Clinical Relevance And Mechanistic Study Of TCR Repertoire Usage As A Biomarker For Immunotherapy In NSCLC Patients

Background:Lung cancer has been the leading cause of morbidity and mortality among all cancers and has become an important disease that endangers the health of the Chinese people.In recent years,many clinical studies have shown that immune checkpoint inhibitors such as PD-1/PD-L1 monoclonal antibody bring efficacy and survival benefits to patients with various cancer such as lung cancer,malignant melanoma,and gastrointestinal cancer.However,only about 20%patients can benefit from immunotherapy,thus exploring biomarkers that effectively predict efficacy is the key to personalized immunotherapy.The current research focuses on PD-L1 expression and biomarkers in tumor tissues such as TMB but has not yet achieved satisfactory clinical predictive efficacy.Integrating the predictive system of tumor internal factors and tumor microenvironment may lead to more accurate prediction of the efficacy of immunotherapy.The immune repertoire is defined as the total number of functionally diverse T cells and B cells possessed by the individual's circulatory system at any given time,covering the diversity and specificity of the individual's immune status.The recognition of tumor antigen by T cell receptors(TCRs)and inducing proliferation of T cell is a key process in the activation of host immune responses against cancer cells.Previous study has revealed that pre-existing PD-1+CD8+T cells represented the functional cytotoxic T cells against cancer and underwent exhaustion induced by immune checkpoint pathway,which could be successfully detected in peripheral blood.Our study intends to explore the prediction role of TCR clone shared in tumor infiltrating lymphocytes and peripheral blood and explain the mechanism at the tumor microenvironment level.T cells play an important role in anti-tumor immune responses.The T-cell receptor(TCR)is a surface specific molecule present on T cells that is responsible for recognizing antigen peptides bound to the major histocompatibility complex(MHC).Based on the attainment and function of the TCR,the TCR repertoire is a mirror of the human immune responses during cancer.Millions of different T-cell clones comprise a diverse TCR repertoire that can be characterized in tissues or in peripheral blood using high-throughput TCR sequencing.The recognition of tumor neoantigen peptides by clonal proliferated T cell receptor(TCR)is a critical process in the activation of host immune response against cancer cells.Previous study on melanoma have revealed that pre-existing PD-1+CD8+T cells represented the functional cytotoxic T cells against cancer and underwent exhaustion induced by immune checkpoint pathway,which could be successfully detected in peripheral blood.Methods:71 patients with advanced NSCLC who received PD-1/PD-L1 immunotherapy were enrolled in our study,among which 31 cases with paired with tumor tissue samples and peripheral blood PD-1+CD8T cells,and 40 cases with peripheral blood PD-1+CD8T cells.We performed TCR sequencing of tumor infiltrating lymphocytes and peripheral blood PD-1+CD8T cells through high-throughput immune library technology,and we performed high-throughput sequencing of tumor tissue DNA and ctDNA based on 1021 Panel.The prediction of the neoantigen is based on the gene mutation and HLA type,neoantigen and wild type were used to stimulate peripheral blood lymphocytes from the patients in vitro.In addition,we analyzed RNA expression,cytokine in plasma and protein expressioni on cell surface to evaluate the tumor miicroenviomennt.Results:In this study,patients with advanced non-small cell lung cancer who received PD-1/PD-L1 immunotherapy in the medical department of the Cancer Hospital of the Chinese Academy of Medical Sciences were enrolled in the group.TCR were sequenced by high-throughput technology in 31 cases with paired tumor tissue samples and peripheral blood PD-1+CD8T cells.A number of T cell clones in tumor infiltrating lymphocytes(TILs)were shared in PD1+CD8 T cell of peripheral blood.The results showed that patients with higher ratio of shared clones in TILs were more likely to benefit from immunotherapy.In order to verify this result,we predicted the neoantigen with the sequencing results of tumor specimens and the HLA type of patients,then we constructed neoantigen peptide and stimulated PBMC from patient.Both results from T cell activity and TCR sequencing showed that shared clones could identify tumor neoantigen.We further detected RNA expression of baseline tumor samples,the results that showed patients with higher ratio of shared clones accompanied by inflammatory tumor microenvironment which is beneficial to immunotherapy.In order to explore of the mechanisms of the resistance to immunotherapy in patients with higher shared clone ratio,we performed multiple immunohistochemical staining and found that the presence of immune checkpoints other than PD-1/PD-L1,such as TIM3 and LAG3,may be the reason why patients fail to benefit from single-agent immune checkpoint inhibitors.Next,we monitor the dynamic changes of frequency in peripheral blood of the ratio of shared clone and find that its dynamic changes are consistent with the radiological evaluation and reflects the disease process.For patients who are unable to obtain pre-treatment biopsies,it is also important to explore non-invasive biomarkers through peripheral blood.We analyzed the diversity of baseline peripheral blood PD-1+CD8T cells in 40 patients who were unable to provide tumor tissue specimens,found that patients with higher TCR diversity at baseline were more likely to benefit from anti PD-1/PD-L1 therapy which achieved a sensitivity of 0.87 and a specificity of 0.94 in stratifying clinical response of ICBs,while non-predictive value were found in peripheral blood T cells without sorting.In addition,neoantigen-specific T cells were expected to experience clonal expansion after response to ICB treatments,which can be reflected by TCR clonality.In the present study,patients with an increased PD-1+CD8+TCR clonality had improved survival outcomes compared to those with decreased TCR clonality.Importantly,PD-1+CD8+TCR clonality displayed a consistent changing positively with tumor burden index.These results indicated that PD-1+CD8+T cells underwent clone expansion after response to anti-PD-1/PD-L1 therapy.Taken together with our study,dynamic monitoring functional T cells or their TCR repertoire may be a useful complementary and potential surrogate biomarker for early differentiation of the patients benefiting from ICBs,once the pre-ICB predictor is not available.Conclusion:In summary,we revealed the characteristics of TCR repertoire and its immune microenvironment in advanced NSCLC patients benefit from PD-1/PD-L1 treatment.Our results showed the TCR clone shared in TILs and peripheral PD-1+CD8+T cells play an important role in immunotherapy and further proven its ability of recognize tumor neoaitgen in vitro.On this basis,we put forward a non-invasive method to stratify NSCLC patients for ICB treatment by detecting diversity and clonality of peripheral PD-1+CD8+TCR.