Genetic regulation on ex vivo differentiated natural killer cells from human umbilical cord blood CD34⁺ cells

Abstract

Natural killer (NK)-cells are a lymphocyte population playing a critical role in the immune surveillance against tumors and virally infected cells. The development of human hematopoietic stem cells (hHSC) into fully differentiated NK-cells pass through discrete stages of differentiation involving a variety of factors such as cytokines, membrane factors, and transcription factors (TFs). Because there is lack of studies in this field, we decided to perform an extended analysis of TFs during in vitro differentiation of NK-cells. At several points of differentiation, cells were characterized by their mRNA expression either for NK-cell cell markers, for a number of mature NK-cell receptors or a large panel of TFs. Our data suggests that some TFs (ID2, EGR-2 and T-BET) play a role in NK-cell commitment, differentiation and maturation. Although delayed on its expression, BLIMP1 also seems to be involved in differentiation and maturation of NK cells, but not in NK-cell commitment. E4BP4 and TOX are more related with initial stages of NK-cell commitment. PU.1, MEF, Ikaros, EGR-1, BCL11B and IRF-2 revealed less involvement in maturation and were more associated with NK-cell commitment and pNK cell production. GATA-3 showed a differential role during the ontogeny of NK-cells. We show that assessment of the transcripts present in the differentiating NK-cells demonstrated, a pattern of preserved and differential gene expression remarkably similar to that seen in mice except for E4BP4 that showed constant downregulation throughout the culture period. A thorough understanding of NK-cell developmental mechanisms is important as it may enable future therapeutic manipulation.

Keywords::

• NK cell ontogeny
• NK cell receptors
• transcription factors

Acknowledgements

We thank the Gynecology and Obstetrics staff of Júlio Dinis Maternity, Porto, Portugal, for human umbilical cord blood collection, the North Regional Division of the Portuguese Institute of Blood, for supplying blood collection bags and the Department of Radiotherapy, St John Hospital, Porto, Portugal, for technical support in radiation experiments. We thank Susana Fernandes (PhD) for the invaluable input and critical review of this manuscript. Maria João Pinho (B.Sc.) conceived and designed the study, performed the research, collected, analyzed and interpreted data, and wrote the first and the final versions of the manuscript; Cristina Joana Marques (PhD) designed the study, supervised the technical work, interpreted and analyzed the data, and reviewed the first and final versions of manuscript; Filipa Carvalho supervised all technical work and reviewed the final version of manuscript, Michael Punzel conceived the study and reviewed the final version of manuscript, Mário Sousa reviewed the final version of manuscript and Alberto Barros supervised all technical work and reviewed the manuscript. Here I state that the material contained in the manuscript has not been published, has not been submitted, or is not being submitted elsewhere for publication. On submission, all authors agreed with the contents and their order of appearance and disclose any potential conflicts of interest, whether of a financial or other nature, with manufacturers of pharmaceuticals, laboratory supplies, and/or medical devices, any financial arrangement with a company whose product is prominent in the submitted manuscript or with a company making a competing product, and any commercial affiliations. We chose to publish this study in Journal of Receptors and Signal Transduction for several reasons: the subject of research is on signal transduction pathways of stem cell differentiation using genetics and molecular biology techniques; it is a characterization of in vitro differentiating Natural Killer cells using CD34+ cells from umbilical cord blood as starting population; the study goes through progressive differentiation steps that are characterized by the appearance of NK cells specific markers and receptors. These differentiation steps are also analyzed concerning Transcription Factors that arise or disappear in each differentiation stage. In summary, this study gives very strong evidence that we can determine whether the switch in the biological properties is accompanied by a change in their gene expression profile. The presented results allows either to facilitate the identification of a variety of necessary components for NK cell development or to provide clues on how NK cell lineage commitment is determined. We sincerely hope that this work might be eligible to be published in Journal of Receptors and Signal Transduction. Thank you in advance for all your attention to us, and do please receive my best compliments.

Declaration of interest

The authors declared no conflict of interest.