IRF4 and IRF8 Act in Cd11c+ Cells to Regulate Terminal Differentiation of Lung Tissue Dendritic Cells

IRF4 and IRF8 Act in CD11c+ Cells To Regulate Terminal Differentiation of Lung Tissue Dendritic Cells

This information is current as Sandra Bajaña, Sean Turner, Jinny Paul, Erola of September 29, 2021. Ainsua-Enrich and Susan Kovats J Immunol 2016; 196:1666-1677; Prepublished online 8 January 2016; doi: 10.4049/jimmunol.1501870

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Supplementary http://www.jimmunol.org/content/suppl/2016/01/07/jimmunol.150187 Material 0.DCSupplemental http://www.jimmunol.org/ References This article cites 49 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/196/4/1666.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IRF4 and IRF8 Act in CD11c+ Cells To Regulate Terminal Differentiation of Lung Tissue Dendritic Cells

Sandra Bajan˜a, Sean Turner, Jinny Paul, Erola Ainsua-Enrich, and Susan Kovats

Dendritic cells (DCs) initiate immune responses in barrier tissues including lung and skin. Conventional DC (cDC) subsets, CD11b2 (cDC1s) or CD11b+ (cDC2s), arise via distinct networks of transcription factors involving IFN regulatory factor 4 (IRF4) and IRF8, and are specialized for unique functional responses. Using mice in which a conditional Irf4 or Irf8 allele is deleted in CD11c+ cells, we determined whether IRF4 or IRF8 deficiency beginning in CD11c+ cDC precursors (pre-cDCs) changed the homeostasis of mature DCs or pre-DCs in the lung, dermis, and spleen. CD11c-cre-Irf42/2 mice selectively lacked a lung-resident CD11chi CD11b+SIRPa+CD24+ DC subset, but not other lung CD11b+ DCs or alveolar macrophages. Numbers of CD11b+CD4+ splenic DCs, but not CD11b+ dermal DCs, were reduced, indicating cDC2s in the lung and dermis develop via different pathways. Irf4 2/2 + a+ deficiency did not alter numbers of cDC1s. CD11c-cre-Irf8 mice lacked lung-resident CD103 DCs and splenic CD8 DCs, yet Downloaded from harbored increased IRF4-dependent DCs. This correlated with a reduced number of Irf82/2 pre-cDCs, which contained elevated IRF4, suggesting that Irf8 deficiency diverts pre-cDC fate. Analyses of Irf4 and Irf8 haploinsufficient mice showed that, although one Irf4 allele was sufficient for lung cDC2 development, two functional Irf8 alleles were required for differentiation of lung cDC1s. Thus, IRF8 and IRF4 act in pre-cDCs to direct the terminal differentiation of cDC1 and cDC2 subsets in the lung and spleen. These data suggest that variation in IRF4 or IRF8 levels resulting from genetic polymorphisms or environmental cues will

govern tissue DC numbers and, therefore, regulate the magnitude of DC functional responses. The Journal of Immunology, 2016, http://www.jimmunol.org/ 196: 1666–1677.

ung resident dendritic cells (DCs) are essential regulators press IRF8 (6, 17). Thus, investigation of the role of IRF4 and of innate and adaptive immune responses to respiratory IRF8 in the differentiation and homeostasis of DC subsets will L pathogens and also promote chronic inflammatory dis- help us to understand human inflammatory responses in peripheral eases such as asthma (1, 2). Tissue DC subsets, including those in tissues. the lung and dermis, are specialized for particular types of func- In mice, conventional DCs (cDCs) in lymphoid organs and tional responses, and their development is governed by specific nonlymphoid tissues are broadly classified as CD11b2 (cDC1s) or networks of transcription factors, such as IFN regulatory factor 4 CD11b+ (cDC2s) (18). The CD11b2 DCs express CD8a+ in the by guest on September 29, 2021 (IRF4) and IRF8, expressed in DC progenitors (3–5). Mature DCs spleen and CD103+ in nonlymphoid tissues, and require IRF8 and continue to express these transcription factors, which specify gene BATF3 for their terminal differentiation (19). CD11c+CD11b+ expression programs that direct their functional responses. IRF4- MHCII+ cells are heterogeneous in nonlymphoid tissues, and the expressing DCs are important for the DC-driven polarization of definition of specific DC subsets has only recently been clarified Th17 responses in the intestine and lung (6, 7), for the induction of by approaches to separate true CD11b+CD24+ cDC2s from mac- Th2 responses in lung allergy and skin parasite models (8–10), rophages and monocyte-derived DCs (20–22). Because of this and for attenuation of Th1 responses (11). In turn, IRF8- diversity, it has been more difficult to discern the transcription expressing DCs are often most important for Th1 and CD8+ factor networks required for terminal differentiation of the cDC2s T cell responses, although the role of specific DC subsets is and other CD11c+CD11b+ subsets. context dependent (12–16). In human blood, CD1c+ DCs prefer- IRF4 is required for differentiation of splenic CD11b+CD4+ entially express IRF4, whereas CD141+ DCs preferentially ex- cDCs (23, 24). However, the role of IRF4 in the development of tissue cDC2s remained unclear. We used mice globally deficient for Irf4 to show that IRF4 is not required for development or skin + Arthritis and Clinical Immunology Program, Oklahoma Medical Research Founda- residence of CD11b dermal DCs (dDCs), but does promote mi- tion, Oklahoma City, OK 73104 gration of dermal CD11b+ DCs to cutaneous lymph nodes (LNs) ORCIDs: 0000-0001-9879-0183 (S.T.); 0000-0002-6863-6406 (J.P.); 0000-0001- (25). More recent work, with mice bearing a conditional Irf4 allele 5479-9952 (S.K.). and a CD11c-cre construct that directs Cre activity in CD11cint Received for publication August 20, 2015. Accepted for publication December 10, cDC precursors (pre-cDCs) and CD11chi mature DCs (CD11c-cre- 2015. Irf42/2 mice), showed that numbers of CD11b+CD24+ lung DCs This work was supported by National Institutes of Health Grant HL119501. were reduced but not completely ablated by Irf4 deficiency; this Address correspondence and reprint requests to Dr. Susan Kovats, Arthritis and was correlated with increased apoptosis in the remaining DCs, Clinical Immunology Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104. E-mail address: [email protected] suggesting an effect on DC survival (6). A similar conclusion was + + The online version of this article contains supplemental material. reached for CD103 CD11b DCs in the small-intestinal lamina Abbreviations used in this article: AM, alveolar macrophage; BM, bone marrow; propria in these mice (6, 7). In contrast, mice bearing a conditional CDP, common DC progenitor; cDC, conventional DC; DC, dendritic cell; dDC, Irf4 allele and a CD11c-cre construct that