Ly-24 (Pgp-1) expression by thymocytes and peripheral T cells.
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Biomedical subjects
R Ceredig
Publications and source records attributed to R Ceredig.
Intracellular pathway of interleukin-2 receptors studied using immunogold electron microscopy.
The fate of internalized interleukin-2 receptors (IL-2R) on proliferating T cell blasts and Lyt-2-/L3T4- thymocytes was studied by immunogold electron microscopy. Immediately following labeling, IL-2R were found in coated pits and with time there was sequential appearance of gold particles in smooth vesicles or endosomes, multivesicular bodies and lysosomes. Thus the IL-2R appears to be a member of cell surface receptors internalized by endocytosis via coated pits.
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Lethal vaccinia infection in cyclophosphamide-suppressed mice is associated with decreased expression of Thy-1, Lyt-2 and L3T4 and diminished IL-2 production in surviving T cells.
Prior treatment of C57BL/6J mice with 300 mg/kg of cyclophosphamide (Cy) converts a subclinical infection with vaccinia virus to a lethal disease. This is accompanied by a loss of more than 80% of spleen cells and a decreased capacity, on a cell-for-cell basis, to develop virus-immune cytotoxic T lymphocytes (CTL), although the frequency of CTL precursors among surviving T cells is not greatly modified. Phenotypically, the surviving T cells express low levels of cell-surface Thy-1, Lyt-2 and L3T4 and, upon stimulation, are less able to produce IL-2 for more than 1 week following Cy treatment. The defect in capacity to generate CTL effectors both in vitro and in vivo can be corrected by providing an exogenous source of IL-2. These experiments indicate that a single dose of Cy induces changes in T cells that persist throughout the development of an immune response. Such effects are in accordance with the known property of Cy to mediate DNA damage.
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Proliferation in vitro of Lyt2-,L3T4- thymocytes shows responsiveness to interleukin 1.
The responsiveness of adult mouse thymocytes to interleukin 1 (IL-1) was investigated. When stimulated with suboptimal concentrations of ionomycin and phorbol myristate acetate (PMA), the immature subpopulation of Lyt2-,L3T4- (2-4-) thymocytes responded to exogenous, purified IL-1 in a dose-dependent manner. In contrast, mature lymph node (LN) T cells were unresponsive to exogenous IL-1 under similar conditions. The proliferation of 2-4- thymocytes, which comprise 2-3% of cells in the adult mouse thymus, was independent of exogenous IL-2 and accounted for essentially all the 3H-thymidine incorporation by unfractionated thymocytes in response to IL-1. Optimally stimulated 2-4- cells did not show responsiveness to exogenous IL-1, and 48 h supernatants from these cultures were found to contain IL-1 like activity. Taken together, the data point to a role for IL-1 in the proliferation of immature 2-4- cells in the thymus.
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Phenotypic analysis of the inflammatory exudate in murine lymphocytic choriomeningitis.
The massive inflammation of the cerebrospinal fluid (CSF) which occurs in adult mice injected with lymphocytic choriomeningitis virus (LCMV) has been analyzed by flow microfluorometry (FMF). The great majority of the T cells detected by direct examination of freshly obtained CSF were found to be Lyt-2+, with an almost total absence of L3T4+ lymphocytes. The Lyt-2/L3T4 ratio of lymphocytes in blood was within normal limits. Predominance of the Lyt-2+ subset was confirmed by culturing the CSF cells after mitogenic stimulation. In addition, the T lymphocytes in CSF of cyclophosphamide-suppressed, virus-infected recipients that had been injected 4 d previously with LCMV-immune spleen cells were almost entirely donor Lyt-2+ cells, while the nonlymphoid elements were exclusively of host origin. However this pattern of donor and host T cell distribution was reversed when the LCMV-infected recipients were not immunosuppressed. The frequency of LCMV-specific CTL precursors in CSF taken immediately before the development of symptoms was as low as 1:3,000 cells. Thus most of the T lymphocytes extravasating into the CSF of mice with LCM are passive participants recruited as a consequence of the function of relatively few LCMV-specific effector T cells. The dominance of the Lyt-2+ T cell subset in the CSF of mice with LCM is intriguing.
