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Hemolytic transfusion reactions (HTRs) due to incompatible red blood cell (RBC) transfusions are a leading cause of transfusion associated death. Although many transfused incompatible RBCs are cleared, some remain in circulation despite the presence of RBC-specific antibodies, potentially due to "antigen modulation." With a goal of better understanding incompatible RBC clearance, we generated a murine model with RBC-specific expression of a clinically significant human antigen (KEL2) known to be involved in antigen modulation as well as in HTRs. Wild-type (WT) recipients transfused with transgenic KEL2 RBCs generated anti-KEL glycoprotein alloantibodies, which fixed complement, led to intravascular hemolysis, and resulted in decreased levels of KEL2 antigen detectable on cells remaining in circulation. Antigen modulation did not appear to solely reflect removal of RBCs with higher antigen expression, because cells continued to display antigen modulation in the absence of significant clearance. Recipients genetically lacking complement exhibited lesser degrees of incompatible RBC clearance and antigen modulation in comparison with WT or FcγR knock-out (KO) animals, suggesting a role for complement in RBC clearance. In summary, this HTR model may serve as a platform to test strategies to downmodulate antigen and inhibit incompatible RBC clearance, thus potentially mitigating transfusion dangers.

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 © 2011 Elsevier Ltd. All rights reserved.)

Purpose of Review: To discuss how inflammation affects humoral alloimmunization to antigens on transfused red blood cells (RBCs)., Recent Findings: Recently, three unique murine models of humoral alloimmunization to transfused RBCs have been described. As in humans, RBC alloimmunization rates in recipient mice are variable, with segregation into responder and nonresponder groups. Because the recipient mice are genetically identical, environmental factors may play a role in regulating alloimmunization. These models have further been used to demonstrate that recipient inflammation has a complex regulatory effect upon alloimmunization. Isolated case reports in humans raise the possibility of a similar role of inflammation in regulating alloimmunization to RBCs., Summary: It is currently unknown why some human transfusion recipients mount strong alloantibody responses, whereas others do not. Human leukocyte antigen immunogenetics likely play a substantial role. However, within groups with the genetic capacity to respond to a given RBC antigen, recipients still segregate into responders and nonresponders. The data reviewed herein indicate that recipient inflammation in mice has the capacity to regulate alloimmunization. Future studies will be required to further understand these effects and to investigate if similar biology regulates alloimmunization to transfused RBCs in humans.

Purpose: Our long-term goal is to determine the optimal methods for inducing allograft tolerance to facilitate transplantation of retinal pigment epithelial cells or stem cells for the treatment of retinal degenerative diseases. These goals have been hampered by the extreme complexity of allograft rejection and the heterogeneity of responding T cells. The current studies were undertaken to develop a simplified transplant model for studying rejection and tolerance in the unique environment of the eye., Methods: To provide a defined transplantation antigen, transgenic C57BL/6 (B6) mice were produced, which express the exogenous chicken egg ovalbumin (OVA) gene under the regulation of the mouse tyrosinase related protein-1 (TRP-1) promoter that is transcriptionally active in retinal pigmented epithelial (RPE) cells. To determine whether the transgene was expressed as a neo-transplantation antigen, RPE from TRP-1-OVA mice were injected into the subretinal space of B6 mice or B6 mice expressing the OVA-specific (OT1) TCR transgenes and examined for inflammatory cell infiltration., Results: The TRP-1-OVA transgenic mice expressed OVA mRNA in the brain and eye but not the heart or kidney. RPE cells from TRP-1-OVA transgenic mice expressed mRNA and protein encoded by the OVA gene and RPE expressing TRP-1-OVA induced an inflammatory response within the subretinal space of OT1 mice but not in B6 mice., Conclusions: OVA serves as a defined, neo-transplantation antigen in RPE that is recognized by mice whose CD8+ T cells recognize OVA peptide. These observations provide new tools for future studies of the mechanisms of rejection and prolongation of RPE transplants in the eye.

