Immunochemical identification and study of interspecies thymus antigen-2

A new interspecific animal thymus antigen (AgT-2) was identified. It was shown that AgT-2 is a microglobulin with a molecular mass about 12 kDa, electrophoretic mobility of alpha 1-globulins and isoelectric point of 4.6. Heating of the protein to 80 degrees C for 30 min did not lead to the loss of its immunochemical activity. AgT-2 was identified in extracts of bovine fetal thymus, spleen and liver. It was discovered in the extracts of the lung and colon of adult animals. Cross-reactions were found between bovine, walrus, deer and chicken AgT-2. This fact confirms that AgT-2 is interspecies-specific. AgT-2 was not identified in men, dogs, mice, rats and rabbits. Immunochemical research of immunocorrecting preparations revealed that AgT-2 is a constant component of T-activin, thymalin, thymosin and TP1-Serono and its concentration varies from 1.6 to 3.2%. It has been stated that immunochemical test for AgT-2 can be used as a marker in the production and standardization of active thymus factors.     

Histological distribution of the 35-KD protein substrate of the epidermal growth factor receptor/kinase in thymus

Rat thymus has been identified as a tissue comparatively enriched in a 35-KD substrate of the epidermal growth factor receptor/kinase (lipocortin-1) (J Biol Chem 261:13784, 1986). A polyclonal antiserum prepared against the 35-KD protein was used to determine histological distribution of the protein in thymus. Frozen sections of rat thymus were examined after indirect labeling of the 35-KD protein with a rhodamine conjugate of secondary antibody. The antigen was localized primarily in the reticular network of the thymic epithelium, with no detectable labeling of resident thymocytes. Immunoblotting (Western blots) of cytosol extracts also demonstrated that thymocytes did not contain detectable amounts of the antigen. Cultured thymic epithelial cells (TEC), however, contained an abundance of two immunologically related protein bands with molecular weights similar but not identical to the antigen from the parental cell line (human A-431 carcinoma). Paraffin sections of rat and human thymus were subjected to an immunoperoxidase staining procedure, and it was observed that Hassall's corpuscles (keratinized epithelial cells) and other cortical and medullary TECs were intensely stained. The demonstration that the antigen is primarily associated with TEC in thymus, in conjunction with its distribution in other tissues, will aid in deducing its physiological role.     

Monoclonal antibodies as probes for differentiation and tumor-associated antigens: a Forssman specificity on teratocarcinoma stem cells

A set of monoclonal antibodies derived by fusing P3-NS1/1-Ag4-1 myeloma cells with spleen cells from a rat immunized with mouse spleen were screened for activity against a tumor cell panel. One of these antibodies was found to react only with mouse embryonal carcinoma cells and no other tumor cell type tested, including differentiated derivatives of teratocarcinomas. In the adult mouse, this antigen is expressed by subpopulations of cells in the spleen, bone marrow, lymph node, brain, kidney and testes, although not in liver and thymus. This antigen has a species and tissue distribution consistent with that of Forssman antigen. The molecules which carry this specificity on the embryonal carcinoma cells appear to be glycolipids.     

Thymic hormones and prostaglandins. I. Lack of stimulation of prostaglandin production by thymic hormones

Prostaglandins (PGs) have been implicated as possible mediators of the biological activity of thymic hormones. It has been shown that type E-PGs are able to mimic the action of several thymic hormones and that indomethacin prevents in vivo or in vitro the appearance of Thy-1+ antigen induced by some of these factors. We thus investigated a possible role for PGs in the mechanism of action of different thymic extracts and peptides. Attempts to modulate prostaglandin production showed that neither thymosin fraction 5 (0.01-100 micrograms/ml), nor thymosin alpha 1 (1-10 micrograms/ml), thymulin (0.001-100 ng/ml), thymopoietin II (10-1000 ng/ml) or TP5 (10-1000 ng/ml) affect PGE2, 6-keto-PGF1 alpha, PGF2 alpha and TXB2 production by spleen cells from control and thymectomized mice. These results do not support the hypothesis that prostaglandins could act as mediators of thymic hormones.     

