Biomedical subjects

R Ceredig

Publications and source records attributed to R Ceredig.

At least 19 recordsLinked to original sources

PubMed1996

Block of T lymphocyte differentiation by activation of the cAMP-dependent signal transduction pathway.

Differentiation of T lymphocytes is a complex and finely tuned process. Here we show that treatment of mouse fetal thymus organ cultures with agents activating the cAMP-dependent signalling pathway results in the block of thymocyte differentiation. This is due to severe impairment of maturation beyond the CD4-/CD8- stage. In addition, rearrangements at the TCR alpha gene locus, but not at the TCR beta locus, are completely inhibited. The cAMP effect is reversible and is restricted to TCR alpha beta+ cells. cAMP acts both by triggering apoptosis and by inducing cell-cycle block in thymocytes. Thus, activation of the cAMP pathway provides a mechanism to modulate thymic function for hormones and ligands whose receptors are coupled to adenylate cyclase.

Animals↗

PubMed1996

Phenotypic and functional analysis of B lymphopoiesis in interleukin-7-transgenic mice: expansion of pro/pre-B cell number and persistence of B lymphocyte development in lymph nodes and spleen.

Transgenic mice in which mouse interleukin (IL)-7 cDNA is expressed under the control of the mouse major histocompatibility complex (MHC) class II (E alpha) promoter develop a lymphoproliferative disease characterized by the early polyclonal expansion of T cells followed in many cases by the development of lymphomas of immature B cells. Here, we have analyzed B cell development in these transgenic mice. Phenotypic analysis using monoclonal antibodies to B220, IgM, IgD, c-kit, IL-7 receptor, MHC class II, AA4.1, CD19, CD23, CD25, CD40 and CD43 shows that B lymphopoiesis in the bone marrow is dramatically altered and the number of pro/pre-B and immature B cells is significantly increased. Interestingly, pro/pre-B and immature B cells persist in the spleens of adult transgenic mice and are also present in lymph nodes and blood. Cell cycle analysis of lymph node cells shows that subpopulations of developing B cells retain the cell cycle profiles of their bone marrow counterparts. Limiting dilution analysis shows that the number of clonable pre-B cells is significantly increased and that at limiting dilution, growth of transgenic pre-B cells is still dependent on exogenous IL-7. Using semiquantitative polymerase chain reaction (PCR) and in situ hybridization, the level of IL-7 transcripts in the spleen was found to decrease between 2 and 4 weeks in control mice with levels in transgenics mice being approximately 50 times greater. These transgenic mice represent an interesting model with which to study the effects of IL-7 overexpression in the bone marrow and raise interesting questions regarding the regulation of B lymphopoiesis in normal mice.

Animals↗

PubMed1996

T cell receptor-gamma, delta-expressing fetal mouse thymocytes are generated without T cell receptor V beta selection.

We investigated whether fetal mouse T cell receptor (TCR) gamma delta cells have been subjected to so-called TCR beta selection at the CD25 stage of thymus development. To this end, we carried out a comparative three-color flow microfluorimetric analysis to TCR gamma delta cells developing in the fetal, neonatal and adult thymus using monoclonal antibodies to CD2, CD8, CD24, CD25 and CD44. Day-15 fetal TCR gamma delta cells were CD2+ suggesting an origin at a post-CD25 stage. Molecular analysis of TCR beta rearrangements were also carried out. Thus, by semi-quantitative polymerase chain reaction (PCR) amplification of V beta 6 and V beta 8 to J beta 2 rearrangements day-15 fetal TCR gamma delta showed extensive TCR beta rearrangements, a finding confirmed by PCR amplification from single micromanipulated cells. Finally, sequencing analysis of 104 PCR-amplified TCR VDJ beta 2 fragments showed that the majority (58%) were rearranged out of frame . Taken together, these phenotypic and molecular analyses suggest that fetal TCR gamma delta cells have not been subject to TCR beta selection.

