Myeloid-derived suppressor cell mitochondrial fitness governs chemotherapeutic efficacy in hematologic malignancies.

Myeloid derived suppressor cells (MDSCs) are key regulators of immune responses and correlate with poor outcomes in hematologic malignancies. Here, we identify that MDSC mitochondrial fitness controls the efficacy of doxorubicin chemotherapy in a preclinical lymphoma model. Mechanistically, we show that triggering STAT3 signaling via ß2-adrenergic receptor (ß2-AR) activation leads to improved MDSC function through metabolic reprograming, marked by sustained mitochondrial respiration and higher ATP generation which reduces AMPK signaling, altering energy metabolism. Furthermore, induced STAT3 signaling in MDSCs enhances glutamine consumption via the TCA cycle. Metabolized glutamine generates itaconate which downregulates mitochondrial reactive oxygen species via regulation of Nrf2 and the oxidative stress response, enhancing MDSC survival. Using ß2-AR blockade, we target the STAT3 pathway and ATP and itaconate metabolism, disrupting ATP generation by the electron transport chain and decreasing itaconate generation causing diminished MDSC mitochondrial fitness. This disruption increases the response to doxorubicin and could be tested clinically.

Consideration of the importance of measuring thermal discomfort in biomedical research.

Core temperature stability is the result of a dynamically regulated balance of heat loss and gain, which is not reflected by a simple thermometer reading. One way in which these changes manifest is in perceived thermal comfort, 'feeling too cold' or 'feeling too hot', which can activate stress pathways. Unfortunately, there is surprisingly little preclinical research that tracks changes in perceived thermal comfort in response to either disease progression or various treatments. Without measuring this endpoint, there may be missed opportunities to evaluate disease and therapy outcomes in murine models of human disease. Here, we discuss the possibility that changes in thermal comfort in mice could be a useful and physiologically relevant measure of energy trade-offs required under various physiological or pathological conditions.

Chronic adrenergic stress and generation of myeloid-derived suppressor cells: Implications for cancer immunotherapy in dogs.

Recent studies have highlighted a key role played by the sympathetic nervous system (SNS) and adrenergic stress in mediating immune suppression associated with chronic inflammation in cancer and other diseases. The connection between chronic SNS activation, adrenergic stress and immune suppression is linked in part to the ability of catecholamines to stimulate the bone marrow release and differentiation of myeloid-derived suppressor cells (MDSC). Rodent model studies have revealed an important role for ß-adrenergic receptor signalling in suppression of cancer immunity in mice subjected to chronic stresses, including thermal stress. Importantly, therapeutic blockade of beta-adrenergic responses by drugs such as propranolol can partially reverse the generation and differentiation of MDSC, and partly restore tumour immunity. Clinical trials in both humans and dogs with cancer have demonstrated that propranolol blockade can improve responses to radiation therapy, cancer vaccines and immune checkpoint inhibitors. Thus, the SNS stress response has become an important new target to relieve immune suppression in cancer and other chronic inflammatory conditions.

Galectin-3 expression in donor T cells reduces GvHD severity and lethality after allogeneic hematopoietic cell transplantation.

Abundant donor cytotoxic T cells that attack normal host organs remain a major problem for patients receiving allogeneic hematopoietic cell transplantation (allo-HCT). Despite an increase in our knowledge of the pathobiology of acute graft versus host disease (aGvHD), the mechanisms regulating the proliferation and function of donor T cells remain unclear. Here, we show that activated donor T cells express galectin-3 (Gal-3) after allo-HCT. In both major and minor histocompatibility-mismatched models of murine aGvHD, expression of Gal-3 is associated with decreased T cell activation and suppression of the secretion of effector cytokines, including IFN-γ and GM-CSF. Mechanistically, Gal-3 results in activation of NFAT signaling, which can induce T cell exhaustion. Gal-3 overexpression in human T cells prevents severe disease by suppressing cytotoxic T cells in xenogeneic aGvHD models. Together, these data identify the Gal-3-dependent regulatory pathway in donor T cells as a critical component of inflammation in aGvHD.

Circadian Rhythm Disruption Increases Tumor Growth Rate and Accumulation of Myeloid-Derived Suppressor Cells.

