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Generation of the natural killer cell repertoire: the sequential vs. the two-step selection model.

Natural killer (NK) cells are lymphocytes which can kill tumor and virus-infected cells, and mediate acute rejection of bone marrow grafts. NK-cell killing is directed primarily at target cells which do not express sufficient levels of self-major histocompatibility complex (MHC) class-I molecules. Inhibition of lysis of self-MHC-expressing target cells is mediated via inhibitory receptors on the NK cell, which bind to MHC molecules. Each NK cell expresses only a subset of all its inhibitory receptor gene repertoire, which may bind to only a subset of the self-MHC molecules expressed by other cells of the organism. Two conceptual models have been proposed to explain the process of NK cell 'education' in which these cells adapt, during their development, to the self-MHC environment: the two-step selection and the sequential models. In this manuscript we develop mathematical and computational models of the process of NK cell development and education, which implement the two conceptual models. We use this theoretical framework to examine the available data on NK cell repertoire compositions, and evaluate the degree these data support either of the two conceptual models. We show that the data published so far on NK cell receptor expression patterns is insufficient to refute either model, since data on NK cell receptor binding affinities to MHC is also needed. However, the models allow us to make predictions on these binding affinities, which can later be tested experimentally.

Animals↗

Neutralized adenovirus-immune complexes can mediate effective gene transfer via an Fc receptor-dependent infection pathway.

Neutralization of adenovirus (Ad) by anti-Ad neutralizing antibodies in serum involves formation of Ad-immune complexes that prevent the virus from interacting with target cells. We hypothesized that Ad-immune complexes likely contain viable Ad vectors which, although no longer capable of gaining access to receptors on target cells, may be able to express transgenes in cells bearing Fc receptors for immunoglobulins, i.e., that antibody-based "neutralization" of Ad vectors may be circumvented by the Fc receptor pathway. To test this hypothesis, we expressed the Fcgamma receptor IIA (FcgammaR) in A549 lung epithelial cells or human dermal fibroblasts and evaluated gene transfer in the presence of human neutralizing anti-Ad serum. FcgammaR-expressing cells bound and internalized copious amounts of Ad, with a distinct population of internalized Ad trafficking to the nucleus. The dose-response curves for inhibition of gene transfer revealed that FcgammaR-expressing cells required a more-than-10-fold higher concentration of anti-Ad serum to achieve 50% inhibition of Ad-encoded beta-galactosidase expression compared with non-FcgammaR-expressing cells. The discrepancy between neutralization of Ad during infection of FcgammaR-expressing cells and neutralization of Ad during infection of non-FcgammaR-expressing cells occurred with either heat-inactivated or non-heat-inactivated sera, was blocked by addition of purified Fc domain protein, and did not require the cytoplasmic domain of FcgammaR, suggesting that immune complex internalization proceeded via endocytosis rather than phagocytosis. FcgammaR-mediated infection by Ad-immune complexes did not require expression of the coxsackie virus-Ad receptor (CAR) since similar data were obtained when CAR-deficient human dermal fibroblasts were engineered to express FcgammaR. However, interaction of the Ad penton base with cell surface integrins contributed to the difference in neutralization between FcgammaR-expressing and non-FcgammaR-expressing cells. The data indicate that complexes formed from Ad and anti-Ad neutralizing antibodies, while compromised with respect to infection of non-FcgammaR-expressing target cells, maintain the potential to transfer genes to FcgammaR-expressing cells, with consequent expression of the transgene. The formation of Ad-immune complexes that can target viable virus to antigen-presenting cells may account for the success of Ad-based vaccines administered in the presence of low levels of neutralizing anti-Ad antibody.

Adenoviridae↗

Regulation of apoptosis and T cell activation by Fas-specific mAb.

