技术资料

Typhoid fever caused by Salmonella enterica serovar (S.) Typhi differs in its clinical presentation from gastroenteritis caused by S. Typhimurium and other non-typhoidal Salmonella serovars. The different clinical presentations are attributed in part to the virulence-associated capsular polysaccharide (Vi antigen) of S. Typhi,which prevents phagocytes from triggering a respiratory burst by preventing antibody-mediated complement activation. Paradoxically,the Vi antigen is absent from S. Paratyphi A,which causes a disease that is indistinguishable from typhoid fever. Here,we show that evasion of the phagocyte respiratory burst by S. Paratyphi A required very long O antigen chains containing the O2 antigen to inhibit antibody binding. We conclude that the ability to avoid the phagocyte respiratory burst is a property distinguishing typhoidal from non-typhoidal Salmonella serovars that was acquired by S. Typhi and S. Paratyphi A independently through convergent evolution. View Publication

Mitochondrial dysfunction has been demonstrated to result in premature aging due to its effects on stem cells. Nevertheless,a full understanding of the role of mitochondrial bioenergetics through differentiation is still lacking. Here we show the bioenergetics profile of human stem cells of embryonic origin differentiating along the hepatic lineage. Our study reveals especially the transition between hepatic specification and hepatic maturation as dependent on mitochondrial respiration and demonstrates that even though differentiating cells are primarily dependent on glycolysis until induction of hepatocyte maturation,oxidative phosphorylation is essential at all stages of differentiation. View Publication

BACKGROUND Transplanting autologous patient-derived enteric neuronal stem/progenitor cells (ENSCs) is an innovative approach to replacing missing enteric neurons in patients with Hirschsprung disease (HSCR). Using autologous cells eliminates immunologic and ethical concerns raised by other cell sources. However,whether postnatal aganglionic bowel is permissive for transplanted ENSCs and whether ENSCs from HSCR patients can be successfully isolated,cultured,and transplanted in vivo remains unknown. METHODS ENSCs isolated from the ganglionic intestine of Ednrb(-/-) mice (HSCR-ENSCs) were characterized immunohistochemically and evaluated for their capacity to proliferate and differentiate in vitro. Fluorescently labeled ENSCs were co-cultured ex vivo with aganglionic Ednrb(-/-) colon. For in vivo transplantation,HSCR-ENSCs were labeled with lentivirus expressing green fluorescent protein (GFP) and implanted into aganglionic embryonic chick gut in ovo and postnatal aganglionic Ednrb(-/-) rectum in vivo. KEY RESULTS HSCR-ENSCs maintain normal capacity self-renewal and neuronal differentiation. Moreover,the Ednrb(-/-) aganglionic environment is permissive to engraftment by wild-type ENSCs ex vivo and supports migratrion and neuroglial differentiation of these cells following transplantation in vivo. Lentiviral GFP-labeled HSCR-ENSCs populated embryonic chick hindgut and postnatal colon of Ednrb(-/-) HSCR,with cells populating the intermuscular layer and forming enteric neurons and glia. CONCLUSIONS & INFERENCES ENSCs can be isolated and cultured from mice with HSCR,and transplanted into the aganglionic bowel of HSCR littermates to generate enteric neuronal networks. These results in an isogenic model establish the potential of using autologous-derived stem cells to treat HSCR and other intestinal neuropathies. View Publication

Continued growth in the cell therapy industry and commercialization of cell therapies that successfully advance through clinical trials has led to increased awareness around the need for specialized and complex materials utilized in their manufacture. Ancillary materials (AMs) are components or reagents used during the manufacture of cell therapy products but are not intended to be part of the final products. Commonly,there are limitations in the availability of clinical-grade reagents used as AMs. Furthermore,AMs may affect the efficacy of the cell product and subsequent safety of the cell therapy for the patient. As such,AMs must be carefully selected and appropriately qualified during the cell therapy development process. However,the ongoing evolution of cell therapy research,limited number of clinical trials and registered cell therapy products results in the current absence of specific regulations governing the composition,compliance,and qualification of AMs often leads to confusion by suppliers and users in this field. Here we provide an overview and interpretation of the existing global framework surrounding AM use and investigate some common misunderstandings within the industry,with the aim of facilitating the appropriate selection and qualification of AMs. The key message we wish to emphasize is that in order to most effectively mitigate risk around cell therapy development and patient safety,users must work with their suppliers and regulators to qualify each AM to assess source,purity,identity,safety,and suitability in a given application. View Publication

