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Abstract

Pancreatic ductal adenocarcinoma (PDAC) is highly aggressive, deadly, and is rarely diagnosed early. Regulatory T cells (Treg) are a multifunctional class of immunosuppressive T cells that help maintain immunologic homeostasis and participate in autoimmune diseases, transplants, and tumors. This cell type mediates immune homeostasis, tolerance, and surveillance and is associated with poor outcomes in PDAC. Tregs remodel the tumor immune microenvironment, mediate tumor immune escape, and promote tumor invasion and metastasis. A promising area of research involves regulating Tregs to reduce their infiltration into tumor tissues. However, the complexity of the immune microenvironment has limited the efficacy of immunotherapy in PDAC. Treg modulation combined with other treatments is emerging. This review summarizes the mechanisms of Tregs activity in tumor immune microenvironments in PDAC and the latest developments in immunotherapy and clinical trials.

Keywords

pancreatic cancer regulatory T cells immunotherapy clinical trial

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a high degree of aggressiveness and a low early detection rate, resulting in poor outcomes. It is the fourth leading cause of cancer-associated death worldwide, with a 5-year overall survival rate < 8% after diagnosis.¹ By 2030, PDAC might become the second leading cause of cancer-associated death.² Currently, the only curative treatment is surgery; however, only 10% of patients are candidates for standard resection. Most patients are diagnosed when PDAC is already metastatic or locally advanced, and both scenarios limit options for surgery.³ First-line chemotherapies for advanced PDAC include albumin paclitaxel with gemcitabine and FOLFIRINOX (irinotecan, leucovorin, oxaliplatin, and fluorouracil).⁴ In recent years, immunotherapy has improved outcomes; these therapies modulate tumor immune microenvironments and circumvent T cell suppression. In PDAC, the tumor microenvironment (TME) is infiltrated by immune suppressor cells, desmoplasia, and fibroblasts, resulting in high degrees of resistance to immunotherapy.⁵ The preinvasive stage of PDAC is characterized by increased Tregs and fewer effector T cells, both of which may enhance tumorigenesis.⁶ Studies demonstrated that Tregs participate in tumor growth and metastasis in lung adenocarcinoma,⁷ basal cell carcinoma,⁸ ovarian cancer,⁹ breast cancer,¹⁰ and colorectal carcinoma.¹¹ This review summarizes Tregs mechanisms in the TME of PDAC and potential immunotherapies involving Tregs.

Definition and classification

Regulatory T cells are multifunctional immunosuppressive cells essential for maintaining immune homeostasis. Tregs modulate immunological events in the TME, mainly by inducing tolerance by inhibiting T cell activation. Tregs abundantly express forkhead box protein P3 (Foxp3), generating CCL5 to recruit intratumoral Tregs in PDAC.¹² Tregs are stable in hostile environments, including low glucose concentrations,¹³ and account for 5–10% of all CD4⁺ T cells.¹⁴ The interleukin-2 receptor αchain (CD25), TNF receptor family, and cytotoxic T cell associated antigen 4 (CTLA-4) are highly expressed on Tregs.¹⁵

Tregs are classified as natural or induced based on anatomical site. The former originate in the thymus and migrate to the periphery; the latter develop in the periphery from naive Foxp3⁻CD4⁺ T cells.¹⁶ Tregs nomenclature changed in 2012. When the anatomical location of differentiation is demonstrated, Tregs are called “Thymus-derived Treg cells” or “Peripherally-derived Treg cells.” “Foxp3+ Treg cells” is used when the anatomical location is unclear.¹⁷

Relationship between Tregs and outcome

Tregs are associated with adverse outcomes. Cheng et al. measured circulating inflammatory factors and Tregs in patients with resectable PDAC and found that numbers of peripheral Tregs were associated with adverse outcomes.¹⁸ Another study of blood samples from patients with unresectable PDAC found that low initial numbers of circulating Tregs predicted long-term overall survival.¹⁹ Immunohistochemistry was used to evaluate intratumoral Tregs in resected PDACs and found that low numbers of intratumoral Tregs were associated with longer survival times.²⁰ Circulating and intratumoral Tregs are potential independent predictors that suggest poor outcomes, and decreased Tregs numbers were found in PDAC patients after surgery and chemotherapy,^18,20,21 suggesting that Tregs depletion may enhance antitumor activities.

The role of Tregs in the TME

Tregs cells remodel the immune TME, mediate immune escape of tumor cells, and promote tumor invasion and metastasis via several mechanisms (Figure 1).

Figure 1.

