技术资料

BACKGROUND Microglia,the principle immune cells of the brain,play important roles in neuronal development,homeostatic function and neurodegenerative disease. Recent genetic studies have further highlighted the importance of microglia in neurodegeneration with the identification of disease risk polymorphisms in many microglial genes. To better understand the role of these genes in microglial biology and disease,we,and others,have developed methods to differentiate microglia from human induced pluripotent stem cells (iPSCs). While the development of these methods has begun to enable important new studies of microglial biology,labs with little prior stem cell experience have sometimes found it challenging to adopt these complex protocols. Therefore,we have now developed a greatly simplified approach to generate large numbers of highly pure human microglia. RESULTS iPSCs are first differentiated toward a mesodermal,hematopoietic lineage using commercially available media. Highly pure populations of non-adherent CD43+ hematopoietic progenitors are then simply transferred to media that includes three key cytokines (M-CSF,IL-34,and TGF$\beta$-1) that promote differentiation of homeostatic microglia. This updated approach avoids the prior requirement for hypoxic incubation,complex media formulation,FACS sorting,or co-culture,thereby significantly simplifying human microglial generation. To confirm that the resulting cells are equivalent to previously developed iPSC-microglia,we performed RNA-sequencing,functional testing,and transplantation studies. Our findings reveal that microglia generated via this simplified method are virtually identical to iPS-microglia produced via our previously published approach. To also determine whether a small molecule activator of TGF$\beta$ signaling (IDE1) can be used to replace recombinant TGF$\beta$1,further reducing costs,we examined growth kinetics and the transcriptome of cells differentiated with IDE1. These data demonstrate that a microglial cell can indeed be produced using this alternative approach,although transcriptional differences do occur that should be considered. CONCLUSION We anticipate that this new and greatly simplified protocol will enable many interested labs,including those with little prior stem cell or flow cytometry experience,to generate and study human iPS-microglia. By combining this method with other advances such as CRISPR-gene editing and xenotransplantation,the field will continue to improve our understanding of microglial biology and their important roles in human development,homeostasis,and disease. View Publication

Natural killer (NK) cell recognition of tumor cells is mediated through activating receptors such as CD226,with suppression of effector functions often controlled by negative regulatory transcription factors such as FOXO1. Here we show that CD226 regulation of NK cell cytotoxicity is facilitated through inactivation of FOXO1. Gene-expression analysis of NK cells isolated from syngeneic tumors grown in wild-type or CD226-deficient mice revealed dysregulated expression of FOXO1-regulated genes in the absence of CD226. In vitro cytotoxicity and stimulation assays demonstrated that CD226 is required for optimal killing of tumor target cells,with engagement of its ligand CD155 resulting in phosphorylation of FOXO1. CD226 deficiency or anti-CD226 antibody blockade impaired cytotoxicity with concomitant compromised inactivation of FOXO1. Furthermore,inhibitors of FOXO1 phosphorylation abrogated CD226-mediated signaling and effector responses. These results define a pathway by which CD226 exerts control of NK cell responses against tumors. View Publication

T-cell responses in the lung are critical for protection against respiratory pathogens. TNFR superfamily members play important roles in providing survival signals to T cells during respiratory infections. However,whether these signals take place mainly during priming in the secondary lymphoid organs and/or in the peripheral tissues remains unknown. Here we show that under conditions of competition,GITR provides a T-cell intrinsic advantage to both CD4 and CD8 effector T cells in the lung tissue,as well as for the formation of CD4 and CD8 tissue-resident memory T cells during respiratory influenza infection in mice. In contrast,under non-competitive conditions,GITR has a preferential effect on CD8 over CD4 T cells. The nucleoprotein-specific CD8 T-cell response partially compensated for GITR deficiency by expansion of higher affinity T cells; whereas,the polymerase-specific response was less flexible and more GITR dependent. Following influenza infection,GITR is expressed on lung T cells and GITRL is preferentially expressed on lung monocyte-derived inflammatory antigen presenting cells. Accordingly,we show that GITR+/+ T cells in the lung parenchyma express more phosphorylated-ribosomal protein S6 than their GITR-/- counterparts. Thus,GITR signaling within the lung tissue critically regulates effector and tissue-resident memory T-cell accumulation. View Publication

We examined the unique contributions of the cytokines IL-21 and IL-4 on germinal center (GC) B cell initiation and subsequent maturation in a murine model system. Similar to other reports,we found T follicular helper cell expression of IL-21 begins prior to T follicular helper cell migration into the B cell follicle and precedes that of IL-4. Consistent with this timing,IL-21 signaling has a greater influence on the perifollicular pre-GC B cell transition to the intrafollicular stage. Notably,Bcl6hi B cells can form in the combined absence of IL-21R- and STAT6-derived signals; however,these nascent GC B cells cease to proliferate and are more prone to apoptosis. When B cells lack either IL-21R or STAT6,aberrant GCs form atypical centroblasts and centrocytes that differ in their phenotypic maturation and costimulatory molecule expression. Thus,IL-4 and IL-21 play nonredundant roles in the phased progression of GC B cell development that can initiate in the combined absence of these cytokine signals. View Publication

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development,however,is restricted by significant off-tumor toxicities associated with Fc$\gamma$R interactions. Here,we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody,1D8N/CEGa1,consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly,treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate Fc$\gamma$R interactions in the 4-1BB-agonist-associated immune abnormalities,and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy. View Publication