Journal: Journal of Biological Chemistry

Article Title: dlk1/FA1 Regulates the Function of Human Bone Marrow Mesenchymal Stem Cells by Modulating Gene Expression of Pro-inflammatory Cytokines and Immune Response-related Factors

doi: 10.1074/jbc.m607530200

Figure Lengend Snippet: FIGURE 5. Dlk1 expression synergizes the effect of LPS-induced cytokines in hMSC cells and stimulates the B cell proliferation. A, control and hMSC-dlk1 cells were stimulated with 100 ng/ml LPS for 24 h. Total RNA was isolated and analyzed for the expression of the indicated genes by real time PCR. Expression of each target gene was normalized to -actin. Data are shown as means S.D. of three independent experiments. *, p 0.05; **, p 0.005 versus control cells (hMSC-TERT) induced with LPS. B, CD19-purified human peripheral blood B lymphocytes were cultured in CM obtained from hMSC-TERT (control cells) or hMSC-dlk1 cells as described under “Experimental Procedures” in the absence and the presence of 2.5 or 10 g/ml CD40L. Cell proliferation was measured with XTT-based assay. Stimulation in the cell proliferation was represented as fold induction over nonstimulated cells cultured in the corresponding CM. The bars are the means S.D. of five independent experiments. *, p 0.05; **, p 0.005 versus cultured cells in control/CM.

Article Snippet: Cells were then resuspended at 0.5 106/ml in RPMI (as a positive control), serum-free conditioned medium from hMSC-TERT (control/CM), or hMSC-dlk1 (dlk1/CM) supplemented with 10% FCS and stimulated with 20 ng/ml recombinant human IL-4 (R&D Systems, Abingdon, UK) and the indicated concentrations of recombinant human CD40L (R&D Systems) or left untreated (nonstimulated).

Techniques: Expressing, Control, Isolation, Real-time Polymerase Chain Reaction, Purification, Cell Culture, XTT Assay

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: Design of the coiled-coil peptide delivery platform. (A) Anti-CD40 antibody constructs of IgG1 or IgG2 isotypes were connected with E domains of various lengths (EIAALEK)3-6 via a peptide linker (4GS)x2to the C-terminal of the light chain, heavy chain or both light and heavy chains. (B). Combination of antibodies connected with E domains with K domains fused with OVA peptides leads to the formation of complexes. Created with BioRender.com.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: Construct

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: K4-OVA peptides strongly bind E4 domains connected to anti-CD40 antibody constructs and aid formation of stable complexes capable of cell binding and CD40 activation. Binding interactions between E and K coiled coil peptides as determined by ELISA and bio-layer interferometry (BLI). ELISA plates were coated with human CD40 antigen followed by addition of antibody constructs of either IgG1 (A) or IgG2 (B) isotype connected with E4 domains. Biotinylated K4 domains fused with OVA peptides were added followed by detection using streptavidin HRP. Representative BLI affinity measurements to determine binding kinetics of (C) mAbLCE4-IgG1 or (D) mAbLCE4-IgG2 antibody constructs interacting with K4 domains fused with OVA peptides. Antibody constructs connected with E domains were captured on anti-fab 2nd generation (FAB2G) sensors and assayed against serially diluted K4-OVA peptides in solution. Antibody-peptide complex formation and binding of the complexes to B16-F10 CD40 expressing and B16-F10 wt cells was assessed by flow cytometry. IgG1 anti-CD40 antibodies connected with E4 were mixed with equimolar amounts of biotynylated K4-OVA to enable complex formation followed by staining using PE streptavidin and flow cytometry analysis. (E) Cells incubated with 5 µg mAbLCE4 + 0.5 µg biotinylated K4-OVA peptides, (F) cells incubated with 5 µg mAbLCE4, (G) cells incubated with 0.5 µg biotinylated K4-OVA peptides, (H) cells without any compounds. Data shown is representative of two independent experiments. (I) a reporter assay was performed to assess the ability of IgG1 anti-CD40 antibody constructs connected with E domains to induce CD40 activation. CD40 reporter cells (promega) were incubated with FcγRI (CD64) CHO expressing cells or wildtype CHO cells in the presence of mAbLCE3 complexed with K4-OVA peptides, mAbLCE3 and mAb control. CD40 activation was measured according to the manufacturer’s instructions.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: Construct, Binding Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Staining, Incubation, Reporter Assay, Control

