The Aryl Hydrocarbon Receptor Antagonist Stemregenin 1 Stimulates Expression of NK Cell Related Transcription Factors, Thereby It Facilitates Generation of Highly Functional NK Cells in Vitro

Introduction

Allogeneic stem cell transplantation (SCT) can be a curative treatment for hematological malignancies. The therapeutic effectiveness is attributed to the graft-versus-tumor (GVT) effect, mediated by alloreactive T cells and natural killer (NK) cells. Although T cells can induce a potent GVT effect, they can also induce graft-versus-host disease (GVHD), causing high morbidity and mortality. Interestingly, after non-myeloablative allogeneic SCT, early NK cell repopulation has been associated with decreased relapse rates, without increasing GVHD incidence, illustrating a possible role for donor NK cell adoptive transfer after allogeneic SCT. However, isolation of sufficient numbers of activated NK cells from donor origin is challenging. Recently, it has been described that antagonizing the aryl hydrocarbon receptor (AhR) using the antagonist StemReginin1 (SR1) promotes expansion of human hematopoietic stem cells. Furthermore, AhR turned out to regulate differentiation of multiple immune cells like dendritic cells, regulatory T cells, γδ T cells, and also NK cells. Therefore, we investigated if SR1 could enhance NK cell generation in a cytokine-based culture system.

Methods

CD34⁺ hematopoietic progenitor cells (HPCs) were isolated using immunomagnetic beads from G-CSF mobilized aphaeresis material. These HPCs were expanded during 14 days in medium containing SCF, Flt3L, TPO, IL-7 and IL-15 and subsequently differentiated into NK cells using IL-15 and IL-2. HPC-NK cell expansions were performed with or without SR1. RNA was collected from the cultures weekly and expression of NK cell related genes was analyzed using qPCR. After 35 days, HPC-NK cells were assessed for their cytolytic functions against acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines and primary AML blasts. In addition, expression levels of typical NK-activating receptors and differentiation markers were analyzed by flow cytometry.

Results

Interestingly, SR1 induced expression of TOX, ID2, EOMES, GATA3 and SH2D1B, which are important factors involved in early and late NK cell differentiation. In addition, SR1 improved the expansion, differentiation, and functionality of the NK cells generated. In the presence of SR1, we were able to expand PB-derived HPCs up to 1000-fold in 5 weeks. The SR1-generated HPC-NK cell products contained 80 – 92% NK cells, which expressed high levels of activating NKG2D and natural cytotoxicity receptors. Furthermore, functional analysis showed marked degranulation and cytokine release upon co-culture with AML and MM cell lines and efficient lysis of primary AML blasts at low NK-target ratios.

Conclusion

Addition of the AHR antagonist SR1 in our culture system induces expression of transcription factors involved in NK cell differentiation and thereby facilitates the generation of high numbers of functional NK cells from G-CSF mobilized CD34⁺ progenitor cells. These NK cells hold great promise for future donor NK cell-mediated therapy after allogeneic SCT.