Comparison of HLA antibody identification methods for the selection of platelet products for HLA-mediated platelet refractory patients.

In an ineffective transfusion context, solid-phase immunoassays using the Luminex platform for the detection and characterization of HLA antibodies are currently used to select HLA-compatible platelet products. A new HLA antibody identification method, the HISTO SPOT® HLA AB test (BAG Health care GmbH, Lich, Germany), based on the detection of antibodies directed against a recombinant single antigen (SA) by colored spots detected by HISTO MATCH HLA AB module software, runs fully automated on the MR.SPOT®. The aim of this study was to compare the ability of the HISTO SPOT HLA AB and C1qScreen™ (C1q SAB) assays with that of the Labscreen single antigen class I (OL SAB) assay to detect anti-HLA class I antibodies in 56 serum samples from 54 platelet refractory acute myeloid leukemia patients who received HLA mismatch platelet concentrates at a single oncohematology center. In total, 1414 class I specificities, 433 HLA-A and 981 HLA-B, were detected by the OL SAB test. The mean fluorescence intensity (MFI) was >5000 for 874 antigens and <5000 for 655 antigens. The HISTO SPOT® HLA AB and C1q SAB tests identified 85% and 79% of OL SA-detected antigens with an MFI >5000, respectively, but did not identify 34% and 44% of OL SAB-detected antigens, highlighting the lower sensitivity of these techniques. Interestingly, the donor-specific antibodies (DSAs) identified by the HISTO SPOT® HLA AB and C1q SAB assays reacted against HLA mismatch platelet concentrates with the same specificity (86%) and positive predictive (77%) value as in the OL SAB test when the MFI threshold was >2000 for DSA detection. Although the HISTO SPOT® HLA AB test is less sensitive than the OL SAB test, this test could be used for the selection of HLA-compatible platelet products.

Low Prevalence of HLA-G Antibodies in Lung Transplant Patients Detected using MAIPA-Adapted Protocol.

Lung transplantation is often complicated by acute and/or chronic rejection leading to graft-function loss. In addition to the HLA donor-specific antibodies (HLA-DSA), a few autoantibodies are correlated with the occurrence of these complications. Recently, antibodies directed against non-classical HLA molecules, HLA-G, -E, and -F have been detected in autoimmune diseases, like systemic lupus erythematosus. Non-classical HLA molecules are crucial in the immunological acceptance of the lung graft, and some of their isoforms, like HLA-G*01:04 and -G*01:06, are associated with a negative clinical outcome. The aim of this study is to determine the frequency of detection of HLA-G antibodies in lung transplant recipients (LTRs) and their impact on the occurrence of clinical complications. After incubating the cell lines SPI-801, with and without three different HLA-G isoform expression, with sera from 90 healthy blood donors and 35 LTRs (before and after transplantation), HLA-G reactivity was revealed using reagents from commercial monoclonal antibody immobilization of platelet antigen assay (MAIPA ApDIA®). Only one serum from one blood donor had specific reactivity against the HLA-G transduced lines. Non-specific reactivity in many sera from LTRs was observed with transduced- and wild-type cell lines, which may suggest recognition of an autoantigen expressed by the SPI-801 cell line. In conclusion, this study allowed the development of a specific detection tool for non-denatured HLA-G antibodies. These antibodies seem uncommon, both in healthy subjects and in complicated LTRs. This study should be extended to patients suffering from autoimmune diseases as well as kidney and heart transplant recipients.

Evaluation of a new complement-dependent lymphocytotoxicity cross match method using an automated cell counter, the NucleoCounter® NC-3000™.

Complement-dependent lymphocytotoxicity cross match (CDC-XM) is the ultimate test of donor/recipient compatibility prior to organ transplantation. This test is based on cell viability, evaluated under fluorescence microscopy by an operator after proper staining. The determination of the positivity threshold may vary depending on the operator. We developed a new method in which the final step of determining cell viability is automated using the NC-3000™ (Chemometec®), an image cytometer able to precisely determine the percentage of dead/live cells in a suspension. After T and B donor cells isolation by negative selection, complement-dependent lysis was performed in macrovolumes in a PCR plate. Then, cell viability was measured by the NC-3000™. The sensitivity and routine CDC-XM results of this new method were compared to those of CDC-XM reference method using Terasaki plates. The sensitivity of CDC-XM expressed in the ASHI scoring system of this method was similar to the reference method results for a dilution range of the positive controls. Similarly, the results of the new method were comparable in a clinical situation to those obtained with the reference method after a study of 10 cross-matches, of which 5 cross-matches with DSA were positive and five cross-matches without DSA were negative. Moreover, ASHI scores were similar to those obtained using the reference method, and the mortality percentage was reproducible (CV < 15%). The assessment of cell viability by the NC-3000™ is easy to perform and highly reproducible but requires CDC-XM to be performed by the macrovolume method. The determination of a precise percentage of viability/mortality by the automation excludes operator variability and allows a better understanding of results close to the decision threshold.

