Targeting MDSC-mediated immunosuppression in melanoma

Immunotherapeutic strategies for malignant melanoma have made significant progress but are still challenged by the development of resistance in a subset of patients. One of the factors contributing to this resistance is the accumulation of myeloid-derived suppressor cells (MDSC) in the melanoma microenvironment. MDSC are a heterogeneous population of myeloid cells that can inhibit anti-tumor T cell responses, thereby promoting immunosuppression and facilitating tumor progression. TLR4 and RAGE signaling are considered as important regulators of MDSC accumulation and acquisition of their immunosuppressive functions. Damage-associated molecular patterns (DAMPs) such as S100A8/9, high mobility group box 1 (HMGB1), and heat shock proteins (HSPs) that act as ligands for TLR4 or RAGE were reported to drive MDSC accumulation and to be significantly expressed in the tumor microenvironment (TME) of solid tumors. However, a precise role of TLR4 and RAGE signaling in the acquisition of immunosuppressive properties by MDSC requires further elucidation. The present study aims to evaluate the impact of endogenous TLR4 ligands and TLR4 signaling on MDSC-mediated immune suppression in malignant melanoma. MDSC were purified from the peripheral blood of late-stage melanoma patients and were generated in vitro from healthy donor-derived monocytes. Normal monocytes were treated with recombinant (r) HSP90a for 24h or with rS100A9 and rHMGB1 in the presence of GM-CSF for 72 h. The immunosuppressive capacity of MDSC and stimulated monocytes were assessed in T cell inhibition assays. In addition, TLR4 inhibitor (Resatorvid) and RAGE inhibitor (FPS-ZM1) were tested in these assays. Expression of immunosuppression related markers and pathways involved in the MDSC stimulation were assessed by flow cytometry, Western Blot, and gene expression profiling. The levels of S100A8/9 and HMGB1 were measured in the plasma of melanoma patients by ELISA. The Cancer Genome Atlas (TCGA) data analysis was performed to evaluate the association between the expression of immunosuppressive markers and S100A9 and HMGB1 in the TME of melanoma. Stimulation of monocytes with HSP90a, S100A9 and HMGB1 resulted in the acquisition of suppressive activity against T cells via increased production of reactive oxygen species (ROS) and nitric oxide (NO) as well as upregulated expression of programmed death ligand-1 (PD-L1) and indoleamine 2,3-dioxygenase (IDO). Increased plasma levels of S100A8/9 was found to be correlated with the expression of immunosuppressive markers on MDSC in the peripheral blood of melanoma patients. Importantly, the blockade of TLR4 signaling and, to a lesser extent RAGE signaling, resulted in a substantial attenuation of T cell inhibition. The supernatant of in vitro generated MDSC contained a significantly higher levels of S100A8/9 and HMGB1 than in that from patient derived MDSC Furthermore, elevated plasma levels of S100A8/9 were found to be associated with a poor progression free survival (PFS) in melanoma patients. In conclusion, this study highlights the crucial role of TLR4 and, to a lesser extent RAGE signaling, as well as TLR4 ligands S100A9 and HMGB1 in the acquisition of immunosuppressive properties by MDSC in malignant melanoma. These findings suggest that targeting TLR4 signaling pathway may represent a promising therapeutic strategy to overcome MDSC-mediated immune suppression and enhance the efficacy of melanoma immunotherapy

Evaluation of T Cell Responses In The Co-Cultures Establıshed Wıth Neutrophıls, Monocytes, and Lung Adenocarcınoma Cells

