Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Protein S variants generated by site-directed mutagenesis

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Generated

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Effect of APC and protein S on thrombin generation. Thrombin generation was performed in protein S–deficient plasma with 100nM inhibitory antibodies against TFPI. Up to 10nM APC had no effect on thrombin generation in the absence of protein S. All concentrations generate lines that are superimposable (A). After addition of 120nM protein S (at 0-10nM APC), an APC dose-dependent effect was observed (B). The top single line represents 0 to 10nM APC in the absence of protein S. Protein S in the presence of no or 2.5nM APC generated lines that were superimposable. Conditions used are noted adjacent to the peaks to which they refer. The anticoagulant effect of 10nM APC and 120nM protein S was inhibited by polyclonal antibodies against protein S (C) or against protein C (D). PS indicates protein S; PC, protein C. Representative experiments are shown (n = 3).

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Generated

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Screening of protein S variants for APC cofactor activity. The APC cofactor activity of protein S was evaluated at 16nM APC and 100nM protein S by CAT. The peak height in the absence of protein S was set to 100%. A high concentration of APC, leading to almost complete inhibition of thrombin generation with 100nM WT protein S, was chosen specifically for screening purposes as this allows widening of the assay window at which mutants with reduced APC cofactor activity are visualized. Results were confirmed by evaluating protein S (at 60 and 90nM) cofactor activity toward 4 or 9nM APC.

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Activity Assay, Concentration Assay, Inhibition

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Effect of WT protein S, D95A, D95N, D78A, and Q79A variants on thrombin generation. Thrombin generation was measured in protein S–deficient plasma supplemented with 9nM APC, 100nM antibodies against TFPI, and 0 to 120nM WT protein S (A), protein S D95A (B), protein S D95N (C), or 90nM purified WT (dashed line) or purified protein S D95A (dotted line; D). Protein S concentrations are positioned adjacent to the peaks to which they refer. The cofactor activity of 60nM WT protein S and protein S variants D95A, D78A, and Q79A was compared at 9nM APC (E). Typical experiments are shown (n = 3). Whereas the cofactor activity of WT protein S is highly dependent on the APC concentration used (Figure 1B), that of protein S D95A is not, explaining the difference in fold activity between WT protein S and protein S D95A in Figures 2 and ​and3.3. Dose-response data from titrations with WT protein S, protein S D95A, and protein S D95N in the presence of 9nM APC are shown in panel F (data are expressed as mean ± SD of 2 independent experiments performed in duplicate). Inset in panel B shows recognition of WT protein S and protein S D95A in media by polyclonal antibodies and a monoclonal antibody recognizing only γ-carboxylated Gla domains. Inset in panel D shows the SeeBlue-prestained marker, plasma-purified protein S from Enzyme Research Laboratories Ltd (lane 1), purified recombinant WT protein S (lane 2), and purified protein S D95A (lane 3) visualized with silver staining.

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Purification, Activity Assay, Concentration Assay, Marker, Recombinant, Silver Staining

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Binding of protein S to phospholipid surfaces. Protein S (0-120nM) was incubated in a plate coated with 25 μg/mL phospholipids. Bound protein S was detected with an HRP-conjugated polyclonal antibody against protein S. A representative experiment is shown. The apparent Kd values, 5.69 ± 1.24 and 9.54 ± 2.26nM for WT protein S and protein S D95A, respectively, were obtained by calculating the mean ± SD of 3 independent experiments performed in duplicate. PL indicates phospholipids.

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Binding Assay, Incubation

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Binding of protein S to phospholipids and domain-specific monoclonal antibodies

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Binding Assay, Mutagenesis

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Protein S enhancement of APC-mediated cleavage of FVa in Arg306. Protein S (0-120nM) in the presence of 0.5nM APC was incubated with 0.8nM FVa R506Q/R679Q in the presence of phospholipids for 10 minutes. The remaining FVa actvity was measured with a prothrombinase assay. Results are plotted as mean ± SD from 3 independent experiments performed in duplicate (A). A time course experiment was performed to calculate the apparent pseudo–first-order rate constants of WT protein S and protein S D95A. It is observed that approximately 6-fold more APC is needed in the presence of protein S D95A to obtain a similar amount of APC-mediated FVa R506Q/R679Q inactivation as with WT protein S (B).

