Effect Of Allogeneic Blood Transfusion On Peripheral Blood Τregs and Cytokine Levels Of Patients That Underwent Joint Replacement Surgery

Clinical and experimental studies have established that allogeneic blood transfusion (ABT) can cause immunosuppression. To identify immune parameters that contribute to this effect, we determined the effect of ABT on peripheral blood (PB) cytokine profiles and Treg numbers and function in a cohort of patients with no underlying pathologies.

Heparinized PB samples were collected from 46 patients (7M/39F, 28-88 yo) that underwent joint replacement surgery. The samples were collected immediately before surgery (BS), and after surgery (AS) on days 0, 7, 1 month, and 3 months to 1 year. Thirty six patients received ABT and 10 did not. PBMC were isolated, and the numbers and % of CD4⁺CD25⁺Foxp3⁺ Tregs and CD4⁺CD25^high/+CD127^low/- Tregs were determined by FACS. Tregs and T effectors (Teff) were isolated from patients on days 0-7 and Treg functional assays were performed by culturing Tregs with PHA-stimulated Teff at different ratios for 72h with CFSE, and analyzed by FACS for proliferation. Cytokine levels were determined in plasma by a cytometric bead array assay for IL-2, IL-4, IL-5, IL-6, IL-10, TNF-á, IFN-ã, and ELISA for TNF-á, TNF-RI(p55/p60) and II(p75/p80), TGF-â1 and â2.

Both, CD4⁺CD25⁺Foxp3⁺ and CD4⁺CD25^high/+CD127^low/- Treg populations increased significantly on d0 AS and decreased on d7 AS to below BS levels, to achieve homeostasis >3 months in transfused patients. In contrast, Treg levels remained the same between BS and AS in non-transfused patients.

Functional assays showed that Tregs were functional post-ABT and could suppress Teff proliferation efficiently. In culture, isolated Tregs secreted TGF-â1.

All cytokines and TNF-RI and II plasma levels increased on d0 AS (IL-6, TNF-RI and II significantly), in transfused patients immediately AS, and decreased by d7 AS. In contrast, TGF-â1 levels decreased on d0 AS and increased by d7. No differences in cytokine or receptor levels were observed in non-transfused patients AS.

ABT induces the immediate production of plasma cytokines, especially pro-inflammatory IL-6 and TNF-RI and II that decreases by d7. In parallel, ABT induces the proliferation of Tregs. These Tregs are functional, and secrete TGF-â1 that reaches BS plasma levels by d7. The Tregs seem to be Th3 inducible Tregs. Homeostasis is reached by 3 months post-ABT.

No relevant conflicts of interest to declare.