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Characterization of Cord Blood Natural Killer and Lymphokine Activated Killer Lymphocytes Following Ex Vivo Cellular Engineering Janet Ayello, Carmella van de Ven, Weiwei Fortino, Cheryl Wade-Harris, Prakash Satwani, Laxmi Baxi, Lynn L. Simpson, Warren Sanger, Diana Pickering, Joanne Kurtzberg, Mitchell S. Cairo Biology of Blood and Marrow Transplantation Volume 12, Issue 6, Pages (June 2006) DOI: /j.bbmt Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions
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Figure 1 A, CD3−/CD16+/CD56+dim/bright subset expansion as determined by flow cytometry. The CD3− lymphocyte population was gated and used as a reference to determine the percentages of CD3−/CD16+/CD56+dim and CD3−/CD16+/CD56+bright expressions of nonadherent UCB MNCs from CTE versus CTCTE versus CTECT after 48 hours in culture with AB/CY versus SF medium alone. Results represent mean ± SEM (n = 3); CD56+dim: *P < .05, CTE, CTECT, CTCTE AB/CY versus medium alone; CD56+bright: **P < .01, CTE, CTECT, CTCTE AB/CY versus medium alone. B, Representative dot plot of the subset of CD3−/CD16+/CD56+dim/bright in nonadherent UCB MNCs from CTECT versus CTCTE after 48 hours in culture in AB/CY as determined by flow cytometry. The CD3− lymphocyte population was gated and used as a reference to determine CD16+/CD56+dim/bright expression. The isotype control was <1%. AIMV indicates serum-free lymphocyte medium. Biology of Blood and Marrow Transplantation , DOI: ( /j.bbmt ) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions
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Figure 2 A, CD94+/NKG2a+ subset expansion was determined by flow cytometry. The CD16+/CD56+ population was gated and used as a reference to determine the percentage of CD94+/NKG2a+ expression of nonadherent UCB MNCs from CTE versus CTCTE versus CTECT after 48 hours in culture with AB/CY versus SF medium alone. Results represent mean ± SEM (n = 3); *P < .012, CTECT AB/CY versus medium alone; **P < .006, CTE AB/CY versus medium alone; ***P < .003, CTCTE AB/CY versus medium alone. B, Representative dot plot of the subset of CD94+/NKG2a+ in nonadherent UCB MNCs from CTECT versus CTCTE after 48 hours in culture in AB/CY as determined by flow cytometry. The CD16+/CD56+ population was gated and used as a reference to determine CD94+/NKG2a+ expression. The isotype control was <1%. Biology of Blood and Marrow Transplantation , DOI: ( /j.bbmt ) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions
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Figure 3 A, CD56+dim/KIR3DL1+ and CD56+bright/KIR3DL1+ subset expansions as determined by flow cytometry. The CD16+/CD56+ population was gated and used as a reference to determine the percentage of CD56+dim/bright/KIR3DL1+ expression of nonadherent UCB MNCs from CTE versus CTCTE versus CTECT after 48 hours in culture with AB/CY versus SF medium alone. Results represent mean ± SEM (n = 3); CD56+dim/KIR3DL1+: **P < .024, CTE AB/CY versus medium alone; ***P < .008, CTECT AB/CY versus medium alone; *P < .04, CTCTE AB/CY versus medium alone; CD56+bright/KIR3DL1+: ††P < .001, CTE AB/CY versus medium alone; †P < .03 CTECT and CTCTE AB/CY versus medium alone. B, Representative dot plot of the subset of CD56+dim/bright/KIR3DL1+ in nonadherent UCB MNCs from CTECT versus CTCTE after 48 hours in culture in AB/CY as determined by flow cytometry. The CD16+/CD56+ population was gated and used as a reference to determine CD56+dim/bright/KIR3DL1+ expression. The isotype control was <1%. Biology of Blood and Marrow Transplantation , DOI: ( /j.bbmt ) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions
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Figure 4 A, CD56+dim/KIR2DL1/S1+ and CD56+bright/KIR2DL1/S1+ subset expansions as determined by flow cytometry. The CD16+/CD56+ population was gated and used as a reference to determine the percentage of CD56+dim/bright/KIR2DL1/S1+ expression of nonadherent UCB MNCs from CTE versus CTCTE versus CTECT after 48 hours in culture with AB/CY versus SF medium alone. Results represent mean ± SEM (n = 3); CD56+dim/KIR2DL1/S1+: **P < .003, CTE, CTECT AB/CY versus medium alone; CD56+bright/KIR2DL1/S1+: ††P < .006, CTE; †P < .017, CTECT; †††P < .009, CTCTE AB/CY versus medium alone; *P < .05, CTE versus CTCTE, CTECT versus CTCTE AB/CY versus medium alone. B, Representative dot plot of the subset of CD56+dim/bright/KIR2DL1/S1+ in nonadherent UCB MNCs from CTECT versus CTCTE after 48 hours in culture in AB/CY as determined by flow cytometry. The CD16+/CD56+ population was gated and used as a reference to determine CD56+dim/bright/KIR2DL1/S1+ expression. The isotype control was <1%. Biology of Blood and Marrow Transplantation , DOI: ( /j.bbmt ) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions
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Figure 5 A, CD56+dim/KIR2DL2+ and CD56+bright/KIR2DL2+ subset expansions as determined by flow cytometry. The CD16+/CD56+ population was gated and used as a reference to determine the percentage of CD56+dim/bright/KIR2DL2+ expression of nonadherent UCB MNCs from CTE versus CTCTE versus CTECT after 48 hours in culture with AB/CY versus SF medium alone. Results represent mean ± SEM (n = 3); CD56+dim/KIR2DL2+: *P < .02, CTE, CTCTE AB/CY versus medium alone; **P < .001, CTECT AB/CY versus medium alone; CD56+bright/KIR2DL2+: †P < .02, CTE AB/CY versus medium alone; ††P < .001, CTECT AB/CY versus medium alone; †††P < .01, CTCTE AB/CY versus medium alone. B, Representative dot plot of the subset of CD56+dim/bright/KIR2DL2+ in nonadherent UCB MNCs from CTECT versus CTCTE after 48 hours in culture in AB/CY as determined by flow cytometry. The CD16+/CD56+ population was gated and used as a reference to determine CD56+dim/bright/KIR2DL2+ expression. The isotype control was <1%. Biology of Blood and Marrow Transplantation , DOI: ( /j.bbmt ) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions
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Figure 6 Effect of UCB ex vivo expanded in medium alone versus AB/CY on survival of NOD/SCID mice that received a xenograft of K562 cells. NOD/SCID mice were injected with 10 × 106 human K562 cells 3 days after tumor cell injection; groups of mice (n = 10) received intraperitoneal injections of CTECT UCB cells (1 × 107 cells/animal) stimulated with medium alone or CTECT UCB cells (1 × 107 cells/animal) stimulated with AB/CY. Parallel sham injections of sterile PBS served as a control group. Injections of ex vivo expanded UCB cells or PBS continued every 7 days for 14 days. Tumor survival was monitored daily and animals were killed when they become moribund with disseminated tumor burden. Biology of Blood and Marrow Transplantation , DOI: ( /j.bbmt ) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions
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