Animals↗

PubMed1995

Quantitative and qualitative changes in CD44 and MEL-14 expression by T cells in C57BL/6 mice during aging.

Aging is associated with a decrease in the functional activity of T cells. We have explored age-related alterations in CD44 and MEL-14 expression by spleen cells bearing the Thy1.2, CD4 or CD8 antigens in C57BL/6 mice at 2, 8, 15 and 23 months of age. The membrane expression of CD44 and MEL-14 molecules can be used to distinguish naive (CD44low, MEL-14high) from preactivated/memory (CD44high, MEL-14low) T cells. Our results show that the proportion of CD4+ splenic cells begins to decrease at an intermediate age (8-month-old mice), whereas the proportion of CD8+ cells remains unaltered. The proportion of CD4+ and CD8+ splenic cells with the CD44high memory phenotype was increased at an early stage of aging (in 8-month-old mice) without a concomitant change in MEL-14 expression. In older mice, MEL-14 expression decreased on CD4+ but not on CD8+ subsets. Recent studies have reported that following activation, the expression of CD44 molecules containing additional, so-called variable exons can be detected. By PCR, we observed an increase in CD44 transcripts containing the v6 or v7 variable exons in murine lymph nodes at the age of 15 months. Our results suggest that v6- or v7-containing variants of CD44 may be involved in the development of memory cells. Taken together, these results suggest that the trafficking of memory T cells in aging may be altered by quantitative and/or qualitative differences in the expression of molecules involved in lymphocyte recirculation.

Aging↗

PubMed1995

IL-7 transgenic mice: analysis of the role of IL-7 in the differentiation of thymocytes in vivo and in vitro.

We have generated a high copy number transgenic mouse line in which expression of mouse IL-7 cDNA is under the control of the mouse MHC class II E alpha promoter. These mice were generated in order to see if IL-7 over-production in the thymus altered either thymocyte differentiation or the process of negative selection. Using in situ hybridization, IL-7 transcripts could be detected in the thymic cortex and medulla as well as the spleen and lymph nodes of transgenic mice but was undetectable in normal controls. Phenotypic and molecular analysis of thymocytes from embryonic and adult transgenic mice failed to reveal a dramatic effect of IL-7 on thymocyte differentiation and negative selection of the TCR V beta repertoire appeared to be intact. In peripheral lymph nodes, there was a massive (30-fold) increase in the number of T cells (CD8+ > CD4+) and simultaneous presence of immature (B220+, Ig-) B cells. TCR repertoire analysis showed that the expansion of peripheral T cells was polyclonal. Using the polymerase chain reaction (PCR), transgene-specific IL-7 transcripts could be detected in the thymus from day 14 of fetal development. However, using semi-quantitative PCR, there was no dramatic increase in the degree of TCR beta or TCR alpha gene rearrangements during thymocyte ontogeny in vivo. Similarly, when fetal mouse thymus lobes were cultured with IL-7 in vitro, there was no dramatic increase in the degree of TCR beta or TCR alpha gene rearrangements. We conclude that IL-7 is probably not an important differentiation factor for immature mouse thymocytes.

Age Factors↗

PubMed1995

Lymphoproliferative disorders in IL-7 transgenic mice: expansion of immature B cells which retain macrophage potential.

Transgenic mice carrying the murine IL-7 gene under the MHC class II (E alpha) promoter are described which develop lymphoid tumours at a high incidence when maintained in conventional or specific pathogen-free environments. Cells obtained from the lesions were relatively monomorphic, expressed a variety of B cell associated markers (BP-1, B220, CD43) but lacked surface Ig. Some mice, showed expanded populations of cells phenotypically similar to the recently reported bipotent B/macrophage stem cell subset (AA4.1high, B220-, Ig-) which could be cloned and maintained in vitro. These cells expressed IL-7 receptors, proliferated in response to IL-7 and in most cases had germline configuration of the Ig heavy chain locus. Cell lines cloned from two such tumours generated macrophages spontaneously in culture, consistent with their bipotent B cell/macrophage phenotype. These results suggest that IL-7 plays a role in very early stages of B cell ontogeny prior to bona fide B cell commitment.

