Journal: PLoS ONE

Article Title: Human and Mouse CD137 Have Predominantly Different Binding CRDs to Their Respective Ligands

doi: 10.1371/journal.pone.0086337

Figure Lengend Snippet: (A) Diagram of hCD137 entire extracellular region and hE, hE2, hE3, hE4, hE300, hE3003 and hE3004 represent the expressed hCD137 segments covering different CD137 CRDs used in this study. (B) Identification of purified expressed series of Fc-fusion proteins with Ponceau S staining of SDS-PAGE gel (left top panel) and ECL (right panel). hE, hE2, hE3, hE4 and hCD137L-hFc (hL-hFc ) were expressed by pCDH and pCDH-L respectively.hE300, hE3003, hE3004 and hCD137L-mFc ( hL-mFc ) were expressed by pCDM-L. (C) Binding of hCD137L to various recombinant hE and subdomains by ELISA assay. Affinity-purified hCD137L-mFc (1 µg/ml) was coated on plastic, followed by addition various hCD137 fragments expressed supernatant, then detected by HRP-goat-anti-human IgG. (D) hCD137L-hFc (1 µg/ml) was coated, followed by addition hE300, hE3003 and hE3004 fragments expressed supernatant and also an unrelated epithelial cell adhesion molecule (Epcam) protein with mFc, the binding detected by HRP-goat-anti-mouse IgG., Similar results were obtained in all of 3 individual experiments. *P/N>2.1.

Article Snippet: In brief, anti-human CD137 mAb h4-1BB-M127 (BD Pharmingen, San Diego, CA, USA) was used as a capture antibody, and a serially diluted solution of hCD137-hFc fusion proteins (Sino Biological Inc, 10041-H03H, China) with a purity over 95% was used as reference standard.

Techniques: Purification, Staining, SDS Page, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Affinity Purification

Journal: PLoS ONE

Article Title: Human and Mouse CD137 Have Predominantly Different Binding CRDs to Their Respective Ligands

doi: 10.1371/journal.pone.0086337

Figure Lengend Snippet: hE3, which is hIgG1-Fc fusion protein expressed by pCDH, containing hCD137 CRD I+II. hCD137L-mFc was coated at 1 µg/ml and a series of decreasing concentration of hE3, 10 µg/ml, 5 µg/ml, 1 µg/ml and 0.2 µg/ml were added, followed by detecting with HRP-goat-anti-human IgG. The experiment was repeated 3 times with different batch supernatants.

Article Snippet: In brief, anti-human CD137 mAb h4-1BB-M127 (BD Pharmingen, San Diego, CA, USA) was used as a capture antibody, and a serially diluted solution of hCD137-hFc fusion proteins (Sino Biological Inc, 10041-H03H, China) with a purity over 95% was used as reference standard.

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Human and Mouse CD137 Have Predominantly Different Binding CRDs to Their Respective Ligands

doi: 10.1371/journal.pone.0086337

Figure Lengend Snippet: mCD137 extracellular region (mE) and hCD137 extracellular region (hE) were run on 10% SDS-PAGE gel without 2-ME, then stained by Ponceau S(left panel) and followed by Western blotting with hCD137L-mFc and HRP-goat-anti-mouse IgG for detection (ECL,right panel).

Article Snippet: In brief, anti-human CD137 mAb h4-1BB-M127 (BD Pharmingen, San Diego, CA, USA) was used as a capture antibody, and a serially diluted solution of hCD137-hFc fusion proteins (Sino Biological Inc, 10041-H03H, China) with a purity over 95% was used as reference standard.

Techniques: SDS Page, Staining, Western Blot

Journal: PLoS ONE

Article Title: Human and Mouse CD137 Have Predominantly Different Binding CRDs to Their Respective Ligands

doi: 10.1371/journal.pone.0086337

Figure Lengend Snippet: Panel A shows the binding of the two mAbs to hE, hE2, hE3, hE4 and control. Panels B, C and D shows an experiment where human CD137L-mFc (hL) was coated (1 µg/ml) on an ELISA plate, blocked with milk and followed by adding hE(1 µg/ml) which had been preincubated with h41BB-M127 (B), 4B4-1 (C) and an anti-human CD3, OKT3 (D) at a concentration of 5 µg/ml or 25 µg/ml. (E) Inhibition of the binding of hCD137L to hCD137 at a series of concentrations of anti-human CD137mAbs.

