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1.
Background
Nerve growth factor (NGF) is a neurotrophin crucial for the development and survival of neurons. It also acts on cells of the immune system which express the NGF receptors TrkA and p75NTR and can be produced by them. However, mouse NK cells have not yet been studied in this context.Methodology/Principal Findings
We used cell culture, flow cytometry, confocal microscopy and ELISA assays to investigate the expression of NGF receptors by NK cells and their secretion of NGF. We show that resting NK cells express TrkA and that the expression is different on NK cell subpopulations defined by the relative presence of CD27 and CD11b. Expression of TrkA is dramatically increased in IL-2-activated NK cells. The p75NTR is expressed only on a very low percentage of NK cells. Functionally, NGF moderately inhibits NK cell degranulation, but does not influence proliferation or cytokine production. NK cells do not produce NGF.Conclusions/Significance
We demonstrate for the first time that mouse NK cells express the NGF receptor TrkA and that this expression is dynamically regulated. 相似文献2.
Nina Müller Katharina Landwehr Kirsten Langeveld Joanna Stenzel Walter Pouwels Menno A.W.G. van der Hoorn Erhard Seifried Halvard Bonig 《Cytotherapy》2018,20(4):532-542
Background aims
For patients needing allogeneic stem cell transplantation but lacking a major histocompatibility complex (MHC)-matched donor, haplo-identical (family) donors may be an alternative. Stringent T-cell depletion required in these cases to avoid lethal graft-versus-host disease (GVHD) can delay immune reconstitution, thus impairing defense against virus reactivation and attenuating graft-versus-leukemia (GVL) activity. Several groups reported that GVHD is caused by cells residing within the naive (CD45RA+) T-cell compartment and proposed use of CD45RA-depleted donor lymphocyte infusion (DLI) to accelerate immune reconstitution. We developed and tested the performance of a CD45RA depletion module for the automatic cell-processing device CliniMACS Prodigy and investigated quality attributes of the generated products.Methods
Unstimulated apheresis products from random volunteer donors were depleted of CD45RA+ cells on CliniMACS Prodigy, using Good Manufacturing Practice (GMP)-compliant reagents and methods throughout. Using phenotypic and functional in vitro assays, we assessed the cellular constitution of CD45RA-depleted products, including T-cell subset analyses, immunological memory function and allo-reactivity.Results
Selections were technically uneventful and proceeded automatically with minimal hands-on time beyond tubing set installation. Products were near-qualitatively CD45RA+ depleted, that is, largely devoid of CD45RA+ T cells but also of almost all B and natural killer cells. Naive and effector as well as γ/δ T cells were greatly reduced. The CD4:CD8 ratio was fivefold increased. Mixed lymphocyte reaction assays of the product against third-party leukocytes revealed reduced allo-reactivity compared to starting material. Anti-pathogen responses were retained.Discussion
The novel, closed, fully GMP-compatible process on Prodigy generates highly CD45RA-depleted cellular products predicted to be clinically meaningfully depleted of GvH reactivity. 相似文献3.
Bin Zhang Ronne Wee Yeh Yeo Ruenn Chai Lai Eugene Wei Kian Sim Keh Chuang Chin Sai Kiang Lim 《Cytotherapy》2018,20(5):687-696
Background aims
The immunomodulatory property of mesenchymal stromal cell (MSC) exosomes is well documented. On the basis of our previous report that MSC exosomes increased regulatory T-cell (Treg) production in mice with allogenic skin graft but not in ungrafted mice, we hypothesize that an activated immune system is key to exosome-mediated Treg production.Methods
To test our hypothesis, MSC exosomes were incubated with mouse spleen CD4+ T cells that were activated with either anti-CD3/CD28 mAbs or allogenic antigen-presenting cell (APC)-enriched spleen CD11c+ cells to determine whether production of mouse CD4+CD25+ T cells or CD4+CD25+Foxp3+ Tregs could be induced. MSC exosomes were also administered to the lethal chimeric human-SCID mouse model of graft-versus-host disease (GVHD) in which human peripheral blood mononuclear cells were infused into irradiated NSG mice to induce GVHD.Results
We report here that MSC exosome–induced production of CD4+CD25+ T cells or CD4+CD25+Foxp3+ Tregs from CD4+ T cells activated by allogeneic APC-enriched CD11C+ cells but not those activated by anti-CD3/CD28 mAbs. This induction was exosome- and APC dose–dependent. In the mouse GVHD model in which GVHD was induced by transplanted human APC-stimulated human anti-mouse CD4+ T cell effectors, MSC exosome alleviated GVHD symptoms and increased survival. Surviving exosome-treated mice had a significantly higher level of human CD4+CD25+CD127low/– Tregs than surviving mice treated with Etanercept, a tumor necrosis factor inhibitor.Conclusions
MSC exosome enhanced Treg production in vitro and in vivo through an APC-mediated pathway. 相似文献4.
