Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: Pembrolizumab does not substantially affect B cell activation and antibody production ex vivo (A-C) Human naïve B cells were isolated from PBMCs and cultured under indicated conditions for 7 days; Pembrolizumab or isotype control IgG4 was added on Day 2, n = 8. (A) Expression of CD38 and CD27 on B cells. Right, percentages of CD27 + CD38 - , CD27 + CD38 + B cells. (B) Expression of CD138 on CD27 + CD38 + B cells. Right, percentage of CD138 + CD27 + CD38 + B cells. (C) Different immunoglobulin isotypes were measured in the culture supernatant of (A-B) by multiplex assay. (D-F) B cells from HuPD-1 mice were isolated and cultured with LPS, IL4, BAFF, or ODN2006, anti-IgM, IL-21, IL-4 or R848, anti-IgM, anti-CD40, IL-21, IFN-γ for 3 days, pembrolizumab or isotype control was added on Day 1. (D) Expression of IgG2c on activated B cells. Right, percentage of IgG2c + B cells, n = 5. (E) Expression of IgG1 on activated B cells. Right, percentage of IgG1 + B cells, n = 5. (F) Different immunoglobulin isotypes in the supernatant of were measured by multiplex assay, n = 5. Data in graphs represent mean ± SEM, Significance was tested by Two-way ANOVA.

Article Snippet: Condition 1: 2.5 μg/mL anti-human Ig (M + G + A) (Jackson Immunoresearch, Cat# 109-006-064), 2.5 μg/mL CpG ODN (Invivogen, Cat# tlrl-2006-1), 10 μg/mL anti-human CD40 (BioXcell, Cat# BE0189), 20 ng/mL rhIL-21 (Peprotech, Cat# 200-21-50UG), 10 ng/mL rhIL-4 (Biolegend, Cat# 574004), 10 ng/mL rhIL-2 (Peprotech, Cat#200-02-250UG).

Techniques: Activation Assay, Ex Vivo, Isolation, Cell Culture, Control, Expressing, Multiplex Assay

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: PD-1 inhibition does not have substantial impact on B cells ex vivo (A) Human naïve B cells were isolated from healthy donor PBMCs and stimulated with different conditions; Pembrolizumab (Keytruda) or isotype control IgG4 was added into the culture media on day 2, mean fluorescence intensity (MFI) of CD86 on B cells was measured, n = 8. (B-F) B cells were isolated from HuPD-1 mice, labeled with CTV, and cultured under 3 different conditions: LPS, rmIL-4, BAFF or Anti-IgM, CpG ODN, rmIL-21, rmIL-4, 100 rhIL-2 or R848, anti-CD40, anti-IgM, rmIL-21, rmIFN-γ for 3 days. Pembrolizumab or isotype control IgG4 was added on day 1, n = 5. (B) Summary of human PD-1 MFI on B cells from different groups. (C) Expression of CD138 on activated B cells. Right, percentage of CD138 + B cells. (D) Summaries of activation marker CD69 and CD86 MFIs on B cells from different groups. (E) Summaries of CD71 and CD98 MFIs on B cells from different groups. (F) Representative flow cytometry plot of CTV dilution on B cells. Right, a summary of the proliferation index of B cells from different groups. Data in graphs represent mean ± SEM, Significance was tested by Two-way ANOVA.

Article Snippet: Condition 1: 2.5 μg/mL anti-human Ig (M + G + A) (Jackson Immunoresearch, Cat# 109-006-064), 2.5 μg/mL CpG ODN (Invivogen, Cat# tlrl-2006-1), 10 μg/mL anti-human CD40 (BioXcell, Cat# BE0189), 20 ng/mL rhIL-21 (Peprotech, Cat# 200-21-50UG), 10 ng/mL rhIL-4 (Biolegend, Cat# 574004), 10 ng/mL rhIL-2 (Peprotech, Cat#200-02-250UG).

