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1.
Clin Dev Immunol ; 2013: 186420, 2013.
Article in English | MEDLINE | ID: mdl-24066003

ABSTRACT

The measurement of vaccine-induced humoral and CD4(+) and CD8(+) cellular immune responses represents an important correlate of vaccine efficacy. Accurate and reliable assays evaluating such responses are therefore critical during the clinical development phase of vaccines. T cells play a pivotal role both in coordinating the adaptive and innate immune responses and as effectors. During the assessment of cell-mediated immunity (CMI) in subjects participating in a large-scale influenza vaccine trial, we identified the expansion of an IFN-γ-producing CD3(+)CD4(-)CD8(-) γδ (+) T cell population in the peripheral blood of 90/610 (15%) healthy subjects. The appearance of CD3(+)CD4(-)CD8(-) γδ (+) T cells in the blood of subjects was transient and found to be independent of the study cohort, vaccine group, subject gender and ethnicity, and ex vivo restimulation conditions. Although the function of this population and relevance to vaccination are unclear, their inclusion in the total vaccine-specific T-cell response has the potential to confound data interpretation. It is thus recommended that when evaluating the induction of IFN-γ-producing CD4(+) and CD8(+) immune responses following vaccination, the CD3(+)CD4(-)CD8(-) γδ (+) T cells are either excluded or separately enumerated from the overall frequency determination.


Subject(s)
Leukocytes, Mononuclear/immunology , T-Lymphocyte Subsets/immunology , Adolescent , Adult , Aged , Aged, 80 and over , CD3 Complex/metabolism , CD4 Antigens/metabolism , CD8 Antigens/metabolism , Cohort Studies , Humans , Immunity, Cellular/immunology , Immunophenotyping , Influenza Vaccines/immunology , Influenza, Human/prevention & control , Leukocytes, Mononuclear/metabolism , Middle Aged , Receptors, Antigen, T-Cell, gamma-delta/metabolism , T-Lymphocyte Subsets/metabolism , Time Factors , Vaccination , Vaccine Potency , Young Adult
2.
Prep Biochem Biotechnol ; 35(2): 119-34, 2005.
Article in English | MEDLINE | ID: mdl-15881594

ABSTRACT

The cancer-testis antigen, NY-ESO-1, has been engineered into a bacterial expression plasmid which incorporates a His6-tag. The plasmid was transfected into E. coli strain BL21 and Master and Working cell banks generated from this expression system. Three 15-litre fermentations were performed under cGMP (code of Good Manufacturing Practice) conditions and the crude NY-ESO-1 tagged protein isolated as solubilised inclusion bodies. A three-step cGMP chromatography process (immobilised metal affinity, anion exchange, and hydrophobic interaction) was utilised to purify the protein. The purified NY-ESO-1 is being used in early stage human cancer vaccine trials in Australia and the U.S.A.


Subject(s)
Antigens, Neoplasm/biosynthesis , Antigens, Neoplasm/isolation & purification , Cancer Vaccines/biosynthesis , Cancer Vaccines/isolation & purification , Membrane Proteins/biosynthesis , Membrane Proteins/isolation & purification , Protein Engineering/methods , Antigens, Neoplasm/genetics , Antigens, Neoplasm/therapeutic use , Australia , Cancer Vaccines/genetics , Drug Industry/standards , Guidelines as Topic , Humans , Membrane Proteins/genetics , Membrane Proteins/therapeutic use , Protein Engineering/standards , Quality Assurance, Health Care/standards , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Reference Standards
3.
Br J Cancer ; 86(6): 979-82, 2002 Mar 18.
Article in English | MEDLINE | ID: mdl-11953832

