The introduction of high throughput technologies has confirmed Otto Warburg’s early observations that tumors consume more glucose than normal tissues and deepened the understanding of the biological role of glucose and lactate in the TME. In this regard, single-cell RNA-sequencing (scRNAseq) data from breast cancer patients showed that tumor cells display a gene signature associated with higher glucose consumption compared to normal epithelial cells (25), and glucose consumption by pancreatic ductal adenocarcinoma (PDAC) cell lines has been reported to impair proliferation of CD8+ T cells (26). Lactate, once considered a mere waste product of glycolytic metabolism, is now well known to play a critical role in establishing the TME and modulating immune evasion mechanisms by altering the phenotype and effector activity of immune cells (27). Li et al. recently published a comprehensive review of the role of lactate in physiologic and pathological processes (20).

In hepatocellular carcinoma (HCC), transcriptomic data revealed a lactate metabolism-related gene signature (LMRGS) based on the expression of six genes (FKTN, PDSS1, PET117, PS1, RARS1, and RNASEH1). HCC cases with high LMRGS were associated with immune cell infiltration characterized by the presence of follicular helper T cells (Tfh), Tregs, and M0 macrophages. In contrast, cases with a low LMRGS were mainly infiltrated by resting NK cells, monocytes, and mast cells (28).

As mentioned earlier, T cells are the most potent mediators of the adaptive antitumor immune response and require high glucose consumption to effectively perform their effector functions. Glucose deprivation decreases the phosphorylation of p28 and JNK induced after TCR stimulation (29), leading to decreased production of IFN-γ, IL-2, and TNF-α, and Th1 differentiation on CD4+ T cells and impaired exocytosis of cytotoxic granules on cytotoxic T lymphocytes (CTLs). Some reports indicate that glucose deprivation allows the glycolytic enzyme GAPDH to bind the 3’UTR region of the IFN-γ transcript and promotes the loss of the open chromatin marks H3K9Ac and H3K27Ac in Th1-related genes, preventing their transcription (30, 31). Additionally, T cells express the lactate transporters SLC5A12 and SLC16A1, and exposure to this metabolite alters some functional activities such as glycolytic activity, motility, the cytotoxic activity of CTLs, and skew CD4+ T cells to Th17 phenotype, characteristic of persistent inflammatory responses (32, 33).

The hostile environmental features of the TME, such as the presence of anti-inflammatory cytokines and metabolic reprogramming, promote the accumulation of Tregs that favor tumor survival and progression. Tregs exert their suppressor activity on other immune cells through membrane-membrane interaction or by producing anti-inflammatory cytokines, such as IL-10 and TGF-β (34). In the glucose deprived, lactate enriched TME, Tregs are able to maintain their suppressor activity, since their metabolism is reprogrammed by the master transcription factor FOXP3 from glycolysis to OXPHOS (35). Interestingly, Watson et al. reported that Tregs display a heterogeneous metabolism of glucose and lactate, in which glucose-avid Tregs show lower suppressor activity than lactate-avid Tregs that promote immune suppression and tumor progression. Moreover, glucose starvation skews Tregs toward lactate consumption, and blockade of the lactate transporter MCT1 promotes the accumulation of glucose-avid Tregs that preserve the antitumor functions of CTLs (36).

These studies demonstrate that glucose depletion and lactate accumulation probably due to the increased metabolism of proliferating immune cells are sufficient to inhibit the effector functions of T cells and induce the accumulation of Tregs. It is tempting to speculate that this process is hijacked and taken one step forward by tumor and stromal cells as an immune evasion mechanism capable of inducing T cell dysfunction in the TME. Dysfunctional T cells can be classified in anergic, exhausted and senescent, and have been described to be induced by the hostile conditions of the TME and fail to eliminate tumor cells (37). As discussed in the next section, metabolic alterations are drivers of ICs expression, which are markers of T cell exhaustion. Furthermore, Tregs and tumor-derived γδ T cells have been reported to induce a senescent phenotype on effector T cells in vitro that differs from exhaustion and anergy. This effect is induced by glucose deprivation-mediated AMPK activation, and glucose supplementation or the blockade of glucose uptake in Tregs reverts the acquisition of the senescent phenotype by effector T cells (38).

