CD8alpha+ and CD11b+ dendritic cell-restricted MHC class II controls Th1 CD4+ T cell immunity

The activation, proliferation, differentiation, and trafficking of CD4 T cells is central to the development of type I immune responses. MHC class II (MHCII)-bearing dendritic cells (DCs) initiate CD4(+) T cell priming, but the relative contributions of other MHCII(+) APCs to the complete Th1 immune response is less clear. To address this question, we examined Th1 immunity in a mouse model in which I-A(beta)(b) expression was targeted specifically to the DCs of I-A(beta)b-/- mice. MHCII expression is reconstituted in CD11b(+) and CD8alpha(+) DCs, but other DC subtypes, macrophages, B cells, and parenchymal cells lack of expression of the I-A(beta)(b) chain. Presentation of both peptide and protein Ags by these DC subsets is sufficient for Th1 differentiation of Ag-specific CD4(+) T cells in vivo. Thus, Ag-specific CD4(+) T cells are primed to produce Th1 cytokines IL-2 and IFN-gamma. Additionally, proliferation, migration out of lymphoid organs, and the number of effector CD4(+) T cells are appropriately regulated. However, class II-negative B cells cannot receive help and Ag-specific IgG is not produced, confirming the critical MHCII requirement at this stage. These findings indicate that DCs are not only key initiators of the primary response, but provide all of the necessary cognate interactions to control CD4(+) T cell fate during the primary immune response.     

Local CD11c+ MHC class II- precursors generate lung dendritic cells during respiratory viral infection, but are depleted in the process

Increases in numbers of lung dendritic cells (DC) observed during respiratory viral infections are assumed to be due to recruitment from bone marrow precursors. No local production has been demonstrated. In this study, we isolated defined populations of murine lung cells based on CD11c and MHC class II (MHC II) expression. After culture for 12 days with GM-CSF, we analyzed cell numbers, DC surface markers, and Ag-presenting capacity. Only CD11c+ MHC II- cells from naive mice proliferated, yielding myeloid DC, which induced Ag-specific proliferation of naive T cells. After respiratory syncytial virus (RSV) infection, numbers of pulmonary CD11c+ MHC II- precursor cells were significantly reduced and DC could not be generated. Moreover, RSV infection prevented subsequent in vivo expansion of pulmonary DC in response to influenza infection or LPS treatment. These results provide direct evidence of local generation of fully functional myeloid DC in the lung from CD11c+ MHC II(-) precursor cells that are depleted by RSV infection, leading to an inability to expand lung DC numbers in response to subsequent viral infection or exposure to bacterial products. This depletion of local DC precursors in respiratory viral infections may be important in explaining complex interactions between multiple and intercurrent pulmonary infections.     

The immune response modifier and Toll-like receptor 7 agonist S-27609 selectively induces IL-12 and TNF-alpha production in CD11c+CD11b+CD8- dendritic cells

IL-12 and TNF-alpha production by dendritic cells (DCs) is a critical step in the initiation of local inflammation and adaptive immune responses. We show in this study that a small molecule immune response modifier that is a Toll-like receptor 7 (TLR7) agonist induces IL-12 and TNF-alpha production from murine CD11c(+)CD11b(+)CD8(-) DCs, a subset not previously known for this activity. Stimulation of these DCs through TLR7 in vivo induces significant cytokine production even 12 h after initial stimulation, as well as migration of the DC into T cell zones of the lymphoid tissue. In contrast, stimulation through TLR4 and TLR9 induced IL-12 production predominantly from CD8(+) DCs, consistent with previously published data. All TLR stimuli induced the increase in surface expression of the activation markers B7-1, B7-2, and class II in both CD8(+) and CD8(-) DCs, demonstrating that CD8(+) DCs do respond to TLR7-mediated stimuli. To date this is the only known stimuli to induce preferential cytokine production from CD8(-) DCs. Given the efficacy of TLR7 agonists as antiviral agents, the data collectively indicate that stimulation of CD8(-) DCs through TLR7 most likely plays a role in the generation of antiviral immune responses.     

