In vitro induction of cytotoxic effector cells with spontaneous killer cell specificity.

The present work shows that stimulation of peripheral blood lymphocytes with autologous or allogeneic B-cell lines leads to a strong induction of cytotoxic effector cells with spontaneous killer (SK) cell specificity, apart from the specific response directed against the particular stimulating cell. To demonstrate this we have determined a relative target cell specificity in the SK system, defined by the short-term 51Cr release assay, and established a relative specificity index (RSI). Using this approach we have been able to show that killer cells induced during a 5-day cocultivation period with B-cell lines have a similar PSI to that of unstimulated SK cells. In addition, we have shown that such killer cells can be induced from several different lymphocyte subpopulations and that they, in contrast to SK cells, do not express Fc receptors. The implications of these findings in relation to the nature, mechanism, and biological significance of the SK cell system is discussed.

Surface markers on human B and T lymphocytes. II. Presence of Epstein-Barr virus receptors on B lymphocytes.

Human peripheral lymphocytes were investigated for receptors binding Epstein-Barr virus (EBV) because of the regular association of this virus with infectious mononucleosis and Burkitt's lymphoma. This was done by a cytoadherence technique where virus-producing cells, displaying fresh viral determinants in their cytoplasmatic membrane, were mixed with lymphocytes. Unfractionated lymphocytes were found to adhere to these cells in contrast to column-purified T lymphocytes. The specificity of the binding was confirmed by blocking experiments that showed that sera containing high titers of antibodies directed against the virus could partially inhibit the adherence in contrast to low-titer sera. It is concluded that B lymphocytes, in contrast to T lymphocytes, have receptors for EBV. In a second line of experiments it was found that established human lymphoblastoid lines that carry the EBV genome had receptors characteristic for B lymphocytes and did not form T-lymphocyte rosettes. In contrast, a line of known T-lymphocyte origin that did not carry the EBV genome had receptors characteristic for T lymphocytes. EBV-transformed simian lymphoblastoid lines had surface markers indicating a B-lymphocyte origin in contrast to HVS-transformed simian lines that lacked surface immunoglobulin but carried receptors for sheep red blood cells.

Recycling and target binding capacity of human natural killer cells.

By combining a newly established single-cell cytotoxicity assay in agarose (16) with estimations of the maximum natural killer (NK) potential (Vmax) by 51Cr release that percentage of target-binding cells (TBC), the fraction of active killers among TBC, the kinetics of single-cell cytotoxicity, and the recycling of effector cells was studied. Using nylon wool-passed peripheral lymphocytes, approximately 10% of the cells will bind to NK- susceptible target cell lines. Most of these have receptors for IgG. Some 50% will go on to kill T cell targets and some 20% to kill the standard target cell K-562. As the individual NK cell is shown to have the capacity to recycle, i.e., to kill more than one target cell in the 3-h test period, and as recycling seems to vary between individuals, there is no consistent correlation between the number of TBC and 51Cr-release values. It seems as if the single-cell cytotoxicity assay, as presently performed in agarose, is a valuable complement to Vmax determinations by 51Cr-release to study the different steps involved in the cytolytic process: recognition, enzyme activation, and effector cell recycling. The discrimination between these steps will probably be necessary to define mechanisms influencing NK cells in different disease states as well as in learning more about the normal function and regulation of the human NK system.

Target-effector interaction in the human and murine natural killer system: specificity and xenogeneic reactivity of the solubilized natural killer-target structure complex and its loss in a somatic cell hybrid.

