Maturation and persistence of the anti-SARS-CoV-2 memory B cell response.

Memory B cells play a fundamental role in host defenses against viruses, but to date, their role has been relatively unsettled in the context of SARS-CoV-2. We report here a longitudinal single-cell and repertoire profiling of the B cell response up to 6 months in mild and severe COVID-19 patients. Distinct SARS-CoV-2 spike-specific activated B cell clones fueled an early antibody-secreting cell burst as well as a durable synchronous germinal center response. While highly mutated memory B cells, including pre-existing cross-reactive seasonal Betacoronavirus-specific clones, were recruited early in the response, neutralizing SARS-CoV-2 RBD-specific clones accumulated with time and largely contributed to the late, remarkably stable, memory B cell pool. Highlighting germinal center maturation, these cells displayed clear accumulation of somatic mutations in their variable region genes over time. Overall, these findings demonstrate that an antigen-driven activation persisted and matured up to 6 months after SARS-CoV-2 infection and may provide long-term protection.

Analysis of mRNA vaccination-elicited RBD-specific memory B cells reveals strong but incomplete immune escape of the SARS-CoV-2 Omicron variant.

The SARS-CoV-2 Omicron variant can escape neutralization by vaccine-elicited and convalescent antibodies. Memory B cells (MBCs) represent another layer of protection against SARS-CoV-2, as they persist after infection and vaccination and improve their affinity. Whether MBCs elicited by mRNA vaccines can recognize the Omicron variant remains unclear. We assessed the affinity and neutralization potency against the Omicron variant of several hundred naturally expressed MBC-derived monoclonal IgG antibodies from vaccinated COVID-19-recovered and -naive individuals. Compared with other variants of concern, Omicron evaded recognition by a larger proportion of MBC-derived antibodies, with only 30% retaining high affinity against the Omicron RBD, and the reduction in neutralization potency was even more pronounced. Nonetheless, neutralizing MBC clones could be found in all the analyzed individuals. Therefore, despite the strong immune escape potential of the Omicron variant, these results suggest that the MBC repertoire generated by mRNA vaccines still provides some protection against the Omicron variant in vaccinated individuals.

Genotypic and phenotypic characteristics of Escherichia coli involved in transfusion-transmitted bacterial infections: implications for preventive strategies.

BACKGROUND: Transfusion-transmitted bacterial infections (TTBIs) are the main residual infectious complications of transfusions. Escherichia coli and platelet (PLT) concentrates may be epidemiologically associated, leading to severe, if not lethal, TTBIs. We investigated the genotypic and phenotypic reasons for this clinically deleterious combination. STUDY DESIGN AND METHODS: We investigated a French national E. coli strain collection related to six independent episodes of TTBIs. Their phenotypic characterizations included antibiotic susceptibility testing, growth testing under different culture conditions, serum survival assays, and virulence in a sepsis mouse model. Their genotypic characterizations included polymerase chain reaction phylotyping, whole genome sequencing, and a subsequent in silico analysis. RESULTS: We highlighted a selection process of highly extraintestinal virulent strains, mainly belonging to the B2 phylogroup, adapted to the hostile environment (high citrate concentration and a bactericidal serum effect) of apheresis-collected platelet concentrates (PCs). Compared to controls, the E. coli TTBI strains grew faster in the PCs due to a superior ability to capture iron. The in vitro growth performances were highly compatible with blood-derived product real-life conditions, including storage conditions and delays. The consistent serum resistance of TTBI strains promotes their survival in both the donor's and the receiver's blood and in the PCs. CONCLUSION: This study pointed out that E. coli strains responsible for TTBI exhibit very specific traits. They belong to the extraintestinal pathogenic phylogroups and have a high intrinsic virulence. They can be resistant to complement, capture iron, and grow in the apheresis-collected PCs. These findings therefore support the reinforcement of the postdonation information.

Phenotypic differences of CD4(+) T cells in response to red blood cell immunization in transfused sickle cell disease patients.

Alloimmunization against red blood cells (RBCs) is the main immunological risk associated with transfusion in patients with sickle cell disease (SCD). However, about 50-70% of SCD patients never get immunized despite frequent transfusion. In murine models, CD4(+) T cells play a key role in RBC alloimmunization. We therefore explored and compared the CD4(+) T-cell phenotypes and functions between a group of SCD patients (n = 11) who never became immunized despite a high transfusion regimen and a group of SCD patients (n = 10) who had become immunized (at least against Kidd antigen b) after a low transfusion regimen. We studied markers of CD4(+) T-cell function, including TLR, that directly control lymphocyte function, and their spontaneous cytokine production. We also tested responders for the cytokine profile in response to Kidd antigen b peptides. Low TLR2/TLR3 expression and, unexpectedly, strong expression of CD40 on CD4(+) T cells were associated with the nonresponder status, whereas spontaneous expression of IL-10 by CD4(+) T cells and weak Tbet expression were associated with the responder status. A Th17 profile was predominant in responders when stimulated by Jb(k) . These findings implicate CD4(+) T cells in alloimmunization in humans and suggest that they may be exploited to differentiate responders from nonresponders.

