Immunoassay for bovine serum thymopoietin: discrimination from splenin by monoclonal antibodies

A polyclonal rabbit anti-bovine thymopoietin antiserum was used to develop a radioimmunoassay and sandwich enzyme-linked immunosorbent (ELISA) assay for the thymic hormone thymopoietin. Both assays showed slightly less sensitivity for the closely related splenic hormone splenin (SP) than thymopoietin (TP) and markedly less sensitivity for the human as compared with the bovine polypeptides. A number of murine monoclonal antibodies specific for bovine thymopoietin were generated; they were unreactive with bovine splenin and were also unreactive with human thymopoietin and splenin. A sandwich ELISA using these monoclonal anti-TP antibodies together with polyclonal rabbit anti-TP was specific for bovine thymopoietin and measured 300-500 ng/ml thymopoietin in bovine serum. Similar approaches are being pursued to develop an immunoassay for thymopoietin in human serum.     

Isolation and characterization of monoclonal antibodies reactive with endothelial cells

Monoclonal antibodies were generated to antigens on cultured human umbilical vein endothelial cells. Spleen cells from BALB/c mice, immunized with low passage cultures of human umbilical vein endothelial cells, were fused with the non-secretory myeloma line, P3 x 63Ag 8.653. Hybridoma supernatants were screened for the desired immunological reactivity using ELISA binding assays. Hybridomas secreting antibodies reacting with the immunizing endothelial cells, but not with peripheral blood mononuclear cells, were cloned by limiting dilution and three stable clones were chosen for study. Further testing by ELISA revealed that each antibody displayed a unique pattern of reactivity. One antibody, 14E5, reacted with the macrophage-like cell line DHL-2, cultured macrophages derived from peripheral blood monocytes, and macrophages derived from malignant effusions. The antibody failed to react with fibroblasts or bovine endothelial cells. The second antibody, 12C6, reacted with human and primate fibroblasts and endothelial cells derived from bovine arteries, but not with mature macrophages. The third clone, 10B9, reacted only with the immunizing endothelial cells and the immature-macrophage line U-937. All three antibodies failed to react with long-term human B or T lymphoblastoid cell lines, leukemic cell lines, or murine macrophage lines. None of the antibodies reacted with a battery of human epithelial derived cell lines or primary cultures of human epithelial cells. Indirect immunofluorescence assays revealed that the antigens were expressed on the cell surface. These antibodies should prove useful as differentiation markers of human endothelial cells and in studies of endothelial cell function.     

Characterization of an antigenic determinant preferentially expressed by type I epithelial cells in the murine thymus.

A hamster monoclonal antibody (MAb), designated 8.1.1, was raised against murine thymic stromal cell lines and was found to react with cell surface molecules expressed by a morphologically distinct population of epithelial cells of the murine thymus comprising the subcapsular environment, cells investing vascular structures throughout the thymus, and some of the cellular elements in the medulla. The epithelial nature of the labeled cells was confirmed with immunoelectron microscopy. Reactivity with MAb 8.1.1 was associated with thymic epithelial cells in contact with basal laminae. Ontological studies of thymic tissue demonstrated that the epitope recognized by this MAb was expressed before Day 14 of gestation, although the restricted subcapsular and medullar expression of 8.1.1 was not apparent until sometime after birth. MAb 8.1.1 also reacted with a number of extra-thymic tissues, including lamina propria of gut, glomeruli and tubules in the kidney, mesothelia covering a number of organs, and the dermis and epidermis of skin. Within the epidermis, reactivity of MAb 8.1.1 was largely restricted to basal epithelial cells. Immunochemical analysis of 8.1.1 reactivity with detergent-soluble extracts of thymic stromal cell lines and thymus tissue indicated that detergent-soluble extracts of thymic stromal cell lines and thymus tissue indicated that the epitope recognized by this MAb was associated with a glycoprotein bearing terminal N-acetylglucosamine residues and possessing an Mr of approximately 36-38 KD under reducing or non-reducing conditions.     