Factors regulating which patients become alloimmunized to red blood cell (RBC) antigens are poorly understood. Using a murine model of transfusion, we recently reported that viral-like inflammation with polyinosinic polycytidylic acid [poly (I:C)] significantly enhances RBC alloimmunization. Herein, we tested the hypothesis that poly (I:C) exerts this effect, at least in part, at the level of antigen-presenting cells (APCs). Using a novel in vivo method, we report that in the noninflamed state, most transfused RBCs were consumed by splenic macrophages, with only trace consumption by splenic dendritic cells (DCs). To a lesser extent, RBCs were also consumed by APCs in the liver. However, unlike soluble antigens, no RBCs were consumed by APCs in the lymph nodes. Inflammation with poly (I:C) induced significant consumption of transfused RBCs by splenic DCs, with a concomitant increase in costimulatory molecule expression. Moreover, this resulted in increased proliferation of CD4(+) T cells specific for the mHEL RBC alloantigen. Finally, splenectomy abrogated the enhancing effects of poly (I:C) on RBC alloimmunization. Together, these data provide additional insight into the nature of transfused RBCs as an immunogen and provide a mechanism by which viral-like inflammation enhances alloimmunization to transfused RBCs.

Administration of anti-RhD immunoglobulin (Ig) to decrease maternal alloimmunization (antibody-mediated immune suppression [AMIS]) was a landmark clinical development. However, IgG has potent immune-stimulatory effects in other settings (antibody-mediated immune enhancement [AMIE]). The dominant thinking has been that IgG causes AMIS for antigens on RBCs but AMIE for soluble antigens. However, we have recently reported that IgG against RBC antigens can cause either AMIS or AMIE as a function of an IgG subclass. Recent advances in mechanistic understanding have demonstrated that RBC alloimmunization requires the IFN-[alpha]/-[beta] receptor (IFNAR) and is inhibited by the complement C3 protein. Here, we demonstrate the opposite for AMIE of an RBC alloantigen (IFNAR is not required and C3 enhances). RBC clearance, C3 deposition, and antigen modulation all preceded AMIE, and both [CD4.sup.+] T cells and marginal zone B cells were required. We detected no significant increase in antigen-specific germinal center B cells, consistent with other studies of RBC alloimmunization that show extrafollicular-like responses. To the best of our knowledge, these findings provide the first evidence of an RBC alloimmunization pathway which is IFNAR independent and C3 dependent, thus further advancing our understanding of RBCs as an immunogen and AMIE as a phenomenon., Introduction Immunoglobulins are most commonly thought of as immune effector molecules. However, it has been known for over a century that Igs can also substantially enhance primary humoral immunity to [...]

Background: Transgenic mice expressing RBC specific antigens are widely used in mechanistic studies of RBC alloimmunization. Existing RBC donor strains have random transgene integration, potentially disrupting host elements that can confound biological interpretation. Study Design and Methods: Integration site and genomic alterations were characterized by both targeted locus amplification and congenic backcrossing in the five most commonly used RBC alloantigen donor strains (KEL‐K2hi, KEL‐K2med, and KEL‐K2lo, and KEL‐K1). A targeted transgenic approach was developed to allow RBC specific transgene expression from a safe harbor locus (ROSA26). Alloimmune responses were assessed by transfusing alloantigen expressing RBCs into wild‐type recipients and measuring alloantibodies by flow cytometry. Results/Findings: Four of the five analyzed strains had at least one gene disrupted by the transgene integration but none of the disrupted genes are known to be involved in RBC biology. The integration of KEL‐K2med potentially altered the immunological properties of RBCs, although the biological significance of the observed changes is unclear. The ROSA26 targeted approach resulted in a single copy of the transgene that maintains RBC specific expression without random disruption of genomic elements. Conclusion: These findings provide a detailed characterization of genomic disruption by transgene integration found in commonly used RBC donor strains that is relevant to numerous previous publications as well as future studies. With the possible exception of KEL‐K2med, transgene integration is not predicted to affect RBC biology in existing models, and new models can avoid this concern using the described targeted transgenic approach. [ABSTRACT FROM AUTHOR]

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.Conclusion: Although storage of RBCs leads to alteration of several lysophospholipids known to be capable of binding CD1D, storage-induced RBC alloimmunization responses are not impacted by recipient CD1D deficiency. [ABSTRACT FROM AUTHOR]

