Inhibition of Ly-6A antigen expression prevents T cell activation.

Antisense oligonucleotides complementary to the 5' end of the mRNA encoding the Ly-6A protein were used to block the expression of that protein. Using this approach we could inhibit the expression of Ly-6A by 60-80% in antigen-primed lymph node (LN) T cells as well as in the D10 T cell clone. Inhibition of Ly-6 expression resulted in the inability to restimulate in vitro, antigen-primed T cells. It also blocked the activation of normal spleen cells by Con A, monoclonal antibody (mAb) to CD3, and mAb to Ly-6. In contrast, stimulation of normal spleen cells with the pharmacological agents PMA + ionomycin were unaffected by the inhibition of Ly-6 expression. Similar results were obtained with the D10 T cell clone; stimulation with Con A + interleukin 1 (IL-1), antigen-presenting cells (APC), or the clonotypic antibody + IL-1 was greatly reduced in the presence of antisense oligonucleotides to Ly-6. Stimulation with PMA + ionomycin was again unaffected. We also studied the effect of antisense oligonucleotides on stimulation of preactivated D10 cells. Preactivation of D10 cells with Con A + IL-1 renders them receptive to secondary stimulation by other lymphokines. In this case, antisense oligonucleotides to Ly-6 had no effect on secondary activation with IL-2, IL-4 + IL-1, or PMA + ionomycin. We conclude from these studies that Ly-6 expression is required for T cell receptor (TCR)-mediated T cell activation.

High mutation rate of a spleen necrosis virus-based retrovirus vector.

Spleen necrosis virus (SNV) is an avian retrovirus that efficiently infects some mammalian cells (e.g., dog and rat cells). We constructed an SNV-based vector, which contains less than 1 kilobase (kb) of the retrovirus sequence, and a number of derivatives containing selectable markers. We obtained high-titer virus stocks, over 10(6) transforming units per ml, with a vector whose genomic RNA consists of 1,850 bases (full-length SNV RNA is 7.7 kb). We also studied two vectors that both carry two genes which should be expressed from a single promoter, one gene from unspliced mRNA and the other gene from spliced mRNA. In one vector, both genes were efficiently expressed as expected. However, in the other vector, expression of the gene 3' to the splice acceptor was inhibited. When we selected for expression of the 3' gene is this latter case, we found that the resistant cells contained mutant proviruses in which the 3' gene could be expressed. Furthermore, we found that mutations were generated during a single round of virus replication (provirus to provirus) at a rate of approximately 0.5% mutations per cycle.

The mouse immunoglobulin heavy-chain gene enhancer contains sequences that inhibit transcription in vitro in HeLa cell extracts.

The effect of the cell-specific mouse immunoglobulin heavy-chain gene (IgH) enhancer on transcription from heterologous promoter elements was studied in vitro with a HeLa whole-cell extract. No stimulation of transcription could be seen under conditions in which an activation was observed with the simian virus 40 enhancer. We found, however, that a specific segment of the IgH enhancer region contains sequences which inhibit transcription in vitro.

Mechanisms of retroviral mutation.

Retroviruses, like other RNA viruses, mutate at very high rates (0.05-1 mutations per genome per replication cycle) and exist as complex genetically heterogeneous populations ('quasispecies') that are ever changing. De novo mutations are generated by inherently error-prone steps in the retroviral life cycle that introduce base substitutions, frame shifts, genetic rearrangements and hypermutations.

A murine model for genetic manipulation of the T cell compartment.

