Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: TNF-α does not impact the levels of TNFR1 and TNFR2 on Th1 and Th17 cells. Purified CD4 + T cell subsets were stimulated with 1 µg/mL of TNF-α for 24 h and stained, for flow cytometry, with anti-human TNFR1 or TNFR2 mAbs. ( A ) Representative histograms showing the expression levels of TNFR1 and TNFR2 on Th1 and Th17 cells at baseline conditions and upon TNF-α stimulus. Solid black line histograms, isotype control; black histograms, Th1 cells; grey histograms, Th17 cells; dashed line histograms, TNF-α-treated Th1 cells; long dashed line histograms, TNF-α-treated Th17 cells. The levels of TNFR1 ( B ) and TNFR2 ( C ) were measured on purified CD4 + T cell subpopulations obtained from two to four healthy controls. Bars represent the mean values ± SD. Statistical analyses were carried out with Kruskal–Wallis followed by Dunn’s multiple comparison tests. ** p < 0.01.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Purification, Staining, Flow Cytometry, Expressing, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: Effect of TNFR1 or TNFR2 blockade on the production of IFN-γ and IL-17 by Th1 and Th17 cells. Purified CD4 + T lymphocyte subpopulations were incubated with neutralizing antibodies to TNFR1 or TNFR2 for 1 h prior to a 4-day stimulus with TNF-α (1 µg/mL). Intracellular staining of IFN-γ and IL-17 was assessed by flow cytometry. ( A ) Representative dot plots of Th1 and Th17 cells treated with anti-TNFR1 or anti-TNFR2 in the presence or absence of TNF-α. An isotype control was used to discard non-specific effects of the neutralizing antibodies. The fold increase in the percentages of IFN-γ ( B , D ) or IL-17 ( C , E ) producers was measured on Th1 and Th17 cells obtained from two to six healthy donors. Bars represent the mean values ± SD. Data were normalized against cells that did not receive treatment with TNF-α or TNF-α plus TNFRs blocking mAbs. For statistical analysis, Kruskal–Wallis and Dunn’s multiple comparison tests were performed. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Purification, Incubation, Staining, Flow Cytometry, Control, Blocking Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: Expression of TNFR1 and TNFR2 on Th1 and Th17 cells present in the peripheral blood of rheumatoid arthritis (RA) patients treated with adalimumab. Cell staining for flow cytometry analysis was performed on PBMC samples from healthy controls ( n = 9) and RA patients ( n = 10) before (PRE) and after (POST) treatment with adalimumab. The levels of TNFR1 ( A ) and TNFR2 ( C ) are expressed in MFI values. The frequency of TNFR1 ( B ) and TNFR2 ( D )-expressing lymphocytes are also shown. Each symbol represents data for one individual. Mean values ± SD are indicated. Significance was assessed with non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparison test (for MFI data) or parametric one-way ANOVA plus Tukey’s post-test (for lymphocyte frequencies data). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Expressing, Staining, Flow Cytometry, Comparison

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

Article Title: Proinflammatory effects of TWEAK/Fn14 interactions in glomerular mesangial cells.

doi: 10.4049/jimmunol.176.3.1889

Figure Lengend Snippet: FIGURE 7. Abs to IL-1 or IL-6 do not inhibit chemokine secretion in response to TWEAK. C57BL/6 mesangial cells were stimulated for 24 h with and without 100 ng/ml TWEAK and 5 g/ml monoclonal rat anti- mouse IL-1 and IL-6. Rat IgG is an IgG2b mAb that was the rat isotype- matched control. A, CCL2/MCP-1; B, CCL5/RANTES; C, CXCL1/KC. Shown here are the mean SD values for duplicate wells; similar results were found in several independent experiments.

Article Snippet: We found that there was significant binding to the surface of murine mesangial cells by hFc-TWEAK (Fig. 1B), but not by a CTLA4-Ig control fusion protein (R&D Systems) or a human IgG1 mAb (PHP010; Serotec), which share the same IgG1 Fc as hFc-TWEAK (data not shown).

Techniques: Control

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

Article Title: Proinflammatory effects of TWEAK/Fn14 interactions in glomerular mesangial cells.

doi: 10.4049/jimmunol.176.3.1889

Figure Lengend Snippet: FIGURE 9. Murine anti-TWEAK mAbs prevent the induction of che- mokines in response to TWEAK stimulation of mesangial cells. The 2.P5.G9 and 2P2.D10 are two IgG2a mouse mAbs against murine TWEAK, derived by immunization of TWEAK knockout mice, as de- scribed in Materials and Methods. P1.17 is an isotype-matched control Ab. C57BL/6 mesangial cells were left untreated, or treated with TWEAK with and without these mAbs at a concentration of 5 g/ml for 24 h. Shown here are the mean SD values for duplicate wells; similar results were found in several independent experiments.

