参考文献

The T cell antigen receptor (TCR) recognizes peptides from pathogenic proteins bound in the major histocompatibility complex (MHC). To convert this binding event into downstream signaling,the TCR complex contains immunoreceptor tyrosine-based activation motifs (ITAMs) that act as docking sites for the cytoplasmic tyrosine kinase ZAP-70. Unique among antigen receptors,the TCR complex uses 10 ITAMs to transduce peptide-MHC binding to the cell interior. Using synthetic,drug-inducible receptor-ligand pairs,it was found that greater ITAM multiplicity primarily enhanced the efficiency with which ligand binding was converted into an intracellular signal. This manifested as an increase in the fraction of cells that became activated in response to antigen,and a more synchronous initiation of TCR-proximal signaling,rather than direct amplification of the intracellular signals. Exploiting these findings,the potency and selectivity of chimeric antigen receptors targeted against cancer were substantially enhanced by modulating the number of encoded ITAMs. View Publication

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a low-grade B-cell neoplasm and ∼2{\%} to 9{\%} patients develop an aggressive lymphoma,most commonly diffuse large B-cell lymphoma (Richter transformation,DLBCL-RT). Programmed death-1 (PD-1) pathway plays a crucial role in tumor host immunity evasion and its blockade has emerged as an effective anti-cancer immunotherapy. PD-L1 and PD-1 expression has shown predictive value in anti-PD cancer immunotherapy; however,it has not been well documented in CLL/SLL and DLBCL-RT. We evaluated PD-1 and PD-L1 expression by immunohistochemistry in 39 CLL/SLL,15 DLBCL-RT,and 26 other DLBCL. In CLL/SLL,neoplastic B-cell PD-1 expression was weak and restricted to prolymphocytes/paraimmunoblasts within proliferation centers (PCs) and accentuated PCs of all sizes. Neoplastic B-cell PD-1 expression was highly prevalent and demonstrated increased intensity in DLBCL-RT,but in contrast was only rarely seen in other DLBCL (12/15 vs. 1/26; P{\textless}0.0001). An excellent correlation (90{\%} concordance) was observed between neoplastic B-cell PD-1 immunohistochemistry positivity and molecularly defined CLL/SLL clonal relatedness in DLBCL-RT. PD-L1 expression was observed on the neoplastic B cells in rare DLBCL-RT and other DLBCL cases (1/15 vs. 1/26; P{\textgreater}0.05) as well as background histiocytes and dendritic cells. Overall survival of DLBCL-RT was significantly inferior to that of the other DLBCL (median,16.9 vs. 106.1 mo; P=0.002). Our findings suggest a biological continuum from prolymphocytes/paraimmunoblasts in CLL/SLL PCs to the neoplastic B-cells in DLBCL-RT. The characteristic PD-1 expression in DLBCL-RT makes it a potential surrogate marker for determining clonal relatedness to CLL/SLL,which may have important prognostic and therapeutic implications. View Publication

Recently,deficiency in the cytosolic DNA sensor RNA Polymerase III was described in children with severe primary varicella-zoster virus (VZV) infection in the CNS and lungs. In the present study we examined adult patients with VZV CNS infection caused by viral reactivation. By whole exome sequencing we identified mutations in POL III genes in two of eight patients. These mutations were located in the coding regions of the subunits POLR3A and POLR3E. In functional assays,we found impaired expression of antiviral and inflammatory cytokines in response to the POL III agonist Poly(dA:dT) as well as increased viral replication in patient cells compared to controls. Altogether,this study provides significant extension on the current knowledge on susceptibility to VZV infection by demonstrating mutations in POL III genes associated with impaired immunological sensing of AT-rich DNA in adult patients with VZV CNS infection. View Publication

Baboons naturally infected with simian T lymphotropic virus (STLV) are a potentially useful model system for the study of vaccination against human T lymphotropic virus (HTLV). Here we expanded the number of available full-length baboon STLV-1 sequences from one to three and related the T cell responses that recognize the immunodominant Tax protein to the tax sequences present in two individual baboons. Continuously growing T cell lines were established from two baboons,animals 12141 and 12752. Next-generation sequencing (NGS) of complete STLV genome sequences from these T cell lines revealed them to be closely related but distinct from each other and from the baboon STLV-1 sequence in the NCBI sequence database. Overlapping peptides corresponding to each unique Tax sequence and to the reference baboon Tax sequence were used to analyze recognition by T cells from each baboon using intracellular cytokine staining (ICS). Individual baboons expressed more gamma interferon and tumor necrosis factor alpha in response to Tax peptides corresponding to their own STLV-1 sequence than in response to Tax peptides corresponding to the reference baboon STLV-1 sequence. Thus,our analyses revealed distinct but closely related STLV-1 genome sequences in two baboons,extremely low heterogeneity of STLV sequences within each baboon,no evidence for superinfection within each baboon,and a ready ability of T cells in each baboon to recognize circulating Tax sequences. While amino acid substitutions that result in escape from CD8+ T cell recognition were not observed,premature stop codons were observed in 7{\%} and 56{\%} of tax sequences from peripheral blood mononuclear cells from animals 12141 and 12752,respectively.IMPORTANCE It has been estimated that approximately 100,000 people suffer serious morbidity and 10,000 people die each year from the consequences associated with human T lymphotropic virus (HTLV) infection. There are no antiviral drugs and no preventive vaccine. A preventive vaccine would significantly impact the global burden associated with HTLV infections. Here we provide fundamental information on the simian T lymphotropic virus (STLV) naturally transmitted in a colony of captive baboons. The limited viral sequence heterogeneity in individual baboons,the identity of the viral gene product that is the major target of cellular immune responses,the persistence of viral amino acid sequences that are the major targets of cellular immune responses,and the emergence in vivo of truncated variants in the major target of cellular immune responses all parallel what are seen with HTLV infection of humans. These results justify the use of STLV-infected baboons as a model system for vaccine development efforts. View Publication

