A Series of Unfortunate Events

CodingELAScience Group Size 2 3 students Target Grades 1 2 Time Required 2 hours Objectives • Learn the process by which living creatures sense, process, and react to information • Connect the sensors on robots to human senses. • Connect computer programs to the way the brain handles information, and learn that those programs are called “event handlers." • Write a short story involving a descriptive flow of events. • Write a blockly program involving events and event handling.

thrombin generation and lengthening of the time to clot. 5 Two important reports in the current issue advance still further our understanding of these important antibodies. There has been controversy over the domain of the ␤ 2 GPI molecule involved in binding of pathogenic antibodies. de Laat and colleagues demonstrate conclusively that it is antibodies directed against an epitope containing Gly40 and Arg43 in domain 1 that have LAC activity and associate strongly with thrombosis, unlike heterogeneous antibodies that bind elsewhere on the molecule. But how are these autoantibodies generated? The report by Kuwana and colleagues offers novel insights into this process. A popular concept to explain why autoreactive lymphocytes are so commonly detected in the absence of disease is that they recognize "cryptic" epitopes. When antigen is taken up by antigen-presenting cells, it is degraded by a series of specific proteases, regulators, and specialized chaperonins 6 so that only a subset of possible epitopes are displayed to interrogating lymphocytes. Cryptic epitopes bind with high affinity to major histocompatibility complex (MHC) class II but are not usually part of the repertoire displayed on the surface of antigen-presenting cells. Kuwana et al show that only ␤ 2 GPI bound to phospholipids can be presented successfully to autoreactive clones. Antiphospholipid antibodies are commonly present in chronic inflammatory disease, such as SLE, but also after acute illness including some infections. In both situations there may be increased circulating phospholipids derived from apoptotic cells 7 or pathogens. The phospholipids are then postulated to "reveal" ␤ 2 GPI to pre-existing autoreactive T-cell clones, allowing autoantibody formation. The process of epitope spreading must then be invoked in the extension of autoantibodies from the domain V epitopes highlighted in the study of Kuwana et al to those directed against domain 1 and thereby thrombosis, as highlighted by de Laat et al. Lupus "anticoagulant" and thrombosis are not so paradoxical after all. ■ 6. Watts C. The exogenous pathway for antigen presentation on major histocompatibility complex class II and CD1 molecules. Nat Immunol. 2004;5:685-692. 7. Bengtsson AA, Sturfelt G, Gullstrand B, Truedsson L. Induction of apoptosis in monocytes and lymphocytes by serum from patients with systemic lupus erythematosus: an additional mechanism to increased autoantigen load? Clin Exp Immunol. 2004;135:535-543.