Red blood cell alloimmunization is influenced by the delay between Toll-like receptor agonist injection and transfusion.

Murine models of red blood cell transfusion show that inflammation associated with viruses or methylated DNA promotes red blood cell alloimmunization. In vaccination studies, the intensity of antigen-specific responses depends on the delay between antigen and adjuvant administration, with a short delay limiting immune responses. In mouse models of alloimmunization, the delay between the injection of Toll-like receptor agonists and transfusion is usually short. In this study, we hypothesized that the timing of Toll-like receptor 3 agonist administration affects red blood cell alloimmunization. Poly(I:C), a Toll-like receptor 3 agonist, was administered to B10BR mice at various time points before the transfusion of HEL-expressing red blood cells. For each time point, we measured the activation of splenic HEL-presenting dendritic cells, HEL-specific CD4(+) T cells and anti-HEL antibodies in serum. The phenotype of activated immune cells depended on the delay between transfusion and Toll-like receptor-dependent inflammation. The production of anti-HEL antibodies was highest when transfusion occurred 7 days after agonist injection. The proportion of HEL-presenting CD8α(+) dendritic cells producing interleukin-12 was highest in mice injected with poly(I:C) 3 days before transfusion. Although the number of early-induced HEL-specific CD4(+) T cells was similar between groups, a high proportion of these cells expressed CD134, CD40 and CD44 in mice injected with poly(I:C) 7 days before transfusion. This study clearly shows that the delay between transfusion and Toll-like receptor-induced inflammation influences the immune response to transfused red blood cells.

Emergence of long-lived autoreactive plasma cells in the spleen of primary warm auto-immune hemolytic anemia patients treated with rituximab.

Primary warm autoimmune hemolytic anemia (wAIHA) is a rare autoimmune disease in which red blood cells are eliminated by IgG autoantibodies. We analyzed the antibody-secreting cells in the spleen and the peripheral blood of wAIHA patients in various contexts of treatment. Plasmablasts were observed in peripheral blood of newly diagnosed wAIHA patients and, accordingly, active germinal center reactions were present in the spleen of patients receiving short-term corticosteroid therapy. Long-term corticosteroid regimens markedly reduced this response while splenic plasma cells were able to persist, a fraction of them secreting anti-red blood cell IgG in vitro. In wAIHA patients treated by rituximab and who underwent splenectomy because of treatment failure, plasma cells were still present in the spleen, some of them being autoreactive. By using a set of diagnostic genes that allowed us to assess the plasma cell maturation stage, we observed that these cells displayed a long-lived program, differing from the one of plasma cells from healthy donors or from wAIHA patients with various immunosuppressant treatments, and more similar to the one of normal long-lived bone-marrow plasma cells. Interestingly, an increased level of B-cell activating factor (BAFF) was observed in the supernatant of spleen cell cultures from such rituximab-treated wAIHA patients. These results suggest, in line with our previous report on primary immune thrombocytopenia, that the B-cell depletion induced by rituximab promoted a suitable environment for the maturation and survival of auto-immune long-lived plasma cells in the spleen.

Red blood cell Thomsen-Friedenreich antigen expression and galectin-3 plasma concentrations in Streptococcus pneumoniae-associated hemolytic uremic syndrome and hemolytic anemia.

