Could the Crosstalk Between Mdscs and Tregs Have a Role in β-thalassemia?

Background: Secondary-iron overload, alloimmunization, and increased risk of infections are common complications in β-thalassemia major (BTM) patients. Tregs and myeloid-derived suppressor cells (MDSCs) play an essential role in preventing excessive immune responses. This research aimed to investigate the interaction between Tregs and MDSCs in BTM patients and their relation with disease severity. Methods: This case-control study included 26 patients with BTM and 23 healthy age- and sex-matched controls. All patients were investigated for complete blood picture, serum ferritin, and ow cytometric analysis of peripheral blood to detect Tregs, MDSCs, and MDSC subsets. Results: A signicant increase was observed in the frequencies of Tregs and MDSCs, particularly MO-MDSCs, in the patients compared with the controls. These cells also showed direct relations with ferritin and TLC and an inverse association with hemoglobin. Furthermore, a positive correlation was seen between Tregs and each of the total MDSCs and MO-MDSCs. Conclusion: Our ndings highlight the role Tregs and MDSCs cooperatively plays in the BTM and their importance in suppressing the high activity of the immune system found in those patients due to repeated blood transfusions and antigenic stimulation. immunomodulatory key Here we have studied the frequency of Tregs and MDSCs in BTM patients and assessed their relation with disease severity. Our results showed increased frequencies of Tregs and total MDSCS, particularly the MO-MDSCs compared to controls. Tregs are a component of the immune system that suppress immune responses of other cells. Earlier studies reported higher Treg levels in TM patients compared with the normal subjects 5,15 . Bozdogan and others suggested that increased Tregs in BTM patients might be due to the chronic exposure to antigenic stimulus because of frequent blood transfusions that trigger Tregs to prevent alloimmunization 5 . In line with our results, a signicant positive correlation was observed between the Tregs and ferritin concentration in thalassemia patients. Consequently, as ferritin increases, it can suppress the immune system by inducing Tregs in these patients 15 . MDSCs are functionally comparable to Tregs. Several studies described a signicant elevation in total MDSCS and MO-MDSCS cells in chronic inammatory diseases and tumors 16,17 as an induced core anti-inammatory mechanism to inhibit unwarranted immune cell activities. They negatively regulate immune function by suppressing the activity of T cells, NK cells, and B cells 18 , and their rise in BTM could be implicated in increased liability to infection in this group.


Introduction
Thalassemia is a genetic disorder in which there is a defect in hemoglobin production. Thalassemia major (TM) patients suffer from severe anemia and require regular blood transfusion 1 . TM is a common health problem in Mediterranean countries, especially Egypt. TM's most common complications are iron overload, heart failure, recurrent infections, and alloimmunization from repeated blood transfusion 2 .
Regulatory T cells (Tregs) are CD4 + CD25 + Hi that express the transcription factor forkhead box protein 3 (Foxp3) and form up to 5-10 % of CD4 + T Cells 3 . Tregs' primary function is to induce and maintain peripheral tolerance, which plays a vital role in preventing excessive immune responses and autoimmunity 4 . The increase in antigenic stimuli that occur due to repeated blood transfusions might change the percentage of Tregs and Foxp3 in β-thalassemia 5 . Previous studies showed that Tregs have a crucial role in the magnitude and frequency of alloimmunization by suppressing the immune system 6 .
Myeloid-derived suppressor cells (MDSCs) are innate immune cells known as a heterogeneous group of immature myeloid cells at a speci c differentiation stage with an immunosuppressive effect. Normally they are present in low numbers and increase in certain disease conditions, psychological stress, or natural aging 7,8 . They include monocytic MDSCs (MO-MDSCs) which are HLA-DR-, CD11b+, CD33+, and CD14 + and polymorphonuclear MDSCs (PMN-MDSCs) which are HLA-DR-, CD11b+, CD33+, and CD15 + 7 . The main function of MDSCs is to suppress immune cells, mainly T-cells, and to a lesser extent, B-cells and Natural killer (NK) cells 9 .
Previous studies showed that chronic in ammatory conditions as in thalassemia, persistent tissue damage as in cancer, autoimmunity, and chronic infections increase the release of damage-associated molecular patterns (DAMPs) and pathogen-associated molecular pattern (PAMPs) and production of various cytokines that in turn increase the release of myeloid cells from marrow and induce the immunosuppressive effect of MDSCs 10 . The possibility that MDSCs play a role in immune dysregulation in hematologic malignancies, BM failure syndromes, and autoimmune disorders and their role as therapeutic targets has attracted the interest to study it in other hematological diseases 11 Interaction between Tregs and MDSCs was studied in various conditions as tumors, allergic disorders,

Patients And Methods
This case-control study was held in the Pediatric Hematology Unit on patients with BTM, Assiut University Children Hospital, Assiut, Egypt. The study was approved by Ethics Committee of Faculty of Medicine, Assiut University, Assiut, Egypt, (Approval No. 17300557). All methods were carried out in accordance with local guidance after written informed consents were obtained from the guardians of participants.
The study included 26 patients with BTM, and 23 healthy age-and sex-matched children were enrolled as controls. Patients were receiving a regular blood transfusion and chelation therapy. Patients with known diabetes, cardiac, renal, infectious, in ammatory, or pulmonary diseases, newly diagnosed and nontransfusion dependent BTM cases were excluded from the study. Any patient with a history of recent infection or any immunosuppressive medications, e.g., steroids, etc. during one month before enrollment, was also excluded.

