Ligation of 4-1BB (CDw137) regulates graft-versus-host disease, graft-versus-leukemia, and graft rejection in allogeneic bone marrow transplant recipients

4-1BB is expressed on activated CD4(+) and CD8(+) T cells; its ligand, 4-1BB ligand is expressed on APCs. Despite expression on both T cell subpopulations, 4-1BB has been reported to predominantly affect CD8(+) T cell responses. By quantifying graft-vs-host disease alloresponses in vivo, we demonstrate that both CD4(+) and CD8(+) T cell-mediated alloresponses are regulated by 4-1BB/4-1BB ligand interactions to approximately the same extent. 4-1BB receptor-facilitated CD4(+) T cell-mediated alloresponses were partly CD28 independent. In two distinct marrow graft rejection systems, host CD8(+) and CD4(+) T cells each separately contributed to host anti-donor T cell-mediated allograft rejection. alpha 4-1BB mAb increased the graft-vs-leukemia effect of a suboptimal number of donor splenocytes given later post bone marrow transplantation by bolstering allogeneic responses resulting in leukemia elimination. In summary, 4-1BB ligation is a potent regulator of CD4(+) and CD8(+) T cell-mediated allogeneic responses in vivo. Modifying the ligation of 4-1BB represents a new approach to altering the graft-vs-host disease and graft-vs-leukemia effects of allogeneic T cells post bone marrow transplantation.     

Activation of human phagocytes through carbohydrate antigens (CD15, sialyl-CD15, CDw17, and CDw65).

The leukocyte carbohydrate (CHO) Ag CD15, sialyl-CD15, and CDw65 have recently been found to function as ligands for CD62 and ELAM-1 cell adhesion molecules on platelets and endothelium, respectively. Cell adhesion ligands also may act as receptors capable of signal transduction. We therefore investigated the possibility that these CHO Ag and CDw17, a glycolipid Ag whose expression is regulated by leukocyte activation, may have receptor-like characteristics. The effects of antibody cross-linking of CHO Ag on phagocyte activation were measured by using flow cytometry and fluorescent indicators for cytoplasmic calcium ions, oxidative burst, and the granule-associated proteins CD11b and CD67. Cross-linking of CD15, sialyl-CD15, CDw65, or CDw17 induced a moderate release of calcium ions into the cytoplasm of granulocytes, a strong activation of oxidative burst, and a low up-regulation of CD11b and CD67 compared to the effects of treatment with 4 microM FMLP. The results suggest a role for CHO Ag in leukocyte signal transduction and support the view that these molecules are involved in phagocyte activation.     

Different isoforms of human FcRII distinguished by CDw32 antibodies

The Third and Fourth International Workshops on Leucocyte Differentiation Antigens identified six mAb, designated CDw32, reacting with human Ig FcR type II (FcRII). We have examined the immunohistochemical and immunocytologic reactivities of these antibodies and find that the antibodies could be divided into three classes of reactivity: 1) antibodies IV.3, CIKM3, and CIKM5 reacted with monocytes, macrophages and neutrophils; 2) antibodies KB61 and 41H.16 gave strong reactions with B lymphocytes, placental and hepatic endothelium, and weaker reactions with monocytes, macrophages, and neutrophils; 3) antibody 2E1 gave an intermediate reaction pattern. Immunoprecipitation from U937 cell lysates showed that antibodies KB61 and 41H.16 recognized Mr 41,000 and Mr 37,000 molecules whereas the other antibodies detected a Mr 42,000 molecule. Preclearing with antibody KB61 removed the Ag recognized by the other five antibodies confirming the identity of the Ag and demonstrating reactivity of KB61 with the Mr 42,000 molecule. Antibodies KB61 and 41H.16 precipitated a Mr 41,000 molecule from B lymphocytes. Flow cytometry and immunoprecipitation studies of cells transfected with cDNA clones coding for two isoforms of FcRII showed that all six of the antibodies react with both transfectants but the only immunoprecipitations were obtained using KB61 and 41H.16 and one of the transfectants. The protein sequence of KB61 Ag isolated from leukemic B cells showed close homology with the proteins encoded by the cDNA clones but diverged in the intracytoplasmic carboxyl-terminal region. It was concluded that preferential recognition of one or more of the numerous isoforms of FcRII underlies the differing reaction patterns of CDw32 antibodies.     

