Data Sheet TIGIT / NFAT Reporter - Jurkat Cell Line Catalog #60538 Background: TIGIT is a co-inhibitory receptor that is highly expressed in Natural Killer (NK) cells, activated CD4+, CD8+ and regulatory T cells. Interaction with the poliovirus receptor (PVR; CD155) on antigen presenting cells, such as dendritic cells, recruits Src homology (SH) domain-containing protein tyrosine phosphatase SHP1 and SHP2 or the inositol phosphatase SHIP1 and SHIP2 to the TIGIT ITIM domain. This increases IL-10 release and suppresses NFkB and NFAT T cell receptor (TCR) signaling, which blocks T cell proliferation and cytokine production. It serves as a competitive inhibitor of CD226, a co-stimulatory receptor for CD155. TIGIT targeting antibodies that block this T cell-intrinsic inhibitory effects have shown enhanced anti-tumor and anti-viral functions in preclinical studies. Description: Recombinant Jurkat cell line constitutively expressing a full length human TIGIT (T cell Immunoreceptor with Immunoglobulin and ITIM domain; VSTM3), and a firefly luciferase gene under the control of nuclear factor of activator T cells (NFAT) response element. Both TIGIT and NFAT constructs have been stably integrated into Jurkat cells. TIGIT expression has been confirmed by Western Blot, and TIGIT-specific NFAT response has been validated using a TIGIT ligand, CD155 (PVR). Application: This cell line is ideal for high throughput screening (HTS) to identify antagonistic monoclonal antibodies targeting either TIGIT or its ligands, such as CD155 in a cellular context. It is also ideal for characterizing TIGIT mediated biological activity and immunological response in T cells upon its interactions with its ligands. Host Cell Human T lymphocyte cell line. Suspension cells. Format Each vial contains ~2 x 10 6 cells in 1mL of 10% DMSO in FBS. Storage Store in liquid nitrogen immediately upon receipt. Culture Medium RPMI (Thermo Fisher Cat. # 11875093), 10% heat-inactivated FBS (Thermo Fisher Cat. #10438026), 1x Penicillin/Streptomycin (Thermo Fisher, Cat. #10378016), 100 µg/ml Hygromycin B (Thermo Fisher, Cat. #10687010) and 1mg/ml Geneticin, G418 Sulfate (Thermo Fisher, Cat. # 10131035).
Culture conditions Frozen Cells: Prepare T-75 culture flask with 20 ml of pre-warmed growth medium. Quickly thaw cells in a 37 C water bath with constant and slow agitation. Clean the outside of the vial with 70% ethanol and immediately transfer the entire contents to the culture media without Hygromycin B and G418. Avoid pipetting up and down, and gently rock the flask to distribute the cells. Incubate the cells in a humidified 37 C incubator with 5% CO 2. 48 hours after incubation, centrifuge cells at 250 x g for 5 minutes and re-suspend to fresh medium without Hygromycin B and G418. Continue to monitor growth for 2-3 days and change medium to remove dead debris. If slow cell growth occurs during resuscitation, increase FBS to 15% for the first week of culture. Begin adding Hygromycin B and G418 to medium after multiple cell colonies (in clumps) start to appear (indicative of healthy cell division). Subculture: When cells reached 90% confluency, transfer cells to a 50 ml conical tube and centrifuge cells at 200 x g for 5 minutes. Wash cells once with PBS (without Magnesium or Calcium) and re-suspend cells in 10 ml pre-warmed medium; gently pipette up and down to dissociate cell clumps. Dispense 2 ml of the cell suspension into a new T-75 flask containing pre-warmed 20 ml medium. Incubate cells in a humidified 37 C incubator with 5% CO 2. Freeze cells in freezing medium (10% DMSO in FBS) when cells reach 90% confluency. Cells have been demonstrated to be stable for at least 15 passages; BPS recommends preparing frozen stocks so cells are not used beyond passage 20. Mycoplasma Testing This cell line has been screened using the MycoAlert Mycoplasma Detection Kit (Lonza, Cat. #LT07-118) to confirm the absence of Mycoplasma contamination. MycoAlert Assay Control Set (Lonza, Cat. #LT07-518) was used as a positive control. Application References 1. Li M et.al (2014) T-cell Immunoglobulin and ITIM Domain (TIGIT) Receptor/ Poliovirus Receptor (PVR) Ligand Engagement Suppresses Interferon-g Production of Natural Killer Cells via b-arrestin 2-mediated Negative Signaling. J Biol Chem. 289: 17647-17657. 2. Stanietsky N. et.al. (2009) The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity. PNAS. 106: 17858-17863. 3. Zhu et.al. (2016) Identification of CD112R as a novel checkpoint for human T cells. J Exp. Med. 213: 167-176. 4. Yu X. et.al. (2008) The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells. Nat.Immunol. 10: 48-57.
