A phage display system for detection of T cell receptor-antigen interactions.

The process of T cell recognition involves a complex set of interactions between the various components of the TCR/MHC/peptide trimolecular complex. We have developed a system for exploring the specific binding interactions contributed by the constituent subunits of TCR complexes for components of their ligands. We utilized an M13 phage display system, designed for multivalent receptor display, to explore specific binding interactions between various TCR alpha chains and specific antigen in the absence of MHC. The multivalent TCR-phage display system was sensitive enough to reveal some TCR alpha chains capable of binding directly to antigen with the same fine specificity shown by the MHC-restricted T cells from which the alpha chains were derived. Cross-specificity analysis using two antigen-binding TCR alpha chains derived from T cells with different polypeptide antigen specificities confirmed the fidelity of this binding. In mixtures of antigen-binding and non-binding TCR alpha-displaying phage, specific selection was achieved at a starting frequency of 1/1000, suggesting that this system can be employed for selection and analysis of TCR-displaying phage libraries. While the binding specificities exhibited by these TCRs are unusual, they provide a novel perspective from which to study the specific binding interactions that constitute TCR antigen binding.

Purification of rat megakaryocyte colony-forming cells using a monoclonal antibody against rat platelet glycoprotein IIb/IIIa.

We recently reported the production and characterization of four monoclonal antibodies (MoAbs) against rat platelet glycoprotein IIb/IIIa (GPIIb/IIIa). In this study we developed a simple and efficient three-step procedure, based on positive selection by immunoadsorption (panning) using one MoAb, P55, to purify rat megakaryocyte colony-forming cells (megakaryocyte colony-forming units, CFU-MK) from normal bone marrow. Cells obtained after each step were assayed for their ability to form megakaryocyte colonies in the presence of Concanavalin A (Con A)-stimulated rat spleen cell-conditioned medium in soft agar cultures. Marrow cells were first separated on discontinuous Percoll gradients. Cells sedimented at densities between 1.063 and 1.082 g/ml were depleted of cells adherent to plastic tissue culture dishes. The nonadherent cells were further incubated on dishes coated with P55 MoAb. CFU-MK were enriched about 50-fold in the adsorbed cell fraction. This sequential fractionation procedure resulted in a 345-fold (range 276 to 412-fold) enrichment of rat CFU-MK over whole bone marrow cells. The average cloning efficiency of CFU-MK in the final fraction was about 7% (range 5%-9.2%) of the nucleated cells. The overall recovery of CFU-MK averaged 20% (range 9%-29%). The panning step provided a 46-fold enrichment of megakaryocyte burst-forming cells (megakaryocyte burst-forming units, BFU-MK), whose average cloning efficiency in the post-panning fraction was 0.14% (range 0.07%-0.2%). In addition, erythroid burst-forming cells (erythroid burst-forming units, BFU-E) were also significantly enriched by panning, but to a lesser degree than BFU-MK and CFU-MK. By contrast, granulocyte-macrophage colony-forming cells (granulocyte-macrophage colony-forming units, CFU-GM) and erythroid colony-forming cells (erythroid colony-forming units, CFU-E) were not enriched by panning. CFU-MK obtained after panning formed megakaryocyte colonies in the presence of recombinant rat interleukin 3 (rIL-3), mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF), or human erythropoietin (hEPO), as has been reported for murine CFU-MK in whole marrow cells. The highly enriched populations of rat CFU-MK should thus provide a basis for the further study of the regulation of megakaryocytopoiesis.

Isolation and expression in Escherichia coli of a cDNA clone encoding porcine pancreatic elastase.