ABSTRACT

Recent evidences suggest that malignant mesothelioma may be sensitive to immunotherapy; however, little is known about malignant mesothelioma-associated tumour antigens. Focusing on cancer/testis antigens, the expression of well-characterised immunogenic tumour-associated antigens was investigated in malignant mesothelioma cells. At variance with MAGE-4 and NY-ESO-1, malignant mesothelioma cells frequently expressed MAGE-1, -2 and -3, GAGE 1-2, GAGE 1-6, SSX-2 and SSX 1-5, and distinct malignant mesothelioma cells concomitantly expressed at least four cancer/testis antigens. Additionally, the tumour-associated antigens RAGE-1 was expressed at high levels in both benign and malignant mesothelial cells. Lastly, treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine induced and up-regulated the expression of the cancer/testis antigen examined in malignant mesothelioma cells. Overall, these findings strongly suggest that cancer/testis antigens-based immunotherapy may represent a suitable therapeutic approach to malignant mesothelioma, and foresee the clinical use of 5-aza-2'-deoxycytidine to design new chemo-immunotherapeutic strategies in malignant mesothelioma patients.


Subject(s)
Antigens, Neoplasm/analysis , DNA Methylation , Membrane Proteins , Mesothelioma/immunology , Testis/immunology , Animals , Azacitidine/pharmacology , Female , Humans , Immunotherapy , Male , Melanoma-Specific Antigens , Mesothelioma/therapy , Neoplasm Proteins/analysis , Proteins/analysis , Rabbits , Repressor Proteins/analysis
4.
J Immunol ; 167(5): 2529-37, 2001 Sep 01.
Article in English | MEDLINE | ID: mdl-11509592

ABSTRACT

This study investigates the differential capacity of TAP-deficient T2 cells, TAP-competent EBV cells, and immature and mature dendritic cells to present peptides to preformed CTL lines. It demonstrates that presentation of exogenous peptides involves peptide uptake and loading onto newly synthesized MHC class I molecules. This mechanism was best demonstrated for low affinity peptides in the presence of irrelevant peptides competing for HLA binding sites. Under these circumstances, inhibition of protein synthesis with cycloheximide or vesicular trafficking with brefeldin A significantly reduced the presentation of low affinity peptides. This was not restored by adding exogenous beta(2)-microglobulin to stabilize the MHC complex on the cell surface. In contrast, presentation of high affinity peptides was not sensitive to cycloheximide or brefeldin A, which suggests that different mechanisms may operate for presentation of high and low affinity peptides by TAP-competent cells. High affinity peptides can apparently compete with peptides in preloaded MHC class I molecules at the cell surface, whereas low affinity peptides require empty MHC molecules within cells. Accordingly, very high concentrations of exogenous low affinity peptides in conjunction with active MHC class I metabolism were required to allow successful presentation against a background of competing intracellular high affinity peptides in TAP-competent cells. These findings have implications for the design of peptide and protein-based vaccines.


Subject(s)
ATP-Binding Cassette Transporters/immunology , ATP-Binding Cassette Transporters/metabolism , Antigen Presentation/physiology , Peptides/immunology , Peptides/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 2 , Amino Acid Sequence , Antigens, Neoplasm , Binding, Competitive , Cell Differentiation , Cell Line , Cell Membrane/immunology , Cell Membrane/metabolism , Dendritic Cells/cytology , Dendritic Cells/immunology , Dendritic Cells/metabolism , HLA-A2 Antigen/metabolism , Histocompatibility Antigens Class I/metabolism , Humans , Interferon-gamma/biosynthesis , Intracellular Fluid/immunology , Intracellular Fluid/metabolism , Kinetics , MART-1 Antigen , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Neoplasm Proteins/metabolism , T-Lymphocytes, Cytotoxic/immunology
5.
Trends Immunol ; 22(1): 41-7, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11286691

ABSTRACT

Different subsets of dendritic cells (DCs) appear to play a role in determining the specific cytokines secreted by T helper (Th) cells. A model is proposed that links together factors such as the pathogen, microenvironment, DCs and T cells in a mechanism that results in a flexible determination of T-cell polarization.


