A novel dual-release scaffold for fluorescent labels improves cyclic immunofluorescence.

Cyclic immunofluorescence is a powerful method to generate high-content imaging datasets for investigating cell biology and developing therapies. This method relies on fluorescent labels that determine the quality of immunofluorescence and the maximum number of staining cycles that can be performed. Here we present a novel fluorescent labelling strategy, based on antibodies conjugated to a scaffold containing two distinct sites for enzymatic cleavage of fluorophores. The scaffold is composed of a dextran decorated with short ssDNA that upon hybridization with complementary dye-modified oligos result in fluorescent molecules. The developed fluorescent labels exhibit specific staining and remarkable brightness in flow cytometry and fluorescence microscopy. We showed that the combination of DNase-mediated degradation of DNA and dextranse-mediated degradation of the dextran as two complementary enzymatic release mechanisms in one molecule, improves signal erasure from labelled epitopes. We envision that such dual-release labels with high brightness and efficient and specific erasure will advance multiplexed cyclic immunofluorescence approaches and thereby will contribute to gaining new insights in cell biology.

Fluorophore multimerization on a PEG backbone as a concept for signal amplification and lifetime modulation.

Fluorescent labels have strongly contributed to many advancements in bioanalysis, molecular biology, molecular imaging, and medical diagnostics. Despite a large toolbox of molecular and nanoscale fluorophores to choose from, there is still a need for brighter labels, e.g., for flow cytometry and fluorescence microscopy, that are preferably of molecular nature. This requires versatile concepts for fluorophore multimerization, which involves the shielding of dyes from other chromophores and possible quenchers in their neighborhood. In addition, to increase the number of readout parameters for fluorescence microscopy and eventually also flow cytometry, control and tuning of the labels' fluorescence lifetimes is desired. Searching for bright multi-chromophoric or multimeric labels, we developed PEGylated dyes bearing functional groups for their bioconjugation and explored their spectroscopic properties and photostability in comparison to those of the respective monomeric dyes for two exemplarily chosen fluorophores excitable at 488 nm. Subsequently, these dyes were conjugated with anti-CD4 and anti-CD8 immunoglobulins to obtain fluorescent conjugates suitable for the labeling of cells and beads. Finally, the suitability of these novel labels for fluorescence lifetime imaging and target discrimination based upon lifetime measurements was assessed. Based upon the results of our spectroscopic studies including measurements of fluorescence quantum yields (QY) and fluorescence decay kinetics we could demonstrate the absence of significant dye-dye interactions and self-quenching in these multimeric labels. Moreover, in a first fluorescence lifetime imaging (FLIM) study, we could show the future potential of this multimerization concept for lifetime discrimination and multiplexing.

Water-Soluble Fluorescent Polymer Dyes with Tunable Emission Spectra for Flow Cytometry Applications.

The application of spectrally unique, bright, and water-soluble fluorescent dyes is indispensable for the analysis of biological systems. Multiparameter flow cytometry is a powerful tool for characterization of mixed cell populations. To discriminate the different cell populations, they are typically stained by a set of fluorescent reagents, e.g., antibody-fluorophore conjugates. The number of parameters which can be studied simultaneously strongly depends on the availability of reagents which can be differentiated by their spectral properties. In this study a series of fluorescent polymer dyes was developed, that can be excited with a single violet laser (405 nm) but distinguished by their unique emission spectra. The polyfluorene-based polymers can be used on their own, or in combination with covalently bound small-molecule dyes to generate energy transfer constructs to red-shift the emission wavelength based on Förster resonance energy transfer (FRET). The polymer dyes were utilized in a biological flow cytometry assay by conjugating several of them to antibodies, demonstrating their effectiveness as reagents. This report represents the first systematic investigation of structure-property relationships for this type of fluorescent dyes.

Artificial Targets: a versatile cell-free platform to characterize CAR T cell function in vitro.

