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The unexplored potential of DC regulation by high dose IFN‐γ, with novel mechanisms of immune regulation within a peak inflammatory environment. Lately, there is increasing evidence that emphasizes the regulatory functions of IFN‐γ, which serve as negative‐feedback mechanisms after, e.g., pathogen clearance, to prevent unnecessary tissue destruction. Inflammatory processes involving Th1 and cytotoxic responses are characterized by high, local IFN‐γ concentrations, followed by resolution and immune silencing. Although this is a well‐known course of events, extensive attempts to address potential differential effects of IFN‐γ in the manner of its availability (quantitatively) in the environment do not exist. We demonstrate that high doses of IFN‐γ do not induce DC maturation and activation but instead, induce specific regulatory characteristics in DCs. Considering their phenotype, high doses of IFN‐γ extensively induce the expression of ILT‐4 and HLA‐G inhibitory molecules. Interestingly, the well‐known priming effect of IFN‐γ for IL‐12p70 production is lost at these conditions, and the DC cytokine profile is switched toward an increased IL‐10/IL‐12p70 ratio upon subsequent stimulation with CD40L. Furthermore, such DCs are capable of silencing cellular immune responses and activation of cytotoxic CD8+T lymphocytes, resulting in reduced cell proliferation and down‐regulation of granzyme B expression. Additionally, we find that in this manner, immune regulation mediated by IFN‐γ is not mainly a result of increased enzymatic activity of IDO in DCs but rather, a result of HLA‐G signaling, which can be reversed by blocking mAb. Altogether, our results identify a novel mechanism by which a Th1‐like environment programs the functional status of DCs to silence ongoing cytotoxic responses to prevent unwanted tissue destruction and inflammation.

DC‐SIGN is shown as critical in the differentiation process of dendritic cells from monocytes. DC‐SIGN is a C‐type lectin selectively expressed by certain types of DCs, including monocyte‐derived DCs. Many reports have described the impact of DC‐SIGN engagement with concomitant TLR signaling in tailoring of the DC maturation process, but so far, none has addressed the importance of DC‐SIGN engagement during their differentiation from blood progenitors. We therefore examined the role of DC‐SIGN engagement limited to the stage of IL‐4‐guided differentiation of DCs from human peripheral blood monocytes but not during maturation. We used two different anti‐DC‐SIGN antibodies with reported DC‐SIGN‐engaging activities. In cultures with DC‐SIGN ligands, the resulting iDCs displayed abrogated expression of differentiation markers CD1a and DC‐SIGN. Without further DC‐SIGN activation, such DCs matured with low CD80/CD86 and high ILT3 expression, along with the appearance of macrophage marker CD14. Additionally, treated DCs indicated a tolerogenic potential by possessing a low, allostimulatory capacity and inducing naïve, allogeneic CD4+T cells to produce low levels of IFN‐γ. Upon activation, IL‐10 production was greatly increased by such DCs; however, the use of IL‐10‐blocking antibodies could not completely reverse alternative DC activation. This suggests an alternative activation response that is a result of a different elementary state of DCs generated with concomitant ligation of DC‐SIGN. During differentiation, IL‐4‐induced pSTAT6 was reduced by DC‐SIGN ligands. Furthermore, during LPS‐induced maturation, treated DCs displayed lowered activation levels of p38 MAPK, STAT1, as well as STAT6, compared with controls. Collectively, evidence is presented confirming a crucial role for DC‐SIGN signaling in DC generation from monocytes.

The maturation status of dendritic cells (DCs) is crucial for effective antigen presentation and initiation of the primary immune response. Maturation stimuli cause the adhesion of immature DCs to the extracellular matrix, which is accompanied by recruitment of the CD11b/CD18 [macrophage antigen‐1 (Mac‐1)] integrin receptor, cytoskeleton reorganization, and podosome formation. Cathepsin X, a cysteine protease expressed in DCs and other APCs, is involved in Mac‐1 activation. We have shown that during maturation, cathepsin X translocates to the plasma membrane of maturing DCs, enabling Mac‐1 activation and consequently, cell adhesion. In mature DCs, cathepsin X redistributes from the membrane to the perinuclear region, which coincides with the de‐adhesion of DCs, formation of cell clusters, and acquisition of the mature phenotype. Inhibition of cathepsin X activity during DC differentiation and maturation resulted in an altered phenotype and function of mature DCs. It reduced surface expression of costimulatory molecules, increased expression of inhibitory Ig‐like transcripts 3 and 4 (ILT3 and ILT4), almost completely abolished cytokine production, diminished migration, and reduced the capacity of DCs to stimulate T lymphocytes. These results stress the importance of cathepsin X in regulating DC adhesion, a crucial event for their maturation and T cell activation.

Antigen‐presenting cells (APC) play a pivotal role in the initiation of the T cell‐mediated and antigen‐specific immune response. The suggested role of endogenous inhibitor cystatin C (CyC) is to modulate cysteine proteases (cathepsins) present in human APC. To test this hypothesis, dendritic cells (DC) were generated in vitro from isolated monocytes, and changes in content, localization, and secretion of CyC and cathepsins S, L, and H (CatS, ‐L, and ‐H, repsectively) were followed in response to interleukin‐4, enabling monocyte differentiation, and to tumor necrosis factor α (TNF‐α), enabling DC maturation. A large increase in intracellular CyC accompanied the differentiation of monocytes to immature DC, also shown by strong immunolabeling of Golgi in immature DC. On DC maturation, intracellular CyC levels decreased, and CyC was mostly absent from the Golgi. On prolonged incubation of mature DC with TNF‐α, CyC was found located in the proximity of the plasma membrane, indicating that the transport of CyC from Golgi was not blocked as the result of the arrested exocytosis in mature DC. The secretion of CyC ceased, consistent with the peak of the surface expression of phenotypic markers (CD40, CD54, CD80, CD83, CD86, and major histocompatibility complex class II), characteristic for the mature DC stage, whereas the secretion of cathepsins did not correlate with the maturation stage. The difference in localization of CyC and of CatS, ‐L, and ‐H in immature and mature DC shows that the regulatory potential of CyC toward CatS, ‐L, and ‐H inside DC is limited. However, these interactions may occur extracellularly in lymph, as suggested by the large excess of CyC over secreted CatS, ‐L, and ‐H, and they may facilitate DC migration to lymph nodes.

Adoptive transfer of effector antigen-specific immune cells is becoming a promising treatment option in allogeneic transplantation, infectious diseases, cancer, and autoimmune disorders. Within this context, the important role of CD8+ cytotoxic T cells (CTLs) is objective of intensive studies directed to their in vivo and ex vivo induction, detection, selection, expansion, and therapeutic effectiveness. Additional questions that are being addressed by the scientific community are related to the establishment and maintenance of their longevity and memory state as well as to defining critical conditions underlying their transitions between discrete, but functionally different subtypes. In this article we review and comment latest approaches and techniques used for preparing large amounts of antigen-specific CTLs, suitable for clinical use.