Subject(s)
Adjuvants, Immunologic/physiology , Dendritic Cells/immunology , Growth Substances/immunology , Animals , Cell Differentiation/immunology , Cell Lineage/immunology , Dendritic Cells/classification , Dendritic Cells/cytology , Humans
6.
Vaccine ; 19(17-19): 2661-5, 2001 Mar 21.
Article in English | MEDLINE | ID: mdl-11257406

ABSTRACT

ISCOMs are typically 40 nm cage-like structures comprising antigen, saponin, cholesterol and phospholipid. ISCOMs have been shown to induce antibody responses and activate T helper cells and cytolytic T lymphocytes in a number of animal species, including non-human primates. Recent clinical studies have demonstrated that ISCOMs are also able to induce antibody and cellular immune responses in humans. This review describes the current understanding of the ability of ISCOMs to induce immune responses and the mechanisms underlying this property. Recent progress in the characterisation and manufacture of ISCOMs will also be discussed.


Subject(s)
ISCOMs/administration & dosage , Animals , Humans , Immunity, Cellular , Mice , Models, Animal , Primates , T-Lymphocytes, Cytotoxic/immunology , Vaccines/administration & dosage
7.
Lancet Oncol ; 2(6): 343-53, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11905751

ABSTRACT

Although the immune system evolved to protect the host from infection, what fires the popular imagination is its potential to recognise and destroy cancer. The immune system can generate potent cytotoxicity (eg transplant rejection), but can these mechanisms be harnessed for therapeutic benefit in patients with cancer? The discovery of an ever-increasing array of tumour antigens shows clearly that the targets exist. The challenge lies in generating a sufficiently potent response towards them. Central to the processes of antigen recognition, processing, and presentation to the immune system are dendritic cells. Understanding of the relation between these and the cellular immune response is crucial to elucidation of how to manipulate immune responses. The past 20 years have witnessed a dramatic expansion in this understanding and led to the first early-phase clinical trials of dendritic cells for the treatment of cancer. These studies have established the safety and feasibility of this approach and have produced encouraging evidence of therapeutic efficacy. This paper reviews the biology of dendritic cells and their use in clinical trials, as well as highlighting issues for future trial design.


Subject(s)
Dendritic Cells , Neoplasms/therapy , Adjuvants, Immunologic/therapeutic use , Antigens, Neoplasm/physiology , Carcinoma, Renal Cell/therapy , Clinical Trials as Topic , Dendritic Cells/physiology , Forecasting , Humans , Kidney Neoplasms/therapy , Lymphoma/therapy , Male , Melanoma/therapy , Multiple Myeloma/therapy , Prostatic Neoplasms/therapy
8.
J Exp Med ; 192(6): 823-33, 2000 Sep 18.
Article in English | MEDLINE | ID: mdl-10993913

ABSTRACT

Interleukin (IL)-12 may be secreted as a bioactive T helper type 1 (Th1) cell-inducing heterodimer, as a monomer, or as an antagonistic homodimer. We analyzed the IL-12 produced by mouse splenic dendritic cells (DCs), human thymic DCs, and cultured human monocyte-derived DCs. IL-12 production required both a microbial or T cell-derived stimulus and an appropriate cytokine milieu. The different IL-12 forms were differentially regulated by the cytokines present rather than the stimulus used. IL-4 alone or together with granulocyte/macrophage colony-stimulating factor or interferon gamma effectively enhanced the production of the bioactive heterodimer and selectively reduced the antagonistic homodimer of IL-12. Therefore, IL-4, the major Th2-driving cytokine, provides a negative feedback causing DCs to produce the major Th1-inducing cytokine, bioactive IL-12.


Subject(s)
Dendritic Cells/immunology , Interleukin-12/genetics , Interleukin-4/pharmacology , Animals , Cells, Cultured , Dendritic Cells/drug effects , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Interferon-gamma/pharmacology , Interleukin-12/biosynthesis , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred C57BL , Monocytes/immunology , Recombinant Proteins/pharmacology , Spleen/immunology , T-Lymphocytes/immunology , Thymus Gland/immunology
9.
Blood ; 96(3): 878-84, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10910900