BACKGROUND: Pneumococcal hemolytic uremic syndrome (P-HUS) is a rare but severe complication of invasive pneumococcal disease (IPD) in young children. Consensual biologic diagnosis criteria are currently lacking. STUDY DESIGN AND METHODS: A prospective study was conducted on 10 children with culture-confirmed IPD. Five presented with full-blown P-HUS, three had an incomplete form with hemolytic anemia and mild or no uremia (P-HA), and two had neither HUS nor HA. Thomsen-Friedenreich (T), Th, and Tk cryptantigens and sialic acid expression were determined on red blood cells (RBCs) with peanut (PNA), Glycine soja (SBA), Bandeiraea simplicifolia II, and Maackia amurensis lectins. Plasma concentrations of the major endogenous T-antigen-binding protein, galectin-3 (Gal-3), were analyzed. RESULTS: We found that RBCs strongly reacted with PNA and SBA lectins in all P-HUS and P-HA patients. Three P-HUS and three P-HA patients showed also concomitant Tk activation. Direct antiglobulin test (DAT) was positive in three P-HUS (one with anti-C3d and two with anti-IgG) and two P-HA patients (one with anti-C3d and one with anti-IgG). RBCs derived from the two uncomplicated IPD patients reacted with PNA but not with SBA lectin. Gal-3 plasma concentrations were increased in all P-HUS patients. CONCLUSIONS: The results indicate high levels of neuraminidase activity and desialylation in both P-HUS and P-HA patients. T-antigen activation is more sensitive than DAT for P-HUS diagnosis. Combining PNA and SBA lectins is needed to improve the specificity of T-antigen activation. High concentrations of Gal-3 in P-HUS patients suggest that Gal-3 may contribute to the pathogenesis of P-HUS.

Red blood cell alloimmunization in sickle cell disease: pathophysiology, risk factors, and transfusion management.

Red blood cell transfusions have reduced morbidity and mortality for patients with sickle cell disease. Transfusions can lead to erythrocyte alloimmunization, however, with serious complications for the patient including life-threatening delayed hemolytic transfusion reactions and difficulty in finding compatible units, which can cause transfusion delays. In this review, we discuss the risk factors associated with alloimmunization with emphasis on possible mechanisms that can trigger delayed hemolytic transfusion reactions in sickle cell disease, and we describe the challenges in transfusion management of these patients, including opportunities and emerging approaches for minimizing this life-threatening complication.

Characteristics and outcome of warm autoimmune hemolytic anemia in adults: New insights based on a single-center experience with 60 patients.

Warm autoimmune hemolytic anemia (wAIHA) is a rare autoimmune disease with poorly known natural history and management remaining mainly empirical. To better describe the characteristics and outcome of wAIHA in adults, we performed a single-center cohort study of patients diagnosed with wAIIHA from 2001 to 2012 in our center. Sixty patients (50% women) were included, the mean age at the time of wAIHA onset was 54 ± 23 years. wAIHA was considered "primary" for 21 patients (35%) and was associated with an underlying disorder in 39 (65%), including mainly lymphoproliferative disorders and systemic lupus. All patients but two needed treatment and received corticosteroids, with an overall initial response rate of 87%. However, 63% of the patients were corticosteroid-dependent and 56% required at least one second-line treatment including mainly rituximab (n = 19). At the time of analysis, after a mean follow-up of 46 months, 28 patients (47%) were in remission and off treatment and 5 (8%) had died. The presence of an underlying lymphoproliferative disorder was associated with reduced response to corticosteroids and increased need for second-line therapy. In conclusion, in the last decade and compared to a previous series from our center, the rate of secondary wAIHA has increased and the use of rituximab has emerged as the preferred second-line treatment and corticosteroid-sparing strategy; the overall mortality has significantly decreased (8 vs. 18%).

Partial dysfunction of Treg activation in sickle cell disease.

Sickle cell disease (SCD) is a chronic inflammatory disease associated with multiple organ damage, chronic anemia, and infections. SCD patients have a high rate of alloimmunization against red blood cells (RBCs) following transfusion and may develop autoimmune diseases. Studies in mouse models have suggested that regulatory T cells (Treg) play a role in alloimmunization against RBC antigens. We characterized the phenotype and function of the Treg cell population in a homogeneous cohort of transfused SCD patients. We found that the distribution of Treg subpopulations differed significantly between SCD patients and healthy blood donors. SCD patients have a particular Treg phenotype, with strong CTLA-4 and CD39 expression and weak HLA-DR and CCR7 expression. Finally, we show that this particular phenotype is related to SCD rather than alloimmunization status. Indeed, we observed no difference in Treg phenotype or function in vitro using autologous feeder cells between strong and weak responders to alloimmunization.

RHCE*cE734C allele encodes an altered c antigen and a suppressed E antigen not detected with standard reagents.

