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Human mitochondrial chaperonin mHsp60 is essential for mitochondrial function by assisting folding of mitochondrial proteins. Unlike the double-ring bacterial GroEL, mHsp60 exists as a heptameric ring that is unstable and dissociates to subunits. The structural dynamics has been implicated for a unique mechanism of mHsp60. We purified active heptameric mHsp60, and determined a cryo-EM structure of mHsp60 heptamer at 3.4 Å. Of the three domains, the equatorial domains contribute most to the inter-subunit interactions, which include a four-stranded β sheet. Our structural comparison with GroEL shows that mHsp60 contains several unique sequences that directly decrease the sidechain interactions around the β sheet and indirectly shorten β strands by disengaging the backbones of the flanking residues from hydrogen bonding in the β strand conformation. The decreased inter-subunit interactions result in a small inter-subunit interface in mHsp60 compared to GroEL, providing a structural basis for the dynamics of mHsp60 subunit association. Importantly, the unique sequences are conserved among higher eukaryotic mitochondrial chaperonins, suggesting the importance of structural dynamics for eukaryotic chaperonins. Our structural comparison with the single-ring mHsp60-mHsp10 shows that upon mHsp10 binding the shortened inter-subunit β sheet is restored and the overall inter-subunit interface of mHsp60 increases drastically. Our structural basis for the mHsp10 induced stabilization of mHsp60 subunit interaction is consistent with the literature that mHsp10 stabilizes mHsp60 quaternary structure. Together, our studies provide structural bases for structural dynamics of the mHsp60 heptamer and for the stabilizing effect of mHsp10 on mHsp60 subunit association., (© 2021. The Author(s).)

Human Chaperonin 10 (hCpn10) was utilised as a novel scaffold for presenting peptides of therapeutic and diagnostic significance. Molecular dynamic simulations and protein sizing analyses identified a peptide linker (P1) optimal for the formation of the quarternary hCpn10 heptamer structure. hCpn10 scaffold displaying peptides targeting Factor VIIa (CE76 -P1 ) and CD44 (CP7) were expressed in E. coli. Functional studies of CE76 -P1 indicated nanomolar affinity for Factor VIIa (3 nM) similar to the E-76 peptide (6 nM), with undetectable binding to Factor X. CE76 -P1 was a potent inhibitor of FX activity (via inhibition of Factor VIIa) and prolonged clot formation 4 times longer than achieved by E-76 peptide as determined by prothrombin time (PT) assays. This improvement in clotting function by CE76 -P1 , highlights the advantages of a heptamer-based scaffold for improving avidity by multiple peptide presentation. In another example of hCPn10 utility as a scaffold, CP7 bound to native CD44 overexpressed on cancer cells and bound rCD44 with high affinity (K D 9.6 nM). The ability to present various peptides through substitution of the hCpn10 mobile loop demonstrates its utility as a novel protein scaffold.

A previously studied immunosuppressive cytokine, Soluble Immune Response Suppressor (SIRS), may have relevance to current studies of immune suppression in a variety of human disease states. Despite extensive efforts using experimental models, mainly in mice, much remains to be discovered as to how autoimmune cells in mice and humans escape normal regulation and, conversely, how tumor cells evade evoking an immune response. It is the contention of this commentary that the literature pre-2000 contain results that might inform current studies. The broadly immunosuppressive protein, SIRS, was studied extensively from the 1970s to 1990s and culminated in the determination of the n-terminal 21mer sequence of this 15kDa protein which had high homology to the short neurotoxins from sea snakes, that are canonical members of the three finger neurotoxin superfamily (3FTx). It was not until 2007 that the prophylactic administration of the synthetic N-terminal peptide of the SIRS 21mer, identical to the published sequence, was reported to inhibit or delay the development of two autoimmune diseases in mice: experimental allergic encephalomyelitis (EAE) and type I diabetes (T1D). These findings were consistent with other studies of the 3FTx superfamily as important probes in the study of mammalian pharmacology. It is the perspective of this commentary that SIRS, SIRS peptide and the anti-peptide mAb, represent useful, pharmacologically-active probes for the study of the immune response as well as in the potential treatment of autoimmune, inflammatory diseases and cancer., (Copyright © 2016 Elsevier Inc. All rights reserved.)

MNSFβ, a ubiquitin-like protein, covalently binds to various target proteins including proapoptotic Bcl-G. During the course of isolation of MNSFβ-conjugating enzyme(s), we identified a novel target protein for MNSFβ. MALDI-TOF MS fingerprinting revealed that the MNSFβ-interacting protein is HSPA8 (heat shock 70-kDa protein 8). We observed that MNSFβ noncovalently binds to HSPA8 in the presence of ATP in vitro. Double knockdown of MNSFβ and HSPA8 strongly inhibited RANKL-induced osteoclastogenesis from Raw264.7 macrophage-like cells. The same treatment inhibited RANKL-induced ERK1/2 and p38 phosphorylation and TNFα production, suggesting that the association of MNSFβ with HSPA8 may promote RANKL-induced osteoclastogenesis. This is the first report that MNSFβ binds to a protein substrate via the noncovalent association and exerts biological effects.

Disease and death caused by bacterial infections are global health problems. Effective bacterial strategies are required to promote survival and proliferation within a human host, and it is important to explore how this adaption occurs. However, the detection and quantification of bacterial virulence factors in complex biological samples are technically demanding challenges. These can be addressed by combining targeted affinity enrichment of antibodies with the sensitivity of liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS). However, many virulence factors have evolved properties that make specific detection by conventional antibodies difficult. We here present an antibody format that is particularly well suited for detection and analysis of immunoglobulin G (IgG)-binding virulence factors. As proof of concept, we have generated single chain fragment variable (scFv) antibodies that specifically target the IgG-binding surface proteins M1 and H of Streptococcus pyogenes. The binding ability of the developed scFv is demonstrated against both recombinant soluble protein M1 and H as well as the intact surface proteins on a wild-type S. pyogenes strain. Additionally, the capacity of the developed scFv antibodies to enrich their target proteins from both simple and complex backgrounds, thereby allowing for detection and quantification with LC-SRM MS, was demonstrated. We have established a workflow that allows for affinity enrichment of bacterial virulence factors.

