In vitro generation of human monocyte-derived dendritic cells

Anais da Academia Brasileira de Ciências (2019) 91(4): e20190310

(Annals of the Brazilian Academy of Sciences)

Printed version ISSN 0001-3765 / Online version ISSN 1678-2690

http://dx.doi.org/10.1590/0001-3765201920190310

www.scielo.br/aabc | www.fb.com/aabcjournal

An Acad Bras Cienc (2019) 91(4)

BIOLOGICAL SCIENCES

In vitro generation of human monocyte-derived dendritic cells

methodological aspects in a comprehensive review

GIOVANA CECHIM and JOSÉ A.B. CHIES

Laboratório de Imunogenética, Departamento de Genética, Instituto de Biociências, Universidade Federal do Rio Grande

do Sul/UFRGS, Av. Bento Gonçalves, 9500, Prédio 43323, Campus do Vale, 90501-930 Porto Alegre, RS, Brazil

Manuscript received on March 15, 2019; accepted for publication on June 1, 2019

How to cite: CECHIM G AND CHIES JAB. 2019. In vitro generation of human monocyte-derived dendritic

cells methodological aspects in a comprehensive review. An Acad Bras Cienc 91: e20190310. DOI 10.1590/0001-

3765201920190310.

Abstract: Dendritic cells (DCs) initiate and shape both innate and adaptive immune responses. They are

specialized in antigen presentation to naive T cells, thereby orchestrating the T cell immune responses.

Human peripheral blood and tissues contain several subsets of phenotypically and functionally distinct

DCs, which promote interactions between the external environment and lymphoid organs. Because of the

diculty in purifying these cells, in vitro studies only became more frequent when Frederica Sallusto and

Antonio Lanzavecchia developed a method to generate DCs from blood monocytes in vitro. Nowadays a

wide range of biotechnological innovations has allowed the study of DCs and their precursors in the most

diverse situations faced by the immune system. As a result of such studies, monocyte-derived dendritic

cells (MDDC) are presently used in clinical protocols for the treatment of a variety of diseases, including

cancer and human immunodeciency virus infection. We summarize recent advances in the understanding

of methodologies and inputs used in protocols to dierentiate DCs from blood monocytes in vitro.

Key words: Cytokines, dendritic cell, differentiation, monocytes.

Correspondence to: José Artur Bogo Chies

E-mail: [email protected]

ORCid:https://orcid.org/0000-0001-7025-0660

INTRODUCTION

In 1973, Ralph M. Steinman and Zanvil A. Cohn

described a new cell type that had a cytoplasm with

pseudopodia structures of various sizes and shapes,

giving the cell a starry aspect, much like neuronal

dendrites (Steinman and Cohn 1973). This work

described the cell currently known as a Dendritic

cell (DC). In 2011, the importance of this discovery

was recognized with the Nobel Prize in Physiology

or Medicine (Steinman 2012). However, the study

of these cells only had significant advances in

the 1990’s, when Frederica Sallusto and Antonio

Lanzavecchia rst developed a method allowing

the generation of DCs from blood monocytes. In

this study, monocytes obtained from peripheral

blood were differentiated into myeloid DCs in

the presence of granulocyte-macrophage colony

stimulating factor (GM-CSF) and interleukin (IL)-

4, (Sallusto and Lanzavecchia 1994).

DCs are the major antigen presenting cells

(APCs) due to their unique ability to activate naive

T cells (Steinman and Witmer 1978, Banchereau

et al. 2000). DCs are bone marrow derived,

originating from both myeloid and lymphoid

precursors and can encompass a range of cell types

with several phenotypes and functions (Ardavin et

al. 1993, Banchereau et al. 2000, Ueno et al. 2007,

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An Acad Bras Cienc (2019) 91(4) e20190310 2 | 21

Merad et al. 2013). They are distributed throughout

the body but are especially present in regions such

as the skin and mucous membranes, promoting

interactions between the external environment and

lymphoid organs (Steinman 1991, Guermonprez

et al. 2002, Granot et al. 2017, Worbs et al. 2017).

Due to its migratory patterns, DCs are called “the

Sentinels” of the immune system (Stockwin et al.

2000, Randolph et al. 2008, Merad et al. 2013,

Worbs et al. 2017).

Generally, these cells are found inside tissues

in a so-called “immature” state characterized by

a high capacity to capture and process antigens

and a limited capacity to stimulate T cells. In the

immature state, DCs exhibit low expression of the

co-stimulatory molecules CD80 and CD86, as well

as a low expression of major histocompatibility

complex (MHC) class II molecules. The presence

of “danger signals” triggers morphological changes

in DCs, inducing a process known as maturation,

which is characterized by a reduction of the DC

endocytic capacity and the increased expression

of MHC class II and co-stimulatory molecules

(Banchereau et al. 2000, Jore et al. 2009, Dalod et

al. 2014, Worbs et al. 2017).

Considering this cell type diversity, DCs may

be classied according to several criteria, including

their location, such as skin, lungs, and lymphoid

organs, since their function is intimately linked to

the compartment where they are located (Wu and

Liu 2007). Within this anatomical classication,

it is also possible to separate DCs in two further

categories: the migratory DCs, which migrate

continuously from peripheral tissues to the

draining lymph nodes via the aerent lymphatic

vessels, and resident DCs, which are found in the

tissues, and when activated, migrate to the draining

lymph nodes (Randolph et al. 2008, Segura et

al. 2012, Merad et al. 2013, Pulendran 2015). In

this regard, DCs are usually classied according

to their different abilities to drive the immune

response by dierential antigen presentation or by

modulating the immune system through cytokine

secretion (Jore et al. 2009, 2012, Kurts et al. 2010,

Pulendran 2015). Nowadays DCs are evaluated

according to their transcriptional proles in order

to understand the regulation of the development

and dierentiation of the distinct DC lineages (Belz

and Nutt 2012, Hammer and Ma 2013, Merad et al.

2013, Collin and Bigley 2018).

Due to its central role in immune responses,

a better understanding of the physiology and

function of the DCs will help us to improve the

use in clinical protocols to treat several diseases

such as cancer and human immunodeciency virus

(HIV) infection (Banchereau et al. 2001, Turville et

al. 2002, Kavanagh and Bhardwaj 2002, Gandhi et

al. 2016, Garg et al. 2017).

This review presents key information

about the generation of human DC from blood

monocytes (MDDC). We will analyze alternatives

for the purification of precursor cells (meaning

the monocytes), the selection of suitable culture

medium, appropriate culture medium supplements,

growth factors, and cytokines. We will also review

the most common molecular markers used to

characterize DCs by ow cytometry. The Figure

1 presents a schematic summary of the steps in a

monocyte-derived dendritic cell culture.

SOURCES OF MONOCYTES

The rst step for monocyte-derived dendritic cell

culture is to choose the monocyte source, since

these cells are the preferred precursors for in vitro

MDDC generation. One possibility is to directly

collect blood by venipuncture. The advantage of

whole blood as a monocyte source is the freshness

of the material. However, the disadvantage of using

whole blood is the low yield of monocytes, since

they represent only 6% of all peripheral blood cells,

so using whole blood requires the processing of

a large blood volume (Meyer et al. 2005, WHO

2010, Gillio-Meina et al. 2013).

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The use of the cells, which are normally

discarded after the processing of donated blood,

is a way to skip such a problem. Commonly, after

blood donation, the blood bag is centrifuged,

which allows the separation of its components into

erythrocytes, plasma, and a bu y coat, which are

collected into separate bags. The bu y coat is a

concentrate of platelets and leukocytes which

can be further processed for platelet puri cation.

The remaining material is rich in leukocytes and

constitutes an excellent source of monocytes, but is

commonly discarded. Processing a 450 mL blood

bag usually generates 30–80 mL of bu y coat with

approximately 1x10

cells (Ito and Shinomiya 2001,

Repnik et al. 2003, Meyer et al. 2005, Strasser and

Eckstein 2010).

When plasmapheresis is performed in order to

obtain distinct blood products, leukocytes are often

collected together with platelets as a byproduct and

must be removed to avoid immune rejection in the

recipient. Therefore, leukocytes are  ltered out by

leukoreduction and collected into leukoreduction

system chambers (LRSC) or leukocyte removal

filters also known as buffy cones (Ebner et al.

2001, Dietz et al. 2006, Strasser et al. 2007). The

bu y cone usually contains 10 mL of a processed

mixture with approximately 1x10

cells.

Another possible source for large quantities

of leukocytes is a leukopak. This is an enriched

leukapheresis product consisting of a variety of

blood cells including monocytes, lymphocytes, and

erythrocytes. There are two types of leukopaks:

one is collected from peripheral blood without

any stimulation on the blood donor and the other

are obtained from donors who were stimulated

with G-CSF (granulocyte colony stimulating

factor) to induce leukocyte production and trigger

migration of stem cells from bone marrow into

the bloodstream. Usually, leukopaks are collected

from healthy donors, but for research purposes it

is possible to obtain leukopaks from donors that

present certain pathologies such as hematological

malignancies and diabetes. Although the production

of leukopaks from such speci c donors is not usual,

this kind of product can be commercially provided

under request. Commercial leukopaks generally

contain 80–200 mL of processed material with

approximately 7x10

peripheral blood mononuclear

cells (PBMC), (more information available on:

www.allcells.com/products/whole-tissue/leuko-

pak). The Table I shows the percentage of cell

types and the amount of cells found in di erent

monocyte sources.

MONOCYTE PURIFICATION

After choosing the source of cells, the next

step in MDDC culture is to process the sample

Source

selection

•Peripheral blood

•Leukoreduction system chambers

•Buffy coat

•Leukopacks

Monocyte

purification

•Density gradient centrifugation

•Adherence to inert surface

•Magnetic-beads/antibody based cell isolation

•Fluorescent antibody based cell sorting

Monocyte-derived

dendritic cell

culture

•Cytokines

•Culture medium

•Supplements of the culture medium

•Culture time

•In vivo activation of immature dendritic cell

Cytometry

characterization

•Immunophenotype markers

Figure 1 - Scheme of the steps of a MDDC culture.

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(Elkord et al. 2005). It is possible to use density

gradient media to separate blood components.

One of the most commonly used density gradient

medium is Ficoll

Paque Plus (GE Healthcare

Life Sciences – Catalog number: 17144003).

Ficoll

Paque Plus is a synthetic sucrose polymer

that ensures the separation of blood components

as follows: erythrocytes and polymorphonuclear

cells (eosinophils and neutrophils) are denser than

Ficoll

Paque Plus and therefore are deposited on

the bottom of the tube. Immediately above the

erythrocytes a density gradient medium Ficoll

Paque Plus layer forms (density of 1.077 G/mL

at 20°C). Above the Ficoll

Paque Plus layer,

PBMCs form a layer of cells similar to a cloud,

while the plasma is the uppermost layer in the tube

(see Figure 2). The PBMC layer includes B and

T lymphocytes, monocytes, NK (Natural Killer)

cells, and dendritic cells (Meyer et al. 2005).

This methodology is highly ecient and recovers

around 95% of the mononuclear cells present in the

original blood sample (Ito and Shinomiya 2001,

Lehner and Holter 2002).

