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Methamphetamine (METH) is a potent stimulant that induces a euphoric state but also causes cognitive impairment, neurotoxicity and neurodevelopmental deficits. Yet, the molecular mechanisms by which METH causes neurodevelopmental defects have remained elusive. Here we utilized human cerebral organoids and single-cell RNA sequencing (scRNA-seq) to study the effects of prenatal METH exposure on fetal brain development. We analyzed 20,758 cells from eight untreated and six METH-treated cerebral organoids and found that the organoids developed from embryonic stem cells contained a diverse array of glial and neuronal cell types. We further identified transcriptionally distinct populations of astrocytes and oligodendrocytes within cerebral organoids. Treatment of organoids with METH-induced marked changes in transcription in multiple cell types, including astrocytes and neural progenitor cells. METH also elicited novel astrocyte-specific gene expression networks regulating responses to cytokines, and inflammasome. Moreover, upregulation of immediate early genes, complement factors, apoptosis, and immune response genes suggests a neuroinflammatory program induced by METH regulating neural stem cell proliferation, differentiation, and cell death. Finally, we observed marked METH-induced changes in neuroinflammatory and cytokine gene expression at the RNA and protein levels. Our data suggest that human cerebral organoids represent a model system to study drug-induced neuroinflammation at single-cell resolution.

Although immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment, many patients do not respond or develop resistance to ICB. N⁶-methylation of adenosine (m⁶A) in RNA regulates many pathophysiological processes. Here, we show that deletion of the m⁶A demethylase Alkbh5 sensitized tumors to cancer immunotherapy. Alkbh5 has effects on m⁶A density and splicing events in tumors during ICB. Alkbh5 modulates Mct4/Slc16a3 expression and lactate content of the tumor microenvironment and the composition of tumor-infiltrating Treg and myeloid-derived suppressor cells. Importantly, a small-molecule Alkbh5 inhibitor enhanced the efficacy of cancer immunotherapy. Notably, the ALKBH5 gene mutation and expression status of melanoma patients correlate with their response to immunotherapy. Our results suggest that m⁶A demethylases in tumor cells contribute to the efficacy of immunotherapy and identify ALKBH5 as a potential therapeutic target to enhance immunotherapy outcome in melanoma, colorectal, and potentially other cancers.

Chronic infection with human immunodeficiency virus (HIV) can cause progressive loss of immune cell function, or exhaustion, which impairs control of virus replication. However, little is known about the development and maintenance, as well as heterogeneity of immune cell exhaustion. Here, we investigated the effects of HIV infection on immune cell exhaustion at the transcriptomic level by analyzing single-cell RNA sequencing of peripheral blood mononuclear cells from four healthy subjects (37,847 cells) and six HIV-infected donors (28,610 cells). We identified nine immune cell clusters and eight T cell subclusters, and three of these (exhausted CD4 + and CD8 + T cells and interferon-responsive CD8 + T cells) were detected only in samples from HIV-infected donors. An inhibitory receptor KLRG1 was identified in a HIV-1 specific exhausted CD8 + T cell population expressing KLRG1, TIGIT, and T-bet dim Eomes hi markers. Ex-vivo antibody blockade of KLRG1 restored the function of HIV-specific exhausted CD8 + T cells demonstrating the contribution of KLRG1 + population to T cell exhaustion and providing an immunotherapy target to treat HIV chronic infection. These data provide a comprehensive analysis of gene signatures associated with immune cell exhaustion during HIV infection, which could be useful in understanding exhaustion mechanisms and developing new cure therapies.

