“What’s in a name?” Clarifying the identity of RORγt⁺ antigen-presenting cells

A study by Sun et al. (2025) in this issue of JEM adds new insights into the rapidly emerging field of tolerogenic APCs, a journey the Gardner group began in 2008 (Gardner et al., 2008, 2013; Wang et al., 2021). A seemingly separate thread arose in 2013 with a report that ILC3s could mediate tolerance to commensal organisms (Hepworth et al., 2013, 2015). More recently, various threads were merged when three copublished studies concluded that APCs expressing retinoic acid receptor–related orphan receptor-γt (RORγt) are indispensable for peripheral regulatory T (pTreg) cell differentiation against commensal antigens (Akagbosu et al., 2022; Kedmi et al., 2022; Lyu et al., 2022). Specifically, Rorc-cre–driven deletion of MHC class II (MHCII), chemokine receptor CCR7, or integrin αvβ8 resulted in loss of RORγt⁺ pTregs. These studies primarily focused on Rorc-expressing cells to identify these APCs (Akagbosu et al., 2022; Lyu et al., 2022). However, it is crucial to recognize that Rorc-cre targets not only cells currently expressing Rorc, but also Rorc-negative cells with past Rorc expression. As such, these strategies may have overlooked key APC populations.

To address this limitation, Sun et al. took an unbiased approach and profiled Rorc-cre–traced APCs by single-cell sequencing. Their analysis revealed three principal populations: ILC3s, DCs, and eTACs (Sun et al., 2025). Notably, this approach captured a substantial population of DCs that do not express Rorc at the time of profiling, consistent with a recent report that DCs with Rorc expression history outnumbered those actively expressing Rorc(γt)-GFP reporter (Narasimhan et al., 2025). Functional studies by Sun et al. demonstrated that MHCII presentation by Rorc-cre–targeted cells is required for pTreg induction in response to food antigens. This requirement was not attributed to ILC3s, as MHCII^ΔRORγt + Il7r^−/− mixed bone marrow chimeras (BMCs) lacking MHCII⁺ ILC3s displayed intact pTreg differentiation. In contrast, diphtheria toxin–treated MHCII^ΔRORγt + AireDTR mixed BMCs, which lack MHCII⁺ eTACs, showed partial impairment of pTreg differentiation (Sun et al., 2025). While the requirement for the AIRE protein is still to be determined, the current study by Sun et al. suggests that eTACs are a key mediator of oral tolerance.

Collectively, these findings affirm that antigen presentation by some Rorc-cre–targeted APCs is necessary for pTreg differentiation in the contexts of commensal (Akagbosu et al., 2022; Kedmi et al., 2022; Lyu et al., 2022; Fu et al., 2025), and dietary and oral tolerance (Parisotto et al., 2024, Preprint; Fu et al., 2025; Rodrigues et al., 2025; Sun et al., 2025). Results from MHCII-ON^RORγt mice suggest that these APCs are not only necessary but also sufficient for pTreg induction in response to microbial antigens (Kedmi et al., 2022; Lyu et al., 2022). In addition to MHCII, other surface proteins such as CCR7 and αvβ8 are also required. One study showed that CCR7 on Rorc-cre–targeted cells is necessary for pTreg induction to a commensal organism (Kedmi et al., 2022). However, migration of CCR7^ΔRORγt Janus cells (JCs) to mesenteric lymph nodes appeared normal (Kedmi et al., 2022). With improved knowledge about these APCs, it will be interesting to revisit whether CCR7 deficiency impairs the migration of other Rorc-cre–traced APCs. αvβ8 is known to activate transforming growth factor β (TGF-β), which is essential for pTreg differentiation (Travis et al., 2007). Multiple studies showed that the ablation of αvβ8 by Rorc-cre abrogated pTreg differentiation in vivo (Akagbosu et al., 2022; Parisotto et al., 2024, Preprint). Itgax-cre (also known as CD11c-cre)–mediated ablation of MHCII, CCR7, or αvβ8 resulted in similar phenotypes, suggesting that the same APCs are likely targeted by both Rorc-cre and Itgax-cre (Travis et al., 2007; Kedmi et al., 2022). Notably, pTreg differentiation was abolished in MHCII^ΔCD11c + Itgav^ΔRORγt mixed BMCs, suggesting the requirement of both antigen presentation and TGF-β activation by the same APC (Kedmi et al., 2022). Together, these findings suggest that pTreg-inducing APCs express MHCII, CCR7, and αvβ8, and are traced by Rorc-cre and Itgax-cre.

