How do B cells differ from T cells in terms of gene expression?

As part of an ongoing program to identify the genes that distinguish B cells from T cells, we have analyzed 910 molecular species of polypeptides detectable in mouse lymphocytes by 2D gel electrophoresis. Of these 910 polypeptides, 488 were present in both B and T cells, 185 in B but not T cells, and 237 in T but not B cells. The detected set of polypeptides accounts for more than 95% of the protein mass of lymphocytes. There are about 2000 other polypeptides that are below the threshold of detection. We have started to identify and to retrieve cDNA clones belonging to the B and T cell subset. Dedicated to Professor J. Šterzl on the occasion of his 70th birthday     

Different expression profiles of human cyclin B1 in normal PHA-stimulated T lymphocytes and leukemic T cells

BACKGROUND: In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes. METHODS: Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results. RESULTS: Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis. CONCLUSIONS: We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle.     

T cell receptor delta gene organization and expression in normal and leukemic natural killer cells

In peripheral blood most NK activity is mediated by CD3- cells with large granular lymphocyte morphology which cannot be assigned to a specific hemopoietic lineage. In accordance with previous studies we have analyzed the organization of the TCR delta gene, which rearranges early in thymic ontogeny, in normal NK cells, and in granular lymphocytes proliferative disorders (GLPD), in an effort to further define their relationship to the T cell differentiation pathway and to identify a possible marker of clonality for CD3- GLPD. The alpha/delta locus was rearranged in five cases of CD3+ GLPD with a biallelic deletion of the C delta region, suggesting V-J alpha rearrangement, whereas CD3- GLPD and normal CD3- NK cells had the delta gene in germ-line configuration, but surprisingly expressed high levels of TCR delta-related mRNA. On the basis of this finding and of the presence of truncated TCR-beta and CD3-epsilon mRNA, we are led to speculate on a possible ontogenic relationship of NK cells to the T cell differentiation pathway at stages preceding TCR gene rearrangement.     

Retinoid-regulated gene expression in normal and leukemic myeloid cells

Physiological concentrations of retinoic acid can induce acute alterations in the expression of the enzyme tissue transglutaminase in cultured macrophages. The induction of this enzyme offers a probe to study the mechanism of retinoid action in both normal and leukemic cells.     

ATRA promotes alpha tocopherol succinate-induced apoptosis in freshly isolated leukemic cells from chronic myeloid leukemic patients

We investigated the in vitro efficacy of all-trans retinoic acid (ATRA) and alpha-tocopherol succinate (α-TS) alone and in combination on the induction of cell death in freshly isolated leukemic cells obtained from chronic myeloid leukemia (CML) patients. In vitro cytotoxicity and induction of lipid peroxidation by ATRA (10 μM) and α-TS (25 or 50 μM) were evaluated in primary leukemic cells by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and malondialdehyde formation respectively. Treatment of leukemic cells with α-TS alone or in combination with ATRA significantly (P < 0.05) decreased the cell viability in a concentration and time dependent manner as compared to peripheral blood mononuclear cells obtained from normal healthy controls. Lipid peroxidation was enhanced by 98% (P < 0.05) on combined treatment of cells with ATRA (10 μM) and α-TS (50 μM). ATRA alone did not enhance the externalization of phosphatidyl serine as studied by annexin-V binding using fluorescence activated cell sorter analysis, whereas in combination with α-TS it increased to 400% at 12 h. The treatment of leukemic cells to combination of ATRA with α-TS significantly decreased (P < 0.05) mitochondrial membrane potential and enhanced lysosomal destabilization. The combination of these drugs also increased mitochondrial and cytosolic reactive oxygen species (ROS) production, nitric oxide levels, and caspase-3 activity significantly and caused DNA fragmentation at 24 h in a concentration dependent manner in the leukemic cells. Our data suggest that ATRA in combination with α-TS efficiently induces apoptosis in leukemic cells, which may be a useful therapeutic modality in CML patients.     

Caveolin-1 knockout mice have altered serum N-glycan profile and sialyltransferase tissue expression

Journal of Physiology and Biochemistry - Caveolin-1 (Cav-1) is a constitutive protein within caveolar membranes. Previous studies from our group and others indicated that Cav-1 could mediate...     

Chronic lymphocytic leukemia (CLL) cells genetically modified to express B7-1, ICAM-1, and LFA-3 confer APC capacity to T cells from CLL patients

In chronic lymphocytic leukemia (CLL), malignant B cells and nonmalignant T cells exhibit dysfunction. We previously demonstrated that infection of CLL cells with modified vaccinia Ankara (MVA) expressing the costimulatory molecules B7-1, ICAM-1, and LFA-3 (designated TRICOM) increased expression of these costimulatory molecules on the surface of CLL cells and thus augmented their antigen-presenting capability. Here, we evaluate the effect of MVA-TRICOM-modified CLL cells on T cells. Following incubation with irradiated MVA-TRICOM-modified CLL cells, allogeneic and autologous CD4⁺ and CD8⁺ T cells expressed significantly higher levels of B7-1, ICAM-1, and LFA-3. We show that this increase was the result of physical acquisition from the antigen-presenting cells (APCs), and that purified T cells that acquired costimulatory molecules from MVA-TRICOM-modified CLL cells were able to stimulate the proliferation of untreated T cells. These results demonstrate for the first time that T cells from CLL patients can acquire multiple costimulatory molecules from autologous CLL cells and can then act as APCs themselves. Given the immunodeficiencies characteristic of CLL, enhancing the antigen-presenting function of CLL cells and T cells simultaneously could be a distinct advantage in the effort to elicit antitumor immune responses. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulated expression of the Shiga toxin B gene induces apoptosis in mammalian fibroblastic cells

