Induced pluripotent stem (iPS) cells can be generated from somatic cells by the introduction of Oct3/4 (also known as Pou5f1), Sox2, Klf4 and c-Myc, in mouse and in human. The efficiency of this ...process, however, is low. Pluripotency can be induced without c-Myc, but with even lower efficiency. A p53 (also known as TP53 in humans and Trp53 in mice) short-interfering RNA (siRNA) was recently shown to promote human iPS cell generation, but the specificity and mechanisms remain to be determined. Here we report that up to 10% of transduced mouse embryonic fibroblasts lacking p53 became iPS cells, even without the Myc retrovirus. The p53 deletion also promoted the induction of integration-free mouse iPS cells with plasmid transfection. Furthermore, in the p53-null background, iPS cells were generated from terminally differentiated T lymphocytes. The suppression of p53 also increased the efficiency of human iPS cell generation. DNA microarray analyses identified 34 p53-regulated genes that are common in mouse and human fibroblasts. Functional analyses of these genes demonstrate that the p53-p21 pathway serves as a barrier not only in tumorigenicity, but also in iPS cell generation.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The generation of tumor-directed cytotoxic T lymphocytes is considered crucial for the induction of antitumor immunity. To activate these CD8+ T cells, antigen-presenting cells (APCs) must initially ...acquire tumor cell-associated antigens. The major source of tumor antigens is dead tumor cells, but little is known about how APCs in draining lymph nodes acquire and crosspresent these antigens. Here we show that CD169+ macrophages phagocytose dead tumor cells transported via lymphatic flow and subsequently crosspresent tumor antigens to CD8+ T cells. Subcutaneous immunization with irradiated tumor cells protects mice from syngenic tumor. However, tumor antigen-specific CD8+ T cell activation and subsequent antitumor immunity are severely impaired in mice depleted with CD169+ macrophages. Neither migratory dendritic cells (DCs) nor lymph node-resident conventional DCs are essential for the crosspresentation of tumor antigens. Thus, we have identified CD169+ macrophages as lymph node-resident APCs dominating early activation of tumor antigen-specific CD8+ T cells.
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► Dead tumor cells in periphery accumulate in the draining lymph node sinus ► CD169+ macrophages phagocytose and crosspresent dead cell-associated antigens ► CD169+ macrophage-depleted mice fail to crossprime tumor-specific CD8 T cells ► CD169+ macrophages link tumor cell death and induction of antitumor immunity
Kaede is a photoconvertible fluorescence protein that changes from green to red upon exposure to violet light. The photoconversion of intracellular Kaede has no effect on cellular function. Using ...transgenic mice expressing the Kaede protein, we demonstrated that movement of cells with the photoconverted Kaede protein could be monitored from lymphoid organs to other tissues as well as from skin to the draining lymph node. Analysis of the kinetics of cellular movement revealed that each subset of cells in the lymph node, such as CD4⁺ T, CD8⁺ T, B, and dendritic cells, has a distinct migration pattern in vivo. Thus, the Kaede transgenic mouse system would be an ideal tool to monitor precise cellular movement in vivo at different stages of immune response to pathogens as well as in autoimmune diseases.
Skin-derived dendritic cells (DCs) play a crucial role in the maintenance of immune homeostasis due to their role in antigen trafficking from the skin to the draining lymph nodes (dLNs). To quantify ...the spatiotemporal regulation of skin-derived DCs in vivo, we generated knock-in mice expressing the photoconvertible fluorescent protein KikGR. By exposing the skin or dLN of these mice to violet light, we were able to label and track the migration and turnover of endogenous skin-derived DCs. Langerhans cells and CD103(+)DCs, including Langerin(+)CD103(+)dermal DCs (DDCs), remained in the dLN for 4-4.5 days after migration from the skin, while CD103(-)DDCs persisted for only two days. Application of a skin irritant (chemical stress) induced a transient >10-fold increase in CD103(-)DDC migration from the skin to the dLN. Tape stripping (mechanical injury) induced a long-lasting four-fold increase in CD103(-)DDC migration to the dLN and accelerated the trafficking of exogenous protein antigens by these cells. Both stresses increased the turnover of CD103(-)DDCs within the dLN, causing these cells to die within one day of arrival. Therefore, CD103(-)DDCs act as sentinels against skin invasion that respond with increased cellular migration and antigen trafficking from the skin to the dLNs.
Distinct subsets of thymic epithelial cells (TECs) support T-cell development and selection. Isolated TECs contain multicellular complexes that enclose many viable thymocytes. However, the functions ...of those TECs, termed thymic nurse cells (TNCs), are unclear and the idea that TNCs are present in vivo is questioned. Here, we show that TNCs represent a fraction of cortical (c)TECs that are defined by the expression of thymoproteasomes. Intravital imaging revealed TNCs in the thymic cortex in situ, whereas TNCs were detected neither during embryogenesis nor in the postnatal thymuses of various “positive-selector” T-cell receptor (TCR)-transgenic mice, indicating that TNCs are not essential for T-cell differentiation, including positive selection. Rather, cells within TNCs were enriched for long-lived CD4 ⁺CD8 ⁺ thymocytes that underwent secondary TCR-Vα rearrangement. Thus, TNC complexes are formed in vivo by persistent cTEC–thymocyte interactions that then provide a microenvironment that optimizes T-cell selection through secondary TCR rearrangement.
