Expression of alpha- and beta-globin genes occurs within different nuclear domains in haemopoietic cells.

The alpha- and beta-globin gene clusters have been extensively studied. Regulation of these genes ensures that proteins derived from both loci are produced in balanced amounts, and that expression is tissue-restricted and specific to developmental stages. Here we compare the subnuclear location of the endogenous alpha- and beta-globin loci in primary human cells in which the genes are either actively expressed or silent. In erythroblasts, the alpha- and beta-globin genes are localized in areas of the nucleus that are discrete from alpha-satellite-rich constitutive heterochromatin. However, in cycling lymphocytes, which do not express globin genes, the distribution of alpha- and beta-globin genes was markedly different. beta-globin loci, in common with several inactive genes studied here (human c-fms and SOX-1) and previously (mouse lambda5, CD4, CD8alpha, RAGs, TdT and Sox-1), were associated with pericentric heterochromatin in a high proportion of cycling lymphocytes. In contrast, alpha-globin genes were not associated with centromeric heterochromatin in the nucleus of normal human lymphocytes, in lymphocytes from patients with alpha-thalassaemia lacking the regulatory HS-40 element or entire upstream region of the alpha-globin locus, or in mouse erythroblasts and lymphocytes derived from human alpha-globin transgenic mice. These data show that the normal regulated expression of alpha- and beta-globin gene clusters occurs in different nuclear environments in primary haemopoietic cells.

Human autologous mixed lymphocyte reactivity is primarily specific for xenoprotein determinants adsorbed to antigen-presenting cells during rosette formation with sheep erythrocytes.

We present evidence that most T cells proliferating in response to autologous sheep erythrocyte (SRBC)-separated non-T cells (NT) cells are not specific for autoantigens but for antigens derived from xenogeneic sources. The conclusion was based on the following three observations. First, we found that NT cells isolated in the absence of xenoproteins by means of density gradient centrifugation on Percoll only weakly stimulated autologous T cells. Because this weak proliferation could not be expanded in restimulation experiments, its significance as an immune recognitive event remains questionable. NT cells isolated by the above method in the absence of xenogeneic determinants readily acquired stimulatory capacity after brief exposure to either SRBC or fetal calf serum. Second, restimulation of T memory cells generated in 1 degree autologous mixed lymphocyte reaction (AMLR) against SRBC-separated autologous NT cells was exclusively seen when NT cells exposed to or separated with xenoproteins were used for restimulation. Third, T memory cells generated against SRBC-separated autologous NT cells were specifically restimulated by autologous Percoll-separated NT cells that had been pulsed with a variety of xenogeneic mammalian sera. These xenogeneic determinants were preferentially recognized in context with autologous HLA-DR+ cells. From these findings and from our previous results that indicated an absolute requirement of HLA-DR+-adherent NT cells (8), we conclude that human AMLR primarily does not represent an autoantigen but a xenoantigen response that is genetically restricted by the HLA-DR type of the antigen-presenting cell.

Sensory adaptation in naive peripheral CD4 T cells.

T cell receptor interactions with peptide/major histocompatibility complex (pMHC) ligands control the selection of T cells in the thymus as well as their homeostasis in peripheral lymphoid organs. Here we show that pMHC contact modulates the expression of CD5 by naive CD4 T cells in a process that requires the continued expression of p56(lck). Reduced CD5 levels in T cells deprived of pMHC contact are predictive of elevated Ca(2)+ responses to subsequent TCR engagement by anti-CD3 or nominal antigen. Adaptation to peripheral pMHC contact may be important for regulating naive CD4 T cell responsiveness.

How many thymocytes audition for selection?

