Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: HA-49K orthologs from the A549-based expression system function comparably to natural E3/49K and exhibit a consistent binding activity to CD45 expressing target cells. Cell supernatants containing individual E3/49K variants were incubated together with target cells and bound E3/49K was measured via flow cytometry. Binding activity of HA-49K of HAdV-D64 from the A549-based producer cell line was compared to E3/49K obtained from cells infected with HAdV-D64, HAdV-D64ΔE3 and HAdV-D64ΔE3 + 49K viruses . Supernatants were incubated with wild-type Jurkat (red), Ramos (blue) and the CD45-deficient Jurkat (orange) and Ramos (light blue) cells, respectively. The grey dashed vertical line separates results obtained from transfected cells (left) from those of infected cell lines (right). Binding of HA-49K and E3/49K was detected with 4D1 mAb (A) . Binding activity to Jurkat and Ramos cell lines of recombinant HAdV-D64 HA-49K of was compared with HA-tagged orthologs of HAdV-D8, -D19 and -D36, respectively. Binding was determined with the target cell system as applied in the previous experiment using α-HA Ab (B) . Contrasting the binding of HA-49K and untagged E3/49K from the A549-based expression system to Jurkat cells reveals no negative influence to the binding activity by the HA-tag. Bound E3/49K versions were detected using HA-specific (blue) or E3/49K-specific (4D1, red) mAbs (C) . The binding specificity of HA-49K orthologs was further characterized by competition with untagged E3/49K of HAdV-D64. Residual binding activity of HA-49K orthologs was monitored by flow cytometry using α-HA Abs and a two-step sequential incubation of Jurkat cells with supernatants, containing E3/49K variants. The order of the individual incubations for competition is indicated within the figure. Significant differences to single incubations were analyzed (D) . Cell supernatants from normal A549 cells were utilized as negative controls. The columns represent the mean-MFIs obtained in independent experiments, each depicted as dots, and error-bars represent the standard deviations. Statistical significance (****P<0.0001) was determined via the two-way ANOVA test and is indicated within the panel (D) .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Expressing, Binding Assay, Activity Assay, Incubation, Flow Cytometry, Infection, Transfection, Recombinant

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Activation of Jurkat T cells is inhibited by HA-49K orthologs to an equal extent. Jurkat cells were previously incubated with cell supernatants (red) containing HA-49K orthologs. Cells were washed and stimulation was conducted via receptor cross-linking using immobilized α-CD3 and soluble α-CD28 Abs for 6h. After cell fixation the activation level was determined by flow cytometry-based monitoring of the cell surface expression of the early activation marker CD69. Relative numbers of CD69 positive cells were normalized to CD3/CD28 stimulation control (not shown). Untreated (unstim.) and isotype control (ISO) treated samples were used as negative controls (grey). To show the efficiency of the CD3/CD28 stimulation we treated the cells also with 50 ng/ml Phorbol-12-myristate-13-acetate and 1 µg/ml ionomycin (PMA/Iono, blue). Administration using CD3/CD28 stimulation and unreactive A549 supernatant was utilized to control the effect of the supernatant on CD3/CD28 stimulation (A549, blue) (A) . pErk1/2 levels were identified by immunoblot analysis upon CD3 stimulation of Jurkat cells with 1 µg/ml for 2 min. Sample loading was controlled by detection of β-actin. One representative blot for Jurkat and CD45-/- Jurkat is shown (B) and the relative expression levels of pErk1/2 to β-actin ratios were normalized to the pos. ctrl. (C) . The columns represent the mean of 3 individual experiments (dots), the error-bars represents the standard deviation for A and (C) Statistical differences compared toPMA/ionomycin treatment in (A) and CD3-stimulation in the presence of A549 supernatants in (C) positive controls were analyzed using the two-way ANOVA test. Only significant results were indicated in the figure.

