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Biomedical subjects

Ken Shortman

Publications and source records attributed to Ken Shortman.

At least 19 recordsLinked to original sources
PubMed2006

Steady-state and inflammatory dendritic-cell development.

The developmental pathways that lead to the production of antigen-presenting dendritic cells (DCs) are beginning to be understood. These are the last of the pathways of haematopoiesis to be mapped. The existence of many specialized subtypes of DC has complicated this endeavour, as has the need to distinguish the DCs formed in steady state from those produced during an inflammatory response. Here we review studies that lead to the concept that different types of DC develop through different branches of haematopoietic pathways that involve different immediate precursor cells. Furthermore, these studies show that many individual tissues generate their own DCs locally, from a reservoir of immediate DC precursors, rather than depending on a continuous flux of DCs from the bone marrow.

Animals↗
PubMed2006

Production of interferons by dendritic cells, plasmacytoid cells, natural killer cells, and interferon-producing killer dendritic cells.

The capacity of mouse spleen conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) to produce interferon-gamma (IFN-gamma) or IFN-alpha was assessed, and compared with that of natural killer (NK) cells and the recently identified interferon-producing killer dendritic cells (IKDCs), both of which are frequent contaminants in DC preparations. Fully developed cDCs or pDCs, if free of NK cells or IKDCs, showed little capacity for IFN-gamma production. However, an early developmental form of the CD4-8+ cDC subtype, and the Ly6C- Ly49Q- pDC subtype, both were able to produce moderate amounts of IFN-gamma, although less than IKDCs. In response to toll-like receptor 9 stimuli, both the Ly6C+ Ly49Q+ and the Ly6C- Ly49Q- pDC subtypes were effective producers of IFN-alpha. However, IKDCs, which efficiently produced IFN-gamma and showed immediate cytotoxicity on NK target cells, did not produce IFN-alpha under these conditions.

Animals↗
PubMed2006

Signal regulatory protein molecules are differentially expressed by CD8- dendritic cells.

A normalized subtracted gene expression library was generated from freshly isolated mouse dendritic cells (DC) of all subtypes, then used to construct cDNA microarrays. The gene expression profiles of the three splenic conventional DC (cDC) subsets were compared by microarray hybridization and two genes encoding signal regulatory protein beta (Sirpbeta1 and Sirpbeta4) molecules were identified as differentially expressed in CD8(-) cDC. Genomic sequence analysis revealed a third Sirpbeta member localized in the same gene cluster. These Sirpbeta genes encode cell surface molecules containing extracellular Ig domains and short intracytoplasmic domains that have a charged amino acid in the transmembrane region which can potentially interact with ITAM-bearing molecules to mediate signaling. Indeed, we demonstrated interactions between Sirpbeta1 and beta2 with the ITAM-bearing signaling molecule Dap12. Real-time PCR analysis showed that all three Sirpbeta genes were expressed by CD8(-) cDC, but not by CD8(+) cDC or plasmacytoid pre-DC. The related Sirpalpha gene showed a similar expression profile on cDC subtypes but was also expressed by plasmacytoid pre-DC. The differential expression of Sirpalpha and Sirpbeta1 molecules on DC was confirmed by staining with mAbs, including a new mAb recognizing Sirpbeta1. Cross-linking of Sirpbeta1 on DC resulted in a reduction in phagocytosis of Leishmania major parasites, but did not affect phagocytosis of latex beads, perhaps indicating that the regulation of phagocytosis by Sirpbeta1 is a ligand-dependent interaction. Thus, we postulate that the differential expression of these molecules may confer the ability to regulate the phagocytosis of particular ligands to CD8(-) cDC.

Amino Acid Sequence↗
PubMed2006

The dominant role of CD8+ dendritic cells in cross-presentation is not dictated by antigen capture.

