<figure><img src="image_25.png" /><figcaption>**Figure 23.** Gating strategy for phenotype analysis of cultured DC subsets. An example is given for cDC2. The same gating strategy can be followed for cDC1 or pDC.</figcaption></figure>

of the DC subsets, as the subsets can cross-activate each other [127–129].

An advantage of FACS sorting over MACS-based isolation techniques is that all three subsets can be isolated simultaneously from the same donor with very high purity. However, to obtain a good yield, the sorting process takes a long time. Furthermore, and perhaps more importantly, FACS sorting can also be stressful for cells, thereby possibly impacting functional behavior. Isolation of all three subsets from a single buffy coat or aphaeresis product by MACS sorting is also possible; then the subsets need to be sorted sequentially, which is also very time consuming (Florez-Grau et al, manuscript in preparation and [130]. Generally, pre-depletion of lineage-positive cells reduces the isolation time and increases the purity.

Another booming field to dissect the functional abilities of DC subsets and to probe their transcriptome is single cell RNA sequencing (scRNAseq). For these types of studies, it is imperative to ensure that the analyzed cell population does not contain any impurities. One approach to overcome this challenge is to make use of index sorting when cells are sorted single cell in single wells by FACS. In this way, the sorted events and the accompanying phenotype can be traced back after transcriptome analysis to warrant cell purity and to exclude the possibility that results are derived from contaminating cells. The number of protocols and approaches to perform scRNAseq is rapidly increasing and the current format precludes to deal with all the pros and cons of that field.

### 2.2.4 Human DC for clinical use

DC for clinical use are classified as Advanced Therapy Medicinal Products (ATMPs). According to European legislation, their manufacturing must therefore be in compliance with good manufac-

turing practice (GMPr). The generation of complex cellular products under full GMPr-compliance is a challenging task. A large number of reagents and substances is used, which all need to be obtained in pharmaceutical quality and all equipment, procedures, and facilities need to be in compliance with the respective regulations as well. Proper documentation is another topic, filling complete books. Hence, we cannot provide any ready-to-use GMPr protocols here since any individual protocol, intended for use in clinical application needs to be discussed and agreed upon with the regulatory authorities, who's advise should be sought early in the establishing process. Hence, the following paragraphs are rather intended as an advise for translational researchers, who intend to develop processes and protocols, which shall later be applied in clinical application and hence need to be transferred to GMPr. Find below some dos and don'ts, which should be adhered to from early on.

All reagents, that may end up in the final product need to have GMPr quality. The easiest source of such reagents, like e.g. cytokines are approved drugs. These are readily available, often cheaper than GMPr-reagents, and usually accepted by the authorities. An example for DC maturation is PGE₂. In opposite, animal-derived substances like fetal bovine serum or BSA are to be avoided due to the issues with transmissible spongiform encephalopathies (TSE). In this context, it is important to arrange with all suppliers of kits and reagents that full content lists are available (if necessary after confidentiality agreements). Any substance that may be contaminating the final product (termed drug product) needs to be either analyzed for, or the maximum possible contamination needs to be evaluated by a risk assessment team and justified to the authorities. For example, when DC are pulsed with peptides, dissolved in DMSO, the final DMSO-contamination of your drug product will be way below any toxic level; nevertheless, it will convince the authorities more easily, if a proper calculation is provided. It is also very helpful if
<table><caption>**Table 14.** Suggested parameters for quality control of DC for clinical use</caption><thead><tr><th>Parameter</th><th>Specification</th></tr></thead><tbody><tr><td><strong>Content</strong></td><td></td></tr><tr><td>Cell count</td><td>Dependent on dose</td></tr><tr><td>Viability</td><td>&gt;80%</td></tr><tr><td><strong>Purity</strong></td><td></td></tr><tr><td>CD11c + HLA-DR on all cells</td><td>&gt;90%</td></tr><tr><td><strong>Impurities</strong></td><td></td></tr><tr><td>Small cells</td><td>&lt;10%</td></tr><tr><td>CD14 on all cells</td><td>&lt;20%</td></tr><tr><td><strong>Identity (phenotype)</strong></td><td></td></tr><tr><td>CD80 on large cells</td><td>&gt;70%</td></tr><tr><td>CD83 on large cells</td><td>&gt;70%</td></tr><tr><td>CD83 on all cells</td><td>&gt;60%</td></tr><tr><td>CD86 on large cells</td><td>&gt;90%</td></tr><tr><td><strong>Potency</strong></td><td></td></tr><tr><td>T cell activation</td><td>Dependent on assay</td></tr><tr><td><strong>Contaminations</strong></td><td></td></tr><tr><td>Sterility</td><td>Sterile</td></tr><tr><td><strong>Endotoxin</strong></td><td>Dependent on volume and dose</td></tr><tr><td><strong>Mycoplasma</strong></td><td>Negative</td></tr></tbody></table>

all providers offer lot-specific certificates of analysis for all goods that enter the production process, otherwise these analyses will have to be performed upon entry.

The second topic relevant for GMPr-production is quality control. All relevant properties of the final product need to be defined, although many values can be set arbitrarily as long as they can be justified and are accepted by the regulatory authorities. A number of surface markers to define mature DC are summarized in Table 14. The specifications in the table are indicative; they vary between DC subtypes and their expression can be dependent on the maturation stimulus used. In addition to phenotype analysis, potency testing is required for DC for clinical use. The potency assay should demonstrate the biological activity of the DC and should be based on the intended biological effect, i.e. T-cell activation. To warrant a stable production process, in-process controls are also strongly advised, i.e. purity of monocytes (when used for DC generation) or freshly isolated blood DC, phenotype of immature DC, etc. In this context clearly quantifiable values are preferable: A “percentage above 80 %” is good, a “typical DC-morphology in the microscope” is poor. However, keep in mind that all assays used need to be validated beforehand, unless described in the European Pharmacopoeia.

