Bu-1 andTh-1, two loci determining surface antigens of B or T lymphocytes in the chicken

Alloantisera were prepared by reciprocal immunizations with bursal or thymic lymphoid cells between chickens of two inbred lines identical at theB major histocompatibility locus. In cytotoxic assays, antibursa sera were specific for donor-line bursa cells without absorption; antithymus sera were similarly specific for donor-line thymus cells. Both types of sera cytolyzed some peripheral lymphoid cells from spleen, bone marrow, and blood. Absorption of either type of serum with peripheral blood leukocytes removed all cytotoxic reactivity for central lymphoid cells. The inheritance of the alloantigens detected by these specific antisera was studied in F₁, F₂, and backcross progeny from the two lines. Phenotypes were determined by a method in which antisera preabsorbed with progeny leukocytes were reacted against⁵¹Cr-labeled bursal or thymic cells from chickens of both lines. The results established two independent autosomal loci-Bu-1 andTh-1-determining antigens expressed, respectively, on bursal cells and peripheral B lymphocytes or on thymic cells and peripheral T lymphocytes. Cytotoxic testing of bursal or thymic cells from chickens of other inbred lines and F₁ populations led to the tentative conclusion that the number of alleles atBu-1 is restricted, whileTh-1 exhibits considerable multiple allelism.     

Immune complexes of E. coli antigens and maternal IgG in the bursa of Fabricius

The bursa of Fabricius of the chicken is known to be both a primary lymphoid organ and a secondary lymphoid tissue. Bursal follicles are equipped with antigen-trapping follicle-associated epithelium. However, bioactive antigens such as protein and bacteria have not been detected in the bursal parenchyma. By immunoperoxidase staining with a polyspecific antibody (Ab) against Escherichia coli, we detected aggregated E. coli antigens in the medulla of bursal follicles after hatching. The distribution of aggregated E. coli antigens is restricted to the medulla of bursal follicles. The antigens are not found in the spleen or the parenchyma of the caecal tonsil. The bursa is thus a trapping site for E. coli antigens from the external environment. Furthermore, two-color immunostaining clarified that these antigens form immune complexes with maternal IgG (MIgG) and are retained by reticular cells. Additionally, immune complexes in the bursa were shown to induce the rapid development of serum IgM Ab for indigenous E. coli. Our results suggest that immune complexes of MIgG and environmental antigens in the medulla of bursal follicles exert positive effects on B-cell differentiation in the bursa in situ.     

Relationship between the expression of class I antigen and reactivity of chicken thymocytes

The expression of major histocompatibility complex (MHC) class I antigens in ontogenesis and the distribution of B-F⁺ cells, defined by means of a monoclonal antibody, were studied by indirect membrane immunofluorescence tests on suspensions of thymus, bursa, spleen, peripheral blood lymphocytes (PBL) and red blood cells (RBC) from 18-day-old chicken embryos and chickens from 1–90 days after hatching. At 18 days of incubation and at the first day after hatching, RBC, PBL, and the cells from bursa and thymus are negative. The percentage of positive PBL and bursal cells increases up to 9 days after hatching. By 2 weeks after hatching almost 100 % of the RBC, PBL, bursa, and spleen cells were positive whereas the thymus showed only 20% positive cells. Analysis on 4-m-thick, frozen acetone-fixed tissue sections of thymus showed that medullary cells are positive, while the cortical area is negative. The graft-versus-host (GvH) competence of these thymus subpopulations was compared after sorting by the fluorescence-activated cell sorter and injection into MHC incompatible embryos. GvH reactivity was associated primarily with the B-F⁺ population. Double staining studies with peanut agglutinin (PNA)-fluorescein isothiocyanate and a rabbit-anti-Ig tetramethyl isothiocyanate-conjugate proved that the PNA^– thymocytes are identical with B-F⁺ thymocytes.Abbreviations used in this paper: FACS fluorescence-activated cell sorter - FCS fetal calf serum - FITC-Ig fluorescein isothiocyanate-conjugated immunoglobulin - GvH graft-versus-host - HAT hypoxanthineaminopterin-thymidine - HBSS Hanks' balanced salt solution - IIF indirect immunofluorescence - MCA monoclonal antibody - MHC major histocompatibility complex - NWL normal white Leghorn - OS Obese strain - PBL peripheral blood lymphocytes - PBS phosphate-buffered saline - PNA peanut agglutinin - RBC red blood cells - TRITC-Ig tetramethyl isothiocyanate-conjugated immunoglobulin     

