Measurement of leukocyte trafficking kinetics in macaques by serial intravascular staining

2021 ◽  
Vol 13 (576) ◽  
pp. eabb4582 ◽  
Author(s):  
E. Lake Potter ◽  
Hannah P. Gideon ◽  
Victor Tkachev ◽  
Giulia Fabozzi ◽  
Alexander Chassiakos ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nonhuman Primates ◽  
Molecular Mechanisms ◽  
Bacterial Pathogen ◽  
Immune Memory ◽  
Multiparameter Flow Cytometry ◽  
Lymphoid Tissues ◽  
Leukocyte Trafficking ◽  
Intravascular Staining

Leukocyte trafficking enables detection of pathogens, immune responses, and immune memory. Dysregulation of leukocyte trafficking is often found in disease, highlighting its important role in homeostasis and the immune response. Whereas some of the molecular mechanisms mediating leukocyte trafficking are understood, little is known about the regulation of trafficking, including trafficking kinetics and its impact on immune homeostasis. We developed a method of serial intravascular staining (SIVS) to measure trafficking kinetics in nonhuman primates using infusions of fluorescently labeled antibodies to label circulating leukocytes. Because antibody infusions labeled only leukocytes in the blood, cells were “barcoded” according to their location at the time of each infusion, providing positional histories that could be used to infer trafficking kinetics. We used SIVS and multiparameter flow cytometry to quantitate cellular trafficking into lymphoid tissues of healthy animals at homeostasis and to identify perivascular cells that could be unique to nonlymphoid organs. To investigate how these parameters could be influenced during disease, SIVS was used to quantify lymphocyte trafficking in macaques infected with the bacterial pathogen Mycobacterium tuberculosis and to enumerate intravascular leukocytes in lung granulomas. We showed that whereas most cells in lung granulomas were localized there for more than 24 hours, granulomas were dynamic with a slow continual cellular influx, the rate of which predicted clearance of M. tuberculosis from the granulomas. SIVS, in combination with intracellular staining and multiparametric flow cytometry, is a powerful method to quantify the kinetics of leukocyte trafficking in nonhuman primates in vivo.

Efficient and Durable Gene Marking of Hematopoietic Progenitor Cells in Nonhuman Primates After Nonablative Conditioning

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2271-2286 ◽  
Author(s):  
M. Rosenzweig ◽  
T.J. MacVittie ◽  
D. Harper ◽  
D. Hempel ◽  
R.L. Glickman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Nonhuman Primates ◽  
Peripheral Blood Stem Cells ◽  
Hematopoietic Stem ◽  
Blood Stem Cells ◽  
High Level

Optimization of mobilization, harvest, and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral blood stem cells and retrovirus transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24 (mCD24), into hematopoietic stem cells in nonhuman primates. Rhesus macaques were treated with Flt3-L (200 μg/kg) and G-CSF (20 μg/kg) for 7 days and autologous CD34+ peripheral blood stem cells harvested by leukapheresis. CD34+ cells were transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321 (50 ng/mL) in serum-free medium. An important and novel feature of this study is that enhanced in vivo engraftment of transduced stem cells was achieved by conditioning the animals with a low-morbidity regimen of sublethal irradiation (320 to 400 cGy) on the day of transplantation. Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flow cytometry and semi-quantitative DNA polymerase chain reaction (PCR). Our data show successful and persistent engraftment of transduced primitive progenitors capable of giving rise to marked cells of multiple hematopoietic lineages, including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% ± 32% (n = 4) of granulocytes expressed mCD24 antigen at the cell surface. Peak in vivo levels of genetically modified peripheral blood lymphocytes approached 35% ± 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks posttransplantation. In addition, naı̈ve (CD45RA+and CD62L+) CD4+ and CD8+cells were the predominant phenotype of the marked CD3+ T cells detected at early time points. A high level of marking persisted at between 10% and 15% of peripheral blood leukocytes for 4 months and at lower levels past 6 months in some animals. A cytotoxic T-lymphocyte response against mCD24 was detected in only 1 animal. This degree of persistent long-lived, high-level gene marking of multiple hematopoietic lineages, including naı̈ve T cells, using a nonablative marrow conditioning regimen represents an important step toward the ultimate goal of high-level permanent transduced gene expression in stem cells.

