In vitro EBV Infection of Mononuclear Cells that Have Been Cryo-preserved

Materials and Reagents

1. Cryo-preserved CBMC
   
2. Cryo-preserved PBMC
   
3. EBV strain B95-8 containing supernatant
   Note: The batch here had a titer of 2.5 x 10⁵ Ramos infectious units. RaIU, which was determined by infection of the EBV negative Burkitts lymphoma B-cell line Ramos, followed by anti-complement immunoflourescent assay (ACIF) to detect the number of infected cells.
   
4. RPMI 1640 (Life Technologies)
   
5. 10% heat-inactivated fetal-calf serum (Hyclone)
   
6. L-glutamine (2 mmol/l)
   
7. Penicillin G-sodium (100 U/ml)
   
8. Streptomycin sulfate (100 mg/ml) (Merck KGaA)
   
9. Complete cell-culture medium (see Recipes)
   

Equipment

1. 48-well flat-bottomed tissue-culture treated plates (Sarstedt AG)
2. Humified incubator with for adjustment of 37 °C and 5% CO₂ for cell culture
   

Procedure

Note about sample handling: Gentle and fast processing of samples during isolation of mononuclear cells contributes to higher B cell viability, which is central for successful in vitro infection.

1. Thaw CBMCs or PBMCs quickly at room temperature and wash two times with incomplete RPMI-1640 at 350-400 x g and 5-10 min at room temperature.
   
2. Determine cell numbers, and if applicable, divide cells to fractions that will be EBV infected and those that will serve as non-infected controls.
   Note: Remember that the cell numbers and concentrations in each well should be matched for the infected and non-infected fractions.
   
3. Wash an additional time with RPMI 1640.
   
4. For non-infected control cultures: Re-suspend cells in complete cell-culture medium to a concentration of 10⁶ cells/ml and allocate to the appropriate wells in the plate. Incubate at 37 °C and 5% CO₂.
   
5. For EBV infection, discard supernatant completely and resuspend 106 cells per 100 μl B95-8 virus–containing supernatant in a small tube. Incubate at 37 °C and 5% CO₂ for 90 min with gentle mixing by swirling the tube every 30 min.
   
6. Wash the cells once with complete cell-culture medium, discard supernatant and re-suspended in complete cell-culture medium to a concentration of 10⁶ cells/ml and then allocate to the appropriate wells in the plate.
   
7. Feed cells with complete medium on a weekly basis. The proportion of EBV-transformed B cells will increase with time and eventually these cells take over the entire culture. As we assessed the dynamics of NK and T cell and their functional capacity (Sohlberg et al., 2013; Saghafian-Hedengren et al., 2013) in our previous experimental settings, we found a 1-2 week-period of time suitable for this purpose.
   
8. The presence of EBV-infected cells can be confirmed by immunofluorescence staining for latent membrane protein-1 and EBV nuclear Ag 2 (Rasul et al., 2012).
   

Representative data

Refer to Figure 1 in Sohlberg et al. (2013) for representative data on B cell characteristics following in vitro EBV infection of B cells.

Notes

1. Careful and fast processing of samples during isolation of mononuclear cells contributes to higher B cell viability, which is central for successful downstream in vitro EBV infection.
   
2. This protocol uses cells from EBV seronegative donors. Deplete T cells (by for instance magnetic-based methods to remove CD3⁺ cells from StemCell^TM or Miltenyi Biotech) prior to EBV in vitro infection in case samples are from EBV seropositive donors.
   

Recipes

1. Complete cell-culture medium
   10% heat-inactivated fetal-calf serum
   L-glutamine (2 mmol/l)
   Penicillin G-sodium (100 U/ml)
   Streptomycin sulfate (100 mg/ml)
   

Acknowledgments

This protocol has been adapted from the previously published paper Sohlberg et al., (2013). At the time of development and implementation of this protocol, the following sources of funding were used: Swedish Research Council Grants 57X-15160-07-03 and 57X-15160-10-4, the Swedish Association for Allergology, the Ragnar Söderberg Foundation, the Ellen, Walter and Lennart Hesselman Foundation, the Konsul Th. C. Bergh Foundation, the Golden Jubilee Memorial Foundation, the Petrus and Augusta Hedlund Foundation, the Crown Princess Lovisa’s/Axel Tielman’s Foundation, the Swedish Cancer Society, and the Karolinska Institute. E.R. and N.N. are recipients of cancer research fellowships from the Cancer Research Institute (New York)/Concern Foundation (Los Angeles). We thank Ehsan Rasul and Eva Klein (Department of Microbiology, Tumor and Cell Biology, KI, Stockholm, Sweden) for their contribution in optimizing the protocol.

References

1. Rasul, A. E., Nagy, N., Sohlberg, E., Adori, M., Claesson, H. E., Klein, G. and Klein, E. (2012). Simultaneous detection of the two main proliferation driving EBV encoded proteins, EBNA-2 and LMP-1 in single B cells. J Immunol Methods 385(1-2): 60-70.
   
2. Saghafian-Hedengren, S., Sohlberg, E., Theorell, J., Carvalho-Queiroz, C., Nagy, N., Persson, J. O., Nilsson, C., Bryceson, Y. T. and Sverremark-Ekström, E. (2013). Epstein-barr virus coinfection in children boosts cytomegalovirus-induced differentiation of natural killer cells. J Virol 87(24): 13446-13455.
   
3. Sohlberg, E., Saghafian-Hedengren, S., Rasul, E., Marchini, G., Nilsson, C., Klein, E., Nagy, N., Sverremark-Ekström, E. (2013). Cytomegalovirus-seropositive children show inhibition of in vitro EBV infection that is associated with CD8⁺CD57⁺ T cell enrichment and IFN-γ. J Immunol 191(11): 5669-5676.