FIGURE 5.

Functional Analysis of naturally occurring DOα variants.

(A and B) HeLa.CIITA cells were transiently transfected with a vector encoding DOB*0101 and vectors encoding each of the individual 21 DOA variants. Cells were analyzed by flow cytometry 72 hours later and the gMFI values for MHCII and MHCII-CLIP (A) and DM and DO (B) levels in mRuby+ AcGFP+ cells were determined as in Fig. 1. Background gMFI for MHCII-CLIP and DO was eliminated by subtracting the gMFI value of the EV-mRuby/DOB*0101-AcGFP control from each sample. Data from 4-5 independent experiments were normalized to the values obtained for DOA*0101/DOB*0101 combination (black bars). The gray zones correspond to the average of DOA*0101 with each of the five common DOB alleles plus or minus two standard deviations: MHCII (1.03 ± 0.06), DM (0.97 ± 0.09), MHCII-CLIP (0.90 ± 0.15), DO (0.95 ± 0.11).

(C) MHCII-CLIP:DO ratio for each of the DOA variants combined with DOB*0101. The ratios were normalized to that of the DOA*0101/DOB*0101 combination (black bar) form 4-5 independent experiments. Constructs transfected are indicated below the bar graphs. The gray zone corresponds to the average plus or minus two standard deviations of the values obtained from DOA*0101 combined with each of the five common DOB alleles (1.10 ± 0.19). DOα variants with altered function (increased-blue bars, decreased-red bars) were defined as possessing a MHCII-CLIP:DO ratio that fell outside this range.