Rehab Alnabhan Ahmed Gaballa Lisa-Mari Mörk Jonas Mattsson Michael Uhlin Isabelle Magalhaes 《Cytotherapy》2018,20(7):941-951
Background
The use of CD19 chimeric antigen receptor (CAR) T cells to treat B-cell malignancies has proven beneficial. Several groups use serum to produce CD19 CAR T cells. Today, ready-to-use serum-free media that require no addition of serum are commercially available. Therefore, it becomes important to evaluate the production of CD19 CAR T cells with and without the addition of serum.Methods
T cells from buffy coats were cultured in AIM-V and TexMACS (TM) supplemented with 5% human serum (A5% and TM5%, respectively), and in TM without serum. Cells were activated with OKT3 and expanded in interleukin (IL)-2. Viral transduction was performed in RetroNectin-coated plates using the spinoculation method. CD19 CAR T cells were tested for their viability, expansion, transduction efficacy, phenotype and cytotoxicity.Results
CD19 CAR T cells expanded in A5% and TM5% showed significantly better viability and higher fold expansion than cells expanded in TM. TM promoted the expansion of CD8+ T cells and effector phenotype of CD19 CAR T cells. The transduction efficacy and the cytotoxic function were comparable between the different media. Higher CD107a+ cells were detected in TM and TM5%, whereas higher IL-2+ and IL-17+ cells were detected in A5%. CD19 CAR exhibited co-expression of inhibitory receptors such as TIM-3+LAG-3+ and/or TIM-3+PD-1+.Conclusion
Our results indicate that serum supplementation promotes better CD19 CAR T-cell expansion and viability in vitro. CD19 CAR T cells produced in TM medium showed lower CD4/CD8 ratio, which warrants further evaluation in clinical settings. Overall, the choice of culture medium impacts CD19 CAR T-cell end product. 相似文献5.
Qingkun Song Jun Ren Xinna Zhou Xiaoli Wang Guohong Song Amy Hobeika Yanhua Yuan Herbert Kim Lyerly 《Cytotherapy》2018,20(1):126-133
Background aims
This study aimed to determine the prognostic value of circulating CD8+CD28? T lymphocytes among breast cancer patients treated with adoptive T-lymphocyte immunotherapy after chemotherapy.Methods
Two hundred and thirty-two breast cancer patients underwent adoptive T-cell immunotherapy. Circulating CD8+CD28? proportion was measured by flow cytometry. Median proportion of CD8+CD28? was 24.2% and set as the categorical cutoff value for further analysis. The median survival was estimated by Kaplan-Meier curve, with difference detection and hazard ratio estimation by log-rank test and Cox hazard proportion regression model.Results
With adoptive T-cell therapy, patients with higher CD8+CD28? levels experienced median progression-free and overall survival of 7.1 months and 26.9 months, respectively—significantly shorter than patients with lower levels (11.8 and 36.2 months). CD8+CD28? proportion >24.2% demonstrated a hazard ratio (HR) of 2.06 (95% confidence interval [CI] 1.31–3.12) for progression and an HR of 1.97 (95% CI 1.06–3.67) for death. Among patients who had received previous first-line chemotherapy, CD8+CD28? proportion >24.2% demonstrated an HR of 2.66 (95% CI 1.45–4.88) for progression. Among patients exposed to previous second-line or higher chemotherapy, CD8+CD28? proportion >24.2% demonstrated a 486% higher risk for death (HR?=?5.86, 95% CI 1.77–19.39). A 1% increase in suppressive T cells was associated with a 5% increased risk of death.Discussion
Elevated peripheral blood CD8+CD28? was associated with poorer prognosis for metastatic breast cancer, especially for higher risk of progression among patients with first-line chemotherapy and higher risk of death among patients with more than second-line chemotherapy. 相似文献6.
