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      Radiation-Induced Lung Injury (RILI)

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          Abstract

          Radiation pneumonitis (RP) and radiation fibrosis (RF) are two dose-limiting toxicities of radiotherapy (RT), especially for lung, and esophageal cancer. It occurs in 5–20% of patients and limits the maximum dose that can be delivered, reducing tumor control probability (TCP) and may lead to dyspnea, lung fibrosis, and impaired quality of life. Both physical and biological factors determine the normal tissue complication probability (NTCP) by Radiotherapy. A better understanding of the pathophysiological sequence of radiation-induced lung injury (RILI) and the intrinsic, environmental and treatment-related factors may aid in the prevention, and better management of radiation-induced lung damage. In this review, we summarize our current understanding of the pathological and molecular consequences of lung exposure to ionizing radiation, and pharmaceutical interventions that may be beneficial in the prevention or curtailment of RILI, and therefore enable a more durable therapeutic tumor response.

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          PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation

          Hypoxia is a common feature of solid tumors (Semenza, 2011). Hypoxic zones in tumors attract immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs; Corzo et al., 2010), tumor-associated macrophages (TAMs; Doedens et al., 2010; Imtiyaz et al., 2010), and regulatory T cells (T reg cells; Clambey et al., 2012). MDSCs are a heterogeneous group of relatively immature myeloid cells and several studies have described mechanisms of MDSC-mediated immune suppression (Gabrilovich et al., 2012). A large body of preclinical and clinical data indicates that antibody blockade of immune checkpoints can significantly enhance antitumor immunity (Pardoll, 2012; West et al., 2013). Recently, antibody-mediated blockade of preprogrammed death 1 (PD-1; Topalian et al., 2012) and its ligand, PD-L1 (Brahmer et al., 2012), was shown to result in durable tumor regression and prolonged stabilization of disease in patients with advanced cancers. PD-1, a cell surface glycoprotein with a structure similar to cytotoxic T lymphocyte antigen 4 (CTLA-4), belongs to the B7 family of co-stimulatory/co-inhibitory molecules and plays a key part in immune regulation (Greenwald et al., 2005). PD-1 has two known ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC). Although hypoxia has been shown to regulate the function and differentiation of MDSCs (Corzo et al., 2010), several major questions remain unresolved. The influence of hypoxia on the regulation of immune checkpoint receptors (PD-1 and CTLA-4) and their respective ligands (PD-L1, PD-L2, CD80, and CD86) on MDSCs remains largely obscure. Furthermore, the potential contribution of these immune checkpoint receptors and their respective ligands on MDSC function under hypoxia is still unknown. In the present study, we showed that hypoxia via hypoxia-inducible factor-1α (HIF-1α) selectively up-regulated PD-L1 on MDSCs, but not other B7 family members, by binding directly to the HRE in the PD-L1 proximal promoter. Blockade of PD-L1 under hypoxia abrogated MDSC-mediated T cell suppression by modulating MDSCs cytokine production. RESULTS AND DISCUSSION Differential expression of PD-L1 on tumor-infiltrating MDSCs versus splenic MDSCs and selective up-regulation of PD-L1 in splenic MDSCs under hypoxic stress We first compared the level of expression of PD-L1 and PD-L2 between splenic MDSCs and tumor-infiltrating MDSCs from tumor-bearing mice. We found that the percentage of PD-L1+ cells was significantly higher on tumor-infiltrating MDSCs as compared with splenic MDSC in B16-F10, LLC (Fig. 1 A), CT26, and 4T1 (Fig. 1 B) tumor models. No significant difference was found in the percentage of PD-L2+ cells in splenic MDSCs as compared with tumor-infiltrating MDSCs in four tumor models tested (Fig. 1 C). We did not observe any significant difference in the expression levels of other members of the B7 family such as CD80, CD86, PD-1, and CTLA-4 on MDSCs from spleen and tumor (unpublished data). Youn et al. (2008) previously observed no significant differences in the percentage of PD-L1+ or CD80+ cells within the splenic MDSCs from tumor-bearing mice and immature myeloid cells from naive tumor-free mice. However, by comparing the expression of immune checkpoint inhibitors between splenic and tumor-infiltrating MDSCs, we showed that there is a differential expression of PD-L1 on tumor-infiltrating MDSCs. Figure 1. Tumor-infiltrating MDSCs differentially express PD-L1 as compared with splenic MDSCs, and hypoxia selectively up-regulates PD-L1 on splenic MDSCs in tumor-bearing mice. Surface expression level of PD-L1 and PD-L2 on Gr1+ CD11b+ cells (MDSCs) from (B16-F10 and LLC; A; CT26 and 4T1; B) in spleens (black dotted line histogram) and tumor (black line histogram) as compared with isotype control (gray-shaded histogram) was analyzed by flow cytometry. (C) Statistically significant differences (indicated by asterisks) between tumor-infiltrating MDSCs and splenic MDSCs are shown (*, P 20 fold for HRE-4), comparable to their binding to an established HRE in VEGF, LDHA, and Glut1 genes. To determine whether this HIF-1α site (HRE-4) was a transcriptionally active HRE, MSC-1 cells were co-transfected with pGL4-hRluc/SV40 vector and pGL3 EV, pGL3 HRE-4, or pGL3 HRE-4 MUT vectors (Fig. 3 M) and grown under normoxia or hypoxia. After 48 h, firefly and renilla luciferase activities were measured. As shown in Fig. 3 N, hypoxia significantly increased the luciferase activity of HRE-4 reporter by more than threefold as compared with normoxia. More interestingly, the luciferase activity of HRE-4 MUT