The promise of inducing immunological tolerance through regulatory T cell (Treg) control of effector T cell function is crucial for developing future therapeutic strategies to treat allograft rejection as well as inflammatory autoimmune diseases. In the current study,we used murine allograft rejection as a model to identify microRNA (miRNA) regulation of Treg differentiation from na{\{i}}ve CD4 cells. We performed miRNA expression array in CD4+ T cells in the draining lymph node (dLN) of mice which received syngeneic or allogeneic grafts to determine the molecular mechanisms that hinder the expansion of Tregs. We identified an increase in miRNA cluster 297-669 (C2MC) after allogeneic transplantation in CD4+ T cells such that 10 of the 27 upregulated miRNAs were all from this cluster with one of its members mmu-miR-466a-3p (miR-466a-3p) targeting transforming growth factor beta 2 (TGF-$\beta$2) as identified through reporter luciferase assay. Transfection of miR-466a-3p in CD4+ T cells led to a decreased inducible FoxP3+ Treg generation while inhibiting miR-466a-3p expression through locked nucleic acid resulting in increased Tregs and a reduction in effector T cells. Furthermore in vivo inhibition of miR-466a-3p in an allogeneic skin-graft model attenuated T cell response against the graft through an increase in TGF-$\beta$2. TGF-$\beta$2 was as effective as TGF-$\beta$1 at both inducing Tregs and through adoptive transfer mitigating host effector T cell response against the allograft. Together the current study demonstrates for the first time a new role for miRNA-466a-3p and TGF-$\beta$2 in the regulation of Treg differentiation and thus offers novel avenues to control inflammatory disorders." View Publication

Chimeric antigen receptors (CARs) link an antigen recognition domain to intracellular signaling domains to redirect T cell specificity and function. T cells expressing CARs with CD28/CD3$\zeta$ or 4-1BB/CD3$\zeta$ signaling domains are effective at treating refractory B cell malignancies but exhibit differences in effector function,clinical efficacy,and toxicity that are assumed to result from the activation of divergent signaling cascades. We analyzed stimulation-induced phosphorylation events in primary human CD8+ CD28/CD3$\zeta$ and 4-1BB/CD3$\zeta$ CAR T cells by mass spectrometry and found that both CAR constructs activated similar signaling intermediates. Stimulation of CD28/CD3$\zeta$ CARs activated faster and larger-magnitude changes in protein phosphorylation,which correlated with an effector T cell-like phenotype and function. In contrast,4-1BB/CD3$\zeta$ CAR T cells preferentially expressed T cell memory-associated genes and exhibited sustained antitumor activity against established tumors in vivo. Mutagenesis of the CAR CD28 signaling domain demonstrated that the increased CD28/CD3$\zeta$ CAR signal intensity was partly related to constitutive association of Lck with this domain in CAR complexes. Our data show that CAR signaling pathways cannot be predicted solely by the domains used to construct the receptor and that signal strength is a key determinant of T cell fate. Thus,tailoring CAR design based on signal strength may lead to improved clinical efficacy and reduced toxicity. View Publication

Somites form during embryonic development and give rise to unique cell and tissue types,such as skeletal muscles and bones and cartilage of the vertebrae. Using somitogenesis-stage human embryos,we performed transcriptomic profiling of human presomitic mesoderm as well as nascent and developed somites. In addition to conserved pathways such as WNT-$\beta$-catenin,we also identified BMP and transforming growth factor $\beta$ (TGF-$\beta$) signaling as major regulators unique to human somitogenesis. This information enabled us to develop an efficient protocol to derive somite cells in vitro from human pluripotent stem cells (hPSCs). Importantly,the in-vitro-differentiating cells progressively expressed markers of the distinct developmental stages that are known to occur during in vivo somitogenesis. Furthermore,when subjected to lineage-specific differentiation conditions,the hPSC-derived somite cells were multipotent in generating somite derivatives,including skeletal myocytes,osteocytes,and chondrocytes. This work improves our understanding of human somitogenesis and may enhance our ability to treat diseases affecting somite derivatives. View Publication

We have recently demonstrated the effectiveness of an influenza A virus (IAV) subunit vaccine based on biodegradable polyanhydride nanoparticles delivery in mice. In the present study,we evaluated the efficacy of ∼200nm polyanhydride nanoparticles encapsulating inactivated swine influenza A virus (SwIAV) as a vaccine to induce protective immunity against a heterologous IAV challenge in pigs. Nursery pigs were vaccinated intranasally twice with inactivated SwIAV H1N2 (KAg) or polyanhydride nanoparticle-encapsulated KAg (KAg nanovaccine),and efficacy was evaluated against a heterologous zoonotic virulent SwIAV H1N1 challenge. Pigs were monitored for fever daily. Local and systemic antibody responses,antigen-specific proliferation of peripheral blood mononuclear cells,gross and microscopic lung lesions,and virus load in the respiratory tract were compared among the groups of animals. Our pre-challenge results indicated that KAg nanovaccine induced virus-specific lymphocyte proliferation and increased the frequency of CD4+CD8$\alpha$$\alpha$+ T helper and CD8+ cytotoxic T cells in peripheral blood mononuclear cells. KAg nanovaccine-immunized pigs were protected from fever following SwIAV challenge. In addition,pigs immunized with the KAg nanovaccine presented with lower viral antigens in lung sections and had 6 to 8-fold reduction in nasal shedding of SwIAV four days post-challenge compared to control animals. Immunologically,increased IFN-$\gamma$ secreting T lymphocyte populations against both the vaccine and challenge viruses were detected in KAg nanovaccine-immunized pigs compared to the animals immunized with KAg alone. However,in the KAg nanovaccine-immunized pigs,hemagglutination inhibition,IgG and IgA antibody responses,and virus neutralization titers were comparable to that in the animals immunized with KAg alone. Overall,our data indicated that intranasal delivery of polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs,with promise to induce cross-protective immunity. View Publication