Mouse spleens contain three populations of conventional (CD11c(high)) dendritic cells (DCs) that play distinct functions. The CD8(+) DC are unique in that they can present exogenous antigens on their MHC class I molecules, a process known as cross-presentation. It is unclear whether this special ability is because only the CD8(+) DC can capture the antigens used in cross-presentation assays, or because this is the only DC population that possesses specialized machinery for cross-presentation. To solve this important question we examined the splenic DC subsets for their ability to both present via MHC class II molecules and cross-present via MHC class I using four different forms of the model antigen ovalbumin (OVA). These forms include a cell-associated form, a soluble form, OVA expressed in bacteria, or OVA bound to latex beads. With the exception of bacterial antigen, which was poorly cross-presented by all DC, all antigenic forms were cross-presented much more efficiently by the CD8(+) DC. This pattern could not be attributed simply to a difference in antigen capture because all DC subsets presented the antigen via MHC class II. Indeed, direct assessments of endocytosis showed that CD8(+) and CD8(-) DC captured comparable amounts of soluble and bead-associated antigen, yet only the CD8(+) DC cross-presented these antigenic forms. Our results indicate that cross-presentation requires specialized machinery that is expressed by CD8(+) DC but largely absent from CD8(-) DC. This conclusion has important implications for the design of vaccination strategies based on antigen targeting to DC.

Animals↗
PubMed2006

Intrasplenic steady-state dendritic cell precursors that are distinct from monocytes.

Immediate precursors of the many subtypes of dendritic cells (DCs) remain obscure. Here we purified a splenic precursor population that produced all splenic CD8+ and CD8- conventional DCs (cDCs) but not plasmacytoid DCs or other lineages. This 'pre-cDC' population included cells 'precommitted' to form either CD8+ or CD8- cDCs. The pre-cDCs, which comprised 0.05% of splenocytes, expressed a CD11c(int) CD45RA(lo) CD43(int) SIRP-alpha(int) CD4- CD8- major histocompatibility complex class II-negative surface phenotype. The pre-cDCs were not monocytes. Monocytes generated few cDCs in steady-state recipient mice. However, when transferred into mice with an inflammatory milieu dependent on granulocyte-macrophage colony-stimulating factor, monocytes produced a distinct type of splenic DC. Thus, the inflammatory status of the host influences the developmental origin and type of DC present in lymphoid tissues.

Animals↗
PubMed2006

Functional comparison of mouse CIRE/mouse DC-SIGN and human DC-SIGN.

CIRE/mDC-SIGN is a C-type lectin we originally identified as a molecule differentially expressed by mouse dendritic cell (DC) populations. Immunostaining with a CIRE/mDC-SIGN-specific mAb revealed that CIRE/mDC-SIGN is indeed on the surface of some CD4+, CD4- 8- DCs and plasmacytoid pre-DCs, but not on CD8+ DCs. It has been proposed that CIRE/mDC-SIGN is the functional orthologue of human DC-SIGN (hDC-SIGN), a molecule that both enhances T cell responses and facilitates antigen uptake. We assessed if CIRE/mDC-SIGN and hDC-SIGN exhibit functional similarities. CIRE/mDC-SIGN is down-regulated upon activation, but unlike hDC-SIGN, incubation with IL-4 and IL-13 did not enhance CIRE/mDC-SIGN expression, indicating differences in gene regulation. Like hDC-SIGN, CIRE/mDC-SIGN bound mannosylated residues. However, we could detect no role for CIRE/mDC-SIGN in T cell-DC interactions and the protein did not bind to pathogens known to interact with hDC-SIGN, including Leishmania mexicana, cytomegalovirus, HIV and lentiviral particles bearing the Ebolavirus glycoprotein. The binding of CIRE/mDC-SIGN to hDC-SIGN ligands was not rescued when CIRE/mDC-SIGN was engineered to express the stalk region of hDC-SIGN. We conclude that there are significant differences in the fine specificity of the C-type lectin domains of hDC-SIGN and CIRE/mDC-SIGN and that these two molecules may not be functional orthologues.

Amino Acid Sequence↗
PubMed2006

The proliferative response of CD4 T cells to steady-state CD8+ dendritic cells is restricted by post-activation death.

