Mus musculus has been a key animal model whereby important discoveries on bacterial pathogenesis of the Lyme disease (LD) pathogen, Borreliella burgdorferi (Bb), were made. In the United States alone, LD affects the lives of about half a million new patients each year, making it the most prevalent tick-borne illness. To date, however, there is no LD vaccine for humans on the market, and therefore, much research effort has focused on identifying protective Bb vaccine candidates. Because murine and human immune responses to Bb significantly differ, we evaluated humanized mice to improve the translatability of preclinical Lyme vaccine studies. Specifically, we assessed two humanized mouse models, UCB-HSC NSG and huNOG-EXL SA, for their capacity to induce protective anti-Bb antibodies. For that, we utilized an antibody-susceptible Bb (∆VlsE), which is effectively cleared by acquired anti-Bb antibodies of immunocompetent mice; and outer surface protein A (OspA), a surface Bb antigen that proved efficacious not only as part of licensed canine vaccines but also as part of a human LD vaccine that was transiently available to the public in the past. The data demonstrated that the tested models could not clear the ∆VlsE infection. Likewise, OspA-vaccinated humanized mouse immune system failed to prevent wild-type challenge. Given that no better alternative preclinical model has been developed, we hope that the inherent translatability limitations of standard LD mouse models will continue to be favorably accepted. Although our results do not encourage the use of the tested humanized models for preclinical LD vaccine efficacy testing, their utility remains valuable for mechanistic studies. IMPORTANCE:In this study, we assessed the ability of mice with humanized immune systems to develop protective antibodies against the bacterial agent of Lyme disease (LD), Borreliella burgdorferi. In contrast to an intact mouse immune system of classical LD models, the humanized immune system could not prevent vaccinated mice from being infected with LD spirochetes. The findings demonstrate that two tested humanized models cannot be used for preclinically testing LD vaccine candidates. Given the results, we hope that the animal-to-human translatability limitations of existing LD mouse models will be more favorably taken as there is no better alternative preclinical model developed.

Autoimmune diseases like multiple sclerosis (MS) and type 1 diabetes (T1D) lack therapies that induce durable, antigen-specific immune tolerance. We investigated whether mRNA lipid nanoparticles (LNPs) encoding disease-relevant autoantigens could re-establish immune homeostasis in preclinical models. While mRNA-LNP microbial vaccines evoke strong effector immune responses, we show that both systemic and intramuscular delivery of MOG27-63 mRNA-loaded LNPs attenuated disease severity in experimental autoimmune encephalomyelitis (EAE). Antigen-specific protection was similarly observed in a T1D adoptive transfer model. Therapeutic efficacy achieved using immunostimulatory LNPs challenges the current assumption that tolerogenic mRNA vaccines require immune-silent LNPs. Furthermore, divergent outcomes between autoantigens and irrelevant antigens suggest that antigen identity determines whether mRNA-LNPs promote immune tolerance or activation. Mechanistically, optimized LNPs efficiently targeted antigen-presenting cells (APCs) in the liver and spleen. This promoted a homeostatic APC phenotype and a hyporesponsive CD4+ T cell phenotype without inducing regulatory T cells (Tregs). Therefore, autoantigen mRNA was co-delivered with "immunoregulatory" mRNAs encoding cytokines (IL-2 mutein) or chemokines (CCL1) known to enhance Treg expansion and recruitment. This co-delivery further improved clinical outcomes in EAE. Together, these findings demonstrate that systemic and intramuscular treatment with mRNA-LNPs encoding autoantigens alongside immunoregulatory molecules represents a promising strategy for antigen-specific immunotherapy in autoimmune diseases.

