Recasting Human Vδ1 Lymphocytes in an Adaptive Role.

γδ T cells are unconventional lymphocytes commonly described as 'innate-like' in function, which can respond in both a T cell receptor (TCR)-independent and also major histocompatibility complex (MHC)-unrestricted TCR-dependent manner. While the relative importance of TCR recognition had remained unclear, recent studies revealed that human Vδ1 T cells display unexpected parallels with adaptive αß T cells. Vδ1 T cells undergo profound and highly focussed clonal expansion from an initially diverse and private TCR repertoire, most likely in response to specific immune challenges. Concomitantly, they differentiate from a Vδ1 T cell naïve (Tnaïve) to a Vδ1 T cell effector (Teffector) phenotype, marked by the downregulation of lymphoid homing receptors and upregulation of peripheral homing receptors and effector markers. This suggests that an adaptive paradigm applies to Vδ1 T cells, likely involving TCR-dependent but MHC-unrestricted responses to microbial and non-microbial challenges.

Effects of the Expression of Random Sequence Clones on Growth and Transcriptome Regulation in Escherichia coli.

Comparative genomic analyses have provided evidence that new genetic functions can emerge out of random nucleotide sequences. Here, we apply a direct experimental approach to study the effects of plasmids harboring random sequence inserts under the control of an inducible promoter. Based on data from previously described experiments dealing with the growth of clones within whole libraries, we extracted specific clones that had shown either negative, neutral or positive effects on relative cell growth. We analyzed these individually with respect to growth characteristics and the impact on the transcriptome. We find that candidate clones for negative peptides lead to growth arrest by eliciting a general stress response. Overexpression of positive clones, on the other hand, does not change the exponential growth rates of hosts, and they show a growth advantage over a neutral clone when tested in direct competition experiments. Transcriptomic changes in positive clones are relatively moderate and specific to each clone. We conclude from our experiments that random sequence peptides are indeed a suitable source for the de novo evolution of genetic functions.

Biological properties of an influenza A virus-specific killer T cell clone. Inhibition of virus replication in vivo and induction of delayed-type hypersensitivity reactions.

We tested two biological properties of a continuously growing mouse cytotoxic T cell line, L4, which is specific for influenza A virus and has been cloned and recloned many times. We previously reported that L4 cells are H-2 restricted and cross-reactive for all type A influenza viruses, whereas they do not recognize type B influenza viruses. They bear Thy-1 and Lyt-2 markers. In the present study, we show that L4 cytotoxic T cells protect mice against a lethal influenza infection on transfer to syngeneic recipients, and reduce virus titers in the lungs of mice challenged with a heterologous type A influenza virus. This provides further support for the active role of cytotoxic T cells in limiting virus replication in influenza infection. We could also demonstrate that the cloned cytotoxic T cells induce a delayed-type hypersensitivity skin reaction in the footpads of mice challenged with live or inactivated influenza virus. This reaction can be observed at 24 h, but has declined by 48 h. A clone of cells derived from L4 that has lost its cytotoxic potential and its ability to recognize infected cells did not induce a delayed-type hypersensitivity reaction in the presence of virus. Thus, cytotoxic T cells actively killing influenza virus-infected cells are able to induce a delayed-type hypersensitivity skin reaction to homologous and heterologous type A influenza viruses.

Anemia- and polycythemia-inducing isolates of Friend spleen focus-forming virus. Biological and molecular evidence for two distinct viral genomes.

Two distinct clones of Friend spleen focus-forming virus (SFFV), differing in their erythroleukemic potential, are described. These isolates have been cloned free of their associated helper viruses and shown to be replication-defective. Both SFFV isolates have been rescued from rat fibroblast nonproducer cell clones with cloned replication-competent viruses, F-MuLVA and F-MuLVP, obtained from the anemia- or polycythemia-inducing isolates of Friend virus complex, respectively. These rescued viruses induce a rapid proliferative disease associated with the appearance of macroscopic spleen foci and splenomegaly. In addition, each is subject to regulation by the W, Steel (Sl), and Fv-2 host gene loci. These two isolates of SFFV can, however, be distinguished by both biological and molecular criteria. Friend SFFVP induces a rapid polycythemia associated with the appearance of large numbers of erythropoietin (EPO)-independent erythroid colony-forming cells in the marrow and spleen. In contrast, SFFVA induces a rapid anemia associated with a progressive decrease in the number of EPO-dependent erythroid colony-forming cells in marrow, and a rapid increase in the number of EPO-dependent erythroid colony-forming cells in spleen. Furthermore, the nature of the disease induced by the two isolates of SFFV is independent of the Friend helper virus: SFFVP, rescued from a nonproducer cell clone with either F-MuLVA or F0MuLVP, induced a polycythemic transformation, whereas SFFVA, rescued with either F-MuLVA or F-MuLVP, induced an anemic transformation. The two Friend SFFV isolates can also be discriminated on the basis of translational products encoded by their gag and env genes: SFFVP encodes the amino-terminal gag-gene protein p15, whereas SFFVA encodes the gag-gene proteins p15, p12, and p30. In addition, the SFFV isolates encode nonidentical 55,000-mol wt env gene-related proteins that can be distinguished by analysis of their methionine-containing tryptic peptides.

Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS.

A cell system was developed for the reproducible detection of human T-lymphotropic retroviruses (HTLV family) from patients with the acquired immunodeficiency syndrome (AIDS) or with signs or symptoms that frequently precede AIDS (pre-AIDS). The cells are specific clones from a permissive human neoplastic T-cell line. Some of the clones permanently grow and continuously produce large amounts of virus after infection with cytopathic (HTLV-III) variants of these viruses. One cytopathic effect of HTLV-III in this system is the arrangement of multiple nuclei in a characteristic ring formation in giant cells of the infected T-cell population. These structures can be used as an indicator to detect HTLV-III in clinical specimens. This system opens the way to the routine detection of HTLV-III and related cytopathic variants of HTLV in patients with AIDS or pre-AIDS and in healthy carriers, and it provides large amounts of virus for detailed molecular and immunological analyses.

Gene Composition as a Potential Barrier to Large Recombinations in the Bacterial Pathogen Klebsiella pneumoniae.

Klebsiella pneumoniae (Kp) is one of the most important nosocomial pathogens worldwide, able to cause multiorgan infections and hospital outbreaks. One of the most widely disseminated lineage of Kp is the clonal group 258 (CG258), which includes the highly resistant "high-risk" sequence types ST258 and ST11. Genomic investigations revealed that very large recombination events have occurred during the emergence of Kp lineages. A striking example is provided by ST258, which has undergone a recombination event that replaced over 1 Mb of the genome with DNA from an unrelated Kp donor. Although several examples of this phenomenon have been documented in Kp and other bacterial species, the significance of these very large recombination events for the emergence of either hypervirulent or resistant clones remains unclear. Here, we present an analysis of 834 Kp genomes that provides data on the frequency of these very large recombination events (defined as those involving >100 kb), their distribution within the genome, and the dynamics of gene flow within the Kp population. We note that very large recombination events occur frequently, and in multiple lineages, and that the majority of recombinational exchanges are clustered within two overlapping genomic regions, which have been involved by recombination events with different frequencies. Our results also indicate that certain lineages are more likely to act as donors to CG258. Furthermore, comparison of gene content in CG258 and non-CG258 strains agrees with this pattern, suggesting that the success of a large recombination depends on gene composition in the exchanged genomic portion.

MAIT cell clonal expansion and TCR repertoire shaping in human volunteers challenged with Salmonella Paratyphi A.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that can detect bacteria-derived metabolites presented on MR1. Here we show, using a controlled infection of humans with live Salmonella enterica serovar Paratyphi A, that MAIT cells are activated during infection, an effect maintained even after antibiotic treatment. At the peak of infection MAIT cell T-cell receptor (TCR)ß clonotypes that are over-represented prior to infection transiently contract. Select MAIT cell TCRß clonotypes that expand after infection have stronger TCR-dependent activation than do contracted clonotypes. Our results demonstrate that host exposure to antigen may drive clonal expansion of MAIT cells with increased functional avidity, suggesting a role for specific vaccination strategies to increase the frequency and potency of MAIT cells to optimize effector function.

Colony formation by simian virus 40-transformed human parapharyngeal cells cultured in semisolid agar.

