Graham3 project protocol

Baseline T cell immune phenotypes in males of 104 F1 hybrid lines of Collaborative Cross strains of mice (CC RIX) predict virologic and disease control upon SARS-CoV infection   (2021)

Graham JB, Baric RS, Lund JM
With: Swarts JL, Leist SR, Schäfer A, Menachery VD, Gralinski LE, Jeng S, Miller DR, Mooney MA, McWeeney SK, Ferris MT, Pardo-Manuel de Villena F, Heise MT

Project protocol - Contents


General Information for All Procedures: F1 hybrid mice (CC RIX) were derived from intercrossing Collaborative Cross strains at UNC. Six to eight week old CC RIX male mice were transferred to the University of Washington. Two weeks later, these mice were used in the baseline flow cytometry studies for T cell subsets (Graham3, this study). Concurrently CC RIX female mice were transferred internally at UNC to a BSL-3 facility for SARS-CoV infection (see Graham2).

Procedure 1: Strain selection criteria


Introductory Comments: F1 hybrid mice (CC RIX) were generated based on the following criteria.

  1. Each CC strain used in an F1 cross had to be certified distributable.
  2. The UNC Systems Genetics Core Facility had to be able to provide a sufficient number of breeding animals.
  3. Each CC RIX had to have one parent with an H2Bb haplotype from either C57BL/6J or 129S1/SvImJ, and one parent with a haplotype from one of the other six CC founder strains. This design was selected such that antigen-specific T cell responses could be examined.
  4. Each CC had to be used at least once (preferably twice) as a dam, and once (preferably twice) as a sire in the relevant CC RIX.
  5. Two CC RIX lines were included which appeared twice in the screen, once measured in the beginning and once towards the end of the five year period to specifically assess and control for batch and seasonal effects.

Procedure 2: Flow cytometry of splenocytes (baseline, no infection)

Equipment, software, and supplies

  • Flow cytometer (Beckton-Dickinson, LSR II)
  • FlowJo software

Reagents and solutions

  • Monoclonal antibody CCR5, clone HM-CCR5
  • Monoclonal antibody CD3e, clone 145-2C11
  • Monoclonal antibody CD4, clone RM4-5
  • Monoclonal antibody CD8a, clone 53-6.7
  • Monoclonal antibody CD25, clone PC61.5
  • Monoclonal antibody CD28, clone 37.51
  • Monoclonal antibody CD44, clone IM7
  • Monoclonal antibody CD73, clone TY/11.8
  • Monoclonal antibody CD127, clone A7R34
  • Monoclonal antibody CTLA-4, clone UC10-4B9
  • Monoclonal antibody CXCR3, clone CXCR3-173
  • Monoclonal antibody Foxp3, clone FJK-16s
  • Monoclonal antibody GITR, clone DTA-1
  • Monoclonal antibody ICOS, clone 7E.17G9
  • Monoclonal antibody IFN-g, clone XMG1.2
  • Monoclonal antibody IL-17a, clone TC11-18H10.1
  • Monoclonal antibody Ki-67, clone SolA15
  • Monoclonal antibody Tbet
  • Monoclonal antibody TNF, clone MP6-XT22
  • Aqua - 405 nm excitation, live/dead gating


Introductory Comments: All monoclonal antibodies were tested using cells from the 8 CC founder strains to confirm that antibody clones were compatible with CC mice prior to being used for testing.

  1. Mice are euthanized and spleens harvested; splenocytes are prepared for staining as previously described (Graham et al., 2015).
  2. The following monoclonal antibodies are used for the Treg panel: CD4, Foxp3, CCR5, CD25, CD44, CD73, CTLA-4, CXCR3, GITR, and ICOS.
  3. The following monoclonal antibodies are used for the T cell panel: CD3, CD4, CD8, CCR5, CD25, CD44, CXCR3, ICOS, and Ki67.
  4. The following monoclonal antibodies are used for intracellular cytokine staining following stimulation with anti-CD3 and anti-CD28: CD3, CD4, CD8, IFNg, IL17, and TNF.

Investigator Notes: SARS-CoV infection of CC mice results in a variety of viral load trajectories and disease outcomes. Overall, a dysregulated, pro-inflammatory signature of circulating T cells at baseline was associated with severe disease upon infection. Our study serves as proof of concept that circulating T cell signatures at baseline can predict clinical and virologic outcomes upon SARS-CoV infection.


Primary References

Graham JB, Swarts JL, Leist SR, Schäfer A, Menachery VD, Gralinski LE, Jeng S, Miller DR, Mooney MA, McWeeney SK, Ferris MT, Pardo-Manuel de Villena F, Heise MT, Baric RS, Lund JM. Baseline T cell immune phenotypes predict virologic and disease control upon SARS-CoV infection in Collaborative Cross mice. PLoS Pathog. 2021 Jan;17(1):e1009287. doi: 10.1371/journal.ppat.1009287. Epub 2021 Jan 29.   PubMed 33513210     FullText

Protocol References

Graham JB, Thomas S, Swarts J, McMillan AA, Ferris MT, Suthar MS, Treuting PM, Ireton R, Gale M Jr, Lund JM. Genetic diversity in the collaborative cross model recapitulates human West Nile virus disease outcomes. MBio. 2015 May 5;6(3):e00493-15. doi: 10.1128/mBio.00493-15.   PubMed 25944860