Assessment of CTLA-4, LAG-3, and TIM-3 Expression in Pediatric Patients with Β-Thalassemia Major and Their Association with Alloimmunization

β-thalassemia major induces chronic immune dysregulation driven by persistent anemia, iron overload, and repeated blood transfusions. It is associated with a complex and dynamic reshaping of immune homeostasis. In this context, immune checkpoints, CTLA-4, LAG-3, and TIM-3, have emerged as critical regulators of the balance between immune activation and tolerance. This study aimed to evaluate their expression and association with alloantibody formation. Pediatric β-thalassemia major patients on regular transfusions, including 22 alloimmunized and 22 nonalloimmunized, along with 22 healthy controls, were recruited for this study. Expression of CTLA-4, LAG-3, and TIM-3 was analyzed using reverse transcription PCR. TIM-3 expression was lower in patients than in controls (P = .003). This reduction was particularly evident in alloimmunized patients compared with the nonalloimmunized group (P = .001). Using random forest machine learning, cross-validated variable importance identified TIM-3 and CTLA-4 as important variables in the alloimmunization process. The pooled receiver operating characteristic (ROC) curve for the 3 studied markers in the patient groups demonstrated good discriminative performance between alloimmunized and non alloimmunized patients, with an area under the curve of 0.779, specificity 90.9%, and sensitivity 68.2%, outperforming conventional statistical analysis. In conclusion, downregulation of TIM-3 and CTLA-4 in pediatric β-thalassemia major patients may contribute to alloantibody formation.