MNSFβ is a ubiquitously expressed member of the ubiquitin-like family that has been involved in various biological functions. Previous studies have demonstrated that MNSFβ covalently binds to various target proteins including Bcl-G, a proapoptotic protein. In this study, we purified a 115 kDa MNSFβ adduct from murine liver lysates by sequential chromatography on DEAE and anti-MNSFβ IgG-conjugated Sepharose in the presence of ATP. MALDI-TOF MS fingerprinting revealed that this MNSFβ adduct consists of an 8.5 kDa MNSFβ and 10-formyltetrahydrofolate dehydrogenase (FDH), an abundant enzyme of folate metabolism. Interestingly, MNSFβ preferably binds to cytosolic but not mitochondrial FDH. Fingerprinting analysis of the MNSFβ adduct demonstrate that MNSFβ conjugates to cytosolic FDH with a linkage between the C-terminal Gly74 and Lys72. The 115 kDa MNSFβ/FDH complex was not expressed in any of the tissues examined, indicating that this adduct formation is not ubiquitous. We found that MNSFβ/FDH complex formation was induced by dexamethasone in thymocytes. Double knockdown of MNSFβ and FDH strongly reduced dexamethasone-induced apoptosis. Collectively, MNSFβ/FDH complex formation may positively regulate apoptosis in thymocytes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Receptor-ligand binding is an essential interaction for biological function. Oxidative stress can modify receptors and/or membrane lipid dynamics, thus altering cell physiological functions. The aim of this study is to analyze how oxidative stress may alter receptor-ligand binding and lipid domain distribution in the case of progesterone-induced blocking factor/progesterone-induced blocking factor-receptor. For membrane fluidity regionalization analysis of MEC-1 lymphocytes, two-photon microscopy was used in individual living cells. Lymphocytes were also double stained with AlexaFluor647/progesterone-induced blocking factor and Laurdan to evaluate -induced blocking factor/progesterone-induced blocking factor-receptor distribution in the different membrane domains, under oxidative stress. A new procedure has been developed which quantitatively analyzes the regionalization of a membrane receptor among the lipid domains of different fluidity in the plasma membrane. We have been able to establish a new tool which detects and evaluates lipid raft clustering from two-photon microscopy images of individual living cells. We show that binding of progesterone-induced blocking factor to progesterone-induced blocking factor-receptor causes a rigidification of plasma membrane which is related to an increase of lipid raft clustering. However, this clustering is inhibited under oxidative stress conditions. In conclusion, oxidative stress decreases membrane fluidity, impairs receptor-ligand binding and reduces lipid raft clustering., (© 2013.)

MNSFbeta (Monoclonal nonspecific suppressor factor beta) is a natural immunosuppressive factor which has been reported to be involved in various biological processes, such as immune responses, cell division, stress response, cell apoptosis, and nuclear transport. However, study on porcine MNSFbeta has been rarely reported. In this study, the full-length sequence of porcine MNSFbeta (GenBank accession number: KF77642500) was predicted in silicon and its cDNA sequence was obtained through RT-PCR from porcine spleen. The nucleic acid and protein sequences were analyzed. Then, the gene was subcloned into pEGFP-C1 to construct a recombinant plasmid pEGFP-MNSFbeta which was transfected into swine umbilical vein endothelial cells (SUVECs) using Lipofectamine 2000. The expression of GFP was detected by fluorescence microscopy, Western blot, and laser confocal fluorescence microscopy. The spatial expression patterns of porcine MNSFbeta were detected by real-time qPCR. Results showed that the full length of porcine MNSFbeta was 402 bp encoding 133 amino acids with only one exon. Bioinformatics analysis showed that porcine MNSFbeta protein was a stable protein consisting of a ubiquitin-like domain fused to the ribosomal protein S30 with no signal peptide. The analyses of homology and phylogenetic tree of porcine MNSFbeta and its homologs in other 18 species showed that the identities of MNSFbeta protein sequence were higher than 91% among different species and the evolutionary distance was less than 0.05. It indicates that MNSFbeta is highly conserved in the process of evolution. Fluorescence signal showed that the fusion protein GFP-MNSFbeta was successfully expressed in SUVECs which was then confirmed by Western blot. Laser confocal fluorescence microscopy showed that MNSFbeta was expressed in both nucleus and cytoplasm. Analysis of spatial expression patterns showed that procine MNSFbeta was widely expressed in immune tissues, but not in lung, suggesting that MNSFbeta may play an important role in immune response.

In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, apoptosis of T cells is mainly seen at inflammation sites of the central nervous system (CNS). Cumulative data suggests that astrocytes might render T cells susceptible to induction of apoptotic cell death. We observed that apoptotic cell death of proteolipid protein (PLP)-reactive T cells was induced by an interferon (IFN)-γ-treated astrocyte cell line. In this study, we have identified and cloned the genes derived from the IFN-γ-treated astrocyte cell line that induce apoptosis of autoreactive T cells. We created subtraction cDNA libraries from the IFN-γ-treated astrocyte cell line and obtained 100 positive clones. After screening of subtracted cDNAs, we found two candidate genes that induced apoptosis of the PLP-reactive T cell line. The first is a previously unknown gene of 726 base pairs that we named astrocyte-derived immune suppressor factor (AdIF). It contained an open reading frame encoding a polypeptide of 228 amino acids. The second was SPARC/osteonectin, a multifunctional glycoprotein secreted in the extracellular matrix. AdIF protein was found at the inflammatory sites of the EAE brain, and bound to the surface of CD4(+) T cells. Purified recombinant AdIF protein inhibited the proliferation of activated PLP-reactive CD4(+) T cells and induced their apoptosis in vitro. Intravenous administration of recombinant AdIF protein to mice with in which acute EAE was induced prevented the incidence of EAE and suppressed the symptoms. The newly discovered molecule AdIF may render auto-reactive T cells susceptible to the induction of apoptotic cell death and could potentially be a new therapeutic agent for multiple sclerosis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Monoclonal nonspecific suppressor factor beta (MNSFbeta) is a ubiquitously expressed member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFbeta covalently binds to the intracellular proapoptotic protein Bcl-G in cells of the macrophage cell line Raw264.7, suggesting involvement of this ubiquitin-like protein in apoptosis. In this study, we purified a 62 kDa MNSFbeta adduct from murine liver lysates by sequential chromatography on DEAE and anti-MNSFbeta IgG-conjugated Sepharose. MALDI-TOF MS fingerprinting revealed that this MNSFbeta adduct consists of an 8.5 kDa MNSFbeta and endophilin II, a member of the endophilin A family. MNSFbeta may conjugate to endophilin II with a linkage between the C-terminal Gly74 and Lys294. We confirmed this result by immunoprecipitation/western blot studies. Endophilin II was ubiquitously expressed in various tissues, although a truncated form was observed in liver. The 62 kDa MNSFbeta-endophilin II was specifically expressed in liver and macrophages. Small interfering RNA-mediated knockdown of endophilin II and/or MNSFbeta promoted phagocytosis of zymosan in Raw264.7 cells. Conversely, cotransfection of endophilin II and MNSFbeta cDNAs inhibited the phagocytosis of zymosan. Such inhibition was not observed in cells expressing a mutant of endophilin II in which Lys294 was replaced by arginine. These results suggest that the post-translational modification of endophilin II by MNSFbeta might be implicated in phagocytosis by macrophages.

