Potential deleterious role of anti-Neu5Gc antibodies in xenotransplantation

International audienceHuman beings do not synthesize the glycolyl form of the sialic acid (Neu5Gc) and only express the acetylated form of the sugar, whereas a diet-based intake of Neu5Gc provokes a natural immunization and production of anti-Neu5Gc antibodies in human serum. However , Neu5Gc is expressed on mammal glycoproteins and glycolipids in most organs and cells. We review here the relevance of Neu5Gc and anti-Neu5Gc antibodies in the context of xenotransplantation and the use of animal-derived molecules and products, as well as the possible consequences of a long-term exposure to anti-Neu5Gc antibodies in recipients of xenografts. In addition, the importance of an accurate estimation of the anti-Neu5Gc response following xenotransplantation and the future contribution of knockout animals mimicking the human situation are also assessed

IL-34 Actions on FOXP3+ Tregs and CD14+ Monocytes Control Human Graft Rejection

International audienceCytokines are major players regulating immune responses toward inflammatory and tolerogenic results. In organ and bone marrow transplantation, new reagents are needed to inhibit tissue destructive mechanisms and eventually induce immune tolerance without overall immunosuppression. IL-34 is a cytokine with no significant homology with any other cytokine but that acts preferentially through CSF-1R, as CSF-1 does, and through PTPζ and CD138. Although IL-34 and CSF-1 share actions, a detailed analysis of their effects on immune cells needs further research. We previously showed that both CD4+ and CD8+ FOXP3+ Tregs suppress effector T cells through the production of IL-34, but not CSF-1, and that this action was mediated through antigen-presenting cells. We showed here by single-cell RNAseq and cytofluorimetry that different subsets of human monocytes expressed different levels of CSF-1R, CD138, and PTPζ and that both CD4+ and CD8+ FOXP3+ Tregs expressed higher levels of CSF-1R than conventional T cells. The effects of IL-34 differed in the survival of these different subpopulations of monocytes and RNAseq analysis showed several genes differentially expressed between IL-34, CSF-1, M0, M1, and also M2 macrophages. Acute graft-vs.-host disease (aGVHD) in immunodeficient NSG mice injected with human PBMCs was decreased when treated with IL-34 in combination with an anti-CD45RC mAb that depleted conventional T cells. When IL-34-differentiated monocytes were used to expand Tregs in vitro, both CD4+ and CD8+ FOXP3+ Tregs were highly enriched and this effect was superior to the one obtained with CSF-1. Human CD8+ Tregs expanded in vitro with IL-34-differentiated allogeneic monocytes suppressed human immune responses in an NSG mouse aGVHD model humanized with hPBMCs. Overall, we showed that IL-34 induced the differentiation of human monocytes with a particular transcriptional profile and these cells favored the development of potent suppressor FOXP3+ Tregs

Characterization of N-glycosylation and amino acid sequence features of immunoglobulins from swine

International audienceThe primary goal of this study was to develop a method to study the N-glycosylation of IgG from swine in order to detect epitopes containing N-glycolylneuraminic acid (Neu5Gc) and/or terminal galactose residues linked in α1-3 susceptible to cause xenograft-related problems. Samples of immunoglobulin were isolated from porcine serum using protein-A affinity chromatography. The eluate was then separated on electrophoretic gel, and bands corresponding to the N-glycosylated heavy chains were cut off the gel and subjected to tryptic digestion. Peptides and glycopeptides were separated by reversed phase liquid chromatography and fractions were collected for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) analysis. Overall no α1-3 galactose was detected, as demonstrated by complete susceptibility of terminal galactose residues to β-galactosidase digestion. Neu5Gc was detected on singly sialylated structures. Two major N-glycopeptides were found, EEQFNSTYR and EAQFNSTYR as determined by tandem MS (MS/MS), as previously reported by Butler et al. (Immu-nogenetics, 61, 2009, 209-230), who found 11 subclasses for porcine IgG. Out of the 11, ten include the sequence corresponding to EEQFNSTYR, and only one codes for EAQFNSTYR. In this study, glycosylation patterns associated with both chains were slightly different, in that EEQFNSTYR had a higher content of galactose. The last step of this study consisted of peptide-mapping the 11 reported porcine IgG sequences. Although there was considerable overlap, at least one unique tryptic peptide was found per IgG sequence. The workflow presented in this manuscript constitutes the first study to use MALDI-TOF-MS in the investigation of porcine IgG structural features

Characterization of immunoglobulins through analysis of N-glycopeptides by MALDI-TOF MS

