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.

The tight interallelic positional coincidence that distinguishes T-cell receptor Jalpha usage does not result from homologous chromosomal pairing during ValphaJalpha rearrangement.

The T-cell receptor (TCR) alpha locus is thought to undergo multiple cycles of secondary rearrangements that maximize the generation of alphabeta T cells. Taking advantage of the nucleotide sequence of the human Valpha and Jalpha segments, we undertook a locus-wide analysis of TCRalpha gene rearrangements in human alphabeta T-cell clones. In most clones, ValphaJalpha rearrangements occurred on both homologous chromosomes and, remarkably, resulted in the use of two neighboring Jalpha segments. No such interallelic coincidence was found for the position of the two rearranged Valpha segments, and there was only a loose correlation between the 5' or 3' chromosomal position of the Valpha and Jalpha segments used in a given rearrangement. These observations question the occurrence of extensive rounds of secondary Valpha-->Jalpha rearrangements and of a coordinated and polarized usage of the Valpha and Jalpha libraries. Fluorescence in situ hybridization analysis of developing T cells in which TCRalpha rearrangements are taking place showed that the interallelic positional coincidence in Jalpha usage cannot be explained by the stable juxtaposition of homologous Jalpha clusters.

Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes.

Individual B lymphocytes normally express immunoglobulin (Ig) proteins derived from single Ig heavy chain (H) and light chain (L) alleles. Allelic exclusion ensures monoallelic expression of Ig genes by each B cell to maintain single receptor specificity. Here we provide evidence that at later stages of B cell development, additional mechanisms may contribute to prioritizing expression of single IgH and IgL alleles. Fluorescent in situ hybridization analysis of primary splenic B cells isolated from normal and genetically manipulated mice showed that endogenous IgH, kappa and lambda alleles localized to different subnuclear environments after activation and had differential expression patterns. However, this differential recruitment and expression of Ig alleles was not typically seen among transformed B cell lines. These data raise the possibility that epigenetic factors help maintain the monoallelic expression of Ig.

Down-regulation of TDT transcription in CD4(+)CD8(+) thymocytes by Ikaros proteins in direct competition with an Ets activator.

Ikaros is a unique regulator of lymphopoiesis that associates with pericentromeric heterochromatin and has been implicated in heritable gene inactivation. Binding and competition experiments demonstrate that Ikaros dimers compete with an Ets activator for occupancy of the lymphocyte-specific TdT promoter. Mutations that selectively disrupt Ikaros binding to an integrated TdT promoter had no effect on promoter function in a CD4(+)CD8(+) thymocyte line. However, these mutations abolished down-regulation on differentiation, providing evidence that Ikaros plays a direct role in repression. Reduced access to restriction enzyme cleavage suggested that chromatin alterations accompany down-regulation. The Ikaros-dependent down-regulation event and the observed chromatin alterations appear to precede pericentromeric repositioning. Current models propose that the functions of Ikaros should be disrupted by a small isoform that retains the dimerization domain and lacks the DNA-binding domain. Surprisingly, in the CD4(+)CD8(+) thymocyte line, overexpression of a small Ikaros isoform had no effect on differentiation or on the pericentromeric targeting and DNA-binding properties of Ikaros. Rather, the small isoform assembled into multimeric complexes with DNA-bound Ikaros at the pericentromeric foci. The capacity for in vivo multimer formation suggests that interactions between Ikaros dimers bound to the TdT promoter and those bound to pericentromeric repeat sequences may contribute to the pericentromeric repositioning of the inactive gene.

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.

Evolutionary conservation, developmental expression, and genomic mapping of mammalian Twisted gastrulation.

The twisted gastrulation gene (tsg) encodes a secreted protein required for the correct specification of dorsal midline cell fate during gastrulation in Drosophila. We report that tsg homologs from human, mouse, zebrafish, and Xenopus share 72-98% identity at the amino acid level and retain all 24 cysteine residues from Drosophila. In contrast to Drosophila where tsg expression is limited to early embryos, expression is found throughout mouse and human development. In Drosophila, tsg acts in synergy with decapentaplegic (dpp), a member of the TGF-beta family of secreted proteins. The vertebrate orthologs of dpp, BMP-2 and -4, are crucial for gastrulation and neural induction, and aberrant signaling by BMPs and other TGF-beta family members results in developmental defects including holoprosencephaly (HPE). Interestingly, human TSG maps to the HPE4 locus on Chromosome 18p11.3, and our analysis places the gene within 5 Mbp of TG-interacting factor (TGIF).

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.

Recessive expression of the H2A-controlled immune response phenotype depends critically on antigen dose.