**Acknowledgements:** This work was supported by the Bavarian State Ministry of Science and Art (Bayresq.Net-IRIS) and the Agence Nationale de la Recherche (ANR) and the DFG

(DU548/6-1), by the DFG (CRC1181-261193037 and GRK2504-401821119) to DD. A.B.K. was supported by the DFG (SFB1054-TPA06 210592381, TRR237-TPB14 369799452 and 391217598). SS was supported by the DFG (SCHE692/6-1, SCHE692/8-1, and 270650915/GRK2158) and the Juergen Manchot Foundation (Manchot Graduate School ‘Molecules of Infection IV’). VB were supported by Wellcome Trust UK (grant number 101155/Z/13/Z) and the Medical Research Council (grant number MR/W01677X/1). Section 1.1.2: we thank K. Götz for support in cell culture, H. Häcker for HoxB8 plasmid and S. Rose-John for hyper-IL-6. HX was supported by a fellowship of China Scholarship Council (grant number 202008080170). MBL, MH, and LC were supported by the IZKF Wuürzburg (A-408); CHKL by intramural funding (Erlangen IZKF-A87) and the DFG (GRK2504-401821119); and SEA by the German Jose Carreras Leukemia Foundation (DJCLS 13 R/2021). Open access funding enabled and organized by Projekt DEAL.

**Conflict of interest:** The authors declare no commercial or financial conflict of interest.

**Author contributions:** Section 1.1.1: Shafaqat Ali, Stefanie Scheu, Manfred B. Lutz and Anne Krug; lead authors Shafaqat Ali and Manfred B. Lutz (Shafaqat.Ali@med.uni-duesseldorf.de and m.lutz@vim.uni-wuerzburg.de).

Section 1.1.2: Martin Zenke, Huaming Xu, Kristin Seré and Anne B. Krug; lead author Martin Zenke (martin.zenke@rwth-aachen.de).

Section 1.2.1: Manfred B. Lutz, Marion Heuer, Laura Cyran, Christian H. K. Lehmann, Diana Dudziak and Stella Autenrieth; lead author Manfred B. Lutz (m.lutz@vim.uni-wuerzburg.de).

Section 1.2.2: Cindy Audiger, Shalin Naik, Anne B. Krug, Martin Zenke and Kristin Seré; lead author Kristin Seré (kristin.sere@rwth-aachen.de).

Section 1.2.3: Gloria J. Godoy, Lucila Giusiano, Christian T. Mayer, Tim Sparwasser and Luciana Berod; lead author Luciana Berod (berod@uni-mainz.de).

Section 1.2.4: Shafaqat Ali and Stefanie Scheu; lead author Shafaqat Ali (Shafaqat.Ali@med.uni-duesseldorf.de).

Section 2.1.1: Elodie Segura, Venetia Bigley, Diana Dudziak and Jan Dörrie; lead authors Elodie Segura (elodie.segura@curie.fr) and Jan Dörrie (jan.doerrie@uk-erlangen.de).

Section 2.1.2: Stella E. Autenrieth, Marc Dalod and Venetia Bigley; lead author Stella E. Autenrieth (stella.autenrieth@dkfz-heidelberg.de).

Section 2.2.1: Elodie Segura, Venetia Bigley, Diana Dudziak and Jan Dörrie; lead authors Elodie Segura (elodie.segura@curie.fr) and Jan Dörrie (jan.doerrie@uk-erlangen.de).

Section 2.2.2: Stella E. Autenrieth, Marc Dalod, Venetia Bigley and Elodie Segura; lead author Venetia Bigley (venetia.bigley@ncl.ac.uk).

Section 2.2.3: Gerty Schreibelt, Jurjen Tel and Georgina Flórez-Grau; lead author Gerty Schreibelt (Gerty.Schreibelt@radboudumc.nl).
Section 2.2.4: Gerty Schreibelt, Jurjen Tel and Jan Dörrie; lead author Jan Dörrie (jan.doerrie@uk-erlangen.de).

**Data availability statement:** The data that support the findings of this study are available from the lead author(s) upon reasonable request.

**Peer review:** The peer review history for this article is available at https://publons.com/publon/10.1002/eji.202249816.

## References

1 Redecke, V., Wu, R., Zhou, J., Finkelstein, D., Chaturvedi, V., High, A. A. and Häcker, H., Hematopoietic progenitor cell lines with myeloid and lymphoid potential. *Nat. Methods* 2013. 10: 795–803.

2 Wang, G. G., Calvo, K. R., Pasillas, M. P., Sykes, D. B., Häcker, H. and Kamps, M. P., Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. *Nat. Methods* 2006. 3: 287–293.

3 Hammerschmidt, S. I., Werth, K., Rothe, M., Galla, M., Permanyer, M., Patzer, G. E., Bubke, A. et al. CRISPR/Cas9 immunoengineering of HoxB8-immortalized progenitor cells for revealing CCR7-mediated dendritic cell signaling and migration mechanisms in vivo. *Front. Immunol.* 2018. 9.

4 Leithner, A., Renkawitz, J., De Vries, I., Hauschild, R., Häcker, H. and Sixt, M., Fast and efficient genetic engineering of hematopoietic precursor cells for the study of dendritic cell migration. *Eur. J. Immunol.* 2018. 48: 1074–1077.

5 Xu, H., Look, T., Prithiviraj, S., Lennartz, D., Cáceres, M. D., Götz, K., Wanek, P. et al. CRISPR/Cas9 editing in conditionally immortalized HoxB8 cells for studying gene regulation in mouse dendritic cells. *Eur. J. Immunol.* 2021. https://doi.org/10.1002/eji.202149482. Online ahead of print.

6 Seré, K. M., Lin, Q., Felker, P., Rehage, N., Klisch, T., Ortseifer, I., Hieronymus, T. et al. Dendritic cell lineage commitment is instructed by distinct cytokine signals. *Eur. J. Cell Biol.* 2012. 91: 515–523.

7 Felker, P., Seré, K., Lin, Q., Becker, C., Hristov, M., Hieronymus, T. and Zenke, M., TGF-β1 accelerates dendritic cell differentiation from common dendritic cell progenitors and directs subset specification toward conventional dendritic cells. *J. Immunol.* 2010. 185: 5326–5335.

8 Fischer, M., Goldschmitt, J., Peschel, C., Brakenhoff, J. P., Kallen, K.-J., Wollmer, A., Grötzinger, J. et al. A bioactive designer cytokine for human hematopoietic progenitor cell expansion. *Nat. Biotechnol.* 1997. 15: 142–145.

