Tatsuo Kina

A DEVELOPMENTAL SWITCH IN THYMIC LYMPHOCYTE MATURATION POTENTIAL OCCURS AT THE LEVEL OF HEMATOPOIETIC STEM-CELLS

Hematopoietic stem cells (HSCs) isolated from mouse fetal liver, like adult HSCs, are Thy-1lo Lin- Sca-1+. Donor-derived V gamma 3+ T cells were detected in fetal thymic lobes repopulated in vitro with fetal liver HSCs, but not in those with adult bone marrow HSCs. Single clonogenic fetal HSCs gave rise to thymic progeny that include V gamma 3+, other gamma delta+, and alpha beta+ T cells. No V gamma 3+ T cells were detected in adult thymus injected intrathymically with either fetal or adult HSCs. These results support the hypothesis that only fetal HSCs have the capacity to differentiate into V gamma 3+ T cells in the fetal thymic microenvironment and that the developmental potential of HSCs may change during ontogeny.

The monoclonal antibody TER-119 recognizes a molecule associated with glycophorin A and specifically marks the late stages of murine erythroid lineage.

The antigen specificity of a rat monoclonal antibody TER‐119 was investigated. In adult mice, TER‐119 reacted with mature erythrocytes, 20–25% of bone marrow cells and 2–3% of spleen cells but not with thymocytes nor lymph node cells. In fetal haematopoietic tissues, 30–40% of d 10 yolk sac cells, 80–90% of d 14 fetal liver cells and 40–50% of newborn liver cells were reactive with TER‐119. TER‐119+ cells in adult bone marrow expressed significant levels of CD45 but not myeloid (Mac‐1, Gr‐1) or B‐cell (B220) markers. Morphological examination and haematopoietic colony‐forming assays for isolated TER‐119+ cells revealed that TER‐119 reacts with erythroid cells at differentiation stages from early proerythroblast to mature erythrocyte, but not with cells showing typical erythroid blast‐forming unit (BFU‐E) and erythroid colony‐forming unit (CFU‐E) activities. Erythroleukaemia cell lines do not express the TER‐119 antigen even after stimulation with dimethylsulphoxide. TER‐119 immunoprecipitated protein bands with molecular masses of 110 kDa, 60 kDa, 52 kDa and 32 kDa from erythrocyte membrane, whereas only a 52‐kDa band was detected by TER‐119 in Western blot analysis. Further molecular and cellular analyses indicated that the TER‐119 antigen is a molecule associated with cell‐surface glycophorin A but not with glycophorin A itself.

Mobilization of hematopoietic primitive and committed progenitor cells into blood in mice by anti-vascular adhesion molecule-1 antibody alone or in combination with granulocyte colony-stimulating factor

OBJECTIVE One of the mechanisms for mobilization of hematopoietic stem cells and progenitor cells is alternation of adhesion molecules. We investigated the mobilization of hematopoietic progenitor cells in blood by administration of anti-vascular cell adhesion molecule (VCAM)-1 antibody (Ab) in mice. MATERIALS AND METHODS Twelve- to 14-week old C57BL/6J mice were injected intravenously with anti-VCAM-1 Ab and anti-very late antigen (VLA)-4 Ab at a dose of 5 mg/kg for 2 days. RESULTS The number of colony-forming cells (CFCs) in blood was increased 11.4-fold after anti-VCAM-1 Ab treatment, but the number of CFCs was not increased after treatment with anti-VLA-4 Ab. The number of colony-forming unit spleen (CFU-S) also was increased 21.6-fold in the peripheral blood by administration of anti-VCAM-1 Ab. The number of CFCs and CFU-S in the bone marrow of mice treated with anti-VCAM-1 Ab was decreased and that in the spleen also was decreased. On administration of recombinant human granulocyte colony-stimulating factor (125 microg/kg twice daily) with anti-VCAM-1 Ab, the numbers of CFCs and CFU-S were increased 141.8-fold and 439-fold, respectively. CONCLUSIONS These observations demonstrated that administration of anti-VCAM-1 Ab induced mobilization of hematopoietic progenitor cells into blood from bone marrow and spleen and that granulocyte colony-stimulating factor has synergistic effects on anti-VCAM-1 Ab-induced mobilization.