A simian virus 40 (SV40)-transformed line of human parapharyngeal cells (SV-TGo) was cultured in semisolid agar to determine its ability to grow in the absence of an anchoring substratum and to evaluate any phenotypic changes that might have resulted during the isolation of sublines specifically selected for anchorage independence. After 2--3 weeks and 14--15 population doublings in culture, SV-TGo plated with over 1,000% higher efficiency than negative controls (F2408 cells). Sublines, 0.3--2.0 mm in diameter, were isolated and transferred to Leighton tubes in which they underwent an additional 0--7 divisions before senescence after 39--44 total population doublings. Subline phenotype was identical to the original parental phenotype, including epithelioid morphology, organized pattern of growth, extreme sensitivity to density-dependent inhibition of growth, and continuous production of infectious SV40 as detected by the combined tests of cocultivation and direct isolation. Limited division potential was within the range observed for the parental line. The ability to grow in agar without identifiable phenotypic changes was therefore confirmed for this line of SV40-transformed human epithelioid cells.

Radiation induction of endogenous type C virus from mouse cells transformed in vitro by murine sarcoma virus.

Cell lines from AKR and BALB/c mouse embryos were compared for their sensitivity to X-ray induction of endogenous type C virus. K-Balb cells, a Balb/3T3 cell line nonproductively transformed by Kirsten murine sarcoma virus, were found to be sensitive to X-irradiation. At a dose as low as 50 R, X-rays induced virus expression in K-Balb cells, and the induction frequency increased with increasing dose of X-rays up to 400 R. Among two classes of inducible endogenous viruses carried by K-Balb cells, only Balb:virus-2 was activated by X-irradiation, whereas both Balb:virus-1 and Balb:virus-2 were activated after the cells were treated with 5-iodo-2'-deoxyuridine. UV light and 4-nitroquinoline 1-oxide were also shown to induce virus expression in K-Balb cells. The virus-induction frequency for these physical and chemical carcinogens was much lower (approximately 3 times 10(-4)) than that for 5-bromo-2'-deoxyuridine (approximately 1 times 10(-1)).

Characterization of an apparently nononcogenic Marek's disease virus.

A new isolate of Marek's disease virus (MDV) was described. This virus, SB, and a clone, SB-1, differed from pathogenic isolates in in vitro growth characteristics as described for other apathogenic isolates. Serologically, as with other apathogenic isolates, SB could be distinguished from pathogenic MDV and the avirulent turkey herpesvirus. SB failed to induce lesions characteristic of Marek's disease (MD) during a 6- to 11-week experimental period. Also, SB was nononcogenic in immunosuppressed chickens or in chickens inoculated with this virus in ovo. However, under those conditions, SB caused a cytolytic infection. The term "nononcogenic" rather than "apopathogenic" was therefore proposed to classify this and similar isolates. SB-1 protected chickens against challenge with either virulent MDV or the non-virus-producing MD tumor transplant, JMV. Possible mechanisms of protection are discussed.

Ectopic expression of genes during chicken limb pattern formation using replication defective retroviral vectors.

A gene transfer method to ectopically express genes during chicken limb pattern formation using replication defective retroviral vectors has been established. Spherical non-proliferating (mitomycin C treated) aggregates of clonal retrovirus producing cells were grafted directly into developing chicken wing buds. The cell aggregates had to be placed in direct contact with the highly proliferative cells of the wing bud to promote efficient in vivo infection of embryonic cells by the released retroviral particles. The majority of grafts resulted in widespread expression of a reporter gene (encoding bacterial beta-galactosidase) during limb pattern formation and early limb bud outgrowth without affecting morphogenesis. This method provides a novel approach to study the effects of ectopic gene expression on limb pattern formation. Possible future applications to study other developmental processes are discussed.

Mouse T cell clones against Mycobacterium avium: identification of clones that modify resistance against atypical mycobacteria infection.

Mouse T cell clones against live Mycobacterium avium were generated from the spleens of BALB/c mice infected with M. avium TMC 702. Eighth clones were of the L3T4+ subset, whereas two were of Lyt2+ subset. Six of the L3T4+ T cell clones were of the TH1 subset whereas two were of the TH2 subset, judged on the profile of cytokine release. One of the Lyt2+ clones exhibited significant cytotoxicity against M. avium-infected mouse macrophages. Transfer of clones to nude BALB/c mice infected with M. avium was associated with insignificant changes in resistance for seven clones. One clone, of the L3T4+/TH2 subset, transferred significant resistance to the infection, also associated with infusion of supernatants from the clone, which was fully inhibited by neutralizing with anti-interleukin 4. By contrast, infusion of one TH1 clone and the cytolytic Lyt2+ led to increased microbial growth in the spleens and livers of infected mice, which was not apparent on infusion with supernatants. Application of clones' supernatants on infected macrophages had marginal effects on M. avium growth and was not correlated with protective or suppressive activity. Overall, these results suggest that T cells may influence M. avium growth in vivo in a bidirectional manner and also suggest that interleukin 4 may be an important factor in host resistance to M. avium.

