Human B cell line deficient in the expression of B cell-specific glycoproteins (GP 27,35).

A human B lymphoid cell line, P3HR-1, expresses only low levels of the 27 000 and 35 000 mol.wt. B cell-specific glycoproteins (GP 27,35). Indirect antibody-binding and quantitative absorption tests with a xenoantiserum against the antigens showed that P3HR-1 cells have on their surface about 1% of the amount found on other human B lymphoblastoid cell lines. The deficit of the glycoproteins on the surface of P3HR-1 cells could be accounted for by a reduced rate of synthesis in these cells. A simple relationship between the reduced expression of GP 27,35 on P3HR-1 cells and their inability to bind Epstein-Barr virus (EBV) or express complement receptors was excluded because other B lymphoid cells which expressed neither virus-binding sites nor complement receptor had normal amounts of GP 27,35 on their surface. However, antibodies against GP 27,35 could block the absorption of EBV by EBV receptor-positive B cells.     

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.     

The predominant heavily glycosylated glycoproteins at the surface of rat lymphoid cells are differentiation antigens.

Rat lymphoid cells have been labeled with sodium 3H-borohydride after periodate oxidation. The labeled glycoproteins were solubilized in detergent and analyzed by fluorography after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Major bands were found at 150 000, 95 000 and 25 000 apparent mol.wt. for thymocytes; at 170 000 and 95 000 mol. wt. for T lymphocytes and at 200 000 mol.wt. for B lymphocytes. Bone marrow cells showed a diffuse band at 100 000 mol.wt. with relatively minor bands around 150 000 mol.wt. With the exception of the 95 000 mol. wt. bands, all these glycoproteins bound to lentil lectin. Using monoclonal or monospecific antibodies in immunoprecipitation and on antibody affinity columns, each of these glycoprotein bands was identified as a previously defined lymphocyte differentiation antigen. The bands at 150 000 mol.wt. on thymocytes, at 170 000 on T lymphocytes, at 200 000 on B lymphocytes, and at 130 000 to 150 000 on bone marrow cells all consist of a leukocyte-common antigen, which has previously been shown to be present on leukocytes but not on other tissues. At least a part of the 95 000 mol.wt. band on thymocytes, T lymphocytes and bone marrow cells is the W3/13 antigen previously shown to be on mature T lymphocytes, polymorphonuclear cells, and in brain. The 25 000 mol.wt. band of thymocytes is the Thy-1 antigen. Similar experiments were carried out on thymocytes labeled with ¹²⁵I by the lactoperoxidase method. An intense band at 150 000 mol. wt. was identified as the leukocyte-common antigen by immunoprecipitation. A labeled band, which did not bind to lentil lectin, was immunoprecipitated at 95 000 mol. wt. with W3/13 antibody. Rat Thy-1 antigen was not labeled with ¹²⁵I.     

Detection and Quantitation of Human Ia-Type Antigens with Iodinated Protein A and Specific Purification of Antibodies Against Ia-Type Alloantigens

Antibodies directed against specific human Ia-type antigens can easily be detected and quantitated by an improved radioimmunoassay using iodinated protein A bound to a specific antibody-Ia-antigen complex on the surface of freshly drawn peripheral human leukocytes, cultured human cell lines, or lymphoid cells fixed with glutardialdehyde or formaldehyde. The same principle can also be used for the detection of Ia alloantigens on human lymphocytes when testing them with specific antisera known to contain antibodies against transplantation antigens. These anti-Ia-alloantigen antibodies had been purified by a two-step procedure involving ion-exchange chromatography on DEAE-cellulose at pH 6.3 and the specific absorption on formaldehyde-fixed Ia-alloantigen-carrying homozygous cell lines, followed by elution of these antibodies with isotonic citrate buffer at pH 3.0. In this way an about 90-fold purification could be achieved. After such a purification the highly enriched antibody fraction still reacted selectively with one specificity of the Ia antigen system.     

