Beta-adrenergic signaling in tumor immunology and immunotherapy

Abstract

Communication between the nervous and immune systems is required for the body to regulate physiological homeostasis. Beta-adrenergic receptors expressed on immune cells mediate the modulation of immune response by neural activity. Activation of Beta-adrenergic signaling results in suppression of anti-tumor immune response and limits the efficacy of cancer immunotherapy. Beta-adrenergic signaling is also involved in regulation of hematopoietic reconstitution, which is critical to Graft-versus-Tumor (GvT) effect and Graft-versus-Host disease (GvHD) following allogeneic hematopoietic cell transplantation (HCT). In this review, the function of Beta-adrenergic signaling in mediating tumor immunosuppression will be highlighted. We will also discuss the implication of targeting Beta-adrenergic signaling to improve the efficacy of cancer immunotherapy including the GvT effect, and to diminish the adverse effects including GvHD.

I. Introduction

Cancer immunotherapy is a new modality of therapies that exploit the immune system to recognize and destroy cancer. It is based on improved understanding of the intricate cellular and molecular mechanisms controlling immune responses and a growing explication of oncology including the discovery of mutation instigated neoantigens.¹ Generally, cancer immunotherapy has been categorized as active immunotherapy, which boosts activation of the immune system to attack cancer cells, such as tumor vaccines^2–3 and dendritic cell (DC) based-immunotherapy,⁴ and passive immunotherapy, which harnesses existing components of immune responses that include using monoclonal antibodies,^5–7 cytokines,⁸ and adoptive transfer of T cells^9–10 and natural killer (NK) cells^11–12 to treat cancer patients. Most recently immune checkpoint inhibitors, which are monoclonal antibodies against the inhibitory signaling pathways of programmed cell death protein 1 (PD-1) and cytotoxic T lymphocytes antigen 4 (CTLA-4), have shown promising efficacy in clinical trials of a broad range of solid and hematological cancers, and therefore led to FDA approvals for clinical use to treat a rapidly rising list of cancers.^13–14 On the other hand, genetically engineered T lymphocytes that express chimeric antigen receptors (CARs) or T cell receptors (TCRs) have emerged as another effective approach of caner immunotherapy.¹⁵ In 2017, two of CAR-T cell therapies, marked as KYMRIAH and YESCARTA, have been approved by FDA for treatment of certain types of B-cell precursor acute lymphoblastic leukemia and certain types of large B-cell lymphoma and non-Hodgkin lymphoma. Furthermore, personalized therapeutic cancer vaccines have also been demonstrated as a potent treatment strategy for cancer patients recently.^16–18 However, there are still limitations in these immunotherapies. Both CAR-T cell therapy and immune checkpoint blockade therapy are restricted to certain types of cancer and a small population of cancer patients.^19–20 Resistance to immune checkpoint blockade and CD19-targeted CAR-T cell therapy was also found in cancer patients.^21–22 Severe and even lethal adverse side effects have recently been reported in many patients treated with PD-1 and CTLA-4 blockade.^23–24 For example, severe diarrhea, colitis, increased alanine aminotransferase levels, inflammation pneumonitis, and interstitial nephritis have been reported in patients with various type of cancer.^25–27 Therefore, to improve the efficacy and safety of cancer immunotherapy, enormous and challenging research work still needs to be performed to further elucidate the complex mechanisms underlying the dysfunction of immune response to cancer, which includes immunosuppression in tumor microenvironment, host systemic immunosuppression, tumor heterogeneity including gene mutations and neoantigens in tumor cells.