Article Snippet: In brief, anti-human CD137 mAb h4-1BB-M127 (BD Pharmingen, San Diego, CA, USA) was used as a capture antibody, and a serially diluted solution of hCD137-hFc fusion proteins (Sino Biological Inc, 10041-H03H, China) with a purity over 95% was used as reference standard.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Inhibition

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: Splenocytes (A-D) and tumor-infiltrating lymphocytes (TIL) (E-H) were isolated from HIS mice 16-18 days after tumor implantation and analyzed by flow cytometry. A, frequency of splenic T cells in tumor-bearing or naïve HIS mice; parent population refers to frequency (%) of CD3 + T cells within human CD45 + cells and CD4 + and CD8 + T cells within CD3 + T cells. B , CD8 + T cell differentiation defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ), T EMRA (CD45RA + CD62L - ) in tumor-bearing or naïve HIS mice. C-D , expression of indicated markers on CD8 + T cells from spleen of tumor-bearing or naïve HIS mice. E, frequency of CD4 + and CD8 + T cells within TILs, gated on total CD3 + T cells. F, CD8 + T cell differentiation within TILs. G-H, expression of indicated markers on CD8 + T cells within TILs. I, expression of CD137 on splenic CD8 + and CD4 + T cells of tumor-bearing or naïve HIS mice. J, expression of PD-1 and CD137 on splenic CD8 + T cells of tumor-bearing mice. K , expression of PD-1 on splenic CD8 + T cells based on CD137 in tumor-bearing HIS mice or bulk CD8 + T cells from naïve HIS mice. L, CD8 + T cell differentiation in spleen of tumor-bearing HIS mice. M-O , expression of indicated markers on splenocyte-derived CD8 + T cell populations based on expression of CD137 and PD-1. Data are pooled from at least 3 independent experiments, n=10-23 mice per group. For each experiment, a different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by paired t-test, one-way ANOVA or 2way ANOVA, as appropriate. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Isolation, Tumor Implantation, Flow Cytometry, Cell Differentiation, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , schematic of generation, expansion and characterization of T cells from HIS mice bearing autologous LCL tumors. B , fold expansion of FACS sorted splenic CD8 + T cells from tumor-bearing HIS mice (CD137 + , CD137 - and CD137 - PD1 - ) or naïve HIS mice (bulk). C , CD8 + T cell differentiation after ex vivo expansion defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ), T EMRA (CD45RA + CD62L - ). D, IFN-ψ ELISpot of expanded T cells in 5:1 (E:T) co-culture with autologous LCL tumor cells for 24 hours. Spot count is normalized to the spots produced by expanded CD8 + bulk T cells from naïve HIS mice. E , TNF⍺ ELISA of supernatant of expanded T cells in co-culture (5:1, E:T) with autologous LCL tumor cells for 24 hours. TNF⍺ concentration is normalized to the TNF⍺ secretion from expanded CD8 + bulk T cells derived from naïve mice. Data are pooled from at least 3 independent experiments, n=6-19 mice per group. For each experiment, a different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by mixed-effects analysis (paired), RM one-way ANOVA or 2way ANOVA, as appropriate. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Cell Differentiation, Ex Vivo, Enzyme-linked Immunospot, Co-Culture Assay, Produced, Enzyme-linked Immunosorbent Assay, Concentration Assay, Derivative Assay

