Immunotherapy with artificial adjuvant vector cells: Harnessing both arms of the immune response.

Both innate and adaptive immunity underpin cancer immunosurveillance. To stimulate both these arms of the immune system, we used allogeneic cells loaded with natural killer T (NKT) cell ligands and expressing tumor-associated antigens, resulting in NKT cell activation, dendritic-cell maturation and ultimately in the elicitation of adaptive T-cell responses. This approach holds great promise for the development of novel anticancer immunotherapies.

Critical roles of a dendritic cell subset expressing a chemokine receptor, XCR1.

Dendritic cells (DCs) consist of various subsets that play crucial roles in linking innate and adaptive immunity. In the murine spleen, CD8α(+) DCs exhibit a propensity to ingest dying/dead cells, produce proinflammatory cytokines, and cross-present Ags to generate CD8(+) T cell responses. To track and ablate CD8α(+) DCs in vivo, we generated XCR1-venus and XCR1-DTRvenus mice, in which genes for a fluorescent protein, venus, and a fusion protein consisting of diphtheria toxin receptor and venus were knocked into the gene locus of a chemokine receptor, XCR1, which is highly expressed in CD8α(+) DCs.

Invariant NKT cells induce plasmacytoid dendritic cell (DC) cross-talk with conventional DCs for efficient memory CD8+ T cell induction.

A key goal of vaccine immunotherapy is the generation of long-term memory CD8(+) T cells capable of mediating immune surveillance. We discovered a novel intercellular pathway governing the development of potent memory CD8(+) T cell responses against cell-associated Ags that is mediated through cross-presentation by XCR1(+) dendritic cells (DCs). Generation of CD8(+) memory T cells against tumor cells pulsed with an invariant NKT cell ligand depended on cross-talk between XCR1(+) and plasmacytoid DCs that was regulated by IFN-α/IFN-αR signals.

The CD4/CD8 lineages: central decisions and peripheral modifications for T lymphocytes.

CD4(+) helper and CD8(+) cytotoxic T cells, two major subsets of αβTCR expressing lymphocytes, are differentiated from common precursor CD4(+)CD8(+) double-positive (DP) thymocytes. Bifurcation of the CD4(+)/CD8(+) lineages in the thymus is a multilayered process and is thought to culminate in a loss of developmental plasticity between these functional subsets. Advances in the last decade have deepened our understanding of the transcription control mechanisms governing CD4 versus CD8 lineage commitment.

RCAI-61 and related 6'-modified analogs of KRN7000: their synthesis and bioactivity for mouse lymphocytes to produce interferon-γ in vivo.

We synthesized ten new analogs of 6'-modified KRN7000 (A): RCAI-58, 61, 64, 83, 85-87, 113, 119, and 125. They could be synthesized by α-selective galactosylation of ceramide 9 with the 6-modified D-galactopyranosyl fluorides (8a-8f) or L-arabinopyranosyl fluoride (17), or by etherification of the known alcohol 19. Bioassay of the ten analogs demonstrated that RCAI-61 (1, 6'-O-methylated analog of A) was the most potent immunostimulant among them, and could induce the production of a large amount of IFN-γ even at a low concentration in mice in vivo.

A pyrrolo-pyrimidine derivative targets human primary AML stem cells in vivo.

Leukemia stem cells (LSCs) that survive conventional chemotherapy are thought to contribute to disease relapse, leading to poor long-term outcomes for patients with acute myeloid leukemia (AML). We previously identified a Src-family kinase (SFK) member, hematopoietic cell kinase (HCK), as a molecular target that is highly differentially expressed in human primary LSCs compared with human normal hematopoietic stem cells (HSCs). We performed a large-scale chemical library screen that integrated a high-throughput enzyme inhibition assay, in silico binding prediction, and crystal structure determination and found a candidate HCK inhibitor, RK-20449, a pyrrolo-pyrimidine derivative with an enzymatic IC50 (half maximal inhibitory concentration) in the subnanomolar range.

Phase responses of oscillating components in a signaling pathway.

Signal transduction pathways control various events in mammalian cells such as growth, proliferation, differentiation, apoptosis, or migration in response to environmental stimuli. Because of their importance, the activity of signaling pathways is controlled by multiple modes of positive and negative feedback regulation. Although negative feedback regulation primarily functions to stabilize a system, it also becomes a source of emerging oscillations.