Further characterization of an antigenic site of HIV-1 gp120 recognized by virus neutralizing human monoclonal antibodies.

OBJECTIVE: The aim of this study is to characterize antigenic sites on HIV-1 gp120 which may be important for the development of active and passive immunization strategies against HIV-1 infection. DESIGN: Two HIV-1-seropositive individuals were selected from the Amsterdam cohort and Epstein-Barr virus (EBV)-transformed B cells were generated from their peripheral blood mononuclear cells, which produce HIV-1-specific human monoclonal antibodies (HuMAb). METHODS: HuMAb were generated and selected based on their reactivities with native gp120. Reactivity with HIV-1 strains from phylogenetically different subfamilies was determined by immunostaining and virus neutralization assays. Specificity for the CD4-binding site was tested by an inhibition enzyme-linked immunosorbent assay and amino acids (aa) involved in the binding of the HuMAb were identified with a set of gp120 molecules with single aa substitutions. RESULTS: Three HuMAb (GP13, GP44, GP68) were generated, all recognizing a conserved conformation dependent epitope within, or topographically near, the CD4-binding site of gp120. HuMAb GP13 and GP68 neutralized a broad range of HIV-1 strains from phylogenetically different subfamilies, whereas HuMAb GP44 exhibited a more restricted pattern of neutralizing activity. The patterns of gp120 aa involved in their binding were unique for each of these HuMAb. CONCLUSIONS: The pattern of reactivities of these three HIV-1-neutralizing HuMAb developed in these studies is similar to, but distinct from other human and rodent MAb that recognize this antigenic site of HIV-1 gp120.

Human epidermal differentiation complex in a single 2.5 Mbp long continuum of overlapping DNA cloned in bacteria integrating physical and transcript maps.

Terminal differentiation of keratinocytes involves the sequential expression of several major proteins which can be identified in distinct cellular layers within the mammalian epidermis and are characteristic for the maturation state of the keratinocyte. Many of the corresponding genes are clustered in one specific human chromosomal region 1q21. It is rare in the genome to find in such close proximity the genes belonging to at least three structurally different families, yet sharing spatial and temporal expression specificity, as well as interdependent functional features. This DNA segment, termed the epidermal differentiation complex, contains 27 genes, 14 of which are specifically expressed during calcium-dependent terminal differentiation of keratinocytes (the majority being structural protein precursors of the cornified envelope) and the other 13 belong to the S100 family of calcium binding proteins with possible signal transduction roles in the differentiation of epidermis and other tissues. In order to provide a bacterial clone resource that will enable further studies of genomic structure, transcriptional regulation, function and evolution of the epidermal differentiation complex, as well as the identification of novel genes, we have constructed a single 2.45 Mbp long continuum of genomic DNA cloned as 45 p1 artificial chromosomes, three bacterial artificial chromosomes, and 34 cosmid clones. The map encompasses all of the 27 genes so far assigned to the epidermal differentiation complex, and integrates the physical localization of these genes at a high resolution on a complete NotI and SalI, and a partial EcoRI restriction map. This map will be the starting resource for the large-scale genomic sequencing of this region by The Sanger Center, Hinxton, U.K.

Variable expression of SV40 large T antigen in CV1 cell clones.

Using immunofluorescence and immunoadsorption, CV1 cell clones MA2, V4, USA3, TR7 and P3 infected with SV40 were found to express variably SV40 large T antigen. The monoclonal antibody used was Pab 419. The results indicate that P3 cells express T antigen to a considerable level as early as 10 h post-infection, while that of TR7 and USA3 cells is minute as judged from their positive nuclei. MA2 and V4 cells did not show any positive nuclei over this period of infection. At 20 h post-infection MA2, V4 and USA3 cells developed a considerable amount of fluorescence in their nuclei while TR7 and P3 cells produced high values. By immunoadsorption of cell extracts for the same periods of infection, similar results were obtained on the electrophoretograms. We also relate these findings with those from induction of heatshock proteins by SV40 infection.

Human astrocytes stimulate HIV-1 expression in a chronically infected promonocyte clone via interleukin-6.

