Selective loss of innate CD4(+) V alpha 24 natural killer T cells in human immunodeficiency virus infection

V alpha 24 natural killer T (NKT) cells are innate immune cells involved in regulation of immune tolerance, autoimmunity, and tumor immunity. However, the effect of human immunodeficiency virus type 1 (HIV-1) infection on these cells is unknown. Here, we report that the V alpha 24 NKT cells can be subdivided into CD4(+) or CD4(-) subsets that differ in their expression of the homing receptors CD62L and CD11a. Furthermore, both CD4(+) and CD4(-) NKT cells frequently express both CXCR4 and CCR5 HIV coreceptors. We find that the numbers of NKT cells are reduced in HIV-infected subjects with uncontrolled viremia and marked CD4(+) T-cell depletion. The number of CD4(+) NKT cells is inversely correlated with HIV load, indicating depletion of this subset. In contrast, CD4(-) NKT-cell numbers are unaffected in subjects with high viral loads. HIV infection experiments in vitro show preferential depletion of CD4(+) NKT cells relative to regular CD4(+) T cells, in particular with virus that uses the CCR5 coreceptor. Thus, HIV infection causes a selective loss of CD4(+) lymph node homing (CD62L(+)) NKT cells, with consequent skewing of the NKT-cell compartment to a predominantly CD4(-) CD62L(-) phenotype. These data indicate that the key immunoregulatory NKT-cell compartment is compromised in HIV-1-infected patients.     

Generation of cloned mice by direct nuclear transfer from natural killer T cells

Cloning mammals by nuclear transfer (NT) remains inefficient. One fundamental question is whether clones have really been derived from differentiated cells rather than from rare stem cells present in donor-cell samples. To date, cells, such as mature lymphocytes, with genetic differentiation markers have been cloned to generate mice only via a two-step NT involving embryonic stem (ES) cell generation and tetraploid complementation [1, 2 and 3]. Here, we show that the genome of a unique T-cell population, natural killer T (NKT) cells, can be fully reprogrammed by a single-step NT. The pups and their placentas possessed the rearranged TCR loci specific for NKT cells. The NKT-cell-cloned embryos had a high developmental potential in vitro: Most (71%) developed to the morula/blastocyst stage, in marked contrast to embryos from peripheral blood T cells (12%; p < 1 x 10(-25)). Furthermore, ES cell lines were efficiently established from these NKT-cell blastocysts. These findings clearly indicate a high level of plasticity in the NKT-cell genome. Thus, differentiation of the genome is not always a barrier to NT cloning for either reproductive or therapeutic purposes, so we can now postulate that at least some mammals cloned to date have indeed been derived from differentiated donor cells.     

Immunotherapy with ligands of natural killer T cells

Natural killer T (NKT) cells are innate lymphocytes that share receptor structures and functions with conventional T cells and natural killer cells. NKT cells are specific for glycolipid antigens bound by the major histocompatibility complex class I-like protein CD1d. One striking property of NKT cells is their capacity to rapidly produce large amounts of cytokines in response to T-cell receptor engagement, suggesting that activated NKT cells can modulate adaptive immune responses. Recent pre-clinical studies have revealed significant efficacy of NKT-cell ligands such as the glycolipid alpha-galactosylceramide for treatment of metastatic cancers and infections, and for prevention of autoimmune diseases. These findings suggest that appropriate stimulation of NKT cells could be exploited for prevention or treatment of human diseases.     

Mitogens match cell numbers to local demand.

Although the control of cell proliferation has been studied intensively at the level of the single cell, less is known about how cell numbers are controlled in developing populations and organs. Often, proliferation provides a pool of cells for organ construction, but the rate of this proliferation must be coordinated with patterning to avoid imbalances in cell numbers. Recent research on the development of the Drosophila eye and the proliferation signals (mitogens) can act to coordinate cell numbers.     

Self-renewal, differentiation or death: regulation and manipulation of hematopoietic stem cell fate.

