And Its Action Mechanism. CD4 <sup> + </ Sup> CD25 <sup> Bright </ Sup> CCR6 <sup> + </ Sup> Regulatory T Cells In Tumor Immune Escape

Tumor immune escape is the important mechanism of tumor development. Recent research has reported that an important T cell subtype- CD4⁺CD25⁺ regulatory T cells (Tregs) probably play a key role in tumor immune escape. Tregs were first reported by Sakaguchi and had immune-regulative function. Tregs could effectively inhibit many immune cells' function mainly through cell-cell contact. It has been shown that Tregs play important roles in tumor immunity, infectious immunity, autoimmunity and transplant immunity, et al.Recent research reported that there are subsets of Treg, displaying different function in immune response, in host. Such as, CD4⁺CD25⁺CCR6⁺Tregs, displaying memory regulatory marker, played an important role in the development of EAE animal model. The study on the function of subsets of Treg could contribute to the exploration of mechanisms of immune regulating and to the interfering of immune response.Recent research has shown that tumor immunity is closely related with different memory T cells subtype. Moreover, researchers found that Tregs expressing CD45RO resided in draining lymph nodes and tumor mass. So, whether memory Tregs subtype exists in tumor, if it does, what's the function of memory Tregs subtype? In the present study, memory Tregs (CD4⁺CD25^brightCCR6⁺ Tregs) were detected in tumor animal model with different genetic background and in clinical breast cancer patients, the function and the mechanism of memory Tregs in tumor immunity was explored, which would provide a new target in tumor immunity.Partâ… The detection of CD4⁺CD25^brightCCR6⁺ Tregs in tumor animal models and in clinical breast cancer patients1. The distribution of CD4⁺CD25^brightCCR6⁺ Tregs in tumor animal models 4T1 tumor model in BALB/c mice was established as previously reported, the distribution of CD4⁺CD25^bright Tregs in tumor-bearing mice and normal mice and CCR6⁺ Tregs in tumor-bearing mice was examined by FACS. The results showed that CCR6⁺ Tregs distributed in peripheral immune organs and lymph nodes both in tumor bearing mice and in normal mice. CCR6⁺ Tregs were rich in tumor infiltrated cells and the percentage was much higher than that in draining lymph nodes and other lymphoid tissues. The same results were in Renca and 3LL tumor-loaded mice.2. The function of CD4⁺CD25^brightCCR6⁺ Tregs in vitroThen CD4⁺CD25^brightCCR6^- T cells and CD4⁺CD25^brightCCR6⁺ T cells were sorted by FACS, their effect on the proliferation of CD4⁺CD25^- T cells was examined. It was found that both CD4⁺CD25^brightCCR6^- and CD4⁺CD25^brightCCR6⁺ T cells could inhibit the proliferation of CD4⁺CD25^-T cells in vitro.It has been reported that CD4⁺CD25⁺ Tregs can inhibit the function of effector T cells through cell-cell contact, so in the present study, the inhibitory ways of CCR6⁺ Tregs in vitro was determined by transwell. The results showed that CCR6⁺ Tregs also exhibited the inhibitory function through cell-cell contact. Since memory T cells had lower stimulatory threshold and weaker dependence to second signal than naive T cells, so the response of CCR6⁺ Tregs to first signal was observed. It showed that under the stimulation of first signal, the proliferation of CCR6⁺Tregs and the level of secreted IL-10 were much higher than that of CCR6^-Tregs. All these showed that CCR6⁺ Tregs had regulatory memory function.3. The detection of CD4⁺CD25^brightCCR6⁺Tregs in clinical breast cancer patientsTo further confirm the enrichment of CCR6⁺ Tregs in tumors, tumor infiltrated lymphocytes and peripheral blood in clinical breast cancer tissues were also examined. The results showed that CCR6⁺ Tregs were also existed in 22 clinical cases, inhibited the proliferation of CD4⁺ effector T cells and enriched in TILs.Partâ…¡The role of CD4⁺CD25^brightCCR6⁺T cells in tumor immune escape1. CD4⁺CD25^brightCCR6⁺Tregs inhibited the function of tumor specific CD8⁺ T cellsIn order to study the role of CCR6⁺ Tregs in tumor immunity, CCR6⁺ and CCR6^-Tregs as well as 4T1 specific CD8⁺ T cells (originated from inactivated-4T1-immunized BALB/c mice) in 4T1 bearing mice were sorted by FACS, then cells were cotransferred into 4T1 bearing BALB/c nude mice, the role of CCR6⁺ and CCR6^- Tregs on CD8⁺ CTL was detected. The results showed that the tumor growth was faster, the survival was significantly decreased and the lung metastasis index was strikingly increased in CCR6⁺Tregs cotransferred group than that in 4T1 specific