Human T cell antigen expression during the early stages of fetal thymic maturation

Human thymus tissue was examined from 7 wk of gestation through birth for the expression of antigens reacting with a panel of anti-T cell monoclonal antibodies. Additionally, the reactivities of reagents against the transferrin receptor, against leukocytes, against low m. w. keratins, and against major histocompatibility complex antigens were studied on human fetal thymic tissue. Frozen tissue sections were evaluated by using indirect immunofluorescence assays. At 7 wk of gestation, no lymphoid cells were identified within the epithelial thymic rudiment; however, lymphoid cells reacting with both antibody 3A1, a pan T cell marker, and antibody T200, a pan leukocyte reagent, were identified in perithymic mesenchyme. After lymphoid colonization of the thymic rudiment at 10 wk of fetal gestation, fetal thymic tissue reacted with antibodies T1, T4, and T8. At 12 wk of gestation, antibodies T3, T6, A1G3 (anti-p80, a marker of mature thymocytes), and 35.1 (anti-E rosette receptor) all reacted with thymic tissue. Our findings indicate that T cell antigens were acquired sequentially on thymocytes at discrete stages during the first trimester of human fetal development. The 3A1 antigen was present on fetal lymphocytes before lymphoid cell colonization of thymic epithelium, suggesting that passage through the thymus was not required for the expression of the 3A1 antigen by T cell precursors. The appearance of mature T cell antigens, T3 and p80, on thymocytes by 12 wk of gestation implies that the T cell antigen repertoire may be established in the thymus during the first trimester. Thus, a critical period of T cell maturation appears to occur between 7 and 12 wk of human fetal gestation.     

Effect of various thymic and nonthymic factors on in vitro antibody formation by spleen cells from nude mice

Thymosin and thymic humoral factor (THF), both prepared from whole thymus tissue, increased the in vitro anti-SRBC response of spleen cells from nu/nu mice. Lymphoid and nonlymphoid control preparations did not influence the response. Standard isolation procedures for thymosin and THF were employed to prepare factors from thymus lymphocytes (TL) separated from calf thymus and from thymus tissue enriched in thymus epithelium (TE) by preirradiation of the calves. The activity of the TE preparations was greater than that of the total thymus (TT) preparations. The TL preparations were marginally effective. However, the activity of the TE preparations could not be attributed exclusively to epithelium-derived factors, since spleen and lymph node extracts from the irradiated calves contained stimulatory material of an unknown origin which may also be present in the TE preparations.     

Studies on the regulation of lymphocyte production in the murine thymus and some effects of a crude thymus extract.

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitiotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0-8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering G0/G1 cells into DNA synthesis, the inhibition of G2 efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.     

Properties and cellular distribution of cyclic AMP-dependent protein kinase from thymus

A protein kinase that catalyzes the phosphorylation of histone was partially purified from rat thymus, and the rate of histone phosphorylation was stimulated three- to fourfold by 1 × 10^?6 M adenosine 3′,5′-monophosphate (cyclic AMP). Thymic protein kinase was more active than the enzyme from spleen. Histone fractions f1, f2a, f2b, and f3 were all capable of serving as phosphate acceptors for the thymic protein kinase, and the rate of phosphorylation of each fraction was stimulated by cyclic AMP. The ability of various 3′,5′-mononucleotides to stimulate protein kinase activity was compared. Inosine 3′,5′-monophosphate (cyclic IMP) was the most effective substitute for cyclic AMP. The cellular distribution of cyclic AMP-dependent protein kinase and adenylate cyclase activities in the thymus was determined. Cyclic AMP-dependent protein kinase activity is present in both small thymocytes and residual thymic tissue. The specific activity of protein kinase from residual tissue, both for basal and cyclic AMP-stimulated enzyme, was greater than that of enzyme from small thymocytes. In contrast to this, adenylate cyclase activity is predominately localized in the thymocytes.     

Lymphocyte plasma membranes. VI. Plasma membrane glycoproteins of thymic and splenic lymphocytes from inbred rats.

Plasma membranes of splenic and thymic lymphocytes from ACI rats were analyzed for their protein and glycoprotein components by surface radioiodination with 125I and SDS-polyacrylamide gel electrophoresis. The glycoproteins were extracted with lithium diiodosalicylate, characterized and assayed with antisera to thymic antigen. Plasma membranes of both cell types showed more than 25 proteins of which 10--15 were glycoproteins. Both cells showed five major glycoproteins but their apparent molecular weights or intensities differed. Surface radioiodination showed a 120 000 daltons component, common to both cell types, and a 27 000 daltons thymus-specific component as the most exposed surface glycoproteins. Lithium diiodosalicylate extracts of the plasma membranes contained almost all of the glycoprotein components and comprised 5-6 percent of the total membrane protein and 40-50 percent of the total membrane carbohydrate, with sialic acid content in thymus twice that of the spleen cells. About 1 percent of the total plasma membrane protein and 7 percent of the total isolated glycoproteins from thymocytes were reactive with rabbit anti-rat thymocyte antiserum and the immune precipitates showed two components with apparent molecular weights of 72 000 and 27 000.     

