ABSTRACT
OBJECTIVE: To explore the stimulation conditions, optimal culture time and infection time of C57BL/6J mice CD3+ T cells in vitro, so as to improve the infection efficiency of CD19 chimeric antigen receptor T cells (mCD19 CAR-T). METHODS: Purified C57BL/6J mice CD3+ T cells were cultured in anti-CD3/CD28 coated, anti-CD3 coated+soluble anti-CD28 and anti-CD3 coated, respectively. The cells were stimulated in above three conditions for 12 h and 24 h, following with 24 h, 48 h and 72 h incubation and then the number of cell clones was recorded. C57BL/6J mice CD3+ T cells were stimulated for 12 h, 24 h, and 36 h under the above three conditions, then interleukin (IL)-2 (100 U/ml) was added. The number of cell clones was recorded under microscope at 24 h, 48 h, and 72 h of culture. After 24 h of stimulation, CD3+ T cells derived from C57BL/6J mice were infected with retrovirus for 48 h to establish mCD19 CAR-T cells, and the percentage of GFP+ CAR-T cells was detected by flow cytometry. RESULTS: The infection efficiency of mCD19 CAR-T cells derived from C57BL/6J mice was only 5.23% under the optimized conditions of mCD19 CAR-T cells derived from BALB/c mice. The number of clones of C57BL/6J mice CD3+ T cells was the highest in anti-CD3 coated+soluble anti-CD28 group after stimulated for 24 h and followed cultured for 48 h. After 24 hours of stimulation under the above conditions and 48 hours of culture with IL-2, the number of T cell proliferating clones in the anti-CD3 coated+soluble anti-CD28 group was significantly increased compared with the same group without IL-2, and the infection efficiency of CAR-T cells in this group reached 17.63%±4.17%. CONCLUSION: The optimal conditions for constructing CAR-T cells from C57BL/6J mice CD3+ T cells are different from those of BABL/c mice. T cells stimulated by anti-CD3 coated+soluble anti-CD28+IL-2 can obtain mCD19 CAR-T cells with the highest efficiency after retrovirus infection.
Subject(s)
Antigens, CD19 , Mice, Inbred C57BL , Receptors, Chimeric Antigen , T-Lymphocytes , Animals , Mice , T-Lymphocytes/immunology , Interleukin-2 , CD3 Complex , Immunotherapy, Adoptive/methods , Receptors, Antigen, T-Cell , CD28 Antigens , RetroviridaeABSTRACT
A 2 × 2 factorial arrangement (rearing room with or without pad-fan cooling × diet with or without 2.5 kg/t organic acid) was used to evaluate the effect of pad-fan cooling and dietary organic acid supplementation during perinatal period on reproductive performance and antioxidant status of sows in hot weather. This study was conducted in a subtropical city in Guangdong Province in South China between August and October, 2015. At day 85 of gestation, a total of 112 sows were randomly assigned to the four treatments with 28 sows per treatment, and maintained until day 21 of lactation, and the feeding trial lasted for 51 days. During the experimental period, room temperature and humidity were recorded hourly. The lactation feed intake of sows (P = 0.109) and stillbirths (P < 0.05) increased when the sows were reared in the room with the pad-fan cooling against the room without pad-fan cooling. The number of weak newborns per litter and the malondialdehyde content in days 14 and 21 milk decreased (P < 0.05), while the lactation feed intake of sows, weaned litter weights, and individual pig weights increased when the sows were fed the organic acid (P < 0.05). In conclusion, pad-fan cooling in rearing room improved the lactation feed intake of sows, and dietary organic acid supplementation improved reproductive performance and milk antioxidant status of sows. Pad-fan cooling is recommended in farrowing room, but not in gestating room.
