Tumor-infiltrating CD8+ T Cell Antitumor Efficacy And Exhaustion: Molecular Insights Part 2

Metabolic factors regulating CD8+ T cell infiltration of tumors

Metabolism is the sum of the biochemical reactions within living cells that provide the necessary energy for vital cellular processes. Cellular metabolism allows T cells to acquire and utilize energy necessary for their survival, proliferation, and function. The alteration of metabolism in the TME significantly affects TIL survival and antitumor responses. Similarly, the survival and effector function of tumor-infiltrating CD8+ T cells is directly associated with metabolic factors in the TME [167–169]. In activated T cells, metabolic reprogramming induces glycolytic flux and lactate, lipid, and protein production [170]. However, impaired glucose metabolism is a major issue that adversely affects tumor-infiltrating CD8+ T cells. Effector T cells require a healthy glucose metabolism; however, the lack of necessary nutrients and other factors impairs glucose metabolism in tumor-infiltrating CD8+ T cells. 

Specifically, metabolic reprogramming can improve the body's immune function. First, it boosts natural immunity. Natural immunity refers to the body's defense mechanisms that protect us from microbes and other pathogens. Through proper diet and exercise, we can boost our natural immunity and make our bodies more resistant to viruses and bacteria.

Second, metabolic reprogramming can enhance adaptive immunity. Adaptive immunity refers to the body's immune response to a specific pathogen. When the body is exposed to a pathogen, adaptive immunity develops resistance to that pathogen, thereby avoiding disease. Adaptive immunity is strengthened through proper diet and exercise, making the body more capable of defending against and destroying pathogens.

In addition, metabolic reprogramming can also reduce inflammatory responses. An inflammatory response is a stress response of the body's immune system to a stimulus. When the body is stimulated by infection or trauma, endogenous mediators trigger the inflammatory response of immune cells to destroy pathogens and promote wound healing. However, long-term chronic inflammation can increase the risk of diseases such as heart disease, diabetes, and cancer. Through proper diet and exercise, we can reduce inflammation and thus reduce the risk of chronic disease.

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In clear cell renal cell carcinoma (ccRCC), tumor-infiltrating CD8+ T cells are abundant. However, they are deficient in their effector functions because of perturbed metabolism. Further analyses suggested that tumor-infiltrating CD8+ T cells in ccRCC were unable to uptake glucose for glycolysis. As a result, the mitochondria in CD8+ T cells were fragmented, hyperpolarized, and unable to generate robust reactive oxygen species (ROS) because of inhibition of mitochondrial superoxide dismutase 2 (SOD2) [171]. In human and mouse melanomas, insufficiency in glycolytic metabolism and oxidative phosphorylation (OXPHOS) in tumor-infiltrating CD8+ T cells is also reported [168].

Rapidly proliferating tumor cells consume most of the nutrients and, therefore, create a nutrient-insufficient environment for tumor-infiltrating CD8+ T cells. In the pro-TME, infiltrating CD8+ T cells also lack or have dysfunctional metabolic enzyme activities. For example, in melanoma, tumor-infiltrating CD8+ T cells had decreased activity of the glycolytic pathway enzymes, ENOLASE 1 (i.e., alpha-enolase) and phosphoenolpyruvate (PEP) [168]. ENOLASE 1 is an upstream enzyme responsible for PEP production in the glycolytic pathway. Furthermore, PEP is a substrate for pyruvate synthesis in glycolysis. Therefore, when ENOLASE1 activity is low, the supply of pyruvate increases CD8+ T cell effector function via the upregulation of glycolysis. Pyruvate is involved in glycolysis and OXPHOS reactions; therefore, the supply of pyruvate increases the function of tumor-infiltrating CD8+ T cells [168]. The active metabolism of PEP promotes calcium ion (Ca2+)- nuclear factor of activated T cells 1 (NFATc1) signaling to induce T cell effector function [172]. 

