CpG-C immunostimulatory oligodeoxyribonucleotide activation of plasmacytoid dendritic cells in rhesus macaques to augment the activation of IFN-gamma-secreting simian immunodeficiency virus-specific T cells

There are two principle subsets of dendritic cells (DCs); CD11c(+)CD123(-) myeloid DCs (MDCs) and CD11c(-)CD123(+) plasmacytoid DCs (PDCs). DC activation via TNF-TNFRs (e.g., CD40L) and TLRs (e.g., immunostimulatory oligodeoxyribonucleotides (ISS-ODNs)) is crucial for maximal stimulation of innate and adaptive immunity. Macaque DC biology is being studied to improve HIV vaccines using the SIV macaque model. Using lineage (Lin) markers to exclude non-DCs, Lin(-)HLA-DR(+)CD11c(+)CD123(-) MDCs and Lin(-)HLA-DR(+)CD11c(-)CD123(+) PDCs were identified in the blood of uninfected macaques and healthy macaques infected with SIV or simian-human immunodeficiency virus. Overnight culture of DC-enriched Lin-depleted cells increased CD80 and CD86 expression. IL-12 production and CD80/CD86 expression by MDC/PDC mixtures was further enhanced by CD40L and ISS-ODN treatment. A CpG-B ISS-ODN increased CD80/CD86 expression by PDCs, but resulted in little IFN-alpha secretion unless IL-3 was added. In contrast, a CpG-C ISS-ODN and aldrithiol-2-inactivated (AT-2) SIV induced considerable PDC activation and IFN-alpha release without needing exogenous IL-3. The CpG-C ISS-ODN also stimulated IL-12 release (unlike AT-2 SIV) and augmented DC immunostimulatory activity, increasing SIV-specific T cell IFN-gamma production induced by AT-2 SIV-presenting MDC/PDC-enriched mixtures. These data highlight the functional capacities of MDCs and PDCs in naive as well as healthy, infected macaques, revealing a promising CpG-C ISS-ODN-driven DC activation strategy that boosts immune function to augment preventative and therapeutic vaccine efficacy.     

Toll-like receptor ligands modulate dendritic cells to augment cytomegalovirus- and HIV-1-specific T cell responses

Optimal Ag targeting and activation of APCs, especially dendritic cells (DCs), are important in vaccine development. In this study, we report the effects of different Toll-like receptor (TLR)-binding compounds to enhance immune responses induced by human APCs, including CD123(+) plasmacytoid DCs (PDCs), CD11c(+) myeloid DCs (MDCs), monocytes, and B cells. PDCs, which express TLR7 and TLR9, responded to imidazoquinolines (imiquimod and R-848) and to CpG oligodeoxynucleotides stimulation, resulting in enhancement in expression of costimulatory molecules and induction of IFN-alpha and IL-12p70. In contrast, MDCs, which express TLR3, TLR4, and TLR7, responded to poly(I:C), LPS, and imidazoquinolines with phenotypic maturation and high production of IL-12 p70 without producing detectable IFN-alpha. Optimally TLR ligand-stimulated PDCs or MDCs exposed to CMV or HIV-1 Ags enhanced autologous CMV- and HIV-1-specific memory T cell responses as measured by effector cytokine production compared with TLR ligand-activated monocytes and B cells or unstimulated PDCs and MDCs. Together, these data show that targeting specific DC subsets using TLR ligands can enhance their ability to activate virus-specific T cells, providing information for the rational design of TLR ligands as adjuvants for vaccines or immune modulating therapy.     

Myeloid and plasmacytoid dendritic cells are susceptible to recombinant adenovirus vectors and stimulate polyfunctional memory T cell responses

