Molecular dynamics of the human RhD and RhAG blood group proteins.

Introduction: Blood group antigens of the RH system (formerly known as "Rhesus") play an important role in transfusion medicine because of the severe haemolytic consequences of antibodies to these antigens. No crystal structure is available for RhD proteins with its partner RhAG, and the precise stoichiometry of the trimer complex remains unknown. Methods: To analyse their structural properties, the trimers formed by RhD and/or RhAG subunits were generated by protein modelling and molecular dynamics simulations were performed. Results: No major differences in structural behaviour were found between trimers of different compositions. The conformation of the subunits is relatively constant during molecular dynamics simulations, except for three large disordered loops. Discussion: This work makes it possible to propose a reasonable stoichiometry and demonstrates the potential of studying the structural behaviour of these proteins to investigate the hundreds of genetic variants relevant to transfusion medicine.

An Oral Carbon Monoxide-Releasing Molecule Protects against Acute Hyper-hemolysis in Sickle Cell Disease.

Acute hyper-hemolysis is a severe life-threatening complication in patients with sickle cell disease (SCD) that may occur during delayed hemolytic transfusion reaction (DHTR), or vaso-occlusive crises associated with multi-organ failure. Here, we developed in vitro and in vivo animal models to mimic endothelial damage during the early phase of hyper-hemolysis in SCD. We then used the carbon monoxide (CO)-releasing molecule CORM-401 and examined its effects against endothelial activation, damage, and inflammation inflicted by hemolysates containing red blood cell membrane-derived particles. The in vitro results revealed that CORM-401: 1) prevented the up-regulation of relevant pro-inflammatory, and pro-adhesion controlled by the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and 2) abolished the expression of the nuclear factor erythroid-2-related factor 2 (Nrf2) that regulates the inducible antioxidant cell machinery. We also show in SCD mice that CORM-401 protects against hemolysate-induced acute damage of target organs such as the lung, liver, and kidney through modulation of NF-kB pro-inflammatory and Nrf2 antioxidant pathways. Our data demonstrate the efficacy of CORM-401 as a novel therapeutic agent to counteract hemolysate-induced organ damage during hyper-hemolysis in SCD. This approach might be considered as possible preventive treatment in high-risk situations such as SCD patients with history of DHTR.

Immunoregulatory molecule expression on extracellular microvesicles in people living with HIV.

Introduction: People living with HIV (PLWH) now benefit from combined antiviral treatments that durably control viral replication. These antiretroviral treatments decrease mortality and improve quality of life in PLWH, but do not completely control the excessive non-specific activation of the immune system in PLWH. This chronic immune activation is a key element of HIV immunopathology that contributes to the pathophysiology of inflammatory comorbid conditions, such as cardiovascular disorders, cancer and autoimmune diseases. Circulating non-exosomal extracellular vesicles, also known as microparticles (MPs) are detected in these diseases and have been linked to immune activation. The objective of this study was to characterize the MPs present in PLWH and to assess their association with chronic immune activation. Methods: We performed flow cytometry for the complete phenotypic characterization of MPs from fresh plasma from PLWH and from people without HIV as the control group. The absolute number, size and cellular origin of MPs were evaluated. The immunoregulatory profile was determined by cell origin, for MPs derived from platelets (PMPs), monocytes (MMPs) and T lymphocytes (LMPs). Results: PLWH had significantly more circulating MPs than controls, for MPs of all sizes originating from T lymphocytes, red blood cells, neutrophils, dendritic cells, B lymphocytes and endothelial cells. PMPs and MMPs were not more numerous in PLWH, but the immunoregulatory phenotypes of these MPs differed between PLWH and controls. These differences in immunoregulatory molecule expression profile were also observed for LMPs. PDL1, ICOSL, CCR5, TGFß1, MHC classes I and II, TRAIL, CXCR4, OX40, DC-SIGN, CTLA4 and PDL2 were more strongly expressed on the surface of MPs from PLWH than on those from controls. Conclusion: MPs are an important element in intercellular communication, making it possible to transfer phenotypes and functions to immune cells. The significantly higher numbers of MPs expressing diverse immunomodulatory molecules in PLWH may make a major contribution to the maintenance and/or the development of immune-cell activation in these individuals.