Membrane antigens of human bronchial epithelial cells identified by monoclonal antibodies

Summary Subpopulations of normal bronchial epithelial cells were identified using a series of murine monoclonal antibodies. These antibodies were used to stain intact bronchial epithelial cells in culture by indirect immunofluorescence. LAM 2 reacted with 80%, LAM 6 with 75%, LAM 7 with 60%, and LAM 8 with 5% of these cells. Sections of human bronchial epithelium were also stained with these antibodies by immunoperoxidase methods. LAM 2 was found to bind with 80%, LAM 6 with 65%, LAM 7 with 50%, and LAM 8 with less than 1% of bronchial epithelial cells. LAM 2 stained both columnar epithelial cells and basal cells; LAM 6 stained mainly basal cells and only a small proportion of columnar cells; LAM 7 showed specificity for basal cells; LAM 8 distinctly stained single cells in the basal cell layer. These antibodies were previously shown to react with the surface membrane of human lung carcinomas, ranging from the broad reactivity of LAM 2 with small cell and non-small cell lung cancers to the highly restricted reactivity of LAM 8 with small cell carcinomas of the lung. Thus, membrane antigens have been identified in bronchial epithelial cells by monoclonal antibodies which exhibit a similar range of cellular reactivity in vitro as in vivo. Inasmuch as these antibodies recognize subsets of cells which could not be easily distinguished by morphologic characteristics, these reagents may be useful in classifying bronchial epithelial cells.     

Monoclonal antibodies reactive with subsets of mouse and human thymic epithelial cells

We describe monoclonal antibodies (MAB) reactive with subsets of mouse and human thymic epithelial cells. Rat MAb CDR1 reacts with mouse but not human cortical epithelial cells. Immunologic staining of thymic nurse cells in suspension indicates the CDR1 antigen is located on the cell surface. Mouse MAb CDR2 reacts with human but not mouse cortical thymic epithelial cells. Rat MAb MD1 and MD2 detect different determinants expressed by most medullary epithelial cells in mouse thymus but fewer such cells in human thymus. In addition, MD1 detects flattened subcapsular cells rarely in mouse thymus but frequently in human thymus. Two-color stains using an anti-keratin antiserum demonstrate the epithelial nature of the cells reactive with these antibodies. The antigens detected by CDR1 and MD1 first appear during the neonatal period, achieving adult distribution by postnatal days 14 and 4, respectively. The extra-thymic staining of these MAb is described. On the basis of their intra- and extra-thymic reactivities, these MAb differ from those previously reported and may permit dissection of the thymic microenvironment.     

A histone 1-like antigen is a component of the nuclear envelope

Rabbit antibodies have been raised against rat liver nuclear envelopes. An enzyme-linked immunosorbent assay (ELISA) demonstrated high titer antiserum specific for the nuclear envelope preparation. Immunocytochemical studies showed that the antiserum stained the nuclear envelopes, but not intra-nuclear components of HEp-2 (human malignant epithelial) cells. When electrophoretically separated peptides were tested by immunoblotting techniques, the rabbit antiserum specifically stained proteins with molecular masses of 26 and 28 kD. These peptides had similar mobilities to purified histone 1 (H1). Indeed purified calf thymus H1 recognized the antiserum. The antigens are not loosely bound to the nuclear envelope, as they could not be extracted with low salt. Therefore, we have established that the 26 and 28 kD nuclear envelope peptides are not contaminants of the nuclear envelope preparation and that they express determinants that are immunologically cross-reactive with purified H1, but not with intra-nuclear H1.     

Various keratin antibodies produce immunohistochemical staining of human myocardium and myometrium

Various polyclonal and monoclonal antibodies to keratins were used to stain different human muscle tissues by paired immunofluorescence and the unlabelled antibody peroxidase-anti-peroxidase method. In the myocardium, distinct coloration of the intercalated discs was produced by two polyclonal reagents to human epidermal keratins but not by two monoclonal antibodies to cytokeratins from pig renal tubular cells. In the myometrium--mainly in the middle layer of the uterine wall--cytoplasmic coloration of a varying fraction of the smooth muscle bundles was produced, especially by one of the polyclonal and by both monoclonal reagents. The staining was often confined to the perinuclear region. The keratin-positive myometrial cells usually coexpressed vimentin and actin in various proportions. These findings indicated that intermediate filaments of the keratin type, or antigenically similar elements, are not restricted to cells of epithelial origin. Other types of muscle cells did not react with keratin antibodies, but keratin-positive macrophages were occasionally found in tongue musculature and in inflamed epicardium. Altogether, our observations emphasize that keratin reactivity cannot be considered specific for epithelial (or mesothelial) cells without reservation.     

