Introduction
Oesophageal adenocarcinoma (OAC) is the predominant subtype of oesophageal cancer in the Western world and its incidence is continuing to increase rapidly (Pera et al.
2005). The cellular and physiological composition of the tumour microenvironment (TME) plays a pivotal role in the development and progression of OAC, including dictating the response to the current standards of care (Davern et al.
2021a). The TME imposes profound metabolic restrictions on anti-tumour T cells, and understanding these insights is important for informing immunotherapeutic anti-cancer strategies (DePeaux and Delgoffe
2021). Therapeutic approaches such as those targeting metabolic restrictions, including low glucose levels and hypoxia have shown promise as combination therapies for different types of cancer (Guo et al.
2019). Both nutrient deprivation and hypoxia have a profound impact on the cellular composition of the TME—subsequently promoting or hindering anti-tumour immune responses. Hypoxia profoundly alters immune cell phenotypes, in particular the myeloid compartment, comprising of macrophages and myeloid-derived suppressor cells, which cooperatively promote immune evasion, tumour cell survival and metastatic dissemination (King et al.
2021). Tumour hypoxia promotes the recruitment of regulatory T cells through induction of the chemokine CC-chemokine ligand 28 which, in turn, promotes tumour tolerance and angiogenesis (Facciabene et al.
2011). In addition, cancer cells grow rapidly, outcompeting anti-tumour immune cells for essential nutrients and producing metabolic by-products, such as lactate which are toxic to immune cells, resulting in a both a nutrient starved and acidic hostile environment for anti-tumour immune cells (Singer et al.
2018).
The TME is often described as a glucose deprived environment, attributed to a poorly vascularised TME and cancer cells sequestering glucose for glycolysis to facilitate tumour progression (Park et al.
2004). These distinct metabolic pathways in tumour cells cause functional impairment in immune cells and contribute to immune evasion (Park et al.
2004). Glucose is also an essential nutrient for the metabolic demands and function of anti-tumour immune cells, particularly effector T cells (Yin et al.
2019). Glucose is an essential nutrient which plays an important role during the early stages of T cell activation in regulating T cell differentiation and maintaining activation states (Yin et al.
2019). T cell activation involves a dramatic increase in nutrient uptake and depletion of glucose or glutamine in cell culture media during the early stages of T cell activation inhibits T cell expansion, cytokine production and suppresses pro-inflammatory T cell differentiation (MacPherson et al.
2018). Conversely, regulatory T cells thrive in a glucose-depleted environment, as these cell types predominantly rely on fatty acid oxidation to fulfil their energy demands (Villa et al.
2021).
Metabolic reprogramming in T cells during their activation and differentiation have led to an emerging concept of “immunometabolism” (Giannone
2020). Considering the recent success of cancer immunotherapy in the treatment of several cancer types, increasing research efforts are elucidating alterations in metabolic profiles of cancer and immune cells in the setting of cancer progression and immunotherapy (Mockler et al.
2014). Therefore, immunometabolism is a key factor in regulating immune responses within the tumour. Immune checkpoint blockade (ICB), in particular programmed death-1 (PD-1) blockade, promotes the metabolic fitness of exhausted immune cells, reinvigorating an exhausted phenotype and enhancing anti-tumour effector T cell responses (Kazemi et al.
2021). However, a substantial number of OAC patients possess tumours refractory to ICB (Power et al.
2020). It is thought that concurrent targeting of immune checkpoints (ICs) and targeting immunometabolism pathways may have a greater effect in restoring effector functions to exhausted T cells (Kazemi et al.
2021). However, more in-depth research is required to study how the nutrient depleted and hypoxic TME might shape immune T cell function and alter responses to ICB in the context of OAC.
Therefore, this study investigates the direct effect of nutrient deprivation (serum deprivation and glucose deprivation) and hypoxia on the function of OAC patient-derived T cells, in particular the expression profile of ICs. Upregulation of inhibitory IC ligands on tumour cells and stromal cells that bind to inhibitory receptors on immune cells is key mechanism of immune suppression and represents a significant barrier for induction of effective anti-tumour immune responses (Toor et al.
