Chan, M

Koh, M

Linn, Y-C

Luah, Y-H

Niam, M

Said, NSB

Sundar Raj, K

Yap, CS

English

St George's Online Research Archive

ESTABLISHING A LOCAL BANK AND COMPARING EFFICACIES OF THIRD PARTY VIRUS-SPECIFIC CYTOTOXIC T CELLS (VST) AND CYTOKINE INDUCED KILLER CELLS (VSCIK)Chui Sun YAP1 , Yen-Hoon LUAH4 , Nur Syafiqah Binte Said1 , Kirubavathy SUNDAR RAJ2 , Madelaine NIAM1, Marieta CHAN1 , Mickey KOH1,3 , Yeh-Ching LINN21. Cell Therapy Facility, BSG, Health Sciences Authority, Singapore.2. Dept of Haematology, Singapore General Hospital, Singapore.3. Haematology, St George's University Hospitals, London, United Kingdom.4. Dept of Public Health, National University of Singapore, Singapore.References1. Population-specific Recombination Sites Within the Human MHC Region. Lam, TH, et al.Heredity 2013. 111(2): 131-138.2. Activity of broad-spectrum T-cells as treatment for AdV, EBV, CMV, BKV and HHV6 infectionsafter HSCT. Papadopoulou, A, et al. Sci. Translational Med. 2014. 6(242):1-1.3. Characterization of the recognition and functional heterogeneity exhibited by cytokine-induced killer cell subsets against acute myeloid leukaemia target cell. Linn, YC, et al.Immunology 2012. 126(3):423-35.Table 2. Number of donor samples that react positively to viralpeptides during screening. A total of 14 donors were screened.Virus No. of positive donorsAdenovirus (AdV) 6BK virus (BKV) 1Cytomegalovirus (CMV) 7Epstein barr virus (EBV) 0Human herpesvirus 6 (HHV6) 6Fig 1. CMV-CIKs are more cytotoxic than CMV-VSTs.VsCIKs consistently lysed more PHA targets loaded with viral peptides compared to VSTs.Results shown are from three separate experiments, each comparing CMV-CIKs with CMV-VSTs generated from the same donor.Table 3. Comparison of functional activities of vsCIKs and VSTs.For AdV, VST cells showed greater responses to viral peptides than the vsCIKs. However,there were incongruities between the cytotoxicity and degranulation comparisons for CMVand HHV6. So far, no donor cells have shown reactivity against EBV peptides. This isdespite a high percentage of EBV carriers among Singaporeans and the successful use ofthe same peptides in published reports.VirusvsCIK > VST VST > vsCIK vsCIK  VSTCytotoxicity Degranulation Cytotoxicity Degranulation Cytotoxicity DegranulationAdV 0 0 3 4 1 0BKV 0 0 0 1 1 0CMV 4 2 0 3 3 2HHV6 2 0 1 4 2 1For the comparisons, differences of 10% or more were considered significant, while valuesbelow 5% were considered negative. For cytotoxicity, the values from wells with a ratio of10:1 (effector:target) were compared.Reactivation of dormant viruses is not uncommon after allogeneichaematopoietic stem cell transplants (allo-HSCT). Antiviral drug therapy islimited by refractory strains and toxicities, while matched donor derived VSTshave been successfully used to treat allo-HSCT patients with viral reactivationand there is promising data that minimally matched third party banked VSTs arealso effective and safe. Here we explore the feasibility of a compact andeffective bank of virus-specific cytotoxic cells for allo-HSCT patients.We (1) reviewed our database of the HLA class I types of 100 consecutive allo-HSCT recipients, (2) screened the local population for response to viruses ofinterest and (3) studied the anti-viral activities of VST and vsCIK cultures todetermine the efficacies of VSTs versus vsCIKs.Cones, which are normally discarded, from plateletpheresis of healthy donorswere collected with consent. The PBMC in the cones were then screened forvirus reactivity and if positive, further cultured following the vsCIK and VSTprotocols.We showed that our local population share common HLA alleles such that acompact third party bank from donors with these alleles can cover mostpatients. We also successfully generated AdV, BKV, CMV and HHV6-specificvsCIKs and VSTs. Interestingly, in the comparison between VSTs and vsCIKs foreach virus, the results of the cytotoxicity assays and the degranulation assaysdo not always agree. Further screening of viral peptides (particularly for EBV)and mechanistic studies will be required to determine the more effective celltype for the proposed bank. We also do not dismiss the possibility that themore effective anti-viral cell type might be specific for each virus.Screen for response to viruses+ viral peptidesovernightFlow cytometry for activation marker, 4-1BB expressionvsCIK Protocol3+ viral peptides+ anti-4-1BB+ IFNγ+ OKT3+ IL-2 VST Protocol2+ viral peptides+ IL-4+ IL-7 1. Cytotoxicity Assay- Cr51-release assay using autologous PHA blasts with/without peptide loading as target cells2. Degranulation Assay - flow cytometry analysis for CD107a expression in response to viral peptides, to quantify antigen-specific degranulation MATERIALS AND METHODSRESULTSCONCLUSIONSAcknowledgementsFunding was obtained from SMART II, National Research Foundation, Singapore.INTRODUCTIONTable 1. Presence of common HLA class I alleles in allo-HSCT patients. Amongst Singaporeans, 96% carry one or more of the 17 HLA class I alleles that have afrequency of > 5% in the local population.1 Analysis of the HLA-A, B and C data of 100consecutive allo-HSCT patients showed that 85% have three or more of these commonalleles, while only 4% do not have any of them. It is, therefore, feasible to establish acompact panel of cytotoxic cells from donors with these common HLA alleles.No. of allelespresentChinese(n= 81)Malay(n= 10)Indian(n= 4)Others(n= 5)Total(n= 100)0 0 3 1 0 41 1 0 2 1 42 5 1 1 0 73 20 4 0 2 264 21 0 0 1 225 20 2 0 0 226 14 0 0 1 15

ESTABLISHING A LOCAL BANK AND COMPARING EFFICACIES OF THIRD PARTY VIRUS-SPECIFIC CYTOTOXIC T CELLS (VST) AND CYTOKINE INDUCED KILLER CELLS (VSCIK)

http://openaccess.sgul.ac.uk/112066/1/ISCT19poster-yapcs.pdf

ESTABLISHING A LOCAL BANK AND COMPARING EFFICACIES OF THIRD PARTY VIRUS-SPECIFIC CYTOTOXIC T CELLS (VST) AND CYTOKINE INDUCED KILLER CELLS (VSCIK)

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