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Incubation

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Location of Asp78, Gln79, and Asp95 within the protein S Gla-TSR-EGF1 model. Domains are labeled on the right side of the model. Residues mutated in the GLA2 variant, Asp78, Gln79, and Asp95, are in light gray on the left side surface model. Residues Asp78, Gln79, and Asp95 are highlighted by the box to show their proximal spatial location. The model is taken from Villoutreix et al.35

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Labeling, Variant Assay

Journal: Scientific Reports

Article Title: Unveiling novel insights into human IL-6 − IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis

doi: 10.1038/s41598-024-69429-w

Figure Lengend Snippet: Affinity kinetics between hIL-6 mutants and hIL-6R − gp130 assayed by SPR. gp130 was immobilized onto a CM5 chips via standard amine coupling and different concentrations of hIL-6 mutants with a saturated concentration of hIL-6R were pre incubated and the mixture were injected. Affinity kinetics were analyzed by the Langmuir’s 1:1 model fit on seven serial dilutions of hIL-6 mutants from 250 nM and flow speed of 30 μL/min. ( A ) Schematic diagram of binding affinity between hIL-6 mutants and hIL-6R − gp130 by SPR; ( B – K ) representative examples of curve fits for the affinity kinetic analysis of hIL-6 mutants.

Article Snippet: The assay was optimized according to the description provided in reference . monomeric gp130 (Sino Biological, Beijing, China, Cat. 10974-HCCH1) was immobilized on CM5 sensor chips (GE Healthcare) by standard amine coupling method, as recommended by the manufacturer.

Techniques: Concentration Assay, Incubation, Injection, Binding Assay

Journal: Scientific Reports

Article Title: Unveiling novel insights into human IL-6 − IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis

doi: 10.1038/s41598-024-69429-w

Figure Lengend Snippet: Affinity of the interaction between hIL-6/mutants and hIL-6R/gp130 by Biacore SPR.

Article Snippet: The assay was optimized according to the description provided in reference . monomeric gp130 (Sino Biological, Beijing, China, Cat. 10974-HCCH1) was immobilized on CM5 sensor chips (GE Healthcare) by standard amine coupling method, as recommended by the manufacturer.

Techniques: Mutagenesis, Binding Assay

Journal: Scientific Reports

Article Title: Unveiling novel insights into human IL-6 − IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis

doi: 10.1038/s41598-024-69429-w

Figure Lengend Snippet: The spatial arrangement of amino acid residues hIL-6 R167 and E171 in the hexameric hIL-6 − hIL-6R − gp130 complex (PDB code: 1p9m). ( A ) Close spatial proximity is observed between hIL-6 R167 and E171 and the hIL-6 AB-loop; ( B ) the potential influence of hIL-6 R167 on the interaction between hIL-6 K53 and hIL-6R Q95 is indicated. The hIL-6 molecule is shown in brown, hIL-6R in purple, and gp130 in green.

Article Snippet: The assay was optimized according to the description provided in reference . monomeric gp130 (Sino Biological, Beijing, China, Cat. 10974-HCCH1) was immobilized on CM5 sensor chips (GE Healthcare) by standard amine coupling method, as recommended by the manufacturer.