2020). The effect of secreted mediators from OAC patient-derived PBMCs cultured under nutrient deprivation and hypoxia on IC expression profiles of OAC tumour cells is also assessed. This will provide a greater insight into how the crosstalk between immune cells and tumour cells under the physiological conditions of the TME alters IC expression profiles of OAC cells. Importantly, the ability of nivolumab to promote anti-tumour T cell-mediated immunity under conditions reflective of the hostile TME is also explored. Collectively, these findings will help guide the clinical development of rational immunotherapeutic strategies to improve immune responses within the inhospitable TME for treating OAC patients.
Methods
Ethical approval
Ethical approval was granted from the St. James’s Hospital/AMNCH Ethical Review Board. All samples were collected with prior informed written consent for sample and data acquisition from patients attending St. James’s Hospital or from healthy donors. This study was carried out in accordance with the World Medical Association’s Declaration of Helsinki guidelines on medical research involving human subjects. Patient samples were pseudonymised in line with GDPR and data protection policies to protect the privacy and rights of the patients.
Specimen collection
All patients involved in this study were enrolled from 2018 to 2020. Treatment-naïve whole blood and tumour tissue biopsies were obtained from OAC patients undergoing endoscopy at St. James’s Hospital at time of diagnosis prior to initiation of chemotherapy or radiotherapy. The group consisted of 16 males and 6 females, with an average age of 66.4 years. The patient demographics are detailed in Table
1.
Table 1
Patient demographic
Age (years) | 66.4 |
Sex ratio (M:F) | 16:6 |
Diagnosis (no. patients) |
OGJ | 11 |
OAC | 11 |
Clinical tumour stage (no. patients) |
T0 | 0 |
T1 | 2 |
T2 | 6 |
T3 | 14 |
T4 | 0 |
Clinical nodal status (no. patients) |
Positive | 11 |
Negative | 11 |
OAC tumour tissue digestion
Biopsies were enzymatically digested to perform OAC cell phenotyping as previously described in Davern et al. (
2021b). Briefly, tissue was minced using a scalpel and digested in collagenase solution (2 mg/ml of collagenase type IV (Sigma) in Hanks Balanced Salt Solution (GE healthcare) supplemented with 4% (v/v) foetal bovine serum) at 37 °C and 1,500 rpm on an orbital shaker. Tissue was filtered and washed with FACs buffer (PBS containing 1% foetal bovine serum and 0.01% sodium azide). Cells were then stained for flow cytometry.
Cell culture
Treatment-naïve OAC donor PBMCs (n = 8) were isolated from whole blood using Ficoll-Paque (GE healthcare) density gradient centrifugation and expanded with plate bound anti-CD3 (10 μg/ml, Biolegend, USA), anti-CD28 (10 μg/ml, Ancell, USA) and recombinant human IL-2 (Immunotools, Germany) for 5 days, followed by 24 h culture of PBMCs in cRPMI, serum-free RPMI, glucose-free RPMI or dual glucose-free and serum-free RPMI under normoxic or hypoxic conditions (0.5% O2) in the absence or presence of nivolumab (10 μg/ml) at 37 °C 5% CO2. Following this 6-day activation up to 85–90% of the lymphocyte population comprise of CD3+ T cells.
The OE33 cell line was established from a poorly differentiated stage IIA adenocarcinoma of the lower oesophagus (Barrett’s metaplasia) of a 73-year-old female patient and was purchased from the European Collection of Cell Cultures and grown in RPMI 1640 medium with 2 mM L-glutamine (Gibco) and supplemented with 1% (v/v) penicillin–streptomycin ((P/S) 50 U/ml penicillin 100 μg/ml streptomycin) and 10% (v/v) foetal bovine serum (Gibco) and maintained in a humidified chamber at 37 °C 5% CO2. Cell lines were tested regularly to ensure mycoplasma negativity.
Nutrient deprivation and hypoxia treatment and co-culture of OAC donor PBMCs with OE33 cells
5-day expanded PBMCs were cultured for an additional 24 h in complete RPMI (cRPMI, 10% FBS, 1% P/S), serum-free RPMI (0% FBS, 1% P/S), glucose-free RPMI (Gibco, 10% FBS, 1% P/S), dual glucose-free and serum deprived RPMI (Gibco, 0% FBS, 1% P/S) under normoxic conditions (37 °C, 5% CO2, 21% atmospheric O2) or hypoxic conditions (37 °C, 5% CO2, 0.5% O2) using the H35 Don Whitley hypoxia station. PBMCs were then harvested for flow cytometry staining.