was significantly decreased (>50%) as compared with HRE-4 under hypoxia (Fig. 3 N). The results presented in Figs. 3 (H–N) demonstrate that PD-L1 is a direct HIF-1α target gene in MSC-1 cells. Thus, we provide evidence here that HIF-1α is a major regulator of PD-L1 mRNA and protein expression, and that HIF-1α regulates the expression of PD-L1 by binding directly to the HRE-4 in the PD-L1 proximal promoter. Blocking PD-L1 decreases MDSC-mediated T cell suppression under hypoxia by down-regulating MDSC IL-6 and IL-10 To directly test the functional consequences of hypoxia-induced up-regulation of PD-L1 in MDSC-mediated T cell suppression, the expression of PD-L1 was blocked on ex vivo MDSCs by using anti–PD-L1 monoclonal antibody. Hypoxia increased the ability of MDSCs to suppress both specific and nonspecific stimuli-mediated T cell proliferation (Fig. 4, A and B). Interestingly, blockade of PD-L1 under hypoxia significantly abrogated the suppressive activity of MDSCs in response to both nonspecific stimuli (anti-CD3/CD28 antibody; Fig. 4 A) and specific stimuli (TRP-2(180–88) peptide; Fig. 4 B). Under hypoxia, MDSCs acquired the ability to inhibit T cell function (Fig. 4, C and D) by decreasing the percentage of IFN-γ+ CD8+ and CD4+ T cells; whereas the percentage of IFN-γ+ CD8+ (Fig. 4 C) and IFN-γ+ CD4+ T cells (Fig. 4 D) significantly increased after PD-L1 blockade under hypoxic conditions. Thus, the immune suppressive function of MDSCs enhanced under hypoxia was abrogated after blocking PD-L1, and hypoxic up-regulation of PD-L1 on MDSCs is involved in mediating the suppressive action of MDSCs, at least in part, as we were not able to completely restore T cell proliferation and function after PD-L1 blockade on MDSCs under hypoxia. Figure 4. Blockade of PD-L1 under hypoxia down-regulates MDSC IL-6 and IL-10 and enhances T cell proliferation and function. MDSCs isolated from spleens of B16-F10 tumor-bearing mice were pretreated for 30 min on ice with 5 µg/ml control antibody (IgG) or antibody against PD-L1 (PDL1 Block) and co-cultured with splenocytes under normoxia and hypoxia for 72 h. (A and B) Effect of MDSC on proliferation of splenocytes stimulated with (A) anti-CD3/CD28 coated beads or (B) TRP-2(180–88) peptide under the indicated conditions. Cell proliferation was measured in triplicates by [3H]thymidine incorporation and expressed as counts per minute (CPM). (C and D) MDSCs were cultured with splenocytes from B16-F10 mice stimulated with anti-CD3/CD28. Intracellular IFN-γ production was evaluated by flow cytometry by gating on (C) CD3+CD8+ IFN-γ+ and (D) CD3+CD4+ IFN-γ+ populations. Statistically significant differences (indicated by asterisks) are shown (**, P 95% as evaluated by FACS analysis. MDSC functional assays. For evaluation of T cell proliferation, splenocytes from B16-F10 mice were plated into U-bottom 96-well plates along with MDSCs at different ratios (50,000 MDSC:200,000 splenocytes/well). Plates were stimulated with either anti-CD3/CD28 beads (Miltenyi Biotec) or TRP-2 180–88 peptide for 72 h at 37°C. Co-cultures were pulsed with thymidine (1 µCi/well; Promega) for 16–18 h before harvesting, and [3H]thymidine uptake was counted using Packard’s TopCount NXT liquid scintillation counter and expressed as counts per minute (CPM). For assessment of T cell functions, MDSCs co-cultured with splenocytes from B16-F10 mice were stimulated with anti-CD3/CD28 beads. After 72 h, intracellular IFN-γ production was evaluated by flow cytometry by gating on CD3+CD8+ IFN-γ+ and CD3+CD4+ IFN-γ+ populations. MDSCs cytokine production (ELISA). MDSCs isolated from spleens of B16-F10 tumor-bearing mice were pretreated for 30 min on ice with 5 μg/ml control antibody (IgG) or Anti-Mouse PD-L1 (B7-H1) Functional Grade Purified antibody 5 µg/ml (clone MIH5; eBioscience; PDL1 Block) and cultured under normoxia and hypoxia for 72 h. Supernatants were collected and the secretion of IL-6, IL-10, and IL-12p70 (eBioscience) was determined by ELISA. ChIP assay. ChIP was performed with lysates prepared from MSC-1 by using SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling Technology). SYBR Green RT-qPCR was performed using the primers detailed in Table S1. Arginase enzymatic activity and NO (nitric oxide) production. Arginase activity was measured in MDSC cell lysates, and for NO production, culture supernatants were mixed with Greiss reagent and nitrite concentrations were determined as described earlier (Youn et al., 2008). Luciferase reporter assay. A 653-bp section corresponding to mouse PD-L1 promoter containing HRE4 sequence was inserted into the NheI–XhoI sites of pGL3-Basic vector (Promega). Mutation of HRE4 was performed by site-directed mutagenesis and verified by sequencing. A 56-bp mouse PD-L1 gene sequence was inserted into the Bgl II site of pGL3-Promoter (Promega). MSC-1 cells were co-transfected with 0.2 µg of pGL4-hRluc/SV40 vector (which contains renilla luciferase sequences downstream of the SV40 promoter) and 1 µg of pGL3 empty vector, pGL3 HRE-4, or pGL3 HRE-4 MUT vectors in 6-well plates with Lipofectamine 2000 (Invitrogen) in OPTIMEM (Invitrogen) medium and grown under normoxia or hypoxia. After 48 h, firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter assay (Promega) and the ratio of firefly/Renilla luciferase was determined. Statistics. Data were analyzed with GraphPad Prism. Student’s t test was used for single comparisons. Online supplemental material. Table S1 shows genomic oligonucleotide primers used for amplification of immunoprecipitated DNA samples from ChIP assays. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20131916/DC1. Supplementary Material Supplemental Material
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            Quantitative Analyses of Normal Tissue Effects in the Clinic (QUANTEC): an introduction to the scientific issues.