Preincubation of natural killer (NK) cells with electrophoresis purified proteins from a variety of NK-sensitive murine and human tumor cells specifically prevented subsequent binding to the intact, homologous target cell. The NK-target structures (NK-TS) consisted of some or all of four characteristic molecular species, tentatively assigned molecular weights of 140K, 160K, 190K, and 240K (+/-10K) based on electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. When these NK-TS molecules were compared in cross-inhibition assays, the large 240K molecule most often carried the unique NK specificity, whereas the smaller 140K molecules cross-reacted between YAC, 136-6 and X-63 in the mouse and between Molt-4 and K562 in the human. Mouse NK cells recognised a different spectrum of NK-TS molecules than human NK cells. The control of NK-TS expression was partially revealed in a cloned, somatic cell hybrid bwtween an NK sensitive (YAC-IR) and insensitive (A9HT) cell line. The hybrid did not express NK-TS and did not bind to NK cells which is in accordance with negative NK cytolytic results previously reported. Although unique specificities are carried by some of the multiple NK-TS protein molecules, cross-reactions were widespread. These observations taken together suggest that the NK cell is polyspecific and has some heterogeneity in the recognition structure although much less than would be expected of an antibody-combining site.

Presence of clonal T cell populations in chronic B lymphocytic leukemia and smoldering myeloma.

Clonality in the non-neoplastic T cell population was investigated in 21 patients with B cell chronic leukemic (B-CLL) or multiple myeloma (MM) by probing for TCR beta chain gene rearrangements using Southern blot analysis. In three patients with a benign form of B-CLL (stage 0), and in one patient with smoldering MM, evidence was found for predominant T cell clones. As cellular immunity against the malignant cells may be important in leukemia, the results are discussed in view of the potential role of T cell immunity in B-CLL and MM.

Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells.

By using the two criteria (a) high density of immunoglobulin determinants on the cell surface and (b) presence of receptors for C'3 on the cell surface for defining bone marrow-derived lymphocytes, it is indirectly shown that all or at least a major population of human thymus-derived lymphocytes under certain conditions will form nonimmune rosettes with sheep red blood cells (SRBC). Almost all thymocytes tested from two different donors formed rosettes. The SRBC rosettes are not formed by virtue of immunoglobulin receptors and form only around living cells. Positive bivalent ions are required for rosette formation since EDTA will block rosette formation. Sodium iodoacetate will also block rosette formation demonstrating the dependence on an intact glycolytic pathway. Rosette formation is temperature dependent and will not appear at 37 degrees C. Trypsin treatment of lymphocytes will abolish their SRBC-binding ability which cannot be restored by treating them with fresh donor serum or fetal calf serum, but which will reappear after culturing the lymphocytes. It is suggested that these rosettes are formed by a rapidly released or metabolized receptor substance on the living cell surface which behaves as a trypsin-sensitive structure produced by the cells themselves.

Killer cells: a functional comparison between natural, immune T-cell and antibody-dependent in vitro systems.

Previous reports have shown that spleen cells from nonimmune adult mice of certain strains do regularly kill Moloney leukemia virus-induced lymphomas in short-term 51Cr release assays. This naturally occuring killer (NK) cell had low adherent properties and had the morphological appearance of a lymphocyte. Still it lacked surface characteristics of mature T or B lymphocytes. In the present report a functional study was carried out, comparing in parallel the NK system, the T-cell killing across an H-2 barrier (anti-P815), and the antibody-dependent cell-mediated chicken red blood cell (CRBC) system. In contrast to the effector cells in the CRBC system, the NK cells were insensitive to erythrocyte antibody complement (EAC) rosette depletion and would pass through nylon wool columns. NK activity was not inhibited by the presence of heat-aggregated human or mouse gamma globulin, in contrast to the strong inhibition noted in the CRBC system. Sensitivity to trypsin pretreatment was noted in the NK system as well as in the immune P815 system, whereas the CRBC system was relatively trypsin resistant. Antitheta plus complement eliminated the anti-P815 activity, but did not touch the NK activity. The present results thus further distinguish the NK cell from cytotoxic T lymphocytes or from antibody-dependent killer cells.

Selective inhibition of human T cell cytotoxicity at levels of target recognition or initiation of lysis by monoclonal OKT3 and Leu-2a antibodies.

