Overexpression of Fibrinogen-Like Protein 2 Promotes Tolerance in a Fully Mismatched Murine Model of Heart Transplantation.

Fibrinogen-like protein 2 (FGL2) is an immunomodulatory protein that is expressed by regulatory T cells (Tregs). The objective of this study was to determine if recombinant FGL2 (rFGL2) treatment or constitutive FGL2 overexpression could promote transplant tolerance in mice. Although rFGL2 treatment prevented rejection of fully mismatched cardiac allografts, all grafts were rejected after stopping treatment. Next, we generated FGL2 transgenic mice (fgl2(Tg) ) that ubiquitously overexpressed FGL2. These mice developed normally and had no evidence of the autoimmune glomerulonephritis seen in fgl2(-/-) mice. Immune characterization showed fgl2(Tg) T cells were hypoproliferative to stimulation with alloantigens or anti-CD3 and anti-CD28 stimulation, and fgl2(Tg) Tregs had increased immunosuppressive activity compared with fgl2(+/+) Tregs. To determine if FGL2 overexpression can promote tolerance, we transplanted fully mismatched cardiac allografts into fgl2(Tg) recipients. Fifty percent of cardiac grafts were accepted indefinitely in fgl2(Tg) recipients without any immunosuppression. Tolerant fgl2(Tg) grafts had increased numbers and proportions of Tregs and tolerant fgl2(Tg) mice had reduced proliferation to donor but not third party antigens. These data show that tolerance in fgl2(Tg) recipients involves changes in Treg and T cell activity that contribute to a higher intragraft Treg-to-T cell ratio and acceptance of fully mismatched allografts.

Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision.

The Cre/loxP system has become an important tool in designing postintegrational switch mechanisms for transgenes in mice. The power and spectrum of application of this system depends on transgenic mouse lines that provide Cre recombinase activity with a defined cell type-, tissue-, or developmental stage-specificity. We have developed a novel mouse line that acts as a Cre reporter. The mice, designated Z/EG (lacZ/EGFP), express lacZ throughout embryonic development and adult stages. Cre excision, however, removes the lacZ gene, which activates expression of the second reporter, enhanced green fluorescent protein. We have found that the double-reporter Z/EG line is able to indicate the occurrence of Cre excision from early embryonic to adult lineages. The advantage of the Z/EG line is that Cre-mediated excision can be monitored in live samples and that live cells with Cre-mediated excision can be isolated using a single-step FACS. It will be a valuable reagent for the increasing number of investigators taking advantage of the powerful tools provided by the Cre/loxP site-specific recombinase system.

A comparison of Notch, Hes and Grg expression during murine embryonic and post-natal development.

Mammalian Hairy/Enhancer-of-split-like (Hes) proteins interact with transcriptional corepressors encoded by the Groucho-related genes (Grg in mice). We have examined the embryonic and postnatal expression of the Hes1 and Hes3 genes as well as all four murine Grgs in order to determine where spatial and temporal overlaps of Hes and Grg corepressors may occur. Both within and between gene families, expression was found to occur in unique but overlapping patterns during embryonic and postnatal development. Areas where the members of these gene families are coexpressed also overlap with areas where Notch signaling is thought to maintain cells in undifferentiated or "precursor" states. Our data suggest that the coordinated transcriptional control of these genes occurs during development and that precise Hes and Grg pairings may play a role in mediating the morphogenesis of CNS and epithelialized structures.

Z/AP, a double reporter for cre-mediated recombination.

The Cre/loxP site-specific recombination system combined with embryonic stem cell-mediated technologies has greatly expanded our capability to address normal and disease development in mammals using genetic approaches. The success of this emerging technology hinges on the production of Cre-expressing transgenic lines that provide cell type-, tissue-, or developmental stage-specific recombination between loxP sites placed in the genome. Here we describe and characterize the production of a double-reporter mouse line that provides a convenient and reliable readout of Cre recombinase activity. Throughout all embryonic and adult stages, the transgenic animal expresses the lacZ reporter gene before Cre-mediated excision occurs. Cre excision, however, removes the lacZ gene, allowing expression of the second reporter, the human alkaline phosphatase gene. This double-reporter transgenic line is able to indicate the occurrence of Cre excision in an extremely widespread manner from early embryonic to adult lineages. It will be a valuable reagent for the increasing number of investigators taking advantage of the powerful tools provided by the Cre/loxP site-specific recombinase system.

