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.     

Regulating the immune system via IL-15 transpresentation

Transpresentation has emerged as an important mechanism mediating IL-15 responses in a subset of lymphocytes during the steady state. In transpresentation, cell surface IL-15, bound to IL-15Rα is delivered to opposing lymphocytes during a cell-cell interaction. The events most dependent on IL-15 include the development and homeostasis of memory CD8 T cells, Natural Killer cells, invariant Natural Killer T cells, and intraepithelial lymphocytes. As lymphocyte development and homeostasis involve multiple steps and mechanisms, IL-15 transpresentation can have diverse roles throughout. Moreover, distinct stages of lymphocyte differentiation require IL-15 transpresented by different cells, which include both hematopoietic and non-hematopoietic cell types. Herein, we will describe the points where IL-15 transpresentation impacts these processes, the specific cells thought to drive IL-15 responses, as well as their role in the course of development and homeostasis.     

Selective activation of immunoregulatory T-cell circuits by an Ia+ antigen-presenting cell line

ORA I-a, a cloned Ia+ monocyte tumor line, interacts with distinct immunoregulatory T-cell subsets. ORA cells present soluble and alloantigen to primed lymph node T cells and alloantigen to antigen-activated T-cell clones. However, they induce dose-dependent suppression during primary mixed lymphocyte cultures. Activation of a mixed lymphocyte response (MLR) suppressor pathway is mediated by Ly 1+ T cells. This T-cell subset proliferates in response to ORA when Ly 2+ cells are depleted. Furthermore, once activated, Ly 1+ T cells induce effectors of suppression within fresh T-cell populations. These studies indicate that antigen presentation to distinct T-cell subsets during different stages of an immune response may be mediated by unique antigen-presenting cell subpopulations. Immune homeostasis may thus be controlled not only by regulatory T cells, but also by unique antigen-presenting cells which are responsible for their selective activation.     

A deficiency in nucleoside salvage impairs murine lymphocyte development, homeostasis, and survival

The homeostasis of the immune system is tightly controlled by both cell-extrinsic and -intrinsic mechanisms. These regulators, not all known to date, drive cells in and out of quiescence when and where required to allow the immune system to function. In this article, we describe a deficiency in deoxycytidine kinase (DCK), one of the major enzymes of the nucleoside salvage pathway, which affects peripheral T cell homeostatic proliferation and survival. As a result of an N-ethyl-N-nitrosourea-induced mutation in the last α helix of DCK, a functionally null protein has been generated in the mouse and affects the composition of the hematopoietic system. Both B and T lymphocyte development is impaired, leading to a state of chronic lymphopenia and to a significant increase in the number of myeloid cells and erythrocytes. In the periphery, we found that mutant lymphocytes adopt a CD44(high)CD62L(low) memory phenotype, with high levels of proliferation and apoptosis. These phenotypes are notably the result of a cell-extrinsic-driven lymphopenia-induced proliferation as wild-type cells transferred into DCK-deficient recipients adopt the same profile. In addition, DCK also regulates lymphocyte quiescence in a cell-intrinsic manner. These data establish dCK as a new regulator of hematopoietic integrity and lymphocyte quiescence and survival.     

Lymphocyte reduction induced by hindlimb unloading： distinct mechanisms in the spleen and thymus

Hindlimb unloading (HU) in rodent is a well-accepted ground-based model used to simulate some of the condi-tions of space flight and reproduce its deleterious effects on the musculoskeletal, cardiovascular and immune systems. In this study, the effects of HU on lymphocyte homeostasis in the spleen and thymus of mice were examined. HU was found to drastically deplete various cell populations in the spleen and thymus. These changes are likely to be mediated by apoptosis, since DNA strand breaks indicative of apoptosis were detected by terminal deoxynucleotidyl transferase-mediated nick end-labeling in both splenocytes and thymocytes. Surprisingly, administration of opioid antagonists or interference with the Fas-FasL interaction was able to block HU-induced reductions of splenocytes, but not thymocytes. On the other hand, steroid receptor antagonists blocked the reduction of lymphocyte numbers in both spleen and thymus. Therefore, the effects of HU on the homeostasis of splenocytes and thymocytes must be exerted through distinct mechanisms.     

