A new set of reagents and related software used for NGS based classical and non-classical HLA typing showing evidence for a greater HLA haplotype diversity.

To evaluate the HLA typing performance of a new Long-Range PCR NGS set of reagents and its dedicated software, a panel of 41 reference homozygous cell lines from the International Histocompatibility Working Group (IHWG) and a panel of 376 volunteer bone marrow donors were analyzed for classical and non-classical HLA class I and class II genes. All results, except HLA-DPB1, were obtained without any ambiguities at the 3rd field level. Based on the high resolution performance of the reagents, a number of new alleles have been described not only for classical but also for non-classical HLA class I genes, leading to a more accurate haplotype definition. Linkage disequilibrium between HLA-A and HLA-G genes has been defined at 4th field level of resolution. Moreover, for the first time, HLA-DQA2 and DQB2 polymorphisms and their linkage disequilibrium with DQB1 were described.

Evidence for a higher resolution of HLA genotyping by a new NGS-based approach.

With more than 16,000 alleles identified, the human leucocyte antigen (HLA) system is one of the most polymorphic regions of the human genome. Regarding the crucial role of HLA compatibility in transplantation and especially in Hematopoietic Stem Cell Transplantation, identification of HLA polymorphisms at a high-resolution level is of major interest. Recently, NGS technology has been proposed which appears to be simpler and more informative than the classical molecular methods such as SSP, SSOr and SBT. In the present report, a new set of NGS reagents and the appropriate associated software for sequence analysis are described. Through different studies, the performances of the system are illustrated and demonstrate that the method herein described overcomes current limitations in performing high-resolution HLA typing in clinical laboratories.

Decrease of blood anti-α1,3 Galactose Abs levels in multiple sclerosis (MS) and clinically isolated syndrome (CIS) patients.

The etiology of multiple sclerosis (MS) remains elusive. Among the possible causes, the increase of anti-Neu5Gc antibodies during EBV primo-infection of Infectious mononucleosis (IMN) may damage the integrity of the blood-brain barrier facilitating the transfer of EBV-infected B cells and anti-EBV T cell clones in the brain. We investigated the change in titers of anti-Neu5Gc and anti-α1,3 Galactose antibodies in 49 IMN, in 76 MS, and 73 clinically isolated syndrome (CIS) patients, as well as age/gender-matched healthy individuals. Anti-Gal and anti-Neu5Gc are significantly increased during IMN (p=0.02 and p<1.10-4 respectively), but not in acute CMV primo-infection. We show that, whereas there was no change in anti-Neu5Gc in MS/CIS, the two populations exhibit a significant decrease in anti-Gal (combined p=2.7.10-3), in contrast with patients with non-MS/CIS central nervous system pathologies. Since anti-Gal result from an immunization against α1,3 Gal, lacking in humans but produced in the gut, our data suggest that CIS and MS patients have an altered microbiota or an altered response to this microbiotic epitope.

Characterization of a new human monoclonal antibody directed against the Vel antigen.

BACKGROUND AND OBJECTIVES: The Vel blood group antigen is a poorly characterized high-prevalence antigen. Until now, anti-Vel antibodies have been observed in only alloimmunized Vel-negative individuals. In this study, we aimed to establish a human hybridoma cell line secreting the first anti-Vel monoclonal antibody (mAb), clone SpG213Dc. MATERIALS AND METHODS: Peripheral blood lymphocytes from a French Vel-negative woman with anti-Vel in her plasma were transformed with Epstein-Barr virus and then hybridized with the myeloma cell line Sp2/O-Ag14 using the polyethylene glycol (PEG) method. A specific anti-Vel mAb was successfully produced and was extensively characterized by serological, flow cytometry and Western blot analyses. RESULTS: One human anti-Vel-secreting clone was produced and the secreted anti-Vel mAb (SpG213Dc) was examined. The specificity of the SpG213Dc mAb was assessed by its reactivity against a panel of nine genotyped RBCs including, respectively, three Vel-negative and six Vel-positive (three wild-type homozygous and three heterozygous) samples using flow cytometry method. Vel-positive RBCs were specifically stained and were subsequently used to perform Western blot and immunoprecipitation analysis of the Vel antigen. CONCLUSION: Serological characterization of the new monoclonal anti-Vel SpG213Dc showed a heterogeneous level of expression of the Vel antigen on the different RBCs. Our results suggest that the mAb SpG213Dc can be reliably used as a blood grouping reagent, thus allowing the mass-scale phenotyping of blood donors to strengthen rare blood banks with Vel-negative RBC units.