Since monocytes are the only circulating

blood cells to show high expression of CD14 on

their membrane, this molecule is widely used as a

biomarker for monocytes and as a target for their

purication (Patel et al. 2017). Another distinct

characteristic of monocytes is the ability to adhere

to inert surfaces like plastic, dierent from other

cells present in the PBMC fraction (Patarroyo

et al. 1988). Protocols that take advantage of

this characteristic usually seed PBMC cells in a

plastic ask with the appropriate culture medium

(presented in the “Culture Medium” topic) and

allow adherence for 2 hours in a humidified

incubator. All monocytes will adhere to the culture

ask while B and T lymphocytes, NK cells, and DCs

will remain non-adherent and can be eliminated as

oating cells.

Another system to isolate monocytes is

the use of magnetic beads coated with specific

antibodies. Such magnetic bead-based cell

isolation methodology can be performed using both

a positive or a negative selection approach. In a

negative selection approach using the Dynabeads™

TABLE I

Percentage of cells in whole blood and blood products in health individuals.

Whole blood (%) Buy coat (%) LRSC (%) Leukopak (%)

CD4

T cells 22.5 24.4 32.39 31,92

CD8

T cells 6.8 10.8 15.04 17.13

B cells 5.2 7.2 11.33 12.52

Monocytes 8.4 10.7 20.28 19.08

NK cells 4.4 4.1 10.21 7.65

Eosinophils 3.2 1.3 1.88 1.07

Neutrophils 53.8 34.4 2.89 3.91

PBMC 2x10

cells/mL 1x10

cells 1x10

cells 7x10

cells/half

LRSC: Leukoreduction System Chamber. NK: Natural Killer. PBMC: Peripheral Blood Mononuclear Cells. Modied from: https://

www.miltenyibiotec.com/US-en/resources/macs-handbook/human-cells-and-organs/human-cell-sources/blood-human.html.

Plasma

Ficoll® Paque Plus

Red blood cells

Mononuclear layer

Figure 2 - Schematic diagram of the separation of the

components of a blood sample from Ficoll

Paque Plus

density gradient medium.

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Untouched™ Human Monocytes Kit, for example,

(Invitrogen, Norway, Catalog number: 11350D),

a mixture of biotinylated monoclonal antibodies,

each one of them specic against a distinct surface

cell marker [anti-CD3 (a typical T lymphocyte

antigen), anti-CD7 (a typical T- and NK-

lymphocyte antigen), anti-CD16a and anti-CD16b

(typical antigens expressed in T lymphocytes, DCs,

NK cells, macrophages and granulocytes), anti-

CD19 (expressed in B lymphocytes), anti-CD56 (a

NK cell surface molecule), anti-CDw123 (a typical

plasmacytoid DCs antigen) and anti-CD235a (a

typical antigen expressed in erythrocytes and stem

cell precursors)] is added to the sample. After that,

beads coupled with streptavidin are added. Of note,

these beads act as superparamagnetic particles,

meaning that they exhibit magnetic properties when

placed in a magnetic eld. After a short incubation

period, the streptavidin-beads will bind to the

biotinylated-antibody-labeled cells. A magnet is

applied to the sample and the cells linked to the

biotinylated-antibody-streptavidin-beads will be

attracted and will be immobilized by the magnet

whereas cells not linked to any of these antibodies

will be free in the supernatant and can be washed

out and collected. Since the mixture of antibodies

encompasses specicities against all PBMCs, but

not against monocytes, this system will capture

all non-monocyte cells. Importantly, no binding

will occur between antibodies and monocytes, and

therefore the isolated cells will be recovered after

a relatively mild handling, without any activation

mediated by antibodies.

Conversely, using a positive selection

approach with anti-human CD14 MicroBeads from

Miltenyi Biotec, Germany, (Catalog number: 130-

050-201), for example, the cell sample is incubated

with an anti-CD14 antibody bound to a magnetic

bead. During this incubation period, the anti-CD14

antibody binds to the CD14-positive cells present

in the sample and the cell suspension is loaded onto

a column which contains a magnet that induces a

high-gradient magnetic eld. Thus, the magnetically

labeled CD14-positive cells are retained within the

column and the unlabeled cells run through. After

removing the column from the magnetic field,

the magnetically retained CD14-positive cells are

eluted. It is worthy of note that the CD14 molecule

belongs to the lipopolysaccharide (LPS) receptor

complex. Recognition through this receptor is

interpreted by the cell as a “danger signal” (Jore et

al. 2009) capable of inducing a maturation process

on immature DCs (more details in “Activation of

Immature Dendritic Cell”). However, according

to the manufacturer, the binding of an antibody to

CD14 does not trigger signal transduction since

CD14 alone lacks a cytoplasmic domain. (Akira

and Takeda 2004, Liew et al. 2005, Napolitani et al.

2005). The advantages of using magnetic selection

are high purity (>95%) and speed (Delirezh et al.

2013).

Another technical alternative for cell

separation involves ow cytometry. Fluorescent

antibody-based cell sorting is a specialized type of

ow cytometry used in the isolation of a specic

cell type from a heterogeneous mixture of cells.

The use of specic antibodies labeled with distinct

uorochromes allied to data about cell size and

complexity allows the sorting of cells based on the

presence or absence of specic molecules on its

surface. Moreover, this technique is the only one

capable of distinguishing and separating cells with

dierent levels of expression of a given molecule

within a population. Thus, through ow cytometry

it is possible, for example, to isolate CD14

high

CD16

cells from a complex sample like total PBMCs, in

a single procedure. As a consequence, cells will

be subjected to less handling and, therefore, will

be exposed to less stress. In this sense, when

the objective is the isolation of a cell population

dened by multiple simultaneous features (meaning

the presence/absence of several surface markers

in distinct expression intensities) cell sorter ow

cytometry is the technique of choice. However,

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drawbacks of this technique include the high cost

of both the equipment and the accessories needed

to ensure operator biosafety due to aerosols formed

during sorting (Holmes et al. 2014).

CYTOKINES AND DCs GENERAL FEATURES

Sallusto and Lanzavecchia (Sallusto and

Lanzavecchia 1994) rst achieved in vitro MDDC

generation through medium supplementation

with distinct cytokines, in this case, through

the combined use of IL-4 and GM-CSF. In the

following years, several works characterized

dierent protocols concerning the use of distinct

cytokines in order to induce and support in vitro

monocyte differentiation and maturation in

MDDCs. It is of crucial importance to note that

dierent combinations of cytokines will generate

MDDCs with distinct characteristics and functions,

so we should choose the cytokines that best match

the purpose of the work. The cytokines most

frequently used for in vitro MDDC dierentiation

and the main characteristics of the generated DCs

will be briey reviewed in the next paragraphs.

Sallusto and Lanzavecchia used the combination

of IL-4 and GM-CSF to promote the dierentiation

of monocytes into DCs. GM-CSF growth factor

seems to down-regulate the expression of the

macrophage colony-stimulating factor (M-CSF)

receptor on monocytes, thereby inhibiting

M-CSF induced dierentiation of monocytes into

macrophages (Suzuki et al. 2004). Similarly, IL-4

exerts its actions in monocytes dierentiation by

inhibiting macrophage colony formation (Jansen et

al. 1989, Relloso et al. 2002). Reports also suggest

that IL-4 can activate some properties of monocytes

and up-regulate MHC class-II molecules, Dendritic

Cell-Specific Intercellular adhesion molecule-

3-Grabbing Non-integrin (DC-SIGN) and co-

stimulatory molecules, and down-regulate CD14

(Ruppert et al. 1991, Ulanova et al. 2001, Relloso

et al. 2002, Sander et al. 2017). The DC generated

by this protocol, after seven days in culture, show

a typical dendritic morphology. The phenotype

of this DC is characterized by high membrane

expression of MHC class I and class II molecules,

CD1a, CD1b, CD1c, FcγRII, ICAM-1, CD11b,

CD11c, CD40, B7, and CD33. In this work, DCs

were also positive for Ii, LFA-1, LFA-3, and CD44.

The expression of CD14 was either low or negative

and these DCs were also negative for FcγRI and

FcγRIII. They also presented a high capacity to

stimulate allogeneic and autologous T cells and a

unique ability to stimulate naive T lymphocytes,

being efficient at presenting soluble antigens

(tetanus toxoid) to a specic T cell (Sallusto and

Lanzavecchia 1994).

Sanarico and colleagues (Sanarico et al. 2006),

described another methodology for inducing

dierentiation of MDDCs in vitro using GM-CSF,

IL-4, and IL-2. The DCs described by this group

show the same morphology and phenotype of the

DCs generated in the presence of GM-CSF and IL-

4. After LPS stimulation, it was observed that there

was an up-regulation of activation markers such as

human leukocyte antigen (HLA)-ABC, HLA-DR,

CD80, CD86, and CD83 but not CD25 (the IL-2

receptor subunit). However, some dierences were

observed regarding the DCs generated with GM-

CSF and IL-4 only, such as a signicantly higher

secretion of IL-1β, Tumor Necrosis Factor (TNF)

-α, and IL-12p70 in response to LPS stimulation.

They also demonstrated the capacity to induce high

interferon (IFN) γ secretion by allogeneic naive T

cells (Sanarico et al. 2006).

Geissmann and coworkers developed a

protocol for generating Langerhans cells (LC) in

vitro, which are specic DCs found on the human

skin (Perussia et al. 1985, Geissmann et al. 1998).

They added GM-CSF, IL-4, and transforming

growth factor (TGF) β to monocyte culture.

The predominant TGF-β isoform expressed

in the immune system, TGF-β1, regulates cell

differentiation and survival during Langerhans

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cell development (Bauer et al. 2012, Esebanmen

and Langridge 2017). TGF-β also induces

immature DCs to present an immunosuppressive

phenotype thereby ensuring system homeostasis by

downregulating the function of these cells (Li et al.

2006, Travis and Sheppard 2014, Esebanmen and

Langridge 2017). The Langerhans cells generated

by this protocol, in an immature state, exhibit

Birbeck granules and express CD1a, E-cadherin,

and CLA, but do not express CD83 and CD86.

However, the addition of TNF-α and IL-1 induces

HLA class II, CD83, and CD86 expression and the

loss of E-cadherin expression. These cells are also

highly ecient in stimulating the proliferation of

allogenous lymphocytes (Geissmann et al. 1998).

A dierent combination of cytokines was used

to produce MDDC by Takahashi et al. (Takahashi

et al. 1997). A combination of GM-CSF and

IL-7 gave rise to floating cells with typical DC

morphology and some adherent cells developed the

appearance of Langerhans cell-like dendrites. The

typical DC presents a membrane phenotype with

high expression of MHC class I and class II, CD1a,

CD11c, CD23, CD40, CD54, CD58, CD80, CD86,

and CD95, and decreased CD14 expression (very

low or absent). DCs induced by this protocol are

also positive for CD21, weakly positive for CD32,

and negative for CD16. Compared to PBMCs, the

DCs generated by this protocol, also called G7 DC

by the authors, are more eective in stimulating

autologous and allogeneic T cell proliferation.

