How to avoid the problem of erythrocyte alloimmunization in sickle cell disease.

Erythrocyte alloimmunization is a major barrier to transfusion in sickle cell disease (SCD) because it can lead to transfusion deadlock and the development of life-threatening hemolytic transfusion reactions (HTRs). Several risk factors have been identified, such as blood group polymorphism in these patients of African ancestry frequently exposed to antigens they do not carry and an inflammatory clinical state of the disease. The most important preventive measure is prophylactic red blood cell antigen matching, and there is a consensus that matching for Rh (D, C, E, c, e) and K antigens should be performed for all SCD patients. However, some patients are high responders and more at risk of developing antibodies and HTRs. For these patients, the extension of matching to other blood groups, including variant antigens of the RH blood group, the use of genotyping rather than serology to characterize significant blood groups, and the prophylactic administration of immunosuppressive treatments remain a matter of debate due to low levels of certainty concerning their effects and the difficulty of determining which patients, other than those already immunized, are at high risk. These issues were recently addressed by a panel of experts established by the American Society of Hematology. Here, we review and stratify the various interventions for preventing alloimmunization, based on the literature and our experience and taking into account the obstacles to their implementation and any future developments required.

Early Myeloid Derived Suppressor Cells (eMDSCs) Are Associated With High Donor Myeloid Chimerism Following Haploidentical HSCT for Sickle Cell Disease.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a widely available curative option for patients with sickle cell disease (SCD). Our original non-myeloablative haplo-HSCT trial employing post-transplant (PT) cyclophosphamide had a low incidence of GVHD but had high rejection rates. Here, we aimed to evaluate immune reconstitution following haplo-HSCT and identify cytokines and cells associated with graft rejection/engraftment. 50 cytokines and 10 immune cell subsets were screened using multiplex-ELISA and flow cytometry, respectively, at baseline and PT-Days 30, 60, 100, and 180. We observed the most significant differences in cytokine levels between the engrafted and rejected groups at PT-Day 60, corresponding with clinical findings of secondary graft rejection. Of the 44 cytokines evaluated, plasma concentrations of 19 cytokines were different between the two groups at PT-Day 60. Factor analysis suggested two independent factors. The first factor (IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, and TGFb1 contributed significantly) was strongly associated with engraftment with OR = 2.7 (95%CI of 1.4 to 5.4), whereas the second factor (GROa and IL-18 contributed significantly) was not significantly associated with engraftment. Sufficient donor myeloid chimerism (DMC) is critical for the success of HSCT; here, we evaluated immune cells among high (H) DMC (DMC≥20%) and low (L) DMC (DMC<20%) groups along with engrafted and rejected groups. We found that early myeloid-derived suppressor cell (eMDSC) frequencies were elevated in engrafted patients and patients with HDMC at PT-Day 30 (P< 0.04 & P< 0.003, respectively). 9 of 20 patients were evaluated for the source of eMDSCs. The HDMC group had high mixed chimeric eMDSCs as compared to the LDMC group (P< 0.00001). We found a positive correlation between the frequencies of eMDSCs and Tregs at PT-Day 100 (r=0.72, P <0.0007); eMDSCs at BSL and Tregs at PT-Day 100 (r=0.63, P <0.004). Of 10 immune regulatory cells and 50 cytokines, we observed mixed chimeric eMDSCs and IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, TGFb1 as potential hits which could serve as prognostic markers in predicting allograft outcome towards engraftment following haploidentical HSCT employing post-transplant cyclophosphamide. The current findings need to be replicated and further explored in a larger cohort.

Effect of lysed and non-lysed sickle red cells on the activation of NLRP3 inflammasome and LTB4 production by mononuclear cells.

