NKp46 expression discriminates porcine NK cells with different functional properties

Abstract

So far little is known about natural killer (NK) cells in the pig due to the lack of NK cell-specific markers. In this study, we identified the activating receptor NKp46 (CD335) in swine with newly developed monoclonal antibodies (mAbs) for more detailed studies on NK cells in this species. The NKp46 mAbs showed a specific reactivity with a distinct population of perforin + CD2 + CD3 − CD8α + CD16 + lymphocytes. In spleen and liver, an additional subset of CD8α dim/− lymphocytes with increased NKp46 expression was observed. Surprisingly, we could identify NKp46 − cells with an NK cell phenotype in all animals analyzed. These lymphocytes showed comparable cytolytic activity against xenogeneic and allogeneic target cells as NKp46 + NK cells. In contrast, NKp46 + NK cells produced several fold higher levels of interferon-γ (IFN-γ) than the NKp46 − cells after cytokine stimulation. Furthermore, an activation-dependent induction of NKp46 expression in formerly NKp46 − cells after stimulation with interleukin-2 (IL-2), IL-12, and IL-18 could be shown. In summary, our data indicate that NKp46 is not expressed by all porcine NK cells and that NKp46 discriminates porcine NK cells differing in regard to cytokine production, which challenges the paradigm of NKp46 as a comprehensive marker for NK cells across different mammalian species.Keywords: NK cell · NKp46 · Swine Supporting Information available online IntroductionNatural killer (NK) cells are a highly specialized subpopulation of lymphocytes that play a crucial role in the early phase of immune responses. So far little is known about NK cells in swine. The phenotype of porcine NK cells was recently described as perforin + CD2 + CD3 − CD4 − CD5 − CD6 − CD8α + CD8ß − CD11b + CD16 + and it was observed that these cells exhibit natural cytotoxicity against NK-susceptible targets [1][2][3][4]. Likewise, the Correspondence: Dr. Armin Saalmüller e-mail: armin.saalmueller@vetmeduni.ac.at involvement of porcine NK cells has been reported in immune responses against parasitic and viral infections [5][6][7][8][9]. But since porcine NK cells share several phenotypic markers with other cell populations such as NKT cells, TCR-γδ T cells, and myeloid cells [3,4], the precise detection, isolation, and more detailed functional characterization of porcine NK cells has been thus far difficult. Thus, the identification of a discrete and unifying marker for this cell population in swine would be highly beneficial.NKp46 (CD335, NCR1), a member of the natural cytotoxicity receptor (NCR) family, is specifically expressed on NK cells, * These authors contributed equally to this work. , the triggering receptor NKp46 might also be a good candidate molecule for the specific identification of NK cells in swine. Porcine NKp46 was previously described as being encoded in the porcine leukocyte receptor complex on chromosome 6 [24]. The first expression analysis at the mRNA level revealed that the overall sequence and tissue distribution of porcine NKp46 was comparable with that of other species [2...

Early Responses of Natural Killer Cells in Pigs Experimentally Infected with 2009 Pandemic H1N1 Influenza A Virus

“…Cells were left on ice overnight for recovery from trypsin-treatment and incubated with different concentrations (40 µg, 20 µg, 10 µg and 5 µg) of a recombinant fusion protein containing the extracellular part of porcine NKp46 and the hinge and Fc part of murine IgG2b [22] for two hours on ice. In addition, cells were stained with anti-H1 mAbs (IgG1, clone C102, AbD Serotec).…”

“…NKp46 − NK cells show a similar ability to kill target cells but produce less IFN-γ as compared to NKp46 + NK cells. In liver and spleen, a third NK cell population with a higher NKp46 expression and a dim or negative CD8α expression has been described [22]. These CD8 −/dim NKp46 high NK cells appear to represent a more activated state [23].…”

“…NK cells in swine blood are identified as CD3 − CD8α + cells [21], and a recent study has found that only about half of the NK cell population in the blood of swine express NKp46 [22]. NKp46 − NK cells show a similar ability to kill target cells but produce less IFN-γ as compared to NKp46 + NK cells.…”

“…Swine are therefore proposed to serve as a valuable model for studying the pathogenesis of respiratory diseases, including influenza, as well as the immune response against it [1], [28]–[30]. They also present a unique model for studying the role of NK cells expressing the NKp46 receptor in vivo , since they have both NKp46 − and NKp46 + NK cells [22].…”

The Optimal Dose of Euphorbia milii Extracts in NKp46 Expression against Mice Infected with Mycobacterium tuberculosis

“…The content of flavonoids in Euphorbia milii flowers gives the possibility to act as an immunomodulator for NK cells. Increased expression of NKp46 of NK cell would increase IFN-γ levels many times higher after cytokine stimulated [12].…”