Characterization of suppressive immunoglobulin-binding factor (IBF). III. Biochemical and immunochemical characteristics of IBF produced by activated T cells.

The biochemical characteristics of immunoglobulin binding factor produced by activated cells (ATC) were investigated. For this purpose, supernatants of ATC were purified by affinity chromatography on insolubilized IgG and the eluted material was iodinated (125I), treated with mercaptoethanol; and run on SDS polyacrylamide gels. The radioactivity was found in two peaks corresponding to m.w. of 38,000 d and 18,000 d. This result extends and confirms our previous findings that IBF produced by ATC is identical to IBF produced by L-5178-Y internally labeled thymoma cells. The effect of various pH, temperatures, and proteolytic and glycolytic enzymes on the binding properties of 3H-leucine-or H-fucose-labeled IBF to IgG and on the polyacrylamide gel profiles was also studied. By all these criteria, IBF appeared to be a glycoprotein in which the presence of the 38,000 to 40,000 d chain is necessary for the binding to IgG. In the attempt to study the relationships between IBF and I-region products, purified IBF produced by ATC was incubated with anti-Ia immunoadsorbent, and the eluted material was iodinated and run on gels. The 38,000 d and 18,000 d chains characteristic of IBF were found to be specifically retained on the relevant immunoadsorbent. These data favor the hypothesis that IBF bears or is associated with Ia determinants.     

Characterization of suppressive immunoglobulin-binding factor (IBF). I. Production of IBF by a theta-positive lymphoma (L-5178-Y).

L-5178-Y, a theta-positive, Fc receptor-bearing mouse thymoma cell line spontaneously releases immunoglobulin-binding factor (IBF) upon short-term incubation in vitro. IBF produced by L-5178-Y cells is identical in its biologic activity with IBF produced by Fc receptor positive alloantigen-activated T cells. It suppresses the in vitro plaque response of mouse spleen cells to sheep erythrocytes by interfering mainly with the late phase of the generation of antibody-forming cells. Therefore, L-5178-Y thymoma affords a homogeneous source of IBF in sufficient quantities for the study of its biochemical nature and the mechanism by which it interferes with cells participating in antibody synthesis.     

Inhibition of the in vitro 19S and 7S antibody response by immunoglobulin-binding factor (IBF) from alloantigen-activated T cells

A soluble factor secreted by alloantigen-activated mouse T cells which binds to the Fc fragment of IgG and inhibits complement activation by IgG (immunoglobulin-binding factor, IBF) suppressed the in vitro 19S and 7S antibody response by mouse spleen cells to T-dependent as well as T-independent antigens. IBF inhibited the 19S plaque response best when it was added late during PFC generation (between 48 and 72 hr). On the other hand, when it was left in cultures for up to 60 hr and then removed, antibody synthesis was not inhibited. However, its presence for only 2 hr starting after 72 hr of incubation was sufficient to inhibit PFC formation. The suppressive activity of IFB could be neutralized by adding aggregated mouse IgG prior to the critical stage around 72 hr. These data favour the view that IBF could be a suppressive T cell factor and point to the possibility that IBF may act on already triggered B cells during their final differentiation to active PFC.     

Human monocyte cytotoxic factor mediates cytolysis of WEHI 164 cells

Human monocytes can be activated to release a 40,000-Da cytostatic protein factor (CF). In this report we have investigated the cytolytic activity of CF on WEHI 164 cells which are sensitive to monocyte-mediated cytolysis. Monocyte supernatants containing CF induced cytolysis of murine WEHI 164 sarcoma cells, as determined in a 51Cr-release assay. Preincubation of WEHI 164 cells with actinomycin D enhanced cytolysis induced by supernatants containing CF, suggesting that CF may be involved in drug-dependent cellular cytotoxicity. The cytolytic activity was profoundly inhibited by a rabbit antiserum raised against purified CF, indicating that the cytolytic activity in the supernatants was in fact mediated by CF. These results indicate that CF may be an important effector molecule in monocyte-mediated cytostatic and cytolytic reactions.     

