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1. A radioactive binding assay for Thy-1.1 alloantigen which functions in the presence of detergents was established by using glutaraldehyde-fixed thymocytes as target cells. Thy-1.1 activity in detergent extracts was then assayed by measuring inhibition of the binding assay. 2. Solubilization of Thy-1.1 from whole thymocytes, and their membranes by a large number of non-ionic detergents and deoxycholate was studied. In the same extracts Ag-B(4) histocompatibility antigenic activities were measured. With the exception of Nonidet P-40, the detergents did not affect the antigenicity of Thy-1.1, but only Lubrol-PX and deoxycholate gave effective solubilization as measured by activity remaining in the supernatant after centrifugation at 200000g for 40min. With Ag-B(4) antigen, Triton X-100, Triton X-67 and Nonidet P-40 gave effective solubilization as well as Lubrol-PX and deoxycholate. Solubilization of Thy-1.1 activity from leukaemia cells and a brain homogenate was also studied, but none of the non-ionic detergents gave satisfactory results with these tissues. 3. Extracts from thymocyte membranes were further examined by gel filtration and sucrose gradient centrifugation. The Thy-1.1 activity behaved as a single component in deoxycholate with a density similar to that of a globular protein, but in Lubrol-PX the antigen was contained in a low-density complex. In Lubrol-PX extracts Ag-B(4) was also found in aggregates not observed in deoxycholate. 4. The s20,w values for Thy-1.1 and Ag-B(4) antigens in deoxycholate were 2.4 and 4.4, and v̄ values were 0.70 and 0.75 respectively. The Stokes radius observed for Thy-1.1 was 3.1nm and for Ag-B(4) 5.3nm. By using these values the molecular weights for the antigen–detergent complexes were calculated to be 28000 for Thy-1.1 and 100000 for Ag-B(4).

Thy-1 antigen, present in large amounts on the brain and thymus of mice, exists in two allelic forms, either Thy-1.1 or Thy-1.2. Previous results have shown that Thy-1.1 alloantigen is expressed on a glycoprotein of molecular weight 25 000, therefore referred to as the Thy-1 glycoprotein. However the presence of Thy-1.2 alloantigen on the purified Thy-1 glycoprotein could not be established unequivocally. The present paper demonstrates that Thy-1.2 alloantigen, identified by F7D5 monoclonal antibody, is localized on the Thy-1 glycoprotein. The limited neutralization by the glycoprotein of the reactivity of AKR anti-C3H allosera to thymocytes is explained by the fact that the antiserum contains antibodies to determinants other than Thy-1.2. The alloantisera are shown to react with viral proteins encoded for by endogenous murine leukemia virus and expressed at the surface of Friend cells and thymocytes.

The present paper describes the characterization of the Thy-1 molecule from rat brain. The molecule was recognized by its antigens, which could be solubilized from brain membrane with deoxycholate. In the solubilized form the Thy-1 antigens were associated with a homogenous component with the following hydrodynamic properties: s20,w=2.2s,v=0.72ml/g and Stokes radius=3.0nm. The mol.wt. of the deoxycholateantigen complex was estimated to be 27000; these values are not significantly different from those obtained thymocyte Thy-1. Brain Thy-1 was further purified by affinity chromatography with lentil lectin coupled to Sepharose 4B, and more than 80% of the antigen was bound. The material eluted with methyl α-D-glucopyranoside was then filtered on a column of Sephadex G-200, and only one glycoprotein was found in the antigenically active fraction. On sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the glycoprotein was very similar to the Thy-1 from thymocytes that binds to lentil lectin. Its apparent mol.wt. on 12.5% acrylamide gels was 24000, and it electrophoresed as a symmetrical band. Brain Thy-1 was antigenically indistinguishable from thymocyte Thy-1 when analysed with rabbit antisera raised against brain or thymocyte Thy-1.

Three antigens similar in tissue distribution can be identified on rat thymocytes; the Thy-1.1 antigen, a rat specific xenoantigen, and a rat-mouse cross-reacting xenoantigen. To determine if these three antigens were on the same molecule their behavior in detergent-solubilized extracts from thymocytes was studied. Membrane fragments containing Thy-1.1 activity were prepared by a rapid method involving the use of Tween-40 detergent, and were solubilized in deoxycholate. The 150 000 × g supernatant from this extract contained aproximately 50 % of the original Thy-1.1 and xenoantigen activity. The supernatant was chromatographed on Sephadex G-200, and subjected to zone sedimentation on sucrose gradients in H2O and 2H2O to determine the hydrodynamic properties of the antigens. The three antigens migrated in identical fashion in all cases, and behaved as a molecule of 28 000 daltons molecular weight. When the antigenically active fraction, recovered after chromatography on Sephadex G-200, was passed through an immunoabsorbent consisting of rabbit antibody to one of the xenoantigens, all three antigens were equally depleted compared with passage through a control column. The results of these experiments suggested that Thy-1.1 antigen and the two xenoantigens were closely associated and most probably all on the Thy-1 molecule.

