In vitro antigen presentation assay
Single cell suspensions were generated from lymph nodes of OT-I or OT-II
mice and T cells were purified as previously described. Cells were
washed with PBS 0.1% bovine serum albumin (BSA) and labeled with
CellTrace Violet (CTV). Splenic wild type or ITD/ITD DC populations were
isolated and cell sorted to purity as previously described. For antigen
presentation with soluble OVA, 2.5 x 104 cDCs were
incubated with 100 µg/mL OVA (Worthington Biochemical) and 0.5 µM CpG at
37ºC 10% CO2. Cells were washed extensively before
plating with 5 x 104 OT-I or OT-II cells in complete
RPMI and 20 ng/mL GM-CSF (Peprotech). For OVA-coated splenocytes, single
cell suspensions were generated from BM-1 (for OT-I) or
IAα-/- (for OT-II) mice. Red blood cells were lysed
and splenocytes were incubated with 10 mg/mL ovalbumin (Sigma) in
KDS-RPMI at 37ºC for 10 mins. Cells were washed to remove excess OVA. 2
x 105 OVA-coated splenocytes were incubated with 2.5 x
104 cDCs and 5 x 104 OT-I or OT-II
cells in complete RPMI and 20 ng/mL GM-CSF (Peprotech). For both soluble
OVA and OVA-coated splenocytes, cells were incubated for three days and
stained with mAbs specific for CD8 (53-6.7, BioLegend) or CD4 (GK1.5;
Walter and Eliza Hall Antibody Facility). The number of divided OT-II
and OT-I was determined as the number of CD4+ or
CD8+ Ly5.1+ cells that had undergone
CTV dilution. To obtain absolute cell numbers, an internal microsphere
counting standard (BD Biosciences) was used.