ABSTRACT

Dendritic cells (DCs) represent a family of ontogenically distinct leukocytes involved in immune response regulation. The ability of DCs to stimulate T-cell immunity has led to their use as vectors for immunotherapy vaccines. However, it is unclear whether and to what degree in vitro-generated DCs are representative of DCs that develop in vivo. Treatment of mice with human Flt3 ligand (FL) dramatically increases the number of DCs. We report here that administration of FL to healthy human volunteers increased the number of circulating CD11c(+ )IL-3Ralpha(low) DC (mean 44-fold) and CD11c(-) IL-3Ralpha(high) DC precursors (mean 12-fold). Moreover, the CD11c(+ )DCs were efficient stimulators of T cells in vitro. Thus, FL can expand the number of circulating, functionally competent human DCs in vivo.


Subject(s)
Dendritic Cells/immunology , Immunity, Cellular , Membrane Proteins/immunology , Adjuvants, Immunologic/administration & dosage , Cell Differentiation/drug effects , Cell Differentiation/immunology , Dendritic Cells/cytology , Humans , Ligands , Membrane Proteins/administration & dosage , T-Lymphocytes/immunology
10.
Int J Cancer ; 86(6): 827-34, 2000 Jun 15.
Article in English | MEDLINE | ID: mdl-10842197

ABSTRACT

A defective function of the antigen-presenting cells may represent one of the ways used by cancer cells to escape the immune response. We have previously shown that human and rat colon carcinomas were infiltrated by dendritic cells that did not express the B7 co-stimulatory molecules required for inducing an efficient T-cell response. Flt3 ligand is a cloned hematopoietic growth factor that markedly augments the number of functional dendritic and NK cells in lymphoid and non-lymphoid tissues and exerts anti-tumor activity in various experimental models. We show here that repeated Flt3 ligand administration delays the s.c. growth of rat colon cancer cells in syngeneic animals without inducing tumor regression. In tumor-bearing animals, Flt3 ligand has a limited stimulatory effect on the antigen-presenting capacity of intra-tumoral and splenic dendritic cells, without restoring the high functional level of dendritic cells from tumor-free animals. Moreover, Flt3 ligand-mediated activation of NK cell cytotoxicity decreases when the tumor mass increases. Our results indicate that Flt3 ligand treatment of tumor-bearing animals does not sufficiently overcome tumor-induced immunosuppression to restore the inhibited functions of dendritic and NK cells and allow complete tumor regression.


Subject(s)
Antineoplastic Agents/pharmacology , Colonic Neoplasms/drug therapy , Dendritic Cells/drug effects , Membrane Proteins/pharmacology , Animals , Antigen Presentation/drug effects , Colonic Neoplasms/immunology , Colonic Neoplasms/pathology , Dendritic Cells/physiology , Killer Cells, Natural/drug effects , Killer Cells, Natural/physiology , Lymphocyte Activation/drug effects , Mice , Neoplasm Transplantation , Rats , Spleen/drug effects , Spleen/immunology
11.
J Immunol ; 165(1): 49-58, 2000 Jul 01.
Article in English | MEDLINE | ID: mdl-10861034

ABSTRACT

Dendritic cells (DC) are potent APCs that can be characterized in the murine spleen as CD11b(high)CD11c(high) or CD11b(low)CD11c(high). Daily injection of mice of Flt3 ligand (FL) into mice transiently expands both subsets of DC in vivo, but the effect of administration of GM-CSF on the expansion of DC in vivo is not well defined. To gain further insight into the role of GM-CSF in DC development and function in vivo, we treated mice with polyethylene glycol-modified GM-CSF (pGM-CSF) which has an increased half-life in vivo. Administration of pGM-CSF to mice for 5 days led to a 5- to 10-fold expansion of CD11b(high)CD11c(high) but not CD11b(low)CD11c(high) DC. DC from pGM-CSF-treated mice captured and processed Ag more efficiently than DC from FL-treated mice. Although both FL- and pGM-CSF-generated CD11b(high)CD11c(high) DC were CD8alpha-, a greater proportion of these DC from pGM-CSF-treated mice were 33D1+ than from FL-treated mice. CD11b(low)CD11c(high) DC from FL-treated mice expressed high levels of intracellular MHC class II. DC from both pGM-CSF- and FL-treated mice expressed high levels of surface class II, low levels of the costimulatory molecules CD40, CD80, and CD86 and were equally efficient at stimulating allogeneic and Ag-specific T cell proliferation in vitro. The data demonstrate that treatment with pGM-CSF in vivo preferentially expands CD11b(high)CD11c(high) DC that share phenotypic and functional characteristics with FL-generated CD11b(high)CD11c(high) DC but can be distinguished from FL-generated DC on the basis of Ag capture and surface expression of 33D1.


