scholarly journals Administration of FVIII-Expressing Human Placental Cells to Juvenile Sheep Yields Multi-Organ Engraftment, Therapeutic Plasma FVIII Levels and Alter Immune Signaling Pathways to Evade FVIII Inhibitor Induction

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3966-3966
Author(s):  
Brady Trevisan ◽  
Martin Rodriguez ◽  
Jacqueline Dizon ◽  
Sunil George ◽  
Jordan E Shields ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Differentiation ◽  
T Cell ◽  
Lymph Nodes ◽  
Signaling Pathways ◽  
Proinflammatory Cytokine ◽  
T Cell Differentiation ◽  
Publicly Traded ◽  
The Past ◽  
Inhibitory Antibodies

Abstract We have previously reported that normal juvenile sheep that received weekly intravenous (IV) infusions of human (n=3) or an expression/secretion-optimized, bioengineered human/porcine hybrid (ET3) FVIII protein (n=3) for 5 weeks (20 IU/kg) developed anti-FVIII inhibitory antibodies (10-116 BU, and IgG titers of 1:20-1:245) by week 3 of infusion. By contrast, the IV infusion, or IP administration, of human placental mesenchymal cells (PLC) transduced with a lentiviral vector encoding a myeloid codon-optimized ET3 transgene (PLC-mcoET3) to produce high levels of ET3 protein (4.9-6IU/10^6 cells/24h) enabled the delivery of FVIII without eliciting antibodies, despite using PLC-mcoET3 doses that provided ~20-60 IU/kg ET3 each 24h to mirror the amount of FVIII protein infused. In addition, we showed that the route of PLC-mcoET3 administration (IP vs IV) did not impact the resultant plasma FVIII levels, with animals in these two groups exhibiting mean increases in FVIII activity (quantified by aPTT) of 30.9% and 34.2%, respectively, at week 15 post-treatment. Here, we investigated whether the sites and levels of PLC-mcoET3 engraftment were dependent upon the route of administration and performed s sheep-specific multiplexed transcriptomic analysis (NanoString) to define the immune signaling pathways that thwarted FVIII/ET3 protein immune response when ET3 was delivered through PLC. Tissue samples were collected from various organs at euthanasia and RT-qPCR performed using primers specific to the mcoET3 transgene, to the human housekeeping transcript GAPDH, and to sheep GAPDH, to quantify PLC-mcoET3 tissue engraftment, and normalize the results. RT-qPCR demonstrated PLC-mcoET3 engrafted, in both IP and IV groups, in all the organs evaluated (liver, lung, lymph nodes, thymus, and spleen). Animals that received PLC-mcoET3 via the IP route displayed higher overall levels of engraftment than their IV counterparts. The spleen was the preferential organ of engraftment for both IP and IV groups (IP:2.41±1.97%; IV: 0.64±0.54%). The IP group exhibited significantly higher engraftment in the left lobe of the liver (IP: 1.36±0.35%; IV: 0.041±0.022%), which was confirmed by immunohisto-chemistry (IHC) with an antibody to the human nuclear antigen Ku80 and ImageJ analysis (IP:5.24±3.36%; IV: 0±0). Of note is that the IP route resulted in higher levels of engraftment in the thymus, while IV infusion yielded higher levels of PLC-mcoET3 in lymph nodes. Analysis of H&E-stained tissues demonstrated they were devoid of any abnormal histologic changes and exhibited no evidence of hyperplasia or neoplasia, supporting the safety of the cell platform, irrespective of the route of administration. To date, NanoString analysis of PBMC collected at day 0, week 1, and week 5 post-infusion demonstrated that animals who received FVIII protein had upregulation of UBA5 and BATF, genes involved in antigen processing and Th17 signaling pathways, respectively. Although both IV and IP recipients of PLC-mcoET3 also had an increase in BATF, the IV group exhibited upregulation of BTLA, a gene involved in immune-tolerance, and downregulation of NOTCH and DDL1, involved in T cell differentiation, as well as MAPK12 and PLCG1, genes involved in proinflammatory cytokine regulation and T signaling within the Th17 signature. In IP recipients, BTLA, NOTCH, and DLL1 were all downregulated. Since ET3-reactive Th 1 cells were not present in any of the treated animals, it is possible that the Th17 cells are responsible for the inhibitory antibodies seen in the juvenile sheep treated with FVIII/ET3 protein, while in animals receiving PLC-mcoET3, downregulation of genes involved in T cell differentiation and proinflammatory cytokine signaling keeps the immune system in check to avoid an immune response. Disclosures Doering: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months. Spencer: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months.

