Mouse Immune System Research Articles

Abstract 1639: Human immune system mouse model of ER+ metastatic breast cancer

Abstract In a recent clinical trial in heavily pretreated estrogen receptor alpha-positive (ER+) metastatic breast cancer (MBC), ~23% of patients reached the primary endpoint of clinical benefit of >24 weeks on Fulvestrant plus the anti-androgen Enzalutamide. ESR1 mutant biopsies had more T regulatory cells (t test p<0.021) and macrophages (p<0.003) than ESR1 wild type (WT), which had more TP53 mutations and higher levels of cytotoxic T effector cells (p<0.01). In addition, PD-L1 increased in post- vs. pre-treatment biopsies (paired t test p<0.002). This increase in PD-L1 post-treatment warrants testing if the addition of a checkpoint inhibitor to the Fulvestrant-Enzalutamide combination will enhance therapy efficacy in ER+ MBC resistant to standard endocrine therapy. However, the lack of immune competent ER+ breast cancer pre-clinical models is a major barrier to these studies. Human immune system (HIS) mouse models were developed as a platform in which to test immune checkpoint inhibitors in other human cancers, and one HIS model was recently shown to support the growth and immune infiltration of ER+ patient-derived xenograft (PDX) primary tumors. We tested whether HIS mice are a robust model in which to define ER+ tumor-immune microenvironment interactions at metastatic sites. We implanted NCG mice (NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl) humanized by engraftment of human umbilical cord blood-derived CD34+ (huCD34+) stem cells with the estrogen-independent HCI-013EI (013EI) PDX, derived from an ER+, invasive lobular MBC which harbors the Y537S ESR1 mutation. Mice were monitored for tumor latency and growth rate, and primary tumors and organs were collected for immunohistochemistry, single-cell RNA sequencing (scRNAseq), and phasor-fluorescence lifetime imaging microscopy (FLIM). The ER mutated PDX 013EI had a longer tumor latency in HIS (median 77 days) vs. age-matched immune deficient control mice (27.5 days, log-rank p=0.043). Once formed, 013EI tumors in HIS mice grew slower than those in controls (mixed-effects analysis, p=0.035). HIS mice had visible lung, liver, and/or gastrointestinal tract metastases despite small primary tumors. Lung metastases from HIS mice retain ER expression and are infiltrated by human CD4+, CD8+, and CD68+ immune cells. Initial scRNAseq analysis also suggests infiltration by diverse immune cell populations in primary tumors from HIS mice. Initial phasor-FLIM for NADH autofluorescence suggests that 013EI tumors are highly glycolytic in HIS mice. In conclusion, our results show that huCD34+ HIS mice effectively support growth, metastasis, and immune infiltration of an ESR1 mutant MBC PDX. Ongoing studies will further define the tumor-immune microenvironment in this and ESR1 WT MBC PDX, focusing on immune cell infiltration, PD-L1 expression, steroid receptor status, metabolic signaling and changes in these parameters following novel combinations of endocrine, immunotherapy and metabolic agents. Citation Format: Ayodeji Olukoya, Shaymaa Bahnassy, Nicole S. Spoelstra, Lu Jin, Suman Ranjit, Anthony Elias, Jennifer K. Richer, Rebecca B. Riggins. Human immune system mouse model of ER+ metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1639.

Abstract 2875: Development of a new tumor-bearing humanized mouse model to evaluate the efficacy and toxicity of bispecific T cell engagers