Hematopoietic stem cells (HSCs) are the rare cells from which all hematopoietic cells are derived. The absence of HSCs is not compatible with life because many essential cells, such as myeloid and erythroid cells, are short lived. The hematopoietic system is the first essential organ system that fails following cytotoxic treatments. It is the vulnerability of HSCs that prevents regeneration following treatment and thus long-term survival. Because HSCs have the capacity to regenerate a functional hematopoietic system, the manipulation of these cells in vitro holds many promises for gene-therapeutic and other applications; however, these are severely curtailed by current difficulties in maintaining and expanding HSCs in culture. This review focuses on recent approaches towards understanding how the HSC compartment is regulated in vivo and discusses how this knowledge might be applied to manipulating HSC numbers.     

Selective loss of natural killer T cells by apoptosis following infection with lymphocytic choriomeningitis virus

Natural killer T (NKT) cells, a unique subpopulation of T cells, coexpress markers also present on NK cells and recognize the major histocompatibility complex class I-like CD1d1 molecule. We studied the effect of an acute virus infection on NKT cells. Mice were infected with the nonhepatotropic Armstrong strain of lymphocytic choriomeningitis virus (LCMV), and at various times postinfection, mononuclear cells from the liver, peritoneum, and spleen were isolated. It was found that within 2 to 3 days, there was a selective loss of NKT cells from the liver with an apparent rapid recovery within 8 to 14 days. There was no increase in peritoneal or splenic NKT cells, indicating that NKT cells did not traffic to these tissues. This loss of NKT cells was independent of gamma interferon (IFN-gamma) and interleukin 12 (IL-12) production, but did occur in mice treated with poly(I-C), a classical inducer of IFN-alpha/beta. The reduction in NKT cells was CD28 and fas/fasL independent and occurred via apoptosis. It was not observed in LCMV-infected DNA fragmentation factor 45-deficient mice, and an increase in active caspase 3-specific staining was found in liver NKT cells from LCMV-infected and poly(I-C)-treated mice compared to uninfected wild-type mice. Interestingly, it was also found that liver NKT cells from LCMV-infected mice were themselves infected. These results suggest that the loss of NKT cells following an acute LCMV infection could be due to the induction of IFN-alpha/beta resulting in NKT-cell apoptosis and is important for the host's immune response to LCMV.     

A STATISTICAL METHOD TO COMPARE THE DEGREE OF MUSCLE CELL MULTIPLICATION IN DIFFERENT CULTURE DISHES

A method was developed for comparing two groups of numbers of cultured muscle cells which were counted under a microscope. Practically important problems for this purpose were: how many fields per dish should be observed, and how many dishes should be prepared under the same conditions, when given test criteria were set.
In the present experiment, 4 dishes were prepared under the same conditions. From each of the dishes, 20 fields were selected, and the numbers of muscle cells *** were counted and separately recorded. Since the purpose was to compare two groups of dishes, the design was a simple case of a nested one. From the experiment, the type of distribution seemed approximately a long-normal distribution with constancy of variance (homoscedasticity). Since the distribution of the cells in dishes belonging to the same group could be considered to be the same, the numbers from each dish could be pooled within a group. Therefore, if the test criteria for Student's t test and the sensitivity to descriminate the ratio of the number of groups are given, the number of fields to be observed per dish times that of dishes can be uniquely determined. This method can be applied for the same purpose to other kinds of cells with log-normal distribution.     

Alpha-galactosylceramide-induced liver injury in mice is mediated by TNF-alpha but independent of Kupffer cells

NKT cells expressing phenotypic markers of both T and NK cells seem to be pivotal in murine models of immune-mediated liver injury, e.g., in Con A-induced hepatitis. Also alpha-galactosylceramide (alpha-GalCer), a specific ligand for invariant Valpha14 NKT cells, induces hepatic injury. To improve the comprehension of NKT-cell mediated liver injury, we investigated concomitants and prerequisites of alpha-GalCer-induced hepatitis in mice. Liver injury induced by alpha-GalCer injection into C57BL/6 mice was accompanied by intrahepatic caspase-3 activity but appeared independent thereof. alpha-GalCer injection also induces pronounced cytokine responses, including TNF-alpha, IFN-gamma, IL-2, IL-4, and IL-6. We provide a detailed time course for the expression of these cytokines, both in liver and plasma. Cytokine neutralization revealed that, unlike Con A-induced hepatitis, IFN-gamma is not only dispensable for alpha-GalCer-induced hepatotoxicity but even appears to exert protective effects. In contrast, TNF-alpha was clearly identified as an important mediator for hepatic injury in this model that increased Fas ligand expression on NKT cells. Whereas intrahepatic Kupffer cells are known as a pivotal source for TNF-alpha in Con A-induced hepatitis, they were nonessential for alpha-GalCer-mediated hepatotoxicity. In alpha-GalCer-treated mice, TNF-alpha was produced by intrahepatic lymphocytes, in particular NKT cells. BALB/c mice were significantly less susceptible to alpha-GalCer-induced liver injury than C57BL/6 mice, in particular upon pretreatment with d-galactosamine, a hepatocyte-specific sensitizer to TNF-alpha-mediated injury. Finally, we demonstrate resemblance of murine alpha-GalCer-induced hepatitis to human autoimmune-like liver disorders. The particular features of this model compared with other immune-mediated hepatitis models may enhance comprehension of basic mechanisms in the etiopathogenesis of NKT cell-comprising liver disorders.     