CDw17: a neutrophil glycolipid antigen regulated by activation

The plasma membrane and intracellular granules of human polymorphonuclear neutrophils (PMN) contain large amounts of the glycolipid, lactosylceramide (LacCer; Gal beta 1----4Glc beta 1----1Cer). Despite its abundance, novel subcellular distribution, and lineage-restricted expression, nothing of PMN LacCer function is known. We examined the relationship between LacCer and PMN activation by assessing binding of anti-LacCer mAb (T5A7; anti-CDw17) to PMN during and after cell stimulation. CDw17 expression markedly decreased after treatment with PMA, dioctanoylglycerol, calcium ionophore, FMLP (with or without cytochalasin B or added Ca2+), TNF-alpha, or lymphotoxin. Depending on the stimulus, CDw17 declined to levels ranging from 70% (TNF, lymphotoxin) to less than 5% (phorbol ester, dioctanoylglycerol) of levels detected on untreated PMN. Loss of CDw17 from PMA-treated PMN followed dose- and temperature-dependent kinetics, with loss being detected after PMA treatment for 1 min. Membrane internalization explained PMA-induced loss of CDw17, as cell-associated 125I-anti-CDw17 became inaccessible to fluorescent anti-Ig after PMA treatment. CDw17 on PMN cytoplasts or retinoic acid-induced HL-60 cells was only slightly affected by stimulation, suggesting that down-regulation of the epitope is associated with granule exocytosis rather than superoxide production. Results with PMN from a patient with chronic granulomatous disease confirmed that normal superoxide production is not required for CDw17 loss induced by PMA or FMLP treatment. The data collectively demonstrate that reduced levels of cell-surface CDw17 are associated with granule exocytosis after PMN activation.     

Immunocytochemical localization of CDw60 antigens on human peripheral T cells

About 25-35% of human T cells display the CDw60 ganglioside (9-O-acetyl-GD3) antigen at the cell surface [E.P. Rieber, in W. Knapp, B. D?rken, W.R. Gilks, E.P. Rieber, R.E. Schmidt, H. Stein, A.E.G.K. von dem Borne (Eds.), Leucocyte Typing IV, Oxford University, Oxford, 1989, p. 361.]. Other leucocytes do not express this antigen on the cell surface. This led us to investigate its presence by flow cytometry and immunoelectron microscopy (IEM). Flow cytometric analysis of isolated peripheral T cells showed 26% of the cell population to have the CDw60 antigen expressed on the cell surface whereas 74% did not. Similarly, IEM analysis of 262 random T cells by the preembedding immunogold labeling technique revealed CDw60 surface expression to be tetrapartite: (a) the majority of 63.7% of the T cells did not show any surface associated gold label; (b) 19.5% were of low CDw60 surface exposition, corresponding to a linear density of 0.05-2.0 gold markers per microm; (c) about 13.4% showed a medium surface exposition with a linear density of 2.1-4.5 gold markers per microm; and (d) a high exposition, ranging from 4.6 to 9.0 gold markers per microm, was seen at 3.4% of the T cells. From postembedding label experiments, which additionally make access to the antigen localized within the cytoplasm, it was found that nearly all T cells contained low levels of intracellular CDw60. Most of it was found to be associated with the cytoplasmic membrane or vesicles, derived from the Golgi. Immunogold conjugates associated with the cytoplasmic membrane showed a linear density up to 0.6 gold markers per microm. The asymmetric expression of the CDw60 antigen on human T cells and its occurrence in nearly all T cells suggests that its surface presentation is tightly regulated.     