Immunotherapy has increased overall survival of metastatic cancer patients,and cancer antigens are promising vaccine targets. To fulfill the promise,appropriate tailoring of the vaccine formulations to mount in vivo cytotoxic T cell (CTL) responses toward co-delivered cancer antigens is essential. Previous development of therapeutic cancer vaccines has largely been based on studies in mice,and the majority of these candidate vaccines failed to induce therapeutic responses in the subsequent human clinical trials. Given that antigen dose and vaccine volume in pigs are translatable to humans and the porcine immunome is closer related to the human counterpart,we here introduce pigs as a supplementary large animal model for human cancer vaccine development. IDO and RhoC,both important in human cancer development and progression,were used as vaccine targets and 12 pigs were immunized with overlapping 20mer peptides spanning the entire porcine IDO and RhoC sequences formulated in CTL-inducing adjuvants: CAF09,CASAC,Montanide ISA 51 VG,or PBS. Taking advantage of recombinant swine MHC class I molecules (SLAs),the peptide-SLA complex stability was measured for 198 IDO- or RhoC-derived 9-11mer peptides predicted to bind to SLA-1(*)04:01,-1(*)07:02,-2(*)04:01,-2(*)05:02,and/or -3(*)04:01. This identified 89 stable (t½ ≥ 0.5 h) peptide-SLA complexes. By IFN-$\gamma$ release in PBMC cultures we monitored the vaccine-induced peptide-specific CTL responses,and found responses to both IDO- and RhoC-derived peptides across all groups with no adjuvant being superior. These findings support the further use of pigs as a large animal model for vaccine development against human cancer. View Publication

The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Using a 3D organoid system,we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes,including TMPRSS2-ERG fusion,SPOP mutation,SPINK1 overexpression,and CHD1 loss. Whole-exome sequencing shows a low mutational burden,consistent with genomics studies,but with mutations in FOXA1 and PIK3R1,as well as in DNA repair and chromatin modifier pathways that have been reported in advanced disease. Loss of p53 and RB tumor suppressor pathway function are the most common feature shared across the organoid lines. The methodology described here should enable the generation of a large repertoire of patient-derived prostate cancer lines amenable to genetic and pharmacologic studies. View Publication

Small-cell lung cancer (SCLC),an aggressive neuroendocrine tumor with early dissemination and dismal prognosis,accounts for 15-20{\%} of lung cancer cases and ∼200,000 deaths each year. Most cases are inoperable,and biopsies to investigate SCLC biology are rarely obtainable. Circulating tumor cells (CTCs),which are prevalent in SCLC,present a readily accessible 'liquid biopsy'. Here we show that CTCs from patients with either chemosensitive or chemorefractory SCLC are tumorigenic in immune-compromised mice,and the resultant CTC-derived explants (CDXs) mirror the donor patient's response to platinum and etoposide chemotherapy. Genomic analysis of isolated CTCs revealed considerable similarity to the corresponding CDX. Most marked differences were observed between CDXs from patients with different clinical outcomes. These data demonstrate that CTC molecular analysis via serial blood sampling could facilitate delivery of personalized medicine for SCLC. CDXs are readily passaged,and these unique mouse models provide tractable systems for therapy testing and understanding drug resistance mechanisms. View Publication

Diverse hematopoietic progenitors,including myeloid populations arising in inflammatory and tumoral conditions and multipotent cells,mobilized by hematopoietic growth factors or emerging during parasitic infections,display tolerogenic properties. Innate immune stimuli confer regulatory functions to various mature B-cell subsets but immature B-cell progenitors endowed with suppressive properties per se or after differentiating into more mature regulatory B cells remain to be characterized. Herein we provide evidence for innate pro-B cells (CpG-proBs) that emerged within the bone marrow both in vitro and in vivo upon Toll-like receptor-9 activation and whose adoptive transfer protected nonobese diabetic mice against type 1 diabetes (T1D). These cells responded to IFN-$\gamma$ released by activated effector T cells (Teffs),by up-regulating their Fas ligand (FasL) expression,which enabled them to kill Teffs through apoptosis. In turn,IFN-$\gamma$ derived from CpG-proBs enhanced IFN-$\gamma$ while dramatically reducing IL-21 production by Teffs. In keeping with the crucial pathogenic role played by IL-21 in T1D,adoptively transferred IFN-$\gamma$-deficient CpG-proBs did not prevent T1D development. Additionally,CpG-proBs matured in vivo into diverse pancreatic and splenic suppressive FasL(high) B-cell subsets. CpG-proBs may become instrumental in cell therapy of autoimmune diseases either on their own or as graft complement in autologous stem cell transplantation. View Publication