Akt Phosphorylation Orchestrates T11TS Mediated Cell Cycle Arrest in Glioma Cells.

The novel anti-neoplastic glycopeptide T11TS retards glioma both in in-vitro clinical samples and in-vivo models. This study investigates the correlation between altering the glioma microenvironment with glioma arrest and death. Flow cytometry, immunoblotting, ELISA, and co-immunoprecipitation were employed to investigate glioma cell arrest and death. Results include a decline in phosphorylation of Akt and attenuation of p21 phosphorylation (Thr145,Ser146) and disassociation of p-Akt-Mdm2 and p-Akt-BAD facilitating death by Akt>BAD. T11TS influence phosphorylation patterns in two focal axes Akt>p21 and Akt>Mdm2>p53. The current article provides crucial insight in deciphering the mechanism of T11TS induced glioma cell arrest and death.

T11TS immunotherapy repairs PI3K-AKT signaling in T-cells: Clues toward enhanced T-cell survival in rat glioma model.

Malignant glioma is the most fatal of astrocytic lineage tumors despite therapeutic advances. Onset and progression of gliomas is accompanied by severe debilitation of T-cell defense and T-cell survival. One of the chief contributors to T-cell survival downstream of activation is the PI3K-AKT pathway. Our prior studies showed that the novel immunotherapeutic molecule T11-target structure (T11TS) blocks T-cell apoptosis in glioma. We also showed activation of immunological synapse components and calcineurin-NFAT pathway following T11TS immunotherapy of glioma-bearing rats. This lead to investigations whether such T-cell activation upon T11TS therapy translates into activation of downstream PI3K/AKT signals which may be related to observed blockade of T-cell apoptosis. For the purpose, we assessed by flowcytometry and immunoblotting, expressions of PI3K, PDK1, AKT, p-AKT, and PTEN in splenic T-cells of normal, experimentally-induced glioma-bearing rats and glioma-bearing rats receiving first, second and third doses of T11TS. We also determined comparative nuclear translocation of NF-κB across groups. We found significant increases in T-cell expressions of PDK1, PI3K, and p-AKT in T11TS-treated animal groups compared to sharp downregulations in glioma. AKT levels remained unchanged across groups. PTEN levels declined sharply after T11TS immunotherapy. T11TS also caused enhanced NF-κB translocation to the T-cell nucleus compared to glioma group. Results showed heightened activation of the PI3K-AKT pathway in glioma-bearing rats following T11TS immunotherapy. These results illustrate the novel role of T11TS immunotherapy in ameliorating the PI3K pathway in T-cells in glioma-bearing animals to enhance T-cell survival, according greater defense against glioma. The study thus has far-reaching clinical outcomes.

Immunotherapy with T11TS / S-LFA-3 specifically induces apoptosis of brain tumor cells by augmenting intracranial immune status.

BACKGROUND: Immunopotentiating agents are the best options in cancer therapeutics because they can specifically destroy tumor cells via immunocytes, which are mostly apoptotic in nature. Previously, immunotherapy with T11TS / SLFA-3 in a ethyl-nitrosourea (ENU)-induced animal model (Druckrey rats) of neural neoplasm showed a significant tumor mass destruction by augmenting the cellular immune status. MATERIALS AND METHODS: The modulations of the peripheral as well as neural immune systems after T11TS administration were monitored by assessing the CD4+ and CD8+ lymphocytes, along with the cytotoxic activity of splenic and brain infiltrating lymphocytes (BIL). The rate of apoptosis of the tumor cells, microglial cells (Mg) and BIL were measured by flow cytometry-based propidium iodide analysis and TUNEL assay. RESULTS: Cell cycle phase distribution analysis by propidium iodide -FACS and TUNEL assay revealed that T11TS administration gradually increased the number of apoptotic brain tumor cells and, at the same time, decreased the number of dividing cells. Up-regulation of the CD4+ and CD8+ lymphocytes were observed after T11TS administration in ENU - induced immunosuppressed animals. A gradual increment of cytotoxicity of splenic and BIL was also demonstrated after successive administration of T11TS. CONCLUSION: These data strongly support the specific apoptosis-inducing role of T11TS in experimental brain tumor cells. Apoptosis of BIL and Mg, that occurred to a much lower level, can be explained in terms of changes in the neural immune system before and after T11TS application.