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Activation Assay, Incubation, Flow Cytometry, Expressing, Marker, Control, Western Blot, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Ramos B cell signaling is inhibited by HA-49K orthologs to comparable levels. Ramos B cell signaling was determined to assess the inhibitory potential of HA-49K orthologs in B cells. Previous incubation of Ramos cells with cell supernatants (red) containing various HA-49K orthologs was performed. Unstimulated cells (unstim.) were used as a negative control (grey), co-incubation with unreactive A549 supernatant (A549) served as a positive control indicated in blue. Cells were washed and stimulated via receptor cross-linking using α-λ Abs. The cellular calcium-response was detected by flow cytometry and the mean peak Ca2+-levels (columns) of 3 individual experiments (dots) including standard deviation are shown. Statistical differences compared to A549 were analyzed using two-way ANOVA test (A) . Immunoblot analysis of pErk1/2 levels upon BCR stimulation of Ramos B cell lines with 1 µg/ml α-λ Abs for 2 min. Unstimulated cells (unstim.) served as negative control while α-λ treated cells (pos. crtl.) and co-incubation with A549 supernatant (A549) served as positive controls. Detection of β-actin levels was used as loading control. One representative blot for Ramos and CD45-/- Ramos cells is shown (B) . The relative detection levels of pErk1/2 to β-actin ratios in Ramos cells were normalized to the positive ctrl. The mean (columns) of 3 individual experiments (dots) including standard deviation is shown. Statistical differences to the positive ctrl. were analyzed using the two-way ANOVA test. Only significant results were indicated in the panel (C) . A two-fold dilution series of cell supernatants containing HA-49K-D64 (D) and purified HA-49K-D64 proteins starting at 8 µg per sample (E) was performed. Samples were either supplemented with 0.5 µg per sample hCD45-Fc (+hCD45-Fc, red) or without (hCD45-Fc, blue). After a 1 h incubation period, the binding of HA-49Ks to Ramos cells was detected using α-HA-based flow cytometry. Undiluted supernatant from untransfected A549 cells (A549) or the 8 µg MT protein were utilized as negative controls and shown as single values at the end of the x-axis separated by the grey dashed line. The mean MFI of 3 individual experiments is displayed for each, including standard deviation in the form of error bars. Erk1/2 phosphorylation was analyzed to investigate the prevention of the inhibitory effect HA 49K-D64 by hCD45-Fc receptors via immunoblotting. The supernatant containing HA-49K-D64 proteins was diluted 1:10 and 4 µg purified HA-49K-D64 proteins were incubated for 1 h with 500 ng hCD45-Fc decoy receptors as indicated in the figure. Subsequently, reagents were incubated with Ramos cells for 1 h Cells were lysed after BCR stimulation with 2 µg/ml α-λ Abs for 2 min. Sample loading was controlled by the detection of β-actin levels. One representative blot is presented (F) .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Incubation, Negative Control, Positive Control, Flow Cytometry, Standard Deviation, Western Blot, Control, Purification, Binding Assay

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Only HAdV-D infected A549 cells bind soluble hCD45-Fc. A549 cells were infected with HAdV-A12, -B7, -B35, -C5, -D8, -D19, -D36, -D64 and -D64ΔE3 and -E4, viruses with an MOI of 5 for 24 h Efficient infection was confirmed by internal hexon protein expression (red) using 2Hx-2 mAbs in comparison to isotype control staining (grey) of infected A549 cells in flow cytometry. Displayed are representative histograms of productive infections (A) . Infected cells were treated with +/- (red/blue) 0.5 µg hCD45-Fc per sample. Bound CD45 molecules were detected by CD45-ECD staining using α-human pan-CD45 MEM-28 in flow cytometry. Mock infected cells as well as cells infected with HAdV-D64ΔE3 virus served as negative controls. As positive control the cell clone stably expressing HA-49K of HAdV-D64 was applied. The mean of 3 individual experiments (columns) from (dots) including standard deviation, presented as error bars, is shown. Significant differences between +/- hCD45-Fc treatment were determined using the two-way ANOVA test and are indicated in the panel (B) .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Infection, Expressing, Comparison, Control, Staining, Flow Cytometry, Virus, Positive Control, Stable Transfection, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Graphical abstract of the putative functional mechanism of E3/49K action in B cells. During BCR antigen ligation, the catalytic activity of CD45 shifts the equilibrium of functional Lyn toward activated Lyn (Lyn Y396). The removal of the inhibitory phosphate group from Y507 sites primes the auto-phosphorylation of Lyn at Y396 sites to induce Lyn kinase activity. Active Lyn promotes phosphorylation of ITAMs and pITAM-attached Syk to facilitate BCR signal transduction. pSyk initiates several signaling pathways, including the MAPK pathway. pErk1/2 and the calcium flux results in transcriptional and cellular activation (A) . E3/49K-mediated dimerization of CD45 molecules prevents the catalytic activity of CD45. As a result, the equilibrium of functional Lyn is shifted toward inactive Lyn (Lyn Y507), increasing the activation threshold during BCR stimulation. As a result of reduced Lyn kinase activity, less pSyk, pErk1/2, and calcium flux are generated, resulting in decreased transcriptional and cellular activation (B) . Dimerization of CD45 by E3/49K may disrupts CD22-CD45 interaction. Active Lyn induces CD22 which enhances its inhibitory effect in reducing BCR signals by affecting pErk1/2 and calcium flux (C) . The figure was created with BioRender.com .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Functional Assay, Ligation, Activity Assay, Transduction, Activation Assay, Generated

