A putative 12 transmembrane domain cotransporter expressed in thymic cortical epithelial cells

We have isolated a full-length cDNA clone (thymic stromal origin (TSO)-1C12) from a SCID thymus library using a probe from a PCR-based subtractive library enriched for sequences from fetal thymic stromal cells. TSO-1C12 mRNA is expressed mainly in the thymic cortex and is highly enriched in SCID thymus. Expression per cell is highest during fetal thymus development and decreases after day 16. Antipeptide Abs immunoprecipitated a hydrophobic, plasma membrane glycoprotein (thymic stromal cotransporter, TSCOT) whose translated sequence has weak homology to bacterial antiporters and mammalian cation cotransporters with 12 transmembrane domains. TSCOT represents a new member of this superfamily that is highly expressed in thymic cortical epithelial cells.     

Epithelial cell-specific laminin 5 is required for survival of early thymocytes

The gene LamC2 encoding the gamma2 chain of laminin 5, an epithelial cell-specific extracellular matrix protein, was identified in a PCR-based subtracted cDNA library from mouse thymic stromal cells. The mRNA existed in two alternative forms (5.1 and 2.4 kb). The full-length message was highly expressed in SCID thymus and in a nurse cell line, but not in other thymic epithelial cell lines, while the short form was more widely expressed. In situ hybridization and immunohistochemical staining revealed laminin 5 expression mostly in the subcapsular region of the adult thymus. Addition to fetal thymic organ cultures of a cell adhesion-blocking mAb to the alpha3 chain of laminin 5 interrupted T cell development. There was a 40% reduction in the total yield of thymocytes, and the most profound decrease (75-90%) was seen in the CD25+CD44+ and CD25+CD44-subsets of the CD4-CD8- double negative fraction. Most of the surviving double negative thymocytes expressed Sca-1, and there were significant increases in the number of cells with CD69 expression and in the fraction of annexin V-stained cells. None of these changes were observed with a nonblocking anti-laminin alpha3 chain mAb. These results suggest that the interaction between double negative thymoctyes and laminin 5 made by subcapsular epithelial cells is required for the survival and differentiation of mouse thymocytes.     

Cloning of a novel MHC-encoded serine peptidase highly expressed by cortical epithelial cells of the thymus.

Antigen presentation by cortical thymic epithelial cells (cTEC) during the positive selection of T cells has been shown to differ from that of other antigen-presenting cells. In the case of MHC class II presentation, cathepsin L as opposed to cathepsin S is responsible at least in part for the degradation of invariant chain. Other proteases, however, must be involved. We have identified a putative serine protease that is specifically expressed in the thymus. Encoded within the class I major histocompatibility complex (MHC) region, this gene has sequence homology with lysosomal prolylcarboxypeptidase, suggesting that it is a serine protease. We have, therefore, designated this gene thymus-specific serine protease (TSSP). In situ hybridization and immunofluorescence staining reveal that TSSP is expressed exclusively by cortical thymic epithelial cells, with the strongest staining noted around vessels and the thymic capsule. The identification of TSSP further supports the theory that MHC class II antigen processing and presentation in the thymic cortex involves a proteolytic milieu that differs from that of other antigen-presenting cells.     

Prss16 is not required for T-cell development

PRSS16 is a serine protease expressed exclusively in cortical thymic epithelial cells (cTEC) of the thymus, suggesting that it plays a role in the processing of peptide antigens during the positive selection of T cells. Moreover, the human PRSS16 gene is encoded in a region near the class I major histocompatibility complex (MHC) that has been linked to type 1 diabetes mellitus susceptibility. The mouse orthologue Prss16 is conserved in genetic structure, sequence, and pattern of expression. To study the role of Prss16 in thymic development, we generated a deletion mutant of Prss16 and characterized T-lymphocyte populations and MHC class II expression on cortical thymic epithelial cells. Prss16-deficient mice develop normally, are fertile, and show normal thymic morphology, cellularity, and anatomy. The total numbers and frequencies of thymocytes and splenic T-cell populations did not differ from those of wild-type controls. Surface expression of MHC class II on cTEC was also similar in homozygous mutant and wild-type animals, and invariant chain degradation was not impaired by deletion of Prss16. These findings suggest that Prss16 is not required for quantitatively normal T-cell development.     