Study of the antigenic characteristics of red blood cells units and their sickle cell disease recipients and the G6PD activity of transfused red blood cells units.

INTRODUCTION: Transfusion has a central place in the treatment of patients with sickle cell disease (SCD). Matching blood groups of red blood cell (RBC) units with the blood groups of the patient is essential to prevent alloimmunization and delayed hemolytic transfusion reaction. African ancestry donors have the best phenocompatibility with patients of the same origin, however their RBCs may present characteristic that can alter quality of the unit such as glucose-6-phosphate dehydrogenase (G6PD) deficiency. The objective is to analyze transfusion protocol, immunization rate and mismatch situations of SCD recipients and to evaluate the frequency of G6PD deficiency in RBCs units from African ancestry donors. METHODS: Samples of units transfused to SCD patients were analyzed. Transfusion data were collected from institutional databases. The activity of G6PD was measured in the segment of the RBC units. RESULTS: A total of 98 segments of units transfused to 37 SCD recipients in 41 transfusions episodes was collected. Among patients, 35.1% (n = 13) had no antibodies; 10.8% (n = 4) had antibodies against Fya/Fyb, Jka/Jkb, M/N, S/s; 21.6% (n = 8) against RH/K antigens. In all cases, the protocols were in line with the recommendations. G6PD deficiency was observed in 9 units, that were all collected from Afro-Caribbean donors. CONCLUSION: The transfusion protocol is established to prevent immunological reactions due to disparities in blood group antigens between donors and SCD recipients. However, the units of African ancestry donors, which allowed the best compatibility, displayed a high rate of G6PD deficiency. The storage and recovery impact of this deficiency must be evaluated.

Alloimmunization and hyperhemolysis in sickle cell disease.

Alloimmunization against red blood cell antigens and delayed hemolytic transfusion reaction (DHTR) are major barriers to transfusion in sickle cell disease (SCD). In SCD, DHTR is a potentially life-threatening. Blood group polymorphism in SCD patients, who are of African ancestry and frequently exposed to antigens they do not carry; an inflammatory clinical state; and occasional transfusion in acute situations are risk factors for alloimmunization and DHTR. In patients at risk, the transfusion indication must be balanced against the risk of developing DHTR. However, when transfusion is absolutely necessary, protocols combining the prevention of exposure to immunogenic antigens with immunosuppressive treatments must be implemented, and patients should be carefully monitored during posttransfusion follow-up. This close monitoring makes it possible to diagnose hyperhemolysis as soon as possible; to avoid retransfusion, which can exacerbate hemolysis; and to administer specific treatments, such as anticomplement therapy, in severe cases. Finally, in patients with severe disease, hematopoietic stem cell transplantation may be indicated. However, transfusion is also required in this context, and its management is complex because these risks must be taken into account.

Prevalence and cost of sickle cell disease in France: real-world analysis using data from the Echantillon Généraliste des Bénéficiaires.

Sickle cell disease (SCD) is a genetic disorder of the hemoglobin resulting in chronic anemia, hemolysis, and vaso-occlusions. Its treatment mostly relies on hydroxycarbamide, transfusions, and stem cell transplantation. This study aimed at describing the epidemiology and management of SCD in adolescent and adult patients in France. This was a retrospective study performed among SCD patients aged ≥12 years between 2016 and 2018 and controls. SCD patients were matched on a 1:3 ratio with a group of individuals with no diagnosis of SCD, referred as control group. The matching of SCD patients and controls was a direct matching based on age, sex, CMU-c status (which corresponds to free-of-charge complementary coverage for people with low resources) and geographical region of residence. SCD patients and their matched controls were followed-up for the same amount of time by adjusting controls' follow-up period to that of the associated patients. This study used claims data from the French representative 1/97th sample of health data system. The main outcomes were the patients' characteristics and treatments received, healthcare consumptions and related costs among SCD cases and controls. Between 2016 and 2018, 151 patients with ≥6 months of follow-up were identified out of the total population of 732,164 individuals. SCD prevalence extrapolated to the entire population [95% CI] was 19,502 [19,230, 19,778] in 2018. The median (Q1-Q3) age at inclusion date was 37.0 (25.0-48.0) years, with 69.5% of patients being female. The mean (SD) reimbursed cost over follow-up was €24,310 (89,167), mostly represented by hospitalization costs accounting for €21,156 (86,402). A switch in SCD management was observed with age, as younger patients presented more frequent hospitalizations and acute procedures, while older ones had more frequent medical visits and paramedical care. Mean (SD) annual costs were €25,680 (91,843) and vs. €3,227 (23,372) for patients and controls, respectively (p < 0.001), representing an extra cost of almost €150 million over the entire SCD population. This study highlighted the important costs related to SCD and the related medical need with treatment alternatives, which could be filled by the emergence of new therapies.

