Purification with monoclonal antibody of a predominant leukocyte-common antigen and glycoprotein from rat thymocytes.

A leukocyte-common (L-C) antigen which can be dominant as an immunogen in rabbit anti-rat thoracic duct lymphocyte serum has been purified from rat thymocytes. Initially, an antigenic fragment of 100,000 apparent mol. wt. was prepared at 400 to 900-fold purification by lentil lectin affinity chromatography and gel filtration in deoxycholate. Mice were then immunized with this fraction, and a hybrid myeloma cell line secreting antibody to the L-C antigen was prepared by cell fusion. This antibody was used for affinity chromatography and gave pure L-C antigen at 1400-fold purification compared with thymocytes.The L-C antigen is a major membrane glyco-protein of rat thymocytes and has an apparent mol. wt. of 150,000 as determined by electrophoresis on polyacrylamide gels in sodium dodecyl sulfate. The antigen constitutes one of the three thymocyte glycoproteins which stain intensely for carbohydrate with periodic acid Schiff stain. It is present on greater than 95% of thymocytes, bone marrow cells and thoracic duct lymphocytes.     

Molecular identification of T cell-specific antigens on human T lymphocytes and thymocytes

We have identified membrane glycoproteins which carry T cell-specific antigens on human T lymphocytes and thymocytes. Purified cells were surface-labeled with NaB³H₄ after treatment with neuraminidase and galactose oxidase. Immunoprecipitations were performed with rabbit anti-human T cell-specific antibodies using co precipitation with protein A-containing staphylococci strain Cowan I. The labeled membrane glycoproteins and the precipitates were subjected to polyacrylamide slab gel electrophoresis and visualized by fluorography. The antibodies specifically precipitated 4 proteins called GP200, GP180, GP165 and GP160 (mol. wts. = 200000, 180000, 165000 and 160000) from surface-labeled T lymphocytes and low-density (medullary) thymocytes. The GP200 and GP180 were not labeled on high-density (cortical) thymocytes. A protein with a mol. wt. of 45000 was precipitated from thymocytes. Another glycoprotein on T lymphocytes and thymocytes with a mol. wt. similar to that of mouse and rat Thy-1 or Θ antigen (mol. wt. 25000) reacted with the antibodies.     

Monoclonal antibody to a human leukocyte-specific membrane glycoprotein probably homologous to the leukocyte-common (L-C) antigen of the rat

The monoclonal antibody (F10–89–4) described in this study recognizes an antigen which by quantitative absorption analysis is absent from human brain, kidney, liver, heart, erythrocytes, platelets and normal serum, but is present on spleen, lymph node, chronic lymphatic leukemia cells, bone marrow, thymus and granulocytes at a ratio of 1 : 1 : 0.8 : 0.3 : 0.3 : 0.1, respectively. Analysis with the fluorescence-activated cell sorter showed that 100% of thymocytes, lymph node lymphocytes, blood mononuclear cells and granulocytes carry the antigen, while 83% of bone marrow cells are positive. There was marked heterogeneity in the amount of labeling of thymocytes, with 3 major peaks. There was also heterogeneity of labeling of blood mononuclear cells and lymph node lymphocytes, with a weakly staining hump containing approximately 20% of the cells in the case of lymph node lymphocytes. Double labeling experiments demonstrated that the weakly staining cells of blood and lymph node were B lymphocytes, while frozen sections of thymus showed that the antigen was expressed most weakly in subcapsular cortical thymocytes, and most strongly on medullary thymocytes. Biochemical studies established that the antigen bound to lentil lectin columns, and sodium dodecyl sulfate polyacrylamide gel electrophoresis studies using NaB³ H₄-labeled blood mononuclear cells established that the antigen was a major glycoprotein of lymphocytes, and that its molecular weight was in the region of 190 000 to 215 000.     

Serological and preliminary biochemical characteristics of a T lymphocyte differentiation antigen detected by rabbit antiserum to rat thymocyte membranes.

