The ubiquitin-like protein monoclonal nonspecific suppressor factor beta conjugates to endophilin II and regulates phagocytosis.

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 influence of TLSFJM on the proportion of Th1/Th2-like cell subsets].

AIM: To explore the influence of TLSF(JM) on the proportion of alloantigen activated Th1 and Th2-like cell subsets. METHODS: TLSF(JM) or IL-4 was added to mixed lymocyte reaction(MLR) system. The influence of TLSF(JM) on the proportion of Th1 and Th2-like cell subsets was analyzed by intracellular immunofluorescence staining and FACS. RESULTS: In the TLSF(JM) group, the proportion of IFN-gamma(+) cells differentiated from activated lymphoblast descended from 49.8% to 43.1%, IL-4(+) cells from 75.4% to 43.7% and IL-6(+) cells from 67.8% to 52.6%. The similar tendency was also observed in the unactivated small lymphocytes. CONCLUSION: TLSF(JM) can inhibit both the Th1 and Th2-like cell subsets, but mainly inhibit the Th2-like cell subset, thereby reducing the proportion of Th2-like cell subsets.

Partial purification of mare early pregnancy factor.

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.

A protein in pig spleen similar to immune suppressive protein of stress.

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.

Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli.

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E. coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding approximately 42 and 36mg EPF from 300ml bacterial and 1L Sf9 cultures, respectively. The preparations were highly purified (#10878;99% purity on SDS-PAGE for the bacterial products and #10878;97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immunosuppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity.

Antibody-binding proteins in human seminal plasma.

PROBLEM: The significance of antibody-mediated infertility is unclear and complicated by the finding of that antisperm antibodies are found both in fertile and infertile couples. Seminal plasma contains immunosuppressive factors, one such factor may be antibody-binding proteins (ABP's). METHOD OF STUDY: Antibody-binding-proteins were purified using human IgG or IgG-Fc affinity chromatography columns. The purified antibody-binding proteins were characterized by their molecular weights, partial amino acid sequences, and immunoreactivities. RESULTS: Three proteins of molecular weight 74, 70 and 55 kDa and other low molecular weight proteins specifically bound to the IgG or IgG-Fc affinity columns demonstrating Fc-binding specificities. These proteins were not FcgammaRIII, IgG, or fragments of these proteins by their behaviors under reducing conditions, Western blot, and partial amino acid sequence analyses. Amino acid sequence data demonstrated some of these proteins to be novel. CONCLUSIONS: We have isolated and partially characterized several ABP's from seminal plasma. The IgG-binding proteins we have identified may protect spermatozoa against antibody-mediated damage by conferring protection to antibody-coated spermatozoa. If this hypothesis holds true, differences in the level or function of these ABP's may alter the status of fertility.

Isolation, purification and partial characterization of early pregnancy factor (EPF) from sera of pregnant women.

Early pregnancy factor (EPF) is a pregnancy protein, which is secreted into the maternal serum 12-16 hours after fertilization. It is thought to be an immunosuppressive molecule. EPF is detected in pregnant woman's serum by the rosette inhibition assay (RIA). In this study, EPF was purified from the pregnant woman's sera by using ion exchange chromatography and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins which showed a positive result with the RIA, were found to be 35 kDa and 17 kDa molecular weights. The biological activities of these proteins were stable upon heat treatment at 56 degrees C for 30 min. Proteins isolated and purified in this study might be of great significance to the field of human reproduction with particular reference to pregnancy and recurrent abortion.

A placenta-derived suppressor factor with a T-cell bias.

PROBLEM: Functional and mechanistic aspects of immunosuppression by murine placental supernatants (MPS) were investigated. METHOD OF STUDY: MPS and a low molecular weight fraction of the supernatant (MPSf) were tested for suppressive action on T-cell reactivity in vitro and in vivo, on B-cell responses and on T-cell activation events. RESULTS: MPS and MPSf suppress mitogen-induced proliferation and mixed lymphocyte reactions of human and murine lymphocytes, antigen-induced proliferation of T cells in vitro and in vivo, proliferation of CD8+ lymphocytes, proliferation induced by cross-linking of surface CD3 and the in vivo response of mice to allogeneic stimuli. MPSf affects cell cycling of activated T cells and blocks interleukin (IL)-2 production. MPSf does not affect antibody production or the induction of MHC class II expression on B cells. CONCLUSIONS: MPSf is a potent inhibitor of T-cell responses in vitro and in vivo, with no demonstrable effect on B-cell function.

