Murine Cytotoxic T-cell Line Research Articles

Zinc signals promote IL‐2‐dependent proliferation of T cells

Zinc signals, i.e. a change of the intracellular concentration of free zinc ions in response to receptor stimulation, are involved in signal transduction in several immune cells. Here, the role of zinc signals in T-cell activation by IL-2 was investigated in the murine cytotoxic T-cell line CTLL-2 and in primary human T cells. Measurements with the fluorescent dyes FluoZin-3 and Zinquin showed that zinc is released from lysosomes into the cytosol in response to stimulation of the IL-2-receptor. Activation of the ERK-pathway was blocked by chelation of free zinc with N,N,N',N'-tetrakis-2(pyridyl-methyl)ethylenediamine, whereas zinc was not required for STAT5 phosphorylation. In addition, the key signaling molecules MEK and ERK were activated in response to elevated free intracellular zinc, induced by incubation with zinc and the ionophore pyrithione. Downstream of ERK activation, ERK-specific gene expression of c-fos and IL-2-induced proliferation was found to depend on zinc. Further experiments indicated that inhibition of MEK and ERK-dephosphorylating protein phosphatases is the molecular mechanism for the influence of zinc on this pathway. In conclusion, an increase of cytoplasmic free zinc is required for IL-2-induced ERK signaling and proliferation of T cells.

In vivo cervical cancer growth inhibition by genetically engineered cytotoxic T cells.

The CD44 v7/8 splice variant that is frequently expressed in cervical carcinoma and rarely expressed in normal tissues displays promising properties as a target antigen for cancer immune therapy. In this study, cytotoxic T lymphocytes (CTLs) were genetically engineered to gain CD44v7/8 target specificity. Clone 96 (Cl96), an established murine cytotoxic T-cell line, and naïve murine T cells were retrovirally transduced with a fusion gene construct encoding for the single chain fragment scFv of the monoclonal antibody VFF17 and for the zeta chain of the T-cell receptor (TCR). The therapeutic potential of genetically engineered T cells was tested in vitro and in vivo. Surface expression of the chimeric TCR on infected Cl96 and naïve T cells was shown by FACS analysis. CD44v7/8-positive target cells were efficiently lysed by transduced Cl96 and naïve T cells, demonstrating the functionality and specificity of the chimeric TCR. In a xenograft BALB/c mouse model, efficient growth retardation of CD44v7/8-positive tumours was mediated by genetically engineered Cl96(VFF17)cyYZ cells. We were able to reprogramme the target specificity of recombinant Cl96 and naïve CTLs resulting in efficient cytolysis of CD44v7/8-positive cervical cancer cells. High transduction rates and the specific cytolysis of CD44v7/8-redirected CTLs are promising tools for an immune gene therapy approach for advanced cervical cancer.

Gentechnische Modifikation der Targetspezifität zytotoxischer T-Zellen - ein additiver Therapieansatz bei gynäkologischen Tumoren

Purpose: The variant epitope CD44v7/8 is frequently expressed on cervical carcinoma but is rarely expressed in normal cervical tissue. Therefore CD44v7/8 is a tumour specific antigen and may play a role as a promising target for tumour-specific immunotherapy. Aim of our study was to develop a selective immunotherapy by using genetically engineered cytotoxic lymphocytes (CTL's) with a v7/8 target specificity. Material and Methods: The genes coding for the single chain fragment scFv of the monoclonal antibody VFF 17 and of the ζ-chain of the TCR (T cell receptor) complex were fused and inserted into a retroviral vector. We retrovirally transduced a well established murine cytotoxic T-cell line (CI96) and primary mouse T-cells. T-cell clones showing surface expression of the chimeric protein were selected. A cytotoxicity assay was performed to investigate the functionality of the chimeric receptor in vitro. In a second step, an animal model of immuno incompetent nude mice bearing CD44v7/8 positive tumours was used to analyse the efficiency of the recombinant T-cell receptor in vivo. Results: Transduced CI96 cells and primary T cells showed MHC independent and efficient killing of CD44v7/8 positive target cells in vitro. The in vivo experiments in the mouse model demonstrated an efficient antitumour activity of recombinant CTL's. We showed that growth of CD44v7/8 expressing tumours can be efficiently inhibited by reprogrammed T cells. Conclusion: The in vivo efficiency and the high transduction rates of CTL's are promising results of this preliminary study. Fur-ther investigations of our group focus on primary autologous human T-cells.