The regulation of immunity by nervous system has been identified in various conditions including inflammatory and autoimmune diseases.^28–32 Activated neurons release neurotransmitters and other regulatory molecules, which engage corresponding receptors, including adrenergic receptors expressed on macrophages, dendritic cells, T cells and other immune cells, and facilitate neural regulation of immune responses.^33–35 In the tumor context, nervous system is also involved in the tumor microenvironment.^36–37 In addition to directly promoting tumor cell growth, invasion and tumor angiogenesis,^38–39 neuronal input also mediates immunosuppression in the tumor microenvironment.^{36, 40} Furthermore, sympathetic nervous system (SNS) is also involved in regulation of hematopoiesis.⁴¹ Here, we summarize the mechanistic insights in the suppressive role of Beta-adrenergic signaling in immune response to cancer and highlight the implication of Beta-adrenergic signaling blockade in the improvement of cancer immunotherapy. The function of Beta-adrenergic signaling in GvT effect and GvHD will also be discussed.

II. Beta-adrenergic signaling in tumor immunosuppression

Several studies have shown that sympathetic nervous system (SNS) activation, which stimulates Beta-adrenergic signaling, is able to regulate cancer cell metastasis and tumor growth by recruiting macrophages into tumor parenchyma.⁴² In a restraint mouse model, chronic stress was able to enhance mammary adenocarcinoma cell metastasis to distant tissues including lung and lymph node but had a negligible effect on the growth of the primary tumor.⁴² The stress-enhanced metastasis was not depending on T lymphocytes, but mediated by promoting infiltration of macrophages, which could induce expression of prometastatic gene such as Tgfb, Arg1 and Csf1, in primary tumor microenvironment.⁴² The Beta-adrenergic antagonist propranolol was able to largely abrogate the stress-induced increase of macrophage infiltration, suggesting that Beta-adrenergic signaling is involved in stress-mediated promotion of tumor metastasis. In another restraint stress mouse model, activation of Beta-adrenergic signaling led to ovarian carcinoma growth by promoting monocytes and macrophages infiltration into tumor tissue. These myeloid cells were recruited by stress-enhanced production of monocyte chemotactic protein 1 from ovarian cancer cells.⁴³ A study analyzing tumors from socially isolated patients demonstrated upregulated expression of genes involved in M2 macrophage polarization and epithelial-mesenchymal transition (EMT) as well as increased density of lymphatic vessels.⁴⁴ Activation of CREB (cAMP response element-binding protein) family transcription factors, which mediate the gene regulatory effects of Beta-adrenergic signaling, was also shown by a TELiS promoter-based bioinformatics analyses.⁴⁴ Bioinformatics analysis of Beta-adrenergic signaling stimulated macrophages showed a transcriptome that locates on the M2 side of the M1-M2 spectrum, but not fit entirely into any pre-defined category of M1 or M2 spectrum.⁴⁵ Beta2-adrenergic receptor activation by social isolation-induced stress also promoted 4T1 breast cancer progression by upregulating macrophage number and enhancing the M2 polarization in the tumor microenvironment.⁴⁶ Given the critical role of macrophages in remodeling tumor microenvironment and facilitating angiogenesis and extracellular matrix breakdown to enhance primary tumor progression and tumor metastasis,^47–49 blockade of macrophage infiltration into tumor microenvironment implies an important strategy for improving cancer immunotherapy.

