CD103+CD11b− salivary gland dendritic cells have antigen cross‐presenting capacity

Dendritic cells (DCs) in lymphoid and non‐lymphoid tissues are professional antigen‐presenting cells that are essential for effective immunity and tolerance. However, the presence and characteristics of DCs in steady‐state salivary glands (SGs) currently remain unknown. We herein identified CD64^?CD11c⁺ classical DCs (cDCs) as well as CD64⁺ macrophages among CD45⁺MHC class II⁺ antigen‐presenting cells in steady‐state murine SGs. SG cDCs were divided into CD103⁺CD11b^? and CD103^?CD11b⁺ cDCs. CD103⁺CD11b^? cDCs expressed XCR1, CLEC9A, and interferon regulatory factor 8, whereas CD103^?CD11b⁺ cDCs strongly expressed CD172a. Both cDC subsets in SGs markedly expanded in response to the Flt3 ligand (Flt3L), were replenished by bone marrow‐derived precursors, and differentiated from common DC precursors, but not monocytes. Furthermore, ovalbumin‐pulsed SG CD103⁺CD11b^? cDCs induced the proliferation of naïve ovalbumin‐specific CD8⁺ T cells and production of interferon‐γ from proliferating T cells. SG CD103⁺CD11b^? cDCs expanded by Flt3L in vivo exhibited the same properties. These results indicate that bona fide cDCs reside in steady‐state murine SGs and cDCs with the CD103⁺CD11b^? phenotype possess antigen cross‐presenting capacity. Moreover, Flt3L enhances protective immunity by expanding cDCs. Taken together, SG cDCs might play an important role in maintaining immune homeostasis in the tissues.     

Dendritic cells from the human female reproductive tract rapidly capture and respond to HIV

Dendritic cells (DCs) throughout the female reproductive tract (FRT) were examined for phenotype, HIV capture ability and innate anti-HIV responses. Two main CD11c⁺ DC subsets were identified: CD11b⁺ and CD11b^low DCs. CD11b⁺CD14⁺ DCs were the most abundant throughout the tract. A majority of CD11c⁺CD14⁺ cells corresponded to CD1c⁺ myeloid DCs, whereas the rest lacked CD1c and CD163 expression (macrophage marker) and may represent monocyte-derived cells. In addition, we identified CD103⁺ DCs, located exclusively in the endometrium, whereas DC-SIGN⁺ DCs were broadly distributed throughout the FRT. Following exposure to GFP-labeled HIV particles, CD14⁺ DC-SIGN⁺ as well as CD14⁺ DC-SIGN⁻ cells captured virus, with ∼30% of these cells representing CD1c⁺ myeloid DCs. CD103⁺ DCs lacked HIV capture ability. Exposure of FRT DCs to HIV induced secretion of CCL2, CCR5 ligands, interleukin (IL)-8, elafin, and secretory leukocyte peptidase inhibitor (SLPI) within 3 h of exposure, whereas classical pro-inflammatory molecules did not change and interferon-α2 and IL-10 were undetectable. Furthermore, elafin and SLPI upregulation, but not CCL5, were suppressed by estradiol pre-treatment. Our results suggest that specific DC subsets in the FRT have the potential for capture and dissemination of HIV, exert antiviral responses and likely contribute to the recruitment of HIV-target cells through the secretion of innate immune molecules.     

Cytokine‐dependent regulation of dendritic cell differentiation in the splenic microenvironment

The DC‐derived chemokine CCL17, a ligand of CCR4, has been shown to promote various inflammatory diseases such as atopic dermatitis, atherosclerosis, and inflammatory bowel disease. Under steady‐state conditions, and even after systemic stimulation with LPS, CCL17 is not expressed in resident splenic DCs as opposed to CD8α^?CD11b⁺ LN DCs, which produce large amounts of CCL17 in particular after maturation. Upon systemic NKT cell activation through α‐galactosylceramide stimulation however, CCL17 can be upregulated in both CD8α^? and CD8α⁺ splenic DC subsets and enhances cross‐presentation of exogenous antigens. Based on genome‐wide expression profiling, we now show that splenic CD11b⁺ DCs are susceptible to IFN‐γ‐mediated suppression of CCL17, whereas LN CD11b⁺CCL17⁺ DCs downregulate the IFN‐γR and are much less responsive to IFN‐γ. Under inflammatory conditions, particularly in the absence of IFN‐γ signaling in IFN‐γRKO mice, CCL17 expression is strongly induced in a major proportion of splenic DCs by the action of GM‐CSF in concert with IL‐4. Our findings demonstrate that the local cytokine milieu and differential cytokine responsiveness of DC subsets regulate lymphoid organ specific immune responses at the level of chemokine expression.     