Exhausted T cells are mainly induced by chronic antigen stimulation, show diminished effector function and high expression of ICs. Interestingly, it has been reported that T cells display characteristics of metabolic dysfunction even before the emergence of the exhausted phenotype. Thus, exhausted T cells show reduced glucose uptake, and transcriptional changes suggestive of glucose deprivation, as well as diminished mitochondrial function related to increased mitochondrial depolarization and production of ROS (39). As a response to TME variations, epigenetic modifications have been suggested as drivers of metabolic exhaustion. Transcription factors TOX and NR4A drive epigenetic changes characteristic of T cell exhaustion that may be promoted by conditions like hypoxia, glucose restriction, methionine deprivation, and ROS accumulation, and in turn promote the expression of some ICs (40, 41).

In comparison, cellular senescence is mainly related to telomere shortening due to repetitive division cycles. Although a senescence-like phenotype has been reported in multiple immune cells (42), it has been mostly studied on T cells that are characterized by loss of surface expression of costimulatory CD28 and telomere shortening due to their elevated proliferative rate (43, 44). Similar to other senescent cells, T cells upregulate their glycolytic metabolism in order to maintain the metabolic requirements of the senescent phenotype, like the senescent-associated secretory phenotype (SASP) (45). Interestingly, senescence in T cells strongly depends on their mitochondrial content. In particular, CD4+ T cells have been reported to better withstand cellular senescence due to higher mitochondrial content and oxidative metabolism, in comparison to CD8+ T cells (46). By contrast, senescent CD8+ T cells are able to acquire a NK cell-like phenotype that might be important for the removal of other senescent cells, and possibly tumor cells (47).

Although changes in the concentrations of glucose and lactate have been shown to inhibit the effector mechanisms and change the phenotype of T cells, more studies are needed to clarify if these metabolic alterations are capable of driving cell exhaustion or senescence in TILs.

Effective antitumor responses require the participation of several other immune cells that may also be impaired by the elevated glucose consumption in the TME. Similar to CD8+ T cells, NK cells require a highly glycolytic metabolism during activation. Increased glucose uptake and mTOR1-mediated glycolytic activity are required for their IFN-γ and granzyme B production. This is illustrated by the fact that some NK cells, especially the CD56^bright subpopulation, depend on glucose uptake through the GLUT1 transporter for IFN-γ production (48). Similarly, high lactate concentrations inhibit the activation of the transcription factor NFAT in both CD8+ T and NK cells, impairing their tumor infiltration and cytolytic effector functions. Brand et al. reported that, in a model of mice engrafted with low Lactate Dehydrogenase-expressing (LDH^low) melanoma, tumor progression was reduced in an IFN-γ-dependent manner. LDH^low tumors showed higher NK and CD8+ T cell infiltration with increased IFN-γ and granzyme B expression, compared to the control group (49). Moreover, CD56^bright NK cells cultured in conditioned media from colorectal cancer metastasis undergo apoptosis induced by lactate-mediated intracellular pH decrease, mitochondrial stress, and accumulation of ROS (50). Based on these findings, we propose that, in addition to promoting immune evasion at the primary tumor, a high lactate production may facilitate the establishment of the premetastatic niche.

Two opposite phenotypes have been described in macrophages. The M1 phenotype shows antitumor activity that depends on glycolytic metabolism, with high oxygen consumption and pentose phosphate pathway (PPP), while the M2 phenotype mainly depends on OXPHOS (51). In this regard, glucose deprivation and lactate accumulation may skew macrophages toward the M2 phenotype, favoring immunosuppression. In fact, the M2 phenotype polarizing cytokine IL-4 inhibits mTOR signaling, which favors oxidative metabolism over glycolytic activity (52). Lactate has been reported to inhibit LPS-induced glycolytic activity on bone marrow-derived macrophages (BMDMs), resulting in lower expression of the cytokines IL-6, IL-12p40, and the co-stimulatory molecule CD40 (53), and to induce the expression of M2-specific markers, such as arginase-1, Mannose Receptor C-Type 1, CD206, and VEGF production, through ERK/STAT3 signaling (54). This effect seems to be dependent on the heterodimer of the odorant receptor Olfr78, and the G-protein-coupled receptor GRP132 since knockdown of Olfr78 prevents M2 polarization and promotes tumor infiltration by M1 macrophages and CD4+ and CD8+ T cells (55).

