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

Journal: OncoImmunology

Article Title: Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism

doi: 10.1080/2162402x.2015.1062967

Figure Lengend Snippet: Figure 1. CD137 mRNA expression is upregulated by hypoxia in mouse tumor cell lines. Quantitative RT-PCR assessment of CD137 mRNA expression under 21% and 1% O2 (hypoxia) in the indicated cell lines cultured under these conditions for 48h. A representative direct immunofluorescence staining for CD137 surface expression ana- lyzed flow cytometry is presented in a corresponding histogram showing the discrepancy between the mRNA levels and the weak or dim surface protein staining. p < 0.05, p < 0.01.

Article Snippet: Serial dilutions of the serum of CT26-bearing CD137¡/¡ mice were incubated in ELISA plates coated with mouse recombinant 4-1BB-Fc chimeric protein (R&D Systems) and the antibodies against CD137 were detected and tittered using a goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Staining, Cytometry

Journal: OncoImmunology

Article Title: Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism

doi: 10.1080/2162402x.2015.1062967

Figure Lengend Snippet: Figure 2. Soluble form of CD137 predominates over membrane-attached forms. (A) Diagram representing the cDNA with the exons of mouse CD137 and RT-PCR products amplified with the indicated primer pairs. (B) Quanti- tative RT-PCR analyses using primers that amplify either transmembrane (primer pair 34) or total CD137 cDNA (primer pair 12) in corresponding cell lines. (C) RT-PCR products of total CD137 cDNA (primer pair 56) amplifi- cation showing that the soluble CD137 (sCD137) predominates over the transmembrane CD137 (tmCD137) form. (D) Sandwich ELISA assessment of the concentration of sCD137 in the tissue culture supernatants of the CT26 cell line. TM: transmembrane domain; MM: molecular weight marker; N: normoxia; H: hypoxia.

Article Snippet: Serial dilutions of the serum of CT26-bearing CD137¡/¡ mice were incubated in ELISA plates coated with mouse recombinant 4-1BB-Fc chimeric protein (R&D Systems) and the antibodies against CD137 were detected and tittered using a goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) according to the manufacturer’s instructions.

Techniques: Membrane, Reverse Transcription Polymerase Chain Reaction, Sandwich ELISA, Concentration Assay, Molecular Weight, Marker

Journal: OncoImmunology

Article Title: Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism

doi: 10.1080/2162402x.2015.1062967

Figure Lengend Snippet: Figure 4. Soluble CD137 produced by tumor cells binds to CD137 ligand. (A) Binding of sCD137 present in the supernatant of CT26 cells cultured under hypoxia to CD137 ligand (CD137L) transfected to 293T cells. Untransfected 293T were used as a specificity control and supernatants from normoxia and hypoxia cultured CT26 cells were tested without dilution. Binding was revealed by an anti-CD137 mAb which does not interfere with ligand binding (1D8 clone) (B) Similar experiment as in A performed with the serum of CT26-bearing Rag2IL2Rg¡/¡ mice as a source of sCD137 or with pre-tumor serum as a control.

Article Snippet: Serial dilutions of the serum of CT26-bearing CD137¡/¡ mice were incubated in ELISA plates coated with mouse recombinant 4-1BB-Fc chimeric protein (R&D Systems) and the antibodies against CD137 were detected and tittered using a goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) according to the manufacturer’s instructions.

Techniques: Produced, Binding Assay, Cell Culture, Transfection, Control, Ligand Binding Assay

Journal: OncoImmunology

Article Title: Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism

doi: 10.1080/2162402x.2015.1062967

Figure Lengend Snippet: Figure 3. Soluble CD137 is produced by in vivo grafted tumors. (A) CT26 tumors (10£10 mm in diameters) excised from CD137¡/¡ mice were analyzed by quantitative RT-PCR for mCD137 encoding the transmembrane and the total CD137 isoforms. (B) The concentration of soluble CD137 (sCD137) assessed by ELISA in the sera of Rag¡/¡ mice grafted with 5£105 CT26 cells for 21 days.

Article Snippet: Serial dilutions of the serum of CT26-bearing CD137¡/¡ mice were incubated in ELISA plates coated with mouse recombinant 4-1BB-Fc chimeric protein (R&D Systems) and the antibodies against CD137 were detected and tittered using a goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) according to the manufacturer’s instructions.

Techniques: Produced, In Vivo, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: OncoImmunology

Article Title: Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism

doi: 10.1080/2162402x.2015.1062967

Figure Lengend Snippet: Figure 5. Soluble CD137 in the supernatant of hypoxia-treated CT26 cells blocks CD137L-mediated T-cell costimu- lation. Cultures of CD8C-purified OT1 WT or OT1 CD137¡/¡ T cells were stimulated with anti-CD3 mAb for 48h prior to seeding in co-cultures with 293T transfected or not with CD137L as indicated. T cells were pre-loaded with Violet 450 fluorescent dye and dilution of the fluorescent dye was used as a surrogate marker of T-cell prolif- eration. Cultures were conditioned with cell culture supernatants (1/16 diluted) of normoxia- or hypoxia-treated CT26 cells. (A) Representative flow cytometry histograms showing the extent of inhibition of proliferation by the sCD137-containing supernatants at 72h of coculture. (B) Pooled data of two independent experiments identically performed. MFI: mean fluorescence intensity; SN: supernatant.