T11TS/S-LFA3 induces apoptosis of the brain tumor cells: a new approach to characterise the apoptosis associated genetic changes by arbitrarily primed-PCR.

Genetic alterations in ethyl nitrosourea (ENU) induced brain tumor model were analysed by simple PCR based technique with arbitrary primers. T11TS/SLFA3 was established previously as a potent immune stimulator with antineoplastic property in experimental glioma model. The goal of this study was to reveal whether T11TS induces apoptosis of the neural neoplastic cell and to decipher the DNA polymorphism level of the cells undergoing apoptosis. The results clearly establish the apoptogenic role of T11TS/S-LFA3 and along with the detection of cancer associated genomic instability, AP-PCR analysis is useful for the detection of DNA level fragmentation, a unique feature of apoptosis.

The MAP kinases are differently utilized by CD28 and CD2 adhesion pathways in superantigen-activated Jurkat T cells.

To mimic the two-signal requirements for T cell activation mediated by ligands, we exposed the superantigens SEA or SEE (signal 1) to T cells incubated with HLA-DR/LFA-3 or HLA-DR/B7-1-CHO transfected cells (signal 2). LFA-3 costimulation was able to induce T cell proliferation as well as IFN-gamma and IL-4 production at similar levels as in cells induced by B7-1. Analysis of the CD28RE of the IL-2 promoter showed specific transcription factor recruitment at the CD28RE element upon induction by B7-1/SEE. Further functional studies with an IL-2 enhancer-promoter carrying either wild type or mutated versions of the CD28RE site revealed that this element is necessary for full activation upon B7-1 costimulation. While both CD28/B7-1 and CD2/LFA-3 costimulation resulted in the up-regulation of IL-4 and IFN-gamma promoters, IL-2 promoter activity and production of IL-2 were only seen after B7-1 costimulation. However, contrary to what has been previously proposed, we show that costimulation with either B7-1 or LFA-3 further enhanced the ERK-2 activity and strongly activated the p38 MAPK pathway, but only B7-1 costimulation induced high levels of JNK-1 activity. These data suggest that the differential effect of CD28 vs. CD2 can be related to the difference in the ability of the two pathways to induce JNK-1 activity.

Vector-driven hyperexpression of a triad of costimulatory molecules confers enhanced T-cell stimulatory capacity to DC precursors.

Activation of T cells requires at least two signals: signal 1, via the T-cell receptor, and signal 2, in which a costimulatory molecule on the antigen presenting cell (APC) interacts with a ligand on the T cell. Dendritic cells (DCs) are the most potent APCs in part due to their expression of costimulatory molecules. DCs, however, constitute only a minor percentage of APCs in the body, and the in vitro preparation of DCs is both costly and time consuming. The studies reported here demonstrate that one can utilize other APCs, such as bone marrow progenitor cells (BMPCs) and make them markedly more effective as APCs; this was accomplished by their infection with recombinant poxviruses (either the replication-defective avipox or vaccinia), which contain transgenes for a triad of costimulatory molecules (B7-1, ICAM-1 and LFA-3, designated TRICOM). APCs infected with TRICOM vectors are shown to significantly enhance the activation of both naive and effector CD4(+) and CD8(+) T-cell populations. The use of TRICOM vectors in vaccine strategies is discussed.

Examination of CD8+ T cell function in humans using MHC class I tetramers: similar cytotoxicity but variable proliferation and cytokine production among different clonal CD8+ T cells specific to a single viral epitope.

