mouse anti human il7r  (R&D Systems)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cytokine receptor–targeting chimera toolbox for expanding extracellular targeted protein degradation

doi: 10.1073/pnas.2524129123

Figure Lengend Snippet: IL4 and IL7 kineTACs enable cell type–specific VEGF internalization in cell lines. ( A ) mRNA normalized TPM of IL4Rα from Human Protein Atlas for Daudi and SiHa cell lines. ( B ) 24 h VEGF-647 internalization at 25 nM as a dose response of IL4-Bevacizumab in Daudi and SiHa cell lines. Curves are three-parameter nonlinear regressions, with dotted lines indicating bell-shaped curve fits. Means ± SEM of three biological replicates are shown. ( C ) mRNA normalized TPM of IL7R from Human protein atlas for Daudi and SiHa cell lines. ( D ) 24 h VEGF-647 internalization at 25 nM as a dose response of IL7-Bevacizumab in Daudi and SiHa cell lines. Curves are best fits for three-parameter nonlinear regressions. Mean values ± SEM are from three biological replicates. ( E ) Diagram of coculture experiments. Daudi cells (gray) and SiHa cells (purple) are coincubated with VEGF-647 and either IL4-Bevacizumab kineTAC (green) or IL7-Bevacizumab kineTAC (blue). Cell type–specific receptor expression allows for cell-specific internalization of VEGF using the respective kineTAC. ( F ) Representative flow cytometry data from coculture experiment. Daudi and SiHa cells in equal amounts were incubated for 24 h with 25 nM VEGF-647, and 0.3 nM IL4-Bevacizumab or 10 nM IL7-Bevacizumab. Gates show thresholds for VEGF positivity, defined as approximately 1% of total cells for VEGF-647 only. Top row: FITC + Daudi GFP . Bottom row: FITC − SiHa. Percentages in gate are displayed. ( G ) Fold change in VEGF-647 median fluorescence intensity in the coculture Daudi and SiHa experiment from F . Mean fold change over 25 nM VEGF-647 alone ± SEM is presented. The asterisk represents a discovery (q < 1%) using the false discovery rate to correct for multiple comparisons. ( H ) Coculture VEGF-647 percent positivity (same gate as in F ) as a dose response of IL4-Bevacizumab. Mean values and ± SEM are from three biological replicates. Curves are three-parameter nonlinear regressions, with dotted lines indicating bell-shaped curve fits. ( I ) same as in H , but for IL7-Bevacizumab.

Article Snippet: Antibodies used included rabbit anti-human EGFR (Cell Signaling Technology, Cat# 4267S, 1:1,000), rabbit anti-human PD-1 (Cell Signaling Technology, Cat# D4W2J, 1:1,000), mouse anti-human IL7R (R&D Systems, Cat# MAB306, 1:1,000), rabbit anti-human phospho NF-κB p65 Ser 536 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human NF-κB p65 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human β-actin (Cell Signaling Technology, Cat# 8H10D10, 1:1,000), and mouse anti-human β-tubulin (Cell Signaling Technology, Cat# DM1a, 1:1,000).

Techniques: Expressing, Flow Cytometry, Incubation, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cytokine receptor–targeting chimera toolbox for expanding extracellular targeted protein degradation

doi: 10.1073/pnas.2524129123

Figure Lengend Snippet: IL4 and IL7 kineTACs enable cell type–specific VEGF internalization in primary lymphocytes and PBMCs. ( A ) Diagram depicting tissue-specific expression of potential kineTAC receptors. ( B ) tSNE analysis of IL4Rα expression from scRNA seq of two PBMC donors, obtained from the Immune Cell Atlas ( 57 ). Notice the prominent naïve B cell cluster of high IL4Rα expressors (green). ( C ) tSNE analysis of IL7R expression from the same dataset. Notice primarily T cell restricted expression of IL7R (blue). ( D ) Immune Cell Atlas cluster annotations for primary cell lineages, colored by lineage as in legend to the right. ( E ) Raw VEGF-647 median fluorescence intensities across two donors for isolated CD19+ cells from four technical replicates. Cells were incubated with 25 nM VEGF-647 and 10 nM of indicated kineTAC or isotype. ( F ) same as E , but using CD3+ T-cells. ( G and H ) VEGF-647 with PBMC components. 10 nM of the indicated kineTAC or isotype was added to 25 nM VEGF-647 along with 200 k cells/well PBMCs and allowed to internalize for 24 h. Percent VEGF positive was defined by the no VEGF-647 condition ( SI Appendix , Fig. S6 ). Analyzed by repeated measures donor-paired one-way ANOVA, with P values for Dunnett’s correction for multiple comparisons to the kineTAC of interest (IL4-Beva for B cells, IL7-Beva for T cells) (* P < 0.05, ** P < 0.01). ( G ) Analysis of B cell gate and h analysis of T cell gate.

