Reconciling the spectrum of RORγt⁺antigen-presenting cells

Abstract

Three recent contemporaneously published papers1–3showed that antigen-presenting cells (APC) expressing the nuclear receptor RORγt instruct peripheral regulatory T (pTreg) cell differentiation and establish tolerance to the gut microbiota. These studies identified a spectrum of RORγt+APCs, distinct from dendritic cells, that included type 3 innate lymphoid cells (ILC3s) as well as novel APC types. Given the discordant conclusions as to the nature of the pTreg-inducing APC and the divergent nomenclature used in these three studies, there is a clear need to reconcile these analyses and the identity of the cell types described. Our reanalysis of the single cell RNA-seq data from these studies revealed the presence of four distinct subsets of non-ILC3 RORγt+APCs, present in all three datasets reported, and confirmed expression of Itgb8, the critical factor for RORγt+APC mediated pTreg induction, in cells synonymous with the non-Aire expressing Thetis cell subset, TC IV.

Cross-species analyses reveal RORγt-expressing dendritic cells are a lineage of antigen presenting cells conserved across tissues

“…Of note, TCII showed highest expression of RORψt + DC signature genes, while TCIV showed lowest expression of these genes (Fig 6a). In line with previous analyses ( 34 , 40 ), TCIII and TCIV showed high expression of the eTAC gene Tnrfsf11a , although Aire expression was only observed in TCI and TCII (Fig. S9c).…”

“…Thetis cells have been described in neonatal mLN and strongly resemble Janus cells although they are apparently absent from skin draining LN ( 34 ). Despite their transcriptional similarities to Janus cells, only a fraction of Thetis cells (TCI and TC III) express AIRE, while TCIV expresses ITGβ8 but lacks AIRE ( 34 ) and it is unclear if Thetis cells even exist in adult mice ( 40 ). While Janus and Thetis cells in mice have been shown to drive T cell tolerance, R-DC-like cells that resemble Thetis cells have been described in human tonsil, where they can activate T cells ( 41 ).…”

Early life imprinting of intestinal immune tolerance and tissue homeostasis

“…Given that TCs were enriched in fetal vs adult lymph node samples in the Human Gut Atlas, 14 RORγt + APCs, may in part be due to studies utilizing the Aire-GFP BAC transgenic ('Adig') mouse 39 to identify and characterize RORγt + Aire + cells. We and others have shown that Aire-GFP expression is not synonymous with Aire protein expression, 37,47 suggesting over-reporting of peripheral Aire expression. Indeed Aire-GFP expression was found in ~50% of ILC3s which do not express Aire protein and are not considered to be part of the spectrum of RORγt + eTACs.…”

“…Our post-hoc analysis of three published datasets alongside our own data resolved the full spectrum of RORγt + APCs, demonstrating transcriptional overlap between all cell types identified across the four studies. 37 In each dataset, RORγt + APCs encompassed ILC3s, distinguished by the expression of canonical ILC3 markers RORα and CXCR6, as well as non-ILC3 cells that mapped to the 4 Thetis cell subsets. Aire-expressing JC1 33 and RORγt + eTAC I 32 were found to be synonymous with Aire + TC I whereas JC2 33 and RORγt + eTAC II 32 encompassed a heterogenous mix of cells spanning TC II, III, and IV cells.…”

“…Indeed Aire-GFP expression was found in ~50% of ILC3s which do not express Aire protein and are not considered to be part of the spectrum of RORγt + eTACs. 37 F I G U R E 1 Phenotype and function of RORγt + APCs. ILC3s that express MHCII are also referred to as LTi or LTi-like cells.…”