Next Article in Journal
Impact of Plasminogen Activator Inhibitor-1 Serum Levels and the -675 4G/5G Variant in the SERPINE1 Gene on Systemic Sclerosis in a Mexican Population
Previous Article in Journal
Growth Characteristics of a Desmodesmus Species from the San Antonio Springs and Its Short-Term Impact on Soil Microbial Dynamics
Previous Article in Special Issue
Age-Dependent Changes in Protist and Fungal Microbiota in a Peruvian Cattle Genetic Nucleus
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Life
Volume 14
Issue 9
10.3390/life14091054
life-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Development of the PD9-9 Monoclonal Antibody for Identifying Porcine Bone Marrow-Derived Dendritic Cells

by
Sang Eun Kim
1,2,
Young Kyu Kim
1,3,
Keon Bong Oh
2,* and
Jeong Ho Hwang
1,3,*
1
Department of Stem Cell and Regenerative Biotechnology, KU Institute of Science and Technology (KIT), Konkuk University, Seoul 05029, Republic of Korea
2
Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Republic of Korea
3
Animal Model Research Group, Korea Institute of Toxicology, 30 Baekhak1-gil, Jeongeup-si 56212, Republic of Korea
*
Authors to whom correspondence should be addressed.
Life 2024, 14(9), 1054; https://doi.org/10.3390/life14091054
Submission received: 3 July 2024 / Revised: 3 August 2024 / Accepted: 19 August 2024 / Published: 23 August 2024
(This article belongs to the Special Issue Feature Papers in Animal Science: 3rd Edition)
Browse Figures
Review Reports Versions Notes

Abstract

:
The purpose of this study was to develop a monoclonal antibody (mAb) that can identify porcine dendritic cells (DCs) that have differentiated from bone marrow progenitor cells. Hybridoma technology was used to obtain mAbs, and bone marrow-derived DCs (BMDCs) were employed as immunogens for producing antibodies. The generated PD9-9 mAbs exhibited considerable reactivity towards porcine BMDCs with applications in flow cytometry and immunostaining. The antibody was composed of heavy immunoglobulin gamma-1 chains and light kappa chains. The PD9-9 mAb recognized fully differentiated porcine BMDCs and cells undergoing DC differentiation. In contrast, bone marrow cells and macrophages were not recognized by PD9-9. In addition, the PD9-9 mAb promoted porcine DC proliferation. Consequently, the PD9-9 mAb may be a biomarker for porcine DCs and will be advantageous for investigating porcine DC biology.

Graphical Abstract

1. Introduction

Pigs are economically essential livestock and crucial large animal models for biomedical research, such as xenotransplantation and influenza infection biology [1,2,3,4,5]. Viral infections in pigs result in substantial economic losses and pose a global threat to the pig industry [6,7,8,9]. Major swine viruses, such as porcine reproductive and respiratory syndrome virus, foot-and-mouth disease virus, African swine fever virus, classical swine fever virus, and porcine circovirus type 2, interact with dendritic cells (DCs) throughout viral infection [10,11,12,13]. Therefore, DCs must be investigated because they play a critical role in primary viral infections. DCs are sentinels of the immune system and are professional antigen-presenting cells that frequently reside in tissues near pathogen entry sites [14,15,16,17]. However, the low frequency of DCs in pig tissues and other species complicates their isolation [18,19,20]. Hence, producing DCs in vitro could be a practical substitute.
DCs in mice and humans exhibit a notable level of CD11c expression; thus, CD11c is frequently used as a DC identification marker [14,21,22]. However, because porcine CD11c is also strongly expressed in monocytes and macrophages, it cannot be used as a marker to distinguish porcine DCs from other cell types [23]. In contrast, the Flt3 ligand, which is essential for DC development, can be a reliable porcine DC marker [24]. Flt3 was abundantly expressed in DC precursors and bone marrow-derived DCs (BMDCs) differentiated via Flt3 ligand stimulation, whereas it was undetectable in monocyte-derived DCs or BMDCs differentiated via GM-CSF supplementation [25]. Therefore, evaluating the expression of surface markers, such as MHC II, CD1, CD172a, and CD16, is essential to distinguish GM-CSF–BMDCs from bone marrow precursor cells or macrophages [26,27]. Porcine DC-pathogen interactions have been extensively studied using DCs differentiated in vitro from bone marrow cells (BMCs) using the granulocyte–macrophage colony-stimulating factor (GM-CSF) [28]. Therefore, this study aimed to develop monoclonal antibodies (mAbs) capable of identifying porcine BMDCs differentiated using the GM-CSF.

