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ABO blood group antigens, which are complex carbohydrate moieties, and the first human polymorphisms identified, are critical in transfusion medicine and transplantation. Despite their discovery over a century ago, significant questions remain about the development of anti-ABO antibodies and the structural features of ABO antigens that cause hemolytic transfusion reactions. Anti-ABO antibodies develop naturally during the first few months of life, in contrast to other red blood cell (RBC) alloantibodies which form after allogeneic RBC exposure. Anti-ABO antibodies are the most common immune barrier to transfusion and transplantation, but the factors driving their formation are incompletely understood. Some studies suggest that microbes that express glycans similar in structure to the blood group antigens could play a role in anti-blood group antibody formation. While the role of these microbes in clinically relevant anti-blood group antibody formation remains to be defined, the presence of these microbes raises questions about how blood group-positive individuals protect themselves against blood group molecular mimicry. Recent studies suggest that galectins can bind and kill microbes that mimic blood group antigens, suggesting a unique host defense mechanism against microbial molecular mimicry. However, new models are needed to fully define the impact of microbes, galectins, or other factors on the development of clinically relevant naturally occurring anti-blood group antibodies., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Red blood cell (RBC) transfusion represents one of the earliest and most widespread forms of cellular therapy. While the primary purpose of RBC transfusions is to enhance the oxygen-carrying capacity of the recipient, RBCs also possess unique properties that make them attractive vehicles for inducing antigen-specific immune tolerance. Preclinical studies have demonstrated that RBC transfusion alone, in the absence of inflammatory stimuli, often fails to elicit detectable alloantibody formation against model RBC antigens. Several studies also suggest that RBC transfusion without inflammation may not only fail to generate a detectable alloantibody response but can also induce a state of antigen-specific non-responsiveness, a phenomenon potentially influenced by the density of the corresponding RBC alloantigen. The unique properties of RBCs, including their inability to divide and their stable surface antigen expression, make them attractive platforms for displaying exogenous antigens with the goal of leveraging their ability to induce antigen-specific non-responsiveness. This could facilitate antigen presentation to the host's immune system without triggering innate immune activation, potentially enabling the induction of antigen-specific tolerance for therapeutic applications in autoimmune disorders, preventing immune responses against protein therapeutics, or reducing alloreactivity in the setting of transfusion and transplantation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Successful pregnancy relies on a coordinated interplay between endocrine, immune, and metabolic processes to sustain fetal growth and development. The orchestration of these processes involves multiple signaling pathways driving cell proliferation, differentiation, angiogenesis, and immune regulation necessary for a healthy pregnancy. Among the molecules supporting placental development and maternal tolerance, the families of pregnancy-specific glycoproteins and galectins are of great interest in reproductive biology. We previously found that PSG1 can bind to galectin-1 (GAL-1). Herein, we characterized the interaction between PSG1 and other members of the galectin family expressed during pregnancy, including galectin-3, -7, -9, and -13 (GAL-3, GAL-7, GAL-9, and GAL-13). We observed that PSG1 binds to GAL-1, -3, and -9, with the highest apparent affinity seen for GAL-9, and that the interaction of PSG1 with GAL-9 is carbohydrate-dependent. We further investigated the ability of PSG1 to regulate GAL-9 responses in human monocytes, a murine macrophage cell line, and T-cells, and determined whether PSG1 binds to both carbohydrate recognition domains of GAL-9. Additionally, we compared the apparent affinity of GAL-9 binding to PSG1 with other known GAL-9 ligands in these cells, Tim-3 and CD44. Lastly, we explored functional conservation between murine and human PSGs by determining that Psg23, a highly expressed member of the murine Psg family, can bind some murine galectins despite differences in amino acid composition and domain structure., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Competing Interests: S.L.M. has served as a paid consultant or advisory board member for Bayer, Takeda, Biomarin, Sanofi, CSL Behring, Genentech, NovoNordisk, Octapharma, Pfizer, Sangamo, Spark/Roche, Sobi, and receives research support from Octapharma and Genentech. S.R.S. has consulted for Novartis, Cellics, Argenx, and Alexion, and has received speaking honoraria from Grifols and research support from Alexion. S.R.P., W.H.B., P.Z., E.T.P., and C.C. declare no conflict of interests.

The United States of America (US) has the highest annual number of human babesiosis cases caused by Babesia microti ( Bm ). Babesia , like malaria-causing Plasmodium, are protozoan parasites that live within red blood cells (RBCs). Both infectious diseases can be associated with hemolysis and organ damage, which can be fatal. Since babesiosis was made a nationally notifiable condition by the Centers for Disease Control and Prevention (CDC) in January 2011, human cases have increased, and drug-resistant strains have been identified. Both the Bm ligand(s) and RBC receptor(s) needed for invasion are unknown, partly because of the difficulty of developing a continuous in vitro culture system. Invasion pathways are relevant for therapies (e.g., RBC exchange) and vaccines. We hypothesize that there is at least one RBC surface antigen that is essential for Bm invasion and that all Bm hosts express this. Because most RBC surface antigens that impact Plasmodium invasion are in human blood group (hBG) systems, which are generated by 51 genes, they were the focus of this study. More than 600 animals with at least one hBG system gene ortholog were identified using the National Center for Biotechnology Information (NCBI) command-line tools. Google Scholar searches were performed to determine which of these animals are susceptible to Bm infection. The literature review revealed 28 Bm non-human hosts (NHH). For 5/51 (9.8%) hBG system genes (e.g., RhD ), no NHH had orthologs. This means that RhD is unlikely to be an essential receptor for invasion. For 24/51 (47.1%) hBG system genes, NHH had 4-27 orthologs. For the ABO gene, 15/28 NHH had an ortholog, meaning that this gene is also unlikely to generate an RBC antigen, which is essential for Bm invasion. Our prior research showed that persons with blood type A, B, AB, O, RhD+, and RhD- can all be infected with Bm , supporting our current study's predictions. For 22/51 (43.1%) hBG system genes, orthologs were found in all 28 NHH. Nineteen (37.3%) of these genes encode RBC surface proteins, meaning they are good candidates for generating a receptor needed for Bm invasion. In vitro cultures of Bm , experimental Bm infection of transgenic mice (e.g., a CD44 KO strain), and analyses of Bm patients can reveal further clues as to which RBC antigens may be essential for invasion.