Role of Tregs in the immune tumor microenvironment in pancreatic cancer. Tregs remodel the tumor immune microenvironment, mediate immune escape, and promote invasion and metastasis in several ways. Direct regulatory function: IL-10, IL-35, transforming growth factor-β, granzyme B, and perforin. Indirect regulatory function: Tregs influence target cells via interactions with cytotoxic T lymphocyte-associated antigen-4, T cell immunoglobulin and mucin domain 3, Programmed cell death 1, lymphocyte activation gene 3, and their ligands.

Direct regulatory function

Interleukin-10 (IL-10) is an immune inhibitory cytokine. Its activity depends on binding its high-affinity and low-affinity receptors.²² Cancer cells and immune cells secrete IL-10.²³ IL-10 inhibits cytokine production and effector immune cells, protecting tumor cells from immune surveillance and promoting proliferation.²⁴ IL-10 blockade enhanced the cytotoxicity of mesothelin-engrafted receptor T cells in the TME of PDAC, suggesting a potential therapeutic target.²⁵ A study found that IL-10 neutralization increased IFN-γ and CD8⁺ T cells to enhance cytotoxic immune responses in tumors. Pegilodecakin, pegylated recombinant human IL-10, and anti-PD-1 inhibitors or tyrosine kinase inhibitors improved outcomes compared to monotherapy in renal cell carcinoma clinical trials.²⁶ Clinical trials of drugs targeting IL-10 in PDAC are in the recruiting phase.

Transforming growth factor-β (TGF-β) is an essential modulator of immune homeostasis, tolerance, and surveillance.²⁷ It promotes the proliferation of Tregs, inhibiting antigen presentation by dendritic cells (DCs),²⁷ reducing natural killer (NK) cell function,²⁸ and promoting type II macrophage development.²⁹ The cytolytic activity of CD8⁺ cells can be inhibited in a TGFβ-dependent manner.³⁰ In this way, early tumor immune responses of CD8⁺ cells can be inhibited by Tregs. A study showed that epithelial-mesenchymal transition genes are regulated by TGFβ signaling, possibly promoting tumor cell motility and invasiveness.³¹ Another study found that TGFβdepletion induced hypoxia and death in tumor cells and remodeled vasculature to inhibit tumor progression.³² In an orthotopic PDAC mouse model, a TGF-β inhibitor (LY364947) showed better antitumor efficacy than an anti-PD-L1 monoclonal antibody.³³ However, because PDAC patients harbor frequent mutations, the effectiveness of this approach is questionable.

Interleukin 35 (IL-35) is an immunosuppressive cytokine secreted by IL-35-Tregs.³⁴ It is a member of the IL-12 family formed by the heterodimerization of p35 and Ebi3 subunits.³⁵ IL-35 is abundantly expressed in rat and human PDAC and inhibits endogenous antitumor T cell responses,³⁶ and mediates expression of intercellular adhesion molecule-1 (ICAM1) to facilitate metastasis.³⁷ When located in the T cell zone, IL-35-Tregs access autogenous T cells more efficiently.³⁴ IL-35 deficiency enhanced anti-PD-1 immunotherapy in a PDAC mouse model, suggesting that IL-35 depletion with anti-PD-1 immunotherapy holds promise for clinical use.³⁶

Granzyme B is a serine protease highly expressed on Tregs in the immune TME.³⁸ It is a cytolytic effector of programmed death in cancer cells. Tregs originating from the TME promote CD8 T+ cell and NK cell apoptosis, inhibiting immune function in the granzyme B and perforin pathways.³⁹ An orthotopic mouse model of PDAC demonstrated that irreversible electroporation ablation increased tumor infiltration of Granzyme B+ cells to relieve immunosuppressive components and enhance the efficacy of dendritic cell vaccination.⁴⁰ Granulomycin B is used as a biomarker to evaluate drug efficacy in clinical trials.

Indirect regulatory function

Cytotoxic lymphocyte-associated antigen-4 (CTLA-4) is expressed on Tregs and activated conventional T cells⁴¹; CD80 and CD86 are B7 ligands that are expressed on antigen-presenting cells (APCs).^42,43 CTLA-4 competes with CD28 by binding to B7 ligands and reduces T cell responses and glucose utilization; both mechanisms help Tregs maintain stable.¹³ Tregs regulate conventional T cell activation⁴⁴ and modulate immunosuppression of APCs via the CTLA-4 pathway.⁴⁵ A mouse model study of PDAC found that Tregs expressing CTLA-4 quickly progressed to the preinvasive stage.⁴⁶ Blockade of CTLA-4/CD80 dependent mechanism accelerated CD4⁺ T cell infiltration into PDAC and regulated T cell exclusion. However, anti-CTLA-4 therapy produced disappointing results in advanced PDAC.⁴⁷ The CTLA-4 inhibitor ipilimumab combined with gemcitabine was suggested to constitute a safe approach in a phase Ib study of advanced PDAC.⁴⁸ Trials of combined anti-CTLA-4 and other therapies are in the recruitment phase.

