scholarly journals Carvedilol, a new antioxidative β-blocker, blocks in vitro human peripheral blood T cell activation by downregulating NF-κB activity

2003 ◽  
Vol 59 (3) ◽  
pp. 776-787 ◽  
Author(s):  
S Yang
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Peripheral Blood ◽  
Cell Activation ◽  
Human Peripheral Blood ◽  
Β Blocker

PBMC-07- Stimulation of human peripheral blood mononuclear cells with immobilized anti-CD3 and soluble anti-CD28 antibodies. v1

2021 ◽  
Author(s):  
Alessia Furgiuele ◽  
Massimilano Legnaro ◽  
Alessandra Luini ◽  
Marco Ferrari ◽  
Emanuela Rasini ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Functional Responses ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

This protocol was designed to activate the lymphocytes T of a population of peripheral blood mononuclear cells (PBMCs), simulating their physiological response to antigen/MHC complex acting on T Cell Receptors-TCR , in order to test their functional responses including cell proliferation and cytokine production. The co-stimulation protocol include: i)anti-CD3 antibody a polyclonal activator specific for invariant framework epitopes on TCR complex (in particular, we use UCHT1 clone an anti-human CD3 antibody that recognizes the ε-chain of CD3 which is used for immobilized option of activation) (http://static.bdbiosciences.com/documents/BD_Tcell_Human_CD3_Activation_Protocol.pdf) ii) anti-CD28 antibody used to cooperate with TCR signals promoting activation of T cells The procedure has been reproduced following the indications contained in the protocol of "EBiooscience" (https://tools.thermofisher.com/content/sfs/manuals/t-cell-activation-in-vitro.pdf). Pilot experiments on PBMC were carried out to determine the best concentrations of anti-CD3 and anti-CD28 to induce optimal proliferation of PBMC and production of cytokines TNF-α and IFN-γ. We found a dose dependent correlation between immobilized anti-CD3 and cells functional responses. The selected amount was 2 µg/mL for both anti-CD3 and anti-CD28 that was the concentration below the maximum response which allows also to test possible modulations by therapeutic agents. References http://static.bdbiosciences.com/documents/BD_Tcell_Human_CD3_Activation_Protocol.pdf https://tools.thermofisher.com/content/sfs/manuals/t-cell-activation-in-vitro.pdf https://www.bdbiosciences.com/ds/pm/tds/555330.pdf https://www.bdbiosciences.com/ds/pm/tds/555726.pdf BEFORE STARTING with this procedure Moreover, work under laminar flow hood when you are processing samples from the beginning to the end of the culture. Make sure you are using,sterile culture mediumand sterile plastic disposable as well.

Effect of CD28 signal transduction on c-Rel in human peripheral blood T cells

1994 ◽  
Vol 14 (12) ◽  
pp. 7933-7942
Author(s):  
R G Bryan ◽  
Y Li ◽  
J H Lai ◽  
M Van ◽  
N R Rice ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Peripheral Blood ◽  
Cell Activation ◽  
Nuclear Translocation ◽  
Human Peripheral Blood ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Cd28 Costimulation

Optimal T-cell activation requires both an antigen-specific signal delivered through the T-cell receptor and a costimulatory signal which can be delivered through the CD28 molecule. CD28 costimulation induces the expression of multiple lymphokines, including interleukin 2 (IL-2). Because the c-Rel transcription factor bound to and activated the CD28 response element within the IL-2 promoter, we focused our study on the mechanism of CD28-mediated regulation of c-Rel in human peripheral blood T cells. We showed that CD28 costimulation accelerated the kinetics of nuclear translocation of c-Rel (and its phosphorylated form), p50 (NFKB1), and p65 (RelA). The enhanced nuclear translocation of c-Rel correlated with the stimulation of Il-2 production and T-cell proliferation by several distinct anti-CD28 monoclonal antibodies. This is explained at least in part by the long-term downregulation of I kappa B alpha following CD28 signalling as opposed to phorbol myristate acetate alone. Furthermore, we showed that the c-Rel-containing CD28-responsive complex is enhanced by, but not specific to, CD28 costimulation. Our results indicate that c-Rel is one of the transcription factors targeted by CD28 signalling.

