Functional Human Immune System Research Articles

Comparative characterization of two monoclonal antibodies targeting canine PD-1.

Monoclonal antibodies targeting immune checkpoints have revolutionized oncology. Yet, the effectiveness of these treatments varies significantly among patients, and they are associated with unexpected adverse events, including hyperprogression. The murine research model used in drug development fails to recapitulate both the functional human immune system and the population heterogeneity. Hence, a novel model is urgently needed to study the consequences of immune checkpoint blockade. Dogs appear to be uniquely suited for this role. Approximately 1 in 4 companion dogs dies from cancer, yet no antibodies are commercially available for use in veterinary oncology. Here we characterize two novel antibodies that bind canine PD-1 with sub-nanomolar affinity as measured by SPR. Both antibodies block the clinically crucial PD-1/PD-L1 interaction in a competitive ELISA assay. Additionally, the antibodies were tested with a broad range of assays including Western Blot, ELISA, flow cytometry, immunofluorescence and immunohistochemistry. The antibodies appear to bind two distinct epitopes as predicted by molecular modeling and peptide phage display. Our study provides new tools for canine oncology research and a potential veterinary therapeutic.

Abstract A005: Preclinical evaluation of novel immune cell therapies, check point inhibitors, and immune cell engagers in humanized mouse models

Abstract Background The preclinical evaluation of novel immune therapies requires humanized mouse models with functional human immune cells. In previous studies we have demonstrated, that either peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T- and NK-cells or hematopoietic stem cells (HSC) can be used to establish a humanized immune system with functional T-, B-, and NK cells, as well as monocytes and dendritic cells in immunodeficient mice. By xenotransplantation of cell-line-derived (CDX) or patient-derived (PDX) tumors on humanized mice, we successfully generated a full human tumor-immune-cell model for different disease entities. Finally, we validated the functionality of these models using checkpoint inhibitors like Ipilimumab (Ipi), Nivolumab (Nivo), Pembrolizumab (Pembro), cell therapies, and immune cell engagers. Methods HSC-humanized mice were generated by i.v. transplantation of CD34+ stem cells to immunodeficient 1st and 2nd generation NOG mice. Engraftment of immune cells was monitored by FACS analysis of blood samples. PBMC or isolated T- or NK-cell preparations were used to humanize mice by single or multiple i.v. injections. CDX and PDX from different entities were s.c. (i.e. lymphoma) and/or i.v. (leukemia) transplanted on those humanized mice. Tumor growth of orthotopic models was followed up by BLI measurement. Blood and tumor samples were analysed by for immune cell infiltration and activation. Results The transplanted HSCs engrafted in mice and established a functional human immune system with proliferation and differentiation. The 2nd generation NOG mice are showing an increased total engraftment and lineage specific differentiation of cells. The selected CDX and PDX tumors successfully engrafted on humanized mice without significant differences in tumor growth compared to non-humanized mice. Check point inhibitor treatments (Ipi, Nivo, or Pembro) induced tumor growth delay in selected models. BLI measurement is a suitable method to follow up systemic disease models and treatment effects over time. We identified a set of CDX and PDX models without interference with parallel injection of PBMC, T- or NK-cell preparations for the evaluation of immune cell engagers and other cell therapies. Conclusions We established human tumor-immune-cell models of different entities using CDX or PDX in combination with different donor derived immune cell subsets as effector cells. We demonstrated successful engraftment of HSC on different immunodeficient mouse strains, generating mice with a functional human hematopoiesis. These models have been successfully used for preclinical evaluation of novel check point inhibitors, cell therapies and immune cell engagers. Our human tumor-immune-cell models allow preclinical translational research on tumor immune biology as well as evaluation of new therapies, drug combinations, as well as biomarker identification and validation in subcutaneous and orthotopic models. Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Joshua Alcaniz, Glenn Smits, Wolfgang Walther, Jens Hoffmann. Preclinical evaluation of novel immune cell therapies, check point inhibitors, and immune cell engagers in humanized mouse models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A005.

A humanized orthotopic mouse model for preclinical evaluation of immunotherapy in Ewing sarcoma.

