Newcastle disease virus expressing a dendritic cell-targeted HIV gag protein induces a potent gag-specific immune response in mice

Viral vaccine vectors have emerged as an attractive strategy for the development of a human immunodeficiency virus (HIV) vaccine. Recombinant Newcastle disease virus (rNDV) stands out as a vaccine vector since it has a proven safety profile in humans, it is a potent inducer of both alpha interferon (IFN-α) and IFN-β) production, and it is a potent inducer of dendritic cell (DC) maturation. Our group has previously generated an rNDV vector expressing a codon-optimized HIV Gag protein and demonstrated its ability to induce a Gag-specific CD8(+) T cell response in mice. In this report we demonstrate that the Gag-specific immune response can be further enhanced by the targeting of the rNDV-encoded HIV Gag antigen to DCs. Targeting of the HIV Gag antigen was achieved by the addition of a single-chain Fv (scFv) antibody specific for the DC-restricted antigen uptake receptor DEC205 such that the DEC205 scFv-Gag molecule was encoded for expression as a fusion protein. The vaccination of mice with rNDV coding for the DC-targeted Gag antigen induced an enhanced Gag-specific CD8(+) T cell response and enhanced numbers of CD4(+) T cells and CD8(+) T cells in the spleen relative to vaccination with rNDV coding for a nontargeted Gag antigen. Importantly, mice vaccinated with the DEC205-targeted vaccine were better protected from challenge with a recombinant vaccinia virus expressing the HIV Gag protein. Here we demonstrate that the targeting of the HIV Gag antigen to DCs via the DEC205 receptor enhances the ability of an rNDV vector to induce a potent antigen-specific immune response.     

一种新型的重组单链三特异抗体的构建与表达

双特异抗体由于其缺乏共刺激信号 ,激活的T细胞往往会导致凋亡。为了更有效地杀伤肿瘤细胞 ,构建了抗卵巢癌单链×抗CD3单链×抗CD2 8单域重组三特异抗体的表达载体。利用大肠杆菌中的分子伴侣FkpA与三特异抗体共表达 ,提高了三特异抗体的在BL2 1Star菌中的可溶性原核表达。ELISA结果显示 ,可溶性的重组单链三特异抗体 (sTRI)与SK OV 3细胞的膜抗原 ,Jurkat细胞上的CD3分子以及重组CD2 8抗原均有较强的结合活性。桥连试验证明该抗体能同时与SK OV 3细胞和Jurkat细胞结合。体外杀伤试验表明该抗体能激活外周血T细胞来杀伤肿瘤细胞。形态学观察也进一步说明该抗体具有良好的结合活性以及体外杀伤活性。这种新型的重组单链三特异抗体为基于T细胞的癌症免疫治疗建立了一个新的技术平台。     

Notch-ligand expression by NALT dendritic cells regulates mucosal Th1- and Th2-type responses

Our previous studies showed that an adenovirus (Ad) serotype 5 vector expressing Flt3 ligand (Ad-FL) as nasal adjuvant activates CD11c(+) dendritic cells (DCs) for the enhancement of antigen (Ag)-specific IgA antibody (Ab) responses. In this study, we examined the molecular mechanism for activation of CD11c(+) DCs and their roles in induction of Ag-specific Th1- and Th2-cell responses. Ad-FL activated CD11c(+) DCs expressed increased levels of the Notch ligand (L)-expression and specific mRNA. When CD11c(+) DCs from various mucosal and systemic lymphoid tissues of mice given nasal OVA plus Ad-FL were cultured with CD4(+) T cells isolated from non-immunized OVA TCR-transgenic (OT II) mice, significantly increased levels of T cell proliferative responses were noted. Furthermore, Ad-FL activated DCs induced IFN-γ, IL-2 and IL-4 producing CD4(+) T cells. Of importance, these APC functions by Ad-FL activated DCs were down-regulated by blocking Notch-Notch-L pathway. These results show that Ad-FL induces CD11c(+) DCs to the express Notch-ligands and these activated DCs regulate the induction of Ag-specific Th1- and Th2-type cytokine responses.     