DAP10 Costimulates Chimeric Receptor-Mediated T Cell Responses to Tumor Antigen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3052-3052
Author(s):  
Bianca Altvater ◽  
Sibylle Pscherer ◽  
Heribert Juergens ◽  
Claudia Rossig
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Adoptive Immunotherapy ◽  
Peripheral Blood ◽  
Cell Activation ◽  
Human Peripheral Blood ◽  
Cytokine Secretion ◽  
Immunotherapy Of Cancer

Abstract Chimeric receptors (chRecs) combining extracellular recognition domains with the T cell receptor ζ an redirect the cellular immune response of primary T-cells to tumor cells. T cell activation by chRec induces efficient cytokine release and cytotoxicity, however, it fails to mediate proliferative responses, limiting the usefulness of chRec-gene-modified T cells for adoptive immunotherapy of cancer. Inclusion of a CD28 costimulatory signaling component in the chRec endodomain enhances antigen-specific proliferation. Whereas the signal mediated by ligation of CD28 is of crucial importance for the activation of resting CD4+ T cells, further molecules with costimulatory functions have contributory roles. NKG2D is a stimulatory receptor that was first identified in NK cells, but is also expressed in cytotoxic T cells and positively modulates CD8+ T cell immune responses. We hypothesized that inclusion of the NKG2D-associated signaling domain DAP10 would enhance the capacity of chRecs to induce tumor-specific activation and proliferation of in vitro expanded effector T cells. Based on a GD2-specific scFv, we generated chRecs containing either the DAP10 signaling chain alone (14.G2a-DAP10) or combined with TCRζ 14.G2a-DAP10ζ), and expressed them in nonspecifically activated human peripheral blood T cells of three individual donors by retroviral gene transfer. As controls, T cells were transduced with 14.G2a-ζ and -CD28ζ chRec. High chRec surface expression was obtained with all four constructs (55±11%, ζ; 85±3, CD28ζ; 68±5%, DAP10; 78±1%; DAP10ζ). Immunophenotypes were dominated by a CD3+CD8+ population in all cell cultures. Whereas DAP10 alone failed to mediate specific tumor cell lysis, 51Cr release assays revealed efficient and comparable lysis of GD2+ tumor targets by T cells transduced with all ζ-containing constructs, with 49±8% (ζ), 52±7% (CD28ζ), and 52±18% (DAP10ζ) cytolysis at an effector-to-target ratio of 40:1. Intracellular cytokine secretion by chRec+ T cells was induced in response to tumor targets by 14.G2a-ζ (up to 37% IFN-γ secreting cells), CD28ζ, and DAPζ (both up to 22%), but not by DAP10 alone (0,2%). Weekly stimulation with tumor cells for 6 weeks induced only limited expansion of T cells transduced with 14.G2a-ζ (7–45fold) or with 14.G2a-DAP10 (14–26-fold). Adding CD28 or DAP10 domains significantly enhanced expansion by a comparable degree (270–483-fold and 126–436-fold, respectively). Thus, while neither CD28 nor DAP10 enhances antigen-specific cytokine secretion and cytolysis, DAP10 signaling can completely replace CD28 signaling in costimulating antigen-specific proliferation of peripheral blood T cells. DAP10-containing chRec may be a powerful new tool for adoptive immunotherapy of cancer.

scholarly journals Effect of CD28 signal transduction on c-Rel in human peripheral blood T cells.

1994 ◽  
Vol 14 (12) ◽  
pp. 7933-7942 ◽  
Author(s):  
R G Bryan ◽  
Y Li ◽  
J H Lai ◽  
M Van ◽  
N R Rice ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Peripheral Blood ◽  
Cell Activation ◽  
Nuclear Translocation ◽  
Human Peripheral Blood ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Cd28 Costimulation

Optimal T-cell activation requires both an antigen-specific signal delivered through the T-cell receptor and a costimulatory signal which can be delivered through the CD28 molecule. CD28 costimulation induces the expression of multiple lymphokines, including interleukin 2 (IL-2). Because the c-Rel transcription factor bound to and activated the CD28 response element within the IL-2 promoter, we focused our study on the mechanism of CD28-mediated regulation of c-Rel in human peripheral blood T cells. We showed that CD28 costimulation accelerated the kinetics of nuclear translocation of c-Rel (and its phosphorylated form), p50 (NFKB1), and p65 (RelA). The enhanced nuclear translocation of c-Rel correlated with the stimulation of Il-2 production and T-cell proliferation by several distinct anti-CD28 monoclonal antibodies. This is explained at least in part by the long-term downregulation of I kappa B alpha following CD28 signalling as opposed to phorbol myristate acetate alone. Furthermore, we showed that the c-Rel-containing CD28-responsive complex is enhanced by, but not specific to, CD28 costimulation. Our results indicate that c-Rel is one of the transcription factors targeted by CD28 signalling.

