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CD8 T cells can play both a protective and pathogenic role in inflammation and autoimmune development. Recent studies have highlighted the ability of CD8 T cells to function as T follicular helper (Tfh) cells in the germinal center in the context of infection. However,whether this phenomenon occurs in autoimmunity and contributes to autoimmune pathogenesis is largely unexplored. In this study,we show that CD8 T cells acquire a CD4 Tfh profile in the absence of functional regulatory T cells in both the IL-2-deficient and scurfy mouse models. Depletion of CD8 T cells mitigates autoimmune pathogenesis in IL-2-deficient mice. CD8 T cells express the B cell follicle-localizing chemokine receptor CXCR5,a principal Tfh transcription factor Bcl6,and the Tfh effector cytokine IL-21. CD8 T cells localize to the B cell follicle,express B cell costimulatory proteins,and promote B cell differentiation and Ab isotype class switching. These data reveal a novel contribution of autoreactive CD8 T cells to autoimmune disease,in part,through CD4 follicular-like differentiation and functionality. View Publication

Chronic myelomonocytic leukemia (CMML) is a clinically heterogeneous neoplasm in which JAK2 inhibition has demonstrated reductions in inflammatory cytokines and promising clinical activity. We hypothesize that annotation of inflammatory cytokines may uncover mutation-independent cytokine subsets associated with novel CMML prognostic features. A Luminex cytokine profiling assay was utilized to profile cryopreserved peripheral blood plasma from 215 CMML cases from three academic centers,along with center-specific,age-matched plasma controls. Significant differences were observed between CMML patients and healthy controls in 23 out of 45 cytokines including increased cytokine levels in IL-8,IP-10,IL-1RA,TNF-alpha$,IL-6,MCP-1/CCL2,hepatocyte growth factor (HGF),M-CSF,VEGF,IL-4,and IL-2RA. Cytokine associations were identified with clinical and genetic features,and Euclidian cluster analysis identified three distinct cluster groups associated with important clinical and genetic features in CMML. CMML patients with decreased IL-10 expression had a poor overall survival when compared to CMML patients with elevated expression of IL-10 (P = 0.017),even when adjusted for ASXL1 mutation and other prognostic features. Incorporating IL-10 with the Mayo Molecular Model statistically improved the prognostic ability of the model. These established cytokines,such as IL-10,as prognostically relevant and represent the first comprehensive study exploring the clinical implications of the CMML inflammatory state. View Publication

Abnormal signaling pathways mediated by N-methyl-d-aspartate receptors (NMDARs) have been implicated in the pathogenesis of various CNS disorders and have been long considered as promising points of therapeutic intervention. However,few efforts have been previously described concerning evaluation of therapeutic modulators of NMDARs and their downstream pathways in human neurons with endogenous expression of NMDARs. In the present study,we assessed expression,functionality,and subunit composition of endogenous NMDARs in human induced pluripotent stem cell (hiPSC)-derived cortical neurons (iCell Neurons and iCell GlutaNeurons). We initially confirmed the expected pharmacological response of iCell Neurons and iCell GlutaNeurons to NMDA by patch-clamp recordings. Subsequent pharmacological interrogation using GluN2 subunit-selective antagonists revealed the predominance of GluN2B in both iCell Neurons and iCell GlutaNeurons. This observation was also supported by qRT-PCR and Western blot analyses of GluN2 subunit expression as well as pharmacological experiments using positive allosteric modulators with distinct GluN2 subunit selectivity. We conclude that iCell Neurons and iCell GlutaNeurons express functional GluN2B-containing NMDARs and could serve as a valuable system for development and validation of GluN2B-modulating pharmaceutical agents. View Publication

Hematotoxicity is a significant issue for drug safety and can result from direct cytotoxicity toward circulating mature blood cell types as well as targeting of immature blood-forming stem cells/progenitor cells in the bone marrow. In vitro models for understanding and investigating the hematotoxicity potential of new test items/drugs are critical in early preclinical drug development. The traditional method,colony forming unit (CFU) assay,is commonly used and has been validated as a method for hematotoxicity screening. The CFU assay has multiple limitations for its application in investigative work. In this paper,we describe a detailed protocol for a liquid-culture,microplate-based in vitro hematotoxicity assay to evaluate lineage-specific (myeloid,erythroid,and megakaryocytic) hematotoxicity at different stages of differentiation. This assay has multiple advantages over the traditional CFU assay,including being suitable for high-throughput screening and flexible enough to allow inclusion of additional endpoints for mechanistic studies. Therefore,it is an extremely useful tool for scientists in pharmaceutical discovery and development. {\textcopyright} 2018 by John Wiley & Sons,Inc. View Publication

Understanding human embryonic ventral midbrain is of major interest for Parkinson's disease. However,the cell types,their gene expression dynamics,and their relationship to commonly used rodent models remain to be defined. We performed single-cell RNA sequencing to examine ventral midbrain development in human and mouse. We found 25 molecularly defined human cell types,including five subtypes of radial glia-like cells and four progenitors. In the mouse,two mature fetal dopaminergic neuron subtypes diversified into five adult classes during postnatal development. Cell types and gene expression were generally conserved across species,but with clear differences in cell proliferation,developmental timing,and dopaminergic neuron development. Additionally,we developed a method to quantitatively assess the fidelity of dopaminergic neurons derived from human pluripotent stem cells,at a single-cell level. Thus,our study provides insight into the molecular programs controlling human midbrain development and provides a foundation for the development of cell replacement therapies. View Publication