Early separation of B and T lymphocyte precursors in chick embryo.

Embryonic chimeras were used to demonstrate an early separation of chicken T and B cell precursors. Genetically polymorphic cell surface antigens, Bu-1 and Ov, which are expressed on cells of the B and T lineage, respectively, are useful markers in adoptive cell transfer studies. Allelic products Bu-1a and Bu-1b can be detected with monoclonal antibodies (mAbs) L22 and 11G2, respectively, and the Ov antigen with mAb 11A9. Chimeric chickens were constructed by reconstituting irradiated 14-d Ov- H.B19 embryos with the sorted Bu-1+ or Bu-1- fractions of spleen cells from age-matched H.B19 Ov+ embryos. Chimeras were analyzed, 3-4 wk after hatching, for the presence of Ov+ cells in the bursa, thymus, spleen, and peripheral blood lymphocytes. T cell precursors giving rise to thymocytes and peripheral T cells were present only in the Bu-1-, but not in the Bu-1+, fraction. We previously demonstrated that, in contrast, all B cell precursors in spleen from 14-d embryos are exclusively present in the Bu-1+ fraction. We also analyzed the immunoglobulin light chain gene rearrangement in these populations by polymerase chain reaction. We show here that VJ recombination occurs in the Bu-1+, but not in the Bu-1-, fraction of spleen. These data demonstrate an early commitment to the B cell lineage, which occurs before the colonization of the bursa of Fabricius. Segregation of B cell precursors from the other hemopoietic precursors, and consequently separation of T and B cell precursors, occurs before the colonization of the primary lymphoid organs.

T cell response to Epstein-Barr virus transactivators in chronic rheumatoid arthritis.

Rheumatoid arthritis is a multistep disorder associated with autoimmune features of yet unknown etiology. Implication of viruses such as Epstein-Barr virus (EBV) in rheumatoid arthritis pathogenesis has been suspected on the basis of several indirect observations, but thus far, a direct link between EBV and rheumatoid arthritis has not been provided. Here we show that a large fraction of T cells infiltrating affected joints from a patient with chronic rheumatoid arthritis recognizes two EBV transactivators (BZLF1 and BMLF1) in a major histocompatibility complex-restricted fashion. Responses to these EBV antigens by synovial lymphocytes from several other chronic rheumatoid arthritis patients were readily detectable. Thus these results suggest a direct contribution of EBV to chronic rheumatoid arthritis pathogenesis. They also demonstrate for the first time the occurrence of T cell responses against EBV transactivating factors, which might be central in the control of virus reactivation.

Exon III splicing switch of fibroblast growth factor (FGF) receptor-2 and -3 can be induced by FGF-1 or FGF-2.

An essential feature of fibroblast growth factor receptors (FGFRs) is the existence of multiple possibilities of alternative splicing. One of these concerns sequences of the mRNA coding for the C-terminal half of Ig domain 3 which corresponds to a part of the ligand-binding site: two alternative exons, IIIb and IIIc, encode the C-terminal half of Ig domain 3. The IIIb/IIIc choice in the FGFR-2 and FGFR-3 is strictly tissue-specific, the IIIb exon being expressed exclusively in epithelial cells. We describe here a reversible switch from IIIb to IIIc for FGFR-2 and FGFR-3 under the influence of exogenous and endogenous FGF-1 or FGF-2. We observed that FGF-induced FGF receptor exon switching (i) occurred as early as 1 h after exposure to FGF (ii) was receptor-mediated (iii) was dependent on cell confluency and showed a link with the cell cycle (iv) was correlated with a reversible loss of epithelial properties. These results support a role for FGF in the regulation of expression of alternatively spliced FGFR mRNA.

Overexpression of vascular endothelial growth factor induces cell transformation in cooperation with fibroblast growth factor 2.

