Engineering antigen-specific NK cell lines against the melanoma-associated antigen tyrosinase via TCR gene transfer.

Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T-lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL-TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off-target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/ß chains, along with CD3 subunits, enables the functional expression of an antigen-specific TCR complex on NK cell lines NK-92 and YTS, demonstrated by using a TCR against the HLA-A2-restricted tyrosinase-derived melanoma epitope, Tyr368-377 . Most importantly, the introduction of a TCR complex to NK cell lines enables MHC-restricted, antigen-specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of "mispairing" in TCR gene therapy.

Bulk and single-cell transcriptomics identify gene signatures of stem cell-derived NK cell donors with superior cytolytic activity.

Allogeneic natural killer (NK) cell therapies are a valuable treatment option for cancer, given their remarkable safety and favorable efficacy profile. Although the use of allogeneic donors allows for off-the-shelf and timely patient treatment, intrinsic interindividual differences put clinical efficacy at risk. The identification of donors with superior anti-tumor activity is essential to ensure the success of adoptive NK cell therapies. Here, we investigated the heterogeneity of 10 umbilical cord blood stem cell-derived NK cell batches. First, we evaluated the donors' cytotoxic potential against tumor cell lines from solid and hematological cancer indications, to distinguish a group of superior, "excellent" killers (4/10), compared with "good" killers (6/10). Next, bulk and single-cell RNA sequencing, performed at different stages of NK differentiation, revealed distinct transcriptomic features of the two groups. Excellent donors showed an enrichment in cytotoxicity pathways and a depletion of myeloid traits, linked to the presence of a larger population of effector-like NK cells early on during differentiation. Consequently, we defined a multi-factorial gene expression signature able to predict the donors' cytotoxic potential. Our study contributes to the identification of key traits of superior NK cell batches, supporting the development of efficacious NK therapeutics and the achievement of durable anti-tumor responses.

Unexpected T-cell recognition of an altered peptide ligand is driven by reversed thermodynamics.

The molecular basis underlying T-cell recognition of MHC molecules presenting altered peptide ligands is still not well-established. A hierarchy of T-cell activation by MHC class I-restricted altered peptide ligands has been defined using the T-cell receptor P14 specific for H-2D(b) in complex with the immunodominant lymphocytic choriomeningitis virus peptide gp33 (KAVYNFATM). While substitution of tyrosine to phenylalanine (Y4F) or serine (Y4S) abolished recognition by P14, the TCR unexpectedly recognized H-2D(b) in complex with the alanine-substituted semiagonist Y4A, which displayed the most significant structural modification. The observed functional hierarchy gp33 > Y4A > Y4S = Y4F was neither due to higher stabilization capacity nor to differences in structural conformation. However, thermodynamic analysis demonstrated that while recognition of the full agonist H-2D(b) /gp33 was strictly enthalpy driven, recognition of the weak agonist H-2D(b) /Y4A was instead entropy driven with a large reduction in the favorable enthalpy term. The fourfold larger negative heat capacity derived for the interaction of P14 with H-2D(b) /gp33 compared with H-2D(b) /Y4A can possibly be explained by higher water entrapment at the TCR/MHC interface, which is also consistent with the measured opposite entropy contributions for the interactions of P14 with both MHCs. In conclusion, this study demonstrates that P14 makes use of different strategies to adapt to structural modifications in the MHC/peptide complex.

Early TRAIL-engagement elicits potent multimodal targeting of melanoma by CD34+ progenitor cell-derived NK cells.

Umbilical cord blood (UCB) CD34+ progenitor cell-derived natural killer (NK) cells exert efficient cytotoxicity against various melanoma cell lines. Of interest, the relative cytotoxic performance of individual UCB donors was consistent throughout the melanoma panel and correlated with IFNγ, TNF, perforin and granzyme B levels. Importantly, intrinsic perforin and Granzyme B load predicts NK cell cytotoxic capacity. Exploring the mode of action revealed involvement of the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46 and most importantly of TRAIL. Strikingly, combinatorial receptor blocking led to more pronounced inhibition of cytotoxicity (up to 95%) than individual receptor blocking, especially in combination with TRAIL-blocking, suggesting synergistic cytotoxic NK cell activity via engagement of multiple receptors which was also confirmed in a spheroid model. Importantly, lack of NK cell-related gene signature in metastatic melanomas correlates with poor survival highlighting the clinical significance of NK cell therapies as a promising treatment for high-risk melanoma patients.

Diagnosis of Alzheimer Disease Using 2D MRI Slices by Convolutional Neural Network.

