Lectin-dependent and anti-CD3 induced cytotoxicity are preferentially mediated by peripheral blood cytotoxic T lymphocytes expressing Leu-7 antigen

Monoclonal antibodies against the CD3 antigen and certain lectins can induce interleukin 2 dependent antigen-specific T cell clones to mediate non-antigen specific cytotoxicity. On the basis of this observation, we predicted that it may be possible to identify cytotoxic T lymphocytes (CTL) in peripheral blood without knowing the antigen specificity of these in vivo primed CTL. By using this strategy, peripheral blood lymphocytes were separated into low and high-density fractions on Percoll gradients and were tested for cytotoxic activity in the presence or absence of concanavalin A (Con A) or anti-Leu-4 antibody. Lectin-dependent cellular cytotoxicity (LDCC) and anti-CD3 induced cytotoxicity against both natural killer (NK)-insensitive and NK-sensitive targets were exclusively mediated by low-density CD3+ T lymphocytes. Additional studies indicated that low-density CD3+ T lymphocytes co-expressing Leu-7 antigen preferentially mediated this activity, although in some individuals, significant activity was also observed in the low-density T cells lacking Leu-7. In contrast, high-density CD3+ T lymphocytes and CD16+ (Leu-11+) NK cells (both Leu-7 and Leu-7+) did not mediate nonantigen-specific cytotoxicity under these conditions. The finding that NK cell-mediated cytotoxicity was unaffected by these lectins refutes the hypothesis that lectin-dependent cellular cytotoxicity is simply a result of effector and target agglutination. T cell-mediated cytotoxicity was both lectin and antibody specific. Phytohemagglutinin, Con A, and pokeweed mitogen induced cytolytic activity in the Leu-7+ T cells, whereas wheat germ agglutinin did not. Of the antibodies against T cell-associated differentiation antigens (anti-Leu-2,3,4, and 5), only anti-Leu-4 induced cytotoxicity. This anti-CD3-induced cytotoxicity was essentially completely inhibited by the presence of anti-LFA-1 or anti-CD2 monoclonal antibodies, implicating these molecules in the triggering process. A proportion of the CD3+, Leu-7+ CTL expressed HLA-DR antigens, indicating possible in vivo activation. Because previous clinical studies have indicated that lymphocytes with this phenotype may be elevated in clinical situations associated with immunosuppression and chronic viral infection, this unique subset of CD3+ T lymphocytes may represent a population of in vivo primed CTL possibly against viral antigens.     

Identification of the Drosophila core 1 beta1,3-galactosyltransferase gene that synthesizes T antigen in the embryonic central nervous system and hemocytes

T antigen (Galbeta1-3GalNAcalpha1-Ser/Thr), the well-known tumor-associated antigen, is a core 1 mucin-type O-glycan structure that is synthesized by core 1 beta1,3-galactosyltransferase (C1beta3GalT), which transfers Gal from UDP-Gal to Tn antigen (GalNAcalpha1-Ser/Thr). Three putative C1beta3GalTs have been identified in Drosophila. However, although all three are expressed in embryos, their roles during embryogenesis have not yet been clarified. In this study, we used P-element inserted mutants to show that CG9520, one of the three putative C1beta3GalTs, synthesizes T antigen expressed on the central nervous system (CNS) during embryogenesis. We also found that T antigen was expressed on a subset of the embryonic hemocytes. CG9520 mutant embryos showed the loss of T antigens on the CNS and on a subset of hemocytes. Then, the loss of T antigens was rescued by precise excision of the P-element inserted into the CG9520 gene. Our data demonstrate that T antigens expressed on the CNS and on a subset of hemocytes are synthesized by CG9520 in the Drosophila embryo. In addition, we found that the number of circulating hemocytes was reduced in third instar larvae of CG9520 mutant. We, therefore, named the CG9520 gene Drosophila core 1 beta1,3-galactosyltransferase 1 because it is responsible for the synthesis and function of T antigen in vivo.     