Article Snippet: Antibodies used included rabbit anti-human EGFR (Cell Signaling Technology, Cat# 4267S, 1:1,000), rabbit anti-human PD-1 (Cell Signaling Technology, Cat# D4W2J, 1:1,000), mouse anti-human IL7R (R&D Systems, Cat# MAB306, 1:1,000), rabbit anti-human phospho NF-κB p65 Ser 536 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human NF-κB p65 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human β-actin (Cell Signaling Technology, Cat# 8H10D10, 1:1,000), and mouse anti-human β-tubulin (Cell Signaling Technology, Cat# DM1a, 1:1,000).

Techniques: Expressing, Fluorescence, Isolation, Incubation

Journal: bioRxiv

Article Title: Antisense modulation of IL7R splicing to control sIL7R expression in human CD4 + T cells

doi: 10.1101/2022.02.22.481529

Figure Lengend Snippet: (A) Schematics of the GFP-IL7R splicing reporter, wherein GFP is only expressed by exclusion of IL7R exon 6. (B) Schematics of approach comparing the effects of mutagenesis of functional cis -acting splicing elements to steric blocking of the corresponding elements with ASOs using the GFP-IL7R reporter. The blue dotted lines expand the exon 6 sequence (gray box), and the relevant cis -acting elements are color-coded: enhancer ESE2 (green), silencers ESS2 and ESS3 (red), and 5’ss (yellow). Mutations to ESE2, ESS3 and the 5’SS are shown underneath each element: Δ ESE2 and Δ ESS3 represent mutations to ESE2 and ESS3, respectively, and 5’Cons and 5’Mut represent consensus and crippling mutations to the 5’ss, respectively. The sequence of ASOs targeting these cis -elements (IL7R-001 – IL7R-005) is shown above the exon sequence: IL7R-001 blocks the 5’ss of exon 6, IL7R-002 blocks ESE2, IL7R-003 blocks ESS2 and ESS3, IL7R-004 blocks the sequence in between ESS2 and ESS3, and IL7R-005 blocks ESS3. (C, E) Percentage (%) of exon 6 exclusion (mean ± S.D.) in transcripts from the reporter in HeLa cells stably expressing wild-type or mutant versions of the reporter ( C ), or cells stably expressing the wild-type reporter and transfected with control (ASO-Ctrl) or experimental (IL7R-001 – IL7R-005) morpholino ASOs ( E ) are shown under the gel images. The top band in the gels represents the exon 6 included product whereas the lower band represents the exon 6 excluded product. (D, F) Mean fluorescence intensity (MFI) of GFP expression in the cells from panel C stably expressing the different versions of the reporter ( D ), or in cells from panel E transfected with ASOs ( F ). The left panels show representative GFP MFI histograms for selected mutants (Wild-type, 5’Cons and 5’Mut) ( D ) and selected ASOs (ASO-Ctrl, IL7R-001 and IL7R-005) ( F ), with quantification shown on the right as Log2(Fold-Change) [Log2(FC)] relative to wild-type for all mutants ( D ) and relative to control for all ASOs ( F ). (***) p < 0.001 .

Article Snippet: Measurements of secreted sIL7R in HeLa cell supernatants were conducted as follows: 96-well plate (R&D Systems) were coated at 4 ° C overnight with a mouse anti-human IL7R monoclonal antibody (R&D Systems, # MAB306).

Techniques: Mutagenesis, Functional Assay, Blocking Assay, Sequencing, Stable Transfection, Expressing, Transfection, Control, Fluorescence

Journal: bioRxiv

Article Title: Antisense modulation of IL7R splicing to control sIL7R expression in human CD4 + T cells

doi: 10.1101/2022.02.22.481529

Figure Lengend Snippet: (A) GFP expression in the wild-type reporter cell line transfected with increasing concentrations of control (ASO-Ctrl) or experimental (IL7R-001, IL7R-004, IL7R-005 and IL7R-006) morpholino ASOs. Overlapped representative histograms are shown on the left for each ASO at 0 μM (light gray), 1 μM (blue), 5 μM (green) and 10 μM (dark gray), with quantification of MFI plotted on the right as dose-response curves of Log2(FC) relative to 0 µM for ASO-Ctrl (black), IL7R-001 (blue), IL7R-004 (green), IL7R-005 (red) and IL7R-006 (yellow). (B-C) Percentage (%) of exon 6 exclusion in transcripts from the GFP-IL7R reporter ( B ) or the endogenous IL7R gene ( C ) Representative gel images for each ASO are shown at the top with quantification of percent exon 6 exclusion (mean ± S.D.) plotted at the bottom as a function of ASO concentration (color-coded as in A ). (*) p < 0.05 , (**) p < 0.01 , and (***) p < 0.001 .

