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P Hugo

Publications and source records attributed to P Hugo.

At least 19 recordsLinked to original sources

Thymic epithelial cell lines that mediate positive selection can also induce thymocyte clonal deletion.

Negative selection of potentially autoreactive thymocytes occurs mainly in the thymus and is thought to be induced primarily by interaction with bone marrow-derived cells. However, some studies have also reported a role for radioresistant thymic cells, which are probably epithelial in origin, in the deletion of thymocytes reacting to endogenous superantigens. We have previously demonstrated that thymic epithelial cell lines could induce thymocyte-positive selection in vivo. In this study, we assessed the potential of these cells to delete thymocytes reacting to the staphylococcal enterotoxin A or B superantigens in vitro. In the presence of staphylococcal enterotoxin A or B we found that all thymic epithelial cell lines used in this study were capable of activating T cell hybrids or deleting CD4+CD8+ thymocytes expressing an appropriate TCR. The extent of superantigen-mediated thymocyte deletion mediated by thymic epithelial cell lines was comparable to that mediated by a thymic macrophage cell line. Similar results were obtained with three phenotypically distinct thymic cell lines, suggesting that the ability to induce thymocyte deletion might be a general feature of various subsets of thymic epithelium. The observations provided in this study, combined with our previous demonstration that the same thymic epithelial cell lines can participate in positive selection, suggest that a given stromal cell population might be capable of taking part both in positive and negative selection of thymocytes.

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Iron overload in beta 2-microglobulin-deficient mice.

The present paper describes the results of a comparative histological and quantitative analysis of iron distribution in tissues of beta 2m-/- and beta 2m+/- mice of different ages. Progressive hepatic iron overload, indistinguishable from that observed in human hemochromatosis, was found only in mice homozygous for the mutated beta 2m gene. Total iron measurements done by flame atomic absorption showed statistically significant differences between liver samples from 5 beta 2m+/- heterozygotes (468 +/- 174 micrograms/g of dry weight) and 9 mice homozygous for the mutated beta 2m gene with average total hepatic iron levels of 1583 +/- 423 micrograms/g of dry weight.

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Fibroblasts can induce thymocyte positive selection in vivo.

During development in the thymus, thymocytes bearing alpha beta T-cell receptors are selected to mature if the receptors they bear are able to interact in some way with major histocompatibility complex (MHC) proteins expressed on thymic stromal cells. It has been shown that thymus cortical epithelial cells are usually the cells presenting the MHC molecules involved in this process of so-called positive selection. Here we tested the ability of fibroblasts to mediate positive selection in vivo. Fibroblasts transfected with the genes for the MHC I-Ab proteins were injected intrathymically into irradiated H-2k animals reconstituted with H-2bxk F1 fetal liver cells. Eight weeks later, the recipient mice were immunized and shown to contain peptide-specific I-Ab-restricted T cells. This demonstrates the ability of I-Ab-transfected fibroblasts to participate in positive selection. Thus a cell type that is not specialized to process and present antigens in the context of MHC class II molecules can mediate positive selection when transfected with an appropriate MHC molecule. The data also support the idea that the ability to mediate positive selection may not be limited to thymic cortical epithelium.

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CD4+8- and CD4-8+ mature thymocytes require different post-selection processing for final development.

Two primary types of TCR-alpha/beta+ T cells are found in the peripheral lymphoid system; CD4+8- T cells, with MHC-class II restricted TCR, and CD4-8+ T cells, which are MHC-class I restricted. Both lineages develop in the thymus from a series of common precursors. However, the precise stage at which they diverge, and the combination of factors that regulates such divergence, are not well defined. The up-regulation of CD3/TCR to high mature levels is thought to be an early event associated with positive selection for self-MHC recognition. Using purified cells from bcl-2 transgenic mice in order to overcome the limitations imposed by cell death on normal thymocytes, we find that a minor subset of CD4+8+ thymocytes expressing high levels of CD3/TCR gives rise to both CD4+8- and CD4-8+ mature cells upon intrathymic transplantation, but only to CD4-8+ in culture. Thus, in addition to demonstrating the dual lineage potential of this subset, these findings show that additional post-selection processing events are required for the production of mature thymocytes, and that CD4+8- and CD4-8+ subsets differ in the types of processing required.

