Generation of natural killer cells from hematopoietic stem cells in vitro for immunotherapy

Abstract

Natural killer (NK) cells are part of the innate immune system and are an alluring option for immunotherapy due to their ability to kill infected cells or cancer cells without prior sensitization. Throughout the past 20 years, different groups have been able to reproduce NK cell development in vitro, and NK cell ontogeny studies have provided the basis for the establishment of protocols to produce NK cells in vitro for immunotherapy. Here, we briefly discuss NK cell development and NK cell immunotherapy approaches. We review the factors needed for NK cell differentiation in vitro, which stem cell sources have been used, published protocols, challenges and future directions for Good Manufacturing Practice protocols.

Figure 1

NK cell immunotherapy. NK cells can be manipulated in order to augment their number and killing capacity. On the left-hand panel, NK cells can be modulated via antibodies or interleukin administration. NK cells are then activated and eliminate infected or cancer cells. The middle panel depicts NK cell adoptive therapy. NK cells can be isolated from healthy donors and undergo expansion or activation in vitro and be subsequently infused into the recipient. Similarly, NK cells can be produced from stem cells in vitro. The options to produce NK cells in vitro include cord blood, bone marrow, mobilized peripheral blood and embryonic stem cells. The right-hand panel shows the gene modification of NK cells. Target genes can be transfected into NK cells in order to re-direct NK cell specificity. Likewise, genes that boost NK cell cytotoxicity can also be transfected. NK, natural killer.

Figure 2

NK cell production in vitro. NK cells can be generated from stem cells. Isolated stem cells are plated over an irradiated feeder layer that creates the microenvironment for NK cell development. The addition of different interleukins (like IL-7, IL-15, SCF, FLT3L among others) drives the differentiation towards the NK cell lineage. Cells undergo different phases; the first stage (pro-NK) is characterized by the expression of important transcription factors like ID2, ID3 an E4BP4. In the second stage, SCF and FLT3L promote the expression of the IL-2/15β receptor, which increases the responsiveness to IL-15. The decreased expression of IL-7Rα and FLT3L are characteristic of the pre-NK stage. Afterwards, the expression of CD161, CD2 and 2B4 reflects the iNK stage. The coordinated acquisition of different activating and inhibitory markers is followed by the increased cytotoxicity capacity that can be enhanced by the addition of IL-12 or IL-2. FLT3L, FLT3 ligand; iNK, immature natural killer; NK, natural killer; SCF, stem cell factor.