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• For human non-RhD RBC alloantigens immunogenicity increases as copy number decreases. • Copy number affects mechanisms of alloimmunization in mouse models. • In humans and mice, very low copy number alloantigens are weakly or nonimmunogenic. • In mice very low copy numbers become tolerogenic, which is untested in humans. • Interpretation of mouse studies must include transgene properties and mouse genetics. RBC alloimmunization remains a significant barrier to ongoing transfusion therapy leading to morbidity, and in extreme cases mortality, due to delayed or insufficient units of compatible RBCs. In addition, the monitoring and characterization of alloantibodies, often with multiple specificities in a single patient, consumes substantial health care resources. Extended phenotypic matching has mitigated, but not eliminated, RBC alloimmunization and is only logistically available for specialized populations. Thus, RBC alloimmunization remains a substantial problem. In recent decades it has become clear that mechanisms of RBC alloimmunization are distinct from other antigens and lack of mechanistic understanding likely contributes to the fact that there are no approved interventions to prevent RBC alloimmunization from transfusion. The combination of human studies and murine modeling have identified several key factors in RBC alloimmunization. In both humans and mice, immunogenicity is a function of alloantigen copy number on RBCs. Murine studies have further shown that copy number not only changes rates of immunization but the mechanisms of antibody formation. This review summarizes the current understanding of quantitative and qualitative effects of alloantigen copy number on RBC alloimmunization. [ABSTRACT FROM AUTHOR]

Abstract: Population-based investigations suggest that red blood cells (RBCs) are therapeutically effective when collected, processed, and stored for up to 42 days under validated conditions before transfusion. However, some retrospective clinical studies have shown worse patient outcomes when transfused RBCs have been stored for the longest times. Furthermore, studies of RBC persistence in the circulation after transfusion have suggested that considerable donor-to-donor variability exists and may affect transfusion efficacy. To understand the limitations of current blood storage technologies and to develop approaches to improve RBC storage and transfusion efficacy, we investigated the global metabolic alterations that occur when RBCs are stored in AS-1 (AS1-RBC). Leukoreduced AS1-RBC units prepared from 9 volunteer research donors (12 total donated units) were serially sampled for metabolomics analysis over 42 days of refrigerated storage. Samples were tested by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry, and specific biochemical compounds were identified by comparison to a library of purified standards. Over 3 experiments, 185 to 264 defined metabolites were quantified in stored RBC samples. Kinetic changes in these biochemicals confirmed known alterations in glycolysis and other pathways previously identified in RBCs stored in saline, adenine, glucose and mannitol solution (SAGM-RBC). Furthermore, we identified additional alterations not previously seen in SAGM-RBCs (eg, stable pentose phosphate pathway flux, progressive decreases in oxidized glutathione), and we delineated changes occurring in other metabolic pathways not previously studied (eg, S-adenosyl methionine cycle). These data are presented in the context of a detailed comparison with previous studies of SAGM-RBCs from human donors and murine AS1-RBCs. Global metabolic profiling of AS1-RBCs revealed a number of biochemical alterations in stored blood that may affect RBC viability during storage as well as therapeutic effectiveness of stored RBCs in transfusion recipients. These results provide future opportunities to more clearly pinpoint the metabolic defects during RBC storage, to identify biomarkers for donor screening and prerelease RBC testing, and to develop improved RBC storage solutions and methodologies. [Copyright &y& Elsevier]

Abstract: Traditionally, alloimmunization to transfused blood products has focused exclusively on recipient antibodies recognizing donor alloantigens present on the cell surface. Accordingly, the immunologic sequelae of alloimmunization have been antibody mediated effects (ie, hemolytic transfusion reactions, platelet refractoriness, anti-HLA and anti-HNA effects, etc). However, in addition to the above sequelae, there is also a correlation between the number of antecedent transfusions in humans and the rate of bone marrow transplant (BMT) rejection—under reduced intensity conditioning with HLA-matched or HLA-identical marrow. Bone marrow transplant of this nature is the only existing cure for a series of nonmalignant hematologic diseases (eg, sickle cell disease, thalassemias, etc); however, rejection remains a clinical problem. It has been hypothesized that transfusion induces subsequent BMT rejection through immunization. Studies in animal models have observed the same effect and have demonstrated that transfusion-induced BMT rejection can occur in response to alloimmunization. However, unlike traditional antibody responses, sensitization in this case results in cellular immune effects, involving populations such as T cell or natural killer cells. In this case, rejection occurs in the absence of alloantibodies and would not be detected by existing immune-hematologic methods. We review human and animal studies in light of the hypothesis that, for distinct clinical populations, enhanced rejection of BMT may be an unappreciated adverse consequence of transfusion, which current blood bank methodologies are unable to detect. [Copyright &y& Elsevier]