Animals↗

PubMed1994

Haemopoiesis and early T-cell differentiation.

Lineage commitment is a fundamental process that is initiated during the early stages of embryogenesis. Ultimately, this leads to the generation of the humoral and cellular arms of the immune system. In a recent workshop, researchers from the diverse fields of embryology, haematology and immunology gathered to address the topic of early T-cell differentiation.

Animals↗

PubMed1994

CD44 in differentiated embryonic stem cells: surface expression and transcripts encoding multiple variants.

Expression of the surface-adhesion molecule CD44 was investigated during the in vitro differentiation of the embryonic stem (ES) cell line D3. By immunofluorescence analysis, totipotent, undifferentiated ES cells did not show surface expression of CD44, although two transcripts of approximately 1.6 and 3.3 kb were detected on Northern blots. Following 1 week of differentiation in either suspension or substrate-attached cultures, CD44 appeared on the surface of some D3 cells, and synthesis of an additional 4.5 kb mRNA species was detected on Northern blots. At this stage, at least three distinct transcripts encoding CD44 variants were induced within the cultures, resulting from alternative splicing of additional exons in the variable domains of CD44. From PCR analysis, they all appeared to contain the variable exon v10, and two of them in addition contained v6. Taken together, these results suggest that CD44 may play a role in cell migration and adhesion in the early development of the mouse embryo.

Alternative Splicing↗

PubMed1994

Influence of the lpr environment on the lymph node cell phenotypes in C57BL/6 nubg and nulpr chimeras.

Mice homozygous for the lpr gene show a marked lymphoproliferative syndrome. Most T cells which accumulate in their lymphoid organs belong to a fairly unusual subpopulation. Although being CD44+ T cells expressing neither CD4 nor CD8, they are CD3 T-cell receptor (TCR) alpha beta positive and express both Thy-1 and B220, the B-cell form of the CD45 marker. To support engraftment and development of transferred lpr lymphomyeloid cells, athymic recipients must be genetically lpr. While nude/beige (nubg) recipients do not allow the development of any lymphoproliferative syndrome, this is variable in nude/lpr (nulpr) recipients, and the genotypic origin of the proliferating lymphocytes in nulpr recipients is unclear. In this study, the surface phenotype of lymph node cells from nulpr recipients of lpr grafts ([lpr-->nulpr] chimeras) was analysed by flow cytometry, and compared with various chimeras and parental (donor and recipient) strains as controls. Abnormal cells of the lpr type were not detectable either in [lpr-->nubg] chimeras or in [wild-->nubg] controls. Absence of lpr cells was also seen in neonatal lpr thymus-grafted nubg mice engrafted previously with lpr haematopoietic cells. In contrast, a substantial emergence of double-positive B220+ Thy-1+ cells occurred in [lpr-->nulpr] chimeras, together with high levels of CD4+ cells, a substantial fraction of which might express B220. Finally, in thymus-grafted nulpr mice, the levels of B220+ Thy-1+ cells were as high as in lpr mice and there was again an expansion of CD4+ (potentially B220+) cells. Abnormality of the nulpr haemopoietic environment was also shown by the low percentages of T cells, particularly CD8+ cells, in short-lived [wild-->nulpr] chimeras. Taken together, our results underline the differences between the nubg and nulpr environments.

Animals↗

PubMed1993

Antioxidant treatment of thymic organ cultures decreases NF-kappa B and TCF1(alpha) transcription factor activities and inhibits alpha beta T cell development.