The yeast Paf1 complex consists of Paf1, Rtf1, Cdc73, Ctr9, and Leo1 and regulates histone H2B ubiquitination, histone H3 methylation, RNA polymerase II carboxy-terminal domain (CTD) Ser2 phosphorylation, and RNA 3' end processing. We provide structural insight into the Paf1 complex with the NMR structure of the conserved and functionally important Plus3 domain of human Rtf1. A predominantly beta-stranded subdomain displays structural similarity to Dicer/Argonaute PAZ domains and to Tudor domains. We further demonstrate that the highly basic Rtf1 Plus3 domain can interact in vitro with single-stranded DNA via residues on the rim of the beta sheet, reminiscent of siRNA binding by PAZ domains, but did not detect binding to double-stranded DNA or RNA. We discuss the potential role of Rtf1 Plus3 ssDNA binding during transcription elongation.

Objective: Ingested type I IFN inhibits clinical attacks, relapses and inflammation in murine chronic relapsing EAE by inhibiting Th1-like cytokines. Type I IFN activates human suppressor T cells that produce SIRS., Methods: We examined whether oral (ingested) SIRS peptide inhibits EAE by decreasing Th1-like cytokines., Results: Parenteral SIRS peptide 1-21 showed a significant inhibition of disease severity in murine EAE. Ingested SIRS peptide at 10 and 100 microg SIRS peptide showed a significant inhibition of disease severity but also a prolonged delay in the onset of disease compared to placebo. There were significantly less inflammatory foci in the SIRS peptide fed group compared to the control mock fed group. Splenocytes from SIRS peptide 1-21 fed mice showed increased production of Th2-like CD30L, IL-13, TCA-3 cytokines/chemokines and decreased production of Th1-like cytokine lymphotactin., Interpretation: Ingested (oral) SIRS peptide significantly inhibits both clinical EAE and inflammation predominately via counter-regulatory type 2-like cytokines/chemokines IL-13, CD30L and TCA-3.

In an attempt to identify new immunoreceptor tyrosine-based activation motif (ITAM)-containing human molecules that may regulate hitherto unknown immune cell functions, we BLAST searched the National Center for Biotechnology Information database for ITAM-containing sequences. A human expressed sequence tag showing partial homology to the murine TJ6 (mTJ6) gene and encoding a putative ITAM sequence has been identified and used to clone the human TJ6 (hTJ6) gene from an HL-60-derived cDNA library. hTJ6 was found to encode a protein of 856 residues with a calculated mass of 98 155 Da. Immunolocalization and sequence analysis revealed that hTJ6 is a membrane protein with predicted six transmembrane-spanning regions, typical of ion channels, and a single putative ITAM (residues 452-466) in a juxtamembrane or hydrophobic intramembrane region. hTJ6 is highly homologous to Bos taurus 116-kDa subunit of the vacuolar proton-translocating ATPase. Over-expression of hTJ6 in HEK 293 cells increased H+ uptake into intracellular organelles, an effect that was sensitive to inhibition by bafilomycin, a selective inhibitor of vacuolar H+ pump. Northern blot analysis demonstrated three different hybridizing mRNA transcripts corresponding to 3.2, 5.0 and 7.3 kb, indicating the presence of several splice variants. Significant differences in hTJ6 mRNA levels in human tissues of different origins point to possible tissue-specific function. Although hTJ6 was found to be a poor substrate for tyrosine-phosphorylating enzymes, suggesting that its ITAM sequence is non-functional in protein tyrosine kinase-mediated signaling pathways, its role in organellar H+ pumping suggests that hTJ6 function may participate in protein trafficking/processing.

Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic antigen-nonspecific suppressive functions. A cDNA clone encoding MNSFbeta, an isoform of the MNSF, has been isolated and characterized. MNSFbeta cDNA encodes a fusion protein consisting of a ubiquitin-like segment (Ubi-L) and ribosomal protein S30. Most recently, we observed that Ubi-L covalently conjugates to Bcl-G, a novel pro-apoptotic protein. In this study, we observed that Ubi-L noncovalently and specifically binds to histone 2A. The maximum binding was observed at a molar ratio equal to 1 for GST-Ubi-L and 2 for histone 2A. Ubi-L formed complex with histone 2A in the presence of 1% Triton X-100. Free Ubi-L was detected in nuclei from unstimulated murine helper T cell line, D10. The increased amounts of free Ubi-L and some Ubi-L adducts were observed in nuclei from mitogen-activated D10 cells. Interestingly, two Ubi-L adducts were unique to the chromatin fraction of nuclei from the activated D10 cells.

Problem: Early pregnancy factor (EPF) is an immunosuppressive protein detected in the serum in early pregnancy. We have already reported the development of the rosette inhibition test for mare EPF and have detected EPF in thoroughbreds and ponies. Here, we attempted to purify equine EPF from pregnant mare serum., Methods of Study: Mare EPF was purified by ultrafiltration and ion-exchange chromatography. Purified EPF was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and a neutralization test. EPF activity was estimated as the rosette inhibition titer (RIT) by the rosette inhibition test., Results: Purified EPF bound to carboxymethyl (CM) sepharose and did not adsorb to diethylaminoethyl (DEAE) sepharose. SDS-PAGE revealed that in the final purified fraction there were many proteins. In the immunoblotting analysis, a protein band of 25.8 kDa was detected as the pregnancy-specific band. Further, antibody gained from the 20 to 30 kDa protein band of the final purified fraction neutralized the RIT activity of pregnant mare serum., Conclusions: Mare EPF was detected in the final purified fraction and had a molecular weight of 25.8 kDa. EPF in the mare is similar to that obtained from the serum of pregnant cows.

Previous studies from our laboratory showed that the immunomodulatory effects of progesterone are mediated by a 34-kDa protein, named the progesterone-induced blocking factor (PIBF). Lymphocytes of women with threatened abortion fail to produce this factor. Via inducing a Th2 biased cytokine production and blocking of NK activity, PIBF prevents induced pregnancy loss in mice, suggesting that substitution therapy with PIBF could be useful as an alternative treatment of certain forms of recurrent spontaneous abortions. Our study was aimed at mapping the sequence and structure of PIBF coding cDNA and characterizing the encoded protein product. Screening of a human liver cDNA library revealed a 2765-bp clone with a 2271-bp open reading frame. The PIBF1 cDNA encodes a protein of 757 amino acid residues with an 89-kDa predicted molecular mass, which shows no significant amino acid sequence homology with any known protein. PIBF produced via recombinant technique is recognized by the Ab specific for the secreted lymphocyte PIBF Ab, and possesses the biological activities of the secreted lymphocyte PIBF. The full-length PIBF is associated with the nucleus, whereas secretion of shorter forms, such a 34-kDa protein is induced by activation of the cell. The 48-kDa N-terminal part of PIBF is biologically active, and the part of the molecule, responsible for modulating NK activity is encoded by exons 2-4. These data provide an initial step for exploiting the possible diagnostic and therapeutic potential of this immunomodulatory molecule.

