cd38 fc chimera protein  (R&D Systems)

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: Generation of CRISPR-engineered CD38 KO /CD38-CAR human primary NK cells using Cas9/RNP and AAV. (A) Schemata of steps for CRISPR/RNP knockout of the CD38 gene and directed insertion of a CD38-CAR encoding DNA delivered by AAV6 vector with homology arms for CD38 targeting site. (B) CD38 (PE) and CAR (APC) expression levels measured by flow cytometry for binding of CD38 antigen, 7 days after stimulation. Constructs contain a 41BB signaling domain, a CD8α transmembrane domain/hinge, a CD3ζ stimulatory domain, and reversed orderings of light and heavy chain orientations. (C) Relative percentage and intensity of CD38-CAR expression (n = 10; mean ± standard deviation [SD]). (D) Fold expansion of WT and CD38-CAR NK cells over 12 days after activation with irradiated, modified mbIL21-K562 cells and IL-2 show no significant change from WT human NK cells (n = 10; mean ± SD). P values were calculated using a 2-way analysis of variance (ANOVA); ∗ P = .0332; ∗∗ P = .0021; ∗∗∗ P = .0002; ∗∗∗∗ P < .0001. (E) Cytotoxicity observed for V3 and V4 CD38 KO /CD38-CAR NK cells against high CD38-expressing MM (H929), BL (Raji), and AML (MV-11) (n = 5; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: CRISPR, Knock-Out, Plasmid Preparation, Expressing, Flow Cytometry, Binding Assay, Construct, Standard Deviation, Activation Assay, Irradiation, Modification

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: CD38-CAR NK cells exhibit enhanced cytotoxic function and cytokine secretion. CD38-CAR NK cells were tested against CD38-expressing AML, MM, and T-cell malignancies collected from patients at baseline. (A) NK and CD38-CAR NK killing of AML-1 (n = 3; mean ± SD). (B) NK and CD38-CAR NK killing of samples from patients with MM (MM-1 and MM-2; n = 1; mean ± SD). (C) NK and CD38-CAR NK killing of T-cell malignancies (hepatosplenic T-cell lymphoma and T-PLL [T-PLL-1 and T-PLL-2]; n = 1; mean ± SD). All cytotoxicity P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001. (D) Bio-Plex Pro Human Cytokine assay was performed on the supernatant of WT and CD38-CAR NK cells cocultured with CD38 + malignancies (n = 9; mean ± SD). P values were calculated using a paired Student t test; ∗ P = .05; ∗∗ P = .01; ∗∗∗ P = .001. GM-CSF, IFN-γ, MCP-1, MIP-1α, TNF-α.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Expressing, Cytokine Assay

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: ATRA upregulates CD38 expression on tumor cells and can enhance antitumor activity. (A) CD38 cell surface expression as measured by flow cytometry across the hematologic malignancies MM, AML, BL, and T-ALL after treatment with 10 nM of ATRA for 48 hours. (B) Mean fluorescence intensity (MFI) of CD38 expression on cell lines with and without ATRA treatment. (C) Cytotoxicity assays performed by coculturing WT and CD38-CAR NK cells against AML, MM, BL, and T-cell malignancies with and without 48-hour, 10-nM ATRA pretreatments. MM1S (n = 4), H929 (n = 4), AML-10 (n = 4), MV4-11 (n = 4), Raji (n = 4), Daudi (n = 4), and primary cells from patients with T-ALL (n = 3; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P <.01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Expressing, Activity Assay, Flow Cytometry, Fluorescence

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: Mass cytometry analysis shows combination ATRA and CD38-CAR NK-cell treatment decreases live AML population. Mass cytometry analysis was performed 24 hours after the coculture of a primary AML cell line (AML-1) with WT or CD38-CAR NK cells with and without 48-hour, 10-nM ATRA pretreatment. Eight distinct conditions were analyzed: WT NK cells alone (A), CD38-CAR NK cells alone (B), AML cells alone (C), AML plus WT NK cells (D), AML plus CD38-CAR NK cells (E), AML cells with ATRA (F), AML plus ATRA plus WT NK cells (G), and AML plus ATRA plus CD38-CAR NK cells (H). (I) Heat map of surface marker appearing on the live AML cells after treatment analyzed by mass cytometry. Original values are ln(x)-transformed. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage using ClustVis.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Mass Cytometry, Marker, Transformation Assay

