There are numerous cell types with scarcely understood functions, whose interactions with the immune system are not well characterized. To facilitate their study, we generated a mouse bearing enhanced green fluorescent protein (EGFP)-specific CD8+ T cells. Transfer of the T cells into EGFP reporter animals can be used to kill EGFP-expressing cells, allowing selective depletion of desired cell types, or to interrogate T-cell interactions with specific populations. Using this system, we eliminate a rare EGFP-expressing cell type in the heart and demonstrate its role in cardiac function. We also show that naive T cells are recruited into the mouse brain by antigen-expressing microglia, providing evidence of an immune surveillance pathway in the central nervous system. The just EGFP death-inducing (Jedi) T cells enable visualization of a T-cell antigen. They also make it possible to utilize hundreds of existing EGFP-expressing mice, tumors, pathogens and other tools, to study T-cell interactions with many different cell types, to model disease states and to determine the functions of poorly characterized cell populations.

To characterize associated reactions (ARs) in the contralateral arm across multiple muscles during unimanual tasks and to identify factors related to ARs in children with cerebral palsy (CP).

This study aimed to investigate useful parameters for estimating gastrocnemius (GCM) muscle volume (MV) using ultrasonography (US) and anthropometry in children with spastic cerebral palsy (CP).

Flavonoids are a diverse family of natural phenolic compounds commonly found in fruits and vegetables. Epidemiologic studies showed that flavonoids also reduce the risk of colon cancer. Tectochrysin is one of the major flavonoids of Alpinia oxyphylla Miquel. However, the anti-cancer effects and the molecular mechanisms of tectochrysin in colon cancer cells have not yet been reported. We investigated whether tectochrysin could inhibit colon cancer cell growth at 1, 5, 10 μg/ml. In in vivo study, we injected a tectochrysin treatment dose of 5 mg/kg to each mouse.

CRISPR pools are being widely employed to identify gene functions. However, current technology, which utilizes DNA as barcodes, permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities, we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate >100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR, we simultaneously analyzed multiple phenotypic markers, including phospho-signaling, on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes, the immunoproteasome component Psmb8 and a chaperone Rtp4, are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution.

The severity and intensity of autoimmune disease in immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) patients and in scurfy mice emphasize the critical role played by thymus-derived regulatory T cells (tTregs) in maintaining peripheral immune tolerance. However, although tTregs are critical to prevent lethal autoimmunity and excessive inflammatory responses, their suppressive mechanism remains elusive. Here, we demonstrate that tTregs selectively inhibit CD27/CD70-dependent Th1 priming, while leaving the IL-12-dependent pathway unaffected. Immunized mice depleted of tTregs showed an increased response of IFN-γ-secreting CD4(+) T cells that was strictly reliant on a functional CD27/CD70 pathway. In vitro studies revealed that tTregs downregulate CD70 from the plasma membrane of dendritic cells (DCs) in a CD27-dependent manner. CD70 downregulation required contact between Tregs and DCs and resulted in endocytosis of CD27 and CD70 into the DC. These findings reveal a novel mechanism by which tTregs can maintain tolerance or prevent excessive, proinflammatory Th1 responses.

Environmental enrichment (EE) is a therapeutic paradigm that consists of complex combinations of physical, cognitive, and social stimuli. The mechanisms underlying EE-mediated synaptic plasticity have yet to be fully elucidated. In this study, we investigated the effects of EE on synaptic vesicle-associated proteins and whether the expression of these proteins is related to behavioral outcomes. A total of 44 CD-1® (ICR) mice aged 6 weeks were randomly assigned to either standard cages or EE (N = 22 each). Rotarod and ladder walking tests were then performed to evaluate motor function. To identify the molecular mechanisms underlying the effects of EE, we assessed differentially expressed proteins (DEPs) in the striatum by proteomic analysis. Quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry were conducted to validate the expressions of these proteins. In the behavioral assessment, EE significantly enhanced performance on the rotarod and ladder walking tests. A total of 116 DEPs (54 upregulated and 62 downregulated proteins) were identified in mice exposed to EE. Gene ontology (GO) analysis demonstrated that the upregulated proteins in EE mice were primarily related to biological processes of synaptic vesicle transport and exocytosis. The GO terms for these biological processes commonly included Synaptic vesicle glycoprotein 2B (SV2B), Rabphilin-3A, and Piccolo. The qRT-PCR and western blot analyses revealed that EE increased the expression of SV2B, Rabphilin-3A and Piccolo in the striatum compared to the control group. Immunohistochemistry showed that the density of Piccolo in the vicinity of the subventricular zone was significantly increased in the EE mice compared with control mice. In conclusion, EE upregulates proteins associated with synaptic vesicle transport and exocytosis such as SV2B, Rabphilin-3A and Piccolo in the striatum. These upregulated proteins may be responsible for locomotor performance improvement, as shown in rotarod and ladder walking tests. Elucidation of these changes in synaptic protein expression provides new insights into the mechanism and potential role of EE.