Inhibition of glycosylation prevents H-2K and D antigen expression on SV40 virus-transformed cells

Tunicamycin, an inhibitor of glycosylation, was used to examine the role of carbohydrate moieties of glycoproteins involved in recognition of Simian virus 40 (SV40)-transformed cells by cytotoxic T lymphocytes (CTL). Tunicamycin treatment renders such cells refractory to lysis by H-2 restricted, SV40-specific CTL as well as by allogeneic CTL directed only against H-2 antigens. Treated cells are no longer susceptible to lysis by anti-H-2 antisera and complement, nor are they stained by monoclonal anti-H-2 antibodies in an indirect immunofluorescence assay. No accumulation of H-2 proteins was detected in immunoprecipitates from cells labeled with [35S]methionine in the presence of tunicamycin. It is concluded that tunicamycin prevents the expression of H-2 glycoproteins in these cells, perhaps due to accelerated degradation of the unglycosylated molecules.     

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.     

HLA antigens: rabbit antisera reacting with all A series or all B series specificities

Papain-solubilized HLA antigens giving only two bands of 34 000 mol.wt. and 11 000 mol.wt. by sodium dodecyl sulfate (SDS) gel electrophoresis and isolated from the cultured human lymphoblastoid cell line RPMI 4265, have been used to prepare antisera in rabbits. Antisera were raised against soluble products of the A (or 1st) series of HLA, bearing the determinant HLA-A2 (A2 substance), or against a mixture of products of the B (or 2nd) series of HLA, bearing the determinants HLA-B7 and HLA-B 12 (B7-12 substances). Rabbit antisera to A2 substance reacted primarily with A2 substance on Ouchterlony analysis, showing an apparent spur of cross-reactivity with B7-12 substances. Rabbit antisera to B7-12 substances reacted primarily with B7-12 substances, giving a spur of cross-reactivity with the A2 material. Neither antiserum precipitated β-₂microglobulin. Both types of sera reacted with membrane molecules of 43 000 mol.wt. and 11 000 mol.wt. by immune precipitation and gel electrophoresis in SDS of detergent-solubilized radiolabeled membranes from cultured cell lines, as predicted for sera directed towards HLA antigens. F(ab′)₂ fragments of the antibodies blocked the complement-mediated cytotoxicity of all HLA alloantisera tested for human peripheral blood lymphocytes. After absorption with B7-12 substances, F(ab′)₂ fragments of antisera to A2 substance only blocked the cytotoxicity of HLA alloantisera to A series specificities. After absorption with A2 substance, F(ab′)₂ fragments of rabbit antisera to B7-12 substances only blocked the cytotoxicity of HLA alloantisera to B series specificities. The results prove the existence of shared antigenic determinants between all members of the same series. These findings support the genetic evidence that A series HLA antigens are allelic products of a single locus, while B series HLA antigens are allelic products of a separate locus, by establishing some invariance of structure, presumably amino acid sequence, between members of the same series. The apparent cross-reactivity between A2 substance and B7-12 substances, and the ability of the unabsorbed F(ab′)₂, preparations to block the cytotoxicity of all HLA alloantisera, suggests that some determinants are common to both HLA loci. This may be considered to support the hypothesis that the two loci arose by gene duplication.     

Analysis of cell-surface markers with staphylococcal protein A: fluorescence studies on mammalian lymphoid determinants.

Membrane antigens including different classes of immunoglobulins, transplantation antigens, beta2-microglobulin, T lymphocyte specific antigens, and virally determined surface components were investigated using fluorescein-labeled Staphylococcal protein A in combination with cytofluorometric studies. Lymphocytes of seven species: mouse, rat, guinea pig, pig, cow, monkey, and human, and of ten human lymphoma-derived lines were tested. Analysis of the differential expression of surface markers revealed a reproducible reaction of protein A with cell-surface Fc of IgG actively produced by lymphoid cells from human, monkey, guinea pig, and pig, and with passively attached IgG molecules in the form of antibodies, directed against cell surface antigens of all lymphoid cells tested. No surface Ig was detected on so-called T lymphocytes. The distribution of cell-bound Ig density among surface Ig-positive cells was found to be different depending upon the origin of the cells with regard to lymphoid organ; it was parallel among the lymphoma lines tested and on peripheral blood cells from human, monkey, and pig, although large variations in fluorescence intensity among individual cells and among the different lines were recorded. Beta2-microglobulin determinants were found equally well on enriched human T and B cells. Transplantation, and T lymphocyte-specific antigens were detected on the majority of the lymphoid cells and on a restricted population respectively.     