Article Snippet: Serial dilutions of the serum of CT26-bearing CD137¡/¡ mice were incubated in ELISA plates coated with mouse recombinant 4-1BB-Fc chimeric protein (R&D Systems) and the antibodies against CD137 were detected and tittered using a goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) according to the manufacturer’s instructions.

Techniques: Transfection, Marker, Cell Culture, Cytometry, Inhibition

Journal: OncoImmunology

Article Title: Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism

doi: 10.1080/2162402x.2015.1062967

Figure Lengend Snippet: Figure 6. CD137 silencing in CT26 tumor cells gives rise to more immunogenic variants. CT26 were stably transfected with lentiviral vectors to express a shRNA targeting CD137 (shCD137) or a scrambled control (shControl). (A) Transfectants were cultured for 36h in normoxia or 1% O2 and analyzed by flow cytometry to quantitatively determine the expression of surface CD137. (B) Quantitative RT-PCR analysis of total and transmembrane CD137 mRNA in the indicated transfectants under hypoxic or normoxic conditions as in A. (C) BALB/c wild type immunocompetent mice were subcutaneously inoculated with 5£105 cells of indicated transfectants and tumor sizes were individually followed over time. Tumor cells had been pre-exposed in every case to 1% O2 for 36h. The fraction of mice spontaneously rejecting their tumors is given in each graph. When indicated, BALB/c mice were mAb-depleted of CD8CT cells. (D) mean§SEM and Statistical comparisons of experiments in C whose results are repre- sentative of two independent experiments. (E) Tumor growth of the indicated transfectants in immunodeficient Rag2IL2Rg¡/¡ mice. Tm: transmembrane.

Article Snippet: Serial dilutions of the serum of CT26-bearing CD137¡/¡ mice were incubated in ELISA plates coated with mouse recombinant 4-1BB-Fc chimeric protein (R&D Systems) and the antibodies against CD137 were detected and tittered using a goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) according to the manufacturer’s instructions.

Techniques: Stable Transfection, Transfection, shRNA, Control, Cell Culture, Cytometry, Expressing, Quantitative RT-PCR

Journal: OncoImmunology

Article Title: Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism

doi: 10.1080/2162402x.2015.1062967

Figure Lengend Snippet: Figure 7. Graphical interpretation of the experimental results. Schematic representation of the postulated mechanism underneath the experimental observations. TM: transmembrane; sCD137: soluble CD137.

Article Snippet: Serial dilutions of the serum of CT26-bearing CD137¡/¡ mice were incubated in ELISA plates coated with mouse recombinant 4-1BB-Fc chimeric protein (R&D Systems) and the antibodies against CD137 were detected and tittered using a goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) according to the manufacturer’s instructions.

Techniques:

Journal: Circulation Research

Article Title: High Levels of Costimulatory Receptors OX40 and 4-1BB Characterize CD4 + CD28 null T Cells in Patients With Acute Coronary Syndrome

doi: 10.1161/circresaha.111.261933

Figure Lengend Snippet: Figure 2. Expression of costimulatory receptors in CD4CD28null T cells. Peripheral blood mononuclear cells (PBMCs) from acute coronary syndrome (ACS, n25) and stable angina (SA, n21) patients were cultured alone (w/o) or activated with anti-CD3 antibodies (aCD3) and the expression of costimulatory receptors (OX40, 4–1BB, inducible costimulator [ICOS]) was analyzed on day 4. A, Illustra- tive histograms show the expression of OX40, 4-1BB, and ICOS (green histograms) and control staining with isotype-matched antibod- ies (red histograms) by activated CD4CD28null (28null) and classical CD4CD28 (28pos) T cells from an ACS patient. B, Scatter plots show the distribution and mean fluorescence intensity (MFI) of cells expressing costimulatory receptors in the ACS group. C, Scatter plots show the distribution and mean MFI of cells expressing costimulatory receptors in the SA group. Probability values (P) were cal- culated by 2-tailed t test. a.u. indicates arbitrary units.

Article Snippet: Stimulation of 4- 1BB and OX40 was done with 10 g/ml stimulating antibodies for 4-1BB (R&D Systems, AF838) or OX40 (AF3388) or with 5 g/ml recombinant ligands 4-1BBL (R&D Systems, 2295-4L) and OX40L (R&D Systems, 1054-OX).