Fas was initially described as a molecule expressed on the surface of certain cell lines that could mediate programmed cell death (apoptosis) subsequent to ligation by specific mAb. To determine whether mAb to other epitopes on the Fas molecule might mediate other functions, we generated a panel of mAb to the extracellular portion of human Fas. Significant lysis of Fas-expressing target cells was only observed when the new mAb were first bound to a solid-phase support and not when the mAb were added in solution. However, several of these mAb inhibited the killing of target cells induced by the prototypic Fas-specific mAb, CH-11. Those mAb that inhibited apoptosis of target cells mediated by the CH-11 mAb also blocked lysis of target cells mediated by cells expressing Fas ligand. Finally, some of the Fas-specific mAb were found to co-stimulate proliferation of peripheral blood T cells in the presence of immobilized CD3 mAb. Thus, the data indicate the existence of a complex set of interactions mediated by Fas in both normal and transformed lymphoid cells that may have important implications regarding the role(s) of this molecule in regulation of immune responses.

Animals↗

Inhibition of heme oxygenase-1 increases responsiveness of pancreatic cancer cells to anticancer treatment.

Heme oxygenase-1 (HO-1) is believed to represent a key enzyme for the protection of cells against "stress." Its overexpression in different types of human cancers supports the notion that HO-1 provides a growth advantage and contributes to cellular resistance against chemotherapy and radiotherapy. Given the poor survival rates of patients with pancreatic cancer due to its aggressive growth behavior and its exceptional resistance to all known forms of anticancer treatment, we have investigated the expression of HO-1 in human pancreatic cancer cells growth behavior and prognosis. Expression of HO-1 was analyzed in human pancreatic cancer samples in comparison with normal pancreas by quantitative PCR, Western blot, and confocal microscopy. The influence of radiotherapy and chemotherapy on HO-1 expression in pancreatic cancer cell lines was evaluated. Furthermore, HO-1 expression was specifically suppressed by small interfering RNA transfection and subsequently the alterations of growth behavior and resistance to anticancer treatment were tested. Human pancreatic cancer showed a 6-fold and 3.5-fold HO-1 up-regulation in comparison to normal pancreas based on mRNA and protein level, respectively (P < 0.05). Cancer tissues revealed marked HO-1 immunoreactivity in tumor cells and in tumor associated immunocytes. Treatment of the pancreatic cancer cell lines with gemcitabine or radiation strongly induced HO-1 expression. Targeted knockdown of HO-1 expression led to pronounced growth inhibition of the pancreatic cancer cells and made tumor cells significantly more sensitive to radiotherapy and chemotherapy. Therefore, specific inhibition of HO-1 expression may be a new option in pancreatic cancer therapy and may be used as sensitizer to chemotherapy and radiotherapy.

Adult↗

Immunochemically purified DR antigens in liposomes stimulate xenogeneic cytolytic T cells in secondary in vitro cultures.

A monoclonal antibody directed against the human class II major histocompatibility antigen DR was generated. Use of this antibody, LB3.1, allowed isolation of large amounts of highly purified DR by immunoaffinity chromatography. The DR was reconstituted into liposomes and shown to stimulate secondary xenogeneic cytolytic T lymphocytes (CTL) specific for targets expressing DR antigens. DR digested with neuraminidase was equally as effective as native DR at stimulating CTL, while denatured DR and other purified membrane proteins were much less effective. The DR liposome-induced CTL lysed only target cells expressing class II antigens. Cytolysis of targets bearing class II antigens was blocked by DR-specific antisera.

Animals↗

A combinatorial network of evolutionarily conserved myelin basic protein regulatory sequences confers distinct glial-specific phenotypes.

Myelin basic protein (MBP) is required for normal myelin compaction and is implicated in both experimental and human demyelinating diseases. In this study, as an initial step in defining the regulatory network controlling MBP transcription, we located and characterized the function of evolutionarily conserved regulatory sequences. Long-range human-mouse sequence comparison revealed over 1 kb of conserved noncoding MBP 5' flanking sequence distributed into four widely spaced modules ranging from 0.1 to 0.4 kb. We demonstrate first that a controlled strategy of transgenesis provides an effective means to assign and compare qualitative and quantitative in vivo regulatory programs. Using this strategy, single-copy reporter constructs, designed to evaluate the regulatory significance of modular and intermodular sequences, were introduced by homologous recombination into the mouse hprt (hypoxanthine-guanine phosphoribosyltransferase) locus. The proximal modules M1 and M2 confer comparatively low-level oligodendrocyte expression primarily limited to early postnatal development, whereas the upstream M3 confers high-level oligodendrocyte expression extending throughout maturity. Furthermore, constructs devoid of M3 fail to target expression to newly myelinating oligodendrocytes in the mature CNS. Mutation of putative Nkx6.2/Gtx sites within M3, although not eliminating oligodendrocyte targeting, significantly decreases transgene expression levels. High-level and continuous expression is conferred to myelinating or remyelinating Schwann cells by M4. In addition, when isolated from surrounding MBP sequences, M3 confers transient expression to Schwann cells elaborating myelin. These observations define the in vivo regulatory roles played by conserved noncoding MBP sequences and lead to a combinatorial model in which different regulatory modules are engaged during primary myelination, myelin maintenance, and remyelination.

