primary antibodies goat anti human cd4  (R&D Systems)

Journal: Nature Communications

Article Title: Smith-specific regulatory T cells halt the progression of lupus nephritis

doi: 10.1038/s41467-024-45056-x

Figure Lengend Snippet: Physical binding affinity assay of HLA-DR15 and 145 synthesized 15-mer peptides derived from a SmB/B’, b SmD1, and c SmD3. X axis shows peptide numbering of the 15-mers sequentially overlapping by an offset of three amino acids starting from the N-terminus of each protein. Y axes showing % binding and stability index, as described in “Methods”. d The top five binding HLA-DR15-restricted Sm peptides from the physical binding assay. e Immunogenicity of SmB/B’ 58-72 , SmB/B’ 1-15 and SmB/B’ 43-57 measured by the proportion of CD4 + CellTrace Violet (CTV) lo T cells following 6-day co-culture of dendritic cells and CTV-labeled CD4 + T cells with or without peptide ( n = 3 independent samples, data are presented as mean with SD) f , representative FACS plots for immunogenicity of SmB/B’ 58-72 -stimulated and un-stimulated CD4 + T cells by CTV dilution. Source data are provided as a Source Data file.

Article Snippet: Immunofluorescent staining was performed on 5 μm snap frozen kidney sections using primary antibodies goat anti-human CD4 (AF379; R&D Systems); rabbit anti-human CD8 (NBP2-29475; Novus Biologicals); and Armenian hamster anti-human CD11c (NB110-97871; Novus Biologicals) and Alexa Fluor-conjugated secondary antibodies (705-545-003, 711-585-152, 127-605-160; Jackson ImmunoLab).

Techniques: Binding Assay, Synthesized, Derivative Assay, Immunopeptidomics, Co-Culture Assay, Labeling

Journal: Nature Communications

Article Title: Smith-specific regulatory T cells halt the progression of lupus nephritis

doi: 10.1038/s41467-024-45056-x

Figure Lengend Snippet: a Experimental timeline of single-cell sequencing experiment from co-culture of cells with the peptide of interest to sorting of cells for sequencing. Created with BioRender.com. b Clonotype numbers of the top 20 SmB/B’58-72-specific TCRs (TCR1 to TCR20) as identified using 10X V(D)J single T-cell sequencing. c – e Volcano plot of genes expressed by CD4 + T cells of interest that express SmB/B’ 58-72 -TCR1-3. P values are derived from a negative binomial exact test with adjustment using Benjamin Hochberg correction for multiple tests. f t-SNE plot of CD4 + T cells that express our TCR of interest, TCR1 (dark blue dots). The majority of the cells expressing this TCR are clustered close together, indicating they are clonally expanded sharing a similar gene expression profile. g t-SNE plot of CD52 expression, a marker of suppressor T cells. The majority of CD52 hi cells are clustered towards the bottom of the plot where cells expressing our TCR1 of interest are. h t-SNE plot of IL9R expression clustered with TCR1 expression. i t-SNE plot of LAIR2 expression clustered with TCR1 expression. Source data are provided as a Source Data file.

Article Snippet: Immunofluorescent staining was performed on 5 μm snap frozen kidney sections using primary antibodies goat anti-human CD4 (AF379; R&D Systems); rabbit anti-human CD8 (NBP2-29475; Novus Biologicals); and Armenian hamster anti-human CD11c (NB110-97871; Novus Biologicals) and Alexa Fluor-conjugated secondary antibodies (705-545-003, 711-585-152, 127-605-160; Jackson ImmunoLab).

Techniques: Sequencing, Co-Culture Assay, Derivative Assay, Expressing, Gene Expression, Marker