Although replication-incompetent recombinant adenovirus (rAd) type 5 is a potent vaccine vector for stimulating T and B cell responses, high seroprevalence of adenovirus type 5 (Ad5) within human populations may limit its clinical utility. Therefore, alternative adenovirus serotypes have been studied as vaccine vectors. In this study, we characterized the ability of rAd5 and rAd35 to infect and induce maturation of human CD11c(+) myeloid dendritic cells (MDCs) and CD123(+) plasmacytoid dendritic cells (PDCs), and their ability to stimulate Ag-specific T cells. Both MDCs and PDCs were found to express the primary receptor for Ad35 (CD46) but not Ad5 (coxsackie-adenovirus receptor; CAR). Both dendritic cell (DC) subsets were also more susceptible to rAd35 than to rAd5. MDCs were more susceptible to both rAd35 and rAd5 than were PDCs. Whereas rAd35 used CD46 for entry into DCs, entry of rAd5 may be through a CAR-independent pathway. Exposure to rAd35 but not rAd5 induced high levels of IFN-alpha in PDCs and phenotypic differentiation in both DC subsets. MDCs and PDCs exposed to either rAd5 or rAd35 encoding for CMV pp65 were able to present pp65 and activate CMV-specific memory CD8(+) and CD4(+) T cells in a dose-dependent manner, but MDCs stimulated the highest frequencies of pp65-specific T cells. Responding T cells expressed multiple functions including degranulation (CD107a surface mobilization) and production of IFN-gamma, IL-2, TNF-alpha, and MIP-1beta. Thus, the ability of rAd35 to naturally target important DC subsets, induce their maturation, and appropriately present Ag to T cells may herald greater in vivo immunogenicity than has been observed with rAd5.     

Differential susceptibility to human immunodeficiency virus type 1 infection of myeloid and plasmacytoid dendritic cells

Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) plays an important role in HIV-1 transmission and pathogenesis. Here, we studied the susceptibility of ex vivo-isolated CD11c+ myeloid DCs (MDCs) and CD123+ plasmacytoid DCs (PDCs) to HIV-1 infection and the function of these cells early after infection. Both DC subsets were susceptible to CCR5- and CXCR4-using HIV-1 isolates (BaL and IIIB, respectively). However, MDCs were more susceptible to HIV-1(BaL) infection than donor-matched PDCs. In addition, HIV-1(BaL) infected MDCs more efficiently than HIV-1(IIIB), whereas PDCs were equally susceptible to both isolates. While exposure to HIV-1 alone resulted in only weak maturation of DCs, Toll-like receptor 7/8 ligation induced full maturation in both infected and uninfected DCs. Maturation did not increase HIV-1 replication in infected DCs, and infected DCs retained their ability to produce tumor necrosis factor alpha after stimulation. Both HIV-1 isolates induced alpha interferon production exclusively in PDCs, irrespective of productive infection. In conclusion, PDCs and MDCs were susceptible to HIV-1 infection, but neither displayed functional defects as a consequence of infection. The difference in susceptibility of PDCs and MDCs to HIV-1 may have implications for HIV-1 transmission and DC-mediated transfer of HIV-1 to T cells.     

Parallel loss of myeloid and plasmacytoid dendritic cells from blood and lymphoid tissue in simian AIDS

The loss of myeloid (mDC) and plasmacytoid dendritic cells (pDC) from the blood of HIV-infected individuals is associated with progressive disease. It has been proposed that DC loss is due to increased recruitment to lymph nodes, although this has not been directly tested. Similarly as in HIV-infected humans, we found that lineage-negative (Lin(-)) HLA-DR(+)CD11c(+)CD123(-) mDC and Lin(-)HLA-DR(+)CD11c(-)CD123(+) pDC were lost from the blood of SIV-infected rhesus macaques with AIDS. In the peripheral lymph nodes of SIV-naive monkeys the majority of mDC were mature cells derived from skin that expressed high levels of HLA-DR, CD83, costimulatory molecules, and the Langerhans cell marker CD1a, whereas pDC expressed low levels of HLA-DR and CD40 and lacked costimulatory molecules, similar to pDC in blood. Surprisingly, both DC subsets were depleted from peripheral and mesenteric lymph nodes and spleens in monkeys with AIDS, although the activation status of the remaining DC subsets was similar to that of DC in health. In peripheral and mesenteric lymph nodes from animals with AIDS there was an accumulation of Lin(-)HLA-DR(moderate)CD11c(-)CD123(-) cells that resembled monocytoid cells but failed to acquire a DC phenotype upon culture, suggesting they were not DC precursors. mDC and pDC from the lymphoid tissues of monkeys with AIDS were prone to spontaneous death in culture, indicating that apoptosis may be a mechanism for their loss in disease. These findings demonstrate that DC are lost from rather than recruited to lymphoid tissue in advanced SIV infection, suggesting that systemic DC depletion plays a direct role in the pathophysiology of AIDS.     