Techniques: Expressing, Cell Culture, Control, Staining

Journal: Circulation Research

Article Title: High Levels of Costimulatory Receptors OX40 and 4-1BB Characterize CD4 + CD28 null T Cells in Patients With Acute Coronary Syndrome

doi: 10.1161/circresaha.111.261933

Figure Lengend Snippet: Figure 5. Analysis of CD4CD28null T cells, costimulatory receptors and ligands in atherosclerotic plaques. A, The frequency of CD4CD28null (CD28neg), CD4CD28 (CD28pos) T cells and the expression of OX40 and 4-1BB costimulatory receptors was quantified in cells isolated from human endarterectomy atherosclerotic plaques using flow cytometry. Representative plots show expression of OX40 and 4-1BB on plaque-derived T cells from atherosclerotic plaques B, Paraffin embedded tissue sections were immunohistochemically labeled

Article Snippet: Stimulation of 4- 1BB and OX40 was done with 10 g/ml stimulating antibodies for 4-1BB (R&D Systems, AF838) or OX40 (AF3388) or with 5 g/ml recombinant ligands 4-1BBL (R&D Systems, 2295-4L) and OX40L (R&D Systems, 1054-OX).

Techniques: Expressing, Isolation, Cytometry, Derivative Assay, Labeling

Journal: Circulation Research

Article Title: High Levels of Costimulatory Receptors OX40 and 4-1BB Characterize CD4 + CD28 null T Cells in Patients With Acute Coronary Syndrome

doi: 10.1161/circresaha.111.261933

Figure Lengend Snippet: Figure 6. Effects of OX40 and 4-1BB blockade on cytokine production by CD4CD28null T cells. Peripheral blood mononuclear cells (PBMCs) from acute coronary syndrome (ACS) patients (n9) were activated with anti-CD3 antibodies alone (aCD3) or in the presence of blocking antibodies against OX40 (aOX40) and 4-1BB (a4–1BB) or control antibodies (ctrl Ig) for 4 days. A, Repre- sentative contour plots and bar graphs display percentage of interferon (IFN)-

Article Snippet: Stimulation of 4- 1BB and OX40 was done with 10 g/ml stimulating antibodies for 4-1BB (R&D Systems, AF838) or OX40 (AF3388) or with 5 g/ml recombinant ligands 4-1BBL (R&D Systems, 2295-4L) and OX40L (R&D Systems, 1054-OX).

Techniques: Blocking Assay, Control

Journal: Circulation Research

Article Title: High Levels of Costimulatory Receptors OX40 and 4-1BB Characterize CD4 + CD28 null T Cells in Patients With Acute Coronary Syndrome

doi: 10.1161/circresaha.111.261933

Figure Lengend Snippet: Figure 7. Effects of OX40 and 4-1BB blockade on perforin and granzyme B in CD4CD28null T cells. Peripheral blood mononuclear cells (PBMCs) from acute coronary syndrome (ACS) patients (n9) were cultured alone (w/o) or acti- vated with anti-CD3 antibodies alone (aCD3) or in the presence of blocking antibodies against OX40 (aOX40) and 4-1BB (a4–1BB) for 4 days. A, Repre- sentative contour plots and bar graphs show surface expression of CD107a in CD4CD28null (28null) T cells. B, Repre- sentative contour plots and bar graphs display percentage of perforin

Article Snippet: Stimulation of 4- 1BB and OX40 was done with 10 g/ml stimulating antibodies for 4-1BB (R&D Systems, AF838) or OX40 (AF3388) or with 5 g/ml recombinant ligands 4-1BBL (R&D Systems, 2295-4L) and OX40L (R&D Systems, 1054-OX).

Techniques: Cell Culture, Blocking Assay, Expressing

Journal: Circulation Research

Article Title: High Levels of Costimulatory Receptors OX40 and 4-1BB Characterize CD4 + CD28 null T Cells in Patients With Acute Coronary Syndrome

doi: 10.1161/circresaha.111.261933

Figure Lengend Snippet: Figure 8. Schematic outline of the effects of costimulatory receptors OX40 and 4-1BB on the functions of CD4CD28null T cells in acute coro- nary syndrome (ACS). Classical CD4CD28 T cells constitutively express the costimulatory receptor CD28. Activation of CD4CD28 T cells induces transient expression of costimu- latory (OX40, 4–1BB) and coinhibitory (cytotoxic T lymphocyte associated antigen-4 [CTLA-4], programmed death [PD]-1) receptors. CD4CD28 T cells produce moderate levels of inflammatory cytokines (interferon [IFN]-, tumor necrosis factor [TNF]-) when activated and do not express cytotoxic molecules (perforin, granzyme B). CD4CD28null T cells from ACS patients lack CD28 and upregulate expression of OX40 and 4-1BB. These receptors drive production of high levels of IFN-, TNF-, and per- forin. We hypothesize that these media- tors could potentially enable CD4CD28null T cells to trigger plaque rupture via macrophage activation and endothelial and vascular smooth muscle cell lysis. VSMC indicates vascular smooth muscle cell.