Ontogenetic development of convulsant action of Ro 5-3663 in the rat

Motor seizures were induced by Ro 5-3663 in 156 male albino rats aged 7,12,18,25, and 90 days. Both minimal and maximal seizures could be elicited in 18-day-old and older animals, whereas only maximal seizures were induced in the two youngest groups. ECoG changes were studied in other 21 young rats. First changes induced by Ro 5-3663 were formed by isolated sharp waves in 7- and 12-day-old rats and by episodes of rhythmic activity in older animals. An imperfect form of this rhythmic activity could be seen even in 12-day-old rats. Ictal ECoG activity exhibited an increase in frequency of individual graphoelements, in generalization and in synchronization of activity among different cortical regions with maturation. Correlation between motor and ECoG phenomena was poor in 7-day-old rats and ameliorated with age but it never reached perfection. The actions of Ro 5-3663 are identical with those induced by metrazol but they differ from those elicited by bicuculline or 3-mercaptopropionic acid.     

Analysis of the inhibition potential of zosuquidar derivatives on selected bacterial and fungal ABC transporters

Abstract

The increasing number of multidrug-resistant pathogenic microorganisms is a serious public health issue. Among the multitude of mechanisms that lead to multidrug resistance, the active extrusion of toxic compounds, mediated by MDR efflux pumps, plays an important role. In our study we analyzed the inhibitory capability of 26 synthesized zosuquidar derivatives on three ABC-type MDR efflux pumps, namely Saccharomyces cerevisiae Pdr5 as well as Lactococcus lactis LmrA and LmrCD. For Pdr5, five compounds could be identified that inhibited rhodamine 6G transport more efficiently than zosuquidar. One of these is a compound with a new catechol acetal structure that might represent a new lead compound. Furthermore, the determination of IC₅₀ values for rhodamine 6G transport of Pdr5 with representative compounds reveals values between 0.3 and 0.9 μM. Thus the identified compounds are among the most potent inhibitors known for Pdr5. For the ABC-type efflux pumps LmrA and LmrCD from L. lactis, seven and three compounds, which inhibit the transport activity more than the lead compound zosuquidar, were found. Interestingly, transport inhibition for LmrCD was very specific, with a drastic reduction by one compound while its diastereomers showed hardly an effect. Thus, the present study reveals new potent inhibitors for the ABC-type MDR efflux pumps studied with the inhibitors of Pdr5 and LmrCD being of particular interest as these proteins are well known model systems for their homologs in pathogenic fungi and Gram-positive bacteria.     

In vivo induced antigen technology (IVIAT)

In vivo induced antigen technology (IVIAT) is a technique that identifies pathogen antigens that are immunogenic and expressed in vivo during human infection. IVIAT is complementary to other techniques that identify genes and their products expressed in vivo. Genes and gene pathways identified by IVIAT may play a role in virulence or pathogenesis during human infection, and may be appropriate for inclusion in therapeutic, vaccine or diagnostic applications.     

Characterization of human K cells by surface antigens and morphology at the single cell level

Human lymphocytes killing bovine erythrocytes in vitro in antibody-mediated reactions were characterized at the effector cell level in the ADCC plaque assay. Five to 10% of highly purified peripheral blood lymphocytes are active K cells in this system. Forty to 50% of these were T gamma cells expressing the T cell-associated surface antigens T3 and Leu-1. These cells also expressed the T8/Leu-2a antigens (approximately 20%) or the T4/Leu-3a antigens. Although approximately 30% of the K cells were T4+ when examined after completion of the ADCC assay (18 hr), only less than 10% were T4+ (and Leu-3a+) when examined before the assay. The results indicated that exposure to antigen/antibody complexes during the assay induced increased T4 expression, probably linked to Fc gamma R modulation on some initially T4-/T3+ lymphocytes. The expression of the other antigens (including Leu-3a) was not affected by exposure of the lymphocytes to antigen/antibody complexes. Two-color fluorescence experiments further demonstrated that a minor fraction (10 to 20%) of the K cells carrying T cell-associated antigens also expressed the monocyte/null cell-associated antigen M1 as detected with the monoclonal antibody OKM1. A second major category of effector cells, composed of at least 25% of the K cells, were large granular lymphocytes (LGL) that lacked detectable T cell-associated antigens but expressed the HNK-1 (Leu-7) as well as the M1 antigen. As seen from the size of the plaques formed by different effector cells, K cells of the LGL type had a greater recycling capacity and/or cytolytic efficiency than those of T gamma type.     

