Cytokine Induced Killer Cells Can Kill Target through Antigen/TCR Dependent and Independent Mechanisms: New Clinical Perspectives of Utilisation.
Authors: Pievani Alice , Borleri Gianmaria , Belussi Camilla , Rambaldi Alessandro , Golay Josee , Introna Martino 
Published: October 2019

Abstract
Abstract 3024
Poster Board II-1000
Cytokine induced killer (CIK) cells cultures can easily be obtained by stimulating PBMCs with monoclonal antibody anti-CD3 OKT3, IFNgamma and IL2. After 3-4 weeks at least 3 separate populations are present in the culture: CD3+/CD56-/CD8+ precursors (40.5 ± 19.9%), CD3-CD56+ NK cells (2.5 ± 1.5%) and CD3+/CD56+/CD8+ CIK cells (56.9 ± 21%) which show a T EMRA phenotype (Franceschetti et al., Exp Hematol. 37, 616-628, 2009). CIK cultures are currently used in allogeneic or autologous settings as potential anti-neoplastic effectors for adoptive transfer clinical approaches. We have further characterised the mechanism of target cell recognition and role of activating receptors and of lytic mediators in the cytotoxicity of purified CD3+/CD56+/CD8+ CIK cells. We have observed that CIK cells can kill targets through at least two distinct mechanisms: the first TCR-dependent and antigen-specific and the second non TCR-dependent. Indeed, upon TCR/CD3 crosslinking in CIK cells we observe ERK-1/2 phosphorylation, IFNgamma production (mean 32.6% of positive cells by intracellular staining) and TNFalpha production (mean 19.6%). CD3 ligation by OKT3 results in a significant increase over time in the percentage of CIK cells undergoing degranulation evaluated as CD107a positive cells (respectively 15.5 ± 2.2% at 60', 24.4 ± 1% at 120', 32,9 ± 8.7% at 180' and 34.2 ± 11.1% at 240'). CD3 ligation on CIK cells can induce cytotoxicity in a reverse Ab-dependent killing assay. Addition of OKT3 enhances also the cytotoxicity of CIK cells against K562 leukaemic target (from 16 ± 5% to 50 ± 4 %, E:T 10:1; p<0.001). The TCR/CD3 mediated activation is blocked by pre-treatment with cyclosporine A, confirming the role of calcium-regulated phosphatase calcineurin in the CD3-linked degranulation pathway. Interestingly, CIK cells retain functional activity as antigen-specific memory T cells. Indeed in case of CIK cultures obtained from CMV-seropositive donors, CMV-specific CD3+/CD56+ CIK cells can be generated. CMV-specific CIK cells immunopurified by HLA-peptide tetramers are able to specifically recognise and kill autologous but not allogeneic PHA-blasts pulsed with pp65495-503 but not with irrelevant peptide (average lysis 63 ± 8%, E:T 10:1) and to produce IFNgamma following antigenic stimulation (26.4 ± 7%). Similar data are obtained with EBV-specific T cells. Moreover, CIK cells also show non-MHC-restricted cytotoxicity against numerous leukemic target cell lines. The same CMV-specific purified CIK population shows to posses non MHC-restricted cytotoxic properties against several leukemic targets (average lysis 44 ± 8%, E:T 10:1). NKG2D, described as receptor that can trigger TCR-independent cytotoxicity in activated T cells, is expressed on all CIK cells (mean 99%, MFI 251). Although we can show that NKG2D is functional in these cells since cross-linking with anti-NKG2D mAb leads to ERK phophorylation, blocking of this receptor with mAb does not affect the cytolysis of leukaemic targets. NKG2D crosslinking in CIK cells is not sufficient to induce granule esocytosis and INFgamma or TNFalpha production. No correlation is found between expression and surface density of NKG2D ligands on leukaemic cell targets and their susceptibility to CIK-mediated lysis. Unrestricted CIK-mediated cytotoxicity occurs in the absence of measurable degranulation, is not affected by cyclosporin A, but is inhibited by 70% in presence of brefeldin A, a surface upregulation of glycopolypeptide molecules inhibitor, suggesting a role for Fas-ligand family molecules. CIK cells indeed express TNFalpha (mean 16%, MFI 56), FasL (mean 34%, MFI 34) and TRAIL (mean 37%, MFI 49). The role of this death ligands in leukaemic cell killing is currently under investigation. These data clearly show that CD3+/CD56+/CD8+ CIK cells are activated T EMRA cells which have retained their TCR/CD3 complex usage and their antigen specificity but have acquired unrestricted anti-leukaemic activity. The identification of the molecules involved in this killing is still under investigation. These data open up the possibility of multiple clinical use of CIK cultures, including anti-leukaemic and anti-infective usage particularly in immunodeficient patients.

Disclosures
No relevant conflicts of interest to declare.