Hematopoietic Stem Cell Quiescence Attenuates DNA Damage Response and Permits DNA Damage Accumulation During Aging

The aging of tissue-specific stem and progenitor cells is believed to be central to the pathophysiological conditions arising in aged individuals. While the mechanisms driving stem cell aging are poorly understood, mounting evidence points to age-dependent DNA damage accrual as an important contributing factor. While it has been postulated that DNA damage may deplete stem cell numbers with age, recent studies indicate that murine hematopoietic stem cell (HSC) reserves are in fact maintained despite the accrual of genomic damage with age. Evidence suggests this to be a result of the quiescent (G0) cell cycle status of HSC, which results in an attenuation of checkpoint control and DNA damage responses for repair or apoptosis. When aged stem cells that have acquired damage are called into cycle under conditions of stress or tissue regeneration however, their functional capacity was shown to be severely impaired. These data suggest that age-dependent DNA damage accumulation may underlie the diminished capacity of aged stem cells to mediate a return to homeostasis after acute stress or injury. Moreover, the cytoprotection afforded by stem cell quiescence in stress-free, steady-state conditions suggests a mechanism through which potentially dangerous lesions can accumulate in the stem cell pool with age.     

Cell population dynamics (apoptosis, mitosis, and cell-cell communication) during disruption of homeostasis

The sequence of events involved in maintenance of homeostasis must encompass mechanisms within single cells as well as interactions between cells within a population. To investigate the interaction among these inter- and intracellular mechanisms, disruption of homeostasis by serum deprivation was performed in WB-F344, a normal diploid epithelial cell line. Changes in cell-cell communication (gap junction function) at the population level and in individual cells were monitored using the scrape load/dye transfer and fluorescence redistribution after photobleaching assays. Apoptosis and mitosis were measured using internucleosomal DNA ladder assays and fluorescence-activated cell sorting. The results indicate that a common element in early apoptosis and early mitosis is sustained gap junction function. As cell life (mitosis) and cell death (apoptosis) progressed, a common process of change in gap junction function occurred. A transient stimulation of mitosis concomitant with increased apoptosis was also observed during serum deprivation. Gap junctions may play a regulatory role during initiation of these opposite yet equally important mechanisms of maintaining homeostasis. This model system is useful for further studies on the relationships among inter- and intracellular mechanisms of homeostasis.     

Homeostatic control of recombination is implemented progressively in mouse meiosis

Humans suffer from high rates of fetal aneuploidy, often arising from the absence of meiotic crossover recombination between homologous chromosomes. Meiotic recombination is initiated by double-strand breaks (DSBs) generated by the SPO11 transesterase. In yeast and worms, at least one buffering mechanism, crossover homeostasis, maintains crossover numbers despite variation in DSB numbers. We show here that mammals exhibit progressive homeostatic control of recombination. In wild-type mouse spermatocytes, focus numbers for early recombination proteins (RAD51, DMC1) were highly variable from cell to cell, whereas foci of the crossover marker MLH1 showed little variability. Furthermore, mice with greater or fewer copies of the Spo11 gene--with correspondingly greater or fewer numbers of early recombination foci--exhibited relatively invariant crossover numbers. Homeostatic control is enforced during at least two stages, after the formation of early recombination intermediates and later while these intermediates mature towards crossovers. Thus, variability within the mammalian meiotic program is robustly managed by homeostatic mechanisms to control crossover formation, probably to suppress aneuploidy. Meiotic recombination exemplifies how order can be progressively implemented in a self-organizing system despite natural cell-to-cell disparities in the underlying biochemical processes.     

Indexation as a novel mechanism of lymphocyte homeostasis: the number of CD4+CD25+ regulatory T cells is indexed to the number of IL-2-producing cells

To fulfill its mission, the immune system must maintain a complete set of different cellular subpopulations that play specific roles in immune responses. We have investigated the mechanisms regulating CD4+CD25+ regulatory T (Treg) cell homeostasis. We show that the expression of the high-affinity IL-2Ralpha endows these cells with the capacity to explore the IL-2 resource, ensuring their presence while keeping their number tied to the number of CD4+ T cells that produce IL-2. We show that such a homeostatic mechanism allows the increased expansion of T cells without causing disease. The indexing of Treg cells to the number of activated IL-2-producing cells may constitute a feedback mechanism that controls T cell expansion during immune responses, thus preventing autoimmune or lymphoproliferative diseases. The present study highlights that maintenance of proportions between different lymphocyte subsets may also be critical for the immune system and are under strict homeostatic control.     

