T cell function and analysis

For analysis of T cell responses, spleens were dissociated and filtered through a 70μm cell strainer. Red blood cells were lysed with Red Blood Cell Lysing Buffer (Sigma, St. Louis, MO). For peptide stimulation assays, splenocytes were stimulated for 4 hours with 1μM OVA_257–264 (SIINFEKL), B8R_20-27, A42R_88–96 or LLO_190–201 peptide in the presence of brefeldin A (GolgiPlug, BD Biosciences, San Jose, CA). Peptides for stimulation were obtained from A&A Labs (San Diego, CA, USA) and reconstituted in DMSO. Unstimulated controls (DMSO only) were used to assess nonspecific protein production for each animal. Cells were stained for surface antigens, and then fixed (Cytofix/Cytoperm buffer, BD Bioscience) and stored at -80C (in Cytofix/Cytoperm buffer) until further analysis. For intracellular cytokine staining, frozen cells were thawed, permeabilized (Perm Wash buffer, BD Biosciences), and stained for intracellular IFN-γ. To assess TipDC/DC activation, splenocytes were processed as described above and incubated 4hs at 37C, 5% CO₂ with or without 10⁷ cfu/ml heat killed L. monocytogenes (HKLm). Cells were stained and iNOS intracellular staining was performed as described above. Samples were acquired on an LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo™ 10.2 (FlowJo LLC, Ashland, OR).

Spleens were harvested from donor mice and either CD8⁺ or total T cells purified by negative selection (EasySep™ Mouse CD8⁺ T cell and EasySep™ Mouse T cell isolation kits, StemCell Technologies, Vancouver, Canada). Prior to adoptive transfer, cells were stained to confirm purity of CD8⁺ T cells and total T cells (>90%).

For in vivo antigen presentation experiments, single-cell suspensions of purified OT-1 CD8⁺ T cells were stained with 5, 6-carboxy-succinimidyl-fluoresceine-ester (CFSE, Molecular Probes, Eugene, OR) 10 minutes at 37C. Reactions were stopped with cold PBS and resuspended in the desired volume. Mice were injected with the CFSE-CD8⁺ T cells and spleens removed and processed after 3 days. Staining was performed as described and analysis conducted with FlowJo 10.2. Mitotic events were determined as described (22).

To evaluate the immune response, 10,000 purified OT-1 CD8⁺ T cells were transferred into Cre^-SOCS1^f/f and CD11c- Cre⁺ SOCS1^f/f mice 1 day prior to immunization with ΔactA-QV. Spleens were harvested and processed for flow cytometry 7 days later.

For reconstitution of RAG1^-/- knockout hosts, 2×10⁷ purified total T cells from Cre^-SOCS1^f/f or CD11c- Cre⁺ SOCS1^f/f spleens and the following day they were infected with ΔactA-QV. Seven days after the immunization, spleens were removed and processed for staining.