Human VISTA-expressing Raji Cells

Human VISTA-expressing B cells

Raji-hVISTA cells were developed from the human Raji cell line, a human B lymphocyte-derived cell line. Raji cells have been successfully used as target cells 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.

V-domain Ig containing suppressor of T cell activation (VISTA), also known as programmed death-1 homolog (PD-1H), is a member of the CD28/B7 protein superfamily [1].  Similar to other immune checkpoints (ICs), VISTA is found to be heightened on immune cells that infiltrate the tumor microenvironment (TME). It is predominantly expressed on antigen-presenting cells (APCs) and directly suppresses T cell activation and proliferation [1,2]. Additionally, VISTA is expressed on a number of T cells subsets and can thus be considered both as a ligand and a receptor, whereby it transmits both extrinsic and intrinsic inhibitory signals to T cells [2]. PSGL-1 has been identified as a receptor for VISTA and interestingly, its engagement suppresses T cells selectively in an acidic pH, such as that found in the TME [3]. It has been suggested that VISTA and PD-1 suppress T cell function in a synergistic manner, and therefore, combined therapy targeting both ICs has been considered extremely promising [4].

Features of Raji-hVISTA cells:

Surface expressed markers and ICs in Raji-hVISTA cells

• Stable overexpression of the human VISTA 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

Applications for Raji-hVISTA 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-hVISTA cells:

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

​References:

1. Nowak, E.C. et al. 2017. Immunoregulatory functions of VISTA. Immunol Rev 276, 66-79.
2. Xu, W. et al. 2018. The structure, expression, and multifaceted role of immune-checkpoint protein VISTA as a critical regulator of anti-tumor immunity, autoimmunity, and inflammation. Cell Mol Immunol 15, 438-446.
3. Johnston, R.J. et al. 2019. VISTA is an acidic pH-selective ligand for PSGL-1. Nature 574, 565-570.
4. Liu, J. et al. 2015. Immune-checkpoint proteins VISTA and PD-1 nonredundantly regulate murine T-cell responses. PNAS112:6682–7.

Figures

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

Comparison of ADCC potency for native and engineered anti-human VISTA antibody isotypes: Raji-hVISTA cells were incubated with gradient concentrations of Anti-hVISTA or Anti-β-galactosidase (β-gal) mAbs for 1 hour. Jurkat-Lucia™ NFAT-CD16 effector cells were then co-incubated with target 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 VISTA expression has been verified by flow-cytometry.
• Induction of antibody-dependent cellular cytotoxicity (ADCC) has been validated using InvivoGen’s anti-hVISTA-hIgG1 antibody and Jurkat-NFAT Lucia™ CD16 reporter cell line.
• The stability for 20 passages following thawing has been verified.
• Raji-hVISTA cells are guaranteed mycoplasma-free.

These products are covered by a Limited Use License (See Terms and Conditions).

Contents

• 3-7 x 10⁶ Raji-hVISTA 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.

IMPORTANT: If cells are shipped frozen (i.e. in a cryovial) and are not frozen upon arrival, contact InvivoGen immediately.

Shipped on dry ice (Europe, USA & Canada)

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