The asparagine-linked oligosaccharide structures of chronic lymphocytic leukemia cells (CLL) were studied by sequential lectin affinity chromatography. Glycopeptides were isolated from a Concanavalin A (ConA)-binding glycoprotein fraction prepared from a soluble CLL extract. This fraction, which contained 1.8% of the proteins present in the soluble extract, greater than 30 polypeptides revealed by SDS-PAGE analysis, and known cell surface antigens such as HLA-DR and p85 glycoprotein, must include a major proportion of the glycoproteins of the CLL cells. Glycopeptides were prepared by digestion with pronase, were separated into four pools (A-D) by Bio-Gel P-6 filtration and were radiolabeled by N-14C-acetylation. Glycopeptide pools C and D (0.45 less than Kd less than 0.77) were 95-100% bound to ConA-Sepharose and 7-12% bound to Lens culinaris (Lens)-Sepharose, but did not interact with any of the other lectins tested, suggesting major amounts of high mannose structures and minor amounts of fucosylated biantennary complex structures with terminal GlcNAc on the Man alpha 1-6 arm. Structures with greater than 3 branches were suggested for pools A and B (0 less than Kd less than 0.44) which were 34-65% unbound to ConA-Sepharose and 37-40% bound to Lens-Sepharose. Analysis of the ConA-unbound glycopeptides on RCA-Agarose before and after acid hydrolysis indicated variable amounts of terminal galactose and sialic acid residues. Major components of pool A were structures with terminal GlcNAc on all branches (22%) and fully sialylated structures (20%). In pool B, 20% of the radioactivity interacted with L-Phaseolus vulgaris agglutinin (PHA)-Agarose and with Ricinus communis (RCA)-Agarose, indicative of a fully galactosylated triantennary structure with branches at C-2 and C-6 of the Man alpha 1-6 arm. Half of the L-PHA-interacting material also bound to Lens-Sepharose, indicative of a core fucose residue. A fully galactosylated biantennary complex structure in pool A (13%) was identified by weak binding to ConA-Sepharose and strong interaction with RCA-Agarose. The presence of the polylactosamine sequence (Gal beta 1-4GlcNAc beta 1-3)n on this structure was suggested by a sialic acid independent interaction with wheat germ agglutinin (WGA)-Agarose. A sialic acid dependent WGA-interaction was observed in the ConA-unbound glycopeptides of pool A (5%). Some of the structures identified in this study may be associated with the malignant CLL phenotype and/or with a distinct stage of B cell differentiation.

Immunization of BALB/c mice with glycoproteins purified from a detergent extract of human chronic lymphocytic leukemia (CLL) cells by affinity to Lens culinaris lectin led to the production of several monoclonal antibodies with similar reactivity. One of the antibodies, 50B4, was purified and the corresponding antigen was isolated from a B-lymphoblastoid cell line extract by affinity chromatography to the 50B4-IgG immunoadsorbent. Co-purification of the antigenic activities associated with five other monoclonal antibodies was achieved. Purified and radiolabelled 50B4 antigen could be specifically immunoprecipitated not only by 50B4 but also by the other five antibodies. SDS-PAGE analysis revealed that all antibodies precipitated the same component, a polypeptide chain of apparent mol. wt 85,000 under reducing conditions. Competitive-binding studies between the purified antibodies indicated the presence of two distinct epitopes on the antigen. The epitopes, each recognized by three different antibodies, were equally accessible on the cell surface of either a B-CLL (3 X 10(5) molecules/cell), a B-lymphoblastoid cell line (11 X 10(5) molecules/cell) or two acute lymphocytic leukemia (ALL) cell lines of pre-B phenotype (5 X 10(5) and 0.8 X 10(5) molecules/cell respectively). Although the antigens purified from the strongly positive ALL cell line gave a gel pattern identical to that of the B-lymphoblastoid cell line, the antigens purified from the B-CLL extract were resolved into two distinct glycosylated polypeptides of mol. wts 85,000 and 77,000 under reducing conditions. The distribution of the antigen(s) is not restricted to cells of the B-lineage as mature T-cells and a variety of non-hematopoietic cell types express both epitopes of the antigen(s).

A human acute lymphoblastic leukemia (ALL) cell line that was transplanted into immune-deficient SCID mice proliferated in the hematopoietic tissues, invaded various organs, and led to the death of the mice. The distribution of leukemic cells in SCID mice was similar to the course of the disease in children. A-1 cells marked with a retrovirus vector showed clonal evolution after the transplant. SCID mice that were injected with bone marrow from three patients with non-T ALL had leukemic cells in their bone marrow and spleen. This in vivo model of human leukemia is an approach to understanding leukemic growth and progression and is a novel system for testing new treatment strategies.

The murine monoclonal antibody (mAb) CB43, raised against the K-562 erythroleukemia line, reacts with monocytes, tissue macrophages, thymocytes and with all the human lines tested but not with resting lymphocytes, large granular lymphocytes, granulocytes and erythrocytes. However, activated lymphocytes and natural killer cells express the CB43 antigen. Embryonic and fetal fibroblasts are positive, while adult fibroblasts are negative. Proximal convoluted tubules in kidney, epithelial cells in esophagus and breast, and sarcolemma in skeletal muscle are reactive with mAb CB43. This antibody can also bind to isolated guinea pig cardiac myocytes, and, furthermore, can induce a transient inotropic effect on isolated atria. The reactivity with different cell and tissue types and the functional effects of the CB43 mAb were reminescent of the 4F2/44D7 antibodies, shown previously to block Na/Ca2+ exchange in heart and skeletal muscle. Co-immunoprecipitation studies with CB43 and 44D7 mAb, using radiolabeled Daudi cells, revealed co-migration of polypeptides of 87 and 38 kDa. However, the epitope recognized by CB43 is not present on the human heavy chain which bears the 44D7/4F2 epitope as demonstrated by the lack of reactivity of CB43 mAb with mouse L cells transfected with the 4F2 heavy chain gene. Thus, CB43 represents a newly described epitope present on the human light chain or dependent on the conformation of the human dimer.

Monoclonal B-cell lines expressing antigen-specific surface IgM or IgD were established by transferring the genes encoding immunoglobulin heavy (mu or delta) and light (kappa) chains specific for the hapten 2,4,6-trinitrophenyl (TNP) into a B-cell lymphoma line. Two IgMTNP and two IgDTNP transformants were selected on the basis of similar levels of anti-TNP idiotype expression by flow microfluorimetric analysis. The IgMTNP and IgDTNP transformants were compared in quantitative assays for their ability to bind TNP-carrier and present TNP-carrier to carrier-specific T cells. Our results indicate that IgMTNP and IgDTNP transformants have an equal capacity to bind and present specific antigen. Thus surface IgM and IgD, when present in equivalent amounts, function similarly as antigen receptors.

