The Effect of Exosomes Isolated from Red Blood Cell Concentrates on T Cell Proliferation and Tregs

Background/Objectives: Transfusion-Related Immunomodulation (TRIM) is a complex biological phenomenon that develops following allogeneic blood transfusions. Exosomes released from stored red blood cell concentrates (RBC) may be a potential mediator of TRIM. This study aims to analyze the effects of exosomes on T cell proliferation and regulatory T cell (Treg) differentiation. Methods: RBC concentrates obtained from six healthy donors were divided into non-leukoreduced (NR) and leukoreduced (LR) units. Exosomes were isolated from these RBCs by ultracentrifugation. Allogeneic and autologous exosome pools were prepared. Peripheral blood mononuclear cells (PBMCs) were cultured with exosome pools (with 0.2, 1, or 5 μg doses) under allogeneic or autologous conditions. T-cell proliferation and Tregs were assessed by flow cytometry. Results: Total exosomal protein content increased during storage, with significantly higher levels in NR-RBC derived exosomes (NR-Exo) than LR-RBC derived exosomes (LR-Exo). NR-Exos significantly enhanced both CD4⁺ and CD8⁺ T cell proliferation, predominantly in allogeneic cultures. The proliferative effect was observed at different doses. In contrast, exosome treatment did not promote expansion of Treg populations. Instead, autologous cultures demonstrated reduced frequencies of CD8⁺ Tregs and CD39-expressing Tregs, whereas allogeneic cultures showed minimal changes. Moreover, leukoreduction attenuated exosome-mediated T-cell proliferation. Conclusions: RBC-Exos preferentially enhance effector T cell proliferation under allogeneic conditions, particularly when isolated from NR-RBC units. Tregs tended to decrease in both the autologous and leukoreduced groups. These findings suggest that RBC-Exos support both immunosuppressive effects and immune activation. However, the characteristics of the RBC and the transfusion (allogeneic/autologous and leukoreduced/non-leukoreduced) are considered the key factors in observing these effects.