Journal: Cell Death & Disease

Article Title: TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcγR-independent agonistic activity

doi: 10.1038/s41419-019-1456-x

Figure Lengend Snippet: a Domain architecture of TNFR2. b HeLa-TNFR2 cells were incubated in triplicates with anti-TNFR2 antibodies recognizing the indicated domain of TNFR2 along with HEK293 cells transfected with empty vector (EV) or a FcγR2B-encoding expression plasmid. One day later, cell supernatants were analyzed for IL8 production. Stimulation with a saturating concentration (200 ng/ml) of the highly potent TNFR2 agonist TNC-scTNF80 served as a positive control. c TNFR2-responsive HeLa-TNFR2 cells and Fn14-responsive WiDr cells were challenged in triplicates with the indicated anti-TNFRSF receptor antibodies and empty vector (EV) or FcγR2B transfected HEK293 cells which have no (TNFR2) or only low (Fn14) endogenous expression of the TNFRSF receptors studied and which only produce limited amounts of IL8. Next day, cell supernatants were analyzed for IL8 production as readout of TNFRSF receptor activation. d HeLa-TNFR2 cells were treated overnight in triplicates with HEK293 cells transfected with empty vector or expression plasmids encoding the indicated FcγR types and 1 µg/ml of IgG1, IgG2, IgG3, IgG4, mIgG1, and mIgG2A variants of the anti-TNFR2 antibody C4. Finally, IL8 production was quantified by ELISA

Article Snippet: For FACS analysis of CD19, CD20, and CD70 expression FITC-labeled antibodies from BD Bioscience, San Jose, CA, USA (anti-CD19 FITC-labeled mouse IgG1, #345776; anti-CD70 FITC-labeled mouse IgG3, #555834), Caprico Biotechnologies, Norcross, GA, USA (anti-CD20 FITC-labeled mouse IgG1, #103714), and Miltenyi Biotec GmbH, Bergisch Gladbach, Germany (anti-murin IgG1 FITC-labeled rat IgG1, #130-095-897) were used.

Techniques: Incubation, Transfection, Plasmid Preparation, Expressing, Concentration Assay, Positive Control, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcγR-independent agonistic activity

doi: 10.1038/s41419-019-1456-x

Figure Lengend Snippet: a Domain architecture and structural organization of C4-IgG1(N297A)-HC:scGITRL, C4-IgG1(N297A)-HC:sc(mu)4-1BBL, C4-IgG1(N297A)-HC:(mu)GITRL, and C4-IgG1(N297A)-HC:muIL2. b HeLa-TNFR2 cells and HEK293 transfectants expressing the indicated cytokine receptors were stimulated with 200 ng/ml of the various C4-IgG1(N297A) cytokine fusion proteins in the presence and absence of 1 µg/ml protein G. Next day, IL8 content of supernatants were determined by ELISA. c Co-cultures of HeLa-TNFR2 cells and the various HEK293 transfectants were stimulated as indicated with the C4-IgG1(N297A) cytokine fusion proteins and IL8 production was again determined the following day by ELISA

Article Snippet: For FACS analysis of CD19, CD20, and CD70 expression FITC-labeled antibodies from BD Bioscience, San Jose, CA, USA (anti-CD19 FITC-labeled mouse IgG1, #345776; anti-CD70 FITC-labeled mouse IgG3, #555834), Caprico Biotechnologies, Norcross, GA, USA (anti-CD20 FITC-labeled mouse IgG1, #103714), and Miltenyi Biotec GmbH, Bergisch Gladbach, Germany (anti-murin IgG1 FITC-labeled rat IgG1, #130-095-897) were used.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcγR-independent agonistic activity