It has long been appreciated that immunoglobulins are not just the effector endpoint of humoral immunity, but rather have a complex role in regulating antibody responses themselves. Donor derived anti-RhD IgG has been used for over 50 years as an immunoprophylactic to prevent maternal alloimmunization to RhD. Although anti-RhD has dramatically decreased rates of hemolytic disease of the fetus and newborn (for the RhD alloantigen), anti-RhD also fails in some cases, and can even paradoxically enhance immune responses in some circumstances. Attempts to generate a monoclonal anti-RhD have largely failed, with some monoclonals suppressing less than donor derived anti-RhD and others enhancing immunity. These difficulties likely result, in part, because the mechanism of anti-RhD remains unclear. However, substantial evidence exists to reject the common explanations of simple clearance of RhD + RBCs or masking of antigen. Donor derived anti-RhD is a mixture of 4 different IgG subtypes. To the best of our knowledge an analysis of the role different IgG subtypes play in immunoregulation has not been carried out; and, only IgG1 and IgG3 have been tested as monoclonals. Multiple attempts to elicit alloimmune responses to human RhD epitopes in mice have failed. To circumvent this limitation, we utilize a tractable animal model of RBC alloimmunization using the human Kell glycoprotein as an antigen to test the effect of IgG subtype on immunoregulation by antibodies to RBC alloantigens. We report that the ability of an anti-RBC IgG to enhance, suppress (at the level of IgM responses), or have no effect is a function of the IgG subclass in this model system. [ABSTRACT FROM AUTHOR]

Background: Anti-red blood cell (RBC) alloantibodies consisting of only the immunoglobulin G (IgG) 4 subtype are typically considered clinically insignificant. A US Food and Drug Administration-approved monoclonal anti-human globulin (16H8) is nonreactive with IgG4, which has been considered a benefit to avoid testing interference from IgG4. However, 16H8 also does not recognize two natural IgG3 variants (IgG3-03 and IgG3-13). Thus, 16H8 may miss clinically significant alloantibodies in some settings.Study Design and Methods: Novel mouse anti-human IgG hybridomas were generated and screened for reactivity with 32 human variants of anti-KEL1 across different IgG subtypes, as well as mutants to allow epitope mapping. Anti-IgG reactivity was determined using KEL1+ RBCs bound by each IgG variant as targets. Binding of anti-IgG was determined by flow cytometry.Results: 16H8 recognized an epitope involving amino acid 419, which is glutamate in IgG4, IgG3-03, and IgG3-13, explaining the lack of 16H8 reactivity with these subtypes/isoallotypes. A new monoclonal antibody (PUMA8) was isolated that, like 16H8, was nonreactive with IgG4 but without blind spots for known variants of IgG1, IgG2, or IgG3. PUMA8 recognized an epitope containing arginine at position 355, which is glutamine in IgG4. However, a recently described new IgG4 variant with an arginine at position 355 results in PUMA8 reactivity.Conclusion: PUMA8 represents an alternative to 16H8 that avoids IgG4 but without blind spots for IgG3 variants. However, PUMA8 reacts with one recently described IgG4 variant. In addition to relevance to immunohematology, these studies highlight the importance of patient variation with regards to assay performance in an era of personalized medicine. [ABSTRACT FROM AUTHOR]

Background: Human immunoglobulin G (hIgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4). Due to genetic variations, each IgG subtype contains different isoallotypes. It was previously shown that a Food and Drug Administration-approved monoclonal anti-IgG failed to recognize 2 of 15 recombinant, human IgG3 anti-Kell (K1) isoallotypes (rIgG3-03 and rIgG3-13) by indirect antiglobulin test (IAT).Study Design and Methods: We expressed and purified 15 recombinant human rIgG3 anti-K1 isoallotypes and investigated their antigen binding and ability to induce phagocytosis using homozygous (KK) and heterozygous (Kk) K1-positive red blood cells (RBCs) by gel IAT, flow cytometry, and a monocyte monolayer assay (MMA) with peripheral blood monocytes and cultured inflammatory (M1) and anti-inflammatory (M2) macrophages.Results: MMA results showed that differences in the Fc region of rIgG3 anti-K1 led to distinctive phagocytic activity with both monocytes and M1 macrophages. rIgG3-18 and rIgG3-19 showed an enhanced ability to induce phagocytosis. Differences in Fc regions also led to variations in the number of antibodies bound to KK RBCs. Despite the differences in phagocytic activity, all 15 rIgG3 clones are predicted to induce clinically significant hemolysis if K1-positive blood was transfused into patients.Conclusion: These results argue that antiglobulin reagents that fail to detect isoallotype rIgG3-03 or rIgG3-13 could present a transfusion risk or lack of detection of a potentially clinically significant anti-K1 in hemolytic disease of the fetus and newborn. [ABSTRACT FROM AUTHOR]