Article Snippet: We found that there was significant binding to the surface of murine mesangial cells by hFc-TWEAK (Fig. 1B), but not by a CTLA4-Ig control fusion protein (R&D Systems) or a human IgG1 mAb (PHP010; Serotec), which share the same IgG1 Fc as hFc-TWEAK (data not shown).

Techniques: Derivative Assay, Knock-Out, Control, Concentration Assay

Journal: Nature Immunology

Article Title: A humanized mouse that mounts mature class-switched, hypermutated and neutralizing antibody responses

doi: 10.1038/s41590-024-01880-3

Figure Lengend Snippet: a , Left, huCD45 + PBMCs reconstitution at indicated time points in THX ( n = 11), huNBSGW ( n = 6) and huNSG ( n = 5) mice grafted with cord blood huCD34 + cells. Engraftment levels depicted as percentage of total (human plus mouse) CD45 + PBMCs. Arrow denotes the beginning of E2 treatment in huNBSGW mice that would later become THX mice (dark navy line starting at 18 weeks of age) and continuing thereafter. Right, Human and mouse CD45 + PBMCs (% total PBMCs), as identified by flow cytometry. Fluorescence-activated cell sorting (FACS) plots are from one THX, one huNBSGW and one huNSG mouse, each representative of five mice. b , huCD45 + mononuclear cell counts in THX, huNBSGW and huNSG mice. c , Total serum huIgM, huIgD, huIgG, huIgA and huIgE (expressed as µg equivalents per ml, µg eq ml −1 ) in THX ( n = 6), huNBSGW ( n = 5) and huNSG ( n = 7) mice—huIgD and huIgE were undetectable in huNBSGW and huNSG mice. d , Survival of THX ( n = 48), huNBSGW ( n = 23) and huNSG ( n = 18) mice through 55 weeks after huCD34 + cell engraftment (Kaplan–Meier curves, THX versus huNSG mice, P = 0.0323; THX versus huNBSGW mice, P = 0.1809, log-rank Mantel–Cox test). e , Number of huB cells (huCD45 + CD19 + ), huT cells (huCD45 + CD3 + ), huDCs (huCD45 + CD3 − CD14 − CD11c + ), huNK cells (huCD45 + CD3 − CD56 + ) and human monocytes (huCD45 + CD3 − CD14 + ) per ml of peripheral blood in THX, huNBSGW and huNSG mice (same mice as in b ). f , Human immune cell profiling of THX mouse ( n = 5) spleen huCD45 + cells analyzed by high-parameter cytometry with time-of-flight (CyTOF) analysis of 30 human markers. THX mouse spleen huCD45 + lymphoid and myeloid cell proportions were similar to those in spleens of humans ( n = 6) who died from accidental death (Supplementary Table ). g , BM huCD34 + cells in THX ( n = 3), huNBSGW ( n = 3) and huNSG mice ( n = 3). In the histograms ( b , c and g ), each dot represents an individual mouse, and the bar depicts the mean with s.e.m. Statistical significance ( c and g ) was assessed using two-sided Student’s unpaired t -test (NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: After washing plates with PBS-Tween 20, bound human antibodies were detected with biotinylated goat anti-huIgM antibody (2020-08, SouthernBiotech; 1:5,000 dilution), goat anti-huIgD antibody (2030-08, SouthernBiotech; 1:5,000 dilution), goat anti-huIgG antibody (2015-08, SouthernBiotech; 1:5,000 dilution), goat anti-huIgG1 monoclonal antibody (555869, BD Pharmingen; 1:5,000), goat anti-huIgG2 monoclonal antibody (555874, BD Pharmingen; 1:5,000 dilution), goat anti-huIgG3 monoclonal antibody (3853-6-250, MABTECH; 1:5,000 dilution), goat anti-huIgG4 monoclonal antibody (555882, BD Pharmingen; 1:5,000 dilution), goat anti-huIgA antibody (2050-08, SouthernBiotech; 1:5,000 dilution) or goat anti-huIgE antibody (9250-08, SouthernBiotech; 1:5,000 dilution; Supplementary Table ), followed by reaction with HRP-labeled streptavidin (405210, BioLegend), development with O -phenylenediamine substrate (P8806-50TAB, Sigma-Aldrich) or 3, 3′, 5, 5′ tetramethyl benzidine substrate (421101, BioLegend), and measurement of converted substrate absorbance at 492 nm or 450 nm, respectively.

