The lysophospholipid‐binding molecule CD1D is not required for the alloimmunization response to fresh or stored RBCs in mice despite RBC storage driving alterations in lysophospholipids

Abstract

Background: Despite the significant adverse clinical consequences of RBC alloimmunization, our understanding of the signals that induce immune responses to transfused RBCs remains incomplete. Though RBC storage has been shown to enhance alloimmunization in the hen egg lysozyme, ovalbumin, and human Duffy (HOD) RBC alloantigen mouse model, the molecular signals leading to immune activation in this system remain unclear. Given that the nonclassical major histocompatibility complex (MHC) Class I molecule CD1D can bind to multiple different lysophospholipids and direct immune activation, we hypothesized that storage of RBCs increases lysophospholipids known to bind CD1D, and further that recipient CD1D recognition of these altered lipids mediates storage-induced alloimmunization responses. Study Design and Methods: We used a mass spectrometry-based approach to analyze the changes in lysophospholipids that are induced during storage of mouse RBCs. CD1D knockout (CD1D-KO) and wild-type (WT) control mice were transfused with stored HOD RBCs to measure the impact of CD1D deficiency on RBC alloimmunization. Results: RBC storage results in alterations in multiple lysophospholipid species known to bind to CD1D and activate the immune system. Prior to transfusion, CD1D-deficient mice had lower baseline levels of polyclonal immunoglobulin (IgG) relative to WT mice. In response to stored RBC transfusion, CD1D-deficient mice generated similar levels of anti-HOD IgM and anti-HOD IgG.

Deuterated Linoleic Acid Attenuates the RBC Storage Lesion in a Mouse Model of Poor RBC Storage

“…Free PUFAs, oxylipins, and lysophospholipids in fresh and stored RBCs were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously ( Fu et al, 2016 ; Fu et al, 2019 ; Medved et al, 2021 ). Briefly, analytes were extracted by 80% methanol (vol/vol) with an internal standard mixture containing arachidonic acid-d8 (AA-d8), docosahexaenoic acid-d5 (DHA-d5), eicosapentaenoic acid-d5 (EPA-d5), dihomo-γ-linolenic acid-d6 (DGLA-d6), α-linolenic acid-d14 (ALA-d14), linoleic acid-d4 (LA-d4), oleic acid-d17 (OA-d17), 15-hydroxyeicosatetraenoic acid-d8 (15-HETE-d8), 5-hydroxyeicosatetraenoic acid-d8 (5-HETE-d8), 12-hydroxyeicosatetraenoic acid-d8 (12-HETE-d8), 9-hydroxyoctadecadienoic acid-d4 (9-HODE-d4), 13-hydroxyoctadecadienoic acid-d4 (13-HODE-d4), 9,10-dihydroxyoctadecenoic acid-d4 (9,10-diHOME-d4), 12,13-dihydroxyoctadecenoic acid-d4 (12,13-diHOME-d4) from Cayman Chemical (Ann Arbor, MI); 17:1 lysophosphatidylcholine (17:1 LPC), 17:1 lysophosphatidylethanolamine (17:1 LPE), 17:1 lysophosphatidylserine (17:1 LPS), and 17:1 lysophosphatidylinositol (17:1 LPI) from Avanti Polar Lipids (Alabaster, AL).…”

Class switching is differentially regulated in RBC alloimmunization and vaccination

“…Measurement of anti-HEL antibodies via ELISA: Antibody responses to the Hen egg lysozyme (HEL) portion of the HOD antigen or HEL-OVA were measured by HEL-specific enzyme-linked immunosorbent assay (ELISA), as previously described. 30,32 Details are available in Supporting Information.…”

“…Data were analyzed using FlowJo software.Murine blood collection and transfusion: Collection and processing of HOD blood was performed as described previously. 30,32 Details are available in Supporting Information.…”

“…28 Multiple studies have shown that transfusion of HOD RBCs into recipient mice after storage leads to a class switched anti-HEL IgG response. [29][30][31][32] Furthermore, we have previously shown that like most infectious stimuli, 5 HOD transfusion into C57BL/6 mice induces an anti-HEL IgG response consisting of all four of the C57BL/6 mouse isotypes: IgG1, IgG2b, IgG2c and IgG3. 32 However, our understanding of the molecular regulators of transfusion-induced class-switching remains limited.…”

Deletion of 12-lipoxygenase normalizes platelet function after storage and transfusion in thrombocytopenic mice

“…As described previously, free PUFAs and oxylipins in platelets were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). [21][22][23] Briefly, analytes were extracted by 80% methanol (vol/vol) with an internal standard mixture. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed using a mass spectrometer (QTrap 6500, AB Sciex) coupled with an ultra-performance liquid chromatographer [UPLC] (Acquity I-Class, Waters).…”