Introduction
Immunological memory is critical for long-term immunity and protection from infection. After naïve T cells are activated by the antigen, naïve T cells differentiate into effector T cells, depending on the anatomical position and phenotypic characteristics; effector T cells display different functions [
1]. However, only a small fraction of effector T cells becomes long-lived memory T cell to provide lifelong protection against the previously encountered pathogens [
2,
3]. With respect to the tissue homing-related molecular expression, memory T cells can be divided into two categories, central memory T cells (Tcm) and effector memory T cells (Tem) [
4]. Recent data revealed that adoptively transferred different subsets of memory T cells have different antitumor activity in mouse models [
5]. The distribution and function of human memory T cells have been identified in healthy subjects [
6], but the physiological distribution and function of human T cell subsets in lung cancer are still limited. Clearly, the understanding of the compartmentalization of memory T cell subsets will provide valuable basis for designing tumor immunotherapy.
Current studies focus on the frequency of the tumor-infiltrating lymphocytes (TILs) to predict the prognosis of cancer patients [
7]. The high frequency of CD4+ T cells in TILs and malignant pleural effusions (MPEs) correlates with a favorable prognosis in lung cancer patients [
8,
9]; however, other studies indicate that the high number of CD8+ T cells, not CD4+ T cells, in TILs has a good clinical outcome [
10‐
12]. The distinct distribution of CD4+ and CD8+ T cell in TILs results in different clinical outcomes in lung cancer patients. The presence of high density of CD3+CD8+CD45RO+ immune cells within tumor region is correlated with favorable clinical outcome in epithelial ovarian cancer [
13], while the number of effector CD8+ T cells in TILs decreased in lung cancer [
14]. In our study, we assessed the expression of memory T cell subsets in non-small cell lung cancer patients, and herein, we show the distinct compartmentalization of naïve T cells (Tn), Tcm, Tem, and effector T cell (Teff) subsets in non-small cell lung cancer (NSCLC). Our results provide further information regarding the distribution and function of CD4+ and CD8+ memory T cell subsets in human NSCLC patients. These results will lead to a better understanding of the biology of lung cancer.
Discussion
In this study, we analyzed the distribution and functional capacity of the CD4+ and CD8+ T cell subsets in the blood and lymph node of human NSCLC patients. The frequencies of the CD8+ T cell subsets were similar for the blood and lymph nodes of the NSCLC patients. The CD8+ Teff cells predominated in both tissue sites, followed by the Tem, Tn, and Tcm cells. However, the composition of the CD4+ T cell subsets differed. The most one cell type was CD4+ Tn, followed by Tem, Tcm, and Teff in the NSCLC-PBMC. The CD4+ Tem cells were the major population in the NSCLC-Ly, followed by the Tcm, Teff, and Tn cells. In the functional analysis, we found that the levels of the IFN-γ-expressing CD8+ Tcm cells were increased in the lymph nodes of the human NSCLC patients. The capacity to express IFN-γ was remarkably reduced in the lymph nodes relative to the blood of the NSCLC patients, even though the CD8+ Teff cells were present at a higher frequency in the NSCLC patients than in the healthy donors. The levels of IFN-γ-expressing, TNF-α-expressing, and IL-17-expressing CD4+ Tem and CD4+ Tcm cells were significantly decreased in the blood of the NSCLC patients compared to healthy donors. We observed that all three of the examined cytokines secreted by the CD4+ and CD8+ T cell subsets were present at lower frequencies in the lymph node than in the blood of the healthy donors. Our results showed differences in the composition and function of the CD4+ and CD8+ T cell subsets in the blood and lymph node of NSCLC patients. The identification of these differences may improve our understanding of the role of the T cell-mediated immune response in antitumor immunity.
Different tissue locations and various types of human cancer possess distinct distributions of T cell subsets. Kuss showed that there was an increase in the effector CD8+ T (CD8+CD27−CD45RA−) population in the peripheral blood from head and neck carcinoma patients [
15]. Another group observed that there were no significant differences between the effector CD8+ T cells (CD8+CD27−CD45RA−) in the peripheral blood of healthy donors and lung adenocarcinoma patients [
16]. However, there was an elevated population of memory (CD45RA−CD45RO+CD27+CD28+) CD8+ T cells and a low proportion of terminally differentiated (CD45RA+CD45RO−CD27−CD28−) CD8+ T cells in the pleural effusions. These results are similar to the data from the TILs of NSCLC patients in whom the CD4+ T cell subpopulation is increased [
10]
. In our analysis of the CD8+ and CD4+ T cell subsets in the blood and lymph node from NSCLC patients, we identified the Tn, Tcm, Tem, and Teff cells according to established surface markers [
1,
6] in eight NSCLC patients. We found that the levels of the Teff CD8+ T cells were significantly elevated in the blood from the NSCLC patients, and these cells were also present in a higher frequency in the lymph node. CD8+ T cells play an important role in the cell-mediated antitumor immune response [
17]. However, the role of the CD8+ Teff cells in the lymph node is unclear. The CD4+ T cell response is essential in preventing the induction of tolerance by tumor antigens, and it helps the CD8+ T cells differentiate into sustainable memory cells [
12] that can initiate antitumor immune responses. Importantly, the numbers of CD4+ T cells are positively correlated with a favorable prognosis in lung cancer patients [
18]. Our results indicate that the subtypes of the CD4+ and CD8+ Tm cells in NSCLC patients are distinct, and a lower proportion of CD8+ Tcm cells, compared to CD4+ Tcm cells, was found in both the peripheral blood and the lymph node from NSCLC patients. In contrast, a lower frequency of the CD8+ Tem cells, compared to the CD4+ Tem cells, was observed only in the lymph node. The mechanism by which CD4+ T cells aid in the formation of CD8+ memory T cells remains unclear. CD4+ effector T cells can mediate direct tumor destruction alone or with the help from CD8+ T cells [
9,
18‐
20]. However, the role of the CD4+ memory T cell subsets in the antitumor response needs to be further clarified. In humans, Tcm cells migrate to lymphoid tissue, while Tem cells circulate to the non-lymphoid tissues [
2,
21,
22]. We found that in the lymph node, the population of the Tem cells was higher than that of the Tcm cells. The high proportion of the Tem cells in the lymph node might be due to a replenishment of the high frequency of recycling Teff cells.