We have cloned a DNA that is complementary to the messenger RNA that encodes porcine pancreatic elastase 1 from pancreas using rat pancreatic elastase 1 cDNA as a probe. This complementary DNA contains the entire protein coding region of 798 nucleotides which encodes an elastase of 266 amino acids, and 22 and 136 nucleotides of the 5' and 3'-untranslated sequences. When this deduced amino acid sequence was compared with known amino acid sequences, a carboxy-terminal 240 amino acids long peptide was found to be identical with a mature form of porcine pancreatic elastase 1, except for two amino acids. The porcine enzyme contains the same number of amino acid residues as the rat enzyme, and their amino acid sequences are 85% homologous. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 240 amino acids including a leader and activation peptide of 26 amino acids. We expressed the cloned porcine pancreatic elastase 1 cDNA in E. coli as a lac-fused protein. The resulting fused protein showed enzymatic activity and immunoreactivity toward anti-elastase serum.

Molecular cloning of complementary DNA encoding one of the human pancreatic protease E isozymes.

We have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with rat and porcine pancreatic elastase 1, although its substrate binding region is homologous with those of the above elastases 1. When this deduced amino acid sequence was compared with known amino acid sequences of pancreatic proteases other than elastases, it was found to contain an amino acid sequence which was highly homologous with the N-terminal amino acid sequence of porcine pancreatic protease E. We also purified human pancreatic protease E isozymes from human pancreatic juice, and determined their N-terminal amino acid sequences. One of the isozymes does not hydrolyze elastin but does hydrolyze a synthetic substrate. Endoglycosidase F digests glycoside bonds of the isozyme. These results suggest that the cDNA cloned by us corresponded to one of the human protease E isozymes.

Molecular cloning and expression in Escherichia coli of a cDNA encoding human pancreatic elastase 2.

We have cloned a DNA that is complementary to the messenger RNA that encodes human pancreatic elastase 2 from a human pancreatic cDNA library using a cloned cDNA for rat pancreatic elastase 2 messenger RNA. This complementary DNA contains the entire protein coding region of 807 nucleotides which encodes preproelastase of 269 amino acids, and 4 and 82 nucleotides of the 5'- and 3'-untranslated sequences, respectively. When this deduced amino acid sequence was compared with known amino acid sequences it showed 82% homology with rat pancreatic elastase 2. This deduced sequence also contains a 16-amino-acid peptide identical with the N-terminal sequence determined for native human pancreatic proelastase 2. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 241 amino acids including 16 and 12 amino acids for a signal peptide and an activation peptide, respectively. Moreover, the predicted key amino acid residues involved in determining the substrate specificity of mammalian pancreatic elastase 2 are retained in the human enzyme. Cloned human pancreatic elastase 2 cDNA was expressed in E. coli as a mature and pro-form protein. Both resulting proteins showed immunoreactivity toward anti-elastase serum and enzymatic activity. We have also cloned and sequenced a porcine pancreatic elastase 2 cDNA.

Conversion of inactive glycosylation inhibiting factor to bioactive derivatives by modification of a SH group.

Escherichia coli-derived recombinant human glycosylation inhibiting factor (rhGIF) contains three cysteine residues (Cys-57, -60, and -81). All SH groups in the cysteine residues are free, and the GIF molecule had no biologic activity. Carboxymethylation of the SH group of Cys-60 in the molecule resulted in the generation of bioactivity, although the activity of the carboxymethylated GIF was 10- to 20-fold less than that of suppressor T cell (Ts)-derived GIF. However, treatment of the inactive rhGIF with ethylmercurithiosalicylate or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in the generation of derivatives whose bioactivity was comparable to that of the Ts-derived bioactive GIF. The activity of these derivatives was lost by treatment with DTT. Isolation and chemical analysis of the DTNB-treated GIF derivative revealed that binding the 5-thio-2-nitrobenzoic acid group with Cys-60 was responsible for the generation of the highly bioactive derivative. Inactive cytosolic GIF from mammalian cells could also be converted to bioactive derivative by treatment with the SH reagent, while Ts-derived bioactive GIF was inactivated by DTT. These results, together with an x-ray crystal structure of GIF molecules, strongly suggest that the generation of bioactivity of GIF in Ts cells is due to posttranslational modifications that result in conformational changes in the molecule.

