Restoration of Peripheral Intermediate and Classical Monocytes Expressing HLA-DR in Patients With Lung Adenocarcinoma After Platinum-Based Chemotherapy.

Introduction

Lung adenocarcinoma is one of the most prevalent subtypes of non-small cell cancer (NSCLC); it has a poor prognosis, with a 5-year life expectancy of less than 30% when detected at an advanced stage (IIIB-IV), 1 which occurs in most cases. 1 The main therapeutic option for those patients who cannot undergo surgical resection is platinum-based chemotherapy. 2 Chemotherapy can improve the quality of life of patients with cancer and prolong their survival time, even in the most advanced clinical stage (stage IV). 3

The immune system has the ability to detect and eliminate neoplastic cells. Some tumors express antigens that can be recognized by T CD8 + cytotoxic lymphocytes, 4 and specific antibodies against tumor antigens can lead to the destruction of tumor cells. 5 , 6 However, in most clinical cases of cancer, the antitumor immune response is limited by the action of immune suppressor cells of lymphoid origin such as regulatory T cells (CD4 + , CD25 + , and FoxP3 + ) 7 and programmed death ligand 1 (PD-1 + ) cells. 8 Furthermore, some macrophages in the inflammatory infiltrate have a pro-tumor effect, promoting neovascularization and tumor metastasis. 9 Macrophages originate from circulating monocytes, which are a heterogeneous myelogenous population. Three subpopulations of monocytes have been described based on the expression of CD14 and CD16: classical (CD14 ++ , CD16 − ), comprising 90% of circulating monocytes, intermediate (CD14 ++ , CD16 + ), and nonclassical monocytes (CD14 Neg/+ , CD16 + ). 10 , 11 These subpopulations of monocytes exhibit different inflammatory, angiogenic, and phagocytic activity. 12

The immunological parameters in neoplastic tissue and peripheral blood that have been associated with a poor prognosis in cancer include the increase in the expression of the PD-1 molecule in lymphocytes of neoplastic tissue 13 , 14 and in T cells of peripheral blood 8 ; they also include an increase in the frequency of regulatory T cells 7 and HLA-DR Low/neg monocytes. 15 – 17 It has been recently reported that CD14 + HLA-DR Neg/Low peripheral cells have immunosuppressive functions in patients with different types of cancer. 16

Furthermore, it has been reported that cisplatin has a pleiotropic immunomodulatory effect. In addition to inhibiting mitosis, this antineoplastic drug increases the migration and proliferation of effector cells such as T CD8 + lymphocytes specific for tumor antigens, as well as migration of macrophages and memory T cells. 18 Moreover, cisplatin increases the lytic activity of cytotoxic lymphocytes and the sensitivity of neoplastic cells to the action of cytotoxic cells; it also upregulates the expression of MHC/HLA class I molecules in macrophages. 18

A recent study proposed the inclusion of systemic and local immunological parameters (Immunoscore) in the criteria used for cancer staging and to predict the outcome of chemotherapy. 19 , 20 Although several experimental studies have pointed out the importance of the immune system in the development of cancer, 21 there have been few studies focused on the usefulness of immune parameters as indicators of the evolution of patients with lung adenocarcinoma, one of the most prevalent types of lung cancer. The present study analyzed the ratio between activation and suppression in immune subpopulations of peripheral blood from patients with pulmonary adenocarcinoma, before and after the third cycle of mainly platinum-based chemotherapy. This was done with the aim of identifying changes in the activation and/or suppression of immune subpopulations of peripheral blood after chemotherapy. A panel of molecules was used to assess the pro-inflammatory context; the panel included cytokines, chemokines, and growth factors present in the plasma of patients and controls.

Methods

Ethics Statement

This study was approved by the Scientific and Bioethics Committee of the National Institute of Respiratory Diseases “Ismael Cosío Villegas.” All participants provided written informed consent.

Patient Recruitment

A total of 9 patients with a diagnosis of lung adenocarcinoma were recruited among those attending the Oncology Pneumology Service and chemotherapy units of INER; 90% of them were female, with a mean age of 56.3 years (35/74). Patients with cardiac comorbidities, renal failure, and uncontrolled diabetes were discarded. The reference values of the populations under study were estimated by recruiting 9 matched healthy controls.

Sample Preparation

Five to 10 mL of peripheral blood from study participants was collected using the Vacutainer system (Becton Dickinson, NJ, USA). Subpopulations of interest were determined by flow cytometry in anticoagulated blood, as described in the following sections. The molecules of interest were measured in plasma recovered from the same blood samples. The plasma was obtained from anticoagulated peripheral blood, which was centrifuged at 1300 g for 10 minutes at 25°C. The plasma collected was frozen at −80°C. Before use, plasma was centrifuged at 10 000 g for 10 minutes at 4°C, to remove lipid components.

Cytometry Determinations

Human regulatory T cells were determined using a commercial kit (BioLegend, San Diego, CA). Mononuclear cells 1 × 10 6 were used; samples were stained according to the manufacturer’s specifications with the following antibodies: CD4-PerCP, CD25-PE, and FOXP3-Alexa Fluor 488.

