Modulation of bone marrow and peripheral blood cytokine levels by age and clonal hematopoiesis in healthy individuals.

Aging is associated with bone marrow (BM) inflammaging and, in some individuals, with the onset of clonal hematopoiesis (CH) of indeterminate potential. In this study conducted on 94 strictly healthy volunteers (18 to 80 yo), we measured BM and peripheral blood (PB) plasma levels of 49 hematopoietic and inflammatory cytokines. With aging, 7 cytokines increased in BM (FLT3L, CXCL9, HGF, FGF-2, CCL27, IL-16, IL-18) and 8 decreased (G-CSF, TNF, IL-2, IL-15, IL-17A, CCL7, IL-4, IL-10). In PB, 10 cytokines increased with age (CXCL9, FLT3L, CCL27, CXCL10, HGF, CCL11, IL-16, IL-6, IL-1 beta, CCL2). CH was associated with higher BM levels of MIF and IL-1 beta, lower BM levels of IL-9 and IL-5 and higher PB levels of IL-15, VEGF-A, IL-2, CXCL8, CXCL1 and G-CSF. These reference values provide a useful tool to investigate anomalies related to inflammaging and potentially leading to the onset of age-related myeloid malignancies or inflammatory conditions.

A New in Silico Antibody Similarity Measure Both Identifies Large Sets of Epitope Binders with Distinct CDRs and Accurately Predicts Off-Target Reactivity.

Developing a therapeutic antibody is a long, tedious, and expensive process. Many obstacles need to be overcome, such as biophysical properties (issues of solubility, stability, weak production yields, etc.), as well as cross-reactivity and subsequent toxicity, which are major issues. No in silico method exists today to solve such issues. We hypothesized that if we were able to properly measure the similarity between the CDRs of antibodies (Ab) by considering not only their evolutionary proximity (sequence identity) but also their structural features, we would be able to identify families of Ab recognizing similar epitopes. As a consequence, Ab within the family would share the property to recognize their targets, which would allow (i) to identify off-targets and forecast the cross-reactions, and (ii) to identify new Ab specific for a given target. Testing our method on 238D2, an antagonistic anti-CXCR4 nanobody, we were able to find new nanobodies against CXCR4 and to identify influenza hemagglutinin as an off-target of 238D2.

Pharmacological Characterization of Low Molecular Weight Biased Agonists at the Follicle Stimulating Hormone Receptor.

Follicle-stimulating hormone receptor (FSHR) plays a key role in reproduction through the activation of multiple signaling pathways. Low molecular weight (LMW) ligands composed of biased agonist properties are highly valuable tools to decipher complex signaling mechanisms as they allow selective activation of discrete signaling cascades. However, available LMW FSHR ligands have not been fully characterized yet. In this context, we explored the pharmacological diversity of three benzamide and two thiazolidinone derivatives compared to FSH. Concentration/activity curves were generated for Gαs, Gαq, Gαi, ß-arrestin 2 recruitment, and cAMP production, using BRET assays in living cells. ERK phosphorylation was analyzed by Western blotting, and CRE-dependent transcription was assessed using a luciferase reporter assay. All assays were done in either wild-type, Gαs or ß-arrestin 1/2 CRISPR knockout HEK293 cells. Bias factors were calculated for each pair of read-outs by using the operational model. Our results show that each ligand presented a discrete pharmacological efficacy compared to FSH, ranging from super-agonist for ß-arrestin 2 recruitment to pure Gαs bias. Interestingly, LMW ligands generated kinetic profiles distinct from FSH (i.e., faster, slower or transient, depending on the ligand) and correlated with CRE-dependent transcription. In addition, clear system biases were observed in cells depleted of either Gαs or ß-arrestin genes. Such LMW properties are useful pharmacological tools to better dissect the multiple signaling pathways activated by FSHR and assess their relative contributions at the cellular and physio-pathological levels.

MAbTope: A Method for Improved Epitope Mapping.