The effect of glucose concentration on DC maturation and the subsequent activation of T cell responses is less clear. On the one hand, maturation of DCs induced by TLR ligands or type I IFN favors a switch from oxidative to glycolytic metabolism. Reports indicate that glucose restriction limits the ability of DCs to produce IL-2 and express the co-stimulatory molecule CD86, which results in impaired CD4+ T cell activation. Moreover, the inhibitory effect of IL-10 has been reported to be due to the blockade of the metabolic shift toward glycolytic metabolism (56, 57). On the other hand, it has been reported that LPS-stimulated DCs under glucose deprivation express higher amounts of co-stimulatory molecules CD80 and CD86 and the cytokine IL-12. Consequently, glucose-restricted DCs exhibit higher capability of activating antigen-specific CD8+ T cells than DCs stimulated in normal glucose concentrations. The restriction of nutrients, such as glucose or amino acids, inhibits mTOR signaling, which prevents HIF-1α activation and the metabolic switch in DCs (58). These seemingly contradictory results may be explained by the dual function of DCs associated with their maturation stage. Immature DCs display high phagocytic activity for antigen capture in tissues, so these cells may require a high nutrient consumption to support this active process. After maturation and migration to the lymph nodes to present antigens to T cells, DCs may reduce their metabolism to avoid T cell inhibition due to nutrient depletion. This is supported by the fact that AKT activation and increased glycolysis are early events of DC activation (59) and that, after migrating to lymph nodes activated T cells deprive DCs of nutrients (58). In this sense, glucose depletion in the TME may impair DC maturation and migration to lymph nodes, preventing efficient T cell activation.

Regarding the effect of lactate on DCs, it has been reported that lactate produced by lung cancer cells inhibits IL-2 and type I IFN production and induces the expression of IL-10. As a result, DCs fail to correctly present antigens to induce efficient antitumor T cell responses (60). In particular, lactate impairs type I IFN production on plasmacytoid DCs (pDC) through GRP81 and MCTs (61). In the TME, granulocyte-macrophage colony stimulating factor (GM-CSF)-induced tolerogenic DCs inhibit glycolytic metabolism on T cells and promote the Treg phenotype through lactate production (62).

As in the case of tumor cells, MDSCs have been reported to exhibit significant metabolic plasticity. Based on dynamic metabolic flux analysis, it has been proposed that MDSCs are mainly dependent on glycolysis while maintaining high tricarboxylic acid (TCA) cycle with minimal PPP and OXPHOS activities (63). In Staphylococcus aureus infection, MDSCs have been shown to require an increased glucose uptake to undergo complete maturation. In contrast, a glucose-deficient environment, such as the TME, promotes the accumulation of immature and highly suppressive MDSCs. Reports indicate that MDSCs cultured in high glucose concentrations display a reduced ability to suppress CD4+ T cell proliferation (64). The dependence of PMN-MDSCs on glucose uptake relies on the expression of glucose transporter GLUT3; knockdown of this transporter reduces glucose uptake and promotes apoptosis, suggesting it as a possible therapeutic target to restore antitumor immunity (65).

Similarly, in vitro exposure of mouse bone marrow cells to IL-6, GM-CSF, and lactate enhances the induction of bone marrow-derived MDSCs that display high capacity to suppress T cell proliferation and cytotoxicity of NK cells (66). Radiotherapy has been reported to promote the Warburg effect inducing lactate secretion in a mouse model of PDAC. Tumor-derived lactate promotes MDSCs induction and activation through the GPR81/HIF-1α/STAT3 signaling pathway, suggesting the role of enhanced lactate secretion as a mechanism of radioresistance (67).

According to this information, progressive glucose deprivation and lactate accumulation in the TME due to increased glycolytic metabolism suppress the effector functions of immune cells and skew them toward anti-inflammatory/pro-resolving phenotypes, which supports the survival of tumor cells. The combination of treatments to inhibit the glycolytic activity of tumor and stromal cells to restore baseline glucose levels and the consequent decrease in lactate accumulation in the TME is a promising therapeutic strategy to reinvigorate the effector functions of tumor-infiltrating cells.

As discussed earlier, metabolic reprogramming in the TME is a hallmark of cancer and is not restricted to the Warburg effect. Rapid growth and multiplication of tumor cells require a high demand for other nutrients, such as amino acids. It has been reported that some cancer cells lack the ability to synthesize some amino acids, so they depend on the exogenous supply of these nutrients to maintain their development and metabolism (68). To meet this metabolic demand, cancer cells increase the expression of transporters and enzymes involved in amino acid synthesis and metabolism (69), which may promote the deprivation of these nutrients in the TME and thus, the impairment of the effector functions of antitumor immune cells.

Multiple studies have demonstrated that glucose deprivation alone or combined with lactate exposure induces ICs expression on immune cells. Although the PD-1/PD-L1 axis is the most studied IC and the main therapeutic target of immunotherapies, alterations in the concentration of metabolites in the TME modulate the expression of many other ICs. Thus, combinatorial therapies that simultaneously target tumor glucose metabolism and the expression of ICs may restore the antitumor state of tumor-infiltrating immune cells.