CD4- plasmacytoid dendritic cells (pDCs) migrate in lymph nodes by CpG inoculation and represent a potent functional subset of pDCs

We have recently identified two groups of plasmacytoid dendritic cells (pDCs) isolated from murine liver based on the expression of CD4 and other cell surface markers uniquely expressed by pDCs. Herein, we describe the identification of both CD4+ and CD4- pDCs that clearly exist in lymph nodes (LNs), spleen, liver, thymus, bone marrow, and lung. Normally, CD4+ pDCs are enriched in LNs. However, after in vivo systemic injection with bacterial CpG, a larger number of CD4- pDCs are recruited to the LNs and local inoculation by CpG drives CD4- pDCs migrating into local sentinel LNs, suggesting that CD4- pDCs are the main subpopulation migrating to the peripheral LNs. Furthermore, although both freshly isolated CD4+ pDCs and CD4- pDCs appear as an immature plasmacytoid cell and develop into a DC morphology following activation, the two subsets have strikingly different immune features, including differences in the production pattern of cytokines stimulated with CpG and in T cell activation.     

The majority of immunogenic epitopes generate CD4+ T cells that are dependent on MHC class II-bound peptide-flanking residues

Peptides bind to MHC class II molecules with a defined periodicity such that the peptide-flanking residues (PFRs) P-1 and P11, which lie outside the core binding sequence (P1-P9), are solvent exposed and accessible to the TCR. Using a novel MHC class II:peptide binding assay, we defined the binding register for nine immunogenic epitopes to formally identify the flanking residues. Seven of the nine epitopes, restricted by H-2A(k), H-2A(g7), or H-2E(k), were found to generate T cells that were completely dependent on either P-1 or P11, with dependency on P-1 favored over P11. Such PFR dependency appears to be influenced by the type of amino acid exposed, in that residues that can form salt bridges or hydrogen bonds are favored over small or hydrophobic residues. Peptides containing alanine substitutions at P-1 or P11 in place of PFRs that mediate dependency were considerably less immunogenic and mediated a substantially reduced in vitro recall response to the native protein, inferring that PFR recognition increases immunogenicity. Our data suggest that PFR recognition is a common event characteristic of all MHC class II-restricted T cell responses. This key feature, which is not shared by MHC class I-restricted responses, may underlie the broad functional diversity displayed by MHC class II-restricted T cells.     

Activation of human plasmacytoid dendritic cells by TLR9 impairs Fc gammaRII-mediated uptake of immune complexes and presentation by MHC class II

Human plasmacytoid dendritic cells (pDCs)(2) exploit Ag uptake receptors like CD32a for internalization of exogenous Ags. Activation of pDC by TLR9 ligand CpG-C induces strong maturation. Surprisingly, we observed that CpG-C-stimulated pDCs showed impaired Ag-specific T cell proliferation whereas the induction of allogeneic T cell proliferation was not affected. We demonstrated that signals from TLR9 caused a rapid down-regulation of the capacity of pDC to take-up Ab-Ag complexes without altering their CD32a expression, thus explaining the reduced Ag presentation. The recent contrasting biological responses that were observed upon TLR9 ligation in pDCs prompted us to study the effect of several TLR9 ligands. We observed that type I IFN-inducer CpG-A, localizing in the early endosomal compartment, did not affect CD32a function, whereas CpGs localizing in the late endosomes and inducing pDC maturation clearly inhibited CD32a-mediated Ag uptake and presentation. We conclude that TLR9 ligands not only determine the type of response, i.e., type I IFN production (innate immunity) or maturation (adaptive immunity), but also directly affect Ag presentation capacity of pDCs. We hypothesize that pDC, once activated via TLR9-ligands reaching the late endosomes, can only present initially sampled Ags and thus are protected from uptake and processing of additional potential self-Ags.     

