Journal: Nature Communications

Article Title: Defective germinal center selection results in persistence of self-reactive B cells from the primary to the secondary repertoire in Primary Antiphospholipid Syndrome

doi: 10.1038/s41467-024-54228-8

Figure Lengend Snippet: A Circos plot depicting the IGHV:IGLV and IGHV:IGKV pairings for a HD and two representative PAPS patients. Pairing between IGHV3-11, 3-21, 3-74, 4-39 , or 4-34 (pink segment), and IGKV (blue segment) or IGLV (green segment) gene are shown by the connecting lines inside the circle with thickness corresponding to pairing frequencies. B Frequencies of IGHV3-21 , 3-74 , 4-39 clonotypes in IGHM and IGHG/A isotypes in PAPS patients ( n = 10) and HD ( n = 7). C Violin plot representing normalized ADT level for B cell signaling and activation markers in IGHV4-39 clonotypes from APS patients (dark red), HD (dark blue), other clonotypes from PAPS patients (light red) and HD (light blue). D Dot plot for expression of mTORC1 and MYC selected genes among naive and memory cells according to clonotypes in PAPS patients and HD. E Dot plot for expression of pre-GC and plasmablast selected genes in naive cells according to clonotypes in PAPS patients and HD. F Violin plot representing pre-GC signature, SELL and CXCR5 gene expression according to clonotypes in patients and HD. G Frequency of plasmablast (CD38 ++ CD27 ++ ) after B cell stimulation with CD40L + IL-21 + IL-4 for 6 days. Two-tailed Mann–Whitney test was used to compare groups. H Representative lineage trees for two clones in APS patients. I Representative amino acid VH alignment for IGHV4-39 clone (clone 1 depicted in H ). J Frequency of positively charged amino acid induced by SHM in VH genes (top) of switched IgG/A B cells from HD ( n = 8191) and PAPS patients ( n = 9406). K Frequency of arginine residues induced by SHM in VH genes CDR of switched IgG/A B cells from HD ( n = 7229) and PAPS patients ( n = 6101). Data are presented as mean values ±SEM. Significance was determined using a Two-tailed Mann–Whitney test. ADT antibody-derived tag, CDR complementary determining region, HD healthy donor, MFI Mean fluorescence intensity, PAPS primary antiphospholipid syndrome, SHM somatic hypermutation.

Article Snippet: A total of 50,000 B cells/well were plated into U96 wells and stimulated with CD40L (rcyec-hcd40l, Invivogen), IL-21 (200-21, Preprotech) and IL-4 (200-04, Preprotech) in Roswell Park Memorial Institute medium (RPMI; Life Technologies) enriched with gentamicine (50 µg/mL), supplemented with 10% FCS.

Techniques: Activation Assay, Expressing, Cell Stimulation, Two Tailed Test, MANN-WHITNEY, Clone Assay, Derivative Assay, Fluorescence

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 1. CD40L expression on SV40 TAg–specific CD8+ T cells during a protective immune response. (A) Splenocytes of WT mice challenged with 16.113 TAg+ cancer cells were stimulated with peptide IV at indicated time points. d, day. The dot plots show the intracellular IFN-γ and CD40L staining of CD3+CD8+CD4−-gated lymphocytes from one representative mouse (n = 4 mice). (B) The diagram summarizes the frequencies of CD40L+IFN-γ+ and CD40L−IFN-γ+CD8+ T cells and (C) the frequency of IL-2– producing cells among CD40L+ and CD40L− tumor-specific CD8+ T cells at the different time points (both means ± SD).

