ALBERT

Description: Mature red blood cells (RBCs) not only account for ~83% of the total host cells in the human body, but they are also exposed to all body tissues during their circulation in the bloodstream. In addition, RBCs are devoid of de novo protein synthesis capacity and, as such, they represent a perfect model to investigate system-wide alterations of cellular metabolism in the context of aging and age-related oxidant stress without the confounding factor of gene expression. In the present study, we employed ultra-high-pressure liquid chromatography coupled with mass spectrometry (UHPLC–MS)-based metabolomics and proteomics to investigate RBC metabolism across age in male mice (6, 15, and 25 months old). We report that RBCs from aging mice face a progressive decline in the capacity to cope with oxidant stress through the glutathione/NADPH-dependent antioxidant systems. Oxidant stress to tryptophan and purines was accompanied by declines in late glycolysis and methyl-group donors, a potential compensatory mechanism to repair oxidatively damaged proteins. Moreover, heterochronic parabiosis experiments demonstrated that the young environment only partially rescued the alterations in one-carbon metabolism in old mice, although it had minimal to no impact on glutathione homeostasis, the pentose phosphate pathway, and oxidation of purines and tryptophan, which were instead aggravated in old heterochronic parabionts.

Description: 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.

Description: Background: Immune-mediated destruction of red blood cells (RBCs) normally occurs following incompatible transfusion, however some RBCs may remain in circulation after initial cellular clearance despite the presence of RBC specific antibodies. Recent studies suggest that antibody-induced “antigen-modulation” may in part be responsible for this cellular resistance to antibody-mediated removal following the initial phase of RBC clearance. However, the factors that dictate whether cells undergo clearance or antigen modulation immediately following incompatible RBC transfusion remain unknown. As previous studies suggest that reticuloendothelial phagocytic capacity is sometimes surpassed upon engagement and removal of antibody-opsonized cells, we hypothesized that rapid change in the phagocytic burden of reticuloendothelial cells may contribute to the development of RBC resistance following incompatible RBC transfusion. Methods: Anti-Fy3 immunized or non-immunizedC57BL/6 recipient mice were first transfused with HOD.FVB RBCs expressing the HEL, OVA and Duffy chimeric antigen (HOD), followed by a second HOD.FVB RBC transfusion at various time intervals following the initial transfusion. Following transfusion, mice were evaluated at 10 min, 1h, 2h and 4h post-transfusion for HOD.FVB RBC survival, detectable HOD antigen, and RBC bound antibody by flow cytometric analysis. To examine the potential impact of RBC age on the sensitivity of HOD.FVB to antibody-induced clearance, HOD.FVB mice were injected with N-hydroxysulfosuccinimide (NHS) biotin 35 days prior to RBC isolation, to aid in differentiation of older from younger transfused RBCs, and then likewise transfused into anti-Fy3 immunized or non-immunizedC57BL/6 recipients and similarly evaluated for clearance, bound antibody and detectable antigen on biotin positive or negative transfused cells using flow cytometric analysis. Results: While HOD.FVB RBCs transfused into anti-Fy3-immunized C57BL/6 recipients displayed a rapid clearance following the initial transfusion, the degree of HOD.FVB RBC clearance significantly decreased following secondary transfusions that occurred within 2 hours of the initial transfusion. However, 2 days following the initial transfusion, recipients cleared HOD.FVB RBCs at the same rate as immunized recipients not previously exposed to HOD.FVB RBCs. Examination of the HOD antigen and bound antibody demonstrated that antigen levels likewise displayed the most significant decreases during the same time period in which RBC clearance decreased. Removal of HOD RBCs during the initial clearance phase did not appear to reflect preferential clearance of older RBCs, as transfused biotinylated and non-biotinylated RBCs harvested 1 day or 35 days following biotinylation displayed equal levels of antibody-induced clearance. Conclusion: Alterations in recipient clearance capacity following incompatible RBC exposure suggests that rapid phagocytic removal of RBCs may temporarily staturate the reticuloendothelial system immediately following RBC transfusion. However, antigen modulation appears to continue even while congestion of the reticuloendothelial system has halted RBC clearance. RBCs that escape the initial wave of clearance appear to become protected from antibody-mediated removal once the phagocytic capability of the recipient is restored, as the level of detectable antigen has dropped below that required for triggering phagocytic removal. The initial phase of clearance does not appear to reflect preferential clearance of older RBCs within the transfused units, but instead likely reflects stochastic removal of antibody bound RBCs, as RBC clearance occurred independent of RBC age. Disclosures No relevant conflicts of interest to declare.

