Line HPB-ALL Research Articles

Recent duplication of the two human CD8 beta-chain genes.

Isolation of cDNA clones encoding the beta-chain of the human T cell surface glycoprotein CD8 revealed the presence of five distinct forms of cDNA resulting from alternative splicing. In the process of analysis of the gene organization, we found that there exist two recently duplicated genes for CD8 beta. These genes, designated CD8 beta 1 and beta 2, consist of nine and seven exons, respectively. The organization of CD8 beta 1 and beta 2 genes is almost identical except in their 3'-ends. There are nine nucleotide differences between the coding regions of the CD8 beta 1 and beta 2 genes in spite of the extremely high similarity of these genes which extends over the entire genes including introns. Pulse field gel analysis demonstrated that CD8 beta 1 and beta 2 genes are located more than 1.5 Mb apart. It was found that the CD8 beta 1 gene is approximately 25 kb upstream from the CD8 alpha gene in the same transcriptional orientation on chromosome 2. Although both CD8 beta 1 and beta 2 genes appear functional from the nucleotide sequence, the five distinct forms of CD8 beta cDNAs and corresponding mRNAs found in thymus, PBL, and leukemic cell line HPB-ALL are all derived by alternative splicing from CD8 beta 1 transcripts.

Intracellular catabolism of radiolabeled anti-CD3 antibodies by leukemic T cells

The endocytosis and intracellular metabolism of radiolabeled anti-CD3 MoAb 64.1 by the malignant human T cell line HPB-ALL were studied using biochemical, morphological, electrophoretic, and Chromatographic techniques. Biosynthetically labeled [ 3H]64.1 and externally radioiodinated 125I-64.1 were similarly internalized and degraded by tumor cells, with approximately 70% of the initially bound radioactivity being released to the culture supernatant as trichloroacetic acid-soluble radioactivity in the first 24 hr of culture. Radiolabeled 64.1 was routed from the cell membrane to endosomes where initial proteolysis began and finally to lysosomes where terminal catabolism to single amino acids occurred. SDS-PAGE demonstrated four major intracellular metabolite species (46, 25, 15, and <10 kDa). Thin-layer chromatography demonstrated that >95% of the trichloroacetic acid-soluble radioactivity in culture supernatants was 125I-monoiodotyrosine, indicating that proteases, not deiodinases, were of primary importance in catabolism of 125I-64.1. In the presence of inhibitors of lysosomal function (leupeptin, monensin, and ammonium chloride), 125I-64.1 degradation was impeded, causing prolonged retention of radioactivity in the lysosomal compartment of cells. However, although the pace of catabolism was markedly diminished by these agents, no major changes in the sizes of intermediate metabolites generated were observed. Our results suggest that judicious administration of lysosomal inhibitors (e.g., chloroquine, verapamil, monensin) may significantly enhance retention of radioimmunoconjugates by lymphoid malignancies, improving radioimmunoscintigraphic and radioimmunotherapeutic efforts.

Biochemical identification of a direct physical interaction between the CD4: p56lck and Ti(TcR)/CD3 complexes

The CD4 and CD8 antigens function in synergy with the TcR/CD3 complex in the generation of intracellular signals leading to T cell proliferation. The association of the protein-tyrosine kinase p56lck with CD4 and CD8 provides a potential mechanism in the generation of intracellular signals. Several studies have shown that CD4 can co-modulate with TcR/CD3 suggesting that these receptor complexes may associated on the surface of the T cell. Nevertheless, it has proven difficult to formally demonstrate a direct physical interaction between the CD4 and TcR/CD3 complexes using biochemical techniques. In this study, we have used the sensitivity of the in vitro kinase assay to show a direct physical linkage between the CD4:p56lck complex and various CD3 subunits. Immunoprecipitation of CD4 from cell lysates derived from the T lymphoblastoid line HPB-ALL results in the co-purification of p56lck with an additional polypeptide at 20 kDa. Re-precipitation analysis and isoelectric focusing demonstrated that this band corresponds to the CD3 epsilon chain. An alternative approach which involves the labeling of microsomal membranes with [gamma-32P]ATP revealed the presence of CD3 epsilon and zeta chains in anti-CD4 immunoprecipitates. By contrast, we were unable to demonstrate the association of the CD4:p56lck and TcR/CD3 complex in resting peripheral blood lymphocytes. These data indicate that the CD4:p56lck and TcR/CD3 complexes have the ability to form stable complexes on the surface of certain T cell lines.

