参考文献

Endothelial cells (ECs) line the luminal surface of blood vessels and have an active role in the recruitment of leukocytes,including immune cell activation. Regulatory T cells (Tregs) are immune suppressor cells that maintain peripheral tolerance and must interact with the endothelium as they traffic into tissue. We hypothesized that human ECs could modulate Tregs and their suppressor function. Cocultures of CD4+ T cells with human umbilical vein ECs (HUVECs) or dermal microvascular ECs (HDMECs) were conducted and analyzed for activation and proliferation after 72 and 120 h using flow cytometry. In monocyte-depleted cultures,human ECs were found to support CD4+ T cell proliferation in the presence of external mitogens phytohemagglutinin or anti-CD3/28 antibodies (aCD3/28). Activation was shown by CD25 expression in these cells that also transiently expressed the Treg transcription factor FOXP3. HUVECs supported the specific concurrent proliferation of both effector T cells and Tregs when cocultured with aCD3/28. Purified Tregs were also functionally activated by prior coculture with EC to suppress effector T (Teff) cell proliferation. Both direct coculture and indirect coculture of EC and Treg showed activation of the Treg suppressive phenotype. However,whereas HUVEC showed enhancement of suppression by both mechanisms,HDMEC only supported Treg suppressive activity via the contact-independent mechanism. In the contact-independent cultures,the soluble mediators IL-6,GM-CSF,or G-CSF released from ECs following interferon-gamma$ activation were not responsible for the enhanced Treg suppressor function. Following direct coculture,Treg expression of inhibitory receptors PD-1 and OX40 was elevated while activated EC expressed the counter ligands programmed death ligand (PD-L)1 and PD-L2. Therefore,human ECs have a role in supporting T cell proliferation and increasing Treg suppressor function. This ability of EC to enhance Treg function could offer novel targets to boost Treg activity during inflammatory disorders. View Publication

Protein glycosylation provides proteomic diversity in regulating protein localization,stability,and activity; it remains largely unknown whether the sugar moiety contributes to immunosuppression. In the study of immune receptor glycosylation,we showed that EGF induces programmed death ligand 1 (PD-L1) and receptor programmed cell death protein 1 (PD-1) interaction,requiring beta$-1,3-N-acetylglucosaminyl transferase (B3GNT3) expression in triple-negative breast cancer. Downregulation of B3GNT3 enhances cytotoxic T cell-mediated anti-tumor immunity. A monoclonal antibody targeting glycosylated PD-L1 (gPD-L1) blocks PD-L1/PD-1 interaction and promotes PD-L1 internalization and degradation. In addition to immune reactivation,drug-conjugated gPD-L1 antibody induces a potent cell-killing effect as well as a bystander-killing effect on adjacent cancer cells lacking PD-L1 expression without any detectable toxicity. Our work suggests targeting protein glycosylation as a potential strategy to enhance immune checkpoint therapy. View Publication

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) grown in engineered heart tissue (EHT) can be used for drug screening,disease modeling,and heart repair. However,the immaturity of hiPSC-CMs currently limits their use. Because mechanical loading increases during development and facilitates cardiac maturation,we hypothesized that afterload would promote maturation of EHTs. To test this we developed a system in which EHTs are suspended between a rigid post and a flexible one,whose resistance to contraction can be modulated by applying braces of varying length. These braces allow us to adjust afterload conditions over two orders of magnitude by increasing the flexible post resistance from 0.09 up to 9.2 mu$N/mu$m. After three weeks in culture,optical tracking of post deflections revealed that auxotonic twitch forces increased in correlation with the degree of afterload,whereas twitch velocities decreased with afterload. Consequently,the power and work of the EHTs were maximal under intermediate afterloads. When studied isometrically,the inotropy of EHTs increased with afterload up to an intermediate resistance (0.45 mu$N/mu$m) and then plateaued. Applied afterload increased sarcomere length,cardiomyocyte area and elongation,which are hallmarks of maturation. Furthermore,progressively increasing the level of afterload led to improved calcium handling,increased expression of several key markers of cardiac maturation,including a shift from fetal to adult ventricular myosin heavy chain isoforms. However,at the highest afterload condition,markers of pathological hypertrophy and fibrosis were also upregulated,although the bulk tissue stiffness remained the same for all levels of applied afterload tested. Together,our results indicate that application of moderate afterloads can substantially improve the maturation of hiPSC-CMs in EHTs,while high afterload conditions may mimic certain aspects of human cardiac pathology resulting from elevated mechanical overload. View Publication

