Paul Thevenot

Subpopulations of myeloid‐derived suppressor cells impair T cell responses through independent nitric oxide‐related pathways

The accumulation of myeloid‐derived suppressor cells (MDSC) in tumor‐bearing hosts is a hallmark of malignancy‐associated inflammation and a major mediator for the induction of T cell suppression in cancer. MDSC can be divided phenotypically into granulocytic (G‐MDSC) and monocytic (Mo‐MDSC) subgroups. Several mechanisms mediate the induction of T cell anergy by MDSC; however, the specific role of these pathways in the inhibitory activity of MDSC subpopulations remains unclear. Therefore, we aimed to determine the effector mechanisms by which subsets of tumor‐infiltrating MDSC block T cell function. We found that G‐MDSC had a higher ability to impair proliferation and expression of effector molecules in activated T cells, as compared to Mo‐MDSC. Interestingly, both MDSC subgroups inhibited T cells through nitric oxide (NO)‐related pathways, but expressed different effector inhibitory mechanisms. Specifically, G‐MDSC impaired T cells through the production of peroxynitrites (PNT), while Mo‐MDSC suppressed by the release of NO. The production of PNT in G‐MDSC depended on the expression of gp91phox and endothelial NO synthase (eNOS), while inducible NO synthase (iNOS) mediated the generation of NO in Mo‐MDSC. Deletion of eNOS and gp91phox or scavenging of PNT blocked the suppressive function of G‐MDSC and induced anti‐tumoral effects, without altering Mo‐MDSC inhibitory activity. Furthermore, NO‐scavenging or iNOS knockdown prevented Mo‐MDSC function, but did not affect PNT production or suppression by G‐MDSC. These results suggest that MDSC subpopulations utilize independent effector mechanisms to regulate T cell function. Inhibition of these pathways is expected to specifically block MDSC subsets and overcome immune suppression in cancer.

l-Arginine Depletion Blunts Antitumor T-cell Responses by Inducing Myeloid-Derived Suppressor Cells

Enzymatic depletion of the nonessential amino acid l-Arginine (l-Arg) in patients with cancer by the administration of a pegylated form of the catabolic enzyme arginase I (peg-Arg I) has shown some promise as a therapeutic approach. However, l-Arg deprivation also suppresses T-cell responses in tumors. In this study, we sought to reconcile these observations by conducting a detailed analysis of the effects of peg-Arg I on normal T cells. Strikingly, we found that peg-Arg I blocked proliferation and cell-cycle progression in normal activated T cells without triggering apoptosis or blunting T-cell activation. These effects were associated with an inhibition of aerobic glycolysis in activated T cells, but not with significant alterations in mitochondrial oxidative respiration, which thereby regulated survival of T cells exposed to peg-Arg I. Further mechanistic investigations showed that the addition of citrulline, a metabolic precursor for l-Arg, rescued the antiproliferative effects of peg-Arg I on T cells in vitro. Moreover, serum levels of citrulline increased after in vivo administration of peg-Arg I. In support of the hypothesis that peg-Arg I acted indirectly to block T-cell responses in vivo, peg-Arg I inhibited T-cell proliferation in mice by inducing accumulation of myeloid-derived suppressor cells (MDSC). MDSC induction by peg-Arg I occurred through the general control nonrepressed-2 eIF2α kinase. Moreover, we found that peg-Arg I enhanced the growth of tumors in mice in a manner that correlated with higher MDSC numbers. Taken together, our results highlight the risks of the l-Arg-depleting therapy for cancer treatment and suggest a need for cotargeting MDSC in such therapeutic settings.

Environmentally persistent free radicals induce airway hyperresponsiveness in neonatal rat lungs.

BackgroundIncreased asthma risk/exacerbation in children and infants is associated with exposure to elevated levels of ultrafine particulate matter (PM). The presence of a newly realized class of pollutants, environmentally persistent free radicals (EPFRs), in PM from combustion sources suggests a potentially unrecognized risk factor for the development and/or exacerbation of asthma.MethodsNeonatal rats (7-days of age) were exposed to EPFR-containing combustion generated ultrafine particles (CGUFP), non-EPFR containing CGUFP, or air for 20 minutes per day for one week. Pulmonary function was assessed in exposed rats and age matched controls. Lavage fluid was isolated and assayed for cellularity and cytokines and in vivo indicators of oxidative stress. Pulmonary histopathology and characterization of differential protein expression in lung homogenates was also performed.ResultsNeonates exposed to EPFR-containing CGUFP developed significant pulmonary inflammation, and airway hyperreactivity. This correlated with increased levels of oxidative stress in the lungs. Using differential two-dimensional electrophoresis, we identified 16 differentially expressed proteins between control and CGUFP exposed groups. In the rats exposed to EPFR-containing CGUFP; peroxiredoxin-6, cofilin1, and annexin A8 were upregulated.ConclusionsExposure of neonates to EPFR-containing CGUFP induced pulmonary oxidative stress and lung dysfunction. This correlated with alterations in the expression of various proteins associated with the response to oxidative stress and the regulation of glucocorticoid receptor translocation in T lymphocytes.

