**5. Monocyte derived DCs**

*Current Cancer Treatment*

CD36<sup>+</sup>

other cDC2 subset (CD5hi or CD32B+

and 8 compared to CD5lo cDC2 and CD32B+

T cells [8, 10, 16].

**4. Plasmacytoid dendritic cells (pDC)**

tion of IDO and TGF-beta [42].

pDCs are recognised as being CD11c<sup>−</sup>/loCD45RA+

dence of this factor for normal human pDC development [46].

and CD8<sup>+</sup>

(also known as STING) in comparison to CD163+

versus CD5lo cDC2 subsets, respectively [8, 28].

CD163<sup>+</sup>

by CD5+

tors of CD4+

isoforms of Clec10A have been found in mice and should be carefully considered when using it across species [27]. Heterogeneity within the human cDC2 subset has been identified using CD5 or CD32B versus CD163 and CD36. The CD5lo or

FL, but also rely on transcription factors *IRF4* and *IRF8*, for development [20*,* 29]. The cDC2 DCs highly express TLR2 and also express a range of cytosolic viral RNA sensors such as RIG-I [30, 31]. Different proposed cDC2 subsets also seem to have different PRR expression patterns. For example, CD5hi cDC2 express high levels of TLR7

Activated cDC2s can drive Th17 immune response and can also produce high levels of IL-12p70, potentially inducing Th1 differentiation [2, 29]*.* However, current data suggests Th17 versus Th1 driven responses may be independently driven

Human cDC2s are able to cross-present *soluble* Ag to naïve and memory CD8<sup>+</sup>

The pDCs constitute ~0.01–0.04% of PBMCs and commonly reside in secondary lymphoid organs localising in the follicular cortex, T cell nodules and around high endothelial venules [36, 37]. As their name suggests, pDCs are similar in morphology to that of plasma cells. Under light microscopy, pDCs are observed to be spherical in shape with a rounded nucleus, often predominant endoplasmic reticulum and present as clusters in T-cell rich regions of lymphoid tissue [36–38]. The pDCs, originally identified as 'natural interferon producing cells' (NIPC), are renowned for their ability to drive immense type I and type III IFN production via TLRs 7 and 9 [39–41]. This IFN production is essential to combat viral infection but pDC-derived IFN is also thought to contribute to disease in autoimmune diseases including systemic lupus erythematosus [42]. They are also thought to play a role in Th2 induction and asthma progression in humans [42]. Conversely, pDC have also been shown to play a major role in tolerance *in vivo*, through their produc-

 and can express CD56 (reviewed in [2]). pDCs may also be identified by their transcription factors including; TCF4 (also known as E2-2), SPIB, ZEB2, IRF8, IRF7 and IRF4 [43–45]. Haploinsufficiency in the *TCF4* gene results in Pitts-Hopkins syndrome, which characteristically generates defective pDCs, illustrating a depen-

The pDCs can be divided into 2 subsets based on CD2 expression [47]. Recent single cell transcriptomic profiling of blood DCs from healthy donors has revealed

segregation of pDCs away from contaminating AS DCs demonstrated potent IFN-α production after TLR9 stimulation and a lack of T cell priming attributes [8]. Whether AS DCs and pDC are 2 distinct cell types or differentiation stages of one

'pDC' also express AXL and SIGLEC6 (known as AS DCs). These AS

cells at comparable levels with cDC1s [32–35]. However, the mechanism of crosspresentation differs between both subsets [35] and cDC2 do not possess the potent ability to cross-present Ags from dead cells. Human cDC2 are also potent stimula-

'cDC2' are transcriptionally more related to monocytes than the

) [8, 28]. Like cDC1, CD1c<sup>+</sup>

CD36+

CD123+

allogeneic T cell proliferation whereas the

CD303+

CD304+

HLA-

cDC2s require

T

cDC2 express higher levels of *TMEM173*

cDC2 subset [8, 28].

