Evaluation ABO-related clinical response to MSCs.

<p>Patient characteristics and evaluation of clinical response to individual MSC-infusions in patients undergoing HSCT (Stockholm, n = 70; and Leiden, n = 35 MSC infusions). Blood type O (containing highest titers of both anti-A/B antibodies) was compared to blood type A, B, and AB (blood containing anti-B, anti-A, or no anti-A/B antibodies, respectively). Abbreviations: HSCT, hematopoietic stem cell transplantation; MSC, mesenchymal stromal cell; BG, blood group; HLA, human leukocyte antigen. Statistics: P-value is calculated using Mann-Whitney rank-sum test (for continuous variables), Fisher’s exact t-test (comparing two categorical variables), or Chi²-test (comparing more than two categorical variables).</p

<i>ABO</i> genotyping and detection of blood group antigens on MSCs.

<p>The <i>ABO</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085040#pone.0085040-HosseiniMaaf1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085040#pone.0085040-Olsson2" target="_blank">[26]</a> and <i>FUT2</i> (secretor) genotype of MSCs was determined with PCR, the ABH histo-blood group phenotype predicted accordingly, and expression of ABH antigens was detected with flow cytometry. The relative fluorescence intensity (RFI) for binding of anti-A, -B, and –H was calculated by dividing the median fluorescence intensity (MFI) obtained for cells labeled with the corresponding antibody, by the MFI obtained for cells labeled with secondary antibody only. Abbreviations: <i>ABO</i>-ASP, <i>ABO</i> allele-specific primer PCR; <i>FUT2</i>-ASP, fucosyltransferase 2 allele-specific primer PCR (nucleotide 428 polymorphism).</p

Adsorption of ABO antigens to MSCs from human AB plasma.

<p>MSCs are often washed and reconstituted in human AB plasma when prepared for systemic infusion. We therefore used flow cytometry, in order to detect potential binding of A/B-antigens to freshly thawed blood type O MSCs (n = 4) after incubation with human plasmas of different blood types (n = 3 each). (<b>A</b>) Histogram overlay for detection of soluble A/B-antigen binding from plasma to MSCs after a 1-hour (pink curve) or 3-hour (blue curve) incubation with 10% or 100% O, A₁B, A₂B, or clinical A_1/2B plasma of unknown A1 or A2 subtype. Upon incubation, cells were washed with serum free media, to remove non-bound plasma components, and A/B-antigen binding was detected with primary mouse-anti-human A/B antibody, followed by incubation with secondary rat-anti-mouse-PE antibody (RAM-PE), and compared to binding of secondary antibody only to untreated cells (black curves). (<b>B</b>) Cells detected positive (%) after two different plasma adsorption times (1 <i>vs</i>. 3 hours). Five AB plasmas of unknowns A subtype, which were previously used for clinical MSC infusion (A_1/2 B), were compared to O, A₁B and A₂B plasma. As controls, cells were labeled with CD14-PE (negative labeling control), CD105-PE (positive control), and anti-paragloboside (PG) + RAM-PE (secondary antibody labeling control), or anti-A/B + RAM-PE, to detect A/B-antigen binding, as indicated below the figure. Mean±SD, <b><i>*</i></b><i>P</i> < 0.05.</p

<i>ABO</i> promoter methylation and <i>ABO</i> transcript expression analysis.

<p>The <i>ABO</i> promoter methylation status (n = 4 clinical grade MSCs, 2 samples each) and the expression of ABO blood group gene transcripts in clinical grade MSCs (n = 2) of known blood group and secretor genotype (K16-<i>A¹/A¹-Se/se</i>, K25-<i>B/O¹-Se/Se</i>) was tested with MagMeDIP Kit and quantitative real time PCR (qPCR), respectively. (<b>A</b>) DNA methylation (%, metDIP/input), of methylated <i>TSH2B</i>, unmethylated <i>GAPDH</i>, and <i>ABO</i> proximal promoter region. (<b>B</b>) <i>ABO</i> transcript analysis (qPCR) on resting MSCs compared to cells subjected to different types of induction treatments. MSCs were either stimulated for 5 days with 5 ng/ml of interferon-gamma (INFg), or activated for 5 days by inflammatory mediators released through a cell-impermeable membrane in trans-well mixed lymphocyte reactions (MLRs). MSCs were also subjected to 14-day <i>in vitro</i> differentiation with adipogenic (ADI) and osteogenic (OST) induction medium, or respective control medium. Adenocarcinoma cell line HPAF-II was used as positive control for <i>ABO</i> transcript expression and distilled H₂O served as negative control. Control genes <i>ICAM1</i> and <i>aP2</i> served as positive controls for cytokine activation and adipogenic induction, respectively. Relative gene expression is shown compared to control gene beta-actin. Adipogenic and osteogenic differentiation was also confirmed with Oil red O staining for lipid rich vacuoles and von Kossa staining for mineralized matrix, respectively. Mean ± SD, **<b><i>*</i></b><i>P</i> < 0.001.</p

Whole blood exposure of MSCs reconstituted in different supplements.

