CD28/CTLA4 double deficient mice demonstrate crucial role for B7 co-stimulation in the induction of allergic lower airways disease.

BACKGROUND: The existence of a third B7-1/B7-2 receptor was postulated in a recent study using a novel mouse strain lacking both CD28 and CTLA4 (CD28/CTLA4-/-). OBJECTIVE: In the present study, it was investigated if T cell co-stimulation via the putative B7-1/B7-2 receptor plays a role in the induction of Th2-mediated asthma manifestations in mice. METHODS: BALB/c wild-type, CD28/CTLA4-/- and B7-1/B7-2-/- mice were sensitized and aerosol challenged with ovalbumin (OVA). RESULTS: At 24 h after the last aerosol, wild-type mice showed airway hyper-responsiveness in vivo and up-regulated levels of serum OVA-specific IgE compared with the situation shortly before OVA challenge. In addition, eosinophil numbers and IL-5 levels in the broncho-alveolar lavage fluid and Th2 cytokine production by lung cells upon OVA re-stimulation in vitro were observed. In agreement with an earlier study, we failed to induce any of the asthma manifestations in B7-1/B7-2-/- mice. Importantly, also CD28/CTLA4-/- mice showed no asthma manifestations upon OVA sensitization and challenge. CONCLUSION: These data clearly demonstrate that T cell co-stimulation via the putative B7-1/B7-2 receptor appears to have no role in the induction of Th2-mediated asthma manifestations in this murine model and, conversely, that CD28 signalling is crucial.

B7-dependent T-cell costimulation in mice lacking CD28 and CTLA4.

To examine whether B7 costimulation can be mediated by a molecule on T cells that is neither CD28 nor CTLA4, we generated mice lacking both of these receptors. CD28/CTLA4(-/-) mice resemble CD28(-/-) mice in having decreased expression of T-cell activation markers in vivo and decreased T-cell proliferation in vitro, as compared with wild-type mice. Using multiple approaches, we find B7-dependent costimulation in CD28/CTLA4(-/-) mice. The proliferation of CD28/CTLA4(-/-) T cells is inhibited by CTLA4-Ig and by the use of antigen-presenting cells lacking both B7-1 and B7-2. CD28/CTLA4(-/-) T-cell proliferation is increased by exposure to Chinese hamster ovary cells transfected with B7-1 or B7-2. Finally, administration of CTLA4-Ig to CD28/CTLA4(-/-) cardiac allograft recipients significantly prolongs graft survival. These data support the existence of an additional receptor for B7 molecules that is neither CD28 nor CTLA4.

p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours.

p73 (ref. 1) has high homology with the tumour suppressor p53 (refs 2-4), as well as with p63, a gene implicated in the maintenance of epithelial stem cells. Despite the localization of the p73 gene to chromosome 1p36.3, a region of frequent aberration in a wide range of human cancers, and the ability of p73 to transactivate p53 target genes, it is unclear whether p73 functions as a tumour suppressor. Here we show that mice functionally deficient for all p73 isoforms exhibit profound defects, including hippocampal dysgenesis, hydrocephalus, chronic infections and inflammation, as well as abnormalities in pheromone sensory pathways. In contrast to p53-deficient mice, however, those lacking p73 show no increased susceptibility to spontaneous tumorigenesis. We report the mechanistic basis of the hippocampal dysgenesis and the loss of pheromone responses, and show that new, potentially dominant-negative, p73 variants are the predominant expression products of this gene in developing and adult tissues. Our data suggest that there is a marked divergence in the physiological functions of the p53 family members, and reveal unique roles for p73 in neurogenesis, sensory pathways and homeostatic control.

The role of CTLA-4 in regulating Th2 differentiation.

