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© The Author(s) 2015.We investigated DNA methylomes of pediatric Bcell acute lymphoblastic leukemias (B-ALLs) using whole-genome bisulfite sequencing and highdefinition microarrays, along with RNA expression profiles. Epigenetic alteration of B-ALLs occurr

The epigenetic changes during B-cell development relevant to both normal function and hematologic malignancy are incompletely understood. We examined DNA methylation and RNA expression status during early B-cell development by sorting multiple replicates o

The phenotypic similarities between natural killer (NK) and T cells have led to the hypothesis that these distinctive lymphocyte subsets may be developmentally related and thus may share a common progenitor (Lanier, L. L., H. Spits, and J. H. Phillips, 1992. Immunol. Today. 13:392; Rodewald, H.-R., P. Moingeon, J. L. Lurich, C. Dosiou, P. Lopez, and E. L. Reinherz. 1992. Cell. 69:139). In this report, we have investigated the potential of human CD34+ triple negative thymocytes ([TN] CD3-, CD4-, CD8-) to generate both T cells and NK cells in murine fetal thymic organ cultures (mFTOC) and in vitro clonogenic assays. CD34+ TN thymocytes, the majority of which express prominent cytoplasmic CD3 epsilon (cytoCD3 epsilon) protein, can be divided into high (CD34Bright) and low (CD34Dim) surface expressing populations. CD34Bright TN thymocytes were capable of differentiating into T and NK cells when transferred into mFTOC, and demonstrated high NK cell clonogenic capabilities when cultured in interleukin (IL)-2, IL-7, and stem cell factor (SCF). Likewise, CD34Bright TN thymocyte clones after 5 d in culture were capable of generating NK and T cells when transferred into mFTOC but demonstrated clonogenic NK cell differentiation capabilities when maintained in culture with IL-2. CD34Dim TN thymocytes, however, possessed only T cell differentiation capabilities in mFTOC but were not expandable in clonogenic conditions containing IL-2, IL-7, and SCF. No significant differentiation of other cell lineage was detected in either mFTOC or in clonogenic assays from CD34+ TN thymocytes. These results represent the first definitive evidence of a common T/NK cell progenitor in the human fetal thymus and delineate the point in thymocyte differentiation where T and NK cells diverge.

Background Cells expressing high levels of CD34 and little or no CD38 comprise a primitive compartment of progenitors, thought to include hematopoietic stem cells. In this study we sought to determine the feasibility of using CD34 and CD38 as markers of hematopoietic differentiation in vitro, using retinoids to induce the expression of CD38. Methods The effects over time of culture, sera and retinoids on the expression of CD34 and CD38 were determined using a base-medium lacking serum. Two early progenitor populations, isolated by FACS from human fetal liver, were studied: CD38 - CD34 ++ and CD38 + CD34 ++ cells. Additionally, HL-60 cells were adapted to grow in serum-deprived medium to study factors that control CD38 expression. Colony forming cell (CFC) assays and short-term expansion cultures were used to measure the effects of all-trans-retinoic acid (ATRA) oil the growth of fetal progenitors. Results Fetal progenitors and HL-60 cells grown under serum-deprived conditions exhibited almost no CD38 expression. However, CD34 expression was observed on fetal progenitors and declined slowly over time. Addition of FBS or human serum restored CD38 expression to cultured cells, but at levels below those found on progenitors in vivo. Addition of ATRA or 9-cis-retinoic acid (9CRA) to cultures of fetal progenitors or HL-60 cells, resulted in a time- and dose-dependent increase in CD38 expression, ATRA being the more potent of the two retinoids. However, ATRA inhibited colony formation, reduced the expansion of CFC and accelerated the loss of CD34 expression at doses required for the induction of CD38 expression. Discussion ATRA-induced CD38 expression on cells to levels comparable to those found on progenitors in vivo. ATRA also inhibited the growth of early progenitors, which was partly due to ATRA accelerating the differentiation of the progenitors. These findings indicate that CD34 and CD38 expression may be followed as markers of hematopoietic differentiation in vitro, but at the cost of culture conditions that are less than optimal for maintaining early progenitors. [ABSTRACT FROM AUTHOR]