Rabbit antiserum against a purified surface glycoprotein decompacts mouse preimplantation embryos and reacts with specific adult tissues

A rabbit antiserum against a purified embryonal carcinoma (EC) cell surface glycoprotein interferes with cell-cell interaction in mouse preimplantation embryos. The 123 kD glycoprotein seems not to be an integral membrane component. The reactivity pattern of the antiserum was studied by immunofluorescence on cryostat sections of post-implantation embryos and of adult tissues. During embryonic development positive reactions were found on all epithelial cells, irrespective of their germ layer origin. Epithelial cells of adult tissues—tongue, uterus, gut, kidney, trachea and liver—react with the antibodies. The results are compared with cell-adhesive molecules previously described on EC cells and preimplantation embryos.     

Epithelial cell components immunoreact with antiserum thymic factor (FTS) antibodies: Possible association with intermediate-sized filaments

By indirect immunofluorescence microscopy, an antiserum raised in rabbit against serum thymic factor (FTS) was found to decorate the epithelial cells not only in the thymus, but also in the kidney, uterus, urinary bladder, prostatic glands, stomach, ileum, colon, submaxillary glands, trachea, epidermis and epidermal appendages of mouse. The staining ability was completely absorbed with an FTS-binding immunoabsorbent, and affinity-purified anti-FTS IgG showed the same staining patterns as the original antiserum. The staining profiles resembled those described for tissues stained with antiprekeratin and antikeratin antibodies in both distribution in tissue and localization in the epithelial cells. In primary-cultured cells from mouse kidney medullae, the anti-FTS antibodies decorated the cytoplasmic fiber network. The fibers were wavy, bundled together and branched. They were dense in the perinuclear cytoplasm and spread in the cytoplasm toward the cell periphery. This decoration was resistant to colchicine and cytochalasin B, but sensitive to pretreatment with formaldehyde. The organization and shape of the fiber network were similar to those of the networks of intermediate-sized filaments containing cytokeratins, keratins and vimentin. However, the antiserum did not give a precipitin band in immunodiffusion test with prekeratin from bovine muzzle, keratin from human epidermis or 3T3 vimentin. Neither tubulin nor actin formed precipitin bands with the antiserum. These results show that the epithelial cells of various mouse tissues contain FTS or substances close to FTS in chemical structure and suggest that they are associated with the intermediate-sized filaments.     

Antibodies to an antigen of human thymus epithelial tissue common to the epidermis of the skin in malignant myasthenia]

It has been demonstrated by the indirect immunofluorecent technique that many of the sera of patients with myasthenia gravis react with the anticells of the human thymus epithelial tissue. Sorption of the sera with the suspension of the epidermis cells and the homogenates of the tissues of other human organs showed that the epithelial cell antigen with which the sera of patients with myasthenia reacted were epidermal heteroorganic thymus antigens, i.e. common for the thymus epithelium and skin epidermis. The presence of antibodies to the cells of the epithelial tissue of the thymus in the sera of patients suffering from myasthenia gravis permits to suppose the existence of an immunopathological process against the thymus tissue antigens (including the heteroorganic structures of its epithelium) in this disease.     

Patterns of expression of trichocytic and epithelial cytokeratins in mammalian tissues II. Concomitant and mutually exclusive synthesis of trichocytic and epithelial cytokeratins in diverse human and bovine tissues (hair follicle, nail bed and matrix, lingual papilla, thymic reticulum)

The hair-forming cells (trichocytes) and the mature hair contain four major trichocytic cytokeratins from each of the subfamilies, basic (Hb1-4) and acidic (Ha1-4); these are related - but not identical - to the epithelial cytokeratins. Here we show, by biochemical methods and immunofluorescence microscopy using antibodies specific for either epithelial or trichocyte cytokeratins, that the same set of hair-type cytokeratins, including two newly identified minor components, designated Hax (type I) and Hbx (type II), are also expressed in cells forming nails, in the filiform papillae of the dorsal surface of human and bovine tongue, and, most surprisingly, in some cells of the epithelial reticulum of bovine and human thymus. By double-label immunofluorescence microscopy, we also show that the expression of the two subsets of cytokeratins, i.e., the epithelial and the trichocytic ones, is not necessarily mutually exclusive, but that certain cells of hair follicles, nail matrix and bed, lingual papillae, and the nonlymphoid cell system of the thymus contain both trichocytic and certain epithelial cytokeratins. This indicates that these cells coexpress representatives of both kinds of cytokeratin. Implications of these findings with respect to problems of regulatory control of cytokeratin synthesis in tissue development and differentiation, and the possible functional meaning of the occurrence of trichocytic cytokeratins in such histologically diverse tissues, are discussed.     

