Therapeutic effects of B and T lymphocyte attenuator extracellular domain and heat shock protein 70 antigen peptide on cervical cancer in mouse model]

To investigate the synergistic therapy effects of B and T lymphocyte attenuator (BTLA) extracellular domain in combination with heat shock protein 70 (HSP70)-TC-1 antigen peptide complex on the mouse model of cervical cancer and the related immunological mechanisms.(1) Detecting the BTLA and herpesvirus entry mediator (HVEM) gene expression in the tumor microenvironment after C57BL/6 mice were inoculated with TC-1 tumor cells by realtime PCR; BTLA, HVEM expression on tumor infiltrating lymphocytes cell surface were detected by flow cytometry (fluorescence intensity). (2) According to different treatments, tumor-bearing mice were divided into 5 groups, which was injected with pcDNA3.1 (empty vector plasmid as control), psBTLA (vector plasmid which expresses BTLA extracellular domain), HSP70 (HSP70-TC-1 cell peptide complex), HSP70 + pcDNA3.1 or HSP70 + psBTLA, respectively. The weight of tumor was recorded. The expression of immunoregulatory genes in tumor microenvironment were detected. The change of lymphocyte amount and cytotoxicity were detected too; lymphocyte proliferation activity was measured by tritium thymidine incorporation assay; the concentration of interleukin (IL)2 and interferon-γ (IFN-γ) in supernatants of spleen lymphocyte were measured by enzyme-linked immunosorbent assay (ELISA).(1) BTLA gene expression was gradually increased after tumor cells inoculation. The highest expression level was 2.83 ± 0.35 at 14th day, which had statistical significance difference with the 7th day expression of 1.66 ± 0.25 (P < 0.05). While HVEM mRNA expression did not change significantly (P > 0.05). The 7th and 14th day after TC-1 cells inoculation, the average fluorescence intensity of BTLA expression on the surface of tumor infiltrating lymphocytes was 33.5 and 51.8, respectively, in which there was statistically significant difference (P < 0.05); while the difference of HVEM expression was not statistically significant (57.2 vs 49.3, P > 0.05). (2) The 28th day after inoculation, tumor inhibition rate of HSP70 + psBTLA group was 88%, which was significantly higher than other treatment groups (P < 0.05). The 28th day after TC-1 cells inoculation, combination therapy not only promoted IFN-γ and IL-2 gene (3.12 ± 0.71, 3.20 ± 0.62) expression but also reduced transforming growth factor-β (TGF-β), Foxp3 and IL-10 expression (0.25 ± 0.03, 0.19 ± 0.03, 0.31 ± 0.04; P < 0.05). It also promoted CD₈(+) T lymphocyte infiltration (52 ± 6)/high power field, cytotoxicity (65.5 ± 2.4)%, proliferation (15.0 × 10³ cpm) and cytokine IL-2, IFN-γ secretion (824 ± 51), (1096 ± 112) pg/ml, which were all significantly higher than other groups (P < 0.05).The effect of immunotherapy on tumor can be augmented by the combination of psBTLA which expresses extracellular domain of BTLA and HSP70-TC-1 tumor antigen peptide complex, which could improve the expression of the related immunoregulatory genes to establish a much better microenvironment in favor of anti-tumor immune response against the mice model of the cervix carcinoma.