Evidence for the presence in calf thymus of a peptidic factor controlling DNA transcription in vitro.

A thymic factor causes a strong inhibition of the DNA-directed RNA polymerase reaction in vitro. The active factor was isolated from aqueous ultrafiltered thymus extracts and purified by means of chromatography on DEAE-cellulose and then on Dowex 50 WX2. The purified thymic factor was characterized as a peptide of low molecular weight (less than 5000). The biological activity of the thymic factor cannot be attributed to the presence of a nuclease or of a histone fragment. The RNA synthesis is controlled by this factor by means of electrostatic interactions between the peptide compound and DNA. Inhibitory activity on RNA synthesis was absent from kidney extracts.     

Characterization of a monoclonal antibody (SBU-1) made to the thymic rudiment of sheep

A monoclonal antibody (SBU-1) was raised to sheep thymic rudiment by fusion of NSI myeloma cells with spleen cells from BALB/c mice immunized with thymic rudiment isolated from fetal sheep between 25-30 days of gestation. By employing the indirect immunoperoxidase technique the antigen recognized by SBU-1 was found to be present in the epithelial reticular cells of the fetal sheep thymus. The intensity of staining decreased as gestation progressed. In the adult thymus the antigen was mainly restricted to Hassall's corpuscles and occasional epithelial cells in the medulla. In addition, the antigen was also shown to be present in epithelial cells of the small intestine, the bronchiole, the keratinized epithelium of the rumen, and the epithelial cells of the kidney tubules. By use of immunofluorescence the antigen was shown to be present in most of the cells of wool follicles and the cortex of developing wool fibers. Western blotting of SBU-1 against the low-sulfur alpha-keratin proteins of wool confirmed that the antigen recognized by SBU-1 belongs to a family of keratins. It was concluded that SBU-1 was raised against alpha-keratin expressed by the epithelial cells of the thymic rudiment and that the expression of this antigen on the reticular network of the thymus declined with advancement of pregnancy.     

The cell surface of mouse dendritic cells: FACS analyses of dendritic cells from different tissues including thymus

The surface of dendritic cells from mouse spleen, thymus, and epidermis has been compared with a panel of monoclonal antibodies and the FACS. A method was first developed to isolate populations of large, adherent, thymic dendritic cells that were greater than 90% pure. These were released by collagenase digestion and separated from adherent macrophages after overnight culture. Enrichment was based on the facts that most macrophages remained plastic adherent and rosetted strongly with antibody-coated erythrocytes. As in spleen, thymic dendritic cells were stellate in shape, had abundant class I and II MHC products, lacked many standard macrophage and lymphocyte markers, and actively stimulated the mixed leukocyte reaction. Most spleen and thymic dendritic cells could be lysed by the 7D4 mAb, to the low-affinity IL-2 receptor, and complement but the levels of 7D4 by FACS were low and sometimes not above background. Differences among dendritic cells from different tissues were noted with other mAb. Adherent dendritic cells from thymus all expressed the J11d "B cell" antigen and the NL145 interdigitating cell marker, but lacked the 33D1 spleen dendritic cell antigen. Eighty to ninety percent of spleen dendritic cells were J11d-, NL145-, 33D1+ but the remainder expressed the J11d+, NL145+, 33D1- thymic phenotype. The latter phenotype also was identical to that of epidermal Langerhans' cells. We postulate that the major 33D1+ cell in spleen represents a migratory stage in which dendritic cells are moving from tissues to lymphoid organs.     

Some endocrine aspects of the thymus gland.

