cd86 fc  (R&D Systems)

Journal: Journal of biomedical science

Article Title: The homodimer interfaces of costimulatory receptors B7 and CD28 control their engagement and pro-inflammatory signaling.

doi: 10.1186/s12929-023-00941-3

Figure Lengend Snippet: Fig. 5 B7-1 and B7-2 dimer interface mimetic peptides attenuate engagement of CD28 by the cognate B7 costimulatory receptor. A In cartoon model of the extracellular domain of costimulatory receptor B7-1 (CD80) (green; 1dr9.pdb), a double beta-barrel, amino acid residues forming pB1-8 are modeled in sticks, with 2 residues making homodimer interface contacts shown in yellow. B, C pB1-8 selectively attenuates intercellular B7-1/CD28 engagement (B) but not B7-2/CD28 engagement (C). Receptor engagement was assayed by flow cytometry as in Fig. 3K for B7-1/CD28 engagement and as in Fig. 3D for B7-2/CD28 engagement. D In the extracellular domain of B7-1, amino acid residues forming pB1-78 are modeled in sticks, with 4 residues making homodimer interface contacts shown in yellow and orange. E, F pB1-78 selectively attenuates intercellular B7-1/ CD28 engagement (E) but not B7-2/CD28 engagement (F). G, H pB2-7 selectively attenuates intercellular B7-2/CD28 engagement (H) but not B7-1/ CD28 engagement (G). Data are mean and SEM of three independent experiments (contour plots: Additional file 1: Fig. S6). Intercellular receptor engagement was compared using the one-tailed unpaired student’s t-test; *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001; n.s, not significant

Article Snippet: Recombinant human B7-2 (CD86) Fc chimera and human CD28 Fc chimera expressed in mouse myeloma NS0 cells (R&D Systems) comprise the extracellular 20–239 and 19–152 amino acid domain, respectively, of the mature human ligands fused to C-terminal human IgG1 Fc and are homodimers, disulfide-linked in the Fc domain.

Techniques: Flow Cytometry, One-tailed Test

Journal: bioRxiv

Article Title: Soluble CTLA-4 mainly produced by Treg cells inhibits and resolves type 1 inflammation but allows type 2 immunity

doi: 10.1101/2023.05.26.542386

Figure Lengend Snippet: a , Schematic representations of the CTLA-4 gene locus of S + M + , S + M - , and S - M - mice. Splice acceptor site (86 bp) in front of exon3 was deleted in S + M - mice. b, Co-staining of CTLA-4 and Foxp3 in CD4 + T cells from indicated groups of mice after stimulation of PMA and ionomycin with monensin for 4 hours. c , Serum concentrations of sCTLA-4 in indicated groups of mice. d, Systemic appearance of the mice in ( a ). e, Survival of S + M - , S - M - , and S + M + mice. Statistical significance between S + M - and S - M - mice was accessed by Log-rank test. f, Body weight of the mice in ( a ). g, Colon lengths of indicated groups of mice. Scale bar represents 5 mm. h , Lung inflammation in S + M - and S - M - mice. Hematoxylin and eosin (HE) staining of the lung tissues of indicated groups of mice. Percentages of leukocyte infiltrated areas (leukocyte foci excluding lymph nodes > 1,000 μm 2 ) among whole areas of lung sections without cavities were determined by computational analysis. Scale bars represent 100 μm in upper figures and 1,000 μm in lower figures. i, Serum titers of anti-dsDNA IgG antibody and anti-parietal cell IgG antibody in indicated groups of mice. j, Increase in CD44 hi CD62L lo CD4 + and CD8 + T cells in S + M - and S - M - mice. k, Suppression of IFNψ- or IL-17A-producing CD4 + T-cell expansion in mesenteric lymph nodes of S + M - mice. l, Serum concentrations of cytokines in indicated groups of mice. Three-week-old mice were used for the experiments in ( a-l ) because of the early mortality of S - M - mice. Error bars denote the mean ± s.e.m. in ( b,c,f-l ). Data are representative or summary of at least three independent experiments with six or more mice ( b-l ). Statistical significances were determined by one-way ANOVA with Holm-Sidak multiple comparisons test in ( b,c,f-l ). dsDNA, double stranded DNA.

Article Snippet: Immunoprecipitation (IP) was performed in binding buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 5% glycerol, 1% BSA, 0.05% Tween-20) containing 1 μg FLAG-tagged sCTLA-4 and 1 μg mCD80 Fc (human IgG1) chimera (R&D, 740-B1), 1 μg mCD86 Fc (human IgG1) chimera (R&D, 741-B2), or 1 μg Human IgG1 Fc (R&D, 110-HG) in the presence or absence of 1 μg mCD152/Fc (CTLA4-Ig, mouse IgG2a) chimera (Sigma-Aldrich, C4358).

