A comparison between isolated blood dendritic cells and monocyte‐derived dendritic cells in pigs

Various dendritic cell (DC) populations exist that differ in phenotype and ability to present antigen to T cells. For example, plasmacytoid DCs (pDCs) are less potent T cell activators compared with conventional DCs (cDCs). Here, we compared porcine blood DCs (BDCs), containing pDCs and cDCs, and monocyte‐derived DCs (MoDC), consisting of cDCs, in their phenotype, ability to uptake antigen, activation and maturation and their ability to present antigen to autologous T cells. Pigs represent an important animal model, whose immune system in many respects closely resembles that of humans. For example, the distribution of Toll‐like receptors is similar to that of humans, in contrast to that of mice. Here we demonstrate that both populations endocytose foreign material. Following lipopolysaccharide stimulation, CD80/86 and chemokine receptor (CCR)7 expression was increased in both populations as was the expression of the chemokine ligands (CCL)‐2, CCL‐4, CCL‐20 and CXCL‐2. Although basal and post‐stimulation protein concentrations of interleukins 6 and 8 and tumour necrosis factor‐α were higher in MoDCs, protein concentrations showed a higher fold increase in BDCs. Antigen‐specific proliferation of autologous T cells was induced by MoDCs and BDCs. Interestingly, while MoDCs induced stronger proliferation in naive T cells, no difference in proliferation was observed when primed T cells were studied. These results demonstrate that isolated porcine BDCs are highly responsive to stimulation with lipopolysaccharide and are functionally able to drive primed T‐cell proliferation to the same extent as MoDCs.     

Differential up-regulation of HLA-DM, invariant chain, and CD83 on myeloid and plasmacytoid dendritic cells from peripheral blood

Two main dendritic cell (DC) subsets have been described in peripheral blood, the myeloid subset or DC1 that is characterized by the presence of CD11c and the plasmacytoid subset or DC2 negative for this marker. The two subsets may perform different functions and have been defined as immunogenic (the myeloid subset) or tolerogenic (the plasmacytoid subset). The expression of human leukocyte antigen (HLA)-DM molecules, which act as peptide editors in the antigen presentation process, was studied in freshly isolated plasmacytoid and myeloid DCs from peripheral blood. The expression of the invariant chain (Ii), the major histocompatibility complex class II (MHC-II) : class II-associated Ii peptide (CLIP) complex, and CD83 was also investigated. The results showed that intracellular expression of HLA-DM and the Ii was significantly higher in the plasmacytoid than in the myeloid DC subset. In contrast, a higher fraction of cell expressing MHC-II : CLIP complex was found in the myeloid than in the plasmacytoid DC subpopulation. CD83 was not detected in any of these two subsets. Following culture of these cells with interleukin-3 (IL-3), tumor necrosis factor-alpha (TNFalpha) and/or heat shock protein-70 (HSP-70), the expression of intracellular HLA-DM was up-regulated in the myeloid DCs to levels similar to those found in the plasmacytoid DCs, whilst the Ii was down-regulated in the plasmacytoid subset to similar levels to those expressed in the myeloid DCs. In addition, CD83 was up-regulated in the myeloid (CD11c+) but not in the plasmacytoid (CD11c-) DCs. The expression pattern of these antigen-processing molecules could be related to the immaturity and function attributed to these DC subsets.     

Antigen processing and CD24 expression determine antigen presentation by splenic CD4+ and CD8+ dendritic cells

To examine heterogeneity in dendritic cell (DC) antigen presentation function, murine splenic DCs were separated into CD4⁺ and CD8⁺ populations and assessed for the ability to process and present particulate antigen to CD4⁺ and CD8⁺ T cells. CD4⁺ and CD8⁺ DCs both processed exogenous particulate antigen, but CD8⁺ DCs were much more efficient than CD4⁺ DCs for both major histocompatibility complex (MHC) class II antigen presentation and MHC class I cross-presentation. While antigen processing efficiency contributed to the superior antigen presentation function of CD8⁺ DCs, our studies also revealed an important contribution of CD24. CD8⁺ DCs were also more efficient than CD4⁺ DCs in inducing naïve T cells to acquire certain effector T-cell functions, for example generation of cytotoxic CD8⁺ T cells and interferon (IFN)-γ-producing CD4⁺ T cells. In summary, CD8⁺ DCs are particularly potent antigen-presenting cells that express critical costimulators and efficiently process exogenous antigen for presentation by both MHC class I and II molecules.     

