Genes 1.2 and 10 of bacteriophages T3 and T7 determine the permeability lesions observed in infected cells of Escherichia coli expressing the F plasmid gene pifA.

Infections of F plasmid-containing strains of Escherichia coli by bacteriophage T7 result in membrane damage that allows nucleotides to exude from the infected cell into the culture medium. Only pifA of the F pif operon is necessary for "leakiness" of the T7-infected cell. Expression of either T7 gene 1.2 or gene 10 is sufficient to cause leakiness, since infections by phage containing null mutations in both of these genes do not result in permeability changes of the F-containing cell. Even in the absence of phage infection, expression from plasmids of either gene 1.2 or 10 can cause permeability changes, particularly of F plasmid-containing cells. In contrast, gene 1.2 of the related bacteriophage T3 prevents leakiness of the infected cell. In the absence of T3 gene 1.2 function, expression of gene 10 causes membrane damage that allows nucleotides to leak from the cell. Genes 1.2 and 10 of both T3 and T7 are the two genes involved in determining resistance or sensitivity to F exclusion; F exclusion and leakiness of the phage-infected cell are therefore closely related phenomena. However, since leakiness of the infected cell does not necessarily result in phage exclusion, it cannot be used as a predictor of an abortive infection.     

Increased synthesis of an Escherichia coli membrane protein suppresses F exclusion of bacteriophage T7.

Increased synthesis of the protein FxsA alleviates the exclusion of T7 in cells harboring the F plasmid. In contrast to wild-type or cells defective in fxsA, overexpression of fxsA+ allows T7 to form plaques at normal efficiency even though the burst size is reduced to about half that obtained on the isogenic F- strain. No defect in DNA synthesis was observed but late protein synthesis remains partially inhibited and a reduced level of cell leakiness, a prominent feature of F+ cells abortively infected by T7, persists. The FxsA protein is shown to be a cytoplasmic membrane protein. How T7 avoids exclusion by F in cells that exhibit increased levels of FxsA is discussed in terms of its membrane localization.     

Recognition of EBV plasma membrane protein expressed on murine cells after gene transfer

The immune response to B lymphocytes infected with Epstein-Barr virus (EBV) prevents their overgrowth in normal humans. A murine model is now described for analyzing the T cell immune response to Epstein-Barr virus genes expressed in murine lymphoblasts by gene transfer. In mice, a 60,000 dalton virus-encoded protein characteristically found in the plasma membrane of latently infected human lymphocytes readily induces both proliferative and cytolytic T lymphocytes specific for both the EBV protein and murine major histocompatibility proteins. Longterm cultures of L3T4+ cells, some of which were cytolytic, were found to be restricted by H-2I-Ed and the latent membrane protein. Similarly, Lyt-2+ cells were cytolytic and were restricted by H-2Ld and the lymphocyte membrane protein gene product. The similarity in murine and human effector cell responses suggests that this is a useful experimental model, and the EBV latent infection membrane protein may be an important antigen in the immune restriction of growth transformed latently infected lymphocytes.     

Mutants of bacteriophage T7 that escape F restriction

Mutants of bacteriophage T7 that escape F restriction have been isolated. Two mutations in gene 10, which codes for the capsid protein, and one mutation in gene 1.2 are required for these phages to grow on F-containing strains. The products of these two genes are the two targets of the exclusion system; the presence of either wild-type product results in an abortive infection. Phages that grow normally in male hosts still lead to membrane dysfunction and nucleotide efflux from the infected cell. This type of membrane damage and the abortive infection are therefore separable phenomena.     

The immunity (imm) gene of Escherichia coli bacteriophage T4.

The immunity (imm) gene of the Escherichia coli bacteriophage T4 effects exclusion of phage superinfecting cells already infected with T4. A candidate for this gene was placed under the control of the lac regulatory elements in a pUC plasmid. DNA sequencing revealed the presence of an open reading frame encoding a very lipophilic 83-residue (or 73-residue, depending on the unknown site of translation initiation) polypeptide which most likely represents a plasma membrane protein. This gene could be identified as the imm gene because expression from the plasmid caused exclusion of T4 and because interruption of the gene in the phage genome resulted in a phage no longer effecting superinfection immunity. It was found that the fraction of phage which was excluded upon infection of cells possessing the plasmid-encoded Imm protein ejected only about one-half of their DNA. Therefore, the Imm protein inhibited, directly or indirectly, DNA ejection.     

T-deficient transmembrane signaling in CD4+ T cells of retroviral-induced immune-deficient mice.