CD8+ CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells

Foxp3(+)CD25(+)CD4(+) regulatory T cells (Treg) mediate immunological self-tolerance and suppress immune responses. A subset of dendritic cells (DCs) in the intestine is specialized to induce Treg in a TGF-beta- and retinoic acid-dependent manner to allow for oral tolerance. In this study we compare two major DC subsets from mouse spleen. We find that CD8(+) DEC-205/CD205(+) DCs, but not the major fraction of CD8(-) DC inhibitory receptor-2 (DCIR2)(+) DCs, induce functional Foxp3(+) Treg from Foxp3(-) precursors in the presence of low doses of Ag but without added TGF-beta. CD8(+)CD205(+) DCs preferentially express TGF-beta, and the induction of Treg by these DCs in vitro is blocked by neutralizing Ab to TGF-beta. In contrast, CD8(-)DCIR2(+) DCs better induce Foxp3(+) Treg when exogenous TGF-beta is supplied. In vivo, CD8(+)CD205(+) DCs likewise preferentially induce Treg from adoptively transferred, Ag-specific DO11.10 RAG(-/-) Foxp3(-)CD4(+) T cells, whereas the CD8(-)DCIR2(+) DCs better stimulate natural Foxp3(+) Treg. These results indicate that a subset of DCs in spleen, a systemic lymphoid organ, is specialized to differentiate peripheral Foxp3(+) Treg, in part through the endogenous formation of TGF-beta. Targeting of Ag to these DCs might be useful for inducing Ag-specific Foxp3(+) Treg for treatment of autoimmune diseases, transplant rejection, and allergy.     

Identification and immunocytochemical analysis of DCNP1, a dendritic cell-associated nuclear protein.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs). Among so-called professional APCs, only DCs can activate naive T cells to initiate immune response. To better understand molecular mechanisms underlying unique functions of DCs, we searched for genes specifically expressed in human DCs, using PCR-based cDNA subtraction in conjunction with differential screening. cDNAs generated from CD34(+) stem cell-derived CD1a(+) DC were subtracted with cDNA from monocytes and used for generation of a cDNA library. The cDNA library was differentially screened to select genes expressed in DCs more abundantly than in monocytes. We identified a gene encoding a protein composed of 244 amino acids, which we designated as DCNP1 (dendritic cell nuclear protein 1). In Northern blot analysis, DCNP1 mRNA was highly expressed in mature DCs and at a lower level in immature DCs. In contrast, monocytes and B cells do not express the gene. In multiple human tissue Northern blot analysis, expression of DCNP1 was detected in brain and skeletal muscle. To examine subcellular localization of DCNP1, we performed immunofluorescence analysis using an anti-DCNP1 polyclonal antibody and found the molecule to be localized mainly in the perinucleus. In an immunohistochemical analysis, we compared the expression of DCNP1 with CD68, a marker for DCs and macrophages, in spleen, lymph node, liver, and brain. While DCNP1-positive cells showed a similar tissue distribution to CD68-positive cells, the number of DCNP1-positive cells was much smaller than that of CD68-positive cells. Our findings are consistent with the proposal that DCNP1 is specifically expressed in DCs.     

Interplay between CD8α+ dendritic cells and monocytes in response to Listeria monocytogenes infection attenuates T cell responses

During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α(+) dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8(+) T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α(+) DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α(+) DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b(+) DCs primarily secrete low levels of TNFα while CD8α(+) DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α(+) DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming.     

Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus.

HNK20 is a mouse monoclonal IgA that binds to the F glycoprotein of respiratory syncytial virus (RSV) and neutralizes the virus, both in vitro and in vivo. The single-chain antibody fragment (scFv) derived from HNK20 is equally active and has allowed us to assess rapidly the effect of mutations on affinity and antiviral activity. Humanization by variable domain resurfacing requires that surface residues not normally found in a human Fv be mutated to the expected human amino acid, thereby eliminating potentially immunogenic sites. We describe the construction and characterization of two humanized scFvs, hu7 and hu10, bearing 7 and 10 mutations, respectively. Both molecules show unaltered binding affinities to the RSV antigen (purified F protein) as determined by ELISA and surface plasmon resonance measurements of binding kinetics (Ka approximately 1x10(9) M-1). A competition ELISA using captured whole virus confirmed that the binding affinities of the parental scFv and also of hu7 and hu10 scFvs were identical. However, when compared with the original scFv, hu10 scFv was shown to have significantly decreased antiviral activity both in vitro and in a mouse model. Our observations suggest that binding of the scFv to the viral antigen is not sufficient for neutralization. We speculate that neutralization may involve the inhibition or induction of conformational changes in the bound antigen, thereby interfering with the F protein-mediated fusion of virus and cell membranes in the initial steps of infection.     