Despite advances in antiretroviral therapy (ART), HIV-1 cure efforts remain hindered by viral persistence in long-lived myeloid cells and immune-privileged tissues that are poorly accessible and therefore unlikely to be assessed in human clinical trials. Consequently, robust research platforms, such as immune-cell humanized mice, are needed to bridge preclinical and clinical HIV research. However, previously described humanized mouse models often show incomplete hematopoietic development, particularly showing low levels of NK or myeloid cells. Herein, we evaluate a humanized FLT3 mouse model that develops NK cells, myeloid progenitors, monocytes, and both conventional (cDCs) and plasmacytoid dendritic cells (pDCs), and assessed its capacity to support HIV-1 infection, persistence, and viral rebound.Human cord blood derived CD34+ hematopoietic stem cells (HSC) were engrafted in FLT3 (Hu-FLT3) and NSG (Hu-NSG) mouse strains for comparison. While Hu-NSG and Hu-FLT3 mice showed comparable human lymphocyte levels, the proportion of myeloid-enriched populations, including monocytes, pDCs and cDCs, was three-fold higher in Hu-FLT3 mice (16.2 %) than in Hu-NSG mice (5.6 %), and the proportion of NK cells was six-fold higher (12.8 % vs. 1.9 %). Both strains supported HIV-1 infection, maintained viral replication for 17 weeks in untreated animals, and had detectable proviral DNA in peripheral blood, bone marrow and spleen. Oral ART reduced plasma HIV-1 RNA to undetectable levels within four weeks in both strains. After treatment discontinuation, viral rebound occurred within three weeks and approached pre-ART levels, consistent with rebound kinetics observed after treatment interruption in people living with HIV (PLWH). Human immune cells and HIV-1 RNA were more abundant in tissues from Hu-FLT3 mice than in Hu-NSG mice.This study establishes Hu‑FLT3 mice as a novel, robust and accessible in vivo platform to investigate potential HIV cure and persistence‑targeting interventions with translational relevance to human therapeutic development thanks to the improved and more complete human immune repertoire in Hu-FLT3.

Monoclonal antibodies (mAbs) are powerful therapeutic tools that are used to treat multiple types of human cancer as well as a diverse set of non-malignant diseases. Humanized NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice implanted with human tumors and with immune cells and tissues are widely used in studies of mAb-based therapeutics. However, due to a gain of function mutation in the Fcgr1 gene in the NOD strain background, NSG mice rapidly clear human IgG1, IgG3, and IgG4. As most mAbs are either IgG1 or IgG4 isotypes, the use of NOD-based mouse models for preclinical testing of therapeutic mAbs is limited by the reduced half-life in vivo. In order to extend the half-life of mAbs in NSG mice and create a more physiologically relevant model, we created NSG Fcγ Receptor I knock out (NSG-Fcgr1null) mice. IgG clearance was measured for three different cancer therapeutic mAbs: rituximab (IgG1), trastuzumab (IgG1), and pembrolizumab (IgG4), by comparing the levels of circulating human IgG over the course of 5 weeks post IV injection in NSG and NSG-Fcgr1null mice. Preliminary pharmacokinetic analyses found significant increases in the half-lives and exposure of each of these mAbs in the NSG-Fcgr1null mice when compared to NSG controls. Additionally, when engrafted with human hematopoietic stem cells (HSCs), NSG-Fcgr1null mice supported higher levels of serum IgG when compared to NSG controls. Overall, the NSG-Fcgr1null mouse presents a more physiologically relevant and translatable model for the in vivo testing of human therapeutic mAbs.

Human kidney organoids derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) have become novel tools for studying various kidney pathologies. Here, we transplanted ESC-derived kidney organoids into humanized mice with a mature human adaptive immune system developed through thymic education. As judged by histology and immunophenotyping, the transplanted HLA-mismatched kidney organoids trigged a robust alloimmune response, characterized by a dense immune cell infiltrate and enhanced memory T cell phenotype in the allograft 30 days post-transplantation. Multiplexed immunofluorescence revealed expression of functional markers of various immune cell infiltrates in response to organoid allografts, mimicking the T cell-mediated rejection process in humans. This validated our model as a novel platform to study various therapeutic strategies to control alloimmunity. Splenocytes isolated from organoid-transplanted hosts showed an alloantigen-specific memory response against 2D kidney organoids ex vivo. Overall, our study indicates that transplanting kidney organoids in humanized mice may be a valuable tool for studying human allogeneic immunity.

