WP8 Modelling of topographic signal: Detailed characterisation of individual structures

This deliverable will describe in detail results obtained during the project for four key areas of the Po Plain, the target area for the INGV contribution to the project. The four area are: 1) Coastal Marche region (southeastern Po Plain – see Section 1) 2) Mantova/Mincio River area (central Po Plain – see Section 2) 3) Mirandola/Secchia-Panaro Rivers area (southern-central Po Plain – see Section 3) 4) Soncino/Oglio River area (western Po Plain – see Section 4

Phasic, nonsynaptic GABA-A receptor-mediated inhibition entrains thalamocortical oscillations.

GABA-A receptors (GABA-ARs) are typically expressed at synaptic or nonsynaptic sites mediating phasic and tonic inhibition, respectively. These two forms of inhibition conjointly control various network oscillations. To disentangle their roles in thalamocortical rhythms, we focally deleted synaptic, γ2 subunit-containing GABA-ARs in the thalamus using viral intervention in mice. After successful removal of γ2 subunit clusters, spontaneous and evoked GABAergic synaptic currents disappeared in thalamocortical cells when the presynaptic, reticular thalamic (nRT) neurons fired in tonic mode. However, when nRT cells fired in burst mode, slow phasic GABA-AR-mediated events persisted, indicating a dynamic, burst-specific recruitment of nonsynaptic GABA-ARs. In vivo, removal of synaptic GABA-ARs reduced the firing of individual thalamocortical cells but did not abolish slow oscillations or sleep spindles. We conclude that nonsynaptic GABA-ARs are recruited in a phasic manner specifically during burst firing of nRT cells and provide sufficient GABA-AR activation to control major thalamocortical oscillations

P09.01 Adoptive cell therapy of hematological malignancies using cytokine-induced killer cells retargeted with monoclonal antibodies

Background Cytokine-Induced Killer (CIK) cells are a population of effector cells that represents a promising tool for adoptive cell therapy. They are easily expandable ex-vivo, safe, and exert cytotoxicity against a broad range of tumor histotypes.1 We recently reported that they have a relevant expression of FcγRIIIa (CD16a), which can be exploited in combination with clinical-grade monoclonal antibodies (mAbs) to redirect their cytotoxicity in an antigen-specific manner, to improve their antitumor activity.2 Indeed, the engagement of CD16a on CIK cells leads to a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian cancer both in vitro and in vivo. Based on this observation, we investigated whether CIK cells can be specifically retargeted against B-cell malignancies by combination with anti-CD20 mAbs, namely Rituximab® (RTX) and Obinutuzumab® (OBI). Materials and Methods CIK cells were obtained from peripheral blood mononuclear cells of healthy donors, and stimulated in vitro with IFN-γ, CD3 mAb and IL-2 for 14 days; fresh IL-2 was provided every 3–4 days. CIK cell phenotype was analyzed by multicolor flow cytometry; cytotoxic activity was assessed by calcein AM-release assay against B-cell lines, primary samples and patient-derived xenografts (PDX) obtained from B-cell lymphoma patients after written informed consent. Results The combination with both RTX and OBI significantly increased specific CIK cells lysis against several CD20-expressing lymphoma B cell lines, primary tumors from B-cell lymphoma patients and an established PDX, compared to the combination with a control mAb (cetuximab, CTX). NK-depletion demonstrated that the mAb-mediated cytotoxicity is accountable to the CIK cells fraction within the bulk population since no difference in the lytic activity was detectd in the absence of NK cells. In addition, these results are further supported by in vivo preliminary experiments where the treatment with CIK cells in combination with OBI extensively reduced the growth of PDX and increased mice survival, compared to CIK cells or OBI administered alone. Conclusions Here we proved that CIK cells can be retargeted with clinical-grade mAbs against CD20-expressing lymphomas. These data indicate that the combination of CIK cells with mAbs can represent a novel approach for the treatment of haematological malignancies. References Franceschetti M, Pievani A, Borleri G, Vago L, Fleischhauer K, Golay J, et al. Cytokine-induced killer cells are terminally differentiated activated CD8 cytotoxic T-EMRA lymphocytes. Exp Hematol 2009;37:616–28. Cappuzzello E, Tosi A, Zanovello P, Sommaggio R, Rosato A. Retargeting cytokine-induced killer cell activity by CD16 engagement with clinical-grade antibodies. Oncoimmunology 2016 Aug;5(8):e1199311. The research leading to these results has received funding from Fondazione AIRC under IG 2018 - ID. 21354 project - P.I. Rosato Antonio Disclosure Information A. Dalla Pieta: None. E. Cappuzzello: None. P. Palmerini: None. R. Sommaggio: None. G. Astori: None. K. Chieregato: None. O. Perbellini: None. M. Tisi: None. C. Visco: None. M. Ruggeri: None. A. Rosato: None

P09.13 Optimization of a GMP-grade large-scale expansion protocol for cytokine-induced killer cells using gas-permeable static culture flasks

