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R Ceredig

Publications and source records attributed to R Ceredig.

At least 91 records · Page 5Linked to original sources
PubMed1984

Immunohistochemical localization of host and donor-derived cells in the regenerating thymus of radiation bone marrow chimeras.

The anatomical distribution of CBA (Thy-1.2) host and AKR (Thy-1.1) donor-derived cells in the regenerating thymus of AKR----CBA radiation bone marrow chimeras was investigated. Cryostat sections of chimeric thymuses were incubated with biotin-conjugated monoclonal anti-Thy-1 antibodies specific for host and donor-derived cells and the distribution of the corresponding Thy-1 antigen revealed by the immunoperoxidase staining technique. The thymus was initially repopulated by Thy-1.2+ host-derived cells, but by 28 days following bone marrow reconstitution the few remaining host cells were found mostly in the thymus medulla. However, occasional Thy-1.2+ cells were still present in extramedullary, primarily cortical, sites. Donor-derived (Thy-1.1+) cells were first seen in the 11-day chimeric thymus as single cells frequently closely associated with blood vessels in medullary areas. By 17 days, the cortex contained many Thy-1.1+ cells, although occasional single positive cells were still present in the medulla. Changes in the anatomical distribution of host and donor-derived cells in the regenerating chimeric thymus appeared to correlate with changes in their Thy-1 fluorescence profile as determined by flow microfluorometry.

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PubMed1983

Precursors of T cell growth factor producing cells in the thymus: ontogeny, frequency, and quantitative recovery in a subpopulation of phenotypically mature thymocytes defined by monoclonal antibody GK-1.5.

In this report, the ontogeny of precursors of T cell growth factor (TCGF)-producing cells in the mouse thymus was investigated using a recently described limiting dilution microculture system. In agreement with previous studies, in the adult thymus TCGF production by cells stimulated by alloantigens was largely the property of the Lyt-2-negative subpopulation. Furthermore, when Lyt-2-negative cells were stained with monoclonal antibody GK-1.5 and sorted according to fluorescence intensity, all precursors of TCGF-producing cells were quantitatively recovered in the GK-1.5-positive subpopulation. During ontogeny, TCGF production by Lyt-2-negative thymocytes was first detectable on the 19th day of embryonic development at which time the precursor frequency was 1/10th that found in the adult thymus. As in the adult thymus, all precursors of TCGF-producing cells had the GK-1.5-positive, Lyt-2-negative phenotype. In parallel to these functional studies, the ontogeny of GK-1.5+, Lyt-2- cells was investigated. In the adult thymus, 80% of cells expressed both GK-1.5 and Lyt-2 antigens, whereas minor subpopulations of 10% and 5% (corresponding to phenotypically mature thymocytes as defined by cortisone-resistant thymocytes [CRT]) expressed GK-1.5 or Lyt-2 exclusively; 3% of cells expressed neither antigen. During ontogeny, thymocytes expressing both GK-1.5 and Lyt-2 first appeared on the 16th day of embryonic development and their proportion increased rapidly thereafter. Interestingly, the GK-1.5+, Lyt-2- subpopulation first appeared in significant numbers on day 19 in parallel with the appearance of functional TCGF activity. Taken together with our previous studies correlating cytolytic T lymphocyte precursor (CTL-P) activity with the Lyt-2+, GK-1.5- subpopulation, these results further emphasize the strict correlation between functional activity and mature surface phenotype of both embryonic and adult thymocytes.

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PubMed1983

Phenotypic and functional properties of murine thymocytes. III. Kinetic analysis of the recovery of intrathymic cytolytic T lymphocyte precursors after in vivo administration of hydrocortisone acetate.

