Using the principle of functional polarization of dendritic cells (DCs), we have developed a novel protocol to generate human DCs combining the three features critical for the induction of type-1 immunity: (a) fully mature status; (b) responsiveness to secondary lymphoid organ chemokines; and (c) high interleukin-12p70 (IL-12p70)-producing ability. We show that IFN-α and polyinosinic:polycytidylic acid (p-I:C) synergize with the “classical” type-1-polarizing cytokine cocktail [tumor necrosis factor α (TNFα)/IL-1β/IFNγ], allowing for serum-free generation of fully mature type-1-polarized DCs (DC1). Such “α-type-1-polarized DC(s)” (αDC1) show high migratory responses to the CCR7 ligand, 6C-kine but produce much higher levels of IL-12p70 as compared to TNFα/IL-1β/IL-6/prostaglandin E₂ (PGE₂)-matured DCs (sDC), the current “gold standard” in DC-based cancer vaccination. A single round of in vitro sensitization with αDC1 (versus sDCs) induces up to 40-fold higher numbers of long-lived CTLs against melanoma-associated antigens: MART-1, gp100, and tyrosinase. Serum-free generation of αDC1 allows, for the first time, the clinical application of DCs that combine the key three features important for their efficacy as anticancer vaccines.

Early stages of viral infections are associated with local recruitment and activation of dendritic cells (DC) and NK cells. Although activated DC and NK cells are known to support each other’s functions, it is less clear whether their local interaction in infected tissues can modulate the subsequent ability of migrating DC to induce T cell responses in draining lymph nodes. In this study, we report that NK cells are capable of inducing stable type 1-polarized “effector/memory” DC (DC1) that act as carriers of NK cell-derived helper signals for the development of type 1 immune responses. NK cell-induced DC1 show a strongly elevated ability to produce IL-12p70 after subsequent CD40 ligand stimulation. NK-induced DC1 prime naive CD4+ Th cells for high levels of IFN-γ, but low IL-4 production, and demonstrate a strongly enhanced ability to induce Ag-specific CD8+ T cell responses. Resting NK cells display stringent activation requirements to perform this novel, DC-mediated, “helper” function. Although their interaction with K562 cells results in effective target cell killing, the induction of DC1 requires a second NK cell-activating signal. Such costimulatory signal can be provided by type I IFNs, common mediators of antiviral responses. Therefore, in addition to their cytolytic function, NK cells also have immunoregulatory activity, induced under more stringent conditions. The currently demonstrated helper activity of NK cells may support the development of Th1- and CTL-dominated type 1 immunity against intracellular pathogens and may have implications for cancer immunotherapy.

In addition to their cytotoxic activities, natural killer (NK) cells can have immunoregulatory functions. We describe a distinct “helper” differentiation pathway of human CD56⁺CD3⁻ NK cells into CD56⁺/CD83⁺/CCR7⁺/CD25⁺ cells that display high migratory responsiveness to lymph node (LN)–associated chemokines, high ability to produce interferon-γ upon exposure to dendritic cell (DC)- or T helper (Th) cell–related signals, and pronounced abilities to promote interleukin (IL)-12p70 production in DCs and the development of Th1 responses. This helper pathway of NK cell differentiation, which is not associated with any enhancement of cytolytic activity, is induced by IL-18, but not other NK cell–activating factors. It is blocked by prostaglandin (PG)E₂, a factor that induces a similar CD83⁺/CCR7⁺/CD25⁺ LN-homing phenotype in maturing DCs. The current data demonstrate independent regulation of the “helper” versus “effector” pathways of NK cell differentiation and novel mechanisms of immunoregulation by IL-18 and PGE₂.

