|Screen||In Vivo NK Cell and CD8+ T Cell Cytotoxicity Screen|
|Posted On||02/18/2010 12:24 PM|
|Science Writer||Eva Marie Y. Moresco|
This in vivo screen is designed to detect mutants with defects in natural killer (NK) cell or CD8+ cytotoxic T lymphocyte (CTL) effector responses. In brief, the screen is based on immunization of mice with apoptotic cells expressing a model antigen, followed by measurement of the killing ability of both NK cells and CD8+ T cells towards class I MHC-deficient splenocytes (NK cell-specific targets) and splenocytes pulsed with antigen-derived peptide (CTL-specific targets).
NK cells and CTLs, components of the innate and adaptive immune systems, respectively, are cytotoxic lymphocytes essential in defense against viruses and intracellular bacteria. Activated NK cells and CTLs kill targets through release of perforin/granzymes, and/or signaling through tumor necrosis factor (TNF) death receptor family proteins, including TRAIL (TNF-related apoptosis inducing ligand), FasL (Fas ligand), and TNFα [reviewed in (1)]. Previous work demonstrated that a strong antigen-specific CD8+ T cell response can be induced in mice by immunization with apoptotic but not with live cells (2). This response is characterized by antigen-stimulated interferon (IFN)-γ production and cytolytic activity, and requires the uptake of apoptotic material by lymphoid dendritic cells (DC), which produce type I interferon (IFN) and efficiently crossprime antigen-specific CD8+ T cells. In addition to ultraviolet- and γ-irradiated apoptotic cells, cells rendered apoptotic by treatment with Fas-activating antibodies are effective stimulators of a strong CTL response, suggesting that NK cells or T cells can initiate the response. Some viruses and bacteria are also capable of inducing apoptosis and may thereby trigger a microbe-specific CTL response. Although the molecular pathway mediating the cell-death induced CTL response is still under investigation, immunization with apoptotic cells provides a means for strong activation of CTLs that is exploited in this screen. The in vivo CD8+ T cell cytolytic activity is measured using blood collected from mice six to eight days after immunization.
C57BL/6J ENU-mutagenized G3 mice are immunized with γ-irradiated (1500 Rad) mouse splenocytes expressing membrane-associated ovalbumin driven by an actin promoter (act-mOVA splenocytes) (Figure 1) (3). Seven to eight days later, the same mice are injected with three cell populations labeled with different intensities of the fluorescent cellular dye CFSE (carboxyfluorescein succinimidyl ester): control C57BL/6J cells (low CFSE), NK specific target cells (syngeneic class I MHC-deficient cells from Tap1-/- mice; intermediate CFSE), and an antigen-specific CTL target population [syngeneic splenocytes externally loaded with an ovalbumin-derived peptide (OVA257-264, SIINFEKL); high CFSE]. After 48 hours, blood samples are collected from the orbital sinus and analyzed by flow cytometry for the number of target cells and control cells. The disappearance of NK target and CTL target populations relative to the low CFSE-containing control population reflects the in vivo cytotoxic ability of NK cells and CTLs (Figure 2).
|Reagents and Solutions|
Iscove’s Modified Dulbecco’s Medium (Gibco)
10% (v/v) heat inactivated fetal bovine serum (Atlanta Biologicals)
200 units penicillin and 200 μg streptomycin (1:50 dilution of PenStrep solution, Gibco)
50 μM β-mercaptoethanol
2 mM L-Glutamine (Gibco)
Red Blood Cell (RBC) Lysis Buffer
Red Blood Cell Lysing Buffer (Sigma)
CFSE (Sigma) in DMSO; 5mM stock solution
Hank’s Balanced Salt Solution (HyClone)
Dulbecco’s Phosphate Buffered Saline (DPBS, Cellgro)
0.1% fetal bovine serum
Dulbecco's Phosphate Buffered Saline (DPBS, Cellgro)
2% (v/v) heat inactivated fetal bovine serum (Atlanta Biologicals)
0.1% sodium azide (Sigma)
BD FACSTM Lysing Solution
Dilute 1:10 ratio with ddH2O for working solution
Immunization of G3 mice
Preparation of control and target cells for cytotoxicity assay
There are three populations of splenocytes to be injected into the immunized G3 mice: Splenocytes from Tap1-/- mice are used as NK cell targets. Splenocytes from syngeneic C57BL/6J mice are used as control cells and for peptide loading to generate CTL targets.
Peptide loading of CTL target cells (only for splenocytes from C57BL/6J mice)
Red blood cell lysis
CFSE labeling of cells
|Critical Parameters and Troubleshooting|
In order to obtain good separation of control and target cell populations on FACS plots (see Figure 2), the populations should ideally be labeled with distinct, non-overlapping intensities of CFSE. To help ensure this, it is important to make accurate cell counts (step 15) and volume measurements during CFSE labeling of cells.
1. Barry, M. and Bleackley, R. C. (2002) Cytotoxic T lymphocytes: all roads lead to death, Nat. Rev. Immunol. 2, 401-409.
2. Janssen, E., Tabeta, K., Barnes, M. J., Rutschmann, S., McBride, S., Bahjat, K. S., Schoenberger, S. P., Theofilopoulos, A. N., Beutler, B., and Hoebe, K. (2006) Efficient T cell activation via a Toll-Interleukin 1 Receptor-independent pathway, Immunity 24, 787-799.