Long-Term Increase in Fetal Hemoglobin Expression in Nonhuman Primates Following Transplantation of Autologous Bcl11a Nuclease-Edited HSCs

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2035-2035 ◽  
Author(s):  
Olivier Humbert ◽  
Hans-Peter Kiem
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Real Time Pcr ◽  
Peripheral Blood ◽  
Nonhuman Primates ◽  
Pcr Analysis ◽  
Beta Globin ◽  
Gamma Globin ◽  
Cd34 Cells

Abstract Elevated levels of fetal hemoglobin (HbF) ameliorate the clinical symptoms of beta-thalassemia and sickle cell anemia. The transcription factor B-cell lymphoma/leukemia 11A (BCL11A) is required for silencing of gamma-globin expression in adult erythroid cells and functions as a switch from fetal to adult hemoglobin production in humans. BCL11A therefore constitutes a therapeutic target for the treatment of hemoglobinopathies. We inactivated BCL11A function by double-strand DNA break-induced mutagenesis using Transcription Activator-Like Effector Nucleases (TALENs). 20 to 30% gene editing could be achieved in vitro in human and nonhuman primate CD34+ cells by TALEN mRNAs electroporation targeting exon 2 of Bcl11a. Colony-forming efficiency was slightly lower in Bcl11a-edited CD34+ cells but lineage differentiation potential was unchanged. Erythroid differentiation of CD34+ cells in culture showed increased Fetal to Beta hemoglobin ratio in both human and primate Bcl11a-modified cells as compared to control cells, thus validating our editing approach to increase HbF production. To determine if Bcl11a-edited hematopoietic stem cells (HSCs) could be engrafted and give rise to HbF-producing erythrocytes, we transplanted a pigtail macaque with autologous CD34+ electroporated with Bcl11a TALEN mRNA following conditioning by total body irradiation. We detected about 1 % gene disruption in vivo early post-transplant and disruption frequency gradually declined to reach a set point of about 0.3% starting at day 28 post-transplantation. In this analysis, which we have so far taken out to 42 days, single clones could be tracked based on their mutation signature, and we found that several clones persisted over time, confirming engraftment of Bcl11a-modified cells. Since the transplantation procedure and chemo-radiotherapy conditioning can raise HbF production, three control animals that were transplanted using similar conditions as with the Bcl11a-edited HSCs and one untransplanted animal were also included in our analysis. Flow cytometry measurement of HbF in peripheral blood showed a rapid increase in F-cell production in all animals, reaching levels that ranged from 10% to 40% by 30 days, while the untransplanted control showed basal HbF expression of about 0.5% (Fig. 1A). The peak for HbF expression lasted for about 140 days and eventually returned to basal levels that averaged 0.5% for all control animals. In comparison, the animal transplanted with Bcl11a-edited cells showed significantly higher HbF levels starting at day 140 post-treatment (1-1.5%), and HbF production has remained constant for at least 150 days. This result was confirmed by hemoglobin mRNA analysis in peripheral blood using real-time PCR. We found a rapid increase in gamma globin expression following transplantation, before returning to near basal levels. As compared to controls, the animal transplanted with Bcl11a-edited cells showed a 5 to 10-fold increase in gamma to beta globin ratio at day 140 and this ratio has remained constant ever since (Fig. 1B). We are currently working on ways to enhance Bcl11a-editing and to select for Bcl11a-modified HSCs using targeted integration of the chemoselection cassette P140K MGMT to ultimately achieve curative HbF production. Potential TALEN off-target sites will also be examined as well as any side effect associated with the inactivation of BCL11A. Overall, our data demonstrate that transplantation of Bcl11a-edited HSCs results in elevated HbF production in nonhuman primates. Furthermore, we show that nonhuman primates can serve as a useful model for novel gene editing strategies toward the treatment of hemoglobinopathies. Figure 1. In vivo monitoring of HbF expression by flow cytometry and real-time PCR. (A) Intracellular HbF staining of peripheral blood measured by flow cytometry. (B) Real-time PCR analysis of hemoglobin transcripts in RNA isolated from peripheral blood. Expression was normalized to GAPDH and %HbG is calculated as HbG/(HbG+HbB). HbG=gamma globin; HbB=beta globin. Black line=Bcl11a transplant; grey line=control transplant; dashed line=untransplanted control. Figure 1. In vivo monitoring of HbF expression by flow cytometry and real-time PCR. (A) Intracellular HbF staining of peripheral blood measured by flow cytometry. (B) Real-time PCR analysis of hemoglobin transcripts in RNA isolated from peripheral blood. Expression was normalized to GAPDH and %HbG is calculated as HbG/(HbG+HbB). HbG=gamma globin; HbB=beta globin. Black line=Bcl11a transplant; grey line=control transplant; dashed line=untransplanted control. Disclosures No relevant conflicts of interest to declare.