Association of the "major histocompatibility complex subregion" I-J determinant with bioactive glycosylation-inhibiting factor.

Murine suppressor T-cell hybridoma cells (231F1) secrete not only bioactive glycosylation-inhibiting factor (GIF) but also an inactive peptide comparable to bioactive GIF peptide in its molecular size and reactivity with anti-GIF; the amino acid sequence of the inactive peptide is identical to that of the bioactive homologue. The inactive GIF peptide in culture supernatant of both the 231F1 cells and a stable transfectant of human GIF cDNA in the murine suppressor T hybridoma selectively bound to Affi-Gel 10, whereas bioactive GIF peptides from the same sources failed to bind to the gel. The inactive cytosolic human GIF from the stable transfectant and Escherichia coli-derived recombinant human GIF also had affinity for Affi-Gel 10. Both the bioactive murine GIF peptide from the suppressor T hybridoma and bioactive recombinant human GIF from the stable transfectant bound to the anti-I-J monoclonal antibody H6 coupled to Affi-Gel. However, bioactive hGIF produced by a stable transfectant of human GIF cDNA in BMT10 cells failed to be retained in H6-coupled Affi-Gel. These results indicate that the I-J specificity is determined by the cell source of the GIF peptide and that the I-J determinant recognized by monoclonal antibody H6 does not represent a part of the primary amino acid sequence of GIF. It appears that the epitope is generated by a posttranslational modification of the peptide.

High-affinity binding of bioactive glycosylation-inhibiting factor to antigen-primed T cells and natural killer cells.

High-affinity binding was demonstrated between suppressor-T-cell-derived bioactive glycosylation-inhibiting factor (GIF) and helper T hybridomas and natural killer cell line cells. Inactive GIF present in cytosol of suppressor T cells and Escherichia coli-derived recombinant human GIF (rhGIF) failed to bind to these cells. However, affinity of rhGIF for the target cells was generated by replacement of Cys-57 in the sequence with Ala or of Asn-106 with Ser or binding of 5-thio-2-nitrobenzoic acid to Cys-60 in the molecule. Such mutations and the chemical modification of rhGIF synergistically increased the affinity of GIF molecules for the target cells. The results indicated that receptors on the target cells recognize conformational structures of bioactive GIF. Equilibrium dissociation constant (Kd) of the specific binding between bioactive rGIF derivatives and high-affinity receptors was 10-100 pM. Receptors for bioactive GIF derivatives were detected on Th1 and Th2 T helper clones and natural killer NK1.1(+) cells in normal spleen but not on naive T or B cells. Neither the inactive rGIF nor bioactive rGIF derivatives bound to macrophage and monocyte lines or induced macrophages for tumor necrosis factor alpha production.

Antigen-binding glycosylation inhibiting factor from a human T-cell hybridoma specific for bee venom phospholipase A2.

We obtained human T-cell hybridomas that are specific for bee venom phospholipase A2 (PLA2) and constitutively secrete glycosylation inhibiting factor (GIF). Upon crosslinking of CD3, the hybridoma produced GIF having affinity for PLA2. When affinity-purified PLA2-binding GIF was used as an immunogen, monoclonal antibodies specific for the antigen-binding GIF were obtained. Monoclonal antibody 110BH3 bound the antigen-binding GIF but failed to bind the 13-kDa nonspecific GIF, as determined by both bioassay and ELISA. In contrast, 388F1, a monoclonal antibody against nonspecific GIF, gave ELISA signals with both the nonspecific GIF and the antigen-binding GIF. Gel filtration of affinity-purified antigen-binding GIF revealed the presence of a 72- to 80-kDa protein which gave ELISA signals with both 110BH3 and 388F1 and contained GIF bioactivity. Upon reduction and alkylation, the antigen-binding GIF dissociated into a 62- to 64-kDa protein which gave positive ELISA with antibody 110BH3 but no signal with antibody 388F1, and a 15-kDa protein, which gave ELISA signal with the 388F1 but not with 110BH3. Immunoblotting of a PLA2-binding GIF preparation revealed that under reducing conditions, the antigen-binding GIF dissociated a 13-kDa peptide which reacted with polyclonal antibodies against recombinant GIF. The results indicate that the 13-kDa nonspecific GIF is a subunit of antigen-binding GIF. The PLA2-binding GIF has affinity for an epitope, representing amino acid residues 19-28 in PLA2 which appears to be an external structure in the antigen.

