Massive clonal expansion of polycytotoxic skin and blood CD8 T cells in patients with toxic epidermal necrolysis.

Toxic epidermal necrolysis (TEN) is a life-threatening cutaneous adverse drug reaction. To better understand why skin symptoms are so severe, we conducted a prospective immunophenotyping study on skin and blood. Mass cytometry results confirmed that effector memory polycytotoxic CD8 T cells (CTLs) are the main leucocytes in TEN blisters at the acute phase.

Involvement of Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-induced Incomplete Cytokinesis in the Polyploidization of Osteoclasts.

Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined.

Real-time tracking of cell cycle progression during CD8+ effector and memory T-cell differentiation.

The precise pathways of memory T-cell differentiation are incompletely understood. Here we exploit transgenic mice expressing fluorescent cell cycle indicators to longitudinally track the division dynamics of individual CD8(+) T cells. During influenza virus infection in vivo, naive T cells enter a CD62L(intermediate) state of fast proliferation, which continues for at least nine generations.

Antibody repertoire diversification through VH gene replacement in mice cloned from an IgA plasma cell.

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 VHQ52(NT); Vκgr32(NT) Ig monoclonal mice reprogrammed from the nucleus of an intestinal IgA(+) plasma cell.

Single-molecule motions of MHC class II rely on bound peptides.

The major histocompatibility complex (MHC) class II protein can bind peptides of different lengths in the region outside the peptide-binding groove. Peptide-flanking residues (PFRs) contribute to the binding affinity of the peptide for MHC and change the immunogenicity of the peptide/MHC complex with regard to T cell receptor (TCR). The mechanisms underlying these phenomena are currently unknown.

Tracking and quantification of dendritic cell migration and antigen trafficking between the skin and lymph nodes.

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.

Contrasting quiescent G0 phase with mitotic cell cycling in the mouse immune system.

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.

Optimizing fluorescence excitation and detection for intravital two-photon microscopy.

Commercial two-photon microscope systems incorporating turnkey ultrafast lasers have made the technology more user-friendly and accessible to nonspecialized biology laboratories. This has been accompanied by the development of an exciting range of new fluorescent proteins and dyes such as near-infra-red fluorescent proteins and optical highlighters. However, the two-photon absorption properties of these fluorescent molecules are not widely available and cannot be reliably predicted from their single photon absorption spectra.

Recirculating memory T cells are a unique subset of CD4+ T cells with a distinct phenotype and migratory pattern.

Several populations of memory T cells have been described that differ in their migration and function. In this study, we have identified a unique subset of memory T cells, which we have named recirculating memory T cells (T(RCM)). By exposing Kaede transgenic mouse skin to violet light, we tracked the fate of cutaneous T cells.

Systemic circulation and bone recruitment of osteoclast precursors tracked by using fluorescent imaging techniques.

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).

Thymic nurse cells provide microenvironment for secondary T cell receptor α rearrangement in cortical thymocytes.

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.

Nuclear transferred embryonic stem cells for analysis of B1 B-lymphocyte development.

The transfer of nuclei of fully differentiated cells into enucleated oocytes is a well-recognized method for the generation of embryonic stem (ES) cells. Here, we demonstrate that nuclear transferred ES (NT-ES) cells can be established with high efficiency using innate-like B lymphocytes as donor cells. We established two mouse lines carrying rearranged immunoglobulin heavy and light chains using NT-ES cells containing nuclei from peritoneal cavity B1 cells.

CD169-positive macrophages dominate antitumor immunity by crosspresenting dead cell-associated antigens.

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.

Vitamin A-dependent transcriptional activation of the nuclear factor of activated T cells c1 (NFATc1) is critical for the development and survival of B1 cells.

B1 cells represent a distinct subset of B cells that produce most of the natural serum IgM and much of the gut IgA and function as an important component of early immune responses to pathogens. The development of B1 cells depends on the nuclear factor of activated T cells c1 (NFATc1), a transcription factor abundantly expressed by B1 cells but not by conventional B2 cells. However, the factors that regulate the expression of NFATc1 in B1 cells remain unknown.

No requirement of trans presentations of IL-15 for human CD8 T cell proliferation.

The trans presentation of IL-15 by cells expressing the specific high-affinity receptor α-chain (IL-15Rα) to cells expressing the signaling receptor β-chain and γ-chain is essential for the generation and maintenance of CD8 memory T cells, NK cells, and NKT cells in an in vivo mouse system. We have also demonstrated in vitro that cell-surface IL-15Rα on cells expressing all the receptor components present IL-15 to receptor β-chain/γ-chain coexpressed on the same cell surface (cis presentation). However, although mouse CD8 T cells express all the IL-15R components, they show no evidence of cis presentation.