Objectives: To assess sex-specific differences in the association between pre-transfusion haemoglobin values and early neurodevelopmental function., Design: Observational follow-up of infants with birth weights <1000 g and gestational ages 22-28 weeks who were enrolled in the NICHD Neonatal Research Network Transfusion of Prematures (TOP) Trial at 19 U.S. sites, 2012-2017., Main Outcome Measures: Pretransfusion haemoglobin values were obtained longitudinally through 36 weeks' postmenstrual age. The infant's mean pretransfusion haemoglobin was used as a marker of degree of anaemia (n=1655 measures). Measures of brain function were obtained at 22-26 months' corrected age using the Bayley Scales of Infant & Toddler Development, third edition (BSID-III) (n=1290 BSID-III scores). Sex-specific estimates for the linear relation between pretransfusion haemoglobin and BSID-III scores were obtained from repeated-measures regression analysis, adjusted for gestational age, birth weight, study site, clinical characteristics, and demographic covariates., Results: The relation of pretransfusion haemoglobin with 24-month BSID-III scores showed significant, independent interactions with both (1) sex (p=0.046) and (2) retinopathy of prematurity (ROP; p=0.004). In 614 males, BSID-III scores were higher by 1.07 points per g/dL (95% CI 1.58 to 4.33; p=0.008), not differing significantly among the three subscales (cognitive, language and motor; p=0.94). In 247 infants with ROP, BSID-III scores were higher by 2.95 points per g/dL (95% CI 0.28 to 1.87; p<0.0001), uniformly across subscales (p=0.73). These associations were non-significant in 676 females (p=0.96) and 1043 infants without ROP (p=0.81)., Conclusions: This study demonstrates sex-specific associations between mean pretransfusion haemoglobin (a marker of the severity of anaemia throughout the neonatal intensive care unit [NICU] hospitalisation) and early neurodevelopmental function at 22-26 months' corrected age., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Crizanlizumab, a monoclonal antibody against P-selectin, has been shown to reduce vaso-occlusive crises (VOCs) compared to placebo in patients ≥ 16 years with sickle cell disease (SCD). However, there have been rare reports of patients experiencing severe pain and subsequent complications within 24 hours of crizanlizumab infusions. These events are defined as infusion-related reactions (IRRs). Informed by current literature and clinical experience, a group of content experts developed clinical guidelines for the management of IRRs in patients with SCD. We used the RAND/University of California, Los Angeles (UCLA) modified Delphi panel method, a valid, reproducible technique for achieving consensus. We present our recommendations for managing IRRs, which depend on patient characteristics including: prior history of IRRs to other monoclonal antibodies or medications, changes to crizanlizumab infusion rate and patient monitoring, pain severity relative to patient's typical SCD crises, and severe allergic symptoms. These recommendations outline how to evaluate and manage IRRs in patients receiving crizanlizumab. Future research should validate this guidance using clinical data and identify patients at risk for these IRRs., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Post-transfusion survival of donor red blood cells (RBCs) is important for effective chronic transfusion therapy in conditions including sickle cell disease (SCD). Biotin labeling RBCs allows direct in vivo measurement of multiple donor RBC units simultaneously post-transfusion., Study Design and Methods: In an observational trial of patients with SCD receiving monthly chronic transfusion therapy, aliquots of RBCs from one transfusion episode were biotin-labeled and infused along with the unlabeled RBC units. Serial blood samples were obtained to measure RBC survival. Donor units were tested for RBC indices, hemoglobin fractionation, and glucose-6-phosphate dehydrogenase (G6PD) enzyme activity. For microcytic donor RBCs (MCV < 70 fL), HBA1 and HBA2 genetic testing was performed on whole blood., Results: We present one recipient, a pediatric patient with SCD and splenectomy who received two RBC units with aliquots from each unit labeled at distinct biotin densities (2 and 18 μg/mL biotin). One donor unit was identified to have microcytosis (MCV 68.5 fL after biotinylation); whole blood sample obtained at a subsequent donation showed 2-gene deletion alpha-thalassemia trait (ɑ- 3.7kb /ɑ- 3.7kb ) and normal serum ferritin. G6PD activity was >60% of normal mean for both. The RBCs with alpha-thalassemia RBC had accelerated clearance and increased surface phosphatidylserine post-transfusion, as compared with the normocytic RBC (half life 65 vs. 86 days, respectively)., Discussion: Post-transfusion RBC survival may be lower for units from donors with alpha-thalassemia trait, although the impact of thalassemia trait donors on transfusion efficacy requires further study., (© 2024 AABB.)

Aims: Formation of red blood cell alloantibodies (RBCAs) complicates transfusion support in liver transplantation (LT). Difficult RBCAs (DAs, >3 antibodies or antibodies for which <25% donors are antigen negative) further challenge care. This study characterises DA outcomes relative to non-difficult RBCAs (NDAs)., Methods: Single-centre, retrospective analysis of LT patients (2002-2021). RBCAs were defined as clinically significant antibodies. DAs were compared with NDAs., Results: 89 patients had clinically significant RBCAs (DA=50, NDA=39). More DAs were anti-Jka, anti-M; fewer were anti-E, anti-K (all p<0.05). DA patients often had multiple antibodies (44% vs 12.8% NDA, p=0.0022). Probability of finding antigen-negative blood was lower for DAs (17.4% vs 68.1% NDA, p<0.0001) as was RBCs received (9.4 vs 14.7 units in NDA, p=0.0036). Although survival was similar, patients with DAs had more adverse reactions (8% vs 0%, p=0.128). Some antibodies appeared to occur with specific liver diseases (such as primary sclerosing cholangitis, alcoholic steatohepatitis and recurrent disease); however, due to low sample size, definitive conclusions cannot be made., Conclusions: DA LT recipients contain >1 RBCA, have a lower probability of finding antigen negative blood and may experience more adverse transfusion event (ATE). Despite this, the incidence of ATEs was still quite low., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Red blood cell (RBC) antibodies are common in multiply transfused patients with sickle cell disease (SCD). Unlike RBC alloantibodies, the potential of autoantibodies to cause post-transfusion hemolysis may be uncertain. Biotin-labeling provides a direct measurement of red cell survival (RCS) over time, thus can be used to assess the clinical significance of RBC antibodies. Antibodies to biotinylated RBC (B-RBC) occasionally are detected after exposure, which may impact B-RBC survival in subsequent RCS studies., Study Design and Methods: Pediatric patients with SCD receiving monthly chronic transfusions underwent RCS studies, receiving aliquots of allogeneic RBC labeled at distinct densities of biotin (2-18 μg/mL). B-RBC survival was followed for 4 months post-transfusion, and B-RBC antibody screening for 6 months. Patients with warm autoantibodies (WAA) or B-RBC antibodies are reported here., Results: RBC antibodies were detected during RCS in four patients: one with WAA, one with WAA followed by B-RBC-specific antibodies, and two with transient B-RBC antibodies within the first 5 weeks of exposure. B-RBC half-lives (T 50 ) ranged 37.6-61.7 days (mean 47.8 days). There was no evidence of increased hemolysis or accelerated B-RBC clearance in the presence of WAA or B-RBC antibodies., Discussion: Biotinylation of allogenic RBC can be used to assess the possible effects of RBC antibodies on transfusion survival in individual cases, particularly when it is uncertain if the detected antibodies may result in hemolysis. In the cases presented here, neither WAA nor B-RBC antibodies were associated with significant shortening of B-RBC survival in individuals with SCD., (© 2024 AABB.)