Cancer immunotherapies using chimeric antigen receptor (CAR) T cells have tremendous potential and proven clinical efficacy against a number of malignancies. Research and development are emerging to deepen the knowledge of CAR T cell efficacy and extend the therapeutic potential of this novel therapy. To this end, functional characterization of CAR T cells plays a central role in consecutive phases across fundamental research and therapeutic development, with increasing needs for standardization. The functional characterization of CAR T cells is typically achieved by assessing critical effector functions, following co-culture with cell lines expressing the target antigen. However, the use of target cell lines poses several limitations, including alterations in cell fitness, metabolic state or genetic drift due to handling and culturing of the cells, which would increase variabilities and could lead to inconsistent results. Moreover, the use of target cell lines can be work and time intensive, and introduce significant background due to the allogenic responses of T cells. To overcome these limitations, we developed a synthetic bead-based platform ("Artificial Targets") to characterize CAR T cell function in vitro. These synthetic microparticles could specifically induce CAR T cell activation, as measured by CD69 and CD137 (4-1BB) upregulation. In addition, engagement with Artificial Targets resulted in induction of multiple effector functions of CAR T cells mimicking the response triggered by target cell lines including cytotoxic activity, as assessed by exposure of CD107a (LAMP-1), expression and secretion of cytokines, as well as cell proliferation. Importantly, in contrast to target cells, stimulation with Artificial Targets showed limited unspecific CAR T cell proliferation. Finally, Artificial Targets demonstrated flexibility to engage multiple costimulatory molecules that can synergistically enhance the CAR T cell function and represented a powerful tool for modulating CAR T cell responses. Collectively, our results show that Artificial Targets can specifically activate CAR T cells for essential effector functions that could significantly advance standardization of functional assessment of CAR T cells, from early development to clinical applications.

Vaccines against the original strain of SARS-CoV-2 provide T cell memory to the B.1.1.529 variant.

BACKGROUND: The SARS-CoV-2 variant B.1.1.529 potentially escapes immunity from vaccination via a heavily mutated Spike protein. Here, we analyzed whether T cell memory towards the B.1.1.529 Spike protein is present in individuals who received two or three doses of vaccines designed against the original Wuhan strain of SARS-CoV-2. METHODS: PBMCs were isolated from two- and three-times vaccinated study participants and incubated in vitro with peptide pools of the Spike protein derived from sequences of the original Wuhan or the B.1.1.529 strains of SARS-CoV-2. Activated antigen-specific T cells were detected by flow cytometry. In silico analyses with NetMHCpan and NetMHCIIpan were used to determine differences in MHC class presentation between the original strain and the B.1.1.529 strain for the most common MHCs in the European-Caucasian population. RESULTS: Here we show, that both CD4 and CD8 responses to the B.1.1.529 Spike protein are marginally reduced compared to the ancestor protein and a robust T cell response is maintained. Epitope analyses reveal minor differences between the two SARS-CoV-2 strains in terms of MHC class presentations for the MHC-alleles being most common in the European-Caucasian population. CONCLUSIONS: The memory T cell response induced via first generation vaccination remains robust and is mostly unaffected by B.1.1.529 mutations. Correspondingly, in silico analyses of MHC presentation of epitopes derived from the B.1.1.529 Spike protein shows marginal differences compared to the ancestral SARS-CoV-2 strain.

Vaccination against SARS-CoV-2 results in the production of proteins called antibodies, that bind and inactivate the virus, and cells that help to eliminate it from the body in a future encounter, such as memory T cells. Both antibodies and memory T cells remain in the body after vaccination with memory T cells being present for longer than antibodies. Here, we determined that even though most of the first generation vaccines were created to prevent infection with the original SARS-CoV-2 virus, the memory T cells generated by this vaccination can also detect the omicron variant.

MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors.

Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.

Dual-labelled antibodies for flow and mass cytometry: A new tool for cross-platform comparison and enrichment of target cells for mass cytometry.