Degradation of fibronectin by human leukocyte elastase. Release of biologically active fragments

We have identified the biological activity of three polypeptides released by limited proteolysis of human plasma fibronectin by leukocyte elastase. A Mr = 140,000 peptide contains cell-spreading activity; a Mr = 60,000 peptide mediates binding to denatured collagen (gelatin), and a Mr = 29,000 peptide contains glutaminyl residues responsible for the transglutaminase (blood coagulation factor XIIIa)-catalyzed incorporation of amines. More extensive proteolysis yielded numerous peptides, including a Mr = 40,000 peptide derived from the Mr = 60,000 peptide which retains gelatin-binding activity. Quantification of the gelatin-binding peptides is consistent with two binding sites per dimeric fibronectin molecule of Mr = 440,000. Both Mr = 60,000 and 40,000 gelatin-binding peptides were enriched with half-cystine residues, containing 28 and 25, respectively, but devoid of cysteine. This, coupled with the electrophoretic behavior of both peptides, was consistent with the presence of intramolecular disulfide bonds in the gelatin-binding domain. Intact fibronectin contains 1 free cysteine residue/monomer, as recently described. This cysteine reacts with 5,5'-dithiobis(2-nitrobenzoic acid) very slowly under nondenaturing conditions but rapidly when fibronectin is denatured. The free cysteine is located in the Mr = 140,000 peptide. While the Mr = 40,000 and 60,000 gelatin-binding peptides bind to gelatin with an affinity about 30-fold and 5-fold less than intact fibronectin (based on a monomeric fibronectin Mr = 220,000), neither gelatin-binding peptide supports spreading of fibronectin-deficient test cells on gelatin or tissue culture plastic substrates. The purified Mr = 140,000 peptide supported cell spreading on plastic, retaining about one-half of the spreading activity of intact fibronectin on a weight basis. These data confirm recent results, suggesting multiple, protease- resistant domains with discrete biological functions within fibronectin. Our results, together with established data, suggest a model for the location of the transglutaminase-reactive glutaminyl residues, gelatin binding, and cell-adhesive domains in fibronectin. The release of univalent, biologically active fibronectin fragments by elastase, a major physiologically released inflammatory protease of human leukocytes, suggests a new potential mechanism for alteration of cell connective tissue interactions at sites of inflammation in vivo.     

Biosynthesis of Rauscher leukemia viral proteins

Antisera to disrupted Rauscher leukemia virus (RLV) or to the purified Rauscher viral 30,000 dalton polypeptide were used to specifically precipitate newly synthesized intracellular viral polypeptides from extracts of infected NIH Swiss mouse cells (JLS-V16). Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of extracts from cells pulse-labeled for 10–20 min with ³⁵S-methionine showed that immune precipitates contained none of the nonglycosylated internal structural polypeptides of mature viruses. The major viral-specific polypeptides labeled in 10 min included polypeptides of 180,000, 140,000, 110,000, 80,000, and 60,000 daltons with minor polypeptides of 65,000, 50,000, and 40,000 daltons. Labeling the intracellular virus-specific polypeptides with ¹⁴C-glucosamine indicated that the 180,000, 110,000, 80,000, and 60,000 dalton polypeptides were glycosylated, and all but the 110,000 dalton polypeptides are contained in the mature virions. Based on pulse-chase experiments, it appears that at least 3 of the large polypeptides (140,000, 65,000, and 50,000 daltons) are precursors to the three major internal structural polypeptides of the mature virions.     

Regulatory factors produced by lymphocytes. II. Inhibition of cellular DNA synthesis associated with a factor inhibiting DNA polymerase alpha activity.

Supernatants of phytohemagglutinin-stimulated human tonsil cells contain two growth inhibitory factors. These factors, called inhibitors of DNA synthesis (IDS), reduce (3)H-thymidine incorporation into mitogen-stimulated lymphocytes and into growing HeLa cells. By Sephadex chromatography, these factors have volumes of distribution corresponding to about 80,000 and 40,000 daltons. Both factors inhibit the activity of calf thymus DNA polymerase alpha in cell-free assays (termed inhibitor of DNA polymerase, IDP). The larger factor, which is chromatographically separable from alpha-lymphotoxin (alpha-LT), is completely inactivated by heating at 70 degrees C for 15 min. This treatment does not destroy alpha-LT. Using supernatants from PHA-stimulated tonsil cells cultured for 5 days in serum-free medium, we attained a 150-fold purification with a succession of molecular sieving, ion exchange, and adsorption chromatographic procedures. Although not purified to homogeneity, the extensive copurification of IDS and IDP activities and their identical heat inactivation profiles suggest that they are the same entity. IDP separated free of alpha-LT inhibits thymidine incorporation into HeLa cells without causing cell death. alpha-LT purified free of IDS does not inhibit thymidine incorporation into HeLa cells, not even at concentrations 7000 times that necessary to kill 50% of growth-inhibited L cell cultures.     

