ABSTRACT
In this report, we describe the characterization of a new monoclonal antibody, named 4H5CR4, against porcine CD9. Its use in combination with antibodies to CD4, CD8α, and 2E3 allows to distinguish at least five main CD4+ T cell subsets. Analysis on these subsets of CD45RA, CD27, CD29, CD95, CCR7, and SLA-DR markers depicts a progressive model of CD4+ T cell development. CD4+ 2E3+ CD8α- CD9- cells are the least differentiated population of naïve cells, whereas the CD4+ 2E3- CD8α+CD9+ and CD4+ 2E3- CD8α+ CD9- cells display phenotypic features of central and effector memory T helper cells, respectively. The latter subsets were able to produce IFN-γ after polyclonal activation with PMA/Ionomycin; however, in vitro virus-specific IFN-γ production of PBMCs collected at 38-44 days after pseudorabies virus vaccination was dominated by cells with a CD9+ phenotype. Therefore, CD9 appears to be a useful marker to investigate CD4+ T cell heterogeneity in swine.
Subject(s)
CD4-Positive T-Lymphocytes , T-Lymphocyte Subsets , Animals , Cell Differentiation , Immunologic Memory , Leukocyte Common Antigens , Phenotype , SwineABSTRACT
CLEC12A has been proposed as a suitable target for delivering antigen to dendritic cells (DCs) to enhance vaccine efficacy both in human and mouse. In this study, we have characterized the porcine homolog of CLEC12A (poCLEC12A). Using new monoclonal antibodies (mAb), raised against its ectodomain, poCLEC12A was found to be expressed on alveolar macrophages, blood conventional type 1 and type 2 DCs and plasmacytoid DCs, but not on monocytes, T cells, B cells or NK cells, in contrast to its human and murine homologs. Western blot analysis showed that in alveolar macrophages this receptor is expressed both as a monomer and a dimer. After binding to DCs, anti- poCLEC12A mAb was efficiently internalized. No significant changes were observed in TNFα or IFNα secretion by plasmacytoid DCs stimulated with either CpGs (ODN2216) or polyinosinic-polycytidylic acid (poly I:C), upon incubation with mAb. These results provide the basis for future investigations aimed to assess the ability of anti-poCLEC12A mAbs to improve vaccine efficacy by targeting antigen to DCs.
Subject(s)
Antibodies, Monoclonal/metabolism , Dendritic Cells/immunology , Lectins, C-Type/metabolism , Leukocytes, Mononuclear/metabolism , Macrophages/metabolism , Animals , Antibodies, Monoclonal/isolation & purification , CHO Cells , Cloning, Molecular , Cricetulus , Lectins, C-Type/genetics , Lectins, C-Type/immunology , Molecular Targeted Therapy , Oligodeoxyribonucleotides/genetics , Poly I-C/immunology , Recombinant Fusion Proteins/genetics , Swine , Toll-Like Receptor 9/agonists , Toll-Like Receptor 9/genetics , TranscriptomeABSTRACT
CLEC12B is a C-type lectin-like receptor expressed on myeloid cells. In this study, we have characterized the porcine homologue of CLEC12B (poCLEC12B). To this end, we have generated constructs encoding a c-myc tagged version of the whole receptor, or its ectodomain fused to the Fc portion of human IgG1, from a cDNA clone obtained from an alveolar macrophage library, and raised monoclonal antibodies (mAb) against this molecule. Using these mAbs, poCLEC12B was found to be expressed on alveolar macrophages and, at lower levels, on blood conventional type 1 dendritic cells (cDC1) and plasmacytoid DCs. No binding was detected on monocytes, monocyte-derived macrophages or monocyte-derived DCs. Engagement of CLEC12B on alveolar macrophages with mAbs had no apparent effect on cytokine production (TNF-α, IL-8) induced by LPS. These results provide the basis for future investigations aimed to assess the role of poCLEC12B in different microbial infections and to evaluate its potential in vaccination strategies targeting DCs.