Beta-adrenergic signaling was also demonstrated to mediate the suppression of anti-tumor immune response. In murine studies, a striking decrease of tumor formation, growth, and metastasis in B16-F10, 4T1, CT26 and pan02 tumor models was observed in mice housed at thermoneutral temperature compared to mildly cold temperature that stimulates Beta2-adrenergic signaling. The enhanced control of tumor growth was dependent on the increased numbers of antigen-specific CD8⁺ T cells and effector phenotype of the CD8⁺ T cells in the tumor microenvironment and a significant reduction of immunosuppressive myeloid-derive suppressive cells (MDSCs) and regulatory T cells in the spleen.⁵⁰ Chronic cold induced a stress response that caused tumor immunosuppression mediated by Beta2-adrenergic signaling in host immune cells.⁵¹ Using physiologic, pharmacologic and genetic blockade of Beta2-adrenergic signaling, the authors demonstrated that reduction of Beta2-adrenergic signaling facilitated an increased frequency of effector CD8⁺ T cells and an elevated effector ratio of CD8⁺ T cells to CD4⁺ regulatory T cells, and a decreased frequency of PD-1 expressing CD8⁺ T cells in the B16 tumor microenvironment.⁵² The accumulation of MDSCs in the spleen of 4T1 tumor-bearing mice was also decreased by blockade of Beta2-adrenergic signaling.⁵² In a B-cell lymphoma mouse model, chronic elevated Beta-adrenergic signaling resulted in less effective control of lymphoma growth, which was mediated by the reduced proliferation, decreased production of IFN-γ and cytotoxic capacity of antigen-specific CD8⁺ T cells.⁵³ The suppressive effect on anti-lymphoma response by chronic Beta-adrenergic signaling was shown to be selective to T cells and independent of innate lymphocyte response to an experimental NKT cell-targeting vaccine.⁵³ In a triple negative breast cancer mouse model, social isolation-induced stress promoted 4T1 tumor progression and caused a reduction of survival in tumor-bearing mice, which was mediated by reducing CD8⁺ and CD3⁺CD69⁺ T cells in the spleen.⁵⁴ Acute and chronic restraint stress also increased the gene expression of granzyme B, a serine protease in cytotoxic T cells and NK cells and CXCL 10, a T cell chemoattractant, in the tumor microenvironment, which were reduced by propranolol, a Beta-adrenergic receptor antagonist.⁵⁴ Beta-adrenergic signaling was involved in suppression of antitumor cytotoxic T cell generation by inhibiting Tumor necrosis factor-α (TNF-α) gene expression.⁵⁵ Recently, a study demonstrated that Beta-adrenergic signaling inhibited CD8⁺ T cells activation by suppressing the required metabolic reprogramming.⁵⁶ Activation of the reward system, which is the dopaminergic neurons in the ventral tegmental area that constitutes a key neuronal network mediating positive emotions, expectations and motivation,⁵⁷ improved the control of B16 melanoma and Lewis lung carcinoma growth in mice, manifested by reduced noradrenergic input to the bone marrow, attenuated the immunosuppressive effect of MDSCs and enhanced Granzyme B expression by CD8⁺ T cells in the tumor.⁵⁸ Similar to the effect of the decreased noradrenergic input, treatment with propranolol, a Beta-adrenergic receptor antagonist, delayed primary tumor growth and development of metastasis in a murine melanoma model, which was mediated by suppressing the infiltration of myeloid cells and prompting cytotoxic lymphocytes including NK and CD8⁺ T cells into the primary and metastatic tumor.⁵⁹

The function of Beta1 and Beta3-adrenergic signaling in immunity still need to be investigated. There are very few reports reveal the role of Beta1 and Beta3-adrenergic receptors in modulating immune response. The Beta1-adrenergic receptor was demonstrated mediating acute cold/restraint stress inhibition of host resistance to Listeria monocytogenes by modifying T cells activation or subsequent T cell function involved in adaptive immunity⁶⁰ and by suppressing cellular immune response.⁶¹

Chronic restraint stress was also able to impair antitumor T cell response, which was mediated by thyroid hormones instead of noradrenaline and corticosterone.⁶² Fluoxetine, an anti-depressant for cancer patients, was shown to inhibit lymphoma growth by modulating antitumor immunity, specifically by enhancing mitogen-induced T-cell proliferation and expression of Tumor Necrosis Factor-α (TNF-α) and Interferon-γ (IFN-γ).^63–64 These studies imply other mechanisms, working in parallel to or in concert with Beta-adrenergic signaling, are also involved in the stress-induced suppression of antitumor immunity.