Background: Kell is a glycoprotein expressed on red blood cells (RBCs). Its K and k variants contain either Met (K antigen) or Thr (k antigen) at Position 193, respectively. Development of anti-K after K-mismatched antigen exposure via blood transfusions or pregnancy can destroy RBCs, leading to hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. The immunogenicity of overlapping 15-mer Kell peptides with M193 or T193 at every possible position was investigated previously. Interestingly, Peptide W179 to M193, with the polymorphic M193T residue at the peptide's C-terminus, was the most effective at stimulating CD4 T cells from a series of K-immunized women.Study Design and Methods: This study investigates the basis for HLA restriction of anti-K immune responses. Major histocompatibility complex Class II (MHCII)-binding prediction algorithms and quantitative peptide-MHCII-binding assays were employed to determine the binding registers; anchor residues; and affinities of wild-type, truncated, and sequence-modified K and k peptides. Predictions were generated using Immune Epitope Database and ProPred algorithms. Competitive peptide-MHCII-binding assays utilized 12 recombinant HLA-DR proteins, K and k peptides, and high-affinity MHCII-restricted reference peptides.Results: The peptide-MHCII-binding assays identified a unique K peptide-binding register (W179-S187) restricted to HLA-DRB1*11:01, in addition to partially overlapping binding registers that included the K/k M193T polymorphic site and that bound promiscuously to multiple HLA-DR proteins.Conclusion: Three partially overlapping MHCII-binding motifs for HLA-DRB1*11:01 result in high-avidity K-peptide binding, which may contribute to HLA-DR11-restricted immunogenicity associated with the K allele. [ABSTRACT FROM AUTHOR]

Intravenous immune globulin (IVIG) was developed initially as an immunoglobulin replacement therapy for primary humoral immunodeficiency, but is now widely used in the treatment of autoinflammatory and autoimmune pathologies. In a small number of patients, hemolytic sequelae have been observed after IVIG administration. The lack of a simple one-to-one correlation between measurable hemagglutinins and hemolysis has led to complicated hypotheses involving coincident necessary variables (e.g., a two-hit hypothesis) and also to the positing of causal factors other than hemagglutinins. One such hypothesis is that immune complexes (ICs) contained within IVIG lead to hemolysis. IVIG-mediated hemolysis was addressed at a recent meeting sponsored by the Food and Drug Administration; the Plasma Protein Therapeutics Association; and the National Heart, Lung, and Blood Institute. The primary literature was reviewed at this meeting followed by detailed discussion. Participants concluded that there is both a theoretical basis by which ICs could contribute to hemolysis after IVIG administration and some published data in support of such a possibility. However, the reported data contain substantial caveats, and the existing evidence does not rise to a level sufficient to either confirm or reject a role for ICs. More detailed and focused human studies will be required to further assess the potential role of ICs in IVIG induced hemolysis. This paper summarizes the relevant literature and expands upon the conclusions of this workshop. [ABSTRACT FROM AUTHOR]

Background Alloimmunization to antigens on transfused red blood cells ( RBCs) represents a major barrier to chronic transfusion. In extreme cases of multiple alloimmunization, clinicians may be faced with the decision of transfusing incompatible RBCs or risking death from lack of transfusion. The disastrous results of hemolytic transfusion reactions are well understood, and major pathways of clearance have been described. However, well described but poorly understood is the survival of a subset of incompatible donor RBCs during hemolysis, despite antibody binding. Study Design And Methods We utilize a tractable murine model of incompatible transfusion in which RBCs from transgenic donor mice expressing human glycophorin A ( hGPA) are transfused into recipients passively immunized with anti- hGPA. Results As in humans, the majority of RBCs are cleared but a subset of incompatible donor RBCs persist in circulation, despite being bound by antibodies. Data contained herein reject the hypothesis that lack of clearance is due to insufficient antibody or overwhelming of phagocytic machinery; rather, we establish that surviving RBCs represent a distinct population resistant to clearance. Conclusions These studies demonstrate that surviving RBCs during incompatible transfusion can represent a population that is resistant to clearance. [ABSTRACT FROM AUTHOR]

