DC-SIGN binds ICAM-3 isolated from peripheral human leukocytes through Lewis x residues

Intercellular adhesion molecule-3 (ICAM-3) binds to the alpha(L)beta(2) integrin and mediates the contact between T cells and antigen-presenting cells. It has been suggested that dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN), a C-type lectin of macrophages and DCs, is an additional ligand of ICAM-3. So far, the glycan structure mediating the interaction of native ICAM-3 with DC-SIGN is undefined. Here, we demonstrate that native ICAM-3 from human peripheral leukocytes binds recombinant DC-SIGN, is recognized by monoclonal Lewis x antibodies, and specifically interacts with DC-SIGN on immature DCs. The presence of Lewis x residues on ICAM-3 was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. Investigations on different peripheral blood cell populations revealed that only ICAM-3 from granulocytes bound DC-SIGN. Cotransfection studies demonstrated that fucosyltransferase (FUT) IX and, to a significantly lesser extent, FUT IV, but not FUTs III and VII, mediate the synthesis of Lewis x residues on ICAM-3. These findings indicate that FUT IX is the main FUT mediating the synthesis of Lewis x residues of ICAM-3 in cells of the myeloid lineage, and that these residues bind DC-SIGN. The results suggest that ICAM-3 assists in the interaction of granulocytes with DC-SIGN of DCs.     

Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses

Contact between dendritic cells (DC) and resting T cells is essential to initiate a primary immune response. Here, we demonstrate that ICAM-3 expressed by resting T cells is important in this first contact with DC. We discovered that instead of the common ICAM-3 receptors LFA-1 and alphaDbeta2, a novel DC-specific C-type lectin, DC-SIGN, binds ICAM-3 with high affinity. DC-SIGN, which is abundantly expressed by DC both in vitro and in vivo, mediates transient adhesion with T cells. Since antibodies against DC-SIGN inhibit DC-induced proliferation of resting T cells, our findings predict that DC-SIGN enables T cell receptor engagement by stabilization of the DC-T cell contact zone.     

Generation of functional dendritic cells for potential use in the treatment of acute lymphoblastic leukemia

Immunotherapy of malignant diseases mediated by dendritic cells (DC) pulsed with tumor antigens ex vivo is a promising new tool in the individual treatment of malignant diseases. The present study focuses on the problem of how to optimize in vitro culture conditions and induce the maturation of DC with the capacity to induce antitumor immunity toward leukemic cells. DC were generated from peripheral mononuclear cells by co-cultivation with granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Tumor antigens were added for 2 h after 7 days in culture. Irradiated leukemic blasts, blast lysate, apoptotic cells from the Jurkat cell line (T ALL) and their lysate were used in various concentrations for antigen pulsing. Harvested DC were phenotyped by flow cytometry, and viability was assessed using trypan blue exclusion (Annexin test). After the cells had been pulsed with tumor antigens and co-cultured with autologous lymphocytes, the production of interferon-gamma (IFN-gamma) and IL-12 was analyzed, and lymphocyte proliferative response and cytotoxicity against the target tumor cell line were assessed. The cultivation of monocytes under the described conditions led to the expression of surface markers typical of DC (i.e. CD83, CD86, HLA-DR, CD11c and CD40). Pulsation by antigens from leukemic cells further increased the cell populations expressing these markers. Antigen pulsation decreased the viability of generated DC depending on the increase in concentration of tumor antigens. Pulsed DC-lymphocyte interaction increased the proliferative response of lymphocytes and IFN-gamma production depending on the type of tumor antigens used for pulsation. The highest proliferative response was detected with DC pulsed with Jurkat cell-line lysate. Similarly to the proliferation assay, cytotoxic testing showed the highest efficiency of DC pulsed with Jurkat cell-line lysate in killing the target malignant cells. Our results show that an appropriate antigen concentration used for DC pulsing is one of the crucial factors in an effective treatment strategy, as high concentrations of tumor antigens induce apoptosis of DC, thereby rendering them non-functional. Under optimal conditions, pulsation by lysate from leukemic blasts induced the maturation of DC and led to an increase in the proliferation of autologous lymphocytes, to the production of Th1-cytokines and to the induction of cytotoxicity toward the leukemic cell line. These results are encouraging for the possible application of pulsed DC in the therapy of acute lymphoblastic leukemia.     