The identification of a "rundlichen Häuflein" by Paul Langerhans more than 150 years ago marked the initiation of a global effort to unravel the mysteries of pancreatic islets, an intricate system of nutrient-sensing, hormone-secreting, and signaling cells. In type 1 diabetes, this interconnected network is vulnerable to malfunction and immune attack, with strategies to prevent or repair islet damage still in their infancy. In 2014, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) established the Human Islet Research Network (HIRN) to accelerate our understanding of the molecular and cellular basis of type 1 diabetes development. In this article, investigators from the HIRN detail pioneering advances, technologies, and systems that contextualize insulin-producing β-cells and other related cells within their physiological environment. Disease models, devices, and therapies are evaluated by the HIRN in light of promising functional and mechanistic data. Collaborative relationships and opportunities within this network are emphasized as a means of enhancing the quality of innovative research and talent in science. Topics are developed through a series of questions, achievements, and milestones, with the 75th anniversary of the NIDDK as an opportunity to reflect on the past, present, and future of type 1 diabetes research. ARTICLE HIGHLIGHTS:The Human Islet Research Network (HIRN) was created by the National Institute of Diabetes and Digestive and Kidney Diseases to accelerate pioneering basic and translational research on the prevention, development, and progression of type 1 diabetes. There are critical knowledge gaps in research on the processes underlying human β-cell protection, loss, and replacement in type 1 diabetes. A multidisciplinary and collaborative research community focused on outstanding biological questions propels the development of innovative models, tools, and technologies and helps contextualize the complexity of this disease. Discoveries arising from the HIRN will profoundly improve our understanding of type 1 diabetes pathogenesis and expedite the development of disease avoidance, diagnosis, and treatment strategies.

Bacterial colonization of tumors is widespread, yet the dynamics during colonization remain underexplored. Here we discover strong variability in the sizes of intratumor bacterial clones and use this variability to infer the mechanisms of colonization. We monitored bacterial population dynamics in murine tumors after introducing millions of genetically barcoded Escherichia coli cells. Results from intravenous injection revealed that roughly a hundred bacteria seeded a tumor and that colonizers underwent rapid, yet highly nonuniform growth. Within a day, bacteria reached a steady-state and then sustained load and clone diversity. Intratumor injections, circumventing colonization bottlenecks, revealed that the nonuniformity persists and that the sizes of bacterial progenies followed a scale-free distribution. Theory suggested that our observations are compatible with a growth model constrained by a local niche load, global resource competition, and noise. Our work provides the first dynamical model of tumor colonization and may allow distinguishing genuine tumor microbiomes from contamination.

Aging and metabolic diseases are accompanied by systemic inflammation, but the mechanisms that induce this state are not known. We developed a human bone-marrow organoid system to explore mechanisms underlying metabolic-disease associated systemic inflammation. We find that a distinct type of hematopoietic stem cell (HSC) develops in the adipose-rich, yellow bone marrow, which is known to gradually replace the hematopoietic red marrow as we age and during metabolic disease. Unlike HSCs derived from the red bone marrow, HSCs derived from the yellow bone marrow have higher proliferation rates, increase myeloid differentiation, skew towards pro-inflammatory M1 macrophage differentiation, and express a distinct transcriptomic profile associated with responsiveness to wounding. Yellow marrow-derived HSCs express higher levels of the leptin receptor, which we find to be further increased in patients with type 2 diabetes. Our work demonstrates that the human long bone yellow marrow is a niche for a distinct class of HSCs which could underlie hematopoietic dysfunction during aging and metabolic disease processes suggesting a shared inflammaging mechanism.

