Boer, J. de (Janneke) (2018) The role of T-cells and immune-checkpoint proteins in diffuse large B-cell lymphoma (DLBCL)A survival analysis and in vitro modulation with a hypomethylating agent. thesis, Medicine.
Full text available on request.Abstract
Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common form of Non-Hodgkin lymphomas. Every year 1100 new patients develop this disease in the Netherlands. The first-line therapy consists of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). However, 40% of the patients remain incurable and their prognosis is poor. In the search to find new therapies for DLBCL focus has been drawn towards the T-cell microenvironment. Tumor cells develop immune evasion strategies to sustain their oncogenic state. Through aberrations in DNA methylation and histone acetylation, tumor cells can over-express ligands of checkpoint receptors and thereby bring T-cells in a state of anergy and exhaustion. As epigenetic silencing is a reversible process, hypomethylating drugs, like decitabine and azacytidine, are developed. The aim of this study is to define the T-cell micro-environment and its influence on patients’ prognosis in DLBCL. Moreover, we want to investigate if we can upregulate the immune-checkpoint proteins herpesvirus entry mediator (HVEM), programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2) with decitabine treatment in DLBCL cell lines. Methods To define the T-cell micro-environment publically-available clinically well annotated gene expression profiles of DLBCL-patients (n=1804) were uploaded to CIBERSORT to define the immune cell fractions within an individual DLBCL patient. Output was uploaded to R2 amc to find an optimal threshold to divide the expression of the T-cells subsets in ‘low fraction or ‘high fraction’. Next, a multivariate survival analysis was performed. To determine the influence of decitabine on DLBCL cell lines, we used an alamar blue assay to determine metabolic activity after decitabine treatment. Moreover, we used flowcytometry to define after decitabine treatment the cell viability, cell cycle arrest and the γH2AX-formation. Moreover, we used flowcytometry to measure the expression of HVEM, PD-L1 and PD-L2 on protein level after decitabine treatment. qPCR was performed to measure the expression of HVEM, PD-L1 and PD-L2 on RNA-level after decitabine treatment. Results CD8+ T-cells were the most prominent T-cell subset in DLBCL (13,5%). Naïve CD4+ T-cells and Memory-resting CD4+ T-cells were least present (<1%). We could only find an optimal cut-off point for T-follicular helper (TFH) cells with R2 amc. Correcting for the international prognostic index (IPI) and cell of origin (COO), patients with a high fraction of TFH-cells (≥12%) had a 2-fold increase in survival compared to patients with a low fraction of TFH-cells (<12%) in the training cohort. (p=0.017, HR= 0.458). We discovered a trend towards a significant difference in the validation cohort (p=0.087). We tested the viability of cell lines after decitabine. WSU-DLCL2, SC-1 and SU-DHL-5 showed the largest decrease in metabolic activity, whereas SU-DHL-2, OCI-LY3 and SUP-HD1 showed barely a decrease in metabolic activity. The cell lines U-2932, SU-DHL-10, KG1a and SU-DHL-4 had a decrease in metabolic activity after treatment, but were not as sensitive 6 as WSU-DLCL2, SC-1 and SU-DHL5. A G1 or G2/M cell cycle arrest and γH2Ax-formation after decitabine treatment occurred in almost all cell lines, except for the three cell lines with barely a decrease in metabolic activity after treatment and the cell line KG1a. Decitabine treatment led to a upregulation of HVEM expression on protein-and RNA-level in the cell lines SC-1 and SU-DHL-4. Moreover, PD-L1 expression was upregulated on protein-and RNA-level in SC-1 and SUP-HD1. Only the cell line SUP-HD1 upregulated PD-L2 expression on protein- and RNA-level after decitabine treatment. Conclusion We conclude that TFH-cells are an independent predictor of survival in DLBCL. Moreover, we showed that DLBCL cell lines vary among their decrease in metabolic activity and cell viability after decitabine treatment. In addition, decitabine causes a G1 or G2/M cell cycle arrest and γH2AX formation in cell lines with a decrease in metabolic activity after treatment. Decitabine treatment cannot upregulate PD-L2. In contrast, decitabine can upregulate HVEM expression in DBLCL cell lines. HVEM interacts with TFH-cells and thereby can play a role in determining patients’ prognosis. Decitabine treatment can also upregulate PD-L1 expression in DBLCL cell lines, suggesting a role for decitabine in combination with specific immune blockade therapy in DBLCL.
| Item Type: | Thesis (Thesis) |
|---|---|
| Supervisor name: | Supervisors: and Meerten, Dr. Tom van and Visser, Dr. Lydia and Collaboration between the Department of Pathology and Hemato and University of Groningen, University Medical Center Groningen |
| Faculty: | Medical Sciences |
| Date Deposited: | 25 Jun 2020 10:58 |
| Last Modified: | 25 Jun 2020 10:58 |
| URI: | https://umcg.studenttheses.ub.rug.nl/id/eprint/1897 |
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