Major histocompatibility complex (MHC) alleles acting as immune response genes are coexpressed in heterozygous individuals and therefore control of immune responses is usually codominant. As an exception to this rule, however, several examples of recessive immune responses have been ascribed to regulatory, e.g. suppressive, interactions. We report here that the recessive phenotype of both antibody and T-cell responses to the mycobacterial 16 000-MW antigen depends critically on a low antigen dose for immunization. On the basis of similar responses in hemi- and heterozygous mice, we suggest that the mechanism of recessive MHC control does not involve regulation by the low-responder allele. We also demonstrated mixed haplotype restriction of peptide recognition for a significant fraction of high-antigen-dose primed T cells. Their paucity under limiting antigen dose conditions may lead to the recessive expression of MHC control. In conclusion, our results suggest that recessive MHC control can be explained as a simple gene dosage effect under conditions where antigen is limiting, without a need for regulatory mechanisms.

Different doses of agonistic ligand drive the maturation of functional CD4 and CD8 T cells from immature precursors.

MHC molecules are normally required for the development of thymocytes from the CD4(+)CD8(+) double-positive to the CD4 or CD8 single-positive stage. Here we show that mitogenic plant lectins can substitute for MHC molecules in driving the differentiation of phenotypically and functionally mature CD4 as well as CD8 T cells. Interestingly, lectin dosage determines whether CD4 or CD8 cells are generated, indicating that variation of cumulative signal strength (not necessarily signal quality) can result in an apparent switching of lineage preference. Thymocyte perception of differentiation-inducing signals is modulated by the cellular context, since stimuli that yield CD8 cells in the context of the thymic microenvironment fail to do so in suspension culture and generate CD4 progeny instead. Finally, we show that lectin-generated single-positive thymocytes retain the ability to respond to the ligands initially used to drive their differentiation. Our results call into question generalizations and predictions made from other experimental systems and reveal that thymocyte selection is considerably more flexible than had been anticipated.

Intrathymic deletion of MHC class I-restricted cytotoxic T cell precursors by constitutive cross-presentation of exogenous antigen.

Cross-priming of cytotoxic T lymphocytes by professional antigen-presenting cells (APC) is a potential hazard to self tolerance because it exposes naive T cells to tissue-specific self antigens in the context of co-stimulatory signals. Here we show that cross-presentation of exogenous material occurs constitutively within the thymus. Although efficient cross-presentation is a property of relatively few APC it results in thymocyte deletion both in vitro and in vivo, suggesting that intrathymic cross-presentation can operate as an effective component of tolerance to circulating self antigens. The capacity of minor cell populations to mediate thymocyte deletion but not positive selection reflects an underlying difference in the biology of these two processes.

Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division.

We show that several transcriptionally inactive genes localize to centromeric heterochromatin in the nucleus of cycling but not quiescent (noncycling) primary B lymphocytes. In quiescent cells, centromeric repositioning of inactive loci was induced after mitogenic stimulation. A dynamic repositioning of selected genes was also observed in developing T cells. Rag and TdT loci were shown to relocate to centromeric domains following heritable gene silencing in primary CD4+8+ thymocytes, but not in a phenotypically similar cell line in which silencing occurred but was not heritable. Collectively, these data indicate that the spatial organization of genes in cycling and noncycling lymphocytes is different and that locus repositioning may be a feature of heritable gene silencing.

Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin.

Ikaros proteins are required for normal T, B, and NK cell development and are postulated to activate lymphocyte-specific gene expression. Here we examined Ikaros distribution in the nucleus of B lymphocytes using confocal microscopy and a novel immunofluorescence in situ hybridization (immuno-FISH) approach. Unexpectedly, Ikaros localized to discrete heterochromatin-containing foci in interphase nuclei, which comprise clusters of centromeric DNA as defined by gamma-satellite sequences and the abundance of heterochromatin protein-1 (HP-1). Using locus-specific probes for CD2, CD4, CD8alpha, CD19, CD45, and lambda5 genes, we show that transcriptionally inactive but not transcriptionally active genes associate with Ikaros-heterochromatin foci. These findings support a model of organization of the nucleus in which repressed genes are selectively recruited into centromeric domains.

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.

Rational primer design greatly improves differential display-PCR (DD-PCR).

Since its conception in 1992, differential display PCR (DD-PCR) has attracted widespread interest. Theoretically an attractive cloning approach, it combines the comparative analysis of several samples with the sensitivity of PCR. Although a large number of studies embracing this technology have been initiated, few novel genes of interest have been identified, suggesting that the method has not realised its potential. The present report shows that by modifying primer design, sampling of differentially expressed genes can be greatly enhanced and relevant genes can be isolated. Using our modified conditions DD-PCR efficiently screens a wide range of gene expression levels, in which differences are represented on a linear scale.

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.

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

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

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.

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.

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.