antibodies against anti il 2 goat polyclonal antibody  (R&D Systems)

Journal: Research in veterinary science

Article Title: Mycoplasma ovipneumoniae-derived lipid-associated membrane proteins induce cytokine secretion in mouse peritoneal macrophages through TLR2 signalling.

doi: 10.1016/j.rvsc.2020.07.022

Figure Lengend Snippet: Fig. 3. TLR2 and NLRP3 stimulate macrophage-induced proinflammatory cytokine secretion after M. ovi or M. ovi-derived LAMPs infection. Wild-type (WT) and corresponding gene-deficient macrophages were infected with M. ovi or M. ovi-derived LAMPs or were uninfected as controls. IL-1β production in the supernatant of macrophage cultures was analysed by ELISA 9 h after infection (a–d). Results are expressed as means ± standard deviations of three independent experiments and were analysed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test or with two-way ANOVA with Bonferroni's post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Rabbit anti-phosphoJNK, anti-phospho-ERK, anti-phospho-p38, and anti-phospho-NF-κB p65 monoclonal antibodies (Cell Signalling Technology, Beverly, MA, USA) were used for protein detection, and corresponding antibodies were used for detection of unphosphorylated molecules; rabbit anti-pro caspase-1 + p10+ p12 monoclonal antibody (1:2000, Abcam, UK) and goat anti-pro-IL-1β polyclonal antibody (1:1000, pIL-1β, R&D Systems, USA) were used for protein detection; rabbit anti-NLRP3 monoclonal antibody (1:1000, 15101S, CST, USA) was used for protein detection.

Techniques: Derivative Assay, Infection, Enzyme-linked Immunosorbent Assay

Journal: Science immunology

Article Title: Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose-tissue immune and metabolic tenors

doi: 10.1126/sciimmunol.aaw3658

Figure Lengend Snippet: (A-D) Cytofluorimetric identification of IL-33-producing cells within eVAT of 8–10-week-old B6 mice using a polyclonal anti-IL-33 Ab. (A) Gating strategy to delineate cell fractions. (B) Representative dot-plots of control-Ab (top panels) or anti-IL-33 staining (bottom panels) of the cell fractions. (C) As per panel B except whole-body IL-33-deficient (II33−/−) mice were assessed. (D) Frequencies of IL-33+ cells in each cell fraction (left) and its contribution to total IL-33+ cells within the tissue (right). n≥7 from at least three experiments. (E-G) Confocal microscopic images of IL-33+ VmSCs. (E) The eVAT depot is circumferenced by a ring of high PDPN positivity, presumably the mesothelium. (F) IL-33+ cells locate in close proximity to CD31+ endothelial cells. (G) PDPN positivity outlines a large blood vessel surrounded by β3-tubulin+ nerves. Color code for Ab staining as indicated on top of each picture. PDPN, podoplanin; DNs, PDGFRα−Sca-1− cells; VmSCs, PDGFRα+Sca-1+ VAT mesenchymal stromal cells.

Article Snippet: Primary Abs were goat anti-mouse IL-33 (R&D Systems, #AF3526, 1:50 dilution), APC-conjugated Syrian Hamster anti-mouse PDPN (Biolegend, #AF3526, 1:100), or rabbit anti-GFP (Abcam, 1:1000), rabbit anti-β3-tubulin (Cell Signalling, #D6584, 1:50) and rat anti-mouse CD31 (BioLegend, #102512, 1:50).

Techniques: Staining

Journal: Science immunology

Article Title: Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose-tissue immune and metabolic tenors

doi: 10.1126/sciimmunol.aaw3658

Figure Lengend Snippet: (A) Two-dimensional tSNE plot of scRNA data from VAT (orange), muscle (green) and lymph node (blue) PDGFRα+Sca-1+ mSCs. VmSC subtypes 1–5 were delineated by k- means clustering; their fractional contributions to total VmSCs are indicated. (B) Heat-map showing genes differentially expressed between the VmSC subtypes (FDR < 5%). k-means clustered. (C) VmSCs dynamics (velocity field). The projected next cell state for each single cell is represented by an arrow and projected on the tSNE plot. (D) Same tSNE plot as in panel A overlain with heat-maps of the density of cells expressing various transcript markers. (E) Heat-map of transcript expression within the combined single-cell data of each VmSC subtype. (F-G) Representative dot-plots delineating the VmSC subtypes (left) and indicating their IL-33 expression levels (right) in PpargTdtIl33Egfp double-reporter mice. (G) Comparison of VmSC subtype frequencies obtained by scRNAseq versus flow cytometry. (H) Matrix of Spearman correlation coefficients in head-to-head comparisons of ≥2-fold differentially expressed genes from the scRNAseq and matching population (pop)-level RNAseq datasets. LN, lymph node.