Preferential localization of IgG memory B cells adjacent to contracted germinal centers.

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 gamma-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.

Naive CD4+ T lymphocytes circulate through lymphoid organs to interact with endogenous antigens and upregulate their function.

Naive T lymphocytes recirculate through the lymph-vascular system and enter and exit lymphoid organs. Using mice expressing the photoconvertible fluorescence protein Kaede, we demonstrated that naive T cells seek to interact with endogenous Ags after migrating to the lymphoid organs. The interaction with endogenous Ags transiently induces CD69 expression on T cells, which prolongs retention in the lymphoid organs.

Adaptive immune regulation in the gut: T cell-dependent and T cell-independent IgA synthesis.

In mammals, the gastrointestinal tract harbors an extraordinarily dense and complex community of microorganisms. The gut microbiota provide strong selective pressure to the host to evolve adaptive immune responses required for the maintenance of local and systemic homeostasis. The continuous antigenic presence in the gut imposes a dynamic remodeling of gut-associated lymphoid tissues (GALT) and the selection of multiple layered strategies for immunoglobulin (Ig) A production.

Epidermal gammadelta T cells sense precancerous cellular dysregulation and initiate immune responses.

Hyperplasia associated with a loss of tissue homeostasis can induce DNA replication stress, leading to precancerous dysregulation. Epidermal gammadelta T cells reside in the primary barrier that protects against diverse environmental insults; however, the functions of these T cells in tissue surveillance are not completely understood. In mice with inducible Notch1 inactivation in keratinocytes that causes epidermal hyperplasia, epidermal gammadelta T cells sensed stressed keratinocytes and migrated into the cutaneous draining lymph nodes.

Activated regulatory T cells are the major T cell type emigrating from the skin during a cutaneous immune response in mice.

Tregs play an important role in protecting the skin from autoimmune attack. However, the extent of Treg trafficking between the skin and draining lymph nodes (DLNs) is unknown. We set out to investigate this using mice engineered to express the photoconvertible fluorescence protein Kaede, which changes from green to red when exposed to violet light.

Critical role of IFN-gamma in CFA-mediated protection of NOD mice from diabetes development.

IFN-gamma signaling-deficient non-obese diabetic (NOD) mice develop diabetes with similar kinetics to those of wild-type NOD mice. However, the immunization of IFN-gamma signaling-deficient NOD mice with CFA failed to induce long-term protection, whereas wild-type NOD mice receiving CFA remained diabetes-free. CFA also failed to protect IFN-gamma receptor-deficient (IFN-gammaR(-/-)) NOD mice from the autoimmune rejection of transplanted islets, as it does in diabetic NOD mice, and from disease transfer by spleen cells from diabetic NOD mice.

Male germline and embryonic stem cell lines from NOD mice: efficient derivation of GS cells from a nonpermissive strain for ES cell derivation.

The nonobese diabetic (NOD) mouse is a valuable model for human type 1 diabetes and the development of humanized mice. Although the importance of this mouse strain is widely recognized, its usefulness is constrained by the absence of NOD embryonic stem (ES) lines with adequate germline transmission competence. In the present study, we established two germline transmission-competent types of cell lines from NOD mice; these cell lines, male germline stem (GS) cells and ES cells, were derived from NOD spermatogonia and blastocysts, respectively.

Time-lapse observation of cellular function with fluorescent probe reveals novel CTL-target cell interactions.

Fluorescent protein that detects caspase-3 activation was used for the time-lapse observation of CTL-target cell interaction. In the target cells transfected with SCAT3.1 (caspase-3-sensitive fusion protein) complementary DNA, caspase-3 activation can be detected significantly earlier than the commonly used annexin-V binding that detects membrane change in apoptotic cells.

Vav links the T cell antigen receptor to the actin cytoskeleton and T cell activation independently of intrinsic Guanine nucleotide exchange activity.

Background: T cell receptor (TCR) engagement leads to formation of signaling microclusters and induction of rapid and dynamic changes in the actin cytoskeleton, although the exact mechanism by which the TCR initiates actin polymerization is incompletely understood. The Vav family of guanine nucleotide exchange factors (GEF) has been implicated in generation of TCR signals and immune synapse formation, however, it is currently not known if Vav's GEF activity is required in T cell activation by the TCR in general, and in actin polymerization downstream of the TCR in particular.

Methodology/principal Findings: Here, we report that Vav1 assembles into signaling microclusters at TCR contact sites and is critical for TCR-initiated actin polymerization.

Suppression of induced pluripotent stem cell generation by the p53-p21 pathway.

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.