Immunohistochemical characterization of the thymic microenvironment

Summary The epithelial framework of the human thymus has been studied in parallel by immunohistochemical methods at the light- and electron-microscopic levels. Different monoclonal antibodies were used, reacting with components of the major histocompatibility complex, keratins, thymic hormones and other as yet antigenically undefined substances, which show specific immunoreactivities with human thymus epithelial cells.The electron-microscopic immunocytochemical observations clearly confirm microtopographical differences of epithelial cells not only between the thymic cortex and medulla, but also within the cortex itself. At least four subtypes of epithelial cells could be distinguished: 1) the cortical surface epithelium; 2) the main cortical epithelial cells and thymic nurse cells; 3) the medullary epithelial cells; and 4) the epithelial cells of Hassall's corpuscles.The various epithelial cell types of the thymus display several common features like tonofilaments, desmosomes and some surface antigens as demonstrated by anti-KiM3. In other respects, however, they differ from each other. The cortical subtype of thymic epithelial cells including the thymic nurse cells shows a distinct pattern of surface antigens reacting positively with antibodies against HLA-DR (anti-HLA-DR) and anti-21A62E. Electron-microscopic immunocytochemistry with these antibodies clearly reveals a surface labeling and a narrow contact to cortical thymocytes particularly in the peripheral cortical regions. An alternative staining pattern is realized by antibodies to some antigens associated with other subtypes of thymic epithelial cells. Medullary epithelial cells as well as the cortical surface epithelium react likewise positively with antibodies to special surface antigens (anti-Ep-1), to special epitopes of cytokeratin (anti-IV/82), and to thymic hormones (anti-FTS). The functional significance of distinct microenvironments within the thymus provided by different epithelial cells is discussed in view of the maturation of T-precursor cells.Glossary of Abbreviations Anti-X anti-X antibody - APUD-cells amine precursor uptake and decarboxylation (gastro-intestinal endocrine cells) - DAB diamino-benzidine - DMSO dimethyl sulfoxide - FTS facteur thymique sérique - HLA-A, B, C human leucocyte antigen, A, B, C-region related - HLA-DR human leucocyte antigen, D-region related - IDC interdigitating cell - MHC major histocompatibility gene complex - PBS phosphate-buffered saline - TNC thymic nurse cell This investigation was supported by grants from the Deutsche Forschungsgemeinschaft, and its Sonderforschungsbereich 111Fellow of the Alexander von Humbold-Stiftung, Institute of Pathology, University of Würzburg, Federal Republic of GermanyThe authors appreciate the contribution of human thymus tissue from Professor Alexander Bernhard, Abteilung kardiovasculäre Chirurgie der Universität Kiel; the gift of monoclonal antibodies from Dr. M.J.D. Anderson, Dr. M. Dardenne and Dr. H.J. Radzun; and the excellent technical assistence of Mrs. O.M. Bracker, Mrs. H. Hansen, Mrs. R. Köpke, Mrs. M. v. Kolszynski, Mrs. J. Quitzau, Mrs. H. Siebke, and Mrs. H. Waluk     

Various keratin antibodies produce immunohistochemical staining of human myocardium and myometrium

Summary Various polyclonal and monoclonal antibodies to keratins were used to stain different human muscle tissues by paired immunofluorescence and the unlabelled antibody peroxidase-anti-peroxidase method. In the myocardium, distinct coloration of the intercalated discs was produced by two polyclonal reagents to human epidermal keratins but not by two monoclonal antibodies to cytokeratins from pig renal tubular cells. In the myometrium — mainly in the middle layer of the uterine wall — cytoplasmic coloration of a varying fraction of the smooth muscle bundles was produced, especially by one of the polyclonal and by both monoclonal reagents. The staining was often confined to the perinuclear region. The keratin-positive myometrial cells usually coexpressed vimentin and actin in various proportions. These findings indicated that intermediate filaments of the keratin type, or antigenically similar elements, are not restricted to cells of epithelial origin. Other types of muscle cells did not react with keratin antibodies, but keratin-positive macrophages were occasionally found in tongue musculature and in inflamed epicardium. Altogether, our observations emphasize that keratin reactivity cannot be considered specific for epithelial (or mesothelial) cells without reservation.Supported by the Norwegian Cancer Society, Jahre's Fund, and the Norwegian Research Council for Science and the Humanities     

