The Relationship Between T-cell Differentiation And Clinical Features And Curative Effect Of Locally Advanced Cervical Cancer And Analysis Of Specific Immune Responses Of MAGE-A1 And MAGE-A3 In Vitro

Objective:1)To analyze the common hematological parameters and their ratios in peripheral blood of patients with locally advanced cercival cancer（LACC）before initial treatment,Including absolute lymphocyte count（ALC）,neutrophil-lymphocyte ratio（NLR）,monocyte-lymphocyte ratio（MLR）and platelet-lymphocyte ratio（PLR）,in the prognosis of patients with locally advanced cervical cancer.2)To analyze the distribution characteristics of naive,central memory and effective memory T cell subsets in peripheral blood and tumor tissues of patients with locally advanced cervical cancer,and its relationship with clinical characteristics of patients,as well as its predictive value for short-term curative effect of patients.3)The frequency of CD4-positive and CD8-positive T cells expressing IFN-γ,TNF-α,IL-2,IL-13 and MIP-1βalone and co-expressing IFN-γ+TNF were detected in peripheral blood mononuclear cells of patients with locally advanced cervical cancer after being stimulated by melanoma antigen-A1（MAGE-A1）and melanoma antigen-A3（MAGE-A3）in vitro.Methods:1)A retrospective analysis was performed on 175 LACC patients who first visited the Tumor Hospital affiliated to Xinjiang Medical University from August 2016 to December 2019.The general information,clinicopathological results,laboratory and radiological examination results of the patients before treatment were reviewed and completely recorded.Firstly,the best cutoff values of ALC,NLR,MLR and PLR in predicting PFS and OS of patients before initial treatment were determined by drawing ROC curve,and the prognosis was analyzed by the best cutoff values and the survival curve was drawn According to the data type,the relationship between each parameter and the clinical characteristics of locally advanced cervical cancer patients was analyzed by chi-square or rank sum test Univariate analysis was performed by Kaplan-Meier method,and multivariate COX regression analysis was used to determine the independent prognostic factors of locally advanced cervical cancer patients.2)The cases in the second part come from the first part.According to the strict inclusion and exclusion criteria,55 patients with locally advanced cervical cancer were included in this study,and 26healthy people who matched the age of the case group in the same period were enrolled.The distribution of naive CD4⁺,CD8⁺,central memory and effective memory T cell subsets in peripheral blood and tumor tissues of patients with locally advanced cervical cancer and healthy control group were detected by flow cytometry.The distribution differences of naive,central memory and effective memory T cell subsets in peripheral blood and tumor tissues of locally advanced cervical cancer group were compared with those of healthy control group and locally advanced cervical cancer group.Univariate analysis was used to screen the related factors for predicting the short-term curative effect of locally advanced cervical cancer,and multivariate analysis was performed by binary Logistic regression to further screen the independent predictors of the short-term curative effect of locally advanced cervical cancer patients.3)The cases in the third part come from the second part.According to the strict inclusion and exclusion criteria,34 newly diagnosed locally advanced cervical cancer patients admitted to the radiotherapy center of the Affiliated Tumor Hospital of Xinjiang Medical University from February 2018 to April 2019 were collected,and 16healthy physical examinees were included in the physical examination center of the Affiliated Tumor Hospital of Xinjiang Medical University in the same period.Patients’peripheral blood mononuclear cells were stimulated with synthetic specific antigen peptides of MAGE-A1 and MAGE-A3.the frequency of CD4⁺and CD8⁺cells secreting IFN-γ,TNF-α,IL-2,IL-13 and MIP-1βwas detected by in vitro cell culture combined with intracellular cytokine staining and multicolor flow cytometry The killing activity of CTL was reflected by detecting the frequency of CD8⁺T cells secreting IFN-γ,and the specific multifunctional T cells induced by MAGE-A1 and MAGE-A3 were reflected by detecting the frequency of CD4⁺and CD8⁺T cells co-expressing IFN-γ+IL-2+and IFN-γ+TNF-α+.Results:1)Before initial treatment,the median progression-free survival time of high NLR group was shortened compared with low NLR group（20.1 months vs.30.7 months）,high MLR group（20.1months）and low MLR group（no outcome event occurred during the follow-up period）;However,before the initial treatment,the median survival time of patients with high ALC（no outcome events in the follow-up time）,low ALC group（33 months）and high PLR group（no outcome events in the follow-up time）was prolonged.Progression-free survival analysis showed that FIGO staging was a risk factor for PFS in patients with locally advanced cervical cancer（HR=2.339,95%CI（1.22-4.48）,P=0.002）,and concurrent chemorotherapy was a protective factor for PFS（HR=0.213,95%CI（0.11-0.43）,P=0.000）.Total