Delta/Jagged-mediated Notch signaling induces the differentiation of agr2-positive epidermal mucous cells in zebrafish embryos

Teleosts live in aquatic habitats, where they encounter ionic and acid-base fluctuations as well as infectious pathogens. To protect from these external challenges, the teleost epidermis is composed of living cells, including keratinocytes and ionocytes that maintain body fluid ionic homeostasis, and mucous cells that secret mucus. While ionocyte progenitors are known to be specified by Delta-Notch-mediated lateral inhibition during late gastrulation and early segmentation, it remains unclear how epidermal mucous cells (EMCs) are differentiated and maintained. Here, we show that Delta/Jagged-mediated activation of Notch signaling induces the differentiation of agr2-positive (agr2⁺) EMCs in zebrafish embryos during segmentation. We demonstrated that agr2⁺ EMCs contain cytoplasmic secretory granules and express muc5.1 and muc5.2. Reductions in agr2⁺ EMC number were observed in mib mutants and notch3 MOs-injected notch1a mutants, while increases in agr2⁺ cell number were detected in notch1a- and X-Su(H)/ANK-overexpressing embryos. Treatment with γ-secretase inhibitors further revealed that Notch signaling is required during bud to 15 hpf for the differentiation of agr2⁺ EMCs. Increased agr2⁺ EMC numbers were also observed in jag1a-, jag1b-, jag2a- and dlc-overexpressing, but not jag2b-overexpressing embryos. Meanwhile, reductions in agr2⁺ EMC numbers were detected in jag1a morphants, jag1b mutants, jag2a mutants and dlc morphants, but not jag2b mutants. Reduced numbers of pvalb8-positive epidermal cells were also observed in mib or jag2a mutants and jag1a or jag1b morphants, while increased pvalb8-positive epidermal cell numbers were detected in notch1a-overexpressing, but not dlc-overexpressing embryos. BrdU labeling further revealed that the agr2⁺ EMC population is maintained by proliferation. Cell lineage experiments showed that agr2⁺ EMCs are derived from the same ectodermal precursors as keratinocytes or ionocytes. Together, our results indicate that specification of agr2⁺ EMCs in zebrafish embryos is induced by DeltaC/Jagged-dependent activation of Notch1a/3 signaling, and the cell population is maintained by proliferation.     

Differential Selection of Cells with Proviral c-myc and c-erbB Integrations after Avian Leukosis Virus Infection

Avian leukosis virus (ALV) infection induces bursal lymphomas in chickens after proviral integration within the c-myc proto-oncogene and induces erythroblastosis after integration within the c-erbB proto-oncogene. A nested PCR assay was used to analyze the appearance of these integrations at an early stage of tumor induction after infection of embryos. Five to eight distinct proviral c-myc integration events were amplified from bursas of infected 35-day-old birds, in good agreement with the number of transformed bursal follicles arising with these integrations. Cells containing these integrations are remarkably common, with an estimated 1 in 350 bursal cells having proviral c-myc integrations. These integrations were clustered within the 3′ half of c-myc intron 1, in a pattern similar to that observed in bursal lymphomas. Bone marrow and spleen showed a similar number and pattern of integrations clustered within 3′ c-myc intron 1, indicating that this region is a common integration target whether or not that tissue undergoes tumor induction. While all tissues showed equivalent levels of viral infection, cells with c-myc integrations were much more abundant in the bursa than in other tissues, indicating that cells with proviral c-myc integrations are preferentially expanded within the bursal environment. Proviral integration within the c-erbB gene was also analyzed, to detect clustered c-erbB intron 14 integrations associated with erythroblastosis. Proviral c-erbB integrations were equally abundant in the bone marrow, spleen, and bursa. These integrations were randomly situated upstream of c-erbB exon 15, indicating that cells carrying 3′ intron 14 integrations must be selected during induction of erythroblastosis.     

