Spleens
were isolated from OT-I and OT-II mice, passed through a cell strainer,
and treated with ammonium chloride to lyse red blood cells. The remaining
white blood cells were sorted for CD8a (Ly-2) and CD4 (L3T4) T-cells
with MicroBeads (Miltenyi, Bergisch Gladbach, Germany). The sorted
T-cells were stained with CFSE (Invitrogen) (20 μM). Then, they
were added to BMDCs activated for 24 h with either PBS, a mixture
of CpG (0.2 μM) and OVA (79 nM), a CpG–OVA conjugate
(equivalent to 0.2 μM CpG and 79 nM OVA), or activated for 3
h with SIINFEKL (2.6 μM) or OVA323-339 (1.4 μM) at a BMDC–T-cell
ratio of 1:10. After 72 h, the cells were stained with LIVE/DEAD Fixable
Near-IR Dead Cell Stain, treated with CD16/CD32 Fc-blocking antibody,
and then labeled with the following antibodies (BioLegend): CD3-PE-CF594
(clone 145-2C11) to distinguish T-cells and CD8α-APC (clone
53–6.7) or CD4-APC (clone RM4-5). Fluorescence was measured
using a Gallios flow cytometer and analyzed using FlowJo software
version 8.8.6 (TreeStar Inc., Ashland, OR). Percent proliferation
of T-cells was calculated using the “Proliferation”
feature in FlowJo, which assesses the proliferation peaks of CFSE-stained
cells. One-way ANOVA with Dunnett’s post hoc test was performed
using GraphPad Prism version 6.0b.
were isolated from OT-I and OT-II mice, passed through a cell strainer,
and treated with ammonium chloride to lyse red blood cells. The remaining
white blood cells were sorted for CD8a (Ly-2) and CD4 (L3T4) T-cells
with MicroBeads (Miltenyi, Bergisch Gladbach, Germany). The sorted
T-cells were stained with CFSE (Invitrogen) (20 μM). Then, they
were added to BMDCs activated for 24 h with either PBS, a mixture
of CpG (0.2 μM) and OVA (79 nM), a CpG–OVA conjugate
(equivalent to 0.2 μM CpG and 79 nM OVA), or activated for 3
h with SIINFEKL (2.6 μM) or OVA323-339 (1.4 μM) at a BMDC–T-cell
ratio of 1:10. After 72 h, the cells were stained with LIVE/DEAD Fixable
Near-IR Dead Cell Stain, treated with CD16/CD32 Fc-blocking antibody,
and then labeled with the following antibodies (BioLegend): CD3-PE-CF594
(clone 145-2C11) to distinguish T-cells and CD8α-APC (clone
53–6.7) or CD4-APC (clone RM4-5). Fluorescence was measured
using a Gallios flow cytometer and analyzed using FlowJo software
version 8.8.6 (TreeStar Inc., Ashland, OR). Percent proliferation
of T-cells was calculated using the “Proliferation”
feature in FlowJo, which assesses the proliferation peaks of CFSE-stained
cells. One-way ANOVA with Dunnett’s post hoc test was performed
using GraphPad Prism version 6.0b.