Durable engraftment after pharmacological pre-transplant immune suppression followed by reduced-toxicity myeloablative haploidentical stem cell transplantation in highly HLA-immunized adults with sickle cell disease.

Allogeneic stem cell transplantation (Allo-SCT) is the only rapidly available curative treatment modality in patients with severe sickle cell disease (SCD). The development of reduced-toxicity myeloablative conditioning (RT-MAC) regimen and the use of partially matched family donors with post-transplantation cyclophosphamide (PT-Cy) have widened the access to Allo-SCT. Antibodies against donor-specific HLA (DSA) increase the risk of engraftment failure in HLA mismatched Allo-SCT. We report the results of five patients with SCD, whereas three with DSA, who underwent an unmanipulated haploidentical stem cell transplantation (Haplo-SCT) after a busulfan-based RT-MAC regimen with PT-Cy. To reduce the risk of engraftment failure, a sequential two courses pharmacological pre-transplant immune suppression (PTIS) phase was added prior to the conditioning regimen. All patients engrafted successfully. The procedure was well tolerated. None of the patients developed acute GVHD, whereas one developed moderate chronic GVHD. After a median follow-up of 5 years (range, 2.2-9), all patients are free of pain with excellent quality of life. Our report shows that Haplo-SCT after a RT-MAC regimen is feasible and safe with stable long-term engraftment and excellent disease control. The risk of graft failure can be abrogated by adding a PTIS phase prior to initiating the conditioning regimen.

Strong humoral response after Covid-19 vaccination correlates with the common HLA allele A*03:01 and protection from breakthrough infection.

Few data exist on the role of genetic factors involving the HLA system on response to Covid-19 vaccines. Moving from suggestions of a previous study investigating the association of some HLA alleles with humoral response to BNT162b2, we here compared the HLA allele frequencies among weak (n = 111) and strong (n = 123) responders, defined as those healthcare workers with the lowest and the highest anti-Spike antibody levels after vaccination. Individuals with clinical history of Covid-19 or positive anti-nucleocapside antibodies were excluded. We found the common HLA-A*03:01 allele as an independent predictor of strong humoral response (OR = 12.46, 95% CI: 4.41-35.21, p < 0.0001), together with younger age of vaccines (p = 0.004). Correlation between antibody levels and protection from breakthrough infection has been observed, with a 2-year cumulative incidence of 42% and 63% among strong and weak responders, respectively (p = 0.03). Due to the high frequency of HLA-A*03:01 and the need for seasonal vaccinations against SARS-CoV-2 mutants, our findings provide useful information about the inter-individual differences observed in humoral response after Covid-19 vaccine and might support further studies on the next seasonal vaccines.

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.

Characterization of 43 novel HLA-H alleles.

Forty-three novel HLA-H alleles, including 32 identified in cohorts of French donors for Hematopoietic Stem Cell Transplantation, have been characterized by Next-Generation Sequencing (NGS) using a long range PCR approach. A phylogenetic study confirms three main HLA-H clades.

A Non-Coding Fc Gamma Receptor Cis-Regulatory Variant within the 1q23 Gene Cluster Is Associated with Plasmodium falciparum Infection in Children Residing in Burkina Faso.

Antibodies play a crucial role in activating protective immunity against malaria by interacting with Fc-gamma receptors (FcγRs). Genetic variations in genes encoding FcγRs can affect immune cell responses to the parasite. In this study, our aim was to investigate whether non-coding variants that regulate FcγR expression could influence the prevalence of Plasmodium falciparum infection. Through bioinformatics approaches, we selected expression quantitative trait loci (eQTL) for FCGR2A, FCGR2B, FCGR2C, FCGR3A, and FCGR3B genes encoding FcγRs (FCGR), in whole blood. We prioritized two regulatory variants, rs2099684 and rs1771575, located in open genomic regions. These variants were identified using RegVar, ImmuNexUT, and transcription factor annotations specific to immune cells. In addition to these, we genotyped the coding variants FCGR2A/rs1801274 and FCGR2B/rs1050501 in 234 individuals from a malaria-endemic area in Burkina Faso. We conducted age and family-based analyses to evaluate associations with the prevalence of malarial infection in both children and adults. The analysis revealed that the regulatory rs1771575-CC genotype was predicted to influence FCGR2B/FCGR2C/FCGR3A transcripts in immune cells and was the sole variant associated with a higher prevalence of malarial infection in children. In conclusion, this study identifies the rs1771575 cis-regulatory variant affecting several FcγRs in myeloid and neutrophil cells and associates it with the inter-individual capacity of children living in Burkina Faso to control malarial infection.

