The generation of abundant inducible pluripotent stem cell derived immunoregulatory dendritic cells to promote antigen-specific immune regulation 2255471

Abstract

Dendritic cells are a rare lineage, consisting of cDC1, cDC2, and the scarce (< 0.3% of blood) DC3. Uniquely, DC3s share monocyte and cDC2 features, marked by co-expression of CD1c and CD14. They promote Th1 and Th17 cells, associate with autoimmunity, and induce CD8+CD103+ tissue-resident memory T-cells that may exacerbate disease. Contrarily, DC3s can be immunosuppressive in a tumor microenvironment. This duality could provide an immunotherapy platform if sufficient-numbers can be obtained. HSPCs were generated from iPSCs via embryoid bodies; HSPCs were cultured for 2 weeks on murine MS5 stromal cells with SCF, FLT3L, and ascorbic acid. To induce regulatory iDC3s (iDC3regs), CD1c+ isolated cells were treated for 24hrs with CHIR99021 (Wnt agonist), L-kyneurine, retinoic acid and IFNγ, IL-10, and/or TGFβ (γ10β), the latter tested alone and in combination. Suppressive function was measured via AlloMLRs. iDC3s emerged with a fold-increase of 1096±123 iDC3s per iPSC in 4 maturation states: CD45RA+CD14- (immature), CD45RA-CD14-, CD45RA-CD14+ CD163-, and CD14+CD163 + (mature). Isolated CD1c+ iDC3s cultured with GM-CSF, known to support DC3s, not FLT3L, gradually promoted maturation to a CD14+CD163+ over 3 days. γ10β-iDC3regs were most suppressive, mediated through PDL1 and IDO1. Interestingly, γ10β rapidly induced two states: CD14+CD163- and CD14+CD163+ that resisted inflammatory exposure (TNF+/IL1β/PGE2, LPS, CpG, R848, or GMCSF/IL4). Bulk RNA-seq of these populations is underway to define the regulatory profile and to compare public DC3 datasets. Studies in humanized mouse graft-versus-host disease models will test iDC3regs in vivo regulatory capacity; Studies examining tumor antigen-loaded and activated iDC3s will test their anti-tumor potential. We demonstrate efficient generation of iDC3s, define their developmental trajectory, and regulatory potential. These findings reveal how DC3s operate across disease contexts. Kidz1stFund, P01CA065493-28 Transplantation Immunology (TRAN)