Abs are very efficient drugs, ∼70 of them are already approved for medical use, over 500 are in clinical development, and many more are in preclinical development. One important step in the characterization and protection of a therapeutic Ab is the determination of its cognate epitope. The gold standard is the three-dimensional structure of the Ab/Ag complex by crystallography or nuclear magnetic resonance spectroscopy. However, it remains a tedious task, and its outcome is uncertain. We have developed MAbTope, a docking-based prediction method of the epitope associated with straightforward experimental validation procedures. We show that MAbTope predicts the correct epitope for each of 129 tested examples of Ab/Ag complexes of known structure. We further validated this method through the successful determination, and experimental validation (using human embryonic kidney cells 293), of the epitopes recognized by two therapeutic Abs targeting TNF-α: certolizumab and golimumab.

Involvement of GPx-3 in the Reciprocal Control of Redox Metabolism in the Leukemic Niche.

The bone marrow (BM) microenvironment plays a crucial role in the development and progression of leukemia (AML). Intracellular reactive oxygen species (ROS) are involved in the regulation of the biology of leukemia-initiating cells, where the antioxidant enzyme GPx-3 could be involved as a determinant of cellular self-renewal. Little is known however about the role of the microenvironment in the control of the oxidative metabolism of AML cells. In the present study, a coculture model of BM mesenchymal stromal cells (MSCs) and AML cells (KG1a cell-line and primary BM blasts) was used to explore this metabolic pathway. MSC-contact, rather than culture with MSC-conditioned medium, decreases ROS levels and inhibits the Nrf-2 pathway through overexpression of GPx3 in AML cells. The decrease of ROS levels also inactivates p38MAPK and reduces the proliferation of AML cells. Conversely, contact with AML cells modifies MSCs in that they display an increased oxidative stress and Nrf-2 activation, together with a concomitant lowered expression of GPx-3. Altogether, these experiments suggest that a reciprocal control of oxidative metabolism is initiated by direct cell-cell contact between MSCs and AML cells. GPx-3 expression appears to play a crucial role in this cross-talk and could be involved in the regulation of leukemogenesis.

n-3 Polyunsaturated fatty acids induce acute myeloid leukemia cell death associated with mitochondrial glycolytic switch and Nrf2 pathway activation.

Acute Myeloid Leukemia (AML) remains a therapeutic challenge and improvements in chemotherapy are needed. n-3 polyunsaturated fatty acids (PUFAs), present in fish oil (FO) at high concentrations, have antitumoral properties in various cancer models. We investigated the effects of two n-3 PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), in AML cell lines and primary AML blasts. EPA and DHA induced a dose-dependent decrease in cell viability in five AML cell lines, which was also observed with FO, but not SO (devoid of n-3 PUFAs) in cell lines and primary leucoblasts. Mitochondrial energy metabolism shifted from oxidative respiration to glycolytic metabolism in the U937, MOLM-13, and HL-60 cell lines. This phenomenon was associated with major disorganization of the mitochondrial network and mitochondrial swelling. Transcriptomic analysis after 6 h and 24 h of exposure to FO revealed a Nrf2 activation signature, which was confirmed by evidence of Nrf2 nuclear translocation in response to oxidative stress, but insufficient to prevent cell death following prolonged exposure. Apoptosis studies showed consistent phosphatidylserine exposition among the AML cell lines tested and a reduced mitochondrial membrane potential. The cell-killing effect of FO was additive with that of cytarabine (AraC), by the Chou and Talalay method, and this combination effect could be reproduced in primary AML blasts. Altogether, our results show deleterious effects of n-3 PUFAs on mitochondrial metabolism of AML cells, associated with oxidative stress and Nrf2 response, leading to cell death. These observations support further investigation of n-3 PUFA addition to standard chemotherapy in AML.

Competing G protein-coupled receptor kinases balance G protein and ß-arrestin signaling.

Seven-transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through ß-arrestins, whose recruitment to the activated receptor is regulated by G protein-coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal-regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT(1A)R) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)-based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well-established function in the desensitization of G-protein activation, GRK2 exerts a strong negative effect on ß-arrestin-dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2-dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT(1A)R, and HEK293 cells expressing other 7TMRs.

Aberrant DNA methylation impacts HOX genes expression in bone marrow mesenchymal stromal cells of myelodysplastic syndromes and de novo acute myeloid leukemia.