Compartmental imbalance and aberrant immune function of blood CD123+ (plasmacytoid) and CD11c+ (myeloid) dendritic cells in atopic dermatitis

Atopic dermatitis (AD) is a pruritic, chronically relapsing skin disease in which Th2 cells play a crucial role in cutaneous and extracutaneous immune reactions. In humans, CD11c+CD123- myeloid dendritic cells (mDC) and CD11c-CD123+ plasmacytoid DC (pDC) orchestrate the decision-making process in innate and acquired immunity. Since the number and function of these blood dendritic cell (DC) subsets reportedly reflect the host immune status, we studied the involvement of the DC subsets in the pathogenesis of AD. Patients with AD had an increased DC number and a low mDC:pDC ratio with pDC outnumbering mDC in the peripheral blood compared with normal subjects and psoriasis patients (a Th1 disease model group). The mDC:pDC ratio was correlated with the total serum IgE level, the ratio of IFN-gamma-producing blood cells:IL-4-producing blood cells, and the disease severity. In vitro allogeneic stimulation of naive CD4+ cells with atopic DC showed that the ability of pDC for Th1 induction was superior or comparable to that of mDC. In skin lesions, pDC infiltration was in close association with blood vessels expressing peripheral neural addressins. Therefore, compartmental imbalance and aberrant immune function of the blood DC subsets may deviate the Th1/Th2 differentiation and thus induce protracted allergic responses in AD.     

A CD91-positive subset of CD11c+ blood dendritic cells: characterization of the APC that functions to enhance adaptive immune responses against CD91-targeted antigens

Dendritic cells (DC) and other APCs rely on a number of specialized receptors to facilitate the uptake and intracellular accumulation of Ags. In this capacity, APCs use receptor-mediated endocytosis to enhance Ag presentation and the stimulation of Ag-specific T cells. Studies have demonstrated that the targeted delivery of Ags in vivo to CD91/the low-density lipoprotein receptor-related protein (CD91/LRP) induces enhanced activation of the adaptive immune system. However, the APC that mediates these augmented, Ag-specific responses remains to be characterized. In this study, we show that a subset of CD11c(+) lineage-negative (lin(-)) DC expresses the scavenger receptor CD91/LRP and that these rare APC are primarily responsible for the T cell activation that occurs following CD91/LRP-mediated Ag uptake in whole blood. The targeting of Ags to CD91/LRP results in enhanced receptor-mediated uptake within both lin(-) DCs and monocytes, and this uptake results in markedly increased T cell activation. Finally, purified cellular populations were used to demonstrate that CD11c(+) lin(-) DC, but not monocytes, are capable of stimulating T cell activation following CD91/LRP-mediated Ag uptake. Therefore, CD11c(+) lin(-) DC use CD91/LRP to facilitate the uptake and subsequent presentation of an array of Ags complexed within the CD91/LRP ligand, the activated form of alpha2-macroglobulin (alpha2M*).     

CD8alpha+ dendritic cells selectively present MHC class I-restricted noncytolytic viral and intracellular bacterial antigens in vivo

CD8alpha(+) dendritic cells (DCs) have been shown to be the principal DC subset involved in priming MHC class I-restricted CTL immunity to a variety of cytolytic viruses, including HSV type 1, influenza, and vaccinia virus. Whether priming of CTLs by CD8alpha(+) DCs is limited to cytolytic viruses, which may provide dead cellular material for this DC subset, or whether these DCs selectively present intracellular Ags, is unknown. To address this question, we examined Ag presentation to a noncytolytic virus, lymphocytic choriomeningitis virus, and to an intracellular bacterium, Listeria monocytogenes. We show that regardless of the type of intracellular infection, CD8alpha(+) DCs are the principal DC subset that initiate CD8(+) T cell immunity.     

CD36 or alphavbeta3 and alphavbeta5 integrins are not essential for MHC class I cross-presentation of cell-associated antigen by CD8 alpha+ murine dendritic cells