Techniques: Inhibition, Ex Vivo, Isolation, Control, Fluorescence, Labeling, Cell Culture, Expressing, Activation Assay, Marker, Flow Cytometry

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: Inflammatory signatures enriched in irAE patients reduce antibody production (A-F) Beads based multiplex assays were used to measure plasma concentration of IL-6, and IL-12p70 (A), TNF-α, IFN-γ and IL-1β (B), HC (n = 19), irAE (n = 34), RAC (n = 45), ICI (n = 9). IP-10 (CXCL10), CXCL11, and CXCL9 (C, HC, n = 17; irAE, n = 33; RAC, n = 46; ICI, n = 17), CCL20 (D), CX3CL1 (E), and CCL2 (F). (G-J) Human naïve B cells were isolated and cultured with 0.5 μg/mL anti-human CD40, 2.5 μg/mL anti-human Ig (M+G+A), and 20 ng/mL rhIL-21 with 100 ng/mL IFN-α, 100 ng/mL IL-6, 100 ng/mL IL-12, control, or the combination of IFN-α, IL-6, and IL-12 for 7 days. Cells and culture supernatants were analyzed. (G) Representative flow plot of CD38 and CD138 expression on CD27 hi CD38 hi ASCs. Right, a summary of the percentage of CD138 + ASCs, n = 6. (H) Expression of CD11c and CD27 on CD27 - IgD - ASCs. Right, percentage of CD11c + IgD - CD27 - B cells, n = 6. (I) Expression of active-caspase-3 in B cells. Right, percentage of active-caspase-3 + B cells from different groups, n = 3. (J) Different immunoglobulin isotype levels in the culture supernatants from G-H were measured by the multiplex assay, n = 6. Data in graphs represent mean ± SEM, Significance was tested by One-way ANOVA (A-I), and paired Student’s t-test (J).

Article Snippet: Condition 1: 2.5 μg/mL anti-human Ig (M + G + A) (Jackson Immunoresearch, Cat# 109-006-064), 2.5 μg/mL CpG ODN (Invivogen, Cat# tlrl-2006-1), 10 μg/mL anti-human CD40 (BioXcell, Cat# BE0189), 20 ng/mL rhIL-21 (Peprotech, Cat# 200-21-50UG), 10 ng/mL rhIL-4 (Biolegend, Cat# 574004), 10 ng/mL rhIL-2 (Peprotech, Cat#200-02-250UG).

Techniques: Multiplex Assay, Clinical Proteomics, Concentration Assay, Isolation, Cell Culture, Control, Expressing

Journal: Molecular Therapy Oncolytics

Article Title: Systemic immunity upon local oncolytic virotherapy armed with immunostimulatory genes may be supported by tumor-derived exosomes

doi: 10.1016/j.omto.2021.02.007

Figure Lengend Snippet: Expression of CD40L and 4-1BBL on Mel526 cells Mel526 cells infected with LOAd700 or LOAd703 express CD40L (upper histograms), while cells infected with LOAd703 also present 4-1BBL (lower histograms), as shown with flow cytometry at day 2 post-infection. Gray and black histograms represent isotype control and transgene staining, respectively.

Article Snippet: Exosomes were then incubated with a primary antibody, either CD40L (AF617, R&D Systems) or 4-1BBL (ab126274, Abcam), followed with goat anti-mouse or goat anti-rabbit as a secondary antibody conjugated with 6-nm gold particles (Jackson ImmunoResearch) in a ratio of 1:80 (40 min incubation each).

Techniques: Expressing, Infection, Flow Cytometry, Control, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Systemic immunity upon local oncolytic virotherapy armed with immunostimulatory genes may be supported by tumor-derived exosomes

doi: 10.1016/j.omto.2021.02.007

Figure Lengend Snippet: Characterization of exosomes (A) Nanoparticle tracking analysis of exosomes isolated from Mel 626 cells using NanoSight NS500. Captures 1–5 represent five captured videos for calculation of size and concentration of exosomes. (B) Exosomal markers (CD63 and CD81) were evaluated on cells (either infected or uninfected) and their exosomes using western blot. (C) Electron micrographs of isolated exosomes stained for CD40L or 4-1BBL. Arrows indicate positive staining. Cells exo, exosomes from uninfected cells; LOAd cells, cells infected with LOAd viruses; LOAd exo, exosomes released by LOAd-infected cells.

