IL-22 mRNA and protein expression during NK development. (A) (Left) Quantitative (Q) RT-PCR analysis of IL-22 expression was performed on fluorescence-activated cell sorting (FACS)–sorted NK stages 1 through 4 from human tonsil after pooling mRNA of each purified stage from 6 to 7 donors to achieve sufficient quantities for cDNA synthesis. Relative quantification was performed using the ΔΔCt method, and gene expression levels were normalized to 18S mRNA. Y-axis indicates fold increase over level of IL-22 mRNA quantified in stage 4 NK cells, arbitrarily normalized to 1. IL-22 was virtually absent from stages 1 and 2, so (right) subsequent RT-PCR measurements were performed using stage 3 iNK and stage 4 NK cells using cDNA from 7 individual donors. The average fold change in IL-22 mRNA present in stage 3 iNK cells compared with stage 4 NK cells is approximately 138. Error bars represent standard error of the mean from n = 7 donors. *P = .001. (B) IL-22 intracellular protein expression during NK development. Total CD3⁻CD19⁻CD34⁻ tonsillar mononuclear cells were stained for surface expression of lineage markers, CD117 and CD94, followed by assessment for intracellular expression of IL-22 protein. Lin⁻CD117⁺CD94⁻ identify stage 3 iNK cells that are then stained for intracellular expression of IL-22 (shaded) as shown in this representative donor, compared with isotype control (clear). Lin⁻CD117⁻CD94⁺ identify stage 4 NK cells that are then stained for intracellular expression of IL-22 (shaded) as shown in this representative donor, compared with isotype control (clear). (C) The average proportion of IL-22⁺ stage 3 iNK cells versus stage 4 NK cells in all donors examined (n = 6). Error bars represent standard error of the mean. *P = .001.