Microenvironment Regulation of IL23R/IL-23 Axis Drives Chronic Lymphocytic Leukemia (CLL) Progression

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 616-616
Author(s):  
Fortunato Morabito ◽  
Giovanna Cutrona ◽  
Anna Grazia Recchia ◽  
Marina Fabbi ◽  
Silvano Ferrini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Biological Effects ◽  
Luciferase Reporter ◽  
Side Chain ◽  
Lymphoid Tissues ◽  
Stat3 Phosphorylation

Abstract Background : CLL displays a considerable degree of clinical heterogeneity, which is in part ascribable to clone-intrinsic biological features and that are also influenced by clone-extrinsic events related to the microenvironment. Among the dynamics-taking place within the CLL microenvironment, those finalized to the induction of an overly inflammatory milieu may significantly impact on the CLL natural history by hijacking the immunological microenvironment at the same time fostering clone fitness. IL-23 acts as a prototypical pro-inflammatory mediator representing a promising therapeutic target. We analyzed the ability of CLL cells to sense IL-23 through the IL-23R complex (consisting of IL12Rß1 and IL23R subunits) expression and correlated this feature with clinical outcome. Moreover, we investigated the synthesis of IL-23 within the CLL microenvironment, and tested the biological effects of the IL-23/IL-23R axis engagement and of its interference in vitro and in vivo. Methods : IL23R complex was detected by quadruple flow cytometry staining with CD19, CD5, IL23R, and IL12Rβ1 in prospectively enrolled CLL cases (O-CLL1 protocol, clinicaltrial.gov identifier NCT00917540). On human tissue specimens, lymph node and bone marrow samples from 16 CLL patients were selected for in situ immunolocalization analyses. NOD/Shi-scid/γcnull (NSG) mice were used for in vivo xenografts, in which activated autologous T cells (AAT), obtained by adding anti-CD3 and CD28 Dynabeads and rIL2 were co-injected with CLL cells. MiRNA analysis was performed by Agilent's Human V2 platform and by quantitative PCR. MirVANA microRNA mimics and inhibitors were purchased from Ambion, Inc. For 3'UTR luciferase reporter experiments, miRNA target reporter vectors were purchased from Origene. Results : By flow cytometry, circulating CLL cells of 281 cases variably expressed IL23R side chain while consistently lacking IL12Rß1 chain expression. The engagement of the uncoupled IL23R complex expression (i.e. IL23R but not IL12Rb1 expression) by IL23 did not activate downstream signaling pathways, such as the up-regulation of pSTAT3. The 3-year TTFT probability of patients with low IL23R expression (IL23R-low) was 91% as compared to 75% of IL23R-high cases [χ2 9.1, P=.003; HR=3.2, 95%CI (1.4-7.1)]; in a multivariate model, IL23R expression still remained independently associated with TTFT. We explored the potential control of IL23R expression in CLL cells by miRNA and found 15 miRNAs inversely associated with IL23R expression, five of which predicted as regulators (miRNA-146b-5p, miRNA-155, miRNA-324-5p, miRNA-532-3p and miRNA-630). Among these, miR-324-3p and miR-146b-5p were demonstrated to functionally regulate the expression of IL23R and IL12Rβ1 proteins in CLL cells, respectively. Within lymphoid tissues, in situ, CLL clones expressing IL23R side chain also showed expression of IL12Rß1, which varied according to the density of CD40L-expressing bystander elements suggesting a microenvironment-driven regulation of the IL-23R complex. To functionally test this hypothesis, CLL cells were co-cultured in the presence of NIH-3T3 transduced with CD40L or with AAT cells. A significant up-regulation was observed for both the IL12Rß1 and IL23R side chains, suggesting the environment co-stimulation as a mechanism of IL-23R complex regulation. Consistently, the IL-23R complex was upmodulated in CLL cells expressing IL-23R but not IL12Rß1, upon xenograft with autologous T cells into NOD-Scid mice. We then investigated the effect of IL-23R engagement by IL-23 in CLL cells and found that IL-23R activity correlated with CLL cell proliferation and survival in vitro via STAT3 phosphorylation. The trophic nature of IL-23-mediated stimuli over CLL cells was further demonstrated in vivo through the adoption of an anti-IL23p19 monoclonal antibody for clinical use, which proved to be effective in eradicating the xenografted CLL clone in the infiltrated tissues (spleen, liver and BM) by inhibiting proliferation and inducing apoptosis. Noteworthy, the therapeutic effect of IL-23 antagonism was demonstrated by histopathology, flow cytometry and BCR clonality. Conclusions : Overall, we demonstrated that IL-23/IL-23R axis is a novel microenvironment-regulated determinant in CLL pathobiology representing a strong prospect in disease prognostication and treatment. Disclosures No relevant conflicts of interest to declare.

