Protocols


Screen (pdf version)
ScreenIn Vivo NK Cell and CD8+ T Cell Cytotoxicity Screen
Posted On02/18/2010 12:24 PM
AuthorPhilippe Krebs
Science WriterEva Marie Y. Moresco
Background
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
IMDM medium
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 solution
CFSE (Sigma) in DMSO; 5mM stock solution
 
HBSS
Hank’s Balanced Salt Solution (HyClone)
 
Wash buffer
Dulbecco’s Phosphate Buffered Saline (DPBS, Cellgro)
0.1% fetal bovine serum
 
FACS buffer
Dulbecco's Phosphate Buffered Saline (DPBS, Cellgro)
2% (v/v) heat inactivated fetal bovine serum (Atlanta Biologicals)
0.1% sodium azide (Sigma)
10mM EDTA
 
BD FACSTM Lysing Solution
Dilute 1:10 ratio with ddH2O for working solution
Method
Immunization of G3 mice
  1. Immediately prior to immunization, irradiate act-mOVA splenocytes (1500 Rad) using a gamma irradiator.
  2. Immunize each mouse by injecting 107 irradiated act-mOVA splenocytes intraperitoneally or subcutaneously.
Preparation of control and target cells for cytotoxicity assay
 
Splenocyte Collection
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.
  1. On day 8 after immunization of G3 mice, harvest spleens from Tap1-/- and C57BL/6J mice and place in IMDM medium. Use one Tap1-/- spleen and two C57BL/6J spleens to obtain enough cells to prepare NK and CTL targets for testing fifteen immunized mice.  Include controls (naïve Tap1-/- recipients and immunized C57BL/6J recipients).
  2. Homogenize the spleens using a cell strainer (70μm, BD Falcon).
  3. Centrifuge the cells at 1500 rpm for 5 minutes, decant the supernatant.
  4. Resuspend in appropriate volume of IMDM medium (1 ml per 2 spleens).
Peptide loading of CTL target cells (only for splenocytes from C57BL/6J mice)
  1. Centrifuge half of the cell suspension from step 4 at 1500 rpm for 5 minutes (this will be the CTL target population), decant the supernatant.  Keep the other half, representing the control population, for later.
  2. Resuspend in IMDM medium (1 ml per 2 spleens) and divide into 15ml conical tubes (1 ml per tube).
  3. Add 0.5 ml of OVA-derived SIINFEKL peptide (stock solution of 10 μM), and incubate the splenocytes for 60 minutes at 37°C; keep the control splenocyte population at 37°C as well.
  4. Mix from time to time.
  5. Add 10 ml of IMDM medium to wash out the peptide and pool all the peptide-pulsed CTL target cells into a 50 ml conical tube.
Red blood cell lysis
  1. Centrifuge the three splenocyte populations at 1500 rpm for 5 minutes, decant the supernatant.
  2. Add 2 ml of RBC Lysis Buffer for each spleen, let sit for 1 minute at room temperature, and then add 10 ml of IMDM media.
  3. Centrifuge again and resuspend the cells in 1 ml per spleen of sterile Wash buffer.
CFSE labeling of cells
  1. Centrifuge the cells at 1500 rpm for 5 minutes and decant the supernatant.
  2. Resuspend the cells in 5 ml of Wash buffer.
  3. Add 20 μl of the cell suspension to 10 ml of Coulter Counter diluent and pass the solution through a Coulter Counter set at cell size 6 μm to count the cells.
  4. Dilute the cell suspension from step 14 to a concentration of approximately 107 cells/ml in Wash buffer.
  5. Add appropriate amount of CFSE solution to 1 ml of Wash buffer. For high CFSE labeling, use 10 μl of CFSE solution for each 107 splenocytes to be labeled. Make two serial dilutions of this solution (140 μl into 860 μl Wash buffer and again 140 μl into 860 μl Wash buffer) to make the intermediate and low CFSE labeling solutions; mix well before each dilution. Protect tubes from light.
  6. Mix equal volumes (typically about 800 μl) of CFSE labeling solution and the appropriate splenocyte suspension (from step 16). Control C57BL/6J splenocytes, not loaded with peptide: low CFSE. Tap1-/- splenocytes (NK cell targets): intermediate CFSE. C57BL/6J splenocytes loaded with peptide (CTL targets): high CFSE.
  7. Incubate the CFSE/cell mixtures for 10 minutes at room temperature.
  8. Add FCS to each mixture at a volume which is 10% the volume of the cell solution to block free CFSE.
  9. Centrifuge the splenocytes at 1500 rpm for 5 minutes and decant the supernatant. Rinse the cells with Wash buffer.
  10. Centrifuge the splenocytes at 1500 rpm for 5 minutes and decant the supernatant and resuspend the cells in HBSS. Amount of HBSS: for each splenocyte population, use the following formula: number of mice to inject x 0.2 μl / 3 ml.
  11. Count the cells from each population: add 20 μl of the cell suspension to 10 ml of Coulter Counter diluent and pass the solution through a Coulter Counter set at cell size 6 μm to count the cells.
  12. Dilute each cell population to a concentration of approximately 2x107 cells/ml in HBSS.
  13. Combine equal volumes of each cell population to make a single cell suspension mixture containing all three labeled populations.
  14. Check the appearance of the three cell populations on a flow cytometer.
Cytotoxicity assay
  1. Inject 200 μl of CFSE-labeled cell solution from step 25 into the tail vein of each immunized mouse (4 x 106 cells/mouse).
  2. Two days after injection of CFSE-labeled cells, collect about 200 μl of blood from the orbital sinus of each mouse in FACS tubes containing FACS buffer.
  3. Centrifuge the blood lymphocytes at 1500 rpm for 5 minutes and aspirate the supernatant.
  4. Resuspend the pelleted cells in 1 ml of BD FACS Lysing Solution, mix and wait until solution becomes transparent.
  5. Centrifuge the blood lymphocytes at 1500 rpm for 5 minutes and decant the supernatant.
  6. Resuspend in FACS buffer and acquire events on flow cytometer.
  7. Calculate the percent NK and CTL cell targets killed (Figure 3):
% targets killed = [1-(target cells/control cells)/(ratio of target cells:control cells in Tap-/-)] x 100
The ratio of target cells:control cells in Tap-/- mice is used to normalize for experiment-to-experiment variations in the numbers of control, CTL target, and NK target cells injected.  Table 1 shows an example of raw data obtained from this screen.
 