OE33 cells were seeded at a density of 1 × 104 cells/100 μl in cRPMI in a flat-bottomed 96-well plate and left to adhere overnight. Following 24 h, 5-day expanded OAC patient-derived PBMCs were cultured alone or co-cultured with OE33 cells at a ratio of 5:1 (PBMCs:OE33 cells) for 48 h at 37 °C 5% CO2. PBMCs were then harvested for flow cytometry staining.
Generation of OAC donor lymphocyte supernatant and co-culture with OE33 cells
Treatment-naïve OAC donor PBMCs were isolated from whole blood using Ficoll-Pacque (GE healthcare) and density gradient centrifugation, expanded for 5 days (using above anti-CD3/28 and IL-2 expansion protocol) and cultured for an additional 24 h under nutrient deprivation ± normoxic/hypoxic conditions (as described above). The supernatant was harvested and stored at − 80 °C for later use.
OE33 cells were seeded at a density of 1 × 104 cells/100 μl in cRPMI in a flat-bottomed 96-well plate and left to adhere overnight. The media was replaced with 100 μl of cRPMI or 100 μl of 1 in 2 diluted supernatant that was collected from OAC donor PBMCs that had been cultured under hypoxia ± nutrient deprivation and cultured for 24 h at 37 °C 5% CO2.
Flow cytometry staining
Fluorochrome-conjugated antibodies were added to 100 µl blood at pre-optimized concentrations and incubated for 15 min at room temperature in the dark. Red cells were lysed using red blood cell lysing solution (Biolegend, USA), according to manufacturer’s recommendations and cells were washed twice with FACs buffer and stained with zombie aqua viability dye (Biolegend, USA). Cells were fixed for 15 min in 1% paraformaldehyde solution (Santa Cruz Biotechnology, USA) prior to flow cytometric analysis.
Tumour tissue biopsies, healthy donor PBMCs or OAC donor PBMCs were stained with zombie aqua viability (Biolegend, USA) dye. Antibodies used for staining included ICOS-PE-eFluor610, LAG-3-FITC, CD160-PerCPCy5.5, PE-1-PE/Cy7, TIGIT-PE/Cy7, CD45RA-PE/Cy7, CD45RO-BV510, CD3-APC, CD3-PerCP, CD4-BV510, CD4-APC (Biolegend, USA), CD69-PE, CD62L-FITC, CD8-BV421 (BD Biosciences, USA), CD27-APC-eFluor780 (eBioscience, USA), TIM-3-AF647, CTLA-4-PE/Cy5, KLRG-1-APC, PD-L1-FITC, PD-L2-PE (BD Bioscience, USA), A2aR-PE (Bio-techne, USA). PBMCs were resuspended in FACs buffer and acquired using BD FACs CANTO II (BD Biosciences) using Diva software and analysed using FlowJo v10 software (TreeStar Inc.). Gating strategy on the lymphocyte population to assess T cell expression profiles of ICs is shown in Figure S1. Gating strategy on the lymphocyte population to assess T cell activation marker expression and T cell differentiation status to differentiate between naïve, central memory, effector memory and terminally differentiated effector memory T cells is shown in Figure S2.,
For intracellular cytokine staining PBMCs were treated with PMA (10 ng/ml) and ionomycin (1 µg/ml) for the last 4 h of the incubation. For the last 3 h of the incubation PBMCs were treated with brefeldin A (10 µg/ml, eBiosciences). Cells were harvested, washed in FACs buffer and intracellular cytokines were assessed using a Fixation/Permeabilisation kit (BD Biosciences), as per manufacturer’s recommendations. Cells were stained with cell surface antibodies (CD8-BV421, CD3-APC or CD3-PerCP, CD4-PerCP, CD4-APC or CD4-BV510 (Biolegend, USA)) washed, permeabilised, and then stained for intracellular cytokines: IFN-γ-BV510, IL-17A-FITC, Granzyme B-PE/Cy7, Perforin-FITC-BV510 (Biolegend, USA) and TNF-α-APC (BD Biosciences, USA). Cells were resuspended in FACs buffer and acquired using BD FACs CANTO II (BD Biosciences). Gating strategy on the lymphocyte population to assess T cell cytokine production is shown in Figure. S3.