International audienceThe aim of this report is to emphasize the role, usefulness and power of matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the analysis of glycoforms of antibodies (Abs) through their proteolytic glycopeptides. Abs are complex biomolecules in which glycans hold determinant properties and thus need to be thoroughly characterized following Ab production by recombinant methods or Ab collection from human/animal serum or tissue. In spite of the great robustness of MALDI-TOF MS in terms of tolerance to impurities, the analysis of Abs and Ab components using this technique requires extensive sample preparation involving all or some of chromatography, solid phase extraction, enzymatic modification, and chemical derivatization. This report focuses on a monoclonal Ab produced in cell culture, as well as on a polyclonal human immunoglobulin (Ig) G obtained commercially and a poly-clonal porcine IgG obtained from serum. A method is first provided to separate Ab protein chain components (light chains, heavy chains) by gel electrophoresis, which is useful for instance for protein-A eluates of Igs either from cell culture or biological samples. This allows for in-gel proteolytic digestion of the protein gel band(s) of choice for further MS characterization. Also discussed is the more conventional in-solution overnight digestion method used here with each of two proteolytic enzymes, i.e. trypsin and chymotrypsin. The overnight method is in turn compared with a much faster approach, that of digesting Abs with trypsin or chymotrypsin through the action of microwave heating. For method comparison, gly-copeptides are fractionated from digestion mixtures using mostly C-18 cartridges for simplicity, although this enrichment procedure is also compared with other published procedures. The advantages of MALDI tandem mass spectrometry are highlighted for glycopeptide analysis, and lastly an esterification method applied to glycopeptides is discussed for retention of sialic acid residues on peptide acidic glycoforms

MicroRNA-29b Modulates Innate and Antigen-Specific Immune Responses in Mouse Models of Autoimmunity

International audienceIn addition to important regulatory roles in gene expression through RNA interference, it has recently been shown that microRNAs display immune stimulatory effects through direct interaction with receptors of innate immunity of the Toll-like receptor family, aggravating neuronal damage and tumour growth. Yet no evidence exists on consequences of microRNA immune stimulatory actions in the context of an autoimmune disease. Using microRNA analogues, we here show that pancreatic beta cell-derived microRNA sequences induce pro-inflammatory (TNFa, IFNa, IL-12, IL-6) or suppressive (IL-10) cytokine secretion by primary mouse dendritic cells in a sequence-dependent manner. For miR-29b, immune stimulation in RAW264.7 macrophages involved the endosomal Toll-like receptor-7, independently of the canonical RNA interference pathway. In vivo, the systemic delivery of miR-29b activates CD11b+B2202 myeloid and CD11b-B220+ plasmacytoid dendritic cells and induces IFNa, TNFa and IL-6 production in the serum of recipient mice. Strikingly, in a murine model of adoptive transfer of autoimmune diabetes, miR-29b reduces the cytolytic activity of transferred effector CD8+ T-cells, insulitis and disease incidence in a single standalone intervention. Endogenous miR-29b, spontaneously released from beta- cells within exosomes, stimulates TNFa secretion from spleen cells isolated from diabetes-prone NOD mice in vitro. Hence, microRNA sequences modulate innate and ongoing adaptive immune responses raising the question of their potential role in the breakdown of tolerance and opening up new applications for microRNA-based immune therapy

Increased IGM and IGG anti-NEU5GC antibodies in infectious mononucleosis (IMN): link with multiple sclerosis (MS)?

