Head and neck squamous cell carcinoma (HNSCC) is characterized by complex relations between stromal, epithelial, and immune cells within the tumor microenvironment (TME). To enable the development of more efficacious therapies, we aim to study the heterogeneity, signatures of unique cell populations, and cell-cell interactions of non-immune and immune cell populations in 6 human papillomavirus (HPV)+ and 12 HPV– HNSCC patient tumor and matched peripheral blood specimens using single-cell RNA sequencing. Using this dataset of 134,606 cells, we show cell type-specific signatures associated with inflammation and HPV status, describe the negative prognostic value of fibroblasts with elastic differentiation specifically in the HPV+ TME, predict therapeutically targetable checkpoint receptor-ligand interactions, and show that tumor-associated macrophages are dominant contributors of PD-L1 and other immune checkpoint ligands in the TME. We present a comprehensive single-cell view of cell-intrinsic mechanisms and cell-cell communication shaping the HNSCC microenvironment.
Immunotherapy can reinvigorate dormant responses to cancer, but response rates remain low. Oncolytic viruses, which replicate in cancer cells, induce tumor lysis and immune priming, but their immune consequences are unclear. We profiled the infiltrate of aggressive melanomas induced by oncolytic Vaccinia virus using RNA sequencing and found substantial remodeling of the tumor microenvironment, dominated by effector T cell influx. However, responses to oncolytic viruses were incomplete due to metabolic insufficiencies induced by the tumor microenvironment. We identified the adipokine leptin as a potent metabolic reprogramming agent that supported antitumor responses. Leptin metabolically reprogrammed T cells in vitro, and melanoma cells expressing leptin were immunologically controlled in mice. Engineering oncolytic viruses to express leptin in tumor cells induced complete responses in tumor-bearing mice and supported memory development in the tumor infiltrate. Thus, leptin can provide metabolic support to tumor immunity, and oncolytic viruses represent a platform to deliver metabolic therapy.
The mosquitoes Aedes aegypti (L.) and Ae. albopictus Skuse are the major vectors of dengue, Zika, yellow fever, and chikungunya viruses worldwide. Wolbachia, an endosymbiotic bacterium present in many insects, is being utilized in novel vector control strategies to manipulate mosquito life history and vector competence to curb virus transmission. Earlier studies have found that Wolbachia is commonly detected in Ae. albopictus but rarely detected in Ae. aegypti. In this study, we used a two‐step PCR assay to detect Wolbachia in wild‐collected samples of Ae. aegypti. The PCR products were sequenced to validate amplicons and identify Wolbachia strains. A loop‐mediated isothermal amplification (LAMP) assay was developed and used for detecting Wolbachia in selected mosquito specimens as well. We found Wolbachia in 85/148 (57.4%) wild Ae. aegypti specimens from various cities in New Mexico, and in 2/46 (4.3%) from St. Augustine, Florida. Wolbachia was not detected in 94 samples of Ae. aegypti from Deer Park, Harris County, Texas. Wolbachia detected in Ae. aegypti from both New Mexico and Florida was the wAlbB strain of Wolbachia pipientis. A Wolbachia‐positive colony of Ae. aegypti was established from pupae collected in Las Cruces, New Mexico, in 2018. The infected females of this strain transmitted Wolbachia to their progeny when crossed with males of Rockefeller strain of Ae. aegypti, which does not carry Wolbachia. In contrast, none of the progeny of Las Cruces males mated to Rockefeller females were infected with Wolbachia.
Integration of signaling and metabolic pathways enables and sustains lymphocyte function. While metabolic changes occurring during T cell activation are well-characterized, the metabolic demands of differentiated T lymphocytes are largely unexplored. Here we defined the bioenergetics of TH17 effector cells generated in vivo. These cells depend on OXPHOS2 for energy and cytokine production. Mechanistically, the essential role of OXPHOS in TH17 cells results from their limited capacity to increase glycolysis in response to metabolic stresses. This metabolic program is observed in mouse and human TH17 cells, including those isolated from Crohn’s disease patients, and is linked to disease, as inhibiting OXPHOS reduces the severity of murine colitis and psoriasis. These studies highlight the importance of analyzing metabolism in effector lymphocytes within in vivo inflammatory contexts and suggest a therapeutic role for manipulating OXPHOS in TH17 driven diseases.