Characterization and partial purification of a non-interferon macrophage activating factor produced by human leukemic T cell line

Culture supernatants from several human leukemic T cell lines were found to contain a macrophage activating factor which enhanced hydrogen peroxide release from human peripheral blood monocyte-derived macrophages. The macrophage activating factor from a T cell line, CCRF-CEM, was characterized biochemically and compared with interferon-gamma, which is also an immunological product of T cells and has a potent macrophage activating activity. In contrast to interferon-gamma, the macrophage activating factor in the culture supernatants bound to an anion exchanger and did not adsorb onto concanavalin A gel. Culture supernatants and active fractions from chromatographies were essentially devoid of anti-viral activity. Anti-human interferon-gamma monoclonal antibody also failed to neutralize the macrophage activating factor from CCRF-CEM. MAF was eluted in the fractions with molecular weight of 40,000 to 60,000 on gel filtration in the presence of a detergent and a salt. MAF was partially purified to about 1,300-fold by the methods described above: chromatography with anion exchangers and gel filtration. It was concluded that MAF from CCRF-CEM was biochemically and immunologically different from interferon-gamma.     

Rapid phosphorylation of MAP-2-related cytoplasmic and nuclear Mr 300,000 protein by serine kinases after growth stimulation in quiescent cells

Antibody against brain microtubule-associated protein 2 (MAP-2) immunoprecipitated Mr 300,000 and 80,000 proteins of cultured fibroblasts and kidney cells. These proteins were not appreciably phosphorylated in quiescent cells, but were rapidly phosphorylated after growth stimulation by insulin, epidermal and fibroblast growth factors, transferrin, phorbol ester and diacylglycerol in the presence of Ca2+, in a manner similar to that of MAP-1-related Mr 350,000 protein (J. Cell Biol. 100, 748-753). A Ca2+ ionophore, which is known to make the quiescent cell competent but not to enter into the growth cycle, did not induce the phosphorylation. In a chase experiment, decay half lives of labeled phosphoproteins were 5 h for Mr 350,000 and 300,000 proteins, and 1.5 h for Mr 80,000 protein. On subcellular fractionation, phosphorylated Mr 350,000 and 300,000 proteins were detected first mainly in the cytoplasm and then in the nucleus, while Mr 80,000 phosphoprotein was consistently detected in the cytoplasm. The phosphorylation of these proteins occurred on serine residues after stimulation with various factors. Thus, the phosphorylation of cytoskeleton-associated Mr 350,000 and 300,000 proteins by serine kinases seems to be a common second process after growth stimulation and to link cytoplasmic and intranuclear events.     

Two forms of membrane-bound sphingosine kinase in Tetrahymena and activity changes during growth and the cell cycle

Sphingosine kinase is responsible for the formation of sphingosine-1-phosphate, a sphingolipid mediator with important roles in numerous physiological processes. The sphingosine kinase activity of Tetrahymena pyriformis was recovered predominantly in the particulate fraction and it could be solubilised in 1% beta-octylglucoside. Anion-exchange chromatography resolved the beta-octylglucoside-solubilised sphingosine kinase activity into two peaks corresponding to proteins of Mr 140,000 and 80,000 respectively, as determined by subsequent size exclusion chromatography on Superdex 200. N,N-dimethylsphingosine did not inhibit the sphingosine kinase activity in either fraction, whereas D,L-threo-dihydrosphingosine inhibited sphingosine phosphorylation by the Mr 80,000 kinase but had no effect on the Mr 140,000 kinase activity. The activities also showed different stimulatory responses to Triton X-100 or NaCl. Overall, the results suggest the existence in Tetrahymena of two distinct membrane-associated sphingosine kinases. The kinase activity determined at the different culture stages showed a transient elevation at the mid-logarithmic phase. Further, the sphingosine kinase activity was examined during the synchronous cell division induced by cyclic heat treatments in T. pyriformis. We report for the first time that the sphingosine kinase activity greatly increased at 30 to 45 min after the end of heat treatment prior to the synchronous cell division (75 min), suggesting that the activity changes were associated with the cell cycle and that the up-regulated sphingosine kinase activity would be required for the initiation of the oncoming synchronous cell division in Tetrahymena cells.     

Association of adenovirus early-region 1A proteins with cellular polypeptides.

