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The microbiome is known to play a role in many human diseases, but identifying key microbes and their functions generally requires large studies due to the vast number of species and genes, and the high levels of intra-individual and inter-individual variation. 16S amplicon sequencing of the rRNA gene is commonly used for large studies due to its comparatively low sequencing cost, but it has poor taxonomic and functional resolution. Deep shotgun sequencing is a more accurate and comprehensive alternative for small studies, but can be cost-prohibitive for biomarker discovery in large populations. Shallow or moderate-depth shotgun metagenomics may serve as a viable alternative to 16S sequencing for large-scale and/or dense longitudinal studies, but only if resolution and reproducibility are comparable. Here we applied both 16S and shallow shotgun stool microbiome sequencing to a cohort of 5 subjects sampled twice daily and weekly, with technical replication at the DNA extraction and the library preparation/sequencing steps, for a total of 80 16S samples and 80 shallow shotgun sequencing samples. We found that shallow shotgun sequencing produced lower technical variation and higher taxonomic resolution than 16S sequencing, at a much lower cost than deep shotgun sequencing. These findings suggest that shallow shotgun sequencing provides a more specific and more reproducible alternative to 16S sequencing for large-scale microbiome studies where costs prohibit deep shotgun sequencing and where bacterial species are expected to have good coverage in whole-genome reference databases. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol-3-kinases (PI3K) γ and δ are preferentially enriched in leukocytes, and defects in these signaling pathways have been shown to impair T cell activation. The effects of PI3Kγ and PI3Kδ on alloimmunity remain underexplored. Here, we show that both PI3Kγ−/− and PI3KδD910A/D910A mice receiving heart allografts have suppression of alloreactive T effector cells and delayed acute rejection. However, PI3Kδ mutation also dampens regulatory T cells (Treg). After treatment with low dose CTLA4-Ig, PI3Kγ−/−, but not PI3ΚδD910A/D910A, recipients exhibit indefinite prolongation of heart allograft survival. PI3KδD910A/D910A Tregs have increased apoptosis and impaired survival. Selective inhibition of PI3Kγ and PI3Kδ (using PI3Kδ and dual PI3Kγδ chemical inhibitors) shows that PI3Kγ inhibition compensates for the negative effect of PI3Kδ inhibition on long-term allograft survival. These data serve as a basis for future PI3K-based immune therapies for transplantation. [ABSTRACT FROM AUTHOR]

Stem cells reside in a specialized regulatory microenvironment or niche, where they receive appropriate support for maintaining self-renewal and multi-lineage differentiation capacity. The niche may also protect stem cells from environmental insults including cytotoxic chemotherapy and perhaps pathogenic immunity. The testis, hair follicle and placenta are all sites of residence for stem cells and are immune-suppressive environments, called immune-privileged sites, where multiple mechanisms cooperate to prevent immune attack, even enabling prolonged survival of foreign allografts without immunosuppression. We sought to determine if somatic stem-cell niches more broadly are immune-privileged sites by examining the haematopoietic stem/progenitor cell (HSPC) niche in the bone marrow, a site where immune reactivity exists. We observed persistence of HSPCs from allogeneic donor mice (allo-HSPCs) in non-irradiated recipient mice for 30?days without immunosuppression with the same survival frequency compared to syngeneic HSPCs. These HSPCs were lost after the depletion of FoxP3 regulatory T (Treg) cells. High-resolution in vivo imaging over time demonstrated marked co-localization of HSPCs with Treg cells that accumulated on the endosteal surface in the calvarial and trabecular bone marrow. Treg cells seem to participate in creating a localized zone where HSPCs reside and where Treg cells are necessary for allo-HSPC persistence. In addition to processes supporting stem-cell function, the niche will provide a relative sanctuary from immune attack. [ABSTRACT FROM AUTHOR]

Here we present methods to longitudinally track islet allograft–infiltrating T cells in live mice by endoscopic confocal microscopy and to analyze circulating T cells by in vivo flow cytometry. We developed a new reporter mouse whose T cell subsets express distinct, 'color-coded' proteins enabling in vivo detection and identification of effector T cells (Teff cells) and discrimination between natural and induced regulatory T cells (nTreg and iTreg cells). Using these tools, we observed marked differences in the T cell response in recipients receiving tolerance-inducing therapy (CD154-specific monoclonal antibody plus rapamycin) compared to untreated controls. These results establish real-time cell tracking as a powerful means to probe the dynamic cellular interplay mediating immunologic rejection or transplant tolerance. [ABSTRACT FROM AUTHOR]