BACKGROUND: KEL1, also known as 'K,' is one of the most immunogenic red blood cell (RBC) antigens. KEL2, also known as 'k,' differs from KEL1 by a single amino acid. Anti-Kell system antibodies can lead to significant adverse clinical outcomes in humans, including hemolytic complications in alloimmunized transfusion recipients or in infants of alloimmunized mothers. To provide a platform for in-depth immunologic studies of alloimmunization and subsequent sequelae, we generated transgenic mice expressing the human KEL1 or KEL2 antigens. STUDY DESIGN AND METHODS: Vectors were created in which cDNAs encoding either KEL1 or KEL2 were regulated by an erythroid specific β-globin promoter and enhancer. Pronuclear microinjections were carried out into a C57BL6 background, and founder pups were identified by polymerase chain reaction and screened for expression by flow cytometry. RBC life span and antigen stability were assessed by dye labeling RBCs, transfusing into agammaglobulinemic (µMT) recipients, and tracking by flow cytometry. RESULTS: The expression of either KEL1 or KEL2 is RBC specific and first occurs on early RBC precursors. Both KEL1 and KEL2 RBCs have a normal circulatory life span and stable antigen expression. Expression of KEL1 or KEL2 does not result in altered levels of murine Kell, and resulting RBCs have normal hematologic variables. CONCLUSION: The KEL1 and KEL2 mice represent the first murine system of RBC immunity with antithetical antigens, allowing a more precise modeling of human RBC immunology in general and also a platform for development of novel therapeutics to prevent or minimize the dangers of RBC alloimmunization to the KEL1 and KEL2 antigens in particular. [ABSTRACT FROM AUTHOR]

BACKGROUND: Increased rates of red blood cell (RBC) alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease. STUDY DESIGN AND METHODS: Transgenic sickle mice, which express human α and βS globin, were transfused with fresh or 14-day-stored RBCs containing the HOD (hen egg lysozyme, ovalbumin, and human Duffyb) antigen; some recipients were inflamed with poly(I : C) before transfusion. Anti-HOD alloantibody responses were subsequently measured by enzyme-linked immunosorbent assay and flow crossmatch; a cohort of recipients had posttransfusion serum cytokines measured by bead array. RESULTS: Both Berkeley and Townes homozygous (SS) and heterozygous (AS) mice had similar rates and magnitude of anti-HOD RBC alloimmunization after fresh HOD RBC transfusion compared with control animals; under no tested condition did homozygous SS recipients make higher levels of alloantibodies than control animals. Unexpectedly, homozygous SS recipients had blunted cytokine responses and lower levels of anti-HOD alloantibodies after transfusion of 14-day stored RBCs, compared with control animals. CONCLUSIONS: In sum, homozygous βS expression and the ensuing disease state are not alone sufficient to enhance RBC alloimmunization to transfused HOD RBCs in two distinct humanized murine models of sickle cell disease under the conditions examined. These data suggest that other factors may contribute to the high rates of RBC alloimmunization observed in humans with sickle cell disease. [ABSTRACT FROM AUTHOR]