G7 DCs are more eective in inducing peptide-

specic CTL activity than the DCs generated with

GM-CSF and IL-4 (Takahashi et al. 1997). The role

of IL-7 in monocyte dierentiation in DCs is not

fully elucidated. Studies on IL-7Rα

-/-

and IL-7

-/-

mice demonstrated that IL-7 may play an important

role in the development of DCs and plasmacytoid

dendritic cells (pDCs) but further investigations are

required (Yang et al. 2005, Vogt et al. 2009).

Two different groups used the combination

of GM-CSF and IFN-α to generate MDDC in

vitro (Santini et al. 2000, Mohty et al. 2003). In

both cases, the DCs generated with GM-CSF and

IFN-α exhibited a typical DC morphology and

show an immature phenotype which demonstrates,

after exposure to maturation stimulus (CD40L or

LPS), up-regulated expression of co-stimulatory

molecules CD40, CD80, CD86, and high

expression of CD83, HLA-DR, and MHC class

I. The prominent ability of these DCs to secrete

IFN-α suggests an ability to promote a Th1

response. Mohty and colleagues also measured the

expression of CD1a, blood dendritic cell antigen

(BDCA) -4, CD54, CD58, and DC lysosome-

associated membrane protein (DC-LAMP) on

MDDC, which showed an increase in expression

after the maturation stimulus with CD40L for 2

days. The DCs generated by Mohty and colleagues

(called as IFN-DCs by the authors) dierentially

secreted, depending on their maturation stage, large

amounts of inammatory cytokines such as IL-1β,

IL-6, IL-10, TNF-α, and especially IL-18 (which

could be detected at both maturational stages).

However, these cells did not secrete IL-12p70.

In the immature state, IFN-DCs induced a potent

autologous antigen-specic immune response. Like

the natural type I IFN-producing plasmacytoid

DCs, the IFN-DCs expressed a several Toll-like

receptors (TLR) including TLR7 and could secrete

IFN-α following viral stimulation or TLR7-specic

stimulation (Ito et al. 2002, Mohty et al. 2003, Liu

2005).

Santini and coworkers observed that, in

response to type I IFN (IFN-α, IFN-α2b and IFN-

β) and GM-CSF treatment, adherent monocytes

became non-adherent cells within three days of

culture. The loss of adherence was associated

with cellular aggregation in large cell clusters.

However, a considerable percentage of the cells

underwent apoptosis after ve days in culture, and

it is important to note that TNF-related apoptosis-

inducing ligand (TRAIL) and TRAIL receptors

(R1 and R2) were upregulated in response to

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LPS stimulus (Wang and El-Deiry 2003). The

DCs obtained by Santini and coworkers showed a

stronger capability to stimulate the proliferation of

allogeneic lymphocytes than DCs generated with

GM-CSF and IL-4. In particular, DCs generated in

the presence of type I IFN and GM-CSF showed

a potent ability to take up, process, and present

inactivated HIV-1 to autologous T lymphocytes in

vitro when compared to DCs generated with GM-

CSF and IL-4 (Santini et al. 2000, Biron 2001, Ito

et al. 2001).

IL-3 cytokines are also used in association

with IFN-β or IL-4 in protocols to produce MDDC.

(Buelens et al. 2002, Ebner et al. 2002). DCs

generated by these protocols acquire a dendritic

morphology and show similarities in phenotype.

The expression of CD14 molecules is low or absent,

but high levels of BDCA-4, and CD11c are found

when compared to DCs generated with GM-CSF

and IL-4. Only CD1a was dierentially expressed,

either at low levels or not at all. After a maturation

stimulus, DCs upregulated the costimulatory

molecules CD40, CD80, CD86, CD83, DC-LAMP,

HLA-DR, and MHC class I (Biron 2001, Buelens

et al. 2002, Ebner et al. 2002).

The IL-15 cytokine is used in two

different protocols to generate MDDC in vitro

(Mohamadzadeh et al. 2001, Saikh et al. 2001).

Saikh and colleagues use only IL-15 to promote

monocyte differentiation into DC. The DCs

generated by Saikh and colleagues show surface

levels of the costimulatory molecules CD86,

CD80, CD40, and CD83 that were equivalent to

DC obtained from cultures of monocytes treated

with GM-CSF plus IL-4 and stimulated with

TNF-α. However, the DCs generated with IL-15

did not express CD1a. Another important feature

of Saikh’s DCs is the ability to stimulate strong

allo-responses and produce signicant amounts of

IFN-γ and IL-12 when compared to DCs generated

with GM-CSF plus IL-4 and stimulated with TNF-α

(Saikh et al. 2001, Dubois et al. 2002, 2005).

Mohamadzadeh’s group differentiated

monocytes in the presence of IL-15 plus GM-CSF

(Mohamadzadeh et al. 2001). The resulting cells,

after six days in culture, had a phenotype positive

for CD1a molecule, negative for CD14, and an

increased in surface expression of HLA-DR,

CD40, CD80, CD86 and CD83. The intracellular

expression of DC-LAMP was observed upon LPS

activation. The interesting features about this DC

are the expression of Langerhans cell markers such

as E-Cadherin, Langerin, and chemokine receptor

(CCR) 6. Nevertheless, they do not express

Birbeck granules like a “genuine” Langerhans cell

(Mohamadzadeh et al. 2001, Dubois et al. 2002,

2005).

TNF-α is used together with GM-CSF by

Iwamoto and coworkers to induce the dierentiation

of monocytes into DCs (called TNF-DC by the

authors), (Iwamoto et al. 2007). The culture of

CD14

monocytes was incubated for seven days

in the presence of GM-CSF and TNF-α, inducing

a spindle-shaped and adherent morphology. Two

days after LPS stimulation, they began to convert

to DC-like oating cells with extended dendrites.

The TNF-DC expressed low levels of CD14 and

substantial levels of HLA-DR, CD40, CD70, CD80,

CD86 and CD83, and produced extremely large

amounts of TNF (>150 ng/mL at peak). They also

produce IL-12/IL-23p40 and IL-23p19/p40, but

very little IL-12p70 compared to DC generated with

IL-4 and GM-CSF. The TNF-DC had the capacity

to induce naive CD4 T cells to produce IFN-γ and

TNF-α and stimulated resting CD4 T cells to secret

IL-17 when compared to DC generated with IL-4

and GM-CSF (Chomarat et al. 2003, Wajant et al.

2003, Iwamoto et al. 2007, Brenner et al. 2015).

The Table II presents a summary of the cytokines,

growth factors and maturation inducing substances

used by the dierent groups described above.

GIOVANA CECHIM and JOSÉ A.B. CHIES HUMAN MONOCYTE-DERIVED DENTRITIC CELLS GENERATION

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TABLE II

Combination of cytokines, grown factors and maturation stimulus for MDDC culture in vitro.

Cytokines Concentration

Time to add

cytokines

Stimulus for maturation

Maturation stimulus

added

Stimulation

period

Total culture

time

References

GM-CSF +

IL-4

50 ng/mL

1000 U/mL

day

TNF-α (10 ng/mL)

CD40L

Not

mentioned

24 hours

Seven days

Sallusto and

Lanzavecchia 1994

GM-CSF +

IL-7

600 U/mL

6 U/mL

day Not

mentioned

Not

mentioned

Not mentioned Seven days Takahashi et al. 1997

GM-CSF +

IL-4 +

TGF-β1

250 ng/mL

100 ng/mL

10 ng/mL

day

TNF-α

IL-1

Not

mentioned

Not mentioned Seven days

Geissmann et al. 1998

GM-CSF +

IFN-α

500 U/mL

1000 U/mL

day

LPS (1 µg/mL)

day 48 hours

Five

days

Santini et al. 2000

GM-CSF +

IFN-α

100 ng/mL

500 IU/mL

day

LPS (10 µg/mL)

Poly(I:C)(15µg/mL)

CD40L

day 48 hours Seven days Mohty et al. 2003

GM-CSF +

IL-15

100 ng/mL

200 ng/mL

day LPS (100 ng/mL)

Not

mentioned

Not mentioned

Six

days

Mohamadzadeh et al.

2001.

IL-15

100 ng/mL

day

Not

mentioned

Not

mentioned

Not

mentioned

Seven

days

Saikh et al. 2001

IL-3 +

IL-4

100 U/mL

1000 U/mL

day

TNF-α (10 ng/mL)

IL-1β (10 ng/mL)

IL-6 (1000U/mL)

PGE2 (1 µg/mL)

day 48 hours

Nine

days

Ebner et al. 2002.

IL-3 +

IFN- β

50 U/mL

1000 U/mL

day

LPS (1 µg/mL)

Inuenza virus

Poly[I:C](20 µg/mL)

CD40L

Not

mentioned

24 hours

48 hours

3 days

Six

days

Buelens et al. 2002

GM-CSF +

IL-4 +

IL-2

50 ng/mL

10 ng/mL

100 U/mL

day

and 5

days

LPS (200 ng/mL)

day 48 hours Seven days Sanarico et al. 2006

GM-CSF +

TNF-α

10 ng/mL

day

LPS (10 µg/mL)

Not

mentioned

Several time

intervals

Seven days Iwamoto et al. 2007

Own lab production.

Reagent supplied by Dr. Manfred Brockhaus (Homann-La Roche, Basel, Switzerland).

A soluble chimeric fusion protein between the mouse CD8 α-chain and the human

CD40 ligand (CD40L) was a generous gift of Dr. Peter Lane (Basel Institute for Immunology).

Pepro Tech Inc. (Rocky Hill, NJ) or provided by Prof. Nicos Nicola (The Walter and Eliza Hall

Institute of Medical Research, Victoria, Australia).

Pepro Tech Inc.

Sandoz AG (Bale, Switzerland).

Genzyme Corp. (Cambridge, MA).

R&D Systems, (Minneapolis, MN).

At days 2 and 4, half

of the medium was removed and an equivalent volume of fresh medium, supplemented with GM-CSF, IL-4 andTGF-β1 was added.

Concentrations not mentioned.

R&D Systems.

CIFN is a

synthetic type I IFN produced from recombinant DNA, whose sequence is based on a consensus derived from the amino acid sequences of the most common types of human IFN- α. (CIFN, specic

activity of 10

U/mG protein), Amgen.

Sigma-Aldrich.

Leucomax, Novartis (Rueil-Malmaison, France).

Introna, Schering-Plough (Levallois-Perret, France).

The medium was replenished

with cytokines every 3 days.

LPS from Escherichia coli, serotype O26: B6, Sigma-Aldrich (St. Quentin Fallavier, France).

Polyriboinosinic polyribocytidylic acid- poly(I:C), (Sigma-Aldrich).

Murine L cells transfected with human CD40L were kindly provided by Schering-Plough (Laboratory for Immunological Research, Dardilly, France), used after 75 Gy irradiation (ratio, 1:10).

Leukine, Immunex.

(R&D Systems or Schering-Plough).

Sigma-Aldrich.

PeproTech Inc (Rocky Hill, NJ).

PeproTech (London, U.K.; sp. act., 10

U/mG).

Genzyme (Cambridge, MA; sp.

act., 5.10

U/mG).

TNF-α (sp. act., 6X10

U/mG) was provided by Dr. G. R. Adolf (Bender, Vienna, Austria).