As mentioned above, a scRNA-seq analysis of breast cancer patients has shown that tumor cells exhibit a gene signature associated with elevated glucose metabolism compared to normal epithelial cells. Interestingly, the highest glucose uptake in TME is displayed by exhausted CD8+ T cells, which displayed increased expression of the ICs PD-1, TIM-3, LAG3, and TIGIT (25). In vitro, culture in low glucose concentration induce the expression of PD-L1 on highly glycolytic PDAC cells and PD-1 on co-cultured CD8+ T cells. Knockdown of the enzyme phosphofructokinase-m (PFK-m) before tumor engraftment on mice reverted PD-1 expression on CD8+ TILs and the corresponding PD-L1 on tumor cells suggesting that this phenomenon was mediated by the high glycolytic activity of PDAC cells (26). Similarly, inhibition of glycolysis in renal cancer cell lines by the knockdown of HIF-1α, PFKFB3, or LDHA, or by culture under glucose deprivation induces PD-L1 expression, and glucose supplementation reverses this effect. Mechanistically, low glucose concentration promotes epithelial growth factor receptor (EGFR) expression, which induces PD-L1 expression via the EGFR/ERK1/2/c-Jun signaling pathway and PD-L1 stabilization through glycosylation (172, 173). Similarly, soluble mediators secreted by tumor cells, such as hyaluronan fragments, induce a metabolic shift toward aerobic glycolysis in tumor-infiltrating monocytes. This metabolic change causes the overexpression of the glycolytic enzyme PFKFB3, promoting the signaling by NF-κB, which increases PD-L1 expression. Inhibition of glycolysis by the glucose analog 2-deoxy-D-glucose (2-DG) reversed this effect (174).

Consequently, the pharmacological inhibition of Akt in esophageal cancer cell lines by ginsenoside Rh4 reduces the expression of crucial glycolytic enzymes, such as GLUT1, HK2, LDHA, PFKL, and PKM2, as well as the lactate production, which results in lower PD-L1 expression (175). Moreover, it has been reported that PKM2 is mandatory for PD-L1 expression in tumor, immune, and stromal cells in the TME and lymph nodes (176). Dimerization and nuclear translocation of PKM2, induced by M2 TAMs-derived TGF-β, allows its interaction with the nuclear factor STAT1 to promote the overexpression of PD-L1, event that impairs NK cell-mediated antitumor immune response (177). Concomitantly, lactate accumulation has been reported to induce PD-L1 expression in lung cancer cell lines in a dose-dependent manner. Lactate represses the activity of PKA, allowing the interaction of TAZ with the transcription factor TEAD, and their recruitment to the PD-L1 promoter (178). Similarly, in absence of lactate, activation of NF-κB prevents the transcription of the IC Galectin-9 (Gal-9) by binding to histone deacetylase HDAC3 in head and neck carcinoma cell lines. Thus, accumulation of PKM2-derived lactate induces the expression and secretion of Gal-9 (179).

Although the main inductor of the IC VISTA is the hypoxic microenvironment, lactate accumulation promotes VISTA-mediated immunosuppression through the acidification of the TME. VISTA suppresses T cell functions at physiologic pH, but this suppression is improved at acidic pH. The extracellular domain of VISTA is enriched with protonated histidine residues at acid pH, which allows its binding to P-selectin glycoprotein ligand-1 (PSGL-1), transmitting an inhibitory signal to T cells (180).

In addition to the direct suppression of the antitumor functions of immune cells by adenosine accumulation, especially due to the overexpression of CD39 and CD73, IC expression is enhanced by adenosine signaling. In biopsies from PDAC patients, CD73 expression on tumor and stromal cells correlates with PD-L1 expression on tumor cells (201). Additionally, in biopsies from NSCLC patients, CD39+ CAFs positively correlate with PD-L1 expression on tumor cells (202). Similarly, the activation of the A2AR induces PD-1 expression, but not CTLA-4, on CD8+ and CD4+ FOXP3+ TILs in vitro, and in a mouse model of colon adenocarcinoma. Consequently, CD8+ TILs from CD73 deficient mice show lower PD-1 expression compared to T cells from wild-type mice (203), and deletion of the A2AR results in lower PD-1 expression in CD8+ TILs in a melanoma mouse model. Nevertheless, the deletion of A2AR in T cells reduces their survival, resulting in enhanced tumor growth (204).

