Active form of Notch imposes T cell fate in human progenitor cells

The crucial role of Notch signaling in cell fate decisions in hematopoietic lineage and T lymphocyte development has been well established in mice. Overexpression of the intracellular domain of Notch mediates signal transduction of the protein. By retroviral transduction of this constitutively active truncated intracellular domain in human CD34+ umbilical cord blood progenitor cells, we were able to show that, in coculture with the stromal MS-5 cell line, depending on the cytokines added, the differentiation toward CD19+ B lymphocytes was blocked, the differentiation toward CD14+ monocytes was inhibited, and the differentiation toward CD56+ NK cells was favored. The number of CD7+cyCD3+ cells, a phenotype similar to T/NK progenitor cells, was also markedly increased. In fetal thymus organ culture, transduced CD34+ progenitor cells from umbilical cord blood cells or from thymus consistently generated more TCR-gammadelta T cells, whereas the other T cell subpopulations were largely unaffected. Interestingly, when injected in vivo in SCID-nonobese diabetic mice, the transduced cells generated ectopically human CD4+CD8+ TCR-alphabeta cells in the bone marrow, cells that are normally only present in the thymus, and lacked B cell differentiation potential. Our results show unequivocally that, in human, Notch signaling inhibits the monocyte and B cell fate, promotes the T cell fate, and alters the normal T cell differentiation pathway compatible with a pretumoral state.     

Cutting edge: CD8+ T cell priming in the absence of NK cells leads to enhanced memory responses

It is uncertain whether NK cells modulate T cell memory differentiation. By using a genetic model that allows the selective depletion of NK cells, we show in this study that NK cells shape CD8(+) T cell fate by killing recently activated CD8(+) T cells in an NKG2D- and perforin-dependent manner. In the absence of NK cells, the differentiation of CD8(+) T cells is strongly biased toward a central memory T cell phenotype. Although, on a per-cell basis, memory CD8(+) T cells generated in the presence or the absence of NK cells have similar functional features and recall capabilities, NK cell deletion resulted in a significantly higher number of memory Ag-specific CD8(+) T cells, leading to more effective control of tumors carrying model Ags. The enhanced memory responses induced by the transient deletion of NK cells may provide a rational basis for the design of new vaccination strategies.     

mTOR Signaling pathway as a master regulator of memory CD8+ T-cells,Th17, and NK cells development and their functional properties

The mammalian target of rapamycin (mTOR) is a member of the evolutionary phosphatidylinositol kinase-related kinases (PIKKs). mTOR plays a pivotal role in the regulation of diverse aspects of cellular physiology such as body metabolism, cell growth, protein synthesis, cell size, autophagy, and cell differentiation. Immunologically, mTOR has a fundamental part in controlling and shaping diverse functions of innate and adaptive immune cells, in particular, T-cell subsets differentiation, survival, and metabolic reprogramming to ultimately regulate the fate of diverse immune cell types. Researchers report that rapamycin, a selective mTOR inhibitor, and immunosuppressive agent, has surprising immunostimulatory effects on inducing both quantitative and qualitative aspects of virus-specific memory CD8⁺ T-cells differentiation and homeostasis in a T-cell-intrinsic manner. The mTOR signaling pathway also plays a critical role in dictating the outcome of regulatory T cells (Treg), T helper 17 (Th17) cells, and natural killer (NK) cells proliferation and maturation, as well as the effector functions and cytotoxic properties of NK cells. Manipulation of mTOR activity is a critical therapeutic approach for pharmacological agents that seek to inhibit mTOR. This approach should enhance specific memory CD8 ⁺ T-cells responses and induce fully functional effector properties of NK cells to provoke their antitumor and antiviral activities.     

Asymmetric cell division during T cell development controls downstream fate

During mammalian T cell development, the requirement for expansion of many individual T cell clones, rather than merely expansion of the entire T cell population, suggests a possible role for asymmetric cell division (ACD). We show that ACD of developing T cells controls cell fate through differential inheritance of cell fate determinants Numb and α-Adaptin. ACD occurs specifically during the β-selection stage of T cell development, and subsequent divisions are predominantly symmetric. ACD is controlled by interaction with stromal cells and chemokine receptor signaling and uses a conserved network of polarity regulators. The disruption of polarity by deletion of the polarity regulator, Scribble, or the altered inheritance of fate determinants impacts subsequent fate decisions to influence the numbers of DN4 cells arising after the β-selection checkpoint. These findings indicate that ACD enables the thymic microenvironment to orchestrate fate decisions related to differentiation and self-renewal.     

