The Characteristics And Clinical Relevance Of CD103~+ ILCs In Human Non-small Cell Lung Cancer Immune Microenvironment

BackgroundNon-small cell lung cancer(NSCLC)is the most common lethal malignant tumor,which is highly immunogenic.The tumor microenvironment of NSCLC contains various immune cells,including tumor-infiltrating lymphocytes,tumor-associated macrophages,and tumor-associated neutrophils.These tumor-infiltrating immune cells play significant roles in cancer genesis and development.Innate lymphoid cells(ILCs)are key members of tumor-infiltrating immune cells and take part in a variety of immune pathways.But the phenotypic and functional characteristics of ILCs in different tumors are widely divergent.There are CD103 positive cell subgroups in ILCs,named CD103~+ILCs.It is believed that CD103~+lymphocytes are closely related to the genesis and development of malignant tumors.However,no studies have reported the microenvironment.In this research,we intend to answer this question and determine pathological characteristics of NSCLC patients.MethodsWith the consent of the Ethics Committee,tissue and blood specimens were obtained from 51 NSCLC patients.Firstly,we used flow cytometry analysis to detect CD103 expression level on ILCs surface and determined the proportion of CD103positive ILCs in total CD45~+lymphocytes and total ILCs respectively.Then,we divided tissue infiltrating ILCs into two groups,CD103~+ILCs and CD103~-ILCs,using primary cell separation technology and multicolor flow cytometry analysis to study the phenotype and cytokines secretion of these two groups.Meanwhile,the classification of CD103~+ILCs and CD103~-ILCs were identified by specific transcription factors and surface markers.Finally,the correlation between tumor-infiltrating CD103~+ILCs and clinical features of NSCLC patients was analyzed.Results1. Identification and characteristics of CD103~+ILCs in NSCLC(1)CD103 expression of tumor-infiltrating ILCs was significantly increased compared to normal tissues-derived ILCs(17%vs 5.2%,P<0.0001),while ILCs in peripheral blood did not express CD103.Besides,PD-1 was significantly over-expressed on(2)NSCLC infiltrating CD103~+ILCs underexpressed the transcription factors T-bet and underexpressed emose;neither of them expresses CRTH2.It was demonstrated that CD103~+ILCs were mainly type 3 ILCs,while CD103~-ILCs were mainly ILC1s.(3)For cell phenotypes,we found that:1)Tumor-infiltrating CD103~+ILCs expressed higher levels of CD69,NKP46,and ICOS;expressed lower levels of CD117,CD57,and CCR5 than normal tissues-derived CD103~+ILCs;2)Tumor-infiltrating CD103~-ILCs express higher levels of CD69,CXCR4,CD127,ICOS;expressed lower levels of CD56and CD57 than normal tissues-derived CD103~-ILCs;3)Tumor-infiltrating CD103~+ILCs express higher levels of CD69,CCR5,ICOS,CD159A,and NKP46;expressed lower levels of KLRG1,CD158D,TLR2,and CD57 than tumor infiltrating CD103~-ILCs.Besides,we also tested CCR3,CXCR5,CXCR6,CXCR7,TLR1,TLR4,TLR6,CD161,TRAIL,and CRTH2,and found these surface markers were not expressed on tissue infiltrating ILCs.(4)For cytokine secretion,we found that tumor-infiltrating CD103~+ILCs and CD103~-ILCs secreted a large amount granzyme B.Besides,1)Tumor-infiltrating CD103~+ILCs secreted higher levels of perforin than normal tissues-derived CD103~+ILCs;2)Tumor-infiltrating CD103~+ILCs secreted higher levels of IFN?and perforin than CD103~-ILCs.(5)We discovered that the frequency of tumor-infiltrating CD103~+ILCs was increased in other invasive tumors,including SCLC and EC,in addition to NSCLC.2. Clinical Relevance of CD103~+ILCs frequency in NSCLCSmoking status and tumor location of NSCLC patients were significantly related to the frequency of tumor-infiltrating CD103~+ILCs.Besides,we found that the frequency of tumor-infiltrating CD103~+ILCs in patients with squamous cell carcinoma,larger tumor size,airway dissemination,positive lymph node metastasis,and higher TNM stage was higher,but the effect was not significant.ConclusionOur study has shown that the functional marker CD103 is overexpressed in tumor-infiltrating ILCs in human NSCLC.And tumor-infiltrating CD103~+ILCs and CD103~-ILCs have specific phenotypes and cytokine secretion characteristics.Besides,the frequency of CD103~+ILCs in NSCLC tumor tissues is related to the clinical and pathological features of patients,which highlights the potential targets of these cells for tumor treatments.