CD4+ and CD8+ T cells are critical mediators in anti-tumor immunity. Together with neutrophils, they have been shown to dominate the immune landscape of the non-small cell lung cancer (NSCLC). A number of studies have estimated the prognostic significance of neutrophil-to-lymphocyte ratio in NSCLC. However, immune regulatory role of neutrophils has not yet been fully elucidated. Hence, this study aims to evaluate T cell responses in the presence of neutrophils, monocytes and lung adenocarcinoma cell lines, in vitro. Peripheral blood neutrophils, CD14+ monocytes, and CD8+ or CD4+ T cells were purified from the healthy volunteers. In order to optimize the co-cultures, different combinations of these cell types were employed under various stimuli. Neutrophils were stimulated with different combinations of IFN-γ, G-CSF, N-acetylcysteine (NAC), and N-Formylmethionine-leucyl-phenylalanine (fMLP). At different ratios, pre-stimulated or freshly isolated neutrophils were co-cultured with NSCLC cell lines media (A549, NCI-H1299, or NCI-H441) or their conditioned with or without monocytes. Soluble anti-human CD3 mAb was added in order to test the antigen-independent influences on T cells. Proliferation, viability, activation, ROS production capacity, cytokine secretion, and expression of co-stimulatory molecules were tested on specific cell types. Based on TIM-3 expression, CD8+ T cells were recovered from the co-cultures and re-stimulated to test whether the TIM-3mod/high subpopulation is exhausted. G-CSF and NAC stimulation promoted the PMN longevity, diminished the ROS production, and enhanced the stimulatory capacity of PMNs on T cell proliferation. The presence of lung cancer cells and monocytes prolonged the survival of neutrophils. In co-cultures, neutrophils swiftly acquired an activated state with decreased CD62L. Besides, ROS production by neutrophils was decreased in the co-cultures. The presence of monocytes, neutrophils, and lung cancer cells enhanced CD8+ T proliferation and the expression of inhibitory receptors. Under certain conditions lacking monocytes as major supporters of T-cell proliferation, the presence of neutrophils together with lung adenocarcinoma cells, barely supported CD8+ T cell responses. TIM-3mod/high CD8+ T cells recovered from NSCLC co-cultures were not exhausted and displayed high proliferation and IFN-γ secretion. NAC stimulation did not modulate the CD8+ T cell proliferation or TIM-3/LAG3 expression in co-cultures. Expression of 4-1BBL, OX40L and B7-2 costimulatory genes were increased in the PMNs co-cultured with lung cancer cells, monocytes and CD8+ T cells. Consequently, in a co-culture setup employing neutrophils, monocytes, CD8+ T cells and lung cancer cells, which was established to partially model the tumor microenvironment, our findings indicate the importance of neutrophils as a critical modulator for CD8+ T cell responses.CONTENTS APPROVAL PAGE iii YAYIMLAMA VE FİKRİ MÜLKİYET HAKLARI BEYANI iv ETHICAL DECLARATION v ACKNOWLEDGEMENTS vi ABSTRACT vii ÖZET viii CONTENTS ix LIST OF ABBREVIATIONS xii FIGURES xv TABLES xviii 1. INTRODUCTION 1 2. LITERATURE OVERVIEW 4 2.1. Polymorphonuclear Leukocytes (PMNs) 4 2.1.1. Origin and Maturation of PMNs 4 2.1.2. Migration of PMNs 11 2.1.3. Functions of PMNs 15 2.1.4. Subsets of PMNs 17 2.1.5. Influence of PMNs on T cell responses 20 2.2. Lung Cancer 22 2.2.1. Classification of Lung Cancer 23 2.3. Immune Response in NSCLC 24 2.3.1. PMNs in NSCLC 27 2.3.2. T Cell Responses in NSCLC 28 2.3.3. Tumor-associated Macrophages in NSCLC 31 2.3.4. Other immune cells in NSCLC 31 3. MATERIALS AND METHODS 35 3.1. Materials Used in This Study 35 3.2. Buffers and Solutions 36 3.3. Cell Culture and Purification 37 3.3.1. Cell Culture 37 3.3.2. Freezing and Thawing of Cell Lines 38 3.3.3. Cell Counting 39 3.3.4. Isolation of the cells and Cell Sorting 40 3.3.5. Establishment of Co-cultures: 45 3.3.6. 