Fabrication of high aspect-ratio polymer microstructures for large-area electronic portal x-ray imagers.

Megavoltage x-ray imaging performed during radiotherapy is the method of choice for geometric verification of patient localization and dose delivery. Presently, such imaging is increasingly performed using electronic portal imaging devices (EPIDs) based on indirect detection active matrix flat panel imagers (AMFPIs). These devices use a scintillating phosphor screen in order to convert incident x-rays into optical photons, which are then detected by the underlying active matrix photodiode array. The use of a continuous phosphor introduces a trade-off between x-ray quantum efficiency and spatial resolution, which limits current devices to use only ∼2% of the incident x-rays. This trade-off can be circumvented by "segmented phosphor screens", comprising a two-dimensional matrix of optically-isolated cell structures filled with scintillating phosphor. In this work we describe the fabrication of millimeter-thick segmented phosphor screens using the MEMS (micro-electro-mechanical-system) polymer SU-8. This method is capable of being extended to large-area substrates.

In vivo generation of human dendritic cell subsets by Flt3 ligand.

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.

Polyethylene glycol-modified GM-CSF expands CD11b(high)CD11c(high) but notCD11b(low)CD11c(high) murine dendritic cells in vivo: a comparative analysis with Flt3 ligand.

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.

Mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells.

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.

Overexpression of Bcl-2 does not rescue impaired B lymphopoiesis in IL-7 receptor-deficient mice but can enhance survival of mature B cells.

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.

A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function.

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.

Developmental pathways of dendritic cells in vivo: distinct function, phenotype, and localization of dendritic cell subsets in FLT3 ligand-treated mice.

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.

Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant rag-1-/- mice.

Signals from cytokine and antigen receptors play crucial roles during lymphocyte development. Mice lacking interleukin-7 receptor are lymphopenic, due to a defect in cell expansion at an early stage of differentiation, and the few mature T cells that develop in IL-7R-/- animals are functionally impaired. Both defects were rescued completely by overexpression of the anti-apoptosis protein Bcl-2. T cell progenitors lacking antigen receptor molecules are also blocked in differentiation and die, presumably because they fail to receive a positive signal via their pre-T cell receptor. Surprisingly, Bcl-2 did not promote survival or differentiation of T cells in rag-1-/- mice. These results provide evidence that blocking apoptosis is the essential function of IL-7R during differentiation and activation of T lymphocytes and that pre-TCR signaling blocks a pathway to apoptosis that is insensitive to Bcl-2.

Impaired survival and proliferation in IL-7 receptor-deficient peripheral T cells.

Mice genetically deficient in IL-7R(alpha) are highly lymphopenic in the peripheral lymphoid organs. The functional competence of T cells that have developed in the absence of an IL-7R signal was investigated. Three important observations were made using several in vitro activation regimens. First, stimulation of T cells from IL-7R -/- mice at limiting dilution with immobilized Abs to CD3, CD4 or CD8, and CD18 revealed a six- to sevenfold reduction in the frequency of clonogenic T cells compared with T cells from IL-7R +/+ mice. IL-7R -/- T cells were also significantly less responsive to alloantigen as well as to receptor-independent stimuli such as PMA and ionomycin. Furthermore, the average clone size of single IL-7R -/- T cells was 50% smaller than that of IL-7R +/+ T cells. These data suggest that the reduced clonogenicity was predominantly due to intrinsic deficiencies in the ability of IL-7R -/- T cells to proliferate upon stimulation. Second, analysis of the kinetics of cell growth of IL-7R -/- T cells revealed that a significant proportion of T cells failed to proliferate within the first 72 h of in vitro stimulation, with the majority undergoing programmed cell death. Third, both clonogenic IL-7 -/- T cells and IL-7R +/+ T cells showed a similar proliferative response in the presence of IL-2 and similar survival kinetics, indicating that a subpopulation of IL-7R -/- T cells is functionally mature. We propose that an absence of IL-7R signaling not only affects T cell development in the thymus, but also results in the accumulation of functionally inactive T cells in the periphery.

Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified.

Dendritic cells (DC) are the most efficient APC for T cells. The clinical use of DC as vectors for anti-tumor and infectious disease immunotherapy has been limited by their trace levels and accessibility in normal tissue and terminal state of differentiation. In the present study, daily injection of human Flt3 ligand (Flt3L) into mice results in a dramatic numerical increase in cells co-expressing the characteristic DC markers-class II MHC, CD11c, DEC205, and CD86. In contrast, in mice treated with either GM-CSF, GM-CSF plus IL-4, c-kit ligand (c-kitL), or G-CSF, class II+ CD11c+ cells were not significantly increased. Five distinct DC subpopulations were identified in the spleen of Flt3L-treated mice using CD8 alpha and CD11b expression. These cells exhibited veiled and dendritic processes and were as efficient as rare, mature DC isolated from the spleens of untreated mice at presenting allo-Ag or soluble Ag to T cells, or in priming an Ag-specific T cell response in vivo. Dramatic numerical increases in DC were detected in the bone marrow, gastro-intestinal lymphoid tissue (GALT), liver, lymph nodes, lung, peripheral blood, peritoneal cavity, spleen, and thymus. These results suggest that Flt3L could be used to expand the numbers of functionally mature DC in vivo for use in clinical immunotherapy.