Secretory IgA mediates bacterial translocation to dendritic cells in mouse Peyer's patches with restriction to mucosal compartment

In addition to fulfilling its function of immune exclusion at mucosal surfaces, secretory IgA (SIgA) Ab exhibits the striking feature to adhere selectively to M cells in the mouse and human intestinal Peyer's patches (PPs). Subsequent uptake drives the SIgA Ab to dendritic cells (DCs), which become partially activated. Using freshly isolated mouse DCs, we found that the interaction with SIgA was tissue and DC subtype dependent. Only DCs isolated from PPs and mesenteric lymph nodes interacted with the Ab. CD11c(+)CD11b(+) DCs internalized SIgA, while CD11c(+)CD19(+) DCs only bound SIgA on their surface, and no interaction occurred with CD11c(+)CD8alpha(+) DCs. We next examined whether SIgA could deliver a sizeable cargo to PP DCs in vivo by administering SIgA-Shigella flexneri immune complexes into a mouse ligated intestinal loop containing a PP. We found that such immune complexes entered the PPs and were internalized by subepithelial dome PP DCs, in contrast to S. flexneri alone that did not penetrate the intestinal epithelium in mice. Dissemination of intraepithelial S. flexneri delivered as immune complexes was limited to PPs and mesenteric lymph nodes. We propose that preexisting SIgA Abs associated with microbes contribute to mucosal defense by eliciting responses that prevent overreaction while maintaining productive immunity.     

Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display

The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces in situ, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers.     

Epidermal Cadm1 expression promotes autoimmune alopecia via enhanced T cell adhesion and cytotoxicity

Autoimmune alopecia is characterized by an extensive epidermal T cell infiltrate that mediates hair follicle destruction. We have investigated the role of cell adhesion molecule 1 (Cadm1; Necl2) in this disease. Cadm1 is expressed by epidermal cells and mediates heterotypic adhesion to lymphocytes expressing class 1-restricted T cell-associated molecule (CRTAM). Using a murine autoimmune alopecia model, we observed an increase in early-activated cytotoxic (CD8-restricted, CRTAM-expressing) T cells, which preferentially associated with hair follicle keratinocytes expressing Cadm1. Coculture with Cadm1-transduced MHC-matched APCs stimulated alopecic lymph node cells to release IL-2 and IFN-γ. Overexpression of Cadm1 in cultured human keratinocytes did not promote cytokine secretion, but led to increased adhesion of alopecic cytotoxic T cells and enhanced T cell cytotoxicity in an MHC-independent manner. Epidermal overexpression of Cadm1 in transgenic mice led to increased autoimmune alopecia susceptibility relative to nontransgenic littermate controls. Our findings reveal that Cadm1 expression in the hair follicle plays a role in autoimmune alopecia.     

1F7, a novel cell surface molecule, involved in helper function of CD4 cells

We have developed a monoclonal antibody, anti-1F7, that inhibits soluble Ag-driven T cell proliferation as well as PWM-driven IgG synthesis. Anti-1F7 antibody reacts with approximately 57% of unfractionated T cells, 62% of CD4+ cells, and 54% of CD8+ cells. Although the 1F7 Ag is widely distributed among lymphoid cells, this Ag on CD4+ cells is preferentially expressed on the CDw29(4B4+) helper population. Moreover, anti-1F7 antibody further subdivides the CD4+CDw29+ cell subset into CDw29+1F7+ and CDw29+1F7- populations. The CD4+CDw29+1F7+ population of cells maximally proliferates to recall Ag such as tetanus toxoid, whereas helper function for PWM-driven IgG synthesis by B cells belongs to both the CD4+CDw29+1F7+ and CD4+CDw29+1F7- population of cells. The most prominent structure defined by this antibody is a 110-kDa molecule that is different from the 135-kDa, 160-kDa, and 185-kDa glycoproteins identified by anti-CDw29 antibody and the 180-kDa glycoprotein identified by UCHL-1 antibody. It is, however, related to the molecule recognized by anti-Ta1, an activation Ag on T cells. Furthermore, although the Ta1 molecule is recognized by anti-1F7 mAb, the 1F7 family of structures also includes molecules distinct from Ta1.     