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Staining

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 2. Prevention of tumor outgrowth is dependent on CD40L expression on CD8+ T cells. (A) RAG1−/− mice were injected subcutaneously with 1 × 106 9.27 TAg+ cancer cells and treated in parallel with intravenously injected CD8+ T cells from WT or CD40L−/− mice and/or with WT CD4+ T cells. The means ± SD of the tumor size of four or five mice per group are shown from one representative of five experiments. (B) The tumor sizes of individual mice in different groups are shown at day 26. (C) Sum- mary of the tumor formation data obtained from five independent experiments. Tumor formation was defined as an established tumor when its volume reached >500 mm3. Statistical analysis: Analysis of variance (ANOVA) with Bonferroni multiple comparisons posttest: *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Injection

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 3. Impaired tumor rejection in nonlymphopenic CD8+ T cell–specific CD40L KO mice. (A) Strategy for the generation of CD40Lfl/fl mice. UTR, untranslated region; FRT, flippase recognition target. (B) E8I-Cre × CD40Lfl/fl, E8I-Cre, and CD40L fl/fl mice as WT control were injected subcutaneously with 1 × 106 9.27 TAg+ cancer cells. Sum- mary of the tumor formation data obtained from three individual experiments, each with 5 to 10 mice per group. Tumor was defined as established when its volume reached >500 mm3. (C and D) WT and E8I-Cre × CD40Lfl/fl mice were subcutaneously injected with 1 × 106 9.27 TAg+ cancer cells, and 7 days later, splenocytes and cells from the draining lymph nodes (dLNs) were isolated and stimulated with peptide IV. (C) The dot plots show the intracellular IFN-γ and CD40L staining of CD3+CD8+CD4−- gated splenocytes from one representative mouse of five mice. (D) The diagram summarizes the frequencies of TAg-specific IFN-γ+CD8+ T cells measured among spleno- cytes and lymph node cells. Statistical analysis: Log-rank test: **P < 0.01.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Control, Injection, Isolation, Staining

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 4. Role of CD40 expression on host and 9.27 cancer cells for tumor rejection. (A) CD40L−/− and CD40−/- mice were injected subcutaneously with 1 × 106 9.27 cancer cells. Summary of the tumor formation data obtained from two individual experiments, each with 6 to 10 mice per group. Tumor was defined as established when its volume reached >500 mm3. FSC-A, forward-scatter-area. (B) Cell surface expression of CD40 was analyzed after culturing 9.27 cancer cells, supplemented with or without TGFβ for 24 hours in three individual experiments. (C and D) The 9.27 cancer cells were treated for 24 hours with TGFβ and subsequently stimulated for further 24 hours with multimeric mouse CD40L. Thereafter, caspase-8 activity was determined with the fluorescence marker FITC-IETD-FMK and costained with annexin V. (C) The representative dot plots show the gating of annexin V and FITC-IETD-FMK–stained cancer cells after triggering CD40. (D) The diagrams summarize the frequencies of ac- tive caspase-8+ cancer cells, measured as triplicates of one of three representative experiments.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Injection, Activity Assay, Fluorescence, Marker, Staining

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 5. CD8+ T cell–mediated CD40 signaling in cancer cells prevents tumor formation. (A) Cell surface expression of CD40 was analyzed after culturing TRAMP-C1 and CD40tg TRAMP-C1 cells with or without TGFβ for 24 hours. (B) Both TRAMP-C1 cancer cell lines were stimulated for 24 hours with multimeric CD40L. Thereafter, cas- pase-8 activity was determined with the fluorescence marker FITC-IETD-FMK. The representative dot plots show the gating of 4′,6-diamidino-2-phenylindole (DAPI) and FITC-IETD-FMK–stained cancer cells after triggering CD40. The diagrams summarize the frequencies of active caspase-8+ cancer cells. (C and D) E8I-Cre × CD40Lfl/fl and E8I-Cre as WT control mice were injected subcutaneously with 5 × 106 TRAMP-C1 (C) or CD40tg TRAMP-C1 (D) cancer cells. In the diagrams [(C) and (D)], the data from one of two representative experiments are shown. Per group, five or six mice were challenged. Tumor was defined as established when its volume reached >500 mm3. Statis- tical analysis: (B) Mann-Whitney U test: **P < 0.01 and [(C) and (D)] log-rank test: *P < 0.05 and **P < 0.01.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Activity Assay, Fluorescence, Marker, Staining, Control, Injection, MANN-WHITNEY

