The C/EBPβ orchestrates dendritic cell maturation and functionality under homeostatic and malignant conditions

Florian Scholza, Michael Graub, Lutz Menzelc, Annika Grabanda,1, Myroslav Zapukhlyakb, Achim Leutzd, Martin Janze,f, Georg Lenzb, Armin Rehmc,2,3, and Uta E. Höpkena,2,3

aDepartment of Microenvironmental Regulation in Autoimmunity and Cancer, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany; bDepartment of Medicine A, University Hospital Münster, 48149 Münster, Germany; cDepartment of Translational Tumorimmunology, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany; dDepartment of Cell Differentiation and Tumorigenesis, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany; eExperimental and Clinical Research Center, Charité, University Hospital Berlin, 13125 Berlin, Germany; and fDepartment of Biology of Malignant Lymphomas, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany

Edited by Kenneth M. Murphy, Washington University in St. Louis School of Medicine, St. Louis, MO, and approved August 31, 2020 (received for review May 12, 2020) Dendritic cell (DC) maturation is a prerequisite for the induction of The CCAAT/Enhancer binding alpha and beta (C/ adaptive immune responses against pathogens and cancer. Tran- EBPα, C/EBPβ) of the basic (bZIP) class (9) are scription factor (TF) networks control differential aspects of early two TFs that are regulated by mTOR. C/EBPβ has been found to DC progenitor versus late-stage DC cell fate decisions. Here, we activate target via the recruitment of the coactivator identified the TF C/EBPβ as a key regulator for DC maturation and CREB-binding /P300 (10). Given that E2F1 is a negative immunogenic functionality under homeostatic and lymphoma- regulator of DC maturation (11), the mTOR–C/EBPβ axis may transformed conditions. Upon cell-specific deletion of C/EBPβ in potentially affect maturation of DC through E2F. + − − CD11c MHCIIhi DCs, expression profiles of splenic C/EBPβ / The functional properties of C/EBP family members (C/ DCs showed a down-regulation of E2F target genes and EBPα−C/EBPζ) comprise the regulation of cellular growth and associated proliferation signaling pathways, whereas maturation differentiation, immune and inflammatory responses, but also signatures were enriched. Total splenic DC cell numbers were mod- metabolism and tumorigenesis (12). In myelomonocytic lineages IMMUNOLOGY AND INFLAMMATION estly increased but differentiation into cDC1 and cDC2 subsets C/EBPβ is highly expressed and regulates cytokines, proliferation + hi + were unaltered. The splenic CD11c MHCII CD64 DC compart- and differentiation markers (13, 14). Moreover, regulated trans- ment was also increased, suggesting that C/EBPβ deficiency favors lation initiation, e.g., downstream of mTOR, controls expression the expansion of monocytic-derived DCs. Expression of C/EBPβ of two long (termed LAP* and LAP) and one truncated C/EBPβ could be mimicked in LAP/LAP* isoform knockin DCs, whereas (LIP) protein isoforms from alternative messenger RNA in the short isoform LIP supported a differentiation program similar to deletion of the full-length TF. In accordance with E2F1 being a − − Significance negative regulator of DC maturation, C/EBPβ / bone marrow- derived DCs matured much faster enabling them to activate and polarize T cells stronger. In contrast to a homeostatic condition, Complex transcription factor networks regulate the develop- lymphoma-exposed DCs exhibited an up-regulation of the E2F ment, maturation, and lineage commitment of dendritic cell transcriptional pathways and an impaired maturation. Pharmaco- (DC) subsets. Here, we demonstrate a previously unexpected β logical blockade of C/EBPβ/mTOR signaling in human DCs abro- role of the transcription factor C/EBP in murine DC matura- gated their protumorigenic function in primary B cell lymphoma tion and immunogenic functionality under homeostatic and cocultures. Thus, C/EBPβ plays a unique role in DC maturation and lymphoma-transformed conditions. Regulated expression of β immunostimulatory functionality and emerges as a key factor of functional C/EBP isoforms enables a controlled maturation of the tumor microenvironment that promotes lymphomagenesis. DCs. In contrast, the presence of lymphoma cells leads to an up- regulation of C/EBPβ in DCs which transforms them into an immature and protumorigenic subtype. This study also shows dendritic cell maturation | transcription factor C/EBPβ | β lymphoma–stroma interaction that inhibition of the C/EBP /mTOR signaling axis abrogates the protumorigenic function of human DCs, suggesting that in- hibitors regulating C/EBPβ activity can be used for blocking tumor- endritic cells (DCs) play a central role in the initiation of promoting functions of DCs in the treatment of hematological Dadaptive immune responses against pathogens (1) and in the neoplasms. immunity against cancer (2, 3). Lineage commitment and mat- uration into specific DC subsets is important for the induction of Author contributions: A.R. and U.E.H. designed research; F.S., M.G., L.M., A.G., M.Z., M.J., immunity and tolerance (4). DCs can modulate their ability to A.R., and U.E.H. performed research; F.S., M.G., L.M., A.G., M.Z., A.L., M.J., G.L., A.R., and prime either effector or regulatory T cells due to their out- U.E.H. analyzed data; F.S., A.R., and U.E.H. wrote the paper; and A.L. and G.L. advised on experiments. standing capability to present antigens together with overlaying The authors declare no competing interest. regulatory elements that promote tolerance or immunity (5). The This article is a PNAS Direct Submission. molecular mechanisms implicated herein involve distinct cyto- Published under the PNAS license. kines and transcription factors (TFs). 1Present address: CECAD Research Center, Cluster of Excellence, University Cologne, TF networks in DC development can be grouped into two major 50931 Cologne. categories: the first consists of TFs that regulate early DC devel- 2 A.R. and U.E.H. contributed equally to this work. opment, i.e., Ikaros, PU.1, and Gfi1. The second group comprises 3To whom correspondence may be addressed. Email: [email protected] or more lineage-restricted TFs regulating late stages of DC cell fate [email protected]. decisions, i.e., RelB, Id2, IRF4, IRF8, Batf3, and Bcl6 (6, 7). The This article contains supporting information online at https://www.pnas.org/lookup/suppl/ mammalian target of rapamycin (mTOR)-mediated signaling doi:10.1073/pnas.2008883117/-/DCSupplemental. pathway also affects DC differentiation and maturation (8).