            Advances in dose-volume/outcome (or normal tissue complication probability, NTCP) modeling since the seminal Emami paper from 1991 are reviewed. There has been some progress with an increasing number of studies on large patient samples with three-dimensional dosimetry. Nevertheless, NTCP models are not ideal. Issues related to the grading of side effects, selection of appropriate statistical methods, testing of internal and external model validity, and quantification of predictive power and statistical uncertainty, all limit the usefulness of much of the published literature. Synthesis (meta-analysis) of data from multiple studies is often impossible because of suboptimal primary analysis, insufficient reporting and variations in the models and predictors analyzed. Clinical limitations to the current knowledge base include the need for more data on the effect of patient-related cofactors, interactions between dose distribution and cytotoxic or molecular targeted agents, and the effect of dose fractions and overall treatment time in relation to nonuniform dose distributions. Research priorities for the next 5-10 years are proposed. Copyright 2010 Elsevier Inc. All rights reserved.
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              Highly efficient generation of airway and lung epithelial cells from human pluripotent stem cells

              The ability to generate lung and airway epithelial cells from human pluripotent stem cells (hPSCs) would have applications in regenerative medicine, drug screening and modeling of lung disease, and studies of human lung development. We established, based on developmental paradigms, a highly efficient method for directed differentiation of hPSCs into lung and airway epithelial cells. Long-term differentiation in vivo and in vitro yielded basal, goblet, Clara, ciliated, type I and type II alveolar epithelial cells. Type II alveolar epithelial cells generated were capable of surfactant protein-B uptake and stimulated surfactant release, providing evidence of specific function. Inhibiting or removing agonists to signaling pathways critical for early lung development in the mouse—retinoic acid, Wnt and BMP—recapitulated defects in corresponding genetic mouse knockouts. The capability of this protocol to generate most cell types of the respiratory system suggests its utility for deriving patient-specific therapeutic cells.
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                Author and article information

                Contributors
                Journal
                Front Oncol
                Front Oncol
                Front. Oncol.
                Frontiers in Oncology
                Frontiers Media S.A.
                2234-943X
                06 September 2019
                2019
                : 9
                Affiliations
                Department of Radiotherapy, GROW School for Oncology Maastricht University Medical Centre , Maastricht, Netherlands
                Author notes

                Edited by: Minesh P. Mehta, Baptist Health South Florida, United States

                Reviewed by: Zhongxing Liao, University of Texas MD Anderson Cancer Center, United States; Yevgeniy Vinogradskiy, University of Colorado Denver, United States

                *Correspondence: Marc A. Vooijs marc.vooijs@ 123456maastrichtuniversity.nl

                This article was submitted to Radiation Oncology, a section of the journal Frontiers in Oncology

                Article
                10.3389/fonc.2019.00877
                6743286
                df8c7f89-bbdd-42db-9829-1aaafc562adc
                Copyright © 2019 Giuranno, Ient, De Ruysscher and Vooijs.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                Page count
                Figures: 1, Tables: 1, Equations: 0, References: 206, Pages: 16, Words: 14750
                Funding
                Funded by: H2020 Marie Skłodowska-Curie Actions 10.13039/100010665
                Categories
                Oncology
                Review

                Oncology & Radiotherapy
                radiotherapy,adverse effects,rili,rilt,pneumonitis,fibrosis,lung
                Oncology & Radiotherapy
                radiotherapy, adverse effects, rili, rilt, pneumonitis, fibrosis, lung

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