Differential contribution of chemotaxis and substrate restriction to segregation of immature and mature thymocytes.

T cell development requires sequential localization of thymocyte subsets to distinct thymic microenvironments. To address mechanisms governing this segregation, we used two-photon microscopy to visualize migration of purified thymocyte subsets in defined microenvironments within thymic slices. Double-negative (CD4(-)8(-)) and double-positive (CD4(+)8(+)) thymocytes were confined to cortex where they moved slowly without directional bias. DP cells accumulated and migrated more rapidly in a specialized inner-cortical microenvironment, but were unable to migrate on medullary substrates. In contrast, CD4 single positive (SP) thymocytes migrated directionally toward the medulla, where they accumulated and moved very rapidly. Our results revealed a requisite two-step process governing CD4 SP cell medullary localization: the chemokine receptor CCR7 mediated chemotaxis of CD4 SP cells towards medulla, whereas a distinct pertussis-toxin sensitive pathway was required for medullary entry. These findings suggest that developmentally regulated responses to both chemotactic signals and specific migratory substrates guide thymocytes to specific locations in the thymus.

Low ligand requirement for deletion and lack of synapses in positive selection enforce the gauntlet of thymic T cell maturation.

Immature double-positive (CD4(+)CD8(+)) thymocytes respond to negatively selecting peptide-MHC ligands by forming an immune synapse that sustains contact with the antigen-presenting cell (APC). Using fluorescently labeled peptides, we showed that as few as two agonist ligands could promote APC contact and subsequent apoptosis in reactive thymocytes. Furthermore, we showed that productive signaling for positive selection, as gauged by nuclear translocation of a green fluorescent protein (GFP)-labeled NFATc construct, did not involve formation of a synapse between thymocytes and selecting epithelial cells in reaggregate thymus cultures. Antibody blockade of endogenous positively selecting ligands prevented NFAT nuclear accumulation in such cultures and reversed NFAT accumulation in previously stimulated thymocytes. Together, these data suggest a "gauntlet" model in which thymocytes mature by continually acquiring and reacquiring positively selecting signals without sustained contact with epithelial cells, thereby allowing them to sample many cell surfaces for potentially negatively selecting ligands.

Space-time considerations for hematopoietic stem cell transplantation.

The mammalian blood system contains a multitude of distinct mature cell lineages adapted to serving diverse functional roles. Mutations that abrogate the development or function of one or more of these lineages can lead to profound adverse consequences, such as immunodeficiency, autoimmunity, or anemia. Replacement of hematopoietic stem cells (HSC) that carry such mutations with HSC from a healthy donor can reverse such disorders, but because the risks associated with the procedure are often more serious than the blood disorders themselves, bone marrow transplantation is generally not used to treat a number of relatively common inherited blood diseases. Aside from a number of other problems, risks associated with cytoreductive treatments that create "space" for donor HSC, and the slow kinetics with which immune competence is restored following transplantation hamper progress. This review will focus on how recent studies using experimental model systems may direct future efforts to implement routine use of HSC transplantation to cure inherited blood disorders.

Engagement of NKG2D by cognate ligand or antibody alone is insufficient to mediate costimulation of human and mouse CD8+ T cells.

CD8+ T cells require a signal through a costimulatory receptor in addition to TCR engagement to become activated. The role of CD28 in costimulating T cell activation is well established. NKG2D, a receptor found on NK cells, CD8+ alphabeta-TCR+ T cells, and gammadelta-TCR+ T cells, has also been implicated in T cell costimulation. In this study we have evaluated the role of NKG2D in costimulating mouse and human naive and effector CD8+ T cells. Unexpectedly, in contrast to CD28, NKG2D engagement by ligand or mAb is not sufficient to costimulate naive or effector CD8+ T cell responses in conventional T cell populations. While NKG2D did not costimulate CD8+ T cells on its own, it was able to modify CD28-mediated costimulation of human CD8+ T cells under certain contitions. It is, therefore, likely that NKG2D acts as a costimulatory molecule only under restricted conditions or requires additional cofactors.

Dynamics of p56lck translocation to the T cell immunological synapse following agonist and antagonist stimulation.

To study the spatio/temporal recruitment of lck during immunological synapse formation, we utilize high-speed time-lapse microscopy to visualize green fluorescent protein (GFP) fusions of lck and CD3zeta following agonist or altered peptide ligand (APL) stimulation. The dynamics of lck and CD3zeta recruitment are comparable; however, lck becomes excluded to the periphery of mature synapses, while most CD3zeta is centrally localized, suggesting a limited time frame within which lck can efficiently phosphorylate CD3 molecules during synapse maturation. Exposure of T cells to specific APLs affects the efficiency of conjugate formation and lck accumulation. Most surprisingly, we find an intracellular pool of lck associated with recycling endosomes that translocates to mature synapses within 10 min of calcium flux. This bolus of lck may contribute to intermediate-late signal transduction.

Linker for activation of T cells, zeta-associated protein-70, and Src homology 2 domain-containing leukocyte protein-76 are required for TCR-induced microtubule-organizing center polarization.

Engagement of the T cell with Ag on an APC results in a series of immediate signaling events emanating from the stimulation of the TCR. These events include the induced phosphorylation of a number of cellular proteins with a subsequent increase in intracellular calcium and the restructuring of the microtubule and actin cytoskeleton within the T cell. This restructuring of the cytoskeleton culminates in the polarization of the T cell's secretory apparatus toward the engaging APC, allowing the T cell to direct secretion of cytokines toward the appropriate APC. This polarization can be monitored by analyzing the position of the microtubule-organizing center (MTOC), as it moves toward the interface of the T cell and APC. The requirements for MTOC polarization were examined at a single-cell level by studying the interaction of a Jurkat cell line expressing a fluorescently labeled MTOC with Staphylococcal enterotoxin superantigen-bound Raji B cell line, which served as the APC. We found that repolarization of the MTOC substantially followed fluxes in calcium. We also used immobilized anti-TCR mAb and Jurkat signaling mutants, defective in TCR-induced calcium increases, to determine whether signaling components that are necessary for a calcium response also play a role in MTOC polarization. We found that zeta-associated protein-70 as well as its substrate adaptor proteins linker for activation of T cells and Src homology 2 domain-containing leukocyte protein-76 are required for MTOC polarization. Moreover, our studies revealed that a calcium-dependent event not requiring calcineurin or calcium/calmodulin-dependent kinase is required for TCR-induced polarization of the MTOC.