www.pnas.org/cgi/doi/10.1073/pnas.2008883117 PNAS Latest Articles | 1of12 Downloaded by guest on September 27, 2021 + frame start sites that exhibit opposing biological functions; these observed a modest increase in total splenic CD11c MHChi DC + + isoforms were previously reported to change monocytic differ- numbers, the proportion of BrdU CD11c MHChi DCs was entiation pathways (14, 15). similar in both genotypes (Fig. 1F). Next, we compared the C/EBPβ can either inhibit or promote cell cycle progression frequencies and the total numbers of the cDC1 and cDC2 sub- + + + during physiologic and neoplastic growth (13). In addition to sets in the spleen. By gating on CD11c MHCII CD64 or − serving as a direct therapeutic target in tumors (16), its impor- CD64 DCs, we observed a significant increase in the percentage and + + − − tant role in the monocytic and MDSC lineage development (17, total numbers of CD11c MHCIIhiCD64 in C/EBPβ / compared + + + 18) suggests that C/EBPβ may also be involved in shaping an to C/EBPβ / mice (Fig. 1G). Since CD64 DCs are described to immunosuppressive tumor microenvironment (19). be monocytic-derived (mo-DCs) rather than classical DCs (cDCs) Here, we analyzed the consequences of C/EBPβ loss on DC (30), we conclude that C/EBPβ deficiency favors the expansion of maturation and function under homeostatic and lymphoma- monocytic-derived DCs. Analysis of cDC1 and cDC2 subset dis- + − transformed conditions. tribution within the CD11c MHCIIhiCD64 compartment revealed that the majority of DCs could be attributed to the cDC2 + Results (CD172a ) subset in both groups (Fig. 1H). Likewise, the fre- C/EBPβ Deficiency Causes Down-Regulation of E2F Target Genes but quencies and expression levels of activation and costimulatory Leads to an Enrichment of Differentiation and Maturation Gene molecules (MHCII, CD40, CD80, and CD86) (SI Appendix,Fig.S1C) −− + + Signatures. We recently showed that lymphoma-exposed DCs were similar in splenic C/EBPβ / compared to C/EBPβ / DCs. In up-regulated C/EBPβ which was associated with a less mature addition, further differentiation into splenic cDC1 and cDC2 subsets and more regulatory phenotype leading to protumorigenic cy- resulted in comparable frequencies (Fig. 1H), DC subset-specific gene tokine secretion (20). signatures, and maturation states (MHCII, CD40, CD80, CD86, CD4, To examine the role of C/EBPβ in the regulation of DC cell CD8, CD24, CD207, CD283; SI Appendix,Fig.S1D and E), inde- fate decision under homeostatic conditions, we employed pendent of C/EBPβ expression. We conclude that lack of C/EBPβ genome-wide expression profiling (GEP) of C/EBPβ-proficient favors DC maturation; however, under homeostatic conditions + and C/EBPβ-deficient murine DCs. Splenic CD11c MHCIIhi in spleen this maturation program does not lead to an altered + + DCs were sorted from CD11c-Cre-eGFP (C/EBPβ / ) and from DC frequency or differentiation state. − − CD11c-Cre-eGFPxC/EBPβflox/flox (C/EBPβ / ) mice (20). Ap- plying gene set enrichment analysis (GSEA), a significant en- Lack of C/EBPβ Facilitates Faster Maturation of DCs In Vitro. Next, we richment of gene sets associated with maturation, proliferation, phenotypically characterized the maturation of DCs differentiated in vitro − − and transcriptional regulation was found in C/EBPβ / DCs from GM-CSF cultured bone marrow (BM) progenitor cells. The fre- + + + + + compared to C/EBPβ / DCs (SI Appendix, Table S1A). Strik- quencies of pre-DCs (CD11c CD172a CD135 ), monocyte-dendritic cell − + + + + ingly, E2F target genes were strongly down-regulated in C/ progenitors (Lin-Sca1 CD117 CD135 CX3CR1 CD115 ), or common − − − + + + EBPβ / DCs, as demonstrated by the enrichment of four dif- dendritic cell precursors (Lin-Sca1 CD117 CD135 CD115 )atday ferent E2F-related gene signatures with the highest enrichment 0 and day 3 of cell culture were similar within BM progenitor cells derived −− ++ score (Fig. 1 A and B and SI Appendix, Table S1A). GSEA data from C/EBPβ / and C/EBPβ / mice (SI Appendix,Fig.S2A and B). + −− were supported by real-time PCR (RT-qPCR) of specific down- At day 7, frequencies of CD11c C/EBPβ / cells were substantially in- regulated E2F target genes, such as timeless, Cdc20, Tacc3, and creased with a concomitant higher MHC class II (I-A/I-E) surface ex- Pik1 (Fig. 1C). The TF E2F1 suppresses DC maturation through pression, indicating a more mature phenotypic status of C/ − − C/EBPβ which directly activates E2F-regulated genes (10, 11). In EBPβ / DCs (Fig. 2 A and B). The overall cell yield of C/ − − + line, we detected down-regulation of a gene set for retinoblas- EBPβ / CD11c MHChi DCs was significantly (2.7-fold) in- − − + + + toma 1 (Rb1) (21) in C/EBPβ / DCs compared to C/EBPβ / creased (Fig. 2C), although the proliferation rate (BrdU )of + DCs. Rb1 can directly interact with E2F and the Rb/E2F path- CD11c MHChi DCs was unaffected by C/EBPβ deficiency (Fig. 2D). + way regulates initiation of DNA replication, a process enabling When CD11c MHCIIhi DCs were further distinguished for DC subset + + cell proliferation. Hence, loss of C/EBPβ most likely impairs Rb1 composition, virtually all DCs were CD11c MHCIIhiCD172a and target . Accordingly, gene signatures related to XCR1-negative (SI Appendix,Fig.S2C). Thus, DCs differentiated proliferation and cell cycle progression (22–24) were down- in vitro from GM-CSF cultured BM progenitors gave rise exclusively − − regulated in C/EBPβ / DCs (SI Appendix, Table S1A). On the to the cDC2 subset. Alternatively, we cultured BM progenitors for contrary, gene signatures for differentiation and maturation (25, 7 d in the presence of Flt3-ligand, a nonredundant cytokine for − − 26) were enriched among genes up-regulated in C/EBPβ / DCs DC development (31, 32). These culture conditions generated + + compared to C/EBPβ / DCs. In addition, GSEA indicated a comparable frequencies of CD11cintMHCIIhi expressing cells in significant enrichment of genes that are up-regulated upon both genotypes (SI Appendix,Fig.S2D). Because of the inter- treatment with IFN-γ, suggesting the induction of a maturation mediate expression level of CD11c, Cre activity is unlikely to be program when C/EBPβ is lost (SI Appendix, Table S1A and Fig. 1 sufficient for C/EBPβ excision and thus, this transgenic mouse D and E). This list contained genes involved in costimulation model is not suitable to analyze the impact of C/EBPβ deletion on (Cd80 and Cd86), chemokine expression (Ccr1), and DC differentiation. toll-like receptors (Tlr2, 4, and 8) known to be regulated by ac- CD11b plays an important role in cell adhesion and migration tivation; functionally, these genes regulate productive innate and and is also involved in the endocytosis of complement-bound adaptive immune responses. Notably, a gene encoding for the antigens. Upon activation of DCs, CD11b is down-regulated homing receptor CCR7 with a leading role in DC maturation, because its expression limits DC migration from peripheral tis- was also significantly up-regulated (P = 0.013). We conclude that sues to secondary lymphoid organs (SLOs) and thus prevents + loss of C/EBPβ in DCs on the one hand led to a profound down- T cell activation (33). Here we found that CD11c MHCIIhi BM- regulation of proliferation signaling pathways, while on the other derived DCs exhibited lower expression of CD11b when C/EBPβ hand a strong enrichment of maturation gene signatures was is lacking (Fig. 2B). Furthermore, surface expression of CD205, a observed. receptor for clathrin-mediated antigen uptake, was profoundly + + To explore the influence of C/EBPβ deletion on proliferation up-regulated during cell culture differentiation in C/EBPβ / + − + − − and cDC1 (CD11c MHCIIhiCD64 XCR1 ) versus cDC2 DCs, but not in C/EBPβ / DCs (Fig. 2B). CD205 facilitates + − + (CD11c MHCIIhiCD64 CD172a ) subset differentiation (27– detection of apoptotic cells and induces tolerance through up- + + − − 29), we first fed C/EBPβ / and C/EBPβ / mice with bromo- take of self-antigens, a feature associated with an intermediate deoxyuridine (BrdU) for 3 consecutive days. Although we degree of DC maturation (34).