Article Snippet: Primary Abs were goat anti-mouse IL-33 (R&D Systems, #AF3526, 1:50 dilution), APC-conjugated Syrian Hamster anti-mouse PDPN (Biolegend, #AF3526, 1:100), or rabbit anti-GFP (Abcam, 1:1000), rabbit anti-β3-tubulin (Cell Signalling, #D6584, 1:50) and rat anti-mouse CD31 (BioLegend, #102512, 1:50).

Techniques: Expressing, Comparison, Flow Cytometry

Journal: Science immunology

Article Title: Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose-tissue immune and metabolic tenors

doi: 10.1126/sciimmunol.aaw3658

Figure Lengend Snippet: (A) Representative plots (top), frequencies and numbers (bottom) of total VmSCs from mice of different ages. (B) Frequencies (top) and numbers (bottom) of total IL-33+ VmSCs. (C and D) Cytofluorometric dot-plots of VmSC subtypes (C) and corresponding fractions (top) and numbers (bottom) (D) from lean B6 males of the indicated ages. Pooled data from three independent experiments. Numbers of cells were normalized per tissue weight. One-way ANOVA analysis was performed to compare three or more groups. For all relevant plots, mean ± SEM. p-values: *, ≤0.05; **, ≤0.01; ***, ≤0.001; ****, ≤0.0001. SVF, stromal vascular fraction. Other abbreviations as per Fig 1.

Article Snippet: Primary Abs were goat anti-mouse IL-33 (R&D Systems, #AF3526, 1:50 dilution), APC-conjugated Syrian Hamster anti-mouse PDPN (Biolegend, #AF3526, 1:100), or rabbit anti-GFP (Abcam, 1:1000), rabbit anti-β3-tubulin (Cell Signalling, #D6584, 1:50) and rat anti-mouse CD31 (BioLegend, #102512, 1:50).

Techniques:

Journal: Science immunology

Article Title: Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose-tissue immune and metabolic tenors

doi: 10.1126/sciimmunol.aaw3658

Figure Lengend Snippet: (A) Frequencies (top) and numbers (bottom) of total IL-33+ VmSCs. (B) Cytofluorimetric dot-plots (left) and corresponding quantification of subtypes thereof (right) in gonadal (g)VAT and iSAT of lean male (M) and female (F) mice aged 18–20 weeks old. Pooled data from at least two independent experiments. (C) Three-dimensional PCA plot of the transcriptomes (population-level RNAseq) from coincidently prepared male and female VmSC subtypes from B6 mice 8–10 weeks old. (D) Esr1 (top) and Ar (bottom) transcripts levels for the individual VmSC subtypes from male (white bars) and female (black bars) mice aged 8–10 weeks old. Each dot represents an individual biological replicate from two or more pooled mice. (E) Correlation curves for numbers of IL-33+ VmSCs versus ST2+ Treg numbers in gVAT using meta-data from all male mice of various ages (6, 16, 18–20 and 32 weeks) and female mice 18–20 weeks old, i.e. all mice of normal physiologic state. In all cases, numbers of cells were normalized to tissue weight. r and p from Pearson’s correlation coefficient. All other statistics and abbreviations as per Fig. 1 and ​and55.

Article Snippet: Primary Abs were goat anti-mouse IL-33 (R&D Systems, #AF3526, 1:50 dilution), APC-conjugated Syrian Hamster anti-mouse PDPN (Biolegend, #AF3526, 1:100), or rabbit anti-GFP (Abcam, 1:1000), rabbit anti-β3-tubulin (Cell Signalling, #D6584, 1:50) and rat anti-mouse CD31 (BioLegend, #102512, 1:50).

