Activation and enhancement of ex vivo (EvE) expanded cryopreserved Cord Blood (CB) Natural Killer (NK) cells, cytolytic potential, and NK receptor (NKR) expression: Potential role for CB NK in Adoptive Cellular Immunotherapy (ACI)

NK subsets exhibit differential NKR profiles including KIR, C-lectin (NKG2), and natural cytotoxicity receptors (NCR) involved with tumor recognition (Farag et al, Blood, 2002). NKRs such as NCR (NKp44, NKp46) and NKG2 surface receptors NKG2D induce NK cell activation and mediated cytolysis (Moretta et al, Curr Opin Immunol, 2004). CB is limited by the absence of available donor effector cells for infusion after UCBT (Cairo et al Transfusion, 2005). We demonstrated the ability to EvE in short term culture with IL-2, IL-7, IL-12, and anti-CD3 (ABCY) cryopreserved, thawed, recryopreserved, rethawed, and EvE (CTCTE) CB with increases in CD3−/16+/56+ bright/dim subsets expressing KIR3DL1, KIR2DL1/S1, KIR2DL2, and CD94/NKG2A (Ayello/Cairo et al, BBMT, 25a, 2004). We now compared short-term with prolonged cultures (4–10 days) on expansion, maturation, and expression of NCR, NKG2, KIR, and cytolytic mechanisms in CTCTE CB. Rethawed CB cells (Kurtzberg/Cairo, Transfuison, 2005) were cultured (2–10 days) in 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). NKR expression (CD94, NKG2C, NKG2D, NKp44, NKp46, KIR2DS4) and intracellular perforin and granzyme B activity were determined by flow cytometry. Europium release assay measured NK and LAK cytotoxicity. NK activating KIR2DS4 was increased day 10 versus 2 in ABCY in both CD3−/16+/56+ dim/bright subsets (16.9 ± 0.4 vs 2.1 ± 0.2% and 22.3 ± 0.3 vs 0.9 ± 0.2%, P < .001, respectively). CD94/NKG2D expression was increased day 7 versus 2 in ABCY EvE (41.4 ± 0.43 vs 23.7 ± 2.0%, P < .001). NK (CD3−/16+/56+dim) KIR3DL1 subset was increased day 10 versus 2 (38.3 ± 2.8 vs 18.9 ± 6.37%, P < .05). CD3−/16+/56+dim NKp44 subset NCR expression was decreased day 10 versu 2 in ABCY (15.2 ± 0.7 vs 27.2 ± 0.7%, P < .001). CD3−/16+/56+ dim NKp46 expression was decreased following day 10 versus 2 (8.5 ± 0.2 vs 23.5 ± 1.2 %, P < .001). Granzyme B was increased from day 2 to 10 (25.8 ± 1.8 vs 45.1 ± 1.7%, P < .0001) yet perforin was decreased in ABCY day 10 versus 2 (55.7 ± 1.8 vs 84.3 ± 1.3%, P < .001). ABCY CB NK and LAK cytotoxicity was increased day 10 versus 2 (NK: 71.5 ± 1.6 vs 53.8 ± 10.3%, P < .001; LAK: 63.2 ± 0.24 vs 31.8 ± 1.8%, P < .001). In summary, CB MNC may be thawed at UCBT, CT at a later date, EvE and activated for 7–10 days to yield viable NK subsets. ABCY 10 day EvE CB yielded increased NK KAR (CD56+dim/bright) and granzyme B expression but decreased NK C-lectin CD94/NKG2D, NCR NKp44 and NKp46, and perforin expression.