Molecular studies in a melanoma model showed that PEP decreases sarco/ER Ca2+- ATPase activity to promote Ca2+-NFATc1 signaling in T cells [172]. Therapeutically, phosphoenolpyruvate carboxykinase 1 has been targeted to increase phosphoenolpyruvate in T cells to increase CD8+ T cell effector function [172]. The aberrant increase in the glycolytic gate-keeping enzyme, pyruvate dehydrogenase kinase 1 (PDK1), was reported to have a protumor role and to adversely regulate the survival and effector functions of tumor-infiltrating CD8+ T cells [169]. In ovarian cancer, PDK1 regulates PD-L1 through the activation of JNK activity. The overexpression of PDK1 can enhance the PD-1–PD-L1 axis in CD8+ T cells and attenuate its effector function [169]. Given the crucial role of glucose metabolism in tumor-infiltrating CD8+ T cell function, several direct or indirect approaches have been proposed to restore normal glucose metabolism in tumor-infiltrating CD8+ T cells. As an example, acetate, a monocarboxylic acid anion, has been suggested to overcome restricted glucose metabolism in tumor-infiltrating CD8+ T cells [173]. The positive influence of acetate treatment has also been reported in acetyl-coenzyme A (acetyl-CoA) metabolism, lipogenesis, and protein acetylation [173,174].

Amino acids have a crucial role in T cell differentiation and functions. For example, glutamine metabolism, an α-amino acid, has a regulatory role in successful adoptive immunotherapy [175]. A study using a tumor-inoculated mouse model showed that the adoptive transfer of CD8+ T cells with attenuated glutamine metabolism promoted tumor regression and overall survival. The attenuation of glutamine metabolism induced CD8+ T cell proliferation and survival via downregulation of PD-1 expression on CD8+ T cells [175]. Another α-amino acid, L-arginine, improves T cell metabolism, tumor infiltration, and antitumor immune responses [176,177]. In a study where ovalbumin-expressing B16 melanoma cells were orthotopically implanted in C57BL/6 mice, adoptive transfer of Larginine-treated OT-1 T cells (specific for ovalbumin antigen) significantly induced antitumor responses and increased the survival of mice [177]. In general, DCs are involved in antitumor responses; however, in a study of the BALB/NeuT mammary carcinoma model, a phenotype (i.e., MHC II+/CD11b+/CD11chigh) of tumor-infiltrating DCs (i.e., TIDCs) decided the suppression of CD8+ T cell-mediated antitumor responses [176]. Interestingly, arginase metabolism by L-arginine regulates TIDC-mediated CD8+ T cell function and survival [176]. Apart from glutamine and L-arginine, several other amino acids are also involved in regulating tumor-infiltrating CD8+ T cell functions. Therefore, a thorough assessment of these amino acids within TME could help strategies for effective cancer immunotherapy.

Fatty acid catabolism has a significant role in tumor-infiltrating CD8+ T cell effector function and T cell-mediated antitumor immune responses [178]. In a mouse model of melanoma, because of concurrent hypoglycemic and hypoxic conditions within tumors, the expression of peroxisome proliferator-activated receptor-α (PPAR-α) and fatty acid catabolism increased in tumor-infiltrating CD8+ T cells [178]. To survive in a hypoxic TME, tumor-infiltrating CD8+ T cells adapt to increase hypoxia-inducible factor (HIF) transcription factors, specifically HIF-1α, but not HIF-2α [179]. The nucleoside, adenosine, is a component of DNA and RNA. Interestingly, its accumulation is involved in tumor growth and progression and impairs the effector function of CD8+ T cells. Further analysis demonstrated that adenosine attenuates central memory T cell generation via the A2A receptor-mediated pathway in peripheral and tumor-infiltrating T cells [180]. Besides the metabolic factors discussed earlier, additional metabolic factors regulate tumor-infiltrating CD8+ T cell survival and function. A detailed understanding of all these metabolic factors could help to restore the antitumor functions of tumor-infiltrating CD8+ T cells.

