CD20-Specific Immunoligands Engaging NKG2D Enhance γδ T Cell-Mediated Lysis of Lymphoma Cells.

Human γδ T cells are innate-like T cells which are able to kill a broad range of tumour cells and thus may have potential for cancer immunotherapy. The activating receptor natural killer group 2 member D (NKG2D) plays a key role in regulating immune responses driven by γδ T cells. Here, we explored whether recombinant immunoligands consisting of a CD20 single-chain fragment variable (scFv) linked to a NKG2D ligand, either MHC class I chain-related protein A (MICA) or UL16 binding protein 2 (ULBP2), could be employed to engage γδ T cells for tumour cell killing. The two immunoligands, designated MICA:7D8 and ULBP2:7D8, respectively, enhanced cytotoxicity of ex vivo-expanded γδ T cells against CD20-positive lymphoma cells. Both Vδ1 and Vδ2 γδ T cells were triggered by MICA:7D8 or ULBP2:7D8. Killing of CD20-negative tumour cells was not induced by the immunoligands, indicating their antigen specificity. MICA:7D8 and ULBP2:7D8 acted in a dose-dependent manner and induced cytotoxicity at nanomolar concentrations. Importantly, chronic lymphocytic leukaemia (CLL) cells isolated from patients were sensitized by the two immunoligands for γδ T cell cytotoxicity. In a combination approach, the immunoligands were combined with bromohydrin pyrophosphate (BrHPP), an agonist for Vδ2 γδ T cells, which further enhanced the efficacy in target cell killing. Thus, employing tumour-directed recombinant immunoligands which engage NKG2D may represent an attractive strategy to enhance antitumour cytotoxicity of γδ T cells.

Fragment molecular orbital study of the cAMP-dependent protein kinase catalyzed phosphoryl transfer: a comparison with the differential transition state stabilization method.

The importance of key residues to the activity of the cAMP-dependent protein kinase catalyzed phosphoryl transfer and to the stabilization of the transition state of the reaction has been investigated by means of the fragment molecular orbital (FMO) method. To evaluate the accuracy of the method and its capability of fragmenting covalent bonds, we have compared stabilization energies due to the interactions between individual residues and the reaction center to results obtained with the differential transition state stabilization method (Szarek, et al., J. Phys. Chem. B, 2008, 112, 11819-11826) and observe, despite a size difference in the fragment describing the reaction center, near-quantitative agreement. We have also computed deletion energies to investigate the effect of virtual deletion of key residues on the activation energy. These results are consistent with the stabilization energies and yield additional information as they clearly capture the effect of secondary interactions, i.e. interactions in the second coordination layer of the reaction center. We find that using FMO to calculate deletion energies is a powerful and time efficient approach to analyze the importance of key residues to the activity of an enzyme catalyzed reaction.

Strong Influence of Coadsorbate Interaction on CO Desorption Dynamics on Ru(0001) Probed by Ultrafast X-Ray Spectroscopy and Ab Initio Simulations.

We show that coadsorbed oxygen atoms have a dramatic influence on the CO desorption dynamics from Ru(0001). In contrast to the precursor-mediated desorption mechanism on Ru(0001), the presence of surface oxygen modifies the electronic structure of Ru atoms such that CO desorption occurs predominantly via the direct pathway. This phenomenon is directly observed in an ultrafast pump-probe experiment using a soft x-ray free-electron laser to monitor the dynamic evolution of the valence electronic structure of the surface species. This is supported with the potential of mean force along the CO desorption path obtained from density-functional theory calculations. Charge density distribution and frozen-orbital analysis suggest that the oxygen-induced reduction of the Pauli repulsion, and consequent increase of the dative interaction between the CO 5σ and the charged Ru atom, is the electronic origin of the distinct desorption dynamics. Ab initio molecular dynamics simulations of CO desorption from Ru(0001) and oxygen-coadsorbed Ru(0001) provide further insights into the surface bond-breaking process.

Indication of non-thermal contribution to visible femtosecond laser-induced CO oxidation on Ru(0001).

We studied CO oxidation on Ru(0001) induced by 400 nm and 800 nm femtosecond laser pulses where we find a branching ratio between CO oxidation and desorption of 1:9 and 1:31, respectively, showing higher selectivity towards CO oxidation for the shorter wavelength excitation. Activation energies computed with density functional theory show discrepancies with values extracted from the experiments, indicating both a mixture between different adsorbed phases and importance of non-adiabatic effects on the effective barrier for oxidation. We simulated the reactions using kinetic modeling based on the two-temperature model of laser-induced energy transfer in the substrate combined with a friction model for the coupling to adsorbate vibrations. This model gives an overall good agreement with experiment except for the substantial difference in yield ratio between CO oxidation and desorption at 400 nm and 800 nm. However, including also the initial, non-thermal effect of electrons transiently excited into antibonding states of the O-Ru bond yielded good agreement with all experimental results.