apparently directs Cre dermal DC; eLC, epidermal DC; IRF, IFN regulatory factor; LN, lymph node; pre- activity only in CD11chi cells showed no reduction in CD11b+ cDC, cDC precursor. CD24+ lung DCs (8). Taken together, these studies suggested that int Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 Irf4 deletion beginning at the CD11c pre-cDC stage diminished www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501870 The Journal of Immunology 1667 in vivo survival (and therefore numbers) of CD11b+ lung DCs, global deletion of Irf8 favors development of neutrophils at the expense of hi DCs (26), we excluded CD11c-cre-Irf8 mice from analysis that had an whereas Irf4 deletion only in mature CD11c DCs did not impact + their numbers. enlarged spleen or BM compartment, or high numbers of Ly-6G cells (identified by mAb Gr-1) in blood; we were unable to develop a PCR assay Despite these advances, it remained unclear whether IRF4 and for this screening in the CD11c-cre-Irf8 mice. Littermate mice of each IRF8 must act in immediate pre-cDCs to promote DC terminal genotype and of both sexes were analyzed at 6–10 wk of age, and we did differentiation and/or survival (19). Irf8 mRNA is present in not find a sex difference in any parameter analyzed. The Oklahoma common DC progenitors (CDPs) and pre-cDCs, whereas Irf4 Medical Research Foundation Institutional Animal Care and Use Com- mittee approved the studies. mRNA is present in pre-cDCs, but not CDPs (reviewed in Ref. 3). Global Irf8 deficiency leads to defects in the formation of the CDP Isolation of cells from tissues and all splenic DC subsets (26), whereas global Irf4 deficiency did Lungs were perfused with PBS before digestion. Lung lobes were digested + + not apparently affect CDPs but did abolish splenic CD11b CD4 for 60 min with collagenase type D (2 mg/ml) and DNAse I (0.2 mg/ml; both cDCs, suggesting effects on pre-cDCs (23, 24). Pre-cDCs [defined from Roche) in 10 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 5 mM KCl, 2 int 2 as (CD19, CD3, Ter119, B220, NK1.1 ) CD11c MHCII 1mMMgCl2, 1.8 mM CaCl2. Spleens were digested to a single-cell sus- int + pension with collagenase type D (1 mg/ml) and DNAse I (0.1 mg/ml) in SIRPa Flt3 ] with the potential to develop into cDCs in lym- 2+ 2+ Ca and Mg containing HBSS at 37˚C for 30 min. BM cells were phoid and nonlymphoid organs have been identified in bone isolated as described previously (40). RBCs in tissues were lysed using marrow (BM), lymphoid organs, and nonlymphoid tissues in- RBC lysis buffer (BD Biosciences). In some experiments, CD11c+ cells in cluding lungs (27–29). The pre-cDC population was initially di- spleen and BM were enriched using a murine CD11c+ selection kit (Stem vided based on low or high CD24 expression into precursors Cell Technologies) to analyze pre-cDCs. 2 precommitted to either the CD11b+ or the CD11b pathways (27). Flow cytometry Downloaded from Recent reports used CD24 and other markers to subdivide the pre- After isolation from tissue, cells were immediately processed for flow cDC population into discrete precursors for the cDC1s and cDC2s cytometry by preincubating with anti-CD16/32 and labeling with optimally that preferentially express Irf8 and Batf3 mRNA or Irf4 mRNA, titered mAbs (obtained from BD Biosciences, eBioscience, or Biolegend) respectively (30, 31). Irf8-deficient mice lacked pre-cDC1s (30). in FACS buffer (PBS, 5% newborn calf serum, 0.1% sodium azide). To However, the effects of Irf4 and Irf8 deficiency or haplo- identify lung DC and macrophage populations, we stained cells with a lymphocyte marker mixture (mAbs specific for CD19, CD3, B220, and insufficiency beginning in pre-cDCs on pre-cDC numbers and http://www.jimmunol.org/ NK1.1 linked to a common fluorochrome) to gate out lymphocytes, in their expression of IRF4 and IRF8 proteins in vivo have not been conjunction with various combinations of fluorochrome-labeled mAbs well characterized. specific for CD11c, MHCII, Siglec F, CD64, CD11b, CD103, SIRPa, CD24, and CD14. Splenic DCs were defined using a combination of mAbs spe- In this study, we have reexamined the effect of Irf4 deficiency on 2 2/2 +/2 cific for CD11c, CD8a, CD11b, CD4, and MHCII. Pre-cDCs (Lin DC differentiation in CD11c-cre-Irf4 and CD11c-cre-Irf4 2 CD11c+ MHCII SIRPalo Flt3+) in BM or spleen were identified using a mice in which Cre activity is present in pre-cDCs and mature DCs. Lineage mixture (mAbs specific for CD19, CD3, B220, NK1.1, Ter119 Similar analyses of CD11c-restricted Irf8 deficiency were done linked to a common fluorochrome) to gate out mature cells, in conjunction 2 2 2 using CD11c-cre-Irf8 / and CD11c-cre-Irf8+/ mice. We de- with mAbs specific for CD11c, MHCII, SIRPa, and Flt3. After surface termined the effect of Irf4 and Irf8 gene dosage on numbers of marker staining, intracellular staining with fluorochrome-conjugated mAbs pre-cDCs and DC subsets and their relative expression of IRF4 specific for IRF4(PE) and IRF8(allophycocyanin) was done using a Foxp3 by guest on September 29, 2021 buffer kit (all from eBioscience). Intracellular staining for activated cas- and IRF8 proteins. Taken together, our data show that changes in pase 3 (mAb from BD Biosciences) was done in conjunction with live/ IRF4 or IRF8 levels beginning in pre-cDCs have profound effects dead fixable Aqua stain (Invitrogen). The data were collected on an LSRII on relative numbers of the cDC1 and cDC2 subsets in the lung and instrument (BD Biosciences) and analyzed with FlowJo (Tree Star) soft- spleen in homeostasis. Thus, variation in IRF4 or IRF8 levels ware. resulting from environmental stimuli or genetic polymorphisms Statistical analyses may regulate tissue DC numbers, and therefore modulate the Significant differences between values measured in wild-type and mutant magnitude of DC functional responses in inflammation. Indeed, mice were determined using an unpaired t test (if two genotypes compared) polymorphisms in human IRF4 genes impart susceptibility to or a one-way ANOVA with Tukey posttests (if three genotypes evaluated) melanoma and lymphocytic leukemia, whereas polymorphisms in in Prism 6 software, as indicated in the figure legends. Differences were IRF8 genes impart susceptibility to systemic lupus erythematosus considered significant when p , 0.05. or lead to deficient antimycobacterial immunity secondary to the absence of select DC subsets (32–36). Results To evaluate the role of IRF4 in the terminal differentiation of tissue Materials and Methods and lymphoid organ DCs, we bred mice with a conditional Irf4 allele to