[Organization for the coverage of the transfusional needs of patients with hemoglobinopathy at the Établissement français du sang Bretagne]. / Organisation de la prise en charge des besoins transfusionnels des patients atteints d'hémoglobinopathie à l'Établissement français du sang Bretagne.

UNLABELLED: Brittany is a low prevalence region for hemoglobinopathies. Despite of that, the number of patients is increasing each year. In 2013, 140 patients were known at the EFS Bretagne, and medical consultations are growing for 50% each year since 2011. The consequence is an increase of needs of 22% of compatible packed red blood cells. To anticipate the announced progress, various actions were implemented as study groups, creation of a new informatic prescription for red blood cells phenotyping, promotion of donation, transfusion organisation. RESULTS: Fifthty-nine percent of the 400 ABO RH-KELL, FY, JK, MNS 3, 4, red blood cells were realised on the basis of this new informatic prescription, as the 99% of the packed red blood cells identified Fy (a- b-). So, 92% of the compatible transfused packed red blood cells were already in stock when the patients needed them. CONCLUSIONS: In Brittany, that organisation leads to assume qualitative and quantitative transfusion for sickle cell disease in more than 90% of cases, with fast distribution. In the same time promotion of donation is done to increase the diversity of donors.

High level of soluble programmed cell death ligand 1 in blood impacts overall survival in aggressive diffuse large B-Cell lymphoma: results from a French multicenter clinical trial.

The dosage of soluble programmed cell death ligand 1 (sPD-L1) protein in the blood of adults with cancer has never been performed in a prospective patient cohort. We evaluated the clinical impact of sPD-L1 level measured at the time of diagnosis for newly diagnosed diffuse large B-cell lymphoma (DLBCL). Soluble PD-L1 was measured in the plasma of 288 patients enrolled in a multicenter, randomized phase III trial that compared R-high-dose chemotherapy with R-CHOP. The median follow-up was 41.4 months. A cutoff of 1.52 ng/ml of PD-L1 level was determined and related to overall survival (OS). Patients with elevated sPD-L1 experienced a poorer prognosis with a 3-year OS of 76% versus 89% (P<0.001). Considering clinical characteristics, the multivariate analysis retained this biomarker besides bone marrow involvement and abnormal lymphocyte-monocyte score as independently related to poor outcome. sPD-L1 was detectable in the plasma and not in the serum, found elevated in patients at diagnosis compared with healthy subjects and its level dropped back to normal value after CR. The intention-to-treat analysis showed that elevated sPD-L1 was associated with a poorer prognosis for patients randomized within the R-CHOP arm (P<0.001). Plasma PD-L1 protein is a potent predicting biomarker in DLBCL and may indicate usefulness of alternative therapeutic strategies using PD-1 axis inhibitors.

Evaluation of CytoDiff™ on cord blood WBC differential.

