Background
Malignant pleural effusions (MPE) are a common consequence of cancer with more than 150,000 cases of MPE per year are diagnosed in the USA [
1]. MPE is found in up to 6% of patients with malignancy. Pleural metastases and/or MPE are mostly of lung cancer (15-30%), breast cancer (10-15%) or lymphomas origin. These patients have a poor prognosis, with a median survival of 3 to 6 months [
1]. Among cancers associated with MPE, malignant pleural mesothelioma (MPM) was previously considered a rare tumor, but its incidence is increasing worldwide due to past (mostly occupational) exposure to asbestos. Outcome of patients with MPM remains poor despite recent treatment improvements [
2]. Spontaneous immune response to mesothelioma seems to be important in counteracting tumor development. The immune system plays an important role in tumor biology, as it can delay mesothelioma progression [
3], and immunotherapy has met with some success in previous and ongoing clinical trials [
4‐
10]. In malignant effusions, the inflammatory processes and the immune responses induce the recruitment of cells into the pleural space [
11]. The ability of T cell subsets at different differentiation stages to migrate to extra-lymphoid tissues as well as the immediate effector capacities of antigen-experienced T cell subsets, are heterogeneous [
12]. Naïve and central memory (T
CM) T cells constitutively express the CC-chemokine receptor (CCR7) permitting their traffic to lymphoid organs. Conversely, effector memory (T
EM) and terminally-differentiated memory cells (T
TD) have lost CCR7 expression, can traffic to peripheral sites, and are characterized by immediate effector capacities such as cytokine secretion and cytotoxic activity [
13]. Depending on antigen load, either early or highly differentiated T-cells may exert the prominent protective capacity [
14‐
17]. Previous studies never compared pleural cell populations from MPM patients with those of healthy subjects, but instead used various control groups such as pleural transudates, tuberculous effusions or ascites, none of which could provide accurate baseline values [
18‐
20].
The aim of this study was to investigate the distribution of all four naïve and memory subsets composing the CD4+ and CD8+ T-cell pools in paired blood and pleural fluid from a large cohort of consecutive patients with MPM before any treatment. To better delineate specific changes in subset composition induced by mesothelioma, control groups included (a) otherwise healthy subjects with severe essential hyperhydrosis, (b) patients with benign lesions associated with asbestos exposure, and (c) patients with pleural metastases of carcinoma.
Discussion
It is widely admitted that T cells and NK cells are key players in anti-tumour immunity, although progressing tumours can ultimately escape immune control. The assessment of T cell subpopulations and NK cells in blood and pleural fluid could be useful for the understanding of the mechanisms involved in leukocyte recruitment in malignant pleural effusions and may help understanding their involvement in anti-tumor immunity. We report here the relative distribution of these populations in patients with MPM and compared it with values obtained from patients with pleural metastases of various carcinomas as done previously [
23,
26], but also from patients with benign pleural involvement related to asbestos exposure and for the first time healthy subjects. This permitted us to more precisely analyze circulating and tumor environment-associated immune cells in patients with malignant pleural effusions. To our best knowledge few studies have specifically reported CD4
+ et CD8
+ T cell subpopulations in malignant pleural effusions [
19,
27,
28].
Our results show for the first time that the pleural fluid from healthy subjects exhibits a significant different CD4
+/CD8
+ T cell ratio not only compared to normal blood value but also with pleural fluid from all groups of patients, as suggested by other authors [
20,
27,
29]. This distribution of the CD4
+/CD8
+ T cells in healthy subjects appeared also very similar to the one observed in peritoneal fluid from healthy women [
30]. By contrast, we found that in all our patients there was a defect in recruiting CD8
+ in the abnormal pleura, as previously reported by several authors [
27,
28,
31,
32]. Interestingly this pleural recruiting defect was slightly less important (but statistically significant) in MPM cases than in patients with pleural metastases (METS) or with benign pleural lesions associated to asbestos exposure (BPLAE).
The first bias to eliminate in order to correctly understand these results is a potential contamination of the pleural samples with blood. This was unlikely because we excluded all hemorrhagic pleural effusions. Furthermore, the distribution of blood T cell subsets in our patients was similar to the one found in healthy subjects of the same age [
26], except the lower percentages of naive CD8
+ T cells in the METS and the MPM groups.