CCR2+CD103− intestinal dendritic cells develop from DC-committed precursors and induce interleukin-17 production by T cells

The identification of intestinal macrophages (mφs) and dendritic cells (DCs) is a matter of intense debate. Although CD103⁺ mononuclear phagocytes (MPs) appear to be genuine DCs, the nature and origins of CD103⁻ MPs remain controversial. We show here that intestinal CD103⁻CD11b⁺ MPs can be separated clearly into DCs and mφs based on phenotype, gene profile, and kinetics. CD64⁻CD103⁻CD11b⁺ MPs are classical DCs, being derived from Flt3 ligand-dependent, DC-committed precursors, not Ly6C^hi monocytes. Surprisingly, a significant proportion of these CD103⁻CD11b⁺ DCs express CCR2 and there is a selective decrease in CD103⁻CD11b⁺ DCs in mice lacking this chemokine receptor. CCR2⁺CD103⁻ DCs are present in both the murine and human intestine, drive interleukin (IL)-17a production by T cells in vitro, and show constitutive expression of IL-12/IL-23p40. These data highlight the heterogeneity of intestinal DCs and reveal a bona fide population of CCR2⁺ DCs that is involved in priming mucosal T helper type 17 (Th17) responses.     

FMS-Like Tyrosine Kinase 3 Ligand Treatment of Mice Aggravates Acute Lung Injury in Response to Streptococcus pneumoniae: Role of Pneumolysin

FMS-like tyrosine kinase-3 ligand (Flt3L) is a dendritic cell (DC) growth and differentiation factor with potential in antitumor therapies and antibacterial immunization strategies. However, the effect of systemic Flt3L treatment on lung-protective immunity against bacterial infection is incompletely defined. Here, we examined the impact of deficient (in Flt3L knockout [KO] mice), normal (in wild-type [WT] mice), or increased Flt3L availability (in WT mice pretreated with Flt3L for 3, 5, or 7 days) on lung DC subset profiles and lung-protective immunity against the major lung-tropic pathogen, Streptococcus pneumoniae. Although in Flt3L-deficient mice the numbers of DCs positive for CD11b (CD11b^pos DCs) and for CD103 (CD103^pos DCs) were diminished, lung permeability, a marker of injury, was unaltered in response to S. pneumoniae. In contrast, WT mice pretreated with Flt3L particularly responded with increased numbers of CD11b^pos DCs and with less pronounced numbers of CD103^pos DCs and impaired bacterial clearance and with increased lung permeability following S. pneumoniae challenge. Notably, infection of Flt3L-pretreated mice with S. pneumoniae lacking the pore-forming toxin, pneumolysin (PLY), resulted in substantially less lung CD11b^pos DCs activation and reduced lung permeability. Collectively, this study establishes that Flt3L treatment enhances the accumulation of proinflammatory activated lung CD11b^pos DCs which contribute to acute lung injury in response to PLY released by S. pneumoniae.     

Dendritic cell homeostasis is maintained by nonhematopoietic and T‐cell‐produced Flt3‐ligand in steady state and during immune responses