OBJECTIVE AND DESIGN: This study tested the hypothesis that sickle red blood cell (SS-RBC) can induce inflammasome NLRP3 components gene expression in peripheral blood mononuclear cells (PBMCs) as well as interleukin-1ß (IL-1ß) and leukotriene B4 (LTB4) production. Additionally, we investigated the effect of hydroxyurea (HU) treatment in these inflammatory markers. METHODS: PBMCs from healthy donors (AA-PBMC) were challenged with intact and lysed RBCs from SCA patients (SS-RBC) and from healthy volunteers (AA-RBC). NLRP3, IL-1ß, IL-18 and Caspase-1 gene expression levels were assessed by quantitative PCR (qPCR). IL-1ß protein levels and LTB4 were measured by ELISA. RESULTS: We observed that lysed SS-RBC induced the expression of inflammasome NLRP3 components, but this increase was more prominent for CASP1 and IL18 expression levels. Moreover, we observed that intact SS-RBC induced higher production of IL-1ß and LTB4 than lysed SS-RBC. Although SCA patients treated with HU have a reduction in NLRP3 gene expression and LTB4 production, this treatment did not modulate the expression of other inflammasome components or IL-1ß production. CONCLUSIONS: Thus, our data suggest that caspase-1, IL-1ß and IL-18 may contribute to the inflammatory status observed in SCA and that HU treatment may not interfere in this inflammatory pathway.

Type I interferon is induced by hemolysis and drives antibody-mediated erythrophagocytosis in sickle cell disease.

Patients with sickle cell disease (SCD) suffer from intravascular hemolysis-associated vascular injury and tissue damage. Classical monocytes (CMo), which are the most abundant of circulating monocytes, are activated in SCD, but the cause and consequences of activation remain incompletely understood. We found a positive correlation between total plasma heme levels and circulating interferon-α (IFN-α) in patients with SCD along with upregulation of the type I IFN (IFN-I) inducible genes in sort-purified SCD patients' CMo by transcriptome analysis. We demonstrated that hemolysis led to IFN-I expression, predominantly by mouse liver monocyte and macrophages (Mⲫ), primarily through Tank kinase binding 1 (TBK1)/IκB kinase-ε (IKKε) but not TLR4. In response to hemolysis-induced IFN-I, mouse CMo migrated to the liver and differentiated into monocyte-derived Mⲫ, increasing their numbers by sixfold with acute hemin treatment. Hemolysis-driven IFN-I activity also led to the induction of Fc receptor CD64 expression on monocyte and Mⲫ populations, enhancing alloantibody-mediated erythrophagocytosis in SCD both in vivo in mice and in in vitro human cultures. Altogether, these data demonstrate IFN-I response to hemolysis as a novel activation pathway in monocytes and Mⲫ in SCD, opening the possibility for development of IFN-I-based diagnostics and therapeutics against alloantibody-mediated erythrophagocytosis.

Antigen matching for transfusion support in Brazilian female patients with sickle cell disease to reduce RBC alloimmunization.

BACKGROUND: Red blood cell (RBC) alloimmunization is a complication of patients with sickle cell disease (SCD) and it has a greater impact on pregnancy, leading to a risk of hemolytic disease of the newborn and reducing blood availability for pregnant women. This study proposed to evaluate antigen matching transfusion protocols, aiming to reduce RBC alloimmunization in Brazilian female patients with SCD. METHODS: Samples from female patients with SCD (153) and self-declared Afro-Brazilian donors (307) were genotyped for RBC antigens and RH variants were investigated. The transfusion needs of patients during 1-year period and the number of compatible donors were assessed using three antigen-matching transfusion protocols: prophylactic CEK antigen-matched RBCs, prophylactic extended antigen-matched RBCs, and extended-matched red blood cells (RBCs) only for alloimmunized patients. In addition, RH molecular matching has been proposed for patients carrying variant RHCE. RESULTS: Provision of CEK antigen-matched donors would have been possible in 92.4% of transfusion events while provision of prophylactic extended antigen-matched RBCs would cover 88.7% of the transfusion events. Extended antigen matching for alloimmunized patients would be efficient in 99% of the cases. The presence of partial D in 10 patients increased the need of D-negative donors. Compatible donors could be enough for four of the five patients with altered RHCE genotypes in both alleles. CONCLUSION: In Brazilians, screening African descent donors allows the implementation of prophylactic CEK and extended antigen-matching transfusion protocols to female patients with SCD to reduce RBC alloimmunization; however, the supply of compatible blood can be impaired for patients with Rh variants.

Screening for new red blood cell alloantibodies after transfusion in patients with sickle cell disease.