Divergent CD4+ T-cell profiles are associated with anti-HLA alloimmunization status in platelet-transfused AML patients.

Introduction: Acute myeloid leukemia (AML) is one of the commonest hematologic disorders. Due to the high frequency of disease- or treatment-related thrombocytopenia, AML requires treatment with multiple platelet transfusions, which can trigger a humoral response directed against platelets. Some, but not all, AML patients develop an anti-HLA immune response after multiple transfusions. We therefore hypothesized that different immune activation profiles might be associated with anti-HLA alloimmunization status. Methods: We tested this hypothesis, by analyzing CD4+ T lymphocyte (TL) subsets and their immune control molecules in flow cytometry and single-cell multi-omics. Results: A comparison of immunological status between anti-HLA alloimmunized and non-alloimmunized AML patients identified differences in the phenotype and function of CD4+ TLs. CD4+ TLs from alloimmunized patients displayed features of immune activation, with higher levels of CD40 and OX40 than the cells of healthy donors. However, the most notable differences were observed in non-alloimmunized patients. These patients had lower levels of CD40 and OX40 than alloimmunized patients and higher levels of PD1. Moreover, the Treg compartment of non-alloimmunized patients was larger and more functional than that in alloimmunized patients. These results were supported by a multi-omics analysis of immune response molecules in conventional CD4+ TLs, Tfh circulating cells, and Tregs. Discussion: Our results thus reveal divergent CD4+ TL characteristics correlated with anti-HLA alloimmunization status in transfused AML patients. These differences, characterizing CD4+ TLs independently of any specific antigen, should be taken into account when considering the immune responses of patients to infections, vaccinations, or transplantations.

Eleven years of alloimmunization in 6496 patients with sickle cell disease in France who received transfusion.

ABSTRACT: Red blood cell (RBC) transfusion is a major therapy for sickle cell disease (SCD). Patients are at risk of forming antibodies to RBC antigens, which can result in the impossibility to find compatible units and can cause hemolytic transfusion reactions. This retrospective study investigates the evolution of RBC consumption and the frequencies, specificities, and chronology of the appearance of antibodies in a population of patients consistently receiving RH (C, D, E, c, e) and K-matched RBC units (RBCus) from a predominantly European donor population. Over the 11-year period in the Paris area, 6496 patients received transfusion at least once for a total of 239 944 units. Antibodies were made by 1742 patients. The first antibodies of a patient were predictive of subsequent immunization. By the 17th RBCu transfused (by the 20th, excluding warm autoantibodies), 75% of the patients who would make antibodies had made their first. By the 16th, 90% who would make antibodies to a high frequency antigen had made their first antibody to these antigens. Females made their first antibodies slightly earlier than males. Patients who received multiple transfusions (>50 units) had a higher immunization prevalence than those who rarely received transfusion (<12 units) but fewer clinically significant antibodies. Patients with SCD and prophylactic RH-K matching not immunized by the 20th RBCu are likely to have a low alloimmunization risk (to antigens other than RH-K), that is, be low responders, especially relative to the most clinically significant antibodies. This number of 20 units is a point before which close monitoring of patients is most important but remains open to future adjustment.

SARS-CoV-2 Omicron BA.1 breakthrough infection drives late remodeling of the memory B cell repertoire in vaccinated individuals.