Influence of genotypes at a non-MHC B lymphocyte alloantigen locus (Bu-1) on expression of Ia (B-L) antigen on chicken bursal lymphocytes

Two inbred lines of chickens known to be identical at the MHC differ in their expression of Ia antigen on cells of the bursa. Line 6 bursas had 23% of intensely staining Ia+ cells while line 7 bursas had a much higher level, 85%. Studies of F4 progeny of line 6(3) X 100 crosses showed that genetic control of the high bursal proportion of Ia+ cells was determined by the Bu-1 alloantigen system. Line 100 is identical to line 7 for the lymphocyte alloantigens identified by the T and B cell reagents used in this study. Tests of F4 heterozygotes at the Bu-1 locus show a dominant effect of the Bu-1b gene in control of a high proportion of strongly staining Ia+ cells in the bursa.     

Immune reconstitution in lymphoid tissue following potent antiretroviral therapy

During untreated HIV-1 infection, a chronic state of immune activation and inflammation develops at the lymphoid tissue sites of viral replication. The early effect of potent combination drug therapy is a reduction in peripheral viral burden and a reduction in the production of inflammatory and type 1 cytokines. Further along in treatment there are trends toward normalization in the frequencies of CD8⁸ T-cells, CD4⁺ CD45RA⁺ cells, as well as CD4⁺ CD45R0⁺ cells. Finally, the CD1a⁺ dendritic cell network is re-established and germinal centers are reformed. Although this restoration of the lymphoid dynamic form is coupled to a reconstitution of peripheral blood T-cell function in vitro and by skin testing, sterilizing immunity to HIV-1 does not develop. Furthermore there is no heightened development of cytotoxic CD8⁺ T-cell function at the site of HIV-1 latency. This is evidenced by a massive recrudescence of HIV-1 viral replication within lymphoid tissue when therapy is stopped. The development of supplemental therapies, which reconstitute anti-HIV-1 immunity, will be required. Specific defects in anti-HIV-1 activity which occur in lymphoid tissue during infection include a downregulation of perform expression by cytotoxic T-cells, the down regulation of the TCR signal transducing chain CD3ζ, and inadequate CD4⁺ T-cell help within the tissue compartment of immune regeneration.     

Immune reconstitution in the immunodeficient chicken by transfer of embryonic lymphoid cells

Embryonic chickens were rendered immunodeficient by in ovo injection of homologous IgM on the 10th embryonic day. The immunodeficient embryos were intravenously given lymphoid cells taken from normal embryonic bursa, spleen or thymus on the 15th embryonic day. Gain of splenic or bursal weight, natural antibodies to sheep red blood cells (SRBC), frequencies of rosette forming cells (RFC) binding to SRBC or dinitrophenyl-SRBC (DNP-SRBC) and of immunoglobulin bearing cells (IBC) in the bursa and the spleen were investigated to assess the effect of transferred cells during the embryonic stage. Transferred bursal and splenic cells showed an ability to restore the frequency of RFC or IBC in the recipients. However, reversion from the immunodeficient state was not observed in the thymic cell transfer. These findings suggested that the cells derived from embryonic bursa and spleen contained stem cells which developed into RFC and also into precursors of IBC.     

Studies of testosterone-induced involution of the bursa of Fabricius

The present investigation deals with the effect of testosterone on each of the tissue components of the bursa of Fabricius: the endodermal epithelium, the mesenchyme, and the hemopoietic stem cells. Tissue combination experiments between testosterone-treated endoderm and normal mesenchyme and vice versa have shown that the androgen damages irreversibly the bursal epithelium. The latter is not seeded by hemopoietic stem cells and cannot undergo follicle formation when treated with high doses of testosterone. This occurs even if it is associated with a nontreated bursal mesenchyme. On the contrary, associations of testosterone-treated mesenchyme with normal endoderm result in normal bursa histogenesis. By using an original test of viability for lymphoid cells based on the application of the quail-chick marker system, we demonstrate that disappearance of hemopoietic cells in the endoderm results from their expulsion from the bursa and not from their death in situ. The conspicuous effect of testosterone on the bursa of Fabricius can be related to the levels of androgen receptors found in the organ. Typical cytosol androgen receptors are demonstrated in both bursal endoderm and mesoderm, although the amount in the former is higher. The concentration of binding sites in the bursa is >10 times higher than that in other organs such as lung and small intestine whose development is not affected by testosterone, contrasting with glucocorticosteroid receptor (measured by labeling with dexamethasone) found in the same concentration in all tissues.     