Despite uncertain nomenclature, a few transcription factors (TFs) can help define pTreg-inducing APCs. Two recent publications demonstrated that RORγt expression, mediated by an enhancer located 7 kb downstream of the Rorγt transcription start site, is required for pTreg induction in a T cell–extrinsic manner (Fu et al., 2025; Rodrigues et al., 2025). Mice lacking this +7-kb enhancer (Δ+7 kb) exhibit reduced frequencies of RORγt⁺ APCs, which coexpress PRDM16 (Fu et al., 2025). This population was completely absent in RORγt^ΔCD11c mice. Moreover, Prdm16^ΔRORγt mice also failed to generate pTregs, phenocopying Δ+7-kb mice (Fu et al., 2025). These findings identify RORγt⁺ PRDM16⁺ APCs as likely candidates for pTreg-inducing APCs. Similar cells have been identified in human secondary lymphoid organs (SLOs) (Ulezko Antonova et al., 2023; Fu et al., 2025). Mouse and human RORγt⁺ PRDM16⁺ APCs exhibit transcriptomic and epigenomic similarities to conventional DCs (cDCs), and express Zbtb46 (Ulezko Antonova et al., 2023; Fu et al., 2025; Narasimhan et al., 2025; Rodrigues et al., 2025), a signature TF gene for cDCs. A subset of these cells expresses Aire and shows accessible chromatin at the conserved noncoding sequence 1 in the Aire locus (Narasimhan et al., 2025). In summary, these data support RORγt⁺ PRDM16⁺Zbtb46⁺ cells as the most likely pTreg-inducing APCs, which are developmentally dependent on PRDM16, RORγt, and the Rorγt +7-kb enhancer.

Diverse nomenclature has been used to describe eTACs and DCs that express RORγt. Previous descriptions of RORγt⁺ eTACs encompass JCs and Thetis cell (TC) subgroups I-III (TC I-III), as reviewed recently (Abramson et al., 2024). RORγt⁺ DCs include TC IV and human RORγt⁺ DC-like cells (Ulezko Antonova et al., 2023; Abramson et al., 2024). Like cDCs and plasmacytoid DCs, RORγt⁺ DCs also depend on the cytokine FLT3L (Narasimhan et al., 2025). The Littman group recently designated the Prdm16-dependent RORγt⁺ PRDM16⁺ APCs as tolerizing DCs without explicitly discussing their relationship to eTACs, although this population does express Aire at high levels and could fall within the eTAC category (Fu et al., 2025). Notably, Sun et al. suggested that tolerogenic DCs and RORγt⁺ eTACs likely represent the same population (Sun et al., 2025).

At present, eTACs are defined by phenotype, but their ontogeny is unknown. eTACs are recognized as AIRE⁺ hematopoietic APCs located in SLOs (Abramson et al., 2024). eTACs and DCs share multiple characteristics, including transcriptomic and chromatin accessibility similarity (Wang et al., 2021; Narasimhan et al., 2025; Rodrigues et al., 2025). eTACs express Zbtb46 and are labeled by Zbtb46-cre (Sun et al., 2025). Some eTACs are traced by Itgax-cre (Kedmi et al., 2022; Lyu et al., 2022). Additionally, flow cytometry shows that 50% of Aire⁺ cells reside within the RORγt⁺ DC gate (Narasimhan et al., 2025). Sun et al. demonstrate that both RORγt⁺ eTACs and cDCs can present soluble antigens to CD4 T cells and induce FOXP3⁺ Treg differentiation in vitro. Given these similarities, eTACs may represent a transcriptional state of cDCs, although a distinct developmental lineage remains possible.

Several questions and opportunities are open in the field of tolerogenic APCs. First, the molecular circuit instructing the development of RORγt⁺ PRDM16⁺ APCs requires elucidation. How are Rorγt and Prdm16 induced? And in which lineage and at what stage? Are these programs developmentally encoded or driven by environmental signals? TF motif analysis of the Rorγt +7-kb enhancer may provide insight. Interestingly, Irf8^fl/flZbtb46-cre mice fail to support pTreg differentiation (Esterházy et al., 2016). A subsequent study showed that Irf8 +32 5’^−/− mice, which lack cDC1s, maintain intact pTreg generation (Russler-Germain et al., 2021). This suggests that IRF8-dependent, Zbtb46-cre–targeted APCs other than cDC1s may be responsible for pTreg induction. It will be interesting to determine whether RORγt⁺ PRDM16⁺ APCs rely on IRF8 for their development or function. Second, beyond antigen presentation and αvβ8-mediated TGF-β activation, what other mechanisms endow this rare population of APCs with their robust tolerogenic capacity? Addressing this question may help harness these cells for therapeutic purposes. Third, do these cells function more broadly in tolerance beyond pTreg induction? For example, earlier studies on RORγt⁺ APCs suggested that ILC3s can facilitate tolerance via negative selection and IL-2 sequestration (Hepworth et al., 2013, 2015). These and other aspects of tolerance warrant revisiting. Finally, RORγt⁺ PRDM16⁺ APCs have been identified in various organs such as the spleen, lung, and tonsil (Ulezko Antonova et al., 2023; Narasimhan et al., 2025). Their functions in these settings remain to be explored.