To examine the role of CTLA-4 in Th cell differentiation, we used two newly generated CTLA-4-deficient (CTLA-4-/-) mouse strains: DO11. 10 CTLA-4-/- mice carrying a class II restricted transgenic TCR specific for OVA, and mice lacking CTLA-4, B7.1 and B7.2 (CTLA-4-/- B7.1/B7.2-/- ). When purified naive CD4+ DO11.10 T cells from CTLA-4-/- and wild-type mice were primed and restimulated in vitro with peptide Ag, CTLA-4-/- DO11.10 T cells developed into Th2 cells, whereas wild-type DO11.10 T cells developed into Th1 cells. Similarly, when CTLA-4-/- CD4+ T cells from mice lacking CTLA-4, B7. 1, and B7.2 were stimulated in vitro with anti-CD3 Ab and wild-type APC, these CTLA-4-/- CD4+ T cells produced IL-4 even during the primary stimulation, whereas CD4+ cells from B7.1/B7.2-/- mice did not produce IL-4. Upon secondary stimulation, CD4+ T cells from CTLA-4-/- B7.1/B7.2-/- mice secreted high levels of IL-4, whereas CD4+ T cells from B7.1/B7.2-/- mice produced IFN-gamma. In contrast to the effects on CD4+ Th differentiation, the absence of CTLA-4 resulted in only a modest effect on T cell proliferation, and increased proliferation of CTLA-4-/- CD4+ T cells was seen only during secondary stimulation in vitro. Administration of a stimulatory anti-CD28 Ab in vivo induced IL-4 production in CTLA-4-/- B7.1/B7.2-/- but not wild-type mice. These studies demonstrate that CTLA-4 is a critical and potent inhibitor of Th2 differentiation. Thus, the B7-CD28/CTLA-4 pathway plays a critical role in regulating Th2 differentiation in two ways: CD28 promotes Th2 differentiation while CTLA-4 limits Th2 differentiation.

CTLA-4 and T cell activation.

The past year has seen significant advances in our understanding of the role of cytotoxic T lymphocyte antigen 4 (CTLA-4) in regulating T cell activation and tolerance. Recent studies indicate that CTLA-4 not only counterbalances CD28 signals but also can inhibit T cell responses independently of CD28. Recent work has also revealed a role for CTLA-4 in regulating Th1/Th2 differentiation. Manipulation of CTLA-4 in animal models of autoimmunity has shown that CTLA-4 regulates both the initiation and the progression of autoimmune diseases.

Cell-fate decisions in early T cell development: regulation by cytokine receptors and the pre-TCR.

During lymphocyte development, cell-fate decisions are determined by a myriad of signals produced by the micro- environment of the thymus and the bone marrow. These yet to be fully defined developmental cues regulate stage-specific gene expression, and the extraordinarily well-characterized stages of T and B cell development have provided attractive model systems for studying regulation of cellular differentiation. In particular, studies on the contribution of both antigen receptors and cytokine receptors to lymphoid development have illuminated essential signalling pathways in early T and B cells. Here, we review investigations supporting an obligatory role for the IL-7 receptor pathway in early T cell development. IL-7 is produced by both thymus and bone marrow stromal cells, and its potential contribution to survival, differentiation and proliferation of pro-T cells is discussed. We also address the contribution of the pre-T cell receptor (pre-TCR) to differentiation past the pro-T cell stage, and recent advances in deciphering the composition and function of the pre-TCR complex are discussed. Finally, we suggest future directions in this field that may serve to reveal whether and how signals initiated by the cytokine receptors and pre-TCR may intersect, and to define which down-stream molecular events are regulated by these receptors.

Induction of TCR gene rearrangements in uncommitted stem cells by a subset of IL-7 producing, MHC class-II-expressing thymic stromal cells.