Relay of Herpes Simplex Virus between Langerhans Cells and Dermal Dendritic Cells in Human Skin

The mechanism by which immunity to Herpes Simplex Virus (HSV) is initiated is not completely defined. HSV initially infects mucosal epidermis prior to entering nerve endings. In mice, epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to encounter HSV, but it is CD103⁺ dermal DCs that carry viral antigen to lymph nodes for antigen presentation, suggesting DC cross-talk in skin. In this study, we compared topically HSV-1 infected human foreskin explants with biopsies of initial human genital herpes lesions to show LCs are initially infected then emigrate into the dermis. Here, LCs bearing markers of maturation and apoptosis formed large cell clusters with BDCA3⁺ dermal DCs (thought to be equivalent to murine CD103⁺ dermal DCs) and DC-SIGN⁺ DCs/macrophages. HSV-expressing LC fragments were observed inside the dermal DCs/macrophages and the BDCA3⁺ dermal DCs had up-regulated a damaged cell uptake receptor CLEC9A. No other infected epidermal cells interacted with dermal DCs. Correspondingly, LCs isolated from human skin and infected with HSV-1 in vitro also underwent apoptosis and were taken up by similarly isolated BDCA3⁺ dermal DCs and DC-SIGN⁺ cells. Thus, we conclude a viral antigen relay takes place where HSV infected LCs undergo apoptosis and are taken up by dermal DCs for subsequent antigen presentation. This provides a rationale for targeting these cells with mucosal or perhaps intradermal HSV immunization.     

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.     

Production and characterization of a CD25-specific scFv-Fc antibody secreted from Pichia pastoris

Antibodies against CD25 would be novel tools for the diagnosis and treatment of adult T cell leukemia lymphoma (ATLL) and many other immune disorders. In our previous work, we successfully produced the single-chain fragment of a variable antibody against CD25, the Dmab(scFv) antibody, using Pichia pastoris. Here, we describe a novel form of an antibody against CD25, the Dmab(scFv)-Fc antibody, also produced by P. pastoris. To construct the Dmab(scFv)-Fc antibody, the Dmab(scFv) antibody was genetically fused to the Fc fragment of a human IgG1 antibody. A fusion gene encoding Dmab(scFv)-Fc antibody was cloned into the pPIC9K plasmid and expressed at high levels, 60–70 mg/l, by P. pastoris under optimized conditions. The Dmab(scFv)-Fc antibody was similar to the Dmab(scFv) antibody in its binding specificity but different in its molecular form and Fc-mediated effector functions. The Dmab(scFv)-Fc antibody and the Dmab(scFv) antibody both bound to CD25-positive MJ cells but not to CD25-negative K562 cells. The Dmab(scFv)-Fc antibody existed as a dimer whereas the Dmab(scFv) antibody was a monomer because it lacks the Fc fragment. The Dmab(scFv)-Fc antibody enhanced the antibody-dependent cellular cytotoxicity of CD25-positive cancer cells, whereas the Dmab(scFv) antibody was inactive in the antibody-dependent cellular cytotoxicity assays. In addition, compared to the Dmab(scFv) antibody, the Dmab(scFv)-Fc antibody showed stronger immunosuppressive activity in the Con A-stimulated lymphocyte proliferation system and in the mixed lymphocyte reaction system. These results demonstrate that the Dmab(scFv)-Fc antibody produced in P. pastoris is functional, and therefore it might be developed as a novel diagnostic and therapeutic tool for ATLL and other immune disorders.     

Obligate multivalent recognition of cell surface tomoregulin following selection from a multivalent phage antibody library

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.     

