Investigation
Characterization of surface receptors expressed in dendritic cells and other swine APC.
We have characterized several members of the family of C-type lectin receptors in the pig. To this end, we have generated cell lines stably expressing soluble or membrane-associated forms of porcine homologues of CLEC4A, CLEC12A and CLEC12B. Using monoclonal antibodies raised against these receptors we have analysed their distribution in different leukocyte subsets. Porcine CLEC12A and CLEC12B show a restricted expression to blood DCs and some tissue macrophage subsets, not being detected on monocytes, granulocytes or lymphocytes.
We have also produced antibodies and their derived scFv fragments specific for CD163 and Siglec-1/CD169. These receptors are expressed on macrophage populations of the subcapsular sinus (SSC) of lymph nodes and the marginal zone of spleen, specialized in trapping lymph and blood-borne antigens, and in some DC subsets.
These mAbs can be useful tools for delivering vaccine antigens to porcine APCs as a strategy to enhance vaccine efficacy. In this regard, we have shown that binding of mAbs to CD163, Siglec-1/CD169 or CLEC12A on these cells led to their internalization. Moreover, pigs inoculated with mAbs to CD163, Siglec-1/CD169 or CLEC12A show stronger anti-mouse Ig humoral responses than control animals, inoculated with an irrelevant isotype-matched mAb.
Permissiveness of bone marrow monocyte subsets to PRRS virus infection
Porcine bone marrow comprises two major monocyte subsets, one of which expresses CD163 and Siglec-1/CD169, two receptors involved in the entry of Porcine reproductive and respiratory síndrome virus (PRRSv) in macrophages. We have investigated the permissiveness of these subsets to PRRSv infection. PRRSv replicates efficiently in bone marrow CD163+ monocytes reaching titers similar to those obtained in alveolar macrophages. The infection of these monocytes by PRRSv may contribute to persistence of the virus and to hematological disorders found in infected animals.
Expression of Siglecs in porcine blood, spleen and lymph node DCs
Cells of immune system express a variety of sialic-acid-binding-immunoglobulin-like lectins (Siglecs) on their surface which can modulate their activation through interaction with sialylated structures expressed by other cells or pathogens. We have analyzed the expression of Siglec-1/CD169, Siglec-3, -5 and -10 in porcine conventional DC subsets (cDC) from blood, spleen and lymph nodes. Siglec-3 and -5 were expressed in type1 cDC1 and, at higher levels, in type 2 cDC2 in all these tissues. On the other hand, Siglec-1 was expressed in cDC1 and, at lower levels, cDC2 of spleen, but not, or at very low levels, in the other tissues. Siglec-10 was not detected in any cDC subset.
Characterization of porcine homologues of CD200R1 and CD200R1L
CD200R1 and CD200R1L are members of a family of paired receptors that contribute to modulate the activation of cells of immune system and prevent excessive responses that may be detrimental for the host. The regulatory role of these molecules appears to be exploited by different pathogens to downregulate host defenses and facilitate their survival into the host.
We have characterized the porcine homologues of CD200R1 and CD200R1L and analyzed their distribution in different leukocyte subsets using mAbs developed by us. These receptors were expressed on monocytes, macrophages and B cells. On the latter, expression of these receptors appears to be tightly regulated, being found on subpopulations of effector/memory B cells and plasmablasts. Besides, they modulate the antibody production induced by the ligand of TLR-7 imiquimoid. The study of the heterogeneity of these cell populations and the role of CD200R1/CD200R1L receptors in the regulation of B cell responses may contribute to a better understanding of memory B cell development in the pig and provide clues to improve vaccination strategies.