2. Materials and Methods

2.1. Animals

Animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals established by the National Institute of Health. Approval was obtained from the Institutional Animal Care and Use Committee (IACUC) of Konkuk University (approval no. KU15183 and KU16168) before the study. Gnotobiotic miniature pigs were housed in an absolute barrier-containing facility at the Bio-Organ Research Center of Konkuk University (Seoul, Republic of Korea) [29]. BMCs were isolated from stillborn piglets. A single 10-week-old male BALB/c mouse was used to generate hybridomas. The mouse was housed in the Animal Facility of the Department of Animal Biotechnology.

2.2. Cells

BMCs were isolated from the humerus, tibia, and femur of stillborn piglets to induce the differentiation of BMCs into DCs. BMDCs were generated as previously described [30]. Non-adherent cells were used for immunization and hybridoma screening.
3D4/2 cells and pulmonary alveolar macrophages were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 U/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco), 1 mM minimal essential medium non-essential amino acids (Gibco). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2.

2.3. Hybridoma Technology

Hybridoma cells were produced as previously described with slight modifications [31,32]. After immunization with BMDCs, the mice were euthanized after three additional boosters. Antibody-producing B lymphocytes were isolated from the spleens of immunized mice. Cell fusion was performed between the antibody-producing B lymphocytes and P3 × 63-Ag8-653 murine myeloma cells using polyethylene glycol 1500 (Roche, Basel, Switzerland). After cell fusion, hypoxanthine–aminopterin–thymidine (Sigma-Aldrich, St. Louis, MO, USA) selection was performed, and hypoxanthine–thymidine (Sigma-Aldrich, St. Louis, MO, USA) was subsequently supplemented. The mAbs produced by the hybridomas were obtained from culture supernatants. A Pierce Rapid ELISA Mouse mAb Isotyping Kit (Invitrogen, Waltham, MA, USA) was used to identify the immunoglobulin isotype of the mAbs.

2.4. Flow Cytometry

The cells were stained with mAbs obtained from the culture supernatant of the hybridomas, including anti-CD1 (76-7-4), anti-CD172a (pan-myeloid marker SWC3; 74-22-15), anti-CD4 (74-12-4), anti-CD8a (76-2-11), anti-CD16 (G7), and anti-MHC II (MSA3) [33]. An FITC- or APC-conjugated goat anti-mouse IgG antibody (BioLegend, San Diego, CA, USA) was used to label the cells. Flow cytometry was conducted with a BD FACSCalibur instrument (BD Biosciences, Franklin Lakes, NJ, USA). Data analysis was conducted using FlowJo 10.10.0 software (BD Biosciences).

2.5. Immunostaining

After fixation in 4% paraformaldehyde in Dulbecco’s phosphate-buffered saline, the cells were stained with cultures derived from the following hybridoma clones PD9-9 and G7 mAbs (anti-CD16). An FITC-conjugated goat anti-mouse IgG antibody (BioLegend) was used to label the cells. Nuclei were stained with TO-PRO-3 iodide (Life Technologies, Carlsbad, CA, USA). Confocal laser microscopy (Carl Zeiss, Oberkochen, Germany) was used to acquire the images.

2.6. Cell Proliferation Assay

The cells were labeled with carboxyfluorescein succinimidyl ester (CFSE; Invitrogen) before incubation with the mAb obtained from the culture supernatant of the PD9-9 hybridoma. Concentration-dependent supplementation of PD9-9 culture supernatant was performed. The cells were collected for analysis after five days of incubation at 37 °C in a humidified atmosphere containing 5% CO2. Cell proliferation was assessed using flow cytometry with a BD FACSCalibur instrument (BD Biosciences). Data analysis was conducted using FlowJo 10.10.0 software (BD Biosciences).

3. Results

3.1. Production of mAbs against Porcine DCs

DCs used as immunogens were differentiated from BMCs to develop mAbs against porcine DCs (Figure 1). mAb-producing hybridomas were obtained by immunizing mice with BMDCs as antigens. Antibodies recognizing porcine DCs were identified within the hybridoma clones. Then, antibodies were screened for reactivity with peripheral blood mononuclear cells. It was subsequently decided to exclude mAbs that exhibited non-specific reactivity against other leukocytes. mAbs that were specifically reactive with porcine BMDCs were chosen, and their isotypes were classified. Each antibody comprised a heavy immunoglobulin G and a light kappa chain (Table 1). The PD9-9 clone was selected and expanded for subsequent mAb screening because of its remarkable capacity to produce mAbs with extremely high reactivity (Figure S1).
PD9-9 mAbs identified porcine DC populations that expressed CD16 and CD1 and had high MHC II expression levels (Figure 2a). PD9-9 mAb bound to porcine DCs, consistent with anti-porcine CD16 (G7 mAb) (Figure 2b). In summary, this study identified the PD9-9 mAb that recognizes porcine DCs. Furthermore, its applicability in flow cytometry and immunocytochemistry was confirmed.

