Immunologic profiles of red blood cells using in vitro models of transfusion

Abstract

Background: Transfusion of packed red blood cells (RBCs) produces a myriad of immunologic derangements, from suppressive to stimulatory. Proliferation of human T cells is suppressed in vitro after exposure to processed red blood cells (PRBCs). We hypothesized that this effect would be mitigated by using fresh RBCs. We also hypothesized that this suppressive effect was a generalized effect on lymphocyte proliferation and would be observed in both CD4+ and CD8+ T-cell subpopulations as well as B cells.

Materials and methods: We isolated human T cells from donor peripheral blood mononuclear cells and exposed them to either blood bank PRBCs or fresh RBCs from volunteer donors and stimulated them with anti-CD3/anti-CD28. Human B cells were stimulated with lipopolysaccharide and exposed to PRBCs or fresh RBCs. We measured proliferation of B cells by thymidine incorporation assays. We also treated RBCs with citrate-phosphate-dextrose (CPD) at different time points before culture them with stimulated T cells to determine the role of this common RBC storage solution in lymphocyte proliferation.

Results: In vitro proliferation of CD4+ and CD8+ T cells was suppressed by blood bank RBCs. This suppression is eliminated when fresh RBCs were used. The B cells showed inhibition of proliferation when exposed to similar conditions, which appeared to be consistent over serial dilutions. Fresh RBCs exposed to CPD did not appear suppressive in the first 6 h after exposure.

Conclusions: T-cell and B-cell proliferation inhibition by blood banked RBCs suggests a generalized effect of RBCs on cellular proliferation. The lack of suppression by fresh RBCs further suggests that something involved in blood banking alters RBC properties such that they attain a suppressive phenotype. One such blood banking component, CPD, does not appear to affect this suppressive phenotype within the first 6 h.

Keywords: Critical care; Immunomodulation; Red blood cell; Transfusion; Transfusion-related immunomodulation.

Fig. 1

Packed red blood cells suppress proliferation of both CD4 and CD8 human T cells. Fresh RBCs restore proliferation of both CD4 and CD8 T cells. T cells were purified from human PBMCs, stained with CFSE, exposed to anti CD3/CD28, and then cultured with no RBCs, PRBCs (100:1), or fresh RBCs (100:1) for 4 d. Cells were then harvested, stained with anti-CD4 or anti-CD8, and analyzed by flow cytometry. Proliferation is assessed by the dilution of CFSE intensity.

Fig. 2

Addition of CPD to RBCs does not suppress T-cell proliferation within the first 6 h after exposure. We added fresh RBCs to anti-CD3/CD28 stimulation cultures of CFSE-labeled T cells after the indicated times of incubation with CPD. T cells were harvested after 3 d and proliferation was measured by CFSE dye dilution using flow cytometry. Initial CFSE-stained T-cell populations are demonstrated by the large peaks at a fluorescence intensity of 10²; the leftward diminution of fluorescence intensity demonstrates continued proliferation. Numbers in each histogram represent the percentage of T-cell division (M2/M1 x 100).

Fig. 3

Red blood cells have a suppressive effect on B-cell proliferation. B cells within human PBMCs were stimulated to proliferate by the addition of LPS at 100 μg/mL. Cells were then exposed to blood bank PRBCs and fresh donor RBCs at an initial concentration of 100 RBCs to one PBMC, followed by serial dilutions. Cells were cultured in 96-well plates with CRPMI media, pulsed with tritiated thymidine at 48 h, harvested, and read by scintillation counter at 72 h. Overall, cells exposed to RBCs showed significant inhibition of proliferation. No difference was appreciated between exposure to PRBCs versus fresh RBCs.