Title: Still Alive and Kicking: In-Vitro-Generated GM-CSF Dendritic Cells!
Abstract: In a recent article (Helft et al., 2015Helft J. Böttcher J. Chakravarty P. Zelenay S. Huotari J. Schraml B.U. Goubau D. Reis e Sousa C. Immunity. 2015; 42: 1197-1211Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar), murine bone marrow (BM) cultures with granulocyte-macrophage colony-stimulating factor (GM-CSF) were analyzed for macrophage ("GM-Mac") and dendritic cell ("GM-DC") subsets, and the GM-DC subset was compared with conventional or classical DC (cDC) subsets isolated from peripheral and lymphoid organs of mice. The article was accompanied by a preview about the usefulness of murine BM-DC cultures in the study of DC biology (Guilliams and Malissen, 2015Guilliams M. Malissen B. Immunity. 2015; 42: 988-990Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), insinuating that BM-DCs are not real DCs. But what is a real DC? To reanimate the "DC-ness" of murine BM-DCs, we collected some additional points that have not been addressed by recent papers or comments. We suggest their consideration for further discussion. In a recent article (Helft et al., 2015Helft J. Böttcher J. Chakravarty P. Zelenay S. Huotari J. Schraml B.U. Goubau D. Reis e Sousa C. Immunity. 2015; 42: 1197-1211Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar), murine bone marrow (BM) cultures with granulocyte-macrophage colony-stimulating factor (GM-CSF) were analyzed for macrophage ("GM-Mac") and dendritic cell ("GM-DC") subsets, and the GM-DC subset was compared with conventional or classical DC (cDC) subsets isolated from peripheral and lymphoid organs of mice. The article was accompanied by a preview about the usefulness of murine BM-DC cultures in the study of DC biology (Guilliams and Malissen, 2015Guilliams M. Malissen B. Immunity. 2015; 42: 988-990Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), insinuating that BM-DCs are not real DCs. But what is a real DC? To reanimate the "DC-ness" of murine BM-DCs, we collected some additional points that have not been addressed by recent papers or comments. We suggest their consideration for further discussion. Helft et al., 2015Helft J. Böttcher J. Chakravarty P. Zelenay S. Huotari J. Schraml B.U. Goubau D. Reis e Sousa C. Immunity. 2015; 42: 1197-1211Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar highlight that murine bone marrow (BM) cultures with granulocyte-macrophage colony-stimulating factor (GM-CSF) contain macrophages and dendritic cells (DCs). Given the name of the cytokine, macrophage generation should not be surprising. For completeness, however, it remains to be considered that granulocytes (in fact, only neutrophils) are also generated in such cultures (Inaba et al., 1992Inaba K. Inaba M. Romani N. Aya H. Deguchi M. Ikehara S. Muramatsu S. Steinman R.M. J. Exp. Med. 1992; 176: 1693-1702Crossref PubMed Scopus (3309) Google Scholar, Lutz et al., 1999Lutz M.B. Kukutsch N. Ogilvie A.L.J. Rössner S. Koch F. Romani N. Schuler G. J. Immunol. Methods. 1999; 223: 77-92Crossref PubMed Scopus (2502) Google Scholar). Further studies showed that murine BM cells cultured in vitro with GM-CSF additionally contained substantial numbers of DCs. Unlike ex-vivo-isolated DCs, millions of DCs could easily be generated by these protocols, which highly promoted and accelerated DC research (Inaba et al., 1992Inaba K. Inaba M. Romani N. Aya H. Deguchi M. Ikehara S. Muramatsu S. Steinman R.M. J. Exp. Med. 1992; 176: 1693-1702Crossref PubMed Scopus (3309) Google Scholar, Lutz et al., 1999Lutz M.B. Kukutsch N. Ogilvie A.L.J. Rössner S. Koch F. Romani N. Schuler G. J. Immunol. Methods. 1999; 223: 77-92Crossref PubMed Scopus (2502) Google Scholar). Helft et al. characterized in detail the potential of different progenitors to contribute to the population of the commonly used BM-DC. They showed that macrophage-DC progenitors (MDPs), common monocyte precursors (cMoPs), common DC precursors (CDPs), and Ly6Chi and Ly6Clo monocytes did differentiate and proliferate to varying degrees in the GM-CSF-supplemented cultures. CDPs expanded only 3-fold (an average of 1.5 divisions per cell in 6 days), resulting in a 0.851% share of standard BM-DC cultures at day 6. Therefore, the contribution of this cDC subset appears rather marginal in comparison to the ∼75-fold expansion of MDPs and almost 50-fold expansion of cMoPs in GM-CSF cultures (Helft et al., 2015Helft J. Böttcher J. Chakravarty P. Zelenay S. Huotari J. Schraml B.U. Goubau D. Reis e Sousa C. Immunity. 