Antibody conjugates applicable in both conventional flow and mass cytometry would offer interesting options for cross-platform comparison, as well as the enrichment of rare target cells by conventional flow cytometry (FC) sorting prior to deep phenotyping by mass cytometry (MC). Here, we introduce a simple method to generate dual fluorochrome/metal-labelled antibodies by consecutive orthogonal labelling. First, we compared different fluorochrome-conjugated antibodies specific for CD4, such as FITC, Vio667, VioGreen or VioBlue for their compatibility with the conventional secondary MAXPAR® labelling protocol. After labelling with 141 Pr, the fluorescence emission spectra of all fluorochromes investigated retained their characteristics, and CD4 dual conjugates (DCs) provided consistent results in immune phenotyping assays performed by FC and MC. The phenotypical composition of CD4+ T-cells was maintained after enrichment by FC sorting using different CD4 DCs. Finally, magnetic cell depletion was combined with FC sorting using CD19-VioBlue-142 Nd, CD20-VioGreen-147 Sm, CD27-Cy5-167 Er and CD38-Alexa488-143 Nd DC to enrich rare human plasmablasts to purities >80%, which allowed a subsequent deep phenotyping by MC. In conclusion, DCs have been successfully established for direct assay comparison between FC and MC, and help to minimise MC data acquisition time for deep phenotyping of rare cell subsets.

Superparamagnetic iron oxide nanoparticles for direct labeling of stem cells and in vivo MRI tracking.

To develop effective stem cell therapies, it is important to track therapeutic cells non-invasively and monitor homing to areas of pathology. The purpose of this study was to design and evaluate the labeling efficiency of commercially available dextran-coated superparamagnetic iron oxide nanoparticles, FeraTrack Direct (FTD), in various stem and immune cells; assess the cytotoxicity and tolerability of the FTD in stem cells; and monitor stem cell homing using FTD-labeled bone-marrow-derived mesenchymal stromal cells (BMSCs) and neural stem cells (NSCs) in a tumor model by in vivo MRI. BMSCs, NSCs, hematopoietic stem cells (HSCs), T-lymphocytes, and monocytes were labeled effectively with FTD without the need for transfection agents, and Prussian blue (PB) staining and transmission electron microscopy (TEM) confirmed intracellular uptake of the agent. The viability, proliferation, and functionality of the labeled cells were minimally or not affected after labeling. When 10(6) FTD-labeled BMSCs or NSCs were injected into C6 glioma bearing nude mice, the cells homing to the tumors were detected as hypointense regions within the tumor using 3 T clinical MRI up to 10 days post injection. Histological analysis confirmed the homing of injected cells to the tumor by the presence of PB positive cells that are not macrophages. Labeling of stem cells or immune cells with FTD was non-toxic, and should facilitate the translation of this agent to clinical trials for evaluation of trafficking of cells by MRI.

Novel bimodal iron oxide particles for efficient tracking of human neural stem cells in vivo.

AIMS: We validated novel bimodal iron oxide particles as substitute of ferumoxides for efficient labeling of human neural stem cells (NSCs). The dextrane-coated FeraTrack Direct (FTD)-Vio particles have additional far-red fluorophores for microscopic cell analysis. METHODS: MR relaxometry, spectrophotometric iron determination and microscopy are used for characterization in vitro and in vivo. RESULTS: Efficient uptake is not transfection agent-dependent. FTD-Vio594 labeling had no influence on viability, proliferation, migration and differentiation capacity. It allows MRI-based tracking of engrafted NSCs in mouse brain up to 11 days, complemented by bioluminescence imaging of firefly luciferase expressed by the engrafted cells. CONCLUSION: Our results highlight the FTD-Vio594 particles as safe and sensitive substitute of ferumoxides for longitudinal tracking of NSCs in preclinical studies.

DNA-templated native chemical ligation of functionalized peptide nucleic acids: a versatile tool for single base-specific detection of nucleic acids.

Single base-specific detection of DNA/RNA sequences is of importance in the diagnosis of disease-associated genetic disorders or early stage cancer. This chapter introduces DNA-templated native chemical PNA ligation as a potentially useful tool for the sequence specific detection of nucleic acids. The template-induced alignment of PNA-thioesters and 1,2-aminothiol-PNAs in close proximity leads to an increase in their effective molarities. This facilitates PNA ligation to proceed at concentrations where no reaction would be possible in absence of the template. Moreover, hybridization of the rather short PNA conjugates with non-complementary DNA/RNA is disfavored, which prevents PNA ligation to occur on single base-mismatched templates. Different readout strategies of the ligation reaction such as HPLC, MALDI-TOF-MS and fluorecence monitoring are discussed, and examples for the detection of a point mutation within single stranded and PCR-amplified double stranded DNA are provided.

