Isolation and in vitro Expansion of Regulatory T Cells from β-Thalassemia Major Kids and in vitro Evaluation of Their Plasticity and Inhibitory Effects in the Presence of Pro-Inflammatory Cytokines

Abstract

Introduction: Graft-versus-host disease (GvHD) is a life-threatening syndrome commonly associated with hematopoietic stem cell transplantation (HSCT). Preventing the incidence of GVHD after HSCT along with minimizing long-term immunosuppression is currently under investigation with regulatory T cells (Tregs). As Tregs are a low-frequency population and the yield of all memory Tregs is not sufficient for clinical application, an initial Treg expansion is essential. Methods: Thirty milliliters of peripheral blood from the β-thalassemia major (beta-TM) patients and healthy controls were obtained and Tregs were isolated using MACS. Isolated cells were cultured in the presence of rapamycin and rIL-2 followed by activated with anti-CD3/CD28-coated beads. To evaluate Treg plasticity, expanded Tregs were cultured in a medium containing IL1β, IL6, TGFβ, and IL2, with or without 500 nM rapamycin for 72 h. To assess the functional properties of Tregs, CFSE dilution assays were performed to evaluate the ability of in vivo expanded Tregs from beta-TM patients. Statistical analysis was performed using paired t-test and independent t-test, with the aid of SPSS version 12.0. p-value ≤0.05 was considered significant. Results: The percentage of Tregs isolated from the control group was significantly higher than the Tregs isolated from patients (p-value = 0.01), which is probably due to the iron overload in beta-TM patients as a result of continuous blood transfusion. Also, the percentage of Tregs after 5 days of expansion had a significant increase in both groups compared to before expansion (p-value = 0.03). Our results also showed that the expansion of Tregs after 72 h in the presence of inflammatory cytokines and in the absence of rapamycin led to the increase in the intracellular expression of IL-17 (p-value = 0.01), while intracellular expression of IL-17 remained low following the addition of 100 nM rapamycin to the culture medium (pvalue = 0.073). The results of the functional evaluation of expanded Tregs showed relatively differences in both patient and control groups. Thus, expanded Tregs inhibited the proliferation of responder T cells in a dose-dependent manner in the control group (p-value = 0.028), while in the patient group this inhibitory effect was not significant (p-value = 0.055). Conclusion: Tregs isolated from beta-TM patients have poorer inhibitory performance than Tregs isolated from healthy individuals. Also, we concluded that rapamycin stabilizes the Treg population by inhibiting the production of IL-17, all necessitating the administration of appropriate immunosuppressive drugs in patients receiving Treg therapy.