Differential presentation of a soluble exogenous tumor antigen,NY-ESO-1, by distinct human dendritic cell populations

Dendritic cells (DCs) play a critical role in initiating antigen-specific immune responses, because they are able to capture exogenous antigens for presentation to naive T cells on both MHC class I and II molecules. As such, DCs represent important elements in the development of vaccine therapy for cancer. Although DCs are known to present antigens from phagocytosed tumor cells or preprocessed peptides, we explored whether they might also present soluble recombinant NY-ESO-1, a well characterized cancer antigen. We compared the abilities of human monocyte-derived DCs and DCs derived in vitro from CD34-positive stem cells to present NY-ESO-1 epitopes to MHC class I-restricted cytotoxic T cells. Although monocyte-derived DCs did not efficiently crosspresent free NY-ESO-1 protein, IgG-immune complexes containing NY-ESO-1 were avidly presented after uptake by Fcgamma receptors (FcgammaRII). In contrast, CD34-derived DCs were unable to process either soluble or immune complexed NY-ESO-1, although they efficiently presented preprocessed NY-ESO-1 peptides. This difference did not necessarily correlate with endocytic capacity. Although monocyte-derived DCs exhibited greater fluid-phase uptake than CD34-derived DCs, the two populations did not differ with respect to their surprisingly limited capacity for Fcgamma receptor-mediated endocytosis. These results indicate that monocyte-derived DCs will be easier to load by using protein antigen in vitro than CD34-derived DCs, and that the latter population exhibits a restricted ability to crosspresent soluble exogenous antigens.     

Human follicular dendritic cells inhibit superantigen-induced T-cell proliferation by distinct mechanisms.

Follicular dendritic cells (FDCs) reside within germinal centers of secondary lymphoid tissue where they play a critical role in antigen-driven immune responses. FDCs express numerous adhesion molecules that facilitate cellular interactions with B and T cells within the germinal center microenvironment. Although human FDCs have been shown to influence B-cell development, very little is known about the ability of FDCs to regulate T-cell responses. To investigate this functional aspect of FDCs, highly enriched preparations were isolated by magnetic cell separation using the FDC-restricted monoclonal antibody HJ2. We found that isolated human FDCs inhibited proliferation of both autologous and allogeneic T cells, and were dependent on the number of FDCs present. Inhibition by FDCs was observed using two serologically distinct superantigens at multiple concentrations (Staphylococcus enterotoxin A and B). In contrast, B cells failed to inhibit, and often augmented superantigen-induced T-cell proliferation. Antibody-blocking studies showed that CD54 and CD106 were involved in the ability of FDC to inhibit T-cell proliferative responses. When FDCs and T cells were separated by a semipermeable membrane, the inhibitory effect was partially abrogated, demonstrating that in addition to cell-cell interactions, a soluble factor(s) was also involved in the process. The addition of indomethicin to cultures improved the proliferative response in the presence of FDCs, indicating that inhibition was mediated, in part, by prostaglandins. These results indicate that FDCs regulate T-cell proliferation by two molecular mechanisms and that FDC:T-cell interactions may play a pivotal role in germinal center development.     

Functionally distinct dendritic cell (DC) populations induced by physiologic stimuli: prostaglandin E(2) regulates the migratory capacity of specific DC subsets

Migration of antigen (Ag)-loaded dendritic cells (DCs) from sites of infection into draining lymphoid tissues is fundamental to the priming of T-cell immune responses. We evaluated monocyte-derived DCs (MoDCs) and peripheral blood DCs (PBDCs) to respond to proinflammatory mediators, CD40L, and intact bacteria. All classes of stimuli induced DC phenotypic maturation. However, for MoDCs, only prostaglandin E(2) (PGE(2))-containing stimuli induced migratory-type DCs. Thus, immature MoDCs that encountered proinflammatory cytokines or CD40L or intact bacteria in the presence of PGE(2) acquired migratory capacity but secreted low levels of cytokines. Conversely, MoDCs that encountered pathogens or CD40L alone become nonmigratory cytokine-secreting cells (proinflammatory type). Interestingly, both migratory- and proinflammatory-type DCs expressed equivalent levels of chemokine receptors, suggesting that the role of PGE(2) was to switch on migratory function. We demonstrate that PGE(2) induces migration via the E-prostanoid 2/E-prostanoid 4 (EP(2)/EP(4)) receptors and the cAMP pathway. Finally, migratory-type MoDCs stimulated T-cell proliferation and predominantly IL-2 secretion, whereas proinflammatory-type MoDCs induced IFN-gamma production. In contrast, CD1b/c(+) PBDC rapidly acquired migratory capacity irrespective of the class of stimulus encountered and secreted low levels of cytokines. This suggests that not all mature stages of DCs are destined to migrate to lymphoid organs and that the sequence in which stimuli are encountered significantly affects which functions are expressed. Thus, certain immature DC subsets recruited from the resting precursor pool may have multiple functional fates that play distinct roles during the induction and effector phases of the immune response. These findings have important implications for the clinical utility of DCs in immunotherapy.     