T-cell receptor Vβ5 usage defines reactivity to a human T-cell receptor monoclonal antibody

The monoclonal antibody 3D6 reacts with the T-cell receptor (Tcr) on the T-leukemic line HPBALL and with 2-13% of human peripheral blood T lymphocytes. In this study V alpha and V beta expression in a panel of T-cell populations and clones expressing the 3D6 epitope was determined by Southern and northern hybridization analysis. The results demonstrate that these 3D6-positive T cells, regardless of CD4/CD8 phenotype or function, express a gene of the V beta 5 family, also expressed by HPBALL. No correlation was found between 3D6 reactivity and expression of the HPBALL V alpha gene. The results demonstrate that 3D6 recognizes an epitope solely on the Tcr beta chain and that the use of this beta chain, together with an appropriate V alpha, can impart a diverse pattern of reactivity to a T cell.

Mechanism of mononuclear cell activation by an anti-CD43 (sialophorin) agonistic antibody.

CD43 (sialophorin, gpL115) is a sialoglycoprotein expressed on a wide variety of blood cells including lymphocytes, monocytes, neutrophils, and platelets. L10, an anti-CD43 mAb, has been shown to induce monocyte-dependent activation and proliferation of human T lymphocytes. We have studied the signaling mechanism involved in this activation process. Treatment of PBMC and purified populations of T cells and monocytes with L10 induced the hydrolysis of phosphoinositides with the resultant generation of the phosphoinositide-derived second messengers diacylglycerol and inositol phosphates. This was associated with the translocation of protein kinase C from cytosol to membrane fractions and an increase in free intracellular Ca2+ in treated cells. In human leukemic T cell lines, the magnitude of signaling via CD43 did not correlate with the density of the TCR/CD3 surface expression nor with the intensity of signaling via the TCR/CD3. Moreover, a mutant derived from the leukemic T cell line HPB-ALL that was severely defective in TCR/CD3 surface expression and signaling nevertheless had normal CD43 surface expression and signaling compared with the parent cell line. It is concluded that CD43 is functionally coupled to the phospholipase C/phosphoinositides signaling pathway. In human T cells, signaling via CD43 proceeds independently of TCR/CD3. The widespread expression of CD43 suggests a potentially important role for this molecule in orchestrating the activation of multiple cell types.

Constitutive endocytosis of HLA class I antigens requires a specific portion of the intracytoplasmic tail that shares structural features with other endocytosed molecules.

HLA class I antigens present in the human leukemia T-cell line HPB-ALL are shown to be endocytosed in the absence of specific antibodies. In 1 hr, approximately 10% of class I molecules initially present at the cell surface are found intracellularly. Genetically engineered mutants of the HLA-A2 gene lacking exon 6 or 7 or both were used to analyze whether the cytoplasmic region contributes to the internalization. The results indicate that amino acids encoded by exon 7 (spanning amino acid residues 323-340) are required for internalization, while deletion of exon 6 had no effect. In addition, a comparison of the cytoplasmic sequences of receptors that are known to be internalized via coated pits and the present data revealed that they share a structural feature that could constitute a specific signal required for endocytosis.

A solubilized T-cell receptor from a human leukemia cell line binds to a ligand in the absence of MHC products

A human T cell alpha beta antigen receptor from the acute lymphoblastoid leukemia line HPB-ALL (also called HPB-MLT) binds and is precipitated in detergent-solubilized form by an antigen present on the surface and secreted by several strains of the gram-positive bacterium Staphylococcus aureus. This binding is completely independent of major histocompatibility complex (MHC) antigens. Receptor/ligand binding is unique to this one cell line (i.e., clonotypic) and furthermore completely blocked by an idiotype-specific monoclonal antibody (mAb) to this receptor, but not by three different nonidiotype-specific mAbs. The nature of this interaction appears more similar to immunoglobulin/antigen binding than to T-cell receptor/antigen/MHC/accessory molecule interactions and would suggest that some T-cell receptors may not require MHC products to interact with antigen.