Following infection by human T cell lymphotrophic virus-I (HTLV-I), high frequencies of polyclonal Tax11-19-reactive CD8(+) T cells can be detected in the peripheral blood. To investigate whether there are differences in the effector functions of these cells, we generated a panel of Tax11-19-reactive T cell clones by single cell sorting of HLA-A2/Tax11-19 tetramer binding CD8(+) T cells followed by repeated stimulation with PHA and IL-2. Examination of the TCRs revealed 17 different T cell clones with unique clonal origins. Nine representative CD8(+) T cell clones showed a similar cytotoxic dose-response activity against Ag-pulsed target cells, even though they express different TCRs. This cytotoxic effector function was not influenced by the engagement of either CD28 or CD2 costimulatory molecules. In contrast to the cytotoxic activity, qualitatively different degrees of proliferative response and cytokine secretion were observed among T cell clones of different clonal origin. The induction of proliferation and cytokine secretion required the engagement of costimulatory molecules, particularly CD2-LFA-3 interaction. These results indicate that functionally diverse, polyclonal CTL populations can be activated specific to a single immunodominant viral epitope; they can manifest virtually identical cytotoxic effector function but have marked differences in proliferation and cytokine secretion.

A dimeric form of soluble recombinant sheep LFA-3(CD58) inhibits human T-cell proliferation by generating regulatory T cells.

We recently observed that the soluble recombinant from of sheep LFA-3, termed sLFA-3 is biologically active as determined by E-rosette inhibition and inhibition of human T-cell proliferation in response to the recall antigen. In the present study, we examined the immunosuppressive properties of a derivative of sLFA-3, a dimeric form of the first domain (D1) of sLFA-3, named sD1Hcys dimer which was made by oxidative binding of the two D1 molecules through disulfide bonds formed between the SH side chains of a cysteine which was added to the C-terminal of the D1 domain. By investigating the suppressive properties of the sD1Hcys dimer, we obtained evidence that antigen-stimulated T-cell proliferation was inhibited by the suppressor T cell, mainly CD4 + CD45RA - CD45RO + and CD8 + CD45RA - CD45RO + T cells, generated by incubating PBLs with a low dose (0.5 microgram/ml) of sD1Hcys dimer in the presence of a low dose of IL-2 and GM-CSF. Flow cytometric analysis showed that the expression of some surface molecules on T cells were modulated by a high dose (5 micrograms/ml) of sD1Hcys dimer such as downregulation of CD3 and upregulation of IL-2R, but were not modulated by a low dose (0.5 microgram/ml) of the sD1Hcys dimer. These findings suggest that the sD1Hcys dimer exerts its suppressive effects on the antigen-induced proliferation assay by generating suppressor T cells. The sD1Hcys dimer might therefore have potential as an immunotherapeutic agent to inhibit and/or anergize antigen-specific T-cell responses.

The role of B7-1 and LFA-3 in costimulation of CD8+ T cells.

This study compares the ability of LFA-3 (CD58) and B7-1 (CD80) ligands to provide costimulatory signals for superantigen (SAg)-stimulated CD8+ and CD4+ T cells. We show that B7-1 and LFA-3 costimulation activate CD8+ T cells to proliferation, cytokine production (IL-2, TNF, and IFN-gamma), and cytotoxicity. A long-lasting proliferative response was observed after combined DR/B7-1/LFA-3 costimulation. Detailed analysis of SEA-activated CD8+ T cells revealed that maximal production of IFN-gamma was seen in LFA-3-costimulated cells, while production of IL-2 was mainly induced after B7-1 costimulation. A fivefold increase in the IFN-gamma production was observed when activated CD8+ T cells were costimulated with Chinese hamster ovary (CHO)-DR/LFA-3 cells compared with the secretion induced by CHO-DR/B7-1. In contrast, SEA-treated CD4+ T cells costimulated with B7-1 or LFA-3 gave rise to a similar production of IFN-gamma, suggesting a preferential function for the CD2/LFA-3 pathway in the regulation of IFN-gamma in CD8+ T cells. Moreover, the generation of CTL was supported similarly by B7-1 and LFA-3 costimulation, but not by CHO-DR cells. We conclude that ligation of the CD28 and CD2 receptors mediate distinct effect on CD8+ and CD4+ T cell effector functions.