Fenlu Zhu Nirav Shah Huiqing Xu Dina Schneider Rimas Orentas Boro Dropulic Parameswaran Hari Carolyn A. Keever-Taylor 《Cytotherapy》2018,20(3):394-406
Background aims
Multiple steps are required to produce chimeric antigen receptor (CAR)-T cells, involving subset enrichment or depletion, activation, gene transduction and expansion. Open processing steps that increase risk of contamination and production failure are required. This complex process requires skilled personnel and costly clean-room facilities and infrastructure. Simplified, reproducible CAR-T-cell manufacturing with reduced labor intensity within a closed-system is highly desirable for increased availability for patients.Methods
The CliniMACS Prodigy with TCT process software and the TS520 tubing set that allows closed-system processing for cell enrichment, transduction, washing and expansion was used. We used MACS-CD4 and CD8-MicroBeads for enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8 TM-41BB-CD3 ζ-cfrag vectors expressing scFv for CD19 or CD20/CD19 antigens for transduction, TexMACS medium-3%-HS-IL2 for culture and phosphate-buffered saline/ethylenediaminetetraacetic acid buffer for washing. Processing time was 13 days.Results
Enrichment (N?=?7) resulted in CD4/CD8 purity of 98?±?4.0%, 55?±?6% recovery and CD3+ T-cell purity of 89?±?10%. Vectors at multiplicity of infection 5–10 resulted in transduction averaging 37%. An average 30-fold expansion of 108 CD4/CD8-enriched cells resulted in sufficient transduced T cells for clinical use. CAR-T cells were 82–100% CD3+ with a mix of CD4+ and CD8+ cells that primarily expressed an effector-memory or central-memory phenotype. Functional testing demonstrated recognition of B-cells and for the CAR-20/19 T cells, CD19 and CD20 single transfectants were recognized in cytotoxic T lymphocyte and interferon-γ production assays.Discussion
The CliniMACS Prodigy device, tubing set TS520 and TCT software allow CAR-T cells to be manufactured in a closed system at the treatment site without need for clean-room facilities and related infrastructure. 相似文献7.
Background
Cell therapy using mesenchymal stromal cells (MSCs) offers new perspectives in the treatment of traumatic brain injury (TBI). The aim of the present study was to assess the impact of platelet-rich plasma scaffolds (PRPS) as support of MSCs in a delayed phase after severe TBI in rats.Methods
TBI was produced by weight-drop impact to the right cerebral hemisphere. Two months after TBI, four experimental groups were established; saline, PRPS, MSCs in saline, or MSCs in PRPS was transplanted into the area of brain lesion through a small hole. All groups were evaluated in the course of the following 12 months after therapy and the animals were then humanely killed.Results
Our results showed that a greater functional improvement was obtained after the administration of MSCs in PRPS compared with the other experimental groups.Discussion
PRPS enhanced the benefit of cell therapy with MSCs to treat chronic brain damage in rats that suffered a severe TBI. The present findings suggest that the use of intralesional MSCs supported in PRPS may be a strategy of tissue engineering for patients with established neurological severe dysfunction after a TBI. 相似文献8.