3.2. mAb PD9-9 Reactivity during DC Differentiation

The reactivity of the PD9-9 mAb to cells was assessed during the in vitro differentiation of BMCs to DCs. mAb reactivity kinetics during DC differentiation were ascertained through comparisons with MHC class II, CD16, CD1, and CD172a molecules (Figure 3). PD9-9 mAb-detectable cells were observed on day six of differentiation, and approximately 73.6% of these cells were positive (Figure 3c). In contrast to MHC II expression, which exhibited two distinct populations distinguished by high and low expressions by day 10, PD9-9 mAbs exhibited consistently high reactivity levels in 95.7% of cells (Figure 3d).

3.3. Distinguishing DCs from Macrophages

DCs and macrophages share numerous characteristics; therefore, the presence of shared markers may make it challenging to distinguish between the two. We examined the reactivity of PD9-9 antibodies towards porcine macrophages. PD9-9 mAbs did not recognize the porcine alveolar macrophage cell line 3D4/2 (Figure 4a). Additionally, primary macrophages isolated from the pulmonary alveoli demonstrated minimal reactivity (Figure 4b). In summary, despite its high reactivity towards DCs, PD9-9 exhibited minimal reactivity towards macrophages. This finding suggests that PD9-9 mAbs could distinguish between DCs and macrophages.

3.4. Effects on DC Proliferation

Certain mAbs modulate or control cellular mechanisms via antigen recognition [34,35]. This study investigated the effect of PD9-9 mAbs on DC proliferation to ascertain their functionality. After dose-dependent treatment with PD9-9 mAbs, DC proliferation was assessed using the CFSE assay. Administering PD9-9 mAb resulted in a dose-dependent increase in DC proliferation in the range of 41.6–64.7% (Figure 5).

4. Discussion

DCs migrate to lymphoid organs, where they present antigens to T cells [36,37]. DCs play a crucial role in priming naïve T cells, thereby initiating an adaptive immune response [38,39]. Thus, DCs are an essential bridge between innate and adaptive immunity during pathogen infection [40,41]. The capacity of DCs to induce antigen-specific immune responses has been employed to develop cell therapy technologies [42,43]. This study developed a mAb specific to porcine DCs, thereby contributing to the advancement of knowledge regarding the biology of porcine DCs. In most studies investigating DCs, monocytes or BMCs were cultured with GM-CSF, with or without IL-4, to induce DC differentiation in vitro. In this study, one of the developed mAbs exhibited remarkable reactivity towards porcine DCs and was designated PD9-9. The PD9-9 mAb application included flow cytometry and immunostaining. Moreover, the mAbs exhibited no reactivity towards porcine macrophages. Distinguishing DCs from macrophages has proven challenging because of the similarity of their properties [44,45]. Hence, the PD9-9 mAb may be a useful tool for distinguishing porcine DCs from macrophages.
Before differentiation, the PD9-9 antibody did not recognize BMCs; however, it exhibited cell reactivity on day 6 after inducing DC differentiation. In addition to being applicable to fully differentiated porcine DCs, the PD9-9 antibody could identify DCs in the process of differentiation. In a previous study, porcine BMDCs were differentiated with GM-CSF on day 10 to distinguish between immature (MHCIIlow) and mature (MHCIIhigh) DCs based on differential MHC II expression [30]. MHCIIhigh and MHCIIlow cells were both possessed the capacity to prime T cells, a signature function of DCs. All BMDCs, including immature DCs with low MHCII expression, were identified by PD9-9 on day 10. PD9-9 was also capable of identifying immature DCs that emerged on day 6 and were characterized by MHCIIlow cells. Because PD9-9 mAbs recognized immature and mature BMDCs, they can be used as a biomarker for porcine BMDCs and in the study of DC ontogeny.
Antibodies are essential components of the immune system that function by circulating within the bloodstream and identifying exogenous substances [46,47]. The emergence of hybridoma technology, which enables manufacturing mAbs, has driven biotechnology forwards [48,49]. This study generated PD9-9 mAb against porcine BMDCs differentiated with GM-CSF using hybridoma technology. PD9-9 mAbs could identify porcine DCs that had completed differentiation and cells in the process of DC differentiation but were inactive against bone marrow progenitor cells. Antibodies generated using hybridoma technology may possess functionalities in addition to antigen recognition [50]. PD9-9 also has the ability to promote cell proliferation in porcine BMDCs when it is administered. Considering the broad roles of DC in immunobiology, the ability of PD9-9 to promote DC proliferation could potentially serve a therapeutic purpose. The immunocytochemistry results indicate that the PD9-9 mAb reactivates the protein that are located on the surface of BMDCs. Hence, it is probable that the antigen corresponds to one of the cell surface proteins present on porcine DCs. The epitope is the specific region of the antigen that the antibody recognizes, and it can be complex for proteinaceous antigen [51]. The binding epitope of an antibody can offer significant mechanistic insights and suggest potential applications for the antibody. Recently, a variety of advanced methods has been developed and is extensively employed to predict antigens and epitopes [52]. Additional research is required to identify the specific protein antigen and epitope that are targeted by the PD9-9 mAb, using these advanced approaches. Consequently, the PD9-9 mAb developed in this research will be the most effective to utilize.