P155 THERAPEUTIC EFFECT OF OPS-2071 ON HEALING OF T CELL-MEDIATED COLITIS, A MURINE MODEL OF CROHN’S DISEASE, WITH ANTI-INFLAMMATORY AND ANTIBACTERIAL ACTIVITY

2020 ◽  
Vol 26 (Supplement_1) ◽  
pp. S7-S8
Author(s):  
Hiroko Takagi ◽  
Masayoshi Sato ◽  
Kazuyuki Fujii ◽  
Isao Shibuya ◽  
Daisuke Oka ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytokine Production ◽  
Human Peripheral Blood ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Inflammatory Index

Abstract Background Crohn’s disease (CD) is a form of chronic inflammatory bowel disease (IBD). Although CD is an immune-mediated condition of unknown etiology, many studies suggested that abnormal immune responses against certain intestinal bacteria may trigger development of chronic inflammation. The T cell-mediated colitis model is a well characterized adaptive transfer murine model of chronic small bowel and colonic inflammation that resembles human CD (e.g., diarrhea, a heavily inflamed colon, loss of mucus from goblet cells, Th1/Th17 dominated cytokine profile). OPS-2071, a novel agent synthesized by Otsuka Pharmaceutical Co., Ltd, has broad and strong antibacterial activity with low systemic absorption. In this study, we have evaluated the therapeutic effect of OPS-2071 on murine T cell-mediated colitis and in vitro activity on T cell activation, cytokine production and antibacterial activity. Methods T cell-mediated colitis was induced via naive T cell (CD4+CD62L+CD44-) injection into T and B cell deficient recipient mice. Two weeks following T cell transfer, OPS-2071 or vehicle solution was administered orally for 3 weeks, followed by harvesting of colon tissue and assessment of efficacy evaluation by histological score and inflammatory index (colon weight/ length). In vitro inhibitory action for TNF-α production was assayed by ELISA using LPS-stimulated human peripheral blood cells. The effect on T cell activation and cytokine production (TNF-α, IFN-γ), was examined using human peripheral blood mononuclear cells and, mouse spleen which were stimulated by anti-CD2/CD3/CD28 antibody-loaded beads. In vitro activity against bacteria considered to be the cause of IBD was also tested. Results In the murine T cell transfer model, OPS-2071 significantly reduced both histological score (control: 9.2, OPS: 2.7, p<0.001) and inflammatory index (control: 54.7±2.2 mg/cm, OPS: 27.8±1.7 mg/cm, p<0.001) at a dose of 10 mg/kg. In the in vitro experiments, OPS-2071 suppressed TNF-a production produced by LPS in human whole blood dose-dependently. OPS-2071 also suppressed human and mouse T cell activation and cytokine production and suppressed T cell proliferation. At high OPS-2071 concentrations, these effects were comparable to prednisolone. The Minimum Inhibitory Concentration against IBD related bacteria for OPS-2071, ciprofloxacin, and metronidazole were 0.015 - 0.5, 0.25 - 8, and 0.03 - >128 mg/mL, respectively. Conclusion OPS-2071 demonstrated significant therapeutic effects on colon inflammation in the murine T cell-medicated colitis model, suppressed TNF-a production in vitro and showed in vitro activity against bacteria related to CD. The dual effect of anti-inflammatory effects and antibacterial activity of the novel agent OPS-2071 demonstrated in this study, provide rationale for exploring the impact of this compound on human CD in clinical trials.

scholarly journals P031 Therapeutic effect of OPS-2071 in a murine model of Crohn’s disease and in in vitro anti-inflammatory assays

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S147-S147
Author(s):  
H Takagi ◽  
M Sato ◽  
F Kazuyuki ◽  
I Shibuya ◽  
D Oka ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Human Peripheral Blood ◽  
In Vitro Activity ◽  
Inflammatory Index ◽  
Histological Score ◽  
Tnf Α