Evaluation of Cytokine Delivery Systems for Cancer Immunotherapy

AbstractCytokines are ideal candidates for controlled release systems; they typically have short half-livesin vivoand may be more effective with localized delivery. A novel microencapsulation technique, phase inversion nanoencapsulation (PIN), was used to encapsulate interleukin-2 or interleukin-12 in biodegradable polymer microspheres. Cytokine was quantified by ELISA and bioactivity analyzed using a murine cytotoxic Tcell line proliferation assay. The release rates of IL-2 was dependant on release buffer components; inclusion of 10% fetal calf serum resulted in significantly higher release rates compared to plain buffer. Encapsulated IL-2 remained stable at all timepoints assayed (up to 4 months) when stored at 4 °C. Results indicated that PIN can be used to encapsulate IL-2 and IL-12, which were released throughout the duration of the release study (up to one month). These microsphere formulations have shown tumor suppressive affectsin vivoand may provide a viable alternative to gene transfer for cancer immunotherapy.

Efficient Lysis of CD44v7/8-Presenting Target Cells by Genetically Engineered Cytotoxic T-Lymphocytes—A Model for Immunogene Therapy of Cervical Cancer

Variant proteins of the CD44 surface glycoprotein family are expressed on many different human tumors and their lymph node metastases. An epitope encoded by sequences of variant exons CD44v7 and v8 and recognized by the monoclonal antibody VFF17 is frequently detected in cervical cancer, whereas the normal cervical epithelium lacks expression of this epitope. We have developed an immunotherapeutic approach for cervical cancer based on the expression of this CD44v7/8 epitope. The single chain antigen-binding fragment of VFF17 was fused to a signal transducing protein (ζ-chain) of the T-cell receptor complex (TCR) and was introduced into a retroviral gene transfer vector. Gene transfer was applied to the murine cytotoxic T-cell line cl96. All recombinant clones expressed the fusion protein on their cell surface. Functionality of the recombinant fusion protein was tested by subjection of several recombinant clones toin vitrocytotoxicity assays. CD44v7/8-expressing target cells were killed efficiently by reprogrammed cl96 in an MHC-independent fashion, whereas CD44v7/8-negative cells were not affected. These transfected T cell lines will now be testedin vivousing immune-deficient mice bearing CD44v7/8-expressing tumors.

Delta-9-tetrahydrocannabinol treatment results in a suppression of interleukin-2-induced cellular activities in human and murine lymphocytes

Delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, has been shownto suppress a variety of interleukin-2-(IL-2)-dependent cellular functions in both murine and human lymphocytes. These effects were examined in both human peripheral blood lymphocytes (hPBL) and the IL-2-dependent murine cytotoxic T-cell line CTLL-2. Interleukin-2-induced thymidine uptake and uridine uptake were suppressed in a dose related manner when cells were co-incubated for 48 h with 100 U rhIL-2/ml and 1 – 10 μg THC/ml. Interleukin-2-induced protein synthesis was also suppressed in a dose related manner over this THC concentration range, with the hPBL being more susceptible to the suppressive effect of THC than the CTLL-2 cells. Autoradiographic analysis of the synthesized proteins from hPBL cell lysates reveals a generalized suppression of all nascent proteins in THC-treated cultures. Human natural killer cell activity is only affected at the highest concentration tested (10 μg THC/ml) while lymphokine-(IL-2)-activated natural killer cell activity is affected throughout the range of 1 – 10 μg THC/ml. Together these results suggest that THC interferes with the IL-2 : IL-2 receptor signaling cascade at one or possibly many points causing a decrease in IL-2-induced metabolic activity and cytolytic function.

Vitamin E Increases Interleukin-2-Dependent Cellular Growth and Glycoprotein Glycosylation in Murine Cytotoxic T-Cell Line

The effect of vitamin E on the modulation of interleukin-2 (IL-2) dependent cellular growth, glycosylation, and expression of certain glycoproteins (GP) was examined in a murine cytotoxic T-cell line (CTLL). CTLL cultures in log phase and growing in 10% IL-2 medium in the presence of 1-16 μg vitamin E/ml showed a dose-dependent 2- to 15-fold stimulation of growth (assessed by 3H-thymidine incorporation) relative to controls. Vitamin E-treated cultures were observed to grow more efficiently at lower levels of IL-2 compared to controls. Glycoconjugate biosynthesis (assessed by 3H-galactose incorporation) was stimulated 5- to 15-fold by the addition of vitamin E to the culture medium. GP glycosylation as determined by wheat germ agglutinin binding GP (50-64 Kd) and concanavalin A binding GP (38-200 Kd) increased significantly in the presence of vitamin E. These results demonstrate that vitamin E stimulates IL-2 dependent cellular growth, glycosylation, and expression of certain GP in CTLL.