Differential regulation of human blood dendritic cell subsets by IFNs

Based on the relative expression of CD11c and CD1a, we previously identified subsets of dendritic cells (DCs) or DC precursors in human peripheral blood. A CD1a(+)/CD11c(+) population (CD11c(+) DCs), also called myeloid DCs, is an immediate precursor of Langerhans cells, whereas a CD1a(-)/CD11c(-) population (CD11c(-) DCs), sometimes called lymphoid DCs but better known as plasmacytoid DCs, is composed of type I IFN (IFN-alpha beta)-producing cells. Here, we investigate the effects of IFN-alpha beta and IFN-gamma as well as other cytokines on CD11c(+) and CD11c(-) DC subsets, directly isolated from the peripheral blood, instead of in vitro-generated DCs. IFN-gamma and IFN-alpha, rather than GM-CSF, were the most potent cytokines for enhancing the maturation of CD11c(+) DCs. Incubation of CD11c(+) DCs with IFN-gamma also resulted in increased IL-12 production, and this IL-12 allowed DCs to increase Th1 responses by alloreactive T cells. In contrast, IFN-alpha did not induce IL-12 but, rather, augmented IL-10 production. IFN-alpha-primed matured CD11c(+) DCs induced IL-10-producing regulatory T cells; however, this process was independent of the DC-derived IL-10. On the other hand, IFN-alpha by itself neither matured CD11c(-) DCs nor altered the polarization of responding T cells, although this cytokine was a potent survival factor for CD11c(-) DCs. Unlike IFN-alpha, IL-3 was a potent survival factor and induced the maturation of CD11c(-) DCs. The IL-3-primed CD11c(-) DCs activated T cells to produce IL-10, IFN-gamma, and IL-4. Thus, CD11c(+) and CD11c(-) DC subsets play distinct roles in the cytokine network, especially their responses to IFNs.     

Flow cytometric detection of circulating dendritic cells in healthy subjects

Dendritic cells (DCs) are the key antigen-presenting cells controlling the initiation of the T cell- dependent immune response. Currently, two peripheral blood DC subsets have been identified on the basis of their CD11c expression. The CD11c-negative (CD11c-) DCs (expressing high levels of CD123) are designated as lymphoid-derived DCs (DC2), whereas the CD11c+/CD123- cells, do identify the myeloid-derived DCs (DC1). A growing number of studies have been conducted in recent years on both the quantitative and functional alterations of DCs and their subsets in different pathological conditions. In the present study we assessed, using two different flow cytometric (FCM) techniques, the normal profile of blood DCs in 50 italian adult healthy subjects (M/F: 25/25, median age 42.5 years, range 20-65). The percentage and the absolute number of DCs and their subsets, were obtained starting from whole blood samples in two ways: 1) by calculating the number of DCs when gated as lineage-negative/ HLA-DR+ and identifing the two subsets as CD11c+ (DC1) and CD123+ (DC2) and 2) by using three specific markers: BDCA.1 (CD11c+ high/CD123+ low, myeloid DCs); BDCA.2 (CD11c-/ CD123+high, lymphoid DCs); BDCA.3 (CD11c+low /CD123-, myeloid DCs). Six parameters, 4-color FCM analysis were perfomed with a BD FACSCanto equipment. The mean values of the percentage and of the absolute number were: 0.5+/-0.2% and 30+/-11 cells/microL for DCs; 0.2+/-0.1% and 15+/-6 cells/microL for DC1; 0.2+/-0.1% and 15+/-7 cells/microL for DC2. The same values were: 0.2+/-0.1% and 16+/-7 cells/microL for BDCA.1; 0.2+/-0.1% and 12+/-7 cells/microL for BDCA.2; 0.02+/-0.01% and 2+/-1 cells/microL for BDCA.3, respectively. Our study confirmes that the two types of FCM analysis are able to identify the DC population. We also provides the first reference values on normal rates and counts of blood DCs in italian adult healthy subjects.     