Article Snippet: Stimulation of 4- 1BB and OX40 was done with 10 g/ml stimulating antibodies for 4-1BB (R&D Systems, AF838) or OX40 (AF3388) or with 5 g/ml recombinant ligands 4-1BBL (R&D Systems, 2295-4L) and OX40L (R&D Systems, 1054-OX).

Techniques: Activation Assay, Expressing, Lysis

Journal: Nature Communications

Article Title: CD137 (4-1BB) costimulation of CD8 + T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation

doi: 10.1038/s41467-021-27613-w

Figure Lengend Snippet: a schematic representation of antibody-coated microbeads used to stimulate CD8 + T cells. b semiquantitative FACS assessment of anti-CD3 and anti-CD137 mAbs coupled into the microbeads was measured by antimouse IgG2a and antimouse IgG1 mAbs staining and detected by FACS. Representative histograms of the geometric mean fluorescence intensity for each condition. c , human primary CD8 + T cells from healthy donors were activated with mAb coated microbeads for 96 h. Representative dot plots of cell-surface CD25 ( n = 15) and d , intracellular Bcl-xL ( n = 13) expression analysed by flow cytometry in the indicated conditions. Summary data are given as delta (Δ), the difference of the value between each costimulation condition subtracted from the anti-CD3 condition in each case. e representative histograms of CD25 ( n = 15) and f , Bcl-xL ( n = 14) expression in CD8 + T cells. Numbers in the FACS histograms represent the geometric mean fluorescence intensity (gMFI). Summary data are given. Concentrations of human IFNγ ( n = 9) ( g ) IL-2 ( n = 8) ( h ) and granzyme B ( n = 4) ( i ) in the culture supernatants of the indicated experimental conditions. j , representative histograms of T-bet and k , Eomes intracellular analyzed by flow cytometry at the indicated conditions (n = 12). Summary data are given as the difference of the value between each costimulation condition subtracted from the anti-CD3 value in each case. l , summary data showing the T-bet:Eomes ratio of CD8 + T cells in cis versus trans costimulation ( n = 12). m , flow cytometry measurement of Mitotracker ( n = 11) and n TMRM (Tetramethylrhodamine, Methyl Ester, Perchlorate) ( n = 16) stainings given as geometric mean fluorescence in FACS-gated CD8 + T cells after cis versus trans costimulation. Data are given as mean ± s.e.m. Statistical significance was determined with the paired t test (two-sided) in b , c and k , Wilcoxon (two-sided) in d , e , i , j , l and m , the Friedman test (one-sided) with Dunn’s correction in f , g and h . Source data are provided as Source Data file.

Article Snippet: The following monoclonal antibodies were used for mAb coated beads to stimulate mouse T cells (Supplementary Table ): antimouse CD3 (100238, Biolegend), antimouse CD137 (BE0239, Bio X cell), antimouse CD137 (3H3, produced in house), rat IgG2a (400533, Biolegend), rat IgG2b (400637, Biolegend).

Techniques: Staining, Fluorescence, Expressing, Flow Cytometry

Journal: Nature Communications

Article Title: CD137 (4-1BB) costimulation of CD8 + T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation

doi: 10.1038/s41467-021-27613-w

Figure Lengend Snippet: a , representative histograms of EpCAM (left panel) and 5T4 (right panel) expression on wild-type HCT116 cells and on two different clones of alternatively silenced EpCAM or 5T4 HCT116 variants attained by CRISPR/Cas9 technology. Numbers indicate geometric mean fluorescence intensity, gMFI. b , schematic illustration of cis CD8 + T cell costimulation using the wild-type HCT116 cell line versus trans costimulation when plating 1:1 mixtures of the alternatively silenced variants (trans1 and trans2) in the presence of CD3-EpCAM and 5T4-CD137 (ALG.APV-527) BsAbs. c , cell-surface CD25 ( n = 9) expression on CD8 + T cells analysed by flow cytometry in the indicated conditions. Concentrations of IFNγ ( n = 8) ( d ), IL-2 ( n = 9) ( e ) and granzyme B ( n = 5) ( f ) in the co-culture supernatants from the indicated conditions. g , flow cytometry measurement of Mitotracker ( n = 5) geometric mean fluorescence in gated CD8 + T cells. Statistical significance was determined with paired t tests (two-sided). Source data are provided as Source Data file.

Article Snippet: The following monoclonal antibodies were used for mAb coated beads to stimulate mouse T cells (Supplementary Table ): antimouse CD3 (100238, Biolegend), antimouse CD137 (BE0239, Bio X cell), antimouse CD137 (3H3, produced in house), rat IgG2a (400533, Biolegend), rat IgG2b (400637, Biolegend).