Viral-induced encephalitis initiates distinct and functional CD103+ CD11b+ brain dendritic cell populations within the olfactory bulb

Dendritic cells (DC) are antigen-presenting cells found in both lymphoid and nonlymphoid organs, including the brain (bDC) of Cd11c/eyfp transgenic C57BL/6 mice. Using an intranasal vesicular stomatitis virus infection, we demonstrated that EYFP(+) cells amass in areas associated with viral antigens, take on an activated morphology, and project their processes into infected neuronal tissue within the olfactory bulb. These bDC separated into three EYFP(+) CD45(+) CD11b(+) populations, all but one being able to functionally promote both T lymphocyte proliferation and T(H)1 cytokine production. One population was shown to emanate from the brain and a second population was peripherally derived. The third population was of indeterminate origin, being both radiosensitive and not replenished by donor bone marrow. Finally, each EYFP(+) population contained CD11b(+) CD103(+) subpopulations and could be distinguished in terms of CD115, Gr-1, and Ly-6C expression, highlighting mucosal and monocyte-derived DC lineages.     

The T-DNA-linked VirD2 protein contains two distinct functional nuclear localization signals.

Agrobacterium tumefaciens causes neoplastic growth in plants by transferring a piece of DNA, called T-DNA, into the nucleus of the plant cell. The virulence protein VirD2 of A. tumefaciens is tightly linked to the T-DNA and is thought to direct it to the plant genome. Here we show that the VirD2 protein contains two nuclear localization signals that are functional both in yeast and in plant cells. One signal is located in the N-terminal part of the protein and resembles a single-cluster-type nuclear localization signal. The second signal is near the C terminus and is a bipartite-type nuclear localization signal. The involvement of these sequences in the entry of the T-DNA into the nucleus is discussed.     

Expression of pTalpha mRNA in a committed dendritic cell precursor in the human thymus.

We have characterized dendritic cell precursors (pre-DC) in the human thymus. These CD1a(-)CD3(-)CD4(+)CD8(-) cells express high levels of interleukin-3Ralpha (IL-3Ralpha) on the membrane and are able to develop into mature DC upon culture with IL-3 and CD40 ligation. The DC precursors are predominantly located in the thymic medulla. Interestingly, the pre-DC express pTalpha mRNA, which is also present in CD1a(+)CD3(-)CD4(+)CD8(-) pre-T cells. Yet, the pre-DC lack expression of recombination activating gene-1 mRNA and fail to develop into T cells in appropriate assays. The thymic pre-DC are very similar to the recently characterized pre-DC found in the T cell areas of the tonsil, and it is suggested that these pre-DC populations are of lymphoid origin.     

Human estrogen receptor beta 548 is not a common variant in three distinct populations

Several isoforms of estrogen receptor (ER) beta (also known as NR3A2) have been reported, including variants with different N-terminal ends. In rodents, two in-frame initiation codons (ATGs) are used to produce proteins of 530 and 549 amino acids, respectively. In humans, the upstream ATG is out of frame in all clones reported, until recently, when human clones with an extra A-T base pair placing the upstream ATG in frame were reported. The authors suggested that this could represent a novel polymorphism in the ERbeta gene. Because human ERbeta548 (hERbeta548) and hERbeta530 display different functional characteristics in vitro, it is of interest to determine if this variant constitutes a polymorphism in human populations. We therefore determined the frequency of this novel isoform in several populations including African (n = 96), Caucasian (n = 100), and Asian (n = 128) subjects using denaturing HPLC. We did not detect any alleles that correspond to hERbeta548 in these samples or in additional samples of heterogeneous origin. It is concluded that hERbeta548 is not a common variant in Africans, Caucasians, or Asians.     