A monoclonal antibody against Igh6-4 determinant

In order to investigate B-cell differentiation in a multicellular experimental system such as a stromal celldependent culture (Whitlock et al. 1984) or a radiation chimera, markers that are able to discriminate the B cells being pursued from other B cells are required. Among such markers, allotypic variations of immunoglobulin M (IgM) molecules are of particular importance, because IgM is the functional molecule that is expressed in all B cells from an early stage of differentiation. Anti-allotype sera are difficult to prepare, however, and used to be contaminated with antibodies of other specificities. Several monoclonal antibodies specific for allotypic determinants of mouse IgM have recently been produced as a solution to this problem (Kincade et al. 1981, Kung et al. 1981, Leptin et al. 1984, Stall and Loken 1984). Here, we report another monoclonal antibody, MB86, the allotypic specificity of which is different from that of other antibodies. MB86 was produced by fusing X63-Ag8/653 myeloma cells with the spleen cells from a BALB/c mouse which had been immunized and boosted with C57BL/6 spleen cells. Because MB86 bound to B1-8 (~t21, IghC b) protein but not to P8-86.9 (~121, IghC b) protein which has the same VH and V L as B1-8 (Reth et al. 1978, Takemori et al. 1982) or to 20.2.267 (Ix21, IghC a) protein (White-Scharf and Imanishi-kari 1981), we concluded that MB86 recognizes an allotypic determinant of mouse IgM. We then tested the allotypic specificity of MB86 on eight mouse strains. Instead of using purified IgM, we used mouse spleen cells as antigens. One million spleen cells were incubated with either 125Ilabeled MB86 or ~SI-labeled rabbit mouse g-specific antibody for 1 h. They were then washed, and the cellbound radioactivity was determined (Table 1). The gspecific antibody bound to the spleen cells of all strains to the same extent, whereas MB86 bound to B6, AKR, NZB, A/J, and CAL/20 cells, but not to BALB/c, DBA/2, or CBA cells. This pattern of reactivity of MB86 suggests that MB86 is specific for an Igh6-4 determinant which was defined by an alloantiserum by Black and co-workers (1978). Interestingly, MB86 further categorizes the Igh6-4-positive lgM into two groups. The first comprises IghC haplotypes b, d, and n which are strongly positive in MB86 binding, and the other includes haplotypes e and o which are weakly reactive. We therefore concluded that at least three allelic forms of the Igh6 (g) gene exist for the Igh6-4 determinant. It is of interest to note that these three allelic forms also determine an Igh6-5 determinant which was defined by monoclonal antibody Bet 1 (Kung et al. 1981). This could simply be a linkage of two determinants at different sites on the ~t chain. A second possibility is that these two specificities are the different manifestations of a p olymorphism of the same region of the g heavy chain. In order to determine the localization of these determinants, we carried out the following two experiments. First, because it was reported that all the currently available monoclonal IgM allotype-specific antibodies recognized the determinants which appear

A monoclonal antibody to the 15,000 Dalton protein associated with porcine pulmonary surfactant

From a surface active fraction of porcine lung lavage fluid, separated by discontinuous sucrose density gradient ultracentrifugation, a protein with a nominal molecular weight (MW) of 15,000 daltons was isolated by sequential extraction with several buffers, including one containing deoxycholate. A monoclonal antibody was prepared from a hybrid cell (8B5E) obtained by fusing a myeloma cell, X63.Ag8.653, with spleen cells of BALB/c mice immunized with the protein. With immunoblotting technique, the antibody was found to be specific to the 15,000 dalton protein and did not react with another surfactant-associated protein with a nominal MW of 38,000 daltons. The antibodys IgG subclass was IgG1 and the light chain was kappa. In immunohistochemical studies using biotinylated antibody, peroxidase reaction products were localized selectively at inclusions of alveolar wall cells which were located chiefly at the alveolar corners. These results strongly suggest that this 15,000 dalton protein was localized in inclusions of alveolar wall cells and did not originate from other larger surfactant-associated proteins degraded after secretion into alveolar space.

Tea polyphenol inhibits allostimulation in mixed lymphocyte culture.

