Against all odds: The road to success in the development of human immune reconstitution mice.

The mouse genome has a high degree of homology with the human genome, and its physiological, biochemical, and developmental regulation mechanisms are similar to those of humans; therefore, mice are widely used as experimental animals. However, it is undeniable that interspecies differences between humans and mice can lead to experimental errors. The differences in the immune system have become an important factor limiting current immunological research. The application of immunodeficient mice provides a possible solution to these problems. By transplanting human immune cells or tissues, such as peripheral blood mononuclear cells or hematopoietic stem cells, into immunodeficient mice, a human immune system can be reconstituted in the mouse body, and the engrafted immune cells can elicit human-specific immune responses. Researchers have been actively exploring the development and differentiation conditions of host recipient animals and grafts in order to achieve better immune reconstitution. Through genetic engineering methods, immunodeficient mice can be further modified to provide a favorable developmental and differentiation microenvironment for the grafts. From initially only being able to reconstruct single T lymphocyte lineages, it is now possible to reconstruct lymphoid and myeloid cells, providing important research tools for immunology-related studies. In this review, we compare the differences in immune systems of humans and mice, describe the development history of human immune reconstitution from the perspectives of immunodeficient mice and grafts, and discuss the latest advances in enhancing the efficiency of human immune cell reconstitution, aiming to provide important references for immunological related researches.

Dclre1c-Mutation-Induced Immunocompromised Mice Are a Novel Model for Human Xenograft Research.

Severe combined immunodeficient (SCID) mice serve as a critical model for human xenotransplantation studies, yet they often suffer from low engraftment rates and susceptibility to graft-versus-host disease (GVHD). Moreover, certain SCID strains demonstrate 'immune leakage', underscoring the need for novel model development. Here, we introduce an SCID mouse model with a targeted disruption of the dclre1c gene, encoding Artemis, which is essential for V(D)J recombination and DNA repair during T cell receptor (TCR) and B cell receptor (BCR) assembly. Artemis deficiency precipitates a profound immunodeficiency syndrome, marked by radiosensitivity and compromised T and B lymphocyte functionality. Utilizing CRISPR/Cas9-mediated gene editing, we generated dclre1c-deficient mice with an NOD genetic background. These mice exhibited a radiosensitive SCID phenotype, with pronounced DNA damage and defective thymic, splenic and lymph node development, culminating in reduced T and B lymphocyte populations. Notably, both cell lines and patient-derived tumor xenografts were successfully engrafted into these mice. Furthermore, the human immune system was effectively rebuilt following peripheral blood mononuclear cells (PBMCs) transplantation. The dclre1c-knockout NOD mice described herein represent a promising addition to the armamentarium of models for xenotransplantation, offering a valuable platform for advancing human immunobiological research.

MicroRNA-29b ameliorates hepatic inflammation via suppression of STAT3 in alcohol-associated liver disease.

Alcohol-associated liver disease (ALD) is induced by chronic excessive alcohol consumption resulting in the clinical manifestations of steatosis, inflammation, and cirrhosis. MicroRNA-29b (miR-29b) is mainly expressed in hepatic nonparenchymal cells, and its expression level varies in different diseases. In this study, we aimed to determine the role of miR-29b in a mouse model of alcohol-associated liver disease. Wild-type (WT) and miR-29b knockout (miR-29b-/-) mice were fed a Lieber-DeCarli liquid diet containing 5% alcohol for 10 days, followed by gavage of a single dose of ethanol (5 g/kg body weight). Histology, immunoblotting, and biochemical analyses were then conducted for comparison. miR-29b expression was decreased in the livers of chronic-plus-binge ethanol-fed mice. Further analysis revealed that alcohol exposure exacerbated hepatic injury by significantly increasing serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, with decreased survival rates for miR-29b-/- mice. Results from the luciferase assay indicated that miR-29b negatively regulated the signal transducer and activator of transcription 3 (STAT3). Depletion of miR-29b led to an increase in STAT3 and more noticeable inflammation in the liver, whereas overexpression of miR-29b downregulated STAT3 and proinflammatory cytokine expression in primary mouse peritoneal macrophages. Taken together, these results demonstrate a novel association between miR-29b and ALD. miR-29b plays a hepatoprotective role in alcohol-induced inflammation and liver injury by targeting STAT3.

Chimeric antigen receptor T cells targeting CD147 for non-small cell lung cancer therapy.