In studies of the mouse thymus, lymphocyte mitoses are seen to be most frequent in the thymus cortex. There is evidence from thymic grafts that a hypothetical factor, thymopoietin, may stimulate mitosis of thymic lymphocytes. It is a factor which is postulated to act in conjunction with the PAS-positive mesenchymal reticular cells and epithelial reticular cells of the cortex. The thymus medulla is necessary for the integrity of thymic grafts, and may also elaborate a secretion for maintaining the cellular functions of the gland. Thymectomy has been used as a gauge for judging normal thymic function and results, in the mouse, in lymphopenia, degeneration of spleen and lymph nodes, delayed rejection of skin allografts, reduced ability of spleen cells to mount the graft versus host reaction, and reduced primary immune response to certain antigens. Correction of these deficiencies offers a means of evaluating various thymic extracts and grafts. Lymphocytosis-stimulating hormone (LSH) is known to maintain the peripheral lymphoid organs and cause lymphocytosis in the thymectomized animal. Diffusion chamber studies of thymic grafts also show restored lymphoid tissue by a cell-free factor (CIF). These two factors may be the same and probably represent the basis of the highly purified lymphocyte-stimulating proteins, LSHr and LSHh, which restore the L/P ratio in thymectomized animals and may stimulate lymphopoiesis in spleen and lymph nodes. LSHr, unlike LSHh, increases the total lymphocyte count. LSHr has been found to increase the humoral antibody response in neonatal mice both by the PFC technique and by direct hemolysis of sheep erythrocytes. Homeostatic thymic hormone (HTH) is a thymic extract of small molecular weight and contains nucleic acid. In the thymectomized guinea pig it has been found to maintain normal levels of lymphocytes in the blood, spleen and lymph nodes, to restore antibody titers to typhoid H antigen and to restore the toxic allergic reaction. Thymic humoral factor (THF) is of smaller molecular weight (less than 1,000) and probably is not a protein. It also enhances lymphoid proliferation in neonatally thymectomized mice. There is evidence that THF participates in humoral antibody formation because it stimulates PFC formation from neonatally thymectomized mice after inoculation with sheep erythrocytes. Its effects on cell-mediated immunity are seen from findings that injection of THF restores the ability of thymectomized mice to reject skin allografts. THF enables spleen cells from thymectomized or neonatal animals to mount the graft versus host reaction, and causes maturation of bone marrow cells and spleen or lymph node cells so that they can participate in the graft versus host reaction. It has been reported to stimulate lymphocytes to kill isogeneic tumor cells in vitro. Thymosin is protein extracted from the thymus. It has been found to alleviate leukopenia slightly and provide some improvement in lymphoid histology in thymectomized mice...     

Methylated DNA-binding protein is present in various mammalian cell types.

A DNA-binding protein from human placenta, methylated DNA-binding protein (MDBP), binds to certain DNA sequences only when they contain 5-methylcytosine (m5C) residues at specific positions. We found a very similar DNA-binding activity in nuclear extracts of rat tissues, calf thymus, human embryonal carcinoma cells, HeLa cells, and mouse LTK cells. Like human placental MDBP, the analogous DNA-binding proteins from the above mammalian cell lines formed a number of different low-electrophoretic-mobility complexes with a 14-bp MDBP-specific oligonucleotide duplex. All of these complexes exhibited the same DNA methylation specificity and DNA sequence specificity. From the extracts of rat and calf tissues, oligonucleotide protein complexes formed that also had the same specificity as human placental MDBP although they had a higher electrophoretic mobility probably due to digestion by proteases in the nuclear extracts. Although MDBP activity was found in various mammalian cell types, it was not detected in extracts of cultured mosquito cells and so may be associated only with cells with vertebrate-type DNA methylation.     

The immunoproliferative response of mice to intravenously or subcutaneously injected BCG

Summary Intravenous injection of BCG caused (1) a transient thymic epithelial hyperplasia with increase of PAS-positive cells in the cortex and medulla which showed the pronounced secretory activity of a substance which could be histochemically identified as an acid mucopolysaccharide; (2) an equally transient increase in the number of pyroninophilic lymphocytes with increased polyribosome content of the cells and mitoses in the thymic cortex; this reached a peak on day 6 following the injection but was unassociated with an increase in thymic weight; and (3) a systemic granulomatous histiocytic reaction in the liver, spleen, lungs, and lymph nodes, but not in the thymus, bone marrow, or Peyer's patches. The significance of the thymic epithelial changes is not clear but it did coincide with increased pyroninophilia and mitotic activity of the thymic cortical cells, suggesting a possible interaction between this secretory product and the thymic cortex. Comparing the thymic changes with the thymus of other animals of the same species injected with i.v. or i.p. LPS, i.v. or s.c. HIU II fraction of BCG, i.v. pertussis vaccine, i.p. complete or incomplete Freund's adjuvant, and killed at the same planned intervals after the injection of the adjuvants, BCG proved to have a unique action on the thymus with regard to both lymphocytic and epithelial changes. Hepatic, pulmonary, and splenic histiocytic granulomas were observed only in those animals injected intravenously with BCG.     