A rabbit antiserum against rat thymocyte membrane was studied using quantitative absorption analysis, and shown to contain a large amount of antibody against antigen(s) on lymphocytes but not liver, erythrocytes, kidney, heart, lung or brain tissue. Among lymphocytes the antigen(s) was at least 20-fold more abundant on thymocytes or T lymphocytes than on bone marrow or B lymphocytes, and is thus referred to as a T lymphocyte antigen. In saturating binding studies with absorbed antiserum 120 000 molecules of antibody were bound per thymocyte. The T lymphocyte antigenic activity was effectively solubilized from thymocyte membrane by deoxycholate, and its hydrodynamic properties suggested that only one molecule (or a group of very similar molecules) express the activity. The S₂₀,_w and v? values of the antigen were 5.7 S and 0.73 ml/g, and the Stoke's radius was 6.2 nm; a molecular weight (including any bound deoxycholate) of 1 50 000 was calculated. All antigenic activity in deoxycholate extracts bound to a lentil lectin affinity column suggesting the antigen may be a glycoprotein. On serological and biochemical grounds the antigen was distinct from Thy-1. In an appendix a method for determining the maximum binding of antibody per cell by absorption analysis is described.     

Monoclonal antibody to a human brain-granulocyte-T lymphocyte antigen probably homologous to the W 3/13 antigen of the rat

The monoclonal antibody (F10–44–2) described in this report recognizes an antigen which by quantitative absorption analysis is found predominantly on spleen, lymph node, bone marrow, thymus, granulocytes and brain, the amount of antigen on these tissues being approximately the same within a factor of 2 or 3. Analysis with the fluorescence-activated cell sorter showed that 29% of thymus cells, 61% of bone marrow cells, 95% of blood mononuclear cells, 98% of lymph node lymphocytes and 100% of granulocytes carried the antigen. With blood mononuclear cells and lymph node lymphocytes, there were two distinct peaks, with one peak labeling very weakly. Double labeling experiments established that the weakly labeled peak contained the B lymphocytes. Studies on frozen sections of thymus established that positive thymocytes were found only in the medulla indicating that the antigen appears late in T lymphocyte maturation. The lymphatic nodules (B lymphocyte areas) of spleen and lymph node appeared virtually negative on frozen sections showing that there was too little antigen on the B lymphocyte surface for confident detection by fluorescence microscopy. Sodium dodecyl sulfate polyacrylamide gel eletrophoresis of NaB³ H₄-labeled blood mononuclear cells established that the antigen was a major glycoprotein of the leukocyte membrane and that its mol. wt. was 105 000. This antigen shows a striking similarity in biochemistry and tissue distribution to the W3/13 antigen of the rat and is likely to be the human homologue of this antigen.     

Characterization of mouse thymocyte and peripheral lymphocyte xenoantigens by two-dimensional electrophoresis

Two-dimensional electrophoresis was used to analyze the surface glycoproteins of murine thymocytes and lymph node cells. Two-dimensional maps of unselected, radioiodinated lymphocyte surface proteins were complex, showing at least 20 different components, but simpler patterns were obtained by using rabbit antibodies directed against the surface proteins of a T lymphoma cell line, to precipitate xenoantigens from lysates of radioiodinated or biosynthetically labeled thymocytes and lymph node cells. These xenoantibodies precipitated 12–13 distinct components from each cell type, of which all but 3 were sialoglycoproteins. Two types of difference between the surface glycoproteins of thymocytes and peripheral lymphocytes could be detected. First, higher mol. wt. glycoproteins and Thy-1 are more acidic in peripheral lymphocytes than in thymocytes, and this difference disappears after neuraminidase treatment. One additional high mol. wt. glycoprotein is also detectable in peripheral lymphocytes, probably reflecting the greater carbohydrate complexity of these molecules, when expressed on such cells. Second, 3 glycoproteins are strongly labeled only on thymocytes, and 3 others only on peripheral lymphocytes. These 6 glycoproteins might represent genuine differentiation antigens.     