Background: Blood collection from donors on testosterone therapy (TT) is restricted to red blood cell (RBC) concentrates to avoid patient exposure to supraphysiological testosterone (T). The objective of this study was to identify TT-related changes in RBC characteristics relevant to transfusion effectiveness in patients., Study Design: This was a two-part study with cohorts of patients and blood donors on TT. In part 1, we conducted longitudinal evaluation of RBCs collected before and at three time points after initiation of T. RBC assays included storage and oxidative hemolysis, membrane deformability (elongation index), and oximetry. In part 2, we evaluated the fate of transfused RBCs from TT donors in immunodeficient mice and by retrospective analyses of NIH's vein-to-vein databases., Results: TT increased oxidative hemolysis (1.45-fold change) and decreased RBC membrane deformability. Plasma free testosterone was positively correlated with oxidative hemolysis (r = .552) and negatively correlated with the elongation index (r = -.472). Stored and gamma-irradiated RBCs from TT donors had lower posttransfusion recovery in mice compared to controls (41.6 ± 12 vs. 55.3 ± 20.5%). Recipients of RBCs from male donors taking T had 25% lower hemoglobin increments compared to recipients of RBCs from non-TT male donors, and had increased incidence (OR, 1.80) of requiring additional RBC transfusions within 48 h of the index transfusion event., Conclusions: TT is associated with altered RBC characteristics and transfusion effectiveness. These results suggest that clinical utilization of TT RBCs may be less effective in recipients who benefit from longer RBC survival, such as chronically transfused patients., (© 2024 AABB.)

Mice with severe immunodeficiencies have become very important tools for studying foreign cells in an in vivo environment. Xenotransplants can be used to model cells from many species, although most often, mice are humanized through the transplantation of human cells or tissues to meet the needs of medical research. The development of immunodeficient mice is reviewed leading up to the current state-of-the-art strains, such as the NOD- scid -gamma (NSG) mouse. NSG mice are excellent hosts for human hematopoietic stem cell transplants or immune reconstitution through transfusion of human peripheral blood mononuclear cells. However, barriers to full hematopoietic engraftment still remain; notably, the survival of human cells in the circulation is brief, which limits overall hematological and immune reconstitution. Reports have indicated a critical role for monocytic cells - monocytes, macrophages, and dendritic cells - in the clearance of xenogeneic cells from circulation. Various aspects of the NOD genetic background that affect monocytic cell growth, maturation, and function that are favorable to human cell transplantation are discussed. Important receptors, such as SIRPα, that form a part of the innate immune system and enable the recognition and phagocytosis of foreign cells by monocytic cells are reviewed. The development of humanized mouse models has taken decades of work in creating more immunodeficient mice, genetic modification of these mice to express human genes, and refinement of transplant techniques to optimize engraftment. Future advances may focus on the monocytic cells of the host to find ways for further engraftment and survival of xenogeneic cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Introduction: Alloimmune responses against platelet antigens, which dominantly target the major histocompatibility complex (MHC), can cause adverse reactions to subsequent platelet transfusions, platelet refractoriness, or rejection of future transplants. Platelet transfusion recipients include individuals experiencing severe bacterial or viral infections, and how their underlying health modulates platelet alloimmunity is not well understood., Methods: This study investigated the effect of underlying inflammation on platelet alloimmunization by modelling viral-like inflammation with polyinosinic-polycytidylic acid (poly(I:C)) or gram-negative bacterial infection with lipopolysaccharide (LPS), hypothesizing that underlying inflammation enhances alloimmunization. Mice were pretreated with poly(I:C), LPS, or nothing, then transfused with non-leukoreduced or leukoreduced platelets. Alloantibodies and allogeneic MHC-specific B cell (allo-B cell) responses were evaluated two weeks later. Rare populations of allo-B cells were identified using MHC tetramers., Results: Relative to platelet transfusion alone, prior exposure to poly(I:C) increased the alloantibody response to allogeneic platelet transfusion whereas prior exposure to LPS diminished responses. Prior exposure to poly(I:C) had equivalent, if not moderately diminished, allo-B cell responses relative to platelet transfusion alone and exhibited more robust allo-B cell memory development. Conversely, prior exposure to LPS resulted in diminished allo-B cell frequency, activation, antigen experience, and germinal center formation and altered memory B cell responses., Discussion: In conclusion, not all inflammatory environments enhance bystander responses and prior inflammation mediated by LPS on gram-negative bacteria may in fact curtail platelet alloimmunization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Tran, Muench, Gaillard, Darst, Tomayko and Jackman.)