A novel murine model for the assessment of human CD2-related reagents in vivo.

CD2 is a T cell surface glycoprotein that mediates both cell-cell adhesion and transmembrane signal transduction. To construct a model for the in vivo evaluation of human (h)CD2 function and hCD2-related reagents, hCD2 transgenic mice and murine (m)CD2 knockout mice were crossed, and the F2 generation selected for mCD2-hCD2+ animals by fluorescent flow cytometry. The mCD2-hCD2+ mice are healthy and have a normal distribution of mCD3, mCD4, and mCD8 in thymus, spleen, and lymph node. Therefore expression of the hCD2 transgene does not appear to disrupt normal T cell development. The functionality of hCD2 was demonstrated by T lymphocyte proliferation upon stimulation by combined anti-CD2 plus anti-CD2R (anti-T11(2) plus anti-T11(3)) mAbs. Anti-T11(2) plus anti-T11(3) anti-human CD2 mAbs also induced proliferation of mCD2+hCD2+ F1 lymphocytes, but not mCD2+hCD2- wild-type murine lymphocytes. Either an anti-murine or the human CD2 specific (anti-T11(1)) mAbs inhibited proliferation in alloantigen, PHA, or anti-CD3 mAb stimulated cultures and inhibited only cells bearing the appropriate cognate CD2. In vivo studies of immune function yielded results consistent with these in vitro assays. Thus, anti-T11(1) mAb suppressed contact sensitivity in vivo in the transgenic/knockout mice. mCD2-hCD2+ mice treated with anti-T11(1) or LFA-3 fusion proteins also showed significant prolongation of cardiac allograft survival. This prolongation was associated both with depletion and down-modulation of CD2 on remaining T cells. These data suggest that the transgenic/knockout mice provide a useful in vivo model for the assessment of hCD2-related reagents and CD2 function, free from any potential interactions with mCD2 and mCD2 ligands.

Molecular interaction between CD58 and CD2 counter-receptors mediates the ability of monocytes to augment T cell activation by IL-12.

IL-12 stimulates both T and NK cells and is pivotal in the development of the Th1 immune response. In this work, we show that an interaction between CD2 and CD58 on activated T cells and monocytes, respectively, regulates the T cell response to IL-12. B cells provide little IL-12-specific costimulation, and this correlates with the low level of CD58 on B cells relative to monocytes and the lack of significant up-regulation in response to IFN-gamma or PHA activation. CHO cell transfectants expressing CD58 at a level comparable with that found on monocytes restore IL-12 responsiveness to APC-depleted T cells. This effect is not observed with CHO cells expressing CD48, a second CD2 ligand with a low avidity for CD2 relative to CD58. Thus, in addition to augmenting adhesion between T cells and their cognate APCs and facilitating TCR-triggered activation, the CD2-CD58 interaction uniquely optimizes the T cell response to IL-12.

CD2 regulates T cell-dependent induction of monocyte IL-1 beta mRNA during anti-CD3 mitogenesis.

The induction of monocyte IL-1 mRNA during T cell activation requires that monocytes receive contact-dependent signals from activated T cells. Furthermore, the ability of T cells to induce IL-1 beta mRNA is not constitutive but rather is rapidly acquired (< or = 30 min) following activation via mechanisms that do not require protein synthesis. The goal of these studies is to identify the T cell signal(s) that mediates the cell contact-dependent induction of monocyte IL-1 beta mRNA. The induction of IL-1 beta mRNA during anti-CD3 mitogenesis was significantly inhibited by anti-CD2 mAb, whereas mAb against CD11a, CD18, CD69, or CD5 molecules had no effect. The inhibition of IL-1 beta mRNA induction by anti-CD2 mAb was restricted to only those mAb that block CD2/CD58(LFA-3) interactions. Furthermore, anti-CD2 blocked the induction of monocyte IL-1 beta mRNA by T cells that were preactivated using either immobilized anti-CD3 or anti-T11(2) plus anti-T11(3) mAb, thereby indicating that the inhibition of IL-1 beta mRNA was not due to negative signaling effects exerted on the T cell by anti-CD2. Finally, although anti-CD69 mAb had no effect on IL-1 beta mRNA induction, it inhibited the generation of soluble IL-1 beta. The combination of anti-CD69 and anti-CD2 mAb exhibited greater inhibition of secreted IL-1 beta than either antibody alone. These results indicate that CD2 is required for T cell induction of IL-1 beta mRNA through interaction with LFA-3 on the monocyte and that the generation of soluble IL-1 beta is regulated by CD69.