Optimal stimulation for CD70 induction on human monocyte‐derived dendritic cells and the importance of CD70 in naive CD4+ T‐cell differentiation

Studies in mice have shown that CD70 on dendritic cells (DCs) is sufficient to convert T‐cell tolerance into immunity and hence induce anti‐tumour immune responses. Therefore, it is important to investigate (i) optimal stimuli to induce CD70 on human monocyte‐derived DCs (MoDCs), which are widely used for tumour immunotherapy, and (ii) the role of CD70 in functional differentiation of naive CD4⁺ and CD8⁺ T cells stimulated with MoDCs. We show that interferon‐α (IFN‐α) is a key cytokine to differentiate monocytes into DCs with the capacity to express CD70 upon maturation. CD70 expression on IFN‐α‐induced MoDCs was elicited by different categories of maturation‐inducing factors (Toll‐like receptor ligands, CD40 ligand and pro‐inflammatory mediators), among which prostaglandin E₂ was most effective. Naive T cells stimulated with MoDCs also expressed CD70. Stimulation with MoDCs promoted naive CD4⁺ T cells to acquire the ability to produce T helper type 1 and 2 cytokines in a CD70‐dependent manner. In contrast, the CD70–CD27 interaction diminished the production of an immunoregulatory cytokine IL‐10. The CD27 signal did not play a dominant role in the induction of effector molecules in naive CD8⁺ T cells during the stimulation with MoDCs. This study adds a novel function to the versatile cytokines, type I IFNs, that is, the induction of CD70 on MoDCs. CD70 promotes naive CD4⁺ T cells to acquire immunostimulatory activity through the DC–T‐cell and T‐cell–T‐cell interactions during the stimulation with MoDCs. Hence, the CD70–CD27 interaction may play an important role in inducing effective immune responses in DC‐based immunotherapy.     

Concerted antigen presentation by dendritic cells and B cells is necessary for optimal CD4 T-cell immunity in vivo

The relative contributions of different types of antigen presenting cells to T-cell activation, expansion and induction of effector functions are still not fully understood. In order to evaluate the roles of dendritic versus B cells during these phases of a CD4 T-cell response in vivo, we adoptively transferred major histocompatibility complex class II restricted, T-cell receptor-transgenic CD4+ T cells into transgenic mice expressing selectively the T-cell restricting class II molecules on either dendritic cells, B cells or both. Upon immunization with peptide antigen, we observed that dendritic cells were sufficient to induce activation, expansion, interleukin-2 production and germinal centre migration of antigen-specific T cells, independently of other antigen-presenting cells. In contrast, neither resting nor activated B cells had similar antigen-presenting capacities in vivo. However, in double transgenic mice where both B cells and dendritic cells were capable of presenting antigen, T cells showed increased proliferation, expansion and cytokine production in vivo. Moreover, higher antigen-specific CD4 T-cell numbers accumulated in germinal centres. Our data demonstrate that dendritic cells are sufficient to activate naive CD4 T cells in vivo, but B cells subsequently can enhance CD4 T-cell expansion further.     

Dual role of monocyte‐derived dendritic cells in Trypanosoma cruzi infection

Pathogens can cause inflammation when inoculated into the skin. The vector‐transmitted protozoan parasite Trypanosoma cruzi induces poor cellular‐infiltration and disseminates, causing high mortality in the experimental model. Here, we characterized the inflammatory foci at the parasite inoculation site and secondary lymphoid organs using a murine model. While no macrophages and few neutrophils and monocytes (Mo) were recruited into the skin, T. cruzi infection elicited the mobilization of Ly6C⁺ Mo to draining lymph nodes and spleen. Over time, this population became enriched in CD11b⁺Ly6C⁺CD11c⁺MHCII⁺CD86⁺ cells resembling inflammatory dendritic cells (DCs). Adoptive transfer of Ly6C⁺ Mo purified from the bone marrow of CD11c‐GFP transgenic mice confirmed the monocytic origin of Ly6C⁺ DCs found in the spleen of infected animals. Isolated Mo‐derived cells not only produced TNF‐α and nitric oxide, but also IL‐10 and displayed a poor capacity to induce lymphoproliferation. Ablation of Mo‐derived cells by 5‐fluorouracil confirmed their dual role during infection, limiting the parasite load by inducible nitric oxide synthase‐related mechanisms and negatively affecting the development of anti‐parasite T‐cell response. This study demonstrated that consistent with their antagonistic properties, these cells not only control the parasite spreading but also its persistence in the host.     