III. Blockade of Beta-adrenergic signaling in cancer immunotherapy

Given the critical role of Beta-adrenergic signaling in suppressing antitumor immune response and regulating immune cells in the tumor microenvironment, blockade of the Beta-adrenergic signaling has broad implication for improving cancer immunotherapy. Indeed, pan-Beta-adrenergic receptor blocker propranolol was able to reverse the immunosuppression caused by cold induced stress, resulting in increased antitumor effector T cells and improved anti-PD-1 efficacy.⁵² A retrospective analysis showed that Beta-adrenergic blocker improved overall survival of metastatic melanoma patients who received immunotherapy (IL-2, anti-CTLA4, or anti-PD1).⁶⁵ This enhanced immunotherapy was further reinforced in a preclinical murine melanoma model showing that Beta-adrenergic blocker combined with anti-PD-1 blockade led to a better control of melanoma growth.⁶⁵

Blockade of Beta-adrenergic signaling was also shown to reduce the immunosuppression caused by surgical excision of primary tumor and may improve immunotherapy when combined with surgery.⁶⁶ Catecholamine and prostaglandin, which are produced abundantly by tumor cells and stromal cells in the tumor microenvironment and host physiologic systems due to tissue trauma of surgery and perioperative stress, are involved in promoting cancer metastasis following tumor surgical resection by suppressing cellular immune response, increasing prometastatic cytokines and inducing inflammation. Therefore, pharmacologic inhibition of Beta-adrenergic receptor signaling and/or prostaglandin synthesis is able to reduce the prometastatic and immunosuppressive effects of tumor surgery and physiologic stress in clinical trials and preclinical studies.⁶⁷ NK cells play an important anti-metastasis role that was suppressed by activation of Beta-adrenergic signaling in animal tumor models,^68–73 whereas this function was able to be recovered by Beta-adrenergic antagonist.^74–75 Recently, administration of the Beta-adrenergic antagonist propranolol and the COX-2 inhibitor etodolac led to multiple favorable effects in a phase-2 randomized trial of breast cancer patients.^76–77 In addition to reducing epithelial-mesenchymal transition (EMT) and decreasing the activity of proinflammatory and prometastatic transcription factors (GATA-1, GATA-2 and STAT-3) and Ki-67 in tumor cells, Beta-adrenergic antagonist treatment suppressed monocytes while promoted B cells infiltrating into the tumor, abrogated postoperative mobilization of CD16⁻ ‘classical’ monocytes and enhanced CD11a expression on circulating NK cells.^76–77 In animal studies, adrenergic nerve input was also shown to control lymphocyte egress from lymph nodes (LNs), but not entry to, through Beta2-adrenergic receptor.^78–79 The retention of lymphocytes in LNs was mediated by physical interaction between Beta2-adrenergic receptors and chemokine receptors CCR7 and CXCR4, and consequently inhibited antigen-primed T cell migration into peripheral tissues in an experimental autoimmune encephalomyelitis mouse model⁷⁸ and enhanced humoral immune response in the LNs.⁷⁹ Therefore, using Beta2-adrenergic blockade to enhance lymphocyte mobilization has substantial significance because infiltration of effector immune cells into tumor microenvironment is critical for improving cancer immunotherapy inlcluding CAT-T cell therapy^80–82 and immune checkpoint blockade therapy.^83–84

IV. Beta-adrenergic signaling in Graft-vs-Tumor effect and Graft-vs-Host disease

Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative adoptive immunotherapy for a variety of hematologic malignancies.⁸⁵ Allogenic HCT is a complicated treatment because of its incorporation of major elements of immunology, oncology, radiobiology and infectious disease. A successfully allogeneic HCT largely depends on GvT effect, which is typically attributed to donor T cells and also likely involves a complex interaction of multiple cell types and cytokines.^86–87 However, the GvT effect is closely connected with GvHD, which is caused by the mismatched major and minor histocompatibility antigens expressed between host and donor.^88–89