Alessandra Micera Barbara Stampachiacchiere Antonio Di Zazzo Roberto Sgrulletta Magdalena Cortes Eduardo Maria Normando Alessandro Lambiase Stefano Bonini 《PloS one》2015,10(11)
Objective
In a previous study, we reported the upregulation of Nerve Growth Factor (NGF) and trkANGFR expression in Ocular Cicatricial Pemphigoid (OCP), an inflammatory and remodeling eye disease. Herein, we hypothesize a potential NGF-driven mechanism on fibroblasts (FBs) during OCP remodeling events. To verify, human derived OCP-FBs were isolated and characterized either at baseline or after NGF exposure.Materials and Methods
Conjunctival biopsies were obtained from 7 patients having OCP and 6 control subjects (cataract surgery). Both conjunctivas and primary FB cultures were characterised for αSMA, NGF and trkANGFR/p75NTR expression. Subcultures were exposed to NGF and evaluated for αSMA, NGF, trkANGFR/p75NTR expression as well as TGFβ1/IL4 release. For analysis, early and advanced subgroups were defined according to clinical parameters.Results
OCP-conjunctivas showed αSMA-expressing FBs and high NGF levels. Advanced OCP-FBs showed higher αSMA expression associated with higher p75NTR and lower trkANGFR expression, as compared to early counterparts. αSMA expression was in keeping with disease severity and correlated to p75NTR. NGF exposure did not affect trkANGFR levels in early OCP-FBs while decreased both αSMA/p75NTR expression and TGFβ1/IL4 release. These effects were not observed in advanced OCP-FBs.Conclusions
Taken together, these data are suggestive for a NGF/p75NTR task in the potential modulation of OCP fibrosis and encourages further studies to fully understand the underlying mechanism occurring in fibrosis. NGF/p75NTR might be viewed as a potential therapeutic target. 相似文献9.
10.
Weiyong Shen Sook H. Chung Mohammad R. Irhimeh Shiying Li So-Ra Lee Mark C. Gillies 《PloS one》2014,9(8)
Objective
Royal College of Surgeons (RCS) rats develop vasculopathy as photoreceptors degenerate. The aim of this study was to examine the effect of erythropoietin (EPO) on retinopathy in RCS rats.Methods
Fluorescein angiography was used to monitor retinal vascular changes over time. Changes in retinal glia and vasculature were studied by immunostaining. To study the effects of EPO on retinal pathology, EPO (5000 IU/kg) was injected intraperitoneally in 14 week old normal and RCS rats twice a week for 4 weeks. Changes in the retinal vasculature, glia and microglia, photoreceptor apoptosis, differential expression of p75 neurotrophin receptor (p75NTR), pro-neurotrophin 3 (pro-NT3), tumour necrosis factor-α (TNFα), pigment epithelium derived factor (PEDF) and vascular endothelial growth factor-A (VEGF-A), the production of CD34+ cells and mobilization of CD34+/VEGF-R2+ cells as well as recruitment of CD34+ cells into the retina were examined after EPO treatment.Results
RCS rats developed progressive capillary dropout and subretinal neovascularization which were accompanied by retinal gliosis. Systemic administration of EPO stabilized the retinal vasculature and inhibited the development of focal vascular lesions. Further studies showed that EPO modulated retinal gliosis, attenuated photoreceptor apoptosis and p75NTR and pro-NT3 upregulation, promoted the infiltration of ramified microglia and stimulated VEGF-A expression but had little effect on TNFα and PEDF expression. EPO stimulated the production of red and white blood cells and CD34+ cells along with effective mobilization of CD34+/VEGF-R2+ cells. Immunofluorescence study demonstrated that EPO enhanced the recruitment of CD34+ cells into the retina.Conclusions
Our results suggest that EPO has therapeutic potentials in treatment of neuronal and vascular pathology in retinal disease. The protective effects of EPO on photoreceptors and the retinal vasculature may involve multiple mechanisms including regulation of retinal glia and microglia, inhibition of p75NTR-pro-NT3 signaling together with stimulation of production and mobilization of bone marrow derived cells. 相似文献11.
