|Screen||LCMV Clearance Screen|
|Posted On||02/18/2010 12:24 PM|
|Author||Owen M. Siggs|
|Science Writer||Nora G. Smart|
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)
Dulbecco’s modified eagle medium (Mediatech Inc., Herndon, VA)
3% (v/v) heat-inactivated fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA)
Dulbecco’s modified eagle medium
10% (v/v) heat-inactivated fetal bovine serum
200 IU/mL penicillin
200 mg/mL streptomycin)
Dissolve 1% (w/v) agarose (Invitrogen UltraPure) in sterile milli-Q water
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)
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.
LCMV plaque-forming assay
1. Day, C. L., Kaufmann, D. E., Kiepiela, P., Brown, J. A., Moodley, E. S., Reddy, S., Mackey, E. W., Miller, J. D., Leslie, A. J., DePierres, C., Mncube, Z., Duraiswamy, J., Zhu, B., Eichbaum, Q., Altfeld, M., Wherry, E. J., Coovadia, H. M., Goulder, P. J., Klenerman, P., Ahmed, R., Freeman, G. J., and Walker, B. D. (2006) PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression, Nature 443, 350-354.
2. Brooks, D. G., Trifilo, M. J., Edelmann, K. H., Teyton, L., McGavern, D. B., and Oldstone, M. B. (2006) Interleukin-10 determines viral clearance or persistence in vivo, Nat. Med. 12, 1301-1309.
3. Ejrnaes, M., Filippi, C. M., Martinic, M. M., Ling, E. M., Togher, L. M., Crotty, S., and von Herrath, M. G. (2006) Resolution of a chronic viral infection after interleukin-10 receptor blockade, J Exp. Med. 203, 2461-2472.
4. Barber, D. L., Wherry, E. J., Masopust, D., Zhu, B., Allison, J. P., Sharpe, A. H., Freeman, G. J., and Ahmed, R. (2006) Restoring function in exhausted CD8 T cells during chronic viral infection, Nature 439, 682-687.