Introduction: Alloimmunization is common following platelet transfusion and can result in negative outcomes for recipients such as refractoriness to subsequent transfusions and rejection of transplants. Healthy people do not receive blood transfusions, and the diseases and therapies that result in a need to transfuse have significant impacts on the immunological environment to which these alloantigens are introduced. Ablative chemotherapies are common among platelet recipients and have potent immunological effects. In this study, we modeled the impact of chemotherapy on the alloresponse to platelet transfusion. As chemotherapies are generally regarded as immunosuppressive, we hypothesized that that they would result in a diminished alloresponse., Methods: Mice were given a combination chemotherapeutic treatment of cytarabine and doxorubicin followed by transfusion of allogeneic platelets, and compared to controls given no treatment, chemotherapy alone, or transfusion alone. Alloantibody responses were measured 2 weeks after transfusion, and cellular responses and growth factors were monitored over time., Results: Contrary to our hypothesis, we found that chemotherapy led to increased alloantibody responses to allogeneic platelet transfusion. This enhanced response was antigen-specific and was associated with increased CD4 + and CD8 + T cell responses. Chemotherapy led to rapid lymphocyte depletion followed by reconstitution, non-specific activation of transitional B cells with the highest levels of activation in the least mature subsets, and increased serum levels of B cell activating factor (BAFF)., Conclusion: These data suggest that ablative chemotherapy can increase the risk of alloimmunization and, if confirmed clinically, that additional measures to protect these patient populations may be warranted., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Jackman, Darst, Gaillard, Tran, Tomayko and Muench.)

Background: Zika virus (ZIKV) epidemics with infections in pregnant women are associated with severe neurological disease in newborns. Although an arbovirus, ZIKV is also blood transfusion-transmitted (TT). Greater knowledge of the efficiency of ZIKV TT would aid decisions on testing and pathogen reduction technologies (PRT)., Study Design and Methods: Plasma units from ZIKV RNA-reactive blood donors were used to study infectivity in vitro, in mice, and in macaques. Furthermore, plasma units were subjected to PRT using amotosalen/ultraviolet light A (A/UVA) before transfusion., Results: In vitro infectivity of ZIKV RNA-reactive plasma varied between 100 and 1000 international units (IU) of ZIKV RNA. Immunodeficient mice were more sensitive with as low as 32 IU sufficient to infect 50% of mice. 50-5500 IU of RNA led to TT in macaques using dose escalation of three different RNA-positive, seronegative plasma units. In contrast, RNA-reactive units collected postseroconversion were not infectious in macaques, even at a dose of 9 million IU RNA. After A/UVA PRT, transfusion of plasma containing up to 18 million IU was no longer infectious in vitro and did not result in ZIKV TT in macaques., Conclusion: Significant risks of ZIKV TT are likely confined to a relatively short viremic window before seroconversion, and that sensitive nucleic acid amplification testing likely identifies the majority of infectious plasma. PRT was demonstrated to be effective at preventing ZIKV TT. Considering that there is no approved ZIKV vaccine, these data are relevant to mitigate the risk of TT during the future ZIKV outbreaks., (© 2023 AABB.)