The biosynthesis and assembly of T cell receptor alpha- and beta-chains with the CD3 complex.

The biosynthesis, processing, and assembly of the TCR alpha- and beta-chains with each other and with the CD3 complex were investigated on both cell surface positive (TCR+CD3-) and negative (TCR-CD3-) cell lines. The results indicate that 1) in cell surface TCR-CD3- cell lines (MOLT 3, CCRF-CEM), TCR-beta, but not alpha-chains are present intracellularly. TCR-beta-CD3 complexes are readily found in these cell lines, but no evidence for final processing or cell surface expression of such incomplete TCR-CD3 complexes is observed. 2) In the cell surface TCR+CD3+ cell line HPB-ALL, both alpha- and beta-chains are present intracellularly. Whereas non-glycosylated forms of TCR-beta chain can be detected, only more mature forms of TCR alpha-chains are detected indicating that the alpha-chains are more rapidly glycosylated than the beta-chains. 3) The large majority of the intracellular alpha- and beta-chains is not disulfide linked and a small fraction of these is associated with CD3. 4) Only small amounts of the total intracellular TCR chains are found as CD3-associated disulfide-linked alpha beta-heterodimers. 5) Final processing of TCR chains for cell surface expression takes place after formation of these TCR-alpha beta-CD3 complexes. Thus, both the TCR alpha- and beta-chains are over-produced and only relatively small amounts of these chains form CD3-associated heterodimers that are processed for cell surface expression. Analogous results were obtained with a non-leukemic CTL clone. Based on these observations, a model for the biosynthesis and assembly of the TCR-CD3 complex is presented.

Complexity of T cell receptor recognition sites for defined alloantigens.

Three monoclonal antibodies react with the T cell receptor on the tumor line HPB-ALL and in addition with 3 to 13% of human peripheral blood T cells of normal donors. These antibodies are shown to react with an epitope encoded by the V beta 5 family of T cell receptor beta-chain variable region gene segments. Cells expressing V beta 5 gene segments can have cytotoxic or helper function, be of the T4+ or T8+ phenotype, and have specificity for either class I or class II major histocompatibility complex alloantigens. Seven T cell clones were generated, which express V beta 5 and are specific for the HLA-A2 molecule. With the use of these clones, we illustrate how isoelectric focusing can be used to analyze T cell receptor alpha- and beta-chain structure. The seven clones recognize five distinct conformational determinants on HLA-A2. They procure different binding sites by the use of different alpha-chains, J beta sequences, or both.

Isolation of anti-idiotypic antibodies to T cells using an anti-framework determinant antibody

A panel of anti-idiotypic antibodies to the T cell line HPB-ALL was produced by screening with a novel enzyme-linked immunoadsorption assay (ELISA). Using the beta framework I (βF1) monoclonal antibody directed at a common determinant on the human T cell receptor β subunit, we were able to specifically capture the receptor molecule from a cell lysate preparation and use this as the basis of an ELISA assay. Hybridoma supernatants were tested for their ability to bind to the receptor thus captured. A total of four antibodies were isolated by this method, and they were shown to immunoprecipitate a disulfide-linked heterodimer composed of alpha (49 kDa) and beta (40 kDa) subunits from HPB-ALL cells, similar to the subunits recognized by the βF1 antibody. Furthermore, all four antibodies blocked the binding of T40/25, an anti-idiotype to HPB-ALL. Three of these antibodies blocked the binding of anti-Leu 4 to a similar degree as did T40/25, while one did not. This suggests that these new anti-idiotypic antibodies recognize distinct but associated idiotypic determinants. The isolation of such antibodies for any particular T cell line or tumor promises to be useful for biological studies of T cell malignancy in humans.