DNA methylation, a major biological process regulating the transcription, contributes to the pathophysiology of hematologic malignancies, and hypomethylating agents are commonly used to treat myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). In these diseases, bone marrow mesenchymal stromal cells (MSCs) play a key supportive role through the production of various signals and interactions. The DNA methylation status of MSCs, likely to reflect their functionality, might be relevant to understand their contribution to the pathophysiology of these diseases. Consequently, the aim of our study was to analyze the modifications of DNA methylation profiles of MSCs induced by MDS or AML. MSCs from MDS/AML patients were characterized via 5-methylcytosine quantification, gene expression profiles of key regulators of DNA methylation, identification of differentially methylated regions (DMRs) by methylome array, and quantification of DMR-coupled genes expression. MDS and AML-MSCs displayed global hypomethylation and under-expression of DNMT1 and UHRF1. Methylome analysis revealed aberrant methylation profiles in all MDS and in a subgroup of AML-MSCs. This aberrant methylation was preferentially found in the sequence of homeobox genes, especially from the HOX family (HOXA1, HOXA4, HOXA5, HOXA9, HOXA10, HOXA11, HOXB5, HOXC4, and HOXC6), and impacted on their expression. These results highlight modifications of DNA methylation in MDS/AML-MSCs, both at global and focal levels dysregulating the expression of HOX genes well known for their involvement in leukemogenesis. Such DNA methylation in MSCs could be the consequence of the malignant disease or could participate in its development through defective functionality or exosomal transfer of HOX transcription factors from MSCs to hematopoietic cells.

Low-Dose Pesticides Alter Primary Human Bone Marrow Mesenchymal Stem/Stromal Cells through ALDH2 Inhibition.

(1) Background: The impact of occupational exposure to high doses of pesticides on hematologic disorders is widely studied. Yet, lifelong exposure to low doses of pesticides, and more particularly their cocktail effect, although poorly known, could also participate to the development of such hematological diseases as myelodysplastic syndrome (MDS) in elderly patients. (2) Methods: In this study, a cocktail of seven pesticides frequently present in water and food (maneb, mancozeb, iprodione, imazalil, chlorpyrifos ethyl, diazinon and dimethoate), as determined by the European Food Safety Authority, were selected. Their in vitro effects at low-doses on primary BM-MSCs from healthy volunteers were examined. (3) Results: Exposure of normal BM-MSCs to pesticides for 21 days inhibited cell proliferation and promoted DNA damage and senescence. Concomitantly, these cells presented a decrease in aldehyde dehydrogenase 2 (ALDH2: mRNA, protein and enzymatic activity) and an increase in acetaldehyde levels. Pharmacological inhibition of ALDH2 with disulfiram recapitulated the alterations induced by exposure to low doses of pesticides. Moreover, BM-MSCs capacity to support primitive hematopoiesis was significantly altered. Similar biological abnormalities were found in primary BM-MSCs derived from MDS patients. (4) Conclusions: these results suggest that ALDH2 could participate in the pathophysiology of MDS in elderly people long exposed to low doses of pesticides.

High prevalence of clonal hematopoiesis in the blood and bone marrow of healthy volunteers.

Clonal hematopoiesis (CH) of indeterminate potential has been described in blood samples from large series of patients. Its prevalence and consequences are still not well understood because sequencing methods vary and because most studies were performed in cohorts comprising individuals with nonhematologic diseases. Here, we investigated the frequency of CH in 82 paired bone marrow and blood samples from carefully selected healthy adult volunteers. Forty-one genes known to be mutated in myeloid malignancies were sequenced with a 1% threshold of detection. In bone marrow samples, clones were found in almost 40% of healthy volunteers more than 50 years old. The most frequent mutations were found in DNMT3A and TET2, with 1 individual carrying 3 variants. Variant allele frequencies were highly concordant between blood and bone marrow samples. Blood parameters were normal except for those in 2 individuals: 1 had a mild macrocytosis and 1 had a mild thrombocytosis. Furthermore, no morphologic abnormalities or dysplasia were detected when bone marrow smears were carefully evaluated. Individuals with CH differed from others by age (62.8 vs 38.6 years; P < .0001) and platelet count (294 vs 241 ×109/L; P = .0208), the latter being no more significant when removing the 2 individuals who carried the JAK2 p.V617F mutation. These results confirm that CH is a very common condition in healthy adults over 50 years old. Consequently, the detection of driver myeloid mutations should be interpreted with caution in the absence of cytologic abnormalities in the blood and/or the bone marrow.