Abstract Background: T cell-based immunotherapies, especially the bispecific T cell engager (BiTE), which recruit T cells to tumor cells, have been developed rapidly for treatment of malignancies. However, the wide application of BiTE therapies is limited by accompanying severe adverse effects such as cytokine release syndrome (CRS). Since human cancer cells are not incorporated in the CRS evaluation, currently used in vivo animal models and in vitro human immune cell activation assays do not reliably evaluate the induction of CRS by immune drugs that is seen in BiTE treated cancer patients. In addition, the differences between human and mouse immune systems hamper the development of new immune-oncology therapeutic drugs. Thus, establishing a predictive in vivo animal model that can screen both efficacy of cancer therapy and risk of developing CRS is critical for improving the safety of immune drug development for treatment of cancer patients. Method: Humanized mice were developed in NSG-MHC class I/II double knock out (DKO) mouse recipients following reconstitution with human peripheral blood mononuclear cells. The MDA-MB-231/Luciferase-2A-GFP stable breast cancer cell line (MDA), which expressed epidermal growth factor receptor (EGFR), were implanted in mice 5 days after PBMC engraftment. Following EGFRxCD3 BiTE treatment whole-body tumor burden was quantified by an in vivo imaging system. To evaluate the CRS levels in response to BiTE therapy, cytokine levels were analyzed by BD cytometric bead array. Clinical observations, CRS scores, body weight changes and human immune cell activation were also monitored for CRS assessment. Results: PBMC humanized models were successfully established with breast cancer cell engraftment. After treatment with EGFRxCD3 BiTE, the mice exhibited elevated serum levels of human IFNgamma, TNF-a, IL-6, IL-10, IL-2 and IL-4 compared to a vehicle control group. This cytokine production was dose-dependent and was associated with an increase of toxic mouse clinical evaluation score. In contrast, there was no significant inflammatory cytokine production in PBMC humanized mice without MDA cell implantation, suggesting T cell activation by BiTE followed binding to the cancer cells. BiTE treatment for 2-4 days significantly suppressed tumor growth in lung as measured by luciferin imaging. With serial doses of BiTE treatment, we observed a dose-dependent inhibition of tumor growth. Conclusion: We developed a novel in vivo PBMC humanized mouse model bearing human tumor cells that can be used to determine the optimal dose of BiTE for inhibition of tumor growth with low toxicity risk. This model provides a growth environment similar to that of patients and has important application values in simultaneously evaluating the efficacy and safety of any T cell-based immunotherapies including BiTE to define optimal approaches for clinical cancer treatment. Citation Format: Guoxiang Yang, Li-Chin Yao, Michael A. Brehm, Dale L. Greiner, Leonard D. Shultz, Danying Cai, MIngshan Cheng, James G. Keck. Development of a new tumor-bearing humanized mouse model to evaluate the efficacy and toxicity of bispecific T cell engagers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2875.

Involvement of MEK1 and MEK2 kinases in the maturation of the mouse immune system

Involvement of MEK1 and MEK2 kinases in the maturation of the mouse immune system

T1D patient-derived hematopoietic stem cells are programmed to generate Tph, Tfh, and autoimmunity-associated B cell subsets in human immune system mice

Interactions between B cells and CD4+ T cells play a central role in the development of Type 1 Diabetes (T1D). Two helper cell subsets, follicular (Tfh) and peripheral (Tph) helper T cells, are increased in patients with T1D but their role in driving B cell autoimmunity is undefined. We used a personalized immune (PI) mouse model to generate human immune systems de novo from hematopoietic stem cells (HSCs) of patients with T1D or from healthy controls (HCs). Both groups developed Tfh and Tph-like cells, and those with T1D-derived immune systems demonstrated increased numbers of Tph-like and Tfh cells compared to HC-derived PI mice. T1D-derived immune systems included increased proportions of unconventional memory CD27−IgD− B cells and reduced proportions of naïve B cells compared to HC PI mice, resembling changes reported for patients with systemic lupus erythematosus. Our findings suggest that T1D HSCs are genetically programmed to produce increased proportions of T cells that promote the development of unconventional, possibly autoreactive memory B cells. PI mice provide an avenue for further understanding of the immune abnormalities that drive autoantibody pathogenesis and T1D.

Sometimes simple is not best: The complex γδ T cell interaction with tumour needs depth

Sometimes simple is not best: The complex γδ T cell interaction with tumour needs depth

Gene editing at position 71 in DRB1*04:01 blocks collagen sensitization and avoids allorecognition.