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 6. CD40L+CD8+ T cells mediate cell death in human CD40+ carcinoma cell lines by caspase-8 activation. (A) Histograms represent the CD40 expression on EJ138 or A704 transfected with nontargeting single guide RNA (black, sgNon) or cells depleted for CD40 by CRISPR-Cas9 and sgRNA against human CD40 (red). Gray-filled his- tograms show isotype staining. (B) sgNon (black) or caspase-8 sgRNA–treated (red) EJ138 and A704 cells were treated with multimeric CD40L for 24 hours, and caspase-8 activation was monitored by FITC-IETD-FMK staining. Gray-filled histograms represent the basal caspase-8 activation of unstimulated WT cells. (C and D) sgNon, CD40 sgRNA-, or caspase-8 (Casp8) sgRNA–treated variants of EJ138 (C) or A704 (D) were cocultivated with CD40L-enriched CD8+ T cells for 24 hours, and apoptosis was de- tected by annexin V and DAPI staining. Shown are representative dot plots with frequencies of technical triplicates (left) and bar graphs summarizing the data from ex- periments with eight different T cell donors. Statistical analysis: [(C) and (D)] Repeated measures ANOVA, followed by Dunnett’s test: **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Activation Assay, Expressing, Transfection, CRISPR, Staining

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 7. Resistance pattern for CD40-mediated cell death and correlations between CD8 and CD40L in different RCC cohorts. (A) In the histograms, the dark gray areas display the CD40 expression, and the light gray areas display the corresponding isotype control on eight different RCC lines. The included numbers represent the mean fluorescence intensity fold change between CD40 and isotype control staining. (B) Percentages of CD40L-induced lysis per RCC line after stimulation for 48 hours are plotted. Representative lysis values after backgorund substraction of each cell line of at least three individual experiments are depicted. (C) The heatmap represents the top 10 differentially expressed genes between CD40-resistant and CD40-sensitive RCC cell lines. (D) In the Kaplan-Meier survival plot, the resistance score of low and high groups of the TCGA-KIRC patient cohort is compared with each other. (E) Partial correlation networks of different patient groups are displayed, and the numbers are the partial correlation coefficients. (F) In the Kaplan-Meier survival plots, the resistance score of low and high groups of the cohorts is compared with all patients treated within the checkmate-009, checkmate-010, and checkmate-025 studies (left) or divided into two arms of the checkmate-025 study (middle nivolumab arm and right everolimus arm). Statistical analysis: (C) Described in Materials and Methods; [(D) and (F)] log-rank test.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Control, Fluorescence, Staining, Lysis

Journal: Antibodies (Basel, Switzerland)

Article Title: Fcγ-Receptor-Independent Controlled Activation of CD40 Canonical Signaling by Novel Therapeutic Antibodies for Cancer Therapy.

doi: 10.3390/antib13020031

Figure Lengend Snippet: Figure 1. Identification of agonistic anti-CD40 antibodies not requiring Fc-effector function. (A) Flow diagram of the anti-CD40 antibody discovery campaign. Wildtype rabbits were immunized with recombinant CD40 protein and monoclonal B-cells were FACS-sorted and cultured. Antibodies secreted into the B-cell supernatant medium were screened for the induction of NFκB signaling in a HEK-Blue-CD40L™cell-based gene reporter assay. Antibody gene sequences were retrieved from B-cells, humanized and cloned as human IgG1-LALA antibodies in HEK-293 FreeStyle™cells. (B) Eighty-eight humanized anti-CD40 hIgG1-LALA antibodies were tested on CD40 expressing HEK 293 cells in the HEK-Blue-CD40L™cell-based gene reporter assay (grey bars; OD@655 = secreted em- bryonic alkaline phosphatase activity) and for the induction of IL-12p40 cytokine release by in vitro differentiated dendritic cells (blue bars; measured by ELISA). Asterisks (*) indicate values exceeding the linear range of the ELISA.

Article Snippet: The agonistic activity of anti-CD40 monoclonal antibodies was tested by stimulating HEK-Blue-CD40L (Invivogen, San Diego, CA, USA, catalogue code: hkb-cd40) cells, which harbor an NF-κB-inducible Secreted Embryonic Alkaline Phosphatase (SEAP) gene construct.