The human thymic microenvironment: Thymic epithelium contains specific keratins associated with early and late stages of epidermal keratinocyte maturation

Normal T-cell development is dependent on interactions with the thymic microenvironment; thymic epithelial cells are thought to play a key role in the induction of thymocyte maturation, both through direct contact and, indirectly, via thymic hormone secretion. It has been postulated that thymic epithelial cells progress through an antigenically defined pathway of differentiation similar to that of epidermal keratinocytes. As keratins vary according to epithelial cell type and the stage of epithelial cell maturation, we used a panel of monoclonal antibodies against keratins to study specific types of keratin intermediate filaments within human thymic epithelium. The demonstration in human thymus of keratins previously shown to be associated with distinct stages of epidermal keratinocytic maturation would support the hypothesis that thymic epithelial cells undergo sequential stages of differentiation. Two-dimensional immunoblot analysis of cytoskeletal extracts from human thymus revealed that thymic epithelium contains the following keratins: 1-2, 5, 6, 7, 8, 10, 13, 14, 15, 16, and 17 (molecular masses, 65-67, 58, 56, 54, 52, 56.5, 51, 50, 50', 48, and 46 kilodaltons, respectively). Thus, in thymic epithelium, we found keratins previously observed in epidermal basal cells (5, 14, 15), as well as keratins specific for terminally differentiated keratinocytes in supra-basal epidermis (1-2, 10). Indirect immunofluorescence (IF) performed on fetal and postnatal human thymus demonstrated that keratin epitopes recognized by antibodies AE-3, 35 beta H11, and RTE-23 are present on epithelial cells of the subcapsular cortex, the cortex, the medulla, and Hassall's bodies. In contrast, antibodies AE-1 and RTE-22 reacted primarily with neuroendocrine thymic epithelium (subcapsular cortex, medulla, Hassall's bodies). The epithelial reactivity of antibody AE-2 was limited to epithelial cells in Hassall's bodies and did not appear until 16 weeks of fetal gestation i.e., when Hassall's bodies first formed. Two-dimensional gel analysis of thymic keratins demonstrated that antibody AE-2 identified only the keratins with molecular masses of 56.6 and 65-67 kilodaltons (10 and 1-2 respectively) in thymus. These data, together with the selective reactivity of AE-2 with Hassall's bodies in fluorescence assays, demonstrate the localization in Hassall's bodies of the high-molecular-weight keratins associated with the late stages of epidermal cell maturation. In summary, we demonstrated that human thymic epithelium contains specific keratins found in multiple epithelial types as well as keratins associated with both early and late stages of epidermal cell differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Myelin-Associated Glycoprotein Shares an Antigenic Determinant with a Glycoprotein of Human Melanoma Cells

A sulfated 100K-dalton glycoprotein has been shown to be released into the culture medium of melanoma cells. Monoclonal antibodies 10C5 and 11B5, which were raised to human melanoma cells, as well as HNK-1 bind to this glycoprotein. It is shown here that mouse anti-myelin-associated glycoprotein (MAG) carbohydrate antibodies raised to human MAG and a human IgM paraprotein associated with neuropathy also bind to the same 100K molecule. However, anti-MAG antibodies recognizing peptide epitopes do not appear to react with this glycoprotein of melanoma cells, a result suggesting that its similarity to MAG is restricted to shared carbohydrate moieties. The anti-melanoma antibodies (10C5 and 11B5) resemble HNK-1 in binding to MAG and to some 19-28K-dalton glycoproteins and sulfated, glucuronic acid-containing sphingoglycolipids of the peripheral nervous system (PNS). In addition, the anti-melanoma antibodies cross-react with neural cell adhesion molecule (N-CAM), an observation emphasizing the shared antigenicity between MAG and other adhesion molecules. The results demonstrate that the anti-melanoma antibodies fall into a class of monoclonal antibodies (including HNK-1, human IgM paraproteins associated with neuropathy, anti-human MAG antibodies, and L2 antibodies) that are characterized by reactivity against related carbohydrate determinants shared by human MAG, N-CAM, and several protein and lipid glycoconjugates of the PNS.     

