Role of Ly-6 in lymphocyte activation. II. Induction of T cell activation by monoclonal anti-Ly-6 antibodies.

The Ly-6 locus controls the expression and/or encodes for alloantigenic specificities found primarily on subpopulations of murine T and B lymphocytes. We have recently identified and characterized a new rat mAb, D7, that recognizes a nonpolymorphic Ly-6 specificity. After crosslinking by anti-Ig reagents or by Fc receptor-bearing accessory cells, mAb D7 could induce IL-2 production from T cell hybridomas, and in the presence of PMA could trigger a vigorous proliferative response in resting peripheral T cells. The addition of mAb D7 to cultures of antigen- and alloantigen-, but not mitogen-stimulated T cells resulted in a marked augmentation of the proliferative response. A number of other well-characterized mAbs to Ly-6 locus products could also stimulate a T cell proliferative response after crosslinking by anti-Ig and in the presence of PMA. These results strongly suggest that Ly-6 molecules may play a critical role in the T cell activation cascade, either as receptors for an unidentified soluble or cell-associated ligand or as transducing molecules that modulate signals initiated by antigen stimulation of the T3-Ti complex.

Functional expression of the murine Thy-1.2 gene in transfected human T cells.

The interaction of certain mAbs with the Thy-1 molecules of murine T lymphocytes leads to cell activation and proliferation. To examine the signal transduction mechanism underlying this process and to determine what, if any, relationship exists between Thy-1-dependent triggering and T cell activation mediated through the T3-antigen receptor (T3-Ti) complex, a genomic clone of murine Thy-1.2 was isolated and transfected into the human T cell tumor, Jurkat. The transfected gene was actively transcribed in these human cells and high levels of Thy-1.2 glycoprotein were found on the cell membrane. Although certain mAbs to Thy-1.2 failed to bind to the Thy-1 transfected Jurkat cells, several known mitogenic anti-Thy-1 mAbs did react, and in the presence of phorbol ester, induced IL-2 secretion. One Thy-1+ transfectant out of five failed to produce IL-2 in response to anti-T3/Ti antibodies even though it retained the ability to increase intracytoplasmic calcium concentration [( Ca2+]i) in response to these ligands. A Thy-1 negative revertant of this cell regained anti-T3/Ti reactivity, suggesting a regulatory defect in signal transmission via T3/Ti in the original transfectant. These data confirm the ability of Thy-1 to act as an activation receptor for T cells. They reveal a potential role for changes in [Ca2+]i in this process, in common with other pathways of T cell activation, but also indicate a more complex series of events is involved.

Utilization of an NF-ATp binding promoter element for EGR3 expression in T cells but not fibroblasts provides a molecular model for the lymphoid cell-specific effect of cyclosporin A.

Cyclosporin A (CsA) mainly exerts its immunosuppressive action by selectively inhibiting Ca2+/calcineurin-dependent gene transcription in lymphoid cells. A model explaining the tissue-specific effect of this drug on gene expression has not been established to date, since none of the known intracellular targets of CsA (e.g., cyclophilins, calcineurin, and NF-AT) is lymphoid cell specific. To investigate this issue, we performed a detailed comparative analysis of the promoter regulating the two-signal-dependent (Ca2+ ionophore plus phorbol myristate acetate [PMA]), CsA-sensitive expression of EGR3 in T cells and the one-signal-dependent (PMA), CsA-insensitive expression of EGR3 in fibroblasts. As a result, we identified a 27-bp promoter element functionally interacting with transcription factors NF-ATp and NF-ATc that is crucial for the CsA-sensitive expression of the EGR3 gene in T cells. In contrast, the same element was without function in fibroblasts, and other, CsA-insensitive promoter regions were found to be responsible for EGR3 gene expression in these cells. The inactivity of the 27-bp element in fibroblasts was apparently due to insufficient expression levels of NF-ATp, since overexpression of NF-ATp, but not NF-ATc, restored the two-signal phenotype and CsA sensitivity of EGR3 promoter induction in these cells. The differential usage of an NF-AT binding site explains the selective effect of CsA on EGR3 gene expression in T cells versus fibroblasts and may represent one of the basic mechanisms underlying the tissue specificity of CsA.

NOT, a human immediate-early response gene closely related to the steroid/thyroid hormone receptor NAK1/TR3.

