Evaluation of the level of LAG-3 gene expression among β-thalassemia major patients with and without alloantibodies in comparison with control group from patient in West Bank, Northern Palestine. رسالة ماجستير

Abstract

Background: The immune system maintains internal homeostasis by defending against pathogens while eliminating potentially harmful self-reactive cells. Among the regulatory molecules involved in this balance, lymphocyte-activation gene 3 (LAG-3) has emerged as a key player in both autoimmunity and tumor immune evasion. In patients reliant on chronic blood transfusions—such as those with β-thalassemia major or sickle cell disease—the development of alloantibodies and immune dysregulation is well documented. However, the specific contribution of LAG-3 expression in this clinical context remains poorly understood. Objectives: The present study aims to evaluate the expression levels of lymphocyte activation gene-3 (LAG-3) in transfusion-dependent patients, including those with β thalassemia major, compared to healthy controls. Additionally, the study seeks to assess LAG-3 expression in thalassemia patients with and without alloantibodies. Methodology: The study enrolled 100 participants: 80 individuals diagnosed with β thalassemia major, 20 with sickle cell disease, and 100 age- and sex-matched healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated, and total RNA was extracted for the assessment of LAG-3 gene expression. Quantitative real-time PCR (qRT PCR) using the SYBR Green method was employed, with β-actin serving as the internal housekeeping gene. Relative expression levels were calculated using the 2−ΔΔCT method. Data were entered and analyzed using IBM SPSS Statistics, with results expressed as medians and interquartile ranges (IQR) unless otherwise specified. VI Results: Transfusion-dependent patients exhibited elevated LAG-3 transcript levels compared to age- and sex-matched healthy controls. However, no statistically significant difference in LAG-3 expression was observed between alloimmunized and non alloimmunized subgroups. Notably, while total white blood cell counts and the number of transfused units approached conventional thresholds for significance, serum ferritin levels and prior splenectomy status showed no discernible effect on LAG-3 expression. Conclusion: Chronic exposure to repeated blood transfusions appears to shift LAG-3 expression away from baseline levels, suggesting that the immune system may be undergoing stress-induced recalibration. Interestingly, the presence of alloantibodies does not correlate with further upregulation of this immune checkpoint marker. Longitudinal studies are warranted to determine whether LAG-3 acts merely as a passive indicator of transfusion-related immune changes or plays an active role in driving sustained immunologic dysregulation