The advent of novel cancer immunotherapy approaches is revolutionizing the treatment for cancer. Current small animal models for most cancers are syngeneic or genetically engineered mouse models or xenograft models based on immunodeficient mouse strains. These models have been limited in evaluating immunotherapy regimens due to the lack of functional human immune system. Development of animal models for bone cancer faces another challenge in the accessibility of tumor engraftment sites. Here, we describe a protocol to develop an orthotopic humanized mouse model for a bone and soft tissue sarcoma, Ewing sarcoma, by transplanting fresh human cord blood CD34+ hematopoietic stem cells into young NSG-SGM3 mice combined with subsequent Ewing sarcoma patient derived cell engraftment in the tibia of the humanized mice. We demonstrated early and robust reconstitution of human CD45+ leukocytes including T cells, B cells, natural killer cells and monocytes. Ewing sarcoma xenograft tumors successfully orthotopically engrafted in the humanized mice with minimal invasive procedures. We validated the translational utility of this orthotopic humanized model by evaluating the safety and efficacy of an immunotherapy antibody, magrolimab. Treatment with magrolimab induces CD47 blockade resulting in significantly decreased primary tumor growth, decreased lung metastasis and prolonged animal survival in the established humanized model. Furthermore, the humanized model recapitulated the dose dependent toxicity associated with the CD47 blockade as observed in patients in clinical trials. In conclusion, this orthotopic humanized mouse model of Ewing sarcoma represents an improved platform for evaluating immunotherapy in bone and soft tissue sarcoma, such as Ewing sarcoma. With careful design and optimization, this model is generalizable for other bone malignancies.

Progress in construction of mouse models to investigate the pathogenesis and immune therapy of human hematological malignancy.

Hematological malignancy is a disease arisen by complicate reasons that seriously endangers human health. The research on its pathogenesis and therapies depends on the usage of animal models. Conventional animal model cannot faithfully mirror some characteristics of human features due to the evolutionary divergence, whereas the mouse models hosting human hematological malignancy are more and more applied in basic as well as translational investigations in recent years. According to the construction methods, they can be divided into different types (e.g. cell-derived xenograft (CDX) and patient-derived xenograft model (PDX) model) that have diverse characteristics and application values. In addition, a variety of strategies have been developed to improve human hematological malignant cell engraftment and differentiation in vivo. Moreover, the humanized mouse model with both functional human immune system and autologous human hematological malignancy provides a unique tool for the evaluation of the efficacy of novel immunotherapeutic drugs/approaches. Herein, we first review the evolution of the mouse model of human hematological malignancy; Then, we analyze the characteristics of different types of models and summarize the ways to improve the models; Finally, the way and value of humanized mouse model of human immune system in the immunotherapy of human hematological malignancy are discussed.

Immune Checkpoint Profiling in Humanized Breast Cancer Mice Revealed Cell-Specific LAG-3/PD-1/TIM-3 Co-Expression and Elevated PD-1/TIM-3 Secretion.

Checkpoint blockade is particularly based on PD-1/PD-L1-inhibiting antibodies. However, an efficient immunological tumor defense can be blocked not only by PD-(L)1 but also by the presence of additional immune checkpoint molecules. Here, we investigated the co-expression of several immune checkpoint proteins and the soluble forms thereof (e.g., PD-1, TIM-3, LAG-3, PD-L1, PD-L2 and others) in humanized tumor mice (HTM) simultaneously harboring cell line-derived (JIMT-1, MDA-MB-231, MCF-7) or patient-derived breast cancer and a functional human immune system. We identified tumor-infiltrating T cells with a triple-positive PD-1, LAG-3 and TIM-3 phenotype. While PD-1 expression was increased in both the CD4 and CD8 T cells, TIM-3 was found to be upregulated particularly in the cytotoxic T cells in the MDA-MB-231-based HTM model. High levels of soluble TIM-3 and galectin-9 (a TIM-3 ligand) were detected in the serum. Surprisingly, soluble PD-L2, but only low levels of sPD-L1, were found in mice harboring PD-L1-positive tumors. Analysis of a dataset containing 3039 primary breast cancer samples on the R2 Genomics Analysis Platform revealed increased TIM-3, galectin-9 and LAG-3 expression, not only in triple-negative breast cancer but also in the HER2+ and hormone receptor-positive breast cancer subtypes. These data indicate that LAG-3 and TIM-3 represent additional key molecules within the breast cancer anti-immunity landscape.

