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: 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

Journal: Scientific reports

Article Title: Distinct gene expression patterns for CD14++ and CD16++ monocytes in preeclampsia.

doi: 10.1038/s41598-022-19847-5

Figure Lengend Snippet: Figure 2. Immunophenotyping of the patient’s monocytes. Representative histograms of gated monocytes stained with antibodies to CD40, CD80, CD86, CD163, CD206 or CX3CR1 (magenta-filled histogram) and control samples (empty contour) for three groups of patients (a). Percents of positively stained cells are indicated above the gate bars. Levels of CD40+, CD80+, CD86+, CD163+, CD206+ and CX3CR1+ monocytes in three groups: the data are listed as mean ± SD (b): eoPE early-onset PE (n = 12), loPE late-onset PE (n = 10), control (n = 11).

Article Snippet: For surface immunophenotype marker staining, the cells we resuspended in autoMACS® Rinsing Solution (1 × 105 cells in 100 μL) with 1% BSA and stained with anti-CD16 (A07766, Beckman Coulter), anti-CD14 (130-110-518, Miltenyi Biotec), anti-CD45 (A07785, Beckman Coulter), anti-CD206 (130-095-131, Miltenyi Biotec), anti-CD86 (130-116-160, Miltenyi Biotec), anti-CD163 (130-097-630, Miltenyi Biotec), anti-HLA-DR (130-111-790, Miltenyi Biotec), CD40 (130-110-947, Miltenyi Biotec), CD80 (130-117- 683, Miltenyi Biotec) and anti-CX3CR1 (12-6099-42, eBioscience).

Techniques: Staining, Control