Background & Aims: Hepatocytic cells found during prenatal development have unique features compared to their adult counterparts, and are believed to be the precursors of pediatric hepatoblastoma. The cell-surface phenotype of hepatoblasts and hepatoblastoma cell lines was evaluated to discover new markers of these cells and gain insight into the development of hepatocytic cells and the phenotypes and origins of hepatoblastoma., Methods: Human midgestation livers and four pediatric hepatoblastoma cell lines were screened using flow cytometry. Expression of over 300 antigens was evaluated on hepatoblasts defined by their expression of CD326 (EpCAM) and CD14. Also analyzed were hematopoietic cells, expressing CD45, and liver sinusoidal-endothelial cells (LSECs), expressing CD14 but lacking CD45 expression. Select antigens were further examined by fluorescence immunomicroscopy of fetal liver sections. Antigen expression was also confirmed on cultured cells by both methods. Gene expression analysis by liver cells, 6 hepatoblastoma cell lines, and hepatoblastoma cells was performed. Immunohistochemistry was used to evaluate CD203c, CD326, and cytokeratin-19 expression on three hepatoblastoma tumors., Results: Antibody screening identified many cell surface markers commonly or divergently expressed by hematopoietic cells, LSECs, and hepatoblasts. Thirteen novel markers expressed on fetal hepatoblasts were identified including ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP-3/CD203c), which was found to be expressed by hepatoblasts with widespread expression in the parenchyma of the fetal liver. In culture CD203c + CD326 ++ cells resembled hepatocytic cells with coexpression of albumin and cytokeratin-19 confirming a hepatoblast phenotype. CD203c expression declined rapidly in culture whereas the loss of CD326 was not as pronounced. CD203c and CD326 were co-expressed on a subset of hepatoblastoma cell lines and hepatoblastomas with an embryonal pattern., Conclusions: CD203c is expressed on hepatoblasts and may play a role in purinergic signaling in the developing liver. Hepatoblastoma cell lines were found to consist of two broad phenotypes consisting of a cholangiocyte-like phenotype that expressed CD203c and CD326 and a hepatocyte-like phenotype with diminished expression of these markers. CD203c was expressed by some hepatoblastoma tumors and may represent a marker of a less differentiated embryonal component., Competing Interests: MM and AB are employed by Vitalant Research Institute and MF, AG, and RG were employed by Vitalant Research Institute when they contributed to this study. Christopher Nosworthy and Gregory Ostolaza were student interns and not employees of Vitalant Research Institute. KM is employed by Omics Data Automation. SC was employed by XenTech during the time that work was performed, and the manuscript was drafted. CK has sponsored research agreements with Eli Lily, Roche-Genentech and Cardiff Oncology as well as research collaborations with Novartis, and is co-founder of Tio Companies. Artisan Biopharma is a wholly-owned subsidiary of cc-TDI. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This study received funding from Vitalant Inc. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication., (Copyright © 2023 Muench, Fomin, Gutierrez, López-Terrada, Gilfanova, Nosworthy, Beyer, Ostolaza, Kats, Matlock, Cairo and Keller.)

Objectives: Cell-surface antigen screening was performed on human fetal liver cells using flow cytometry. The goal was to provide proteomic expression data on a number of human fetal liver cell populations that can inform studies on developmental hepatology and hematology., Data Description: A 21 weeks' gestation liver was depleted of erythrocytes prior to antibody staining. Screening was performed using phycoerythrin-labelled antibodies against 332 antigens. In addition to these antibodies, all samples were stained for CD14, CD45, CD235a, and CD326 (epithelial cell adhesion molecule - EpCAM). Subpopulations of fetal liver cells were identified using the co-stained antigens. Hematopoietic cells were identified by their expression of CD45 and CD235a; non-hematopoietic cells were further subdivided based on CD14 and CD326 expression. CD326 ++ CD14 low hepatoblasts and CD14 ++ liver sinusoidal endothelial cells were analyzed for the frequency and intensity of antigen expression. Analyzed flow cytometry data are presented for the expression of the antigens on hematopoietic cells and on non-hematopoietic cells in the context of CD14 and CD326 expression., (© 2022. The Author(s).)

Respiratory viruses such as influenza do not typically cause viremia; however, SARS-CoV-2 has been detected in the blood of COVID-19 patients with mild and severe symptoms. Detection of SARS-CoV-2 in blood raises questions about its role in pathogenesis as well as transfusion safety concerns. Blood donor reports of symptoms or a diagnosis of COVID-19 after donation (post-donation information, PDI) preceded or coincided with increased general population COVID-19 mortality. Plasma samples from 2,250 blood donors who reported possible COVID-19-related PDI were tested for the presence of SARS-CoV-2 RNA. Detection of RNAemia peaked at 9%-15% of PDI donors in late 2020 to early 2021 and fell to approximately 4% after implementation of widespread vaccination in the population. RNAemic donors were 1.2- to 1.4-fold more likely to report cough or shortness of breath and 1.8-fold more likely to report change in taste or smell compared with infected donors without detectable RNAemia. No infectious virus was detected in plasma from RNAemic donors; inoculation of permissive cell lines produced less than 0.7-7 plaque-forming units (PFU)/mL and in susceptible mice less than 100 PFU/mL in RNA-positive plasma based on limits of detection in these models. These findings suggest that blood transfusions are highly unlikely to transmit SARS-CoV-2 infection.