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Fig. 1. C/EBPβ deletion in splenic DCs leads to down-regulation of E2F target genes and up-regulation of DC activation gene signatures. (A and D) Heatmaps displaying differential gene expression of E2F targets (A) and up-regulation of DC activation genes (D) in splenic C/EBPβ−/− (n = 3) in comparison to C/EBPβ+/+ DCs (n = 6). Gene expression changes are color coded. Enrichment of the E2F target gene signature is depicted in B.(C) Differential expression of specific E2F − − + + target genes was validated by qRT-PCR in C/EBPβ / (n = 3 mice) compared to C/EBPβ / (n = 6) splenic DCs. The expression of timeless, Cdc20, Tacc3, and Plk1 was assessed by TaqMan RT-PCR. Bars represent mean ± SEM; a Student’s t test was used; *P < 0.05; ***P < 0.001. Enrichment of the DC activation gene + + − − signature is depicted in E.(F) C/EBPβ / (n = 4) and C/EBPβ / (n = 3) mice were injected intraperitoneally on 3 consecutive days with 100 μL BrdU (10 mg/mL) in + PBS. After 3 d, proliferation of splenic CD11c MHCIIhi DCs was determined via BrdU staining. Representative dot plots are shown and the mean frequencies of proliferating BrdU+CD11c+MHCIIhi DCs (Lower). Upper bars depict the total numbers of splenic CD11c+MHCIIhi DCs (C/EBPβ+/+ [n = 10] and C/EBPβ−/− [n = 8] mice); a Student’s t test was used; *P < 0.05. (G) Splenic CD11c+MHCIIhi cells from C/EBPβ+/+ and C/EBPβ−/− mice were stained for CD64 and (H) further analyzed for CD64-XCR1+ (cDC1) and CD64-CD172a+ (cDC2) DC subsets. Representative dot plots are shown and bars represent means of n = 6micepergroup.Upper bars + + + − + − + + − + depict mean frequencies of CD11c MHCIIhiCD64 and CD11c MHCIIhiCD64 cells (G) and of CD11c MHCIIhiCD64 XCR1 and CD11c MHCIIhiCD64 CD172a cells (H). Lower bars in G and H show the respective total numbers of all depicted DC subsets; a Student’s t test was used; *P < 0.05; **P < 0.01; n.s., not significant.

Scholz et al. PNAS Latest Articles | 3of12 Downloaded by guest on September 27, 2021 + + − − Fig. 2. Loss of C/EBPβ induces rapid maturation of BM-derived DCs during development. (A) A total of 1 to 2 × 106 BM cells/mL from C/EBPβ / and C/EBPβ / + + − − mice were cultured with 20 ng/mL murine GM-CSF over 7 d. Representative dot plots of C/EBPβ / and C/EBPβ / BM cell cultures show the frequency of + + + − − CD11c DCs and their corresponding MHC class II (MHCII) expression after 7 d. (B) BM-derived DCs from C/EBPβ / (n = 4) and C/EBPβ / mice (n = 4) were analyzed for the frequencies of CD11c+ DCs, and the intensity of the maturation and stimulatory markers, MHCII, CD11b, and CD205 on days 3, 5, and 7 of culturing in four independent experiments. Bars represent mean ± SEM; a Mann–Whitney U test was used. *P < 0.05. Histograms show the fluorescence + intensity as geometric mean fluorescent intensity (gMFI) normalized against a fluorescent minus one (FMO) sample for all surface markers from CD11c C/ + + − − + + + − − EBPβ / (black curve) and C/EBPβ / (solid blue curve) DCs. (C) Total numbers of BM-derived CD11c MHCIIhi C/EBPβ / (open bar) and C/EBPβ / (blue bar) DCs were analyzed at day 7 of culture. Bars represent mean ± SEM; a Mann–Whitney U test was used. **P < 0.01. (D) To compare the proliferation rate of GM-CSF + + + + + − − CD11b CD11c DCs and their CD11b progenitors from C/EBPβ / (n = 4) with those from C/EBPβ / mice (n = 4), BM-derived DCs were cultured for 3 d with GM-CSF (20 ng/mL). Twenty-four hours before analysis, culture medium was supplemented with BrdU (10 μM). Bars represent the mean percentages of proliferative CD11b+BrdU+ progenitors or CD11b+CD11c+BrdU+ DCs of both groups; for statistical analysis a Mann–Whitney U test was used; n.s., not sig- nificant. (E)C/EBPβ gene expression levels in sorted BM-derived CD11c+MHCII−/+C/EBPβ+/+ cells versus CD11c+MHCIIhiC/EBPβ+/+ DCs at day 7 of culture with GM-CSF (P value 0.006; unpaired two-sample t test).

+ Next, at day 7 of culture we sorted CD11c MHCIIlo/negC/ upon DC maturation and homing to SLOs (35). Expression of + + + + + + EBPβ / immature and CD11c MHCIIhiC/EBPβ / mature DCs these molecules was compared between CD11c MHCIIhiC/ − − + + − + and performed RT-qPCR analysis. A significant 1.6-fold down- EBPβ / , CD11c MHCIIhiC/EBPβ / , and CD11c MHCIIhiC/ + + regulation (P = 0.006) of C/EBPβ expression was observed upon EBPβ / DCs at day 7 of culture (Fig. 3A). The proportion of + + DC maturation (Fig. 2E) and confirmed that C/EBPβ down- CD40 and CD86 DCs as well as the expression levels of CD80 − − regulation is tightly linked to terminal DC differentiation. increased significantly in C/EBPβ / DCs as compared with C/ These results suggest that in the absence of microbial or in- EBPβ-proficient DCs. C/EBPβ heterozygosity had an interme- flammatory stimulation loss of C/EBPβ in DCs accelerates diate effect in this regard, suggesting a dose-dependent effect of maturation. this TF (Fig. 3A). C/EBPβ is encoded by a single exon but can be translated into Loss of C/EBPβ and Its Functional Isoform Leads to an Up-Regulation three different polypeptides. The longer isoforms, termed LAP* of Costimulatory Molecules. Up-regulation of costimulatory mol- and LAP, have an N-terminal transactivation domain (TAD), ecules, including CD40, CD80, and CD86, is usually observed that is missing in the short isoform LIP. It is thought that LIP