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: Transcriptome analysis and pathway analysis of expanded CD8 + T cells from tumor-bearing HIS mice (CD137 + , CD137 - and CD137 - PD1 - ) or naïve HIS mice (bulk). A, Upset plot (intersect) showing number of differentially expressed genes between groups in bulk RNAseq. p(FDR) < 0.05, log2 FC > 1.5. B , PCA plot of RNAseq showing PC1 and PC2. C, Top 50 upregulated and D, downregulated genes of T cell subsets based on the DEG between CD137 + vs. CD137 - PD1 - CD8 + T cells. p(FDR) < 0.05, log2 FC > 1.5. E, Volcano plot showing DEG between CD137 + and CD137 - PD1 - CD8 + T cells with genes of interest highlighted in yellow. F , differential expression of genes of interest between groups. G , Overrepresentation analysis (ORA) of upregulated pathways in CD137 + CD8 + T cells. H , Gene set enrichment analysis (GSEA) of signatures described on the y-axis. Gene ratio (# genes related to GO term / total number of sig genes) is displayed on the x-axis. Signatures with an adjusted p-value <0.05 are highlighted with a red box. Shown are data from 5 individual experiments, each experiment with different human HPC donor for reconstitution of HIS mice and autologous tumor.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Quantitative Proteomics

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , total number of individual clonotypes found per population. Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. B, TCR (CDR3 of TRA , TRB , TRG and TRD ) sequence sample diversity estimation using Hill numbers method, with Q=1 describing the Shannon diversity. C, rare clonal proportion showing the occupied repertoire space by clonotypes with defined counts (1, 2-3, 4-10, etc.). Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. D, relative abundance of clonotypes with defined frequencies (size). Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. E, repertoire overlap analysis cross-comparing every population from every experiment (each with different donor). F , repertoire overlap comparing the repertoire of bulk CD8 + T cells from naïve HIS mice to the populations from tumor-bearing HIS mice from individual experiments (each with different donor; data from individual experiments are indicated by different symbols). Mixed effects analysis with Tukey’s multiple comparisons test. G , tracking of clonotypes over populations. The top 10 most abundant clonotypes of the TCR repertoire of CD137 + CD8 + T cells from one representative experiment are shown. H , proportion of the top 10 most abundant clonotypes (from repertoires of CD137 + CD8 + T cells) in the repertoire of all populations, correlated with the spot count of IFN-ψ ELISpot. Shown are data from 4 individual experiments, each experiment with different human HPC donor for reconstitution of HIS mice and autologous tumor.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Sequencing, Enzyme-linked Immunospot

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , schematic of generation of tumor-reactive T cells and subsequent ACT. NSG mice were injected with 2 x 10 LCL s.c. in the flank and after three days, 10 x 10 ex vivo expanded T cells were adoptively transferred intravenously. Transferred T cells and LCL tumors were autologous to each other. B , tumor volume on the day of sacrifice in NSG recipient mice after ACT of the indicated cell populations. C, waterfall plot of tumor size in NSG recipient mice of ACT on the day of sacrifice relative to the tumor volume of control mice (no ACT). Bars depict individual mice. D , frequency of CD3 + T cells (% of human CD45 + cells) in TIL from NSG mice after ACT of the indicated cell populations, measured by flow cytometry. E , frequency of CD8 + T cells (% of total cells) in tumors of NSG mice after ACT of the indicated cell populations, measured by immunohistochemistry. F , differentiation of CD8 + T cells in TIL of NSG mice after ACT of the indicated cell populations defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ) and T EMRA (CD45RA + CD62L - ). G, correlation between tumor volume and infiltration of CD8 + T cells (measured by IHC) in tumors of NSG mice after adoptive transfer of CD137 + CD8 + T cells. H , schematic of ACT. CD137 + CD8 + T cells, CD137 - CD8 + T cells and CD137 - PD-1 - CD8 + T cells were isolated from spleen of tumor-bearing HIS mice or bulk CD8 + T cells from spleen of naïve HIS mice and expanded ex vivo . Recipient HIS mice were injected with 2 x 10 LCL s.c. in the flank and after three days, ex vivo expanded T cells were adoptively transferred intravenously. Tumor-bearing HIS recipient mice received 2 x 10 T cells without prior conditioning/lymphodepletion. Donor and recipient HIS mice as well as LCL were autologous to each other. I, tumor volume on the day of sacrifice in HIS recipient mice after ACT of the indicated cell populations. J, waterfall plot of tumor size on the day of sacrifice of HIS mice receiving ACT relative to the tumor volume of control HIS mice (no ACT). Bars depict individual mice. B-G: Data are pooled from 2-3 independent experiments, n=6-13 per group. I, data are pooled from 2-3 independent experiments (n=6-13 per group); J, data are from 1-3 independent experiments (n=3-13 per group). For each experiment, a with different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by one-way ANOVA. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Injection, Ex Vivo, Control, Flow Cytometry, Immunohistochemistry, Adoptive Transfer Assay, Isolation