Background Aims: The cryopreservation of hematopoietic stem cells (HSCs) in dimethyl sulfoxide (DMSO) is used widely, but DMSO toxicity in transplant patients and the effects of DMSO on the normal function of cryopreserved cells are concerns. To address these issues, in vitro and clinical studies have explored using reduced concentrations of DMSO for cryopreservation. However, the effect of reducing DMSO concentration on the efficient cryopreservation of HSCs has not been directly measured., Methods: Cryopreservation of human bone marrow using 10%, 7.5% and 5% DMSO concentrations was examined. Cell counting, flow cytometry and colony assays were used to analyze different cell populations. The recovery of stem cells was enumerated using extreme limiting dilution analysis of long-term multi-lineage engraftment in immunodeficient mice. Four different methods of analyzing human engraftment were compared to ascertain stem cell engraftment: (i) engraftment of CD33 + myeloid, CD19 + B-lymphoid, CD235a + erythroid and CD34 + progenitors; (ii) engraftment of the same four populations plus CD41 + CD42b + platelets; (iii) engraftment of CD34 ++ CD133 + cells; and (iv) engraftment of CD34 ++ CD38 - cells., Results: Hematopoietic colony-forming, CD34 ++/+ , CD34 ++ CD133 + and CD34 ++ CD38 - cells were as well preserved with 5% DMSO as they were with the higher concentrations tested. The estimates of stem cell frequencies made in the xenogeneic transplant model did not show any significant detrimental effect of using lower concentrations of DMSO. Comparison of the different methods of gauging stem cell engraftment in mice led to different estimates of stem cell numbers, but overall, all measures found that reduced concentrations of DMSO supported the cryopreservation of HSCs., Conclusion: Cryopreservation of HSCs in DMSO concentrations as low as 5% is effective., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

The cryopreservation of hematopoietic cells using dimethyl sulfoxide (DMSO) and serum is a common procedure used in transplantation. However, DMSO has clinical and biological side effects due to its toxicity, and serum introduces variation and safety risks. Inspired by natural antifreeze proteins, a novel class of ice-interactive cryoprotectants was developed. The corresponding DMSO-, protein-, and serum-free cryopreservation media candidates were screened through a series of biological assays using human cell lines, peripheral blood cells, and bone marrow cells. XT-Thrive-A and XT-Thrive-B were identified as lead candidates to rival cryopreservation with 10% DMSO in serum based on post-thaw cell survival and short-term proliferation assays. The effectiveness of the novel cryopreservation media in freezing hematopoietic stem cells from human whole bone marrow was assessed by extreme limiting dilution analysis in immunodeficient mice. Stem cell frequencies were measured 12 weeks after transplant based on bone marrow engraftment of erythroid, myeloid, B-lymphoid, and CD34 + progenitors measured by flow cytometry. The recovered numbers of cryopreserved stem cells were similar among XT-Thrive A, XT-Thrive B, and DMSO with serum groups. These findings show that cryoprotectants developed through biomimicry of natural antifreeze proteins offers a substitute for DMSO-based media for the cryopreservation of hematopoietic stem cells., (© 2021. The Author(s).)

Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates of diverse mixtures of thousands of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors or from immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in under 3 months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin, we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Obesity is a global pandemic characterized by multiple comorbidities, including cardiovascular and metabolic diseases. The aim of this study was to define the associations between blood donor body mass index (BMI) and RBC measurements of metabolic stress and hemolysis., Study Design and Methods: The associations between donor BMI (<25 kg/m 2 , normal weight; 25-29.9 kg/m 2 , overweight; and ≥30 kg/m 2 , obese) and hemolysis (storage, osmotic, and oxidative; n = 18 donors) or posttransfusion recovery (n = 14 donors) in immunodeficient mice were determined in stored leukocyte-reduced RBC units. Further evaluations were conducted using the National Heart, Lung, and Blood Institute RBC-Omics blood donor databases of hemolysis (n = 13 317) and metabolomics (n = 203)., Results: Evaluations in 18 donors revealed that BMI was significantly (P < 0.05) and positively associated with storage and osmotic hemolysis. A BMI of 30 kg/m 2 or greater was also associated with lower posttransfusion recovery in mice 10 minutes after transfusion (P = 0.026). Multivariable linear regression analyses in RBC-Omics revealed that BMI was a significant modifier for all hemolysis measurements, explaining 4.5%, 4.2%, and 0.2% of the variance in osmotic, oxidative, and storage hemolysis, respectively. In this cohort, obesity was positively associated (P < 0.001) with plasma ferritin (inflammation marker). Metabolomic analyses on RBCs from obese donors (44.1 ± 5.1 kg/m 2 ) had altered membrane lipid composition, dysregulation of antioxidant pathways (eg, increased oxidized lipids, methionine sulfoxide, and xanthine), and dysregulation of nitric oxide metabolism, as compared to RBCs from nonobese (20.5 ± 1.0 kg/m 2 ) donors., Conclusions: Obesity is associated with significant changes in RBC metabolism and increased susceptibility to hemolysis under routine storage of RBC units. The impact on transfusion efficacy warrants further evaluation., (© 2020 AABB.)