Using electrophoretic mobility shift assays (EMSA), we have recently shown that nuclear extracts of 14-day mouse fetal thymocytes contain abundant NF-kappa B transcription factor activity. To determine the functional role of NF-kappa B in early thymocyte development, we have exposed fetal thymus organ cultures to inhibitors of NF-kappa B activation, namely the antioxidants N-acetyl-L-cysteine and butylated hydroxyanisole. Both compounds caused a dose-dependent arrest of thymocyte differentiation toward alpha beta, but not gamma delta, T cells. This was associated with a profound decrease in nuclear content of NF-kappa B and TCF1(alpha) transcription factor activity, as determined by EMSA. In contrast, NF-Y was affected less strongly, and cyclic AMP-response-element-binding protein levels remained essentially unchanged by antioxidants. To test the idea that alpha beta T cell development is correlated with NF-kappa B and TCF1(alpha) activity, we conducted additional experiments in a submersion culture system in which the generation of alpha beta T cells can be manipulated. Standard submersion culture supports gamma delta but alpha beta T cell development. Under these conditions, EMSA showed that transcription factor activities were similar to those seen in the presence of antioxidants. Importantly, when the generation of alpha beta T cells in submersion culture was restored by elevating oxygen concentrations, there was a dramatic increase in TCF1(alpha) activity, and both NF-kappa B and NF-Y returned to control levels. Taken together, these results strongly suggest that NF-kappa B and TCF1(alpha), presumably in concert with other transcription factors, play an important role in the development of alpha beta T cells.

Acetylcysteine↗

PubMed1993

Analysis of intraepithelial lymphocytes from major histocompatibility complex (MHC)-deficient mice: no evidence for a role of MHC class II antigens in the positive selection of V delta 4+ gamma delta T cells.

Three-color flow cytometric analysis was carried out with intraepithelial lymphocytes from mice deficient in expression of major histocompatibility complex (MHC) antigens. These experiments were done to address the possible role of MHC class II molecules in the positive selection of V delta 4+ gamma delta T cells. By analyzing mice deficient MHC class II antigens alone or in combination with MHC class I antigens, no evidence was found for positive selection of V delta 4+ cells among CD8 alpha + or CD4-CD8- subpopulations of gamma delta T cell receptor-positive cells. Because V delta 4+, CD8 alpha + cells were reported to be positively selected on I-Ek and hybrid I-Ek/b molecules, class II-deficient animals were crossed with I-Ek transgenic mice and progeny examined for V delta 4 expression. Again, no evidence for positive selection was found. Interestingly, in MHC class I-deficient animals, the total number of gamma delta T cells was about twofold higher than in control and MHC class II-deficient mice and the proportion of V delta 4-expressing cells was correspondingly decreased. Taken together, these results cast doubt on a major role for conventional MHC antigens in shaping the gamma delta T cell repertoire of intraepithelial lymphocytes.

Animals↗

PubMed1993

Activated mouse astrocytes and T cells express similar CD44 variants. Role of CD44 in astrocyte/T cell binding.

The CD44 adhesion molecule is expressed by astrocytes, glial-type cells which exhibit features of accessory cells for immune responses in the central nervous system. In primary cultures of mouse astrocytes, we have observed that surface expression and mRNA levels of CD44 are induced following stimulation with either PMA, or tumor necrosis factor alpha plus gamma interferon. Comparison of CD44 splice variants expressed by astrocytes and a T cell hybridoma shows that upon activation, both cell types express a similar pattern of CD44 transcripts. Thus, in both cell types, CD44 transcripts are produced which contain additional exons, including the exon v6 (known to be expressed by in vivo activated lymphocytes and by metastatic variants of tumor cells) as well as variants of larger size. In the autoimmune disease multiple sclerosis, activated T cells cross the blood-brain barrier and lead to inflammation in the central nervous system. Analysis of mice with experimental allergic encephalomyelitis, frequently used as an animal model of multiple sclerosis, shows that CD44 is induced in vivo on glial cells surrounding inflammatory lesions. Using an in vitro model for adhesion between T cells and astrocytes, we have found a correlation between the activation state of these cells and their adhesion potential. Dose-dependent inhibition of adhesion by hyaluronate and by anti-CD44 monoclonal antibody KM81 shows that CD44 is involved in the adhesive interactions between T cells and astrocytes.