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.

HLA-F and LILRB1 Genetic Polymorphisms Associated with Alloimmunisation in Sickle Cell Disease.

Red blood cell (RBC) transfusion remains a critical component in caring for the acute and chronic complications of sickle cell disease (SCD). Patient alloimmunisation is the main limitation of transfusion, which can worsen anaemia and lead to delayed haemolytic transfusion reaction or transfusion deadlock. Although biological risk factors have been identified for immunisation, patient alloimmunisation remains difficult to predict. We aimed to characterise genetic alloimmunisation factors to optimise the management of blood products compatible with extended antigen matching to ensure the self-sufficiency of labile blood products. Considering alloimmunisation in other clinical settings, like pregnancy and transplantation, many studies have shown that HLA Ib molecules (HLA-G, -E, and -F) are involved in tolerance mechanism; these molecules are ligands of immune effector cell receptors (LILRB1, LILRB2, and KIR3DS1). Genetic polymorphisms of these ligands and receptors have been linked to their expression levels and their influence on inflammatory and immune response modulation. Our hypothesis was that polymorphisms of HLA Ib genes and of their receptors are associated with alloimmunisation susceptibility in SCD patients. The alloimmunisation profile of thirty-seven adult SCD patients was analysed according to these genetic polymorphisms and transfusion history. Our results suggest that the alloimmunisation of SCD patients is linked to both HLA-F and LILRB1 genetic polymorphisms located in their regulatory region and associated with their protein expression level.

Characterization of the novel HLA-A*33:246 allele using next-generation sequencing.

HLA-A*33:246 differs from HLA-A*33:01:01:01 allele by one nucleotide substitution in codon 135 in exon 3.

Characterization of the novel HLA-B*07:482 allele using next-generation sequencing methods.

HLA-B*07:482 differs from HLA-B*07:02:01:01 allele by one nucleotide substitution in codon 285 in exon 5.

The novel HLA-DQA1*03:01:12 allele identified using next-generation sequencing in a Melanesian individual.

HLA-DQA1*03:01:12 differs from HLA-DQA1*03:01:01:01 by one nucleotide substitution in codon 21 in exon 2.

Targeted Central Nervous System Irradiation with Proton Microbeam Induces Mitochondrial Changes in Caenorhabditis elegans.

Fifty percent of all patients with cancer worldwide require radiotherapy. In the case of brain tumors, despite the improvement in the precision of radiation delivery with proton therapy, studies have shown structural and functional changes in the brains of treated patients with protons. The molecular pathways involved in generating these effects are not completely understood. In this context, we analyzed the impact of proton exposure in the central nervous system area of Caenorhabditis elegans with a focus on mitochondrial function, which is potentially implicated in the occurrence of radiation-induced damage. To achieve this objective, the nematode C. elegans were micro-irradiated with 220 Gy of protons (4 MeV) in the nerve ring (head region) using the proton microbeam, MIRCOM. Our results show that protons induce mitochondrial dysfunction, characterized by an immediate dose-dependent loss of the mitochondrial membrane potential (ΔΨm) associated with oxidative stress 24 h after irradiation, which is itself characterized by the induction of the antioxidant proteins in the targeted region, observed using SOD-1::GFP and SOD-3::GFP strains. Moreover, we demonstrated a two-fold increase in the mtDNA copy number in the targeted region 24 h after irradiation. In addition, using the GFP::LGG-1 strain, an induction of autophagy in the irradiated region was observed 6 h following the irradiation, which is associated with the up-regulation of the gene expression of pink-1 (PTEN-induced kinase) and pdr-1 (C. elegans parkin homolog). Furthermore, our data showed that micro-irradiation of the nerve ring region did not impact the whole-body oxygen consumption 24 h following the irradiation. These results indicate a global mitochondrial dysfunction in the irradiated region following proton exposure. This provides a better understanding of the molecular pathways involved in radiation-induced side effects and may help in finding new therapies.

HLA-F transcriptional and protein differential expression according to its genetic polymorphisms.