BACKGROUND: The RH blood group system has many RHCE variant alleles that have arisen through gene conversion or nucleotide changes. Two probands, with red blood cells (RBCs) that were D+C+E-c+(w) e+ were sent to our laboratories to resolve the weak c expression. STUDY DESIGN AND METHODS: Hemagglutination tests were performed by automated and manual procedures. Genomic DNA analysis was performed by sequencing of Exons 1 to 10 of RHCE and RHD. RESULTS: The probands' RBCs did not react with standard monoclonal anti-E reagents from Bio-Rad, Diagast, DiaMed, Immucor, Ortho, and Quotient. The RBCs reacted variably with anti-c reagents from Diagast, DiaMed, Immucor, or Ortho and did not react with the Quotient anti-c reagent. Surprisingly, sequencing results of RHCE showed the presence of C/G at Position 676 (E/e polymorphism) and the association of the E polymorphism with a 734T>C transition in Exon 5 of the RHCE, encoding a Leu245Pro amino acid substitution in the mature RhcE polypeptide. Replacement of leucine 245 by proline in the eighth transmembrane domain of the RhcE protein may have a steric effect on the protein such that most anti-E reagents do not bind and the interaction between anti-c and c antigen is also affected. CONCLUSION: We report a novel RHCE*cE allele, RHCE*cE734C, which was assigned the provisional ISBT allele name RHCE*cE.14 or RHCE*03.14. It was found in two probands whose RBCs had weakened c expression and typed E- with conventional anti-E reagents. These data, once again, highlight the fact that the genotype does not always reflect the phenotype.

IgA-mediated human autoimmune hemolytic anemia as a result of hemagglutination in the spleen, but independent of complement activation and FcαRI.

Autoimmune hemolytic anemia (AIHA) due to warm-acting IgA autoantibodies is rare. We explored the pathogenic mechanisms underlying destruction of red blood cells (RBCs) in a patient with severe AIHA mediated exclusively by polymeric immunoglobulin A (pIgA) anti-Band 3 autoantibodies. The follow-up period was 17 months. RBCs were not destroyed by complement activation as no deposition of complement was observed on the patient's RBCs. pIgA eluted from the patient's RBCs did not induce RBC destruction through phagocytosis by monocytes or antibody-dependent cell-mediated cytotoxicity by natural killer cells. Induction of eryptosis (ie, RBC apoptosis) due to direct alteration of the RBC membrane by pIgA autoantibodies was also excluded. By contrast, upon incubation with pIgA-opsonized RBCs, substantial RBC membrane transfers (ie, trogocytosis) to monocytes were observed that might contribute to RBC immune destruction. This effect was poorly inhibited by blockers of Fc receptors, excluding a major contribution of FcαRI to this process. Histologic analysis revealed a massive accumulation of agglutinated RBCs with little sign of erythrophagocytosis in the spleen. These results, together with the efficacy of splenectomy 17 months after AIHA onset, suggest that the trapping and subsequent sequestration of agglutinated RBCs in the spleen are the principal pathogenic mechanisms of pIgA-mediated AIHA.

Delayed hemolytic transfusion reaction in children with sickle cell disease.

BACKGROUND: Transfusion is a cornerstone of the management of sickle cell disease but carries a high risk of hemolytic transfusion reaction, probably because of differences in erythrocyte antigens between blood donors of European descent and patients of African descent. Patients may experience hemolytic transfusion reactions that are delayed by from a few days to two weeks and manifest as acute hemolysis (hemoglobinuria, jaundice, and pallor), symptoms suggesting severe vaso-occlusive crisis (pain, fever, and acute chest syndrome), and profound anemia, often with reticulocytopenia. This case-series study aims to describe the main characteristics of this syndrome, to discuss its pathophysiology, and to propose a management strategy. DESIGN AND METHODS: We identified 8 pediatric cases of delayed hemolytic transfusion reactions between 2006 and 2009 in the database of the Necker Hospital, France. All patients had received cross-matched red cell units compatible in the ABO, RH, and KEL systems. We reviewed the medical charts in the computerized blood transfusion databases. All patients were admitted to the intensive care unit. We progressively adopted the following strategy: intravenous immunoglobulins, and darbopoietin alpha when the reticulocyte count was below 150×10(9)/L, without further blood transfusion during the acute episode unless absolutely necessary. RESULTS: The median time between the transfusion and the diagnosis of delayed hemolytic transfusion reaction was six days. All patients had severe bone pain; all but one had a high-grade fever. Five patients had hemoglobin levels less than than 4 g/dL and 3 had reticulocytopenia. In 5 patients, no new antibody was found; one patient had weakly reactive antibodies. Only 2 patients had new allo-antibodies possibly responsible for the delayed hemolytic reaction. CONCLUSIONS: The initial symptoms of delayed hemolytic transfusion reaction were complex and mimicked other complications of sickle cell disease. In most of our cases, no new antibody was identified, which underlines the complexity of the pathophysiology of this syndrome.

