The transcription factor 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 proteins alpha and beta (C/ adaptive immune responses against pathogens and cancer. Tran- EBPα, C/EBPβ) of the basic leucine zipper (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 E2F target genes via the recruitment of the coactivator identified the TF C/EBPβ as a key regulator for DC maturation and CREB-binding protein/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, gene expression profiles of splenic C/EBPβ / The functional properties of C/EBP family members (C/ DCs showed a down-regulation of E2F cell cycle 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