Abstract DRB1*04:01 is strongly associated with susceptibility to rheumatoid arthritis (RA). Our previous work demonstrated that changing lysine (K) at position 71 in DRB1*04:01 to glutamic acid (E), an amino acid found in resistant alleles, abrogated binding of the immunodominant collagen258–272 peptide. The purpose of this study was to determine if the K71E mutation in DRB1*04:01 would prevent sensitization to collagen and be tolerated by the host immune system in mice. DRB1*04:01 and DRB1*04:01K71E transgenic mice were created on the murine MHC class II knockout background (B6.129S2-H2dlAb1-Ea/J). DRB1*04:01 and DRB1*04:01K71E mice were immunized twice with bovine type II collagen in complete Freund’s adjuvant on days 0 and 21. On day 56, lymph node cells were harvested and stimulated in vitro with collagen258–272 peptide. Introduction of the K71E mutation in DRB1*04:01 resulted in a >90% reduction in proliferation as compared to CD4+ cells from collagen-immunized DRB1*04:01 mice. Next, skin grafts were transplanted from DRB1*04:01K71E mice onto DRB1*04:01 recipients to test for rejection. The skin grafts from DRB1*04:01K71E mice were accepted by the recipient DRB1*04:01 mice with no signs of rejection out to day 70. Our results demonstrate that introducing the K71E mutation in DRB1*04:01 blocks collagen sensitization and avoids allorecognition suggesting that this mutation could safely halt progression of RA by blocking autoimmune T cell responses to collagen. Supported by IOS-2103367

Gene editing at position 71 in DRB1*04:01 blocks collagen sensitization and avoids allorecognition.

Abstract DRB1*04:01 is strongly associated with susceptibility to rheumatoid arthritis (RA). Our previous work demonstrated that changing lysine (K) at position 71 in DRB1*04:01 to glutamic acid (E), an amino acid found in resistant alleles, abrogated binding of the immunodominant collagen258-272 peptide. The purpose of this study was to determine if the K71E mutation in DRB1*04:01 would prevent sensitization to collagen and be tolerated by the host immune system in mice. DRB1*04:01 and DRB1*04:01K71E transgenic mice were created on the murine MHC class II knockout background (B6.129S2-H2dlAb1-Ea/J). DRB1*04:01 and DRB1*04:01K71E mice were immunized twice with bovine type II collagen in complete Freund’s adjuvant on days 0 and 21. On day 56, lymph node cells were harvested and stimulated in vitro with collagen258-272 peptide. Introduction of the K71E mutation in DRB1*04:01 resulted in a >90% reduction in proliferation as compared to CD4+ cells from collagen-immunized DRB1*04:01 mice. Next, skin grafts were transplanted from DRB1*04:01K71E mice onto DRB1*04:01 recipients to test for rejection. The skin grafts from DRB1*04:01K71E mice were accepted by the recipient DRB1*04:01 mice with no signs of rejection out to day 70. Our results demonstrate that introducing the K71E mutation in DRB1*04:01 blocks collagen sensitization and avoids allorecognition suggesting that this mutation could safely halt progression of RA by blocking autoimmune T cell responses to collagen.

Nicotine and THC Effects on Rodents

Nicotine is a highly addictive stimulant that causes an increase in blood pressure, heart rate, and inflammation in the lungs. The use of vaping devices has a grand effect on the youth today. Vapes and e‐cigarettes are used for recreational purposes, yet this may affect adolescents by weakening their respiratory and immune systems. Nicotine and THC both have a strong impact on vapes. Experimental models in rodents suggest differences in behavior and physiological conditions when exposed to nicotine with or without THC. These changes can include differences in weight, bone density, bone strength, and respiratory rates. The aim of this study is to determine the effects of nicotine in the presence and absence of THC on the respiratory and immune systems in mice. Over the course of 12 weeks, both groups were treated with glycerin and propylene glycol. The 19 mice in the experimental group received nicotine on top of glycerin and propylene glycol. Animals were exposed to respective environments for 5 min/day, 2 days/week for a total of 12 weeks. Respiratory, minute volume, tidal volume, bone scans, fat percentage, and weights of the mice were collected weekly during the 12‐week period. The vital organs were harvested for further examination: these tissues included serum, lung, brain, and femur. Data analysis showed significance in weight and fat percentage. Conclusions upon THC in addition to nicotine are in the process of being completed.

