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