Purification and IgE-binding epitopes of a major allergen in the gastropod Turbo cornutus.

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).

Herpesvirus saimiri transformation of HIV type 1 suppressive CD8+ lymphocytes from an HIV type 1-infected asymptomatic individual.

CD8+ T lymphocytes from HIV+ individuals can potently suppress HIV-1 replication in a noncytolytic manner. This suppression appears to be multifactorial and the molecules contributing have not been fully elucidated. As an approach to this question we used herpesvirus saimiri (HVS) to transform CD8+ T lymphocytes from an HIV+ asymptomatic donor to a continuously growing, activation-independent, IL-2-dependent phenotype. The transformed cell population, termed CD8(HVS), had an activated phenotype, contained HVS sequences, did not shed infectious HVS virus, and was polyclonal. The CD8(HVS) cells, despite the absence of detectable CTL activity, potently suppressed HIV-1 production by both autologous and heterologous CD4+ cells from infected donors. The CD8(HVS) cells in coculture also suppressed virus production from PBMCs acutely infected with syncytium-inducing (SI) strains or NSI primary isolates of HIV-1. The supernatants from the CD8(HVS) cells and their concentrates derived from these supernatants were suppressive to NSI primary isolates of HIV-1 but not to SI strains. Fractionation of these concentrates showed that the suppressive activity was associated with low molecular mass (6500- to 19,300-Da) protein species. Western blotting and ELISA indicated that the CC chemokines MIP-1alpha, MIP-1beta, and RANTES were present in these fractions. Antibody-blocking studies with antibodies to the CC chemokines indicated that a significant portion of the soluble HIV-suppressive activity was due to these molecules. However, these experiments also suggested the inhibitory activity of the CD8(HVS) cells in coculture is not due exclusively to the CC chemokines. The HVS-transformed cells provide a useful tool for the study of noncytolytic CD8+ T lymphocyte-mediated suppression of HIV-1.

Isolation and purification of an early pregnancy factor-like molecule from culture supernatants obtained from lymphocytes of pregnant women.

PURPOSE: Our purpose was to determine whether lymphocytes synthesize proteins during pregnancy, to observe whether one of the proteins synthesized has early pregnancy factor (EPF)-like activity and to isolate and purify this molecule from culture supernatants obtained from stimulated lymphocytes of pregnant women. METHODS: Lymphocyte proliferation assay and 35S-methionine labeling were done to study de novo synthesis of proteins followed by autoradiography to confirm synthesis of proteins. The rosette inhibition assay was used for detection of the EPF-like molecule. Gel filtration on Sephadex G-100 and RPHPLC were used for purification of the EPF-like molecule. RESULTS: The rate of incorporation of 35S-methionine was significantly higher in the lymphocytes of pregnant women compared to those of the control, and autoradiography confirmed the synthesis of proteins during pregnancy. There is a total protein enhancement trend observed during the first trimester that declines toward term. The EPF-like molecule is observed to be synthesized during all the trimesters of pregnancy. This molecule, when purified, showed a single homogeneous biologically active peak. CONCLUSIONS: The results indicated that there is an enhancement of existing protein or synthesis of new proteins during pregnancy. The EPF-like molecule is one of the many proteins synthesized and secreted by lymphocytes during pregnancy that, when purified, is biologically active.

Preliminary characterization of an immunosuppressive inducer factor secreted by the JEG-3 choriocarcinoma cell line: in vitro and in vivo studies.