Estimation of the amount and tissue distribution of rat Thy-1.1 antigen

The distribution and amount of Thy-1.1 antigen expressed in rat and mouse tissues have been studied. When anti-Thy-1.1 antisera were absorbed with various tissues and assayed by cytotoxicity or radioactive binding assays, it was found that antigen was expressed in similar amounts on thymocytes and brain of both species. Lymphocytic leukemia cells from PVG/c rats also expressed as much Thy-1.1 as rat thymocytes. In contrast, Thy-1.1 was only detected in very small amounts on rat lymph node cells or thoracic duct lymphocytes; the level was 20-fold less than the AKR mouse equivalent. Rat spleen cells had larger amounts than other peripheral rat lymphocytes, and had only 2–3 times less antigen than AKR spleen cells. These results were confirmed by direct binding assays which showed that AKR mouse and PVG/c rat thymocytes bound more than 500000 molecules of anti-Thy-1.1 antibody per cell. Autoradiographic analysis showed 90 % of thymocytes, 12 % of spleen cells, and 2 % of lymph node or thoracic duct lymphocytes of the rat to be specifically labeled.     

Identification of Ia glycoproteins in rat thymus and purification from rat spleen.

A fraction of la-like glycoproteins was prepared from rat thymocytes by lentil lectin affinity chromatography and gel filtration in deoxycholate. Spleen cells from mice immunized with this preparation were fused with myeloma cells to produce antibody-secreting hybrid cell lines. Antibody from four lines called MRC OX, 3, 4, 5, 6 reacted with the la-like glycoproteins, and MRC OX 3 antibody recognized an antigenie determinant polymorphic in the rat. All four antibodies also bound to mouse spleen cells and all detected polymorphisms. Studies on recombinant mouse strains suggest that the determinants are coded by the I-A subregion of the H-2 complex. MRC OX 3 correlates with Ia specificity 9, while MRC OX 4, 5, 6 correlate with specificity 17 or 18. MRC OX 4 monoclonal antibody was used for affinity chromatography to purify Ia glycoproteins from rat spleen. The rat Ia glycoprotein complex was composed of two noncovalently linked polypeptide chains of apparent mol. wt. (unreduced) 30 000 and 24 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The purified Ia glycoprotein partially inhibited the binding to thoracic duct lymphocytes of an alloantiserum which detects Ia antigens linked to the major histocompatibility complex. The monoclonal anti-la antibodies bound to the majority of peripheral B lymphocytes and 18% of thymocytes, but did not significantly bind to peripheral T lymphocytes. There were on average 150000 molecules of Ia glycoprotein per la-positive B lymphocyte, and 45 000 molecules per la-positive thymocyte. From the same fusion, another cell line was prepared called MRC OX 2 which secretes monoclonal antibody to a previously undefined thymus glycoprotein of apparent mol. wt. 60000. Preliminary studies showed that the antigen was expressed on all thymocytes and on peripheral B lymphocytes in smaller amounts. It was also present in brain, but not liver or kidney homogenate.     

Many cells in rat bone marrow have cell-surface Thy-1 antigen.

Thy-1.1 and Thy-1 xenoantigenic determinants were detected at the cell surface of many rat bone marrow cells. The absorptive capacity of bone marrow cells was 6-10% of that of thymocytes for Thy-1 antigenic determinants, and 30-45% of rat bone marrow cells were specifically labeled with anti-Thy-1 antibody as detected by autoradiography. Thus, while mice and rats are similar in having large amounts of Thy-1 in brain and thymocytes, they differ in that the rat lacks the antigen in most peripheral T cells and expresses it in a large number of bone marrow cells; the opposite is true in the mouse.     

In rat bone marrow Thy-1 antigen is present on cells with membrane immunoglobulin and on precursors of peripheral B lymphocytes.