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Fig. 3. C/EBPβ-deficient and LIP knockin DCs exhibit an up-regulation of costimulatory molecules. (A) BM-derived DCs from C/EBPβ+/+, C/EBPβ+/−, and C/ EBPβ−/− LIP and LAP/LAP* mice were cultured for 7 d in the presence of GM-CSF (20 ng/mL). Dot plots represent expression of the maturation markers by C/ + + + − − − EBPβ / (n = 11), C/EBPβ / (n = 6), and C/EBPβ / (n = 15) DCs in 11 independent experiments. Bars represent mean ± SEM; a Student’s t test was used. *P < 0.05; **P < 0.01; ***P < 0.001. (B) BM-derived DCs from LIP (n = 4) and LAP/LAP* (n = 5) mice were analyzed for surface markers as in A in four independent + + + + − − experiments. Bars represent mean ± SEM; a Mann–Whitney U test was used. *P < 0.05. (C–F)BMofRFP -C/EBPβ / and CD11c-eGFP -C/EBPβ / mice were + + + mixed in a 1:1 ratio and the cell suspension was injected i.v. into lethally irradiated Ly 5.1 wild-type mice (n = 6). After 8 wk, the proportion of RFP -C/EBPβ / + − − + + + + + + − − and CD11c-eGFP -C/EBPβ / BM (CD45.2 ) was determined (D), and the distribution of CD11c -MHCIIhi RFP -C/EBPβ / and CD11c-eGFP MHCIIhi-C/EBPβ / DCs (CD45.2+) within the CD172a and XCR1 DC subset of the organs indicated was quantitated (E and F) in two independent experiments. E shows representative dot plots of the gating strategy for the splenic DC subsets. Statistical analysis was performed comparing the distribution of % GFP+CD45.2+ and % + + RFP CD45.2 cells within the DC subsets in spleen, LN, and lung by a paired Student’s t test; *P < 0.05; **P < 0.01.

Scholz et al. PNAS Latest Articles | 5of12 Downloaded by guest on September 27, 2021 mostly acts as a repressor of its targets, whereas LAP and LAP* of migratory skin DCs to the draining LNs independent of C/ represent activators of gene expression (14). We generated DCs EBPβ expression. + from GM-CSF cultured BM of LIP and C/EBPβΔuORF (LAP/ CD103 lung DCs are important for transporting intact anti- + LAP*) knockin (KI) mice to determine selectively the role of the gens to draining LNs and priming tumor-specific CD8 T cells. various isoforms on maturation (36, 37). LIP DCs exhibited a mature Here, we found that frequencies and total numbers of − − + + phenotype comparable to that of C/EBPβ / DCs with enhanced CD11c MHCIIhiCD103 resident lung DCs under steady-state + + + + − − frequencies of CD11c , CD11c MHCIIhi, CD11c MHCIIhiCD40 , conditions were profoundly reduced in C/EBPβ / mice. In con- + + + and CD11c MHCIIhiCD86 cells. In contrast, DCs from LAP/LAP* trast, numbers of resident CD11b cDC1 DCs were unaltered + + mice developed in a similar manner to C/EBPβ / DCs, character- and showed a similar distribution in situ (SI Appendix, Fig. S3 D + − ized by lower frequencies of aforementioned subpopulations (Fig. and E). Notably, CD11c MHCIIintCD11b alveolar macro- − − 3B). Hence, the long LAP/LAP* and the truncated LIP isoforms had phages were almost completely deleted in C/EBPβ / mice, which opposing effects on DC maturation, most likely by repressing C/ emphasized previous data (39) demonstrating that in a ubiqui- EBPβ target genes involved in differentiation. tous C/EBPβ knockout model reduced peritoneal and alveolar − − To study whether C/EBPβ / DCs had different growth and macrophages occur (SI Appendix, Fig. S3G). + + differentiation dynamics in vivo compared to C/EBPβ / DCs, + + + we generated mixed BM chimera of RFP C/EBPβ / and C/EBPβ Regulates Antigen Processing in DCs and Their Capability to + − − + CD11c-eGFP C/EBPβ / BM (both CD45.2 ) in CD45.1 con- Induce Antigen-Specific Proliferative T Cell Responses. Next, we ex- genic recipient mice (Fig. 3C). Eight weeks after transfer, mixed- plored the impact of C/EBPβ expression on immunogenic DC + BM chimera were fully reconstituted (98.87 ± 0.21% CD45.2 ; functions. Antigen uptake is facilitated by clathrin-mediated + − n = 6), and the proportion of transplanted RFP to RFP BM endocytosis via the macrophage mannose receptor CD206, cells was 51.80 ± 5.55 to 48.23 ± 5.55, respectively (Fig. 3D). We which is predominantly expressed on immature DCs (40). On + + further analyzed the proportion of RFP CD11c MHCIIhiC/ BM-derived DCs at day 7, CD206 was partially down-regulated + + + + − − + − EBPβ / to eGFP CD11c MHCIIhiC/EBPβ / DCs within distinct on C/EBPβ / and even more strongerly down-regulated on C/ + + − − + + cDC1 (XCR1 ), cDC2 (CD172a ), and plasmacytoid DC (pDC) EBPβ / compared to C/EBPβ / DCs (Fig. 4A). CD206 down- + (CD11cintSiglecH ) subsets (Fig. 3 E and F and SI Appendix,Fig. regulation had a profound impact on the uptake and processing − − S3A). The small proportion of pDCs only expressed intermediate of ovalbumin (OVA) as C/EBPβ / DCs exhibited a twofold + levels of CD11c (CD11cintSiglecH ) and hence, CD11c promoter reduction (Fig. 4B). LIP DCs also showed decreased OVA up- activity is too low to activate Cre expression and C/EBPβ down- take and processing compared to LAP/LAP* DCs (Fig. 4C). As regulation. Accordingly, a near complete lack of GFP positivity on DCs mature, they develop a higher metabolic need of iron (41, + CD11cintSiglecH cells (SI Appendix, Fig. S3 A and B) resulted 42). Iron uptake is mediated through internalization of iron- and made further analysis impossible. The majority of loaded transferrin complexes via CD71 (43). Homozygous C/ + CD11c MHCIIhi DCs in the spleen, lymph nodes (LNs), and EBPβ deletion caused a 1.5-fold up-regulation of CD71 surface + − − lung belonged to the CD172a cDC2 subset (spleen: 80.13 ± expression (Fig. 4D); in agreement, C/EBPβ / and LIP DCs had 3.21%; LN: 70.45 ± 2.06%; lung: 76.1 ± 1.9%) and only a small a more efficient uptake of iron-loaded transferrin complexes + + fraction to the cDC1 subset (spleen: 5.88 ± 0.54%; LN: 20.42 ± compared to C/EBPβ / or LAP/LAP* DCs (Fig. 4 E and F). 1.59%; lung: 9.84 ± 0.94%). In spleen, about two-thirds of cDC2 Immature DCs efficiently endocytose antigens, but they are + − − DCs were derived from the eGFP C/EBPβ / (eGFP: 60.80 ± less capable of generating class II peptide complexes for surface 14.91 compared to RFP: 33.78 ± 3.58; P = 0.04) precursor pool, display (44). MHC class II αβ heterodimers associate early dur- + whereas cDC1 cells were equally recruited from the eGFP C/ ing biosynthesis with the invariant chain Ii. A nonameric complex − − + + + EBPβ / and the RFP C/EBPβ / pool (eGFP: 46.6 ± 6.8 of αβIi (45) is redirected to a late endosomal/lysosomal com- compared to RFP: 53.23 ± 6.85; nonsignificant) (Fig. 3 E and F). partment where class II molecules bind antigenic peptides at the + + + In LNs the proportion of RFP C/EBPβ / DCs compared to expense of invariant chain degradation (46). Here, DCs from C/ + − − + + − − eGFP C/EBPβ / DCs (cDC2 eGFP: 43.93 ± 3.0; cDC2 RFP: EBPβ / and C/EBPβ / mice were pulse labeled and chased up 55.97 ± 2.99; P = 0.04) was not profoundly different within the to 240 min, followed by immunoprecipitation of class II αβ di- cDC2 and the cDC1 subset (cDC1 eGFP: 42.4 ± 3.72; cDC1 RFP: mers and αβIi complexes (Fig. 4G). Half of all samples were 57.27 ± 3.79). In the lungs, the major proportion of cDC2 as well analyzed under mildly denaturing conditions to visualize SDS- + + + as of cDC1 cells originated from RFP C/EBPβ / precursors stable peptide-loaded αβ dimers. The other half was analyzed (cDC2 eGFP: 24.95 ± 3.19; cDC2 RFP: 74.62 ± 3.26; P < 0.01 and under fully denaturing conditions to visualize Ii breakdown in- cDC1 eGFP: 23.93 ± 2.64; cDC1 RFP: 75.45 ± 2.67; P < 0.01) termediates. Concomitant with the loss of the p41 Ii form, the (Fig. 3F). SDS-stable αβ-peptide complexes became visible after 60 min − − Overall, C/EBPβ / DCs have no overall growth advantage and further increased after 240 min. Differences between both + + compared to C/EBPβ / DCs, as evidenced by similar BrdU genotypes were not observed, suggesting that antigen presenta- uptake (Figs. 1H and 3D) and bone marrow engraftment. tion by class II molecules as a readout for the confluence of the However, in a competitive situation various tissues affect the biosynthetic route with the endocytic pathway is seemingly un- − − distribution of mature DCs depending on C/EBPβ expression. disturbed in C/EBPβ / DCs. We conclude that C/EBPβ governs DC fate decision or recruit- Next, we investigated whether C/EBPβ deficiency alters the ment depending on microenvironmental factors. capability of DCs to stimulate antigen-specific T cell prolifera- − − + + Upon antigen uptake, DCs differentiate into mature DCs as- tion. C/EBPβ / and C/EBPβ / DCs were pulsed with OVA + sociated with the up-regulation of CCR7 that enables DC mi- protein and subsequently cocultured with CD4 OT-II trans- gration from peripheral tissues into SLOs to induce adoptive genic T cells. C/EBPβ deficiency endowed DCs with a 2-fold immune responses (38). Here, we performed a skin-painting enhanced stimulatory effect on OT-II cell proliferation and a experiment applying Rhodamine B on the back flank of mice 2.7-fold increased IL-2 secretion (Fig. 4 H and I). We also and determined migrated DCs within the draining LNs. No sig- addressed differentiation of T cell subsets upon stimulation with + − − + + nificant differences regarding the number of Rhodamine B - either C/EBPβ / or C/EBPβ / OVA-pulsed DCs. We observed + + migrated DCs were detected in the draining LNs of C/ that the frequencies of FOXP3 CD25 regulatory T cells (Tregs) − − + + EBPβ / compared to C/EBPβ / mice (SI Appendix, Fig. S3C), were 2-fold decreased when T cells were stimulated with C/ − − + + suggesting that a strong inflammatory stimulus mobilizes homing EBPβ / compared to C/EBPβ / DCs (Fig. 4J), suggesting that