Vascular endothelial growth factor (VEGF) is a family of homodimeric proteins produced from a single gene by alternative splicing of the VEGF transcript. VEGF induces in vivo angiogenesis and vascular permeability. We have recently demonstrated that VEGF is an autocrine growth factor for retinal pigment epithelial (RPE) cells. To further understand the role of VEGF, we overexpressed VEGF in rat RPE cells. The transfected cells exhibited a growth advantage in vitro and an increased response to the mitogenic effect of fibroblasts growth factor-2 (FGF-2), and formed colonies in soft agar upon FGF-2 addition. Moreover, analysis of FGF-receptors evidenced a dramatic increase in FGFR-1 mRNA and protein level, supporting the hypothesis that this receptor mediates the transforming effect of FGF-2. These results reveal that the oncogenic role of VEGF is exerted through a cross regulation between VEGF and FGF signal transduction pathways.

Multiple mRNAs code for proteins related to the BEK fibroblast growth factor receptor.

The BEK transmembrane protein tyrosine kinase is a receptor for both acidic and basic fibroblast growth factors. We identify several different transcripts which code for BEK-related proteins. These proteins differ from BEK in regions expected to control receptor activity. Thus, some of the proteins have altered extracellular, ligand-binding domains, and others an altered carboxy-terminal tail. Still other forms of BEK differ only in their juxtamembrane domains. Sequencing of parts of the BEK gene shows that alternative splicing of the premessenger can account for at least some of this diversity. In particular, an apparently tissue specific, mutually exclusive splicing of two internal exons permits both the previously described K-SAM mRNA and the BEK mRNA to be derived from the same premessenger.

The choice between alternative IIIb and IIIc exons of the FGFR-3 gene is not strictly tissue-specific.

An essential feature of fibroblast growth factor receptors (FGFR) is the existence of multiple possibilities for alternative splicing. One of these concerns sequences of the mRNA coding for the C-terminal half of Ig domain 3 which corresponds to a part of the ligand-binding site. Two alternative exons, IIIb and IIIc, encode the C-terminal half of Ig domain 3. We show here that the alternative splicing choice between IIIb and IIIc exons of the FGFR-3 is not strictly tissue-specific: epithelial cells show exclusively IIIb transcripts while fibroblastic cells show a mixture of IIIb and IIIc transcripts. This is in contrast with the strictly exclusive alternative choice between IIIb or IIIc exons of the FGFR-2 gene: epithelial cells make only the IIIb choice while fibroblastic cells make only the IIIc choice.

Cytoadherence of lymphocytes from type I diabetic subjects to insulin-secreting cells. Marker of anti-beta-cell cellular immunity.

We studied the ability of lymphocytes from type I (insulin-dependent) diabetic patients to adhere to murine beta-cells. Lymphocytes from 17 recent-onset type I diabetic subjects (less than 6 mo) displayed enhanced ability to form rosettes with RINm5F cells (P less than .001) compared with lymphocytes from 27 healthy subjects forming background rosettes, whereas the number of RIN cytoadherent lymphocytes was unimpaired in 12 type II (non-insulin-dependent) diabetic subjects. This phenomenon tended to decline in 21 subjects with long-standing diabetes (greater than 1 yr) who taken as a group presented a normal number of RIN rosetting lymphocytes. The islet specificity of these diabetic rosettes was confirmed because, compared with controls, lymphocytes from recent-onset type I diabetic subjects also displayed a greater intensity of adherence to normal mouse islets but not to unrelated K562 and TS cell lines. As demonstrated by indirect immunofluorescence studies, these diabetic rosettes contained 54% of T-lymphocytes (OKT3+, OKT4+, or OKT8+), whereas only 20% of T-lymphocytes were found in background rosettes. The high percentage (66%) of la+ cells found in diabetic rosettes suggests that at least some of the cytoadherent T-lymphocytes from recent-onset type I diabetic subjects are activated. Natural killer (NK) cells do not seem to be the major cell type implicated in this phenomenon, because Leu 11+ cells were less represented in diabetic rosettes (25%) than in background rosettes (53%).(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro relationship of CD4 cells from type I diabetic patients and xenogeneic beta-cell membranes.