Two novel acute lymphoblastic leukemia (ALL) cell lines, HOON and HYON, have been established from patients with non-T, non-B ALL. The cell lines have been characterized and shown to express phenotypic markers on non-T, non-B ALL. By indirect immunofluorescence and flow cytometry they express Ia and common ALL (CALLA) antigens and are reactive with monoclonal antibodies BA-1, BA-2 and OKT-9. However, the cells do not express detectable amounts of B1 antigen or of cytoplasmic mu chain, which are markers of pre-B cells. Quantitation of Ia and CALLA antigens on HOON and HYON cell lines using a cellular radioimmunoassay revealed that both cells bind high levels of anti-Ia antibodies, 110 X 10(4) molecules per cell, and low levels of anti-CALLA antibodies, 7 X 10(4) molecules per cell. Although both HOON and HYON carry equivalent amounts of Ia on their surface, only the former is a good stimulator of the one-way mixed lymphocyte reaction.

A monoclonal antibody 77.34, reactive with polymorphic HLA class II molecules, was produced. The allotype specificity of this IgG2a antibody was analyzed by cytotoxicity, flow cytometry, and cellular radioimmunoassay. Cytotoxic reactivity on a panel of B cells from 88 unrelated individuals was concordant with the MT1 (DC1) allospecificity (r = 0.83). Immunoanalysis by flow cytometry showed that cells from MT1 + homozygous cell lines were reactive, whereas MT2 + and MT3 + homozygous cells were not. A cellular radioimmunoassay performed under saturating conditions indicated that three MT1 + cell lines bound 14–45 × 10 5 molecules of antibody per cell representing 30–40% of the amount detected with monomorphic anti-DR monoclonal antibody 21w4. The subset of molecules bearing the MT1 allospecificity was purified with a 77.34 IgG immunoadsorbent. The purified molecules were antigenically reactive with several antibodies directed at DQw1 molecules but were devoid of reactivity to monomorphic anti-DR antibodies. Two-dimensional gel electrophoresis showed that the α subunit is composed of several acidic spots of Mr 32,000 whereas the β subunit was seen as a single spot of Mr 25,000, corresponding to DQw1 molecules. DR molecules purified by monoclonal antibody affinity were unreactive with 77.34 antibody. All of the 77.34 reactivity was observed with the fractions depleted of DR molecules. Two-dimensional gel analysis showed marked differences between the purified DR and DQw1 molecules. Two-dimensional gel analysis showed marked differences between the purified DR and DQw1 molecules. The presence of the MT1 determinant on Ia molecules referred to as the DQw1 molecules and distinct from those bearing the DR epitopes was confirmed on two DR1, MT1 homozygous cell lines. Thus, DQw1 molecules can be purified away from DR1 molecules by affinity chromatography to 77.34 IgG, specifically reactive with the MT1 (DQw1) allospecificity. The binding of 77.34 IgG to MT1 + cells was not inhibited by all monoclonal antibodies reported to be correlated with the MT1 allospecificity suggesting that the latter might be comprised of more than a single epitope.

The concentration of a glycoprotein reactive with monoclonal antibody 44D10 was studied during development and aging in 19 normal brains. Little change in the concentration of the antigen was found in white matter of brains ranging from the age of 1 to 54 years. However, a linear increase in the concentration of antigen was observed between the ages of 54 and 80 years. By the age of 80 years, the concentration of the glycoprotein had increased 2–3-fold. In contrast to white matter, gray matter did not contain detectable levels of antigen at ages from 1 to 54 years. Low levels of antigen were detected in brains of all older individuals examined. However, the relative concentration of this glycoprotein in gray matter was less than 5% of that in white matter. An examination of white matter homogenates from guinea pig and bovine brain showed that monoclonal antibody 44D10 was not reactive in these species.

The level of Ia antigens present on a murine B cell leukemia, passaged in BALB/c mice and designated BCL 1 , was measured in the cellular radioimmunoassay. Leukemic cells obtained from spleens of mice inoculated 4 weeks previously with the tumor were shown to express, on a per cell basis, 14 times more Ia than control BALB/c spleen cells. The wet weight of a leukemic spleen was approximately 18 times more than that of a control spleen. Several preparations yielded, on the average, 110 times more Ia per leukemic mouse spleen than per control spleen. This augmentation suggests that BCL 1 leukemic spleens are an abundant source for the purification of murine Ia antigens. Contrary to a previous report, I-A d antigens could be demonstrated on BCL 1 cells. I b cells, which do not express I-E antigens on their surface shared with BCL 1 cells Ia specificities recognized by A.TH anti-A.TL serum. Furthermore, a monoclonal antibody to Ia.15-like specificity of I-A subregion was reactive with BCL 1 cells. I-E d antigens, as reported previously, were also present on BCL 1 cells: 0.7 × 10 6 molecules of RAM-Fc were bound per BCL 1 cell incubated with either of two independently derived monoclonal antibodies to Ia.7-like specificity. Immunoprecipitation of 125 I-labelled Ia enriched fractions with either A.TH anti-A.TL serum or with monoclonal antibodies directed at I-A d or I-E d antigens yielded a major radioactive band of mol. wt 28,000 under non-reducing conditions and of mol. wt 34,000 under reducing conditions.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a variety of phenotypic changes on normal and malignant cells. In chronic and acute lymphocytic leukemia we have observed a marked augmentation in mixed lymphocyte reaction stimulatory capacity (MLRs) following pretreatment of leukemia blasts with TPA. We have now characterized the effects of TPA on MLRs utilizing the non-T, non-B lymphoblastic leukemia-cell line, HOON. Following pretreatment with TPA (optimal concentration, 1.6 X 10(-10) M), but not with other phorbol esters, there was a marked increase in MLRs, particularly at lower stimulator-cell concentrations. This effect was maximal following a 44-hr preincubation period and the enhancement in MLRs was not accompanied by changes in levels of Ia expression. This cell line provides a model for determining the molecular basis for the TPA-induced augmentation of stimulatory capacity in the mixed lymphocyte reaction.