6of12 | www.pnas.org/cgi/doi/10.1073/pnas.2008883117 Scholz et al. Downloaded by guest on September 27, 2021 IMMUNOLOGY AND INFLAMMATION

+ + + − Fig. 4. Differential C/EBPβ expression determines functional DC activity. (A) CD206 expression was analyzed in C/EBPβ / (n = 6), C/EBPβ / (n = 4), and C/ − − − − + + EBPβ / DCs (n = 6). DQ-OVA uptake by (B)C/EBPβ / DCs (n = 4) compared to C/EBPβ / DCs (n = 3) and by (C) LIP (n = 4) compared to LAP/LAP* DCs (n = 4) was + + + − − − assessed in three to four independent experiments. (D) CD71 expression was analyzed in C/EBPβ / (n = 6), C/EBPβ / (n = 4), and C/EBPβ / DCs (n = 6). Transferrin-647 uptake and processing of (E) C/EBPβ+/+ (n = 4) and C/EBPβ−/− DCs (n = 4), and of (F) LIP (n = 5) and LAP/LAP* DCs (n = 5) was determined in four independent experiments. gMFI values were normalized against a control without fluorophore-coupled transferrin; bars and lines represent mean ± SEM; a Mann–Whitney U test or Student’s t test was used depending on the sample number; *P < 0.05; **P < 0.01; ***P < 0.001. (G)C/EBPβ-deficiency does not alter MHC class II trafficking and assembly in DCs. Equal numbers of in vitro generated DCs were labeled with [35S] methionine/cysteine for 20 min and chased for 1 and 4 h. MHC class II-Ii complexes were immunoprecipitated and analyzed by 12.5% SDS/PAGE under mildly denaturing conditions (nonboiled, nonreduced) (Left) and denaturing conditions (boiled, reduced) (Right); n = 3 independent experiments. Shown are representative gels. (H) Dot plots and bars represent the antigen-specific proliferation rate of eFluor 670 labeled CD4+ T cells after coculturing with C/EBPβ+/+ (n = 6) and C/EBPβ−/− (n = 6) DCs. Bars represent mean ± SEM; a Mann–Whitney U test was used; **P < 0.01. (I) IL-2 secretion of the cell cultures described in H was assessed by ELISA; bars represent x-fold of IL-2 secretion relative to control (C/EBPβ+/+), set arbitrarily to 1 (n = 6; mean ± SEM; a one-sample t test was used; **P < 0.01. (J) Representative dot plots and + + + + + − − bars show the frequency of antigen-specific expansion of CD4 CD25 FOXP3 T cells after 5 d of cocultivitation with C/EBPβ / (n = 4) and C/EBPβ / (n = 3) DCs; n = 2 independent experiments. Bars represent mean ± SEM; a Student’s t test was used; **P < 0.01.

Scholz et al. PNAS Latest Articles | 7of12 Downloaded by guest on September 27, 2021 Fig. 5. Splenic tumor-exposed DCs up-regulate genes that targets of E2F transcription. (A) Heatmap displaying differential gene expression in Eμ--challenged DCs (n = 5) versus control DCs (n = 6). Gene expression changes are color coded. Displayed are genes of the signatures “KONG_E2F3_TARGETS” and “ISHIDA_E2F_TARGETS.” (B) Enrichment plots of both gene expression signatures are shown. (C) RT-qPCR arrays were performed + + − − + + − − with RNA extracted from splenic DCs isolated from tumor-naive C/EBPβ / and C/EBPβ / animals, or from C/EBPβ / and C/EBPβ / lymphoma cell recipients 10 and 13 d after Eμ-Myc lymphoma challenge (n = 3 to 5 mice per group; n = 3 to 5 independent experiments). Altered gene expression levels are depicted as x-fold expression normalized to DCs from untreated WT mice set arbitrarily at 1 (solid horizontal line). Bars represent means ± SEM; a Mann–Whitney U test was used; *P < 0.05.

8of12 | www.pnas.org/cgi/doi/10.1073/pnas.2008883117 Scholz et al. Downloaded by guest on September 27, 2021 C/EBPβ could function in programming an immunosuppressive effect of DCs relies on the expression of the long functional potential in DCs. isoforms.