Cross-presentation of cell-associated Ag is thought to involve receptor-mediated uptake of apoptotic cells by dendritic cells (DC), and studies with human DC strongly implicate the endocytic receptor CD36 and the integrins alpha(v)beta(3) and/or alpha(v)beta(5) in this process. In the mouse, cross-presentation was recently shown to be a function of CD8alpha(+) DC. Here we report that CD36 is expressed on CD8alpha(+), but not on CD8alpha(-), DC. To address the role of CD36 in cross-presentation we compared CD36(-/-) and CD36(+/+) H-2(b) DC for their ability to stimulate naive OT-1 T cells specific for OVA plus H-2K(b) in the presence of OVA-loaded MHC-mismatched splenocytes as a source of cell-associated Ag for cross-presentation. Surprisingly, no difference was seen between CD36(-/-) and CD36(+/+) CD8alpha(+) DC in their ability to cross-present cell-associated OVA or to capture OVA-bearing cells. Furthermore, the proliferation of CFSE-labeled OT-1 cells in response to OVA cross-presentation in vivo was normal in CD36(-/-) bone marrow chimeras, also arguing against a necessary role for CD36 in cross-presentation by DC or other APC. DC doubly deficient for beta(3) and beta(5) integrins were similarly unimpaired in their ability to cross-present OVA-bearing cells in vitro. These data demonstrate that in the mouse, receptors other than CD36 or beta(3) and beta(5) integrins can support the specialized cross-presenting function of CD8alpha(+) DC.     

Distinct CpG DNA and polyinosinic-polycytidylic acid double-stranded RNA, respectively, stimulate CD11c- type 2 dendritic cell precursors and CD11c+ dendritic cells to produce type I IFN

Two classes of nucleic acids, bacterial DNA containing unmethylated CpG motifs and dsRNA in viruses, induce the production of type I IFN that contributes to the immunostimulatory effects of these microbial molecules. Thus, it is important to determine which cells produce type I IFN in response to CpG DNA and dsRNA. CD4(+)CD11c(-) type 2 dendritic cell precursors (pre-DC2) were identified as the main producers of type I IFN in human blood in response to viruses. Here we asked whether pre-DC2 also produce type I IFN in response to CpG DNA and dsRNA. Oligodeoxynucleotides containing particular palindromic CpG motifs induced pre-DC2, but not CD11c(+) blood DC or monocytes, to produce IFN-alpha. In contrast, a synthetic dsRNA, polyinosinic polycytidylic-acid, induced CD11c(+) DC, but not pre-DC2 or monocytes, to produce IFN-alphabeta. These data indicate that CpG DNA and polyinosinic-polycytidylic acid stimulate different types of cells to produce type I IFN and that it is important to select oligodeoxynucleotides containing particular CpG motifs to induce pre-DC2 to produce type I IFN, which may play a key role in the strong adjuvant effects of CpG DNA.     

CD4+CD25- T cells transduced to express MHC class I-restricted epitope-specific TCR synthesize Th1 cytokines and exhibit MHC class I-restricted cytolytic effector function in a human melanoma model

Cytolytic T cell-centric active specific and adoptive immunotherapeutic approaches might benefit from the simultaneous engagement of CD4(+) T cells. Considering the difficulties in simultaneously engaging CD4(+) and CD8(+) T cells in tumor immunotherapy, especially in an Ag-specific manner, redirecting CD4(+) T cells to MHC class I-restricted epitopes through engineered expression of MHC class I-restricted epitope-specific TCRs in CD4(+) T cells has emerged as a strategic consideration. Such TCR-engineered CD4(+) T cells have been shown to be capable of synthesizing cytokines as well as lysing target cells. We have conducted a critical examination of functional characteristics of CD4(+) T cells engineered to express the alpha- and beta-chains of a high functional avidity TCR specific for the melanoma epitope, MART-1(27-35), as a prototypic human tumor Ag system. We found that unpolarized CD4(+)CD25(-) T cells engineered to express the MART-1(27-35) TCR selectively synthesize Th1 cytokines and exhibit a potent Ag-specific lytic granule exocytosis-mediated cytolytic effector function of comparable efficacy to that of CD8(+) CTL. Such TCR engineered CD4(+) T cells, therefore, might be useful in clinical immunotherapy.     

Survival, maturation, and function of CD11c- and CD11c+ peripheral blood dendritic cells are differentially regulated by cytokines.