IL-1β (sp. act., 5X10

U/mG), Genzyme.

IL-6 (sp. act., 1X10

U/mG), Genzyme.

PGE2 (prostine E), Pharmacia & Upjohn (Buurs, Belgium).

R&D Systems Europe (Oxon, United Kingdom).

Ares Serono Europe (London, United Kingdom).

Provided by N. Kuehm,

Aventis, Pasteur Merieux (Val de Reuil, France).

Sigma.

DCs were activated by coculture with irradiated 3T6 broblasts transfected with the human CD40L gene (CD40L transfectants) (5x10

mL).

EuroClone.

R&D Systems (Minneapolis, MN).

Sigma-Aldrich (St. Louis, MO).

Diaclone Research.

LPS from Escherichia coli (L4516), Sigma-Aldrich.

GIOVANA CECHIM and JOSÉ A.B. CHIES HUMAN MONOCYTE-DERIVED DENTRITIC CELLS GENERATION

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CULTURE MEDIUM

The importance of the culture medium in the in

vitro induction of DCs is undeniable since it should

provide a suitable environment (an appropriate pH

and all the necessary nutrients and factors) for cell

maintenance, growth, and dierentiation. There are

many dierent culture media described and used

in the extensive literature on MDDC. To make the

best choice, it is necessary to consider the general

and specic objectives of the experiments, as well

to take into consideration the cost of the dierent

supplements to be used (Duperrier et al. 2000, Peng

et al. 2005, Janetzki et al. 2010).

If the main objective encompasses the

clinical (therapeutic grade) use of the MDDCs

generated in vitro, such as vaccines, the only

Food and Drug Administration (FDA) approved

culture medium is the AIM-V (GIBCO, Catalog

number: 087-0112DK). The complete AIM-V

composition is not informed by the manufacturer

(GIBCO), although the manufacturer provides

some important information (in this sense, it is

reported that this medium does not contain serum

but contains L-glutamine and antibiotics - 50 µg/

mL streptomycin sulfate and 10 µg/mL gentamicin

sulfate).

For research purposes, PromoCell, for

example, oers three dierent medium types: DC

Generation Medium (Catalog number: C-28050),

DC Generation MediumDXF (Catalog number:

C-28052), and Monocyte Attachment Medium

(Catalog number: C-28051). As in the previous

example, the complete composition is not disclosed.

The only information available states that the DC

Generation Medium DXF is free from animal-

derived components, although it contains human

serum albumin. An important point, according to

the manufacturer, is that the Monocyte Attachment

Medium allows an ecient selection of monocytes

from freshly isolated mononuclear cells through

differential adherence and immunomagnetic

purication steps. For all media, supplementation

with growth factors and cytokines is needed

(Challagundla et al. 2015).

Another culture medium used to generate

MDDC for research purposes is X-VIVO 15.

The complete composition is not reported by the

manufacturer (LONZA), so the only information

available is that it is chemically dened and serum-

free. There are versions with or without L-glutamine,

gentamicin, recombinant transferrin and phenol red.

As previously stated, all media requires cytokine

and growth factor supplementation, depending on

the specic goal of the experiment and the initial

cell source.

A commonly used medium in cell culture

experiments is RPMI-1640. It was developed

by Moore and colleagues at the Roswell Park

Memorial Institute, hence the acronym RPMI

(Moore et al. 1967). The exact RPMI-1640

composition is available and well established,

which allows the production of quite similar

media by several manufacturers. There are several

modied versions of RPMI, some of them lacking

specic components, others already supplemented

with certain growth factors or other molecules.

Nevertheless, as a general rule, supplementation

with serum, cytokines, and/or growth factors is

required.

SUPPLEMENTS FOR THE CULTURE MEDIUM

The widely used culture medium RPMI-1640

is also the only commercial medium for which

the complete formula is available. This enables

each researcher to direct the supplementation

specically according to the particularities of each

experiment. The most commonly used culture

medium supplements (biological or chemical

factors) for in vitro production of MDDCs will be

briey presented below.

An essential function of culture medium

consists in providing cells an environment with a

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physiological pH, and thus, buering agents are

essential. Sodium bicarbonate (NaHCO

) is a

commonly used pH regulating agent, since it can

be used in cell culture without increased toxicity

to the cells. Sodium bicarbonate chemically reacts

either as acid or as a base due to its amphoteric

characteristic. In solution, sodium bicarbonate

produces bicarbonate ions, while the metabolism

of cells in culture produces carbon dioxide (CO

decreasing the culture medium pH (if the culture

medium contains phenol red, the color of the

medium will become yellowish). In addition,

the incubator also injects carbon dioxide into its

atmosphere, thereby increasing the carbon dioxide

concentration in the microenvironment and lowering

the culture medium pH. Thus, within the incubator

microenvironment, a sodium bicarbonate-carbon

dioxide buer balance is established. The carbon

dioxide dissolved in the culture medium, either

from the incubator or from the cell metabolism,

equilibrates with the bicarbonate ions, buering

the medium. The sodium bicarbonate concentration

in the medium should be in equilibrium with the

carbon dioxide level in the incubator atmosphere.

Thus, for media containing 1.5–2.2 g/L of sodium

bicarbonate, the incubator must inject 5% of

carbon dioxideinto its microenvironment. For

media containing 3.7 g/L of sodium bicarbonate,

the incubator should inject 10% of carbon dioxide.

If the sodium bicarbonate concentration is too high

in relation to the carbon dioxide level injected by

the incubator, the culture medium will alkalize, and

its color will turn pink (Barker 1998).

HEPES (4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid) is a buering agent

which is membrane impermeable, chemically

and enzymatically stable, demonstrates a limited

eect on biochemical reactions, and has very low

visible and UV light absorbance. HEPES provides

extra buering capacity when cell cultures require

extended periods of manipulation outside of a

incubator. The HEPES concentration in cell

culture media may vary from 10 mM to 25 mM.

Although HEPES-buffered medium exposed to

uorescent light was shown to be cytotoxic, due

to the formation of free radicals, this effect is

preventable by manipulating the cells under light-

shielded conditions.

Serum is a quite ubiquitous supplement in

cell cultures. This component will supply mainly

growth factors and cytoines to the cells, although

generally it is not possible to dene the exact types

and concentrations of these components in the

serum. Many studies use human serum from an AB

serum pool to supplement cell cultures. The nal

concentrations of serum used vary from 1 - 10%.

It is also possible to use plasma or even umbilical

cord blood serum. Nevertheless, if the cells will

not be used in clinical applications, fetal bovine

serum is the least expensive choice, and is also

easier to obtain. However, it is crucial to observe

if the serum was inactivated. This inactivation is

necessary due to the presence of proteins from the

complement system which could cause cell death

(Anton et al. 1998).

Although cell culture procedures are

conducted in aseptic environments, any biological

contaminant can devastate the cell culture, and

therefore the use of antibiotics and antimycotics is

sometimes required. The most common antibiotics

used in cell cultures are penicillin-streptomycin and

gentamicin. Penicillin-streptomycin acts on Gram-

positive and Gram-negative bacteria whereas

gentamicin is also active against mycoplasma. The

recommended concentration for use in cell culture

for Penicillin-streptomycin are 50 to 100 I.U./

mL penicillin and 50 to 100 μg/mL streptomycin

(Catalog number: ATCC® 30-2300), and 5 to 50

μg/mL gentamicin (Barker 1998). In culturing cells

that are intended for clinical use, the FDA does not

recommend the use of penicillin or β-lactams due to

the possibility of severe hypersensitivity reactions

(Vatsan et al. 2013).

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A widely used antifungal agent is amphotericin

B. It acts against both fungi and yeasts. The

recommended concentration for use in cell culture

is 0.25-2.5 μg/mL (GIBCO, Catalog number:

15290026).

A component often added to culture medium is

the amino acid glutamine. This amino acid can lead

to citrate production, which is exported to the DC’s

cytoplasm and acts as a substrate for fatty acids

synthesis, an essential process for the induction

of DC activation by TLR ligands. Due to its rapid

degradation and instability in solution, glutamine

supplementation must be performed after careful

consideration. The recommended concentration for

use in cell culture generally falls in the range of 2-6

mM (GIBCO, Catalog number: 21051024).

Free radicals are a natural side product of

the increased metabolism in monocytes and

in immature DCs during differentiation and

maturation. To avoid the toxic effects of the

accumulation of these reactive oxygen species in

the culture medium, 2-mercaptoethanol (2-ME)

is used as a reducing agent. Due to its instability

in solution, 2-mercaptoethanol should be added

daily. It is recommended to dilute 2-ME to 50 mM

in phosphate-buered saline (PBS), and add this

solution to the cell medium at a nal concentration

of 50 µM. (GIBCO, Catalog Number: 21985023),

(Click 2014).

CULTURE TIME

The time needed to establish a cell line or to induce

cell dierentiation in vitro is quite variable, and

several aspects should be taken into consideration.

The majority of studies consider that the

dierentiation process from monocyte to immature

DC is completed in ve days with an additional

period of 48 hours for maturation. However,

several works have been demonstrated that in

vitro development of mature DCs from monocyte

precursors does not require more than 2 days of

culture, dened by some as fast DCs (fDC), (Dauer

et al. 2003, Obermaier et al. 2003, Ramadan et al.

2004, Massa and Seliger 2013). The data about

phenotypic characteristics, immune properties, and

the pattern of responses induced for the fDC are

still poorly understood (Tanaka et al. 2006, Rojas-

Canales et al. 2012, Pavlović et al. 2015).

In vitro MATURATION OF IMMATURE

DENDRITIC CELL

In the majority of MDDCs generation protocols,

the resulting DCs are in an immature state. An

immature dendritic cell (iDC) shows a high

capacity to capture and process antigens but has

limited capacity to stimulate T cells due to the

low expression of the co-stimulatory molecules

CD40, CD80, CD86, and HLA-class II. The in

vitro maturation process is characterized by several

events and although dierent groups may dene

distinct features, the main events include the

downregulation of endocytic/phagocytic receptors,

the upregulation of costimulatory molecules CD40,

CD80 and CD86, the upregulation of CD58 and

CD83, the shift in lysosomal compartments with

downregulation of CD68, the upregulation of DC-

LAMP, and the changes in HLA-class II molecules

(Banchereau et al. 2000, Lutz and Schuler 2002,

Iwasaki and Medzhitov 2004).

Some important morphological changes

also occur during the DC in vitro maturation

process, including the loss of adhesive structures,

cytoskeleton reorganization, and the acquisition of

high cellular motility (see Table III). These changes

allow the DCs a better capacity for lymphocyte

stimulation (Banchereau et al. 2000, Dalod et al.

2014).

To induce the DC maturation process in vitro it

is necessary to give a stimulus that mimics a danger

signal (Matzinger 1994, Gallucci et al. 1999, Jore

et al. 2009, Castiello et al. 2011). It is important

to note that in a seven days protocol for MDDC

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generation, the stimulus is usually given on day

5 and is maintained for 48 hours. In the case of

fDCs, the dierentiation cocktail already contains

substances that will induce cell maturation. The

main substances used to promote DC maturation in

MDDC cultures are described below (Haenssle et

al. 2008, Castiello et al. 2011, Li et al. 2012).