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Expression of Pgp-1 (or Ly24) by subpopulations of mouse thymocytes and activated peripheral T lymphocytes.
The expression of Pgp-1 (Ly24) by subpopulations of thymocytes was investigated and a subpopulation of Lyt-2-/L3T4-/J11d- thymocytes was identified which contained significant numbers (80%) of Pgp-1+ cells. Among freshly isolated lymph node T cells but not cortisone-resistant thymocytes, Pgp-1 expression was heterogeneous. Stimulation of T lymphocytes with either concanavalin A or the combination of phorbol myristate acetate plus calcium ionophore resulted in increased Pgp-1 expression which was found to be regulated independently of DNA synthesis and interleukin 2 receptor expression. T cells in the cerebrospinal fluid exudate of mice infected with lymphocytic choriomeningitis virus were also found to be Pgp-1+.
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Phenotypic properties, interleukin 2 production, and developmental origin of a "mature" subpopulation of Lyt-2- L3T4- mouse thymocytes.
Two-color flow microfluorometry using monoclonal antibodies to cell surface determinants has shown that a subpopulation of mouse Lyt-2- L3T4- thymocytes, comprising 18% of Lyt-2- L3T4- cells in adult C57BL/6 mice, appears in the thymus late during fetal development. These Lyt-2- L3T4- cells are characterized by lack of expression of determinants recognized by monoclonal antibody J11d, a phenotype characteristic of more "mature" functional T cells. This J11d- subpopulation of Lyt-2- L3T4- thymocytes has now been shown to produce significant quantities of interleukin 2 following mitogen stimulation and to express T-cell receptor molecules recognized by monoclonal antibodies KJ-16 and F23.1. Furthermore, culturing of fetal thymus lobes has shown that precursors of this subpopulation of Lyt-2- L3T4- thymocytes are already present in thymus at 14 days of embryonic development and are thus derived from an intrathymic precursor cell. So, within the mouse thymus, phenotypic changes and acquisition of mature T-cell characteristics occur within a subpopulation of cells originally thought of as exclusively "immature."
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Changes in the expression of potassium channels during mouse T cell development.
In this report we have combined the whole-cell electrophysiological recording technique with flow microfluorometry to isolate phenotypically defined thymocytes and T lymphocytes. Results obtained showed that J11d-/Lyt-2-/L3T4- cells express none or very few delayed rectifier K+ channels, whereas most other Lyt-2-/L3T4- cells, as well as typical cortical thymocytes (Lyt-2+/L3T4+), do express K+ channels. Mature (Lyt-2+/L3T4- or Lyt-2-/L3T4+) thymocytes, which are heterogeneous for J11d expression, were also found to be heterogeneous for K+ channel expression. Consistent with this finding was the observation that the cortisone-resistant subpopulation of thymocytes, which express low levels of J11d, were enriched for cells expressing low levels of K+ channels. Mature phenotype peripheral T lymphocytes expressed very low levels of K+ channels, but upon activation with Con A were found to express high levels of K+ channels. The results suggest that K+ channel expression in T cells is developmentally regulated. Increased expression of the channel is induced in response to mitogenic signals throughout the T cell lineage. Expression of the channel, therefore, serves as a useful marker in defining steps in the T cell differentiation pathway.
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Effect of antioxidants on primary alloantigen-induced T cell activation and proliferation.
