Immunology Letters 261 (2023) 13–16
Contents lists available at ScienceDirect
Immunology Letters
journal homepage: www.elsevier.com/locate/immlet
Human toll-like receptor 8 (TLR8) in NK cells: Implication for
cancer immunotherapy
Irene Veneziani #, Claudia Alicata #, Lorenzo Moretta *, Enrico Maggi
Tumor Immunology Unit, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
Adjuvants
NK cells subsets
Endosomal toll like receptors
Toll like receptors agonists
Tumor immunotherapy
Toll-like receptors (TLR)s are homo- or heterodimeric proteins, whose structure and function were widely
described in the antigen presenting cells (APC), such as Dendritic cells (DC). Recently, the expression and the role
of TLRs in fighting against pathogens, was described also in NK cells. Their activation and functional properties
can be directly and indirectly modulated by agonists for TLRs. In particular CD56bright NK cells subset, that is the
most abundant NK cell subset in tissues and tumor microenvironment (TME), was mostly activated in terms of
pro-inflammatory cytokine production, proliferation and cytotoxicity, by agonists specific for endosomal TLR8.
The interplay between DC and NK, that depends on both cell-to-cell contact and soluble factors such as cytokines,
promote both DC maturation and NK cell activation. Based on this concept, a TLR based immunotherapy aimed
to activate NK-DC axis, may modulate TME by inducing a pro-inflammatory phenotype, thus improving DC
ability to present tumor-associated antigens to T cells, and NK cell cytotoxicity against tumor cells. In this minireview, we report data of recent literature about TLRs on human NK cells and their application as adjuvant in
cancer vaccines or in combined tumor immunotherapy.
1. Introduction
Cells of the innate immunity recognize microbial-associated or
pathogen-associated molecular patterns (MAMPs/PAMPs) through their
pattern recognition receptors (PRRs) including, NOD-like receptors
(NLRs), C-type lectin receptors (CLRs), RIG-I-like receptors (RLRs) and
Toll-like receptors (TLRs) [1,2]. Similar to the antigen presenting cells
(APC), TLR-mediated signaling pathways represent the first-line of de­
fense against bacterial, viral, and fungal pathogens also in Natural Killer
(NK) cells [3–5]. Moreover, TLRs are engaged to favor the cross-talk
between NK cells and APC. In particular, plasmacytoid dendritic cells
(pDC), which are activated through TLR7/8, promote the release of type
I interferon (IFN) [6,7], induce an increased expression of CD69 and
potentiate the cytolytic activity of NK cells [8]; on the other hand,
activated NK cells stimulate DC maturation, upon the production of
TNF-α [8].
This mini-review reports data of recent literature on the expression
and function of TLRs on human NK cells and their application in cancer
immunotherapy. It will be particularly focused on TLR8 agonists
employed as adjuvant in cancer vaccines or in combined tumor
immunotherapy, aimed to re-activate intra-tumoral dysfunctional NK
cells [9].
2. Toll-like receptors: features and function
TLRs are the most well-defined PRRs expressed on both the cell
surface and endosome membranes of immune cells [10,11]. The human
TLRs expressed at the cell surface are TLR1, 2, 4, 5, 6, 10 while the
endosomal ones are TLR3, 7, 8, 9 [12]. TLRs recognize conserved
PAMPs, which serve as TLR agonists/ligands (TLRL), with differences in
terms of timing, binding affinity and conformational site of interaction
[13,14]. Besides PAMPs, some endogenous components, such as
fibrinogen, heat shock proteins, RNA and DNA, also provide signals
through TLRs [14,15]. TLRs are expressed not only on cells of innate
immunity but also on some cells of the adaptive immunity (as Treg and
activated T cells), thus widely contributing to immune response against
pathogens [15]. TLRs are type I transmembrane proteins with ectodo­
mains containing leucine-rich repeats (LRRs) that mediate PAMP
recognition, transmembrane domains, and a conserved region of ~200
aa intracellular Toll-interleukin 1 (IL-1) receptor (TIR) domain required
* Corresponding author at: Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy.
E-mail address: Lorenzo.moretta@opbg.net (L. Moretta).
#
IV and CA equally contributed to the paper.
https://doi.org/10.1016/j.imlet.2023.07.003
Received 21 June 2023; Received in revised form 6 July 2023; Accepted 8 July 2023
Available online 13 July 2023
0165-2478/© 2023 The Author(s). Published by Elsevier B.V. on behalf of European Federation of Immunological Societies. This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
I. Veneziani et al.
Immunology Letters 261 (2023) 13–16
for downstream signal transduction [1,16].