There are many kinds of brain abnormalities that cause changes in different parts of the brain. Alzheimer's disease is a chronic condition that degenerates the cells of the brain leading to memory asthenia. Cognitive mental troubles such as forgetfulness and confusion are one of the most important features of Alzheimer's patients. In the literature, several image processing techniques, as well as machine learning strategies, were introduced for the diagnosis of the disease. This study is aimed at recognizing the presence of Alzheimer's disease based on the magnetic resonance imaging of the brain. We adopted a deep learning methodology for the discrimination between Alzheimer's patients and healthy patients from 2D anatomical slices collected using magnetic resonance imaging. Most of the previous researches were based on the implementation of a 3D convolutional neural network, whereas we incorporated the usage of 2D slices as input to the convolutional neural network. The data set of this research was obtained from the OASIS website. We trained the convolutional neural network structure using the 2D slices to exhibit the deep network weightings that we named as the Alzheimer Network (AlzNet). The accuracy of our enhanced network was 99.30%. This work investigated the effects of many parameters on AlzNet, such as the number of layers, number of filters, and dropout rate. The results were interesting after using many performance metrics for evaluating the proposed AlzNet.

Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency.

The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.

Natural Killer Cell-Based Therapies Targeting Cancer: Possible Strategies to Gain and Sustain Anti-Tumor Activity.

Natural killer (NK) cells were discovered 40 years ago, by their ability to recognize and kill tumor cells without the requirement of prior antigen exposure. Since then, NK cells have been seen as promising agents for cell-based cancer therapies. However, NK cells represent only a minor fraction of the human lymphocyte population. Their skewed phenotype and impaired functionality during cancer progression necessitates the development of clinical protocols to activate and expand to high numbers ex vivo to be able to infuse sufficient numbers of functional NK cells to the cancer patients. Initial NK cell-based clinical trials suggested that NK cell-infusion is safe and feasible with almost no NK cell-related toxicity, including graft-versus-host disease. Complete remission and increased disease-free survival is shown in a small number of patients with hematological malignances. Furthermore, successful adoptive NK cell-based therapies from haploidentical donors have been demonstrated. Disappointingly, only limited anti-tumor effects have been demonstrated following NK cell infusion in patients with solid tumors. While NK cells have great potential in targeting tumor cells, the efficiency of NK cell functions in the tumor microenvironment is yet unclear. The failure of immune surveillance may in part be due to sustained immunological pressure on tumor cells resulting in the development of tumor escape variants that are invisible to the immune system. Alternatively, this could be due to the complex network of immune-suppressive compartments in the tumor microenvironment, including myeloid-derived suppressor cells, tumor-associated macrophages, and regulatory T cells. Although the negative effect of the tumor microenvironment on NK cells can be transiently reverted by ex vivo expansion and long-term activation, the aforementioned NK cell/tumor microenvironment interactions upon reinfusion are not fully elucidated. Within this context, genetic modification of NK cells may provide new possibilities for developing effective cancer immunotherapies by improving NK cell responses and making them less susceptible to the tumor microenvironment. Within this review, we will discuss clinical trials using NK cells with a specific reflection on novel potential strategies, such as genetic modification of NK cells and complementary therapies aimed at improving the clinical outcome of NK cell-based immune therapies.

The use of novel drugs can effectively improve response, delay relapse and enhance overall survival in multiple myeloma patients with renal impairment.

BACKGROUND: Renal impairment is a common feature in multiple myeloma and is considered a poor prognostic factor. AIM: To determine the impact of novel drugs (i.e. bortezomib, lenalidomide and thalidomide) in the treatment of myeloma patients with renal impairment. The primary endpoint was overall survival and secondary endpoints were time to next treatment and response. METHODS: The study population included all patients diagnosed with treatment-demanding multiple myeloma January 2000 to June 2011 at 15 Swedish hospitals. Renal impairment was defined as an estimated glomerular filtration rate under 60 mL/min/1.73 m2. RESULT: The study population consisted of 1538 patients, of which 680 had renal impairment at diagnosis. The median overall survival in patients with renal impairment was 33 months, which was significantly shorter than 52 months in patients with normal renal function (P<0.001). Novel agents in first line improved overall survival (median 60 months) in non-high-dose treated patients with renal impairment (n = 143) as compared to those treated with conventional cytotoxic drugs (n = 411) (median 27 months) (P<0.001). In the multivariate analysis up front treatment with bortezomib was an independent factor for better overall survival in non-high-dose treated renally impaired patients. High-dose treated renally impaired patients had significantly better median overall survival than non-high-dose ones (74 versus 26 months) and novel drugs did not significantly improve survival further in these patients. Patients with renal impairment had both a shorter median time to next treatment and a lower response rate than those with normal renal function. However, novel drugs and high dose treatment lead to a significantly longer time to next treatment and the use of novel agents significantly improved the response rate of these patients. CONCLUSION: High dose treatment and novel drugs, especially bortezomib, can effectively overcome the negative impact of renal impairment in patients with multiple myeloma.

Inflammation-associated nitrotyrosination affects TCR recognition through reduced stability and alteration of the molecular surface of the MHC complex.