A model of sequential branching in hierarchical cell fate determination

Multipotent stem or progenitor cells undergo a sequential series of binary fate decisions, which ultimately generate the diversity of differentiated cells. Efforts to understand cell fate control have focused on simple gene regulatory circuits that predict the presence of multiple stable states, bifurcations and switch-like transitions. However, existing gene network models do not explain more complex properties of cell fate dynamics such as the hierarchical branching of developmental paths. Here, we construct a generic minimal model of the genetic regulatory network controlling cell fate determination, which exhibits five elementary characteristics of cell differentiation: stability, directionality, branching, exclusivity, and promiscuous expression. We argue that a modular architecture comprising repeated network elements reproduces these features of differentiation by sequentially repressing selected modules and hence restricting the dynamics to lower dimensional subspaces of the high-dimensional state space. We implement our model both with ordinary differential equations (ODEs), to explore the role of bifurcations in producing the one-way character of differentiation, and with stochastic differential equations (SDEs), to demonstrate the effect of noise on the system. We further argue that binary cell fate decisions are prevalent in cell differentiation due to general features of the underlying dynamical system. This minimal model makes testable predictions about the structural basis for directional, discrete and diversifying cell phenotype development and thus can guide the evaluation of real gene regulatory networks that govern differentiation.     

Cell Fate Reprogramming by Control of Intracellular Network Dynamics

Identifying control strategies for biological networks is paramount for practical applications that involve reprogramming a cell’s fate, such as disease therapeutics and stem cell reprogramming. Here we develop a novel network control framework that integrates the structural and functional information available for intracellular networks to predict control targets. Formulated in a logical dynamic scheme, our approach drives any initial state to the target state with 100% effectiveness and needs to be applied only transiently for the network to reach and stay in the desired state. We illustrate our method’s potential to find intervention targets for cancer treatment and cell differentiation by applying it to a leukemia signaling network and to the network controlling the differentiation of helper T cells. We find that the predicted control targets are effective in a broad dynamic framework. Moreover, several of the predicted interventions are supported by experiments.     

A Virtual Culture of CD4+ T Lymphocytes

The CD4+ T cell lineages Th1, Th2, Th17, and Treg, are mammalian cell types that differentiate from the common precursor naive CD4+ T cell. While there is a wealth of experimental data regarding the molecular and cellular signals involved in the differentiation of CD4+ T cells in vitro, there is still no consensus regarding the structure of the network of interactions at the molecular and cellular levels controlling this differentiation process. In this work, a virtual culture of cells is constructed by interconnecting several instances of an updated version of the regulatory network controlling the differentiation process of CD4+ T cells in mice. The virtual culture is a multi-compartment model with an instance of a regulatory network inside each compartment, thus simulating a simplified version of a cell culture in a well-stirred reactor. The virtual culture is able to describe the stable molecular expression patterns described for fully differentiated CD4+ T cells in mice, as well as the differentiation process from a precursor to a given effector cell in response to specific molecular stimuli.     

Pluripotency,Differentiation, and Reprogramming: A Gene Expression Dynamics Model with Epigenetic Feedback Regulation

Embryonic stem cells exhibit pluripotency: they can differentiate into all types of somatic cells. Pluripotent genes such as Oct4 and Nanog are activated in the pluripotent state, and their expression decreases during cell differentiation. Inversely, expression of differentiation genes such as Gata6 and Gata4 is promoted during differentiation. The gene regulatory network controlling the expression of these genes has been described, and slower-scale epigenetic modifications have been uncovered. Although the differentiation of pluripotent stem cells is normally irreversible, reprogramming of cells can be experimentally manipulated to regain pluripotency via overexpression of certain genes. Despite these experimental advances, the dynamics and mechanisms of differentiation and reprogramming are not yet fully understood. Based on recent experimental findings, we constructed a simple gene regulatory network including pluripotent and differentiation genes, and we demonstrated the existence of pluripotent and differentiated states from the resultant dynamical-systems model. Two differentiation mechanisms, interaction-induced switching from an expression oscillatory state and noise-assisted transition between bistable stationary states, were tested in the model. The former was found to be relevant to the differentiation process. We also introduced variables representing epigenetic modifications, which controlled the threshold for gene expression. By assuming positive feedback between expression levels and the epigenetic variables, we observed differentiation in expression dynamics. Additionally, with numerical reprogramming experiments for differentiated cells, we showed that pluripotency was recovered in cells by imposing overexpression of two pluripotent genes and external factors to control expression of differentiation genes. Interestingly, these factors were consistent with the four Yamanaka factors, Oct4, Sox2, Klf4, and Myc, which were necessary for the establishment of induced pluripotent stem cells. These results, based on a gene regulatory network and expression dynamics, contribute to our wider understanding of pluripotency, differentiation, and reprogramming of cells, and they provide a fresh viewpoint on robustness and control during development.     