Human group X secreted phospholipase A2 induces dendritic cell maturation through lipoprotein-dependent and -independent mechanisms

ObjectiveIncreased secreted phospholipase A₂ (sPLA₂) activity has been documented in several inflammatory disorders. Among sPLA₂s, the human group X (hGX)-sPLA₂ has the highest catalytic activity towards phosphatidylcholine (PC), the major phospholipid of cell membranes and blood lipoproteins. hGX-sPLA₂ has been detected in human atherosclerotic lesions, indicating that sPLA₂s are an important link between lipids and inflammation, both involved in atherosclerosis. The presence of dendritic cells (DC), the most potent antigen presenting cells, in atherosclerotic lesions has raised the question about their role in disease progression.Methods and resultsIn this study, we show that hGX-sPLA₂-treated LDL induces human monocyte-derived DC maturation, resulting in a characteristic mature DC phenotype and enhanced DC ability to activate IFNγ secretion from T cells. hGX-sPLA₂ phospholipolysis of LDL produces high levels of lipid mediators, such as lysophosphatidylcholine (LPC) and free fatty acids (FFAs), which also modulate DC maturation. The major molecular species of LPC containing a palmitic or stearic acid esterified in the sn-1 position induce DC maturation, whereas the FFAs can positively or negatively modulate DC maturation depending on their nature. hGX-sPLA₂ added alone can also activate DC in vitro through the hydrolysis of the DC membrane phospholipids leading, however, to a different cytokine profile secretion pattern than the one observed with hGX-sPLA₂-phospholipolysed LDL.ConclusionhGX-sPLA₂ secreted in inflamed tissues can contribute to local DC maturation, resulting in pro-Th1 cells, through the production of various lipid mediators from hydrolysis of either LDL and/or cell plasma membrane.     

Intravital imaging reveals distinct responses of depleting dynamic tumor-associated macrophage and dendritic cell subpopulations

Tumor-infiltrating inflammatory cells comprise a major part of the stromal microenvironment and support cancer progression by multiple mechanisms. High numbers of tumor myeloid cells correlate with poor prognosis in breast cancer and are coupled with the angiogenic switch and malignant progression. However, the specific roles and regulation of heterogeneous tumor myeloid populations are incompletely understood. CSF-1 is a major myeloid cell mitogen, and signaling through its receptor CSF-1R is also linked to poor outcomes. To characterize myeloid cell function in tumors, we combined confocal intravital microscopy with depletion of CSF-1R–dependent cells using a neutralizing CSF-1R antibody in the mouse mammary tumor virus long-terminal region-driven polyoma middle T antigen breast cancer model. The depleted cells shared markers of tumor-associated macrophages and dendritic cells (M-DCs), matching the phenotype of tumor dendritic cells that take up antigens and interact with T cells. We defined functional subgroups within the M-DC population by imaging endocytic and matrix metalloproteinase activity. Anti–CSF-1R treatment altered stromal dynamics and impaired both survival of M-DCs and accumulation of new M-DCs, but did not deplete Gr-1⁺ neutrophils or block doxorubicin-induced myeloid cell recruitment, and had a minimal effect on lung myeloid cells. Nevertheless, prolonged treatment led to delayed tumor growth, reduced vascularity, and decreased lung metastasis. Because the myeloid infiltrate in metastatic lungs differed significantly from that in mammary tumors, the reduction in metastasis may result from the impact on primary tumors. The combination of functional analysis by intravital imaging with cellular characterization has refined our understanding of the effects of experimental targeted therapies on the tumor microenvironment.Cells of the myeloid lineage, including macrophages, monocytes, neutrophils, mast cells, and immature myeloid cells, are major components of the complex stromal microenvironment of solid tumors (1, 2). Abundant evidence from human and experimental tumor types shows that myeloid cells support tumor growth and progression by a wide range of mechanisms, including stimulation of angiogenesis, secretion of factors inducing tumor growth, survival and cell migration, remodeling of the extracellular matrix to facilitate growth and invasion, recruitment of additional support cells, and suppression of the antitumor immune response (3–5). In human breast cancer, as in most other solid tumors, large numbers of myeloid cells, characterized as tumor-associated macrophages (TAMs), correlate with poor prognosis, as does high expression of the myeloid cell mitogen colony-stimulating factor-1 (CSF-1, M-CSF) or its receptor CSF-1R (CD115, c-fms), and gene-expression profiles reflecting myeloid involvement or CSF-1 signaling (3, 5–8).The marked effects of myeloid cells on tumor growth, invasion, and metastasis identify them and the signaling pathways mediating cancer cell–myeloid cell interactions as potentially attractive targets for treatment of aggressive cancer. TAM depletion by clodronate-encapsulated liposomes reduces tumor growth in various grafted tumors (3, 5, 9). Genetic ablation, antisense and antibody approaches for inhibition of CSF-1/CSF-1R signaling lead to macrophage depletion and reduce malignancy in several models, with abrogated tumor angiogenesis in most cases (3, 5, 9, 10, and references therein). A null mutation in CSF-1, which leads to the lifelong absence of the majority of macrophages, results in delayed tumor progression in the transgenic mouse mammary tumor virus long terminal region-driven polyoma middle T antigen (MMTV-PyMT) model of aggressive breast cancer, evidenced by more differentiated histology and decreased lung metastasis, apparently because of decreased tumor angiogenesis (11–13).Our understanding of the properties and functions of myeloid cell types in primary and metastatic tumors is still incomplete, because of the broad specificity and varying use of cellular markers, as well as myeloid cell plasticity and contrasting results obtained from a wide variety of tumor models (4, 14–16). We and others have used intravital microscopy to define real-time dynamics of several distinct subpopulations of stromal cells in different stages of mammary tumor progression and tumor response to chemotherapy, or relapse thereafter (17–20). In this study, we sought to define the characteristics and importance of CSF-1–dependent myeloid cells in the MMTV-PyMT breast cancer model by using the neutralizing monoclonal CSF-1R antibody M279 (21) to deplete this population at different stages of tumor progression. We then analyzed the tumor response, phenotype of the depleted cells, and cell dynamics in the tumor microenvironment.     