Programmed cell death 1 (PD-1) contains an immunoreceptor tyrosine-based inhibition site and an immunoreceptor tyrosine-based switch motif.^49,50 Members of the B7 superfamily are the primary ligands of PD-1, and they include PD-L1 (B7-H1) and PD-L2 (B7-DC).⁵¹ PD-1 is abundantly expressed on intratumoral Tregs that correlate with lymph node metastasis in PDAC.⁵² In PDAC patients who underwent surgery, PD-L1 expression negatively correlated with CD8⁺ T cells and outcome.⁵³ Loos et al. found that intratumoral Tregs positively correlated with PD-L1 expression in PDAC.^54,55 Through the PD-1/PD-L1 pathway, Tregs enhance immune suppression and compromise antitumor effects, resulting in accelerated tumor development. PD-1/PD-L1 pathway blockade followed by Tregs depletion represents a possible mechanism of promoting antitumor responses.⁵⁶ Currently, pembrolizumab and nivolumab are FDA-approved PD-L1 inhibitors used in clinical trials.⁵⁷ Preclinical and clinical trials primarily focus on PD-L1 combined with chemotherapy, radiotherapy, and immunotherapy.

The galectins family of proteins act by binding to β-galactose residues.⁵⁸ Galectin-1 (Gal-1) is not found in normal pancreatic tissue but is abundantly expressed in pancreatitis and pancreatic tumors on pancreatic satellite cells,⁵⁹ where it participates in tumor aggressiveness and progression.⁶⁰ Gal-1 is also abundantly expressed on Tregs in mice and humans⁶¹; gal-1 downregulation is accompanied by decreased Treg activity. Gal-1 facilitated Treg differentiation via the NF-κB/RelBIL-27 pathway.⁶² In current clinical trials, medications targeted to Gal-1 increased the sensitivity of chemotherapy in advanced tumors; nevertheless, the tumor-related pathways remain under investigation.⁶³

T cell immunoglobulin-3 (Tim-3) is expressed by CD8⁺ T cytotoxic cells,⁶⁴ Tregs, DC, and NK cells.⁴⁴ Tim-3 inhibits by binding to C-Type lectin galectin-9.^65,66 TIM-3 is abundantly expressed in PDCA tissues and is strongly associated with tumor progression and recurrence.⁶⁷ Tumor-infiltrating Tregs increase Tim-3 and PD-1 expression to inhibit the antitumor effect of cytotoxic T lymphocytes in PDAC.^68,69 In a preclinical study of hematologic and solid tumors, combination blockade of the Tim-3 and PD-1 pathways showed better antitumor function than monotherapy.⁶⁵ Recent clinical trials showed similar results in advanced metastatic melanoma,⁷⁰ non-small cell lung cancer,⁷¹ and follicular B cell non-Hodgkin lymphoma.⁷² Treg depletion combined with PD-1 and Tim-3 blockade may become a treatment for PDAC.⁶⁹

Lymphocyte activation gene 3 (Lag-3) has a structure similar to the CD4 co-receptor and a higher affinity for major histocompatibility complex-II expressed on APCs.⁷³ It is abundantly expressed on murine and human circulating Tregs. Lag-3+ FoxP3+ Treg cells are found in large numbers in PBMC, para-cancer lymph nodes, and tumor sites.⁷⁴ Lag-3+ FoxP3+ Tregs secrete IL-10 and TGF-β, inhibiting CD4⁺ T-cell activation, increasing Treg suppressor activity, and decreasing cytotoxic CD8⁺ T cell function.^73,75 Co-blockade of the Lag-3 and PD-1 signaling pathway inhibited tumor growth, enhanced antitumor responses, and maintained immune homeostasis. A study found that high levels of LAG-3 correlated with poor survival and early tumor recurrence in PDAC.⁷⁶ The combination of IMP321, a soluble LAG-3 Ig fusion protein and MHC Class II agonist, and gemcitabine therapy was well-tolerated. It was associated with few immune-related adverse events in a phase I study of PDAC.⁷⁷

Tregs as therapeutic targets

Currently approved chemotherapeutic regimens have disappointingly poor effects on PDAC survival rates. Immunotherapy is gaining attention from researchers and has shown promising results in several cancers. The primary treatment strategy is to reduce Tregs infiltration into tumor tissues, followed by lower immunosuppression function. However, because the immune microenvironment is complex, immunotherapy in PDAC has returned less satisfactory results. Treg modulation combined with other treatments has become an emerging strategy for PDAC therapy.

