Tumor infiltrated CD8⁺ T cells (also called cytotoxic T lymphocytes; CTLs) are immune effector cells that can kill cancer cells using perforin and granzyme molecules. Analysis in peripheral blood has revealed significantly decreased circulating CTLs and lower perforin expression levels in pancreatic cancer patients compared with healthy controls (15). Immunohistochemistry on pancreatic cancer samples showed a higher cellular infiltration compared to normal pancreas and survival studies have shown that higher levels of tumor infiltrating CD4⁺ and CD8⁺ T cells are associated with longer survival (16).

Shortly after T-cell activation, cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) is translocated to the plasma membrane. This co-receptor molecule binds to B7 ligand with a higher affinity than does the co-receptor CD28, leading to inhibition of the T-cell activation. Furthermore, PDAC cells express PD-L1, which binds to PD1 expressed on activated T cells (17). Interaction between these molecules leads to T-cell anergy or death and consequently promotes tumor progression (18).

The restoration of exhausted CD8⁺ T cells and recovery of their effector role represent one of the main therapeutic objectives toward the destruction of cancer cells.

CD4⁺ T cells (T helper cells) play an important role in the immune response by secreting several cytokines that modulate the function of B and CD8⁺ T cells. Their peripheral blood levels are reduced in patients with pancreatic cancer compared to healthy controls (19). Naive CD4⁺ T cells can differentiate into the following two main subsets: Th1 cells, which support cell-mediated immune responses by secreting IL-2 and IFN-γ (activate macrophages and CD8⁺ T-cell proliferation), and Th2 cells, which induce humoral immune responses by secreting IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 (stimulate B-cell proliferation and induce B-cell antibody class switching) (20). In PDAC cancer, the shift from Th1 to Th2 cells is correlated to reduced survival.

Natural killer cells are cytotoxic lymphocytes of the innate immune system. Activation of these cells is determined by the balance between activating and inhibitory receptor stimulation. Analysis of peripheral blood mononuclear cells has revealed reduced levels of NK cells in patients with PDAC compared to healthy controls (19). Patients were also found to have significantly lower levels of two activating receptors (CD226 and CD96) on their circulating NK cells compared to healthy controls (21). The decrease in the level of activating receptors on NK cells could indicate dysfunction of these cells and may represent a factor promoting PDAC progression. These data suggest that reactivation of NK cells via these activator receptors could be a new target for cancer immunotherapy.

Monocytes recruited to the tumor site can differentiate into TAMs. In the majority of solid tumors such as in PDAC, TAMs represent the most abundant immune population in the TME. Tumor cells express many factors including CCL2 (under hypoxic conditions), M-CSF or GM-CSF, IL-10, TGF-β, and IL-6, all of which favor the recruitment and generation of TAMs (22). At early cancer stages, TAMs can be polarized into an anti- (M1) or pro- (M2) tumor phenotype (23, 24), whereas at advanced stages, they are mainly present as the M2 subtype (CD14⁺ CD163⁺) (23) and their presence is associated with bad prognosis in PDAC (25, 26). As reported by Cui et al, TAMs play large roles in the promotion of tumor growth and development of an immunosuppressive microenvironment. They do this by secreting angiogenic factors (IL-6, VEGF, and MMP), as well as immunosuppressive factors (IL-10 TGF-β), that promote the generation of an immunosuppressive cell population and inhibit effector T cells, and also other factors such as chemokines and cytokines that promote metastasis and epithelial–mesenchymal transition (27). Therefore, TAMs represent an important therapeutic target for inhibition at the level of their activation, recruitment, and survival or for the reprogramming of polarization (27, 28). Shibuya et al. showed that multimodal neoadjuvant chemotherapy could decrease the number of immunosuppressive infiltration cells such as myeloid cells (29).

Analogous to the M1 and M2 dichotomy for TAMs, TANs exhibit a pro-tumor N2 profile with pro-tumor function through the influence of TGF-β (30). Furthermore, pancreatic cancer cells attract neutrophils through the secretion of chemokines, such as CXCL8 and CXCL16 (31). Few studies have evaluated the function of TANs; however, those with the N2 profile have been shown to produce matrix metallopeptidases including MMP-8, MMP-9, neutrophil elastase, reactive oxygen species (ROS), and VEGF and some inflammatory cytokines including TNFα and GM-CSF, which promote tumor and immune cell proliferation (metastatic potential) and favor chronic inflammation (31, 32).

Myeloid-derived suppressor cells are a population of cells defined by their immature state, myeloid origin and capacity to suppress the immune response. Through factors in the TME, they can acquire phenotypic and functional characteristics of TAMs and TANs and are thus called mononuclear (Mo-) or granulocytic (G-) MDSCs (14, 31). They are strongly immunosuppressive by their ability to inhibit T-cell proliferation, IFNγ production, and effector T-cell function and to favor Treg generation through the secretion of ROS, Arg1, and iNOS (33, 34). They also promote tumor growth by VEGF and MMP9 secretions. High concentration of MDSCs in the peripheral blood is associated with poor prognosis in PDAC (35).

Mast cells can release cytotoxic granules and have the capacity to recruit other immune cell populations by chemokine secretion. They have been found in significantly higher numbers in PDAC compared to those in normal pancreatic tissue (36), where they support tumor growth and angiogenesis and inhibit anti-tumor immunity. The MCs accumulate within the TME, along with macrophages, through the action of tumor-derived chemoattractants such as MCP-1 and RANTE and by tumor-secreted VEGF and FGF (37). MC accumulation correlates with higher tumor grade, diminished survival, and lymph node metastasis.

In vitro, MCs induce PDAC cell proliferation and migration (angiogenesis and metastasis) by secreting factors including secretin, VEGF, and IL-8 and tumor growth factors including PDGF and proteases (38, 39).

