In peripheral blood of OC patients, the expression level of mTOR factor in CD4 + Tregs was increased and higher than that in CD4+ Teffs
We used fluorescence-activated cell sorting (FACS) to investigate the expression level of mTOR factor in CD4+ Tregs in peripheral blood, CD4+ Tregs were identified as CD4+ CD25+ CD127− T cells and CD4+ Teffs were CD4+ CD25− CD127+ T cells. As shown in Fig. 1A, we found that the expression level of mTOR factor in CD4+ Tregs in OC patients was higher than healthy control (HC) patients, but there was no significant difference compared with BOT patients ((77.4% ± 8.12%) vs. (74.6% ± 9.25%) vs. (64.19% ± 9.7%); P > 0.05, P < 0.01, respectively). In addition, in peripheral blood of OC patients, the expression level of mTOR factor in CD4+ Tregs were higher than that in CD4+ Teffs (77.4% ± 8.12% vs. 72.61% ± 6.1%, P < 0.05, Fig. 1B). These results suggested that the mTOR factor in CD4+ Tregs was highly expressed in peripheral blood of patients with ovarian cancer and was higher than in CD4+ Teffs.
Analysis of the prognosis and immune infiltration of mTOR gene expression in patients with ovarian cancer
We used the TIMER and Kaplan-Meier plotter database to make a clinical analysis for mTOR gene in patients with ovarian cancer. As shown in Fig. 2A, B, we found that the expression level of mTOR mRNA was positively correlated with CD4+ T cells and Tregs (P = 4.58E-04, Rho = 0.22, P = 1.32E-03, Rho = 0.202) infiltration level in patients with ovarian cancer. Furthermore, the expression level of mTOR mRNA was related to the prognosis of patients with ovarian cancer, the high expression of mTOR mRNA was related to the poor prognosis in patients with OC (HR = 1.44, P = 0.013, Fig. 2C), so we analyzed the relationship between mTOR mRNA and prognosis in ovarian cancer patients with different Tregs infiltration status. As shown in Fig. 2D, E, we found that OC patients had poor prognosis with the high expression of mTOR mRNA in the state of high infiltration level of Tregs, but the expression level of mTOR mRNA was not associated with the prognosis of patients in the state of low infiltration level of Tregs ((HR = 2.38, P = 0.036) vs. (HR = 1.31, P = 0.077)). These results indicate that mTOR and Tregs are closely related and affect the prognosis of patients with OC.
The activation of TLR8 signal can down-regulate the expression of mTOR in CD4 + Tregs in the growth environment of OC cells
4E-BP1 is a downstream molecule of mTOR signal pathway, which controls cell proliferation and function. We explored the expression levels of mTOR and 4E-BP1 factors in CD4+ Tregs co-cultured with ovarian serous adenocarcinoma cell line SKOV3. As shown in Fig. 3A, B, the expression levels of mTOR and 4E-BP1 factors in CD4+ Tregs co-cultured with SKOV3 was significantly increased compared with normal cultured group ((78.73% ± 5.30%) vs. (86.55% ± 2.08%), P < 0.05; (26.50% ± 1.84%) vs. (51.08% ± 6.70%), P < 0.01)). At the same time, in the SKOV3 growth environment, we compared the expression levels of mTOR and 4E-BP1 factors in CD4+ Tregs and in CD4+ Teffs. As shown in Fig. 3C, D, we found that the expression levels of mTOR and 4E-BP1 factors in CD4+ Tregs were higher than in CD4+ Teffs ((78.73% ± 5.30%) vs. (62.75% ± 5.79%), P < 0.01; (26.50% ± 1.84%) vs. (42.20% ± 6.46%), P < 0.05). We activated the TLR8 signal pathway of CD4+ Tregs and observed the changes in the expression levels of mTOR and 4E-BP1 factors. We found the activation of TLR8 signal pathway could down-regulate the expression levels of mTOR and 4E-BP1 factors from CD4+ Tregs ((78.73% ± 5.30%) vs. (54.83% ± 7.47%), P < 0.01; (26.50% ± 1.84%) vs. (22.70% ± 3.71%), P < 0.01, Fig. 3E, F). These results suggested that the expression level of mTOR in CD4+ Tregs was increased in the growth environment of SKOV3 and the activation of TLR8 signal could reverse the high expression of mTOR in CD4+ Tregs.
