Evaluation Of The Immune Status Of Patients And The Clinical Efficacy Of Cellular Immunotherapy Combined With Chemotherapy In Lung Cancer

Background and purpose:Lung cancer is the most commonly diagnosed cancer and also the leading cause of cancer death globally. Even with more advanced treatment methods, the prognosis of this disease remains poor with only 17% surviving to 5 years. More effective therapies should be developed to improve its prognosis. Previously, lung cancer is considered as a tumor with weak immunogenicity. With the discovery of immune checkpoint molecules, such as cytotoxic T lymphocyte antigen-4(CTLA-4), programmed cell death protein-1(PD-1), and its ligand PD-L1, immunotherapy of lung cancer achieves new breakthroughs, indicated that immunotherapy will open a new era in the treatment of lung cancer. Studies show that the immune status of patients with lung cancer has a close association with their prognosis. Decreased expression of MHC class I molecules and increased expression of PD-L1 have been reported in lung cancer, which may be the mechanisms of lung cancer cells to escape CTL attack. The expression of major histocompatibility complex class I-related chain A(MICA) and MICB is upregulated in lung cancer, MICA and MICB can be released from the surface of tumor, yielding soluble MICA(s MICA) and MICB(s MICB) in serum. By binding to NKG2 D receptor, soluble MICA/B molecules may block the activation of effector lymph cells by MICA/B, thereby facilitate the escape of tumor from immunosurveillance. The proportion of tumor infiltrating lymphocytes(TIL) could change in lung cancer, such as the regulatory T(Treg) cells. Besides, tumor cells and immune cells could release somes cytokines to evade immune surveillance. Immunotherapy could effectively improve patients’ immunosuppression status and exert anti-tumor effects. Patients with lung cancer have often been found to have functional deficiency in a variety of immunocytes, therefore cellular immunotherapy(CIT) with ex vivo-activated and expanded immunocytes may be feasible and effective in lung cancer patients. CIT has been shown to be effective for lung cancer in some studies. But the way CIT combined with chemotherapy need to be explored. Besides, there is no ideal markers for predicting response to CIT and assessing immune response after CIT. We performed the following experiments to evaluate the immune status of patients and the clinical efficacy of CIT combined with chemotherapy in lung cancer.Methods:1. Tissue samples of patients with pathological diagnosis of non-small cell lung cancer(NSCLC) were collected in this study. Lung cancer surface molecules such as PD-L1, MICA/B and CD8+ TIL in lung cancer tissue were detected with immunohistochemistry, and analysed the association between their expression levels and patients’ clinical features and prognosis.2. Serum samples of patients with newly diagnosed NSCLC in our hospital were collected in this study. The cytokines such as IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α levels in serum were detected with Luminex. The s MICA and s MICB levels were examined by ELISA assays. The association between their expression levels and patients’ clinical features and prognosis was detected.3. In order to explore the possible mechanism of cisplatin enhanced NK cells cytotoxicity effect, NKG2 D ligands(MICA and MICB), CD226 ligands(CD112 and CD155) and TRAIL receptor(DR4 and DR5) on lung cancer cells after cisplatin treatment were evaluated with flow cytometry. Calcein-AM release assays were performed to detect the cytotoxicity of NK cells to lung cancer cells in the presence or absence of cisplatin.4. We retrospectively analysed the clinical data of patients with CIT combined with chemotherapy and chemotherapy alone in our hospital. Progression-free survival(PFS) and overall survival(OS) examined to see whether CIT combined with chemotherapy could have a positive effect on patients prognosis. To assess of the impact of CIT on the immune system, proportion of the immune cells in peripheral blood before and after CIT were detected with flow cytometry. The cytokines levels(IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α) in the serum before and after CIT were detected with Luminex.Results:1. MHC-I was down-expressed in lung cancer, and its expression level was related to patients’ sex, smoking history, histological type, TNM stage and tumor differentiation. Male patients with smoking index 3 400, squamous cell carcinoma, later TNM stage and worse tumor differentiation tended to have lower MHC-I expression. PFS and OS were longer in patients with high MHC-I expression compared with patients with low MHC-I expression(P < 0.05). The expression