Development of a humanized HLA-A30 transgenic mouse model

Background:There are remarkable genetic differences between animal major histocompatibility complex(MHC)systems and the human leukocyte antigen(HLA)system.HLA transgenic humanized mouse model systems offer a much better method to study the HLA-A-related principal mechanisms for vaccine development and HLA-Arestricted responses against infection in human.Methods:A recombinant gene encoding the chimeric HLA-A30 monochain was constructed.This HHD molecule contains the following:α1-α2 domains of HLA-A30,α3 and cytoplasmic domains of H-2D~b,linked at its N-terminus to the C-terminus of humanβ2m by a 15-amino-acid peptide linker.The recombinant gene encoding the chimeric HLA-A30 monochain cassette was introduced into bacterial artificial chromosome(BAC)CH502-67J3 containing the HLA-A01 gene locus by Red-mediated homologous recombination.Modified BAC CH502-67J3 was microinjected into the pronuclei of wild-type mouse oocytes.This humanized mouse model was further used to assess the immune responses against influenza A virus(H1N1)pdm09 clinically isolated from human patients.Immune cell population,cytokine production,and histopathology in the lung were analyzed.Results:We describe a novel humanβ2m-HLA-A30(α1α2)-H-2D~b(α3 transmembrane cytoplasmic)(HHD)monochain transgenic mouse strain,which contains the intact HLA-A01 gene locus including 49 kb 5’-UTR and 74 kb 3’-UTR of HLA-A01*01.Five transgenic lines integrated into the large genomic region of HLA-A gene locus were obtained,and the robust expression of exogenous transgene was detected in various tissues from A30-18#and A30-19#lines encompassing the intact flanking sequences.Flow cytometry revealed that the introduction of a large genomic region in HLA-A gene locus can influence the immune cell constitution in humanized mice.Pdm09 infection caused a similar immune response among HLA-A30 Tg humanized mice and wild-type mice,and induced the rapid increase of cytokines,including IFN-γ,TNF-α,and IL-6,in both HLA-A30 humanized Tg mice and wild-type mice.The expression of HLA-A30 transgene was dramatically promoted in tissues from A30-9#line at 3 days post-infection(dpi).Conclusions:We established a promising preclinical research animal model of HLA-A30 Tg humanized mouse,which could accelerate the identification of novel HLA-A30-restricted epitopes and vaccine development,and support the study of HLA-A-restricted responses against infection in humans.

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

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 tran-scriptional 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 expres-sion 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 expres-sion of HLA-DPA1/DPB1 in those mice.A model of S aureus-induced pneumonia in the HLA-DP401-H2-Aβ1^(-/-)humanized mice was further developed,and S aureus in-fection 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.

Ablation of NTPDase2+ cells inhibits the formation of filiform papillae in tongue tip

Background: Lingual epithelia in the tongue tip are among the most rapidly regenerating tissues, but the mechanism of cell genesis in this tissue is still unknown. Previous study has suggested the existence of multiple stem cell pools in lingual epithelia and papillae. Like K14+ and Sox2+ cells, NTPDase2+ cells have characteristics of stem cells.Methods: We employed a system using doxycycline to conditionally ablate NTPDase2+ cells in lingual epithelia and papillae by regulated expression of the diphtheria toxin A(DTA) gene. Transgenic lines, which expressed the rtTA gene in NTPDase2+ cells, were produced by pronuclear injection of zygotes from C57 BL/6 mice using the BAC clone RP23-47 P18. The NTPDase2-rtTA transgenic mice were crossed with the TetO-DTA transgenic animals. The double transgenic mice were treated with doxycycline. Doxycycline(Dox) was diluted in 5% sucrose in water to a final concentration of 0.3-0.5 mg/mL and supplied as drinking water.Results: After 15 days of Dox induction, the expression of NTPDase2, Sox2 and K14 was ablated from lingual epithelia. DTA expression in NTPDase2+cells did not inhibit the turnover of GNAT3+ or PLCb2+ cells in taste buds,nor the expression of S100 b beneath lingual epithelia and papillae. After35 days ablation of NTPDase2+ cells, the basic structure of lingual epithelia and papillae remained intact. However, the ratio of cell to total tissue area was decreased in lingual epithelia and circumvallate(CV) papillae. DTA expression also inhibited the regeneration of filiform papillae on the dorsal surface of the tongue tip.Conclusions: These studies provide important insights into the understanding of dynamic equilibrium among the multiple stem cell populations present in the lingual epithelia and papillae.