Regulation of apoptosis and replicative senescence in CD8+ T cells from patients with viral infections

Activated T lymphocytes are generated during an immune response. The induction of T lymphocyte proliferation is one way in which cell numbers can be controlled. However, once generated, the increased numbers of cells must be removed in order to re-establish cellular homoeostasis within the immune system. In this paper we describe how the numbers of activated T cells can be regulated by two distinct mechanisms, namely apoptosis and replicative senescence. In addition, we suggest that the regulation of cell clearance, as opposed to cell persistence, after an immune response is intimately involved in the generation of immune memory.     

Generation and Fates of Supernumerary Centrioles in Dividing Cells

The centrosome is a subcellular organelle from which a cilium assembles. Since centrosomes function as spindle poles during mitosis, they have to be present as a pair in a cell. How the correct number of centrosomes is maintained in a cell has been a major issue in the fields of cell cycle and cancer biology. Centrioles, the core of centrosomes, assemble and segregate in close connection to the cell cycle. Abnormalities in centriole numbers are attributed to decoupling from cell cycle regulation. Interestingly, supernumerary centrioles are commonly observed in cancer cells. In this review, we discuss how supernumerary centrioles are generated in diverse cellular conditions. We also discuss how the cells cope with supernumerary centrioles during the cell cycle.     

Monoclonal B-cell hyperplasia and leukocyte imbalance precede development of B-cell malignancies in uracil-DNA glycosylase deficient mice

Ung-deficient mice have reduced class switch recombination, skewed somatic hypermutation, lymphatic hyperplasia and a 22-fold increased risk of developing B-cell lymphomas. We find that lymphomas are of follicular (FL) and diffuse large B-cell type (DLBCL). All FLs and 75% of the DLBCLs were monoclonal while 25% were biclonal. Monoclonality was also observed in hyperplasia, and could represent an early stage of lymphoma development. Lymphoid hyperplasia occurs very early in otherwise healthy Ung-deficient mice, observed as a significant increase of splenic B-cells. Furthermore, loss of Ung also causes a significant reduction of T-helper cells, and 50% of the young Ung(-/-) mice investigated have no detectable NK/NKT-cell population in their spleen. The immunological imbalance is confirmed in experiments with spleen cells where the production of the cytokines interferon gamma, interleukin 6 and interleukin 2 is clearly different in wild type and in Ung-deficient mice. This suggests that Ung-proteins, directly or indirectly, have important functions in the immune system, not only in the process of antibody maturation, but also for production and functions of immunologically important cell types. The immunological imbalances shown here in the Ung-deficient mice may be central in the development of lymphomas in a background of generalised lymphoid hyperplasia.     

Evaluating quantitative methods for measuring plasmid copy numbers in single cells

The life of plasmids is a constant battle against fluctuations: failing to correct copy number fluctuations can increase the plasmid loss rate by many orders of magnitude, as can a failure to more evenly divide the copies between daughters at cell division. Plasmids are therefore long-standing model systems for stochastic processes in cells, much thanks to the efforts of Kurt Nordstr?m to whose memory this issue is dedicated. Here we analyze a range of experimental methods for measuring plasmid copy numbers in single cells, focusing on challenges, trade-offs, and necessary experimental controls. In particular we analyze published and unpublished strategies to infer copy numbers from expression of plasmid-encoded reporters, direct labeling of plasmids with fluorescent probes or DNA binding proteins fused to fluorescent reporters, PCR based methods applied to single cell lysates, and plasmid-specific replication arrest. We conclude that no method currently exists to measure plasmid copy numbers in single cells, and that most methods are overwhelmed by various types of experimental noise. We also discuss how accurate methods can be developed.     