A family of T-cell receptor molecules expressed on T-cell clones with different specificities for allomajor histocompatibility antigens

A monoclonal antibody, 3D6, identifies a public idiotope or allotope on the human T-cell receptor for antigen, since it not only reacts with the tumor line HPB-ALL, against which it has been raised, but also with 3–13% of peripheral blood T lymphocytes of normal donors. 3D6 + cells have been isolated from an allogeneic mixed lymphocyte culture and cloned by limiting dilution. In this way, allospecific clones were obtained both of the T4 + T8 − and the T4 − T8 + phenotype, which included proliferative as well as cytotoxic cells. Within a panel of 20 cytotoxic clones, different specificities for both class I and class II MHC antigens were found. The clones were tested for their reactivities with four additional anti-T-cell receptor antibodies raised against HPB-ALL. Two of these, 1C1 and 1C2, reacted with all 3D6 + clones. By means of two other antibodies, 2D4 and 65, the 3D6 + receptor family could be divided into four structurally distinct subfamilies. Biochemical analysis suggested that the 1C1, 1C2, 2D4, and 3D6 antibodies define epitopes on the beta chain of the receptor. Isoelectric focusing of receptor molecules isolated from cytotoxic clones with different specificites indicated that there are extensive structural differences in both alpha and beta chains of the receptors. No correlation could be found between the antigenic specificity of a clone and the structure of its receptor in this analysis. It is postulated that the 1C1, 1C2, and 3D6 epitopes may be encoded by a particular germline V beta segment, in analogy with similar, previously described findings in both the human and the murine system.

Properties of a panel of monoclonal antibodies which react with the human T cell antigen receptor on the leukemic line HPB-ALL and a subset of normal peripheral blood T lymphocytes.

Five Mab raised against the T cell antigen receptor of the human T cell line HPB-ALL which react with a subpopulation of normal peripheral blood T cells are described. Three Mab, 3D6, 1C1, and 1C2, react with 3 to 5% of normal PBL and stimulate proliferation of the cells with which they react. An increase in the number of cells which react with all five Mab occurs. Two Mab, 2D4 and 65, react with subsets of the cells which bind 1C1, 1C2, and 3D6 and divide the family into four subgroups, 2D4+ 65+, 2D4+ 65-, 2D4- 65+, and 2D4- 65-. Functional T cell clones in all four subfamilies have been observed. Cytolytic function can be correlated with the TcR phenotype expressed because all of the Mab which react with a particular clone inhibit its ability to lyse a specific target. The epitopes recognized by the panel are closely related because all five block each other's binding to HPB-ALL. In addition, the determinants recognized by 3D6, 1C1, and 1C2 on normal lymphocytes are probably very closely related because all clones examined react with all three Mab.

Secretion of HLA-A and -B antigens via an alternative RNA splicing pathway.

Human class I major histocompatibility antigens (HLA-A, -B and -C) are integral membrane protein heterodimers, which are anchored in the membrane via a stretch of hydrophobic amino acids near the carboxyl terminus of the heavy chain. It has previously been shown that a mutagenized cell line secretes a water soluble form of the HLA-A2 antigen, due to a pattern of RNA splicing that removes exon 5 (encoding the transmembrane hydrophobic amino acids) from mature, HLA-A2--encoding transcripts. The present study was undertaken to assess whether a similar process might be operative in nonmutagenized cells. It is shown that water soluble class I molecules (primarily HLA-A24) are secreted by the T leukemic cell line HPB-ALL, and that alternative splicing removes exon 5 from a fraction of HLA-A24--encoding transcripts. It is further shown that class I molecules are secreted, possibly in an allele-specific fashion, from a variety of tumor cells and normal cells. The possible relationship between these findings and previous reports of HLA-A and -B antigens in human serum is discussed.

Human T cell lines differing in phenotype and specificity are reactive with the same anti-idiotypic antibody.

