Abstract 499

Introduction:

CD56+ NK subsets exhibit differential NK receptors (NKR) such as cytotoxicity profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytotoxicity receptors (NCR) involved with tumor target recognition, which, in part, may play a role in adoptive cellular immunotherapy (ACI) for malignancies (Farag et al Blood, 2002). NK cell activation and NK mediated cytolysis is induced by triggering receptors such as NCR (i.e. NKp46), and NKG2 surface receptors like NKG2D (Moretta et al, Curr Opin in Immunol, 2004, Marcenaro et al, Eur J Immunol, 2003). The major limitations of the use of NK cells in ACI include lack of tumor recognition and/or limited numbers of viable and functionally active NK cells (Shereck/Cairo et al. PBC, 2007). To circumvent these limitations, methods to expand and activate PB NK cells by genetic reengineering have been developed (Imai/Campana et al. Blood, 2005). It has been demonstrated that PB NK cells expanded with modified K562 cells expressing membrane bound IL-15 and 4-1BBL (K562-mb15-41BBL; Imai et al Blood, 2005) are significantly increased in number and maintain heterogeneous KIR expression (Fusaki/Campana et al, BJH, 2009) .We have previously reported the ex-vivo expansion, activation and cytolytic activity of CB NK cells with a cocktail of antibody and cytokines (Ayello/Cairo et al, BBMT, 2006; Ayello/Cairo, Exp Hem, 2009, In Press).

Objective:

In this study, we compared CB NK expansion and activation following stimulation with genetically engineered K562 cells (K562-mb15-41BBL, generously supplied by D.Campana, St Jude's Children's Hospital, Memphis, TN) with wild-type (WT) K562 cells and NK cell characterization expressing inhibiting and activating KIRs, c-lectin, NCRs and NK cytolytic activation. Methods: Following irradiation with 100Gy, K562-mb15-41BBL or WTK562 were incubated at a 1:1 ratio with fresh CB MNCs at 37C, 5% CO2 for 7 days in RPMI-1640+10IU IL-2. NKR expression (KIR2DS4, NKG2D, NKG2A, CD94, KIR3DL1, KIR2DL2, Nkp46) and LAMP-1 (CD107a) receptor expression and NK cell phenotype (CD56 dim and bright subsets) were determined by flow cytometry. Results: On Day 0, NK cells population was 3.9±1.3%. After 7 days in culture, CB NK cells were significantly increased compared to WTK562 and media alone (72±3.9 vs 43±5.9 vs 9±2.4%, p<0.01). This represented a 35-fold or 3374±385% increase of the input NK cell number. This was significantly increased compared to WTK562 (1771±300%, p<0.05). Concomitantly, there was a significant decrease in CB T cells vs WTK562 or media alone (15±2 vs 36±2 vs 51±7%, p<0.001),respectively. There was a significant increase in CD56bright vs CD56dim populations (67 vs 33%, p<0.01) following stimulation with K562-mb15-41BBL. Also, there was a 10-fold increase in CB NK cells expressing KIR3DL1 following stimulation with K562-mb15-41BBL vs WTK562 (p<0.01) and a 5-fold increase in NK KIR2DS4 expression (p<0.05), respectively. There was a significant increase in the expression of NK activation marker, CD107a, compared to WTK562 (51±0.7 vs 32±1.1,p<0.05). There was no change in CB NK cell expression of the c-lectin receptor, CD94/NKG2A and CD94/NKG2D after stimulation with K562-mb15-41BBL. A standard cryopreserved CB unit (25 ml) contains approximately 750×106 MNC. By using the smaller 5-ml aliquot (20%) of a two-aliquot bag (150×106 MNCs × 3.9%=5.8×106 NK cells), this expansion method would hypothetically yield 200×106 CB NK cells after 7 days stimulation with K562-mb15-41BBL.

Conclusion:

These results suggest that CB MNC can be ex-vivo expanded with K562-mb15-41BBL resulting in specific expansion of CB NK cells with increased NK KIR expression (KIR2DS4 and KIR3DL1) and NK activation (CD107a), along with a significant decrease in CB T cells. This expansion provides a means to enhance specific CB NK cell expansion for possible use for adoptive cellular immunotherapy in the post UCBT setting

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.

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