Extracts from adenovirus-transformed human 293 cells were immunoprecipitated with monoclonal antibodies specific for the early-region 1A (E1A) proteins. In addition to the E1A polypeptides, these antibodies precipitated a series of proteins with relative molecular weights of 28,000, 40,000, 50,000, 60,000, 80,000, 90,000, 110,000, 130,000, and 300,000. The two most abundant of these polypeptides are the 110,000-molecular-weight protein (110K protein) and 300K protein. Three experimental approaches have suggested that the 110K and 300K polypeptides are precipitated because they form stable complexes with the E1A proteins. The 110K and 300K polypeptides do not share epitopes with the E1A proteins, they copurify with a subset of the E1A proteins, and they bind to the E1A proteins following mixing in vitro. The 110K and 300K polypeptides are not adenoviral proteins, but are encoded by cellular DNA. Both the 12S and the 13S E1A proteins bind to the 110K and 300K species, and these complexes are found in adenovirus-transformed and -infected cells.     

Thymocyte differentiation activity from the cloned monocyte/macrophage cell line RAW 264.7. Alterations in the expression of immature thymocyte surface antigens

The cloned monocyte/macrophage cell line RAW 264.7 was previously shown to produce thymocyte mitogenic and co-mitogenic activity that eluted from a Sephadex G-75 column not only at approximately 16,000 daltons, the m.w. described for interleukin 1 (IL 1), but also at 30,000 to 40,000 daltons. The studies reported here indicate that the 30,000 to 40,000 dalton molecule has thymic differentiating activity. Thymocytes from A/J mice were fractionated on discontinuous BSA gradients, which yielded populations of cells enriched for immature and mature cells. The cells found at the interface between 35 and 29% BSA (band 1 cells), which are the most immature, were cultured for 48 hr with highly purified IL 1, with the 30,000 to 40,000 dalton form of thymocyte co-mitogenic activity obtained after Sephadex G-75 chromatography and chromatofocusing chromatography, or with media alone. The surface antigens TL-3, H-2Kk, Thy-1.2, Lyt-1, and Lyt-2 were examined by immunofluorescence. It was found that the highly purified 30,000 to 40,000 dalton species of co-mitogenic activity induced a significant increase in the content of surface H-2Kk, a decrease in TL-3, and a very small decrease in Thy-1.2 on the cell surface, whereas IL 1 was not capable of inducing a change in these surface antigens. There was no change in Lyt-1 on the surface of band 1 thymocytes after incubation with either IL 1 or the 30,000 to 40,000 dalton species. The 30,000 to 40,000 dalton species caused a significant decrease in the percentage of cells staining positive for Lyt-2, whereas IL 1 caused a smaller but significant decrease in Lyt-2. These changes in the surface markers TL-3, H-2Kk, and Thy-1.2 are consistent with changes that occur during thymocyte differentiation. It was also observed that the proliferative response to the 30,000 to 40,000 dalton form and IL 1 increased with increasing functional maturity of each band of thymocytes when used in the thymocyte mitogenic assay. However, only the 30,000 to 40,000 dalton form was capable of inducing a proliferative response in the immature band 1 thymocytes in the thymocyte co-mitogenic assay. These results indicate that the RAW 264.7 cells produce a factor that has, in addition to thymocyte co-mitogenic activity, thymocyte differentiation activity, and this factor is distinct from IL 1.     

A novel gene IBF1 is required for the inhibition of brown pigment deposition in rice hull furrows

The role of flavonoids as the major red, blue, purple and brown pigments in plants has gained these secondary products a great deal of attention over the years. In this study, we characterized a rice inhibitor for brown furrows1 (ibf1) mutant. In the ibf1 mutant, brown pigments specifically accumulate in hull furrows during seed maturation and reach a maximum level in dry seeds. Higher amounts of total flavonoids and anthocyanin in hull may be responsible for the brown pigmentation of ibf1. The IBF1 gene, which encodes a similar kelch repeat-containing F-box protein, was isolated by map-based cloning approach. Real-time RT-PCR and GUS activity assays revealed that IBF1 specifically expressed in reproductive tissues. GFP-IBF1 fusion protein mainly localized in cytoplasm. The expression of some major structural enzymatic genes involved in flavonoids biosynthesis could be up- or down-regulated to some different extent in ibf1 mutant. Our data suggested that IBF1 as a suppressor could inhibit the brown pigmentation of rice hull furrows.     

