| BackgroundBreast cancer is one of the most common female malignancies. In the developed countries and in the metropolis of China such as Shanghai and Beijing, breast cancer incidence is ranking first in women’s cancers. The global number of new cases in breast cancer was more than1.2million each year, death toll was about300000. It was so serious a threat to women health.In recent years, a major breakthrough in the field of tumor immunology was the discovery of regulatory T cells (Regulatory T Cell, Treg) which was significantly higher in peripheral blood of cancer patients than that of health people. And the Treg and other immune suppressive cells revealed the nature of the tumor immune microenvironment. Treg cells used to be a protector of the normal immune balance have turned into the tumor immune barrier, which made the killer cells (CD8+T cells, etc.) unable to remove the tumor cells. Therefore, it has more recently been recognized as an important immunotherapy target at tumor-related immunosuppression.Several distinct subsets of Treg have already been described specifically in published papers. However, it has yet to be clarified for the redundant function and a "division of labor" between these subtypes. Valmori et al. has reported the identification and analysis of a distinct subset of Tregs in adult peripheral blood, which were described as naive natural Tregs (nTreg), CD4+CD25+CCR7+CD62L+CTLA-4+Foxp3+cells contained in the CD45RA+/RO" naive fraction. Subsequently, Nabila seddiki et al. has further demonstrated the existence and the function of this population. He found two subsets of Tregs:naive CD45RA+regulatory T cells and memory CD45RO+phenotype in peripheral blood, lymph node and spleen. And he identified that the naive CD45RA+phenotype expressed CTLA-4markers, the transcription factors Foxp3, T-bet and GATA3, and had suppressive activity. Furthermore, it was revealed in one literature that the methylation level of Treg cells transcription factor promoter region was similar to the undifferentiated naive T cells.Objective1. Whether naive Treg cells existed in tumors, and had the quantity change following tumor growing.2. During surgery and chemotherapy, whether the naive subtype Treg cells in peripheral blood had the correlation with tumor burden status.MethodsThis study recruited20healthy volunteers and70patients with early stage breast cancer. All patients received surgical treatment and postoperative adjuvant chemotherapy. We collected peripheral venous blood samples at the time of physical examination, pre-and post-operation, pre-and post-each cycle of chemotherapy, then isolated peripheral blood mononuclear cells. Tumor tissues in situ were obtained by ultrasound guided biopsy or surgery. We collected tumor infiltrating lymphocytes by primary culture technique. Based on phenotypic markers of naive T cell:CD45RA and CD62L, by immunofluorescence antibody staining techniques and Flow Cytometry method, we analyzed CD4+CD25highCD45RA+and CD4+CD25highCD62L+naive Treg in peripheral blood of healthy people and patients, tumor in situ, during surgery and adjuvant chemotherapy. Furthermore, we explored the relations of different clinical characteristics with naive Treg cells.Results1. The percentage of CD45RA+and CD62L+naive Tregs in healthy volunteers’PBMC were significant lower than both naive Tregs of breast cancer patients.2. In patients with breast cancer, CD45RA+and CD62L+naive Tregs in the TILs were also higher than in PBMC.3. Significant difference was observed in the CD45RA+circulating Tregs before and after operation. The comparison of CD62L+naive Treg between pre-and post-operation did not show statistical significance.4. During the treatment of adjuvant chemotherapy, the percentage of CD45RA+or CD62L+naive Tregs was decreased, especially during the initial cycles of chemotherapy. And it retained stable during the last cycles of chemotherapy.5. The percentage of CD45RA+and CD62L+naive Treg in patients with later clinical stage was higher than that with earlier clinical stage; The percentage of CD45RA+and CD62L+naive Treg in patients with molecular classification of Her-2type was higher than that with luminal A or luminal B; The percentage of CD45RA+and CD62L+naive Treg in patients with higher level of Ki-67was higher than that of lower level of Ki-67.Conclusions1. Naive Treg cells existed in cancer patients, and regardless of the tumor in situ or in peripheral blood, the percentage was higher than in peripheral blood of healthy people. And naive Treg in tumor in situ of cancer patients were higher than that in peripheral blood.2. After treatment with surgery which removed the tumor burden, CD45RA+naive Treg decreased in peripheral blood. During chemotherapy, especially in the2nd,3rd cycles, naive Treg cells decreased in peripheral blood.3. And there was a relation between naive Treg with different clinical features of patients. The percentage of CD45RA+and CD62L+naive Treg in patients with higher level of Ki-67was higher than that of lower level of Ki-67. |
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