T cell maturation requires the rearrangement of clonotypic T cell receptors (TCR) capable of interacting with major histocompatibility complex (MHC) ligands to initiate positive and negative selection. Only 3-5% of thymocytes mature to join the peripheral T cell pool. To investigate the basis for this low success rate, we have measured the frequency of preselection thymocytes capable of responding to MHC. As many as one in five MHC-naive thymocytes show upregulation of activation markers on exposure to MHC-expressing thymic stroma in short-term reaggregate culture. The majority of these cells display physiological changes consistent with entry into the selection process within 24 h. By exposing TCR transgenic thymocytes to a range of MHC-peptide complexes, we show that CD69 induction is indicative of thymocyte selection, positive or negative. Our data provide evidence that the fraction of thymocytes that qualify to enter the thymic selection process far exceeds the fraction that successfully complete it, and suggest that most MHC-reactive thymocytes are actively eliminated in the course of selection.

T cell development: new approaches.

The differentiation of T lymphocyte precursors into functionally mature progeny proceeds in distinct stages. Since these are identified by characteristic constellations of phenotypic markers, the effects of experimental manipulations on T cell development can be readily monitored. In order to complete their developmental program, thymocytes must interact with stromal elements, which positively and negatively select the functional T cell receptor (TCR) repertoire. This process normally assures self tolerance and immunocompetence. Disturbances are of practical importance for clinical disorders including immunodeficiencies and autoimmune phenomena, raising a particular interest in human T cell development and repertoire formation. Here, we discuss results and possible applications of a culture system for human thymocytes. Further, we describe an in vitro approach addressing the requirements for a crucial step in T cell development; the transition from the immature CD4 CD8 double positive (DP) to the mature CD4 single positive (SP) stage.

In vitro construction of graded thymus chimeras.

A simple in vitro approach is described for constructing chimeric thymi in which the degree of chimeric contribution can be tightly controlled. This is achieved by protease-assisted dissociation of fresh fetal thymic tissue, mixing the resulting cell suspensions at the desired ratios, and subsequent reaggregation. Analysis of these graded chimeras shows that stromal elements become evenly interspersed and form a microenvironment able to support the maturation of endogenous T cell precursors. Chimeras between normal and MHC-deficient thymi have been used to demonstrate the rescue of mutant, allotype-marked thymocytes by wild-type stromal cells. Other potential applications of the method include quantitative studies on positive and negative thymic selection, the functional role of defined stromal cell types in these processes, and the analysis of mutants (either natural or engineered) by complementation.

Establishment of efficient reaggregation culture system for gene transfection into immature T cells by retroviral vectors.

To overcome low efficiency of retroviral infection into immature T cells, we modified reaggregation fetal thymus organ culture by closely packed co-culture with virus-producing cells (VPC). The viral vector was constructed in chimeric vector, pMX, with IRES and tailless-rat CD2 as a surface marker of infected cells. A rearranged TCR beta gene (Vbeta8.2) was further inserted into the construct for investigating effect of the introduced gene in T cell development. Using this system, we succeeded to transfer the viral vector into immature thymocytes at a remarkably higher efficiency compared to conventional methods using medium containing retrovirus. Moreover, the introduced TCR beta gene was expressed on thymocytes of RAG2-deficient mice to induce in the transition of CD4-CD8- double-negative (DN) into CD4+CD8+ double-positive (DP) cells by transducing beta-selection signaling. Thus, our modified reaggregation culture system is useful for studying the molecular mechanism of T cell development due to a highly efficient gene transfer into immature T cells.

Progress in T cell biology.

We outline recent work in our laboratories on thymus progenitors, lineages within the thymus, interactions between regulatory and effector lymphocytes, splitting the CD4 (T4) T cell subset, and Ir and Is genes. We highlight the possibilities for future research opened up by the demonstration that certain marrow-derived cell lines can repopulate thymic lobes in culture, and also the deep insight into the logical structure of the lymph node provided by our ability to make an exact comparison between two-cell-type and three-cell-type immunoregulatory clusters.

Attempts to localize the defect in dysgammaglobulinemia of UM-B19 chickens by studying the effect of immunomodulating substances on immunoglobulin and antibody production.