Conditional genome alteration in mice.

The recent ability to inactivate specific genes in mice has significantly accelerated our understanding of molecular, cellular, and even behavioral aspects of normal and disease processes. However, this ability has also demonstrated the extreme complexity of genetic determination in mammals, in particular, that genes in the same family or pathway can be functionally redundant and that a given gene often has multiple roles. Thus, inactivation of a gene often does not indicate its complete spectrum of functions. To circumvent this problem, many new tools and novel applications of classic techniques have been developed to place spatial and temporal restrictions on the genomic alterations. These approaches include chimera and mosaic studies, organ transplantation, complementation assays, dominant negative mutants, conditional gene knockouts, and lineage-specific gene rescue. Not only has this opened up more sophisticated ways to make genomic alterations, but it has provided the opportunity to create animal models for sporadic human genetic diseases.

Expression of the helix-loop-helix factor, Hes3, during embryo development suggests a role in early midbrain-hindbrain patterning.

The Hes gene family members are mammalian homologues of the Drosophila hairy and Enhancer of split genes. hairy and Enhancer of split function in both segmentation and in the Notch neurogenic pathway during Drosophila embryo development. Previous expression data suggested a conserved role for the Hes genes in the Notch signalling pathway, but not in segmentation. Here, Hes3 expression during mouse embryogenesis is described. During early development of the central nervous system, Hes3 is expressed specifically in the region of the midbrain/hindbrain boundary, and in rhombomeres 2, 4, 6 and 7. This pattern suggests that Hes3 may have a conserved role as a segmentation gene. Later in development, Hes3 is co-expressed with other neurogenic gene homologues in the developing central nervous system and epithelial cells undergoing mesenchyme induction.

Grg3, a murine Groucho-related gene, is expressed in the developing nervous system and in mesenchyme-induced epithelial structures.

We have isolated cDNAs representing multiple members of murine groucho homologues, designated Grg for groucho-related genes. Among them, Grg3 appears to produce two transcripts. One of the Grg3 transcripts contains coding sequence for a complete Groucho protein homologue. The second transcript contains coding sequence for only the two amino-terminal domains of the Groucho protein, followed by a hydrophobic tail and a stop codon. We analyzed the expression of both transcripts in mouse embryos using RNase protection and in situ hybridization. Expression was detected during cell determination in the nervous system and in somitic mesoderm, overlapping Notch1 expression and adjacent to Mash1, MyoD and Myf5 expression. Thus, the expression pattern of Grg3 suggests a conserved role in the Notch signalling pathway to regulate expression of basic helix-loop-helix proteins and cell determination. Grg3 expression was also consistently detected in epithelial structures undergoing mesenchyme induction.

Products of the grg (Groucho-related gene) family can dimerize through the amino-terminal Q domain.

The murine grg (Groucho-related gene) products are believed to interact with transcription factors and repress transcription, thereby regulating cell proliferation and differentiation. Most proteins in the grg family contain all of the domains found in the Drosophila Groucho protein, including the S/P (Ser-Pro-rich) domain required for interaction with transcription factors and the WD40 domain, which is thought to interact with other proteins. However, at least two Grg proteins contain only the amino-terminal Q (glutamine-rich) domain. We examined whether the Q domain is used for dimerization between Grg proteins, using the yeast two-hybrid system and binding assays with glutathione S-transferase fusion proteins. We found that Grg proteins are able to dimerize through the Q domain and that dimerization requires a core of 50 amino acids. Surprisingly, the dimerization does not require the leucine zipper located within the Q domain.

Transcripts of Grg4, a murine groucho-related gene, are detected in adjacent tissues to other murine neurogenic gene homologues during embryonic development.

