Ex vivo expansion of canine cytotoxic large granular lymphocytes exhibiting characteristics of natural killer cells

Abstract

Canine NK cells still are not well-characterized due to the lack of information concerning specific NK cell markers and the fact that NK cells are not an abundant cell population. In this study, we selectively expanded the canine cytotoxic large granular lymphocytes (CLGLs) that exhibit morphologic, genetic, and functional characteristics of NK cells from normal donor PBMCs. The cultured CLGLs were characterized by a high proportion of CD5(dim) expressing cells, of which the majority of cells co-expressed CD3 and CD8, but did not express TCRαβ and TCRγδ. The phenotype of the majority of the CLGLs was CD5(dim)CD3+CD8+ TCRαβ−TCRγδ−CD4−CD21−CD11c+/−CD11d+/−CD44+. The expression of mRNAs for NK cell-associated receptors (NKG2D, NKp30, NKp44, Ly49, perforin, and granzyme B) were highly upregulated in cultured CLGLs. Specifically, NKp46 was remarkably upregulated in the cultured CLGLs compared to PBMCs. The mRNAs for the NKT-associated iTCRα gene in CLGLs was present at a basal level. The cytotoxic activity of the CLGLs against canine NK cell-sensitive CTAC cells was remarkably elevated in a dose-dependent manner, and the CLGLs produced large amounts of IFN-γ. The antitumor activity of CLGLs extended to different types of canine tumor cells (CF41.Mg and K9TCC-pu-AXC) without specific antigen recognition. These results are consistent with prior reports, and strongly suggest that the selectively expanded CLGLs represent a population of canine NK cells. The results of this study will contribute to future research on canine NK cells as well as NK cell-based immunotherapy.

NCR1 Expression Identifies Canine Natural Killer Cell Subsets with Phenotypic Similarity to Human Natural Killer Cells

“…For human IL-2 comparison expansions, human IL-2 was added at 100 IU/mL in place of canine IL-2. The concentration of human IL-2 was based on the dose of IL-2 used in previous publications on canine NK-like cells (21, 22, 39). …”

The anti-canine distemper virus activities of ex vivo-expanded canine natural killer cells

“…Furthermore, studies seeking to correlate NK activities with resistance or susceptibility to CDV infections have yielded conflicting results (Ho and Babiuk, 1979;Ho et al, 1978;Ringler and Krakowka, 1985;Shek et al, 1980). Canine NK cells are not well-characterized, and this may explain the limited data gathered to date on correlations between the severity of CDV infection and NK cell activities, and among-study inconsistencies in results (Bonkobara et al, 2005(Bonkobara et al, , 2007Huang et al, 2008;Shin et al, 2013). No NK cell-restricted marker, or receptor, allowing canine NK cells to be distinguished from B and T cells, has yet been identified, and canine NK cells are simply defined as non-B, non-T, large granular lymphocytes (Michael et al, 2013).…”

“…Cells were stained as described previously (Shin et al, 2013). Briefly, fluorescence-activated cell sorting (FACS) analysis was performed with the aid of an FITC-labeled mAb against CD3, RPE-labeled mAbs against CD21, and an APC-labeled mAb against CD5 (Serotec, Oxford, UK).…”

“…For NKrelated genes, extraction of total RNAs, synthesis of firststrand cDNAs, and real-time PCR, were performed as described previously (Shin et al, 2013). To determine viral copy numbers, viral RNAs were extracted from cell-free culture supernatants of CDV-infected Vero cells using a viral RNA extraction kit (Bioneer, Daejeon, Korea), according to the manufacturer's instructions.…”

“…This cRNA was synthesized via in vitro transcription of a linearized pGEM T-Easy vector (Promega, Madison, WI) carrying the full-length CDV N gene, with the aid of T7 RNA Polymerase (TaKaRa, Tokyo, Japan). The thermal cycling conditions for quantitative PCR were 95 8C for 15 min, followed by 45 cycles of 94 8C for 30 s; 53 8C for 30 s; and 72 8C for 30 s. The sequences of the oligonucleotide primers used to amplify NK cell-associated genes have been described previously (Shin et al, 2013). The sequences of oligonucleotide primers for the CDV N gene amplified will be made available upon request.…”