Aim: To purify a protein in pig spleens, which was similar to immune suppressive protein of stress (ISPS), and characterize its properties and functions., Methods: 1) Pig spleen was extracted in dilute hydrochloric acid. 2) The extract was ultra-filtrated for having high molecular weight proteins (Mr>30 000). 3) The filtrates were purified with FPLC affinity chromatography. 4) The elute from FPLC was used for T-lymphocyte proliferation and ELISA test. 5) Lastly, SDS-PAGE was used to determine the molecular weight and purity of the final product., Results: A protein purified from pig spleen (the pig ISPS homologue) inhibited concanavalin A (Con A)-induced mouse lymphocyte proliferation. The molecular weight of this protein was about Mr 190 000. It has a stronger selectivity against T-lymphocyte line such as Jurkat cell line and mastocyte line (P8l5) and has a weaker inhibitory activity on macrophage line (U937)., Conclusion: A protein similar to rat/mouse ISPS was found in pig spleen. This may provide an opportunity to study its roles in tumors and autoimmune diseases.

A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-gamma, TNF-alpha, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft-versus-host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins.

Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses a pleiotropic antigen-nonspecific suppressive function. We have shown that 70 kDa MNSF comprises an 8 kDa ubiquitin-like polypeptide (Ubi-L) and 62 kDa T cell receptor (TCR) alpha-like molecule. Ubi-L binds specifically to its 82 kDa receptor protein on target cells. In the current study, we have further characterized the biochemical nature of the TCR(alpha)-like molecule. The 62 kDa protein was separated into two species of 46 kDa and 16 kDa on reverse-phase HPLC. Anti-TCR(alpha) monoclonal antibody recognized the 46 kDa, but not the 16 kDa protein. Anti-TCRbeta monoclonal antibody failed to recognize these proteins. Ubi-L conjugated to the 46 kDa protein, whereas Ubi-L lacking its C-terminal Gly-Gly did not. Although Ubi-L was labile both to heating at 56 degrees C and to acidification to pH 4, the Ubi-L-46 kDa protein complex was unaffected by these treatments. In addition, the 46 kDa protein elongated the Ubi-L-induced protein tyrosine phosphorylation in a concanavalin A-activated murine T helper type 2 clone, D10 cells. One of the four tryptic peptide sequences derived from the 46 kDa protein was in alignment with a related sequence found in the J(alpha) region of the TCR(alpha), including the highly conserved motif F-G-X-G-T-X-L.

Patients with gliomas exhibit deficient in vitro and in vivo T cell immune activity, and human glioblastoma culture supernatants (GCS) inhibit in vitro T lymphocyte responses. Because APC are essential for initiating and regulating T cell responses, we investigated whether GCS would affect cytokines produced by monocytes and T cells from healthy donors of PBMC. Incubation of PBMC with GCS decreased production of IL-12, IFN-gamma, and TNF-alpha, and increased production of IL-6 and IL-10. The GCS-induced changes in IL-12 and IL-10 occurred in monocytes, and involved changes in IL-12 p40 and IL-10 mRNA expression. Incubation with GCS also resulted in reduced expression of MHC class II and of CD80/86 costimulatory molecules on monocytes. The immunosuppressive effects were not the result of IL-6 or TGF-beta1 that was detected in GCS. However, it was due to a factor(s) that is resistant to pH extremes, differentially susceptible to temperature, susceptible to trypsin, and has a minimum molecular mass of 40 kDa. Our findings show that glioblastoma-generated factors that are known to suppress T cell responses alter the cytokine profiles of monocytic APC that, in turn, inhibit T cell function. This model indicates that monocytes can serve as an intermediate between tumor-generated immune-suppressive factors and the T cell responses that are suppressed in gliomas.

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.

The major allergen (Tur c 1) in the muscle of the gastropod, Turbo cornutus, was isolated by Sephacryl S-300, Mono Q HR 5/5 and TSKgel Phenyl-5PW RP column chromatography. ELISA showed Tur c 1 to react strongly with sera from three individuals sensitive to both mollusks and crustaceans. SDS-PAGE showed Tur c 1 to produce a major band corresponding to a molecular mass of 35 kDa under the reduced condition. Its amino acid composition was characterized by the abundance of Glx, followed by Leu, Ala and Lys in decreasing abundance, and the absence of Trp. In addition to these properties, the determined partial amino acid sequence identified Tur c 1 to be a tropomyosin, as in the case of the known mollusk and crustacean allergens. However, the results of competitive ELISA inhibition experiments suggest that Tur c 1 has an IgE-binding epitope in the C-terminal region which is dissimilar to those proposed for Cra g 1 (the oyster Crassostrea gigas allergen) and Pen i 1 (the shrimp Penaeus indicus allergen).

Although existence of suppressor T cells is a controversial issue in cellular immunology, several lines of evidence indicate that T-cell-receptor alpha-chain (TCR-alpha) is a critical component of suppressor factors produced by these cells. Monoclonal non-specific suppressor factor (MNSF), a lymphokine produced by murine T-cell hybridoma, possesses pleiotrophic antigen-non-specific suppressive functions. Recently, we have shown that the 70,000-MW MNSF comprises an 8000-MW ubiquitin-like polypeptide and other subunit(s). Here we report that the 8000-MW ubiquitin homologue is associated with an intracellular TCR-alpha (but not TCR-beta)-like molecule and released from the cells. The affinity eluates obtained from the culture supernatants of E17 cells and concanavalin A (Con A)-activated splenocytes with anti-TCR-alpha monoclonal antibody (mAb) showed an antigen-non-specific, major histocompatibility complex (MHC)-non-restricted suppression. Immunoblot analysis demonstrated that anti-TCR-alpha, but not anti-TCR-beta, mAb recognizes native 70,000-MW MNSF. In addition, we found the dissociation of the 8000-MW polypeptide from the 62,000-MW TCR-alpha cross-reactive protein by hydrolase which cleaves isopeptide bonds. Thus the covalent attachment of ubiquitin-like protein(s) may be involved in the underlying mechanism of suppressor T-cells and TCR-alpha-like molecule(s) might be a main link between antigen-specific and non-specific suppression.

The infection of human peripheral blood B cells with Epstein-Barr Virus (EBV), induced the production of suppressor factor(s) which were released into the supernatant of the B-cell cultures. The induction of suppressive activity was independent of T-cell presence. The suppression was exhibited both against T-cell activity (MLR and mitogenic stimulation) as well as against B-cell mitogenic stimulation of human or murine B lymphocytes. The suppressive factor(s) was of a low molecular weight (equal or less than 5,000), resistant to trypsin and heating at 80 degrees C and its activity was partially inhibited by neuraminidase treatment. These findings indicate that the suppressive factor(s) is not correlated to immunoglobulin production, is not apparently of a protein nature, and might be of ganglioside or siaylated glycoprotein structure. Our present findings suggest that, in addition to T cells, B cells might also play an immunoregulatory role in the expression of immune response potential.

A relationship is described between TS--a human suppressive lymphokine produced by hybridoma T cells--and alpha-enolase from the U937 monocyte human cell line. A strong homology (92%) was observed by comparing the internal amino acid sequences of 6 TS peptides, corresponding to 52 residues, with the complete sequence of alpha-enolase. The molecular masses of these two molecules were found to be of about 47 kDa and both were detected with the same monoclonal antibody to TS. In contrast, no immunosuppressive activity was detected with the purified enolase fraction, whereas TS did not show any enolase activity. A suppressive monokine secreted by U937 cells was found to be unrelated with TS. Theses results suggest that the TS immunosuppressive factor despite its absence of enzyme activity, belongs to the enolase family.