Animals↗

The malignant capacity of skin tumours induced by expression of a mutant H-ras transgene depends on the cell type targeted.

BACKGROUND: . Pinpointing the cells from which tumours arise is a major challenge n tumour biology. Previous work has shown that the targeted expression of a mutant ras gene within the interfollicular cell compartment of mouse skin induces the formation of benign papillomas, but these do not spontaneously progress to malignancy. We have investigated the carcinogenic effects of expressing the same oncogene in a different population of epidermal cells. RESULTS: Expression of mutant ras from a truncated keratin 5 gene promoter, which directs expression to the follicular and interfollicular cells of newborn mice and the hair follicle cells of adults, stimulated the development of acanthotic areas in newborn mice. Within one week of birth, the acanthotic skin developed areas of carcinoma in situ and adult mice developed papillomas and keratoacanthomas, the latter having a high frequency of spontaneous malignant transformation to squamous and occasionally spindle carcinomas. The benign tumours that arose had several hallmarks of tumours at a high risk of malignant progression, including suprabasal cell proliferation and heterogeneous expression of keratin 13. In contrast to tumours induced by expressing mutant ras under the control of the keratin 10 or keratin 1 gene promoters, the formation of these lesions was not dependent on wounding or a tumour promoter. CONCLUSIONS: Benign tumours that are at a risk of malignant conversion are primarily derived from cells located within the hair follicle, and the nature of the cell in which tumour initiation occurs is a major determinant of malignant potential.

Animals↗

A novel DNA vaccine based on ubiquitin-proteasome pathway targeting 'self'-antigens expressed in melanoma/melanocyte.

Cancer vaccine that targets 'self'-antigens expressed at high levels in tumor cells is a potentially useful immunotherapy, but immunological tolerance often defeats this strategy. Here, we describe the use of a naked DNA vaccine encoding a self tumor antigen, tyrosinase-related protein 2, to whose N-terminus ubiquitin is fused in a 'nonremovable' fashion. Unlike conventional DNA vaccines, this vaccine broke the tolerance and induced protective immunity to melanoma in C57BL/6 mice, as evaluated by tumor growth, survival rate and lung metastasis. The protective immunity was cancelled in the proteasome activator PA28alpha/beta knockout mice. Moreover, this vaccination exhibited therapeutic effects on melanoma implanted before vaccination. Our findings provide evidence for the first time that naked DNA vaccines encoding a ubiquitin-fused self-antigen preferentially induce the main effector CD8+ T cells through efficient proteolysis mediated by the ubiquitin-proteasome pathway, and lead the way to strategies aimed at targeting tissue differentiation antigens expressed by tumors.

Animals↗

Identification of naturally processed CD8 T cell epitopes from prostein, a prostate tissue-specific vaccine candidate.

Prostein is a prostate tissue-specific protein that is uniquely and abundantly expressed in normal and cancerous prostate tissues. Due to this expression profile, we examined the immunogenicity of prostein as a potential vaccine candidate for prostate cancer. To determine the presence of CD8 T cells specific for naturally processed prostein-derived epitopes in healthy individuals, we developed and applied an in vitro stimulation protocol. Using this protocol, we identified CD8 T cells specific for prostein in the peripheral blood of a male and a female donor. Prostein-specific CD8 T cell clones specifically recognized prostein-expressing targets, including prostate tumor cell lines expressing the relevant HLA alleles. CD8 T cell clones isolated from the male donor were significantly less effective in recognizing target cells compared to cells isolated from the female donor and appeared to recognize subdominant epitopes. The identification of a prostein-specific CD8 T cell repertoire supports the development of prostein in vaccination strategies against prostate cancer. Furthermore, the naturally processed peptide epitopes identified provide tools for the development of peptide-based vaccination strategies against prostate cancer and for monitoring of prostein-specific responses in vaccinated patients.

