HIGHGLY EFFICIENT ACTIVATION AND EXPANSION OF NATURAL KILLER CELLS FOR CLINICAL USE IN CANCER IMMUNOTHERAPY

Natural killer (NK) cells can detect and kill tumor cells and infusion of NK cells to cancer patients may be a promising option to treat cancer. In this context, ex vivo expansion is used to produce large quantities of activated NK cells, because sufficient numbers of these effector cells are essential for successful NK cell based adoptive cancer immunotherapy. The development of efficient NK cell expansion protocols and the transfer of these protocols to clinically applicable methods represent a major challenge. To overcome this issue, the aim of my project was to develop a clinically applicable method that yields large numbers of highly functional NK cells. First, a fully automated technical process was developed to activate and expand NK cells with (interleukin) IL-2 and irradiated clinical-grade feeder cells (EBV-LCL). In comparison to the manual procedure, the automated process yielded similar NK cells in terms of cell numbers, surface marker profile, gene expression and in vitro effector functions. Upon expansion, NK cells up-regulated functional surface molecules, such as TRAIL, FasL, NKG2D and DNAM-1, they increased the production of interferon (IFN)-g and tumor necrosis factor (TNF)-a and they became more cytotoxic against tumor cell lines. Next, because in the used protocol NK cell expansion was restricted to a period of 2-4 weeks, a more efficient protocol for long-term expansion was developed. Manual NK cell expansion with EBV-LCL and IL-2 induced a 22– fold mean NK cell expansion after one week that was significantly increased to 53–fold by addition of IL-21. Furthermore, repeated stimulation with irradiated EBV-LCL and IL-2 and addition of IL-21 at the initiation of the culture allowed sustained NK cell proliferation with 1011–fold NK cell expansion after six weeks, which is an unprecedented high expansion rate not achieved by any other method so far. Most importantly, adoptive transfer of NK cells expanded with this optimized protocol led to significant inhibition of tumor growth in a melanoma xenograft mouse model, proofing the therapeutic efficacy of the ex vivo generated NK cells. This anti-tumor efficacy was superior over that from conventionally IL-2 activated NK cells, demonstrating that the improved NK cell expansion method enhanced not only the quantity but also the therapeutic quality of NK cells. In conclusion, the outcome of this project is a fully automated process for ex vivo production of NK cells and an optimized protocol for NK cell expansion with unparalleled efficacy. The expanded NK cells possess potent anti-tumor features and showed therapeutic efficacy in a preclinical melanoma xenograft model. Thereby, the project serves clinical needs and makes it possible to generate high cell doses of functional NK cells for the use in cancer immunotherapy

Survival of newly formed particles in haze conditions

Intense new particle formation events are regularly observed under highly polluted conditions, despite the high loss rates of nucleated clusters. Higher than expected cluster survival probability implies either ineffective scavenging by pre-existing particles or missing growth mechanisms. Here we present experiments performed in the CLOUD chamber at CERN showing particle formation from a mixture of anthropogenic vapours, under condensation sinks typical of haze conditions, up to 0.1 s(-1). We find that new particle formation rates substantially decrease at higher concentrations of pre-existing particles, demonstrating experimentally for the first time that molecular clusters are efficiently scavenged by larger sized particles. Additionally, we demonstrate that in the presence of supersaturated gas-phase nitric acid (HNO3) and ammonia (NH3), freshly nucleated particles can grow extremely rapidly, maintaining a high particle number concentration, even in the presence of a high condensation sink. Such high growth rates may explain the high survival probability of freshly formed particles under haze conditions. We identify under what typical urban conditions HNO3 and NH3 can be expected to contribute to particle survival during haze.Peer reviewe

An intercomparison study of four different techniques for measuring the chemical composition of nanoparticles

Currently, the complete chemical characterization of nanoparticles (< 100 nm) represents an analytical challenge, since these particles are abundant in number but have negligible mass. Several methods for particle-phase characterization have been recently developed to better detect and infer more accurately the sources and fates of sub-100 nm particles, but a detailed comparison of different approaches is missing. Here we report on the chemical composition of secondary organic aerosol (SOA) nanoparticles from experimental studies of α-pinene ozonolysis at −50, −30, and −10 ∘C and intercompare the results measured by different techniques. The experiments were performed at the Cosmics Leaving OUtdoor Droplets (CLOUD) chamber at the European Organization for Nuclear Research (CERN). The chemical composition was measured simultaneously by four different techniques: (1) thermal desorption–differential mobility analyzer (TD–DMA) coupled to a NO$^-_3$ chemical ionization–atmospheric-pressure-interface–time-of-flight (CI–APi–TOF) mass spectrometer, (2) filter inlet for gases and aerosols (FIGAERO) coupled to an I$^−$ high-resolution time-of-flight chemical ionization mass spectrometer (HRToF-CIMS), (3) extractive electrospray Na$^+$ ionization time-of-flight mass spectrometer (EESI-TOF), and (4) offline analysis of filters (FILTER) using ultra-high-performance liquid chromatography (UHPLC) and heated electrospray ionization (HESI) coupled to an Orbitrap high-resolution mass spectrometer (HRMS). Intercomparison was performed by contrasting the observed chemical composition as a function of oxidation state and carbon number, by estimating the volatility and comparing the fraction of volatility classes, and by comparing the thermal desorption behavior (for the thermal desorption techniques: TD–DMA and FIGAERO) and performing positive matrix factorization (PMF) analysis for the thermograms. We found that the methods generally agree on the most important compounds that are found in the nanoparticles. However, they do see different parts of the organic spectrum. We suggest potential explanations for these differences: thermal decomposition, aging, sampling artifacts, etc. We applied PMF analysis and found insights of thermal decomposition in the TD–DMA and the FIGAERO