A common substance used to mimic DC

activation when provoked by bacteria is LPS,

a characteristic component of the wall of Gram-

negative bacteria (Raetz and Whiteld 2002). The

LPS molecule bis bound by the CD14 molecule

present on the cell surface and activates the TLR4/

MD-2 complex pathway, promoting the secretion

of pro-inammatory cytokines by DCs (Iwasaki

and Medzhitov 2004, Park and Lee 2013).

Nevertheless, due to its potential toxicity, LPS is

only used in research protocols. The majority of

protocols aiming for DC maturation use LPS in

a concentration range of 10 ng/mL to 10 µg/mL

(InvivoGen, Catalog code: tlrl-eklps).

Flagellin is another substance that mimics the

danger signal triggered by the presence of Gram-

negative and/or Gram-positive bacteria. Flagellin is

generally detected by TLR5 whereas intracellular

agellin is detected by NOD-like receptors (NLRs)

NLRC4 and NAIP5. The process of DC maturation

induced by agellin results in the activation of the

NF-κB signaling pathway and cytokine production

(Akira et al. 2001, Vicente-Suarez et al. 2009, Miao

and Warren 2010). It was already been suggested

that agellin could be used as a vaccine adjuvant

(Coman et al. 2010). As in the previous case of

LPS use, the agellin concentrations used in DC

maturation protocols widely vary, ranging from 10

ng/mL to 10 µg/mL (Vicente-Suarez et al. 2009),

(InvivoGen, Catalog code:tlrl-sta).

To mimic virus-mediated activation during

DC maturation it is possible to use commercially

available synthetic polynucleotide sequences such

as the double stranded RNA analog polyinosinic-

polycytidylic acid [poly(I:C)], the single-stranded

RNA analog poly-uridine (polyU), or the single-

stranded RNA analog guanosine- and uridine-rich

(GU-rich). Whereas poly(I:C) binds to TLR3, the

remaining two types of sequences bind to TLR7/

TLR8 (Heil et al. 2004, Schlee and Hartmann

2016). Alternatively, the use of compounds

derived from imidazoquinoline such as the analog

imiquimod can be used to activate immature DCs.

Imiquimod is a commercially available compound

which selectively binds to TLR7 (Xiao et al. 2016).

Poly(I:C) and imiquimod are also used as adjuvants

in protocols to develop cancer vaccines (Coman

et al. 2010), (InvivoGen, Catalog code: vac-pic).

Short synthetic single-stranded DNA molecules

that contain unmethylated CpG dinucleotides

(CpG) are also used as TLR9 agonists to activate

TABLE III

Characteristics of DCs according to dierent maturation stages. The characteristics described refers to the expression in

the plasma membrane.

Immature DC Mature DC

High intracellular expression of HLA-II High membrane expression of HLA-II

High endocytic/phagocytic capacity Low endocytic/phagocytic capacity

High membrane expression of CCR1, CCR5 and CCR6 Low membrane expression of CCR1, CCR5, and CCR6

Low membrane expression of CCR7 High membrane expression of CCR7

Low membrane expression of CD40, CD54 and CD58 High membrane expression of CD40, CD54, and CD58

Absent/low membrane expression of DC-LAMP High membrane expression of DC-LAMP

Low membrane expression of CD80, CD86 and CD83 High membrane expression of CD80, CD86, and CD83

High membrane expression of CD68 Low membrane expression of CD68

Induce T cell anergy/tolerance Great capacity to lymphocyte stimulation

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iDCs (Jakob et al. 1998, Hemmi et al. 2000, Akira

et al. 2001, Iwasaki and Medzhitov 2004). A

modied version of CpGs called CpG-ODN has

been developed for clinical use as an adjuvant

(Cella et al. 1999, Bauer et al. 2001, Coman et

al. 2010).

The CD40 is a costimulatory molecule

expressed on DCs and the interaction with its

ligand CD40L (CD154) leads to an activation of

the NF-κB signaling pathway, a key transducer

of inflammatory signals in DCs (Schulz et al.

2000, Aggarwal 2003, O’Sullivan and Thomas

2003, Ma and Clark 2009). Transfected cells

expressing the CD40L protein can be used in co-

culture assays to induce the maturation of iDCs

(Buelens et al. 2002, Mohty et al. 2003), whereas,

to promote DC activation via CD40 receptor,

some commercial alternatives are available. For

example, CD40L recombinant protein, which

should be used in conjunction with an enhancer for

better performance, is already available (Enzo Life

Sciences, Catalog number: ALX-850-064).

Interleukins and several other molecules can

concomitantly be used to induce DC maturation.

A “maturation cocktail” can, for example, contain

IL-6, IL-1β, TNF-α, IFN-γ, and Prostaglandin E2

(PGE2), (Jonuleit et al. 1997). In the cited case, this

cocktail enhances the pro-inammatory eects of

TNF-α by creating an inammatory environment

that induces DC maturation. This cocktail generates

mature DCs that strongly stimulate the proliferation

of allogeneic lymphocytes (Castiello et al. 2011).

TYPICAL MDDC IMMUNOPHENOTYPE

MARKERS

The phenotype characterization of the obtained

DCs is crucial to demonstrate the success of the

MDDC dierentiation. However, due to the absence

of a consensus concerning which molecules

characterize a DC and which markers are expressed

in dierent DC subsets, it is quite common to nd

a wide variety of markers used in distinct studies.

Nevertheless, the expression of certain molecules

is consistently used to indicate successful MDDC

differentiation and/or maturation. For example,

since a peripheral blood monocyte, the main source

for in vitro DC generation, expresses high levels

of CD14 on its membrane, and MDDC lacks the

expression of this same molecule, CD14 is a marker

for DC dierentiation. Additionally, to determine

the success of the iDC maturation process, it is

essential to monitor the increased expression of

co-stimulatory molecules such as CD40, CD80,

and CD86. These molecules are constitutively

expressed at low levels in DCs, however, their

expression can be considerably increased after

induction of maturation by LPS (Banchereau et al.

2000, Ueno et al. 2007).

The HLA-II molecule is also constitutively

expressed at low levels both in monocytes and DCs.

After a maturation stimulus, its expression on DCs

is increased. Thus, like co-stimulatory molecules,

HLA-II can be used as an indicator of the success

of the iDC maturation process (Al-daccak et al.

2004, Neefjes et al. 2011).

CD1 represents a group of antigen-presenting

molecules which are responsible for the

presentation of lipid antigens and their derivatives.

There are ve isoforms of CD1 proteins [CD1a,

CD1b, CD1c, CD1e (group 1) and CD1d (group

2)]. Myeloid DCs express all ve proteins whereas

Langerhans cells express only CD1a and CD1e

(Mori et al. 2016). In blood monocytes, CD1a,

CD1b, and CD1c (BDCA-1) can be upregulated

on the cell surface by cytokine cocktails designed

to drive DC dierentiation in vitro (Moody 2006).

A strategy for characterizing a pDC, for

example, would encompasses the measurement

of the CD123 (IL-3Rα), CD303 (BDCA-2), and

CD304 (BDCA-4) expression levels. This strategy

depends on the fact that pDCs present high

expression of such molecules in the membrane

GIOVANA CECHIM and JOSÉ A.B. CHIES HUMAN MONOCYTE-DERIVED DENTRITIC CELLS GENERATION

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(Dzionek et al. 2000, Colonna et al. 2004, Swiecki

and Colonna 2015).

Other potential markers are CD207 (Langerin),

which is used to characterize Langerhans cells

(Merad et al. 2008, Nestle et al. 2009), and

CD209 (DC-SIGN), which is expressed on pDCs

and MDDCs and is highly expressed on DCs in

mucosal tissues. The expression of this last marker

is increased after LPS-mediated DC maturation

induction (Belz and Nutt 2012).

Finally, in the analyses performed by flow

cytometry it is of great importance to evaluate the

cellular viability. Substances used to determine

cell viability can be divided into two broad groups:

substances used in live cells or xed cells. Calcein

AM (BD Biosciences, Catalog Number 564061) is a

dye used for staining live cells. The hydrophobicity

of the acetomethoxy (AM) derivative of Calcein

allows this dye to enter on viable cells. Once inside,

intracellular esterases cleave the AM groups o

allowing Calcein to uoresce within the cell. Only

viable cells are stained, since dead cells lack esterase

activity. Calcein AM is optimally excited at the 495

nm wavelength of light and emits maximally at 515

nm (Chung et al. 2017).

7-Amino-actinomycin D, or 7-AAD, is a

nucleic acid dye used to indicate cell viability

in flow cytometric assays. The fluorescence is

detected in the far red range of the spectrum with a

650 nm long-pass lter (Schmid et al. 1992).

For fixed cells, an alternative may be the

reagents BD Horizon FVS from BD Biosciences

(Catalog Number 564406). BD Horizon FVS are

amine-reactive dyes used to discriminate viable

from non-viablecells based on fluorescence

intensity. These dyes react by covalently binding

the cell surface and intracellular amines, resulting

in live cells poorly stained and cells with permeable

membranes exhibiting highly fluorescent.

Typically, dead cells exhibit a uorescence intensity

10 to 20-fold greater than live cells stained with

the same amount of dye. The advantage of these

dyes is that they can be used in cells xed with

formaldehyde, and can be used in protocols where

cell permeabilization occurs (Mahalingaiah et al.

2018).

CONCLUSIONS

Despite several technical challenges, signicant

progress has been made in recent years concerning

the generation of human DCs in vitro. Much of the

recently acquired knowledge was due to studies

using blood monocytes as in vitro precursors of

DCs. The incorporation of knowledge concerning

cellular biology, cytokines, growth factors,

transcription profiles of these cells in different

stages of maturation with the characterization

of the patterns of expression of surface markers

has allowed a more comprehensive and accurate

description of the DCs present in distinct tissues

both in pathological, as well as in healthy situations.

The appropriate combination of cytokines and

growth factors that will generate MDDCs with the

most suitable characteristics for a given study is

already possible. The development of protocols that

allow us to dierentiate MDDCs into a particular

DC subtype as well as to simulate pathological

microenvironments is a reality that has not only

broadened our knowledge but also allows us to

manipulate these cells for use in clinical protocols.

The increasing amounts of reagents available to

induce maturation of iDCs via a specic receptor

also contributes to the production of DCs with

specific characteristics, membrane markers,

specic prole of cytokine secretion, or a given

lymphocyte polarization state.

Another important advance in the study

of MDDCs was the use of multiparametric ow

cytometry (Hasan et al. 2015). The careful selection

of antibodies and the optimal combination of the

uorochromes used to label such specic reagents

are key steps in the establishment of a robust

panel to analyze MDDCs (Maecker et al. 2004,

GIOVANA CECHIM and JOSÉ A.B. CHIES HUMAN MONOCYTE-DERIVED DENTRITIC CELLS GENERATION

An Acad Bras Cienc (2019) 91(4) e20190310 16 | 21

Byrd et al. 2015). To succeed in this step, several

companies provide specic programs to help set

up the analysis panels and protocols to identify the

dierent DC subtypes. International consortia have

also been working for the increased standardization

of flow cytometry protocols including the

EuroFlowConsortium, the Human Immunology

Project Consortium (HIPC), the European

Network for Translational Immunology Research

and Education (ENTIRE) and the Association for

Cancer Immunotherapy (CIMT).