Animals↗

Postnatal anti-interferon-gamma treatment prevents pancreatic inflammation in transgenic mice with beta-cell expression of interferon-gamma.

beta-Cell-targeted expression of interferon-gamma (IFN-gamma) leads to pancreatitis and immune sensitization to beta-cells. This transgenic model is used to explore the possible role of locally produced IFN-gamma in loss of tolerance to beta-cell-specific antigens in insulin-dependent diabetes mellitus (IDDM). The aim of the present study was to test if postnatal treatment with antibodies against IFN-gamma could inhibit morphological changes in the IFN-gamma transgenic pancreas, even though the transgene is expressed during embryogenesis. Treatment with a monoclonal rat anti-mouse IFN-gamma antibody for 6 weeks, starting from 5 to 7 days of age, completely inhibited IFN-gamma-induced morphological changes in the pancreas, and only a modest inflammatory reaction emerged after prolonged treatment for 12 weeks. The lack of morphological changes may reflect the ability of nonterminally differentiated neonatal pancreatic cells to compensate for transgene-induced pathological alterations occurring in utero prior to the antibody treatment. We conclude that inflammation and altered pancreas morphology in the transgenic mice is the result of the biological actions of IFN-gamma and not by disrupted islet development due to transgene overexpression in the pancreatic beta-cells. Furthermore, our treatment schedule can serve as a model for future intervention studies in the transgenic mice, elaborating the role of IFN-gamma in localized inflammatory reactions, IDDM in particular.

Animals↗

Smell and taste perception in Drosophila melanogaster larva: toxin expression studies in chemosensory neurons.

GAL4-driven targeted expression of tetanus toxin light chain (UAS-TeTxLC) in a subset of chemosensory neurons of the larval antennomaxillary complex (AMC) and pharynx causes abnormal chemosensory behavior in Drosophila melanogaster. Consistent with strongest staining in the dorsal organ (DO), the presumed olfactory organ of the AMC, tetanus toxin-expressing larvae subjected to an olfactory preference assay show anosmic behavior to most volatile substances tested. Furthermore, we observed reduced responses to sodium chloride, fructose, and sucrose in gustatory plate assays. Surprisingly, the entire subset of labeled sensory neurons from the terminal (maxillary) organ (TO) of the AMC was found to project via the antennal nerve to the larval antennal lobe region. The maxillary nerve remained completely unstained. Hence, a subset of neurons from the TO builds an anatomical entity with projections from the DO. Our results suggest that the AMC contains both olfactory and gustatory sensilla, and that the DO is the main olfactory organ in larvae.

Animals↗

Dependence on target configuration of express saccade-related activity in the primate superior colliculus.

To help understand how complex visual stimuli are processed into short-latency saccade motor programs, the activity of visuomotor neurons in the deeper layers of the superior colliculus was recorded while two monkeys made express saccades to one target and to two targets. It has been shown previously that the visual response and perimotor discharge characteristic of visuomotor neurons temporally coalesce into a single burst of discharge for express saccades. Here we seek to determine whether the distributed visual response to two targets spatially coalesces into a command appropriate for the resulting saccade. Two targets were presented at identical radial eccentricities separated in direction by 45 degrees. A gap paradigm was used to elicit express saccades. Express saccades were more likely to land in between the two targets than were saccades of longer latency. The speeds of express saccades to two targets were similar to those of one target of similar vector, as were the trajectories of saccades to one and two targets. The movement fields for express saccades to two targets were more broad than those for saccades to one target for all neurons studied. For most neurons, the spatial pattern of discharge for saccades to two targets was better explained as a scaled version of the visual response to two spatially separate targets than as a scaled version of the perimotor response accompanying a saccade to a single target. Only the discharge of neurons with large movement fields could be equally well explained as a visual response to two targets or as a perimotor response for a one-target saccade. For most neurons, the spatial properties of discharge depended on the number of targets throughout the entire saccade-related burst. These results suggest that for express saccades to two targets the computation of saccade vector is not complete at the level of the superior colliculus for most neurons and an explicit process of target selection is not necessary at this level for the programming of an express saccade.