Article Snippet: Exosomes were then incubated with a primary antibody, either CD40L (AF617, R&D Systems) or 4-1BBL (ab126274, Abcam), followed with goat anti-mouse or goat anti-rabbit as a secondary antibody conjugated with 6-nm gold particles (Jackson ImmunoResearch) in a ratio of 1:80 (40 min incubation each).

Techniques: Isolation, Concentration Assay, Infection, Western Blot, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Systemic immunity upon local oncolytic virotherapy armed with immunostimulatory genes may be supported by tumor-derived exosomes

doi: 10.1016/j.omto.2021.02.007

Figure Lengend Snippet: CD40L and 4-1BBL protein content of cells and exosomes (A and B) Exosomes derived from Mel526 cells infected with LOAd viruses or left uninfected were investigated for the presence of CD40L (A) and 4-1BBL (B) with ELISA. Each group was tested in duplicate. Error bars represent standard error and median values are displayed in the graphs.

Article Snippet: Exosomes were then incubated with a primary antibody, either CD40L (AF617, R&D Systems) or 4-1BBL (ab126274, Abcam), followed with goat anti-mouse or goat anti-rabbit as a secondary antibody conjugated with 6-nm gold particles (Jackson ImmunoResearch) in a ratio of 1:80 (40 min incubation each).

Techniques: Derivative Assay, Infection, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy Oncolytics

Article Title: Systemic immunity upon local oncolytic virotherapy armed with immunostimulatory genes may be supported by tumor-derived exosomes

doi: 10.1016/j.omto.2021.02.007

Figure Lengend Snippet: Surface expression of CD40L and 4-1BBL on exosomes from LOAd-infected cells using flow cytometry (A) Total percentage of CD40L + and 4-1BBL + exosomes. (B) Mean fluorescence intensity (MFI) of CD40L and 4-1BBL expression on CD63 + bead-coupled exosomes. ∗p ≤ 0.05, n = 3. Error bars represent standard error and median values are displayed in the graphs.

Article Snippet: Exosomes were then incubated with a primary antibody, either CD40L (AF617, R&D Systems) or 4-1BBL (ab126274, Abcam), followed with goat anti-mouse or goat anti-rabbit as a secondary antibody conjugated with 6-nm gold particles (Jackson ImmunoResearch) in a ratio of 1:80 (40 min incubation each).

Techniques: Expressing, Infection, Flow Cytometry, Fluorescence

Journal: Molecular Therapy Oncolytics

Article Title: Systemic immunity upon local oncolytic virotherapy armed with immunostimulatory genes may be supported by tumor-derived exosomes

doi: 10.1016/j.omto.2021.02.007

Figure Lengend Snippet: TMZ-CD40L and 4-1BBL mRNA in infected cells and exosomes (copy number per picogram) (A and B) LOAd-infected or uninfected Mel526 cells and cell-derived exosomes were evaluated for their TMZ-CD40L (A) or 4-1BBL (B) mRNA content using quantitative real-time PCR. Samples were analyzed in triplicates (cells) or duplicates (exosomes). Statistical analyses were done using a Kruskal-Wallis test for non-parametric samples with a Dunn’s multi-comparison test. A p value of ≤0.05 was considered significant. n = 3.Error bars represent standard error and median values are displayed in the graphs.

Article Snippet: Exosomes were then incubated with a primary antibody, either CD40L (AF617, R&D Systems) or 4-1BBL (ab126274, Abcam), followed with goat anti-mouse or goat anti-rabbit as a secondary antibody conjugated with 6-nm gold particles (Jackson ImmunoResearch) in a ratio of 1:80 (40 min incubation each).