The two different receptors for tumor necrosis factor mediate distinct cellular responses.

The individual roles of the murine type 1 and type 2 tumor necrosis factor (TNF) receptors (TNF-R1 and TNF-R2) were investigated utilizing (i) the strong species specificity of TNF-R2 for murine TNF compared to human TNF and (ii) agonistic rabbit polyclonal antibodies directed against the individual TNF receptors. Proliferation of mouse thymocytes and the murine cytotoxic T-cell line CT-6 is stimulated by murine TNF but not by human TNF. Consistent with this observation, polyclonal antibodies directed against TNF-R2 induced proliferation in both of these cell types, whereas polyclonal antibodies directed against TNF-R1 had no effect. In contrast, cytotoxicity in murine LM cells (which are sensitive to murine and human TNF) was induced by antibodies against TNF-R1 but not by antibodies against TNF-R2. Also, the steady-state level of manganous superoxide dismutase mRNA in the murine NIH 3T3 cell line was induced by murine TNF, human TNF, and anti-TNF-R1 but not by anti-TNF-R2. These results suggest that TNF-R2 initiates signals for the proliferation of thymocytes and cytotoxic T cells, whereas TNF-R1 initiates signals for cytotoxicity and the induction of the protective activity, manganous superoxide dismutase. The nonredundant signaling observed for the two TNF receptors cannot be explained simply by the differential expression of the two TNF receptors in the various cell types, because LM cells express on their surface higher levels of TNF-R2 than TNF-R1, and LM cells, NIH 3T3 cells, and thymus cells all express mRNA corresponding to both receptor types. It is therefore likely that the two receptors initiate distinct signaling pathways that result in the induction of different cellular responses.

Structure and differential mechanisms of regulation of expression of a serine esterase gene in activated human T lymphocytes.

A cDNA clone encoding a polypeptide resembling proteolytic serine esterases (from cytotoxic T-cells; SECT) was isolated from human peripheral blood lymphocytes which had been cultured in the presence of the T-cell mitogen phytohemagglutinin for 72 h. The cDNA encodes a polypeptide of 247 amino acids which show homology of 99% with the protein sequence encoded by a cDNA clone (1-3E) isolated from staphylococcal enterotoxin A-stimulated human peripheral blood lymphocytes and 68% with the protein sequence (cytotoxic cell protease type I) derived from a cDNA clone (C11) encoding a serine esterase isolated from a murine cytotoxic T-cell line. The overall nucleotide sequence homology between the SECT cDNA and 1-3E was 99% and 73% between SECT and C11. Comparing the coding regions of SECT and C11 showed 75% homology, whereas the 5'- and 3'-untranslated regions showed 67% homology. Phytohemagglutinin stimulation results in 30-, 60-, and 370-fold increases in cytoplasmic SECT mRNA with respect to unstimulated cells after 6-, 24-, and 72-h cultures, respectively. At 6 h, the increase in SECT mRNA occurs in the absence of increases in SECT gene transcription and cytoplasmic mRNA stabilization. A 5-fold increase in SECT nuclear RNA seen at this time suggests that stabilization of SECT nuclear RNA transcript is responsible for early increases in SECT mRNA levels. At 24 and 72 h, the increased cytoplasmic SECT mRNA levels can be accounted for by increased transcriptional activity of the SECT gene.

Expression of UDP-N-acetylgalactosamine: beta-galactose beta 1,4-N-acetylgalactosaminyltransferase in functionally defined T-cell clones.

