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Transplanting bone marrow from wild-type mice into MECP2-lacking mice results in wild-type microglial engraftment, extends lifespan and reduces symptoms of disease such as breathing and locomotor abnormalities, implicating microglia in the pathophysiology of Rett syndrome. Marrow implants in Rett syndrome The X-linked autism spectrum disorder known as Rett syndrome is predominantly linked to mutations in the MECP2 gene. It is typically associated with neuronal dysfunction, almost exclusively in girls, but new evidence suggests that restoring MECP2 function in other cell types may also arrest disease development. Here, the authors show in a mouse model that transplanting bone marrow from wild-type mice into mice lacking Mecp2 results in an invasion of donor-derived microglial cells into the brain, accompanied by increased lifespan and reduced signs of disease, including improved breathing and locomotion. The donor cells expressed normal MECP2 and high levels of the neurotrophic factor IGF-1. These results point to a crucial role for microglia in Rett syndrome, and open the possibility that bone-marrow implants might be of therapeutic benefit. Rett syndrome is an X-linked autism spectrum disorder. The disease is characterized in most cases by mutation of the MECP2 gene, which encodes a methyl-CpG-binding protein 1 , 2 , 3 , 4 , 5 . Although MECP2 is expressed in many tissues, the disease is generally attributed to a primary neuronal dysfunction 6 . However, as shown recently, glia, specifically astrocytes, also contribute to Rett pathophysiology. Here we examine the role of another form of glia, microglia, in a murine model of Rett syndrome. Transplantation of wild-type bone marrow into irradiation-conditioned Mecp2 -null hosts resulted in engraftment of brain parenchyma by bone-marrow-derived myeloid cells of microglial phenotype, and arrest of disease development. However, when cranial irradiation was blocked by lead shield, and microglial engraftment was prevented, disease was not arrested. Similarly, targeted expression of MECP2 in myeloid cells, driven by Lysm cre on an Mecp2 -null background, markedly attenuated disease symptoms. Thus, through multiple approaches, wild-type Mecp2 -expressing microglia within the context of an Mecp2 -null male mouse arrested numerous facets of disease pathology: lifespan was increased, breathing patterns were normalized, apnoeas were reduced, body weight was increased to near that of wild type, and locomotor activity was improved. Mecp2 +/− females also showed significant improvements as a result of wild-type microglial engraftment. These benefits mediated by wild-type microglia, however, were diminished when phagocytic activity was inhibited pharmacologically by using annexin V to block phosphatydilserine residues on apoptotic targets, thus preventing recognition and engulfment by tissue-resident phagocytes. These results suggest the importance of microglial phagocytic activity in Rett syndrome. Our data implicate microglia as major players in the pathophysiology of this devastating disorder, and suggest that bone marrow transplantation might offer a feasible therapeutic approach for it.

Pediatric high-grade gliomas (pHGGs) are aggressive diseases with poor outcomes. The diverse molecular heterogeneity in these rare tumors and inadequate tumor models have limited the development of effective therapies. In this issue of the JCI, Haase et al. produced a genetically engineered mouse model of H3.3-G34R-mutant pHGG to help identify vulnerabilities in DNA repair pathways. The authors designed a therapy that combined radiation with DNA damage response inhibitors to induce an adaptive immune response and extend survival. These findings suggest that combinations of small-molecule therapies with immunotherapies could drive a more durable response and improve mortality for patients with pHGG.