The extreme decreased levels of IgG in dysgammaglobulinemic UM-B19 chickens are linked with decreased antibody activity. Antibody activity to T-dependent (although diminished) and T-independent antigens is present but is restricted to IgM and IgA antibodies. Complete Freund's adjuvant enhances the existing isotype pattern of serum immunoglobulins and antibodies. The antibody response to a "T-independent" antigen (B. abortus) is greatly increased by CFA in dysgammaglobulinemic chickens. Beside the appearance of high levels of IgG in dysgammaglobulinemic chickens during the first weeks of life and in transitory dysgammaglobulinemia, remarkable IgG synthesis can be temporarily induced by the mitogenic activity of LPS and even more by the regulatory function of Levamisole. Furthermore, LPS and Levamisole induce IgG antibody synthesis to concomitantly administered antigen, the IgG antibodies appearing within the normal time. Contrary to a missing feedback inhibition from total IgG to IgM serum immunoglobulins, a feedback inhibition from IgG to IgM antibodies is found. No correlation can be found between Levamisole-induced IgG immunoglobulin concentrations, and IgG antibodies. Germfree rearing for one week or longer prevents the dysgammaglobulinemic defect. The following conclusions are drawn: Early antigenic stimulation seems to be the inducing factor for dysgammaglobulinemia in UM-B19 chickens. A BG cell pool is still present in adult dysgammaglobulinemic chickens. This BG cell pool is probably diminished to a varying extent. T cell helper functions seem to be present (albeit they may be disturbed) and can be stimulated. IgG specific T cell suppression is probable. From these conclusions the etiology of the dysgammaglobulinemia in UM-B19 chickens is hypothesized to be primarily due to delayed bursal development: Immature BG cells are eliminated by environmental antigens during the neonatal period in a process similar to tolerance induction. This event, in turn, induces suppressor mechanism(s) or disturbance in helper mechanism(s).

Tracing interactions of thymocytes with individual stromal cell partners.

Cellular interactions in T cell development can be analyzed using thymus chimeras prepared in vitro, in which stromal cells and T cell precursors are manipulated separately. In an earlier study, we showed that for optimal T cell maturation most--if not all--stromal cells must display appropriate (selecting) major histocompatibility complex (MHC) molecules: the substitution of selecting by nonselecting stromal cells leads to a proportional decrease in mature T cell production. These data imply that the availability of selecting stromal micro-environments is rate limiting for positive selection, and that in positive selection each thymocyte engages only one (rather than multiple) stromal cell partners. To test this hypothesis, we developed a tracing system for thymocyte/stromal cell interactions, based on the acquisition by thymocytes of stroma-derived MHC class II determinants. When MHC class II-deficient precursors are placed in H-2b x k F1 environments (where all stromal cells co-express H-2b and H-2k), individual thymocytes acquire class II determinants of both haplotypes. In striking contrast, when placed in mosaic stromal environments (where stromal cells express either H-2b or H-2k evenly interspersed), individual thymocytes preferentially acquire MHC class II determinants of one or the other haplotypes, but rarely both. This provides strong evidence that thymocytes have intimate interactions with individual stromal cells: having engaged one stromal cell niche, thymocytes do not (or only rarely) have promiscuous liaisons with others.

Evidence for differential expression of CD45 isoforms by precursors for memory-dependent and independent cytotoxic responses: human CD8 memory CTLp selectively express CD45RO (UCHL1).

On the basis of differential CD45 expression, human T cells can be separated into approximately reciprocal populations. The mAb UCHL1 detects a 180 kd molecular mass isoform, CD45R0. The CD45RA cluster of mAbs reacts with CD45 isoforms of 205 and 220 kd molecular mass. These reagents subdivide both CD4 and CD8 T cell populations. CD4 T cells that proliferate in response to memory-dependent (recall) antigens have been shown to selectively express CD45R0. We extend these observations to a model for cytotoxic responses that allows the functional analysis of CD8 T cells with differential CD45 expression. Precursors for allo-specific CTL responses are readily detectable in CD45R0 as well as CD45RA populations. In contrast, we find memory CTLp greatly enriched among CD45R0 cells. In combination with earlier work, these results suggest that differential expression of CD45 isoforms is associated with memory formation for different classes of immune responses in both major T cell lineages.

CD45 isoform switching precedes the activation-driven death of human thymocytes by apoptosis.