Correction: Disruption of gap junctions attenuates acute myeloid leukemia chemoresistance induced by bone marrow mesenchymal stromal cells.

The original version of this Article omitted the following from the Acknowledgements: This research was also supported by grants to KZ (UL and L-CNRS). This has now been corrected in both the PDF and HTML versions of the Article.

Disruption of gap junctions attenuates acute myeloid leukemia chemoresistance induced by bone marrow mesenchymal stromal cells.

The bone marrow (BM) niche impacts the progression of acute myeloid leukemia (AML) by favoring the chemoresistance of AML cells. Intimate interactions between leukemic cells and BM mesenchymal stromal cells (BM-MSCs) play key roles in this process. Direct intercellular communications between hematopoietic cells and BM-MSCs involve connexins, components of gap junctions. We postulated that blocking gap junction assembly could modify cell-cell interactions in the leukemic niche and consequently the chemoresistance. The comparison of BM-MSCs from AML patients and healthy donors revealed a specific profile of connexins in BM-MSCs of the leukemic niche and the effects of carbenoxolone (CBX), a gap junction disruptor, were evaluated on AML cells. CBX presents an antileukemic effect without affecting normal BM-CD34+ progenitor cells. The proapoptotic effect of CBX on AML cells is in line with the extinction of energy metabolism. CBX acts synergistically with cytarabine (Ara-C) in vitro and in vivo. Coculture experiments of AML cells with BM-MSCs revealed that CBX neutralizes the protective effect of the niche against the Ara-C-induced apoptosis of leukemic cells. Altogether, these results suggest that CBX could be of therapeutic interest to reduce the chemoresistance favored by the leukemic niche, by targeting gap junctions, without affecting normal hematopoiesis.

The role of platelet/endothelial cell adhesion molecule 1 (CD31) and CD38 antigens in marrow microenvironmental retention of acute myelogenous leukemia cells.

In acute myelogenous leukemia (AML), leukemic cell-microenvironment interactions within various niches (stromal/osteoblastic or sinusoidal endothelial cell niches) have a role in leukemia cell survival and drug resistance. The AML leukemic cells express platelet/endothelial cell adhesion molecule-1 (CD31) and CD38, two adhesion molecules that could interact with microenvironmental elements, i.e., CD31 on the surface of marrow endothelial cells (CD31/CD31 and CD38/CD31 interactions) and hyaluronate (CD38/hyaluronate interactions). We report a physical association of these two antigens on the plasma membrane of myeloid leukemic cells. In this context, in vitro experiments done using interaction-blocking anti-CD31 and anti-CD38 monoclonal antibodies (CLB-HEC75 and OKT10, respectively) indicate that an excess of CD31 on the cell membrane of leukemic cells (CD31/CD38 MFI ratio >1) promotes a homotypic interaction with marrow endothelial cells, resulting in higher transendothelial migration. Conversely, an excess of CD38 (CD31/CD38 MFI ratio <1) allows leukemic cells to be entrapped within the bone marrow microenvironment through hyaluronate adhesion. The results obtained in vitro using fluorescence resonance energy transfer, co-capping, and co-immunoprecipitation experiments, and hyaluronate adhesion and transendothelial migration assays, are supported by immunophenotypic characterization of marrow leukemic cells from 78 AML patients on which CD38 expression levels were found to be positively correlated with those of CD31. Importantly, the excess of CD31 in those samples was associated with a higher peripheral WBC count. These findings indicate that bone marrow retention of AML cells depends on CD31 and CD38 coexpression levels.