BackgroundGlioblastoma (GBM) is the most common and aggressive type of cancer in the central nervous system, characterized by a poor prognosis and limited treatment efficacy. Consistent with findings in other cancer types, the high expression of the transmembrane protein B7-H3 in GBM correlates with enhanced tumor progression and poor prognosis. However, chimeric antigen receptor (CAR) T cell therapy—an effective strategy in hematologic malignancies—has emerged as a promising approach that could complement and enhance conventional treatments for GBM. Previously, we observed that a single intravenous dose of a novel B7-H3-targeted CAR T cell (Y111) significantly extended the survival of mice intracerebrally implanted with the human glioblastoma cell line GBM01, compared to those treated with untransduced T cells.In this study, we aimed to evaluate the efficacy Y111 CAR T cells via three distinct routes of administration: intravenous (IV), intratumoral (IT), and intracerebroventricular (ICV), in a human GBM orthotopic model using the GBM01 cell line.MethodsHuman GBM01 GFP-Luc cells were stereotaxically injected into the brain of NRG immunocompromised mice. The tumor growth was monitored weekly using in vivo imaging systems (IVIS), and the general condition of mice was evaluated daily. Once tumors became detectable, the animals were randomized into groups to receive Y111 CAR T or untransduced T cells via different routes of administration: a single Intratumoral (IT) dose, a single Intracerebroventricular (ICV) dose, or two Intravenous (IV) doses.ResultsIT treatment markedly reduced tumor size and, in most animals, eliminated detectable tumor signal, resulting in significantly extended survival compared to the control group. IV treatment also slowed tumor growth and improved survival relative to controls; however, no significant difference was observed using two doses in comparison to a single dose. Although ICV administration reduced tumor growth and prolonged survival compared to the control group, its efficacy was less than IT treatment.ConclusionsWe are investigating Y111 CAR T cells, a novel immunotherapy targeting B7-H3, for the treatment of glioblastoma. Our results demonstrate that intratumoral (IT) administration of Y111 CAR T cells achieves near-complete preclinical efficacy, establishing this approach as a promising alternative to conventional therapies. Although ICV and two-dose IV treatment were less effective than IT administration, all routes of administration reduced tumor growth and prolonged survival, suggesting Y111 CAR-T cells may have future potential for clinical translation.Ethics ApprovalMouse experiments were reviewed and approved by the Animal Care and Use Committee (ACUC) at the National Institutes of Health.

BackgroundMyeloid cells are found in virtually all tumors and play diverse roles in the tumor microenvironment (TME), with the ability to contribute to both pro-tumor and anti-tumor functions depending on their phenotype and environment. During tumor progression, bone marrow cells are activated and mobilized to tumor and pre-metastatic sites where they rapidly differentiate into immunosuppressive myeloid cells. We hypothesized that we can take advantage of myeloid biology and infiltration into tumors to deliver therapeutic cargo to reprogram the TME in favor of anti-tumor immunity. In mouse models, we demonstrated that Genetically Engineered Myeloid cells (GEMys) engineered to express interleukin 12 (IL-12) reverse the immunosuppressive signature and activate effective anti-tumor immunity.1 In combination with cyclophosphamide/fludarabine (Cy/Flu) pre-conditioning, IL-12 GEMys cure mice of established solid tumors and provide protection from subsequent rechallenge with the same tumor line, indicating formation of immunological memory against the tumor. We are translating these promising pre-clinical findings into the clinic and have developed a manufacturing protocol for human GEMys which will provide a platform for modular cell therapy development.MethodsWe generated a lentiviral vector encoding truncated human epidermal growth factor receptor (tEGFR) and single chain human IL-12. The tEGFR serves as a marker of transduction efficiency for product release and can also be targeted using anti-EGFR antibodies for depletion of transduced cells in vivo in case of toxicity. We obtained granulocyte colony-stimulating factor mobilized apheresis product from three healthy donors and isolated CD34+ cells using the CliniMACS Plus system. Cryopreserved CD34+ cells were thawed, transduced, expanded, and differentiated. We characterized the final IL-12 GEMy cell product by measuring transduction efficiency, IL-12 secretion, IFN-gamma production in co-culture with donor matched T cells, and described the cell phenotype in vitro and in vivo in immunocompromised mouse models.ResultsWe successfully manufactured human CD34+ cell-derived IL-12 GEMys that express tEGFR on the surface and secrete IL-12. The phenotype of the final cell product is consistent with a myeloid progenitor phenotype that rapidly differentiates into myeloid cells following injection into mice or additional time in culture.ConclusionsWe have demonstrated the feasibility and reproducibility of manufacturing human IL-12 GEMys. These data will enable an NCI-sponsored first-in-human clinical trial of IL-12 GEMys in patients with relapsed and refractory solid tumors. Patients will be mobilized, apheresed for cell product manufacturing, treated with a standard preconditioning regimen of Cy/Flu, and receive IL-12 GEMys intravenously in a 3+3 dose escalation trial design.ReferenceKaczanowska S, Beury DW, Gopalan V, Tycko AK, Qin H, Clements ME, Drake J, Nwanze C, Murgai M, Rae Z, Ju W, Alexander KA, Kline J, Contreras CF, Wessel KM, Patel S, Hannenhalli S, Kelly M, Kaplan RN. Genetically engineered myeloid cells rebalance the core immune suppression program in metastasis. Cell. 2021;184:1-20.