5. Conclusions

In conclusion, PD9-9 mAbs recognized fully differentiated porcine BMDCs and cells that were in the process of undergoing DC differentiation. In order to distinguish between macrophages, the PD9-9 mAb can be employed as an additional porcine DC marker. Additionally, in vitro DC proliferation is promoted by the PD9-9 mAb. Thus, the PD9-9 mAb could be a novel tool for investigating the immune ontogeny of porcine DC cell types and studying the biology of porcine DCs.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/life14091054/s1, Figure S1: Monoclonal antibodies (mAbs) that react to porcine dendritic cells.

Author Contributions

Conceptualization, J.H.H. and S.E.K.; validation, Y.K.K. and S.E.K.; formal analysis, S.E.K.; investigation, Y.K.K.; resources, J.H.H.; data curation, K.B.O.; writing—original draft preparation, S.E.K.; writing—review and editing, J.H.H. and K.B.O.; visualization, Y.K.K.; supervision, J.H.H. and K.B.O.; project administration, J.H.H. and K.B.O.; funding acquisition, J.H.H. and K.B.O. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Korea Institute of Toxicology grant funded by the Ministry of science and ICT (Project number: KK-2403) and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (grant number: HI23C041702).

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University (KU15183/15 February 2016 and KU16168/14 October 2016).

Data Availability Statement

Data are contained within the article or the Supplementary Materials.