Action Potentials↗

Studies of the mechanism of cytolysis by HIV-1-specific CD4+ human CTL clones induced by candidate AIDS vaccines.

Vaccine-induced, virus-specific CTLs may rapidly eliminate the host cells that first become infected after virus exposure, thereby preventing disseminated infection. Thus, there is much interest in the ability of candidate AIDS vaccines to elicit CTLs. All HIV-1 envelope (env) protein-based vaccines tested to date in seronegative humans induce CTLs from the CD4+ subset. Because the mechanism of cytolysis by CD4+ CTLs is controversial, a detailed study of the cytolytic reactions mediated by vaccine-induced, HIV-1-specific human CD4+ CTL clones was conducted. CD4+ CTL clones induced rapid destruction of Ag-pulsed target cells. Lysis was readily detectable within 15 min. Lysis was not a result of syncytium formation between CD4+ effector cells and env-expressing targets. Target cell destruction was not dependent upon de novo RNA or protein synthesis in either the effector or the target cell. Expression of perforin mRNA was detected by Northern blotting and reverse-transcriptase-PCR in CD4+ CTL clones but not in autologous B lymphoblastoid cell lines. Immunohistochemical studies demonstrated perforin protein in cytoplasmic granules in CD4+ CTL clones. Lysis by CD4+ CTLs was strictly dependent upon extracellular Ca2+ and was highly specific, with no lysis of innocent bystander cells. DNA fragmentation was detectable in target cells, but did not precede 51Cr release. Taken together, these results provide a dramatically different view of cytolysis by human CD4+ CTLs. Target cells are lysed by a rapid and efficient mechanism that involves a preformed mediator and that is functionally similar to the mechanism used by CD8+ CTLs.

AIDS Vaccines↗

Insect renal tubules constitute a cell-autonomous immune system that protects the organism against bacterial infection.

Innate immunity is a widespread and important defence against microbial attack, which in insects is thought to originate mainly in the fat body. Here we demonstrate that the fluid-transporting Malpighian (renal) tubule of Drosophila melanogaster constitutes an autonomous immune-sensing tissue utilising the nitric oxide (NO) signalling pathway. Reverse transcriptase PCR (RT-PCR) shows that tubules express those genes encoding components of the Imd pathway. Furthermore, isolated tubules bind and respond to lipopolysaccharide (LPS), by upregulating anti-microbial peptide (diptericin) gene expression and increased bacterial killing. Excised, LPS-challenged tubules, as well as tubules from LPS-infected flies, display increased NO synthase (NOS) activity upon immune challenge. Targetted expression of a Drosophila NOS (dNOS) transgene to only principal cells of the tubule main segment using the GAL4/UAS system increases diptericin expression. In live flies, such targetted over-expression of dNOS to tubule principal cells confers increased survival of the whole animal upon E. coli challenge. Thus, we describe a novel role of Malpighian tubules in immune sensing and insect survival.

Animals↗

Major histocompatibility complex class I-dependent cell binding to isolated Ly-49A: evidence for high-avidity interaction.

Ly-49A molecules negatively regulate a subset of mouse natural killer (NK) cells, preventing lysis of H-2Dd-expressing target cells. In the present report, we immunoaffinity-purified Ly-49A from the EL4 lymphoma using the A1 monoclonal antibody (mAb) and examined cell adhesion to immobilized Ly-49A. Adhesion was observed by cells expressing relatively high levels of H-2Dd, but not cells expressing very low or no cell surface Dd, while antibodies specific for Dd or Ly-49A inhibited the cell binding, indicating that Dd and Ly-49A mediate the observed adhesion. The density of immobilized Ly-49A was varied and confirmed by ELISA. Cell binding exhibited a threshold Ly-49A density requirement, and above this threshold, increases in Ly-49A density resulted in substantial increases in cell adhesion to a high maximum cell binding. The density of Ly-49A homodimers required to mediate cell adhesion was found to be quite low: 140-250 molecules/microm2. These results suggest that the avidity of Ly-49A for Dd is relatively high and indicate that small changes in Ly-49A density near the threshold result in large changes in stable Ly-49A receptor engagement. The relatively sharp threshold and marked density dependence presented here for Ly-49A receptor engagement may explain the observation that relatively small differences in Ly-49A expression level on NK cells result in significant differences in functional outcome, i.e. whether a target cell expressing a low level of Dd is spared from lysis or not.