The high rate of recurrence after radiation therapy in triple-negative breast cancer (TNBC) indicates that novel approaches and targets are needed to enhance radiosensitivity. Here, we report that neuropilin-2 (NRP2), a receptor for vascular endothelial growth factor (VEGF) that is enriched on subpopulations of TNBC cells with stem cell properties, is an effective therapeutic target for sensitizing TNBC to radiotherapy. Specifically, VEGF/NRP2 signaling induces nitric oxide synthase 2 (NOS2) transcription by a mechanism dependent on Gli1. NRP2-expressing tumor cells serve as a hub to produce nitric oxide (NO), an autocrine and paracrine signaling metabolite, which promotes cysteine-nitrosylation of Kelch-like ECH-associated protein 1 (KEAP1) and, consequently, nuclear factor erythroid 2-related factor 2-mediated (NFE2L2-mediated) transcription of antioxidant response genes. Inhibiting VEGF binding to NRP2, using a humanized mAb, results in NFE2L2 degradation via KEAP1, rendering cell lines and organoids vulnerable to irradiation. Importantly, treatment of patient-derived xenografts with the NRP2 mAb and radiation resulted in significant tumor necrosis and regression compared with radiation alone. Together, these findings reveal a targetable mechanism of radioresistance, and they support the use of NRP2 mAb as an effective radiosensitizer in TNBC.

Relapse in T-cell acute lymphoblastic leukemia (T-ALL) may signify the persistence of leukemia-initiating cells (L-ICs). Ectopic TAL1/LMO expression defines the largest subset of T-ALL, but its role in leukemic transformation and its impact on relapse-driving L-ICs remain poorly understood. In TAL1/LMO mouse models, double negative-3 (DN3; CD4-CD8-CD25+CD44-) thymic progenitors harbored L-ICs. However, only a subset of DN3 leukemic cells exhibited L-IC activity, and studies linking L-ICs and chemotolerance are needed. To investigate L-IC heterogeneity, we used mouse models and applied single-cell RNA-sequencing and nucleosome labeling techniques in vivo. We identified a DN3 subpopulation with a cell cycle-restricted profile and heightened TAL1/LMO2 activity, that expressed genes associated with stemness and quiescence. This dormant DN3 subset progressively expanded throughout leukemogenesis, displaying intrinsic chemotolerance and enrichment in genes linked to minimal residual disease. Examination of TAL/LMO patient samples revealed a similar pattern in CD7+CD1a- thymic progenitors, previously recognized for their L-IC activity, demonstrating cell cycle restriction and chemotolerance. Our findings substantiate the emergence of dormant, chemotolerant L-ICs during leukemogenesis, and demonstrate that Tal1 and Lmo2 cooperate to promote DN3 quiescence during the transformation process. This study provides a deeper understanding of TAL1/LMO-induced T-ALL and its clinical implications in therapy failure.

Omnipresent suppressive myeloid populations in the tumor microenvironment limit the efficacy of T-cell-directed immunotherapies, become more inhibitory after administration of T-cell checkpoint inhibitors, and are overall associated with worse survival of cancer patients. In early clinical trials, positive outcomes have been demonstrated for therapies aimed at repolarizing suppressive myeloid populations in the tumor microenvironment. We have previously described the key role of P-selectin glycoprotein ligand-1 (PSGL-1) in maintaining an inhibitory state of tumor-associated macrophages (TAMs), most of which express high levels of PSGL-1. Here we describe a novel, first-in-class humanized high-affinity monoclonal antibody VTX-0811 that repolarizes human macrophages from an M2-suppressive phenotype towards an M1 inflammatory phenotype, similar to siRNA-mediated knockdown of PSGL-1. VTX-0811 binds to PSGL-1 of human and cynomolgus macaque origins without inhibiting PSGL-1 interaction with P- and L-Selectins or VISTA. In multi-cellular assays and in patient-derived human tumor cultures, VTX-0811 leads to the induction of pro-inflammatory mediators. RNAseq data from VTX-0811 treated ex vivo tumor cultures and M2c macrophages show similar pathways being modulated, indicating that the mechanism of action translates from isolated macrophages to tumors. A chimeric version of VTX-0811, consisting of the parental murine antibody in a human IgG4 backbone, inhibits tumor growth in a humanized mouse model of cancer. VTX-0811 is exceptionally well tolerated in NHP toxicology assessment and is heading into clinical evaluation after successful IND clearance.