An orthotopic murine experimental model showed combined blockade of CD25, and TGFβ significantly reduced peripheral and peritumor regulatory T cells and increased intratumor CD8⁺ TILs levels compared to monotherapy.⁷⁸ PD-1, CD25, and TGFβ triple combined therapy more effectively restricted tumor proliferation and progression than monotherapy. Tregs inhibition via multiple pathways more effectively delayed tumor progression. Azad et al. employed several mathematical orthotopic models. They found that radiosensitization after PD-L1 blockade arrested tumor progression in KPC and Pan02 murine models that rely on increasing intratumoral CD8⁺ T cells and decreasing CD11b+Gr1+ myeloid cells.⁷⁹ Monotherapy is insufficient to inhibit growth, progression, and metastasis of PDAC; combination with radiation therapy may hold promise. DC and tumor cell fusion hybrid vaccine combined with anti-CD25 antibody enhanced antitumor responses in NK cells, increased IFN-γsecretion, and reduced Tregs proliferation, synergistically improving immunotherapy in an orthotopic tumor setting.⁸⁰ IFN-a adenovirus vector intratumoral injection increased the infiltration of CD4⁺ and CD8⁺ T cells into tumors. Anti-GITR mAb decreased Tregs and synergistically reinforced the efficacy of IFN-a gene therapy in an orthotopic PDAC model.⁸¹ These findings suggest that combined treatment can remodel the TME and strengthen systemic antitumor effects. The combination of GVAX and TGFβ inhibitors enhanced antitumor immunoreactions to vaccine therapy and depleted intratumoral Tregs in an orthotopic tumor model.⁸² These authors found that combination therapy increased the cure rate in PDAC-bearing mice through increased intratumoral CD8⁺ T cells and dampened intratumoral Tregs. TGFβ blockade has little effect on Treg depletion or improvement of the cure rate in this model, suggesting that targeted therapy to Tregs alone is insufficient to modulate the TME in PDAC mice successfully. The same result may apply to clinical trials in PDAC.

Clinical trials of immunotherapy against Tregs in PDAC are ongoing (Table 1). The TME can be disrupted by irreversible electroporation (IRE), reducing circulating Tregs.⁸³ A recent phase I b trial found that IRE combined with nivolumab had an excellent safety profile; there were no toxicity findings and favorable overall survival in PDAC.⁸⁴ CTLA-4 expressed on Tregs regulates immunosuppression, and anti-CTLA-4 treatment contributed to tumor regression and long-term survival. Ipilimumab is a CTLA-4 inhibitor used to treat melanoma; however, it was not effective against advanced PDAC.⁴⁷ Nevertheless, gemcitabine and ipilimumab combined therapy had a better overall safety profile in advanced PDAC than monotherapy.⁴⁸ BL-8040 is a small synthetic peptide that can associate with CXCR4 to decrease circulating Tregs and increase intratumoral CD8⁺ T cells.⁸⁵ Clinical trials showed that BL-8040 combined with pembrolizumab co-blockade reinforced the efficacy of chemotherapy in PDAC, and triple therapy may be a potential treatment.⁸⁶ Clinical trials in PDAC are currently recruiting patients. These include NCT04324307, NCT04117087, NCT03953235, NCT03816358, NCT03373188, NCT03058289, and NCT03058289. In previous clinical trials, mono-immunotherapy failed to decrease the mortality rate in PDAC. Clinical trials of combination therapy are ongoing.

Table 1.

Clinical trials of immunotherapy against Tregs in pancreatic cancer.

NCT Number	Phase	Intervention/treatment	Status	Conditions
NCT02720666	II	Biological: GVAX pancreas CRS-207 Drug: Cyclophosphamide	Completed	Metastatic Pancreatic cancer
NCT03621982	I	Drug: ADCT-301 Biological: Pembrolizumab	Recruiting	Advanced solid Tumors
NCT02720666	I	Drug: K-001	Completed	Pancreatic cancer
NCT04324307	I/II	Drug: Programmed cell death-ligand 1/CTLA-4 BsAb Combination Product: FOLFIRINO, GP	Recruiting	Locally advanced and Metastatic Pancreatic cancer
NCT04117087	I	Drug: KRAS peptide vaccine Drug: Nivolumab Drug: Ipilimumab	Recruiting	Colorectal cancer Pancreatic cancer
NCT03816358	I/II	Biological: Anetumab ravtansine Drug: Gemcitabine Hydrochloride Biological: Ipilimumab Biological: Nivolumab	Recruiting	Pancreatic cancer