To measure UDP-N-acetylgalactosamine: beta-galactose beta 1,4-N-acetylgalactosaminyltransferase (beta 1,4-GalNActransferase) in crude cell and tissue extracts we designed an assay containing UDP-[3H]N-acetylgalactosamine as donor and biotinylated human glycophorin A as an acceptor. After incubation the labelled acceptor was separated by the use of avidin-agarose from extract-derived endogenous acceptors. This assay permitted one to measure specifically the beta 1,4-GalNActransferase in crude extracts. This glycosyltransferase has previously been shown to be involved in the biosynthesis of Vicia villosa (hairy winter vetch)-lectin (VV)-binding sites of the murine cytotoxic T-cell line B6.1. Since VV-binding sites are a distinct marker for the cytotoxic subclass of murine T-lymphocytes, we used this assay to determine enzyme levels in a panel of functionally defined murine T-cell clones. Non-cytolytic T-cell lines generally have low activity, whereas most cytotoxic lines have high levels of activity. However, one cytotoxic T-cell line does not express the enzyme, although it has large numbers of VV-binding sites. This suggests the existence of another type of VV-binding sites which is independent of the beta 1,4-GalNActransferase in some cytotoxic-T-lymphocyte lines. The enzyme was also assayed in a variety of other tissues and found to have a very high activity in the intestine but a low activity in most other tissues. This was in considerable contrast with the ubiquitously high expression of UDP-GalNAc:peptide alpha 1-GalNActransferase. Therefore, the beta 1,4-GalNActransferase seems to be regulated during differentiation.

Immunoglobulin production in cultures of pokeweed mitogen stimulated human peripheral blood mononuclear cells requires interaction of interleukin 2 with the B cells

Immunoglobulin (Ig) production in cultures of pokeweed mitogen (PWM)-stimulated human peripheral blood mononuclear cells (PBMC) is dependent on the presence of T cells. The latter can be replaced by a crude supernatant of PWM-stimulated PBMC, a conditioned medium containing interleukin 2 (IL-2) and presumably other helper factors for B cells. The present study examines the role of IL-2 among these helper factors. Anti-Tac, a monoclonal antibody that blocks the human IL-2 receptor, was found to suppress PWM-induced Ig production in cultures of human PBMC. The inhibition by anti-Tac was at least partly exerted at the level of B cells. Indeed, Ig production in cultures of T-cell-depleted PBMC or highly enriched B-cell preparations supplemented with PWM-induced conditioned medium was also inhibited by anti-Tac (82 ± 13% inhibition of IgG and 83 ± 12% inhibition of IgM production). When IL-2 was removed from the PWM-induced conditioned medium by absortion with an IL-2-dependent murine cytotoxic T-cell line, CTLL, the conditioned medium was unable to support PWM-induced Ig production by T-cell-depleted PBMC. On the other hand, recombinant IL-2 alone at a concentration similar to that of IL-2 in the PWM-induced conditioned medium was not able to support Ig production by PWM-stimulated B cells. We conclude that IL-2 acts on PWM-activated B cells in conjunction with other helper factors to induce Ig production and that anti-Tac inhibits PWM-induced Ig production by blocking IL-2 receptors on activated B cells.

Only high-affinity receptors for interleukin 2 mediate internalization of ligand.

Interleukin 2 (IL-2) receptors are expressed on activated T cells and in select T-cell leukemias. Recently, it has been demonstrated that at least two classes of receptor for IL-2 exist with markedly different affinities for ligand. All known biological actions of IL-2 have been correlated with occupancy of high-affinity sites; the function of the low-affinity sites remains unknown. Receptor-mediated endocytosis is the primary means of internalization of cell-surface receptors and their ligands. The internalization of IL-2 bound to high- and low-affinity receptor sites was studied in a human T-cell lymphotrophic virus type 1 (HTLV-1)-infected human T-cell leukemia cell line and in a cloned murine cytotoxic T-cell line (CTLL). Internalization of IL-2 occurred only when bound to high-affinity sites. In addition, an anti-receptor antibody (anti-Tac), which binds equally well to high- and low-affinity sites, demonstrated no detectable internalization. The implications of these findings as they relate to IL-2 receptor structure and function are discussed.

Inhibition of Lymphocyte Proliferation by a Synthetic Peptide Homologous to Retroviral Envelope Proteins

The retroviral transmembrane envelope protein p15E is immunosuppressive in that it inhibits immune responses of lymphocytes, monocytes, and macrophages. A region of p15E has been conserved among murine and feline retroviruses; a homologous region is also found in the transmembrane envelope proteins of the human retroviruses HTLV-I and HTLV-II and in a putative envelope protein encoded by an endogenous C-type human retroviral DNA. A peptide (CKS-17) was synthesized to correspond to this region of homology and was examined for its effects on lymphocyte proliferation. CKS-17 inhibited the proliferation of an interleukin-2-dependent murine cytotoxic T-cell line as well as alloantigen-stimulated proliferation of murine and human lymphocytes. Four other peptides, representing different regions of virus proteins, were inactive. These results suggest that the immunosuppressive portion of retroviral transmembrane envelope proteins may reside, at least in part, in a-conserved sequence represented by the CKS-17 peptide.