Techniques: Recombinant, Cell Culture, Reporter Assay, Clone Assay, Expressing, Activity Assay, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Antibodies (Basel, Switzerland)

Article Title: Fcγ-Receptor-Independent Controlled Activation of CD40 Canonical Signaling by Novel Therapeutic Antibodies for Cancer Therapy.

doi: 10.3390/antib13020031

Figure Lengend Snippet: Figure 2. Differentiation of novel Fcγ-receptor-independent anti-CD40 antibodies from other clinical antibodies. (A) The activity of the four most active CD40 hIgG1-LALA antibodies, MAB 271, 273, 276 and 278, were tested for the activation of in vitro differentiated DCs in direct comparison to different Fc-variants of CP-870,893. IL-12p40, released by DCs, was measured by ELISA. The statistical significance for difference between MAB 273, MAB 271 and the Fc-variants of CP-870,893 was determined by an unpaired t-test. * p < 0.05, ** p < 0.01, ns = not significant. (B) CD40 antibodies were incubated with CD40-expressing HEK-Blue-CD40L™cells, and interference with CD40L binding to CD40 was tested by the addition of increasing concentrations of recombinant CD40L-mIgG2a-Fc- fusion protein. The binding of CD40L was detected by flow cytometry using a fluorescently labeled anti-mouse IgG antibody. (C) The competition of anti-CD40 antibodies for binding to human CD40 was tested in a sandwich ELISA. Each antibody was tested both as a coating (vertical label) and as a detection antibody (horizontal label) in combination with any other anti-CD40 antibody. Error bars in (A,C) indicate the standard deviation (mean) of triplicates.

Article Snippet: The agonistic activity of anti-CD40 monoclonal antibodies was tested by stimulating HEK-Blue-CD40L (Invivogen, San Diego, CA, USA, catalogue code: hkb-cd40) cells, which harbor an NF-κB-inducible Secreted Embryonic Alkaline Phosphatase (SEAP) gene construct.

Techniques: Activity Assay, Activation Assay, In Vitro, Comparison, Enzyme-linked Immunosorbent Assay, Incubation, Expressing, Binding Assay, Recombinant, Flow Cytometry, Labeling, Sandwich ELISA, Standard Deviation

Journal: Antibodies (Basel, Switzerland)

Article Title: Fcγ-Receptor-Independent Controlled Activation of CD40 Canonical Signaling by Novel Therapeutic Antibodies for Cancer Therapy.

doi: 10.3390/antib13020031

Figure Lengend Snippet: Figure 3. Induction of costimulatory receptors and cytokine release by Fc-silenced anti-CD40 an- tibodies in dendritic cells. Human PBMC-derived moDCs were treated for 2 days with anti-CD40 hIgG1-LALA antibodies MAB 271 and MAB 273, CP-870,893 hIgG2, APX hIgG1-S276E or isotype control antibodies. The expression of CD80, CD86 and HLA-DR was measured by flow cytometry (A,B), and cytokine concentrations in culture supernatants were determined by a cytometric bead array (C,D). MFIs were normalized to isotype control antibody treatments. In (A,C) a dose response analysis with DCs of an exemplary donor is shown; (B,D) summarize the mean of indicated donors treated with saturating concentrations of 1.25 or 2 µg/mL. Error bars (B,D) show SEM. Significant differences according to paired t-test are indicated in the bar diagrams. * p < 0.05, ** p < 0.01.

Article Snippet: The agonistic activity of anti-CD40 monoclonal antibodies was tested by stimulating HEK-Blue-CD40L (Invivogen, San Diego, CA, USA, catalogue code: hkb-cd40) cells, which harbor an NF-κB-inducible Secreted Embryonic Alkaline Phosphatase (SEAP) gene construct.