Great progress has been made in the

understanding of the development, dierentiation,

and function of DCs. However, the major challenge

will be to transpose the acquired knowledge into in

vivo situations to elucidate the DCs physiological

behavior. This knowledge will be useful in the design

of novel vaccines and DC-based immunotherapies

for the prevention and treatment of several human

diseases.

ACKNOWLEDGMENTS

This work was supported by the Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior

(CAPES).

AUTHOR CONTRIBUTIONS

Both authors wrote and revised the manuscript.

REFERENCES

AGGARWAL BB. 2003. Signalling pathways of the TNF

superfamily: a double-edged sword. Nat Rev Immunol 3:

745-756.

AKIRA S AND TAKEDA K. 2004. Toll-like receptor

signalling. Nat Rev Immunol 4: 499-511.

AKIRA S, TAKEDA K AND KAISHO T. 2001. Toll-like

receptors: critical proteins linking innate and acquired

immunity. Nat Immunol 2: 675-680.

AL-DACCAK R, MOONEY N AND CHARRON D. 2004.

MHC class II signaling in antigen-presenting cells. Curr

Opin Immunol 16: 108-113.

ANTON D, DABADGHAO S, PALUCKA K, HOLM G AND

YI Q. 1998. Generation of dendritic cells from peripheral

blood adherent cells in medium with human serum. Scand

J Immunol 47: 116-121.

ARDAVIN C, WU L, LI CL AND SHORTMAN K. 1993.

Thymic dendritic cells and T cells develop simultaneously

in the thymus from a common precursor population.

Nature 362: 761-763.

BANCHEREAU J, BRIERE F, CAUX C, DAVOUST

J, LEBECQUE S, LIU YJ, PULENDRAN B AND

PALUCKA K. 2000. Immunobiology of dendritic cells.

Annu Rev Immunol 18: 767-811.

BANCHEREAU J ET AL. 2001. Immune and clinical

responses in patients with metastatic melanoma to

CD34(+) progenitor-derived dendritic cell vaccine. Cancer

Res 61: 6451-6458.

BARKER K. 1998. At the Bench: A Laboratory Navigator.

Cold Spring Harbor Laboratory Press, 465p.

BAUER M, REDECKE V, ELLWART JW, SCHERER B,

KREMER JP, WAGNER H AND LIPFORD GB. 2001.

Bacterial CpG-DNA triggers activation and maturation of

human CD11c-, CD123+ dendritic cells. J Immunol 166:

5000-5007.

BAUER T, ZAGÓRSKA A, JURKIN J, YASMIN N, KÖFFEL

R, RICHTER S, GESSLBAUER B, LEMKE G AND

STROBL H. 2012. Identification of Axl as a downstream

effector of TGF-β1 during Langerhans cell differentiation

and epidermal homeostasis. J Exp Med 209: 2033-2047.

BELZ GT AND NUTT SL. 2012. Transcriptional programming

of the dendritic cell NETWORK. Nat Rev Immunol 12:

101-113.

BIRON CA. 2001. Interferons alpha and beta as immune

regulators-a new look. Immunity 14: 661-664.

BRENNER D, BLASER H AND MAK TW. 2015. Regulation

of tumour necrosis factor signalling: live or let die. Nat

Rev Immunol 15: 362-374.

BUELENS C, BARTHOLOMÉ EJ, AMRAOUI Z,

BOUTRIAUX M, SALMON I, THIELEMANS K,

WILLEMS F AND GOLDMAN M. 2002. Interleukin-3

and interferon beta cooperate to induce differentiation of

monocytes into dendritic cells with potent helper T-cell

stimulatory properties. Blood 99: 993-998.

BYRD T, CARR KD, NORMAN JC, HUYE L AND HEGDE

M. 2015. Polystyrene microspheres enable 10-color

compensation for immunophenotyping of primary human

leukocytes. Cytometry A 87: 1038-1046.

CASTIELLO L, SABATINO M, JIN P, CLAYBERGER C,

MARINCOLA FM, KRENSKY AM AND STRONCEK

DF. 2011. Monocyte-derived DC maturation strategies and

related pathways: a transcriptional view. Cancer Immunol

Immunother 60: 457-466.

CELLA M, SALIO M, SAKAKIBARA Y, LANGEN

H, JULKUNEN I AND LANZAVECCHIA A. 1999.

Maturation, activation, and protection of dendritic cells

GIOVANA CECHIM and JOSÉ A.B. CHIES HUMAN MONOCYTE-DERIVED DENTRITIC CELLS GENERATION

An Acad Bras Cienc (2019) 91(4) e20190310 17 | 21

induced by double-stranded RNA. J Exp Med 189: 821-

829.

CHALLAGUNDLA KB ET AL. 2015. Exosome-mediated

transfer of microRNAs within the tumor microenvironment

and neuroblastoma resistance to chemotherapy. J Natl

Cancer Inst 7: djv135.

CHOMARAT P, DANTIN C, BENNETT L, BANCHEREAU

J AND PALUCKA AK. 2003. TNF skews monocyte

differentiation from macrophages to dendritic cells. J

Immunol 171: 2262-2269.

CHUNG S, NGUYEN V, LIN YL, KAMEN L AND SONG A.

2017. Thaw-and-use target cells pre-labeled with calcein

AM for antibody-dependent cell-mediated cytotoxicity

assays. J Immunol Methods 447: 37-46.

CIMT - ASSOCIATION FOR CANCER IMMUNOTHERAPY.

http://www.cimt.eu/workgroups/cip/proficiency

(Accessed 12 June 2018).

CLICK RE. 2014. Review: 2-mercaptoethanol alteration of

in vitro immune functions of species other than murine. J

Immunol Methods 402: 1-8.

COFFMAN RL, SHER A AND SEDER RA. 2010. Vaccine

adjuvants: putting innate immunity to work. Immunity 33:

492-503.

COLLIN M AND BIGLEY V. 2018. Human dendritic cell

subsets: an update. Immunology 154: 3-20.

COLONNA M, TRINCHIERI G AND LIU YJ. 2004.

Plasmacytoid dendritic cells in immunity. Nat Immuno l5:

1219-1226.

DALOD M, CHELBI R, MALISSEN B AND LAWRENCE T.

2014. Dendritic cell maturation: functional specialization

through signaling specificity and transcriptional

programming. EMBO J 33: 1104-1116.

DAUER M, OBERMAIER B, HERTEN J, HAERLE C,

POHL K, ROTHENFUSSER S, SCHNURR M, ENDRES

S AND EIGLER A. 2003. Mature dendritic cells derived

from human monocytes within 48 hours: a novel strategy

for dendritic cell differentiation from blood precursors. J

Immunol 170: 4069-4076.

DELIREZH N, SHOJAEEFAR E, PARVINP AND ASADI B.

2013. Comparison the effects of two monocyte isolation

methods, plastic adherence and magnetic activated cell

sorting methods, on phagocytic activity of generated

dendritic cells. Cell J 15: 218-223.

DIETZ AB, BULUR PA, EMERY RL, WINTERS JL, EPPS

DE, ZUBAIR AC AND VUK-PAVLOVIĆ S. 2006. A

novel source of viable peripheral blood mononuclear cells

from leukoreduction system chambers. Transfusion 46:

2083-2089.

DUBOIS S, MARINER J, WALDMANN TA AND TAGAYA

Y. 2002. IL-15Ralpha recycles and presents IL-15 In trans

to neighboring cells. Immunity 17: 537-547.

DUBOIS SP, WALDMANN TA AND MÜLLER JR. 2005.

Survival adjustment of mature dendritic cells by IL-15.

Proc Natl Acad Sci USA 102: 8662-8667.

DUPERRIER K, ELJAAFARI A, DEZUTTER-

DAMBUYANT C, BARDIN C, JACQUET C, YONEDA

K, SCHMITT D, GEBUHRER L AND RIGALD. 2000.

Distinct subsets of dendritic cells resembling dermal DCs

can be generated in vitro from monocytes, in the presence

of different serum supplements. J Immunol Methods 238:

119-131.

DZIONEK A, FUCHS A, SCHMIDT P, CREMER S, ZYSK

M, MILTENYI S, BUCK DW AND SCHMITZ J. 2000.

BDCA-2, BDCA-3, and BDCA-4: three markers for

distinct subsets of dendritic cells in human peripheral

blood. J Immunol 165: 6037-6046.

EBNER S, HOFER S, NGUYEN VA, FÜRHAPTER C,

HEROLD M, FRITSCH P, HEUFLER C AND ROMANI

N. 2002. A novel role for IL-3: human monocytes cultured

in the presence of IL-3 and IL-4 differentiate into dendritic

cells that produce less IL-12 and shift Th cell responses

toward a Th2 cytokine pattern. J Immunol 168: 6199-6207.

EBNER S, NEYER S, HOFER S, NUSSBAUMER W,

ROMANI N AND HEUFLER C. 2001. Generation of

large numbers of human dendritic cells from whole blood

passaged through leukocyte removal filters: an alternative

to standard buffy coats. J Immunol Methods 252: 93-104.

ELKORD E, WILLIAMS PE, KYNASTON H AND

ROWBOTTOM AW. 2005. Human monocyte isolation

methods influence cytokine production from in vitro

generated dendritic cells. Immunology 114: 204-212.

ESEBANMEN GE AND LANGRIDGE WHR. 2017. The role

of TGF-beta signaling in dendritic cell tolerance. Immunol

Res 65: 987-994.

EuroFlow. 2018. https://euroflow.org/usr/pub/prlogin.php.

(accessed 03 May 2018).

ENTIRE - EUROPEAN NETWORK FOR

TRANSLATIONAL IMMUNOLOGY RESEARCH

AND EDUCATION. 2018. http://www.cost.eu/COST_

Actions/bmbs/BM0907?parties (accessed 03 May 2018).

GALLUCCI S, LOLKEMA M AND MATZINGER P. 1999.

Natural adjuvants: endogenous activators of dendritic

cells. Nat Med 5: 1249-1255.

GANDHI RT ET AL. 2016. Immunization of HIV-1-Infected

Persons with Autologous Dendritic Cells Transfected

With mRNA Encoding HIV-1 Gag and Nef: Results of a

Randomized, Placebo-Controlled Clinical Trial. J Acquir

Immune Defic Syndr 71: 246-253.

GARG AD, COULIE PG, VAN DEN EYNDE BJ AND

AGOSTINIS P. 2017. Integrating Next-Generation

Dendritic Cell Vaccines into the Current Cancer

Immunotherapy Landscape. Trends Immunol 38: 577-593.

GEISSMANN F, PROST C, MONNET JP, DY M, BROUSSE

N AND HERMINE O. 1998. Transforming growth factor

GIOVANA CECHIM and JOSÉ A.B. CHIES HUMAN MONOCYTE-DERIVED DENTRITIC CELLS GENERATION

An Acad Bras Cienc (2019) 91(4) e20190310 18 | 21

beta 1, in the presence of granulocyte/macrophage colony-

stimulating factor and interleukin 4, induces differentiation

of human peripheral blood monocytes into dendritic

Langerhans cells. J Exp Med 187: 961-966.

