Human CTLA4-expressing Raji Cells

Human CTLA-4-expressing B cells

Raji-hCTLA4 cells were developed from the Raji cell line, a human B lymphocyte-derived cell line. Raji cells have been successfully used as target cells in CAR-T toxicity assays as well as in human effector studies such as antibody-dependent cellular cytotoxicity (ADCC), either with peripheral blood mononuclear cells, natural killer (NK) cells, or T cell-derived Jurkat reporter cells.

CTLA-4 (cytotoxic T cell lymphocyte antigen-4; also known as CD152) is a type I transmembrane protein expressed at the cell surface of activated conventional T cells and constitutively on immunosuppressive regulatory T cells (Tregs). CTLA-4 is an inhibitory immune checkpoint that prevents T-cell overstimulation and host damage. It exerts competitive binding for stimulatory CD28 ligands (CD80/CD86) [1].

Features of Raji-hCTLA4 cells:

Surface expressed markers and ICs in Raji-hCTLA4 cells

• Stable overexpression of the human CTLA-4 gene 
• Characterized by a number of cell-surface expressed markers including the B cell receptor (BCR), CD19, and CD20
• Constitutive expression of various immune checkpoints (ICs) such as CD27, CD70, CD80, PD-L1, and 4-1BBL
• The stability for 20 passages following thawing has been verified

Applications for Raji-hCTLA4 cells:

• Target cell line for ADCC assays using InvivoGen's Jurkat-Lucia™ NFAT-CD16 cells
• Target cell line for cell toxicity assays using NK or CAR-T cells
• For use in the development of other functional assays

Validation of Raji-hCTLA4 cells:

• Overexpression of CTLA-4 verified by flow cytometry
• Functionally tested as target cells in ADCC assays using anti-human CTLA-4 monoclonal Abs and Jurkat-Lucia™ NFAT-CD16 cells
• Guaranteed mycoplasma-free

References:

1. Ribas A. and Wolchock J.D., 2018. Cancer immunotherapy using checkpoint blockade. Science. 359:1350-55.

Figures

Validation of the expression of human CTLA-4 by Raji-hCTLA4 cells. Raji-Null (A) and Raji‑hCTLA4 (B) cells were incubated with a PE‑conjugated Anti-hCTLA4 mAb for 30 minutes. The binding affinity was then measured using flow cytometry.

Comparison of ADCC potency for native and engineered anti-human CTLA-4 antibody isotypes: Raji-hCTLA4 cells were incubated with gradient concentrations of Anti-hCTLA4 or Anti-β-galactosidase (β-gal) mAbs for 1 hour. Jurkat-Lucia™ NFAT-CD16 effector cells were then co-incubated with targets cells for 6 hours. NFAT activation, reflecting the induced ADCC response, was assessed by determining Lucia luciferase activity in the supernatant using QUANTI-Luc™. Percentages of the maximal response normalized to the IgG1 isotype are shown.

Specifications

Antibiotic resistance: Blasticidin

Growth medium: IMDM, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum (FBS; 30 min at 56 °C), 10 μg/ml Blasticidin, Pen-Strep (100 U/ml-100 µg/ml)

Test medium: IMDM, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated FBS, Pen-Strep (100 U/ml-100 µg/ml)

Quality control:

• Human CTLA-4 expression has been verified by flow-cytometry.
• Induction of antibody-dependent cellular cytotoxicity (ADCC) has been validated using InvivoGen’s anti-hCTLA4-hIgG1 antibody and Jurkat-NFAT Lucia™ CD16 reporter cell line.
• The stability for 20 passages following thawing has been verified.
• Raji-hCTLA4 cells are guaranteed mycoplasma-free.

Contents

• 3-7 x 10⁶ Raji-hCTLA4 cells in a cryovial or shipping flask.
• 1 ml of Blasticidin (10 mg/ml). Store at 4 °C or at -20 °C.
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria and fungi. Store at -20 °C.

Shipped on dry ice (Europe, USA & Canada)

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