Protocols


Screen (pdf version)
ScreenLCMV Clearance Screen
Posted On02/18/2010 12:24 PM
AuthorOwen M. Siggs
Science WriterNora G. Smart
Background
The clone 13 LCMV clearance screen was designed to detect mutations that enhance the efficiency of virus clearance in a chronic infection model.
 
While the immune system provides sterilizing immunity against some viruses, chronic persistent infection is a common outcome, witnessed, for example, in hepatitis C or HIV infections.  The clone 13 variant of lymphocytic choriomeningitis virus (LCMV), a negative single stranded RNA virus belonging to the family Arenaviridae (which also includes Lassa fever virus), can establish chronic infection in mice accompanied by functional impairment of effector T cells, also known as immune exhaustion.  Mutations in either of two host genes, programmed cell death 1 (Pdcd1) and interleukin-10 (Il-10), are known to restrict persistent infection of mice with clone 13 LCMV (1-3). 
 
Expression of PD-1, an inhibitory receptor of the CD28 family, positively correlates with immune exhaustion during chronic HIV infection of humans (1) or LCMV infection of mice (4).  Treatment of chronically infected mice with anti-PD-1 antagonist antibody can lead to viral clearance, whereas chronic infection of mice deficient in the PD-1 ligand, PD-L1, results in pronounced immunopathology and subsequent death, despite a normal capacity to control acute LCMV infection.  Unlike PD-L1-deficient mice, those lacking the cytokine IL-10 can clear chronic LCMV infection without any immunopathology (2;3).  IL-10 deficiency provides protection from several other chronic murine pathogens as well (5). 
 
Upon infection with clone 13 LCMV, wild type C57BL/6 mice typically yield serum titers in the order of 104-105 PFU at day 9.  The same titers can be seen in serum at day 42 (2).  By contrast, Il10-/- mice have serum titers below the assay detection limit of 2x102 PFU.  The LCMV screen is designed to identify mutations that prevent chronic infection and immune exhaustion.  Three wild type and three Il10-/- mice are used as controls in every experiment along with roughly 30 mutant G3 mice. 
 
G3 mutants with low serum titers to LCMV will be identified, as well as mice that do not survive infection, since these will likely have died due to immunopathology.
Reagents and Solutions
EDTA (Greiner Bio-One, San Diego, CA)
 
Vero 76 cells (ATCC #CLR-1587)
 
1.2 ml FACS tubes (Costar)
 
LCMV medium
Dulbecco’s modified eagle medium (Mediatech Inc., Herndon, VA)
3% (v/v) heat-inactivated fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA)
 
Vero medium
Dulbecco’s modified eagle medium
10% (v/v) heat-inactivated fetal bovine serum
200 IU/mL penicillin
200 mg/mL streptomycin)
 
1% agarose
Dissolve 1% (w/v) agarose (Invitrogen UltraPure) in sterile milli-Q water
Autoclave
 
2x 199 medium
100 ml of medium 199 2x with Earle’s salts (Sigma)
10 ml FBS
2 ml 100x L-glutamine (Gibco)
2 ml 100x penicillin/streptomycin (Gibco)
Note: If medium becomes yellow, add 4.4g/l Sodium Bicarbonate (pH)
 
Fixation solution
Dilute 37% formaldehyde stock (Sigma) to 25% with milli-Q water.
 
Cell staining solution
1% crystal violet (w/v) in 20% ethanol (store up to 1 yr at RT)
Before use, dilute 1 part with 9 parts milli-Q water.
Method
LCMV infection
  1. Inject 2x106 PFU clone 13 LCMV into the lateral tail vein in a volume of 200μL LCMV medium.
  2. Collect blood from the retro-orbital plexus nine days after infection in EDTA-coated tubes, and collect serum by centrifugation.
  3. Store serum in 96 well plates at -70oC until use in plaque forming assay. Thaw on ice before use and avoid repeated freeze-thaw cycles.
LCMV plaque-forming assay
  1. Plate Vero cells on 6-well plates in Vero medium at 2x106 cells/plate, in a volume of 2mL/well. Incubate O/N. Cells should be 50-60% confluent immediately prior to assay. Overconfluence will prevent formation of defined plaques.
  2. Prepare FACS tubes for dilution: add 630μl of Vero medium to each of two FACS tubes per sample.
  3. Add 70μL LCMV infected mouse serum (or LCMV stock to be titered) to 630μL Vero medium. To titer stock: make 7 1/10 dilutions starting from 10-2. To titer serum: 10-2 and 10-3.
     
  4. Remove medium from Vero cells. Plate 500ul of dilutions or media alone.
  5. Incubate 1h at 37ºC. Rock every 10 minutes.
  6. Prepare agarose overlay: microwave 1% agarose and cool down to ~50ºC in a water bath. Warm the 2x 199 medium to 37 ºC, and immediately before applying mix the two. Note: The temperature of this mix should be between 40 and 44ºC. If too high, Vero cells will be killed. If too low, it will solidify too soon and be impossible to transfer into the well.
  7. After 1h, aspirate the inoculum, and add 3 ml of molten overlay mix to each well. Allow it to solidify (~10min) before returning the plate into the incubator. Incubation: 4 days.
  8. Fix and stain cells:
    a.       Add 800μL fixation solution/well. Incubate for 2hrs at RT, then drain off and carefully remove the agarose plug.
    b.       Add 800μL cell staining solution. Incubate 1 to 2hrs at RT.
    c.       Wash plates with running tap water.
  9. PFU count: if 36 plaques at 10-6 dilution. Concentration is: 36x2x106 PFU/mL.
Alleles Identified
rogue
References