The expression of exogenous genes in long-lived primary T cells is potentially beneficial for the treatment of various diseases including cancer, AIDS, genetic defects of the lymphoid compartment, and systemic enzyme deficiencies such as hemophilia. One approach for genetic modification of T cells is to introduce therapeutic genes into hematopoietic stem cells that would give rise to cells of the lymphoid lineage. Efficient gene transfer and expression in stem cells is often problematic, however. A more direct approach is to introduce the genes into mature primary T lymphocytes since the transferred genes can be maintained and expressed for long periods by long-lived peripheral T cells. In this report, we describe the adoptive transfer into SCID mice of both murine and human primary T cells that have been efficiently transduced with exogenous genes. Primary murine T cells transduced with a retroviral vector containing the human adenosine deaminase (ADA) gene persisted for at least 5 months in lymphoid organs of SCID mice, continuously expressing the exogenous gene. Primary human T cells were also used as target cells for transfer of the beta-galactosidase (lacZ) gene. Expression of the lacZ gene could be detected in over 20% of the transduced primary T cells before adoptive transfer into SCID mice. Transduced human T cells were injected into SCID mice intraperitoneally (ip), and the beta-galactosidase activity could be detected in cells collected from peritoneal exudate washes of recipient mice 6 weeks post-injection. These results demonstrate the availability of a murine model in which the long-term effects of expression of exogenous genes in both murine and human T cells can be tested.

N2- and C8-substituted oligodeoxynucleotides with enhanced thrombin inhibitory activity in vitro and in vivo.

2'-Deoxyguanosine (G) analogues carrying various hydrophobic substituents in the N2 and C8 positions were synthesized and introduced through solid-phase synthesis into 15-mer oligodeoxynucleotide, GGTTGGTGTGGTTGG, which forms a chairlike structure consisting of two G-tetrads and is a potent thrombin inhibitor. The effects of the substitutions at N2 and C8 of the G-tetrad-forming G residues on the thrombin inhibitory activity are relatively small, suggesting that these substitutions cause relatively small perturbations on the chairlike structure formed by the oligodeoxynucleotide. Introduction of a benzyl group into N2 of G6 and G11 and naphthylmethyl groups into N2 of G6 increased the thrombin inhibitory activity, whereas other substituents in these positions had almost no effect or decreased the activity. Particularly, the oligodeoxynucleotide carrying a 1-naphthylmethyl group in the N2 position of G6 showed an increase in activity by about 60% both in vitro and in vivo. Substitutions on the N2 position of other G residues had little effect or decreased the activity. Introduction of a relatively small group, such as methyl and propynyl, into the C8 positions of G1, G5, G10, and G14 increased the activity, presumably due to the stabilization of a chairlike structure, whereas introduction of a large substituent group, phenylethynyl, decreased the activity, probably due to the steric hindrance.

Synergy between encephalitogenic T cells and myelin basic protein-specific antibodies in the induction of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an experimentally induced demyelinating disease mediated by CD4+ T cells specific for various myelin proteins including myelin basic protein (MBP) and myelin proteolipid protein (PLP). Although myelin- and other CNS-specific antibodies are produced in EAE, B cells and antibodies are thought by most not to play a decisive role in the induction of EAE. In this report we show that B cells serve as the major antigen-presenting cells (APC) during the T cell activation stage in lymph nodes, and that MBP-specific antibodies can greatly enhance the induction of EAE. The role of B cells as APC is demonstrated in B cell-depleted mice. EAE cannot be induced by antigen/complete Freund's adjuvant immunization unless these mice are locally reconstituted with B cells prior to immunization. The enhancing effect of antibodies is demonstrated in experiments in which EAE is induced by the adoptive transfer of encephalitogenic T cells. The adoptive transfer of large numbers of encephalitogenic T cells induces EAE in 90% of normal recipient mice, but only 33% of B cell-depleted mice get EAE at the same cell dose. The efficiency of EAE induction in B cell-depleted mice can be enhanced if MBP-specific antibodies are simultaneously administered. A similar enhancement is also seen in normal mice when the number of adoptively transferred T cells is limiting. We propose that MBP-specific antibodies enhance the presentation of myelin-derived antigens by APC in the CNS to the adoptively transferred encephalitogenic T cells.

Both anti-CD11a (LFA-1) and anti-CD11b (MAC-1) therapy delay the onset and diminish the severity of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system (CNS) induced in rodents by activated CD4+ T cells specific for various myelin proteins such as myelin basic protein and proteolipid protein. The disease is characterized by breach of the blood-brain barrier, perivascular infiltration of leukocytes into the CNS, local inflammation and demyelination in the form of plaques. In this study, we evaluated the effect of administration of antibodies to two members of the beta 2 integrin sub-family of adhesion molecules, CD11a and CD11b, on the onset and progression of EAE. CD11a and CD11b are involved in cell-cell interactions leading to T cell and macrophage extravasation to inflammatory sites and T cell activation. Our results show that anti-CD11a antibodies could completely block the induction of EAE and anti-CD11b antibodies significantly delayed the onset and diminished the severity of clinical signs of EAE even when injections were initiated at the first appearance of clinical signs.