Dendritic cells, but not macrophages or B cells, activate major histocompatibility complex class II-restricted CD4+ T cells upon immune-complex uptake in vivo

Professional antigen-presenting cells (APC) are able to process and present exogenous antigen leading to the activation of T cells. Antigen-immunoglobulin (Ig)G complexes (IC) are much more efficiently processed and presented than soluble antigen. Dendritic cells (DC) are known for their ability to take up and process immune complex (IC) via FcgammaR, and they have been shown to play a crucial role in IC-processing onto major histocompatibility complex (MHC) class I as they contain a specialized cross-presenting transport system required for MHC class I antigen-processing. However, the MHC class II-antigen-processing pathway is distinct. Therefore various other professional APC, like macrophages and B cells, all displaying FcgammaR, are thought to present IC-delivered antigen in MHC class II. Nonetheless, the relative contribution of these APC in IC-facilitated antigen-presentation for MHC class II in vivo is not known. Here we show that, in mice, both macrophages and DC, but not B cells, efficiently capture IC. However, only DC, but not macrophages, efficiently activate antigen-specific MHC class II restricted CD4(+) T cells. These results indicate that mainly DC and not other professional APC, despite expressing FcgammaR and MHC class II, contribute significantly to IC-facilitated T cell activation in vivo under steady-state conditions.     

CD1a expression defines an interleukin‐12 producing population of human dendritic cells

Human and murine dendritic cell (DC) subsets are often defined by phenotypic features that predict their functional characteristics. In humans and mice, DC have been shown to have the ability to polarize naive CD4 T cells to a T helper type 1 (Th1) or Th2 phenotype. However, human myeloid DC generated from monocytes (monocyte-derived DC) have often been regarded as a homogeneous population, both phenotypically and functionally. Monocytes give rise to subpopulations of DC in vitro that can be separated on the basis of their expression of CD1a, a well-described DC subset marker. Importantly, we show that the CD1a⁺ DC subset produces significant quantities of interleukin-12p70 (IL-12p70) upon stimulation and, similar to the murine CD8α⁺ DC subset, can polarize naive CD4⁺ T cells to a Th1 phenotype. In contrast, CD1a⁻ DC, similar to murine CD8α⁻ DC, do not produce significant amounts of IL-12p70 upon stimulation or polarize T cells to a Th1 phenotype. Like monocyte-derived DC, CD1a⁺ and CD1a⁻ DC subsets obtained from CD34⁺ haematopoietic progenitors under distinct culture conditions were found to have these same features, suggesting that CD1a expression is a marker for myeloid DC that are a major source of IL-12 and Th1 CD4⁺ T cell polarization in man.     

CD8α+ DC are not the sole subset cross‐presenting cell‐associated tumor antigens from a solid tumor

One of the clear paradoxes in tumor immunology is the fact that cross‐presentation of cell‐associated tumor antigens to CD8⁺ T cells is efficient, yet CTL generation is weak, and tumors continue to grow. We examined, for the first time whether this may be due to alterations in the phenotype or function of cross‐presenting DC using a solid tumor model expressing a membrane bound neo‐antigen (hemagglutinin, HA). Tumor antigen was constitutively cross‐presented in the tumor‐draining LN throughout tumor progression by CD11c⁺ DC. Further analysis revealed that both CD8α⁺ and CD8α^? DC subsets, but not plasmacytoid DC, were effective at cross‐presenting HA tumor antigen. The proportions of DC subsets in the tumor‐draining LN were equivalent to those seen in the LN of naïve mice; however, a significant increase in the expression of the potential inhibitory B7 molecule, B7‐DC, was noted and appeared to be restricted to the CD8α^– DC subset. Therefore LN resident CD8α⁺ DC are not the sole DC subset capable of cross‐presenting cell‐associated tumor antigens. Migratory tumor DC subsets with altered co‐stimulatory receptor expression may contribute to induction and regulation of tumor‐specific responses.     