Immunosuppressive Effect of the Infectious Bursal Agent in the Chicken

IT is well established that in the chicken humoral antibody formation depends on the bursa of Fabricius, whereas delayed hypersensitivity and other manifestations of cellular immunity depend on the thymus for their development^1,2. Surgical bursectomy^3,4 and the administration of testosterone^5–7, cortisone acetate⁸ or cyclophosphamide^9–11 have been found to limit the bursa-dependent antibody system. Infectious bursal disease (IBD), formerly known as Gumboro disease, is a naturally occurring virus disease of young chickens¹², characterized by the destruction of the lymphoid tissue in the bursa without repopulation¹³. The disease has been reported from many countries in Europe and in North America. The effect of IBD on the course of other infections in the chicken is therefore of interest. We report here that the primary and secondary serological responses to Newcastle disease vaccine were reduced significantly in chickens which were experimentally inoculated with the infectious bursal agent (IBA) at one day of age.     

Maturation of bursal stem cells within allogeneic or syngeneic bursal microenvironment: acquisition of postbursal maturity.

Differentiation of bursal stem cells in an allogeneic or syngeneic bursal microenvironment was compared. Bursal stem cells were transplanted into CY-treated 4-day-old recipients and permitted to differentiate in these hosts for 6 weeks. Their maturity degree was thereafter assessed by transplanting them into secondary recipients by using morphologic and functional criteria. As the secondary recipients 4-day-old CY-treated or CY-treated and surgically bursectomized chicks were used. The results obtained demonstrate that bursal stem cells develop to mature postbursal cells also within an allogeneic bursa. They also indicate that although the interaction of different lymphoid cells requires histocompatibility, the interaction between stromal cells in the bursa and lymphoid progenitors is not genetically restricted.     

Chimeric Thymus Rudiments: A Strategy for the Study of Tolerance Induction in Vitro

The techniques of fetal thymus organ culture have been widely utilized for the study of thymocyte differentiation under carefully controlled conditions. Recent results suggesting a role for dendritic cells (DC) in selection of a competent T-cell repertoire have prompted attempts to construct chimeric thymus rudiments in vitro. Here, we describe a novel approach based on the migratory properties of mature lymphoid DC. Purified DC from adult thymus or spleen were found to migrate into fetal thymus rudiments in culture and localize specifically within the medulla. This distribution closely mirrors the situation in vivo, underlining the physiological relevance of the resulting microenvironment. Recolonization was shown to be selective, excluding cell types not normally represented in the thymus. To assess the extent of repertoire selection in recolonized fetal thymi, chimeric rudiments in which I-E determinants were expressed exclusively on the surface of immigrant DC were constructed. The failure of such rudiments to recruit a population of CD4⁺8⁻Vβ6⁺ thymocytes, restricted to antigen recognition in the context of I-E, argued against a role for DC in positive selection, in contrast, the widespread deletion of potentially autoreactive CD4⁺8⁻Vβ17a⁺ cells suggested an active role for DC in negative selection of the T-cell repertoire. These conclusions are consistent with the findings of various in vivo studies, endorsing the suitability of such a model for the study of tolerance induction in vitro.     

