Jurkat-Lucia™ NFAT Cells for ADCC & ADCP Assays

ADCC and ADCP reporter T-cell lines

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). 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 immunoglobulin G (IgG). 

 –  Jurkat-Lucia™ NFAT-CD16 cells, featuring the high-affinity CD16 allotype (V158)
 –  Jurkat-Lucia™ NFAT-CD16-Low cells, featuring the low-affinity CD16 allotype (F158)
 –  Jurkat-Lucia™ NFAT-CD32 cells, featuring the high-affinity CD32 allotype (H131)

ADCC and ADCP are initiated when multiple IgG molecules are bound simultaneously to FcγRs. The outcome depends on the preferential IgG affinity of each FcγR and the balance in FcγR signaling. The IgG-FcγR interaction is regulated by the antibody 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].

Intracellular signaling in Jurkat-Lucia™ NFAT cells for ADCC/ADCP assays

 More details

Jurkat-Lucia™ NFAT-CD16 and Jurkat-Lucia™ NFAT-CD16-Low cells have been engineered to express a high-affinity CD16 (V158 allotype) and low-affinity CD16 (F158), respectively [3]. They may be used as effector reporter cells for InvivoGen’s ADCC assays.
Jurkat-Lucia™ NFAT-CD32 cells have been engineered to express a high-affinity CD32 (H131 allotype) [3]. They may be used as effector reporter cells for InvivoGen’s ADCP assays.
These three cell lines 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 or F158 allotype) expression
• Stable CD32A (FcgRIIA; H131 allotype) expression
• Readily assessable Lucia luciferase reporter activity for NFAT activation

Applications:

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

   

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1. Shaw J-P. et al., 1998. Identification of a putative regulator of early T cell activation genes. Science. 241:202.
2. Leibson P.J., 1997. Signal transduction during natural killer cell activation: inside the mind of a killer. Immunity. 6:655.
3. Nagelkerke S.Q. et al., 2019. Genetic variation in low-to-medium-affinity Fcγ receptors: functional consequences, disease associations, and opportunities for personalized medicine. Front. Immunol. 10:2237.

Figures

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.

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-Low 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.

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.

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® for Jurkat-Lucia™ NFAT-CD16 and Jurkat-Lucia™ NFAT-CD32 cells
• Blasticidin, Hygromycin, and Zeocin® for Jurkat-Lucia™ NFAT-CD16-Low cells

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 IgG isotypes and Raji-Null 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™

OR

• 3-7 x 10⁶ Jurkat-Lucia™ NFAT-CD16-Low cells in a cryovial or shipping flask
• 1 ml of Blasticidin (10 mg/ml)
• 1 ml of Hygromycin B Gold (100 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 & ADCP

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. Human IgGs bind to activatory (FcγRI, FcγRIIA (CD32A), FcγRIIa (CD16A), and inhibitory (FcγRIIb) receptors. The IgG-FcγR interaction is regulated by the Ab isotype and glycosylation [1, 2]. FcγRs differ in their cellular distribution and are often co-expressed. FcgRIIA (CD32A) is expressed myeloid cells including monocytes, macrophages, and dendritic cells (DCs). FcgRIIIa (CD16A) is expressed on macrophages and Natural Killer (NK) cells [3].

ADCC and ADCP are initiated when multiple IgG molecules bind simultaneously to FcγRs. The binding of antibody-antigen complexes to activatory and inhibitory FcγRs induces their cross-linking and subsequent signaling through immunoreceptor tyrosine-based activation motifs (ITAMs) and inhibition motifs (ITIMs), respectively. 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].

Balance in FcγR signaling

The balance in FcγR signaling controls the immune outcome.

• No response: inhibiting signals counterbalance activating signals.

• ADCC: an excess of engaged CD16A (FcγRIIIA) at the surface of Natural Killer (NK) cells induces the release of cytotoxic granules which kill the target [1].

• ADCP: an excess of engaged CD32A (FcγRIIA) at the surface of monocytes, macrophages, and dendritic cells induces 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].

CD16A and CD32A allelic polymorphism

Single nucleotide polymorphisms (SNPs) in human Fc receptors affect interactions with antibody Fc. Allelic variants of the same FcR can display lower or higher affinities for antibody-antigen immune complexes.

• CD16A features allelic polymorphisms among the human population, notably at position 158 in the mature protein (or position 161 in the full protein) [3]. The V158 allotype is reported to have a higher affinity for monoclonal immunoglobulin G (IgGs) than the F158 allotype [3, 4].

• CD32A features allelic polymorphisms among the human population, notably at position 131 in the mature protein (or position 166 in the full protein) [3]. The H131 allotype is reported to have a higher affinity for monoclonal immunoglobulin G (IgGs) than the R131 allotype [3].

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.
3. Nagelkerke S.Q. et al., 2019. Genetic variation in low-to-medium-affinity Fcγ receptors: functional consequences, disease associations, and opportunities for personalized medicine. Front. Immunol. 10:2237. 
4. Bruhns P. et al., 2009. Specificity and affinity of human Fcγ receptors and their polymorphic variants for human IgG subclasses. Blood. 113(16):3716. 

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