Biomedical subjects

Christine Trumpfheller

Publications and source records attributed to Christine Trumpfheller.

10 recordsLinked to original sources

PubMed2007

DEC-205 receptor on dendritic cells mediates presentation of HIV gag protein to CD8+ T cells in a spectrum of human MHC I haplotypes.

Optimal HIV vaccines should elicit CD8+ T cells specific for HIV proteins presented on MHC class I products, because these T cells contribute to host resistance to viruses. We had previously found that the targeting of antigen to dendritic cells (DCs) in mice efficiently induces CD8+ T cell responses. To extend this finding to humans, we introduced the HIV p24 gag protein into a mAb that targets DEC-205/CD205, an endocytic receptor of DCs. We then assessed cross-presentation, which is the processing of nonreplicating internalized antigen onto MHC class I for recognition by CD8+ T cells. Low doses of alphaDEC-gag, but not control Ig-gag, stimulated proliferation and IFN-gamma production by CD8+ T cells isolated from the blood of HIV-infected donors. alphaCD205 fusion mAb was more effective for cross-presentation than alphaCD209/DC-SIGN, another abundant DC uptake receptor. Presentation was diverse, because we identified eight different gag peptides that were recognized via DEC-205 in 11 individuals studied consecutively. Our results, based on humans with highly polymorphic MHC products, reveal that DCs and DEC-205 can cross-present several different peptides from a single protein. Because of the consistency in eliciting CD8+ T cell responses, these data support the testing of alphaDEC-205 fusion mAb as a protein-based vaccine.

Amino Acid Sequence↗

PubMed2007

Differential antigen processing by dendritic cell subsets in vivo.

Dendritic cells (DCs) process and present self and foreign antigens to induce tolerance or immunity. In vitro models suggest that induction of immunity is controlled by regulating the presentation of antigen, but little is known about how DCs control antigen presentation in vivo. To examine antigen processing and presentation in vivo, we specifically targeted antigens to two major subsets of DCs by using chimeric monoclonal antibodies. Unlike CD8+ DCs that express the cell surface protein CD205, CD8- DCs, which are positive for the 33D1 antigen, are specialized for presentation on major histocompatibility complex (MHC) class II. This difference in antigen processing is intrinsic to the DC subsets and is associated with increased expression of proteins involved in MHC processing.

Amino Acid Sequence↗

PubMed2006

Intensified and protective CD4+ T cell immunity in mice with anti-dendritic cell HIV gag fusion antibody vaccine.

Current human immunodeficiency virus (HIV) vaccine approaches emphasize prime boost strategies comprising multiple doses of DNA vaccine and recombinant viral vectors. We are developing a protein-based approach that directly harnesses principles for generating T cell immunity. Vaccine is delivered to maturing dendritic cells in lymphoid tissue by engineering protein antigen into an antibody to DEC-205, a receptor for antigen presentation. Here we characterize the CD4+ T cell immune response to HIV gag and compare efficacy with other vaccine strategies in a single dose. DEC-205-targeted HIV gag p24 or p41 induces stronger CD4+ T cell immunity relative to high doses of gag protein, HIV gag plasmid DNA, or recombinant adenovirus-gag. High frequencies of interferon (IFN)-gamma- and interleukin 2-producing CD4+ T cells are elicited, including double cytokine-producing cells. In addition, the response is broad because the primed mice respond to an array of peptides in different major histocompatibility complex haplotypes. Long-lived T cell memory is observed. After subcutaneous vaccination, CD4+ and IFN-gamma-dependent protection develops to a challenge with recombinant vaccinia-gag virus at a mucosal surface, the airway. We suggest that a DEC-targeted vaccine, in part because of an unusually strong and protective CD4+ T cell response, will improve vaccine efficacy as a stand-alone approach or with other modalities.

AIDS Vaccines↗

PubMed2006

HIV-1 selectively infects a subset of nonmaturing BDCA1-positive dendritic cells in human blood.