Recently,many hurdles and limitations for production of clinically applicable iPSC derivatives have been overcome. Transgene-free iPSCs can be efficiently derived from easily accessible cell sources such as blood. Here we describe the generation of transgene-free hiPS cells from cord blood derived CD34+ cells,reprogrammed using CytoTune™ Sendai reprogramming vectors. CD34+ cell isolation,cultivation,reprogramming and establishment of resulting hiPSC lines were performed under the exclusive usage of animal-derived component-free (ADCF) materials and components. View Publication

A small subset of human cord blood CD34+ cells express endothelial protein C receptor (EPCR/CD201/PROCR) when exposed to the hematopoietic stem cell (HSC) self-renewal agonist UM171. In this article,we show that EPCR-positive UM171-treated cells,as opposed to EPCR-negative cells,exhibit robust multilineage repopulation and serial reconstitution ability in immunocompromised mice. In contrast to other stem cell markers,such as CD38,EPCR expression is maintained when cells are introduced in culture,irrespective of UM171 treatment. Although engineered overexpression of EPCR fails to reproduce the effects of UM171 on HSC activity,its expression is required for the repopulating activity of human HSCs. Altogether,our results indicate that EPCR is a reliable and cell culture-compatible marker of UM171-expanded human cord blood HSCs. View Publication

With increasing worldwide demand for safe blood,there is much interest in generating red blood cells in vitro as an alternative clinical product. However,available methods for in vitro generation of red cells from adult and cord blood progenitors do not yet provide a sustainable supply,and current systems using pluripotent stem cells as progenitors do not generate viable red cells. We have taken an alternative approach,immortalizing early adult erythroblasts generating a stable line,which provides a continuous supply of red cells. The immortalized cells differentiate efficiently into mature,functional reticulocytes that can be isolated by filtration. Extensive characterization has not revealed any differences between these reticulocytes and in vitro-cultured adult reticulocytes functionally or at the molecular level,and importantly no aberrant protein expression. We demonstrate a feasible approach to the manufacture of red cells for clinical use from in vitro culture. View Publication

Background: Local ischemia is the main pathological performance in osteonecrosis of the femoral head (ONFH). There is currently no effective therapy to promote angiogenesis in the femoral head. Recent studies revealed that exosomes secreted by induced pluripotent stem cell-derived mesenchymal stem cells (iPS-MSC-Exos) have great therapeutic potential in ischemic tissues,but whether they could promote angiogenesis in ONFH has not been reported,and little is known regarding the underlying mechanism. Methods: iPS-MSC-Exos were intravenously injected to a steroid-induced rat osteonecrosis model. Samples of the femoral head were obtained 3 weeks after all the injections. The effects were assessed by measuring local angiogenesis and bone loss through histological and immunohistochemical (IHC) staining,micro-CT and three-dimensional microangiography. The effects of exosomes on endothelial cells were studied through evaluations of proliferation,migration and tube-forming analyses. The expression levels of angiogenic related PI3K/Akt signaling pathway of endothelial cells were evaluated following stimulation of iPS-MSC-Exos. The promoting effects of exosomes were re-evaluated following blockade of PI3K/Akt. Results: The in vivo study revealed that administration of iPS-MSC-Exos significantly prevented bone loss,and increased microvessel density in the femoral head compared with control group. We found that iPS-MSC-Exos significantly enhanced the proliferation,migration and tube-forming capacities of endothelial cells in vitro. iPS-MSC-Exos could activate PI3K/Akt signaling pathway in endothelial cells. Moreover,the promoting effects of iPS-MSC-Exos were abolished after blockade of PI3K/Akt on endothelial cells. Conclusions: Our findings suggest that transplantation of iPS-MSC-Exos exerts a preventative effect on ONFH by promoting local angiogenesis and preventing bone loss. The promoting effect might be attributed to activation of the PI3K/Akt signaling pathway on endothelial cells. The data provide the first evidence for the potential of iPS-MSC-Exos in treating ONFH. View Publication

Retinal degenerative diseases are among the leading causes of blindness worldwide,and cell replacement is considered as a promising therapeutic. However,the resources of seed cells are scarce. To further explore this type of therapy,we adopted a culture system that could harvest a substantial quantity of retinal progenitor cells (RPCs) from human embryonic stem cells (hESCs) within a relatively short period of time. Furthermore,we transplanted these RPCs into the subretinal spaces of Royal College of Surgeons (RCS) rats. We quantified the thickness of the treated rats' outer nuclear layers (ONLs) and explored the visual function via electroretinography (ERG). It was found that the differentiated cells expressed RPC markers and photoreceptor progenitor markers. The transplanted RPCs survived for at least 12 weeks,resulting in beneficial effects on the morphology of the host retina,and led to a significant improvement in the visual function of the treated animals. These therapeutic effects suggest that the hESCs-derived RPCs could delay degeneration of the retina and partially restore visual function. View Publication

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration,but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs),endothelial cells (hiPSC-ECs),and smooth muscle cells (SMCs) are often limited by low yield,purity,and/or poor phenotypic stability. Here,we present novel protocols for generating hiPSC-CMs,-ECs,and -SMCs that are substantially more efficient than conventional methods,as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells,by sealing the needle track to prevent the cells from being squeezed out of the myocardium,and cell survival,by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury,the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch,and treatment with both the cells and the patch,but not with the cells alone,was associated with significant improvements in cardiac function and infarct size. View Publication