9 Bozzacco, L., Trumpfheller, C., Huang, Y., Longhi, M. P., Shimeliovich, I., Schauer, J. D., Park, C. G. et al. HIV gag protein is efficiently cross-presented when targeted with an antibody toward the DEC-205 receptor in Flt3 ligand-mobilized murine DC. *Eur. J. Immunol.* 2010. 40: 36–46.

10 Landázuri, N., Krishna, D., Gupta, M. and Doux, J. M. L., Retrovirus-polymer complexes: Study of the factors affecting the dose response of transduction. *Biotechnol. Progr.* 2007. 23: 480–487.

11 Burgess, A. W. and Metcalf, D., The nature and action of granulocyte-macrophage colony stimulating factors. *Blood* 1980. 56: 947–958.

12 Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S. et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. *J. Exp Med.* 1992. 176: 1693–1702.

13 Lutz, M. B., Strobl, H., Schuler, G. and Romani, N., GM-CSF monocyte-derived cells and langerhans cells as part of the dendritic cell family. *Front Immunol* 2017. 8: 1388.

14 Talmor, M., Mirza, A., Turley, S., Mellman, I., Hoffman, L. A. and Steinman, R. M., Generation of large numbers of immature and mature dendritic cells from rat bone marrow cultures. *Eur. J. Immunol.* 1998. 28: 811–817.

15 Berger, T. G., Schulze-Koops, H., Schäfer, M., Müller, E. and Lutz, M. B., Immature and maturation-resistant human dendritic cells generated from bone marrow require two stimulations to induce T cell anergy in vitro. *PLoS One* 2009. 4: e6645.

16 Zanna, M. Y., Yasmin, A. R., Omar, A. R., Arshad, S. S., Mariatulqabtiah, A. R., Nur-Fazila, S. H. and Mahiza, M. I. N., Review of dendritic cells, their role in clinical immunology, and distribution in various animal species. *Int J Mol Sci* 2021. 22: 8044.

17 Menges, M., Baumeister, T., Rossner, S., Stoitzner, P., Romani, N., Gessner, A. and Lutz, M. B., IL-4 supports the generation of a dendritic cell subset from murine bone marrow with altered endocytosis capacity. *J Leukoc Biol* 2005. 77: 535–543.

18 Helft, J., Bottcher, J., Chakravarty, P., Zelenay, S., Huotari, J., Schraml, B. U., Goubau, D. et al. GM-CSF mouse bone marrow cultures comprise a heterogeneous population of CD11c(+)MHCII(+) macrophages and dendritic cells. *Immunity* 2015. 42: 1197–1211.

19 Yanez, A., Coetzee, S. G., Olsson, A., Muench, D. E., Berman, B. P., Hazelett, D. J., Salomonis, N. et al. Granulocyte-monocyte progenitors and monocyte-dendritic cell progenitors independently produce functionally distinct monocytes. *Immunity* 2017. 47: 890–902 e894.

20 Liu, Z., Gu, Y., Chakarov, S., Bleriot, C., Kwok, I., Chen, X., Shin, A. et al. Fate mapping via Ms4a3-expression history traces monocyte-derived cells. *Cell* 2019. 178: 1509–1525 e1519.

21 Kwok, I., Becht, E., Xia, Y., Ng, M., Teh, Y. C., Tan, L., Evrard, M. et al. Combinatorial Single-Cell Analyses of Granulocyte-Monocyte Progenitor Heterogeneity Reveals an Early Uni-potent Neutrophil Progenitor. *Immunity* 2020.

22 Lutz, M. B., Inaba, K., Schuler, G. and Romani, N., Still alive and kicking: in-vitro-generated GM-CSF dendritic cells! *Immunity* 2016. 44: 1–2.

23 Helft, J., Bottcher, J. P., Chakravarty, P., Zelenay, S., Huotari, J., Schraml, B. U., Goubau, D. et al. Alive but confused: heterogeneity of CD11c(+) MHC class II(+) cells in GM-CSF mouse bone marrow cultures. *Immunity* 2016. 44: 3–4.

24 Nikolic, T., de Bruijn, M. F., Lutz, M. B. and Leenen, P. J., Developmental stages of myeloid dendritic cells in mouse bone marrow. *Int Immunol* 2003. 15: 515–524.

25 Ryu, S. E., Granulocyte macrophage-colony stimulating factor produces a splenic subset of monocyte-derived dendritic cells that efficiently polarize T helper type 2 cells in response to blood-borne antigen. *Front Immunol.* 2022.

26 Naik, S. H., Proietto, A. I., Wilson, N. S., Dakic, A., Schnorrer, P., Fuchsberger, M., Lahoud, M. H. et al. Cutting edge: generation of splenic CD8+ and CD8- dendritic cell equivalents in Fms-like tyrosine kinase 3 ligand bone marrow cultures. *J. Immunol.* 2005. 174: 6592–6597.

27 Cheong, C., Matos, I., Choi, J.-H., Dandamudi, D. B., Shrestha, E., Longhi, M. P., Jeffrey, K. L. et al. Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(+) dendritic cells for immune T cell areas. *Cell* 2010. 143: 416–429.

28 Bosteels, C., Fierens, K., De Prijck, S., Van Moorleghem, J., Vanheerswylsghels, M., De Wolf, C., Chalon, A. et al. CCR2- and Flt3-dependent inflammatory conventional type 2 dendritic cells are necessary for the
induction of adaptive immunity by the human vaccine adjuvant system AS01. *Front Immunol.* 2020. 11: 606805.

29 Plantinga, M., Guilliams, M., Vanheerswynghels, M., Deswarte, K., Branco-Madeira, F., Toussaint, W., Vanhoutte, L. et al. Conventional and monocyte-derived CD11b(+) dendritic cells initiate and maintain T helper 2 cell-mediated immunity to house dust mite allergen. *Immunity* 2013. 38: 322–335.

30 Tang-Huau, T. L., Gueguen, P., Goudot, C., Durand, M., Bohec, M., Baulande, S., Pasquier, B. et al. Human in vivo-generated monocyte-derived dendritic cells and macrophages cross-present antigens through a vacuolar pathway. *Nat. Commun.* 2018. 9: 2570.