Techniques: Derivative Assay, Control, Expressing, Flow Cytometry, Analysis

Journal: Antibodies (Basel, Switzerland)

Article Title: Fcγ-Receptor-Independent Controlled Activation of CD40 Canonical Signaling by Novel Therapeutic Antibodies for Cancer Therapy.

doi: 10.3390/antib13020031

Figure Lengend Snippet: Figure 4. Fc-silenced anti-CD40 antibodies induce costimulatory receptors on human B-cells. B-cells were isolated from human PBMCs and incubated with anti-CD40 hIgG1-LALA antibod- ies MAB 271 and MAB 273, CP-870,893-hIgG2, APX hIgG1-S276E or isotype control antibodies. The expression of CD80 and CD86 was measured by flow cytometry. MFIs were normalized to isotype control antibody treatments. In (a), a dose response analysis with B-cells of an exemplary donor is shown; (b) summarizes the mean of four donors treated with saturating concentrations of 312 µg/mL (1 donor) or 500 µg/mL (3 donors). Error bars indicate SEM from four donors; the respective differences between anti-CD40 antibody treatments are not statistically significant according to paired t-test.

Article Snippet: The agonistic activity of anti-CD40 monoclonal antibodies was tested by stimulating HEK-Blue-CD40L (Invivogen, San Diego, CA, USA, catalogue code: hkb-cd40) cells, which harbor an NF-κB-inducible Secreted Embryonic Alkaline Phosphatase (SEAP) gene construct.

Techniques: Isolation, Incubation, Control, Expressing, Flow Cytometry, Analysis

Journal: Antibodies (Basel, Switzerland)

Article Title: Fcγ-Receptor-Independent Controlled Activation of CD40 Canonical Signaling by Novel Therapeutic Antibodies for Cancer Therapy.

doi: 10.3390/antib13020031

Figure Lengend Snippet: Figure 5. Changes in the body weight and body temperature of human-stem-cell-transplanted mice after anti-CD40 antibody injection. Antibodies were injected on day 0, and measurement was performed on different days after treatment. Depicted is the change in body weight (mean +/−SD) and body temperature (mean +/−SD) following the injection of the indicated antibodies and isotype controls. The injection of 4–6 mice per group with MAB 273 (n = 6; 10 mg/kg), IgG1-LALA (n = 6; 10 mg/kg) and CP-870,893 (n = 4; 3 mg/kg). Statistically significant differences between indicated groups were determined using two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.005.

Article Snippet: The agonistic activity of anti-CD40 monoclonal antibodies was tested by stimulating HEK-Blue-CD40L (Invivogen, San Diego, CA, USA, catalogue code: hkb-cd40) cells, which harbor an NF-κB-inducible Secreted Embryonic Alkaline Phosphatase (SEAP) gene construct.

Techniques: Stem Cell, Injection

Journal: Antibodies (Basel, Switzerland)

Article Title: Fcγ-Receptor-Independent Controlled Activation of CD40 Canonical Signaling by Novel Therapeutic Antibodies for Cancer Therapy.

doi: 10.3390/antib13020031

Figure Lengend Snippet: Figure 6. Effect of anti-CD40 antibody injection on human immune cell numbers and cytokine production in a human-stem-cell-transplanted mouse model. (a) Shown are changes in human immune cells in the peripheral blood of humanized mice injected with MAB 273, CP-870,893 or IgG1-LALA control antibodies as determined by flow cytometry. Bar graphs (mean + SD) with individual data points represent B-cell, T-cell and NK cell numbers as per cent of total human CD45+ cells in the peripheral blood of mice treated with the respective antibodies. Symbols depict individual mice. The statistical assessment of the data was performed with a one-way ANOVA test. * indicates a significant difference between the marked value and day 0. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001. (b) The bar graphs (mean + SD) depict the concentration of human IL-12p70, TNF-α, IL-6 and IFN-γ at the indicated time points after the injection of MAB 273, CP-870,893 and IgG1-LALA control antibodies. Symbols indicate individual mice. Cytokine concentrations in murine serum were determined using a LEGENDplexTM Multi-Analyte Flow Assay. Values between groups did not reach statistical significance as determined by a one-way ANOVA.

Article Snippet: The agonistic activity of anti-CD40 monoclonal antibodies was tested by stimulating HEK-Blue-CD40L (Invivogen, San Diego, CA, USA, catalogue code: hkb-cd40) cells, which harbor an NF-κB-inducible Secreted Embryonic Alkaline Phosphatase (SEAP) gene construct.