We have previously shown that the In(Lu) gene down-regulates expression of an erythrocyte protein antigen identified by murine monoclonal antibody (MoAb) A3D8. In the present study we have examined In(Lu) Lu(a- b-) erythrocytes for expression of additional epitopes on the erythrocyte 80 kilodalton protein (p80) bearing the A3D8 antigen. Using a total of seven additional MoAbs that recognize three epitopes on erythrocyte p80, we have shown that In(Lu) Lu(a-b-) erythrocytes exhibit down-regulation of expression of all three epitopes. In(Lu) erythrocytes also showed a reduction in their reactivity to rabbit antibodies produced against purified p80 from either erythrocytes or lymphocytes. Furthermore the reactivity of the MoAbs was not altered by treatment of the cells with neuraminidase but was substantially reduced by treatment of cells with trypsin or chymotrypsin. The polyclonal anti- p80 sera were shown to react with a fragment of 50,000 daltons, still associated with erythrocyte ghosts, following treatment of the cells with trypsin or chymotrypsin. Treatment of erythrocytes with the thiol- reactive reagent AET decreased their reactivity with the MoAbs but had a variable effect on their reactivity with polyclonal antibodies. Erythrocyte p80 is a glycoprotein with N-linked oligosaccharides, as demonstrated by its binding to concanavalin A (Con A) and Len culinaris lectins. Following Endoglycosidase F treatment, erythrocyte p80 underwent a reduction in apparent mol wt of 11,000. The presence of a reduced amount of an intact p80 glycoprotein, seen by a decrease in reactivity with MoAbs directed at three distinct epitopes and with two different polyclonal antibodies, suggests that the In(Lu) gene interferes with expression by erythrocytes of the entire p80 glycoprotein.

Seven purified IgG monoclonal antibodies reactive with different epitopes on DQw1, DR, HLA-A3 or p85 glycoprotein of human lymphocytes have each been shown to inhibit pokeweed mitogen (PWM)-induced IgG and IgM secretion in a dose-dependent manner. Binding of these antibodies to their target antigens was required for the suppression. Antibodies of IgG1, IgG2 alpha, and IgG2b subclasses were able to inhibit both IgG and IgM secretion in the PWM system. The mechanisms by which two of the monoclonal antibodies (MoAbs)-77.34, specific for the class II antigen DQw1, and GAP A3, specific for the class I antigen HLA-A3-caused inhibition--were analyzed. The suppressive effects of 77.34 and GAP A3 were maximal when added at the initiation of the culture period. No inhibition of IL-2 production or cellular proliferation was detected. Supernatants obtained from inhibited cultures were not themselves suppressive. The F(ab')2 fragments of either 77.34 or GAP A3 failed to influence PWM-Ig secretion, indicating that intact IgG molecules were required. This suggests that the observed inhibition might be mediated via Fc receptors. Together F(ab')2 fragments of either 77.34 or GAP A3 and a control IgG2a protein did not reconstitute the inhibitory effects of intact 77.34 or GAP A3. Suppression, therefore, required intact Fc portions on the same IgG molecules as those that bound to DQw1 or HLA-A3. These studies suggest that populations of IgG molecules that crosslink sufficient numbers of Fc receptors with other cell surface antigens on peripheral blood mononuclear cells (PBMs) during the early stages of B-cell activation can inhibit Ig secretion. Crosslinking of B-cell Fc receptors with SIg has been proposed by others to act as a negative signal for Ig production; our data raise the possibility that crosslinking of FcR with B-cell plasma membrane components other than SIg can also suppress Ig secretion.

Publisher Summary This chapter explores the θ antigen, which is designated as Thy-1 antigen. The two alleles of the Thy-1 gene are referred to as Thy-1a and Thy-1b. The product encoded by the Thy-1a allele is the Thy-l.1 alloantigen (θ-AKR) and the product of the Thy-1b allele is the Thy-l.2 alloantigen (θ-C3H). It discusses the distribution of Thy-l.l and Thy-l.2 alloantigens in different murine strains. The chapter reviews that congenic lines differing at the Thy-1 locus have been developed: A- Thy-1a and A.AL-old carrying the Thy-1a allele on A background and B10.PL carrying the Thy-1a allele on the B10 background. Such congenic lines have established the Thy-1.1 and Thy-l.2 allospecificities of allosera and of monoclonal antibodies. It discusses that Thy-1.1 and Thy-l.2 antigens are present in large amounts in brain and thymus of mice but in smaller amounts on thymus-derived peripheral T cells. Lymph node, spleen, and bone marrow cell suspension have anti-Thy-1 absorptive capacities of 14, 7, and 1% relative to thymocytes. It also reviews that Thy-1 is a general T cell marker only in the murine species. The appearance of Thy-1 on mouse lymphocytes is generally associated with maturation in the thymus. Thymectomized and nude mice show a marked reduction in the number of Thy-1 bearing cells in lymph nodes and spleen. In thymic nude mice, however, immunocompetent cells expressing low levels of Thy-1 are present.

Publisher Summary This chapter reviews the Thy-1 antigens, which have been identified in mice, rats, humans, and dogs. Their tissue distribution varies among species. Thy-1 antigens are abundant in adult brain and thymus of rat and mouse. It is also present on mouse peripheral T cells and constitutes the T cell marker of that species. The chapter explores that the existence of antibodies to species cross-reactive determinants led to the identification of the canine and human homologs of rat Thy-l. Purification and characterization of the Thy-1 antigens of thymus and brain have been achieved primarily in the rat. The purification of Thy-1 molecules is monitored by a cellular radioimmunoassay using glutaraldehyde-fixed target cells. It also discusses the amount of Thy-1 antigenic activity, which is present at different stages of the isolation procedure and is measured by inhibition of the binding of anti- Thy-1 antibody to glutaraldehyde-fixed mouse or rat thymocytes.