Rat bone marrow cells carrying Thy-1 antigen were studied morphologically, and tested for their independence of the thymus and their relationship to the B lymphocyte lineage. Using a fluorescence-activated cell sorter to separate Thy-1⁺ and Thy-1^- fractions, it has been confirmed that up to 50 % of all nucleated bone marrow cells are Thy-1⁺, most of which have the morphology of small lymphocytes. Thy-1^-cells were mainly neutrophils and erythroid. Thy-1⁺ cells were found also in the marrow of B rats (rats thymectomized as adults, irradiated and reconstituted with syngeneic bone marrow from thymectomized donors drained of recirculating lymphocytes), though at a lower frequency (roughly half) than of normal rats. In both normal and B rats about 1/4 of the Thy-1⁺ cells also bore lymphocyte surface immunoglobulin (sIg), and these doubly labeled cells accounted for the majority (～ 2/3) of marrow cells carrying large amounts of sIg. Therefore, unlike mice, Thy-1 is not a marker of thymus-dependent lymphocytes in rats. The B precursor activity of marrow fractions was measured in a long-term re-constitution assay counting sIg⁺ cells in the thoracic duct of lethally irradiated recipients. Virtually all the precursors were in the Thy-1⁺ or sIg^- fractions, and were barely detectable among Thy-1^- or sIg⁺ cells. Thus, in the rat peripheral B lymphocytes descend from precursors bearing Thy-1 antigen but lacking sIg.     

Four distinct antigen systems on human thymus and T cells defined by monoclonal antibodies: immunohistological and immunochemical studies.

Human thymus and T cell antigens were identified by using four distinct monoclonal antibodies (MoAb), designated 2D5, 5B3, 7A5 and 9D4. 2D5 antibody reacted with most human thymocytes and a few peripheral lymphocytes as well as with a subpopulation (20%) of bone marrow cells, and precipitated a 45K molecular weight (mol. wt.) component from 125I-labelled thymus cell lysate. 7A5 antibody also reacted with the majority (80%) of thymocytes but neither with peripheral lymphocytes nor with bone marrow cells. The antigen detected by 7A5 was a glycoprotein consisting of 48K and 12K mol. wt. components, which were non-covalently associated with each other. 5B3 reacted with virtually all of human thymus and T cells but not with the majority of B cells and bone marrow cells. This reagent precipitated a 72K mol. wt. glycoprotein from thymus and T cells. An additional 65K mol. wt. glycoprotein was precipitated by 5B3 together with the 72K mol. wt. component, but with poor reproducibility. 9D4 antibody, on the other hand, reacted with a 200K mol. wt. component from thymus and T cells as well as 220K and 210K components from the non-T cell fraction of tonsil lymphocytes. Whereas antigens detected by 2D5 and 7A5 appeared to be highly expressed on cortical thymocytes, the antigen defined by 5B3 occurred much more abundantly on medullary thymocytes and peripheral T cells than on cortical thymocytes. Based on the data described above, it is suggested that 7A5, 5B3 and 9D4 MoAb recognize human homologues of mouse TL, Ly-1 and Ly-5 antigens, respectively, whereas 2D5 antibody seems to resemble OKT10, as described by others.     

The role of hematogenous and intrinsic precursor cells in lymphocyte production in murine bone marrow and thymus