Statistical analysis
Data were analysed using GraphPad Prism version 5 (GraphPad Prism, San Diego, CA, USA) software and was expressed as mean ± SEM. Statistical differences between treatments within cancer donors or within healthy donors were analysed using paired non-parametric t test and statistical differences between treatments between healthy donors and cancer donors were analysed using unpaired non-parametric t tests. Statistical significance was determined as p ≤ 0.05. Spearman correlations were performed to analyse correlation data between clinical characteristics and flow data and visualised using the R package ‘corrplot’.
Discussion
It is well known that the TME has profound effects on tumour progression and response to treatments by mediating immune suppression (Munn and Bronte
2016). Several mechanisms within the TME are responsible for this and include cellular components such as M2-like macrophages, neutrophils, myeloid-derived suppressor cells, regulatory T cells that produce immunosuppressive factors such as transforming growth factor-β, IL-10, indoleamine 2,3-dioxygenase, arginase, vascular endothelial growth factor, prostaglandins. Collectively these factors promote pro-tumorigenic processes such as angiogenesis, hypoxia and suppression of anti-tumour immunity, which ultimately facilitates immune escape and tumour progression (Gajewski et al.
2006). Importantly, the TME is infamously characterised as a nutrient depleted milieu as a result of rapidly growing tumour cells, which outcompete anti-tumour immune cells for essential nutrients required for their optimal function (Li et al.
2021). The well-known Warburg effect depicts the metabolic hard-wiring of tumour cells to carry out aerobic glycolysis providing tumour cells with an immediate source of fuel but in parallel contributes to rapid depletion of essential nutrient such as glucose which is essential for effector T cell function (Warburg et al.
1927). Similarly, arginase and indoleamine 2,3-dioxygenase selectively deplete arginine and tryptophan, which are essential amino acid required by effector T cells (Mondanelli et al.
2017). Oxygen is also consumed by rapidly growing tumour cells generating hypoxic ‘pockets’ within the tumour and in conjunction with a nutrient depleted TME these features promote angiogenesis and potential metastatic dissemination (Muz et al.
2015). Pro-angiogenic processes, hypoxia and nutrient depletion have profound effects on the metabolism of not just cancer cells but also stromal and tumour-infiltrating immune cells. Such metabolic reprogramming in immune cells ultimately promotes pro-tumorigenic immune cell phenotypes (Li et al.
2021).
Cham et al. demonstrated that glucose deprivation or inhibition of glycolysis by 2-deoxy-D-glucose inhibited the production of IFN-γ, GM-CSF, cytotoxic granule proteins and cell cycle progression by T cells derived from healthy donors (Cham et al.
2008). Similarly, the findings from this study showed that conditions recapitulating the hostile TME, such as dual glucose and serum deprivation under hypoxic conditions significantly decreased IFN-γ production in OAC patient-derived T cells. In addition to a reduction in IFN-γ production by T cells a decrease in the production of immunosuppressive IL-10 was also observed under nutrient deprivation and hypoxic conditions. This may likely be attributed to the depletion of nutrients which are essential ‘building blocks’ required by T cells to synthesize proteins and subsequent cytokines. Cohen et al., reported that nutrient depletion significantly reduces T cell survival and proliferation and in particular cytokine production (Cohen et al.
2017).
Our results indicate a particularly critical role for glucose in regulating specific effector functions of CD8
+ T cells. Similarly, our study found that glucose deprivation and serum deprivation, which mimics both a glucose and amino acid deprived TME had the greatest effect in altering T cell activation status and cytokine production, highlighting the immunosuppressive effects of nutrient deprivation within the TME. These findings highlight a key role of a glucose and amino acid depleted TME in driving T cell dysfunction, which likely confers resistance to ICB and other immunotherapies in the OAC setting. These findings also strengthen the rationale for implementing therapeutic approaches that target metabolic restrictions, such as nutrient depletion and hypoxia which have shown promise as combination therapies for different types of cancer to improve T cell metabolic fitness and bolster the anti-tumor immune response (Guo et al.
2019). Glucose consumption by antigenic tumours can metabolically restrict T cells, directly dampening their effector function and allowing tumour progression (Chang et al.
2015). ICB therapy may correct this resource imbalance through a direct effect on tumour cells (Chang et al.
2015). In particular, PD-L1 blockade on melanoma and lung cancer cells inhibited glycolysis resulting in an increased availability of glucose in the TME and subsequently promoted anti-tumour T cell function (Kim et al.
2019).