The infection of cultured monocyte-derived dendritic cells (DCs) with HIV-1 involves CD4 and CCR5 receptors, while transmission to T cells is enhanced at least in part by the lectin DC-SIGN/CD209. In the present study, we studied BDCA-1+ myeloid DCs isolated directly from human blood. These cells express CD4 and low levels of CCR5 and CXCR4 coreceptors, but not DC-SIGN. The myeloid DCs replicate two R5 viruses, BaL and YU2, and transfer infection to activated T cells. The virus productively infects a small fraction of the blood DCs that fail to mature in culture, as indicated by the maturation markers CD83 and DC-LAMP/CD208, and the expression of high CD86 and MHC class II, in contrast to many noninfected DCs. A greater proportion of BDCA-1+ DCs are infected when the virus is pseudotyped with the vesicular stomatitis envelope VSV-G (5-15%), as compared with the R5 virus (0.3-3.5%), indicating that HIV-1 coreceptors may limit the susceptibility of DCs to become infected, or the endocytic route of viral entry used by HIV/vesicular stomatitis virus enhances infectivity. When infected and noninfected cells are purified by cell sorting, the former uniformly express HIV p24 gag and are virtually inactive as stimulators of the allogeneic MLR, in contrast to potent stimulation by noninfected DCs from the same cultures. These results point to two roles for a small fraction of blood DCs in HIV-1 pathogenesis: to support productive infection and to evade the direct induction of T cell-mediated immunity.

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↗

PubMed2004

HIV-1-infected monocyte-derived dendritic cells do not undergo maturation but can elicit IL-10 production and T cell regulation.

Dendritic cells (DCs) undergo maturation during virus infection and thereby become potent stimulators of cell-mediated immunity. HIV-1 replicates in immature DCs, but we now find that infection is not accompanied by many components of maturation in either infected cells or uninfected bystanders. The infected cultures do not develop potent stimulating activity for the mixed leukocyte reaction (MLR), and the DCs producing HIV-1 gag p24 do not express CD83 and DC-lysosome-associated membrane protein maturation markers. If different maturation stimuli are applied to DCs infected with HIV-1, the infected cells selectively fail to mature. When DCs from HIV-1-infected patients are infected and cultured with autologous T cells, IL-10 was produced in 6 of 10 patients. These DC-T cell cocultures could suppress another immune response, the MLR. The regulation was partially IL-10-dependent and correlated in extent with the level of IL-10 produced. Suppressor cells only developed from infected patients, rather than healthy controls, and the DCs had to be exposed to live virus rather than HIV-1 gag peptides or protein. These results indicate that HIV-1-infected DCs have two previously unrecognized means to evade immune responses: maturation can be blocked reducing the efficacy of antigen presentation from infected cells, and T cell-dependent suppression can be induced.

Coculture Techniques↗

PubMed2004

Quantitative histology by multicolor slide-based cytometry.

BACKGROUND: In lymphatic organs, the quantitative analysis of the spatial distribution of leukocytes by tissue cytometry would give relevant information about alterations during diseases (leukemia, HIV, AIDS) and their therapeutic regimen, as well as in experimental settings. METHODS: We have developed a semiautomated analysis method for laser scanning cytometry (LSC) termed "multiple thresholding," which is suitable for archived or fresh biopsy material of human lymph nodes and tonsils. Sections are stained with PI for nuclear DNA and up to four antigens using direct or indirect immunofluorescence staining. Measurement is triggered on DNA-fluorescence (argon laser, Ar) or on specific cell labeling. Due to the heterogeneity of cell density, measurements are performed repeatedly at different threshold levels (low threshold: regions of low cellular density, germinal center; high threshold: dense regions, mantle zone). Data are acquired by single- (Ar) or dual-laser excitation (Ar-HeNe) in order to analyze single- (FITC) up to four-color (FITC/PE/PECy5/APC) stained specimen. RESULTS: Percentage and cellular density of cell-subsets is quantified in different microanatomical regions of the specimen. These data were highly correlated with manual scoring of identical specimens (r(2) = 0.96, P < 0.0001). With LSC, semiautomated operator-independent immunophenotyping in tissue sections of lymphatic organs with up to three antibodies simultaneously is possible. CONCLUSIONS: We expect this tissue cytometric approach to yield new insight into processes during diseases and help to quantify the success of therapeutic interventions.