3D6, a monoclonal antibody selected for reactivity with the T cell antigen receptor on the T leukemic cell line HPB-ALL, was found to react with 3 to 13% of peripheral blood T lymphocytes of 10 out of 15 normal donors. Peripheral T cells of two donors were stimulated with allogeneic cells, and the 3D6+ cells were enriched by rosetting 3D6-coated cells with goat anti-mouse-coupled human red blood cells and were expanded in interleukin 2-containing medium. In this way, 90 to 100% 3D6+ cell lines were obtained that were cytotoxic for the allogeneic stimulator cells. 3D6 antibody could block antigen-specific cytotoxicity, as well as induce nonspecific cytotoxicity toward target cells that could not be killed in the absence of the 3D6 antibody. The 3D6+ cell populations contained T4+, as well as T8+ cells, indicating that 3D6 antibody defined a T cell receptor population that might harbor various antigenic specificities. One 3D6+ cell line was separated into T4+ T8- and T4- T8+ populations. 3D6 reactive T cell receptors isolated from HPB-ALL and normal cell lines were analyzed biochemically by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and V8 protease peptide mapping. Isoelectric focusing analysis provided additional evidence for the idea that 3D6 antibody detected a number of structurally distinct T cell receptors, because the T cell receptor alpha-chain was homogeneous in charge after desialation on the clonal tumor line HPB-ALL, but remained heterogeneous in charge on the 3D6+ normal cell lines. No great differences in charge were found between T cell receptors isolated from T4+ and T8+ 3D6+ lines, but their isoelectric focusing patterns were not identical. V8 protease peptide mapping revealed structural differences between the T cell receptor alpha-chain isolated from HPB-ALL on one hand and from the normal 3D6+ lines on the other, whereas the beta-chains did not differ greatly in primary structure according to this analysis. In addition, the peptide mapping suggested differences in primary structure between T cell receptors present on the T4+ population vs those present on the T8+ populations.

T cell clones which share T cell receptor epitopes differ in phenotype, function and specificity

Recently, we described a monoclonal antibody (3D6) that reacts with the T cell receptor (Ti) of the T leukemic cell line HPB-ALL and that cross-reacts with 2-10% of the T cells of normal healthy individuals. In this study we report the establishment of T cell clones that are 3D6+ but that differ in function and phenotype. These clones were established according to two different protocols: T cells of donor HY (10% 3D6+) were stimulated with the Epstein-Barr virus-transformed cell line JY. The proliferating 3D6+ T cells were enriched using a rosetting technique and cloned. T cells of donor HY were stimulated with the 3D6 antibody and subsequently expanded in recombinant interleukin 2-containing medium. This yielded 70% 3D6+ T cells which after activation with either Daudi cells or with TT in the presence of autologous non-T cells, followed by cloning, resulted in antigen-specific 3D6+ T cell clones. The 3D6+ T cell clones were also tested on their reactivity with 4 other monoclonal antibodies (1C1, 1C2, 2D4, 65) specific for the Ti of HPB-ALL. The antibodies 1C1 and 1C2 reacted with all 3D6+ T cell clones and recognize probably the same epitope as 3D6. The antibodies 2D4 and 65 reacted with two mutually exclusive subsets of T cell clones. All the anti-Ti antibodies reacted with functional epitopes, since they were able to block the function of the T cell clones. The specificity of the clones was investigated by blocking studies using monoclonal antibodies specific for different major histocompatibility complex antigens. No correlation was found between the expression of the different Ti epitopes and the specificity, the CD4/CD8 phenotype or function of the clones.

Stimulation and Expansion of a Human T-Cell Subpopulation by a Monoclonal Antibody to T-Cell Receptor Molecule

A murine monoclonal antibody (MAb) was obtained that showed unique specificity for the immunizing T-cell line HPB-ALL. This antibody, C37 (an IgG1,K) also reacted with a small (2-5%) population of normal peripheral blood T (PBL-T) cells. These C37-positive (C37+) cells were found in both the T4/Leu3+ and T8/Leu2+ subsets. Like OKT3 antibody, C37 induced T-cell mitogenesis with a peak proliferative response at day 3. In long-term cultures containing irradiated autologous feeder cells and IL-2, C37 antibody caused the selective expansion of C37+ T cells. On HPB-ALL cells C37 induced comodulation of the T3 molecule. C37 precipitated a disulfide-linked dimer characteristic of the T-cell antigen receptor consisting of an alpha-subunit (45-48 kD) and a beta-subunit (38-42 kD) from both C37+ T-cell blasts of a normal individual and HPB-ALL cells that were surface radioiodinated. However, the precipitated molecule isolated from C37 antibody-activated T-cell blasts exhibited a different pI from that isolated from HPB-ALL cells. Our studies indicate that C37 recognizes an epitope on the T-cell receptor molecule that is shared by a subpopulation of human T cells, which raises the possibility that multiple variable-region associated and/or framework-like determinants of the T-cell antigen receptor can be defined serologically and used in functional and molecular studies of T-cell subsets.