Two types of dendritic cells (DC) are circulating in human blood and can be identified by their differential expression of the myeloid Ag CD11c. In this study, we show that CD11c- peripheral blood (PB)-DC correspond to plasmacytoid DC of lymphoid tissue not only by their surface Ag expression profile but, more impressively, by their peculiar ultramorphology. We also demonstrate that CD11c- and CD11c+ DC differ in the quality of their response to and in their requirement for certain cytokines. Freshly isolated CD11c- cells depend on IL-3 for survival and use autocrine or exogenous TNF-alpha as maturation signal, leading to the appearance of a highly dendritic phenotype, the up-regulation and redistribution of MHC class II from lysosomal compartments to the plasma membrane, the increased expression of costimulatory molecules, and the switch from a high Ag-processing to a low Ag-processing/potent accessory cell mode. Surprisingly, IL-4 efficiently killed freshly isolated CD11c- PB-DC, but did not impair the viability of CD11c+ PB-DC and, together with GM-CSF, induced maturation of these cells. A direct functional comparison revealed that neo-Ag-modified and subsequently matured CD11c- but to a lesser extent CD11c+ DC were able to prime naive Ag-specific CD4+ T cells. Our findings show that two diverse DC types respond to certain T cell-derived cytokines in a differential manner and, thus, suggest that suppression or activation of functionally diverse DC types may be a novel mechanism for the regulation of the quantity and quality of immune responses.     

Expansion and function of CD8+ T cells expressing Ly49 inhibitory receptors specific for MHC class I molecules

MHC class I-specific Ly49 inhibitory receptors regulate NK cell activation, thereby preventing autologous damage to normal cells. Ly49 receptors are also expressed on a subset of CD8+ T cells whose origin and function remain unknown. We report here that, despite their phenotypic and cytolytic similarities, Ly49+CD8+ T cells and conventional Ly49-CD44high memory-phenotype CD8+ T cells present strikingly distinct features. First, under steady state conditions Ly49+CD8+ T cells are poor cytokine producers (TNF-alpha and IFN-gamma) upon TCR triggering. Second, Ly49+CD8+ T cells are not induced upon various settings of Ag immunization or microbial challenge. However, Ly49 can be induced on a fraction of self-specific CD8+ T cells if CD4+ T cells are present. Finally, the size of the Ly49+CD8+ T cell subset is selectively reduced in the absence of STAT1. These results indicate that Ly49 expression is associated with a differentiation program of cytolytic CD8+ T cells triggered upon chronic antigenic exposure. They further suggest that the size of the Ly49+CD8+ T cell subset marks a history of CD8+ T cell activation that might preferentially result from endogenous inducers of inflammation rather than from microbial infections.     

BCG vaccine mediated reduction in the MHC-II expression of macrophages and dendritic cells is reversed by activation of Toll-like receptors 7 and 9

Tuberculosis is a major cause of death in mankind and BCG vaccine protects against childhood but not adult tuberculosis. BCG avoids lysosomal fusion in macrophages decreasing peptides required for activating CD4 T cells and Th1 immunity while suppressing the expression of MHC-II by antigen presenting cells (APCs). An in vitro model of antigen presentation showed that ligands for TLR-9, 7, 4 and 1/2 increased the ability of APCs to present antigen-85B of BCG to CD4 T cells, which correlated with an increase in MHC-II expression. TLR-activation led to a down-regulation of MARCH1 ubiquitin ligase which prevents the degradation of MHC-II and decreased IL-10 also contributed to an increase in MHC-II. TLR-activation induced up-regulation of MHC-II was inhibited by the blockade of IRAK, NF-kB, and MAPKs. TLR-7 and TLR-9 ligands had the most effective adjuvant like effect on MHC-II of APCs which allowed BCG vaccine mediated activation of CD4 T cells.     

Coordinated expression of Ig-like inhibitory MHC class I receptors and acquisition of cytotoxic function in human CD8+ T cells

MHC class I-specific inhibitory receptors are expressed by a subset of memory-phenotype CD8(+) T cells. Similar to NK cells, MHC class I-specific inhibitory receptors might subserve on T cells an important negative control that participates to the prevention of autologous damage. We analyzed here human CD8(+) T cells that express the Ig-like MHC class I-specific inhibitory receptors: killer cell Ig-like receptor (KIR) and CD85j. The cell surface expression of Ig-like inhibitory MHC class I receptors was found to correlate with an advanced stage of CD8(+) T cell maturation as evidenced by the reduced proliferative potential of KIR(+) and CD85j(+) T cells associated with their high intracytoplasmic perforin content. This concomitant regulation might represent a safety mechanism to control potentially harmful cytolytic CD8(+) T cells, by raising their activation threshold. Yet, KIR(+) and CD85j(+) T cells present distinct features. KIR(+)CD8(+) T cells are poor IFN-gamma producers upon TCR engagement. In addition, KIR are barely detectable at the surface of virus-specific T cells during the course of CMV or HIV-1 infection. By contrast, CD85j(+)CD8(+) T cells produce IFN-gamma upon TCR triggering, and represent a large fraction of virus-specific T cells. Thus, the cell surface expression of Ig-like inhibitory MHC class I receptors is associated with T cell engagement into various stages of the cytolytic differentiation pathway, and the cell surface expression of CD85j or KIR witnesses to the history of qualitatively and/or quantitatively distinct T cell activation events.     