Mucosal and peripheral Lin- HLA-DR+ CD11c/123- CD13+ CD14- mononuclear cells are preferentially infected during acute simian immunodeficiency virus infection

Massive infection of memory CD4 T cells is a hallmark of early simian immunodeficiency virus (SIV) infection, with viral infection peaking at day 10 postinfection (p.i.), when a majority of memory CD4 T cells in mucosal and peripheral tissues are infected. It is not clear if mononuclear cells from the monocyte and macrophage lineages are similarly infected during this early phase of explosive HIV and SIV infections. Here we show that, at day 10 p.i., Lin(-) HLA-DR(+) CD11c/123(-) CD13(+) CD14(-) macrophages in the jejunal mucosa were infected, albeit at lower levels than CD4 memory T cells. Interestingly, Lin(-) HLA-DR(+) CD11c/123(-) CD13(+) CD14(-) macrophages in peripheral blood, like their mucosal counterparts, were preferentially infected compared to Lin(-) HLA-DR(+) CD11c/123(-) CD13(+) CD14(+) monocytes, suggesting that differentiated macrophages were selectively infected by SIV. CD13(+) CD14(-) macrophages expressed low levels of CD4 compared to CD4 T cells but expressed similar levels of CCR5 as lymphocytes. Interestingly, CD13(+) CD14(-) macrophages expressed Apobec3G at lower levels than CD13(+) CD14(+) monocytes, suggesting that intracellular restriction may contribute to the differential infection of mononuclear subsets. Taken together, our results suggest that CD13(+) CD14(-) macrophages in mucosal and peripheral tissues are preferentially infected very early during the course of SIV infection.     

Natural alpha interferon-producing cells respond to human immunodeficiency virus type 1 with alpha interferon production and maturation into dendritic cells

Natural alpha interferon (IFN-alpha)-producing cells (IPCs) are now recognized as identical to plasmacytoid dendritic cell (DC) precursors in human blood and are thought to play an important role in antiviral immunity. In the present study, we examined the susceptibility as well as the cellular responses of IPCs to human immunodeficiency virus type 1 (HIV-1) infection. HLA-DR(+) CD11c(-) lineage-negative cells (IPCs) were purified from peripheral blood mononuclear cells by magnetic-bead separation and cell sorting. We substantiated that IPCs expressing the major HIV-1 coreceptors, CXCR4 and CCR5, are susceptible to infection of both T-cell-line-tropic NL4-3 and macrophage-tropic JR-CSF HIV-1 by quantification of HIV-1 p24 in the culture supernatants and by provirus integration assay using human conserved Alu-HIV-1 long terminal repeat PCR. To evaluate the cellular response of IPCs to HIV-1, we examined IFN-alpha production and their differentiation into DCs. After incubation with either NL4-3 or JR-CSF, IPCs produced a large amount of IFN-alpha and at the same time underwent morphological differentiation into DCs with upregulation of CD80 and CD86. Heat inactivation of the supernatants containing HIV-1 did not affect the IFN-alpha production and maturation, whereas removal of virions by ultracentrifugation completely nullified both biological effects, indicating that these cellular responses do not require actual HIV-1 infection but are elicited by interaction with HIV-1 virions or certain viral components. In conclusion, these data strongly suggest that IPC can directly recognize and respond to HIV-1 with IFN-alpha production, which is crucial for preventing progress of HIV-1 infection and occurrence of opportunistic infection.     

Functional specializations of intestinal dendritic cell and macrophage subsets that control Th17 and regulatory T cell responses are dependent on the T cell/APC ratio, source of mouse strain, and regional localization

