Generation and expression analysis of BAC humanized mice carrying HLA-DP401 haplotype

Abstract

Background: Human leukocyte antigen (HLA)-DP is much less studied than other HLA class II antigens, that is, HLA-DR and HLA-DQ, etc. However, the accumulating data have suggested the important roles of DP-restricted responses in the context of cancer, allergy, and infectious disease. Lack of animal models expressing these genes as authentic cis-haplotypes blocks our understanding for the role of HLA-DP haplotypes in immunity.

Methods: To explore the potential cis-acting control elements involved in the transcriptional regulation of the HLA-DPA1/DPB1 gene, we performed the expression analysis using bacterial artificial chromosome (BAC)-based transgenic humanized mice in the C57BL/6 background, which carried the entire HLA-DP401 gene locus. We further developed a mouse model of Staphylococcus aureus pneumonia in HLA-DP401 humanized transgenic mice, and performed the analysis on the expression pattern of HLA-DP401 and immunological responses in the model.

Results: In this study, we screened and identified a BAC clone spanning the entire HLA-DP gene locus. DNA from this clone was analyzed for integrity by pulsed-field gel electrophoresis and then microinjected into fertilized mouse oocytes to produce transgenic founder animals. Nine sets of PCR primers for regional markers with an average distance of 15 kb between each primer were used to confirm the integrity of the transgene in the five transgenic lines carrying the HLA-DPA1/DPB1 gene. Transgene copy numbers were determined by real-time PCR analysis. HLA-DP401 gene expression was analyzed at the mRNA and protein level. Although infection with S aureus Newman did not alter the percentage of immune cells in the spleen and thymus from the HLA-DP401-H2-Aβ1 humanized mice. Increased expression of HLA-DP401 was observed in the thymus of the humanized mice infected by S aureus.

Conclusions: We generated several BAC transgenic mice, and analyzed the expression of HLA-DPA1/DPB1 in those mice. A model of Saureus-induced pneumonia in the HLA-DP401-H2-Aβ1^-/- humanized mice was further developed, and S aureus infection upregulated the HLA-DP401 expression in thymus of those humanized mice. These findings demonstrate the potential of those HLA-DPA1/DPB1 transgenic humanized mice for developing animal models of infectious diseases and MHC-associated immunological diseases.

Keywords: HLA‐DP4; Staphylococcus aureus pneumonia; bacterial artificial chromosome (BAC); gene expression; humanized mice.

FIGURE 1

Schematic diagram of the HLA‐DP4‐haplotype‐containing BAC. A, The original BAC CH501‐138A21 is ~124 kb in size, harboring the full HLA‐DP4 locus. Nine sets of PCR primers for regional markers with an average distance of 15 kb between each primer were used to identify the HLA‐DP BAC transgenes. The transgenic lines carrying the different fragments are indicated. BAC transgene integrity assayed by these primers is shown in B. Copy number is estimated by real‐time PCR, and shown in C. M 5Kb DNA marker

FIGURE 2

Expression level of the transgene is analyzed by real‐time PCR. A and D PBMC, B and F spleen, C and G lung. Labeling is as shown in Fig

FIGURE 3

Representative western blot revealed the tissue specificity of transgene expression with antibody against HLA‐DPA1/DPB1. The band at 29 kDa for HLA‐DPA1 is indicated: A, spleen; B, kidney. The band at 29 kDa for HLA‐DPB1 is indicated: C, spleen; D, kidney; E, lung; F, gut

FIGURE 4

Immunofluorescence staining reveal the expression of HLA‐DPA1/DPB1 in transgenic mice. A‐C HLA‐DPA1 in spleen from transgenic mice DP3‐3#, D‐F spleen from WT mice, G‐I HLA‐DPB1 in spleen from transgenic mice DP3‐3#, J‐L spleen from WT mice. Scale bar 50 µm

FIGURE 5

Immunofluorescence staining reveal the expression of HLA‐DPB1 in transgenic mice. A‐C HLA‐DPB1 in lung from transgenic mice DP3‐3#, D‐F lung from WT mice, G‐I HLA‐DPB1 in gut from transgenic mice DP3‐3#, D‐F gut from WT mice. Scale bar 50 µm

FIGURE 6

Positive and negative selection of CD4⁺ T cells in HLA‐DP401 humanized mice. A‐C, Splenic T cells from mice with the indicated genotypes were double‐stained with CD3, CD4 and CD8. D, Splenic B cells from mice with the indicated genotypes were CD19. Splenic cells were stained with antibodies against CD11c, CD11b, F4/80. Subsequently, cells were gated on CD11b⁺CD11c⁺ dendritic cells (E) or F4/80⁺ CD11b⁺ macrophage (F). (G), (H) and (I) thymic T cells from mice with the indicated genotypes were double‐stained with CD3, CD4 and CD8. *P < .05

FIGURE 7

HLA‐DP expression on spleen and thymus cells from HLA‐DP401 humanized mice. HLA‐DP‐H2Aβ1^−/− and H2Aβ1^−/− mice were intranasally infected with Staphylococcus aureus. Spleen and thymus were collected 3 d later, HLA‐DP expression were quantified by FACS analysis. Y‐axis represents percentages in total spleen and thymus, each bar represents mean ± SD from three mice. The expression of HLA‐DP in thymus significantly increased in Staphylococcus aureus pneumonia of HLA‐DP humanized mice. A, The percentage of DP⁺ cells in total thymus. B, The percentage of DP⁺CD4⁺ T cells in total thymus. C, The percentage of DP⁺CD8⁺ T cells in total thymus. D, The percentage of DP⁺ cells in total spleen. E, The percentage of DP⁺CD19⁺ B cells in total spleen. F, The percentage of DP⁺CD4⁺ T cells in total spleen. G, The percentage of DP⁺CD8⁺ T cells in total spleen. H, The percentage of DP⁺CD11c⁺ dendritic cells. I, The percentage of DP⁺F4/80⁺ macrophage. *P < .05