Augmentation of lymphoid tissues in the human immune system mouse to advance host directed therapies for HIV

Abstract The study of HIV infection and pathogenicity in physical reservoirs requires a biologically relevant model which resembles the human immune system and physiology. The human immune system (HIS) mouse is well established as a model of HIV, but defects in immune tissue reconstitution remain a challenge for examining pathology in tissues. Herein we show that exogenous injection of the human cytokine FLT-3L into the HSC cord blood HIS mouse model significantly expanded the total area of axial lymph nodes and the circulating percentage of human T cells, thus enabling us to visualize and quantify HIV infectivity in the secondary lymphoid tissues of the spleen and axial node. Further, we detected cell death and T cell depletion in tissues, consistent with HIV pathogenesis. Treatment with the caspase-1 inhibitor VX-765 significantly decreased viral load as measured by gag qPCR, and apoptosis as measured by TUNEL assay, in the spleen. A significant restoration of CD4+ T cells in the spleen due to VX-765 treatment was observed via flow cytometry. In situ hybridization further demonstrated a significant decrease in viral RNA in both the spleen and axial lymph nodes. Transcriptomic analysis further revealed an upregulation in host HIV restriction factors such as APOBR, SAMHD1 and APOBEC3A as a result of VX-765 administration. These findings demonstrate FLT-3L as a mechanism whereby the human immune environment in HIS mice can be enhanced to support investigations of HIV pathogenesis and immune outcomes in tissue compartments. Preliminary results indicate that targeting inflammasome pathways with VX-765 in HIS mice treated with FLT-3L, and infected with HIV, preserved T cell populations and decreased viral load. Supported by R01 A1HL129881, NIH/NHLBI

Developing a model of autoimmune diseases with human tonsil organoids

Abstract Mouse models have contributed significantly to our knowledge of the pathogenesis of autoimmune diseases. However, differences in mouse and human immune systems limit the translation of the knowledge in mechanism and therapeutic outcomes from mouse to human. Therefore, a human model of autoimmune diseases is urgently needed. Recently, our lab has developed a functional tonsil organotypic system that can recapitulate key T cells responses and germinal center response to influenza vaccine in vitro. We now want to utilize this tonsil system to study autoimmunity. Regulatory T cells (Tregs) have been suggested to play a central role in maintaining self-tolerance. Reduced Treg cell frequencies and impaired suppressive function have been reported in a wide range of autoimmune diseases. In this study, we show that we can efficiently knock out FOXP3 in T cells from human tonsil organoids by using CRISPR technology. We also show that tonsil organoids with FOXP3 knocked-out T cells are able to induce humoral immune responses towards autoantigens stimulation and influenza vaccination. This system could be useful for studying underlying mechanisms in the functions of Tregs in the autoimmune condition in humans and provides insights into the therapeutic strategy for autoimmune diseases.

Long-term administration of Tetragenococcus halophilus No. 1 over generations affects the immune system of mice

Japanese people have been consuming miso soup over generations; it is beneficial for health and longevity. In this study, Tetragenococcus halophilus No. 1 in miso was found to possess salient immunomodulatory functions. Recently, we also demonstrated its effect on boosting immunological robustness. Although the consumption of miso is suggested to affect health over generations, such a long-term experiment has not been conducted until now. Thus, we evaluated the effects of miso-derived T. halophilus No. 1 over generations on the immune system of mice. As the generations increase, the proportion of germinal center B cells tends to increase. Furthermore, we found that CD4+ T cells expressing CD69, an activation marker, were increased in the third generation of mice. In addition, the proportion of follicular helper T cells and regulatory T cells tended to increase. Among the subsets of CD4+ T cells in the fourth generation, effector T cells and effector memory T cells tended to increase. In contrast, central memory T cells and naive T cells decreased. Moreover, autoimmunity was suppressed by long-term administration of T. halophilus No. 1. Based on these findings, we believe that the long-term administration of T. halophilus No. 1 over generations promotes immune activation and tolerance and enhances immunological robustness.

Defects in Long-Term APC Repopulation Ability of Adult Human Bone Marrow Hematopoietic Stem Cells (HSCs) Compared with Fetal Liver HSCs.