Mouse mammary tumor virus and murine leukemia virus cell surface antigens on virus producer and nonproducer mammary epithelial tumor cells.

Mouse mammary tumor virus (MMTV)- and murine leukemia virus (MuLV)- specific cell surface antigens (CSA) on virus producer and nonproducer mammary epithelial tumor cells were studied using the techniques of lactoperoxidase catalyzed iodination of cell surface proteins followed by radioimmune precipitation with monospecific antisera to the major MMTV proteins gp52, gp36, p27, and p10 and to the major MuLV proteins gp70 and p30. The incorporation of iodinated CSA into extracellular virus was determined by analyzing labeled proteins in purified virus. On cells producing only MMTV both gp52 and gp70 were present on the cell surface. Furthermore, gp52 was the only labeled protein in extracellular MMTV produced by these cells. On cells producing both MMTV and MuLV, both gp52 and gp70 were present on the cell surface, and were the only labeled proteins present in their respective extracellular viruses indicating that gp70 and gp52 are present on mutually exclusive cellular viral budding sites. In addition, MuLV anti-p30 serum precipitated two iodinated proteins with molecular weights of 85,000 and 95,000 daltons, analogous to the Gross cell surface antigen (GCSA). Labeled gp52 and gp70 represent true CSA as demonstrated by the fact that they were also present on the surface of cells producing no virus, but producing large amounts of MMTV glycoproteins and nonglyco-proteins. These results further demonstrate that the precursor to the MMTV glycoproteins (gPr75-MMTV env) is cleaved prior to the appearance of gp52 on the cell surface.     

Characterization and expression of the HLA-DC antigens defined by anti-Leu 10

The expression of HLA-DC antigens on peripheral blood mononuclear cells, in tonsil and lymph node tissue sections, on tumor cell lines, and on activated T cells was studied using monoclonal antibody, anti-Leu 10. Anti-Leu 10 reacts with HLA-DC molecules on homozygous B cell lines expressing HLA-DR 1,2,4,5,6,8, and 9. It reacts with heterozygous B lymphocytes expressing DR7 and DRw10, suggesting it also recognizes HLA-DC molecules linked to DRw10. The HLA-DC molecules detected by anti-Leu 10 are expressed on all Ig-positive and DR-positive peripheral B lymphocytes and an apparent subpopulation of DR-positive periperal blood monocytes. Two-color immunofluorescence experiments using phycoerythrin-anti-HLA-DR (L243) and FITC-anti-Leu 10 demonstrated a correlation of the amounts of HLA-DR and DC antigens expressed on B lymphocytes. Cells expressing relatively low or high amounts of one Class II molecule express respectively low or high amounts of the other Class II molecule. Anti-Leu 10 reacted with all B lymphocyte derived tumor cell lines not with lines of myeloid or erythroid origin, and with only one T cell derived line, HUT-78 which has an activated T cell phenotype. Consistent with this result, anti-Leu 10 binding suggested the presence of HLA-DC on activated T cells in lymphoid tissue, in addition to staining B cells. HLA-DC was also detected on mitogen and MLC activated T cells by anti-Leu 10 binding. Anti-Leu 10 is, therefore, a useful reagent for further studies of the role of HLA-DC in T cell activation and in normal B cell and monocyte functions.     

