Discussion
Immune surveillance is the process in which precancerous and malignant cells are detected by the immune system as damaged cells and are consequently targeted for elimination. Cytotoxic T cells, NK and NK-like T cells are indispensable factors in the body’s ongoing defense against these transformed tumor cells. This Cytotoxic function is a highly regulated, multi-factorial immune process that is carried out via using a granule exocytosis pathway to induce granzyme-dependent death in targeted cells. For this reason, in order for lung cancer cells to survive, they must hold certain properties that enable them to evade and resist these cytotoxic cell attacks. In this regard, previous studies have shown that malignant tumor cells are able to escape immune surveillance and thrive [16]. Lung cancer cells produce mediators such as prostaglandin E2 (PGE2) and transforming growth factor (TGF)-β that further support tumor cell proliferation, anti-apoptotic properties and chemotherapeutic resistance [17] . Therefore, a broader understanding of the different immunosuppressive strategies which foster immune evasion, preventing host immune cells from carrying out an effective anti-tumor immune response is needed for the identification of new therapeutic strategies that can potentially improve treatment outcomes in lung cancer patients. The tumor microenvironment (TME) in lung cancer has been a subject of intensive and rigorous studies. The capacity of tumor cells to reshape the constitution and function of stromal and immune cells at the tumor site eventually creating an immunosuppressive milieu. Many studies have reported that this local impairment of immune cells renders them less tumoricidal and thereby supporting the progression of cancer. For example, intratumoral NK cells exhibited profound defects in their ability to produce interferon-gamma and activate degranulation[5]. In a similar manner, both CD4+ and CD8+ cells from lung cancer tissue demonstrated a significant decrease in intracellular levels of granzyme B compared to non-cancerous tissue [18] . On the other hand, cytokines and chemokines that promote angiogenesis, chemoresistance and activate immune suppressive cells (T-regs and MDSCs) have found to exhibit an alteration in their expression within the TME. Collectively, the critical role the TME plays in altering the biological behaviors of tumors has been well established. However, information regarding the systemic effect that tumor cells exert in lung cancer patients remains limited. As increasing evidence suggests that tumors are able to manipulate the systemic immune components, in our study we aimed to clarify the changes in the peripheral immune environment in terms of numbers and functionality of immune cells as well as cytokine levels present in peripheral blood of lung cancer patients[19] .
This study was conducted on 15 patients diagnosed with lung cancer at different stages before (n=5), during (n=5) and after (n=5) induction of chemotherapy as they were compared to healthy donors (n=5). All patients were in the age group ranging between 35 to 65 years and showed an obvious male predominance with a male: female ratio of 85:15. In addition, all male patients were found to be long-term positive smokers which confirms that smoking is a strong prognostic and predictive patient characteristic in lung cancer [20]
Selection of patients must be with a clear history of treatment with board spectrum antibiotics especially before chemotherapy treatment, the effect of inducing antibiotics make a loss of functionality by affecting the normal lymphocyte activation mechanisms of the immune response this was proved when using immune activation in-vitro or applying immunotherapy [21].
The aim was to determine the functionality of CD3, CD8, CD4, NK, NKT, NKCD4 and NKCD8 cells in lung cancer patients before, during and after induction of chemotherapy. We hypothesized that the peripheral immune components are affected to create a pro-tumor peripheral immune environment decreasing the number and functionality of immune cells. Therefore, we investigated the effect of culturing these immune cells inactivation conditions and comparing the results with our initial findings. As our previous study concluded that the numbers of circulating T, NK and NKT cells and their levels of GzB expression are decreased in lung cancer patients, The findings of these study suggest that lung cancer cells might decrease the proportion of peripheral of all the cell populations (CD3, CD8, CD4, NK, NKT, NKCD4 and NKCD8) cells by yet unidentified mechanisms. Notably, the present study also observed a decrease in the levels of GzB before and during induction of chemotherapy when compared to CTRL, while increasing to some extent after induction of chemotherapy. This combined deficiency in the count and functionality of the various lymphocyte subsets can be explained by the effect of the previously mentioned soluble factors produced from the tumor cells, which is in line with the previous studies of Zhao et al., who that these mediators are responsible for inhibition of CTL and NK function as secretors of GzB [10, 22] On the other hand, these findings may be also attributed to the effect of immune suppressive cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Treg) which are correlated with the stages and cancer progression excreting their effect on immune cells [23, 24]
However, after cultivating PBMCs in the stimulatory media, immense changes were observed in most populations of cells, reflecting an increase in the numbers of cells in the before, during and after chemotherapy stages. There was more evident in the early diagnosed lung cancer group of patients before induction of chemotherapy and in the CD3, CD8 and NKCD4 cells in specific. Likewise, the fold change in the expression of GzB on CD3, CD8, CD4 and NKT cells after culture were recorded to be significantly increased in lung cancer patients before induction of chemotherapy with clearly elevated percentages when compared to CTRL and other lung cancer patients. This can be explained by the effect of adding Con-A and IL-2 to the culturing media. Con-A is known for its effect as a stimulator of lymphocyte blastogenesis and mitosis, it is also known to induce the uptake of nucleotides, sugars, amino acids [25] . As of IL-2, it has been considered to be a key growth for antigen-activated T lymphocytes since it also maintains self-tolerance and causes a massive expansion of CD8+cells [26] . Extensive studies on IL-2 have led to the clinical utilization of this molecule in patients with advanced cancer however this was limited due to the significant systemic side effects associated with its use. For this reason, attempting to overcome this obstacle without decreasing the antitumor potency current research is oriented towards developing combination regimens that have additive or synergistic antitumor effects with fewer toxicities [27].Our study demonstrated that the combined use of IL-2 and Con-A promoted synergistic in-vitro expansion and activation of peripheral immune cells, specifically CD8+ cells. Therefore, the combined use of IL-2 and Con-A should be considered a practical strategy for the generation of antitumoral responses in-vivo and for future clinical applications.
On the contrary, the NK population of cells was not affected by this stimulatory effect in terms of either numbers or functionality. This suggests that since NK cells haven’t been activated compared to CTL, CTL are the main secretors of GzB and that they have the ability to be activated to express GzB in abundance. However, NK cells have been previously identified as the main secretors of IFN-γ which may explain why their expression levels of GzB were not potently affected