Long-time correlations in company profit fluctuations: presence of extreme events.

The accuracy of earnings predictions is hampered by the several predominantly unpredictable effects due to the complex evolution of economy. Finding out which are the dominant market features embracing uncertainty is therefore the key to get beyond present state-of-art earnings forecasts. The analysis of annual revenues and earnings data (1954-2008) from the 500 largest-revenue U.S. companies suggests a linear relation between company expected mean profit and revenue. Annual profit fluctuations are then obtained as difference between actual annual profits and expected mean values. It is found that the temporal evolution of profit fluctuations for a single company displays a slowly decaying autocorrelation, yielding Hurst exponents in the range H=0.75+/-0.17 . The study of profits cross correlations between companies suggests a way to distinguish typical earnings years from anomalous ones by looking at minimal information structures contained within the space defined by the associated covariance metric.

Quantitative detection of small molecule/DNA complexes employing a force-based and label-free DNA-microarray.

Force-based ligand detection is a promising method to characterize molecular complexes label-free at physiological conditions. Because conventional implementations of this technique, e.g., based on atomic force microscopy or optical traps, are low-throughput and require extremely sensitive and sophisticated equipment, this approach has to date found only limited application. We present a low-cost, chip-based assay, which combines high-throughput force-based detection of dsDNA.ligand interactions with the ease of fluorescence detection. Within the comparative unbinding force assay, many duplicates of a target DNA duplex are probed against a defined reference DNA duplex each. The fractions of broken target and reference DNA duplexes are determined via fluorescence. With this assay, we investigated the DNA binding behavior of artificial pyrrole-imidazole polyamides. These small compounds can be programmed to target specific dsDNA sequences and distinguish between D- and L-DNA. We found that titration with polyamides specific for a binding motif, which is present in the target DNA duplex and not in the reference DNA duplex, reliably resulted in a shift toward larger fractions of broken reference bonds. From the concentration dependence nanomolar to picomolar dissociation constants of dsDNA.ligand complexes were determined, agreeing well with prior quantitative DNAase footprinting experiments. This finding corroborates that the forced unbinding of dsDNA in presence of a ligand is a nonequilibrium process that produces a snapshot of the equilibrium distribution between dsDNA and dsDNA.ligand complexes.

Cyclic pyrrole-imidazole polyamides targeted to the androgen response element.

Hairpin pyrrole-imidazole (Py-Im) polyamides are a class of cell-permeable DNA-binding small molecules that can disrupt transcription factor-DNA binding and regulate endogenous gene expression. The covalent linkage of antiparallel Py-Im ring pairs with an gamma-amino acid turn unit affords the classical hairpin Py-Im polyamide structure. Closing the hairpin with a second turn unit yields a cyclic polyamide, a lesser-studied architecture mainly attributable to synthetic inaccessibility. We have applied our methodology for solution-phase polyamide synthesis to cyclic polyamides with an improved high-yield cyclization step. Cyclic 8-ring Py-Im polyamides 1-3 target the DNA sequence 5'-WGWWCW-3', which corresponds to the androgen response element (ARE) bound by the androgen receptor transcription factor to modulate gene expression. We find that cyclic Py-Im polyamides 1-3 bind DNA with exceptionally high affinities and regulate the expression of AR target genes in cell culture studies, from which we infer that the cycle is cell permeable.

DNA sequence selectivity of hairpin polyamide turn units.

A class of hairpin polyamides linked by 3,4-diaminobutyric acid, resulting in a beta-amine residue at the turn unit, showed improved binding affinities relative to their alpha-amino-gamma-turn analogs for particular sequences. We incorporated beta-amino-gamma-turns in six-ring polyamides and determined whether there are any sequence preferences under the turn unit by quantitative footprinting titrations. Although there was an energetic penalty for G.C and C.G base pairs, we found little preference for T.A over A.T at the beta-amino-gamma-turn position. Fluorine and hydroxyl substituted alpha-amino-gamma-turns were synthesized for comparison. Their binding affinities and specificities in the context of six-ring polyamides demonstrated overall diminished affinity and no additional specificity at the turn position. We anticipate that this study will be a baseline for further investigation of the turn subunit as a recognition element for the DNA minor groove.