It is well recognized that the bone marrow contains cells that can repopulate a depleted thymus as well as cells that can be induced to express phenotypic markers characteristic of T cells. It is not known, however, to what extent thymocytopoiesis in the normal thymus relies on immigrant, bone marrow-derived cells, nor whether some T cell precursors have entered the bone marrow from the circulation. We used the parabiotic system to test whether thymocytopoiesis relies on progenitors intrinsic to the thymus or on cells that enter the organ from the circulation. In the same system, we have also investigated whether Thy-1^? bone marrow lymphocytes that respond to phytohemagglutinin (PHA) by proliferation and Thy-1 expression are produced by my-elogenous or hematogenous progenitors. Syngeneic CBA/HT6 and CBA/CaJ mice were joined in parabiotic union at 4–6 weeks of age. Cross circulation between the two partners was verified by the equilibration of Evans' blue dye injected into one partner and by the equilibration of PHA-responsive T cells in the spleen of the parabionts. Chromosome spreads were prepared from the PHA-stimulated T cell-depleted bone marrow and from spontaneously proliferating thymocytes as well as from thymocytes stimulated by PHA or Concanavalin A (Con A). The exchange of spleen colony-forming units (CFU-S) in the femoral marrow was assessed by karyotyping individual spleen colonies. Regardless of the length of parabiotic union, ranging from 4 to 20 weeks, Thy-1^?, PHA-responsive bone marrow lymphocytes remained predominantly of the host type with only 3% being derived from the opposite partner. The same held true for CFU-S in the femur; only around 5% of this cell population were of the nonhost type. Thus, although some Thy-1^?, PHA-responsive lymphocytes in the bone marrow may be derived from hematogenous stem cells, the majority of them are generated by precursors resident in the bone marrow. Likewise, regardless of the length of parabiotic union, at least 95% of spontaneously proliferating cells in the thymus of each partner possessed the karyotype of the host, and this held true also for PHA- or Con A-stimulated thymocytes, indicating that the small population of spontaneously proliferating immigrant cells cannot account for the production of the large number of postmitotic (mitogen-responsive) thymocytes. Our findings, therefore, demonstrate a high degree of self-maintenance for Thy-1^?, PHA-reponsive lymphocytes in the bone marrow and also for intrathymic T cell precursors. In the unperturbed, postnatal thymus, thymoctye production does not rely on cell input from the circulation; the vast majority of thymoctyes are generated by an intrathymic precursor pool that is independent of immigrant myelogenous T cell precursors.     

Characterization of a novel bovine leukocyte protein involved in cell-cell adhesion

Abstract: Preliminary characterization of an apparently novel bovine leukocyte adhesion protein is described. Two IgG₁ monoclonal antibodies, UC-C1 and UC-H5, raised against established cultures of IL-2-dependent bovine peripheral blood lymphocytes (PBL) were found to react with an antigen expressed by the majority of bovine peripheral blood leukocytes. Immunoprecipitation and polyacrylamide gel electrophoresis of the antigen produced a distinct protein band of molecular weight 160 000, and additional diffuse protein bands of approximate molecular weight 180 000, 175 000 and 150 000. Two-color flow cytometric analyses showed that the antigen was expressed at low density on a small proportion of circulating B lymphocytes, but was highly expressed on all circulating T lymphocytes. The majority of monocytes and all granulocytes expressed the antigen at a density lower than that of T lymphocytes. Peripheral blood lymphocytes stimulated with concanavalin A had an approximately 3-fold increased expression of the antigen, which was apparent within 18 h and remained stable in long-term cultures. Expression of the antigen in thymus, analyzed by the immunoperoxidase technique, was predominantly restricted to thymocytes in the immediate subcapsular cortex and medulla; expression in lymph nodes and spleen was predominantly confined to lymphocytes in T-cell areas. Flow-cytometric analysis demonstrated that thymocytes and the majority of peripheral and mesenteric lymph node-derived T cells had relatively low surface density of antigen compared to circulating T cells. Binding of UC-C1 or UC-H5 to the antigen on lymphocytes induced homotypic aggregation. UC-C1 completely blocked binding of FITC-conjugated UC-H5 to blood mononuclear cells, suggesting that the antibodies recognize the same epitope or proximal epitopes.     

Lymphocyte cell surface glycoproteins which bind to soybean and peanut lectins.