The phenotypic and functional properties of cells in the C57BL/6 mouse thymus regenerating after a single dose of 100 mg/kg hydrocortisone acetate (H/C) are described. Functionally, the frequency of anti-H-2d cytolytic T lymphocyte precursors (CTL-P) in thymuses from individual mice was determined by limit dilution analysis of mixed leukocyte microcultures. The initial increase in CTL-P frequency, seen 48 hr post-H/C, was followed at 6 to 8 days by a phase of rapid decrease. The CTL-P frequency returned to a normal level by 28 days post-H/C. Analysis of the results from individual mice suggested that changes in total thymic CTL-P content were independent of the kinetics of thymus regeneration. Phenotypically, whereas the thymus 48 hr after H/C was considerably depleted of Lyt-2+ cells, there followed a rapid increase in the proportion of such cells to normal levels by 14 days post-H/C. In addition, as measured by FLS, a subpopulation of larger, predominantly Lyt-2+ cells was found during the phase of rapid thymic regeneration. With the use of a monoclonal anti-Thy-1.2 antibody, the weakly Thy-1-staining subpopulation of cells was absent from the thymus at 14 days post-H/C. These changes in the phenotypic properties of the post-H/C regenerating thymus were correlated with changes in their functional properties.

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PubMed1983

Stimulator cell requirements for allospecific T cell subsets: specialized accessory cells are required to activate helper but not cytolytic T lymphocyte precursors.

Murine cortisone-resistant thymocytes were separated by staining with monoclonal anti-Lyt-2 antibody and FMF into Lyt-2- and Lyt-2+ subsets in order to analyze the nature of stimulator accessory cells required to activate each of these functionally distinct T cell subpopulations. The Lyt-2- fraction was able to proliferate but not to generate cytotoxic cells when stimulated by irradiated allogeneic spleen cells. Fractionation of the stimulator population showed that low numbers of dendritic cells and splenic macrophages, but not equivalent numbers of whole spleen cells or peritoneal macrophages, were able to stimulate the Lyt-2- population. On the other hand, the Lyt-2+ population, which showed little if any proliferation in response to irradiated spleen cells, contained all the precursors of cytolytic T lymphocytes. In contrast to the highly specific stimulator requirement of the Lyt-2- fraction, allospecific cytotoxic cells were generated from Lyt-2+ cells by any alloantigen-bearing stimulator cell provided interleukin 2 was present. This was confirmed by limiting dilution analysis: alloreactive CTL-P frequencies in spleen and thymus were not influenced by the nature of the stimulator cell. These data collectively indicate that heterogeneous Ia+ accessory cells are required to stimulate helper but not cytolytic T cell precursors.

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PubMed1983

Generation of thymocyte subpopulations in organ culture: correlated analysis of Lyt-2 phenotype and cell cycle status by flow microfluorometry.

We have investigated the cell cycle kinetics and the proliferative activity of Lyt-2-defined subpopulations in organ-cultured thymic rudiments. Fetal thymuses that were removed at 13 days of gestation and cultured for 4 to 7 days were incubated with bromodeoxyuridine and were then stained with Hoechst 33342 to quantitate the duration of the cell cycle and the number of cycling cells. The total duration of the cell cycle was 30 hr. The sorting of organ culture cells according to their Lyt-2 phenotype and their forward light scatter, followed by Hoechst fluorescence and propidium iodide analysis, indicated that small Lyt-2+ cells that are themselves noncycling were derived through mitosis from another cell population. The combination of the Hoechst-BrdU substitution technique with monoclonal antibodies and the organ culture should provide a powerful tool for the study of lineage pathways in the thymus.

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PubMed1982

Phenotypic and functional properties of murine thymocytes. I. Precursors of cytolytic T lymphocytes and interleukin 2-producing cells are all contained within a subpopulation of "mature" thymocytes as analyzed by monoclonal antibodies and flow microfluorometry.