scholarly journals EXTH-45. DELTA-24-RGDOX ACTIVATION OF THE IDO-KYN-AHR CASCADE IN GLIOBLASTOMA: OLD TARGETS FOR A NEW THERAPY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii96-ii97
Author(s):  
Teresa Nguyen ◽  
Dong Ho Shin ◽  
Hong Jiang ◽  
Derek Wainwright ◽  
Sagar Sohoni ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Oncolytic Viruses ◽  
Immune Memory ◽  
Tryptophan Depletion

Abstract Immune enhancement of virotherapy by reshaping the tumor immune landscape may improve its success rates. IDO, an IFNγ inducible tryptophan catabolizing enzyme, is upregulated in glioblastoma, correlating with poor prognoses. IDO-mediated tryptophan depletion in the tumor-microenvironment decreases proliferation and induces apoptosis of surrounding effector T-cells. Kynurenine, a metabolite of tryptophan, induces T-cell differentiation into immunosuppressive Tregs. Excess kynurenine elicits AhR-mediated lymphocyte dysfunction and immunosuppression. The immune stimulating effect of oncolytic-virus, Delta-24-RGDOX, triggers IFNγ production contributing to a positive IDO-Kynurenine-AhR feedback loop. We hypothesized that combining Delta-24-RGDOX with IDO inhibitors will synergize to effectively treat glioblastoma. We characterized IDO and AhR in Delta-24-RGDOX infected cancers using immunofluorescence, qRT-PCR, and flow cytometry and found increased expression of both proteins in vitro and in vivo. We also observed induction of AhR in CD4+ and CD8+ T-cells by Delta-24-RGDOX in vivo. Delta-24-RGDOX also increased activity of AhR in cancer cells as indicated by an AhR responsive elements transcription assay. We used a murine glioblastoma model to test the efficacy of combining Delta-24-RGDOX with IDO inhibitor, 1MT/indoximod; the combination produced 30% more long-term survivors compared Delta-24-RGDOX alone (P=0.03), which we showed, through lymphocytic depletion studies, was dependent on CD4+ T-cell activation. We observed 100% survival in the re-challenged long-term glioblastoma survivors, indicating the establishment of immune memory by the combination. Functional studies showed significant increases in anti-tumor activity of splenocytes from combination-treated mice compared to Delta-24-RGDOX-treated mice (P=0.009). Flow cytometry studies revealed that combination-treated mice yielded the highest levels of chronically activated T-cells and lowest levels of Tregs and myeloid derived suppressor cells compared to Delta-24-RGDOX single treatment (P≤0.05). This microenvironment remodeling correlated with complete tumor elimination. Altogether, Delta-24-RGDOX activates the IDO-Kyn-AhR cascade in gliomas, identifying new targets, which when inhibited have the potential to enhance the anti-glioma effect of oncolytic-viruses by reversing tumor immunosuppression.

