Journal: bioRxiv

Article Title: Differentiation of human T follicular helper cells in vitro requires co-operation between STAT3 and SMAD signaling cytokines that is restrained by IL-2

doi: 10.1101/2025.01.12.632620

Figure Lengend Snippet: (A) Naïve CD4 T cells were isolated from peripheral blood and cultured as displayed. Cells were harvested at day 6. (B) Representative plots of PD-1 and CXCR5 expression by naïve CD4 T cells, control CD4 and in vitro TFH cultured as in (A). Numbers represent percent of cells in each gate. (C) CXCR5 MFI, PD1 MFI and (D) proportion of CXCR5+ PD1+ cells generated (as gated in (B)). (E) Representative histogram and quantification of BCL6 expression. (F) IL-21 production was quantified after cells were stimulated with PMA/Ionomycin in the presence of Brefeldin A. (G) Expression of core TFH-associated transcripts was quantified, in relation to expression of housekeeping gene ACTB and GAPDH transcripts. (H) Ratio of BCL6:PRDM1 transcript was quantified from data in (G). (I) Heterologous naïve B cells were cultured with either 100ng/mL IL-21 and 5μg/mL αCD40 or in vitro TFH for 72 hours. (J) BCL6, CXCR5 and PD1 upregulation was quantified daily on a subset of in vitro TFH cultures. For (C-I) Tukey’s multiple comparison test was used.

Article Snippet: 5×10 4 heterologous B cells were plated per well and then cultured with either 100ng/mL IL-21 (R&D Systems) and 5μg/mL αCD40 (Invitrogen) or 2.5 x 10 4 in vitro TFH.

Techniques: Isolation, Cell Culture, Expressing, Control, In Vitro, Generated, Comparison

Journal: Journal for Immunotherapy of Cancer

Article Title: Brain tumors induce immunoregulatory dendritic cells in draining lymph nodes that can be targeted by OX40 agonist treatment

doi: 10.1136/jitc-2025-011548

Figure Lengend Snippet: Treatment with an anti-OX40 agonist enhances immune responses in cervical TdLN. Schematic illustration of the experimental layout ( A ) used to obtain data shown in ( B–F ): groups of 5–7 mice were inoculated with the glioma cell line SB28-OVA, followed by i.p. administration of either an anti-OX40 agonist or IgG control on days 4, 7, and 10 post-tumor inoculation. On day 12 post-tumor inoculation, the TdLN-c were harvested, and single-cell suspensions were collected for immune profile analysis using flow cytometry. ( B ) H2b-SIINFEKL-positive DCs in TdLN on day 12 after treatment with OX40 agonist or control antibody. ( C ) Activation markers, CD40 and CD86, expressed by DCs. ( D ) Regulatory T cells (CD4+CD25+ Foxp3+). ( E ) Regulatory T cell (CD127+/-). ( F ) Memory T-cell response for CD4+ and CD8+ central memory (CM, CD44+CD62L+) and effector memory (EFM, CD44+CD62L-). ( G, I ) Tumor growth was evaluated using bioluminescence imaging. ( H ) H&E staining of brain tissue from the endpoint. ( J ) Kaplan–Meier survival curves of tumor-bearing mice treated with OX40 agonist or control IgG. The graph shows combined survival curves obtained from two independent experiments. Bar graphs show the mean±SD. One-way ANOVA multiple comparisons test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; i.p., intraperitoneally; TdLN, tumor-draining lymph nodes.

Article Snippet: The following stimuli were added: Poly-I:C 5 μg/mL (Adipogen Life Sciences), αCD40 (FGK45), αIL-6 (MP5-20F3), αIL-4 (BVD6-24G2), αOX40 (OX-86), αCD200 (OX-90), and αPDL-1 (10F.9G2TM) (BioXcell) at a concentration of 100 ng/mL.