BackgroundA significant challenge to overcome in pancreatic ductal adenocarcinoma (PDAC) is the profound systemic immunosuppression that renders this disease non-responsive to immunotherapy. Our supporting data provide evidence that CD200, a regulator of myeloid cell activity, is expressed in the PDAC microenvironment. Additionally, myeloid-derived suppressor cells (MDSC) isolated from patients with PDAC express elevated levels of the CD200 receptor (CD200R). Thus, we hypothesize that CD200 expression in the PDAC microenvironment limits responses to immunotherapy by promoting expansion and activity of MDSC.MethodsImmunofluorescent staining was used to determine expression of CD200 in murine and human PDAC tissue. Flow cytometry was utilized to test for CD200R expression by immune populations in patient blood samples. In vivo antibody blocking of CD200 was conducted in subcutaneous MT-5 tumor-bearing mice and in a genetically engineered PDAC model (KPC-Brca2 mice). Peripheral blood mononuclear cells (PBMC) from patients with PDAC were analyzed by single-cell RNA sequencing. MDSC expansion assays were completed using healthy donor PBMC stimulated with IL-6/GM-CSF in the presence of recombinant CD200 protein.ResultsWe found expression of CD200 by human pancreatic cell lines (BxPC3, MiaPaca2, and PANC-1) as well as on primary epithelial pancreatic tumor cells and smooth muscle actin+ stromal cells. CD200R expression was found to be elevated on CD11b+CD33+HLA-DRlo/− MDSC immune populations from patients with PDAC (p=0.0106). Higher expression levels of CD200R were observed in CD15+ MDSC compared with CD14+ MDSC (p<0.001). In vivo studies demonstrated that CD200 antibody blockade limited tumor progression in MT-5 subcutaneous tumor-bearing and in KPC-Brca2 mice (p<0.05). The percentage of intratumoral MDSC was significantly reduced in anti-CD200 treated mice compared with controls. Additionally, in vivo blockade of CD200 can also significantly enhance the efficacy of PD-1 checkpoint antibodies compared with single antibody therapies (p<0.05). Single-cell RNA sequencing of PBMC from patients revealed that CD200R+ MDSC expressed genes involved in cytokine signaling and MDSC expansion. Further, in vitro cytokine-driven expansion and the suppressive activity of human MDSC was enhanced when cocultured with recombinant CD200 protein.ConclusionsThese results indicate that CD200 expression in the PDAC microenvironment may regulate MDSC expansion and that targeting CD200 may enhance activity of checkpoint immunotherapy.

CXCR4 is a key regulator of the development of NK cells and DCs, both of which play an important role in early placental development and immune tolerance at the maternal-fetal interface. However, the role of CXCR4 in pregnancy is not well understood. Our study demonstrates that adult-induced global genetic CXCR4 deletion, but not uterine-specific CXCR4 deletion, was associated with increased pregnancy resorptions and decreased litter size. CXCR4-deficient mice had decreased NK cells and increased granulocytes in the decidua, along with increased leukocyte numbers in peripheral blood. We found that CXCR4-deficient mice had abnormal decidual NK cell aggregates and NK cell infiltration into trophoblast areas beyond the giant cell layer. This was associated with low NK cell expression of granzyme B, a NK cell granule effector, indicative of NK cell dysfunction. Pregnancy failure in these mice was associated with abnormalities in placental vascular development and increased placental expression of inflammatory genes. Importantly, adoptive BM transfer of WT CXCR4+ BM cells into CXCR4-deficient mice rescued the reproductive deficits by normalizing NK cell function and mediating normal placental vascular development. Collectively, our study found an important role for maternal CXCR4 expression in immune cell function, placental development, and pregnancy maintenance.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States with 5-year survival rates below 10%. PDAC is commonly diagnosed after metastasis has occurred and treatment options are limited. Immune checkpoint inhibitor (ICI) monoclonal antibody (mAb) therapy has shown great promise in other cancers, however little efficacy has been observed in patients with PDAC. The protein responsible for recycling IgG based mAb therapeutics like ICIs in the bloodstream, as well as processing peptides for antigen presentation, is the neonatal Fc receptor (FcRn). Little is known about FcRn in cancer, and to our knowledge no characterization of host FcRn, or FcRn extrinsic to tumor cells exists in PDAC patients. We hypothesized that PDAC patients and tumor-bearing animals would have altered FcRn expression by their immune populations compared to their healthy counterparts.MethodsC57BL/6 mice were orthotopically injected with KPC-luc (KrasLSL-G12D, Trp53LSL-R270H, Pdx1-cre) pancreatic tumor cells, and FcRn expression in myeloid-derived splenocytes were analyzed by fluorescence cytometry. Time-of-flight mass cytometry (CyTOF) was utilized to immunophenotype peripheral blood mononuclear cells (PBMCs) of PDAC or non-cancer patients for expression levels of FcRn within these immune populations.ResultsPDAC tumor-bearing mice exhibit altered FcRn expression among myeloid immune cell populations. Mice with pancreatic tumors had elevated expression of FcRn on migratory cDC2 (CD8-CD11b+CD103+CD24++; p = 0.017), monocytic MDSC (CD11b+Ly6G-Ly6C+; p = 0.0023), granulocytic MDSC (CD11b+Ly6G+Ly6C±; p = 0.0542), and cDC2 (CD8-CD11b+CD103-CD24±; p=0.036) cells. PBMCs from non-cancer obese patients (healthy control samples; n=8) and PDAC patients prior to surgical resection (n=13) were subjected to CyTOF analyses. The majority of FcRn expression was concentrated to monocyte (p=0.017), DCs (p=0.017) and MDSC (p=0.012) immune populations. Overall, we observed increased expression of FcRn on myeloid-derived immune populations from patients with PDAC. FcRn expression was elevated in both monocytes and DC populations in PDAC relative to non-cancer PBMCs. Monocytic and granulocytic MDSC from patients with PDAC had significantly elevated FcRn positivity compared to healthy controls (p = 0.034, p = 0.026, respectively).ConclusionsFcRn is upregulated in monocytes, dendritic cells and MDSC immune populations in patients and mice with pancreatic tumors. Future investigations into FcRn function in preclinical models and PDAC patients will hopefully elucidate new mechanisms of ICI resistance and possible alternative approaches for improving immunotherapy efficacy in these patients.Ethics ApprovalAll patients provided voluntary written informed consent (Institutional Review Board protocol: 2010C0051) to participate. The protocols and subsequent amendments were approved by The Ohio State University Institutional Review Board. All animal protocols were approved by the Ohio State University Institutional Animal Care and Use Committee (IACUC) at The Ohio State University (Approved IACUC protocols 2009A0178-R4 and 2017A00000117-R1) and mice were treated in accordance with institutional guidelines for animal care. The Ohio State University Laboratory Animal Shared Resource is an Association for Assessment and Accreditation of Laboratory Animal Care International accredited program that follows Public Health Service policy and guidelines. All other experiments were completed under the research protocols (2014R00000086; 2013R00000056) approved by the Ohio State University Institutional Biosafety Committee.