SARS-CoV-2 Omicron (B.1.1.529) shows minimal neurotropism in a double-humanized mouse model

Although severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) initially infects the respiratory tract, it also directly or indirectly affects other organs, including the brain. However, little is known about the relative neurotropism of SARS-CoV-2 variants of concern (VOCs), including Omicron (B.1.1.529), which emerged in November 2021 and has remained the dominant pathogenic lineage since then. To address this gap, we examined the relative ability of Omicron, Beta (B.1.351), and Delta (B.1.617.2) to infect the brain in the context of a functional human immune system by using human angiotensin-converting enzyme 2 (hACE2) knock-in triple-immunodeficient NGC mice with or without reconstitution with human CD34+ stem cells. Intranasal inoculation of huCD34+-hACE2-NCG mice with Beta and Delta resulted in productive infection of the nasal cavity, lungs, and brain on day 3 post-infection, but Omicron was surprisingly unique in its failure to infect either the nasal tissue or brain. Moreover, the same infection pattern was observed in hACE2-NCG mice, indicating that antiviral immunity was not responsible for the lack of Omicron neurotropism. In independent experiments, we demonstrate that nasal inoculation with Beta or with D614G, an ancestral SARS-CoV-2 with undetectable replication in huCD34+-hACE2-NCG mice, resulted in a robust response by human innate immune cells, T cells, and B cells, confirming that exposure to SARS-CoV-2, even without detectable infection, is sufficient to induce an antiviral immune response. Collectively, these results suggest that modeling of the neurologic and immunologic sequelae of SARS-CoV-2 infection requires careful selection of the appropriate SARS-CoV-2 strain in the context of a specific mouse model.

Liujunzi decoction exerts potent antitumor activity in oesophageal squamous cell carcinoma by inhibiting miR-34a/STAT3/IL-6R feedback loop, and modifies antitumor immunity

BackgroundLiujunzi decoction (LJZD), a traditional herbal formula and one of the most commonly used adjuvant medications for the treatment of oesophageal squamous cell carcinoma (ESCC), exerts good antitumor and immunomodulatory activity. However, its specific mechanism of action remains largely unclear. PurposeIn order to examine the potential primary and adjuvant antitumor mechanisms of LJZD, both in vitro and in vivo. MethodsIL-6 and miR-34a inhibitors were used to activate the miR-34a/STAT3/IL-6R feedback loop to observe the effects of LJZD. A humanised mouse model with a functional human immune system was constructed to evaluate the antitumor efficacy of LJZD in vivo on xenograft tumours, which was compared to that of the positive control drug anti-PD-1 monoclonal antibodies (mAb). Finally, a co-culture system of peripheral blood mononuclear and tumour cells in vitro was used to analyse the cytotoxic activity of LJZD on T cells. ResultsLJZD significantly interfered with IL-6-induced activation of the miR-34a/STAT3/IL-6R feedback loop in ESCC by restoring the expression of the tumour suppressor miR-34a, and inhibited the proliferation of EC109 oesophageal cancer cells in a dose-dependant manner. Furthermore, LJZD effectively suppressed oesophageal tumour growth in vivo and alleviated organ injury and visceral index. Furthermore, LJZD boosted antitumor immunity by increasing IFN-γ expression and CD8+tumour-infiltrating lymphocytes (TILs) infiltration in the peripheral blood and tumour tissues, respectively, which may be related to a decrease in PD-1, but not PD-L1 expression. Finally, we confirmed that LJZD strengthens the killing ability of T cells by suppressing PD-1 expression in a co-culture system in vitro. ConclusionLJZD exerts excellent antitumor effect by interfering with the miR-34a/STAT3/IL-6R feedback loop and augmenting antitumor immune responses. Which provides new insights into mechanisms for LJZD and sheds light on the multifaceted role of phytomedicine in cancer.

Human neutrophil development and functionality are enabled in a humanized mouse model.

Mice with a functional human immune system serve as an invaluable tool to study the development and function of the human immune system invivo. A major technological limitation of all current humanized mouse models is the lack of mature and functional human neutrophils in circulation and tissues. To overcome this, we generated a humanized mouse model named MISTRGGR, in which the mouse granulocyte colony-stimulating factor (G-CSF) was replaced with human G-CSF and the mouse G-CSF receptor gene was deleted in existing MISTRG mice. By targeting the G-CSF cytokine-receptor axis, we dramatically improved the reconstitution of mature circulating and tissue-infiltrating human neutrophils in MISTRGGR mice. Moreover, these functional human neutrophils in MISTRGGR are recruited upon inflammatory and infectious challenges and help reduce bacterial burden. MISTRGGR mice represent a unique mouse model that finally permits the study of human neutrophils in health and disease.