Techniques: Injection, Immune Cell, Stem Cell, Control, Flow Cytometry, Concentration Assay, FACS

Journal: The Journal of Clinical Investigation

Article Title: Combined FXIII-C3 autoantibodies elicit bleeding and complement dysfunction in autoimmune FXIII deficiency

doi: 10.1172/JCI192619

Figure Lengend Snippet: ( A and B ) Quantification of platelets’ activation in 8 different groups of mice via CD62p surface expression analyzed by flow cytometry. A representative dot plot ( A ) and statistical chart of activated platelets ( B ) are shown. ( C ) Platelet activation upon in vitro co-culture of platelets with C . albicans . CD62p expression (left) and GPIIbIIIa expression (right) on platelet surfaces were analyzed by flow cytometry upon in vitro co-culture of platelets (1 × 10 7 /sample) with different doses of C . albicans (1 × 10 5 , 1 × 10 6 and 1 × 10 7 /sample) in the presence of Gly-Pro-Arg-Pro (Pefa 6003) to prevent platelet aggregation. ( D ) Soluble CD40L levels in plasma of 8 groups of mice on day 7 after infection. ( E ) Soluble CD40L levels in plasma of patients with FXIII deficiency and in healthy control donors. ( F ) CD40L release in the platelet- C . albicans co-culture supernatant. Data are shown as mean ± SD; 1-way ANOVA was used for statistical analysis. ** P < 0.01, *** P < 0.001. FSC-A, forward scatter area; PLA, proximity ligation assay; APC-A, allophycocyanin area.

Article Snippet: Purified CD40L (Elabscience, PKSH032214) was used as a positive control.

Techniques: Activation Assay, Expressing, Flow Cytometry, In Vitro, Co-Culture Assay, Clinical Proteomics, Infection, Control, Proximity Ligation Assay

Journal: The Journal of Clinical Investigation

Article Title: Combined FXIII-C3 autoantibodies elicit bleeding and complement dysfunction in autoimmune FXIII deficiency

doi: 10.1172/JCI192619

Figure Lengend Snippet: ( A ) Binding of CD40L to B cells analyzed by flow cytometry. ( B ) Co-localization CD40L and CD40 on the surface of B cells. Memory B cells isolated from P1 (1 × 10 6 /sample) were incubated with the platelet– C . albicans culture supernatant. Co-localization of CD40L and CD40 was examined by confocal microscopy. Representative images (out of 3) are shown. Scale bar: 10 μm. ( C ) Combined autoantibody release detected by ELISA. ( D ) Specificity of released autoantibody against C3 analyzed by ELISA. ( E ) Specificity of released autoantibody against FXIII analyzed by Far Western blot. ( F ) Neutralization of CD40L blocked autoantibody release by memory B cells. ( C – F ) CD40L-containing supernatant was added to the isolated memory B cells in the presence or absence of anti-CD40L. Purified CD40L was used as a positive control. Data are shown as mean ± SD; 1-way ANOVA was used for statistical analysis. *** P < 0.001. NC, negative control; PC, positive control; HB, memory B cells isolated from healthy donors; PB, memory B cells isolated from patient 1.

Article Snippet: Purified CD40L (Elabscience, PKSH032214) was used as a positive control.

Techniques: Binding Assay, Flow Cytometry, Isolation, Incubation, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Far Western Blot, Neutralization, Purification, Positive Control, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: Combined FXIII-C3 autoantibodies elicit bleeding and complement dysfunction in autoimmune FXIII deficiency

doi: 10.1172/JCI192619

Figure Lengend Snippet: A combined FXIII-C3 autoantibody was identified in patients with autoimmune FXIII deficiency. The combined autoantibody, by forming a complex with C3, blocks C3 cleavage by C3 convertase, thereby inhibiting complement activation and complement mediated T cell activation, which increases host susceptibility to pathogenic C . albicans infections. Uncontrolled exogenous infections, by triggering platelets’ activation and platelet-related CD40L release, further promote autoantibody generation, resulting in progressive consumption of FXIII and C3. This process likely forms a self-enhanced amplification loop for autoantibody generation and progressive consumption of FXIII and C3 antigens.

Article Snippet: Purified CD40L (Elabscience, PKSH032214) was used as a positive control.

Techniques: Activation Assay, Amplification