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: Stability characterization of anti-CD40 antibody constructs connected with E domains. (A) Ability of the constructs to withstand shear stress was determined by measuring A280 before and after rapid agitation. (B) Constructs were incubated in human serum or BSA for seven days after which a Mono ELISA was performed. (C) Stability of the constructs in un optimized buffer (PBS) was analyzed by incubation at room temperature (RT) or 40 °C for 2 weeks followed by SEC-HPLC analysis. Data is presented as changes in high molecular weight species (Δ HMWS) before and after stability studies. (D) Constructs were subjected to three rounds of freeze (-80 °C)-thawing (37 °C) at 24, 48, and 72 hours followed by SEC-HPLC analysis. Data is presented as changes in high molecular weight species (Δ HMWS) before and after stability studies.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: Construct, Shear, Incubation, Enzyme-linked Immunosorbent Assay, High Molecular Weight

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: Combining the coiled coil platform with potential antigen carrying nanocarriers. (A) An illustration of formation of antibody-peptide-bead complexes from interactions between antibody-peptide conjugate constructs, biotinylated K6-OVA peptides and streptavidin labeled beads (utilized to mimic antigen loaded nanocarriers). Antibody-peptide-bead complexes were prepared first before addition to cells. (B) B16-F10 CD40 and B16-F10 wt cells incubated with antibody-peptide-bead complexes and stained with both PE anti-streptavidin and anti-human lgG Fc-PE. (C) B16-F10 CD40 and B16-F10 wt cells incubated with antibody-peptide-bead complexes stained with only PE anti-streptavidin. (D) B16-F10 CD40 and B16-F10 wt cells incubated with antibody-peptide-bead complexes stained with only anti-human lgG Fc-PE. (E) Unstained B16-F10 CD40 and B16-F10 wt cells without any compounds. All stainings were followed by FACS analysis. The data depicted here is representative of two independent experiments. was created with BioRender.com.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: Construct, Labeling, Incubation, Staining

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: Coiled-coil technology enables efficient OVA peptide cross-presentation and activation of OVA-specific CD8+ T cells. (A) Mice treatment plan. On day 0 and day 7, non-tumor bearing male hCD40tg mice were subcutaneously treated with different compound formulations. Seven days after the second treatment (day 14), mice were sacrificed and their inguinal lymph nodes closest to the treatment site (iLN) were removed for FACS analysis of the percentage of OVA peptide-specific CD8+ T cells. (B) Mice were treated with vehicle (PBS), or molar equivalent amounts of the peptides alone or in combination with mAbLCE4 IgG1 construct. (C) a similar treatment set up as that in (B) was used and additional molecules were included (mAbLCE4 -IgG2 + K4-OVA, mAbLCE4 -IgG2+ OVA peptides, mAb wt + OVA peptides). Four to five mice were used per group in each experiment. The graphs show the mean ± SD of data from each experiment. Statisctical analysis was performed using a Mann-Whitney test, * p < 0.05; ** p < 0.01. (D) a postulated mode of action of the technology. After internalization of the antigen-peptide complexes, DCs process and cross-present antigenic peptides to T-cells that are activated to kill tumor cells. (E) An example of a potential application of the technology could be in personalized neoantigen cancer vaccines. From patient tumors, neoantigens are identified and characterized. Neoantigens fused with K domains are manufactured and combined with off-the-shelf anti-CD40 antibodies connected with E domains. Finally, patients are vaccinated with the mixture. were created with BioRender.com.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: Activation Assay, Construct, MANN-WHITNEY, Vaccines, Immunopeptidomics

