To discriminate between CD8 T cells in tissue parenchyma versus tissue vasculature, i.v. injected Ab was used as previously described (28 (link)). Briefly, 3μg of anti-CD8α Ab (53-6.7, Biolegend, San Diego, CA) was injected i.v. and allowed to circulate for three minutes prior to mouse sacrifice.
Organs were harvested and digested as previously described (29 (link)). For isolation of small intestinal intraepithelial lymphocytes (IEL), Peyer’s patches were removed, the small intestine was cut longitudinally and then laterally into small pieces. Pieces were incubated for 30 minutes with stirring at 37°C with 0.154mg/mL dithioerythritol (Sigma-Aldrich, St. Louis, MO) in 10% HBSS/HEPES. Female reproductive tract (FRT), lung and salivary gland (SG) tissues were cut into small pieces in RPMI 1640 containing 5% FBS, 2 mM MgCl2, 2 mM CaCl2 and 0.5mg/mL type IV collagenase for FRT (Sigma-Aldrich, St. Louis, MO) or 100 U/mL type I collagenase for lung and SG (Worthington, Lakewood, NJ) and incubated for 1 hr at 37°C with stirring. After enzymatic digestion, the remaining tissue pieces were mechanically disrupted using a gentleMACs dissociator (Miltenyi Biotec, San Diego, CA). The liver was mechanically dissociated by pushing the tissue through a 70μm-cell strainer. Single cell suspensions of IEL, FRT, lung, liver and SG were further separated using a 44/67% Percoll (GE Healthcare Life Sciences, Pittsburgh, PA) density gradient. Spleen and lymph nodes (LN) were dissociated mechanically. Splenocytes and blood were treated with ACK lysis buffer to lyse red blood cells.
The following antibodies were used for flow cytometry: anti-KLRG1 (2F1), anti-Eomes (Dan11ma), anti-T-bet (4B10), anti-CD44 (IM7), anti-CD122 (TM-b1), anti-CD27 (LG.7F9), anti-CD69 (H1.2F3) (all from eBioscience, San Diego, CA), anti-CD8α (53-6.7, eBioscience, Biolegend, San Diego, CA), anti-CD103 (M290), anti-CD25 (PC61), anti-Bcl-2 (Bcl-2/100) and anti-CD127 (SB/199) (BD Biosciences, San Jose, CA). Cell viability was determined using Ghost DyeTM Red 780 (Tonbo Biosciences, San Diego, CA). Kb-SIINFEKL-specific CD8 T cells were identified using H-2Kb tetramers made in house containing the SIINFEKL peptide (New England Peptide, Gardener, MA). The BD Biosciences intracellular kit for cytokine staining and the eBioscience FoxP3 kit for transcription factor staining were used in accordance with manufacturer’s directions. Peptide stimulation was performed as previously described (30 (link)). Briefly, splenocytes were plated in RPMI 1640 containing 10% FBS, 1× NEAA, 2mM L-glutamine, 1mM sodium pyruvate, 1× penicillin/streptomycin and 0.05mM β-mercaptoethanol and incubated with 1ug/mL SIINFEKL peptide and 1ug/mL GolgiPlug (BD Biosciences, San Jose, CA) for four hours at 37°C. Cells were washed and stained with fixable LIVE/DEAD aqua dead cell stain (Life Technologies, San Diego, CA) before surface and intracellular staining. Samples were acquired on an LSRII flow cytometer (BD Biosciences, San Diego, CA).
Organs were harvested and digested as previously described (29 (link)). For isolation of small intestinal intraepithelial lymphocytes (IEL), Peyer’s patches were removed, the small intestine was cut longitudinally and then laterally into small pieces. Pieces were incubated for 30 minutes with stirring at 37°C with 0.154mg/mL dithioerythritol (Sigma-Aldrich, St. Louis, MO) in 10% HBSS/HEPES. Female reproductive tract (FRT), lung and salivary gland (SG) tissues were cut into small pieces in RPMI 1640 containing 5% FBS, 2 mM MgCl2, 2 mM CaCl2 and 0.5mg/mL type IV collagenase for FRT (Sigma-Aldrich, St. Louis, MO) or 100 U/mL type I collagenase for lung and SG (Worthington, Lakewood, NJ) and incubated for 1 hr at 37°C with stirring. After enzymatic digestion, the remaining tissue pieces were mechanically disrupted using a gentleMACs dissociator (Miltenyi Biotec, San Diego, CA). The liver was mechanically dissociated by pushing the tissue through a 70μm-cell strainer. Single cell suspensions of IEL, FRT, lung, liver and SG were further separated using a 44/67% Percoll (GE Healthcare Life Sciences, Pittsburgh, PA) density gradient. Spleen and lymph nodes (LN) were dissociated mechanically. Splenocytes and blood were treated with ACK lysis buffer to lyse red blood cells.
The following antibodies were used for flow cytometry: anti-KLRG1 (2F1), anti-Eomes (Dan11ma), anti-T-bet (4B10), anti-CD44 (IM7), anti-CD122 (TM-b1), anti-CD27 (LG.7F9), anti-CD69 (H1.2F3) (all from eBioscience, San Diego, CA), anti-CD8α (53-6.7, eBioscience, Biolegend, San Diego, CA), anti-CD103 (M290), anti-CD25 (PC61), anti-Bcl-2 (Bcl-2/100) and anti-CD127 (SB/199) (BD Biosciences, San Jose, CA). Cell viability was determined using Ghost DyeTM Red 780 (Tonbo Biosciences, San Diego, CA). Kb-SIINFEKL-specific CD8 T cells were identified using H-2Kb tetramers made in house containing the SIINFEKL peptide (New England Peptide, Gardener, MA). The BD Biosciences intracellular kit for cytokine staining and the eBioscience FoxP3 kit for transcription factor staining were used in accordance with manufacturer’s directions. Peptide stimulation was performed as previously described (30 (link)). Briefly, splenocytes were plated in RPMI 1640 containing 10% FBS, 1× NEAA, 2mM L-glutamine, 1mM sodium pyruvate, 1× penicillin/streptomycin and 0.05mM β-mercaptoethanol and incubated with 1ug/mL SIINFEKL peptide and 1ug/mL GolgiPlug (BD Biosciences, San Jose, CA) for four hours at 37°C. Cells were washed and stained with fixable LIVE/DEAD aqua dead cell stain (Life Technologies, San Diego, CA) before surface and intracellular staining. Samples were acquired on an LSRII flow cytometer (BD Biosciences, San Diego, CA).