Acknowledgments

This study was supported by the 2024 RDA Fellowship Program of the National Institute of Animal Science of the Rural Development Administration of the Republic of Korea.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Xi, J.; Zheng, W.; Chen, M.; Zou, Q.; Tang, C.; Zhou, X. Genetically engineered pigs for xenotransplantation: Hopes and challenges. Front. Cell Dev. Biol. 2022, 10, 1093534. [Google Scholar] [CrossRef]
  2. Vintrych, P.; Al-Obeidallah, M.; Horák, J.; Chvojka, J.; Valešová, L.; Nalos, L.; Jarkovská, D.; Matějovič, M.; Štengl, M. Modeling sepsis, with a special focus on large animal models of porcine peritonitis and bacteremia. Front. Physiol. 2022, 13, 1094199. [Google Scholar] [CrossRef]
  3. Kim, S.W.; Gormley, A.; Jang, K.B.; Duarte, M.E. Invited Review—Current status of global pig production: An overview and research trends. Anim. Biosci. 2024, 37, 719–729. [Google Scholar] [CrossRef] [PubMed]
  4. Netzley, A.H.; Pelled, G. The Pig as a Translational Animal Model for Biobehavioral and Neurotrauma Research. Biomedicines 2023, 11, 2165. [Google Scholar] [CrossRef] [PubMed]
  5. Paslawski, R.; Kowalczyk, P.; Paslawska, U.; Wiśniewski, J.; Dzięgiel, P.; Janiszewski, A.; Kiczak, L.; Zacharski, M.; Gawdzik, B.; Kramkowski, K.; et al. Analysis of the Model of Atherosclerosis Formation in Pig Hearts as a Result of Impaired Activity of DNA Repair Enzymes. Int. J. Mol. Sci. 2024, 25, 2282. [Google Scholar] [CrossRef]
  6. Wang, J.; Zhu, X.; Cai, C.; Pan, X.; Wang, C. Risk Analysis of Viral Diseases in Infected Pig Farms during the Lockdown Period in China, January to May 2020. Int. J. Environ. Res. Public Health 2022, 19, 3215. [Google Scholar] [CrossRef]
  7. Cai, H.; Zhang, H.; Cheng, H.; Liu, M.; Wen, S.; Ren, J. Progress in PRRSV Infection and Adaptive Immune Response Mechanisms. Viruses 2023, 15, 1442. [Google Scholar] [CrossRef]
  8. Lim, J.-W.; Vu, T.T.H.; Le, V.P.; Yeom, M.; Song, D.; Jeong, D.G.; Park, S.-K. Advanced Strategies for Developing Vaccines and Diagnostic Tools for African Swine Fever. Viruses 2023, 15, 2169. [Google Scholar] [CrossRef] [PubMed]
  9. Bergmann, H.; Dups-Bergmann, J.; Schulz, K.; Probst, C.; Zani, L.; Fischer, M.; Gethmann, J.; Denzin, N.; Blome, S.; Conraths, F.J.; et al. Identification of Risk Factors for African Swine Fever: A Systematic Review. Viruses 2022, 14, 2107. [Google Scholar] [CrossRef]
  10. Crisci, E.; Fraile, L.; Montoya, M. Cellular Innate Immunity against PRRSV and Swine Influenza Viruses. Vet. Sci. 2019, 6, 26. [Google Scholar] [CrossRef]
  11. Medina, G.N.; Segundo, F.D.-S.; Stenfeldt, C.; Arzt, J.; de los Santos, T. The Different Tactics of Foot-and-Mouth Disease Virus to Evade Innate Immunity. Front. Microbiol. 2018, 9, 2644. [Google Scholar] [CrossRef] [PubMed]
  12. Franzoni, G.; Graham, S.P.; Dei Giudici, S.; Oggiano, A. Porcine Dendritic Cells and Viruses: An Update. Viruses 2019, 11, 445. [Google Scholar] [CrossRef]
  13. Parra-Sánchez, H.; Bustamante-Córdova, L.; Reséndiz, M.; Mata-Haro, V.; Pinelli-Saavedra, A.; Hernández, J. Analysis of Swine Conventional Dendritic Cells, DEC205+CD172a+/−CADM1+, from Blood and Spleen in Response to PRRSV and PEDV. Viruses 2019, 11, 1001. [Google Scholar] [CrossRef]
  14. Merad, M.; Sathe, P.; Helft, J.; Miller, J.; Mortha, A. The dendritic cell lineage: Ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu. Rev. Immunol. 2013, 31, 563–604. [Google Scholar] [CrossRef] [PubMed]
  15. Steinman, R.M. The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 1991, 9, 271–296. [Google Scholar] [CrossRef] [PubMed]
  16. Zanna, M.Y.; Yasmin, A.R.; Omar, A.R.; Arshad, S.S.; Mariatulqabtiah, A.R.; Nur-Fazila, S.H.; 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. [Google Scholar] [CrossRef]
  17. Tong, C.; Liang, Y.; Han, X.; Zhang, Z.; Zheng, X.; Wang, S.; Song, B. Research Progress of Dendritic Cell Surface Receptors and Targeting. Biomedicines 2023, 11, 1673. [Google Scholar] [CrossRef]
  18. Edwards, J.C.; Everett, H.E.; Pedrera, M.; Mokhtar, H.; Marchi, E.; Soldevila, F.; Kaveh, D.A.; Hogarth, P.J.; Johns, H.L.; Nunez-Garcia, J.; et al. CD1− and CD1+ porcine blood dendritic cells are enriched for the orthologues of the two major mammalian conventional subsets. Sci. Rep. 2017, 7, 40942. [Google Scholar] [CrossRef]