FCGR2A-HH Gene Variants Encoding the Fc Gamma Receptor for the C-Reactive Protein Are Associated with Enhanced Monocyte CD32 Expression and Cardiovascular Events' Recurrence after Primary Acute Coronary Syndrome.

Fcγ receptors (FcγRs) interact with the C-reactive protein (CRP) and mediate activation of inflammation-related pathogenic mechanisms affecting cardiovascular health. Our study evaluated whether FcγRIIA and FcγRIIIA profiles are associated with the recurrence of adverse cardiovascular events during the first year after a primary acute coronary syndrome (ACS). The primary endpoint was the recurrence of cardiovascular events (RCE), identified as a composite outcome comprising acute heart failure (AHF) and major adverse cardiovascular events (MACE). We obtained blood samples of 145 ACS patients to measure hsCRP circulating levels, to identify FcγRIIA-131RH rs1801274 and FcγRIIIA-158FV rs396991 polymorphisms, to analyze circulating monocytes and NK cell subsets expressing CD16 and CD32, and to detect serum-mediated FCGR2A-HH activation by luciferase reporter assays. The hsCRP, CD32-expression, and Fc-R mediated activation levels were similar in all patients regardless of their MACE risk. In contrast, the hsCRP levels and the proportion of CD14+ circulating monocytes expressing the CD32 receptor for CRP were significantly higher in the patients who developed AHF. The FCGR2A rs1801274 HH genotype was significantly more common in patients who developed RCE and MACE than in RCE-free patients and associated with an enhanced percentage of circulating CD32+CD14+ monocytes. The FCGR2A-HH genotype was identified as an independent predictor of subsequent RCE (OR, 2.7; p = 0.048; CI, 1.01-7.44) by multivariate analysis. These findings bring preliminary evidence that host FCGR2A genetic variants can influence monocyte CD32 receptor expression and may contribute to the fine-tuning of CD32-driven chronic activating signals that affect the risk of developing RCEs following primary ACS events.

HLA-H*02:07 Is a Membrane-Bound Ligand of Denisovan Origin That Protects against Lysis by Activated Immune Effectors.

The biological relevance of genes initially categorized as "pseudogenes" is slowly emerging, notably in innate immunity. In the HLA region on chromosome 6, HLA-H is one such pseudogene; yet, it is transcribed, and its variation is associated with immune properties. Furthermore, two HLA-H alleles, H*02:07 and H*02:14, putatively encode a complete, membrane-bound HLA protein. Here we thus hypothesized that HLA-H contributes to immune homeostasis similarly to tolerogenic molecules HLA-G, -E, and -F. We tested if HLA-H*02:07 encodes a membrane-bound protein that can inhibit the cytotoxicity of effector cells. We used an HLA-null human erythroblast cell line transduced with HLA-H*02:07 cDNA to demonstrate that HLA-H*02:07 encodes a membrane-bound protein. Additionally, using a cytotoxicity assay, our results support that K562 HLA-H*02:07 inhibits human effector IL-2-activated PBMCs and human IL-2-independent NK92-MI cell line activity. Finally, through in silico genotyping of the Denisovan genome and haplotypic association with Denisovan-derived HLA-A*11, we also show that H*02:07 is of archaic origin. Hence, admixture with archaic humans brought a functional HLA-H allele into modern European and Asian populations.

Qualitative and quantitative comparison of cell-free DNA and cell-free fetal DNA isolation by four (semi-)automated extraction methods: impact in two clinical applications: chimerism quantification and noninvasive prenatal diagnosis.