Development and functional specialization of CD103+ dendritic cells

Summary: CD103 (α_E) integrin expression distinguishes a population of dendritic cells (DCs) that can be found in many if not all lymphoid and non-lymphoid organs. CD103⁺ DCs display distinct functional activities. Migratory CD103⁺ DCs derived from skin, lung, and intestine efficiently present exogenous antigens in their corresponding draining lymph nodes to specific CD8⁺ T cells through a mechanism known as cross-presentation. On the T cells they prime, intestinal CD103⁺ DCs can drive the induction of the chemokine receptor CCR9 and α₄β₇ integrin, both known as gut-homing receptors. CD103⁺ DCs also contribute to control inflammatory responses and intestinal homeostasis by fostering the conversion of naive T cells into induced Foxp3⁺ regulatory T cells, a mechanism that relies on transforming growth factor-β and retinoic acid signaling. This review discusses recent findings that identify murine CD103⁺ DCs as important regulators of the immune response.     

Increased PD‐L1 expression and PD‐L1/CD86 ratio on dendritic cells were associated with impaired dendritic cells function in HCV infection

Impaired hepatitis C virus (HCV)‐specific T cell immunity was associated with the persistence of HCV infection. Dysfunction of dentritic cells (DCs) was believed to be involved in T cell exhaustion, but the mechanisms were rarely understood. In this study, surface costimulatory marker (CD83, CD86, and CD40), coinhibitory marker (PD‐L1) expression and allostimulatory capacity of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were evaluated in HCV‐infected patients. Results showed that the expression of both costimulatory and coinhibitory markers was increased in HCV‐infected patients compared with healthy controls. PD‐L1/CD86 ratio was increased and positively correlated with PD‐L1 expression on DCs in HCV‐infected patients. Allostimulatory capacity of DCs was impaired and inversely correlated with PD‐L1 expression and PD‐L1/CD86 ratio. These findings suggested that the effect of inhibitory marker PD‐L1 overwhelmed the effect of costimulatory markers and down regulated DC‐T activation in HCV‐infected patients. The results will be helpful to understand the mechanism of dysfunction of DCs in HCV infection and shed light on the DC‐based immunotherapeutic strategy. J. Med. Virol. 82: 1152–1159, 2010. © 2010 Wiley‐Liss, Inc.     

Lentiviral calnexin-modified dendritic cells promote expansion of high-avidity effector T cells with central memory phenotype

Dendritic cells (DCs) are key immune mediators for the education and activation of effector cytotoxic T lymphocytes (CTLs). Ex vivo manipulation of DCs is an attractive strategy in immunotherapy. The chaperone proteins are known to hold the keys to proper protein folding and antigen processing. However, little is known of the role of molecular chaperones in DC and T-cell functions. We report that DCs expressing supraphysiological levels of calnexin, a chaperone protein, via lentiviral gene transfer stimulated the expansion of high-avidity CTLs with increased central memory phenotype. Microarray RNA profiling and analyses of protein expression with flow cytometry and multiplex enzyme-linked immunosorbent assay indicated that calnexin had a global effect on DCs with up-regulation of immune modulatory signals including costimulatory molecules, cytokines, chemokines and adhesion molecules. Compared with unmodified DCs, calnexin-DCs were capable of activating T cells to exhibit increased functional avidity associated with up-regulation of CCR7 and costimulatory tumour necrosis factor receptor superfamily molecules. These findings demonstrate a prominent role of calnexin in optimizing DC immunity with potential for improving immunotherapy.     