Although immune tolerance evolved to reduce reactivity with self, it creates a gap in the adaptive immune response against microbes that decorate themselves in self-like antigens. This is particularly apparent with carbohydrate-based blood group antigens, wherein microbes can envelope themselves in blood group structures similar to human cells. In this study, we demonstrate that the innate immune lectin, galectin-4 (Gal-4), exhibits strain-specific binding and killing behavior towards microbes that display blood group-like antigens. Examination of binding preferences using a combination of microarrays populated with ABO(H) glycans and a variety of microbial strains, including those that express blood group-like antigens, demonstrated that Gal-4 binds mammalian and microbial antigens that have features of blood group and mammalian-like structures. Although Gal-4 was thought to exist as a monomer that achieves functional bivalency through its two linked carbohydrate recognition domains, our data demonstrate that Gal-4 forms dimers and that differences in the intrinsic ability of each domain to dimerize likely influences binding affinity. While each Gal-4 domain exhibited blood group-binding activity, the C-terminal domain (Gal-4C) exhibited dimeric properties, while the N-terminal domain (Gal-4N) failed to similarly display dimeric activity. Gal-4C not only exhibited the ability to dimerize but also possessed higher affinity toward ABO(H) blood group antigens and microbes expressing glycans with blood group-like features. Furthermore, when compared to Gal-4N, Gal-4C exhibited more potent antimicrobial activity. Even in the context of the full-length protein, where Gal-4N is functionally bivalent by virtue of Gal-4C dimerization, Gal-4C continued to display higher antimicrobial activity. These results demonstrate that Gal-4 exists as a dimer and exhibits its antimicrobial activity primarily through its C-terminal domain. In doing so, these data provide important insight into key features of Gal-4 responsible for its innate immune activity against molecular mimicry., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

While glycans are among the most abundant macromolecules on the cell with widespread functions, their role in immunity has historically been challenging to study. This is in part due to difficulties assimilating glycan analysis into routine approaches used to interrogate immune cell function. Despite this, recent developments have illuminated fundamental roles for glycans in host immunity. The growing field of glycoimmunology continues to leverage new tools and approaches to uncover the function of glycans and glycan-binding proteins in immunity. Here we utilize clinical vignettes to examine key roles of glycosylation in allergy, inborn errors of immunity, and autoimmunity. We will discuss the diverse functions of glycans as epitopes, as modulators of antibody function, and as regulators of immune cell function. Finally, we will highlight immune modulatory therapies that harness the critical role of glycans in the immune system., (Copyright © 2023 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

RBC transfusion remains a cornerstone in the treatment of sickle cell disease (SCD). However, as with many interventions, transfusion of RBCs is not without risk. Allogeneic RBC exposure can result in the development of alloantibodies, which can make it difficult to find compatible RBCs for future transfusion and increases the likelihood of life-threatening complications. The development of RBC alloantibodies occurs when a patient's immune system produces alloantibodies against foreign alloantigens present on RBCs. Despite its longstanding recognition, RBC alloimmunization has increasingly become a challenge when caring for patients with SCD. The growing prominence of alloimmunization can be attributed to several factors, including expanded indications for transfusions, increased lifespan of patients with SCD, and inadequate approaches to prevent alloimmunization. Recognizing these challenges, recent observational studies and preclinical models have begun to elucidate the immune pathways that underpin RBC alloimmunization. These emerging data hold promise in paving the way for innovative prevention strategies, with the goal of increasing the safety and efficacy of RBC transfusion in patients with SCD who are most vulnerable to alloimmunization., (Copyright © 2023. Published by Elsevier Masson SAS.)

Plasmodium knowlesi ( Pk ) causes zoonotic malaria and is known as the "fifth human malaria parasite". Pk malaria is an emerging threat because infections are increasing and can be fatal. While most infections are in Southeast Asia (SEA), especially Malaysia, travelers frequently visit this region and can present with Pk malaria around the world. So, clinicians need to know (1) patients who present with fever after recent travel to SEA might be infected with Pk and (2) Pk is often misdiagnosed as P. malariae (which typically causes less severe malaria). Here we review the history, pathophysiology, clinical features, diagnosis, and treatment of Pk malaria. Severe disease is most common in adults. Signs and symptoms can include fever, abdominal pain, jaundice, acute kidney injury, acute respiratory distress syndrome, hyponatremia, hyperparasitemia, and thrombocytopenia. Dengue is one of the diseases to be considered in the differential. Regarding pathophysiologic mechanisms, when Pk parasites invade mature red blood cells (RBCs, i.e., normocytes) and reticulocytes, changes in the red blood cell (RBC) surface can result in life-threatening cytoadherence, sequestration, and reduced RBC deformability. Since molecular mechanisms involving the erythrocytic stage are responsible for onset of severe disease and lethal outcomes, it is biologically plausible that manual exchange transfusion (ET) or automated RBC exchange (RBCX) could be highly beneficial by replacing "sticky" parasitized RBCs with uninfected, deformable, healthy donor RBCs. Here we suggest use of special Pk -resistant donor RBCs to optimize adjunctive manual ET/RBCX for malaria. "Therapeutically-rational exchange transfusion" (T-REX) is proposed in which Pk -resistant RBCs are transfused (instead of disease-promoting RBCs). Because expression of the Duffy antigen on the surface of human RBCs is essential for parasite invasion, T-REX of Duffy-negative RBCs-also known as Fy(a-b-) RBCs-could replace the majority of the patient's circulating normocytes with Pk invasion-resistant RBCs (in a single procedure lasting about 2 h). When sequestered or non-sequestered iRBCs rupture-in a 24 h Pk asexual life cycle-the released merozoites cannot invade Fy(a-b-) RBCs. When Fy(a-b-) RBC units are scarce (e.g., in Malaysia), clinicians can consider the risks and benefits of transfusing plausibly Pk -resistant RBCs, such as glucose-6-phosphate dehydrogenase deficient (G6PDd) RBCs and Southeast Asian ovalocytes (SAO). Patients typically require a very short recovery time (<1 h) after the procedure. Fy(a-b-) RBCs should have a normal lifespan, while SAO and G6PDd RBCs may have mildly reduced half-lives. Because SAO and G6PDd RBCs come from screened blood donors who are healthy and not anemic, these RBCs have a low-risk for hemolysis and do not need to be removed after the patient recovers from malaria. T-REX could be especially useful if (1) antimalarial medications are not readily available, (2) patients are likely to progress to severe disease, or (3) drug-resistant strains emerge. In conclusion, T-REX is a proposed optimization of manual ET/RBCX that has not yet been utilized but can be considered by physicians to treat Pk malaria patients.

Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of human angiotensin converting enzyme 2 (hACE-2) binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using Wuhan-WT (vaccine strain), delta (B.1.167.2), omicron BA1 and BA2 variant viral strains showed strong correlation with cell-based pseudovirus neutralization activity (PNA) and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta and omicron variant resistance to neutralization in samples with paired vaccine strain and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. Importantly, this completely automated assay can be performed in 4 h to measure neutralizing antibody titers for 16 samples over 8 serial dilutions or, 128 samples at a single dilution with replicates. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels., Competing Interests: N.C., H.V., S.P., K.B.M., W.H.Os., H.W., A.S.N., and J.D.R. are co-inventors of BoAb assay technology. Emory University filed a patent on this technology., (© 2023 The Authors.)

Red blood cell (RBC) transfusion is a common clinical intervention used to treat patients with acute and chronic anemia. The decision to transfuse RBCs in the acute setting is based on several factors but current clinical studies informing optimal RBC transfusion decision making (TDM) are largely based upon hemoglobin (Hb) level. In contrast to transfusion in acute settings, chronic RBC transfusion therapy has several different purposes and is associated with distinct transfusion risks such as iron overload and RBC alloimmunization. Consequently, RBC TDM in the chronic setting requires optimizing the survival of transfused RBCs in order to reduce transfusion exposure over the lifespan of an individual and the associated transfusion complications mentioned. This review summarizes the current medical literature addressing optimal RBC-TDM in the acute and chronic transfusion settings and discusses the current gaps in knowledge which need to be prioritized in future national and international research initiatives., Competing Interests: Declaration of Competing Interest RMF serves on medical advisory boards for Global Blood Therapeutics, Forma Therapeutics, and Cerus, and receives research funding from Forma Therapeutics and Cerus. RMF also serves as a consultant for REDSIV-Pediatric which is funded by the NIH/NHLBI. AD has no conflicts relevant to this publication. SRS serves as a consultant for Novartis, Alexison, Cereus, Grifols, Cellics, and Argenx. PCS serves as a consultant for Hemanext. JLC serves as the principal investigator of the NIH sponsored Myocardial Ischemia and Transfusion (MINT) trial, and is the Chair of DSMB for Cerus. CLM has no conflicts relevant to this publication. CDJ serves as a consultant for Westat, Immuocr, and has an unrestricted grant through Medtronics. DJT serves on the medical advisory board for Fresenius Kabi., (Copyright © 2023 Elsevier Inc. All rights reserved.)

While the COVID-19 pandemic has impacted many aspects of healthcare, including routine blood donations, the impact of COVID-19 on the donation of source plasma critical to many aspects of patient care, including apheresis procedures, has been more difficult to define. As production of plasma-derived medicinal products (PDMPs) can take up to a year, shortages in source plasma donations may not be immediately appreciated. Given current shortages in PDMPs, in particular albumin, we examined the impact of COVID-19 on source plasma donations. Our data demonstrate that source plasma donations were disproportionately impacted by COVID-19 and that these shortages remained until the latter half of 2022. Given the time delay in PDMP manufacturing, these results suggest that while source plasma donation levels are returning to pre-pandemic levels, shortages in PDMPs may not be quickly overcome. These results also highlight the unique vulnerabilities in plasma sourcing that may continue to manifest as PDMP shortages for years to come., (© 2023 Wiley Periodicals LLC.)

Antibodies against fetal red blood cell (RBC) antigens can cause hemolytic disease of the fetus and newborn (HDFN). Reductions in HDFN due to anti-RhD antibodies have been achieved through use of Rh immune globulin (RhIg), a polyclonal antibody preparation that causes antibody-mediated immunosuppression (AMIS), thereby preventing maternal immune responses against fetal RBCs. Despite the success of RhIg, it is only effective against 1 alloantigen. The lack of similar interventions that mitigate immune responses toward other RBC alloantigens reflects an incomplete understanding of AMIS mechanisms. AMIS has been previously attributed to rapid antibody-mediated RBC removal, resulting in B-cell ignorance of the RBC alloantigen. However, our data demonstrate that antibody-mediated RBC removal can enhance de novo alloimmunization. In contrast, inclusion of antibodies that possess the ability to rapidly remove the target antigen in the absence of detectable RBC clearance can convert an augmented antibody response to AMIS. These results suggest that the ability of antibodies to remove target antigens from the RBC surface can trigger AMIS in situations in which enhanced immunity may otherwise occur. In doing so, these results hold promise in identifying key antibody characteristics that can drive AMIS, thereby facilitating the design of AMIS approaches toward other RBC antigens to eliminate all forms of HDFN., (© 2023 by The American Society of Hematology.)

Patients with sickle cell disease (SCD) are considered to be immunocompromised, yet data on the antibody response to SARS-CoV-2 vaccination in SCD is limited. We investigated anti-SARS-CoV-2 IgG titres and overall neutralizing activity in 201 adults with SCD and demographically matched non-SCD controls. Unexpectedly, patients with SCD generate a more robust and durable COVID-19 vaccine IgG response compared to matched controls, though the neutralizing activity remained similar across both cohorts. These findings suggest that patients with SCD achieve a similar antibody response following COVID-19 vaccination compared to the general population, with implications for optimal vaccination strategies for patients with SCD., (© 2023 British Society for Haematology and John Wiley & Sons Ltd.)

Transfusion of allogeneic human red blood cell (hRBCs) is limited by supply and compatibility between individual donors and recipients. In situations where the blood supply is constrained or when no compatible RBCs are available, patients suffer. As a result, alternatives to hRBCs that complement existing RBC transfusion strategies are needed. Pig RBCs (pRBCs) could provide an alternative because of their abundant supply, and functional similarities to hRBCs. The ability to genetically modify pigs to limit pRBC immunogenicity and augment expression of human 'protective' proteins has provided major boosts to this research and opens up new therapeutic avenues. Although deletion of expression of xenoantigens has been achieved in genetically-engineered pigs, novel genetic methods are needed to introduce human 'protective' transgenes into pRBCs at the high levels required to prevent hemolysis and extend RBC survival in vivo. This review addresses recent progress and examines future prospects for clinical xenogeneic pRBC transfusion., Competing Interests: Declaration of Competing Interest DKCC and SCR are consultants to eGenesis Bio, Cambridge, MA, but the opinions expressed in the article are those of the authors and do not necessarily represent those of eGenesis. SCR is also a scientific founder of PURINOMIA and consults for SynLogic. No other author reports a conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Among the risk factors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), ABO(H) blood group antigens are among the most recognized predictors of infection. However, the mechanisms by which ABO(H) antigens influence susceptibility to COVID-19 remain incompletely understood. The receptor-binding domain (RBD) of SARS-CoV-2, which facilitates host cell engagement, bears significant similarity to galectins, an ancient family of carbohydrate-binding proteins. Because ABO(H) blood group antigens are carbohydrates, we compared the glycan-binding specificity of SARS-CoV-2 RBD with that of galectins. Similar to the binding profile of several galectins, the RBDs of SARS-CoV-2, including Delta and Omicron variants, exhibited specificity for blood group A. Not only did each RBD recognize blood group A in a glycan array format, but each SARS-CoV-2 virus also displayed a preferential ability to infect blood group A-expressing cells. Preincubation of blood group A cells with a blood group-binding galectin specifically inhibited the blood group A enhancement of SARS-CoV-2 infection, whereas similar incubation with a galectin that does not recognize blood group antigens failed to impact SARS-CoV-2 infection. These results demonstrated that SARS-CoV-2 can engage blood group A, providing a direct link between ABO(H) blood group expression and SARS-CoV-2 infection., (© 2023 by The American Society of Hematology.)