Subpopulations of monocytes and T CD4 and CD8 lymphocytes were determined by staining whole blood samples. The antibody panels used were titered and were validated using FMO controls, which were also used as gating controls. In addition, compensation controls were used. Each of the following antibody panels were added to 100 µL of anticoagulated blood: T CD4 lymphocytes (CD4 FITC, HLA-DR PE, CD3APC/Cy7, and CD279 APC), T CD8 lymphocytes (CD8 FITC, HLA-DR PE, CD3 APC/Cy7, and CD279 APC), and monocytes (CD16 Alexa Fluor 488, CD14 PE, HLA-DR PE/Cy7, and CD15 PerCP/Cy5.5). Samples were incubated for 20 minutes at room temperature, protected from light. Afterward, erythrocytes were lysed with a commercial solution (BioLegend) according to the manufacturer’s instructions. Subsequently, samples were centrifuged at 350 g for 5 minutes and washed with 2 mL of staining buffer. Finally, cells were washed and resuspended in 300 µL of staining buffer. All samples were acquired using an Accuri C6 flow cytometer (Becton Dickinson, San Jose, California; 2-2 configuration) and analyzed using the FlowJo X software (TreeStar, San Carlos, California). For human regulatory T cells, we used the 3-1 cytometry configuration.

Determination of Plasma Inflammatory Molecules

The concentration of inflammatory molecules was determined using the Luminex system (Bio-Plex Pro Human Cytokine 17-plex Assay kit; M5000031YV). The sample was processed according to the manufacturer’s instructions and read using a Bio-plex 200 system (BioRad, CA). The results were analyzed using the Bioplex Manager software (v6.1).

Statistical Analysis

Statistical analysis was performed using the SPSS software (La Jolla, California). Continuous variables are expressed in medians ± standard error in the case of cell subpopulations and in means in the case of cytokine concentrations. We used the Shapiro-Wilk test to analyze the normality of all the variables. This test is designed for a small sample size with an n of up to 50. Then, nonparametrical variables ( P ≤ .05 on the Shapiro-Wilk test) were analyzed using the Wilcoxon test, as indicated in the tables and figures. The homogeneity of variances was confirmed by Levene test. Variables with normal distribution were analyzed using Student t test. Differences were considered significant when P ≤ .05.

The data on the concentration of inflammatory molecules in plasma were divided into the following groups: pretreatment patients, posttreatment patients, and healthy controls. A t test for independent samples was used to compare the control group with patients before chemotherapy and patients after chemotherapy. A t test for dependent samples was used to compare the groups of patients before and after chemotherapy. On the determination of inflammatory molecules, we had missing values. We performed an analysis of missing values using SPSS. In the corresponding figure, the n value of each group is indicated. Differences were considered significant when P ≤ .05. The survival rate of patients was analyzed using Kaplan-Meier curves over a period of 33 months.

Results

The study group included 9 patients with a confirmed diagnosis of pulmonary adenocarcinoma, of which 78% were female and had a mean age of 56.3 (35/74) years. Their clinical characteristics are detailed in Table 1 . Nine healthy individuals with similar characteristics of age and sex were recruited as controls ( Table 1 ).

In order to estimate the patients’ survival rate after treatment with chemotherapeutic drugs, Kaplan-Meier tests were performed during the study period. To estimate differences between the different chemotherapy regimens, the log-rank test was used and a χ 2 value = 2395, gl = 3, and P ≤ 0.495 were obtained. The overall survival rate of the patients was 44% with an average survival of 13 months ( Figure 1 ). No differences were found in survival between chemotherapy regimens.

Subpopulations of lymphocytes and peripheral monocytes associated with the activation or suppression of the immune system were analyzed, as well as a panel of inflammatory molecules. Regarding the populations of suppressor cells, there were no statistically significant differences in T regulatory cells (CD4 + , CD25 + , FoxP3 + ) and CD4 + PD-1 + T cells between patients and controls ( Table 2 ), but the proportion of CD4 + PD-1 + T cells was higher in patients after treatment with chemotherapy, from 0.29% to 2.42%, representing a fold increase of 8.3 ( Figure 2 ).

Monocyte subpopulations were analyzed based on the expression profile of CD14 and CD16 as follows: classical monocytes (CD14 ++ , CD16 − ), intermediate monocytes (CD14 ++ , CD16 + ), and nonclassical monocytes (CD14 Neg/+ , CD16 ++ ). Patients before chemotherapy showed a 2.77-fold increase in the relative percentage of intermediate monocytes compared to control individuals (5.03% vs 13.96%, P ≤ .017; Figure 3 ). No statistically significant differences were found in the proportion of monocyte subpopulations before and after chemotherapy ( Table 3 ). Subsequently, the expression of HLA-DR was evaluated on monocyte subpopulations.

The expression of HLA-DR in the monocyte population is very heterogeneous; for this reason, we first analyzed the percentage of HLA-DR − and HLA-DR + g cells (Supplemental Figure 1). After gating HLA-DR + g cells, we analyzed whether between study groups, HLA-DR-positive cells had differences between positive and highly positive cells. In the analysis of this parameter, cells were divided into positive and highly positive for HLA-DR; the threshold was set at the MFI (1 × 104). Thus, this study reports HLA-DR − , globally positive HLA-DR cells (g), HLA-DR + , and HLA-DR ++ .

