Abstract The immune milieu within tumors, consisting of diverse cell types including adaptive immune cells as well as macrophages, dendritic cells, natural killer and other innate immune cells, is critical to determining cancer outcome. However, the immune tumor microenvironment (TME) has been challenging to model, owing to inherent inter-species differences. While humanized mice can support human immune cells, the hematopoietic stem and progenitor cells (HSPCs) used for transplantation have been largely limited to fetal or neonatal stem cell sources, necessitating allogeneic experiments with limited applicability. We sought to develop a method to pre-clinically model an individual adult cancer patient, capturing the unique features of an individual such as germline genetic determinants of immune function and somatic tumor heterogeneity, and creating an autologous system. MISTRG6 may be engrafted with low numbers of HSPCs. When engrafted with equivalent numbers of CD34+ cells from human fetal liver (FL), neonatal cord blood (CB), adult mobilized peripheral blood (MPB), or adult bone marrow (BM), MISTRG6 mice harbored greatly increased human hematopoietic cells as a proportion of total hematopoietic cells in peripheral blood compared with NOD-scid-gamma (NSG) and MISTRG mice (p<0.0001). We found that MISTRG6 mice could be engrafted with as few as 1,000 human HSPCs, arguably 100x more efficient than other models, and achieve robust hematopoietic transplantation after 10-12 weeks, indicating the efficiency of this strain in supporting the growth of hematopoietic cells. To better elucidate the mechanism responsible for this enhanced human engraftment, we enumerated human and mouse hematopoietic progenitors in BM of NSG, MISTRG, and MISTRG6 mice. Human progenitors, including CD34+ and CD34+CD38+ cells, were significantly increased in both frequency and absolute numbers in MISTRG and MISTRG6 mice compared with NSG mice (p<0.001), and mouse hematopoietic lin(-)cKit+ (LK) and lin(-)Sca1+cKit+ (LSK) progenitor populations were significantly diminished (p<0.0001), suggesting that the enhanced hematopoietic engraftment observed in MISTRG6 is, in part, a consequence of increased human progenitor frequency and reduced mouse competition. MISTRG6 allows efficient engraftment of patient derived HSPCs. We sought to apply this improved engraftment prospectively to model individual patients’ TME through collection of BM-derived CD34+ cells from patients under active treatment along with tumor tissue from the same patient. At two cancer centers, we enrolled patients with melanoma, NSCLC, PDAC, and HNSCC to provide BM aspirate, peripheral blood, and tumor tissue. CD34+ cells were isolated from BM aspirates and tumor tissue was utilized to generate PDXs. Overall, 71 patients were enrolled, 46 melanoma, 19 NSCLC, 4 PDAC, 2 HNSCC, ages 22-85, 39% females. These yielded autologous, immune-reconstituted MISTRG6 hosts from 14 melanoma, 5 NSCLC, 2 PDAC, and 1 HNSCC patients. Autologously engrafted MISTRG6 mice displayed the gamut of human immune cells of adaptive and innate types in PB at 7 weeks of age. Notably, this included CD33+ myeloid cells such as CD14+CD16− classical, CD14+CD16+ intermediate, and CD14−CD16+ non-classical monocytes. Moreover, human dendritic cells (DCs), key innate immune cells for initiation of anti-tumor responses were readily detected by flow cytometry in spleens of autologously-engrafted mice, including cDC1, cDC2, and pDC cells. MISTRG6 mice bearing a patient’s hematopoietic cells support autologous PDX growth. Having achieved successful engraftment of patient hematopoietic systems in MISTRG6 hosts, we next subcutaneously introduced the patient’s matched PDX tumor tissue to generate autologously engrafted PDX mice. For most patients, tumors grown in autologous HSPC-engrafted hosts were significantly larger than in non-engrafted hosts. Multicolor immunofluorescence staining of PDX tumors demonstrated that human immune cells, including CD3+ T cells, CD14+ and HLA-DR+ myeloid cells, penetrated deeply into the tumor and co-localized with tumor cells as well as with other engrafted immune cells. Indeed, HLA-DR+CD14+macrophages and HLA-DR+CD14(-) dendritic cells were present, and direct physical interaction between T cells and macrophages was evident. Using whole-exome sequencing, we found that 225 somatic changes were shared between patient Mel738’s surgical resection sample, two PDX tumors from non-engrafted mice lacking human immune cells, and two PDX tumors from mice with autologous engraftment. 