Can next-generation humanized mice that reconstituted with both functional human immune system and hepatocytes model the progression of viral hepatitis to hepatocarcinogenesis?

Hepatitis B virus (HBV) and Hepatitis C virus (HCV) chronic infections cause liver immunopathological diseases such as hepatitis, fibrosis, cirrhosis, and hepatocellular carcinomas, which are difficult to treat and continue to be major health problems globally. Due to the species-specific hepato-tropism of HBV and HCV, conventional rodent models are limited in their utility for studying the infection and associated liver immunopathogenesis. Humanized mice reconstituted with both functional human immune system and hepatocytes (HIS-HuHEP mice) have been extremely instrumental for in vivo studies of HBV or HCV infection and human-specific aspects of the progression of liver immunopathogenesis. However, none of the current HIS-HuHEP mice can model the progression of viral hepatitis to hepatocarcinogenesis which may be a notorious result of HBV or HCV chronic infection in patients, suggesting that they were functionally compromised and that there is still significant space to improve and establish next-generation of HIS-HuHEP mice with more sophisticated functions. In this review, we first summarize the principal requirements to establish HIS-HuHEP mice. We then discuss the respective protocols for current HIS-HuHEP mice and their applications, as well as their advantages and disadvantages. We also raise perspectives for further improving and establishing next-generation HIS-HuHEP mice.

Improvement of human myeloid and natural killer cell development in humanized mice via hydrodynamic injection of transposon plasmids containing multiple human cytokine genes.

Humanized mice reconstituted with a functional human immune system (HIS) are instrumental in studying human immunity and immune disorders in vivo. The poor or lack of cross-reactivity between mouse cytokines and human cells limits the development and/or function of human immune cell subsets including human myeloid, natural killer and B cells. Here we explored the potential to achieve long-term production of human cytokines in immunodeficient mice using a transposon-plasmid-based hydrodynamic injection approach. We constructed a transposon-plasmid carrying five human cytokine coding sequences (named PB-5F), and observed that four of the cytokines (granulocyte-macrophage colony-stimulating factor, interleukin (IL)-15, IL-6 and IL-3) were detectable in sera and three (granulocyte-macrophage colony-stimulating factor, IL-15 and IL-6) showed long-term production in immunodeficient mice that received a single hydrodynamic injection of PB-5F plus the transposase plasmid (Super PB). Furthermore, a single injection of PB-5F/Super PB markedly enhanced the reconstitution of human myeloid cells and natural killer cells, and promoted human B-cell maturation in HIS mice. Taken together, our data revealed that hydrodynamic injection of the PB-5F/Super PB vectors may serve as a convenient and efficacious means to promote human immune function in HIS mice.

Abstract 1650: Humanized mouse models for preclinical evaluation of novel immune cell therapies, checkpoint inhibitors, and immune cell engagers

