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. The composite and dynamic gut environment also necessitates heterogeneous, versatile, and convertible T cells, capable of inhibiting (Foxp3(+) T cells) or helping (T(FH) cells) local immune responses. In this review, we describe recent advances in our understanding of dynamic pathways that lead to IgA synthesis, in gut follicular structures and in extrafollicular sites, by T cell-dependent and T cell-independent mechanisms. We discuss the finely tuned regulatory mechanisms for IgA production and emphasize the role of mucosal IgA in the selection and maintenance of the appropriate microbial composition that is necessary for immune homeostasis.

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. 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.

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. In VHQ52(NT) mice, IgA replaced IgM to drive early B-cell development and peripheral B-cell maturation. In VHQ52(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 VH germ-line gene. VH replacement was also identified in 5% of peripheral B cells of mice inheriting a different productive VH 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.

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. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation.

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. The molecular flexibility of the peptide/MHC complex may be an important determinant of the structures recognized by certain T cells. We used single-molecule x-ray analysis (diffracted x-ray tracking (DXT)) and fluorescence anisotropy to investigate these mechanisms. DXT enabled us to monitor the real-time Brownian motion of the peptide/MHC complex and revealed that peptides without PFRs undergo larger rotational motions than peptides with PFRs. Fluorescence anisotropy further revealed that peptides without PFRs exhibit slightly larger motions on the nanosecond timescale. These results demonstrate that peptides without PFRs undergo dynamic motions in the groove of MHC and consequently are able to assume diverse structures that can be recognized by T cells.

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. One population of memory CD4(+) T cells remained in the skin. A second population migrated from the skin into draining lymph nodes (LNs) in a CCR7-dependent manner. These migrating CD4(+) T cells expressed a novel cell surface phenotype (CCR7(int/+)CD62L(int)CD69(-)CD103(+/-) E-selectin ligands(+)) that is distinct from memory T cell subsets described to date. Unlike memory T cell subsets that remain resident within tissues long-term, or that migrate either exclusively between lymphoid tissues or into peripheral nonlymphoid sites, CD4(+) T(RCM) migrate from the skin into draining LNs. From the draining LNs, CD4(+) T(RCM) reenter into the circulation, distal LNs, and sites of non-specific cutaneous inflammation. In addition, CD4(+) T(RCM) upregulated CD40L and secreted IL-2 following polyclonal stimulation. Our results identify a novel subset of recirculating memory CD4(+) T cells equipped to deliver help to both distal lymphoid and cutaneous tissues.

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). 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.

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. 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.

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. 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.

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. Analysis of B1 clone lines revealed that the B1-cell generation critically depends on the interaction between antigen (possibly self-antigen) and surface immunoglobulin, while the B1-cell maintenance requires the peritoneal environment. The B1-cell expansion takes place in spleen, and is held in check by competitor B2 cells. The results indicate that the NT-ES method could replace the transgenic or knock-in mouse approaches currently used to study the biology of cells that undergo somatic rearrangements of their antigen receptor genes.

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. Here we show that a vitamin A-deficient diet results in reduction of NFATc1 expression in B1 cells and almost complete loss of the B1 cell compartment. As a consequence, vitamin A-deficient mice have reduced serum IgM and are unable to mount T cell-independent antibody responses against bacterial antigens. We demonstrate that injection of all-trans retinoic acid induces the expression of NFATc1, particularly from the constitutive P2 promoter, and leads to the increase of the B1 cells. Thus, the retinoic acid-dependent pathway is critical for regulating NFATc1 expression and for maintenance of the natural memory B cell compartment.

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. 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.

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. Cells that fail to express CD69 or lose CD69 expression migrate to other lymphoid organs. Functionally, CD69(+)-naive CD4(+) T cells exhibit faster and greater cytokine production than do CD69(-) naive CD4(+) T cells. These results indicate that CD4(+) T cells continuously migrate to interact with endogenous Ags, and such an interaction plays an important role in the Ag reactivity of naive CD4(+) T cells.

