Method for enhancing a function of a t cell
A cell and functional technology, applied in the direction of animal cells, vertebrate cells, cell culture active agents, etc., can solve problems such as incomplete understanding
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Embodiment 1
[0083] Example 1 Confirmation of the expression of PD-L1 in CD8 positive T cells
[0084] Preparation of HLA-A24 restricted MAGE-A 4 according to WO 2007 / 032255 143-151 - Specific cytotoxic CD8-positive T cell clone 2-28 (Miyahara Y, and 13 others, Clin. Cancer Res., Vol. 11, pp. 5581-5589 (2005), hereinafter, referred to as CD8-positive T cell clone 2-28) and MAGE-A4 143-151 peptide. The CD8-positive T cell clone 2-28 was co-cultured at 37°C with an autologous lymphoblastoid cell line (LCL) that had been irradiated with 80Gly X-rays and had been treated with MAGE-A4 143-151 After peptide pulse, PD-L1 on the cell surface was stained with an anti-human PD-L1 antibody (manufactured by BD bioscience) after 0 day, 1 day, 2 days and 3 days, and analyzed by flow cytometry. figure 1 The results of analysis by flow cytometry are shown. The expression of PD-L1 was the highest after 1 day of culture.
Embodiment 2
[0085] Example 2 Inhibiting the Expression of PD-L1 and PD-L2 of CD8 Positive T Cells by RNA Interference
[0086] The siRNA specific to PD-L1 having the sequence described in SEQ ID NO: 1, the siRNA specific to PD-L2 having the sequence described in SEQ ID NO: 2, or the negative control siRNA (all manufactured by Invitrogen) were introduced by electroporation CD8 positive T cell clones 2-28.
[0087] The cells into which siRNA against PD-L1 was introduced were cultured for 3 days, and then irradiated with 80Gly X-rays and treated with MAGE-A4 at 37°C. 143-151 Peptide-pulsed autologous LCL co-culture. The next day, they were stained with an anti-human PD-L1 antibody, and the expression of PD-L1 on the cell surface was analyzed by flow cytometry.
[0088] Regarding cells into which siRNA against PD-L2 was introduced, 2 days and 3 days after the introduction, the cells were stained with an anti-human PD-L2 antibody (manufactured by BD bioscience) and PD in the cells was analyz...
Embodiment 3
[0090] Example 3 Promoting CD8-positive T cells expressing PD-L1 and PD-L2 to produce IFN-γ by RNA interference
[0091] The negative control siRNA, the siRNA specific to PD-1 (manufactured by Invitrogen) having the sequence described in SEQ ID NO: 3, the siRNA specific to PD-L1 having the sequence described in SEQ ID NO: 1 or the siRNA specific to PD-1 having the sequence described in SEQ ID NO: 1 were electroporated. The siRNA specific to PD-L2 with the sequence described in ID NO: 2 was introduced into CD8 positive T cell clone 2-28. After culturing for 3 days, the cells were mixed with MAGE-A4 143-151 Peptide-pulsed cells of autologous LCL were mixed at a ratio of 4:1 or 2:1, the mixture was co-cultured at 37°C, and the concentration of IFN-γ in each culture supernatant was measured by ELISA method on the next day.
[0092] Figure 4 The concentration of IFN-γ in the supernatant of each cell is shown in . In cells into which siRNA specific for PD-1 was introduced (siPD-...
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