Alloimmunization against platelet-rich plasma (PRP) transfusions can lead to complications such as platelet refractoriness or rejection of subsequent transfusions and transplants. In mice, pathogen reduction treatment of PRP with UVB light and riboflavin (UV+R) prevents alloimmunization and appears to induce partial antigen-specific tolerance to subsequent transfusions. Herein, the in vivo responses of antigen-presenting cells and T cells to transfusion with UV+R-treated allogeneic PRP were evaluated to understand the cellular immune responses leading to antigen-specific tolerance. Mice that received UV+R-treated PRP had significantly increased transforming growth factor β (TGF-β) expression by CD11b+ CD4+ CD11cHi conventional dendritic cells (cDCs) and CD11bHi monocytes (P < .05). While robust T-cell responses to transfusions with untreated allogeneic PRP were observed (P < .05), these were blocked by UV+R treatment. Mice given UV+R-treated PRP followed by untreated PRP showed an early significant (P < .01) enrichment in regulatory T (Treg) cells and associated TGF-β production as well as diminished effector T-cell responses. Adoptive transfer of T-cell-enriched splenocytes from mice given UV+R-treated PRP into naive recipients led to a small but significant reduction of CD8+ T-cell responses to subsequent allogeneic transfusion. These data demonstrate that pathogen reduction with UV+R induces a tolerogenic profile by way of CD11b+ CD4+ cDCs, monocytes, and induction of Treg cells, blocking T-cell activation and reducing secondary T-cell responses to untreated platelets in vivo., (© 2020 by The American Society of Hematology.)

A critical barrier to the development of a human immunodeficiency virus (HIV) cure is the lack of a scalable animal model that enables robust evaluation of eradication approaches prior to testing in humans. We established a humanized mouse model of latent HIV infection by transplanting "J-Lat" cells, Jurkat cells harboring a latent HIV provirus encoding an enhanced green fluorescent protein (GFP) reporter, into irradiated adult NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice. J-Lat cells exhibited successful engraftment in several tissues including spleen, bone barrow, peripheral blood, and lung, in line with the diverse natural tissue tropism of HIV. Administration of tumor necrosis factor (TNF)-α, an established HIV latency reversal agent, significantly induced GFP expression in engrafted cells across tissues, reflecting viral reactivation. These data suggest that our murine latency ("μ-Lat") model enables efficient determination of how effectively viral eradication agents, including latency reversal agents, penetrate, and function in diverse anatomical sites harboring HIV in vivo., (© 2020 Federation of American Societies for Experimental Biology.)

Animal models are vital to the study of transfusion and development of new blood products. Post-transfusion recovery of human blood components can be studied in mice, however, there is a need to identify strains that can best tolerate xenogeneic transfusions, as well as to optimize such protocols. Specifically, the importance of using immunodeficient mice, such as NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, to study human transfusion has been questioned. In this study, strains of wild-type and NSG mice were compared as hosts for human transfusions with outcomes quantified by flow cytometric analyses of CD235a+ erythrocytes, CD45+ leukocytes, and CD41+CD42b+ platelets. Complete blood counts were evaluated as well as serum cytokines by multiplexing methods. Circulating human blood cells were maintained better in NSG than in wild-type mice. Lethargy and hemoglobinuria were observed in the first hours in wild-type mice along with increased pro-inflammatory cytokines/chemokines such as monocyte chemoattractant protein-1, tumor necrosis factor α, keratinocyte-derived chemokine (KC or CXCL1), and interleukin-6, whereas NSG mice were less severely affected. Whole blood transfusion resulted in rapid sequestration and then release of human cells back into the circulation within several hours. This rebound effect diminished when only erythrocytes were transfused. Nonetheless, human erythrocytes were found in excess of mouse erythrocytes in the liver and lungs and had a shorter half-life in circulation. Variables affecting the outcomes of transfused erythrocytes were cell dose and mouse weight; recipient sex did not affect outcomes. The sensitivity and utility of this xenogeneic model were shown by measuring the effects of erythrocyte damage due to exposure to the oxidizer diamide on post-transfusion recovery. Overall, immunodeficient mice are superior models for xenotransfusion as they maintain improved post-transfusion recovery with negligible immune-associated side effects., Competing Interests: The authors have declared that no competing interests exist.