Antigen-specific lymphocyte sequestration in lymphoid organs: lack of essential roles for alphaL and alpha4 integrin-dependent adhesion or Galphai protein-coupled receptor signaling

Selective lymphocyte sequestration was described over 30 years ago as the transient withdrawal of Ag-specific lymphocytes from the circulation as a result of their activation in secondary lymphoid organs. We used a TCR-transgenic adoptive transfer system to further characterize the Ag and adjuvant dependence of this process in mice. In addition, we examined the contribution of the alpha(L) and alpha(4) integrin chains as well as Galpha(i) protein-coupled receptor signaling to the retention of Ag-specific T cells in peripheral lymph nodes. Our results demonstrate that selective lymphocyte sequestration is T cell autonomous and adjuvant independent, and that the duration of sequestration is not controlled by the continued presence of Ag in secondary lymphoid organs. This process is not critically dependent on the alpha(L) and alpha(4) integrin chains or Galpha(i) protein-coupled receptor signaling. Selective lymphocyte sequestration may be mediated by redundant mechanisms and/or controlled by novel or nonclassical adhesion or trafficking molecules.     

Accessory cells reduce lipoprotein suppression of lymphocyte activation

Plasma lipoproteins of d less than or equal to 1.063 g/ml suppress lymphocyte activation triggered in vitro by polyclonal T cell mitogens. The extent of suppression decreases as the number of accessory cells per culture increases. Accessory cells isolated by glass adherence and by counter-flow centrifugation reduce lipoprotein suppression to the same extent. Modulation of lipoprotein suppression by accessory cells is independent of the amount and type of polyclonal activator. Reduction of lipoprotein suppression requires viable accessory cells and that they be present with lymphocytes, mitogen and lipoproteins during the initial 24-h culture period. It is within this same time period that lipoproteins exert their suppressive effect. Accessory cells isolated from a patient with the homozygous form (receptor-defective) of familial hypercholesterolemia also reduce the extent of lipoprotein suppression, suggesting that modulation is not mediated by the classic low density lipoprotein receptor. There appear to be at least two mechanisms by which accessory cells may alter lipoprotein suppression of T lymphocyte activation: by secretion of a soluble factor, probably not interleukin 1, that decreases the extent of suppression and by direct modification of the population of suppressive lipoproteins. Neither mechanism accounts for the lipoprotein-enhanced activation that occurs when cultures contain approximately equal numbers of T lymphocytes and accessory cells.     

Characterization of cultured epithelial cells using a novel technique not requiring enzymatic digestion for subculturing

Our laboratory had developed a methodology to expand epithelial cells in culture by growing keratinocyte monolayers, under large volumes of medium that produces large numbers of keratinocytes that leave the monolayer and move into suspension. The cells have been defined as epithelial Pop Up Keratinocytes or ePUKs cells and appear to be highly suitable for clinical applications. In this publication we extend the characterization of the cells with a detailed analysis of the capabilities of the monolayer of a single culture flask to produce, over time, ePUK cells. The cells were characterized using standard epithelial markers for proliferation and differentiation. Analysis of morphology of the monolayer formed and total number of cells produced is presented for a variety of human epithelial cell strains. These keratinocytes provide an additional controlled human cell system for investigation of the mechanisms regulating epithelia cell growth and differentiation and since they are produced in large numbers, they are highly suitable for use in epithelial cell banking.     

Microbe-induced T cell apoptosis: subversion of the host defense system?

Cells of multicellular organisms are equipped with a self destruction program called apoptosis to ensure homeostasis of the organism. Contraction of the lymphocyte compartment following recovery from an infection is controlled by this mechanism. But apoptosis of lymphocytes might be an Achilles tendon accessible to microbes to subvert the immune system. Evidence is cumulating that microbes use this mechanism to destroy microbe-specific T cells. We present an overview of microbe-induced T cell apoptosis discussing the consequences for the pathogenesis of microbial infection. The conventional role of lymphocytes during infection is to impose apoptotic threat to infected cells, the subject of this review highlights the opposite, lymphocytes as targets of microbe-induced death.     

Why does inflammation persist: a dominant role for the stromal microenvironment?

Inflammatory responses occur within tissue microenvironments, with functional contributions from both haematopoietic (lymphocytic) cells and stromal cells (including macrophages and fibroblasts). These environments are complex--a compound of many different cell types at different stages of activation and differentiation. Traditional models of inflammatory disease highlight the role of antigen-specific lymphocyte responses and attempt to identify causative agents. However, recent studies have indicated the importance of tissue microenvironments and the innate immune response in perpetuating the inflammatory process. The prominent role of stromal cells in the generation and maintenance of these environments has begun to challenge the primacy of the lymphocyte in regulating chronic inflammatory processes. Sensible enquiries into factors regulating the persistence of inflammatory disease necessitate an understanding of the mechanisms regulating tissue homeostasis and remodelling during inflammation. This article highlights recent insights into the factors regulating dynamic aspects of inflammation, focusing particularly on mononuclear cell infiltrates, their interactions with stromal cells in tissues and the relevance of these interactions to existing and possible future therapies. A key feature of current research has been a growing appreciation that disordered spatial and temporal interactions between infiltrating immune cells and resident stromal cells lie at the heart of disease persistence.     