Transcription factor Foxp3 is critical for generating regulatory T cells (Treg cells). Transforming growth factor-β (TGF-β) induces Foxp3 and suppressive Treg cells from naive T cells, whereas interleukin 6 (IL-6) inhibits the generation of inducible Treg cells. Here we show that IL-4 blocked the generation of TGF-β-induced Foxp3+ Treg cells and instead induced a population of T helper cells that produced IL-9 and IL-10. The IL-9+IL-10+ T cells demonstrated no regulatory properties despite producing abundant IL-10. Adoptive transfer of IL-9+IL-10+ T cells into recombination-activating gene 1–deficient mice induced colitis and peripheral neuritis, the severity of which was aggravated if the IL-9+IL-10+ T cells were transferred with CD45RBhi CD4+ effector T cells. Thus IL-9+IL-10+ T cells lack suppressive function and constitute a distinct population of helper-effector T cells that promote tissue inflammation. [ABSTRACT FROM AUTHOR]

On activation, naive T cells differentiate into effector T-cell subsets with specific cytokine phenotypes and specialized effector functions. Recently a subset of T cells, distinct from T helper (TH)1 and TH2 cells, producing interleukin (IL)-17 (TH17) was defined and seems to have a crucial role in mediating autoimmunity and inducing tissue inflammation. We and others have shown that transforming growth factor (TGF)-β and IL-6 together induce the differentiation of TH17 cells, in which IL-6 has a pivotal function in dictating whether T cells differentiate into Foxp3+ regulatory T cells (Treg cells) or TH17 cells. Whereas TGF-β induces Foxp3 and generates Treg cells, IL-6 inhibits the generation of Treg cells and induces the production of IL-17, suggesting a reciprocal developmental pathway for TH17 and Treg cells. Here we show that IL-6-deficient (Il6-/-) mice do not develop a TH17 response and their peripheral repertoire is dominated by Foxp3+ Treg cells. However, deletion of Treg cells leads to the reappearance of TH17 cells in Il6-/- mice, suggesting an additional pathway by which TH17 cells might be generated in vivo. We show that an IL-2 cytokine family member, IL-21, cooperates with TGF-β to induce TH17 cells in naive Il6-/- T cells and that IL-21-receptor-deficient T cells are defective in generating a TH17 response. [ABSTRACT FROM AUTHOR]

Treatment with ex vivo–generated regulatory T cells (T-reg) has been regarded as a potentially attractive therapeutic approach for autoimmune diseases. However, the dynamics and function of T-reg in autoimmunity are not well understood. Thus, we developed Foxp3gfp knock-in (Foxp3gfp.KI) mice and myelin oligodendrocyte glycoprotein (MOG)35–55/IAb (MHC class II) tetramers to track autoantigen-specific effector T cells (T-eff) and T-reg in vivo during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. MOG tetramer–reactive, Foxp3+ T-reg expanded in the peripheral lymphoid compartment and readily accumulated in the central nervous system (CNS), but did not prevent the onset of disease. Foxp3+ T cells isolated from the CNS were effective in suppressing naive MOG-specific T cells, but failed to control CNS-derived encephalitogenic T-eff that secreted interleukin (IL)-6 and tumor necrosis factor (TNF). Our data suggest that in order for CD4+Foxp3+ T-reg to effectively control autoimmune reactions in the target organ, it may also be necessary to control tissue inflammation. [ABSTRACT FROM AUTHOR]

Contrary to the proinflammatory role of mast cells in allergic disorders, the results obtained in this study establish that mast cells are essential in CD4+CD25+Foxp3+ regulatory T (TReg)-cell-dependent peripheral tolerance. Here we confirm that tolerant allografts, which are sustained owing to the immunosuppressive effects of TReg cells, acquire a unique genetic signature dominated by the expression of mast-cell-gene products. We also show that mast cells are crucial for allograft tolerance, through the inability to induce tolerance in mast-cell-deficient mice. High levels of interleukin (IL)-9—a mast cell growth and activation factor—are produced by activated TReg cells, and IL-9 production seems important in mast cell recruitment to, and activation in, tolerant tissue. Our data indicate that IL-9 represents the functional link through which activated TReg cells recruit and activate mast cells to mediate regional immune suppression, because neutralization of IL-9 greatly accelerates allograft rejection in tolerant mice. Finally, immunohistochemical analysis clearly demonstrates the existence of this novel TReg–IL-9–mast cell relationship within tolerant allografts. [ABSTRACT FROM AUTHOR]