Table 1. Example of data from in vivo NK cell and CD8+ T cell cytotoxicity screen
Mouse
# control cells
# CTL targets
# NK targets
ratio of CTL target cells:control cells
ratio of NK target cells:control cells
TAP KO 1
125.00
228.00
62.00
1.82
0.50
TAP KO 2
186.00
250.00
56.00
1.34
0.30
TAP KO 3
186.00
240.00
68.00
1.29
0.37
TAP KO 4
163.00
231.00
56.00
1.42
0.34
 
 
 
mean
1.47
0.38
 
 
 
 
 
 
Mouse
# control cells
# CTL targets
# NK targets
% CTL killing
% NK killing
TAP KO no1
125.00
228.00
62.00
-24.17
-31.72
TAP KO no2
186.00
250.00
56.00
8.50
20.05
TAP KO no3
186.00
240.00
68.00
12.16
2.91
TAP KO no4
163.00
231.00
56.00
3.52
8.76
B6 immunized 1
171.00
31.00
2.00
87.66
96.89
B6 immunized 2
139.00
19.00
2.00
90.69
96.18
B6 immunized 3
158.00
32.00
14.00
86.21
76.47
B6 immunized 4
169.00
27.00
4.00
89.12
93.71
B6 immunized 5
171.00
31.00
3.00
87.66
95.34
B6 immunized 6
8.00
1.00
0.00
91.49
100.00
A2083
61.00
7.00
1.00
92.19
95.65
A2086
111.00
47.00
44.00
71.17
-5.27*
A2087
60.00
4.00
1.00
95.46
95.57
A2088
78.00
3.00
0.00
97.38
100.00
A2089
105.00
19.00
7.00
87.68
82.30
A2080
144.00
38.00
2.00
82.03
96.31
A2081
137.00
32.00
5.00
84.10
90.31
A2082
159.00
36.00
6.00
84.59
89.98
A2095
106.00
22.00
8.00
85.87
79.96
A2096
73.00
13.00
2.00
87.88
92.72
A2077
140.00
11.00
2.00
94.65
96.21
A2078
104.00
32.00
1.00
79.05
97.45
A2079
58.00
7.00
0.00
91.78
100.00
A2074
152.00
29.00
2.00
87.01
96.51
A2075
172.00
28.00
6.00
88.92
90.74
A2076
114.00
26.00
1.00
84.47
97.67
10601
111.00
85.00
1.00
47.87*
97.61
10617
158.00
38.00
8.00
83.63
86.55
10618
127.00
64.00
5.00
65.69
89.54
10619
140.00
50.00
8.00
75.69
84.82

*Mouse A2086 and mouse 10601 are putative NK- and CTL-defective mutants, respectively.

 
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.

 

Alleles Identified
43jl_nk
elektra
Endeka
ganymede
hebe
iconoclast
Incognito
Joker
king
Leopard
panr2
poison
salt_and_pepper
sphinx
styx
triaka
zeitgeist
References