Method for proliferation of antigen-specific T cells
A specific and antigenic technology, applied in the field of immunology and cancer therapy, can solve the problems of unpredictable in vivo efficacy and poor effect
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Embodiment 1
[0084] Materials and methods : In tissue culture flasks, gamma-irradiated PBMCs from healthy blood donors were compared with those from allogeneic donors (relative to healthy donors) at a ratio of 1:1 in serum-free X-VIVO 15 medium. Unirradiated PBMC were co-cultured for 5-7 days to generate allo-sensitized allogeneic lymphocytes (ASAL) in a standard one-way mixed lymphocyte reaction (MLR). For proliferation of immature DCs, peripheral blood mononuclear cells (PBMCs) obtained from healthy blood donors were isolated on a density gradient (Lymphoprep, Nycomed, Oslo, Norway). Separated PBMCs were resuspended on AIM-V (Invitrogen, Paisley, UK) medium, and 2.5 × 10 6 Cells were plated per well in a 24-well plastic culture plate and allowed to adhere for 2 hours. Non-adherent cells were removed, and the remaining adherent monocytes were incubated in AIM-V medium supplemented with recombinant human GM-CSF and IL-4 (R&D Systems, Abingdon, UK; both 1,000 U / mL). Cultured in medium f...
Embodiment 2
[0092] Materials and methods : ASAL was produced during conventional MLR for 7 days using irradiated allogeneic PBMCs as stimuli (see Example 1). After harvesting and irradiation, ASAL ("MLR") or depleted CD4 + , CD8 + or CD56 + Mixed ASAL populations of (NK / NKT) were co-cultured with mature allogeneic monocyte-derived DCs (autologous to the PBMCs used to elicit ASAL). Co-culture supernatants were collected after 24 hours and subsequently assayed for IL-2, IL-2 and IFN-γ production.
[0093] result : The production of IL-2 was found to be strictly CD4-dependent ( Figure 6 A), while IL-12 produces ( Figure 6 B) shows complete absence of ASAL-dependence and IFN-γ production ( Figure 6 C) Shows partial dependence on co-cultured and allogeneic challenged CD4 in the ASAL population + , CD8 + and CD56 + (NK / NKT).
Embodiment 3
[0095] Materials and methods : Generation of immature DCs by plastic-adhered monocytes. After adding 1000U / mL of IL-4 and GM-CFS Monocytes were cultured in DC for 7 days. During the last 2 days of incubation, DCs were induced to mature by adding 50 ng / mL TNF-α, 25 ng / mL IL-1β, 50 ng / mL IFN-γ, 3000 U / mL IFN-α and 20 μg / mL poly I:C.
[0096] ASAL was produced in a one-way mixed lymphocyte reaction by co-culturing gamma-irradiated PBMCs with non-irradiated autologous PBMCs relative to DC donors at a 1:1 ratio in X-VIVO 15 for 7 days.
[0097] From adding 50ng / mL IL-15 (final concentration is 0.5×10 6 Lymphocytes / mL) CD8 was isolated by positive selection from autologous PBMC cultured in X-VIVO15 for 7 days + T lymphocytes. Centrifuge PBMCs and resuspend in PBS-0.5% BSA-2M EDTA to a final concentration of 1 x 10 7 / 80 μL. Combine PBMCs with CD8 + Microbeads (Miltenyi Biotec) were incubated at 4°C for 15 minutes, washed, resuspended and placed on an LS MACS column. Unlab...
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