31 Moutel, S., Beugnet, A., Schneider, A., Lombard, B., Loew, D., Amigorena, S., Perez, F. et al. Surface LSP-1 is a phenotypic marker distinguishing human classical versus monocyte-derived dendritic cells. *iScience* 2020. 23: 100987.

32 Erra Diaz, F., Ochoa, V., Merlotti, A., Dantas, E., Mazzitelli, I., Gonzalez Polo, V., Sabatte, J. et al. Extracellular acidosis and mTOR inhibition drive the differentiation of human monocyte-derived dendritic cells. *Cell Rep.* 2020. 31: 107613.

33 Lutz, M. B., Kukutsch, N., Ogilvie, A. L., Rossner, S., Koch, F., Romani, N. and Schuler, G., An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. *J. Immunol. Methods* 1999. 223: 77–92.

34 Zal, T., Volkmann, A. and Stockinger, B., Mechanisms of tolerance induction in major histocompatibility complex class II-restricted T cells specific for a blood-borne self-antigen. *J. Exp. Med.* 1994. 180: 2089–2099.

35 Jiang, A., Bloom, O., Ono, S., Cui, W., Unternaehrer, J., Jiang, S., Whitney, J. A. et al. Disruption of E-cadherin-mediated adhesion induces a functionally distinct pathway of dendritic cell maturation. *Immunity* 2007. 27: 610–624.

36 Woltman, A. M., van der Kooij, S. W., de Fijter, J. W. and van Kooten, C., Maturation-resistant dendritic cells induce hyporesponsiveness in alloreactive CD45RA+ and CD45RO+ T-cell populations. *Am J Transplant* 2006. 6: 2580–2591.

37 Steinbrink, K., Wolfl, M., Jonuleit, H., Knop, J. and Enk, A. H., Induction of tolerance by IL-10-treated dendritic cells. *J. Immunol.* 1997. 159: 4772–4780.

38 Hackstein, H. and Thomson, A. W., Dendritic cells: emerging pharmacological targets of immunosuppressive drugs. *Nat Rev Immunol.* 2004. 4: 24–34.

39 Lutz, M. B., Differentiation stages and subsets of tolerogenic dendritic cells. In Lutz, M. B., Romani, N. and Steinkasserer, A. (Eds.) *Handbook of Dendritic Cells. Biology, Diseases and Therapy*. VCH-Wiley, Weinheim 2006, pp 517–543.

40 Svajger, U., Obermajer, N. and Jeras, M., Novel findings in drug-induced dendritic cell tolerogenicity. *Int. Rev. Immunol.* 2010. 29: 574–607.

41 Thomson, A. W. and Ezzelarab, M. B., Regulatory dendritic cells: profiling, targeting, and therapeutic application. *Curr. Opin. Organ. Transplant.* 2018. 23: 538–545.

42 Divito, S. J. and Morelli, A. E., Method of generating tolerogenic maturation-resistant dendritic cells and testing for their immune-regulatory functions in vivo in the context of transplantation. *Methods Mol. Biol.* 2019. 1899: 181–193.

43 Zahorchak, A. F., Kean, L. S., Tokita, D., Turnquist, H. R., Abe, M., Finke, J., Hamby, K. et al. Infusion of stably immature monocyte-derived dendritic cells plus CTLA4Ig modulates alloimmune reactivity in rhesus macaques. *Transplantation* 2007. 84: 196–206.

44 Mou, H. B., Lin, M. F., Cen, H., Yu, J. and Meng, X. J., TGF-beta1 treated murine dendritic cells are maturation resistant and down-regulate Toll-like receptor 4 expression. *J Zhejiang Univ Sci* 2004. 5: 1239–1244.

45 Lutz, M. B., Suri, R. M., Niimi, M., Ogilvie, A. L., Kukutsch, N. A., Rossner, S., Schuler, G. et al. Immature dendritic cells generated with low doses of GM-CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo. *Eur. J. Immunol.* 2000. 30: 1813–1822.

46 Abdi, K., Thomas, L. M., Laky, K., Abshari, M., Matzinger, P. and Long, E. O., Bone marrow-derived dendritic cell cultures from RAG(-/-) mice include IFN-gamma-producing NK cells. *Immunohorizons* 2020. 4: 415–419.

47 Oriss, T. B., Krishnamoorthy, N., Ray, P. and Ray, A., Dendritic cell c-kit signaling and adaptive immunity: implications for the upper airways. *Curr. Opin. Allergy Clin. Immunol.* 2014. 14: 7–12.

48 Rossner, S., Voigtlander, C., Wiethe, C., Hanig, J., Seifarth, C. and Lutz, M. B., Myeloid dendritic cell precursors generated from bone marrow suppress T cell responses via cell contact and nitric oxide production in vitro. *Eur. J. Immunol.* 2005. 35: 3533–3544.

49 Lutz, M. B., Schnare, M., Menges, M., Rossner, S., Rollinghoff, M., Schuler, G. and Gessner, A., Differential functions of IL-4 receptor types I and II for dendritic cell maturation and IL-12 production and their dependency on GM-CSF. *J. Immunol.* 2002. 169: 3574–3580.

50 Lutz, M. B. and Rößner, S., Factors influencing the generation of murine dendritic cells from bone marrow: The special role of fetal calf serum. *Immunobiology* 2008. 212: 855–862.

51 Rößner, S., Zinser, E., Menges, M., Wiethe, C., Littmann, L., Hänig, J., Steinkasserer, A. et al. Minor role of bystander tolerance to fetal calf serum in a peptide-specific dendritic cell vaccine model against autoimmunity: comparison with serum-free cultures. *J. Immunother.* 2008. 31: 656–664.

52 Lu, L., McCaslin, D., Starzl, T. E. & Thomson, A. W. Bone marrow-derived dendritic cell progenitors (NLDC 145+, MHC class II+, B7-1dim, B7-2-) induce alloantigen-specific hyporesponsiveness in murine T lymphocytes. *Transplantation* 1995. 60: 1539–1545.

53 Inaba, K., Steinman, R. M., Pack, M. W., Aya, H., Inaba, M., Sudo, T., Wolpe, S. et al., Identification of proliferating dendritic cell precursors in mouse blood. *J. Exp. Medicine* 1992. 175: 1157–1167.