Lymphoid‐tissue dendritic cells (DCs) are short‐lived and need to be continuously replenished from bone marrow‐derived DC progenitor cells. Fms‐related tyrosine kinase 3 is expressed during cellular development from hematopoietic progenitors to lymphoid‐tissue DCs. Fms‐related tyrosine kinase 3 ligand (Flt3L) is an essential, nonredundant cytokine for DC progenitor to lymphoid tissue DC differentiation and maintenance. However, which cells contribute to Flt3L production and how Flt3L cytokine levels are regulated in steady state and during immune reactions remains to be determined. Here we demonstrate that besides nonhematopoietic cells, WT T cells produce Flt3L and contribute to the generation of both classical DCs (cDCs) and plasmacytoid DCs in Flt3L^?/? mice. Upon stimulation in vitro, CD4⁺ T cells produce more Flt3L than CD8⁺ T cells. Moreover, in vivo stimulation of naïve OT‐II CD4⁺ T cells with OVA leads to increase of pre‐cDCs and cDCs in draining lymph nodes of Flt3L^?/? mice in a partially Flt3L‐dependent manner. Thus, Flt3L‐mediated lymphoid tissue DC homeostasis is regulated by steady‐state T cells as well as by proliferative T cells, fostering local development of lymphoid organ resident DCs.     

Crosstalk between human DC subsets promotes antibacterial activity and CD8+ T‐cell stimulation in response to bacille Calmette‐Guérin

To date, little is known about the unique contributions of specialized human DC subsets to protection against tuberculosis (TB). Here, we focus on the role of human plasmacytoid (p)DCs and myeloid (m)DCs in the immune response to the TB vaccine bacille Calmette‐Guérin (BCG). Ex vivo DC subsets from human peripheral blood were purified and infected with BCG expressing GFP to distinguish between infected and noninfected cells. BDCA‐1⁺ myeloid DCs were more susceptible than BDCA‐3⁺ mDCs to BCG infection. Plasmacytoid DCs have poor phagocytic activity but are equipped with endocytic receptors and can be activated by bystander stimulation. Consequently, the mutual interaction of the two DC subsets in response to BCG was analyzed. We found that pDCs were activated by BCG‐infected BDCA‐1⁺ mDCs to upregulate maturation markers and to produce granzyme B, but not IFN‐α. Reciprocally, the presence of activated pDCs enhanced mycobacterial growth control by infected mDCs and increased IL‐1β availability. The synergy between the two DC subsets promoted BCG‐specific CD8⁺ T‐cell stimulation and the role of BCG‐infected BDCA‐1⁺ mDCs could not be efficiently replaced by infected BDCA‐3⁺ mDCs in the crosstalk with pDCs. We conclude that mDC–pDC crosstalk should be exploited for rational design of next‐generation TB vaccines.     

Mesenteric lymph node CD11b− CD103+ PD‐L1High dendritic cells highly induce regulatory T cells

Dendritic cells (DCs) in mesenteric lymph nodes (MLNs) induce Foxp3⁺ regulatory T cells to regulate immune responses to beneficial or non‐harmful agents in the intestine, such as commensal bacteria and foods. Several studies in MLN DCs have revealed that the CD103⁺ DC subset highly induces regulatory T cells, and another study has reported that MLN DCs from programmed death ligand 1 (PD‐L1) ‐deficient mice could not induce regulatory T cells. Hence, the present study investigated the expression of these molecules on MLN CD11c⁺ cells. Four distinct subsets expressing CD103 and/or PD‐L1 were identified, namely CD11b⁺ CD103⁺ PD‐L1^High, CD11b⁻ CD103⁺ PD‐L1^High, CD11b⁻ CD103⁺ PD‐L1^Low and CD11b⁺ CD103⁻ PD‐L1^Int. Among them, the CD11b⁻ CD103⁺ PD‐L1^High DC subset highly induced Foxp3⁺ T cells. This subset expressed Aldh1a2 and Itgb8 genes, which are involved in retinoic acid metabolism and transforming growth factor‐β (TGF‐β) activation, respectively. Exogenous TGF‐β supplementation equalized the level of Foxp3⁺ T‐cell induction by the four subsets whereas retinoic acid did not, which suggests that high ability to activate TGF‐β is determinant for the high Foxp3⁺ T‐cell induction by CD11b⁻ CD103⁺ PD‐L1^High DC subset. Finally, this subset exhibited a migratory DC phenotype and could take up and present orally administered antigens. Collectively, the MLN CD11b⁻ CD103⁺ PD‐L1^High DC subset probably takes up luminal antigens in the intestine, migrates to MLNs, and highly induces regulatory T cells through TGF‐β activation.     