Techniques: Expressing, Clone Assay, CRISPR, Fluorescence, Flow Cytometry, Co-Culture Assay

Journal: Nature Communications

Article Title: CD137 (4-1BB) costimulation of CD8 + T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation

doi: 10.1038/s41467-021-27613-w

Figure Lengend Snippet: a , CD137-Jurkat NF-κB reporter cell line was incubated with CD137 antibody-coated microbeads ( n = 7 technical replicates) or b , CD137Ligand-Fc coated microbeads ( n = 4 technical replicates). Luciferase activity was measured after 6 h. Data are representative of at least three independent experiments. c , activity of CD137-Jurkat NF-κB reporter cell line was assessed by cis-costimulation with wild-type HCT116 cells or trans costimulation with combinations of the alternatively silenced variants in the presence of CD3-EpCAM and ALG.APV-527 BsAbs ( n = 3). Data are representative of at least three independent experiments. Data are given as mean ± s.e.m . d , Western blot analyses of IκBα (representative of CD8 + T cells from two independent donors) on nuclear extracts from human primary human donors stimulated for 48 h with mAb coated beads. e , representative immunofluorescence images of p65 localization in human primary CD8 + T cells stimulated with mAb coated beads for 48 h. Cells are labeled with antibodies against p65 (red) and CD8 (green), DNA is visualized by Hoescht (scale 10 µm). Bars graph displays nuclear to total signal ratio ( n = 30 cells each). Shown data are representative of two independent donors with similar results. Representative Western blot of p65 ( f ) and p52 ( g ) in nuclear extracts of human primary human donors stimulated for 48 h with mAb coated beads. Numbers below Western blot indicate relative expression normalized to the total expression of β-actin for d and to C23 for f and g as analysed by densitometry. Data are representative of at least two independent donors. h , schematic layout of the experiment in i and j . Wild-type HCT116 cells and a mixture of 1:1 ratio of HCT116 KO EpCAM (clon3) together with HCT116 KO 5T4 (clon1) were subcutaneously engrafted in the left flank or in the right flank of Rag2 −/− IL-2Rγc −/− , respectively. Tumors were established for 9 days before intratumoral injection of CD137-Jurkat-4 NF-κB reporter cell line co-injected with CD3-EpCAM and ALG.APV-527 BsAbs. The relative presence of the tumor cell clones in the xenografted tumors are shown in an inset in the experimental layout. Photon emission was measured with a PhotonIMAGER from the anesthetized living animals. In vivo imaging ( i ) and quantification ( j ) ( n = 8) of luciferase activity from CD137-Jurkat NF-κB reporter cell line intratumorally transferred into cis or trans HCT116 paired tumors of each mouse in the presence of CD3-EpCAM and ALG.APV-527 BsAbs 6 h postreporter cell injection. Summary data are given as the bioluminescence photon flux subtracted from the tumors prior to the experiment (background). As negative controls mice whose tumors were injected with the reporter CD137-Jurkat transfectants and PBS are shown. Each pair of dots represents a single mouse bearing a cis tumor and a trans tumor in the left and right flank, respectively. RLU relative luciferase units. Scale bars: 10 μM. In d and f - g samples derive from the same experiment and loading control gels were processed in parallel. Statistical significance was determined with one-way Anova with Tukey’s multiple comparison test for a , b and e (one-sided), the Friedman test with Dunn’s correction for c and paired t test for j (two-sided). Source data are provided as Source Data file.

Article Snippet: The following monoclonal antibodies were used for mAb coated beads to stimulate mouse T cells (Supplementary Table ): antimouse CD3 (100238, Biolegend), antimouse CD137 (BE0239, Bio X cell), antimouse CD137 (3H3, produced in house), rat IgG2a (400533, Biolegend), rat IgG2b (400637, Biolegend).

Techniques: Incubation, Luciferase, Activity Assay, Western Blot, Immunofluorescence, Labeling, Expressing, Injection, Clone Assay, In Vivo Imaging, Control, Comparison

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: Sequences.

Article Snippet: 100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

Techniques:

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: Activation of 4-1BB signaling promotes the secretion of pro-fibrotic mediators by MH-S cells. MH-S cells treated with or without agonist 4-1BB mAb (10 µg/mL) or IgG (10 µg/mL) for 2 h prior to exposure to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) The levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E) Western blots analysis of IκBα and phospho-IκBα. (F) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (G–I) Real-time polymerase chain reaction analysis of MMP12, MMP9, and monocyte chemoattractant protein-1 mRNA expression ( n = 4). (J–L) ELISA analysis was used to quantify the secretion of IL-1β, IL-6 and tumor necrosis factor-α ( n = 4). The results were representative of three independent experiments. Results were graphed as the mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Article Snippet: 100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

Techniques: Activation Assay, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: Blockade of 4-1BB signaling attenuates the secretion of pro-fibrotic mediators by MH-S cells. Transfected (Len-cont. and sh-4-1BB) MH-S cells were treated with or without NQDI 1 (10 µM), 4-1BBIg (10 µg/mL), or IgG1 (10 µg/mL) for 2 h, then exposed to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) Levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E,G) Western blots analysis of IκBα and phospho-IκBα. (F,H) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (I–K) The expressions of MMP12, MMP9, and monocyte chemoattractant protein-1 were detected by real-time polymerase chain reaction analysis ( n = 4). ELISA analysis of cytokines in the culture supernatants. (L) IL-1β, (M) IL-6, (N) tumor necrosis factor-α ( n = 4). Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant). The data were representative of three independent experiments.