Interestingly, extremely harsh conditions of combined hypoxia with both glucose and serum deprivation significantly decreased the frequency of naive CD8
+ T cells and subsequently increased the frequency of effector memory CD8
+ T cells compared with cells cultured in normoxic cRPMI conditions. It is unclear if these harsh conditions are promoting differentiation of naïve T cells into an effector memory-like state or if these conditions are specifically inducing naïve T cell death and perhaps effector memory-like T cells may be more resilient to these extremely harsh conditions that recapitulate the TME. It has been demonstrated that effector memory-like T cells exhibit high expression levels of anti-apoptotic proteins Bcl-2 (Elyaman et al.
2008) and Mcl-1 (Kim et al.
2016).
Malignant tissue consists not only of tumour cells but also tumour-associated stromal immune cells which are thought to have important roles in tumour growth, disease progression and drug resistance in a context-dependent manner (Yoshihara et al.
2013). An important mechanism by which tumours avoid clearance by the immune system is by inducing the upregulation of inhibitory IC ligands and receptors on tumour cells, immune cells and stromal cells. O’Malley et al. (
2018) demonstrated that the soluble inflammatory TME in colorectal cancer induces upregulation of PD-L1 on the surface of stromal cells. PD-L1 expression was identified on both tumour cells and on the immune stroma of 12% and 42% of gastric adenocarcinoma patients undergoing surgical tumour resection, respectively (Thompson et al.
2017). Although higher levels of PD-L1 expression on the tumour cells or immune stroma was associated with an increase in CD8
+ T cell infiltration, reflecting an ongoing anti-tumour immune response, these patients had reduced progression-free and overall survival. This suggests that PD-L1 plays an important role in mediating immune escape and dampening anti-tumour CD8
+ effector T cell function (Thompson et al.
2017). Tumour cells upregulate IC ligands on their surface, which negatively regulate T cell activation pathways involved in physiological immune responses against specific antigens, representing significant barriers for induction of effective anti-tumour immune responses. However, the majority of studies to date in OAC focus on assessing the effect of cancer cells on immune cell function and T cell IC expression profiles, while the effect of immune cells on the IC expression profile of cancer cells is often overlooked. Importantly, our study demonstrated that the secreted factors from OAC patient-derived PBMCs, cultured in full nutrient conditions, significantly upregulated PD-L1 and PD-L2 on the surface of OE33 OAC cells in vitro. Similarly, the secretome from OAC patient-derived PBMCs cultured under nutrient deprivation and hypoxia upregulated PD-L1 and PD-L2 on the surface of OE33 cells in vitro. These data suggest that the PD-1 axis may play important role in dampening effector T cell function in OAC by binding to PD-1 on tumour-infiltrating antigen-specific T cells. These findings reaffirm the rationale for administering PD-1 ICB to OAC patients and indicates that the PD-1 axis may play an important role in immune escape. Chen
et al., performed a meta-analysis for clinical trials testing the efficacy of anti-PD-1 and anti-PD-L1 ICBs in advanced gastric cancers and oesophagogastric cancers, which demonstrated that the addition of ICBs to the second- and third-line settings for treating GOCs improves some, but not all survival endpoints (Chen et al.
2019). The objective response rates were 9.9% and 12.0%, respectively, and the disease control ratios were 33.3% and 34.7%, respectively (Chen et al.
2019). The median progression-free survival (mPFS) was 1.6 months for both ICBs and the median overall survival was 6.0 and 5.4 months, respectively (Chen et al.
2019). Impressive findings from the CheckMate 577 trial demonstrated a substantial therapeutic benefit for administering nivolumab to OAC patients in the adjuvant setting in patients who had residual disease post-surgery in the first-line setting. A doubling in progression-free survival was observed between the nivolumab arm vs. the placebo arm (22 vs. 11 months) (Kelly et al.
2021).
In this study, several ICs were significantly upregulated on the surface of OAC patient-derived T cells following dual glucose deprivation and hypoxia treatment, including PD-1, PD-L1, PD-L2 and CTLA-4 ICs. IC intrinsic signalling in T cells has profound effects on T cell metabolism, PD-1 intrinsic signalling in T cells inhibits glycolysis and promotes lipolysis and fatty acid oxidation (Patsoukis et al.
2015), similarly, CTLA-4 intrinsic signalling in T cells inhibits glycolysis (Patsoukis et al.