Oncogenic programs regulate the proliferation and maintenance of cancer stem cells, and can define pharmacologic dependencies. In acute myeloid leukemia (AML) with the chromosome inversion 16 (inv(16)), the fusion oncoprotein CBFβ::MYH11 regulates pathways associated with leukemia stem cell activity. Here we demonstrate that expression of Neuropilin-1 (NRP1) is regulated by the fusion oncoprotein, and promotes AML expansion. Mechanistically, we show that the NRP1 locus has open chromatin in inv(16) AML, and that CBFβ::MYH11 modulates the local function of the transcription factors ERG, GATA2 and RUNX1 to sustain NRP1 levels. We found that ERG activates NRP1 expression, and that CBFβ::MYH11 knockdown represses ERG expression, thereby allowing the repressive activity of GATA2/RUNX1 at three NRP1 enhancers. Functionally, we demonstrate that NRP1 enhances the expansion of leukemic cells in vitro and in mice, and that this activity is dependent on its VEGFR-associated FV/FVIII domain. Finally, we show that treatment with VEGF inhibitor axitinib reduces AML cell growth and delays median leukemia latency in vivo. Our findings reveal that the NRP1/VEGF axis mediates proliferation in inv(16) AML blasts, and suggest that targeting NRP1 function could be promising in combination AML therapy.

ABSTRACTSalmonella serovars Typhi and Paratyphi cause a prolonged illness known as enteric fever, whereas other serovars cause acute gastroenteritis. Mechanisms responsible for the divergent clinical manifestations of nontyphoidal and enteric fever Salmonella infections have remained elusive. Here, we show that S. Typhi and S. Paratyphi A can persist within human macrophages, whereas S. Typhimurium rapidly induces apoptotic macrophage cell death that is dependent on Salmonella pathogenicity island 2 (SPI2). S. Typhi and S. Paratyphi A lack 12 specific SPI2 effectors with pro-apoptotic functions, including nine that target nuclear factor κB (NF-κB). Pharmacologic inhibition of NF-κB or heterologous expression of the SPI2 effectors GogA or GtgA restores apoptosis of S. Typhi-infected macrophages. In addition, the absence of the SPI2 effector SarA results in deficient signal transducer and activator of transcription 1 (STAT1) activation and interleukin 12 production, leading to impaired TH1 responses in macrophages and humanized mice. The absence of specific nontyphoidal SPI2 effectors may allow S. Typhi and S. Paratyphi A to cause chronic infections.IMPORTANCESalmonella enterica is a common cause of gastrointestinal infections worldwide. The serovars Salmonella Typhi and Salmonella Paratyphi A cause a distinctive systemic illness called enteric fever, whose pathogenesis is incompletely understood. Here, we show that enteric fever Salmonella serovars lack 12 specific virulence factors possessed by nontyphoidal Salmonella serovars, which allow the enteric fever serovars to persist within human macrophages. We propose that this fundamental difference in the interaction of Salmonella with human macrophages is responsible for the chronicity of typhoid and paratyphoid fever, suggesting that targeting the nuclear factor κB (NF-κB) complex responsible for macrophage survival could facilitate the clearance of persistent bacterial infections.

AbstractPurpose: Brain metastases are associated with high morbidity and are often resistant to immune checkpoint inhibitors. We evaluated whether CDK4/6 inhibitor (CDKi) abemaciclib can sensitize intracranial tumors to programmed cell death protein 1 (PD-1) inhibition in mouse models of melanoma and breast cancer brain metastasis. Experimental Design: Treatment response was evaluated in vivo using immunocompetent mouse models of brain metastasis bearing concurrent intracranial and extracranial tumors. Treatment effect on intracranial and extracranial tumor–immune microenvironments (TIME) was evaluated using immunofluorescence, multiplex immunoassays, high-parameter flow cytometry, and T-cell receptor profiling. Mice with humanized immune systems were evaluated using flow cytometry to study the effect of CDKi on human T-cell development. Results: We found that combining abemaciclib with PD-1 inhibition reduced tumor burden and improved overall survival in mice. The TIME, which differed on the basis of anatomic location of tumors, was altered with CDKi and PD-1 inhibition in an organ-specific manner. Combination abemaciclib and anti–PD-1 treatment increased recruitment and expansion of CD8+ effector T-cell subsets, depleted CD4+ regulatory T (Treg) cells, and reduced levels of immunosuppressive cytokines in intracranial tumors. In immunodeficient mice engrafted with human immune systems, abemaciclib treatment supported development and maintenance of CD8+ T cells and depleted Treg cells. Conclusions: Our results highlight the distinct properties of intracranial and extracranial tumors and support clinical investigation of combination CDK4/6 and PD-1 inhibition in patients with brain metastases. See related commentary by Margolin, p. 257