Clinically significant signs and symptoms of hemolysis can result from the transfusion of crossmatch incompatible blood, the development of anti–red blood cell (RBC) alloantibodies posttransfusion, or the development of autoantibodies to RBCs (ie, autoimmune hemolytic anemia). However, a less understood and poorly appreciated phenomenon is when an antibody induces the loss of its target antigen without significant damage to the cells themselves. This has been referred to as “depressed antigen,” “antigen suppression,” “weakened antigenicity,” and “antigen loss.” This phenomenon has been observed for multiple blood group antigens on human RBCs, in addition to antigens on leukocytes, platelets, neurons, and neoplastic cells. This review discusses the published human case reports of antigen loss in each of the contexts described above and describes several experimental models, including whole animal models and in vitro culture systems. Our current understanding of the cellular and molecular mechanisms is discussed, and the role of antigen loss phenomena in both normal immune function and in disease states is reviewed. [Copyright &y& Elsevier]

Currently, more than 50 years after its apparent early recognition in case reports, and more than 20 years after its name was coined to denote a distinct entity of pulmonary transfusion reactions, transfusion-related acute lung injury (TRALI) has emerged as a serious cause of transfusion-associated morbidity and the subject of an exponentially growing scientific literature. However, review articles, clinical case reports, and case series continue to dominate the published literature on the topic and experimental studies aimed at modeling and elucidating TRALI mechanisms are less frequent. This article reviews the current status of the known experimental models of TRALI, with particular emphasis on efforts to establish in vivo animal models of this important pulmonary transfusion reaction. [Copyright &y& Elsevier]

Unique issues in blood donation and blood transfusion regarding African Americans (AA) in the United States span the donation process, manufacturing of products, and hospital transfusion service. As AAs become a growing population, a constant supply of blood donated by AAs is necessary to support this growth. Nationally, AAs are underrepresented in blood collection, which may be secondary to AAs having higher rates of anemia and other deferrable conditions or unique motivators as well as other barriers to blood donation. When investigating blood transfusion practices, blood utilization for different races and ethnicities is unknown. AAs may receive more red blood cell (RBC) transfusions because they have a higher proportion of diseases that require transfusion. Patients with sickle cell disease are at increased risk of RBC alloimmunization likely due to the predominance of RBC units from white donors in the existing blood supply, but it is not known if all AA recipients experience increased alloimmunization rates compared with whites. In conclusion, there is a need to increase donation by AAs, which can only be achieved by conducting studies to understand racial differences in donor recruitment and to better understand blood utilization and adverse events as a factor of race and ethnicity. [Copyright &y& Elsevier]

Transfusion-transmitted cytomegalovirus (CMV) infection (TT-CMV) continues to complicate blood transfusion therapy, which can lead to severe morbidity or mortality in immunocompromised or immuno-immature recipients. The biological mechanisms that underlie TT-CMV (eg, viral latency in donor monocytes or stimulatory signals in the transfusion recipient leading to cytomegalovirus reactivation) are difficult to study in humans, but can be addressed in animal models. In this review, we discuss a mouse blood transfusion model, which can be used to investigate these issues as well as to validate methods to prevent TT-CMV in at-risk patients. [Copyright &y& Elsevier]

Abstract: In this report, we provide evidence that muscarinic receptors play a role in the generation of CD8+ cytolytic T lymphocytes. Analysis of mice with targeted deletions of each of the known muscarinic receptors (M1–M5) showed that CD8+ T cells from M1 receptor-deficient mice had a defect in the ability to differentiate into cytolytic T lymphocytes. Additional pharmacological experiments support the role of muscarinic receptors in wild type mice and suggest that acetylcholine may be involved. Together, these findings suggest that the M1 muscarinic receptor is involved in CTL development, thus providing novel insights into CD8+ T cell biology and the potential role of cholinergic signaling in immune regulation. [Copyright &y& Elsevier]