Laser-excited atomic-fluorescence spectrometry in an electrothermal atomizer with Zeeman background correction.

A pulsed excimer-pumped dye laser was used to excite atomic flourescence in graphite tube electrothermal atomizer. A 60-Hz ac magnitude field was applied around the atomizer and parallel to the excitation beam, for Zeeman background correction. The correction system was found to degrade the detection limits for silver, cobalt, indium, manganese, lead, and thallium by a factor of between 1 and 10. An increase in magnetic field strength, or a decrease in laser linewidth, should improve the detection limits, but was not possible here. For copper, the application of Zeeman background correction was unsuccessfull because the instrumentation was unable to resolve the sigma components from the laser emission profile sufficiently during the background correction measurement. For elements that exhibit sufficient Zeeman splitting, the linear dynamic range was the same with or without background correction Zeeman background correction was used to correct for scatter, in the resonance flourescence determination of manganese in a zinc chloride matrix and in mouse brain tissue.

Determination of the rate of base-pair substitution and insertion mutations in retrovirus replication.

We recently described a protocol for determination of retrovirus mutation rates, that is, the mutation frequency in a single cycle of retrovirus replication (J.P. Dougherty and H.M. Temin, Mol. Cell. Biol. 6:4378-4395, 1987; J.P. Dougherty and H.M. Temin, p. 18-23, in J. H. Miller and M. P. Calos, ed., Gene Transfer Vectors for Mammalian Cells, 1987). We used this protocol to determine the mutation rates for defined mutations in a replicating retrovirus by using a spleen necrosis virus-based vector. We determined that the mutation rate for a single base pair substitution during replication of this avian retrovirus is 2 x 10(-5) per base pair per replication cycle and the insertion rate is 10(-7) per base pair per replication cycle. It will be possible to use this protocol to determine mutation rates for other retroviruses.

A promoterless retroviral vector indicates that there are sequences in U3 required for 3' RNA processing.

We constructed a retrovirus vector in which all viral transcriptional signals are deleted during provirus formation. This vector would be safer and more useful in gene-transfer experiments. Construction of this vector involved the deletion of all of the U3 sequence except for 10 base pairs, required for integration, from the right-side long terminal repeat. We found that this deletion resulted in an inability to propagate virus efficiently. When we inserted sequences containing all of the signals for polyadenylylation of simian virus 40 late mRNA at the end of our vector, we were able to propagate virus efficiently. This result indicates that there are sequences in U3 upstream from AAUAAA that are essential for 3' processing of viral RNA.

In vivo antigen presentation by both brain parenchymal cells and hematopoietically derived cells during the induction of experimental autoimmune encephalomyelitis.

A fundamental issue in the etiology of autoimmune diseases of the central nervous system such as multiple sclerosis and its animal counterpart experimental autoimmune encephalomyelitis (EAE) concerns the identity of cells capable of presenting autoantigen to the T cells that mediate these diseases. The prevailing dogma is that only bone marrow-derived cells function as APC during EAE induction. We have addressed this issue by studying EAE induction in mouse bone marrow chimeras, and have found that although bone marrow-derived APC such as macrophages and brain microglial cells are more efficient at presenting autoantigen, brain parenchymal cells such as astrocytes and endothelial cells are also capable of inducing disease. EAE was induced in these chimeras by the adoptive transfer of encephalitogenic T cell lines designed to be MHC-histocompatible with APC contained either within the hematopoietic system of the chimera or with APC resident to the brain of the chimera. The subsequent development of EAE in these chimeras then indicated which population of cells served as in vivo APC during EAE pathogenesis. Possible effects of alloreactivity between the host chimera and the adoptively transferred T cells were eliminated by using encephalitogenic T cell lines made tolerant to the haplotype(s) of the recipient chimera.