Ligands for the receptor tyrosine kinases hek and elk: isolation of cDNAs encoding a family of proteins.

Hek is a member of the eph subfamily of receptor tyrosine kinases whose members include elk, hek2, sek, eph and eck among others. Using a soluble form of hek consisting of the extracellular region of the receptor fused to the Fc domain of human IgG1 and an expression cloning strategy, we have isolated two different but related cDNAs from the human T-lymphoma line HSB-2 that encode ligands for hek. The cDNAs encode proteins of 238 and 201 amino acids (44% amino acid identity) that are anchored to the membrane by glycosylphosphatidylinositol (GPI)-linkage. The proteins encoded by these cDNAs are bound by hek with affinity constants of 2 x 10(8) M-1. These proteins also bind the elk tyrosine kinase receptor. These cDNAs are related to other cDNAs that we have recently isolated from a human placental library that encode ligands for both hek and elk and define an emerging family of ligands for eph-related kinases (LERKs).

cMpl ligand is a humoral regulator of megakaryocytopoiesis.

Megakaryocytopoiesis is the cellular developmental process that leads to platelet production. At least two humoral growth factors may be necessary for megakaryocyte proliferation and maturation. One is a megakaryocyte-colony stimulating factor (MK-CSF) which induces the proliferation and differentiation of megakaryocyte progenitors, and the second, thrombopoietin, is a megakaryocyte maturation factor. Neither of these factors has been fully characterized. The proto-oncogene c-mpl, an orphan member of the haematopoietin receptor family, is specifically involved in megakaryocyte regulation. Here we present evidence that the c-mpl-encoded receptor binds a ligand (c-Mpl ligand) which is a humoral factor implicated in platelet homeostasis. Our results suggest that c-Mpl ligand, thrombopoietin and MK-CSF might be the same molecule.

Recombinant murine steel factor stimulates in vitro production of granulocyte-macrophage progenitor cells.

The ability of murine Steel factor to promote the in vitro production of granulocyte-macrophage progenitor cells (CFU-GM) was examined in short-term liquid cultures. Bone marrow from C57BL/6J or Sl/Sld mice was placed in culture for seven days with either Steel factor alone or in the presence of IL-3. CFU-GM responsive to GM-CSF, IL-3, and CSF-1 were measured in the input population and again after 3 or 7 days in culture. Steel factor alone increased the number of all CFU-GM types as early as 3 days after culture initiation, with further increases at day 7. This effect was potentiated by the addition of IL-3. Production of CFU-GM by C57BL/6J or Sl/Sld marrow was comparable except for enhanced production of CSF-1 responsive progenitors by Sl/Sld marrow. A recombinant Sld protein was also shown to be equivalent to the wild-type protein in its capacity to promote CFU-GM production from normal bone marrow.

Ex vivo expansion of peripheral blood progenitor cells with recombinant cytokines.

Peripheral blood mononuclear cells were isolated and cultured in the presence of Steel factor and/or PIXY321, a GM-CSF/IL-3 fusion protein. We compared the number of colony forming cells (CFC) per culture on day zero to the number of CFC after liquid culture in the presence of these cytokines. After a four day incubation with PIXY321 and Steel factor the number of CFU-GM increased 5.6-fold and the number of BFU-E increased 2.2-fold in four separate experiments. The expansion on day 8 post incubation was 16.1-fold for myeloid colony formation and 9.7-fold for erythroid colony formation. These studies demonstrate the potential to expand CFC from peripheral blood with a simple ex vivo culture procedure.

Alternative forms of the human G-CSF receptor function in growth signal transduction.

Two forms of the human granulocyte colony-stimulating factor (G-CSF) receptor (HuG-CSFR), differing only at the carboxyl terminus, were recently identified by cDNA cloning. In this report we show that transfection and subsequent expression of either cDNA clone in the interleukin-3 (IL-3)-dependent murine cell line BAF/BO3 converts the cells to G-CSF-responsiveness. The transfected cells bound HuG-CSF in a manner indistinguishable from the native receptors. Expression of a mutant form of the HuG-CSFR, with a deletion in the cytoplasmic domain, in BAF/BO3 cells failed to convert the cells to HuG-CSF-responsiveness. In a similar manner, expression of these two HuG-CSFRs in the interleukin-6 (IL-6)-dependent murine hybridoma B9 resulted in the ability of these cells to grow in HuG-CSF [corrected]. These results strongly suggest that sequences in the first 96 amino acids of the cytoplasmic domain of the HuG-CSFR are required for signal transduction in response to ligand binding.