The blocking of mTOR signal pathway can down-regulate the glucose metabolism level of CD4+ Tregs
In our previous studies, we found that TLR8-mediated reversal of CD4+ Tregs immunosuppressive function had a causal relationship with glucose metabolism. So we explored the relationship between mTOR signal pathway and glucose metabolism of CD4+ Tregs in SKOV3 growth environment. Rapamycin was a specific inhibitor of mTOR signal pathway[24], we detected glucose metabolism level of CD4+ Tregs after rapamycin treatment in SKOV3 growth environment. Compared with cells without rapamycin treatment, rapamycin significantly decreased the expression levels of genes related to glucose metabolism (Fig. 4A). Meanwhile, proteins had the same results, rapamycin could also decreased the expression levels of proteins related to glucose metabolism (Fig. 4B). The levels of glucose uptake (OD value: (3.31 ± 0.19) vs. (3.04 ± 0.21), P < 0.01; 2-DG6P value: (193.49 ± 13.28) vs. (174.05 ± 14.58), P < 0.01) and glycolysis (OD value: (1.82 ± 0.07) vs. (0.39 ± 0.13), P < 0.001; L-Lactate value: (21.97 ± 0.87) vs. (4.85 ± 1.54), P < 0.001) were also down-regulated (Fig. 4C, D). As shown in Fig. 4E and 4F, we found that rapamycin could also down-regulate levels of genes and proteins related to glucose metabolism in CD4+ Teffs. The levels of glucose uptake and glycolysis were also down-regulated. These results indicated the blocking of mTOR signal pathway could down-regulate the glucose metabolism level of CD4+ Tregs and Teffs.
Inhibition of mTOR signal and activation of TLR8 signal had a synergistic inhibitory effect on the glucose metabolism and immune function of CD4 + Tregs
In order to explore the relationship between mTOR signal, TLR8 signal, glucose metabolism and immunosuppressive function of CD4+ Tregs in SKOV3 growth environment, we blocked the mTOR signal of CD4+ Tregs and then activated its TLR8 signal to observe the changes in the glucose metabolism and immune function of CD4+ Tregs. As shown in Fig. 5A and 5B, we found that compared with the rapamycin or ssRNA40 treatment group alone, the glycolysis level of CD4+ Tregs in the rapamycin and ssRNA40 combined treatment group was significantly reduced (OD value: (0.41 ± 0.14) vs. (1.08 ± 0.14) vs. (0.15 ± 0.06), P < 0.01, P < 0.001; L-Lactate value: (5.14 ± 1.71) vs. (13.15 ± 1.66) vs. (1.79 ± 0.53), P < 0.01, P < 0.001). Compared with the rapamycin treatment group alone, the glucose uptake level of CD4+ Tregs in the rapamycin and ssRNA40 combined treatment group were significantly reduced, but compared with the ssRNA40 treatment group alone, the glucose uptake level did not change (OD value: (2.75 ± 0.38) vs. (2.74 ± 0.51) vs. (2.65 ± 0.47), P < 0.05, P > 0.05; 2-DG6P value: (154.28 ± 26.76) vs. (153.07 ± 35.64) vs. (146.6 ± 33.33), P < 0.05, P > 0.05). We could find that the inhibition of mTOR signal and activation of TLR8 signal has a synergistic inhibitory effect on the glucose metabolism of CD4+ Tregs.
In addition, we used CFSE to test the immunosuppressive function of CD4+ Tregs in SKOV3 growth environment, we showed that the mTOR signal pathway inhibitor rapamycin markedly abolished CD4+ Tregs activities of suppression on CD4+ Teffs proliferation. Furthermore, combined treatments with rapamycin and ssRNA40 significantly enhanced TLR8-mediated reversal of CD4+ Tregs suppression (Fig. 5C). These results suggested that mTOR signal pathway was correlated with the TLR8-mediated reversal of CD4+ Tregs suppression by glucose metabolism.
mTOR signaling pathway played an important role in regulating TLR8-mediated reversal of CD4+ Tregs immunosuppression
To further clarify the role of mTOR signal pathway in the reversal of immunosuppressive function of CD4+ Tregs mediated by TLR8 in SKOV3 growth environment, we used SB203580 to block p38 to test the involvement of p38 in regulating mTOR signaling of CD4+ Tregs. We observed that blockage of p38 signaling in CD4+ Tregs reversed the downregulated phosphorylation of mTOR signaling mediated by TLR8 (Fig. 6A-E). Furthermore, we performed a functional rescue experiment with activation of the mTOR signal pathway by MHY1485, Figs. 6F suggested that mTOR signaling activation prevented the TLR8-mediated reversal of CD4+ Tregs immunosuppressive activities on responder CD4+ Teffs proliferation, the proliferation level of CD4+ Teffs is restored. These results indicated that the mTOR signaling pathway was important and involved in regulating TLR8-mediated reversal of CD4+ Tregs immunosuppressive in SKOV3 growth environment.