Bellanger C Dubanet L Lise MC Fauchais AL Bordessoule D Jauberteau MO Troutaud D 《PloS one》2011,6(11):e27213
Background
Diffuse large B-cell lymphoma (DLBCL) is a common and often fatal malignancy. Immunochemotherapy, a combination of rituximab to standard chemotherapy, has resulted in improved survival. However a substantial proportion of patients still fail to reach sustained remission. We have previously demonstrated that autocrine brain-derived neurotrophic factor (BDNF) production plays a function in human B cell survival, at least partly via sortilin expression. As neurotrophin receptor (Trks) signaling involved activation of survival pathways that are inhibited by rituximab, we speculated that neurotrophins may provide additional support for tumour cell survival and therapeutic resistance in DLBCL.Methodology/Principal Findings
In the present study, we used two DLBCL cell lines, SUDHL4 and SUDHL6, known to be respectively less and more sensitive to rituximab. We found by RT-PCR, western blotting, cytometry and confocal microscopy that both cell lines expressed, in normal culture conditions, BDNF and to a lesser extent NGF, as well as truncated TrkB and p75NTR/sortilin death neurotrophin receptors. Furthermore, BDNF secretion was detected in cell supernatants. NGF and BDNF production and Trk receptor expression, including TrkA, are regulated by apoptotic conditions (serum deprivation or rituximab exposure). Indeed, we show for the first time that rituximab exposure of DLBCL cell lines induces NGF secretion and that differences in rituximab sensitivity are associated with differential expression patterns of neurotrophins and their receptors (TrkA). Finally, these cells are sensitive to the Trk-inhibitor, K252a, as shown by the induction of apoptosis. Furthermore, K252a exhibits additive cytotoxic effects with rituximab.Conclusions/Significance
Collectively, these data strongly suggest that a neurotrophin axis, such NGF/TrkA pathway, may contribute to malignant cell survival and rituximab resistance in DLBCL. 相似文献12.
Pavlína Ptáčková Jan Musil Martin Štach Petr Lesný Šárka Němečková Vlastimil Král Milan Fábry Pavel Otáhal 《Cytotherapy》2018,20(4):507-520
Background aims
Clinical-grade chimeric antigenic receptor (CAR)19 T cells are routinely manufactured by lentiviral/retroviral (LV/RV) transduction of an anti-CD3/CD28 activated T cells, which are then propagated in a culture medium supplemented with interleukin (IL)-2. The use of LV/RVs for T-cell modification represents a manufacturing challenge due to the complexity of the transduction approach and the necessity of thorough quality control.Methods
We present here a significantly improved protocol for CAR19 T-cell manufacture that is based on the electroporation of peripheral blood mononuclear cells with plasmid DNA encoding the piggyBac transposon/transposase vectors and their cultivation in the presence of cytokines IL-4, IL-7 and IL-21.Results
We found that activation of the CAR receptor by either its cognate ligand (i.e., CD19 expressed on the surface of B cells) or anti-CAR antibody, followed by cultivation in the presence of cytokines IL-4 and IL-7, enables strong and highly selective expansion of functional CAR19 T cells, resulting in >90% CAR+ T cells. Addition of cytokine IL-21 to the mixture of IL-4 and IL-7 supported development of immature CAR19 T cells with central memory and stem cell memory phenotypes and expressing very low amounts of inhibitory receptors PD-1, LAG-3 and TIM-3.Conclusions
Our protocol provides a simple and cost-effective method for engineering high-quality T cells for adoptive therapies. 相似文献13.
