טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKhsheibun Rana
SubjectB Cells in Multiple Sclerosis: Gene Expression Profile and
Pathway Analysis of the Response to
IFN-beta
DepartmentDepartment of Medicine
Supervisors Professor Ariel Miller
Dr. Tamar Paperna
Full Thesis textFull thesis text - English Version


Abstract

Multiple sclerosis (MS) is a chronic immune-mediated neurological disease of the central nervous system. Recently, there is accumulating evidence for the central role of B cells in MS pathogenesis and their potential as a therapeutic target. Interferon beta (IFN-β) is a widely used treatment in MS; however, up to 50% of the patients are poor responders to the drug. Scarce information is available on IFN-β response in B cells. Lymphoblastoid cell lines (LCLs) are Epstein bar virus transferred B cell lines.


The overall study goals were to characterize the B cell response to IFN-β using LCLs as a surrogate B cell model, and to identify genes that can potentially serve as biomarkers of the clinical response to IFN-β.

 

To verify that B cell subset composition of LCLs is not a confounding factor when comparing gene expression patterns; multicolor flow cytometry analysis was performed. Results showed that LCLs express the four main B cell subsets as in peripheral blood and that the B cell subsets composition is highly similar between healthy controls and different MS patient groups including excellent and poor responders.


To investigate the genome wide transcriptional response to IFN-β, LCLs derived from MS patients classified as excellent responders and poor responders to IFN-β (n= 8 in each group) were treated with 100 IU/ml IFN-β for 4hr, and RNA was extracted for microarray analysis. More than 115 genes were more than two fold differentially upregulated following IFN-β, with over 50 genes previously unrecognized as IFN-β response genes. Pathway analysis highlighted several novel genes and functional pathways that could explain IFN-β's mechanism of action in B cells, such as antigen presentation; chemokine receptors and ligands; TLR signaling; antimicrobial pathway and B cell receptor signaling. Selected novel IFN-β response genes of statistical and B cell functional relevance (i.e. NEXN,HAPLN3,DDX60L, IGFBP4, CD79B, SYK, LAG3, IL27RA, and KLF2) were validated using REAL TIME PCR in a larger set of LCLs samples and in primary B cells.


Furthermore, preliminary data showed that LCLs derived from excellent and poor responders differed in the baseline gene expression pattern with a higher expression of IFN related genes in the excellent responders group. In addition, the two groups differed in the magnitude of response to IFN-β exposure with a higher magnitude of induction of classic IFN-β genes in poor responders. Finally, the study identified several potential biomarkers of clinical response that showed a differential expression in response to IFN-β in one group but not the other, such as NCOA7 and TBXAS1. These results suggest that a dysregulated IFN signaling pathway might be the underlying factor behind clinical unresponsiveness to IFN-β.


In conclusion, the study provided a proof of concept for the use of LCLs for identifying novel B cell-specific IFN-β-induced gene expression patterns and pathways and gene expression signatures associated with the mode of clinical response to IFN-β.  Further investigation and validation of the genes in larger cohorts may aid in the development of IFN-β response biomarkers for MS and treatment optimization for the individual patient.