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A cell line that can induce thymocyte positive selection.

The thymus positively selects thymocytes that bear T-cell receptors which recognize antigen presented by self major histocompatibility complex (MHC) proteins. Positive selection is usually driven by MHC products on radiation-resistant cortical epithelial cells. It is unknown whether positive selection is mediated by all thymic epithelial cells or by some specialized subsets. Here we introduce an H-2b-expressing thymic epithelial cell line into the thymuses of lethally irradiated H-2k animals reconstituted with H-2b/k F1 BM or fetal liver cells. I-Ab-restricted T cells are found in these animals, demonstrating that selection occurs on the introduced epithelial cells.

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Treatment of fetal thymic organ culture with IL-1 leads to accelerated differentiation of subsets of CD4-CD8- cells.

Using fetal thymic organ culture (FTOC), we describe the effects of IL-1 on T cell differentiation, particularly within the CD4-CD8- subset. While treatment of FTOC with IL-1 led to a modest reduction in total thymocyte yield, it induced an increase in the percentage of CD4-CD8- cells that express IL-2R early in culture and a decrease in the number of their precursors (CD44+IL-2R- cells). The increase in the percentage of cells expressing IL-2R was not accompanied by an increase in the number of these cells. At later time points these IL-2R+ cells (and their precursors) were reduced relative to controls. The total number of CD4-CD8-CD3- precursor cells in IL-1-treated cultures was reduced to approximately half that in controls at Day 12 of culture. However, only minor inhibition of total cell number was observed, which, taken together with the greater frequency of IL-2R+ precursors, suggests that this depletion of the pool of precursors may have been due to the induction of premature differentiation rather than to its inhibition.

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Thymic shared antigen-1. A novel thymocyte marker discriminating immature from mature thymocyte subsets.

In a previous study, we raised a mAb (MTS 35) reacting with a plasma membrane Ag expressed on both cortical thymocytes and a subset of thymic medullary epithelial cells. In view of the shared expression of this molecule, we have defined it as thymic shared Ag-1 (TSA-1). Considering its selective reactivity with cortical, but not medullary thymocytes, the relevance of TSA-1 as a marker of immature T cells was investigated in detail in this study, using multicolor flow cytometric analysis. TSA-1 was found on all immature thymocyte subsets (CD3-4-8-, CD3-4+8-, CD3-4-8+, CD3-4+8+, CD3low4+8+). Conversely, CD3high4+8- and CD3high4-8+ thymocytes, early thymic migrants and peripheral T cells were TSA-1-. More refined gating and analysis of the transitional CD3intermediate/high4+8+ thymocytes, proposed candidates for negative selection, demonstrated that approximately one half were TSA-1-. In fact, there was a directly inverse relationship between TSA-1 and CD3 expression on thymocytes. In the periphery, TSA-1 was detected on B lymphocytes. TSA-1 is PI-linked and has a molecular mass of 17 kDa nonreduced, or 12 to 13 kDa reduced. Through cross-correlation analysis, this molecule was distinct from H-2K, PNA-R, CD5, CD11a/18, Thy-1, HSA, Ly6A/E, Ly6C, ThB, CD25, CD44. Hence TSA-1 appears to be a unique marker which exquisitely separates mature from immature thymocytes.

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Characterization of immature CD4+CD8-CD3- thymocytes.

Previously we have described (Hugo, P. et al., Int. Immunol. 1990. 2: 209) an immature CD4+CD8-CD3- thymocyte subset which is thought to be the counterpart of the CD4-CD8+CD3- subset. In this study we show that the ontogeny of these two subsets is parallel in fetal thymic organ culture. Extensive phenotypic characterization of CD4+CD8-CD3- cells reveals that they closely resemble CD4-CD8+CD3- thymocytes being: HSAhigh, Thy-1high, interleukin 2 receptor alpha chain negative, CD44-, H-2K+/-, CD5low, MEL-14low/intermediate, CD2+, LFA-1+ and MTS 35+. Finally, we show that the proportion of CD4+CD8-CD3- thymocytes is highly variable between mouse strains.