Functional evaluation of DC-SIGN monoclonal antibodies reveals DC-SIGN interactions with ICAM-3 do not promote human immunodeficiency virus type 1 transmission

DC-SIGN, a type II membrane-spanning C-type lectin that is expressed on the surface of dendritic cells (DC), captures and promotes human and simian immunodeficiency virus (HIV and SIV) infection of CD4(+) T cells in trans. To better understand the mechanism of DC-SIGN-mediated virus transmission, we generated and functionally evaluated a panel of seven monoclonal antibodies (MAbs) against DC-SIGN family molecules. Six of the MAbs reacted with myeloid-lineage DC, whereas one MAb preferentially bound DC-SIGNR/L-SIGN, a homolog of DC-SIGN. Characterization of hematopoietic cells also revealed that stimulation of monocytes with interleukin-4 (IL-4) or IL-13 was sufficient to induce expression of DC-SIGN. All DC-SIGN-reactive MAbs competed with intercellular adhesion molecule 3 (ICAM-3) for adhesion to DC-SIGN and blocked HIV-1 transmission to T cells that was mediated by THP-1 cells expressing DC-SIGN. Similar but less efficient MAb blocking of DC-mediated HIV-1 transmission was observed, indicating that HIV-1 transmission to target cells via DC may not be dependent solely on DC-SIGN. Attempts to neutralize DC-SIGN capture and transmission of HIV-1 with soluble ICAM-3 prophylaxis were limited in success, with a maximal inhibition of 60%. In addition, disrupting DC-SIGN/ICAM-3 interactions between cells with MAbs did not impair DC-SIGN-mediated HIV-1 transmission. Finally, forced expression of ICAM-3 on target cells did not increase their susceptibility to HIV-1 transmission mediated by DC-SIGN. While these findings do not discount the role of intercellular contact in facilitating HIV-1 transmission, our in vitro data indicate that DC-SIGN interactions with ICAM-3 do not promote DC-SIGN-mediated virus transmission.     

Identification of different binding sites in the dendritic cell-specific receptor DC-SIGN for intercellular adhesion molecule 3 and HIV-1.

The novel dendritic cell (DC)-specific human immunodeficiency virus type 1 (HIV-1) receptor DC-SIGN plays a key role in the dissemination of HIV-1 by DC. DC-SIGN is thought to capture HIV-1 at mucosal sites of entry, facilitating transport to lymphoid tissues, where DC-SIGN efficiently transmits HIV-1 to T cells. DC-SIGN is also important in the initiation of immune responses by regulating DC-T cell interactions through intercellular adhesion molecule 3 (ICAM-3). We have characterized the mechanism of ligand binding by DC-SIGN and identified the crucial amino acids involved in this process. Strikingly, the HIV-1 gp120 binding site in DC-SIGN is different from that of ICAM-3, consistent with the observation that glycosylation of gp120, in contrast to ICAM-3, is not crucial to the interaction with DC-SIGN. A specific mutation in DC-SIGN abrogated ICAM-3 binding, whereas the HIV-1 gp120 interaction was unaffected. This DC-SIGN mutant captured HIV-1 and infected T cells in trans as efficiently as wild-type DC-SIGN, demonstrating that ICAM-3 binding is not necessary for HIV-1 transmission. This study provides a basis for the design of drugs that inhibit or alter interactions of DC-SIGN with gp120 but not with ICAM-3 or vice versa and that have a therapeutic value in immunological diseases and/or HIV-1 infections.     