Although several subsets of intestinal APCs have been described, there has been no systematic evaluation of their phenotypes, functions, and regional localization to date. In this article, we used 10-color flow cytometry to define the major APC subsets in the small and large intestine lamina propria. Lamina propria APCs could be subdivided into CD11c(+)CD11b(-), CD11c(+)CD11b(+), and CD11c(dull)CD11b(+) subsets. CD11c(+)CD11b(-) cells were largely CD103(+)F4/80(-) dendritic cells (DCs), whereas the CD11c(+)CD11b(+) subset comprised CD11c(+)CD11b(+)CD103(+)F4/80(-) DCs and CD11c(+)CD11b(+)CD103(-)F4/80(+) macrophage-like cells. The majority of CD11c(dull)CD11b(+) cells were CD103(-)F4/80(+) macrophages. Although macrophages were more efficient at inducing Foxp3(+) regulatory T (T(reg)) cells than DCs, at higher T cell/APC ratios, all of the DC subsets efficiently induced Foxp3(+) T(reg) cells. In contrast, only CD11c(+)CD11b(+)CD103(+) DCs efficiently induced Th17 cells. Consistent with this, the regional distribution of CD11c(+)CD11b(+)CD103(+) DCs correlated with that of Th17 cells, with duodenum > jejunum > ileum > colon. Conversely, CD11c(+)CD11b(-)CD103(+) DCs, macrophages, and Foxp3(+) T(reg) cells were most abundant in the colon and scarce in the duodenum. Importantly, however, the ability of DC and macrophage subsets to induce Foxp3(+) T(reg) cells versus Th17 cells was strikingly dependent on the source of the mouse strain. Thus, DCs from C57BL/6 mice from Charles River Laboratories (that have segmented filamentous bacteria, which induce robust levels of Th17 cells in situ) were more efficient at inducing Th17 cells and less efficient at inducing Foxp3(+) T(reg) cells than DCs from B6 mice from The Jackson Laboratory. Thus, the functional specializations of APC subsets in the intestine are dependent on the T cell/APC ratio, regional localization, and source of the mouse strain.     

Coevolution of markers of innate and adaptive immunity in skin and peripheral blood of patients with erythema migrans

We used multiparameter flow cytometry to characterize leukocyte immunophenotypes and cytokines in skin and peripheral blood of patients with erythema migrans (EM). Dermal leukocytes and cytokines were assessed in fluids aspirated from epidermal suction blisters raised over EM lesions and skin of uninfected controls. Compared with corresponding peripheral blood, EM infiltrates were enriched for T cells, monocytes/macrophages, and dendritic cells (DCs), contained lower proportions of neutrophils, and were virtually devoid of B cells. Enhanced expression of CD14 and HLA-DR by lesional neutrophils and macrophages indicated that these innate effector cells were highly activated. Staining for CD45RO and CD27 revealed that lesional T lymphocytes were predominantly Ag-experienced cells; furthermore, a subset of circulating T cells also appeared to be neosensitized. Lesional DC subsets, CD11c(+) (monocytoid) and CD11c(-) (plasmacytoid), expressed activation/maturation surface markers. Patients with multiple EM lesions had greater symptom scores and higher serum levels of IFN-alpha, TNF-alpha, and IL-2 than patients with solitary EM. IL-6 and IFN-gamma were the predominant cytokines in EM lesions; however, greater levels of both mediators were detected in blister fluids from patients with isolated EM. Circulating monocytes displayed significant increases in surface expression of Toll-like receptor (TLR)1 and TLR2, while CD11c(+) DCs showed increased expression of TLR2 and TLR4; lesional macrophages and CD11c(+) and CD11c(-) DCs exhibited increases in expression of all three TLRs. These results demonstrate that Borrelia burgdorferi triggers innate and adaptive responses during early Lyme disease and emphasize the interdependence of these two arms of the immune response in the efforts of the host to contain spirochetal infection.     

A subset of myeloid dendritic cells derived from peripheral blood monocytes represented a predominant subset characterized by their potential tumor-inhibiting activity

Besides their role as potent antigen-presenting cells, myeloid dendritic cells (MDCs), but not plasmacytoid dendritic cells (PDCs), have been reported to have cytotoxic or cytostatic activity on some tumor cells. In this article, we analyzed the tumoristatic potential of a distinct peripheral blood monocyte-derived MDC subset which co-expressed PDC-specific marker CD123. CD123⁺ MDCs represented a subset of small-sized DCs and accounted for 45–60% of peripheral blood monocytes cultured with granulocyte-macrophage colony-stimulating factor and interleukine-4 (IL-4) for 7 d. They exhibited more significant antiproliferative activity toward hematological tumor cell lines of Jurkat, HL60, and myelodysplastic syndromes over-leukemia than CD123⁻ MDCs even at a low effecter/target ratio. Pretreatment of MDC and their supernatant with TRAIL-R2:Fc significantly reduced the tumoristatic effect of CD123⁺ MDCs but not of CD123⁻ MDCs and their supernatant. CD123⁺ MDCs expressed higher level of cytoplasmic TNF-α-related apoptosis-inducing ligand (TRAIL) than CD123⁻ MDCs, whereas both expressed very little surface and soluble TRAIL. These results reveal that CD123⁺ cells represented a predominant subset of MDCs generated from peripheral blood monocytes in vitro, characterized by their potential tumoristic activity partially via cytoplasmic TRAIL.     