Circadian rhythm disruption is implicated in the initiation and progression of many diseases, including cancer. External stimuli, such as sunlight, serve to synchronize physiological processes and cellular functions to a 24-h cycle. The immune system is controlled by circadian rhythms, and perturbation of these rhythms can potentially alter the immune response to infections and tumors. The effect of circadian rhythm disruption on the immune response to tumors remains unclear. Specifically, the effects of circadian disruption (CD) on immunosuppressive cell types within the tumor, such as myeloid-derived suppressor cells (MDSCs), are unknown. In this study, a shifting lighting schedule is used to disrupt the circadian rhythm of mice. After acclimation to lighting schedules, mice are inoculated with 4T1 or B16-F10 tumors. Tumor growth is increased in mice housed under circadian disrupting lighting conditions compared to standard lighting conditions. Analysis of immune populations within the spleen and tumor shows an increased accumulation of MDSCs within these tissues, suggesting that MDSC mediated immunosuppression plays a role in the enhanced tumor growth caused by circadian disruption. This paves the way for future studies of the effects of CD on immunosuppression in cancer.

Isolation of human and mouse myeloid-derived suppressor cells for metabolic analysis.

Metabolic reprogramming is associated with myeloid-derived suppressor cell (MDSC) immunosuppressive function. Here, we outline the process for acquiring MDSCs from human and murine sources for subsequent analysis of fatty acid oxidation, oxidative phosphorylation, and glycolysis using the Seahorse XFe 96 Analyzer. Murine MDSCs can be isolated directly from tumor-bearing mice or derived through IL-6 and GM-CSF culture of bone marrow cells from non-tumor-bearing mice. To generate human MDSCs, peripheral blood mononuclear cells (PBMCs) can be cultured with IL-6 and GM-CSF. For complete details on the use and execution of this protocol, please refer to Mohammadpour et al. (2021).

ß2-adrenergic receptor signaling regulates metabolic pathways critical to myeloid-derived suppressor cell function within the TME.

Myeloid-derived suppressor cells (MDSCs) impede antitumor immunity; however, the precise mechanisms that regulate their suppressive function remain unresolved. Identifying these mechanisms could lead to therapeutic interventions to boost cancer immunotherapy efficacy. Here, we reveal that ß2 adrenergic receptor (ß2-AR) expression on MDSCs increases with tumor growth and that the ß2-AR stress pathway drives the immune suppressive activity of MDSCs by altering their metabolism. We show that ß2-AR signaling decreases glycolysis and increases oxidative phosphorylation and fatty acid oxidation (FAO). It also increases expression of the fatty acid transporter CPT1A, which is necessary for the FAO-mediated immunosuppressive function of MDSCs. Moreover, we show that ß2-AR signaling increases autophagy and activates the arachidonic acid cycle, both required for increasing the release of the immunosuppressive mediator, PGE2. Our data reveal that ß2-AR signaling triggered by stress is an important physiological regulator of key metabolic pathways in MDSCs, driving their immunosuppressive function.

Psychosocial stress and immunosuppression in cancer: what can we learn from new research?

It is generally believed that the physiological consequences of stress could contribute to poor outcomes for patients being treated for cancer. However, despite preclinical and clinical evidence suggesting that stress promotes increased cancer-related mortality, a comprehensive understanding of the mechanisms involved in mediating these effects does not yet exist. We reviewed 47 clinical studies published between 2007 and 2020 to determine whether psychosocial stress affects clinical outcomes in cancer: 6.4% of studies showed a protective effect; 44.6% showed a harmful effect; 48.9% showed no association. These data suggest that psychosocial stress could affect cancer incidence and/or mortality, but the association is unclear. To shed light on this potentially important relationship, objective biomarkers of stress are needed to more accurately evaluate levels of stress and its downstream effects. As a potential candidate, the neuroendocrine signalling pathways initiated by stress are known to affect anti-tumour immune cells, and here we summarise how this may promote an immunosuppressive, pro-tumour microenvironment. Further research must be done to understand the relationships between stress and immunity to more accurately measure how stress affects cancer progression and outcome.

Chronic Adrenergic Stress Contributes to Metabolic Dysfunction and an Exhausted Phenotype in T Cells in the Tumor Microenvironment.