The proportion of HLA-DR − classical monocytes was found to be moderately increased in patients before chemotherapy in relation to control individuals, from 22.7% (±2.95%) to 28.05% (±3.85%), representing 1.2-fold increase; however, these differences were not statistically significant. After chemotherapy, the proportion of HLA-DR − classical monocytes decreased from 28.05% (±3.85%) to 11.38% (±3.78%), P ≤ .010 ( Table 3 ); similarly, the proportion of HLA-DR − intermediate monocytes was increased in patients before chemotherapy in relation to control individuals, from 9.77% (±3.23%) to 18.29% (±3.13%), which represents an increase of 1.87 times. Subsequent to chemotherapy, patients presented a statistically significant reduction in the proportion of HLA-DR − intermediate monocytes, from 18.29% to 8.10% (±1.20%), P ≤ .004.

On the other hand, the proportion of classical monocytes HLA-DR + g was increased after chemotherapy from 77.66% (±3.88%) to 88.35% (±3.94%), P ≤ .011, while in control individuals, the proportion of classical monocytes was 77.64%, similar to values found in patients before chemotherapy ( Table 3 and Figure 4 ). In addition, the proportion of intermediate monocytes HLA-DR + g was also increased in patients after chemotherapy compared to patients before chemotherapy, from 81.34% (±3.14%) to 92.44% (±1.29%), respectively ( P ≤ .004), reaching values similar to those found in control individuals (90.16% ± 3.30%). We found that although HLA-DR + monocytes are increased after chemotherapy, there are no differences regarding their HLA-DR expression pattern before and after chemotherapy, since we did not find differences in the percentage of HLA-DR + and HLA-DR ++ monocytes.

The following molecules were analyzed in the plasma of the study participants: G-CSF, GM-CSF, INF-γ, interleukin (IL)-1β, IL-2, IL-4 to IL-8, IL-12 (p70), IL-13, IL-17, MCP-1, MIP-1β, and TNF-α. The concentrations of these molecules were compared between the following groups: pretreatment patients, posttreatment patients, and healthy controls. Patients with pulmonary adenocarcinoma showed an increase in the plasma concentration of the chemokine MIP-1β compared to the control group, with values of 76.87 ± 12.91 pg/mL and 35.42 ± 5.12 pg/mL, respectively, representing a 2.24-fold increase ( P ≤ .02). It is worth noting that the concentration of MIP-1β decreased to 23.06 ± 4.44 pg/mL after chemotherapy (approaching the value observed in the control individuals, P ≤ .005). Other cytokines were also higher in the patient’s plasma after chemotherapy, compared to pretreatment values. Interleukin-1β increased from 0.17 ± 0.08 pg/mL to 0.85 ± 0.33 pg/mL ( P ≤ .042), IL-5 increased from 0.63 ± 0.07 pg/mL to 2.19 ± 0.36 pg/mL ( P ≤ .017), and IL-12 increased from 2.02 pg/mL ± 0.71 to 12.59 ± 2.4 pg/mL ( P ≤ .011; Figure 5 ).

Discussion

Evidence accumulated for more than 20 years indicates that the immune system plays an important role in the evolution of neoplasms. 21 Thus, it has recently been proposed that the evaluation of immune subpopulations could be useful in the clinical management of patients with cancer, as well as in the staging of the disease. 19 Only a limited number of health institutions have sufficient financial and infrastructure resources to support such studies, and thus, the inclusion of this type of parameters in the staging of the disease and in the clinical control of the patients could represent a great challenge. For this reason, we evaluated the potential utility of determination of peripheral immune subpopulations that met the following criteria: (1) they had been previously associated with the evolution of neoplasms, (2) they can be quantified with relative ease, and (3) the cost of quantifying them is relatively low.

Among the lymphoid subpopulations that suppress the immune response, the present study evaluated the classical regulatory T cells (CD4 + CD25 + FoxP3 + ). These cells inhibit the action of effector T cells through mechanisms that include the secretion of the cytokine IL-10. 22 Although it has been widely reported that the levels of classical regulatory T cells are increased in several types of cancer such as cell hepatocarcinoma and multiple myeloma, 23 , 24 in our study, no significant differences were found in the frequency of classical regulatory T cells between patients and control individuals; this result coincide with a study by Kotsakis et al that included a group of 156 patients with advanced and untreated NSCLC and 31 healthy controls. 25 That study did not find differences in Tregs (defined as CD4 + , CD25 + , and FoxP3 + T cells) until they included additional markers. Currently, there is controversy about the markers that should be used to study regulatory T cells. It has been proposed that CD127, CD152, and CD45 RO should also be included to define the Treg cells, according to their differentiation and activation state. When Kotsakis et al used these markers, patients showed an increase in naive Treg cells defined as CD25 high , CD127 −/low , CD152 − , FoxP3 low , and CD45RO − . 25