Green tea polyphenols are known to protect allogenic donor tissues from acute rejection by their recipients. This immunosuppressive effect may be generated by a unique chemical property of the major component, epigallocatechin-o-gallate (EGCG), which can block specific cell surface molecules of the donor tissues. To test this hypothesis, we examined the effects of EGCG on the murine mixed lymphocyte reactions. EGCG treatment of stimulator cells significantly attenuated the proliferation of responder T cells. The proliferation did not recover upon the secondary stimulations by fresh untreated cells or exogenous IL-2. Flow cytometric analyses showed that EGCG treatment decreased the staining intensities of various cell surface molecules including MHC II, which plays a major role in antigen presentation, and B7.1, B7.2, and their ligand, CD28, which are required for costimulatory signals in T-cell activation. These results suggest that an anergic state of alloreactive T cells may be induced by either weakening of antigen signaling or blockage of costimulatory signals with EGCG. Other possible mechanisms behind the immunosuppressive effect and a potential use of EGCG treatment of donor tissues in transplantation medicine are discussed.

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. I. Preferential induction of tolerance to mls antigens and resistance to allo-MHC antigens

Neonatal tolerance inducibility of self-major histocompatibility complex (MHC)-class II-associated antigens was compared with that of allo-class II antigens. BALB/c (H-2d, Mlsb) mice, less than 24 hr after birth, were intravenously injected with bone marrow cells of either (BALB/c X DBA/2)F1 (H-2d, Mlsb/a, semiallogeneic at the Mls locus) or (BALB/c X B10.BR)F1 (H-2d/k, Mlsb; semiallogeneic at the MHC), as antigens. The mice were tested for in vivo immune activity of class II-reactive T cells by means of the popliteal lymph node-swelling assay. They developed tolerance, irrespective of type of antigens, showing profoundly suppressed host-versus-graft reaction, and those tolerized to the allo-MHC antigens accepted skin grafts of the corresponding allogeneic mice. In the thymus and spleen of the Mls-tolerant mice, antigen-specific class II-reactive T-cell activity was completely abolished, without the apparent involvement of suppressor cells. In contrast, the activity in allo-MHC-tolerant mice was not reduced in either thymus or peripheral lymphoid organs, suggesting that systemic hyporesponsiveness is attributable to reversible suppression of immune competent cells. The resistance for cell-level tolerance induction to allo-class II antigens may not be ascribed to the active participation of allo-MHC antigens in prevention of or in escape from tolerance induction or both, since an injection of bone marrow cells of both Mls and H-2-semiallogeneic (DBA/2 X B10.BR)F1 (H-2d/k, Mlsa/b) mice could induce tolerance to Mlsa-H-2d antigens in newborn thymus cells.

Glycophorin A requirement for expression of O‐linked antigens on the erythrocyte membrane

Background:  Glycophorin A (GPA) has a large number of sialic acid‐containing oligosaccharide chains. GPA is highly conserved among vertebrates, mice with a GPA deletion have not been reported and GPAs physiologic role remains uncertain.

Enhancement of Activation-Induced Cell Death by Fibronectin in Murine CD4[+]CD8[+] Thymocytes

extracellular matrix molecule in the thymus, in the cell death induced by activation via the T‐cell antigen receptor. FN alone did not increase cell death in murine thymocytes above the baseline level, but it significantly enhanced the cell death induced by fixed anti‐CD3 monoclonal antibody (mAb), especially when a high concentration of anti‐CD3 mAb was used. DNA fragmentation increased in parallel with cell death, indicating that cell death was a result of the apoptosis. Fluorescence‐activated cell sorter (FACS) analysis revealed that the activation‐induced cell death (AICD) caused by anti‐CD3 mAb alone, or by a combination of anti‐CD3 mAb and FN, occurred selectively in CD4+ CD8+ thymocytes. Very late activation antigen (VLA)‐4 and VLA‐5 are two major ligands to FN on thymocytes. The expression of both ligands was investigated at different stages of thymocyte development. VLA‐4 was predominantly expressed at the CD4− CD8− stage, and thereafter the expression was reduced, whereas VLA‐5 was constantly expressed during maturation. Furthermore, the enhancing effect by FN was inhibited in the presence of the Gly‐Arg‐Gly‐Asp‐Ser‐Pro (GRGDSP) peptide but not in the presence of the connecting segment‐1 (CS‐1) peptide, suggesting that enhancement of AICD observed in CD4+ CD8+ thymocytes is mediated through VLA‐5.

Modulation of cell surface lectin receptors on K562 human erythroleukemia cells induced by transfection with annexin IV cDNA

© 1997 Federation of European Biochemical Societies.