RBC-Derived Microparticles Evolve Over RBC Storage Time and Boost T Cell Mitogen Responses

It is widely believed that transfusion of younger blood carries less risk of adverse reactions compared to older blood, attributed to a red cell “storage lesion”. One potential effect of RBC transfusion is immune modulation, and we tested whether cell-derived microparticles (MPs) could modulate T cell responses. MPs are small double membrane vesicles with an approximate size of 0.1–1.2 μm, released into circulation during activation, stress, necrosis or apoptosis or can be generated during the storage of blood products. MPs express different surface markers depending on their cell of origin and may contain RNA, DNA or cellular proteins. Here we tracked the cells of origin for MPs found in RBC units throughout the storage period. Furthermore, we hypothesized that RBC-derived MPs would suppress T cell immune responses.

RBC units were split into 35 ml in replicate small transfer bags and aged at 4°C. MPs were isolated using differential centrifugation with a final speed of 100,000g. Panels including 17 fluorochrome-conjugated antibodies were used identify MPs derived from RBCs, platelets, lymphocytes subpopulations, monocytes and granulocytes for four different RBC units. CFSE staining was used to measure T cell proliferation after stimulation with PHA plus or minus MP addition using MPs from six different units.

Two platelet markers in our panel (CD41a and CD42a) increased from day 0 through 21 of storage. The mean percentage of CD41a⁺ MPs increased from 3.9% at day 0 to 20% at day 21 (P<0.05) and decreased to 4.3% at day 42. CD42a⁺ MPs increased from 3.3% at day 0 to 13% at day 21 (P<0.05) and decreased to 1.9% at day 42. The percentages of CD14⁺ MPs (monocyte marker) ranged from 7.0% at day 0 to 12% at Day 42 and CD108a⁺ MPs (RBC marker) showed a trend to increasing from 1.7% at day 0 to 27.1% at day 42 (P>0.05). The percentage of CD3 (T lymphocyte marker) and CD16 (from NK cells and other cell types), CD19 (B cell marker), CD152 and CD154 (costimulatory molecules) percentages were <5% at baseline and remained low throughout storage.

Contrary to our hypothesis, CD4⁺ and CD8⁺ T lymphocytes showed augmented proliferation when PBMCs were stimulated with MPs plus PHA relative to PHA alone. Mean percentage of CFSE^low (proliferated) CD4⁺ T cells was 42% for PHA stimulated vs. 62% for PHA+MP stimulated PBMCs (p=0.018). CD8⁺ T cells showed similar differences (52 vs. 75% CFSE^low, p=0.03). In the three samples studied longitudinally, the MP-induced augmentation was not significantly different for day 0 vs. day 42 MPs. Addition of MPs to PBMCs without mitogen did not induce any de novo proliferation.

RBC-derived MPs appear to be predominantly derived from RBCs, platelets, and monocytes, and their composition can change with RBC unit storage time. Surprisingly, RBC-derived MPs augmented mitogen-stimulated CD4⁺ and CD8⁺ T cell responses. Further work will be required to determine the mechanism of enhancement and requirement of antigen presenting cells in the MP-induced enhancement of T cell responses. These findings illustrate how complicated and pleiotropic effects of transfusion related immune modulation can be.

No relevant conflicts of interest to declare.