2015; 42: 1197-1211Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). Given this comparatively poor expansion of CDPs, it is hard to conceive that they contribute substantially to the subset of CD115−CD135+ "GM-DCs" in standard BM-DC cultures. The above-mentioned concerns regarding low numbers of CDP progeny lead us to draw attention to earlier BM-DC work in which major histocompatibility complex class II (MHC II)lo cells were shown to contain progenitors for both macrophages (which downregulate MHC II from their surface) and immature DCs (which further upregulate MHC II on their surface, indicating maturation) (Menges et al., 2005Menges M. Baumeister T. Rössner S. Stoitzner P. Romani N. Gessner A. Lutz M.B. J. Leukoc. Biol. 2005; 77: 535-543Crossref PubMed Scopus (35) Google Scholar). Endocytic capacity and expression of the surface markers E-cadherin, scavenger receptor CD204, CD11b, and Gr-1 were then used as the discriminating markers between progenitor cells of macrophages and two DC subtypes within the MHC IIlo cells closely resembling what Helft et al. described as "GM-Macs" (MHC IIint). We would therefore hesitate to uniformly name this bulk population "GM-Macs," because the described observations strongly suggest that besides macrophages, MHC IIlo DCs are also present in this fraction. Cytokine and chemokine production and gene-expression profiling after lipopolysaccharide (LPS) treatment of already MHC IIhi mature DCs ("GM-DCs") might appear as a secondary maturation stimulus resulting in findings similar to what we found by subsequent tumor necrosis factor (TNF) and LPS maturation of BM-DCs. It is not unexpected that such cells appear different from MHC IIlo macrophage or immature DC mixtures ("GM-Macs") treated with LPS in regard to secretion patterns and gene profiles. Helft et al. touch on another interesting issue, namely as to what extent BM-DCs can mirror any of the DCs subsets in vivo. To this end, the authors used microarray technology to compare the 20% subset of GM-CSF-dependent, CD135-expressing cells among the MHC IIhi cells (i.e., "GM-DCs"), thus representing activated or mature BM-DCs with different DC subsets from the ImmGen databank. These ex-vivo-isolated DCs were obtained from different organs of mice as resident or immature DCs and steady-state migratory or semi-mature DCs, which mostly depend on Flt3L to develop. The authors found large differences in gene expression between the different maturation stages of BM-DCs and ex vivo DCs and concluded that an in vivo correlate of BM-DCs could not be identified and that they were only "distantly related to macrophages and DCs found in vivo" (Guilliams and Malissen, 2015Guilliams M. Malissen B. Immunity. 2015; 42: 988-990Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Although it is indeed important to always keep in mind and emphasize that, like any other model, the model of BM-DCs also has its limitations, we would like to hint at some data that do suggest possible in vivo "relatives." It was proposed that GM-CSF-derived BM-DCs might reflect the in vitro counterparts of monocyte-derived DCs (MoDCs) in vivo. MoDCs in the tissues are found mostly under inflammatory conditions, and they become the dominant DC population in vivo after certain infections (Cheong et al., 2010Cheong C. Matos I. Choi J.-H. Dandamudi D.B. Shrestha E. Longhi M.P. Jeffrey K.L. Anthony R.M. Kluger C. Nchinda G. et al.Cell. 2010; 143: 416-429Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). Unfortunately, the authors included neither MoDCs nor the 80% majority population of CD135− MHC IIhi mature DCs, termed "GM-DNs," in the comparative microarray analyses. The dissection of BM-DC cultures by Helft et al. adds some interesting and important facets to our knowledge about this widely used cellular model. However, major critical questions are left open. We therefore feel that strong and potentially misleading statements on the nomenclature, such as "GM-DN" for a subset of mature DCs or "GM-Macs" for mixed macrophages and immature DCs, or the even more reductionistic proposal of "monocyte-derived cells (MCs)" as suggested earlier (Guilliams et al., 2014Guilliams M. Ginhoux F. Jakubzick C. Naik S.H. Onai N. Schraml B.U. Segura E. Tussiwand R. Yona S. Nat. Rev. 2014; 14: 571-578Google Scholar), need to be taken with a grain of salt. They do not necessarily contribute to a deeper understanding of the complexity of the origins, subsets, and maturation stages of BM-DCs in GM-CSF-derived BM cultures. We would therefore rather replace the "death notice" (Guilliams and Malissen, 2015Guilliams M. Malissen B. Immunity. 2015; 42: 988-990Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) with the announcement "still alive and kicking"—and add the urgent plea to all of us working with BM-DCs to optimally and maximally characterize the populations that we have in our hands.