PROBLEM: The direct immunosuppressive and suppressive inducing capacities of supernatants from human trophoblastic choriocarcinoma cell lines (HCS) are well investigated in several former studies. The responsible factor is not yet determined. METHOD OF STUDY: We first confirmed those data and we purified a 3-5-kDa suppressor-inducer factor from HCS by using high performance liquid chromatography (HPLC) on both DEAE and gel filtration columns, followed by ultrafiltration. We then tested the activities of such isolated fractions on in vitro immune responses from human cells and in vivo by its effects in a murine local graft-versus-host (GVH) assay (popliteal lymph node assay, PLN). RESULTS: A single fraction induces both "direct suppression" in vitro as well as in vitro suppressor cell activation/development in human peripheral blood lymphocyte cultures as assessed by suppression of cells cultured in such a fraction containing culture medium of the mixed lymphocyte reaction. Furthermore the very same fraction suppresses in vivo murine allogeneic immune responses as assessed by a local GVH reaction (PLN assay). CONCLUSIONS: We have isolated a suppressive fraction, whose activities suggest that it might be of interest not only in reproductive immunology, but also in transplantation systems.

Influence of a DLE-extracted lymphocytic suppressor factor on CsA-induced immunosuppression.

From dialyzable leucocyte extracts (DLE) we have purified a hydrophilic low-mol. wt. factor (about 1 kDa) which we have named lymphocytic suppressor factor (LSF) as it is able to suppress antigen- and mitogen-induced lymphocyte transformation and to prolong allograft survival in C57b/6N mice (H-2b) transplanted with fully mismatched skin from C3H/HeN mice (H-2k). At the molecular level LSF acts by inhibiting DNA replicational and transcriptional processes in activated lymphocytes, isolated rat hepatocyte nuclei, and cell-free systems. Amino acid analysis indicates that LSF is a peptide composed of Asp, Glu, Ser, Thr, Ala, Gly, Arg and probably Met, with the N-terminus blocked, possibly by pyroglutamic acid. When combined "in vitro" with cyclosporine A (CsA), LSF increased about 20 times the potency of CsA in inducing suppression of mitogen-stimulated lymphocytes. In C57b/6N mice with skin graft from C3H/HeN mice and undergoing immunosuppression with CsA (50 mg/kg/day), the splenocyte LSF content increased about 5 times. However, LSF values returned to normal in mice recovering normal responsiveness due to progressive withdrawal of CsA. These data show that LSF has an important role in the development and maintenance of CsA-induced immunosuppression. We suggest that, by influencing DNA replicational and transcriptional processes of lymphocytes, LSF may play a role also in the onset and progression of AIDS induced by retroviruses.

Human nonspecific suppressor factor (hNSF): cell source and effects on T and B lymphocytes.

The human nonspecific suppressor factor (hNSF), the probable counterpart of the murine monoclonal nonspecific suppressor factor (MNSF), has been isolated from the ascitic fluid of a patient with systemic lupus erythematosus and characterized. hNSF presents an inhibitory activity on the proliferation and IgG production of mitogen stimulated human PBMC. In the present study, we demonstrate that hNSF can be isolated from the supernatants of ConA-activated T cells, but not from CD8-depleted T cells, indicating the CD8+ T cells are the major source of the factor. We also studied the effects of hNSF on purified human B and T cells; hNSF strongly inhibited the proliferation and Ig secretion by highly purified B cells induced by SAC plus IL-2, as well as the proliferation of T cells activated by Con A plus IL-2. These results indicate that hNSF is a CD8+ T cell product with strong antigen-nonspecific immunoregulatory action on both lymphocyte populations.

Stabilization and characterization of antigen-specific T suppressor inducer and T suppressor effector molecules.

H-2 specific T suppressor inducer (Tsfi) and T suppressor effector (Tsfe) factors show a dose-dependent inhibition of one-way mixed lymphocyte responses (MLR) between CBA/J responder spleen cells and C57BL/6 mitomycin C-treated stimulator spleen cells. The hydrophobic proteins Tsfi and Tsfe purified by ammonium sulfate precipitation and affinity methods were stabilized by the addition of Tris-saline pH 8 buffered octylglucopyranoside solution. The stabilized Tsfi and Tsfe fractions stored at 4 degrees C for 3-7 months retained a significant (> 72%) amount of their ability to inhibit MLR. Tsfi and Tsfe purified by salt precipitation and affinity methods were analyzed by SDS-PAGE. Enzyme-linked immunoassay (ELISA) and Western blots indicated that these molecules had T cell receptor (TcR) alpha chain, I-J, and IL-10 epitopes, but not TcR beta chain epitopes.