Processing of a fusion protein by endoprotease in COS-1 cells for secretion of mature peptide by using a chimeric expression vector.

The subtilisin-related proprotein convertase furin is expressed in various mammalian tissues. Expecting that COS-1 cells have a furin-like endoprotease, we constructed a fusion expression vector for production of a recombinant foreign protein having no signal peptide or a protein in truncated form into secreted mature protein. A cDNA fragment encoding N-terminal procalcitonin (pro-CT) of human calcitonin precursor was inserted into the mammalian expression vector pME18S. We used PCR techniques to generate four kinds of cDNAs encoding the C terminus of the pro-CT with Arg residues at P4 (Arg-Xaa-Lys-Arg), P6 (Arg-Xaa-Xaa-Xaa-Lys-Arg), or both (Arg-Xaa-Arg-Xaa-Lys-Arg), in addition to the Lys-Arg motif at the cleavage site, in order to determine the conditions for efficient processing in nonendocrine cells, such as COS-1 cells. The cDNA coding for the Fc fragment of human immunoglobulin G1 was fused in-frame to the cDNA encoding pro-CT at its C terminus. Upon transfection of the chimeric plasmids into COS-1 cells, almost all of the fusion protein with the Arg residues at both P4 and P6 were processed into secreted Fc product, even without cotransfection of furin. These results indicate that COS-1 cells have a furin-like endoprotease and suggest that pro-CT, with the Arg residues at both P4 and P6, can be used as a carrier peptide for expression of a foreign protein having no signal peptide or a protein in truncated form in COS-1 cells.

Deficiency of the macrophage migration inhibitory factor gene has no significant effect on endotoxaemia.

By targeted disruption of the MIF gene, we have established a mouse strain deficient in macrophage (Mphi) migration inhibitory factor (MIF). Despite previous reports indicating an essential role of MIF in endotoxaemia, an injection of lipopolysaccharide (LPS) into the MIF-deficient mice (maintained under specific pathogen-free conditions) caused shock. No significant difference was detected between the MIF-deficient mutant and normal mice in susceptibility to LPS for endotoxaemia or tumour necrosis factor-alpha (TNF-alpha) formation upon LPS injection. Peritoneal Mphi from the two strains produced TNF-alpha in response to LPS with similar dose responses. Dexamethasone suppressed the LPS-induced TNF-alpha response of Mphi, but no difference was detected between the Mphi from the two strains. These results suggest that endogenous MIF has no significant effect on the LPS-induced TNF-alpha production and no effect on suppression of the response by glucocorticoids. Thus, MIF is not crucial for LPS-induced immune responses leading to shock.

The crystal structure of human glycosylation-inhibiting factor is a trimeric barrel with three 6-stranded beta-sheets.

Glycosylation-inhibiting factor (GIF) is a cytokine that is involved in the regulation of IgE synthesis. The crystal structure of recombinant human GIF was determined by the multiple isomorphous replacement method. The structure was refined to an R factor of 0.168 at 1.9 angstrom resolution. The overall structure is seen to consist of three interconnected subunits forming a barrel with three 6-stranded beta-sheets on the inside and six alpha-helices on the outside. There is a 5-angstrom-diameter "hole" through the middle of the barrel. The barrel structure of GIF in part resembles other "trefoil" cytokines such as interleukin 1 and fibroblast growth factor. Each subunit has a new class of alpha + beta sandwich structure consisting of two beta-alpha-beta motifs. These beta-alpha-beta motifs are related by a pseudo-twofold axis and resemble both interleukin 8 and the peptide binding domain of major histocompatibility complex protein, although the topology of the polypeptide chain is quite different.