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 ß-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 ß-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. In this study, we demonstrate that increased expression of mouse IL-15Rα in mouse CD8 T cells by retrovirus-mediated gene transfer changes the ability of the T cell to use cis presentation on the cell surface, indicating that cis presentation requires high expression of mouse IL-15Rα on the cell surface. Using cell lines expressing human or mouse receptors, we demonstrate that cis presentation occurs more efficiently in the human receptor-ligand combination than in that of the mouse system. Moreover, we found that primary human CD8 T cells do not require trans presentation of human IL-15 in vitro. These findings raise the possibility that the maintenance and generation of memory CD8 T cells are achieved via distinct mechanisms in humans and mice. Therefore, careful study of the human immune system, rather than extrapolation from the murine model, is necessary to achieve more complete understanding of memory CD8 T cell development in humans.

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. Simultaneous induction of beta-galactosidase (beta-Gal) as a putative antigen expressed in the process of precancerous dysregulation and Notch1 ablation in the epidermis resulted in elevated beta-Gal-specific IgG2a production. Epidermal gammadelta T cells were found to have the capacity to express chemokine (C-C motif) receptor 7 and migrate into the lymph nodes. Cutaneous draining lymph node cells in Notch1-inactivated mice expressed high levels of IFN-gamma upon anti-CD3 plus anti-CD28 stimulation. Furthermore, induced expression of beta-Gal in mice that lacked epidermal gammadelta T cells failed to induce anti-beta-Gal IgG. These results suggest that epidermal gammadelta T cells play an essential role in the initiation process of epidermal antigen-specific humoral immune responses and demonstrate the importance of epidermal gammadelta T cells in sensing precancerous dysregulation and activating adaptive immunity.

Production of healthy cloned mice from bodies frozen at -20 degrees C for 16 years.

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 degrees 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.

Monitoring cellular movement in vivo with photoconvertible fluorescence protein "Kaede" transgenic mice.

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.

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. Deep T cell receptor (TCR) repertoire sequencing identified massive expansion of unique CDR3 clonotypes in blister cells. The same clones were highly expanded in patient's blood, and the degree of their expansion showed significant correlation with disease severity. By transducing α and ß chains of the expanded clonotypes into a TCR-defective cell line, we confirmed that those cells were drug specific. Collectively, these results suggest that the relative clonal expansion and phenotype of skin-recruited CTLs condition the clinical presentation of cutaneous adverse drug reactions.

Interferon-producing cells fail to induce proliferation of naive T cells but can promote expansion and T helper 1 differentiation of antigen-experienced unpolarized T cells.

Interferon-producing cells (IPCs) secrete high levels of type I interferon in response to certain viruses. The lack of lineage markers, the expression of major histocompatibility complex (MHC) class II and the capacity to stimulate allogeneic T cells have led these cells to be classified as a subset of dendritic cells (DCs), called plasmacytoid DCs (PDCs). However, the role of IPCs/PDCs in initiating primary immune responses remains elusive. Here we examined the antigen presenting capacity of murine IPCs in antigen specific systems. While CD8alpha+ and CD11b+ DCs induced logarithmic expansion of naive CD4 and CD8 T cells, without conferring T helper commitment at a first encounter, primary IPCs lacked the ability to stimulate naive T cells. However, when antigen-experienced, nonpolarized T cells expanded by classical DC subsets, were restimulated by IPCs, they proliferated and produced high amounts of IFN-gamma. These data indicate that IPCs can effectively stimulate preactivated or memory-type T cells and exert an immune-regulatory role. They also suggest that expansion of naive T cells and acquisition of effector function during antigen-specific T cell responses may involve different antigen-presenting cell (APC) types. Independent and coordinated control of T cell proliferation and differentiation would provide the immune system with greater flexibility in regulating immune responses.

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. Moreover, during the cytolytic interaction between OE4 CTL and W3 tumor target cells, detachment of CTL from the target cells occurred prior to caspase-3 activation and death of the target cells, indicating very early sensing of apoptotic target cells by CTL. This early detachment of OE4 CTL from W3 target cells was inhibited by the expression of CD80 co-stimulatory molecule on the target cells. Taken together, time-lapse observation of cellular interaction with functional probe, SCAT3.1 provides new kinetic information and demonstrates that co-stimulatory molecules regulate the kinetics of CTL-target cell interaction.