Tolerogenic dendritic cells, methods for their production and uses thereof
A dendritic cell, tolerance technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve problems such as harmful treatment results
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Embodiment 1
[0194] This example demonstrates that tolerogenic DCs generated with dexamethasone and vitamin D2 are phenotypically and functionally stable even after stimulation with a variety of biologically relevant inflammatory stimuli in the absence of tolerizing factors of.
[0195] 6.1 Materials and methods:
[0196] Reagents and Antibodies: Flow cytometry: Commercial antibodies against CD86-FITC, CD274(PD-L1)-FITC, CD273(PD-L2)-PE, HLA-DR-PE-Cy7, IFNγ-FITC were purchased from BD Biosciences; CD83-PerCP- Cy5.5 was purchased from BeckmanCoulter; CD80-FITC, CD40-PerCP-eFluor710, CD1a-PE-Cy7, CD4-PE-Cy7, FoxP3-AF488 were purchased from eBiosciences; TLR2-FITC, CD25-PerCP-Cy5.5, IL- 10-PE was purchased from BioLegend; TIM-3-PE, CD14-PE-DL594, CD11c-APC, CD3-AF700, CD8-PE-Dy590 were purchased from Exbio; CD85k(IL-T3)-PE, CD85d(IL-T4 )-FITC was purchased from R&D Systems.
[0197] For western blot, anti-p-ERK1 / 2, anti-p-JNK / SAPK, anti-p-IкB-α, anti-IDO, anti-p-mTOR, anti-p-STAT3, anti-...
Embodiment 2
[0271] This example demonstrates that tolerogenic DC (tDC) generated using dexamethasone and vitamin D2 are phenotypically and even when stimulated with various biologically relevant inflammatory stimuli in the absence of tolerizing factors functionally stable. This example further demonstrates that the stability of tDC in an inflammatory environment is regulated by various signaling pathways.
[0272] 7.1 Materials and methods:
[0273] Reagents and Antibodies:Flow cytometry: commercial antibodies against CD86-FITC (clone 2231FUN-1), CD274(PD-L1)-FITC (clone MIH1), CD273(PD-L2)-PE (clone MIH-18), HLA-DR- PE-Cy7 (clone L243), IFN-γ-FITC (clone 4SB3) was purchased from BD Biosciences; CD83-PerCP-Cy5.5 (clone HB15a) was purchased from BeckmanCoulter; CD80-FITC (clone MAB104), CD40-PerCP-eFluor710 (clone 5C3), CD1a-PE-Cy7 (clone HI149) and CD4-PE-Cy7 (clone RPA-T4) were purchased from eBioscience; TLR2-FITC (clone T2.5), TIM-3-PE (clone F38-2E2 ), IL-10-PE (clone JES3-9D7), ...
Embodiment 3
[0310] This example demonstrates that monocyte-derived tolerogenic DC (tDC) generated from type 1 diabetes (T1D) patients using dexamethasone and vitamin D2 are phenotypically and functionally stable. Furthermore, this example demonstrates that tDCs derived from patients with glycated hemoglobin (Hb) A1c ("HbA1c") levels less than or equal to 60 mmol / mol Hb induce stable hyporesponsiveness of GAD-specific T cells. This example further demonstrates the feasibility of using cryopreserved tDCs to re-establish tolerance in future clinical trials.
[0311] 8.1 Materials and methods:
[0312] Subject: Anticoagulated blood samples (50-60 ml, EDTA) were obtained from a total of 71 T1D children after signing informed consent forms / or their legal representatives (where required). None of the enrolled patients had other comorbidities other than T1D-related co-morbidities such as thyroiditis or celiac disease (15.5%, 18% of subjects enrolled). In this study, patients with different du...
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