The defective virus found in the LP-BM5 mixture of murine leukemia viruses induces a severe immune deficiency disease in C57BL/6 mice that is characterized by the activation and expansion of T and B cells that become unresponsive to normal immune stimuli. The nature of the biochemical lesion in these defective lymphocyte populations remains unknown. Flow cytometric analysis of the T cell population in infected animals has demonstrated expansion of both CD4+ and CD8+ subsets. Despite chronic expansion in vivo, CD4+ T cells by wk 4 postinfection failed to up-regulate cell surface IL-2R expression, produced IL-2, or proliferate in vitro in response to either Con A, Staphylococcal enterotoxin super-antigens, or anti-CD3 stimulation. Exogenous IL-2 did not restore the proliferative response and also failed to up-regulate IL-R expression on CD4+ T cells from infected mice, even though basal IL-2R expression was initially elevated compared to normals. In contrast, CD4+ T cells from infected mice could be induced to proliferate by stimulation with PMA and ionomycin resulting in IL-2R up-regulation, IL-2 production, and proliferation. Moreover, proliferation could also be induced by anti-CD3 plus PMA, although anti-CD3 plus ionomycin was without effect. These studies suggest that chronic expansion of CD4+ T cells in infected mice is probably not maintained by normal TCR signaling, which appears defective in these cells. In addition, the lesion in biochemical signaling appears to result in defective activation of protein kinase C, which can be overcome by direct activation with PMA.     

F-Factor-mediated restriction of bacteriophage T7: protein synthesis in cell-free systems from T7-infected Escherichia coli F- and F+ cells.

A characteristic phenomenon in the F-factor-mediated inhibition of T7 phage is a virtual absence of T7 late protein synthesis in T7-infected Escherichia coli male cells, in spite of the presence of T7 late mRNA which is translatable in vitro when isolated from the cell. To determine whether the translational defect in T7-infected F+ cells is due to a T7 late mRNA-specific translational block, or to a general decrease of F+ cell translational activity, we compared the activities of cell-free, protein-synthesizing systems prepared from isogenic F- and F+ cells harvested at different times of T7 infection. The cell-free systems from uninfected F- and F+ cells translated T7late mRNA equally as well as MS2 RNA and T7early mRNA. The activity of cell-free systems from T7-infected F+ cells to translate MS2 RAN, T7 early mRNA, and T7 late mRNA decreased concomitantly at a much faster rate than that of T7-infected F- cells. Therefore, the abortive infection of F+ cells by T7 does not result from a T7 late mRNA-specific translational inhibition, although a general reduction of the translational activity appears to be a major factor for the inability of the F+ cells to produce a sufficient amount of T7 late proteins.     

Diagnostic surface expression of SWAP-70 on HIV-1 infected T cells

Following immunization with HIV-1 infected cells, a hybridoma cell line termed 9F11 was established from the P3U-1 myeloma line fused with lymphocytes from a trans-chromosome (TC) mouse, that harbors human chromosomes containing immunoglobulin genes. The 9F11 human IgM monoclonal antibody (9F11 Ab) reacts with HIV-1 infected MOLT4 cells but not with uninfected MOLT4 cells, and causes immune cytolysis with homologous human complement at a concentration as low as 0.4 microg/ml. This Ab was used to perform immunoscreening of a cDNA expression library derived from HIV-1 infected cells. All positive cDNA clones contained SWAP-70 cDNA. SWAP-70 RNA and protein expression are much stronger in HIV-1 infected cells. SWAP-70 was also detected on the surface of HIV-1 infected cells by flow cytometric analysis. The monocyte cell line U937 cells expresses SWAP-70 on its cell surface regardless of whether it was infected with HIV-1. Furthermore, among PBMCs surface expression of SWAP-70 was detected on CD21+, CD56+ and CD14+ cells. Although CD3+ cells scarcely express SWAP-70 on their surface, once activated, they become positive. SWAP-70 may therefore serve as a marker for T cell differentiation as well as for HIV-1 infection.     

Preferential usage of the V beta 8 gene family by CD4-CD8-T cell lines derived from spleen

Receptors encoded by the V beta 8 gene family and identified by the F23.1 antibody are commonly expressed amongst the CD4-CD8-T cell lines isolated from spleen cells infected in vitro with the RadLV retrovirus. All but one out of 12 cell lines showed between 50 and 85% F23.1+ cells in the uncloned cell population which is noticably higher than the approximately 13% level amongst the Ig- normal spleen cell population. There was a high frequency (approximately 50%) of F23.1+ clones from five of these cell lines. The frequency of F23.1 binding cells in the Ig-, CD4/CD8-depleted spleen population is only 0.2%, which gives a precursor frequency in spleen of less than 0.002%. This reflects selective isolation of CD4-CD8- alpha beta+ cells which express V beta 8 gene products by this culture scheme. The requirement for RadLV in induction of these cell lines has been established, suggesting that this retrovirus may selectively stimulate CD4-CD8-F23.1+ T cells. These cells may represent an autoimmune subset present in peripheral lymphoid tissue.     