Subject(s)
Dendritic Cells/cytology , Dendritic Cells/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Integrin alphaXbeta2/biosynthesis , Macrophage-1 Antigen/biosynthesis , Membrane Proteins/physiology , Polyethylene Glycols/pharmacology , Animals , Antigen Presentation , B7-1 Antigen/biosynthesis , Biomarkers , CD40 Antigens/biosynthesis , Cell Division/immunology , Dendritic Cells/metabolism , Dextrans/immunology , Dextrans/metabolism , Female , Granulocyte-Macrophage Colony-Stimulating Factor/administration & dosage , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacokinetics , Half-Life , Hematopoiesis/immunology , Histocompatibility Antigens Class II/biosynthesis , Injections, Intravenous , Intracellular Fluid/immunology , Intracellular Fluid/metabolism , Ligands , Lymphocyte Activation/immunology , Membrane Proteins/administration & dosage , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout , Mice, Transgenic , Ovalbumin/immunology , Ovalbumin/metabolism , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/deficiency , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Recombinant Proteins , T-Lymphocytes/immunology
12.
Blood ; 95(11): 3489-97, 2000 Jun 01.
Article in English | MEDLINE | ID: mdl-10828034

ABSTRACT

The ligand for the receptor tyrosine kinase fms-like tyrosine kinase 3 (flt3), also referred to as fetal liver kinase-2 (flk-2), has an important role in hematopoiesis. The flt3 ligand (flt3L) is a growth factor for hematopoietic progenitors and induces hematopoietic progenitor and stem cell mobilization in vivo. In addition, when mice are treated with flt3L immature B cells, natural killer (NK) cells and dendritic cells (DC) are expanded in vivo. To further elucidate the role of flt3L in hematopoiesis, mice lacking flt3L (flt3L-/-) were generated by targeted gene disruption. Leukocyte cellularity was reduced in the bone marrow, peripheral blood, lymph nodes (LN), and spleen. Thymic cellularity, blood hematocrit, and platelet numbers were not affected. Significantly reduced numbers of myeloid and B-lymphoid progenitors were noted in the BM of flt3L-/- mice. In addition a marked deficiency of NK cells in the spleen was noted. DC numbers were also reduced in the spleen, LN, and thymus. Both myeloid-related (CD11c(++) CD8alpha(-)) and lymphoid-related (CD11c(++) CD8alpha(+)) DC numbers were affected. We conclude that flt3L has an important role in the expansion of early hematopoietic progenitors and in the generation of mature peripheral leukocytes.


Subject(s)
B-Lymphocytes/cytology , Dendritic Cells/cytology , Hematopoiesis/physiology , Hematopoietic Stem Cells/immunology , Killer Cells, Natural/cytology , Membrane Proteins/physiology , Animals , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Bone Marrow/immunology , Colony-Forming Units Assay , Dendritic Cells/drug effects , Dendritic Cells/immunology , Genomic Library , Hematopoiesis/genetics , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Humans , Interleukin-7/pharmacology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Kinetics , Leukocytes/cytology , Ligands , Lymph Nodes/immunology , Lymphocyte Culture Test, Mixed , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , Poly I-C/pharmacology , Recombinant Proteins/pharmacology , Spleen/immunology , Thymus Gland/immunology
13.
Nat Med ; 5(4): 405-11, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10202929

ABSTRACT

Cytotoxic T lymphocytes and natural killer cells are essential effectors of anti-tumor immune responses in vivo. Dendritic cells (DC) 'prime' tumor antigen-specific cytotoxic T lymphocytes; thus, we investigated whether DC might also trigger the innate, NK cell-mediated anti-tumor immunity. In mice with MHC class I-negative tumors, adoptively transferred- or Flt3 ligand-expanded DC promoted NK cell-dependent anti-tumor effects. In vitro studies demonstrated a cell-to-cell contact between DC and resting NK cells that resulted in a substantial increase in both NK cell cytolytic activity and IFN-gamma production. Thus, DC are involved in the interaction between innate and adaptive immune responses.