There is an urgent need to develop new tumor biomarkers for early cancer detection, but the variability of tumor-derived antigens has been a limitation. Here we demonstrate a novel anti-Tn antibody microarray platform to detect Tn+ glycoproteins, a near universal antigen in carcinoma-derived glycoproteins, for broad detection of cancer. The platform uses a specific recombinant IgG1 to the Tn antigen (CD175) as a capture reagent and a recombinant IgM to the Tn antigen as a detecting reagent. These reagents were validated by immunohistochemistry in recognizing the Tn antigen using hundreds of human tumor specimens. Using this approach, we could detect Tn+ glycoproteins at subnanogram levels using cell lines and culture media, serum, and stool samples from mice engineered to express the Tn antigen in intestinal epithelial cells. The development of a general cancer detection platform using recombinant antibodies for detection of altered tumor glycoproteins expressing a unique antigen could have a significant impact on cancer detection and monitoring., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The galectin family consists of carbohydrate (glycan) binding proteins that are expressed by a wide variety of cells and bind to galactose-containing glycans. Galectins can be located in the nucleus or the cytoplasm, or can be secreted into the extracellular space. They can modulate innate and adaptive immune cells by binding to glycans on the surface of immune cells or intracellularly via carbohydrate-dependent or carbohydrate-independent interactions. Galectins expressed by immune cells can also participate in host responses to infection by directly binding to microorganisms or by modulating antimicrobial functions such as autophagy. Here we explore the diverse ways in which galectins have been shown to impact immunity and discuss the opportunities and challenges in the field., (© 2023. Springer Nature Limited.)

Objective: To improve our understanding of the immune response, including the neutralization antibody response, following COVID-19 vaccination in pregnancy., Methods: This was a prospective cohort study comprising patients with PCR-confirmed SARS-CoV-2 infection and patients who received both doses of mRNA COVID-19 vaccine (mRNA-1273, BNT162b2) in pregnancy recruited from two hospitals in Atlanta, GA, USA. Maternal blood and cord blood at delivery were assayed for anti-receptor binding domain (RBD) IgG, IgA and IgM, and neutralizing antibody. The detection of antibodies, titers, and maternal to fetal transfer ratios were compared., Results: Nearly all patients had detectable RBD-binding IgG in maternal and cord samples. The vaccinated versus infected cohort had a significantly greater proportion of cord samples with detectable neutralizing antibody (94% vs. 28%, P < 0.001) and significantly higher transfer ratios for RBD-specific IgG and neutralizing antibodies with a transfer efficiency of 105% (vs. 80%, P < 0.001) and 110% (vs. 90%, P < 0.001), respectively. There was a significant linear decline in maternal and cord blood RBD-specific IgG and neutralizing antibody titers as time from vaccination to delivery increased., Conclusions: Those who receive the mRNA COVID-19 vaccine mount an immune response that is equivalent to-if not greater than-those naturally infected by SARS-CoV-2 during pregnancy., (© 2023 International Federation of Gynecology and Obstetrics.)

Antibodies against red blood cell (RBC) alloantigens can increase morbidity and mortality among transfusion recipients. However, alloimmunization rates can vary dramatically, as some patients never generate alloantibodies after transfusion, whereas others not only become alloimmunized but may also be prone to generating additional alloantibodies after subsequent transfusion. Previous studies suggested that CD4 T-cell responses that drive alloantibody formation recognize the same alloantigen engaged by B cells. However, because RBCs express numerous antigens, both internally and externally, it is possible that CD4 T-cell responses directed against intracellular antigens may facilitate subsequent alloimmunization against a surface RBC antigen. Here, we show that B cells can acquire intracellular antigens from RBCs. Using a mouse model of donor RBCs expressing 2 distinct alloantigens, we demonstrate that immune priming to an intracellular antigen, which would not be detected by any currently used RBC compatibility assays, can directly influence alloantibody formation after exposure to a subsequent distinct surface RBC alloantigen. These findings suggest a previously underappreciated mechanism whereby transfusion recipient responders may exhibit an increased rate of alloimmunization because of prior immune priming toward intracellular antigens., (© 2023 by The American Society of Hematology.)

Background: We examined the relationship between placental histopathology and transplacental antibody transfer in pregnant patients after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection., Methods: Differences in plasma concentrations of anti-receptor biding domain (RBD) immunoglobulin (Ig)G antibodies in maternal and cord blood were analyzed according to presence of placental injury., Results: Median anti-RBD IgG concentrations in cord blood with placental injury (n = 7) did not differ significantly from those without injury (n = 16) (median 2.7 [interquartile range {IQR}, 1.8-3.6] vs 2.7 [IQR, 2.4-2.9], P = 0.59). However, they were associated with lower transfer ratios (median 0.77 [IQR, 0.61-0.97] vs 0.97 [IQR, 0.80-1.01], P = 0.05)., Conclusions: SARS-CoV-2 placental injury may mediate reduced maternal-fetal antibody transfer., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For cipermissions, please e-mail: journals.permissions@oup.com.)

The pathogenesis of multi-organ dysfunction associated with severe acute SARS-CoV-2 infection remains poorly understood. Endothelial damage and microvascular thrombosis have been identified as drivers of COVID-19 severity, yet the mechanisms underlying these processes remain elusive. Here we show alterations in fluid shear stress-responsive pathways in critically ill COVID-19 adults as compared to non-COVID critically ill adults using a multiomics approach. Mechanistic in-vitro studies, using microvasculature-on-chip devices, reveal that plasma from critically ill COVID-19 adults induces fibrinogen-dependent red blood cell aggregation that mechanically damages the microvascular glycocalyx. This mechanism appears unique to COVID-19, as plasma from non-COVID sepsis patients demonstrates greater red blood cell membrane stiffness but induces less significant alterations in overall blood rheology. Multiomics analyses in pediatric patients with acute COVID-19 or the post-infectious multi-inflammatory syndrome in children (MIS-C) demonstrate little overlap in plasma cytokine and metabolite changes compared to adult COVID-19 patients. Instead, pediatric acute COVID-19 and MIS-C patients show alterations strongly associated with cytokine upregulation. These findings link high fibrinogen and red blood cell aggregation with endotheliopathy in adult COVID-19 patients and highlight differences in the key mediators of pathogenesis between adult and pediatric populations., (© 2023. The Author(s).)