Animals↗

PubMed1993

Lymphoproliferative disorders in an IL-7 transgenic mouse line.

A high incidence of severe lymphoproliferative disease was observed in a newly generated strain of mice carrying murine IL-7 as a transgene under the control of the E alpha (MHC class II) promoter. An analysis of the cells from lesions in these mice shows the selective expansion of cells at an early stage of B cell development and, more interestingly, expansion of cells phenotypically identical to the recently reported bipotent (B/macrophage) stem cell populations described in midgestation embryonic liver. Such cells can be propagated (and remain dependent upon) bone marrow feeder cell lines obtained from IL-7 transgenic mice. A molecular analysis of fresh and cultured cells reveals that the lesions are oligoclonal, or in rare cases monoclonal, and include clones of cells with unrearranged Ig heavy chain loci. These data suggest that IL-7 acts at multiple stages of B cell development. Furthermore cell lines derived from IL-7 transgenic mice may provide a novel source of rare factor-dependent bipotent stem cells.

Animals↗

PubMed1992

Selection of murine T cell receptor alpha beta and gamma delta cells in organ cultures established from 14-day embryos.

The expression of minor lymphocyte stimulatory locus (Mls) determinants in combination with murine major histocompatibility complex (MHC) class II molecules, leads to the destruction of lymphocytes bearing specific V region-encoded T cell receptor (TcR) products. A much studied example is the elimination of V beta 6+ cells in IE+/Mls-1a mice, in which deletion can be detected 7-10 days after birth but is not fully operational earlier in embryonic life. Here we investigate this transitional period in development and show that selective deletion of V beta 6 occurs in vitro, approximately 1 week after organ cultures are established from 14 day embryos. These unmanipulated organ cultures receive no additional cell immigrants after day 14, suggesting that the cellular elements mediating negative selection (or their direct precursors), are already resident in the fetal thymus by day 14 of gestation. Hence, the developmental timing of the outset of rigorous negative selection of V beta 6 is not dictated by the postnatal entry of deleting elements into the thymus, but perhaps by the maturation of the pre-existing environment. Using a parallel organ-culture approach we have looked at the development of V delta 4 and V gamma 3, TcR gamma delta+ cells in a variety of mouse strains. These receptors have recently been reported to be subject of MHC and non-MHC linked selection, respectively. We find that after an initial period of expansion, the number of V gamma 3-expressing cells dramatically declines. However, this selective loss of V gamma 3 cells is not contingent on the C57BL/6 mouse strain (in contrast to a previous report). These findings are discussed in the context of current models of ontogeny and repertoire selection.

Animals↗

PubMed1992

Transcription factors in mouse fetal thymus development.

T cell precursors from murine fetal liver enter the fetal thymus where they proliferate, differentiate, and mature. These processes are accompanied by changes in the pattern of transcription factors known to control the expression of specific genes. We have monitored the expression of five different transcription factors during mouse fetal thymus ontogeny: nuclear factor (NF)-kappa B, cAMP-response-element binding protein (CREB), NF-IL-2A, msNF-AT1, and hNF-AT1. NF-kappa B binding activity was not detected in extracts from fetal liver but was present in the thymus at day 14 of embryogenesis. Thereafter, NF-kappa B expression was biphasic, being maximal at 14-16 days gestation and in newborn mice, and decreased during the intermediate gestational stages and in the adult. An inverse correlation was observed between NF-kappa B binding activity in the nuclei and levels of its inactive precursor in the cytoplasm of all samples analyzed. In contrast, CREB activity was uniform throughout thymus development. Similarly, NF-IL-2A activity was detected in fetal liver and thymic extracts from different gestational stages, in approximately equivalent amounts. However, band shift experiments revealed three distinct NF-IL-2A-DNA complexes, whose relative abundance is altered during thymic ontogeny. Likewise, NF-AT1 transcription factor appears to be heterogeneous and includes representatives which are differentially (msNF-AT1) or stably (hNF-AT1) expressed during thymic development. These results are discussed in the context of present knowledge about T cell development within the thymus.