Transforming growth factor (TGF)-beta 2-related-decidual suppressor factor (DSF) and TJ6 protein are both immunosuppressive molecules present in murine and human pregnancy. Treatment of mice with either anti-TJ6 or anti-TGF-beta 2 neutralizing antibodies results in increased fetal loss. Western blots of supernatants from pregnant mouse decidua probed with anti-TJ6 (soluble form) showed a doublet at a similar molecular size as when the blot was probed with anti-TGF-beta 2 antibody. The problem is to determine whether TJ6 and DSF are the same protein. In order to determine if TJ6 and DSF are the same or different proteins, we used affinity column purified TGF-beta 2-DSF and stained Western blots with anti-TJ6. The TGF-beta 2-monoclonal antibody affinity column-purified DSF that stained with anti TGF-beta 2 was not reactive with anti-TJ6 antibody. TJ6 has only a 30% gene sequence homology and a 13% amino acid homology to TGF-beta 2. TJ6 and TGF-beta 2-related DSF appear to be different immunosuppressive proteins in decidua.

We have previously demonstrated that sonic extracts of Fusobacterium nucleatum FDC 364 were capable of inhibiting human T-cell responses to mitogens and antigens. The purified F. nucleatum immunosuppressive protein (FIP) is composed of two subunits of 44 and 48 kDa. Furthermore, FIP inhibits T-cell activation by arresting cells in the middle of the G(1) phase of the cell cycle; the data available to date suggest that FIP impairs the expression of the proliferating-cell nuclear antigen. To initiate delineation of FIP structure-function relationships, molecular cloning of the FIP gene was carried out. A DNA library of F. nucleatum FDC 364 was constructed by partial digestion of genomic DNA with Sau3A and screened for the production of FIP with polyclonal antibody. Twelve immunoreactive clones were identified. One of these clones contained a 3.1-kbp insert and was chosen for further study. Cell lysates were found to contain an immunoreactive band that comigrated with the 44-kDa subcomponent of the native FIP. Sequencing of the 3.1-kpb insert revealed the presence of three open reading frames (ORFs). One ORF extends from nucleotides 415 to 1620, encodes 402 amino acids, and is preceded by a ribosome-binding site. Deletion analysis and antibody elution analysis showed that this ORF encodes the 44-kDa subunit (FipA) of native FIP. A second ORF is situated upstream of fipA. However, Northern (RNA) analysis suggested that fipA is not transcribed as part of an operon but transcribed from its own promotor. Finally, the partially purified recombinant FipA protein was capable of impairing T-cell activation in a manner consistent with the native protein. These results indicate that the two components that form the native protein are most probably distinct gene products and suggest that the 44-kDa FipA polypeptide is sufficient to mediate the immunosuppressive activities of the native protein complex.

These studies have demonstrated that TJ6m protein can be measured in women prior to a spontaneous abortion based on expression of TJ6 on CD56-positive NK cells. This suggested to us a possible regulator function for TJ6 during pregnancy. We have shown that in T cells only crosslinking of the T-cell receptor can upregulate TJ6 expression and the other activators, such as mitogens, do not. A clear discrepancy in the pattern of expression of TJ6 on CD56 and CD19-positive cells was noted between successful and unsuccessful pregnancies. A successful pregnancy is denoted by TJ6 expression on B cells, whereas in a potential spontaneous abortion there is TJ6 expression on both NK and B cells.

We isolated a unique TCR alpha-chain derived from the OVA-specific Ts hybridoma, 231F1. The TR alpha-chain consists of V alpha 11.3, a unique J alpha and a complete sequence of C alpha region. Transfection of the TCR-alpha cDNA into a TCR-alpha-, TCR-beta+ T cell line, 175.2, resulted in the expression of TCR-alpha beta, and the transfectant contained a 35-kDa peptide having the TCR- alpha-specific antigenic determinant. However, the stable transfectant failed to release a peptide with the TCR-alpha determinant upon stimulation with anti-CD3. In contrast, overexpression of the cDNA in the 231 F1 cells markedly increased the formation of the 55-kDa peptide, which reacted with both anti-glycosylation-inhibiting factor (GIF) and the mAb H28-710. Definitive evidence for the relationship between the 55-kDa peptide and the TCR alpha-chain was obtained by transfection of the cDNA of the TCR alpha-chain with histidine tag into the 231F1 cells. The 55 kDa GIF peptide formed by stable transfectants of the TCR-alpha-tag cDNA bound to Ni+-nitrilotriacetic acid-agarose. Upon stimulation with anti-CD3, a stable transfectant of the TCR-alpha cDNA formed OVA-specific GIF which contained the 55-kDa GIF peptide, and bound not only to anti-TCR-alpha column but also to anti-TCR-beta column. The results indicate that the OVA-specific GIF consists of the TCR-alpha+ 55-kDa GIF and another peptide with TCR-beta determinant. It was found that the association of the TCR-beta+ peptide with the 55-kDa GIF is required for binding of the factor to OVA, but not essential for the formation and release of the latter peptide.

Stimulation of OVA-specific suppressor T cell (Ts) hybridoma and bee venom phospholipase A2 (PLA2)-specific Ts hybridoma with Ag-pulsed APC or by cross-linking of CD3 resulted in the formation of Ag-specific glycosylation-inhibiting factor (GIF). Affinity-purified Ag-specific GIF preparations, obtained by using Ag-coupled Sepharose or anti-TCR alpha-chain-coupled Affi-Gel, contained a 55-kDa peptide, which bound both polyclonal anti-GIF Abs and anti-TCR-alpha mAb in immunoblotting. The same hybridomas constitutively secrete 13-kDa bioactive GIF peptide that has no affinity for homologous Ag, but neither the Ag-specific GIF activity nor 55-kDa GIF peptide was detectable in culture supernatants of unstimulated cells. Northern blot analysis of mRNA from the anti-CD3-stimulated hybridoma with 32P-labeled GIF cDNA revealed only 0.6 kb mRNA, which encodes the 13-kDa nonspecific GIF. No mRNA of the 55-kDa GIF was detectable. A representative OVA-specific Th hybridoma, DO 11.10 cells contain the 0.6 kb GIF mRNA and constitutively secrete inactive GIF peptide. However, the Th hybridoma failed to secrete the 55-kDa peptide or any peptide with the TCR-alpha determinant upon stimulation with anti-CD3. It appears that the formation of the 55-kDa peptide with the TCR-alpha determinant is unique for a subset of T cells including Ts cells that form bioactive GIF.

Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic Ag-nonspecific suppressive functions. Most recently, a cDNA clone encoding MNSF beta (a subunit of MNSF) was isolated and characterized. The MNSF beta cDNA encodes a 14.5-kDa fusion protein with MNSF-like activity consisting of an 8-kDa ubiquitin-like segment (ubi-L) and the ribosomal protein S30 (6.5 kDa). To determine whether ubi-L itself has biologic activity, cDNA encoding the ubi-L region was expressed in bacteria and the recombinant product was tested for the activity. Ubi-L showed MNSF-like biologic activity without any cytotoxic action. Interestingly, the addition of ubiquitin to the assay inhibited ubi-L-induced suppression. IFN-gamma, which is known to enhance the expression of MNSF receptor, induced splenocytes to secrete ubi-L by increasing mRNA. Ubi-L has species-specific action and the ability to selectively inhibit the B cell proliferative response stimulated by soluble but not by Sepharose-bound anti-Ig Ab. In addition, okadaic acid, a serine/threonine phosphatase 1 and 2A inhibitor, showed a synergistic inhibitory action with ubi-L, indicative of the possible involvement of phosphatase(s) in the regulation of ubi-L action.

Non-adherent cells from PPD+ tuberculosis patients (TBP PPD+) and from healthy individuals treated with whole tuberculosis anergic immune sera or with its protein A-Sepharose IgG fraction, or with sera fraction separated by PPD-Sepharose chromatography, were submitted to immunofluorescence assays. Anti-human IgG or IgM FITC-conjugate were used to reveal the assays, and results were expressed by a fluorescence percentage or fluorescence index. The presence of IgG over the surface of PPD+ non-adherent cells was detected. High fluorescence percentages were observed only in those PPD+ cells treated with whole anergic serum or with its IgG fraction. Positive fluorescence index values were obtained only in those PPD+ cells treated with anergic serum, meanwhile fluorescence index was always negative when non-bound fractions from PPD-Sepharose were used. Results suggest that non-adherent population are the cell targets for the serum inhibitory factor, which previously has been detected to inhibit antigen response in PPD reactive cells and, point out the specific behavior of this factor, since it was eliminate by PPD-Sepharose chromatography. The IgG nature of the factor was demonstrated by SDS-PAGE and immunoelectrophoresis.

Problem: To study whether embryo associated immunosuppressor factor (EASF) is synthesized at the maternal-fetal interface., Method: Anti-EASF monoclonal antibody H5D12 was used to identify EASF. Paraffin-embedded sections were prepared from placental and fetal tissues and immunohistochemistry was done by the avidin-biotin-peroxidase technique. EASF was affinity purified using H5D12-Sepharose 4B from culture media of placental villi and analyzed for immunosuppressive activity (by Concanavalin A-induced lymphocyte proliferation assay) and molecular weight identity (by metabolic labeling studies with 35S-methionine followed by immunoprecipitation and SDS-PAGE)., Results: Immunohistochemical studies demonstrates intense immunostaining of villous syncytiotrophoblast and cytotrophoblast cells of first trimester placental tissues. Hoffbauer cells and decidual cells stained positive. The same cells in second and third trimester placental tissues stained weakly. However, the endothelium and smooth muscle cells of fetal blood vessels, fetal ovarian stroma and primordial follicles, kidney epithelium, cerebral neurons, and glial cells all stained negative. The affinity-purified EASF from the conditioned media of placental villi (less than 12 wk gestational age) was identified as a 37-kDa molecule with immunosuppressive activity. Metabolic labeling studies revealed that placental villi from early gestational age secretes a major factor of 37-kDa and minor factors of 41-kDa and 47-kDa molecular weight., Conclusions: Monoclonal antibody H5D12 identifies a factor that is produced by the pre-implantation embryo and also synthesized by decidua and trophoblast cells.

Secretion of bioactive glycosylation-inhibiting factor (GIF) appears to be restricted to suppressor T (Ts) cells, although various human and murine cell line cells secrete the 13-kDa peptide that reacts with anti-GIF. Nucleotide sequences of GIF cDNA from the Ts and non-Ts cells are identical, indicating that bioactive GIF and inactive GIF have an identical amino acid sequence. A stable transfectant of human GIF (hGIF) cDNA in BMT10 cells secretes inactive GIF peptide, whereas transfection of a chimeric cDNA encoding a fusion protein consisting of the N-terminal region of procalcitonin precursor and hGIF into the same cells results in secretion of bioactive GIF. Evidence was obtained that the fusion protein goes into the endoplasmic reticulum and is cleaved for the secretion of mature GIF peptide, whereas the inactive 13-kDa peptide synthesized by the former transfectant does not go through the endoplasmic reticulum. However, a stable transfectant of hGIF cDNA in mouse Ts hybridoma contains inactive GIF in the cytosol and secretes bioactive hGIF without participation of the endoplasmic reticulum-Golgi system. Heterogeneity of the 13-kDa hGIF from the transfectant was detected in two-dimensional electrophoresis. The results suggested that Ts cells have a machinery that converts a portion of inactive cytosolic GIF peptide to bioactive GIF during secretion.

Contrasuppression is a regulatory T cell activity that acts on helper and contact sensitivity effector T cells to protect them from the action of suppressor T cells. In this study, we examined a monoclonal Ag-specific T cell-secreted contrasuppressor factor (TcsF) with Ag specificity for the hapten TNP (trinitrophenyl). This factor positively influences the adoptive cell transfer of contact sensitivity in the presence of active suppression. Results presented in this report demonstrate a relationship between TcsF and the alpha,beta-T cell receptor (TCR). We found that TcsF bound to mAb anti-TCR-alpha (H28) and to mAb anti-TCR-beta (H57) immunoaffinity columns, but not to a mAb anti-TCR-gamma,delta (UC7) column. The bound contrasuppressor activity could be recovered from these affinity columns by base elution. Reduction of TcsF with DTT followed by alkylation with methylmethanethiosulfonate (MMTS), demonstrated that the active subunit of TcsF bound to and eluted from anti-TCR-alpha, but not to anti-TCR-beta columns. The active TcsF that bound to anti-TCR-alpha but not to the anti-TCR-beta column was shown subsequently to have the ability to bind to specific Ag columns TNP-BSA and TNP-BGG Sepharose 4B. The TcsF could be successfully recovered later from Ag columns; thus suggesting that the Ag-binding domain for mAb, and the biologic activity, resides on the TCR-alpha chain of TcsF. Separation of TcsF on SDS-PAGE under reducing conditions, followed by elution and renaturation of proteins from the gel slices, showed that a 35 to 40 kDa protein had the T cell contrasuppressive activity. Western blot analysis of nonreduced TNP-binding TcsF revealed TCR-alpha,beta determinants on an 80-kDa native molecule similar to TCR-alpha,beta from a helper T cell clone. In summary, Ag-specific, Ag-binding T cell-derived contrasuppressor factor has serologic determinants of TCR-alpha and TCR-beta chains, but the TCR-alpha chain and not the TCR-beta chain is required for biologic function. This TCR-alpha determinant-containing factor is absorbed by specific Ag (TNP) columns. SDS-PAGE analysis under reducing conditions suggested a molecular weight of about 35 to 40 kDa for the TNP-specific TcsF. The mechanism of action of the TCR-alpha-containing contrasuppressor factor in the regulation contact sensitivity is discussed.