By using mixed lymphocyte reactions with T cells and DCs derived from mouse, it has been shown that A2AR activation by an agonist reduces T cell proliferation, while inducing PD-1 and CTLA-4 expression (205). Moreover, A2AR stimulation induces the expansion of Tregs in the TME, which further increases adenosine accumulation, since A2AR agonist-induced Tregs to express CD39 and CD73. These A2AR-induced Tregs show high expression of CTLA-4, which plays a crucial role in their inhibitory activity (206). Contrary to the effect of adenosine on ICs expression, ATP binds to its P2X7R on monocytes to inhibit the shedding of soluble HLA-G (207). In the context of the TME, adenosine accumulation due to overexpression of CD39 and CD73 ectonucleotidases may inhibit the stimulatory effect of extracellular ATP, promoting an increased production of both membrane and soluble HLA-G to limit antitumor immune responses.

Regarding combinatorial therapies that target adenosine signaling to improve the response to immunotherapies, oral administration of A2AR antagonists reduces PD-1 and LAG-3 expression on activated CD8+CD44+ T cells within tumor-draining lymph nodes (dLNs) and Tregs infiltrating tumors in a mouse melanoma model (208).

Metabolically, PD-1 signaling inhibits T cell activation by altering metabolic reprogramming induced by TCR-mediated antigen recognition. It has been demonstrated that, when activation occurs in presence of PD-1 interacting with recombinant PD-L1, T cells show diminished glucose uptake and glycolytic capability due to a reduction in the expression of Glut1 transporter and HK2. Furthermore, PD-1 inhibits T cell capability of uptaking and utilizing amino acids such as valine and glutamine while promoting FAO by enhancing the expression of the carnitine pamitoyltransferase (CPT1A) and desnutrin/adipose triglyceride lipase (ATGL) (209). Exposition of PD-1 positive esophageal adenocarcinoma cell lines to the anti-PD-1 pembrolizumab augments glycolytic reserve by upregulating the expression of GLUT-1.

Similarly, PD-1 blockade increases GLUT1 expression on CD8+ T cells from B cell lymphoblastic leukemia-bearing animals. However, PD-1 blockade was insufficient to restore the antitumor functions of T cells, suggesting the existence of additional immunosuppressive or compensatory mechanisms that impair antitumor immunity (210). In this regard, it has been reported that resistance to anti-PD-1 treatment is due to increased lactic acid in TME, which promotes PD-1 expression of Tregs in intrahepatic tumors. Furthermore, resistance to anti-PD-1 treatment can be overcome by hindering lactate metabolism through inhibition of LDHA or MCT1 on Tregs (211). Moreover, knockdown of the glycolytic enzyme PKM2 in PDAC cells promotes NK cell infiltration, production of IFN-γ, granzyme B, and NKp46, and response to anti-PD-1 treatment (177).

These results show that metabolic modulations in TME improve the efficacy of ICIs, suggesting that IC blockade induces metabolic changes that impact the use of combination therapy. In addition, it has been suggested that PD-1 engagement impairs metabolic functions beyond glycolytic activity. RNA-seq and Gene Ontology analysis revealed that PD-1 signaling triggers a specific transcriptional program in CD8+ T cells involved in altered amino acid, nucleotide, and carbohydrate metabolism, as well as altered TCA cycle and OXPHOS. Furthermore, PD-1 affects the expression of genes involved in the structure and function of mitochondria, resulting in reduced mitochondria number and cristae length (212). Metabolic alterations driving T cell exhaustion after PD-1 engagement may be due to PD-1-mediated inhibition of the peroxisome proliferator-activated receptor-gamma co-activator (PGC)-1α, acting as a regulator of genes involved in energy metabolism and mitochondrial biogenesis (213). Moreover, PD-1 inhibitory signaling shifts metabolism away from aerobic glycolysis and glutaminolysis and forces T cell to events of anaplerotic input to the TCA cycle, mainly at acetyl-CoA and succinyl-CoA, also preventing the de novo nucleoside phosphate synthesis accompanied by decreased mTORC1 signaling (214).