Human promonocyte cells chronically infected with human immunodeficiency virus type (HIV-1) (clone U1.1.5) were grown in the presence of media conditioned by human astrocytes and glioma cell lines U251 and 253. HIV-1 expression was assessed by measuring reverse transcriptase activity. All media conditioned by unstimulated and lipopolysaccharide (LPS) stimulated glial cells induced HIV-1 expression and contained detectable levels of interleukin-6 (IL-6) but not tumor necrosis factor-alpha (TNF-alpha). An antibody against IL-6, but not against TNF-alpha, reduced the induction of HIV-1 by the conditioned media in a concentration-dependent manner. The magnitude of HIV-1 induction by the conditioned media was proportional to the concentration of IL-6 in them. The data indicate that normal and transformed human astrocytes are capable of stimulating HIV-1 expression in chronically infected promonocytic cells by secreting IL-6. The results demonstrate that cytokines secreted by neural cells could play an important role in regulating HIV-1 expression in the brain.

A unique human immunodeficiency virus culture secreting soluble gp160.

A clone of the HUT78 cell line, chronically infected with the HIV-1 isolate HTLV-III451, has been demonstrated to secrete unprocessed HIV-1 envelope precursor protein gp160 as well as mature gp120. Further, when grown in serum-free defined medium these cells released approximately five times the amount of virus compared with cultures in normal medium. These proteins corresponded in their immunologic reactivities with the respective envelope proteins of the HTLV-IIIB isolate. They formed high-affinity soluble complexes with the CD4 antigen and inhibited the syncytium formation induced by HTLV-IIIB on CD4-positive cells. This is the first description of an HIV-1 culture system capable of shedding into the medium native gp160 that is soluble in the absence of detergents.

Characterization of cell markers in type B retroviral-induced thymic lymphomas--II. Surface antigen phenotype, karyotype and proviral integration pattern in cultured lymphoma cells and cloned lines.

This study examined the surface antigen phenotype, karyotype and proviral integration patterns of cultured cells from murine thymic lymphomas induced by injecting neonatal mice with the type B leukemogenic retrovirus (DMBA-LV). Cells from the primary thymic lymphomas were established in mass culture and from these, clonal tumor cell lines were derived. During in vitro culture of lymphoma cells, Lyt 1-2+ cells predominated with an apparent selection against cells of the Lyt 1+2- and Lyt 1+2+ phenotypes. Of 21 cloned lines established, five had a diploid chromosome complement and expressed the Lyt 1-2+ phenotype. The other 16 clones lines were characterized by trisomy of chromosome 15 and expressed the Lyt 1+2+ or Lyt 1-2+ phenotype. Cells characterized by either a diploid or trisomy chromosome complement were capable of growth in vivo. Southern blot analyses showed that during growth in culture, cells from the mass cultures and cloned lines continued to acquire low numbers of new integrated DMBA-LV proviral copies while maintaining the basic proviral integration pattern present in the DNA from cells of the primary lymphomas. These findings support the notion that the acquisition of new genetic information in cells from DMBA-LV-induced thymic lymphomas may contribute to the continual generation of tumor heterogeneity.

Enhancement of endogenous xenotropic murine retrovirus expression by tuftsin.

The exposure of a high-passage clone of Kirsten sarcoma virus transformed Balb/c (K-Balb) mouse cells to tuftsin (Thr-Lys-Pro-Arg) enhanced the expression of endogenous xenotropic retrovirus. The tetrapeptide increased the expression of virus that was infectious for rat, but not mouse, cells in a concentration-dependent fashion (0.001-1000 micrograms/ml). Increased virus expression could be achieved during short-term incubations (3-4 hr), with maximum enhancement occurring over longer time periods (16-18 hr). The enhancement of virus expression by tuftsin was proportional to the spontaneous release of virus. The infectivity of the enhanced virus was neutralized by goat anti-RLV gp 70 serum. Actinomycin D inhibited the induction of virus, suggesting that enhanced expression required de novo RNA synthesis. Tuftsin stimulated DNA, RNA, and protein synthesis in K-Balb cells during 16-hr incubations. Increased cellular proliferation was also seen at various time periods. The effects observed using K-Balb cells offer an opportunity to study the modulation of gene expression by tuftsin in a fibroblast culture system.

Clones of MRC-C cells may be superior to the parent line for the culture of 229E-like strains of human respiratory coronavirus.

A clone was selected from the MRC-C continuous heteroploid cell line which was significantly better than MRC-C for the culture of human respiratory coronavirus (HCV) 229E, strain LP. Such clones could prove generally useful for the isolation of HCV-229E from clinical specimens, and for the propagation and assay of laboratory adapted strains.