5 additional changes were shared among the tumor samples and absent from the cell line, with 36 additional mutations being specific to the cell line. These data underscore the capacity of the autologous PDX method to recapitulate the somatic heterogeneity that the patient tumor possesses. Autologous MISTRG6 mice display diverse human immune cell populations and recapitulate an immunosuppressive TME. To fully characterize the autologous MISTRG6 model and investigate mechanisms by which autologous human immune cells enhance tumor growth, we performed single cell transcriptomics on hCD45+-enriched cells from blood and tumor isolated from autologous mice. This revealed 16 distinct cell subtypes, including 3 myeloid, 2 NK cell, 2 CD8 T cell, 3 CD4 T cell, 2 cycling lymphocyte, 1 B cell, and 3 melanoma cell clusters. Subclustering of myeloid cells revealed 9 distinct clusters including 4 monocyte, 4 macrophage, and 1 DC cluster. Comparing CD8 T cells present in blood versus tumor revealed that the most differentially expressed genes (DEGs) found in blood were characteristic of naïve T cells, while genes present in the TME were consistent with activated T cell phenotypes. In addition, sub-clustering revealed 3 distinct CD8 T cell types that included two activated-like populations, with one of these populations also expressing an activated/exhausted program typified by expression of PDCD1, LAG3, and GZMA. Naïve-like T cells were most highly represented in the blood, while activated and activated/exhausted-like genes were more present in the TME. Inhibiting the actions of human VEGF-A blocks the enhanced tumor growth in autologously engrafted mice. Notably, IPA Upstream Regulator Analysis identified VEGFA, a central player in tumor growth and vascularization, as a key upstream inducer of genes in the TME (FDR p= 5.65 × 10−13). Indeed, expression VEGFA itself was nearly absent in blood but induced in the TME, especially in macrophages and VEGFA targets were highly represented among the DEGs between tumor and blood.To test the relevance of VEGF-A in the TME, we selectively blocked human VEGF-A by treating autologous mice humanized from Mel2 with the anti-hVEGF-A antibody bevacizumab that has high affinity for human VEGF-A yet low affinity for mouse VEGF-A. PDXs grown in untreated autologously engrafted MISTRG6 mice grew significantly larger than those in non-engrafted littermate control hosts (p<0.05). When treated with bevacizumab, the enhanced tumor growth was significantly abrogated, with bevacizumab-treated mice bearing significantly smaller tumors compared with controls (p<0.001). Future Directions: Thus, these in silico and in vivo results suggest that human VEGF-A production in the autologous TME enhances tumor growth in MISTRG6 PDX models and underscores the utility of the MISTRG6 system for pre-clinical testing of drugs that act on human immune components of the TME. By engrafting mice with bone marrow derived stem cells followed by implantation of tumor derived from the same donor, we have demonstrated that autologous MISTRG6 models recapitulate important features of the human TME, including sufficient immunosuppression to prevent tumor clearance, presence of activated/exhausted T cells, and innate immune cells including DCs, monocytes, NK cells, and macrophages, the latter especially relevant to the production of VEGF-A. Citation Format: Michael Chiorazzi, Jan Martinek, Bradley Krasnick, Yunjiang Zheng, Keenan Robbins, Rihao Qu, Gabriel Kaufmann, Zachary Skidmore, Laura Henze, Frederic Brösecke, Adam Adonyi, Jun Zhao, Liang Shan, Esen Sefik, Jacqueline Mudd, Ye Bi, S Peter Goedegebuure, Malachi Griffith, Obi Griffith, Abimbola Oyedeji, Sofia Fertuzinhos, Roland Garcia-Milian, Daniel Boffa, Frank Detterbeck, Andrew Dhanasopon, Justin Blasberg, Benjamin Judson, Scott Gettinger, Katerina Politi, Yuval Kluger, A Karolina Palucka, Ryan Fields, Richard A. Flavell. Autologous humanized PDX modeling for immuno-oncology recapitulates the human tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr NG11.