Techniques:

Journal: Science immunology

Article Title: Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose-tissue immune and metabolic tenors

doi: 10.1126/sciimmunol.aaw3658

Figure Lengend Snippet: (A and B) Fractions (top) and numbers (bottom) of total IL-33+ VmSCs (A) and VmSC subtypes (B) subsequent to 4 or 16 weeks of high-fat feeding of 14-week-old B6 males. Data from at least two independent experiments. (C and D). Effects of IL-33 administration to 8–12-week-old B6 males. Frequency (top) and numbers (bottom) of total IL-33+ VmSCs (C) or of individual VmSC subtypes (D). Data shown corresponds to at least two pooled independent experiments. Numbers of cells were tissue weight-normalized in all cases. LFD, low-fat diet; HFD, high-fat diet; PBS, phosphate-buffered saline. All other statistics and abbreviations as per Figs. 1 and ​and55.

Article Snippet: Primary Abs were goat anti-mouse IL-33 (R&D Systems, #AF3526, 1:50 dilution), APC-conjugated Syrian Hamster anti-mouse PDPN (Biolegend, #AF3526, 1:100), or rabbit anti-GFP (Abcam, 1:1000), rabbit anti-β3-tubulin (Cell Signalling, #D6584, 1:50) and rat anti-mouse CD31 (BioLegend, #102512, 1:50).

Techniques: Saline

Journal: Science immunology

Article Title: Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose-tissue immune and metabolic tenors

doi: 10.1126/sciimmunol.aaw3658

Figure Lengend Snippet: (A and B) IL-33 was administered to wild-type B6 males aged 8–12 weeks. Frequencies (top) and numbers (bottom) of total (left) and ST2+ (right) Tregs (A) or ILC2s (B). (C) Confocal images (group of left panels) showing Tregs and IL-33-expressing cells within eVAT at low-magnification (left), corresponding delineation of adipocyte edges based on autofluorescence of DAPI channel (middle) and high-magnification of the squared indicated area (right). Distance quantification between Foxp3+ Tregs and the nearest IL-33- expressing cell (group of right panels) from whole eVAT tissue sections taken from male VAT-Treg TCR- transgenic mice at 7 (top) and 17 (bottom) weeks of age. Arrows in panel C depict Treg:IL-33+ cell proximity. Color code for Ab staining as indicated within the picture. (D, E and F) IL- 33 was injected into mice lacking ST2 expression specifically by Tregs vs wild-type littermate controls. Frequencies (top) and numbers (bottom) corresponding to total and KLRG1+ Tregs (D), ILC2s (E) and IL-33+ VmSCs (F). All numbers were calculated relative to total tissue weight. (G) Graphic scheme of the proposed VmSC:Treg negative regulatory loop model. All other abbreviations and statistics as per Figs. 1, ​,55 and ​and77.

Article Snippet: Primary Abs were goat anti-mouse IL-33 (R&D Systems, #AF3526, 1:50 dilution), APC-conjugated Syrian Hamster anti-mouse PDPN (Biolegend, #AF3526, 1:100), or rabbit anti-GFP (Abcam, 1:1000), rabbit anti-β3-tubulin (Cell Signalling, #D6584, 1:50) and rat anti-mouse CD31 (BioLegend, #102512, 1:50).

Techniques: Expressing, Transgenic Assay, Staining, Injection

Journal: Science translational medicine

Article Title: Targeted antibody and cytokine cancer immunotherapies through collagen affinity

doi: 10.1126/scitranslmed.aau3259

Figure Lengend Snippet: (A) Schematic of conjugation of a collagen-binding domain (CBD), the recombinant VWF A3 domain, to checkpoint inhibitor (CPI) antibody, resulting in affinity for collagen. CBD-fused IL-2 was recombinantly expressed, with the CBD on the N-terminus of IL-2 using a (GGGS)2 linker. (B) Dissociation constants (KD values) of CBD- and unmodified aPD-L1, αCTLA4, and IL-2 against collagen type I and collagen type III, recombinant mouse (rm)CTLA4, rmPD-L1, and/or rmIL-2Ra were measured by ELISA. N.D.= not determined because of low signals. Graphs of concentrations vs signals are shown in fig. S2. (C) 5 × 105 MMTV-PyMT cells were inoculated in the mammary fat pad. When the tumor volume reached 500 mm3, 300 μg of DyLight 800-labeled CBD was injected i.v.. A pie chart represents the biodistribution of CBD protein 48 hr after injection as determined by fluorescence analysis of each organ (n = 4). (D) Intratumoral imaging was performed on MMTV-PyMT tumors when they reached 200 mm3 by injecting 100 μg of DyLight 594-labeled CBD-αPD-L1 or (E) 100 μg of DyLight 594-labeled αPD-L1 i.v. 30 min after injection. The tumor was then harvested, and fluorescence was analyzed by microscopy. Top panels: images of whole tumors, scale bar = 500 μm. Bottom panels: images of enlarged yellow squares within upper panels, scale bar = 50 μm. Representative images of 2 tumors each. (F, G) Binding of (F) CBD-IL-2 or (G) unmodified IL-2 to human melanoma cryosections was imaged by fluorescence microscopy. Scale bar = 100 μm. Two experimental replicates. Statistical analyses were done using ANOVA with Tukey’s test. **p < 0.01.