Subject(s)
Cytotoxicity, Immunologic , Dendritic Cells/immunology , Killer Cells, Natural/immunology , Neoplasms, Experimental/immunology , Adoptive Transfer , Animals , Cell Communication , Coculture Techniques , DNA-Binding Proteins , Ligands , Lymphocyte Activation , Major Histocompatibility Complex/immunology , Membrane Proteins/immunology , Mice , Mice, Inbred Strains , Mice, Mutant Strains , Neoplasms, Experimental/classification
14.
Proc Natl Acad Sci U S A ; 96(3): 1036-41, 1999 Feb 02.
Article in English | MEDLINE | ID: mdl-9927689

ABSTRACT

Dendritic cells (DCs) are unique in their ability to stimulate T cells and initiate adaptive immunity. Injection of mice with the cytokine Flt3-ligand (FL) dramatically expands mature lymphoid and myeloid-related DC subsets. In contrast, injection of a polyethylene glycol-modified form of granulocyte/macrophage colony-stimulating factor (GM-CSF) into mice only expands the myeloid-related DC subset. These DC subsets differ in the cytokine profiles they induce in T cells in vivo. The lymphoid-related subset induces high levels of the Th1 cytokines interferon gamma and interleukin (IL)-2 but little or no Th2 cytokines. In contrast, the myeloid-related subset induces large amounts of the Th2 cytokines IL-4 and IL-10, in addition to interferon gamma and IL-2. FL- or GM-CSF-treated mice injected with soluble ovalbumin display dramatic increases in antigen-specific antibody titers, but the isotype profiles seem critically dependent on the cytokine used. Although FL treatment induces up to a 10, 000-fold increase in ovalbumin-specific IgG2a and a more modest increase in IgG1 titers, GM-CSF treatment favors a predominantly IgG1 response with little increase in IgG2a levels. These data suggest that distinct DC subsets have strikingly different influences on the type of immune response generated in vivo and may thus be targets for pharmacological intervention.


Subject(s)
Dendritic Cells/immunology , Membrane Proteins/immunology , Adoptive Transfer , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , CHO Cells , Cricetinae , Crosses, Genetic , Dendritic Cells/classification , Dendritic Cells/drug effects , Flow Cytometry , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Lymphoid Tissue/cytology , Lymphoid Tissue/immunology , Male , Membrane Proteins/pharmacology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , Recombinant Proteins/immunology , Recombinant Proteins/pharmacology , Species Specificity , T-Lymphocytes/immunology
16.
J Exp Med ; 188(11): 2075-82, 1998 Dec 07.
Article in English | MEDLINE | ID: mdl-9841921

ABSTRACT

Injections of soluble proteins are poorly immunogenic, and often elicit antigen-specific tolerance. The mechanism of this phenomenon has been an enduring puzzle, but it has been speculated that tolerance induction may be due to antigen presentation by poorly stimulatory, resting B cells, which lack specific immunoglobulin receptors for the protein. In contrast, adjuvants, or infectious agents, which cause the release of proinflammatory cytokines such as tumor necrosis factor alpha and interleukin 1beta in vivo are believed to recruit and activate professional antigen-presenting cells to the site(s) of infection, thereby eliciting immunity. Here we show that administration of Flt3 ligand (FL), a cytokine capable of inducing large numbers of dendritic cells (DCs) in vivo, (a) dramatically enhances the sensitivity of antigen-specific B and T cell responses to systemic injection of a soluble protein, through a CD40-CD40 ligand-dependent mechanism; (b) influences the class of antibody produced; and (c) enables productive immune responses to otherwise tolerogenic protocols. These data support the hypothesis that the delicate balance between immunity and tolerance in vivo is pivotally controlled by DCs, and underscore the potential of FL as a vaccine adjuvant for immunotherapy in infectious disease and other clinical settings.