Intestinal CD103− dendritic cells migrate in lymph and prime effector T cells

Intestinal dendritic cells (DCs) continuously migrate through lymphatics to mesenteric lymph nodes where they initiate immunity or tolerance. Recent research has focused on populations of intestinal DCs expressing CD103. Here we demonstrate, for the first time, the presence of two distinct CD103⁻ DC subsets in intestinal lymph. Similar to CD103⁺ DCs, these intestine-derived CD103⁻ DCs are responsive to Flt3 and they efficiently prime and confer a gut-homing phenotype to naive T cells. However, uniquely among intestinal DCs, CD103⁻ CD11b⁺ CX₃CR1^int lymph DCs induce the differentiation of both interferon-γ and interleukin-17-producing effector T cells, even in the absence of overt stimulation. Priming by CD103⁻ CD11b⁺ DCs represents a novel mechanism for the rapid generation of effector T-cell responses in the gut. Therefore, these cells may prove to be valuable targets for the treatment of intestinal inflammation or in the development of effective oral vaccines.     

Functional specialization of intestinal dendritic cell subsets during Th2 helminth infection in mice

Dendritic cells (DCs) are essential in dictating the nature and effectiveness of immune responses. In the intestine DCs can be separated into discrete subsets, defined by expression of CD11b and CD103, each with different developmental requirements and distinct functional potential. Recent evidence has shown that different intestinal DC subsets are involved in the induction of T helper (Th)17 and regulatory T cell responses, but the cells that initiate Th2 immune responses are still incompletely understood. We show that in the Th2 response to an intestinal helminth in mice, only CD11b⁺ and not CD11b^? DCs accumulate in the local lymph node, upregulate PDL2 and express markers of alternative activation. An enteric Th1 response instead activated both CD11b⁺ and CD11b^? DCs without eliciting alternative activation in either population. Functionally, only CD11b⁺ DCs activated during helminth infection supported Th2 differentiation in naive CD4⁺ T cells. Together our data demonstrate that the ability to prime Th2 cells during intestinal helminth infection, is a selective and inducible characteristic of CD11b⁺ DCs.     

Enrichment of IL-12–Producing Plasmacytoid Dendritic Cells in Donor Bone Marrow Grafts Enhances Graft-versus-Leukemia Activity in Allogeneic Hematopoietic Stem Cell Transplantation

A critical question in the field of allogeneic hematopoietic stem cell transplantation (HSCT) is how to enhance graft-versus-leukemia (GVL) activity while limiting graft-versus-host-disease (GVHD). We have previously reported that donor bone marrow (BM) precursors of plasmacytoid dendritic cells (pre-pDCs) can polarize donor T cells toward Th1 immunity and augment the GVL activity of donor T cells while attenuating their GVHD activity in a murine model of allogeneic HSCT. Clinical data on the role of donor pre-pDCs and conventional DCs (cDCs) on transplantation outcomes has been conflicting. To test the effect of increasing the proportion of pre-pDCs versus cDCs in a BM graft, we enriched CD11b⁻ pDCs by selectively depleting the CD11b⁺ myeloid DC (mDC) population from BM using FACS sorting in a murine model of allogeneic BM transplantation. Donor T cell expansion and GVL activity were greater in mice that received BM depleted of mDCs compared with mice that received undepleted BM. GVHD was not increased by depleting mDCs. To examine the mechanism through which mDC depletion enhances the GVL activity of donor T cells, we used BM and pDCs from IL-12p40KO mice, and found that the increased GVL activity of mDC-depleted BM was IL-12–dependent. This study indicates that a clinically translatable strategy of engineering the DC content of grafts can improve clinical outcomes in allogeneic HSCT through the regulation of donor T cell activation and GVL activity.     

Differentiation of human dendritic cell subsets for immune tolerance induction

Objectives

Since no further progress was achieved, in order to improve the long-term organ transplantation outcome, the immune tolerance appears as an interesting therapeutic goal. Dendritic cells (DCs) are specialized cells participating in the homeostasis of the immune response. Moreover, subsets of DCs, identified in humans, appear to have their respective competences in immune response modulation. Our objective is to purify from PBMC or to differentiate DC subsets from monocytes using several strategies and evaluate their IL10 secretion.