BackgroundThe immunosuppressive tumor microenvironment (TME) remains a significant barrier to solid tumor immunotherapy. Tumor immunosuppression is primarily driven by myeloid cells, including both tumor associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC). We previously demonstrated that interleukin 12-secreting genetically engineered myeloid cells (IL-12 GEMys) prolong survival in a syngeneic mouse model of rhabdomyosarcoma and become curative when combined with cyclophosphamide and fludarabine (Cy/Flu) preconditioning. IL-12 GEMy therapy was dependent upon an anti-tumor CD8 T cell response. However, the mechanisms of why Cy/Flu is required for cures and how this therapy overcomes tumor myeloid immunosuppression remains unexplored. Further, we have only applied IL-12 GEMys in a limited number of tumor models, therefore testing in additional malignancies is needed to support future clinical translation. Osteosarcoma is a difficult to treat malignant bone tumor with high rates of metastasis, making it an important disease for continued studies of IL-12 GEMy therapy.MethodsF42010 murine syngeneic osteosarcoma cells were orthotopically injected into the tibia of C57BL/6 mice and tissues were analyzed by flow cytometry and multiplex immunofluorescence. For treatment experiments, tumors were palpable prior to treatment (typically 10 days). Cy/Flu was given two days prior to GEMy injection by tail vein. GEMys were generated from bone marrow hematopoietic progenitor and stem cells harvested by magnetic bead isolation (StemCell) and transduced with lentivirus during a five day culture in Stemspan SFEM media (StemCell) supplemented with growth factors.ResultsOrthotopic F42010 osteosarcoma tumors were dominated by an immunosuppressive myeloid infiltrate. Treatment with Cy/Flu depleted most circulating immune cells and TAMs. Cy/Flu plus IL-12 GEMy therapy activated intratumoral CD8+ T cells and was curative. Six days after IL-12 GEMy treatment there was pronounced systemic reprogramming of monocytes and macrophages to a major histocompatibility complex class II (MHCII) positive phenotype. In vitro skewing of bone marrow derived macrophages (BMDM) revealed that pretreatment with tumor conditioned media impaired IFNg-mediated MHCII expression, whereas pretreatment with IFNg prior to TCM exposure overcame suppression of MHCII.ConclusionsThese results provide preclinical support for IL-12 GEMys in osteosarcoma and shed light on how Cy/Flu plus IL-12 GEMy therapy overcomes tumor immunosuppression by depleting TAMs in the tumor and reprogramming myeloid cells to an MHCII-high phenotype. Our results suggest that exposure to IFNg prior to tumor factors facilitates robust and complete anti-tumor skewing, which may be a key function of Cy/Flu preconditioning by eliminating tumor-skewed myeloid cells and inducing production of naive monocytes.Ethics ApprovalMouse experiments were reviewed and approved by the Animal Care and Use Committee (ACUC) at the National Institutes of Health.

B7-H3 is a cell surface protein overexpressed in many solid tumors and an attractive target for chimeric antigen receptor (CAR) T cell therapy. The most clinically advanced B7-H3 CARs are derived from murine monoclonal antibodies (mAbs) 376.96 and MGA271, which are now in phase I/II trials. However, non-human mAb sequences can provoke immune responses, leading to CAR T-cell rejection and therapeutic failure. Although scFv humanization reduces this risk, residual foreign residues within the variable domains remain. To overcome this limitation, we used in vitro phage display to generate fully human B7-H3-specific scFvs for CAR design. In pancreatic cancer, neuroblastoma, and glioblastoma xenograft models, CAR T cells incorporating the lead human binder Y111 were well tolerated and demonstrated superior antitumor activity compared with 376.96- and MGA271-based CARs. Y111 CAR treatment induced complete responses, tumor rejection, and significant survival benefits, identifying Y111 as a promising fully human B7-H3 CAR for solid tumors.