Type IV collagen contains two distinct chains in separate molecules

Type IV collagen from the EHS sarcoma, a basement membrane producing tumor, was characterized. Acid extracts of tumor grown in lathyritic animals contain proteins with two predominant collagen chains of apparent molecular weights of 160,000 and 140,000 daltons, designated α1(IV) and α2(IV). In addition, dimers and larger polymers formed by covalent crosslinking of the α1(IV) and α2(IV) chains are also present. These two chains were found to be distinct from one another in amino acid composition and in the peptide patterns obtained following treatment with cyanogen bromide and S. aureus V8 protease. Precipitation of native collagen from acid solutions by the stepwise addition of NaCl yielded mixtures of the two chains with varying ratios suggesting that the two chains reside in different macromolecules.     

Development of thymic and splenic dendritic cell populations from different hemopoietic precursors.

The antigen-presenting dendritic cells (DCs) found in mouse lymphoid tissues are heterogeneous. Several types of DCs have been identified on the basis of the expression of different surface molecules, including CD4, CD8alpha, and DEC-205. Previous studies by the authors showed that the mouse intrathymic lymphoid-restricted precursors (lin(-)c-kit(+)Thy-1(low)CD4(low)) can produce DCs in the thymus and spleen upon intravenous transfer, suggesting a lymphoid origin of these DCs. In the current study, the potential for DC production by the newly identified bone marrow (BM) common lymphoid precursors (CLPs), common myeloid precursors (CMPs), and committed granulocyte and macrophage precursors was examined. It was found that both the lymphoid and the myeloid precursors had the potential to produce DCs. All the different DC populations identified in mouse thymus and spleen could be produced by all these precursor populations. However, CLPs produced predominantly the CD4(-)CD8alpha(+) DCs, whereas CMPs produced similar numbers of CD4(-)CD8alpha(+) and CD4(+)CD8alpha(-) DCs, although at different peak times. On a per cell basis, the CLPs were more potent than the CMPs at DC production, but this may have been compensated for by an excess of CMPs over CLPs in BM. Overall, this study shows that the expression of CD8alpha does not delineate the hemopoietic precursor origin of DCs, and the nature of the early precursors may bias but does not dictate the phenotype of the DC product.     

Toll-like receptor ligands synergize through distinct dendritic cell pathways to induce T cell responses: implications for vaccines

Toll-like receptors (TLRs) may need to cooperate with each other to be effective in detecting imminent infection and trigger immune responses. Understanding is still limited about the intracellular mechanism of this cooperation. We found that when certain TLRs are involved, dendritic cells (DCs) establish unidirectional intracellular cross-talk, in which the MyD88-independent TRIF-dependent pathway amplifies the MyD88-dependent DC function through a JNK-dependent mechanism. The amplified MyD88-dependent DC function determines the induction of the T cell response to a given vaccine in vivo. Therefore, our study revealed an underlying TLR mechanism governing the functional, nonrandom interplay among TLRs for recognition of combinatorial ligands that may be dangerous to the host, providing important guidance for design of novel synergistic molecular vaccine adjuvants.     