CD8(+) splenic dendritic cells (DCs) from steady-state mice are less effective than the CD8(-) DC subset in their capacity to stimulate CD4 T cell proliferation in culture. However, we found that the two DC subtypes were equally potent at activating CD4 T cells, based on up-regulation of CD69 and CD25 expression. Also, we found no difference in the rate of T cell death prior to entry into the first division. We then tracked carboxyfluorescein diacetate succinimidyl ester-labeled T cells and employed a quantitative model to assess in detail the CD4 T cell expansion process in response to stimulation with CD8(+) or with CD8(-) DCs. The time required for most T cells to replicate their DNA prior to the first division was similar in both DC cultures. However, progression of the CD4 T cell population through subsequent divisions was reduced in CD8(+) DCs compared with CD8(-) DC culture. This was associated with an increased loss of viable T cells at each division. Post-activation, division-associated T cell death is therefore a major factor in the reduced response of CD4 T cells to CD8(+) DCs.

Animals↗
PubMed2006

Migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient CTL priming.

Skin dendritic cells (DCs) are thought to act as key initiators of local T cell immunity. Here we show that after skin infection with herpes simplex virus (HSV), cytotoxic T lymphocyte (CTL) activation required MHC class I-restricted presentation by nonmigratory CD8(+) DCs rather than skin-derived DCs. Despite a lack of direct presentation by migratory DCs, blocking their egress from infected skin substantially inhibited class I-restricted presentation and HSV-specific CTL responses. These results support the argument for initial transport of antigen by migrating DCs, followed by its transfer to the lymphoid-resident DCs for presentation and CTL priming. Given that relatively robust CTL responses were seen with small numbers of skin-emigrant DCs, we propose that this inter-DC antigen transfer functions to amplify presentation across a larger network of lymphoid-resident DCs for efficient T cell activation.

Animals↗
PubMed2006

Gene structure and transcript analysis of the human and mouse EGF-TM7 molecule, FIRE.

The epidermal growth factor-transmembrane seven (EGF-TM7) family are proteins that express EGF-like domains at their extracellular N-terminus coupled to a classical seven transmembrane (TM7) cassette. Recently, we identified, in mice, a novel member of this family termed FIRE (EMR-4). Here, we present the structure of the mouse and human FIRE genes. The structures of the two genes are strikingly similar, with the positions of the introns, relative to the deduced protein sequences, highly conserved. Moreover, the gene structures are typical of other members of the EGF-TM7 family. Other researchers have identified a point deletion in exon eight of the human FIRE gene, which introduces a frame shift into the cDNA leading to a premature stop codon. Thus, human FIRE is predicted to be expressed only as a soluble protein; even though sequence potentially encoding the TM7 cassette is found in a separate open reading frame of the same mRNA transcript. We explored the possibility that a cell surface expressed form of human FIRE did exist, either as an allelic variant, or as an alternatively spliced transcript. Although, we did identify two alternatively spliced human FIRE transcripts, neither are predicted to express the TM7 cassette. Thus if human FIRE exists, it is likely to be expressed as a soluble secreted molecule.

Alternative Splicing↗
PubMed2005

Distinct roles for the NF-kappaB1 and c-Rel transcription factors in the differentiation and survival of plasmacytoid and conventional dendritic cells activated by TLR-9 signals.