Nitrotyrosination of proteins, a hallmark of inflammation, may result in the production of MHC-restricted neoantigens that can be recognized by T cells and bypass the constraints of immunological self-tolerance. Here we biochemically and structurally assessed how nitrotyrosination of the lymphocytic choriomeningitis virus (LCMV)-associated immunodominant MHC class I-restricted epitopes gp33 and gp34 alters T cell recognition in the context of both H-2D(b) and H-2K(b). Comparative analysis of the crystal structures of H-2K(b)/gp34 and H-2K(b)/NY-gp34 demonstrated that nitrotyrosination of p3Y in gp34 abrogates a hydrogen bond interaction formed with the H-2K(b) residue E152. As a consequence the conformation of the TCR-interacting E152 was profoundly altered in H-2K(b)/NY-gp34 when compared to H-2K(b)/gp34, thereby modifying the surface of the nitrotyrosinated MHC complex. Furthermore, nitrotyrosination of gp34 resulted in structural over-packing, straining the overall conformation and considerably reducing the stability of the H-2K(b)/NY-gp34 MHC complex when compared to H-2K(b)/gp34. Our structural analysis also indicates that nitrotyrosination of the main TCR-interacting residue p4Y in gp33 abrogates recognition of H-2D(b)/gp33-NY complexes by H-2D(b)/gp33-specific T cells through sterical hindrance. In conclusion, this study provides the first structural and biochemical evidence for how MHC class I-restricted nitrotyrosinated neoantigens may enable viral escape and break immune tolerance.

Simultaneous EEG/fMRI analysis of the resonance phenomena in steady-state visual evoked responses.

The stability of the steady-state visual evoked potentials (SSVEPs) across trials and subjects makes them a suitable tool for the investigation of the visual system. The reproducible pattern of the frequency characteristics of SSVEPs shows a global amplitude maximum around 10 Hz and additional local maxima around 20 and 40 Hz, which have been argued to represent resonant behavior of damped neuronal oscillators. Simultaneous electroencephalogram/functional magnetic resonance imaging (EEG/fMRI) measurement allows testing of the resonance hypothesis about the frequency-selective increases in SSVEP amplitudes in human subjects, because the total synaptic activity that is represented in the fMRI-Blood Oxygen Level Dependent (fMRI-BOLD) response would not increase but get synchronized at the resonance frequency. For this purpose, 40 healthy volunteers were visually stimulated with flickering light at systematically varying frequencies between 6 and 46 Hz, and the correlations between SSVEP amplitudes and the BOLD responses were computed. The SSVEP frequency characteristics of all subjects showed 3 frequency ranges with an amplitude maximum in each of them, which roughly correspond to alpha, beta and gamma bands of the EEG. The correlation maps between BOLD responses and SSVEP amplitude changes across the different stimulation frequencies within each frequency band showed no significant correlation in the alpha range, while significant correlations were obtained in the primary visual area for the beta and gamma bands. This non-linear relationship between the surface recorded SSVEP amplitudes and the BOLD responses of the visual cortex at stimulation frequencies around the alpha band supports the view that a resonance at the tuning frequency of the thalamo-cortical alpha oscillator in the visual system is responsible for the global amplitude maximum of the SSVEP around 10 Hz. Information gained from the SSVEP/fMRI analyses in the present study might be extrapolated to the EEG/fMRI analysis of the transient event-related potentials (ERPs) in terms of expecting more reliable and consistent correlations between EEG and fMRI responses, when the analyses are carried out on evoked or induced oscillations (spectral perturbations) in separate frequency bands instead of the time-domain ERP peaks.

Triticum durum metallothionein. Isolation of the gene and structural characterization of the protein using solution scattering and molecular modeling.

A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been identified in durum wheat Triticum durum cv. Balcali85 genomic DNA. Multiple alignment analyses on the cDNA and the translated protein sequences showed that T. durum MT (dMT) can be classified as a type 1 MT. dMT has three Cys-X-Cys motifs in each of the N- and C-terminal domains and a 42-residue-long hinge region devoid of cysteines. dMT was overexpressed in Escherichia coli as a fusion protein (GSTdMT), and bacteria expressing the fusion protein showed increased tolerance to cadmium in the growth medium compared with controls. Purified GSTdMT was characterized by SDS- and native-PAGE, size exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was shown that the recombinant protein binds 4 +/- 1 mol of cadmium/mol of protein and has a high tendency to form stable oligomeric structures. The structure of GSTdMT and dMT was investigated by synchrotron x-ray solution scattering and computational methods. X-ray scattering measurements indicated a strong tendency for GSTdMT to form dimers and trimers in solution and yielded structural models that were compatible with a stable dimeric form in which dMT had an extended conformation. Results of homology modeling and ab initio solution scattering approaches produced an elongated dMT structure with a long central hinge region. The predicted model and those obtained from x-ray scattering are in agreement and suggest that dMT may be involved in functions other than metal detoxification.