Radical-Containing Ultrafine Particulate Matter Initiates Epithelial-to-Mesenchymal Transitions in Airway Epithelial Cells

Environmentally persistent free radicals (EPFRs) in combustion-generated particulate matter (PM) are capable of inducing pulmonary pathologies and contributing to the development of environmental asthma. In vivo exposure of infant rats to EPFRs demonstrates their ability to induce airway hyperresponsiveness to methacholine, a hallmark of asthma. However, the mechanisms by which combustion-derived EPFRs elicit in vivo responses remain elusive. In this study, we used a chemically defined EPFR consisting of approximately 0.2 μm amorphrous silica containing 3% cupric oxide with the organic pollutant 1,2-dichlorobenzene (DCB-230). DCB-230 possesses similar radical content to urban-collected EPFRs but offers several advantages, including lack of contaminants and chemical uniformity. DCB-230 was readily taken up by BEAS-2B and at high doses (200 μg/cm(2)) caused substantial necrosis. At low doses (20 μg/cm(2)), DCB-230 particles caused lysosomal membrane permeabilization, oxidative stress, and lipid peroxidation within 24 hours of exposure. During this period, BEAS-2B underwent epithelial-to-mesenchymal transition (EMT), including loss of epithelial cell morphology, decreased E-cadherin expression, and increased α-smooth muscle actin (α-SMA) and collagen I production. Similar results were observed in neonatal air-liquid interface culture (i.e., disruption of epithelial integrity and EMT). Acute exposure of infant mice to DCB-230 resulted in EMT, as confirmed by lineage tracing studies and evidenced by coexpression of epithelial E-cadherin and mesenchymal α-SMA proteins in airway cells and increased SNAI1 expression in the lungs. EMT in neonatal mouse lungs after EPFR exposure may provide an explanation for epidemiological evidence supporting PM exposure and increased risk of asthma.

Model Combustion-Generated Particulate Matter Containing Persistent Free Radicals Redox Cycle to Produce Reactive Oxygen Species

Particulate matter (PM) is emitted during thermal decomposition of waste. During this process, aromatic compounds chemisorb to the surface of metal-oxide-containing PM, forming a surface-stabilized environmentally persistent free radical (EPFR). We hypothesized that EPFR-containing PM redox cycle to produce ROS and that this redox cycle is maintained in biological environments. To test our hypothesis, we incubated model EPFRs with the fluorescent probe dihydrorhodamine (DHR). Marked increases in DHR fluorescence were observed. Using a more specific assay, hydroxyl radicals ((•)OH) were also detected, and their level was further increased by cotreatment with thiols or ascorbic acid (AA), known components of epithelial lining fluid. Next, we incubated our model EPFR in bronchoalveolar lavage fluid (BALF) or serum. Detection of EPFRs and (•)OH verified that PM generate ROS in biological fluids. Moreover, incubation of pulmonary epithelial cells with EPFR-containing PM increased (•)OH levels compared to those in PM lacking EPFRs. Finally, measurements of oxidant injury in neonatal rats exposed to EPFRs by inhalation suggested that EPFRs induce an oxidant injury within the lung lining fluid and that the lung responds by increasing antioxidant levels. In summary, our EPFR-containing PM redox cycle to produce ROS, and these ROS are maintained in biological fluids and environments. Moreover, these ROS may modulate toxic responses of PM in biological tissues such as the lung.

