Quantification of T-cell progenitors during ontogeny: thymus colonization depends on blood delivery of progenitors.
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An in vivo thymus reconstitution assay based on intrathymic injection of hematopoietic progenitors into irradiated chicks was used to determine the number of T-cell progenitors in peripheral blood, paraaortic foci, bone marrow (BM), and spleen during ontogeny. This study allowed us to analyze the regulation of thymus colonization occurring in three waves during embryogenesis. It confirmed that progenitors of the first wave of thymus colonization originate from the paraaortic foci, whereas progenitors of the second and the third waves originate from the BM. The analysis of the number of T-cell progenitors indicates that each wave of thymus colonization is correlated with a peak number of T-cell progenitors in peripheral blood, whereas they are almost absent during the periods defined as refractory for colonization. Moreover, injection of T-cell progenitors into the blood circulation showed that they homed into the thymus without delay during the refractory periods. Thus, thymus colonization kinetics depend mainly on the blood delivery of T-cell progenitors during embryogenesis. (+info)
Requirement of type III TGF-beta receptor for endocardial cell transformation in the heart.
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Transforming growth factor-beta (TGF-beta) signaling is mediated by a complex of type I (TBRI) and type II (TBRII) receptors. The type III receptor (TBRIII) lacks a recognizable signaling domain and has no clearly defined role in TGF-beta signaling. Cardiac endothelial cells that undergo epithelial-mesenchymal transformation express TBRIII, and here TBRIII-specific antisera were found to inhibit mesenchyme formation and migration in atrioventricular cushion explants. Misexpression of TBRIII in nontransforming ventricular endothelial cells conferred transformation in response to TGF-beta2. These results support a model where TBRIII localizes transformation in the heart and plays an essential, nonredundant role in TGF-beta signaling. (+info)
Inhibition of myosin light chain kinase by p21-activated kinase.
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p21-activated kinases (PAKs) are implicated in the cytoskeletal changes induced by the Rho family of guanosine triphosphatases. Cytoskeletal dynamics are primarily modulated by interactions of actin and myosin II that are regulated by myosin light chain kinase (MLCK)-mediated phosphorylation of the regulatory myosin light chain (MLC). p21-activated kinase 1 (PAK1) phosphorylates MLCK, resulting in decreased MLCK activity. MLCK activity and MLC phosphorylation were decreased, and cell spreading was inhibited in baby hamster kidney-21 and HeLa cells expressing constitutively active PAK1. These data indicate that MLCK is a target for PAKs and that PAKs may regulate cytoskeletal dynamics by decreasing MLCK activity and MLC phosphorylation. (+info)
Expression of functional selectin ligands on Th cells is differentially regulated by IL-12 and IL-4.
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Immune responses may be qualitatively distinct depending on whether Th1 or Th2 cells predominate at the site of Ag exposure. T cell subset-specific expression of ligands for vascular selectins may underlie the distinct patterns of recruitment of Th1 or Th2 cells to peripheral inflammatory sites. Here we examine the regulation of selectin ligand expression during murine T helper cell differentiation. Large numbers of Th1 cells interacted with E- and P-selectin under defined flow conditions, while few Th2 and no naive T cells interacted. Th1 cells also expressed more fucosyltransferase VII mRNA than naive or Th2 cells. IL-12 induced expression of P-selectin ligands on Ag-activated naive T cells, even in the presence of IL-4, and on established Th2 cells restimulated in the presence of IL-12 and IFN-gamma. In contrast, Ag stimulation alone induced only E-selectin ligand. Interestingly, restimulation of established Th2 cells in the presence of IL-12 and IFN-gamma induced expression of P-selectin ligands but not E-selectin ligands; IFN-gamma alone did not enhance expression of either selectin ligand. In summary, functional P- and E-selectin ligands are expressed on most Th1 cells, few Th2 cells, but not naive T cells. Furthermore, selectin ligand expression is regulated by the cytokine milieu during T cell differentiation. IL-12 induces P-selectin ligand, while IL-4 plays a dominant role in down-regulating E-selectin ligand. (+info)
T cell repertoire alterations of vascularized xenografts.
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The role of T cells in the rejection of vascularized xenografts has been little explored. Because of the high potential diversity of xenoantigens, it has been suggested that xenotransplantation could induce a strong cellular response that could contribute to delayed rejection. Alternatively, alterations in molecular interactions could impair the T cell response. Because the analysis of TCR repertoire in vivo indirectly reflects the nature and the magnitude of T cell xenorecognition, we took advantage of the possibility of obtaining long term survival of hamster heart xenografts in rat recipients treated with a combination of cobra venom factor and cyclosporin A (CsA), to analyze T cell infiltration and, for the first time, V beta TCR usage, at the complementarity-determining region 3 level, in accommodated and rejected xenografts, compared with allografts. After withdrawal of CsA (on day 40), the analysis of V beta family expression and corresponding complementarity-determining region 3 lengths in rejected xenografts revealed a Gaussian pattern, in contrast to a much more restricted pattern in rejected allografts (p = 0.002), suggesting that, after withdrawal of CsA, all the underrepresented T cell clones are rapidly expanded in xenografts. These results correlate with the rapid kinetics of rejection (4 +/- 1 days), the high number of T cells, the rapid expression of markers of activation (IL-2 receptor alpha-chain and class II receptor), and the strong deposit of IgG Abs in rejected xenografts. Taken together, these results suggest that the intensity and diversity of the T cell response to xenografts could be stronger than the response to allografts in vivo. (+info)
Effect of fever-like whole-body hyperthermia on lymphocyte spectrin distribution, protein kinase C activity, and uropod formation.
