Ex Vivo Generation Of Functional Plasmacytoid and Myeloid Dendritic Cells Is Strongly Promoted By The Aryl Hydrocarbon Receptor Antagonist Stemregenin 1
Authors: Thordardottir Soley , Basav N. Hangalapura , Hutten Tim , Cossu Marta , Spanholtz Jan , Schaap Nicolaas PM , Radstake Timothy , Voort Robbert van der , Dolstra Harry 
Published: October 2019

Abstract
The prominent role of dendritic cells (DCs) in T cell activation is the rational for DC-based immunotherapy of cancer and infectious diseases. In cancer, DC therapy aims to induce tumor-specific effector T cell responses that can reduce or eliminate the tumor, and to develop immunological memory to control tumor relapse. So far, the vast majority of DC vaccination studies have been performed with DCs differentiated from monocytes (Mo-DCs) that are loaded with tumor-associated antigens (TAAs) or minor histocompatibility antigens (MiHA). This strategy has been reported to induce the expansion of antigen-specific CD4+ and/or CD8+ T cells in the majority of patients, however only a fraction of the patients develop clinical responses. Strategies to improve the potency of DC-based vaccines are to increase the stimulatory and migratory capacity of Mo-DCs, or to use alternative DC subtypes, such as naturally circulating plasmacytoid DCs (pDCs), BDCA1+ myeloid DCs (mDCs) or BDCA3+ mDCs. These DC subsets are potent inducers of antigen-specific T cell responses, and are therefore attractive cells to exploit for DC-based therapy. However, since their frequency in blood is very low, it is a challenge to obtain high enough numbers for immunotherapy. It would be advantageous if DCs, which are phenotypically and functionally similar to blood pDCs and mDCs, could be generated from CD34+ hematopoietic progenitor cells (HPCs). Interestingly, recent findings have indicated that the aryl hydrocarbon receptor (AhR) not only regulates toxic effects of environmental contaminants, but also plays a role in modulating hematopoiesis and the immune system. For instance, it has been reported that StemRegenin 1 (SR1), a small molecule inhibitor of AhR, promotes the ex vivo expansion of human CD34+ HPCs that are able to effectively engraft immunodeficient mice. Furthermore, differentiation of Langerhans cells and monocytes in vitro from HPCs can be inhibited by the addition of the AhR agonist VAF347. In light of these data, we investigated if we could generate DC subsets from CD34+ HPCs by supplementing SR1. Therefore, we cultured CD34+ HPCs in medium containing SCF, Flt3L, IL-6, TPO supplemented with 1 μM SR1 or DMSO as control. Interestingly, addition of SR1 explicitly promoted the emergence of pDCs (CD11c-HLA-DR+CD123hiBDCA2+BDCA4+ cells), BDCA1+ mDCs (Lin1-HLA-DR+BDCA1+BDCA3- cells) and BDCA3+ mDCs (Lin1-HLA-DR+BDCA1-BDCA3+ cells). After three weeks of culture, the frequency of these DC subsets was significantly higher in cultures with SR1 compared to control conditions; 2.9% vs. 0.04% for pDCs, 4.6% vs. 0.5% for BDCA1+ mDCs and 1.1% vs. 0.1% for BDCA3+ mDCs (n=3-5 donors). The average yield after three weeks of culture with SR1 starting from 105 CD34+ UCB cells was 3.8x106 pDCs, 5.3x106 BDCA1+ mDCs and 1.2x106 BDCA3+ mDCs (n=3-5 donors). Furthermore, SR1 also promoted the differentiation of DC subsets from CD34+ cells obtained from peripheral blood of G-CSF-mobilized donors. The average frequency of DCs in these SR1-cultures was 4.7%, 3.8% and 0.9% for pDCs, BDCA1+ and BDCA3+ mDCs, respectively (n=3 donors), which is comparable to the frequency obtained from UCB CD34+ cells. But the expansion potential of G-CSF-mobilized blood CD34+ HPCs was lower than that of UCB CD34+ cells, resulting in average DC yields of 0.6x106, 0.5x106 and 0.1x106 from 105 CD34+ cells (n=3). Flow cytometry analysis demonstrated that the SR1-induced pDCs and mDCs are phenotypically comparable to their naturally occurring counterpart in blood. Furthermore, the ex vivo-generated pDCs potently responded to stimulation with TLR7 and TLR9 ligands by secreting high amounts of IFN-α and upregulating CD83, CD80, CD86 and CCR7. The HPC-mDC subsets also upregulate CD80 and CD83 upon TLR3, TLR4 or TLR7/8 ligation. Finally, both the ex vivo-generated pDCs and mDCs induced potent allogeneic T cell responses and activated CD8+ effector T cells against hematopoietic-restricted MiHA. These findings demonstrate that our SR1 culture system not only allows detailed study of DC differentiation and molecular regulations in vitro, but it also offers the opportunity to evaluate the in vivo efficacy of cultured DC subsets upon vaccination into patients with cancer and viral infections.

Disclosures:
Spanholtz: Glycostem Therapeutics: Employment.