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Ralph M Steinman

Publications and source records attributed to Ralph M Steinman.

At least 37 records · Page 2Linked to original sources
PubMed2005

Network of dendritic cells within the muscular layer of the mouse intestine.

Dendritic cells (DCs) are located at body surfaces such as the skin, respiratory and genital tracts, and intestine. To further analyze intestinal DCs, we adapted an epidermal sheet separation technique and obtained two intestinal layers, facing the lumen and serosa. Unexpectedly, immunolabeling of the layer toward the serosa revealed a regular, dense, planar network of cells with prominent dendritic morphology within the external muscular layer and with increasing frequency along the length of the intestine. Direct examination of the serosal-disposed layers showed a significant fraction of the DCs to express DEC-205/CD205, CD11c, Langerin/CD207, Fcgamma receptor/CD16/32, CD14, and low levels of activation markers, CD25, CD80, CD86, and CD95. By more sensitive FACS analyses, cells from this layer contained two CD11c(+) populations of CD45(+) CD205(+), CD19(-) leukocytes, MHC II(+) and MHC II(-). When ovalbumin conjugated to an anti-DEC-205 antibody was injected into mice, the conjugate targeted to these DCs, which upon isolation were able to stimulate ovalbumin-specific, CD4(+) and CD8(+) T cell antigen receptor-transgenic T cells. In vivo, these DCs responded to two microbial stimuli, systemic LPS and oral live bacteria, by up-regulating CD80, CD86, DEC-205, and Langerin within 12 h. This network of DCs thus represents a previously unrecognized antigen-presenting cell system in the intestine.

Animals↗
PubMed2005

Innate NKT lymphocytes confer superior adaptive immunity via tumor-capturing dendritic cells.

If irradiated tumor cells could be rendered immunogenic, they would provide a safe, broad, and patient-specific array of antigens for immunotherapies. Prior approaches have emphasized genetic transduction of live tumor cells to express cytokines, costimulators, and surrogate foreign antigens. We asked if immunity could be achieved by delivering irradiated, major histocompatibility complex-negative plasmacytoma cells to maturing mouse dendritic cells (DCs) within lymphoid organs. Tumor cells injected intravenously (i.v.) were captured by splenic DCs, whereas subcutaneous (s.c.) injection led only to weak uptake in lymph node or spleen. The natural killer T (NKT) cells mobilizing glycolipid alpha-galactosyl ceramide, used to mature splenic DCs, served as an effective adjuvant to induce protective immunity. This adjuvant function was mimicked by a combination of poly IC and agonistic alphaCD40 antibody. The adjuvant glycolipid had to be coadministered with tumor cells i.v. rather than s.c. Specific resistance was generated both to a plasmacytoma and lymphoma. The resistance afforded by a single vaccination lasted >2 mo and required both CD4+ and CD8+ T cells. Mature tumor capturing DCs stimulated the differentiation of P1A tumor antigen-specific, CD8+ T cells and uniquely transferred tumor resistance to naive mice. Therefore, the access of dying tumor cells to DCs that are maturing to activated NKT cells efficiently induces long-lived adaptive resistance.

Adjuvants, Immunologic↗
PubMed2005

Dendritic cells expand Epstein Barr virus specific CD8+ T cell responses more efficiently than EBV transformed B cells.

Adoptive transfer of Epstein Barr virus (EBV) specific cytotoxic T lymphocytes (CTLs) has been successfully applied in the treatment of EBV associated post-transplant lymphoproliferative disease (PTLD). In most studies EBV transformed B cells (LCLs) have been used for the induction of EBV specific T cell lines. Application of this approach to other EBV associated tumors is difficult, because LCLs focus T cell expansion toward immunodominant EBV antigens that are not expressed in EBV associated Hodgkin's lymphoma and nasopharyngeal carcinoma. Therefore, we compared dendritic cells (DCs) with LCLs for CD8+ T cell stimulation against dominant and subdominant EBV antigens. DCs expanded tenfold more EBNA3A and LMP2 specific CD8+ T cells than LCL and also stimulated EBV specific CTL from PTLD patients. Both, DCs and LCLs stimulations led to the expansion of high affinity T cells, capable to target EBV transformed B cells. While LCLs and DCs expressed MHC class I and II products at similar levels, DCs showed a higher expression of costimulatory and adhesion molecules. This resulted in more efficient T cell conjugate formation with DCs than with LCLs. We propose the use of DCs for stimulation of EBV specific T cells in active or passive immunotherapy of EBV associated malignancies.