Journal: Nature Communications

Article Title: Smith-specific regulatory T cells halt the progression of lupus nephritis

doi: 10.1038/s41467-024-45056-x

Figure Lengend Snippet: Healthy human Tregs were transduced with TCR1 and expanded 10 days in vitro. a Representative flow cytometry dot plot of the CD4 + Treg cells expressing GFP and TCR Vβ21.3, the antibody specific for the variable gene TRBV11-2 of TCR1 showing a mean co-expression of 20.68%. b Treg surface marker phenotype by representative dot plots of CD25 and CD127 expression on CD4+ cells. c Sm-Treg transcription factor phenotype by representative dot plots of forkhead box P3 (FOXP3) and Helios expression. d Treg expression of GFP and Vβ21.3 by flow cytometry from four separate experiments on healthy donor Tregs. e methylation of the Treg-cell specific demethylated region (TDSR) of the FOXP3 locus of Treg and Tconv cells at day 0 (before transduction and expansion) and 3 weeks after transduction and expansion. f , g IL-17A and IFN-γ expression after stimulation with PMA and ionomycin of Tconv (brown), mock-transduced Tregs (blue), the un-transduced (GFP-) portion of Tregs that underwent lentiviral transduction (purple) and Sm-TCR1-transduced Tregs after 10 days of expansion culture in vitro, measured by intracellular flow cytometry ( n = 2 biologically independent samples), data are presented as mean with SD. Source data are provided as a Source Data file.

Article Snippet: Immunofluorescent staining was performed on 5 μm snap frozen kidney sections using primary antibodies goat anti-human CD4 (AF379; R&D Systems); rabbit anti-human CD8 (NBP2-29475; Novus Biologicals); and Armenian hamster anti-human CD11c (NB110-97871; Novus Biologicals) and Alexa Fluor-conjugated secondary antibodies (705-545-003, 711-585-152, 127-605-160; Jackson ImmunoLab).

Techniques: Transduction, In Vitro, Flow Cytometry, Expressing, Marker, Methylation

Journal: Cell Reports Medicine

Article Title: B cell depletion attenuates CD27 signaling of T helper cells in multiple sclerosis

doi: 10.1016/j.xcrm.2023.101351

Figure Lengend Snippet: BCDTs alter the peripheral CD4 + T cell landscape of patients with MS (A) Heatmap showing median expression of lineage markers across identified CD4 + T cell subsets defined from the combined dataset signature including all samples. (B–F) Untreated/treated PBMC samples of 12 patients with MS, resulting in 24 samples (before and after treatment) analyzed longitudinally. (B) Violin plots showing changes in the frequency across CD4 + T cell subsets in BCDT-treated MS. Paired Wilcoxon rank-sum test and Benjamini-Hochberg correction were applied. (C) Violin plot showing changes in the naïve-to-memory ratio within the CD4 + T cell compartment. Paired Wilcoxon rank-sum test was applied. (D) Radar plots showing changes in the marker expression of CD4 + T cell subsets in BCDT-treated MS. Values correspond to −log10(p value) and were adjusted with the Benjamini-Hochberg correction. Inner circle color annotations denote each respective subset. Bar color denotes the upregulation or downregulation of the respective marker. Marker order is consistent across subsets. Dashed line denotes −log10(0.05) cutoff for p value. (E) Violin plots showing changes in CD27 expression across CD4 + T cell subsets in BCDT-treated MS. p values correspond to the paired Wilcoxon rank-sum test applying a Benjamini-Hochberg correction. (F) Violin plot showing changes in CD27 expression in the CD4 + T cell compartment in BCDT-treated MS. Paired Wilcoxon rank-sum test was applied. (G) Median CD27 expression along CD4 + T cell subsets of the patients with MS. Color code denotes patient values before or after treatment. (H) Percentage of fold change in CD27 expression along CD4 + T cell subsets of the patients with MS, depicted in percentage of changes. Shaded area represents the 95% confidence interval.

Article Snippet: The following primary antibodies were used: Rabbit anti-CD27 (abcam, clone EPR8569, dilution 1:500) was combined with mouse anti-CD3 (Leica Microsystems Ltd, clone LN10, dilution 1:500) and goat anti-CD4 (R&D, polyclonal, dilution 1:50) to co-label CD4 + T cells.