Immune-related adverse events

In addition to survival benefits, Treg depletion is associated with immune-related adverse events (irAE). However, the overall incidence of irAE was lower than that of chemotherapy. IrAE are well-tolerated and self-limiting and are primarily characterized by visceral toxicity.^87–89 The most common immune-related adverse reactions are induced by immune checkpoint inhibitors, namely CTLA-4 and PD-1/PD-L1 inhibitors. The incidence of adverse reactions for CTLA-4 inhibitors is higher than PD-1.⁹⁰ CTLA-4 inhibitors are associated with maculopapular rashes, hypophysitis, and colitis. The most common irAEs associated with PD-1/PD-L1 inhibitors are vitiligo, hypothyroidism, pneumonitis, and arthralgia. Different types of tumors treated with the same immune checkpoint inhibitor may respond with specific immune-related adverse reactions.⁹⁰ Clinical trials showed that PD-1 inhibitors manifest different irAE in renal cell carcinoma (rash, pruritus, diarrhea, dyspnea), non-small-cell lung cancer (pneumonitis), and melanoma (colitis). In oral tumors, irAEs included Sicca syndrome.⁹¹ After analyzing tens of thousands of adverse reactions in several clinical trials of pembrolizumab, investigators found that infections significantly enhanced the incidence of irAE during immunotherapy.⁹² Several clinical trials are ongoing to identify a balance of immune safety and antitumor efficacy.

Treg depletion may accelerate tumor progression

Although Tregs mediate immunologic tolerance, their influences in the TME are incompletely understood. Previous studies demonstrated Treg depletion could be a potential treatment for PDAC. However, a few reports suggested that Tregs depletion may accelerate tumor progression, challenging the dominant belief that Treg cells enable tumor progression.

The most novel finding in an orthotopically transplanted mice model was that Treg depletion did not alleviate immunosuppression and accelerated tumor progression.⁹³ Tregs accumulated in mouse and human PanIN and PDAC.^94,95 Tregs depletion can increase CD8⁺ T cells to enhance antitumor immune responses.⁹⁶ However, a recent study showed that increased CD8⁺ T cells after Treg depletion was offset by compensatory increases in other CD4⁺ T cells, immunosuppressive myeloid cells, and low levels of expression of α-smooth muscle actin (SMA) fibroblasts.⁹³ Ccl3, Ccl6, and Ccl8 increased upon Treg depletion, resulting in increased immunosuppressive myeloid cells and promotion of PDAC. Tregs secrete TGFβ and induce differentiation of fibroblasts into tumor-restricting and predominantly SMA-expressing fibroblasts.^97,98 SMA^high fibroblasts are defined as “myofibroblastic CAFs” (myCAFs). Treg depletion reduced TGFβ levels and induced a pathologic Th2 response and fibroblast population reprogramming of myCAFs to tumor-promoting fibroblasts, helping to mediate carcinogenesis in PDAC.⁹⁹ These mechanisms need to be further investigated and may reverse the unsatisfactory results of Treg cell-targeted therapy.

Conclusion

Pancreatic ductal adenocarcinoma is characterized by a high degree of aggressiveness, poor early diagnosis rates, and short overall survival times. Tregs are crucial components of the remodeled tumor immune microenvironment. The mechanisms and functions of Tregs in the TME of PDAC require further investigation. Interventions targeting Tregs through several approaches might reduce the degree of infiltration around solid tumors and their immunosuppressive functions. The emergence of immune checkpoint suppression coupled with currently available treatments suggests that Treg depletion might emerge as a practical therapeutic pathway. Tregs depletion reinforces the efficacy of treatments such as chemotherapy and radiotherapy. A thorough understanding of Treg immunobiology, particularly related to the TME and interactions of Tregs with tumor-infiltrating cell subsets, will provide a theoretical basis for further clinical research.

Footnotes

Appendix

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Zhejiang Natural Fund Project (grant no. LQ21H090001), Ningbo Clinical Medicine Research Center Project (grant no. 2019A21003).

ORCID iD

Yufei Song

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Advance of T regulatory cells in tumor microenvironment remodeling and immunotherapy in pancreatic cancer

Abstract

Keywords

Introduction

Definition and classification

Relationship between Tregs and outcome

The role of Tregs in the TME

Direct regulatory function

Indirect regulatory function

Tregs as therapeutic targets

Immune-related adverse events

Treg depletion may accelerate tumor progression

Conclusion

Footnotes

Appendix

Declaration of conflicting interests

Funding

ORCID iD

References