Blood microparticles are a component of immune modulation in red blood cell transfusion

Abstract

Patients may display alloimmunization following transfusion. Microparticles (MPs) released into the blood are present in transfusion products. We show that MPs can modulate the immune system, CD4+ T-cell, and humoral responses, through their concentration, cellular origin and phenotype, and should therefore be considered to reduce the immune impact of transfusion. Extracellular microvesicles (EVs) bud off the cell membrane into the blood. There are two types of EVs: exosomes (40-100 nm across, from intracellular membrane compartments) and microparticles (MPs, 200-900 nm across, produced by plasma membrane budding). MPs have physiological and immunomodulatory effects, potentially affecting health [1, 2]. Packed red blood cell (pRBC) transfusion has been reported to induce immunomodulations independently of residual leukocytes and cytokines [3]. MPs are produced by various cell types, are in pRBCs, are transferred during transfusion and may affect transfusion outcomes [3, 4]. CD4+ T lymphocytes (LTs), which express immunomodulatory molecules, are key regulators of alloimmunization [5]. MPs inherit the properties of their cells of origin [1, 2]. Thus, LT MPs (LMPs) would be likely to induce immunomodulations during transfusion. Many blood factors affect MP characteristics, potentially accounting for variation between HDs [1, 2]. We therefore investigated the variability of MPs in fresh pRBCs. We used calibration beads to select MPs and exclude exosomes (Supporting Information Fig. S1). Total MP and LMP numbers varied considerably between HDs (Supporting Information, Table 1). Many factors contribute to this in pathophysiological conditions [1, 2]. The preparation and storage of pRBCs may favor MP production [3, 6]. The variability of MP numbers, particularly for LMPs, may be a key feature of immunomodulatory effects after transfusions. As characteristics of LMPs have never been studied, we first investigated their phenotype. All the immunomodulatory molecules studied were detected, with variations between HDs (Fig. 1A, Supporting Information Fig. S2). We found no correlation of immunomodulatory molecules expression between LTs and LMPs (Supporting Information Fig. S3). This lack of correlation suggests that pRBC preparation and storage may affect the expression of these molecules [3-6]. LMPs expressed several molecules, consistent with coexperession. We therefore analyzed the coexpression of the most frequently immunomodulatory molecules (Fig. 1B). CTLA-4 and TGFβ were the most coexpressed, suggesting that the LMPs concerned may have been derived from Tregs (Fig. 1B) [7]. Cytokines are a major key of immunomodulation. We studied purified LMPs by flow cytometry sorting, to investigate their cytokine content and functions (Supporting Information Fig. S4). Few cytokines were found in total MPs and in TGFβ−LMPs (Fig. 1C). However, all the cytokines tested were present, some at high levels in TGFβ+LMPs (Fig. 1C and D). The presence of proinflammatory cytokines (TNFα, IL-22, IL-1RA, IL-27, IL-31) and chemokines (MCP1, SDF1), suggest a role of these MPs in immune functions [8, 9]. IL-27+LMPs suggest a regulatory function, consistent with a Treg origin of TGFβ+LMPs [8]. We then investigated whether the phenotype and concentrations of purified TGFβ+LMPs affected LTs. We also compared these data with those for purified total MPs, regardless of origin. Purified MPs were cocultured with PBMCs, to assess their effects on the lymphoproliferation of Tregs and Tfh cells (Supporting Information Fig. S5). As the numbers of MPs varied between HDs, we investigated whether MPs number affected lymphoproliferation. TGFβ+LMPs appeared to facilitate lymphoproliferation more effectively than total MPs (Fig. 2A). Furthermore, at high concentrations, MPs decreased lymphoproliferation without mortality (Fig. 2A and B). It remains unknown how MPs induce lymphoproliferation. MPs may interact with cells in two ways: by internalization or fusion [1]. A large increase in LT size after interaction with MPs may mimic morphological changes occurring in senescent cells and stop lymphoproliferation [10]. MPs modulate lymphoproliferation and, may indirectly affect immunoglobulin production. We investigated the effect of purified MPs on cocultures of purified LTs and B lymphocytes (LBs) (Supporting Information Fig. S6). Antibody production was significantly upregulated by MPs, in a dose–dependent manner (Fig. 2C). MPs may interact directly with LBs to promote antibody production or indirectly [1]. Indeed, MPs may contain mitochondria, and their CpG methylation may promote TLR9 expression [1]. These findings concerning humoral activation in vitro raise questions about the role of MPs in transfusion. We addressed this issue by purifying total MPs from mice and transfusing them into mice with an antigen (HEL) (Supporting Information Fig. S7). The mice cotransfused with allogeneic MPs and antigen had higher anti-HEL antibody levels than mice transfused with antigen alone (Fig. 2D). MPs from blood donors thus have immunoregulatory functions. These results suggest that the phenotype and number of MPs may be associated with immunomodulations during transfusions, as shown in our in vivo model. As the phenotype of MPs may change during pRBC storage, a prospective study should be conducted before generalizing these results to humans [3]. This work was supported by the Etablissement Français du Sang, Inserm, and Université Paris-Est. We would like to thank the Flow cytometry facility of Institut Cochin and Denis Clay from the flow cytometry facility of Institut André Lwoff, Villejuif, France. We declare no commercial or financial conflict of interest. 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