Toll-like receptor agonists in cancer immunotherapy

TLRs are involved in the regulation of immune activation upon pathogenic encounters. TLRs can act as natural immune modulators and, therefore, their potential could be exploited in cancer immunotherapy [181]. TLRs recognize pathogen-associated molecular patterns (PAMPs), which are expressed by pathogens and also associated with endogenous damage-associated molecular patterns (DAMPs), which are released from dying and stressed cells [182]. Given the immune-stimulatory nature of TLRs, TLR agonists have been implicated in cancer immunotherapy to boost host immunity against tumors [183]. TLR agonists are reported to promote immune cell activation in TME and to attenuate tolerance and immune inhibitory signaling [184]. The role of several TLRs, including TLR1/2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 ligands, are well established in enhancing CD8+ T cell antitumor efficacy [185–190]. TLR1/2 and 7 decrease PD1 expression on CD8+ T cells, leading to increased antitumor efficacy and tumor suppression [185]. The regulatory function of TLR1/2 has been reported in 4-1BB-mediated activation of CD8+ T cells and antitumor responses [191]. The agonist of TLR1/2, diplodocid, improved antitumor CD8+ T cell efficacy during anti-PD-L1-based cancer immunotherapy [192]. In a murine model of leukemia, small-molecule agonist 23 (SMU-Z1) induced TLR2 via association with TLR1, which promoted the proliferation of CD8+ T cells [193]. This TLR2 agonist was also associated with the human DC phenotype CD141+, required to induce antitumor effects by CD8+ T cells [194]. TLR3 agonists have also been reported to improve CD8+ T cell effector function along with antibody-mediated cancer immunotherapy [186].

Irreversible T-cell exhaustion is associated with the failure of checkpoint inhibitors. Targeting the CD40–TLR4 axis restored T cell effector function in a preclinical cancer model [195]. TLR4 agonists and other cancer therapeutics have been reported to successfully promote CD8+ T cell antitumor efficacy [187,196]. CBLB502, an agonist of TLR5, promoted CD8+ T cell-mediated antitumor responses in graft-versus-tumor models [197]. In colon and mammary metastatic preclinical in vivo models, TLR5 agonists showed organ-specific immuno-adjuvant function independent of tumor antigens [188]. The intratumoral treatment with a TLR7 agonist promoted M1 macrophages in TME and induced CD8+ T cell effector function by increasing IFN-γ expression [198]. An agonist of TLR7/8 (i.e., MEDI9197) promoted localized effects, which led to enhanced CD8+ T cell effector function and antitumor effects [199]. The bispecific agonist of TLR7/8 has been reported in melanoma, bladder, and RCC tumor models. The TLR7/8 bispecific agonist increased DC activation and expansion in lymph nodes, leading to enhanced priming and expansion of cytotoxic CD8+ T cells [200]. TLR9 has been reported to regulate the accumulation, maturation, and lymph node migration of antigen-loaded tumor DCs, which ultimately expand cytotoxic CD8+ T cells, leading to the enhanced killing of tumor cells [201]. Together, these results suggest TLR agonists as potential therapeutic tools to enhance anticancer efficacy in cancer immunotherapy.

STING in cancer immunotherapy

The stimulator of interferon genes (STING) is an intracellular receptor of the endoplasmic reticulum (ER), which is crucial for DNA-mediated immunological reactions [202]. It is also a cytosolic DNA-sensing pathway. The DNA sensor in the cytosol is cyclic GMP-AMP Synthase (cGAS), which activates the innate immune system mainly via IFN-1 expression [203]. STING-induced IFN-1 production promotes DC activation, which ultimately activates CD8+ T cells against tumor antigens [204]. The differential expression of STING has been reported in CRC. Higher STING expression is observed in the early stages of cancer and is associated with higher tumor infiltration of CD8+ T cells. The higher expression of STING in CRC is also associated with increased overall survival in patients with cancer. By contrast, lower STING expression is associated with lower infiltration and lower survival of patients [205]. Cyclic dinucleotides (CDNs) are STING agonists that could induce an immune response [206]. In an in vivo study, colon cancer tumors treated with a STING agonist showed a decreased tumor burden and increased expression of ICOS and IFNγ on CD8+ T cells [205]. STING also modulates chemotherapeutic drug efficacy. For example, in BRCA-deficient models of TNBC, the efficacy of PARP-1 inhibitors was dependent on STINGmediated CD8+ T cell antitumor effects [207]. The combined efficacy of STING-based immunotherapy with VEGFR2, PD-1, and CTLA-4 blockade has shown promising outcomes in cancer therapy by improving CD8+IFN-γ + T cell population and function [208].