International audienceIntroduction: Multiple Sclerosis (MS) is a chronic inflammatory and demyelinating disease which displays an autoimmune component at its overt clinical stage. Among etiological hypotheses, the potential initiating role of EBV in the disease has been raised following works linking EBNA levels and the risk of MS occurrence [1] showing a linkage between the occurrence of IMN and MS. Our hypothesis is that a vigorous production of anti-Neu5Gc in IMN (as indirectly measured by Paul-Bunnell Davidson reaction) may induce brain blood barrier alterations when the EBV primo-infection is contemporary of an extraordinary high frequency of EBV committed T cells and of circulating EBV+ memory B cells[2]. Our aim was to measure anti-Neu5Gc IgG and IgM levels during the IMN and to investigate whether levels of anti-NeuGc could be also increased in MS. Material and Methods: Sera from 49 patients with overt IMN were collected from the University Hospital of Grenoble and Nantes. The gender ratio was 22F/ 27M. The age average was 24 years (range: 7- 65). Two cohorts of EBV negative individuals (n= 45) and healthy individuals (n= 120) were used as controls. A second cohort of 46 patients with proven MS was collected from the University Hospital of Nantes. The gender ratio was 30F/ 16M. The age average was 48 years (range: 22-73). This cohort contained 30 patients presenting a Remitting Relapsing (RR) form (65.2%) and 16 with a Secondary or Primary Progressive (SP-PP) one (34.7%). A cohort of healthy individuals (n= 37) matched for gender and age was used as controls. On these samples, we performed two Elisa [3][4] to quantify anti-Neu5Gc IgG and IgM antibodies. Results: We showed a highly significant increase in anti-Neu5Gc IgM in IMN patients in comparison to EBV negative controls (0.82±0.16 vs. 0.15±0.05 ng/ul; p<0.0001). Anti-Neu5Gc IgG were also, though less dramatically, increased (3.64±0.6 vs. 2.78±0.57ng/ul; p=0.04). The values obtained in IMN differed significantly to healthy individuals (0.82±0.16 vs. 0.2±0.03 ng/ul;p<0.0001 for IgM and 3.63±0.6 vs. 2.74±0.3 ng/ul; p= 0.01 for IgG). The levels of anti-α1-3Gal epitopes, an other type of «xeno» antibody against a sugar not synthesized by normal individuals were also increased during IMN (12.4±1.1 vs. 9.67±1.46 ng/ul for EBV negative controls and vs. 11.7±3.4 ng/ul for healthy individuals; p<0.0001 for both). We found neither increase in anti-Neu5gc antibodies in this small MS patient cohort (p=0.8 for IgM and p=0.84 for IgG), nor in RR or SP-PP subgroups. We found no correlation between the anti-Neu5Gc titers and age or gender of IMN or MS patients, or of healthy individuals. Conclusion: Despite the fact that EVB does not express Neu5Gc, anti-Neu5Gc antibodies are strongly increased in IMN patients, suggesting that uptake of Neu5Gc from dietary sources by EBV infected cells induces them to become more immunogenic during the acute disease. However our data did not evidence higher anti-Neu5Gc antibodies in established MS

Anti-Gal and Anti-Neu5Gc Responses in Nonimmunosuppressed Patients After Treatment With Rabbit Antithymocyte Polyclonal IgGs

International audienceBackground. Polyclonal antihuman thymocyte rabbit IgGs (antithymocyte globulin [ATG]) are popular immunosuppressive drugs used to prevent or treat organ or bone-marrow allograft rejection, graft versus host disease, and autoimmune diseases. However, animal-derived glycoproteins are also strongly immunogenic and rabbit ATG induces serum sickness disease in almost all patients without additional immunosuppressive drugs, as seen in the Study of Thymoglobulin to arrest Type 1 Diabetes (START) trial of ATG therapy in new-onset type 1 diabetes.Methods. Using enzyme-linked immunosorbent assay, we analyzed serial sera from the START study to decipher the various anti-ATG specificities developed by the patients in this study: antitotal ATG, but also antigalactose-α1-3-galactose (Gal) and anti-Neu5Gc antibodies, 2 xenocarbohydrate epitopes present on rabbit IgG gly-cans and lacking in humans.Results. We show that diabetic patients have substantial levels of preexisting antibodies of the 3 specificities, before infusion, but of similar levels as healthy individuals. ATG treatment resulted in highly significant increases of both IgM (for anti-ATG and anti-Neu5Gc) and IgG (for anti-ATG,-Gal, and-Neu5Gc), peaking at 1 month and still detectable 1 year postinfusion. Conclusions. Treatment with rabbit polyclonal IgGs in the absence of additional immunosuppression results in a vigorous response against Gal and Neu5Gc epitopes, contributing to an inflammatory environment that may compromise the efficacy of ATG therapy. The results also suggest using IgGs lacking these major xenoantigens may improve safety and efficacy of ATG treatment

Extracellular hemoglobin combined with an O 2 ‐generating material overcomes O 2 limitation in the bioartificial pancreas: Extracellular hemoglobin combined with an O-2-generating material overcomes O-2 limitation in the bioartificial pancreas