Background Cytokine-Induced Killer (CIK) cells are ex vivo expanded T cells with NK cell phenotype. They express both CD3 and CD56 antigens, and exert a potent antitumor activity against a variety of tumors. Several clinical trials demonstrated the safety and the feasibility of CIK cell therapy, with very low side effects and minimal graft-versus-host toxicity. In this study, we developed a GMP-compliant protocol for robust large-scale expansion of CIK cells using G-Rex® gas-permeable static culture flasks. Materials and Methods CIK cells were obtained by stimulating healthy donor PBMCs with GMP-grade IFN-γ, IL-2 and CD3 mAbs, and were cultured in G-Rex6® or G-Rex®6M well plates. CIK cells in G-Rex6® were split only once at day 7 to reduce cell density, whereas the number of CIK cells culterd in G-Rex®6M was not adjusted. In both culture conditions, fresh IL-2 was provided every 3–4 days. We compared these two culture protocols with the culture in standard flasks. Phenotype was analyzed by flow cytometry and cytotoxicity was assessed against several tumor cell lines by calcein-release assay. Results CIK cells cultured in G-Rex6® well plates showed an outstanding cell expansion compared to G-Rex®6M well plates or standard culture flasks, with a 400-fold expansion and a mean of 109 total cells obtained per single well in 14 days, starting from just 2.5 × 106 cells per well. Moreover, the cultures in G-Rex6® were characterized by an higher percentage of CD3+CD56+ cells, as compared to G-Rex®6M or standard culture flasks. Cells cultured in all devices had a comparable expression of NKG2D, NKp30, NKp44, 2B4 receptors. Importantly, CIK cells expanded in G-Rex®6 were as cytotoxic as cells expanded in standard culture flasks. Conversely, CIK cells cultured in G-Rex®6M showed a remarkable reduction of cytotoxicity against tumor cell targets, thus suggesting that cell density during expansion could affect CIK cell activity. Conclusions We propose a GMP-compliant protocol for robust large-scale production of CIK cells. G-Rex® system allows to obtain large amounts of CIK cells highly enriched in the CD3+CD56+ subset and endowed with high cytotoxic activity; this can be accomplished with just a single cell culture split at day 7, which dramatically reduces the culture manipulation as compared to the standard culture flasks. Notably, this strategy can be further and easily scalable to produce CIK cells for clinical immunotherapy applications. Disclosure Information A. Ventura: None. P. Palmerini: None. A. Dalla Pieta: None. R. Sommaggio: None. G. Astori: None. K. Chieregato: None. M. Tisi: None. C. Visco: None. O. Perbellini: None. M. Ruggeri: None. E. Cappuzzello: None. A. Rosato: None

Developmental regulation of CB1-mediated spike-time dependent depression at immature mossy fiber-CA3 synapses

Early in postnatal life, mossy fibres (MF), the axons of granule cells in the dentate gyrus, release GABA which is depolarizing and excitatory. Synaptic currents undergo spike-time dependent long-term depression (STD-LTD) regardless of the temporal order of stimulation (pre versus post and viceversa). Here we show that at P3 but not at P21, STD-LTD, induced by negative pairing, is mediated by endocannabinoids mobilized from the postsynaptic cell during spiking-induced membrane depolarization. By diffusing backward, endocannabinoids activate cannabinoid type-1 (CB1) receptors probably expressed on MF. Thus, STD-LTD was prevented by CB1 receptor antagonists and was absent in CB1-KO mice. Consistent with these data, in situ hybridization experiments revealed detectable level of CB1 mRNA in the granule cell layer at P3 but not at P21. These results indicate that CB1 receptors are transiently expressed on immature MF terminals where they counteract the enhanced neuronal excitability induced by the excitatory action of GABA

Gene Transcription and Splicing of T-Type Channels Are Evolutionarily-Conserved Strategies for Regulating Channel Expression and Gating

T-type calcium channels operate within tightly regulated biophysical constraints for supporting rhythmic firing in the brain, heart and secretory organs of invertebrates and vertebrates. The snail T-type gene, LCav3 from Lymnaea stagnalis, possesses alternative, tandem donor splice sites enabling a choice of a large exon 8b (201 aa) or a short exon 25c (9 aa) in cytoplasmic linkers, similar to mammalian homologs. Inclusion of optional 25c exons in the III–IV linker of T-type channels speeds up kinetics and causes hyperpolarizing shifts in both activation and steady-state inactivation of macroscopic currents. The abundant variant lacking exon 25c is the workhorse of embryonic Cav3 channels, whose high density and right-shifted activation and availability curves are expected to increase pace-making and allow the channels to contribute more significantly to cellular excitation in prenatal tissue. Presence of brain-enriched, optional exon 8b conserved with mammalian Cav3.1 and encompassing the proximal half of the I–II linker, imparts a ∼50% reduction in total and surface-expressed LCav3 channel protein, which accounts for reduced whole-cell calcium currents of +8b variants in HEK cells. Evolutionarily conserved optional exons in cytoplasmic linkers of Cav3 channels regulate expression (exon 8b) and a battery of biophysical properties (exon 25c) for tuning specialized firing patterns in different tissues and throughout development

Cervical human papillomavirus infection and squamous intraepithelial lesions in rural Gambia, West Africa: viral sequence analysis and epidemiology

The development of effective strategies against cervical cancer in Africa requires accurate type specific data on human papillomavirus (HPV) prevalence, including determination of DNA sequences in order to maximise local vaccine efficacy. We have investigated cervical HPV infection and squamous intraepithelial lesions (SIL) in an unselected cohort of 1061 women in a rural Gambian community. Squamous intraepithelial lesions was diagnosed using cytology and histology, HPV was typed by PCR-ELISA of DNA extracts, which were also DNA sequenced. The prevalence of cervical HPV infection was 13% and SIL were observed in 7% of subjects. Human papillomavirus-16 was most prevalent and most strongly associated with SIL. Also common were HPV-18, -33, -58 and, notably, -35. Human papillomavirus DNA sequencing revealed HPV-16 samples to be exclusively African type 1 (Af1). Subjects of the Wolof ethnic group had a lower prevalence of HPV infection while subjects aged 25–44 years had a higher prevalence of cervical precancer than older or younger subjects. This first report of HPV prevalence in an unselected, unscreened rural population confirms high rates of SIL and HPV infection in West Africa. This study has implications for the vaccination of Gambian and other African populations in the prevention of cervical cancer