Activation Of Dendritic Cells In The Umbilical Cord Blood Can Improve The Autologous NK Cytotoxic Activity

Recipients of umbilical cord blood transplantation (UCBT) face a high risk of morbidity and mortality related to opportunistic infections and leukemic relapse. NK cells are the major lymphocyte population of the innate immune response which early reconstruct after UCBT. The activation of NK cells relies on a combination of interactions with cytokine receptors, adhesion molecules and activating receptors ligated via interactions with tumors, pathogen-infected cells or other immune cells. Dendritic cells (DCs) are key players in the immune system as they bridge innate and adaptive immunity. In this study, we co-cultured the selected CD56⁺NK cells with activated mature DCs(include myeloid DCs and plasmacytoid DCs) from the autologous umbilical cord blood purposed to investigate their change of biological activity.

Umbilical cord blood samples were obtained from the donors(n=5). Mononuclear cells were separated on a Ficoll density gradient centrifugation (1.077 g/Ml). DCs and NK cells were isolated respectively using the indirect NK cells and dendritic cells isolation Kit (Miltenyi Biotec Inc).DCs cultured with IL-3 (30ng/ml),GM-CSF (30ng/ml),LPS(100ng/ml),CpG₂₀₀₆OND(5umol/l)in X-vivo20 medium. After 24 hours of incubation, cell phenotype of CD40 and CD80 was analyzed by FACS. Supernatants were tested for secreted cytokines of IFN-a, IFN-γ, IL-12 and TNF-a by ELISA assays. NK and activated DCs were resuspended in X-vivo20 medium and cultured alone or together at 5:1 NK:DC cell ratio for 48 hours. Supernatants were tested for secreted cytokines and the NK cells phenotype of CD62L, NKG2D, NKG2A, NKp44, NKp46, granzyme B and perforin was analyzed by FACS. The cytotoxicity of NK cells against K562 cell line was detected by WST-1 method,and the E:T ratio was 5:1. We used WinMDI 2.9 to analyze the phenotype and two-tailed paired Student’s t-tests to determine the statistical significance.

1. The purity of CD56⁺NK cell populations were >95%.CD11c⁺ and CD123⁺DCs populations were over >95% too.

2.DCs maturation led to the up-regulation of CD40 /CD80 from the original less than 10% up to almost 75%, as well as produce more INF-a(292±74pg/ml vs. 56±12pg/ml) and IL-12(168±54pg/ml vs. 62±27pg/ml) than control.

3.After 48 hours co-culture with mature induced DCs, the expressing of CD62L, NKG2A, NKp44 on CD56⁺NK cells were decreased significantly(P<0.05). However, the expressing of NKG2D, granzyme B and perforin were increased (P<0.05).The IFN-γ and TNF-a production in the supernatant were significantly enhanced (P<0.01). NK cell cytotoxicity against K562 cell line was enhanced (71.8±8.9% vs. 94.6±7.2%, P<0.01).

The results showed DCs maturation led to the up-regulation of co-stimulatory molecules, as well as to the release of IFN-a and IL-12 cytokines. Those observations suggest that DCs from umbilical cord blood enhanced autologous NK cells cytotoxicity through the alter of surface stimulatory or inhibitory receptors and perforin/granzyme B molecules on NK cells and release of cytokines. This vitro research may be provided help for the early cell therapy to reduce the opportunistic infections and leukemia relapse risk after UCBT.

Correspondence: Professor Z-M Sun, Department of Hematology, Anhui Provincial Hospital, Anhui Medical University, 17 Lujiang Road, Hefei 230001, Anhui, China. E-mail: zmsun_vip@163.com

No relevant conflicts of interest to declare.