of PD-L1 in NSCLC tissues was significantly higher than that in adjacent tissues, and its expression level was related to patients’ smoking history and histological type. Patients with smoking index 3 400 and squamous cell carcinoma had higher PD-L1 expression. PFS was longer in patients with low PD-L1 expression compared with patients with high PD-L1 expression(P < 0.05). OS in low PD-L1 expression group was also longer than that of high PD-L1 expression group, but this difference was not significant(P > 0.05). MICA/B expression was also up-regulated in lung cancer, but is had no correlation with patients’ clinical features and prognosis. CD8+ TIL could be seen in tumor stoma and nest, and the number of CD8+ TILs in tumor stroma was higher than in tumor nest. The number of CD8+ TILs was related to patients’ histological type. Patients with squamous cell carcinoma had more CD8+ TILs. PFS was longer in patients with more CD8+ TILs in stroma than those with less CD8+ TILs(P < 0.05), OS also tented to be longer, but this difference was not significant(P > 0.05). There was no difference in PFS and OS between patients with more and less CD8+ TILs in tumor nest.2. TNF-α and IFN-γ of lung cancer were significantly decreased compared with healthy volunteers, IL-6 was significantly increased in lung cancer patients(P < 0.05). IL-2 tended to be decreased, and IL-10 tended to be elevated in lung cancer, but there was no significant difference(P > 0.05). There were no connections between the cytokines levels with patients clinical features and prognosis(P > 0.05). Serum s MICA levels were significantly higher in NSCLC patients than in healthy controls(P < 0.05). s MICB tented to be elevated in lung cancer, but there was no significant difference(P > 0.05). There were no connections between the s MICA and s MICB levels with patients clinical features(P > 0.05). Patients with low s MICA expression had longer PFS than high s MICA expression(P < 0.05). There was no difference in PFS between patients with high and low s MICB levels.3. Expression of MICA, MICB, CD112, CD155, DR4, DR5 were increased after treatment with cisplatin in a time-dependent manner, which may increase the cytotoxicity effect of NK cells. Cisplatin combined with NK cells also showed a synergistic anti-tumor effect to A549 and H446 cells in a time-dependent manner after treatment with cisplatin.4. PFS and OS were significantly longer in the CIT combination group than the control(P < 0.05). Among patients in I-IIIA stage, there was no difference in PFS between the groups(P > 0.05), OS was longer in the CIT group compared to the control, but there was no difference between two groups(P > 0.05). Among patients in the IIIB- IV stage, both PFS and OS were longer in the CIT group than the control(P < 0.05). There were more CD3+CD8+ cells and less Treg cells after CIT(P < 0.05). CD3+CD4+, CD3-CD56+ and CD3+CD56+ cells showed no significant change after CIT(P > 0.05). The cytokine levels showed no difference before and after CIT(P > 0.05).Conclusions:1. MHC-I is down-expressed, PD-L1 and MICA/B was up-expressed in lung cancer. The down-expression of MHC-I and up-expression of PD-L1 had a association with poor prognosis of patients. MICA/B expression had no correlation with patients’ prognosis. CD8+ TILs could be seen in tumor stoma and nest, and the number of CD8+ TILs in tumor stroma was higher than in tumor nest. Stromal CD8+ TILs density has independent prognostic impact in resected NSCLC. The number of CD8+ TILs in tumor nest had no connections with patients’ prognosis.2. TNF-α and IFN-γ of lung cancer were significantly decreased, IL-6 was significantly increased in lung cancer patients. There were no connections between the cytokines levels with patients prognosis. Serum s MICA levels were significantly higher in NSCLC patients than in healthy controls. High s MICA expression had had a association with poor prognosis.3. Cisplatin could increase the expression of MICA, MICB, CD112, CD155, DR4, DR5 in lung cancer cells, and increase the cytotoxicity effect of NK cells to lung cancer cells.4. CIT in combination with chemotherapy could improve the prognosis of patients with lung cancer. There were more CD3+CD8+ cells and less Treg cells after CIT, CD3+CD8+ and Treg cells might be the markers to assess the immune response after CIT.In this study, we found that the immune status of patients with lung cancer has a close association with their prognosis. CIT in combination with chemotherapy can improve the prognosis of patients with lung cancer. The results of this study will provide the theoretical basis for the application of CIT in patients. CIT combined with chemotherapy may be a novel strategy for lung cancer therapy.