In cellular immunology, peanut (Arachis hypogaea) lectin has been used to selectively agglutinate immature lymphoid cells and soybean (Glycine max-lectin to agglutinate B lymphocytes. We have used affinity chromatography to study the surface glycoproteins of rat and mouse lymphoid cells which bind to these lectins. Thymocyte and T and B lymphocyte glycoproteins were analysed either without modification (native) or after the removal of sialic acid with neuraminidase (asialo). The only native glycoprotein which was seen to bind to peanut lectin was the 95,000 mol. wt sialoglycoprotein from thymocytes. The equivalent molecules from T lymphocytes bound to peanut lectin only after neuraminidase digestion. Thus the selective agglutination of thymocytes by peanut lectin would seem to be due to a partial lack of sialic acid residues on the O-glycosidically-linked oligosaccharides of the thymocyte sialoglycoprotein. The B lymphocyte form of the leucocyte-common antigen was the only prominent native glycoprotein which was seen to bind to soybean lectin and this probably accounts for the specific binding of this lectin to B cells. The leucocyte-common antigens, in their asialo forms, from thymocytes and B and T lymphocytes differed in their binding to the lectins and this establishes that these glycoproteins which share antigenic determinants differ in their carbohydrate structures.     

Molecular nature of the W3/25 and MRC OX-8 marker antigens for rat T lymphocytes: comparisons with mouse and human antigens

The mouse monoclonal antibodies W3/25 and MRC OX-8 have been used to distinguish rat T lymphocyte subpopulations. In the peripheral T lymphocyte population, W3/25 antibody recognizes an antigen on the Thelper (Th) subset, while MRC OX-8 antibody recognizes an antigen on the Tsuppressor/cytotoxic (Ts/c) subset. To determine the nature of these antigens, rat thymocytes were either metabolically labeled with [35S]L-methionine or surface-labeled at sialic acid residues by periodate oxidation followed by [3H]NaBH4 reduction. Thymocytes were solubilized with nonionic detergent, the antigens immunoprecipitated and the molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Using metabolically labeled cells, W3/25 antibody immunoprecipitated an antigen that electrophoresed under reducing conditions as a broad band between 48 000-53 000 Mr. Unreduced samples migrated between 46 000-50 000 Mr. Surface-labeled W3/25 antigen, electrophoresed under reducing conditions, separated into two bands of 44 000 and 52 000 Mr. Metabolically labeled MRC OX-8 antigen was identified as a protein of at least two chains of 39 000 and 34 000 Mr. There was also a fainter band at approximately 67 000 Mr. Unreduced samples indicated a more complex structure with bands at 70 000, 110 000 and 165 000 Mr. Surface-labeled MRC OX-8 antigen was of similar nature. These data, when considered with functional and tissue distribution data, suggest that W3/25 antigen is equivalent to T4 in man and MRC OX-8 antigen is equivalent to T8 in man, and Lyt-2 in mouse.     

Rat lymphocyte differentiation antigens detected by rabbit antiserum to thymocytes and leukaemic cells.

The lymphocyte differentiation antigens detected by rabbit antisera to rat thymocytes and leukaemic cells have been investigated. Thy-1 was the only such antigen detected by the anti-thymocyte serum, which was predominately directed to two xenoantigenic determinants rather than the Thy-1.1 determinant on this molecule. Two additional antigens were recognized by the anti-leukaemia serum. One of these was found on thymocytes and peripheral lymphocytes, but only on 1 per cent of bone marrow cells. The other was found not only on lymphocytes, but also on the nucleated myeloid and erythroid cells of bone marrow.     

Molecular and antigenic heterogeneity of the rat leukocyte-common antigen from thymocytes and T and B lymphocytes