scholarly journals The NAD-dependent deacetylase SIRT2 regulates T cell differentiation involved in tumor immune response

2020 ◽  
Vol 16 (15) ◽  
pp. 3075-3084
Author(s):  
Cui Jiang ◽  
Jingwei Liu ◽  
Min Guo ◽  
Xiaoxin Gao ◽  
Xuan Wu ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Differentiation ◽  
T Cell ◽  
T Cell Differentiation

scholarly journals 2-Methoxyestradiol Alleviates Experimental Autoimmune Uveitis by Inhibiting Lymphocytes Proliferation and T Cell Differentiation

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Linxinyu Xu ◽  
Tianshu Yang ◽  
Shaobo Su ◽  
Fang Wang
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
Lymph Nodes ◽  
Lymphocyte Proliferation ◽  
T Cell Differentiation ◽  
Control Group ◽  
Experimental Autoimmune Uveitis ◽  
Autoimmune Uveitis ◽  
Draining Lymph Nodes ◽  
Experimental Autoimmune

Purpose. To investigate the effect of 2-Methoxyestradiol (2ME2) on experimental autoimmune uveitis (EAU) and the mechanism.Method. C57BL/6 male mice were used to establish the EAU model. 2ME2 was abdominal administrated in D0–D13, D0–D6, and D7–D13 and control group was given vehicle from D0–D13. At D14, pathological severity was scored. Lymphocyte reaction was measured using MTT assay. T cell differentiation in draining lymph nodes and eye-infiltrating cells was tested by flow cytometry. Proinflammatory cytokines production from lymphocytes was determined by ELISA.Result. The disease scores from 2ME2 D0–D13, 2ME2 D0–D6, 2ME2 D7–D13, and vehicle groups were0.20±0.12,1.42±0.24,2.25±0.32, and2.42±0.24. Cells from all 2ME2 treated groups responded weaker than control (p<0.05). The inhibitory effect of 2ME2 on lymphocyte proliferation attenuated from 2ME2 D0–D13 to 2ME2 D0–D6 and to 2ME2 D7–D13 groups (p<0.05). 2ME2 treated mice developed fewer Th1 and Th17 cells both in draining lymph nodes and in eyes than control (p<0.05). Lymphocytes from 2ME2 group secreted less IFN-γand IL-17A than those from control (p<0.05).Conclusion. 2ME2 ameliorated EAU progression and presented a better effect at priming phase. The possible mechanism could be the inhibitory impact on IRBP specific lymphocyte proliferation and Th1 and Th17 cell differentiation.

scholarly journals In Utero Transplantation of FVIII-Expressing Human Placental Cells Upregulates Gene Pathways Associated with Immune Tolerance, Altering the Response to Postnatal FVIII Infusion

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1863-1863
Author(s):  
Martin Rodriguez ◽  
Brady Trevisan ◽  
Sunil George ◽  
Jordan E Shields ◽  
Jorge Figueroa ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Signaling ◽  
Immune Tolerance ◽  
In Utero ◽  
Publicly Traded ◽  
The Past ◽  
In Utero Transplantation ◽  
Placental Cells ◽  
Inhibitory Antibodies ◽  
Human Placental Cells