Isolation and characterization of a partial cDNA clone for heparin cofactor II1

A human fetal liver cDNA library constructed in lambda gt11 was screened with affinity-purified rabbit antibodies raised against heparin cofactor II. One positive clone was plaque purified and the cDNA insert was completely sequenced. The clone encodes the C-terminal 167 amino acid residues of heparin cofactor II as well as the entire 3'-untranslated region of the message. Proline and leucine were identified in the P2 and P1 positions of the protease cleavage site, providing a possible explanation for the ability of heparin cofactor II to inhibit both thrombin and chymotrypsin-like proteases. The coding sequence is identical to that of the recently published human leuserpin 2 (Ragg (1986) Nucl. Acids Res. 14, 1073).     

Isolation and chromosomal localization of a novel nonerythroid ankyrin gene.

Immunoreactive isoforms of erythrocyte ankyrin have been shown to be present in a variety of nonerythroid tissues. Isolation of the genes that encode these isoforms will clarify their relationship to erythrocyte ankyrin. Using an erythrocyte ankyrin cDNA clone as a hybridization probe, we screened a human genomic library and isolated a clone that hybridizes with the probe at low stringency but not at high stringency. Partial nucleotide sequence of the clone revealed the presence of a 99-bp segment that is homologous to an exon of the erythrocyte ankyrin gene. Northern analysis showed that a labeled fragment of the clone hybridized to a 7-kb message in RNA of fetal brain but not of erythroid cells, suggesting that this clone is part of a novel gene that is expressed predominantly in nonerythroid tissue. Comparison of the sequence of the genomic clone with that of a recently isolated cDNA clone for brain ankyrin (Otto et al., 1989) showed identity of 96 of 99 bp between the putative exon and a segment of the cDNA clone (V. Bennett, personal communication, 1991), suggesting that the genomic clone is part of a gene for nonerythroid ankyrin, which we have designated ANK2. By analysis of somatic cell hybrids and fluorescence in situ hybridization, we assigned ANK2 to human chromosome 4 at a position equivalent to bands 4q25-q27.     

Molecular and biochemical characterization of a serine proteinase predominantly expressed in the medulla oblongata and cerebellar white matter of mouse brain

A full-length cDNA clone of a serine proteinase, mouse brain serine proteinase (mBSP), was isolated from a mouse brain cDNA library. mBSP, which has been recently reported to be expressed in the hair follicles of nude mice, is most similar (88% identical) in sequence to rat myelencephalon-specific protease. The mBSP mRNA was steadily expressed in the brain of adult mice with a transient expression in the early fetal stage during development. The genomic structure of the mouse gene for mBSP was determined. The gene, which is mapped to chromosome 7B4-B5, is about 7.4 kilobases in size and contains 7 exons. Interestingly, the 5'-untranslated region of the mBSP gene was interrupted by two introns. In situ hybridization analyses revealed that mBSP is expressed in the white matter of the cerebellum, medulla oblongata, and capsula interna and capsula interna pars retrolenticularis of mouse brain. Further, mBSP was immunolocalized to the neuroglial cells in the white matter of the cerebellum. Recombinant mBSP was produced in the bacterial expression system and activated by lysyl endopeptidase digestion, and the activated enzyme was purified for characterization. The enzyme showed amidolytic activities preferentially cleaving Arg-X bonds when 4-methylcoumaryl-7-amide-containing peptide substrates were used. Typical serine proteinase inhibitors, such as diisopropyl fluorophosphates, phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, leupeptin, antipain, and benzamidine, strongly inhibited the enzyme activity. The recombinant mBSP effectively hydrolyzed fibronectin and gelatin, but not laminin, collagens I and IV, or elastin. These results suggest that mBSP plays an important role in association with the function of the adult mouse brain.     

Establishment of a mouse thymic epithelial cell line, IT-76MHC and a brief review on cultured thymic epithelial cells.