Antigens, Surface↗
PubMed2005

Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin/CD209 is abundant on macrophages in the normal human lymph node and is not required for dendritic cell stimulation of the mixed leukocyte reaction.

The C-type lectin dendritic cell-specific ICAM 3-grabbing nonintegrin (DC-SIGN)/CD209 efficiently binds several pathogens, including HIV-1. DC-SIGN is expressed on monocyte-derived DCs in culture, and importantly, it is able to sequester HIV-1 within cells and facilitate transmission of virus to CD4+ T cells. To investigate DC-SIGN function, we have generated new mAbs. We report in this study that these and prior anti-DC-SIGN mAbs primarily label macrophages in the medullary sinuses of noninflamed human lymph node. In contrast, expression is not detected on most DCs in the T cell area, except for occasional cells. We also noted that IL-4 alone can induce expression of DC-SIGN in CD14+ monocytes and circulating blood DCs. However, blockade of DC-SIGN with Abs and DC-SIGN small interfering RNA did not result in a major reduction in the capacity of these DCs to transfer HIV to T cells, confirming significant DC-SIGN-independent mechanisms. The blocking approaches did reduce HIV-1 transmission by DC-SIGN-transfected cells by >90%. DC-SIGN blockade also did not reduce the ability of DCs to stimulate T cell proliferation in the MLR. These results indicate that DC-SIGN has the potential to contribute to macrophage function in normal human lymph node, and that DCs do not require DC-SIGN to transmit HIV or to initiate T cell responses.

Animals↗
PubMed2005

Dendritic cell maturation by innate lymphocytes: coordinated stimulation of innate and adaptive immunity.

Pathogen recognition by Toll-like receptors (TLRs) on dendritic cells (DCs) leads to DC maturation and the initiation of adaptive immunity. Recent studies have shown that innate lymphocytes--natural killer (NK), natural killer T (NKT), and gammadelta T cells--also trigger DC maturation. This interaction in turn expands and activates innate lymphocytes and initiates adaptive T cell immunity. Here, we comment on the evidence that these pathways are TLR independent and have the potential to respond to infection, malignancy, and immunotherapy.

Animals↗
PubMed2005

Association of SIGNR1 with TLR4-MD-2 enhances signal transduction by recognition of LPS in gram-negative bacteria.

SIGNR1, a member of a new family of mouse C-type lectins, is expressed at high levels in macrophages (Mphi) within the splenic marginal zone, lymph node medulla, and in some strains, in peritoneal cavity. We previously reported that SIGNR1 captures gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium, as well as Candida albicans. We have now investigated the precise ligands and innate responses that involve SIGNR1. The interaction of SIGNR1 with FITC-dextran and E. coli was completely inhibited by LPS from E. coli and Salmonella minnesota. Using LPS from various types of rough mutants of Salmonella, we found that SIGNR1 primarily recognizes oligosaccharides in the non-reductive end of the LPS core region. In transfectants, expression of SIGNR1 enhanced the oligomerization of Toll-like receptor (TLR) 4 molecules as well as the degradation of IkappaB-alpha after stimulation with E. coli under low-serum conditions. The enhanced TLR4 oligomerization was inhibited by pre-treatment of the cells with anti-SIGNR1 mAb or with mannan. A physical association between SIGNR1 and the TLR4-MD-2 complex was also observed by immunoprecipitation. Finally, we found that transfection of SIGNR1 into the macrophage-like RAW264.7 cells resulted in significant augmentation of cytokine production. These results suggest that SIGNR1 associates with TLR4 to capture gram-negative bacteria and facilitate signal transduction to activate innate M responses.

Animals↗
PubMed2005

Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity.

Langerhans cells (LC) form a unique subset of dendritic cells (DC) in the epidermis but so far their in vivo functions in skin immunity and tolerance could not be determined, in particular in relation to dermal DC (dDC). Here, we exploit a novel diphtheria toxin (DT) receptor (DTR)/DT-based system to achieve inducible ablation of LC without affecting the skin environment. Within 24 h after intra-peritoneal injection of DT into Langerin-DTR mice LC are completely depleted from the epidermis and only begin to return 4 wk later. LC deletion occurs by apoptosis in the absence of inflammation and, in particular, the dDC compartment is not affected. In LC-depleted mice contact hypersensitivity (CHS) responses are significantly decreased, although ear swelling still occurs indicating that dDC can mediate CHS when necessary. Our results establish Langerin-DTR mice as a unique tool to study LC function in the steady state and to explore their relative importance compared with dDC in orchestrating skin immunity and tolerance.

