RORγt-expressing dendritic cells are functionally versatile and evolutionarily conserved antigen-presenting cells

Abstract

Conventional dendritic cells (cDCs) are potent antigen-presenting cells (APCs) that integrate signals from their environment allowing them to direct situation-adapted immunity. Thereby they harbor great potential for being targeted in vaccination, autoimmunity, and cancer. Here, we use fate mapping, functional analyses, and comparative cross-species transcriptomics to show that RORγt⁺ DCs are a conserved, functionally versatile, and transcriptionally distinct type of DCs. RORγt⁺ DCs entail various populations described in different contexts including Janus cells/RORγt-expressing extrathymic Aire-expressing cells (eTACs), subtypes of Thetis cells, RORγt⁺-DC (R-DC) like cells, cDC2C and ACY3⁺ DCs. We show that in response to inflammatory triggers, RORγt⁺ DCs can migrate to lymph nodes and in the spleen can activate naïve CD4⁺ T cells. These findings expand the functional repertoire of RORγt⁺ DCs beyond the known role of eTACs and Thetis cells in inducing T cell tolerance to self-antigens and intestinal microbes in mice. We further show that RORγt⁺ DCs with proinflammatory features accumulate in autoimmune neuroinflammation in mice and men. Thus, our work establishes RORγt⁺ DCs as immune sentinel cells that exhibit a broad functional spectrum ranging from inducing peripheral T cell tolerance to T cell activation depending on signals they integrate from their environment.

Keywords: AIRE; RORγt; antigen presenting cells; dendritic cells; innate lymphocytes.

Fig. 1.

RORγt⁺ DCs exist in the murine spleen across age. (A and B) Splenocytes from Clec9a^CreRosa^Tom or Clec9a^CreRosa^TomRORγt^GFP mice at the indicated ages were analyzed by flow cytometry. (A) Live CD90^-CD127^-CD11c⁺MHCII⁺ cells were gated and RORγt⁺ cells revealed by GFP or anti-RORC intranuclear staining (2-wk time point). GFP⁺ or RORC⁺ cells were further analyzed for CD11b and CD24 expression. (B–D) Expression of the indicated surface markers in cDC1 (gray), cDC2 (red), MHCII⁺ ILC3 (green), and CD11c⁺MHCII⁺RORγt⁺ cells (blue) from spleens of mice at the indicated ages. Gating strategy SI Appendix, Fig. S1A. (E) CD11c⁺MHCII⁺ cells from RORγt^GFP mice were stained using an anti-RORC antibody to demonstrate that anti-RORC staining and RORγt driven GFP are largely congruent. (F) Frequency of RORγt⁺ cells within CD11c⁺MHCII⁺ cells and number of RORγt⁺ DCs in spleens of the indicated ages (1-wk-old n = 10; 2-wk-old, n = 9; 3-wk-old n = 8, 4-wk-old n = 3; 8 to 12-wk-old n = 6; 15-wk-old n = 3). Data are pooled from 1 to 2 independent experiments; each data point represents a biological replicate. (G and H) MHCII⁺ ILC3 and RORγt⁺ DCs were quantified in spleens from 2-wk-old (G) and adult Rag2^−/−γc^−/− mice (H) and littermate controls. Each data point represents an individual mouse from two independent experiments. (I) Quantification of cDC1, cDC2, RORγt⁺ DC, and MHCII⁺ ILC3 in spleens from 1-wk-old Flt3l^−/− and Flt3l^+/− littermate controls. RORγt⁺ DC quantified by intranuclear staining against RORC. Each dot represents one mouse, horizontal bars represent mean, error bars represent SD. **P (0.0021) ***P (0.0002). Statistical analyses in (G–I) were performed using two-tailed Welch’s t test.

Fig. 2.