Many specificities single out HLA-F: its structure, expression regulation at cell membrane and function. HLA-F mRNA is detected in the most cell types and the protein is localized in the ER and Golgi apparatus. When expressed at cell surface, HLA-F may be associated to ß2-microglobulin and peptide or expressed as an open-conformer molecule. HLA-F reaches the membrane upon activation of different primary cell types and cell-lines. HLA-F has its highest affinity for the KIR3DS1-activating NK receptor, but also binds inhibitory immune receptors. Some studies reported that HLA-F expression is associated with its genotype. Higher HLA-F mRNA expression associated with F*01:01:02, and 3 noncoding SNPs, rs1362126, rs2523405, and rs2523393, located in HLA-F-AS1 or upstream the HLA-F sequence were associated with HLA-F mRNA expression. Given the implication of HLA-F in many clinical setting, and the undisclosed process of its expression regulation, we aim to confirm the effect of the aforementioned SNPs with HLA-F transcriptional and protein expression. We analyzed the distribution, frequency and linkage disequilibrium of these SNPs at worldwide scale in the 1000 Genomes Project samples. Influence on the genotype of each SNP on HLA-F expression was explored using RNAseq data from the 1000 Genomes Project, and using Q-PCR and intracellular cytometry in PBMC from healthy individuals. Our results show that the SNPs under studied displayed remarkably different allelic proportion according to geography and confirm that rs1362126, rs2523405, and rs2523393 displayed the most concordant results, with the highest effect size and a double-dose effect.

Clinical relevance of cell-free DNA quantification and qualification during the first month after lung transplantation.

Background: Many studies have reported the relevance of donor-derived cfDNA (dd-cfDNA) after lung transplantation (LTx) to diagnose and monitor acute rejection (AR) or chronic rejection or infection (INF). However, the analysis of cfDNA fragment size has not been studied. The aim of this study was to determine the clinical relevance of dd-cfDNA and cfDNA size profiles in events (AR and INF) during the first month after LTx. Methods: This prospective, single-center study includes 62 LTx recipients at the Marseille Nord Hospital, France. Total cfDNA quantification was performed by fluorimetry and digital PCR, dd-cfDNA by NGS (AlloSeq cfDNA-CareDX®), and the size profile by BIABooster (Adelis®). A bronchoalveolar lavage and transbronchial biopsies at D30 established the following groups: not-injured and injured graft (AR, INF, or AR+INF). Results: Quantification of total cfDNA was not correlated with the patient's status at D30. The percentage of dd-cfDNA was significantly higher for injured graft patients at D30 (p=0.0004). A threshold of 1.72% of dd-cfDNA correctly classified the not-injured graft patients (negative predictive value of 91.4%). Among recipients with dd-cfDNA >1.72%, the quantification of small sizes (80-120bp) >3.70% identified the INF with high performance (specificity and positive predictive value of 100%). Conclusion: With the aim of considering cfDNA as a polyvalent non-invasive biomarker in transplantation, an algorithm combining the quantification of dd-cfDNA and small sizes of DNA may significantly classify the different types of allograft injuries.

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.

Therapeutic efficacy of platelet transfusion treated with amotosalen/UVA pathogen inactivation technology (INTERCEPTTM Blood System) in acute myeloid leukemia patients undergoing chemotherapy with curative intent: a single center experience.

BACKGROUND: The INTERCEPTTM Blood System (Intercept Blood System, Cerus Europe BV, Amersfoort, the Netherlands) has been used to reduce or inactivate pathogen load in platelet concentrates in France for three years. MATERIALS AND METHODS: After comparing the transfusion efficiency between pathogen-reduced platelets (PR_PLT) and untreated platelet products (U_PLT), our single-center observational study assessed the effectiveness of PR_PLT for the prevention of bleeding and for therapeutic treatment of WHO grade 2 bleeding in 176 patients undergoing chemotherapy with curative intent for acute myeloid leukemia (AML). The main endpoints were the 24-hour (h) corrected count increment (24h_CCI) after each transfusion, and time to next transfusion. RESULTS: Whereas the transfused doses tended to be higher in the PR_PLT group compared to U_PLT, there was a significant difference in intertransfusion interval (ITI) and 24h_CCI. In prophylactic transfusions, PR_PLT transfusions of >0.65×1011/10 kg, regardless of the age of the product (day 2 to day 5), resulted in a 24h_CCI similar to that of the untreated platelet product; this meant the patient could be transfused at least every 48h. In contrast, most PR_PLT transfusions of <0.55×1011/10 kg did not achieve a transfusion interval of 48h. In the context of WHO grade 2 bleeding, PR_PLT transfusions >0.65×1011/10 kg and storage of less than 4 days seems more effective in stopping bleeding. DISCUSSION: These results, which must be confirmed by prospective studies, indicate the need for vigilance regarding the quantity and quality of PR_PLT products used to treat patients at risk of bleeding crisis. Future prospective studies are needed to confirm these findings.