2015). Glycolysis is essential for effector T cell function; therefore, upregulation of PD-1 and CTLA-4 could be detrimental to anti-tumour immunity and may reflect the skewing of T cells toward an altered phenotype facilitated by PD-1 and CTLA-4 metabolic reprogramming. However, a fatty acid oxidative phenotype that could be promoted by PD-1 and CTLA-4 signalling is utilised by both regulatory T cells (Raud et al.
2018) and tissue resident memory T cells, which are known for their anti-tumour functions (Lin, et al.
2020). CTLA-4 expression on regulatory T cells plays a pivotal role in hindering anti-tumour immunity by promoting regulatory T cell function, which suppresses antigen-presenting cells by depleting immune stimulating cytokines, producing immunosuppressive cytokines and constitutively expressing CTLA-4 (Sobhani
2021). Preclinical murine models have demonstrated that CTLA-4 blockade promotes cancer regression by increasing the frequency of effector T cells within the TME and selectively depleting intra-tumoral regulatory T cells via an Fc-dependent mechanism (Sharma et al.
1233).
In contrast, certain conditions also decreased specific ICs, dual hypoxia and serum deprivation significantly decreased LAG-3, A2aR and CD160 on the surface of CD8+ T cells compared with cells cultured in cRPMI and dual hypoxia and glucose deprivation significantly decreased TIGIT expression on the surface of CD8+ T cells compared with cells cultured in cRPMI. These particular conditions are very harsh and may be a reflection of reduced protein synthesis, which subsequently translated into a reduction in the production and surface expression of IC proteins.
Interestingly, this study demonstrated that nivolumab treatment significantly reduced the production of IL-10 by T cells under glucose deprived hypoxic conditions compared with untreated cells under glucose deprived hypoxic conditions, suggesting that PD-1 blockade may help skew T cells toward an anti-tumour phenotype within the OAC TME. However, nivolumab did not decrease IL-10 production by T cells under complete nutrient conditions, highlighting that nivolumab was more effective at promoting an anti-tumour T cell phenotype under ‘stressful’ glucose deprived and hypoxic conditions which are more reflective of the TME than full nutrient conditions.
Surprisingly, nivolumab significantly decreased the surface expression of CD69 on the surface of T cells under glucose deprived hypoxic conditions compared with untreated cells under glucose deprived hypoxic conditions. CD69 is an important co-stimulatory molecule that sustains T cell activation, proliferation and cytolytic activity (Ma et al.
2017). However, studies have implicated a role for CD69 in promoting exhaustion in tumour-infiltrating T cells perhaps as a result of prolonged T cell activation promoted by CD69 signalling in the T cells (Blackburn et al.
2009). Mita et al. demonstrated in
CD69–/– mice using 4T1-luc2 murine breast cancer models, a significant reduction in tumour growth and metastasis, increased levels of tumour-infiltrating lymphocytes and a significant reduction in T cell exhaustion and enhanced IFN-γ production compared with wild-type controls (Blackburn et al.
2009). Additionally, anti-CD69 monoclonal antibody treatment attenuated the T-cell exhaustion and tumour progression in tumour-bearing mice (Blackburn et al.
2009). Considering our findings in the context of the study by Mita et al. (Blackburn et al.
2009), nivolumab-induced downregulation of CD69 under glucose deprived hypoxic conditions may be a benefit to anti-tumour T cell function. Furthermore, nivolumab has currently been FDA approved for the treatment of OAC and several other IC inhibitors targeting the PD-1 axis and CTLA-4 axis are currently in clinical trials (Davern and Lysaght
2020; Smyth and Thuss-Patience
2018).
However, tumour cells are able to counteract the activity of PD-1 and CTLA-4 ICBs and can commission additional inhibitory pathways via expression of other ICs/ligands within the TME (Lee et al.
2021). Of particular clinical relevance regarding mechanisms for development of acquired resistance to therapeutics targeting the conventional PD-1 and CTLA-4 axes, the A2aR IC receptor was also upregulated on the surface of T cells under nutrient deprivation and hypoxic conditions. Furthermore, co-culturing OAC cells with PBMCs upregulated LAG-3 on the surface of T cells. These novel ICs have been shown to have profound immunosuppressive effects on effector T cells. Therefore, co-blockade of multiple ICs may be a better strategy to enhance effector T cell function in OAC. Clinical trials are ongoing in other cancer types targeting LAG-3 and A2aR (Braun et al.
