CD11b-CD27-NK Cells Are Associated With The Progression Of Lung Carcinoma

Lung cancer is one of the leading causes of cancer death worldwide. Identification of innovative and efficacious therapeutic strategies, including immunotherapies, is therefore urgently needed. To achieve this goal requires a thorough understanding of anti-tumor immune responses and specifically of the role of NK cells in this process. NK cells are innate lymphocytes that have the natural ability to recognize and kill aberrant cells, including cancer cells. However, elegant studies have provided evidence that NSCLC-infiltrating NK cells exhibit profound defects in degranulation and IFN-y production. These defects are associated with coordinated changes in the NK-cell receptor repertoire, suggesting a tumor-induced local impairment of NK cells. These findings suggest that it is not the quantity but the quality of intratumoral NK cells that account for their dysfunction in NSCLC. Notably, this study also revealed that intratumoral NK cells expressed dramatically lower levels of killer-cell immunoglobulin-like receptor (KIR) than did peripheral blood NK cells from the same patients. Similarly, another study demonstrated the loss of KIR expression on intratumoral NK cells when compared with NK cells from peritumoral lung tissues. Interestingly, the disappearance of KIR on tumor-infiltrating NK (TINK) cells is also observed in human breast tumors, whereas KIR is present on NK cells infiltrating healthy mammary tissue (Mt-NK cells), confirming that TINK cells could not exert cytotoxic functions like non-educated cells. In addition, the terminal differentiation of NK cells is characterized by the acquisition of KIRs and decreases in NKG2A. Accordingly, the study describing the loss of KIR expression on TINK cells from patients with breast cancer suggests that a strong inhibitory environment, such as the tumor microenvironment, can reorient or reverse the transcriptional program of NK cell maturation toward a nonreactive self-tolerant profile. Several recent studies showing that NK-cell developmental programming is not entirely fixed and that mature NK cells can be reeducated by their environment support this hypothesis. In light of the aforementioned studies, it is likely that the tumor microenvironment has a negative impact on NK-cell maturation.Based on these considerations, our study is to investigate the phenotypic profile of TINK cells based on the expression of the NK-cell maturation markers CD lib and CD27. Through these studies we hope to find out the relationship between NK-cell development and function in the context of cancer. The major findings and conclusions of our study are shown as follows:1. DN NK cells are present within the TINK-cell populationTo determine the maturation status of TINK cells, we compared the expression of CD lib and CD27on TINK cells with that on pNK cells from autologous patients and healthy control subjects. There was no significant difference in CD27expression among these three types of NK cells. However, TINK cells exhibited an evident reduction in CDllb expression when compared with the other two types of NK cells. Furthermore, we detected a DN (CDllb"CD27-) NK-cell subset that accounted for approximately35%of the TINK population. By contrast, the proportion of CDllb+SP (CDllb+CD27") NK cells was lower within the TINK population than within the pNK population. These data provide the first demonstration that a large population of DN NK cells exists within the TINK-cell population in human tumors.2. DN NK cells exist mainly within the CD16" TINK-cell populationPrevious reports have demonstrated that NK cells infiltrating human non-small-cell lung cancer are enriched in CD16" cells. Similarly, we found that the expression of CD16(Fey receptor Ⅲ) was dramatically reduced on TINK cells from NSCLC patients as compared with pNK cells from autologous patients and healthy control subjects. Given that CD16not only facilitates antibody-dependent cellular cytotoxicity and killing but also serves as an NK-cell maturation marker, we analyzed the expression of CDllb and CD27on CD16" and CD16+TINK cells. Interestingly, approximately90%of CD16+TINK cells were CDllb+SP NK cells. However, among CD16’TINK cells, approximately60%were DN NK cells. Thus, among the four subsets defined by CDllb and CD27, DN NK cells comprise the majority of the CD16" TINK-cell subpopulation, indicating that TINK cells are phenotypically immature.3. TINK cells display an immature and inactive phenotypeTo investigate the maturation status of TINK cells, we studied other NK-cell receptors related to NK-cell maturation. Downregulation of CD57, which is expressed on highly mature NK cells, was observed on TINK cells, whereas no change in CD57expression was observed on pNK cells from autologous patients and healthy control subjects. Conversely, CD127and