In vivo effects of interleukin-6 on thrombopoiesis in healthy and irradiated primates.

We have studied the in vivo effects of recombinant human interleukin-6 (rhIL-6) on hematopoiesis in eight healthy and nine irradiated cynomolgus monkeys. Of the healthy animals, three received rhIL-6 alone (10 micrograms/kg/d, subcutaneously [SC]), one received rhIL-6 in combination with rhIL-3 (10 micrograms/kg/d, SC), one received rhIL-6 in combination with recombinant cynomolgus granulocyte-macrophage colony-stimulating factor (rcGM-CSF; 10 micrograms/kg/d, SC), two received rhIL-6 in combination with recombinant human granulocyte-CSF (rhG-CSF; 10 micrograms/kg/d, SC), and one received rhIL-6 in combination with recombinant human leukemia inhibitory factor (rhLIF; 10 micrograms/kg/d, SC). All animals were treated for at least 2 weeks with rhIL-6 or the above mentioned combinations. rhIL-6 alone significantly increased the peripheral blood platelet counts (2- to 3.5-fold). The platelets reached a plateau between days 10 and 15 of treatment. No synergistic effects on platelet numbers were observed when rhIL-6 was combined with rhIL-3, rcGM-CSF, rhG-CSF, or rhLIF. In addition to rhIL-6, only rhLIF increased the platelet numbers when administered alone. To test whether rhIL-6 might also protect the animal from thrombocytopenia or shorten the time of thrombocytopenia after irradiation, we treated nine animals with total body irradiation (3.8 Gy). Six of the animals were additional treated with rhIL-6 (4 with 10 micrograms/kg/d; and 2 with 100 micrograms/kg/d) from day -1 or +1 to day 28 post irradiation. In these animals, rhIL-6 at the same dose effective in healthy animals (10 micrograms/kg/d) was not capable of protecting the animals from platelet nadir. However, when pegylated rhIL-6 was used at a dosage of 100 micrograms/kg/d post irradiation, the mean of the nadirs was 71,000/microL as compared with 39,000/microL in control animals and the time of thrombocytopenia was shorter (3 v 5 days). In all animals (healthy and irradiated), rhIL-6 did not increase the number of bone marrow megakaryocytes but induced a right shift of DNA ploidy in megakaryocytes. These data suggest that IL-6 acts as "thrombopoietin"-like activity, but not as "megakaryocyte-CSF"-like activity.

A highly enhanced thrombopoietic activity by monomethoxy polyethylene glycol-modified recombinant human interleukin-6.

Interleukin-6 (IL-6) is a novel cytokine with activities that can affect hematopoietic cells including those of the megakaryocytic lineage. We have examined the effects of monomethoxy polyethylene glycol-modified recombinant human interleukin-6 (Peg-IL-6) on thrombopoiesis in vivo. To compare the thrombopoietic activity between Peg-IL-6 and unmodified IL-6, each was administered subcutaneously to mice every 24 hours at various doses. A dose-response experiment showed that Peg-IL-6 and IL-6 increased platelet counts in a dose-dependent fashion at a plateau stimulation level of 0.5 micrograms/day and 10 micrograms/day, respectively. This dose of Peg-IL-6 and IL-6 induced the elevated platelet counts by approximately 140% to 160% and 50% to 60%, respectively. Peg-IL-6 treatment (0.5 micrograms/day) for x-ray (6.0 Gy) irradiated mice induced an increase in the rate of platelet recovery, and a higher dosage (5 micrograms/day) completely blocked the induction of thrombocytopenia in this model. In contrast, IL-6 (10 micrograms/day) could not protect the animals from platelet nadir but reduced the period of thrombocytopenia after x-ray irradiation. Furthermore, when administered to 5-fluorouracil-treated mice, 5 micrograms/day of Peg-IL-6 diminished the platelet nadir and increased platelet counts on individual days during the recovery phase. The potent thrombopoietic activity of Peg-IL-6 were due to prolongation of circulating IL-6 levels that were reverted from Peg-IL-6 in vivo. These findings indicate that reduction of total body clearance of IL-6 induces potent thrombopoiesis and that Peg-IL-6 may be a useful thrombopoietic agent.