Glucocorticoid Therapy Targets Proliferation, Differentiation and Bcl-2 Dependent Survival Signaling in ALL Blasts.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1841-1841
Author(s):  
Peter Rhein ◽  
Stefanie Scheid ◽  
Richard Ratei ◽  
Christian Hagemeier ◽  
Karl Seeger ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Induction Therapy ◽  
Molecular Mechanisms ◽  
Leukemia Cell ◽  
Apoptotic Pathway ◽  
Therapy Targets ◽  
Cell Cycling ◽  
Induced Apoptosis

Abstract In the multicentric ALL-BFM (Berlin-Frankfurt-Munster) study, all patients are uniformly treated during the first week of induction therapy which uses glucocorticoids (GC) as the principal therapeutic agent. The GC response assessed at day 8 of therapy provides one of the basic parameters for further risk stratification. In spite of the clinical significance, molecular mechanisms of GC action in vivo are largely unknown. Our recent genome-wide analysis of gene expression in blasts persisting during induction therapy identified a common set of genes differentially expressed in blasts at day 8 (d8) and at diagnosis (d0) (n=457, false discovery rate <0.05). Expression changes indicated therapy-induced inhibition of cell cycling, expression shift towards normal mature B cells and downregulation of the apoptosis regulator Bcl-2. In the current study, we validated the key differences between d8 and d0 blasts at protein and cellular levels. DNA distribution and percentage of cycling blasts was determined by flow cytometry in a series of matched d8 and d0 samples (13 pts) and demonstrated the decreased proliferative activity of d8 cells (4.3-fold, p=0.014). Protein expression, investigated by flow cytometry in a total of 84 pts, demonstrated statistically significant expression decrease of the progenitor cell antigenes CD10, CD34 and TdT and expression increase of the B-cell antigene CD20 and the inflammatory response molecules CD11b and IFNGR1 (p<0.05). We were also able to confirm the lower expression values of the Bcl-2 protein in d8 blasts (p<0.05, n=57). Investigation of GC-sensitive B-lineage leukemia cell lines demonstrated that BCL-2 downregulation by GC was a pre-requisite of GC-induced apoptosis. Moreover, we found a considerable cross-correlation between viability, cell cycling and Bcl-2 expression levels. Upregulation of the Bcl-2 expression via IL-7 receptor signaling prevented GC-induced apoptosis in these cell lines. Collectively, GC therapy interferes with differentiation and proliferation programs in leukemic blasts which are closely related to the Bcl-2 specific apoptotic pathway.

scholarly journals CD137 and PD-L1 targeting with immunovirotherapy induces a potent and durable antitumor immune response in glioblastoma models

2021 ◽  
Vol 9 (7) ◽  
pp. e002644
Author(s):  
Montserrat Puigdelloses ◽  
Marc Garcia-Moure ◽  
Sara Labiano ◽  
Virginia Laspidea ◽  
Marisol Gonzalez-Huarriz ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tumor Microenvironment ◽  
Antitumor Effect ◽  
Immune Memory ◽  
Programmed Death ◽  
Long Term Survivors ◽  
Murine Glioma