The red blood cell (RBC)-Omics study, part of the larger NHLBI-funded Recipient Epidemiology and Donor Evaluation Study (REDS-III), aims to understand the genetic contribution to blood donor RBC characteristics. Previous work identified donor demographic, behavioral, genetic, and metabolic underpinnings to blood donation, storage, and (to a lesser extent) transfusion outcomes, but none have yet linked the genetic and metabolic bodies of work. We performed a genome-wide association (GWA) analysis using RBC-Omics study participants with generated untargeted metabolomics data to identify metabolite quantitative trait loci in RBCs. We performed GWA analyses of 382 metabolites in 243 individuals imputed using the 1000 Genomes Project phase 3 all-ancestry reference panel. Analyses were conducted using ProbABEL and adjusted for sex, age, donation center, number of whole blood donations in the past 2 years, and first 10 principal components of ancestry. Our results identified 423 independent genetic loci associated with 132 metabolites (p < 5x10-8). Potentially novel locus-metabolite associations were identified for the region encoding heme transporter FLVCR1 and choline and for lysophosphatidylcholine acetyltransferase LPCAT3 and lysophosphatidylserine 16.0, 18.0, 18.1, and 18.2; these associations are supported by published rare disease and mouse studies. We also confirmed previous metabolite GWA results for associations, including N(6)-methyl-L-lysine and protein PYROXD2 and various carnitines and transporter SLC22A16. Association between pyruvate levels and G6PD polymorphisms was validated in an independent cohort and novel murine models of G6PD deficiency (African and Mediterranean variants). We demonstrate that it is possible to perform metabolomics-scale GWA analyses with a modest, trans-ancestry sample size. [ABSTRACT FROM AUTHOR]

• Mouse IgG (mIgG) subclasses cross-react with human Fc gamma receptors (hFcγRs). • Exact affinities were determined for the full spectrum of mIgG-hFcγR interactions. • Antibody deglycosylation decreases mIgG binding to hFcγRs. • Antibody deglycosylation only eliminates mIgG2b binding to hFcγRs. Immunoglobulin G (IgG) antibodies are important for protection against pathogens and exert effector functions through binding to IgG-Fc receptors (FcγRs) on myeloid and natural killer cells, resulting in destruction of opsonized target cells. Despite interspecies differences, IgG subclasses and FcγRs show substantial similarities and functional conservation between mammals. Accordingly, binding of human IgG (hIgG) to mouse FcγRs (mFcγRs) has been utilized to study effector functions of hIgG in mice. In other applications, such as immunostaining with mouse IgG monoclonal antibodies (mAbs), these cross-reactivities are undesired and prone to misinterpretation. Despite this drawback, the binding of mouse IgG (mIgG) subclasses to human FcγR (hFcγR) classes has never been fully documented. Here, we report detailed and quantifiable characterization of binding affinities for all mIgG subclasses to hFcγRs, including functional polymorphic variants. mIgG subclasses show the strongest binding to hFcγRIa, with relative affinities mIgG2a = mIgG2c > mIgG3 >> mIgG2b, and no binding by mIgG1. hFcγRIIa/b showed general low reactivities to all mIgG (mIgG1> mIgG2a/c > mIgG2b), with no reactivity to mIgG3. A particularly high affinity was observed for mIgG1 to the hFcγRIIa-R131 polymorphic variant. hFcγRIIIa showed lower binding (mIgG2a/c > mIgG3), slightly favouring binding to the hFcγRIIIa-V158 over the F158 polymorphic variant. No binding was observed of mIgG to hFcγRIIIb. Deglycosylation of mIgG1 did not abrogate binding to hFcγRIIa-R131, nor did deglycosylation of mIgG2a/c and mIgG3 prevent hFcγRIa binding. Importantly, deglycosylation of the least cross-reactive mIgG subclass, mIgG2b, abrogated reactivity to all hFcγRs. Together, these data document for the first time the full spectrum of cross-reactivities of mouse IgG to human FcγRs. [ABSTRACT FROM AUTHOR]

