Gene Expression Profiling of Natural Killer Cell Subsets Isolated Using Novel MACS Methods.

Natural Killer cells are now regarded as an important (Hokland, Mol Immunol 2005) but heterogenous (Cooper, Blood 2001) lymphocyte subset. Different Natural Killer cell subsets have different functions (Cooper, Blood 2001; Mavilio, PNAS 2005; Trotta, Blood 2005) and further research will be required to identify the role of those NK cell subsets in immune responses. We have developed novel methods, based on magnetic cell sorting, for purification of several NK cell subsets and have evaluated the separation performance of these kits using peripheral blood mononuclear cells from 10 donors. More than 1x10⁵ CD56^brightCD16⁻ cells from 1x10⁸ PBMC can be isolated with >70% purity (>85% CD56⁺CD16⁻), and more than 1x10⁶ CD56^dimCD16⁺ cells with >94% purity. Additional subsets have been identified based on CD8 expression: >2x10⁶ CD8⁺ NK cells can be purified with >90% purity.

Only limited data has previously been published on gene expression profiling of NK cell subsets using DNA microarrays (Koopman, J Exp Med 2003). We thus have used these MACS sorted NK cell subsets from 3 donors for an immune system specific microarray analysis (“PIQOR Immunology”). Usually gene expression profiling experiments are combined with flow cytometry characterization and functional tests. We used a novel amplification method to perform microarray analysis from mRNA isolated from as little as 1000 cells. Genes for surface molecules known to be differently expressed on NK cell subsets and immune effector molecules produced preferentially by single subsets served as control for the combination of amplification and microarray analysis (CD56, CD16, CD8, CD62L, CD122, Perforin, Granzyme B). Results of the analysis will be shown.