Abstract Background The preclinical evaluation of novel immune therapies demands humanized mouse models with functional human immune cells. In previous studies we have demonstrated, that either peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T- and NK-cells or hematopoietic stem cells (HSC) can be used to establish a humanized immune system with functional T-, B-, and NK cells, as well as monocytes and dendritic cells in immunodeficient mice. By transplantation of cell-line-derived (CDX) or patient-derived (PDX) tumor xenografts on humanized mice, we successfully generated a full human tumor-immune-cell model for different tumor entities. Finally, we validated the functionality of these models using checkpoint inhibitors like Ipilimumab (Ipi), Nivolumab (Nivo), Pembrolizumab (Pembro), cell therapies and immune cell engagers. Methods HSC-humanized mice were generated by i.v. transplantation of CD34+stem cells to immunodeficient NOG mice. PBMC or isolated T- or NK-cell preparations were used to humanize mice by single or multiple i.v. injections. CDX and PDX from different entities (i.e. lymphoma, neuroblastoma, and breast cancer) were transplanted on those humanized mice. These models were used to evaluate novel immune therapies. Blood and tumor samples were analysed by FACS for immune cell infiltration and activation. Results The transplanted HSCs engrafted in mice and established a functional human immune system with proliferation and differentiation. 14 weeks after HSC inoculation up to 20% of the human immune cells in the blood were functional T-cells, characterized by a high PD-1 expression. The selected CDX and PDX tumors successfully engrafted on humanized mice without significant differences in tumor growth compared to non-humanized mice. Checkpoint inhibitor treatments induced tumor growth delay in selected models. FACS analysis of tumors revealed an increased percentage of tumor infiltrating T-cells. We identified a set of CDX and PDX models without interference with parallel injection of PBMC, T- or NK-cell preparations for the evaluation of immune cell engagers and other cell therapies. Conclusions We established human tumor-immune-cell models of different entities using CDX or PDX in combination with different donor derived immune cell subsets as effector cells. We demonstrated successful engraftment of HSC on immunodeficient mouse strains, generating mice with a functional human hematopoiesis. These models have been employed for preclinical evaluation of novel checkpoint inhibitors, cell therapies and immune cell engagers. Our models allow preclinical, translational studies on tumor immune biology as well as evaluation of new therapies, drug combinations and biomarker identification and validation. Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Wolfgang Walther, Jens Hoffmann. Humanized mouse models for preclinical evaluation of novel immune cell therapies, checkpoint inhibitors, and immune 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 1650.

Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2.HLA-DR4.Rag1KO.IL-2RγcKO.NOD) for COVID-19

ABSTRACT We report a Human Immune System (HIS)-humanized mouse model (“DRAGA”: HLA-A2.HLA-DR4.Rag1KO.IL-2 RγcKO.NOD) for COVID-19 research. DRAGA mice express transgenically HLA-class I and class-II molecules in the mouse thymus to promote human T cell development and human B cell Ig-class switching. When infused with human hematopoietic stem cells from cord blood reconstitute a functional human immune system, as well as human epi/endothelial cells in lung and upper respiratory airways expressing the human ACE2 receptor for SARS-CoV-2. The DRAGA mice were able to sustain SARS-CoV-2 infection for at least 25 days. Infected mice showed replicating virus in the lungs, deteriorating clinical condition, and human-like lung immunopathology including human lymphocyte infiltrates, microthrombi and pulmonary sequelae. Among the intra-alveolar and peri-bronchiolar lymphocyte infiltrates, human lung-resident (CD103+) CD8+ and CD4+ T cells were sequestered in epithelial (CD326+) lung niches and secreted granzyme B and perforin, suggesting anti-viral cytotoxic activity. Infected mice also mounted human IgG antibody responses to SARS-CoV-2 viral proteins. Hence, HIS-DRAGA mice showed unique advantages as a surrogate in vivo human model for studying SARS-CoV-2 immunopathological mechanisms and testing the safety and efficacy of candidate vaccines and therapeutics.

Inducing Long-Term HIV-1 Latency in the TKO-BLT Mouse Model.

Multiple humanized mouse models have been produced for the study of HIV-1 infection and treatment. Humanized mice produced using the bone marrow, liver, thymus (BLT) method particularly have well-reconstituted and functional human immune systems, providing an excellent model for HIV-1 cure strategies that aim to harness the human immune system as part of the cure approach. The TKO-BLT humanized mouse model is especially useful for long-term studies as it is highly resistant to the wasting syndrome and graft-versus-host disease (GVHD ) that can limit the use of other BLT-models. Here we describe the methods used to induce latency in TKO-BLT mice, using both injectable and free-fed combination antiretroviral therapy (cART) regimens, for use in the study of HIV-1 latency and evaluation of HIV-1 cure interventions.