A recently described chimaeric organ culture system is employed to investigate the effects of antibodies to the CD3 and CD2 cell surface structures on the fate of developing human thymocyte populations. Engagement of CD3 as well as CD2 with reagents that stimulate peripheral human T cell results in the activation-induced death of human thymocytes in organ culture. Death is preceded by a characteristic phenotypic change; thymocytes that bear CD45RA (determinants associated with high-molecular-mass isoforms of the leukocyte common antigen family, CD45) are first induced to co-express CD45RO (the low-molecular-mass isoform of CD45), and subsequently lose CD45RA expression. This antigenic change is followed by cellular DNA fragmentation, characteristic of apoptosis. We conclude that engagement of CD3 as well as CD2 can recruit human CD45RA+ thymocytes into the CD45RO+ population, where cell death occurs. Our results suggest that this phenotype conversion is a marker for thymocytes destined for programmed cell death.

Human postnatal thymocytes generate phenotypically immature CD3dim, CD5dim, CD1abright progeny in organ culture.

We previously described an organ culture system that supports the in vitro development of human fetal thymocytes in murine alymphoid thymic rudiments without addition of exogenous factors. This approach to study human T cell precursors is limited by the requirement for fetal tissue. We show that human thymocytes from pediatric sources can be expanded under similar conditions. Organ cultures generated predominantly CD4 CD8 double positive cells, most of which maintained the phenotype CD3dim, CD5dim, and CD1abright, characteristic for double positive thymocytes ex vivo. This culture system should facilitate in vitro studies on human thymocyte development and repertoire selection.

MHC control of the naive TCR alpha-chain repertoire.

The naive T cell repertoire is shaped by interactions between developing thymocytes and thymic stroma. Both positive and negative selection involve the clonotypic TCR and MHC molecules carrying self-peptides. Except for the MHC-dependent effects of superantigens on TCR V beta usage; there has been little evidence that the TCR structure of naive T cell varies with the selecting MHC products. To examine this point from another angle, in particular the TCR alpha-chain, we have analyzed alpha-chain usage in a system in which the vast majority of T cells express a transgene-encoded TCR beta-chain, compatible with efficient T cell development on a wide range of MHC haplotypes. Endogenous TCR alpha-chains are thus selected without interference from the forces known to act on TCR-beta, permitting us to observe MHC influences on alpha-chain selection. We have used V alpha-specific Abs to quantitate alpha-chain usage in MHC congenic, MHC recombinant, and MHC transgenic mice and provide evidence that the naive TCR alpha-chain repertoire is under MHC control. The data demonstrate a direct impact of known MHC class II products but also reflect more complex influences, apparently involving other gene products within the MHC. Sequence analysis of differentially selected TCR suggests that selection acts on the entire alpha-chain, including V alpha, J alpha, and the junctional region.

Evidence for a single-niche model of positive selection.

Thymocyte maturation depends on interactions with thymic stromal elements expressing major histocompatibility complex (MHC) molecules. Mutant mouse strains lacking MHC class I (beta 2-microglobulin-null) or class II (A beta-null) expression fail to generate normal CD8 or CD4 T-cell populations and provide model systems for reconstitution experiments. We have constructed in vitro chimeras between normal and MHC-deficient thymi to evaluate the efficiency of positive selection. Unexpectedly, the generation of mature single-positive thymocytes was proportional to the fraction of wild-type (i.e., MHC-expressing) stroma over a wide range of chimerism. Similar results were obtained for the development of T-cell receptor-transgenic thymocytes in graded chimeras expressing selecting and nonselecting MHC alleles. These findings are best explained by hypothesizing that positive selection involves a rate-limiting step at which each thymocyte can interact with only one stromal cell niche.

[The ionized calcium in the blood of domestic chickens: dependence on age and sex]. / Das ionisierte Calcium im Blut des Haushuhnes: Abhängigkeit von Alter und Geschlecht.