Lymphoma-Exposed DCs Up-Regulate Gene Signatures That Negatively Discussion Correlate with DC Maturation. Loss of C/EBPβ resulted in a We define a role of the TF C/EBPβ in the regulation of DC strongly accelerated maturation of DCs under homeostatic maturation and differentiation under homeostatic and lymphoma- conditions. The opposite effect was observed when we exposed induced conditions. C/EBPβ controlled maturation states were DCs to lymphoma cells in the murine aggressive Myc-driven B intimately linked to the functional activity of DCs, as evidenced cell lymphoma model (20). Based on these data, we analyzed by their T cell priming capacity and their role in tumor promotion. whole genome transcriptional changes in DCs upon exposure to We further prove that C/EBPβ orchestrates DC maturation lymphoma cells. Gene expression profiling was performed from depending on the microenvironmental stimuli that affect its + hi sorted splenic CD11c MHCII DCs isolated from Eμ-Myc expression (49). lymphoma-challenged and nonchallenged (control) mice 8 to 15 Conventional DCs derive from committed DC precursors d after adoptive transfer of lymphoma cells. Lymphoma-exposed (pre-DCs) in the BM and then migrate into the blood and pe- DCs showed an enrichment of several E2F signatures (Fig. 5 A ripheral tissues where they develop into lineage-specific subsets B SI Appendix and and , Table S1B), gene sets associated with cell (28). BM-derived DC precursors were unaltered in mice with proliferation and mitosis, an Rb1 gene set for Rb1 targets, and an IL-6 inflammatory gene signature (SI Appendix, Table S1B). These gene signatures are in line with our previous data derived from quantitative real-time PCR (qPCR) arrays, in which we found up-regulated gene expression for Ifnγ, Il6, Il10, Ccl1, Ccl5, and C3 in lymphoma-exposed wild-type (WT) DCs, but not in C/ − − EBPβ / DCs (20). We revisited these qPCR data and identified in whole genome GEP datasets a profound lymphoma-induced up- regulation of CCR1. This chemokine receptor is expressed on monocytes and DCs and binds to the inflammatory chemokines CCL3,CCL5,andCCL7.Similarly,lymphotoxinalpha(LTα) was significantly up-regulated in WT DCs upon lymphoma-challenge, − − but not in C/EBPβ / DCs (Fig. 5C). IMMUNOLOGY AND INFLAMMATION Collectively, E2F target genes and E2F-associated proliferation gene sets were significantly up-regulated in tumor-experienced C/ − − EBPβ-proficient DCs (Fig. 5 A and B), as compared with C/EBPβ / DCs (Fig. 1). Hence, gene signatures linked to a more immature and immunosuppressive DC status are enriched in lymphoma-exposed DCs, whereas loss of C/EBPβ shows opposite effects regarding proliferation and immunoregulation.

Therapeutic Implications of the Role of the mTOR–C/EBPβ Signaling Axis in the Stromal Response to Rapamycin. Given the observed role of DC-associated C/EBPβ expression in Myc-driven lymphoma disease progression (20), we hypothesized a therapeutical con- sequence of C/EBPβ targeting in protumorigenic DCs. Because the mTOR pathway acts upstream of C/EBPβ, we used the mTOR inhibitor rapamycin, that exerts potent immunosuppres- sive properties, including inhibition of DC maturation and function in vitro and in vivo (8, 47, 48). In a human DC-primary lymphoma cell coculture system, we studied whether rapamycin pretreatment could alleviate the growth-promoting functions of DCs by modulating their C/EBPβ transcriptional activity (Fig. 6A). Cocultures of patient-derived xenograft (PDX) tumor cells derived from mantle cell (MCL) (#44685, #96069), and diffuse large B cell lymphoma (DLBCL) (#86381) patients with human DCs enhanced lymphoma B cell proliferation rates substantially (2.2-fold, 1.4-fold, and 2.6-fold) and increased the proportion of viable Annexin/7AAD-negative tumor cells (6.1-fold, 3.6-fold, and 1.5-fold). This protumorigenic effect was almost completely abolished in two of the three cases Fig. 6. Pretreatment of human DCs with rapamycin reverses their protu- when human DCs were pretreated with 10 ng/mL of rapamycin morigenic function toward PDX-derived human MCL and DLBCL samples. (A) (Fig. 6 B and C). We conclude that human DCs confer a pro- The proliferation or viability rate of primary PDX-derived B-NHL cells found proliferative and antiapoptotic effect on primary B-NHL cocultured with or without human DCs, which were pretreated or not with samples, most likely via the C/EBPβ/mTOR signaling axis. To rapamycin for 24 h, was determined. (B) For proliferation analysis, DCs and substantiate this conclusion, we additionally cocultured PDX- PDX cells were cocultivated in a 1:3.5 ratio with or without BrdU for pro- lymphoma cells with murine BM-derived DCs generated from liferation analysis for 48 h. (C) The viability of PDX cells cocultured with or LAP/LAP* or LIP transgenic mice (Fig. 6D). In two of the three without human DCs was determined with annexin and 7-AAD staining. Bars represent the percentage of proliferative or of viable primary MCL #44685, cocultures, we obtained a clear B-NHL prosurvival effect #96069, and DLBCL #86381 cells. (D) The viability of primary PDX-derived B- through coculturing them with LAP/LAP* DCs, but not with LIP NHL cells cocultured with or without murine LAP/LAP* or LIP DCs in a 1:3.5 DCs that mimick C/EBPβ deficiency (Fig. 6E). This result sup- ratio was determined after 48 h. (E) Bars represent the percentage of viable ported our notion that the C/EBPβ-mediated protumorigenic primary B-NHL cells #44685, #96069, and #86381.