BACKGROUND: Patients with sickle cell disease (SCD) are frequent recipients of red blood cell (RBC) transfusions and are at risk for RBC alloimmunization. RBC alloimmunization is diagnosed by identifying RBC alloantibodies as part of pre-transfusion testing, but this testing fails to detect alloantibodies that have evanesced. It may be beneficial to screen for new RBC alloantibody development after transfusion before possible antibody evanescence. STUDY DESIGN AND METHODS: Our institution started a new initiative for episodically transfused patients with SCD to obtain at least one antibody screen 2-6 months after transfusion as part of their clinical care. A database was created to prospectively track all transfused patients for 1 year and their post-transfusion antibody screen results. Patients received prophylactically CEK-matched RBC units. RESULTS: During the study year, 138 patients with SCD received a total of 242 RBC transfusions. Patients with a history of an RBC alloantibody (n = 13, 9.4%) had previously received more RBC units than non alloimmunized patients (median 11 vs. 2 RBC units, p = .0002). A total of 337 post-transfusion antibody screens were obtained in 127 patients (92.0%) with 110 patients (79.7%) having at least one antibody screen 2-6 months post-transfusion. With this prospective testing, two new RBC alloantibodies (anti-C and -M) were identified in two patients. CONCLUSION: It is feasible to test for new RBC alloantibody development in most episodically transfused patients with SCD as part of their routine care. The yield of this screening appears low with CEK matching, but it could still provide important information for individual patients.

Agonistic Anti-CD40 Antibody Triggers an Acute Liver Crisis With Systemic Inflammation in Humanized Sickle Cell Disease Mice.

Sickle cell disease (SCD) is an inherited hemolytic disorder, defined by a point mutation in the ß-globin gene. Stress conditions such as infection, inflammation, dehydration, and hypoxia trigger erythrocyte sickling. Sickled red blood cells (RBCs) hemolyze more rapidly, show impaired deformability, and increased adhesive properties to the endothelium. In a proinflammatory, pro-coagulative environment with preexisting endothelial dysfunction, sickled RBCs promote vascular occlusion. Hepatobiliary involvement related to the sickling process, such as an acute sickle hepatic crisis, is observed in about 10% of acute sickle cell crisis incidents. In mice, ligation of CD40 with an agonistic antibody leads to a macrophage activation in the liver, triggering a sequence of systemic inflammation, endothelial cell activation, thrombosis, and focal ischemia. We found that anti-CD40 antibody injection in sickle cell mice induces a systemic inflammatory and hemodynamic response with accelerated hemolysis, extensive vaso-occlusion, and large ischemic infarctions in the liver mimicking an acute hepatic crisis. Administration of the tumor necrosis factor-α (TNF-α) blocker, etanercept, and the heme scavenger protein, hemopexin attenuated end-organ damage. These data collectively suggest that anti-CD40 administration offers a novel acute liver crisis model in humanized sickle mice, allowing for evaluation of therapeutic proof-of-concept.

Immunological Hallmarks of Inflammatory Status in Vaso-Occlusive Crisis of Sickle Cell Anemia Patients.

Sickle Cell Anemia (SCA) is the most common genetic disorder around the world. The mutation in the ß-globin gene is responsible for a higher hemolysis rate, with further involvement of immunological molecules, especially cytokines, chemokines, growth factors, and anaphylatoxins. These molecules are responsible for inducing and attracting immune cells into circulation, thus contributing to increases in leukocytes and other pro-inflammatory mediators, and can culminate in a vaso-occlusive crisis (VOC). This study aimed to characterize the levels of these molecules in SCA patients in different clinical conditions in order to identify potential hallmarks of inflammation in these patients. An analytical prospective study was conducted using the serum of SCA patients in steady-state (StSt; n = 27) and VOC (n = 22), along with 53 healthy donors (HD). Samples from the VOC group were obtained on admission and on discharge, in the convalescent phase (CV). Levels of chemokines (CXCL8, CXCL10, CL2, CLL3, CCL4, CL5, and CCL11), cytokines (IL-1ß, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p70, IL-13, IL-17A, TNF-α, and IFN-γ) and growth factors (VEGF, FGFb, PDGF-BB, GM-CSF, and G-CSF) were measured using a Luminex assay, and anaphylatoxins (C3a, C4a, and C5a) were measured using Cytometric Bead Array. SCA patients in StSt showed a pro-inflammatory profile, and were indicated as being higher producers of CCL2, IL-1ß, IL-12p70, IFN-γ, IL-17A, and GM-CSF, while VOC is highlighted by molecules IL-4 and IL-5, but also IL-2, IL-7, PDGF-BB, and G-CSF. PDGF-BB and IL-1ra seemed to be two important hallmarks for the acute-to-chronic stage, due to their significant decrease after crisis inflammation and statistical difference in VOC and CV groups. These molecules show higher levels and a strong correlation with other molecules in VOC. Furthermore, they remain at higher levels even after crisis recovery, which suggest their importance in the role of inflammation during crisis and participation in immune cell adhesion and activation. These results support a relevant role of cytokines, neutrophil and monocytes, since these may act as markers of VOC inflammation in SCA patients.