Because oxygen-centered free radicals are involved in cell-mediated immune responses, we examined the possibility that these reactive species could also have a role in the lymphoproliferative response to alloantigens in the mixed lymphocyte culture (MLC). Three classes of agents that prevent the formation, or damaging effects, of oxygen radicals were tested: the non-permeant electron acceptor ferricyanide; the iron chelators desferrioxamine, desferrithiocin, octanohydroxamic acid, and pyridoxal isonicotinoyl hydrazone, which are thought to be capable of preventing the iron-catalyzed reduction of H2O2 to more reactive species; and the lipid-soluble free radical scavenger butylated hydroxyanisole. These compounds inhibited the proliferation of all potential responder cells in the MLC in a dose-dependent manner. Inhibition was observed only if these agents were present early (less than 40 hr) in the MLC; if added later, their effects were significantly reduced. In contrast, the interleukin 2 (IL 2)-dependent proliferation of CTLL-2 cells or Con A blasts was not affected by these compounds at concentrations that inhibited proliferation in MLC by greater than 90%. Interleukin 1 (IL 1) production by peritoneal exudate cells and IL 2 and IL 1 levels in MLC were not affected by any of the agents tested. By flow microfluorometry, the expression of IL 2 receptors on stimulated T cells was found to be inhibited in the presence of these drugs. Taken together, the data point to an important role for free radical-mediated processes during the early stages of T lymphocyte activation, before IL 2 receptor expression.
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Functional status of interleukin 2 receptors expressed by immature (Lyt-2-/L3T4-) thymocytes.
A subpopulation of phenotypically immature (Lyt-2-/L3T4-) thymocytes express receptors for the polypeptide hormone interleukin 2 (IL 2); however, these cells do not proliferate in vitro in response to IL 2. In investigating this phenomenon in greater detail, we observed that the IL 2 receptors (IL 2-R) on freshly isolated immature thymocytes bound IL 2 with about fivefold lower affinity (Kd approximately 100 pM) than IL 2-R on activated mature T cells and T cell lines (Kd approximately 20 pM). Furthermore, in contrast to activated T cells, Lyt-2-/L3T4- thymocytes did not endocytose bound IL 2. When stimulated in short-term culture with a combination of phorbol ester (PMA) and calcium ionophore, Lyt-2-/L3T4- thymocytes proliferated in a largely IL 2-dependent fashion. IL 2-R expression on these activated cells initially disappeared (at 24 hr) and subsequently reappeared (at 48 to 72 hr). Reexpressed IL 2-R on activated thymocytes resembled those on mature T lymphocytes in that they bound IL 2 with high affinity (Kd = 15 to 25 pM) and were capable of endocytosing IL 2. Taken together, these data place certain constraints on the putative physiologic role of IL 2 in intrathymic growth regulation.
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Proliferation in vitro and interleukin production by 14 day fetal and adult Lyt-2-/L3T4- mouse thymocytes.
When 14 day fetal mouse thymocytes, which are phenotypically Lyt-2-/L3T4-(2-4-), were stimulated in vitro with a combination of phorbol myristate acetate (PMA) and the calcium ionophore ionomycin, they proliferated without addition of exogenous interleukins and/or growth factors. Addition of exogenous IL 2 resulted in a slight enhancement of fetal thymocyte proliferation. By using factor-dependent indicator cell lines, this proliferation was shown to be accompanied by the production of IL 2 and IL 3. However, phenotypic analysis by using flow microfluorometry and monoclonal antibodies to Lyt-2 and L3T4 showed little differentiation among proliferating 2-4-fetal thymocytes. Interestingly, the in vitro growth of PMA + ionomycin-stimulated fetal thymocytes appeared to be IL 2 dependent in that it was inhibited by a monoclonal antibody to the IL 2 receptor. The results obtained with fetal thymocytes were compared with those obtained when using 2-4- thymocytes from adult mice.
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High antigen concentration inhibits T cell proliferation but not interleukin 2 production: examination of limiting dilution microcultures and T cell clones.
The effect of high antigen dose on the activation of cytochrome c peptide-primed lymph node cells was determined in several strains of mice by a limiting dilution analysis. It was found that proliferation of cytochrome c peptide-specific T cells was completely inhibited at high antigen concentration in C57BL/6 but only partially in DBA mice and had no effect in SJL mice. Clones derived from DBA mice showed a differential capacity to be inhibited by high antigen dose. On the other hand, interleukin 2 production by these clones was not impaired regardless of the antigen concentrations used.
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Major histocompatibility-restricted cytolytic T-lymphocyte precursors from the thymus of in vivo primed mice: increased frequency and resistance to anti-Lyt-2 antibody inhibition.
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Intrathymic differentiation: some unanswered questions.