Abstract Background OPS-2071 is a novel fluoroquinolone that is currently in Phase 2 clinical trials for the evaluation of Crohn’s disease (CD) as an add-on therapy to standard of care. OPS-2071 has broad and potent antibacterial activity with low systemic absorption. It was previously investigated for the treatment of enteric infections, including Clostridium difficile. CD is an immune-mediated condition of unknown aetiology; however, studies suggest that an abnormal immune response against certain intestinal bacteria may trigger the development of chronic inflammation. In this study, we have evaluated the effect of OPS-2071 on both the murine T-cell-mediated colitis as well as its in vitro effect on the activity of immune cells. Methods Transfer of naïve T cells (CD4+CD62L+CD44−) into immunodeficiency mice induces autoimmune colitis. Two weeks following T-cell transfer, OPS-2071, sulfasalazine and anti-IL12/23 p40 antibody were administered for 3 weeks, followed by assessment of efficacy evaluation by histological score and inflammatory index. In vitro inhibitory action for TNF-α production was assayed using LPS-stimulated human peripheral blood cells and THP-1 macrophages. The effect on T-cell activation and cytokine production (TNF-α, IFN-γ) was examined using anti-CD2/CD3/CD28 antibody-loaded beads-stimulated human peripheral blood mononuclear cells. In vitro activity against bacteria considered to be a potential cause of IBD such as M. avium sub. paratuberculosis, Klebsiella pneumonia, etc., was also tested. Results OPS-2071 and anti-IL12/23 p40 antibody significantly reduced both histological score (control: 9.0, OPS: 4.7, anti-IL12: 4.3, p < 0.01) and inflammatory index (control: 58.3 ± 3.7 mg/cm, OPS: 25.9 ± 1.4 mg/cm, anti-IL12: 41.1 ± 2.4 mg/cm, p < 0.01) at a dose of 10 and 25 mg/kg, respectively. OPS-2071 suppressed TNF-α production in T cells, as well as macrophage activity in a dose-dependent fashion. OPS-2071 also suppressed human T-cell activation and proliferation. At high OPS-2071 concentrations, these effects were comparable to prednisolone. The minimum inhibitory concentration against IBD-related bacteria for: OPS-2071, ciprofloxacin, and metronidazole were 0.015–0.5, 0.25–8 and 0.03–>128 mg/ml, respectively. Conclusion OPS-2071 demonstrated significant therapeutic effect on colonic inflammation, suppressed TNF-α production in vitro and showed in vitro activity against bacteria related to CD. From these in vitro results, it can be concluded that OPS-2071 has anti-inflammatory activity, independent of its antibacterial activity. The dual effects demonstrated by this novel agent, OPS-2071, provided a rationale for exploring the impact of this compound on human CD in clinical trials.

scholarly journals Anti-CD6 Monoclonal Antibody Itolizumab Efficiently Inhibits T Cell Proliferation after in Vitro TCR Stimulation in the Setting of Acute Graft Versus Host Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4517-4517 ◽  
Author(s):  
Benedetta Rambaldi ◽  
Carol Reynolds ◽  
Sharmila Chamling Rai ◽  
Takeru Asano ◽  
Yohei Arihara ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Cell Activation ◽  
T Cell Proliferation