Out of a panel of seven monoclonal antibodies with affinity for human lymphoid cells, three were shown to prevent cytotoxic T cell activity, whereas none affected natural killer cell activity when applied without complement. Anti-OKT3 and anti-Leu-2a, with affinity for all T cells and the cytotoxic/suppressive subset, respectively were both shown to inhibit T killing by their interaction with the effector cell. For anti-OKT3, the inhibition remained after free antibody was washed away. Anti-Leu-2a, in contrast, induced a rapidly reversible inhibition. Using a single cell assay, anti-OKT3 was shown to reduce the lytic ability without affecting target cell binding, whereas anti-Leu-2a prevented the effectors from binding target cells.

TLR9 activation increases TAP-independent vesicular MHC class I processing in vivo.

Cross-presentation of soluble protein antigens on major histocompatibility complex (MHC) class I by dendritic cells (DC) can occur in vesicular, endolysosomal compartments and be either dependent or independent of TAP peptide transporters. Here we investigate if an immunostimulatory CpG oligodeoxynucleotide can increase the activity in a TAP-independent endolysosomal vesicular pathway (el-VP) in vivo as we have earlier found in in vitro cultured DC. We use the in vivo response of CFSE labelled OT-1 T cells, transgenic for a T-cell receptor (TCR) that recognizes an ovalbumin (OVA)-derived peptide (SIINFEKL) presented by H-2K(b), transferred into TAP1(-/-) mice, as a functional read-out for activity in the el-VP. We have found a poor OT-1 T-cell response to soluble OVA which, however, could be strongly enhanced by the simultaneous administration of CpG. This increased responsiveness required both the endolysosomal cathepsin S (CatS) and Toll like receptor (TLR)9, the CpG receptor, both of which are present in the el-VP. Confocal microscopy demonstrated a co-localization of H-2K(b)/SIINFEKL and the endolysosomal marker LAMP1 in CD11c positive DC which was markedly increased by CpG administration. No complexes were found in the ER and cis-Golgi compartments in TAP1(-/-) mice, indicating the lack of classical MHC-I processing. In DC isolated from CatS(-/-) mice the opposite was found, complexes were present in the ER but not in the el-VP. We conclude that in vivo activation of TLR9 by CpG increases the efficiency of TAP independent el-VP and that this might contribute to the potent adjuvant activity of this type of compound. The cellular mechanisms remain to be established.

Regulation of calcium influx across the plasma membrane of the human T-leukemic cell line, JURKAT: dependence on a rise in cytosolic free calcium can be dissociated from formation of inositol phosphates.

A rise in the cytosolic free Ca2+ concentration due to both mobilization of Ca2+ from internal stores and influx of extracellular Ca2+ across the plasma membrane through 'second messenger-operated Ca2+ channels' is one of the first transmembrane signals detected following activation of CD2 or CD3 receptors on T-cells. In this study, we have further elucidated the regulation of these channels in the human T-leukemic cell line, JURKAT. Stimulation with either OKT3 or PHA induced a prompt influx of Ca2+ as assessed by MN2+ quenching of intracellular fura-2 fluorescence. When cytosolic free Ca2+ transient was partially buffered by loading the cells with BAPTA, neither agonist could induce Ca2+ entry into the cells as depicted by the lack of quenching of the fluorescence signal by Mn2+. This is in good agreement with our previous data on agonist-induced 45Ca2+ influx demonstrating that a rise in cytosolic free Ca2+ due to agonist-induced mobilization of Ca2+ from intracellular stores, could, directly or indirectly via the inositol cycle, initiate Ca2+ influx in these cells. Further support of this idea comes from the data demonstrating that agonist-induced mobilization of Ca2+ precedes the influx of Ca2+ across the plasma membrane. The present findings show that agonist-stimulation significantly increased the levels of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 after only 5 s, indicating that one or both of these substances could play a role in the regulation of Ca2+ influx. However, when agonist-induced Mn2+ influx was totally abolished, by partially buffering the cytosolic free Ca2+ rise, the formation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 was not affected. Consequently, the dependence of an initial rise in cytosolic free Ca2+ for the subsequent regulation of Ca2+ influx across the plasma membrane, can be dissociated from the formation of both Ins(1,4,5)P3 and Ins(1,3,4,5)P4.