IL-10-producing B220+CD11c- APC in mouse spleen

APC acting at the early stages of an immune response can shape the nature of that response. Such APC will include dendritic cells (DCs) but may also include populations of B cells such as marginal zone B cells in the spleen. In this study, we analyze APC populations in mouse spleen and compare the phenotype and function of B220(+)CD11c(-) populations with those of CD11c(+) spleen DC subsets. Low-density B220(+) cells had morphology similar to DCs and, like DCs, they could stimulate naive T cells, and expressed high levels of MHC and costimulatory molecules. However, the majority of the B220(+) cells appeared to be of B cell lineage as demonstrated by coexpression of CD19 and surface Ig, and by their absence from RAG-2(-/-) mice. The phenotype of these DC-like B cells was consistent with that of B cells in the marginal zone of the spleen. On bacterial stimulation, they preferentially produced IL-10 in contrast to the DCs, which produced IL-12. Conventional B cells did not produce IL-10. The DC-like B cells could be induced to express low levels of the DC marker CD11c with maturational stimuli. A minority of the B220(+)CD11c(-) low-density cells did not express CD19 and surface Ig and may be a DC subset; this population also produced IL-10 on bacterial stimulation. B220(+) APC in mouse spleen that stimulate naive T cells and preferentially produce IL-10 may be involved in activating regulatory immune responses.     

Depletion of invariant NKT cells reduces inflammation-induced preterm delivery in mice

This study sought to determine whether invariant NKT (iNKT) cells play an essential role in inflammation-induced preterm delivery. Preterm delivery and fetal death rates were determined in wild-type (WT) C57BL/6 mice and iNKT cell-deficient Jα18(-/-) mice injected i.p. with LPS. The percentages of decidual immune cells, including activated subsets, and costimulatory molecule expression were analyzed by flow cytometry. Th1 and Th2 cytokine production in the culture supernatants of decidual mononuclear cells was measured by ELISA. To some extent, Jα18(-/-) mice were resistant to LPS-induced preterm delivery. The proportions of decidual CD3(+) and CD49b(+) cells were slightly lower in Jα18(-/-) mice than in WT Jα18(+/+) mice, whereas almost no CD3(+)CD49b(+) cells could be found in Jα18-null mice. The percentages of activated decidual DCs, T cells, and NK cells were significantly lower in LPS-treated Jα18(-/-) mice than in WT mice. The CD40, CD80, and CD86 expression levels on decidual CD11c(+) cells from Jα18(-/-) mice were also significantly lower than in WT mice. Mean concentrations of Th1 cytokines IFN-γ and IL-12p70 in the culture supernatants of decidual mononuclear cells from LPS-treated Jα18(-/-) mice were apparently lower than those of LPS-induced WT mice. Additionally, the proportions of activated CD11c(+) cells, CD3(+) cells, and CD49b(+) cells in LPS-induced preterm delivery mice were strikingly higher in both WT and null mice when compared with the control PBS group and LPS-injected but normally delivered mice. Our results suggest that iNKT cells may play an essential role in inflammation-induced preterm birth.     

Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: methodology and new insights.

BACKGROUND: The need to accurately discriminate dendritic cells (DCs) and macrophages (Mphs) in mouse lungs is critical given important biological differences. However, a validated flow cytometry-based method is still lacking, resulting in much confusion between both cell types. METHODS: Single-cell suspensions freshly obtained from collagenase-digested lung tissue were stained with a CD11c-specific monoclonal antibody, detected using a PE-Cy5 or APC-conjugated secondary reagent. Cellular immunophenotype was simultaneously explored using a panel of PE-conjugated markers. The FL1 or FITC-detection channel was reserved for the assessment of autofluorescence. RESULTS: CD11c-bright cells were heterogeneous and displayed a bimodal distribution with regard to autofluorescence (AF). CD11c+/low-AF cells were lineage-negative and showed features compatible with myeloid DCs. This was confirmed by morphology, potent T-cell stimulatory function in a mixed-leukocyte reaction, surface expression of MHCII and costimulatory molecules, and further immunophenotypical criteria, including the expression of Mac-1 and absence of CD8alpha. In contrast, CD11c+/high-AF cells displayed the features of pulmonary Mphs, including typical Mph morphology, very weak induction of T-cell proliferation, low to absent expression of MHCII and costimulatory molecules, and very low levels of Mac-1 as well as F4/80. We also show that only CD11c+/high-AF cells strongly expressed the macrophage marker MOMA-2, while interestingly Mac-3 was expressed at high levels by CD11c+/high-AF and low-AF alike. CONCLUSIONS: This study shows that the combination of CD11c-expression and autofluorescence is necessary and sufficient to accurately separate DCs from macrophage subpopulations in mouse lungs.     

