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