Other cells that appear to play a central role in the antitumor immune response are PD-1 + T lymphocytes. These cells are susceptible to inhibition by the binding of a PD-1L molecule, expressed by neoplastic cells. 26 Similar to other types of cancer, in pulmonary adenocarcinoma, the expression of PD-1L in neoplastic tissue has been associated with poor patient survival. 27 Patients showed an increase in the proportion of CD4 + PD-1 + T lymphocytes after chemotherapy, but not in CD8 + PD-1 + T lymphocytes. Since CD4 + lymphocytes can collaborate with the antitumor immune response through the release of IL-2 (an important cytokine in the clonal expansion of cytotoxic CD8 + T lymphocytes and in the maintenance of CD8 + memory cells), a rise in the proportion of CD4 + PD-1 + T cells could increase the antitumor activity of CD8 + T lymphocytes. 28 Nevertheless, we did not find a change in IL-2 levels in the plasma of patients after chemotherapy (data not shown). We also analyzed the expression of the HLA-DR. Unlike other cell types such as monocytes, in which this molecule is constitutively expressed, HLA-DR is only expressed in activated T lymphocytes, and its expression is considered a marker of activation. Previous studies in patients with systemic lupus erythematosus found that the HLA-DR expression of CD8 + T lymphocytes is a predictor of periods of disease activity. 29 In the present study, no differences were found in the lymphocyte expression of HLA-DR before and after chemotherapy.

The percentage of monocytes can vary in various pathological conditions: an increase in monocytes is mainly related to an active inflammatory process, which is compatible with a neoplastic process. However, it is now known that monocyte subpopulations have different properties, 30 and the balance between subpopulations of classical, intermediate, and nonclassical monocytes and their activation state could be relevant in pathological conditions. Recent studies show that classical monocytes are specialized in phagocytosis and have low pro-inflammatory potential. In contrast, nonclassical and intermediate monocytes have the ability to produce high quantities of cytokines. Intermediate monocytes also have a higher expression of HLA-DR and have been described as being angiogenic. 12 After activating intermediate monocytes with LPS, intracellular cytometry showed that these cells produce the anti-inflammatory cytokine IL-10, while nonclassical monocytes produce the pro-inflammatory cytokines IL-1β and TNF-α. 12 We found higher levels of intermediate monocytes in patients compared to healthy individuals. After treatment, the percentage of intermediate monocytes was not significantly reduced. Since intermediate monocytes are important producers of IL-10 and are angiogenic, the increase in these cells could potentially contribute to immunosuppression and metastasis. An increase in the proportion of intermediate monocytes was previously reported in gastric cancer, in which it was associated with a higher metastatic potential. 31 We did not find significant changes in the proportion of nonclassical monocytes in postchemotherapy patients.

The expression of HLA-DR is highly variable in monocytes and its decrease has been reported in situations of systemic inflammation, sepsis, and surgical procedures, among others. 32 An increase in peripheral monocyte subpopulations (CD14 + , HLA-DR Neg/low ) has been previously reported in several pathological conditions, as in acute liver failure and in infections with the respiratory syncytial virus. 33 , 34 In the latter, the decrease in HLA-DR expression correlated with the severity of the disease. 34 It has also been reported as a marker of immunosuppression in patients with some types of cancer, such as squamous cell carcinoma of the head and neck, 16 bladder carcinoma, 35 and NSCLC. 36 Comparing different monocyte subpopulations regarding HLA-DR expression, patients showed a decrease in the proportion of classical and intermediate monocytes (HLA-DR + ) compared to healthy individuals. This is compatible with the reports of immunosuppression associated with the reduction of HLA-DR + expression in other neoplasms. After chemotherapy, monocyte expression of HLA-DR + showed a statistically significant increase, with values similar to those found in control individuals.

The robust monocyte expression of HLA-DR has been linked to a better prognosis in colorectal cancer, 37 while a decrease in circulating CD14 + and HLA-DR + cells is associated with a poor prognosis in small cell lung cancer. 38 , 39 The size of the sample used in this study does not allow us to establish a correlation between an increase in the levels of classical and intermediate HLA-DR + monocytes and the survival rate of patients. However, the recovery of the monocyte expression of HLA-DR + found in patients with adenocarcinoma after chemotherapy could not be indicative of a protection in the progression of the disease since the patients died.

The observed increase in intermediate and classical HLA-DR + monocytes after chemotherapy could indicate an immunosuppression, since only phagocytic and IL-10 producing monocytes were increased, while monocytes that produce pro-inflammatory cytokines (nonclassical) did not change. Pharmacological treatment could modify the expression of HLA. Previous studies indicated that cisplatin upregulates the expression of MHC/HLA class I molecules in macrophages. 18 Additionally, the expression of HLA-DR could be affected in patients being treated with tyrosine kinase inhibitors, because this molecule signals through tyrosine kinases. 40 , 41 Therefore, a compensatory mechanism could increase the expression of this molecule in the patients treated with gefitinib.