Charlotta Dagnell Johan Grunewald Marija Kramar Helga Haugom-Olsen G?ran P Elmberger Anders Eklund Caroline Olgart H?glund 《Respiratory research》2010,11(1):156
Background
Pulmonary sarcoidosis is an inflammatory disease, characterized by an accumulation of CD4+ lymphocytes and the formation of non-caseating epithelioid cell granulomas in the lungs. The disease either resolves spontaneously or develops into a chronic disease with fibrosis. The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been suggested to be important mediators of inflammation and mediate tissue remodelling. In support of this, we have recently reported enhanced NGF levels in the airways of patients with pulmonary sarcoidosis. However, less is known about levels of BDNF and NT-3, and moreover, knowledge in the cellular sources of neurotrophins and the distribution of the corresponding neurotrophin receptors in airway tissue in sarcoidosis is lacking.Methods
The concentrations of NGF, BDNF and NT-3 in bronchoalveolar lavage fluid (BALF) of 41 patients with newly diagnosed pulmonary sarcoidosis and 27 healthy controls were determined with ELISA. The localization of neurotrophins and neurotrophin receptors were examined by immunohistochemistry on transbronchial lung biopsies from sarcoidosis patients.Results
The sarcoidosis patients showed significantly enhanced NT-3 and NGF levels in BALF, whereas BDNF was undetectable in both patients and controls. NT-3 levels in BALF were found higher in patients with non-Löfgren sarcoidosis as compared to patients with Löfgren''s syndrome, and in more advanced disease stage. Epithelioid cells and multinucleated giant cells within the sarcoid granulomas showed marked immunoreactivity for NGF, BDNF and NT-3. Also, immunoreactivity for the neurotrophin receptor TrkA, TrkB and TrkC, was found within the granulomas. In addition, alveolar macrophages showed positive immunoreactivity for NGF, BDNF and NT-3 as well as for TrkA, TrkB and TrkC.Conclusions
This study provides evidence of enhanced neurotrophin levels locally within the airways of patients with sarcoidosis. Findings suggest that sarcoid granuloma cells and alveolar macrophages are possible cellular sources of, as well as targets for, neurotrophins in the airways of these patients. 相似文献14.
Background aims
Umbilical cord blood (UCB) provides an alternative source for hematopoietic stem/progenitor cells (HSPCs) in the treatment of hematological malignancies. However, clinical usage is limited due to the low quantity of HSPCs in each unit of cord blood and defects in bone marrow homing. Hyperbaric oxygen (HBO) is among the more recently explored methods used to improve UCB homing and engraftment. HBO works by lowering the host erythropoietin before UCB infusion to facilitate UCB HSPC homing, because such UCB cells are not directly exposed to HBO. In this study, we examined how direct treatment of UCB-CD34+ cells with HBO influences their differentiation, proliferation and in vitro transmigration.Methods
Using a locally designed HBO chamber, freshly enriched UCB-CD34+ cells were exposed to 100% oxygen at 2.5 atmospheres absolute pressure for 2?h before evaluation of proliferative capacity, migration toward a stromal cell–derived factor 1 gradient and lineage differentiation.Results
Our results showed that HBO treatment diminishes proliferation and in vitro transmigration of UCB-CD34+ cells. Treatment was also shown to limit the ultimate differentiation of these cells toward an erythrocyte lineage. As a potential mechanism for these findings, we also investigated HBO effects on the relative concentration of cytoplasmic and nucleic reactive oxygen species (ROS) and on erythropoietin receptor (Epo-R) and CXCR4 expression. HBO-treated cells showed a relative increase in nucleic ROS but no detectable differences in the level of Epo-R nor CXCR4 expression were established compared with non-treated cells.Discussion
In summary, HBO amplifies the formation of ROS in DNA of UCB-CD34+ cells, potentially explaining their reduced proliferation, migration and erythrocytic differentiation. 相似文献15.
Marina V. Kovina Michael E. Krasheninnikov Tatiana G. Dyuzheva Michael I. Danilevsky Ilya D. Klabukov Maxim V. Balyasin Olga K. Chivilgina Alexey V. Lyundup 《Cytotherapy》2018,20(3):361-374
Background
Menstrual blood is only recently and still poorly studied, but it is an abundant and noninvasive source of highly proliferative mesenchymal stromal cells (MSCs). However, no appropriate isolation method has been reported due to its high viscosity and high content of clots and desquamated epithelium.Methods
We studied three different isolation approaches and their combinations: ammonium-containing lysing buffer, distilled water and gradient-density centrifugation. We tested the proliferative capacity, morphology, surface markers and pluripotency of the resulting cells.Results
Our isolation method yields up to four million nucleated cells per milliliter of initial blood, of which about 0.2–0.3% are colony-forming cells expressing standard mesenchymal markers CD90, CD105 and CD73, but not expressing CD45, CD34, CD117, CD133 or HLA-G. The cells have high proliferative potential (doubling in 26?h) and the ability to differentiate into adipocytes and osteocytes. Early endometrial MSCs (eMSCs) express epithelial marker cytokeratin 7 (CK7). CK7 is easily induced in later passages in a prohepatic environment. We show for the first time that a satisfactory and stable yield of eMSCs is observed throughout the whole menstrual period (5 consecutive days) of a healthy woman.Discussion
The new cost/yield adequate method allows isolation from menstrual blood a relatively homogenous pool of highly proliferative MSCs, which seem to be the best candidates for internal organ therapy due to their proepithelial background (early expression of CK7 and its easy induction in later passages) and for mass cryobanking due to their high yield and availability. 相似文献16.