Natural killer (NK) cells play a pivotal role in the immune response against infections and malignant transformation,and adopted transfer of NK cells is thought to be a promising therapeutic approach for cancer patients. Previous reports describing the phenotypic features of canine NK cells have produced inconsistent results. Canine NK cells are still defined as non-B and non-T (CD3-CD21-) large granular lymphocytes. However,a few reports have demonstrated that canine NK cells share the phenotypic characteristics of T lymphocytes,and that CD3+CD5dimCD21- lymphocytes are putative canine NK cells. Based on our previous reports,we hypothesized that phenotypic modulation could occur between these two populations during activation. In this study,we investigated the phenotypic and functional differences between CD3+CD5dimCD21- (cytotoxic large granular lymphocytes) and CD3-CD5-CD21- NK lymphocytes before and after culture of peripheral blood mononuclear cells isolated from normal dogs. The results of this study show that CD3+CD5dimCD21- lymphocytes can be differentiated into non-B,non-T NK (CD3-CD5-CD21-TCRalpha$beta$-TCRgamma$delta$-GranzymeB+) lymphocytes through phenotypic modulation in response to cytokine stimulation. In vitro studies of purified CD3+CD5dimCD21- cells showed that CD3-CD5-CD21- cells are derived from CD3+CD5dimCD21- cells through phenotypic modulation. CD3+CD5dimCD21- cells share more NK cell functional characteristics compared with CD3-CD5-CD21- cells,including the expression of T-box transcription factors (Eomes,T-bet),the production of granzyme B and interferon-gamma$,and the expression of NK cell-related molecular receptors such as NKG2D and NKp30. In conclusion,the results of this study suggest that CD3+CD5dimCD21- and CD3-CD5-CD21- cells both contain a subset of putative NK cells,and the difference between the two populations may be due to the degree of maturation. View Publication

Retinal ganglion cells (RGCs) are the projection neurons of the retina and transmit visual information to postsynaptic targets in the brain. While this function is shared among nearly all RGCs,this class of cell is remarkably diverse,comprised of multiple subtypes. Previous efforts have identified numerous RGC subtypes in animal models,but less attention has been paid to human RGCs. Thus,efforts of this study examined the diversity of RGCs differentiated from human pluripotent stem cells (hPSCs) and characterized defined subtypes through the expression of subtype-specific markers. Further investigation of these subtypes was achieved using single-cell transcriptomics,confirming the combinatorial expression of molecular markers associated with these subtypes,and also provided insight into more subtype-specific markers. Thus,the results of this study describe the derivation of RGC subtypes from hPSCs and will support the future exploration of phenotypic and functional diversity within human RGCs. View Publication

Expression of miR-143 and miR-145 is reduced in hematopoietic stem/progenitor cells (HSPCs) of myelodysplastic syndrome patients with a deletion in the long arm of chromosome 5. Here we show that mice lacking miR-143/145 have impaired HSPC activity with depletion of functional hematopoietic stem cells (HSCs),but activation of progenitor cells (HPCs). We identify components of the transforming growth factor beta$ (TGFbeta$) pathway as key targets of miR-143/145. Enforced expression of the TGFbeta$ adaptor protein and miR-145 target,Disabled-2 (DAB2),recapitulates the HSC defect seen in miR-143/145-/- mice. Despite reduced HSC activity,older miR-143/145-/- and DAB2-expressing mice show elevated leukocyte counts associated with increased HPC activity. A subset of mice develop a serially transplantable myeloid malignancy,associated with expansion of HPC. Thus,miR-143/145 play a cell context-dependent role in HSPC function through regulation of TGFbeta$/DAB2 activation,and loss of these miRNAs creates a preleukemic state. View Publication

Targeted cancer immunotherapy with irradiated,granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting,allogeneic cancer cell lines has been an effective approach to reduce tumor burden in several patients. It is generally assumed that to be effective,these cell lines need to express immunogenic antigens coexpressed in patient tumor cells,and antigen-presenting cells need to take up such antigens then present them to patient T cells. We have previously reported that,in a phase I pilot study (ClinicalTrials.gov NCT00095862),a subject with stage IV breast cancer experienced substantial regression of breast,lung,and brain lesions following inoculation with clinical formulations of SV-BR-1-GM,a GM-CSF-secreting breast tumor cell line. To identify diagnostic features permitting the prospective identification of patients likely to benefit from SV-BR-1-GM,we conducted a molecular analysis of the SV-BR-1-GM cell line and of patient-derived blood,as well as a tumor specimen. Compared to normal human breast cells,SV-BR-1-GM cells overexpress genes encoding tumor-associated antigens (TAAs) such as PRAME,a cancer/testis antigen. Curiously,despite its presumptive breast epithelial origin,the cell line expresses major histocompatibility complex (MHC) class II genes (HLA-DRA,HLA-DRB3,HLA-DMA,HLA-DMB),in addition to several other factors known to play immunostimulatory roles. These factors include MHC class I components (B2M,HLA-A,HLA-B),ADA (encoding adenosine deaminase),ADGRE5 (CD97),CD58 (LFA3),CD74 (encoding invariant chain and CLIP),CD83,CXCL8 (IL8),CXCL16,HLA-F,IL6,IL18,and KITLG. Moreover,both SV-BR-1-GM cells and the responding study subject carried an HLA-DRB3*02:02 allele,raising the question of whether SV-BR-1-GM cells can directly present endogenous antigens to T cells,thereby inducing a tumor-directed immune response. In support of this,SV-BR-1-GM cells (which also carry the HLA-DRB3*01:01 allele) treated with yellow fever virus (YFV) envelope (Env) 43-59 peptides reactivated YFV-DRB3*01:01-specific CD4+ T cells. Thus,the partial HLA allele match between SV-BR-1-GM and the clinical responder might have enabled patient T lymphocytes to directly recognize SV-BR-1-GM TAAs as presented on SV-BR-1-GM MHCs. Taken together,our findings are consistent with a potentially unique mechanism of action by which SV-BR-1-GM cells can act as APCs for previously primed CD4+ T cells. View Publication