Molecular cloning and functional expression of a cDNA encoding glycosylation-inhibiting factor.

By using probes based on partial amino acid sequence of glycosylation-inhibiting factor (GIF) from a mouse T-cell hybridoma, a full-length cDNA encoding mouse GIF was isolated. A cDNA clone encoding human GIF was isolated from cDNA libraries of a GIF-producing human T-cell hybridoma by using mouse GIF cDNA as a probe. The cDNAs encode a putative 12.5-kDa peptide of 115 amino acids. Northern blot analysis demonstrated a single, 0.6-kb transcript. Polyclonal rabbit antibodies against the Escherichia coli-derived recombinant 13-kDa peptide bound hybridoma-derived GIF. Although the peptide did not contain a signal peptide sequence, transfection of the cDNA into COS-1 cells resulted in secretion of 13-kDa peptide, but the peptide had substantially less bioactivity than the hybridoma-derived GIF. However, expression of a chimeric cDNA encoding a fusion protein consisting of the N-terminal pro region of calcitonin precursor and human GIF and cotransfection with furin cDNA to allow intracellular cleavage of the fusion protein resulted in secretion of 13-kDa peptide that was comparable to hybridoma-derived GIF in its bioactivity. Both the 13-kDa peptide and GIF bioactivity in the transfected COS-1 supernatant bound to a monoclonal antibody against hybridoma-derived human GIF. These results indicate that the 13-kDa peptide represents recombinant GIF, but posttranslational modification of the peptide is important for generation of the bioactivity. The GIF cDNA had high homology with the cDNA encoding macrophage migration inhibitory factor. However, the recombinant GIF failed to inhibit migration of human monocytes, and recombinant human macrophage migration inhibitory factor did not have GIF bioactivity.

Immunosuppressive activities of recombinant glycosylation-inhibiting factor mutants.

We have shown previously that glycosylation-inhibiting factor (GIF) in culture supernatants of suppressor T cell (Ts) hybridomas had bioactivity, while the same cells contained a substantial quantity of inactive GIF in cytosol. Mass-spectrometric analysis of GIF in the culture supernatant and cytosol of a Ts hybridoma provided direct evidence that GIF protein was posttranslationally modified in the Ts cells, and that the GIF bioactivity is associated with the posttranslationally modified species. Assuming that conformational changes induced by the posttranslational modifications are responsible for generation of bioactivity, we constructed cysteine mutants of human rGIF (rhGIF) in which cysteine at position 57, 60, or 81 was replaced with Ala, and the mutants were expressed in Escherichia coli. Replacement of Cys57 or Cys60 with Ala resulted in generation of bioactivity, while replacement of Cys81 with Ala failed to do so. It was also found that replacement of Cys57 with Ala and carboxymethylation of a sulfhydryl group in Cys60 synergistically increased the GIF bioactivity of the GIF derivatives. A mutated GIF protein, in which Cys57 and Asn106 in the rhGIF were replaced with Ala and Ser, respectively, had immunosuppressive effects on the IgE and IgG1 Ab responses of BDF1 mice to DNP-OVA, while wild-type rhGIF did not. Evidence was obtained that the mutated GIF suppressed Ag priming of Th cells for the Ab responses and proliferative response.