IL-1, ICAM-1, LFA-3, and hydrocortisone differentially regulate cytokine secretion by human fetal thymic epithelial cells.

Human fetal thymic epithelial cells (TEC) were cultured under serum-free conditions. The TEC were analyzed for the secretion of 14C-labelled peptides and of IL-3, IL-6 IL-7, IL-8, GM-CSF, LIF, fibronectin and thymosin alpha 1 by ELISA tests. IL-3 and IL-7 were not detected from these TEC. Lack of IL-7 and presence of thymosin alpha 1 and of the surface molecule TE-4 depicts these cells as subcapsular/medullary TEC. TEC secreted constitutively IL-6, IL-8, fibronectin and thymosin alpha 1 but not GM-CSF of LIF. Stimulants included recombinant IL-1 and monoclonal antibodies to the surface adhesion molecules ICAM-1 and LAF-3. In addition, hydrocortisone (2.7 x 10(-7) M) was used to dissect secretion patterns. Recombinant IL-1, anti ICAM-1, and anti LFA-3 alone and collectively induced modest but significant increases in the secretions of 14C-labelled peptides and of IL-6 and IL-8 which were not inhibited by HC. Recombinant IL-1 but not anti ICAM-1 and anti LFA-3 induced GM-CSF and LIF. HC inhibited the secretion of GM-CSF and LIF induced by IL-1. None of the stimulants augmented the constitutive secretion of fibronectin or thymosin alpha 1 and HC inhibited thymosin alpha 1 secretion. TEC secretion of cytokines but not thymosin alpha 1 and fibronectin appear to be regulated in a more complex manner than heretofore recognized.

Immunomodulation by LFA3TIP, an LFA-3/IgG1 fusion protein: cell line dependent glycosylation effects on pharmacokinetics and pharmacodynamic markers.

LFA3TIP, a fusion protein comprised of the first extracellular domain of LFA-3 fused to the hinge, CH2, and CH3 domains of human IgG1 inhibits responses of human and non-human primate T cells in vitro. In seeking to optimize the expression efficiency to prepare large quantities of LFA3TIP for primate studies, the protein was produced in both the CHO (Chinese hamster ovary) and murine NS-0 myeloma cell lines. Although LFA3TIP derived from these cell lines performs identically in vitro in CD2 receptor binding and T cell assays, examination of a pharmacodynamic marker-the reduction in CD2+ lymphocyte numbers-following the administration of equal doses of NS-0 or CHO derived LFA3TIP to baboons, suggested that the effect of the NS-0 derived material was less sustained. Pharmacokinetic analysis of the materials in baboons and mice shows that LFA3TIP produced by NS-0 cells is rapidly cleared from circulation relative to the product derived from CHO cells. The disparate clearance profiles correlate with distinct glycosylation patterns, with LFA3TIP derived from NS-0 cells being less extensively sialylated than that from CHO cells due in part to alpha-galactosyl capping of selected lactosamine moieties in the N-linked glycans of NS-0 derived LFA3TIP. Moreover, enzymatic desialylation of CHO derived LFA3TIP results in a glycoprotein with an evanescent serum profile when administered to mice and baboons. These results correlate the extent of N-acetylneuraminic acid capping with the clearance rates of LFA3TIP derived from the two distinct cell lines, and underscore the importance of evaluating glycosylation dependent PK parameters when choosing production cell lines for recombinant immunotherapeutics.