CD3-dependent increase in cyclic AMP in human T-cells following stimulation of the CD2 receptor.

We have previously shown that stimulation of the Ti/CD3 receptor complex on human T-cells potentiates adenylate cyclase activation by adenosine or forskolin. Anti-CD2 receptor antibodies shared with anti-CD3 antibodies the ability to potentiate dose dependently the adenosine- and forskolin-stimulated cyclic adenosine monophosphate (cAMP) accumulation, whereas stimulation of the CD45 receptor had no effect on cyclase activity. Modulation of the CD3 complex with anti-CD3 antibodies was found to decrease the CD2 receptor effect on adenylate cyclase activity greatly. The possible involvement of CD3-stimulated phospholipase C (PLC) activation on the cAMP potentiation was examined using HPB-ALL cells that express a CD3 complex with a defect coupling to PLC. Stimulation of the CD3 complex on HPB-ALL cells had only slight effects on adenosine-stimulated cAMP formation, whereas the effect on forskolin-stimulated cAMP was virtually unchanged. The CD3 effect was further analyzed in Jurkat cell membranes. In contrast to the results obtained after stimulation of intact cells, it was found that OKT3 stimulation of membranes did not potentiate the forskolin response. Finally, we tested whether inhibition of endogenous adenylate cyclase agonist production affected the CD3 effect. Inhibition of adenosine production or adenosine breakdown with 8-p-sulphophenyl theophylline (8-PST) or adenosine deaminase (ADA), respectively, did not alter the CD3 effects. Indometacin, which inhibits prostaglandin production, also had no effect. Together, these data show that stimulation of the CD2 receptor potentiates adenylate cyclase responses by a mechanism that is dependent on CD3 expression. Furthermore, the CD3 effect on cAMP appears to be mediated by two different mechanisms, one which is, and one which is not dependent on PLC. Finally, this effect is not due to an endogenous production of adenylate cyclase agonists.

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase.

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca2+ concentration ([Ca2+]i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and alpha-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n-6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n-3 fatty acids (alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n-6 fatty acids (linoleic acid and arachidonic acid), the total n-3 fatty acyl content was reduced in all the phospholipids examined. In n-3 and n-6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n-9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appears to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca2+]i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n-3 and n-6 PUFA but not in n-9 monounsaturated fatty acid modified cells. In Ca2+ free medium, OKT3-induced transient increase in [Ca2+]i representing Ca2+ release from the inositol 1,4,5-trisphosphate-sensitive Ca2+ stores, were similar in control and UFA modified cells. Using Mn2+ entry as an index of plasma membrane Ca2+ permeability, the rate of fura-2 fluorescence quenching as a result of Mn2+ influx stimulated by OKT3 in n-9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n-3 and n-6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca2+ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and monounsaturated fatty acids appears to be important for the maintenance of a functional Ca2+ influx mechanism.

Regulation of receptor-mediated calcium influx across the plasma membrane in a human leukemic T-cell line: evidence of its dependence on an initial calcium mobilization from intracellular stores.

