goat anti cd4  (R&D Systems)

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: PLoS ONE

Article Title: Screening of Human Tumor Antigens for CD4 + T Cell Epitopes by Combination of HLA-Transgenic Mice, Recombinant Adenovirus and Antigen Peptide Libraries

doi: 10.1371/journal.pone.0014137

Figure Lengend Snippet: A , Composition of the TRP-2-specific library peptide pools X1 to X8 and Y1 to Y8 used for combinatorial screening of specific T cell responses ex vivo . Individual library peptides determined by combinatorial screening are highlighted. B , Spleen cells from HLA-DR3tg mice injected i.p. with 5×10 8 pfu Ad5.TRP-2 or Ad5.EGFP (2 mice per group) were screened ex vivo by IFN-γ ELISpot assay for recognition of TRP-2-specific library peptide pools. T cell responses of two control mice (Ad5.EGFP) and two TRP-2-immunized mice (Ad5.TRP-2) are represented as individual columns in the diagram. Reactivity against two controls, the H2-K b -restricted CD8 + T cell epitope TRP-2 180-188 and the HLA-DRB1*0301-restricted CD4 + T cell epitope TRP-2 60–74 was tested in addition. Error bars show standard error of the mean. Experiments were performed four times, yielding similar results.

Article Snippet: Two days later, T cell responses against the cognate peptides were analyzed by intracellular IFN-γ staining, using a cytokine staining kit (BD Pharmingen) according to the manufacturer's instructions in combination with goat anti-mouse CD4- and CD8-specific monoclonal antibody conjugates (BD Pharmingen).

Techniques: Ex Vivo, Injection, Enzyme-linked Immunospot