Differential Effects Of Taxol On Treg And Teff Cells And Possible Mechanisms

Taxol, one of the most widely used clinical anti-tumor drugs in cancer chemotherapy, is mainly applied in ovarian cancer, breast cancer, non-small cell lung cancer therapy. It binds to P-subunit of the tubulin heterodimer, accelerating the polymerization of tubulin, and stabilizes the resultant microtubules, thereby inhibiting their depolymerization. Its inhibition of microtubule depolymerization results in the arrest of the cell division cycle, eventually leading to apoptosis of the cancer cells. Though the molecular conformation varies differently, Taxol can mimic the activity of LPS to trigger TLR4 and the downstream signaling molecules, which leads to a series of activities including inflammatory cytokines secretion. The activities are confirmed in innate immune cells which express TLR4, such as macrophages, mononuclear cells and so on. Recent studies have indicated that TLRs (TLR2, TLR4,TLR6,TLR7, TLR8, TLR9) are not only expressed on the innate immune cell, but also on the adaptive immune cells, such as CD4+CD25+Treg cells. The interaction of adaptive immune cells and TLRs can also trigger the signal pathway.The previous data of our lab showed that TLRs were expressed on Treg cell, and Taxol could reduce the percentage of Treg cells in vivo. It suggested the possible mechanism of Taxol is via TLR4 signaling pathway. Based on the hypothesis, we firstly detected the expression of TLR4 on Treg/Teff cells and the effect of Taxol on these cell respectively. TLR4 mutation mice and siRNA interference technique were used in further confirmatory studies which demonstrated whether the different action of Taxol on the two subsets of T cells is dependent on TLR4 pathway. In view of the negative results, we explored the role of the other target Bcl-2 to clarify the mechanisms in this process. Our research may illustrate the mechanisms of Taxol's effect on adaptive immune system, and provide a new strategy for clinical cancer therapy. Part I The differential effects of Taxol on Treg and TeffTaxol is widely used in clinical therapy because of its satisfying anti-tumor effect, but the major side effect is impairing the immune system by diminishing the number of lymphocytes in patients. In this study, we established the C57BL/6 tumor bearing model by subcutaneously inoculating 3LL tumor cells in right groin of C57BL/6 mice. Once the size of tumor reached 25mm2, the experimental group was intraperitoneally injected with 10mg/kg Taxol, while the control group was injected with PBS. Mice were sacrificed for detecting the configuration of immune system on day 4, and data showed that CD4+CD25+Foxp3+T cells (Treg cells) of the experimental group were decreased sharply compared with the control group. In vitro, after stimulated with CD3/28 mAb for 48h, T cells were sorted by Nylon wool fiber column and treated with different doses of Taxol for another 24h. The percentage of Foxp3+T cells declined compared with the control group by FACS. These results demonstrated that Taxol can selectively decrease the ratio of Foxp3+T cells.In order to exclude the effects of contaminated dentritic cells (DCs), we purified Treg and Teff cells by Magnetic Cell Seperation(MACS) technology, and treated them with Taxol respectively. Results showed that the cell viability of Treg cells declined significantly compared with Teff cells under the same dosage of Taxol. In further study, we found that the suppressive cytokine secreted by Treg cells decreased sharply while Teff s secretion had no changes; in addtion, we found that the fluorescence of Foxp3 expression attenuated after treating with Taxol. Meanwhile we got the similar results when the same experiment was carried out on the normal C57BL/6 mice. Based on the studies above, function of Taxol on Treg cells may offer a new method for biological treatment after chemotherapy.PartⅡThe possible mechanisms involved in the differential effect of Taxol on Treg and Teff cellsFrom the data above, we observed the differential effect of Taxol on Treg and Teff cells.The next step is to explore the possible mechanisms behind it. It was observed that TLRs are also expressed on the adaptive immune cells including Treg and Teff cells; Besides, Taxol can mimic the activity of TLR4 ligand LPS to trigger the TLR4 signal pathway. Therefore, we further investigated whether the expression of TLR4 is similar between the two subsets of T cells and the possible role of TLR4 in the effect of Taxol. Firstly, we detected TLR4 expression after sorting