The correlation between surface phenotype and function in subpopulations of murine thymocytes has been investigated using flow microfluorometry (FMF). C57BL/6 thymocytes stained with monoclonal antibodies directed against Lyt-2, H-2K(b), and Thy-l.2 and passed on an FACS II flow cytometer could be resolved into at least four distinct subpopulations on the basis of fluorescence and forward light scatter (FLS) measurements. (a) Medium-sized Lyt-2(+) cells that stained strongly with H-2K(b) and weakly with Thy-l.2 (5 percent of total cells); (b) medium-sized Lyt-2(-) cells with other properties as in (a) (10 percent); (c) small Lyt-2(+) cells that stained weakly with H-2K(b) and strongly with Thy-l.2 (60 percent); and (d) large Lyt-2(+) cells that stained weakly with H-2K(b) and very strongly with Thy- 1.2 (23 percent). Cortisone-resistant thymocytes (CRT) were found to correspond phenotypically to populations (a) and (b). The distribution of cytolytic T lymphocyte precursors (CTL-P) directed against H-2(d) alloantigens in subpopulations of C57BL/6 thymocytes that had been sorted according to the phenotypic criteria described above was then investigated. CTL-P in sorted and control populations were quantitated by limiting dilution analysis of mixed leukocyte microcultures established in an excess of interleukin 2 (IL-2). These studies established that all thymus CTL-P could be quantitatively recovered in a subpopulation of cells that was cortisone-resistant, medium-sized, Lyt-2(+), H-2K(b+), and weakly stained with Thy-l.2. In parallel studies, the production of IL-2 by subpopulations of C57BL/6 thymocytes was quantitatively assessed using a recently developed sensitive microassay system. Graded numbers of sorted or control thymocytes were stimulated with irradiated T cell-depleted allogeneic cells and assayed for their ability to support the growth of an IL-2-dependent cytolytic T lymphocyte clone. Using this method, IL-2 production was found to reside entirely in a subpopulation of cortisone-resistant, medium-sized Lyt-2(-) thymocytes. Further phenotypic analysis of this subpopulation of cells indicated that it was homogeneously H-2K(b+) and weakly staining with Thy- 1.2. Taken together with the CTL-P results, these data directly demonstrate that a subpopulation of thymocytes with a mature phenotype (i.e., cortisone- resistant, medium-sized, H-2K(b+), and weakly staining with Thy-l.2) accounts for all the functional activity in the thymus. Reasons for the apparent discrepancy between these results and other recent studies will be discussed.

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PubMed1982

Development of cytolytic T lymphocyte precursors in organ-cultured mouse embryonic thymus rudiments.

The appearance of immunologically competent cells in the organ-cultured mouse fetal thymic rudiment has been investigated. Fetal thymuses removed at 14 d of gestation and cultured for 7-21 d were assayed for their content of cytolytic T lymphocyte precursors (CTL-P) directed against H-2d alloantigens. Whereas CTL-P were undetectable within fetal thymus until 18-19 d of gestation, their frequency in the organ-cultured fetal thymus was similar to, or greater than that found in the normal adult thymus. This direct demonstration of the appearance of alloreactive CTL-P in a closed in vitro system should provide an accessible model for the investigation of interactions between developing T cells and the thymic microenvironment.

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PubMed1981

Cells mediating graft rejection in the mouse. I. Lyt-1 cells mediate skin graft rejection.

The Ly phenotype of cells mediating skin graft rejection was determined using monoclonal anti-Lyt-1.1 and Lyt-2.1 antibodies in CBA mice that received CBA lymphoid cells from mice sensitized to C57BL/6; i.e., alloantigenic differences arising from the H-2 and non-H-2 loci. It was clear that graft rejection was due wholly to the presence of Lyt-1 cells in the inoculum and that Lyt-123 or Lyt-23 cells had no effect. Furthermore, no synergism was noted between Lyt-1 and Lyt-2 cells. In this model, both the cytotoxic T cell and cytotoxic lymphocyte precursors were shown to be Lyt-123 and these could be depleted from sensitized Lyt-1 populations that mediated graft rejection. Thus cytotoxic T cells are not responsible for skin graft rejection, but rather, this is mediated by an Lyt-1 cell. Whether this T cell is distinct from other Lyt-1 cells (T helper, T cells mediating delayed hypersensitivity) is not clear at present, but other evidence, and traditional concepts, link graft rejection and delayed type hypersensitivity as being different manifestations of the same mechanism.

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PubMed1981

Growth-regulatory control of human cell hybrids in nude mice.