How infection by a viral variant showing antigenic drift impacts a preformed mature human memory B cell (MBC) repertoire remains an open question. Here, we studied the MBC response up to 6 months after SARS-CoV-2 Omicron BA.1 breakthrough infection in individuals previously vaccinated with three doses of the COVID-19 mRNA vaccine. Longitudinal analysis, using single-cell multi-omics and functional analysis of monoclonal antibodies from RBD-specific MBCs, revealed that a BA.1 breakthrough infection mostly recruited pre-existing cross-reactive MBCs with limited de novo response against BA.1-restricted epitopes. Reorganization of clonal hierarchy and new rounds of germinal center reactions, however, combined to maintain diversity and induce progressive maturation of the MBC repertoire against common Hu-1 and BA.1, but not BA.5-restricted, SARS-CoV-2 Spike RBD epitopes. Such remodeling was further associated with a marked improvement in overall neutralizing breadth and potency. These findings have fundamental implications for the design of future vaccination booster strategies.

Alloimmunisation against red blood cells in sickle cell disease: transfusion challenges in high-income and low-income countries.

Sickle cell disease is the most frequent inherited disorder in sub-Saharan Africa and in many high-income countries (HICs). Transfusion is a key element of treatment, but it results in high rates of alloimmunisation against red blood cell antigens and post-transfusion haemolysis, which can be life-threatening in severe cases. The prevention of alloimmunisation is, therefore, an important issue in both HICs and in low-income countries (LICs). In HICs, the main reason for high alloimmunisation rates is blood group disparity between blood donors, who are mostly of European descent, and the patients, who are mostly of African descent. However, alloimmunisation rates also remain high in sub-Saharan Africa despite the homogeneity of blood group antigen frequencies between donors and patients; this occurrence is probably due to matching strategies limited to ABO blood group and RhD. However, other possible underlying causes of alloimmunisation have also been suggested, with each cause affecting HICs and LICs in different ways-eg, the immunogenetic and inflammatory status of the patient and the characteristics of the red blood cell products. In this Viewpoint, we discuss the available data and hypotheses that potentially account for the association of sickle cell disease with high rates of alloimmunisation in both settings, HICs and LICs (focusing particularly on sub-Saharan Africa), and the challenges faced by HICs and LICs to improve prevention of alloimmunisation.

CD27+ microparticle interactions and immunoregulation of CD4+ T lymphocytes.

Introduction: Aplasia and hematological malignancies are treated with platelet transfusions, which can have major immunomodulatory effects. Platelet concentrates (PCs) contain many immunomodulatory elements, including the platelets themselves, residual leukocytes, extracellular vesicles, such as microparticles (MPs), cytokines and other soluble elements. Two of these components, MPs and a soluble form of CD27 (sCD27), have been shown to play a particularly important role in immune system modulation. The loss of CD27 expression is an irreversible marker of terminal effector CD3+ T-lymphocyte (TL) differentiation, and the CD27+ MPs present in PCs may maintain CD27 expression on the surface of TLs, and, thus, the activation of these cells. Methods: In this study, we phenotyped the CD27-expressing MPs present in PCs by microscale flow cytometry and investigated the interaction of these particles with CD4+ TLs. We cocultured MPs and PBMCs and determined the origin of the CD27 expressed on the surface of CD4+ TLs with the aid of two fluorochromes (BV510 for CD27 originating from MPs and BV786 for cellular CD27). Results: We showed that the binding of CD27- expressing MPs involved the CD70 molecule, which was also present on these MPs. Finally, the maintenance of CD27 expression on the surface of TLs by sorted CD27+ MPs led to activation levels lower than those observed with other types of MPs. Discussion: These results for CD27-expressing MPs and their CD70-mediated targeting open up new possibilities for immunotherapy based on the use of MPs to maintain a phenotype or to target immune cells, for example. Moreover, decreasing the levels of CD27-expressing MPs in transfused platelets might also increase the chances of success for anti-CD27 monoclonal immunotherapy.

Near-Infrared Spectroscopy Demonstrates the Benefit of Erythracytapheresis in Sickle Cell Disease Adult Patients with Cerebral Vasculopathy.

