ABSTRACT
HLA class I (HLA-I) transgenic mice have proven to be useful models for studying human MHC-related immune responses over the last two decades. However, differences in the processing and presentation machinery between humans and mice may have profound effects on HLA-I restricted antigen presentation. In this study, we generated a novel human TAP-LMP (hTAP-LMP) gene cluster transgenic mouse model carrying an intact human TAP complex and two human immunoproteasome LMP subunits, PSMB8/PSMB9. By crossing the hTAP-LMP strain with different HLA-I transgenic mice, we found that the expression levels of human HLA-I molecules, especially the A3 supertype members (e.g., A11 and A33), were remarkably enhanced in corresponding HLA-I/hTAP-LMP transgenic mice. Moreover, we found that humanized processing and presentation machinery increased antigen presentation of HLA-A11-restricted epitopes and promoted the rapid reduction of hepatitis B virus (HBV) infection in HLA-A11/hTAP-LMP mice. Together, our study highlights that HLA-I/hTAP-LMP mice are an improved model for studying antigen presentation of HLA-I molecules and their related CTL responses.
Subject(s)
Antigen Presentation/immunology , Histocompatibility Antigens Class I/immunology , Amino Acid Sequence , Animals , Antigen Presentation/drug effects , Antiviral Agents/pharmacology , Epitopes, T-Lymphocyte/immunology , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/metabolism , Humans , Immunity/drug effects , Membrane Transport Proteins/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Models, Animal , Peptides/chemistry , Proteasome Endopeptidase Complex/metabolism , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
Wnt/ß-catenin signaling pathway plays essential roles in mammalian development and tissue homeostasis. MicroRNAs (miRNAs) are a class of regulators involved in modulating this pathway. In this study, we screened miRNAs regulating Wnt/ß-catenin signaling by using a TopFlash based luciferase reporter. Surprisingly, we found that miR-142 inhibited Wnt/ß-catenin signaling, which was inconsistent with a recent study showing that miR-142-3p targeted Adenomatous Polyposis Coli (APC) to upregulate Wnt/ß-catenin signaling. Due to the discordance, we elaborated experiments by using extensive mutagenesis, which demonstrated that the stem-loop structure was important for miR-142 to efficiently suppress Wnt/ß-catenin signaling. Moreover, the inhibitory effect of miR-142 relies on miR-142-3p rather than miR-142-5p. Further, we found that miR-142-3p directly modulated translation of Ctnnb1 mRNA (encoding ß-catenin) through binding to its 3' untranslated region (3' UTR). Finally, miR-142 was able to repress cell cycle progression by inhibiting active Wnt/ß-catenin signaling. Thus, our findings highlight the inhibitory role of miR-142-3p in Wnt/ß-catenin signaling, which help to understand the complex regulation of Wnt/ß-catenin signaling.
Subject(s)
Gene Expression Regulation , MicroRNAs/genetics , Wnt Signaling Pathway , 3' Untranslated Regions , Animals , Base Sequence , Binding Sites , Cell Line , Cell Proliferation , Humans , Mice , MicroRNAs/chemistry , Nucleic Acid Conformation , Protein Biosynthesis/genetics , Wnt Proteins/metabolism , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
MicroRNA-155 (miR-155) plays significant role in various physiological processes involving both innate and adaptive immunity. miR-155 expression level changes dynamically during various immune responses. However, current approaches for miR-155 detection at the RNA level do not precisely reflect the real-time activity. Herein, we generated a transgenic mouse line (R26-DTR-155T) for determination of miR-155-5p activity in vivo by inserting miR-155-5p target sequence downstream of a reporter transgene comprising Diphtheria Toxin Receptor and TagBlue fluorescence protein. Using this approach, R26-DTR-155T mice were able to measure variation in levels of miR-155-5p activity in specific cell types of interest. The DTR expression levels were inversely correlated with the endogenous miR-155 expression pattern as detected by quantitative RT-PCR. Our data demonstrate a novel transgenic mouse line which could be useful for tracing miR-155-5p activity in specific cell types through measurement of miR-155-5p activity at single cell level.