Osteosarcoma is the most common malignant bone tumor in young patients and remains a significant clinical challenge, particularly in the context of metastatic disease. Despite extensive documentation of genomic alterations in osteosarcoma, studies detailing the immunosuppressive mechanisms within the metastatic osteosarcoma microenvironment are lacking. Our objective was to characterize the spatial transcriptional landscape of metastatic osteosarcoma to reveal these immunosuppressive mechanisms and identify promising therapeutic targets. Here, we performed spatial transcriptional profiling on a cohort of osteosarcoma pulmonary metastases from pediatric patients. We reveal a conserved spatial gene expression pattern resembling a foreign body granuloma, characterized by peripheral inflammatory signaling, fibrocollagenous encapsulation, lymphocyte exclusion, and peritumoral macrophage accumulation. We also show that the intratumoral microenvironment of these lesions lack inflammatory signaling. Additionally, we identified CXCR4 as an actionable immunomodulatory target that bridges both the intratumoral and extratumoral microenvironments and highlights the spatial heterogeneity and complexity of this pathway. Collectively, this study reveals that metastatic osteosarcoma specimens are comprised of multiple regionally distinct immunosuppressive microenvironments.

Rett syndrome is a devastating central nervous system (CNS) developmental disorder, which is caused in the majority of cases by mutation of the X‐linked epigenetic factor methyl‐CpG‐binding protein (MeCP) 2. The disease, which affects primarily girls, features severe neurological sequelae, including seizures, stereotypies, impaired breathing, mental retardation, and locomotor deficits. A majority of patients also develop somatic symptoms including intestinal pathology, impaired growth, and osteopenia. Rett syndrome was long thought to be a disease solely involving neuronal dysfunction. However, we recently showed a prominent role for the immune cells, microglia, in the pathophysiology of the disease. In this chapter, we give a broad overview of Rett syndrome, and recent findings in microglial biology as they relate to CNS development and pathology. We further focus on microglia, the tissue‐resident macrophages of the brain, as possible therapeutic targets in the amelioration of Rett syndrome.

The Cape flora of South Africa grows in a continental area with many diverse and endemic species 1 , 2 , 3 , 4 . We need to understand the evolutionary origins and ages of such ‘hotspots’ to conserve them effectively 5 . In volcanic islands the timing of diversification can be precisely measured with potassium–argon dating. In contrast, the history of these continental species is based upon an incomplete fossil record and relatively imprecise isotopic palaeotemperature signatures. Here we use molecular phylogenetics and precise dating of two island species within the same clade as the continental taxa to show recent speciation in a species-rich genus characteristic of the Cape flora. The results indicate that diversification began approximately 7–8 Myr ago, coincident with extensive aridification caused by changes in ocean currents. The recent origin of endemic species diversity in the Cape flora shows that large continental bursts of speciation can occur rapidly over timescales comparable to those previously associated with oceanic island radiations 6 , 7 .

Phylogenetic trees based upon DNA sequence data, when calibrated with a dimension of time, allow inference of: (i) the pattern of accumulation of lineages through time; (ii) the time of origin of monophyletic groups; (iii) when lineages arrived in different geographical areas; (iv) the time of origin of biome-specific morphologies. This gives a powerful new view of the history of biomes that in many cases is not provided by the incomplete plant fossil record. Dated plant phylogenies for angiosperm families such as Leguminoaceae (Fabaceae), Melastomataceae sensu stricto, Annonaceae and Rhamnaceae indicate that long-distance, transoceanic dispersal has played an important role in shaping their distributions, and that this can obscure any effect of tectonic history, previously assumed to have been the major cause of their biogeographic patterns. Dispersal from other continents has also been i mportant in the assembly of the Amazonian rainforest flora and the Australian flora. Comparison of dated biogeographic patterns of plants and animals suggests that recent long-distance dispersal might be more prevalent in plants, which has major implications for community assembly and coevolution. Dated plant phylogenies also reveal the role of past environmental changes on the evolution of lineages in species-rich biomes, and show that recent Plio-Pleistocene diversification has contributed substantially to their current species richness. Because of the critical role of fossils and morphological characters in assigning ages to nodes in phylogenetic trees, future studies must include careful morphological consideration of fossils and their extant relatives in a phylogenetic context. Ideal study systems will be based upon DNA sequence data from multiple loci and multiple fossil calibrations. This allows cross-validation both of age estimates from different loci, and from different fossil calibrations. For a more complete view of biome history, future studies should emphasize full taxon sampling in ecologically important groups, and should focus on geographical areas for which few species-level phylogenies are available, such as tropical Africa and Asia. These studies are urgent because understanding the history of biomes can both inform conservation decisions, and help predict the effects of future environmental changes at a time when biodiversity is being impacted on an unprecedented scale.