mice bearing Cre recombinase driven by the CD11c Mice promoter. The CD11c-cre-Irf4 mice bear 0, 1, or 2 copies of Irf4 Mice (purchased from The Jackson Laboratory) bearing a conditional allele in CD11c+ cells and are hereafter designated as CD11c-cre-Irf42/2, of Irf4 (B6.129S1-Irf4,tm1Rdf./J) (37) were bred to mice bearing Cre CD11c-cre-Irf4+/2,orCD11c-cre-Irf4+/+, respectively. CD11c+ recombinase driven by the CD11c promoter pB6.Cg-Tg(Itgax-cre)1-1Reiz/ J] (38) and then interbred to yield CD11c-cre-Irf42/2, CD11c-cre-Irf4+/2, cells include pre-cDCs, mature DCs, NK cells, and some macro- or CD11c-cre-Irf4+/+ mice. Mice used as “wild-type” were either Cre+ but phage populations, including alveolar macrophages (AMs). The bearing two wild-type alleles of Irf4, or Cre2 and bearing either wild-type CD11c-specific Irf4 deletion did not significantly alter numbers of or conditional Irf4 alleles; we did not note any differences between these CD11c2 cell types as the +/2 and 2/2 mice had normal numbers two groups. As noted in a prior report, the Cre activity may randomly act + 2 of lung (CD45 ), spleen, and BM cells (Supplemental Fig. 1). In in CD11c cells in the line of CD11c-Cre mice, leading to mice that have the conditional allele deleted in many tissues (7). Therefore, we screened these mice, the deletion of Irf4 places an Egfp minigene in frame, + for mice bearing a global deletion of Irf4 using PCR primers (forward: resulting in GFP expression in CD11c cells that deleted the Irf4 59-CAGGATGTTGCCGTCCTCCTTG-39 and reverse: 59-CCTGCAGC- gene in +/2 and 2/2 mice; wild-type mice do not express any CAATAAGCTTATAAC-39), and excluded those mice from this study. GFP. Based on GFP expression, ∼100% of CD11chi DCs have Similarly, mice bearing a conditional allele of Irf8 [B6(Cg)-Irf8, . deleted the Irf4 gene (Fig. 1G, Supplemental Fig. 1). However, a tm1.1Hm /J) (39) were purchased from the Jackson Laboratory, bred to int + B6.Cg-Tg(Itgax-cre)1-1Reiz/J mice, and then interbred to produce CD11c- subset (∼30%) of CD11c cells is not clearly GFP , and thus may cre-Irf82/2, CD11c-cre-Irf8+/2,orCD11c-cre-Irf8+/+ mice. Because a not have deleted the Irf4 gene (Supplemental Figs. 1, 2D). Back- 1668 IRF4 AND IRF8 REGULATE TERMINAL DIFFERENTIATION OF LUNG DC Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. IRF4 expression in CD11c+ cells is required for development of a CD11chiMHCIIhiCD11b+CD24hiSIRPa+ DC subset in the lung. (A–C) Definition of DC populations in CD11c-cre-Irf4 +/+ mice. (A) CD11c+ myeloid cells displaying distinct levels of SiglecF are identified in the Lineage- negative (CD192CD32B2202 NK1.12) fraction of lung cells. The R1 gate defines CD11chiSiglecFlo DCs that are (B) CD642 and (C) MHCIIhi. The R2 gate defines CD11cintSiglecF2 cells, some of which are (B) CD642 and MHCII+ (see Supplemental Fig. 2). The numbers within these plots from +/+ mice indicate the percentage of cells within each gate. (D) CD11chiMHCIIhi cells in gate R1 are divided into CD11b+ and CD103+ subsets; a comparison of +/+, +/2, and 2/2 mice is shown. (E) The total numbers of CD11b+ and CD103+ DC subsets in multiple +/+, +/2, and 2/2 mice are compiled; shown are values for individual mice, n = 7 per genotype. (F) CD11chiMHCIIhi cells in gate R1 are divided into P1, P2, and P3 subsets based on SIRPa and CD24; a comparison of +/+, +/2, and 2/2 mice is shown. (G) The CD11chiMHCIIhi DC subsets SIRPa+CD24hi (P1), SIRPa+CD24int (P2), and SIRPaloCD24hi (P3) are GFP+ in +/2 mice. (H) The total numbers of P1, P2, and P3 DC subsets in multiple +/+, +/2, and 2/2 mice are compiled, n = 4–7 per genotype. (I) DCs within the CD11chi P1, P2, and P3 subsets display lower levels of CD14 than the CD11clo CD64hi macrophages. (J) Shown is the percentage of DCs in P2 and P3 subsets that contain activated caspase 3 in +/+, +/2, and 2/2 mice, n = 8–11 per genotype. The significance of the data in (E), (H), and (J) was evaluated using a one-way ANOVA with a Tukey multiple-comparisons test: **p , 0.01, ***p , 0.001, ****p , 0.0001. gating of GFP+ and GFP2 CD11chi cells in the lung shows that To also evaluate the role of IRF8 in the terminal differentiation all CD11chi MHCII+ cells are GFP+, whereas only a subset of of lung tissue DCs, we bred mice bearing a conditional Irf8 allele to CD11cint cells are GFP+ (Supplemental Fig. 1C, 1D). GFP2 the CD11c-cre mice; these mice do not bear a Gfp reporter. Total CD11chi cells are primarily AMs. numbers of BM cells, lung (CD45+) cells, and splenocytes were The Journal of Immunology 1669 not altered in CD11c-cre-Irf8+/2 and CD11c-cre-Irf82/2 mice cDC subsets differ in expression of IRF4 and IRF8, and those (Supplemental Fig. 1). cDCs that develop in CD11c-cre-Irf42/2 mice do not alter Through comparisons of CD11c-cre-Irf4 and CD11c-cre-Irf82/2, their expression of IRF8 +/2 +/+ and mice, we determined whether a reduction or absence of We next determined IRF4 and IRF8 protein levels in these distinct Irf4 and Irf8 gene expression in pre-cDCs and cDCs regulates the lung DC subsets using specific Abs for intracellular flow cytometry. numbers and IRF4 or IRF8 expression of DCs and pre-cDCs in The P1 DCs harbored the highest level of IRF4 and no IRF8, lymphoid organs and lung tissue during homeostasis. whereas P3 DCs expressed very low levels of IRF4 and high levels IRF4 expression in CD11c+ cells is required for development of IRF8 (Fig. 2A, 2B). The P2 population also expressed IRF4 and +/2 of a CD11chi MHCIIhi CD11b+ CD24hi SIRPa+ DC subset in very little IRF8. Analyses of CD11c-cre-Irf4 mice showed that the lung a single copy of the Irf4 gene led to a reduced level of IRF4 protein, ∼75% of the level in +/+ mice, in the P1 subset (Fig. 2B). To determine whether IRF4 were required for the development or In CD11c-cre-Irf42/2 and CD11c-cre-Irf4+/2 mice, the IRF8 survival of specific lung DC subsets, we analyzed the CD11chi lo 2 hi protein levels in the P2 and P3 subsets were not different from SiglecF CD64 MHCII population in perfused lungs (Fig. 1A– CD11c-cre-Irf4+/+ mice (Fig. 2A, 2B). Thus, a single copy of the C). Combined with a lineage gate to exclude CD19+, CD3+, and + Irf4 gene leads to a reduced amount of IRF4 protein in DCs, yet NK1.1 cells, this marker combination also facilitates exclusion Irf4 deficiency or haploinsufficiency did not alter IRF8 expression of CD11chiSiglecFhiCD64+ AMs and helps to clearly delineate hi int in the P2 and P3 DC subsets. Furthermore, although the P2 DCs CD11c (R1 gate) versus CD11c (R2 gate) cells. Once gated, express IRF4, Irf4 deficiency did not alter their numbers.