BACKGROUND: Non-invasive molecular analysis of cell-free DNA (cfDNA) became a sensitive biomarker for monitoring organ transplantation or for detection of fetal DNA (cffDNA) in noninvasive prenatal test. In this study, we compared the efficiencies of four (semi)-automated cfDNA isolation instruments using their respective isolation kit: MagNA Pure 24 (Roche®), IDEAL (IDSolution®), LABTurbo 24 (Taigen®) and Chemagic 360 (Perkin Elmer®). The cfDNA was isolated from 5 plasma samples and the Rhesus D (RhD)-cffDNA from 5 maternal plasmas. The cfDNA were quantified by digital droplet PCR (ddPCR), BIABooster system and QUBIT fluorometer. The cfDNA fragment size profiles were assessed by BIABooster system. Chimerism were quantified by home-made ddPCR and Devyser NGS kit. RhD-cffDNA in maternal plasma were detected between weeks 14 and 24 of amenorrhea using free DNA Fetal RHD Kit® (Biorad®). RESULTS: Statistical tests have shown differences in DNA yield depending on the isolation procedure and quantification method used. Magna Pure isolates smaller cfDNA fragment size than other extraction methods (90% ± 9% vs. 74% ± 8%; p = 0.009). Chimerism was only reliable from LABTurbo 24 extractions using the NGS but not with ddPCR whatever extraction methods. RhD-cffDNA were detected by all isolation methods, although IDEAL and LABTurbo 24 systems seemed more efficient. CONCLUSIONS: This comparative study showed a dependency of cfDNA yield depending on isolation procedure and quantification method used. In total, these results suggest that the choice of pre-analytical isolation systems needs to be carefully validated in routine clinical practice.

Soluble HLA-G Expression Inversely Correlates With Fetal Microchimerism Levels in Peripheral Blood From Women With Scleroderma.

Women with scleroderma (SSc) maintain significantly higher quantities of persisting fetal microchimerism (FMc) from complete or incomplete pregnancies in their peripheral blood compared to healthy women. The non-classical class-I human leukocyte antigen (HLA) molecule HLA-G plays a pivotal role for the implantation and maintenance of pregnancy and has often been investigated in offspring from women with pregnancy complications. However data show that maternal HLA-G polymorphisms as well as maternal soluble HLA-G (sHLA-G) expression could influence pregnancy outcome. Here, we aimed to investigate the underlying role of maternal sHLA-G expression and HLA-G polymorphisms on the persistence of FMc. We measured sHLA-G levels by enzyme linked immunosorbent assay in plasma samples from 88 healthy women and 74 women with SSc. Male Mc was quantified by DYS14 real-time PCR in blood samples from 58 women who had previously given birth to at least one male child. Furthermore, eight HLA-G 5'URR/3'UTR polymorphisms, previously described as influencing HLA-G expression, were performed on DNA samples from 96 healthy women and 106 women with SSc. Peripheral sHLA-G was at lower concentration in plasma from SSc (76.2 ± 48.3 IU/mL) compared to healthy women (117.5 ± 60.1 IU/mL, p < 0.0001), independently of clinical subtypes, autoantibody profiles, disease duration, or treatments. Moreover, sHLA-G levels were inversely correlated to FMc quantities (Spearman correlation, p < 0.01). Finally, women with SSc had lower sHLA-G independently of the eight HLA-G 5'URR/3'UTR polymorphisms, although they were statistically more often homozygous than heterozygous for HLA-G polymorphism genotypes -716 (G/T), -201 (G/A), 14 bp (ins/del), and +3,142 (G/A) than healthy women. In conclusion, women with SSc display less sHLA-G expression independently of the eight HLA-G polymorphisms tested. This decreased production correlates with higher quantities of persisting FMc commonly observed in blood from SSc women. These results shed some lights on the contribution of the maternal HLA-G protein to long-term persistent fetal Mc and initiate new perspectives in this field.

Soluble CD146 boosts therapeutic effect of endothelial progenitors through proteolytic processing of short CD146 isoform.

