In this study, we found that tumor infiltrating lymphocytes were an independent predictive factor of PD-1 blockade treatment response, and we presented a cutoff value for tumor infiltrating lymphocytes to discriminate between responder and non-responder groups. In addition, we demonstrated that tumor infiltrating lymphocytes status evaluated in biopsy samples and archival tissues is a feasible approach for predicting therapeutic response.

Examination of the tumor microenvironment in patients treated with immune checkpoint inhibitors has revealed an association between the tumor infiltrating lymphocytes count both before and after treatment and therapy response [8, 22]. Recently, in addition to evaluation of tumor infiltrating lymphocytes based on H&E or immunohistochemical staining, studies were conducted to evaluate tumor infiltrating lymphocytes using a developed a multiplex technique. [23] However, this method cannot be routinely used for evaluation of tumor infiltrating lymphocytes in most pathology laboratories because of the high cost of equipment and the complicated technology, and there is still a need technical verification. Therefore, we tried to evaluate the number of tumor infiltrating lymphocytes as objectively as possible using a method that can be applied in routine practice and used this method as an index to predict PD-1 blockade response. As a result, CD3+ T cells greater than 120 per HPF, and CD8+/FOXP3+ T cell ratios greater than 4:1 were found to be associated with clinical benefit in PD-1 blockade treated patients.

The binding of PD-1 to PD-L1 causes T cell to become anergy and contributes to the immune escape of tumor cells [24,25,26]. CD8+ cytotoxic tumor infiltrating lymphocytes can kill tumor cell directly and general T-cells expressing pan T cell marker, CD3, interplay with different subsets of immune cells via cytokine such as INFγ [24, 27]. Our study results are in line with the results of Schalper et al. [23], who noted that the presence of elevated CD3+ and CD8+ T cells is consistently associated with survival and reported that CD8 was the only independent prognostic factor in non-small cell lung cancer. Taken together, these tumor specific CD3+ and CD8+ subsets of T cells were involved in adaptive immunity and might be important not only as a prognostic marker but also a predictive biomarker for PD-1 blockade in non-small cell lung cancer.

In addition, we should note the role of FOXP3+ tumor infiltrating lymphocytes in immunotherapy; the CD8+/FOXP3+ T cell ratio was a more important factor in predicting immune checkpoint inhibitor response than the absolute number of CD8+ T cells. Accumulating studies have demonstrated a large number of tumor-infiltrating FOXP3+ Tregs in various tumors. In breast [28], gastric [29], and ovarian cancer [30]; in the majority of solid tumors in the cervix, kidney, and breast; and in melanoma, decreased ratios of tumor-infiltrating CD8+ T cells to FOXP3+ Tregs or increased tumor-infiltrating FOXP3+ cells were shown to be correlated with poor prognosis [28]. In contrast, FOXP3+ tumor infiltrating lymphocytes in colorectal cancer were heterogeneously present in both the suppressive and non-suppressive forms, and the effect of FOXP3+ tumor infiltrating lymphocytes on prognosis is controversial [31,32,33]. Based on our results, FOXP3+ tumor infiltrating lymphocytes in non-small cell lung cancer might have a suppressive function similar to that found in other solid tumors and may inhibit the cytotoxic activity of the accompanying CD8+ T cells and affect the immunotherapy response [34]. In vitro coexistence of Tregs suppresses nivolumab‐induced release of interferon‐γ from effector T cells [35]. In short, Tregs may interfere with the immunostimulatory effects of PD‐L1 inhibitors in non-small cell lung cancer.

In most cases (31/38, 82%), tumors were positive for PD-L1, and the PD-L1-positive tumors were more likely to contain tumor infiltrating lymphocytes than PD-L1-negative tumors. Several studies have reported that PD-L1 is more frequently expressed in such a favorable immune microenvironment [36,37,38], which supports the notion that PD-L1 expression by tumors is a mechanism of adaptive immune resistance. However, PD-L1 expression was not a predictor of PD-1 blockade response. This finding was similar to the results of the checkmate 017 and 057 trials, suggesting that the majority of patients in our cohort used nivolumab, and thus, PD-L1 expression had little effect on immune checkpoint inhibitor response [17, 39]. We think that the lack of an association between PD-L1 expression and drug response is likely not related to performance of the PD-L1 assay but is rather a function of the complex interactions between tumors and the immune system. In this context, the evaluation of tumor infiltrating lymphocytes is a more important key factor in accurately predicting immune checkpoint inhibitor response in patients with PD-L1 expression and could compensate for the limitations of PD-L1 expression.