New methods for the quantification of mixed chimerism in transplantation.

Background: Quantification of chimerism showing the proportion of the donor in a recipient is essential for the follow-up of hematopoietic stem cell transplantation but can also be useful to document an immune tolerance situation after solid organ transplantation. Historically, chimerism has been quantified from genomic DNA, but with technological advances, chimerism from donor-derived cell-free DNA seems particularly relevant in solid organ transplantation. Methods: The reference method was until recently the short tandem repeat technique, but new innovative techniques as digital PCR (dPCR) and NGS, have revolutionized the quantification of chimerism, such as the so-called microchimerism analysis. After a short review of chimerism methods, a comparison of chimerism quantification data for two new digital PCR systems (QIAcuity™ dPCR (Qiagen®) and QuantStudio Absolute Q (ThermoFisher®) and two NGS-based chimerism quantification methods (AlloSeq HCT™ (CareDx®) and NGStrack™ (GenDX®)) was performed. Results: These new methods were correlated and concordant to routinely methods (r²=0.9978 and r²=0.9974 for dPCR methods, r²=0.9978 and r²=0.9988 for NGS methods), and had similar high performance (sensitivity, reproductibility, linearity). Conclusion: Finally, the choice of the innovative method of chimerism within the laboratory does not depend on the analytical performances because they are similar but mainly on the amount of activity and the access to instruments and computer services.

TWEAK and TNFα, Both TNF Ligand Family Members and Multiple Sclerosis-Related Cytokines, Induce Distinct Gene Response in Human Brain Microvascular Endothelial Cells.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the TNF ligand family involved in various diseases including brain inflammatory pathologies such as multiple sclerosis. It has been demonstrated that TWEAK can induce cerebrovascular permeability in an in vitro model of the blood-brain barrier. The molecular mechanisms playing a role in TWEAK versus TNFα signaling on cerebral microvascular endothelial cells are not well defined. Therefore, we aimed to identify gene expression changes in cultures of human brain microvascular endothelial cells (hCMEC/D3) to address changes initiated by TWEAK exposure. Taken together, our studies highlighted that gene involved in leukocyte extravasation, notably claudin-5, were differentially modulated by TWEAK and TNFα. We identified differential gene expression of hCMEC/D3 cells at three timepoints following TWEAK versus TNFα stimulation and also found distinct modulations of several canonical pathways including the actin cytoskeleton, vascular endothelial growth factor (VEGF), Rho family GTPases, and phosphatase and tensin homolog (PTEN) pathways. To our knowledge, this is the first study to interrogate and compare the effects of TWEAK versus TNFα on gene expression in brain microvascular endothelial cells.

Mobilisation of HLA-F on the surface of bronchial epithelial cells and platelets in asthmatic patients.

Uncontrolled inflammation of the airways in chronic obstructive lung diseases leads to exacerbation, accelerated lung dysfunction and respiratory insufficiency. Among these diseases, asthma affects 358 million people worldwide. Human bronchial epithelium cells (HBEC) express both anti-inflammatory and activating molecules, and their deregulated expression contribute to immune cell recruitment and activation, especially platelets (PLT) particularly involved in lung tissue inflammation in asthma context. Previous results supported that HLA-G dysregulation in lung tissue is associated with immune cell activation. We investigated here HLA-F expression, reported to be mobilised on immune cell surface upon activation and displaying its highest affinity for the KIR3DS1-activating NK receptor. We explored HLA-F transcriptional expression in HBEC; HLA-F total expression in PBMC and HBEC collected from healthy individuals at rest and upon chemical activation and HLA-F membrane expression in PBMC, HBEC and PLT collected from healthy individuals at rest and upon chemical activation. We compared HLA-F transcriptional expression in HBEC from healthy individuals and asthmatic patients and its surface expression in HBEC and PLT from healthy individuals and asthmatic patients. Our results support that HLA-F is expressed by HBEC and PLT under healthy physiological conditions and is retained in cytoplasm, barely expressed on the surface, as previously reported in immune cells. In both cell types, HLA-F reaches the surface in the inflammatory asthma context whereas no effect is observed at the transcriptional level. Our study suggests that HLA-F surface expression is a ubiquitous post-transcriptional process in activated cells. It may be of therapeutic interest in controlling lung inflammation.