Techniques:

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Cross-talk Between Glia, Neurons and Mast Cells in Neuroinflammation Associated with Parkinson’s Disease

doi: 10.1007/s11481-017-9766-1

Figure Lengend Snippet: MPP+ and mast cell proteases-induce neuronal degeneration. Primary mouse neurons from Wt mice and GMF-KO mice fetal brains were cultured for 2 weeks. These cells were incubated with MPP+ (10 μM) or mast cell proteases (MMCP-6 or MMCP-7 at 100 ng/ml) for 24 h. MAP-2 ICC was performed to analyze the neuronal morphology. (A) MPP+, MMCP-6 and MMCP-7-induced significant neuronal degeneration (arrows) in the neurons obtained from Wt mice fetal brains as compared to non-treated control neuronal cells. (B) GMF-KO neurons showed less neuronal degeneration when compared to the extent of neurodegeneration observed in the neurons grown from Wt mice fetal brains. Control neuronal cells did not show any degeneration. Scale bar = 100 μm. (C, D) The total neuronal outgrowth length was measured in the whole photomicrographs using MetaMorph software to determine the neuronal degeneration. MPP+, MMCP-6 and MMCP-7 significantly reduced the total neurite length as compared with control cells as shown in the bar graphs (One-way ANOVA and Tukey-Kramer post hoc, n=3, *p<0.05). Neurodegeneration is relatively higher in Wt neurons than in GMF-KO neurons.

Article Snippet: Recombinant mouse mast cell protease-6/Mcpt6, Recombinant mouse tryptase beta-1/Mcpt7, tryptase/BSSP-4, Enzyme-linked immunosorbent assay (ELISA) kits for mouse CCL2, MMP-3, tryptase/BSSP-4, monoclonal anti-mouse CD40L/TNFSF5 Phycoerythrin conjugated Rat IgG2A antibodies and flow cytometry reagents were purchased from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Incubation, Software

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Cross-talk Between Glia, Neurons and Mast Cells in Neuroinflammation Associated with Parkinson’s Disease

doi: 10.1007/s11481-017-9766-1

Figure Lengend Snippet: MMCP-6 -induces CCL2 release from Wt mouse glia/neurons. (A) Wt mouse glia/neurons were incubated with MMCP-6 for dose-response (5 to 200 ng/ml) and time-course studies (6 to 48 h), and CCL2 levels in the culture media were determined by ELISA (n=3). MMCP-6 induced significant CCL2 release from 6 h (at 25 ng/ml concentration) onwards from glia/neurons. Results were presented as mean ± SEM (*p<0.05 control vs MMCP-6 treated cells, One-way ANOVA and Tukey-Kramer post hoc). (B) Wt glia/neurons were incubated with MMCP-7 (100 ng/ml) for 24 h and CCL2 release was measured by ELISA (n=5). MMCP-7 induced significant CCL2 release from glia/neurons as compared to untreated control cells (*p<0.05 control vs cells incubated with MMCP-7, t-test).

Article Snippet: Recombinant mouse mast cell protease-6/Mcpt6, Recombinant mouse tryptase beta-1/Mcpt7, tryptase/BSSP-4, Enzyme-linked immunosorbent assay (ELISA) kits for mouse CCL2, MMP-3, tryptase/BSSP-4, monoclonal anti-mouse CD40L/TNFSF5 Phycoerythrin conjugated Rat IgG2A antibodies and flow cytometry reagents were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Cross-talk Between Glia, Neurons and Mast Cells in Neuroinflammation Associated with Parkinson’s Disease

doi: 10.1007/s11481-017-9766-1

Figure Lengend Snippet: MMCP-6 on CCL2 release from GMF-KO mouse glia/neurons. Glia/neurons obtained from GMF-KO mice were incubated with MMCP-6 (100 ng/ml) for 6, 24 and 48 h. CCL2 release was measured in the culture media by ELISA (n=4). MMCP-6 significantly released CCL2 only at 48 h when compared to untreated control cells. Results were presented as mean ± SEM (*p<0.05 control vs MMCP-6 treated cells, t-test).