Rescue of Notch 1 signaling in antigen-specific CD8+ T cells overcomes tumor-induced T cell suppression and enhances immunotherapy in cancer

Sierra and colleagues show that myeloid-derived suppressor cells (MDSC) blocked Notch expression in T cells via nitric oxide–dependent mechanisms, and overexpression of the Notch 1 intracellular active domain rendered the CD8+ T cells resistant to the MDSC-induced tolerogenic effect. An impaired antitumor immunity is found in patients with cancer and represents a major obstacle in the successful development of different forms of immunotherapy. Signaling through Notch receptors regulates the differentiation and function of many cell types, including immune cells. However, the effect of Notch in CD8+ T-cell responses in tumors remains unclear. Thus, we aimed to determine the role of Notch signaling in CD8+ T cells in the induction of tumor-induced suppression. Our results using conditional knockout mice show that Notch-1 and Notch-2 were critical for the proliferation and IFNγ production of activated CD8+ T cells and were significantly decreased in tumor-infiltrating T cells. Conditional transgenic expression of Notch-1 intracellular domain (N1IC) in antigen-specific CD8+ T cells did not affect activation or proliferation of CD8+ T cells, but induced a central memory phenotype and increased cytotoxicity effects and granzyme B levels. Consequently, a higher antitumor response and resistance to tumor-induced tolerance were found after adoptive transfer of N1IC-transgenic CD8+ T cells into tumor-bearing mice. Additional results showed that myeloid-derived suppressor cells (MDSC) blocked the expression of Notch-1 and Notch-2 in T cells through nitric oxide–dependent mechanisms. Interestingly, N1IC overexpression rendered CD8+ T cells resistant to the tolerogenic effect induced by MDSC in vivo. Together, the results suggest the key role of Notch in the suppression of CD8+ T-cell responses in tumors and the therapeutic potential of N1IC in antigen-specific CD8+ T cells to reverse T-cell suppression and increase the efficacy of T cell–based immunotherapies in cancer. Cancer Immunol Res; 2(8); 800–11. ©2014 AACR.

Early-life exposure to combustion-derived particulate matter causes pulmonary immunosuppression

Elevated levels of combustion-derived particulate matter (CDPM) are a risk factor for the development of lung diseases such as asthma. Studies have shown that CDPM exacerbates asthma, inducing acute lung dysfunction and inflammation; however, the impact of CDPM exposure on early immunological responses to allergens remains unclear. To determine the effects of early-life CDPM exposure on allergic asthma development in infants, we exposed infant mice to CDPM and then induced a mouse model of asthma using house dust mite (HDM) allergen. Mice exposed to CDPM+HDM failed to develop a typical asthma phenotype including airway hyper-responsiveness, T-helper type 2 (Th2) inflammation, Muc5ac expression, eosinophilia, and HDM-specific immunoglobulin (Ig) compared with HDM-exposed mice. Although HDM-specific IgE was attenuated, total IgE was twofold higher in CDPM+HDM mice compared with HDM mice. We further demonstrate that CDPM exposure during early life induced an immunosuppressive environment in the lung, concurrent with increases in tolerogenic dendritic cells and regulatory T cells, resulting in the suppression of Th2 responses. Despite having early immunosuppression, these mice develop severe allergic inflammation when challenged with allergen as adults. These findings demonstrate a mechanism whereby CDPM exposure modulates adaptive immunity, inducing specific antigen tolerance while amplifying total IgE, and leading to a predisposition to develop asthma upon rechallenge later in life.

T cells conditioned with MDSC show an increased anti-tumor activity after adoptive T cell based immunotherapy

The success of adoptive T cell-based immunotherapy (ACT) in cancer is limited in part by the accumulation of myeloid-derived suppressor cells (MDSC), which block several T cell functions, including T cell proliferation and the expression of various cytotoxic mediators. Paradoxically, the inhibition of CD8+ T cell differentiation into cytotoxic populations increased their efficacy after ACT into tumor-bearing hosts. Therefore, we aimed to test the impact of conditioning CD8+ T cells with MDSC on their differentiation potential and ACT efficacy. Our results indicate that MDSC impaired the progression of CD8+ T cells into effector populations, without altering their activation status, production of IL-2, or signaling through the T cell receptor. In addition, culture of CD8+ T cells with MDSC resulted in an increased ACT anti-tumor efficacy, which correlated with a higher frequency of the transferred T cells and elevated IFNγ production. Interestingly, activated CD62L+ CD8+ Tcells were responsible for the enhanced anti-tumor activity showed by MDSC-exposed T cells. Additional results showed a decreased protein synthesis rate and lower activity of the mammalian/mechanistic target of rapamycin (mTOR) in T cells conditioned with MDSC. Silencing of the negative mTOR regulator tuberous sclerosis complex-2 in T cells co-cultured with MDSC restored mTOR activity, but resulted in T cell apoptosis. These results indicate that conditioning of T cells with MDSC induces stress survival pathways mediated by a blunted mTOR signaling, which regulated T cell differentiation and ACT efficacy. Continuation of this research will enable the development of better strategies to increase ACT responses in cancer.