CD6 is a co-stimulatory receptor expressed on T cells that binds activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presenting cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation and trafficking and is central to inflammation. Early studies by Soiffer et al. demonstrated that ex vivo depletion of CD6+ donor cells prior to hematopoietic cell transplantation (HCT) decreased the incidence of acute graft versus host disease (aGVHD), highlighting the importance of CD6+ cells in GVHD pathogenesis. Itolizumab, a humanized anti-CD6 monoclonal antibody, has been shown to modulate T cell activation and proliferation. The aim of this study was to characterize: (1) expression of CD6 and ALCAM, and (2) activity of itolizumab on T cell responses in peripheral blood from HCT patients pre- and post-aGvHD. We analyzed immune reconstitution in 31 adult patients who underwent HLA matched donor HCT for hematological malignancies. Patients received peripheral blood stem cell grafts and GVHD prophylaxis with tacrolimus and methotrexate. Twelve of 31 patients developed aGVHD at a median of 58 days, range 27-208, after HCT and systemic treatment was started in 83% of these cases. aGVHD grade severity was 25%, 58.3% and 16.7% of grade I, II and IV, respectively. Patient samples were collected at 1, 2 and 3 months after HCT and analyzed using multi-color flow cytometry. Nine healthy donors (HD) were analyzed as controls. Suppressive activity of itolizumab was tested using peripheral blood mononuclear cells (PBMC) obtained from HD and patients before (preGVHD) and after (postGVHD) aGvHD onset (within 30 days). PBMC were stimulated with antiCD3/CD2/CD28 coated beads in the presence of itolizumab or isotype control (cetuximab) for 72 hours. T cell proliferation was measured by CFSE dilution, while T cell activation and maturation was measured by expression of CD25 and CD45RO, respectively. For statistical analysis, non-parametric unpaired (Mann-Whitney) or paired (Wilcoxon matched-pairs signed rank) test were used. CD6+ T cells reconstituted early after transplant, accounting for 95% of positive CD3 T cells, range 57-100 at 1 month. Similar to HD PBMC, in the first 3 months after HCT, CD4 Tcon had the highest CD6 expression, while CD4 Treg had a lower CD6 expression compared to both CD4 Tcon and CD8 T cells (Fig 1A and 1B). To characterize the expression of CD6 on different T cell subsets, we used a t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm and visualized the data using a viSNE map (Fig 1C). Within the Tcon compartment, there were no differences in expression of CD6 between HD and patients at all 3 time points. Within CD4 Treg and CD8 T cells, CD6 expression was reduced in naïve CD8 T cells and CM Treg after transplant compared to HD. In HD, ALCAM expression was detected in 35% of CD14+ monocytes, 23% of CD19+ B cells, 20% of myeloid (CD11c+ CD123-) DCs and 97% of plasmacytoid (CD11c-CD123+) DCs. After HCT, expression of ALCAM in DC compartments was similar to HD. In functional studies, itolizumab inhibited CD4 and CD8 T cell proliferation in preGVHD samples, similar to HD controls. This effect was less prominent in samples collected from patients who had developed GVHD and were already receiving immunosuppressive medications, potentially confounding the ability to assess the effect of itolizumab in this assay (Fig 2A). Similar results were observed for CD25 (Fig 2B) and CD45RO (Fig 2C) expression pre- and post-aGVHD. Finally, itolizumab did not increase rates of cell death in samples from HCT patients as assessed by Annexin V expression, suggesting that itolizumab-mediated T cell inhibition was not due to increased T cell apoptosis. There was a slight increase in Annexin V expression in HD vs isotype control (21%, range 10-43 vs 15%, range 11-31, p= 0.0273). In conclusion, we demonstrate for the first time that CD6+ T cells reconstitute rapidly in peripheral blood after HCT and that CD6 expression is highest in Tcon while lowest in Treg (Tcon>CD8>Treg). Itolizumab efficiently inhibits T cell proliferation and activation after in vitro TCR stimulation of PBMC from aGvHD patients, thus representing a potential therapeutic for treating aGvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Rambaldi: Equillium: Research Funding. Koreth:Amgen: Consultancy; Cugene: Consultancy; Equillium: Consultancy. Cutler:Pharmacyclics: Consultancy; Omeros: Consultancy; Kadmon: Consultancy; BiolineRx: Other: DSMB; Cellect: Other: DSMB; Kalytera: Other: DSMB; ElsaLys: Consultancy; Genentech: Consultancy; BMS: Consultancy; Jazz: Consultancy; Incyte: Consultancy; Fate Therapeutics: Consultancy. Nikiforow:Kite/Gilead: Honoraria; Novartis: Honoraria; NKarta: Honoraria. Ho:Omeros Corporation: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy. Soiffer:Jazz: Consultancy; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Mana therapeutic: Consultancy; Kiadis: Other: supervisory board. Ampudia:Equillium: Employment. Ng:Equillium: Employment, Equity Ownership. Connelly:Equillium: Employment, Equity Ownership. Ritz:Equillium: Research Funding; Merck: Research Funding; Kite Pharma: Research Funding; Aleta Biotherapeutics: Consultancy; Celgene: Consultancy; Avrobio: Consultancy; LifeVault Bio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy.

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