hi hi + Downloaded from the CD11c MHCII cells in gate R1 were divided into CD11b IRF4 was reported to regulate CIITA, a transcription factor that and CD103+ fractions (Fig. 1D). CD11c-cre-Irf42/2 mice showed hi promotes expression of MHCII (23). Despite the reduction in IRF4 a significant reduction of the percentage and number of CD11c in the P1 subset of +/2 mice, the MHCII level was not reduced MHCIIhi CD11b+ DCs (Fig. 1D, 1E). In an alternate marker hi hi (Fig. 2C). In the P2 subset, IRF4 deficiency, but not hemizygosity, scheme, the CD11c MHCII DCs were divided into three sub- led to decreased MHCII expression. This suggests that the amount sets based on expression of CD24 and SIRPa (CD172a) (Fig. 1F). 2 + hi lo of IRF4 in +/ DCs is sufficient for induction of CIITA. MHCII Previous reports showed that the CD103 DCs are CD24 SIRPa , levels in the P3 subset were not affected by loss of IRF4. http://www.jimmunol.org/ whereas CD11b+ DCs are CD24intSIRPahi. We identified two + populations of SIRPa+ DCs, differing in the level of expression of Deletion of Irf4 in CD11c cells does not alter numbers of int CD24. Notably, Irf4 deficiency results in a near-absolute reduction CD11c DCs or macrophage subsets in the lung + hi in the SIRPa CD24 DCs (designated the P1 population), We also determined whether IRF4 were required for the devel- + int whereas the numbers of the SIRPa CD24 cells (P2 population) opment of the CD11cintSiglecFlo CD642MHCIIhi subset, which lo hi + and the SIRPa CD24 cells (P3 population; also CD103 ) are appears to represent a distinct population of DCs (R2 gate in present in normal numbers (Fig. 1F, 1H). The P1, P2, and P3 Fig. 1A; Supplemental Fig. 2A). This population is CD11b+, with populations express high levels of GFP, reporting the deletion of a SIRPa+CD24int phenotype similar to the CD11chi P2 population

+ by guest on September 29, 2021 Irf4 (Fig. 1G). Because a fraction of CD11b tissue DCs is derived described earlier (Supplemental Fig. 2B), but only ∼50% of the from monocytes, and thus would not be reduced by CD11c-Cre cells in +/2 mice express GFP (Supplemental Fig. 2D), suggest- hi + activity (41), it is possible that the CD11c MHCII P2 cells ing that not all cells bear a deletion of Irf4. Numbers of this represent this monocyte-derived fraction. However, the P2 cells do CD11cintCD11b+MHCIIhi population do not differ among CD11c- lo not express high levels of CD14 or CD64, relative to the CD11c cre-Irf42/2, CD11c-cre-Irf4+/2, and CD11c-cre-Irf4+/+ mice, even + CD64 macrophage population (Fig. 1B, 1I). when gating only upon the GFP+ population in +/2 and 2/2 mice +/2 CD11c-cre-Irf4 heterozygotes showed a trend to reduced (Supplemental Fig. 2C, 2D). The CD11cintCD11b+MHCIIhi DCs + + hi percentages and numbers of the CD11b SIRPa CD24 pop- express low levels of IRF4 and IRF8 (Supplemental Fig. 2E). ulation relative to +/+ mice, but this reduction did not reach sta- CD11chiSiglecFhiCD64+ AMs also will have deleted Irf4; tistical significance (Fig. 1H). This suggests that one copy of the however, numbers of these cells did not differ among CD11c- Irf4 gene leads to decreased levels of IRF4, which is nearly suf- cre-Irf42/2, CD11c-cre-Irf4+/2,andCD11c-cre-Irf4+/+ mice ficient for complete lung cDC2 development. (Supplemental Fig. 2F, 2G). This is consistent with low expression hi hi + hi Thus, separation of the CD11c MHCII SIRPa DCs into CD24 of Irf4 in AMs (http://www.immgen.org). int and CD24 cells revealed that only one of two distinct DC subsets + + + IRF4 expression in CD11c cells is required for development within the CD11b SIRPa population is dependent upon Irf4 for + + development. Prior reports of the CD11c-cre-Irf42/2 mice showed a of splenic CD11b CD4 DCs reduction, but not absence, of the numbers of CD11b+ lung resident Consistent with previous reports in globally Irf4-deficient mice, in DCs and CD11b+CD103+ DCs in the small-intestinal lamina propria CD11c-cre-Irf42/2 and CD11c-cre-Irf4+/2 mice, we observed an (6, 7). Based on this reduction and other data showing increased Irf4 dose-dependent reduction in the percentages and numbers of apoptosis of the CD11b+ subset, it was suggested that Irf4 was not splenic CD4+CD82 DCs; these DCs are also CD11b+ (Fig. 3A– required for development of the CD11b+ subset, but rather was im- C). Numbers of the CD42CD82 DCs and the IRF8-dependent portant for regulation of its survival. To address DC survival in view CD42CD8+ DCs were not altered by Irf4 deficiency. These data of our finding that the CD11c-cre-Irf42/2 mice do lack the SIRPa+ show that a reduction of IRF4 activity beginning at the pre-cDCs CD24hi (P1) DC subset, we determined activated caspase 3 levels in stage preferentially reduces the development of splenic CD11b+ the SIRPa+CD24int (P2) subset directly ex vivo. In CD11c-cre-Irf42/2 CD4+CD82 DCs. Consistent with this finding, in +/+ mice, the mice, the P2 subset did not show a significant difference in the low CD4+CD82 DCs express IRF4 but little IRF8, whereas the CD42 fraction of cells bearing activated caspase 3 (Fig. 1J), suggesting that CD8+ DCs express IRF8, but not IRF4 (Fig. 3D). CD42CD82 these cells are not undergoing higher levels of apoptosis than in +/+ DCs express IRF4, but little IRF8, but their numbers and ex- mice. Taken together, these data show that Irf4 is required for the pression of IRF8 were not altered by Irf4 deficiency. Levels of development of the P1 DC subset, without an effect on the numbers IRF4 protein in CD4+CD82 DCs in CD11c-cre-Irf4+/2 mice were or survival of the P2 and P3 DC subsets. reduced. In CD11c-cre-Irf42/2 mice, the levels of IRF8 were 1670 IRF4 AND IRF8 REGULATE TERMINAL DIFFERENTIATION OF LUNG DC Downloaded from http://www.jimmunol.org/