doi: 10.1038/s41419-019-1456-x

Figure Lengend Snippet: a Domain architecture and structural organization of C4-IgG1(N297A)-HC:scFvCD19, C4-IgG1(N297A)-HC:scFvCD20, and C4-IgG1(N297A)- HC:scFvCD70. b Analysis of CD19, CD20, and CD70 expression of Jurkat and BJAB cells by FACS. c HeLa-TNFR2 cells and BJAB (CD19 + CD20 + CD70 + ) or Jurkat cells were cocultured and stimulated overnight with the indicated concentration of the various C4-IgG1(N297A) scFv fusion proteins. IL8 content of supernatants were determined by ELISA. d HeLa-TNFR2 cells alone or in combination with the indicated HEK293 transfectants were preincubated with 20 µM MLN4924 for 30 min and afterwards stimulated for the different times with C4-IgG1(N297A)-HC:scFvCD70 or TNC-sc(mu)TNF80, a potent TNFR2 agonist as a positive control. MLN4924 was used to prevent proteasomal IκBα degradation to rule out underestimation of IκBα phosphorylation. MLN4924 is an inhibitor which interferes with the activity of the E3 ligase complex responsible for K48 ubiquitination of IκBα

Article Snippet: For FACS analysis of CD19, CD20, and CD70 expression FITC-labeled antibodies from BD Bioscience, San Jose, CA, USA (anti-CD19 FITC-labeled mouse IgG1, #345776; anti-CD70 FITC-labeled mouse IgG3, #555834), Caprico Biotechnologies, Norcross, GA, USA (anti-CD20 FITC-labeled mouse IgG1, #103714), and Miltenyi Biotec GmbH, Bergisch Gladbach, Germany (anti-murin IgG1 FITC-labeled rat IgG1, #130-095-897) were used.

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Positive Control, Phospho-proteomics, Activity Assay, Ubiquitin Proteomics

Journal: Cell Death & Disease

Article Title: TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcγR-independent agonistic activity

doi: 10.1038/s41419-019-1456-x

Figure Lengend Snippet: a CD40- (HT1080-CD40), 4-1BB- (HT1080-4-1BB), CD95- (HT1080), and Fn14-responsible cells (HeLa) were cocultured with HEK293-EV and HEK293-CD20 cells. Co-cultures were stimulated with the indicated concentrations of anti-4-1BB-IgG1(N297A)-HC:scFvCD20, anti-CD95-IgG1(N297A)-HC:scFvCD20, anti-Fn14-IgG1(N297A)-HC:scFvCD20, and anti-CD40-IgG1(N297A)-HC:scFvCD20. Next day, TNFRSF receptor activation was evaluated by measuring IL8 production. The CD95-specific antibody was applied in the presence of 20 µM ZVAD to prevent apoptosis. b Co-cultures were performed as described in “A” with the difference that HEK293-EV cells have been replaced by Jurkat cells and HEK293-CD20 cells by BJAB cells. c Co-cultures of Jurkat or BJAB cells with the indicated TNFRSF receptor-responsive cell lines were stimulated overnight in the presence and absence of 50 µg/ml anti-CD20-IgG1 with 100 ng/ml of the various IgG1(N297A)-HC:scFvCD20 fusion proteins and IL8 production was finally quantified by ELISA. In case of anti-CD40-IgG1(N297A)-HC:scFvCD20 cells were treated with only 20 ng/ml

Article Snippet: For FACS analysis of CD19, CD20, and CD70 expression FITC-labeled antibodies from BD Bioscience, San Jose, CA, USA (anti-CD19 FITC-labeled mouse IgG1, #345776; anti-CD70 FITC-labeled mouse IgG3, #555834), Caprico Biotechnologies, Norcross, GA, USA (anti-CD20 FITC-labeled mouse IgG1, #103714), and Miltenyi Biotec GmbH, Bergisch Gladbach, Germany (anti-murin IgG1 FITC-labeled rat IgG1, #130-095-897) were used.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: RNA polymerase II pausing factor NELF in CD8 + T cells promotes antitumor immunity

doi: 10.1038/s41467-022-29869-2

Figure Lengend Snippet: a , b Percentages of CD62L + CD45RA + cells within CD4 + ( a ) and CD8 + ( b ) populations of 28z or 28z-hNELFB CAR-T cells after 10 days of in vitro expansion, n = 4/group. c Survival curves of Raji tumor-bearing mice receiving PBS or mock T cells, CD19 CAR-28z T cells, or CD19 CAR-28z-hNELFB T cells. d Model of enhancer–promoter looping mediated by NELFB, Pol II, and TCF1. Mean differences were compared using Student’s t -test. Log-rank (Mantel–Cox) tests were used for survival analyses. Source data are provided as a Source Data file.