Bone marrow oxidative stress and specific antioxidant signatures in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders with an inherent tendency for transformation in secondary acute myeloid leukemia. This study focused on the redox metabolism of bone marrow (BM) cells from 97 patients compared with 25 healthy controls. The level of reactive oxygen species (ROS) was quantified by flow cytometry in BM cell subsets as well as the expression level of 28 transcripts encoding for major enzymes involved in the antioxidant cellular response. Our results highlight increased ROS levels in BM nonlymphoid cells and especially in primitive CD34posCD38low progenitor cells. Moreover, we identified a specific antioxidant signature, dubbed "antioxidogram," for the different MDS subgroups or secondary acute myeloblastic leukemia (sAML). Our results suggest that progression from MDS toward sAML could be characterized by 3 successive molecular steps: (1) overexpression of enzymes reducing proteic disulfide bonds (MDS with <5% BM blasts [GLRX family]); (2) increased expression of enzymes detoxifying H2O2 (MDS with 5% to 19% BM blasts [PRDX and GPX families]); and finally (3) decreased expression of these enzymes in sAML. The antioxidant score (AO-Score) defined by logistic regression from the expression levels of transcripts made it possible to stage disease progression and, interestingly, this AO-Score was independent of the revised International Scoring System. Altogether, this study demonstrates that MDS and sAML present an important disturbance of redox metabolism, especially in BM stem and progenitor cells and that the specific molecular antioxidant response parameters (antioxidogram, AO-Score) could be considered as useful biomarkers for disease diagnosis and follow-up.

Molecular profile of mouse stromal mesenchymal stem cells.

We determined a transcriptional profile specific for clonal stromal mesenchymal stem cells from adult and fetal hematopoietic sites. To identify mesenchymal stem cell-like stromal cell lines, we evaluated the adipocytic, osteoblastic, chondrocytic, and vascular smooth muscle differentiation potential and also the hematopoietic supportive (stromal) capacity of six mouse stromal cell lines from adult bone marrow and day 14.5 fetal liver. We found that two lines were quadripotent and also supported hematopoiesis, BMC9 from bone marrow and AFT024 from fetal liver. We then ascertained the set of genes differentially expressed in the intersection set of AFT024 and BMC9 compared with those expressed in the union set of two negative control lines, 2018 and BFC012 (both from fetal liver); 346 genes were upregulated and 299 downregulated. Using Ingenuity software, we found two major gene networks with highly significant scores. One network contained downregulated genes that are known to be implicated in osteoblastic differentiation, proliferation, or transformation. The other network contained upregulated genes that belonged to two categories, cytoskeletal genes and genes implicated in the transcriptional machinery. The data extend the concept of stromal mesenchymal stem cells to clonal cell populations derived not only from bone marrow but also from fetal liver. The gene networks described should discriminate this cell type from other types of stem cells and help define the stem cell state.

CXCL12 polymorphism and malignant cell dissemination/tissue infiltration in acute myeloid leukemia.

Stromal cell-derived factor 1 (SDF-1), a chemokine abundantly produced by the bone marrow microenvironment, and its receptor CXCR4 have crucial roles in malignant cell trafficking. In acute myeloid leukemia (AML), blasts invade the bloodstream and may localize in extramedullar sites, with variations from one patient to another. We hypothesized that a polymorphism in the SDF-1 coding gene (CXCL12 G801A) could influence blast dissemination and tissue infiltration in AML. CXCL12 G801A polymorphism was determined in 86 adult patients and 100 healthy volunteers. The allelic status and CXCR4 expression on bone marrow blasts were analyzed in relation to peripheral blood blast (PBB) counts and frequency of extramedullar tumor sites. 801A carrier status (801G/A, 801A/A) was found to be associated with a higher PBB count compared with 801G/G homozygous patients (P=0.031) and higher frequency of extramedullar tumor sites (odds ratio 2.92, 95% confidence interval 1.18-7.21, P=0.018). Moreover, the PBB count was correlated with CXCR4 expression (correlation coefficient 0.546, P=0.001) when considering 801A carriers. In conclusion, a polymorphism in the SDF-1 gene is shown for the first time to be associated with the clinical presentation of a malignant hematological disease and more generally with the risk of distant tissue infiltration by tumor cells.

Phosphorylation of ß-arrestin2 at Thr383 by MEK underlies ß-arrestin-dependent activation of Erk1/2 by GPCRs.

In addition to their role in desensitization and internalization of G protein-coupled receptors (GPCRs), ß-arrestins are essential scaffolds linking GPCRs to Erk1/2 signaling. However, their role in GPCR-operated Erk1/2 activation differs between GPCRs and the underlying mechanism remains poorly characterized. Here, we show that activation of serotonin 5-HT2C receptors, which engage Erk1/2 pathway via a ß-arrestin-dependent mechanism, promotes MEK-dependent ß-arrestin2 phosphorylation at Thr383, a necessary step for Erk recruitment to the receptor/ß-arrestin complex and Erk activation. Likewise, Thr383 phosphorylation is involved in ß-arrestin-dependent Erk1/2 stimulation elicited by other GPCRs such as ß2-adrenergic, FSH and CXCR4 receptors, but does not affect the ß-arrestin-independent Erk1/2 activation by 5-HT4 receptor. Collectively, these data show that ß-arrestin2 phosphorylation at Thr383 underlies ß-arrestin-dependent Erk1/2 activation by GPCRs.