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: Evaluating the potential of the technology to evoke anti-tumor responses in vivo . (A) Human CD40 transgenic mice were subcutaneously (s.c) administered with (0.2x10 6 ) MB49-EpCam-OVA tumor cells on day 0 followed by s.c. treatment on the right outer flank with either vehicle (PBS), 1.5 µM free OVA-peptides, 0.4 µM of mAbLCE4, 1.5 µM K4-OVA peptides, 0.4 µM of mAbLCE4 in combination with 1.5 µM K4-OVA peptides or 0.4 µg of mAbLCE4 in combination with 1.5 µM OVA peptides on day 10 and 17. (B) Tumor growth and (C) survival over time were monitored. The graphs show the mean (+SEM) of 7–10 mice per group in one experiment. Statistical analysis of tumor volumes was performed on days 21–34 using a Mann-Whitney test. Survival analysis was conducted using Kaplan-Meier log-rank test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns- not statistically significant.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: In Vivo, Transgenic Assay, MANN-WHITNEY

Journal: Science Advances

Article Title: STAT3 haploinsufficiency is associated with autosomal dominant hyper-IgE syndrome

doi: 10.1126/sciadv.adw2464

Figure Lengend Snippet: ( A ) STAT3 expression was evaluated by Western blot in Stat3 +/+ and Stat3 +/− cells derived from the spleen, thymus, and bone marrow (BM). Tubulin was used as loading control. Each lane represents one mouse. ( B ) A quantification of the STAT3/loading control ratio after normalization is shown for independently performed experiments as in (A). The mean ± SD is plotted. ( C ) IgE serum levels in Stat3 +/+ and Stat3 +/− mice were measured by ELISA. ( D ) Flow cytometry to identify IgG1 + and IgE + cells from IgM − IgD − cells 4 days after culturing Stat3 +/+ and Stat3 +/− B220 + splenocytes with IL-4 (12.5 ng/ml) and anti-CD40 antibody (62.5 ng/ml) in the absence or presence of IL-21 (25 ng/ml). Numbers in dot plots represent cell frequencies. ( E ) Mean (±SD) frequencies of the IgG1 + and IgE + cells as gated in (D). ( F ) Levels of IgG1 and IgE in the supernatant of cell cultures as done in (D) were measured by ELISA. B220 + splenocytes from Stat3 +/+ and Stat3 +/− mice were stimulated for different time points with IL-21 (50 ng/ml) ( G ), IL-10 (50 ng/ml) ( H ), and IL-6 (50 ng/ml) ( I ), and the phosphorylation of STAT3 (p-STAT3) was evaluated by flow cytometry. Mean (±SD) of mean fluorescent intensity (MFI) is plotted. Data are representative of at least three independent experiments. P values by unpaired t test (C) or two-way ANOVA followed by Bonferroni’s or Tukey’s multiple comparisons test (B and E to I).

Article Snippet: Cells were cultured in complete B cell medium [RPMI with 10% FBS, 10 mM Hepes, GlutaMAX, penicillin-streptomycin (Gibco), and 50 μM β-mercaptoethanol (Sigma-Aldrich), supplemented with IL-4 (12.5 ng/ml; PeproTech) and anti-CD40 antibody (62.5 ng/ml; Miltenyi Biotec), in the absence or presence of IL-21 (25 ng/ml; PeproTech)].