Depletion of CD11c+ cells in the CD11c.DTR model drives expansion of unique CD64+ Ly6C+ monocytes that are poised to release TNF‐α

Dendritic cells (DCs) play a vital role in innate and adaptive immunities. Inducible depletion of CD11c⁺ DCs engineered to express a high‐affinity diphtheria toxin receptor has been a powerful tool to dissect DC function in vivo. However, despite reports showing that loss of DCs induces transient monocytosis, the monocyte population that emerges and the potential impact of monocytes on studies of DC function have not been investigated. We found that depletion of CD11c⁺ cells from CD11c.DTR mice induced the expansion of a variant CD64⁺ Ly6C⁺ monocyte population in the spleen and blood that was distinct from conventional monocytes. Expansion of CD64⁺ Ly6C⁺ monocytes was independent of mobilization from the BM via CCR2 but required the cytokine, G‐CSF. Indeed, this population was also expanded upon exposure to exogenous G‐CSF in the absence of DC depletion. CD64⁺ Ly6C⁺ monocytes were characterized by upregulation of innate signaling apparatus despite the absence of inflammation, and an increased capacity to produce TNF‐α following LPS stimulation. Thus, depletion of CD11c⁺ cells induces expansion of a unique CD64⁺ Ly6C⁺ monocyte population poised to synthesize TNF‐α. This finding will require consideration in experiments using depletion strategies to test the role of CD11c⁺ DCs in immunity.     

Signal regulatory protein alpha (SIRPα) regulates the homeostasis of CD103+CD11b+ DCs in the intestinal lamina propria

Signal regulatory protein alpha (SIRPα/CD172a) is a conserved transmembrane protein thought to play an inhibitory role in immune function by binding the ubiquitous ligand CD47. SIRPα expression has been used to identify dendritic cell subsets across species and here we examined its expression and function on intestinal DCs in mice. Normal mucosa contains four subsets of DCs based on their expression of CD103 and CD11b and three of these express SIRPα. However, loss of SIRPα signaling in mice leads to a selective reduction in the CD103⁺CD11b⁺ subset of DCs in the small intestine, colon, and among migratory DCs in the mesenteric lymph node. In parallel, these mice have reduced numbers of T_H17 cells in steady‐state intestinal mucosa, and a defective T_H17 response to Citrobacter infection. Identical results were obtained in CD47KO mice. DC precursors from SIRPα mutant mice had an enhanced ability to generate CD103⁺CD11b⁺ DCs in vivo, but CD103⁺CD11b⁺ DCs from mutant mice were more prone to die by apoptosis. These data show a previously unappreciated and crucial role for SIRPα in the homeostasis of CD103⁺CD11b⁺ DCs in the intestine, as well as providing further evidence that this subset of DCs is critical for the development of mucosal T_H17 responses.     

Intestinal CD103+CD11b− dendritic cells restrain colitis via IFN-γ-induced anti-inflammatory response in epithelial cells

A crosstalk between commensals, gut immune cells, and colonic epithelia is required for a proper function of intestinal mucosal barrier. Here we investigated the importance of two distinct intestinal dendritic cell (DC) subsets in controlling intestinal inflammation. We show that Clec9A–diphtheria toxin receptor (DTR) mice after depletion of CD103⁺CD11b⁻ DCs developed severe, low-dose dextran sodium sulfate (DSS)-induced colitis, whereas the lack of CD103⁺CD11b⁺ DCs in Clec4a4-DTR mice did not exacerbate intestinal inflammation. The CD103⁺CD11b⁻ DC subset has gained a functional specialization that able them to repress inflammation via several epithelial interferon-γ (IFN-γ)-induced proteins. Among others, we identified that epithelial IDO1 and interleukin-18-binding protein (IL-18bp) were strongly modulated by CD103⁺CD11b⁻ DCs. Through its preferential property to express IL-12 and IL-15, this particular DC subset can induce lymphocytes in colonic lamina propria and in epithelia to secrete IFN-γ that then can trigger a reversible early anti-inflammatory response in intestinal epithelial cells.     