STUDIES ON THE REGULATION OF LYMPHOCYTE PRODUCTION IN THE MURINE THYMUS AND SOME EFFECTS OF A CRUDE THYMUS EXTRACT

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0–8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering Go/Gi cells into DNA synthesis, the inhibition of G₂ efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.     

Differentiation of human T lymphocytes. I. Acquisition of a novel human cell surface protein (p80) during normal intrathymic T cell maturation

The thymus is thought to be the primary central lymphoid organ in which T cells mature. Although thymic cortical and medullary compartments are distinct histologically, few antigens have been described that are absolutely acquired during the presumed intrathymic maturation pathway from cortical to medullary thymocytes. In this paper, we describe the acquisition during human intrathymic T cell maturation of a novel protein (p80) defined by a monoclonal antibody (A1G3). Although the p80-A1G3 antigen is distributed throughout the body and is not T cell specific, our study demonstrates that expression of p80-A1G3 antigen in normal human thymus is associated with thymocyte functional maturity and location in the thymus medulla. Moreover, in contrast to other markers of mature human T cells, the p80-A1G3 cell surface protein is not expressed on T6+ cortical thymocytes, and, therefore, is absolutely acquired by medullary thymocytes during T cell maturation. Thus, the p80-A1G3 antigen and the A1G3 antibody provide a heretofore unavailable system for the study of molecular events that transpire during the maturation of thymocytes.     

Lipolysis induction in adipocytes by a protein from tumor cells

Extracts of thymic lymphoma that are obtained from AKR mice and are kept in the cold for at least several days can induce lipolytic activity in rat adipocyte suspensions. Freshly prepared extracts have low activity but contain a low molecular weight material of less than 10,000 daltons that aggregates on standing in the cold and becomes active. Treatment of aged extracts with trypsin causes a loss in activity indicating that the active material is a protein. It has been obtained in partially purified form, is relatively heat stable, and is not a lipase. Activity was also demonstrated in AKRXDBA/2 lymphoma (induced by AKR SL3-3 virus) and in transplanted lymphomas from a Friend-virus-induced erythroleukemia cell line in DBA/2 mice, but was not detected in normal thymus, spleen, liver, or other tissues. The partially purified material produced a massive fat mobilization when injected into normal mice.     

Atrial natriuretic peptide in lymphoid organs of various species

1. Evidence for the occurrence of atrial natriuretic peptide (ANP) in various lymphoid organs of different species (rat, mouse, pig, chicken) is provided. 2. ANP precursor material (1-126) as well the physiologically active ANP (99-126), were identified by chromatographic analysis and RIA in extracts of thymus, spleen and lymph nodes of rat, mouse and pig. 3. mRNA coding for ANP was demonstrated both in the thymus and in isolated thymocytes of these species. Furthermore, mRNA for ANP was detected in spleen and lymph nodes (rat and pig). 4. The bursa of Fabricius, thymus glands and spleen of chickens were also shown to express mRNA coding for ANP. 5. These findings provide a firm basis for a link of ANP to the immune system, a novel aspect of possible biological functions of this peptide.     

Thymic hormones and prostaglandins. I. Lack of stimulation of prostaglandin production by thymic hormones

Prostaglandins (PGs) have been implicated as possible mediators of the biological activity of thymic hormones. It has been shown that type E-PGs are able to mimic the action of several thymic hormones and that indomethacin prevents in vivo or in vitro the appearance of Thy-1⁺ antigen induced by some of these factors. We thus investigated a possible role for PGs in the mechanism of action of different thymic extracts and peptides. Attempts to modulate prostaglandin production showed that neither thymosin fraction 5 (0.01 – 100 μg/ml), nor thymosin α 1 (1–10 μg/ml), thymulin (0.001–100 ng/ml), thymopoietin II (10 – 1000 ng/ml) or TP5 (10 – 1000 ng/ml) affect PGE2, 6-keto-PGF1 α, PGF2 α and TXB2 production by spleen cells from control and thymectomized mice. These results do not support the hypothesis that prostaglandins could act as mediators of thymic hormones.