Incomplete entry of bacteriophage T7 DNA into F plasmid-containing Escherichia coli.

The penetration of bacteriophage T7 DNA into F plasmid-containing Escherichia coli cells was determined by measuring Dam methylation of the entering genome. T7 strains that cannot productively infect F-containing cells fail to completely translocate their DNA into the cell before the infection aborts. The entry of the first 44% of the genome occurs normally in an F-containing cell, but the entry of the remainder is aberrant. Bypassing the normal mode of entry of the T7 genome by transfecting naked DNA into competent cells fails to suppress F exclusion of phage development. However, overexpression of various nontoxic T7 1.2 alleles from a high-copy-number plasmid or expression of T3 1.2 from a T7 genome allows phage growth in the presence of F.     

TIRC7 deficiency causes in vitro and in vivo augmentation of T and B cell activation and cytokine response

The membrane protein T cell immune response cDNA 7 (TIRC7) was recently identified and was shown to play an important role in T cell activation. To characterize the function of TIRC7 in more detail, we generated TIRC7-deficient mice by gene targeting. We observed disturbed T and B cell function both in vitro and in vivo in TIRC7(-/-) mice. Histologically, primary and secondary lymphoid organs showed a mixture of hypo-, hyper-, and dysplastic changes of multiple lymphohemopoietic compartments. T cells from TIRC7(-/-) mice exhibited significantly increased proliferation and expression of IL-2, IFN-gamma, and IL-4 in response to different stimuli. Resting T cells from TIRC7(-/-) mice exhibited decreased CD62L, but increased CD11a and CD44 expression, suggesting an in vivo expansion of memory/effector T cells. Remarkably, activated T cells from TIRC7(-/-) mice expressed lower levels of CTLA-4 in comparison with wild-type cells. B cells from TIRC7-deficient mice exhibited significantly higher in vitro proliferation following stimulation with anti-CD40 Ab or LPS plus IL-4. B cell hyperreactivity was reflected in vivo by elevated serum levels of various Ig classes and higher CD86 expression on B cells. Furthermore, TIRC7 deficiency resulted in an augmented delayed-type hypersensitivity response that was also reflected in increased mononuclear infiltration in the skin obtained from TIRC7-deficient mice food pads. In summary, the data strongly support an important role for TIRC7 in regulating both T and B cell responses.     

Evidence for the Presence of Nontranslated T7 Late mRNA in Infected F′(PIF+) Episome-Containing Cells

The inability of T7 to develop in cells of Escherichia coli containing F(+) or substituted F' episomes is a result of the failure to synthesize late proteins; no in vivo translation of mRNA species synthesized by the T7 RNA polymerase occurs. Further experiments have been performed to measure the amount of late mRNA in T7-infected F'(PIF(+)) cells. (We have designated the property of phage inhibition of F factors as PIF; the wild-type episome is therefore F'[PIF(+)].) T7 late proteins were synthesized in vitro by using a system programed with RNA extracted from T7-infected F(-) and F'(PIF(+)) cells. The T7 lysozyme, product of gene 3.5, and the gene 10 head protein were assayed. The following results were obtained: (i) mRNA capable of supporting in vitro synthesis of lysozyme and the gene 10 head protein is present in T7-infected F'(PIF(+)) cells; (ii) lysozyme mRNA extracted from T7-infected F'(PIF(+)) cells is present at 70 to 75% of the level found in T7-infected F(-) cells; (iii) gene 10 mRNA is present at 35 to 78% of the level found in T7-infected F(-) cells. No in vivo synthesis of either lysozyme or gene 10 protein can be detected in T7-infected F'(PIF(+)) cells although normal synthesis of these proteins occurs in F(-) cells. These findings confirm that the block in T7 development in F'(PIF(+)) cells results from the failure to translate late classes of T7 RNA.     