Human cervicovaginal lavage fluid contains an inhibitor of HIV binding to dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin

A small percentage of women at high risk for human immunodeficiency virus (HIV) exposure remain uninfected for long periods, protected by unknown mechanisms. We hypothesized that one mechanism could be inhibition of interactions between HIV and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) in the genital tract. In an analysis of 95 cervicovaginal lavage samples, we found that 12 (12.6%) strongly inhibited the binding of laboratory-adapted and primary HIV-1 isolates to B-THP-1/DC-SIGN cells in a dose-dependent manner, independently of the donor's risk of exposure. Three of 5 primary isolates were also blocked from binding to primary DCs. The inhibitor has a high molecular weight, is heat stable, and is resistant to trypsin. It is sensitive to pronase and periodate, indicating that it is likely a glycoprotein. Mannosidase digestion and concanavalin A adsorption indicate that the terminal residues of the carbohydrate are not mannose. Mechanistic experiments indicate that the inhibitor acts via binding to DC-SIGN. Further study of such inhibitors may help to elucidate the role played by DC-SIGN in HIV transmission.     

Human lung small-cell carcinoma contains bombesin.

The presence of immunoreactive bombesin in a human lung small-cell carcinoma grown in nude mice was established by several criteria: (i) Radioimmunoassay of tissue extracts for bombesin revealed approximately 6.5 pmol/g of tissue; (ii) bombesin was found in 12-14% of the tumor cells by immunohistochemical localization; (iii) gel filtration of small-cell carcinoma extract on Sephadex G-75 and Bio-Gel P-4 gave only a single peak of immunoreactivity, which occurred at the elution volume of bombesin; and (iv) reverse-phase HPLC of acid-solubilized extracts separated the immunoreactive material into three discrete peaks, one of which eluted with a retention time identical to that of synthetic bombesin. The presence of bombesin may represent the ectopic expression of this peptide in small-cell carcinoma, because immunoreactive bombesin was found in human fetal and neonatal lung but apparently not in adult lung tissue [Wharton, J., Polak, J. M., Bloom, S. R., Ghatei, M. A., Solcia, E., Brown, M. R. & Pearse, A. G. E. (1978) Nature (London) 273, 769-770]. The immunoreactive bombesin previously found in mammalian tissues is considerably larger than amphibian bombesin; these data substantiate the presence of a mammalian form of bombesin in a human tumor that may have a structure similar to that of the amphibian peptide.     

Presence of two distinct acinar cell populations in human pancreas based on their antigenicity

The immunohistochemical localization of ABH- and Lewis (Le)-related blood group antigens, including CA 19-9, a sialylated Lea antigen, was examined using monoclonal antibodies (MoAbs) in 18 normal human pancreases and compared with ABH blood group antigenicity of the individuals. Acinar cells expressed ABH, Leb, Ley, and in some cases, Lex antigen in various proportions, but not Lea and CA 19-9. The reactivity of Leb and Ley was similar with regard to cellular localization and specificity. In all specimens but one, the distribution of Leb (and Ley) and H antigens on the one hand, and of A or B antigens on the other, showed a reciprocal relationship, in that one group of acini expressed Leb (and Ley) and H antigens, but lacked any A or B antigens (type 1 acinar cell); another group of acinar cells had A or B antigens, but expressed neither Leb (Ley) or H antigens (type 2 acinar cell). In ductal cells, four of eight individuals with blood group A, two of three with blood group B, and five of six with blood group O expressed the appropriate antigen, while the remainder did not. Lea antigen was expressed primarily by centroacinar and terminal ductular and ductal cells of medium-sized ducts of all specimens, whereas Leb was present in the cells of small and large ducts in all but four cases. The reactivity of ductal and ductular cells to Lex was negative, except for one case. MoAb-Ley and MoAb 19-9 reacted only with a few ductal cells in six (33%) and 12 cases (67%), respectively. There was no relationship in the expression of Le-related antigens between acinar and ductal/ductular cells; nor were there any sex difference with regard to the binding patterns of any antibodies. However, age appeared to influence the reactivity of some antibodies with acinar cells. Islet cells did not react with any of the antibodies. The results indicate that, although the antigenicity of epithelial cells can be affected by the host blood group types, there might be several regulatory systems for expression of blood group antigens in a cell-specific pattern.