Article Snippet: The tissues were stained with hamster anti-human CD31 antibody (Abcam), goat anti-mouse IL-2 antibody (R and D systems), and rabbit anti-collagen I antibody (Abcam) for 1 hour at room temperature.

Techniques: Conjugation Assay, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Labeling, Injection, Fluorescence, Imaging, Microscopy

Journal: Science translational medicine

Article Title: Targeted antibody and cytokine cancer immunotherapies through collagen affinity

doi: 10.1126/scitranslmed.aau3259

Figure Lengend Snippet: Adverse events were studied in mice bearing B16F10 melanomas. 5 × 105 B16F10 cells were inoculated on day 0. (A-F) CBD- or unmodified αCTLA4 and αPD-L1 (100 μg each/injection) were injected i.v. on day 4 and 7. (A) On day 8, serum concentrations of TNFα in blood plasma were measured (mean ± SEM). (B, C) On day 10, the numbers of lymphocytic infiltration spots in histologic (B) lung and (C) liver sections were counted and divided by area (mean ± SEM). (D, E) On day 10, blood serum (D) ALT and (E) AST activities were measured (mean ± SEM). (F) On day 10, the liver was harvested and weighed. Water content in the liver was determined by weighing before and after lyophilization and was normalized to dry tissue weight (mean ± SEM). (G, H) 5 × 105 B16F10 cells were inoculated on day 0. CBD-IL-2 (6 μg for IL-2 basis) or unmodified IL-2 (6 μg) was injected i.v. on day 7, 8, and 9. On day 10, (G) spleen and (H) lung were harvested and weighed. Water content in the lung was determined as described above (mean ± SEM). Statistical analyses were done using ANOVA with Tukey’s test. Kruskal-Wallis test followed by Dunn’s multiple comparison was used in (C) due to nonparametric data. Two experimental replicates. *p < 0.05; **p < 0.01; N.S. = not significant.

Article Snippet: The tissues were stained with hamster anti-human CD31 antibody (Abcam), goat anti-mouse IL-2 antibody (R and D systems), and rabbit anti-collagen I antibody (Abcam) for 1 hour at room temperature.

Techniques: Injection

Journal: Science translational medicine

Article Title: Targeted antibody and cytokine cancer immunotherapies through collagen affinity