The role of natural killer (NK) cells in the control of growth of human cell hybrids in nude mice was evaluated. Both nontumorigenic and tumorigenic HeLa-fibroblast hybrids were highly sensitive to NK-mediated cytotoxicity, but neither hybrid induced such activity when injected into nude mice. Furthermore, tumorigenic hybrids grew in mice which had high levels of NK activity induced by i.p. inoculation of Corynebacterium parvum vaccine. Histological examination of the nontumorigenic and tumorigenic populations inoculated s.c. into nude mice indicated that both populations initially divide actively for the first 3 to 4 days. After this time, the nontumorigenic cells showed a dramatic decline in mitotic activity accompanied by a morphological shift to a more fibroblastoid appearance. The cells remained in the animal as a viable nondividing tissue. The tumorigenic population continued to actively divided and produced a large progressively growing tumor. This series of events determined from histological examination was supported by kinetic studies. These results suggest that NK cells play no role in the suppression of growth of the nontumorigenic hybrid cells and that host-mediated growth-regulatory control is responsible for the shutdown of mitotic activity of these cells without causing their death.

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PubMed1980

Frequencies of alloreactive cytotoxic T-lymphocyte precursors responding to H-2 antigens alone.

The experiments reported here were designed to investigate the role of major histocompatibility antigens alone in primary allogeneic responses in vitro. Mixed lymphocyte cultures were established using small numbers of responder spleen cells and semiallogeneic or fully allogeneic stimulator spleen cells. The cytotoxicity generated in individual cultures was then detected by a sensitive 51Cr-release assay and the frequencies of responding cytotoxic T lymphocyte precursors (CTL-P) determined by limit dilution analysis. To investigate the responses of spleen cells to major histocompatibility (H-2) antigens alone, strains of BALB/c mice congenic at the H-2 locus were used as responder and stimulator cells. The frequencies of CTL-P responding in such congenic combinations were compared with those obtained using mouse strain combinations differing at minor histocompatibility (minor-H) loci in addition to H-2. A similar fraction of spleen cells responded regardless of whether the stimulator cells differed at the H-2 locus alone or at multiple minor-H loci in addition to H-2. It was concluded that minor-H antigenic differences do not contribute significantly to allogeneic responses.

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PubMed1979

Murine cutaneous leishmaniasis: disease patterns in intact and nude mice of various genotypes and examination of some differences between normal and infected macrophages.

The course of the disease, cutaneous leishmaniasis, caused by the intracellular protozoan parasite Leishmania tropica, differs markedly amongst various common inbred mouse strains. After intradermal injection of 1 x 10(6) promastigotes to young female specific pathogen-free (SPF) derived mice, persistent infection characterized by an expanding ulcerous lesion is seen in BALB/c and DBA/2 mice. In the strains CBA/H, C3H/He and A/J, lesions resolve within 8 weeks, and in C57B1/6 mice no real lesion typical of cutaneous leishmaniasis develops at the injection site. NZB mice are highly resistant. Macrophages harvested from the thioglycollate-stimulated peritoneal cavity of NZB and C57B1/6 mice appear to differ from macrophages of the other mouse strains in not supporting multiplication of L. tropica organisms in vitro. Nevertheless, hypothymic nude (nu/nu) mice of C57B1/6 genotype, as well as CBA/H-nu/nu and BALB/c-nu/nu mice, develop large lesions with metastases to other cutaneous and visceral locations. In the intact mice in which infection resolves spontaneously, resistance to reinfection is complete. Using mouse antipromastigote sera and an indirect fluorescent antibody test in carefully controlled experiments, L. tropica antigens were detected on in vitro infected macrophages of both highly susceptible BALB/c and relatively resistant CBA/H genotypes. After incubation with a crude soluble antigen preparation from cultured promastigotes, infected BALB/c macrophages differed from infected CBA/H macrophages (and uninfected macrophages of both genotypes) in being unable to sensitize syngeneic recipients for a delayed-type hypersensitivity response to that antigen. When infected and uninfected macrophages were used as "blocking cells" in an in vitro alloreactive cytotoxic T cell system involving cells from congenic mice, evidence was obtained for reduced H-2d expression on infected macrophages of the susceptible mouse strains, BALB/c. The data in this model system of cutaneous leishmaniasis raise the possibility that genetic susceptibility is associated with both a permissive macrophage and defective T cell recognition of parasite antigens on infected macrophages. Defective recognition may be the result of reduced functional expression of H-2d antigens on infected BALB/c macrophages required for efficient recognition by syngeneic T cells of one or more subpopulations.

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