Novel molecular mechanisms of dendritic cell-induced T cell activation.

In this study we have re-examined the molecular mechanisms involved in activation of T cells by dendritic cells (DC). Human peripheral blood DC (PBDC) were derived by 2 h adhesion followed by 7 day culture in a combination of granulocyte macrophage colony stimulating factor and IL-4, and depletion of residual T and B cells. These PBDC were used to induce autologous T cell proliferation in a CD3-dependent response, and antibodies against CD11a/18 and CD86 were used as control inhibitors of accessory function. Antibodies against five of the cell surface molecules that we have recently identified on the surface of DC, CD13, CD87, CD98, CD147 and CD148, and an antibody which recognizes a molecule that has not as yet been identified, all inhibited the CD3-induced T cell proliferation. These findings were observed not only when antibodies were present throughout the culture, but also when they were prepulsed on to the surface of the DC, suggesting the inhibition was mediated via the antigen-presenting cells rather than the T cell. The same set of antibodies also inhibited an allospecific mixed lymphocyte reaction, confirming that the inhibitory effect was not dependent on the use of a CD3 antibody as the stimulating agent. All the antibodies of known specificity inhibited both CD4 and CD8 T cells equally. Unlike CD87, CD98 and CD147 antibodies, which inhibited activation of both CD45RA (naive) T cells and CD45RO (memory) T cells, CD13 and CD148 appeared to be involved in activation of naive cells only. The molecules identified in this study have not previously been demonstrated to play a role as accessory molecules on DC, the cells that are pivotal for immune induction. Therefore they may provide new potential targets for modulation of the immune response at the APC level.

The monoclonal antibody 97A6 defines a novel surface antigen expressed on human basophils and their multipotent and unipotent progenitors.

Basophils (Ba) and mast cells (MC) are important effector cells of inflammatory reactions. Both cell types derive from CD34(+) hematopoietic progenitors. However, little is known about the cell subsets that become committed to and give rise to Ba and/or MC. We have generated a monoclonal antibody (MoAb), 97A6, that specifically detects human Ba, MC (lung, skin), and their CD34(+) progenitors. Other mature hematopoietic cells (neutrophils, eosinophils, monocytes, lymphocytes, platelets) did not react with MoAb 97A6, and sorting of 97A6(+) peripheral blood (PB) and bone marrow (BM) cells resulted in an almost pure population (>98%) of Ba. Approximately 1% of CD34(+) BM and PB cells was found to be 97A6(+). Culture of sorted CD34(+)97A6(+) BM cells in semisolid medium containing phytohemagglutinin-stimulated leukocyte supernatant for 16 days (multilineage assay) resulted in the formation of pure Ba colonies (10 of 40), Ba-eosinophil colonies (7 of 40), Ba-macrophage colonies (3 of 40), and multilineage Ba-eosinophil-macrophage and/or neutrophil colonies (12 of 40). In contrast, no Ba could be cultured from CD34(+)97A6(-) cells. Liquid culture of CD34(+) PB cells in the presence of 100 ng/mL interleukin (IL)-3 (Ba progenitor assay) resulted in an increase of 97A6(+) cells, starting from 1% of day-0 cells to almost 70% (basophils) after day 7. Culture of sorted BM CD34(+)97A6(+) cells in the presence of 100 ng/mL stem cell factor (SCF) for 35 days (mast cell progenitor assay) resulted in the growth of MC (>30% on day 35). Anti-IgE-induced IgE receptor cross-linking on Ba for 15 minutes resulted in a 4-fold to 5-fold upregulation of 97A6 antigen expression. These data show that the 97A6-reactive antigen plays a role in basophil activation and is expressed on multipotent CD34(+) progenitors, MC progenitors, Ba progenitors, as well as on mature Ba and tissue MC. The lineage-specificity of MoAb 97A6 suggests that this novel marker may be a useful tool to isolate and analyze Ba/MC and their progenitors.

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.

Irradiation induces up-regulation of E9 protein (CD105) in human vascular endothelial cells.