TLRs are homo- or heterodimeric proteins. In particular, TLR8 is
present on endosomal membranes, both as homodimer or as hetero­
dimer in combination with TLR7 [17]. TLR8, as well as TLR7, binds
single-stranded RNA (ssRNA)-derived from RNA viruses (as vesicular
stomatitis, influenza A, and HIV) and imidazoquinoline derivatives as
Resiquimod (R848), or guanine analogs as Loxoribine [12,18]. While
human TLR7 recognizes GU-rich sequences of ssRNA, human TLR8
binds both AU- and GU-rich sequences thanks to its ability to form
ligand-specific secondary structures [9]. Notably, the stimulation of a
TLR may modulate the expression of other endosomal TLRs. For
instance, TLR8 triggering leads to downregulation of TLR7 and TLR9
and the increase of TLR2 in monocyte/macrophages [19]. This obser­
vation was reproduced in Tlr8− /− mice, in which the absence of TLR8
led to higher levels of expression of TLR7 and interferon-stimulated
genes [20].
The intracellular signaling of all TLRs starts with phosphorylation of
myeloid differentiation primary response protein88- (MyD88-); the only
exception is TLR3 which stimulates the TIR domain-containing adapterinducing interferon-β (TRIF)-dependent pathways [1,16]. Both path­
ways lead to activation of transcription factors as NF-kB, AP1, CREB,
IRF3/7, which, in turn, promote the transcription of genes for type I and
III interferons (IFN), inflammatory cytokines/chemokines, cos­
timulatory and adhesion molecules and antimicrobial mediators [3].
Clear-cut data indicate that, even though NK cells can be directly
activated by some agonists of the endosomal TLRs, both the cross-talk
with DCs and the cytokine microenvironment are crucial for activation
of NK cell effector function.
4. NK cell subsets are mostly activated through TLR8
When compared to other endosomal TLRL as TLR3L (Poly I:C) and
TLR9L (ODN2395), TLR7/8L (R848) resulted the most effective in
inducing NK cell activation. Indeed, the proportion of CD69+ and CD25+
NK cells was highly increased upon R848 stimulation, significantly less
upon Poly I:C and nothing upon ODN2395 stimulation. In addition, the
cytotoxic activity and the cytokine production (IFN-γ, TNF-α, and GMCSF) were improved only with Resiquimod. Importantly, R848 was
the only endosomal TLRL able to activate and induce IFN-γ production
by CD56brightCD16− and, to a lesser extent, by CD56dimCD16+NK cell
subset, while Poly I:C triggered only weakly CD56dimCD16+ subset.
Notably, R848 did not induce the increase of CD57 expression on NK
cells, suggesting a marginal ability to induce their maturation. Besides
induction of activation markers, R848, but not the other TLRL, induced
CD56brightCD16− NK cell proliferation (peaking at 4 day) and cytotox­
icity with upregulation of CD107a, Perforin and Granzymes expression
[32].
Previous reports underscored the ability of different TLR7/8 agonists
(including R848) to stimulate monocytes/macrophages, DC and NK
cells; our recent findings indicated that R848 triggered NK cells exclu­
sively through TLR8 [32]. Data from several reports would support this
notion, particularly: (i.) TLR8 but not TLR7, is expressed in a dimeric
(active) form in a ligand-independent way in NK cells, perhaps favored
by DC-driven cytokines [35], (ii.) the IFN− γ produced by NK cells upon
different stimuli, is a strong inducer (in an autocrine way) of the
expression of TLR8 (much more than TLR7) [36], (iii.) the miRNAs
contained in exosomes derived from NK, tumor and tumor environ­
mental cells mainly bind TLR8, impacting on NK cell function [37,38],
(iv.) many functions of CD56brightCD16− NK cells are upregulated by the
agonists specific for TLR8 (TL8–506) but not for TLR7 (Imiquimod,
Loxoribin and modified 8-hydroxy Adenine) [32]. Notably, similar to
NK cells, TLR8, but not TLR7, has been recently shown to be functionally
active on other innate cells as neutrophils [39,40]. It is relevant to un­
derscore that TLR8 is the only TLR capable of distinguishing the host
RNA by nucleoside modifications and of activating its signaling pathway
when a microbial RNA enters into the cell [41]. In addition, it is
important to underline that TLR8 recognizes microbial structures from
viable microbes [42], as well as poly(A)/T sequences and small chemical
molecules with antiviral function [43].