BACKGROUND: Fourteen-day stored red blood cells (RBCs) containing an RBC-specific transgenic antigen (HOD) induce a recipient proinflammatory cytokine storm and are significantly more immunogenic compared to fresh RBCs. Given that recipient mice clear transfused stored RBCs more rapidly than fresh RBCs, we hypothesized that rapid RBC clearance was associated with adverse transfusion outcomes. STUDY DESIGN AND METHODS: HOD RBCs were treated by two distinct methods known to lead to rapid posttransfusion RBC clearance: phenylhydrazine or heat. HOD antigen expression was analyzed on the treated cells before transfusion, and RBC recovery, recipient cytokine response, and recipient anti-HOD alloimmunization response were measured after transfusion. RESULTS: Phenylhydrazine and heat treatment each led to near complete RBC clearance in recipients by 24 hours posttransfusion, without significantly altering HOD antigen expression on the transfused RBCs. Recipients of phenylhydrazine- or heat-treated RBCs had elevated circulating levels of keratinocyte-derived chemokine/CXCL-1, monocyte chemoattractant protein-1, and interleukin-6 after transfusion. Furthermore, phenylhydrazine- or heat-treated RBCs were significantly more immunogenic than control RBCs, with a mean 25.1- and 10.3-fold enhancement, respectively, of anti-HOD alloimmunization magnitude by flow cytometric crossmatch. CONCLUSIONS: Three separate insults to RBCs (storage, phenylhydrazine, or heat treatment) result in rapid posttransfusion clearance, with a recipient proinflammatory cytokine storm and enhanced alloimmunogenicity. These data are consistent with the hypothesis that rapid clearance of RBCs is causally involved in these outcomes and suggest that human donor RBCs with favorable posttransfusion clearance profiles may be less immunogenic. [ABSTRACT FROM AUTHOR]

BACKGROUND: Red blood cell (RBC) alloimmunization can be a serious complication of blood transfusion, but factors influencing the development of alloantibodies are only partially understood. Within FDA-approved time limits, RBCs are generally transfused without regard to length of storage. However, recent studies have raised concerns that RBCs stored for more than 14 days have altered biologic properties that may affect medical outcomes. To test the hypothesis that storage time alters RBC immunogenicity, we utilized a murine model of RBC storage and alloimmunization. STUDY DESIGN AND METHODS: Blood from transgenic HOD donor mice, which express a model antigen (hen egg lysozyme [HEL]) specifically on RBCs, was filter leukoreduced and stored for 14 days under conditions similar to those used for human RBCs. Fresh or 14-day-stored RBCs were transfused into wild-type recipients. The stability of the HOD antigen and posttransfusion RBC survival were analyzed by flow cytometry. RBC alloimmunization was monitored by measuring circulating anti-HEL immunoglobulin levels. RESULTS: Transfusion of 14-day-stored, leukoreduced HOD RBCs resulted in 10- to 100-fold higher levels of anti-HEL alloantibodies as detected by enzyme-linked immunosorbent assay than transfusion of freshly collected, leukoreduced RBCs. RBC expression of the HOD antigen was stable during storage. CONCLUSIONS: These findings demonstrate that HOD murine RBCs become more immunogenic with storage and generate the rationale for clinical trials to test if the same phenomenon is observed in humans. Length of storage of RBCs may represent a previously unappreciated variable in whether or not a transfusion recipient becomes alloimmunized. [ABSTRACT FROM AUTHOR]

Blood bank serology has identified hundreds of red blood cell (RBC) antigens contained within numerous blood group systems. Although most blood group antigens are defined by amino acid polymorphisms in the extracellular domain of membrane proteins, it is also possible that additional nonexofacial polymorphisms (NEPs) may exist within cytoplasmic or transmembrane domains. To assess this possibility, we analyzed several blood group molecules by searching the SNPper database for nonsynonymous single-nucleotide polymorphisms. We report the identification of a number of NEPs in the Kell, Kidd, and Duffy molecules. Because the identified NEPs are not exposed on the surface of intact RBCs and are, thus, not accessible to recipient antibodies, they would neither be detected by blood bank serology in vitro, nor would they be recognized targets in hemolytic transfusion reactions in vivo. The presentation of peptides containing NEPs by recipient MHC Class II molecules, however, would nevertheless produce helper T-cell epitopes. In addition to identifying NEPs in human blood group molecules, we explore a novel hypothesis that the presence of NEPs contributes to the immunogenicity of blood group antigens. We further hypothesize that NEPs provide a mechanism by which transfusion can lead to anti-RBC autoantibodies, which are known to occur in humans after transfusion. The scientific basis, existing evidence, approaches to testing, and predicted biology of this hypothesis are presented. [ABSTRACT FROM AUTHOR]

The author presents views on a study which reports mapping out the peptide determinants of alloimmunization to the K antigen. He presents several problems associated with the study's approach including risk of inadvertently inducing an immune response and polymorphism related problems in K/k systems. The study presents advancement in the understanding of human immunology in the area of immunization to the K antigen on red blood cells (RBCs).