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CD4+CD8+CD3high thymocytes appear transiently during ontogeny: evidence from phenotypic and functional studies.

During T cell development thymocyte subsets emerge in a defined order, reflective of their maturational stage. In this study we determined the timing of appearance of CD4+CD8+CD3high thymocytes during in vivo and in vitro embryonic development, and thymic reconstitution after cortisone treatment. In these models, CD4+CD8+CD3high cells followed CD4+CD8+CD3low and preceded mature CD4+CD8-CD3high/CD4-CD8+CD3high thymocytes, while cortisone resistance was first seen among CD4+CD8+CD3high cells. CD4+CD8+CD3high thymocytes were also shown to display a pattern of antigen receptor-mediated calcium influx intermediate between that induced in other CD4+CD8+ cells and mature thymocytes. These results are consistent with a precursor-progeny relationship between CD4+CD8+CD3low and CD4+CD8+CD3high thymocytes, the latter developing to mature thymocytes (Hugo, P. et al., Int. Immunol. 1991. 3: 265).

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Towards an integrated view of thymopoiesis.

One prediction from the complex series of steps in intrathymic T-cell differentiation is that to regulate it the stroma controlling the process must be equally complex: the attraction of precursors, commitment to the T-cell lineage, induction of T-cell receptor (TCR) gene rearrangement, accessory molecule expression, repertoire expansion, major histocompatibility complex (MHC) molecule-based selection (positive and negative), acquisition of functional maturity and migratory capacity must all be controlled. In this review, Richard Boyd and Patrice Hugo combine knowledge of T-cell differentiation with thymic stromal cell heterogeneity to offer an integrated view of thymopoiesis within the thymic microenvironment.

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Timing of deletion of autoreactive V beta 6+ cells and down-modulation of either CD4 or CD8 on phenotypically distinct CD4+8+ subsets of thymocytes expressing intermediate or high levels of T cell receptor.

In this paper we describe a differentiation sequence amongst adult murine thymocytes which goes from CD4+8+3lo(low) to CD4+8+3int(intermediate) to CD4+8+3hi(high) and then to mature single positive CD3hi thymocytes. Phenotypic characterization of CD4+8+3int/hi cells for a number of other surface markers is consistent with them being in transition from CD4+8+3lo phenotype to mature phenotype. The same observation was made for sensitivity towards ionomycin-mediated apoptosis. In the thymus of Mls-1a mice, where autoreactive TCR-V beta 6+ cells are negatively selected, deletion of TCR-V beta 6+ cells was first detected in the CD4+8+3int subset, and was complete by the CD4+8+3hi stage, suggesting that up-regulation of the TCR/CD3 complex is required for deletion of Mls-1a autoreactive thymocytes. No sign of apoptosis was detected among any fresh thymocyte subsets suggesting that apoptotic cells are rapidly cleared from the thymus. The CD4+8+3int/CD4+8+3hi cells are therefore populations in transit from the typical cortical thymocytes to the mature T-cells.

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Lineage relationships and developmental kinetics of immature thymocytes: CD3, CD4, and CD8 acquisition in vivo and in vitro.