Involvement of PPAR-γ and p53 in DHA-induced apoptosis in Reh cells

Dendritic cells (DCs) are professional antigen-presenting cells and have come to be appreciated as critical controllers of the immune response, especially T cell responses. Apart from presenting antigens to T cells, DCs carry out many other functions in regulating immunity. DC-specific intercellular adhesion molecule (ICAM)-3 grabbing non-integrin (DC-SIGN) is a novel receptor that plays an important role in DC migration and adhesion, the inflammatory response, T cell activation, initiating the immune response, and immune escape of pathogens and tumors. DC-SIGN mediates DC binding to ICAM-3 on the T cell surface and ICAM-2 on the endothelial cell (EC) surface, and takes part in the initial interaction between DC and T cells or vascular ECs. The procedure of systematic evolution of ligands by exponential enrichment (SELEX) is a method in which single-stranded oligonucleotides are selected from a wide variety of sequences, based on their interaction with a target molecule. In this study, we selected DNA aptamers against DC-SIGN protein by SELEX, and measured their binding affinity for DC-SIGN. Finally, an appropriate aptamer with high affinity for DC-SIGN was obtained, and it blocked DC adhesion to ECs as effectively as anti-DC-SIGN monoclonal antibody.     

Regulated expression of the pathogen receptor dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin in THP-1 human leukemic cells, monocytes, and macrophages

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) is a type II C-type lectin that functions as an adhesion receptor and mediates binding and internalization of pathogens such as virus (human immunodeficiency virus, hepatitis C), bacteria (Mycobacterium), fungi, and parasites. DC-SIGN expression in vivo is primarily restricted to interstitial dendritic cells (DC) and certain tissue macrophages. We now report that leukemic THP-1 cells, widely used as a model for monocyte-macrophage differentiation, express very low basal levels of DC-SIGN and that DC-SIGN expression in THP-1 cells is regulated during differentiation. Differentiation-inducing agents (phorbol ester, bryostatin) conveyed THP-1 cells with the ability to up-regulate DC-SIGN mRNA levels and cell surface expression in response to interleukin-4 (IL-4) or IL-13. DC-SIGN up-regulation required a functional JAK-STAT signaling pathway, was inhibited in the presence of lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha), and conferred THP-1 cells with increased pathogen recognition and T cell stimulatory capabilities. The up-regulation of DC-SIGN on THP-1 cells resembles its inducible expression on monocytes and macrophages, where DC-SIGN expression is also induced by IL-4/IL-13 and negatively regulated by TNF-alpha, LPS, and vitamin D(3). These results point to THP-1 cells as a useful cellular system to characterize the pathogen-binding capabilities of DC-SIGN and to dissect the molecular mechanisms that control its regulated and tissue-specific expression in myeloid dendritic cells, and the results suggest that DC-SIGN constitutes a marker for both DC and alternatively activated macrophages.     

Expression of cell surface Lewis X and Y antigens and FUT4 mRNA is increased in Jurkat cells undergoing apoptosis

Cell surface molecules undergo specific changes during apoptosis, including the expression of phosphatidylserine (PS) and some proteins and alterations in sugar chains. Among the various sugar chains on the cell surface, Lewis X (Le(X)) and Lewis Y (Le(Y)) antigens are key determinants for a variety of biological processes. We studied the changes in Le(X) and Le(Y) expression in Jurkat cells, a human T cell line, during apoptosis. Flow cytometry showed that Le(X) and Le(Y) antigen expression was enhanced on the cell surface during apoptosis induced by anti-Fas antibody. To clarify the mechanism of enhanced Le(X) and Le(Y) expression, we assessed the expression levels of fucosyltransferase (FUT1, 2, 3-5-6, 4, and 9) mRNAs that are predominantly expressed in Jurkat cells and which are considered to form Le(X) and Le(Y). The expression of FUT4 mRNA was up-regulated after exposing cells to anti-Fas antibody. Moreover, the increase in Le(X) and Le(Y) antigen levels was significantly suppressed by caspase 3 or 8 inhibitors. These results indicated that the induction of FUT (mainly FUT4), the gene expression of which is mediated by signals downstream of caspase 3, increases Le(X) and Le(Y) expression in apoptotic cells.     