The second major outcome of our study was demonstration of the feasibility of using biopsy specimens to evaluate tumor infiltrating lymphocytes. In examining biopsy specimens from tumors, the representativeness of a small biopsy and its ability to adequately reflect the heterogeneous tumor microenvironment has always been an issue. Obeid et al. evaluated CD8+ tumor infiltrating lymphocytes density using eight sampling strategies and examined which method resulted in measures that were most similar to those obtained with the whole tumor [40]. Although different tumor sampling strategies may yield discordant tumor infiltrating lymphocyte density results, sampling 10–20 small areas randomly, sampling the tumor center, or taking large core biopsies (10 × 1 mm) may best represent the whole tumor [40]. Based on this result, we included only biopsy tissues in which the size of the specimen was more than 10 mm and the peripheral normal tissue was nearly absent, which were most likely to be obtained from the center of the tumor. As a result, biopsy tissues showed less CD3+ and CD8+ tumor infiltrating lymphocyte density than resected tissues, but the difference was not statistically significant and consequently did not affect treatment response prediction. Considering that many lung cancer patients receiving immunotherapy have advanced stage disease, a biopsy specimen may be the only sample type that can be used to assess the patient tumor microenvironment status. Therefore, obtaining biopsy tissue of appropriate size from the center of the tumor at the time of diagnosis can be great help for predicting a patient’s immunotherapy response.

PD-1 blockade therapy seems induce a definite change in tumor infiltrating lymphocytes infiltration and PD-L1 expression in tumor cells. Tumeh et al. previously reported that a greater increase in CD8+ density from baseline to post-dosing biopsy was significantly correlated with immune checkpoint inhibitor in responding malignant melanoma patients [8]. Similar to these results, our study also showed a significant decrease in CD8+ tumor infiltrating lymphocytes in paired samples from patients who did not respond to PD-1 blockade. Another non-responder had increased CD8+ T cells after treatment, but it was difficult to make a direct comparison because the sampling sites before and after treatment were different. Nevertheless, in the pretreatment sample, the CD3+ tumor infiltrating lymphocytes number and FOXP3+/ CD8+ tumor infiltrating lymphocyte ratio were lower than the cutoff value, and the patient did not respond to the PD-1 blockade regardless of PD-L1 status. As a result, tumor infiltrating lymphocytes were confirmed to be a major predictor of PD-1 blockade response that can complement PD-L1 assessment.

There are limitations and possible bias in this study. The sample number was not large, and because of the nature of a retrospective study, it is possible that patient selection was biased according to sample availability. In addition, 4 out of 5 metastatic lesions in our cohort were in the lymph nodes, and this might have affected the proportion of tumor infiltrating lymphocytes. Although we attempted to analyze only the tumor cells and the tumor microenvironment while excluding the normal lymph node portion, there was still the possibility of bias caused by lymph node specimens. Despite these limitations, we found that tumor infiltrating lymphocyte quantitation using an immunohistochemical technique, which can be used in routine practice in patients with lung cancer treated with immune checkpoint inhibitors, is an important factor in predicting therapeutic response.

In conclusion, we evaluated tumor infiltrating lymphocytes in 38 pretreatment non-small cell lung cancer samples from immune checkpoint inhibitor-treated patients. A high number of CD3+ T cells and a low FOXP3+/CD8+ T cell ratio were identified as independent factors predicting the response to PD-1 blockade. In addition, properly sampled biopsy tissue and well-preserved archival specimens can both be useful in evaluating tumor infiltrating lymphocyte status. Based on our results, tumor infiltrating lymphocytes might become a promising biomarker that may also guide therapeutic decisions.