Co-expression of an epitope on human free kappa-light chains and on a cytoplasmic component in activated T cells

K-1-21 is a murine monoclonal antibody that reacts with human kappa-light chains in free form but not when they are associated with immunoglobulin heavy chains. K-1-21 was unexpectedly shown to bind to a determinant, STA (Sezary T cell antigen), detected by immunofluorescence in the cytoplasm but not on the surface of Sezary T cells isolated from peripheral blood (4/4 cases) and in Sezary T cells from lymph node and bone marrow (one patient). STA was detected in F2/F7, CCRF-CEM, Molt-4, and CCRF-HSB (four human T ALL cell lines), in JURKAT (a human T cell leukemia line), and in MLA144 (a Gibbon T cell lymphoma line). It also occurred in Leu-3a+ antigen-specific T cell clones (6/6 tested). Moreover, although STA was absent from freshly isolated normal T cells, its expression could be evoked in E+ cells from peripheral blood by in vitro culture with phytohemagglutinin. Thus, STA appears to be a cytoplasmic marker for activated T cells. Cytoplasmic inhibition immunofluorescence studies indicated that K-1-21 binding to STA in Sezary cells or T cell lines was inhibited by preincubation of the K-1-21 antibody with purified kappa-Bence Jones protein. STA from radiolabeled MLA144 cell lysates was immunoprecipitated by K-1-21 and was identified on polyacrylamide gel electrophoresis under reducing conditions as a protein of m.w. 57,000. Additional experiments are underway to define the molecular basis of the interesting cross-reactivity between a determinant in T cells and the K-1-21 reactive epitope on free kappa-light chains.     

Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene.

The nef gene, which encodes related cytoplasmic proteins in both human (HIV) and simian (SIV) immunodeficiency viruses is dispensable for viral replication in vitro. In contrast, in vivo experiments have revealed that SIV nef is required for efficient viral replication and development of AIDS in SIV infected rhesus monkeys, thus indicating that nef plays an essential role in the natural infection. We show that expression of the Nef protein from the HIV-1 NL43 isolate in transgenic mice perturbs development of CD4+ T cells in the thymus and elicits depletion of peripheral CD4+ T cells. Thymic T cells expressing NL43 Nef show altered activation responses. In contrast, Nef protein of the HIV-1 HxB3 isolate does not have an overt effect on T cells when expressed in transgenic animals. The differential effects of the two HIV-1 nef alleles in transgenic mice correlate with down-regulation of CD4 antigen expression on thymic T cells. The differential interactions of the NL43 and HxB3 nef alleles with CD4 were reproduced in a transient assay in human CD4+ CEM T cells. Down-regulation of CD4 by nef in both human and transgenic murine T cells indicates that the relevant interactions are conserved in these two systems and suggests that the consequences of Nef expression on the host cell function can be analyzed in vivo in the murine system. Our observations from transgenic mice suggest that nef-elicited perturbations in T cell signalling play an important role in the viral life cycle in vivo, perhaps resulting in elimination of infected CD4+ T cells.     

Evaluation of human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte responses utilizing B-lymphoblastoid cell lines transduced with the CD4 gene and infected with HIV-1.

Analysis of major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) capable of killing human immunodeficiency virus type 1 (HIV-1)-infected targets is essential for elucidating the basis for HIV-1 disease progression and the potential efficacy of candidate vaccines. The use of primary CD4+ T cells with variable infectivity as targets for such studies has significant limitations, and immortal autologous cells with high levels of CD4 expression that can be consistently infected with HIV-1 would be of much greater utility. Therefore, we transduced Epstein-Barr-virus-transformed B-lymphoblastoid cell lines (LCL) with a retroviral vector, LT4SN, containing the human CD4 gene. Stable LCL in which more than 95% of cells expressed membrane CD4 were obtained. Aliquots were infected with HIV-1, and, after 4 to 7 days, nearly all of the cells contained cytoplasmic gag and produced high levels of p24 antigen. The ability of major histocompatibility complex-restricted CD8+ CTL to lyse such HIV-1-infected CD4-transduced LCL (LCL-CD4HIV-1) was evaluated. These autologous targets were lysed by CTL generated from an HIV-1-uninfected vaccinee over a broad range of effector-to-target ratios. Similarly, the LCL-CD4HIV-1 were efficiently lysed by fresh circulating CTL from HIV-1-infected individuals, as well as by CTL activated by in vitro stimulation. Both HIV-1 env- and gag-specific CTL effectors lysed LCL-CD4HIV-1, consistent with the cellular expression of both HIV-1 genes. The LCL-CD4HIV also functioned as stimulator cells, and thus are capable of amplifying CTL against multiple HIV-1 gene products in HIV-1-infected individuals. The ability to produce HIV-1-susceptible autologous immortalized cell lines that can be employed as target cells should enable a more detailed evaluation of vaccine-induced CTL against both homologous and disparate HIV-1 strains. Furthermore, the use of LCL-CD4HIV-1 should facilitate the analysis of the range of HIV-1 gene products recognized by CTL in seropositive persons.     