Techniques: Flow Cytometry, Fluorescence, FACS, Cytometry

Journal: Nature Immunology

Article Title: A humanized mouse that mounts mature class-switched, hypermutated and neutralizing antibody responses

doi: 10.1038/s41590-024-01880-3

Figure Lengend Snippet: a – f , THX ( n = 7), huNBSGW ( n = 7) and JAX NSG huCD34 ( n = 4) mice were injected i.p. with NP 16 -CGG (100 μg in 100 μl alum) on day 0, boosted (100 μg in 100 μl PBS) on day 14 and euthanized on day 28. a , Total serum human immunoglobulin and NP 4 -specific human antibodies measured by ELISAs. Total human immunoglobulin concentrations expressed as μg eq ml −1 and NP 4 -specific human antibodies expressed as relative units (RUs). Fewer than seven data points were derived for human immunoglobulins other than NP 4 -specific huIgM, huIgG and huIgG1. b , Left, spleen huIgM + , huIgG + and huIgA + B cells, class-switched memory huCD27 + IgD − B cells and huCD27 + CD38 + PBs/PCs. Right, NP-specific huCD19 + B cells and memory huCD19 + CD27 + IgG + B cells (identified by binding of PE-labeled NP 16 ), and MZ huCD19 + IgM + IgD + CD27 + B cells in THX mouse spleen. c , Spleen huB cell intracellular AID and BLIMP-1 expression in THX and huNBSGW mice. d , Left, point mutation frequencies (change/base) in spleen huB cell huV H DJ H -C H transcripts of THX 406, 407, 408 and 409 mice depicted as scatterplots. Each dot represents a single sequence and the bar depicts the mean with s.e.m. Right, means of total, S and R huV3 mutation frequencies in FR1, CDR1, FR2, CDR2 and FR3 of huV H DJ H -C H transcripts depicted as histograms. e , In the SHM pie charts, slices depict proportions of huV H DJ H -C H transcripts carrying given numbers of point mutations; slice gray gradients depict increasing numbers of point mutations; the overall mutation frequency is listed below each pie chart. Spectrum of point mutations depicted as donut charts (same mice as in d ). f , huV1DJ H -Cγ1 B cell clones and intraclonal diversification in THX mice (same mice as in d ) depicted by TreeMaps and phylogenetic trees. Individual rectangle or square (unique color) area reflects huB cell clone size. In THX 406, 407, 408 and 409 mice, the three largest huV1DJ H -Cγ1 clones accounted for 42.9%, 26.6%, 32.0% and 23.4% of huV1DJ H -Cγ1 B cells. g – p , THX ( n = 7), huNBSGW ( n = 7) and JAX NSG huCD34 ( n = 4) mice were injected i.p. with DNP-CpG (50 μg in 100 μl PBS) on day 0, boosted (50 μg in 100 μl PBS) on day 14 and euthanized on day 28. g , Total serum immunoglobulin concentration (μg eq ml −1 ) and DNP 5 -specific human antibodies (RUs) measured by specific ELISAs. Fewer than seven data points were derived for DNP-specific huIgE. h , Spleen huB cells, huMBCs and huPBs/PCs as in b . i , huCD45 + huCD19 + B cells, huCD3 + T cells, huCD11c + DCs, huCD14 + monocytes, memory huCD19 + CD27 + B cells, huCD27 + CD38 + PBs/PCs as well as huIgM + , huIgD + , huIgG + and huIgA + B cells in THX mouse mesenteric LNs and spleen. j , Blood and spleen MZ huCD19 + IgM + IgD + CD27 + B cells (8.1% ± 0.3% and 12.9% ± 0.2% huB cells, respectively) in THX ( n = 6) and huNBSGW ( n = 6) mice. FACS plots are from one THX and one huNBSGW mouse, each representative of six mice. k , Total and DNP 5 -specific huIgM, huIgG and huIgA ASCs in THX mouse spleen and BM, as analyzed by specific ELISPOTs. l , Spleen huB cell intracellular AID and BLIMP-1 expression in THX and huNBSGW mice. m , Total human immunoglobulin (μg eq ml −1 ) in the BALF of THX ( n = 5) and huNBSGW ( n = 5) mice. n , huCD45 + cells, huCD19 + B cells, huCD3 + T cells, and huIgM-, huIgD- and huIgA-producing cells in THX mouse lamina propria (immunofluorescence; scale bar, 100 μm). Different pseudocolors denote different cells. o , p , Free and bacteria-bound huIgD and huIgA in feces of THX ( n = 5) and huNBSGW ( n = 5) mice measured by specific ELISA (μg eq/g, THX versus huNBSGW mice: huIgD, P < 0.0001; huIgA, P = 0.007, two-sided Student’s unpaired t -test) and identified by flow cytometry (% total bacterial cells). Flow cytometry plots ( b , c , h , i and l ) are from one THX, one huNBSGW or one JAX NSG huCD34 mouse, each representative of three mice. huCD45 + cells were pre-gated in all FACS analyses. ELISPOT images ( k ) and micrographs ( n ) are from one THX mouse representative of five mice. In the histograms ( a , g , j , m and o ), each dot represents an individual mouse and the bar depicts the mean with s.e.m. Statistical significance ( a , g , j and m ) was assessed by two-sided Student’s unpaired t -test (NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: After washing plates with PBS-Tween 20, bound human antibodies were detected with biotinylated goat anti-huIgM antibody (2020-08, SouthernBiotech; 1:5,000 dilution), goat anti-huIgD antibody (2030-08, SouthernBiotech; 1:5,000 dilution), goat anti-huIgG antibody (2015-08, SouthernBiotech; 1:5,000 dilution), goat anti-huIgG1 monoclonal antibody (555869, BD Pharmingen; 1:5,000), goat anti-huIgG2 monoclonal antibody (555874, BD Pharmingen; 1:5,000 dilution), goat anti-huIgG3 monoclonal antibody (3853-6-250, MABTECH; 1:5,000 dilution), goat anti-huIgG4 monoclonal antibody (555882, BD Pharmingen; 1:5,000 dilution), goat anti-huIgA antibody (2050-08, SouthernBiotech; 1:5,000 dilution) or goat anti-huIgE antibody (9250-08, SouthernBiotech; 1:5,000 dilution; Supplementary Table ), followed by reaction with HRP-labeled streptavidin (405210, BioLegend), development with O -phenylenediamine substrate (P8806-50TAB, Sigma-Aldrich) or 3, 3′, 5, 5′ tetramethyl benzidine substrate (421101, BioLegend), and measurement of converted substrate absorbance at 492 nm or 450 nm, respectively.