Non-small cell lung cancer (NSCLC) is a highly malignant tumor, with a significant mortality and morbidity. With the development of tumor immunotherapy, chimeric antigen receptor T cells (CART) gets increasingly attention and achieves prominent contributions in the treatment of hematologic malignancies. However, CART therapy for NSCLC proceeds slowly and further researches need to be investigated. In our study, we performed bioinformatics analysis to evaluate the significant role of CD147 in NSCLC. The expression level of CD147 was detected in human NSCLC cell lines and NSCLC tissues. Meanwhile, CD147-CART was constructed and identified. Cell cytotoxicity and cytokine secretion were performed to evaluate the efficacy of CD147-CART. We also constructed cell-derived xenograft (CDX) model and patient-derived xenograft (PDX) model, which was used to further investigate the safety and efficacy of CD147-CART in vivo. Our observations show that CD147 is a specific tumor antigen of NSCLC and plays an essential role in NSCLC progression, which can be used as a target for CART therapy in NSCLC. CD147-CART cells exhibit robust cytotoxicity and cytokine production in vitro, suggesting a strong anti-tumor activity against NSCLC tumor cells. Importantly, CD147-CART cells have strong anti-tumor activity against NSCLC cells in vivo in both CDX and PDX models and no adverse side effects. Our findings show that CD147-CART immunotherapy for NSCLC is safe and effective, which is an ideal and promising medical patch for treating NSCLC.

Ligand-based discovery of small molecules suppressing cancer cell proliferation via autophagic flux inhibition.

Autophagy is a conserved self-degradation system closely related to cancer progression. Small molecule inhibitors of autophagy have proven to be efficient tools in cancer therapy and are in high demand. Here we report the discovery of two compounds (LZ02/01) capable of suppressing cancer cell proliferation via inhibiting autophagy flux and promoting apoptosis. Potential autophagy inhibitors were selected based on the pharmacophore model derived from the structures of known autophagy inhibitors. LZ02/01-mediated autophagy flux disruption and apoptosis promotion in breast and hepatocellular carcinoma cells (MCF-7 and Hep3B) were examined using a combination of molecular methods in vitro and in vivo. The synergistic tumor-suppressing effects of LZ02 and chloroquine were validated by adopting a xenograft mice model of human breast cancer. Two potential inhibitors (LZ02/01) targeting an autophagy pathway were discovered from the Enamine database. In both MCF-7 and Hep3B cells, LZ02 and LZ01 had the effect of causing the co-occurrence of autophagic flux inhibition and apoptosis induction, robustly suppressing the growth, proliferation, and cell cycle progression. Further tests revealed that FoxO3a and its downstream target genes regulating autophagy, apoptosis, and cell cycle progression were activated and overexpressed, suggesting such effects of LZ02/01 on autophagy and apoptosis were associated with the activation and overexpression of FoxO3a. In addition, LZ02/01-mediated apoptosis is not independent; it was verified to be promoted by autophagic flux inhibition. Meanwhile, synergistic effects on tumor growth reduction were detected in the xenograft mice model of human breast cancer simultaneously treated with LZ02 and chloroquine. Our findings suggest that LZ01 and LZ02 are potent in suppressing cancer cell proliferation and tumor growth through autophagic flux inhibition and apoptosis promotion. The synergistic anti-cancer effects of LZ02 with chloroquine may provide a rational basis for prospective cancer therapy. KEY MESSAGES: A ligand-based pharmacophore model of high quality is constructed to query hits and two novel scaffold lead compounds LZ01/02 were identified by high-throughput virtual screening. LZ01/02 works to inhibit autophagic flux by attenuating lysosome function. LZ01/02 induces apoptosis through autophagic flux inhibition and apoptosis is the main mechanism to inhibit MCF-7 and Hep3B cancer cell proliferation. The synergistic antitumor growth effects of LZ02 and chloroquine are verified in human xenograft model.

Biological Characteristics of Severe Combined Immunodeficient Mice Produced by CRISPR/Cas9-Mediated Rag2 and IL2rg Mutation.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 is a novel and convenient gene editing system that can be used to construct genetically modified animals. Recombination activating gene 2 (Rag2) is a core component that is involved in the initiation of V(D)J recombination during T- and B-cells maturation. Separately, the interleukin-2 receptor gamma chain gene (IL2rg) encoded the protein-regulated activity of natural killer (NK) cells and shared common receptors of some cytokines. Rag2 and IL2rg mutations cause immune system disorders associated with T-, B-, and NK cell function and some cytokine activities. In the present study, 2 single-guide RNAs (sgRNAs) targeted on Rag2 and IL2rg genes were microinjected into the zygotes of BALB/c mice with Cas9 messenger RNA (mRNA) to create Rag2/IL2rg -/- double knockout mice, and the biological characteristics of the mutated mice were subsequently analyzed. The results showed that CRISPR/Cas9-induced indel mutation displaced the frameshift of Rag2 and IL2rg genes, resulting in a decrease in the number of T-, B-, and NK cells and the destruction of immune-related tissues like the thymus and spleen. Mycobacterium tuberculosis 85B antigen could not induce cellular and humoral immune response in mice. However, this aberrant immune activity compromised the growth of several tumor heterogenous grafts in the mutated mice, including orthotopic and subcutaneous transplantation tumors. Thus, Rag2/IL2rg -/- knockout mice possessed features of severe combined immunodeficiency (SCID), which is an ideal model for human xenograft.