Automation↗

PubMed2004

Quantitative histology by multicolor slide-based cytometry.

BACKGROUND: In lymphatic organs, the quantitative analysis of the spatial distribution of leukocytes by tissue cytometry would give relevant information about altercations during diseases (leukemia, HIV, AIDS) and their therapeutic regime, as well as in experimental settings. METHODS: We have developed a semiautomated analysis method for laser scanning cytometry (LSC) termed "multiple thresholding," which is suitable for archived or fresh biopsy material of human lymph nodes and tonsils. Sections are stained with PI for nuclear DNA and up to four antigens using direct and indirect immunofluorescence (argon laser, Ar) or on specific cell labeling. Due to the heterogeneity of cell density, measurements are performed repeatedly at different threshold levels (low threshold: regions of low cellular density, germinal center; high threshold: dense regions, mantle zone). Data are acquired by single-(Ar) or dual-laser excitation (Ar-HeNe) in order to analyze single-(FITC) up to fourcolor (FITC/PE/PECy5/APC) stained specimen. RESULTS: Percentage and cellular density of cell-subsets is quantified in different microanatomical regions of the specimen. These data were highly correlated with manual scoring a identical specimens (r(2) = 0.96, P < 0.0001). With LSC, semiautomated operator-independent immunophenotyping in tissue sections of lymphatic organs with up to three antibodies simultaneously is possible. CONCLUSIONS: We expect this tissue cytometric approach to yield new insight into processes during diseases and help to quantify the success of therapeutic interventions.

Automation↗

PubMed2003

DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells.

Dengue virus is a single-stranded, enveloped RNA virus that productively infects human dendritic cells (DCs) primarily at the immature stage of their differentiation. We now find that all four serotypes of dengue use DC-SIGN (CD209), a C-type lectin, to infect dendritic cells. THP-1 cells become susceptible to dengue infection after transfection of DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN), or its homologue L-SIGN, whereas the infection of dendritic cells is blocked by anti-DC-SIGN antibodies and not by antibodies to other molecules on these cells. Viruses produced by dendritic cells are infectious for DC-SIGN- and L-SIGN-bearing THP-1 cells and other permissive cell lines. Therefore, DC-SIGN may be considered as a new target for designing therapies that block dengue infection.

Antibodies, Monoclonal↗

PubMed2003

Cell type-dependent retention and transmission of HIV-1 by DC-SIGN.

DC-SIGN (CD209) is a C-type lectin expressed by several groups of dendritic cells (DC), including those derived from blood monocytes and DC found beneath genital epithelium. DC-SIGN binds the envelope glycoprotein of HIV-1 and facilitates transmission of infectious virus to permissive CD4(+) T cells. We have compared the capacity of DC-SIGN in different cell types to bind, retain and transmit infectious HIV-1 to T cells. The analyzed cells included monocyte-derived DC, and three different DC-SIGN-expressing transfectants termed THP, 293 and HOS. Our results show that DC-SIGN transfectants were able to bind HIV-1 virions comparably to DC. However, only the THP monocytic cell line shared with DC the capacity to retain for several days virus that was infectious for T cells. In both THP-DC-SIGN transfectants and DC, but not in 293 cells, HIV-1 was localized to intracellular compartments that did not double label for endosomal and lysosomal markers or for DC-SIGN itself. Virus remained detectable in these compartments for at least 2 days. Anti-DC-SIGN antibodies blocked the binding and transmission of HIV-1 in DC-SIGN transfectants, as monitored by PCR for HIV LTR/gag and p24 ELISA. However anti-DC-SIGN antibodies did not block virus binding and transmission to T cells as well in DC as in THP-DC-SIGN transfectants. Thus, the function of DC-SIGN in HIV-1 transmission depends on its cellular context, since only DC and the THP monocyte cell line, but not 293 and HOS, are able to use DC-SIGN to retain HIV-1 in a highly infectious state for several days.

CD4 Antigens↗