ABO compatibility is important for kidney transplantation, with longer waitlist times for blood group B kidney transplant candidates. However, kidneys from non-A 1 (eg, A 2 ) subtype donors, which express less A antigen, can be safely transplanted into group B recipients. ABO subtyping is routinely performed using anti-A 1 lectin, but DNA-based genotyping is also possible. Here, we compare lectin and genotyping testing. Lectin and genotype subtyping was performed on 554 group A deceased donor samples at 2 transplant laboratories. The findings were supported by 2 additional data sets of 210 group A living kidney donors and 124 samples with unclear lectin testing sent to a reference laboratory. In deceased donors, genotyping found 65% more A 2 donors than lectin testing, most with weak lectin reactivity, a finding supported in living donors and samples sent for reference testing. DNA sequencing and flow cytometry showed that the discordances were because of several factors, including transfusion, small variability in A antigen levels, and rare ABO∗A2.06 and ABO∗A2.16 sequences. Although lectin testing is the current standard for transplantation subtyping, genotyping is accurate and could increase A 2 kidney transplant opportunities for group B candidates, a difference that should reduce group B wait times and improve transplant equity., (Copyright © 2022 American Society of Transplantation & American Society of Transplant Surgeons. Published by Elsevier Inc. All rights reserved.)

Microbial glycan microarrays (MGMs) populated with purified microbial glycans have been used to define the specificity of host immune factors toward microbes in a high throughput manner. However, a limitation of such arrays is that glycan presentation may not fully recapitulate the natural presentation that exists on microbes. This raises the possibility that interactions observed on the array, while often helpful in predicting actual interactions with intact microbes, may not always accurately ascertain the overall affinity of a host immune factor for a given microbe. Using galectin-8 (Gal-8) as a probe, we compared the specificity and overall affinity observed using a MGM populated with glycans harvested from various strains of Streptococcus pneumoniae to an intact microbe microarray (MMA). Our results demonstrate that while similarities in binding specificity between the MGM and MMA are apparent, Gal-8 binding toward the MMA more accurately predicted interactions with strains of S. pneumoniae, including the overall specificity of Gal-8 antimicrobial activity. Taken together, these results not only demonstrate that Gal-8 possesses antimicrobial activity against distinct strains of S. pneumoniae that utilize molecular mimicry, but that microarray platforms populated with intact microbes present an advantageous strategy when exploring host interactions with microbes., (© 2023. The Author(s).)

Introduction: The impact of blood storage on red blood cell (RBC) alloimmunization remains controversial, with some studies suggesting enhancement of RBC-induced alloantibody production and others failing to observe any impact of storage on alloantibody formation. Since evaluation of storage on RBC alloimmunization in patients has examined antibody formation against a broad range of alloantigens, it remains possible that different clinical outcomes reflect a variable impact of storage on alloimmunization to specific antigens., Methods: RBCs expressing two distinct model antigens, HEL-OVA-Duffy (HOD) and KEL, separately or together (HOD × KEL), were stored for 0, 8, or 14 days, followed by detection of antigen levels prior to transfusion. Transfused donor RBC survival was assessed within 24 h of transfusion, while IgM and IgG antibody production were assessed 5 and 14 days after transfusion., Results: Stored HOD or KEL RBCs retained similar HEL or KEL antigen levels, respectively, as fresh RBCs, but did exhibit enhanced RBC clearance with increased storage age. Storage enhanced IgG antibody formation against HOD, while the oppositive outcome occurred following transfusion of stored KEL RBCs. The distinct impact of storage on HOD or KEL alloimmunization did not appear to reflect intrinsic differences between HOD or KEL RBCs, as transfusion of stored HOD × KEL RBCs resulted in increased IgG anti-HOD antibody development and reduced IgG anti-KEL antibody formation., Conclusions: These data demonstrate a dichotomous impact of storage on immunization to distinct RBC antigens, offering a possible explanation for inconsistent clinical experience and the need for additional studies on the relationship between RBC storage and alloimmunization., (© 2023 AABB.)

Background: Nucleocapsid antigenemia in adults has demonstrated high sensitivity and specificity for acute infection, and antigen burden is associated with disease severity. Data regarding SARS-CoV-2 antigenemia in children are limited., Methods: We retrospectively analyzed blood plasma specimens from hospitalized children with COVID-19 or MIS-C. Nucleocapsid and spike were measured using ultrasensitive immunoassays., Results: We detected nucleocapsid antigenemia in 62% (50/81) and spike antigenemia in 27% (21/79) of children with acute COVID-19 but 0% (0/26) and 15% (4/26) with MIS-C from March 2020-March 2021. Higher nucleocapsid levels were associated with radiographic infiltrates and respiratory symptoms in children with COVID-19., Conclusions: Antigenemia lacks the sensitivity to diagnose acute infection in children but is associated with signs and symptoms of lower respiratory tract involvement. Further study into the mechanism of antigenemia, its association with specific organ involvement, and the role of antigenemia in the pathogenesis of COVID-19 is warranted., Competing Interests: CAR’s institution has received funds to conduct clinical research unrelated to this manuscript from BioFire Inc, GSK, MedImmune, Micron, Janssen, Merck, Moderna, Novavax, PaxVax, Pfizer, Regeneron, Sanofi-Pasteur. She is co-inventor of patented RSV vaccine technology unrelated to this manuscript, which has been licensed to Meissa Vaccines, Inc. EJA has consulted for Pfizer, Sanofi Pasteur, Janssen, and Medscape, and his institution receives funds to conduct clinical research unrelated to this manuscript from MedImmune, Regeneron, PaxVax, Pfizer, GSK, Merck, Sanofi-Pasteur, Janssen, and Micron. He also serves on a safety monitoring board for Kentucky BioProcessing, Inc. and Sanofi Pasteur. His institution has also received funding from NIH to conduct clinical trials of Moderna and Janssen COVID-19 vaccines. GLD, HPV, KRVH, KM, AL, MAP, LH, SRS, JDR, and WAL report no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

While red blood cell (RBC) transfusion is the most common medical intervention in hospitalized patients, as with any therapeutic, it is not without risk. Allogeneic RBC exposure can result in recipient alloimmunization, which can limit the availability of compatible RBCs for future transfusions and increase the risk of transfusion complications. Despite these challenges and the discovery of RBC alloantigens more than a century ago, relatively little has historically been known regarding the immune factors that regulate RBC alloantibody formation. Through recent epidemiological approaches, in vitro-based translational studies, and newly developed preclinical models, the processes that govern RBC alloimmunization have emerged as more complex and intriguing than previously appreciated. Although common alloimmunization mechanisms exist, distinct immune pathways can be engaged, depending on the target alloantigen involved. Despite this complexity, key themes are beginning to emerge that may provide promising approaches to not only actively prevent but also possibly alleviate the most severe complications of RBC alloimmunization.