survival analysis showed that:before chemoradiation and initial treatment MLR is affecting the prognosis of patients with locally advanced cervical cancer OS independent factors（P<0.05）,concurrent chemoradiotherapy for the prognosis of patients with locally advanced cervical cancer protection factor（HR=0.229,95%CI（0.07-0.81）,P=0.001）,and before initial treatment high MLR as its prognostic risk factors（HR=4.933,95%CI（1.39-17.54）,P=0.000）.2)CD4⁺naive T cells（34.99±18.51 vs.24.13±17.16）and CD4⁺central memory T cells（34.99±18.51 vs.23.97±16.35）in patients with locally advanced cervical cancer were higher than those in healthy controls.However,CD4⁺effector memory T cells（12.66±10.40 vs.47.16±22.52）and CD8⁺effector memory T cells（11.95±12.18 vs.45.52±22.78）were lower than those in healthy controls（P<0.05）.CD4⁺naive T cells（34.99±18.5 vs.1.62±3.01）and CD4⁺central memory T cells（32.44±13.64 vs.8.83±14.86）in peripheral blood of patients with locally advanced cervical cancer were higher than those in tumor tissues（P<0.001）.However,CD4⁺effector memory T cells（12.66±10.40 vs.38.14±32.47）and CD8⁺effector memory T cells（11.95±12.18 vs.59.11±27.61）in tumor tissues were lower than those in peripheral blood（P<0.001）.The ratio of CD8⁺effector memory t cells in FIGO stage ⅢA-ⅢB patients was lower than that in IB₂-IIB patients（49.69±34.36 vs.69.50±27.36）,while the ratio of CD8⁺central memory T cells was higher than that in IB₂-IIB patients（9.09±15.56 vs.1.96±3.06）The ratio of CD8⁺effect memory T cells in tumor tissues of patients with HPV infection,regional lymph node metastasis and SCC-Ag increased（P<0.05）.Univariate analysis showed:histological grade（OR:1.57,95%CI（0.97-2.55）,P=0.048）,regional lymph node metastasis（OR:2.00,95%CI（1.20-3.33）,P=0.002）,TSGF（OR:1.73,95%CI 0.94-3.16,P=0.035）,The variables with P<0.1 in the above univariate analysis were included in Logistic regression analysis,and the results showed that the naive CD8⁺T cells in tumor tissues were clinically complete remission（Clinical complete remission,c CR）and（Clinical partial remission,c PR）are two independent predictors of short-term response.Before initial treatment,patients with low level of naive CD8⁺T in tumor tissue are more likely to achieve clinical complete remission.3)Among 34 patients,CD4⁺T cells in peripheral blood of 8 patients and CD8⁺T cells in peripheral blood of 7patients produced immune response to MAGE-A1 specific antigen polypeptide,with positive rates of 23.5%and 20.6%respectively.The level of IL-13 secretion of MAGE-A1-specific CD8⁺T cells in LACC group was（1.12±2.13）%higher than that in healthy control group（0.48±1.05）%,while the level of MIP-1βsecretion was（1.57±3.70）%lower than that in healthy control group（6.96±9.22）%;Among 22 patients stimulated by MAGE-A3,CD4⁺T cells in peripheral blood of 2 patients and CD8⁺T cells in peripheral blood of 3 patients produced immune response to MAGE-A3 specific antigen polypeptide,with positive rates of 9.1%and 13.6%respectively.The level of IL-2+secretion of MAGE-A3 specific CD8⁺T cells in LACC group was（1.77 2.06）%higher than that in healthy control group（10.12 13.11）%,P<0.05.The TNF-α+secretion level of MAGE-A3 specific CD8⁺T cells in LACC group was（2.17±2.78）%,higher than that in healthy control group（0.67±0.68）%,P<0.05).The level of IL-13 secretion of MAGE-A3 specific CD8⁺T cells in LACC group was（1.17±1.58）%higher than that in healthy control group（0.26±0.65）%,P<0.05).The MIP-1βsecretion level of MAGE-A3specific CD8⁺T cells in LACC group was（2.03±4.57）%higher than that in healthy control group（10.04±12.08）%,P<0.05).The frequency of CTL reaction in 22 patients with MAGE-A3 stimulation was（1.49±0.32）%,which was higher than that in the unstimulated group（1.28±0.27）%,P<0.05)。After receiving MAGE-A1 stimulation,the proportion of cells secreting IFN-γand IL-2 in CD4⁺T cell subsets of LACC patients was（3.30±2.06）%higher than that of healthy control group（0.6±0.39）%,while the proportion of cells secreting IFN-γand TNF-αwas（1.60±1.87）%higher than that of healthy control group After receiving MAGE-A3 stimulation,the proportion of cells secreting IFN-γand IL-2 in CD4⁺T cell subsets of LACC patients was（3.91±4.75）%higher than that of healthy control group（0.58±0.39）%,while the proportion of cells secreting IFN-γand TNF-αwas（1.71±1.82）%higher than that of healthy control group.Conclusion:1)FIGO staging and concurrent chemoradiotherapy are independent prognostic factors of PFS in patients with LACC;Concurrent chemoradiotherapy and MLR can be regarded as independent prognostic factors of OS.MLR is expected to be a potential biomarker for monitoring recurrence and metastasis after LACC treatment.2)Naive CD8⁺T cells in tumor tissue before initial treatment can be used as an independent predictor of clinical complete remission in locally advanced cervical cancer patients after treatment.Patients with low level of naive CD8⁺T cells in tumor tissue during initial treatment are more likely to have clinical complete remission.3)Tumor antigen polypeptides MAGE-A1 and MAGE-A3 can be used to prepare ideal tumor antigen peptide vaccines for patients with locally advanced cervical cancer.