By analyzing the early genetic response of human T cells following mitogenic activation we have identified NOT, a member of the steroid/thyroid hormone family of receptors. NOT has all structural features of steroid/thyroid hormone receptors (C2C2 zinc-finger domain, ligand binding domain), but is rapidly and only very transiently expressed after cell activation, which is clearly at variance with classical steroid receptors such as glucocorticoid or estrogen receptors. NOT gene induction is independent of de novo protein synthesis, defining NOT as an immediate-early response gene. Short-lived NOT mRNA (4.2 kilobases) expression could be observed in vitro in a greater number of tissue types following activation by a variety of distinct stimuli. In vivo, NOT mRNA expression was detected exclusively in the brain, where a very strong signal was observed. By immunoblot analysis of human T cell lysates with NOT specific antisera two activation-dependent protein bands (66 and 59 kilodaltons) could be detected. NOT gene was localized to human chromosome 2q22-q23. Sequence comparison revealed that NOT is the human homolog of the murine NURR1 and rat RNR-1. Moreover NOT is closely related to NAK1/TR3, a previously identified human orphan steroid receptor. Several lines of evidence indicate that NOT and NAK1/TR3 form a distinct and exclusive subgroup of orphan steroid receptors, whose expression characteristics in vitro and in vivo resemble the expression of nonsteroid immediate-early transcription factors such as jun and fos. NOT and NAK1/TR3 thus may function as general coactivators of gene transcription rather than participate in the induction of specific target genes, as is the case with classical steroid receptors.

Lymph-borne CD8α+ dendritic cells are uniquely able to cross-prime CD8+ T cells with antigen acquired from intestinal epithelial cells.

Cross-presentation of cellular antigens is crucial for priming CD8(+) T cells, and generating immunity to intracellular pathogens--particularly viruses. It is unclear which intestinal phagocytes perform this function in vivo. To address this, we examined dendritic cells (DCs) from the intestinal lymph of IFABP-tOVA 232-4 mice, which express ovalbumin in small intestinal epithelial cells (IECs). Among lymph DCs (LDCs) only CD103(+) CD11b(-) CD8α(+) DCs cross-present IEC-derived ovalbumin to CD8(+) OT-I T cells. Similarly, in the mesenteric lymph nodes (MLNs), cross-presentation of IEC-ovalbumin was limited to the CD11c(+) MHCII(hi) CD8α(+) migratory DCs, but absent from all other subsets, including the resident CD8α(hi) DCs. Crucially, delivery of purified CD8α(+) LDCs, but not other LDC subsets, into the MLN subcapsular lymphatic sinus induced proliferation of ovalbumin-specific, gut-tropic CD8(+) T cells in vivo. Finally, in 232-4 mice treated with R848, CD8α(+) LDCs were uniquely able to cross-prime interferon γ-producing CD8(+) T cells and drive their migration to the intestine. Our results clearly demonstrate that migrating CD8α(+) intestinal DCs are indispensable for cross-presentation of cellular antigens and, in conditions of inflammation, for the initial differentiation of effector CD8(+) T cells. They may therefore represent an important target for the development of antiviral vaccinations.

Induction of IL-2 receptor expression in vivo. Response to allogeneic cells.

Although the requirements for the induction of IL-2 receptor expression following lymphocyte stimulation in vitro have been well characterized, little information is available on the role of IL-2 and the IL-2 receptor in lymphocyte activation in vivo. We have established a quantitative assay for the measurement of IL-2-receptor-positive cells in the draining popliteal lymph node following the injection of allogeneic cells in the footpad. At the peak of the response more than 20% of the lymph node cells were IL-2-receptor-positive, and the majority of the IL-2-receptor-positive cells were Ly-2+ T cells and B cells. IL-2 receptor expression in vivo was closely associated with an increase in cell size and spontaneous and IL-2-driven proliferation, as well as with the induction of CTL activity specific for the donor strain. The model system described here should be useful in the further characterization of the mechanism of action in vivo of immunosuppressive drugs, antibodies, or lymphokines.

Activated platelets induce tissue factor expression on human umbilical vein endothelial cells by ligation of CD40.