54 Scheicher, C., Mehlig, M., Zecher, R. and Reske, K. Dendritic cells from mouse bone marrow: in vitro differentiation using low doses of recombinant granulocyte-macrophage colony-stimulating factor. *J. Immunol. Methods* 1992. 154: 253–264.

55 Naik, S. H., Metcalf, D., van Nieuwenhuijze, A., Wicks, I., Wu, L., O’Keeffe, M., Shortman, K. et al., Intrasplenic steady-state dendritic cell precursors that are distinct from monocytes. *Nat. Immunol.* 2006. 7: 663–671.

56 Xu, Y., Zhan, Y., Lew, A. M., Naik, S. H. and Kershaw, M. H. Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking. *J. Immunol.* 2007. 179: 7577–7584.

57 Audiger, C. & Lesage, S., Flt3 Ligand Is Dispensable for the Final Stage of Type 1 Conventional Dendritic Cell Differentiation. *J. Immunol.* 2020. 205: 2117–2127.

58 Durai, V., Bagadia, P., Briseño, C. G., Theisen, D. J., Iwata, A., Davidson, J. T., Gargaro, M. et al., Altered compensatory cytokine signaling underlies the discrepancy between Flt3(-/-) and Flt3I(-/-) mice. *J. Exp. Med.* 2018. 215: 1417–1435.

59 Karsunky, H., Merad, M., Cozzio, A., Weissman, I. L. & Manz, M. G. Flt3 ligand regulates dendritic cell development from Flt3+ lymphoid and myeloid-committed progenitors to Flt3+ dendritic cells in vivo. *J. Exp. Medicine* 2003. 198: 305–313.
60 D'Amico, A. & Wu, L. The early progenitors of mouse dendritic cells and plasmacytoid predendritic cells are within the bone marrow hemopoietic precursors expressing Flt3. *J. Exp. Medicine* 2003. **198**: 293–303.

61 Lin, D. S., Tian, L., Tomei, S., Amann-Zalcenstein, D., Baldwin, T. M., Weber, T. S., Schreuder, J. et al., Single-cell analyses reveal the clonal and molecular aetiology of Flt3L-induced emergency dendritic cell development. *Nat. Cell Biol.* 2021. 23: 219–231.

62 Gilliet, M., Boonstra, A., Paturel, C., Antonenko, S., Xu, X. L., Trinchieri, G., O'Garra, A. et al. The development of murine plasmacytoid dendritic cell precursors is differentially regulated by FLT3-ligand and granulocyte/macrophage colony-stimulating factor. *J. Exp. Med.* 2002. **195**: 953–958.

63 Naik, S. H., O'Keeffe, M., Proietto, A., Shortman, H. H. & Wu, L. CD8+, CD8-, and plasmacytoid dendritic cell generation in vitro using flt3 ligand. *Methods Mol. Biology* 2010. **595**: 167–176.

64 Naik, S. H., Sathe, P., Park, H. Y., Metcalf, D., Proietto, A. I., Dakic, A., Carotta, S. et al., Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo. *Nat. Immunol.* 2007. 8: 1217–1226.

65 Brawand, P., Fitzpatrick, D. R., Greenfield, B. W., Brasel, K., Maliszewski, C. R. and De Smedt, T., Murine Plasmacytoid Pre-Dendritic Cells Generated from Flt3 Ligand-Supplemented Bone Marrow Cultures Are Immature APCs. *J. Immunol.* 2002. **169**: 6711–6719.

66 Perez, C. R. & Palma, M. D. Engineering dendritic cell vaccines to improve cancer immunotherapy. *Nat. Commun.* 2019. 10: 5408.

67 Lin, Q., Chauvistré, H., Costa, I. G., Gusmao, E. G., Mitzka, S., Hänzelmann, S., Baying, B. et al. Epigenetic program and transcription factor circuitry of dendritic cell development. *Nucleic. Acids. Res.* 2015. **43**: 9680–9693.

68 Macri, C., Pang, E., Patton, T. and O'Keeffe, M., Dendritic cell subsets. *Semin. Cell Dev. Biol.* 2018. **84**: 11–21. Available at: https://pubmed.ncbi.nlm.nih.gov/29246859/ [Accessed September 16, 2021].

69 Guilliams, M., Ginhoux, F., Jakubzick, C., Naik, S., Onai, N., Schraml, B., Segura, E. et al., Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny. *Nat. Rev. Immunol.* 2014. 14: 571–578. Available at: https://pubmed.ncbi.nlm.nih.gov/25033907/ [Accessed September 29, 2021].

70 Del Rio, M, Bernhardt, G, Rodriguez-Barbosa, J. and Förster, R., Development and functional specialization of CD103+ dendritic cells. *Immunol. Rev.* 2010. 234: 268–281. Available at: https://pubmed.ncbi.nlm.nih.gov/20193025/ [Accessed September 16, 2021].

71 Mayer, C. T., Ghorbani, P., Nandan, A., Dudek, M., Arnold-Schrauf, C., Hesse, C., Berod, L. et al., Selective and efficient generation of functional Batf3-dependent CD103+ dendritic cells from mouse bone marrow. *Blood*. 2014. **124**: 3081–3091. Available at: http://www.bloodjournal.org/cgi/doi/10.1182/blood-2013-12-545772 [Accessed February 1, 2019].

72 Cossarizza, A., Chang, H. D., Radbruch, A., Acs, A., Adam, D., Adam-Klages, S., Agace, W. W. et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). *Eur. J. Immunol.* 2019. 49: 1457–1973.

73 Tai, L. H., Goulet, M. L., Belanger, S., Toyama-Sorimachi, N., Fodil-Cornu, N., Vidal, S. M., Troke, A. D. et al. Positive regulation of plasmacytoid dendritic cell function via Ly49Q recognition of class I MHC. *J. Exp. Med.* 2008. 205: 3187–3199.

74 Steinman, R. M., Cohn, Z. A., Identification of a novel cell type in peripheral lymphoid organs of mice. *I. Morphology, quantitation, tissue distribution. J. Exp. Med.* 1973. **137**: 1142–1162.