Rac1-GTPase activation plays a key role in the development and progression of cardiac remodeling. Therefore, we engineered a transgenic mouse model by overexpressing cDNA of a constitutively active form of Zea maize Rac gene (ZmRacD) specifically in the hearts of FVB/N mice. Echocardiography and MRI analyses showed cardiac hypertrophy in old transgenic mice, as evidenced by increased left ventricular (LV) mass and LV mass-to-body weight ratio, which are associated with relative ventricular chamber dilation and systolic dysfunction. LV hypertrophy in the hearts of old transgenic mice was further confirmed by an increased heart weight-to-body weight ratio and histopathology analysis. The cardiac remodeling in old transgenic mice was coupled with increased myocardial Rac-GTPase activity (372%) and ROS production (462%). There were also increases in α(1)-integrin (224%) and β(1)-integrin (240%) expression. This led to the activation of hypertrophic signaling pathways, e.g., ERK1/2 (295%) and JNK (223%). Pravastatin treatment led to inhibition of Rac-GTPase activity and integrin signaling. Interestingly, activation of ZmRacD expression with thyroxin led to cardiac dilation and systolic dysfunction in adult transgenic mice within 2 wk. In conclusion, this is the first study to show the conservation of Rho/Rac proteins between plant and animal kingdoms in vivo. Additionally, ZmRacD is a novel transgenic model that gradually develops a cardiac phenotype with aging. Furthermore, the shift from cardiac hypertrophy to dilated hearts via thyroxin treatment will provide us with an excellent system to study the temporal changes in cardiac signaling from adaptive to maladaptive hypertrophy and heart failure.
Background Aedes aegypti mosquitoes are vectors of a variety of emerging viral pathogens, including yellow fever, dengue, chikungunya, and Zika virus. This species has established endemic populations in all cities across southern New Mexico sampled to date. Presently, control of Aedes -borne viruses relies on deployment of insecticides to suppress mosquito populations, but the evolution of insecticide resistance threatens the success of vector control programs. While insecticide resistance is quite common in Ae . aegypti field populations across much of the U.S., the resistance status of this species in populations from New Mexico has not previously been assessed. Results First, we collected information on pesticide use in cities in southern New Mexico and found that the most commonly used active ingredients were pyrethroids. The use of insecticides with the same mode-of-action over multiple years is likely to promote the evolution of resistance. To determine if there was evidence of resistance in some cities in southern New Mexico, we collected Ae . aegypti from the same cities and established laboratory strains to assess resistance to pyrethroid insecticides and, for a subset of populations, to organophosphate insecticides. F2 or F4 generation mosquitoes were assessed for insecticide resistance using bottle test bioassays. The majority of the populations from New Mexico that we analyzed were resistant to the pyrethroids permethrin and deltamethrin. A notable exception to this trend were mosquitoes from Alamogordo, a city that did not report using pyrethroid insecticides for vector control. We screened individuals from each population for known knock down resistance (kdr) mutations via PCR and found a strong association between the presences of the F1534C kdr mutation in the para gene of Ae . aegypti (homologue to F1534C in Musca domestica L .) and pyrethroid resistance. Conclusion High-level pyrethroid resistance is common in Ae . aegypti from New Mexico and geographic variation in such resistance is likely associated with variation in usage of pyrethroids for vector control. Resistance monitoring and management is recommended in light of the potential for arbovirus outbreaks in this state. Also, alternative approaches to mosquito control that do not involve insecticides should be explored.
Objectives: We sought to describe the genetic complexity of 14 UM-SCC oral cavity cancer cell lines that have remained uncharacterized despite being used as model systems for decades. Materials and Methods: We performed exome sequencing on 14 oral cavity UM-SCC cell lines and denote the mutational profile of each line. We used a SNP array to profile the multiple copy number variations of each cell line and use immunoblotting to compare alterations to protein expression of commonly amplified genes (EGFR, PIK3CA, etc.). RNA sequencing was performed to characterize the expression of genes with copy number alterations. Results: The cell lines displayed a highly complex network of genetic aberrations that was consistent with alterations identified in the HNSCC TCGA project including PIK3CA amplification, CDKN2A deletion, as well as TP53 and CASP8 mutations, enabling genetic stratification of each cell line in the panel. Copy number FISH and spectral karyotyping analysis demonstrate that cell lines retain chromosomal heterogeneity. Conclusions: Collectively, we developed an important resource for future oral cavity HNSCC cell line studies and highlight the complexity of genomic aberrations in cell lines.
SUMMARY Regulatory T cells (Treg cells) are critical mediators of self-tolerance, but they can also limit effective anti-tumor immunity. Although under homeostasis a small fraction of Treg cells in lymphoid organs express the putative checkpoint molecule Tim-3, this protein is expressed by a much larger proportion of tumor-infiltrating Treg cells. Using a mouse model that drives cell-type-specific inducible Tim-3 expression, we show that expression of Tim-3 by Treg cells is sufficient to drive Treg cells to a more effector-like phenotype, resulting in increases in suppressive activity, effector T cell exhaustion, and tumor growth. We also show that T-reg-cell-specific inducible deletion of Tim-3 enhances anti-tumor immunity. Enhancement of Treg cell function by Tim-3 is strongly correlated with increased expression of interleukin-10 (IL-10) and a shift to a more glycolytic metabolic phenotype. Our data demonstrate that Tim-3 + Treg cells may be a relevant therapeutic target cell type for the treatment of cancer.
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