AIMS: Endothelial colony-forming cells (ECFC) constitute an endothelial progenitor fraction with a promising interest for the treatment of ischaemic cardiovascular diseases. As soluble CD146 (sCD146) is a new factor promoting angiogenesis, we examined whether sCD146 priming could improve the therapeutic potential of ECFC and defined the involved mechanism. METHODS AND RESULTS: We investigated the effects of sCD146 priming on regenerative properties of ECFC in vivo. In a mouse model of hindlimb ischaemia, the homing of radiolabelled cells to ischaemic tissue was assessed by SPECT-CT imaging. Soluble CD146 priming did not modify the number of engrafted ECFC but improved their survival capacity, leading to an enhanced revascularization. The mechanism of action of sCD146 on ECFC was studied in vitro. We showed that sCD146 acts in ECFC through a signalosome, located in lipid rafts, containing angiomotin, the short isoform of CD146 (shCD146), VEGFR1, VEGFR2, and presenilin-1. Soluble CD146 induced a sequential proteolytic cleavage of shCD146, with an extracellular shedding followed by an intramembrane cleavage mediated by matrix metalloprotease (MMP)/ADAM and presenilin-1, respectively. The generated intracellular part of shCD146 was directed towards the nucleus where it associated with the transcription factor CSL and modulated the transcription of genes involved in cell survival (FADD, Bcl-xl) and angiogenesis (eNOS). This effect was dependent on both VEGFR1 and VEGFR2, which were rapidly phosphorylated by sCD146. CONCLUSIONS: These findings establish that activation of the proteolytic processing of shCD146, in particular by sCD146, constitutes a promising pathway to improve endothelial progenitors' regenerative properties for the treatment of cardiovascular diseases.

Sphingosine kinase 1 expressed by endothelial colony-forming cells has a critical role in their revascularization activity.

AIMS: Cell therapy based on endothelial colony-forming cells (ECFCs) is a promising option for ischaemic cardiovascular diseases. A better understanding of the mechanisms by which these cells promote revascularization remains a critical challenge to improving their therapeutic potential. We aimed to identify the critical mechanisms involved in the revascularization activity of ECFCs by using the paracrine properties of mesenchymal stem cells (MSC). METHODS AND RESULTS: Conditioned medium from human bone marrow-derived MSCs (MSC-CM) increased the angiogenic activity of cord blood ECFCs in vitro (proliferation, migration, and pseudo-tube formation), the survival of ECFCs in mice (Matrigel Plug assay), and the capacity of ECFCs to promote the recovery of blood perfusion in mice with hindlimb ischaemia. Furthermore, the capillary density in ischaemic gastrocnemius muscle was significantly increased in mice transplanted with the ECFCs pre-treated with the MSC-CM. The enhancement of ECFCs activity involved the up-regulation of sphingosine kinase 1 (SphK1) expression and activity. The inhibition of SphK1 in ECFCs by using an inhibitor or a siRNA knockdown of SphK1 prevented the stimulation of the ECFCs induced by the MSC-CM. The improvement of ECFC activity by MSC-CM also involved the up-regulation of sphingosine-1-phosphate receptor 1 (S1P1) and a S1P/S1P1/3-dependent mechanism. Finally, we showed that the stimulation of ECFCs with exogenous S1P increased angiogenesis and promoted blood perfusion in hindlimb ischaemia. CONCLUSION: The up-regulation of SphK1 and S1P-dependent pathways is critical for the angiogenic/vasculogenic activity of ECFCs. The identification of this pathway provides attractive targets to optimize cell-based therapy for revascularization in ischaemic diseases.

Soluble melanoma cell adhesion molecule (sMCAM/sCD146) promotes angiogenic effects on endothelial progenitor cells through angiomotin.

The melanoma cell adhesion molecule (CD146) contains a circulating proteolytic variant (sCD146), which is involved in inflammation and angiogenesis. Its circulating level is modulated in different pathologies, but its intracellular transduction pathways are still largely unknown. Using peptide pulldown and mass spectrometry, we identified angiomotin as a sCD146-associated protein in endothelial progenitor cells (EPC). Interaction between angiomotin and sCD146 was confirmed by enzyme-linked immunosorbent assay (ELISA), homogeneous time-resolved fluorescence, and binding of sCD146 on both immobilized recombinant angiomotin and angiomotin-transfected cells. Silencing angiomotin in EPC inhibited sCD146 angiogenic effects, i.e. EPC migration, proliferation, and capacity to form capillary-like structures in Matrigel. In addition, sCD146 effects were inhibited by the angiomotin inhibitor angiostatin and competition with recombinant angiomotin. Finally, binding of sCD146 on angiomotin triggered the activation of several transduction pathways that were identified by antibody array. These results delineate a novel signaling pathway where sCD146 binds to angiomotin to stimulate a proangiogenic response. This result is important to find novel target cells of sCD146 and for the development of therapeutic strategies based on EPC in the treatment of ischemic diseases.