Enzymes catalyze biochemical reactions and play critical roles in human health and disease. Enzyme variants and deficiencies can lead to variable expression of glycans, which can affect physiology, influence predilection for disease, and/or directly contribute to disease pathogenesis. Although certain well-characterized enzyme deficiencies result in overt disease, some of the most common enzyme deficiencies in humans form the basis of blood groups. These carbohydrate blood groups impact fundamental areas of clinical medicine, including the risk of infection and severity of infectious disease, bleeding risk, transfusion medicine, and tissue/organ transplantation. In this review, we examine the enzymes responsible for carbohydrate-based blood group antigen biosynthesis and their expression within the human population. We also consider the evolutionary selective pressures, e.g. malaria, that may account for the variation in carbohydrate structures and the implications of this biology for human disease., Competing Interests: The authors declare no competing interests., (© 2022.)

Detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is essential for diagnosis, treatment, and infection control. Polymerase chain reaction (PCR) fails to distinguish acute from resolved infections, as RNA is frequently detected after infectiousness. We hypothesized that nucleocapsid in blood marks acute infection with the potential to enhance isolation and treatment strategies. In a retrospective serosurvey of inpatient and outpatient encounters, we categorized samples along an infection timeline using timing of SARS-CoV-2 testing and symptomatology. Among 1860 specimens from 1607 patients, the highest levels and frequency of antigenemia were observed in samples from acute SARS-CoV-2 infection. Antigenemia was higher in seronegative individuals and in those with severe disease. In our analysis, antigenemia exhibited 85.8% sensitivity and 98.6% specificity as a biomarker for acute coronavirus disease 2019 (COVID-19). Thus, antigenemia sensitively and specifically marks acute SARS-CoV-2 infection. Further study is warranted to determine whether antigenemia may aid individualized assessment of active COVID-19., Competing Interests: Potential conflicts of interest. C. A. R.’s institution has received funds to conduct clinical research unrelated to this work from BioFire Inc, GSK, MedImmune, Micron, Janssen, Merck, Moderna, Novavax, PaxVax, Pfizer, Regeneron, and Sanofi-Pasteur; she is coinventor of patented RSV vaccine technology unrelated to this work, which has been licensed to Meissa Vaccines, Inc. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: From 2008 to 2017, 28.8 % fewer United States allopathic medical students (MD seniors) applied to pathology residency in the Main Residency Match (MRM) and 27.5 % fewer matched. This study is a 5-year follow-up., Methods: MRM data from 2018 to 2022 were reviewed to determine the numbers of MD seniors that applied and matched to pathology residency and other major medical specialties., Results: From 2018 to 2022, the number of MD seniors applying to pathology increased 4.6 % from 237 to 248, while MD seniors matching to pathology increased 5.0 % from 220 to 231. For the 4 years from 2018 to 2021, there was a slight decline in MD seniors filling pathology positions, followed by a substantial 16.7 % spike in 2022. For the entire 5-year interval, because the number of filled pathology residency positions increased by 9.0 %, the percentage of filled positions taken by MD seniors declined from 38.7 % to 37.3 %. Of the 15 major medical specialties evaluated, pathology now has the 14th lowest percentage of filled positions taken by MD seniors., Conclusions: The number of MD seniors applying and matching to pathology residency increased over the past 5-years, in contrast to the timespan of 2008 to 2017. However, the percentage of pathology residency positions taken by MD seniors continued to decline and remains low compared to other major medical specialties. MRM data should be continually monitored to study trends in MD seniors filling pathology residency positions in the context of new recruitment efforts and the pandemic., Competing Interests: Declaration of competing interest Dr. Ryan Jajosky is the CEO and part-owner of Biconcavity Inc. Dr. Philip Jajosky is CMO and part-owner of Biconcavity Inc. Biconcavity Inc. is a biotechnology research and development company exploring drug-linked-erythrocytes. Biconcavity does not have any interest in the Main Residency Match. Visit www.biconcavity.com for more information. Dr. Audrey Jajosky and Dr. Sean Stowell do not have any disclosures., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Passive immunization with anti-D can prevent maternal alloimmunization to RhD thereby preventing hemolytic disease of the fetus and newborn. Unexpectedly, anti-D fails in some cases and some monoclonal anti-D preparations paradoxically enhances alloimmunization. The underlying mechanisms modulating humoral alloimmunization by anti-D are unknown. We previously reported that IgG antibody subclasses differentially regulate alloimmunity in response to red blood cell (RBC) transfusions in a mouse model; in particular, IgG2c significantly enhanced RBC alloantibody responses. Initial mechanistic studies revealed that IgG2c:RBC immune complexes were preferentially consumed by the splenic dendritic cell (DC) subsets that play a role in RBC alloimmunization. The deletion of activating Fc-gamma receptors (FcγRs) (i.e., FcγRI, FcγRIII, and FcγRIV) on DCs abrogated IgG2c-mediated enhanced alloimmunization. Because DCs express high levels of FcγRIV, which has high affinity for the IgG2c subclass, we hypothesized that FcγRIV was required for enhanced alloimmunization. To test this hypothesis, knockout mice and blocking antibodies were used to manipulate FcγR expression. The data presented herein demonstrate that FcγRIV, but not FcγRI or FcγRIII, is required for IgG2c-mediated enhancement of RBC alloantibody production. Additionally, FcγRI is alone sufficient for IgG2c-mediated RBC clearance but not for increased alloimmunization, demonstrating that RBC clearance can occur without inducing alloimmunization. Together, these data, combined with prior observations, support the hypothesis that passive immunization with an RBC-specific IgG2c antibody increases RBC alloantibody production through Fc γ RIV ligation on splenic conventional DCs (cDCs). This raises the question of whether standardizing antibody subclasses in immunoprophylaxis preparations is desirable and suggests which subclasses may be optimal for generating monoclonal anti-D therapeutics., Competing Interests: Although unrelated to the contents of this manuscript, KH has a sponsored research agreement with Alpine Immune Sciences. JZ is a consultant for Rubius Therapeutics and is the founder and CSO of Svalinn Therapeutics. 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., (Copyright © 2022 Qiu, Miller, Zotti, Santhanakrishnan, Hendrickson, Tredicine, Stowell, Luckey, Zimring and Hudson.)

