Korean J Physiol Pharmacol.  2012 Jun;16(3):167-174. 10.4196/kjpp.2012.16.3.167.

Generation of CD2+CD8+ NK Cells from c-kit+ Bone Marrow Cells in Porcine

• Lim KH
• Han JH
• Roh YS
• Kim B
• Kwon JK
• You MJ
• Han HJ
• Ejaz S
• Kang CW
• Kim JH

• KMID: 1768008
• DOI: http://doi.org/10.4196/kjpp.2012.16.3.167

Abstract

Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. Because bone marrow-derived hematopoietic stem cells (HSCs), lymphoid protenitors, can give rise to NK cells, NK ontogeny has been considered to be exclusively lymphoid. Here, we show that porcine c-kit+ bone marrow cells (c-kit+ BM cells) develop into NK cells in vitro in the presence of various cytokines [interleukin (IL)-2, IL-7, IL-15, IL-21, stem cell factor (SCF), and fms-like tyrosine kinase-3 ligand (FLT3L)]. Adding hydrocortisone (HDC) and stromal cells greatly increases the frequency of c-kit+ BM cells that give rise to CD2+CD8+ NK cells. Also, intracellular levels of perforin, granzyme B, and NKG2D were determined by RT-PCR and western blotting analysis. It was found that of perforin, granzyme B, and NKG2D levels significantly were increased in cytokine-stimulated c-kit+ BM cells than those of controls. And, we compared the ability of the cytotoxicity of CD2+CD8+ NK cells differentiated by cytokines from c-kit+ BM cells against K562 target cells for 28 days. Cytokines-induced NK cells as effector cells were incubated with K562 cells as target in a ratio of 100:1 for 4 h once a week. In results, CD2+CD8+ NK cells induced by cytokines and stromal cells showed a significantly increased cytotoxicity 21 days later. Whereas, our results indicated that c-kit+ BM cells not pretreated with cytokines have lower levels of cytotoxicity. Taken together, this study suggests that cytokines-induced NK cells from porcine c-kit+ BM cells may be used as adoptive transfer therapy if the known obstacles to xenografting (e.g. immune and non-immune problems) were overcome in the future.

Keyword

Hematopoietic stem cells; Natural killer cells; Differentiation; Cytokines; Cytotoxicity

MeSH Terms

Adoptive Transfer
Blotting, Western
Bone Marrow
Bone Marrow Cells
Cytokines
Granzymes
Hematopoietic Stem Cells
Hydrocortisone
Interleukin-15
Interleukin-7
Interleukins
K562 Cells
Killer Cells, Natural
Perforin
Stem Cell Factor
Stromal Cells
Transplantation, Heterologous
Tyrosine
Cytokines
Granzymes
Hydrocortisone
Interleukin-15
Interleukin-7
Interleukins
Perforin
Stem Cell Factor
Tyrosine

Figure

• 

  Fig. 1 Phenotyping of porcine bone marrow derived c-kit+ cells. Porcine c-kit+ BM cells were isolated from bone marrow using a ficoll and magnetic activated cell sorter (MACS) and confirmed by fluorescence cell sorting (A). Histogram of isotype unstained control is shown (A: the part shown in dotted line area). Total number of CD117+ (c-kit+) BM cells is shown (A: the part shown in black area). Panel 1; Respective isotype control in unstained c-kit+ BM cells gate is shown. Panel 2; Frequency of CD117+ cells after staining with monoclonal antibodies CD117-FITC is shown in Q4 region: (Q4=98.9%). Porcine c-kit+ cell colony obtained from porcine BM cells was photographed in stem cell medium (B, Bar: 50 µm).

• 

  Fig. 2 Phenotypic analysis of CD2+CD8+ cells subpopulations in length of time for 28 days. There was a significant difference in expression of CD2 and CD8 molecules three weeks later from the treatment of cytokines compared to control (*p<0.01).

• 

  Fig. 3 The expression of perforin, granzyme B, and NKG2D mRNA in length of time. Normal control (N/C) is the cytokines-not treated cells. Before 7 days the treatment of cytokines, perforin and granzyme B mRNAs were not expressed and but 14 days later, these cells expressed perforin and granzyme B mRNA (A, B, *p<0.01). Whereas, NKG2D mRNA was expressed 21 days later (C, *p<0.01). Interestingly, perforin, granzyme B, and NKG2D mRNAs were not expressed within a week (A~C, p>0.05). All expression is presented as the fold relative to the N/C. Results are representative of five independent experiments.

• 

  Fig. 4 The analysis of perforin, granzyme B, and NKG2D protein in length of time. The expressions of perforin, granxyme B, and NKG2D proteins were determined by Western blotting. Densitometric analysis of each protein is shown. Within a week after the treatment of cytokines, perforin and granzyme B proteins were not expressed but two weeks later, these cells expressed perforin and granzyme B mRNAs (A, B, *p<0.01). Whereas, NKG2D mRNA was significantly expressed 21 days later treatment of cytokines (C, *p<0.01). All expression is presented as the fold relative to the N/C. Results are representative of five independent experiments.

• 

  Fig. 5 Cytotoxic activity of CD2+CD8+ NK cells compared with normal control. Cytokines-activated porcine CD2+CD8+ NK cells were incubated with K562 cells as target in a ratio of 100:1 for 4 h. Cytotoxicity was measured by 51Cr release assay for 18 h. Mean cytotoxicity percent of K562 cells are shown (sample number=5, *p<0.01).

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