Techniques: Expressing, Western Blot, Derivative Assay, Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Phospho-proteomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics 1

doi: 10.4049/jimmunol.1600782

Figure Lengend Snippet: PBMCs of subjects were recovered from liquid nitrogen and stimulated with CD40L at concentrations ranging from 0 to1000ng/mL and phosphorylation of P65, P38, ERK and JNK were monitored at different time points by instant fixation, permeabilization and staining with antibody master mixes containing cell surface markers and phospho-epitope antibodies. Single cells were then analyzed by flow cytometry. A) Gating strategy to identify cell subtypes within PBMCs. Cells were first gated on FSC-A and SSC-A to exclude cell debris. After singlet gating, cells were gated on CD3 versus CD4 to identify CD4 T cells (CD3+CD4+), CD8 T cells (CD3+CD4−) and monocytes (CD3dimCD4dim with higher SSC-A than lymphocytes). B cells were then identified in the CD3−CD4− population based on the presence of CD20 expression. Memory B cells (CD20+CD27+) and naïve B cells (CD20+CD27−) were further identified in total B cell pools based on the presence or absence of CD27 expression. B–C) Phosphorylation of P65 was analyzed in indicated cell subsets. The median fluorescent intensity (MFI) was based on 2000–40,000 single-cell measurements depending on cell type. B) Overlaid histograms show the time course of P65 phosphorylation in memory and naïve B cells of a healthy donor after stimulation with 1µg/mLCD40L. C) Overlaid histograms show the dose-response of P65 phosphorylation in memory and naïve B cells at 15min after stimulation with indicated concentrations of CD40L. Black histograms show the unstimulated samples corresponding to basal phosphorylation levels. Data are representative of 9 healthy individuals tested.

Article Snippet: Human CD40 ligand (Cell Signaling Technology, Danvers, MA) or human TNFα (R&D Systems, Minneapolis, MN) in complete medium was added at indicated concentrations.

Techniques: Phospho-proteomics, Staining, Flow Cytometry, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics 1

doi: 10.4049/jimmunol.1600782

Figure Lengend Snippet: A) PBMCs from treatment-naïve RRMS patients (RRMS_TN) and healthy donors (HD) were analyzed by multi-parameter phosflow at different time points post-stimulation with 2ng/mL CD40L, gating on memory and naïve B cell populations, as indicated. Scatterplots of the MFI for p-P65, p-P38, p-ERK, and p-JNK in memory and naïve B cells are shown. A) Data shown are phosflow of PBMCs isolated from leukopheresis pack of treatment-naïve RRMS patients (n=12, closed circles) and healthy donors (n=9, open circles). B) Western blot analysis of CD40-induced pP65 and pP38 in purified CD19+ B cells from healthy donors and RRMS patients. CD19+ B cells were magnetically isolated from PBMCs from treatment-naive RRMS patients (RRMS-TN, n=6) and healthy donors (HD, n=6) using CD19 microbeads. Isolated CD19+ B cells were left unstimulated (labeled as "-") or stimulated with 2ng/mL CD40L for 15 minutes (labeled as "+"). After stimulation, the lysates were harvested and analyzed by Western blot with antibodies recognizing the phosphorylated form of P65 and P38 Antibodies. Level of β-actin was shown as a loading control. C) Mean Pixel Values (band density) of Western blot data in B were acquired using Image Studio software. The levels of pP65 and pP38 were then normalized to β-actin level. Data shown were fold induction of pP65 and pP38 after CD40L stimulation compared to unstimulated condition.

Article Snippet: Human CD40 ligand (Cell Signaling Technology, Danvers, MA) or human TNFα (R&D Systems, Minneapolis, MN) in complete medium was added at indicated concentrations.

Techniques: Isolation, Western Blot, Purification, Labeling, Control, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics 1

doi: 10.4049/jimmunol.1600782

Figure Lengend Snippet: CD40 stimulation and phosflow were performed as described in Figure 2 on PBMCs isolated from blood of treatment-naïve SPMS patients (n=12, closed squares), treatment-naïve NMO patients (n=5, inverted triangles) and their controls isolated from blood of healthy donors (n=9, open circles). Data shown is representative of two repeated experiments. Significant differences in MFI values were calculated by Student’s t test (P<0.05).