Partial C antigen in sickle cell disease patients: clinical relevance and prevention of alloimmunization.

BACKGROUND: Partial Rh antigens have been widely described in black individuals. Carriers are prone to immunization when exposed to the normal antigens. In sickle cell disease (SCD), patient alloimmunization is a major cause of transfusion failure. The potential of individuals with partial C antigen to make anti-C has not been investigated. We sought partial C status and anti-C production in a cohort of SCD patients with the C+ phenotype, to determine whether exposure to normal C antigen should be avoided. STUDY DESIGN AND METHODS: We constituted a cohort of 177 randomly selected SCD patients expressing C antigen. We screened for (C)ce(s) and R(N) haplotypes, presumably associated with partial C antigen in Afro-Caribbeans, and we recorded the number of transfused C+ red blood cell (RBC) units, immunization status, and extended phenotype. RESULTS: Forty-nine patients carried abnormal C antigen, deduced from the presence of (C)ce(s) and/or R(N), not compensated by a normal RHC allele in trans. Among patients with partial C phenotype exposed repeatedly to C+ RBCs, 30% produced anti-C. Two patients experienced hemolysis. In our hospital, with 22% of SCD patients expressing C, prevention of anti-C immunization for all individuals with partial C antigen would require a 7% increase in the use of C- RBC units. These RBCs are already in short supply for SCD patients who are C-. CONCLUSION: This study demonstrates the need to detect partial C within C+ SCD patients and to prevent immunization. A larger number of Afro-Caribbeans donors is needed to provide these patients with C- RBCs.

Delayed hemolytic transfusion reaction in sickle cell disease patients: evidence of an emerging syndrome with suicidal red blood cell death.

BACKGROUND: Delayed hemolytic transfusion reaction (DHTR) is a life-threatening complication in sickle cell disease (SCD) characterized by recurrence of disease complications, recipient red blood cell (RBC) destruction, and frequently no detectable antibody. Phosphatidylserine (PS) exposure signs suicidal RBC death or eryptosis and is involved in vasoocclusive crisis (VOC). STUDY DESIGN AND METHODS: Transfusion was monitored in 48 SCD patients for up to 20 days. PS exposure was evaluated in vivo on patient RBCs (PS-RBCs) at five time points and in vitro after incubation of donor RBCs with pretransfusion plasma. RESULTS: Three VOC patients displayed DHTR with recurrent SCD features and no detectable antibody in two cases. In vitro, PS-RBC percentage was significantly increased by incubating donor RBCs with pretransfusion plasma samples from DHTR patients with no detectable antibody. No such increase was observed with samples from other patients. This result indicates that donor RBCs may be damaged by the environment of SCD patients, increasing the physiologic clearance of apoptotic RBCs. In vivo, PS-RBC percentage increased in all three cases after destruction of transfused RBCs, indicating that DHTR induces PS-RBCs and, possibly, subsequent VOC and autologous RBC destruction. CONCLUSION: This study clearly demonstrates that DHTR can occur in the absence of detectable antibody. In these cases, a mechanism of excessive eryptosis is proposed.

Biological parameters predictive of percent dense red blood cell decrease under hydroxyurea.

BACKGROUND: Dense red blood cells (DRBCs) are associated with chronic clinical manifestations of sickle-cell-disease (SCD). Hydroxyurea (HU) decreases the percent (%) DRBCs, thereby improving its therapeutic benefits, especially the prevention of SCD clinical complications, but parameters influencing %DRBCs remain unknown. The purpose of this study was to determine predictive biological parameters of %DRBC decline under HU. METHODS: Factors affecting the %DRBC decrease in SCD patients HU-treated for ≥6 months were analyzed. Biological parameters and the %DRBCs were determined before starting HU and after ≥6 months of HU intake. Bivariate analyses evaluated the impact of each biological parameter variation on %DRBC changes under treatment. Multivariate analyses assessed the correlations between the decreased %DRBCs and biological parameters. RESULTS: The %DRBCs declined by 40.95% after ≥6 months on HU. That decrease was associated with less hemolysis, however in several analyses on this group of patients we did not find a statistically significant correlation between decrease in %DRBCs and increase in HbF. Initial %DRBC values were the most relevant parameter to predict %DRBC decline. CONCLUSION: Our results strengthen the known HU efficacy in SCD management statistically independently of the classical HbF biological response. Decreasing %DRBCs is essential to limiting chronic SCD symptoms related to DRBCs and predictive factors might help prevent those manifestations. The results of this study provide new perspectives on indication for HU use, i.e., to prevent SCD-induced organ damage.