A new epitope of the T200 molecule family defined by the 3A35 monoclonal antibody and expressed by macrophages and activated T lymphocytes

A monoclonal antibody (mAb), 3A35, produced against mouse macrophages (M phi) was found to react against certain activated T cells. This mAb, a rat IgM, resulted from a cell fusion between a mouse plasmacytoma and rat lymphocytes immunized against mouse M phi. It bound more avidly to activated than to resident M phi. It did not react against B cells and resting T lymphocytes but recognized certain dividing T cells like EL4 lymphoma, concanavalin A-activated and interleukin 2-expanded spleen cells, and helper T cell hybridomas. By contrast, other T lymphocyte-derived cell lines such as YAC-1 and CTLL2 were unreactive. No clear relationship was found between the binding of 3A35 to cells and the expression of L3T4 and Lyt-2 antigens. The specific stimulation of T cell clones with antigen rapidly induced a strong reactivity with 3A35 mAb which declined thereafter to a low (helper clones) or non-reactivity (cytotoxic clones) after 10 days of culture. Immunoprecipitation experiments, performed with M phi derived from bone marrow cell cultures, surface iodinated with 125I or metabolically labeled with [35S]methionine, showed that 3A35 bound to a 200-kDa molecule, shifting to 175 kDa under reducing conditions. In peritoneal M phi activated in vivo, in addition to the 175-kDa band, new bands migrating at 140, 120 and 85 kDa were identified by 3A35 and could be absorbed on a commercial anti-T200 mAb bound to Sepharose beads. After strengthening the cell binding of 3A35 to EL4 lymphoma cells by a cross-linking agent, only a 85-kDa molecule was immunoprecipitated. Thus, 3A35 identifies a new epitope of the T200 molecule family which is expressed on M phi and activated T cells.     

Antisera against leukaemia-associated antigens on human lymphocytes.

Antisera were raised in rabbits against leukaemic lymphosarcoma (LSL) cells which carried surface markers of both thymus-derived T lymphocytes (T cells) and bone marrow-derived B lymphocytes (B cells). After absorption with leucocytes, erythrocytes and serum proteins from normal individuals, the antisera demonstrated significant complement-dependent cytotoxicity against leukaemic cells from patients with acute lymphoblastic leukaemia (ALL) (9/11), LSL (7/9) and chronic lymphocytic leukaemia (CLL) (9/12), with an antibody titre of 1:64 or greater. The antisera did not react with: (a) blood lymphocytes from clinically healthy individuals (0/23), patients with ono-lymphoproliferative disorders (0/8) and normal umbilical cords (0/3), (b) normal lymphocytes stimulated by pokeweed mitogen (0/7), allogeneic lymphocytes (0/3), fetuin (0/1), purified protein derivative (PPD) (0/2), and candida antigen (0/1); (C) normal marrow cells (0/3), (D) normal thymocytes (0/2) and (E) leukaemic cells from patients with acute myeloblastic (AML) (0/10) and chronic granulocytic leukaemia (CGL) (0/3). However, the antisera did react with lymphoblastoid cells from continuous B-cell lines derived from an AML patient and from a non-leukaemic individual and, to a lesser extent, with lymphocytes from patients with infectious mononucleosis. The antisera also reacted with lymphocytes from chronically infected tonsils. Cytotoxicity of the antisera against lymphoblastoid and tonsillar cells was inhibited by ALL and CLL cell-lysates; and, conversely, cytotoxicity against ALL cells was inhibited by the lymphoblastoid cell extract. In contrast, a cell lysate or extract from normal inhibited by the lymphoblastoid cell extract. In contrast, a cell lysate or extract from normal lymphocytes did not inhibit cytotoxicity toward lymphoblastoid, tonsillar or ALL cells. Cytotoxicity of the antisera was neutralized by a goat anti-rabbit IgG (GAR IgG). These results suggest that the antisera contained antibodies reactive with antigens possibly common to neoplastic lymphocytes, tonsillar cells, lymphoblastoid cells and some lymphocytes from patients with infectious mononucleosis.     

Stimulation of lymphocytes by allogeneic lymphocytes and lymphoblasts in the presence of anti-HLA antisera.