The embryonic thymic microenvironment provides the necessary elements for T cell lineage commitment, but the precise role of individual stromal cell components remains to be determined. Here we address the question of which stromal cell types are required for initiation of V-DJ rearrangements of the TCR-beta and TCR-delta locus in CD117+CD45+ uncommitted fetal liver progenitors. We show that fetal thymic stroma alone is necessary and sufficient for induction of TCR-beta and TCR-delta rearrangements. Furthermore, the ability to induce this T cell commitment step is confined to a subset of MHC class II-positive epithelial cells. Thymic stroma derived from mice with a targeted deletion in the IL-7 gene, however, lacks this ability. These findings set the stage for a further definition of the nature of the thymic stromal cell support in the regulation of T cell commitment.

Expression of tyrosine kinase gene in mouse thymic stromal cells.

Amongst the most important signal transduction molecules involved in regulating growth and differentiation are the protein tyrosine kinases (PTK). Since T cell development is a consequence of interactions between thymic stromal cells (TSC) and thymocytes, identification of the PTK in both compartments is required to dissect the mechanisms that control this process. Here we report a search for PTK in mouse TSC, using RT-PCR to survey the repertoire of PTK mRNAs expressed in a freshly isolated TSC preparation. We identified 10 different PTK cDNAs among the 216 cDNAs sequenced, and demonstrate that transcripts of three of those (ufo, fyn and fer) are widely expressed among a large panel of immortalized thymic epithelial cell lines (TEC) and in primary cultures of TSC. Of the other seven, none were expressed in established TEC lines but, instead, displayed distinct expression patterns in cell types likely to have contaminated the fresh TSC preparation, i.e., macrophages, B cells, T cells and fibroblasts. Among the three PTK expressed in TEC lines, only one, ufo, exhibited expression exclusively in cells of non-hemopoietic origin. Although expression of ufo (also known as tyro 7, axl or ark) is not thymic-specific, in that it is also expressed in cell types of mesodermal origin in other tissues, its presence in TEC suggests a role for ufo in differentiation of the TSC compartment. Consistent with this notion, high-level expression of this receptor PTK at the protein level could be documented in every TEC line investigated, as well as in fresh thymus tissue sections. These data provide the first example of a receptor PTK in TSC and open new approaches to study the regulation of TSC differentiation.

XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos.

XTcf-3 is a maternally expressed Xenopus homolog of the mammalian HMG box factors Tcf-1 and Lef-1. The N-terminus of XTcf-3 binds to beta-catenin. Microinjection of XTcf-3 mRNA in embryos results in nuclear translocation of beta-catenin. The beta-catenin-XTcf-3 complex activates transcription in a transient reporter gene assay, while XTcf-3 by itself is silent. N-terminal deletion of XTcf-3 (delta N) abrogates the interaction with beta-catenin, as well as the consequent transcription activation. This dominant-negative delta N mutant suppresses the induction of axis duplication by microinjected beta-catenin. It also suppresses endogenous axis specification upon injection into the dorsal blastomeres of a 4-cell-stage embryo. We propose that signaling by beta-catenin involves complex formation with XTcf-3, followed by nuclear translocation and activation of specific XTcf-3 target genes.

Defects in cardiac outflow tract formation and pro-B-lymphocyte expansion in mice lacking Sox-4.

A striking example of the relationship between regulation of transcription and phenotype is the central role of the Y-chromosomal gene Sry in mammalian sex determination. Sry is the founding member of a large family of so-called Sox genes. During murine embryogenesis, the transcriptional activator Sox-4 is expressed at several sites, but in adult mice expression is restricted to immature B and T lymphocytes. Using targeted gene distruption, we have found that SOX-4(-/-) embryos succumb to circulatory failure at day E14. This was a result of impaired development of the endocardial ridges (a specific site of Sox-4 expression) into the semilunar valves and the outlet portion of the muscular ventricular septum. The observed range of septation defects is known as 'common arterial trunk' in man. We studied haemopoiesis in lethally irradiated mice reconstituted with SOX-4(-/-) fetal liver cells and found that a specific block occurred in B-cell development at the pro-B cell stage. In line with this, the frequency and proliferative capacity of IL-7-responsive B cell progenitors in fetal liver were severely decreased in vitro.