On activation, T cells undergo distinct developmental pathways, attaining specialized properties and effector functions. T-helper (TH) cells are traditionally thought to differentiate into TH1 and TH2 cell subsets. TH1 cells are necessary to clear intracellular pathogens and TH2 cells are important for clearing extracellular organisms. Recently, a subset of interleukin (IL)-17-producing T (TH17) cells distinct from TH1 or TH2 cells has been described and shown to have a crucial role in the induction of autoimmune tissue injury. In contrast, CD4+CD25+Foxp3+ regulatory T (Treg) cells inhibit autoimmunity and protect against tissue injury. Transforming growth factor-β (TGF-β) is a critical differentiation factor for the generation of Treg cells. Here we show, using mice with a reporter introduced into the endogenous Foxp3 locus, that IL-6, an acute phase protein induced during inflammation, completely inhibits the generation of Foxp3+ Treg cells induced by TGF-β. We also demonstrate that IL-23 is not the differentiation factor for the generation of TH17 cells. Instead, IL-6 and TGF-β together induce the differentiation of pathogenic TH17 cells from naive T cells. Our data demonstrate a dichotomy in the generation of pathogenic (TH17) T cells that induce autoimmunity and regulatory (Foxp3+) T cells that inhibit autoimmune tissue injury. [ABSTRACT FROM AUTHOR]

Tim-3 is a T helper type 1 (TH1)–specific cell surface molecule that seems to regulate TH1 responses and the induction of peripheral tolerance. However, the identity of the Tim-3 ligand and the mechanism by which this ligand inhibits the function of effector TH1 cells remain unknown. Here we show that galectin-9 is the Tim-3 ligand. Galectin-9-induced intracellular calcium flux, aggregation and death of TH1 cells were Tim-3-dependent in vitro, and administration of galectin-9 in vivo resulted in selective loss of interferon-γ-producing cells and suppression of TH1 autoimmunity. These data suggest that the Tim-3–galectin-9 pathway may have evolved to ensure effective termination of effector TH1 cells. [ABSTRACT FROM AUTHOR]

The newly identified TIM family of proteins is associated with regulation of T helper type 1 (TH1) and TH2 immune responses. TIM-1 is genetically linked to asthma and is a receptor for hepatitis A virus, but the endogenous ligand of TIM-1 is not known. Here we show that TIM-4, which is expressed by antigen-presenting cells, is the ligand for TIM-1. In vivo administration of either soluble TIM-1-immunoglobulin (TIM-1-Ig) fusion protein or TIM-4-Ig fusion protein resulted in hyperproliferation of T cells, and TIM-4-Ig costimulated T cell proliferation mediated by CD3 and CD28 in vitro. These data suggest that the TIM-1-TIM-4 interaction is involved in regulating T cell proliferation. [ABSTRACT FROM AUTHOR]

Type 1 diabetes mellitus results from autoimmune destruction of the insulin-secreting cells in the pancreas. Daily treatment with exogenous insulin is required, but because of difficulties in achieving physiological control of blood-glucose concentrations, chronic and degenerative complications still occur in a marked fraction of patients. Islet transplantation can normalize metabolic control in a way that has been virtually impossible to achieve with exogenous insulin, but life-long immunosuppression of the recipients is required, limiting the procedure to the most severe forms of diabetes. This article outlines the history of and recent progress in the field, as well as the present immunological challenges and possible strategies for tolerance induction that are crucial to make clinical islet transplantation more widely available. [ABSTRACT FROM AUTHOR]

T helper type 1 (TH1) immune responses are central in cell-mediated immunity, and a TH1-specific cell surface molecule called Tim-3 (T cell immunoglobulin domain, mucin domain) has been identified. Here we report the identification of a secreted form of Tim-3 that contains only the immunoglobulin (Ig) variable (V) domain of the full-length molecule. Fusion proteins (Tim-3-Ig) of both Tim-3 isoforms specifically bound CD4+ T cells, indicating that a Tim-3 ligand is expressed on CD4+ T cells. Administration of Tim-3-Ig to immunized mice caused hyperproliferation of TH1 cells and TH1 cytokine release. Tim-3-Ig also abrogated tolerance induction in TH1 cells, and Tim-3-deficient mice were refractory to the induction of high-dose tolerance. These data indicate that interaction of Tim-3 with Tim-3 ligand may serve to inhibit effector TH1 cells during a normal immune response and may be crucial for the induction of peripheral tolerance. [ABSTRACT FROM AUTHOR]