Immune response plays an important role in pleural tumours, and the development of a CD8
+ mediated immune response have been associated with treatment success in animal models as well as in humans [
33‐
36]. Here we show that this less abundant CD8
+ T cells compartment is not of the effector-memory phenotype, as in healthy subjects. Prado-Gracia et al. previously reported that only a low percentage of this T CD8
+ cell population expressed perforin, an important mediator of cell-mediated cytotoxicity [
28]. In our study, the percentages of total CD8
+ T cells or of CD8
+ T cell subsets in blood or pleural effusion assessed before any treatment were not associated with survival in a Cox proportional hazard model (data not shown). However, it would be very interesting to study the kinetics of CD8
+ T cell populations in patients with malignant pleural effusions of all types under different therapies, and its potential relation with patient outcome as previously reported in animal tumor models [
29,
30].
In all groups of patients, the enrichment of pleural fluid with CD4
+ T cells was due to an increase in the percentages of central memory and naive CD4
+ T cell subsets. This was an unexpected finding since these cells should theoretically rather be found circulating from blood to the lymphoid organs. The same T cells subpopulations were predominant among the CD8
+ T cells in the pleural fluid while one would normally expect an increase in effector memory and terminally differentiated CD8
+ T cells as we found in healthy subjects. Compared to benign pleurisies associated with asbestos exposure, MPE were associated with a decrease in CD8
+ naïve and central memory T cells. But this was not associated with an increase of effector memory and terminally differentiated CD8
+ populations. These results are in accordance with the results published by Prado-Garcia et al. and by Atanackovic et al. [
27,
28] who reported an increase of non effector CD4
+ and CD8
+ subsets as well as with the data of Aguiar et al. [
19] who found a diminishing Naïve T cell population along with an increase of memory T cells in pleura of patients with MPE.
Possible explanations for these findings could relate to different mechanisms. First a defect in local recruitment of effector T cells in target organs or MPE may be due to changes of chemo-attractant signals by the tumor [
37,
38]. A second hypothesis could be that effector cells rapidly undergo apoptosis which generate a higher T cell turnover with accumulation of central memory cells and a depletion in effector cells compartment [
19,
39]. A third hypothesis would be a deficit in the expression of the ζ chain [
40‐
43] which is important in the signal transduction via the T cell receptor. This could results in a defect in activating T cells and thus a generation of a lower number of effectors.
We also found in normal pleural fluid a lower proportion of natural regulatory T cells (T
reg) than in the pathological pleural effusions. These results are consistent with previous reports [
44‐
47]. However this accumulation of T
reg seems to be extremely moderate and related more to the pleura itself than to the type of pleural disease. However, firm conclusions are difficult to extract due to the very limited number of patients in each subgroup. It has also to be taken into account that human malignant mesothelioma tissue contains not only regulatory T cells but also cytokines and chemokines suppressing an efficient anti-tumor immune response and promoting angiogenesis [
38,
47‐
49].
Natural killer cells are also a very important component of the anti-tumor immune response. In accordance with previous reports [
27,
50], we found that despite a higher percentage of circulating NK cells in patients with pleural malignancies (or even in BPLAE patients) than in healthy subjects, there was a defect in recruiting NK cells in the malignant pleural effusions. This defect seemed to be less important in MPM patients. In the literature, NK cells which express the low affinity Fc receptor, FcγRIII (with a CD56
+(low)CD16
+ phenotype) have mostly a cytotoxic function [
51] while the CD56
+(high)CD16
- NK cells mostly produce various cytokines [
52]. In our patients, the defect of pleural NK cells mainly concerned mainly the CD16
neg cells.
There are two crucial and still unsolved questions. Are these findings in MPE correlated with local T cell populations in tumor tissue? Can these changes be therapeutically manipulated in order to obtain a better response to treatment by transforming local immunologic tolerance into anti-tumor immune response? Recently CD8+ tumor infiltrating lymphocytes (TIL) have been associated with a better survival in resected MPM patients [
53].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AS conceived the design and coordinated the study, and wrote the manuscript. BDG participated in the design of the study, performed the statistical analysis, and helped to draft the manuscript. MN participated in the patients recruitment and the samples collection. TG, BC, and SB collected clinical data from patients, and helped to draft the manuscript. VM. JT carried out the immunoassays. MCC and JPD revised the manuscript critically for important intellectual content. HP participated in the patients’ recruitment, and helped to draft the manuscript. ML coordinated the biological part of the study, and helped to draft the manuscript. All authors read and approved the final manuscript.