Article Snippet: Human CD40 ligand (Cell Signaling Technology, Danvers, MA) or human TNFα (R&D Systems, Minneapolis, MN) in complete medium was added at indicated concentrations.

Techniques: Isolation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics 1

doi: 10.4049/jimmunol.1600782

Figure Lengend Snippet: A–B) PBMCs from RRMS patients (n=12 with 1 data point from 12 RRMS patients, closed circles) and healthy donors (n=12 with 2 data points from 6 healthy individuals, open circles) were analyzed by multi-parameter phosflow at different time points post-CD40L (2ng/mL) stimulation, gating on memory (A) and naïve (B) B cell populations, as indicated. Scatterplots of the MFI for p-IKKα/β are shown. Data shown is representative of two repeated experiments. Significant differences in MFI values were calculated by Student’s t test (P<0.05). (C–F) PBMCs from RRMS patients were stimulated with 2ng/mL CD40L in the presence or absence of TCPA-1 inhibitor at indicated concentrations. The cells were instantly fixed 15 minutes after the stimulation and analyzed by phosflow to detect p-P65 and p-P38. Cells stimulated with CD40L in the presence of DMSO served as a vehicle control. Data shown is from one RRMS patient as a representative of experiments from 3 individual RRMS patients.

Article Snippet: Human CD40 ligand (Cell Signaling Technology, Danvers, MA) or human TNFα (R&D Systems, Minneapolis, MN) in complete medium was added at indicated concentrations.

Techniques: Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics 1

doi: 10.4049/jimmunol.1600782

Figure Lengend Snippet: A–D) Following CD40 stimulation for 15 minutes, the level of p-P65 and p-P38 in memory B (A and C) and naïve B cells (B and D) from healthy donors (n=9, open circles), RRMS patients (n=8) before (RRMS_TN (T0), closed circles) and after (RRMS_A+C (T1), closed triangles) the therapy were included. The levels of p-P65 and p-P38 were detected at 15 minutes after stimulation with CD40L at 2ng/mL. P values were calculated by Student’s t test and were shown in the figure. E–F) EDSS scores were calculated before (T0) and after (T1) the combination treatment with Avonex and Cellcept. Changes in EDSS score (ΔEDSS) were assessed by subtracting the EDSS at the time of sampling (EDSST1) with the baseline EDSS before any treatments (EDSST0). The peak MFI values for p-P65 (15 minutes after CD40 stimulation) in memory (A) and naïve B cells (B), were plotted relative to ΔEDSS as indicated.

Article Snippet: Human CD40 ligand (Cell Signaling Technology, Danvers, MA) or human TNFα (R&D Systems, Minneapolis, MN) in complete medium was added at indicated concentrations.

Techniques: Sampling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics 1

doi: 10.4049/jimmunol.1600782

Figure Lengend Snippet: PBMCs isolated from blood of treatment-naïve RRMS patients (n=12, closed circles) and GA-treated RRMS patients (n=8, closed triangle) were analyzed by multi-parameter phosflow at 15 minutes post-CD40L (2ng/mL) stimulation, gating on memory and naïve B cell. A and C) P65 phosphorylation of memory B (upper panel) and naïve B cells (lower panel). B and D) P38 phosphorylation of memory B (upper panel) and naïve B cells (lower panel). P values were calculated by Student’s t test and were shown in the figure.

Article Snippet: Human CD40 ligand (Cell Signaling Technology, Danvers, MA) or human TNFα (R&D Systems, Minneapolis, MN) in complete medium was added at indicated concentrations.