ISI Document Delivery No.: HR1SX Times Cited: 1 Cited Reference Count: 61 Moure, Anne Bacou, Elodie Bosch, Steffi Jegou, Dominique Salama, Apolline Riochet, David Gauthier, Olivier Blancho, Gilles Soulillou, Jean-Paul Poncelet, Denis Olmos, Eric Bach, Jean-Marie Mosser, Mathilde Bosch, Steffi/A-1557-2009 Bosch, Steffi/0000-0002-0995-2775; Moure, Anne/0000-0002-1891-3698 Agence Nationale de la Recherche (ECTIS IHU program, France) [ANR-10-IBHU-005]; Pays de la Loire Region (Xenothera program, Nantes, France) [2011-1296]; University of Nantes (Interdisciplinary program, Nantes, France) [2017-2203] Agence Nationale de la Recherche (ECTIS IHU program, France), Grant/Award Number: ANR-10-IBHU-005; Pays de la Loire Region (Xenothera program, Nantes, France), Grant/Award Number: 2011-1296; University of Nantes (Interdisciplinary program, Nantes, France), Grant/Award Number: 2017-2203 1 4 15 Wiley Hoboken 1097-0290International audienceThe bioartificial pancreas encapsulating pancreatic islets in immunoprotective hydrogel is a promising therapy for Type 1 diabetes. As pancreatic islets are highly metabolically active and exquisitely sensitive to hypoxia, maintaining O-2 supply after transplantation remains a major challenge. In this study, we address the O-2 limitation by combining silicone-encapsulated CaO2 (silicone-CaO2) to generate O-2 with an extracellular hemoglobin O-2-carrier coencapsulated with islets. We showed that the hemoglobin improved by 37% the O-2-diffusivity through an alginate hydrogel and displayed antioxidant properties neutralizing deleterious reactive O-2 species produced by silicone-CaO2. While the hemoglobin alone failed to maintain alginate macroencapsulated neonate pig islets under hypoxia, silicone-CaO2 alone or combined to the hemoglobin restored islet viability and insulin secretion and prevented proinflammatory metabolism (PTGS2 expression). Interestingly, the combination took the advantages of the two individual strategies, improved neonate pig islet viability and insulin secretion in normoxia, and VEGF secretion and PDK1 normalization in hypoxia. Moreover, we confirmed the specific benefits of the combination compared to silicone-CaO2 alone on murine pseudo-islet viability in normoxia and hypoxia. For the first time, our results show the interest of combining an O-2 provider with hemoglobin as an effective strategy to overcome O-2 limitations in tissue engineering

Stimulation of the TLR-7 pathway by miR-29b in murine RAW264.7 macrophages.

<p>(A) 2′-O-methyl modifications were introduced in all uracil residues of the miR-29b reverse strand as indicated. RAW264.7 cells were plated four hours before stimulation with DOTAP-embedded miR-29b, 2′-O-Me-modified miR-29b, or the control miR-127 (750 nM working concentration). TNFa was quantified in supernatants eighteen hours later. 2′-O-Me modifications were introduced in the miR-29b reverse strand before annealing to the unmodified guide strand. Results are represented as individual values of cytokine concentrations (pg/ml). Data from one representative experiment out of three is shown. *<i>P</i><0.05 (Mann-Whitney). (B) Intracellular distribution of Alexa-488-labelled miR-29b 1hour after transfection of RAW264.7 cells was observed with a confocal fluorescence microscope. Top row: IF EEA-1 on fixed cells Bottom row: lysotracker in living cells. Scale bar = 20 µm for fluorescence images and overlays with differential interference contrast (DIC) (a–d, f–i) except for enlarged single cell images scale bar = 5 µm (e, j). (C) Chloroquine (CQ) was added to the plated RAW264.7 cells, at a final concentration of 10 nM, 30 minutes before stimulation with miR-29b or the miR-127 control (750 nM). Supernatants were harvested eighteen hours later for TNFa quantification. Results are represented as individual values of cytokine concentrations (pg/ml) compiled from two independent experiments. **<i>P</i><0.01 (Mann-Whitney) (D) RAW264.7 cells were stimulated with miR-29b, miR-127 (750 nM), the positive controls TLR-7-ligand imiquimod and R848, or were left untreated (NT), and were cultured eighteen hours with or without the TLR-7 antagonist IRS661. TNFa was quantified in supernatants. Results are presented as mean cytokine concentration of replicates (pg/ml) ± SEM. Data from one representative experiment out of three is shown.</p

Cytokine profile in BALB/c mice serum after intravenous miRNA delivery.

<p>Cytokine content in serum from BALB/c mice was analysed by a BD Cytometric Bead Array two and seven hours following intravenous injection of miR29b, the immune-silent miR-127 or positive (R848) or negative (HBS) controls. Results are presented as mean concentration (pg/ml) ± SEM from two experiments (n = 4 total mice); nd: not detected.</p><p>Cytokine profile in BALB/c mice serum after intravenous miRNA delivery.</p