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Graphical representation about E3/49K functions. CD45 modulation via binding of E3/49K proteins to its ECD is a common feature of HAdVs of species D. E3/49K ECDs are shed from infected cells and bind to and inhibit CD45 positive target cells. Based on the current hypothesis, inhibition is mediated by enforced dimerization of CD45 , which inhibits leukocyte receptor signaling. B cells are here identified as a new target for E3/49K-mediated immune evasion. Since there are more CD45 expressing leukocytes existing, it is hypothesized that they serve as targets for E3/49K as well. The figure was created with BioRender.com .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Binding Assay, Infection, Inhibition, Expressing

Journal: Science translational medicine

Article Title: SynNotch-CAR T cells overcome challenges of specificity, heterogeneity, and persistence in treating glioblastoma

doi: 10.1126/scitranslmed.abe7378

Figure Lengend Snippet: (A) In vitro killing by T cells constitutively expressing α-EphA2/IL13Rα2 tandem CAR (relative to nontransduced control). (B) Flow cytometry plots distinguishing naïve-like cells (CD45RA+CD62L+), central memory cells (CD45RA−CD62L+), effector memory cells (CD45RA−CD62L−), and effector memory RA cells (CD45RA+CD62L−) (representative of three experiments from different donors). Percentages of cells in the naïve/memory stem cell state and effector memory state are highlighted. T cells were rested for 10 days in vitro after transfection before phenotypic analysis. (C) Percentage of CD62L+CD45RA+ T cells in indicated CAR T cells and synNotch-CAR T cells (n = 3 per group). (D) Expression of exhaustion markers by indicated CAR and synNotch-CAR T cells, both without and with stimulation by target cells. Pie chart shows percentage of cells that express 0, 1, 2, or 3 exhaustion markers (PD1, LAG3, or TIM3), average of three different donors. (E) Tonic signaling in constitutive versus α-EGFRvIII-synNotch induced α-EphA2/IL13Rα2 CAR T cells, measured by percent CD25+ cells (one-way ANOVA followed by a Dunnett’s test, P < 0.05, n = 5 different donors). Boxes represent min to max with median center line. (F) SynNotch-CAR T cells show improved persistence in vivo compared to constitutive CAR T cells. GBM6 tumor–bearing mice were euthanized 6 days after infusion of either the constitutive tandem CAR T cells (left) or α-EGFRvIII synNotch–α-EphA2/IL13Rα2 CAR T cells (right). Representative confocal fluorescent microscopy of brain slices and single-cell suspensions of tumor-bearing brain show few T cells (CD3+CD45+) in constitutive CAR T cell–treated tumors. In contrast, mice treated with synNotch-CAR T cells reveal high number of T cells in tumors.