Diffuse Large B Cell Pdtx in Humanized Mice Are Valuable Models to Study Host-Lymphoma Interactions and Immune-Modulating Agents

Introduction: Immunotherapy is a promising therapeutic intervention for cancer treatment. Activation of the immune system via checkpoint blockade has been shown to produce antitumor responses in patients with both solid and hematological tumors. However, many patients do not respond to checkpoint inhibitors, and additional therapies are needed to treat these patients. Testing immunotherapies requires a functional human immune system; thus, it is difficult to evaluate their effectiveness using conventional experimental models. For this reason, establishing in vivo models that closely reproduce not only human tumors, but also their interactions with the human immune system, has become mandatory.Methods: We developed a humanized mouse model and combined it with a patient-derived tumor xenograft (PDTX). Humanized mice (HuMice) were generated by transplantation of cord blood or mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells into preconditioned immunodeficient mice. We compared human engraftment in 3 different mouse strains: NSG (NOD.Cg-Prkdc scidIl2rg tm1Wjl/SzJ), NSGS (NOD.Cg-Prkdc scidIl2rg tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ) and NBSGW (NOD.Cg-Kit W-41J Tyr + Prkdc scid Il2rg tm1Wjl/ThomJ). Immune cell profiling and distribution was performed using flow cytometry and immunohistochemistry. The B cell receptor (BCR) repertoire was evaluated using an RNA-based NGS assay. To evaluate the maturation and functionality of T cells developing in HuMice we performed proliferation, degranulation and intracellular cytokine staining.Results: Two months after CD34+ cell transplantation, we observed high levels of human hematopoietic chimerism in all the 3 strains. NSGS mice supported high-level chimerism as early as 1 month after transplantation, with more than 25% of human CD45+ cells in the blood. In all mice the majority of human circulating leukocytes were CD19+ B cells. An early appearance of CD3+ human T cells was detected in NSGS mice as compared to the other strains. Notably, the T cell expansion correlated with a decrease in relative B cell abundance while the myeloid cell contribution to the graft remained steady. We documented the differentiation of CD4+ and CD8+ human T cells at a 2:1 ratio. The characterization of the T cell subsets revealed that the majority was represented by CD45RA-CCR7- effector memory cells in both the spleen and the blood of HuMice. Nevertheless, recipient mice did not exhibit overt signs of graft-versus-host disease. We also evaluated the cytotoxic potential of T cells isolated from the spleen of HuMice: ex vivo peptide antigen (i.e. EBV) presentation let to generation of effective and specific cytotoxic T-cells.After assessing a functional human immune system reconstitution in HuMice, we challenged them in vivo with low-passage tumor fragments from a diffuse large B cell lymphoma (DLBCL) PDTX. All tumor implants were successfully engrafted in both HuMice and non-humanized controls. Remarkably, all the 3 HuMice strains showed a significant reduction in the tumor volume and/or eradication compared to matched non-humanized controls. Flow cytometry analysis of the peripheral blood of humanized PDTX revealed that the tumor engraftment elicited a significant expansion of CD3+ T cells and cytotoxic CD8+ lymphocytes. Moreover, tumors developing in HuMice exhibited intermediate to high levels of tumor infiltrating T lymphocytes commingling with the neoplastic B cells, as determined by immunohistochemistry. Large areas of necrosis were often observed in PDTX of HuMice. Infiltrating CD3+ cells were TIGIT, PD-1 and Lag-3 positive, and did not efficiently proliferate ex vivo: all features consistent with an exhaustion phenotype. PDTX of HuMice often displayed larger areas of necrosis.Conclusions: Collectively, our data demonstrate that a robust reconstitution can be achieved in different strains of immunocompromised mice and that HuMice elicit effective anti-lymphoma responses. PDTX HuMice represent a powerful platform to study host-tumor interactions, and to test novel immune-based strategies (CAR-T, bifunctional Abs) and new pharmacological approaches to counteract T-cell exhaustion. [Display omitted] DisclosuresScandura: Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Constellation: Research Funding; MPN-RF (Foundation): Research Funding; CR&T (Foudation): Research Funding; European Leukemia net: Honoraria, Other: travel fees . Roth: Janssen: Consultancy; Merck: Consultancy.