FIGURE 2. cDC subsets in CD11c-cre-Irf4 mice differ in expression of IRF4 and IRF8, whereas heterozygotes show reduced levels of protein. (A)In CD11c-cre-Irf4 mice, lung cDCs were subdivided into P1, P2, and P3 subsets as in Fig. 1F, and intracellular IRF4 and IRF8 levels were determined using flow cytometry. For each subset in +/+, +/2, and 2/2 mice, the binding of anti-IRF4 (left panels) and anti-IRF8 (right panels) is shown. The CD11c-cre- Irf42/2 and CD11c-cre-Irf82/2 mice were used to determine the nonspecific level of anti-IRF4 and anti-IRF8 Ab binding, respectively, to DCs. (B) The mean fluorescence intensity (MFI) of anti-IRF4 and anti-IRF8 binding is shown for each cDC subset (P1, P2, P3) in CD11c-cre-Irf4+/+, CD11c-cre-Irf4+/2, and CD11c-cre-Irf42/2 mice. The number sign (#) indicates the nonspecific binding of the anti-IRF4 Ab on Irf42/2 cells. The multiplication sign (3) indicates that the P1 subset is absent in the 2/2 mouse. (C) The relative MHCII expression (normalized MFI) on the P1, P2, and P3 subsets present in CD11c-cre-Irf4+/+, CD11c-cre-Irf4+/2, and CD11c-cre-Irf42/2 mice is shown. The multiplication sign (3) indicates that the P1 subset is absent in the 2/2 mouse. (B and C) The significance of the data were evaluated using an unpaired t test (P1) or a one-way ANOVA (P2, P3), n = 4 per genotype: *p , 0.05, by guest on September 29, 2021 **p , 0.01, ****p , 0.0001. reproducibly increased in CD4+CD82 and CD42CD8+ DCs, whereas CD103+ dDCs expressed high levels of IRF8 and little suggesting that Irf4 deficiency increases expression of IRF8 in the IRF4 (Fig. 4E). eLCs expressed low levels of both IRF4 and IRF8. DCs that develop (Fig. 3D). Taken together, these data show that the IRF4-expressing tissue resident CD11b+ DC populations in the lung and dermis show a Irf4 deficiency in CD11c+ cells does not affect skin DC differential dependence on IRF4 for their development and tissue development but reduces migration of CD11b+ dDCs to local residence. draining lymph nodes + We previously published that CD11b+ dDCs and epidermal DCs IRF4 deficiency in CD11c cells does not alter numbers or (eLCs) developed normally and were present in the skin of IRF8 expression of pre-cDCs in BM or spleen globally Irf4-deficient mice (25). However, these Irf42/2 CD11b+ The immediate pre-cDCs are CD11cint pre-cDCs, defined as DC subsets failed to migrate to LN in homeostasis and inflam- lineage-negative CD11c+MHCII2SIRPaloFlt3+ (Fig. 5A). In mation, consistent with the failure to upregulate CCR7. To de- CD11c-cre-Irf42/2 and CD11c-cre-Irf4+/2 mice, in which GFP termine whether CD11c-specific Irf4 deficiency led to the same expression can be used to monitor Irf4 deletion, 60–85% of the phenotype, we analyzed skin and cutaneous LN DC populations, pre-cDCs were GFP+ (Fig. 5B). Numbers of total and GFP+ pre- gating as in Bajanã et al. (25). The percentages of total DCs and cDCs in the BM and spleen did not differ in CD11c-cre-Irf42/2, DC subsets in the dermis were similar in CD11c-cre-Irf42/2, CD11c-cre-Irf4+/2, and CD11c-cre-Irf4+/+ mice (Fig. 5B, 5C), CD11c-cre-Irf4+/2, and CD11c-cre-Irf4+/+ mice, and tended to- suggesting that Irf4 deficiency does not affect pre-cDC numbers. ward being increased in the CD11c-cre-Irf42/2 mice as if they had Intracellular staining with IRF4- and IRF8-specific mAbs showed accumulated (Fig. 4A, 4B). Although the total number of cells in that BM pre-cDCs in +/+ mice express fairly uniform levels of the cutaneous LN was not different, we detected an Irf4 gene IRF4 and IRF8; a small percentage of pre-cDCs are IRF82,but dosage-dependent reduction in numbers of CD11b+ dDCs in the their IRF4 expression level is unchanged (Fig. 5D, 5I). Thus, the cutaneous LN of CD11c-cre-Irf42/2 and CD11c-cre-Irf4+/2 mice pre-cDCs cannot be divided into cells expressing variable levels of (Fig. 4C, 4D). However, unlike the global Irf42/2 mice, the IRF4. In pre-cDCs of +/2 mice, IRF4 was present at a reduced numbers of migrating eLCs were not significantly reduced in the level relative to +/+ mice (Fig. 5D, 5E). These IRF4 levels were CD11c-cre-Irf42/2 and CD11c-cre-Irf4+/2 mice (Fig. 4D). Ex- similar to levels on mature cDCs in BM of the same +/+ and +/2 pression of GFP, indicating Irf4 deletion, was equivalent in the mice (Fig. 5F). Despite the incomplete Irf4 deletion predicted by three skin DC subsets (Supplemental Fig. 1). As in the lung, the GFP expression, we did not identify two levels of anti-IRF4 CD11b+ dDCs expressed high levels of IRF4 and little IRF8, binding in 2/2 pre-cDCs that would be indicative of IRF4 ex- The Journal of Immunology 1671 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. IRF4 expression in CD11c+ cells is required for development of splenic CD11b+CD4+DCs. (A) Definition of CD11chiMHCIIhi cDCs in the spleen of CD11c-cre-Irf4+/+ mice. (B) CD11chiMHCIIhi cells are divided into CD4+CD82, CD42CD8+, and CD42CD82 DC subsets; a comparison of +/+, +/2, and 2/2 mice is shown. (C) The total numbers of CD4+CD82, CD42CD8+, and CD42CD82 DC subsets in multiple +/+, +/2, and 2/2 mice are compiled; shown are values for individual mice, n = 6–8 per genotype. The significance of the data were evaluated using a one-way ANOVAwith a Tukey multiple-comparisons test: **p , 0.01, ***p , 0.001, ****p , 0.0001. (D) The mean fluorescence intensity (MFI) of anti-IRF4 and anti-IRF8 binding is shown for the CD4+CD82, CD42CD8+, and CD42CD82 DC subsets in +/+, +/2, and 2/2 mice. The number sign (#) indicates the background binding of the anti-IRF4 Ab on Irf42/2 cells. The binding of anti-IRF8 to Irf82/2 splenic DCs was an MFI of 195. The significance of the data were evaluated using a one-way ANOVA, n = 4 per genotype: *p , 0.05, **p , 0.01, ****p , 0.0001. pression stemming from intact Irf4 genes versus nonspecific IRF8 deficiency in CD11c+ cells alters numbers of both binding of the mAb. Indeed, a similar level of nonspecific binding IRF8 - and IRF4-dependent lung DCs 2 2 2 2 2 of the anti-IRF4 mAb to / mature BM cDCs, which were In CD11c-cre-Irf8 / , CD11c-cre-Irf8+/ ,andCD11c-cre-Irf8+/+ ∼ + 2 88% GFP , was observed (Fig. 5F). We could not directly cor- mice, we analyzed the CD11chiSiglecFloCD64 MHCIIhi lung DC relate IRF4 expression with GFP (indicating Irf4 deficiency) in population (gated as in Fig. 1). Unexpectedly, the CD103+ DC subset pre-cDCs, because GFP leaks out of cells permeabilized for in- was absent in both CD11c-cre-Irf82/2 and CD11c-cre-Irf8+/2 mice tracellular staining. IRF8 levels on pre-cDCs did not change in + +/2 2/2 (Fig. 6A, 6B). The numbers of CD11b DCs increased in both CD11c-cre-Irf4 or CD11c-cre-Irf4 mice (Fig. 5G, 5H). 2 2 2 CD11c-cre-Irf8 / and CD11c-cre-Irf8+/ mice (Fig. 6A, 6B). The Binding of the anti-IRF8 mAb to pre-cDCs of Irf8-deficient mice same results were obtained when CD11chiMHCIIhi DCs were defined was used to show background levels of mAb binding, and this by CD24 and SIRPa: numbers of SIRPaloCD24hi (P3) DCs were revealed that not all pre-cDCs in the Irf8-deficient mice have reduced whereas SIRPa+CD24hi (P1) DCs were increased in both deleted Irf8 (Figs. 5G). 