Modulating HIV-1 replication by RNA interference directed against human transcription elongation factor SPT5

Background

Dendritic cell (DC) transmission of human immunodeficiency virus (HIV) to CD4+ T cells occurs across a point of cell-cell contact referred to as the infectious synapse. The relationship between the infectious synapse and the classically defined immunological synapse is not currently understood. We have recently demonstrated that human B cells expressing exogenous DC-SIGN, DC-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing nonintegrin, efficiently transmit captured HIV type 1 (HIV-1) to CD4+ T cells. K562, another human cell line of hematopoietic origin that has been extensively used in functional analyses of DC-SIGN and related molecules, lacks the principal molecules involved in the formation of immunological synaptic junctions, namely major histocompatibility complex (MHC) class II molecules and leukocyte function-associated antigen-1 (LFA-1). We thus examined whether K562 erythroleukemic cells could recapitulate efficient DC-SIGN-mediated HIV-1 transmission (DMHT).

Results

Here we demonstrate that DMHT requires cell-cell contact. Despite similar expression of functional DC-SIGN, K562/DC-SIGN cells were inefficient in the transmission of HIV-1 to CD4+ T cells when compared with Raji/DC-SIGN cells. Expression of MHC class II molecules or LFA-1 on K562/DC-SIGN cells was insufficient to rescue HIV-1 transmission efficiency. Strikingly, we observed that co-culture of K562 cells with Raji/DC-SIGN cells impaired DMHT to CD4+ T cells. The K562 cell inhibition of transmission was not directly exerted on the CD4+ T cell targets and required contact between K562 and Raji/DC-SIGN cells.

Conclusions

DMHT is cell type dependent and requires cell-cell contact. We also find that the cellular milieu can negatively regulate DC-SIGN transmission of HIV-1 in trans.     

Low-dose anisomycin is sufficient to alter the bio-behaviors of Jurkat T cells

Anisomycin is a pyrrolidine antibiotic isolated from Streptomyces griseolus. It has been found that a quite low dose of anisomycin is sufficient to block proliferation of primary T lymphocytes. The focus of this study is to explore the possibility of anisomycin to treat human acute leukemia Jurkat T cells in vitro. The results indicated that the low dose of anisomycin could significantly inhibit the colony formation of Jurkat T cells and elevate the inhibition rate of Jurkat T cell growth along with its increasing concentrations. Jurkat T cell cycle was blocked into S-phase by anisomycin. Consistent with the increased proportion of sub-G1 phase, anisomycin promoted Jurkat T cell apoptosis. The CD69 and CD25 expression on the surface of Jurkat T cells was also down-regulated prominently along with the enhancing concentrations of anisomycin, followed by the decreased production of IL-4, IL-10, IL-17, TGF-β and IFN-γ, and the down-regulated expression of phosphorylated-ERK1/2. The results suggest that the suppressive effect of anisomycin on Jurkat T cell growth may be related to inhibiting TGF-β production and ERK1/2 activation, arresting the cell cycle at S-phase and promoting the apoptosis of Jurkat T cells.     

DC-SIGN points the way to a novel mechanism for HIV-1 transmission

Dendritic cell (DC)-specific intercellular adhesion molecule 3 (ICAM-3) grabbing nonintegrin (DC-SIGN), a recently discovered type II transmembrane protein on DCs with a C-type lectin extracellular domain, is capable of binding ICAM-3 on resting T cells in the secondary lymphoid organs, providing the initial contact between these cells during the establishment of cell-mediated immunity. DC-SIGN also binds the HIV-1 envelope glycoprotein gp120 but does not function as a receptor for viral entry into DCs. Instead, DC-SIGN allows DCs in the peripheral mucosa to carry HIV-1 through the lymphatics in a "Trojan horse" fashion, where it is eventually delivered to the T cells. Also, the period of infectivity of HIV-1 is increased by several days as a result of DC-SIGN-gp120 binding, allowing for efficient trans-infection of T cells on DC arrival. The discovery of a cluster of related genes colocalized with DC-SIGN on chromosome 19p13.2-3, all displaying complex alternative splicing patterns, has led to a reexamination of the mechanisms underlying both the interactions between antigen-presenting cells (APCs) and T cells and the pathogenesis of HIV-1 infection.     