Reticuloendotheliosis viral oncogene homolog/nuclear factor of kappa light polypeptide gene enhancer in B cells 1 (Rel/NF-kappaB) activation is a ubiquitous outcome of engaging Toll-like receptors (TLRs), yet the cell-type-specific functions of this pathway in response to particular microbial signals remain poorly defined. Here we show that NF-kappaB1 and C-Rel, Rel/NF-kappaB proteins induced in conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) by cytosine-phosphate-guanosine (CpG) DNA, a TLR-9 ligand, serve markedly different functions in these DC subsets. With the exception of impaired interleukin-12 (IL-12) production, cultured Nfkb1(-/-)C-Rel(-/-) cDCs responded relatively normally to CpG DNA. In contrast, CpG-treated Nfkb1(-/-)C-Rel(-/-) pDCs, which were still able to produce type I interferon and regulated on activation normal T-cell expressed and secreted (RANTES), but not IL-6 or IL-12, failed to acquire an activated dendritic phenotype and underwent apoptosis. Although the TLR-9-mediated death of Nfkb1(-/-)C-Rel(-/-) pDCs, which coincided with a failure to up-regulate the prosurvival proteins B-cell lymphoma apoptosis regulator xL (Bcl-x(L)) and A1, was blocked by Bcl-2 transgene expression, this inhibition of apoptosis still failed to rescue the differentiation defects. This indicated that these NF-kappaB transcription factors independently regulate TLR-9-mediated pDC morphogenesis and survival. Collectively, these findings establish that NF-kappaB1 and c-Rel, while largely dispensable for TLR-9-induced cDC activation, are critical for regulating differentiation and survival programs during pDC activation.

Animals↗
PubMed2005

CD8alpha+ dendritic cells selectively present MHC class I-restricted noncytolytic viral and intracellular bacterial antigens in vivo.

CD8alpha(+) dendritic cells (DCs) have been shown to be the principal DC subset involved in priming MHC class I-restricted CTL immunity to a variety of cytolytic viruses, including HSV type 1, influenza, and vaccinia virus. Whether priming of CTLs by CD8alpha(+) DCs is limited to cytolytic viruses, which may provide dead cellular material for this DC subset, or whether these DCs selectively present intracellular Ags, is unknown. To address this question, we examined Ag presentation to a noncytolytic virus, lymphocytic choriomeningitis virus, and to an intracellular bacterium, Listeria monocytogenes. We show that regardless of the type of intracellular infection, CD8alpha(+) DCs are the principal DC subset that initiate CD8(+) T cell immunity.

Animals↗
PubMed2005

Cutting edge: generation of splenic CD8+ and CD8- dendritic cell equivalents in Fms-like tyrosine kinase 3 ligand bone marrow cultures.

We demonstrate that functional and phenotypic equivalents of mouse splenic CD8(+) and CD8(-) conventional dendritic cell (cDC) subsets can be generated in vitro when bone marrow is cultured with fms-like tyrosine kinase 3 (flt3) ligand. In addition to CD45RA(high) plasmacytoid DC, two distinct CD24(high) and CD11b(high) cDC subsets were present, and these subsets showed equivalent properties to splenic CD8(+) and CD8(-) cDC, respectively, in the following: 1) surface expression of CD11b, CD24, and signal regulatory protein-alpha; 2) developmental dependence on, and mRNA expression of, IFN regulatory factor-8; 3) mRNA expression of TLRs and chemokine receptors; 4) production of IL-12 p40/70, IFN-alpha, MIP-1alpha, and RANTES in response to TLR ligands; 5) expression of cystatin C; and 6) cross-presentation of exogenous Ag to CD8 T cells. Furthermore, despite lacking surface CD8 expression, the CD24(high) subset contained CD8 mRNA and up-regulated surface expression when transferred into mice. This culture system allows access to bona fide counterparts of the splenic DC subsets.

Animals↗
PubMed2005

Fms-like tyrosine kinase 3 ligand administration overcomes a genetically determined dendritic cell deficiency in NOD mice and protects against diabetes development.

A dendritic cell (DC) imbalance with a marked deficiency in CD4- 8+ DC occurs in non-obese diabetic (NOD) mice, a model of human autoimmune diabetes mellitus. Using a NOD congenic mouse strain, we find that this CD4- 8+ DC deficiency is associated with a gene segment on chromosome 4, which also encompasses non-MHC diabetes susceptibility loci. Treatment of NOD mice with fms-like tyrosine kinase 3 ligand (FL) enhances the level of CD4- 8+ DC, temporarily reversing the DC subtype imbalance. At the same time, fms-like tyrosine kinase 3 ligand treatment blocks early stages of the diabetogenic process and with appropriately timed administration can completely prevent diabetes development. This points to a possible clinical use of FL to prevent autoimmune disease.