Although T helper (TH) cell-mediated immunity is required to effectively eliminate pathogens, unrestrained TH activity also contributes to tissue injury in many inflammatory and autoimmune diseases. We report here that the TH type 1 (TH1)-specific Tim-3 (T cell immunoglobulin domain, mucin domain) protein functions to inhibit aggressive TH1-mediated auto- and alloimmune responses. Tim-3 pathway blockade accelerated diabetes in nonobese diabetic mice and prevented acquisition of transplantation tolerance induced by costimulation blockade. These effects were mediated, at least in part, by dampening of the antigen-specific immunosuppressive function of CD4+CD25+ regulatory T cell populations. Our data indicate that the Tim-3 pathway provides an important mechanism to down-regulate TH1-dependent immune responses and to facilitate the development of immunological tolerance. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-2 and IL-15 are redundant in stimulating T-cell proliferation in vitro. Their precise role in vivo in governing T-cell expansion and T-cell homeostasis is less clear. Each may have distinct functions and regulate distinct aspects of T-cell activation. The functional receptors for IL-2 and IL-15 consist of a private a-chain, which defines the binding specificity for IL-2 or IL-15, and shared IL-2 receptor ?- and ?-chains. The ?-chain is also a critical signaling component of IL-4, IL-7 and IL-9 receptors. Thus, the ?-chain is called the common ? or ?-c. As these receptor subunits can be expressed individually or in various combinations resulting in the formation of receptors with different affinities, distinct signaling capabilities or both, we hypothesized that differential expression of IL-2 and IL-15 receptor subunits on cycling T cells in vivo may direct activated T cells to respond to IL-2 or IL-15, thereby regulating the homeostasis of T-cell response in vivo. By observing in vivo T-cell divisions and expression of IL-2 and IL-15 receptor subunits, we demonstrate that IL-15 is a critical growth factor in initiating T cell divisions in vivo, whereas IL-2 limits continued T-cell expansion via downregulation of the ?-c expression. Decreased ?-c expression on cycling T cells reduced sustained Bcl-2 expression and rendered cells susceptible to apoptotic cell death. Our study provides data that IL-2 and IL-15 regulate distinct aspects of primary T-cell expansion in vivo. [ABSTRACT FROM AUTHOR]

The alloimmune response against fully MHC-mismatched allografts, compared with immune responses to nominal antigens, entails an unusually large clonal size of alloreactive T cells. Thus, induction of peripheral allograft tolerance established in the absence of immune system ablation and reconstitution is a challenging task in transplantation. Here, we determined whether a reduction in the mass of alloreactive T cells due to apoptosis is an essential initial step for induction of stable allograft tolerance with non-lymphoablative therapy. Blocking both CD28-B7 and CD40-CD40 ligand interactions (co-stimulation blockade) inhibited proliferation of alloreactive T cells in vivo while allowing cell cycle-dependent T-cell apoptosis of proliferating T cells, with permanent engraftment of cardiac allografts but not skin allografts. Treatment with rapamycin plus co-stimulation blockade resulted in massive apoptosis of alloreactive T cells and produced stable skin allograft tolerance, a very stringent test of allograft tolerance. In contrast, treatment with cyclosporine A and co-stimulation blockade abolished Tcell proliferation and apoptosis, as well as the induction of stable allograft tolerance. Our data indicate that induction of T-cell apoptosis and peripheral allograft tolerance is prevented by blocking both signal 1 and signal 2 of T-cell activation. [ABSTRACT FROM AUTHOR]

The mechanisms of allograft tolerance have been classified as deletion, anergy, ignorance and suppression/regulation. Deletion has been implicated in central tolerance, whereas peripheral tolerance has generally been ascribed to clonal anergy and/or active immunoregulatory states. Here, we used two distinct systems to assess the requirement for T-cell deletion in peripheral tolerance induction. In mice transgenic for Bcl-x[sub L], T cells were resistant to passive cell death through cytokine withdrawal, whereas T cells from interleukin-2-deficient mice did not undergo activation-induced cell death. Using either agents that block co-stimulatory pathways or the immunosuppressive drug rapamycin, which we have shown here blocks the proliferative component of interleukin-2 signaling but does not inhibit priming for activation-induced cell death, we found that mice with defective passive or active T-cell apoptotic pathways were resistant to induction of transplantation tolerance. Thus, deletion of activated T cells through activation-induced cell death or growth factor withdrawal seems necessary to achieve peripheral tolerance across major histocompatibility complex barriers. [ABSTRACT FROM AUTHOR]