GILLIO-MEINA C, CEPINSKAS G, CECCHINI EL AND

FRASER DD. 2013. Translational research in pediatrics

II: blood collection, processing, shipping, and storage.

Pediatrics 131: 754-766.

GRANOT ET AL. 2017. Dendritic Cells Display Subset and

Tissue-Specific Maturation Dynamics over Human Life.

Immunity 46: 504-515.

GUERMONPREZ P, VALLADEAU J, ZITVOGEL L, THÉRY

C AND AMIGORENA S. 2002. Antigen presentation and

T cell stimulation by dendritic cells. Annu Rev Immunol

20: 621-667.

HAENSSLE H, BUHL T, KNUDSEN S, KRUEGER U,

ROSENBERGER A, REICH K AND NEUMANN C.

2008. CD40 ligation during dendritic cell maturation

reduces cell death and prevents interleukin-10-induced

regression to macrophage-like monocytes. Exp Dermatol

17: 177-187.

HAMMER GE AND MA A. 2013. Molecular control of steady-

state dendritic cell maturation and immune homeostasis.

Annu Rev Immunol 31: 743-791.

HASAN M ET AL. 2015. Semi-automated and standardized

cytometric procedures for multi-panel and multi-

parametric whole blood immunophenotyping. Clin

Immunol 157: 261-276.

HEIL F, HEMMI H, HOCHREIN H, AMPENBERGER F,

KIRSCHNING C, AKIRA S, LIPFORD G, WAGNER H

AND BAUER S. 2004. Species-specific recognition of

single-stranded RNA via toll-like receptor 7 and 8. Science

303: 1526-1529.

HEMMI H ET AL. 2000. A Toll-like receptor recognizes

bacterial DNA. Nature 408: 740-745.

HOLMES KL, FONTES B, HOGARTH P, KONZ R,

MONARD S, PLETCHER CHJR, WADLEY RB,

SCHMID I AND PERFETTO SP. 2014. International

Society for the Advancement of Cytometry cell sorter

biosafety standards. Cytometry A 85: 434-453.

ITO T, AMAKAWA R, INABA M, IKEHARA S, INABA K

AND FUKUHARA S. 2001. Differential regulation of

human blood dendritic cell subsets by IFNs. J Immunol

166: 2961-2969.

ITO T, AMAKAWA R, KAISHO T, HEMMI H, TAJIMA K,

UEHIRA K, OZAKI Y, TOMIZAWA H, AKIRA S AND

FUKUHARA S. 2002. Interferon-alpha and interleukin-12

are induced differentially by Toll-like receptor 7 ligands

in human blood dendritic cell subsets. J Exp Med 195:

1507-1512.

ITO Y AND SHINOMIYA K. 2001. A new continuous-flow

cell separation method based on cell density: principle,

apparatus, and preliminary application to separation of

human buffy coat. J Clin Apher 16: 186-191.

IWAMOTO S, IWAI S, TSUJIYAMA K, KURAHASHI C,

TAKESHITA K, NAOE M, MASUNAGA A, OGAWA

Y, OGUCHI K AND MIYAZAKI A. 2007. TNF-alpha

drives human CD14+ monocytes to differentiate into

CD70+ dendritic cells evoking Th1 and Th17 responses. J

Immunol 179: 1449-1457.

IWASAKI A AND MEDZHITOV R. 2004. Toll-like receptor

control of the adaptive immune responses. Nat Immunol

5: 987-995.

JAKOB T, WALKER PS, KRIEG AM, UDEY MC AND

VOGEL JC. 1998. Activation of cutaneous dendritic cells

by CpG-containing oligodeoxynucleotides: a role for

dendritic cells in the augmentation of Th1 responses by

immunostimulatory DNA. J Immunol 161: 3042-3049.

JANETZKI S ET AL. 2010. Performance of serum-

supplemented and serum-free media in IFN gamma Elispot

Assays for human T cells. Cancer Immunol Immunother

59: 609-618.

JANSEN JH, WIENTJENS GJ, FIBBE WE, WILLEMZE

R AND KLUIN-NELEMANS HC. 1989. Inhibition of

human macrophage colony formation by interleukin 4. J

Exp Med 170: 577-582.

JOFFRE O, NOLTE MA, SPÖRRI R AND REIS E SOUSA C.

2009. Inflammatory signals in dendritic cell activation and

the induction of adaptive immunity. Immunol Rev 227:

234-247.

JOFFRE OP, SEGURA E, SAVINA A AND AMIGORENA

S. 2012. Cross-presentation by dendritic cells. Nat Rev

Immunol 12: 557-569.

JONULEIT H, KÜHN U, MÜLLER G, STEINBRINK K,

PARAGNIK L, SCHMITT E, KNOP J AND ENK AH.

1997. Pro-inflammatory cytokines and prostaglandins

induce maturation of potent immunostimulatory dendritic

cells under fetal calf serum-free conditions. Eur J Immunol

27: 3135-3142.

KAVANAGH DG AND BHARDWAJ N. 2002. A division

of labor: DC subsets and HIV receptor diversity. Nat

Immunol 3: 891-893.

KURTS C, ROBINSON BW AND KNOLLE PA. 2010. Cross-

priming in health and disease. Nat Rev Immunol 10: 403-

414.

LEHNER M AND HOLTER W.2002. Endotoxin-free

purification of monocytes for dendritic cell generation

via discontinuous density gradient centrifugation based

on diluted ficoll-paque plus. Int. Arch Allergy Immunol

128: 73-76.

LI DY, GU C, MIN J, CHU ZH AND OU QJ. 2012. Maturation

induction of human peripheral blood mononuclear cell-

derived dendritic cells. Exp Ther Med 4: 131-134.

LI MO, WAN YY, SANJABI S, ROBERTSON AK AND

FLAVELL RAL. 2006. Transforming growth factor-beta

GIOVANA CECHIM and JOSÉ A.B. CHIES HUMAN MONOCYTE-DERIVED DENTRITIC CELLS GENERATION

An Acad Bras Cienc (2019) 91(4) e20190310 19 | 21

regulation of immune responses. Annu RevImmunol 24:

99-146.

LIEW FY, XU D, BRINT EK AND O’NEILL LA. 2005.

Negative regulation of toll-like receptor-mediated immune

responses. Nat Rev Immunol 5: 446-458.

LIU YJ. 2005. IPC: professional type 1 interferon-producing

cells and plasmacytoid dendritic cell precursors. Annu Rev

Immunol 23: 275-306.

LUTZ MB AND SCHULER G. 2002. Immature, semi-mature

and fully mature dendritic cells: which signals induce

tolerance or immunity? Trends Immunol 23: 445-449.

MA DY AND CLARK EA. 2009. The role of CD40 and

CD154/CD40L in dendritic cells. Semin Immunol 21:

265-272.

MAECKER HT, FREY T, NOMURA LE AND TROTTER J.

2004. Selecting fluorochrome conjugates for maximum

sensitivity. Cytometry A 62: 169-173.

MAHALINGAIAH PK, PALENSKI T AND VAN VLEET

TR. 2018. An In Vitro Model of Hematotoxicity:

Differentiation of Bone Marrow-Derived Stem/Progenitor

Cells into Hematopoietic Lineages and Evaluation of

Lineage-Specific Hematotoxicity. Curr Protoc Toxicol 1:

e45.

MASSA C AND SELIGER B. 2013. Fast dendritic cells

stimulated with alternative maturation mixtures induce

polyfunctional and long-lasting activation of innate and

adaptive effector cells with tumor-killing capabilities. J

Immuno l190: 3328-3337.

MATZINGER P. 1994. Tolerance, danger, and the extended

family. Annu Rev Immunol 12: 991-1045.

MERAD M, GINHOUX F AND COLLIN M. 2008. Origin,

homeostasis and function of Langerhans cells and other

langerin-expressing dendritic cells. Nat Rev Immunol 8:

935-947.

MERAD M, SATHE P, HELFT J, MILLER J AND MORTHA

A. 2013. The dendritic cell lineage: ontogeny and function

of dendritic cells and their subsets in the steady state and

the inflamed setting. Annu Rev Immunol 31: 563-604.

MEYER TP, ZEHNTER I, HOFMANN B, ZAISSERER J,

BURKHART J, RAPP S, WEINAUER F, SCHMITZ

J AND ILLERT WE. 2005. Buffy Coats (FBC): a

source of peripheral blood leukocytes recovered from

leukocytedepletion filters. J Immunol Methods 20: 150-

166.

MIAO EA AND WARREN SE. 2010. Innate immune

detection of bacterial virulence factors via the NLRC4

inflammasome. J Clin Immunol 30: 502-506.

MOHAMADZADEH M, BERARD F, ESSERT G,

CHALOUNI C, PULENDRAN B, DAVOUST J,

BRIDGES G, PALUCKA AK AND BANCHEREAU J.

2001. Interleukin 15 skews monocyte differentiation into

dendritic cells with features of Langerhans cells. J Exp

Med 194: 1013-1020.

MOHTY M, VIALLE-CASTELLANO A, NUNES JA,

ISNARDON D, OLIVE D AND GAUGLER B. 2003.

IFN-alpha skews monocyte differentiation into Toll-like

receptor 7-expressing dendritic cells with potent functional

activities. J Immunol 171: 3385-3393.

MOODY DB. 2006. TLR gateways to CD1 function. Nat

Immunol 7: 811-817.

MOORE GE, GERNER RE AND FRANKLIN HA. 1967.

Culture of normal human leukocytes. JAMA 199: 519-

524.

MORI L, LEPORE M AND DE LIBERO G. 2016. The

Immunology of CD1- and MR1-Restricted T Cells. Annu

Rev Immunol 34: 479-510.

NAPOLITANI G, LEPORE M AND DE LIBERO G. 2005.

The Immunology of CD1- and MR1-Restricted T Cells.

Nat Immunol 6: 769-776.

NEEFJES J, JONGSMA ML, PAUL P AND BAKKE O. 2011.

Towards a system understanding of MHC class I and MHC

class II antigen presentation. Nat Rev Immunol 11: 823-

836.

NESTLE FO, DI MEGLIO P, QIN JZ AND NICKOLOFF BJ.

2009. Skin immune sentinels in health and disease. Nat

Rev Immunol 9: 679-691.

OBERMAIER B, DAUER M, HERTEN J, SCHAD K,

ENDRES S AND EIGLER A. 2003. Development of a

new protocol for 2-day generation of mature dendritic cells

from human monocytes. Biol Proced Online 5: 197-203.

O’SULLIVAN B AND THOMAS R. 2003. CD40 and dendritic

cell function. Crit Rev Immunol 23: 83-107.

PARK BS AND LEE JO. 2013. Recognition of

lipopolysaccharide pattern by TLR4 complexes. Exp Mol

Med 6: 45-66.

PATARROYO M, PRIETO J, BEATTY PG, CLARK EA AND

GAHMBERG CG. 1988. Adhesion-mediating molecules

of human monocytes. Cell Immunol 113: 278-289.