BACKGROUND: Cerebral vasculopathy can induce chronic cerebral hypoperfusion leading to stroke in patients with sickle cell disease (SCD) and is treated by blood exchange transfusion (BET). However, no prospective clinical study has demonstrated the benefit of BET in adults with SCD and cerebral vasculopathy. Near Infrared Spectroscopy (NIRS) is a recent non-invasive method complementary to Magnetic Resonance Imaging (MRI). We evaluated cerebral perfusion using NIRS during erythracytapheresis in patients with SCD with and without steno-occlusive arterial disease. METHODS: We conducted a monocentric, prospective study in 16 adults with SCD undergoing erythracytapheresis in 2014. Among them, 10 had cerebral steno-occlusive arterial disease. NIRS measured the relative amounts of oxyhemoglobin (OxyHb), deoxyhemoglobin (DeoxyHb) and total hemoglobin (Total Hb) in brain tissue and in muscle. RESULTS: In cerebral hemispheres associated with steno-occlusive arterial disease, we observed a significant increase of OxyHb and Total Hb during BET, without modification of DeoxyHb. CONCLUSION: Using NIRS during BET showed that BET improves cerebral perfusion in adult patients with SCD with cerebral vasculopathy.

Inflammatory markers and auto-Abs to type I IFNs in COVID-19 convalescent plasma cohort study.

BACKGROUND: COVID-19 convalescent plasma (CCP) contains neutralising anti-SARS-CoV-2 antibodies that may be useful as COVID-19 passive immunotherapy in patients at risk of developing severe disease. Such plasma from convalescent patients may also have additional immune-modulatory properties when transfused to COVID-19 patients. METHODS: CCP (n = 766) was compared to non-convalescent control plasma (n = 166) for soluble inflammatory markers, ex-vivo inflammatory bioactivity on endothelial cells, neutralising auto-Abs to type I IFNs and reported adverse events in the recipients. FINDINGS: CCP exhibited a statistically significant increase in IL-6 and TNF-alpha levels (0.531 ± 0.04 vs 0.271 ± 0.04; (95% confidence interval [CI], 0.07371-0.4446; p = 0.0061) and 0.900 ± 0.07 vs 0.283 ± 0.07 pg/mL; (95% [CI], 0.3097-0.9202; p = 0.0000829) and lower IL-10 (0.731 ± 0.07 vs 1.22 ± 0.19 pg/mL; (95% [CI], -0.8180 to -0.1633; p = 0.0034) levels than control plasma. Neutralising auto-Abs against type I IFNs were detected in 14/766 (1.8%) CCPs and were not associated with reported adverse events when transfused. Inflammatory markers and bioactivity in CCP with or without auto-Abs, or in CCP whether or not linked to adverse events in transfused patients, did not differ to a statistically significant extent. INTERPRETATION: Overall, CCP exhibited moderately increased inflammatory markers compared to the control plasma with no discernible differences in ex-vivo bioactivity. Auto-Abs to type I IFNs detected in a small fraction of CCP were not associated with reported adverse events or differences in inflammatory markers. Additional studies, including careful clinical evaluation of patients treated with CCP, are required in order to further define the clinical relevance of these findings. FUNDING: French National Blood Service-EFS, the Association "Les Amis de Rémi" Savigneux, France, the "Fondation pour la Recherche Médicale (Medical Research Foundation)-REACTing 2020".

Delayed haemolytic transfusion reaction in paediatric patients with sickle cell disease: A retrospective study in a French national reference centre.

Delayed haemolytic transfusion reaction (DHTR) is a life-threatening haemolytic anaemia following red blood cell transfusion in patients with sickle cell disease, with only scarce data in children. We retrospectively analysed 41 cases of DHTR in children treated between 2006 and 2020 in a French university hospital. DHTR manifested at a median age of 10.5 years, symptoms occurred a median of 8 days after transfusion performed for an acute event (63%), before surgery (20%) or in a chronic transfusion programme (17%). In all, 93% of patients had painful crisis. Profound anaemia (median 49 g/L), low reticulocyte count (median 140 ×109 /L) and increased lactate dehydrogenase (median 2239 IU/L) were observed. Antibody screening was positive in 51% of patients, and more frequent when there was a history of alloimmunisation. Although no deaths were reported, significant complications occurred in 51% of patients: acute chest syndrome (12 patients), cholestasis (five patients), stroke (two patients) and kidney failure (two patients). A further transfusion was required in 23 patients and corticosteroids were used in 21 to reduce the risk of additional haemolysis. In all, 13 patients subsequently received further transfusions with recurrence of DHTR in only two. The study affords a better overview of DHTR and highlights the need to establish guidelines for its management in children.

