Prolonged Ex Vivo Expansion (7–10 Days) of Cord Blood (CB) Recryopreserved and Rethawed Aliquots with Anti-CD3, IL-2, IL-12 Significantly Expands NKT Cells, NKT KIR Substes and NK Cytotoxicity Compared to Short-Term Expansion: Potential for CB Adoptive Cellular Immunotherapy (ACI).
Authors: Ayello Janet , Satwani Prakash , van de Ven Carmella , Roman Elizabeth , O’Neill Allison , Shutran Max , Greenhawt Matthew , Baxi Laxmi , Simpson Lynn , Kurtzberg Joanne , Cairo Mitchell S. 
Published: April 2020

Abstract
CD56+ NK subsets exhibit differential receptor profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytotoxicity receptors involved with tumor target recognition which may play a role in ACI for malignancies (Farag et al, Blood 2002). UCB is limited by the absence of available donor effector cells (NK, CTL, LAK and NKT cells) for infusion after transplantation for the treatment of minimal residual resistant hematological relapse and/or PTLD (Barker et al, Blood 2001; Locatelli, et al Blood 1999). We have demonstrated the ability to ex-vivo expand (EVE) CB in short term culture (48hrs) with IL-2, IL-7, IL-12 and anti-CD3 (AB/CY) cryopreserved, thawed, recryopreserved, rethawed and ex vivo expanded (CTCTE) with a significant increase in CD3−/16+/56+ bright/dim subsets expressing KIR3DL1, KIR2DL1/S1, KIR2/DL2, CD94/NKG2A (Ayello/Cairo et al, BBMT 25a 2004). Timeus et al (Hematologica 2003) demonstrated the ability to sequentially freeze-thaw UCB twice without a detrimental effect on UCB clonogenic potential or viability (Hematologica 2003). In this study we compared previous short term (48hr) cultures with prolonged cultures (48–240hrs) on expansion, maturation, NK and LAK cytolytic ability, KIR inhibitory and C-lectin NK subsets in CTCTE UCB utilizing the same AB/CY cocktail. UCB were cryopreserevd and thawed by the NHBLI/COBLT method (Fraser/Cairo et al, J Hemat 1998). After rethawing, nonadherent UCB cells were cultured in serum-free media alone or with anti-CD3 (50 ng/ml), IL-2 (5 ng/ml), IL-7 (10 ng/ml) and IL-12 (10 ng/ml) [ABCY] for 4 time periods: 48, 96, 168 and 240 hrs. NK subsets (CD94+, CD16+, CD56+) and NK receptor (KIR3DL1, KIR2DL1/S1, KIR2DL2 and NKG2a) expression were analyzed by flow cytometry using CD3, CD16, CD56, NKBL1, CD158a, CD158b, CD94 and NKG2a mAbs. Additionally, NK and LAK cytotoxicity was measured by europium release assay. There was a significant increase in NKT (CD3+/16+/56+) UCB with AB/CY from 48–240 hrs vs media (2.97±.3 vs 66.7±7.1 vs 35.8±5.9%, p&lt;.001; p&lt;.01), NKT KIR2DL1/S1 from 48–168 hrs vs media (2.76±.4 vs 33.6±8.5 vs 12.1±0.9%, p&lt;.001, p&lt;.05), NKT KIR3DL1 at 48 vs 240 hrs (1.27±0.5 vs 22.2±4.6%, p&lt;.05) and NK (CD3−/16+/56+) KIR3DL1 48 vs 240 hrs vs media (18.8±2.8 vs 38.3±6.3 vs 12.1±4.2%, p&lt;.05, p&lt;.001). A significant increase was shown in UCB NK cytotoxicity on Day 7 (168hrs) [48 vs 96 vs168 hrs] AB/CY vs media alone (168hrs): 53.8±3.9 vs 56±1.6 vs 71.5±.8 vs 40.7±2.0%, p&lt;.001). Similarly, there was a significant maximal increase in UCB LAK cytotoxicity at 168 hrs [48 vs 96 vs 168 hrs] AB/CY vs media: 31.8±1.8 vs 5.47±1.4 vs 63.95±.74 vs 41.6±1.1%, p&lt;.001. We observed a significant enhancement in proliferation at 48 vs 96 vs 168 vs 240 hrs in ABCY vs media (p&lt;.001) and maximal proliferation in ABCY vs media at 240 hrs:.41±.098 vs 3.93±.014, p&lt;.001. This data suggests that UCB cryopreserved cells may be thawed at the time of UCBT, recryopreserved, rethawed at a later date, EVE and activated to yield viable NK and NKT KIR subsets with enhanced cytolytic ability for ACI to induce GVHM effects after UCBT.