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. Author manuscript; available in PMC: 2019 Sep 1.
Published in final edited form as: Cell Immunol. 2018 May 14;331:38–48. doi: 10.1016/j.cellimm.2018.05.003

FIGURE 1.

FIGURE 1

Downregulation of Aire-dependent genes and immune cell distribution in Aire-deficient HLA-DR2b and HLA–DR4 tg mice. Genomic DNA extracted from off-springs of Aire+/− HLA-DR2b or Aire+/− HLA-DR4 breeders were genotyped by PCR with primers for Aire, Ia, HLA-DR2b and HLA-DR4 genes as described in Materials and Methods. (A) Shown is the representative PCR result run on 1.8% agarose gel. Arrows indicate Aire knockout band of 750 bp and Aire Wt band of 1200 bp respectively. All the mice are either positive for HLA-DR2b or HLA-DR4 and show absence of Ia. M, marker (100 bp low scale DNA ladder); −/−, Aire knockout; +/+, Aire Wt; +/−, Aire heterozygous. (B and C) Flow cytometry analysis of splenocytes from C57BL/6 Wt, HLA-DR2b, and HLA-DR4 tg mice using fluorochrome conjugated anti-CD45, anti-Ia and anti-HLA-DR mAb. (D) Aire+/+ and Aire−/− HLA-DR2b tg or (E) Aire+/+ and Aire−/− HLA-DR4 tg mice were euthanized, their thymi were harvested and RNA was extracted and used for performing qPCR against a panel of Aire-dependent genes as described in Materials and Methods. (F – J) Thymus and spleens of HLA-DR2b or HLA–DR4 mice were procured and stained with fluorochrome conjugated anti-CD4, CD8, CD11c, CD11b, CD25, Foxp3 mAbs followed by flow cytometry analysis as described in Materials and Methods. (F) Shown are representative flow plots of CD4+, CD8+ and double positive thymocytes in (F) Aire+/+ HLA-DR4 vs Aire−/− HLA-DR4 mice. (G, H) Quantification for percentages of CD4+, CD8+ and double positive thymocytes in (G) Aire−/− HLA-DR2b and (H) Aire−/− HLA-DR4 mice in comparison with their Aire+/+ HLA-DR littermates. (I, J) Distribution of CD4+, CD8+, CD11c+, CD11b+ cells in the spleens of Aire−/− HLA-DR2b (I) and Aire−/− HLA-DR4 mice (J) in comparison with their respective Aire+/+ HLA-DR littermates. Data are representative for two independent experiments (n= 3 – 4 mice/groups), (Mean ± SEM) * indicates significant difference between HLA-DR2b group and C57BL/6 group, # indicates significant difference between HLA-DR4 group and C57BL/6 group; * indicates significant difference between Aire−/− group and Aire+/+ group.

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