It has been repeatedly shown that stimulation of a human leukemic T-cell line, JURKAT, by lectins such as phytohaemagglutinin and anti-T3 antibody (OKT3) leads to an elevation in the concentration of cytosolic free Ca2. This Ca2+ transient results from both an intracellular mobilization and an influx of Ca2+ through specific membrane channels. The objective of this study was to investigate the mechanism by which receptor-mediated influx of Ca2+ is regulated in JURKAT cells, which demonstrably lack 'voltage-dependent calcium channels'. It was found that upon increased loading with quin2 or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) there was a pronounced decline of both phytohaemagglutinin-stimulated and OKT3-stimulated influx of 45Ca2+. Using 15 microM quin2/AM or 30 microM BAPTA/AM, agonist-stimulated 45Ca2+ influx was almost totally abolished. At these concentrations of both quin2/AM or BAPTA/AM, phytohaemagglutinin and OKT3 could still induce a rise of cytosolic free Ca2+ above 200 nM. In the presence of La3+ (200 microM), which completely inhibited the agonist-induced 45Ca2+ influx, both phytohaemagglutinin and OKT3 were able to raise the concentrations of cytosolic free Ca2+ to well above 200 nM by merely mobilizing Ca2+ from intracellular stores alone. The data suggest that an agonist-induced increase in the concentration of cytosolic free Ca2+, due to mobilization from intracellular stores, could either directly or indirectly, initiate receptor-mediated Ca2+ influx across the plasma membrane in JURKAT cells.

Mitogen stimulation of T-cells increases c-Fos and c-Jun protein levels, AP-1 binding and AP-1 transcriptional activity.

We have analysed the effect of mitogenic lectins on c-Fos and c-Jun protein levels as well as on activator protein-1 (AP-1) binding and enhancer activity in Jurkat T-cells. Both c-Fos and c-Jun protein levels were increased after Con A and PHA stimulation. Since T-cell stimulation increases both intracellular Ca2+ and cAMP levels and activates protein kinase C (PKC), the possible involvement of these intracellular messengers in c-Fos and c-Jun induction was tested. PMA, which directly activates PKC, mimicked the effect of the lectins on c-Fos and c-Jun, but elevation of either intracellular Ca2+ or cAMP levels had little or no effect. The mitogen-induced increase of c-Fos and c-Jun immunoreactivity was inhibited by H-7, a kinase inhibitor with relatively high specificity for PKC, and less efficiently by H-8, a structurally related kinase inhibitor less active on PKC, but more active on cyclic nucleotide-dependent kinases. Con A stimulation was found to increase both binding of AP-1 to the AP-1 consensus sequence, TRE, and AP-1 enhancer activity, in Jurkat cells. PMA was also found to increase the AP-1 enhancer activity, whereas elevation of Ca2+ or cAMP had only minor effects. We conclude that stimulation with mitogenic lectins is sufficient to increase both c-Fos and c-Jun protein levels, AP-1 binding and AP-1 enhancer activity in Jurkat cells and that they act via mechanisms that could involve the activation of PKC.

Stimulation of T-cells with OKT3 antibodies increases forskolin binding and cyclic AMP accumulation.

It has recently been shown that elevation of cAMP by adenosine receptor stimulation may be potentiated by stimulation of the T-cell receptor/CD3 complex on human T-cells with the monoclonal antibody OKT3, and that this is mimicked by activation of protein kinase C [Kvanta, A. et al. (1989) Naunyn-Schmeideberg's Arch. Pharmac. 340, 715-717]. In this study the diterpene forskolin, which binds to and activates the adenylate cyclase, has been used to examine further how the CD3 complex may influence the adenylate cyclase pathway. Stimulation with OKT3 alone was found to cause a small dose-dependent increase in basal cAMP accumulation. When combining OKT3 with a concentration of forskolin (10 microM), which by itself had little effect on the cyclase activity, the cAMP accumulation was markedly potentiated. This potentiation was paralleled by an increase in [3H]forskolin binding to saponine permeabilized Jurkat cells from 24 to 41 fmol/10(6) cells. The OKT3 effect on cAMP was blocked by chelating extracellular Ca2+ with EGTA or intracellular Ca2+ with BAPTA and also by W-7, an inhibitor of calmodulin, but was unaffected by H-7, an inhibitor of protein kinase C. Even though OKT3 caused an increase in inositolphosphate turnover, and activated protein kinase C, neither phorbol 12,13 dibutyrate (PDBu) nor the Ca2(+)-ionophore A23187 could mimic the OKT3 effect, whereas a combination of PDBu and A23187 at high concentrations could potentiate forskolin stimulated cyclase activity. Together, these results indicated that stimulation of the CD3 complex could influence the adenylate cyclase by two different mechanisms, one involving activation of protein kinase C and another which does not.