The groucho-related genes (Grg) of the mouse comprise at least four family members. In Drosophila, groucho is one of the neurogenic genes that participates in the Notch signalling pathway. The Groucho protein interacts with Hairy-related transcription factors to regulate segmentation, neurogenesis and sex determination. Thus, by analogy to the Drosophila proteins, murine Grg proteins may interact with mammalian Hairy and E(spl) homologues (Hes proteins) and take part in a signalling pathway downstream of murine Notch. We have isolated murine Grg4 cDNAs and examined Grg4 expression during embryogenesis. Transcripts of Grg4 were detected in proliferating epithelial tissues undergoing mesenchymal induction, overlapping with Grg3, Notch1 and Hes1 expression. Grg4 was also expressed in the central nervous system and somites, but in cells adjacent to Grg3-, Notch1-, and Hes1-expressing cells. This distinct pattern of expression suggests a role for Grg4 in later stages of cell differentiation than for the other mouse neurogenic gene homologues.

Activation of Hox gene expression by Hoxa-5.

The Hox genes are expressed during embryonic development and have a role in specifying antero-posterior positional information. The genes are arranged in four clusters and a colinear relation exists between a gene's position in the cluster and its anterior boundary of expression. Genes with more anterior boundaries are also expressed earlier than genes with more posterior boundaries. Hox genes encode transcription factors; therefore, a model for the coordinate regulation of the genes within the Hox clusters is that Hox gene products regulate their own expression. To test this model, an inducible promoter was used to direct expression of exogenous Hoxa-5 in F9 embryonal carcinoma cells and the effect on endogenous Hox gene expression was measured using RNase protection assays. The production of Hoxa-5 from the expression vector activated a transient and simultaneous expression of other upstream and downstream genes of the same Hox cluster and genes from other clusters. There was an 8-hr delay between the peak of expression from the Hox vector and the endogenous Hox gene response, suggesting that Hox proteins activate other Hox genes indirectly or require additional factors which, in F9 cells, they also induce.

Identification and promoter activity of DNase I hypersensitive sites in the region of the Hox-1.3 gene.

The Hox genes encode transcriptional regulatory proteins that play a critical role in rostrocaudal specification in the developing embryo. The genes lie in four clusters in the mouse and human genome and are arranged such that a colinear relation exists between a gene's position in the cluster and the time of activation of the gene's expression. We have analyzed the Hox-1.3 region within the Hox-1 gene cluster for DNase I hypersensitive sites to identify putative regulatory sequences. Fragments identified in this way were then analyzed for transcriptional activity using gene transfer experiments in embryonal carcinoma (EC) cells. Three DNase I hypersensitive sites were identified, one of which includes the Hox-1.3 promoter and another, located 550 bp upstream, which enhances the Hox-1.3 promoter activity. The third occurs in the intron and may represent a Hox binding site. Significantly, the DNase I hypersensitive site pattern of this region of the Hox-1 cluster is not altered when F9 stem cells are differentiated with retinoic acid, suggesting that sequential activation of Hox genes by retinoic acid is not due to a sequential opening of the chromatin structure in the Hox gene region.

Mouse versions of fly developmental control genes: legitimate or illegitimate relatives?

Embryo development requires a complex order of events that must occur at the correct time and in the correct space. A series of decisions takes place as cells divide and become committed to increasingly specialized and limited domains of the embryo. Many genes that are important in orchestrating this process were originally identified in the fruitfly Drosophila melanogaster. Conserved sequences from the fly genes have been used to clone homologous genes in vertebrates, including mice. Studies of the pattern of expression of these genes during murine development in conjunction with the use of new functional assays suggest that not only DNA sequences, but also functional roles during embryogenesis, have been conserved. Thus, we may have the tools in hand to begin to understand how vertebrate development and cell differentiation take place.

Transcriptional regulation of two cytotoxic T lymphocyte-specific serine protease genes.

The expression of two serine proteases is induced by antigenic stimulation in cytotoxic T lymphocytes. Using nuclear run-on analysis the increase in steady state mRNA level has been shown to correspond to transcriptional activation. However, the two genes appear to be sequentially rather than coordinately induced. Both genes were shown to be more sensitive to DNase I digestion than a beta-globin gene in cytotoxic T cells. In addition, for the cytotoxic cell protease 1 gene the 5' region of the gene was more sensitive than the 3' end. Two DNaseI hypersensitive sites were seen in the 5' flanking sequences of both genes. The DNA sequences of the upstream regions of both genes were determined and compared. Although the two flanking sequences are overall quite dissimilar, there are short regions which are shared between the two CTL-protease genes. A number of these have been implicated in regulating the expression of other T cell genes.