Techniques: Expressing, Marker

Journal: Cell Reports Medicine

Article Title: B cell depletion attenuates CD27 signaling of T helper cells in multiple sclerosis

doi: 10.1016/j.xcrm.2023.101351

Figure Lengend Snippet: The altered CD4 + T cell landscape is consistent across cohorts (A and B) Untreated/treated PBMC samples of 14 patients with MS, resulting in 28 samples (before and after treatment) analyzed longitudinally. (A) Violin plot showing the frequency in CD4 + Tfh-like cells in B cell-depleted patients with MS of the validation cohort. Paired Wilcoxon rank-sum test was applied. (B) Violin plots showing changes in CD27 expression across CD4 + memory and Tfh-like cells in B cell-depleted patients with MS of the validation cohort. Paired Wilcoxon rank-sum test was applied. (C and D) Untreated/treated PBMC samples of 8 patients with MG, resulting in 16 samples (before and after treatment) analyzed longitudinally. (C) Violin plot showing the frequency in CD4 + Tfh-like cells in B cell-depleted patients with MG of the validation cohort. Paired Wilcoxon rank-sum test was applied. (D) Violin plots showing changes in CD27 expression across CD4 + memory and Tfh-like cells in B cell-depleted patients with MG of the validation cohort. Paired Wilcoxon rank-sum test was applied.

Article Snippet: The following primary antibodies were used: Rabbit anti-CD27 (abcam, clone EPR8569, dilution 1:500) was combined with mouse anti-CD3 (Leica Microsystems Ltd, clone LN10, dilution 1:500) and goat anti-CD4 (R&D, polyclonal, dilution 1:50) to co-label CD4 + T cells.

Techniques: Expressing

Journal: Cell Reports Medicine

Article Title: B cell depletion attenuates CD27 signaling of T helper cells in multiple sclerosis

doi: 10.1016/j.xcrm.2023.101351

Figure Lengend Snippet: CD70 expression in CNS of patients with MS (A–C) Single-cell RNA sequencing data from Schafflick et al. derived from the CSF of patients with MS. (A) UMAP displaying the cellular composition of the CSF in MS. The color code corresponds to the clusters as defined in the study. (B) Dot plot illustrating specific marker genes across distinct clusters. Dot size correlates with the percentage of cells within each cluster expressing the chosen marker, while the color code represents the average marker expression level within the respective cluster. (C) Dot plot illustrating CD70 gene across distinct clusters. Dot size correlates with the percentage of cells within each cluster expressing CD70, while the color code represents the average CD70 expression level within the respective cluster. (D and E) Brain tissue from a patient with relapsing MS with tumefactive brain lesions (n = 3 formalin-fixed, paraffin-embedded brain biopsy samples were examined). (D) Representative immunostaining of CD20 + B cells and CD4 + CD3 + T cells in proximity in inflammatory MS lesions. Scale bar, 20 μm. (E) Representative immunofluorescence (IF) images of CD27-expressing CD4 + CD3 + T cells (a–a′′′) and CD70-expressing CD20 + B cells in or close to inflammatory infiltrates in MS brain biopsies (b–b′′, c, and c′). Insets show enlargement of areas outlined in main images. Arrowheads show co-localization; nuclear DAPI staining in blue. Image labels (a), (b), and (c) denote distinct brain lesion sites of the representative patient with MS. Scale bars, 40 μm.

Article Snippet: The following primary antibodies were used: Rabbit anti-CD27 (abcam, clone EPR8569, dilution 1:500) was combined with mouse anti-CD3 (Leica Microsystems Ltd, clone LN10, dilution 1:500) and goat anti-CD4 (R&D, polyclonal, dilution 1:50) to co-label CD4 + T cells.

Techniques: Expressing, RNA Sequencing Assay, Derivative Assay, Marker, Formalin-fixed Paraffin-Embedded, Immunostaining, Immunofluorescence, Staining

Journal: Cell Reports Medicine

Article Title: B cell depletion attenuates CD27 signaling of T helper cells in multiple sclerosis

doi: 10.1016/j.xcrm.2023.101351

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used: Rabbit anti-CD27 (abcam, clone EPR8569, dilution 1:500) was combined with mouse anti-CD3 (Leica Microsystems Ltd, clone LN10, dilution 1:500) and goat anti-CD4 (R&D, polyclonal, dilution 1:50) to co-label CD4 + T cells.