The use of MAb E-9 raised against tissue-cultured endothelial cells (EC) has shown marked heterogeneity in vascular EC lining the blood vessels of normal and tumour tissues. MAb E-9 is human EC-specific and the protein recognized by it is a homodimer with a molecular mass of 97 kDa. The E-9 protein is resistant to treatment by 3 mM sodium periodate, but is sensitive to 10% trichloroacetic acid and 70% ethanol. E-9 protein has been assigned to a new cluster, CD105, and mapped to human chromosome 9q3. It has approximately 70% homology with type-III cell-surface receptor for transforming growth factor beta (TGF-beta). Recently CD105 has been reported to be the gene in patients with hereditary haemorrhagic telangiectasia. We have examined the effects of radiation on its expression in normal human umbilical-vein endothelial cells (HUVEC) and brain-tumour-derived endothelial cells (BTEC). Irradiation induced dose- and time-dependent up-regulation in the expression of the E-9 protein on the plasma membranes of EC, and also resulted in greater increase in the expression of the E-9 protein in semi-confluent (proliferating) as compared with confluent (non-proliferating) EC. It may well be that, following radiotherapy in cancer patients, E-9 protein is also up-regulated. The presence of increased amounts of E-9 protein in EC makes it an attractive target in the control of angiogenesis, especially after radiotherapy in cancer patients. The time scale involved in the up-regulation of E-9 protein following irradiation has led us to suggest that it may be a secondary event, the primary being the production and release of mitogenic factors (such as basic fibroblast growth factor) from irradiated EC.

Selective expression of V beta families by T cells in the blood and salivary gland infiltrate of patients with primary Sjögren's syndrome.

OBJECTIVE: The T cell infiltration of the salivary gland of patients with Sjögren's syndrome (SS) has been implicated in the pathogenic process of the disease. We examined the representation of V beta subsets in the blood and salivary tissue of patients with SS. METHODS: Circulating T cells from 12 patients and paired samples of blood and labial salivary glands obtained from 8 patients were studied. A panel of monoclonal antibodies directed against the variable region of the T cell receptor was used to enumerate the cells expressing V beta families in the peripheral blood by flow cytometry, and in tissue sections by immunofluorescence. RESULTS: We found an increase of cells bearing V beta 2 family gene products in the circulation, and an increase in both V beta 2 and V beta 8 in the salivary gland infiltrate of patients with SS. No significant difference was noted between the 5 DR3+ patients and 7 DR3- patients studied with regard to the V beta families seen. CONCLUSION: Our data are consistent with a role for specific T cell families in the pathogenesis of SS.

A monoclonal antibody detects heterogeneity in vascular endothelium of tumours and normal tissues.

A new murine monoclonal antibody (MAb), E-9, has been raised using tissue-cultured human umbilical vein endothelial cells. The antigen recognized by this MAb is a peptide of 170 kDa under non-reducing conditions and 96 kDa under reducing conditions. MAb E-9 showed marked heterogeneity in its distribution in various tissues. The antigen recognized by it was present in vascular endothelial cells of all tumours, foetal organs and in regenerating and inflamed tissues. It stained a few normal tissues. However, with the exception of tonsils, staining tended to be weak and limited to a few blood vessels, as revealed by double staining using pan-endothelial antibody (CD31) and antibody to von Willebrand factor, another marker of vascular endothelium. Surprisingly, blood vessels within the placental villi were completely negative. The function of the antigen recognized by MAb E-9 is not known, but its evaluation and use should increase our understanding of angiogenesis.

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.

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.

Modulation of expression of class II histocompatibility antigens by secretion of a cellular inhibitor in K562 leukemic cells.

In this report we show that it is possible to induce the expression of HLA-DR antigens on K562 cells, previously reported to be unresponsive to interferon-gamma (IFN-gamma). However, only low cell concentrations and a high dose of IFN-gamma allowed the induction of HLA-DR antigens. Furthermore, the recombinant glycosylated IFN-gamma is 100-fold more efficient than the unglycosylated form. This induction of HLA-DR antigens on K562 was not related to a stage of differentiation or to the presence of cells subsets specifically sensitive to IFN-gamma, since repeated sorting of K562 HLA-DR-positive and negative cells did not lead to the selection of a cell subset with a different potential of induction for HLA-DR. The difficulty in obtaining induction is due to the production of a soluble endogenous inhibitor of proteic nature, whose action is not restricted to the K562 cell line since it operates also on both epithelial and fibroblastic cells. Treatment of normal human epithelial and fibroblastic cells with conditioned medium from K562 cultures caused a marked decrease in the expression of HLA class II antigens (DR and DP) induced by IFN-gamma (10,000 U/ml), but had no effect on cell growth; however, it also affected expression of HLA class I antigens. This inhibition is not mediated by prostaglandin or an IFN-alpha or IFN-beta-dependent mechanism. Production of this inhibitor by pluripotent human leukemic cells could cause an unbalance in the complex control exerted by the immunological system during hematopoietic differentiation or leukemic progression.