Integrative transcriptional analyses align RORγt⁺ DCs with Janus cells, RORγt⁺ eTACs and Thetis cells. (A–E) Single-cell multiomic profiling (paired scATAC and scRNA-seq) of RORγt⁺ DCs, RORγt^negCD11c⁺MHCII⁺ cells, and MHCII⁺ ILC3 from spleens of two-week-old (n = 2) or adult (n = 3) RORγt^GFP mice was performed. (A) RNA-based and ATAC-based UMAP of 11,980 nuclei annotated by cell type (see also SI Appendix, Fig. S2). (B) Expression of indicated genes on the RNA-based UMAP. (C) RNA-based UMAP depicting enrichment score for genes that distinguished RORγt⁺ cDC2-like cells in the neonatal spleen (10). (D) Bubble plot of select cell type defining genes. (E) Pearson’s correlation to show the similarity between clusters identified based on RNA and ATAC profiles. (F) Staining of the indicated surface markers on RORγt⁺ DCs (blue), cDC2 (red), and CD24⁺ DCs (gray). (G) Chromatin accessibility at the CNS1 region of Aire locus in the indicated cell types. (H) AIRE expression in RORγt⁺CD11c⁺MHCII⁺ cells revealed by anti-hCD2 staining in the spleen of adult Aire^hCD2 mice. (I) The indicated murine datasets were integrated. UMAP of the integrated datasets colored by dataset and by Leiden clusters. (J) Zoomed-in display of the RORγt⁺ DC cluster from the UMAP in (I) with all cells contributing to the cluster in gray. The contribution of the specified populations from the indicated publications is shown.

Fig. 3.

Comparative transcriptomics reveals RORγt⁺ DCs are evolutionarily conserved. (A) Annotated UMAP of 4,717 cells from scRNA-seq dataset of human splenic DCs. (B) Enrichment scores for the RORγt⁺ DC and ILC3 signatures from the murine multiome dataset were calculated for each cluster. (C) Annotated UMAP of 262 cells from scRNA-seq dataset of human spleen. (D) Enrichment scores for the RORγt⁺ DC signature or ILC3 signature from the murine multiome dataset were calculated for each cluster. (E and F) Annotated UMAP (E) of integrated scRNA-seq datasets generated using Seurat Integration of scRNA-seq datasets pipeline, and colored by dataset (F). (G) Venn diagram showing the overlap of genes distinguishing RORγt⁺ DCs in the indicated mouse and human scRNA-seq datasets. (H and I) Bubble plots of selected genes deduced from comparative gene expression analyses that distinguish RORγt⁺ DCs in various human (H) and murine (I) scRNA-seq datasets. Genes are ordered according to the species and organs they were found in to differentiate RORγt⁺ DCs from other cell types. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical analysis was performed using the Wilcoxon nonparametric ranked sum test.

Fig. 4.

RORγt⁺ DCs exist across lymphoid and nonlymphoid tissues in mice and humans. (A–C) RORγt⁺ DCs were quantified in mLN (A), siLP (B) colon LP (C), lung (D), and skin draining lymph nodes (E) of RORγt^GFP reporter mice (representative gating in SI Appendix, Fig. S7 and S8). (A) Frequency of RORγt⁺ cells within CD11c⁺MHCII⁺ cells (Upper plot) and number of RORγt⁺ cells (Lower plot) in mLN of mice of indicated ages (1 wk n = 5; 2 wk n = 3; 3 wk n = 8, 4 wk n = 4; Adult n = 6). (B–D) Frequency of RORγt⁺ cells within CD11c⁺MHCII⁺CD64^- cells (Top) and number (Bottom) in siLP (1-wk-old n = 4 to 8; 2-wk-old n = 4; 3-wk-old n = 6, 4-wk-old n = 3; Adult n = 6), colon (1-wk-old n = 4; adult n = 4), and lung (1-wk-old n = 11; 2-wk-old n = 4; 3-wk-old n = 7, 4-wk-old n = 6; Adult n = 6) from mice of indicated ages. (E) RORγt⁺ DCs from axial, brachial, cervical, and inguinal skin draining LN were gated as in SI Appendix, Fig. S8D and quantified. The frequency of RORγt⁺ cells within CD11c⁺MHCII⁺ cells (Top) and number of RORγt⁺ DCs (Bottom) is shown.

Fig. 5.