75 Aarntzen, E. H., Figdor, C. G., Adema, G. J., Punt, C. J., de Vries, I. J., Dendritic cell vaccination and immune monitoring. *Cancer Immunol. Immunother.* 2008. 57: 1559–1568.

76 Banchereau, J. and Steinman, R. M., Dendritic cells and the control of immunity. *Nature*. 1998. **392**: 245–252.

77 Palucka, K. and Banchereau, J., Cancer immunotherapy via dendritic cells. *Nat Rev Cancer.* 2012. **12**: 265–277.

78 Sallusto, F. and Lanzavecchia, A., Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. *J. Exp Med.* 1994. **179**: 1109–1118.

79 Bender, A., Sapp, M., Schuler, G., Steinman, R. M. and Bhardwaj, N., Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood. *J Immunol Methods.* 1996. **196**: 121–135.

80 Coillard, A. and Segura, E., In vivo Differentiation of Human Monocytes. *Front Immunol.* 2019. 10: 1907.

81 Erdmann, M., Dorrie, J., Schaft, N., Strasser, E., Hendelmeier, M., Kampgen, E. et al., Effective clinical-scale production of dendritic cell vaccines by monocyte elutriation directly in medium, subsequent culture in bags and final antigen loading using peptides or RNA transfection. *J. Immunother.* 2007. **30**: 663–674.

82 Hordyjewska, A., Popiolek, Ł. and Horecka, A., Characteristics of hematopoietic stem cells of umbilical cord blood. *Cytotechnology*. 2015. 67: 387–396.

83 Breton, G., Lee, J., Liu, K. and Nussenzweig, M. C., Defining human dendritic cell progenitors by multiparametric flow cytometry. *Nat. Protoc.* 2015. 10: 1407–1422.

84 Balan, S. and Dalod, M., Dendritic Cell Protocols. *Methods Mol Biology*. 2016. 1423: 19–37.

85 Goudot, C., Coillard, A, Villani, A. C., Gueguen, P., Cros, A., Sarkizova, S. et al., Aryl hydrocarbon receptor controls monocyte differentiation into dendritic cells versus macrophages. *Immunity*. 2017. 47: 582–596 e6.

86 Caux, C., Vanbervliet, B., Massacrier, C., Dezutter-Dambuyant, C., Saint-Vis, B., Jacquet, C., Yoneda, K. et al., CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNF alpha. *J. Exp. Medicine* 1996. 184: 695–706.

87 Klechevsky, E., Morita, R., Liu, M., Cao, Y., Coquery, S., Thompson-Snipes, L., Briere, F. et al., Functional specializations of human epidermal langerhans cells and CD14+ dermal dendritic cells. *Immunity*. 2008. 29: 497–510.

88 Balan, S., Kale, V. P. and Limaye, L. S., A large number of mature and functional dendritic cells can be efficiently generated from umbilical cord blood-derived mononuclear cells by a simple two-step culture method. *Transfusion (Paris).* 2010. 50: 2413–2423.

89 Balan, S., Ollion, V., Colletti, N., Chelbi, R., Montanana-Sanchis, F., Liu, H., Manh, T. P. V. et al., Human XCR1+ dendritic cells derived in vitro from CD34+ progenitors closely resemble blood dendritic cells, including their adjuvant responsiveness, contrary to monocyte-derived dendritic cells. *J. Immunol.*. 2014. **193**: 1622–1635.

90 Poulin, L. F., Salio, M., Griessinger, E., Anjos-Afonso, F, Craciun, L, Chen, J.-L., Keller, A. M. et al., Characterization of human DNGR-1+ BDCA3+ leukocytes as putative equivalents of mouse CD8alpha+ dendritic cells. *J. Exp. Med.* 2010. 207: 1261–1271.

91 Chen, W., Antonenko, S., Sederstrom, J. M., Liang, X., Chan, A. S. H., Kanzler, H., Blom, B. et al., Thrombopoietin cooperates with FLT3-ligand in
the generation of plasmacytoid dendritic cell precursors from human hematopoietic progenitors. *Blood*. 2004. 103: 2547–2553.

92 Proietto, A. I., Mittag, D., Roberts, A. W., Sprigg, N., Wu, L., The equivalents of human blood and spleen dendritic cell subtypes can be generated in vitro from human CD34+ stem cells in the presence of fms-like tyrosine kinase 3 ligand and thrombopoietin. *Cellular and Molecular Immunology*. 2012. 9: 446–454.

93 Lee, J., Zhou, Y. J., Ma, W., Zhang, W., Aljoufi, A., Luh, T., Lucero, K. et al., Lineage specification of human dendritic cells is marked by IRF8 expression in hematopoietic stem cells and multipotent progenitors. *Nat. Immunol*. 2017. 18: 877–888. Available at: message: %3C1F0EEA2B-D8D7-41B4-9A37-DEAFC1A56D03@wehi.edu.au%3E.

94 Lee, J., Breton, G., Oliveira, T. Y. K., Zhou, Y. J., Aljoufi, A., Puhr, S., Cameron, M. J. et al., Restricted dendritic cell and monocyte progenitors in human cord blood and bone marrow. *J. Exp. Med*. 2015. 212: 385–399.

95 Balan, S., Arnold-Schrauf, C., Abbas, A., Couespel, N., Savoret, J., Imperatore, F., Villani, A.-C. et al., Large-scale human dendritic cell differentiation revealing notch-dependent lineage bifurcation and heterogeneity. *Cell Rep*. 2018. 24: 1902–1915.e6

96 Kirkling, M. E., Cytlak, U., Lau, C. M., Lewis, K. L., Resteu, A., Khodadadi-Jamayran, A., Siebel, C. W. et al., Notch signaling facilitates in vitro generation of cross-presenting classical dendritic cells. *Cell Rep*. 2018. 23: 3658–3672.e6.

97 Thordardottir, S., Hangalapura, B. N., Hutten, T., Cossu, M., Spanholtz, J., Schaap, N., Radstake, T. R. D. J. et al., The aryl hydrocarbon receptor antagonist StemRegenin 1 promotes human plasmacytoid and myeloid dendritic cell development from CD34+ hematopoietic progenitor cells. *Stem Cells Dev*. 2014. 23: 955–967.