Immunodeficient mice reconstituted with immune systems from patients, or personalized immune (PI) mice, are powerful tools for understanding human disease. Compared with immunodeficient mice transplanted with human fetal thymus tissue and fetal liver-derived CD34+ cells administered i.v. (Hu/Hu mice), PI mice, which are transplanted with human fetal thymus and adult bone marrow (aBM) CD34+ cells, demonstrate reduced levels of human reconstitution. We characterized APC and APC progenitor repopulation in human immune system mice and detected significant reductions in blood, bone marrow (BM), and splenic APC populations in PI compared with Hu/Hu mice. APC progenitors and hematopoietic stem cells (HSCs) were less abundant in aBM CD34+ cells compared with fetal liver-derived CD34+ cell preparations, and this reduction in APC progenitors was reflected in the BM of PI compared with Hu/Hu mice 14-20 wk posttransplant. The number of HSCs increased in PI mice compared with the originally infused BM cells and maintained functional repopulation potential, because BM from some PI mice 28 wk posttransplant generated human myeloid and lymphoid cells in secondary recipients. Moreover, long-term PI mouse BM contained functional T cell progenitors, evidenced by thymopoiesis in thymic organ cultures. Injection of aBM cells directly into the BM cavity, transgenic expression of hematopoietic cytokines, and coinfusion of human BM-derived mesenchymal stem cells synergized to enhance long-term B cell and monocyte levels in PI mice. These improvements allow a sustained time frame of 18-22 wk where APCs and T cells are present and greater flexibility for modeling immune disease pathogenesis and immunotherapies in PI mice.

Host Immunity Influences the Composition of Murine Gut Microbiota.

The influence of gut microbiota on host immunity is widely studied, and its disturbance has been linked to several immune-mediated disorders. Conversely, whether and how inherently disturbed canonical Th1 (pro-inflammatory) and/or Th2 (anti-inflammatory) immune pathways modify the host microbiome is not sufficiently investigated. Here, we characterized the humoral, cellular, and cytokine immunity, and associated alterations in gut microbiota of naïve wild-type mice (C57BL/6 and BALB/c), and mice with deficiencies in Th2 responses (IL-4Rα and IL-33 knockout mice) or in both Th1 and Th2 responses (NOD scid gamma, NSG mice). A global analysis by de novo clustering of 16S rRNA profiles of the gut microbiota independently grouped wild-type immunocompetent (C57BL/6 and BALB/c), Th2-deficient (IL-4Rα-/- and IL-33-/-), and severely immunodeficient (NSG) mice; where wild-type mice, but not Th2 or severely immunodeficient mice, were enriched in gut bacteria that produce short-chain fatty acids. These include members of phyla Firmicutes, Verrucomicrobia, and Bacteroidetes such as Lactobacillus spp., Akkermansia muciniphila, and Odoribacter spp. Further comparison of the two naïve wild-type mouse strains showed higher microbial diversity (Shannon), primarily linked to higher richness (Chao1), as well as a distinct difference in microbial composition (weighted UniFrac) in BALB/c mice compared to C57BL/6. T-cell and blood cytokine analyses demonstrated a Th1-polarization in naïve adaptive immunity in C57BL/6 animals compared to BALB/c mice, and an expected Th2 deficient cellular response in IL-4Rα-/- and IL-33-/- mice compared to its genetic background BALB/c strain. Together, these data suggest that alterations in the Th1/Th2 balance or a complete ablation of Th1/Th2 responses can lead to major alterations in gut microbiota composition and function. Given the similarities between the human and mouse immune systems and gut microbiota, our finding that immune status is a strong driver of gut microbiota composition has important consequences for human immunodeficiency studies.

Effect of Triatoma infestans saliva on mouse immune system cells: The role of the pore-forming salivary protein trialysin

Triatoma infestans is one of the most important vectors of Trypanosoma cruzi in the Americas. While feeding, they release large amounts of saliva that will counteract the host's responses triggered at the bite site. Despite the various activities described on T. infestans saliva, little is known about its effect on the modulation of the host's immune system. This work aimed to describe the effects of T. infestans saliva on cells of the mouse immune system and access the role in hematophagy. The effect of saliva or salivary gland extract (SGE) was evaluated in vivo and in vitro by direct T. infestans feeding on mice or using different biological assays. Mice that were submitted to four bites by three specimens of T. infestans had their anti-saliva IgG serum levels approximately 2.4 times higher than controls, but no change in serum IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α levels was observed. No macroscopic alterations were seen at the bite site, but an accumulation of mononuclear and polymorphonuclear cells shortly after the bite and 24 h later were observed in histological cuts. At low concentrations (up to ∼5 μg/well), SGE induced TNF-α production by macrophages and spleen cells, IFN-γ and IL-10 by spleen cells and NO by macrophages. However, at higher concentrations (10 and 20 μg/well), viability of macrophages and spleen cells was reduced by SGE, reducing the production of NO and cytokines (except TNF-α). The salivary trialysin was the main inducer of cell death as macrophage viability and NO production was restored in assays carried out with SGE from trialysin knockdown insects. The reduction of the salivary trialysin by RNAi affected the total ingestion rate, the weight gain, and retarded the molt from second to the fifth instar of T. infestans nymphs fed on mice. The results show that T. infestans saliva modulates the activity of cells of the host immune system and trialysin is an important salivary molecule that reduces host cells viability and impacts the feeding performance of T. infestans feeding on live hosts.