BackgroundGlioblastoma (GBM) is a devastating primary brain tumor with a highly immunosuppressive tumor microenvironment, and treatment with oncolytic viruses (OVs) has emerged as a promising strategy for these tumors. Our group constructed a new OV named Delta-24-ACT, which was based on the Delta-24-RGD platform armed with 4-1BB ligand (4-1BBL). In this study, we evaluated the antitumor effect of Delta-24-ACT alone or in combination with an immune checkpoint inhibitor (ICI) in preclinical models of glioma.MethodsThe in vitro effect of Delta-24-ACT was characterized through analyses of its infectivity, replication and cytotoxicity by flow cytometry, immunofluorescence (IF) and MTS assays, respectively. The antitumor effect and therapeutic mechanism were evaluated in vivo using several immunocompetent murine glioma models. The tumor microenvironment was studied by flow cytometry, immunohistochemistry and IF.ResultsDelta-24-ACT was able to infect and exert a cytotoxic effect on murine and human glioma cell lines. Moreover, Delta-24-ACT expressed functional 4-1BBL that was able to costimulate T lymphocytes in vitro and in vivo. Delta-24-ACT elicited a more potent antitumor effect in GBM murine models than Delta-24-RGD, as demonstrated by significant increases in median survival and the percentage of long-term survivors. Furthermore, Delta-24-ACT modulated the tumor microenvironment, which led to lymphocyte infiltration and alteration of their immune phenotype, as characterized by increases in the expression of Programmed Death 1 (PD-1) on T cells and Programmed Death-ligand 1 (PD-L1) on different myeloid cell populations. Because Delta-24-ACT did not induce an immune memory response in long-term survivors, as indicated by rechallenge experiments, we combined Delta-24-ACT with an anti-PD-L1 antibody. In GL261 tumor-bearing mice, this combination showed superior efficacy compared with either monotherapy. Specifically, this combination not only increased the median survival but also generated immune memory, which allowed long-term survival and thus tumor rejection on rechallenge.ConclusionsIn summary, our data demonstrated the efficacy of Delta-24-ACT combined with a PD-L1 inhibitor in murine glioma models. Moreover, the data underscore the potential to combine local immunovirotherapy with ICIs as an effective therapy for poorly infiltrated tumors.

scholarly journals Disulfiram and copper combination therapy targets NPL4, cancer stem cells and extends survival in a medulloblastoma model

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0251957
Author(s):  
Riccardo Serra ◽  
Tianna Zhao ◽  
Sakibul Huq ◽  
Noah Leviton Gorelick ◽  
Joshua Casaos ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Cancer Stem Cells ◽  
Western Blotting ◽  
Molecular Mechanisms ◽  
Protein Localization ◽  
Mortality And Morbidity ◽  
Group 3

Background Medulloblastoma (MB) is the most common brain malignancy in children, and is still responsible for significant mortality and morbidity. The aim of this study was to assess the safety and efficacy of Disulfiram (DSF), an FDA-approved inhibitor of Aldehyde-Dehydrogenase (ALDH), and Copper (Cu++) in human SSH-driven and Group 3 MB. The molecular mechanisms, effect on cancer-stem-cells (CSC) and DNA damage were investigated in xenograft models. Methods The cytotoxic and anti-CSC effects of DSF/Cu++ were evaluated with clonogenic assays, flow-cytometry, immunofluorescence, western-blotting. ONS76, UW228 (SHH-driven with Tp53m), D425med, D283 and D341 (Group 3) cell-lines were used. In vivo survival and nuclear protein localization protein-4 (NPL4), Ki67, Cleaved-Caspase-3, GFAP and NeuN expression were assessed in two Group 3 MB xenografts with immunohistochemistry and western-blotting. Results Significant in vitro cytotoxicity was demonstrated at nanomolar concentrations. DSF/Cu++ induced cell-death through NPL4 accumulation in cell-nucleus and buildup of poly-ubiquitylated proteins. Flow-cytometry demonstrated a significant decrease in ALDH+, Nestin+ and CD133+ following treatment, anti-CSC effect was confirmed in vitro and in vivo. DSF/Cu++ prolonged survival, and increased nuclear NPL4 expression in vivo. Conclusions Our data suggest that this combination may serve as a novel treatment, as monotherapy or in combination with existing therapies, for aggressive subtypes of pediatric MB.

scholarly journals Autophagy in T cells from aged donors is maintained by spermidine, and correlates with function and vaccine responses

2020 ◽  
Author(s):  
Ghada Alsaleh ◽  
Isabel Panse ◽  
Leo Swadling ◽  
Hanlin Zhang ◽  
Alain Meyer ◽  
...  
Keyword(s):  
Older Adults ◽  
T Cells ◽  
Molecular Mechanisms ◽  
Cell Function ◽  
Degradation Pathway ◽  
Immune Memory ◽  
Vaccine Responses ◽  
Human T Cells