In the present study concentration of ionized calcium and the correlation between total and ionized calcium in blood of domestic fowl and its dependence on age and sex were investigated. Furthermore a possible influence of blood sampling procedure on the measured values was assessed. Leghorn chickens of either sex, aged 3, 9, 25, and 80 weeks, respectively, were used. Blood sampling was performed by vein-puncture and indwelling catheters, respectively, of both ulnaris vein and artery of animals which had been fasting for 12 hours. Determinations of ionized calcium concentration was carried out by use of an ion-selective electrode (ICA1), and of total calcium concentration in plasma using atomic absorption, respectively. After attainment of sexual maturity total calcium in roosters rose from 2.73 to 2.85 mmol/l, whereas ionized calcium fell from 1.45 to 1.40 mmol/l and per cent fraction of ionized/total calcium (extent of ionization) from 53.1 to 49.1%, respectively. After attainment of sexual maturity in hens, the content of total calcium in venous blood rose from 2.78 to 5.78 mmol/l, and of ionized calcium from 1.45 to 1.59 mmol/l, respectively, whereas the extent of ionization was significantly reduced from 52.2 to 27.1%. On comparison of the vein-puncture and indwelling catheter technique a marked influence of the blood sampling procedure on calcium concentrations measured in blood of chicken became evident. On attainment of sexual maturity both the arterial and the venous blood (sampled from indwelling catheters) of hens showed levels of ionized calcium about 8% higher as compared to roosters. The extent of ionization amounted to 39% in sexually mature females as opposed to 51% in males. Concentrations of calcium ions in venous blood averaged 0.07 to 0.08 mmol/l higher compared to arterial blood in either sex. Concentration of ionized calcium did not exhibit any correlation to total calcium in adult animals. However, the concentration of ionized calcium was inversely correlated to blood pH.

[The ionized calcium in the blood of domestic chickens: dependence on the laying cycle]. / Das ionisierte Calcium im Blut des Haushuhnes: Abhängigkeit vom Legezyklus.

In the present study the plasma concentration of both ionized and total calcium in arterial as well as in venous blood of Leghorn hens and its dependence on the ovulatory cycle were investigated. During the ovulatory cycle, the concentration of ionized calcium in blood of hens follows the course of a sine curve. Maximum calcium ion concentrations (1.58 mmol/l in venous blood and 1.48 mmol/l in arterial blood, respectively) were observed 3 to 6 hours after oviposition, and minimum concentrations (1.26 and 1.19 mmol/l, respectively) at 18 to 21 hours after oviposition. Changes in total and ionized calcium concentration went parallel. Six to 9 hours after oviposition maximum values (4.24 and 4.15 mmol/l respectively) and 21 to 24 hours after oviposition minimum values (3.25 and 2.99 mmol/l, respectively) were measured. The arterio-venous difference was -0.22 mmol/l on average.

Selective manipulation of the human T-cell receptor repertoire expressed by thymocytes in organ culture.

A recently described organ culture system for human thymocytes is shown to support the generation of a diverse T-cell receptor repertoire in vitro: thymocytes of the alpha beta lineage, including representatives of the V beta families 5.2/5.3, 6.7, and 8, accounted for the majority of T-cell receptor-positive cells throughout a 3-week culture period. Thymocytes bearing gamma delta receptors were also identified, particularly among the CD4 CD8 double-negative subset. The T-cell receptor repertoire expressed in organ culture responded to experimental manipulation with staphylococcal enterotoxins. Staphylococcal enterotoxin D (a powerful activator of human peripheral T cells expressing V beta 5.2/5.3 receptors) caused a marked reduction of V beta 5.2/5.3 expression, as determined with the V beta-specific antibody 42/1C1. Evidence is presented that this loss of V beta 5.2/5.3 expression resulted from the selective deletion of activated thymocytes by apoptosis, in concert with T-cell receptor modulation. These effects of staphylococcal enterotoxin D were specific (since staphylococcal enterotoxin E did not influence V beta 5.2/5.3 expression) and V beta-selective (since expression of V beta 6.7 remained unaffected by staphylococcal enterotoxin D). On the basis of these observations, we suggest that thymic organ culture provides a powerful approach to study the generation of the human T-cell repertoire.

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.