Erythropoietin receptor on cDC1s dictates immune tolerance

Abstract

Type 1 conventional dendritic cells (cDC1s) are unique in their efferocytosis¹ and cross-presenting abilities², resulting in antigen-specific T cell immunity³ or tolerance^4-8. However, the mechanisms that underlie cDC1 tolerogenic function remain largely unknown. Here we show that the erythropoietin receptor (EPOR) acts as a critical switch that determines the tolerogenic function of cDC1s and the threshold of antigen-specific T cell responses. In total lymphoid irradiation-induced allograft tolerance^9,10, cDC1s upregulate EPOR expression, and conditional knockout of EPOR in cDC1s diminishes antigen-specific induction and expansion of FOXP3⁺ regulatory T (T_reg) cells, resulting in allograft rejection. Mechanistically, EPOR promotes efferocytosis-induced tolerogenic maturation^7,11 of splenic cDC1s towards late-stage CCR7⁺ cDC1s characterized by increased expression of the integrin β8 gene¹² (Itgb8), and conditional knockout of Itgb8 in cDC1s impairs tolerance induced by total lymphoid irradiation plus anti-thymocyte serum. Migratory cDC1s in peripheral lymph nodes preferentially express EPOR, and their FOXP3⁺ T_reg cell-inducing capacity is enhanced by erythropoietin. Reciprocally, loss of EPOR enables immunogenic maturation of peripheral lymph node migratory and splenic CCR7⁺ cDC1s by upregulating genes involved in MHC class II- and class I-mediated antigen presentation, cross-presentation and costimulation. EPOR deficiency in cDC1s reduces tumour growth by enhancing anti-tumour T cell immunity, particularly increasing the generation of precursor exhausted tumour antigen-specific CD8⁺ T cells¹³ in tumour-draining lymph nodes and supporting their maintenance within tumours, while concurrently reducing intratumoural T_reg cells. Targeting EPOR on cDC1s to induce or inhibit T cell immune tolerance could have potential for treating a variety of diseases.

Extended Data Fig. 1 |. XCR1⁺CD8α⁺ cDC1s in the spleen following TLI/ATS are bona fide cDC1s.

a, Total splenocyte frequency per spleen (UNT n=5; TLI/ATS n=10). b, Gating of CD11c^highMHCII^high cDCs from the splenocytes. Summary graph of the frequency of cDCs in total splenocytes (UNT, n=6; TLI/ATS, n=7). c, Gating of CD8α⁺CD11b⁻ cDC1s and CD8α⁻CD11b⁺ cDC2s; frequencies in cDCs (UNT, n=6; TLI/ATS, n=6). d, XCR1⁺ and e, Ki67⁺ cDC1 frequencies (UNT, n=5; TLI/ATS, n=5). f, qPCR of Batf3 and Irf8 in XCR1⁺CD8α⁺ cDC1s (n=5/group). g, IRF8 and h, Zbtb46-GFP expression (UNT, n=6; TLI/ATS, n=5). i-k, MFI of MafB-mCherry expression and % of MafB-mCherry⁺ cells in XCR1⁺CD8α⁺ cDC1s (i), cDC2s (j) and red pulp macrophages (RPMΦs) (k). Day 0 (UNT), n = 4; TLI/ATS Day 5, n = 5; TLI/ATS Day 9, n = 5 (i,j,k). Data are shown from one experiment, representative of at least two independent experiments with similar results (a-k). Statistical analysis was performed using unpaired two-tailed Student’s t-test (a,b,c,d,e,h), or one-way ANOVA with Tukey’s multiple-comparison test (f,g,i,j,k). Data are mean ± s.e.m. (a-k).

Extended Data Fig. 2 |. TLI/ATS leads to widespread apoptosis and extramedullary erythropoiesis in the spleen and a marked rise in serum EPO.

a, TUNEL staining of spleen sections, UNT vs. TLI/ATS (scale bar = 200 μm). b, Spleen cell composition after ATS, TLI, or TLI/ATS; pie chart shows mean frequencies of indicated populations (n = 3). T cells (TCRβ⁺ CD19⁻NK1.1⁻), B cells (CD19⁺TCRβ⁻NK1.1⁻), erythroid progenitors (CD11c⁻TER119⁺CD71⁺), cDCs (CD3ε⁻B220⁻SiglecH⁻PDCA-1⁻CD11c^highMHCII^high), other myeloid cells are subdivided into CD11b⁺Ly6C ⁻Ly6G⁺, CD11b⁺Ly6G ⁻Ly6C⁺ and CD11b^intF4/80⁺. c, Serum EPO levels over time after TLI/ATS (ELISA, n = 8). d, CD71⁺TER119⁺ erythroid progenitors in spleen (day 6) (upper) and in spleen/BM (lower) over time after TLI/ATS (n = 3). e, Co-detection by indexing (CODEX) imaging of WT C57BL/6 spleen (UNT vs. 1 day after TLI; scale bar = 500 μm). f, Scheme of EPO treatment in Epor-tdTomato-Cre mice (i.p. × 5 days). g,h, Flow cytometry showing splenic cDC1 frequency among cDCs (g) and Epor-tdT⁺ cDC1 frequency/MFI (h) (+PBS, n = 5; +EPO, n = 5). i-j, Frequencies of Epor-tdT⁺ and Epor-tdT ⁻ TER119⁺ erythroid cells (i) and Epor-tdT⁺CD11b^intF4/80⁺Epor-tdT⁺ RPMΦs (j), (+PBS, n = 5; +EPO, n = 5). k, CCR7 vs. Epor-tdT expression in XCR1⁺CD8α⁺ cDC1s that were gated as live-dead aqua⁻CD3ε⁻CD19⁻B220⁻ SiglecH⁻PDCA-1⁻CD11c^highMHCII^high; histogram overlay for CCR7⁺ cDC1s (UNT, n = 5; TLI/ATS, n = 5). Data are shown from one experiment, representative of at least two independent experiments with similar results (a,b,c,d,g,h,i,j,k) or from one experiment (e). Statistical analysis was performed using unpaired two-tailed Student’s t-test (g,h,i,j,k). Data are mean ± s.e.m. (d,g-k). The diagram in f was created with BioRender by Xiangyue Zhang (2025).