Background and Objectives: Alloimmunization targeting major histocompatibility (MHC) antigens is common following platelet transfusion. Pathogen reduction of platelets can block alloimmunization to MHC in mice and induce partial antigen-specific tolerance to subsequent transfusions. This study utilized small allelic variants to evaluate the relative contributions of class I and class II MHC to the alloresponse against untreated or pathogen-reduced platelets., Materials and Methods: C57BL/6 (B6) K bm1 and B6 IA bm12 mice with small variants in the class I K b and class II IA b alleles, respectively, were used as platelet donors for wild-type B6 recipients. Both untreated and pathogen-reduced platelet-rich plasma (PRP) transfusions were evaluated for immunogenicity by measuring antibody responses and ex vivo cytokine production., Results: Both the K bm1 and IA bm12 alleles induced antibody responses, though the response to K bm1 was greater. Pathogen reduction blocked the antibody responses to IA bm12 , but not to K bm1 . Both the K bm1 and IA bm12 alleles primed ex vivo cytokine responses that were blocked with pathogen reduction, though responses to IA bm12 were broader and larger (K bm1 responses: IFN-γ, TNFα, and MIP-1β; IA bm12 responses: IFN-γ, TNFα, IL-1β, IL-10, IL-13, and GM-CSF). Pathogen-reduced K bm1 PRP did not appear to induce any tolerance to subsequent untreated K bm1 PRP transfusions., Conclusion: Minor allelic variants in both the class I and class II MHC are capable of inducing an alloresponse to transfusion. The K bm1 PRP induced alloantibodies even with pathogen reduction and did not show signs of inducing the partial tolerance to subsequent transfusions observed with a larger MHC mismatch., (© 2020 International Society of Blood Transfusion.)

Background: Alloimmunization to platelet-rich plasma (PRP) transfusions can cause adverse reactions such as platelet refractoriness or transplant rejection. Pathogen reduction treatment with ultraviolet light and riboflavin (UV + R) of allogeneic PRP was shown to reduce allogeneic antibody responses and confer partial antigen-specific immune tolerance to subsequent transfusions in mice. Studies have shown that UV + R was effective at both rapidly killing donor white blood cells (WBCs) and reducing their ability to stimulate an allogeneic response in vitro. However, the manner in which UV + R induces WBC death and its associated role in the immune response to treated PRP is unknown., Methods and Materials: This study evaluates whether UV + R causes WBC apoptosis by examining phosphatidylserine exposure on the plasma membrane, membrane asymmetry, caspase activity, and chromatin condensation by flow cytometry. The immunogenicity of WBCs killed with UV + R versus apoptotic or necrotic pathways was also examined in vivo., Results: WBCs after UV + R exhibited early apoptotic-like characteristics including phosphatidylserine exposure on the outer leaflet of the plasma membrane and loss of membrane asymmetry, but unlike canonical apoptotic cells, caspase activity and chromatin condensation were not apparent. However, in vivo studies demonstrated, unlike untreated or necrotic WBCs, both apoptotic WBCs and UV + R-treated WBCs failed to prime alloantibody responses to subsequent untreated transfusions., Conclusion: Overall, the mechanism of WBC death following UV + R treatment shares some membrane characteristics of early apoptosis but is distinct from classic apoptosis. Despite these differences, UV + R-treated and apoptotic WBCs both offer some protection from alloimmunization., (© 2019 AABB.)