Journal: bioRxiv

Article Title: High-resolution profiling of neoantigen-specific T cell receptor activation signatures links moderate stimulation patterns to resilience and sustained tumor control

doi: 10.1101/2022.09.23.508529

Figure Lengend Snippet: A, Schematic experiment setting. B, Increase in total number of cells of known (KIF-P1 and -P2, SYT-T1, -T2, -P1, -P2) and newly identified (KIF-sc1 and -sc2) TCRs upon antigen-specific stimulation and CD137-enrichment with dominance of KIF-P1 and -P2 harboring high precursor frequency. The two newly identified KIF2C-TCRs were selected based on fold change of TCR frequency and highest absolute frequency in the stimulated sample. C, Assessment of antigen-specific IFN-γ-secretion for the two newly identified TCRs KIF-sc1 and -sc2 in comparison to the known TCR KIF-P2. Cytokine secretion was measured by IFN-γ-ELISA upon 24h of co-culture of TCR-tg T cells from one representative donor with Mel15-LCLs transgenic for the mutated KIF2C P13L minigene (mut mg) and the wildtype KIF2C minigene (wt mg) as well as pulsed for 2h at 37°C with the mutated and wildtype peptide (mut pep and wt pep). An irrelevant peptide (irr peptide), target cells (LCL only) or T cells alone (T cell only) served as negative controls. D, Frequency of KIF-sc1 and -sc2 in relation to the previously identified TCR-sequences identified by deep sequencing of the TCR-β-chain in intestinal (MInt) and lung metastases (MLung) as well as corresponding non-malignant draining lymph nodes (MInt-LN1, MInt-LN2 and MLung-LN) of patient Mel15. Non-td: non-transduced.

Article Snippet: Another 24h later, reactive T cells were separated using magnetic labelling and positive selection with the CD137 MicroBead Kit (Miltenyi).

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Transgenic Assay, Sequencing

Journal: bioRxiv

Article Title: High-resolution profiling of neoantigen-specific T cell receptor activation signatures links moderate stimulation patterns to resilience and sustained tumor control

doi: 10.1101/2022.09.23.508529

Figure Lengend Snippet: A-C, Mel15 LCLs were pulsed with titrated peptide concentrations (2h, 37°C) and co- incubated with TCR-tg T cells with subsequent ELISA-based assessment of IFN-γ-secretion within 24h of co-culture (A). The cellular activation level was determined after 24h by FACS staining of the extracellular level of CD137 (B) and PD-1 (C) expression (reflected by geometric mean of all CD3 + CD8 + /TCRmu + cells). The mean for ELISA data is depicted for technical triplicates of one donor; triplicates from the same donor have been pooled prior to EC FACS- staining. E:T = 1:1 (15.000 tg T cells:15.000 tumor cells). D-G, EC FACS staining at different timepoints after co-culture setup displays temporal dynamics of T cell activation marker CD137 (D, E) and inhibitory receptor LAG-3 (F, G) for TCR-tg T cells upon co-culture with JJN3-B27 peptide-pulsed target cells. A weak (0.01 µM for peptide pulsing; D, F) versus a strong (1 µM for peptide pulsing; E, G) stimulus were compared. E:T = 1:1 (10.000 tg T cells:10.000 tumor cells). H, Annexin-V/PI-staining was employed for detection of activation induced cell death (AICD) after 20h of co-culture upon strong stimulation with 1µM mut-peptide pulsed Mel15 LCLs (early apoptotic = AnnexinV + PI - , late apoptotic = AnnexinV + PI + ). E:T = 1:1 (30.000 tg T cells:30.000 tumor cells). I, Representative FACS plot of a healthy donor of CTV-analysis for all TCRmu + cells depicted after 4 days of co-culture with 1µM mut-peptide pulsed Mel15 LCLs (colors were chosen according to Figure B-E; representative wt mg-control depicted in grey). E:T = 1:1 (30.000 tg T cells:30.000 tumor cells). For all co-cultures in D-I technical triplicates per donor were pooled prior to staining; the mean and SD for biological replicates from three different human donors are shown.

