Jurkat-Lucia™ NFAT Cells for ADCC & ADCP Assays

ADCC and ADCP reporter T-cell lines

Intracellular signaling in Jurkat™-Lucia NFAT-CD16 (or CD32) cells

InvivoGen offers a series of Jurkat-Lucia™ NFAT cell lines, specifically designed to assess the potency of specific immunoglobulin for ADCC (antibody-dependent cellular cytotoxicity) and ADCP (antibody-dependent cell-mediated phagocytosis).

 –  Jurkat-Lucia™ NFAT-CD16 cells for ADCC reporter assays
 –  Jurkat-Lucia™ NFAT-CD32 cells for ADCP reporter assays

These cells derive from the human T-lymphocyte Jurkat cell line and stably express CD16 or CD32, two Fc-gamma receptors (FcγR) for the constant region of immunoglobulins. ADCC and ADCP outcomes depend on the preferential affinity of each antibody (Ab) for one FcγR. The Ab-FcγR interaction is regulated by the Ab isotype and glycosylation. Jurkat cells naturally express a functional NFAT (nuclear factor of activated T cells) transcription factor, which is involved in the early signaling events of ADCC and ADCP [1, 2].

More details
 

Jurkat-Lucia™ NFAT-CD16 and Jurkat-Lucia™ NFAT-CD32 cells have been designed as effector reporter cells for InvivoGen’s ADCC and ADCP assays, respectively. These cells stably express the Lucia luciferase reporter gene under the control of an ISG54 minimal promoter fused to six NFAT response elements. ADCC or ADCP induction is measured as a bioluminescent signal produced by the Lucia luciferase upon the addition of the appropriate detection reagent QUANTI-Luc™. 

Key Features:

• Endogenous NFAT expression
• Stable CD16A(FcgRIIIA; V158 allotype [3]) expression
• Stable CD32A(FcgRIIA; H131 allotype [4])expression
• Readily assessable Lucia luciferase reporter activity for NFAT activation
• Resistance to Blasticidin and Zeocin™

Applications:

• Screening of engineered monoclonal antibodies (mAbs) potency for ADCC using Jurkat-Lucia™ NFAT-CD16 cells
• Screening of engineered mAbs potency for ADCP using Jurkat-Lucia™ NFAT-CD32 cells

    

Read our review on Immune Checkpoint Blockade.

Learn more about Immune Checkpoint Antibodies.

References:

1. Shaw J-P. et al., 1998. Identification of a putative regulator of early T cell activation genes. Science. 241:202-205.
2. Leibson P.J., 1997. Signal transduction during natural killer cell activation: inside the mind of a killer. Immunity. 6:655-61.
3. Quast I. et al., 2016. Regulation of antibody effector functions through IgG Fc N-glycosylation. Cell. Mol. Life. Sci. 74(5):837-47.
4. Tay M.Z. et al.., 2019. Antibody-Dependent Cellular Phagocytosis in Antiviral Immune Responses. Front Immunol. 10:332.

Figures

Comparison of ADCC potency for native and engineered anti-human CD20 isotypes: Raji-Null cells were incubated with gradient concentrations of Anti-hCD20 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.

Increased ADCC activity mediated by IgG1 compared to IgG1fut (non-fucosylated): Raji-hCTLA4, Raji-hPD-1, and Raji-hPD-L1 cells were incubated with Jurkat-Lucia™ NFAT-CD16 effector cells and corresponding IgG1 or IgG1fut specific mAbs. The data represent the EC50 for each antibody.

ADCP potency of Anti-hCD20 hIgG1.
Raji-Null cells (expressing hCD20) were incubated with gradient concentrations of Anti-hCD20 hIgG1 (featuring the variable region of Rituximab) or Anti-β-galactosidase (β-gal) hIgG1 for 1 hour. Jurkat-Lucia™ NFAT-CD32 effector cells were then co-incubated with target cells for 6 hours. NFAT activation, reflecting the induced ADCP response, was assessed by determining Lucia luciferase activity in the supernatant using QUANTI-Luc™. Relative light units (RLUs) are shown.

ADCP potency of Anti-hPD-L1 hIgG2.
Raji-hPD-L1 cells were incubated with gradient concentrations of Anti-hPD-L1 hIgG2 or Anti-β-galactosidase (β-gal) hIgG2 for 1 hour. Jurkat-Lucia™ NFAT-CD32 effector cells were then co-incubated with target cells for 6 hours. NFAT activation, reflecting the induced ADCP response, was assessed by determining Lucia luciferase activity in the supernatant using QUANTI-Luc™. Relative light units (RLUs) are shown.

Specifications

Growth medium: IMDM, 2 mM L-glutamine, 25 mM HEPES, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml Normocin™

Antibiotic resistance: Blasticidin and Zeocin™

Quality Control:

• Human CD16A and CD32A expression have been verified by flow-cytometry.
• Induction of antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) have been validated using InvivoGen’s anti-hCD20-hIgG1, anti-hPD-L1-hIgG2, Raji-Null cells, and Raji-hPD-L1 cells.
• The stability for 20 passages following thawing has been verified.
• These cells are guaranteed mycoplasma-free.

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

Contents

Please note: Each cell line is sold separately. See TDS for the exact contents of each cell line. 

• 3-7 x 10⁶ Jurkat-Lucia™ NFAT-CD16 cells OR Jurkat-Lucia™ NFAT-CD32 cells in a cryovial or shipping flask
• 1 ml of Blasticidin (10 mg/ml)
• 1 ml of Zeocin™ (100 mg/ml)
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.
• 1 pouch of QUANTI-Luc™ 

 Shipped on dry ice  (Europe, USA & Canada).

Details

ADCC and ADCP are immune mechanisms through which Fc receptor-bearing effector cells can recognize and clear antibody (Ab)-coated microbes and target cells expressing specific antigens on their surface. The binding of antibody-antigen complexes to activatory FcγRs induces their cross-linking and subsequent signaling through immunoreceptor tyrosine-based activation motifs (ITAMs). Cytoplasmic signaling includes an increase in intracellular calcium concentration and calcineurin/calmodulin-mediated dephosphorylation of NFAT (nuclear factor of activated T cells), allowing its nuclear translocation and binding to promoter regions of ADCC and ADCP relevant genes [1, 2].
The ADCC and ADCP outcomes depend on the preferential affinity of each Ab for one FcgR. The Ab-FcgR interaction is regulated by the Ab isotype and glycosylation.

Antibody-dependent cellular cytotoxicity (ADCC)

ADCC is triggered by the cross-linking between antigen-bound Abs and the Fc receptor CD16A (FcγRIIIA)at the surface of Natural Killer (NK) cells [3]. Activated NK cells release cytotoxic granules which kill the microbe or target cells [1].

Antibody-dependent cell-mediated phagocytosis (ADCP)

ADCP is triggered by the cross-linking between antigen-bound Abs and the Fc receptor CD32A (FcγRIIA) at the surface of monocytes, macrophages, and dendritic cells. These interactions induce the phagocytosis of the microbe or target cells. This internalization is followed by phagolysosomal degradation, thus facilitating antigen presentation and stimulating inflammatory cytokine secretion [2].

References:

1. Quast I. et al., 2016. Regulation of antibody effector functions through IgG Fc N-glycosylation. Cell. Mol. Life. Sci. 74(5):837-47.
2. Tay M.Z. et al., 2019. Antibody-Dependent Cellular Phagocytosis in Antiviral Immune Responses. Front Immunol. 10:332

FAQ

Any questions about our cell lines ? Visit our frequently asked questions page