We report that SHIP(-/-) mice,compared to SHIP(+/+) mice,are Th2 skewed with elevated serum IgE and twice as many splenic CD4(+) Th2 cells that,when stimulated with anti-CD3,produce more IL-4 and less IFN-$\gamma$. Exploring the reason for this Th2 skewing,we found that freshly isolated SHIP(-/-) splenic and bone marrow basophils are present in elevated numbers and secrete far more IL-4 in response to IL-3 or to Fc$\epsilon$RI stimulation than do WT basophils. These SHIP(-/-) basophils markedly skew wild-type macrophage colony stimulating factor-derived macrophages toward an M2 phenotype,stimulate OT-II CD4(+) Th cells to differentiate into Th2 cells,and trigger SHIP(+/+) B cells to become IgE-producing cells. All these effects are completely abrogated with neutralizing anti-IL-4 Ab. Exploring the cell signaling pathways responsible for hyperproduction of IL-4 by SHIP(-/-) basophils,we found that IL-3-induced activation of the PI3K pathway is significantly enhanced and that PI3K inhibitors,especially a p110$\alpha$ inhibitor,dramatically suppresses IL-4 production from these cells. In vivo studies,in which basophils were depleted from mast cell-deficient SHIP(+/+) and SHIP(-/-) mice,confirmed the central role that basophils play in the Th2 skewing of naive SHIP-deficient mice. Taken together,these studies demonstrate that SHIP is a potent negative regulator of IL-4 production from basophils and thus may be a novel therapeutic target for Th1- and Th2-related diseases. View Publication

Long-lived multipotent stem cells (ISCs) at the base of intestinal crypts adjust their phenotypes to accommodate normal maintenance and post-injury regeneration of the epithelium. Their long life,lineage plasticity,and proliferative potential underlie the necessity for tight homeostatic regulation of the ISC compartment. In that context,the guanylate cyclase C (GUCY2C) receptor and its paracrine ligands regulate intestinal epithelial homeostasis,including proliferation,lineage commitment,and DNA damage repair. However,a role for this axis in maintaining ISCs remains unknown. Transgenic mice enabling analysis of ISCs (Lgr5-GFP) in the context of GUCY2C elimination (Gucy2c -/- ) were combined with immunodetection techniques and pharmacological treatments to define the role of the GUCY2C signaling axis in supporting ISCs. ISCs were reduced inGucy2c -/- mice,associated with loss of active Lgr5+cells but a reciprocal increase in reserve Bmi1+cells. GUCY2C was expressed in crypt base Lgr5+cells in which it mediates canonical cyclic (c) GMP-dependent signaling. Endoplasmic reticulum (ER) stress,typically absent from ISCs,was elevated throughout the crypt base inGucy2c -/- mice. The chemical chaperone tauroursodeoxycholic acid resolved this ER stress and restored the balance of ISCs,an effect mimicked by the GUCY2C effector 8Br-cGMP. Reduced ISCs inGucy2c -/- mice was associated with greater epithelial injury and impaired regeneration following sub-lethal doses of irradiation. These observations suggest that GUCY2C provides homeostatic signals that modulate ER stress and cell vulnerability as part of the machinery contributing to the integrity of ISCs. View Publication

Traumatic brain injury (TBI) is soon predicted to become the third leading cause of death and disability worldwide. After the primary injury,a complex set of secondary injuries develops hours and days later with prolonged neuroinflammation playing a key role. TBI and other inflammatory conditions are currently being treated in preclinical and clinical trials by a number of cellular therapies. Mesenchymal stem cells (MSC) are of great interest due to their widespread usage,safety,and relative ease to isolate and culture. However,there has been a wide range in efficacy reported using MSC clinically and in preclinical models,likely due to differences in cell preparations and a significant amount of donor variability. In this study,we seek to find a correlation between in vitro activity and in vivo efficacy. We designed assays to explore the responsiveness of MSC to immunological cues to address the immunomodulatory properties of MSC,one of their primary modes of therapeutic activity in TBI. Our results showed intrinsic differences in the immunomodulatory capacity of MSC preparations from different bone marrow and amniotic fluid donors. This difference mirrored the therapeutic capacity of the MSC in an experimental model of TBI,an effect confirmed using siRNA knockdown of COX2 followed by overexpressing COX2. Among the immunomodulatory factors assessed,the therapeutic benefit correlated with the secretion of prostaglandin E2 (PGE2 ) by MSC prior to treatment,suggesting that measurement of PGE2 could be a very useful potency marker to create an index of predicted efficacy for preparations of MSC to treat TBI. Stem Cells 2017;35:1416-1430. View Publication