98 Thordardottir, S., Schaap, N., Louer, E., Kester, M. G. D., Falkenburg, J. H. F., Jansen, J., Radstake, T. R. D. et al., Hematopoietic stem cell-derived myeloid and plasmacytoid DC-based vaccines are highly potent inducers of tumor-reactive T cell and NK cell responses ex vivo. *Oncoimmunology* 2017. 6: 00–00.

99 van Eck van der Sluijs, J., van Ens, D., Thordardottir, S., Vodegel, D., Hermens, I., van der Waart, A. B., Falkenburg, J. H. F. et al., Clinically applicable CD34+-derived blood dendritic cell subsets exhibit key subset-specific features and potently boost anti-tumor T and NK cell responses. *Cancer Immunol. Immunother*. 2021. 70: 3167–3181.

100 Luo, X.-L. and Dalod, M., The quest for faithful in vitro models of human dendritic cells types. *Mol. Immunol*. 2020. 123: 40–59.

101 Cytlak, U., Resteu, A., Pagan, S., Green, K., Milne, P., Maisuria, S., McDonald, D. et al., Differential IRF8 Transcription Factor Requirement Defines Two Pathways of Dendritic Cell Development in Humans. *Immunity* 2020. 53: 353–370.e8.

102 Wolins, N. E., Quaynor, B. K., Skinner, J. R., Tzekov, A., Park, C., Choi, K., and Bickel, P. E., OP9 mouse stromal cells rapidly differentiate into adipocytes: characterization of a useful new model of adipogenesis. *J Lipid Res*. 2006. 47: 450–460.

103 Murray, L. J., Young, J. C., Osborne, L. J., Luens, K. M., Scollay, R., Hill, B. L., Thrombopoietin, flt3, and kit ligands together suppress apoptosis of human mobilized CD34+ cells and recruit primitive CD34+Thy-1+ cells into rapid division. *Exp. Hematol*. 1999. 27: 1019–1028.

104 Sander, J., Schmidt, S. V., Cirovic, B., McGovern, N., Papantonopoulou, O., Hardt, A.-L., Aschenbrenner, A. C. et al., Cellular differentiation of human monocytes is regulated by time-dependent interleukin-4 signaling and the transcriptional regulator NCOR2. *Immunity* 2017. 47: 1051–1066.e12.

105 Robbins, S. H., Walzer, T., Dembélé, D., Thibault, C., Defays, A., Bessou, G., Xu, H. et al., Novel insights into the relationships between dendritic

cell subsets in human and mouse revealed by genome-wide expression profiling. *Genome Biol*. 2008. 9: R17.

106 Carpentier, S., Manh, T.-P. V., Chelbi, R., Henri, S., Malissen, B., Haffa, M., Ginhoux, F. et al., Comparative genomics analysis of mononuclear phagocyte subsets confirms homology between lymphoid tissue-resident and dermal XCR1+ DC in mouse and human and distinguishes them from Langerhans cells. *J. Immunol. Methods* 2016. 432: 35–49.

107 See, P., Dutertre, C.-A., Chen, J., Günther, P., McGovern, N., Irac, S. E., Gunawan, M. et al., Mapping the human DC lineage through the integration of high-dimensional techniques. *Science*. 2017. 356: eaag3009–15.

108 Villani, A.-C., Satija, R., Reynolds, G., Sarkizova, S., Shekhar, K., Fletcher, J., Griesbeck, M. et al., Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. *Science* 2017. 356: eaah4573–14.

109 Leylek, R., Alcántara-Hernández, M., Lanzar, Z., Lüdtke, A., Perez, O. A., Reizis, B. and Idoyaga, J., Integrated cross-species analysis identifies a conserved transitional dendritic cell population. *Cell Rep*. 2019. 29: 3736–3750.e8. Available at: https://reader.elsevier.com/reader/sd/pii/S2211124719315244?token=CFE31696767128AE70DC803E1611F08743B55FB0B8C8F6ED9E7389350205278FA7AC8674D36244665DADD3864C6DED F9.

110 Mulder, K., Patel, A. A., Kong, W. T., Piot, C., Halitzki, E., Dunsmore, G., Khalilnezhad, S. et al., Cross-tissue single-cell landscape of human monocytes and macrophages in health and disease. *Immunity* 2021. 54: 1883–1900.e5.

111 Dutertre, C.-A., Becht, E., Irac, S. E., Khalilnezhad, A., Narang, V., Khalilnezhad, S., Ng, P. Y. et al., Single-cell analysis of human mononuclear phagocytes reveals subset-defining markers and identifies circulating inflammatory dendritic cells. *Immunity* 2019. 51: 573–589.e8.

112 Bourdely, P., Anselmi, G., Vaivode, K., Ramos, R. N., Missolo-Koussou, Y., Hidalgo, S., Tosselo, J. et al., Transcriptional and functional analysis of CD1c+ human dendritic cells identifies a CD163+ subset priming CD8+CD103+ T cells. *Immunity*. 2020. 53: 335–352.e8.

113 Ruffin, N., Gea-Mallorquí, E., Brouiller, F., Jouve, M., Silvin, A., See, P., Dutertre, C.-A. et al., Constitutive Siglec-1 expression confers susceptibility to HIV-1 infection of human dendritic cell precursors. *Proc. National Acad. Sci*. 2019. 116: 21685–21693.

114 Alcántara-Hernández, M., Leylek, R., Wagar, L. E., Engleman, E. G., Keler, T., Marinkovich, M. P., Davis, M. M. et al., High-dimensional phenotypic mapping of human dendritic cells reveals interindividual variation and tissue specialization. *Immunity* 2017. 47: 1037–1050.e6.

115 Alculumbre, S. G., Saint-André, V., Domizio, J. D., Vargas, P., Sirven, P., Bost, P., Maurin, M. et al., Diversification of human plasmacytoid dendritic cells in response to a single stimulus. *Nat. Immunol*. 2018. 19: 63–75.

116 Segura, E., Touzot, M., Bohineust, A., Cappuccio, A., Chiocchia, G., Hosmalin, A., Dalod, M. et al., Human inflammatory dendritic cells induce Th17 cell differentiation. *Immunity* 2013. 38: 336–348. Available at: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=23352235&retmode=ref&cmd=prlinks

117 Coillard, A. and Segura, E., Antigen presentation by mouse monocyte-derived cells: Re-evaluating the concept of monocyte-derived dendritic cells. *Mol. Immunol*. 2021. 135: 165–169.