  19. Maisonnasse, P.; Bouguyon, E.; Piton, G.; Ezquerra, A.; Urien, C.; Deloizy, C.; Bourge, M.; Leplat, J.J.; Simon, G.; Chevalier, C.; et al. The respiratory DC/macrophage network at steady-state and upon influenza infection in the swine biomedical model. Mucosal Immunol. 2016, 9, 835–849. [Google Scholar] [CrossRef]
  20. Parra-Sánchez, H.; Puebla-Clark, L.; Reséndiz, M.; Valenzuela, O.; Hernández, J. Characterization and expression of DEC205 in the cDC1 and cDC2 subsets of porcine dendritic cells from spleen, tonsil, and submaxillary and mesenteric lymph nodes. Mol. Immunol. 2018, 96, 1–7. [Google Scholar] [CrossRef]
  21. Schraml, B.U.; Reis e Sousa, C. Defining dendritic cells. Curr. Opin. Immunol. 2015, 32, 13–20. [Google Scholar] [CrossRef]
  22. Haniffa, M.; Collin, M.; Ginhoux, F. Chapter One—Ontogeny and Functional Specialization of Dendritic Cells in Human and Mouse. In Advances in Immunology; Murphy, K.M., Merad, M., Eds.; Academic Press: Cambridge, MA, USA, 2013; Volume 120, pp. 1–49. [Google Scholar]
  23. Summerfield, A.; McCullough, K.C. The porcine dendritic cell family. Dev. Comp. Immunol. 2009, 33, 299–309. [Google Scholar] [CrossRef]
  24. Guzylack-Piriou, L.; Alves, M.P.; McCullough, K.C.; Summerfield, A. Porcine Flt3 ligand and its receptor: Generation of dendritic cells and identification of a new marker for porcine dendritic cells. Dev. Comp. Immunol. 2010, 34, 455–464. [Google Scholar] [CrossRef]
  25. Xu, Y.; Zhan, Y.; Lew, A.M.; Naik, S.H.; 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. [Google Scholar] [CrossRef] [PubMed]
  26. Carrasco, C.P.; Rigden, R.C.; Schaffner, R.; Gerber, H.; Neuhaus, V.; Inumaru, S.; Takamatsu, H.; Bertoni, G.; McCullough, K.C.; Summerfield, A. Porcine dendritic cells generated in vitro: Morphological, phenotypic and functional properties. Immunology 2001, 104, 175–184. [Google Scholar] [CrossRef]
  27. Summerfield, A.; Guzylack-Piriou, L.; Schaub, A.; Carrasco, C.P.; Tâche, V.; Charley, B.; McCullough, K.C. Porcine peripheral blood dendritic cells and natural interferon-producing cells. Immunology 2003, 110, 440–449. [Google Scholar] [CrossRef] [PubMed]
  28. Martelet, L.; Lacouture, S.; Goyette-Desjardins, G.; Beauchamp, G.; Surprenant, C.; Gottschalk, M.; Segura, M. Porcine Dendritic Cells as an In Vitro Model to Assess the Immunological Behaviour of Streptococcus suis Subunit Vaccine Formulations and the Polarizing Effect of Adjuvants. Pathogens 2017, 6, 13. [Google Scholar] [CrossRef]
  29. Hwang, J.H.; Gupta, M.K.; Park, C.K.; Kim, Y.B.; Lee, H.T. Establishment of major histocompatibility complex homozygous gnotobiotic miniature swine colony for xenotransplantation. Anim. Sci. J. 2015, 86, 468–475. [Google Scholar] [CrossRef] [PubMed]
  30. Kim, S.E.; Hwang, J.H.; Kim, Y.K.; Lee, H.T. Heterogeneity of porcine bone marrow-derived dendritic cells induced by GM-CSF. PLoS ONE 2019, 14, e0223590. [Google Scholar] [CrossRef]
  31. Hwang, J.H.; Kim, S.E.; Jung, S.H.; Kim, Y.K.; Kim, Y.B.; Lee, H.T. Characterization of monoclonal antibodies against porcine pulmonary alveolar macrophages of gnotobiotic miniature swine. Biochem. Biophys. Res. Commun. 2015, 461, 427–434. [Google Scholar] [CrossRef]
  32. Kim, E.-J.; Im, G.-M.; Lee, C.-S.; Kim, Y.-G.; Ko, B.J.; Jeong, H.-J.; Kim, B.-G. Generation of Monoclonal Antibodies for Sensitive Detection of Pro-Inflammatory Protein S100A9. Appl. Sci. 2021, 11, 4659. [Google Scholar] [CrossRef]
  33. Dawson, H.D.; Lunney, J.K. Porcine cluster of differentiation (CD) markers 2018 update. Res. Vet. Sci. 2018, 118, 199–246. [Google Scholar] [CrossRef] [PubMed]
  34. Lan, H.; Li, W.; Jiang, H.; Yang, Y.; Zheng, X. Intracellular signaling transduction pathways triggered by a well-known anti-GHR monoclonal antibody, Mab263, in vitro and in vivo. Int. J. Mol. Sci. 2014, 15, 20538–20554. [Google Scholar] [CrossRef] [PubMed]
  35. Chiu, M.L.; Goulet, D.R.; Teplyakov, A.; Gilliland, G.L. Antibody Structure and Function: The Basis for Engineering Therapeutics. Antibodies 2019, 8, 55. [Google Scholar] [CrossRef]