Abstract We have previously reported that in utero transplantation (IUTx) of sheep fetuses (n=14) with human placental cells (PLC) transduced with a lentiviral vector encoding mcoET3, an expression/secretion-optimized, bioengineered fVIII transgene (PLC-mcoET3) increased plasma FVIII activity levels by 57%, 42%, and 35% at 1, 2, and 3 years post-IUTx, respectively, without the development of FVIII/ET3 inhibitors. We also demonstrated that immune tolerance to the cell/gene product was maintained after postnatal administration of PLC-mcoET3 (cells producing 20 IU/kg/24h were administered i.v. for 3 consecutive weeks). However, when IUTx-treated animals received weekly i.v. infusions of purified ET3 protein (20IU/kg) for 5 weeks, all recipients developed a robust ET3-specific IgG response that appeared at week 3 of infusion at titers ranging from 1:70 to 1:857 and inhibitory antibodies that ranged from 3-36 BU. Here, we investigated differences in the immune responses of animals that received IUTx with PLC-mcoET3 and were boosted postnatally with PLC-mcoET3 (IUTx-PLC-mcoET3) vs. ET3 protein (IUTx-ET3) to define the pathways by which the immune system differentially responds to protein vs. cell-secreted ET3. A sheep-specific multiplex gene expression analysis with 165 genes involved in immune cell signaling pathways (NanoString) was used to evaluate mRNA isolated from peripheral blood mononuclear cells collected at Weeks (W) 0, 1, and 5 of postnatal infusions. Significant fold-change expression in these mRNA targets was determined using NanoString nSolver 4.0 software. Animals in the IUTx-PLC-mcoET3 group (known to be devoid of inhibitors to ET3 post-boosting) showed that immunoregulation and immune tolerance gene clusters were among the top three clusters that increased expression from W0 to W5 (adj. p-value&lt;0.01). Differential expression of genes in pathways involved in Th1, Th2, and Th17 responses was also found, at differing levels, in the IUTx-PLC group, suggesting a balance between immunity and tolerance was maintained. Surprisingly, the IUTx-ET3 group, which developed inhibitory antibodies after ET3 boosting, also showed significantly increased expression of immune tolerance genes, and downregulation of Th1 and Th17 cell signaling, when evaluated by direct global significance score. Nevertheless, 66% of these animals had a significant upregulation of Th2 cell signaling by W1 vs W0. To determine if the increase in expression of immune tolerance genes was due to the IUTx treatment, we also evaluated a group of aged-matched, non-transplanted sheep that received ET3 protein under the same dose and schedule. Results from Gene Set Analysis (GSA) demonstrated significant upregulation of genes involved in interferon signaling, class I MHC antigen processing, and Th17 signaling in these animals, suggesting the potential involvement of Th17 cells in the immune response in this group. In conclusion, IUTx with PLC-mcoET3 induces the upregulation of genes associated with immune tolerance, providing an explanation for the long-lasting elevation in plasma FVIII levels in these animals in the absence of inhibitors. Nevertheless, despite the continued expression of tolerogenic genes, administration of purified ET3 protein to these IUTx recipients induced upregulation of Th2 signaling, a pathway that was not observed in animals that only received ET3 protein, demonstrating that the mechanism by which tolerance is broken in IUTx recipients differs from that by which an immune response to ET3 occurs in animals with no prior exposure. Of note is that animals that develop inhibitors by the Th17 pathway had considerably higher inhibitor titers than the IUTx recipients that responded to ET3 infusion by the Th2 pathway. These studies underscore the need for a more complete understanding of the mechanisms by which immune tolerance to FVIII develops during ontogeny. Disclosures Doering: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months. Spencer: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months.

Electron microscope study of the secretory activity of epithelial cells in embryonic thymus

1990 ◽  
Vol 48 (3) ◽  
pp. 382-383
Author(s):  
H. Alasam
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
Epithelial Cells ◽  
Electron Density ◽  
Secretory Activity ◽  
T Cell Differentiation ◽  
High Electron ◽  
Transmission Electron ◽  
Medullary Epithelial Cells ◽  
Thymic Factor

The possibility that intrathymic T-cell differentiation involves stem cell-lymphoid interactions in embryos led us to study the ultrastructure of epithelial cell in normal embryonic thymus. Studies in adult thymus showed that it produces several peptides that induce T-cell differentiation. Several of them have been chemically characterized, such as thymosin α 1, thymopoietin, thymic humoral factor or the serum thymic factor. It was suggested that most of these factors are secreted by populations of A and B-epithelial cells.Embryonic materials were obtained from inbred matings of Swiss Albino mice. Thymuses were disected from embryos 17 days old and prepared for transmission electron microscopy. Our studies showed that embryonic thymus at this stage contains undifferentiated and differentiated epithelial cells, large lymphoblasts, medium and few small lymphocytes (Fig. 5). No differences were found between cortical and medullary epithelial cells, in contrast to the findings of Van Vliet et al,. Epithelial cells were mostly of the A-type with low electron density in both cytoplasm and nucleus. However few B-type with high electron density were also found (Fig. 7).

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