Leukemic cells from progressive B-CLL respond strongly to growth stimulation in vitro.

Isolated leukemic B cells from patients with B-chronic lymphocytic leukemia (B-CLL) were tested for proliferative response in vitro to Staphylococcus Aureus strain Cowan 1 (SAC), IL-2, and low molecular weight (MW) BCGF. Patients were classified according to clinical stage and progressiveness. Ten of eighteen cell populations from patients with progressive B-CLL responded in vitro with a stimulation index (SI) > 20. Only 1/16 non-progressive patients had a proliferative but low response. Normal unfractionated tonsillar B cells responded to SAC and BCGF, whereas normal high buoyant density B cells were unresponsive. After 3 days of stimulation, responding B-CLL cells had multiplied and the B cells expressed CD5, CD19, and weakly CD21. No cells in the responding cultures exhibited CD3 or the EBV nuclear antigen EBNA-1. Cell maturation, measured as IgM secretion, was found in some, but not in all responding B-CLL cultures. Thus, B-CLL cells from patients with progressive disease have the capacity to respond to signaling through surface Ig receptors and to certain T-cell factors which was not the case for B-CLL cells from non-progressive patients. The pattern of in vitro response may be related to disease progression, reflecting a dependency of normal immunoregulatory mechanisms and/or a dysregulation of the growth control in the leukemic cells.

Sensitization to TRAIL-induced apoptosis and modulation of FLICE-inhibitory protein in B chronic lymphocytic leukemia by actinomycin D.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent activator of the cell death pathway and exerts tumoricidal activity in vivo with minimal toxicity. In order to investigate the therapeutic potential of TRAIL in B chronic lymphocytic leukemia (B-CLL) we have analyzed the expression of TRAIL receptors (TRAIL-Rs) in leukemic cells from B-CLL patients and their in vitro sensitivity to apoptosis induced by recombinant human TRAIL. We have found TRAIL-R1 and -R2 death receptor, and TRAIL-R3 and -R4 decoy receptor mRNA expression in most of the 57 B-CLL patients studied (R1 82%, R2 100%, R3 96% and R4 82%). TRAIL-R1 and R2 proteins were expressed on the surface and within the cells, whereas R3 and R4 decoy receptors were almost exclusively expressed in the cytoplasm. Despite TRAIL death receptor expression, B-CLL cells were relatively resistant to induction of apoptosis by recombinant human TRAIL (300 ng/ml). However, the susceptibility to TRAIL-induced apoptosis was increased by treatment of B-CLL cells with actinomycin D (Act D). Western blot analysis showed higher constitutive expression of the long form of FLICE-inhibitory protein (FLIP(L)) in B-CLL as compared to normal tonsillar B cells. Act D treatment down-regulated both long and short FLIP expression, which was correlated with the increase in B-CLL sensitivity to TRAIL. Although the surface TRAIL death receptor expression was up-regulated both by cell culture and by Act D treatment, the changes were not correlated with a gain in susceptibility to TRAIL. In addition, neither decoy receptors nor Bcl-2 expression were affected by Act D. Our findings suggest the possible involvement of FLIP in regulating TRAIL-mediated apoptosis in B-CLL.

Expression and transcriptional regulation of functionally distinct Bmf isoforms in B-chronic lymphocytic leukemia cells.