Mustafa H. Ghanem Sara Bolivar-Wagers Barna Dey Agnes Hajduczki Diego A. Vargas-Inchaustegui David T. Danielson Virgilio Bundoc Li Liu Edward A. Berger 《Cytotherapy》2018,20(3):407-419
Background aims
Chimeric antigen receptors (CARs) offer great potential toward a functional cure of human immunodeficiency virus (HIV) infection. To achieve the necessary long-term virus suppression, we believe that CARs must be designed for optimal potency and anti-HIV specificity, and also for minimal probability of virus escape and CAR immunogenicity. CARs containing antibody-based motifs are problematic in the latter regard due to epitope mutation and anti-idiotypic immune responses against the variable regions.Methods
We designed bispecific CARs, each containing a segment of human CD4 linked to the carbohydrate recognition domain of a human C-type lectin. These CARs target two independent regions on HIV-1 gp120 that presumably must be conserved on clinically significant virus variants (i.e., the primary receptor binding site and the dense oligomannose patch). Functionality and specificity of these bispecific CARs were analyzed in assays of CAR-T cell activation and spreading HIV-1 suppression.Results
T cells expressing a CD4-dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DCSIGN) CAR displayed robust stimulation upon encounter with Env-expressing targets, but negligible activity against intercellular adhesion molecule (ICAM)-2 and ICAM-3, the natural dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin ligands. Moreover, the presence of the lectin moiety prevented the CD4 from acting as an entry receptor on CCR5-expressing cells, including CD8+ T cells. However, in HIV suppression assays, the CD4-DCSIGN CAR and the related CD4-liver/lymph node-specific intercellular adhesion molecule-3-grabbing non-integrin CAR displayed only minimally increased potency compared with the CD4 CAR against some HIV-1 isolates and reduced potency against others. By contrast, the CD4-langerin and CD4-mannose binding lectin (MBL) CARs uniformly displayed enhanced potency compared with the CD4 CAR against all the genetically diverse HIV-1 isolates examined. Further experimental data, coupled with known biological features, suggest particular advantages of the CD4-MBL CAR.Discussion
These studies highlight features of bispecific CD4-lectin CARs that achieve potency enhancement by targeting two distinct highly conserved Env determinants while lacking immunogenicity-prone antibody-based motifs. 相似文献17.
RYAN R. KELLY LINDSAY T. MCDONALD VINCENT D. PELLEGRINI JAMES J. CRAY AMANDA C. LARUE 《Cytotherapy》2018,20(11):1371-1380
Background aims
Previous studies identified a circulating human osteoblastic population that expressed osteocalcin (OCN), increased following fracture and pubertal growth, and formed mineralized colonies in vitro and bone in vivo. A subpopulation expressed CD34, a hematopoietic/endothelial marker. These findings led to our hypothesis that hematopoietic-derived CD34+OCN+ cells exist in the circulation of mice and are modulated after fracture.Methods
Flow cytometry was used to identify CD34+OCN+ cells in male B6.SJL-PtprcaPepcb/BoyJ and Vav-Cre/mTmG (VavR) mice. Non-stabilized tibial fractures were created by three-point bend. Fractures were longitudinally imaged by micro-computed tomography, and immunofluorescent staining was used to evaluate CD34+OCN+ cells within fracture callus. AMD3100 (10 mg/kg) was injected subcutaneously for 3 days and the CD34+OCN+ population was evaluated by flow cytometry.Results
Circulating CD34+OCN+ cells were identified in mice and confirmed to be of hematopoietic origin (CD45+; Vav1+) using two mouse models. Both circulating and bone marrow-derived CD34+OCN+ cells peaked three weeks post-non-stabilized tibial fracture, suggesting association with cartilage callus transition to bone and early mineralization. Co-expression of CD34 and OCN in the fracture callus at two weeks post-fracture was observed. By three weeks, there was 2.1-fold increase in number of CD34+OCN+ cells, and these were observed throughout the fracture callus. AMD3100 altered CD34+OCN+ cell levels in peripheral blood and bone marrow.Discussion
Together, these data demonstrate a murine CD34+OCN+ circulating population that may be directly involved in fracture repair. Future studies will molecularly characterize CD34+OCN+ cells, determine mechanisms regulating their contribution, and examine if their number correlates with improved fracture healing outcomes. 相似文献18.