Cell-to-cell communication is essential for the organization, coordination, and development of cellular networks and multi-cellular systems. Intercellular communication is mediated by soluble factors (including growth factors, neurotransmitters, and cytokines/chemokines), gap junctions, exosomes and recently described tunneling nanotubes (TNTs). It is unknown whether a combination of these communication mechanisms such as TNTs and gap junctions may be important, but further research is required. TNTs are long cytoplasmic bridges that enable long-range, directed communication between connected cells. The proposed functions of TNTs are diverse and not well understood but have been shown to include the cell-to-cell transfer of vesicles, organelles, electrical stimuli and small molecules. However, the exact role of TNTs and gap junctions for intercellular communications and their impact on disease is still uncertain and thus, the subject of much debate. The combined data from numerous laboratories indicate that some TNT mediates a long range gap junctional communication to coordinate metabolism and signaling, in relation to infectious, genetic, metabolic, cancer, and age-related diseases. This review aims to describe the current knowledge, challenges and future perspectives to characterize and explore this new intercellular communication system and to design TNT-based therapeutic strategies.

Dendritic cells (DCs) activated by CD40L-expressing CD4+ T cells act as mediators of “T helper (Th)” signals for CD8+ T lymphocytes, inducing their cytotoxic function and supporting their long-term activity. Here, we show that the optimal activation of DCs, their ability to produce high levels of bioactive interleukin (IL)-12p70 and to induce Th1-type CD4+ T cells, is supported by the complementary DC-activating signals from both CD4+ and CD8+ T cells. Cord blood– or peripheral blood–isolated naive CD8+ T cells do not express CD40L, but, in contrast to naive CD4+ T cells, they are efficient producers of IFN-γ at the earliest stages of the interaction with DCs. Naive CD8+ T cells cooperate with CD40L-expressing naive CD4+ T cells in the induction of IL-12p70 in DCs, promoting the development of primary Th1-type CD4+ T cell responses. Moreover, the recognition of major histocompatibility complex class I–presented epitopes by antigen-specific CD8+ T cells results in the TNF-α– and IFN-γ–dependent increase in the activation level of DCs and in the induction of type-1 polarized mature DCs capable of producing high levels of IL-12p70 upon a subsequent CD40 ligation. The ability of class I–restricted CD8+ T cells to coactivate and polarize DCs may support the induction of Th1-type responses against class I–presented epitopes of intracellular pathogens and contact allergens, and may have therapeutical implications in cancer and chronic infections.

Natural killer (NK) cells and dendritic cells (DCs), two important components of the immune system, can exchange bidirectional activating signals in a positive feedback. Myeloid DCs, the cell type specialised in the presentation of antigen and initiation of antigen-specific immune responses, have recently been documented to be involved in supporting innate immunity, promoting the production of cytokines and cytotoxicity of NK cells, and enhancing their tumouricidal activity. Natural interferon-producing cells/plasmacytoid DCs (IPCs/PDCs) play an additional role in NK cell activation. Reciprocally, NK cells, traditionally considered to be major innate effector cells, have also recently been shown to play immunoregulatory 'helper' functions, being able to activate DCs and to enhance their ability to produce pro-inflammatory cytokines, and to stimulate T helper (Th) 1 and cytotoxic T lymphocyte (CTL) responses of tumour-specific CD4+ and CD8+ T cells. Activated NK cells induce the maturation of myeloid DCs into stable type-1 polarised DCs (DC1), characterised by up to a 100-fold enhanced ability to produce IL-12p70 in response to subsequent interaction with Th cells. In addition, the ability of NK cells to kill tumour cells may facilitate the generation of tumour-related antigenic material, further accelerating the induction of tumour-specific immunity. DC1, induced by NK cells or by NK cell-related soluble factors, are stable, resistant to tumour-related suppressive factors, and demonstrate a strongly enhanced ability to induce Th1 and CTL responses in human in vitro and mouse in vivo models. Compared with the standard mature DCs that are used in clinical trials at present, human NK cell-induced DC1s act as superior inducers of anticancer CTL responses during in vitro sensitisation. This provides a strong rationale for the combined use of NK cells and DCs in the immunotherapy of patients with cancer and patients with chronic infections that are resistant to standard forms of treatment. Stage I/II clinical trials that are being implemented at present should allow evaluation of the immunological and clinical efficacy of combined NK-DC therapy of melanoma and other cancers.