Selective ultrafast probing of transient hot chemisorbed and precursor states of CO on Ru(0001).

We have studied the femtosecond dynamics following optical laser excitation of CO adsorbed on a Ru surface by monitoring changes in the occupied and unoccupied electronic structure using ultrafast soft x-ray absorption and emission. We recently reported [M. Dell'Angela et al. Science 339, 1302 (2013)] a phonon-mediated transition into a weakly adsorbed precursor state occurring on a time scale of >2 ps prior to desorption. Here we focus on processes within the first picosecond after laser excitation and show that the metal-adsorbate coordination is initially increased due to hot-electron-driven vibrational excitations. This process is faster than, but occurs in parallel with, the transition into the precursor state. With resonant x-ray emission spectroscopy, we probe each of these states selectively and determine the respective transient populations depending on optical laser fluence. Ab initio molecular dynamics simulations of CO adsorbed on Ru(0001) were performed at 1500 and 3000 K providing insight into the desorption process.

Markers of operational immune tolerance after pediatric liver transplantation in patients under immunosuppression.

A prospective identification of the estimated 20-50% of pediatric LTX recipients developing operational tolerance would be of great clinical advantage. So far markers of immune tolerance - T-cell subpopulations or gene expression profiles - have been investigated only retrospectively in successfully weaned patients. Fifty children aged 8-265 months (median 89) were investigated 1-180 months (median 44) after LTX under ongoing immunosuppression. T-cell subpopulations were measured during regular post-transplant visits using FACS (Vδ1- vs. Vδ2-γδ-T cells and Tregs). A Vδ1/Vδ2-γδ-T-cell ratio ≥1.42 previously reported in operational tolerance was found in 12 of 50 (24%) patients. In analogy, a Treg count ≥44 per µL was found in 35 of 50 (70%) patients and a Treg proportion ≥2.23% of CD3(+) -T cells in 39 of 50 (78%) patients. Only 9 of 50 patients (18%) fulfilled both criteria. The parameters Vδ1/Vδ2-γδ-T-cell ratio and Tregs were not significantly correlated to each other or with donor type or immunosuppression. Vδ1/Vδ2-γδ-T-cell ratio was more stable in serial examinations compared with Treg analyses. The observed proportion of 18% pediatric LTX patients with potential operational tolerance is in accordance with previous reports. However, clinical experience shows that rejections may happen even after long-time weaning of immunosuppression. This suggests that operational tolerance is a dynamic process, with uncertain prediction by Vδ1/Vδ2-γδ-T-cell ratio and/or Tregs under immunosuppression.

X-ray emission spectroscopy and density functional study of CO/Fe(100).

We report x-ray emission and absorption spectroscopy studies of the electronic structure of the predissociative α(3) phase of CO bound at hollow sites of Fe(100) as well as of the on-top bound species in the high-coverage α(1) phase. The analysis is supported by density functional calculations of structures and spectra. The bonding of "lying down" CO in the hollow site is well described in terms of π to π∗ charge transfer made possible through bonding interaction also at the oxygen in the minority spin-channel. The on-top CO in the mixed, high-coverage α(1) phase is found to be tilted due to adsorbate-adsorbate interaction, but still with bonding mainly characteristic of "vertical" on-top adsorbed CO similar to other transition-metal surfaces.

Functional expression of NOD2 in freshly isolated human peripheral blood γδ T cells.

γδ T cells play an important role in anti-infective immunity. The major subset of human γδ T cells selectively recognizes phosphorylated bacterial metabolites of the isoprenoid biosynthesis pathway, so-called phosphoantigens. The activation of γδ T cells is modulated by functionally expressed innate immune receptors, notably Toll-like receptor 2 and 3. It was also reported that in vitro expanded γδ T cells respond to muramyl dipeptide (MDP), the minimal peptidoglycan motif activating the nucleotide-binding oligomerization domain containing 2 (NOD2) receptor, although it is unknown whether ex vivo isolated human γδ T cells express functional NOD2. Here, we report that freshly isolated, highly purified peripheral blood γδ T cells express NOD2 mRNA and detectable amounts of NOD2 protein. The biologically active MDP L-D isomer but not the inactive D-D isomer augmented the interferon-γ (IFN-γ) secretion in phosphoantigen-stimulated peripheral blood mononuclear cells. Moreover, a moderate but reproducible and statistically significant increase in IFN-γ secretion was also observed when highly purified peripheral blood γδ T cells were activated by T cell receptor cross-linking in the presence of MDP. Taken together, our results indicate that in addition to the T cell receptor and Toll-like receptors, circulating human γδ T cells express NOD2 as a third class of pattern recognition receptor for sensing bacterial products.