More than 90% of allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients receive red blood cell (RBC) or platelet transfusions in the peritransplantation period. We tested the hypothesis that transfusions are associated with the development of severe (grade III-IV) acute graft-versus-host disease (aGVHD) or mortality after allo-HSCT in a retrospective study of 322 consecutive patients receiving an allogeneic bone marrow or granulocyte colony-stimulating factor-mobilized blood stem cell graft for a hematologic malignancy. Counting transfused RBC and platelet units between day −7 pretransplantation and day +27 post-transplantation, but excluding transfusions administered after a diagnosis of aGVHD, yielded medians of 5 RBC units and 2 platelet units transfused. Sixty-three patients (20%) developed a maximal grade III-IV aGVHD with onset up to day +150 post-transplantation (median aGVHD onset of 28 days). HLA mismatch (hazard ratio [HR], 2.4; 95% confidence interval [CI], 1.2 to 4.7; P  = .01), and transfusion of more than the median number of RBC units (HR, 2.1; 95% CI, 1.1 to 3.7; P  = .02) were independently associated with greater risk of grade III-IV aGVHD in a multivariable analysis model. Disease risk strata (HR, 1.7; 95% CI, 1.2 to 2.4 for high risk versus low risk; P  = .005) and transfusion of more than the median number of RBC units (HR, 1.4; 95% CI, 1.0 to 2.0; P  = .054) were independently associated with inferior overall survival. These data support our hypothesis that peritransplantation RBC transfusions are associated with the risk of developing severe aGVHD and worse overall survival following allo-HSCT, and suggest that strategies to reduce routine RBC transfusion may favorably reduce the incidence and severity of GVHD. [ABSTRACT FROM AUTHOR]

Methods designed to monitor humoral immune responses, in a variety of settings, typically use a broadly reactive detection reagent ( e.g. polyclonal anti-Ig (immunoglobulin)) in order to characterize antibody responses. In the context of murine models of immunity, which are widely used, this would typically be antisera to mouse Ig or mouse IgG. However, there are 4 different subtypes of mouse IgG; thus, the validity of the above approach, as a general screen for humoral immune responses, depends upon the assumption that the antisera recognize all IgG subtypes. This seems like a reasonable assumption, since polyclonal antisera recognize multiple epitopes; however, herein we report that two commercial sources of goat anti-mouse Ig are hyporeactive with IgG3. Given that relative IgG3 levels are different in distinct types of immune response, these findings demonstrate a potential for misinterpretation, and suggest a need to modify immunological methods in this context. [ABSTRACT FROM AUTHOR]

Abstract: Generation of alloantibodies to transfused RBCs can be a serious medical problem for patients who require chronic RBC transfusion therapy. Patients with sickle cell disease have a substantially increased rate of alloimmunization compared to other chronically transfused populations. A recent study has forwarded the hypothesis that a polymorphism in an immunoregulatory gene in close proximity to beta-globin (TRIM21 rs660) plays a role in the increased rates of RBC alloimmunization in sickle cell patients. In particular, it was hypothesized that rs660C/T decreases expression of TRIM21, resulting in loss of a negative feedback pathway in immune responses and increased RBC alloimmunization. To test the effects of TRIM21 expression on alloimmunization, we analyzed antibody responses to alloantigens on RBCs and platelets transfused into wild-type and TRIM21 KO mice. No significant increases were seen in the frequency or magnitude of humoral immunization to alloantigens on transfused RBCs or platelets in adult or juvenile TRIM21 KO recipients compared to wild-type controls. Moreover, recipient inflammation with poly (I:C) enhanced RBC alloimmunization to similar degrees in both TRIM21 KO and wild-type control recipients. Together, these data rule out the hypothesis that decreased TRIM21 expression enhances transfusion induced humoral alloimmunization, in the context of a reductionist murine model. [Copyright &y& Elsevier]

Abstract: A number of studies have demonstrated that non-neuronal acetylcholine can play a role in the regulation of T cell function. Recently, we reported that CD8+ T cells, from mice with a targeted deletion of the M1 muscarinic receptor, had a defect in differentiating into cytolytic T lymphocytes when stimulated in vitro. In the current report, we analyze the in vivo function of CD8+ T cells from mice with targeted deletions of either M1 or M5 muscarinic receptors. M1 or M5 knockout mice were infected with either lymphocytic choriomeningitis virus or vesicular stomatitis virus. Expansion of anti-viral CD8+ T cells was monitored by staining with tetramer reagents specific for the immunodominant peptides of the viruses. No defect in expansion of CD8+ T cells was observed in either M1 or M5 knockout mice. The extent to which one can draw a generalized conclusion that M1 and M5 are not involved in anti-viral immunity depends upon issues of antigen strength, genetic background, induction of redundant receptors, and the potential for qualitative defects in the expanded CD8+ T cells. [Copyright &y& Elsevier]