Description: Background: Factors influencing rates of alloimmunization to antigens on transfused red blood cells (RBC alloimmunization) are poorly defined. In particular, the role of the spleen with respect to alloantibody formation is unclear, with conflicting clinical reports in the literature. Moreover, the complexities of multiply mismatched antigens and antigen priming due to transfusions prior to splenectomy make human studies difficult to interpret. To better define the role of the spleen in RBC alloimmunization, we utilized a murine model of transfusion medicine (with the model RBC antigen mHEL (membrane bound hen egg lysozyme)). Methods: Cohorts of splenectomized and non-splenectomized mice (C57BL/6 × B10.BR) were transfused with the human dose equivalent of 1 unit of leukoreduced mHEL RBCs. RBC alloimmunization was assessed by anti-HEL IgG specific ELISA. The role of antigen-specific CD4+ T cells was studied by the adoptive transfer of 1.5 × 106 HEL-specific CFSE-labeled CD4+ T cells from 3A9 TCR transgenic donors. Adoptively transferred cells were visualized using a congenic marker (Thy1.1); enumeration and division of these cells were monitored by flow cytometry in liver, spleen (if applicable), and lymph node preparations. Results: Splenectomy dramatically decreased RBC alloimmunization; 14 of 14 splenectomized mice (from 3 experiments) had undetectable to very low levels of anti-HEL IgG following transfusion with mHEL RBCs (average 6.3 fold less than non-splenectomized mice, 95% C.I. 4.6). Moreover, ten of ten splenectomized mice failed to make detectable levels of anti-HEL IgG even following the adoptive transfer of HEL-specific CD4+ T cells. In comparison, elevating the precursor frequency of HEL-specific CD4+ T cells increased RBC alloimmunization by 10,000 fold in non-splenectomized mice. Proliferation and division of CD4+ T cells were detectable in both spleen and liver preparations of non-splenectomized mice by day 3 following transfusion; in contrast, no expansion nor division of CD4+ T cells was seen in liver nor lymphatic preparations of splenectomized animals. Conclusions: The low level of RBC alloimmunization seen in non-splenectomized mice in this system is limited by existing CD4+ helper T cell responses, as increasing the naive helper T cell precursor frequency dramatically increased RBC alloimmunization. Furthermore, the spleen itself is critical to CD4+ helper T cell function during alloimmunization given that, in splenectomized mice, adoptively transferred HEL-specific CD4+ T cells fail to expand, divide, or stimulate production of detectable alloantibody. Ongoing studies are investigating the phenotype of HEL-specific CD4+ T cells (as effector cells, anergic cells, or regulatory cells) in splenectomized and non-splenectomized mice. These studies have implications for preventing alloimmunization in transfusion-dependent patient populations.

Description: Background Patients requiring repeat transfusion often develop RBC specific alloantibodies that decrease the therapeutic efficacy of transfused cells and limit the availability of compatible RBCs for future transfusion. However, not all RBC antigens possess equal ability to induce alloantibodies. While many factors likely influence this process, several studies suggest that antigen density may independently influence rates of RBC alloimmunization. To directly examine this, we generated transgenic founders with normal or lower levels of the human KEL antigen specifically on RBCs and examined the impact of RBC antigen levels on the development of anti-KEL antibodies following transfusion. Materials and methods Transgenic C57BL/6 founders expressing the human KEL antigen specifically on RBCs were generated using the β-globin promoter and screened for levels of KEL antigen using monoclonal anti-KEL antibodies by flow cytometric analysis. The number of KEL antigens on RBCs isolated from different founders was estimated using QIFIKIT beads. The molecular weight of KEL on RBCs isolated from each founder was assessed by Western blot analysis. C57BL/6 recipients were transfused with RBCs that expressed normal levels of KEL (KEL RBCs) or reduced levels of KEL (KELlo RBCs), followed by harvesting blood on days 3, 5, 7, 14, 21 and 28 following transfusion and analysis of serum for anti-KEL antibodies by indirect immunofluorescence using flow cytometry with KEL and control C57BL/6 RBCs as targets. In addition, C57BL/6 recipients were transfused with KELlo RBCs followed by KEL RBCs and similar analysis for anti-KEL antibody formation on days 3, 5, 7, 14, 21 and 28 following KEL RBC transfusion. All experiments were completed at least three times with 3–5 recipients per group per experiment. Results While KEL RBCs express approximately 1200 antigens per cell, KELlo RBCs express fewer than 200 KEL antigens. However, each KEL transgenic expressed a KEL protein of the predicted molecular weight (83 kD) as assessed by Western blot analysis. Transfusion of KEL RBCs induced IgM anti-KEL antibodies as early as 3 days post transfusion followed by peak IgG anti-KEL antibody levels 14 days following transfusion. In contrast, transfusion of KELlo RBCs failed to induce detectable IgM or IgG anti-KEL antibody formation following transfusion. Similarly, while antibodies could be detected on the surface of KEL RBCs following the development of detectable anti-KEL antibodies in the serum, no antibodies could be detected on KELlo RBCs following transfusion, although anti-KEL generated following KEL RBC transfusion readily bound KELlo RBCs in vitro. Although subsequent KEL RBC exposure following initial KEL RBC transfusion induced considerable increases in anti-KEL antibody formation, KEL RBC transfusion following initial KELlo RBCs transfusion completely failed to induce detectable IgM or IgG anti-KEL antibody formation. (All the above differences achieved a p value of