Aside from T cells, PD-1 signaling causes metabolic dysfunctions in monocytes and macrophages. Monocytes isolated from Chronic Lymphocytic Leukemia (CLL) show diminished glucose uptake and lactate production. As discussed above, M1 Macrophages rely on aerobic glycolysis for their antitumoral functions. CLL monocytes show enhanced PD-1 expression compared to healthy donors, and ligation with recombinant PD-L1 diminishes monocytes switch to aerobic glycolysis, while anti-PD-L1 blockade reverts this effect, promoting phagocytosis of tumor cells (215). Interestingly, it is known that PD-1 expression on TAM of the TME can be induced by CAFs (216). In addition to PD-1-mediated metabolic alterations described in T cells and monocytes, in a melanoma mouse model it has been shown that PD-1 knockout reduces the accumulation of granulocyte and monocyte precursors, as well as the immunosuppressive activity of MDSCs, in part through metabolic alterations. In PD-1 knockout tumor-bearing mice, glucose uptake and mitochondrial biogenesis were elevated in myeloid progenitors, and it was suggested that, in PD-1 deficiency, glycolytic activity is progressively switched to mitochondrial metabolism. PD-1-deficient myeloid progenitors display increased metabolic intermediates of glycolysis, PPP, TCA cycle, and elevated cholesterol, resulting in enhanced differentiation toward effector monocytic/macrophage and DCs, thus promoting antitumor responses (217). Whereas PD-1/PD-L1 axis-mediated immune suppression is commonly attributed to PD-1 signaling, PD-L1 is known to mediate intracellular signaling that promotes cancer progression, immune escape, and metabolic reprogramming (218).

In patients with NSCLC, 2-Deoxy-2-[fluorine-18] fluoro-D-glucose (2-FDG) uptake has been reported to be higher in tumors with high PD-L1 expression, suggesting an interplay between PD-L1 expression and glucose uptake. Moreover, it has been suggested that an elevated glycolytic metabolism might be used as a prognostic biomarker for ICI treatment (219). In cervical cancer cell lines, it has been reported that PD-L1 promotes glucose metabolism and lactate secretion by interacting with integrin β4 (ITGB4), and suppressing SIRT3, resulting in the augmented expression of glycolytic enzymes HK2 and LDHA and of transporters GLUT1 and GLUT4 (220). Similar results have been reported in AML cell lines, where genes and the corresponding proteins associated with glucose metabolisms, such as ALDOA, PGK1, LDHA, and HK2, are highly expressed when PD-L1 is overexpressed (221). In a mouse model of sarcoma, it has been shown that monoclonal antibody blockade of CTLA-4, PD-1, or PD-L1 augments glucose availability in the TME and glycolytic metabolism in T cells by restoring mTOR-mediated signaling, as well as the activity of the enzyme glutamate dehydrogenase (Glud1). PD-L1 knockdown and antibody blockade diminish glucose metabolism and Akt/mTOR signaling in tumor cells without affecting proliferation in vitro or tumor growth in RAG^-/- mice. These results suggest that ICIs might revert metabolic alterations in tumor cells, allowing an enhanced nutrient availability in the TME to restore the functionality of antitumor immune cells (222). Mechanistically, PD-L1 might promote glycolysis by enhancing the expression of the glycolytic enzyme PFKFB3 since PD-L1 knockdown by a small interfering RNA also diminishes the expression of this enzyme (172).

As discussed earlier, lactate accumulation in the TME also impairs immune cell function and response to immunotherapy. For instance, lung cancer cells A549 exposed to high lactate concentration inhibit IFN-γ production and induce apoptosis of co-cultured Jurkat T cells. Interestingly, treatment with PD-L1-blocking antibody reverses the indicated effects (178), suggesting that anti-PD-L1 treatment modulates the inhibitory effect of tumor-derived lactate on immune cells. In addition, the inhibition of glutamine metabolism by a competitive antagonist of transmembrane glutamine flux or a GLS inhibitor, in combination with anti-PD-1 or anti-PD-L1 antibodies, has been shown to reduce tumor growth in breast and colon cancer mouse models to a greater extent than monotherapies (181, 187).

Interestingly, IDO-mediated tryptophan metabolism in the TME has been proposed as a major mechanism for resistance to ICIs. High IDO expression in macrophages and endothelial cells in TME are related to anti-PD-1 non-responsiveness in metastatic renal cell carcinoma and sarcomas patients undergoing anti-PD-1 treatment with pembrolizumab or nivolumab antibodies (223, 224). As mentioned earlier, the accumulation of IDO-derived kynurenine in the TME is associated with the upregulation of multiple immune checkpoints that may contribute to anti-PD-1 resistance (188). Combined checkpoint blockade therapy is becoming increasingly important, especially regarding cancers in advanced stages or associated with poor prognosis. For instance, a recent study in HER2-overexpressing cancers showed that using a bispecific antibody, simultaneously targeting PD-1 and HER2, represents a new promising approach for treating late-stage metastatic HER2-positive cancers (225).