CD40 is a type I member of the tumour necrosis factor (TNF) receptor superfamily of proteins, and is present on a wide variety of cells including vascular endothelial cells. Ligation of this receptor on endothelial cells is known to increase expression of inflammatory adhesion molecules. We have recently demonstrated that platelets express the ligand of CD40 (CD154) within seconds of exposure to agonist, and interact with endothelial cells to participate directly in the induction of an inflammatory response. Here we show that activated platelets induce tissue factor (TF) expression on endothelial cells in a CD40/CD154-dependent manner, and that the magnitude of this response can equal that induced by TNFe. Moreover, CD40 ligation on endothelial cells downregulates the expression of thrombomodulin. We also show that CD40-mediated TF expression is less sensitive to inhibition with the oxidative radical scavenger pyrrolidine dithiocarbamate than is that mediated by TNFalpha, indicating that CD40 has a distinct signalling pathway. Tissue factor is a cell membrane protein which functions as the main trigger of the extrinsic pathway of blood coagulation, and its expression on endothelial cells is implicated in wound healing and angiogenesis. Since platelets are among the first cells involved in haemostasis following tissue injury, our data showing that ligation of CD40 by CD154 induces a procoagulant phenotype on vascular endothelial cells suggests that platelets may play an important role in the induction of wound healing.

Ontogeny of CD40L [corrected] expression by activated peripheral blood lymphocytes in humans.

The CD40 ligand (CD40L) is a molecule expressed by activated T cells which plays a critical role in the regulation of B-cell responses, including differentiation into Ig-producing cells. Using the specific monoclonal antibody TRAP1 we have evaluated the ontogeny of CD40L expression in 97 normal individuals between birth and 50 years of age. The expression of CD40L is a function of age; it is severely reduced at birth, progressively increases during the first months of life, and reaches a plateau in the second decade. This progressive attainment of the ability to express CD40L is due to a process of maturation of the CD4 + subset, being significantly correlated with the expression of the CD45RO antigen.

Co-stimulatory molecules as potential targets for therapeutic intervention in allergic airway disease.

Airway inflammation is a characteristic feature of allergic asthma. Central to the initiation and progression of the inflammatory process are allergen-specific T lymphocytes that attract eosinophils, mast cells, and B cells to the airways by the secretion of specific cytokines. The direction of T cell responses is influenced by co-stimulatory signals that modulate the antigen-specific signal delivered by the T cell receptor. In addition to the prototypic co-stimulatory molecule, CD28, a number of newly identified co-stimulatory molecules and their ligands have now been characterized. Over the past 5 years, the role of these molecules in the pathophysiology of allergen-mediated sensitization and airway inflammation has been extensively studied in animal models of allergic asthma. The aim of this review is to provide a detailed overview on recent studies in mice and preliminary findings in man and to discuss the potential therapeutic and preventive treatment strategies offered by interactions with co-stimulatory molecules for patients with allergic airway diseases.

Chemokine-receptor expression on T cells in lung compartments of challenged asthmatic patients.

BACKGROUND: The interaction of chemokines with their receptors strongly influences the migration of leucocytes. OBJECTIVE: In order to assess the contribution of these molecules to the local recruitment of T cells in bronchial asthma, we analysed the expression of 14 chemokine receptors on lung-derived T cells. METHODS: Chemokine-receptor expression by T cells derived from the peripheral blood, the bronchoalveolar lavage fluid and the bronchial mucosa was analysed by flow cytometry and immunohistochemistry. Expression profiles in healthy and mildly asthmatic individuals were compared, the latter prior and after segmental allergen provocation. RESULTS: Compared with peripheral blood, alveolar T cells expressed significantly more CCR2, CCR5, CCR6, CXCR3 and CCR4. However, no differences were observed between healthy controls and unchallenged asthmatics. In patients developing significant inflammatory responses following specific allergen challenge, a marked increase in the percentage of CCR4+ and CCR7+, and reduced numbers of CXCR3-bearing alveolar T cells were detected. Following specific allergen challenge, chemokine-receptor expression profiles of T cells from the alveolar space and the mucosa or the submucosa were similar, excluding a particular subcompartmentalization of the chemokine/chemokine-receptor system. CONCLUSION: The expression of certain chemokine receptors by lung T cells suggests a contribution to the physiological recruitment of T cells to the lungs, both in healthy controls and unchallenged mild asthmatics. However, strong allergen-induced airway responses were associated with a specific chemokine-receptor profile, suggesting the involvement of certain chemokine receptors in the pathogenesis of allergic bronchial inflammation.

House dust mite-specific T cells in healthy non-atopic children.

