Research paperCanine CD8 T cells showing NK cytotoxic activity express mRNAs for NK cell-associated surface molecules
Introduction
Natural killer (NK) cells are important effector cells involved in innate immune responses. They have been described on a functional basis as capable of killing tumors or virally infected cells without previous stimulation (Cerwenka and Lanier, 2001, Kiessling et al., 1976). The function of NK cells is regulated by a balance between signals transmitted via activating receptors, which recognize ligands on tumors and virus-infected cells, and signals transmitted via inhibitory receptors, which recognize major histocompatibility complex (MHC) class I molecules (Backstrom et al., 2004). Thus, NK cells preferentially kill target cells lacking, or with low levels of, surface MHC class I antigens, the so-called missing self-hypothesis (Ljunggren and Karre, 1990), which shifts the balance towards activation. Many types of inhibitory and activating NK cell receptors, such as KIR, NCRs, ILT/ILR, 2B4, CD94/NKG2, NKG2D, Ly49, and NKR-P1, have been identified in mammals (Lanier et al., 1998, McQueen and Parham, 2002, Sawicki et al., 2001), but there is only limited information on such receptors in the dog. In 1993, it was reported that CD18 and CD44 might be associated with canine NK cell activity (Ishiyama et al., 1993, Loughran et al., 1993). Pende et al. (1999) discovered that a canine genomic DNA segment was similar to that coding for human NKp30. The genes for the NK cell-related molecules Ly49 and CD56 have been reported in the dog (Bonkobara et al., 2005, Gagnier et al., 2003).
NK cells are large granular lymphocytes and represent about 5–15% of the lymphocyte population (Timonen et al., 1981). They are generally defined as a lymphocyte population lacking T or B cell-specific surface antigens and were designated as non-T non-B cells before NK cell-specific cell markers were well-defined. More recently, the phenotype of canine NK cells has been reported to be CD4− CD6− CD8− (Guenther et al., 1994, Knapp et al., 1995). Leukemic cells isolated from a dog which were able to kill CTAC cells (canine NK target cells) carried no CD4 or CD8 (Helfand et al., 1995). Our recent study showed that, after IL-2 stimulation of PBLs, strong NK activity is seen in the CD5lo cells, which also express CD8 and CD3 (Huang et al., 2008). This supports the notion that one of the canine NK cell subpopulations may be of the T cell lineage. In addition, CD8 has been shown to be expressed on a subpopulation of NK cells in other mammals. Human NK cells express CD8α (Moretta et al., 2002). In cattle, 4–15% of the NKp46+ cells express CD8 and the proportion of CD8+ cells increases after IL-2 culture (Storset et al., 2004). In swine, CD8+ cells show NK cytotoxicity (Yang and Parkhouse, 1997). CD8 T cells from rat lymph nodes or spleen almost exclusively express the alpha/beta isoform, while NK cells only carry the CD8α chain (Torres-Nagel et al., 1992).
In this study, we carried out an in-depth study on a canine CD8+ subpopulation in the peripheral blood that showed NK cytotoxic activity. We demonstrated that this subpopulation of cells expressed mostly TCR α/β and contained much higher levels of mRNAs for NKp30 and other NK cell-related molecules than the CD8− cells. When purified from IL-2 stimulated PBLs (LAKs), this subset showed significantly higher NK cell cytotoxicity than the corresponding purified CD8− cells. The phenotypic and morphologic characteristics of this subset were also studied.
Section snippets
Preparation of canine peripheral blood lymphocytes (PBLs)
Peripheral blood was obtained from six healthy 1- to 2-year-old beagles. All the dogs had been dewormed and vaccinated on a regular basis. All experiments were performed according to the University Animal Experimental Ethics Committee guidelines. Peripheral blood mononuclear cells (PBMCs) were purified from sterile heparinized whole blood as described previously (Hsiao et al., 2004). Briefly, the whole blood was layered onto Ficoll-Hypaque (density: 1.077) (Amersham Pharmacia Biotech,
Cytotoxicity of PBLs and LAKs
We first examined differences in the NK cytotoxic activity of PBLs and LAKs. As anticipated, LAKs were significantly more effective than PBLs in killing CTAC cells (p < 0.01) (Fig. 1). We then analyzed phenotypic subpopulations in the PBLs and LAKs. The percentage of non-T/non-B cells (CD3− CD21− or CD4− CD8− CD21− cells) was not higher in the LAKs cells than in the PBLs. Instead, the percentage (Fig. 2) of CD8+ cells were increased in the LAK cells (p < 0.01). This led us to further explore the
Discussion
Although canine NK cells have not been defined as CD8+ cells, the first study suggesting that canine NK cells express cell surface antigen of the T cell lineage was performed in 1985 (Loughran et al., 1985). These authors found that NK cytotoxic activity was decreased when canine PBMCs were treated with complement and a monoclonal antibody functionally equivalent to anti-human CD8+ antibody. In the present study, we provided several lines of evidence indicating that an IL-2-activated population
Acknowledgements
This research was supported by grants COA 97AS-1.2.1-AD-U1 from the Council of Agriculture, Taipei, Taiwan, the Republic of China.
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