Article Snippet: Recombinant mouse mast cell protease-6/Mcpt6, Recombinant mouse tryptase beta-1/Mcpt7, tryptase/BSSP-4, Enzyme-linked immunosorbent assay (ELISA) kits for mouse CCL2, MMP-3, tryptase/BSSP-4, monoclonal anti-mouse CD40L/TNFSF5 Phycoerythrin conjugated Rat IgG2A antibodies and flow cytometry reagents were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Cross-talk Between Glia, Neurons and Mast Cells in Neuroinflammation Associated with Parkinson’s Disease

doi: 10.1007/s11481-017-9766-1

Figure Lengend Snippet: MMCP-6 and MMCP-7-induce CCL2 release from Wt mouse astrocytes. Wt mouse astrocytes were grown and stimulated with MMCP-6 and MMCP-7 for 6 h and 24 h, and CCL2 release was measured in the culture media by ELISA (n=3–8). Both (A) MMCP-6 and (B) MMCP-7 induced CCL2 release in a dose-dependent manner from mouse astrocytes when compared with non-treated control cells. Results were presented as mean ± SEM (*p<0.05 control vs MMCP-6 or MMCP-7 treated cells, One-way ANOVA and Tukey-Kramer post hoc).

Article Snippet: Recombinant mouse mast cell protease-6/Mcpt6, Recombinant mouse tryptase beta-1/Mcpt7, tryptase/BSSP-4, Enzyme-linked immunosorbent assay (ELISA) kits for mouse CCL2, MMP-3, tryptase/BSSP-4, monoclonal anti-mouse CD40L/TNFSF5 Phycoerythrin conjugated Rat IgG2A antibodies and flow cytometry reagents were purchased from R&D Systems (Minneapolis, MN).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Cross-talk Between Glia, Neurons and Mast Cells in Neuroinflammation Associated with Parkinson’s Disease

doi: 10.1007/s11481-017-9766-1

Figure Lengend Snippet: MMCP-6, MMCP-7, and MPP+ -induce CCL2 release from mouse neurons. We examined whether MMCP-6, MMCP-7 and MPP+ activate mouse neurons to release CCL2. Mouse neuronal cells grown from Wt mice as well as from GMF-KO mice were incubated with MMCP-6 (200 ng/ml), MMCP-7 (200 ng/ml) or MPP+ (20 μM) for 24 h and the CCL2 release was assayed in the culture media by ELISA (n=3). Neurons obtained from Wt mice significantly released CCL2 after incubation with MMCP-6, MMCP-7, and MPP+ when compared to control non-treated cells. However, only MPP+ significantly released CCL2 from neuronal cultures obtained from GMF-KO mice. Results were presented as mean ± SEM (*p<0.05 control vs treated cells, One-way ANOVA and Tukey-Kramer post hoc).

Article Snippet: Recombinant mouse mast cell protease-6/Mcpt6, Recombinant mouse tryptase beta-1/Mcpt7, tryptase/BSSP-4, Enzyme-linked immunosorbent assay (ELISA) kits for mouse CCL2, MMP-3, tryptase/BSSP-4, monoclonal anti-mouse CD40L/TNFSF5 Phycoerythrin conjugated Rat IgG2A antibodies and flow cytometry reagents were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Cross-talk Between Glia, Neurons and Mast Cells in Neuroinflammation Associated with Parkinson’s Disease