Description: Key Points Transfused murine RBCs expressing the KEL2 antigen induce polyclonal anti-KEL glycoprotein antibodies capable of fixing complement. Complement plays a role in incompatible RBC clearance and modulation of the KEL2 antigen on transfused RBCs.

Description: Abstract 843 Introduction: Alloantibodies generated against red blood cell (RBC) antigens can lead to severe and potentially fatal outcomes such as hemolytic transfusion reactions (HTRs) or hemolytic disease of the fetus and newborn (HDFN). Several mouse models have been generated in efforts to study the alloimmune response to RBC antigens; however, few express clinically significant human RBC antigens. As a result, we developed a novel transgenic mouse model with RBC specific expression of the human KEL antigen, one of the most common non-ABO(H) antigens implicated in HTRs and HDFN. Using this model, we examined the rate, magnitude and consequence of alloimmunization following transfusion of RBCs expressing the human KEL2 antigen in moderate copy number (approximately 1200/RBC). In addition, we evaluated the potential involvement of marginal zone B (MZ B) cells, a B cell population involved in antibody-mediated immunity toward blood borne antigens, in the RBC alloimmunization process. Materials and Methods: C57BL/6 or KEL2 transgenic control recipient mice were transfused with KEL2 and C57BL/6 RBCs labeled with distinct lipophilic dyes to facilitate detection of cells following transfusion. To determine the role of MZ B cells, an additional group of C57BL/6 mice was treated with a-LFA-1 and a- CD49d blocking antibodies, four days prior to transfusion, in order to selectively deplete B cells from the marginal zone; additional animals were treated with an isotype matched control antibody or were surgically splenectomized. Post-transfusion survival and RBC bound antibody levels were determined by flow cytometry at specific timepoints after transfusion utilizing the fluorescent lypophilic dyes, anti-IgM, and anti-IgG. Serum antibodies were also analyzed by indirect immunofluorescence using flow cytometry with KEL2 and control C57BL/6 RBCs as targets. All experiments were completed at least three times with 3–5 recipients per group per experiment. Results: In C57BL/6 recipient mice, anti-KEL glycoprotein IgM was detectable within 2 days after KEL2 RBC transfusion, and peaked at day 5 post transfusion (compilation data of 3 experiments: adjusted MFI of IgM bound to KEL2 RBCs was 24.5 +/− 8.2 (mean +/− SD) on D5). Anti-KEL glycoprotein IgG was detectable on the transfused KEL2 RBCS and in the serum by day 5 and peaked between 14 and 21 days post transfusion (compilation data of 3 experiments: adjusted MFI of IgG bound to KEL2 RBCs was 19.7 +/− 16.7 on D14). Clearance of KEL2 RBCs began as early as 2 days following transfusion and continued to largely parallel the formation of anti-KEL alloantibodies. In contrast, KEL2 recipients failed to make anti-KEL alloantibodies (adjusted MFI of IgM bound to KEL2 RBCs was 2.1 +/− 2.7 on D5; adjusted MFI of IgG bound to KEL2 RBCs was 1.2 +/− 0.6 on D14, p

Description: Transfusion of red blood cells (RBCs) into patients with anti–donor RBC antibodies (crossmatch-incompatible transfusion) can result in lethal antibody-mediated hemolysis. Less well appreciated is the ability of anti-RBC antibodies to specifically remove their target antigen from donor RBCs without compromising cell survival or adversely affecting the transfusion recipient. In an effort to elucidate the mechanistic details of this process, we describe the first animal model of nonhemolytic antibody-induced RBC antigen loss. RBCs from transgenic mHEL mice express surface hen egg lysozyme (HEL) as a transmembrane protein. Transfusion of mHEL RBCs into mice immunized with HEL results in selective loss of HEL antigen from donor RBCs without affecting other blood group antigens or reducing the circulatory life span of the transfused RBCs. While this process does not require the presence of a spleen, it requires both anti-RBC immunoglobulin G (IgG) antibodies and the FcγIII receptor. These studies provide mechanistic insight into the phenomenon of antigen loss during incompatible transfusion in humans.