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Morphological and phenotypical characterization of bone marrow-derived dendritic Thy-1-positive epidermal cells of the mouse.
Increasing evidence exists that the spectrum of dendritic cells within the epidermis is more complex than previously thought. In addition to Langerhans cells, Merkel cells, and melanocytes, the murine epidermis contains a dendritic cell population whose most prominent phenotypic feature is the Thy-1 antigen. These cells are now generally referred to as dendritic Thy-1+ epidermal cells (dThy-1+EC). The ultrastructural features of these cells do not resemble those of other resident epidermal cells (EC). In particular, their cytoplasm contains abundant intermediate-sized filaments of the vimentin type as well as membrane-limited organelles with a central granular core. The bone marrow derivation of dThy-1+EC is now well established: dThy-1+EC carry Ly-5 determinants whose expression is restricted to cells of the hemopoietic differentiation pathway, and studies using Thy-1-disparate radiation bone marrow chimeras have revealed the presence of donor-type Thy-1+ cells within the epidermis; by immunoelectron microscopy, these cells represent dThy-1+EC. dThy-1+EC repopulate the epidermis at a slower rate than Langerhans cells as evidenced by a direct comparison of the repopulation kinetics of both cell systems in radiation bone marrow chimeras, and by experiments studying the emergence of either Ia+- or dThy-1+EC in an epidermis which had been previously depleted of either Langerhans cells (glucocorticosteroids) or of dThy-1+EC (PUVA). The phenotypical features of dThy-1+EC differ from those of thymus-derived lymphocytes, B cells, dendritic cells, and mononuclear phagocytes. The surface marker repertoire of dThy-1+EC (Thy-1, Ly-5, asialo-GM1) resembles certain members of the rather heterogeneous natural killer (NK) cell system but functional studies are needed to ascertain this contention.
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Therapeutic effect of anti-L3T4 monoclonal antibody GK1.5 on cutaneous leishmaniasis in genetically-susceptible BALB/c mice.
The effect of in vivo treatment with anti-L3T4 monoclonal antibody (mAb) on the course of experimentally-induced cutaneous leishmaniasis was studied in genetically susceptible BALB/c mice. Administration of anti-L3T4 mAb resulted in a pronounced therapeutic effect as reflected by both resolution of lesions and a dramatic reduction in the parasite load in treated mice. This effect was specific for anti-L3T4 mAb and correlated with a selective depletion of L3T4+ T cells in the lymphoid tissues of treated mice. Moreover, both the therapeutic effect of anti-L3T4 mAb on cutaneous leishmaniasis and the concurrent depletion of L3T4+ T cells in the lymphoid tissues of treated mice were dependent upon the isotype of the anti-L3T4 mAb employed.
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Functional analysis of T cell subsets from mice bearing the lpr gene.
The autosomal recessive lpr (lymphoproliferation) gene is responsible for a thymus-dependent massive lymphoproliferation associated with the development of lupus-like autoimmune disease. Phenotypic analysis of adult lpr/lpr lymph nodes has demonstrated accumulation of a dull Lyt-1+, Thy-1+ population that expresses neither Lyt-2 nor L3T4 antigens. With the use of a depletion method based on complement-mediated lysis with an anti-Lyt-2 monoclonal antibody (31 M) and a new anti-L3T4 monoclonal antibody (RL 172.4), we have purified the Lyt-2- L3T4- subset from lymph nodes or spleens of C57BL/6-lpr/lpr mice and determined whether they are immunologically functional in vitro. Production of neither interleukin 2 nor interferon-gamma was detected by the double-negative subset after stimulation with concanavalin A and/or phorbol myristate acetate. The frequencies of allospecific cytotoxic T lymphocyte (CTL) precursors and lectin-induced antigen-nonspecific CTL precursors were diminished to almost undetectable levels, whereas the Lyt-2+ population from lpr/lpr mice had CTL-precursor frequencies comparable with that of +/+ mice. These results show that the major cell subset of adult lpr/lpr lymph nodes or spleens is composed of lymphocytes with markedly limited potential for lymphokine production or antigenic stimulation.
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Development and localization of 'T-like' cells in the nude mouse.
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