A new subset of CD103+CD8alpha+ dendritic cells in the small intestine expresses TLR3, TLR7, and TLR9 and induces Th1 response and CTL activity

CD103(+) dendritic cells (DCs) are the major conventional DC population in the intestinal lamina propria (LP). Our previous report showed that a small number of cells in the LP could be classified into four subsets based on the difference in CD11c/CD11b expression patterns: CD11c(hi)CD11b(lo) DCs, CD11c(hi)CD11b(hi) DCs, CD11c(int)CD11b(int) macrophages, and CD11c(int)CD11b(hi) eosinophils. The CD11c(hi)CD11b(hi) DCs, which are CD103(+), specifically express TLR5 and induce the differentiation of naive B cells into IgA(+) plasma cells. These DCs also mediate the differentiation of Ag-specific Th17 and Th1 cells in response to flagellin. We found that small intestine CD103(+) DCs of the LP (LPDCs) could be divided into a small subset of CD8α(+) cells and a larger subset of CD8α(-) cells. Flow cytometry analysis revealed that CD103(+)CD8α(+) and CD103(+)CD8α(-) LPDCs were equivalent to CD11c(hi)CD11b(lo) and CD11c(hi)CD11b(hi) subsets, respectively. We analyzed a novel subset of CD8α(+) LPDCs to elucidate their immunological function. CD103(+)CD8α(+) LPDCs expressed TLR3, TLR7, and TLR9 and produced IL-6 and IL-12p40, but not TNF-α, IL-10, or IL-23, following TLR ligand stimulation. CD103(+)CD8α(+) LPDCs did not express the gene encoding retinoic acid-converting enzyme Raldh2 and were not involved in T cell-independent IgA synthesis or Foxp3(+) regulatory T cell induction. Furthermore, CD103(+)CD8α(+) LPDCs induced Ag-specific IgG in serum, a Th1 response, and CTL activity in vivo. Accordingly, CD103(+)CD8α(+) LPDCs exhibit a different function from CD103(+)CD8α(-) LPDCs in active immunity. This is the first analysis, to our knowledge, of CD8α(+) DCs in the LP of the small intestine.     

Human immunodeficiency virus type 1 activates plasmacytoid dendritic cells and concomitantly induces the bystander maturation of myeloid dendritic cells

In this study, we analyzed the phenotypic and physiological consequences of the interaction of plasmacytoid dendritic cells (pDCs) with human immunodeficiency virus type 1 (HIV-1). pDCs are one cellular target of HIV-1 and respond to the virus by producing alpha/beta interferon (IFN-alpha/beta) and chemokines. The outcome of this interaction, notably on the function of bystander myeloid DC (CD11c+ DCs), remains unclear. We therefore evaluated the effects of HIV-1 exposure on these two DC subsets under various conditions. Blood-purified pDCs and CD11c+ DCs were exposed in vitro to HIV-1, after which maturation markers, cytokine production, migratory capacity, and CD4 T-cell stimulatory capacity were analyzed. pDCs exposed to different strains of infectious or even chemically inactivated, nonreplicating HIV-1 strongly upregulated the expression of maturation markers, such as CD83 and functional CCR7, analogous to exposure to R-848, a synthetic agonist of toll-like receptor-7 and -8. In addition, HIV-1-activated pDCs produced cytokines (IFN-alpha and tumor necrosis factor alpha), migrated in response to CCL19 and, in coculture, matured CD11c+ DCs, which are not directly activated by HIV. pDCs also acquired the ability to stimulate na?ve CD4+ T cells, albeit less efficiently than CD11c+ DCs. This HIV-1-induced maturation of both DC subsets may explain their disappearance from the blood of patients with high viral loads and may have important consequences on HIV-1 cellular transmission and HIV-1-specific T-cell responses.     