HIV-1 infection of human T lymphocytes results in enhanced alpha 5 beta 1 integrin expression.

Altered T cell adherence after human immunodeficiency virus 1 (HIV-1) infection may contribute to viral pathogenesis in the acquired immune deficiency syndrome. To address this hypothesis, we assessed mechanisms of T cell adherence to extracellular matrix proteins in vitro. We found that after HIV-1 infection, both chronically infected H9 CD4+ T cells and acutely infected primary peripheral blood lymphocytes acquired the ability to adhere to the extracellular matrix glycoprotein fibronectin, to a lesser extent to type IV collagen and laminin, but not to type I collagen. H9 cells chronically infected with two of the three HIV-1 strains studied showed approximately a sevenfold increase in attachment to fibronectin, while the same cells infected with the human retrovirus HIV-2 did not. Adhesion was accompanied by changes in morphology, including marked spreading and increased filopodia. These alterations were not blocked by the protein kinase C inhibitor H-7, which did inhibit TPA-induced T cell attachment to fibronectin. Monoclonal antibodies against both the alpha 5 and the beta 1 subunits of the classical fibronectin receptor as well as an Arg-Gly-Asp (RGD) peptide inhibited attachment, whereas anti-alpha 4 monoclonal antibodies and the CS1 peptide did not. Binding to collagen IV was also inhibited by the anti-beta 1 monoclonal antibody, but not the other antibodies. Cells metabolically labeled with [35S]methionine and analyzed by immunoprecipitation with polyclonal anti-beta 1 integrin antibody showed a 2.5-fold increase in integrin synthesis in infected cells compared to uninfected controls. This increase in synthesis was associated with an increase in cell surface expression of both alpha 5 and beta 1 integrins by FACS (registered trademark of Becton Dickinson for a fluorescence-activated cell sorter) analysis. Enhanced expression of integrins such as alpha 5 beta 1 may cause T cell adherence to a variety of tissues, where released viral gene products may induce some of the tissue-specific manifestations of HIV-1 infection.     

Heterologous antibody responses in mice with chronic T. cruzi infection: depressed T helper function restored with supernatants containing interleukin 2

Antibody production to sheep erythrocytes (SRBC) or hapten-conjugated SRBC (TNP-SRBC) was studied in mice with chronic Trypanosoma cruzi infections. Studies in vivo demonstrated that both IgM and IgG anti-SRBC responses were suppressed during chronic infection. Secondary IgG responses were suppressed regardless of whether the primary immunization was given before or after infection. The ability of cells from infected mice to provide help for antibody production was examined in vitro. Anti-SRBC responses were restored to cultures of whole spleen cells from infected mice by the addition of interleukin 2 (IL 2)-rich supernatants, indicating that these cells were capable of antibody production when sufficient help was provided. T cells from SRBC-primed infected mice were unable to provide significant help to normal B cell/M phi cultures for in vitro anti-TNP or anti-SRBC responses. The percentages of Thy-1+, Lyt-1+, and Lyt-2+ spleen cells were not significantly different between normal and infected mice. Anti-TNP and anti-SRBC responses were restored to cultures that contained T cells from infected mice and normal B cell/M phi by the addition of IL 2-rich spleen cell supernatants. The suppression of in vitro antibody responses in mice with chronic T. cruzi infections was associated with a lack of T cell help, which was provided by exogenous spleen cell supernatant.     

Differentiated S100A7 expression in infected tonsils and tonsils from allergic individuals

Palatine tonsils are continuously exposed to microorganisms and antigens and secrete antimicrobial peptides as a first line of defense. S100A7 is a protein with antimicrobial and chemotactic properties. Our aim was to investigate how the expression of S100A7 in human palatine tonsils is affected by inflammatory processes. Tonsils obtained from 109 patients undergoing tonsillectomy were divided into groups of infected and noninfected as well as allergic and nonallergic, based on the results from tonsillar core culture tests and Phadiatop analysis, respectively. Western blot and immunohistochemistry were used to assess protein expression and real-time PCR was used to quantify mRNA levels. To explore the induction of S100A7, tonsils were stimulated with lipopolysaccharide in vitro. The immunohistochemical staining for S100A7 was most intense in the tonsillar epithelium, but the protein was also detected in B- and T-cell regions, which was confirmed with Western blot on isolated B and T cells. The S100A7 expression appeared to be the highest in CD8+ T cells. Reduced mRNA levels of S100A7 were detected in infected tonsils as well as in tonsils from allergic individuals. In vitro stimulation of tonsils with lipopolysaccharide did not have any effect on the expression. The results suggest a role for S100A7 in recurrent tonsillitis and allergic disease.