TCR complex-activated CD8 adhesion function by human T cells

The CD8 receptor plays a central role in the recognition and elimination of virally infected and malignant cells by cytolytic CD8(+) T cells. In conjunction with the TCR, the CD8 coreceptor binds Ag-specific class I MHC (MHC-I) molecules expressed by target cells, initiating signaling events that result in T cell activation. Whether CD8 can further function as an adhesion molecule for non-Ag MHC-I is currently unclear in humans. In this study, we show that in human CD8(+) T cells, TCR complex signaling activates CD8 adhesion molecule function, resulting in a CD8 interaction with MHC-I that is sufficient to maintain firm T cell adhesion under shear conditions. Secondly, we found that while CD8 adhesive function was triggered by TCR complex activation in differentiated cells, including in vitro generated CTL and ex vivo effector/memory phenotype CD8(+) T cells, naive CD8(+) T cells were incapable of activated CD8 adhesion. Lastly, we examine the kinetics of, and signaling for, activated CD8 adhesion in humans and identify notable differences from the equivalent CD8 function in mouse. Activated CD8 adhesion induced by TCR signaling may contribute to the more rapid and robust elimination of pathogen-infected cells by differentiated CD8(+) T cells.     

Characterization of myeloid and plasmacytoid dendritic cells in human lung

Dendritic cells (DCs) are bone marrow-derived mononuclear cells that play a central role in the initiation of immune responses. Because human lung DCs have been incompletely characterized, we enumerated and phenotyped mononuclear cell populations from excess lung tissue obtained at surgery. Myeloid DCs (MDCs) were identified as CD1c(+)CD11c(+)CD14(-)HLA-DR(+) cells and comprised approximately 2% of low autofluorescent (LAF) mononuclear cells. Plasmacytoid DCs (PDCs) were characterized as CD123(+)CD11c(-)CD14(-)HLA-DR(+) cells and comprised approximately 1.0% of the LAF mononuclear cells. Cells enriched in MDCs expressed CD86, moderate CD80, and little CD40, but cells enriched in PDCs had little to no expression of these three costimulatory molecules. CD11c(+)CD14(-) lineage-negative (MDC-enriched) LAF cells were isolated and shown to be much more potent in stimulating an alloreaction than CD11c(+)CD14(+) lineage-negative (monocyte-enriched) LAF cells. PDC-enriched cells were more capable of responding to a TLR-7 agonist by secreting IFN-alpha than MDC-enriched cells. MDC-enriched cells were either CD123(+) or CD123(-), but both subsets secreted cytokines and chemokines typical of MDC upon stimulation with a TLR-4 agonist and both subsets failed to secrete IFN-alpha upon stimulation with a TLR-7 agonist. By immunohistochemistry, we identified MDCs throughout different anatomical locations of the lung. However, our method did not allow the localization of PDCs with certainty. In conclusion, in the human lung MDCs were twice as numerous and expressed higher levels of costimulatory molecules than PDCs. Our data suggest that both lung DC subsets exert distinct immune modulatory functions.     

Effect of plasma viremia on apoptosis and immunophenotype of dendritic cells subsets in acute SIVmac239 infection of Chinese rhesus macaques

Non-human primates such as Chinese rhesus macaques (Ch Rhs) provide good animal models for research on human infectious diseases. Similar to humans, there are two principal subsets of dendritic cells (DCs) in the peripheral blood of Ch Rhs: myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). In this study, two-color fluorescence-activated cell sorting (FACS) analyses were used to identify the main DC subsets, namely CD1c(+) mDCs and pDCs from Ch Rhs. Then, the apoptosis and immunophenotype changes of DCs subsets were first described during the acute phase of SIVmac239 infection. Both the DCs subsets showed decreased CD4 expression and enhanced CCR5 expression; in particular, those of pDCs significantly changed at most time points. Interestingly, the plasma viral loads were negatively correlated with CD4 expression, but were positively correlated with CCR5 expression of pDCs. During this period, both CD1c(+) mDCs and pDCs were activated by enhancing expressions of co-stimulatory molecules, accompanied with increase in CCR7. Either CD80 or CD86 expressed on CD1c(+) mDCs and pDCs was positively correlated with the plasma viral loads. Our analysis demonstrates that the pDCs were more prone to apoptosis after infection during the acute phase of SIVmac239 infection, which may be due to their high expressions of CD4 and CCR5. Both DCs subsets activated through elevating the expression of co-stimulatory molecules, which was beneficial in controlling the replication of SIV. However, a mere broad immune activation initiated by activated DCs may lead to tragic AIDS progression.