Techniques: Infection, Derivative Assay, Real-time Polymerase Chain Reaction, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Exosomal CD40, CD25, and Serum CA19-9 as Combinatory Novel Liquid Biopsy Biomarker for the Diagnosis and Prognosis of Patients with Pancreatic Ductal Adenocarcinoma

doi: 10.3390/ijms26041500

Figure Lengend Snippet: Validation of exosomal CD40 as a novel biomarker for pancreatic cancer and pancreatitis. ( A ) Normalized signal intensities of exosomal surface protein CD40 in plasma exosomes of controls (n = 51), patients with pancreatitis (n = 21), and patients with PDAC (n = 48) (Mann–Whitney test, *** p = 0.0010, **** p < 0.0001). ( B , C ) Correlations between normalized signal intensities of exosomal surface protein CD40 with nodal category and perineural invasion status in patients with PDAC. ( D ) Kaplan–Meier curves displaying survival analysis of patients with PDAC (Gehan–Breslow–Wilcoxon). Data are mean ± s.d.

Article Snippet: Serum CA19-9, CD40 and CD25 protein levels in analyzed groups were assessed using the Cancer Antigen CA19-9 Human ELISA Kit (Catalog No. ab108642, Abcam), Human CD40 Quantikine ELISA Kit (Catalog No. DCCD40 R&D systems, Minneapolis, MN, USA) and Human CD25/IL-2R alpha Quantikine ELISA Kit (Catalog No. DR2A00 R&D systems), according to the manufacturer’s protocol.

Techniques: Biomarker Discovery, Clinical Proteomics, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Exosomal CD40, CD25, and Serum CA19-9 as Combinatory Novel Liquid Biopsy Biomarker for the Diagnosis and Prognosis of Patients with Pancreatic Ductal Adenocarcinoma

doi: 10.3390/ijms26041500

Figure Lengend Snippet: Receiver operating characteristic (ROC) curves analysis of exosome-surface markers and CA19-9. ( A ) ROC curves discriminating the PDAC group from clinical controls. Each ROC curve shows the single markers exo-CD40, exo-CD25, and CA19-9 and the combination with the highest AUC. ( B ) Table representing the cut-off values, sensitivity, specificity for exo-CD40, exo-CD25, and CA19-9 surface markers and the selected combination. ( C ) ROC curves discriminating the PDAC group from pancreatitis. ( D ) Table representing the cut-off values, sensitivity, specificity for exo-CD40, exo-CD25, and CA19-9 surface markers and the selected combination.

Article Snippet: Serum CA19-9, CD40 and CD25 protein levels in analyzed groups were assessed using the Cancer Antigen CA19-9 Human ELISA Kit (Catalog No. ab108642, Abcam), Human CD40 Quantikine ELISA Kit (Catalog No. DCCD40 R&D systems, Minneapolis, MN, USA) and Human CD25/IL-2R alpha Quantikine ELISA Kit (Catalog No. DR2A00 R&D systems), according to the manufacturer’s protocol.

Techniques:

Journal: Stem Cells

Article Title: Microglia in the spinal cord stem cell niche regulate neural precursor cell proliferation via soluble CD40 in response to myelin basic protein

doi: 10.1093/stmcls/sxae076

Figure Lengend Snippet: Soluble CD40 is the microglia-derived inhibitory factor. (A) Cytokine array analysis reveals 13 factors in the 10-30 kDa molecular weight range that are more highly expressed in MG-spCM with MBP compared to MG - spCM grown in the absence of MBP. The differential intensity was determined by subtracting cytokine intensities of MG-spCM with MBP from MG-spCM minus MBP. ( n = 3 independent experiments) (B) Dose response of mResistin shows inhibition of neurosphere formation at 2000 and 4000 pg/mL ( n = 4 independent experiments, * P < .05, ** P < .01) (C) Dose response of sCD40 shows an inhibitory effect on neurosphere formation at all concentration examined from the spinal cord ( n = 4 independent experiments, **** P < .001). (D) qPCR of CD40 receptor (CD40) and ligand (CD40L) expression in brain and spinal cord derived neurospheres ( n = 3 independent experiments, *** P < .001, * P < .05). (E) Dose response of sCD40 on brain neurosphere formation ( n = 4 independent experiments, **** P < .0001).

Article Snippet: Enzyme-linked immunosorbent assays (ELISAs) were purchased from R&D Systems for CD40 (Catalog #MCCD40) and used for the analysis of microglia-derived CM samples as per the manufacturer's instructions for cell culture supernatant samples.

Techniques: Derivative Assay, Molecular Weight, Inhibition, Concentration Assay, Expressing