Subject(s)
Dendritic Cells/immunology , Immune Tolerance , Membrane Proteins/immunology , Adjuvants, Immunologic/administration & dosage , Animals , Antigen Presentation , CD40 Antigens/immunology , Cell Communication/immunology , Membrane Proteins/administration & dosage , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL
17.
Int Immunol ; 10(9): 1367-75, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9786436

ABSTRACT

IL-7 receptor-deficient (IL-7R(-/-)) mice are lymphopenic as a result of defective cell production at early steps in both B and T lymphopoiesis. In the bone marrow, there is an incomplete block in B cell development at the transition from the pro-B to the pre-B cell stage. As a consequence, peripheral lymphoid organs of IL-7R(-/-) mice contain abnormally low numbers of mature surface (s) Ig-expressing B cells and this is accompanied by a relative increase in immature sIg- B cells. Transgenic expression of the anti-apoptotic protein Bcl-2 in IL-7R(-/-) mice rescues the defect in T cell development and in mature T cell function. The present report shows that constitutive expression of Bcl-2 is incapable of rescuing B lymphopoiesis in IL-7R(-/-) mice but can enhance survival of those mature B cells which escape the developmental arrest. Thus the essential role of IL-7R signaling in B lymphoid cells cannot be replaced by Bcl-2, indicating that in B lymphopoiesis IL-7R signaling is necessary for promoting cell division and/or for inhibiting a Bcl-2-insensitive pathway to apoptosis.


Subject(s)
B-Lymphocytes/cytology , Hematopoiesis/physiology , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Receptors, Interleukin-7/deficiency , Animals , Apoptosis/physiology , B-Lymphocytes/drug effects , Cell Differentiation/physiology , Cell Survival/physiology , Female , Gene Expression , Lymphoid Tissue/cytology , Lymphoid Tissue/metabolism , Male , Mice , Mitogens/pharmacology , Receptors, Interleukin-7/physiology , Signal Transduction/physiology , Stimulation, Chemical , Transgenes
18.
Nature ; 390(6656): 175-9, 1997 Nov 13.
Article in English | MEDLINE | ID: mdl-9367155

ABSTRACT

Dendritic cells are rare haematopoietic cells that reside in a number of organs and tissues. By capturing, processing and presenting antigens to T cells, dendritic cells are essential for immune surveillance and the regulation of specific immunity. Several members of the tumour necrosis factor receptor (TNFR) superfamily are integral to the regulation of the immune response. These structurally related proteins modulate cellular functions ranging from proliferation and differentiation to inflammation and cell survival or deaths. The functional activity of dendritic cells is greatly increased by signalling through the TNFR family member CD40. Here we report the characterization of RANK (for receptor activator of NF-kappaB), a new member of the TNFR family derived from dendritic cells, and the isolation of a RANK ligand (RANKL) by direct expression screening. RANKL augments the ability of dendritic cells to stimulate naive T-cell proliferation in a mixed lymphocyte reaction, and increases the survival of RANK+ T cells generated with interleukin-4 and transforming growth factor (TGF)-beta. Thus RANK and RANKL seem to be important regulators of interactions between T cells and dendritic cells.