Human Luteinizing Hormone and Chorionic Gonadotropin Display Biased Agonism at the LH and LH/CG Receptors.

Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) have been considered biologically equivalent because of their structural similarities and their binding to the same receptor; the LH/CGR. However, accumulating evidence suggest that LH/CGR differentially responds to the two hormones triggering differential intracellular signaling and steroidogenesis. The mechanistic basis of such differential responses remains mostly unknown. Here, we compared the abilities of recombinant rhLH and rhCG to elicit cAMP, ß-arrestin 2 activation, and steroidogenesis in HEK293 cells and mouse Leydig tumor cells (mLTC-1). For this, BRET and FRET technologies were used allowing quantitative analyses of hormone activities in real-time and in living cells. Our data indicate that rhLH and rhCG differentially promote cell responses mediated by LH/CGR revealing interesting divergences in their potencies, efficacies and kinetics: rhCG was more potent than rhLH in both HEK293 and mLTC-1 cells. Interestingly, partial effects of rhLH were found on ß-arrestin recruitment and on progesterone production compared to rhCG. Such a link was further supported by knockdown experiments. These pharmacological differences demonstrate that rhLH and rhCG act as natural biased agonists. The discovery of novel mechanisms associated with gonadotropin-specific action may ultimately help improve and personalize assisted reproduction technologies.

A Comprehensive View of the ß-Arrestinome.

G protein-coupled receptors (GPCRs) are membrane receptors critically involved in sensing the environment and orchestrating physiological processes. As such, they transduce extracellular signals such as hormone, neurotransmitters, ions, and light into an integrated cell response. The intracellular trafficking, internalization, and signaling ability of ligand-activated GPCRs are controlled by arrestins, adaptor proteins that they interact with upon ligand binding. ß-arrestins 1 and 2 in particular are now considered as hub proteins assembling multiprotein complexes to regulate receptor fate and transduce diversified cell responses. While more than 400 ß-arrestin interaction partners have been identified so far, much remains to be learnt on how discrimination between so many binding partners is accomplished. Here, we gathered the interacting partners of ß-arrestins through database mining and manual curation of the literature to map the ß-arrestin interactome (ß-arrestinome). We discussed several parameters that determine compatible (AND) or mutually exclusive (XOR) binding of ß-arrestin interactors, such as structural constraints, intracellular abundance, or binding affinity.

Eculizumab epitope on complement C5: Progress towards a better understanding of the mechanism of action.

Eculizumab is an anti-complement C5 monoclonal antibody which has greatly improved the prognosis and outcomes of nocturnal paroxysmal hemoglobinuria and atypical hemolytic and uremic syndromes. It is also known to be very species-specific for human C5, despite an important degree of conservation of the targeted macroglobulin domain, MG7, with that of other primates. However, the published eculizumab linear epitope does not explain this species specificity. Sequence analysis, in silico docking and reverse phase protein array were implemented to fully characterize the eculizumab epitope on human complement C5. Several residues potentially involved in the species specificity were identified outside the known epitope by sequence analysis. In silico docking confirmed the implication of a beta-hairpin located between residues 913 and 922, outside the known epitope, in the binding of eculizumab to C5. This beta-hairpin spreads from S913 to I922 and contains a tryptophan residue on position 917 which is unique to humans. The contribution of both this peptide and the already known one epitope, which spreads between residues C883 and S891, was validated by reverse phase protein assay, clearly demonstrating the discontinuous nature of the epitope. Two residues in particular, Arg885 and Trp917, were defined as major participants in the interaction of C5 and eculizumab. Their important role was confirmed by the recent publication of a crystal structure of eculizumab Fab bound to C5. The beta-hairpin not only explains the fine species specificity of eculizumab but is also an important site at the C5/C5 convertase interface, revealing how eculizumab acts as a competitor of C5 convertases.