The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.

Monoclonal antibodies, 21w4 and 44H10, against human MHC class II determinants, were analysed for their reactivity with rabbit lymphoid cells. Both MAb bind to all human B lymphoblastoid lines, irrespective of their HLA-DR phenotype. HLA-DR antigens, purified by affinity to 21W4 IgG Sepharose, can be precipitated with 44H10 MAb, indicating that both antibodies react with the same molecules. Competitive inhibition studies with purified MAb IgG show that 44H10 and 21w4 recognize different epitopes of HLA-DR molecules. The 21w4 MAb, previously shown to cross-react with cells of pig, mouse and sheep, does not react with rabbit lymphoid cells. The 44H10 MAb binds to lymphoid cell suspensions prepared from rabbit appendix, spleen and mesenteric lymph nodes. Its reactivity correlates with that of RABELA, a polyclonal antibody specific for rabbit B cells. Mesenteric lymph node and spleen cell suspensions, depleted of sIg + cells, are devoid of 44H10 + cells, while similarly-treated appendix B cells still contain a subset of B cells which are slg − , RABELA + and 44H10 + . These studies thus report on the presence of MHC class II determinants on rabbit B cells, cross-reacting with human HLA-DR.

The exchange of Na+ for Ca2+ across the plasma membrane is mediated by a carrier transport system known as the Na+–Ca2+ exchanger. We have recently reported the specific inhibition of Na+–Ca2+ exchanger activity in cardiac and skeletal muscle sarcolemmal vesicles by monoclonal antibody 44D7. In this review, we summarize the properties of the 44D7 monoclonal antibody and the antigenic complex reacting with this antibody. The 44D7 antibody was produced against human acute lymphocytic cells and recognizes a molecular complex composed of two subunits of the apparent molecular weights 95 000 and 38 000, linked by disulfide bonds. Two other monoclonal antibodies react with the same complex: 4F2 which binds to the same epitope as 44D7 and specifically inhibits the Na+–Ca2+ exchanger activity, and 44H7 which reacts with a distinct epitope and does not inhibit exchanger activity. The 44D7 antibody reacts with nerve fibers in brain and proximal convoluted tubules of kidney, both known to possess Na+–Ca2+ exchanger activity. Reactivity of 44D7 antibody with tonsil and thymus sections is restricted to certain subpopulations of cells. The reactivity of the antibody is very weak with resting lymphocytes in suspension; however, activated T lymphocytes and leukemic cells show increased binding to 44D7 antibody. Several malignant cell lines express high levels of the 44D7 antigen. The reactivity of a human hepatoma with 44D7 antibody is much greater than that observed with normal hepatocytes. The inhibition by monoclonal antibody 44D7 of the Na+–Ca2+ exchanger activity and the similarity in tissue distribution of the 44D7 antigenic complex and the exchanger system suggests that these two molecules might be related. Demonstration of a direct correlation between these two entities will require the isolation of a molecule expressing both antigenic and exchanger activities.

Spleen cells from RNC-nu/nu and RNC-nu/+ mice were separated into populations either enriched or depleted for cells bearing Fc-receptors. These populations were tested for the presence of Thy-1 surface antigen by a sensitive cellular radioimmunoassay and for the capacity to respond in the one-way mixed leukocyte reaction. The population depleted of Fc-receptorbearing cells was found to contain the majority of the cells with Thy-1 surface antigen and cells from this population also gave the highest responses in the mixed leukocyte reaction. Thus, we conclude that the nu/nu spleen cells that respond in the mixed leukocyte reaction are Fc-receptor negative and likely carry Thy-1 surface antigen. These cells may be identical with certain classes of helper T cells or with pre-T cells.

The ability of the tumor promotor 12–0-tetradecanoylphorbol 13-acetate (TPA) to induce differentiation of leukemic cells was studied in 10 cases of chronic lymphocytic leukemia (CLL). An increase in modal volume and an enhancement of the capacity of te leukemic cells to stimulate in mixed lymphocyte reaction (MLR) was seen in the majority of cases. A significant increase in Ia expression was observed upon culture of leukemic cells with TPA in 6 of the 10 cases; 5 of these cases also showed an induction of cytoplasmic IgM production. Correlations between the phenotypic markers of the leukemic cells and their ability to respond to TPA were evaluated. CLL cells with low amounts to surface Ig. a volume less than or equal to 165 fl. and relatively low la expression responded well to TPA. Cells with bright surface Ig. a volume greater than or equal to 178 fl. and elevated amounts of Ia responded poorly to TPA. These results suggest that differences in the response of B leukemic cells to TPA reflect the underlying heterogeneity of the leukemic cells and might be correlated with their stage of maturation.

Monoclonal antibodies 44D7 and 4F2 inhibited specifically the Na+-dependent Ca2+ fluxes characteristic of the Na+/Ca2+ exchanger in cardiac and skeletal muscle sarcolemmal vesicles. Preincubation of membrane vesicles with monoclonal antibody 44D7 inhibited 90% of the Na+-dependent Ca2+ uptake measured in the first 10 s of the reaction and 50% of that measured after 60 s. Ca2+/calmodulin-dependent ATPase activity and ATP-dependent Ca2+ uptake by sarcolemmal vesicles were not affected by monoclonal antibody 44D7 whereas the Na+-dependent release of accumulated Ca2+ was inhibited. In the presence of the 44D7 antigen isolated from human kidney, monoclonal antibody 44D7 could no longer inhibit Na+-dependent Ca2+ fluxes. The distribution of 4F2 antigenic activity in the isolated muscle membrane fractions correlated with that of Na+/Ca2+ exchanger activity; cardiac and skeletal muscle sarcolemmal vesicles expressed higher levels of the antigen than skeletal muscle transverse tubule membrane, while no antigen could be detected in sarcoplasmic reticulum membranes. Our results suggest that monoclonal antibodies 44D7 and 4F2 interact either directly with the Na+/Ca2+ exchanger molecules or with some other protein(s) responsible for the regulation of this activity in the heart and skeletal muscle.