3. Expression of TLRs and their role in NK cell activation
The expression of TLRs (especially of endosomal TLRs) in human NK
cells is influenced by: (i.) variable degree of activation of freshly-isolated
TLR+ NK cells in different donors, (ii.) DC-NK cell interactions mediated
by different surface and soluble signals (cytokines) which, in turn, may
favor or impair, the expression and triggering of endosomal TLRs in NK
cells, (iii.) cross-modulation of such receptors after triggering of one of
them, iv. interference of DAMPs and miRNAs with the expression and
activation of some TLRs (mainly TLR4 and TLR7/8) [21–25].
The majority of reports agrees that NK cells express functional TLR1
[26], TLR2 [24], TLR3 [22], TLR4 [27], TLR5 [26], TLR7/8 [28,29] and
TLR9 [30], regardless their state of activation. mRNA analysis demon­
strated that, in purified NK cells, TLR1 was the most expressed, followed
by moderate levels of TLR2, TLR3, TLR5 and TLR6, and low levels of
other endosomal TLRs [26,30]. However, there was uncertainty on the
expression of different TLRs and activity in the two main NK cell subsets:
the CD56dimCD16+ cells which prevail (>90%) in peripheral blood and
are specialized in cytotoxic activity, and the CD56brightCD16− cells,
which are less cytolytic, produce high levels of cytokines and are less
represented in peripheral blood (< 10%), while they are prevalent in
tissues (e.g. T-cell areas of lymph nodes) and tumor microenvironment
(TME) [31]. A recent study by our group has established that, the
endosomal TLRs are consistently expressed in CD56brightCD16− and
CD56dimCD16+ NK cell subsets at both the mRNA and protein levels
[32].
The response to TLRL occurs in vitro in both activated and freshly
isolated NK cells, in which suboptimal doses of inflammatory cytokines,
primarily IL-2 and IL-12, generate an increased TLR-mediated activa­
tion. In particular, the agonists of endosomal TLRs can activate NK cell
function either directly or indirectly through accessory cells, cytokines
or cell-to-cell contact [23]. Recently, we provided definitive evidence
that resting NK cells can be activated by endosomal TLRL in the presence
of co-stimuli such as suboptimal doses of cytokines, including IL-2,
IL-12, IL-15, IL-18 (alone or in combination) [23]. Indeed, some
studies indicated that NK cells are activated in vitro by DC-derived IL-12
and T cell-derived IL-2 [22,29,30,33,34].
Taken together, these data reinforce the concept that the direct and
indirect impacts of TLR engagement, facilitates the interaction between
DC and NK cells which, in turn, may affect downstream other cells of
innate and adaptive immunity.
5. Cancer immunotherapy: targeting TLR8 in NK cells
Preclinical and clinical studies have provided evidence that the
downstream inflammatory response after TLR7 and TLR8 engagement,
results in an initial efficacy for the treatment of cancer and viral in­
fections, as well as for its action as vaccine adjuvant. This explains why
in the last decade, there has been significant progress in the optimization
of novel TLR7 and TLR8 small molecule agonists with more than 70 new
patents. These novel compounds are currently analyzed to evaluate their
ability to desired immune responses, to improve their safety and to act
synergistically with checkpoint inhibitors in combination therapies
[44].
When administered intratumorally, TLR8 agonists, in addition to NK
cell activation, promote the interplay between NK and macrophages, the
M2 to M1 shift, leading to an increased MHC class II expression, and the
differentiation of naïve CD4+ T cells into type 1 profile [45]. Moreover,
TLR8 agonists mediate the reversal of the suppressive function of human
Treg cells through the TLR8-MyD88-IRAK4 signaling pathway and
contribute to the follicular helper T cell differentiation [9,42].