Non-myeloablative human leukocyte antigen-matched related donor transplantation in sickle cell disease: outcomes from three independent centres.

Non-myeloablative haematopoietic progenitor cell transplantation (HPCT) from matched related donors (MRD) has been increasingly utilized in sickle cell disease (SCD). A total of 122 patients received 300 cGy of total body irradiation (TBI), alemtuzumab, unmanipulated filgrastim-mobilized peripheral blood HPC and sirolimus. The median follow-up was four years; median age at HPCT was 29 years. Median neutrophil and platelet engraftment occurred on day 22 and 19 respectively; 41 patients required no platelet transfusions. Overall and sickle-free survival at one and five years were 93% and 85% respectively. Age, sex, pre-HPCT sickle complications, ferritin and infused HPC numbers were similar between graft failure and engrafted patients. Mean donor myeloid chimaerism at one and five years post HPCT were 84% and 88%, and CD3 was 48% and 53% respectively. Two patients developed grade 1 and 2 skin graft-versus-host disease (GVHD) with no chronic GVHD. Median days of recipients taking immunosuppression were 489; 83% of engrafted patients have discontinued immunosuppression. Haemoglobin, haemolytic parameters and hepatic iron levels improved post HPCT. Pulmonary function testing, hepatic histology and neurovascular imaging remained stable, suggesting cessation of further sickle-related injury. Fourteen patients had children. In this largest group of adult SCD patients, this regimen was highly efficacious, well-tolerated despite compromised organ functions pre HPCT, and without clinically significant GVHD.

Allogeneic hematopoietic stem cell transplantation from sibling and unrelated donors in pediatric patients with sickle cell disease-A single center experience.

HSCT is curative in SCD. Patients with HLA-identical sibling donor have an excellent outcome ranging from 90%-100% overall and event-free survival. However, due to the lack of matched sibling donors this option is out of reach for 70% of patients with SCD. The pool of potential donors needs to be extended. Transplantations from HLA-matched unrelated donors were reported to be less successful with shorter event-free survival and higher incidences of complications including graft-vs-host disease, especially in patients with advanced stage SCD. Here we report transplantation outcomes for 25 children with SCD transplanted using HLA-matched grafts from related or unrelated donors. Overall survival was 100% with no severe (grade III-IV) graft-vs-host disease and a 12% rejection rate. Mixed donor chimerisms only occurred in transplantations from siblings, while transplantations from unrelated donors resulted in either complete donor chimerism or rejection. Despite the small patient number, overall and disease-free survival for unrelated donor transplantations is excellent in this cohort. The advanced disease state, higher alloreactive effect and stronger immunosuppression in unrelated donor transplantations raises patient risk, for which possible solutions could be found in optimization of transplant preparation, graft manipulation or haploidentical transplantation using T cell receptor α/ß-depleted grafts.

Endothelial Barrier Integrity Is Disrupted In Vitro by Heme and by Serum From Sickle Cell Disease Patients.