Anti-HLA alloantisera inhibit mixed lymphocyte responses in which normal lymphocytes are used as stimulator cells. These same antisera are unable to inhibit lymphocyte proliferative responses stimulated by lymphoblastoid cells from cultured lymphoid cell lines. They also fail to inhibit either the generation of cytotoxic effector cells by lymphoblastoid cells or lymphocyte-mediated cytotoxicity against the lymphoblasts. Although the number of HLA antigens on the surface of lymphoblasts is reported to be greater than on normal lymphocytes, the failure of alloantisera to inhibit lymphoblast-induced responses in vitro does not appear to be due to insufficient amounts of antiserum to react with the antigenic sites. Rather, the data are interpreted to suggest that antigens which are not HLA and are not closely associated with HLA on the lymphocyte membrane are responsible for the stimulation of allogeneic lymphocytes by lymphoblastoid cells. Although lymphoid cell lines are known to contain the genome of the Epstein-Barr virus, antisera against products of the viral genome fail to inhibit proliferative responses to lymphoblastoid cells, suggesting that these antigens do not directly participate in lymphocyte activation.     

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.     

Identification and partial characterization of surface antigens specific for human normal and leukemic T cells

Rabbit antisera to membrane preparations of human thymocytes (anti-HTLA), T lymphocytes of the MOLT 4 cell line or B lymphocytes of the LIK cell line, have been serologically characterized and used for the identification of surface membrane antigens specifically expressed by human lymphocyte subpopulations. By immunoprecipitation of surface ¹²⁵I- or NaB (³H)₄-labeled protein followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis of the precipitates, the anti-HTLA antiserum was shown to detect on thymocytes three glycoproteins of apparent molecular weights 160000, 110000 and 45000. The first two only appeared to be sialylated. A similar pattern of antigens could be detected on cultured T lymphocytes, except for the 45 kDalton glycoprotein which could not be detected on the surface of these cells. None of these proteins were detected on ¹²⁵I-labeled Raji cells. The serological specificity of the anti-MOLT 4 antiserum for T lymphocytes was more restricted since it only precipitated the 160 kDalton antigen. In addition, the 45 kDalton protein was shown to be sensitive to trypsin treatment as was the sheep red blood cell (SRBC) receptor. The results shown suggest a possible activity as SRBC receptor for this particular protein. In contrast, the anti-LIK antiserum which behaves as an anti-HLA-DR reagent in cytotoxicity testing, only precipitates from cultured B lymphocyte lysates the typical Ia-like 28 and 33 kDalton polypeptides.     

Identification of A T lineage antigen in the catfish

A murine monoclonal antibody produced against catfish thymocytes and immunoglobulin-negative lymphocytes in the blood identified a catfish T cell antigen designated Cf T1. The Cf T1 antigen was found to be expressed on thymocytes, a subpopulation of the lymphoid cells in blood and other lympho-hemopoietic tissues, and a T cell line, but was not expressed by erythrocytes, thrombocytes, myeloid cells, B cells or macrophage cell lines. Stimulation of blood mononuclear cells with the T cell mitogen, concanavalin A, resulted in an increased frequency of Cf T1⁺ cells. Conversely, lipopolysaccharide stimulation increased the number of IgM⁺ B cells and decreased the frequency of Cf T1⁺ cells. The Cf T1 antigen was defined as a single chain protein of M_r 35 000 lacking N- and O-linked sugars. The Cf T1 molecule thus provides a T lineage-specific marker in this bony fish representative.     

Membrane characteristics of established human T- and B-cell lines. Cross-reactivity of human T-antigenic determinants with peripheral lymphocytes of non-human primates and the presence of MLC antigens on cultured T-cell lines.

Human T-cell lines (MOLT-4 and SOMMER-T) were injected into rabbits and monkeys (stumptail and squirrel monkeys). Rabbit anti-MOLT serum was absorbed with human liver and cultured B cells. Absorbed anti-MOLT serum was cytotoxic to lymphocytes of baboons and stumptail monkeys. Rabbit anti-MOLT and SOMMER-T sera after absorption with liver and B cell showed florescent ring formation in baboon and stumptail lymphocytes by using immunoflurescence techniques. On the other hand, antisera against MOLT and SOMMER-T cells in stumptail and squirrel monkeys were not only cytotoxic to MOLT and SOMMER-T cells, but also to other T-cell lines, CCRF-CEM and CCRF-HSB-2 cells. Cultured B-cell lines stimulated allogeneic and xenogeneic (rabbit and monkeys) lymphocytes far better than cultured T-cells lines did. T-cell lines, CCRF-HSB-2 and SOMMER-T (number 8402), gave small but significant stimulation to allogenic lymphocytes especially in the presence of foetal calf serum. MOLT-4B failed to stimulatie allogeneic lymphocytes. When lymphocytes of non-human primates and rabbit were cultured with human B- and T-cell lines in the presence of foetal calf serum, CCRF-HSB-2 and SOMMER-T cells stimulated xenogeneic lymphocytes of rabbit, squirrel monkey and stumptail monkey. MOLT-4 cells stimulated lymphocytes of baboon to some extent. These results indicate that although cultured B-cell lines had more mixed lyphocyte reaction (MLR) stimulating structure than T-cell lines, the latter still maintained some stimulating structure on the membrances.     