Multiple immunoregulatory drugs, capable of constraining cell activation, differentiation, and/or proliferation, and each with distinct side effects, are currently used in the clinic to facilitate organ engraftment. Cyclosporine, azathioprine, corticosteroids, FK506 (tacrolimus), and RS61443 (mycophenolate mofetil) have already been approved by the United States Food and Drug Administration. Rapamycin (sirolimus), mizoribine, and 15-deoxyspergualin are being explored for their clinical efficacy. Based on their subcellular site of action, the immunosuppressants can be considered as: inhibitors of transcription (cyclosporine, tacrolimus), inhibitors of nucleotide synthesis (azathioprine, mycophenolate mofetil, mizoribine), inhibitors of growth factor signal transduction (sirolimus), and inhibitors of differentiation (15-deoxyspergualin). Clearly, the transplant clinician now has a greater choice in the selection and application of immunosuppressants in the clinic for the fine regulation of anti-allograft immunity. The existing hypothesis regarding the mechanisms of action of immunosuppressants is that they all function to prevent allograft rejection by preventing/inhibiting cell activation, cytokine production, differentiation, and/or proliferation. A complementary supposition is that some of the immunosuppressants might regulate the anti-allograft repertory by stimulating the expression of immunosuppressive molecules and/or cells. [ABSTRACT FROM AUTHOR]

Percutaneous transluminal angioplasty was performed on a right common iliac artery stenosis presumed to be causing renovascular hypertension in a patient with a renal allograft anastomosis to the right external iliac artery. This was complicated by an obstructive dissection resulting in acute threatening renal allograft ischemia. Renal blood flow was restored by means of a transluminal reperfusion catheter until corrective surgery could be performed. This case is reported because such catheters can be acutely helpful to the interventionalist, and they have not been described in the radiology literature. [ABSTRACT FROM AUTHOR]

Activated mucosal T lymphocytes correlate with the intestinal inflammation of inflammatory bowel disease. Activated T cells elaborate interferon-gamma (IFN-gamma) and express high-affinity interleukin-2 (IL-2) receptors. The IL-2/diphtheria toxin fusion protein (DAB389IL-2) has been shown to specifically kill high affinity IL-2 receptor-bearing cells. We tested whether DAB389IL-2 could specifically target activated lamina propria lymphocytes. Lymphocytes were activated in vitro with phytohemagglutinin and IL-2 for 24-48 hr. Toxin efficacy was determined by the [14C]leucine incorporation, IFN-gamma ELISA, and flow cytometry. DAB389IL-2 (10(-11) M) inhibited protein synthesis by 80% in activated lamina propria lymphocytes. This inhibition was blocked by coculture of either excess IL-2 or a nonfunctional IL-2 diphtheria toxin mutant protein. DAB389IL-2 (10(-12) M) also significantly reduced the numbers of activated helper T cells and IFN-gamma levels in 24-hr cultures. DAB389IL-2 specifically targets activated IL-2 receptor-positive lamina propria lymphocytes and is a potential new therapeutic agent for patients with active inflammatory bowel disease. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor about the symptoms of Crohn's disease.

The article reports on the findings of a study which used a noninvasive, easy-to-perform biomarker-based assay to detect cellular rejection in transplanted kidney. It notes that the study determined that renal biopsy is not considered as the gold standard for detection of allograft response until evidence of renal dysfunction is apparent. Moreover, the study revealed the diagnostic and prognostic role of urinary cells in acute cellular rejection.

Focuses on the improvement of bone marrow transplantation by facilitating cells. Promotion of hematopoietic stem cell engraftment by the facilitating cells; Role of facilitating cells in solid organ transplantation; Identification of facilitating cells in bone marrow by cell surface makers and flow cytometric cell sorting.

Replies to a comment on the authors' paper 'Failure of combined costimulatory blockade in animal transplant model.' Point about the ability of costimulation blockade using anti-CD154 and CTLA4-Ig to produce permanent engraftment in allograft models.