Induced MHCII expression in vivo and in vitro. (A-B) LN cells of Aα^-/-, RFX5^-/-, and CIITA^-/- mice that did (TTX) and did not undergo transplantation and control C57BL/6 mice were analyzed for I-A^b, B220, and CD11c expression. The dot plots show I-A^b expression on gated B220⁺CD11c^- cells. The percentage of MHCII⁺ cells is indicated in the respective windows. Parts A and B are from independent experiments. (C) I-A^b-expressing and I-Ab-negative B cells and DCs of CIITA^-/- animals that underwent transplantation were sorted by FACS, and the genotype of the cells was assessed by a 3-point PCR for the WT allele (bottom row, top band) and KO allele (bottom row, bottom band). The products of the PCR reaction using primers for only the WT fragment were hybridized with a CIITA-specific probe (top row). Shown are results from 2 independent sorting experiments (left). The PCR reactions were standardized based on cell number, and the sensitivity of amplification was determined by dilution of WT cells in cells from CIITA^-/- animals at ratios of 1:1 to 1:10 000 as indicated (right). (D) Splenocytes of Aα^-/-, RFX5^-/-, CIITA^-/-, CR^-/-, and C57BL/6 mice that did not undergo transplantation were cultured O/N in the presence (bold line) or absence (thin line) of anti-CD40. The histograms show I-A^b expression on CD19⁺ cells of the indicated mouse strains. The Aα^-/- control is shown as a dotted line in each histogram. (E) Collagenase-digested spleen cells of the same mouse strains as shown in panel D were incubated O/N in medium without additional stimuli. Cells were stained with anti-CD11c and anti-I-A^b and gated on CD11c⁺ DCs. Percent of MHCII⁺ cells in the indicated gates is given in the figure. The Aα^-/- control is shown as dotted line. (F) The expression of I-A^b α- and β-chain in magnetically purified splenic B cells of the indicated mouse strains was analyzed by qRT-PCR after overnight culture in the presence or absence of 10 μg/mL LPS. Analysis of β-actin expression was used for standardization. Shown are expression levels relative to the ones of untreated B cells from C57BL/6 mice. The error bars indicate the standard deviation. LPS indicates lipopolysaccharide.