Article Snippet: Measurements of secreted sIL7R in HeLa cell supernatants were conducted as follows: 96-well plate (R&D Systems) were coated at 4 ° C overnight with a mouse anti-human IL7R monoclonal antibody (R&D Systems, # MAB306).

Techniques: Expressing, Transfection, Control, Concentration Assay

Journal: bioRxiv

Article Title: Antisense modulation of IL7R splicing to control sIL7R expression in human CD4 + T cells

doi: 10.1101/2022.02.22.481529

Figure Lengend Snippet: (A) Representative gel image of exon 6 splicing in the endogenous IL7R transcripts from human primary CD4 + T cells transfected with control (ASO-Ctrl) or experimental (IL7R-001, IL7R-004, IL7R-005 and IL7R-006) morpholino ASOs. Percent (%) exon 6 exclusion (mean ± S.D.) is shown for each ASO under the gel image. (B) Levels of secreted sIL7R (mean ± S.D.) in supernatants from cells in panel A . (C) Relative MFI values (mean ± S.D.) of mIL7R cell surface expression in cells from panel A . (*) p < 0.05 , (**) p < 0.01 , and (***) p < 0.001 .

Article Snippet: Measurements of secreted sIL7R in HeLa cell supernatants were conducted as follows: 96-well plate (R&D Systems) were coated at 4 ° C overnight with a mouse anti-human IL7R monoclonal antibody (R&D Systems, # MAB306).

Techniques: Transfection, Control, Expressing

Journal: bioRxiv

Article Title: Antisense modulation of IL7R splicing to control sIL7R expression in human CD4 + T cells

doi: 10.1101/2022.02.22.481529

Figure Lengend Snippet: Control (ASO-Ctrl) or lead anti-sIL7R morpholino ASOs IL7R-005 and IL7R-006 were transfected at 10 µM with Endo-Porter into HeLa cells stably expressing versions of the GFP-IL7R reporter containing either the protective ‘T’ allele or the risk ‘C’ allele of the MS SNP rs6897932. (A) Representative gel image of exon 6 splicing in transcripts from the GFP-IL7R reporter containing either the protective ‘T’ allele or the risk ‘C’ allele of the MS SNP rs6897932, and transfected with control (ASO-Ctrl) or lead anti-sIL7R (IL7R-005 and IL7R-006) morpholino ASOs. Percentage of exon 6 exclusion (mean ± S.D.) for each condition is plotted below the gel image. (B) GFP MFI (mean ± S.D.) from cells in panel A shown relative to the T reporter treated with ASO-Ctrl. (*) p < 0.05 , (**) p < 0.01 , and (***) p < 0.001 .

Article Snippet: Measurements of secreted sIL7R in HeLa cell supernatants were conducted as follows: 96-well plate (R&D Systems) were coated at 4 ° C overnight with a mouse anti-human IL7R monoclonal antibody (R&D Systems, # MAB306).

Techniques: Control, Transfection, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: Antisense modulation of IL7R splicing to control sIL7R expression in human CD4 + T cells

doi: 10.1101/2022.02.22.481529

Figure Lengend Snippet: (A) Representative gel image of exon 6 splicing in transcripts from the GFP-IL7R reporter containing either the MS risk ‘C’ allele or the protective ‘T’ allele of the IL7R SNP rs6897932, and transfected with control (ASO-Ctrl) or lead pro-sIL7R (IL7R-001 and IL7R-004) morpholino ASOs. Percentage of exon 6 exclusion (mean ± S.D.) for each condition is plotted below the gel image. (B) GFP MFI (mean ± S.D.) from cells in panel A shown relative to the C reporter treated with ASO-Ctrl. (***) p < 0.001 .

Article Snippet: Measurements of secreted sIL7R in HeLa cell supernatants were conducted as follows: 96-well plate (R&D Systems) were coated at 4 ° C overnight with a mouse anti-human IL7R monoclonal antibody (R&D Systems, # MAB306).

Techniques: Transfection, Control