Perinatal deletion of B cells expressing surface Ig molecules that lack V(D)J-encoded determinants in the bursa of Fabricius is not due to intrafollicular competition

During embryonic development, the avian bursa of Fabricius selects B cell precursors that have undergone productive V(D)J recombination for expansion in oligoclonal follicles. During this expansion, Ig diversity is generated by gene conversion. We have used retroviral gene transfer in vivo to introduce surface Ig molecules that lack V(D)J-encoded determinants into B cell precursors. This truncated mu heavy chain supports both B cell expansion within embryo bursal lymphoid follicles and gene conversion. We show that individual follicles can be colonized exclusively by cells expressing the truncated mu chain and lacking endogenous surface IgM, ruling out a requirement for V(D)J-encoded determinants in the establishment of bursal lymphoid follicles. In striking contrast to their normal development in the embryo, bursal cells expressing the truncated mu-chain exhibit reduced rates of cell division and increased levels of apoptosis after hatching. The level of apoptosis in individual follicles reflects the proportion of cells within the follicle that express the truncated mu-chain. In particular, high levels of apoptosis are associated with follicles containing exclusively cells expressing the truncated micro receptor. Thus, apoptotic elimination of such cells is not due to competition within the follicle by cells expressing endogenous surface IgM receptors. This provides the first direct demonstration that the regulation of B cell development in the avian bursa after hatching differs fundamentally from that seen in the embryo. The requirement for intact IgM expression when the bursa is exposed to exogenous Ag implicates a role for Ag in avian B cell development after hatching.     

Allelic exclusion of surface IgM allotypes on spleen and bursal B cells in the chicken

The expression of IgM-1a and IgM-1b (C) allotypes on the surface of splenic and bursal cells of B14 line chickens was examined by the triple-layer immunfluorescence technique. We have demonstrated that all surface Ig⁺ cells in both spleen and bursa expressed sIgM-1 which was subject to allelic exclusion in M-l^a/M-l^b heterozygous chickens. Furthermore, a phenotypic equilibrium in the proportion of cells carrying the products of the twoM-1 alleles was observed.Abbreviations used in this paper Ig immunoglobulin - cIg cytoplasmic immunoglobulin - sIg surface immunoglobulin - RAC rabbit anti-chicken Ig serum - PBS phosphate buffered saline - NCS normal chicken serum     

A novel method to bursectomize avian embryos and obtain quail----chick bursal chimeras. II. Immune response of bursectomized chicks and chimeras and post-natal rejection of the grafted quail bursas

Two methods to bursectomize chick embryos before hemopoietic cell seeding of the bursa of Fabricius were compared in this work: section of the tail region at E3 including the presumptive bursal territory, and selective removal of the bursa at E5. Hatching ability is better with the former method, but survival rate and effectiveness of bursectomy are favored with the second, novel technique. Moreover, selective removal of the bursa at E5 can be followed by in situ engraftment of a quail bursa and construction of quail-chick bursal chimeras. The immune response of bursaless birds and bursal chimeras has been studied. Total absence of the bursa does not prevent a few B cells from differentiating and nonspecific Ig (IgM and/or IgG) from being secreted. As reported previously, bursaless birds, however, are unable to mount an immune response by producing specific antibodies. This immune function is restored by the graft of a quail bursa. The microenvironment of the bursa, although heterospecific, allows the expansion of the B cell population and generates the repertoire of the B cell antigen receptors. This process takes place during late embryonic and early postnatal life because the grafted quail bursal stroma is subjected to immune rejection from 2 to 3 wk after birth in all chimeras, which are, however, perfectly immunocompetent.     

NKp46+ Innate Lymphoid Cells Dampen Vaginal CD8 T Cell Responses following Local Immunization with a Cholera Toxin-Based Vaccine

Innate and adaptive immune cells work in concert to generate efficient protection at mucosal surface. Vaginal mucosa is an epithelial tissue that contains innate and adaptive immune effector cells. Our previous studies demonstrated that vaginal administration of Cholera toxin -based vaccines generate antigen-specific CD8 T cells through the stimulation of local dendritic cells (DC). Innate lymphoid cells (ILC) are a group of lymphocytes localized in epithelial tissues that have important immune functions against pathogens and in tissue homeostasis. Their contribution to vaccine-induced mucosal T cell responses is an important issue for the design of protective vaccines. We report here that the vaginal mucosa contains a heterogeneous population of NKp46⁺ ILC that includes conventional NK cells and ILC1-like cells. We show that vaginal NKp46⁺ ILC dampen vaccine-induced CD8 T cell responses generated after local immunization. Indeed, in vivo depletion of NKp46⁺ ILC with anti-NK1.1 antibody or NKG2D blockade increases the magnitude of vaginal OVA-specific CD8 T cells. Furthermore, such treatments also increase the number of DC in the vagina. NKG2D ligands being expressed by vaginal DC but not by CD8 T cells, these results support that NKp46⁺ ILC limit mucosal CD8 T cell responses indirectly through the NKG2D-dependent elimination of vaginal DC. Our data reveal an unappreciated role of NKp46⁺ ILC in the regulation of mucosal CD8 T cell responses.     