PATEL AA ET AL. 2017. The fate and lifespan of human

monocyte subsets in steady state and systemic

inflammation. J Exp Med 214: 1913-1923.

PAVLOVIĆ B, TOMIĆ S, ĐOKIĆ J, VASILIJIĆ S, VUČEVIĆ

D, LUKIĆ J, GRUDEN-MOVSESIJAN A, ILIĆ N,

MARKOVIĆ M AND ČOLIĆ M. 2015. Fast dendritic

cells matured with Poly (I:C) may acquire tolerogenic

properties. Cytotherapy 17: 1763-1776.

PENG JC, THOMAS R ANDNIELSEN LK. 2005. Generation

and maturation of dendritic cells for clinical application

under serum-free conditions. J Immunother 28: 599-609.

PERUSSIA B, FANNING V AND TRINCHIERI G. 1985. A

leukocyte subset bearing HLA-DR antigens is responsible

for in vitro alpha interferon production in response to

viruses. Nat Immun Cell Growth Regul 4: 120-137.

PULENDRAN B. 2015. The Varieties of Immunological

Experience: Of Pathogens, Stress, and Dendritic Cells.

Annu Rev Immunol 33: 563-606.

GIOVANA CECHIM and JOSÉ A.B. CHIES HUMAN MONOCYTE-DERIVED DENTRITIC CELLS GENERATION

An Acad Bras Cienc (2019) 91(4) e20190310 20 | 21

RAETZ CR AND WHITFIELD C. 2002. Lipopolysaccharide

endotoxins. Annu Rev Biochem 71: 635-700.

RAMADAN G, KONINGS S, KURUP VP AND KEEVER-

TAYLOR CA. 2004. Generation of Aspergillus- and

CMV- specific T-cell responses using autologous fast DC.

Cytotherapy 6: 223-234.

RANDOLPH GJ, OCHANDO J AND PARTIDA-SÁNCHEZ

S.2008. Migration of dendritic cell subsets and their

precursors. Annu Rev Immunol 26: 293-316.

RELLOSO M, PUIG-KRÖGER A, PELLO OM,

RODRÍGUEZ-FERNÁNDEZ JL, DE LA ROSA G,

LONGO N, NAVARRO J, MUÑOZ-FERNÁNDEZ

MA, SÁNCHEZ-MATEOS P AND CORBÍ AL. 2002.

DC-SIGN (CD209) expression is IL-4 dependent and

is negatively regulated by IFN, TGF-beta, and anti-

inflammatory agents. J Immunol 168: 2634-2643.

REPNIK U, KNEZEVIC M AND JERAS M. 2003. Simple

and cost-effective isolation of monocytes from buffy coats.

J Immunol Methods 278: 283-292.

ROJAS-CANALES D, KRISHNAN R, JESSUP CF AND

COATES PT. 2012. Early exposure of interferon-γ inhibits

signal transducer and activator of transcription-6 signalling

and nuclear factor κB activation in a short-term monocyte-

derived dendritic cell culture promoting ‘FAST’ regulatory

dendritic cells. Clin Exp Immunol 167: 447-458.

RUPPERT J, FRIEDRICHS D, XU H AND PETERS JH.

1991. IL-4 decreases the expression of the monocyte

differentiation marker CD14, paralleled by an increasing

accessory potency. Immunobiology 182: 449-464.

SAIKH KU, KHAN AS, KISSNER T AND ULRICH RG.

2001. IL-15-induced conversion of monocytes to mature

dendritic cells. Clin Exp Immunol 126: 447-455.

SALLUSTO F AND LANZAVECCHIA A. 1994. Efficient

presentation of soluble antigen by cultured human dendritic

cells is maintained by granulocyte/macrophage colony-

stimulating factor plus interleukin 4 and downregulated by

tumor necrosis factor alpha. J Exp Med 179: 1109-1118.

SANARICO N, CIARAMELLA A, SACCHI A,

BERNASCONI D, BOSSÙ P, MARIANI F, COLIZZI

V AND VENDETTI S. 2006. Human monocyte-derived

dendritic cells differentiated in the presence of IL-2

produce proinflammatory cytokines and prime Th1

immune response. J Leukoc Biol 80: 555-562.

SANDER J ET AL. 2017. Cellular Differentiation of Human

Monocytes Is Regulated by Time-Dependent Interleukin-4

Signaling and the Transcriptional Regulator NCOR2.

Immunity 47: 1051-1066.

SANTINI SM, LAPENTA C, LOGOZZI M, PARLATO S,

SPADA M, DI PUCCHIO T AND BELARDELLI F. 2000.

Type I interferon as a powerful adjuvant for monocyte-

derived dendritic cell development and activity in vitro and

in Hu-PBL-SCID mice. J Exp Med 191: 1777-1788.

SCHLEE M AND HARTMANN G. 2016. Discriminating self

from non-self in nucleic acid sensing. Nat Rev Immunol

16: 566-580.

SCHMID I, KRALL WJ, UITTENBOGAART CH, BRAUN

J AND GIORGI JV. 1992. Dead cell discrimination with

7-amino-actinomycin D in combination with dual color

immunofluorescence in single laser flow cytometry.

Cytometry 13: 204-208.

SCHULZ O, EDWARDS AD, SCHITO M, ALIBERTI J,

MANICKASINGHAM S, SHER A AND REIS E SOUSA

C. 2000. CD40 triggering of heterodimeric IL-12 p70

production by dendritic cells in vivo requires a microbial

priming signal. Immunity 13: 453-462.

SEGURA E, VALLADEAU-GUILEMOND J, DONNADIEU

MH, SASTRE-GARAU X, SOUMELIS V AND

AMIGORENA S. 2012. Characterization of resident and

migratory dendritic cells in human lymph nodes. J Exp

Med 209: 653-660.

STEINMAN RM. 2012. Decisions about dendritic cells: past,

present, and future. Annu Rev Immunol 30: 1-22.

STEINMAN RM AND COHN ZA. 1973. Identification of a

novel cell type in peripheral lymphoid organs of mice. I.

Morphology, quantitation, tissue distribution. J Exp Med

137: 1142-1462.

STEINMAN RM AND WITMER MD. 1978. Lymphoid

dendritic cells are potent stimulators of the primary mixed

leukocyte reaction in mice. Proc Natl Acad Sci USA 75:

5132-5136.

STOCKWIN LH, MCGONAGLE D, MARTIN IG AND

BLAIR GE. 2000. Dendritic cells: immunological

sentinels with a central role in health and disease. Immunol

Cell Biol 78: 91-102.

STRASSER EF AND ECKSTEIN R. 2010. Optimization of

leukocyte collection and monocyte isolation for dendritic

cell culture. Transfus Med Rev 24: 130-139.

STRASSER EF, WEIDINGER T, ZIMMERMANN R,

RINGWALD J AND ECKSTEIN R. 2007. Recovery

of white blood cells and platelets from leukoreduction

system chambers of Trima Accel and COBE Spectra

plateletpheresis devices. Transfusion 47: 1943-1944

SUZUKI H ET AL. 2004. Activities of granulocyte-

macrophage colony-stimulating factor and interleukin-3

on monocytes. Am J Hematol 75: 179-189.

SWIECKI M AND COLONNA M. 2015. The multifaceted

biology of plasmacytoid dendritic cells. Nat Rev Immunol

15: 471-485.

TAKAHASHI K, HONEYMAN MC AND HARRISON

LC. 1997. Dendritic cells generated from human blood

in granulocyte macrophage-colony stimulating factor and

interleukin-7. Hum Immunol 55: 103-116.

TANAKA F, YAMAGUCHI H, HARAGUCHI N, MASHINO

K, OHTA M, INOUE H AND MORI M. 2006. Efficient

induction of specific cytotoxic T lymphocytes to tumor

rejection peptide using functional matured 2 day-cultured

GIOVANA CECHIM and JOSÉ A.B. CHIES HUMAN MONOCYTE-DERIVED DENTRITIC CELLS GENERATION

An Acad Bras Cienc (2019) 91(4) e20190310 21 | 21

dendritic cells derived from human monocytes. Int J Oncol

29: 1263-1268.

TRAVIS MA AND SHEPPARD D. 2014. TGF-β activation

and function in immunity. Annu Rev Immunol 32: 51-82.

TURVILLE SG, CAMERON PU, HANDLEY A, LIN G,

PÖHLMANN S, DOMS RW AND CUNNINGHAM AL.

2002. Diversity of receptors binding HIV on dendritic cell

subsets. Nat Immunol 3: 975-983.

UENO H ET AL. 2007. Dendritic cell subsets in health and

disease. Immunol Rev 219: 118-142.

ULANOVA M, TARKOWSKI A, HAHN-ZORIC M AND

HANSON LA. 2001. The Common vaccine adjuvant

aluminum hydroxide up-regulates accessory properties

of human monocytes via an interleukin-4-dependent

mechanism. Infect Immun 69: 1151-1159.

VATSAN RS, BROSS PF, LIU K, THEORET M, DE CLARO

AR, LU J, HELMS W, NILAND B, HUSAIN SR AND

PURI RK. 2013. Regulation of immunotherapeutic

products for cancer and FDA’s role in product development

and clinical evaluation. J Immunother Cancer 29: 1-5.

VICENTE-SUAREZ I, BRAYER J, VILLAGRA A, CHENG

F AND SOTOMAYOR EM. 2009. TLR5 ligation by

flagellin converts tolerogenic dendritic cells into activating

antigen-presenting cells that preferentially induce T-helper

1 responses. Immunol Lett 125: 114-118.

VOGT TK, LINK A, PERRIN J, FINKE D AND LUTHER

SA. 2009. Novel function for interleukin-7 in dendritic cell

development. Blood 113: 3961-3968.

WAJANT H, PFIZENMAIER K AND SCHEURICH P. 2003.

Tumor necrosis factor signaling. Cell Death Differ 10: 45-65.

WANG S AND EL-DEIRY WS. 2003. TRAIL and apoptosis

induction by TNF-family death receptors. Oncogene 22:

8628-8633.

WHO - WORLD HEALTH ORGANIZATION. 2010.

Guidelines on Drawing Blood: Best Practices in

Phlebotomy. http://www.euro.who.int/__data/assets/pdf_

file/0005/268790/WHO-guidelines-on-drawing-blood-

best-practices-in-phlebotomy-Eng.pdf?ua-1 (accessed 12

April 2018).

WORBS T. 2017. Dendritic cell migration in health and

disease. Nat Rev Immunol 17: 30-48.

WU L AND LIU YJ. 2007. Development of dendritic-cell

lineages. Immunity 26: 741-750.

XIAO Q, LI X, SUN D, YI H, LU X AND NIAN H. 2016. TLR7

Engagement on Dendritic Cells Enhances Autoreactive

Th17 Responses via Activation of ERK. J Immunol 197:

3820-3830.

YANG GX, LIAN ZX, KIKUCHI K, MORITOKI Y, ANSARI

AA, LIU YJ, IKEHARA S AND GERSHWIN ME. 2005.

Plasmacytoid dendritic cells of different origins have

distinct characteristics and function: studies of lymphoid

progenitors versus myeloid progenitors. J Immunol 175:

7281-7287.