Next generation hairpin polyamides with (R)-3,4-diaminobutyric acid turn unit.

The characterization of a new class of pyrrole-imidazole hairpin polyamides with beta-amino-gamma-turn units for recognition of the DNA minor groove is reported. A library of eight hairpins containing ( R)- and ( S)-3,4-diaminobutyric acid (beta-amino-gamma-turn) has been synthesized, and the impact of the molecules on DNA-duplex stabilization was studied for comparison with the parent gamma-aminobutyric acid (gamma-turn) and standard ( R)-2,4-diaminobutyric acid (alpha-amino-gamma-turn)-linked eight-ring polyamides. For some, but not all, sequence compositions, melting temperature analyses have revealed that both enantiomeric forms of the beta-amino-gamma-turn increase the DNA-binding affinity of polyamides relative to the ( R)-alpha-amino-gamma-turn. The ( R)-beta-amine residue may be an attractive alternative for constructing hairpin polyamide conjugates. Biological assays have shown that ( R)-beta-amino-gamma-turn hairpins are able to inhibit androgen receptor-mediated gene expression in cell culture similar to hairpins bearing the standard ( R)-alpha-amino-gamma-turn, from which we infer they are cell-permeable.

Single nucleotide specific detection of DNA by native chemical ligation of fluorescence labeled PNA-probes.

DNA-directed chemical ligations provide the opportunity to diagnose DNA sequences with very high sequence specificity. Fluorescent labels have been attached to reactive probes to enable the homogeneous detection of DNA and RNA. However, it has frequently been found that the attachment of fluorescent labels results in decreases of ligation fidelity. Herein we describe the development of a fluorogenic ligation reaction that provides for 10(2)-fold to perfect sequence selectivity. The reaction is based on the isocysteine-mediated native chemical PNA ligation. It is shown that DNA-induced rate accelerations of approximately 43.000-fold can be obtained through subtle variations of the ligation conditions. PNA-thioesters and isocysteine-PNA conjugates were labeled with FAM and TMR fluorophores, respectively. For gaining rapid synthetic access, a convenient on-resin labeling approach was developed. A new PNA monomer featuring an Alloc-protected lysine side chain was synthesized and coupled in solid-phase PNA synthesis. In the event of a ligation reaction the two fluorophores are brought into proximity. It is shown that fluorescence resonance energy transfer provides a positive fluorescence signal which is specific for product formation rather than for loss of starting materials. Single base mutations can be detected within minutes and with very high sequence selectivity at optimized conditions.

As fast and selective as enzymatic ligations: unpaired nucleobases increase the selectivity of DNA-controlled native chemical PNA ligation.

DNA-controlled reactions offer interesting opportunities in biological, chemical, and nanosciences. In practical applications, such as in DNA sequence analysis, the sequence fidelity of the chemical-ligation reaction is of central importance. We present a ligation reaction that is as fast as and much more selective than enzymatic T4 ligase-mediated oligonucleotide ligations. The selectivity was higher than 3000-fold in discriminating matched from singly mismatched DNA templates. It is demonstrated that this enormous selectivity is the hallmark of the particular ligation architecture, which is distinct from previous ligation architectures designed as "nick ligations". Interestingly, the fidelity of the native chemical ligation of peptide nucleic acids was increased by more than one order of magnitude when performing the ligation in such a way that an abasic-site mimic was formed opposite an unpaired template base. It is shown that the high sequence fidelity of the abasic ligation could facilitate the MALDI-TOF mass-spectrometric analysis of early cancer onset by allowing the detection of as little as 0.2 % of single-base mutant DNA in the presence of 99.8 % wild-type DNA.

Convergent synthesis of peptide nucleic acids by native chemical ligation.

[reaction: see text] A convergent strategy for synthesizing long contiguous PNA by a native chemical ligation-like technique of PNA segment couplings is presented. This approach required the synthesis of a new PNA-monomer featuring a 1-amino-2-thiol group. It is shown that the additional mercaptomethyl group leaves the hybridization properties of PNA ligation products unaffected. Furthermore, rapid and efficient fluorescence labeling of the ligation products is demonstrated.