Apart from immunotherapeutic agents, STING agonists, and other chemotherapeutic drugs have shown promising results. For example, in a preclinical in vivo tumor model, combined treatment with a STAT3 inhibitor and STING agonist promoted CD8+ T cell infiltration, and decreased Tregs and MDSCs in TME, leading to an antitumor response [209]. CDNs have some limitations, including barriers to drug delivery and their rapid clearance; therefore, an improved version of CDNs has also been reported. STING-activating nanoparticles (STINGNPs), along with immune checkpoint inhibitors, increase STING signaling in TME and sentinel lymph nodes, leading to enhanced immunogenic and tumoricidal microenvironments [206]. The emerging and promising role of STING agonists might have a promising future in improving CD8+ T cell effector functions, leading to a robust antitumor response.

Concluding remarks

In summary, cancer-induced immunosuppressive environments in the host and TME are key reasons for the failure of numerous antineoplastic therapeutics and, subsequently, drug resistance. Current approaches to cancer treatment are based on treating tumor cells to induce cell death. However, the discovery of checkpoint inhibitors and their efficacies have forced us to consider tumor immunity for a successful treatment plan. Emerging trends suggest that targeting tumors and immune systems has promising therapeutic outcomes, especially in hematological cancer; however, the results of several ongoing clinical trials in patients with solid malignancies are still anticipated. Chemotherapeutic agent-induced TME plasticity is crucial for CD8+ T cell-mediated antitumor responses and, thus, a clear understanding of the drug-induced alterations in TME will be essential to overcome drug resistance. Therefore, basic knowledge of antineoplastic therapeutic-induced changes in cytokines/chemokines, protein kinases, and metabolic factors in the TME will be vital to restoring CD8+ T cell-mediated antitumor responses.

Acknowledgments

The authors acknowledge Enrico Benedetti for providing access to departmental resources and financial support, Arnav Rana for editing the manuscript, and funding support from the Department of Surgery, Veterans Affairs Career Scientist Award (BX004855), and National Cancer Institute Award (CA 216410) to A.R. B.R. is supported by NCI Award (CA 219764) and Veterans Affairs Merit Award (BX003296).

Author Biographies

Sandeep Kumar

Sandeep Kumar has a broad background in immunology and cancer biology, with specific training and expertise in T cell-mediated antitumor responses. During his postdoctoral training at Roswell Park Cancer Institute, Dr. Kumar explored the role of cytokines/chemokines associated with apoptosis resistance in cancer cells. Currently, Dr. Kumar is exploring the role of various MAPK upstream and downstream family members in T cell biology and the implications of these findings for immunotherapy in breast and pancreatic cancers.

Basabi Rana

Basabi Rana received her PhD in biochemistry from the University of Calcutta (now Kolkata), and postdoctoral training from Boston University School of Medicine. She has worked extensively on elucidating the signaling mechanisms that govern WNT/β-catenin signaling and on the effect of TRAIL-PPARγ ligand combination therapies to ameliorate resistance. Current research in her laboratory focuses on understanding the signaling mechanisms of therapy resistance and designing strategies to overcome these, with specific emphasis on sorafenib resistance in hepatocellular carcinoma.

Ajay Rana

Ajay Rana completed his Ph.D. in biochemistry at the Indian Institute of Chemical Biology and his postdoctoral training at the Harvard Medical School in cell signaling and cancer biology. Dr. Rana is well-recognized in the cell signaling and breast cancer fields. The current focus of his laboratory is to delineate MAPKs and other related proteins in breast, pancreatic, and liver cancer, with particular emphasis on identifying mechanism-based targeted therapies.

Sunil K. Singh

Sunil Singh received his Ph.D. in life sciences from Jawaharlal Nehru University, New Delhi, India. Currently, he is studying the regulatory function of MAPK upstream regulators in pancreatic and breast malignancies.

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