Extended Data Fig. 3 |. EPO-EpoR downstream signaling is activated in cDC1s following TLI/ATS.

a-c, Gene Set Enrichment Analysis (GSEA) of transcriptional profiles using the Hallmark gene set of MSigDB. NES, normalized enrichment score; FDR, false discovery rate. Red: upregulated; Blue: downregulated. TLI/ATS vs. UNT. b, Upregulated gene sets. c, Downregulated gene sets. d-e, Intracellular phospho-flow cytometric analysis of EPO-EpoR downstream signaling in live-dead blue⁻Lin⁻SiglecH⁻PDCA-1⁻CD11c^highMHCII^high. Spleens were harvested on the next day following the last dose of TLI or TLI/ATS. UNT (n=4) vs. TLI (n=4) vs. TLI/ATS (n=4). d, XCR1⁺CD8α⁺ cDC1s and e, XCR1⁻CD8α⁻ cDC2s. f,g, Histograms and MFI of the indicated EPO-EpoR downstream signaling molecules with fluorescence minus one (FMO) as controls by intracellular phospho-flow staining on the next day following the last dose of TLI/ATS treatment. Epor^flox/flox (n=4) vs. Epor^ΔXcr1 (n=5). cDC1s (f) and cDC2s (g). h, Ex vivo analysis of EPO-EpoR downstream signaling in splenic cDC1s. Splenic cDCs were MACS-purified with a pan-DC isolation kit and cultured at 5 × 10⁶ cells/ml, then rested overnight. Cells were isolated from UNT or TLI/ATS-treated Epor^flox/flox (n=4; n=4) and Epor^ΔXcr1 (n=4; n=4) mice. cDCs from TLI/ATS-treated mice were stimulated ex vivo with EPO (10 IU/200 μl) or PBS (control) overnight. Phosphorylation of downstream signaling molecules was assessed by flow cytometry, after gating on XCR1⁺SIRPα⁻ splenic cDC1s. Data are shown from one experiment, representative of at least two independent experiments with similar results (d-h). Statistical analysis was performed using unpaired two-tailed Student’s t-test (f,g), one-way ANOVA Tukey’s multiple-comparison test (d, e and h left) or paired two-tailed Student’s t-test (h right). Data are mean ± s.e.m. (d-h).

Extended Data Fig. 4 |. FOXP3⁺ T_regs play an indispensable role in TLI/ATS-induced cDC1 EpoR-dependent immune tolerance.

a, Representative pseudocolor plots showing FOXP3⁺ T_reg depletion efficiency in recipient mice on day 6 after DT treatment (DT injections on days 0, 2, and 4). b,c, Representative pseudocolor plots of C57BL/6, Batf3^−/−, or Epor^ΔXcr1 recipient conventional CD4⁺ T cell percentages and FOXP3⁺ T_reg percentages in CD4⁺ T cells. d,e, Absolute cell number of indicated cell populations. b,d, Day 0 (UNT, n=5; n=5; n=5 and TLI/ATS, n=6; n=5; n=4) and c,e, Day 14 of UNT or TLI/ATS-treated groups post allo-BM infusion (UNT, n=11; n=6; n=9 and TLI/ATS, n=3; n=11; n=10). f, MHCII expression on cDC1s and cDC2s from MHCII^flox/flox and MHCII^ΔXcr1 spleens. g,h,i, MHCII^flox/flox (n=6) and MHCII^ΔXcr1 (n=6) recipients were given TLI/ATS and i.v. infused with BALB/c donor BM cells. 14 days post BM infusion, the percentages of donor type (H2K^d+) cells among leukocyte populations were determined in the peripheral blood of hosts. g,i, Recipient MHCI (H-2K^b)⁺TCRβ⁺CD4⁺ T cell frequency among total live cells and FOXP3⁺ frequency among CD4⁺ T cells were analyzed on day 14. j, CD45.2⁺Foxp3^WTEpor^flox/flox (+PBS/without DT, n = 8; +DT, n = 8) or Epor^ΔXcr1 (+PBS/ without DT, n = 8; +DT, n = 8) mice were injected with 30 million CD45.1⁺FOXP3^DTR CD4⁺ T cells isolated by MACS. Two consecutive doses of DT or PBS were given on each of the following 2 days. Subsequently, the mice were treated with TLI/ATS, and 2W1S-BALB/c donor BM cells were infused i.v., and 14 days later, 2W1S-tetramer⁺CD44⁺H-2K^b+TCRβ⁺CD4⁺ T cells from the spleens were analyzed for FOXP3 expression by flow cytometry. FOXP3 expression in CD45.1⁺ or CD45.2⁺2W1S-tetramer⁺ CD4⁺ T cells is shown. One experiment (j) or one of two independent experiments with similar results are shown (a-i). Statistical analysis was performed using unpaired two-tailed Student’s t-test (g,h,i), or two-way ANOVA with Tukey’s multiple-comparison test (d,e,j). Data are mean ± s.e.m. (d,e,g,i,j). The diagram in j was created with BioRender by Xiangyue Zhang (2025).