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Regional inflammation and systemic fever are hallmarks of host immune responses to pathogenic stimuli. Although the thermal element of fever is thought to enhance the activity of immune effector cells, it is unclear what the precise role of increased body temperatures is on the activation state and effector functions of lymphocytes. We report here that mild, fever-like whole body hyperthermia (WBH) treatment of mice results in a distinct increase in the numbers of tissue lymphocytes with polarized spectrin cytoskeletons and uropods, as visualized in situ. WBH also induces a coincident reorganization of protein kinase C (PKC) isozymes and increased PKC activity within T cells. These hyperthermia-induced cellular alterations are nearly identical with the previously described effects of Ag- and mitogen-induced activation on lymphocyte spectrin and PKC. Immunoprecipitation studies combined with dual staining and protein overlay assays confirmed the association of PKC beta and PKC theta with spectrin following its reorganization. The receptor for activated C kinase-1 was also found to associate with the spectrin-based cytoskeleton. Furthermore, all these molecules (spectrin, PKC beta, PKC theta, and receptor for activated C kinase-1) cotranslocate to the uropod. Enhanced intracellular spectrin phosphorylation upon WBH treatment of lymphocytes was also found and could be blocked by the PKC inhibitor bisindolylmaleimide I (GF109203X). These data suggest that the thermal element of fever, as mimicked by these studies, can modulate critical steps in the signal transduction pathways necessary for effective lymphocyte activation and function. Further work is needed to determine the cellular target(s) that transduces the signaling pathway(s) induced by hyperthermia. (+info)
CD86 (B7-2) can function to drive MHC-restricted antigen-specific CTL responses in vivo.
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Activation of T cells requires both TCR-specific ligation by direct contact with peptide Ag-MHC complexes and coligation of the B7 family of ligands through CD28/CTLA-4 on the T cell surface. We recently reported that coadministration of CD86 cDNA along with DNA encoding HIV-1 Ags i.m. dramatically increased Ag-specific CTL responses. We investigated whether the bone marrow-derived professional APCs or muscle cells were responsible for the enhancement of CTL responses following CD86 coadministration. Accordingly, we analyzed CTL induction in bone marrow chimeras. These chimeras are capable of generating functional viral-specific CTLs against vaccinia virus and therefore represent a useful model system to study APC/T cell function in vivo. In vaccinated chimeras, we observed that only CD86 + Ag + MHC class I results in 1) detectable CTLs following in vitro restimulation, 2) detectable direct CTLs, 3) enhanced IFN-gamma production in an Ag-specific manner, and 4) dramatic tissue invasion of T cells. These results support that CD86 plays a central role in CTL induction in vivo, enabling non-bone marrow-derived cells to prime CTLs, a property previously associated solely with bone marrow-derived APCs. (+info)
Structural deficiencies in granuloma formation in TNF gene-targeted mice underlie the heightened susceptibility to aerosol Mycobacterium tuberculosis infection, which is not compensated for by lymphotoxin.
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TNF and lymphotoxin-alpha (LT alpha) may act at various stages of the host response to Mycobacterium tuberculosis. To dissect the effects of TNF independent of LT alpha, we have used C57BL/6 mice with a disruption of the TNF gene alone (TNF-/-). Twenty-one days following aerosol M. tuberculosis infection there was a marked increase in the number of organisms in the lungs of TNF-/- mice, and by 28-35 days all animals had succumbed, with widespread dissemination of M. tuberculosis. In comparison with the localized granulomas containing activated macrophages and T cells in lungs and livers of C57BL/6 wild-type (wt) mice, cellular infiltrates in TNF-/- mice were poorly formed, with extensive regions of necrosis and neutrophilic infiltration of the alveoli. Phenotypic analysis of lung homogenates demonstrated similar numbers of CD4+ and CD8+ T cells in TNF-/- and wt mice, but in TNF-deficient mice the lymphocytes were restricted to perivascular and peribronchial areas rather than colocated with macrophages in granulomas. T cells from TNF-/- mice retained proliferative and cytokine responses to purified protein derivative, and delayed-type hypersensitivity to purified protein derivative was demonstrable. Macrophages within the lungs of TNF-/- and wt mice showed similar levels of MHC class II and inducible nitric oxide synthase expression, and levels of serum nitrite were comparable. Thus, the enhanced susceptibility of TNF-/- is not compensated for by the presence of LT alpha, and the critical role of TNF is not in the activation of T cells and macrophages but in the local organization of granulomas. (+info)