Animals↗

PubMed1992

T cell receptor repertoire of gamma delta cells generated from the 14-day embryonic mouse thymus.

Whole, undisrupted 14 day mouse fetal thymus lobes cultured in the presence of 10 U/ml IL-2 generate a heterogeneous population of gamma delta-expressing T cells. Phenotypic analysis has shown that the majority of gamma delta T cells in such cultures stain with the anti-V gamma 3-specific mAb 536. To investigate the V gamma T-cell receptor diversity of cultured fetal thymocytes, cDNA was prepared and amplified using the polymerase chain reaction. The DNA fragments obtained were subsequently cloned and sequenced and compared with those obtained from fresh and organ-cultured 14 day fetal thymus lobes. Results obtained tend to support a positive selection model of gamma delta T cell differentiation.

Animals↗

PubMed1991

Transcripts encoding mouse CD44 (Pgp-1, Ly-24) antigen: strain variation and induction by mitogen.

The cell surface molecule CD44, thought to be a cell surface receptor for hyaluronic acid, is expressed by thymocytes and peripheral T cells. Following T cell activation with mitogens or antigens, surface expression of CD44 increases. This increase in CD44 cell surface expression has been used as a means of identifying memory T cells in vivo. In this report, using Northern analysis, we have quantitated mouse CD44 transcripts in thymocytes and a cloned T hybridoma cell line. In contrast to a recent report by Nottenburg et al. (Proc. Natl. Acad. Sci. USA 1989. 86: 8521), we show that in all instances CD44 transcripts are polyadenylated. In all cells studied, three major mRNA species of approximately 4.5, 3.5 and 1.6 kb are detected. After stimulation with phorbol 12-myristate 13-acetate (PMA), there is a de novo protein synthesis-dependent increase in the level of CD44 transcripts. The increase in CD44 transcripts precedes the increase in cell surface expression. In contrast to CD44, PMA stimulation results in a decrease in the level of Thy-1 transcripts. Thymocytes from different mouse strains vary in their level of CD44 cell surface expression and Northern analysis indicates that this mouse strain variation correlates with a corresponding difference in the level of CD44 transcripts.

Animals↗

PubMed1991

Culture conditions dictate whether mouse fetal thymus lobes generate predominantly gamma/delta or alpha/beta T cells.

Thymus lobes from 14 day-old mouse embryos cultured submerged in r-IL-2 generated a mixture of CD8 alpha+/CD4- and CD8-/CD4- gamma delta TcR expressing cells (Ceredig et. al. 1989). Based upon Northern analysis with TcR constant region probes, no alpha beta T cells could be identified in these cultures. Submerged lobes also showed responsiveness to IL-7. In contrast, when cultured at an air liquid interface as organ cultures (OC), most cells appeared to express alpha beta TcR (Ceredig 1988). Thus depending on the mode of culture, fetal thymus lobes generate predominantly gamma delta or alpha beta T cells; it is unclear how this difference is regulated. Previous phenotypic and functional experiments suggested that gamma delta T cells may be present in OC. In order to study gamma delta T cells in both submerged lobe and OC, we have carried out three colour flow microfluorimetric analysis of gamma delta TcR, abTcR, CD3, J11d and CD8 beta expression by subpopulations of CD8 alpha and CD4 defined thymocytes. In addition, using V gamma-specific oligonucleotides and the polymerase chain reaction, we have begun identifying and sequencing the V gamma repertoire of gamma delta T cells in these mouse fetal thymus cultures.

Animals↗