Article Snippet: In vitro expanded cells were stained by FITC-labeled human CD19 (ACROBiosystems, CD9HF2H225UG) and subsequently purified by Anti-FITC MicroBeads (Miltenyi Biotec, 130-048-701) before the detection of NELFB overexpression by western blot or CAR expression by FITC-labeled human CD19.

Techniques: In Vitro

Journal: Cytometry. Part B, Clinical cytometry

Article Title: Expression and function of DNAM-1 on human B-lineage cells.

doi: 10.1002/cyto.b.21859

Figure Lengend Snippet: FIGURE 1 Conjugation of platelets to B cells. Human PBMCs were stained with anti-CD41/61, anti-CD19 and anti-DNAM-1 antibodies and analyzed by imaging flow cytometry. (a) Gating strategy of DNAM-1+ CD19+ cells (left). The frequency of CD41/61+ cells in DNAM-1+ CD19+

Article Snippet: FITC-conjugated anti-human CD3 (UCHT1) and Horizon V450-conjugated anti-human CD19 (H1B19) mAbs were purchased from Tonbo Biosciences.

Techniques: Conjugation Assay, Staining, Imaging, Flow Cytometry

Journal: Cytometry. Part B, Clinical cytometry

Article Title: Expression and function of DNAM-1 on human B-lineage cells.

doi: 10.1002/cyto.b.21859

Figure Lengend Snippet: FIGURE 2 DNAM-1 expression on human B-lineage cells before and after exclusion of CD41/61+ cells. (a–f) Human PBMCs were stained with the indicated antibodies and analyzed for DNAM-1 expression on CD19+ B cells by flow cytometry before (a, c, e) and after (b, d, f) exclusion of CD41/61+ cells. The CD19+ B-cell population was gated as in a, b (c–f). The percentages indicate the proportion of DNAM-1 positive B cells. Open and shaded histograms indicate staining with anti-DNAM-1 antibody and isotype control, respectively. Representative data from more than five donors are shown. (g) The mean proportion of DNAM-1+ B cells in each subpopulation before and after CD41/61+ cells is shown (n = 5). Error bars indicate SD. **p < .01. NS, not significant

Article Snippet: FITC-conjugated anti-human CD3 (UCHT1) and Horizon V450-conjugated anti-human CD19 (H1B19) mAbs were purchased from Tonbo Biosciences.

Techniques: Expressing, Staining, Flow Cytometry, Control

Journal: Cytometry. Part B, Clinical cytometry

Article Title: Expression and function of DNAM-1 on human B-lineage cells.

doi: 10.1002/cyto.b.21859

Figure Lengend Snippet: FIGURE 3 Involvement of DNAM-1 in IL-10 production by CD19+ B cells. (a) CD19+ B cells purified from human PBMCs were stimulated or not with CpG-ODN for 5 days and analyzed for DNAM-1 expression by flow cytometry. Open and shaded histograms indicate staining with anti- DNAM-1 antibody and isotype control, respectively. Representative data from more than six donors are shown. (b–d) CD19+ B cells purified from human PBMCs were pretreated with control antibody or an anti-DNAM-1 neutralizing antibody and then stimulated or not with CpG-ODN for 5 (b), or 7 (c, d) days. IL-10 concentration in the culture supernatants was measured by flow cytometry (b). Representative data from more than six donors are shown. The IgM (n = 4) and IgG (n = 3) concentration in the culture supernatants was analyzed by ELISA (c, d). Error bars indicate SD. *p < .05, **p < .01

Article Snippet: FITC-conjugated anti-human CD3 (UCHT1) and Horizon V450-conjugated anti-human CD19 (H1B19) mAbs were purchased from Tonbo Biosciences.