Pattern recognition receptor expression and maturation profile of dendritic cell subtypes in human tonsils and lymph nodes

Dendritic cells (DCs) with capacity of antigen cross-presentation are of key interest for immunotherapy against cancer as they can induce antigen-specific cytotoxic T lymphocyte (CTL) responses. This study describes frequencies of DC subtypes in human tonsils and lymph nodes, and phenotypic aspects that may be targeted by adjuvant measures.From human tonsils and neck lymph nodes, DCs were identified through flow cytometry, and subsets of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were investigated. Maturity status was assessed and surface receptors with CTL-promoting potentials were studied.CD123⁺ pDCs as well as CD1c⁺, CD141⁺, and CD1c^-CD141^- mDCs were detected in tonsils and lymph nodes. Both sites featured a similar presence of DC subsets, with CD123⁺ pDC being dominant and CD141⁺ mDCs least frequent. Based on CD80/CD86 expression, all DC subtypes featured a low degree of maturation. Expression of pattern recognition receptors (PRRs) CD206, CD207, DC-SIGN, TLR2, and TLR4, as well as the chemokine receptor XCR1, indicated DC subset-specific receptor profiles.We conclude that tonsils and lymph nodes share common features in terms of DC subset frequency and maturation as well as PRR and XCR1 expression pattern. Our work suggests that both sites may be considered for vaccine deposition in DC-mediated immunotherapy.     

Mammalian target of rapamycin controls dendritic cell development downstream of Flt3 ligand signaling

Dendritic cells (DCs) comprise distinct functional subsets including CD8? and CD8(+) classical DCs (cDCs) and interferon-secreting plasmacytoid DCs (pDCs). The cytokine Flt3 ligand (Flt3L) controls the development of DCs and is particularly important for the pDC and CD8(+) cDC and their CD103(+) tissue counterparts. We report that mammalian target of rapamycin (mTOR) inhibitor rapamycin impaired Flt3L-driven DC development in vitro, with the pDCs and CD8(+)-like cDCs most profoundly affected. Conversely, deletion of the phosphoinositide 3-kinase (PI3K)-mTOR negative regulator Pten facilitated Flt3L-driven DC development in culture. DC-specific Pten targeting in vivo caused the expansion of CD8(+) and CD103(+) cDC numbers, which was reversible by rapamycin. The increased CD8(+) cDC numbers caused by Pten deletion correlated with increased susceptibility to the intracellular pathogen Listeria. Thus, PI3K-mTOR signaling downstream of Flt3L controls DC development, and its restriction by Pten ensures optimal DC pool size and subset composition.     

BDCA3 expression is associated with high IFN‐λ production by CD34+‐derived dendritic cells generated in the presence of GM‐CSF,IL‐4, and/or TGF‐β

High BDCA3 expression is associated with a specific human IFN‐λ‐producing dendritic cell (DC) subset. However, BDCA3 has also been detected on other DC subsets. Thus far, development and function of BDCA3 expression on DCs remains poorly understood. Human Langerhans cells (LCs) and interstitial DCs (intDCs) can be generated in vitro by differentiation of CD34⁺ hematopoietic progenitors via distinct precursor DCs (preDCs), CD1a⁺ preDCs, and CD14⁺ preDCs, respectively. Here, we identified BDCA3 expression in this well‐known GM‐CSF/TNF‐α‐driven culture system and described the effect of IL‐4 and/or TGF‐β on induction of BDCA3 expression. In control or TGF‐β cultures, BDCA3 was only detected on CD14⁺ preDC‐derived intDCs. IL‐4 induced BDCA3 expression in both CD14⁺‐derived and CD1a⁺‐derived cultures. TGF‐β and IL‐4 together further increased CD14⁺‐derived and CD1a⁺‐derived BDCA3⁺ DC frequencies, which partly expressed CLEC9A, but were not identical to the BDCA3^highCLEC9A⁺ DC subset in vivo. Importantly, BDCA3⁺ cells, but not BDCA3^? cells, in this system produced high IFN‐λ levels upon polyinosinic:polycytidylic acid (polyI:C) stimulation. This culture system, in which BDCA3 expression is preferentially associated with the intDC lineage and IFN‐λ‐producing capacity, will greatly contribute to further research on the function and regulation of BDCA3 expression and IFN‐λ production by DCs.