Techniques: Control, Flow Cytometry, Activation Assay, Imaging, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Brain tumors induce immunoregulatory dendritic cells in draining lymph nodes that can be targeted by OX40 agonist treatment

doi: 10.1136/jitc-2025-011548

Figure Lengend Snippet: CCR7+OX40+ dendritic cells (DCs) in cervical lymph nodes correlate with anti-OX40 agonist immunotherapy. DCs from TdLNs were isolated and cultured in the presence of the labeled peptide DQ-OVA, which allowed for quantification of antigen uptake, processing, and presentation. (A, B) Representative histograms and percentages of DQ-OVA signals in different DC populations. (C) DCs from TdLN were cultured as in (A) and incubated in the presence of anti-OX40 agonist, Poly-I:C or IgG control. The percentages of CD40 (C), CD86 (D), and PDL-1 (E) were calculated. (F) The percentage of CCR7+OX40+ DCs was calculated from TdLNs harvested 12 days post-tumor inoculation. (G) Representative immunofluorescence staining images of brain sections showing LAMP3+ (*), CD4+ (+), and OX40+ (¤). (H) The graph shows the average number of positive cells across different regions of interest (ROIs, n=392). (I, J) Kaplan-Meier survival curves of EG7-OVA tumor-bearing mice treated with agonistic OX40 antibody or IgG control, either alone or combined with FTY720, were analyzed using the log-rank test (n=5–7 mice/group test). Statistical analyses were conducted using the Mann-Whitney U test. Bar graphs show the mean±SD; two-way ANOVA with Tukey’s correction: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance TdLNs, tumor-draining lymph nodes.

Article Snippet: The following stimuli were added: Poly-I:C 5 μg/mL (Adipogen Life Sciences), αCD40 (FGK45), αIL-6 (MP5-20F3), αIL-4 (BVD6-24G2), αOX40 (OX-86), αCD200 (OX-90), and αPDL-1 (10F.9G2TM) (BioXcell) at a concentration of 100 ng/mL.

Techniques: Isolation, Cell Culture, Labeling, Incubation, Control, Immunofluorescence, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: IL-21 Shapes the B Cell Response in a Context-Dependent Manner

doi: 10.1101/2024.07.13.600808

Figure Lengend Snippet: (a) Illustration of activation protocol of naïve primary B cells ex vivo . Naïve splenic B cells were cultured with αCD40 (1 μg/mL), IL-4 (12.5 ng/mL), or IL-21 (50 ng/mL). (b) Changes in cell numbers in culture over time. The y-axis represents changes in cell numbers relative to the starting point (0 hours). (c) Percentage of active caspase-3 + B cells among total live B cells. (d, h) Changes in cell numbers over time under various stimulation conditions. The y-axis represents the (d) absolute cell number or (h) changes in cell numbers relative to the starting point (0 hours). (e, i) Percentage of active caspase-3 + B cells among the total live B cell population in different stimulation conditions at 72 hours after starting culture. (f) Geometric MFI of Bim in STAT3-deficient or control B cells stimulated as indicated for 8 hours. (g) Splenic B cells were pre-stimulated with αCD40 + IL-4 for 48 hours and FACS-sorted IgM+ B cells were re-stimulated with either αCD40 + IL-4 or αCD40 + IL-21 for 30 minutes. Chromatin binding of STAT3 was subsequently assessed by CUT&RUN and spike-in scaled signals were averaged for track visualization. Blue highlight denotes a significant peak called by MACS2 algorithm for pooled samples of WT IL-21 condition. Data were pooled from two or three independent experiments with n = 2 or 3 per experiment. (a-c) Different stimulation conditions are represented by distinct colors: αCD40 (black), αCD40 + IL-4 (red), αCD40 + IL-21 (blue), and αCD40 + IL-4 + IL-21 (green). (d-h) Different experimental groups are represented by distinct colors: Bcl2l11 +/+ (black), Bcl2l11 -/- (red), Stat3 fl/fl (blue), and Stat3 fl/fl ::Cd23-cre (green). For (b), the 96-hour timepoint data were subject to ANOVA in conjunction with Tukey’s multiple comparisons test. Data in (c) were analyzed with two-way ANOVA in conjunction with Tukey’s multiple comparisons test. Data in (d), (e), (f), (h), and (i) were analyzed with Student’s unpaired t-test, with multiple comparisons correction applied using the Holm-Šídák method.

Article Snippet: B cells were stimulated under various conditions as listed below, unless otherwise specified: αCD40 mAb (Invitrogen or BioLegend, Clone HM40-3, 1 μg/mL), IL-4 (R&D Systems, 12.5 ng/mL), IL-21 (R&D Systems, 50 ng/mL), αIgM mAb (F(ab’)2-Goat anti-Mouse IgM, Invitrogen, 1 μg/mL).

