Biochemical analysis of human T lymphocyte differentiation antigens T4 and T5.

Two major functionally distinct T cell subsets in man have been defined with heteroantiserums and monoclonal antibodies directed against stable cell surface antigens that appear during thymic ontogeny. A monoclonal antibody to T4 antigen (anti-T4) is reactive with the peripheral inducer T cell population while a monoclonal antibody to T5 antigen (anti-T5) is reactive with the cytotoxic and suppressor population. Immunoprecipitation and electrophoresis on sodium dodecyl sulfate polyacrylamide gel were used to show that on human thymocytes or peripheral T cells the T4 antigen is a single 62,000-dalton glycoprotein while the T5 antigen is a complex of two glycoproteins, one being 30,000 daltons and the other 32,000 daltons. Similar glycoproteins have been isolated with antibodies to murine Lyt 1 and Lyt 2,3 antigens. Both the antigens defining the phenotypes of inducer and suppressor populations in man and mouse are structurally homologous.

Endoglin is expressed on a subpopulation of immature erythroid cells of normal human bone marrow.

A monoclonal antibody (1G2) was raised by immunization of a Balb/c mouse with the leukemic blasts from a patient suffering from chronic myelogenous leukemia blast crisis (CML-BC). Sequential immunoprecipitation of the protein from human umbilical vein endothelial cells with 1G2 and the endoglin-specific monoclonal antibody 44G4 indicated that both antibodies react with the same molecule, a homodimer of molecular mass 180,000. This protein was first identified on acute lymphoblastic leukemia and was shown to be primarily associated with endothelial cells. In addition, 1G2 and 44G4 identified the same subpopulation of human bone marrow mononuclear cells (BMMNC), as established by two colour immunofluorescence analysis. By cell sorting and colony assays it could be demonstrated that endoglin is not expressed on hemopoietic precursor cells (CFU-G, CFU-GM, CFU-GEMM, BFU-E). May-Grünwald-Giemsa staining of sorted BMMNC revealed that 1G2 recognized immature proerythroblasts and double-fluorescence analysis showed that endoglin is present on a subset of glycophorin A-positive BMMNC. 1G2 was not reactive on bone marrow B-cells (CD19, CD20), T-cells (CD3, CD7), natural killer cells (CD56), myeloid cells (CD13, CD14, CD15, CD33), and on CD34-positive cells. Endoglin contains an arginine-glycine-aspartic acid sequence, a feature generally associated with extracellular matrix proteins which interact with integrins. It is suggested that proerythroblasts may utilize endoglin to interact with integrins in cell-cell adhesion events in the stromal or hemopoietic compartment of the bone marrow.

Identification of a previously unrecognized polypeptide associated with lymphocyte function associated antigen one (LFA-1).

In lymphocyte function associated antigen one (LFA-1) preparations from metabolically labeled lymphocytes we have observed a new polypeptide component of 86-kilodalton additional to the already described alpha- and beta-chains. This chain is cosynthesized with the alpha- and beta-chains and can be covalently cross-linked with them, resulting in a three-chain complex. This complex is recognized by the H35-89.9 anti-LFA-1 monoclonal antibody. Cleveland peptide mapping analysis indicates that the new chain is structurally different from the alpha- and beta-chains of the LFA-1 complex. The chain has been observed in B-cells as well as in T-cells. Labeling properties of the 86-kilodalton chain suggest that this molecule is not exposed on the membrane.

Transformation of LMTK- cells with purified HLA class I genes--IV. A determinant on beta 2-microglobulin is controlled by HLA heavy chain in a mouse-human hybrid complex: a biochemical analysis.

Transformation of LMTK- murine fibroblast cells with purified HLA class I heavy chain genes resulted in the expression of serologically detectable HLA-A3 molecules. Surprisingly, such cells also react with a murine monoclonal antibody specific for a serological determinant notably not expressed by murine but by human beta 2-microglobulin. The human HLA molecules expressed by the transformed cells were characterized on two-dimensional gels. The heavy chain was shown to be associated with a murine beta 2-microglobulin molecule, which could be distinguished from human beta 2-microglobulin by its higher isoelectric point. This heterodimer molecule was immunoprecipitated with the mouse anti-human beta 2-microglobulin monoclonal antibody showing that indeed the complex of mouse beta 2-microglobulin and human heavy chain expresses a human beta 2-microglobulin determinant.