An HMG-box-containing T-cell factor required for thymocyte differentiation.

Two candidate genes for controlling thymocyte differentiation, T-cell factor-1 (Tcf-1) and lymphoid enhancer-binding factor (Lef-1), encode closely related DNA-binding HMG-box proteins. Their expression pattern is complex and largely overlapping during embryogenesis, yet restricted to lymphocytes postnatally. Here we generate two independent germline mutations in Tcf-1 and find that thymocyte development in (otherwise normal) mutant mice is blocked at the transition from the CD8+, immature single-positive to the CD4+/CD8+ double-positive stage. In contrast to wild-type mice, most of the immature single-positive cells in the mutants are not in the cell cycle and the number of immunocompetent T cells in peripheral lymphoid organs is reduced. We conclude that Tcf-1 controls an essential step in thymocyte differentiation.

Transcription factors in early T-cell development.

Characterization of the transcription control regions for most genes encoding the TCR-CD3 complex has been followed by identification of DNA-binding factors, some of which are restricted to lymphoid or T-lineage cells. The sequential expression of these factors is linked with the appearance of stage-specific T-cell markers, thus controlling the commitment of the elusive lymphoid progenitor cell and its progression along the T-lineage. Here, Hans Clevers, Mariëtte Oosterwegel and Katia Georgopoulos discuss recent progress made in the recognition of transcription factors that regulate these early events in T-cell ontogeny.

Sox-4, an Sry-like HMG box protein, is a transcriptional activator in lymphocytes.

Previous studies in lymphocytes have described two DNA-binding HMG box proteins, TCF-1 and LEF-1, with affinity for the A/TA/TCAAAG motif found in several T cell-specific enhancers. Evaluation of cotransfection experiments in non-T cells and the observed inactivity of an AACAAAG concatamer in the TCF-1/LEF-1-expressing T cell line BW5147, led us to conclude that these two proteins did not mediate the observed enhancer effect. We therefore searched for additional HMG box proteins. By a PCR-aided strategy, we cloned Sox-4, a gene with homology to the HMG box region of the sex determining gene SRY. Sox-4 was expressed in T and pre-B lymphocyte lines and in the murine thymus. Significantly, BW5147 T cells did not express Sox-4. Recombinant Sox-4 bound with high affinity (Kd 3 x 10(-11) M) to the minor groove of the AACAAAG motif, most likely contacting all seven base pairs. In contrast with observations on TCF-1 and LEF-1, cotransfection with Sox-4 unveiled a transactivating capacity, which mapped to its serine-rich C terminus. This region remained functional upon grafting onto a GAL4 DNA-binding domain. Sox-4 is thus the first 'classical' transcription factor in the Sox gene family with separable DNA-binding and transactivation domains. Our observations indicate that a detailed understanding of T cell-specific gene control must integrate the concerted activity of at least three tissue-specific HMG box genes.

HMG box proteins in early T-cell differentiation.

The central theme of this review is the molecular basis for commitment of cells to the T-cell lineage. Principles of transcriptional regulation are illustrated by two examples; the role of GATA-1 during erythroid differentiation and the function of MyoD-like proteins in myogenesis. Several regulatory proteins have been described in the T-cell lineage. Here, we focus attention on the HMG box family of DNA binding proteins. This recently defined family can be divided in two subfamilies: the HMG/UBF and the TCF/SOX group. The first group contains at least two HMG boxes and binds DNA non-specifically, while the other group of proteins has one HMG box and interacts with DNA sequence-specifically. Characteristics of the most prominent members of both subfamilies will be discussed. In particular, we will address the role of HMG box proteins in controlling the expression of T-cell specific proteins during differentiation.

Differential expression of the HMG box factors TCF-1 and LEF-1 during murine embryogenesis.