Techniques: Isolation, Phospho-proteomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics 1

doi: 10.4049/jimmunol.1600782

Figure Lengend Snippet: CD40-CD40L interaction recruits TRAFs (TRAF2, 3, 5 and 6) which bind to the CD40 cytoplasmic domain and mediates the activation of multiple signaling pathways including canonical and non-canonical NFκB pathways, PI3K pathway, as well as phosphorylation of MAP kinases including P38, ERK and JNK. For canonical NFκB activation, TRAF2 or TRAF6 mediates signaling activation and phosphorylates the subunits (IKKα, IKKβ and IKKγ) in the IKK complex, which further induces the phosphorylation of IκB. The phosphorylation of IκB induces its degradation and this process leads to phosphorylation and nuclear translocation of NFκB, where it acts as a transcription activator. In normal B cells (left), the CD40 signaling plays an essential role in B cell proliferation, survival and cytokine production. In B cells of RRMS patients (right), we found enhanced CD40-mediated canonical NFκB signaling while the MAPK pathway remains unaffected. A prediction of the model is that dysregulation of CD40-mediated NFκB signaling leads to the hyper-proliferation of MS B cells upon CD40 engagement as we described previously. In addition, our data points to the potential of therapeutic interventions including Avonex/Cellcept combination therapy and GA therapy to correct this signaling dysregulation.

Article Snippet: Human CD40 ligand (Cell Signaling Technology, Danvers, MA) or human TNFα (R&D Systems, Minneapolis, MN) in complete medium was added at indicated concentrations.

Techniques: Activation Assay, Protein-Protein interactions, Phospho-proteomics, Translocation 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: 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: Science advances

Article Title: High-throughput functional screening for next-generation cancer immunotherapy using droplet-based microfluidics.

doi: 10.1126/sciadv.abe3839

Figure Lengend Snippet: Fig. 4. Screening CD40 agonist from a spike-in library using a microfluidic system. (A) RFP-positive hexameric CD40L protein–secreting cells were spiked into a 10-fold excess of BFP-positive anti-HEL antibody–secreting cells, and the mixture of cells was coencapsulated with the reporter cells. After incubation, droplets containing activated reporter cells were sorted. The proportion of droplets containing RFP- or BFP-positive cells before and after sorting was analyzed. (B) Bright-field and fluores- cence images of droplets before and after sorting.

Article Snippet: CD40 antibody selectivity Human CD40 (ACROBiosystems), GITR (ACROBiosystems), OX40 (ACROBiosystems), 4-1BB (ACROBiosystems), or bovine serum albumin (BSA) (Solarbio) was plated onto a microtiter plate at 4°C overnight.

Techniques: Incubation

Journal: Science advances

Article Title: High-throughput functional screening for next-generation cancer immunotherapy using droplet-based microfluidics.

doi: 10.1126/sciadv.abe3839

Figure Lengend Snippet: Fig. 5. Screening CD40 agonist antibody from a monoclonal antibody library. HEK293FT cells were infected with a lentivirus antibody library and individually coen- capsulated with Jurkat/NF-B-GFP-hCD40 reporter cells and fluorescence-labeled secondary antibodies in droplets. Droplets containing reporter cells activated by anti- bodies secreted by the coencapsulated antibody–expressing cells were sorted. The sorted cells were expanded for the second round of selection, and enriched antibodies were identified by next-generation sequencing. (A) Schematic of possible time traces. (B) Proportions of different types of droplets for each round of selection were analyzed. (C) Bright-field and fluorescence images of the sorted droplets after the second round of selection. (D) Bar plot for the top 20 scFv clusters and their fre- quencies during the selection process. (E) The change in frequencies of the selected antibodies during the selection process. (F) Agonist activity of the selected antibodies was determined using the CD40 reporter cell line in the presence or absence of the cross-linking secondary antibody.

Article Snippet: CD40 antibody selectivity Human CD40 (ACROBiosystems), GITR (ACROBiosystems), OX40 (ACROBiosystems), 4-1BB (ACROBiosystems), or bovine serum albumin (BSA) (Solarbio) was plated onto a microtiter plate at 4°C overnight.

Techniques: Infection, Fluorescence, Labeling, Expressing, Selection, Next-Generation Sequencing, Activity Assay