On the other hand, although the proportion of HLA-DR + in classical and intermediate monocytes varies widely in patients without treatment, it is important to emphasize that HLA-DR + monocytes showed homogeneous values after chemotherapy. This is important, since a recurrent problem in the use of cellular subpopulations as pathological prognostic indicators is the high variability between individuals. Thus, these subpopulations could be relevant in the study of the clinical evolution of patients with pulmonary adenocarcinoma.

In order to assess the inflammatory context, the concentration of a panel of molecules was determined in the plasma of patients before and after treatment, as well as in control individuals. Previous studies have described that cytokines such as IL-1β, IL-6, IL-8, and IL-17 promote the expression of transcription factors, whose target genes are involved in pro-neoplastic responses, such as resistance to apoptosis, cell migration, and chemoresistance 42 ; other cytokines have effects on the antitumor response. In NSCLC, previous studies in the plasma of patients with squamous cell lung cancer and adenocarcinoma suggest that there are no differences in the cytokines of patients with lung cancer and control individuals. 43 Of the 17 cytokines determined in this study, the only significant differences between controls and untreated patients were in the concentration of MIP-1β, which was 2.24 (34.22-76.87 pg/mL) times higher in the plasma of the patients. It is known that this chemokine is important in the selective migration of activated CD4 + T lymphocytes. 44 Therefore, an increase in the plasma concentration of MIP-1β in patients with adenocarcinoma could be part of a compensatory response to cancer-associated immunosuppression. The levels of MIP-1β decreased markedly after chemotherapy (from 76.87 to 23.06 pg/mL) and showed a tendency to approach values found in control individuals. Furthermore, the plasma concentration of IL-12 significantly increased after chemotherapy; this cytokine is known to mediate its antitumor effect through the activation of NK, CD4 + , and CD8 + T cells. 45

Cytokines with pro-tumoral activity (IL-1β and IL-5) showed an increase after chemotherapy, compared to the levels found in patients before treatment. Interleukin-1β is a pro-inflammatory cytokine that induces the transcription of NF-κB-driven genes, which has been correlated with a poor prognosis in breast cancer. 46 A recent report showed that the chemotherapeutic action of gemcitabine could be limited by its induction of the secretion of IL-1-β by MDSCs, thus promoting the secretion of IL-17 by CD4 lymphocytes. 47 Interleukin-17 is a cytokine with potent pro-angiogenic effects, 48 which has been reported to increase in several types of cancer, including pulmonary adenocarcinoma. 49 In the present study, we did not find a statistically significant increase in IL-17 after chemotherapy, but with a larger study sample the differences could become significant. Another chemokine that was increased after chemotherapy is IL-5; it is produced mainly by CD4 + and CD8 + T lymphocytes and has been reported to increase the metastatic potential of various types of cancer, including colorectal carcinoma, metastatic liver cancer, and lung cancer 50 ; in the latter, IL-5 is also important for the survival of tumor cells. 50 The changes found in the plasma concentration of IL-1β and IL-5 before and after chemotherapy could be explained in part by the side effects of chemotherapy and by the time of evolution of the disease.

Conclusion

Before chemotherapy, patients with lung adenocarcinoma have lower levels of classical and intermediate HLA-DR + monocytes, compared to those present in healthy individuals. After chemotherapy, the percentage of classical and intermediate monocytes increased, approaching the values found in individuals without neoplasia, whereas nonclassical (pro-inflammatory) monocytes were not modified.

The observed increase in intermediate and classical HLA-DR + monocytes after chemotherapy could indicate an immunosuppression in advanced lung adenocarcinoma, since only phagocytic- and IL-10-producing monocytes were increased, while monocytes producing pro-inflammatory cytokines did not change; and this change was accompanied by an increase in peripheral CD4 + PD-1 + T cells and low patient survival.

In the peripheral inflammatory context, an increase in IL-5 was found, which could potentially favor the migration of CD4 + PD-L1 + T cells to tissue and thus favor immunosuppression. In the postchemotherapy group, the cytokines IL-1β and IL-12 were also increased, while MIP-1β was decreased.

Supplemental Material

Supplementary_figures - Restoration of Peripheral Intermediate and Classical Monocytes Expressing HLA-DR in Patients With Lung Adenocarcinoma After Platinum-Based Chemotherapy

Click here for additional data file.

Supplementary_figures for Restoration of Peripheral Intermediate and Classical Monocytes Expressing HLA-DR in Patients With Lung Adenocarcinoma After Platinum-Based Chemotherapy by Selma Rivas-Fuentes, Anjarath Higuera Iglesias, Ana García Trejo, Daniel Yair Chavarría Castro, Norma Inclán Figueroa, Teresa Aguirre Pérez, Renata Báez Saldaña, and Patricia Gorocica Rosete in Technology in Cancer Research & Treatment

Authors’ Note: S. R. F. contributed to the experimental and data analysis design, procedures, manuscript design, and writing. A. H. contributed to the clinical and experimental data statistic (design and analysis). A. G. T., Y. C. C., and N. I. F. contributed to the performance sample processing, FlowJo analysis, and collection of clinical information. T. A. P., R. B. S. contributed to diagnostic and following of patients enrolled and collected clinical information. P. G. contributed to study design, founding, supervision of procedures, and approval of the final version. S. R. F. and A. H. contributed equally to the work presented.

