The Expression Of TSLP In Lung Carcinoma And The Relationship Between Tslp And The Number Of Regulatory T Cells In Tumor Tissue

Objective:1.To investigate the expression of hTSLP in human lung cancer tissue and the correlation between TSLP expression with clinic pathological index and the number of regulatory T cells（Tregs）in situ.2.To examine the phenotype and cytokines secreting level of TSLP conditioned monocytes origined dendritic cells of lung cancer patients.To investigate the role of TSLP in the differentiation of CD₄⁺CD25+foxp3+T cells from CD₄⁺CD25 T cells and the migration of CD₄⁺CD25+T cells.Method:1.The expression of TSLP mRNA and protein were detected by Q-RT-PCR and immunohistochemistry respectively.To analyze the expression of TSLP in different pathological changes of lung tissues,the expression of TSLP protein in different pathological type of lung tumor was analyzed by immunohistochemistry approach.Foxp3+ Tregs were detected by immunohistochemistry method.The correlation of TSLP and clinical characteristics,and the number of Tregs was analyzed.2.Peripheral blood mononuclear cells（PBMCs）were collected from lung cancer patients（n=13）.PBMCs were used for the follow two steps of experiment.DC were subcultured from PBMCs every 3 days in GM-CSF and IL-4 containing medium.On day 6,DCs were stimulated with different factors including human TSLP and LPS.On Day 7,the phenotype was detected with FCM and the cytokines secreting was analyaed with ELISA after 24hr co-culture.3.Isolation of CD₄⁺CD25+ T cells and CD₄⁺CD₂₅^-T cells were performed in a two-step procedure by the magnetic bead separation system CD₄⁺CD25+ Regulatory T Cell Isolation Kit（Miltenyi Biotec,USA）.The percentage of CD₄⁺CD25+ foxp3+T cells was examined by FCM method after culturing in triplicate at a 1:10 ratio of dendritic cells:CD₄⁺CD₂₅^-T cells.The function of TSLP-DC in the migration of CD₄⁺CD25+ cells was analyzed with transwell methods.Results:1.TSLP protein expressed in cytoplasm.The expression rate of TSLP protein was 69.57%in tumor tissues,13.33%of benign lesion and 30.00%of non-tumor lung tissue,which had siginificant difference（P<0.05）.The expression of TSLP protein was correlated with tumor size（P=0.000）and lymphonode metastasis（P=0.018）.The number of Tregs in TSLP positive group was increased comparing with that of TSLP negative group（P<0.05）.2.TSLP-DCs expressed intermediate levels of CD83 which is a characteristic of mature DC.TSLP-DCs secreted low levels of IL-6,IL-12,IL-10,TNF-α,IFN-γ and high levels of TGF-β,MDC which showed the feature of tolerated DCs.3.The ratio of CD₄⁺CD25+foxp3+T cells in the co-culture system of different cytokines conditioned DCs and CD₄⁺CD₂₅^-T cells was examined by FCM method.The ratio of Tregs in TSLP-DCs group was（9.70%± 3.686%）,which was statistically higher than that of im-DC（4.144%±0.954%）and non-DC（2.560%±1.138%）.The ratio of TSLP-DCs was also higher than LPS-DCs（4.563%±2.308%）,but there was no statistically difference.4.We examined the function of TSLP-DC in the migration of CD₄⁺CD25+ cells.The number of CD₄⁺CD25+ cells migrated into the underlayer of TSLP-DCs group was 46.60±34.54,which was statistically more than imDC group（14.83±7.86）and non-DC group（0.33±0.82）（P<0.05）,slightly more than LPS-DC group（44.20±29.21）（P>0.05）.Conclusion:Our study demonstrates that tumor tissue secreting TSLP could instruct the DCs to induce foxp3（+）regulatory T cell differentiation and migration into tumor site.