Animals↗

Novel CNBP- and La-based translation control systems for mammalian cells.

Throughout the development of Xenopus, production of ribosomal proteins (rp) is regulated at the translational level. Translation control is mediated by a terminal oligopyrimidine element (TOP) present in the 5' untranslated region (UTR) of rp-encoding mRNAs. TOP elements adopt a specific secondary structure that prevents ribosome-binding and translation-initiation of rp-encoding mRNAs. However, binding of CNBP (cellular nucleic acid binding protein) or La proteins to the TOP hairpin structure abolishes the TOP-mediated transcription block and induces rp production. Based on the specific CNBP-TOP/La-TOP interactions we have designed a translation control system (TCS) for conditional as well as adjustable translation of desired transgene mRNAs in mammalian cells. The generic TCS configuration consists of a plasmid encoding CNBP or La under control of the tetracycline-responsive expression system (TET(OFF)) and a target expression vector containing a TOP module between a constitutive P(SV40) promoter and the human model product gene SEAP (human secreted alkaline phosphatase) (P(SV40)-TOP-SEAP-pA). The TCS technology showed excellent SEAP regulation profiles in transgenic Chinese hamster ovary (CHO) cells. Alternatively to CNBP and La, TOP-mediated translation control can also be adjusted by artificial phosphorothioate anti-TOP oligodeoxynucleotides. Confocal laser-scanning microscopy demonstrated cellular uptake of FITC-labeled oligodeoxynucleotides and their localization in perinuclear organelles within 24 hours. Besides their TOP-based translation-controlling capacity, CNBP and La were also shown to increase cap-independent translation from polioviral internal ribosomal entry sites (IRES) and La alone to boost cap-dependent translation initiation. CNBP and La exemplify for the first time the potential of RNA-binding proteins to exert translation control of desired transgenes and to increase heterologous protein production in mammalian cells. We expect both of these assets to advance current gene therapy and biopharmaceutical manufacturing strategies.

Animals↗

Inducible gene targeting in mice using the Cre/lox system.

Molecular techniques now allow the design of precise genetic modifications in the mouse. Not only can defined nucleotide changes be engineered into the genome of the mouse, but genetic switches can be designed to target expression or ablation of any gene (for which basic molecular information is available) to any tissue at any defined time. These strategies promise to contribute substantially to an increased understanding of individual gene function in development and pathogenesis. A powerful tool, both for the design of such genetic switches and for speeding the creation of gene-modified animals, is the Cre site-specific DNA recombinase of bacteriophage P1. Precise DNA rearrangements and genetic switches can be efficiently generated in a straightforward manner using Cre recombinase. In conjunction with inducible systems for controlling Cre expression and function, these recombination-based strategies are likely to have a profound impact on developmental biology and the generation of useful animal models of human disease.

Animals↗

Tissue-specific and developmental regulation of rod opsin chimeric genes in transgenic mice.

Chimeric gene fusions between 4.4 kb of rod opsin 5' flanking sequence fused to a diphtheria toxin gene and 4.4 kb or 500 bp of rod opsin 5' flanking sequence fused to the E. coli IacZ gene were used to generate transgenic mice for analysis of cell type-specific expression and temporal and spatial distribution of reporter gene product during retinal development. Opsin-diphtheria toxin transgene expression evoked photoreceptor-specific cell death. The 4.4 kb opsin-IacZ transgene followed temporal and spatial gradients of expression that approximate opsin expression. The 500 bp opsin fragment targeted expression to photoreceptors, but expression was weaker and nonuniform, suggesting that elements located upstream may be required for enhanced and uniform spatial expression.

Animals↗