CD8⁺ T cells alone group or in CCR6^- Tregs cotransferred group. To detect the function of CD8⁺ T cells, 4T1 specific CD8⁺ T cells were labeled by CFSE and the data showed that CCR6^+sTregs could significantly inhibit the proliferation of CD8⁺ T cells in vivo, whereas CCR6^- Tregs showed little inhibitory effect on the proliferation of CD8⁺T cells in vivo.3. The enrichment of CD4⁺CD25^brightCCR6⁺ Tregs in the tumorTregs play an immune inhibitory role in tumor immunity, since the cell number of Tregs is the important factor, the hypothesis was presented that the difference between the function of CCR6⁺Tregs and CCR6^- Tregs in vivo was associated with the different cell numbers in tumor. So, the cell number of Tregs in tumor in 4T1 -loaded nude mice was detected using immunofluorescence. The results showed that there were more Tregs in tumor in CCR6⁺Tregs transferred mice than that in CCR6^- Tregs transferred group. The same results were found in the tumor draining lymph nodes. All these indicate that the extent of the inhibitory function of CCR6⁺ Tregs in vivo was correlated with Tregs cell number.Partâ…¢The mechanism of the role of CD4⁺CD25^brightCCR6⁺ T cells in tumor immune escape1. The proliferation of CD4⁺CD25^brightCCR6⁺Tregs in tumor immunityMemory T cells have better proliferative activity than naive T cells, so whether the increase of the infiltration in tumor of CCR6⁺ Tregs was associated with cells proliferation. So, in the present study, the proliferation of CCR6⁺Tregs and CCR6^-Tregs in tumor-loaded mice and in normal mice was detected using Brdu incorporation.The results showed that in normal mice, the proliferative ability of CCR6⁺ Tregs was better than that of CCR6^- Tregs and in tumor bearing mice, the proliferation of CCR6⁺ Tregs was strikingly increased. To further confirm the proliferation of CCR6⁺ Tregs in vivo, CCR6⁺Tregs and CCR6^-Tregs labeled with CFSE were transferred into 4T1 bearing nude mice and the proliferation was detected. The similar results were obtained. It indicates that CCR6⁺ Tregs had better proliferation ability in tumor bearing mice.2. The effect of the proliferation on the inhibitory function of CD4⁺CD25^brightCCR6⁺ Tregs in tumor immunityTo confirm the relationship of the proliferation of CCR6⁺ Tregs in vivo and the cell number as well as the inhibitory function, 4T1 bearing mice were pretreated with vinblastine, CCR6⁺ Tregs were sorted and were cotransferred with 4T1 specific CD8⁺ T cells into 4T1 bearing nude mice. It showed that the inhibitory effect of CCR6⁺ Tregs on the anti-tumor function of CD8⁺ T cells was abrogated and in the draining lymph nodes, the percentage of CCR6⁺ Tregs was significantly decreased. Brdu incorporation assay also detected that in vinblastine pretreated CCR6⁺ Tregs cotransferred group, the proliferation of CD8⁺ T cells was increased and the tumor bearing mice survived longer. While the proliferation of CCR6⁺ Tregs pretreated with vinblatine was obviously decreased. These results suggest that the proliferation of CCR6⁺ Tregs closely related to the effect on cell number and the inhibitory function of Tregs.3. DCs induced the proliferation of CD4⁺CD25^brightCCR6⁺ TregsIt has been reported that DCs can promote the proliferation of Tregs in vivo through TGF-β, to explore the mechanism of the proliferation of CCR6⁺ Tregs in vivo, the percentage of DCs in tumor was detected. The results showed that the percentage was increased and DCs were immature. Further studies showed that DCs could promote the proliferation of CCR6⁺ Tregs in vitro, and anti-TGF-βblocking antibody or anti-MHC-â…¡antibody could effectively inhibit this effect. Moreover, CCR6⁺ Tregs after proliferation promoted by DCs maintained the expression of CCR6 and Foxp3, and maintained the inhibitory function. The in vivo studies also showed that DCs could significantly promote the proliferation of CCR6⁺ Tregs.In conclusion, in the present study, CD4⁺CD25^brightCCR6⁺Tregs were confirmed in tumor immunity, had memory function, and enriched in tumor. CD4⁺CD25^brightCCR6⁺ Tregs could inhibit the function of CD8⁺ T cells in vivo. Most importantly, the inhibitory function was closely related with CD4⁺CD25^brightCCR6⁺ Tregs proliferation. Moreover, immature DCs in tumor immunity played important roles in the proliferation of CD4⁺CD25^brightCCR6⁺Tregs in vivo. The study has not only provided the experimental basis for the exploration of the mechanism of tumor escape and the amelioration of the strategy of tumor therapy, but also provided a new target for tumor therapy targeting Treg.