Effect upon mitogenic stimulation of calcium-dependent phosphorylation of cytoskeleton-associated 350,000- and 80,000-mol-wt polypeptides in quiescent 3Y1 cells

Rabbit antiserum raised against highest molecular weight microtubule-associated protein (MAP-1) of brain immunoprecipitated 350,000-, 300,000-, and 80,000-mol-wt phosphoproteins of rat embryo fibroblasts (3Y1-B). The 350,000-mol-wt protein was sensitive to heat as was brain MAP-1, but the 300,000- and 80,000-mol-wt proteins were not. These polypeptides were hardly phosphorylated in cells in the quiescent G0 phase but were rapidly phosphorylated after addition of serum, epidermal growth factor, phorbol ester, insulin, or transferrin in the presence of calcium ions. All these agents also induced incorporation of [3H]-thymidine into DNA. These polypeptides were detected in isolated microtubules and cold-resistant filaments by immunoblotting. Since the 350,000-mol-wt polypeptide was detected in the membrane, the cytoskeletons, and the nucleus, and has been suggested to function as a linker, its rapid phosphorylation might represent an early process in transduction of the signal of mitogenic stimulation to the nucleus.     

T cell replacing factor for steroids (TRF-S): a 40,000 dalton protein produced by a T4+ T cell

The induction of polyclonal immunoglobulin (Ig) synthesis by glucocorticosteroids (GCS) in human peripheral blood lymphocytes is dependent on both T cells and monocytes. T cells can be replaced by a cytokine, T cell replacing factor for steroids (TRF-S), which promotes GCS-induced Ig production. T cells produce the cytokine when cultured with intact monocytes, with 24 hr monocyte supernatants, or with small quantities (0.1 U/ml or more) of highly purified interleukin 1 (IL 1). TRF-S was produced by isolated T4+ cells, whereas isolated T8+ cells were unable to help GCS-induced Ig synthesis. High pressure liquid chromatography with a gel permeation column revealed a single locus of activity that corresponded to an apparent m.w. of 40,000. At the dilutions utilized in culture, supernatants containing optimal TRF-S activity (3 U/ml final concentration in culture) were found to have less than 0.2 U/ml (final concentration) of interleukin 2 (IL 2) activity. Neither recombinant IL 2 nor recombinant interferon-gamma (IFN-gamma) over a broad range of concentrations was able to reproduce the capacity of TRF-S to induce the development of Ig-secreting cells with GCS. Thus, we report that TRF-S is synthesized primarily by T4+ T cells, and that its production is stimulated by small concentrations of IL 1. The apparent m.w. of TRF-S is 40,000, and its biological activity is distinct from that of IL 1, IL 2, and IFN-gamma.     

Functional characteristics of histamine receptor-bearing mononuclear cells. II. Identification and characterization of two histamine-induced human lymphokines that inhibit lymphocyte migration

Although functional histamine receptors have generally been restricted to those human T lymphocytes expressing suppressor cell functions, more recent evidence suggests that histamine receptor-bearing human T lymphocytes are functionally heterogeneous and capable of other immunomodulatory activities. Lymphocyte chemoattractant factor (LCF) is a cationic sialoprotein with an apparent m.w. of 56,000, whose production is limited to histamine-type 2 receptor-bearing human T cells. LCF is selectively chemokinetic for T lymphocytes, and presumably contributes to the recruitment of unsensitized effector lymphocytes at inflammatory sites. In addition to LCF, Sephadex G-100 gel filtration of histamine-induced lymphocyte supernatants revealed two regions of migration inhibitory activity for human blood T and rat splenic lymphocytes. These regions corresponded to m.w. of 70,000 to 80,000 (LyMIF75K) and 30,000 to 40,000 (LyMIF35K). LyMIF75K had a single pI of 7.5 to 8.0, and its biologic activity was sensitive to trypsin but not to neuraminidase or heat (56 degrees C). LyMIF35K had a single pI of 8.5 to 8.8, and its biologic activity was sensitive to neuraminidase and heat but not to trypsin. These LyMIFs therefore appeared to be distinct from one another and physicochemically different from other migration inhibitory lymphokines. All three lymphokine activities appeared within 4 hr of incubation. The minimum concentration of histamine required to stimulate production of the LyMIF was 10(-6) M. Lymphocytes that did not adhere to a histamine affinity matrix were unable to produce either LyMIF upon subsequent stimulation with histamine or concanavalin A (Con A). Lymphocytes incubated with histamine and diphenhydramine produced LCF but neither LyMIF, whereas cells incubated with histamine in the presence of cimetidine produced both LyMIF but not LCF. These data suggest that a subset of lymphocytes defined by the presence of histamine-type 1 receptors are capable of producing two distinct species of lymphocyte migration inhibitory activity. These cells may contribute to the immobilization of effector T lymphocytes chemokinetically attracted to certain inflammatory sites.     