<p>Freshly harvested or freeze-thawed MSCs (15,000 cells per ml, n = 18), washed and reconstituted in buffer containing 10% human AB plasma (ABP) or 10% human serum albumin (HSA), were tested for triggering of the instant blood mediated inflammatory reaction (IBMIR) by exposing them to non-anticoagulated whole blood in the chandler blood loop model. (<b>A</b>) Representative photographs of clot formation after a 60-min incubation of blood with MSCs, or buffer as negative control. (<b>B</b>) Percentage (% relative to PBS, 30 minutes time point) of coagulation and complement activation markers after treatment of blood with fresh or thawed MSCs (MSC-F or MSC-T, respectively, n = 18 each): free platelets, and ELISA quantification of thrombin-anti-thrombin complex (TAT), and complement C3 activation fragment a (C3a). Box plot whiskers Tukey: <b><i>*’</i></b><i>P</i><0.1, <b><i>*</i></b><i>P</i><0.05, and *<b><i>*</i></b><i>P</i><0.01.</p

HSCT patient ABO antibody titers at the time of MSC infusion.

<p>Abbreviations: MSC, mesenchymal stromal cell; HSC, hematopoietic stem cell; R/D, donor/recipient; Anti-A or anti-B IgM/IgG, agglutination titers of anti blood group antigen A or B antibodies of immunoglobulin M or G type; GvHD, graft versus host disease; and clinical response to MSC treatment expressed as: CR, complete response; PR, partial response; SD, stable disease; and PD, progressive disease.</p

Adsorption of ABO antigens to MSCs from human AB culture serum.

<p>Clinical MSCs can be grown with three different culture supplements at our centers: 10% fetal calf serum (FCS), 5% human blood type AB serum (ABS), and 5% human platelet rich plasma (PRP), which may potentially contain antigens recognized by ABO-antibodies in patient blood <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085040#pone.0085040-Galili1" target="_blank">[8]</a>. We therefore used flow cytometry in order to detect the binding of anti-A/B antibodies to low passage MSCs (P1-4, n = 8 donors), expanded with either supplement. (<b>A</b>) Representative histogram overlays for binding of anti-A (red curve), anti-B (blue), and anti-H (green) antibodies to MSCs grown with different supplements, as compared to respective isotype controls (black curves). (<b>B</b>) Quantification of relative fluorescence intensity (RFI) compared to respective isotype controls, showing expression intensity of typical MSC surface antigens; Negative controls: CD45 (leukocyte common antigen) and CD14 (LPS receptor on myeloid cells); Positive controls: CD44 (Hyaluronic acid receptor), CD73 (Ecto-5’-nucelotidase), CD90 (Thy-1 antigen), CD105 (Endoglin); and binding of anti-A, anti-B, and anti-H antibodies. Mean ± SD, <b><i>*</i></b><i>P</i> < 0.05.</p

Image_1_Immunological signatures unveiled by integrative systems vaccinology characterization of dengue vaccination trials and natural infection.pdf

IntroductionDengue virus infection is a global health problem lacking specific therapy, requiring an improved understanding of DENV immunity and vaccine responses. Considering the recent emerging of new dengue vaccines, here we performed an integrative systems vaccinology characterization of molecular signatures triggered by the natural DENV infection (NDI) and attenuated dengue virus infection models (DVTs).Methods and resultsWe analyzed 955 samples of transcriptomic datasets of patients with NDI and attenuated dengue virus infection trials (DVT1, DVT2, and DVT3) using a systems vaccinology approach. Differential expression analysis identified 237 common differentially expressed genes (DEGs) between DVTs and NDI. Among them, 28 and 60 DEGs were up or downregulated by dengue vaccination during DVT2 and DVT3, respectively, with 20 DEGs intersecting across all three DVTs. Enriched biological processes of these genes included type I/II interferon signaling, cytokine regulation, apoptosis, and T-cell differentiation. Principal component analysis based on 20 common DEGs (overlapping between DVTs and our NDI validation dataset) distinguished dengue patients by disease severity, particularly in the late acute phase. Machine learning analysis ranked the ten most critical predictors of disease severity in NDI, crucial for the anti-viral immune response. ConclusionThis work provides insights into the NDI and vaccine-induced overlapping immune response and suggests molecular markers (e.g., IFIT5, ISG15, and HERC5) for anti-dengue-specific therapies and effective vaccination development. </p

Table_1_Immunological signatures unveiled by integrative systems vaccinology characterization of dengue vaccination trials and natural infection.xlsx

IntroductionDengue virus infection is a global health problem lacking specific therapy, requiring an improved understanding of DENV immunity and vaccine responses. Considering the recent emerging of new dengue vaccines, here we performed an integrative systems vaccinology characterization of molecular signatures triggered by the natural DENV infection (NDI) and attenuated dengue virus infection models (DVTs).Methods and resultsWe analyzed 955 samples of transcriptomic datasets of patients with NDI and attenuated dengue virus infection trials (DVT1, DVT2, and DVT3) using a systems vaccinology approach. Differential expression analysis identified 237 common differentially expressed genes (DEGs) between DVTs and NDI. Among them, 28 and 60 DEGs were up or downregulated by dengue vaccination during DVT2 and DVT3, respectively, with 20 DEGs intersecting across all three DVTs. Enriched biological processes of these genes included type I/II interferon signaling, cytokine regulation, apoptosis, and T-cell differentiation. Principal component analysis based on 20 common DEGs (overlapping between DVTs and our NDI validation dataset) distinguished dengue patients by disease severity, particularly in the late acute phase. Machine learning analysis ranked the ten most critical predictors of disease severity in NDI, crucial for the anti-viral immune response. ConclusionThis work provides insights into the NDI and vaccine-induced overlapping immune response and suggests molecular markers (e.g., IFIT5, ISG15, and HERC5) for anti-dengue-specific therapies and effective vaccination development. </p