The Influence Of DC From Axillary Lymph Node Of Breast Cancer To The Killing Activity Of The Specific CTL In Vitro

Objective: Dendritic cells(DCs), as the professional antigen processing cells, are in the center of immune reaction. The im-matured DCs will be activated to matured DCs after captured antigen, which express MHC classⅠmolecule,MHC classⅡmolecule,co-stimulatory molecule B7-1(CD80) and B7-2 (CD86),adhesion molecule CD54(ICAM-1) and CD50 (ICAM-3) in the molecular surface. After presenting the antigen peptide to CD4+ and CD8+ T cells, DCs induce the T cells to specific cytotoxicity T cells,also induce CD4+ T cells to produce Th1 type immune response to educe the anti-tumor effect by excrete cytokine. In this study, the mononuclear cells were isolated from axillary draining lymph nodes of women with breast cancer, and the adherent cells were cultured with rhGM-CSF and rhIL-4 to induce DCs. DCs stimulated by the auto-breast cancer freeze-thawing antigen and T lymph cells cultured with IL-2 were co-cultured to derivation into tumor antigen specific CTLs. At last, the killing activity of the CTLs to auto-breast cancer cells and MCF-7 cells were detected. The function of DCs after being stimulated and killing specificity of the CTLs were analyzed, to expected to establish a kind of individual treatment method to breast cancer.Methods: 1. To select 1 to 2 lymphonodes strictly with asepsis, harvesting lymphocyte suspension, then separate single-nucleus cells in albuginea rete, re-suspend and culture them with 10% FCS RPMI-1640.2. After adherencing 2 hours, the attached cells were cultured with rhGM-CSF(1000u/ml), rhIL-4(200u/ml) and TNF-α(200u/ml) to be induced into DCs. And the unattached cells were cultured with rhIL-2(200u/ml) into tumor draining lymph node cells(TDLNCs).3. To separate breast cancer cells from auto-breast tumor by enzyme digestion. DCs stimulated by the auto-breast cancer freeze-thawing antigen in order to load the tumor antigen were co-cultured with TDLNCs to derivation tumor antigen specific CTL.4. The PE-CD1a,PE-CD83,FITC-CD86 MoAb were added into DCs suspension harvested separately in the 1st day and the 7th day, the cytophenotype was detected with FCS. The FITC-CD3 MoAb and FITC-CD4/PE-CD8 bigeminy antibody were added into TDLNCs harvested separately in the 1st day and the 10th day, the cytophenotype of TDLNCs was detected.5. The killing activity of the CTLs to auto-breast cancer cells and MCF-7 cells were determined with non-radioaction cytotoxic analytical reagent kite to analysis the function of the differentiated DCs and the killing specificity of the CTLs.Results: 1. The mononuclear cells from axillary draining lymph node could be induced to typical maturated DCs in cell appearance after being stimulated by cytokine (rhGM-CSF,rhIL-4 and TNF-α).2. The percentage of specific surface marker CD1a,CD83,CD86 in DCs from mononuclear cells in axillary draining lymph nodes in the first day were 10.98±2.38,26.55±5.24,32.96±6.09 respectively; They were obviously heightened after cultured with rhGM-CSF and rhIL-4 and induced by autoallergic breast cancer freeze-thawing antigen and TNF-α, percent rate were 50.17±5.68,60.48±16.46,56.22±16.38 respectively, P<0.01.3. The percentage of CD3+ and CD8+ T cells in TDLNCs before induced were 73.93±2.18 and 32.78±3.21. The percentage after induced with rhIL-2 and Ag-DCs were 82.67±2.79 and 62.54±2.51. The percentage of CD3+ and CD8+ T cell could be stimulated to proliferate by the Ag-DCs, P<0.01. The percentage of CD4+ in TDLNCs before induced were 27.3±2.58,however the percentage in DC-Ag-TDLNCs and DC-TDLNCs group after induced were 17.49±4.21 and 19.49±2.12. The percentage of CD4+ after induced wasn't heighten by comparison,P>0.05。4. The ratio of the CTLs activated by the DCs loaded the auto-breast cancer freeze-thawing antigen in killing the auto-breast cancer cells (67.64%) was higher than the CTLs activated by the DCs no-loaded the auto-breast cancer freeze-thawing antigen (31.25%) and the TDLNCs (26.36%) in killing the auto-breast cancer cells (P<0.001), and the ratio of the CTLs activated by the DCs no-loaded the auto-breast cancer freeze-thawing antigen in killing the auto-breast cancer cells was higher than the TDLNCs (P < 0.05). There was no significant difference in killing activity of the three group cells on the MCF-7 cells (P>0.05).Conclusion: 1. The mononuclear cells from axillary draining lymph nodes can be induced to typical maturated DCs in cell appearance after being stimulated by cytokines (rhGM-CSF,rhIL-4 and TNF-α).2. The matured DCs express highly specific surface marker and possess stronger angtigen presentation capacity, and can stimulate TDLNCs to proliferate and differentiate into CTLs which possess highly killing effect.3. The DCs induced by autoallergic breast cancer freeze-thawing antigen can stimulate TDLNCs to proliferate and differentiate into tumor antigen specific CTLs. The CTLs possess stronger killing capacity to autoallergic breast cancer cells, however they have no the stronger capacity to other line of tumor cells.4. The DCs haven't been induced by autoallergic breast cancer freeze-thawing antigen can stimulate TDLNCs to proliferate and differentiate into CTLs also, but the CTLs have no the specific killing capacity to autoallergic breast cancer cells. The killing capacity to autoallergic breast cancer cells of them are similar to TDLNCs cultured with rhIL-2.5. The mathod , which co-stimulate and induce DCs with cytokines and autoallergic breast cancer freeze-thawing antigen to stimulate TDLNCs into tumor specificity killing function CTLs in order to killing autoallergic breast cancer cells, is a feasible method.