Genes, pathways and checkpoints in lymphocyte development and homeostasis

A plethora of genes involved in murine B and T cell development have been identified, and developmental pathways within the primary lymphoid tissues have been well delineated. The generation of a functional, but non-self reacting lymphocyte repertoire results from the completion of several checkpoints during lymphocyte development and competition for survival factors in the periphery. Improved knowledge of these developmental checkpoints and homeostatic mechanisms is critical for understanding human immunodeficiency, leukaemia/lymphoma and autoimmunity, which are conditions where checkpoints and homeostasis are likely to be deregulated.     

Spatial dynamics of feedback and feedforward regulation in cell lineages

Feedback mechanisms within cell lineages are thought to be important for maintaining tissue homeostasis. Mathematical models that assume well-mixed cell populations, together with experimental data, have suggested that negative feedback from differentiated cells on the stem cell self-renewal probability can maintain a stable equilibrium and hence homeostasis. Cell lineage dynamics, however, are characterized by spatial structure, which can lead to different properties. Here, we investigate these dynamics using spatially explicit computational models, including cell division, differentiation, death, and migration / diffusion processes. According to these models, the negative feedback loop on stem cell self-renewal fails to maintain homeostasis, both under the assumption of strong spatial restrictions and fast migration / diffusion. Although homeostasis cannot be maintained, this feedback can regulate cell density and promote the formation of spatial structures in the model. Tissue homeostasis, however, can be achieved if spatially restricted negative feedback on self-renewal is combined with an experimentally documented spatial feedforward loop, in which stem cells regulate the fate of transit amplifying cells. This indicates that the dynamics of feedback regulation in tissue cell lineages are more complex than previously thought, and that combinations of spatially explicit control mechanisms are likely instrumental.     

Dynamic control of lymphocyte trafficking by fever-range thermal stress

Migration of blood-borne lymphocytes into tissues involves a tightly orchestrated sequence of adhesion events. Adhesion molecules and chemokine receptors on the surface of circulating lymphocytes initiate contact with specialized endothelial cells under hemodynamic shear prior to extravasation across the vascular barrier into tissues. Lymphocyte–endothelial adhesion occurs preferentially in high endothelial venules (HEV) of peripheral lymphoid organs. The continuous recirculation of naïve and central memory lymphocytes across lymph node and Peyer’s patch HEV underlies immune surveillance and immune homeostasis. Lymphocyte–endothelial interactions are markedly enhanced in HEV-like vessels of extralymphoid organs during physiological responses associated with acute and chronic inflammation. Similar adhesive mechanisms must be invoked for efficient trafficking of immune effector cells to tumor sites in order for the immune system to have an impact on tumor progression. Here we discuss recent evidence for the role of fever-range thermal stress in promoting lymphocyte–endothelial adhesion and trafficking across HEV in peripheral lymphoid organs. Findings are also presented that support the hypothesis that lymphocyte–endothelial interactions are limited within tumor microenvironments. Further understanding of the molecular mechanisms that dynamically promote lymphocyte trafficking in HEV may provide the basis for novel approaches to improve recruitment of immune effector cells to tumor sites.     

Recent advances in understanding the relationship between adenosine metabolism and the function of T and B lymphocytes in diabetes

Adenosine plays an important role in physiology of several organs. Its turnover inside and outside of the cell is controlled by several enzymes and transport processes. The action of extracellular adenosine is mediated via at least four receptors named A(1), A(2A), A(2B), and A(3). Recent studies have reported that adenosine is a significant mediator of regulatory lymphocyte function. Numerous data indicates that adenosine affects T lymphocyte activation, proliferation and lymphocyte-mediated cytolysis. Impaired lymphocyte functioning and enhanced susceptibility to infections is a common feature of human diabetes. This review collects data bringing us closer to understanding the disturbances in lymphocytes adenosine homeostasis in diabetes. Adenosine receptors and nucleoside transporters are targets for potential drugs in many pathophysiological situations. Therefore, action of adenosine on lymphocyte function in diabetes may be important target for modulation of immune responses and understanding of mechanisms leading to several pathologies of immune cells observed in diabetes.