H Fluxes in Excised Samanea Motor Tissue : II. Rhythmic Properties

Homogeneous groups of cells were excised at regular intervals from opposing (extensor and flexor) motor tissue of Samanea saman (Jacq) Merrill maintained in white light for 34 hours. H⁺ fluxes between the tissue and bathing solution were then monitored during 30 minutes of darkness. Flux rates in both cell types vary with circadian rhythms. Flexor cells secrete H⁺ to the medium during two-thirds of the circadian cycle and take up H⁺ during the remainder of the cycle, while extensor cells take up H⁺ from the medium during the entire cycle.     

Analysis of the myogenic lineage in chick embryos: I. Studies on the terminal cell division

An antibody prepared against the MM isozyme of creatine phosphokinase (M-CK) stained multinucleated myotubes and post-mitotic mononucleated myoblasts in mass cultures of myogenic cells taken from the breast muscles of 11-day chick embryos. No cycling cells bound the antibody. Single cells isolated either directly from the embryo or from mass cultures were seeded at clonal density and allowed to undergo one division. The resulting pairs of cells were stained with the antibody and were scored as (a) both members of the pair M-CK⁺; (b) both M-CK^?; or (c) mixed (one M-CK⁺ and one M-CK^?). No mixed pairs were observed. Conditioned medium did not induce all myogenic pairs to differentiate and growth medium did not keep myogenic pairs in the cell cycle. About 10% of clonal pairs established from 10 h cultures were M-CK⁺, while about 27% of pairs established from 30 h mass cultures were M-CK⁺. These results indicate that (1) the myogenic lineage ends in a symmetrical division whose products are two post-mitotic M-CK⁺ cells; (2) the expression of the muscle phenotype is not determined exclusively by the environment; (3) the terminal cells are the product of an intrinsic program or cell lineage in which only the last cells can synthesize muscle-specific proteins.     

Vaginal Immunization to Elicit Primary T-Cell Activation and Dissemination

Primary T-cell activation at mucosal sites is of utmost importance for the development of vaccination strategies. T-cell priming after vaginal immunization, with ovalbumin and CpG oligodeoxynucleotide adjuvant as model vaccine formulation, was studied in vivo in hormone-synchronized mice and compared to the one induced by the nasal route. Twenty-four hours after both vaginal or nasal immunization, antigen-loaded dendritic cells were detected within the respective draining lymph nodes. Vaginal immunization elicited a strong recruitment of antigen-specific CD4⁺ T cells into draining lymph nodes that was more rapid than the one observed following nasal immunization. T-cell clonal expansion was first detected in iliac lymph nodes, draining the genital tract, and proliferated T cells disseminated towards distal lymph nodes and spleen similarly to what observed following nasal immunization. T cells were indeed activated by the antigen encounter and acquired homing molecules essential to disseminate towards distal lymphoid organs as confirmed by the modulation of CD45RB, CD69, CD44 and CD62L marker expression. A multi-type Galton Watson branching process, previously used for in vitro analysis of T-cell proliferation, was applied to model in vivo CFSE proliferation data in draining lymph nodes 57 hours following immunization, in order to calculate the probabilistic decision of a cell to enter in division, rest in quiescence or migrate/die. The modelling analysis indicated that the probability of a cell to proliferate was higher following vaginal than nasal immunization. All together these data show that vaginal immunization, despite the absence of an organized mucosal associated inductive site in the genital tract, is very efficient in priming antigen-specific CD4⁺ T cells and inducing their dissemination from draining lymph nodes towards distal lymphoid organs.