A transgenic mouse line expressing Fucci (fluorescent ubiquitination-based cell-cycle indicator) probes allows us to monitor the cell cycle in the hematopoietic system. Two populations with high and ...low intensities of Fucci signals for Cdt1(30/120) accumulation were identified by FACS analysis, and these correspond to quiescent G0 and cycling G1 cells, respectively. We observed the transition of immune cells between quiescent and proliferative phases in lymphoid organs during differentiation and immune responses.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Osteoclasts are bone-resorbing polykaryons differentiated from monocyte/macrophage-lineage hematopoietic precursors. It remains unclear whether osteoclasts originate from circulating blood monocytes ...or from bone tissue-resident precursors. To address this question, we combined two different experimental procedures: 1) shared blood circulation "parabiosis" with fluorescently labeled osteoclast precursors, and 2) photoconversion-based cell tracking with a Kikume Green-Red protein (KikGR). In parabiosis, CX(3)CR1-EGFP knock-in mice in which osteoclast precursors were labeled with EGFP were surgically connected with wild-type mice to establish a shared circulation. Mature EGFP(+) osteoclasts were found in the bones of the wild-type mice, indicating the mobilization of EGFP(+) osteoclast precursors into bones from systemic circulation. Receptor activator for NF-κB ligand stimulation increased the number of EGFP(+) osteoclasts in wild-type mice, suggesting that this mobilization depends on the bone resorption state. Additionally, KikGR(+) monocytes (including osteoclast precursors) in the spleen were exposed to violet light, and 2 d later we detected photoconverted "red" KikGR(+) osteoclasts along the bone surfaces. These results indicate that circulating monocytes from the spleen entered the bone spaces and differentiated into mature osteoclasts during a certain period. The current study used fluorescence-based methods clearly to demonstrate that osteoclasts can be generated from circulating monocytes once they home to bone tissues.
Cloning animals by nuclear transfer provides an opportunity to preserve endangered mammalian species. However, it has been suggested that the "resurrection" of frozen extinct species (such as the ...woolly mammoth) is impracticable, as no live cells are available, and the genomic material that remains is inevitably degraded. Here we report production of cloned mice from bodies kept frozen at -20 °C for up to 16 years without any cryoprotection. As all of the cells were ruptured after thawing, we used a modified cloning method and examined nuclei from several organs for use in nuclear transfer attempts. Using brain nuclei as nuclear donors, we established embryonic stem cell lines from the cloned embryos. Healthy cloned mice were then produced from these nuclear transferred embryonic stem cells by serial nuclear transfer. Thus, nuclear transfer techniques could be used to "resurrect" animals or maintain valuable genomic stocks from tissues frozen for prolonged periods without any cryopreservation.
It has long been presumed that after leaving the germinal centers (GCs), memory B cells colonize the marginal zone or join the recirculating pool. Here we demonstrate the preferential localization of ...nitrophenol-chicken γ-globulin-induced CD38⁺IgG1⁺ memory B cells adjacent to contracted GCs in the spleen. The memory B cells in this region proliferated after secondary immunization, a response that was abolished by depletion of CD4⁺ T cells. We also found that these IgG1⁺ memory B cells could present antigen on their surface, and that this activity was required for their activation. These results implicate this peri-GC region as an important site for survival and reactivation of memory B cells.
Significance Antibodies produced by B cells provide a protective barrier to our organism against the penetration and dissemination of microorganisms. Each antibody recognizes a specific antigen ...through variable (V) region domains of pairs of immunoglobulin (Ig) heavy (H) and light (L) chains. In mammals, VDJ recombination generates a highly diversified preimmune pool of V H and V L domains. Acquisition of a functional V H rearrangement is thought to prevent further VDJ recombination at the IgH locus. Instead, mice cloned from a terminally differentiated B cell unravel the ability of VDJ recombination to revise a functionally rearranged V H gene through VH replacement. We show that up to 20% of the antibody V gene repertoire of mature B-lymphocytes can be generated through VH replacement.
In mammals, VDJ recombination is responsible for the establishment of a highly diversified preimmune antibody repertoire. Acquisition of a functional Ig heavy (H) chain variable (V) gene rearrangement is thought to prevent further recombination at the IgH locus. Here, we describe V HQ52 ᴺᵀ; Vκgr32 ᴺᵀ Ig monoclonal mice reprogrammed from the nucleus of an intestinal IgA ⁺ plasma cell. In V HQ52 ᴺᵀ mice, IgA replaced IgM to drive early B-cell development and peripheral B-cell maturation. In V HQ52 ᴺᵀ animals, over 20% of mature B cells disrupted the single productive, nonautoimmune IgH rearrangement through VH replacement and exchanged it with a highly diversified pool of IgH specificities. VH replacement occurred in early pro-B cells, was independent of pre–B-cell receptor signaling, and involved predominantly one adjacent V H germ-line gene. VH replacement was also identified in 5% of peripheral B cells of mice inheriting a different productive V H rearrangement expressed in the form of an IgM H chain. In summary, editing of a productive IgH rearrangement through VH replacement can account for up to 20% of the IgH repertoire expressed by mature B cells.
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