Use of single-cycle analysis to study rates and mechanisms of retroviral mutation.

Retroviruses evolve at rapid rates. This allows them to escape immune surveillance, thwarts vaccine development, and leads to rapid emergence of drug-resistant virus. Information regarding the retroviral mutation rates and the underlying mechanisms of mutagenesis will undoubtedly expedite the development of strategies to combat retroviral-mediated diseases. In this review, we discuss how the unique retroviral life cycle can be adapted such that retroviral variation can be studied in a single cycle of replication. By limiting replication to a single cycle, retroviral mutation rates can be directly measured, and the consequences of mutations can be observed. In addition, retroviral recombination rates as well as the nature of primer strand transfer during reverse transcription can be studied using this system. Molecular analysis of the spectrum of mutations arising during a single cycle of virus replication also sheds light on the mechanisms of mutagenesis and retroviral replication.

Regulated lentiviral packaging cell line devoid of most viral cis-acting sequences.

Packaging cell lines derived from human immunodeficiency virus-1 (HIV-1) are promising tools for in vivo somatic cell gene therapy protocols due to the ability of lentiviruses to infect nondividing cells. We describe here the generation of a safe, stable HIV-1 packaging cell line capable of expressing all of the HIV-1 structural, enzymatic, and regulatory proteins but lacking majority of the cis-acting sequences. The use of an inducible expression system circumvented the cytotoxic and cytostatic effects associated with the expression of some of the HIV-1 viral proteins. Reverse transcriptase activity was detectable in the supernatant from the stable packaging line 1 day after induction, while vector titers peaked 5 days postinduction. Vector titers of up to 3.5 x 10(4) infectious units/ml (IU/ml) were maintained through 8 months following the establishment of the cell line. Lineage-specific gene delivery can be achieved from this established cell line as viral stocks obtained specifically infect CD4(+) target cells. Moreover, this cell line provides a safe and easy to use system for screening of drugs that inhibit HIV-1 replication.

Synthesis of (2'-5')(A)n from ATP. Characteristics of the reaction catalyzed by (2'-5')(A)n synthetase purified from mouse Ehrlich ascites tumor cells treated with interferon.

The treatment of Ehrlich ascites tumor cells with mouse interferon increases the level of the latent enzyme (2'-5')(A)n synthetase. If activated by double-stranded RNA, this catalyzes the synthesis from ATP of a series of 2'-5'-oligoadenylates: (2'-5')(A)n where n extends from 2 to about 15. We isolated (2'-5')(A)n synthetase in a homogeneous state. In the presence of double-stranded RNA, the purified enzyme can convert the large majority (about 97%) of the ATP into (2'-5')(A)n and pyrophosphate, although it does not cleave the pyrophosphate. The stoichiometry of the reaction can be formulated as: (n + I) ATP leads to (2'-5') pppA(pA)n + n pyrophosphate. Added pyrophosphate does not inhibit the synthesis of (2'-5')(A)n. The extent of the reverse reaction, i.e. the pyrophosphorolysis of (2'-5')(A)n, was below the level of detection under our conditions. The affinity of the enzyme for ATP is low: the rate of the reaction increases by about 10% when the concentration of ATP is increased from 5 mM to 10 mM. The optimal concentration of double-stranded RNA increases with the concentration of the enzyme. As tested at 0.4, 2, and 10 micrograms/ml of enzyme concentrations, close to maximal (2'-5')(A)n synthesis can be obtained if reovirus double-stranded RNA or poly(I) . poly(C) are used at about half the concentration (in w/v) of the enzyme. The plot of the reaction rate versus enzyme concentration is sigmoidal. It remains to be seen if this reflects on a cooperative behavior of the enzyme.

New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors.