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) cell viability assay: 46 3.4. Immunological Techniques 47 3.4.1. Flow Cytometry Analysis 47 3.5. Molecular Techniques 51 3.5.1. Total RNA isolation and spectrophotometric analysis 51 3.5.2. cDNA synthesis 51 3.5.3. Polymerase chain reaction (PCR) 52 3.5.4. Real-time PCR (RT-PCR) 54 3.5.5. Agarose gel electrophoresis 56 3.6. Statistical analysis 57 4. RESULTS 58 4.1. Preliminary analyses on PMN for the establishment of co-culture conditions 58 4.1.1. Assessment of the PMN viability 58 4.1.2. Assessment of PMN activation 66 4.1.3. The influence of PMNs on T cell proliferation 71 4.2. Assessment of CD8+ T cell responses in the co-cultures of PMNs, monocytes and NSCLC cells 77 4.2.1. CD8+ T cell proliferation in the co-cultures 77 4.2.2. CD8+ T cell activation in the co-cultures 78 4.2.3. CD8+ T cell-related cytokine production in the co-cultures 83 4.2.4. CD8+ T cell exhaustion in the co-cultures 85 4.3. Potential influence of PMN in the co-cultures 88 4.3.1. ROS production and CD8+ T cell responses 88 4.3.2. The costimulatory gene expression in PMNs 91 5. DISCUSSION 93 6. RESULTS AND RECOMMENDATION 106 7. REFERENCES 108 8.APPENDICES APPENDIX 1: Ethics Committee Approval APPENDIX 2: Scientific meetings where the data of this thesis were presented. APPENDIX 3: Thesis Orijinality Report. APPENDIX 4: Digital Receipt 9. CURRICULUM VITAECD4+ ve CD8+ T hücreleri, anti-tümör immün yanıtlarında kritik aracılardır. Nötrofillerle beraber, küçük hücreli dışı akciğer kanserinin (KHDAK) tümör mikroçevresinde fazla miktarlarda bulundukları gösterilmiştir. Ayrıca, bir dizi çalışma, akciğer kanserinde nötrofil/lenfosit oranının prognostik önemini göstermiştir. Bununla birlikte, nötrofillerin immün düzenleyici rolü henüz tam olarak açıklanamamıştır. Bu nedenle, bu çalışma, nötrofiller, monositler ve akciğer adenokarsinom hücrelerinin varlığında T hücre yanıtlarını değerlendirmeyi amaçlamaktadır. Periferik kan nötrofilleri, CD14+ monositler ve CD8+ ve CD4+ T hücreleri sağlıklı bireylerden toplanan kanlardan izole edilmiştir. Ko-kültürleri optimize etmek için, bu hücre tiplerinin farklı kombinasyonları ile ve çeşitli uyaranlar varlığında ko-kültürler oluşturulmuştur. Nötrofiller IFN-γ, G-CSF, NAC ve fMLP ile uyarılmıştır. Farklı oranlarda, önceden uyarılmış veya yeni izole edilmiş nötrofiller, KHDAK hücre hatları (A549, NCI-H1299 veya NCI-H441 ve/veya monositler) ile birlikte ko-kültür edilmiştir. T hücreleri üzerindeki antijenden bağımsız etkileri test etmek için çözünür anti-human CD3 monoklonal antikoru eklenmiştir. İmmün hücrelerin proliferasyonu, canlılığı, aktivasyonu, ROS üretim kapasitesi, sitokin üretimi, ve kostimülatör gen ekspresyonu belirli hücre tiplerinde test edilmiştir. TIM-3mod/high alt popülasyonun yorulmuş olup olmadığını anlamak için, TIM-3 ekspresyonuna göre, CD8+ T hücreleri ko-kültürlerden toplanarak yeniden uyarılmıştır. G-CSF ve NAC ile uyarılan nötrofillerde, ROS üretimi düşerek canlılık süresi artmıştır ve bu nötrofillerin T hücre proliferasyonunu da desteklediği görülmüştür. Kanser hücrelerinin ve monositlerin varlığı, nötrofillerin yaşam sürelerini uzatmış, üzerlerindeki CD62L ekspresyonunu düşürmüş ve ROS üretim kapasitelilerini azaltmıştır. Monositlerin, nötrofillerin ve akciğer kanseri hücrelerinin varlığı, CD8+ T hücre çoğalmasını ve üzerlerindeki inhibitör reseptörlerinin ekspresyonunu arttırmıştır. T hücre proliferasyonunun ana destekçileri olarak monositlerden yoksun olan belirli koşullar altında, akciğer kanseri hücreleri ile birlikte nötrofillerin varlığında CD8+ T hücre proliferasyonu ve aktivasyonu engellenmemiştir. KHDAK ko-kültürlerinden toplanan TIM-3mod/high CD8+ T hücreleri yüksek proliferasyon ve IFN-γ üretimi kapasitesine sahip olup yorulmamış hücre oldukları gösterilmiştir. NAC eklenen ko-kültürlerde CD8+ T hücre proliferasyonu veya TIM-3/LAG3 ekspresyonu değişmemiştir. Akciğer kanseri hücreleri, monositler ve CD8+ T hücreler ile birlikte kültürlenen nötrofillerde 4-1BBL, OX40L ve B7-2 kostimülatör genlerinin ekspresyonu artmıştır. Nötrofiller, monositler, CD8+ T hücreleri ve akciğer kanseri hücrelerini kullanarak tümör mikroçevresini kısmen modellemek için kurulan bir ko-kültür düzeninde, bulgularımız nötrofillerin CD8+ T hücre yanıtları için kritik bir modülatör olarak önemini göstermektedir