Extended Data Fig. 5 |. Differentially expressed genes (DEGs) in cDC1s in scRNA-seq analysis and ex vivo TGFβ-dependent Ag-specific FOXP3⁺ T_reg induction by CCR7⁺ cDC1s.

a, UMAP of splenic cDC1 gene expression by sample identity. b, Dot plots of top condition-specific DEGs in Epor^flox/flox and Epor^ΔXcr1 mice (TLI/ATS vs. UNT). c, Absolute cDC1 numbers per spleen in UNT vs. TLI/ATS-treated Epor^flox/flox (n=5/condition) and Epor^ΔXcr1 (n=5/condition) mice. d, UMAPs of cDC1 subtypes in Epor-tdT⁺ and Epor-tdT⁻ cells. e-g, Dot plots of top condition-specific DEGs in Epor-tdT⁺ and Epor-tdT⁻ cDC1s (TLI/ATS) and in Epor^flox/flox and Epor^ΔXcr1 mice (TLI/ATS vs. UNT). Dot color = expression, size = % of indicated gene expressed cells (b,e-g). h, Bar charts showing cDC subtype (d) proportions in Epor-tdT⁺ and EpoR-tdT⁻ cDC1s following TLI/ATS. i, Role of TGFβ in FOXP3⁺ T_reg induction by CCR7⁺ cDC1s: 12 h after apoptotic Act-mOVA injection, CCR7⁺ cDC1s (1×10⁴) were cocultured with CD45.1⁺ CTV-labeled naïve OT-II cells ± anti-TGFβ; FOXP3 expression was analyzed by flow cytometry (n=5/group). j,k, Representative flow cytometry analysis and l,m, Absolute cell number of indicated cell populations of Fig. 3j, Itgb8^ΔXcr1 vs. littermate controls. j,l, Day 0 and k,m, Day 14 of UNT (n=5; n=5) or TLI/ATS-treated (n=5; n=5) groups post allo-BM infusion. n,o, Aldh1a2^ΔCD11c: Batf3^−/− (n=6) vs. Aldh1a2^flox/flox: Batf3^−/− (n=7) BM chimeric recipient mice (CD45.1⁺) were given TLI/ATS. 1 day after the last dose of TLI/ATS, 2W1S-BALB/c donor BM cells were infused i.v., and 14 days later, the percentages of donor type (H2K^d+) cells among leukocyte populations in the peripheral blood of hosts were determined (n) and 2W1S-tetramer⁺CD44⁺H-2K^b+TCRβ⁺CD4⁺ T cells from the spleens were analyzed for FOXP3 expression by flow cytometry and FOXP3⁺ T_regs were counted (o). Data are representative of at least three independent experiments with similar results (c,i) or one experiment (j-o). Statistical analysis was performed using unpaired two-tailed Student’s t-test (c,i,n,o), or two-way ANOVA followed by Tukey’s multiple-comparison test with p-value adjusted (l,m), or propeller test, two-sided, no multiple-comparison correction (b), or Wilcoxon rank sum test, two-sided, Bonferroni correction (h). Data are mean ± s.e.m. (c,i-o). The diagram in i was created with BioRender by Xiangyue Zhang (2025).

Extended data Fig. 6 |. Absence of EpoR on cDC1s gives rise to immunogenic cDC1s that promote both CD8⁺ T cell cross-priming and CD4⁺ T cell priming to cell-associated Ags.

a, MFI of indicated molecules on gated cDC1s with fluorescence minus one (FMO) as controls. b, MFI of indicated molecules on gated cDC2s with fluorescence minus one (FMO) as controls. c, Percentages of cDC2s in splenic cDCs. d, Representative flow gating of CCR7⁺XCR1⁺SIRPα⁻ cDC1s in splenic cDC1s (Upper), and percentages and absolute numbers of CCR7⁺ cDC1s (Lower). a-d, Epor^flox/flox (n=5) vs. Epor^ΔXcr1 (n=5) mice. e, MFI of indicated molecules on CCR7⁺ vs. CCR7⁻ cDC1s. CD40 and PD-L1: Epor^flox/flox (n=5); Epor^ΔXcr1 (n=5). CD80 and CD86: Epor^flox/flox (n=6); Epor^ΔXcr1 (n=6). f, Cross-presentation assay: apoptotic Act-mOVA thymocytes injected into Epor^flox/flox (n=5) or Epor^ΔXcr1 (n=5) mice 1 day after transfer of CTV-labeled naïve CD45.1⁺ naïve OT-I cells; spleens analyzed on day 4 for OT-I expansion and proliferation. g, Same setup with OT-II cells; percentages and absolute numbers of OT-II cells and proliferating OT-II cells were assessed. Ag-specific CD4⁺ T cell response: Ag-specific CD4⁺ T cell immune response following i.v. injection of apoptotic Act-mOVA thymocytes 1 day after i.v. injection of CTV-labeled naïve CD45.1⁺ naïve OT-II cells. Spleens were analyzed at day 4 for OT-II expansion and proliferation. Epor^flox/flox (n=5) and Epor^ΔXcr1 (n=5) mice. Data are shown from one experiment, representative of at least three independent experiments with similar results (a-g). Statistical analysis was performed using unpaired two-tailed Student’s t-test (a,b,c,d,f,g) and two-way ANOVA followed by Tukey’s multiple-comparison test (e). Data are mean ± s.e.m. (a-g). The diagrams in f,g were created with BioRender by Xiangyue Zhang (2025).