Oxidation of Pt(111) under near-ambient conditions.

The oxidation of Pt(111) at near-ambient O2 pressures has been followed in situ using x-ray photoelectron spectroscopy (XPS) and ex situ using x-ray absorption spectroscopy (XAS). Polarization-dependent XAS signatures at the O K edge reveal significant temperature- and pressure-dependent changes of the Pt-O interaction. Oxide growth commences via a PtO-like surface oxide that coexists with chemisorbed oxygen, while an ultrathin α-PtO2 trilayer is identified as the precursor to bulk oxidation. These results have important implications for understanding the chemical state of Pt in catalysis.

Low O2 dissociation barrier on Pt(111) due to adsorbate-adsorbate interactions.

O(2) dissociation on Pt(111) has been followed at low and saturation coverage using temperature-programmed x-ray photoelectron spectroscopy and simulated with mean-field kinetic modeling, yielding dissociation (E(a)) and desorption (E(d)) barriers of 0.32 and 0.36 eV, respectively. Density functional theory calculations show that E(a) is strongly influenced by the O-O interatomic potential in the atomic final state: of the supercells considered, that which maximizes attractive third-nearest-neighbor interactions in the atomic final state yields both the lowest computed dissociation barrier (0.24 eV) and the best agreement with experiment. It is proposed that the effect of adsorbate-adsorbate interactions must be considered when modeling catalytic processes involving dissociative steps.

Toll-like receptor expression and function in subsets of human gammadelta T lymphocytes.

Two subsets of human gammadelta T cells can be identified by T cell receptor (TCR) V gene usage. Vdelta2Vgamma9 T cells dominate in peripheral blood and recognize microbe- or tumour-derived phosphoantigens. Vdelta1 T cells are abundant in mucosal tissue and recognize stress-induced MHC-related molecules. Toll-like receptors (TLRs) are known to co-stimulate interferon-gamma (IFN-gamma) production in peripheral blood gammadelta T cells and in Vdelta2Vgamma9 T cell lines. By microarray analysis, we have identified a range of genes differentially regulated in freshly isolated gammadelta T cells by TCR versus TCR plus TLR3 stimulation. Furthermore, we have investigated TLR expression in freshly isolated Vdelta1 and Vdelta2 subsets and cytokine/chemokine production in response to TLR1/2/6, 3 and 5 ligands. TLR1,2,6,7 RNA was abundantly expressed in both subsets, whereas TLR3 RNA was present at low levels, and TLR5 and 8 RNA only marginally in both subsets. Despite abundant RNA expression, TLR1 was rarely detectable by flow cytometry. In contrast, TLR2 and TLR6 proteins were detected in purified Vdelta1 and Vdelta2 T cells, and TLR3 protein was detected intracellularly in both subsets. TLR1/2/6, 3 and 5 ligands co-stimulated the IFN-gamma and chemokine secretion in TCR-activated Vdelta1 and Vdelta2 subsets, although the levels of IFN-gamma secreted by Vdelta1 T cells were much lower than those produced by Vdelta2 T cells. Our results reveal comparable expression of TLRs and functional responses to TLR ligands in freshly isolated Vdelta1 and Vdelta2 T cells and underscore the intrinsically different capacity for IFN-gamma secretion of Vdelta1 versus Vdelta2 T cells.

Real-Time Elucidation of Catalytic Pathways in CO Hydrogenation on Ru.

The direct elucidation of the reaction pathways in heterogeneous catalysis has been challenging due to the short-lived nature of reaction intermediates. Here, we directly measured on ultrafast time scales the initial hydrogenation steps of adsorbed CO on a Ru catalyst surface, which is known as the bottleneck reaction in syngas and CO2 reforming processes. We initiated the hydrogenation of CO with an ultrafast laser temperature jump and probed transient changes in the electronic structure using real-time X-ray spectroscopy. In combination with theoretical simulations, we verified the formation of CHO during CO hydrogenation.

Regulation of T-cell death-associated gene 51 (TDAG51) expression in human T-cells.