In a rosette assay, 63 patients with recent-onset type I (insulin-dependent) diabetes mellitus had a higher (P less than .001) number of lymphocytes adhering to rat insulinoma RINm5F cells (diabetic rosettes) than 153 healthy control (background rosettes) or 20 nondiabetic subjects with other organ-specific autoimmune diseases. Furthermore, lymphocytes from diabetic patients displayed a highly correlated (r = .97, P less than .001) binding on two different xenogeneic beta-cell lines (RIN and hamster insulinoma HIT cells). This phenomenon was not found on a panel of seven non-beta-cell lines (e.g., exocrine pancreatic cells, endocrine cells). By increasing lymphocyte-to-RIN ratios (0.25:1 to 30:1), the supernumerary RIN-adherent lymphocytes from diabetic patients, expressed as the percentage of lymphocytes involved conjugates, were only detectable at lower ratios (0.25:1 to 4:1), and their binding efficiency was two times higher than that of control lymphocytes. This efficiency fell at higher ratios (greater than 4:1) to the level of background rosettes that remained constant through the ratio scale. This specific RIN-rosette formation was abrogated when lymphocytes from diabetic patients were preabsorbed on beta-cells (either HIT or RIN) but not on non-beta-cells, whereas preabsorption of control lymphocytes did not modify the number of background rosettes. In addition, diabetic rosettes, but not background rosettes, were inhibited by competition with RIN membrane extracts but not by non-beta-cell extracts. Moreover, diabetic rosettes were inhibited during blocking experiments with anti-CD3 monoclonal antibody (MoAb) but not with unrelated MoAbs.(ABSTRACT TRUNCATED AT 250 WORDS)

The follicle-associated epithelium in the bursa of Fabricius cell origin studied by means of quail-chick chimeras and monoclonal antibodies.

The cell origin of the follicle-associated epithelium (FAE) of the bursa of Fabricius was studied by two different technical approaches. Precolonized quail bursal rudiments were grafted into chick embryos and the grafts were recovered 2 wk after hatching of the recipients. By taking advantage of the distinct nuclear characteristics of chick and quail cells, it could be shown that the specialized FAE consists of a mixture of epithelial cells, with special features, among which hemopoietic cells, originating from the host, are dispersed. Staining of chicken bursas with different monoclonal antibodies reacting either with the epithelial component (BEP-1) or with the hemopoietic cells of the bursa (L22, L17) confirmed that hemopoietic cells, presumably macrophages, are mixed with the epithelial cells at the level of FAE.

Identification of a gp100 epitope recognized by HLA-A3 restricted melanoma infiltrating lymphocytes.

The large majority of known melanoma-associated antigenic peptides presented by MHC class I molecules are presented by the most frequent allele, HLA-A*0201. Thus although a significant percentage of Caucasians express HLA-A3, no melanoma-associated antigenic peptide presented by this allele has yet been identified. We show here that the T cell clone M45-10 isolated from tumor infiltrating lymphocytes recovered from a melanoma biopsy recognizes the gp100-derived peptide ALLAVGATK presented by HLA-A*0301. Since gp100 is expressed on most melanoma cells, our results imply that the gp100-based anti-melanoma strategies developed for individuals expressing HLA-A2 will also be applicable to those expressing HLA-AS (about one Caucasian in four). gp100 is therefore a particularly promising melanoma antigen, as different peptides derived from it can be presented by at least two different frequently encountered HLA class I molecules.

Oncoprotein fos activation in epithelial-cells induces an epitheliomesenchymal conversion and changes the receptor encoded by the fgfr-2 messenger-RNA from k-sam to bek.

Activation of c-Fos, by using an inducible c-Fos estrogen receptor fusion protein, triggers the epitheliofibroblastoid cell conversion of mouse mammary epithelial cells. We show that this change in phenotype is accompanied by a definitive switch of the fibroblast growth factor receptor 2 from K-SAM to BEK. This splicing switch occurs a few hours after estrogen stimulation. Our data suggest that Fos proteins could be important in modulating the FGFR-2 splicing choice. Moreover, these observations reinforce previous evidence that the BEK/K-SAM choice is strictly tissue-specific: the K-SAM exon is expressed exclusively in epithelial cells, the BEK exon in cells of the fibroblastic type.