  36. Mellman, I.; Steinman, R.M. Dendritic cells: Specialized and regulated antigen processing machines. Cell 2001, 106, 255–258. [Google Scholar] [CrossRef]
  37. Klein, L.; Hinterberger, M.; Wirnsberger, G.; Kyewski, B. Antigen presentation in the thymus for positive selection and central tolerance induction. Nat. Rev. Immunol. 2009, 9, 833–844. [Google Scholar] [CrossRef] [PubMed]
  38. Mempel, T.R.; Henrickson, S.E.; Von Andrian, U.H. T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 2004, 427, 154–159. [Google Scholar] [CrossRef] [PubMed]
  39. Reddi, D.; Durant, L.; Bernardo, D.; Noble, A.; English, N.R.; Hendy, P.; Clark, G.C.; Prior, J.L.; Williamson, E.D.; Knight, S.C. In Vitro Priming of Human T Cells by Dendritic Cells Provides a Screening Tool for Candidate Vaccines for Burkholderia pseudomallei. Vaccines 2021, 9, 929. [Google Scholar] [CrossRef]
  40. Banchereau, J.; Steinman, R.M. Dendritic cells and the control of immunity. Nature 1998, 392, 245–252. [Google Scholar] [CrossRef]
  41. Palucka, K.; Banchereau, J. Dendritic cells: A link between innate and adaptive immunity. J. Clin. Immunol. 1999, 19, 12–25. [Google Scholar] [CrossRef]
  42. Kurilin, V.; Alshevskaya, A.; Sennikov, S. Development of Cell Technologies Based on Dendritic Cells for Immunotherapy of Oncological Diseases. Biomedicines 2024, 12, 699. [Google Scholar] [CrossRef] [PubMed]
  43. Khan, F.U.; Khongorzul, P.; Raki, A.A.; Rajasekaran, A.; Gris, D.; Amrani, A. Dendritic Cells and Their Immunotherapeutic Potential for Treating Type 1 Diabetes. Int. J. Mol. Sci. 2022, 23, 4885. [Google Scholar] [CrossRef]
  44. Guilliams, M.; Ginhoux, F.; Jakubzick, C.; Naik, S.H.; Onai, N.; Schraml, B.U.; Segura, E.; Tussiwand, R.; Yona, S. Dendritic cells, monocytes and macrophages: A unified nomenclature based on ontogeny. Nat. Rev. Immunol. 2014, 14, 571–578. [Google Scholar] [CrossRef]
  45. Lee, M.; Du, H.; Winer, D.A.; Clemente-Casares, X.; Tsai, S. Mechanosensing in macrophages and dendritic cells in steady-state and disease. Front. Cell Dev. Biol. 2022, 10, 1044729. [Google Scholar] [CrossRef] [PubMed]
  46. Marshall, J.S.; Warrington, R.; Watson, W.; Kim, H.L. An introduction to immunology and immunopathology. Allergy Asthma Clin. Immunol. 2018, 14, 49. [Google Scholar] [CrossRef]
  47. Malik, B.; Ghatol, A. Understanding How Monoclonal Antibodies Work. In StatPearls; StatPearls Publishing LLC: Treasure Island, FL, USA, 2024. [Google Scholar]
  48. Forthal, D.N. Functions of Antibodies. Microbiol. Spectr. 2014, 2, 1–17. [Google Scholar] [CrossRef]
  49. Mitra, S.; Tomar, P.C. Hybridoma technology; advancements, clinical significance, and future aspects. J. Genet. Eng. Biotechnol. 2021, 19, 159. [Google Scholar] [CrossRef]
  50. Schroeder, H.W., Jr.; Cavacini, L. Structure and function of immunoglobulins. J. Allergy Clin. Immunol. 2010, 125, S41–S52. [Google Scholar] [CrossRef] [PubMed]
  51. Nilvebrant, J.; Rockberg, J. An Introduction to Epitope Mapping. Methods Mol. Biol. 2018, 1785, 1–10. [Google Scholar] [CrossRef]
  52. Zeng, X.; Bai, G.; Sun, C.; Ma, B. Recent Progress in Antibody Epitope Prediction. Antibodies 2023, 12, 52. [Google Scholar] [CrossRef]
Life 14 01054 g001
Figure 1. Diagram illustrating the production process of monoclonal antibodies (mAbs) against porcine dendritic cells (DCs). Porcine bone marrow cells (BMCs) were isolated and differentiated into DCs. B lymphocytes from mice were obtained after four immunizations with porcine bone marrow-derived DCs (BMDCs). Cell fusion was performed between murine B lymphocytes and myeloma cells. Antibody-producing hybridomas were obtained via hypoxanthine–aminopterin–thymidine (HAT) selection and hypoxanthine–thymidine (HT) supplementation.
Figure 1. Diagram illustrating the production process of monoclonal antibodies (mAbs) against porcine dendritic cells (DCs). Porcine bone marrow cells (BMCs) were isolated and differentiated into DCs. B lymphocytes from mice were obtained after four immunizations with porcine bone marrow-derived DCs (BMDCs). Cell fusion was performed between murine B lymphocytes and myeloma cells. Antibody-producing hybridomas were obtained via hypoxanthine–aminopterin–thymidine (HAT) selection and hypoxanthine–thymidine (HT) supplementation.