T-cell death-associated gene 51 (TDAG51) has been described to regulate T-cell receptor/CD3-dependent induction of CD95/Fas and subsequent activation-induced cell death (AICD) in a murine T-cell hybridoma. Using well-defined pharmacological inhibitors, we investigated the regulation of TDAG51 expression in human T-cells and the correlation with cell death. TDAG51 was induced in resting T-cells, lymphoid cell lines and AICD-susceptible as well as AICD-resistant T-cell clones, and induction was inhibited by MAP-kinase inhibitors and PKC inhibitor Gö6983. No correlation between the effects of inhibitors on TDAG51 expression and cell death was observed. The constitutive TDAG51 expression in five pancreatic carcinoma cell lines was reduced by MAP-kinase inhibitors but not by Gö6983. Furthermore, the inducible overexpression of TDAG51 in TetOn Jurkat cells did not modulate cellular proliferation, phorbolester/ionomycin-induced growth arrest, or the expression of various cell surface molecules. Our results indicate that the expression of TDAG51 in human T-cells does not correlate with AICD.

Differential role of tyrosine phosphorylation in the induction of apoptosis in T cell clones via CD95 or the TCR/CD3-complex.

Activated T cells undergo apoptosis when the Fas-antigen (APO-1, CD95) is ligated by Fas Ligand (FasL) or agonistic anti-Fas antibodies. Repeated stimulation of T lymphocytes via the TCR/CD3-complex induces activation-induced cell death (AICD) associated with FasL surface expression. FasL binding to Fas molecules triggers the Fas-dependent death signaling cascade. Since it is still controversial whether Fas-induced cell death is associated with tyrosine kinase activity, we investigated the tyrosine kinase activation requirements in anti-Fas antibody-induced cell death and AICD in human T cell clones. We report that cell death triggered by anti-Fas antibody is not accompanied by an increase in tyrosine phosphorylation and cannot be blocked by inhibitors of protein tyrosine kinases (PTK). Under the same conditions, AICD of T cell clones is clearly associated with tyrosine kinase activation. In fact, semiquantitative RT-PCR analysis of FasL mRNA expression triggered in T cell clones via the TCR/CD3-complex revealed that tyrosine phosphorylation is required for functional FasL mRNA and surface expression.

Surface chemistry. Probing the transition state region in catalytic CO oxidation on Ru.

Femtosecond x-ray laser pulses are used to probe the carbon monoxide (CO) oxidation reaction on ruthenium (Ru) initiated by an optical laser pulse. On a time scale of a few hundred femtoseconds, the optical laser pulse excites motions of CO and oxygen (O) on the surface, allowing the reactants to collide, and, with a transient close to a picosecond (ps), new electronic states appear in the O K-edge x-ray absorption spectrum. Density functional theory calculations indicate that these result from changes in the adsorption site and bond formation between CO and O with a distribution of OC-O bond lengths close to the transition state (TS). After 1 ps, 10% of the CO populate the TS region, which is consistent with predictions based on a quantum oscillator model.

Apoptosis of mature T lymphocytes: putative role in the regulation of cellular immune responses and in the pathogenesis of HIV infection.

In this chapter, some aspects of programmed cell death, or apoptosis, of T lymphocytes are discussed. It has been recognized that transformed T cells and immature T lymphocytes can be triggered to undergo apoptosis. As in other cell systems, apoptosis is characterized by cell shrinkage, nuclear condensation, and DNA fragmentation that displays the characteristic "ladder" pattern of approximately 180-200 bp fragments. More recently, however, it has become clear that apoptosis is not restricted to immature thymocytes or transformed T lymphocytes, but can also occur in mature peripheral T cells. This raises the question of whether apoptosis plays a role as a mechanism in regulating cellular immune responses, which will be discussed in the following sections. We will also address the issue of the potential role of T cell apoptosis in pathophysiology. Here, we will concentrate on the infection with human immunodeficiency virus (HIV), where apoptosis is thought to contribute to the continuous decline in CD4+ T cells.

The association of kidney graft outcome with pretransplant serum IgG-anti-F(ab')2 gamma activity.