Humanized Mouse Models of Systemic Lupus Erythematosus: Opportunities and Challenges.

Animal models have played a crucial role in the understanding of the mechanisms and treatments of human diseases; however, owing to the large differences in genetic background and disease-specific characteristics, animal models cannot fully simulate the occurrence and progression of human diseases. Recently, humanized immune system mice, based on immunodeficient mice, have been developed that allow for the partial reconstruction of the human immune system and mimic the human in vivo microenvironment. Systemic lupus erythematosus (SLE) is a complex disease characterized by the loss of tolerance to autoantigens, overproduction of autoantibodies, and inflammation in multiple organ systems. The detailed immunological events that trigger the onset of clinical manifestations in patients with SLE are still not well known. Two methods have been adopted for the development of humanized SLE mice. They include transferring peripheral blood mononuclear cells from patients with SLE to immunodeficient mice or transferring human hematopoietic stem cells to immunodeficient mice followed by intraperitoneal injection with pristane to induce lupus. However, there are still several challenges to be overcome, such as how to improve the efficiency of reconstruction of the human B cell immune response, how to extend the lifespan and improve the survival rate of mice to extend the observation period, and how to improve the development of standardized commercialized models and use them. In summary, there are opportunities and challenges for the development of humanized mouse models of SLE, which will provide novel strategies for understanding the mechanisms and treatments of SLE.

Exploration of Human Lung-Resident Immunity and Response to Respiratory Viral Immunization in a Humanized Mouse Model.

There are urgent needs for humanized mouse models of viral respiratory diseases to study immunopathogenesis and therapeutic interventions. Although human immune system (HIS) mice permit analysis in real time of human immune responses in vivo, evolutionary divergences preclude their usefulness for the respiratory viruses that do not infect mouse lungs. In this study, we sought to use HIS mice with human lung (HL) tissue xenografts (HISL mice) to address this issue. The grafted HL tissue maintained histologically normal structure, and populated with human tissue-resident immune cells, including CD11c+ dendritic cells and CD4+ and CD8+ tissue-resident memory T cells. HISL mice showed a marked expansion of tissue-resident memory T cells and generation of viral Ag-specific T cells in the HL xenografts, and production of antiviral IgM and IgG Abs upon immunization of the HL xenograft by H1N1 influenza viruses. RNA-seq analysis on H1N1-infected and control HL xenografts identified a total of 5089 differentially expressed genes with enrichments for genes involved in respiratory diseases, viral infections, and associated immune responses. Furthermore, prophylactic viral exposures resulted in protection against subsequent lethal challenge by intranasal viral inoculation. This study supports the usefulness of this preclinical model in exploring the immunopathology and therapies of respiratory viral diseases.

Mouse Models of Food Allergy in the Pursuit of Novel Treatment Modalities.

The prevalence of IgE-mediated food allergies has increased dramatically in the past three decades, now affecting up to 10% of the US population. IgE-mediated food allergy is an immunologic disease, involving a variety of cells, including B and T cells, mast cells, basophils, ILC2s, and epithelial cells. Mouse models of food allergy mimic the overall immunologic processes known to exist in humans. Due to the limitations of invasive sampling of human tissue and the similarities of the human and mouse immune systems, comprehensive pathogenesis studies of food allergy have been performed in mouse models. Mouse models have been effective in elucidating the roles of non-oral routes of sensitization and identifying key cells and molecules involved in allergic sensitization. Furthermore, the development of novel therapeutic approaches for food allergy has been accelerated through the use of pre-clinical mouse models. Despite the groundbreaking findings stemming from research in mice, there are continued efforts to improve the translational utility of these models. Here, we highlight the achievements in understanding food allergy development and efforts to bring novel treatment approaches into clinical trials.