Methamphetamine (METH) and tetrahydrocannabinol (THC) are drugs that a segment of pregnant women may be exposed to, however, their effect on the development of the fetal brain is not completely understood. Research has been done on the effects of these drugs, but these studies are typically done in mice models which may not be completely representative of the human brain. Analyzing the effects of these drugs through the human cerebral organoid model can provide valuable insights into the human fetal brain and the factors that affect its development. We use single-cell RNA-sequencing analysis of human cerebral brain organoids to study the changes in gene expression caused by these drugs and to determine their effects on the developing brain. Exposure of human cerebral organoids to METH led to the upregulation of genes associated with stress and inflammation suggesting that METH leads to neuroinflammation. Furthermore, genes associated with neural development were downregulated suggesting that METH hinders the development of the brain. The upregulated and downregulated pathways as a result of METH exposure indicate that there is likely a negative effect on fetal brain development and that METH exposure may lead to neuroinflammation. Analysis of THC organoids suggests that THC exposure may be altering glutamatergic neurons with enhanced expression of CNR1 and leading to preferential differentiation toward glutamatergic fate via NEUROD6. These results indicate that human cerebral organoids may provide valuable insights into the effects of drugs on the development and gene expression in the human fetal brain.

Substance use disorders (SUD) and drug addiction are major threats to public health, impacting not only the millions of individuals struggling with SUD, but also surrounding families and communities. One of the seminal challenges in treating and studying addiction in human populations is the high prevalence of co-morbid conditions, including an increased risk of contracting a human immunodeficiency virus (HIV) infection. Of the ~15 million people who inject drugs globally, 17% are persons with HIV. Conversely, HIV is a risk factor for SUD because chronic pain syndromes, often encountered in persons with HIV, can lead to an increased use of opioid pain medications that in turn can increase the risk for opioid addiction. We hypothesize that SUD and HIV exert shared effects on brain cell types, including adaptations related to neuroplasticity, neurodegeneration, and neuroinflammation. Basic research is needed to refine our understanding of these affected cell types and adaptations. Studying the effects of SUD in the context of HIV at the single-cell level represents a compelling strategy to understand the reciprocal interactions among both conditions, made feasible by the availability of large, extensively-phenotyped human brain tissue collections that have been amassed by the Neuro-HIV research community. In addition, sophisticated animal models that have been developed for both conditions provide a means to precisely evaluate specific exposures and stages of disease. We propose that single-cell genomics is a uniquely powerful technology to characterize the effects of SUD and HIV in the brain, integrating data from human cohorts and animal models. We have formed the Single-Cell Opioid Responses in the Context of HIV (SCORCH) consortium to carry out this strategy.