doi: 10.1007/s11481-017-9766-1

Figure Lengend Snippet: GMF, MPP+, MMCP-6, MMCP-7 and tryptase release MMP-3 from Wt brain cells or mast cells. (A, B) BMMCs grown from Wt mice were incubated with GMF (100 ng/ml) or MPP+ (20 μM) for 24 h and MMP-3 release in the cell culture media was measured by ELISA. (A) GMF (n=3) and (B) MPP+ (n=4) significantly increased the release of MMP-3 when compared with non-treated control BMMCs (*p<0.05; t test, control vs treated). (C) Wt mouse BMMCs plus glia/neurons incubated with MPP+ significantly induced MMP-3 release as compared to non-treated control cells (n=3, *p<0.05; t test, control vs treated). (D) Wt mouse glia/neurons were then incubated with MPP+ (15 μM) or GMF (100 ng/ml) for 24 h and the release of MMP-3 was measured in the culture media (n=3). MPP+ and GMF significantly induced the release of MMP-3 from glia/neurons. Wt glia/neurons and BMMCs were co-cultured and then stimulated with GMF. GMF augments the release of MMP-3 in this co-culture system as compared to single glia/neurons culture condition (*p<0.05; t test, control vs treated). (E, F, G) Wt mouse astrocytes or glia/neurons were incubated with MMCP-6, MMCP-7, MPP+ or tryptase/BSSP-4 and the release of MMCP-3 in the culture media was measured by ELISA. MMCP-7 (n=6), MMP-6 (n=6), MPP+ (n=6) and tryptase/BSSP-4 (n=3)-induced significantly increased MMP-3 release as compared to control cells. Results were presented as mean ± SEM (*p<0.05 control vs treated cells, One-way ANOVA and Tukey-Kramer post hoc).

Article Snippet: Recombinant mouse mast cell protease-6/Mcpt6, Recombinant mouse tryptase beta-1/Mcpt7, tryptase/BSSP-4, Enzyme-linked immunosorbent assay (ELISA) kits for mouse CCL2, MMP-3, tryptase/BSSP-4, monoclonal anti-mouse CD40L/TNFSF5 Phycoerythrin conjugated Rat IgG2A antibodies and flow cytometry reagents were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

Journal: Nature Communications

Article Title: Improved safety of chimeric antigen receptor T cells indirectly targeting antigens via switchable adapters

doi: 10.1038/s41467-024-53996-7

Figure Lengend Snippet: a Schematic diagram of the murine CD40 CAR construct. EC, extracellular; TM, transmembrane; Cyt, cytoplasmic domain. b Representative flow cytometry plot of CD40 CAR expression on mouse T cells 4 days after transduction. c Cytotoxicity of CD40 CAR-T cells against A20 cells. d IFN-γ production after co-culture of control T cells or CD40 CAR-T cells with A20 cells for 24 h, measured in triplicate. Results are representative of three independent experiments ( c , d ). e – g Balb/C mice were injected i.v . with A20-Luc cells (1 × 10 6 ) on day 0, irradiated (2.5 Gy) for lymphodepletion on day 6, and injected i.v . with control T or CD40 CAR-T cells (5 × 10 6 ) on day 7. Body weight change ( e , n = 5) and survival ( f , n = 10) were measured. Serum levels of IL-6 were measured three days after T cell injection ( g , n = 9). Each dot represents the value of a single mouse. h – j The same experiments as in e – g were performed including the groups not injected with A20-Luc cells. Body weight change ( h , n = 5), survival ( i , n = 5), and serum levels of IL-6 ( j , n = 7) were measured. k – m Wild-type and CD40 knockout B6 mice were irradiated (3 Gy) on day 6 and injected i.v . with CD40 CAR-T cells (5 × 10 6 ) on day 7. Body weight change ( k , n = 5), survival ( l , n = 10), and serum levels of IL-6 ( m , n = 9) were measured. Measurements of body weight were halted when the mice began to die (red asterisks in e , h , k ). Data in ( e , h , i , and k ) are representative of at least two independent experiments. Data in ( f , g , j , l , and m ) are pooled from two replicate experiments. Data in ( d , e , g , h , j , k , and m ) are presented as mean ± SEM. Statistical significance was determined by either the log-rank (Mantel-Cox) test ( f , i , l ) or the unpaired two-tailed t test ( d , g , j , m ). p : p -value, n.s.: not significant. Source data are provided in the Source Data file.