A rapid method for isolation of antigenically active human cell surface antigens associated with beta 2-microglobulin using a monoclonal antibody.

Serologically and biochemically pure HLA antigens and other beta 2-microglobulin-associated proteins were isolated using a rapid immunoprecipitation technique with a monoclonal anti-beta 2-microglobulin reagent A88. Elution of the immunoprecipitate with beta 2-microglobulin isolated from kidney patients proved to be efficient. Analysis of the eluate showed that the antigen preparation does not contain any immunoglobulins, retains antigenic activity and can be used for protein micelle formation. This method of preparation of human beta 2-microglobulin-associated membrane antigens has several advantages over classical immunoadsorbent techniques.

How should CD34+ cells be analysed? A study of three classes of antibody and five leucocyte preparation procedures.

For patients undergoing stem cell transplantation after intensive marrow ablative therapy it is important to enumerate the CD34+ stem cells in peripheral blood so that the harvest can be timed in order to maximize the number of cells collected by leucophoresis for subsequent haematopoietic reconstitution. The use of rapid flow cytometric techniques for the determination CD34+ leucocyte numbers has been advocated, although there is no consensus as to the best method. In this study, we have examined the effects of preparation procedures for flow cytometry on the binding of four CD34 antibodies (Immu-133, QBEND-10, HPCA2 and BIRMA-K3) to the three classes of epitopes on leucocytes. Whole blood, bone marrow and leucophoresis samples were analysed either directly after labelling with a vital nuclear dye (LDS-751) and fluorochrome-conjugated antibodies or after additional erythrocyte lysis and leucocyte fixation using four commercially available reagents (Q-Prep, OptiLyse B, OptiLyse C and FACS Lysing Solution). By comparison with the results obtained from viable leucocytes in unmanipulated samples, it was found that the binding of all four antibodies could be affected by lysis and fixation procedures and that the binding of the class I antibody Immu-133 was most markedly decreased. We conclude that CD34+ cells are best analysed using a whole blood procedure in which nucleated cells are identified by their side light scatter and the fluorescence associated with a vital nuclear dye (in this instance LDS-751) and the CD34+ cells are detected with fluorescein isothiocyanate- or phycoerythrin-conjugated antibodies.

Clonal origin of the Philadelphia translocation in chronic myeloid leukemia demonstrated in somatic cell hybrids using an adenylate kinase-1 polymorphism.

Hybrid cell lines were derived from fusion of rodent cells with leukocytes from a t(9q+; 22q-)-positive chronic myeloid leukemia (CML) patient carrying a chromosome No. 9-linked adenylate kinase-1 (AK1) polymorphism (AK1 1-2). The AK1*2 allele was consistently expressed when 9q+ was present, whereas the AK1*1-coded isozyme was formed when the normal chromosome No. 9 was present. These results provide additional data confirming the clonal origin of the Ph1 translocation in CML.

Is the chromosomal region 9q34 always involved in variants of the Ph1 translocation?

Six variants of the Ph1 translocation are described. The clinical diagnoses were chronic myeloid leukemia (CML) in 5 cases (patients 1-5) and acute lymphocytic leukemia (ALL) in patient 6. Three Ph1 variants were clear complex translocations, involving chromosomes #9, #22, and a third chromosome, i.e., #16, #11, or #14. The other three Ph1 variants appeared as "simple" translocations between chromosome #22 and chromosome #19, #4, or #12 when G- or Q-banding were used. When studied with high resolution R-banding, a small deletion of the terminal part of one chromosome #9 was visible, strongly suggesting that these variants were also complex translocations, i.e., t(9;19;22)(q34;p13;q11),t(4;9;22) (p16;q34;q11), and t(9;12;22)(q34;p13;q11). In the latter two cases, using in situ hybridization techniques, we demonstrated the presence of c-abl sequences on the Ph1 chromosome. This proved the involvement of 9q34 in these two variants. Our proposal is that most, and probably all, variants of Ph1 are complex translocations involving part of 9q34 and that the conjunction of a specific region of 22q11 with a specific segment of 9q34 (carrying the c-abl protooncogene) is essential for the development of Ph1 + CML.