A joining-diversity-joining complex generated by inversion mechanism and a variable-diversity complex in the beta-chain gene of the human T-cell receptor.

We have analysed an inactive allele of the beta-chain gene of the T-cell receptor in a human T-cell line HPB-ALL. Comparison with germline sequences showed that HPB-ALL has a joining (J)-diversity (D)-J complex recombined in head-to-head configuration and a variable (V)-D complex in tail-to-tail configuration. These results demonstrate that the inversion mechanism functions in the beta-chain gene of the T-cell receptor. The presence of the V-D complex suggests that V-D recombination could occur prior to D-J recombination although there is no definite proof that the V-D complex is an intermediate to form the V-D-J complex.

Characteristics of a monoclonal antibody (WT-31) that recognizes a common epitope on the human T cell receptor for antigen.

We describe a monoclonal antibody, WT-31, that reacted with all human T lymphocytes. Electrophoretic analysis of the material reacting with WT-31 revealed that it precipitated predominantly an 80-kD disulfide-linked heterodimer from the cell surface-labeled T leukemic cell line HPB-ALL. This heterodimer was identical to the one precipitated with a recently described monoclonal reagent, T40/25, which recognizes a clonotypic structure on HPB-ALL. The target antigen of WT-31 comodulated with T3 after incubation of T cells with excess anti-T3 antibody, indicating that the WT-31 target antigen is associated with T3. We also found that anti-T3 reagents, but not the clonotypic reagent T40/25, blocked binding of FITC-labeled WT-31 to HPB-ALL cells. This indicates that the T cell receptor epitope recognized by WT-31 is located close to the epitopes recognized by the anti-T3 reagents anti-Leu-4 and SPV-T3b but distal from the clonotypic T40/25 epitope. Functional studies showed that WT-31 reacts similar to anti-T3 antibodies. It is mitogenic for resting T cells, blocks cytolysis mediated by alloantigen-specific CTL clones, and induces antigen-nonspecific cytolysis by CTL clones against Daudi target cells. WT-31 did not inhibit the formation of conjugates, but it blocked cytolysis just before or during the Ca2++-dependent programming for lysis. We conclude that WT-31 is an antibody that recognizes a common determinant on the T cell receptor for antigen. The present results support the notion that the two chains of the T cell receptor (alpha and beta) form a functional protein ensemble with the three invariable T3 polypeptide chains (T3-gamma-, delta-, epsilon).

Stimulation of the T3-T cell receptor complex induces a membrane-potential-sensitive calcium influx

Three monoclonal antibodies selected for their recognition of parts of the T3-T cell receptor complex on human T lymphocytes were found to induce an increase in cytoplasmic free Ca 2+ (Ca 1 2+) in the T cell leukemia line HPB-ALL as measured by Quin2 fluorescence. These reagents are directed against T3 (OKT3), a non-variable T3-associated structure (WT-31) and the variable region of the T3-associated antigen receptor (T40/25) of this cell line. The rise in Ca 1 2+ was dependent on the presence of extracellular Ca 2+, occurred within 30 sec of stimulation, and was sustained for at least 10 min. Fab fragments of OKT3 also caused a rapid increase in Ca 1 2+, indicating that cross-linking is not necessary to induce a Ca 2+ response. Alterations in plasma membrane potential and La 3+ blocked the Ca 2+ influx induced by OKT3 and T40/25. These data suggest that the T3-T cell receptor complex of human T lymphocytes may be an antigen-regulated Ca 2+ channel.

Partial Primary Structure of the Alpha and Beta Chains of Human Tumor T-Cell Receptors

The T-cell receptor for antigen (Ti) was purified from the human tumor cell line HPB-ALL. Amino-terminal sequence analysis of an acid-cleaved peptide of the Ti alpha chain showed that it is highly homologous to a putative murine alpha chain recently described. Amino-terminal sequence analysis of the Ti beta chain revealed that it shares 50 percent homology with the Ti beta chain amino acid sequences from two other human T-cell tumors. Nucleotide sequence analysis of a complementary DNA clone encoding the Ti beta chain from the HPB-MLT cell line showed that this chain represents a second human constant region gene segment and suggested that it arises from direct joining of the variable and joining gene segments without any intervening D region sequences.