Animals↗
PubMed2005

Antigen delivery via two molecules on the CD8- dendritic cell subset induces humoral immunity in the absence of conventional "danger".

Targeting antigen to dendritic cells (DC) in vivo might be an effective method of modulating immune responses. Given the functional specializations among DC subsets, we investigated how targeting different receptors on different DC subsets may influence antibody (Ab) production. We show here that targeting FIRE (F4/80-like receptor) or CIRE (C-type lectin receptor), two molecules expressed on the surface of immature CD8- DC in the mouse, increases Ab production 100-1000-fold over a non-targeted control. This response was equivalent to that achieved with CpG adjuvant. In contrast, targeting CD205, which is primarily expressed on CD8+ DC, did not elicit an Ab response unless an adjuvant was added. Strong Ab responses in FcRgamma-/- mice, and with the use of F(ab')2 fragments, confirmed that FIRE and CIRE targeting was due to specific rather than FcR or complement binding. Our findings may reflect differences in the ability of CD8+ and CD8- DC subsets to stimulate immune responses in vivo. Although the consensus view is that Ag presentation on DC in their steady state leads to tolerance, the Ab enhancement from FIRE and CIRE targeting in the apparent absence of any "danger" or inflammatory signal would suggest that targeting certain DC molecules can supplant the need for external adjuvants for eliciting immune responses.

Animals↗
PubMed2005

Switching from a restricted to an effective CD4 T cell response by activating CD8+ murine dendritic cells with a Toll-like receptor 9 ligand.

Freshly isolated quiescent splenic dendritic cell (DC) subtypes differ in their capacity to activate naive CD4 T cells in culture. The CD8+ DC showed a reduced capacity to stimulate T cell proliferation compared to either of the CD8- DC subsets, regardless of antigen and DC dose. In contrast to CD8- DC, the quiescent CD8+ DC did not induce IFN-gamma production from CD4 T cells. The difference between the DC subtypes appeared to be at the level of initial surface molecule interactions, but could not be attributed to differences in expression of MHC class II or B7 family molecules, or to the expression of Fas ligand on DC. However, when activated by inclusion of the Toll-like receptor 9 ligand CpG in culture, CD8+ DC became potent stimulators of both CD4 T cell proliferation and IFN-gamma production. In contrast, similar activation of CD8- DC produced a more modest increase in capacity to stimulate CD4 T cell proliferation and no increase in capacity to stimulate IFN-gamma production. The difference between a quiescent and an activated state is therefore more extreme for CD8+ than for CD8- DC. The especially tight regulation of the activity of CD8+ DC may be essential for the maintenance of self tolerance.

Animals↗
PubMed2005

Heterogeneity of thymic dendritic cells.

Thymus is the site of generation and selection of T-lymphocytes. It also contains phenotypically and functionally distinct dendritic cell (DC) populations, including conventional DC (cDC) and plasmacytoid DC (pDC). Thymic cDC are heterogeneous and contain two subsets: a major subset derived from the precursors within thymus, and a minor subset presumably of extrathymic origin. Increasing evidence suggest that thymic cDC can cross-present self-antigens to developing thymocytes and play an important role in thymocyte negative selection and central tolerance induction. Thymic pDC can produce type-I interferon upon appropriate activation. However, their role in a steady state thymus is currently unclear.

Animals↗
PubMed2005

Disruption of the langerin/CD207 gene abolishes Birbeck granules without a marked loss of Langerhans cell function.

Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin(-/-) mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin(-/-) mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin(-/-) LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin(-/-) mice were not impaired in their capacity to process native OVA protein for I-A(b)-restricted presentation to CD4(+) T lymphocytes or for H-2K(b)-restricted cross-presentation to CD8(+) T lymphocytes. langerin(-/-) mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin(-/-) and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin(-/-) C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG.

9,10-Dimethyl-1,2-benzanthracene↗