Dendritic cells in the human decidua

Dendritic cells (DCs) in the pregnant human uterine mucosa have been poorly characterized, although they are likely to regulate immune responses to both placental trophoblast cells and uterine infections. In this study an HLA-DR+, CD11c+ lin- (CD3-, CD19-, CD56-, CD14-) population has been identified by three-color flow cytometry. The cell isolates were prepared either by collagenase digestion or mechanically from first-trimester decidual tissue. The decidual DCs comprised approximately 1.7% of CD45+ cells in the isolates and had the phenotype of immature myeloid DCs. No CD1a+ Langerhans cells or CD123+ plasmacytoid DCs were detected. The decidual DCs were DC-SIGN-, DEC-205+, CD40+. Two subsets could be distinguished on the basis of relative expression of HLA-DR, which also differed in expression of DC-activation markers. The DCs were identified in situ by immunohistology by DEC-205 staining. Cells with dendritic processes were found scattered through both the decidua basalis (in which trophoblast cells are infiltrating) and the decidua parietalis. They were also visible in endothelial-lined spaces. This is the first study to identify and describe the phenotype and distribution of human decidual DCs.     

Heterogeneity of freshly isolated human tonsil dendritic cells demonstrated by intracellular markers,phagocytosis, and membrane dye transfer

BACKGROUND: Heterogeneity within human dendritic cells (DCs) has been described but its functional relationships to cells of macrophage lineage and its role in human immunodeficiency virus (HIV) infection in vivo remain unclear. METHODS: Tonsil macrophages and DCs were isolated from low-density cells by negative selection and DCs were sorted into myeloid and plasmacytoid populations using antibodies to CD11c or CD123. Phagocytosis of latex beads and uptake of dye-labeled target cells were compared by flow cytometry and CD68 and S-100 by immunofluorescence on cytospins of sorted cells. RESULTS: Bead uptake and membrane dye transfer were found in both blood and tonsil CD11c(+) DCs and in CD14(+) cells particularly from blood monocytes. CD11c(-) DCs were poorly phagocytic but took up fluorescent dye from intact, necrotic or apoptotic cells. Tonsil DCs and macrophages expressed both CD68 and S-100 but CD11c(-) DCs expressed CD68 only. CONCLUSIONS: Freshly isolated CD11c(+) tonsil DCs are similar to CD14(+) macrophages in phagocytic function but the poorly phagocytic CD11c(-) DCs can also take up membrane from target cells. The intracellular markers commonly used to identify DCs and macrophages in situ do not identify accurately the CD11c(-) DC subset nor do they distinguish tonsil macrophages from DCs.     

Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo

Dendritic cells (DCs) have a unique ability to stimulate naive T cells. Recent evidence suggests that distinct DC subsets direct different classes of immune responses in vitro and in vivo. In humans, the monocyte-derived CD11c+ DCs induce T cells to produce Th1 cytokines in vitro, whereas the CD11c- plasmacytoid T cell-derived DCs elicit the production of Th2 cytokines. In this paper we report that administration of either Flt3-ligand (FL) or G-CSF to healthy human volunteers dramatically increases distinct DC subsets, or DC precursors, in the blood. FL increases both the CD11c+ DC subset (48-fold) and the CD11c- IL-3R+ DC precursors (13-fold). In contrast, G-CSF only increases the CD11c- precursors (>7-fold). Freshly sorted CD11c+ but not CD11c- cells stimulate CD4+ T cells in an allogeneic MLR, whereas only the CD11c- cells can be induced to secrete high levels of IFN-alpha, in response to influenza virus. CD11c+ and CD11c- cells can mature in vitro with GM-CSF + TNF-alpha or with IL-3 + CD40 ligand, respectively. These two subsets up-regulate MHC class II costimulatory molecules as well as the DC maturation marker DC-lysosome-associated membrane protein, and they stimulate naive, allogeneic CD4+ T cells efficiently. These two DC subsets elicit distinct cytokine profiles in CD4+ T cells, with the CD11c- subset inducing higher levels of the Th2 cytokine IL-10. The differential mobilization of distinct DC subsets or DC precursors by in vivo administration of FL and G-CSF offers a novel strategy to manipulate immune responses in humans.