Metabolic dysfunction and exhaustion in tumor-infiltrating T cells have been linked to ineffectual antitumor immunity and the failure of immune checkpoint inhibitor therapy. We report here that chronic stress plays a previously unrecognized role in regulating the state of T cells in the tumor microenvironment (TME). Using two mouse tumor models, we found that blocking chronic adrenergic stress signaling using the pan ß-blocker propranolol or by using mice lacking the ß2-adrenergic receptor (ß2-AR) results in reduced tumor growth rates with significantly fewer tumor-infiltrating T cells that express markers of exhaustion, with a concomitant increase in progenitor exhausted T cells. We also report that blocking ß-AR signaling in mice increases glycolysis and oxidative phosphorylation in tumor-infiltrating lymphocytes (TIL), which associated with increased expression of the costimulatory molecule CD28 and increased antitumor effector functions, including increased cytokine production. Using T cells from Nur77-GFP reporter mice to monitor T-cell activation, we observed that stress-induced ß-AR signaling suppresses T-cell receptor (TCR) signaling. Together, these data suggest that chronic stress-induced adrenergic receptor signaling serves as a "checkpoint" of immune responses and contributes to immunosuppression in the TME by promoting T-cell metabolic dysfunction and exhaustion. These results also support the possibility that chronic stress, which unfortunately is increased in many patients with cancer following their diagnoses, could be exerting a major negative influence on the outcome of therapies that depend upon the status of TILs and support the use of strategies to reduce stress or ß-AR signaling in combination with immunotherapy.

ß2-Adrenergic receptor activation on donor cells ameliorates acute GvHD.

Acute graft versus host disease (aGvHD) remains a major impediment to successful allogeneic hematopoietic cell transplantation (allo-HCT). To solve this problem, a greater knowledge of factors that regulate the differentiation of donor T cells toward cytotoxic cells or Tregs is necessary. We report that the ß2-adrenergic receptor (ß2-AR) is critical for regulating this differentiation and that its manipulation can control aGvHD without impairing the graft-versus-tumor (GvT) effect. Donor T cell ß2-AR expression and signaling is associated with decreased aGvHD when compared with recipients of ß2-AR-/- donor T cells. We determined that ß2-AR activation skewed CD4+ T cell differentiation in vitro and in vivo toward Tregs rather than the T helper 1 (Th1) phenotype. Treatment of allo-HCT recipients with a selective ß2-agonist (bambuterol) ameliorated aGvHD severity. This was associated with increased Tregs, decreased cytotoxic T cells, and increased donor BM-derived myeloid-derived suppressor cells (MDSCs) in allogeneic and humanized xenogeneic aGvHD models. ß2-AR signaling resulted in increased Treg generation through glycogen synthase kinase-3 activation. Bambuterol preserved the GvT effect by inducing NKG2D+ effector cells and central memory T cells. These data reveal how ß-AR signaling can be targeted to ameliorate GvHD severity while preserving GvT effect.

ß2 adrenergic receptor-mediated signaling regulates the immunosuppressive potential of myeloid-derived suppressor cells.

Catecholamines released by sympathetic nerves can activate adrenergic receptors present on nearly every cell type, including myeloid-derived suppressor cells (MDSCs). Using in vitro systems, murine tumor models in wild-type and genetically modified (ß2-AR-/-) mice, and adoptive transfer approaches, we found that the degree of ß2-AR signaling significantly influences MDSC frequency and survival in tumors and other tissues. It also modulates their expression of immunosuppressive molecules such as arginase-I and PD-L1 and alters their ability to suppress the proliferation of T cells. The regulatory functions of ß2-AR signaling in MDSCs were also found to be dependent upon STAT3 phosphorylation. Moreover, we observed that the ß2-AR-mediated increase in MDSC survival is dependent upon Fas-FasL interactions, and this is consistent with gene expression analyses, which reveal a greater expression of apoptosis-related genes in ß2-AR-/- MDSCs. Our data reveal the potential of ß2-AR signaling to increase the generation of MDSCs from both murine and human peripheral blood cells and that the immunosuppressive function of MDSCs can be mitigated by treatment with ß-AR antagonists, or enhanced by ß-AR agonists. This strongly supports the possibility that reducing stress-induced activation of ß2-ARs could help to overcome immune suppression and enhance the efficacy of immunotherapy and other cancer therapies.