While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying extracellular gene functions, particularly in the tissue context. Here, we developed an approach for spatial functional genomics called Perturb-map. We applied Perturb-map to knock out dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Moreover, we paired Perturb-map and spatial transcriptomics for unbiased analysis of CRISPR-edited tumors. We found that in Tgfbr2 knockout tumors, the tumor microenvironment (TME) was converted to a fibro-mucinous state, and T cells excluded, concomitant with upregulated TGFβ and TGFβ-mediated fibroblast activation, indicating that TGFβ-receptor loss on cancer cells increased TGFβ bioavailability and its immunosuppressive effects on the TME. These studies establish Perturb-map for functional genomics within the tissue at single-cell resolution with spatial architecture preserved and provide insight into how TGFβ responsiveness of cancer cells can affect the TME.

To find factors affecting hip and femoral deformities in children with spastic cerebral palsy (CP) by comparing various clinical findings with imaging studies including plain radiography and computed tomography (CT) imaging.

The aim of this study was to determine if botulinum toxin type A (BoNT-A) injection into the medial hamstring can improve gait kinematics and muscle-tendon length in spastic cerebral palsy (CP) with a flexed knee gait (FKG). Twenty-nine children with spastic CP (Gross Motor Function Classification System I-III) with FKG were recruited for this prospective study. BoNT-A was injected into the semitendinosus and semimembranosus (SM) muscles under ultrasonography guidance. Assessments included Gross Motor Function Measure (GMFM), Modified Ashworth Scale (MAS), Modified Tardieu Scale (MTS), 3-dimensional computerized gait analysis, calculated SM muscle-tendon length and lengthening velocity during gait using musculoskeletal modeling at baseline, 4 and 16 weeks after the injection. Compared to baseline data, significant improvements in GMFM, MAS, and MTS were demonstrated at weeks 4 and 16, and also a significant increase in maximum knee extension during the stance phase was observed at week 4. In addition, the mean lengthening velocity during the swing phase was increased at week 16 without a change in the SM muscle length. Furthermore, there was a significant increase in anterior pelvic tilt at week 4, compared to baseline data. The significant decrease in hip internal rotation after injection was observed only in children with excessive hip internal rotation at initial contact before injection. BoNT-A injection into hamstrings leads to a significant increase in knee extension and anterior pelvic tilt with an increase in lengthening velocity of SM in spastic CP with FKG.

Stem cells are critical for the maintenance of many tissues, but whether their integrity is maintained in the face of immunity is unclear. Here we found that cycling epithelial stem cells, including Lgr5+ intestinal stem cells, as well as ovary and mammary stem cells, were eliminated by activated T cells, but quiescent stem cells in the hair follicle and muscle were resistant to T cell killing. Immune evasion was an intrinsic property of the quiescent stem cells resulting from systemic downregulation of the antigen presentation machinery, including MHC class I and TAP proteins, and is mediated by the transactivator NLRC5. This process was reversed upon stem cell entry into the cell cycle. These studies identify a link between stem cell quiescence, antigen presentation, and immune evasion. As cancer-initiating cells can derive from stem cells, these findings may help explain how the earliest cancer cells evade immune surveillance.