CD117, which are barely detectable on traditional mature NK cells, were expressed on TINK cells. Collectively, these results demonstrate that TINK cells display an immature phenotype. We also examined the expression of NK-cell receptors that have been reported to be involved in NK-cell activation. As expected, CD226and NKp30were expressed at significantly lower levels in TINK cells than in pNK cells from autologous patients and healthy control subjects. These results support our observation that CD16expression was downregulated on TINK cells. In contrast, NKG2A, an inhibitory receptor reported to be highly expressed by immature NK cells, was slightly upregulated on TINK cells when compared with pNK cells from autologous patients and healthy control subjects. Together, these data confirm that TINK cells exhibit an inactive state, supporting previous findings that they have poor cytotoxic capacity. Based on these findings, we hypothesized that TINK-cell dysfunction is associated with their immature phenotype, as the TINK-cell population contains a large subset of DN NK cells.4. DN NK cells are responsible for the immature phenotype of TINK cellsTo determine whether the appearance of DN NK cells has any impact on TINK-cell maturation, we evaluated the phenotypes of the DN NK-cell subset and the CDllb+SP NK-cell subset of the larger TINK-cell population separately. The proportions of the DN NK-cell subset expressing CD16and CD57were significantly lower than those within the CDllb+SP NK-cell subset. Moreover, CD127, a key marker of immature NK cells, was expressed at slightly higher levels on DN NK cells than on CDllb+SP NK cells. These results confirmed that DN NK cells within the TINK-cell population display an immature phenotype.To further dissect the roles of the DN NK-cell and CDllb+SP NK-cell subsets of TINK cells, we assessed the expression of activating NK receptors on cells of the two subsets. By comparing the median percentages of NK cells positive for CD226and NKp30, we found that DN NK cells expressed lower levels of NKp30and even more obviously lower levels of CD226. Hence, the DN NK-cell subset displays an inactive phenotype. Together, our observations concerning the DN NK-cell subset within tumors are consistent with the immature developmental status of human DN NK cells reported in recent studies. Thus, we hypothesized that the presence of DN NK cells influences the phenotypic and functional attributes of TINK-cell population.5. The appearance of DN NK cells is associated with tumor progression in humansTo elucidate the significance of the presence of DN NK cells in tumors, we evaluated the effect of DN NK cells on clinical outcomes. The NSCLC patients included in this study were classified into3groups (Ⅰa/b, Ⅱa/b and Ⅲa/b) based on tumor node metastasis (TNM). Remarkably, the frequency of DN (CD1lb"CD27") NK cells within the TINK-cell population increased as tumors progressed. We next searched for correlations between the frequency of DN NK cells and the size of the tumors. Notably, the frequency of DN NK cells within the TINK-cell population positively correlated with the maximum diameter of the resected tumors. These results show that DN NK cells might play an important role in tumor staging.6. The kinetics of DN NK-cell accumulation in lung tissue are associated with tumor progression in vivoTo further assess whether tumor-infiltrating DN NK cells are associated with tumor progression, we examined the expression of CDllb and CD27on TINK cells from lung tissue in a intrapleurally implanted murine Lewis lung cancer (LLC) model. Consistent with previous reports, the frequency of TINK cells in the lung tissue gradually decreased over time after LLC injection. Analysis of the frequency of each NK-cell subset within the TINK-cell population at different time-points revealed several findings. We observed striking differences in the frequency of each subset within the lung TINK-cell population at each time-point (here depicted as0d,10d and20d). The injection of LLC induced a reduction in the proportion of CDllb+SP NK cells within the lung tissue and a concomitant increase in the proportion of DN NK cells over time. Collectively, our findings suggest that the frequency of DN NK cells within the TINK-cell population is associated with the progression of lung carcinoma.In conclusion, our study has shed light on NK-cell maturation within human tumors. To our knowledge, this study is the first to report that a significant immature CDllb"CD27"(DN) NK-cell subset exists within the tumor-infiltrating NK-cell population and the first to demonstrate the correlation between DN NK-cell frequency and tumor progression. More importantly, our data provide evidence that the tumor microenvironment impedes the functional maturation of NK cells locally, rendering them less tumoricidal and thereby supporting cancer progression.