Sheree Bailey

ProNGF mediates death of Natural Killer cells through activation of the p75NTR–sortilin complex

The common neurotrophin receptor P75NTR, its co-receptor sortilin and ligand proNGF, have not previously been investigated in Natural Killer (NK) cell function. We found freshly isolated NK cells express sortilin but not significant amounts of P75NTR unless exposed to interleukin-12 (IL-12), or cultured in serum free conditions, suggesting this receptor is sequestered. A second messenger associated with p75NTR, neurotrophin-receptor-interacting-MAGE-homologue (NRAGE) was identified in NK cells. Cleavage resistant proNGF123 killed NK cells in the presence of IL-12 after 20h and without IL-12 in serum free conditions at 48h. This was reduced by blocking sortilin with neurotensin. We conclude that proNGF induced apoptosis of NK cells may have important implications for limiting the innate immune response.

Cytometry‐acquired calcium‐flux data analysis in activated lymphocytes

Flow cytometry enables the sequential determination of calcium levels in millions of stimulated lymphocytes over a short period of time. Current algorithms available are not suitable for the statistical analysis of this large amount of data. The authors aimed to develop a robust algorithm that fits a function to median values of measured data and provides an opportunity for statistical comparison between different calcium‐flux measurements. The alteration of calcium signal was monitored in CD4+ cells loaded with calcium binding fluorescent dyes and stimulated with phytohemagglutinin; the alteration of calcium signal was monitored for 10 minutes. The authors also reanalyzed published calcium‐flux data of CD3+ cells and Jurkat cells stimulated with different concentrations of anti‐CD3 and thapsigargin. The authors fitted different functions to the medians of data per time unit and identified hormesis function as the best fitting one. On the basis of the optimally fitting function, the authors calculated the most relevant biological descriptors such as starting value, peak, time to reach the maximum, and time to reach 50% of maximum before and after the peak. Statistically significant differences in cell activation kinetics at different stimulatory concentrations were also demonstrated. This approach enables us to characterize the kinetics and distribution of calcium‐flux data derived by flow cytometry and may be a reliable tool for the characterization of lymphocyte activation (for details see: http://calciumflux.intralab.eu).

FcγRIIb expression on human germinal center B lymphocytes

IgG antibody can specifically suppress the antibody response to antigen. This has been explained by the hypothesis that signaling through the B cell antigen receptor is negatively modulated bythe co‐ligation of immunoglobulin with the receptor for IgG, FcγRIIb. We hypothesized that inhibitory signaling through FcγRIIb would be counter‐productive in germinal center cells undergoing selection by affinity maturation, since these cells are thought to receive a survival/proliferative signal by interacting with antigen displayed on follicular dendritic cells. We have identified and characterized a population of B lymphocytes with low/negative FcγRIIb expression that are present in human tonsil. Phenotypically these cells correspond to germinal center B cells and comprise both centroblast and centrocyte populations. In examining expression at the molecular level we determined that these B cells do not express detectable mRNA for FcγRIIb. We examined several culture conditions to induce expression of FcγRIIb on germinal center cells but could not determine conditions that altered expression. We then examined the functional consequence of cross‐linking membrane immunoglobulin and the receptor for IgG on human B lymphocytes. Our results cast some doubt on the value of anti‐IgG as a model for antigen–antibody complexes in studying human B cell regulation.

Clinical utility of molecular and flow cytometric markers in chronic lymphocytic leukaemia.

Background: Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous disease. While immunoglobulin variable region heavy chain (IgVH) mutational status remains the ‘gold standard’ in molecular prognostication, a range of additional markers is increasingly being used in clinical trials. As awareness of trial data increases, requests to determine these prognostic markers for new CLL patients are becoming more prevalent in Australia.

Development of locus specific sub-clone separation by fluorescence in situ hybridization in suspension in chronic lymphocytic leukemia: Development of Locus Specific Sub-Clone Separation

Intra‐tumor genetic heterogeneity is a hallmark of cancer. The ability to monitor and analyze these sub‐clonal cell populations can be considered key to successful treatment, particularly in the modern era of targeted therapies. Although advances in sequencing technologies have significantly improved our ability to analyze the mutational landscape of tumors, this utility is reduced when considering small, but clinically significant sub‐clones, that is, those representing <10% of the tumor burden. We have developed a high‐throughput method that utilizes a 17‐probe labeled bacterial artificial chromosome contig to quantify sub‐clonal populations of cells based on deletion of a single locus. Chronic lymphocytic leukemia (CLL) cells harboring deletion of the short arm of chromosome 17 (del17p), an important prognostic marker for CLL were used to demonstrate the technique. Sub‐clones of del17p cells were quantified and isolated from heterogeneous CLL populations using fluorescence in situ hybridization in suspension (FISH‐IS) and the locus specific probe set. Using the combination of FISH‐IS with the locus‐specific probe set enables automated analysis of tens of thousands of cells, accurately quantifying and isolating cells carrying a del17p. Based on the fluorescence intensity of 17p probes, 17p (TP53) deleted cells were identified and sorted using flow cytometric techniques, and enrichment was demonstrated using single nucleotide polymorphism analysis. The ability to separate sub‐clones of cells based on genetic heterogeneity, independent of the clone size, highlights the potential application of this method not only in the diagnostic and prognostic setting, but also as an unbiased approach to enable further detailed genetic analysis of the sub‐clone with deep sequencing approaches.

Magnetic and Fluorescence Encoded Polystyrene Microparticles for Cell Separation

Materials assisting with the efforts of cell isolation are attractive for numerous biomedical applications including tissue engineering and cell therapy. Here, we have developed surface modification methods on microparticles for the purposes of advanced cell separation. Iron oxide nanoparticles were incorporated into 200 ìm polystyrene microparticles for separation of particle-bound cells from non-bound cells in suspension by means of a permanent magnet. The polystyrene microparticles were further encoded with fluorescent quantum dots (QD) as identification tags to distinguish between specific microparticles in a mixture. Cluster of differentiation (CD) antibodies were displayed on the surface of the microparticles through direct adsorption and various methods of covalent attachment. In addition, a protein A coating was used to orientate the antibodies on the microparticle surface and to maximise accessibility of the antigen-binding sites. Microparticles which carried CD antibodies via covalent attachment showed greater cell attachment over those modifications that were only adsorbed to the surface through weak electrostatic interactions. Greatest extent of cell attachment was observed on microparticles modified with protein A - CD antibody conjugates. B and T lymphocytes were successfully isolated from a mixed population using two types of microparticles displaying B and T cell specific CD antibodies, respectively. Our approach will find application in preparative cell separation from tissue isolates and for microcarrier-based cell expansion.

Chapter 57 – Preparation of Monoclonal Antibodies

Publisher Summary This chapter presents procedures for the preparation of monoclonal antibodies. Their preparation depends on a series of steps that require attention to detail and careful laboratory management. Several myeloma cell lines are commonly used for fusion with murine spleen cells. Animals can make antibodies to a wide range of molecular structures except that they will not usually make antibodies to self-antigen and will not usually recognize small molecules. Small molecules need to be conjugated to a carrier protein. Commonly used carrier proteins include keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Six-week-old female BALB/c mice are preferred for immunization as the myeloma lines used for fusion were derived from that strain. Analysis of a test bleed can allow the fusion to be postponed and the animals reimmunized if the antibody titer is low or nonexistent. Test bleeds should only be carried out by experienced personnel. HAT medium is used to selectively grow hybrids following fusion. Aminopterin selects against unfused myeloma cells and myeloma:myeloma-fused cells by blocking the main synthetic pathway for DNA.