Article Snippet: Briefly, serial diluents of anti-CD40 scFv-Cκ or irrelevant scFv -Cκ proteins were loaded into the wells of the 96-well plates coated with mouse CD40-Fc (1215-CD; R&D Systems, USA) or human CD40-Fc (1493-CDB; R&D Systems) proteins.

Techniques: Construct, Flow Cytometry, Expressing, Transduction, Co-Culture Assay, Control, Injection, Irradiation, Knock-Out, Two Tailed Test

Journal: Nature Communications

Article Title: Improved safety of chimeric antigen receptor T cells indirectly targeting antigens via switchable adapters

doi: 10.1038/s41467-024-53996-7

Figure Lengend Snippet: a Production of IL-6 or IL-1β after co-culture of CD40 CAR-T cells with A20 or peritoneal macrophages (Mφ) for 24 h, measured in triplicate. Results are representative of three independent experiments. b – f Balb/C mice were irradiated (2.5 Gy) for lymphodepletion on day -1 and injected with 5 × 10 6 ( b , d , f ) or 1 × 10 6 ( c , e ) control T cells or CD40 CAR-T cells on day 0. IL-6 or IL-1β neutralization ( b , c ) and phagocyte depletion ( d – f ) were performed as described in “Methods”. Body weight change ( b – e , n = 5) and serum levels of IL-6 ( f , n = 5) were measured. Each dot in ( f ) represents the value of a single mouse. *: p = 0.0211 on day 5, p = 0.0187 on day 8 ( c ), and p = 0.0029 on day 8 ( e ), compared to CD40 CAR‐T group. g , h BM chimeras were established (e.g., CD40-/-»WT denotes the transfer of donor BMs from CD40-knockout mice to B6 wild-type recipients). After 8 weeks, chimeric mice were irradiated (2.5 Gy) on day -1 and injected with CD40 CAR-T cells (5 × 10 6 ) on day 0. Body weight change ( g , n = 5) and survival ( h , n = 10) were measured. p for WT»CD40-/- versus WT»WT ( h ). i Balb/C mice were irradiated (2.5 Gy) on day -1 and injected with GFP-efflux T (control-Luc-T) or CD40 CAR-efflux T cells (CD40 CAR-Luc-T) (5 or 1 × 10 6 ) on day 0. In vivo, the distribution of injected T cells was visualized by bioluminescence imaging. j Histology of lung and liver was examined by H&E staining 3 and 10 days after CAR-T (1 × 10 6 ) injection (100× magnification, scale bar: 200 μm). Measurements of body weight were halted when the mice began to die (red asterisks in b , d , g ). Data in ( h ) are pooled from two replicate experiments. All other data are representative of at least two independent experiments. Data in a – g are presented as mean ± SEM. Statistical significance was determined by 1-way ANOVA ( c , e ), unpaired two-tailed t test ( f ), or log-rank (Mantel-Cox) test ( h ). p : p -value, n.s.: no t signi f icant. Source data are provided in the Source Data file.

Article Snippet: Briefly, serial diluents of anti-CD40 scFv-Cκ or irrelevant scFv -Cκ proteins were loaded into the wells of the 96-well plates coated with mouse CD40-Fc (1215-CD; R&D Systems, USA) or human CD40-Fc (1493-CDB; R&D Systems) proteins.

Techniques: Co-Culture Assay, Irradiation, Injection, Control, Neutralization, Knock-Out, In Vivo, Imaging, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Improved safety of chimeric antigen receptor T cells indirectly targeting antigens via switchable adapters