INTRODUCTION: An umbilical cord blood bank was recently opened in our institution as an alternative source of hematopoietic stem cells. Before inclusion of a cord blood in an international register, a WBC with differential is requested, among others. Currently, the reference method is the microscopic manual count, and we sought to evaluate the routine flow cytometric method (CytoDiff™) as an alternative. METHODS: A total of 161 cord bloods were analyzed between November 2010 and February 2011. WBC differentials were determined for each sample, by (i) the cell counter (DxH800), (ii) a manual review, and (iii) the flow cytometry using the CytoDiff™ antibody cocktail. RESULTS: Correlation coefficients between flow cytometry and microscopic count were satisfying for neutrophils, lymphocytes, and immature granulocytes and acceptable for eosinophils. On the other hand, we found lower correlation coefficient for basophils and monocytes. Monocytes' correlation was better when comparing flow cytometry with cell counter. CONCLUSION: The flow cytometric approach is suitable to realize cord blood WBC differential and allows for the identification of additional cell subsets.

Characterization of intratumoral follicular helper T cells in follicular lymphoma: role in the survival of malignant B cells.

Accumulating evidences indicate that the cellular and molecular microenvironment of follicular lymphoma (FL) has a key role in both lymphomagenesis and patient outcome. Malignant FL B cells are found admixed to specific stromal and immune cell subsets, in particular CD4(pos) T cells displaying phenotypic features of follicular helper T cells (T(FH)). The goal of our study was to functionally characterize intratumoral CD4(pos) T cells. We showed that CXCR5(hi)ICOS(hi)CD4(pos) T cells sorted from FL biopsies comprise at least two separate cell populations with distinct genetic and functional features: (i) CD25(pos) follicular regulatory T cells (T(FR)), and (ii) CD25(neg) T(FH) displaying a FL-B cell supportive activity without regulatory functions. Furthermore, despite their strong similarities with tonsil-derived T(FH), purified FL-derived T(FH) displayed a specific gene expression profile including an overexpression of several genes potentially involved directly or indirectly in lymphomagenesis, in particular TNF, LTA, IL4 or CD40LG. Interestingly, we further demonstrated that these two last signals efficiently rescued malignant B cells from spontaneous and rituximab-induced apoptosis. Altogether, our study demonstrates that tumor-infiltrating CD4(pos) T cells are more heterogeneous than previously presumed, and underlines for the first time the crucial role of T(FH) in the complex set of cellular interactions within FL microenvironment.

[Platelet transfusion: products, indications, dose, threshold and efficacy]. / Transfusion de plaquettes: produits, indications, dose, seuil, efficacité

The quality of platelet concentrates had been greatly improved since the implementation of processing techniques based on the use of the buffy-coat in the years 1980. More recently, in the last 10 years, it is in the domain of safety that the greatest advances have been done, by the introduction of platelet additive solutions, on one hand, and by the progressive availability of pathogen reduction techniques, on the other hand. These developments in quality and safety of platelet components are important, as they lead to the conclusion that nowadays, apheresis platelet concentrates and pooled random donor platelet concentrates can be considered as equivalent, the only specific indication of the former being the care of HLA or HPA allo-immunized patients. This review covers the physiological basis of prophylactic and curative platelet transfusions, and the means to evaluate their efficacy. The main investigations that are necessary to perform in the event of an inefficient prophylactic transfusion are also mentioned. Platelet transfusion is an essential part of the transfusion support in case of central thrombocytopenia, and more indications in other medical and surgical situations are well defined. The vast majority of clinical situations in which platelet transfusions are indicated have been defined in the national guidelines published in 2003 by the AFSSAPS French authority, and are still fully valid today. Therefore, only some specific domains for which recent published data are questioning our present practices are discussed, such as the use of platelet concentrates in massive transfusion. Finally, three critical factors for establishing a coherent platelet transfusion strategy are developed: the transfusion trigger for prophylactic platelet transfusion, the platelet dose, and the impact of ABO compatibility between the product and the recipient.

Patients suffering from acute graft-versus-host disease after bone-marrow transplantation have functional CD4+CD25hiFoxp3+ regulatory T cells.