Article Snippet: Another 24h later, reactive T cells were separated using magnetic labelling and positive selection with the CD137 MicroBead Kit (Miltenyi).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Activation Assay, Staining, Expressing, Marker, Control

Journal: bioRxiv

Article Title: High-resolution profiling of neoantigen-specific T cell receptor activation signatures links moderate stimulation patterns to resilience and sustained tumor control

doi: 10.1101/2022.09.23.508529

Figure Lengend Snippet: A, Schematic experiment setting of xenograft neoTCR-tumor rejection experiment with newly transduced T cells. B, Tumor growth kinetics are displayed as tumor area (in cm 2 ) for mut mg-U698M-tumor- bearing NSG-mice comparing neoTCR-tg T cells to the irrelevant TCR 2.5D6 until day 20. Mean values and SDs for each group of mice display rejection dynamics (n=6). Parts of this dataset were already published before . In this renewed version, tumor rejection kinetics of KIF-sc1 and -sc2 analyzed in the same experiment are included. C, Kaplan-Meier-survival curve is displayed until day 20 for tumor-bearing mice injected with different neoTCR-tg T cells. D, schematic experiment setting of xenograft neoTCR-TIL-rechallenge experiment. E-G, Ex vivo restimulation of T cells derived from TIL products on day 21 after tumor explant (TIL-P) compared to newly transduced (NEW) TCR-tg T cells from the same human donor stained for CD137 (EC; E), IFN-γ (IC; F) and GzmB (IC; G); expression was analyzed using geometric mean of all CD3 + CD8 + /TCRmu + cells after 18h of co-culture. Mut-mg and wt-mg U698M cells were used as target cells in E:T = 1:1 (50.000 tg T cells:50.000 tumor cells). Mean and SD are shown for three experimental replicates. Statistical significance is calculated with one-way ANOVA and Tukey’s multiple comparison test (*p≤0.05, ****p≤0.0001). H, Tumor growth kinetics are displayed as tumor area (in cm 2 ) for NSG-mice continuing the experiment until day 17 after second injection of in total 5x10 6 neoTCR-tg T cells (transduction rate of 55% equalized for all groups). For the TIL- P-groups, TIL-P from two mice per TCR (highest numbers of expanded neoTCR-tg T cells) were pooled. Mean values and SEMs for each group of mice display rejection dynamics (n=5 for experimental groups, n=3 for 2.5D6; due to achievement of humane endpoint criteria one 2.5D6- control mouse was sacrificed on day 13 and excluded from this graph). Statistical significance is calculated for the tumor area on day 17 with one-way ANOVA and Tukey’s multiple comparison test (***p≤0.001, ****p≤0.0001). I , Kaplan-Meier-survival curve is displayed for tumor-bearing mice injected with different TCR-tg T cells (n=5 for experimental groups, n=4 for 2.5D6 control). Survival of mice receiving TIL-P-KIF-P2 compared to TIL-P-KIF-sc1 was significantly prolonged (p=0.0019, Mantel-Cox test).

Article Snippet: Another 24h later, reactive T cells were separated using magnetic labelling and positive selection with the CD137 MicroBead Kit (Miltenyi).

Techniques: Injection, Ex Vivo, Derivative Assay, Staining, Expressing, Co-Culture Assay, Comparison, Transduction, Control

Journal: Science Advances

Article Title: Uncovering receptor-ligand interactions using a high-avidity CRISPR activation screening platform

doi: 10.1126/sciadv.adj2445

Figure Lengend Snippet: ( A ) FC was used to monitor binding of FITC-labeled 4-1BB magnetic beads to activator cells following up to four rounds of selection using streptavidin–4-1BB magnetic beads. ( B ) FC was used to monitor 4-1BBL expression in activator cells following up to four rounds of enrichment with streptavidin–4-1BB magnetic beads. ( C ) The gRNA enrichment following first, second, third, or fourth round of selection using streptavidin–4-1BB magnetic beads was monitored using −log 10 (score). For each library, the percentages indicate the total gRNA counts for the gene of interest divided by the total number of gRNA counts for all genes (×100). ( D ) Immunoblotting was used to detect myc in 293 cells transfected with siglec-4–myc or 4-1BB–myc. β-Actin (β-act) was included as a loading control. ( E ) FC was used to measure the level of siglec-4 or 4-1BBL on the surface of 293 cells following transfection with pCMV EV (control), pCMV–siglec-4 (siglec-4), or pCMV–4-1BBL (4-1BBL). ( F ) Binding of 4-1BB–HIS to 293 cells transfected with EV (negative control), pCMV–4-1BB (positive control), or pCMV–siglec-4. Data in (A), (B), (D), (E), and (F) were representative of three experiments.