doi: 10.1038/s41467-024-53996-7

Figure Lengend Snippet: a Scheme of conventional CAR-T and switchable CAR-T cells. b Schematic diagram of the murine Cot CAR construct and a representative flow cytometry plot of Cot CAR expression on mouse T cells 4 days after transduction. c Chemical structure of carboxy cotinine (top left). Schematic diagram of cotinine-labeled anti-mouse CD40 scFv-Cκ (C1C02-Cot) (top right). Representative flow cytometry plot of binding of C1C02-Cot to A20 cells (bottom). d Cytotoxicity of Cot CAR-T cells against A20 cells preincubated with C1C02-Cot. e IFN-γ production after co-culture of Cot CAR-T cells with A20 cells preincubated with C1C02-Cot for 24 h, measured in triplicate. Data are presented as mean ± SEM. Statistical significance was determined by an unpaired two-tailed t test. Results are representative of three independent experiments ( d , e ). f CD40 expression levels in A20 cells and F4/80 (+) peritoneal macrophages (Mφ) as determined by staining with a commercially available anti-mouse CD40 antibody. g , h Comparison of dose-dependent cell binding affinity of C1C02-Cot between A20 and macrophage. Mean fluorescence intensities (MFIs) of binding are shown as values within the plot ( g ) and as a graph ( h ). i For toxicity readouts, macrophages were preincubated with various concentrations of C1C02-Cot and co-cultured with Cot CAR-T or CD40 CAR-T cells for 24 h. The amount of IL-6 in culture supernatants was measured (mean ± SEM, left axis and blue lines). For efficacy readouts, A20 cells (target) were preincubated with various concentrations of C1C02-Cot and then co-cultured with Cot CAR-T cells (effector) at an E:T ratio of 5:1 for 6 h. CD40 CAR-T cells were co-cultured with target cells not treated with C1C02-Cot. Percent cytotoxicity was calculated from flow cytometry-based viable cell counting (right axis and red lines). Results in g – i are representative of at least two independent experiments. p : p -value. Source data are provided in the Source Data file.

Article Snippet: Briefly, serial diluents of anti-CD40 scFv-Cκ or irrelevant scFv -Cκ proteins were loaded into the wells of the 96-well plates coated with mouse CD40-Fc (1215-CD; R&D Systems, USA) or human CD40-Fc (1493-CDB; R&D Systems) proteins.

Techniques: Construct, Flow Cytometry, Expressing, Transduction, Labeling, Binding Assay, Co-Culture Assay, Two Tailed Test, Staining, Comparison, Fluorescence, Cell Culture, Cell Counting

Journal: Nature Communications

Article Title: Improved safety of chimeric antigen receptor T cells indirectly targeting antigens via switchable adapters

doi: 10.1038/s41467-024-53996-7

Figure Lengend Snippet: a Experimental scheme for the treatment of murine B-cell lymphoma using syngeneic Cot CAR-T cells. Balb/C mice were injected i.v . with A20-Luc cells (1 × 10 6 ) on day 0, irradiated (2.5 Gy) for lymphodepletion on day 6, and injected with Cot CAR-T cells (5 × 10 6 ) on day 7. From the day of CAR-T cell injection, C1C02-Cot (20 μg/head) was injected i.v . every other day for a total of 8 times. b Body weight change ( n = 5) and survival ( n = 5) were measured. Measurements of body weight were halted when the mice began to die (red asterisk). c Serum levels of IL-6 were measured 3 days after CAR-T injection ( n = 5). Each dot represents the value of a single mouse. Statistical significance was determined by log-rank (Mantel-Cox) test ( b ) and unpaired two-tailed t test ( c ). d , e Bioluminescence imaging of tumor burden at indicated time points after A20-Luc cell injection ( d ). Bioluminescence intensity is calculated as the mean flux (p/s/cm 2 /sr) of a region of interest (ROI) in an individual mouse. Statistical significance between groups at each time point ( n = 5) was determined by the nonparametric Kruskal-Wallis test ( e ). *: p = 0.0172 on day 14, p = 0.011 on day 21, and p = 0.0115 on day 28, compared to A20 only group. f In vivo CAR-T tracing using luciferase-expressing CAR-T cells and bioluminescence imaging. Balb/C mice were injected s.c . on the back with A20 cells. When tumor mass was detectable at the injection site, mice were irradiated (2.5 Gy) and injected the next day with CD40 CAR-Luc-T cells or Cot CAR-Luc-T cells (5 × 10 6 ) with or without C1C02-Cot injection every other day. Bioluminescence imaging was performed at the indicated time points after CAR-T cell injection. Data in ( b , c , and e ) are presented as mean ± SEM. Results are representative of at least three ( b , d , e ) or two ( c , f ) independent experiments. p : p -value. Source data are provided in the Source Data file.