Adrenergic Receptor Signaling Regulates the Response of Tumors to Ionizing Radiation.

While ionizing radiation is a major form of cancer therapy, radioresistance remains a therapeutic obstacle. We have previously shown that the mandated housing temperature for laboratory mice (∼22°C) induces mild, but chronic, cold stress resulting in increased circulating norepinephrine, which binds to, and triggers activation of, beta-adrenergic receptors (ß-AR) on tumor and immune cells. This adrenergic signaling increases tumor cell intrinsic resistance to chemotherapy and suppression of the anti-tumor immune response. These findings led us to hypothesize that adrenergic stress signaling increases radioresistance in tumor cells in addition to suppressing T-cell-mediated anti-tumor immunity, thus suppressing the overall sensitivity of tumors to radiation. We used three strategies to test the effect of adrenergic signaling on responsiveness to radiation. For one strategy, mice implanted with CT26 murine colon adenocarcinoma were housed at either 22°C or at thermoneutrality (30°C), which reduces physiological adrenergic stress. For a second strategy, we used a ß-AR antagonist ("beta blocker") to block adrenergic signaling in mice housed at 22°C. In either case, tumors were then irradiated with a single 6 Gy dose and the response was compared to mice whose adrenergic stress signaling was not reduced. For the third strategy, we used an in vitro approach in which several different tumor cell lines were treated with a ß-AR agonist and irradiated, and cell survival was then assessed by clonogenic assay. Overall, we found that adrenergic stress significantly impaired the anti-tumor efficacy of radiation by inducing tumor cell resistance to radiation-induced cell killing and by suppression of anti-tumor immunity. Treatment using beta blockers is a promising strategy for increasing the anti-tumor efficacy of radiotherapy.

An overview of the role of sympathetic regulation of immune responses in infectious disease and autoimmunity.

Stress in patients and pre-clinical research animals plays a critical role in disease progression Activation of the sympathetic nervous system (SNS) by stress results in secretion of the catecholamines epinephrine (Epi) and norepinephrine (NE) from the adrenal gland and sympathetic nerve endings. Adrenergic receptors for catecholamines are present on immune cells and their activity is affected by stress and the accompanying changes in levels of these neurotransmitters. In this short review, we discuss how this adrenergic stress impacts two categories of immune responses, infections and autoimmune diseases. Catecholamines signal primarily through the ß2-adrenergic receptors present on innate and adaptive immune cells which are critical in responding to infections caused by pathogens. In general, this adrenergic input, particularly chronic stimulation, suppresses lymphocytes and allows infections to progress. On the other hand, insufficient adrenergic control of immune responses allows progression of several autoimmune diseases.

ß-Adrenergic Signaling in Mice Housed at Standard Temperatures Suppresses an Effector Phenotype in CD8+ T Cells and Undermines Checkpoint Inhibitor Therapy.

The immune context of tumors has significant prognostic value and is predictive of responsiveness to several forms of therapy, including immunotherapy. We report here that CD8+ T-cell frequency and functional orientation within the tumor microenvironment is regulated by ß2-adrenergic receptor (ß-AR) signaling in host immune cells. We used three strategies-physiologic (manipulation of ambient thermal environment), pharmacologic (ß-blockers), and genetic (ß2-AR knockout mice) to reduce adrenergic stress signaling in two widely studied preclinical mouse tumor models. Reducing ß-AR signaling facilitated conversion of tumors to an immunologically active tumor microenvironment with increased intratumoral frequency of CD8+ T cells with an effector phenotype and decreased expression of programmed death receptor-1 (PD-1), in addition to an elevated effector CD8+ T-cell to CD4+ regulatory T-cell ratio (IFNγ+CD8+:Treg). Moreover, this conversion significantly increased the efficacy of anti-PD-1 checkpoint blockade. These data highlight the potential of adrenergic stress and norepinephrine-driven ß-AR signaling to regulate the immune status of the tumor microenvironment and support the strategic use of clinically available ß-blockers in patients to improve responses to immunotherapy. Cancer Res; 77(20); 5639-51. ©2017 AACR.