Recently, several studies demonstrated that Beta-adrenergic signaling is involved in the GvT effect and GvHD. In a mouse allogeneic HCT study, host Beta2-adrenergic signaling was shown to inhibit GvT activity by suppressing donor T cell reconstitution.⁹⁰ Blockade of Beta2-adrenergic signaling was able to induce increased CD11c⁺ DC development and render DC more immunogenic, thereby improving donor T cell reconstitution.⁹⁰ The improved reconstitution of T cells, including CD8⁺, CD4⁺, and CD4⁺Foxp3⁺ regulatory T cells led to an enhanced GvT effect without increasing GvHD. In another study, GvHD was suppressed by cold temperature-induced stress through Beta2-adrenergic signaling.⁹¹ Host-derived, but not donor T cell-derived, Beta2-adrenergic receptor signaling is essential for inhibiting GvHD.⁹¹ Through MLR-based in vitro T cell activation and tumor cell-killing experiments, it was demonstrated that Beta2-adrenergic receptor signaling deficiency in DCs enhances the alloreactive CD8⁺ T cell response in the tumor setting. However, the other study showed that Beta2-adrenergic receptor inhibition exacerbated GvHD induced by total T cells, in which CD4⁺ T cells presumably play a dominant role. Overall, these two studies may appear to contradict each other. However, these findings suggest that Beta2-adrenergic signaling plays more complicated and differential roles in GvT and GvHD involving CD4⁺ versus CD8⁺ T cell responses and possibly other immune cells including DCs and MDSCs. Further studies will be required to delineate the underlying mechanisms.

After performing total body irradiation and allogeneic HCT, the recovery of hematopoietic homeostasis is critical to the generation of GvT and GvHD. Hematopoietic stem cells (HSCs) are the only cell type in the bone marrow (BM) that is able to differentiate to all blood cell lineages, and their behavior is tightly regulated by the HSC niche in the BM microenvironment.^92–93 The function and regenerative capacity of HSC can be diminished by the damaging effects of radiation injury or chemotherapy, and their recovery to homeostasis is highly dependent on the BM microenvironment.^94–95 Neural regulation of hematopoiesis has been reported recently.⁴¹ The sympathetic nervous system is a critical regulatory component of the BM microenvironment, and sympathetic nerve fibers and neural crest-derived cells serve as major niche constituents and are essential to maintain HSC homeostasis and restore normal function from stress.^96–97 Sympathetic neuronal activation was revealed to promote HSC proliferation by modulating Beta-adrenergic signaling. Starting from the observation of increased leukocytes, including neutrophils, monocytes and lymphocytes in patient blood, the authors further found that chronic variable stress activated proliferation of primitive hematopoietic progenitors, and led to decreased CXCL12 levels through Beta3-adrenergic signaling in BM niche cells, which was signaled by surplus noradrenaline released from nerve fibers.⁹⁸ Most recently, myeloid cell numbers in blood of diabetic patients were shown strongly correlated with concentration of norepinephrine in the plasma.⁹⁹ Consistent with the observation in diabetic patients, myeloid cell number was increased in blood and spleen of experimental diabetic mice, in which proliferation and differentiation of splenic granulocyte macrophage progenitors (GMPs) are activated by sympathetic neuronal activity through Beta2-adrenergic receptors expressed on the splenic granulocyte macrophage progenitors (GMPs).⁹⁹ Beta-adrenergic signaling also plays an important role in modulating mobilization of HSCs from BM. In UDP-galactose ceramide galactosyltransferase-deficient mice, which exhibit aberrant nerve conduction, hematopoietic stem and progenitor cell (HSPC) egress from BM was significantly decreased when induced by granulocyte colony-stimulating factor (G-CSF).⁹⁶ Specifically, Beta2-adrenergic receptor signaling is required for granulocyte colony-stimulating factor (G-CSF)-induced HSPC mobilization, osteoblast suppression and bone CXCL12 downregulation.⁹⁶ Further study also showed that cooperation of Beta2- and Beta3-adrenergic receptor signaling is essential for granulocyte colony-stimulating factor (G-CSF)-induced HSPC egress from BM.¹⁰⁰ The recruitment of leukocytes to tissues under homeostasis exhibited circadian oscillation, which is orchestrated by molecular clock via adrenergic nerves.¹⁰¹ Beta2- and Beta3-adrenergic signaling is impaired in sympathetic denervation that causes decreased expression of HSC-regulating genes in osteoblasts,^{93, 95} which is an important HSPC niche and mediator of HSPC mobilization.¹⁰²