Scholz et al. PNAS Latest Articles | 9of12 Downloaded by guest on September 27, 2021 + + CD11c cell-type-specific deletion of C/EBPβ. In the periphery, detected that loss of C/EBPβ in splenic CD11c DCs led to a + total numbers of splenic CD11c MHCIIhi DCs were modestly modest up-regulation of TFs that regulate late stages of DC cell increased but the frequencies of conventional splenic cDC1 and fate decisions, such as STAT3, Id2, Bcl6,andIRF4. TFs regulating cDC2 subsets were unaltered upon C/EBPβ deficiency, suggest- the development of early DC progenitors were unaltered. The ing that C/EBPβ is not a lineage-defining TF for the differenti- strongest effect of C/EBPβ deficiency was observed for the down- ation of conventional cDC1 and cDC2 subsets. regulation of E2F target genes related to proliferation and cell However, when we compared the frequencies of splenic cycle progression, whereas gene sets for differentiation and mat- + + + − CD11c MHCIIhiCD64 and CD11c MHCIIhiCD64 DCs un- uration were profoundly up-regulated. These data together with der steady-state conditions we observed an expansion of the the observed down-regulation of C/EBPβ in mature DCs support + monocyte-derived CD64 DC subset when C/EBPβ is lacking. the conclusion that C/EBPβ is a key TF in the control of temporal + Notably, all CD64 DCs coexpress CD172a (30). These data DC maturation under physiological conditions. pointed toward a regulatory role of C/EBPβ in a balanced ex- Conversely, microenvironmental cues during lymphomagenesis pansion of monocyte-derived DCs (mo-DCs) in the spleen, up-regulated C/EBPβ expression in DCs associated with an imma- whereas no influence on conventional DCs was seen. In agree- ture phenotype and immunosuppressive features (20). Hence, in − − ment, in BM mixed chimera, enhanced expansion of C/EBPβ / contrast to homeostatic conditions, we hypothesized that expres- + + + compared to C/EBPβ / DCs within the splenic CD172a subset sion of E2F gene sets with the functional annotation proliferation − − was obtained. In lung, C/EBPβ / DCs were reduced compared might be enriched upon lymphoma-induced up-regulation of C/ + + + + to C/EBPβ / DCs in both CD172a as well as in XCR1 sub- EBPβ. Indeed, we found E2F-associated proliferation gene sets sets. We conclude that lung DCs are poorly replenished by C/ increased in splenic DCs upon exposure to lymphoma cells in vivo. EBPβ-deficient DCs. This supports the notion that in lymphoma-challenged DCs gene A reduction of frequencies and total numbers of lung-resident signatures linked to a more immature phenotype are enriched, − − C/EBPβ / CD103hi cDCs under steady-state conditions con- whereas C/EBPβ deletion results in decreased DC proliferation firmed this conclusion because CD103hi and CD11bhi DCs in signatures, but conversely, leads to positively enriched maturation lung continuously emigrate to the bronchial draining LNs, re- signatures. quiring their continuous replenishment (50). As on their devel- Consistent with a tumor-induced C/EBPβ up-regulation and opmental origin, blood monocyte subsets are likely to be the loss of immunocompetence of DCs, the expression levels of C/ + precursor population that gives rise to replenished CD103hi and EBPβ in cell culture favored their capacity to trigger FoxP3 Treg − − CD11bhi lung DCs (51, 52). More recently, four major lung DC expansion, as compared to C/EBPβ / DCs. We conclude that C/ subsets were distinguished according to the expression of characteristic EBPβ and LAP/LAP* versus LIP isoform ratios are crucial for DC surface markers: conventional CD11chiMHCIIhiCD103hicDCs, maturation kinetics and that C/EBPβ expression can be modulated + − CD11chiMHCIIhiCD11bhicDCs, moDCs (CD11bhiCD64 CD103 ), by polarizing microenvironmental factors. − and pDCs (CD11cloMHCIIloCD103 CD11blo) (53). Here, we found The concept of cancer immunoediting characterized a final that the occurrence of all CD11bhi cells was unaffected in C/ phase in which an immunosuppressive tumor microenvironment − − EBPβ / mice, suggesting that C/EBPβ plays no role in the develop- is established that facilitates tumor outgrowth (55, 56). Based on + ment of pulmonary CD11b cDCs or moDCs. On the other hand, an enhanced understanding of the tumor-promoting role of the lung resident-alveolar macrophages that are CD11chigh CD11blow were immune system, novel targeted immunotherapies may evolve. − − largely absent in C/EBPβ / mice, supporting the notion that C/ Accordingly, we found that rapamycin inhibits the protumori- EBPβ plays an intrinsic role in the generation of resident alveolar genic function of DCs in vitro. This observation needs to be macrophages (39). reconciled with a proposed inhibitory effect of the drug on DC + Peripheral CD11c CD103hi DCs are particularily important maturation and functionality; however, this outcome was ob- + for priming CD8 T cell responses in SLO during acute infec- served in stimulations of syngeneic T cells (47, 48). Rapamycin tions; however, they are also involved in the generation of Tregs treatment inhibited DC’s ability to take up antigen and to pro- and thus, important for the maintenance of tolerance (53, 54). duce inflammatory cytokines such as IL-12 and TNFα. It remains Because we found that the frequencies of Tregs were twofold to be seen in vivo how the drug’s activity during direct DC’s − − decreased when OT-II T cells were stimulated with C/EBPβ / lymphoma support competes with T cell immunosuppression. + + compared to C/EBPβ / OVA-pulsed DCs, we conclude that C/ In conclusion, impaired DC functionality during lymphoma- EBPβ may support homeostatic immune tolerance by maintain- genesis is critically influenced by the TF C/EBPβ. We propose ing the balance of DC subset composition. In view of the more that the TF C/EBPβ is a key modulator of tolerogenic versus advanced homeostatic maturation program in C/EBPβ-deficient immunogenic states during homeostatic DC maturation. B cell DCs from mice, it could have been expected that under steady- lymphoma that provides microenvironmental stimuli can exploit state conditions loss of C/EBPβ endowed DCs with higher im- the C/EBPβ transcriptional switch in DCs directly for their own munogenic potential. However, immune tolerance was not com- growth and survival advantage. DCs harboring this TF orches- promised as indicated by a lack of autoimmune disease symptoms trate a tolerogenic T cell differentiation and hence tumor im- − − in C/EBPβ / or LIP KI mice. We suggest that C/EBPβ activity is a mune escape. Selective inhibitors regulating C/EBPβ activity may decisive negative signal for driving homeostatic maturation. This be useful for blocking the protumorigenic function of DCs in TF defines an endogenous threshold for an immunogenic versus tumor patients. tolerogenic DC fate decision. The definition of DC maturation comprises phenotypic and Materials and Methods functional terms; a leading property of mature DCs is their capacity Mice. Eμ-Myc (C57BL/6) transgenic (57), C57BL/6 Ly5.1 (CD45.1) congenic, and to stimulate effector T cell priming. The deletion of C/EBPβ in C57BL/6 mice were bred at the animal facility of the MDC. CD11c.Cre × C/ flox/flox −/− GM-CSF-cultured BM-derived cDC2-like DCs accelerated their EBPβ mice, referred to as C/EBPβ , were generated as described (20). βΔ maturation substantially, as indicated by an up-regulation of The LIP and C/EBP uORF knockin mice have been described earlier (36, 37) and are referred to as LAP*/LAP knockin mice. Studies were performed costimulatory and MHC class II molecules. Functionally, these according to the institutional and Berlin State guidelines (Landesamt für DCs had typical features of a mature subset characterized by Gesundheit und Soziales, Berlin; LaGeSo 0373/13 and 0052/12). down-regulation of CD206-mediated endocytosis, enhanced CD71- mediated iron uptake, and twofold more productive T cell expan- Flow Cytometry and Cell Sorting. Cells were blocked with CD16/32 antibody sion (40–43). The control of DC commitment and maturation is followed by staining with antibodies listed under SI Appendix, Supplemental mediated by temporal expression of TF networks (6, 11). Here, we Methods. Human cells were blocked with fluorescence-activated cell sorting

