In vitro preparation and functional assessment of human monocyte-derived dendritic cells—potential antigen-specific modulators of in vivo immune responses

Abstract

Dendritic cells (DCs) are highly specialized professional antigen presenting cells that have a potent capacity for stimulating naïve, memory and effector T-cells. They are located in lymphoid organs as well as in almost all nonlymphoid tissues. Immature DCs, residing in the host microenvironment, respond to danger signals with maturation, a differentiation process along which they acquire the ability to direct the extent and the type of primary immune responses according to the type of danger perceived.

In this review we present some of our approaches and experiences regarding the isolation of human monocytes from peripheral blood and the in vitro preparation of, first, immature and then mature DCs by applying several maturation factors: bacterial lipopolysaccharide (LPS), a defined mixture of recombinant pro-inflammatory cytokines, monocyte conditioned medium (MCM) and TNF-α alone. The assessment of DC phenotypes and their functional capabilities as well as some of the techniques used for tumour associated antigen loading are also presented. The results of such studies represent a basis for optimal in vitro preparation of DCs, which could be clinically used to modulate immune responses in cancer, autoimmune diseases and in the planned onset of tolerance to disparate major histocompatibilty complex (MHC) antigens prior to tissue or organ transplantation.

Introduction

Dendritic cells (DCs) were originally discovered in 1973 [1] and represent a heterogeneous cell population that is scarce in human peripheral blood. They are professional antigen presenting cells (APCs) and they arise from bone marrow residing progenitors in response to growth and differentiation factors, fms-like tyrosine kinase-3 ligand (Flt3L) and granulocyte–macrophage colony-stimulating factor (GM-CSF) [2]. According to their differentiation stage they can be divided in three groups: precursors, immature and mature DCs. Human DC precursors and immature DCs are lineage negative (CD3⁻ CD14⁻ CD19⁻ CD56⁻), HLA-DR⁺ mononuclear cells that constitute two distinct populations, according to their reaction patterns with anti-CD11c and anti-CD123 antibodies. While myeloid DCs are CD11c⁺ CD123^lo, plasmacytoid DCs are CD11c⁻ CD123^hi [2]. The two types of cells differ morphologically as well as in their tissue distribution, cytokine production and growth requirements. Plasmacytoid DCs play an important role in innate antiviral immunity and are found in blood and lymphoid organs. They are the most important interferon alpha (IFN-α)-producing cells in the body and can mount anti-tumour and antiviral antigen responses [2]. There are two major subtypes of myeloid DCs: Langerhans cells, which reside in the epidermis and oral, respiratory and genital mucosa, and dermal or interstitial or submusocal DCs, which reside in the dermis and interstitia of most organs [3].

Once they emerge from bone marrow, DCs migrate into blood and peripheral tissues, where they reside in an immature state. The immature phenotype is associated with a low MHC class II, and lack of co-stimulatory molecule expression (CD80, CD86), but on the other hand with a very efficient antigen uptake. The latter encompasses phagocytosis, endocytosis and pinocytosis. Upon capture by DCs, antigens are processed and presented as peptides in the context of MHC molecules on the DC surface. Danger signals [4], such as microbial components (LPS, dsRNA, CpG motif) or host molecules released by damaged tissues (inflammatory cytokines TNF-α, IL-1β, heat shock proteins Hsp60, Hsp70) deliver to DCs a maturation signal. Many microbial molecules signal through distinct Toll-like receptors (TLRs). These belong to the innate immune system and are called pattern-recognition receptors (PRRs). So far, 11 TLRs were described, each with different expression patterns and each recognizing different sets of molecules [2]. By maturation signals DCs are induced, through a change of their chemokine receptor profiles, to migrate via the afferent lymphatic vessels to secondary lymphoid organs, such as regional lymph nodes. They acquire the so-called mature phenotype with a strong T-cell attracting and activating potential which is characterized by a high expression of MHC class I and II, co-stimulatory (CD80, CD86), adhesion (ICAM-1, ICAM-2, LFA-1, LFA-3) and DC “specific” molecules (DC-SIGN (CD209), DC-LAMP, CD83), as well as by a concomitant loss of their antigen uptake activity.

It was long assumed that only mature DCs migrate to secondary lymphoid organs. Later, immature-like DCs (also termed semi-mature DCs) were observed in afferent lymph and these findings led to the idea that DCs are also involved in the induction and maintenance of peripheral tolerance to self-antigens [5]. This linear DC paradigm, implying that a single cell is involved in a peripheral antigen acquisition, migration, maturation and presentation, has lately been challenged by a model of inter-DC antigen transfer, which suggests transfer of antigen between tissue-derived, migrating DCs and lymph-node resident DC populations [6].

There is ample evidence that DCs play a key role in initiation, regulation and modulation of immune responses. Mature DCs are extremely effective in evoking specific reactiveness of naïve T cells as well as in determining the qualitative properties (Th1, Th2), extent and duration of their immune responses, while the immature DCs are deeply involved in inducing and maintaining the tolerance to self-antigens [2], [3]. The T-cell interaction with immature DCs results in initial proliferation of T-cells but subsequently in their short-term survival (abortive proliferation), while mature DCs cause long-term T-cell survival and their differentiation into effector and memory cells [7]. There are other immune cells whose functions are importantly influenced by their interactions with DCs. Activated myeloid DCs can induce B-cell proliferation, immunoglobulin isotype switching and plasma cell differentiation, through direct interactions as well as through production of B-cell activation and survival enhancing molecules BAFF and APRIL [2]. Activated plasmacytoid DCs too, are able to induce differentiation of CD40-activated B-cells into plasma cells, in this case by secreting IFN-α/β and IL-6 [2]. DCs can also cause the expansion of resting natural killer (NK) cells, following direct cell to cell interactions and production of different soluble factors. Activated NK cells can in turn kill immature, but not mature DCs and can stimulate DCs to induce protective CD8⁺ T-cell based immune responses [2]. It was also shown that activated NKT cells can mediate DC maturation which leads to a potent anti-tumour immune response [8].

Since the advent of techniques, invented and designed for the in vitro preparation of high numbers of human DCs, either from CD34⁺ haematopoietic stem cells [9], [10] or easily accessible peripheral blood monocytes [11], [12], numerous studies have been conducted, addressing their potential clinical use in a form of antigen specific cellular vaccines, functioning either as enhancive or suppressive modulators of immune responses. These different effects are largely dependant on the microenvironmental conditions, immature DCs are exposed to and affect their activation state. Therefore precise phenotypic and functional characterization of in vitro generated DCs should be performed before using them in a given immunomodulation/immunotherapy protocol [13], [14].

Cancer and autoimmune diseases are promoted and sustained, the former by ineffective and the latter by autoaggressive immune responses, facilitated by complex pathophysiological regulatory mechanisms, very often with a clear involvement of DCs as well. Although a very promising approach to cancer treatment, the clinical use of tumour associated antigen (TAA)-loaded mature DCs, designed to specifically target immune responses against various tumours, has not yet fulfilled, perhaps somewhat overoptimistic expectations and should therefore still be considered at its early stage. Different, well-established methods are available for the efficient loading of in vitro generated DCs with TAA: transfection with tumour cell derived genetic material (DNA, cDNA, RNA, mRNA), incubation with apoptotic tumour cells (TCs), TC lysates or synthetic TAA peptides, electro-fusion of TCs and DCs, resulting in immunohybrids, etc. The efficiency of such TAA presenting DCs can further be increased by transfecting them with genes, coding for various anti-tumour immunity enhancing factors, like IL-18 or CD40-L [15].