Targeting the cross-talk between DC and NK cells may represent a
14
I. Veneziani et al.
Immunology Letters 261 (2023) 13–16
Fig. 1. Potential mechanisms of TLR8 agonists in tumor immunotherapy.
promising approach to the treatment of infections and cancer. However,
it is well-known that the clinical efficacy of TLRL delivered systemically
is limited by their solubility, systemic toxicity and possible induction of
autoimmunity [46,47]. To overcome these limitations in the use of
TLR7/8 agonists in cancer therapy, biomaterial-based drug delivery
systems have been developed. These devices can be directly adminis­
tered at the tumor site and are passively accumulated in lymphoid tis­
sues. In addition, they may be triggered by environmental stimuli, and
may target specific cell populations. In addition, recent studies reveled
the potential benefits of TLR7/8 agonist co-delivery with other thera­
pies, particularly checkpoint inhibitors, experimental cancer vaccines,
and chemotherapy, leading to relevant anti-cancer effects [48].
In murine models, the local infusion of endosomal TLR agonists
enhanced both the recruitment and the activation of immune cells at the
tumor site with more effective NK cell activity, type 1 profile of T cell
responses and the efficacy improvement of checkpoint inhibitors [49].
In particular, TLR7/8 agonists have been found to delay tumor growth
and even to induce tumor regression in Acute Myeloid Leukemia models
[50,51]. Recent studies also showed that some novel TLR7/8 agonists
induce robust pro-inflammatory cytokine secretion and enhance NK
cell-mediated ADCC in vitro as well as the enhanced efficacy of mono­
clonal antibodies in two different in vivo tumor mouse models [52].
Based on these data, pretreatment of NK cells with endosomal TLRL,
or intra-tumoral (IT) therapy with these molecules, may indeed repre­
sent a novel potential strategy of solid tumor therapy [53]. In addition,
dermal application of Imiquimod (TLR7 agonist) or Resiquimod
(TLR7/8 agonist) to treat human skin tumors, by inducing mainly local
activation of NK cells, greatly limits systemic involvement and un­
wanted side-effects [54,55]. Of note, these findings highlight the po­
tential role of TLR8 engagement of NK cells infiltrating TME as a novel
strategy of tumor immunotherapy. The predominant activity of TLR8
agonists on CD56brightCD16− NK cells, which are predominant in tissues
and in tumor lesions, makes the local infusion of TLR8 agonists more
than a potential candidate of cancer therapy. In this context, our recent
data showed that TLR8 targeting improved both IFN-γ production and
cytotoxic activity of NK cells derived from the ascitic fluid of patients
with metastatic ovarian carcinoma (mainly represented by
CD56brightCD16− NK cells) [32]. In this tumor, TLR8 signaling activates
NK cells which, in turn, may produce cytokines and chemokines which
amplify the effect on NK cells, by inducing proliferation and recruitment
of other effector cells [32]. In the TME of solid tumors, TLR8 agonists
have been reported to induce tumor cell apoptosis and to favor matu­
ration (and loss of function) of myeloid-derived suppressor cells [56]
and prevent T cell senescence/exhaustion [57]. Moreover, TLR8 ago­
nists have been reported to exert a metabolic inhibitory effect on CD4+ T
regulatory cells present in ovarian cancer cell microenvironment [58].
Lastly, in humans, TLR8 agonists have been locally assayed in clinical
phase Ib-II trials in patients with different metastatic solid tumors [49,
59,60].
6. Concluding remarks
In conclusion, recently, different results have suggested that the ef­
fects referred to TLR8 agonists in some in vivo models are consistently
mediated by activated NK cells [61,62]. Future research efforts should
be devoted to establish which interactions occur between NK cells
activated by such agonists and other cells present in the TME, also
focusing on drug safety, efficiency and specificity [63]. The findings on
TLR8-mediated rescue of NK cell reactivity in ascites of patients with
ovarian cancer, can also be exploited as a new in vitro assay to determine
which patients could obtain relevant benefits by the therapy with
infused TLR8 agonists in the peritoneal cavity (as adjuvants of tumor
vaccines or combined chemo- or immuno-therapies) and/ or the adop­
tive transfer of autologous or allogenic NK cells expanded in vitro with
TLR8 ligands (Fig. 1).
15
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Immunology Letters 261 (2023) 13–16
Declaration of Competing Interest
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The authors declare that they have no conflicts of interest.
Acknowledgements
This work was supported by the Italian Ministry of Health with
“Current Research funds” (grant no. RC-2020 OPBG to L.M. and E.M.)
and from Associazione Italiana per la Ricerca sul Cancro (project no.
5×1000 2018 Id 21147 and project no. IG 2017 Id 19920 to L.M.). I.V.
was supported by FIRC-AIRC fellowship for Italy; C.A.is a recipient of a
grant awarded by Fondazione Umberto Veronesi.
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