Article Snippet: Briefly, serial diluents of anti-CD40 scFv-Cκ or irrelevant scFv -Cκ proteins were loaded into the wells of the 96-well plates coated with mouse CD40-Fc (1215-CD; R&D Systems, USA) or human CD40-Fc (1493-CDB; R&D Systems) proteins.

Techniques: Injection, Irradiation, Two Tailed Test, Imaging, In Vivo, Luciferase, Expressing

Journal: mBio

Article Title: The GPI sidechain of Toxoplasma gondii inhibits parasite pathogenesis

doi: 10.1128/mbio.00527-24

Figure Lengend Snippet: Enhanced CD36 binding and slight macrophage tropism 3 hours after infection. Mice (C57BL/6J) were injected i.p. with 10 6 parasites of CEP, CEP Δ pigj, or CEP Δpigj + PIGJ and PECs were analyzed 3 hours later by flow cytometry. ( A ) Gating strategy for panels B–G. ( B ) Total GFP+ signal in the peritoneal exudate; GFP frequency of total events. ( C ) GFP+ events were separated by size using FSC and SSC parameters to determine extracellular or “non-cell-associated” and intracellular or “cell-associated” GFP+ parasites. ( D ) Frequency of PI+ (non-viable) cells of the indicated GFP+ category. ( E ) Frequency of cell-associated GFP+ events that were GR-1 hi Cd11b + neutrophils. ( F ) Frequency of cell-associated GFP+ events that were MHCII hi F4/80 lo “BMDM,” or ( G ) MHCII lo F4/80 hi “yolk sack-derived macrophages” (YSM). Plotted are seven total experiments; each dot represents a single mouse (mice; n = 12 CEP, n = 12 CEP Δ pigj , n = 8 CEP Δ pigj + PIGJ ). Statistics performed were one-way ANOVAs with multiple comparisons and a Tukey correction; none were significant, and the values were indicated. ( H ) In vitro differentiated BMDMs were plated on coverslips overnight and infected with 100 and 300 GFP+ parasites per well, and parasites were allowed 20 minutes to invade cells before being fixed and stained for fluorescence microscopy. No permeabilization was used, and SAG1 staining was performed. Intracellular parasites were not stained with SAG1 but were GFP+, while extracellular parasites are SAG1+GFP+ . The fraction of intracellular parasites is plotted, dots represent independent experiments; n = 3 experiments. ( I ) Parasites were incubated for 1 hour with recombinant CD36-Fc and CD36 binding was measured with anti-human-IgG-Daylight-550 via flow cytometry. Representative histogram and ( J ) average + SD MFI of CD36 binding from four experiments is plotted with each experiment represented as a dot, and an unpaired t-test is shown, ** P < 0.01. For I and J, also plotted are IgG Fc staining controls of the CEP strain (Fc control).

Article Snippet: Parasites (10 7 ) were incubated with 0.5 µg recombinant human IgG Fc (R&D Systems # 110-HG) or recombinant CD36-Fc (R&D Systems, #2519 CD) in 50 µL binding buffer (0.14 M NaCl, 2.5 mM CaCl 2 , 0.01 M HEPES [pH 7.4], 3% BSA) for 1 hour at 15°C.

Techniques: Binding Assay, Infection, Injection, Flow Cytometry, Derivative Assay, In Vitro, Staining, Fluorescence, Microscopy, Incubation, Recombinant, Control