The monoclonal nonspecific suppressor factor (MNSF) is a lymphokine produced by a murine T cell hybridoma capable of suppressing Ab production by LPS-stimulated B cells. The existence of a human counterpart of MNSF, designated as the human nonspecific suppressor factor (hNSF), was likely because the anti-MNSF mAb (MO6) recognizes a similar suppressive activity in supernatants of Con A-stimulated human PBMC. By using the MO6 mAb, we investigated the presence of hNSF in the ascitic fluid of a patient with SLE. A small amount of cross-reactive hNSF was isolated from concentrated ascitic fluid fractionated with the MO6-affinity column, and a specific anti-hNSF mAb (P2) was produced. The hNSF eluted from the P2-affinity column could suppress up to 80% of the PWM-induced IgG production of human PBMC in a dose-dependent manner, even when added in late culture periods. Moreover, hNSF could inhibit proliferation of PBMC triggered by either PWM or Con A, which also implies an effect on T cells. On SDS-PAGE, the isolated hNSF resolved as a single peak of about 66 kDa and probably represents an aggregate of hydrophobic subunits. On reverse-phase HPLC, the bioactivity could be recovered from a single peak at 18.3 min. The suppression of IgG production induced by hNSF could be partly neutralized by preincubation with an anti-TCR-alpha mAb, whereas an anti-TCR-beta did not have any effect. Anti-TCR-alpha could also directly bind to the isolated nNSF, demonstrating some serologic relationship, as has been reported for several Ag-specific suppressor systems.

Ovalbumin-specific, H-2Kd restricted, CD8+ Ts cells of clone 17.2 were shown to produce an OVA-specific Ts cell factor (TsF17.2) possessing the same Ag specificity and MHC restriction as those of the intact Ts cells. The Ts cell clone was generated from a single cell of the spleen of a mouse which had been immunosuppressed by injection of tolerogenic OVA(mPEG)12 conjugate. For the elucidation of the nature of TsF17.2, it was characterized by serologic, physicochemical, and Western blot analyses. It was found that 1) the OVA-specific suppression of in vitro antibody production by TsF17.2 could be blocked by mAb H28-710 which binds to an epitope of the constant region of the alpha-chain of TCR; 2) the TsF17.2 could be sequestered by, and eluted from, immunosorbents prepared by coupling to Affi-Gel Hz the H28-710 mAb or the mAb H57-597 and F23.1 which are specific, respectively, for an epitope of the constant region of the beta-chain and an epitope of the V beta 8 region of the TCR; and 3) the TsF17.2 had a pl of 7.0, m.w. of 84,000, and consisted of two disulfide-linked subunits of 42,000 each. After electroelution from the SDS-PAGE gel, the m.w. 84,000 molecule retained its capacity to suppress in vitro antibody production in an OVA-specific manner. From all these results it was concluded that this Ts cell factor may represent a soluble form of the alpha beta heterodimer of TCR of cloned Ts cells.

A nonspecific suppressor factor has been identified in serum of newborn rats and calves. This factor, designated SUF-s, was shown to interfere--across species barriers--with lymphocyte responses in vitro and in vivo. SUF-s interferes in vitro with T- and B-cell proliferation induced by different mitogens and IL-2. Our findings indicate that the activity of SUF-s in vitro, which is of a reversible nature, is directed at an early event in the cascade of T-cell activation. SUF-s does not affect intrinsically regulated proliferation, such as that of tumor cells or established cell lines. In vivo, SUF-s prevents graft-vs-host disease induced by transplantation of allogeneic bone marrow cells in lethally irradiated mice. Using of affinity chromatography, hydrophobic interaction chromatography, and gel filtration, a 15,000-fold purification of the suppressive factor was attained. The moiety engaged in suppression was identified as a 20- to 40-kDa protein. The biological activity is destroyed at temperatures above 70 degrees C, by proteolytic enzyme digestion and under alkaline conditions but was resistant to acidic and reducing conditions. Judged by its biological activity and some of its physical properties, SUF-s is most likely distinct from other described suppressor factors or known cytokines with suppressor activity, such as IL-4, IL-10, interferon-gamma, transforming growth factor-beta or alpha-fetoprotein.

Anti-mouse melanoma suppressor T cells clone (F12) secreted a functional factor (TsF) which suppresses the generation of cytotoxic T lymphocytes (CTL). Ts clone F12 secreted TsF into culture supernatant by stimulating for more than 12 hours with anti-CD3 monoclonal Ab. In syngeneic CTL induction system, TsF suppresses the generation of both anti-melanoma and anti-lymphoma CTL. Moreover, TsF suppresses allogeneic CTL not only from C57BL/6 mice but also from C3H mice. Analysis of the biochemical and physiochemical character of TsF shows that TsF is stable for variance of pH and freeze and thawing, but unstable for heat in region of 100 degrees C. and in acetonitorile. It is also suggested that the molecular weight of TsF is in the vicinity of 12,000 daltons on the analysis in high performance liquid chromatography. F12 clone secretes other suppressive cytokine, TGF-beta and INF-gamma. However, TsF seems different from these suppressive cytokine, because anti-TGF-beta serum which blocks the suppressive function of TGF-beta 1 for CTL generation does not block TsF function. Moreover, recombinant INF-gamma does not suppress the generation of CTL.

In the picryl chloride contact sensitivity system in mice, i.v. injections of trinitrobenzene sulfonic acid (TNBSA) prevents elicitation of delayed-type hypersensitivity reactions. This suppression is due in part to a non-specific, PG-dependent factor (TNBSA-F) that is induced by i.v. injection of TNBSA and is produced by pooled spleen and lymph node cells in vitro. Inasmuch as a role for lymphokines such as IL-2 has been postulated in delayed-type hypersensitivity, we determined the in vitro effects of TNBSA-F on the responsiveness of HT-2 target cells to IL-2. TNBSA-F induced a dose-dependent unresponsiveness of HT-2 cells to IL-2. The inhibitory activity was not present in supernatants from lymphoid cells of sham-treated mice. In the presence of indomethacin, spleen, and lymph node cells from TNBSA-immunized mice produced a factor whose activity was much reduced compared to TNBSA-F. This suggested that PG were required for TNBSA-F activity. However, PG alone did not induce the unresponsiveness because TNBSA-F but not sham-treated mice had inhibitory activity despite containing similar levels of PGE2. Rather, the combination of i.v. TNBSA injections and PG synthesis during production of TNBSA-F were required to produce a suppressive TNBSA-F. The inhibitory effect of TNBSA-F was not due to the presence of transforming growth factor-beta, soluble immune-response suppressor, INF-gamma, or JE in the factor preparation. Partial characterization showed a single peak of in vitro TNBSA-F activity (molecular mass approximately 35-55 kDa) by Sephadex G-200 gel filtration chromatography and by HPLC. In addition, TNBSA-F retained its activity after multiple cycles of freeze-thaw and heating for 1 h at 56 degrees C. The inhibitory effects of TNBSA-F on IL-2-induced proliferation suggest that suppression of delayed type hypersensitivity after i.v. administration of TNBSA may, in part, be due to a PG-dependent suppressor factor that inhibits the responsiveness of target cells to IL-2.