Shiga toxins (Stxs) produced by enterohaemorrhagic Escherichia coli may induce colonic ulceration, bloody diarrhoea and acute renal failure. The A subunit (StxA) is known to inhibit protein synthesis, whereas the B subunits (StxB) bind to Gb3 on the cell surface. However, the mechanisms by which Stxs kill target cells remain unclear. Stx1A or Stx1B genes were introduced into pcDNA3.1 vectors and transfected into NIH3T3 and HeLa cells. The Stx1B gene-transfected cells became apoptotic with accompanying DNA fragmentation, whereas the Stx1A gene-transfected cells were found to be necrotic and no DNA fragmentation occurred. The HeLa/C4 cells integrated with the Stx1B gene with a tetracycline-inducible promoter eventually produced cytoplasmic Stx1B, leading to DNA fragmentation on the addition of doxycycline. These apoptotic changes were abrogated by pretreatment with Z-VAD-fmk. These results suggest that the transfected Stx1B gene induces apoptosis by activating the caspase cascade after Stx1B expression in the cytoplasm.     

Late signals from CD27 prevent Fas-dependent apoptosis of primary CD8+ T cells

The role of costimulation has previously been confined to the very early stages of the CD8+ T cell response. In this study, we demonstrate the requirement for CD27 costimulation during the later phase, but not programming of the primary CD8+ T cell response to influenza virus and reveal a novel mechanism of action for CD27 costimulation. CD27 signals, during the later phase of the primary CD8+ T cell response, prevent apoptosis of Ag-specific CD8+ T cells. Blocking CD27L (CD70) on days 6 and 8 after infection reduces the number of NP(366-374)-specific CD8+ T cells, increases their sensitivity to CD95/Fas-mediated apoptosis, and up-regulates FasL on CD4+ T cells. This reduction of NP(366-374)-specific CD8+ T cells requires the presence of CD4+ T cells and Fas signaling. Lack of CD27 signals also decreases the quality of memory CD8+ T cell responses. Memory CD8+ T cells, which express surface CD27 similar to naive cells, however, do not require CD27 costimulation during a secondary response. Thus, CD27 acts indirectly to regulate primary Ag-specific CD8+ T cell responses by preventing apoptosis of CD8+ T cells during the later phase of the primary response and is required for optimal quality of memory cells, but is not required during normally primed secondary CD8+ T cell responses.     

Interference with gene expression induces rapid apoptosis in p53-null T lymphoma cells

Two p53-null T lymphoma cell lines proved to be highly sensitive to inhibition of gene expression. With either actinomycin D or cycloheximide, apoptosis commenced within 2 h, as indicated by loss of membrane integrity, degradation of certain proteins (including the phosphatase calcineurin) and DNA fragmentation. These effects were ablated by co-expression of Bcl-2 or co-incubation with the caspase inhibitor Z-VAD-fmk. These results suggest that the apoptotic machinery is in place in these cells but held in check by an unknown labile protein, which probably acts upstream of Bcl-2. Although cycloheximide can activate the JNK or p38 MAP kinases in some cells, neither was implicated here. However, disruption of phosphoinositide 3-kinase signaling may be involved, because the cells were also sensitive to wortmannin. The high sensitivity of the p53-null lymphoma cells to inhibitors of gene expression suggests that such inhibitors might prove useful in the cytotoxic therapy of certain tumors.     

Cutting edge: CD25+ regulatory T cells prevent expansion and induce apoptosis of B cells specific for tissue autoantigens

To study the role of CD25(+) regulatory T cells (T(regs)) in peripheral B cell tolerance, we generated transgenic rat insulin promoter RIP-OVA/HEL mice expressing the model Ags OVA and HEL in pancreatic islet beta cells (where RIP is rat insulin promoter and HEL is hen egg lysozyme). Adoptively transferred transgenic OVA-specific CD4(+) and CD8(+) T cells proliferated only in the autoantigen-draining pancreatic lymph node (PLN), demonstrating pancreas-specific Ag expression. Transferred HEL-specific transgenic B cells (IgHEL cells) disappeared within 3 wk from transgenic but not from nontransgenic mice immunized with autoantigen. Depletion of CD25(+) FoxP3(+) cells completely restored IgHEL cell numbers. T(reg) exerted an analogous suppressive effect on endogenous HEL-specific autoreactive B cells. T(regs) acted by inhibiting the proliferation of IgHEL cells in the spleen and PLN and by systemic induction of their apoptosis. Furthermore, they reduced BCR and MHC II surface expression on IgHEL cells in the PLN. These findings demonstrate that autoreactive B cells specific for a nonlymphoid tissue autoantigen are controlled by T(regs).