Multiple Tolerization Subtractive Immunization (MTSI) Protocol: Effects on Mice and Monoclonal Antibody Specificity.

Monoclonal antibodies (mAbs) have been a valuable tool to elucidate several biological processes, such as stem cell differentiation and cancer, and contributed to virtually all areas of biomedical sciences. Yet, it remains a challenge to obtain mAbs specific to poorly expressed epitopes, or to epitopes that are actually involved in important biological phenomena, such as cell differentiation and metastasis. Drug-induced subtractive immunization, and recently the multiple tolerization subtractive immunization (MTSI) technique, reported by our group, have the potential to level up the field, as they direct the host´s immune response towards these epitopes. However, due to cyclophosphamide (CY) treatment, high mice mortality can be observed, and only a few data are available on how these techniques affect the immune system of mice. Tolerogen and immunogen cells, RWPE-1 and PC-3 cells, respectively, were individually seeded at 2 × 104 cells/cm2, and then adjusted to 2 × 106 cells per mouse before immunization, which was conducted in a subtractive approach (MTSI) with CY. Immunosuppression of mice was recorded via total white blood counting, as well the reactivity of circulating polyclonal antibodies (pAbs). General parameters, including weight, physical appearance, and behavior on mice subjected to three different concentrations of CY were recorded. mAbs were obtained using classical hybridoma techniques, using the spleen of immunized mice. After purification, antibodies were characterized by Western blotting, and Indirect immunofluorescence. In conclusion, all CY dosage were efficient in creating an immunosuppression state, but only the 100 mg/kg body weight was feasible, as the others resulted in extensive mice mortality. pAbs obtained in the peripheral blood of mice showed more reactivity towards tumor cells. MAbs 2-7A50 and 2-5C11 recognized antigens from tumor cells, but not from their non-tumor counterparts, as shown in western blotting and immunofluorescence assays. MTSI technique was successful in generating mAbs that recognize tumor-specific antigens.

Effect of inactivated nature-derived microbial composition on mouse immune system.

IntroductionThe hygiene hypothesis suggests that decrease in early life infections due to increased societal‐level hygiene standards subjects one to allergic and autoimmune diseases. In this report, we have studied the effect of sterilized forest soil and plant‐based material on mouse immune system and gut microbiome.MethodsInbred C57Bl/6 mice maintained in normal sterile environment were subjected to autoclaved forest soil‐derived powder in their bedding for 1 h a day for 3 weeks. Immune response was measured by immune cell flow cytometry, serum cytokine enzyme‐linked immunoassay (ELISA) and quantitative polymerase chain reaction (qPCR) analysis. Furthermore, the mouse gut microbiome was analyzed by sequencing.ResultsWhen compared to control mice, mice treated with soil‐derived powder had decreased level of pro‐inflammatory cytokines namely interleukin (IL)−17F and IL‐21 in the serum. Furthermore, splenocytes from mice treated with soil‐derived powder expressed less IL‐1b, IL‐5, IL‐6, IL‐13, and tumor necrosis factor (TNF) upon cell activation. Gut microbiome appeared to be stabilized by the treatment.ConclusionsThese results provide insights on the effect of biodiversity on murine immune system in sterile environment. Subjecting mice to soil‐based plant and microbe structures appears to elicit immune response that could be beneficial, for example, in type 2 inflammation‐related diseases, that is, allergic diseases.

Efficient adoptive transfer of autologous modified B cells: a new humanized platform mouse model for testing B cells reprogramming therapies

Here, we report a novel experimental setup to perform adoptive transfer of gene-edited B cells using humanized immune system mice by infusing autologous HIS mouse-derived human B cells “educated” in a murine context and thus rendered tolerant to the host. The present approach presents two advantages over the conventional humanized PBMC mouse models: (i) it circumvents the risk of xenogeneic graft-versus-host reaction and (ii) it mimics more closely human immune responses, thus favoring clinical translation. We show that the frequencies and numbers of transduced B cells in recipient’s spleens one week post-transfer are within the range of the size of the pre-immune B cell population specific for a given protein antigen in the mouse. They are also compatible with the B cell numbers required to elicit a sizeable immune response upon immunization. Altogether, our findings pave the way for future studies aiming at assessing therapeutic interventions involving B cell reprogramming for instance by an antibody transgene in a “humanized” hematopoietic setting.