Extended data Fig. 7 |. Phenotypes of T cells in the spleens of Epor^flox/flox vs. Epor^ΔXcr1 mice and role of EpoR in cDC1-mediated cell-associated Ag-specific CD4⁺ T cell priming and proliferation and FOXP3⁺ T_reg induction.

a-e, Percentages and absolute numbers of CD4⁺ T cells (a), FOXP3⁺CD25⁺ T_regs in CD4⁺ T cells (b), CD44^highCD62L^low effector cells and CD44^low CD62L^high naïve cells in CD4⁺ T cells (c), CD8⁺ T cells (d), and CD44^highCD62L^low effector cells and CD44^lowCD62L^high naïve cells in CD8⁺ T cells (e) in the spleens of Epor^ΔXcr1 and littermate Epor^flox/flox control mice with representative flow cytometric plots. a-e, Epor^flox/flox, n=5; Epor^ΔXcr1, n=5. f,g, Flow cytometry-based measurement of cell-associated Ag-specific CD4⁺ T cell immune response in the spleen following i.v. injection of apoptotic Act-mOVA thymocytes into mice of the indicated genotypes 1 day after i.v. injection of CTV-labeled naïve CD45.1⁺ OT-II cells. f, WT C57BL/6 (n=5) and Batf3^−/− (n=7). g, FOXP3⁺ T_reg induction in Epor^flox/flox and Epor^ΔXcr1 mice. Recombinant EPO or PBS was administered daily, from Day −3 to Day 4. +PBS: Epor^flox/flox (n = 5), Epor^ΔXcr1 (n = 5); +EPO: Epor^flox/flox (n = 5), Epor^ΔXcr1 (n = 5) mice. Data are shown from one experiment, representative of at least three independent experiments with similar results (a-e), or two independent experiments with similar results (f,g). Statistical analysis was performed using unpaired two-tailed Student’s t-test (a-f), or two-way ANOVA followed by Tukey’s multiple-comparison test (g). Data are mean ± s.e.m. (a-g). The diagrams in f,g were created with BioRender by Xiangyue Zhang (2025).

Extended Data Fig. 8 |. Epor-tdT expression on XCR1⁺ cDC1s in selected organs and tolerogenic phenotype of Epor-tdT⁺ migratory cDC1s in pLNs.

a,b,c CCR7- and Batf3-dependent Epor-tdT expression on migratory cDCs in pLNs. Migratory cDCs were gated as CD11c^intMHCII^high from live-dead aqua⁻Lin⁻SiglecH⁻PDCA-1⁻EpCAM⁻ cells, and resident cDCs were gated as CD11c^highMHCII^int from live-dead aqua⁻Lin⁻SiglecH⁻PDCA-1⁻ cells. pLNs including inguinal, axillary, brachial, and superficial cervical LNs were combined for analysis by flow cytometry (a,b). Ccr7^−/−Epor^tdT/+, Batf3^−/−Epor^tdT/+ and WT C57BL/6 mice (a). Histogram overlay of Epor-tdT expression on migratory or resident cDCs from individual mouse strains (b). EpoR-tdT expression on migratory cDCs from individual pLNs of Epor^tdT/+ mice (c). d, Epor-tdT expression on cDC1s obtained from the indicated organs in Zbtb46^GFP/+Epor^tdT/+ mice. cDCs were gated in CD45⁺ cells as CD11c⁺Zbtb46-GFP⁺, in which cDC1s were further gated as XCR1⁺ CD103⁺. e, Flow cytometric analysis of tolerance associated cell-surface molecules on pLN Epor-tdT⁺ migratory cDC1s compared with Epor-tdT^- cDCs and resident cDCs with FMO serving as controls (n=8). Data are representative of at least two independent experiments with similar results (a-e). Statistical analysis was performed using one-way ANOVA Tukey’s multiple-comparison test (e). Data are mean ± s.e.m. (e). The diagrams in a,e were created with BioRender by Xiangyue Zhang (2025).

Extended Data Fig. 9 |. Induction of Ag-specific CD4⁺FOXP3⁺ T_regs by pLN migratory EpoR-tdT⁺ cDC1s.

a, FACS-sorted pLN Epor-tdT⁺ (n=4) or EpoR-tdT⁻ (n=4) XCR1⁺ migratory cDC1s from Epor-tdT mice were cocultured for 5 days with CTV-labeled naïve OT-II cells + DEC205-OVA (ratio 1:5); FOXP3 expression in OT-II cells was analyzed by flow cytometry. b, Same setup as (a) but with apoptotic Act-mOVA thymocytes (ratio 1:5:2) ± EPO (20 IU per well per day for 5 days); FOXP3 expression in OT-II cells was measured. Epor-tdT⁺ (+PBS or w/o, n=4; +EPO, n=4) or EpoR-tdT⁻ (+PBS or w/o, n=4; +EPO, n=3). c, FACS-sorted pLN Epor-tdT⁺ or EpoR-tdT⁻ migratory cDC1s were cocultured for 12 h with apoptotic CD45.1⁺ thymocytes ± EPO; MFIs of surface markers were analyzed. Epor-tdT⁺: n=2. Epor-tdT⁻: n=2. d, Migratory cDC1s from Epor^flox/flox or Epor^ΔXcr1 mice were cocultured with naïve CTV-labeled OT-II cells and Act-mOVA thymocytes (1:5:2) ± EPO (20 IU per well per day for 5 days); FOXP3 induction was assessed. n=6/group. e, Efferocytosis of PKH67-labeled apoptotic thymocytes by migratory cDC1s and cDC2s in dLNs 12 h post-injection. f, Act-mOVA^{CD45.1/CD45.2} mice were reconstituted with either Epor^flox/flox (n=5) or Epor^ΔXcr1 (n=6) BM cells after lethal irradiation. 8 weeks post-reconstitution, naïve CTV-labeled OT-II cells were i.v. infused (day 0), and EPO was administered on days −2 to 2. FOXP3 induction in OT-II cells was assessed in inguinal LNs on day 9. Data are shown from one experiment, representative of two independent experiments with similar results (a-e) or one (f) independent experiment. Statistical analysis was performed using unpaired two-tailed Student’s t-test (a,b,f), or two-way ANOVA with Tukey’s multiple-comparison test (d). Data are mean ± s.e.m. (a,b,c,d,f). The diagrams in a,b,c,e,f were created with BioRender by Xiangyue Zhang (2025).

Extended Data Fig. 10 |. Phenotypes of T cells in the pLNs of Epor^flox/flox vs. Epor^ΔXcr1 mice.

a-e, Percentages and absolute numbers of CD4⁺ T cells (a), FOXP3⁺CD25⁺ T_regs in CD4⁺ T cells (b), CD44^highCD62L^low effector cells and CD44^lowCD62L^high naïve cells in CD4⁺ T cells (c), CD8⁺ T cells (d), and CD44^highCD62L^low effector cells and CD44^lowCD62L^high naïve cells in CD8⁺ T cells (e) in the pLNs of Epor^ΔXcr1 and littermate Epor^flox/flox control mice with representative flow cytometry plots. a-e, Epor^flox/flox (n=9) and Epor^ΔXcr1 (n=9). Data are shown from one experiment, representative of at least three independent experiments with similar results (a-e). Statistical analysis was performed using unpaired two-tailed Student’s t-test (a-e). Data are mean ± s.e.m. (a-e).

Extended Data Fig. 11 |. EpoR expression on tumor-infiltrating leukocytes (TILs), tumor Ag-carrying migratory cDC1s in tdLNs and tumors, and the correlation of tumor growth with systemic EPO levels.

a, Zbtb46^GFP/+ Epor^tdT/+ mice were implanted s.c. with MC38 or B16F10 tumors, or EO771 tumors in the mammary fat pad. On day 12, tumors were harvested for flow cytometric analysis of Epor-tdT on cDCs (live-dead aqua⁻CD45⁺Zbtb46-GFP⁺CD11c⁺); cDC1s were gated as XCR1⁺ and non-cDC1s as XCR1^-. b-d, Mice implanted s.c. with MC38-OVA^dim or B16F10-OVA; on day 10, tumors were analyzed for Epor-tdT expression in TILs. b, Representative gating strategy of individual live-dead blue^- TIL populations. c,d, Histogram overlay showing Epor-tdT expression in individual cell populations. e, Zbtb46^GFP/+Epor^tdT/+ mice with MC38-OVA^dim tumors (day 12) were analyzed for EpoR-tdT on tumor-infiltrating cDCs; CCR7⁺ (population I) and CCR7⁻ (populations II/III by Ly6A) subsets were gated, with XCR1/CD103 staining to define cDC1s and cDC2s. f, Quantification of Epor-tdT expression on individual tumor infiltrating cDC subsets. MC38-OVA^dim (n=4) and B16F10-OVA (n=4) tumors were harvested on day 12 post-s.c. implantation for flow cytometry. Gating strategy as in Fig. 5a and Extended Data Fig. 11e. g, Flow cytometry analysis of Epor-tdT expression on tdLN migratory cDC1s. Overlay of migratory cDC1s with Lin⁻ live cells to show Epor-tdT expression levels. h, Serum EPO levels were measured by ELISA on the indicated days after s.c. implantation of MC38-OVA^dim (n=6) or B16F10-OVA (n=5) tumors in WT mice. i,j,k, B16F10-OVA-ZsGreen cells were s.c. implanted into Epor^tdT/+ mice, and tdLN and tumor were analyzed on day 9 after inoculation. j, Flow cytometry analysis of Epor-tdT expression on tdLN ZsGreen⁺ migratory and resident XCR1⁺ cDC1s. Overlay of migratory ZsGreen⁺ cDC1s or resident ZsGreen⁺ cDC1s with Lin^-live cells to show Epor-tdT expression levels. k, Flow cytometry analysis of Epor-tdT expression on tumor infiltrating ZsGreen⁺ cDC1s. Data are shown from one experiment, representative of at least two independent experiments with similar results (a-e,f,g,h,j,k). Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple-comparison test (f). Data are mean ± s.e.m. (f,h). The diagrams in a,b,e,g,i were created with BioRender by Xiangyue Zhang (2025).

Extended Data Fig. 12 |. Loss of EpoR in cDC1s limits tumor growth and promotes immunogenic function of tumor Ag-carrying cDC1s in both tdLNs and tumor.

a, Growth of MC38-OVA^dim tumor cells implanted s.c. into Epor^flox/flox (n=8) and Epor^ΔXcr1 mice (n=9). b, Growth of B16F10-OVA tumor cells implanted s.c. into Epor^flox/flox (n=6) and Epor^ΔXcr1 mice (n=7). c, Experimental design for phenotyping tumor-Ag carrying ZsGreen⁺ cDC1s in tdLN and tumors in Epor^flox/flox vs. Epor^ΔXcr1 mice. B16F10-OVA-ZsGreen cells were implanted s.c. into Epor^tdT/+ mice, and tdLNs and tumors were analyzed on day 9 after implantation. d, Flow cytometry analysis of Epor-tdT expression on ZsGreen⁺ migratory XCR1⁺SIRPα⁻ cDC1s in tdLNs with summary graph of statistical quantification. Epor^flox/flox (n=7) and Epor^ΔXcr1 (n=8). e, Flow cytometry analysis of CD40, CD80 and CD86 expression on tumor infiltrating ZsGreen⁺ cDC1s with summary graph of statistical quantification. Epor^flox/flox (n=7) and Epor^ΔXcr1 (n=8) mice. (f-l) MC38-OVA^dim tumors were s.c. implanted into Epor^flox/flox (n=8) and Epor^ΔXcr1 (n=7) and 10 days later TILs were analyzed. f. Percentages of CD45⁺ live immune cells and CD8⁺ or CD4⁺ T cells in CD45⁺ TILs. g, Frequency of OVA_257–264-dextramer⁺CD8⁺ T cells among CD8⁺ T cells. h, Representative flow plots and quantification of CD8⁺ T cells expressing TIM-3 and PD-1. i, Representative flow plots and quantification of TCF1⁺TIM-3⁻CD8⁺ T cells. j, Representative histograms and quantification of perforin, granzyme-B, IFNγ and TNFα expression in tumor-infiltrating CD8⁺ T cells. k, Percentage of FOXP3⁺ T_regs in CD4⁺ T cells with representative flow plots (Left). Absolute number of T_regs (Right). l, Representative flow plots and percentages of T-bet⁺CXCR3⁺ T_regs in CD4⁺ FOXP3⁺ T_regs. (f-l). Data are shown from one experiment, representative of at least two independent experiments with similar results (a,b,d,e,f-l). Statistical analysis was performed using two-way ANOVA with Šídák’s multiple comparison test (a,b), or two-tailed unpaired Student’s t-test (d,e,f,g,i,j.k,l), or two-way ANOVA with Tukey’s multiple-comparison test (h). Data are mean ± s.e.m. (a,b,d,e-l). The diagram in c was created with BioRender by Xiangyue Zhang (2025).

Fig. 1 |. Efferocytotic tolerance-inducing cDC1s upregulate Epor following TLI.

a, Schematic of TLI/ATS treatment, allo-BM infusion, and heart transplantation in C57BL/6 and Batf3^−/− mice. b, Donor-type (H2Kᵈ⁺) leukocytes (B220⁺, TCRβ⁺, Ly6G⁺, CD64⁺) in peripheral blood 28 days post-BM infusion. WT (n=10) vs. Batf3^−/− (n=7). c, Heart allograft survival in C57BL/6 (n=10) vs. Batf3^−/− (n=7). d-e, RNA-seq of splenic cDC1s from UNT or TLI/ATS-treated mice. d, Enriched GO biological processes. e, Volcano plot of differentially expressed genes. f, qPCR of selected genes (UNT, n=8; TLI/ATS, n=6). g-i, Flow cytometry analysis of Epor-tdTomato (tdT) on cDC1s/cDC2s from UNT, TLI, and TLI/ATS-treated mice; summary of cDC1 frequency (h) and EpoR-tdT⁺ cells (i), (n=5/group). j, tdT expression in Epor⁺ cDC1s: histogram (left) and MFI (right) (UNT, n=5; TLI/ATS, n=4). k-l, RNA-seq of splenic Epor-tdT⁺ vs. EpoR-tdT⁻ cDC1s post-TLI/ATS (n=2/group, pooled from 15 mice). k, Volcano plot of upregulated genes. l, Heatmap of genes enriched in EpoR-tdT⁺ cDC1s. Data are pooled from 2 independent experiments (b). Data are shown from one experiment, representative of at least three independent experiments with similar results (c,g,h,i,j). Statistical analysis was performed using unpaired two-tailed Student’s t-test (b,f,j), one-way ANOVA (h) or two-way ANOVA with Tukey’s multiple-comparison test (i), or Kaplan-Meier survival analysis with Mantel-Cox test (c). P-values were calculated using hypergeometric tests with Benjamini-Hochberg correction (d) or two-sided generalized linear model (GLM) likelihood ratio tests with Benjamini-Hochberg correction (e,k). Data are mean ± s.e.m. (b,f,h,i,j). The diagram in a was created with BioRender by Xiangyue Zhang (2025).