The levels of a glycoprotein reactive with monoclonal antibody (MAb) 44D10 in white and gray matter from brains of victims of several neurological diseases, including Multiple Sclerosis, Alzheimer's, Parkinson's and Huntington's diseases, were compared to that of normal individuals. The concentration of antigen reactive with MAb 44D10 was elevated in both gray and white matter of all MS brains examined, but not in brains with other neurological diseases. The increase in the concentration of antigen varied amongst the MS brains, such that the levels of antigen were only slightly increased in 2 of the 6 MS brains whereas 2 to 4 fold higher levels were found in the other 4 brains. Increased levels of antigen were detected in gray matter of MS brains, whereas this antigen was either not detected or present in very low levels in gray matter homogenates prepared from age-matched normal brains. MAb Leu 1, which reacts with T lymphocytes, was not absorbed by normal and MS brain tissue suggesting the increase in antigen reactive with MAb 44D10 in MS brain homogenates was not associated with non-specific infiltration by T lymphocytes. Comparison of the purified antigen from MS gray matter and normal white matter by gel electrophoresis demonstrated that MAb 44D10 was reacting with a similar protein in both tissues with an apparent molecular weight of 80K. We have named this molecule P-80 glycoprotein.

A.TH anti-A.TL alloserum has previously been shown to react with monomorphic determinants of human Ia molecules. In an attempt to produce monoclonal antibodies detecting human-mouse cross-reacting epitopes, A.TH mice (I s ) which lack I-E antigens were immunized with human Ia glycoproteins. Five hybridoma lines were established which recognize monomorphic human Ia determinants. Only one of the lines, 21w4, is reactive with murine cells and also with pig and sheep cells but not with rat cells. Binding studies to murine target cells show a strain distribution analogous to that of an Ia.7-like specificity. The binding of 21w4 antibody to human and murine cells was compared to that of two other monoclonal antibodies directed to the murine Ia.7-like specificity. Although all three antibodies had a similar pattern of reactivity with cells of different mouse strains, only 21w4 bound to the human cells tested. Competitive inhibition studies on murine cells have revealed that the epitopes were spatially related but not identical. Our results support the view that Ia.7, the putative species cross-reacting specificity, might be composed of clusters of epitopes with variable degrees of conservation throughout evolution.

We have used antigen-specific human T-cell clones to study the relationship between MHC and antigen recognition specificities expressed by T cells. Tetanus toxoid (TT)-specific T-lymphocyte clones were derived from a immunized HLA-DR2,7 heterozygous donor by limiting dilution from peripheral blood mononuclear cells (PBM) restimulated with TT in vitro. Clones were screened for MHC-restricted antigen recognition against antigen-presenting cells (APC) from a panel of HLA-typed donors, using an in vitro T-cell proliferation assay. Several distinct patterns of antigen recognition were identified. In addition to T cells that recognized TT in association with donor class II MHC antigens, we found clones that simultaneously expressed self-restricted antigen recognition and alloreactivity, and clones with specificity for antigen in the context of MHC antigens not expressed by the T-cell donor. This was confirmed in inhibition studies using well-characterized monoclonal antibodies against class II MHC antigens to block specific proliferative responses. We propose a possible structure for the determinant recognized by two of the clones. These results suggest that the T-cell antigen receptor undergoes random or antigen-dependent changes in vitro, and that this may be a mechanism for somatic diversification of the T-cell repertoire.

The number of Ia molecules accessible at the surface of a cell could be related to the stage of differentiation and to the functional status of that cell. We have measured the level of Ia expressed on various normal and leukemic human lymphocytes using monoclonal antibody 21w4 which recognizes a non-polymorphic epitope of human Ia (1,2). Our results with ALL and CLL do not support a correlation between stage of differentiation and level of Ia. Furthermore PHA-activated T cells and tonsillar B cells express the highest amounts of Ia.

We have previously isolated a cDNA clone encoding the common acute lymphoblastic leukemia antigen (CALLA) from a normal human kidney library. Analysis of the amino acid sequence deduced from nucleotide sequencing of this cDNA established that CALLA is identical to the recently cloned human neutral endopeptidase (NEP; E.C. 3.4.24.11). Southern blot analysis of SacI fragments of DNA from human—rodent somatic cell hybrids using a 1.6-kb CALLA cDNA probe showed that the CALLA-NEP gene is located on human chromosome 3.

Publisher Summary This chapter discusses glycoprotein antigens of murine lymphocytes. It discusses the techniques presently utilized to identify and quantitate cell surface antigens and summarizes the biochemical data available on H-2 and Ia, the antigens of the major histocompatibility (MHC) gene complex, and on the T-cell differentiation antigens, Thy-1, Tla, and Ly-2,3. To remain brief, the chapter is restricted to murine antigen that is partially characterized biochemically. The nature of the antigens coded for by the MHC has also been investigated various species, particularly in humans and guinea pigs; from preliminary biochemical data currently available, it seems that both transplantation antigens and Ia-like antigens show interspecies homology. Evidence accumulated so far for H-2, Thy-1, and Ia favors the localization of antigenic determinants in the polypeptide portion of these molecules. The evidence is, however, not conclusive and does not exclude the possibility that the carbohydrate portion of these molecules is important in certain immunological mechanisms in which the antigens participate. The biochemical and functional characterization of murine glycoprotein antigens clarify the relationship between the structure of membrane proteins and their immunological significance.