Immunodominant CD8 T cell response to Epstein-Barr virus.

Epstein-Barr virus (EBV) provides one of the most informative systems for analysing cytotoxic T lymphocyte responses in humans. The viral infection and its persistence are the results of an alternation of lytic and latent phases that are controlled by the immune response. Using a transient COS transfection assay that permits semi-quantitative estimation of CD8 T cell responses against a large number of HLA/viral protein combinations, we analyzed responses to EBV within a large number of polyclonal T cell lines. This allowed a rapid identification of major epitopes and the demonstration that EBV-specificT cells were mainly directed against a restricted set of immunodominant epitopes, primarily generated during the early lytic cycle. Knowledge of the antigen specificity of CDB T cell responses against EBV should help generate cytotoxic T cell lines to this herpesvirus, and more generally to study the molecular basis of immunodominance.

[Development of the immune system]. / Le développement du système immunitaire.

Immune protection in vertebrates is provided by a dual system: the cellular immune response, mediated by T lymphocytes and the humoral immune response, mediated by B lymphocytes. These lymphocytes develop immunocompetence in the primary lymphoid organs: thymus, for the T lymphocytes and bursa of Fabricius (in birds), on its equivalent (in mammals), for B lymphocytes. The crucial period during which thymus and bursa influence the immunological development is the embryonic and early postnatal life. Hemopoietic stem cells home to these primary lymphoid organs during well-defined periods of colonization. Under the influence of thymic and bursal microenvironments, they become oriented respectively toward the T or B cell differentiation pathway. They acquire various membrane antigens and an antigen receptor. T lymphocytes also learn to recognize self-antigens (antigens encoded by the Major Histocompatibility Complex). T and B lymphocytes then colonize respectively T-dependent and B-dependent areas of secondary lymphoid organs where they become functional. Some immune deficiencies result from a defect in development which can affect selectively T or B lymphocytes or both systems in case of severe combined immunodeficiency disorders.

CD4+ T-cell responses to Epstein-Barr virus (EBV) latent-cycle antigens and the recognition of EBV-transformed lymphoblastoid cell lines.

There is considerable interest in the potential of Epstein-Barr virus (EBV) latent antigen-specific CD4+ T cells to act as direct effectors controlling EBV-induced B lymphoproliferations. Such activity would require direct CD4+ T-cell recognition of latently infected cells through epitopes derived from endogenously expressed viral proteins and presented on the target cell surface in association with HLA class II molecules. It is therefore important to know how often these conditions are met. Here we provide CD4+ epitope maps for four EBV nuclear antigens, EBNA1, -2, -3A, and -3C, and establish CD4+ T-cell clones against 12 representative epitopes. For each epitope we identify the relevant HLA class II restricting allele and determine the efficiency with which epitope-specific effectors recognize the autologous EBV-transformed B-lymphoblastoid cell line (LCL). The level of recognition measured by gamma interferon release was consistent among clones to the same epitope but varied between epitopes, with values ranging from 0 to 35% of the maximum seen against the epitope peptide-loaded LCL. These epitope-specific differences, also apparent in short-term cytotoxicity and longer-term outgrowth assays on LCL targets, did not relate to the identity of the source antigen and could not be explained by the different functional avidities of the CD4+ clones; rather, they appeared to reflect different levels of epitope display at the LCL surface. Thus, while CD4+ T-cell responses are detectable against many epitopes in EBV latent proteins, only a minority of these responses are likely to have therapeutic potential as effectors directly recognizing latently infected target cells.

Cell lineage segregation during bursa of Fabricius ontogeny.