Techniques: Purification, Expressing, Flow Cytometry, Staining, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: B cell-intrinsic epigenetic modulation of antibody responses by dietary fiber-derived short-chain fatty acids

doi: 10.1038/s41467-019-13603-6

Figure Lengend Snippet: After weaning, C57BL/6 mice were fed a fiber diet (regular chow) or no-fiber diet. Two weeks later, these mice were started on SCFAs water (SCFAs) or plain water (Nil). All mice were then administered OVA together with CT via intragastric gavage once a week for 4 weeks starting at 8 weeks of age, and killed a week after the last OVA and CT administration. a Total and OVA-binding IgM, IgG1, IgA, and IgE concentrations in serum of fiber or no-fiber-fed mice on SCFAs water or plain water, as measured by ELISA. Each symbol represents an individual mouse ( n = 5 per group, pooled from two experiments). The bars represent the mean ± SD. b OVA-binding IgM, IgG1, IgA, and IgE concentrations in the feces of fiber-fed mice on SCFAs water or plain water, as measured by ELISA. Each symbol represents an individual mouse ( n = 5 per group, pooled from two experiments). c IgM, IgA, and IgD-positive cells in the lamina propria, PPs, and MLNs of fiber or no-fiber-fed mice on SCFAs water or plain water as visualized by fluorescence microscopy. d ELISPOT analysis of IgM, IgG1, and IgA AFCs in MLNs, PPs, spleen, and bone marrow of fiber-fed mice on SCFAs water or plain water. e Proportion of surface CD19 + IgG1 + and CD19 + IgA + B cells, germinal center B cells (CD19 + GL-7 + ), and plasmablasts/plasma cells (CD138 + ) in MLNs, PPs, and the spleen of fiber-fed mice given SCFAs water or plain water, as analyzed by flow cytometry. Data in c – e are representative of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant (unpaired t test). Scale bar = 100 μm. The source data are provided in Source Data file.

Article Snippet: For intracellular staining, cells were stained with anti-CD19 mAb (Clone 1D3; Tonbo) and fixable viability dye eFluor ® 450 (FVD 450, eBiosciences) followed by incubation with the BD Cytofix/Cytoperm buffer at 4 °C for 20 min. After washing twice with the BD Perm/Wash buffer, cells were resuspended in HBSS with 1% BSA and stored overnight at 4 °C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Microscopy, Enzyme-linked Immunospot, Clinical Proteomics, Flow Cytometry

Journal: Nature Communications

Article Title: B cell-intrinsic epigenetic modulation of antibody responses by dietary fiber-derived short-chain fatty acids

doi: 10.1038/s41467-019-13603-6

Figure Lengend Snippet: a – c C57BL/6 mice on plain water (Nil), SCFAs “lower dose” water (pH = 7.4, 20 mM sodium butyrate, and 30 mM sodium propionate) or SCFAs “higher dose” water (pH = 7.4, 20 mg ml −1 tributyrin emulsion, 140 mM sodium butyrate and 150 mM sodium propionate, SCFAs higher dose. The SCFAs water is referred here as SCFAs “higher dose” as opposed to the “lower dose” used in these experiments for the sake of clarify) were i.p. injected with NP-LPS. a Total and NP 3 -binding IgM, IgG2b, and IgG3 induced by low dose of SCFAs, as measured by ELISA. Each symbol represents an individual mouse ( n = 4–5 per group, pooled from two experiments). Bars represent mean ± SD. b AID and Blimp1 expression in spleen B cells from the same mice, as analyzed by intracellular staining followed flow cytometry. c IgG3 + , IgG2b + B cells, and CD19 low CD138 + plasmablasts/plasma cells in the spleen, as analyzed by flow cytometry. Data in b and c are representative of three independent experiments. d – i NSG/B mice on plain water or SCFAs water (pH = 7.4, 20 mg ml −1 tributyrin emulsion, 140 mM sodium butyrate, and 150 mM sodium propionate) were injected with NP-LPS. d Titers of total and NP 3 -binding serum IgM, IgG3, IgG2b, and IgA, as analyzed by ELISA. Each symbol represents an individual mouse ( n = 5 per group from one experiments). e NP 3 -binding IgM, IgG3, IgG2b, and IgA AFCs in spleen as analyzed by ELISPOT. f Viable (7-AAD − ) spleen CD19 + B cells as analyzed by flow cytometry. g Aicda, Prdm1 , germline I H -C H , and post-recombination Iμ-C H transcripts, as analyzed by qRT-PCR and normalized to Gapdh . Data are ratios to B cells cultured with nil (set as 1; means ± SEM of three independent experiments). h Proportion of IgG3 + and IgG2b + B cells, and CD138 + plasmablasts/plasma cells in the spleen, as analyzed by flow cytometry. i Somatic point mutations in the V H region of V H DJ H -Cγ2b transcripts. Mutations are in pooled sequences from two out of five mice per group. Pie charts depict the proportions of sequences that carry different numbers of point mutations; listed below the pie charts is the overall mutation frequency. Donut charts depict the spectrum of point mutations. Histograms depict frequencies of silent and replacement mutations in FR and CDR regions. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant (unpaired t test). The source data are provided in Source Data file.