Subject(s)
Carrier Proteins , Dendritic Cells/immunology , Receptors, Tumor Necrosis Factor/immunology , T-Lymphocytes/immunology , Amino Acid Sequence , Animals , CD40 Ligand , Cell Line , Cells, Cultured , Chromosome Mapping , Chromosomes, Human, Pair 13 , Chromosomes, Human, Pair 18 , Humans , Ligands , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , Receptors, Tumor Necrosis Factor/analysis , Receptors, Tumor Necrosis Factor/genetics , Sequence Homology, Amino Acid , Transfection
19.
J Immunol ; 159(5): 2222-31, 1997 Sep 01.
Article in English | MEDLINE | ID: mdl-9278310

ABSTRACT

We have recently shown that Flt3 ligand administration dramatically increases dendritic cell (DC) numbers in various mouse tissues. This has enabled the identification of distinct mature DC subpopulations. These have been designated: population C (CD11c(bright) CD11b(bright)), D (CD11c(bright) CD11b(dull)), and E (CD11c(bright) CD11b(negative)) This report demonstrates that the mature DC subsets (C, D, and E) from Flt3 ligand-treated mice differ with respect to phenotype, geographic localization, and function. The myeloid Ags CD11b, F4/80, and Ly-6C are predominantly expressed by population C, but not D or E. In addition, a subset of population C-type DC expresses 33D1 and CD4. In contrast, DC within population D and E selectively express the lymphoid-related DC markers CD8alpha, DEC 205, CD1d, as well as CD23, elevated levels of CD117 (c-kit), CD24 (HSA), CD13, and CD54. Immunohistology indicates that the different DC subsets reside in distinct microenvironments, with populations D and E residing in the T cell areas of the white pulp, while DC within population C localize in the marginal zones. These DC subpopulations showed different capacities to phagocytose FITC-zymosan and to secrete IL-12 upon stimulation with Staphylococcus aureus cowan I strain + IFN-gamma + granulocyte-macrophage-CSF. Population C-type DC were more phagocytic but secreted little inducible IL-12 while population D- and E-type DC showed poor phagocytic capacity and secreted considerably higher levels of IL-12. These results underscore the importance of viewing DC development in vivo, as an interplay between distinct lineages and a maturational dependence on specific microenvironmental signals.


Subject(s)
Dendritic Cells/cytology , Membrane Proteins/pharmacology , Animals , Antigens, Differentiation/analysis , Cell Differentiation/drug effects , Cell Lineage , Dendritic Cells/classification , Dendritic Cells/immunology , Female , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Immunophenotyping , Interleukin-12/biosynthesis , Interleukin-4/pharmacology , Mice , Mice, Inbred C57BL , Models, Immunological , Phagocytosis , Recombinant Proteins/pharmacology , Specific Pathogen-Free Organisms , Spleen/cytology
20.
Cell Immunol ; 179(2): 174-84, 1997 Aug 01.
Article in English | MEDLINE | ID: mdl-9268501

ABSTRACT

FLT3 ligand (FL) is a recently described hematopoietic growth factor that stimulates the proliferation and differentiation of hematopoietic progenitors. We have investigated the effect of FL on murine hematopoiesis and dendritic cell (DC) generation and accumulation in lymphoid tissues and liver in vivo and in vitro evaluating the morphologic, phenotypic, and functional characteristics of these DC. We have observed extramedullary hematopoiesis in the mouse spleen with all lineages of hematopoietic cells represented after the administration of FL. Injection of FL results in a time-dependent and reversible accumulation of DC in the spleen, bone marrow, lymph nodes, and liver. Both flow cytometry and immunohistochemistry revealed a significant accumulation of DC in these tissues. Results of mixed leukocyte reaction suggested that these cells, isolated from murine bone marrow or spleen, were active as antigen presenting cells. Furthermore, cultivation of splenic and marrow cells with GM-CSF and IL-4 gave rise to large numbers of functionally active mature DC. Thus, the results of this study suggest that FL is a promising growth factor that stimulates the generation of large number of DC and may be a useful cytokine for the immunotherapy of cancer.


Subject(s)
Dendritic Cells/cytology , Dendritic Cells/immunology , Hematopoiesis, Extramedullary/drug effects , Hematopoiesis, Extramedullary/immunology , Membrane Proteins/pharmacology , Animals , Antigen-Presenting Cells/cytology , Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/immunology , Cell Differentiation/drug effects , Cell Differentiation/immunology , Dendritic Cells/drug effects , Female , Injections, Subcutaneous , Ligands , Membrane Proteins/administration & dosage , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL
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