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...

Differentiation of porcine T helper cells is still poorly investigated, partly due to a lack of monoclonal antibodies (mAbs) specific for molecules involved in this process. Recently, we identified a mAb specific for porcine CD27 and showed that CD27 is expressed by all naïve CD8α- T helper cells but divides CD8α+ T helper cells into a CD27+ and a CD27- subset. In the present study, detailed phenotypical and functional analyses of these T-helper cell subpopulations were performed. Naïve CD8α-CD27+ T helper cells predominantly resided in various lymph nodes, whereas higher proportions of CD8α+CD27+ and CD8α+CD27- T helper cells were found in blood, spleen and liver. Both CD8α+CD27+ and CD8α+CD27- T helper cells were capable of producing IFN-γ upon in vitro polyclonal stimulation and antigen-specific restimulation. Experiments with sorted CD8α-CD27+, CD8α+CD27+ and CD8α+CD27- T-helper cell subsets following polyclonal stimulation revealed the lowest proliferative response but the highest ability for IFN-γ and TNF-α production in the CD8α+CD27- subset. Therefore, these cells resembled terminally differentiated effector memory cells as described in human. This was supported by analyses of CCR7 and CD62L expression. CD8α+CD27- T helper cells were mostly CCR7- and had considerably reduced CD62L mRNA levels. In contrast, expression of both homing-receptors was increased on CD8α+CD27+ T helper cells, which also had a proliferation rate similar to naïve CD8α-CD27+ T helper cells and showed intermediate levels of cytokine production. Therefore, similar to human, CD8α+CD27+ T helper cells displayed a phenotype and functional properties of central memory cells.

Natural Killer (NK) cells play a crucial role in the early phase of immune responses against various pathogens. In swine so far only little information about this lymphocyte population exists. Phenotypical analyses with newly developed monoclonal antibodies (mAbs) against porcine NKp46 recently revealed that in blood NKp46- and NKp46+ cells with NK phenotype exist with comparable cytotoxic properties. In spleen a third NKp46-defined population with NK phenotype was observed that was characterised by a low to negative CD8α and increased NKp46 expression. In the current study it is shown that this NKp46high phenotype was correlated with an increased expression of CD16 and CD27 compared to the CD8α+NKp46- and NKp46+ NK-cell subsets in spleen and blood. Additionally NKp46high NK cells expressed elevated levels of the chemokine receptor CXCR3 on mRNA level. Functional analyses revealed that splenic NKp46high NK cells produced much higher levels of Interferon-γ and Tumor Necrosis Factor-α upon stimulation with cytokines or phorbol-12-myristate-13-acetate/Ionomycin compared to the other two subsets. Furthermore, cross-linking of NKp46 by NKp46-specific mAbs led to a superior CD107a expression in the NKp46high NK cells, thus indicating a higher cytolytic capacity of this subset. Therefore porcine splenic NKp46high NK cells represent a highly activated subset of NK cells and may play a profound role in the immune surveillance of this organ.

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