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.

Analysis of B Cell Repertoire Dynamics Following Hepatitis B Vaccination in Humans, and Enrichment of Vaccine-specific Antibody Sequences.

Generating a diverse B cell immunoglobulin repertoire is essential for protection against infection. The repertoire in humans can now be comprehensively measured by high-throughput sequencing. Using hepatitis B vaccination as a model, we determined how the total immunoglobulin sequence repertoire changes following antigen exposure in humans, and compared this to sequences from vaccine-specific sorted cells. Clonal sequence expansions were seen 7 days after vaccination, which correlated with vaccine-specific plasma cell numbers. These expansions caused an increase in mutation, and a decrease in diversity and complementarity-determining region 3 sequence length in the repertoire. We also saw an increase in sequence convergence between participants 14 and 21 days after vaccination, coinciding with an increase of vaccine-specific memory cells. These features allowed development of a model for in silico enrichment of vaccine-specific sequences from the total repertoire. Identifying antigen-specific sequences from total repertoire data could aid our understanding B cell driven immunity, and be used for disease diagnostics and vaccine evaluation.

A multimerized form of recombinant human CD40 ligand supports long-term activation and proliferation of B cells.

BACKGROUND AIMS: CD40-activated B cells have long been studied as potent antigen-presenting cells that can potentially be used for cancer immunotherapy. Nevertheless, their use in human clinical trials has been limited by the lack of a Good Manufacturing Practice-grade soluble human CD40 ligand that is able to induce activation and proliferation of primary B cells. We describe an in vitro method to effectively generate and expand B cells through the use of a multimerized form of human recombinant CD40 ligand (rCD40L). METHODS: Human B cells were isolated from healthy donors and cultivated with either rCD40L or on a monolayer of murine NIH3T3 cells stably expressing human CD40L (NIH3T3/tCD40L) as a widely used standard method. Morphology, expansion rate, immune phenotype and antigen presentation function were assessed. RESULTS: B cells efficiently proliferated in response to rCD40L over 14 days of culture in comparable amounts to NIH3T3/tCD40L. B-cell division in response to CD40L was also confirmed by carboxyfluorescein succinimidyl ester dilution. Moreover, rCD40L induced on B cells upregulation of co-stimulatory molecules essential for antigen presentation. Additionally, proliferation of T cells from allogeneic healthy volunteers confirmed the immunostimulatory capacities of CD40-activated B cells. CONCLUSIONS: We demonstrated that B cells with potent antigen presentation capacity can be generated and expanded by use of a non-xenogeneic form of CD40L that could be implemented in future human clinical settings.

Genome-wide association study identifies a psoriasis susceptibility locus at TRAF3IP2.

Psoriasis is a multifactorial skin disease characterized by epidermal hyperproliferation and chronic inflammation, the most common form of which is psoriasis vulgaris (PsV). We present a genome-wide association analysis of 2,339,118 SNPs in 472 PsV cases and 1,146 controls from Germany, with follow-up of the 147 most significant SNPs in 2,746 PsV cases and 4,140 controls from three independent replication panels. We identified an association at TRAF3IP2 on 6q21 and genotyped two SNPs at this locus in two additional replication panels (the combined discovery and replication panels consisted of 6,487 cases and 8,037 controls; combined P = 2.36 × 10⁻¹° for rs13210247 and combined P = 1.24 × 10⁻¹6 for rs33980500). About 15% of psoriasis cases develop psoriatic arthritis (PsA). A stratified analysis of our datasets including only PsA cases (1,922 cases compared to 8,037 controls, P = 4.57 × 10⁻¹² for rs33980500) suggested that TRAF3IP2 represents a shared susceptibility for PsV and PsA. TRAF3IP2 encodes a protein involved in IL-17 signaling and which interacts with members of the Rel/NF-κB transcription factor family.

Candidate genes controlling pulmonary function in mice: transcript profiling and predicted protein structure.