Translocation of c-abl to "masked" Ph in chronic myeloid leukemia.

In two patients with chronic myeloid leukemia (CML), the nature of the chromosomal rearrangement giving rise to "masked" Ph has been studied by in situ hybridization of human c-abl sequences. The c-abl probes hybridized to the 22q11 region of the "masked" Ph, demonstrating that translocation of sequences from 9q34 to the Ph did occur exactly as in standard Ph or in other types of variants previously studied. These results provide additional evidence for the occurrence of a constant molecular rearrangement in Ph-positive CML.

Structural analysis of differentiation antigens Mo1 and Mo2 on human monocytes.

Mo1 and Mo2 are membrane differentiation antigens expressed by human monocytes, and in the case of Mo1, by granulocytes and null cells. Mo1 antigen expression is resistant to protease treatment of intact monocytes while Mo2 is protease sensitive; Mo2 antigenic activity is regenerated by trypsin-treated monocytes during culture under conditions that favor protein synthesis. Further biochemical analysis reveals that Mo1 is associated with a glycoprotein consisting of two subunits of 94,000 and 155,000 MW. Mo2 resides on a glycoprotein of 55,000 MW. Immunoprecipitation of Mo1- and Mo2-bearing structures from lysates of internally labeled cells indicates that these proteins are synthesized by human monocytes.

The monoclonal antibody 11G7 recognizes a novel differentiation antigen expressed on hemopoietic precursor cells.

A monoclonal antibody (11G7) detecting a novel antigen on human hemopoietic progenitor cells (named 11G7R = 11G7 receptor) was raised by immunization of a Balb/c mouse with the leukemic blasts of a patient suffering from chronic myelogenous leukemia blast crisis (CML-BC). The antigen is expressed on most of MHC class II bearing peripheral blood leucocytes (PBL) and on a subpopulation of bone marrow mononuclear cells (BMMNC). By FACS-sorting and colony assays, it could be demonstrated that 11G7R is expressed on myelo-monocytic and myelo-granulocytic bone marrow precursor cells (GM-CFC, G-CFC, M-CFC) but is absent from erythroid precursor cells (BFU-E) and on cells exhibiting the capacity to form mixed colonies (GEMM-CFC). Double-fluorescence analysis on BMMNC revealed that 11G7R is expressed on a subset of B-cells, myeloid cells and cells carrying the HPCA-1 antigen (CD34). It has a similar distribution pattern to the myeloid antigens CD13 and CD33. However, in contrast to these antigens, 11G7R is also expressed on the blasts of several lymphoid leukemias (4/9 B-ALL, 1/2 T-ALL) and therefore it is not restricted to the myeloid lineage.

Lymphocyte function-associated antigens one (LFA-1) on B and on T lymphocytes bind a monoclonal antibody with different affinities.

The kinetics of the binding of an anti-LFA-1 monoclonal antibody to lymphocytes of the B and T lineages was studied. A Kd approximately ten times lower was observed when binding the antibody to B splenocytes compared to T splenocytes. Using Fab fragments and measuring the dissociation rate constants gave a similar difference in binding kinetics of anti-LFA-1 between the different splenocytes. This difference of cell-binding affinity was independent of the state of stimulation of the cells by lipopolysaccharide or concanavalin A. Thymocytes reacted with the antibody with the same Kd as T splenocytes. The combined results indicate that LFA-1 from B and from T lymphocytes have a different conformation. Binding with a higher affinity to B and a lower affinity to T lymphocytes could also be demonstrated using liposomes coated with anti-LFA-1 antibody.

Further biochemical characterization of the human thymocyte differentiation antigen T6.