Survival of newly formed particles in haze conditions

Intense new particle formation events are regularly observed under highly polluted conditions, despite the high loss rates of nucleated clusters. Higher than expected cluster survival probability implies either ineffective scavenging by pre-existing particles or missing growth mechanisms. Here we present experiments performed in the CLOUD chamber at CERN showing particle formation from a mixture of anthropogenic vapours, under condensation sinks typical of haze conditions, up to 0.1 s(-1). We find that new particle formation rates substantially decrease at higher concentrations of pre-existing particles, demonstrating experimentally for the first time that molecular clusters are efficiently scavenged by larger sized particles. Additionally, we demonstrate that in the presence of supersaturated gas-phase nitric acid (HNO3) and ammonia (NH3), freshly nucleated particles can grow extremely rapidly, maintaining a high particle number concentration, even in the presence of a high condensation sink. Such high growth rates may explain the high survival probability of freshly formed particles under haze conditions. We identify under what typical urban conditions HNO3 and NH3 can be expected to contribute to particle survival during haze.Peer reviewe

Molecular understanding of the suppression of new-particle formation by isoprene

Nucleation of atmospheric vapours produces more than half of global cloud condensation nuclei and so has an important influence on climate. Recent studies show that monoterpene (C10H16) oxidation yields highly oxygenated products that can nucleate with or without sulfuric acid. Monoterpenes are emitted mainly by trees, frequently together with isoprene (C5H8), which has the highest global emission of all organic vapours. Previous studies have shown that isoprene suppresses new-particle formation from monoterpenes, but the cause of this suppression is under debate. Here, in experiments performed under atmospheric conditions in the CERN CLOUD chamber, we show that isoprene reduces the yield of highly oxygenated dimers with 19 or 20 carbon atoms - which drive particle nucleation and early growth - while increasing the production of dimers with 14 or 15 carbon atoms. The dimers (termed C-20 and C-15, respectively) are produced by termination reactions between pairs of peroxy radicals (RO2 center dot) arising from monoterpenes or isoprene. Compared with pure monoterpene conditions, isoprene reduces nucleation rates at 1.7 nm (depending on the isoprene = monoterpene ratio) and approximately halves particle growth rates between 1.3 and 3.2 nm. However, above 3.2 nm, C-15 dimers contribute to secondary organic aerosol, and the growth rates are unaffected by isoprene. We further show that increased hydroxyl radical (OH center dot) reduces particle formation in our chemical system rather than enhances it as previously proposed, since it increases isoprene-derived RO2 center dot radicals that reduce C-20 formation. RO2 center dot termination emerges as the critical step that determines the highly oxygenated organic molecule (HOM) distribution and the corresponding nucleation capability. Species that reduce the C-20 yield, such as NO, HO2 and as we show isoprene, can thus effectively reduce biogenic nucleation and early growth. Therefore the formation rate of organic aerosol in a particular region of the atmosphere under study will vary according to the precise ambient conditions.Peer reviewe

Role of iodine oxoacids in atmospheric aerosol nucleation

Iodic acid (HIO₃) is known to form aerosol particles in coastal marine regions, but predicted nucleation and growth rates are lacking. Using the CERN CLOUD (Cosmics Leaving Outdoor Droplets) chamber, we find that the nucleation rates of HIO₃ particles are rapid, even exceeding sulfuric acid–ammonia rates under similar conditions. We also find that ion-induced nucleation involves IO₃⁻ and the sequential addition of HIO₃ and that it proceeds at the kinetic limit below +10°C. In contrast, neutral nucleation involves the repeated sequential addition of iodous acid (HIO₂) followed by HIO₃, showing that HIO₂ plays a key stabilizing role. Freshly formed particles are composed almost entirely of HIO₃, which drives rapid particle growth at the kinetic limit. Our measurements indicate that iodine oxoacid particle formation can compete with sulfuric acid in pristine regions of the atmosphere

Highly efficient IL-21 and feeder cell-driven ex vivo expansion of human NK cells with therapeutic activity in a xenograft mouse model of melanoma

Interior, fabric store; Mumbai was a formally a textile center; Mumbai (also known as Bombay, renamed in 1996) is the capital city of the Indian state of Maharashtra. It is the most populous city in India and the fifth most populous city in the world, with an estimated city population of 18.4 million and metropolitan area population of 20.7 million as of 2011. Mumbai lies on the west coast of India and has a deep natural harbor. Mumbai is the financial, commercial and entertainment capital of India. Dharavi, Asia's second largest slum is located in central Mumbai and houses between 800,000 to one million people in 2.39 square kilometers, however the literacy rate in the Mumbai slums is 69%. Source: Wikipedia; http://en.wikipedia.org/wiki/Main_Page (accessed 8/4/2014