Interactions between thymocytes and thymic stromal cells are essential for thymocyte differentiation, but little evidence has been presented to directly show in vivo functions or interactions of the stromal cells. Among the stromal cells, the thymic epithelial cell has been considered to have profound effect on thymocyte differentiation and maturation. The calcium-depleted medium, originally developed for the culture of mouse epidermal cells, was applied for the culture of the mouse thymic epithelial cells, and successfully, an epithelial cell line, IT-76MHC was obtained from the mouse thymus. IT-76MHC cells were identified as distinct mouse thymic epithelial cells by 1/ mosaic-like arrangement, 2/ presence of well-developed desmosome and 3/ tonofilaments, 4/ positivity for cytokeratin, and 5/ induced expression of MHC class I and II by IFN-gamma treatment. IGF-1, IGF-2, oxytocin and vasopressin were also detected immunohistochemically in IT-76MHC cells. Furthermore, the IT-76MHC thymic epithelial cells, when injected intrathymically in the allogeneic mouse, prolonged the survival of skin graft from the same donor strain that IT-76MHC cells were derived. These results demonstrate that the thymic epithelial cell line IT-76MHC produces modest thymocyte survival factors as well as a growth suppressor, and that IT-76MHC cells have the ability to induce transplantation tolerance probably through their expression of MHC class I and II molecules. Taken altogether, the IT-76MHC thymic epithelial cells have been proved to be useful tools to better understand the in vivo functions of thymic epithelial cells, and to gain a deep insight into their involvement in the critical selection process of thymocytes which still remains obscure. Finally and additionally, literatures so far reported on thymic epithelial cells in culture, especially lines and clones, are reviewed and their identity as well as their functions are discussed.     

Shedding of membrane epithin is blocked without LDLRA4 and its protease activation site

Epithin, a mouse type II transmembrane serine protease, is processed at Gly149 and released from the membrane. Here, we report the identification of an epithin isoform, epithin(Delta), containing a 66 amino acid deletion from the full-length epithin, which is missing the 4th LDLRA domain and the protease activation sequence. This truncated isoform showed the same characteristic N-terminal processing at Gly149 as the full-length form, however, no protease activity was detected. The N-terminal processed epithin(Delta) short form (Epi(Delta)-S) was not released into the medium under conditions in which the processed epithin short form (Epi-S) is released. This type of epithin shedding was also prevented when serine protease inhibitors were added to cells expressing the full-length form. These results strongly suggest that the serine protease activity is involved in the shedding process. The presence of epithin(Delta) message was detected in multiple tissues and its significance is discussed.     

Expression and characterization of a novel mesophilic protease from metagenomic library derived from Antarctic coastal sediment

A metagenomic cosmid library was constructed, in which the insert DNA was derived from the coastal sediment near Antarctic China Zhongshan Station. One clone (ACPRO001) expressing protease activity was isolated from the library using milk agar plates. Sequencing of the clone revealed a novel protease gene. The amino acid sequence comparison and phylogenetic analysis indicated that it could be classified as a subtilisin-like serine protease, though the highly conserved residue Asp was replaced by Ala. The ACPRO001 protease gene was expressed in pET-His and purified for characterization. The optimal temperature and pH for the activity of the ACPRO001 protease were 60°C and pH 9.0, respectively. The enzyme retained about 73% of residual activity after 2 h incubation at 50°C in the presence of Ca²⁺. The presence of Ca²⁺ increased the thermostability of ACPRO001 protease obviously. The enzymatic activity was inhibited by 1 mM phenylmethyl sulfonylfluoride (PMSF) and hydrochloride 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), indicating that it was a serine protease.     