Acute Graft-Versus-Host Disease (aGVHD), mediated by CD4(+) and CD8(+) effector T cells, is a life-threatening complication in hematopoietic stem cell (HSC) transplantation. Naturally-occurring CD4(+)CD25(hi)(Foxp3(+)) regulatory T cells (T(reg)) have been shown to modulate tolerance to aGVHD in murine graft models. In this report, we investigated their role in the prevention of aGVHD in patients transplanted with bone-marrow-derived HSC. When CD4(+)CD25(hi)Foxp3(+) T cells were isolated from bone-marrow grafts, they showed no suppressive activity. The analysis of their function in patients suffering from aGVHD after transplantation revealed a gain of suppressive activity indicating their inability to control the aGVHD induction. Thus, our findings clearly demonstrate that CD4(+)CD25(+) and CD4(+)CD25(hi)Foxp3(+) T cells, when administered in steady-state physiological conditions, do not influence the outcome of aGVHD after bone-marrow transplantation.

HLA-DRB1*15 allele influences the later course of relapsing remitting multiple sclerosis.

Most of the published works so far have aimed at finding genes associated with multiple sclerosis (MS) susceptibility. Very few studies have attempted to correlate disease features with DNA variants. In a well-characterized sample (651 patients) representative of multiple sclerosis natural history, we engaged a comprehensive study of the role of human leukocyte antigen (HLA) in the course of the disease. We investigated the role of HLA-DRB1*15 allele in samples stratified according to severity evaluated by the Multiple Sclerosis Severity Score (MSSS), time to reach EDSS 6.0 and disease type. We found that HLA-DRB1*15 genotype does not influence MS severity even among patients presenting with a given type of the disease. However, we show for the first time that HLA-DRB1*15 allele modulates the course of MS for relapsing-remitting (RR) onset patients likely by precipitating the secondary progressive (SP) phase.

IL2RA and IL7RA genes confer susceptibility for multiple sclerosis in two independent European populations.

Multiple sclerosis (MS) is the most common chronic inflammatory neurologic disorder diagnosed in young adults and, due to its chronic course, is responsible for a substantial economic burden. MS is considered to be a multifactorial disease in which both genetic and environmental factors intervene. The well-established human leukocyte antigen (HLA) association does not completely explain the genetic impact on disease susceptibility. However, identification and validation of non-HLA-genes conferring susceptibility to MS has proven to be difficult probably because of the small individual contribution of each of these genes. Recently, associations with two single nucleotide polymorphisms (SNPs) in the IL2RA gene (rs12722489, rs2104286) and one SNP in the IL7RA gene (rs6897932) have been reported by several groups. These three SNPs were genotyped in a French and a German population of MS patients using the hME assay by the matrix-assisted laser desorption/ionization time of flight technology (Sequenom, San Diego, CA, USA). We show that these SNPs do contribute to the risk of MS in these two unrelated European MS patient populations with odds ratios varying from 1.1 to 1.5. The discovery and validation of new genetic risk factors in independent populations may help toward the understanding of MS pathogenesis by providing valuable information on biological pathways to be investigated.

Soluble human leucocyte antigen-G molecules in peripheral blood haematopoietic stem cell transplantation: a specific role to prevent acute graft-versus-host disease and a link with regulatory T cells.

Haematopoietic stem cell transplantation is often complicated by the life-threatening graft-versus-host disease (GVHD) which consists of an allogeneic reaction of the graft cells against the host organs. The aim of this study was to investigate the putative involvement of soluble human leucocyte antigen (sHLA) class I molecules, and particularly sHLA-G molecules, in the occurrence and/or prevention of acute GVHD (aGVHD) in allogeneic peripheral blood stem cell (PSC) transplantation. Whole sHLA class I molecules seem to be involved in aGVHD pathogenesis because detection of a high concentration of these molecules in the first month post allograft is correlated with aGVHD occurrence. Conversely, a high level of sHLA-G molecules before and after allograft could indicate good prognosis in PSC allograft transplantation. sHLA-G molecules seem to be involved in aGVHD prevention, not only because they are enriched in plasma of patients without aGVHD, but also because: (i) a positive correlation has been found between sHLA-G level and CD4+ CD25+ CD152+ natural regulatory T cell (T(reg)) frequency in the blood of transplanted patients; and (ii) the presence of CD4+ CD25+ CD152+ natural T(reg) is correlated with increased sHLA-G expression in in vitro mixed leucocyte reaction cultures. Altogether, these results support the immunomodulatory function of sHLA-G molecules that might create a regulatory network together with the natural T(reg) to foster the induction of a tolerogenic environment and improve PSC transplantation favourable outcome.

