Characterization of the Myeloid Cell Populations’ Resident in the Porcine Palatine Tonsil

Abstract

The palatine tonsil is the portal of entry for food and air and is continuously subjected to environmental challenges, including pathogens, which use the tonsil and pharynx as a primary site of replication. In pigs, this includes the viruses causing porcine respiratory and reproductive syndrome, and classical and African swine fever; diseases that have impacted the pig production industry globally. Despite the importance of tonsils in host defense, little is known regarding the phenotype of the myeloid cells resident in the porcine tonsil. Here, we have characterized five myeloid cell populations that align to orthologous populations defined in other mammalian species: a CD4+ plasmacytoid dendritic cell (DC) defined by expression of the conserved markers E2.2 and IRF-7, a conventional dendritic cell (cDC1) population expressing CADM1highCD172alow and high levels of XCR1 able to activate allogeneic CD4 and CD8 T cells; a cDC2 population of CADM1dim cells expressing FLT3, IRF4, and CSF1R with an ability to activate allogeneic CD4 T cells; CD163+ macrophages (Mϴs) defined by high levels of endocytosis and responsiveness to LPS and finally a CD14+ population likely derived from the myelomonocytic lineage, which showed the highest levels of endocytosis, a capacity for activation of CD4+ memory T cells, combined with lower relative expression of FLT3. Increased knowledge regarding the phenotypic and functional properties of myeloid cells resident in porcine tonsil will enable these cells to be targeted for future vaccination strategies to current and emerging porcine viruses.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…In porcine tonsils, plasmacytoid DC (CD172a low CD4 + MHCII low ) and conventional DC (cDC1, CD172a −/low CADM1 ++ MHCII ++ and cDC2, CD172a ++ CD14 − CD163 − MHCII ++ CADM1 low ) but also monocyte‐derived cells (CD172a ++ CD163 − CD14 + MHCII ++ ) and macrophage‐like cells (CD172a ++ , CD163 + CD14 − MHCII ++ ) have been identified . Compared to blood, not all tonsil‐resident cDC1 and cDC2 cells express the C‐type lectin receptor DEC205 .…”

Development of Pig Conventional Dendritic Cells From Bone Marrow Hematopoietic Cells in vitro

“…Taken together, and considering that CADM1 is a cross-species, evolutionarily-conserved DC-specific marker that is not present in moDCs ( 38 , 39 ), classification of the CD14 + subset as cDCs or moDCs cannot be entirely precise. In the work of Soldevila et al ( 20 ), a similar CD14 + subset was also identified in porcine tonsils, which displayed an intermediate attribute between cDCs and moDCs in terms of phenotype, transcriptomics, and pathogen clearance. To note, morphologically cDC2 were produced as clusters of 5–15 cells, while CD14 + cells were seen as single cells, but both possessed typical DC dendritic and veiled extensions and lack of plastic adherence (data not shown).…”

“…This was compatible with the expression pattern of CD11b on blood cDC1 ( 22 ) but in contrast with the findings for lung cDC1 ( 14 ). In mice, CD11b is considered a specific marker of cDC2 ( 29 ); in pigs, according to the current result, CD11b expression on cDCs may depend on the cell type, as well as on the location of the cells, namely, blood or tissues; (2) in addition to FLT3 and ZBTB46, these cells highly expressed XCR1, which is a factor strictly associated with cDC1 ( 30 ) as well as IRF8, a driving factor of cDC1 development in human and mice ( 31 ); (3) they showed antigen presentation capability, particularly in activating allogenic CD8 + CD4 + T cells, indicating the potential of inducing CD8 + effector/memory T cells; (4) they secreted a substantial amount of IL-12p40 following poly I:C stimulation, similarly to what was found in tonsil cDC1 ( 20 ) and, in lung cDC1 after influenza A virus infection ( 14 ). However, this differed from blood cDC1 where no IL-12p40 was detected by multiplex cytokine assay or qRT-PCR.…”

“…The use of Flt3L alone to stimulate BMHCs produced three main populations, which shared a CADM1 + MHC-II + phenotype and a variable expression of CD14 and CD172a. The first population was CADM1 + CD14 – MHC-II + CD172a –/ lo , which presented full characteristics of cDC1: (1) it was further phenotyped as CD1 – , CD163 – , CD11R3 lo , CD11R1 + , and DEC205 + , equal to the cDC1 described in porcine lymphoid tissues ( 15 , 20 ), skin ( 21 ), lung ( 14 ), and blood ( 22 ). Of note, CD11b was positive on our cDC1, as revealed by the staining of CD11R3 and CD11R1 and the Quantitative Real-Time PCR (RT-qPCR) of ITGAM.…”

“…In pigs, Guzylack-Piriou et al ( 19 ) showed that addition of Flt3 ligand (Flt3L) to bone marrow cell cultures produced cells with features of bona fide DCs. In the past years, DCs from pig lymphoid (spleen, tonsil, and lymph nodes) ( 15 , 20 ) and non-lymphoid organs (skin and lung) ( 14 , 21 ) as well as blood ( 22 ) have been successfully identified and characterized. From the abovementioned studies, consensus has been reached on the phenotype of cDC1 (CADM1 + CD14 – MHC-II + CD172a –/ lo ), cDC2 (CADM1 + CD14 – MHC-II + CD172a + ), and pDC (CADM1 – CD14 – CD172a + CD4 + ).…”