**104**

DR+

that CD2<sup>+</sup>

DCs can stimulate CD4<sup>+</sup>

another is yet to be defined.

Monocyte derived DC (moDC) refers to DCs induced from monocytes with GM-CSF *in vitro.* These tissue culture systems originated in the early 1990s based on work showing varying combination of cytokines with GM-CSF could induce the acquisition of antigen presentation capacity in stem cells and CD34<sup>+</sup> blood precursors [53–56], and this was optimised with the addition of IL-4 [57]. These systems have been an immensely popular tool for more than two decades for *in vitro* research pertaining to conventional DC biology and immunological function. They have been particularly useful in human research due to the difficulties in obtaining large numbers of *ex vivo* primary human DC for research. However, the feasibility of these models has recently been questioned, detailed analyses of GM-CSF induced DC cultures reveal a heterogeneous population of macrophages and conventional DCs, with the MHCIIhi cells the most DC-like [58*–*61].

It still remains unclear whether the moDC actually represent an *in viv*o equivalent cell subset. They potentially represent an *in vitro* equivalent of an inflammatory monocyte known as TNF/iNOS producing DCs (TipDCs), based on their surface phenotype [62], cytokine profile and a shared precursor [62]. Importantly, high intra-tumoral expression of CD40L, TNF-α and iNOS, key phenotypes of TipDCs, were strongly correlated with substantially higher long term disease free survival rates over 10 years in patients with colorectal cancer [63]. Therefore, moDCs may represent a useful and relevant *in vitro* model of inflammatory DCs.

#### **5.1 MoDC and cancer vaccines**

While the *ex vivo* induced moDC do not recapitulate *bona fide* DC subsets, the ease of isolation and culture has made the moDC a popular vaccine candidate in human clinical trials since the late 1990s. However, results from clinical trials using moDC in cancer immunotherapies for various cancer types have been modest at best [64*,* 65]. In a more recent phase II trial of patients with surgically resectable liver metastatic colon adenocarcinoma, vaccination of patients with autologous tumour lysate pulsed moDC conferred interim protection, demonstrating a 3-fold increase in the median disease free survival compared to the control arm of the study [66]. The continued refinement of moDC preparations and the choice of antigens, may see future improvements of DC cancer vaccines.

The ability to present Ag and activate the adaptive immune response makes DCs an attractive target to re-invigorate anti-cancer immunity. There are different types of DC vaccines, with the most common type involving the *ex vivo* maturation of autologous DCs. In this method, DCs are isolated from patient peripheral blood mononuclear cells (PBMCs) obtained via leukapheresis, incubated with maturation stimuli and tumour Ags, and vaccinated back into the patient. Because this method requires a large number of DCs, and naturally circulating blood DCs are rare, the majority of clinical trials have previously used moDCs for this type of DC vaccine and have been extensively characterised [67*,* 68].

Thus far, a wide variety of moDC vaccine strategies have been trialled [68]. moDCs have been differentiated and matured using monocyte conditioned medium with various supplements of cytokines (TNF-α, GM-CSF, IL-4, IFN-α), TLR agonists (LPS) and other factors such as prostaglandin E2 [67–69]. There is also variety in the type of Ags loaded into DCs such as peptides from tumour-associated Ags (TAA), TAA-encoding mRNA and whole tumour lysates [67]. More recently, the electroporation of synthetic mRNA encoding DC-maturation factors such as CD40 ligand, constitutively active TLR4 and CD70 together with fusion proteins DC-LAMP and melanoma-associated Ags into autologous moDCs (TriMixDC-MEL) have proven safe and immunogenic in phase 1 clinical trials in metastatic melanoma [70]. However, the variation in the aforementioned vaccine factors as well as the route of DC administration (intranodal, i.v.) and lack of standardised method of moDC generation has shown variable efficacies of moDC vaccines in clinical outcomes.