Techniques: Staining

Journal: bioRxiv

Article Title: Soluble CTLA-4 mainly produced by Treg cells inhibits and resolves type 1 inflammation but allows type 2 immunity

doi: 10.1101/2023.05.26.542386

Figure Lengend Snippet: a , Schematic representation of the CTLA-4 allele in S + M - mice. A targeting vector was designed to delete splicing acceptor site (86 bp) in front of the exon3. After generation of chimera mice, the mice were bred with FLPeR mice harboring FLP1 recombinase gene to delete a neo cassette. The offspring were the S + M - mice. b, Constitutional exon3 skipping in CTLA-4 mRNA in S + M - mice. Relative mRNA expression of sCTLA-4 (exon2-4 junction spanning primers) and mCTLA-4 (exon2-3 or exon3-4 junction spanning primers) in CD4 + T cells of indicated groups of mice purified from spleens and lymph nodes. c, HE staining of colons of indicated groups of mice (n = 4 to 7 per group). Scale bars represent 100 μm. Colitis were scored histologically. The data were assessed by Kruskal-Wallis test followed by Dunn’s test. d, HE staining of indicated organs of S + M - , S - M - , and S + M + mice (n = 6 per group). Scale bars represent 100 μm. Arrows show leukocyte infiltration in tissues. e, Blood chemistry analysis for assessment of organ functions and inflammation. The data of ALT was accessed by Kruskal-Wallis test followed by Dunn’s test. AST, aspartate aminotransferase; ALT, alanine transaminase. f, Decrease in serum hemoglobin concentration and red blood cell counts in S + M - and S - M - mice. g, Serum concentrations (μg/mL) of IgA, IgE, IgM, IgG1, IgG2a, IgG2b, and IgG3 immunoglobulins in indicated groups of mice. h, Representative data of CD44/CD62L expression by CD4 + or CD8 + T cells in S + M - , S - M - , and S + M + mice shown in . i, Splenomegaly and lymphadenopathy in S + M - and S - M - mice. j, HE staining of spleens of S + M - , S - M - , and S + M + mice (n = 6 per group). White pulps in S + M - and S - M - mice are shown by white dot lines because of obscure borders by massive leukocyte infiltration into red pulps. WP, white pulp; RP, red pulp; TB, trabecular vessel. Scale bars represent 100 μm. k, Ratios and numbers of CD4 + and CD8 + T cells in spleens/lymph nodes of indicated groups of mice. l, Ratios and numbers of Foxp3 + CD4 + T cells in spleens/lymph nodes of indicated groups of mice. Three-week-old mice were used for the experiments ( b-l ) because of the early mortality of S - M - mice. Error bars denote the mean ± s.e.m. in ( e-g,i,k,l ) or the mean ± s.d. in ( b ). Data are representative or summary of at least two independent experiments with three or more mice ( b-l ). Statistical significances were determined by one-way ANOVA with Holm-Sidak multiple comparisons test in ( e-g,i,k,l ). Neo, Neomycin resistance cassette; FRT, Flippase Recognition Target; BV421, Brilliant Violet 421.

Article Snippet: Immunoprecipitation (IP) was performed in binding buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 5% glycerol, 1% BSA, 0.05% Tween-20) containing 1 μg FLAG-tagged sCTLA-4 and 1 μg mCD80 Fc (human IgG1) chimera (R&D, 740-B1), 1 μg mCD86 Fc (human IgG1) chimera (R&D, 741-B2), or 1 μg Human IgG1 Fc (R&D, 110-HG) in the presence or absence of 1 μg mCD152/Fc (CTLA4-Ig, mouse IgG2a) chimera (Sigma-Aldrich, C4358).

Techniques: Plasmid Preparation, Expressing, Purification, Staining, Concentration Assay

Journal: bioRxiv

Article Title: Soluble CTLA-4 mainly produced by Treg cells inhibits and resolves type 1 inflammation but allows type 2 immunity

doi: 10.1101/2023.05.26.542386

Figure Lengend Snippet: a , Schematic representation of bacterial artificial chromosome (BAC) CTLA-4 allele for preparation of S - M + mice and a representative picture of the mice. The BAC vector RP23-146J17 was modified to delete the intron between exon3 and exon4. The BAC transgenic mice were crossed with S - M - mice to generate S - M + mice. b, Histology of the adipose tissue (n = 10 mice per group) and the lung (n = 6 mice per group) in S - M + and S + M + mice. Scale bars represent 100 μm. c-e, Serum concentrations of IgE, IL-6, TNF-α, anti-dsDNA IgG antibody, and anti-parietal cell IgG antibody in indicated groups of mice. The data were accessed by Mann-Whitney test. f, Relative expression of mRNA for iNOS (Nos2), IL-6, CD80, CD206 (Mrc1), and Fizz1 (Retnla) in CD11b + F4/80 + peritoneal macrophages purified from indicated groups of mice. g-k, Co-staining of IFNγ and Eomes in CD8 + T cells ( g,h ), and IFNγ and IL-17A in CD4 + T cells ( i-k ) of indicated groups of mice. Cells prepared from spleen, mesenteric lymph nodes, or surface lymph nodes were stimulated in vitro with PMA/Ionomycin/Monensin for 5 hours. l,m, Increase in Tfh cells as Bcl-6 + CXCR5 + Foxp3 - CD44 + CD4 + B220 - cells in mesenteric lymph nodes and Peyer’s patches from S - M + mice. n, Relative expression of mRNA for major lineage transcription factors in CD11c + splenic DCs purified from indicated groups of mice. Thirty-week-old mice were used for all the experiments ( a-n ). All error bars denote the mean ± s.e.m. Data are representative or summary of at least two independent experiments with five or more mice per group ( b-n ). Statistical significances were determined by unpaired two-tailed t -test in ( f,h,j,k,m,n ) or Mann-Whitney test ( c-e ). AF647, Alexa Fluor 647.

