| Objective To investigate the association of Sema4C expression with the number of infiltrating CD4+T cells, CD8+T cells, CD68+macrophages and CD4/CD8ratio in primary invasive ductal carcinoma, and the relationship of these index with clinicopathological features. To study how Sema4C affects the activation, proliferation and differentiation of T cell by regulating the expression of MHC-I molecules, MHC-II molecules and costimulatory molecule, and the secretion of cytokines in macrophages in vitro.Methods Formalin-fixed, paraffin-embedded primary invasive ductal carcinoma (n=22) were obtained from the department of pathology of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Tissue morphology was observed by H&E staining. The expression of Sema4C and immune cells of CD4+T cells, CD8+T cells and CD68+macrophages were analyzed by immunohistochemistry and imaging analysis software. The correlation of Sema4C expression with the number of CD4+T cells, CD8+T cells, CD68+macrophages and CD4/CD8ratio in invasive ductal carcinoma, and the association of these index with clinicopathological features were statistically by SPSS. Peripheral blood mononuclear cells (PBMCs) from normal healthy blood donors were prepared by Ficoll-Hypaque gradient centrifugation. After adhered, macrophages were treated with Sema4C-siRNA or Sema4C recombinant protein for3days. Morphological changes of cells was observed under ordinary optical microscope; The expression of HLA-A, B, C, HLA-DR, DP, DQ and costimulatory molecules such as CD58, CD54, CD86, CD80, CD40was detected by flow cytometry; The secretion of cytokines such as TGF-β1, IL-6, IL-2, IL-4, IL-10and IFN-y was determined by ELISA. After treated macrophages were co-cultured with CD3+T cells sorted from PBMCs by flow cytometry for7days, proliferation and differentiation of T cells were detected by flow cytometry.Results The results of immunohistochemistry of Primary invasive ductal carcinoma (n=22) showed that a majority of these infiltrating immune cells were distributed in cancer tissues; higher expression of Sema4C, decreasing number of infiltrating CD4+T and CD8+T cells, and lower CD4/CD8ratio (<1) were relative with lymph node metastasis (P<0.05); there was no correlation between these index with other clinicopathological features (P>0.05). Significant negative correlation were observed between Sema4C and infiltrating CD4+T cells, CD8+T cells, lower CD4/CD8ratio; there were no relationship between the number of CD68+macrophages with other index and all the clinicopathological features. Adherent cells from culture owned typical characteristics of macrophages, and the percentage of CD11b+cells was (89.47±1.10)%, in which CD68+cells were accounted for (92.37±2.11)%. Sema4C and its receptor plexin B2were expressed in macrophages. Liposome2000mediated siRNA transfection could inhibite the expression of Sema4C in macrophages, and the transfection efficiency was (96.17±2.45)%. After treated with Sema4C-siRNA or Sema4C recombinant proteins for3days, In the control group, Sema4C-siRNA group, Sema4C group (100ng) and Sema4C group (800ng), the expression of CD58was (87.00±1.13)%,(87.40±1.41)%,(81.70±0.14)%and (20.20±6.51)%, respectively; CD54was (94.90±0.71)%,(92.25±0.92)%,(92.85±1.20)%and (46.70±8.62)%, respectively; CD86was (35.15±0.64)%,(37.85±1.63)%,(32.75±5.44)%and (4.45±0.72)%, respectively; there was significant statistical difference between Sema4C group (800ng) and the other three groups (P<0.05). In the control group, Sema4C-siRNA group and Sema4C group (800ng), the concentration of TGF-β1was (1.54±0.35) ng/mL,(1.27±0.26) ng/mL and (2.01±0.16) ng/mL, respectively; the concentration of IL-6was (1.04±0.06) ng/μL,(0.61±0.11) ng/μL and (1.89±0.10) ng/μL, respectively; there was significant statistically difference between Sema4C group with other groups (P<0.05). Macrophages and CD3+T lymphocytes were co-cultured for7days, compared with the control group (proliferation index=5.19±1.07), the proliferation of T cells was inhibited significantly in Sema4C group (proliferation index=2.21±0.35, P<0.05), while there was no significant differences control group between Sema4C-siRNA group (proliferation index=5.55±1.02, P<0.05). In the control group, Sema4C-siRNA group and Sema4C group, IL17+CD4+cells accounted for (11.40±1.04)%,(11.98±2.40)%and (32.99±5.43)%of CD4+cells, respectively; there was significant statistically difference between Sema4C group and the other two two groups (P<0.05).Conclusion Our study showed that Sema4C could impair activation and proliferation of T cells by reducing the expression of CD58, CD54and CD86in macrophage, weaken the antigen presenting ability, and reducing activation and proliferation index of T cells. At the same time, Sema4C could promote Th17differentiation by increasing the secretion of IL-6and TGF-P in macrophages. Thus, Sema4C may promote breast caner development by attenuating the immune response. |
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