Article Snippet: For intracellular staining, cells were stained with anti-CD19 mAb (Clone 1D3; Tonbo) and fixable viability dye eFluor ® 450 (FVD 450, eBiosciences) followed by incubation with the BD Cytofix/Cytoperm buffer at 4 °C for 20 min. After washing twice with the BD Perm/Wash buffer, cells were resuspended in HBSS with 1% BSA and stored overnight at 4 °C.

Techniques: Emulsion, Injection, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Flow Cytometry, Clinical Proteomics, Enzyme-linked Immunospot, Quantitative RT-PCR, Cell Culture, Mutagenesis

Journal: Nature Communications

Article Title: B cell-intrinsic epigenetic modulation of antibody responses by dietary fiber-derived short-chain fatty acids

doi: 10.1038/s41467-019-13603-6

Figure Lengend Snippet: a – f Mouse B cells were stimulated in the presence of Nil, butyrate (500 μM, But), propionate (2000 μM, Prop), or butyrate (500 μM) plus propionate (2000 μM). Proportions of surface CD19 + IgG1 + B cells and CD138 + plasmablasts/plasma cells ( a , b ), surface CD19 + IgA + B cells ( c , d ), intracellular CD19 + IgE + B cells ( e ), and surface CD19 + IgM – IgD + B cells ( f ) as analyzed by flow cytometry 96 h after stimulation. g , h CD19 + B cells were labeled with CFSE and stimulated for 96 h with LPS plus IL-4 in the presence of nil, butyrate (500 μM), or propionate (2000 μM). CFSE intensity and surface IgG1 expression as analyzed by flow cytometry. Progressive left shift of fluorescence intensity indicates CD19 + B-cell division. Data are representative of three independent experiments yielding comparable results ( g ). Proportion of surface IgG1 + B cells at each cell division. Data are mean and SE from three independent experiments ( h ). i Titers of IgG1 and IgA in culture fluids as analyzed by ELISA. Data are from three independent experiments (mean and SE). j Aicda, Prdm1 , Irf4, Xbp1, Sdc1 , post-recombination Iμ-Cγ1, Iμ-Cε, and Iμ-Cα transcripts as well as germline Iγ1-Cγ1, Iε-Cε, and Iα-Cα transcripts in B cells stimulated with LPS plus IL-4, or CD154 plus IL-4, IL-5, TGF-β, anti-δ mAb-dextran in the presence of butyrate, propionate, or butyrate plus propionate for 60 h, as analyzed by qRT-PCR and normalized to Gapdh transcripts. Data are ratios to stimulated B cells cultured with nil (set as 1; means ± SEM of three independent experiments). k , l B cells were stimulated with LPS plus IL-4 in the presence of nil, butyrate, propionate, or butyrate plus propionate for 72 h. The B-cell expression of AID and Blimp1 as determined by intracellular staining and flow cytometry ( k ). AID, Blimp1, and β-Actin proteins, as detected by immunoblotting ( l ). Data are one representative of three independent experiments yielding comparable results. * p < 0.05, **p < 0.01, ***p < 0.001 (unpaired t test). The source data are provided in Source Data file.