Impaired development and reduced lung capacity are risk factors of asthma and chronic obstructive pulmonary disease. Previously, our genomewide linkage analysis of C3H/HeJ (C3H) and JF1/Msf (JF1) mouse strains identified quantitative trait loci (QTLs) associated with the complex traits of dead space volume (Vd), total lung capacity (TLC), lung compliance (CL), and diffusing capacity for CO (D(CO)). We assessed positional candidate genes by comparing C3H with JF1 lung transcript levels by microarray and by comparing C3H, BALB/cByJ, C57BL/6J, A/J, PWD/PhJ, and JF1 strains, using exon sequencing to predict protein structure. Microarray identified >900 transcripts differing in C3H and JF1 lungs related to lung development, function, and remodeling. Of these, three genes localized to QTLs associated with differences in lung function. C3H and JF1 strains differed in transcript and protein levels of superoxide dismutase 3, extracellular [SOD3; mouse chromosome (mCh) 5: VD] and transcript of trefoil factor 2 (TFF2; mCh 17: TLC and D(CO)), and ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2; mCh 15: TLC and CL). Nucleotide sequencing of Sod3, Tff2, and previously identified Relaxin 1 (Rln1; mCh 19: CL) uncovered polymorphisms that could lead to nonsynonymous amino acid changes and altered predicted protein structure. Gene-targeted Sod3(-/-) mice had increased conducting airway volume (Vd/TLC) compared with strain-matched control Sod3(+/+) mice, consistent with the QTL on mCh 5. Two novel genes (Tff2 and Enpp2) have been identified and two suspected genes (Sod3 and Rln1) have been supported as determinants of lung function in mice. Findings with gene-targeted mice suggest that SOD3 is a contributing factor defining the complex trait of conducting airway volume.

Instillation of six different ultrafine carbon particles indicates a surface area threshold dose for acute lung inflammation in mice.

Increased levels of particulate air pollution are associated with increased respiratory and cardiovascular mortality and morbidity. Some epidemiologic and toxicologic research suggests ultrafine particles (UFPs) (< 100 nm) to be more harmful per unit mass than larger particles. Our study was aimed at a quantitative comparison of acute adverse effects of different types of carbonaceous UFPs at a dose range that causes a moderate inflammatory response in lungs. We used six different particle types (primary particle size 10-50 nm, specific surface area 30-800 m2/g, and organic content 1-20%): PrintexG, Printex90, flame soot particles with different organic content (SootL, SootH), spark-generated ultrafine carbon particles (ufCP), and the reference diesel exhaust particles (DEP) SRM1650a. Mice were instilled with 5, 20, and 50 microg of each particle type, and bronchoalveolar lavage was analyzed 24 hr after instillation for inflammatory cells and the level of proinflammatory cytokines. At respective mass-doses, particle-caused detrimental effects ranked in the following order: ufCP > SootL > or = SootH > Printex90 > PrintexG > DEP. Relating the inflammatory effects to the particle characteristics--organic content, primary particle size, or specific surface area--demonstrates the most obvious dose response for particle surface area. Our study suggests that the surface area measurement developed by Brunauer, Emmett, and Teller is a valuable reference unit for the assessment of causative health effects for carbonaceous UFPs. Additionally, we demonstrated the existence of a threshold for the particle surface area at an instilled dose of approximately 20 cm2, below which no acute proinflammatory responses could be detected in mice.

Role of the IBD5 susceptibility locus in the inflammatory bowel diseases.

The field of inflammatory bowel disease genetics plays a leading role in the genetics of complex traits. One of the first genetic loci for a complex trait to be identified by genome-wide linkage scans and confirmed by multiple studies was IBD1 for Crohn's disease. Shortly after this initial success, a second susceptibility locus, the IBD5 risk haplotype, was discovered and unequivocally replicated. In this review, we examine the genetics and potential functional implications of the IBD5 locus on disease susceptibility, prognosis, classification, and treatment. In addition, we discuss the challenges faced when the region identified by association contains multiple genes that are not easily separated by recombination-the primary tool of the human geneticist.