Background: Membranes surrounding the fetus play a crucial role in providing a physical and immunological barrier between a semiallogeneic fetus and mother during pregnancy. In this study, we tested whether cotransplantation of fetal membranes (FMs) and allogeneic donor cells would improve the retention and function of allografts in mice., Methods: Intact and enzyme-digested membranes obtained from E18-E19 pregnant mice were subcutaneously cotransplanted with 10F7MN hybridoma cells that are of BALB/cByJ (Balb) origin and secrete anti-human CD235a antibody. Cells were transplanted into C57BL/6J (B6, allogeneic), Balb (syngeneic), and FVB/NJ (third-party) mice. Serum was collected after 1 and 3 weeks of cell transplantation and tested using flow cytometry for the presence of anti-human CD235a antibody. Immunosuppressive functions of membranes were further investigated by analyzing the cytokine profile of supernatants collected from allo-reactive mixed lymphocyte reactions (MLRs) using a multiplex cytokine assay., Results: B6 mice transplanted with 10F7MN cells along with membranes syngeneic to the host had significantly higher levels of CD235a antibody when compared to B6 mice that received cells without membranes, allogenic membranes, or third-party membranes. Syngeneic membranes significantly inhibited T-cell proliferation in the presence of allogeneic stimuli and suppressed the release of Th1-cytokines such as IFNγ, TNFα, and IL-2 in MLRs. Additionally, increases in the levels of Th2-cytokines were found in MLRs containing membrane-derived cells., Conclusions: Our study highlights the potential use of syngeneic FMs to act as potent cell-carriers that could improve graft retention as well as graft-specific immunoprotection during allograft transplantation., Competing Interests: The authors declare no conflicts of interest.

Background and Objectives: Alloimmunization is common following transfusion with platelet-rich plasma (PRP) and can cause complications such as platelet refractoriness or transplant rejection. It has previously been shown that pathogen reduction of PRP with riboflavin and UV light (UV+R) can protect against alloimmunization in mice and induce partial tolerance to subsequent transfusions., Materials and Methods: Using B6 H2 d congenic mice, this study evaluated the relative contributions of major histocompatibility complex (MHC) antigens and minor antigens to both the alloresponse to PRP transfusion and the partial tolerance induced by UV+R treatment., Results: Both total and MHC-specific alloantibody responses were highest when both MHC and minor antigens were mismatched, with lower alloantibody responses observed with MHC mismatch alone, demonstrating that allogeneic minor antigens can enhance the response to allogeneic MHC. There was a weak, but significant alloantibody response to minor antigens only. UV+R treatment protected against both major and minor antigen alloimmunization. Both allogeneic MHC and minor antigens primed an enhanced cytokine response ex vivo, though this was weaker with minor antigens, and both responses were blocked with UV+R treatment., Conclusion: Allogeneic MHC is both necessary and sufficient to induce the partial tolerance associated with UV+R treatment., (© 2019 International Society of Blood Transfusion.)

Background: Babesia microti is a parasite that infects red blood cells (RBCs) in mammals. It is transmitted to humans by tick bites, transfusion, organ transplantation, and congenital acquisition. Although the Babesia natural history and seroprevalence in donors have been well described, gaps in knowledge relevant to transfusion remain., Study Design and Methods: Mice were infected with dilutions of parasitized blood to address the minimal infectious dose and the kinetics of parasitemia by quantitative polymerase chain reaction (qPCR) and of antibodies by enzyme immunoassay., Results: In immunocompetent DBA/2 mice infected with 100 parasitized RBCs (pRBCs) and in immunodeficient NSG mice infected with 63 pRBCs, parasitemia was detectable in five of five mice each. Peak parasitemia up to 2 × 10 7 pRBCs/mL at 2 to 3 weeks or 5 × 10 8 pRBCs/mL at 6 weeks was observed for DBA/2 and NSG mice, respectively. Protracted fluctuating parasitemia was observed for 8 months in DBA/2 mice, whereas NSG mice exhibited a high-plateau parasitemia. Antibody titers continued to increase until 6 to 18 weeks in DBA/2 mice and remained high through 6 months. This study also investigated the analytical performance of Babesia assays that detect parasite DNA or RNA using a blinded panel. A Babesia assay targeting parasite RNA was approximately 10-fold more sensitive compared to qPCR targeting DNA., Conclusion: The mice in this study were highly susceptible to Babesia infection using as few as 1 to 2 log pRBCs and maintained chronic parasitemia. If the infectious dose in human transfusion recipients is comparably low, a highly sensitive assay targeting parasite RNA may safeguard the blood supply, particularly before antibody detection., (© 2018 AABB.)