Jurkat-Lucia™ NFAT(-CD28) Cells

NFAT-Luc Reporter T Lymphocytes (expressing or not human CD28)

Intracellular signaling in Jurkat™-Lucia NFAT(-CD28) cells

InvivoGen offers a choice of two cell lines derived from the human T Lymphocyte Jurkat cell line and specifically designed to assess activation of the NFAT (nuclear factor of activated T cells) transcription factor:

 –  Jurkat-Lucia™ NFAT cells express endogenous levels of CD3, but no CD28.
 –  Jurkat-Lucia™ NFAT-CD28 cells express endogenous levels of CD3 and feature a stable expression of CD28.

Jurkat cells naturally express functional NFAT proteins, a family of transcription factors involved in T cell activation, differentiation, and self-tolerance [1, 2]. They regulate the expression of many genes, either alone or in cooperation with other transcription factors  [1]. The current paradigm is that full activation of T cells requires at least 2 signals upon contact with antigen-presenting cells (APCs) [3, 4]. Most NFAT proteins are controlled by calcium influx upon T cell receptor (TCR) stimulation (signal 1) [1, 2]. The co-engagement of CD28, the prototypical T cell costimulatory receptor, triggers the activation of the AKT kinase, which contributes to enhancing NFAT translocation into the nucleus [1, 2].

Several therapeutic approaches have focused on targeting the NFAT signaling to control T cell responses in autoimmune diseases and graft rejection [1]. In vitro, NFAT activation can be achieved without APCs; for example, using:
 – cell-permeable small molecules triggering the calcium signaling (e.g. ionomycin), or
 – antibody-mediated cross-linking of the receptors implicated in signal 1 and signal 2 (e.g. anti-CD3 and anti-CD28 monoclonal antibodies).

 More details

Jurkat-Lucia™ NFAT and Jurkat-Lucia™ NFAT-CD28 cells stably express the Lucia luciferase reporter gene under the control of an ISG54 minimal promoter fused to six NFAT response elements. NFAT activation can be readily measured as a bioluminescent signal produced by the Lucia luciferase using the appropriate detection reagent QUANTI-Luc™ 4 Lucia/Gaussia.  These cells were engineered from a Jurkat clone deficient for CD28 expression.  Jurkat-Lucia™ NFAT-CD28 cells were obtained by stable transfection of Jurkat-Lucia™ NFAT cells with the gene encoding human CD28.

Key Features:

• Endogenous NFAT and CD3 expression for Jurkat-Lucia™ NFAT and Jurkat-Lucia™ NFAT-CD28 cells
• Stable hCD28 expression for Jurkat-Lucia™ NFAT-CD28 cells
• Readily assessable Lucia luciferase reporter activity for NFAT activation

Applications:

• Screening of antibodies or small molecules mimicking the TCR stimulation and/or inducing the calcium signaling pathway
• Screening of NFAT targeting drugs

1. Lee J-U., et al., 2018. Revisiting the Concept of Targeting NFAT to Control T Cell Immunity and Autoimmune Diseases. Front Immunol. DOI: 10.3389/fimmu.2018.02747.
2. Macian F., 2005. NFAT proteins: key regulators of T-cell development and function. Nat Rev Immunol. 5(6):472-484.
3. Budd R.C. & Fortner K.A., 2017. Chapter 12 - T Lymphocytes. Kelley and Firestein's Textbook of Rheumatology (Tenth Edition). pages 189-206.
4. Smith-Garvin J.E. et al., 2009. T Cell Activation. Ann. Rev. Immunol. 27:591-619.

Figures

Validation of human CD3 expression and absence of CD28 expression by Jurkat-Lucia™ NFAT cells. Jurkat-Lucia™ NFAT cells were incubated with a cocktail of APC-Cy7-conjugated anti-hCD3 and PE-conjugated anti-hCD28 antibodies for 30 minutes. The binding affinity was then measured using flow-cytometry. (A) CD3 and CD28 co-staining. (B) CD3 and (C) CD28 expression compared to unstained cells.

Validation of NFAT activation in Jurkat-Lucia™ NFAT cells. Jurkat-Lucia™ NFAT cells were incubated with increasing concentrations of concanavalin A (Con A) (A), phytohaemagglutinin P (PHA-P) (B), or Ionomycin with 50 ng/ml phorbol myristate acetate (PMA) (C) for 24 hours. NFAT activation was assessed by determining Lucia luciferase activity in the supernatant using QUANTI-Luc™. Fold change over non-induced cells is shown.