V. Conclusion

Effective immunotherapy is not only dependent on activating antitumor immune response, but also needs to reverse tumor-induced immunosuppression. Beta-adrenergic receptors expressed on immune cells engage with neurotransmitters released by sympathetic nervous system and mediate immunosuppression in the tumor microenvironment by recruiting tumor-associated macrophage and dampening cytotoxic T cells and NK cells (Fig. 1). Therefore, blockade of the Beta-adrenergic signaling implies a promising strategy to improve current immunotherapy.

Figure 1.

The complex role of Beta-adrenergic signaling in immunosuppression in tumor microenvironment and hematopoiesis. Stress caused by surgery and irradiation can activate sympathetic nervous system (SNS) and induce production of neurotransmitters including norepinephrine and epinephrine. These neurotransmitters engage Beta-adrenergic receptors expressed on various innate and adaptive immune cells and may differentially regulate their function. Beta-adrenergic signaling can also stimulate hematopoietic stem cells (HSCs) and granulocyte macrophage progenitors (GMPs) and promote their proliferation in bone marrow and spleen, respectively. The neurotransmitters can enter the tumor microenvironment and induce immunosuppressive cells including macrophages, FoxP3⁺ T cells and myeloid-derived suppressive cells (MDSCs) to suppress the function of cytotoxic CD8⁺ T cells and natural killer (NK) cells. Beta-adrenergic signaling may also be manipulated to improve the Graft-versus-Tumor effect (GvT) and inhibit Graft-versus Host Disease (GvHD) via modulating T cell reconstitution and dendritic cell (DC) function during allogeneic hematopoietic cell transplantation.

Given the limitation of current immunotherapies, combination immunotherapy or combination therapy between immunotherapy and chemotherapy, surgical therapy or radiotherapy, would be considered prospective treatment strategies for cancer patients in the near future.^103–105 Immunotherapy combining with hematopoietic cell transplantation (HCT) also potentially provides synergistic effect for treating cancer patients.^106–108 Physical and psychosocial stress caused by these treatment strategies is involved in promoting tumor progression and reducing life quality and overall survival of cancer patients.^109–112 Beta-adrenergic signaling is one of the suppressive mechanisms induced by stress (Fig. 1). Tumor cells expressing Beta-adrenergic receptors are also able to be regulated by activated neural system and thereby increase tumorigenesis, angiogenesis, proliferation and metastasis.¹¹³ Indeed, some cancer patients are shown to benefit from taking the Beta-blocker propranolol.^114–117 Propranolol has also been shown as beneficial for treating multiple myeloma patients during HCT.¹¹⁸ However, the complete mechanisms underlying the suppressive function of Beta-adrenergic signaling still need to be fully understood. As discussed in this review, combination immunotherapy with Beta-adrenergic signaling blockade would be promising treatment strategy for cancer patients.

Abbreviations:

GvHD

Graft-versus-Host disease

GvT

Graft-versus-Tumor

HCT

Hematopoietic cell transplantation

PD-1

Programmed cell death protein 1

CTLA-4

Cytotoxic T lymphocytes antigen 4

MDSCs

Immunosuppressive myeloid-derive suppressive cells

NK

Natural killer cell

DC

Dendritic cell

BM

Bone marrow

HSPC

Hematopoietic stem and progenitor cell