| OBJECTIVEBased on the previous results, this study was designed to investigate the functional changes in peripheral blood lymphocytes after high intensity focused ultrasound (HIFU) treatment of H22 implanted tumor in mice, and to determine the local expression and specific antitumor immunity of HIFU-activated cytotoxic T lymphocytes (CTLs) within the implanted tumor region after adoptive immunotherapy for the syngeneic tumor-bearing mice. It will provide more evidences involved in HIFU-triggered antitumor immune response against the homogeneous tumor cells.MATERIALS AND METHODS1. Changes in the cellular immune functions of peripheral blood lymphocytes after HIFU treatment in tumor-bearing miceSeventy-two normal C57BL/6J mice were randomly divided into three groups: HIFU group (n =24), sham-HIFU group (n =24), and control group (n=24). The mice in both HIFU group and sham-HIFU group received H22 hepatocellular carcinoma implantation using the hypodermic injection of H22 cells suspension in the back of each mouse. Seven days after the tumor implantation, tumor mass was obviously detected in the local region. Subsequently, the mice in HIFU group underwent HIFU ablation for the implanted tumor, but those in sham-HIFU group received a sham therapy procedure with no acoustic energy deposition in the tumor. Those in control group were normal mice, without either tumor implantation or HIFU treatment. Fourteen days after HIFU, the peripheral blood was collected in each mouse, and flow cytometry technique was employed to observe changes in the positive expression of CTLs stained with recombinant MHC peptide fluorescence in each group, respectively. Using lymphocyte density gradient centrifugation, lymphocytes were extracted and purified from the peripheral blood of each mouse, and LDH assay was performed to measure the cytotoxic activity of the lymphocytes against H22 tumor cells in vitro in the three groups respectively. In addition, both IFN-γand TNF-αlevels were determined by the ELISA method in the supernatant after a 24-hour culture of the lymphocytes with H22 cells. 2. Local expression of HIFU-activated CTLs after adoptive immunotherapy in the homogeneous tumor-bearing miceSeven days after after the H22 tumor implantation, 24 tumor-bearing C57BL/6J mice were randomly divided into three groups: HIFU group (n =8), sham-HIFU group (n =8), and control group (n=8). They underwent the intravenous injection of 1×107 lymphocytes stained with recombinant MHC peptide fluorescence via the tail vein. The lymphocytes were obtained from the syngeneic tumor-bearing mice treated by either HIFU or sham-HIFU procedure, and the normal syngeneic mice, respectively. Twenty-four hours after the injection, the tumor and normal tissues surrounding the tumor were harvested in each mouse, and then sliced into histological sections using a freeze section cutter. The local expression of the positive-stained CTLs was examined under a laser confocal microscope in the tumor, and Software Image-Pro Version 5.0 was used to analyze the amount of the positive-stained CTLs in each group. Furthermore, the slices were stained with H & E method, and were observed to determine the distribution characteristic of the CTLs using optical microscopy.3. The functional change of HIFU-activated CTLs after adoptive immunotherapy in the homogeneous tumor-bearing miceSeven days after H22 tumor implantation, 30 tumor-bearing C57BL/6J mice were randomly divided into three groups: HIFU group (n =10), sham-HIFU group (n =10), and control group (n=10). They received the intravenous injection of 1×107 lymphocytes via the tail vein. The lymphocytes were obtained from the syngeneic tumor-bearing mice treated by either HIFU or sham-HIFU procedure, and the normal syngeneic mice, respectively. Seven days after adoptive immunotherapy, surgical procedure was performed to harvest the implanted tumor in each mouse. Viable tumor tissue was minced and dispersed to a single-cell suspension by grinding in a loose-fitting ground glass homogenizer and filtrating through a sterile steel mesh. The ELISpot assay was used to measure the number of IFNγ-secreting lymphocytes.RESULTS1. Changes in the cellular immune functions of peripheral blood lymphocytes after HIFU treatment in the tumor-bearing miceFourteen days after HIFU treatment, the positive expression rates (%) of CTLs in the HIFU group, sham-HIFU group and control group were 1.9±0.08, 1.42±0.09 and 1.27±0.05 respectively. Compared to the values in the sham-HIFU and control groups, a statistical increase of the positive CTLs was significantly observed in the HIFU group (P < 0.01). The cytotoxic activity of the lymphocytes against H22 tumor cells gradually enhanced with an increase of effector/target cell ratios in each group. The cytotoxicity of the lyphocytes was 3.01±1.02, 13.76±2.21, 33.38±3.68 and 51.59±3.74 in the HIFU group while effector/target cell ratios were 1:1, 2.5:1, 5:1 and 10:1 respectively. Compared to the sham-HIFU and control groups, the cytotoxic activity was significantly increased in the HIFU group, and statistical differences were observed among them while effector/target cell ratios were 2.5:1, 5:1 and 10:1. The concentration of the IFN-γand TNF-αwas increased in the supernatant with an increase of effector/target cell ratios in each group. The IFN-γlevels were 141.17±19.24, 178.46±16.56, 443.98±13.26 and 743.43±18.18 pg/ml in the HIFU group respectively while effector/target cell ratios were 1:1, 10:1, 20:1 and 40:1. The TNF-αlevels were 242.41±21.74, 510.07±16.63, 764.86±15.86 and 1212.83±89.21 pg/ml in the HIFU group respectively while effector/target cell ratios were 1:1, 10:1, 20:1 and 40:1. Compared to the sham-HIFU and control groups, there were significant increases of the IFN-γand TNF-αlevels in the HIFU group while effector/target cell ratios were 1:10, 1:20 and 1:40 (P <0.01).2. Local expression of HIFU-activated CTLs after adoptive immunotherapy in the homogeneous tumor-bearing miceOne day after adoptive immunotherapy with the HIFU-activated lymphocytes stained by Pro5 Recombinant MHC peptide fluorescence, the positive-stained CTLs were observed in 7 mice in the HIFU group, 6 in the sham-HIFU group, and 2 in the control group. These positive cells mainly distributed along the marginal region between the tumor and normal tissues, with no appearance in the central tumor. The number of the positive-stained CTL was 17.13±8.29 in the HIFU group, 10.37±6.47 in the sham-HIFU group, and 0.83±0.74 in the control group. Compared to the values in the sham-HIFU group and control groups, there was a significant increase of the activated-CTL infiltration within the local tumor in the HIFU group (P < 0.05). 3. The functional change of HIFU-activated CTLs after adoptive immunotherapy in the homogeneous tumor-bearing miceSeven days after adoptive immunotherapy with the HIFU-activated lymphocytes, the number of INF-γsereting lymphocyte frequencies was 110±18.04 in the HIFU group, 38±9.41 in the sham-HIFU group, and 23±1.87 in the control group respectively. Compared to the sham-HIFU and control groups, there was a significant increase of INF-γsereting lymphocytes in the HIFU group (P < 0.01).CONCLUSION1. Peripheral blood lymphocytes were significantly activated after HIFU treatment of implanted H22 hepatocellualr carcinoma in mice, with an increase of the positive CTLs. The activated lymphocytes had a stronger cytotoxicity against H22 tumor cells in vitro.2. After adoptive immunotherapy with the HIFU-activated lymphocytes, the local expression of the HIFU-activated CTLs was significantly increased within the implanted tumor in the homogeneous tumor-bearing mice, suggesting that the activated CTLs could move from peripheral blood into the local tumor.3. After adoptive immunotherapy with the HIFU-activated lymphocytes, local CTLs presented a stronger specific antitumor immune response within the tumor in the homogeneous tumor-bearing mice.
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