Genetics, evolution, and expression of the 68,000-mol-wt neurofilament protein: isolation of a cloned cDNA probe

A 1.2-kilobase (kb) cDNA clone (NF68) encoding the mouse 68,000-mol-wt neurofilament protein is described. The clone was isolated from a mouse brain cDNA library by low-stringency cross-hybridization with a cDNA probe encoding mouse glial fibrillary acidic protein (Lewis et al., 1984, Proc. Natl. Acad. Sci. USA., 81:2743-2746). The identity of NF68 was established by hybrid selection using mouse brain polyA+ mRNA, and cell-free translation of the selected mRNA species. The cell-free translation product co-migrated with authentic 68,000-mol-wt neurofilament protein on an SDS/polyacrylamide gel, and was immunoprecipitable with a monospecific rabbit anti-bovine neurofilament antiserum. In addition, DNA sequence analysis of NF68 showed 90% homology at the amino acid level compared with the sequence of the porcine 68,000-mol-wt neurofilament protein. At high stringency, NF68 detects a single genomic sequence encoding the mouse 68,000-mol-wt neurofilament protein. Two mRNA species of 2.5 kb and 4.0 kb are transcribed from the single gene in mouse brain. The level of expression of these mRNAs remains almost constant in postnatal mouse brains of all ages and, indeed, in the adult. At reduced stringency, NF68 detects a number of mRNAs that are expressed in mouse brain, one of which encodes the 150,000-mol-wt neurofilament protein. The NF68 probe cross-hybridizes at high stringency with genomic sequences in species as diverse as human, chicken, and (weakly) frog, but not with DNA from Drosophila or sea urchin.     

Human natural regulatory T cell development, suppressive function, and postthymic maturation in a humanized mouse model

CD4(+) regulatory T cells (Tregs) control adaptive immune responses and promote self-tolerance. Various humanized mouse models have been developed in efforts to reproduce and study a human immune system. However, in models that require T cell differentiation in the recipient murine thymus, only low numbers of T cells populate the peripheral immune systems. T cells are positively selected by mouse MHC and therefore do not function well in an HLA-restricted manner. In contrast, cotransplantation of human fetal thymus/liver and i.v. injection of CD34(+) cells from the same donor achieves multilineage human lymphohematopoietic reconstitution, including dendritic cells and formation of secondary lymphoid organs, in NOD/SCID mice. Strong Ag-specific immune responses and homeostatic expansion of human T cells that are dependent on peripheral human APCs occur. We now demonstrate that FOXP3(+)Helios(+) "natural" Tregs develop normally in human fetal thymic grafts and are present in peripheral blood, spleen, and lymph nodes of these humanized mice. Humanized mice exhibit normal reversal of CD45 isoform expression in association with thymic egress, postthymic "naive" to "activated" phenotypic conversion, and suppressive function. These studies demonstrate the utility of this humanized mouse model for the study of human Treg ontogeny, immunobiology and therapy.     

Isolation,Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.     

Use of a cDNA recombinant for the gamma-subunit of mouse nerve growth factor to localize members of this multigene family near the TAM-1 locus on chromosome 7.

The gamma-subunit of mouse 7S nerve growth factor (gamma-NGF) is a member of a family of closely related serine proteases that includes kallikreins and tamases. We have isolated from a DBA/2J male submaxillary gland cDNA library a clone, pSM676, which codes for gamma-NGF. Sequence analysis of the clone shows that it codes for the C-terminal 138 amino acids of the protein plus 23 bases of the 3'-nontranslated portion of the message. The predicted amino acid sequence agrees with that determined by Thomas et al. (1) for the gamma-subunit of nerve growth factor from Swiss Webster mice except for the single, conservative substitution of glutamate for aspartate at amino acid 175. When used as a probe for Southern blot analysis, pSM676 hybridizes to at least twelve fragments of restricted mouse genomic DNA which correspond to several different serine protease genes. Using mouse-hamster hybrid cell lines and recombinant inbred strains of mice, we have demonstrated that all of the genes which show homology to pSM676 are located on mouse chromosome 7, clustered at or near the Tam-1 locus.     