Positioning of the NOR-bearing chromosomes in relation to nucleoli in daughter cells after mitosis

It is known that chromosomes occupy non-random positions in the cell nucleus. However, it is not clear to what extent their nuclear positions, together with their neighborhood, are conserved in daughter cells. To address specific aspects of this problem, we used the model of the chromosomes carrying ribosomal genes that are organized in clusters termed Nucleolus Organizer Regions (NORs). We compared the association of chosen NOR-bearing chromosomes (NOR-chromosomes) with nucleoli, as well as the numbers of nucleoli, in the pairs of daughter cells, and established how frequently the daughter cells had equal numbers of the homologs of certain NOR-chromosomes associated with individual nucleoli. The daughter cells typically had different numbers of nucleoli. At the same time, using immuno-FISH with probes for chromosomes 14 and 15 in HeLa cells, we found that the cell pairs with identical combinations appeared significantly more frequently than predicted by the random model. Thus, although the total number of chromosomes associated with nucleoli is variable, our data indicate that the position of the NOR-bearing chromosomes in relation to nucleoli is partly conserved through mitosis.     

Delta1 expression, cell cycle exit, and commitment to a specific secretory fate coincide within a few hours in the mouse intestinal stem cell system

The stem cells of the small intestine are multipotent: they give rise, via transit-amplifying cell divisions, to large numbers of columnar absorptive cells mixed with much smaller numbers of three different classes of secretory cells - mucus-secreting goblet cells, hormone-secreting enteroendocrine cells, and bactericide-secreting Paneth cells. Notch signaling is known to control commitment to a secretory fate, but why are the secretory cells such a small fraction of the population, and how does the diversity of secretory cell types arise? Using the mouse as our model organism, we find that secretory cells, and only secretory cells, pass through a phase of strong expression of the Notch ligand Delta1 (Dll1). Onset of this Dll1 expression coincides with a block to further cell division and is followed in much less than a cell cycle time by expression of Neurog3 – a marker of enteroendocrine fate – or Gfi1 – a marker of goblet or Paneth cell fate. By conditional knock-out of Dll1, we confirm that Delta-Notch signaling controls secretory commitment through lateral inhibition. We infer that cells stop dividing as they become committed to a secretory fate, while their neighbors continue dividing, explaining the final excess of absorptive over secretory cells. Our data rule out schemes in which cells first become committed to be secretory, and then diversify through subsequent cell divisions. A simple mathematical model shows how, instead, Notch signaling may simultaneously govern the commitment to be secretory and the choice between alternative modes of secretory differentiation.     

Aurora-A overexpression reveals tetraploidization as a major route to centrosome amplification in p53-/- cells

Aberrations in centrosome numbers have long been implicated in aneuploidy and tumorigenesis, but their origins are unknown. Here we have examined how overexpression of Aurora-A kinase causes centrosome amplification in cultured cells. We show that excess Aurora-A does not deregulate centrosome duplication but gives rise to extra centrosomes through defects in cell division and consequent tetraploidization. Over expression of other mitotic kinases (Polo-like kinase 1 and Aurora-B) also causes multinucleation and concomitant increases in centrosome numbers. Absence of a p53 checkpoint exacerbates this phenotype, providing a plausible explanation for the centrosome amplification typical of p53-/- cells. We propose that errors during cell division, combined with the inability to detect the resulting hyperploidy, constitute a major cause for numerical centrosome aberrations in tumors.     

