Nutrient deprivation and hypoxia alter T cell immune checkpoint expression: potential impact for immunotherapy

Abstract

Aim Use of immune checkpoint blockade to enhance T cell-mediated immunity within the hostile tumour microenvironment (TME) is an attractive approach in oesophageal adenocarcinoma (OAC). This study explored the effects of the hostile TME, including nutrient deprivation and hypoxia, on immune checkpoint (IC) expression and T cell phenotypes, and the potential use of nivolumab to enhance T cell function under such conditions. Methods and Results ICs were upregulated on stromal immune cells within the tumour including PD-L2, CTLA-4 and TIGIT. OAC patient-derived PBMCs co-cultured with OE33 OAC cells upregulated LAG-3 and downregulated the co-stimulatory marker CD27 on T cells, highlighting the direct immunosuppressive effects of tumour cells on T cells. Hypoxia and nutrient deprivation altered the secretome of OAC patient-derived PBMCs, which induced upregulation of PD-L1 and PD-L2 on OE33 OAC cells thus enhancing an immune-resistant phenotype. Importantly, culturing OAC patient-derived PBMCs under dual hypoxia and glucose deprivation, reflective of the conditions within the hostile TME, upregulated an array of ICs on the surface of T cells including PD-1, CTLA-4, A2aR, PD-L1 and PD-L2 and decreased expression of IFN-γ by T cells. Addition of nivolumab under these hostile conditions decreased the production of pro-tumorigenic cytokine IL-10. Conclusion Collectively, these findings highlight the immunosuppressive crosstalk between tumour cells and T cells within the OAC TME. The ability of nivolumab to suppress pro-tumorigenic T cell phenotypes within the hostile TME supports a rationale for the use of immune checkpoint blockade to promote anti-tumour immunity in OAC. Graphical abstract Study schematic: (A) IC expression profiles were assessed on CD45+ cells in peripheral whole blood and infiltrating tumour tissue from OAC patients in the treatment-naïve setting. (B) PBMCs were isolated from OAC patients and expanded ex vivo for 5 days using anti-CD3/28 + IL-2 T cell activation protocol and then co-cultured for 48 h with OE33 cells. T cell phenotypes were then assessed by flow cytometry. (C) PBMCs were isolated from OAC patients and expanded ex vivo for 5 days using anti-CD3/28 + IL-2 T cell activation protocol and then further cultured under conditions of nutrient deprivation or hypoxia for 48 h and T cell phenotypes were then assessed by flow cytometry. Key findings: (A) TIGIT, CTLA-4 and PD-L2 were upregulated on CD45+ immune cells and CTLA-4 expression on CD45+ cells correlated with a subsequent decreased response to neoadjuvant regimen. (B) Following a 48 h co-culture with OE33 cells, T cells upregulated LAG-3 and decreased CD27 co-stimulatory marker. (C) Nutrient deprivation and hypoxia upregulated a range of ICs on T cells and decreased IFN-γ production by T cells. Nivolumab decreased IL-10 production by T cells under nutrient deprivation-hypoxic conditions.

Multifaceted roles of PD‐1 in tumorigenesis: From immune checkpoint to tumor cell‐intrinsic function

“…Additionally, PD‐1 expression is intricately regulated by factors including cytokine signaling, Costimulatory and coinhibitory receptors, and elements within the tumor microenvironment (TME) (Table 2). Interestingly, while hypoxia alone did not affect PD‐1 levels on T cells in triple negative breast cancer, 58 combined oxygen and glucose deprivation significantly increased PD‐1 expression in oesophageal adenocarcinoma 55 . This pathway enhanced tumor cell survival and chemoresistance.…”

“…Interestingly, while hypoxia alone did not affect PD-1 levels on T cells in triple negative breast cancer, 58 combined oxygen and glucose deprivation significantly increased PD-1 expression in oesophageal adenocarcinoma. 55 This pathway enhanced tumor cell survival and chemoresistance. Conversely, in CD8+ T cells lactic acid accumulation has an inhibitory effect, repressing PD-1 transcription.…”

The future of combination immunotherapy in oesophageal adenocarcinoma

“…These well-characterized functions of immune checkpoint proteins have formed the central dogma for many years (13). Following T cell activation, inhibitory immune checkpoint receptors are upregulated on the surface of T cells (14). Due to chronic antigen stimulation and the inhospitable hypoxic and nutrient deprived tumor microenvironment several inhibitory immune checkpoint receptors such as PD-1, TIM-3, LAG-3 and CTLA-4 are upregulated on T cells that cooperatively inhibit T cell proliferation, cytokine production and function creating an exhausted T cell phenotype (15, 16).…”

Simultaneous targeting of Tim3 and A2a receptors modulates MSLN-CAR T cell antitumor function in a human cervical tumor xenograft model

“…Although the use of CAR T cells as a type of adoptive T cell therapy has achieved unprecedented success in hematologic malignancy, however, its success in solid tumors has been marginal more likely due to the presence of a hostile tumor microenvironment (TME). Several factors, including immunosuppressive cells and molecules (such as Tregs; regulatory T cells, MDSCs; Myeloid-derived suppressor cells, and TGF-b; Transforming growth factor-b), nutrient deprivation and hypoxia in the TME, induce the expression of inhibitory receptors such as Tim3, PD-1 (Programmed cell death protein 1), LAG3 (Lymphocyte-activation gene 3), TIGIT (T cell immunoreceptor with Ig and ITIM domains) and A2aR on the surface of T cells where they home into such a harsh microenvironment (2,3). Hypoxia in the TME activates the HIF-1a (Hypoxia inducible factor-1a) pathway, which upregulates the expression of the ectonucleotidases CD39 and CD73 on tumor cells and immune cells.…”