Acknowledgments

The authors thank Tania Cázares who participated in the quantification of regulatory T lymphocytes and Joel Nava Palacios who assisted in the compilation of the data produced in the present study and participated in the collection of clinical data.

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The Department of Biochemistry was partially funded by Grants 180516 from the Consejo Nacional de Ciencia y Tecnología (CONACyT), Mexico, through the Fondo Sectorial en Salud.

Supplemental Material: Supplementary material for this article is available online.

Abbreviation

interleukin

non-small cell cancer

programmed death ligand 1

References

1 Ellis PM Vandermeer R Delays in the diagnosis of lung cancer . J Thorac Dis . 2011 ; 3 ( 3 ): 183 – 188 . 22263086 10.3978/j.issn.2072-1439.2011.01.01 PMC3256519

2 Coate LE Shepherd FA Maintenance therapy in advanced non-small cell lung cancer: evolution, tolerability and outcomes . Ther Adv Med Oncol . 2011 ; 3 ( 3 ): 139 – 157 . 21904577 10.1177/1758834011399306 PMC3150062

3 Du L Morgensztern D Chemotherapy for advanced-stage non-small cell lung cancer . Cancer J . 2015 ; 21 ( 5 ): 366 – 370 . 26389760 10.1097/PPO.0000000000000141

4 Sato N Nabeta Y Kondo H Human CD8 and CD4 T cell epitopes of epithelial cancer antigens . Cancer Chemother Pharmacol . 2000 ; 46 ( suppl ): S86 – S90 . 10950155 10.1007/pl00014057

5 Reuschenbach M von Knebel Doeberitz M Wentzensen N A systematic review of humoral immune responses against tumor antigens . Cancer Immunol Immunother . 2009 ; 58 ( 10 ): 1535 – 1544 . 19562338 10.1007/s00262-009-0733-4 PMC2782676

6 Scott AM Wolchok JD Old LJ Antibody therapy of cancer . Nat Rev Cancer . 2012 ; 12 ( 4 ): 278 – 287 . 22437872 10.1038/nrc3236

7 Zhuo C Xu Y Ying M FOXP3+ Tregs: heterogeneous phenotypes and conflicting impacts on survival outcomes in patients with colorectal cancer . Immunol Res . 2015 ; 61 ( 3 ): 338 – 347 . 25608795 10.1007/s12026-014-8616-y

8 Waki K Yamada T Yoshiyama K PD-1 expression on peripheral blood T-cell subsets correlates with prognosis in non-small cell lung cancer . Cancer Sci . 2014 ; 105 ( 10 ): 1229 – 1235 . 25117757 10.1111/cas.12502 PMC4462362

9 Santoni M Massari F Amantini C Emerging role of tumor-associated macrophages as therapeutic targets in patients with metastatic renal cell carcinoma . Cancer Immunol Immunother . 2013 ; 62 ( 12 ): 1757 – 1768 . 24132754 10.1007/s00262-013-1487-6 PMC11029754

10 Ziegler-Heitbrock L Ancuta P Crowe S Nomenclature of monocytes and dendritic cells in blood . Blood . 2010 ; 116 ( 16 ): e74 – 80 . 20628149 10.1182/blood-2010-02-258558

11 Wong KL Tai JJ Wong WC Gene expression profiling reveals the defining features of the classical, intermediate, and nonclassical human monocyte subsets . Blood . 2011 ; 118 ( 5 ): e16 – e31 . 21653326 10.1182/blood-2010-12-326355

12 Mukherjee R Kanti Barman P Kumar Thatoi P Tripathy R Kumar Das B Ravindran B Non-classical monocytes display inflammatory features: validation in sepsis and systemic lupus erythematous . Sci Rep . 2015 ; 5 : 13886 . 26358827 10.1038/srep13886 PMC4566081

13 Mu CY Huang JA Chen Y Chen C Zhang XG High expression of PD-L1 in lung cancer may contribute to poor prognosis and tumor cells immune escape through suppressing tumor infiltrating dendritic cells maturation . Med Oncol . 2011 ; 28 ( 3 ): 682 – 688 . 20373055 10.1007/s12032-010-9515-2

14 Nakanishi J Wada Y Matsumoto K Azuma M Kikuchi K Ueda S Overexpression of B7-H1 (PD-L1) significantly associates with tumor grade and postoperative prognosis in human urothelial cancers . Cancer Immunol Immunother . 2007 ; 56 ( 8 ): 1173 – 1182 . 17186290 10.1007/s00262-006-0266-z PMC11029839

15 Vuk-Pavlovic S Bulur PA Lin Y Immunosuppressive CD14+HLA-DRlow/- monocytes in prostate cancer . Prostate . 2010 ; 70 ( 4 ): 443 – 455 . 19902470 10.1002/pros.21078 PMC2935631

16 Chikamatsu K Sakakura K Toyoda M Takahashi K Yamamoto T Masuyama K Immunosuppressive activity of CD14+ HLA-DR- cells in squamous cell carcinoma of the head and neck . Cancer Sci . 2012 ; 103 ( 6 ): 976 – 983 . 22360618 10.1111/j.1349-7006.2012.02248.x PMC7685071