Rat seminiferous tubule segments in defined stages of the epithelial cycle were isolated by transillumination-assisted microdissection. The segments were cultured together with ConA-stimulated peripheral blood lymphocytes (PBL) and incorporation of 3H-labelled thymidine was measured. Tubule segments in stages II-VIII of the seminiferous epithelial cycle inhibited PBL proliferation significantly more than stages IX-I. Inhibition was lowest in stages IX-XII and increased progressively to reach a maximum in stages II-VIII. In a more detailed analysis, tubules in stages V and VI inhibited PBL proliferation significantly less than stage II tubules. No significant difference was observed between stages II and VII. The immunosuppressive activity had molecular weights of approximately 25 kDa and approximately 65 kDa in stage II-VIII seminiferous tubules. In stage II-VI seminiferous tubules activity was present also at approximately 10 kDa. The results suggest that the seminiferous tubules produce high-molecular weight immunosuppressive activity in a stage-dependent way. In addition to its contribution to the immunologically privileged milieu of the testis this activity may also be involved in the physiological regulation of DNA synthesis in the seminiferous epithelium.

Maternal bovine EPF activity can be reduced to one single polypeptide enriched and identified from serum of cows in early pregnancy. The relative molecular weight of this active polypeptide was estimated at 67 kDa. This bovine EPF was labelled by 125I and peroxidase. In parallel investigations of non-pregnant animals a 67-kDa polypeptide was additionally identified in the last purification step, but without EPF activity in the rosette inhibition test. This indicated occurrence of an inactive pre-compound (or carrier protein) of the EPF in the non-pregnant state. On pre-incubation of lymphocytes with EPF analogues (inactive polypeptide from nonpregnancy serum) EPF retained its optimal activity, its lymphocyte receptors being unaffected. Monoclonal antibodies produced against HPLC-enriched EPF were reactive to the 67-kDa polypeptide in pregnancy material as well as in nonpregnancy material and were not able to differentiate between 'pregnant' and 'nonpregnant'. A mouse anti-EPF serum produced against highly purified EPF isolated from SDS PAGE showed reactivity only against the 67-kDa polypeptide of pregnancy serum but not against that of non-pregnancy serum. This is the first evidence for a difference in antigenic determinants of the two 67-kDa proteins found in pregnancy and non-pregnancy serum. Furthermore, a second higher molecular weight protein could be identified by this antiserum in pregnancy and non-pregnancy serum.

We report a new suppressor function of CD8+ CD57+ lymphocytes from HIV-seropositive patients recipients, on the cytolytic activity of allospecific CTL, NK and LAK cells. This inhibitory effect is mediated by a non-antigen specific soluble factor distinct from PGE2, TGF beta and TNF alpha and beta. Biochemical characterization indicates that the CD8+ CD57+ inhibitory activity: 1) is heat and trypsin resistant but remains sensitive to pronase E hydrolyse, 2) specifically bind to concanavalin A-sepharose column, 3) is mediated by a 20-30 kdaltons glycoprotein.

The production of soluble forms of low-affinity Fc gamma R by cells expressing recombinant or natural membrane Fc gamma RII, and the structural relationships between these soluble receptors and membrane Fc gamma RII are described. We show that 37-40 kD soluble Fc gamma RII, corresponding to the two N-terminal domains of Fc gamma RII and binding to IgG, are spontaneously produced in vitro by cleavage of membrane Fc gamma RII. Moreover, we describe methods to produce and purify to homogeneity large quantities of endotoxin-free recombinant IgG-binding factor (rIgG-BF) from the culture medium of a cell line transfected with a mutated Fc gamma RII cDNA. These methods include the use of bioreactors for culturing transfected fibroblasts and the purification of rIgG-BF by ion-exchange chromatography and hydrophobic-interaction chromatography. By using such procedures, about 2.4 mg of rIgG-BF were purified from 1 liter of culture medium of transfected fibroblasts. Like natural IgG-BF, the 95-99% pure rIgG-BF suppressed, in a dose-dependent manner, secondary in vitro IgG antibody responses to sheep red blood cells.

Pregnancy serum contains a factor or factors which suppress T lymphocyte proliferation, although the identity of the factor(s) is still unclear. We have demonstrated that the immunosuppressive activity of pregnancy sera can be destroyed by treatment with periodate which oxidises protein-linked oligosaccharides. Similar effects have been noted with uromodulin, a potent immunosuppressive glycoprotein initially isolated from pregnancy urine. We find, however, that uromodulin is present in both pregnancy and non-pregnancy sera, and that removal of uromodulin from pregnancy serum by lectin affinity chromatography is not associated with loss of activity, ruling out this glycoprotein as the immunosuppressive factor. The possible role of protein-linked oligosaccharides of other serum glycoproteins in causing the pregnancy-related immunosuppression is discussed.

Monoclonal nonspecific suppressor factor (MNSF), a product of a murine T cell hybridoma, suppresses the antibody response to lipopolysaccharide. In an attempt to clarify the N-terminal sequence, MNSF was prepared and purified by affinity chromatography with the use of an anti-MNSF monoclonal antibody (MO6), and reverse-phase high-pressure liquid chromatography. On the SDS-PAGE, the purified MNSF showed a single band with a molecular weight of 12,000. The N-terminal amino acid sequence of the protein was determined and showed no strong homology to any of the sequences of known biologically active proteins. However, the sequence revealed significant (60%) amino acid identity to transforming growth factor beta 2 (TGF beta 2).

In order to study circulating substances which could be involved in uremic immunodeficiency, the activity of plasma and plasmatic fractions of different molecular weight MW (A and B) from 12 patients with chronic renal failure (CRF) and 12 normal subjects (N) was assayed in vitro on PHA stimulated normal lymphocyte cultures. Plasma from CRF patients inhibited lymphoproliferation compared to normal plasma activity (mean +/- SD: 9,990 +/- 3,980 cpm vs. 22,163 +/- 3,054 cpm; p < 0.001). Nevertheless, the corresponding plasmatic fractions failed to induce similar effects. Both normal fractions showed inhibitory effects on proliferation while most of the CRF fractions allowed greater cellular proliferation than the former. The dose-response curves showed that all the normal fractions contained inhibitor(s) whose effect decreased with increasing dilution. Most of the B normal fractions also produced stimulatory effects when they were diluted between 1:5 and 1:25. Variable dose-response curves were obtained in the presence of CRF fractions. However, the lack of inhibitory activity in 9 of 12 patients and the stimulatory effects produced by several A-CRF fractions suggest qualitative differences between CRF and normal fractions. Present findings demonstrate inhibitory and stimulatory activities in the normal fractions which might be due to immunomodulator substances. Disorders in this immunomodulator system could be responsible for the immunodeficiency described in CRF patients.