TNF-α-expressing and IFN-γ-expressing naïve, memory, and effector T cells were observed with low frequencies in the blood of the NSCLC patients. IL-17 production was only observed in the CD4+ Tcm and Tem cells of NSCLC subjects.
IFN-γ is the hallmark cytokine of Th1 cells and CD8+ T cells, and it is critical for immune surveillance [
23]. The function of the CD8+ T cells from lung cancer patients was impaired with respect to both Th1 cytokine production and cytotoxic potential [
23]. However, our results showed that the proportion of the IFN-γ-producing CD8+ Tcm cells was increased in the lymph node from the NSCLC patients. IFN-γ production by the CD8+ Teff cells was significantly decreased in the blood from the NSCLC patients, yet the number of CD8+ Teff cells was increased. Tcm cells can differentiate into Tem and Teff cells. It is possible that the CD8+ Tcm cells replenish the Teff cells, leading to the high frequency of circulating CD8+ Teff cells observed in the blood of the lung cancer patients. In addition to IFN-γ, CD8+ Teff cells also express the perforin, granzyme to kill tumor cells. Studies have found that the IFN-γ-producing Th1 and CD8+ T cells are more prone to apoptosis and are involved in the reduction of the Teff cell populations [
24,
25]. The IFN-γ-deficient CD8+ T cells that expressed high levels of IL-7r were shown to be the precursors of the memory cells. These cells block the IFN-γ signaling pathway that contributes to the memory responses involved in tumor vaccination [
26‐
28]. The role of the IFN-γ-producing CD8+ Teff cells during the formation of the CD8+ T memory cells in human lung cancer is less clear.
A role for inflammation in tumorigenesis is now generally accepted, and it has become evident that an inflammatory microenvironment is an essential component of all tumors [
29]. The cytokines in the tumor microenvironment can either promote antitumor immunity (IL-12, IFN-γ), enhance tumor development and progression (IL-6, IL-17, IL-23) [
30], or influence the cancer cell growth and survival (TRAIL, FasL, TNF-α, TGF-β, IL-6). TNF-α in the bloodstream may have oncogenic effects through several pathways, such as the stimulation of the production of reactive oxygen species (ROS), which can induce DNA damage and genomic instability; the stimulation of stem cell-like tumor progenitors by promoting β-catenin entry into the nucleus in inflammation-associated gastric cancer [
31]; and the promotion of MMP expression, the invasiveness, and the survival of circulating metastatic seeds via NF-κB and STAT3 [
32,
33]. We found that the frequency of TNF-α production in the Tcm, Tem, Tn, and Teff cells from the blood of lung cancer patients is lower than that of healthy donors. The low circulating levels of TNF-α in the bloodstream might have beneficial effects in lung cancer patients. Whether these low levels are one of the protective mechanisms in human lung cancer needs to be verified.
The role of IL-17 in antitumor processes remains controversial. Some studies have reported that the proportion of Th17-producing cells was higher in multiple human cancers and that these cells have a potent antitumor effect. This effect might be related to the polyfunctional effector cytokines induced by the IL-17 cells, specifically the induction of TNF-α, IL-2, IFN-γ, and chemokines and the recruitment of NK cells into the tumor microenvironment to target the tumor [
34‐
36]. Other reports have shown that IL-17 induces tumor angiogenesis [
37,
38] and that a high level of IL-17 is correlated with advanced cancer [
39]. Our current data revealed that IL-17 was produced primarily by the CD4+ Tcm and CD4+ Tem cells, not by other CD4+ and CD8+ T cell subsets, which is consistent with the reports that 99% of the tumor-infiltrating IL-17 T cells were IL-17 CD4+ (Th17) cells [
40,
41]. In addition, the proportion of the IL-17 CD4+ memory T cells was decreased in the blood from the NSCLC patients relative to the healthy donors. The number of cells positive for dual cytokines, including IL-17/IFN-γ and IL-17/TNF-α in the blood of the NSCLC patients, was lower than in the healthy donors. The effect of IL-17 production on the CD4+ memory T cells in human lung cancer requires further investigation.