The population dynamics of myeloid and lymphoid lineages during bursa of Fabricius ontogeny were analyzed by immunofluorescence by using two monoclonal antibodies (mAb). CL-1 mAb reacts with all chicken hemopoietic cells, except mature erythrocytes. L22 mAb reacts with bursa and bursa-derived lymphocytes, with a minor subset of macrophages and with some cells of the thymic medulla. The staining of embryonic bursas by these antibodies helps to distinguish between two different lineages of hemopoietic cells: CL-1+/L22+ cells represent B lymphocytes and a minor subset of macrophages, while CL-1+/L22- cells correspond to most of the macrophages and to the granulocytes, which disappear at the end of the embryonic life. CL-1+/L22- as well as CL-1+/L22+ cells were first observed outside the bursal rudiment. This indicates that there is a pre-bursal segregation between these two hemopoietic lineages and that two different kinds of precursors colonize the bursal rudiment at about the same time (day 9 for CL-1+/L22- cells and days 9 or 10 for CL-1+/L22+ cells). Moreover our data show that the colonization of the bursal epithelium by hemopoietic precursors is a two-step phenomenon. The first cells which enter belong to the CL-1+/L22- lineage, express Ia-like antigens at a high level, are dendritic in morphology, and represent cells of the macrophage/dendritic cell lineage. They are responsible for the formation of the epithelial bud which are then colonized by a small number of lymphoid precursors which belong to the CL-1+/L22+ lineage. Quail-chick bursa grafting experiments were also performed and the grafts were examined for CL-1 (restricted to chicken hemopoietic cells) and L22 reactivity. These observations confirmed our previous findings about the kinetics of the colonization of bursal rudiment by hemopoietic precursors and give support for a pre-bursal segregation between two hemopoietic pathways.

Differentiation of the mouse hepatic primordium. II. Extrinsic origin of the haemopoietic cell line.

The whole hepatic primordium (endoderm + mesenchyme of the septum transversum) was isolated from mouse embryos at various developmental stages, from 8 to 10 days of gestation, and was either grafted into chick or quail embryo or cultivated in vitro. Haemopoiesis developed only if the liver rudiment had been explanted after the 28- to 30-somite stage, but not if explanted prior to this stage, despite normal differentiation of the hepatocytes. However, when the liver rudiment, isolated before the 28-somite stage in in vitro culture, was supplied with exogenous haemopoietic stem cells, haemopoiesis developed in the hepatic tissue. These data show that foetal hepatic haemopoiesis depends on migration of haemopoietic cells which home the liver rudiment at the 28- to 30-somite stage.

[Reversibility of the morphological and functional dedifferentiation occurring in cultures of avian embryonic liver cells (author's transl)]. / Réversibilité de la dédifférenciation observée dans les cultures cellulaires de foie embryonnaire d'oiseau

Primary cell cultures are established from 8-day quail embryo livers. During the first three days the culture is made up of areas of epithelial-like cells and scattered fibroblasts. The cytoplasm of the epithelial cells shows a high glycogen content as detected by the PAS reaction controlled with salivary amylase digestion. During the following days an important increase in the number of fibroblastic cells is observed. After 6-7 days of cultivation, the epithelial cells have disappeared and the culture is entirely fibroblastic. PAS technique does not show any trace of glycogen in these cultures which have been prolonged up to 45 days. Six-to 45-day primary cultures entirely made up of fibroblasts were associated with hepatic or pulmonary mesenchyme in organotypic culture for 3-4 days. In some cases the explant was first cultivated in vitro for 2 days and then grafted into a 5-day-old chick embryo on the chorioallantoic membrane for 6 days. In the secondary cultures hepatocytes showing an epithelial arrangement and a high glycogen content were observed. It appears from this observation that some of the primary culture fibroblasts are in fact dedifferentiated parenchymal cells. Such a dedifferentiation is a reversible phenomenon since the cells retain the ability to express their initial determination if they are placed in convenient environmental conditions. The role of the specific tissular arrangement in the stability of the differentiated state is discussed.

The role of thymic epithelium in the acquisition of tolerance.

There are two separate mechanisms of induction of T-cell tolerance in the thymus. First, MHC molecules expressed on bone-marrow-derived cells can cause clonal deletion of autoreactive cells. Second, as discussed here by Elisabeth Houssaint and Martin Flajnik, thymic epithelial cells can generate a form of tolerance that does not eliminate self-reactive clones. This nondeletional mechanism, which is also a feature of the other MHC class-II-bearing epithelia, may contribute to the establishment of tolerance-maintaining regulatory networks.