Free extracellular heme has been shown to activate several compartments of innate immunity, acting as a danger-associated molecular pattern (DAMP) in hemolytic diseases. Although localized endothelial barrier (EB) disruption is an important part of inflammation that allows circulating leukocytes to reach inflamed tissues, non-localized/deregulated disruption of the EB can lead to widespread microvascular hyperpermeability and secondary tissue damage. In mouse models of sickle cell disease (SCD), EB disruption has been associated with the development of a form of acute lung injury that closely resembles acute chest syndrome (ACS), and that can be elicited by acute heme infusion. Here we explored the effect of heme on EB integrity using human endothelial cell monolayers, in experimental conditions that include elements that more closely resemble in vivo conditions. EB integrity was assessed by electric cell-substrate impedance sensing in the presence of varying concentrations of heme and sera from SCD patients or healthy volunteers. Heme caused a dose-dependent decrease of the electrical resistance of cell monolayers, consistent with EB disruption, which was confirmed by staining of junction protein VE-cadherin. In addition, sera from SCD patients, but not from healthy volunteers, were also capable to induce EB disruption. Interestingly, these effects were not associated with total heme levels in serum. However, when heme was added to sera from SCD patients, but not from healthy volunteers, EB disruption could be elicited, and this effect was associated with hemopexin serum levels. Together our in vitro studies provide additional support to the concept of heme as a DAMP in hemolytic conditions.

CD4+ T Cell Profile and Activation Response in Sickle Cell Disease Patients with Osteonecrosis.

Recent evidence suggests that abnormalities involving CD4+T lymphocytes are associated with the pathophysiology of osteonecrosis (ON); however, few studies have addressed the CD4+T cells in ON related to sickle cell disease (SCD/ON). In addition, T cells producing multiple cytokines simultaneously are often present in the inflammatory milieu and may be implicated in the immune response observed in SCD/ON. In the present study, we aimed to characterize the functional status of CD4+T cells in SCD by simultaneously determining the frequency of IFN-γ +, IL-4+, and IL-17+ CD4+T in cell cultures under exogenous stimuli. Peripheral blood mononuclear cells (PB-MNCs) from 9 steady-state SCD patients, 15 SCD/ON patients, and 19 healthy controls had functional status of CD4+T cells analyzed. Bone marrow mononuclear cells (BM-MNCs) from 24 SCD/ON patients (SCD BM) and 18 patients with ON not related to SCD (non-SCD BM) were also analyzed. We found that PB-MNC of SCD patients with or without ON presented significantly reduced TCD4+, TCD8+, and TCD4+ naïve cell frequencies and increased frequency of circulating CD4+T cells able to simultaneously produce IFN-γ +/IL4+ and IL-17+/IL4+ compared to healthy controls. Conversely, the polyclonal stimulation of BM-MNC induced an increased frequency of CD4+IFN-γ + and CD4+IL-17+ in SCD BM compared to non-SCD BM. The increased proportion of CD4+ T cells able to produce a broad spectrum of proinflammatory cytokines after a strong stimulus indicates that the immune system in SCD/ON patients presents an expressive pool of partially differentiated cells ready to take on effector function. It is possible that this increased subpopulation may extend to inflammatory sites of target organs and may contribute to the maintenance of inflammation and the pathophysiology of osteonecrosis in sickle cell disease.

CXCR4hi effector neutrophils in sickle cell anemia: potential role for elevated circulating serotonin (5-HT) in CXCR4hi neutrophil polarization.

Leukocyte recruitment and heterocellular aggregate formation drive the inflammatory vaso-occlusive processes associated with sickle cell anemia (SCA). We characterized neutrophils in a population of patients with SCA and investigated whether platelet-derived molecules can induce phenotypic alterations in this cell type. Imaging flow cytometry analysis demonstrated that the frequency of circulating CXCR4hi neutrophils was significantly higher in steady-state SCA individuals than in healthy control individuals and that these cells presented increased CD11b activation and toll-like receptor-4 expression. SCA neutrophils display increased neutrophil-platelet aggregation, and CXCR4hi neutrophils demonstrated augmented neutrophil-platelet aggregate frequency with a higher mean number of platelets adhered per neutrophil. Importantly, incubation of neutrophils with platelets significantly elevated their CXCR4 expression, while SCA plasma was found to induce CXCR4hi neutrophil polarization significantly more than control plasma. SCA individuals had significantly increased plasma levels of serotonin (5-HT), and serotonin molecule and SCA plasma induced neutrophil CXCR4 expression in a serotonin-receptor-dependent manner. Thus, the augmented CXCR4hi neutrophil population may contribute to mechanisms that promote vaso-occlusion in SCA; furthermore, circulating serotonin, derived from platelet activation, may play a role in the polarization of neutrophils, suggesting that serotonin-receptor antagonists or serotonin reuptake inhibitors could represent therapeutic approaches to reduce neutrophil activation in SCA.