The recent identification of a number of T lymphocyte-specific enhancers has allowed the cloning of several novel transcription factors. Two of these, TCF-1 and LEF-1, contain a virtually identical DNA-binding domain of the High Mobility Group (HMG-1) box type. TCF-1 and LEF-1 originate from a recent gene duplication event as evidenced by comparison with the chicken homologue, chTCF. We have now analyzed the differential expression of these two transcription factors. In a panel of lymphoid cell lines, TCF-1 was exclusively expressed in the T cell lineage. In contrast, LEF-1 mRNA was detected at equivalent levels in pro- and pre-B cells and in all T lineage cells. In situ hybridization on murine embryos revealed that TCF-1 and LEF-1 were widely expressed at day 7.5 of gestation. At later stages, the expression patterns were complex and only partially overlapping. The expression of TCF-1 and LEF-1 coincided until day 10.5, when mRNAs were detected in limb buds, neural crest, pharyngeal arches and nasal process. At later time points (day 13.5 to 14.5), sites of overlapping expression included lung, the urogenital system, tooth buds, thymus and choroid plexus. Unique expression sites for TCF-1 included Reichert's membrane and trophectoderm-derived cells, the ribs and thoracic prevertebrae, craniofacial structures, the adrenal gland and meninges. Unique LEF-1 expression was observed in the tail prevertebrae, brain and inner ear. Postnatally, expression of both genes could only be detected in lymphoid tissues. These observations suggest that TCF-1 and LEF-1 exert differential functions during murine embryogenesis.

The human T cell transcription factor-1 gene. Structure, localization, and promoter characterization.

We have recently isolated cDNA clones representing four alternative splice forms of a T cell-specific transcription factor, TCF-1. Here we report the characterization of the human gene encoding this factor. The TCF-1 gene is contained in 10 exons including an untranslated first exon. The DNA-binding high mobility group (HMG) box of TCF-1 is encoded by the closely spaced exons VI and VII. Differential splicing involves an alternative exon (IX) and three splice acceptor sites in exon X. Based on comparison of sequence and on the placement of an alternative exon, TCF-1 appears closely related to the recently characterized HMG box transcription factor TCF-1 alpha/LEF. In particular, the HMG boxes encoded by the two TCF genes are virtually identical. The TCF-1 gene resides on chromosome 5 band q31.1. The TCF-1 promoter coincides with a CpG island. As determined by chloramphenicol acetyltransferase analysis, the promoter is preferentially active in T cells. The promoter does not contain TCF-1/TCF-1 alpha binding sites and is therefore not autoregulated. This observation implies the existence of yet uncharacterized T cell transcription factors that are active during early T cell differentiation.

Expression of GATA-3 during lymphocyte differentiation and mouse embryogenesis.

The GATA family of C4 zinc-finger transcription factors has been implicated in tissue-specific gene regulation in birds and mammals. One of the members of this family, GATA-3, is reportedly expressed specifically in the T-cell lineage, where it interacts with GATA motifs in the TCR-alpha, TCR-beta, and TCR-delta enhancers, thereby controlling the T-cell phenotype. To evaluate the differentiation control properties of GATA-3, we have now documented its expression pattern during lymphoid differentiation and murine embryogenesis. The onset of GATA-3 expression in the lymphoid lineage was studied in a panel of lymphoid (precursor) cell lines by Northern blot analysis. GATA-3 was uniquely expressed in T-lineage lymphocytes expressing TCR and CD3 genes; it was absent from TCR/CD3 mRNA-negative prothymocytes and from all B-lineage cells. In order to obtain information on the expression of GATA-3 outside the immune system, in situ hybridization was performed on mouse embryos on day 11.5-14.5 of gestation. GATA-3 mRNA was detected in fetal thymus and in erythroid cells. Outside the haemopoietic system, we detected GATA-3 mRNA throughout the central nervous system, in kidney, in the epidermis, lens fibers, the inner ear, whisker follicles, and in the primary palate. These data provide new clues about the potential role of GATA-3 during mouse development, and will aid the interpretation of currently ongoing gene knockout experiments.