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: CD38 KO /CD38-CAR T cells generated by Cas9/RNP and AAV6 show antitumor activity. (A) CD38 (PE) and CAR (APC) expression levels measured by flow cytometry. (B) Relative percentage of CD38-CAR expression (n = 5; mean ± SD). (C) Ten-day fold expansion of generated CD38-CAR T cells with matched donors (n = 4; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P = .0332; ∗∗ P = .0021; ∗∗∗ P = .0002; ∗∗∗∗ P < .0001. (D) Cytotoxicity of CD38 KO /CD38-CAR T cells toward H929, Raji, and MV4-11 cell lines (n = 4; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Generated, Activity Assay, Expressing, Flow Cytometry

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: AAVS1KO/CD38-CAR NK cells avoid fratricide. (A) CD38 (PE) and CAR (APC) expression levels measured by flow cytometry for the CD38-CAR inserted into different loci. All CAR NK cells were generated with matched donors. (B) Fold expansion of WT and CD38-CAR NK cells over 12 days (n = 6; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001. (C) Relative percentage of CD38-CAR expression (n = 6; mean ± SD). (D) Reverse-transcription qPCR was performed using CD38 primer probes to detect transcription of the CD38 gene in the WT and AAVS1KO/CD38-CAR NK cells. (E) CD38 expression measured by flow cytometry using a polyclonal anti-CD38 antibody.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Expressing, Flow Cytometry, Generated, Reverse Transcription

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: AAVS1KO/CD38-CAR NK cells display enhanced cytotoxicity and metabolism. CD38-CAR NK cells were tested against CD38-expressing AML, MM, and BL. (A) CD38-CAR NK-cell killing of CD38-expressing cell lines (n = 3; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001. (B) Oxygen consumption rate (OCR) for CD38-CAR NK cells. (C) Glycolytic capacity, measured by the extracellular acidification rate (ECAR), observed in CD38-CAR NK cells. (D) Spare respiratory capacity, a measure of the cell's ability to produce adenosine triphosphate (ATP) in response to stress, measured in CAR NK cells. (E) CAR NK-cell maximal respiration rates compared with WT.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Expressing

Journal: Arthritis research & therapy

Article Title: Abatacept downregulates Fcγ receptor I on circulating monocytes: a potential therapeutic mechanism in patients with rheumatoid arthritis.

doi: 10.1186/s13075-022-02886-8

Figure Lengend Snippet: Fig. 2 Downregulation of CD64/FcγRI on monocytes after exposure to abatacept, as shown by immunoblotting. Peripheral blood samples were obtained from 11 patients with RA and 5 controls and used for immunoblotting. The protein expression of CD64/FcγRI, CD80, CD86, CD32a/FcγRIIa, CD32b/FcγRIIb, and CD16/FcγRIII in whole-cell extracts of monocytes cultured for 24 h in the absence (mock) or presence of abatacept or CD28-Ig. A representative immunoblot image showing a patient with RA in the right panel with the positions of molecular weight markers (A). The signal intensity of individual protein bands was quantified using the ImageJ software and is shown as the intensity ratio, which was calculated by dividing the intensity of the band of interest by the intensity of the β-actin band (B). The results are shown in boxplots, and the statistical comparisons between the two groups were made using the Mann–Whitney U test

Article Snippet: Highly purified CD14+ monocytes were resuspended in RPMI 1640 (Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum and were cultured in 5% CO2 for 24 h in the absence (mock) or presence of abatacept (10 μg/mL; BristolMyers Squibb, NJ, USA) or recombinant human CD28 Fc chimera (CD28-Ig) (10 μg/mL; R&D systems, MN, USA).

Techniques: Western Blot, Expressing, Cell Culture, Molecular Weight, Software, MANN-WHITNEY