IL-2 induces expression of serine protease enzymes and genes in natural killer and nonspecific T killer cells.

The expression of serine protease genes was examined in murine NK cells that were purified by panning spleen cells with PMA. Although unstimulated NK cells were cytolytic, they were found not to express the C11 (chymotrypsin-like) mRNA. Culturing these cells in IL-2 (500 to 800 U/ml) for 5 to 7 days induced both the lytic activities and the protease enzymes by 20- to 30-fold. Concomitant to these activation events, the total steady state mRNA of both C11 and HF (trypsin-like) genes were also elevated. The activation of lysis, serine protease enzymes, and C11 and HF mRNA all peaked around day 5 in culture and was dose dependent. In order to exclude the possibility that PMA synergizes with IL-2 in this system, spleen cells from SCID mice, which contained mainly NK cells, were cultured under the same conditions (800 U/ml IL-2, with or without PMA) and PMA did not appear to enhance the expression of these mRNA. Similarly, IL-2 also induced the lytic activities, enzyme levels, and mRNA in the non-Ag-specific T killer cells isolated from spleens of normal mice. Lytic activity of T killer cells was not as high as the NK cells, however, the addition of PHA into the lytic assay resulted in enhanced lysis comparable to that of NK cells. These results showed that lytic activity increased along with protease enzyme levels and mRNA expression in both NK and resting T cells. Therefore, elevated levels of the protease enzymes could be one mechanism involved in optimal lytic activity of IL-2-induced lymphokine activated killer cells.

Organization of two genes encoding cytotoxic T lymphocyte-specific serine proteases CCPI and CCPII.

The genes encoding two recently described cytotoxic T cell proteases, CCPI and CCPII, have been isolated and sequenced. The organizations of the coding and noncoding portions of the two genes are very similar to each other and also to the gene encoding rat mast cell protease type II. Similarly to other serine protease genes, each of the active-site residues is contained on a separate exon; however, two introns were found in particularly interesting positions. One occurs within the postulated activation dipeptide and the other in a position close to the active-site Asp residue. This latter intron interrupts the amino acid sequence in the invariant core region of the protein. We believe that these genes represent a new subfamily of serine protease genes.

Isolation of two cDNA sequences which encode cytotoxic cell proteases.

Two cDNAs which cross-hybridized with cytotoxic cell protease genes were identified in a library generated from a cytotoxic T cell line. Sequence analysis revealed that the two new members of the family contained the three catalytic triad residues which characterize the active sites of serine proteases. A comparison of the protein sequences revealed not only a high degree of homology but also the conservation of some unusual structural features. These include the lack of a disulphide bond which spans the active site serine, the presence of a signal sequence and the inference of a dipeptide activation sequence.

A high proportion of T lymphocytes that infiltrate H-2-incompatible heart allografts in vivo express genes encoding cytotoxic cell-specific serine proteases, but do not express the MEL-14-defined lymph node homing receptor.

The role of cytotoxic cells in in vivo immune functions such as allograft rejection is unknown. To begin to assess the function of cytolytic cells in vivo we have begun with cytolytic cell-specific functional molecules: we have isolated and characterized cytolytic cell-specific cDNA clones from cytolytic T cell clones, both encoding distinct serine esterases. The HF gene encodes a trypsin-like enzyme while the C11 gene encodes an enzyme with likely specificity for acidic residues. Here we demonstrate, using in situ hybridization with RNA probe, that both genes are expressed selectively in a subset of T lymphocytes that have infiltrated cardiac allografts. The phenotype of these cells is consistent with the most frequent phenotype of active CTL raised in vitro: they are predominantly CD4-, CD8+, MEL-14- T cell blasts. Thus the expression of these genes, each of which encodes serine esterase found in killer cell granules in vitro, is a valid marker for these cells in vivo as well. The kinetics of their accumulation is consistent with, but not proof of, a putative role in allograft rejection. It is likely that HF and C11 gene expression will be of diagnostic value.