Monoclonal antibodies 50B4 and 50E6 recognize two distinct epitopes of human p85 glycoprotein (CDw44). Both epitopes are destroyed by reduction of the purified gycoprotein as demonstrated by inhibition of cellular radioimmunoassay and Western blot analysis. Endoglycosidase F treated p85 glycoprotein, with an apparent molecular weight of 73,000 is still reactive with both monoclonal antibodies. Thus both epitopes are conformational determinats of the polypeptide chain. A rabbit antibody produced against purified native p85 glycoprotein also reacted only with the non-reduced form of p85. Repeated immunizations with SDS-dissociated and reduced p85 yielded a polyclonal antibody reactive by Western blot analysis with reduced and non-reduced forms of p85 glycoprotein. When a HOON leukemia cell line cDNA expression library was screened with this polyclonal antibody, two cDNA clones were isolated which reacted specifically with the antiserum and not with the control non-immune serum. Preliminary characterization of these clones indicates that they are p85-related.

The Ia antigens and the common acute lymphoblastic leukemia antigens (CALLA) accessible on the cell surface were quantitated in newly diagnosed patients with non-T, non-B ALL using monoclonal antibodies and a cellular radioimmunoassay. The levels of both antigens expressed in molecules of RAM-Fc bound per cell exhibited a wide range of variation amongst patients. Ia levels, measured with monoclonal antibody 21w4 which recognizes a monomorphic epitope of human Ia molecules, were between 5.0 X 10(4) and 87 X 10(4) molecules per cell in 37 patients. CALLA levels measured with BA-3 or J-5 antibody varied from 3.4 X 10(4) to 22 X 10(4) molecules per cell in 13 patients. In 12/13 cases for which Ia and CALLA were quantitated simultaneously, the amount of Ia was found to be higher than that of CALLA. A positive correlation (p less than 0.02) between the levels of these two antigens was observed suggesting that ALL cells with the highest levels of Ia also had the highest levels of CALLA. In addition, our results suggest a possible correlation (0.05 less than p less than 0.1) between the amount of Ia expressed on the leukemic cells and the white blood cell count of the patient at the time of diagnosis. The data indicate that non-T, non-B ALL are heterogeneous with respect to their expression of Ia and CALLA antigens. A longitudinal study of non-T, non-B ALL patients will allow us to assess if the levels of Ia and CALLA at diagnosis are correlated with prognosis of the disease in these patients.

Monoclonal antibodies 50D6 and 2Ir5, reactive with human class II molecules, were analyzed quantitatively by flow cytometry and cellular radioimmunoassay for their binding to cells of different HLA-DR types. Monoclonal antibody 50D6 bound equally to cells of all DR types tested except DR7, where no reactivity was observed. Monoclonal antibody 2Ir5 was reactive with all cells. However, the percentage of DR molecules at the cell surface expressing 2Ir5 epitope varied with the DR type and increased as follows: DR3 = DR7 < DR2 < DR5 < DR4 < DR1. MAb 50D6 reacted with an epitope spatially related to but distinct from the 2lw4 epitope present on all DR molecules. The 50D6 epitope was shown to be present on isolated DR1 molecules.

Erythrocyte membrane antigens have been detected on induced Friend erythroleukemic cells with a rabbit antiserum raised against mouse erythrocyte membranes. The antibody specificities of this antiserum have been quantitatively analyzed using a cellular radioimmunoassay. After absorption with thymocytes, the rabbit anti-erythrocyte membrane serum bound to dimethylsulfoxide (DMSO)-induced Friend erythroleukemic cells and to mouse erythrocytes but not to uninduced Friend cells or thymocytes. Reciprocal inhibition studies demonstrated that, following complete thymocyte absorption, the antiserum detected similar antigenic specificities, termed erythrocyte membrane antigens (EMA), on both mature erythrocytes and induced Friend cells. The expression of these erythrocyte membrane antigens was also induced on Friend cells by other agents, such as ouabain and dimethylacetamide (DMA). In contrast, exogenous hematin, which did not induce hemoglobin synthesis in the Friend cell clones used in this study, also did not induce erythrocyte membrane antigen expression. Two independently derived variant clones which do not produce hemoglobin in reponse to DMSO were analyzed for their ability to produce erythrocyte membrane antigens in response to various inducers of Friend cell differentiation. Clone TG-13 is not inducible by DMSO or hematin but is weakly induced by DMA for both hemoglobin production and erythrocyte membrane antigen expression. Another variant clone, M18, was also analyzed. This clone does not synthesize detectable hemoglobin when grown in either DMSO or hematin alone, but undergoes extensive hemoglobin synthesis when grown in medium containing both DMSO and hematin. M18 does, however, express erythrocyte membrane antigens when grown in DMSO alone: the presence of hematin and DMSO together in the growth medium does not enhance expression of these antigens. Thus M18 appears to be defective for hemoglobin inducibility, and this defect can be overcome by exogenous hematin; however, the expression of erythrocyte membrane antigens is not affected by this block in hemoglobin synthesis. The results with the variant clones are discussed in terms of a program for Friend cell differentiation in which the induction of hemoglobin synthesis and erythrocyte membrane antigen expression are under both co-ordinate and separate controls.