Purification and characterization of a pregnancy-associated protein: TJ6s.

PROBLEM: Characterization of the soluble form of a novel protein, TJ6 (TJ6s) with immune suppressive activity from murine fetoplacental units. METHOD: Preferential ammonium sulfate precipitation, gel filtration and ion-exchange chromatography were employed to purify the protein TJ6s from murine fetoplacental units using an anti-peptide antibody as a detection tool. Biological activity of the purified protein was studied in lymphocyte proliferation assays. RESULTS: Purified TJ6s has a M(r) of approximately 18 kDa as evidenced by SDS-PAGE in both reducing and non reducing conditions. It exerted a strong anti-proliferative activity in both anti-CD3 and Con A proliferation lymphocyte proliferation assays but not in a PHA assay, suggesting that the anti-proliferative effects on T cells are exerted only on cells specifically activated directly through T cell receptor complex. CONCLUSION: The results indicate that TJ6s is a novel anti-proliferative protein that has many of the characteristics that are considered necessary for survival of the fetal allograft.

Antigen-specific suppressor factor: missing pieces in the puzzle.

Few areas of immunologic research have endured such strident criticism or engendered such fainthearted support as the study of antigen-specific suppression of the immune response. Although enjoying a modest resurgence as a means of promoting or maintaining peripheral tolerance to autoantigens, the study of antigen-specific suppression is not mainstream immunology. The field of immune regulation has, in fact, shifted focus toward explaining the data in terms of the Th1/Th2 paradigm. Indeed, the term suppression has been coopted, by those willing to use it, to describe the bioactivity of conventional cytokines, such as IL-4, IL-10 or TGF beta, which can be inhibitory in certain experimental models. In a very real sense, those who performed much of the early work in the field bear responsibility for the outcast status of suppression. With the increasing number of soluble mediators and cascades of interacting T cells, which populated reviews of the subject in the 1980s, the concept of antigen-specific suppression and suppressor factors simply became too complicated and was dismissed as artifact. Several laboratories have in the past few years made significant advances in the molecular characterization of antigen-specific TsF. Their work, as well as that of our own laboratory have established certain minimal molecular requirements for the expression of TsF bioactivity.

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.

Isolation and characterization of a human nonspecific suppressor factor from ascitic fluid of systemic lupus erythematosus. Evidence for a human counterpart of the monoclonal nonspecific suppressor factor and relationship to the T cell receptor alpha-chain.

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.

Identification of the predominant proteins in uterine fluids of unilaterally pregnant ewes that inhibit lymphocyte proliferation.

Uterine fluid from unilaterally pregnant ewes contains activity inhibitory to lymphocyte proliferation. The molecules responsible for this activity may thereby regulate uterine immune responses during pregnancy. The purpose of the experiment described here was to identify the major protein in uterine fluid responsible for this activity. When uterine fluid was fractionated by a combination of cation exchange chromatography, gel filtration, and lectin affinity chromatography, the majority of the lymphocyte activity co-migrated with a pair of proteins previously identified as related, serpin-like glycoproteins. Together, this pair of proteins, called the uterine milk proteins (UTM-proteins), are the predominant endometrial secretory protein produced under the influence of progesterone. Preparations of uterine protein highly enriched for the UTM-proteins inhibited lymphocyte proliferation induced by phytohemagglutinin, concanavalin A, and mixed lymphocyte reactions but did not inhibit proliferation induced by pokeweed mitogen. In some experiments, UTM-proteins also reduced viability of cultured lymphocytes. Another previously described lymphocyte-inhibitory factor, megasuppressin, was also observed. Megasuppressin, which eluted at an apparent molecular weight of greater than 4 x 10(6) even after treatment with urea, guanidine-HCl, and beta-mercaptoethanol, was a more potent inhibitor of lymphocyte proliferation than UTM-proteins. Megasuppressin is not very abundant, however, and probably is responsible for only a small fraction of the lymphocyte inhibitory activity in uterine fluid. The majority of lymphocyte-inhibitory activity is caused by the UTM-proteins or by a molecule that co-purifies in trace amounts with UTM-proteins.