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. 2010 May;24(5):1059-62.
doi: 10.1038/leu.2010.19. Epub 2010 Feb 18.

Identifying candidate allogeneic NK-cell donors for hematopoietic stem-cell transplantation based on functional phenotype

L V Hurton, R I Siddik, H Singh, S Olivares, B A Rabinovich, R Tian, D Mojsilovic, W Hildebrand, D A Lee, S S Kelly, R Champlin, E J Shpall, M Fernandez-Viña, L J N Cooper

Identifying candidate allogeneic NK-cell donors for hematopoietic stem-cell transplantation based on functional phenotype

L V Hurton et al. Leukemia. 2010 May.
No abstract available

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Figures

Figure 1
Figure 1
Schematic of alloreactivity generated between NK cells that are KIR-ligand mismatched with targets. HLA-B*5801, C*0702 and C*0401 alleles were amplified from cDNA using 5UTCE (5’GGG CGA ATTC GCC CGA GAT GCG GGT CAT GGC GCC CC 3’) and 3UTCH (5’CCG CAA GCT T TC GGG GAG GGA ACA CAG GTC AGT GTG GGG AC 3’) primers. The resulting PCR amplicon which encodes the full length classical HLA class I antigen was cloned into the mammalian expression vector pcDNA3.1- (Invitrogen). A clone was isolated and the fidelity of the HLA class I gene was confirmed by DNA sequencing. DNA for each class I allele was transfected by electroporation into the HLA class I deficient human B-cell line, 721.221. Antibiotic selection with G418 identified positive transfectants and class I expressing subclones were subsequently screened by flow cytometry using anti-HLA class I clone W6/32 as a primary antibody and PE-conjugated goat anti-mouse IgG as a secondary antibody. Following flow cytometric identification of a population of transfected cells expressing class I HLA, the HLA-expressing 721.221 cells were subjected to single-cell sorting to obtain stable transfectants of a clonal nature. Three stable/clonal HLA class I transfectants (B*5801, Cw*0702 and Cw*0401) were established by this approach. The B*5801 transfectant was produced in a similar manner in the laboratory of Dr. Jennifer Gumperz (University of Wisconsin, Madison, WI) and the other transfectants were produced in the laboratory of Dr. William Hildebrand (University of Oklahoma Health Sciences Center, Oklahoma City, OK).
Figure 2
Figure 2
Inhibition of NK-cell mediated lysis of HLA+ 721.221 transfectants, relative to the HLAneg 721.221 parental target. The star symbol indicates predicted inhibition based on presence/absence of KIR ligand in donor NK cells. Error bars are ± SD. The cytolytic activity of NK-cell populations from each donor was determined in a 4-hour chromium release assay (CRA) using 51Cr-labeled 721.221 parental target and a panel of three 721.221 transfectants expressing HLA-B*5801, HLA-Cw*0401, and HLA-Cw*0702. The isolated NK cells were washed and plated in 96-well V-bottom microtiter plates (Costar, Cambridge, MA) at 37°C for 4 hours in triplicate at 105/well with 5 × 103 target cells. After centrifugation and incubation, 100 μL aliquots of cell-free supernatant were transferred to LumiPlates, dried, and read on a Perkin Elmer Gamma Counter (Perkin Elmer, Waltham, MA). The percent specific cytolysis (SC) was calculated from the release of 51Cr as follows: [(CPSexperimental – CPScontrol)/(CPSmaximal – CPScontrol)]*100. Control wells contained target cells incubated in media. The maximal 51Cr was determined by measuring the 51Cr content released by target cells lysed with 2% SDS. Data are reported as an average ± standard deviation (SD). Percent relative inhibition (RI) to the 721.221 parental target was determined by: RI = 100 – (target cell SL/parental SL)*100.
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References

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    1. NCT00698009, http://clinicaltrials.gov

    1. NCT009419285, http://clinicaltrials.gov

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