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Neutral killer cells
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+
NK cells are a subpopulation of CD3 CD56 lymphatic cells characterized by their ability to kill tumor- or
virus-infected cells. Although NK cells are also known for their activity in recognizing and killing cancer
cells, few studies have been performed in CCA. NKs can carry out their cytotoxicity through two pathways,
one antigen-nonspecific, exploiting the release of enzymes such as perforin, proteases, and granzymes, and a
direct one, through the activation of the Fas cell surface death receptor ligand (FasL)/TNF-related
apoptosis-inducing ligand signal pathway . The responsiveness of NK cells to the Fas/FasL pathway also
[50]
has a drawback; in fact, a recent study demonstrated that, in vitro, iCCA cells express high levels of Fas and
FasL, which induce apoptosis of NK cells, as an immune escape mechanism . Conversely, the
[51]
overexpression in CCA tumor cells of CXCL9, a ligand of C-X-C motif chemokine receptor (CXCR) 3,
[52]
induces the recruitment of NK cells that infiltrate the tumor and positively correlated with postoperative
overall survival in a cohort of 70 patients . Similarly, using a xenograft model in which iCCA-derived
[53]
HuCCT-1 cells were xenotransplanted into non-immunocompetent NCr athymic nude mice, infusion of
NK cells (SMT01) induced significant inhibition of tumor growth . Furthermore, in vitro treatment of
[54]
HuCCT-1 and NK co-cultures with cetuximab, an EGFR inhibitor, demonstrated a significant increase in
NK cytolytic activity against tumor cells . These data suggest a potential use of NK in the treatment of
[55]
CCA. NK cells are characterized by the expression of natural killer group 2 member D (NKG2D) receptor, a
receptor whose polymorphisms are linked to the susceptibility to cancer development . A study on 82
[56]
patients with eCCA who underwent surgical resection showed that overexpression of the NKG2D receptor
[57]
on NK cells and its ligands in the cancer cells correlated with a better patient prognosis .
Tumor-associated neutrophils
[58]
Despite their importance in the immune response , there are very few studies on the involvement of
neutrophils in the pathogenesis of CCA. Similar to TAMs, TANs are divided into two subcategories, N1,
which is endowed with antitumor function, and N2, which has protumoral activity . Circulating
[59]
neutrophils are recruited to the tumor site by cells of the TME, such as CAFs [that secrete granulocyte-
macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), VEGF,
and IL-1β], TAMs (IL-6 and IL-8), T lymphocytes (CXCL1, CXCL2, interferon γ, and TNFα) . The
[60]
neoplastic cells themselves secrete CXCL5, which induces neutrophils recruitment by activating the
phosphatidylinositol 3-kinases/Akt and ERK1/2 pathways . Once they reach the tumor site, TANs secrete
[61]
a vast set of factors potentially involved in the biology of CCA, as mentioned above (such as MMP-8, MMP-
9, CXCL1, CXCL2, CXCL6, CXCL8, CCL7, and VEGF) [16,58] . It has been shown that, in both iCCA and
eCCA, the accumulation of TANs leads to a worse overall and disease-free survival of tumoral patients [62-64] .
Unfortunately, only a few studies have evaluated the impact of TANs on CCA and they are mostly
observational.

Dendritic cells
DCs are antigen-presenting cells and are usually found in small numbers in healthy tissues. It should be
noted that the TME often has fewer DCs than the surrounding healthy tissue . Similar to TANs, the
[41]
studies regarding DCs in CCA are also rather sporadic. From a topographical point of view, mature DCs
tend to accumulate on the tumor invasion front, while immature DCs are found in the tumor bulk . A
[65]
more recent study also demonstrated in a cohort of 350 patients with iCCAs that accumulation of DCs in
the peritumoral tissue, but not in the CCA, is associated with a worse outcome . A strong interaction
[66]
between DCs and T cells has also been demonstrated; in fact, the inhibition of IL-10 and TGFβ receptors
and the overexpression of cAMP-dependent protein kinase type I-alpha regulatory subunit (PRKAR1A) on
DCs stimulates the antitumor activity of T cells against CCA [67,68] . Finally, a recent work with four different
mouse models of iCCA showed that anti-CD40/PD-1 treatments, accompanied by gemcitabine/cisplatin, is