One of the first relationships reported between ICs and metabolism was the influence of CTLA-4 on IDO expression (226, 227). In vitro stimulation of DCs with recombinant CTLA-4 showed an increased tryptophan metabolism to kynurenine, and IDO inhibitor 1-MT reversed this effect. Moreover, CTLA-4 stimulation induces IDO expression in DCs similarly to IFN-γ (228). Consequently, mice with a Treg-specific CTLA-4 knockdown show reduced IDO expression in CD11c+ mesenteric DC and reduced kynurenine production by spleen-isolated DCs (229). In the context of the TME, CTLA-4 expressing Tregs induced by metabolic alterations, may promote IDO expression in tumor-infiltrating DCs, reinforcing immune suppression. In T cells, CTLA-4 signaling inhibits glycolysis without inducing FAO; in contrast to PD-1 signaling, the authors suggest that CTLA-4 does not induce metabolic alterations related to T cell exhaustion but preserves the metabolic profile of unstimulated T cells (209). In addition, IDO activity in the peripheral blood of melanoma patients increases PD-L1 expression in circulating CTLs. This IDO activity is associated with a CTLA-4 increase in Tregs. This IDO/PD-L1/CTLA-4 interplay is associated with a negative prognosis of cancer patients, showing that the expression of IDO, PD-L1, and CTLA-4 is strongly interconnected (230).

Regarding ICIs, ipilimumab-mediated CTLA-4 blockade promotes immune cells metabolic fitness and infiltration. Moreover, ipilimumab treatment induces the functional destabilization of tumor-infiltrating Tregs by impairing cell glycolysis and CD28 signaling (231). It has been reported that anti-CTLA-4 monotherapy produces durable responses in many cancers, mainly in melanoma (232). However, combining anti-CTLA-4 antibodies with other therapeutic strategies, such as chemotherapy or radiotherapy, increases their efficacy (233–236). In fact, the modulation of TME by propranolol increases tumor T cell infiltration and the efficacy of anti-CTLA-4 treatment (237).

Research is still in progress to enhance anti-CTLA-4 therapy response. Notably, the modulation of the metabolic conditions in the TME may enhance response to ICIs in cancer patients. For instance, PD-1 or CTLA-4 inhibition has been reported to synergize with the blockade of adenosine metabolism to inhibit tumor growth in colon cancer and sarcoma mouse models. Targeting adenosine production by CD73 blockade combined with PD-1 or CTLA-4 inhibition promotes tumor regression in a manner dependent on IFN-γ and CD8+ T cells (203).

LAG-3 expression has been reported to impair the metabolic shift of mouse T cells toward glycolysis, and its deficiency has been shown to augment metabolic fitness by promoting oxygen consumption and glycolytic activity. Conversely, LAG-3 expression in CD4+ T cells impairs mitochondrial biogenesis by altering the AMPK/Sirt-1 pathway, resulting in a diminished proliferation of CD4+ T cells and high IL-7 dependence (238). Similarly, LAG-3 deletion in mouse BMDCs has been reported to increase their glycolytic activity and FAO, which can be counteracted by IL-10. This enhanced metabolic switch in LAG3^-/-BMDCs increases their ability to induce Th1-like responses, promoting antitumor immunity (239).

In Jurkat T cells, TIM-3 overexpression has been reported to diminish glucose consumption and lactate production. TIM-3 expression downregulates Glut-1, while TIM-3 knockdown has the opposite effect. Interestingly, TIM-3 did not affect glutamine consumption, glutamate release, mitochondrial mass, ROS production, or membrane potential (240). Conversely, in Tregs, TIM-3 induces a metabolic shift from OXPHOS toward glycolysis while decreasing mitochondrial mass and membrane potential. TIM-3 expression in Tregs promotes tumor progression and exhaustion of CD8+ T cells enhancing their suppressive activity and IL-10 production (241).

Regarding myeloid cells, Gal-9 or antibody-mediated TIM-3 stimulation in THP-1 cells has been reported to induce mTOR phosphorylation, HIF-1α expression, as well as enhanced glycolytic activity and VEGF secretion by activating the PLC-1/PI3K/mTOR signaling pathway (242). Conversely, stimulation with a TIM-3 agonist in a mouse macrophage cell line has been reported to inhibit glucose uptake and lactate production by inhibiting HK2 expression, resulting in diminished production of TNF-α and IL-1β (243). As mentioned earlier, macrophages depend on glycolysis to differentiate into the antitumoral M1 phenotype, and TIM-3 expression may prevent M1 differentiation and promote the pro-tumoral M2 phenotype.