BACKGROUND: T-helper type 2 (Th2) cells play an important role in the pathogenesis of allergic diseases. Recent studies have demonstrated that allergen-specific T cells can also be found in the blood of healthy individuals. Both IL-10 and IFN-gamma might modulate the induction and maintenance of allergen-specific tolerance. AIM: To study the phenotype and functional characteristics of allergen-specific T cells in healthy non-atopic children. METHODS: Peripheral blood mononuclear cells (PBMC) from 13 symptomatic house dust mite (HDM)-allergic children and from nine matched healthy control children were stimulated with recombinant (r)Der p 2, a major allergen from HDMs. RESULTS: Stimulation with rDer p 2 resulted in Th2 cytokine production in cultures of PBMC from allergic but not from healthy children. In contrast, IL-10 and IFN-gamma were induced in PBMC cultures from both healthy and HDM-allergic children. Intracellular staining revealed that IL-10 and IFN-gamma are largely produced by the same T cells. Stimulation of T cells from healthy children with rDer p 2 also induced expression of inducible costimulator (ICOS) on a small T cell subset. CONCLUSION: Allergen-specific memory T cells from healthy non-atopic children produce IL-10 and IFN-gamma (but not Th2 cytokines) and express ICOS upon stimulation. These cells might be responsible for a normal immune balance after allergen encounter in non-atopics.

Southern and northern analysis.

This review on Southern and Northern analysis, rather than providing step-by-step protocols, focuses on a critical evaluation of the existing experimental methods and modifications thereof. Principal parameters influencing electrophoresis of DNA/RNA in agarose gels are outlined and at the same time alterations in these parameters for optimal resolution of DNA of varying length are discussed. Further, methods for evaluating the quality of DNA/RNA size separation in agarose gels are described. Since efficient transfer of DNA/RNA from the gel onto a membrane support is critical in both methods, several experimental approaches for transfer are compared. Also discussed in this review are alternative methods for radioactive and non-radioactive labeling of DNA probes. Finally, detailed protocols are provided for an effective hybridization of Southern and Northern blots.

Congenital chyloperitoneum: direct comparison of medium-chain triglyceride treatment with total parenteral nutrition.

A severe case of congenital chyloperitoneum was managed over a prolonged period by permanent drainage and replacement of lymphatic loss by fresh frozen plasma, resulting in normal development of the infant. The average daily drainage of 360 ml during a period of a medium-chain-triglyceride (MCT) diet could be reduced to 228 ml (1.58 ml/kg per h) with total parenteral nutrition, representing apparently the basal flow rate of the intestinal lymphatics. Reduction in leakage, however, did not influence the observed lymphocytopenia. Healing of the lesion within the intestinal lymphatic system occurred after a brief period of bacterial peritonitis. The rationale for treatment with an MCT diet and for the application of total parenteral nutrition in infants with chyloperitoneum is discussed.

Optimization of northern analysis by vacuum-blotting, RNA-transfer visualization, and ultraviolet fixation.

We have optimized Northern analysis at several steps. Overnight electrophoresis was replaced by short gel runs and overnight capillary transfer by rapid vacuum-blotting adapted to Northern analysis. Short uv irradiation was used as a substitute for the usual RNA fixation by baking. Direct staining of RNA before electrophoresis made it possible to check RNA integrity and to evaluate the quality of the size separation immediately after electrophoresis. In this system, RNA transfer onto the membrane support could also be quickly assessed after the blotting step. The net result of all modifications was a doubling of the autoradiography signal compared with that obtained by modern Northern protocols. At the same time, the duration of the procedure was shortened drastically, allowing an autoradiography signal to be obtained within 24 h.

Cloverleaf skull associated with Pfeiffer syndrome: pathology and management.

The clinical and radiological findings in an infant with severe cloverleaf skull associated with a Pfeiffer syndrome are presented. The skull anomaly resulted in raised intracranial pressure and proptosis of the right eye within several weeks of birth. The child died at 3 months of age after subtotal craniectomy. At autopsy a detailed macroscopical and histological analysis of the skull base was conducted. The timing and value of surgery are discussed as well as the pathology of the cloverleaf skull anomaly.

CD154 stimulation of interleukin-12 synthesis in human endothelial cells.