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.

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.

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.

Clonospecific structural heterogeneity in the Thy-1 molecule from mouse T lymphocytes.

The Thy-1 molecule immunoprecipitated from detergent-solubilized, 125I-labeled cell-surface proteins was shown to be processed in two distinct ways by mouse T lymphocytes: one leading to the expression by thymocytes, concanavalin A-activated spleen blasts, and six of nine T-cell clones of a molecule of 25-28 kd, and another, observed in three other T-cell clones, leading to the expression at their surface of a so far undescribed low Mr (23 kd) form of Thy-1. The results of two-dimensional gel electrophoresis and neuraminidase, endoglycosidase H, and endoglycosidase F treatment revealed that the observed heterogeneity of Thy-1 molecules from peripheral cloned T cells was due to major differences in the maturation and sialylation of their N-linked complex-type oligosaccharide residues. It was also found that a given T-cell clone could express T200, LFA.1, and transferrin receptor molecules with a low or high Mr. Furthermore, and in contrast to previously reported results, this study revealed that the differences in cell-surface glycoprotein profiles could not be correlated with the Lyt-2,3/T4 phenotypes, the specificity for allo-H-2, allo-I-A, allo-I-E, or GAT + I-Ak determinants, nor with the cytolytic or helper/amplifier potential of the various T-cell clones examined. The possible implications of these findings are discussed.

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.

A rat anti-mouse T4 monoclonal antibody (H129.19) inhibits the proliferation of Ia-reactive T cell clones and delineates two phenotypically distinct (T4+, Lyt-2,3-, and T4-, Lyt-2,3+) subsets among anti-Ia cytolytic T cell clones.

Hybridoma H129 .19 was derived by fusion between spleen cells of a Lou / Ws1 rat immunized with an Lyt-1+,2- anti-I-Ak cytolytic T lymphocyte (CTL) clone and the nonsecreting myeloma X63-Ag8.653. The monoclonal antibody (mAb) H129 .19 (IgG2a, kappa) was selected for its capacity to inhibit the lytic potential of the immunizing clone. H129 .19 identified a monomorphic determinant on a 55 m.w. murine T cell differentiation antigen, which appeared to be homologous to the human T4 molecule in that: 1) H129 .19 reacted with 80% adult thymocytes, with a subset of splenic T cells, and with the interleukin 2 (IL 2)-producing EL4 thymoma; 2) The mAb bound to and inhibited the IL 2 production and the proliferation of various allo- or soluble antigen-reactive T cell clones that recognized restriction or activating determinants on the I-A or I-E molecules, respectively; 3) H129 .19 did not inhibit the proliferation and/or cytolysis of Lyt-2,3+ T cells specific for class I MHC antigen; and 4) Among six anti-Iak CTL clones examined in this study, the mAb H129 .19 reacted with two I-Ak-specific, Lyt-2,3- clones on which it exerted strong cytolysis inhibiting effect at the effector cell level. By contrast, two other anti-I-Ak and two anti-I-Ek CTL clones were found to express the Lyt-2,3+,T4- cell surface phenotype. The cytolytic potential of the latter clones was not inhibited by anti-Lyt-2,3 mAb. These studies strongly suggest that the mouse T4 molecule facilitates the recognition of class II MHC antigen by most but not all T cells.

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.

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.

Localization of a gene controlling the expression of the human transferrin receptor to the region q12 leads to qter of chromosome 3.

A monoclonal antiserum, 66-IG10, raised against human thymocytes was found to be directed against the human transferrin receptor. A panel of human X Chinese hamster somatic cell hybrids, in conjunction with the 66-IG10 reagent, was used to assign the gene(s) coding for the transferrin receptor to the q12 leads to qter region of human chromosome 3.