17 Xiu B Lin Y Grote DM IL-10 induces the development of immunosuppressive CD14(+)HLA-DR(low/-) monocytes in B-cell non-Hodgkin lymphoma . Blood Cancer J . 2015 ; 5 : e328 . 26230952 10.1038/bcj.2015.56 PMC4526782

18 de Biasi AR Villena-Vargas J Adusumilli PS Cisplatin-induced antitumor immunomodulation: a review of preclinical and clinical evidence . Clin Cancer Res . 2014 ; 20 ( 21 ): 5384 – 5391 . 25204552 10.1158/1078-0432.CCR-14-1298 PMC4216745

19 Galon J Pages F Marincola FM Cancer classification using the Immunoscore: a worldwide task force . J Transl Med . 2012 ; 10 : 205 . 23034130 10.1186/1479-5876-10-205 PMC3554496

20 Galon J Pages F Marincola FM The immune score as a new possible approach for the classification of cancer . J Transl Med . 2012 ; 10 : 1 . 22214470 10.1186/1479-5876-10-1 PMC3269368

21 de Visser KE Eichten A Coussens LM Paradoxical roles of the immune system during cancer development . Nat Rev Cancer . 2006 ; 6 ( 1 ): 24 – 37 . 16397525 10.1038/nrc1782

22 Mosmann TR Moore KW The role of IL-10 in crossregulation of TH1 and TH2 responses . Immunol Today . 1991 ; 12 ( 3 ): A49 – A53 . 1648926 10.1016/S0167-5699(05)80015-5

23 Ormandy LA Hillemann T Wedemeyer H Manns MP Greten TF Korangy F Increased populations of regulatory T cells in peripheral blood of patients with hepatocellular carcinoma . Cancer Res . 2005 ; 65 ( 6 ): 2457 – 2464 . 15781662 10.1158/0008-5472.CAN-04-3232

24 Beyer M Kochanek M Giese T In vivo peripheral expansion of naive CD4+CD25high FoxP3+ regulatory T cells in patients with multiple myeloma . Blood . 2006 ; 107 ( 10 ): 3940 – 3949 . 16410445 10.1182/blood-2005-09-3671

25 Kotsakis A Koinis F Katsarou A Prognostic value of circulating regulatory T cell subsets in untreated non-small cell lung cancer patients . Sci Rep . 2016 ; 6 : 39247 . 27976733 10.1038/srep39247 PMC5157012

26 Hersey P Gallagher S A focus on PD-L1 in human melanoma . Clin Cancer Res . 2013 ; 19 ( 3 ): 514 – 516 . 23251000 10.1158/1078-0432.CCR-12-3312

27 Shimoji M Shimizu S Sato K Clinical and pathologic features of lung cancer expressing programmed cell death ligand 1 (PD-L1) . Lung Cancer . 2016 ; 98 : 69 – 75 . 27393509 10.1016/j.lungcan.2016.04.021

28 Zippelius A Batard P Rubio-Godoy V Effector function of human tumor-specific CD8 T cells in melanoma lesions: a state of local functional tolerance . Cancer Res . 2004 ; 64 ( 8 ): 2865 – 2873 . 15087405 10.1158/0008-5472.can-03-3066

29 Viallard JF Bloch-Michel C Neau-Cransac M HLA-DR expression on lymphocyte subsets as a marker of disease activity in patients with systemic lupus erythematosus . Clin Exp Immunol . 2001 ; 125 ( 3 ): 485 – 491 . 11531958 10.1046/j.1365-2249.2001.01623.x PMC1906149

30 Wong KL Yeap WH Tai JJ Ong SM Dang TM Wong SC The three human monocyte subsets: implications for health and disease . Immunol Res . 2012 ; 53 ( 1-3 ): 41 – 57 . 22430559 10.1007/s12026-012-8297-3

31 Eljaszewicz A Jankowski M Gackowska L Gastric cancer increase the percentage of intermediate (CD14++CD16+) and nonclassical (CD14+CD16+) monocytes . Cent Eur J Immunol . 2012 ; 37 : 355 – 361 .

32 Kim OY Monsel A Bertrand M Coriat P Cavaillon JM Adib-Conquy M Differential down-regulation of HLA-DR on monocyte subpopulations during systemic inflammation . Crit Care . 2010 ; 14 ( 2 ): R61 . 20385017 10.1186/cc8959 PMC2887183

33 Abeles RD McPhail MJ Sowter D CD14, CD16 and HLA-DR reliably identifies human monocytes and their subsets in the context of pathologically reduced HLA-DR expression by CD14(hi)/CD16(neg) monocytes: Expansion of CD14(hi) /CD16(pos) and contraction of CD14(lo)/CD16(pos) monocytes in acute liver failure . Cytometry A . 2012 ; 81 ( 10 ): 823 – 834 . 22837127 10.1002/cyto.a.22104

