The identification of the mechanisms of lrba deficiency dependent defects in regulatory t-cell function

Zahedimaram, Pegah (2022) The identification of the mechanisms of lrba deficiency dependent defects in regulatory t-cell function. [Thesis]

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Primary immunodeficiencies are a very diverse group of disorders characterized by recurring infections. Loss of function mutations in the gene encoding LRBA (lipopolysaccharide-responsive beige like anchor protein) were recently identified to cause PID which is associated with autoimmunity. Several studies have shown that the LRBA molecule regulates CTLA-4 cell surface expression. Besides CTLA-4, LRBA has been claimed to have an effect on the vesicular trafficking of epidermal growth factor receptor (EGFR) and cell surface expression of FasL in the plasma membrane. In this study, the main objective was to discover the defects caused by LRBA deficiency in T-cells. For this purpose, the Jurkat leukemic T-cell line was used as the model cell line and the CRISPR/Cas9 system was used to knock-out (KO) this protein in the Jurkat cell line. The differentially expressed proteins on the surface of WT and KO Jurkat cells were assessed by the means of cell surface biotinylation coupled with mass spectrometry (MS) analysis. CD3, CD4, PVRIG, NOTCH3, CD1d, and Sema7A were found to be downregulated and CD53, CD148, CD154, CD134, A33, and CD70 to be upregulated in LRBA negative cell lines. We identify the decrease in CD3 and CD4 seemed to be most significant and relevant to the loss of LRBA. As expected, endogenous CTLA-4 expression was also decreased in our LRBA KO cell lines. Additionally, CTLA-4 protein overexpression could not rescue CTLA4 expression to WT levels in LRBA KO Jurkat iv cell lines indicating the dominant phenotype obtained by LRBA loss. This study identifies alternative targets of the LRBA protein and delineates the mechanism of surface CTLA4 expression, potentially identifying new targets that can be used to cure immunodeficiencies resulting from LRBA loss.
Item Type: Thesis
Uncontrolled Keywords: lipopolysaccharide-responsive beige like anchor protein (LRBA). -- Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4). -- CRISPR/Cas9. -- Biotinylation. -- Mass Spectrometry. -- lipopolisakkarit duyarlı bej benzeri çapa proteini (LRBA). -- Sitotoksik T-lenfosit ile ilişkili protein-4 (CTLA-4). -- CRISPR/Cas9. -- Biyotinilasyon. -- Kütle Spektrometrisi.
Subjects: T Technology > TA Engineering (General). Civil engineering (General) > TA164 Bioengineering
Divisions: Faculty of Engineering and Natural Sciences > Academic programs > Biological Sciences & Bio Eng.
Faculty of Engineering and Natural Sciences
Depositing User: Dila Günay
Date Deposited: 04 Jul 2023 15:29
Last Modified: 13 Nov 2023 15:07

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