We prepared retrovirus packaging cell lines containing gag-pol genes from spleen necrosis virus (expressed from a cytomegalovirus promoter and the simian virus 40 (SV40) polyadenylation sequences) and, on a separate vector, either the env gene from spleen necrosis virus (expressed from the Rous sarcoma virus promoter and the SV40 polyadenylation sequences) or the env gene from amphotropic murine leukemia virus (expressed from a cytomegalovirus promoter and the SV40 polyadenylation sequences). The nucleotide sequences in these packaging cell lines have almost no homology to the retrovirus vectors we used. Retrovirus vectors were produced from these new helper cell lines without any genetic interactions between the vectors and sequences in the helper cells and without transfer of the packaging sequences.

Discontinuous plus-strand DNA synthesis in human immunodeficiency virus type 1-infected cells and in a partially reconstituted cell-free system.

Human immunodeficiency virus type 1 (HIV-1) replication requires conversion of viral RNA to double-stranded DNA. To better understand the molecular mechanisms of this process, we examined viral DNA synthesis in a simple cell-free system that uses the activities of HIV-1 reverse transcriptase to convert regions of single-stranded HIV-1 RNA to double-stranded DNA in a single incubation. This system recapitulated several of the required intermediate steps of viral DNA synthesis: RNA-templated minus-strand polymerization, preferential plus-strand initiation at the central and 3' HIV-1 polypurine tracts, and DNA-templated plus-strand polymerization. Secondary sites of plus-strand initiation were also observed at low frequency both in the cell-free system and in cultured virus. Direct comparison of viral and cell-free products revealed differences in the precision and selectivity of plus-strand initiation, suggesting that the cell-free system lacks one or more essential replication components. These studies provide clues about mechanisms of plus-strand initiation and serve as a starting point for the development of more complex multicomponent cell-free systems.

A peripheral blood-derived monolayer supports long-term cultures of human CD4+ and CD8+ T lymphocytes.

An in vitro culture system has been developed for the long-term maintenance of primary, human peripheral blood and umbilical cord blood T lymphocytes, which does not rely on the use of stimulatory cytokines, antigen, or mitogens. In these cultures, a monolayer of adherent cells, some spindle-shaped and some resembling macrophages, developed within a week. All adherent cells were positive for the extracellular matrix proteins laminin and fibronectin, the intermediate filament vimentin, and for the surface markers major histocompatibility complex class II, platelet-endothelial cell adhesion molecule l (CD31), and E-Selectin (ELAM-1; CD62E). They were negative for the leukocyte common antigen (CD45), the macrophage marker MO-2 (CD14), muscle-specific actin, and Factor VIII-related antigen. These monolayers supported the maintenance of nonadherent, resting, mature T cells for up to 3 months, and these cells retained their ability to respond to mitogens and allogeneic cells. Both CD4+ and CD8+ cells were supported. The proportion of CD4+ and CD8+ cells remained unchanged after 3 months in culture. We have also used T cells from 2-month-old cultures as target cells for retroviral vector-mediated gene transfer. Up to 30% of the long-term T cells expressed the transferred lacZ gene after infection with a retroviral vector. The infection efficiency was similar to that obtained for fresh peripheral blood T cells, indicating that the long-term-cultured cells might be suitable for certain gene therapy applications.

High rate of genetic rearrangement during replication of a Moloney murine leukemia virus-based vector.

A protocol was designed to measure the forward mutation rate over an entire gene replicated as part of a Moloney murine leukemia virus-based vector. For these studies, the herpes simplex virus thymidine kinase (tk) gene under the control of the spleen necrosis virus U3 promoter was used as target sequence since it allows selection for either the functional or the inactivated gene. Our results indicate that after one round of retroviral replication, the tk gene is inactivated at an average rate of 0.08 per cycle of replication. Southern blotting revealed that the majority of the mutant proviruses resulted from gross rearrangements and that deletions of spleen necrosis virus and tk sequences were the most frequent cause of the gene inactivation. Sequence analysis of the mutant proviruses suggested that homologous as well as nonhomologous recombination was involved in the observed rearrangements. Some mutations consisted of simple deletions, and others consisted of deletions combined with insertions. The frequency at which these mutations occurred during one cycle of retroviral replication provides evidence indicating that Moloney murine leukemia virus-based vectors may undergo genetic rearrangement at high rates. The high rate of rearrangement and its relevance for retrovirus-mediated gene transfer are discussed.