Article Snippet: For intracellular staining, cells were stained with anti-CD19 mAb (Clone 1D3; Tonbo) and fixable viability dye eFluor ® 450 (FVD 450, eBiosciences) followed by incubation with the BD Cytofix/Cytoperm buffer at 4 °C for 20 min. After washing twice with the BD Perm/Wash buffer, cells were resuspended in HBSS with 1% BSA and stored overnight at 4 °C.

Techniques: Clinical Proteomics, Flow Cytometry, Labeling, Expressing, Fluorescence, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Cell Culture, Staining, Western Blot

Journal: Nature Communications

Article Title: B cell-intrinsic epigenetic modulation of antibody responses by dietary fiber-derived short-chain fatty acids

doi: 10.1038/s41467-019-13603-6

Figure Lengend Snippet: a Human B cells purified from PBMCs of healthy subjects were stimulated with CD154 plus IL-4 and IL-21, in the presence of nil, butyrate (250, 500, or 1000 μM), propionate (2000 or 4000 μM), or butyrate (500 μM) plus propionate (2000 μM). The proportions of CD19 + IgA + and CD19 + IgG + as well as viable (7-AAD – ) CD19 + B cells were analyzed 120 h post stimulation by flow cytometry. Data are representative of three independent experiments yielding comparable results. b – d Human B cells were stimulated with CD154 plus IL-4 and IL-21, in the presence of nil, butyrate (500 μM), propionate (2000 μM), or butyrate (500 μM) plus propionate (2000 μM). CD19 lo CD138 + plasma cells were analyzed 120 h post stimulation by flow cytometry ( b ). IgG and IgA titers in culture fluids of these B cells were analyzed 120 h post stimulation by ELISA. Data are from three independent experiments (mean and SE) ( c ). Expression of AICDA and PRDM1 , germline Iγ1-Cγ1, Iα-Cα, and Iε-Cε, as well as mature V H DJ H -Cγ1, V H DJ H -Cε, and V H DJ H -Cα transcripts were analyzed 72 h post stimulation by qRT-PCR and normalized to the expression of HRPT . Data are ratios to stimulated B cells cultured with nil (set as 1; means ± SEM of three independent experiments) ( d ). e Human B cells were labeled with CFSE and stimulated with CD154 plus IL-4 and IL-21 in the presence of nil, butyrate (500 μM), or propionate (2000 μM) for 96 h. CFSE intensity were analyzed by flow cytometry. Progressive left shift of fluorescence intensity indicates CD19 + B-cell division. Data are representative of three independent experiments yielding comparable results. ** p < 0.01, *** p < 0.001 (unpaired t test). The source da t a are provided in Source Data file.

Article Snippet: For intracellular staining, cells were stained with anti-CD19 mAb (Clone 1D3; Tonbo) and fixable viability dye eFluor ® 450 (FVD 450, eBiosciences) followed by incubation with the BD Cytofix/Cytoperm buffer at 4 °C for 20 min. After washing twice with the BD Perm/Wash buffer, cells were resuspended in HBSS with 1% BSA and stored overnight at 4 °C.

Techniques: Purification, Flow Cytometry, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Cell Culture, Labeling, Fluorescence

Journal: Nature Communications

Article Title: B cell-intrinsic epigenetic modulation of antibody responses by dietary fiber-derived short-chain fatty acids

doi: 10.1038/s41467-019-13603-6

Figure Lengend Snippet: a , b Human and mouse B cells were stimulated with CD154 plus IL-4 and IL-21, or CD154 plus IL-4, respectively, in the presence of nil, butyrate (500 μM), propionate (2000 μM), butyrate (500 μM) plus propionate (2000 μM) alone, or butyrate (2000 μM) plus propionate (2000 μM) together with increasing doses of GPR43 inhibitor GLPG0974. Human B cell AICDA and PRDM1 transcripts as well as mouse B cell Aicda and Prdm1 transcripts were analyzed 72 h post stimulation by qRT-PCR and normalized to HRPT and Gapdh expression, respectively. Data are ratios to stimulated B cells cultured with nil (set as 1; means ± SEM of three independent experiments) ( a ). Proportion of CD19 + IgG + B cells were analyzed 120 h post stimulation by flow cytometry. Data are representative of three independent experiments yielding comparable results ( b ). c – f Mouse B cells were stimulated with LPS plus IL-4, in the presence of increasing doses of butyrate, propionate, palmitate, and SAHA. Expression of Aicda and Prdm1 transcripts were analyzed 72 h post stimulation by qRT-PCR and normalized to Gapdh expression. Data are ratios to stimulated B cells cultured with nil (set as 1; means ± SEM of three independent experiments) ( c ). The proportion of CD19 + IgG1 + B cells and CD138 + plasmablasts/plasma cells were analyzed 120 h post stimulation by flow cytometry. Data are representative of three independent experiments yielding comparable results ( d – f ). * p < 0.05, ** p < 0.01, *** p < 0.001 (unpaired t test). The source data are provided in Source Data file.