Early Effects of HTLV-1 Infection on the Activation, Exhaustion, and Differentiation of T-Cells in Humanized NSG Mice.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive malignancy of CD4+ T-cells associated with HTLV-1 infection. In this study, we used the model of immunodeficient NSG mice reconstituted with a functional human immune system (HIS) to investigate early events in HTLV-1 pathogenesis. Upon infection, human T-cells rapidly increased in the blood and lymphoid tissues, particularly CD4+CD25+ T-cells. Proliferation of CD4+ T-cells in the spleen and mesenteric lymph nodes (MLN) correlated with HTLV-1 proviral load and CD25 expression. In addition, splenomegaly, a common feature of ATLL in humans, was also observed. CD4+ and CD8+ T-cells predominantly displayed an effector memory phenotype (CD45RA−CCR7−) and expressed CXCR3 and CCR5 chemokine receptors, suggesting the polarization into a Th1 phenotype. Activated CD8+ T-cells expressed granzyme B and perforin; however, the interferon-γ response by these cells was limited, possibly due to elevated PD-1 expression and increased frequency of CD4+FoxP3+ regulatory T-cells in MLN. Thus, HTLV-1-infected HIS-NSG mice reproduced several characteristics of infection in humans, and it may be helpful to investigate ATLL-related events and to perform preclinical studies. Moreover, aspects of chronic infection were already present at early stages in this experimental model. Collectively, we suggest that HTLV-1 infection modulates host immune responses to favor viral persistence.

Advances in Human Immune System Mouse Models for Personalized Treg-Based Immunotherapies.

Immunodeficient mice engrafted with a functional human immune system [Human immune system (HIS) mice] have paved the way to major advances for personalized medicine and translation of immune-based therapies. One prerequisite for advancing personalized medicine is modeling the immune system of individuals or disease groups in a preclinical setting. HIS mice engrafted with peripheral blood mononuclear cells have provided fundamental insights in underlying mechanisms guiding immune activation vs. regulation in several diseases including cancer. However, the development of Graft-vs.-host disease restrains relevant long-term studies in HIS mice. Alternatively, engraftment with hematopoietic stem cells (HSCs) enables mimicking different disease stages, however, low frequencies of HSCs in peripheral blood of adults impede engraftment efficacy. One possibility to overcome those limitations is the use of patient-derived induced pluripotent stem cells (iPSCs) reprogrammed into HSCs, a challenging process which has recently seen major advances. Personalized HIS mice bridge research in mice and human diseases thereby facilitating the translation of immunomodulatory therapies. Regulatory T cells (Tregs) are important mediators of immune suppression and thereby contribute to tumor immune evasion, which has made them a central target for cancer immunotherapies. Importantly, studying Tregs in the human immune system in vivo in HIS mice will help to determine requirements for efficient Treg-targeting. In this review article, we discuss advances on personalized HIS models using reprogrammed iPSCs and review the use of HIS mice to study requirements for efficient targeting of human Tregs for personalized cancer immunotherapies.

Development of a huBLT Mouse Model to Study HCMV Latency, Reactivation, and Immune Response.

Immunodeficient mice engrafted with human tissues provide a robust model for the in vivo investigation of human-restricted viruses such as human cytomegalovirus (HCMV). Several humanized mouse models have been developed and improved over the last 30years. Here, we describe a protocol for the transplant of human hematopoietic stem cells with autologous fetal liver and thymic tissues into NOD.Cg-PrkdcscidIL2rγtm1Wjl mice to create a humanized bone marrow-liver-thymus model (huBLT) that can be infected with HCMV. The presence of human thymus allows the development of a functional human immune system, including HLA-restricted human T-cells and B-cells. Indeed, following infection, huBLT mice generate virus-specific CD4+ and CD8+ T-cell responses. Additionally, both HCMV-specific IgM and IgG B-cell responses can be detected. This huBLT model provides the first animal model to explore the adaptive human immune response to HCMV infection.

Human Immune System Mice With Autologous Tumor for Modeling Cancer Immunotherapies.

Mouse models are the most commonly used in vivo system for biomedical research, in which immune-related diseases and therapies can be investigated in syngeneic and immunologically intact hosts. However, because there are significant differences between rodent and human, most findings from conventional mouse models cannot be applied to humans. The humanized mouse with a functional human immune system, also referred to as human immune system (HIS) mouse, is the only model available to date for in vivo studies in real-time of human immune function under physiological and pathological conditions. HIS mice with human tumor xenografts are considered an emerging and promising in vivo model for modeling human cancer immunotherapy. In this review, we briefly discuss the protocols to construct HIS mice and elaborate their pros and cons. Particular attention is given to HIS mouse models with human tumor that is autologous or genetically identical to the human immune system, which are discussed with examples of their usefulness in modeling human cancer immunotherapies.