Although RBC transfusion can result in the development of anti-RBC alloantibodies that increase the probability of life-threatening hemolytic transfusion reactions, not all patients generate anti-RBC alloantibodies. However, the factors that regulate immune responsiveness to RBC transfusion remain incompletely understood. One variable that may influence alloantibody formation is RBC alloantigen density. RBC alloantigens exist at different densities on the RBC surface and likewise exhibit distinct propensities to induce RBC alloantibody formation. However, although distinct alloantigens reside on the RBC surface at different levels, most alloantigens also represent completely different structures, making it difficult to separate the potential impact of differences in Ag density from other alloantigen features that may also influence RBC alloimmunization. To address this, we generated RBCs that stably express the same Ag at different levels. Although exposure to RBCs with higher Ag levels induces a robust Ab response, RBCs bearing low Ag levels fail to induce RBC alloantibodies. However, exposure to low Ag-density RBCs is not without consequence, because recipients subsequently develop Ag-specific tolerance. Low Ag-density RBC-induced tolerance protects higher Ag-density RBCs from immune-mediated clearance, is Ag specific, and occurs through the induction of B cell unresponsiveness. These results demonstrate that Ag density can potently impact immune outcomes following RBC transfusion and suggest that RBCs with altered Ag levels may provide a unique tool to induce Ag-specific tolerance. [ABSTRACT FROM AUTHOR]

Exposure to nonself red blood cell (RBC) antigens, either from transfusion or pregnancy, may result in alloimmunization and incompatible RBC clearance. First described as a pregnancy complication 80 years ago, hemolytic disease of the fetus and newborn (HDFN) is caused by alloimmunization to paternally derived RBC antigens. Despite the morbidity/mortality of HDFN, women at risk for RBC alloimmunization have few therapeutic options. Given that alloantibodies to antigens in the KEL family are among the most clinically significant, we developed a murine model with RBC-specific expression of the human KEL antigen to evaluate the impact of maternal/fetal KEL incompatibility. After exposure to fetal KEL RBCs during successive pregnancies with KEL-positive males, 21 of 21 wild-type female mice developed antl-KEL alloantibodies; intrauterine fetal anemia and/or demise occurred in a subset of KEL-positive pups born to wild type, but not agammaglobulinemic mothers. Similar to previous observations in humans, pregnancy-associated alloantibodies were detrimental in a transfusion setting, and transfusion-associated alloantibodies were detrimental in a pregnancy setting. This is the first pregnancy-associated HDFN model described to date, which will serve as a platform to develop targeted therapies to prevent and/or mitigate the dangers of RBC alloantibodies to fetuses and newborns. [ABSTRACT FROM AUTHOR]

Bone marrow transplantation (BMT) represents a cure for nonmalignant hematological disorders. However, compared with the stringent conditioning regimens used when performing BMT to treat hematological malignancies, the reduced intensity conditioning regimen used in the context of nonmalignant hematological disorders leads to substantially higher rates of BMT rejection, presumably due to an intact immune system. The relevant patient population typically receives transfusion support, often including platelets, and the frequency of BMT rejection correlates with the frequency of transfusion. Here, we demonstrate that immunity to transfused platelets contributes to subsequent BMT rejection in mice, even when the BMT donor and recipient are MHC matched. We used MHC-matched bone marrow because, although immunity to transfused platelets is best characterized in relation to HLA-specific antibodies, such antibodies are unlikely to play a role in clinical BMT rejection that is HLA matched. However, bone marrow is not matched in the clinic for minor histocompatibility antigens, such as those carried by platelets, and we report that transfusion of minor histocompatibility antigen-mismatched platelets induced subsequent BMT rejection. These findings indicate previously unappreciated sequelae of immunity to platelets in the context of transplantation and suggest that strategies to account for minor histocompatibility mismatching may help to reduce the chance of BMT rejection in human patients. [ABSTRACT FROM AUTHOR]