118 Crozat, K., Guiton, R., Guilliams, M., Henri, S., Baranek, T., Schwartz-Cornil, I., Malissen, B. et al. Comparative genomics as a tool to reveal functional equivalences between human and mouse dendritic cell subsets. *Immunol. Rev*. 2010. 234: 177–198.

119 Reizis, B., Plasmacytoid Dendritic Cells: Development, Regulation, and Function. *Immunity* 2019. 50: 37–50.
120 Nizzoli, G., Krietsch, J., Weick, A., Steinfelder, S., Facciotti, F., Gruarin, P., Bianco, A. et al. Human CD1c+ dendritic cells secrete high levels of IL-12 and potently prime cytotoxic T-cell responses. Blood 2013. 122: 932–942.

121 Lauterbach, H., Bathke, B., Gilles, S., Traidl-Hoffmann, C., Luber, C. A., Fejer, G., Freudenberg, M. A. et al. Mouse CD8alpha+ DC and human BDCA3+ DCs are major producers of IFN-lambda in response to poly IC. J. Exp. Med. 2010. 207: 2703–2717.

122 Jongbloed, S. L., Kassianos, A. J., McDonald, K. J., Clark, G. J., Ju, X., Angel, C. E., Chen, C. J. et al. Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens. J. Exp. Med. 2010. 207: 1247–1260.

123 Segura, E. and Amigorena, S., Cross-presentation by human dendritic cell subsets. Immunol. Lett. 2014. 158: 73–78.

124 MacDonald, K. P., Munster, D. J., Clark, G. J., Dzionek, A., Schmitz, J. and Hart, D. N., Characterization of human blood dendritic cell subsets. Blood 2002. 100: 4512–4520.

125 Schreibelt, G., Tel, J., Sliepen, K. H., Benitez-Ribas, D., Figdor, C. G., Adema, G. J. and de Vries, I. J., Toll-like receptor expression and function in human dendritic cell subsets: implications for dendritic cell-based anti-cancer immunotherapy. Cancer Immunol. Immunother. 2010. 59: 1573–1582.

126 Sittig, S. P., Bakdash, G., Weiden, J., Skold, A. E., Tel, J., Figdor, C. G., de Vries, I. J. et al. A Comparative Study of the T Cell Stimulatory and Polarizing Capacity of Human Primary Blood Dendritic Cell Subsets. Mediators Inflamm. 2016. 2016: 3605643.

127 Skold, A. E., Mathan, T. S. M., van Beek, J. J. P., Florez-Grau, G., van den Beukel, M. D., Sittig, S. P., Wimmers, F. et al. Naturally produced type I IFNs enhance human myeloid dendritic cell maturation and IL-12p70 production and mediate elevated effector functions in innate and adaptive immune cells. Cancer Immunol. Immunother. 2018. 67: 1425–1436.

128 Piccioli, D., Sammicheli, C., Tavarini, S., Nuti, S., Frigimelica, E., Manetti, A. G., Nuccitelli, A. et al. Human plasmacytoid dendritic cells are unresponsive to bacterial stimulation and require a novel type of cooperation with myeloid dendritic cells for maturation. Blood 2009. 113: 4232–4239.

129 van Beek, J. J., Gorris, M. A., Skold, A. E., Hatipoglu, I., Van Acker, H. H., Smits, E. L., de Vries, I. J. et al. Human blood myeloid and plasmacytoid

dendritic cells cross activate each other and synergize in inducing NK cell cytotoxicity. Oncoimmunology 2016. 5: e1227902.

130 Worah, K., Mathan, T. S. M., Vu Manh, T P., Keerthikumar, S., Schreibelt, G., Tel, J. et al. Proteomics of Human Dendritic Cell Subsets Reveals Subset-Specific Surface Markers and Differential Inflammasome Function. Cell Rep. 2016. 16: 2953–2966.

**Abbreviations:** ANR: Agence Nationale de la Recherche · ATMPs: Advanced Therapy Medicinal Products · BDCA: Blood Dendritic Cell Antigen · BM MNC: BM mononuclear cells · cDC: conventional DC · CDP: common dendritic cell progenitor · cMoP: common monocyte progenitors · CSC: Chondroitin sulfate C · CSC: Chondroitin sulfate sodium salt from shark cartilage · CTV: Cell Trace Violet · DPBS: Dulbecco's Phosphate Buffered Saline · FACS: Fluorescence Activated Cell Sorting · FC: flow cytometry · FLT3L: fms-like tyrosine kinase 3 ligand · FSC-A and SSC-A: forward and side scatter area · GMPr: good manufacturing practice · GMP: granulocyte macrophage progenitors · HoxB8 MPP: HoxB8 multipotent progenitors · HoxB8-ER: HoxB8 estrogen receptor fusion gene · HSC: hematopoietic stem cells · HSPC: hematopoietic stem/progenitor cells · LC: Langerhans cells · MDP: monocyte DC progenitors · MDSC: monocytic myeloid-derived suppressor cells · moDC: monocyte-derived DC · MPP: Multipotent progenitors · MSC: mesenchymal stem cells · NAF: non-adherent fraction · OP9-DL1: OP9 stromal cells expressing Delta-like 1 · PB: Polybrene · PB/CSC: polybrene/chondroitin sulfate C · pDC: plasmacytoid DC · PFA: Paraformaldehyde · SCF: Stem Cell Factor · scRNAseq: single cell RNA sequencing · SIRP-α: signal regulatory protein α · SSC-A and SSC-H: area vs the height of SSC · TLR: Toll-like receptor · TSEs: transmissible spongiform encephalopathies · WEHI: Walter and Eliza Hall Institute · β-Me: β-mercaptoethanol

**Full correspondence:** Prof. Manfred B. Lutz, Institute for Virology and Immunobiology, University of Würzburg, 97078 Würzburg, Germany e-mail: m.lutz@vim.uni-wuerzburg.de

Received: 14/1/2022

Revised: 28/3/2022

Accepted: 10/8/2022