Article Snippet: For the m4-1BB protein binding assay, HEK293 cells transfected with EV, pCMV–m-siglec-4 (SinoBiological, cat. no. MG51398-CM) or pCMV–m4-1BBL (SinoBiological, cat. no. MG50067-CM) were incubated with 1 μg of m4-1BB–Fc protein (SinoBiological, cat. no. 50811-M02H) followed by staining with AF647 AffiniPure Donkey Anti-Human IgG (H + L) (Jackson ImmunoResearch, cat. no. 709-605-149).

Techniques: Binding Assay, Labeling, Magnetic Beads, Selection, Expressing, Western Blot, Transfection, Negative Control, Positive Control

Journal: Science Advances

Article Title: Uncovering receptor-ligand interactions using a high-avidity CRISPR activation screening platform

doi: 10.1126/sciadv.adj2445

Figure Lengend Snippet: ( A to C ) FC was used to monitor (A) 4-1BB expression and 4-1BBL–Fc or siglec-4–Fc binding to activated T cells, (B) binding of siglec-4–Fc to stimulated T cells transfected with nonspecific (NS) or 4-1BB knockdown (KD) siRNAs, or (C) binding of siglec-4–Fc to stimulated T cells in the presence of increasing amounts of soluble 4-1BB–HIS protein. MFI, mean fluorescence intensity. Statistical analysis: unpaired t test. ( D ) ELISA was used to measure IFN-γ produced by activated T cells mixed with 293 cells overexpressing EV, siglec-4, 4-1BBL, or siglec-4 plus 4-1BBL. Statistical analysis: unpaired t test; P values correspond to comparisons between groups with or without siglec-4. ( E ) Luciferase assays were used to measure the viability of eGFP-FFLuc–labeled 293 target cells 24 hours after mixing with anti–TEM8–CAR-T cells. TEM8 knockout control cells (293/T8KO) were included as a specificity control. E:T, effector:target cell ratio. Statistical analysis: unpaired t test; P values correspond to comparisons between 293 and 293–Siglec-4 at each E:T cell ratio. ( F to H ) Immunoblotting was used to assess (F) p-c-Jun and c-Jun levels in 293 cells or 293–4-1BB cells following transient transfection with full-length siglec-4–myc or 4-1BB–myc, (G) p-c-Jun and c-Jun levels in unstimulated (U) or stimulated (S) T cells derived from two independent donors, and (H) p-c-Jun and c-Jun levels in T cells cocultured for 1 hour at a ratio of 1:1 with 293 cell transfected with EV (E) or siglec-4 (Sig4). Note that Siglec-4 expression can mediate the down-regulation of c-Jun only if 4-1BB is also present. β-Actin was used as a loading control in (F), (G), and (H). All data or images in (A) to (H) were representative of at least three independent experiments. For (C) to (E), n > = 3 biologically independent samples per group. ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: For the m4-1BB protein binding assay, HEK293 cells transfected with EV, pCMV–m-siglec-4 (SinoBiological, cat. no. MG51398-CM) or pCMV–m4-1BBL (SinoBiological, cat. no. MG50067-CM) were incubated with 1 μg of m4-1BB–Fc protein (SinoBiological, cat. no. 50811-M02H) followed by staining with AF647 AffiniPure Donkey Anti-Human IgG (H + L) (Jackson ImmunoResearch, cat. no. 709-605-149).

Techniques: Expressing, Binding Assay, Transfection, Fluorescence, Enzyme-linked Immunosorbent Assay, Produced, Luciferase, Labeling, Knock-Out, Western Blot, Derivative Assay