T lymphocytes develop in the thymus from immunologically naive bone marrow precursors. Based on T cell receptor rearrangement and transcription, and thymic reconstitution potential, we have deduced a developmental sequence among immature thymocytes, before the acquisition of the lineage markers CD3, CD4, and CD8. In the current study, we have followed the ontogenic progression of the latter stages in this sequence, using two different systems: (a) in vivo, by direct injection into the thymus of nonirradiated, congenic recipients; and (b) in vitro, using culture medium without mitogens or cytokines. In vivo, the less mature Pgp-1- interleukin 2 receptor alpha-positive (IL-2R alpha+) CD3-4-8- subset (also heat-stable antigen high) requires 3 d before becoming predominantly IL-2R alpha- CD3lo4+ 8+ typical cortical-type cells, and at least 5 d before the appearance of any mature single-positive cells (CD3hi4+ 8- or CD3hi4-8+). However, these Pgp-1- IL-2R alpha+ precursors do not differentiate further in unstimulated culture. The more mature Pgp-1- IL-2R alpha- CD3-4-8- subset becomes primarily CD3lo4+ 8+ within 1 d after transplantation, and some mature single-positive progeny are evident by day 3. By 5 d, most of these Pgp-1-IL-2R alpha- precursor cells have become CD3hi, and have lost or are downregulating either CD4 or CD8. In culture, these Pgp-1- IL-2R alpha- cells also acquire high levels of CD4 and CD8 within 1 d, and low levels of CD3 by 2 d. However, they do not progress further to mature single positives in vitro, and most of them die by day 3. These experiments directly confirm our previously proposed developmental sequence, and demonstrate the kinetics of T lymphocyte production in a low-stress, steady-state environment.

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Selection of CD4+CD8+ thymocytes by complex formation with medulla-derived epithelial cells.

The role of lymphostromal complexes in T-cell differentiation is far from elucidated, mainly because a clear association of a particular stromal cell type with a distinct thymocyte subset has never been identified. Using an in vitro system, detecting the adherence of thymocytes to a thymic medullary epithelial cell line (E-5), we showed that the phenotype of these thymocytes was that of cortical type: Thy-1hi, LFA-1+, PNAhi, CD4+CD8+, MEL-14-/lo, IL-2R-, CD3-/lo, and TcR V beta 8-/lo. They were enriched in cells in G2/M at the time of complex formation, showed a higher basal proliferation in culture, and did not respond to PHA, IL-2 and only marginally to Con A. These data show that complex formation with mouse thymic medullary epithelium selects for CD4+CD8+ thymocytes, as shown by the marked decrease in CD4+CD8-/CD4-CD8+ thymocytes, and the incapacity of CD4-CD8- thymocytes to adhere.

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Ontogeny of a novel CD4+CD8-CD3- thymocyte subpopulation: a comparison with CD4- CD8+ CD3- thymocytes.

We have studied the ontogeny of a novel thymocyte subset, CD4+CD8-CD3-. Three-colour flow cytometric analysis demonstrated that these cells constituted approximately 1% of the total thymocyte content in adult CBA mice, and were not present in lymph nodes. They were mainly blastic, cortisone-sensitive, and localized in the outer thymic cortex. During foetal life they were first observed at day 15 and reached a maximum (6%) at day 17, beyond which they decreased to the adult level. This kinetic profile was similar to that of the CD4-CD8+CD3- subpopulation, except that the CD4+CD8-CD3- cells appeared slightly earlier and their percentage was lower. Both these populations appeared after the CD4-CD8-CD3- cells but before the CD4+CD8+CD3- cells. Similar observations were made during thymic reconstitution following dexamethasone treatment. In this case, both CD4+CD8-CD3- and CD4-CD8+CD3- thymocytes disappeared 48 h after the treatment. While their absolute number increased up to 14 days post-treatment, their percentage was maximal at day 7 post-treatment and returned to normal values by day 10 post-treatment. These results argue strongly that not only the CD4-CD8+CD3- population but also the CD4+CD8-CD3- population can be considered an intermediate precursor in CBA thymuses.

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Thymic lymphoma cells as a model for complex-formation between thymocytes and thymic medullary epithelial cells.

The formation of complexes between thymocytes and thymic stromal elements is known to be involved in T cell differentiation. We have previously described one type of lympho-stromal interaction involving CD4+ CD8+ thymocytes and a medullary epithelial cell line (E-5). In this study we report the potential for complex formation of two different thymic lymphoma cell lines (Ti-6 and RDM-4). Ti-6 cells were shown to adhere to the E-5 cells, while RDM-4 cells were totally incompetent. Phenotypic characterization of these cell lines suggests that immature thymocytes are not capable of forming complexes with medullary epithelium and that a certain level of differentiation is required to do so. Comparison of their phenotypes showed that the possibility of some classical T cell surface markers being the receptor for the E-5 ligand can be dismissed.

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