Flow cytometric detection of regulatory T cells:
Regulatory T cells were enumerated using uorescein isothiocyanate (FITC)-conjugated Foxp3 (IQ Product the Netherland), phycoerythrin (PE) conjugated CD25 (Bioscience, USA), and Peridiniumchlorophyll-protein (Per-CP)-conjugated CD4 (Becton Dickinson (BD) Bioscience, CA, USA). Five µl of CD4 and CD25 were incubated with 50 µl of the blood sample for 15 minutes at 4 C in the dark. Following incubation, red blood cell lysis and washing with phosphate-buffered saline (PBS) were done. Then xing solution was added, and incubation for 10 minutes was done. Afterward, cells were washed with PBS, and then the permeabilizing solution and 5 µl of Foxp3 were added and incubated for 20 minutes at 4C . After one wash, the cells were resuspended in PBS and analyzed by FACSCalibur ow cytometry with CellQuest software (Becton Dickinson Biosciences, USA). An isotype-matched negative control was used for each sample. Lymphocytes were detected according to their forward and side scatters. Then CD4 + cells were gated. Total CD4 + CD25+, CD4 + CD25 + low , CD4 + CD25 + Hi T cells and CD4 + CD25 + Hi Foxp3+ Tregs were evaluated as percentages from CD4 + cells as shown in gure (1)

Statistical Analysis
Data analysis was done using the Statistical Package for Social Sciences (SPSS 22, IBM, USA). Data were expressed as the mean ± standard deviation of the mean (SD) or standard error (SE). The differences between the groups were examined for statistical signi cance using the Independent t-test.
The correlation coe cient was generated by Pearson's correlation. Statistical signi cance was de ned as p < 0.05.

Results
This study was conducted on 26 BTM patients with a mean age of 8.7 ± 5, and 54% were males. The healthy individuals' mean age was 8.2 ± 3, and 52% were males. Demographic data and laboratory investigations of all patients are presented in table (1). Results showed a signi cant increase in total leukocyte count (TLC) and ferritin than the reference ranges. On the other hand, there was a decrease in HB and platelet (PLT) count.  The correlations between the frequencies of Treg cells, MDSCs, and laboratory parameters: As illustrated in gure (3) Pathogenesis is not fully understood 1 . Alloimmunization was found to depend on many factors as RBC antigen discrepancy between donor and recipient, immune status of the recipient, and the immunomodulatory effects of allogeneic blood transfusion 15 . The immune system is, therefore, a key player in the clinical features accompanying thalassemia.
Here we have studied the frequency of Tregs and MDSCs in BTM patients and assessed their relation with disease severity. Our results showed increased frequencies of Tregs and total MDSCS, particularly the MO-MDSCs compared to controls. Tregs are a component of the immune system that suppress immune responses of other cells. Earlier studies reported higher Treg levels in TM patients compared with the normal subjects 5,15 . Bozdogan and others suggested that increased Tregs in BTM patients might be due to the chronic exposure to antigenic stimulus because of frequent blood transfusions that trigger Tregs to prevent alloimmunization 5 . In line with our results, a signi cant positive correlation was observed between the Tregs and ferritin concentration in thalassemia patients. Consequently, as ferritin increases, it can suppress the immune system by inducing Tregs in these patients 15 .
MDSCs are functionally comparable to Tregs. Several studies described a signi cant elevation in total MDSCS and MO-MDSCS cells in chronic in ammatory diseases and tumors 16,17 as an induced core antiin ammatory mechanism to inhibit unwarranted immune cell activities. They negatively regulate immune function by suppressing the activity of T cells, NK cells, and B cells 18 , and their rise in BTM could be implicated in increased liability to infection in this group.
Little is known about MDSCs in BTM. Siriworadetkun  Our ndings demonstrated that in the BTM group, both Tregs and MDSCs, particularly the MO-MDSCs, were signi cantly higher than healthy control subjects. Also, these cells had shown direct relations with ferritin and TLC and an inverse association with hemoglobin. Furthermore, a positive correlation was seen between Tregs and each of the total MDSCs and MO-MDSCs.

Conclusion
Altogether, our ndings highlight the role Tregs and MDSCs cooperatively plays in the BTM and their importance in suppressing the high activity of the immune system that is found in those patients due to repeated blood transfusions and antigenic stimulation.