FcγR2B B2.4 haplotype predicts increased risk of red blood cell alloimmunization in sickle cell disease patients.

BACKGROUND: Red blood cell (RBC) alloimmunization is an important transfusion complication which is prevalent among sickle cell disease (SCD) patients. Autoimmune diseases are a known risk factor for RBC alloimmunization, suggesting that autoimmunity and post-transfusion alloantibody development occur through similar physiopathological pathways. Polymorphisms in the FcγR2B gene have already been associated with several autoimmune disorders and hypothetically could be associated with RBC alloimmunization. Our goal was to evaluate if important polymorphisms of FcγR2B have an impact on the risk of RBC alloimmunization among SCD patients. STUDY DESIGN AND METHODS: This was a case-control study in which alloimmunized and non-alloimmunized SCD patients were compared in terms of the genotype frequency of the FcγR2B polymorphisms -386G/C, -120 T/A, and 695C/T, genotyped through direct Sanger sequencing. RESULTS: A total of 237 patients met the eligibility criteria, 120 cases (alloimmunized) and 117 controls (non-alloimmunized). RBC alloimmunization was associated with female sex (p < 0.001), lifetime number of RBC units transfused (p = 0.002) and 120 T/A FcγR2B genotype (p = 0.031). The FcγR2B promoter region haplotype 2B.4 (386C120A) was positively associated with RBC alloimunization (p = 0.045). The logistic regression (LR) model identified female sex (OR 10.03, CI 95% 5.16-19.49; p < 0.001) and FcγR2B 2B.4 haplotype (OR 4.55, CI95% 1.1118.65; p = 0.035) as independent predictors of RBC alloimmunization in SCD patients. CONCLUSION: SCD patients with the FcγR2B 2B.4 haplotype had over a fourfold higher risk for RBC alloimmunization. This highlights the role played by FcγR2B on RBC alloimmunization and may be helpful in identifying the immune responders.

Utilising red cell antigen genotyping and serological phenotyping in sickle cell disease patients to risk-stratify patients for alloimmunisation risk.

BACKGROUND: Alloimmunisation and haemolytic transfusion reactions (HTRs) can occur in patients with sickle cell disease (SCD) despite providing phenotype-matched red blood cell (RBC) transfusions. Variant RBC antigen gene alleles/polymorphisms can lead to discrepancies in serological phenotyping. We evaluated differences between RBC antigen genotyping and phenotyping methods and retrospectively assessed if partial antigen expression may lead to increased risk of alloimmunisation and HTRs in SCD patients at a tertiary centre in Canada. METHODS: RBC antigen phenotyping and genotyping were performed by a reference laboratory on consenting SCD patients. Patient demographic, clinical and transfusion-related data were obtained from a local transfusion registry and chart review after research ethics board approval. RESULTS: A total of 106 SCD patients were enrolled, and 91% (n = 96) showed additional clinically relevant genotyping information when compared to serological phenotyping alone. FY*02N.01 (FY*B GATA-1) (n = 95; 90%) and RH variant alleles (n = 52, 49%; majority accompanied by FY*02N.01) were common, the latter with putative partial antigen expression in 25 patients. Variability in genotype-phenotype antigen prediction occurred mostly in the Rh system, notably with the e antigen (kappa: 0.17). Fifteen (14.2%) patients had a history of alloimmunisation, with five having HTR documented; no differences in clinical outcomes were found in patients with partial antigen expression. Genotype/extended-phenotype matching strategies may have prevented alloimmunisation events. CONCLUSION: We show a high frequency of variant alleles/polymorphisms in the SCD population, where genotyping may complement serological phenotyping. Genotyping SCD patients before transfusion may prevent alloimmunisation and HTRs, and knowledge of the FY*02N.01 variant allele increases feasibility of finding compatible blood.