Quality Assurance and Functional Analysis Figure 1. Human TIGIT Expression in TIGIT/NFAT-Luciferase cells. Cells were seeded at semi-confluency in 24 well plates overnight and were lysed by RIPA lysis buffer containing protease inhibitors. Human TIGIT protein level was assessed by Western blot using a TIGIT antibody (181-194) (Sigma Aldrich, Cat. #SAB1103801). β-actin (8H10D10) (Cell Signaling Technology, Cat. #3700) was used as a loading control. Figure 2. NFAT Signaling in TIGIT overexpressed reporter Jurkat cells is suppressed by CD155/HEK293 cells. CD155 (PVR) HEK293 Stable Cell Line (BPS Cat. #60537) or naïve HEK293 cells were seeded overnight at ~ 4x10 4 cells/well, and TIGIT-expressing NFATluciferase (TIGIT NFAT; TIGIT+ ) or NFAT-luciferase (NFAT; TIGIT- ) Jurkat T cells were seeded overnight at ~ 2x10 4 cells/well at 100 µl in a white 96 well plate. The next day, NFAT Jurkat T cells were activated with Human T-activator CD3/CD28 Dynabeads (Thermo Fisher,
Cat. #111.61D) at ~ 4:1 bead-to-cell ratio and were subsequently incubated with CD155/HEK293 or HEK293 for 24 hours before the addition of ONE-Step Luciferase Assay substrates (BPS, Cat. #60690) for luminescence detection. The % NFAT-Luciferase activity represents relative luminescence units released by TIGIT or NFAT T cells incubated with CD155/HEK293 vs. HEK293. Error bar = S.E.M. n=3, ***P<0.001, Student s two-tailed unpaired t-test. Figure 3. NFAT signal in TIGIT overexpressed Jurkat cells is inhibited by soluble recombinant human CD155 protein. Cells were seeded in 96 well at 2x10 4 cells per well at 100 µl overnight in a white 96 well plate. The next day, TIGIT expressing NFAT Jurkat cells (TIGIT NFAT) and NFAT Jurkat cells (NFAT) were activated with Human T-activator CD3/CD28 Dynabeads (Thermo Fisher, Cat. #111.61D) at ~ 4:1 bead-to-cell ratio, and subsequently treated with various concentrations of CD155 (BPS Cat. #71181) for 24 hours. IC 50 = 98.42 µg/ml. (Left). Activated TIGIT NFAT (TIGIT+) or NFAT (TIGIT-) Jurkat cells were treated with soluble recombinant human CD155 (BPS Cat. #71181) at 100 µg/ml for 24 hours (Right) before the addition of ONE-Step Luciferase Assay substrates (BPS, Cat. #60690) for luminescence detection. The % NFAT-Luciferase activity represents relative luminescence units released by TIGIT or NFAT T cells incubated with CD155/HEK293 vs. HEK293. Error bar = S.E.M. n = 3, ***P < 0.001, ns = not significant. Student s two-tailed unpaired t-test.
Figure 4. A CD155 human monoclonal antibody blocks TIGIT-CD155 mediated NFAT signal inhibition. CD155/HEK293 (BPS Cat. #60537) cells were seeded in 96 well at 4x10 5 cells per well at 100 µl, and TIGIT/NFAT cells were seeded at 2x10 5 cells per well overnight in a white 96 well plate. The next day, TIGIT/NFAT were activated with Human T-activator CD3/CD28 Dynabeads (Thermo Fisher, Cat. #111.61D) at ~ 2:1 bead-to-cell ratio. TIGIT/NFAT cells were added to each well containing HEK293 cells. Human anti-cd155 monoclonal antibody (TX21, MBL International, Cat. #D174-3) were added (at 50 µg/ml) to co-cultures and incubated for 20 hours before the addition of ONE-Step Luciferase Assay substrates (BPS, Cat. #60690) for luminescence detection. Relative Luminescence Units (RLUs) released by activated TIGIT/NFAT cells were normalized to cells without CD3 activation. Error bar = S.E.M. n = 5. ****P<0.0001, *** P< 0.001 One-way ANOVA, Tukey s multiple comparison test. Vector and sequence NFAT-Luciferase was cloned into the MCS of pcdna3.1 (+) vector (Invitrogen, Cat. #V79020). Human TIGIT (NP_776160.2; Accession BC026692) was cloned into the MCS of pireshyg3 vector (Clontech, Cat. # 631620). MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQD QLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLE SSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSA PSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG
Related Products Product Cat. # Size CD155 (PVR) HEK293 Stable Cell Line 60537 2 vials ONE-Step TM Luciferase Assay System 60690-1 10 ml ONE-Step TM Luciferase Assay System 60690-2 100 ml Human CD155 (PVR) His-tag Protein 71181 100 μg Mouse CD155 (PVR) His-tag Protein 71167 100 μg Mouse CD155, His-tag, Biotin-labeled 71168 50 μg Human CD226, Fc fusion 71252 100 μg Human CD226, Fc fusion, Biotin-labeled 71253 50 μg Human CD112, Fc fusion 71197 100 μg Human CD112, Fc fusion, Biotin-labeled 71234 50 μg Human TIGIT, Fc fusion 71186 100 μg Human TIGIT, Fc fusion, Biotin-labeled 71251 50 μg TIGIT:CD155 Homogenous Assay Kit 72029 384 reactions CD226: CD155 Homogenous Assay Kit 72052 384 reactions TIGIT:CD112 Homogenous Assay Kit 72030 384 reactions CD226: CD112 Homogenous Assay Kit 72051 384 reactions NFAT Reporter (Luc) Jurkat Cell Line 60621 2 vials PD-1 / NFAT Reporter Jurkat Cell Line 60535 2 vials TCR Activator / PD-L1 Cho Recombinant Cell Line 60536 2 vials