Tracking the immunoregulatory mechanisms active during allograft tolerance

Immunoregulatory mechanisms dependent on regulatory CD4+ T cells are believed to be critical in the maintenance of peripheral tolerance to allografts. However, a detailed characterization of the effects of these regulatory T cells has been hampered by the absence of a simple means to track and study them. In this work we provide evidence that in a murine model of islet transplantation the interactions between alloaggressive and regulatory T cells can be studied in vitro and in vivo at the single-cell level. The observations made in both an in vitro coculture system and an in vivo CFSE-based adoptive transfer model indicate that lymphocytes from tolerant allograft recipients 1) proliferate weakly to donor strain allogeneic cells but vigorously to third-party strain cells; and 2) suppress the proliferation of naive syngeneic CD4+ and CD8+ T cells to donor tissue in a cell dose- and Ag-specific manner. These effects depend on the presence of CD4+CD25+ T cells and are neutralized by anti-CTLA4 mAb or rIL-2. The principal effect of anti-CTLA4 is directed against the naive, not regulatory, T cell population. These results can be replicated in vivo by transferring lymphocyte populations into transplant recipients, proving that the graft-protecting actions of regulatory T cells are blunted by a rise in the number of allodestructive T cells (pool size model) and depend on the presence of CD4+CD25+ T cells and the integrity of the CTLA4/B7 pathway.     

Reference ranges and age-related changes of peripheral blood lymphocyte subsets in Chinese healthy adults

This study was performed to build region-specific reference ranges of peripheral blood lymphocyte subsets for Chinese healthy adults from the young to the elderly and analyze the trends of changes in lymphocyte subsets for evaluating the impact of age on the values.151 healthy adults aged 19—86 were recruited based on the SENIEUR protocol.Three sets of reference ranges were finally built applicable for the healthy young(19—44 years),middle-aged(45—64 years) and elder adults(≥65).Comparisons in parameters am...     

Essential Role of NK Cells in IgG Therapy for Experimental Autoimmune Encephalomyelitis

Intravenous immunoglobulin has long been used in treating autoimmune diseases, although mechanisms remain uncertain. Activating Fcγ receptors are receptors of IgG and reported to be essential in intravenous immunoglobulin (IVIG) therapy. Therefore, we hypothesized natural killer (NK) cells, which express abundant activating Fcγ receptors, are the potential cellular target. In experimental autoimmune encephalomyelitis (EAE), we demonstrated that IgG suppressed disease development in intact, but not in NK cell depleted mice. Adoptive transfer of IgG-treated NK cell could protect mice against EAE, and suppressed interferon γ and interleukin 17 production. The percentage of CD4⁺Foxp3⁺ regulatory T cells was significantly increased. The increase of regulatory T cells was also observed in IgG-treated EAE mice but not in NK cell depleted mice. In vitro experiments confirmed that IgG-treated NK cells enhanced regulatory T cell induction from naïve CD4⁺ T cells. Interestingly, cells from draining lymph nodes produced more interleukin 2 after the adoptive transfer of IgG-treated NK cells. We neutralized interleukin 2 and the induction of CD4⁺Foxp3⁺ T cells by IgG-treated NK cells was significantly reduced. To our knowledge, we identified for the first time the critical role of NK cells in the mechanism of IgG-induced induction of Treg cells in treatment of autoimmunity.     

Characterization of developmental pathway of natural killer cells from embryonic stem cells in vitro

In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34(+) EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34(+) EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1-transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34(+) EB cells, NK and T cell potentials were detected in a CD45(-) subset, whereas CD45(+) EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34(+)CD45(-) EB cells showed that CD45(+)Mac-1(-)Ter119(-) ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45(-)CD34(+) EB cells have lymphoid potential, and they differentiate into more mature CD45(+)Lin(-) hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122(-) and they rapidly acquire CD122 as they differentiate along the NK lineage.