doi: 10.1126/scitranslmed.aau3259

Figure Lengend Snippet: We used three tumor models to study efficacy of targeted immunotherapy, namely the B16F10 melanoma model, the CT26 colon carcinoma model, and the MMTV-PyMT breast cancer model. (A, F) 5 × 105 B16F10 cells were inoculated on back skin, (B, G) 5 × 105 CT26 cells were inoculated on back skin, (C, D, H-K) 5 × 105 MMTV-PyMT cells were inoculated on the right mammary fat pad day 0. (A-D) CBD-αCTLA4 + CBD-PD-L1 (CBD-CPI), αCTLA4 + αPD-L1 (CPI) or PBS was administered on (A) day 4, (B) day 5, (C, D) day 7. (A) CBD- and unmodified CPI were injected i.v., and PlGF-2123–144-CPI was injected peritumorally (p.t.). Antibody doses per administration are indicated on the figure. (A-C) Graphs depict tumor volume until the first mouse died and (D) survival rate. (E) 30 days after the first tumor inoculation in the right mammary gland fat pad, 5 × 105 MMTV-PyMT cells were again inoculated into the left mammary gland fat pad in CBD-CPI treated tumor-free survivors or in naïve mice. Numbers indicate how many mice remain tumor-free among total mice after 40 days of tumor re-challenge. (F) 5 × 105 B16F10 cells were inoculated on the back skin, (G) 5 × 105 CT26 cells were inoculated on the back skin, (H-K) 5 × 105 MMTV-PyMT cells were inoculated on the right mammary fat pad day 0. (F-I) 6 μg IL-2 or equimolar CBD-IL-2 was injected i.v. on (F) day 4, (G) day 5, (H, I) day 7. (F-H) Graphs depict tumor volume until the first mouse died and (I) survival rate. (J, K) After 7, 14, and 21 days from tumor inoculation, CBD-αCTLA4 + CBD-αPD-L1 (CBD-CPI) + CBD-IL-2, αCTLA4 + αPD-L1 (CPI) + IL-2 or PBS were injected i.v.. (J) Graphs depict tumor volume until the first mouse died and (K) survival rate. Numbers indicate how many mice remained tumor-free among total mice 100 days after tumor inoculation. (A) PBS, n = 9; CPI 100 μg and CBD-CPI 25 μg, n = 8; CBD-CPI 100 μg and PlGF-2123–144-CPI 100 μg, n = 7. (B) PBS and CPI 25 μg, n = 11; CPI 100 μg and CBD-CPI 25 μg, n = 10; CBD-CPI 100 μg, n = 9. (C, D) CBD-CPI, n = 12; other groups, n = 11. (E-G) n = 6. (H, I) PBS, n = 10; other groups, n = 11. (J, K) PBS, n = 10; other groups, n = 13. Tumor volumes are presented as mean ± SEM. Two experimental replicates. Statistical analyses were done using ANOVA with Tukey’s test for tumor size and Log-rank (Mantel-Cox) test for survival curves. *p < 0.05; **p < 0.01.

Article Snippet: The tissues were stained with hamster anti-human CD31 antibody (Abcam), goat anti-mouse IL-2 antibody (R and D systems), and rabbit anti-collagen I antibody (Abcam) for 1 hour at room temperature.

Techniques: Injection

Journal: Science translational medicine

Article Title: Targeted antibody and cytokine cancer immunotherapies through collagen affinity

doi: 10.1126/scitranslmed.aau3259

Figure Lengend Snippet: We used the B16F10 model to study T cell behavior in tumors with targeted immunotherapy. 5 × 105 B16F10 cells were inoculated on day 0. CBD-αCTLA4 + CBD-αPD-L1 (CBD-CPI), αCTLA4 + αPD-L1 (CPI), or PBS was administered on day 4. CPI was injected i.v. at 100 μg each. Tumors were collected on day 8, followed by flow cytometric analysis. Frequency of (A) CD8+CD3+ and (B) CD4+CD3+ tumor-infiltrating T cells within CD45+ lymphocytes and (C) Treg (Foxp3+CD25+) of CD4+CD3+ tumor-infiltrating T cells. (D) The ratio of cytotoxic T lymphocytes (CTL; CD62L−CD44+CD8+CD3+) versus Treg (Foxp3+CD25+CD4+). (E-G) T cells were extracted from the tumors and stimulated with aCD28 and aCD3 for 6 hours. Graphs depict the % of (E) IL-2+, (F) TNFα+, (G) IFNγ+ of CD8+CD3+ T cells. (H-K) CBD-IL-2, IL-2, or PBS was administered on day 7, 8 and 9. Lymphocytes were extracted from the tumor on day 10, followed by flow cytometric analysis. Graphs depict the number of (H) CD8+CD3+ T cells per tumor weight (mg), the frequency of (I) CD8+CD3+ T cells within total CD45+ lymphocytes, (J) CD4+CD3+ T cells within total CD45+ lymphocytes, and (K) NK1.1+CD3− NK cells within total CD45+ lymphocytes. Lines represent mean ± SEM. Two experimental replicates. Statistical analyses were done using ANOVA with Tukey’s test. Kruskal-Wallis test followed by Dunn’s multiple comparison was used in (H) due to nonparametric data. *p < 0.05; **p < 0.01.

Article Snippet: The tissues were stained with hamster anti-human CD31 antibody (Abcam), goat anti-mouse IL-2 antibody (R and D systems), and rabbit anti-collagen I antibody (Abcam) for 1 hour at room temperature.

Techniques: Injection