High-affinity vanadate transport system in the cyanobacterium Anabaena variabilis ATCC 29413

High-affinity vanadate transport systems have not heretofore been identified in any organism. Anabaena variabilis, which can fix nitrogen by using an alternative V-dependent nitrogenase, transported vanadate well. The concentration of vanadate giving half-maximum V-nitrogenase activity when added to V-starved cells was about 3 x 10(-9) M. The genes for an ABC-type vanadate transport system, vupABC, were found in A. variabilis about 5 kb from the major cluster of genes encoding the V-nitrogenase, and like those genes, the vupABC genes were repressed by molybdate; however, unlike the V-nitrogenase genes the vanadate transport genes were expressed in vegetative cells. A vupB mutant failed to grow by using V-nitrogenase unless high levels of vanadate were provided, suggesting that there was also a low-affinity vanadate transport system that functioned in the vupB mutant. The vupABC genes belong to a family of putative metal transport genes that include only one other characterized transport system, the tungstate transport genes of Eubacterium acidaminophilum. Similar genes are not present in the complete genomes of other bacterial strains that have a V-nitrogenase, including Azotobacter vinelandii, Rhodopseudomonas palustris, and Methanosarcina barkeri.     

B7, a B-cell-restricted antigen that identifies preactivated B cells

After activation with antigen or mitogen, a number of cell surface proteins appear that are not expressed on resting B cells. To date, a number of B lineage restricted and associated activation antigens have been reported that appear at distinct intervals after in vitro activation. In this report, we describe a new B lineage restricted activation antigen (B7) that appears within 24 hr of in vitro stimulation. The expression of B7 antigen, which is detected on a minor subpopulation of B cells isolated from peripheral blood and lymphoid tissues, is strongly induced following stimulation with either anti-immunoglobulin or Epstein-Barr virus. In contrast, B7 was not detected on resting or activated T cells or monocytes. The B7 antigen was expressed on a subset of B cell lines and B cell neoplasms, but was not detected on leukemias and lymphomas of T cell or myeloid origin. B7 was distinguished from other B cell restricted and associated activation antigens by its unique pattern of expression on a variety of hemopoietic cell lines. The biochemical characterization of B7, that it is a single chain protein of 60 kDa, further distinguishes it from other B cell activation antigens. The functional importance of the B7 antigen was demonstrated when splenic B cells were fractionated into the B7+ and B7- populations. The peak of proliferation in response to anti-Ig, appeared earlier within the B7+ population. These studies suggest that B7 antigen identifies a subpopulation of B cells that are preactivated or primed in vivo, and have an accelerated response to subsequent activation via cross-linking of surface Ig.     

Characterization of the Rhizobium (Sinorhizobium) meliloti high- and low-affinity phosphate uptake systems.

Genetic studies have suggested that Rhizobium (Sinorhizobium) meliloti contains two distinct phosphate (Pi) transport systems, encoded by the phoCDET genes and the orfA-pit genes, respectively. Here we present data which show that the ABC-type PhoCDET system has a high affinity for Pi (Km, 0.2 microM) and that Pi uptake by this system is severely inhibited by phosphonates. This high-affinity uptake system was induced under Pi-limiting conditions and was repressed in the presence of excess Pi. Uptake via the OrfA-Pit system was examined in (i) a phoC mutant which showed increased expression of the orfA-pit genes as a result of a promoter-up mutation and (ii) a phoB mutant (PhoB is required for phoCDET expression). Pi uptake in both strains exhibited saturation kinetics (Km, 1 to 2 microM) and was not inhibited by phosphonates. This uptake system was active in wild-type cells grown with excess Pi and appeared to be repressed when the cells were starved for Pi. Thus, our biochemical data show that the OrfA-Pit and PhoCDET uptake systems are differentially expressed depending on the state of the cell with respect to phosphate availability.     

