Figure 3.

Rapamycin upregulates IL-27 production of UECC and IL-27R expression on NK cells. (A) After treatment with rapamycin (100 nM) for 48 hours, the secretion levels of IL-27 from UEC cells UECC (Ishikawa, RL95-2, and KLE cells) were evaluated by ELISA. (B, C) The expression of IL-27 in cytokeratin (CK)7⁺UECC of cancer lesions from Ishikawa-xenografted nude mice (n = 8 mice/group) after intraperitoneal injection of rapamycin (2 mg/kg) or PBS for 3 weeks was analyzed by FCM. (D-F) The mock or IL-27⁺ UECC (Ishikawa, RL95-2, and KLE cells) were prestimulated with or without rapamycin (100 nM) for 48 hours and co-cultured with NK cells for 24 hours, and then the IL-27 receptors (gp130 and WSX-1) in these NK cells were evaluated by FCM. (G-I) Nude mice of 4 to 5 weeks of age were inoculated subcutaneously under the scruff on day 0 with 200 μl of Ishikawa cells (2 × 10⁶) (IL-27^over Ishikawa cells on the right or NC Ishikawa cells on the left). After 7 days, Ishikawa-xenografted nude mice (n = 8 mice/group) were intraperitoneally injected rapamycin (2 mg/kg) or PBS for 3 weeks. Moreover, human NK cells from peripheral blood labeled with PKH67 were adoptively transferred to Ishikawa-xenografted nude mice every 7 days for the first 2 weeks. After 3 weeks, the tumors were collected for FCM, TEM and IHC. (H, I) FCM was used to analyze the IL-27 receptors on PKH-67–labeled NK cells in cancer lesions of Ishikawa-xenografted nude mice. NC: Ishikawa cells were transfected with negative control lentivirus; IL-27^over: Ishikawa was transfected with the lentiviruses expressing human IL-27. Data are expressed as mean ± SEM. *P < .05, **P < .01, ***P < .001, and ****P < .0001.