Animals↗
PubMed2005

CpG oligonucleotides induce strong humoral but only weak CD4+ T cell responses to protein antigens in rhesus macaques in vivo.

Oligonucleotides containing CpG motifs (CpG ODN) are strong adjuvants for humoral immune responses but data on cellular immune responses in primates are scarce. Rhesus macaque blood contained similar numbers of plasmacytoid dendritic cells and B cells, the key sensors of CpG ODN, as human blood, and these cells were activated by CpG-A and CpG-B in vitro. In vivo, both ODNs induced equal plasma levels of interferon-inducible protein 10 and similarly enhanced antibody responses following i.m. injections of the ODNs, protein antigen, and aluminium hydroxide into rhesus macaques, whereas antigen-specific CD4(+) T cell responses were only slightly increased by CpG ODN.

Adjuvants, Immunologic↗
PubMed2005

Sustained expansion of NKT cells and antigen-specific T cells after injection of alpha-galactosyl-ceramide loaded mature dendritic cells in cancer patients.

Natural killer T (NKT) cells are distinct glycolipid reactive innate lymphocytes that are implicated in the resistance to pathogens and tumors. Earlier attempts to mobilize NKT cells, specifically, in vivo in humans met with limited success. Here, we evaluated intravenous injection of monocyte-derived mature DCs that were loaded with a synthetic NKT cell ligand, alpha-galactosyl-ceramide (alpha-GalCer; KRN-7000) in five patients who had advanced cancer. Injection of alpha-GalCer-pulsed, but not unpulsed, dendritic cells (DCs) led to >100-fold expansion of several subsets of NKT cells in all patients; these could be detected for up to 6 mo after vaccination. NKT activation was associated with an increase in serum levels of interleukin-12 p40 and IFN-gamma inducible protein-10. In addition, there was an increase in memory CD8+ T cells specific for cytomegalovirus in vivo in response to alpha-GalCer-loaded DCs, but not unpulsed DCs. These data demonstrate the feasibility of sustained expansion of NKT cells in vivo in humans, including patients who have advanced cancer, and suggest that NKT activation might help to boost adaptive T cell immunity in vivo.

Adult↗
PubMed2005

Selective blockade of inhibitory Fcgamma receptor enables human dendritic cell maturation with IL-12p70 production and immunity to antibody-coated tumor cells.

The final differentiation or maturation of dendritic cells (DCs) in response to environmental stimuli influences their ability to both initiate immunity and determine the quality of the response to antigens. Circulating immune complexes and cell-bound immunoglobulins present in normal human sera represent a potential stimulus for inadvertent DC activation in the steady state and during autoimmunity. Here, we show that selective blockade of the inhibitory Fcgamma receptor (FcgammaR) FcgammaRIIb with recently developed monoclonal antibodies leads to maturation of human monocyte-derived DCs, which depends on the presence of IgG in normal human plasma. Plasma, in the presence of an FcgammaRIIb blockade, caused the DCs to up-regulate the expression of costimulatory molecules and to produce the inflammatory mediator IL-12p70. FcgammaRIIb blockade of DCs loaded with tumor cells led to increased tumor-specific T cell immunity without the need for exogenous stimuli other than human plasma. Therefore, the activation status of DCs in the presence of normal human serum depends on the balance between activating and inhibitory FcgammaRs and can be enhanced by new antibodies that react selectively with FcgammaRIIb. These data suggest an approach for modifying this balance to enhance immunity to immune complexes and antibody-coated tumor cells and to silence DC activation by immune complexes in autoimmune states.

Antibodies, Monoclonal↗
PubMed2005

CD8+ T cells from most HIV-1-infected patients, even when challenged with mature dendritic cells, lack functional recall memory to HIV gag but not other viruses.