Techniques: Injection, Derivative Assay, Binding Assay, Labeling, Expressing, Mutagenesis, Sequencing, Clone Assay, Concentration Assay, Immunofluorescence, Bacteria, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Enzyme-linked Immunospot

Journal: Nature Immunology

Article Title: A humanized mouse that mounts mature class-switched, hypermutated and neutralizing antibody responses

doi: 10.1038/s41590-024-01880-3

Figure Lengend Snippet: a – c , Naïve huIgM + IgD + B cells isolated from blood of healthy humans ( n = 3, HS 14, 15, 16) and spleens of non-intentionally immunized THX mice ( n = 3, THX 442, 443, 444) were cultured for 120 h upon stimulation with: ( a ) CD154 (3 U/ml), huIL-2 (100 ng/ml), huIL-4 (20 ng/ml) and huIL-21 (50 ng/ml), ( b ) CpG ODN2395 (2.5 mg/ml), huIL-2, huIL-21, TGF-β (4 ng/ml) and retinoic acid (4 ng/ml), or ( c ) CpG ODN2395, huIL-2, huIL-4 and huIL-21. Identification of huIgM + , huIgD + , huIgG + , huIgA + or huIgE + B cells, huCD27 + IgD – class-switched memory-like B cells (huMB) and huCD27 + CD38 + PBs by flow cytometry. huCD45 + CD19 + cells were pre-gated in all FACS analyses. d , AICDA , PRDM1 , V H DJ H -Cμ, V H DJ H -Cγ1, V H DJ H -Cα1 and V H DJ H -Cε transcript expression in HS and THX mice huB cell microcultures ( n = 3 biological replicates for each different microculture), as measured by qPCR and normalized to HPRT1 expression (2 −ΔCt method). In histograms, each dot represents transcript expression from one human or one THX mouse huB cell microculture and the bar depicts the mean with s.e.m. Statistical significance ( d ) was assessed by two-sided Student’s unpaired t- test (NS, not significant).

Article Snippet: After washing plates with PBS-Tween 20, bound human antibodies were detected with biotinylated goat anti-huIgM antibody (2020-08, SouthernBiotech; 1:5,000 dilution), goat anti-huIgD antibody (2030-08, SouthernBiotech; 1:5,000 dilution), goat anti-huIgG antibody (2015-08, SouthernBiotech; 1:5,000 dilution), goat anti-huIgG1 monoclonal antibody (555869, BD Pharmingen; 1:5,000), goat anti-huIgG2 monoclonal antibody (555874, BD Pharmingen; 1:5,000 dilution), goat anti-huIgG3 monoclonal antibody (3853-6-250, MABTECH; 1:5,000 dilution), goat anti-huIgG4 monoclonal antibody (555882, BD Pharmingen; 1:5,000 dilution), goat anti-huIgA antibody (2050-08, SouthernBiotech; 1:5,000 dilution) or goat anti-huIgE antibody (9250-08, SouthernBiotech; 1:5,000 dilution; Supplementary Table ), followed by reaction with HRP-labeled streptavidin (405210, BioLegend), development with O -phenylenediamine substrate (P8806-50TAB, Sigma-Aldrich) or 3, 3′, 5, 5′ tetramethyl benzidine substrate (421101, BioLegend), and measurement of converted substrate absorbance at 492 nm or 450 nm, respectively.

Techniques: Isolation, Cell Culture, Flow Cytometry, Expressing