DC-SIGN on B lymphocytes is required for transmission of HIV-1 to T lymphocytes

Infection of T cells by HIV-1 can occur through binding of virus to dendritic cell (DC)-specific ICAM-3 grabbing nonintegrin (DC-SIGN) on dendritic cells and transfer of virus to CD4+ T cells. Here we show that a subset of B cells in the blood and tonsils of normal donors expressed DC-SIGN, and that this increased after stimulation in vitro with interleukin 4 and CD40 ligand, with enhanced expression of activation and co-stimulatory molecules CD23, CD58, CD80, and CD86, and CD22. The activated B cells captured and internalized X4 and R5 tropic strains of HIV-1, and mediated trans infection of T cells. Pretreatment of the B cells with anti-DC-SIGN monoclonal antibody blocked trans infection of T cells by both strains of HIV-1. These results indicate that DC-SIGN serves as a portal on B cells for HIV-1 infection of T cells in trans. Transmission of HIV-1 from B cells to T cells through this DC-SIGN pathway could be important in the pathogenesis of HIV-1 infection.     

Dendritic cell-specific ICAM-3-grabbing nonintegrin expression on M2-polarized and tumor-associated macrophages is macrophage-CSF dependent and enhanced by tumor-derived IL-6 and IL-10

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN; CD209) is a human pathogen-attachment C-type lectin with no obvious murine ortholog and for which ligation leads to enhanced anti-inflammatory cytokine release and altered proinflammatory cytokine production. Although induced by IL-4 in monocytes and considered as a DC marker, DC-SIGN expression on human APCs under homeostatic conditions is so far unexplained. We report in this study that M-CSF enhances DC-SIGN expression on in vitro derived anti-inflammatory macrophages and that M-CSF mediates the induction of DC-SIGN by fibroblast- and tumor cell-conditioned media. The M-CSF-inducible DC-SIGN expression along monocyte-to-macrophage differentiation is dependent on JNK and STAT3 activation, potentiated by STAT3-activating cytokines (IL-6, IL-10), and abrogated by the M1-polarizing cytokine GM-CSF. In pathological settings, DC-SIGN expression is detected in tumor tissues and on ex vivo-isolated CD14(+) CD163(+) IL-10-producing tumor-associated macrophages. Importantly, DC-SIGN Abs reduced the release of IL-10 from macrophages exposed to Lewis(x)-expressing SKBR3 tumor cells. These results indicate that DC-SIGN is expressed on both wound-healing (IL-4-dependent) and regulatory (M-CSF-dependent) alternative (M2) macrophages and that DC-SIGN expression on tumor-associated macrophages might help tumor progression by contributing to the maintenance of an immunosuppressive environment.     

DC-SIGN facilitates fusion of dendritic cells with human T-cell leukemia virus type 1-infected cells

Interactions between the oncogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) and dendritic cells (DCs) are poorly characterized. We show here that monocyte-derived DCs form syncytia and are infected upon coculture with HTLV-1-infected lymphocytes. We examined the role of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a C-type lectin expressed in DCs, in HTLV-1-induced syncytium formation. DC-SIGN is known to bind with high affinity to various viral envelope glycoproteins, including human immunodeficiency virus (HIV) and hepatitis C virus, as well as to the cellular receptors ICAM-2 and ICAM-3. After cocultivating DCs and HTLV-1-infected cells, we found that anti-DC-SIGN monoclonal antibodies (MAbs) were able to decrease the number and size of HTLV-1-induced syncytia. Moreover, expression of the lectin in epithelial-cell lines dramatically enhanced the ability to fuse with HTLV-1-positive cells. Interestingly, in contrast to the envelope (Env) glycoproteins of HIV and other viruses, that of HTLV-1 does not bind directly to DC-SIGN. The facilitating role of the lectin in HTLV-1 syncytium formation is mediated by its interaction with ICAM-2 and ICAM-3, as demonstrated by use of MAbs directed against these adhesion molecules. Altogether, our results indicate that DC-SIGN facilitates HTLV-1 infection and fusion of DCs through an ICAM-dependent mechanism.     