the purified Treg and Teff cells by MACS. The fluorescence of TLR4 expressed on Treg cells is stronger than that on Teff cells in both normal C57BL/6 mice and 3LL tumor bearing mice, which is consist with the reports of other research groups. Then, we stimulated purified Treg and Teff cells with plated CD3/28 mAb for 48h before Taxol treatment. Cells were harvested at 12h and 24h for TLR4 detection by FACS. The results showed that there was no alteration of TLR4 between the two subsets on mRNA and protein levels at different time points. Experiments were carried out in both normal C57BL/6 mice and 3LL tumor bearing mice.Further, we used the TLR4 mutation C3H/HeJ mice whose TLR4 could not be activated by LPS to confirm the results above. The experimental group was intraperitoneally injected with 10mg/kg Taxol, while the control group was injected with PBS. Mice were sacrificed for detecting the configuration of immune system on day 4, and CD4+CD25+Foxp3+T cells (Treg cells) of the experimental group were decreased sharply compared with the control group in C3H/HeJ mice. In addition, experiment results in vivo including cell viability and cytokines secretion were similar with that of C57BL/6 mice. We then silenced the TLR4 expression by transfecting PEI mixed si-TLR4 plasmid in C57BL/6 mice. Their TLR4 was silenced after 48h compared with mice injected with PEI mixed si-control plasmid. The silence efficacy of TLR4 could be stable when the plasmid was injected intraperitoneally twice a week. The TLR4 silenced mice were intraperitoneally injected with 10mg/kg Taxol, while the control group was injected with PBS. And we still get the same results in both C57BL/6 and C3H/HeJ mice. In a word, it indicated that TLR4 may not be involved in the differential effect of Taxol on Treg and Teff cells.The results mentioned above lead us to find another mechanism for the phenomenon. We sorted the purified Treg and Teff cells, and stimulated them with plated CD3/CD28 mAb for 48h. FACS detection for apoptosis of Treg and Teff cells treated with Taxol for another 24h demonstrated that the percentage of Treg cells apoptosis was much higher than that of Teff cells. The results were confirmed by the nuclear staining of DAPI. The nuclear fragmentation was obvious in Taxol treated Treg group compared with Taxol treated Teff group.Till now the anti-tumor effect of Taxol is considered to cause mitotic arrest in cancer cells, leading to apoptosis through inhibition of the depolymerization of microtubules. So here we explored whether the apoptosis of Treg is partially through the microtubules. First of all, we sorted Treg and Teff cells from C57BL/6 mice and stimulated them by plated CD3/CD28 mAb for 48h. The two subsets of cells were respectively cocultued with Taxol for another 24h. Cell smear were prepared for immunoflorecence assay by staining with FITC-anti-a-tubulin and DAPI after cell docking. We observed the structure of microtubules under the confocal microscopy, and found that theβ-tubulin did not change after Taxol treatment.As is known to all, Taxol can bind to the sites of Bcl-2, a key molecule of anti-apoptotic family. Therefore we analyzed its effect on Bcl-2 expression, and data showed Taxol could lead to significant decrease of Bcl-2 on Treg cells but not on Teff cells. In addition, we studied another molecule of Bcl-2 family, Bax, which can promote apoptosis. It was found that Bax was upregulated in Treg cells after Taxol treatment compared with Teff cells.In order to confirm the function of Bcl-2 on Taxol's effect on Treg, we blocked the Bcl-2 signal pathway by using the inhibitor of Bcl-2(ABT-737). Taxol lost its selectively apoptotic effect on Treg cells after ABT-737 treatment, suggesting its important role in Taxol's differential effect on Treg and Teff cells. Taxol can selectively downregulate the expression of Bcl-2 to increase the apoptosis of Treg cells; while have no effect on Teff cells.In conclusion, our study demonstrated that chemotherapeutic drug Taxol can selectively down regulate the function of Treg cells and induce apoptosis, while has little effect on Teff cells. The differential effects are not caused by the difference of TLR4 expression, and independent on inhibition of microtubules depolymerization. Taxol can downregulate the anti-apoptotic molecule Bcl-2 and upregulate apoptotic molecule Bax of Treg cells, while have little effects on Teff cells as we shown in this paper. Thus, to clarify the mechanisms of Taxol's effect on Treg and Teff cells may provide a new target for the biological therapy.