via blockade of PD‐1 alone. ⁹⁰ Some of the impact of IL‐10 blockade is most likely the result of a return of CD28 signalling, which induces mTOR activity and glycolysis and is directly altered by IL‐10; ⁹¹ however, studies have yet to show a direct link between IL‐10 expression and CD8 T‐cell metabolism. A potential alternative approach to reinvigorating exhausted CD8 T‐cells, beyond efforts to directly enhance helper‐cell activity, is to provide a substitute for the output of helper cells. For example, treatment of herpes virus with IL‐2 complexes targeting IL‐2 receptor (IL‐2R) has been shown to substantially improve T‐cell cytotoxicity in the context of suppressed immunity. ⁹² While this would not address the problem of helper‐cell dysfunction, it could help enhance the CD8 T‐cell response to clear the CVIs by enhancing the PI3K pathway.

In addition to directly targeting inhibitory factors that drive exhaustion, more precise approaches for reversing inhibitory pathways could be based on targeting transcription factors that control expression of these inhibitory factors. The functions of T‐bet, FoxO1 and TOX include controlling the expression of inhibitory receptors that induce CD8 T‐cell exhaustion. A possible mechanism to enhance T‐bet expression would be to use miRNA‐155, which has been shown to upregulate T‐bet expression and increase the CD8 T‐cell effector response to LCMV. ⁹³ Additionally, miR‐155 is upregulated by anti‐PD‐1 treatment, and is associated with increased cytokine production and tumour control by CD8 T‐cells in melanoma. ⁹⁴ However, miR‐155 has also been shown to promote exhaustion in CD8 T‐cells in a LCMV model. ⁹⁵ Inhibition of FoxO1 or TOX represent additional potential therapies for reversing T‐cell exhaustion; however, these strategies will be difficult to carry out, as inhibition of these multi‐functional proteins could have off‐target effects. It is clear that therapies targeting transcription factors that could reinvigorate TCR signalling have potential, but more work must be done to elucidate the mechanisms through which these transcription factors function, in order to better understand the pathways that they effect.

While therapies aimed at reducing the expression of inhibitors, or blocking them completely, are capable of enhancing the immune response, they can render CD8 T‐cells more susceptible to overstimulation by viral antigen. An approach that could potentially achieve the same goal while avoiding this drawback is to directly target pathways that enhance CD8 T‐cell glycolysis. Upregulating the mTOR pathway via blockade of AMPK signalling, enhancing AKT activity, or upregulating the transcription factors HIF1α and MYC, could reinvigorate T‐cells and reverse exhaustion by upregulating the expression of proteins that promote an optimized glycolytic state. In addition, blocking AMPK activity would not only upregulate mTOR activity, but would also help shift the metabolic state by reducing dependence on FAO and glutamine metabolism, as AMPK activity has been shown to enhance both of these processes and reduce IFNγ production in effector CD8 T‐cells. ⁹⁶ Another metabolic pathway to potentially target is glutamine metabolism, through glutaminase 1 (GLS1) blockade. In a recent study, reduced glutamine metabolism through blockade of GLS1 was shown to induce a highly activated and long‐lived state in tumour‐infiltrating CD8 T‐cells. ⁹⁷ Additionally, glutaminase deficiency has been linked to increased T‐bet activity, and promoting the differentiation and effector function of effector CD8 T‐cells through sensitization of cells to IL‐2‐mediated mTOR signalling. ⁹⁸ Alternative metabolism‐targeting approaches include enhancing production of particular glycolytic metabolites to promote glycolysis, such as phosphoenolpyruvate (PEP). PEP is associated with enhanced CD8 T‐cell antitumour activity, and has been shown to alter CD8 T‐cell metabolism by maintaining nuclear factor of activated T‐cells (NFAT) signalling, which enhances expression of glycolytic transcription factors. ⁹⁹ , ¹⁰⁰ This could be achieved by enhancing the activity of the glycolytic enzyme phosphoenolpyruvate carboxykinase‐1, or the concentration of its substrate oxaloacetate.

If such treatments were successful in reinvigorating glycolysis, this restoration of normal effector metabolism could produce the metabolites and energy necessary to restore effector functions. While the above strategies demonstrate the strong potential of metabolism‐based therapies to reinvigorate exhausted T‐cells, further research into the effects of such treatments as well as methods of delivery and length of treatment is needed before they can be confirmed as effective in a CVI model. However, based on our current knowledge, direct targeting of pathways to upregulate mTOR signalling and thereby upregulate glycolysis could provide a successful therapeutic strategy.

Summary

While many of the markers and phenotypes that identify exhausted CD8 T‐cell populations are well known, the mechanisms underlying the development of T‐cell exhaustion are poorly understood. Thus, any therapeutic options that target suspected mechanisms must be considered. Targeting metabolism in particular holds great promise for reversing CD8 T‐cell exhaustion and treating CVIs. There is robust evidence that reversal of the metabolic dysfunction characteristic of T‐cell exhaustion enhances effector function sufficiently to overcome CVIs in different models. Therefore, therapeutic strategies that directly target metabolic dysfunction should be strongly considered in the treatment of CVIs.

Disclosures

None.