The molecular forms and antigenic heterogeneity of the leukocyte-common antigen (L-CA) of rat lymphocytes have been analyzed. Thymocytes show one main band at 180 kDa, T cells four bands at 180, 190, 200 and 220 kDa and B cells one broad band at about 240 kDa. T helper and T cytotoxic cell subsets show the same four bands with some differences in the proportion of each. Four mouse monoclonal antibodies (MRC OX-1, 28, 29 and 30) reacted with all molecular forms of L-CA and fell into two sets that were noncompetitive in binding to L-CA (MRC OX-1, 28, 29 vs. OX-30). The antigenic determinants seen by all these antibodies were lost when L-CA was reduced and alkylated. Three antibodies (MRC OX-22, 31 and 32) reacted selectively with B cells, T cytotoxic cells and about 2/3 of T helper cells. OX-22 and OX-31 competed for binding but were noncompetitive with OX-32. All these antibodies bound to a subfraction of the 190, 200 and 220-kDa forms of T cell L-CA but not at all to the 180-kDa form of T cells or thymocytes. One antibody bound to B cells only (MRC OX-33) and precipitated a subfraction of B cell L-CA. With all the antibodies that did not label thymocytes the antigenic determinants survived reduction and alkylation. Subsequent proteolysis with trypsin then destroyed all determinants except the one reacting with MRC OX-22 antibody. In this case tryptic peptides retained full antigenic activity which was, however, destroyed by further proteolysis with pronase.     

Isolation and characterization of a molecular complex containing Thy-1 antigen from the surface of murine thymocytes and T cells

A complex containing the Thy-1 (θ) antigen has been isolated from the surface of murine thymocytes and T cells by cell surface radioiodination, lysis by freezing and thawing, and immunoprecipitation. The specificity of the immunoprecipitation was firmly established using both congenic and noncongenic anti-Thy-1 sera and thymocytes from congenic and noncongenic mice. Unlike cell surface Ig and the TL and H-2 alloantigens, the antigenicity of Thy-1 was abolished by treatment with nonionic detergent. Thy-1 was localized exclusively in a complex of cell surface molecules which has a density lower than that of protein. Moreover, Thy-1 could be readily labeled by incubating thymocytes with [³H]galactose, but not ³H-labeled amino acids. These observations suggest that the antigenicity of Thy-1 may reside in a glycolipid.     

Further chemical characterization of guinea pig Ia molecules derived from the three major classes of immunocompetent cells.

The guinea pig system is unique in that Ia molecules are readily demonstrable on the three major classes of immunocompetent cells. We were, therefore, able to characterize and compare the structures of Ia molecules from B and T lymphocytes and macrophages. T lymphocyte Ia molecules which bound to Lens culinaris (lentil) lectin co-electrophoresed on SDS-PAGE with lymph node cell Ia molecules (predominantly B lymphocyte Ia molecules) which also bound to lentil lectin. T lymphocyte and macrophage Ia molecules which did not bind to lentil lectin had a slower mobility on SDS-PAGE than lymph node cell Ia molecules which did bind to lentil lectin. One dimensional isoelectric focussing of Ia molecules derived from lymph nodes, T lymphocytes, or macrophages revealed discrete banding patterns for all Ia molecules examined. This result indicates that the Ia molecules which can be chemically isolated from B cells, T cells and macrophages do not have variable regions and probably do not possess antigen recognition capability analogous to immunoglobulin.     

Surface antigens, SBU-T4 and SBU-T8, of sheep T lymphocyte subsets defined by monoclonal antibodies.

Monoclonal antibodies have been used to characterize molecules found on the surfaces of T cells of sheep. SBU-T4 and STU-T8 are present on 80-85% of thymocytes, absent from B lymphocytes, and present on two-thirds and one-third of lymph node T lymphocytes, respectively. Double-labelling of T lymphocytes shows that the populations recognized are mutually exclusive. Immunoprecipitation of the SBU-T4 antigen from thymocytes and T lymphocytes reveals a molecule which migrates on SDS-PAGE as a single band of 56,000 MW under both reducing and non-reducing conditions. Under reducing conditions, the SBU-T8 molecule migrates as two bands of 33,000 and 36,000 MW when immunoprecipitated from thymocytes, but only the higher MW band is present following immunoprecipitation from peripheral lymphocytes. Under non-reducing conditions, SBU-T8 migrates as dimers and other multiples. Based on immunofluorescent, immunochemical and immunohistological data, these molecules are considered to be the structural analogues of the marker molecules used to delineate T-helper and T-cytotoxic/suppressor lymphocyte subsets of other species.