Type 1 diabetes mellitus (T1DM) is a growing global health concern that affects approximately 8.5 million individuals worldwide. T1DM is characterized by an autoimmune destruction of pancreatic β cells, leading to a disruption in glucose homeostasis. Therapeutic intervention for T1DM requires a complex regimen of glycaemic monitoring and the administration of exogenous insulin to regulate blood glucose levels. Advances in continuous glucose monitoring and algorithm-driven insulin delivery devices have improved the quality of life of patients. Despite this, mimicking islet function and complex physiological feedback remains challenging. Pancreatic islet transplantation represents a potential functional cure for T1DM but is hindered by donor scarcity, variability in harvested cells, aggressive immunosuppressive regimens and suboptimal clinical outcomes. Current research is directed towards generating alternative cell sources, improving transplantation methods, and enhancing cell survival without chronic immunosuppression. This Review maps the progress in cell replacement therapies for T1DM and outlines the remaining challenges and future directions. We explore the state-of-the-art strategies for generating replenishable β cells, cell delivery technologies and local targeted immune modulation. Finally, we highlight relevant animal models and the regulatory aspects for advancing these technologies towards clinical deployment. Type 1 diabetes mellitus affects 8.5 million people globally and is characterized by autoimmune destruction of pancreatic β cells. This Review discusses cell replacement therapies for T1DM and outlines the challenges and future directions

Antitumor effects of anti-PSGL-1 in a humanized mouse model. A, Schematic of the NGS-SGM3-BLT humanized mouse melanoma model. B, Tumor volume of melanoma tumors in NGS-SGM3-BLT mice treated with anti-PSGL-1 or an Isotype control (n = 8–9 mice per group). C, MFI of MHCII and the ratio of the MFI of CD163 to MHCII was assessed on CD11b+CD14+ macrophages within the tumor measured by flow cytometry. D, The percent of total CD3+ T cells in the tumor and the activation status of T cells in both the tumor and spleen measured by the MFI CD69 on CD3+ T cells. Two-way ANOVA with multiple comparisons in Graph Prism software was used to assess significance in B and a one-way ANOVA in Graph Prism was used to assess significance in C and D.

Staining panels for the humanized mouse model experiment.

Prostate cancers are largely unresponsive to immune checkpoint inhibitors (ICIs), and there is strong evidence that programmed death-ligand 1 (PD-L1) expression itself must be inhibited to activate antitumor immunity. Here, we report that neuropilin-2 (NRP2), which functions as a vascular endothelial growth factor (VEGF) receptor on tumor cells, is an attractive target to activate antitumor immunity in prostate cancer because VEGF-NRP2 signaling sustains PD-L1 expression. NRP2 depletion increased T cell activation in vitro. In a syngeneic model of prostate cancer that is resistant to ICI, inhibition of the binding of VEGF to NRP2 using a mouse-specific anti-NRP2 monoclonal antibody (mAb) resulted in necrosis and tumor regression compared with both an anti–PD-L1 mAb and control immunoglobulin G. This therapy also decreased tumor PD-L1 expression and increased immune cell infiltration. We observed that the NRP2 , VEGFA , and VEGFC genes are amplified in metastatic castration-resistant and neuroendocrine prostate cancer. We also found that individuals with NRP2 High PD-L1 High metastatic tumors had lower androgen receptor expression and higher neuroendocrine prostate cancer scores than other individuals with prostate cancer. In organoids derived from patients with neuroendocrine prostate cancer, therapeutic inhibition of VEGF binding to NRP2 using a high-affinity humanized mAb suitable for clinical use also diminished PD-L1 expression and caused a substantial increase in immune-mediated tumor cell killing, consistent with the animal studies. These findings provide justification for the initiation of clinical trials using this function-blocking NRP2 mAb in prostate cancer, especially for patients with aggressive disease.