Draining lymph nodes and spleen were used to gather activated T cells. Ovalbumin (Sigma-Aldrich, St. Louis, MO) or HSVgB (produced in the lab using baculovirus expression) were decontaminated of lipopolysaccharide (LPS) using a Triton X-114 LPS detoxification method 70 (link) were used in combination with polyI:C (GE Healthcare), pam3cys [N-palmitoyl-S-2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteine-(S)serine-(S)lysine 4] (InvivoGen, San Diego, CA), anti-CD40 (clone FGK4.5, BioXCell, West Lebanon, NH) or the TLR7a agonist, 3M012, conjugated to Ovalbumin 32 (link),33 (link). For pathogen challenge, female B6 mice were injected with either 1×107 pfu/mouse of Vaccinia Virus Western Reserve Strain expressing the Ovalbumin protein (VV-ova) or 1×105 Listeria Monocytogenes (Lm) expressing whole Ovalbumin protein (Lm-ova).
Protocol detail
Generating Antigen-Specific T Cells
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Baculovirus
Clone
Cysteine
Detoxification
Female
Lipopolysaccharide
Listeria monocytogenes
Lymph nodes
Lysine
Mouse
Ovalbumin
Pam3cys
Pathogen
Protein
Serine
Spleen
Strain
T cells
Triton x 114
Vaccinia virus
Corresponding Organization :
Other organizations : University of Colorado Denver
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Variable analysis
independent variables
- Ovalbumin (Sigma-Aldrich, St. Louis, MO) or HSVgB (produced in the lab using baculovirus expression)
- PolyI:C (GE Healthcare)
- Pam3cys [N-palmitoyl-S-2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteine-(S)serine-(S)lysine 4] (InvivoGen, San Diego, CA)
- Anti-CD40 (clone FGK4.5, BioXCell, West Lebanon, NH)
- The TLR7a agonist, 3M012, conjugated to ovalbumin
- Vaccinia Virus Western Reserve Strain expressing the ovalbumin protein (VV-ova)
- Listeria Monocytogenes (Lm) expressing whole Ovalbumin protein (Lm-ova)
- Activated T cells
- Female B6 mice
- Positive control: Ovalbumin (Sigma-Aldrich, St. Louis, MO) or HSVgB (produced in the lab using baculovirus expression)
- Negative control: Not explicitly mentioned
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