Chronically HIV-1-infected patients fail to contain their viremia despite high frequencies of HIV-1-specific, IFN-gamma-producing CD8(+) T cells. However, these cells are known to exhibit both phenotypic and functional defects. We tested if mature dendritic cells (DC) could correct defective HIV-1 gag-specific T cell responses and if responses to other viral antigens were comparably affected. The circulating gag-specific CD8(+) T cells in fresh blood reliably produced IFN-gamma but lacked IL-2 and high perforin levels and failed to expand significantly during culture with mature DC presenting HIV-1 gag peptides. In contrast, CD8(+) T cells from long-term nonprogressors contained gag-specific IFN-gamma and IL-2 double producers, and the numbers of IFN-gamma producers expanded approximately 15-fold during culture with DC. DC from chronically infected patients could expand IFN-gamma- and IL-2-producing cells specific for influenza, cytomegalovirus and Epstein Barr virus, and the expansions were comparable to those in healthy donors. When the proliferative capacity of CD8(+) T cells from progressor patients was assessed by CFSE dilution, proliferation to other viral antigens was more vigorous than to HIV-1 gag. Therefore, monocyte-derived DC from HIV patients present viral antigens effectively, but there is a selective inability to expand CD8(+) IFN-gamma-producing and IFN-gamma and IL-2 double-producing T cells when challenged with HIV-1 gag.

Antigen Presentation↗
PubMed2005

Harnessing the immune system against human glioma.

Our studies are directed toward immune resistance to glial tumors, particularly in children. Initial experiments indicate that it is possible to boost both innate natural killer T lymphocytes and adaptive CD4+ and CD8+ T cells by targeting the appropriate antigens to dendritic cells. Our hypothesis is that optimal vaccination approaches against cancer will need to recruit both innate and adaptive defense mechanisms.

Brain Neoplasms↗
PubMed2004

Induction of neutralising antibodies restricts the use of human granulocyte/macrophage colony stimulating factor for vaccine studies in rhesus macaques.

Granulocyte/macrophage-colony stimulating factor (GM-CSF) is a valuable adjuvant to enhance induction of cellular immune responses in rodents. Less information is available regarding its use as an adjuvant in primates or humans. We explored recombinant human GM-CSF for potential vaccine studies in rhesus macaques and focused on its effect on peripheral monocytes as progenitors of dendritic cells and its potential immunogenicity. Application of human GM-CSF to nine animals led to an average 32-fold increase in monocyte numbers. This was not observed upon re-treatment, which coincided with GM-CSF-specific neutralising antibodies. These also neutralised the activity of rhesus macaque GM-CSF. The data underscore the need to use species-specific GM-CSF for immunomodulation in primates.

Animals↗
PubMed2004

Immunotherapy: bewitched, bothered, and bewildered no more.

The field of immunotherapy holds clear promise not only for the development of new approaches to cancer and other diseases, but also for providing fundamental insight into the human immune response. In order for this promise to be realized, however, the scientific community must overcome an array of challenges. These challenges reflect not only the difficulties inherent in conducting investigations in human patients, but also difficulties created by the culture and practice of our own institutions, reward structure, and funding mechanisms. We suggest steps to be taken to reinvigorate basic research in human subjects as part of the mainstream of science.

Animals↗
PubMed2004

The linkage of innate to adaptive immunity via maturing dendritic cells in vivo requires CD40 ligation in addition to antigen presentation and CD80/86 costimulation.

Dendritic cell (DC) maturation is an innate response that leads to adaptive immunity to coadministered proteins. To begin to identify underlying mechanisms in intact lymphoid tissues, we studied alpha-galactosylceramide. This glycolipid activates innate Valpha14(+) natural killer T cell (NKT) lymphocytes, which drive DC maturation and T cell responses to ovalbumin antigen. Hours after giving glycolipid i.v., tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were released primarily by DCs. These cytokines induced rapid surface remodeling of DCs, including increased CD80/86 costimulatory molecules. Surprisingly, DCs from CD40(-/-) and CD40L(-/-) mice did not elicit CD4(+) and CD8(+) T cell immunity, even though the DCs exhibited presented ovalbumin on major histocompatibility complex class I and II products and expressed high levels of CD80/86. Likewise, an injection of TNF-alpha up-regulated CD80/86 on DCs, but CD40 was required for immunity. CD40 was needed for DC interleukin (IL)-12 production, but IL-12p40(-/-) mice generated normal ovalbumin-specific responses. Therefore, the link between innate and adaptive immunity via splenic DCs and innate NKT cells has several components under distinct controls: antigen presentation in the steady state, increases in costimulatory molecules dependent on inflammatory cytokines, and a distinct CD40/CD40L signal that functions together with antigen presentation ("signal one") and costimulation ("signal two") to generate functioning CD4(+) T helper cell 1 and CD8(+) cytolytic T lymphocytes.

Animals↗