A glycoprotein of apparent mol. wt 85,000 isolated from human cells of B lineage by affinity to 50B4-IgG immunoadsorbent was shown previously to express two spatially distinct epitopes identified with monoclonal antibodies 50B4 and 50E6 [Letarte et al. (1985) Molec. Immun. 22, 113-124]. It is now demonstrated that the p85 glycoprotein is homologous to the F10-44-2 antigen, defined initially as a T-lymphocyte-granulocyte-brain antigen [Dalchau et al. (1980) Eur. J. Immun. 10, 745-749], the A1G3 human medullary thymocyte antigen [Haynes et al. (1983) J. Immun. 131, 1195-1200] and the A3D8 antigen defined on erythrocytes [Telen et al. (1983) J. clin. Invest. 71, 1878-1886]. The 50B4 antigen purified either from a B lymphoblastoid cell line or a leukemic cell line, at a concn ranging from 0.25 to 2.0 micrograms protein/ml, could block the binding of either F10-44-2 and F-10-62-1 (a related antibody) or A1G3 and A3D8 antibodies to human leukemic cells. All of these antibodies immunoprecipitated, from the purified antigenic preparation, a single glycosylated polypeptide chain of apparent mol. wt 85,000. Competitive binding studies indicated that these antibodies define at least three epitopes. The F10-44-2 and A3D8 antibodies blocked the reactivity of monoclonal 50E6-IgG with human leukemic cells and thus bind to an epitope identical or spatially related to the 50E6 epitope. Antibody F10-62-1 competed for the binding of 50B4-IgG to leukemic cells and thus recognizes the 50B4-like epitope. The A1G3 antibody blocked 30% of the binding of 50E6-IgG but did not inhibit the binding of 50B4-IgG: it is thus reacting with a third epitope on the p85 glycoprotein distinct from but close to the 50E6 epitope.

• Available data suggest that ionized calcium may interact with a cell surface "sensor" or "receptor" to produce changes in one or more intracellular second messengers that ultimately regulate the release of parathyroid hormone (PTH). Recently, we developed a series of monoclonal antibodies directed toward specialized differentiation antigens expressed on endocrine cells. Since many of these monoclonal antibodies displayed exquisite specificity for cell surface molecules on the parathyroid cell, we used these reagents as probes to investigate signal recognition/transduction mechanisms associated with abnormal calcium-regulated PTH secretion. Depending on their binding site on the respective target antigen molecules, these monoclonal antibodies either stimulated or inhibited hormone secretion. Thus, defects in membraneassociated structures may contribute to deranged calciumregulated PTH secretion in abnormal parathyroid cells. ( Arch Surg 1987;122:436-442)

Endoglin is an homodimeric membrane antigen with capacity to bind transforming growth factor-beta (TGF-beta) and whose expression is up-regulated on myeloid cells upon differentiation to macrophages. We have isolated full-length cDNA clones from a lambda gt 10 library, prepared from phorbol 12-myristate 13-acetate-differentiated HL60 cells by screening with an endoglin-specific cDNA probe from endothelial cells. Sequencing of the largest clone (3073 bp), revealed that the leader sequence contains 25 residues and that the 586 amino acids of the extracellular and transmembrane domains were identical to those described for endothelial endoglin. However, the cytoplasmic tail encoded by this cDNA clone contains only 14 amino acids as opposed to the 47 residues previously reported, suggesting the existence of two alternative endoglin variants. The expression of these isoforms was demonstrated by polymerase chain reaction analyses on endothelial cells, myelomonocytic cell lines HL-60 and U-937, and placenta. Independent cDNA constructs corresponding to both forms were transfected into mouse fibroblasts leading to the expression of two distinct endoglin molecules. Both forms were shown to bind TGF-beta 1 and, when overexpressed in transfected mouse fibroblasts, to form disulfide-linked homodimers, indicating that the cysteine residues present in the extracellular domain are responsible for the dimerization.

Endoglin is an endothelial homodimeric membrane antigen containing the tripeptide arginine-glycine-aspartic acid (RGD), which is a recognition motif for adhesion receptors of the integrin family. We have investigated the expression of endoglin by monocyte/macrophage cells from different tissue compartments and at different stages of cell differentiation. Although endoglin is absent from peripheral blood monocytes, it is expressed by in vitro differentiated monocytes as determined by flow cytometry using the endoglin-specific monoclonal antibody 44G4 and 8E11. Furthermore, Northern blot analyses revealed a correlation between the presence of endoglin mRNA and the surface expression of the antigen by in vitro differentiated monocytes. Immunostaining of frozen tissue sections with the 8E11 monoclonal antibody demonstrated the presence of endoglin not only in the endothelium of all the tissues studied, but also on the interstitial macrophages present in the red pulp of the spleen. Using as a model of macrophage differentiation monocytic cell lines treated with phorbol esters, we found that the reactivity of the 8E11 monoclonal antibody is greatly increased on U-937 and HL-60 cells during their PMA-induced differentiation. These findings clearly demonstrate for the first time the regulated expression of the putative adhesion molecule endoglin by macrophages.

Hereditary haemorrhagic telangiectasia (HHT) is a genetic vascular disorder characterised by epistaxis, telangiectases, and visceral manifestations. The two known disease types, HHT1 and HHT2, are caused by mutations in the endoglin (ENG) and ALK-1 genes, respectively. A higher frequency of pulmonary arteriovenous malformations (AVMs) has been reported for HHT1 while HHT2 is thought to be associated with a lower penetrance and milder disease manifestations. In this study, we present 10 families with an ALK-1 genotype. Visceral manifestations were detected in 24 (26%) of the 93 HHT2 patients from nine of the families and included gastrointestinal bleeding (14%), intrahepatic shunts (6%), and AVMs in the lung (4%) and brain (3%). Gastrointestinal bleeding, the most frequent visceral manifestation, was reported in six of the 10 families, mostly in patients over the age of 50. These patients also had frequent epistaxis and suffered from anaemia, often requiring blood transfusions. The identification of ALK-1 mutations in subjects with a suspected diagnosis and without clinical signs of HHT argue in favour of a molecular diagnosis. We also analysed the data published on 44 families with HHT2 and conclude that visceral manifestations occur in 26 of these families and affect 30% of HHT2 patients. This is considered an underestimate given incomplete and variable screening for lung, brain, and/or liver involvement in different clinical centres. These findings, however, stress the need for an early diagnosis of HHT that can be useful for the early control of associated visceral involvement.