2/2 +/2 Taken together, these data show that Irf4 deficiency in CD11c+ cells CD11c-cre-Irf8 and CD11c-cre-Irf8 mice (Fig. 6C, 6D). + int (as judged by GFP expression) did not alter numbers of GFP+ pre- Numbers of SIRPa CD24 (P2) DCs did not change in the CD11c- 2/2 +/2 cDCs or their expression of IRF8. This is consistent with the absence cre-Irf8 and CD11c-cre-Irf8 mice (Fig. 6D). Thus, one copy of hi hi + of an effect of Irf4 deficiency on numbers of lung and splenic IRF8- the Irf8 gene is insufficient to promote lung CD11c MHCII CD103 dependent DCs, and suggests that IRF4 is not needed for mainte- DC development, and zero or one copy of Irf8 leads to increased nance of normal numbers of pre-cDCs. Furthermore, our data using differentiation of CD11b+SIRPa+CD24hi DCs. Interestingly, in 2 2 2 this intracellular staining approach did not reveal a relationship be- CD11c-cre-Irf8 / and CD11c-cre-Irf8+/ mice, IRF4 protein levels tweenlevelsofIRF4andIRF8inindividualpre-cDCsintheBM. were increased in the P2, but not the P1, DC subset (Fig. 6E), indi- 1672 IRF4 AND IRF8 REGULATE TERMINAL DIFFERENTIATION OF LUNG DC

FIGURE 4. Irf4 deﬁciency in CD11c+ cells does not affect skin DC development but reduces migration of CD11b+ dDCs to local draining lymph nodes. (A and B) The percentages of total dDCs and distinct DC subsets (eLC, CD103+, CD11bhi) in the skin of multiple CD11c-cre-Irf4+/+, CD11c-cre-Irf4+/2, and CD11c-cre-Irf42/2 mice, n = 2–3. (C and D) Numbers of LN cells and migratory DC subsets in CD11c-cre-Irf4+/+, CD11c-cre-Irf4+/2, and CD11c-cre- Irf42/2 mice, n = 4–7. (E) The MFI of anti-IRF4 and anti- IRF8 binding is shown for the eLC, CD103+, and CD11bhi DC subsets in +/+, +/2 and 2/2 mice. The number sign (#) indicates the background binding of the anti-IRF4 Ab on 2/2 2/2 hi Irf4 cells. The binding of anti-IRF8 to Irf8 CD11b Downloaded from DCs was an MFI of 102. The signiﬁcance of the data was evaluated with a one-way ANOVA with a Tukey’s multiple comparisons test, **p , 0.01. http://www.jimmunol.org/

cating that the increased numbers of the IRF4-dependent P1 DCs is the +/2 and 2/2 mice harbored an IRF8 dose-dependent decrease in not due to their significantly increased IRF4 levels in mature DCs. the numbers of pre-cDCs in the spleen (Fig. 7B, 7C). Intracellular CD11c-cre-Irf82/2 and CD11c-cre-Irf8+/2 mice did not show staining with IRF4- and IRF8-specific mAbs showed that BM pre- 2 differences in the numbers of lung CD11cintSiglecFloCD64 cDCs in +/+ mice expressed uniform levels of IRF4 and IRF8, by guest on September 29, 2021 MHCIIhi DCs (Supplemental Fig. 2I), yet the Irf82/2 DCs which did not permit identification of pre-cDC subsets (Fig. 7D–G). expressed elevated levels of IRF4 (Supplemental Fig. 2J), as ob- In the 2/2 mice, not all of the pre-cDCs appeared to have deleted served for the CD11chi P2 population (Fig. 6E). Numbers of the Irf8 gene, as approximately half of the cells expressed IRF8 CD11chiSiglecFhiCD64+ AMs also were unaffected by Irf8 defi- (Fig. 7D). Notably, gating on the IRF82 pre-cDCs in the BM of 2/2 ciency (Supplemental Fig. 2K). However, in CD11c-cre-Irf82/2 mice revealed an increase in IRF4 expression relative to the IRF8+ mice, the AMs displayed reduced levels of CD64 (FcgRI); pre-cDCs (Fig. 7D–G). Taken together, these data show that IRF8 this was not observed on AMs in CD11c-cre-Irf42/2 mice deficiency reduces numbers of splenic pre-cDCs, and the remaining (Supplemental Fig. 2H, 2L). This suggests that the Irf82/2 mac- pre-cDCs express elevated IRF4 compared with +/+ pre-cDCs. This rophages may be functionally impaired. elevated IRF4 in pre-cDCs likely explains the increased development of IRF4-dependent CD11b+ DCs in the lung and spleens of + IRF8 deficiency in CD11c cells alters both IRF8- and CD11c-cre-Irf82/2 mice (Fig. 6). IRF4-dependent splenic DC differentiation Consistent with previous reports in globally Irf8-deficient or Discussion mutant mice (42), in CD11c-cre-Irf82/2 and CD11c-cre-Irf8+/2 Through studies of CD11c-cre-Irf42/2 and CD11c-cre-Irf4+/2 mice, we observed a reduction in the percentages and numbers of mice, we determined whether IRF4 deficiency or haplo- splenic CD42CD8+ (CD11b2) DCs (Fig. 6F, 6G). These data insufficiency in CD11c+ cells altered the numbers of pre-cDCs show that a reduction of IRF8 activity beginning at the pre-cDC and specific DC subsets in the lung, dermis, and spleen. In this stage decreases the development of splenic CD42CD8+ DCs. The study, we have shown that CD11c-cre-Irf42/2 mice selectively CD11c-cre-Irf82/2 mice also showed a significant increase in the lacked a lung resident CD11b+CD11chiSIRPa+CD24hiMHCIIhi numbers of CD4+CD82 (CD11b+) DCs (Fig. 6G). This increment DC subset, whereas other lung CD11b+ CD642 (CD11chi or in CD4+CD82 DCs was not in proportion to the reduction in CD11cint) DC subsets and CD11chi AMs were present in normal CD42CD8+ DCs, but suggests that the absence of IRF8 expression numbers. Consistent with prior studies of globally Irf42/2 mice, potentiates the development of IRF4-dependent DCs, as we ob- the CD11c-cre-Irf42/2 and CD11c-cre-Irf4+/2 mice also showed a served in the lung. Numbers of CD42CD82 (CD11b+) DCs were gene dose-dependent reduction in splenic CD4+CD11b+ DCs, but not affected by IRF8 deficiency (Fig. 6G). no defect in numbers of CD11b+ dDCs. Measurement of IRF4 and

+ IRF8 protein levels revealed that IRF4 is expressed at the highest IRF8 deﬁciency in CD11c cells leads to decreased numbers of levels in CD11b+ DCs and less so in Irf8-dependent DCs in the splenic pre-cDCs expressing elevated levels of IRF4 spleen and lung, whereas IRF8 is expressed almost exclusively in Although numbers of pre-cDCs in the BM of CD11c-cre-Irf82/2, Irf8-dependent DCs. A reduced amount of IRF4 protein was CD11c-cre-Irf8+/2,andCD11c-cre-Irf8+/+ mice were similar (Fig. 7A), present in +/2 CD11b+CD11chi DCs, but this amount was ∼75% The Journal of Immunology 1673 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. IRF4 deficiency in CD11c+ cells does not alter numbers or IRF8 expression of pre-cDCs in BM or spleen. (A) Definition of pre-cDCs in the BM of CD11c-cre-Irf4+/+ mice as lineage-negative (Lin2, CD192CD32NK1.12Ter1192B2202) CD11c+MHCII2SIRPaloFlt3+.