Michael B. Jordan CINCINNATI CHILDREN'S HOSPITAL
Cytokine levels are broadly deranged in patients with hemophagocytic syndromes. Untangling the "Who, What, Where, When, and Why" of these abnormalities will be essential to understanding this fatal disorder.
T he profound elevation of multiple serum cytokine levels, including interferon-␥ (IFN-␥), tumor necrosis factor ␣ (TNF-␣), interleukin 6 (IL-6), and IL-10, has long intrigued investigators studying patients with hemophagocytic syndromes. While it is clear that T cells and macrophages are involved in this disease process, the sequence of events and the source(s) of the these cytokines remain unclear. While investigators have previously found that circulating CD8 ϩ T cells in these patients are secreting IFN-␥ and that monocytes from these patients can secrete IL-6 and TNF-␣ in vitro, 1 these observations have not been repeated in a diverse patient population. In this issue of Blood, Billiau and colleagues have added to our understanding of hemophagocytic syndromes by staining tissue samples for the presence of each of these cytokines. They examined a series of liver biopsy specimens from a diverse group of patients with what is variably termed hemophagocytic lymphohistiocytosis, hemophagocytic syndrome, or macrophage activation syndrome. Despite the diversity of this group, they consistently found that CD8 ϩ T cells prominently contained A theorized sequence of events in the development of hemophagocytic syndromes. In patients with hemophagocytic syndromes, antigen presentation (1) stimulates CD8 ؉ T cells to secrete IFN-␥ (2), which activates macrophages to produce multiple, toxic inflammatory mediators (3) and causes hemophagocytosis (4).
IFN-␥ and that macrophages contained IL-6 and TNF-␣ in situ. These findings are notable for 2 reasons. First, they confirm the idea that despite a variety of clinical and etiologic features, patients with hemophagocytic syndromes have similar disease pathophysiology. Second, this study directly demonstrates which cell type is producing each cytokine.
While the observations of Billiau et al do not establish causality, they are consistent with the findings of a murine model of hemophagocytic lymphohistiocytosis, which clearly demonstrated that IFN-␥ was essential for disease development. 2 The findings of this model and the current paper by Billiau and colleagues are consistent with the theory that hemophagocytic syndromes develop when CD8 ϩ T cells secrete excessive amounts of IFN-␥ and thereby drive macrophages to toxic levels of activation. How the T-cell response spirals out of control and precisely how IFN-␥ is linked to the clinical phenotype of hemophagocytic syndromes are not yet known. Future investigators will need to establish a clear, causal sequence of events that leads to the unfortunate and fatal immune activation seen in the hemophagocytic syndromes.
T wo articles in this issue address how Notch ligands induce T-cell development. Mammalian Notch ligands comprise 2 families, serrate-like (Jagged1, Jagged2) and Delta-like (Dll1, 3, and 4). Both families encode transmembrane proteins whose extracellular domain contains epidermal growth factor (EGF)-like repeats and an N-terminal DSL domain (for Delta, Serrate, Lag2) that binds Notch receptors and short, poorly conserved intracellular domains that have important but poorly understood functions. Notch receptor binding by ligand initiates a series of proteolytic cleavages in the receptor, causing release of the intracellular domain of Notch from the plasma membrane and translocation into the nucleus, where it creates a short-lived complex that activates transcription.
Specificity of Notch ligand-receptor interactions is poorly understood. In some assays, such as inhibition of myocyte development, Delta and Jagged function equivalently. In vivo, Delta and Jagged often show overlapping expression, providing limited insight into specificity. Knockout studies demonstrate that Notch ligands are not equivalent, at least for marginal zone B-cell development. These results show that Dll1 is the important partner for Notch2 in marginal zone B-cell development, as conditional deletion of either leads to loss of these B cells. 1 The precise Notch ligands in T-cell development are less certain. Notch1 signals are uniquely required among the 4 receptors for both T-cell commitment from a multipotent progenitor and proper development to the double-positive (DP) stage. 2 Conditional deletion of either Jagged1 or Dll1 leaves T-cell development unaffected, suggesting that neither uniquely signals T-cell development and that conditional deletion of multiple family members will be necessary. 1,3 A major breakthrough in identifying Notch ligands relevant to T-cell lymphopoiesis was provided by establishing stromal cell cultures that recapitulate many aspects of T-cell development. 4 Using either OP9 or S17 stromal cells engineered to express Dll1, Schmitt and Zuniga-Pflucker 5 and Jaleco et al 6 succeeded in establishing a cell culture-based assay that recapitulates T-cell development, a process previously thought to require intact thymic organ cultures. In the absence of Delta-induced Notch signals, these cultures generate B cells. OP9-Dl1 cells are particularly useful as they efficiently generate DP and even mature single-positive CD8 cells from murine hematopoietic progenitors derived from embryonic stem cells, fetal liver, and adult bone marrow. In addition, these cultures have provided important mechanistic insights into murine T-cell development. Jaleco et al 6 also showed that CD34 ϩ human cord blood cells were capable, albeit inefficiently, of generating DP T cells on S17-Dl1 cells. In this issue, La Motte-Mohs and colleagues extend the previous studies by showing that culture of human cord blood on OP9-Dl1 cells leads to efficient T-cell commitment, expansion, and generation of T-cell receptor ␣␤ (TCR␣␤)-expressing cells. Whether the latter are functionally mature remains to be determined. Similar results were obtained using human adult bone marrow progenitors. 7 Thus, it should now be possible to obtain a detailed understanding of early human T-cell development and use this information to manipulate and expand T-cell progenitors for therapeutic purposes.
The ability of OP9-Dl1 cells to induce T-cell development raises the question of whether Jagged ligands have the equivalent ability. In this issue, Lehar and colleagues engineered OP9 cells to express increased levels of Jagged1 (OP9-Jag1). As Jagged1, unlike Dll1, is expressed on parental OP9 cells, this appears to be a quantitative rather than qualitative change. In contrast to Dll1, OP9-Jag1 cells failed to induce T-cell development and induced only weak expansion of committed T-cell progenitors. Nevertheless, Jagged had some effects, as thymic DN1 cells, a heterogeneous population, were unable to form B cells when cultured on OP9-Jag1, as opposed to OP9 control cells. This study and previous work from Jaleco et al 6 demonstrate that Jagged and Delta signals are not equivalent in the context of T-cell development. Whether these differences are quantitative or qualitative is unknown. For example, Fringe-induced Notch modification could lead to Jagged insensitivity. Alternatively, ligand density may play a role, a parameter that has not yet been assessed.
Although much remains to be learned about the mechanism of Notch-induced T-cell development, the ability to efficiently generate human T cells in culture holds great therapeutic promise, such as in improving T-cell generation after myeloablative therapy. The culture systems described to date are an excellent start and understanding the functions of the ligands in producing specific signals will be important. This hope must be tempered with caution, as it is not yet known whether the T cells derived from For personal use only. on August 23, 2017. by guest www.bloodjournal.org From