Single-cell multiomic profiling reveals the unique identity of RORγt⁺ DCs. (A) RORγt⁺ DCs and RORγt⁺ eTACs from the multiome analysis in Fig. 2 A–E were reclustered. The resulting UMAPs based on RNA and ATAC profiles are shown and annotated by timepoint. (B) Expression of Rorc, Aire, Itgav, and Itgb8 on the RNA-based UMAP from (A). (C and D) Spleen (C), mLN, siLP and lung (D) from 2-week-old and adult RORγt^CreRosa^YFPRORγt^GFP/wt mice were analyzed by flow cytometry. YFP (RORγt-expression history) versus GFP (active RORγt) expression in CD11c⁺MHCII⁺ cells were plotted and the ratio of YFP⁺ to GFP⁺ cells was calculated. Each dot represents one mouse. (E) UMAP display of dimensionality reduction based on target genes and region enrichment scores generated using SCENIC+. (F) UMAP colored by target gene activity of the indicated eRegulons predicted to regulate specific cell types (SI Appendix, Fig. S9F). (G) Visualization of the gene regulatory network formed by Prdm16, Thrb, and Rorc in RORγt⁺ DCs. Genes that determine the identity of RORγt⁺ DCs are colored in blue.

Fig. 6.

RORγt⁺ DCs are bona fide dendritic cells (DCs). (A–H) 250 cDC2, MHCII⁺ ILC3, and RORγt⁺ DCs from spleens of 2-wk-old (A–D) or adult (E–H) RORγt^GFP mice were pulsed with OVA_323-339 and cocultured with 2500 CTV-labeled naïve OTII cells as indicated. 3.5 d later, proliferation (CTV dilution), cytokine production, and FOXP3 expression in proliferated T cells were quantified. (A) CTV-trace of OTII cells cocultured with cDC2, RORγt⁺ DCs, MHCII⁺ ILC3, or cDC2 without OVA_323-339 (gray) under nonpolarizing conditions (Th0). Right: Quantification of proliferated cells after culture with cDC2, RORγt⁺ DCs, MHCII⁺ ILC3, or cDC2 without OVA_323-339 (open circle) under the indicated conditions. (B–D) Proliferated OTII cells were analyzed for TNF and IFNγ production (B), FOXP3 expression (C), or TNF, IL-17A, and IL-17F production (D). (E–H) OTII T cells cocultured with cDC2, MHCII⁺ ILC3, and RORγt⁺ DCs from adult mice were analyzed as in A–D above. (I) CLEC4A4 expression on the indicated populations. (J–L) Adult RORγt^GFP mice were injected i.p. with anti-CLEC4A4-OVA or isotype-OVA control antibody plus CpG-B. After 12 h 300 cDC2, cDC1, MHCII⁺ILC3, and RORγt⁺ DCs were sorted and cocultured with 3000 naïve CTV-labeled OTII cells. (J) Experimental setup. (K) CTV dilution and (L) number of proliferated OTII cells after coculture with the indicated populations (n ≥ 5). (M) Expression of the indicated markers on cDC1 (gray), cDC2 (red), and RORγt⁺ DCs (blue) 12 h after i.p injection of CpG-B. (N) Frequency and absolute number of cDC1, MHCII⁺ILC3, and RORγt⁺ DCs in mLN of 11-d-old RORγt^GFP reporter mice 24 h after oral administration of R848. Each dot represents one biological replicate pooled from two independent experiments. Horizontal bars represent mean, error bars represent SD. *P (0.0332), **P (0.0021) ***P (0.0002), ****P < 0.0001. Statistical analysis: two-tailed Welch’s t test (B–D, F–H, and L) or one-way ANOVA with Tukey’s multiple comparisons (A and E). Only statistically significant comparisons are indicated.

Fig. 7.

RORγt⁺ DCs in autoimmune neuroinflammation. (A) Bubble plots depicting expression of select genes that distinguished RORγt⁺ DCs from other APCs in at least two of the indicated human CSF datasets. (B and C). Quantification of total leukocytes, frequency and number of MHCII⁺ ILC3, cDC1, and RORγt⁺ DCs in the spinal cord (B) and brain (C) of mice during peak EAE (day 16, n = 5/6) compared to healthy controls (n = 6). Each dot represents one mouse, horizontal bars represent mean, error bars represent SD. *P (0.0332), **P (0.0021) ***P (0.0002), ****P < 0.0001. Statistical analysis was performed using two-tailed Welch’s t test comparing cells from healthy mice vs EAE mice for each cell type. Only statistically significant comparisons are indicated.