Techniques: Purification, Recombinant, Electron Microscopy, Blocking Assay, Polymer, Mass Cytometry, RNA Sequencing Assay, Software, Imaging, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Transcriptome analysis reveals genes associated with stem cell activation by physical exercise in the dentate gyrus of aged p16Ink4a knockout mice

doi: 10.3389/fcell.2023.1270892

Figure Lengend Snippet: Cellular and gene expression analyses of stem cell activation elicited by sequential running stimuli in p16 KO and p16 WT dentate gyrus. (A) Timeline of the experiments performed on p16 WT and p16 KO 1-year-old mice: i) no run; ii) single run of 7 days; iii) single run of 12 days followed by a sedentary period; iv) double run of 12 and 7 days spaced 21 days apart. (B) Representative images by confocal microscopy (×40 magnification) showing that the proliferating type-1 stem cells (Ki67 + /Sox2 + /GFAP + , labeled in red, blue and green, respectively) in the dentate gyrus of p16 KO mice increase, relative to p16 WT, after a single run of 12 days followed by a sedentary period, whereas they do not increase after a double run of 12 and 7 days spaced 21 days apart. The white dashed line labels the boundaries of the dentate gyrus. Arrowheads: Ki67 + /Sox2 + /GFAP − cells, arrows: Ki67 + /Sox2 + /GFAP + cells. Scale bar 50 μm. White boxes: area at higher magnification (×2), shown in the panels on the right. (C) Graph showing the changes induced by running on the proliferation of stem cells (type-1 cells, Ki67 + /Sox2 + /GFAP + ) in p16 WT and in p16 KO dentate gyrus, in the different groups indicated. A single RUN of 12 days induces the proliferation of p16 KO stem cells for an extended period (28 days; group 12d RUN +28d), but a second running added (7 days; group 12d RUN +21d + 7d RUN) does not stimulate p16 KO stem cell proliferation. (D) Similar changes were observed analyzing stem and progenitor cells together (i.e., type-1 and type-2a cells, respectively; Ki67 + /Sox2 + cells). (C,D) Analysis of simple effects: NS p > 0.05, ** p < 0.01, or **** p < 0.0001, PLSD ANOVA test. Cell numbers in the dentate gyrus are means ± SEM of the analysis of five animals per group. (E) Real-time PCR analysis of the differential expression response to the second running stimulus for the Set A_Set E_GLM genes whose activation by running had been validated, i.e., Tfap2c , Lepr , Top2a , Slc18a2 , Lpin2 , Rgs14 , Ramp3 , Chrm5 . The expression of these genes was activated in the p16 knockout dentate gyrus by a single run of 12 days but was not changed by a second running, relative to p16 wild-type. The figure shows the mean mRNA expression fold increases ±SEM from two independent experiments. Four biological replicates were used for mice that run 12 days and five biological replicates for mice that underwent the double run. TBP was used to normalize data. The statistical analysis of real-time PCR data was performed by non-parametric Kruskal-Wallis test to detect main effects (resulting with p -value <0.05 for all the genes tested), followed by Mann-Whitney U post hoc test for single Set comparison. * p < 0.05, ** p < 0.01, *** p < 0.001, Mann-Whitney U post hoc test; n = at least 24, i.e., total number of data for each gene analyzed for all four groups (WT RUN and KO RUN, WT 12d RUN +21d + 7d RUN or KO 12d RUN +21d + 7d RUN).

Article Snippet: Proliferating stem and progenitor cells were visualized by means of a rabbit monoclonal antibody against Ki67 (Invitrogen, San Diego, CA, USA; MA514520; 1:200), a goat polyclonal antibody against Sox2 (Abcam, Cambridge, UK; Ab239218; 1:300), and a mouse monoclonal antibody against GFAP (Sigma-Aldrich, St Louis, MO; G6171; 1:200).

Techniques: Expressing, Activation Assay, Confocal Microscopy, Labeling, Real-time Polymerase Chain Reaction, Knock-Out, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: Transcriptome analysis reveals genes associated with stem cell activation by physical exercise in the dentate gyrus of aged p16Ink4a knockout mice

doi: 10.3389/fcell.2023.1270892

Figure Lengend Snippet:

Article Snippet: Proliferating stem and progenitor cells were visualized by means of a rabbit monoclonal antibody against Ki67 (Invitrogen, San Diego, CA, USA; MA514520; 1:200), a goat polyclonal antibody against Sox2 (Abcam, Cambridge, UK; Ab239218; 1:300), and a mouse monoclonal antibody against GFAP (Sigma-Aldrich, St Louis, MO; G6171; 1:200).

Techniques: Translocation Assay, Real-time Polymerase Chain Reaction, Knock-Out, Activity Assay