Charge heterogeneity of the human thymocyte antigen T6 was studied by isoelectric focusing and two-dimensional gel electrophoresis. The larger subunit of T6 (a 49,000 m.w. glycoprotein) contained several oligosaccharide side chains bearing up to 12 terminal sialic acids. When T6 antigens from 19 individual thymuses were analyzed, no differences in the isoelectric focusing patterns of the larger subunit could be detected. The larger subunit of the T6 antigen from MOLT-4 cells (52,000 m.w.) contained an extra oligosaccharide if compared with the T6 antigen from thymus or three other T leukemic cell lines. Two types of small subunits of T6 were found. In addition to a protein of m.w. 12,000, pI 5.5 identified as beta 2-microglobulin, a (m.w. 12,000, pI 7.0) nonglycosylated protein, was detected on two dimensional gels. This protein does not cross-react with beta 2-microglobulin, and its amount varied in different T6 preparations. The tissue distribution, the m.w. the association with beta 2-microglobulin, and the limited structural heterogeneity of T6 support the idea that T6 is the human homologue of the murine thymus leukemia antigen (TL).

Isolation and characterization of the acidic phosphoproteins of 60-S ribosomes from Artemia salina and rat liver.

Eucaryotic L7/L12-type proteins are present in ethanol/salt extracts (P1 protein) of ribosomes from Artemia salina and rat liver. These proteins are partially phosphorylated and occur in two forms of closely related structure: a major form eL12 having methionine at the N-terminal position and a minor form of eL12 (eL12') which seems slightly elongated and contains a blocked N terminus. Purification of the four different forms of this protein, eL12, eL12-P, eL12' and eL12'-P, was performed by ion-exchange chromatography on carboxymethyl-cellulose and DEAE-cellulose. Using a radioimmuno assay, 1.8 copies of eL12 and 0.9 of eL12' were found on the 80-S A. salina ribosome. In ribosomes of both rat liver and A. salina, eL12 is present in a larger quantity than eL12'. 40-S and 60-S ribosomal subunits extracted with ethanol/salt were essentially free of eL12 proteins. A large pool of eL12 was found in the cytosol after removal of the ribosomes by centrifugation or molecular sieving. The proteins of rat liver and A. salina are similar with regard to their isoelectric points and molecular weights. Sedimentation equilibrium studies indicated that the isolated protein eL12 occurs as a dimer.

Characterization of T cell surface glycoproteins T 1 and T 3 present on all human peripheral T lymphocytes and functionally mature thymocytes.

The monoclonal antibodies, anti-T1 and anti-T3, both react with all human peripheral thymus-derived lymphocytes and with 10% of thymocytes; each, however, recognizes different cell surface structures. It was determined that the target antigen of anti-T is a 69 000 molecular weight cell surface glycoprotein and that the T3 antigen is a 19 000 mol. wt. glycoprotein.

Structural characteristics of the mouse transferrin receptor.

Rat monoclonal antibodies against mouse transferrin receptor have been used to isolate and characterize the mouse receptor molecule. The molecule is a dimeric glycoprotein of Mr 200 000 resembling its human homolog of Mr 190 000. Receptor molecules prepared from different lymphoid cell populations show structural differences which can be explained by variations in the carbohydrate moiety of the molecule. Both the antibody-binding site and the transferrin-binding site are located on tryptic fragments of Mr 80 000 on the extracellular part of the molecule. After trypsin treatment, these fragments are partially retained at the cell surface, probably non-covalently bound to one intact receptor subunit, but they are released at higher trypsin concentrations. The soluble fragments retain their ability to bind transferrin and appear to exist as dimers. In this fragment, there are no disulfide bonds present. Disulfide bonds are located near the plasma membrane. Studies using a cleavable cross-linker indicated the presence of cross-linking sites at the intramembranous or the cytoplasmic part of the molecule.

Biochemical studies of the human thymocyte cell-surface antigens T6, T9 and T10.

Three human thymic cell-surface antigens T6, T9 and T10, previously defined by monoclonal antibodies, were analyzed using immunoprecipitation techniques. The antigen T6 was found to be a 49,000 dalton glycoprotein, which is associated with beta 2-microglobulin, the small subunit (12,000 daltons) of the HLA-A, -B, and -C antigens. The target antigen for the monoclonal reagent anti-T9 was found to be a glycoprotein of 94,000 daltons, which appears as a disulfide-linked dimer of 190,000 daltons on the cell surface. The antigen precipitated by the anti-T10 antibody is a 45,000 dalton glycoprotein. We present preliminary evidence that all three cell-surface proteins may be integral membrane proteins. These findings, in addition to the distribution patterns, suggest that the T6 antigen is the human homolog of the murine thymus leukemia (TL) antigen.