Article Snippet: 100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

Techniques: Transfection, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: Expression of 4-1BB (CD137) and 4-1BBL (CD137L) on pulmonary macrophages. After 7-days exposure to crystalline silica, mice were sacrificed. The lungs were prepared as single-cell suspensions for flow cytometric analyses ( n = 3–4). (A) Representative plots of flow cytometric analyses for 4-1BB and 4-1BBL on alveolar macrophages (AMs) and interstitial macrophages (IMs). (B,C) The percentage of AMs expressing 4-1BB and 4-1BBL. (D,E) The frequency of AMs and IMs in CD45+ cells from the lungs. (F,G) The percentage of IMs expressing 4-1BB and 4-1BBL. The experiments were performed twice with similar results. Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant).

Article Snippet: 100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: Expression of 4-1BB on CD4+ T cells. (A) Representative plots of flow cytometric analyses for 4-1BB on effector and naïve T cells. (B) The percentage of effector T cells expressing 4-1BB. (C,D) The frequency of effector and naïve T cells in CD4+ T cells from the lungs. (E) The percentage of naïve T cells expressing 4-1BB. The experiments were performed twice with similar results. Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; ns, not significant).

Article Snippet: 100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: 4-1BB expression on the mouse alveolar macrophage cell line, MH-S. MH-S cells were treated with crystalline silica (50 µg/cm 2 ) or saline for 12 h. (A,B) The percentage of MH-S cells expressing 4-1BB ( n = 4). (C) Western blot analysis of 4-1BB protein from whole cell lysates. (D) Quantification of the 4-1BB protein level relative to that of β-actin is shown ( n = 3). (E) Total RNA was isolated to analyze 4-1BB mRNA expression ( n = 4) relative to GAPDH. The data were representative of three independent experiments. Data were expressed as mean ± SEM (*** p ≤ 0.001).

Article Snippet: 100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

Techniques: Expressing, Western Blot, Isolation

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: The secretion of pro-fibrotic mediators is reduced in the lungs from crystalline silica (CS)-injured mice, upon inhibition of 4-1BB signaling. C57BL/6 mice were administered a CS suspension or saline, respectively; 4-1BBIg or isotype control (IgG1) were injected intraperitoneally (i.p.; n = 3–4). (A–D) Quantification of MMP9 and MMP12 protein levels by western blot, which were normalized to those of β-actin in lungs. Shown as bar graph. (E–H) ELISA analysis of cytokines in lung tissues. (E) IL-1β, (F) IL-6, (G) tumor necrosis factor-α, (H) monocyte chemoattractant protein-1. Experiments were performed three times. C57BL/6 mice were administered a CS suspension or saline, respectively; NQDI 1 or isotype control were injected i.p. ( n = 10). (I) Immunohistochemical staining of paraffin-embedded lung tissue sections at 7 and 56 days showed CD68, MMP9, and MMP12 expression. Nuclei were stained by hematoxylin (blue). (J–L) Identification of MMP9 and MMP12 protein levels in mouse lung tissues at 7 and 56 days by western blot. The levels of MMP9 and MMP12 were normalized to those of β-actin. (M) Representative images for the immunohistochemical staining of collagen I in paraffin-embedded lung tissue sections 56 days after CS instillation. Nuclei were stained by hematoxylin (blue). (I,M) Scale bar, 50 µm. Experiments were performed three times. Data are shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant).

Article Snippet: 100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

Techniques: Inhibition, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: A model for alveolar macrophages (AMs) expressing 4-1BB in the regulation of pulmonary fibrosis through secreting pro-fibrotic mediators. The expression of 4-1BB increases in AMs in response to crystalline silica, which leads to elevated secretion of pro-inflammatory and pro-fibrotic cytokines, chemokines, and MMPs. These pro-fibrotic mediators promote pulmonary alveoli injury, the accumulation of monocytes, lymphocytes, and fibrocytes, and collagen deposition, resulting in pulmonary fibrosis.