Pretransplant sera of 474 kidney graft recipients were tested for IgG-anti-F(ab')2 gamma activity. The patients had significantly higher IgG-anti-F(ab')2 gamma activity than healthy controls (P = 0.0004). Serum lymphocytotoxic antibodies were correlated with IgG-anti-F(ab')2 gamma (P = 0.004), whereas CMV infection and blood transfusions were not. We found a significant association between pretransplant IgG-anti-F(ab')2 gamma activity and early and 1-year kidney graft outcome. This association was pronounced in recipients with no lymphocytotoxic antibodies. Recipients with immediately functioning grafts and a creatinine < 130 mumol/L at 1 year had strikingly higher pretransplant IgG-anti-F(ab')2 gamma activity than patients with graft failure (P < 0.0001).

Induction of cell death via Fas (CD95, Apo-1) may be associated with but is not dependent on Fas-induced tyrosine phosphorylation.

Cross-linking of the Fas-antigen (CD95, Apo-1) triggers apoptosis in activated T cells and transformed T cell lines. Fas-induced apoptosis has been previously reported to require Fas-triggered tyrosine phosphorylation of various proteins. In the present study, we have compared the protein tyrosine phosphorylation pattern and the apoptosis sensitivity in a set of Jurkat variants selected for the absence or presence of T cell receptor (TCR)/CD3 expression and resistance or sensitivity to Fas-mediated apoptosis. While tyrosine phosphorylation upon Fas-ligation was readily apparent in wild-type Jurkat cells (which are sensitive to anti-Fas-induced apoptosis), drastically reduced tyrosine phosphorylation was observed in Fas-resistant Jurkat subclones (which still express CD95 on their surface). More importantly, TCR/CD3-negative Jurkat variants which expressed normal levels of CD95 and were fully susceptible to Fas-triggered cell death, did not show any protein tyrosine phosphorylation upon Fas-ligation. Taken together, our data demonstrate that Fas-induced cell death can be associated with but is not dependent on protein tyrosine phosphorylation.

Pharmacologically elevated levels of endogenous kynurenic acid prevent nicotine-induced activation of nigral dopamine neurons.

Previous studies have shown that systemically administered nicotine is associated with an activation of rat midbrain dopamine neurons. The aim of the present electrophysiological study was to investigate if manipulation of brain kynurenic acid, an endogenous excitatory amino acid receptor antagonist, can affect the response of nigral dopamine neurons to nicotine. A potent inhibitor of kynurenine 3-hydroxylase, PNU 156561A (40 mg/kg, i.v., 4-7 h), was utilized to increase the levels of kynurenic acid in rat brain. This treatment, which caused a fourfold increase in brain kynurenic acid levels, abolished the increase in firing rate and burst activity of nigral dopamine neurons as induced by nicotine (25-400 microg/kg, i.v.). It is proposed that the excitation of dopamine neurons in the substantia nigra following nicotine administration is an indirect effect, mediated by glutamate release. In addition, our data highlight the role of brain kynurenic acid as a potentially important modulator of basic glutamatergic responses in brain.

Adipogenic differentiation potential of rat adipose tissue-derived subpopulations of stromal cells.

Adipose-derived stromal cells (ASCs) are mostly isolated by enzymatic digestion, centrifugation and adherent growth resulting in a very heterogeneous cell population. Therefore, other cell types in the cell culture can comprise the differentiation and proliferation potential of the ASC population. Recent studies indicated that an antibody-aided isolation of distinct ASC subpopulations provides advantages over the conventional method of ASC isolation. The aim of this study was to investigate the adipogenic differentiation potential of CD29-, CD71-, CD73- and CD90-selected ASCs in vitro. The stromal vascular fraction (SVF) was obtained from rat adipose tissue by enzymatic digestion and centrifugation. Subsequently, CD29(+)-, CD71(+)-, CD73(+)- and CD90(+) cells were isolated by magnetic activated cell sorting (MACS), seeded into culture plates and differentiated into the adipogenic lineage. ASCs isolated by adherent growth only served as controls. Adipogenic differentiation was assessed by Oil Red O staining and quantification of the adiponectin and leptin concentrations in the cell culture supernatants. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by the Scheffe's post hoc procedure. The results showed that different subpopulations with different adipogenic differentiation potentials can be isolated by the MACS procedure. The highest adipogenic differentiation potential was determined in the CD29-selected ASC population followed by the unsorted ASC population. The CD71-, CD73- and CD90-selected cells exhibited significantly the lowest adipogenic differentiation potential. In conclusion, the CD29-selected ASCs and the unsorted ASCs exhibited a similar adipogenic differentiation potential. Therefore, we do not see a clear advantage in the application of an anti-CD29-based isolation of ASCs over the conventional technique using adherent growth. However, the research on isolation/purification methods of adipogenic ASCs should continue in order to make this stem cell source even more attractive for future adipose tissue engineering applications.