In recent years, immunotherapy has made great progress in the treatment of NSCLC, especially the PD-1 and PD-L1 inhibitors. In order to more accurately screen patients who could benefit from immunotherapy, many studies have reported some biomarkers related to the efficacy of immune checkpoint inhibitors [6–9, 18, 29]. Among them, PD-L1 expression on tumor cells was the most widely used. However, several studies have found that some patients whose tumor cells without PD-L1 expressing could also achieve durable response from immunotherapy [30, 31]. Moreover, PD-L1 has been observed not only to be expressed on tumor cells, but also broadly expressed on immune cells, which have an important role in immunotherapy. In addition to the expression of PD-L1, the tumor microenvironment was also inextricably linked to the efficacy of ICI , and the tumor-infiltrating lymphocytes (TILs) could effectively stratify patients with NSCLC [33, 34]. Therefore, a better understanding of the expression of PD-1 and PD-L1 distribute over different cells and their impact on clinical outcomes, which could provide insights for the treatment of NSCLC patient. To achieve this objective, we first conducted a detailed retrospective study of the tumor tissue of 174 patients with NSCLC. The results indicate that immune cells expressing PD-1 or PD-L1 were able to predict the clinical prognosis of patients with NSCLC.
A large number of mechanistic research data showed that PD-1 was expressed primarily on T cells and PD-L1 was mainly expressed on tumor cells. Combination of PD-1 and PD-L1 results in tumor immune evasion. In the present study, we found that PD-1 and PD-L1 were expressed on T cells, NK cells and macrophages using multiplex IHC detection. The average positive rates of PD-1 expression on CD8 + T cells, CD57 + NK cells, CD68 + macrophages and CD163 + M2 macrophages were 14.00%, 4.17%, 5.96% and 9.61% in SA, and 12.27%, 2.64%, 3.07% and 4.89% in TA, respectively. The average positive rates of PD-L1 expression on CD8 + T cells, CD57 + NK cells, CD68 + macrophages and CD163 + M2 macrophages were 13.30%, 5.61%, 12.23% and 8.5% in SA, and 6.86%, 2.88%, 6.22% and 2.5% in TA, respectively. PD-L1 distribution in immune cells was mainly expressed on macrophages and T cells. This type of macrophages (CD68 + PD-L1+) were probably related to the promotion of immune escape and belonged to tumor associated macrophages (TAM) . However, there were few studies about PD-L1 expression on T cells, which is needed further explore.
According to the positive rates of immune profiling, the NSCLC cohort were divided into a strong immune feature group (high) and a weak immune feature group (low). Interestingly, we observed a better survival rate in patients from the low group, suggesting that tumor microenvironment in the low group may be biased towards the activated state, while the high group was the opposite. The ratio analysis of CD8/PD-1 and CD8/PD-L1 in TA and SA revealed that the low group had higher ratio of CD8/PD-1 and CD8/PD-L1, especially the CD8/PD-L1 ratio was significantly different between two groups. Meanwhile, multivariate analysis of random forest trees in two groups indicated that CD68 + PD-L1+, CD57 + PD-L1+, CD8 + PD-L1+, CD68 + in SA and CD68 + PD-L1 + in TA had a major effect on survival rate. These results demonstrated that more immune cells expressing PD-1 or PD-L1 in the high group cause the suppression of immune cells, leading to decrease of survival rate.
The immune profiling had shown a significant impact on the survival outcome of patients with NSCLC. Effects of the single immune characteristic on the survival were further explored. In the study, 16 immune characteristics (including CD57+, CD68+, CD8 + PD-L1+, CD57 + PD-L1+, CD68 + PD-1 + and CD68 + PD-L1 + in TA, and CD57+, CD68+, CD8 + PD-1+, CD8 + PD-L1+, CD57 + PD-1+, CD57 + PD-L1+, CD68 + PD-1+, CD68 + PD-L1+, CD163 + PD-1 + and CD163 + PD-L1 + in SA) according to the optimum cutoff values could effectively distinguish the difference of survival rate in the NSCLC cohort. Results of the Kaplan-Meier survival analysis of these categories were consistent with conclusion of the immune feature analysis, which the more immune cell infiltration exhibited the worse of survival rate in the NSCLC cases. However, several studies discovered that increased NK cells (CD57+) and macrophages (CD68+) infiltration in lung cancer were associated with good clinical outcome [36, 37]. Expression of PD-1/PD-L1 on immune cells was a key factor causing the different results. Owing to the retrospective NSCLC cohort untreated with immunotherapy, association of PD-1/PD-L1 expression on immune cells and outcomes of immunotherapy was not discussed. Taken together, these demonstrations indicated that immune cells expressing PD-1 or PD-L1 could predict the clinical outcome of patients with NSCLC.