[What's new in multiple sclerosis genetics?]. / Où en sont les études génétiques de la SEP?

INTRODUCTION: Multiple Sclerosis (MS) is a multifactorial disorder caused by the interaction of environmental factors with a genetic predisposition. BACKGROUND: The chromosomal region comprising MHC contains one or several genes which contributes from 20 to 50 p. 100 to MS genetic predisposition. Other genes are unknown but are likely to have an individual contribution less than MHC. PERSPECTIVES AND CONCLUSION: Large DNA collections, high output genotyping facilities, a precise knowledge of the human genome and adequate statistical methods should allow the identification of MS predisposition genes.

Use of random versus apheresis platelet concentrates.

The respective use of random (RPC) and apheresis (APC) platelet concentrates is highly heterogeneous among countries, ranging from 10 to 98% RPC in countries supposed to provide a similar transfusion service to patients. Moreover, when considering each country in the past 10 years, one can observe that some have changed their policy, switching from a majority of APC to RPC or vice versa. This presentation intends to analyse which factors may impact such decisions. For many years, the only available platelet component was a RPC obtained from whole blood donation by a two centrifugation steps process, the "platelet rich plasma" or PRP method. Since the beginning of the 1970s, APCs became available, with in fact many different techniques leading to many APCs that may not be equivalent. Since the end of the 1980s, a new method of RPC preparation was developed, using the buffy-coat (BC-PC), providing a blood component with highly preserved platelet functions as compared to RPCs prepared by the PRP technique. Finally, the use of each of these components either native, or leuco-reduced, or suspended in a storage solution, or processed with a pathogen inactivation technique adds new layers of complexity to compare them. Innumerable references can be found in the literature describing in vitro functional parameters of platelet concentrates. Although it is clear that BC-RPC retain much more their in vitro functions than PRP-RPC, indicating that no one should use the latter any more, it is much more difficult to distinguish differences between other PCs. Conversely, only a very few studies have been published related to a comparison of clinical efficacy of RPC versus APC, the endpoints being mainly CCI. Similarly to the in vitro studies, although RPC prepared with the PRP method show the lowest CCIs, no clear difference exists between "modern" RPC and APC. Another factor that may impact policy decision is the occurrence of adverse reactions in recipients. When considering only comparable data, for example leuco-reduced RPC versus leuco-reduced APC, there is now evidence that the latter is more associated with adverse reactions in recipients: data from hemovigilance in France show that, although no difference is noted for febrile non haemolytic transfusion reactions, nor for bacteria contamination, the incidence of allergic adverse reactions is about four times higher with APC as compared with RPC. Other aspects may impact the decision: the fact that using APC in place of RPC reduces the total donor exposure of patients was considered critical in some countries to reduce the risk of transmission of blood transmissible disease. Finally, the cost of the components, much higher for APC may be considered.

A second-generation genomic screen for multiple sclerosis.