AbstractOlder adults are at high risk for infectious diseases such as the recent COVID-19 and vaccination seems to be the only long-term solution to the pandemic. While most vaccines are less efficacious in older adults, little is known about the molecular mechanisms that underpin this. Autophagy, a major degradation pathway and one of the few processes known to prevent aging, is critical for the maintenance of immune memory in mice. Here, we show induction of autophagy is specifically induced in human vaccine-induced antigen-specific T cells in vivo. Reduced IFNγ secretion by vaccine-induced T cells in older vaccinees correlates with low autophagy. We demonstrate in human cohorts that levels of the endogenous autophagy-inducing metabolite spermidine, fall with age and supplementing it in vitro recovers autophagy and T cell function. Finally, our data show that endogenous spermidine maintains autophagy via the translation factor eIF5A and transcription factor TFEB. With these findings we have uncovered novel targets and biomarkers for the development of anti-aging drugs for human T cells, providing evidence for the use of spermidine in improving vaccine immunogenicity in the aged human population.

461 Improving specific targeting of tumors through bispecific SNIPER antibodies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A489-A489
Author(s):  
Amy Erbe ◽  
Daniel Gerhardt ◽  
Reinier Hernandez ◽  
Bonnie Hammer ◽  
Mildred Felder ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tumor Antigens ◽  
Nerve Cells ◽  
External Beam Radiation ◽  
Immune Memory ◽  
Human Neuroblastoma ◽  
Beam Radiation ◽  
Multiple Tumor ◽  
Normal Tissues

BackgroundDisialoganglioside 2 (GD2) is expressed on neuroblastomas as well as melanomas, small cell lung cancers, and sarcomas. Anti-GD2 mAb (Dinutuximab) can be used to treat these cancers and is part of the standard care for neuroblastoma. While GD2 is expressed minimally on most normal tissues, it is expressed on some nerve cells, and anti-GD2 treatment can cause neuropathic pain. A separate tumor-antigen, B7H3, is overexpressed on multiple tumor types, including those listed above, with minimal expression on most normal cells and no expression on nerve cells. We developed a bispecific SNIPER antibody, INV721, to simultaneously target these 2 tumor antigens, with one arm specific to GD2 and the other arm to B7H3. The individual Fab arms targeting GD2 and B7H3 are each low to moderate affinity, such that INV721 will only bind with high affinity when both arms bind to their antigens on the same cell, resulting in high-specificity of the SNIPER to tumor cells.MethodsINV721 binding to GD2/B7H3-expressing tumors was confirmed by flow cytometry, as well as in tumor-bearing mice injected with 89Zr-labeled to monitor in vivo biodistribution via positron emission tomography imaging. Antibody-dependent cellular cytotoxicity (ADCC) testing of INV721 was performed on human neuroblastoma and melanoma cell lines with an Incucyte spheroid-killing-assay. In vivo efficacy studies were carried out in mice bearing GD2/B7H3-expressing melanoma tumors to test our in situ vaccine (ISV) regimen, which included testing combinations of external beam radiation therapy (RT, 12Gy) ± INV721 (40 ug/dose) ± IL2 (75K U/dose).ResultsINV721 showed binding by flow cytometry to tumors that express both GD2 and B7H3 but minimal binding to cells that don’t express both antigens. 89Zr-INV721 showed elevated and persistent accumulation in the tumor with minimal uptake in normal tissues. Incucyte spheroid-killing assays revealed that INV721 was capable of ADCC. The ISV combination of RT+INV721+IL2 was capable of curing mice bearing ~57 mm3 melanoma tumors (12/12 mice tumor free), with >70% of these mice exhibiting long-term immune memory.ConclusionsINV721 binds to cells that express both GD2 and B7H3, and these preliminary studies show that INV721 is effective in our ISV regimen at curing mice bearing tumors that express these antigens. We are continuing our efforts to determine if INV721 is associated with less pain than Dinutuximab. The goal of this SNIPER-antibody is to enhance the tumor-specific delivery of therapeutic mAbs, which may decrease toxicity and improve efficacy for cancers expressing both GD2 and B7H3.

Sign in / Sign up

Export Citation Format

Share Document