Red blood cell alloimmunisation in sickle cell disease patients in the Democratic Republic of the Congo.

OBJECTIVES: To determine the prevalence of red blood cell (RBC) alloimmunisation and alloantibody specificity in sickle cell disease (SCD) patients in Kisangani, Democratic Republic of Congo (DRC) in comparison with those followed at the Centre Hospitalier Régional (CHR) de la Citadelle of Liège (Belgium). BACKGROUND: Data regarding RBC alloimmunisation (immune response of the organism to foreign erythrocyte antigens, antigens that lack on its own RBC) in SCD patients are scarce in sub-Saharan Africa. METHODS: We conducted a multi-site-based cross-sectional study among 125 SCD patients at Kisangani and 136 at the CHR de la Citadelle of Liège. The diagnosis of SCD was confirmed by high-performance liquid chromatography. Alloantibodies were screened using the agglutination technique on gel cards and their specificity determined using 11 and/or 16 cell panels. Statistical analyses were carried out using SPSS. RESULTS: The prevalence of RBC alloimmunisation was 9.6% among SCD patients in Kisangani versus 22.8% in those of Liège. At Kisangani as well as at Liège, the median age of alloimmunised patients was higher than that of non-alloimmunised patients, 15.5 years (IQR:4.8-19.8) and 24 years (IQR:14-31) versus 10 years (IQR: 6.5-17) and 17 years (IQR:12-24), respectively. The median number of blood units was higher in both Kisangani and Liège immunised patients compared to non-immunised patients, 8 (IQR:5-11) versus 5 (IQR:3-13) and 41(IQR:6-93) versus 6.5(3-37) respectively. At Kisangani (N = 14), the most frequent antibodies were anti-D (28.6%) and anti-C versus anti-E (13.6%), anti-S (13.6%) and anti-Lea (11.4%) at Liège (N = 44). CONCLUSIONS: These findings stated that alloimmunisation is a common complication in SCD patients in the DRC. In the resource-limited setting of this country, blood transfusion with minimal ABO, D, C and E antigen matching in addition to the use of compatibility test could significantly reduce the incidence of this complication.

Anti-CD38 monoclonal antibody interference with blood compatibility testing: Differentiating isatuximab and daratumumab via functional epitope mapping.

BACKGROUND: There are two FDA-approved anti-CD38 monoclonal antibodies for treatment of multiple myeloma: isatuximab and daratumumab. Owing to expression of CD38 on reagent red blood cells (RBCs), these antibodies interfere with indirect antiglobulin tests (IATs). We sought to understand differences in such interference by performing binding experiments. STUDY DESIGN AND METHODS: In vitro experiments to compare the binding to RBCs of isatuximab and daratumumab alone or in the presence of a mouse anti-human CD38 antibody (HB-7 or AT13/5) or a nicotinamide adenine dinucleotide-analog CD38 inhibitor were performed and quantified by flow cytometry, imaging, mass spectrometry, surface plasmon resonance, and LigandTracer technologies. Serologic testing was performed on plasma samples spiked with isatuximab or daratumumab. RESULTS: CD38 expressed on RBCs can be directly bound by daratumumab, whereas isatuximab requires a co-factor, such as HB-7, AT13/5, or a CD38 inhibitor, suggesting that the isatuximab epitope on RBCs is masked in vitro. Daratumumab samples more frequently showed interference and had stronger reactions than isatuximab samples. Dithiothreitol treatment was equally effective in mitigating the interference caused by either drug. DISCUSSION: Both isatuximab and daratumumab interfere with IATs but at different magnitudes, reflecting distinct binding to CD38 on RBCs. From the binding studies, we conclude that the isatuximab epitope on RBCs is masked in vitro and binding requires a certain CD38 conformation or co-factor. This circumstance may explain why interference is seen only in a subset of patients receiving isatuximab when compared with interference seen in most patients on daratumumab therapy.