Detection of T and B cell-specific heteroantigens on electrophoretically separated lymphocytes of the mouse

Mouse spleen lymphocytes were electrophoretically separated into pools of T and B lymphocytes. Heteroantisera were raised in rabbits against these cell pools, the IgG fractions were isolated and their lymphocytotoxicity tested. After appropriate absorption, the antisera were specifically directed against lymphocyte antigens. Further cross-absorption with T and B lymphocytes made the antisera specific for antigens which were mutually exclusively present on T and B cells and were related to neither alloantigens nor immuno-globulins. These anti-B and anti-T cell antisera killed antibody-forming cells and graft-versus-host reactive cells, respectively, in vitro. Furthermore, anti-B cell antiserum was cytotoxic to lymphocytes of low electrophoretic mobility in bone marrow and peripheral organs, except in the thymus, whereas anti-T cell antiserum was cytotoxic to lymphocytes of high electrophoretic mobility in all organs and, in addition, killed all thymocytes of low electrophoretic mobility, which had not been affected by anti-B cell antiserum. This distribution of lymphocytes in the electrophoretic distribution profiles, together with the described properties, gives evidence that heteroantigens were found to be exclusively present on T and B cells. They were designated “mouse B cell-specific” and “mouse T cell-specific” antigens and are part of mouse lymphocyte-specific antigen.     

Relationship between mouse lymphocyte receptors for peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA)

The relationship between mouse lymphocyte receptors for peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA) has been investigated by immunofluorescence (cocapping) and radiolabeling. In neuraminidase-treated and untreated thymocytes there are two groups of glycoproteins which bind roughly equivalent amounts of PNA. One group also carries all the detectable receptors for HPA, the other binds only PNA. Binding inhibition experiments suggest that PNA and HPA receptors are in close proximity on the shared glycoproteins. The same two groups of receptors are present on 35–10% of neuraminidase-treated spleen lymphoid cells, mainly immunoglobulin (Ig)-negative lymphocytes. Almost all B cells have only PNA-specific receptors. Five–12% of the untreated spleen cells appreciably bind PNA and only a few bind HPA. Solubilized glycoproteins specific for PNA or HPA were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The major PNA-specific radioiodinated glycoproteins of neuraminidase-treated thymocytes, as isolated by affinity chromatography, consist of the 185-kDa and 195-kDa components of the T200 antigen and of two (diffuse) components of about 140 and 120–125 kDa. All these molecules also bind to HPA-Sepharose, with the exception of the 185 kDa component, which is probably the main constituent of the “pure” PNA receptors on the intact thymocytes. In gels directly labeled with radioactive lectins, the only band strongly labeled by PNA and HPA is the diffuse 140-kDa band. The band at 120 kDa is well labeled by PNA, but all the other components are weakly labeled. The mobility of the 140- and 120-kDa bands depends strongly on neuraminidase-treatment. These bands cannot be detected in gels of untreated thymocytes, but a major HPA-and PNA-specific band of lower molecular weight can be labeled after treating the gels with neuraminidase. The factors determining the differences in labeling pattern obtained by different methods as well as the nature of PNA and HPA binding sites are discussed. The same major PNA- and HPA-binding glycoproteins (apart from minor differences) are present on neuraminidase-treated Ig-negative spleen lymphocytes. The major PNA-binding protein of B lymphocytes appears to correspond to the 225-kDa (“B220”) antigen specific for these cells.