CCL9 is secreted by the follicle-associated epithelium and recruits dome region Peyer's patch CD11b+ dendritic cells

The follicle-associated epithelium (FAE) secretes chemokines important in the recruitment of various cell types including CCL20 (MIP-3alpha). CCL20 is chemotactic to the CD11b(+) dendritic cells (DCs) distributed in the subepithelial dome regions of the Peyer's patches, and mice deficient in the receptor for CCL20, CCR6, have been reported to be devoid of the CD11b(+) DCs in the dome regions. Here, we describe another chemokine specifically secreted from the FAE of mouse Peyer's patches, CCL9 (MIP-1gamma, CCF18, MRP-2). By in situ hybridization, we demonstrated that CCL9 mRNA was expressed by the FAE but not by the villus epithelium. At the protein level, CCL9 was detected on the FAE and on extracellular matrix structures within the dome regions of the Peyer's patches. By RT-PCR, we demonstrated that one of the putative receptors for CCL9, CCR1, was expressed by the Peyer's patch CD11b(+) DCs and in a chemotaxis assay, CD11b(+) DCs migrated toward CCL9. To compare the abilities of the chemokines CCL20 and CCL9 to recruit CD11b(+) DCs to the dome regions, we examined the in vivo distribution of these cells in CCR6-deficient, CCL9-blocked wild type, or CCL9-blocked CCR6-deficient mice. To our surprise, using a sensitive immunofluorescence analysis, we observed that CD11b(+) DCs were present in the dome regions of the CCR6-deficient mice. In contrast, Ab neutralization of CCL9 in vivo resulted in significant reduction of the CD11b(+) DC number in the subepithelial dome regions of Peyer's patches of both wild type and CCR6 -/- mice. Taken together, these results demonstrate an important role of CCL9 in CD11b(+) DC recruitment to the dome regions of mouse Peyer's patches.     

Lung CD103+ dendritic cells efficiently transport influenza virus to the lymph node and load viral antigen onto MHC class I for presentation to CD8 T cells

The uptake, transport, and presentation of Ags by lung dendritic cells (DCs) are central to the initiation of CD8 T cell responses against respiratory viruses. Although several studies have demonstrated a critical role of CD11b(low/neg)CD103(+) DCs for the initiation of cytotoxic T cell responses against the influenza virus, the underlying mechanisms for its potent ability to prime CD8 T cells remain poorly understood. Using a novel approach of fluorescent lipophilic dye-labeled influenza virus, we demonstrate that CD11b(low/neg)CD103(+) DCs are the dominant lung DC population transporting influenza virus to the posterior mediastinal lymph node as early as 20 h postinfection. By contrast, CD11b(high)CD103(neg) DCs, although more efficient for taking up the virus within the lung, migrate poorly to the lymph node and remain in the lung to produce proinflammatory cytokines instead. CD11b(low/neg)CD103(+) DCs efficiently load viral peptide onto MHC class I complexes and therefore uniquely possess the capacity to potently induce proliferation of naive CD8 T cells. In addition, the peptide transporters TAP1 and TAP2 are constitutively expressed at higher levels in CD11b(low/neg)CD103(+) DCs, providing, to our knowledge, the first evidence of a distinct regulation of the Ag-processing pathway in these cells. Collectively, these results show that CD11b(low/neg)CD103(+) DCs are functionally specialized for the transport of Ag from the lung to the lymph node and also for efficient processing and presentation of viral Ags to CD8 T cells.