In CD8+ T cells, the downregulation of GLUT1, HK1, HK2, GAPDH, PKM2, and HIF-1α metabolism-associated genes correlates with the expression of TIGIT. In addition, CD8+ T cells expressing TIGIT show reduced activation of the Akt/mTOR signaling pathway. Co-culture of gastric cancer cells with CD8+ T cells induces TIGIT expression and metabolic impairment, while the blockade of the TIGIT/CD155 axis restores normal metabolic functions in T cells and promotes antitumor immune response (244). Similarly, T cell dysfunction in colorectal cancer has been related to diminished glucose metabolism since TIGIT expression, and metabolic alterations induced by colorectal cancer cells are restored by antibody-mediated TIGIT blockade (245). Anti-TIGIT monotherapy has shown encouraging results in the treatment of diverse cancers, and combining TIGIT blockade with the inhibition of adenosine production, restores NK cell-mediated AML cell killing, which might enhance treatment efficacy (246).

Once we have analyzed current immunometabolism studies that comprehend the local impact of metabolic reprogramming on the antitumor immune response, it is important to highlight that systemic alterations of nutrients availability may also modify the functions of immune cells and impact the effectiveness of immunotherapies. Aging and obesity are two processes in which the systemic availability of nutrients is modified, and the normal functions of immune cells may be altered in consequence. For instance, aging has been related to changes in blood concentrations of amino acids and lipids (263), while obesity is related to metabolic alterations such as hyperglycemia, dyslipidemia, and insulin resistance (264). In fact, both aging and obesity have been reported to involve a low-grade chronic inflammation that alters the anti-tumor innate and adaptive immune responses (265). Excellent reviews have addressed the metabolic impact of these conditions on immune cells (266, 267).

Aging is a physiological process in which the biological functions of an individual slowly deteriorate with age. Immunosenescence is the term that describes the alterations on the immune system related to aging that impair its ability to respond against pathogens and cancer cells. Alterations on the metabolism and effector functions of multiple immune cells that may alter the antitumor response have been related to aging (266). For instance, a predisposition toward myelopoiesis has been suggested in aged mice, while lymphopoiesis is reduced (268). Regarding the metabolic changes related to immunosenescence, an increased glycolytic metabolism has been reported on monocytes, macrophages, and T cells from elderly mice. This effect is mediated by ROS accumulation that drives HIF-1α and NF-κB activation (269, 270). In addition, in adipose tissue macrophages from elderly mice it has been shown a decrease in FAO, driven by decreased PPARγ expression, which promotes an increased secretion of proinflammatory factors (271).

Systemic changes in the availability and metabolism of amino acids have also been related with aging, which may impair the antitumor immune responses as discussed above. On the one hand, the concentration of glutamine is reduced with ageing, while accumulation of ROS impairs the activity of glutamine synthetase (272).On the other hand, ageing increases IDO activity, which is reflected in increased kynurenine and reduced tryptophan concentrations in elderly (273). Additionally, the expression of the asparagine transporter SLC7A2 has been reported to decrease in macrophages from elderly mice (274).

Similarly, obesity-induced inflammation has been regarded as an important risk factor for cancer development, and secondary conditions such as insulin resistance, hyperglycemia and dyslipidemia have been related to tumor growth (264). Paradoxically, obesity has been related to enhanced response to ICB in tumor-bearing mice and cancer patients (275).

At the metabolic level, the two main adipokines altered in obesity display opposing roles in the activation of T cells. On the one hand, leptin is necessary for normal T cell proliferation, glycolytic metabolism, and production of IFN-γ, and IL-2 (276), and it has been reported to promote M1 polarization on macrophages, reduce MDSCs, and increase the effectiveness of ICB (277). On the other hand, adiponectin has been reported to impair the glycolytic metabolism of Th1 and Th17 cells (278). In addition, a mouse model revealed that diet-induced dyslipidemia impairs mTOR1 signaling in Tregs, which results in reduced glycolytic metabolism and increased FAO (279). Moreover, high fat diet consumption has been related to induction of an exhausted phenotype on T cells from the white adipose tissue, and overexpression of the IC BTLA (280).

At the TME, high fat diet has been reported to alter the expression of activation markers on T cell, probably related to a reduction on GLUT1 expression, while promoting fatty acid metabolism on tumor cells (281). This effect may be due to adipocyte derived leptin that impairs glycolytic metabolism and promotes FAO on TILs through activation of STAT3. In fact, tumor-infiltrating CD8+ T cells from obese breast cancer patients display lower expression of granzyme B compared to T cells from lean patients (282).