RNA helicase DHX9 has been extensively characterized as a transcriptional regulator, which is consistent with its mostly nucleic localization. It is also involved in recognizing RNA viruses in the cytoplasm. However, there is no in vivo data to support the antiviral role of DHX9; meanwhile, as a nuclear protein, if and how nucleic DHX9 promotes antiviral immunity remains largely unknown. Here, we generated myeloid-specific and hepatocyte-specific DHX9 knockout mice and confirmed that DHX9 is crucial for host resistance to RNA virus infections in vivo. By additional knockout MAVS or STAT1 in DHX9-deficient mice, we demonstrated that nucleic DHX9 plays a positive role in regulating interferon-stimulated gene (ISG) expression downstream of type I interferon. Mechanistically, upon interferon stimulation, DHX9 is directly bound to STAT1 and recruits Pol II to the ISG promoter region to participate in STAT1-mediated transcription of ISGs. Collectively, these findings uncover an important role for nucleic DHX9 in antiviral immunity.

Hepatocytes, the major metabolic hub of the body, execute functions that are human-specific, altered in human disease, and currently thought to be regulated through endocrine and cell-autonomous mechanisms. Here, we show that key metabolic functions of human hepatocytes are controlled by non-parenchymal cells (NPCs) in their microenvironment. We developed mice bearing human hepatic tissue composed of human hepatocytes and NPCs, including human immune, endothelial, and stellate cells. Humanized livers reproduce human liver architecture, perform vital human-specific metabolic/homeostatic processes, and model human pathologies, including fibrosis and non-alcoholic fatty liver disease (NAFLD). Leveraging species mismatch and lipidomics, we demonstrate that human NPCs control metabolic functions of human hepatocytes in a paracrine manner. Mechanistically, we uncover a species-specific interaction whereby WNT2 secreted by sinusoidal endothelial cells controls cholesterol uptake and bile acid conjugation in hepatocytes through receptor FZD5. These results reveal the essential microenvironmental regulation of hepatic metabolism and its human-specific aspects.

BACKGROUND:Interactions between immune and tumor cells are critical to determining cancer progression and response. In addition, preclinical prediction of immune-related drug efficacy is limited by interspecies differences between human and mouse, as well as inter-person germline and somatic variation. To address these gaps, we developed an autologous system that models the tumor microenvironment (TME) from individual patients with solid tumors. METHOD:With patient-derived bone marrow hematopoietic stem and progenitor cells (HSPCs), we engrafted a patient's hematopoietic system in MISTRG6 mice, followed by transfer of patient-derived xenograft (PDX) tissue, providing a fully genetically matched model to recapitulate the individual's TME. We used this system to prospectively study tumor-immune interactions in patients with solid tumor. RESULTS:Autologous PDX mice generated innate and adaptive immune populations; these cells populated the TME; and tumors from autologously engrafted mice grew larger than tumors from non-engrafted littermate controls. Single-cell transcriptomics revealed a prominent vascular endothelial growth factor A (VEGFA) signature in TME myeloid cells, and inhibition of human VEGF-A abrogated enhanced growth. CONCLUSIONS:Humanization of the interleukin 6 locus in MISTRG6 mice enhances HSPC engraftment, making it feasible to model tumor-immune interactions in an autologous manner from a bedside bone marrow aspirate. The TME from these autologous tumors display hallmarks of the human TME including innate and adaptive immune activation and provide a platform for preclinical drug testing.