[Role of adaptor molecule Gab2 in mast cell-mediated allergy response].

Mast cells are major players in allergic responses. IgE-dependent activation through Fc epsilon RI leads to degranulation and cytokine production, both of which require Gab2. To clarify how the signals diverge at Gab2, we established Gab2 knock-in mice that express Gab2 mutated at either the PI-3K- or SHP-2-binding sites.

Epigenetic Thpok silencing limits the time window to choose CD4(+) helper-lineage fate in the thymus.

CD4(+) helper and CD8(+) cytotoxic T cells differentiate from common precursors in the thymus after T-cell receptor (TCR)-mediated selection. Commitment to the helper lineage depends on persistent TCR signals and expression of the ThPOK transcription factor, whereas a ThPOK cis-regulatory element, ThPOK silencer, represses Thpok gene expression during commitment to the cytotoxic lineage. Here, we show that silencer-mediated alterations of chromatin structures in cytotoxic-lineage thymocytes establish a repressive state that is epigenetically inherited in peripheral CD8(+) T cells even after removal of the silencer.

Zinc signal: a new player in osteobiology.

Disturbed zinc (Zn) homeostasis in mammals is mainly characterized by impaired bone generation accompanied with growth retardation. However, the underlying mechanisms that determine how Zn controls bone homeostasis remain to be defined. Zn homeostasis is tightly controlled by Zn transporter families.

Synthesis and biological activity of hydroxylated analogues of KRN7000 (α-galactosylceramide).

KRN7000 is one of the α-galactosylceramides, which has a 2-hexacosanoylamino-3,4-dihydroxyoctadecyl group. This compound, known as a ligand for the activation of CD1d mediated invariant natural killer T cells (iNKT cells) which release both T helper 1 (Th1) cytokines such as IFNγ and Th2 cytokines such as IL-4, has been anticipated as an antitumor drug, because of its strong secretion of IFNγ. This time, we focused on the hydroxylated analogues of KRN7000 which could be thought of as increasing hydrophilicity and showing bias to Th2 cytokine (IL-4) secretion.

High levels of AID cause strand bias of mutations at A versus T in Burkitt's lymphoma cells.

Ig gene somatic hypermutation in the germinal center (GC) B cells occurs at C and G at roughly the same frequency. In contrast, there is a 2-fold increase of mutations at A relative to T on the non-transcribed strand of the V genes but it is unclear what triggers such strand bias. Using an efficient mutagenesis system that recapitulates characteristic features of Ig gene hypermutation in the GC B cells, we found that low levels of AID induced similar frequency of mutations at A and T.

Human CD300C delivers an Fc receptor-γ-dependent activating signal in mast cells and monocytes and differs from CD300A in ligand recognition.

CD300C is highly homologous with an inhibitory receptor CD300A in an immunoglobulin-like domain among the human CD300 family of paired immune receptors. To clarify the precise expression and function of CD300C, we generated antibodies discriminating between CD300A and CD300C, which recognized a unique epitope involving amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Notably, CD300C was highly expressed in human monocytes and mast cells.

Impaired selection of IgA and intestinal dysbiosis associated with PD-1-deficiency.

A major function of immunoglobulin A (IgA) is to maintain balanced bacterial communities in the gut. We have previously shown that diversification of IgA upon somatic hypermutation (SHM) is critical for IgA function yet the principles governing the selection of IgA in the gut have remained elusive. Here we discuss recent progress in understanding this process as revealed by our studies in mice that lack the inhibitory co-receptor programmed cell death-1 (PD-1).

Cutting edge: fine-tuning of Thpok gene activation by an enhancer in close proximity to its own silencer.

Differentiation of MHC class II-selected thymocytes toward the CD4(+) helper lineage depends on function of the transcription factor ThPOK, whose expression is repressed in CD8(+) cytotoxic lineage cells by a transcriptional silencer activity within the distal regulatory element (DRE) in the Thpok gene. Interestingly, the DRE also functions as a transcriptional enhancer. However, how the DRE exerts such dual functionality remains obscure.