Numerous studies have highlighted the utility of glycan microarray analysis for the elucidation of protein-glycan interactions. However, most current glycan microarray studies analyze glycan binding protein (GBP)-glycan interactions at a single protein concentration. While this approach provides useful information related to a GBP's overall binding capabilities, extrapolation of true glycan binding preferences using this method fails to account for printing variations or other factors that may confound relative binding. To overcome this limitation, we examined glycan array binding of three galectins over a range of concentrations to allow for a more complete assessment of binding preferences. This approach produced a richer data set than single concentration analysis and provided more accurate identification of true glycan binding preferences. However, while this approach can be highly informative, currently available data analysis approaches make it impractical to perform binding isotherms for each glycan present on currently available platforms following GBP evaluation. To overcome this limitation, we developed a method to directly optimize the efficiency of assessing association constants following multi-GBP concentration glycan array analysis. To this end, we developed programs that automatically analyze raw array data (kdMining) to generate output graphics (kaPlotting) following array analysis at multiple doses. These automatic programing methods reduced processing time from 32.8 h to 1.67 min. Taken together, these results demonstrate an effective approach to glycan array analysis that provides improved detail and efficiency when compared to previous methods., 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., (Copyright © 2022 Ho, Wu, Kamili, Blenda, Cummings, Stowell and Arthur.)

The development of anti-drug antibodies represents a significant barrier to the utilization of protein-based therapies for a wide variety of diseases. While the rate of antibody formation can vary depending on the therapeutic employed and the target patient population receiving the drug, the antigen-specific immune response underlying the development of anti-drug antibodies often remains difficult to define. This is especially true for patients with hemophilia A who, following exposure, develop antibodies against the coagulation factor, factor VIII (FVIII). Models capable of studying this response in an antigen-specific manner have been lacking. To overcome this challenge, we engineered FVIII to contain a peptide (323-339) from the model antigen ovalbumin (OVA), a very common tool used to study antigen-specific immunity. FVIII with an OVA peptide (FVIII-OVA) retained clotting activity and possessed the ability to activate CD4 T cells specific to OVA 323-339 in vitro. When compared to FVIII alone, FVIII-OVA also exhibited a similar level of immunogenicity, suggesting that the presence of OVA 323-339 does not substantially alter the anti-FVIII immune response. Intriguingly, while little CD4 T cell response could be observed following exposure to FVIII-OVA alone, inclusion of anti-FVIII antibodies, recently shown to favorably modulate anti-FVIII immune responses, significantly enhanced CD4 T cell activation following FVIII-OVA exposure. These results demonstrate that model antigens can be incorporated into a therapeutic protein to study antigen-specific responses and more specifically that the CD4 T cell response to FVIII-OVA can be augmented by pre-existing anti-FVIII antibodies.

Adaptive immunity can target a nearly infinite range of antigens, yet it is tempered by tolerogenic mechanisms that limit autoimmunity. Such immunological tolerance, however, creates a gap in adaptive immunity against microbes decorated with self-like antigens as a form of molecular mimicry. Our results demonstrate that the innate immune lectin galectin-7 (Gal-7) binds a variety of distinct microbes, all of which share features of blood group-like antigens. Gal-7 binding to each blood group expressing microbe, including strains of Escherichia coli , Klebsiella pneumoniae , Providencia alcalifaciens, and Streptococcus pneumoniae , results in loss of microbial viability. Although Gal-7 also binds red blood cells (RBCs), this interaction does not alter RBC membrane integrity. These results demonstrate that Gal-7 recognizes a diverse range of microbes, each of which use molecular mimicry while failing to induce host cell injury, and thus may provide an innate form of immunity against molecular mimicry., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Humoral immunity to factor VIII (FVIII) represents a significant challenge for the treatment of patients with hemophilia A. Current paradigms indicate that neutralizing antibodies against FVIII (inhibitors) occur through a classical CD4 T cell, germinal center (GC) dependent process. However, clinical observations suggest that the nature of the immune response to FVIII may differ between patients. While some patients produce persistent low or high inhibitor titers, others generate a transient response. Moreover, FVIII reactive memory B cells are only detectable in some patients with sustained inhibitor titers. The determinants regulating the type of immune response a patient develops, let alone how the immune response differs in these patients remains incompletely understood. One hypothesis is that polymorphisms within immunoregulatory genes alter the underlying immune response to FVIII, and thereby the inhibitor response. Consistent with this, studies report that inhibitor titers to FVIII differ in animals with the same F8 pathogenic variant but completely distinct backgrounds; though, how these genetic disparities affect the immune response to FVIII remains to be investigated. Given this, we sought to mechanistically dissect how genetics impact the underlying immune response to FVIII. In particular, as the risk of producing inhibitors is weakly associated with differences in HLA, we hypothesized that genetic factors other than HLA influence the immune response to FVIII and downstream inhibitor formation. Our data demonstrate that FVIII deficient mice encoding the same MHC and F8 variant produce disparate inhibitor titers, and that the type of inhibitor response formed associates with the ability to generate GCs. Interestingly, the formation of antibodies through a GC or non-GC pathway does not appear to be due to differences in CD4 T cell immunity, as the CD4 T cell response to an immunodominant epitope in FVIII was similar in these mice. These results indicate that genetics can impact the process by which inhibitors develop and may in part explain the apparent propensity of patients to form distinct inhibitor responses. Moreover, these data highlight an underappreciated immunological pathway of humoral immunity to FVIII and lay the groundwork for identification of biomarkers for the development of approaches to tolerize against FVIII., Competing Interests: SM serves on the advisory board for Genentech, Sanofi Genzyme, Takeda, Biomarin, and CSL Behring. SM consults for Pfizer and Spark, as well as receives research funding from Octapharma, Genentech. RJ is the CEO and part-owner of Biconcavity Inc, as well as owns stock in BioMarin Pharmaceutical Inc. 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., (Copyright © 2022 Patel, Lundgren, Baldwin, Cox, Parker, Healey, Jajosky, Zerra, Josephson, Doering, Stowell and Meeks.)

Background: Alloimmunization can be a significant barrier to red blood cell (RBC) transfusion. While alloantigen matching protocols hold promise in reducing alloantibody formation, transfusion-dependent patients can still experience RBC alloimmunization and associated complications even when matching protocols are employed. As a result, complementary strategies capable of actively preventing alloantibody formation following alloantigen exposure are warranted., Study Design and Methods: We examined whether pharmacological removal of macrophages using clodronate may provide an additional strategy to actively inhibit RBC alloimmunization using two preclinical models of RBC alloimmunization. To accomplish this, mice were treated with clodronate, followed by transfusion of RBCs expressing the HOD (HEL, OVA, and Duffy) or KEL antigens. On days 5 and 14 post transfusion, anti-HOD or anti-KEL IgM and IgG antibodies were evaluated., Results: Low dose clodronate effectively eliminated key marginal zone macrophage populations from the marginal sinus. Prior treatment with clodronate, but not empty liposomes, also significantly inhibited IgM and IgG anti-HOD alloantibody formation following transfusion of HOD RBCs. Similar exposure to clodronate inhibited IgM and IgG antibody formation following KEL RBC transfusion., Conclusions: Clodronate can inhibit anti-HOD and anti-KEL antibody formation following RBC transfusion in preclinical models. These results suggest that clodronate may provide an alternative approach to actively inhibit or prevent the development of alloantibodies following RBC transfusion, although future studies will certainly be needed to fully explore this possibility., (© 2022 AABB.)