Life 14 01054 g001
Life 14 01054 g002
Figure 2. Application of PD9-9 mAbs to identify porcine DCs. (a) The identification of porcine DCs stained with PD9-9 mAb in combination with MHC II, CD16, and CD1 was confirmed using flow cytometry. Blue-filled histograms indicate cells that are stained with each mAb, whereas gray-filled histograms represent isotype controls. (b) The binding of PD9-9 mAb to porcine DCs is confirmed using immunocytochemistry. The FITC-labeled antibodies that recognize the antigens are denoted in green. Topro-3-stained nuclei exhibit a red luminescence.
Figure 2. Application of PD9-9 mAbs to identify porcine DCs. (a) The identification of porcine DCs stained with PD9-9 mAb in combination with MHC II, CD16, and CD1 was confirmed using flow cytometry. Blue-filled histograms indicate cells that are stained with each mAb, whereas gray-filled histograms represent isotype controls. (b) The binding of PD9-9 mAb to porcine DCs is confirmed using immunocytochemistry. The FITC-labeled antibodies that recognize the antigens are denoted in green. Topro-3-stained nuclei exhibit a red luminescence.
Life 14 01054 g002
Life 14 01054 g003
Figure 3. PD9-9 mAb reactivity to cells during BMDC differentiation. Cells were harvested and examined on days 0 (a), 3 (b), 6 (c), and 10 (d) using flow cytometry during BMDC differentiation. Cells were stained with the indicated mAb.
Figure 3. PD9-9 mAb reactivity to cells during BMDC differentiation. Cells were harvested and examined on days 0 (a), 3 (b), 6 (c), and 10 (d) using flow cytometry during BMDC differentiation. Cells were stained with the indicated mAb.
Life 14 01054 g003
Life 14 01054 g004
Figure 4. Evaluation of PD9-9 mAb reactivity towards porcine macrophages. (a) The reactivity of PD9-9 mAbs with 3D4/2 cells is evaluated. (b) The reactivity of PD9-9 mAb against pulmonary alveolar macrophages is evaluated. Red histograms represent cells stained with the indicated antibody, whereas gray histograms denote the isotype control.
Figure 4. Evaluation of PD9-9 mAb reactivity towards porcine macrophages. (a) The reactivity of PD9-9 mAbs with 3D4/2 cells is evaluated. (b) The reactivity of PD9-9 mAb against pulmonary alveolar macrophages is evaluated. Red histograms represent cells stained with the indicated antibody, whereas gray histograms denote the isotype control.
Life 14 01054 g004
Life 14 01054 g005
Figure 5. The application of PD9-9 mAbs to promote porcine DC proliferation. Porcine DCs are labeled with the carboxyfluorescein succinimidyl ester (CFSE) and incubated for 5 days in the presence of PD9-9 mAbs in a ranging dose. Cell proliferation is verified using the CFSE assay and assessed via flow cytometry.
Figure 5. The application of PD9-9 mAbs to promote porcine DC proliferation. Porcine DCs are labeled with the carboxyfluorescein succinimidyl ester (CFSE) and incubated for 5 days in the presence of PD9-9 mAbs in a ranging dose. Cell proliferation is verified using the CFSE assay and assessed via flow cytometry.
Life 14 01054 g005
Table 1. Isotype of monoclonal antibodies against porcine dendritic cells.
Table 1. Isotype of monoclonal antibodies against porcine dendritic cells.
CloneHeavy ChainLight Chain
PD9-7IgG1ϰ
PD9-9IgG1ϰ
PD10-3IgG1ϰ
G7IgG1ϰ
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Kim, S.E.; Kim, Y.K.; Oh, K.B.; Hwang, J.H. Development of the PD9-9 Monoclonal Antibody for Identifying Porcine Bone Marrow-Derived Dendritic Cells. Life 2024, 14, 1054. https://doi.org/10.3390/life14091054

AMA Style

Kim SE, Kim YK, Oh KB, Hwang JH. Development of the PD9-9 Monoclonal Antibody for Identifying Porcine Bone Marrow-Derived Dendritic Cells. Life. 2024; 14(9):1054. https://doi.org/10.3390/life14091054

Chicago/Turabian Style

Kim, Sang Eun, Young Kyu Kim, Keon Bong Oh, and Jeong Ho Hwang. 2024. "Development of the PD9-9 Monoclonal Antibody for Identifying Porcine Bone Marrow-Derived Dendritic Cells" Life 14, no. 9: 1054. https://doi.org/10.3390/life14091054

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop