CD11b+ dendritic cells (DCs) seem to be specialized for presenting antigens via major histocompatibility (MHC) class II complexes to stimulate helper T cells, but the genetic and regulatory basis for this is not established. Conditional deletion of Irf4 resulted in loss of CD11b+ DCs, impaired formation of peptide–MHC class II complexes and defective priming of helper T cells but not of cytotoxic T lymphocyte (CTL) responses. Gene expression and chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analyses delineated an IRF4-dependent regulatory module that programs enhanced MHC class II antigen presentation. Expression of the transcription factor IRF4 but not of IRF8 restored the ability of IRF4-deficient DCs to efficiently process and present antigen to MHC class II–restricted T cells and promote helper T cell responses. We propose that the evolutionary divergence of IRF4 and IRF8 facilitated the specialization of DC subsets for distinct modes of antigen presentation and priming of helper T cell versus CTL responses.
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Integrated supplementary information
(a) Splenic DC populations of wild-type mice were analyzed by flow cytometry for intracellular expression of IRF4 and IRF8 protein. Filled grey histograms are isotype controls. IRF4 and IRF8 expression was similarly analyzed for MHCIIhi DC populations in cutaneous lymph nodes (CLN) (d) and mesenteric lymph nodes (MLN) (g). (b) Left, DC-enriched cells from spleens of Cd11c-Cre and Irf4fl/fl Cd11c-Cre mice were analyzed by flow cytometry for expression of CD11c and MHCII. Numbers indicate percentage of cells within the CD11c+MHCIIint gate. Right, CD11c+MHCIIint DC subpopulations were distinguished by expression of CD11b and CD103. Numbers indicate percentage of cells within each gate. (c) Quantitative analysis of DCs described in (b). Data represent mean ± s.e.m. from 3-5 independent experiments, each with pooled splenic tissue from 5 animals. NS = not significant; * = P < 0.05, two-tailed t-test. (e) Left, DC-enriched cells from CLNs of Cd11c-Cre and Irf4fl/fl Cd11c-Cre mice were analyzed by flow cytometry for expression of CD11c and MHCII. Numbers indicate percentage of cells within the CD11c+MHCIIhi gate. Right, CD11c+MHCIIhi migratory DC subpopulations were distinguished by expression of CD11b and CD103. Numbers indicate percentage of cells within each gate. (f) Quantitative analysis of DCs described in (e). Data represent mean ± s.e.m. from at least 3 independent experiments, each with pooled CLN tissue from 5 animals. NS = not significant; * = P < 0.05, two-tailed t-test. (h, i) Migratory MHCIIhi DCs from MLN of Cd11c-Cre and Irf4fl/fl Cd11c-Cre mice were analyzed as in panels e and f. Data represent mean ± s.e.m. from at least 3 independent experiments, each with pooled CLN tissue from 5 animals. NS = not significant; * = P < 0.05, two-tailed t-test.
Supplementary Figure 2 IRF4-dependent CD11b+ DCs preferentially prime helper T cell responses to cell-associated antigen.
CellTrace violet-labeled OT-I and OT-II T cells were co-transferred into Cd11c-Cre and Irf4fl/fl Cd11c-Cre mice. Recipient mice were subsequently immunized by footpad injection with the indicated numbers of necrotic ovalbumin-expressing MC38 (MC38-OVA) tumor cells. Histograms show OT-I and OT-II proliferation measured by dye dilution, 3 days after antigen administration. For each dose, paired OT-II and OT-I data from the same mouse is displayed. Data are representative of 2 independent experiments, n = 2 mice per dose in each experiment.
(a, b) Cd11c-Cre and Irf4fl/fl Cd11c-Cre mice were immunized with TNP-OVA and serum antibody titers for IgG1 (a) and IgE (b) were determined by ELISA on the indicated days. Data represent antibody titers from 4 individual mice per group with horizontal bars indicating mean values. Data are representative of 2 independent experiments. (c, d) Cd11c-Cre and Irf4fl/fl Cd11c-Cre mice were sensitized with TNP-OVA. 35 days after sensitization mice were challenged by administration of nebulized TNP-OVA into the airways for 7 consecutive days. 24 h after the last antigen challenge lungs were removed. Lungs from naïve mice served as controls. Lung infiltrating CD4+ T cells (c) and neutrophils (d) were enumerated by flow cytometry. Data represent cell counts from individual mice (naïve n = 5, challenged n = 8) mean ± s.d.; one experimental replicate. (e, f) Cd11c-Cre and Irf4fl/fl Cd11c-Cre mice were infected with LCMV. Uninfected (UI) wild-type (WT) C56BL/6 mice served as controls. (e) 8 days after infection splenocytes were stimulated in vitro with LCMV gp33 peptide. CD8+ CTLs were analyzed by intracellular flow cytometry for expression of IFN-γ. Representative data from individual mice are shown. Numbers indicate percentage of gated CD8+IFN-γ+ splenocytes. (f) Viral titers in livers of infected mice were determined at day 8 after infection by pfu assay. Data depict titers determined for individual mice, mean indicated by horizontal bars; NS = not significant, one-tailed Mann-Whitney. Data are representative of 2 independent experiments.
Supplementary Figure 4 IRF4 as a regulatory determinant of enhanced pMHCII formation in murine and human DCs.
(a) Efficiency of pMHCII formation by MHCIIhiCD11b+CD103– (red) or MHCIIhiCD11b–CD103+ (blue) CLN migratory DC subsets was assessed after subcutaneous administration of the indicated doses of Eα antigen in wild-type (WT) C57BL/6 mice. DCs were enriched and then stained, 18 h after injection of Eα, with Y-Ae antibody and analyzed by flow cytometry. Data are from two independent experiments, n = 2 mice per dose, and represent geometric mean fluorescence intensity (gMFI) ± s.e.m.; * = P < 0.05, one-tailed t-test. (b) Differential gene expression analysis in paired mouse and human DC subsets. Mouse resident DCs are represented by splenic CD4+CD8– (CD4) and CD4–CD8+ (CD8) subsets. Mouse migratory DCs are represented by cutaneous lymph node MHCIIhiCD103–CD11b+ (CD11b) and MHCIIhiCD103+CD11b– (CD103) subsets. Human DCs isolated from blood are represented by BDCA1+ and BDCA3+ subsets. The murine data is from the ImmGen database17 whereas the human data is from E-TABM-34 (ref. 36). Expression of indicated genes is displayed as fold-increase (red) or decrease (blue) relative to its average expression in paired DC subsets.
Supplementary Figure 5 Flt3L-generated BMDCs express abundant IRF8 but not IRF4 and are not a suitable model for IRF4-dependent CD11b+ DCs.
BMDCs were differentiated from Cd11c-Cre (a) and Irf4fl/fl Cd11c-Cre (b) hematopoietic progenitors by culture in the presence of Flt3L. BMDCs were analyzed by flow cytometry for their expression of CD11c and MHCII. Numbers indicate percentages of cells with the CD11c+MHCII+ DC gate. CD11c+MHCII+ DC subpopulations were distinguished by their expression of CD11b. Numbers indicate percentage of cells within each gate. CD11b+ (red) and CD11b– (blue) DC populations were analyzed by flow cytometry for intracellular expression of IRF4 or IRF8 protein. Filled grey histograms are isotype controls. Data are representative of 2 independent experiments.
(a-c) BMDCs were differentiated from Cd11c-Cre hematopoietic progenitors by culture in the presence of GM-CSF and IL-4. (a) CD11c+ DC populations were analyzed by flow cytometry for intracellular expression of IRF4 or IRF8 protein. Filled grey histograms are isotype controls. Data are representative of 3 independent experiments. (b) BMDCs derived from Cd11c-Cre or Irf4fl/fl Cd11c-Cre mice were left unstimulated (US, dashed histrograms) or stimulated overnight with LPS (solid histograms), then analyzed by flow cytometry for expression of the indicated markers (see also Fig. 4a). Isotype controls are shown in filled grey histograms. Data are representative of at least 3 independent experiments. (c) Cd11c-Cre and Irf4fl/fl Cd11c-Cre BMDCs were loaded with the indicated concentrations of ovalbumin (OVA) then stimulated with LPS. Antigen-loaded CD11c+ DCs were isolated by magnetic separation and used to stimulate OT-II T cells for 3 days. Histograms show OT-II proliferation as measured by dilution of CellTrace violet dye. Data are representative of at least 3 independent experiments.
(a-c) Wild-type C56BL/6 BMDCs differentiated with GM-CSF and IL-4 were stimulated for 6 h with LPS. IRF4, PU.1, BATF, or JunB-bound chromatin fragments were immunoprecipitated and subjected to high-throughput sequencing (ChIPseq). Sequence tracks displaying peaks for each of the above transcription factors are shown for Ctss (a), H2-Dmb2 (b), and Ciita (c) genes. Boxed peaks containing Ets-IRF (EICE) or AP-1-IRF (AICE) composite motifs are highlighted. Two independent ChIPseq replicates showed greater than 95% overlap in called IRF4 binding sites.
(a) Irf4fl/fl Cd11c-Cre hematopoietic progenitors were transduced with MSCV-IRES-huCD4 (MSCV) or its IRF4- or IRF8-expressing derivatives and then differentiated into DCs with GM-CSF and IL-4 (see Fig. 5a). Complemented BMDCs were analysed by flow cytometry for expression of IRF4, IRF8, CD80, and CD86. Histograms represent staining for MSCV control (black), MSCV-IRF4 (red), and MSCV-IRF8 (blue) complemented BMDCs. Data are representative of at least 3 independent experiments. (b) Complemented BMDCs were loaded with the indicated concentrations of ovalbumin (OVA) protein and then stimulated overnight with LPS. Transduced CD11c+ cells were purified by flow cytometry and used to stimulate labelled OT-I T cells for 3 days. T cell proliferation was analysed by dye dilution. Data are representative of 2 independent experiments.
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Vander Lugt, B., Khan, A., Hackney, J. et al. Transcriptional programming of dendritic cells for enhanced MHC class II antigen presentation. Nat Immunol 15, 161–167 (2014). https://doi.org/10.1038/ni.2795
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