Skip to main content
. Author manuscript; available in PMC: 2021 Aug 15.
Published in final edited form as: J Immunol. 2020 Jul 20;205(4):923–935. doi: 10.4049/jimmunol.2000476

FIGURE 3.

FIGURE 3.

Analysis of DOA and DOB allelic variations and their natural haplotype combinations.

(A) Schematic of the DOB common alleles relative to conical DOB*0101 gene. DOB contains the following domains as indicated: a signal sequence (SS), a MHCII beta-like (beta-like), an immunoglobulin-like (Ig), a transmembrane (TM), and a cytoplasmic tail (Ct). The four other common alleles have single missense mutations that result in single amino acid changes which are shown. Numbering indicates amino acid number starting before (negative) or after signal sequence cleavage and the amino acid encoded by the DOB*0101 gene is shown in the rectangle domains followed by an arrow showing amino acid changes for DOB*0102, DOB*0103, DOB*0104, and DOB*0105 alleles.

(B) Association of DOA and DOB allelic variants into haplotypes and their corresponding frequencies shown as a pie charts for all individuals (ALL) covered by the 1000GP, and the corresponding subpopulations (AFR = African; AMR: American; EAS: East Asians; EUR: European; SAS: South Asian). Enlarged pie pieces show frequencies of DOA*0102. The table on the right side depicts the color code used for each haplotype in the pie charts and the frequency of homozygous (homo) and heterozygous allotypes containing DOA allelic variants different than the canonical DOA*0101 is given for all individuals and each subpopulation. Combinations that were not found in the 1000GP database are left blank. The following SNPs were used to search 1000GP DOA*0102, (rs11575906); DOA*0103 (rs41542323), DOA*0104 (rs41541116), DOB*0102 (rs2071554), DOB*0103 (rs2621330), DOB*0104 (rs2070121), and DOB*0105 (rs11575907). The two SNPs resulting in substitutions for the DOA*0103 and DOA*0104 allelic variants in the IMGT-HLA are not found in the 1000GP dataset and are not shown.

(C) To determine if natural DOA-DOB haplotype combinations described in (B) resulted in DO proteins with altered function, HeLa.CIITA cells were transiently transfected with vectors encoding DOA*0101 and DOB*0101, DOB*0102, DOB*0103, DOB*0104, or DOB*0105 or DOA*0102 and DOB*0101, DOB*0102, DOB*0104, or DOB*0105 and the gMFI values for MHCII, DM, MHCII-CLIP and DO 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/EV-AcGFP control from each sample. Data from 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 (1.00 ± 0.04), DM (0.86 ± 0.16), MHCII-CLIP (1.02 ± 0.05), DO (0.96 ± 0.11)). Blue bars indicate haplotype combinations with reduced DO levels.

(D) DO proteins resulting from DOA*0102 paired with four DOB allotypes have altered function. MHCII-CLIP:DO ratios for each of the natural DOA-DOB combinations were calculated and normalized to that of the DOA*0101/DOB*0101 combination (black bar). The gray zone corresponds to the average of DOA*0101 with each of the five common DOB alleles plus or minus two standard deviations (1.09 ± 0.19). Blue bars indicate haplotype combinations with function.