Regeneration of human tumor antigen-specific T cells from iPSCs derived from mature CD8(+) T cells.

Antigen-specific T cells represent a potential therapeutic avenue for a variety of conditions, but current approaches for generating such cells for therapeutic purposes are limited. In this study, we established iPSCs from mature cytotoxic T cells specific for the melanoma epitope MART-1. When cocultured with OP9/DLL1 cells, these iPSCs efficiently generated TCRβ(+)CD4(+)CD8(+) double positive (DP) cells expressing a T cell receptor (TCR) specific for the MART-1 epitope.

Toward an understanding of immune cell sociology: real-time monitoring of cytokine secretion at the single-cell level.

The immune system is a very complex and dynamic cellular system, and its intricacies are considered akin to those of human society. Disturbance of homeostasis of the immune system results in various types of diseases; therefore, the homeostatic mechanism of the immune system has long been a subject of great interest in biology, and a lot of information has been accumulated at the cellular and the molecular levels. However, the sociological aspects of the immune system remain too abstract to address because of its high complexity, which mainly originates from a large number and variety of cell-cell interactions.

Tollip-induced down-regulation of MARCH1.

In addition to their classical antigen presenting functions, MHC class II molecules potentiate the TLR-triggered production of pro-inflammatory cytokines. Here, we have addressed the effect of Tollip and MARCH1 on the regulation of MHC II trafficking and TLR signaling. Our results show that MARCH1-deficient mice splenocytes are impaired in their capacity to produce pro-inflammatory cytokines in response to poly(I:C) and that TLR3 and MHC II molecules interact in the endocytic pathway.

Stochastic simulation of structured skin cell population dynamics.

The epidermis is the outmost skin tissue. It operates as a first defense system to process inflammatory signals and responds by producing inflammatory mediators that promote the recruitment of immune cells. Various skin diseases such as atopic dermatitis occur as a result of the defect of proper skin barrier function and successive impaired inflammatory responses.

Spermatogenesis arrest caused by conditional deletion of Hsp90α in adult mice.

It is controversial whether a functional androgen receptor (AR) on germ cells, including spermatogonia, is essential for their development into sperm and, thus, initiation and maintenance of spermatogenesis. It was recently shown that many spermatocytes underwent apoptosis in the testes of Hsp90α KO mice. We had generated Hsp90α KO mice independently and confirmed this phenotype.

M cell differentiation: distinct lineage or phenotypic transition? Salmonella provides answers.

Whether M cells arise from a distinct lineage or result from phenotypic transition is a matter of debate. In this issue of Cell Host & Microbe, Tahoun et al. (2012) provide evidence that SopB, a virulence factor of Salmonella enterica serovar Typhimurium, can induce phenotypic transition of lymphoid follicle-associated enterocytes into M cells.

A Dual Reporter Splicing Assay Using HaloTag-containing Proteins.

To evaluate the effects of genetic variations on mRNA splicing, we developed a minigene-based splicing assay using reporter genes encoding luciferase and the multifunctional HaloTag protein. In addition to conventional RT-PCR analysis, splicing events can be monitored in this system using two parameters: luciferase activity and signals derived from HaloTag-containing proteins bound to a fluorescent ligand following SDS-PAGE. The luciferase activity reflects the accumulated amounts of successfully spliced HaloTag-luciferase fusion products, whereas the amounts and sizes of HaloTag-containing proteins provide quantitative insights into precursor, correctly spliced, and aberrantly spliced mRNA species.

Vaccination with antigen-transfected, NKT cell ligand-loaded, human cells elicits robust in situ immune responses by dendritic cells.

Both innate and adaptive immunity are crucial for cancer immunosurveillance, but precise therapeutic equations to restore immunosurveillance in patients with cancer patients have yet to be developed. In murine models, α-galactosylceramide (α-GalCer)-loaded, tumor antigen-expressing syngeneic or allogeneic cells can act as cellular adjuvants, linking the innate and adaptive immune systems. In the current study, we established human artificial adjuvant vector cells (aAVC) consisting of human HEK293 embryonic kidney cells stably transfected with the natural killer T (NKT) immune cell receptor CD1d, loaded with the CD1d ligand α-GalCer and then transfected with antigen-encoding mRNA.