Publication Date:
2004-11-16
Description:
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
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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