Advertisement

Stage 3 immature human natural killer cells found in secondary lymphoid tissue constitutively and selectively express the TH17 cytokine interleukin-22

Tiffany Hughes, Brian Becknell, Susan McClory, Edward Briercheck, Aharon G. Freud, Xiaoli Zhang, Hsiaoyin Mao, Gerard Nuovo, Jianhua Yu and Michael A. Caligiuri

Data supplements

  • Supplemental materials for: Hughes et al

    Files in this Data Supplement:

    • Figure S1. Immunohistochemical staining for IL-22 and CD117 in tissue sections from human tonsil (JPG, 62 KB) -
      Immunohistochemistry was performed as previously described1. Briefly, the Ultraview Universal system (DAB, CD117 and fast red, IL-22) was employed. The paraffin-embedded tonsillar tissue sections were digested in Protease 1 for 4 minutes and then incubated with the anti-human IL-22 (R&D systems, Minneapolis, MN) antibody (1:100). The slides were photographed at room temperature, the coverslip removed, and the slides then tested with the anti-human CD117 (1:1000, DakoCytomation, Glostrup, Denmark) after antigen retrieval for 30 minutes in CC1. Images were digitally acquired using the following equipment, all from Olympus (Olympus Imaging America Inc., Center Valley, PA): DP 12 camera, BX50 microscope, and UPLANF1 objectives at 400× magnification power. Images were analyzed using Adobe Photoshop CS3 software (Adobe Systems, Inc., San Jose, CA). The different colored arrows indicate the exact same cell staining for IL-22 (left) and co-labeling with IL-22 and CD117 (right) from serial sections of the same region. Cells with red staining are IL-22(+). Cells with brown staining are CD117(+). Staining indicated that CD117(+)IL-22(+) immature NK cells reside in the epithelia and lamina propria as well as the parafollicular T-cell�rich regions of SLT.





      REFERENCE:
      Blood

Article Figures & Data

  • Figure 1

    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 CD3CD19CD34 tonsillar mononuclear cells were stained for surface expression of lineage markers, CD117 and CD94, followed by assessment for intracellular expression of IL-22 protein. LinCD117+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). LinCD117CD94+ 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.

  • Figure 2

    Surface phenotype of IL-22+ stage 3 iNK cells. Total CD3CD19CD34 resting tonsillar mononuclear cells were stained for surface expression of lineage markers, CD117, CD94, followed by intracellular expression of IL-22, and events were gated on total LinCD117+CD94IL-22+ stage 3 iNK cells. (A) Representative histograms show expression for each indicated surface marker (shaded) in a donor, compared with isotype control (clear). (B) Graphic summary of the mean proportion of IL-22+ stage 3 iNK cells expressing various surface markers from all (n = 4) donors is summarized. Error bars represent standard error of the mean.

Supplementary Materials

  • Figure S1

    Supplementary PDF file available online.