Article Snippet: 100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

Techniques: Expressing

Journal: Cellular Oncology

Article Title: Discover personalized drug opportunity and optimized tumor-infiltrating lymphocyte therapy for rare cancer: an organoid-based whole-journey clinical mapping study in a neuroendocrine cancer patient

doi: 10.1007/s13402-026-01172-y

Figure Lengend Snippet: Rebuilding autologous tumor antigen microenvironment using PTOs in TIL production generates optimized populations with enhanced anti-tumor ability. a Schematic diagram illustrating the optimization process of TIL production using PTOs. The different colors of TILs represent distinct TCR clones. b Representative bright-field and fluorescent-field images of M3 PTOs after co-culture with unsorted, CD137 − -sorted, or CD137 + -sorted TILs for 48 h. Scale bar: 100 μm. c Representative bright-field and fluorescent-field images showing disintegration of organoid structure (Upper panel), cell apoptosis (Middle panel), and immune cell infiltration (Lower panel) in M3 PTOs after co-culture with CD137 + -sorted TILs for 48 h, compared to the untreated group and the MHC-Blocker (MHC-B) rescue group. Scale bar: 100 μm. d Live cell flow cytometry analysis of residual M3 PTOs after co-culture with unsorted, CD137 − -sorted, and CD137 + -sorted TILs for 24 h, compared to the untreated group and the MHC-Blocker rescue group (MHC-B rescue). Data are shown as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (ANOVA). e Intracellular cytokine staining (ICS) flow cytometry analysis of unsorted, CD137 − -sorted, and CD137 + -sorted TILs against matched M3 PTOs. f ICS flow cytometry analysis of unsorted, CD137 − -sorted, and CD137 + -sorted TILs against autologous M3 tumor cells. g Schematic diagram of tumor-specific TIL molecular characterization strategy based on single-cell transcriptomics and TCR analysis. h Unsupervised UMAP plot displaying T cells from fresh tumor, preREP TILs, and TIL product of the NECC patient based on single-cell RNA sequencing. i Distribution of putative tumor-specific TILs in fresh tumor, preREP TILs, and TIL product of the NECC patient based on TCR sequencing analysis. j Immune cell phenotype of tumor-specific TILs in PTO-trained TILs, fresh tumor, preREP TILs, and TIL product of the NECC patient. k Number of tumor-specific TCR β-chains in TIL product and in the patient’s peripheral blood before and after TIL infusion. l Proportion of tumor-specific TCR β-chains in TIL product and in the patient’s peripheral blood before and after TIL infusion

Article Snippet: On day 2, PTO-specific CD137 expression was confirmed via flow cytometric analysis, and CD137 + cells were isolated using a CD137 MicroBead kit according to the manufacturer’s instructions (Miltenyi).

Techniques: Clone Assay, Co-Culture Assay, Flow Cytometry, Staining, Single-cell Transcriptomics, Single Cell, RNA Sequencing, Sequencing

Journal: Cell

Article Title: Intratumoral CD4 + T Cells Mediate Anti-tumor Cytotoxicity in Human Bladder Cancer

doi: 10.1016/j.cell.2020.05.017

Figure Lengend Snippet: Flow Cytometry and Immunofluorescence Validation of T Cell Phenotypes in Bladder Tumors, Related to , , , , and (A) Schematic of processing for paired tumor and adjacent non-malignant tissue from either anti-PD-L1-treated, or standard-of-care (untreated/chemotherapy-treated) cystectomy patients. FACS-sorted CD4 + or CD8 + T cells were subjected to droplet-based single-cell RNA sequencing (dscRNA-seq) with paired T cell receptor (TCR) sequencing as described in the text. (B) Parallel flow cytometry data from the same single-cell digest used for dscRNA-seq from 4 anti-PD-L1-treated tumors, showing the percentage of CD4 + or CD8 + T cells from total CD3 + cells. (C) Gating strategy for flow cytometric analysis of populations in CD4 + and CD8 + T cells from RNA-seq. CD4 + and CD8 + populations were gated out of CD3 + CD45 + single live cells. CD4 + cells were further gated as FoxP3 - and FoxP3 + . Treg cells are gated as FOXP3 + CD25 + cells. FOXP3 - CD4 + and CD8 + cells were gated into central memory (CM, CCR7 + CD45RA - ), and CCR7 - cells (a combination of effector memory CCR7 - CD45RA - and effector CCR7 - CD45RA + ). Boolean gating of CCR7 - cells was used to obtain GZMK + , GZMB + and Ki67 + populations for further marker analysis. Plots are shown here to demonstrate the presence of these populations. (D) Representative gates shown for each marker for CD4 + and CD8 + T cells were used for Boolean gating for the populations described above. (E) Flow cytometry staining of GZMB, GZMK, or perforin versus CD3 in CCR7 - CD8 + T cells. Gates used for Boolean analysis are shown. (F) Flow cytometry staining of GZMB or GZMK co-expression with perforin in CCR7 - CD8 + T cells. (G) Percentage of cells expressing GZMB, GZMK, or perforin from CCR7 - CD8 + T cells by flow cytometry (left), and the percentage of cells co-expressing perforin within GZMB + or GZMK + CCR7 - CD8 + T cells (right), are shown (N = 7 tumors, mean + SEM). (H) Percentages of cells expressing IFNγ, TNFα, or both from GZMB + or GZMK + CCR7 - CD8 + T cells with and without stimulation (N = 11 tumors, mean + SEM). (I) Multiplex immunofluorescent staining of DAPI (blue), CD4 (red), GZMK (green), GZMB (white) and overlay without DAPI are shown from a cystectomy tumor region from an additional patient with parallel scRNA-seq and TCR-seq data (anti-PD-L1 D). CD4 + cells that co-express GZMK (arrows) or GZMB (arrowhead) are indicated. Scale bar, 10 μm. (J) Percentage of cells co-expressing Ki67 and either GZMB or GZMK from CCR7 - CD4 + FOXP3 - T cells (left), or Ki67 and CD25 from CD4 + FOXP3 + T cells (right), by flow cytometry are shown, with dots for values from individual tumors (N = 7 tumors, mean ± SEM). (K) Flow cytometry staining showing co-expression of GZMB and Ki67, or GZMK and Ki67, from CCR7 - CD8 + T cells.