CDw 60 antibodies bind to acetylated forms of ganglioside GD3.

Four monoclonal antibodies, M-T21, M-T32, M-T41 and UM4D4, which belong to the new CDw 60 cluster of antibodies specific for a subpopulation of human T-lymphocytes, were found to bind mainly to acetylated forms of ganglioside GD3. After O-deacetylation of the antigen, binding was reduced ("M-T"-antibodies) or abolished (UM4D4).     

Quantitative analysis of LacCer/CDw17 in human myelogenous leukaemic cells

LacCer/CDw17 is the most abundant GSL in neutrophils. The cell-surface and intracellular presence of LacCer was determined quantitatively using anti-CDw17 mAbs in a flow cytometry assay. The quantified alterations in the level of CDw17 antigen expression are consistent with alterations in LacCer content, determined chemically. Our results show that CDw17 antigen expression defines successive stages in the maturation of the myeloid cell. The assessment of cell-surface and intracellular CDw17 expression may be useful in evaluating neutrophil physiological status.     

CDw60: an antigen expressed in many normal tissues and in some tumours

CDw60 is a recently described T-cell antigen, which functionally delivers a costimulatory signal in T-cell activation. In addition, CDw60 has been regarded as a melanoma-associated antigen. To date, only limited information exists on the distribution of CDw60 in other normal and pathologically altered tissues in human. In the present study, the expression of CDw60 was analysed immunohistologically in a large panel of formalin-fixed and paraffin-embedded normal and pathological human tissues. The antigen was detected in several normal tissues, such as epithelia of the reproductive system, exocrine and endocrine glands, glial cells and neurons of the central and peripheral nervous systems, and lymphoid cells. These showed different subcellular distribution patterns, i.e. (1) cell surface labelling of peripheral lymphocytes and lymphocytes of the lymph node and thymus, (2) diffuse cytosolic staining in lymphocytes, subpial glial processes, and the outer plexiform layer of the retina, (3) granular cytoplasmic staining associated with the Golgi apparatus in epithelial cells of certain endocrine and exocrine glands, of the ductus epididymis and deferens, neurons of the peripheral and central nervous system, and lymphocytes and megakaryocytes of the bone marrow.In exocrine glands, e.g. of the prostate and uterine corpus, CDw60-positive Golgi fields were located in the juxtaluminal cell compartment, thus reflecting a polarized distribution. In some malignant tumours, the neoplastic cells contained CDw60-immunolabelled Golgi complexes, which were disorderly distributed throughout the cytoplasm, thus reflecting a loss of epithelial polarity. Only in mammary carcinomas was abnormal cell surface labelling detected. A putative de novo expression of CDw60 was observed in pleomorphic adenoma and mucoepidermoid carcinoma of the parotid gland, seminoma, embryonal and teratocarcinoma of the testis, small cell carcinoma of the lung, and malignant melanoma. These results define the CDw60 determinant as a broadly distributed antigen within a large panel of normal human tissues. The antigen is also detectable in some previously undescribed benign and malignant tumours, which may give importance to CDw60 as a possible diagnostic marker.     

Contents of volume 137 1991

The halophyte, Suaeda salsa, was grown in saline soil in pots and watered with a NaCl solution containing 0.2 g L^-1 Na-ions. S. salsa accumulated Na during a 120-day growing period and caused a net reduction in the Na content of the soil. S. salsa also decreased the Na content of saline soil in a field experiment. The Na content of the soil at depth 20–30 cm was reduced by 4.5% with S. salsa at a density of 15 plants m^-2 and by 6.7% with a density of 30 plants m^-2. In contrast, the Na content was decreased by only 1% with Medicago sativa at 15 plant m^-2 and increased by 3.8% with bare soil. The results confirm that S. salsa is an effective salt absorber in saline soils.