In PDAC and solid tumors, CD4⁺ CD25⁺ Foxp3⁺ Tregs are strongly associated with poor prognosis and inversely correlated to the presence of CD8⁺ T cells, with more advanced disease presentation, a lower chance of surgical resection and a poorer survival after resection (36, 40). Patients with PDAC have increased numbers of Tregs. They produce IL-10 and TGF-β and express CTLA-4; thus, they inhibit effector T cells and induce M2 profile TAMs and N2 profile TANs (41).

The role of Th17 cells in cancer is highly controversial. Their function seems to depend on the type of cancer, the tumor stage, and the localization (42). In PDAC, while some evidence favors a higher level of Th17 cells in advanced stage tumors, other data in a murine model of pancreatic cancer support Th17 induction increasing survival (43, 44). This inconsistency can be explained by the plasticity of Th17 cells and their ability to promote smoldering inflammation at early stages (45–47). Indeed, Th17 cells are polarized on the one hand by IL-6, IL-23, and Il-1β with pro-inflammatory functions (impact on smoldering inflammation and recruitment of inflammatory immune population) and on the other hand by TGF-β, which induces anti-inflammatory functions (impact on tumor growth, immunosuppressive microenvironment, and angiogenesis) (46, 48). Furthermore, the shift of Th17 to Treg, explained by the plasticity of these cells, is important. In PDAC, patients were shown to exhibit Th17/Treg disorders with higher Treg and lower Th17 cells (49).

Th2 cells (GATA-3⁺ IL-13⁺ IL-4⁺), in contrast to Th1 cells, are anti-inflammatory T cells. In PDAC, the TME and CAFs were shown to induce the polarization of Th2 cells by IL-13-mediated dendritic cell secretion in vitro (50). The Th2 cells produce IL-13 and IL-4 and thereby induce M2 macrophages or TAMs, which further increases the anti-inflammatory TME. Furthermore, via an amplification loop and T-cell plasticity, Th2 cells inhibit Th1-cell polarization and induce themselves. In tumor tissue, Th2 T-cell infiltrates are a predictive marker of poor prognosis, confirmed by the shift of Th1 to Th2 cells within the TME (13).

ɣδ T cells are “unconventional” T cells. Unlike αβ T cells, these lymphocytes do not require antigen processing and major histocompatibility complex presentation of peptide epitopes. In contrast to current dogma, one study using a mouse model and human samples showed that ɣδ T cells have no anti-cancer properties in pancreatic cancer (51). In vivo deletion of ɣδ T cells using a neutralizing antibody resulted in a robust protection against oncogenic progression. The analysis also revealed that infiltrating ɣδ T cells express high levels of T-cell exhaustion ligands (PD-L1 and Galectin-9) and may block the immune response by immune checkpoint inhibition. Altogether these data suggest that, in PDAC, ɣδ T cells promote pancreatic oncogenesis and that their deletion or reactivation could be a novel therapeutic strategy. Surprisingly, the key regulator of Vγ9Vδ2 function BTN3A1 was found to act as a critical marker of PDAC prognosis and is detectable either by IHC or by its soluble receptor sBTN3A1 (52).

Pancreatic cancer stroma is composed of several main components: CAFs, immune cells and associated cytokines, adipocytes, and endothelial cells. These stromal components are involved in the production of highly toxic conditions including low pH and low oxygen environment (hypoxia). To define the hypoxic status of pancreatic cancer, one study measured tissue oxygenation of the tumor and normal adjacent pancreas during pancreaticoduodenectomy surgery (53). Results of this study showed that PDAC are highly hypoxic compared to normal pancreas.

Cancer cells under hypoxic conditions are more resistant to radiation and chemotherapy (54, 55). This ability to survive is mainly conferred by the hypoxia-inducible pathway involving transcription factors able to induce the expression of several genes controlling cell survival, glycolysis, and other cellular metabolism events. Recent evidence supports the hypothesis of hypoxia being one cause of radioresistance. Indeed, Hajj et al. showed that radiation therapy in combination with TH-302 (a hypoxia-activated pro-drug) allowed tumor growth delay in an orthotopic model of PDAC by comparison with the outcome following these two treatments given separately (56). This TH-302 compound is currently being tested in a pancreatic cancer Phase I clinical trial in combination with Nab-paclitaxel and gemcitabine.

Despite the high levels of hypoxia found in pancreatic cancer, which would be expected to promote angiogenesis, PDAC remains poorly vascularized. This poor vascularization limits blood flow to the tumor and is associated with prominent desmoplasia, which prevents drug delivery and could impede the immune response (57). This hypoxia seems to impact on several escape mechanisms and could therefore be a relevant target for next generation therapeutic options.

In non-inflamed pancreas, PSCs are resident cells involved in maintaining tissue homeostasis by regulating extracellular matrix turnover (58). During pancreatic injury, quiescent PSCs are activated and transform into myofibroblast-like cells. These activated PSCs secrete extracellular matrix proteins, which generate fibrosis and limit drug delivery to cancer cells (59). Inordinate secretion of extracellular matrix proteins is also linked to hypoxia (see paragraph above) and promotes cancer cell proliferation.

Pancreatic stellate cells can also modulate immune cells via their secretion of cytokines. Indeed, secretion of CXCL12 by activated PSCs reduces the migration of CD8⁺ and CD4⁺ T cells, NK cells, and Tregs to the juxtatumoral compartment within close proximity to the tumor (60). Another study showed that PSCs secreted Galectin-1, which mediated immunosuppression of CD8⁺ T cells and promoted T-cell apoptosis (61). All these data suggest that PSCs could be a good target to enhance immunotherapy for PDAC.