10 of 12 | www.pnas.org/cgi/doi/10.1073/pnas.2008883117 Scholz et al. Downloaded by guest on September 27, 2021 + + (FACS) buffer/5% (vol/vol) human serum. Data were acquired on a FACS (CD11c MHCclassII ) from C57BL/6, CD11c.Cre and CD11c.Cre x C/EBPβflox/flox mice, Canto II, FACS Fortessa, or a FACS LSR II (BD Biosciences). All cell sorting steps or from Eμ-Myc transfer mice (10 to 36% splenic tumor load of all lymphocytes) were carried out on a FACSAria or a FACSAria Fusion (BD Biosciences) and was performed as described in SI Appendix, Supplemental Methods. analyzed with FlowJo software version 10 (BD Biosciences). Cell sorting (purity >95%) was performed on a FACSAria (BD Biosciences). GSEA. GSEA was performed as described (59) against an integrated database containing the Molecular Signature Database v5.1 (60), the GeneSigDB v4 − − BrdU Treatment and Analysis. In vivo, CD11c.Cre and C/EBPβ / mice were (61), and the Staudt Lab library (StaudtSigDB_dNov2012) (62). Signatures of injected intraperitoneally (i.p.) with 100 μL (10 mg/mL Dulbecco’s phosphate- human genes were translated via gene homology. Only gene signatures that buffered saline [DPBS]) BrdU solution on 3 consecutive days. After 4 d, displayed a significant enrichment (P < 0.005, false discovery rate [FDR] < splenic cell suspension was prepared and analyzed by flow cytometry 5%) as well as those that contained at least 20% significantly differentially according to the manufacturer’s instructions. In vitro, proliferation of cells expressed genes (P < 0.05 by two-sample t tests when comparing two groups was analyzed according to the BrdU Flow Kit protocol (APC BrdU Flow Kit, by paired t tests when comparing measured with hypothetical mixtures) BD Biosciences). were considered to represent differentially regulated pathways. Signatures with fewer than 10 genes were filtered out. Generation of Murine BM-Derived DCs. GM-CSF-derived BM-DCs were gen- RNA isolation, reverse transcription, and quantitative real-time PCR (RT-qPCR). + + erated as described (58). Briefly, 1 to 2 × 106/mL BM progenitor cells were CD11c MHCclassII DCs were purified by FACS (>95% purity). Total RNA was seeded in RPMI 1640/10% fetal calf serum (FCS), β-mercaptoethanol (50 μM) extracted using the RNeasy Mini Kit (Qiagen), transcribed into cDNA with and 20 ng/mL GM-CSF (Peprotech). On day 3, 90% of the supernatant was the SuperScript VILO cDNA Synthesis Kit (Life Technologies), and analyzed discarded. On day 6, half of the medium was renewed. On day 7, the DC cell employing the TaqMan based qRT-PCR gene expression assay (Applied culture was harvested. Biosystems). Alternatively, flt3-ligand-derived BM-DCs were generated (https://www. Antigen uptake and processing studies with transferrin-647 and DQ-ovalbumin. DCs 6 jimmunol.org/content/179/11/7577). Briefly, 1 to 2 × 10 /mL BM progenitor were resuspendend in 7 mL (1 × 106 DCs/mL) RPMI medium/0.2% bovine cells were seeded in RPMI 1640 with 10% FCS, β-mercaptoethanol (50 μM), serum albumin (BSA), 25 μg/mL of fluorophore-coupled transferrin-647 Hepes buffer (2.5 mM), glutamine (5 mM), 100 U/mL penicillin, 100 μg (Thermo Fisher Scientific) or 20 μg/mL of DQ-ovalbumin was added, and streptomycin, and 200 ng/mL Flt3-ligand (Peprotech). On day 7, the DC cell DCs were incubated for 10 min at 37 °C to ensure metabolic activity. To stop culture was harvested and analyzed. endocytosis, cold PBS was added. After centrifugation at 4 °C, the cell pellet was resuspended in 7 mL of 37 °C RPMI medium 1640/10% FCS, supple- Rhodamine B Skin Painting Assay. Fifty microliters of Rhodamine B (Sigma- mented with 250 μg/mL human holo-transferrin, or 200 μg ovalbumin to Aldrich) solution (Rhodamine B; 5 mg/mL in 1:1 dibutylphthalate/acetone replace fluorophore-labeled antigen on the cell surface. For analysis, ali- (Sigma-Aldrich and Carl Roth) was applied onto the shaved abdomen of C/ quots were washed in low pH buffer, fixed with glutaraldehyde solution, +/+ −/− EBPβ and C/EBPβ mice. After 24 h, draining LNs and control LNs were and analyzed by flow cytometry. digested by incubation with collagenase D medium (Roche) at 37 °C for 30 IMMUNOLOGY AND INFLAMMATION μ + + min to increase DC numbers. After cell filtration with a 100- m cell strainer, Determination of T Cell Proliferation and Differentiation. Cultured C/EBPβ / and − − cell suspension was counted and analyzed by flow cytometry. C/EBPβ / DCs were pulsed with 37.5 μg/mL ovalbumin for 24 h. Nonpulsed + cells served as a control. Splenic OT-II T cells were enriched with CD4 MACS Isolation and Differentiation of PBMCs into Human DCs. Human DCs were beads. For the analysis of proliferation, cells were additionally stained with generated from 60 to 100 mL peripheral blood mononuclear cells (PBMCs) or eFluor dye 670 (Life Technologies). Then, cell suspension was washed and × 7 from a buffy coats (DRK, Berlin). A total of 1 to 2 10 PBMCs, separated by a 1 × 105 DCs were cocultured with 1 × 106 of either eFluor dye 670 labeled Biocoll density gradient were resuspended in RPMI medium 1640/2% FCS or unlabeled OT-II T cells in 2 mL culture medium. After 3 or 5 d, T cell and incubated for 3 h at 37 °C. Nonadherent cells were removed and the proliferation rates or differentiation were analyzed, respectively. remaining adherent cells were cultured in medium containing 100 ng/mL human GM-CSF and 20 ng/mL human IL-4 (Peprotech). After 48 h, medium Statistical Analysis. The statistical software GraphPad Prism Version 6 was renewed and 10 ng/mL TNF-α (Peprotech) was added. On day 3, human (GraphPad Software, Inc.) was employed. Values of P < 0.05 were considered DC cell suspension was harvested. statistically significant. Normally distributed data were analyzed by using Student’s unpaired, a paired two-tailed t test, or a one-sample t test. Non- In Vitro Coculture Assays. DC:tumor cell cocultures were performed with PDX normally distributed values were evaluated with the unpaired Mann–Whitney B-NHL cells and human DCs, or with murine BM-derived DCs from LIP or LAP/ U test or Wilcoxon signed-rank test. Permutation tests were performed for LAP* mice in a 1:3.5 ratio in RPMI 1640/10% FCS. PBMC-derived human DCs GSEA. All results were presented as arithmetic mean ± SEM. were non- or pretreated with rapamycin for 24 h and washed twice before coculturing with tumor cells for 24 to 48 h. The study was performed Data Availability. Microarray data are available at the Gene Expression according to the declaration of Helsinki, and in accordance with local ethical Omnibus database under accession number GSE123593. All other study data guidelines. are included in the article and SI Appendix. + + + − − Mixed BM Chimera Experiments. BM cells of RFP C/EBPβ / and C/EBPβ / mice + ACKNOWLEDGMENTS. This work was funded by the Wilhelm Sander- (Ly5.2 ) were mixed in a 1:1 ratio and injected i.v. into lethally irradiated Stiftung (Grant 213.100.02) and in part by grants from the Deutsche congenic Ly5.1 mice. Krebshilfe (Grant 107749) and from the Berliner Krebsgesellschaft (to A.R. and U.E.H.). We thank Kerstin Krüger, Kerstin Gerlach, Brigitte Wollert-Wulf, Gene Expression Profiling. The GeneChip Mouse Gene 2.0 ST Array (Thermo and Heike Schwede for excellent technical assistance and the core facility Fisher Scientific) was employed. Isolation of RNA from splenic DCs “Preparative Flow Cytometry” for help with FACS.

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