Mice with a functional human immune system serve as an invaluable tool to study the development and function of the human immune system in vivo. 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.

Cancer immunotherapy has emerged as a promising therapeutic intervention. However, complete and durable responses are only seen in a fraction of patients who have cancer. A key factor that limits therapeutic success is the infiltration of tumors by cells of the myeloid lineage. The inhibitory receptor signal regulatory protein-α (SIRPα) is a myeloid-specific immune checkpoint that engages the "don't eat me" signal CD47 expressed on tumors and normal tissues. We therefore developed the monoclonal antibody KWAR23, which binds human SIRPα with high affinity and disrupts its binding to CD47. Administered by itself, KWAR23 is inert, but given in combination with tumor-opsonizing monoclonal antibodies, KWAR23 greatly augments myeloid cell-dependent killing of a collection of hematopoietic and nonhematopoietic human tumor-derived cell lines. Following KWAR23 antibody treatment in a human SIRPA knockin mouse model, both neutrophils and macrophages infiltrate a human Burkitt's lymphoma xenograft and inhibit tumor growth, generating complete responses in the majority of treated animals. We further demonstrate that a bispecific anti-CD70/SIRPα antibody outperforms individually delivered antibodies in specific types of cancers. These studies demonstrate that SIRPα blockade induces potent antitumor activity by targeting multiple myeloid cell subsets that frequently infiltrate tumors. Thus, KWAR23 represents a promising candidate for combination therapy.

The inflammasome receptor Nlrp9b defends against enteric viruses by interacting with double-stranded viral RNA-bound helicase Dhx9, triggering gasdermin-D-dependent pyroptotic cell death of infected cells and secretion of Il-18. Rotaviruses cause severe and often fatal gastroenterological illnesses in young children. The mechanism by which such enteric viruses are detected and restricted in vivo is largely unknown. Richard Flavell and colleagues report how the inflammasome receptor Nlrp9 helps to defend against enteric viruses, via RNA helicase Dhx9, by interacting with double-stranded viral RNA. This interaction triggers gasdermin-D-dependent pyroptotic cell death of infected cells and secretion of IL-18. This innate immune signalling functions uniquely in intestinal epithelial cells and could reveal useful targets in the modulation of viral defences. Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide1. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling2,3,4,5, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

Although different autoimmune diseases show discrete clinical features, there are common molecular pathways intimately involved. Here we show that miR-125a is downregulated in peripheral CD4(+) T cells of human autoimmune diseases including systemic lupus erythematosus and Crohn's disease, and relevant autoimmune mouse models. miR-125a stabilizes both the commitment and immunoregulatory capacity of Treg cells. In miR-125a-deficient mice, the balance appears to shift from immune suppression to inflammation, and results in more severe pathogenesis of colitis and experimental autoimmune encephalomyelitis (EAE). The genome-wide target analysis reveals that miR-125a suppresses several effector T-cell factors including Stat3, Ifng and Il13. Using a chemically synthesized miR-125a analogue, we show potential to re-programme the immune homeostasis in EAE models. These findings point to miR-125a as a critical factor that controls autoimmune diseases by stabilizing Treg-mediated immune homeostasis.

Nlrp6 keeps gut infections in check Most viruses infect only certain cells of the body. Enteric viruses, such as norovirus and rotavirus, specifically infect the gut. Wang et al. now show that the response to such viruses is tissue-specific, too. Antiviral immunity to enteric but not systemic viral infections in mice required Nlrp6, a member of the NOD-like receptor family of proteins that play important roles in host defense. Together with the RNA helicase protein Dhx15, Nlrp6 bound viral RNA and elicited downstream antiviral immune responses necessary for viral clearance. These included the production of type I and type III interferons and the expression of interferon-stimulated genes. Science , this issue p. 826