Cytotoxic T lymphocytes (CTL) rapidly destroy their targets. Here we show that although target cell death occurs within 5 min of CTL-target cell contact, an immunological synapse similar to that seen in CD4 cells rapidly forms in CTL, with a ring of adhesion proteins surrounding an inner signaling molecule domain. Lytic granule secretion occurs in a separate domain within the adhesion ring, maintaining signaling protein organization during exocytosis. Live and fixed cell studies show target cell plasma membrane markers are transferred to the CTL as the cells separate. Electron microscopy reveals continuities forming membrane bridges between the CTL and target cell membranes, suggesting a possible mechanism for this transfer.

Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the α subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash). Cytotoxicity and secretion assays show that ash/ash CTLs are unable to kill target cells or to secrete granzyme A and hexosaminidase. By immunofluorescence and electron microscopy, we show polarization but no membrane docking of ash/ash lytic granules at the immunological synapse. In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity. Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A. However, only some of the granules polarize to the immunological synapse, and many remain dispersed around the periphery of the CTLs. These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.

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Neural stem cells (NSCs) are uncommitted cells of the CNS defined by their multipotentiality and ability to self renew. We found these cells to not be present in substantial numbers in the CNS until after embryonic day (E) 10.5 in mouse and E5 in chick. This coincides with the induction of SOX9 in neural cells. Gain- and loss-of-function studies indicated that SOX9 was essential for multipotent NSC formation. Moreover, Sonic Hedgehog was able to stimulate precocious generation of NSCs by inducing Sox9 expression. SOX9 was also necessary for the maintenance of multipotent NSCs, as shown by in vivo fate mapping experiments in the adult subependymal zone and olfactory bulbs. In addition, loss of SOX9 led ependymal cells to adopt a neuroblast identity. These data identify a functional link between extrinsic and intrinsic mechanisms of NSCs specification and maintenance, and establish a central role for SOX9 in the process.

Key Points• AML blasts have an arginase-dependent ability to inhibit T-cell proliferation and hematopoietic stem cells.• AML blasts have an arginase-dependent ability to modulate the polarization of monocytes.Acute myeloid leukemia (AML) is the most common acute leukemia in adults and the second most common frequent leukemia of childhood. Patients may present with lymphopenia or pancytopenia at diagnosis. We investigated the mechanisms by which AML causes pancytopenia and suppresses patients' immune response. This study identified for the first time that AML blasts alter the immune microenvironment through enhanced arginine metabolism. Arginase II is expressed and released from AML blasts and is present at high concentrations in the plasma of patients with AML, resulting in suppression of T-cell proliferation. We extended these results by demonstrating an arginase-dependent ability of AML blasts to polarize surrounding monocytes into a suppressive M2-like phenotype in vitro and in engrafted nonobese diabetic-severe combined immunodeficiency mice. In addition, AML blasts can suppress the proliferation and differentiation of murine granulocyte-monocyte progenitors and human CD34 1 progenitors. Finally, the study showed that the immunosuppressive activity of AML blasts can be modulated through smallmolecule inhibitors of arginase and inducible nitric oxide synthase, suggesting a novel therapeutic target in AML. The results strongly support the hypothesis that AML creates an immunosuppressive microenvironment that contributes to the pancytopenia observed at diagnosis. (Blood. 2013;122(5):749-758)