Human lymphocyte differentiation antigens HB-10 and HB-11. I. Ontogeny of antigen expression

T, B, and NK cells appear to represent separate lymphocyte lineages, but indirect evidence suggests that they may be related via a common lymphoid precursor cell. We have produced two monoclonal antibodies, HB-10 (IgM) and HB-11 (IgG1), by fusing spleen cells from mice immunized with the human B cell line SB, and have shown that both antibodies react with lymphocyte-specific cell surface antigens present on T, B, and NK cells, but not on other types of blood cells. The antibodies were reactive with most cell lines and malignancies of B cell origin and with some of T and NK cell lineage. Although the populations of cells expressing these two antigens were virtually identical, the HB-10 and HB-11 antibodies identified separate protease-sensitive determinants on the cell surface. The HB-11 antigenic determinant was also sensitive to neuraminidase and periodate treatments, but the HB-10 determinant was not. Antigen expression by lymphocytes from fetal, newborn, and adult tissues was examined. Within the B cell lineage, these antigens were expressed by most pre-B cells in bone marrow (88% +/- 5) and almost all B cells, but were not expressed by mature plasma cells. Virtually all of the granular lymphocytes in blood marked by the Leu-7 and Leu-11 (anti-Fc receptor) antibodies were HB-10+ and 11+. Among T lineage cells, the HB-10 and 11 antigens were expressed by a subset of relatively mature T3+ thymocytes and by greater than 90% of the T cells in newborn blood. In adults, however, only 65% of blood T cells and 24 to 30% of splenic or tonsillar T cells expressed the HB-10 and HB-11 antigens. The postnatal emergence of T cells which, like plasma cells, do not express these antigens suggests that post-thymic T lymphocyte maturation occurs and may be an activation-dependent process.     

Interleukin-15 rescues tolerant CD8+ T cells for use in adoptive immunotherapy of established tumors

CD8+ T cells can mediate eradication of established tumors, and strategies to amplify tumor-reactive T-cell numbers by immunization or ex vivo expansion followed by adoptive transfer are currently being explored in individuals with cancer. Generating effective CD8+ T cell-mediated responses to tumors is often impeded by T-cell tolerance to relevant tumor antigens, as most of these antigens are also expressed in normal tissues. We examined whether such tolerant T cells could be rescued and functionally restored for use in therapy of established tumors. We used a transgenic T-cell receptor (TCR) mouse model in which peripheral CD8+ T cells specific for a candidate tumor antigen also expressed in liver are tolerant, failing to proliferate or secrete interleukin (IL)-2 in response to antigen. Molecular and cellular analysis showed that these tolerant T cells expressed the IL-15 receptor alpha chain, and could be induced to proliferate in vitro in response to exogenous IL-15. Such proliferation abrogated tolerance and the rescued cells became effective in treating leukemia. Therefore, high-affinity CD8+ T cells are not necessarily deleted by encounter with self-antigen in the periphery, and can potentially be rescued and expanded for use in tumor immunotherapy.     

T Cell Responses Induced by Adenoviral Vectored Vaccines Can Be Adjuvanted by Fusion of Antigen to the Oligomerization Domain of C4b-Binding Protein