Fig. 2 |. Absence of EpoR on cDC1s abrogates T_reg-mediated alloantigen-specific tolerance following TLI/ATS, resulting in allograft rejection.

a, Schematic of TLI/ATS treatment, allo-BM infusion, and heart transplantation in Epor^flox/flox and Epor^ΔXcr1 mice. b, Donor-type (H2Kᵈ⁺) leukocytes (B220⁺, TCRβ⁺, Ly6G⁺, CD64⁺) in peripheral blood 28 days post-BM infusion. Epor^flox/flox (n=10) vs. Epor^ΔXcr1 (n=10). c, Heart allograft survival in Epor^flox/flox (n=10) vs. Epor^ΔXcr1 mice (n=8). d,e, Foxp3-DTR mice conditioned with TLI/ATS; Group A received DT from day 1 to day 14 post-BM infusion (n=8; n=8; n=8), Group B received DT from day 15 after confirmation of BM chimerism (n=8; n=5; n=8). BM chimerism was assessed on the indicated days. e, Summary of BM chimerism on the indicated days post-BM infusion. f, Epor-tdT⁺ or Epor-tdT⁻ cDC1s cocultured with CTV-labeled naïve OT-II cells and EPO for 5 days or with PBS (w/o); FOXP3 expression on OT-II cells was assessed by flow cytometry (Epor-tdT⁺ cDC1s: +PBS, n = 5; +EPO, n = 6; Epor-tdT⁻ cDC1s: +PBS, n = 4; +EPO, n = 6). g, C57BL/6, Batf3 ^−/−, or Epor^ΔXcr1 recipients treated with TLI/ATS and infused with BALB/c BM; frequencies of recipient H-2K^b+ TCRβ⁺CD4⁺ and FOXP3⁺ CD4⁺ T cells analyzed on day 0 (UNT, n=5; n=5; n=5 and TLI/ATS, n=6; n=5; n=5) or 14 days post BM infusion (UNT, n=5; n=5; n=5 and TLI/ATS, n=5; n=5; n=5). h, Epor^flox/flox and Epor^ΔXcr1 recipients given TLI/ATS or UNT; 14 days post-2W1S-BALB/c donor BM infusion, 2W1S-tetramer⁺CD44⁺H-2K^b+TCRβ⁺CD4⁺ T cells from the spleens were analyzed for FOXP3 expression (Epor^flox/flox, n=7; Epor^ΔXcr1, n=7). Data are pooled from 2 independent experiments (b) or shown from one experiment, representative of at least two independent experiments with similar results (c,e,f,g,h). Statistical analysis was performed using unpaired two-tailed Student’s t-test (b,h), or two-way ANOVA with Tukey’s multiple-comparison test (e,f,g), or Kaplan-Meier survival analysis with Mantel-Cox test (c). Data are mean ± s.e.m. (b,e,f,g,h,). The diagrams in a,d,g,h were created with BioRender by Xiangyue Zhang (2025).

Fig. 3 |. scRNA-seq analysis reveals that TLI/ATS promotes EpoR-dependent, efferocytosis-triggered tolerogenic maturation of splenic cDC1s.

a, UMAP of splenic cDC1s colored by subtype with dot plot of marker genes. b, cDC1 subtype proportions in UNT vs. TLI/ATS-treated Epor^flox/flox; box highlights increased mature cDC1s after TLI/ATS. c, UMAP by sample identity with violin plots of Itgae, Lgals3, Apol7c. d, Log₂ fold-change of cDC1 subtypes post-TLI/ATS in Epor^flox/flox and Epor^ΔXcr1 mice; box shows EpoR-dependent differences in mature cDC1s. e, Flow cytometry showing cDC1% in splenic Lin⁻SiglecH⁻PDCA-1⁻CD11c^highMHCII^high cDCs (Epor^flox/flox: UNT, n=5; TLI/ATS, n=6; Epor^ΔXcr1: UNT, n=5; TLI/ATS, n=6). f, UMAP with violin plots of Cd83, Rel, Dnase1l3 in cDC1s from Epor^flox/flox and Epor^ΔXcr1 mice post-TLI/ATS. g, Bar charts of cDC1 subtype proportions and h, UMAP and violin plots of Dnase1l3, Xcr1, Cd274 in Epor^flox/flox and Epor^ΔXcr1 mice at baseline (UNT). i, qPCR of selected genes in CCR7⁺XCR1⁺SIRPα⁻ cDC1s from Epor^flox/flox (n=5/condition) and Epor^ΔXcr1 mice (n=5/condition) (UNT or TLI/ATS). j, Itgb8^ΔXcr1 vs. control recipients UNT or treated with TLI/ATS, and infused with BALB/c BM; frequencies of recipient H-2K^b+TCRβ⁺CD4⁺ and FOXP3⁺ T_regs in CD4⁺ T cells were analyzed on day 0 (UNT, n=5; n=5 and TLI/ATS, n=5;n=5) or 14 days post-BM infusion (UNT, n=5; n=5 and TLI/ATS, n=5; n=5). k,l, Itgb8^ΔXcr1 (n=8) vs. controls (n=5) and infused with 2W1S-BALB/c BM. Donor-type leukocyte percentages (k) and the frequency and absolute number of FOXP3⁺2W1S-tetramer⁺CD44⁺CD4⁺ T_regs in spleens (l) were analyzed 14 days later. Data are representative of two (e,i) or one (j,k,l) independent experiments. Statistical analysis was performed using unpaired two-tailed Student’s t-test (i,k,l), or two-way ANOVA followed by Tukey’s multiple-comparison test (e,j), or propeller test, two-sided, no multiple-comparison correction (b.g), or Wilcoxon rank sum test, two-sided, with Bonferroni correction (c,f,h). Data are mean ± s.e.m. (e,i,g,k,l)