Ontogeny of T lymphocytes in the pig

Mononuclear cells isolated from pig fetal thymus (and thymus region), spleen and cord blood were examined for their reactivity with polyclonal sheep anti-pig T cell antiserum. First immunofluorescence-positive cells were detected after 28 d of gestation in the thymus region, cord blood and the liver.     

Epithelial heterogeneity in the murine thymus: a cell surface glycoprotein expressed by subcapsular and medullary epithelium

A monoclonal antibody (MAb), G8.8, was raised against glycoconjugates isolated from a cloned line of murine medullary thymic epithelial cells. Flow cytometric analysis of the reactivity of this MAb with cultured thymic epithelium demonstrated that the ligand was expressed on the cell surface. Immunohistochemical examination of normal murine thymus revealed labeling of cells in the subcapsular and medullary areas. Immunoelectron microscopy revealed surface labeling restricted to cells possessing ultrastructural features of epithelium (desmosomes, tonofilaments, and cytoplasmic cysts). During thymic ontogeny, G8.8+ cells predominated in fetal development at the earliest time point examined (Day 14 of gestation). There was an expansion of the cortical epithelial component so that by Day 18 cortical and medullary compartments could be clearly distinguished. Immunoprecipitation of radioiodinated thymic stroma with MAb G8.8 detected a molecule with an apparent Mr of approximately 38 KD under non-reducing conditions. When reduced, the apparent Mr was slightly increased (42 KD). This MAb also exhibited reactivity with gut and epidermal epithelium and some tubular epithelium in the kidney, but did not react with epithelial parenchymal cells of the liver.     

Unique subset of thymic epithelial cells defined by the expression of a novel neuroendocrine antigen

Murine monoclonal antibodies (MoAbs) were produced against a rat medullary thyroid carcinoma to identify neuroendocrine differentiation antigens. One of these antibodies (1D4) identified a novel 62- to 69-kDa antigen expressed by subsets of immune system epithelial and neuroendocrine cells. This antigen is expressed by distinct subsets of thymic epithelial and splenic reticular cells and is shared by discrete subsets of anterior pituitary and thyroid neuroendocrine cells. In the thymus, 1D4 expression identified a unique subset of stellate-shaped Ia+ medullary epithelial cells which did not react with thymosin alpha-1 antisera nor with the MoAb A2B5 specific for a GQ ganglioside expressed by thymic hormone-producing cells. The availability of the 1D4 MoAb should facilitate further characterization of 1D4+ immune system epithelial cells and may advance our understanding of neuroendocrine-immune system interactions.     

Cross-reactivity of antibodies on thymic epithelial cells from humans and marmosets by flow-cytometry

Callithrix jacchus, the common marmoset, is particularly suitable for immunological studies in vivo and in vitro since many antibodies directed against epitopes of human cells do also react with their analogues from this non-human primate. We studied the reactivity of antibodies against human epitopes on primary cultures of thymic epithelial cells from marmosets and humans by flow-cytometry after different culture periods. The antibodies against integrins, including CD61, reacted with thymic epithelial cells from both humans and marmosets, as did anti-CD44 and anti-CD106. Antibodies specific for thymic epithelial cells (TE-3, TE-4, TE-8, TE-15, TE-16, TE-19) also bound to cells from marmosets but expression of all epitopes was not observed in all cultures studied. The expression of CD51, CD54, CD58 and CD106 on human cells declined after 4 weeks of culture. Our findings indicate that marmosets are a valuable model for immunological studies of effects of xenobiotics on the thymic epithelium.     

Cooperativity of thymopoietin 32-36 (the active site) and thymopoietin 38-45 in receptor binding

Thymopoietin is a 49 amino acid polypeptide hormone of the thymus whose biological activity is reproduced by the synthetic pentapeptide thymopentin, corresponding to amino acids 32-36. Thymopentin requires the addition of an octapeptide corresponding to thymopoietin 38-45 for full competition with native thymopoietin in a radioreceptor assay with receptor derived from the human T-cell line CEM. Thus thymopoietin appears to bind to its receptor on T-cells by two regions (32-36 and 38-45). Thymopentin alone is biologically active and induces elevations of intracellular cyclic GMP. Whilst occupancy of the adjacent site by thymopoietin 37-45 does not of itself cause an elevation of intracellular cyclic GMP this peptide is not biologically silent as it does enhance the potency of thymopentin.