Abstract 1632: Long-term maintenance of human mature NK cells from hCD34+HSCs engraftment supported by immune humanized B-NDG hIL15 mice

Abstract Human HSC (hematopoietic stem cells) engrafted humanized mice are a powerful model that allows researchers to examine the human immune system and evaluate cellular immunotherapy in the preclinical stage. Successful reconstitution of the human immune system in B-NDG mice (a complete immunodeficient mouse model previously developed by Biocytogen) has been accomplished using CD34+ hematopoietic stem cells. However, the species barrier between human immune cells and the mouse microenvironment can obstruct human immunity acquisition, as well as innate immune function development, in the mice upon the reconstitution of immune system. To address this issue, Biocytogen has developed and validated the B-NDG hIL15 mice that combines a B-NDG mouse background and expresses human IL15 cytokine, which plays important roles in the innate and adaptive cell homeostasis, as well as peripheral immune function. The targeting strategy stipulates the insertion of CDS region of the human IL15 gene after the 5′UTR of the mouse Il15. In this work, human CD34+ hematopoietic stem cells have been successfully grafted in B-NDG hIL15 mice, to reconstitute a functional human immune system. At 4 weeks post CD34+ cell implantation, the percentage of hCD45+ cells reached over 25%, containing human T, B, and NK cells. In summary, these CD34+ reconstituted B-NDG hIL15 mice are validated to be promising tools for the efficacy study of clinically relevant therapeutic agents given the mice's long-term maintenance of human NK cells upon the reconstitution of immune system. Citation Format: Qingcong Lin, Yuting Hu, Xiaofei Zhou, Youhong Su, Yuelei Shen. Long-term maintenance of human mature NK cells from hCD34+HSCs engraftment supported by immune humanized B-NDG hIL15 mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1632.

Abstract 5631: Leukaemia and lymphoma patient-derived xenografts (PDX) engraft on humanized mice and respond to immune therapy with check point inhibitors

Abstract Background The preclinical evaluation of novel immune therapies for cancer remains a challenge, as models require both, engraftment of human tumor cells and a matching human immune cell population. In previous experiments, we have demonstrated, that we can use either peripheral blood mononuclear cells (PBMC) or hematopoietic stem cells (HSC) to establish a humanized immune system on highly immunodeficient mice. In this study, we transplanted patient-derived xenografts (PDX) from leukaemia and lymphoma on these humanized mice. Further, we validated the models by evaluating treatments with checkpoint inhibitors Nivolumab (Nivo) and Ipilimumab (Ipi) or standard of care therapies. Methods HSC were transplanted i.v. on immunodeficient mice. Engraftment of human immune cells was monitored by FACS analysis of blood samples. Three lymphoma PDX and one acute myeloid leukaemia (AML) model were s.c. transplanted on humanized mice. To model systemic leukaemia, three AML models were transplanted i.v. on humanized mice. Engraftment of the PDX was compared to growth on non-humanized mice. After successful engraftment, mice were treated with Nivo, Ipi or in combination with standard therapies and irradiation. Results We confirmed successful engraftment of HSC in the humanized mice. Within 14 weeks after HSC inoculation, FACS analysis revealed an established functional human immune system with up to 20% T-cells, characterized by a high PD-1 expression. The s.c. transplanted leukaemia and lymphoma PDX engrafted on the humanized mice and tumor growth was measured with calipers. Engraftment of i.v. transplanted AML models was confirmed by FACS measurement of human CD33+ AML cells in the blood of the humanized mice. Treatment with Ipi or Nivo alone or in combination led to a minor growth delay. FACS analysis confirmed an increased percentage of activated T-cells in the blood and in the s.c. tumors. Response to checkpoint inhibitors was in correlation to PD-L1 expression on the lymphoma and AML cells. Combination of check point inhibitors with radiotherapy provided additive effects in our models. Conclusions We developed a humanized immune-PDX model for different blood cancers enabling appropriate preclinical translational research on tumor immune biology and the evaluation of new therapies and combinations, as well as the identification and validation of biomarkers for immune therapy. These novel model are currently optimized for the preclinical evaluation of new bispecific immune cell engagers (BITE) and cell therapies (CART). Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Antje Siegert, Bernadette Brzezicha, Anja Sterner-Kock, Wolfgang Walther, Jens Hoffmann. Leukaemia and lymphoma patient-derived xenografts (PDX) engraft on humanized mice and respond to immune therapy with check point inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5631.