Fig. 4 |. EPO-activated cDC1 EpoR supports Ag-specific FOXP3⁺ T_reg induction in pLN and restrains the immunogenic maturation of CCR7⁺ cDC1s.

a,b, Epor-tdT expression on cDCs in pLNs (n=5) and mLNs (n=5) of Zbtb46^GFP/+Epor^tdT/+ mice (a) and statistical graph comparison (b). c, Epor-tdT⁺ cells were identified in migratory XCR1⁺ cDC1s or XCR1⁻ cDC2s (upper) and in migratory vs. resident cDCs (lower) in pLN Zbtb46-GFP⁺CD11c⁺ cDCs (n=5). d, Epor-tdT expression in pLNs and e, mammary fat pad cDC1s of Epor^tdT/+mice (n=8). f, Efferocytosis of PKH67-labeled CD45.1⁺ apoptotic thymocytes by migratory or resident cDCs in the dLN of CD45.2⁺Epor^tdT/tdT mice (n=7) 12 h after injection of the apoptotic cells into the 3^rd mammary fat pad. g, Effect of EPO on CD45.1⁺ OT-II T_reg induction after Act-mOVA thymocyte injection into the 3^rd mammary fat pad (Epor^flox/flox: +PBS, n=5; +EPO, n=5; Epor^ΔXcr1: +PBS, n=5; +EPO n=5). h, Frequency and absolute number and i, XCR1 MFI of migratory cDC1s per pLN (Epor^flox/flox, n= 15; Epor^ΔXcr1, n=15). j,k, scRNA-seq of pLN migratory cDC1s from Epor^flox/flox and Epor^ΔXcr1; heatmap of DEGs and UMAP colored by cluster identity, gene expression. The violin plots represent module score of CD4⁺ T helper licensing gene signature (259 genes) in migratory cDC1s from Epor^flox/flox (n = 6,890 cells) and Epor^ΔXcr1 (n = 7,225 cells) pLNs. The boxes inside the violin plots show the median (centre line) and the interquartile range (25% to 75%; box limits). l, MFI of indicated molecules on pLN migratory cDC1s from Epor^flox/flox (n=6) and Epor^ΔXcr1 mice (n=6). m, Heatmap of top shared DEGs in pLN migratory and splenic CCR7⁺ cDC1s from bulk RNA-seq (Epor^flox/flox vs. Epor^ΔXcr1). Data are shown from one experiment, representative of at least three independent experiments with similar results (a-h, l). Statistical analysis was performed by using unpaired two-tailed Student’s t-test (b,c,e,f,h,i,l), or two-way ANOVA with Tukey’s multiple-comparison test (g), or uncorrected Wilcoxon rank-sum test, one-sided (k). Data are mean ± s.e.m. (b,c,e,f,h,i,l). The diagrams in b,c,d,e,f,g were created with BioRender by Xiangyue Zhang (2025).

Fig. 5 |. EpoR expression on cDC1s hinders Ag-specific anti-tumor T cell immunity and the loss of EpoR in cDC1s leads to tumor reduction.

a, B16F10-OVA tumor cells were implanted s.c. into the flanks of Zbtb46^GFP/+Epor^tdT/+ mice, and 10 days later, Epor-tdT expression on tumor-infiltrating cDC subsets (CCR7⁺ vs. CCR7⁻ populations) was determined. b, MC38-OVA^dim tumor size and weight on day 14 following implantation. Epor^flox/flox (n=8) vs. Epor^ΔXcr1 (n=7). c, CTV-labeled naïve CD45.1⁺ OT-I cells were i.v. transferred 4 days after B16F10-OVA implantation; tdLNs were analyzed 6 days later for OT-I proliferation. Representative flow plots are shown for each marker expressed on OT-I cells vs. CTV dilution. Proliferating CD44⁺CTV^lowOT-I cells were quantified. Epor^flox/flox (n=5) vs. Epor^ΔXcr1 (n=7). d-j, B16F10-OVA tumors implanted in Epor^flox/flox (n=6) or Epor^ΔXcr1 (n=6) mice; TILs analyzed on day 12: d, Frequencies of CD45⁺ TILs, CD8⁺ and CD4⁺ T cells. e, OVA_257–264-dextramer⁺ CD8⁺ T cells. f,g, Representative flow plots and quantification of CD8⁺ T cells expressing TIM-3 and PD-1 (f) and TCF1⁺TIM-3⁻CD8⁺ T cells (g). h, Representative histograms and quantification of perforin, granzyme-B, IFNγ and TNFα expression in CD8⁺ T cells. i, Frequencies and absolute cell numbers of FOXP3⁺ T_regs in CD4⁺ T cells with representative flow contour. j, Frequencies of T-bet⁺CXCR3⁺ T_regs in CD4⁺FOXP3⁺ T_regs with representative flow contour. k, B16F10-OVA tumor growth in Epor^flox/flox vs. Epor^ΔXcr1 treated with anti-PD-1 (n=6 and n=6) or an IgG2a isotype control (n=6 and n=6). Data are shown from one experiment, representative of at least three independent experiments with similar results (a-k). Statistical analysis was performed using unpaired two-tailed Student’s t-test (b,c,d,e,g,h, i,j), or two-way ANOVA followed by Tukey’s multiple-comparison test (f), or Šídák’s multiple-comparison test (k). Data are mean ± s.e.m. (b-k). The diagram in c was created with BioRender by Xiangyue Zhang (2025).