Article Snippet: Primary antibodies used were the following: CD45 (D9M8I) XP Rabbit mAb (Cell Signaling Technology; 1:100), Anti-EGFRvIII, clone DH8.3 (MilliporeSigma; 1:100), EphA2 (D4A2) XP Rabbit mAb (Cell Signaling Technology; 1:100), Anti-IL13 receptor alpha 2 antibody (Abcam; 1:100), and Cleaved Caspase 3 (Asp175) Rabbit mAb (Cell Signaling Technology; 1:400).

Techniques: In Vitro, Expressing, Flow Cytometry, Transfection, In Vivo, Microscopy

Journal: International journal of molecular medicine

Article Title: Exosomal microRNA‑146a derived from mesenchymal stem cells increases the sensitivity of ovarian cancer cells to docetaxel and taxane via a LAMC2‑mediated PI3K/Akt axis.

doi: 10.3892/ijmm.2020.4634

Figure Lengend Snippet: Figure 1. Identification of hUCMSCs and their derived exosomes. (A) Expression of MSCs surface markers, including CD29, CD44, CD73, CD90, CD105, CD14, CD34, CD45 and HLA‑DR, analyzed by flow cytometry. (B) hUCMSC morphology at passage 3 observed under a light microscope. Scale bar, 100 µm. (C) Representative images of osteogenic differentiation of hUCMSCs using Alizarin Red staining. Scale bar, 100 µm. (D) Representative images of adipogenic differentiation of hUCMSCs using Oil red O staining. Scale bar, 100 µm. (E) Morphological analysis of hUCMSC by transmission electronic microscopy. Scale bar, 200 nm. (F) Detection of exosomal marker expression in hUCMSC‑released exosomes by western blotting. (G) Internalization of PKH26‑labeled exosomes by SKOV3 and A2780 cells was observed under a fluorescence microscope. Scale bar, 50 µm. hUCMSCs, human umbilical cord mesenchymal stem cells.

Article Snippet: The following antibodies were used to identify hUcMSc surface markers: FITc-conjugated anti-cd90 (cat. no. 13801), anti-cd14 (cat. no. 29943), anti-cd73 (cat. no. 13160) and anti-cd105 (cat. no. 14606), and phycoerythrin (PE)-conjugated anti-cd34 (cat. no. 79253), anti-cd29 (cat. no. 34971), anti-cd44 (cat. no. 38200), anti-cd45-Pc7 (cat. no. 28418) and anti-human leukocyte antigen dR-1 (HLA-dR; cat. no. 17634) (all cell Signaling Technology, Inc.).

Techniques: Derivative Assay, Expressing, Flow Cytometry, Light Microscopy, Staining, Transmission Assay, Microscopy, Marker, Western Blot, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: Treatment with gelsolin reduces brain inflammation and apoptotic signaling in mice following thermal injury

doi: 10.1186/1742-2094-8-118

Figure Lengend Snippet: Gelsolin affects migration of myeloid-derived cells into brain . CD45 + cells from gelsolin-treated mice 8 h postburn (A) and quantification of infiltrating CD45 + (B) cells in 10 high power fields (HPF) of the periventricle region following gelsolin treatment. Gelsolin positive cells were seen in medial habenular nucleus (MHb), stria medullaris (sm), hippocampal CA field (CA2) and blood vessel (BV). Magnifications are × 400. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sham-injured mice; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. placebo mice; ++p < 0.01, and +++p < 0.001 vs. Gsn-L mice by ANOVA, Newman-Keuls post-hoc test. Data are means ± SD for n = 6-8.

Article Snippet: Sections used for immunocytochemistry were incubated in 0.3% hydrogen peroxide (H 2 O 2 ) for 10 min, and incubated free-floating in antibodies (Abs) of polyclonal anti-mouse ionized calcium-binding adapter molecule 1 (Iba-1, 1:1000; Wako, Osaka, Japan), monoclonal anti-mouse CD11b (Mac-1, 1:1000; EuroBioScience, Lund, Sweden), monoclonal anti-mouse CD45 (1:1000; EuroBioScience), or rabbit anti-cleaved caspase-3 (1:50; Cell Signaling, Danvers, MA, USA) with 3% normal goat serum, 0.05%Triton-X in PBS, for 24-48 h rotating at 4°C.

Techniques: Migration, Derivative Assay