The interaction of proinflammatory type 1 T helper (Th1) cells expressing the CD40 ligand (CD154) with endothelial cells expressing the corresponding receptor (CD40) may play an important role in chronic inflammation including arteriosclerosis. Here we demonstrate that activation of CD40 in human cultured endothelial cells (e.g. by interaction with freshly isolated human T cells) not only up-regulates expression of various adhesion molecules, chemokines and cytokines, but within 12-24 h also causes the release of bioactive interleukin-12 (IL-12 p70) through induction of IL-12 p40 synthesis. IL-12 p35, on the other hand, appears to be constitutively expressed in these cells. Despite enhancing expression of the other gene products, cytokines such as interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha, alone or in combination, failed to induce IL-12 p40 expression, whereas IFN-gamma markedly augmented CD154-induced IL-12 p40 and p70 release. Of note was that the magnitude of CD154-induced IL-12 synthesis in the cultured endothelial cells was comparable to that evoked in freshly isolated human monocytes. This CD40-mediated induction of endothelial IL-12 synthesis may thus lead to an enhanced activation of the adherent CD154-expressing Th1 cells, thereby fuelling the proinflammatory response.

Induction of IL-2 receptor expression in vivo: response to concanavalin A.

The requirements for the induction of IL-2 receptor expression following lymphocyte stimulation in vitro are well understood but little is known about the role of IL-2 and the IL-2 receptor in lymphocyte activation in vivo. Following the subcutaneous injection of concanavalin A, cells from popliteal and lumbar lymph nodes draining the footpad became enlarged and expressed the IL-2 receptor. At the peak of the response, 9-15 hr after injection, more than 70% of all cells were IL-2 receptor positive; the majority were T cells of both Lyt-2+ and L3T4+ subsets. IL-2 receptor expression was closely associated with both spontaneous and IL-2-driven proliferation. Analysis of the biodistribution of 125I-labeled concanavalin A revealed that much of the injectate remains in the footpad injection site but the mitogen also accumulates in the draining lymph nodes and eventually in the major organs. This model of lymphocyte activation was used to demonstrate that in vivo cyclosporin A inhibits both IL-2 receptor expression and the induction of spontaneous proliferation.

Upregulation of CD40 and CD40 ligand expression in IgE-associated cutaneous diseases.

In order to better understand the immunological processes connected with IgE-associated cutaneous disease, we have examined the expression of CD40 and its ligand CD40L, required for the induction of IgE synthesis in B-cells, as well as of IgE and its receptors in various dermatoses (atopic dermatitis (AD), scabies, chronic recurrent urticaria) versus normal skin, and in one dermopathic lymph node versus normal lymphatic tissue by immunohistochemistry. Compared to normal skin, cells expressing IgE, Fc epsilon RI, Fc epsilon RII, CD40, CD40L and L26 were increased in the dermis, partly also in the epidermis, from patients with AD and scabies, but not in chronic urticaria. CD40 and CD40L were detected on numerous cells in lymphatic tissue from both normal donors and a patient with AD, whereas large numbers of IgE- and Fc epsilon RI-positive cells were only found in the dermopathic lymph node from the AD patient, in contrast to very few in normal lymphatic tissue. These results with selectively increased IgE/Fc epsilon RI and associated CD40/CD40L expression in the skin of AD and scabies suggest that cutaneous tissue, in addition to dermopathic lymphatic tissue, might contribute to IgE synthesis.

Induction, regulation, and function of soluble TRAP (CD40 ligand) during interaction of primary CD4+ CD45RA+ T cells with dendritic cells.

To assess the induction, regulation, and the relative roles of cell surface tumor necrosis factor-related activation protein (TRAP; CD40 ligand) and the soluble form of TRAP (sTRAP) in the initial phase of T cell activation, primary CD4+ CD45RA+ (naive) T cells were co-cultured with mature Langerhans' cells (mLC) in the presence of superantigen. In this cell system, TRAP was very efficiently induced in T cells at both the mRNA and protein levels. After appearing on the cell surface, TRAP was rapidly down-regulated by a mechanism triggered through interaction of TRAP with CD40 on mLC. Co-culture of T cells with mLC led to the release of sTRAP, an 18-kDa protein capable of binding to CD40. Experimental data strongly suggest that sTRAP is not released by proteolytic cleavage of TRAP on the cell surface, but is generated in an intracellular compartment. Release of sTRAP and induction of TRAP cell surface expression were found to be regulated independently. In terms of function, sTRAP cannot compete with cell surface TRAP for ligation of CD40 on mLC, indicating that sTRAP release is not a mechanism for termination of the TRAP/CD40 interaction. However, sTRAP on its own rapidly down-regulates CD40 expression on mLC and has long-lasting anti-apoptotic effects on dendritic cells. Thus, we infer from our results obtained in vitro that primary activation of CD4+ T cells by dendritic cells in the lymphoid tissues leads to release of sTRAP, which may act on CD40+ bystander cells in a cytokine-like fashion.