TCF-1, a T cell-specific transcription factor of the HMG box family, interacts with sequence motifs in the TCR beta and TCR delta enhancers.

We have recently identified and cloned TCF-1, a T cell-specific transcription factor with specificity for the AACAAAG motif in the CD3 epsilon enhancer and for the TTCAAAG motif in the TCR alpha enhancer. TCF-1 belongs to the family of transcription-regulating proteins which share a region of homology termed the HMG-box. Here, we show by gel retardation analysis that TCF-1 specifically recognizes the T beta 5 element of the TCR beta enhancer and the T delta 7 element of the TCR delta enhancer. Comparison of the sequences of all elements recognized by TCF-1 defines a consensus motif A/T A/T C A A/G A G. These observations imply that TCF-1 is involved in the control of several T cell-specific genes and might thus play an important role in the establishment and maintenance of the mature T cell phenotype.

Cloning of murine TCF-1, a T cell-specific transcription factor interacting with functional motifs in the CD3-epsilon and T cell receptor alpha enhancers.

CD3-epsilon gene expression is confined to the T cell lineage. We have recently identified and cloned a human transcription factor, TCF-1, that binds to a functional element in the T lymphocyte-specific enhancer of CD3-epsilon. In a panel of human cell lines, TCF-1 expression was restricted to T lineage cells. TCF-1 belonged to a novel family of genes that contain the so-called high mobility group 1 (HMG) box. Here we report the cloning of murine TCF-1. Two splice alternatives were identified that were not previously observed in human TCF-1. Murine and human TCF-1 displayed a 95.5% overall amino acid homology. Recombinant murine and human TCF-1 recognized the same sequence motif in the CD3-epsilon enhancer as judged by gel retardation and methylation interference assays. With the murine cDNA clones several aspects of TCF-1 were analyzed. First, deletion analysis revealed that a region of TCF-1 containing the HMG box was sufficient for sequence-specific binding. Second, by high stringency Northern blotting and in situ hybridization, TCF-1 expression was shown to be confined to the thymus and to the T cell areas of the spleen. Third, TCF-1 bound specifically to a functional T cell-specific element in the T cell receptor alpha (TCR-alpha) enhancer. The T lineage-specific expression and the affinity for functional motifs in the TCR-alpha and CD3-epsilon enhancers imply an important role for TCF-1 in the establishment of the mature T cell phenotype.

Absence of preferential homologous H/L chain association in hybrid hybridomas.

Bispecific mAb contain two Ag-combining sites each composed of a different combination of H and L chains. The resulting ability to react with and cross-link two different Ag makes these molecules a novel tool for application in biology and medicine. Intact bispecific mAb can be made only by biologic means, e.g., by fusion of two established hybridomas. Appropriate assembly of bispecific mAb by these hybrid cells depends on H = L chain behavior: strong preferential homologous H-L pairing would benefit the yield of bispecific antibodies. We have analyzed the Ig species produced by eight hybrid hybridomas (quadromas). Quadroma-produced IgG was fractionated and characterized for H and L chain content. The Ag reactivities were verified by using ELISA and immunofluorescence. Preferential homologous pairing was seen only with a minority of H-L chain pairs; L chains associated on average in a random fashion with H chains. This indicates that in the B cells from which the parental hybridomas were obtained, no strong selection had occurred on H-L recombination. Our results extend recent biochemical competitive H-L reassociation experiments, where on average an at random association of L chains with H chains was found; evidently this random association occurs in our biologic system as well. For the biologic production of bispecific antibodies this means that only in a small number of cases the "ideal" producer will be met. From the viewpoint of generation of antibody diversity, our results favor a large freedom for combinatorial binding of H and L chains during B cell ontogeny.