2021). A2aR elicits profound immunosuppressive effects within the TME. Regulatory T cells secrete adenosine within the TME, which potently inhibits production of IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-12, IL-13, TNF-α, granulocyte–macrophage colony-stimulating factor, CCL3, and CCL4 in effector T cells upon binding to its cognate receptor A2aR (Romio et al.
2011). Increased expression of A2aR on tumour infiltrating immune cells correlated with advanced pathological grade, larger tumour size and positive lymph node status in head and neck squamous cell carcinoma (HNSCC) (Ma et al.
2017). Interestingly, the expression of A2AR was found to significantly correlate with HIF-1α, CD73, CD8 and Foxp3. Furthermore, the increased population of CD4
+Foxp3
+ regulatory T cells (Tregs), which partially expressed A2aR, was observed in an immunocompetent mouse model that spontaneously develops HNSCC (Ma et al.
2017). Pharmacological blockade of A2aR by SCH58261 delayed the tumour growth in the HNSCC mouse model and significantly decreased the population of CD4
+Foxp3
+ Tregs and enhanced the anti-tumour response of CD8
+ T cells (Ma et al.
2017). These studies highlight the important role A2aR plays in the TME for promoting tumour progression and the pharmacologic impact of A2aR inhibition for promoting anti-tumour responses.
Similarly, LAG-3 is expressed on the surface of activated T cells and is emerging as an important IC in supressing several arms of the anti-tumour repertoire of immune cells and is garnering a lot of attention as a therapeutic target to reinvigorate exhausted T cells (Blackburn et al.
2009). Grosso et al. (
2007), also demonstrated that LAG-3 knockout adoptively transferred antigen-specific CD8 T cells in mice bearing their cognate antigen, as either a self or a tumour antigen, showed enhanced proliferation and cytokine production. Furthermore, expression of LAG-3 on regulatory CD4 T cells identified a more immunosuppressive phenotype, which subsequently hindered CD8 T cell function in Hodgkin’s lymphoma (Gandhi et al.
2006), melanoma and colorectal cancer (Camisaschi et al.
2010). Collectively, these studies identify a pivotal role for LAG-3 in dampening anti-tumour immunity and highlight that LAG-3 blockade can induce durable responses in pre-clinical models. Of particular clinical relevance for designing combination ICB therapies, co-targeting LAG-3 in combination with PD-1 inhibition is thought to achieve synergistic responses. To date at least 13 agents that target LAG-3 have been developed and are under clinical trials for various cancers (Maruhashi et al.
2020). In phase I/II study evaluating the safety and efficacy of relatlimab (anti-LAG-3) in combination with nivolumab in patients with advanced melanoma that had progressed during previous anti-PD-1 or anti-PD-L1 immunotherapy (NCT0198609), the combination of relatlimab and nivolumab was well tolerated and the objective response rate (ORR) was 11.5% in 61 patients. ORR was at least 3.5-fold higher in patients with LAG-3 expression in at least 1% of tumour-associated immune cells within the tumor margin (
n = 33) than that in the patients with less than 1% LAG-3 expression (
n = 22) (18% and 5%, respectively) (Ascierto et al.
2017). Similarly, LAG525 in combination with anti-PD-1 (spartalizumab) exhibited a durable response in 9.9% of patients (
n = 121) with a variety of solid tumors, including mesothelioma (two of eight patients) and triple-negative breast cancer (two of five patients) in phase I/II study (NCT02460224) (Hong et al.
2018).
Taken together, these pre-clinical and clinical studies investigating A2aR and LAG-3 demonstrate that targeting these novel ICs to enhance anti-tumour immunity is a viable strategy for boosting the efficacy of conventional PD-1 ICBs. However, A2aR and LAG-3 IC pathways remain under-investigated in the context of OAC and acquiring a deeper insight into the expression profiles of ICs in OAC patients and understanding how features of the hostile TME affect IC expression profiles will help guide rational therapeutic design of appropriate immunotherapeutic strategies to overcome features of the immunosuppressive TME in OAC patients.
Overall, this study highlights that multiple ICs are expressed on circulating immune cells and tumour-infiltrating stromal immune cells. In addition, nutrient deprivation had the greatest effect on upregulating several ICs on T cell surfaces, which likely cooperate in tandem to suppress anti-tumour immunity within the TME.