Ateşle emeğin birleştiği yer : Paşabahçe

Ankara : İhsan Doğramacı Bilkent Üniversitesi İktisadi, İdari ve Sosyal Bilimler Fakültesi, Tarih Bölümü, 2013.This work is a student project of the The Department of History, Faculty of Economics, Administrative and Social Sciences, İhsan Doğramacı Bilkent University.by Berna Kamay.Kamay, Berna. HIST 200-20KAMAY HIST 200-20/2 2012-1

Melanoma patients with immune-related adverse events after immune checkpoint inhibitors are characterized by a distinct immunological phenotype of circulating T cells and M-MDSCs

ABSTRACTTreatment with immune checkpoint inhibitors (ICIs) has improved the prognosis of melanoma patients. However, ICIs can cause an overactivation of the immune system followed by diverse immunological side effects known as immune-related adverse events (irAE). Currently, the toxicity of irAE is limiting the usage of ICIs. Here, we studied circulating monocytic myeloid-derived suppressor cells (M-MDSCs) and T cells in course of irAE after the ICI therapy. Our longitudinal study involved 31 melanoma patients with and without adverse events during anti-PD-1 monotherapy or anti-CTLA-4/PD-1 combination therapy. Peripheral blood samples were analyzed before ICI start, during ICI treatment, at the time point of irAE and during immunosuppressive treatment to cure irAE. We observed an enhanced progression-free survival among patients with irAE. In patients with irAE, we found an upregulation of CD69 on CD8+ T cells and a decreased frequency of regulatory T cells (Tregs). Moreover, lower frequencies of Tregs correlated with more severe side effects. Patients treated with immunomodulatory drugs after irAE manifestation tend to show an elevated number of M-MDSCs during an immunosuppressive therapy. We suggest that an activation of CD8+ T cells and the reduction of Treg frequencies could be responsible for the development of irAE

Identification Of Circulating Mog-Specific B Cells In Patients With Mog Antibodies

Objective To identify circulating myelin oligodendrocyte glycoprotein (MOG)-specific B cells in the blood of patients with MOG antibodies (Abs) and to determine whether circulating MOG-specific B cells are linked to levels and epitope specificity of serum anti-MOG-Abs. Methods We compared peripheral blood from 21 patients with MOG-Abs and 26 controls for the presence of MOG-specific B cells. We differentiated blood-derived B cells in vitro in separate culture wells to Ab-producing cells via engagement of Toll-like receptors 7 and 8. We quantified the anti-MOG reactivity with a live cell–based assay by flow cytometry. We determined the recognition of MOG epitopes with a panel of mutated variants of MOG. Results MOG-Ab–positive patients had a higher frequency of MOG-specific B cells in blood than controls, but MOG-specific B cells were only detected in about 60% of these patients. MOG-specific B cells in blood showed no correlation with anti-MOG Ab levels in serum, neither in the whole group nor in the untreated patients. Epitope analysis of MOG-Abs secreted from MOG-specific B cells cultured in different wells revealed an intraindividual heterogeneity of the anti-MOG autoimmunity. Conclusions This study shows that patients with MOG-Abs greatly differ in the abundance of circulating MOG-specific B cells, which are not linked to levels of MOG-Abs in serum suggesting different sources of MOG-Abs. Identification of MOG-specific B cells in blood could be of future relevance for selecting patients with MOG-Abs for B cell–directed therapy.PubMedWoSScopu