(B and C) The numbers of total pre-cDCs and GFP+ pre-cDCs in the BM and spleen in multiple +/+, +/2, and 2/2 mice are compiled, n = 4–6 per genotype. (D) IRF4 protein levels were determined in +/+, +/2, and 2/2 BM pre-cDCs by intracellular staining, and (E) the anti-IRF4 MFI values were compiled from multiple mice. (F) IRF4 protein levels were determined in CD11c+MHCII+SIRPa+ cDCs in BM of +/+, +/2, and 2/2 mice by intracellular staining, and the anti-IRF4 MFI values were compiled from multiple mice. (G) IRF8 protein levels were determined in +/+, +/2, and 2/2 pre-cDCs by intracellular staining, and (H) the anti-IRF8 MFI values were compiled from multiple mice. Binding of the anti-IRF8 mAb to pre-cDCs of Irf8-deficient mice was used to show background levels of mAb binding, and this revealed that not all pre-cDCs in the Irf8-deficient mice have deleted Irf8 (see Fig. 7). (I) Binding of anti-IRF4 and anti- IRF8 mAbs to pre-cDCs in +/+ mice. of the amount in +/+ DCs, consistent with near-normal numbers of ment. In contrast, the spleens of CD11c-cre-Irf82/2 and CD11c- the IRF4-dependent DCs in +/2 mice. IRF4 reduction or absence cre-Irf8+/2 mice showed an Irf8 dose-dependent reduction in led to minimal changes in IRF8 expression. Irf4 deficiency (as CD42CD8+ DCs. Irf8 deficiency led to a reduced number of judged by GFP expression) did not alter numbers of pre-cDCs in splenic pre-cDCs with increased IRF4 expression relative to the BM and spleen, or pre-cDC expression of IRF8; this was +/+ mice, which correlated with increased numbers of IRF4- consistent with normal numbers of IRF8-dependent DCs in the dependent DCs in the spleen and lung. Taken together, these CD11c-cre-Irf42/2 and CD11c-cre-Irf4+/2 mice. data from CD11c-cre-Irf82/2 and CD11c-cre-Irf42/2 mice show Similar experiments were done with CD11c-cre-Irf82/2 and that IRF4 and IRF8 are critically required in pre-cDCs to direct CD11c-cre-Irf8+/2 mice, in which Irf8 is deleted in CD11c+ cells. the terminal differentiation of select cDC subsets in the lung and CD11c-cre-Irf82/2 and CD11c-cre-Irf8+/2 mice both lacked lung- spleen. resident CD103+ DCs, indicating that a single copy of Irf8 leads to Adoptive transfer and lineage tracing studies have shown that insufficient amounts of IRF8 required to promote their develop- lung resident CD11b+ DCs arise from both pre-cDCs and mono- 1674 IRF4 AND IRF8 REGULATE TERMINAL DIFFERENTIATION OF LUNG DC Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. IRF8 deficiency in CD11c+ cells alters numbers of both IRF8- and IRF4-dependent lung and spleen DCs. (A) CD11chiMHCIIhi DCs (gate R1) in the lungs of CD11c-cre-Irf8 mice were gated as in Fig. 1A and divided into CD11b+ and CD103+ subsets; a comparison of +/+, +/2, and 2/2 mice is shown. (B) The total numbers of CD11b+ and CD103+ DC subsets in multiple +/+, +/2, and 2/2 mice are compiled; shown are values for individual mice, n = 10–15 per genotype. (C) Lung CD11chiMHCIIhi cells in gate R1 (see Fig. 1A) are divided into subsets based on SIRPa and CD24; a comparison of +/+, +/2, and 2/2 mice is shown. (D) The total numbers of SIRPa+CD24hi (P1), SIRPa+CD24int (P2), and SIRPaloCD24hi (P3) DC subsets in multiple +/+, +/2, and 2/2 mice are compiled, n = 7–8 per genotype. (E) The mean fluorescence intensity (MFI) of anti-IRF4 and anti-IRF8 binding is shown for each lung cDC subset (P1, P2, P3) in CD11c-cre-Irf8+/+, CD11c-cre-Irf8+/2, and CD11c-cre-Irf82/2 mice. The number sign (#) indicates the nonspecific binding of the anti-IRF8 Ab to Irf82/2 cells. The multiplication signs (3) indicate that the P3 subset is absent in the +/2 and 2/2 mice. The significance of the data was evaluated using a one-way ANOVA (P1, P2), n = 3 per genotype: *p , 0.05, **p , 0.01. (F) Splenic CD11chiMHCIIhi cells (gated as in Fig. 3A) are divided into CD4+ and CD8a+ subsets; a comparison of +/+, +/2, and 2/2 mice is shown. (G) The total numbers of CD4+ and CD8a+ splenic DC subsets in multiple +/+, +/2, and 2/2 mice are compiled; shown are values for individual mice, n = 5–11 per genotype. The significance of the data in (B), (D), and (G) was evaluated using a one-way ANOVAwith a Tukey multiple-comparisons test: *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. cytes in homeostasis (28, 29, 41, 43, 44). The CD11b+ population differentiation pathway. This latter possibility is supported by is heterogeneous, and these initial studies did not clarify whether studies showing that a significant proportion of lung CD11b+ DCs distinct subsets of lung resident CD11b+MHCIIhi DCs were de- originate from monocytes (41, 43). rived from different precursors. In this study, we have subdivided A prior report using the same CD11c-cre-Irf42/2 mice showed the CD11b+MHCIIhi DCs to show that the CD11chiSIRPa+CD24hi that numbers of lung resident CD11b+ tissue DCs were reduced, subset is uniquely dependent on IRF4 expression in CD11c+ cells, but not absent (6). Based on evidence for increased apoptosis of whereas two other CD642 subsets, CD11chiSIRPa+CD24int and these CD11b+ DCs, the conclusion was made that IRF4 was re- CD11cintSIRPa+CD24int, are not. This suggests that the Irf4-de- quired in mature CD11b+ DCs for their survival, but not their pendent lung CD11b+ DCs arise from CD11c+ pre-cDCs, whereas development from pre-cDCs. In contrast, our more detailed sep- the Irf4-independent CD11b+ DCs arise from CD11c2 monocytes. aration of the CD11chi CD11b+ tissue DC subset in the lung However, it is formally possible that Irf4-dependent CD11b+ DCs revealed that in fact one CD11b+ DC subset (SIRPa+CD24hi)is also arise from monocytes and are deficient in these mice because absent, whereas a second CD11b+ DC subset (SIRPa+CD24int)is IRF4 is required at a later CD11c+ stage of the monocyte-to-DC present in normal numbers. Furthermore, levels of activated cas- The Journal of Immunology 1675 Downloaded from http://www.jimmunol.org/