Hepatitis C virus targets DC-SIGN and L-SIGN to escape lysosomal degradation

Hepatitis C virus (HCV) is a major health problem. However, the mechanism of hepatocyte infection is largely unknown. We demonstrate that the dendritic cell (DC)-specific C-type lectin DC-SIGN and its liver-expressed homologue L-SIGN/DC-SIGNR are important receptors for HCV envelope glycoproteins E1 and E2. Mutagenesis analyses demonstrated that both HCV E1 and E2 bind the same binding site on DC-SIGN as the pathogens human immunodeficiency virus type 1 (HIV-1) and mycobacteria, which is distinct from the cellular ligand ICAM-3. HCV virus-like particles are efficiently captured and internalized by DCs through binding of DC-SIGN. Antibodies against DC-SIGN specifically block HCV capture by both immature and mature DCs, demonstrating that DC-SIGN is the major receptor on DCs. Interestingly, internalized HCV virus-like particles were targeted to nonlysosomal compartments within immature DCs, where they are protected from lysosomal degradation in a manner similar to that demonstrated for HIV-1. Lewis X antigen, another ligand of DC-SIGN, was internalized to lysosomes, demonstrating that the internalization pathway of DC-SIGN-captured ligands may depend on the structure of the ligand. Our results suggest that HCV may target DC-SIGN to "hide" within DCs and facilitate viral dissemination. L-SIGN, expressed by THP-1 cells, internalized HCV particles into similar nonlysosomal compartments, suggesting that L-SIGN on liver sinusoidal endothelial cells may capture HCV from blood and transmit it to hepatocytes, the primary target for HCV. We therefore conclude that both DCs and liver sinusoidal endothelial cells may act as reservoirs for HCV and that the C-type lectins DC-SIGN and L-SIGN, as important HCV receptors, may represent a molecular target for clinical intervention in HCV infection.     

Glycosyltransferases involved in type 1 chain and Lewis antigen biosynthesis exhibit glycan and core chain specificity

Sialyl Lewis A (SLe(a)), Lewis A (Le(a)), and Lewis B (Le(b)) have been studied in many different biological contexts, for example in microbial adhesion and cancer. Their biosynthesis is complex and involves beta1,3-galactosyltransferases (beta3Gal-Ts) and a combined action of alpha2- and/or alpha4-fucosyltransferases (Fuc-Ts). Further, O-glycans with different core structures have been identified, and the ability of beta3Gal-Ts and Fuc-Ts to use these as substrates has not been resolved. Therefore, to examine the in vivo specificity of enzymes involved in SLe(a), Le(a), and Le(b) synthesis, we have transiently transfected CHO-K1 cells with relevant human glycosyltransferases and, on secreted reporter proteins, detected the resulting Lewis antigens on N- and O-linked glycans using western blotting and Le-specific antibodies. beta3Gal-T1, -T2, and -T5 could synthesize type 1 chains on N-linked glycans, but only beta3Gal-T5 worked on O-linked glycans. The latter enzyme could use both core 2 and core 3 precursor structures. Furthermore, the specificity of FUT5 and FUT3 in Le(a) and Le(b) synthesis was different, with FUT5 fucosylating H type 1 only on core 2, but FUT3 fucosylating H type 1 much more efficient on core 3 than on core 2. Finally, FUT1 and FUT2 were both found to direct alpha2-fucosylation on type 1 chains on both N- and O-linked structures. This knowledge enables us to engineer recombinant glycoproteins with glycan- and core chain-specific Lewis antigen substitution. Such tools will be important for investigations on the fine carbohydrate specificity of Le(b)-binding lectins, such as Helicobacter pylori adhesins and DC-SIGN, and may also prove useful as therapeutics.     