Techniques: Activation Assay, Ex Vivo, Cell Culture, Control, Binding Assay

Journal: bioRxiv

Article Title: IL-21 Shapes the B Cell Response in a Context-Dependent Manner

doi: 10.1101/2024.07.13.600808

Figure Lengend Snippet: (a) Changes in cell numbers over time under various stimulation conditions. (b) Percentage of active caspase-3 + B cells among the total live B cells. (c) Schematic representation of pre-activated B cell culture. Naïve splenic B cells were cultured with αCD40 + IL-4 for 48 hours, followed by secondary stimulation with αCD40 + IL-4, αCD40 + IL-21, or αCD40 + IL-4 + IL-21 for an additional 48 hours. Concentration for stimuli: αCD40 (1 μg/mL), IL-4 (12.5 ng/mL), IL-21 (50 ng/mL). (d) Changes in cell numbers at 72 and 96 hours of culture compared to the 48-hour time point. The y-axis represents changes in cell numbers relative to the beginning of secondary stimulation (48 hour). (e) Schematic depiction of the iGB culture setup. Naïve splenic B cells were cultured with the 40LB feeder cells and IL-4 (1 ng/mL) for 96 hours. Subsequently, secondary stimulation was performed with fresh 40LB feeder cells in the presence of IL-4 (1 ng/mL) or IL-21 (10 ng/mL). (f) Changes in cell numbers over time. The y-axis represents changes in cell numbers relative to the start of cell culture (0 hours). Data were pooled from three independent experiments with n = 3 or 4 per experiment. Data in (a), (b), and (f) were analyzed with Student’s unpaired t-test, with multiple comparisons correction applied using the Holm-Šídák method. Data in (d) were statistically analyzed using ANOVA in conjunction with Tukey’s multiple comparisons test.

Article Snippet: B cells were stimulated under various conditions as listed below, unless otherwise specified: αCD40 mAb (Invitrogen or BioLegend, Clone HM40-3, 1 μg/mL), IL-4 (R&D Systems, 12.5 ng/mL), IL-21 (R&D Systems, 50 ng/mL), αIgM mAb (F(ab’)2-Goat anti-Mouse IgM, Invitrogen, 1 μg/mL).

Techniques: Cell Culture, Concentration Assay

Journal: bioRxiv

Article Title: IL-21 Shapes the B Cell Response in a Context-Dependent Manner

doi: 10.1101/2024.07.13.600808

Figure Lengend Snippet: (a, c) Representative flow cytometry plot displaying IgM + and IgG1 + B cell gating after 96 hours of ex vivo culture for (a) naïve B cell or (c) pre-activated B cells. Gating was adjusted for different stimulation conditions. (b, d) Percentage of IgG1-positive cells among live B cells after 96 hours of ex vivo culture for (b) naïve B cell or (d) pre-activated B cells. (e) Percentage of IgG1 + B cells among the live B cell population in the iGB culture. (f-g) Quantitative RT-PCR analysis for (f) germline transcript or (g) Aicda mRNA 48 hours after the culture of naïve primary B cells with the indicated combinations of stimulation. Each dot represents a biological replicate, and each biological replicate is the average of three technical replicates. The y-axis represents the expression of germline transcripts relative to that of B cells from the same animal stimulated with αCD40. Germline transcript expression for each sample was calculated by subtracting the (f) β-actin or (g) Ubc C T (cycle threshold) value from the sample C T value. For (b), (f), and (g), data were pooled from two independent experiments with n = 3 per experiment. For (d) and (e), data were pooled from three independent experiments with n = 3 or 4 per experiment. Data in (b), (d), (f) and (g) were analyzed with ANOVA in conjunction with Tukey’s multiple comparisons test. For (f), αCD40 was used as the control for statistical test. For (g), pairwise comparison for every condition was performed. Data in (e) were analyzed with Student’s unpaired t-test, with multiple comparisons correction applied using the Holm-Šídák method.

Article Snippet: B cells were stimulated under various conditions as listed below, unless otherwise specified: αCD40 mAb (Invitrogen or BioLegend, Clone HM40-3, 1 μg/mL), IL-4 (R&D Systems, 12.5 ng/mL), IL-21 (R&D Systems, 50 ng/mL), αIgM mAb (F(ab’)2-Goat anti-Mouse IgM, Invitrogen, 1 μg/mL).

Techniques: Flow Cytometry, Ex Vivo, Quantitative RT-PCR, Expressing, Control, Comparison