Article Snippet: Dynabeads Human T-Activator CD3/CD28/CD137 , GIBCO , Cat# 11162D.

Techniques: Flow Cytometry, Immunofluorescence, RNA Sequencing Assay, Sequencing, Marker, Staining, Expressing, Multiplex Assay

Journal: Cell

Article Title: Intratumoral CD4 + T Cells Mediate Anti-tumor Cytotoxicity in Human Bladder Cancer

doi: 10.1016/j.cell.2020.05.017

Figure Lengend Snippet: Bladder Cancer Contains Canonical CD8 + T Cell States (A) Uniform manifold approximation and projection (UMAP) plots of 10,762 single sorted CD3 + CD8 + T cells obtained from bladder tumors and adjacent non-malignant tissue (N = 7 patients). Phenotypic clusters are represented in distinct colors. (B) Relative intensity of expression of select genes superimposed on the UMAP projections in (A). (C) Violin plots showing the relative expression of select differentially expressed genes (columns) for each cluster shown in (A) (rows) (all P adj < 0.05). (D) The frequency of cells expressing MAIT-associated TRAV1-2/TRAJ33 + TCRs within each defined CD8 + phenotypic cluster. (E) The frequency of cells in individual clusters shown as a proportion of total CD8 + cells within tumor or non-malignant compartments across all patients (orange, tumor; blue, non-malignant). For each cluster, a box and whisker plot is shown with the median, interquartile range (IQR, a box with lower and upper bounds representing 25 th and 75 th percentiles, respectively), and 1.5 times the IQR (whiskers). Outlier points are shown if more than 1.5 times the IQR beyond the lower and upper quartiles. Statistical testing was done using an exact permutation test. (F) Density plots showing distribution of cells in tumor or non-malignant samples.

Article Snippet: Dynabeads Human T-Activator CD3/CD28/CD137 , GIBCO , Cat# 11162D.

Techniques: Expressing, Whisker Assay

Journal: Cell

Article Title: Intratumoral CD4 + T Cells Mediate Anti-tumor Cytotoxicity in Human Bladder Cancer

doi: 10.1016/j.cell.2020.05.017

Figure Lengend Snippet: CD4 + T Cells in Bladder Tumors Are Composed of Multiple Distinct Functional States (A) UMAP plots of 19,842 single sorted CD3 + CD4 + T cells obtained from bladder tumors and adjacent non-malignant tissue (N = 7 patients). Each distinct phenotypic cluster identified using Leiden clustering is identified with a distinct color. Annotation of each unbiased cluster was performed by manual inspection of the highest-ranked differentially expressed genes for each cluster and using reference signature-based correlation methods (SingleR) as described in the text. (B) Relative intensity of expression of select genes superimposed on the UMAP projections shown in (A). (C) Violin plot showing relative expression of select differentially expressed genes (columns) for each cluster shown in (A) (rows) (all P adj < 0.05). (D) Density plots showing distribution of cells in tumor or non-malignant samples. (E) The frequency of cells in individual CD4 + T cell states defined by scRNA-seq clustering is shown as a proportion of total CD4 + cells within either tumor or non-malignant compartments across all patients (orange, tumor; blue, non-malignant). A box and whisker plot is shown with formatting as in E. ∗ p < 0.05, ∗∗ p < 0.01 by exact permutation test.

Article Snippet: Dynabeads Human T-Activator CD3/CD28/CD137 , GIBCO , Cat# 11162D.

Techniques: Functional Assay, Expressing, Whisker Assay

Journal: Cell

Article Title: Intratumoral CD4 + T Cells Mediate Anti-tumor Cytotoxicity in Human Bladder Cancer

doi: 10.1016/j.cell.2020.05.017

Figure Lengend Snippet:

Article Snippet: Dynabeads Human T-Activator CD3/CD28/CD137 , GIBCO , Cat# 11162D.

Techniques: Recombinant, Staining, Cell Culture, Cell Stimulation, Multiplex Assay, Sequencing, Software