A novel 2,6-diisopropylphenyl-docosahexaenoamide conjugate induces apoptosis in T cell acute lymphoblastic leukemia cell lines

We have previously characterized the effects of 2,6-diisopropylphenyl–docosahexaenoamide (DIP–DHA) conjugates and their analogs on the proliferation and progression of breast cancer cell lines. For this study, we investigated the effects of the DIP–DHA conjugate on 2 representative T cell acute lymphoblastic leukemia (T-ALL) cell lines: CEM and Jurkat. Treatment of both cell lines with DIP–DHA resulted in significantly greater inhibition of proliferation and induction of apoptosis than that of parent compounds, 2,6-diisopropylphenol (DIP) or docosahexaenoate (DHA). Treatment of the cells with DIP–DHA resulted in increased activation of caspase-3, and caspase-7. Furthermore, induction of apoptosis in both cell lines was reversed in the presence of a caspase family inhibitor. Treatment with DIP–DHA reduced mitochondrial membrane potential. These observations suggest that the effects are driven by intrinsic apoptotic pathways. DIP–DHA treatment also downregulated surface CXCR4 expression, an important chemokine receptor involved in cancer metastasis that is highly expressed in both CEM and Jurkat cells. In conclusion, our data suggest that the DIP–DHA conjugate exhibits significantly more potent effects on CEM and Jurkat cells than that of DIP or DHA alone. These conjugates have potential use for treatment of patients with T cell acute lymphoblastic leukemia.     

Differential transmission of human immunodeficiency virus type 1 by distinct subsets of effector dendritic cells

Dendritic cells (DC) support human immunodeficiency virus type 1 (HIV-1) transmission by capture of the virus particle in the mucosa and subsequent transport to the draining lymph node, where HIV-1 is presented to CD4(+) Th cells. Virus transmission involves a high-affinity interaction between the DC-specific surface molecule DC-SIGN and the viral envelope glycoprotein gp120 and subsequent internalization of the virus, which remains infectious. The mechanism of viral transmission from DC to T cells is currently unknown. Sentinel immature DC (iDC) develop into Th1-promoting effector DC1 or Th2-promoting DC2, depending on the activation signals. We studied the ability of these effector DC subsets to support HIV-1 transmission in vitro. Compared with iDC, virus transmission is greatly upregulated for the DC1 subset, whereas DC2 cells are inactive. Increased transmission by DC1 correlates with increased expression of ICAM-1, and blocking studies confirm that ICAM-1 expression on DC is important for HIV transmission. The ICAM-1-LFA-1 interaction is known to be important for immunological cross talk between DC and T cells, and our results indicate that this cell-cell contact is exploited by HIV-1 for efficient transmission.     

Functional and antigenic characterization of human, rhesus macaque, pigtailed macaque, and murine DC-SIGN

DC-SIGN, a type II membrane protein with a C-type lectin binding domain that is highly expressed on mucosal dendritic cells (DCs) and certain macrophages in vivo, binds to ICAM-3, ICAM-2, and human and simian immunodeficiency viruses (HIV and SIV). Virus captured by DC-SIGN can be presented to T cells, resulting in efficient virus infection, perhaps representing a mechanism by which virus can be ferried via normal DC trafficking from mucosal tissues to lymphoid organs in vivo. To develop reagents needed to characterize the expression and in vivo functions of DC-SIGN, we cloned, expressed, and analyzed rhesus macaque, pigtailed macaque, and murine DC-SIGN and made a panel of monoclonal antibodies (MAbs) to human DC-SIGN. Rhesus and pigtailed macaque DC-SIGN proteins were highly similar to human DC-SIGN and bound and transmitted HIV type 1 (HIV-1), HIV-2, and SIV to receptor-positive cells. In contrast, while competent to bind virus, murine DC-SIGN did not transmit virus to receptor-positive cells under the conditions tested. Thus, mere binding of virus to a C-type lectin does not necessarily mean that transmission will occur. The murine and macaque DC-SIGN molecules all bound ICAM-3. We mapped the determinants recognized by a panel of 16 MAbs to the repeat region, the lectin binding domain, and the extreme C terminus of DC-SIGN. One MAb was specific for DC-SIGN, failing to cross-react with DC-SIGNR. Most MAbs cross-reacted with rhesus and pigtailed macaque DC-SIGN, although none recognized murine DC-SIGN. Fifteen of the MAbs recognized DC-SIGN on DCs, with MAbs to the repeat region generally reacting most strongly. We conclude that rhesus and pigtailed macaque DC-SIGN proteins are structurally and functionally similar to human DC-SIGN and that the reagents that we have developed will make it possible to study the expression and function of this molecule in vivo.