Antigen-specific T cells conditioned with MDSC display a surprising increased anti-tumor activity after adoptive T cell-based immunotherapy

The success of adoptive T cell-based immunotherapy (ACT) in cancer is limited in part by the accumulation of myeloid-derived suppressor cells (MDSC), which block several T cell functions, including proliferation and expression of effector mediators. Paradoxically, inhibition of CD8+ T cell differentiation during the pre-ACT phase also showed to increase anti-tumor efficacy. Thus, we aimed to determine the effect of MDSC on T cell differentiation and on ACT efficacy after in vitro conditioning of CD8+ T cells. Results indicate that MDSC block the differentiation of CD8+ T cells into effector cells, without altering their activation status, production of IL-2, or signaling through the T cell receptor. Moreover, culturing of CD8+ T cells in the presence of MDSC resulted in an increased ACT anti-tumor activity and elevated frequency and IFNγ production after transfer onto tumor-bearing mice. Additional findings confirmed that undifferentiated CD62L+ T cells mediated the enhanced anti-tumor activity triggered by MDSC-exposed T cells. Mechanistic studies showed that MDSC restricted de novo protein synthesis and activity of mechanistic target of rapamycin (mTOR) in T cells. Silencing of the negative mTOR regulator tuberous sclerosis complex 2 restored mTOR activity in T cells co-cultured with MDSC, but resulted in T cell apoptosis. Thus, our results indicate that culturing of CD8+ T cells in the presence of MDSC improves their anti-tumor efficacy, frequency, and function after ACT possibly through inhibition of mTOR signaling. Continuation of this research will enable the development of novel strategies to enhance the efficacy of ACT in cancer.

Tumor derived stress triggers C/EBPβ homologous protein (Chop) expression in myeloid derived suppressor cells (MDSC) and mediates immunosuppressive activity

Suppression of anti-tumor T cell responses by MDSC remains a significant barrier in cancer immunotherapy. Although several pathways have been characterized as critical for MDSC-induced suppression, there are currently no therapies to globally and specifically inhibit MDSC function. We postulate that identifying and inhibiting the central mediators of MDSC-regulatory activity will overcome T cell suppression and increase the efficacy of T cell-based immunotherapy in cancer. We aimed to determine the role of the common stress sensor C/EBP-homologous-stress-related protein (Chop), a downstream product of integrated stress responses, as a master regulator of MDSC-suppressive activity. Our results show that Chop is preferentially expressed in malignant cells and MDSC in s.c. mouse tumors. Selective expression of Chop was also detected in tumor-infiltrating MDSC from colon carcinoma patients. Interestingly, injection of tumor cells having functional Chop into systemic Chop -/- mice or Chop null bone marrow chimeric mice resulted in a significant antitumor effect mediated by CD8 + T cells, suggesting the importance of MDSC-Chop in tumor-induced tolerance. In fact, deletion of Chop in MDSC increased the efficacy of T cell-based immunotherapy. MDSC isolated from tumor-bearing Chop null mice had decreased ability to block T cell responses; impaired expression of major MDSC-inhibitory pathways; and a surprising ability to prime T cell proliferation and induce anti-tumor effects. Accordingly, depletion of Gr-1 + MDSC restored tumor growth in Chop -/- mice, while it prevented tumor growth in wild type mice, confirming functional differences in MDSC from wild type and Chop -/- mice. To therapeutically block Chop in tumors, we used a specific liposomal-encapsulated siRNA, which successfully blocked Chop expression and induced anti-tumor effects. We next examined the effects of Chop on C/EBPb and STAT-3, both master regulators of MDSC function. MDSC from Chop -/- mice had elevated expression of C/EBPb inhibitory isoform LIP, low C/EBPb binding to IL-6-promoter, decreased IL-6 production, and impaired expression of IL-6 target phospho-STAT-3. Also, Chop -/- MDSC expressed higher levels of miR-142-3p, a mi-RNA that promotes C/EBPb LIP over LAP and LAP*. Ectopic expression of IL-6 in tumors restored tumor growth, MDSC suppression, and C/EBPb and phospho-STAT-3 levels in Chop -/- mice, suggesting the role of this pathway in the effects induced by Chop deletion. Collectively, this data suggests the role of Chop as a master regulator of the immune inhibitory activity of MDSC and justify the potential targeting of Chop as a way to restore protective immunity in cancer. Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.