The host environment regulates the function of CD8+ graft-versus-host-reactive effector cells

We have examined how the host environment influences the graft-vs-leukemia (GVL) response following transfer of donor T cells to allogeneic chimeras. Donor T cells induce significant GVL when administered in large numbers to established mixed chimeras (MC). However, when using limiting numbers of T cells, we found that late transfer to MC induced less GVL than did early transfer to freshly irradiated allogeneic recipients. Late donor T cell transfer to MC was associated with marked accumulation of anti-host CD8 cells within the spleen, but delayed kinetics of differentiation, reduced expression of effector molecules including IFN-gamma, impaired cytotoxicity, and higher rates of sustained apoptosis. Furthermore, in contrast to the spleen, we observed a significant delay in donor CD8 cell recruitment to the bone marrow, a key location for hematopoietic tumors. Increasing the numbers of T cells transferred to MC led to the enhancement of CTL activity and detectable increases in absolute numbers of IFN-gamma(+) cells without inducing graft-vs-host disease (GVHD). TLR-induced systemic inflammation accelerated differentiation of functional CTL in MC but was associated with severe GVHD. In the absence of inflammation, both recipient T and non-T cell populations impeded the full development of GVHD-inducing effector function. We conclude that per-cell deficits in the function of donor CD8 cells activated in MC may be overcome by transferring larger numbers of T cells without inducing GVHD.     

From cells to colonies: at what levels of body organization does the ‘temperature‐size rule’ apply?

SUMMARY An inverse relationship between temperature during ontogeny and final body size is widespread in ectotherms, but poorly understood. Evidence suggests that within organs, this “temperature‐size rule” (TSR) may also apply to cell size with no change in numbers. So how closely do reductions in size and number of cells and other repeated structures correlate with size reduction at higher levels of organization? We examine this in the context of a proposal that size and/or number changes at various organizational levels are adaptive responses to temperature‐ and size‐dependent oxygen supply. We subjected two clones of the modular colonial bryozoan, Celleporella hyalina, to orthogonal combinations of two temperatures and two oxygen concentrations during ontogeny, observing effects on sizes of colonies and larvae, and sizes and numbers of cells, tentacles, and modules (autozooids). We found that the size:number responses varied among cell types and among structures at different levels of organization, with the inverse temperature‐size relationship applying only to larval parenchymal cells and colony modules. Using our findings and other evidence we propose a unifying adaptive hypothesis that predicts how temperature affects the sizes of mitochondria, cells, organs, modules and organisms, and their relationships with processes that determine the functional capacity of aerobic metabolism.     

Developmental Changes in the in Vitro Activated Regenerative Activity of Primitive Mammary Epithelial Cells

Many normal adult tissues contain rare stem cells with extensive self-maintaining regenerative potential. During development, the stem cells of the hematopoietic and neural systems undergo intrinsically specified changes in their self-renewal potential. In the mouse, mammary stem cells with transplantable regenerative activity are first detectable a few days before birth. They share some phenotypic properties with their adult counterparts but are enriched in a subpopulation that displays a distinct gene expression profile. Here we show that fetal mammary epithelial cells have a greater direct and inducible growth potential than their adult counterparts. The latter feature is revealed in a novel culture system that enables large numbers of in vitro clonogenic progenitors as well as mammary stem cells with serially transplantable activity to be produced within 7 days from single fetal or adult input cells. We further show that these responses are highly dependent on novel factors produced by fibroblasts. These findings provide new avenues for elucidating mechanisms that regulate normal mammary epithelial stem cell properties at the single-cell level, how these change during development, and how their perturbation may contribute to transformation.     

Glycolipid antigen drives rapid expansion and sustained cytokine production by NK T cells

NKT cells are enigmatic lymphocytes that respond to glycolipid Ags presented by CD1d. Although they are key immunoregulatory cells, with a critical role in immunity to cancer, infection, and autoimmune diseases, little is known about how they respond to antigenic challenge. Current theories suggest that NKT cells die within hours of stimulation, implying that their direct impact on the immune system derives from the initial cytokine burst released before their death. Here we show that NKT cell disappearance results from TCR down-regulation rather than apoptosis, and that they expand to many times their normal number in peripheral tissues within 2-3 days of stimulation, before contracting to normal numbers over subsequent days. This expansion is associated with ongoing cytokine production, biased toward a Th1 (IFN-gamma(+) IL-4(-)) phenotype, in contrast to their initial Th0 (IFN-gamma(+)IL-4(+)) phenotype. This study provides critical new insight into how NKT cells can have such a major impact on immune responses, lasting many days beyond the initial stimulation of these cells.