MHC class II-restricted-presentationMHC class II and DM molecules in chickens: different yet again

Abstract

The chicken MHC is simple and compact, with strong genetic associations for resistance to infectious disease, due at least in part to high expression of a single classical class I gene, BF2. The closely-linked TAP and tapasin genes are highly polymorphic, and co-evolve with the dominantly-expressed BF2 gene, due to the rarity of recombination between genes of this system. The classical class II system has some similarities to the class I system, with two class II B (BLB), one DMA and two DMB genes, all of which are closely-linked and polymorphic. Like β2-microglobulin, the class II A gene is monomorphic and located distant to the MHC. Only BLB2 is well-expressed in spleen and blood. The objective is to determine to what extent the chicken class II system follows the same principles as the class I system. RNA levels were determined by RT-PCR and RT-qPCR, proteins were detected by Western blot, flow cytometry and tissue section staining, and genes were knocked out (KO) by CRISPR-Cas9. RNAs for BLB2 and DMB2 are widely expressed, while BLB1 and DMB1 are only strongly expressed in gut tissues. Tissue staining shows very high DMB1 (and BLB1) staining in intestinal epithelial cells (IECs), while sorted IECs show similar levels for DMB1 and DMB2 by RT-PCR, but only DMB1 by western blot. Both BLB1 and BLB2 class II molecules mostly bear CLIP peptides in DMA KO cells, but some alleles are exquisitely DM-dependent and others less so. Both BLB1 and BLB2 are affected in DMB1 and DMB2 KO cells. The presence of high level DMB1 (and BLB1) expression in IECs but not elsewhere is unprecedented for class II molecules, and the exact ways in which the different DM loci and alleles affect the two class II molecules remain interesting questions still to answer.