Joana M. Murad Susanne H. Baumeister Lillian Werner Heather Daley Hélène Trébéden-Negre Jake Reder Charles L. Sentman David Gilham Frederic Lehmann Sarah Snykers Marie-Louise Sentman Terri Wade Adam Schmucker Michael W. Fanger Glenn Dranoff Jerome Ritz Sarah Nikiforow 《Cytotherapy》2018,20(7):952-963
Background aims
Adoptive cell therapy employing natural killer group 2D (NKG2D) chimeric antigen receptor (CAR)-modified T cells has demonstrated preclinical efficacy in several model systems, including hematological and solid tumors. We present comprehensive data on manufacturing development and clinical production of autologous NKG2D CAR T cells for treatment of acute myeloid leukemia and multiple myeloma (ClinicalTrials.gov Identifier: NCT02203825). An NKG2D CAR was generated by fusing native full-length human NKG2D to the human CD3ζ cytoplasmic signaling domain. NKG2D naturally associates with native costimulatory molecule DAP10, effectively generating a second-generation CAR against multiple ligands upregulated during malignant transformation including MIC-A, MIC-B and the UL-16 binding proteins.Methods
CAR T cells were infused fresh after a 9-day process wherein OKT3-activated T cells were genetically modified with replication-defective gamma-retroviral vector and expanded ex vivo for 5 days with recombinant human interleukin-2.Results
Despite sizable interpatient variation in originally collected cells, release criteria, including T-cell expansion and purity (median 98%), T-cell transduction (median 66% CD8+ T cells), and functional activity against NKG2D ligand-positive cells, were met for 100% of healthy donors and patients enrolled and collected. There was minimal carryover of non–T cells, particularly malignant cells; both effector memory and central memory cells were generated, and inflammatory cytokines such as granulocyte colony-stimulating factor, RANTES, interferon-γ and tumor necrosis factor-α were selectively up-regulated.Conclusions
The process resulted in production of required cell doses for the first-in-human phase I NKG2D CAR T clinical trial and provides a robust, flexible base for further optimization of NKG2D CAR T-cell manufacturing. 相似文献19.
Keren Ettinger Shimon Lecht Hadar Arien-Zakay Gadi Cohen Shlomit Aga-Mizrachi Nurit Yanay H. Uri Saragovi Hinyu Nedev Cezary Marcinkiewicz Yoram Nevo Philip Lazarovici 《Cellular signalling》2012,24(12):2378-2388
The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75NTR, α9β1 integrin, and its regulator ADAM12, but not trkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and βNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9β1 integrin; and (c) p75NTR antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4 days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60–70%, 85–90% and 60–80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of βNGF, added either 1 h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and βNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75NTR and α9β1 integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9β1 in NGF-induced signaling in skeletal muscle model, resembling the role of trkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets. 相似文献
20.
Zhisheng Her Kylie Su Mei Yong Kathirvel Paramasivam Wilson Wei Sheng Tan Xue Ying Chan Sue Yee Tan Min Liu Yong Fan Yeh Ching Linn Kam Man Hui Uttam Surana Qingfeng Chen 《Journal of hematology & oncology》2017,10(1):162