A mouse serine protease TESP5 is selectively included into lipid rafts of sperm membrane presumably as a glycosylphosphatidylinositol-anchored protein

We have previously indicated that at least in mouse, sperm serine protease(s) other than acrosin probably act on the limited proteolysis of egg zona pellucida to create a penetration pathway for motile sperm, although the participation of acrosin cannot be ruled out completely. A 42-kDa gelatin-hydrolyzing serine protease present in mouse sperm is a candidate enzyme involved in the sperm penetration of the zona pellucida. In this study, we have PCR-amplified an EST clone encoding a testicular serine protease, termed TESP5, and then screened a mouse genomic DNA library using the DNA fragment as a probe. The DNA sequence of the isolated genomic clones indicated that the TESP5 gene is identical to the genes coding for testicular testisin and eosinophilic esp-1. Immunochemical analysis using affinity-purified anti-TESP5 antibody revealed that 42- and 41-kDa forms of TESP5 with the isoelectric points of 5.0 to 5.5 are localized in the head, cytoplasmic droplet, and midpiece of cauda epididymal sperm probably as a membranous protein. Moreover, these two forms of TESP5 were selectively included into Triton X-100-insoluble microdomains, lipid rafts, of the sperm membranes. These results show the identity between TESP5/testisin/esp-1 and the 42-kDa sperm serine protease. When HEK293 cells were transformed by an expression plasmid carrying the entire protein-coding region of TESP5, the recombinant protein produced was released from the cell membrane by treatment with Bacillus cereus phosphatidylinositol-specific phospholipase C, indicating that TESP5 is glycosylphosphatidylinositol-anchored on the cell surface. Enzymatic properties of recombinant TESP5 was similar to but distinguished from those of rat acrosin and pancreatic trypsin by the substrate specificity and inhibitory effects of serine protease inhibitors.     

Cloning of the cDNA for the serine protease homolog CAP37/azurocidin, a microbicidal and chemotactic protein from human granulocytes

Human cationic antimicrobial protein (CAP37) is a neutrophil granule protein with monocyte chemotactic and antibacterial activity. A CAP37 cDNA clone of 899 bp was isolated from an HL-60 cDNA library using degenerate oligonucleotide probes based on partial N-terminal sequence of the CAP37 protein. The cDNA sequence predicts an open reading frame of 753 bp encoding a protein of 251 amino acids. A 26-residue eukaryotic signal peptide and a potential 7 amino acid pro-peptide are present at the N-terminus of the protein. The cDNA sequence also predicts three N-linked glycosylation attachment sites and eight intramolecular cysteines. The deduced amino acid sequence of CAP37 shows 44, 42, and 32% homology at the amino acid level to neutrophil elastase, myeloblastin, and cathepsin G, respectively, suggesting that CAP37 is a member of the serine protease gene family. CAP37 does not possess serine protease activity probably due to mutations in two of three residues in the catalytic triad of the "charge relay system." Whereas CAP37 is expressed in undifferentiated HL-60 cells no message is detected in mature neutrophils.     

TSCOT+ thymic epithelial cell-mediated sensitive CD4 tolerance by direct presentation

Although much effort has been directed at dissecting the mechanisms of central tolerance, the role of thymic stromal cells remains elusive. In order to further characterize this event, we developed a mouse model restricting LacZ to thymic stromal cotransporter (TSCOT)-expressing thymic stromal cells (TDLacZ). The thymus of this mouse contains approximately 4,300 TSCOT+ cells, each expressing several thousand molecules of the LacZ antigen. TSCOT+ cells express the cortical marker CDR1, CD40, CD80, CD54, and major histocompatibility complex class II (MHCII). When examining endogenous responses directed against LacZ, we observed significant tolerance. This was evidenced in a diverse T cell repertoire as measured by both a CD4 T cell proliferation assay and an antigen-specific antibody isotype analysis. This tolerance process was at least partially independent of Autoimmune Regulatory Element gene expression. When TDLacZ mice were crossed to a novel CD4 T cell receptor (TCR) transgenic reactive against LacZ (BgII), there was a complete deletion of double-positive thymocytes. Fetal thymic reaggregate culture of CD45- and UEA-depleted thymic stromal cells from TDLacZ and sorted TCR-bearing thymocytes excluded the possibility of cross presentation by thymic dendritic cells and medullary epithelial cells for the deletion. Overall, these results demonstrate that the introduction of a neoantigen into TSCOT-expressing cells can efficiently establish complete tolerance and suggest a possible application for the deletion of antigen-specific T cells by antigen introduction into TSCOT+ cells.