34 Ahout IM Jans J Haroutiounian L Reduced expression of HLA-DR on monocytes during severe respiratory syncytial virus infections . Pediatr Infect Dis J . 2016 ; 35 ( 3 ): e89 – e96 . 26658377 10.1097/INF.0000000000001007

35 Yuan XK Zhao XK Xia YC Zhu X Xiao P Increased circulating immunosuppressive CD14(+)HLA-DR(-/low) cells correlate with clinical cancer stage and pathological grade in patients with bladder carcinoma . J Int Med Res . 2011 ; 39 ( 4 ): 1381 – 1391 . 21986138 10.1177/147323001103900424

36 Huang A Zhang B Wang B Zhang F Fan KX Guo YJ Increased CD14(+)HLA-DR (-/low) myeloid-derived suppressor cells correlate with extrathoracic metastasis and poor response to chemotherapy in non-small cell lung cancer patients . Cancer Immunol Immunother . 2013 ; 62 ( 9 ): 1439 – 1451 . 23760662 10.1007/s00262-013-1450-6 PMC11028777

37 Matsushita K Takenouchi T Shimada H Strong HLA-DR antigen expression on cancer cells relates to better prognosis of colorectal cancer patients: possible involvement of c-myc suppression by interferon-gamma in situ . Cancer Sci . 2006 ; 97 ( 1 ): 57 – 63 . 16367922 10.1111/j.1349-7006.2006.00137.x PMC11158856

38 Novellino PS Trejo YG Beviacqua M Bordenave RH Rumi LS Cisplatin containing chemotherapy influences HLA-DR expression on monocytes from cancer patients . J Exp Clin Cancer Res . 1999 ; 18 ( 4 ): 481 – 484 . 10746974

39 Tian T Gu X Zhang B Increased circulating CD14(+)HLA-DR-/low myeloid-derived suppressor cells are associated with poor prognosis in patients with small-cell lung cancer . Cancer Biomark . 2015 ; 15 ( 4 ): 425 – 432 . 25792471 10.3233/CBM-150473 PMC12965079

40 Kuhn U Brand P Willemsen J Induction of tyrosine phosphorylation in human MHC class II-positive antigen-presenting cells by stimulation with contact sensitizers . J Immunol . 1998 ; 160 ( 2 ): 667 – 673 . 9551901

41 Matsuoka T Tabata H Matsushita S Monocytes are differentially activated through HLA-DR, -DQ, and -DP molecules via mitogen-activated protein kinases . J Immunol . 2001 ; 166 ( 4 ): 2202 – 2208 . 11160273 10.4049/jimmunol.166.4.2202

42 Sethi G Shanmugam MK Ramachandran L Kumar AP Tergaonkar V Multifaceted link between cancer and inflammation . Biosci Rep . 2012 ; 32 ( 1 ): 1 – 15 . 21981137 10.1042/BSR20100136

43 Almatroodi SA McDonald CF Collins AL Darby IA Pouniotis DS Blood classical monocytes phenotype is not altered in primary non-small cell lung cancer . World J Clin Oncol . 2014 ; 5 : 1078 – 1087 . 25493244 10.5306/wjco.v5.i5.1078 PMC4259935

44 Taub DD Conlon K Lloyd AR Oppenheim JJ Kelvin DJ Preferential migration of activated CD4+ and CD8+ T cells in response to MIP-1 alpha and MIP-1 beta . Science . 1993 ; 260 ( 5106 ): 355 – 358 . 7682337 10.1126/science.7682337

45 Nastala CL Edington HD McKinney TG Recombinant IL-12 administration induces tumor regression in association with IFN-gamma production . J Immunol . 1994 ; 153 ( 4 ): 1697 – 1706 . 7913943

46 Vikhreva P Petrova V Gokbulut T TAp73 upregulates IL-1beta in cancer cells: potential biomarker in lung and breast cancer? Biochem Biophys Res Commun . 2017 ; 482 ( 3 ): 498 – 505 . 28212736 10.1016/j.bbrc.2016.10.085 PMC5243147

47 Bruchard M Mignot G Derangere V Chemotherapy-triggered cathepsin B release in myeloid-derived suppressor cells activates the Nlrp3 inflammasome and promotes tumor growth . Nat Med . 2013 ; 19 ( 1 ): 57 – 64 . 23202296 10.1038/nm.2999

48 Li Y Cao ZY Sun B Effects of IL-17A on the occurrence of lung adenocarcinoma . Cancer Biol Ther . 2011 ; 12 ( 7 ): 610 – 616 . 21785272 10.4161/cbt.12.7.16302

49 Bao Z Lu G Cui D Yao Y Yang G Zhou J IL-17A-producing T cells are associated with the progression of lung adenocarcinoma . Oncol Rep . 2016 ; 36 ( 2 ): 641 – 650 . 27277161 10.3892/or.2016.4837 PMC4933549

50 Zaynagetdinov R Sherrill TP Gleaves LA Interleukin-5 facilitates lung metastasis by modulating the immune microenvironment . Cancer Res . 2015 , 75 ( 8 ): 1624 – 1634 . 25691457 10.1158/0008-5472.CAN-14-2379 PMC4401663