Antigen-presenting capacity of macrophages and dendritic cells in the peritoneal cavity of patients treated with peritoneal dialysis

In this study the antigen-presenting capacity of human peritoneal cells and the influence of continuous ambulant peritoneal dialysis (CAPD) were studied. On average 6% of the peritoneal cells were dendritic cells (DC), with no difference between CAPD and control peritoneal cells. DC were enriched by selecting for non-adherent, Fc receptor-negative, low density cells. A typical spot-like CD68 positivity was seen in DC, in contrast to the pancytoplasmic staining pattern in macrophages. Peritoneal DC morphologically and functionally showed features of cells belonging to the DC lineage. Peritoneal DC were superior antigen-presenting cells for both allo-antigen, and Candida albicans antigen or purified protein derivative. CAPD peritoneal macrophages were two- to threefold better stimulator cells for allogeneic T cells compared with control macrophages. The level of integrins/adhesins or MHC class I or II, as measured semi-quantitatively on the FACS, could not account for this phenomenon. In addition, a double chamber system showed that dialysate-activated macrophages produced soluble factors that could enhance DC-induced allogeneic T cell proliferation. In conclusion, human peritoneal cells contain a relatively high percentage of classical DC. CAPD treatment does not impair the antigen-presenting capacity of peritoneal cells, but instead up-regulates the allo-antigcn-presenting capacity of peritoneal macrophages.     

Highly efficient antigen targeting to M-DC8+ dendritic cells via FcgammaRIII/CD16-specific antibody conjugates

Conjugates of peptide antigens with antibodies specifically recognizing surface molecules on dendritic cells (DC) represent an attractive approach to target antigens to antigen-presenting cells (APC) for the induction of specific T cell responses. The present study evaluates the potential of M-DC8(+) DC, a sub-population of professional APC in the blood, for an antibody-based vaccination strategy. We prepared, by chemical cross-linking, conjugates of peptide model antigens with antibodies directed against different cell surface molecules of DC. Antigen-peptide conjugates using an anti-CD16 (FcgammaRIII) antibody were most potent in inducing in vitro activation of a specific CD4(+) T cell response. They were at least 300 times more efficient than two other antibody-antigen conjugates and approximately 500 times more efficient than unconjugated antigen peptides. Our data demonstrate that specific antigen targeting via CD16 on M-DC8(+) DC is a promising vaccination approach for the efficient induction of specific CD4(+) T cell responses ex vivo, and perhaps in vivo.     

An improved enrichment method for functionally competent, highly purified peripheral blood dendritic cells and its application to HIV-infected blood samples.

Dendritic cells (DC) were purified from human peripheral blood using a rapid and simple method based on magnetic depletion of phagocytes with carbonyl iron, followed by centrifugation of nonphagocytic cells on a Percoll density gradient and depletion of lymphocytes and macrophages/monocytes with a panel of MoAbs and immunomagnetic beads. Enriched DC were obtained with > 99% purity as judged by non-specific esterase (NSE) staining. After isolation, these cells, representing 0.4% of the starting mononuclear cell population, still function as potent antigen-presenting cells for purified T lymphocytes. The present results confirm the ability of human peripheral blood DC to present soluble antigens to T cells including microbial antigens and show, further, that DC are more potent soluble antigen-presenting cells than monocytes. The method was successfully applied to the purification of DC from the blood of HIV-infected individuals. We could not detect decreased numbers of DC in four individuals with early HIV infection and no replicating HIV was detected by in situ hybridization in the DC.     

Morphologically and functionally intact dendritic cells can be derived from the peripheral blood of aged individuals

Dendritic cells are antigen-presenting cells (APC), which are crucial for the initiation of an immune response. In spite of the well known decline of immune function in old age, no information is yet available on whether dendritic cells are also affected by the ageing process. It was therefore the aim of this study to compare peripheral blood dendritic cells (DC) from old and young healthy individuals. Using granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4, DC were propagated from peripheral blood mononuclear cells (PBMC). The obtained cell populations had a typical dendritic morphology and expressed HLA class I and class II, CD23, CD32, CD40, CD44 and CD54, but not CD3 and CD19. Larger numbers of DC were obtained from old individuals than from young ones in spite of a similar expression pattern of surface molecules. DC from aged persons also survived better under in vitro culture conditions. When tested for their antigen-presenting capacity, DC from young and old individuals were equally effective in inducing the proliferation of tetanus toxoid-specific T cell clones after antigenic stimulation. Peripheral blood DC from aged individuals may thus still function as powerful APC. They may represent useful tools for immunotherapy in the aged.