Validation of human CD3 and CD28 cell surface expression by Jurkat-Lucia™ NFAT-CD28 cells. Jurkat-Lucia™ NFAT-CD28 cells were incubated with a cocktail of PE-conjugated anti-hCD28 and APC-Cy7-conjugated anti-hCD3 antibodies for 30 minutes. The binding affinity was then measured using flow cytometry. (A) Co-expression of CD3 and CD28, (B) CD3 and (C) CD28 expression compared to unstained cells.

Validation of NFAT activation in Jurkat-Lucia™ NFAT-CD28 cells. Jurkat-Lucia™ NFAT-CD28 cells were incubated with increasing concentrations of anti-hCD3 mAb only (black) or with 0.5 µg/ml anti-hCD28 mAb (red) for 6 hours. NFAT activation was assessed by determining Lucia luciferase activity in the supernatant using QUANTI-Luc™. Fold change over non-induced cells is shown (mean±SEM).

Specifications

Jurkat-Lucia™ NFAT cells

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: Zeocin®

Quality Control:

• Human CD3 expression has been verified by flow cytometry.
• The activation of NFAT has been confirmed following the induction of Jurkat-Lucia™ NFAT cells with PMA/ionomycin and concanavalin A (ConA) by measuring the levels of Lucia™ luciferase secreted. 
• The stability for 20 passages following thawing has been verified.
• These cells are guaranteed mycoplasma-free.

Jurkat-Lucia™ NFAT-CD28 cells

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 CD3 and human CD28 expression have been verified by flow-cytometry.
• The activation of NFAT has been confirmed following CD3 and CD28 cross-linking, by measuring the levels of Lucia™ luciferase secreted.
• 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 cells in a cryovial or shipping flask
• 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 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)

OR

• Jurkat-Lucia™ NFAT-CD28 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 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)

 Shipped on dry ice (Europe, USA, Canada and some areas in Asia)

Details

The current paradigm is that full activation of T cells requires at least two signals upon contact with APCs [1, 2].  Signal 1 is delivered through the interaction of the TCR and a specific antigenic peptide associated with an MHC (major histocompatibility complex) molecule on APCs. Signal 2 is delivered through the interaction of CD28, the prototypical T cell co-stimulatory molecule, and its ligands, CD80 or CD86, expressed by the APC. 

Signal 1: TCR and [HLA::peptide]

The 'classical' and most represented TCR is an 80 to 90 kDa heterodimer composed of one α chain and one β chain. The αβTCR is a transmembrane protein expressed by developing and mature T cells. It features an extracellular ligand-binding pocket and a short cytoplasmic tail. Each αβTCR is restricted to a specific complex made of an antigenic peptide and a class I or class II MHC molecule. Human MHC molecules are also known as HLA (human leukocyte antigen). Because of its short cytoplasmic tail, the TCR, once engaged,  lacks the ability to signal and requires non-covalent association with the CD3 to trigger downstream intracellular signaling and T cell activation [1, 2]. Importantly, signal 1 without co-stimulation results in T cell unresponsiveness or 'anergy', a tolerance mechanism that guards against premature activation.

Signal 2: CD28 and CD80/86

CD28 is a homodimeric and transmembrane protein expressed by T cells. Nearly all human CD4+ T cells and 50% of human CD8+ T cells express CD28. The CD28 interaction with CD80 (aka B7-1) or CD86 (aka B7-2) on APCs, in conjunction with TCR engagement, triggers a co-stimulation signal (signal 2). It results in T cell proliferation, cytokine production, cell survival and cellular metabolism [1, 2].

NFAT activation:

Most NFAT proteins are controlled by calcium influx upon TCR stimulation. Calcium binds calmodulin, which in turn activates calcineurin, a calmodulin-dependent phosphatase. Calcineurin dephosphorylates NFAT proteins, leading to their translocation into the nucleus, where they regulate the expression of many genes, either alone or in cooperation with other transcription factors [3, 4]. The co-engagement of CD28 triggers the activation of the AKT kinase, which contributes to enhancing NFAT translocation into the nucleus [4].

References:

1. Budd R.C. & Fortner K.A., 2017. Chapter 12 - T Lymphocytes. Kelley and Firestein's Textbook of Rheumatology (Tenth Edition). pages 189-206.
2. Smith-Garvin J.E. et al., 2009. T Cell Activation. Ann. Rev. Immunol. 27:591-619.
3. Lee J-U., et al., 2018. Revisiting the Concept of Targeting NFAT to Control T Cell Immunity and Autoimmune Diseases. Front Immunol. DOI: 10.3389/fimmu.2018.02747.
4. Macian F., 2005. NFAT proteins: key regulators of T-cell development and function. Nat Rev Immunol. 5(6):472-484.
 

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