Article Snippet: Immunoprecipitation (IP) was performed in binding buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 5% glycerol, 1% BSA, 0.05% Tween-20) containing 1 μg FLAG-tagged sCTLA-4 and 1 μg mCD80 Fc (human IgG1) chimera (R&D, 740-B1), 1 μg mCD86 Fc (human IgG1) chimera (R&D, 741-B2), or 1 μg Human IgG1 Fc (R&D, 110-HG) in the presence or absence of 1 μg mCD152/Fc (CTLA4-Ig, mouse IgG2a) chimera (Sigma-Aldrich, C4358).

Techniques: Plasmid Preparation, Modification, Transgenic Assay, MANN-WHITNEY, Expressing, Purification, Staining, In Vitro, Two Tailed Test

Journal: bioRxiv

Article Title: Soluble CTLA-4 mainly produced by Treg cells inhibits and resolves type 1 inflammation but allows type 2 immunity

doi: 10.1101/2023.05.26.542386

Figure Lengend Snippet: a , sCTLA-4-FLAG recombinant protein. Purified protein was detected by SDS-PAGE and CBB staining. Baculovirus expression system was used for expression of the recombinant protein. b, Staining of FLAG in LPS-stimulated CD11b hi or CD11c hi cells pre-incubated with sCTLA-4-FLAG (1 μg/mL, for 20 minutes at 4°C). The CD11b hi and CD11c hi cells were prepared from WT or CD80/86 KO mice. c, Immunofluorescence microscopy of splenic CD11c + cells with or without CD80/86 expression pre-incubated with sCTLA-4-FLAG and then stained for FLAG (Alexa fluor 555), CD11c (Alexa fluor 647), and DAPI. Scale bar, 5 μm. d, Weaker affinity of sCTLA-4 for CD80/86 compared with CTLA-4 Fc chimera. 1 μg FLAG-tagged sCTLA-4 and 1 μg CD80 or CD86 Fc chimera protein (Fc: human IgG1) were immunoprecipitated with anti-human Fc antibody-coated Dynabeads in the presence or absence of 1 μg CTLA-4 Fc chimera (Fc: mouse IgG2a). Recombinant Fc (Fc: human IgG1) was used as a negative control. The immunoprecipitates were analyzed by immunoblotting with anti-FLAG M2 antibody. Data are representative of two independent experiments ( a-d ). e,f, Comparison of Nur77 (Nr4a1) and CD69 expression kinetics in Th1 and Th2 cells skewed in the presence of αCD80/86 antibodies. g, Comparison of Ki-67 expression in Nur77 positive cells in Th1 and Th2 differentiation 24 hours after TCR stimulation. h, Ki-67 expression kinetics under Th1 and Th2 differentiation during 0-24 hours after TCR stimulation. Each symbol indicates the presence or absence of αCD80 or αCD86 antibody, or both. i,j, Quantification of IL-2 by PEA-qPCR (lower limit of quantification, 0.064 pg/mL, as shown in the dotted line) in the presence of αCD80/CD86 antibodies for 0-24 hours in Th1/Th2 polarization experiments shown in e-h ( i ), and quantification of IL-2 levels at 72 and 120 hours after TCR stimulation ( j ). Two-way ANOVA with Holm-Sidak multiple comparisons test ( f-i ). Kruskal-Wallis test with Dunn’s multiple comparisons test ( j ). Data points show results of three independent experiments with cells isolated from different mice (n = 3 to 6 mice per experiment) ( f-j ). PEA, Proximity Extension Assay.

Article Snippet: Immunoprecipitation (IP) was performed in binding buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 5% glycerol, 1% BSA, 0.05% Tween-20) containing 1 μg FLAG-tagged sCTLA-4 and 1 μg mCD80 Fc (human IgG1) chimera (R&D, 740-B1), 1 μg mCD86 Fc (human IgG1) chimera (R&D, 741-B2), or 1 μg Human IgG1 Fc (R&D, 110-HG) in the presence or absence of 1 μg mCD152/Fc (CTLA4-Ig, mouse IgG2a) chimera (Sigma-Aldrich, C4358).

Techniques: Recombinant, Purification, SDS Page, Staining, Expressing, Incubation, Immunofluorescence, Microscopy, Immunoprecipitation, Negative Control, Western Blot, Isolation