Bmf is a BH3-only Bcl-2 family member that is normally sequestered to myosin V motors by binding to the dynein light chain 2 (DLC2). Certain damage signals release Bmf, which then binds prosurvival Bcl-2 proteins and triggers apoptosis. Here, two novel isoforms of human Bmf, Bmf-II and Bmf-III, were identified and cloned from cDNA derived from B-chronic lymphocytic leukemia (B-CLL) cells. Bmf-II and Bmf-III were characterized as two splice variants, lacking the BH3 domain but retaining the DLC2 binding domain. Bmf (here called Bmf-I) expression in HeLa cells induced apoptosis and reduced colony formation in contrast to Bmf-II and Bmf-III, which had no effect on apoptosis and instead increased colony formation. While bmf-I mRNA was expressed in many cell types, expression was higher in B lymphoid cells and bmf-II and bmf-III were mainly detected in B-CLL and normal B cells. bmf-I mRNA was upregulated in normal and leukemic B cells, while bmf-III mRNA was downregulated only in B-CLL cells by serum deprivation. We show that Bmf is regulated by transcriptional activation and alternative splicing and conclude that the relative levels of Bmf isoforms may have a role in regulating growth and survival in B cells and leukemic B-CLL cells.

Diagnostic implication of specific immunoglobulin G patterns of children born to HIV-infected mothers.

We analysed HIV-specific immunoglobulin G (IgG) responses to gag and env peptides in infants born to HIV-positive mothers. Questions of interest were whether there are early specific markers for prognosis, and whether any specific IgG is related to the prevention of vertical transmission of infection. Fifty-three children, 0-24 months old and born to HIV-1-infected mothers, were retrospectively divided into two groups based on HIV seroreactivity or non-reactivity at 15 months of age. Their sera were used to find reactivities important in diagnosis and/or prediction of the putative HIV disease. Three important findings emerged. First, a low IgG titer against the very immunodominant penv9 in newborns was found to be associated with rapid progression to AIDS. This difference was clearly reflected in the reactivity to a small peptide representing amino acid (aa) 598-606. The second interesting finding was the putative hypervariable loop on gp120 (especially aa 324-338), reactivity to which was found only in the uninfected group, and was seen in six out of 19 uninfected children under 6 months of age. This specific response was not caused by a generally high total anti-HIV reactivity, and may indicate a role of protective antibodies against vertical transmission. The response to this region in the infected group, on the other hand, was directed to the amino terminal half of the putative loop, in particular peptide 53, aa 304-318. Finally, response to a part of the amino terminal end of P17 was seen in seven out of eight infected children over 6 months of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ entry in T cells is activated by emptying the inositol 1,4,5-triphosphate sensitive Ca2+ pool.

Using alpha-linolenic acid (ALA), one of several polyunsaturated fatty acids (PUFAs) that have previously been shown to both mobilize intracellular Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool independently of IP3 production and inhibit Ca2+ influx, the relationship between Ca2+ mobilization from intracellular stores and Ca2+ influx in T cells (JURKAT) was studied. JURKAT cells were treated with 30 microM ALA to deplete the IP3-sensitive Ca2+ pool. When the intracellular free Ca2+ concentration [( Ca2+]i) returned to basal level, fatty acid free bovine serum albumin (BSA) was added to remove extracellular and membrane bound ALA. This resulted in a sustained increase in [Ca2+]i in the absence of inositol phosphates' formation. This sustained increase in [Ca2+]i was insensitive to protein kinase C activation but was inhibited by Ni2+ ions. The extent of Ca2+ influx was found to be correlated to the amount of Ca2+ initially discharged from the IP3-sensitive Ca2+ pool by sub-optimal concentrations of ALA. Ligation of the CD3 complex of the T cell antigen receptor with an anti-CD3 antibody (OKT3) during the sustained [Ca2+]i increased (induced by a sub-optimal concentration of ALA), produced a greater response. No increase in the sustained response was observed when the CD3 complex was activated in cells pretreated with an optimal concentration of ALA. In summary, Ca2+ entry in T cells is activated by emptying of the IP3-sensitive Ca2+ pool which can be dissociated from inositol phosphate production. The rate of Ca2+ influx appears to be closely correlated to the initial discharge of Ca2+ from the IP3-sensitive Ca2+ pool, suggesting that Ca2+ may first enter the depleted pool and then is released into the cytosol.