Multiple sclerosis (MS) is a debilitating neuroimmunological and neurodegenerative disorder. Despite substantial evidence for polygenic inheritance of the disease, the major histocompatibility complex is the only region that clearly and consistently demonstrates linkage and association in MS studies. The goal of this study was to identify additional chromosomal regions that harbor susceptibility genes for MS. With a panel of 390 microsatellite markers genotyped in 245 U.S. and French multiplex families (456 affected relative pairs), this is the largest genomic screen for MS conducted to date. Four regions met both of our primary criteria for further interest (heterogeneity LOD [HLOD] and Z scores >2.0): 1q (HLOD=2.17; Z=3.38), 6p (HLOD=4.21; Z=2.26), 9q (HLOD; Z=2.71), and 16p (HLOD=2.64; Z=2.05). Two additional regions met only the Z score criterion: 3q (Z=2.39) and 5q (Z=2.17). Further examination of the data by country (United States vs. France) identified one additional region demonstrating suggestive linkage in the U.S. subset (18p [HLOD=2.39]) and two additional regions generating suggestive linkage in the French subset (1p [HLOD=2.08] and 22q [HLOD=2.06]). Examination of the data by human leukocyte antigen (HLA)-DR2 stratification identified four additional regions demonstrating suggestive linkage: 2q (HLOD=3.09 in the U.S. DR2- families), 6q (HLOD=3.10 in the French DR2- families), 13q (HLOD=2.32 in all DR2+ families and HLOD=2.17 in the U.S. DR2+ families), and 16q (HLOD=2.32 in all DR2+ families and HLOD=2.13 in the U.S. DR2+ families). These data suggest several regions that warrant further investigation in the search for MS susceptibility genes.

Intercellular adhesion molecule-1: a protective haplotype against multiple sclerosis.

Intercellular adhesion molecule-1 (ICAM-1) and its receptors are adhesion molecules that play a key role in the transmigration of inflammatory cells through the blood-brain barrier, one of the earliest events in multiple sclerosis (MS), which leads to demyelination in the central nervous system. To investigate the role of genes encoding ICAM-1 and its receptors, we used a strategy of genetic linkage and association in 439 case-parent MS families of French origin, well characterized according to HLA status and severity. We demonstrate that the genes encoding ICAM-1 receptors do not influence MS susceptibility or severity. ICAM-1 had a modest, but significant effect on MS genetic susceptibility, independent of HLA and disease severity. We observed a rare, and an as yet unreported, ICAM-1 gene haplotype defined by amino acids K469 and R241 that was never transmitted to patients suggesting a protective effect against MS in our population.

Genetic and functional studies in multiple sclerosis patients from Martinique attest for a specific and direct role of the HLA-DR locus in the syndrome.

Among candidate genes involved in multiple sclerosis (MS) genetic susceptibility, MHC genes and particularly HLA-DRB1*1501-DQB1*0602 haplotype play a major role. Based on the strong linkage disequilibrium observed in Caucasians between DRB1*1501 and DQB1*0602 alleles, it is still impossible to draw a firm conclusion about the DRB1 or DQB1 locus involvement. In order to address this issue a strategy associating a genetic and a functional approach was conducted in a population of-non-Caucasian MS patients. We observed that in Martinicans (55 MS and 100 controls), the DRB1*15 and DRB1*07 alleles were positively associated with the disease. However in Martinicans the most common DRB1*15 subtype was *1503 and not *1501. Moreover, in Martinicans, the frequency of DQB1*0602, found in association with other DRB1 alleles than DRB1*15 (42% of DQB1*0602 haplotypes), was not increased in DRB1*15-negative MS patients, suggesting a neutral role of DQB1*0602 in MS genetics. In a second step, we demonstrated the capability of the DRB1*1503 allele associated with MS in Martinicans to present the immunodominant autoantigen MBP 85-99 peptide to a DRB1*1501 restricted MBP specific T cell line. Interestingly, structural features of DRB1*1501 or DRB1*1503 molecules are in good fit with the hypothesis that *1501 and *1503 molecules may act similarly in MS development by presenting the same immunodominant MBP peptide. On the whole, our results show a prominent role of the DRB1 locus (DRB1*1501 and/or DRB1*1503 alleles) in the immunodominant MBP 85-99 peptide presentation to genetically different MS patients and suggest a neutral role of the DQB1 encoded molecule in MS susceptibility.