Cross-linking of human T cell receptor proteins: association between the T cell idiotype beta subunit and the T3 glycoprotein heavy subunit

Bifunctional cross-linking reagents DSP, DSS, and BSOCOES were used to cross-link 125I-surface-labeled viable T lymphocytes. The cross-linked cells were solubilized in Nonidet-P40, immunoprecipitated with anti-Ti (monoclonal antibody T40/25) or anti-T3 (monoclonal antibodies UCHT-1 or OKT3), and analyzed by SDS-PAGE. With all three cross-linkers, the intact cross-linked products obtained with monoclonal antibody T40/25 from HPB-ALL cells were 20-30 kd heavier than the Ti dimer (Mr 80,000). When the DSP cross-linked product was isolated using either anti-Ti or anti-T3 monoclonal antibodies and then cleaved, bands having molecular weights identical with both the Ti and T3 subunits were obtained. The two-dimensional SDS-PAGE analysis (nonreducing followed by reducing conditions) of the DSS and BSOCOES cross-linked products revealed the specifically cross-linked bands to have Mr 40,000 and Mr 28,000. These data indicate that the Ti molecule and the T3 molecule are spatially associated on the cell surface and suggest the predominant association is between the Ti beta subunit (Mr 40,000) and the T3 heavy subunit (Mr 28,000).     

Regulatory proteins for the activated third and fourth components of complement (C3b and C4b) in mice. II. Identification and properties of complement receptor type 1 (CR1)

We identified on the membrane of mouse spleen cells a polypeptide of Mr 190,000 (S190), with binding affinity for the mouse third component of the complement system (C3). S190, purified by affinity chromatography on C3-Sepharose, has properties resembling those of the human C3 receptor type 1 (CR1). Thus, S190, like CR1, served as a cofactor for the C3b inactivator (I)-mediated cleavage of fluid-phase C3b into iC3b, and had cofactor activity comparable to that of serum factor H (H). S190 also acted as a cofactor for the cleavages of membrane-bound C3b or membrane-bound iC3b into C3c (Mr 140,000) and C3dg (Mr 40,000) by serum factor I. As is the case with CR1, the specific activity of S190 for the cleavages leading to C3c-C3dg formation was approximately 100-fold greater than that of H. We therefore conclude that S190 and CR1 are analogous proteins.     

Effect of thymocyte-stimulating factor-containing supernatants on the surface antigens of murine thymocytes.

Incubation of murine thymocytes in thymocyte-stimulating factor (TSF)-containing supernatants causes a four- to fivefold increase in the expression of the H-2^k and H-2^d antigens and a similar decrease in the expression of the TL antigen (in TL⁺ strains) on the surface of these cells. Experiments with antisera directed toward the private H-2K and H-2D antigens showed that TSF-containing supernatants cause approximately the same increase in the expression of the H-2K and H-2D antigens of thymocytes of the d and b haplotypes. With thymocytes of the k haplotype, only an increase in the expression of the H-2D antigens takes place, while no significant increase was found for the H-2K antigens. TSF-containing supernatants cause no significant change in the expression of the following antigens on the surface of thymocytes: Thy-1.1, Thy-1.2, Ly-1.2, Ly-2.2, Ly-6.2, Th-B, Ia-1,2,3,7, and G_IX. A factor similar to murine TSF, produced by human peripheral blood leukocytes, does not affect appreciably the expression of the H-2 antigens on the surface of murine thymocytes. The factor(s) causing the increased expression of the H-2 antigens on the surface of murine thymocytes appears to be produced by T lymphocytes. The factor(s) is eluted from a Sephadex G-100 column in at least two broad peaks with molecular weights of 300,000 and 90,000-25,000. Most of the activity enhancing the expression of the H-2 antigens is lost at pH 2, while most of it is maintained at pH 11.5 and at 56 °C. On the basis of these properties, it is concluded that the factor under study is probably different from the factor enhancing the phytohemagglutinin responsiveness of thymocytes.