FIGURE 7. IRF8 deficiency in CD11c+ cells leads to decreased numbers of splenic pre-cDCs expressing elevated levels of IRF4. (A) The numbers of pre- cDCs in the BM of multiple CD11c-cre-Irf8+/+, CD11c-cre-Irf8+/2, and CD11c-cre-Irf82/2 mice are compiled, n = 4–9 per genotype. (B) Shown is the gating of SIRPa2Flt3+ pre-cDCs within the lineage-negative (CD192CD32NK1.12Ter1192B2202) CD11c+MHCII2 fraction of splenocytes in CD11c- cre-Irf8+/+, CD11c-cre-Irf8+/2,andCD11c-cre-Irf82/2 mice. (C) The numbers of pre-cDCs in the spleens of multiple CD11c-cre-Irf8+/+, CD11c-cre-Irf8+/2, and CD11c-cre-Irf82/2 mice are compiled, n = 5–7 per genotype. (A and C) The significance of the data was evaluated using a one-way ANOVAwith a Tukey by guest on September 29, 2021 multiple-comparisons test, **p , 0.01. (D) IRF8 protein levels were determined in +/+, +/2,and2/2 BM pre-cDCs by intracellular staining. Binding of the anti-IRF8 mAb to pre-cDCs of Irf8-deficient mice revealed that not all pre-cDCs in the Irf8-deficient mice have deleted Irf8.(E) The anti-IRF8 MFI values were compiled from multiple mice (n = 2–3). In 2/2 mice, the IRF8 MFI is reported separately for the IRF8+ and IRF82 populations. (F) IRF4 protein levels were determined in +/+, +/2, and 2/2 pre-cDCs by intracellular staining, and (G) the anti-IRF4 MFI values were compiled from multiple mice (n = 2–3). In 2/2 mice, the IRF4 MFI is reported separately for the IRF8+ and IRF82 populations. pase 3 in the remaining CD11b+ DCs were low in CD11c-cre- inability to migrate to LN, as we and others observed previously Irf42/2 mice, suggesting that their survival was not impaired. (11, 25). Although both pre-cDCs and monocytes were reported to Thus, we favor the interpretation that IRF4 is selectively required give rise to lung and dermal CD11b+ DCs, it is notable that lung for the terminal differentiation of the CD11chiSIRPa+CD24hi CD11b+ DCs were found to be much more dependent on Flt3L subset of lung CD11b+ DCs from pre-cDCs. (29). Thus, our data suggest that the Flt3L-driven, IRF4-dependent Our finding is consistent with a recent report that a develop- pathway for pre-cDC development into CD11b+ DCs proceeds to mental requirement for KLF4 subdivides IRF4-expressing CD11b+ a greater extent in the lung than in the dermis. Development of DCs. KLF4 is required for the differentiation of lung CD11chi splenic CD4+CD11b+ DCs also was significantly impaired with SIRPa+CD24hi DCs that promote Th2 responses, but not CD11chi Irf4 deficiency restricted to CD11c+ cells, consistent with prior SIRPa+CD24int DCs that promote Th17 responses (10). This work showing that most splenic DCs develop from pre-cDCs (45). may help to explain observations in CD11c-cre-Irf42/2 mice that The distinct developmental pathways of CD11b+ DCs in different IRF4-dependent DCs are required for both Th2 and Th17 re- peripheral tissues may arise because of differences in homeostatic sponses. The deficit of Th17 responses upon Aspergillus fumigatus tissue microenvironments, such as levels of Flt3L, M-CSF, or GM- challenge of CD11c-cre-Irf42/2 mice (6) is likely due to IRF4 CSF directing pre-cDC–derived versus monocyte-derived DC deficiency in the CD11chiSIRPa+CD24int DCs, which are present differentiation, or the production of chemokines that preferentially in the lungs but may lack capacity to produce IL-23 or migrate to attract monocytes or pre-cDCs. LNs. Recent reports demonstrated the subdivision of the pre-cDC It is surprising that Irf4 deficiency restricted to CD11c+ cells led population into discrete precursors for cDC1s and cDC2s (30, to differential effects on CD11c+CD11b+MHCIIhi DCs in the lung 31). Pre-cDC1s preferentially express Irf8 and Batf3 mRNA, and the dermis. Whereas a subset of CD11b+ lung DCs expresses whereas pre-cDC2s preferentially express Irf4 mRNA. Our own and depends on IRF4 expression as outlined earlier, our data show attempts to subdivide the pre-cDCs into discrete populations based that numbers of CD11b+ DCs in the dermis were not reduced by on relative IRF4 and IRF8 protein expression were unsuccessful. this same Irf4 deficiency. In fact, numbers of dermal CD11b+ DCs This could be because of incomplete deletion of the Irf4 or Irf8 were increased in CD11c-cre-Irf42/2 mice, consistent with their genes because use of the GFP reporter showed that the CD11c-cre 1676 IRF4 AND IRF8 REGULATE TERMINAL DIFFERENTIATION OF LUNG DC did not act in all CD11cint pre-cDCs in the CD11c-cre-Irf42/2 immunity (36). Identified polymorphisms in the human IRF4 mice (Fig. 5B). Alternately, Grajales-Reyes et al. (30) reported promoter change transcription factor binding and gene expression that pre-cDC2s do express IRF8 directly ex vivo, and the IRF8 is levels (34, 35). Interestingly, Irf4 RNA levels also are increased or lost as the pre-cDC2s differentiate in vitro. decreased by physiological cues such as prostaglandins and es- We investigated the effect of Irf4 and Irf8 deficiency beginning trogens (40, 49). Thus, consistent with our data obtained from in the pre-cDCs, thus bypassing an effect on CDPs. Irf4 deficiency these murine models during homeostasis, variable IRF4 or IRF8 (as judged by GFP expression) did not apparently change numbers expression resulting from genetic polymorphisms or environ- of total pre-cDCs or their expression of IRF8, consistent with mental stimuli may govern tissue DC numbers, and therefore unchanged numbers of IRF8-dependent DCs in the lung and regulate the magnitude of DC functional responses during in- spleen. Thus, IRF4 is apparently not required for maintenance of flammation. pre-cDC2s, although we cannot rule this out definitively because we were unable to assess IRF4 expression and GFP indicating Acknowledgments Irf4 deletion at the same time using intracellular flow cytometry. We thank Dr. Jose´ Alberola-Ila for helpful discussions and Mike Jones Furthermore, the absence of IRF4 does not significantly alter IRF8 for assistance with the animal colony. expression or divert pre-cDC2s into the cDC1 lineage. In contrast, Irf8 deficiency led to normal numbers of BM pre-cDCs expressing Disclosures higher levels of IRF4 and a reduction in splenic pre-cDC numbers, The authors have no financial conflicts of interest. which correlated with increased numbers of IRF4-dependent lung and splenic DCs. This suggests that IRF8 is required for mainte- Downloaded from nance of a pre-cDC1 population in the spleen, and that the absence References of IRF8 allows greater expression of IRF4 in the remaining 1. Braciale, T. J., J. Sun, and T. S. Kim. 2012. Regulating the adaptive immune 2/2 response to respiratory virus infection. Nat. Rev. Immunol. 12: 295–305. pre-cDC2s. Indeed, in Batf3 mice lacking appropriate IRF8 2. Lambrecht, B. N., and H. Hammad. 2012. Lung dendritic cells in respiratory autoactivation, the pre-cDC1s did not commit to the CD8+ DC viral infection and asthma: from protection to immunopathology. Annu. Rev. lineage and were diverted into the CD4+CD11b+ lineage (30). 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