Generation and characterization of dickkopf3 mutant mice.

dickkopf (dkk) genes encode a small family of secreted Wnt antagonists, except for dkk3, which is divergent and whose function is poorly understood. Here, we describe the generation and characterization of dkk3 mutant mice. dkk3-deficient mice are viable and fertile. Phenotypic analysis shows no major alterations in organ morphology, physiology, and most clinical chemistry parameters. Since Dkk3 was proposed to function as thyroid hormone binding protein, we have analyzed deiodinase activities, as well as thyroid hormone levels. Mutant mice are euthyroid, and the data do not support a relationship of dkk3 with thyroid hormone metabolism. Altered phenotypes in dkk3 mutant mice were observed in the frequency of NK cells, immunoglobulin M, hemoglobin, and hematocrit levels, as well as lung ventilation. Furthermore, dkk3-deficient mice display hyperactivity.

Genomewide linkage analysis identifies novel genetic Loci for lung function in mice.

RATIONALE: Pulmonary function, including lung volumes and compliance, may be genetically determined, but few genetic polymorphisms have been identified that control these traits. We used an experimental approach and performed the first whole genome scan for pulmonary function in mice. OBJECTIVES AND METHODS: To identify novel chromosomal regions contributing to lung function, quantitative trait locus linkage analysis was applied in N(2) backcross and F(2) intercross mice derived from two inbred strains-C3H/HeJ and JF1/Msf-with extremely divergent phenotypes. MAIN RESULTS: Significant linkages to total lung capacity with LOD (logarithm of the odds) scores up to 6.0 were detected on chromosomes 15 and 17, to dead space volume and lung compliance on chromosomes 5 and 15 (LOD scores higher than 4.0), to lung compliance also on chromosome 19 (LOD score of 5.8), and to diffusing capacity on chromosomes 15 and 17 (LOD scores up to 5.0). The region of interest on chromosome 17 near D17Mit133 contains a syntenic region to human chromosome 6q27, which was recently identified to be linked to lung function in humans. The identified intervals harbor valuable candidate genes, such as the relaxin1 and transforming growth factor beta receptor 3 gene, which revealed missense polymorphisms between the parental strains. CONCLUSION: The study provides evidence for linkage of different measures of lung function on murine chromosomes 5, 15, 17, and 19 and suggests novel candidate genes that may also affect the expression of human pulmonary function.

Inbred strain variation in lung function.

The purpose of the present study was to determine the strain-specific phenotype variance of lung function parameters among common inbred laboratory mouse strains. In accordance with the "Mouse Phenome Project" run by The Jackson Laboratory (http://www.jax.org/phenome), lung volumes, lung mechanics, and diffusing capacity of 16 males and 16 females of the strains C3H/HeJ, BALB/cByJ, C57B1/6J, A/J, FVB/J, 129SV/ImJ, and SWR/J were determined in a standardized manner. The defined respiratory maneuvers for lung function testing were performed with a custom-made, computer-controlled servo-ventilator in anesthetized animals. Sex differences within the strains were found in most (83%) of the absolute lung function parameters. Usually, normalization to body or lung size completely compensates for the observed gender differences. There was great diversity between strains for all of the lung function parameters studied; for example, the total lung capacity as well as the pulmonary diffusing capacity for carbon monoxide varied by 50% and the static lung compliance by a factor of almost two among the strains. Little, but statistically significant variability was detectable for the dead space volume and the respiratory system resistance. There was no clear-cut evidence for any strain exhibiting either the smallest or the largest values for all parameters studied, suggesting that there were no simple allometric relationships of lung size between the strains. Well-established genealogical relationships among strains were not constantly reflected in phenotype similarities of pulmonary function. Therefore, these data strongly support heritable genetic traits for pulmonary function. Moreover, it constitutes a basis for further genetic lung function-related studies.