Viral vectored vaccines have been shown to induce both T cell and antibody responses in animals and humans. However, the induction of even higher level T cell responses may be crucial in achieving vaccine efficacy against difficult disease targets, especially in humans. Here we investigate the oligomerization domain of the α-chain of C4b-binding protein (C4 bp) as a candidate T cell “molecular adjuvant” when fused to malaria antigens expressed by human adenovirus serotype 5 (AdHu5) vectored vaccines in BALB/c mice. We demonstrate that i) C-terminal fusion of an oligomerization domain can enhance the quantity of antigen-specific CD4⁺ and CD8⁺ T cell responses induced in mice after only a single immunization of recombinant AdHu5, and that the T cells maintain similar functional cytokine profiles; ii) an adjuvant effect is observed for AdHu5 vectors expressing either the 42 kDa C-terminal domain of Plasmodium yoelii merozoite surface protein 1 (PyMSP1₄₂) or the 83 kDa ectodomain of P. falciparum strain 3D7 apical membrane antigen 1 (PfAMA1), but not a candidate 128kDa P. falciparum MSP1 biallelic fusion antigen; iii) following two homologous immunizations of AdHu5 vaccines, antigen-specific T cell responses are further enhanced, however, in both BALB/c mice and New Zealand White rabbits no enhancement of functional antibody responses is observed; and iv) that the T cell adjuvant activity of C4 bp is not dependent on a functional Fc-receptor γ-chain in the host, but is associated with the oligomerization of small (<80 kDa) antigens expressed by recombinant AdHu5. The oligomerization domain of C4 bp can thus adjuvant T cell responses induced by AdHu5 vectors against selected antigens and its clinical utility as well as mechanism of action warrant further investigation.     

Specific Schistosoma mansoni rat T cell clones. I. Generation and functional analysis in vitro and in vivo

In an attempt to determine the role of schistosome-specific T cells in the immune mechanisms developed during schistosomiasis, Schistosoma mansoni-specific T cells and clones were generated in vitro and some of their functions analyzed in vitro and in vivo in the fischer rat model. The data presented here can be summarized as follows: a) Lymph node cells (LNC) from rats primed with the excretory/secretory antigens-incubation products (IPSm) of adult worms proliferate in vitro only in response to the homologous schistosome antigens and not to unrelated antigens (Ag) such as ovalbumin (OVA) or Dipetalonema viteae and Fasciola hepatica parasite extracts. b) After in vitro restimulation of the primed LNC population with IPSm in the presence of antigen-presenting cells (APC) and maintenance in IL 2-containing medium, the frequency of IPSm-specific T cells is increased and the T cells can be restimulated only in the presence of APC possessing the same major histocompatibility complex (MHC) antigens. c) Following appropriate limiting dilution assays (LDA) (1 cell/well), 10 IPSm-specific T cell clones were obtained, and two of four maintained in culture were tested for their helper activity because they expressed only the W3/13+ W3/25+ surface phenotypes. d) The two highly proliferating IPSm-specific T cell clones (G5 and E23) exhibit an IPSm-dependent helper activity, as shown by the increase in IgG production by IPSm-primed B cells. e) IPSm-T cell clone (G5) as well as IPSm-T cell lines when injected in S. mansoni-infested rats can exert an in vivo helper activity, which is characterized by an accelerated production of IgG antibodies specific for the previously identified 30 to 40 kilodaltons (kd) schistosomula surface antigens (Ag). As recent studies have demonstrated that rat monoclonal antibodies recognize some incubation products of adult S. mansoni as well as one of the 30 to 40 kd schistosomula surface antigens, and taking into account the fact that the T cell clones here studied were restimulated either with IPSm or with schistosomulum Ag, it appears that such IPSm-specific T cell clones could be involved in the concomitant immunity mechanisms.     

Extracellular complementation of a developmental mutation implicates a small sporulation protein in aerial mycelium formation by S. coelicolor

The filamentous bacterium S. coelicolor differentiates by forming aerial hyphae, which protrude into the air and metamorphose into chains of spores. Aerial hyphae formation is associated with the production of a small, abundant protein, SapB, which is present in a zone around colonies of differentiating bacteria. Production of SapB is impaired in bld mutants, which are blocked in aerial hyphae formation, but not in whi mutants in which spore formation is prevented. We report that aerial hyphae formation by a newly identified bld mutant is restored by juxtaposition of the mutant near colonies of SapB-producing bacteria or by the application of the purified protein near mutant colonies. These observations implicate SapB in aerial mycelium formation and suggest that SapB is a morphogenetic protein that enables hyphae on the surface of colonies to grow into the air.