Article Snippet: For intracellular staining, cells were stained with anti-CD19 mAb (Clone 1D3; Tonbo) and fixable viability dye eFluor ® 450 (FVD 450, eBiosciences) followed by incubation with the BD Cytofix/Cytoperm buffer at 4 °C for 20 min. After washing twice with the BD Perm/Wash buffer, cells were resuspended in HBSS with 1% BSA and stored overnight at 4 °C.

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Flow Cytometry, Clinical Proteomics

Journal: Nature Communications

Article Title: B cell-intrinsic epigenetic modulation of antibody responses by dietary fiber-derived short-chain fatty acids

doi: 10.1038/s41467-019-13603-6

Figure Lengend Snippet: a Primary mouse B cells, CH12F3 B cells, and human B cells were stimulated with indicated stimuli for 60 h in the presence of nil, butyrate (500 μM), or propionate (2000 μM). Ex vivo B cells were isolated from mice injected with NP-LPS and on plain water or SCFA water for 21 days. Acetylated-histone H3 (H3K9ac), histone H3, and β-actin proteins as detected by immunoblotting. Data are one representative of three independent experiments. b Relative densities of the Ac-histone H3 bands normalized to histone H3 and β - actin levels. c , d B cells stimulated with LPS plus IL-4 in the presence of nil or butyrate for 60 h. Expression of the Aicda- and Prdm1- targeting miRNAs, and irrelevant miRNAs as analyzed by qRT-PCR ( c ). Relative abundance of H3K9ac/K14ac in miRNA host genes (HGs) as analyzed by ChIP-qPCR ( d ). e Schematic diagram of the luciferase reporter constructs-containing 3′UTRs of Aicda and Prdm1 mRNAs and their mutant (mut) counterparts. f Surface CD19 and IgA on CH12F3 B cells stimulated for 96 h in the presence of nil, butyrate (500 μM), propionate (2000 μM), or VPA (500 μM) as analyzed by flow cytometry. Data are one representative of 3 independent experiments yielding comparable results. g , miRNA expression in CH12F3 cells stimulated for 24 h in the presence of nil or butyrate, as analyzed by qRT-PCR. Data in c , d , and g are ratios to stimulated B cells cultured with nil (set as 1; means ± SEM of three independent experiments). h Luciferase activity in CH12F3 cells transfected with luciferase reporter vectors-containing wild-type or mutated Aicda or Prdm1 3′UTRs after 24 h treatment with nil or butyrate. Luciferase activity was measured 6 h after transfection, and normalized relative to luciferase activity in B cells cultured with nil. Data in h are ratios to transfected B cells cultured with nil (set as 100%; means ± SEM of three independent experiments). * p < 0.05, ** p < 0.01, ns: not significant (unpaired t test). The source da t a are provided in Source Data file.

Article Snippet: For intracellular staining, cells were stained with anti-CD19 mAb (Clone 1D3; Tonbo) and fixable viability dye eFluor ® 450 (FVD 450, eBiosciences) followed by incubation with the BD Cytofix/Cytoperm buffer at 4 °C for 20 min. After washing twice with the BD Perm/Wash buffer, cells were resuspended in HBSS with 1% BSA and stored overnight at 4 °C.

Techniques: Ex Vivo, Isolation, Injection, Western Blot, Expressing, Quantitative RT-PCR, ChIP-qPCR, Luciferase, Construct, Mutagenesis, Flow Cytometry, Cell Culture, Activity Assay, Transfection